Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

PubMed Central

Burke, Russell T.; Orth, James D.

2016-01-01

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls. PMID:27213923

Cells respond to distinct nanoparticle properties with multiple strategies as revealed by single-cell RNA-Seq

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mitchell, Hugh D.; Markillie, Lye Meng; Chrisler, William B.

The impact of distinct nanoparticle (NP) properties on cellular response and ultimately human health is unclear. This gap is partially due to experimental difficulties in achieving uniform NP loads in the studied cells, creating heterogeneous populations with some cells “overloaded” while other cells are loaded with few or no NPs. Yet gene expression studies have been conducted in the population as a whole, identifying generic responses, while missing unique responses due to signal averaging across many cells, each carrying different loads. In this paper, we applied single-cell RNA-Seq to alveolar epithelial cells carrying defined loads of aminated or carboxylated quantummore » dots (QDs), showing higher or lower toxicity, respectively. Interestingly, cells carrying lower loads responded with multiple strategies, mostly with up-regulated processes, which were nonetheless coherent and unique to each QD type. In contrast, cells carrying higher loads responded more uniformly, with mostly down-regulated processes that were shared across QD types. Strategies unique to aminated QDs showed strong up-regulation of stress responses, coupled in some cases with regulation of cell cycle, protein synthesis, and organelle activities. In contrast, strategies unique to carboxylated QDs showed up-regulation of DNA repair and RNA activities and decreased regulation of cell division, coupled in some cases with up-regulation of stress responses and ATP-related functions. Finally, together, our studies suggest scenarios where higher NP loads lock cells into uniform responses, mostly shutdown of cellular processes, whereas lower loads allow for unique responses to each NP type that are more diversified proactive defenses or repairs of the NP insults.« less

Cells respond to distinct nanoparticle properties with multiple strategies as revealed by single-cell RNA-Seq

DOE PAGES

Mitchell, Hugh D.; Markillie, Lye Meng; Chrisler, William B.; ...

2016-10-27

The impact of distinct nanoparticle (NP) properties on cellular response and ultimately human health is unclear. This gap is partially due to experimental difficulties in achieving uniform NP loads in the studied cells, creating heterogeneous populations with some cells “overloaded” while other cells are loaded with few or no NPs. Yet gene expression studies have been conducted in the population as a whole, identifying generic responses, while missing unique responses due to signal averaging across many cells, each carrying different loads. In this paper, we applied single-cell RNA-Seq to alveolar epithelial cells carrying defined loads of aminated or carboxylated quantummore » dots (QDs), showing higher or lower toxicity, respectively. Interestingly, cells carrying lower loads responded with multiple strategies, mostly with up-regulated processes, which were nonetheless coherent and unique to each QD type. In contrast, cells carrying higher loads responded more uniformly, with mostly down-regulated processes that were shared across QD types. Strategies unique to aminated QDs showed strong up-regulation of stress responses, coupled in some cases with regulation of cell cycle, protein synthesis, and organelle activities. In contrast, strategies unique to carboxylated QDs showed up-regulation of DNA repair and RNA activities and decreased regulation of cell division, coupled in some cases with up-regulation of stress responses and ATP-related functions. Finally, together, our studies suggest scenarios where higher NP loads lock cells into uniform responses, mostly shutdown of cellular processes, whereas lower loads allow for unique responses to each NP type that are more diversified proactive defenses or repairs of the NP insults.« less

Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses.

PubMed

Pardi, Norbert; Hogan, Michael J; Naradikian, Martin S; Parkhouse, Kaela; Cain, Derek W; Jones, Letitia; Moody, M Anthony; Verkerke, Hans P; Myles, Arpita; Willis, Elinor; LaBranche, Celia C; Montefiori, David C; Lobby, Jenna L; Saunders, Kevin O; Liao, Hua-Xin; Korber, Bette T; Sutherland, Laura L; Scearce, Richard M; Hraber, Peter T; Tombácz, István; Muramatsu, Hiromi; Ni, Houping; Balikov, Daniel A; Li, Charles; Mui, Barbara L; Tam, Ying K; Krammer, Florian; Karikó, Katalin; Polacino, Patricia; Eisenlohr, Laurence C; Madden, Thomas D; Hope, Michael J; Lewis, Mark G; Lee, Kelly K; Hu, Shiu-Lok; Hensley, Scott E; Cancro, Michael P; Haynes, Barton F; Weissman, Drew

2018-06-04

T follicular helper (Tfh) cells are required to develop germinal center (GC) responses and drive immunoglobulin class switch, affinity maturation, and long-term B cell memory. In this study, we characterize a recently developed vaccine platform, nucleoside-modified, purified mRNA encapsulated in lipid nanoparticles (mRNA-LNPs), that induces high levels of Tfh and GC B cells. Intradermal vaccination with nucleoside-modified mRNA-LNPs encoding various viral surface antigens elicited polyfunctional, antigen-specific, CD4 + T cell responses and potent neutralizing antibody responses in mice and nonhuman primates. Importantly, the strong antigen-specific Tfh cell response and high numbers of GC B cells and plasma cells were associated with long-lived and high-affinity neutralizing antibodies and durable protection. Comparative studies demonstrated that nucleoside-modified mRNA-LNP vaccines outperformed adjuvanted protein and inactivated virus vaccines and pathogen infection. The incorporation of noninflammatory, modified nucleosides in the mRNA is required for the production of large amounts of antigen and for robust immune responses. © 2018 Pardi et al.

The Role of B Cells for in Vivo T Cell Responses to a Friend Virus-Induced Leukemia

NASA Astrophysics Data System (ADS)

Schultz, Kirk R.; Klarnet, Jay P.; Gieni, Randall S.; Hayglass, Kent T.; Greenberg, Philip D.

1990-08-01

B cells can function as antigen-presenting cells and accessory cells for T cell responses. This study evaluated the role of B cells in the induction of protective T cell immunity to a Friend murine leukemia virus (F-MuLV)-induced leukemia (FBL). B cell-deficient mice exhibited significantly reduced tumor-specific CD4^+ helper and CD8^+ cytotoxic T cell responses after priming with FBL or a recombinant vaccinia virus containing F-MuLV antigens. Moreover, these mice had diminished T cell responses to the vaccinia viral antigens. Tumor-primed T cells transferred into B cell-deficient mice effectively eradicated disseminated FBL. Thus, B cells appear necessary for efficient priming but not expression of tumor and viral T cell immunity.

Nano- and microstructured materials for in vitro studies of the physiology of vascular cells

PubMed Central

Chen, Hao; Biela, Sarah A; Kaufmann, Dieter

2016-01-01

The extracellular environment of vascular cells in vivo is complex in its chemical composition, physical properties, and architecture. Consequently, it has been a great challenge to study vascular cell responses in vitro, either to understand their interaction with their native environment or to investigate their interaction with artificial structures such as implant surfaces. New procedures and techniques from materials science to fabricate bio-scaffolds and surfaces have enabled novel studies of vascular cell responses under well-defined, controllable culture conditions. These advancements are paving the way for a deeper understanding of vascular cell biology and materials–cell interaction. Here, we review previous work focusing on the interaction of vascular smooth muscle cells (SMCs) and endothelial cells (ECs) with materials having micro- and nanostructured surfaces. We summarize fabrication techniques for surface topographies, materials, geometries, biochemical functionalization, and mechanical properties of such materials. Furthermore, various studies on vascular cell behavior and their biological responses to micro- and nanostructured surfaces are reviewed. Emphasis is given to studies of cell morphology and motility, cell proliferation, the cytoskeleton and cell-matrix adhesions, and signal transduction pathways of vascular cells. We finalize with a short outlook on potential interesting future studies. PMID:28144512

Comparative Transcriptome Profiling of an SV40-Transformed Human Fibroblast (MRC5CVI) and Its Untransformed Counterpart (MRC-5) in Response to UVB Irradiation

PubMed Central

Chang, Cheng-Wei; Chen, Chaang-Ray; Huang, Chao-Ying; Shu, Wun-Yi; Chiang, Chi-Shiun; Hong, Ji-Hong; Hsu, Ian C.

2013-01-01

Simian virus 40 (SV40) transforms cells through the suppression of tumor-suppressive responses by large T and small t antigens; studies on the effects of these two oncoproteins have greatly improved our knowledge of tumorigenesis. Large T antigen promotes cellular transformation by binding and inactivating p53 and pRb tumor suppressor proteins. Previous studies have shown that not all of the tumor-suppressive responses were inactivated in SV40-transformed cells; however, the underlying cause is not fully studied. In this study, we investigated the UVB-responsive transcriptome of an SV40-transformed fibroblast (MRC5CVI) and that of its untransformed counterpart (MRC-5). We found that, in response to UVB irradiation, MRC-5 and MRC5CVI commonly up-regulated the expression of oxidative phosphorylation genes. MRC-5 up-regulated the expressions of chromosome condensation, DNA repair, cell cycle arrest, and apoptotic genes, but MRC5CVI did not. Further cell death assays indicated that MRC5CVI was more sensitive than MRC-5 to UVB-induced cell death with increased caspase-3 activation; combining with the transcriptomic results suggested that MRC5CVI may undergo UVB-induced cell death through mechanisms other than transcriptional regulation. Our study provides a further understanding of the effects of SV40 transformation on cellular stress responses, and emphasizes the value of SV40-transformed cells in the researches of sensitizing neoplastic cells to radiations. PMID:24019915

Decipher the Molecular Response of Plant Single Cell Types to Environmental Stresses

DOE PAGES

Nourbakhsh-Rey, Mehrnoush; Libault, Marc

2016-01-01

The analysis of the molecular response of entire plants or organs to environmental stresses suffers from the cellular complexity of the samples used. Specifically, this cellular complexity masks cell-specific responses to environmental stresses and logically leads to the dilution of the molecular changes occurring in each cell type composing the tissue/organ/plant in response to the stress. Therefore, to generate a more accurate picture of these responses, scientists are focusing on plant single cell type approaches. Several cell types are now considered as models such as the pollen, the trichomes, the cotton fiber, various root cell types including the root hairmore » cell, and the guard cell of stomata. Among them, several have been used to characterize plant response to abiotic and biotic stresses. Lastly, in this review, we are describing the various -omic studies performed on these different plant single cell type models to better understand plant cell response to biotic and abiotic stresses.« less

Decipher the Molecular Response of Plant Single Cell Types to Environmental Stresses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nourbakhsh-Rey, Mehrnoush; Libault, Marc

The analysis of the molecular response of entire plants or organs to environmental stresses suffers from the cellular complexity of the samples used. Specifically, this cellular complexity masks cell-specific responses to environmental stresses and logically leads to the dilution of the molecular changes occurring in each cell type composing the tissue/organ/plant in response to the stress. Therefore, to generate a more accurate picture of these responses, scientists are focusing on plant single cell type approaches. Several cell types are now considered as models such as the pollen, the trichomes, the cotton fiber, various root cell types including the root hairmore » cell, and the guard cell of stomata. Among them, several have been used to characterize plant response to abiotic and biotic stresses. Lastly, in this review, we are describing the various -omic studies performed on these different plant single cell type models to better understand plant cell response to biotic and abiotic stresses.« less

Dual Modifications of α-Galactosylceramide Synergize to Promote Activation of Human Invariant Natural Killer T Cells and Stimulate Anti-tumor Immunity.

PubMed

Chennamadhavuni, Divya; Saavedra-Avila, Noemi Alejandra; Carreño, Leandro J; Guberman-Pfeffer, Matthew J; Arora, Pooja; Yongqing, Tang; Koay, Hui-Fern; Godfrey, Dale I; Keshipeddy, Santosh; Richardson, Stewart K; Sundararaj, Srinivasan; Lo, Jae Ho; Wen, Xiangshu; Gascón, José A; Yuan, Weiming; Rossjohn, Jamie; Le Nours, Jérôme; Porcelli, Steven A; Howell, Amy R

2018-05-17

Glycosylceramides that activate CD1d-restricted invariant natural killer T (iNKT) cells have potential therapeutic applications for augmenting immune responses against cancer and infections. Previous studies using mouse models identified sphinganine variants of α-galactosylceramide as promising iNKT cell activators that stimulate cytokine responses with a strongly proinflammatory bias. However, the activities of sphinganine variants in mice have generally not translated well to studies of human iNKT cell responses. Here, we show that strongly proinflammatory and anti-tumor iNKT cell responses were achieved in mice by a variant of α-galactosylceramide that combines a sphinganine base with a hydrocinnamoyl ester on C6″ of the sugar. Importantly, the activities observed with this variant were largely preserved for human iNKT cell responses. Structural and in silico modeling studies provided a mechanistic basis for these findings and suggested basic principles for capturing useful properties of sphinganine analogs of synthetic iNKT cell activators in the design of immunotherapeutic agents. Copyright © 2018 Elsevier Ltd. All rights reserved.

A systems analysis of the chemosensitivity of breast cancer cells to the polyamine analogue PG-11047

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuo, Wen-Lin; Das, Debopriya; Ziyad, Safiyyah

2009-11-14

Polyamines regulate important cellular functions and polyamine dysregulation frequently occurs in cancer. The objective of this study was to use a systems approach to study the relative effects of PG-11047, a polyamine analogue, across breast cancer cells derived from different patients and to identify genetic markers associated with differential cytotoxicity. A panel of 48 breast cell lines that mirror many transcriptional and genomic features present in primary human breast tumours were used to study the antiproliferative activity of PG-11047. Sensitive cell lines were further examined for cell cycle distribution and apoptotic response. Cell line responses, quantified by the GI50 (dosemore » required for 50% relative growth inhibition) were correlated with the omic profiles of the cell lines to identify markers that predict response and cellular functions associated with drug sensitivity. The concentrations of PG-11047 needed to inhibit growth of members of the panel of breast cell lines varied over a wide range, with basal-like cell lines being inhibited at lower concentrations than the luminal cell lines. Sensitive cell lines showed a significant decrease in S phase fraction at doses that produced little apoptosis. Correlation of the GI50 values with the omic profiles of the cell lines identified genomic, transcriptional and proteomic variables associated with response. A 13-gene transcriptional marker set was developed as a predictor of response to PG-11047 that warrants clinical evaluation. Analyses of the pathways, networks and genes associated with response to PG-11047 suggest that response may be influenced by interferon signaling and differential inhibition of aspects of motility and epithelial to mesenchymal transition.« less

Radiation induces an antitumour immune response to mouse melanoma.

PubMed

Perez, Carmen A; Fu, Allie; Onishko, Halina; Hallahan, Dennis E; Geng, Ling

2009-12-01

Irradiation of cancer cells can cause immunogenic death. We used mouse models to determine whether irradiation of melanoma can enhance the host antitumour immune response and function as an effective vaccination strategy, and investigated the molecular mechanisms involved in this radiation-induced response. For in vivo studies, C57BL6/J mice and the B16F0 melanoma cell line were used in a lung metastasis model, intratumoural host immune activation assays, and tumour growth delay studies. In vitro studies included a dendritic cell (DC) phagocytosis assay, detection of cell surface exposure of the protein calreticulin (CRT), and small interfering RNA (siRNA)-mediated depletion of CRT cellular levels. Irradiation of cutaneous melanomas prior to their resection resulted in more than 20-fold reduction in lung metastases after systemic challenge with untreated melanoma cells. A syngeneic vaccine derived from irradiated melanoma cells also induced adaptive immune response markers in irradiated melanoma implants. Our data indicate a trend for radiation-induced increase in melanoma cell surface exposure of CRT, which is involved in the enhanced phagocytic activity of DC against irradiated melanoma cells (VIACUC). The present study suggests that neoadjuvant irradiation of cutaneous melanoma tumours prior to surgical resection can stimulate an endogenous anti-melanoma host immune response.

Two photon microscopy intravital study of DC-mediated anti-tumor response of NK cells

NASA Astrophysics Data System (ADS)

Caccia, Michele; Gorletta, Tatiana; Sironi, Laura; Zanoni, Ivan; Salvetti, Cristina; Collini, Maddalena; Granucci, Francesca; Chirico, Giuseppe

2010-02-01

Recent studies have demonstrated that dendritic cells (DCs) play a crucial role in the activation of Natural Killer cells (NKs) that are responsible for anti-tumor innate immune responses. The focus of this report is on the role of pathogen associated molecular pattern (PAMP) activated-DCs in inducing NK cell-mediated anti-tumor responses. Mice transplanted sub-cute (s.c.) with AK7 cells, a mesothelioma cell line sensitive to NK cell responses, are injected with fluorescent NK cells and DC activation is then induced by s.c. injection of Lipopolysaccharide (LPS). Using 4 dimensional tracking we follow the kinetic behavior of NK cells at the Draining Lymph-Node (DLN). As control, noninflammatory conditions are also evaluated. Our data suggest that NK cells are recruited to the DLN where they can interact with activated-DCs with a peculiar kinetic behavior: short lived interactions interleaved by rarer longer ones. We also found that the changes in the NK dynamic behavior in inflammatory conditions clearly affect relevant motility parameters such as the instantaneous and average velocity and the effective diffusion coefficient. This observation suggests that NK cells and activated-DCs might efficiently interact in the DLN, where cells could be activated. Therefore the interaction between activated-DCs and NK cells in DLN is not only a reality but it may be also crucial for the start of the immune response of the NKs.

Suppressed plasmablast responses in febrile infants, including children with Kawasaki disease

PubMed Central

Martin, Meghan; Wrotniak, Brian H.

2018-01-01

Background Kawasaki disease (KD), the leading cause of acquired heart disease in children, primarily affects infants and toddlers. Investigations on immune responses during KD are hampered by a limited understanding of normal immune responses in these ages. It’s well known that Infants have poorer vaccine responses and difficulty with maintaining prolonged serum immunity, but there are few studies on human infants detailing immune deficiencies. Limited studies propose an inability to maintain life-long bone marrow plasma cells. Plasmablasts are a transitional cell form of B cells that lead to long-term Plasma cells. Plasmablasts levels rise in the peripheral blood after exposure to a foreign antigen. In adult studies, these responses are both temporally and functionally well characterized. To date, there have been few studies on plasmablasts in the predominant age range of KD. Methods Children presenting to an urban pediatric emergency room undergoing laboratory evaluation, who had concern of KD or had fever and symptoms overlapping those of KD, were recruited. Peripheral blood mononuclear cells were isolated and evaluated utilizing flow cytometry with specific B cell markers from 18 KD subjects and 69 febrile controls. Results Plasmablast numbers and temporal formation are similar between infectious disease controls and KD subjects. In both groups, infants have diminished plasmablast responses compared to older children. Conclusion In this single-time point survey, infants have a blunted peripheral plasmablast response. Overall, similar plasmablast responses in KD and controls support an infectious disease relationship to KD. Future time-course studies of plasmablasts in infants are warranted as this phenomenon may contribute to observed immune responses in this age group. PMID:29579044

Study of the Genes and Mechanism Involved in the Radioadaptive Response

NASA Technical Reports Server (NTRS)

Dasgupta, Pushan R.

2009-01-01

The radioadaptive response is a phenomenon where exposure to a prior low dose of radiation reduces the level of damage induced by a subsequent high radiation dose. The molecular mechanism behind this is still not well understood. Learning more about the radioadaptive response is critical for long duration spaceflight since astronauts are exposed to low levels of cosmic radiation. The micronucleus assay was used to measure the level of damage caused by radiation. Although cells which were not washed with phosphate buffered saline (PBS) after a low priming dose of 5cGy did not show adaptation to the challenge dose, washing the cells with PBS and giving the cells fresh media after the low dose did allow radioadaptation to occur. This is consistent with the results of a previous publication by another research group. In the present study, genes involved in DNA damage signaling and the oxidative stress response were studied using RT PCR techniques in order to look at changes in expression level after the low dose with or without washing. Our preliminary results indicate that upregulation of oxidative stress response genes ANGPTL7, NCF2, TTN, and SRXN1 may be involved in the radioadaptive response. The low dose of radiation alone was found to activate the oxidative stress response genes GPR156 and MTL5, whereas, washing the cells alone caused relatively robust upregulation of the oxidative stress response genes DUSP1 and PTGS2. Washing after the priming dose showed some changes in the expression level of several DNA damage signaling genes. In addition, we studied whether washing the cells after the priming dose has an effect on the level of nitric oxide in both the media and cells, since nitric oxide levels are known to increase in the media of the cells after a high dose of radiation only if the cells were already exposed to a low priming dose. Based on this preliminary study, we propose that washing the cells after priming exposure actually eliminates some factor secreted by the cells that inhibits radioadaptation leading to the upregulation of some genes which initiates the response.

Metabolic responses induced by DNA damage and poly (ADP-ribose) polymerase (PARP) inhibition in MCF-7 cells

PubMed Central

Bhute, Vijesh J.; Palecek, Sean P.

2015-01-01

Genomic instability is one of the hallmarks of cancer. Several chemotherapeutic drugs and radiotherapy induce DNA damage to prevent cancer cell replication. Cells in turn activate different DNA damage response (DDR) pathways to either repair the damage or induce cell death. These DDR pathways also elicit metabolic alterations which can play a significant role in the proper functioning of the cells. The understanding of these metabolic effects resulting from different types of DNA damage and repair mechanisms is currently lacking. In this study, we used NMR metabolomics to identify metabolic pathways which are altered in response to different DNA damaging agents. By comparing the metabolic responses in MCF-7 cells, we identified the activation of poly (ADP-ribose) polymerase (PARP) in methyl methanesulfonate (MMS)-induced DNA damage. PARP activation led to a significant depletion of NAD+. PARP inhibition using veliparib (ABT-888) was able to successfully restore the NAD+ levels in MMS-treated cells. In addition, double strand break induction by MMS and veliparib exhibited similar metabolic responses as zeocin, suggesting an application of metabolomics to classify the types of DNA damage responses. This prediction was validated by studying the metabolic responses elicited by radiation. Our findings indicate that cancer cell metabolic responses depend on the type of DNA damage responses and can also be used to classify the type of DNA damage. PMID:26478723

The Yin and Yang aspects of IL-27 in induction of cancer-specific T-cell responses and immunotherapy.

PubMed

Li, Ming-Song; Liu, Zhenzhen; Liu, Jin-Qing; Zhu, Xiaotong; Liu, Zhihao; Bai, Xue-Feng

2015-01-01

Accumulating evidences from animal studies have indicated that both endogenous and exogenous IL-27, an IL-12 family of cytokine, can increase antitumor T-cell activities and inhibit tumor growth. IL-27 can modulate Treg responses, and program effector T cells into a unique T-effector stem cell (TSEC) phenotype, which enhances T-cell survival in the tumor microenvironment. However, animal studies also suggest that IL-27 induces molecular pathways such as IL-10, PD-L1 and CD39, which may downregulate tumor-specific T-cell responses. In this review paper, we will discuss the Yin and Yang aspects of IL-27 in the induction of tumor-specific T-cell responses, and the potential impacts of these functions of IL-27 in the design of cancer immunotherapy.

T cell-independent and T cell-dependent immunoglobulin G responses to polyomavirus infection are impaired in complement receptor 2-deficient mice.

PubMed

Szomolanyi-Tsuda, Eva; Seedhom, Mina O; Carroll, Michael C; Garcea, Robert L

2006-08-15

Polyomavirus (PyV) infection induces protective T cell-independent (TI) IgM and IgG antibody responses in T cell-deficient mice, but these responses are not generated by immunization with viral proteins or virus like particles. We hypothesized that innate signals contribute to the generation of isotype-switched antiviral antibody responses. We studied the role of complement receptor (CR2) engagement in TI and T cell-dependent (TD) antibody responses to PyV using CR2-deficient mice. Antiviral IgG responses were reduced by 80-40% in CR2-/- mice compared to wild type. Adoptive transfer experiments demonstrated the need for CR2 not only in TD, but also in TI IgG responses to PyV. Transfer of CR2-/- B lymphocytes to SCID mice resulted in TI antiviral IgG responses that corresponded to 10% of that seen in wild-type B cell-reconstituted mice. Thus, our studies revealed a profound dependence of TI and TD antiviral antibody responses on CR2-mediated signals in PyV-infected mice, where the viral antigen is abundant and persistent.

Comprehensive longitudinal analysis of hepatitis C virus (HCV)-specific T cell responses during acute HCV infection in the presence of existing HIV-1 infection.

PubMed

van den Berg, C H S B; Ruys, T A; Nanlohy, N M; Geerlings, S E; van der Meer, J T; Mulder, J-W; Lange, J A; van Baarle, D

2009-04-01

The aim of this study was to study the development of HCV-specific T cell immunity during acute HCV infection in the presence of an existing HIV-1 infection in four HIV-1 infected men having sex with men. A comprehensive analysis of HCV-specific T cell responses was performed at two time points during acute HCV infection using a T cell expansion assay with overlapping peptide pools spanning the entire HCV genome Three patients with (near) normal CD4+ T cell counts (range 400-970 x 10(6)/L) either resolved (n=1) or temporary suppressed HCV RNA. In contrast, one patient with low CD4+ T cell counts (330 x 10(6)/L), had sustained high HCV RNA levels. All four patients had low HCV-specific CD8+ T cell responses, and similar magnitudes of CD4+ T cell responses. Interestingly, individuals with resolved infection or temporary suppression of HCV-RNA had HCV-specific CD4+ T cell responses predominantly against nonstructural (NS) proteins. While the individual with high HCV RNA plasma concentrations had CD4+ T cell responses predominantly directed against Core. Our data show that an acute HCV infection in an HIV-1 infected person can be suppressed in the presence of HCV-specific CD4+ T cell response targeting non-structural proteins. However further research is needed in a larger group of patients to evaluate the role of HIV-1 on HCV-specific T cell responses in relation to outcome of acute HCV infection.

Escape is a more common mechanism than avidity reduction for evasion of CD8+ T cell responses in primary human immunodeficiency virus type 1 infection

PubMed Central

2011-01-01

Background CD8+ T cells play an important role in control of viral replication during acute and early human immunodeficiency virus type 1 (HIV-1) infection, contributing to containment of the acute viral burst and establishment of the prognostically-important persisting viral load. Understanding mechanisms that impair CD8+ T cell-mediated control of HIV replication in primary infection is thus of importance. This study addressed the relative extent to which HIV-specific T cell responses are impacted by viral mutational escape versus reduction in response avidity during the first year of infection. Results 18 patients presenting with symptomatic primary HIV-1 infection, most of whom subsequently established moderate-high persisting viral loads, were studied. HIV-specific T cell responses were mapped in each individual and responses to a subset of optimally-defined CD8+ T cell epitopes were followed from acute infection onwards to determine whether they were escaped or declined in avidity over time. During the first year of infection, sequence variation occurred in/around 26/33 epitopes studied (79%). In 82% of cases of intra-epitopic sequence variation, the mutation was confirmed to confer escape, although T cell responses were subsequently expanded to variant sequences in some cases. In contrast, < 10% of responses to index sequence epitopes declined in functional avidity over the same time-frame, and a similar proportion of responses actually exhibited an increase in functional avidity during this period. Conclusions Escape appears to constitute a much more important means of viral evasion of CD8+ T cell responses in acute and early HIV infection than decline in functional avidity of epitope-specific T cells. These findings support the design of vaccines to elicit T cell responses that are difficult for the virus to escape. PMID:21635736

NK cells and poxvirus infection

PubMed Central

Burshtyn, Deborah N.

2013-01-01

In recent years, our understanding of the role of natural killer (NK) cells in the response to viral infection has grown rapidly. Not only do we realize viruses have many immune-evasion strategies to escape NK cell responses, but that stimulation of NK cell subsets during an antiviral response occurs through receptors seemingly geared directly at viral products and that NK cells can provide a memory response to viral pathogens. Tremendous knowledge has been gained in this area through the study of herpes viruses, but appreciation for the significance of NK cells in the response to other types of viral infections is growing. The function of NK cells in defense against poxviruses has emerged over several decades beginning with the early seminal studies showing the role of NK cells and the NK gene complex in susceptibility of mouse strains to ectromelia, a poxvirus pathogen of mice. More recently, greater understanding has emerged of the molecular details of the response. Given that human diseases caused by poxviruses can be as lethal as smallpox or as benign as Molluscum contagiosum, and that vaccinia virus, the prototypic member of the pox family, persists as a mainstay of vaccine design and has potential as an oncolytic virus for tumor therapy, further research in this area remains important. This review focuses on recent advances in understanding the role of NK cells in the immune response to poxviruses, the receptors involved in activation of NK cells during poxvirus infection, and the viral evasion strategies poxviruses employ to avoid the NK response. PMID:23372568

The in vitro mitogenic response to intact bacteria by murine B cells does not predict in vivo susceptibility to Salmonella typhimurium.

PubMed

Elkins, K; Metcalf, E S

1986-05-01

We are interested in developing in vitro culture systems that will permit immune responses to intact Salmonella typhimurium, since these systems would have certain advantages over in vivo infection models for the characterization of the host's responding cell types. In this report, the in vitro proliferative response of nonimmune murine spleen cells to four different killed preparations of S. typhimurium, strain TML (TML), are examined. These studies show that UV-killed TML, heat-killed TML, glutaraldehyde-killed TML, and acetone-killed and dried TML, all elicit a nonspecific mitogenic spleen cell response in vitro, as does a live, avirulent, temperature-sensitive mutant of TML, TS27. This response reaches a maximum on day 2 after initiation of culture, which is similar to the time course of a conventional lipopolysaccharide (LPS) response. Unlike the LPS response, little 3H-thymidine incorporation is observed in low-density cultures (2 X 10(5) cells/well), which suggests a critical role for accessory cells. The responding cell types include, but are not necessarily limited to, the B-cell population. The response cannot be readily inhibited by polymyxin B, which binds specifically to the lipid A portion of LPS. Thus, the bacterial components required for mitogenicity are not yet definitively identified. A survey of the mitogenic responses of lymphocytes from various inbred mouse strains, including the C3H/HeJ LPS hyporesponsive strain, indicates that all B cells tested are capable of proliferating vigorously in response to intact TML, regardless of the in vivo susceptibility to virulent infection. These results also emphasize the importance of assessing the nonspecific components of the immune response when studying the specific immune response to intact S. typhimurium.

The effect of incorporation of SDF-1alpha into PLGA scaffolds on stem cell recruitment and the inflammatory response.

PubMed

Thevenot, Paul T; Nair, Ashwin M; Shen, Jinhui; Lotfi, Parisa; Ko, Cheng-Yu; Tang, Liping

2010-05-01

Despite significant advances in the understanding of tissue responses to biomaterials, most implants are still plagued by inflammatory responses which can lead to fibrotic encapsulation. This is of dire consequence in tissue engineering, where seeded cells and bioactive components are separated from the native tissue, limiting the regenerative potential of the design. Additionally, these interactions prevent desired tissue integration and angiogenesis, preventing functionality of the design. Recent evidence supports that mesenchymal stem cells (MSC) and hematopoietic stem cells (HSC) can have beneficial effects which alter the inflammatory responses and improve healing. The purpose of this study was to examine whether stem cells could be targeted to the site of biomaterial implantation and whether increasing local stem cell responses could improve the tissue response to PLGA scaffold implants. Through incorporation of SDF-1alpha through factor adsorption and mini-osmotic pump delivery, the host-derived stem cell response can be improved resulting in 3X increase in stem cell populations at the interface for up to 2 weeks. These interactions were found to significantly alter the acute mast cell responses, reducing the number of mast cells and degranulated mast cells near the scaffold implants. This led to subsequent downstream reduction in the inflammatory cell responses, and through altered mast cell activation and stem cell participation, increased angiogenesis and decreased fibrotic responses to the scaffold implants. These results support that enhanced recruitment of autologous stem cells can improve the tissue responses to biomaterial implants through modifying/bypassing inflammatory cell responses and jumpstarting stem cell participation in healing at the implant interface. Copyright 2010 Elsevier Ltd. All rights reserved.

The Effect of Incorporation of SDF-1α into PLGA Scaffolds on Stem Cell Recruitment and the Inflammatory Response

PubMed Central

Thevenot, Paul; Nair, Ashwin; Shen, Jinhui; Lotfi, Parisa; Ko, Cheng Yu; Tang, Liping

2010-01-01

Despite significant advances in the understanding of tissue responses to biomaterials, most implants are still plagued by inflammatory responses which can lead to fibrotic encapsulation. This is of dire consequence in tissue engineering, where seeded cells and bioactive components are separated from the native tissue, limiting the regenerative potential of the design. Additionally, these interactions prevent desired tissue integration and angiogenesis, preventing functionality of the design. Recent evidence supports that mesenchymal stem cells (MSC) and hematopoietic stem cells (HSC) can have beneficial effects which alter the inflammatory responses and improve healing. The purpose of this study was to examine whether stem cells could be targeted to the site of biomaterial implantation and whether increasing local stem cell responses could improve the tissue response to PLGA scaffold implants. Through incorporation of SDF-1α through factor adsorption and mini-osmotic pump delivery, the host-derived stem cell response can be improved resulting in 3X increase in stem cell populations at the interface for up to 2 weeks. These interactions were found to significantly alter the acute mast cell responses, reducing the number of mast cells and degranulated mast cells near the scaffold implants. This led to subsequent downstream reduction in the inflammatory cell responses, and through altered mast cell activation and stem cell participation, increased angiogenesis and decreased fibrotic responses to the scaffold implants. These results support that enhanced recruitment of autologous stem cells can improve the tissue responses to biomaterial implants through modifying/bypassing inflammatory cell responses and jumpstarting stem cell participation in healing at the implant interface. PMID:20185171

Cellular injury evidenced by impedance technology and infrared microspectroscopy

NASA Astrophysics Data System (ADS)

le Roux, K.; Prinsloo, L. C.; Meyer, D.

2015-03-01

Fourier Transform Infrared (FTIR) spectroscopy is finding increasing biological application, for example in the analysis of diseased tissues and cells, cell cycle studies and investigating the mechanisms of action of anticancer drugs. Cancer treatment studies routinely define the types of cell-drug responses as either total cell destruction by the drug (all cells die), moderate damage (cell deterioration where some cells survive) or reversible cell cycle arrest (cytostasis). In this study the loss of viability and related chemical stress experienced by cells treated with the medicinal plant, Plectranthus ciliatus, was investigated using real time cell electronic sensing (RT-CES) technology and FTIR microspectroscopy. The use of plants as medicines is well established and ethnobotany has proven that crude extracts can serve as treatments against various ailments. The aim of this study was to determine whether FTIR microspectroscopy would successfully distinguish between different types of cellular injury induced by a potentially anticancerous plant extract. Cervical adenocarcinoma (HeLa) cells were treated with a crude extract of Pciliatus and cells monitored using RT-CES to characterize the type of cellular responses induced. Cell populations were then investigated using FTIR microspectroscopy and statistically analysed using One-way Analysis of Variance (ANOVA) and Principal Component Analysis (PCA). The plant extract and a cancer drug control (actinomycin D) induced concentration dependent cellular responses ranging from nontoxic, cytostatic or cytotoxic. Thirteen spectral peaks (915 cm-1, 933 cm-1, 989 cm-1, 1192 cm-1, 1369 cm-1, 1437 cm-1, 1450 cm-1, 1546 cm-1, 1634 cm-1, 1679 cm-1 1772 cm-1, 2874 cm-1 and 2962 cm-1) associated with cytotoxicity were significantly (p value < 0.05, one way ANOVA, Tukey test, Bonferroni) altered, while two of the bands were also indicative of early stress related responses. In PCA, poor separation between nontoxic and cytostatic responses was evident while clear separation was linked to cytotoxicity. RT-CES detected morphological changes as indicators of cell injury and could distinguish between viable, cytostatic and cytotoxic responses. FTIR microspectroscopy confirmed that cytostatic cells were viable and could still recover while also describing early cellular stress related responses on a molecular level.

A cell-based systems biology assessment of human blood to monitor immune responses after influenza vaccination.

PubMed

Hoek, Kristen L; Samir, Parimal; Howard, Leigh M; Niu, Xinnan; Prasad, Nripesh; Galassie, Allison; Liu, Qi; Allos, Tara M; Floyd, Kyle A; Guo, Yan; Shyr, Yu; Levy, Shawn E; Joyce, Sebastian; Edwards, Kathryn M; Link, Andrew J

2015-01-01

Systems biology is an approach to comprehensively study complex interactions within a biological system. Most published systems vaccinology studies have utilized whole blood or peripheral blood mononuclear cells (PBMC) to monitor the immune response after vaccination. Because human blood is comprised of multiple hematopoietic cell types, the potential for masking responses of under-represented cell populations is increased when analyzing whole blood or PBMC. To investigate the contribution of individual cell types to the immune response after vaccination, we established a rapid and efficient method to purify human T and B cells, natural killer (NK) cells, myeloid dendritic cells (mDC), monocytes, and neutrophils from fresh venous blood. Purified cells were fractionated and processed in a single day. RNA-Seq and quantitative shotgun proteomics were performed to determine expression profiles for each cell type prior to and after inactivated seasonal influenza vaccination. Our results show that transcriptomic and proteomic profiles generated from purified immune cells differ significantly from PBMC. Differential expression analysis for each immune cell type also shows unique transcriptomic and proteomic expression profiles as well as changing biological networks at early time points after vaccination. This cell type-specific information provides a more comprehensive approach to monitor vaccine responses.

T cell-derived Lymphotoxin is Essential for anti-HSV-1 Humoral Immune Response.

PubMed

Yang, Kaiting; Liang, Yong; Sun, Zhichen; Xue, Diyuan; Xu, Hairong; Zhu, Mingzhao; Fu, Yang-Xin; Peng, Hua

2018-05-09

B cell-derived lymphotoxin (LT) is required for the development of follicular dendritic cell clusters for the formation of primary and secondary lymphoid follicles, but the role of T cell-derived LT in antibody response has not been well demonstrated. We observed that lymphotoxin-β-receptor (LTβR) signaling is essential for optimal humoral immune response and protection against an acute HSV-1 infection. Blocking the LTβR pathway caused poor maintenance of germinal center B (GC-B) cells and follicular helper T (Tfh) cells. Using bone marrow chimeric mice and adoptive transplantation, we determined that T cell-derived LT played an indispensable role in the humoral immune response to HSV-1. Up-regulation of IFNγ by the LTβR-Ig blockade impairs the sustainability of Tfh-like cells, thus leading to an impaired humoral immune response. Our findings have identified a novel role of T cell-derived LT in the humoral immune response against HSV-1 infection. IMPORTANCE Immunocompromised people are susceptible for HSV-1 infection and lethal recurrence, which could be inhibited by anti-HSV-1 humoral immune response in the host. This study sought to explore the role of T cell-derived LT in the anti-HSV-1 humoral immune response using LT-LTβR signaling deficient mice and the LTβR-Ig blockade. The data indicate that the T cell-derived LT may play an essential role in sustaining Tfh-like cells and ensure Tfh-like cells' migration into primary or secondary follicles for further maturation. This study provides insights for vaccine development against infectious diseases. Copyright © 2018 American Society for Microbiology.

Sarcoptes scabiei Mites Modulate Gene Expression in Human Skin Equivalents

PubMed Central

Morgan, Marjorie S.; Arlian, Larry G.; Markey, Michael P.

2013-01-01

The ectoparasitic mite, Sarcoptes scabiei that burrows in the epidermis of mammalian skin has a long co-evolution with its hosts. Phenotypic studies show that the mites have the ability to modulate cytokine secretion and expression of cell adhesion molecules in cells of the skin and other cells of the innate and adaptive immune systems that may assist the mites to survive in the skin. The purpose of this study was to identify genes in keratinocytes and fibroblasts in human skin equivalents (HSEs) that changed expression in response to the burrowing of live scabies mites. Overall, of the more than 25,800 genes measured, 189 genes were up-regulated >2-fold in response to scabies mite burrowing while 152 genes were down-regulated to the same degree. HSEs differentially expressed large numbers of genes that were related to host protective responses including those involved in immune response, defense response, cytokine activity, taxis, response to other organisms, and cell adhesion. Genes for the expression of interleukin-1α (IL-1α) precursor, IL-1β, granulocyte/macrophage-colony stimulating factor (GM-CSF) precursor, and G-CSF precursor were up-regulated 2.8- to 7.4-fold, paralleling cytokine secretion profiles. A large number of genes involved in epithelium development and keratinization were also differentially expressed in response to live scabies mites. Thus, these skin cells are directly responding as expected in an inflammatory response to products of the mites and the disruption of the skin’s protective barrier caused by burrowing. This suggests that in vivo the interplay among these skin cells and other cell types, including Langerhans cells, dendritic cells, lymphocytes and endothelial cells, is responsible for depressing the host’s protective response allowing these mites to survive in the skin. PMID:23940705

Listeria Monocytogenes: A Model Pathogen Continues to Refine Our Knowledge of the CD8 T Cell Response.

PubMed

Qiu, Zhijuan; Khairallah, Camille; Sheridan, Brian S

2018-06-16

Listeria monocytogenes ( Lm ) infection induces robust CD8 T cell responses, which play a critical role in resolving Lm during primary infection and provide protective immunity to re-infections. Comprehensive studies have been conducted to delineate the CD8 T cell response after Lm infection. In this review, the generation of the CD8 T cell response to Lm infection will be discussed. The role of dendritic cell subsets in acquiring and presenting Lm antigens to CD8 T cells and the events that occur during T cell priming and activation will be addressed. CD8 T cell expansion, differentiation and contraction as well as the signals that regulate these processes during Lm infection will be explored. Finally, the formation of memory CD8 T cell subsets in the circulation and in the intestine will be analyzed. Recently, the study of CD8 T cell responses to Lm infection has begun to shift focus from the intravenous infection model to a natural oral infection model as the humanized mouse and murinized Lm have become readily available. Recent findings in the generation of CD8 T cell responses to oral infection using murinized Lm will be explored throughout the review. Finally, CD8 T cell-mediated protective immunity against Lm infection and the use of Lm as a vaccine vector for cancer immunotherapy will be highlighted. Overall, this review will provide detailed knowledge on the biology of CD8 T cell responses after Lm infection that may shed light on improving rational vaccine design.

Radiosensitivity of cancer-initiating cells and normal stem cells (or what the Heisenberg uncertainly principle has to do with biology).

PubMed

Woodward, Wendy Ann; Bristow, Robert Glen

2009-04-01

Mounting evidence suggests that parallels between normal stem cell biology and cancer biology may provide new targets for cancer therapy. Prospective identification and isolation of cancer-initiating cells from solid tumors has promoted the descriptive and functional identification of these cells allowing for characterization of their response to contemporary cancer therapies, including chemotherapy and radiation. In clinical radiation therapy, the failure to clinically eradicate all tumor cells (eg, a lack of response, partial response, or nonpermanent complete response by imaging) is considered a treatment failure. As such, biologists have explored the characteristics of the small population of clonogenic cancer cells that can survive and are capable of repopulating the tumor after subcurative therapy. Herein, we discuss the convergence of these clonogenic studies with contemporary radiosensitivity studies that use cell surface markers to identify cancer-initiating cells. Implications for and uncertainties regarding incorporation of these concepts into the practice of modern radiation oncology are discussed.

Anti-Donor Immune Responses Elicited by Allogeneic Mesenchymal Stem Cells and Their Extracellular Vesicles: Are We Still Learning?

PubMed Central

Lohan, Paul; Treacy, Oliver; Griffin, Matthew D.; Ritter, Thomas; Ryan, Aideen E.

2017-01-01

Mesenchymal stromal cells (MSC) have been used to treat a broad range of disease indications such as acute and chronic inflammatory disorders, autoimmune diseases, and transplant rejection due to their potent immunosuppressive/anti-inflammatory properties. The breadth of their usage is due in no small part to the vast quantity of published studies showing their ability to modulate multiple immune cell types of both the innate and adaptive immune response. While patient-derived (autologous) MSC may be the safer choice in terms of avoiding unwanted immune responses, factors including donor comorbidities may preclude these cells from use. In these situations, allogeneic MSC derived from genetically unrelated individuals must be used. While allogeneic MSC were initially believed to be immune-privileged, substantial evidence now exists to prove otherwise with multiple studies documenting specific cellular and humoral immune responses against donor antigens following administration of these cells. In this article, we will review recent published studies using non-manipulated, inflammatory molecule-activated (licensed) and differentiated allogeneic MSC, as well as MSC extracellular vesicles focusing on the immune responses to these cells and whether or not such responses have an impact on allogeneic MSC-mediated safety and efficacy. PMID:29225601

Emerging concepts in T follicular helper cell responses to malaria.

PubMed

Hansen, Diana S; Obeng-Adjei, Nyamekye; Ly, Ann; Ioannidis, Lisa J; Crompton, Peter D

2017-02-01

Antibody responses to malaria and candidate malaria vaccines are short-lived in children, leaving them susceptible to repeated malaria episodes. Because T follicular helper (T FH ) cells provide critical help to B cells to generate long-lived antibody responses, they have become the focus of recent studies of Plasmodium-infected mice and humans. The emerging data converge on common themes, namely, that malaria-induced T H1 cytokines are associated with the activation of (i) T-like memory T FH cells with impaired B cell helper function, and (ii) pre-T FH cells that acquire Th1-like features (T-bet expression, IFN-γ production), which impede their differentiation into fully functional T FH cells, thus resulting in germinal center dysfunction and suboptimal antibody responses. Deeper knowledge of T FH cells in malaria could illuminate strategies to improve vaccines through modulating T FH cell responses. This review summarizes emerging concepts in T FH cell responses to malaria. Copyright © 2016. Published by Elsevier Ltd.

Reproducible fashion of the HSP70B' promoter-induced cytotoxic response on a live cell-based biosensor by cell cycle synchronization.

PubMed

Migita, Satoshi; Wada, Ken-Ichi; Taniguchi, Akiyoshi

2010-10-15

Live cell-based sensors potentially provide functional information about the cytotoxic effect of reagents on various signaling cascades. Cells transfected with a reporter vector derived from a cytotoxic response promoter can be used as intelligent cytotoxicity sensors (i.e., sensor cells). We have combined sensor cells and a microfluidic cell culture system that can achieve several laminar flows, resulting in a reliable high-throughput cytotoxicity detection system. These sensor cells can also be applied to single cell arrays. However, it is difficult to detect a cellular response in a single cell array, due to the heterogeneous response of sensor cells. The objective of this study was cell homogenization with cell cycle synchronization to enhance the response of cell-based biosensors. Our previously established stable sensor cells were brought into cell cycle synchronization under serum-starved conditions and we then investigated the cadmium chloride-induced cytotoxic response at the single cell level. The GFP positive rate of synchronized cells was approximately twice as high as that of the control cells, suggesting that cell homogenization is an important step when using cell-based biosensors with microdevices, such as a single cell array. Copyright 2010 Wiley Periodicals, Inc.

Transcriptional Profiling Confirms the Therapeutic Effects of Mast Cell Stabilization in a Dengue Disease Model.

PubMed

Morrison, Juliet; Rathore, Abhay P S; Mantri, Chinmay K; Aman, Siti A B; Nishida, Andrew; St John, Ashley L

2017-09-15

There are no approved therapeutics for the treatment of dengue disease despite the global prevalence of dengue virus (DENV) and its mosquito vectors. DENV infections can lead to vascular complications, hemorrhage, and shock due to the ability of DENV to infect a variety of immune and nonimmune cell populations. Increasingly, studies have implicated the host response as a major contributor to severe disease. Inflammatory products of various cell types, including responding T cells, mast cells (MCs), and infected monocytes, can contribute to immune pathology. In this study, we show that the host response to DENV infection in immunocompetent mice recapitulates transcriptional changes that have been described in human studies. We found that DENV infection strongly induced metabolic dysregulation, complement signaling, and inflammation. DENV also affected the immune cell content of the spleen and liver, enhancing NK, NKT, and CD8 + T cell activation. The MC-stabilizing drug ketotifen reversed many of these responses without suppressing memory T cell formation and induced additional changes in the transcriptome and immune cell composition of the spleen, consistent with reduced inflammation. This study provides a global transcriptional map of immune activation in DENV target organs of an immunocompetent host and supports the further development of targeted immunomodulatory strategies to treat DENV disease. IMPORTANCE Dengue virus (DENV), which causes febrile illness, is transmitted by mosquito vectors throughout tropical and subtropical regions of the world. Symptoms of DENV infection involve damage to blood vessels and, in rare cases, hemorrhage and shock. Currently, there are no targeted therapies to treat DENV infection, but it is thought that drugs that target the host immune response may be effective in limiting symptoms that result from excessive inflammation. In this study, we measured the host transcriptional response to infection in multiple DENV target organs using a mouse model of disease. We found that DENV infection induced metabolic dysregulation and inflammatory responses and affected the immune cell content of the spleen and liver. The use of the mast cell stabilization drug ketotifen reversed many of these responses and induced additional changes in the transcriptome and immune cell repertoire that contribute to decreased dengue disease. Copyright © 2017 Morrison et al.

Effects of direct current electric fields on lung cancer cell electrotaxis in a PMMA-based microfluidic device.

PubMed

Li, Yaping; Xu, Tao; Chen, Xiaomei; Lin, Shin; Cho, Michael; Sun, Dong; Yang, Mengsu

2017-03-01

Tumor metastasis is the primary cause of cancer death. Numerous studies have demonstrated the electrotactic responses of various cancer cell types, and suggested its potential implications in metastasis. In this study, we used a microfluidic device to emulate endogenous direct current electric field (dcEF) environment, and studied the electrotactic migration of non-small cell lung cancer cell lines (H460, HCC827, H1299, and H1975) and the underlying mechanisms. These cell lines exhibited greatly different response in applied dcEFs (2-6 V/cm). While H460 cells (large cell carcinoma) showed slight migration toward cathode, H1299 cells (large cell carcinoma) showed increased motility and dcEF-dependent anodal migration with cell reorientation. H1975 cells (adenocarcinoma) showed dcEF-dependent cathodal migration with increased motility, and HCC827 cells (adenocarcinoma) responded positively in migration speed and reorientation but minimally in migrating directions to dcEF. Activation of MAPK and PI3K signaling pathways was found to be associated with the realignment and directed migration of lung cancer cells. In addition, both Ca 2+ influx through activated stretch-activated calcium channels (SACCs) (but not voltage-gated calcium channels, VGCCs) and Ca 2+ release from intracellular storage were involved in lung cancer cell electrotactic responses. The results demonstrated that the microfluidic device provided a stable and controllable microenvironment for cell electrotaxis study, and revealed that the electrotactic responses of lung cancer cells were heterogeneous and cell-type dependent, and multiple signals contributed to lung cancer cells electrotaxis.

Responses of single cells in cat visual cortex to prolonged stimulus movement: neural correlates of visual aftereffects.

PubMed

Vautin, R G; Berkley, M A

1977-09-01

1. The activity of single cortical cells in area 17 of anesthetized and unanesthetized cats was recorded in response to prolonged stimulation with moving stimuli. 2. Under the appropriate conditions, all cells observed showed a progressive response decrement during the stimulation period, regardless of cell classification, i.e., simple, complex, or hypercomplex. 3. The observed response decrement was shown to be largely cortical in origin and could be adequately described with an exponential function of the form R = Rf +(R1-Rf)e-t/T. Time constants derived from such calculations yielded values ranging from 1.92 to 12.45 s under conditions of optimal-stimulation. 4. Most cells showed poststimulation effects, usually a brief period of reduced responsiveness that recovered exponentially. Recovery was essentially complete in about 5-35 s. 5. The degree to which stimuli were effective at inducing response was shown to have significant effects on the magnitude of the response decrement. 6. Several cells showed neural patterns of response and recovery that suggested the operation of intracortical inhibitory mechanisms. 7. A simple two-process model that adequately describes the behavior of all the studied cells is presented. 8. Because the properties of the cells studied correlate well with human psychophysical measures of contour and movement adaptation and recovery, a causal relationship to similar neural mechanisms in humans is suggested.

Four-point bending protocols to study the effects of dynamic strain in osteoblastic cells in vitro.

PubMed

Galea, Gabriel L; Price, Joanna S

2015-01-01

Strain engendered within bone tissue by mechanical loading of the skeleton is a major influence on the processes of bone modeling and remodeling and so a critical determinant of bone mass and architecture. The cells best placed to respond to strain in bone tissue are the resident osteocytes and osteoblasts. To address the mechanisms of strain-related responses in osteoblast-like cells, our group uses both in vivo and in vitro approaches, including a system of four-point bending of the substrate on which cells are cultured. A range of cell lines can be studied using this system but we routinely compare their responses to those in primary cultures of osteoblast-like cells derived from explants of mouse long bones. These cells show a range of well-characterized responses to physiological levels of strain, including increased proliferation, which in vivo is a feature of the osteogenic response.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kateley, J.R.; Patel, C.B. Friedman, H.

The immune response at the level of individual immunocytes to the somatic lipopolysaccharide antigen derived from whole Vibrio cholerae and to the purified protein exotoxin from this organism were studied in terms of the role of T- and B-lymphocytes. By adoptive cell transfer studies with irradiated recipient mice, it was shown that normal spleen cells from normal syngeneic mice could readily transfer the capability of responding to both types of cholera antigens. However, when the spleen cells were depleted of T-cells with anti-theta serum and complement, antibody responsiveness to the LPS antigen, but not the exotoxin, could be achieved inmore » recipients. Furthermore, by appropriate transfer of either bone marrow, thymus, or thymus-marrow cell mixtures to irradiated mice, it was shown that the response to the cholera somatic antigen was relatively independent of thymus cells, whereas the response to exotoxin required ''helper'' T-cells.« less

Yellow fever vaccination elicits broad functional CD4+ T cell responses that recognize structural and nonstructural proteins.

PubMed

James, Eddie A; LaFond, Rebecca E; Gates, Theresa J; Mai, Duy T; Malhotra, Uma; Kwok, William W

2013-12-01

Yellow fever virus (YFV) can induce acute, life-threatening disease that is a significant health burden in areas where yellow fever is endemic, but it is preventable through vaccination. The live attenuated 17D YFV strain induces responses characterized by neutralizing antibodies and strong T cell responses. This vaccine provides an excellent model for studying human immunity. While several studies have characterized YFV-specific antibody and CD8(+) T cell responses, less is known about YFV-specific CD4(+) T cells. Here we characterize the epitope specificity, functional attributes, and dynamics of YFV-specific T cell responses in vaccinated subjects by investigating peripheral blood mononuclear cells by using HLA-DR tetramers. A total of 112 epitopes restricted by seven common HLA-DRB1 alleles were identified. Epitopes were present within all YFV proteins, but the capsid, envelope, NS2a, and NS3 proteins had the highest epitope density. Antibody blocking demonstrated that the majority of YFV-specific T cells were HLA-DR restricted. Therefore, CD4(+) T cell responses could be effectively characterized with HLA-DR tetramers. Ex vivo tetramer analysis revealed that YFV-specific T cells persisted at frequencies ranging from 0 to 100 cells per million that are detectable years after vaccination. Longitudinal analysis indicated that YFV-specific CD4(+) T cells reached peak frequencies, often exceeding 250 cells per million, approximately 2 weeks after vaccination. As frequencies subsequently declined, YFV-specific cells regained CCR7 expression, indicating a shift from effector to central memory. Cells were typically CXCR3 positive, suggesting Th1 polarization, and produced gamma interferon and other cytokines after reactivation in vitro. Therefore, YFV elicits robust early effector CD4(+) T cell responses that contract, forming a detectable memory population.

Yellow Fever Vaccination Elicits Broad Functional CD4+ T Cell Responses That Recognize Structural and Nonstructural Proteins

PubMed Central

James, Eddie A.; LaFond, Rebecca E.; Gates, Theresa J.; Mai, Duy T.; Malhotra, Uma

2013-01-01

Yellow fever virus (YFV) can induce acute, life-threatening disease that is a significant health burden in areas where yellow fever is endemic, but it is preventable through vaccination. The live attenuated 17D YFV strain induces responses characterized by neutralizing antibodies and strong T cell responses. This vaccine provides an excellent model for studying human immunity. While several studies have characterized YFV-specific antibody and CD8+ T cell responses, less is known about YFV-specific CD4+ T cells. Here we characterize the epitope specificity, functional attributes, and dynamics of YFV-specific T cell responses in vaccinated subjects by investigating peripheral blood mononuclear cells by using HLA-DR tetramers. A total of 112 epitopes restricted by seven common HLA-DRB1 alleles were identified. Epitopes were present within all YFV proteins, but the capsid, envelope, NS2a, and NS3 proteins had the highest epitope density. Antibody blocking demonstrated that the majority of YFV-specific T cells were HLA-DR restricted. Therefore, CD4+ T cell responses could be effectively characterized with HLA-DR tetramers. Ex vivo tetramer analysis revealed that YFV-specific T cells persisted at frequencies ranging from 0 to 100 cells per million that are detectable years after vaccination. Longitudinal analysis indicated that YFV-specific CD4+ T cells reached peak frequencies, often exceeding 250 cells per million, approximately 2 weeks after vaccination. As frequencies subsequently declined, YFV-specific cells regained CCR7 expression, indicating a shift from effector to central memory. Cells were typically CXCR3 positive, suggesting Th1 polarization, and produced gamma interferon and other cytokines after reactivation in vitro. Therefore, YFV elicits robust early effector CD4+ T cell responses that contract, forming a detectable memory population. PMID:24049183

Different responses of Fe transporters in Caco2/HT29-MTX cocultures than in independent Caco-2 cell cultures

USDA-ARS?s Scientific Manuscript database

The human intestinal epithelium is composed of several cell types; mainly enterocytes and globet (mucin-secreting) cells. This study compares the cellular response for Fe transporters in Caco-2, HT29-MTX, and Caco-2/HT29-MTX coculture models for Fe bioavailability studies. Under culture, Caco-2 cell...

Flow Cytometric and Cytokine ELISpot Approaches To Characterize the Cell-Mediated Immune Response in Ferrets following Influenza Virus Infection

PubMed Central

DiPiazza, Anthony; Richards, Katherine; Batarse, Frances; Lockard, Laura; Zeng, Hui; García-Sastre, Adolfo; Albrecht, Randy A.

2016-01-01

ABSTRACT Influenza virus infections represent a significant socioeconomic and public health burden worldwide. Although ferrets are considered by many to be ideal for modeling human responses to influenza infection and vaccination, efforts to understand the cellular immune response have been severely hampered by a paucity of standardized procedures and reagents. In this study, we developed flow cytometric and T cell enzyme-linked immunosorbent spot (ELISpot) approaches to characterize the leukocyte composition and antigen-specific T cell response within key lymphoid tissues following influenza virus infection in ferrets. Through a newly designed and implemented set of serological reagents, we used multiparameter flow cytometry to directly quantify the frequency of CD4+ and CD8+ T cells, Ig+ B cells, CD11b+ myeloid-derived cells, and major histocompatibility complex (MHC) class II-positive antigen-presenting cells (APCs) both prior to and after intranasal infection with A/California/04/09 (H1N1). We found that the leukocyte composition was altered at 10 days postinfection, with notable gains in the frequency of T cells and myeloid cells within the draining lymph node. Furthermore, these studies revealed that the antigen specificity of influenza virus-reactive CD4 and CD8 T cells was very broad, with recognition of the viral HA, NA, M1, NS1, and NP proteins, and that total reactivity to influenza virus postinfection represented approximately 0.1% of the circulating peripheral blood mononuclear cells (PBMC). Finally, we observed distinct patterns of reactivity between individual animals, suggesting heterogeneity at the MHC locus in ferrets within commercial populations, a finding of considerable interest in efforts to move the ferret model forward for influenza vaccine and challenge studies. IMPORTANCE Ferrets are an ideal animal model to study transmission, diseases, and vaccine efficacies of respiratory viruses because of their close anatomical and physiological resemblances to humans. However, a lack of reagents has limited our understanding of the cell-mediated immune response following infection and vaccination. In this study, we used cross-reactive and ferret-specific antibodies to study the leukocyte composition and antigen-specific CD4 and CD8 T cell responses following influenza A/California/04/09 (H1N1) virus infection. These studies revealed strikingly distinct patterns of reactivity between CD4 and CD8 T cells, which were overlaid with differences in protein-specific responses between individual animals. Our results provide a first, in-depth look at the T cell repertoire in response to influenza infection and suggest that there is considerable heterogeneity at the MHC locus, which is akin to that in humans and an area of intense research interest. PMID:27356897

Flow Cytometric and Cytokine ELISpot Approaches To Characterize the Cell-Mediated Immune Response in Ferrets following Influenza Virus Infection.

PubMed

DiPiazza, Anthony; Richards, Katherine; Batarse, Frances; Lockard, Laura; Zeng, Hui; García-Sastre, Adolfo; Albrecht, Randy A; Sant, Andrea J

2016-09-01

Influenza virus infections represent a significant socioeconomic and public health burden worldwide. Although ferrets are considered by many to be ideal for modeling human responses to influenza infection and vaccination, efforts to understand the cellular immune response have been severely hampered by a paucity of standardized procedures and reagents. In this study, we developed flow cytometric and T cell enzyme-linked immunosorbent spot (ELISpot) approaches to characterize the leukocyte composition and antigen-specific T cell response within key lymphoid tissues following influenza virus infection in ferrets. Through a newly designed and implemented set of serological reagents, we used multiparameter flow cytometry to directly quantify the frequency of CD4(+) and CD8(+) T cells, Ig(+) B cells, CD11b(+) myeloid-derived cells, and major histocompatibility complex (MHC) class II-positive antigen-presenting cells (APCs) both prior to and after intranasal infection with A/California/04/09 (H1N1). We found that the leukocyte composition was altered at 10 days postinfection, with notable gains in the frequency of T cells and myeloid cells within the draining lymph node. Furthermore, these studies revealed that the antigen specificity of influenza virus-reactive CD4 and CD8 T cells was very broad, with recognition of the viral HA, NA, M1, NS1, and NP proteins, and that total reactivity to influenza virus postinfection represented approximately 0.1% of the circulating peripheral blood mononuclear cells (PBMC). Finally, we observed distinct patterns of reactivity between individual animals, suggesting heterogeneity at the MHC locus in ferrets within commercial populations, a finding of considerable interest in efforts to move the ferret model forward for influenza vaccine and challenge studies. Ferrets are an ideal animal model to study transmission, diseases, and vaccine efficacies of respiratory viruses because of their close anatomical and physiological resemblances to humans. However, a lack of reagents has limited our understanding of the cell-mediated immune response following infection and vaccination. In this study, we used cross-reactive and ferret-specific antibodies to study the leukocyte composition and antigen-specific CD4 and CD8 T cell responses following influenza A/California/04/09 (H1N1) virus infection. These studies revealed strikingly distinct patterns of reactivity between CD4 and CD8 T cells, which were overlaid with differences in protein-specific responses between individual animals. Our results provide a first, in-depth look at the T cell repertoire in response to influenza infection and suggest that there is considerable heterogeneity at the MHC locus, which is akin to that in humans and an area of intense research interest. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Proteomic profiling reveals DNA damage, nucleolar and ribosomal stress are the main responses to oxaliplatin treatment in cancer cells.

PubMed

Ozdian, Tomas; Holub, Dusan; Maceckova, Zuzana; Varanasi, Lakshman; Rylova, Gabriela; Rehulka, Jiri; Vaclavkova, Jana; Slavik, Hanus; Moudry, Pavel; Znojek, Pawel; Stankova, Jarmila; de Sanctis, Juan Bautista; Hajduch, Marian; Dzubak, Petr

2017-06-06

Oxaliplatin is widely used to treat colorectal cancer in both palliative and adjuvant settings. It is also being tested for use in treating hematological, esophageal, biliary tract, pancreatic, gastric, and hepatocellular cancers. Despite its routine clinical use, little is known about the responses it induces in cancer cells. Therefore the whole-cell proteomics study was conducted to characterize the cellular response induced by oxaliplatin. Chemosensitive CCRF-CEM cells were treated with oxaliplatin at 29.3μM (5×IC 50 ) for 240min (half-time to caspase activation). The proteomes of un-/treated cells were then compared by high-resolution mass spectrometry, revealing 4049 proteins expressed over 3 biological replicates. Among these proteins, 76 were significantly downregulated and 31 significantly upregulated in at least two replicates. In agreement with the DNA-damaging effects of platinum drugs, proteins involved in DNA damage responses were present in both the upregulated and downregulated groups. The downregulated proteins were divided into three subgroups; i) centrosomal proteins, ii) RNA processing and iii) ribosomal proteins, which indicates nucleolar and ribosomal stress. In conclusion, our data supported by further validation experiments indicate the initial cellular response to oxaliplatin is the activation of DNA damage response, which in turn or in parallel triggers nucleolar and ribosomal stress. We have performed a whole-cell proteomic study of cellular response to oxaliplatin treatment, which is the drug predominantly used in the treatment of colorectal cancer. Compared to its predecessors, cisplatin and carboplatin, there is only a small fraction of studies dedicated to oxaliplatin. From those studies, most of them are focused on modification of treatment regimens or study of oxaliplatin in new cancer diagnoses. Cellular response hasn't been studied deeply and to our best knowledge, this is the first whole-cell proteomics study focused exclusively to this important topic, which can help to understand molecular mechanisms of action. Copyright © 2017 Elsevier B.V. All rights reserved.

Activation requirements and responses to TLR ligands in human CD4+ T cells: comparison of two T cell isolation techniques.

PubMed

Lancioni, Christina L; Thomas, Jeremy J; Rojas, Roxana E

2009-05-15

Direct regulation of T cell function by microbial ligands through Toll-like receptors (TLR) is an emerging area of T cell biology. Currently either immunomagnetic cell sorting (IMACS) or fluorescence-activated cell sorting (FACS), are utilized to isolate T-cell subsets for such studies. However, it is unknown to what extent differences in T cell purity between these isolation techniques influence T cell functional assays. We compared the purity, response to mitogen, activation requirements, and response to TLR ligands between human CD4(+) T cells isolated either by IMACS (IMACS-CD4(+)) or by IMACS followed by FACS (IMACS/FACS-CD4(+)). As expected, IMACS-CD4(+) were less pure than IMACS/FACS-CD4(+) (92.5%+/-1.4% versus 99.7%+/-0.2%, respectively). Consequently, IMACS-CD4(+) proliferated and produced cytokines in response to mitogen alone and had lower activation requirements compared to IMACS/FACS-CD4(+). In addition IMACS-CD4(+) but not IMACS/FACS-CD4(+) responses were upregulated by the TLR-4 ligand lipopolysaccharide (LPS). On the other hand, TLR-2 and TLR-5 engagement induced costimulation in both IMACS-CD4(+) and highly purified IMACS-/FACS-CD4(+). Altogether these results indicate that small differences in cell purity can significantly alter T cell responses to TLR ligands. This study stresses the importance of a stringent purification method when investigating the role of microbial ligands in T cell function.

Preserving HIV-specific T cell responses: does timing of antiretroviral therapy help?

PubMed

Macatangay, Bernard J C; Rinaldo, Charles R

2015-01-01

HIV-specific T cell responses are likely to have an important role in HIV cure strategies that aim for long-lasting viral control without antiretroviral therapy (ART). An important issue in enhancing virus-specific T cell responses is whether timing of ART can influence their magnitude and breadth. Early ART is associated with lower T cell activation, preservation of T cell numbers, smaller DNA and RNA reservoir size, and, in a single study (VISCONTI), control of plasma viremia after treatment interruption. The prevention of T cell destruction by early ART is associated with relatively low anti-HIV CD8⁺ T cell responses but stronger CD4⁺ T helper function. The relatively lower CD8⁺T cell response, which is presumably due to rapid lowering of HIV antigen burden after early ART, appears sufficient to control residual viral replication as well as viral rebound upon treatment interruption. Available evidence of starting ART during acute or early HIV infection has shown benefit in both virologic and immunologic parameters despite the lower HIV-specific CD8⁺ T cell responses observed. Encouraging as this is, more extensive data are necessary to evaluate its role in combination with immunotherapeutic and latency activation strategies that are being assessed in various HIV cure-related studies.

HIV-1 Antibody Neutralization Breadth Is Associated with Enhanced HIV-Specific CD4+ T Cell Responses

PubMed Central

Soghoian, Damien Z.; Lindqvist, Madelene; Ghebremichael, Musie; Donaghey, Faith; Carrington, Mary; Seaman, Michael S.; Kaufmann, Daniel E.; Walker, Bruce D.

2015-01-01

ABSTRACT Antigen-specific CD4+ T helper cell responses have long been recognized to be a critical component of effective vaccine immunity. CD4+ T cells are necessary to generate and maintain humoral immune responses by providing help to antigen-specific B cells for the production of antibodies. In HIV infection, CD4+ T cells are thought to be necessary for the induction of Env-specific broadly neutralizing antibodies. However, few studies have investigated the role of HIV-specific CD4+ T cells in association with HIV neutralizing antibody activity in vaccination or natural infection settings. Here, we conducted a comprehensive analysis of HIV-specific CD4+ T cell responses in a cohort of 34 untreated HIV-infected controllers matched for viral load, with and without neutralizing antibody breadth to a panel of viral strains. Our results show that the breadth and magnitude of Gag-specific CD4+ T cell responses were significantly higher in individuals with neutralizing antibodies than in those without neutralizing antibodies. The breadth of Gag-specific CD4+ T cell responses was positively correlated with the breadth of neutralizing antibody activity. Furthermore, the breadth and magnitude of gp41-specific, but not gp120-specific, CD4+ T cell responses were significantly elevated in individuals with neutralizing antibodies. Together, these data suggest that robust Gag-specific CD4+ T cells and, to a lesser extent, gp41-specific CD4+ T cells may provide important intermolecular help to Env-specific B cells that promote the generation or maintenance of Env-specific neutralizing antibodies. IMPORTANCE One of the earliest discoveries related to CD4+ T cell function was their provision of help to B cells in the development of antibody responses. Yet little is known about the role of CD4+ T helper responses in the setting of HIV infection, and no studies to date have evaluated the impact of HIV-specific CD4+ T cells on the generation of antibodies that can neutralize multiple different strains of HIV. Here, we addressed this question by analyzing HIV-specific CD4+ T cell responses in untreated HIV-infected persons with and without neutralizing antibodies. Our results indicate that HIV-infected persons with neutralizing antibodies have significantly more robust CD4+ T cell responses targeting Gag and gp41 proteins than individuals who lack neutralizing antibodies. These associations suggest that Gag- and gp41-specific CD4+ T cell responses may provide robust help to B cells for the generation or maintenance of neutralizing antibodies in natural HIV-infection. PMID:26656715

HLA-DR4-associated T and B cell responses to specific determinants on the IA-2 autoantigen in type 1 diabetes.

PubMed

McLaughlin, Kerry A; Gulati, Kavita; Richardson, Carolyn C; Morgan, Diana; Bodansky, H Jonathan; Feltbower, Richard G; Christie, Michael R

2014-11-01

Autoantibodies to IA-2 in type 1 diabetes are associated with HLA-DR4, suggesting influences of HLA-DR4-restricted T cells on IA-2-specific B cell responses. The aim of this study was to investigate possible T-B cell collaboration by determining whether autoantibodies to IA-2 epitopes are associated with T cell responses to IA-2 peptides presented by DR4. T cells secreting the cytokines IFN-γ and IL-10 in response to seven peptides known to elicit T cell responses in type 1 diabetes were quantified by cytokine ELISPOT in HLA-typed patients characterized for Abs to IA-2 epitopes. T cell responses were detected to all peptides tested, but only IL-10 responses to 841-860 and 853-872 peptides were associated with DR4. Phenotyping by RT-PCR of FACS-sorted CD45RO(hi) T cells secreting IL-10 in response to these two peptides indicated that these expressed GATA-3 or T-bet, but not FOXP3, consistent with these being Th2 or Th1 memory T cells rather than of regulatory phenotype. T cell responses to the same two peptides were also associated with specific Abs: those to 841-860 peptide with Abs to juxtamembrane epitopes, which appear early in prediabetes, and those to peptide 853-872 with Abs to an epitope located in the 831-862 central region of the IA-2 tyrosine phosphatase domain. Abs to juxtamembrane and central region constructs were both DR4 associated. This study identifies a region of focus for B and T cell responses to IA-2 in HLA-DR4 diabetic patients that may explain HLA associations of IA-2 autoantibodies, and this region may provide a target for future immune intervention to prevent disease. Copyright © 2014 by The American Association of Immunologists, Inc.

The frequencies of IFNγ+IL2+TNFα+ PPD-specific CD4+CD45RO+ T-cells correlate with the magnitude of the QuantiFERON® gold in-tube response in a prospective study of healthy indian adolescents.

PubMed

Jenum, Synne; Grewal, Harleen M S; Hokey, David A; Kenneth, John; Vaz, Mario; Doherty, Timothy Mark; Jahnsen, Frode Lars

2014-01-01

QuantiFERON-TB Gold In-Tube (QFT) is an IFNγ-release assay used in the diagnosis of Mycobacterium tuberculosis (MTB) infection. The risk of TB progression increases with the magnitude of the MTB-specific IFNγ-response. QFT reversion, also associated with low Tuberculin Skin Test responses, may therefore represent a transient immune response with control of M. tuberculosis infection. However, studies at the single cell level have suggested that the quality (polyfunctionality) of the T-cell response is more important than the quantity of cytokines produced. To explore the quality and/or magnitude of mycobacteria-specific T-cell responses associated with QFT reversion and persistent QFT-positivity. Multi-color flowcytometry on prospectively collected peripheral blood mononuclear cells was applied to assess mycobacteria-specific T-cell responses in 42 QFT positive Indian adolescents of whom 21 became QFT negative (reverters) within one year. Ten QFT consistent negatives were also included as controls. There was no difference in the qualitative PPD-specific CD4+ T-cell response between QFT consistent positives and reverters. However, compared with QFT consistent positives, reverters displayed lower absolute frequencies of polyfunctional (IFNγ+IL2+TNFα+) CD4+ T-cells at baseline, which were further reduced to the point where they were not different to QFT negative controls one year later. Moreover, absolute frequencies of these cells correlated well with the magnitude of the QFT-response. Whereas specific polyfunctional CD4+ T-cells have been suggested to protect against TB progression, our data do not support that higher relative or absolute frequencies of PPD-specific polyfunctional CD4+ T-cells in peripheral blood can explain the reduced risk of TB progression observed in QFT reverters. On the contrary, absolute frequencies of these cells correlated with the QFT-response, suggesting that this readout reflects antigenic load.

CD4(+) T-cell responses to Epstein-Barr virus (EBV) latent membrane protein 1 in infectious mononucleosis and EBV-associated non-Hodgkin lymphoma: Th1 in active disease but Tr1 in remission.

PubMed

Marshall, Neil A; Culligan, Dominic J; Johnston, Peter W; Millar, Colin; Barker, Robert N; Vickers, Mark A

2007-10-01

Primary infection with Epstein-Barr virus (EBV) in childhood is usually asymptomatic, whereas infection in adolescence may result in infectious mononucleosis (IM) often followed by a fatigue syndrome. EBV latent membrane protein 1 (LMP1) is expressed in latency and in many EBV-associated tumours, including non-Hodgkin lymphoma (NHL). Given the regulatory nature of the CD4(+) T-cell response against LMP1 previously reported in healthy donors, we investigated whether patients with active EBV-driven disease can nevertheless mount effector [T-helper cell, type 1 (Th1)] anti-LMP1 responses. We therefore performed a longitudinal study of the nature of CD4(+) T-cell responses to LMP1 in four patients with IM, and five patients with NHL. In both groups, responses changed with time. During symptomatic infection or active tumour growth, responses were dominated by a Th1 effector phenotype, but switched to a regulatory interleukin-10 response upon recovery. In addition, the fine specificities of the T cells driving these responses evolved. This study showed the dynamic nature of CD4(+) T-cell responses to LMP1, and demonstrated that, although patients can mount Th1 effector responses, recovery from IM and NHL is associated with regulatory responses.

Improved Academic Performance and Student Perceptions of Learning through Use of a Cell Phone-Based Personal Response System

ERIC Educational Resources Information Center

Ma, Sihui; Steger, Daniel G.; Doolittle, Peter E.; Stewart, Amanda C.

2018-01-01

Personal response systems, such as clickers, have been widely used to improve the effectiveness of teaching in various classroom settings. Although hand-held clicker response systems have been the subject of multiple prior studies, few studies have focused on the use of cell phone-based personal response system (CPPRS) specifically. This study…

High hydrostatic pressure and the cell membrane: stress response of Saccharomyces cerevisiae.

PubMed

Bravim, Fernanda; de Freitas, Jéssica M; Fernandes, A Alberto R; Fernandes, Patricia M B

2010-02-01

The brewing and baking yeast Saccharomyces cerevisiae is a useful eukaryotic model of stress response systems whose study could lead to the understanding of stress response mechanisms in other organisms. High hydrostatic pressure (HHP) exerts broad effects upon yeast cells, interfering with cell membranes, cellular architecture, and the processes of polymerization and denaturation of proteins. In this review, we focus on the effect of HHP on the S. cerevisiae cell membrane and describe the main signaling pathways involved in the pressure response.

Pancreatic Endoderm-Derived From Diabetic Patient-Specific Induced Pluripotent Stem Cell Generates Glucose-Responsive Insulin-Secreting Cells.

PubMed

Rajaei, Bahareh; Shamsara, Mehdi; Amirabad, Leila Mohammadi; Massumi, Mohammad; Sanati, Mohammad Hossein

2017-10-01

Human-induced pluripotent stem cells (hiPSCs) can potentially serve as an invaluable source for cell replacement therapy and allow the creation of patient- and disease-specific stem cells without the controversial use of embryos and avoids any immunological incompatibility. The generation of insulin-producing pancreatic β-cells from pluripotent stem cells in vitro provides an unprecedented cell source for personal drug discovery and cell transplantation therapy in diabetes. A new five-step protocol was introduced in this study, effectively induced hiPSCs to differentiate into glucose-responsive insulin-producing cells. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, primitive gut-tube endoderm, posterior foregut, pancreatic endoderm, and endocrine precursor. Each stage of differentiation were characterized by stage-specific markers. The produced cells exhibited many properties of functional β-cells, including expression of critical β-cells transcription factors, the potency to secrete C-peptide in response to high levels of glucose and the presence of mature endocrine secretory granules. This high efficient differentiation protocol, established in this study, yielded 79.18% insulin-secreting cells which were responsive to glucose five times higher than the basal level. These hiPSCs-derived glucose-responsive insulin-secreting cells might provide a promising approach for the treatment of type I diabetes mellitus. J. Cell. Physiol. 232: 2616-2625, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Interleukin-6 Reduces β-Cell Oxidative Stress by Linking Autophagy With the Antioxidant Response.

PubMed

Marasco, Michelle R; Conteh, Abass M; Reissaus, Christopher A; Cupit V, John E; Appleman, Evan M; Mirmira, Raghavendra G; Linnemann, Amelia K

2018-05-21

Production of reactive oxygen species (ROS) is a key instigator of β-cell dysfunction in diabetes. The pleiotropic cytokine IL-6 has previously been linked to β-cell autophagy but has not been studied in the context of β-cell antioxidant response. We used a combination of animal models of diabetes and analysis of cultured human islets and rodent β-cells to study how IL-6 influences antioxidant response. We show that IL-6 couples autophagy to antioxidant response to reduce β-cell and human islet ROS. β cell-specific loss of IL-6 signaling in vivo renders mice more susceptible to oxidative damage and cell death by the selective β-cell toxins streptozotocin and alloxan. IL-6-driven ROS reduction is associated with an increase in the master antioxidant factor NRF2, which rapidly translocates to the mitochondria to decrease mitochondrial activity and stimulate mitophagy. IL-6 also initiates a robust transient drop in cellular cAMP, likely contributing to the stimulation of mitophagy for ROS mitigation. Our findings suggest that coupling autophagy to antioxidant response in the β cell leads to stress adaptation that can reduce cellular apoptosis. These findings have implications for β-cell survival under diabetogenic conditions and present novel targets for therapeutic intervention. © 2018 by the American Diabetes Association.

B cell-intrinsic TLR7 signaling is required for optimal B cell responses during chronic viral infection

PubMed Central

Clingan, Jonathan M.; Matloubian, Mehrdad

2013-01-01

The importance for activation of innate immunity by pattern recognition receptors in forming an effective adaptive immune response is well known. Toll-like receptors (TLRs) have been demonstrated to be critical for antibody responses to a variety of immunizations. In particular, recent evidence suggests that B cell-intrinsic TLR signaling is required for optimal responses to virus-like antigens, but mechanisms by which TLR signaling impacts antibody responses during infection in vivo is unclear. In the present study, we demonstrate that deficiency of TLR7 in B cells alone is sufficient to significantly impact antibody responses in mice during chronic viral infection. This effect was independent of T follicular helper cells, and resulted in a loss of plasma cells generated later, but not early, in the response. The defect in plasma cell formation appeared to be secondary to a qualitative effect of TLR signaling on the germinal center (GC) B cell response. GC B cells in TLR7-deficient mice proliferated to a lesser extent and had a greater proportion of cells with phenotypic characteristics of light zone, relative to dark zone GC B cells. These results suggest that B cell-intrinsic TLR signaling in vivo likely affects plasma cell output by altered selection of antigen-specific B cells in the germinal center. PMID:23761632

Sweet taste transduction in hamster: sweeteners and cyclic nucleotides depolarize taste cells by reducing a K+ current.

PubMed

Cummings, T A; Daniels, C; Kinnamon, S C

1996-03-01

1. The gigaseal voltage-clamp technique was used to record responses of hamster taste receptor cells to synthetic sweeteners and cyclic nucleotides. Voltage-dependent currents and steady-state currents were monitored during bath exchanges of saccharin, two high-potency sweeteners, 8-chlorophenylthio-adenosine 3',5'-cyclic monophosphate (8cpt-cAMP), and dibutyryl-guanosine 3',5'-cyclic monophosphate (db-cGMP). 2. Of the 237 fungiform taste cells studied, only one in eight was sweet responsive. Outward currents, both voltage-dependent and resting, were reduced by all of the sweeteners tested in sweet-responsive taste cells, whereas these currents were unaffected by sweeteners in sweet-unresponsive taste cells. 3. In every sweet-responsive cell tested, 8cpt-cAMP and db-cGMP mimicked the response to the sweeteners, but neither nucleotide elicited responses in sweet-unresponsive cells. Thus there was a one-to-one correlation between sweet responsivity and cyclic nucleotide responsivity. 4. Sweet responses showed cross adaptation with cyclic nucleotide responses. This indicates that the same ion channel is modulated by sweeteners and cyclic nucleotides. 5. The sweetener- and cyclic nucleotide-blocked current had an apparent reversal potential of -50 mV, which was close to the potassium reversal potential in these experiments. In addition, there was no effect of sweeteners and cyclic nucleotides in the presence of the K+ channel blocker tetraethylammonium bromide (TEA). These data suggest that block of a resting, TEA-sensitive K+ current is the final common step leading to taste cell depolarization during sweet transduction. 6. These data, together with data from a previous study (Cummings et al. 1993), suggest that both synthetic sweeteners and sucrose utilize second-messenger pathways that block a resting K+ conductance to depolarize the taste cell membrane.

Molecular Responses of the Spiral Ganglion to Aminoglycosides

ERIC Educational Resources Information Center

Balaban, Carey D.

2005-01-01

Aminoglycosides are toxic to both the inner ear hair cells and the ganglion cells that give rise to the eighth cranial nerve. According to recent studies, these cells have a repertoire of molecular responses to aminoglycoside exposure that engages multiple neuroprotective mechanisms. The responses appear to involve regulation of ionic homeostasis,…

The Pseudopod System for Axon-Glia Interactions: Stimulation and Isolation of Schwann Cell Protrusions that Form in Response to Axonal Membranes.

PubMed

Poitelon, Yannick; Feltri, M Laura

2018-01-01

In the peripheral nervous system, axons dictate the differentiation state of Schwann cells. Most of this axonal influence on Schwann cells is due to juxtacrine interactions between axonal transmembrane molecules (e.g., the neuregulin growth factor) and receptors on the Schwann cell (e.g., the ErbB2/ErbB3 receptor). The fleeting nature of this interaction together with the lack of synchronicity in the development of the Schwann cell population limits our capability to study this phenomenon in vivo. Here we present a simple Boyden Chamber-based method to study this important cell-cell interaction event. We isolate the early protrusions of Schwann cells that are generated in response to juxtacrine stimulation by sensory neuronal membranes. This method is compatible with a large array of current biochemical analyses and provides an effective approach to study biomolecules that are differentially localized in Schwann cell protrusions and cell bodies in response to axonal signals. A similar approach can be extended to different kinds of cell-cell interactions.

Secondary allergic T cell responses are regulated by dendritic cell-derived thrombospondin-1 in the setting of allergic eye disease.

PubMed

Smith, R E; Reyes, N J; Khandelwal, P; Schlereth, S L; Lee, H S; Masli, S; Saban, D R

2016-08-01

Allergic eye disease, as in most forms of atopy, ranges in severity among individuals from immediate hypersensitivity to a severe and debilitating chronic disease. Dendritic cells play a key role in stimulating pathogenic T cells in allergen re-exposure, or secondary responses. However, molecular cues by dendritic cells underpinning allergic T cell response levels and the impact that this control has on consequent severity of allergic disease are poorly understood. Here, we show that a deficiency in thrombospondin-1, a matricellular protein known to affect immune function, has subsequent effects on downstream T cell responses during allergy, as revealed in an established mouse model of allergic eye disease. More specifically, we demonstrate that a thrombospondin-1 deficiency specific to dendritic cells leads to heightened secondary T cell responses and consequent clinical disease. Interestingly, whereas thrombospondin-1-deficient dendritic cells augmented activity of allergen-primed T cells, this increase was not recapitulated with naïve T cells in vitro. The role of dendritic cell-derived thrombospondin-1 in regulating secondary allergic T cell responses was confirmed in vivo, as local transfer of thrombospondin-1-sufficient dendritic cells to the ocular mucosa of thrombospondin-1 null hosts prevented the development of augmented secondary T cell responses and heightened allergic eye disease clinical responses. Finally, we demonstrate that topical instillation of thrombospondin-1-derived peptide reduces T cell activity and clinical progression of allergic eye disease. Taken together, this study reveals an important modulatory role of dendritic cell-derived thrombospondin-1 on secondary allergic T cell responses and suggests the possible dysregulation of dendritic cell-derived thrombospondin-1 expression as a factor in allergic eye disease severity. © Society for Leukocyte Biology.

Secondary allergic T cell responses are regulated by dendritic cell-derived thrombospondin-1 in the setting of allergic eye disease

PubMed Central

Smith, R. E.; Reyes, N. J.; Khandelwal, P.; Schlereth, S. L.; Lee, H. S.; Masli, S.; Saban, D. R.

2016-01-01

Allergic eye disease, as in most forms of atopy, ranges in severity among individuals from immediate hypersensitivity to a severe and debilitating chronic disease. Dendritic cells play a key role in stimulating pathogenic T cells in allergen re-exposure, or secondary responses. However, molecular cues by dendritic cells underpinning allergic T cell response levels and the impact that this control has on consequent severity of allergic disease are poorly understood. Here, we show that a deficiency in thrombospondin-1, a matricellular protein known to affect immune function, has subsequent effects on downstream T cell responses during allergy, as revealed in an established mouse model of allergic eye disease. More specifically, we demonstrate that a thrombospondin-1 deficiency specific to dendritic cells leads to heightened secondary T cell responses and consequent clinical disease. Interestingly, whereas thrombospondin-1-deficient dendritic cells augmented activity of allergen-primed T cells, this increase was not recapitulated with naïve T cells in vitro. The role of dendritic cell-derived thrombospondin-1 in regulating secondary allergic T cell responses was confirmed in vivo, as local transfer of thrombospondin-1-sufficient dendritic cells to the ocular mucosa of thrombospondin-1 null hosts prevented the development of augmented secondary T cell responses and heightened allergic eye disease clinical responses. Finally, we demonstrate that topical instillation of thrombospondin-1-derived peptide reduces T cell activity and clinical progression of allergic eye disease. Taken together, this study reveals an important modulatory role of dendritic cell-derived thrombospondin-1 on secondary allergic T cell responses and suggests the possible dysregulation of dendritic cell-derived thrombospondin-1 expression as a factor in allergic eye disease severity. PMID:26856994

Comparative proteomic analysis of Listeria monocytogenes exposed to enterocin AS-48 in planktonic and sessile states.

PubMed

Caballero Gómez, Natacha; Abriouel, Hikmate; Ennahar, Said; Gálvez, Antonio

2013-10-15

Enterocin AS-48 is a cyclic peptide of great interest for application in food preservation and sanitation. In the present study, the proteome response of Listeria monocytogenes to purified enterocin AS-48 was studied under two different conditions: planktonic cells and sessile cells grown on polystyrene plates. Ten different proteins were differentially expressed in planktonic L. monocytogenes cells treated with 0.1 μg/ml enterocin AS-48 compared to the untreated controls. Overexpressed proteins were related to stress response (DnaK) or carbohydrate transport and metabolism, while underexpressed and unexpressed proteins were related to metabolism (such as glyceraldehyde-3-phosphate dehydrogenase, pyruvate oxidase, glutamate dehydrogenase or glutamate decarboxylase) or stress (GroEL). In the sessile state, L. monocytogenes cells tolerated up to 10 μg/ml bacteriocin, and the treated biofilm cells overexpressed a set of 11 proteins, some of which could be related to stress response (DnaK, GroEL), protein synthesis and carbohydrate metabolism, while glyceraldehyde-3-phosphate dehydrogenase was the only unexpressed protein. Some of the overexpressed proteins (such as elongation factor Tu and GroEL) could also be implicated in cell adhesion. These results suggest different cell responses of L. monocytogenes to enterocin AS-48 in the planktonic and in the sessile state, including stress response and cell metabolism proteins. While in the planktonic state the bacterium may tend to compensate for the cytoplasmic cell permeability changes induced by AS-48 by reinforcing carbohydrate transport and metabolism, sessile cells seem to respond by shifting carbohydrate metabolism and reinforcing protein synthesis. Stress response proteins also seem to be important in the response to AS-48, but the stress response seems to be different in planktonic and in sessile cells. © 2013.

The role of accessory cells in polyclonal T cell activation. I. Both induction of interleukin 2 production and of interleukin 2 responsiveness by concanavalin A are accessory cell dependent.

PubMed

Hünig, T; Loos, M; Schimpl, A

1983-01-01

Recent studies from other laboratories have shown that concanavalin A (Con A) acts at two separate steps in polyclonal T cell activation: interleukin 2 (IL2) production, and induction of responsiveness to IL2. Using a combination of techniques for the depletion of accessory cells from lymph node T cells, we have investigated which of these steps, if not both, is responsible for the known requirement for accessory cells in the Con A response. It was found that with increasing T cell purification, first the ability is lost to produce sufficient levels of endogenous IL2, whereas induction of IL2 responsiveness can still take place. Further removal of accessory cells however yields a population of resting T cells that cannot be induced by Con A to become IL2-reactive. It was concluded that both IL2 production and induction of reactivity to IL2 are accessory cell-dependent events.

CD8 single-cell gene coexpression reveals three different effector types present at distinct phases of the immune response

PubMed Central

Peixoto, António; Evaristo, César; Munitic, Ivana; Monteiro, Marta; Charbit, Alain; Rocha, Benedita; Veiga-Fernandes, Henrique

2007-01-01

To study in vivo CD8 T cell differentiation, we quantified the coexpression of multiple genes in single cells throughout immune responses. After in vitro activation, CD8 T cells rapidly express effector molecules and cease their expression when the antigen is removed. Gene behavior after in vivo activation, in contrast, was quite heterogeneous. Different mRNAs were induced at very different time points of the response, were transcribed during different time periods, and could decline or persist independently of the antigen load. Consequently, distinct gene coexpression patterns/different cell types were generated at the various phases of the immune responses. During primary stimulation, inflammatory molecules were induced and down-regulated shortly after activation, generating early cells that only mediated inflammation. Cytotoxic T cells were generated at the peak of the primary response, when individual cells simultaneously expressed multiple killer molecules, whereas memory cells lost killer capacity because they no longer coexpressed killer genes. Surprisingly, during secondary responses gene transcription became permanent. Secondary cells recovered after antigen elimination were more efficient killers than cytotoxic T cells present at the peak of the primary response. Thus, primary responses produced two transient effector types. However, after boosting, CD8 T cells differentiate into long-lived killer cells that persist in vivo in the absence of antigen. PMID:17485515

Perturbation of B Cell Gene Expression Persists in HIV-Infected Children Despite Effective Antiretroviral Therapy and Predicts H1N1 Response.

PubMed

Cotugno, Nicola; De Armas, Lesley; Pallikkuth, Suresh; Rinaldi, Stefano; Issac, Biju; Cagigi, Alberto; Rossi, Paolo; Palma, Paolo; Pahwa, Savita

2017-01-01

Despite effective antiretroviral therapy (ART), HIV-infected individuals with apparently similar clinical and immunological characteristics can vary in responsiveness to vaccinations. However, molecular mechanisms responsible for such impairment, as well as biomarkers able to predict vaccine responsiveness in HIV-infected children, remain unknown. Following the hypothesis that a B cell qualitative impairment persists in HIV-infected children (HIV) despite effective ART and phenotypic B cell immune reconstitution, the aim of the current study was to investigate B cell gene expression of HIV compared to age-matched healthy controls (HCs) and to determine whether distinct gene expression patterns could predict the ability to respond to influenza vaccine. To do so, we analyzed prevaccination transcriptional levels of a 96-gene panel in equal numbers of sort-purified B cell subsets (SPBS) isolated from peripheral blood mononuclear cells using multiplexed RT-PCR. Immune responses to H1N1 antigen were determined by hemaglutination inhibition and memory B cell ELISpot assays following trivalent-inactivated influenza vaccination (TIV) for all study participants. Although there were no differences in terms of cell frequencies of SPBS between HIV and HC, the groups were distinguishable based upon gene expression analyses. Indeed, a 28-gene signature, characterized by higher expression of genes involved in the inflammatory response and immune activation was observed in activated memory B cells (CD27 + CD21 - ) from HIV when compared to HC despite long-term viral control (>24 months). Further analysis, taking into account H1N1 responses after TIV in HIV participants, revealed that a 25-gene signature in resting memory (RM) B cells (CD27 + CD21 + ) was able to distinguish vaccine responders from non-responders (NR). In fact, prevaccination RM B cells of responders showed a higher expression of gene sets involved in B cell adaptive immune responses ( APRIL, BTK, BLIMP1 ) and BCR signaling ( MTOR, FYN, CD86 ) when compared to NR. Overall, these data suggest that a perturbation at a transcriptional level in the B cell compartment persists despite stable virus control achieved through ART in HIV-infected children. Additionally, the present study demonstrates the potential utility of transcriptional evaluation of RM B cells before vaccination for identifying predictive correlates of vaccine responses in this population.

Perturbation of B Cell Gene Expression Persists in HIV-Infected Children Despite Effective Antiretroviral Therapy and Predicts H1N1 Response

PubMed Central

Cotugno, Nicola; De Armas, Lesley; Pallikkuth, Suresh; Rinaldi, Stefano; Issac, Biju; Cagigi, Alberto; Rossi, Paolo; Palma, Paolo; Pahwa, Savita

2017-01-01

Despite effective antiretroviral therapy (ART), HIV-infected individuals with apparently similar clinical and immunological characteristics can vary in responsiveness to vaccinations. However, molecular mechanisms responsible for such impairment, as well as biomarkers able to predict vaccine responsiveness in HIV-infected children, remain unknown. Following the hypothesis that a B cell qualitative impairment persists in HIV-infected children (HIV) despite effective ART and phenotypic B cell immune reconstitution, the aim of the current study was to investigate B cell gene expression of HIV compared to age-matched healthy controls (HCs) and to determine whether distinct gene expression patterns could predict the ability to respond to influenza vaccine. To do so, we analyzed prevaccination transcriptional levels of a 96-gene panel in equal numbers of sort-purified B cell subsets (SPBS) isolated from peripheral blood mononuclear cells using multiplexed RT-PCR. Immune responses to H1N1 antigen were determined by hemaglutination inhibition and memory B cell ELISpot assays following trivalent-inactivated influenza vaccination (TIV) for all study participants. Although there were no differences in terms of cell frequencies of SPBS between HIV and HC, the groups were distinguishable based upon gene expression analyses. Indeed, a 28-gene signature, characterized by higher expression of genes involved in the inflammatory response and immune activation was observed in activated memory B cells (CD27+CD21−) from HIV when compared to HC despite long-term viral control (>24 months). Further analysis, taking into account H1N1 responses after TIV in HIV participants, revealed that a 25-gene signature in resting memory (RM) B cells (CD27+CD21+) was able to distinguish vaccine responders from non-responders (NR). In fact, prevaccination RM B cells of responders showed a higher expression of gene sets involved in B cell adaptive immune responses (APRIL, BTK, BLIMP1) and BCR signaling (MTOR, FYN, CD86) when compared to NR. Overall, these data suggest that a perturbation at a transcriptional level in the B cell compartment persists despite stable virus control achieved through ART in HIV-infected children. Additionally, the present study demonstrates the potential utility of transcriptional evaluation of RM B cells before vaccination for identifying predictive correlates of vaccine responses in this population. PMID:28955330

Theoretical analysis of insulin-dependent glucose uptake heterogeneity in 3D bioreactor cell culture.

PubMed

Magrofuoco, Enrico; Elvassore, Nicola; Doyle, Francis J

2012-01-01

Three-dimensional (3D) cell cultures in bioreactors are becoming relevant as models for biological and physiological in vitro studies. In such systems, mathematical models can assist the experiment design that links the macroscopic properties to single-cell responses. We investigated the relationship between biochemical stimuli and cell response within a 3D cell culture in scaffold with heterogeneous porosity. Specifically, we studied the effect of insulin on the local glucose metabolism as a function of 3D pore size distribution. The multiscale mathematical model combines the mass transport within a 3D scaffold and a signaling pathways model. It considers the scaffold heterogeneity, and it describes spatiotemporal concentration of metabolites, biochemical stimuli, and cell density. The signaling model was integrated into this model, linking the local insulin concentration at cell membrane to the glucose uptake rate through glucose transporter type 4 (GLUT4) translocation from the cytosol to the cell membrane. The integrated model determines the cell response heterogeneities in a single channel, hence the biological response distribution in a 3D system. It also provides macroscopic outcomes to evaluate the feasibility of an experimental measurement of the system response. From our analysis, it became apparent that the flow rate is the most important operative variable, and that an optimum value ensures a fast and detectable cell response. This model on insulin-dependent glucose consumption rate offers insight into the cell metabolism physiology, which is a fundamental requirement for the study metabolic disorder such as Type 2 diabetes mellitus, in which the physiological insulin-dependent glucose metabolism is impaired. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

Definition of epitopes and antigens recognized by vaccinia specific immune responses: their conservation in variola virus sequences, and use as a model system to study complex pathogens.

PubMed

Sette, Alessandro; Grey, Howard; Oseroff, Carla; Peters, Bjoern; Moutaftsi, Magdalini; Crotty, Shane; Assarsson, Erika; Greenbaum, Jay; Kim, Yohan; Kolla, Ravi; Tscharke, David; Koelle, David; Johnson, R Paul; Blum, Janice; Head, Steven; Sidney, John

2009-12-30

In the last few years, a wealth of information has become available relating to the targets of vaccinia virus (VACV)-specific CD4(+) T cell, CD8(+) T cell and antibody responses. Due to the large size of its genome, encoding more than 200 different proteins, VACV represents a useful model system to study immunity to complex pathogens. Our data demonstrate that both cellular and humoral responses target a large number of antigens and epitopes. This broad spectrum of targets is detected in both mice and humans. CD4(+) T cell responses target late and structural antigens, while CD8(+) T cells preferentially recognize early antigens. While both CD4(+) and CD8(+) T cell responses target different types of antigens, the antigens recognized by T(H) cells are highly correlated with those recognized by antibody responses. We further show that protein abundance and antibody recognition can be used to predict antigens recognized by CD4(+) T cell responses, while early expression at the mRNA level predicts antigens targeted by CD8(+) T cells. Finally, we find that the vast majority of VACV epitopes are conserved in variola virus (VARV), thus suggesting that the epitopes defined herein also have relevance for the efficacy of VACV as a smallpox vaccine.

Integration of Immune Cell Populations, mRNA-Seq, and CpG Methylation to Better Predict Humoral Immunity to Influenza Vaccination: Dependence of mRNA-Seq/CpG Methylation on Immune Cell Populations

PubMed Central

Zimmermann, Michael T.; Kennedy, Richard B.; Grill, Diane E.; Oberg, Ann L.; Goergen, Krista M.; Ovsyannikova, Inna G.; Haralambieva, Iana H.; Poland, Gregory A.

2017-01-01

The development of a humoral immune response to influenza vaccines occurs on a multisystems level. Due to the orchestration required for robust immune responses when multiple genes and their regulatory components across multiple cell types are involved, we examined an influenza vaccination cohort using multiple high-throughput technologies. In this study, we sought a more thorough understanding of how immune cell composition and gene expression relate to each other and contribute to interindividual variation in response to influenza vaccination. We first hypothesized that many of the differentially expressed (DE) genes observed after influenza vaccination result from changes in the composition of participants’ peripheral blood mononuclear cells (PBMCs), which were assessed using flow cytometry. We demonstrated that DE genes in our study are correlated with changes in PBMC composition. We gathered DE genes from 128 other publically available PBMC-based vaccine studies and identified that an average of 57% correlated with specific cell subset levels in our study (permutation used to control false discovery), suggesting that the associations we have identified are likely general features of PBMC-based transcriptomics. Second, we hypothesized that more robust models of vaccine response could be generated by accounting for the interplay between PBMC composition, gene expression, and gene regulation. We employed machine learning to generate predictive models of B-cell ELISPOT response outcomes and hemagglutination inhibition (HAI) antibody titers. The top HAI and B-cell ELISPOT model achieved an area under the receiver operating curve (AUC) of 0.64 and 0.79, respectively, with linear model coefficients of determination of 0.08 and 0.28. For the B-cell ELISPOT outcomes, CpG methylation had the greatest predictive ability, highlighting potentially novel regulatory features important for immune response. B-cell ELISOT models using only PBMC composition had lower performance (AUC = 0.67), but highlighted well-known mechanisms. Our analysis demonstrated that each of the three data sets (cell composition, mRNA-Seq, and DNA methylation) may provide distinct information for the prediction of humoral immune response outcomes. We believe that these findings are important for the interpretation of current omics-based studies and set the stage for a more thorough understanding of interindividual immune responses to influenza vaccination. PMID:28484452

Cell Wall Ultrastructure of Stem Wood, Roots, and Needles of a Conifer Varies in Response to Moisture Availability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pattathil, Sivakumar; Ingwers, Miles W.; Victoriano, Olivia L.

The composition, integrity, and architecture of the macromolecular matrix of cell walls, collectively referred to as cell wall ultrastructure, exhibits variation across species and organs and among cell types within organs. Indirect approaches have suggested that modifications to cell wall ultrastructure occur in response to abiotic stress; however, modifications have not been directly observed. Glycome profiling was used to study cell wall ultrastructure by examining variation in composition and extractability of non-cellulosic glycans in cell walls of stem wood, roots, and needles of loblolly pine saplings exposed to high and low soil moisture. Soil moisture influenced physiological processes and themore » overall composition and extractability of cell wall components differed as a function of soil moisture treatments. The strongest response of cell wall ultrastructure to soil moisture was increased extractability of pectic backbone epitopes in the low soil moisture treatment. The higher abundance of these pectic backbone epitopes in the oxalate extract indicate that the loosening of cell wall pectic components could be associated with the release of pectic signals as a stress response. The increased extractability of pectic backbone epitopes in response to low soil moisture availability was more pronounced in stem wood than in roots or needles. Additional responses to low soil moisture availability were observed in lignin associated carbohydrates released in chlorite extracts of stem wood, including an increased abundance of pectic arabinogalactan epitopes. Overall, these results indicate that cell walls of loblolly pine organs undergo changes in their ultrastructural composition and extractability as a response to soil moisture availability and that cell walls of the stem wood are more responsive to low soil moisture availability compared to cell walls of roots and needles. In conclusion, to our knowledge, this is the first direct evidence, delineated by glycomic analyses, that abiotic stress affects cell wall ultrastructure. This study is also unique in that glycome profiling of pine needles has never before been reported.« less

Cell Wall Ultrastructure of Stem Wood, Roots, and Needles of a Conifer Varies in Response to Moisture Availability.

PubMed

Pattathil, Sivakumar; Ingwers, Miles W; Victoriano, Olivia L; Kandemkavil, Sindhu; McGuire, Mary Anne; Teskey, Robert O; Aubrey, Doug P

2016-01-01

The composition, integrity, and architecture of the macromolecular matrix of cell walls, collectively referred to as cell wall ultrastructure, exhibits variation across species and organs and among cell types within organs. Indirect approaches have suggested that modifications to cell wall ultrastructure occur in response to abiotic stress; however, modifications have not been directly observed. Glycome profiling was used to study cell wall ultrastructure by examining variation in composition and extractability of non-cellulosic glycans in cell walls of stem wood, roots, and needles of loblolly pine saplings exposed to high and low soil moisture. Soil moisture influenced physiological processes and the overall composition and extractability of cell wall components differed as a function of soil moisture treatments. The strongest response of cell wall ultrastructure to soil moisture was increased extractability of pectic backbone epitopes in the low soil moisture treatment. The higher abundance of these pectic backbone epitopes in the oxalate extract indicate that the loosening of cell wall pectic components could be associated with the release of pectic signals as a stress response. The increased extractability of pectic backbone epitopes in response to low soil moisture availability was more pronounced in stem wood than in roots or needles. Additional responses to low soil moisture availability were observed in lignin-associated carbohydrates released in chlorite extracts of stem wood, including an increased abundance of pectic arabinogalactan epitopes. Overall, these results indicate that cell walls of loblolly pine organs undergo changes in their ultrastructural composition and extractability as a response to soil moisture availability and that cell walls of the stem wood are more responsive to low soil moisture availability compared to cell walls of roots and needles. To our knowledge, this is the first direct evidence, delineated by glycomic analyses, that abiotic stress affects cell wall ultrastructure. This study is also unique in that glycome profiling of pine needles has never before been reported.

Cell Wall Ultrastructure of Stem Wood, Roots, and Needles of a Conifer Varies in Response to Moisture Availability

DOE PAGES

Pattathil, Sivakumar; Ingwers, Miles W.; Victoriano, Olivia L.; ...

2016-06-24

The composition, integrity, and architecture of the macromolecular matrix of cell walls, collectively referred to as cell wall ultrastructure, exhibits variation across species and organs and among cell types within organs. Indirect approaches have suggested that modifications to cell wall ultrastructure occur in response to abiotic stress; however, modifications have not been directly observed. Glycome profiling was used to study cell wall ultrastructure by examining variation in composition and extractability of non-cellulosic glycans in cell walls of stem wood, roots, and needles of loblolly pine saplings exposed to high and low soil moisture. Soil moisture influenced physiological processes and themore » overall composition and extractability of cell wall components differed as a function of soil moisture treatments. The strongest response of cell wall ultrastructure to soil moisture was increased extractability of pectic backbone epitopes in the low soil moisture treatment. The higher abundance of these pectic backbone epitopes in the oxalate extract indicate that the loosening of cell wall pectic components could be associated with the release of pectic signals as a stress response. The increased extractability of pectic backbone epitopes in response to low soil moisture availability was more pronounced in stem wood than in roots or needles. Additional responses to low soil moisture availability were observed in lignin associated carbohydrates released in chlorite extracts of stem wood, including an increased abundance of pectic arabinogalactan epitopes. Overall, these results indicate that cell walls of loblolly pine organs undergo changes in their ultrastructural composition and extractability as a response to soil moisture availability and that cell walls of the stem wood are more responsive to low soil moisture availability compared to cell walls of roots and needles. In conclusion, to our knowledge, this is the first direct evidence, delineated by glycomic analyses, that abiotic stress affects cell wall ultrastructure. This study is also unique in that glycome profiling of pine needles has never before been reported.« less

Pre-vaccination inflammation and B-cell signalling predict age-related hyporesponse to hepatitis B vaccination

PubMed Central

Fourati, Slim; Cristescu, Razvan; Loboda, Andrey; Talla, Aarthi; Filali, Ali; Railkar, Radha; Schaeffer, Andrea K.; Favre, David; Gagnon, Dominic; Peretz, Yoav; Wang, I-Ming; Beals, Chan R.; Casimiro, Danilo R.; Carayannopoulos, Leonidas N.; Sékaly, Rafick-Pierre

2016-01-01

Aging is associated with hyporesponse to vaccination, whose mechanisms remain unclear. In this study hepatitis B virus (HBV)-naive older adults received three vaccines, including one against HBV. Here we show, using transcriptional and cytometric profiling of whole blood collected before vaccination, that heightened expression of genes that augment B-cell responses and higher memory B-cell frequencies correlate with stronger responses to HBV vaccine. In contrast, higher levels of inflammatory response transcripts and increased frequencies of pro-inflammatory innate cells correlate with weaker responses to this vaccine. Increased numbers of erythrocytes and the haem-induced response also correlate with poor response to the HBV vaccine. A transcriptomics-based pre-vaccination predictor of response to HBV vaccine is built and validated in distinct sets of older adults. This moderately accurate (area under the curve≈65%) but robust signature is supported by flow cytometry and cytokine profiling. This study is the first that identifies baseline predictors and mechanisms of response to the HBV vaccine. PMID:26742691

The effects of cyclic tensile strain on the organisation and expression of cytoskeletal elements in bovine intervertebral disc cells: an in vitro study.

PubMed

Li, S; Jia, X; Duance, V C; Blain, E J

2011-06-20

It is still relatively unclear how intervertebral disc (IVD) cells sense a mechanical stimulus and convert this signal into a biochemical response. Previous studies demonstrated that the cytoskeletal elements are mechano-responsive in many cell types and may contribute to mechano-signalling pathways. The objective of this study was to determine the response of cells from the outer annulus fibrosus (OAF) to physiological levels of cyclic tensile strain; further, cells from the nucleus pulposus (NP) were also subjected to an identical loading regime to compare biological responses across the IVD populations. We determined whether the organisation and expression of the major cytoskeletal elements and their associated accessory proteins are responsive to mechanical stimulation in these cells, and whether these changes correlated with either a catabolic or anabolic phenotype. OAF and NP cells from immature bovine IVD were seeded onto Flexcell® type I collagen coated plates. Cells were subjected to cyclic tensile strain (10 %, 1 Hz) for 60 minutes. Post-loading, cells were processed for immunofluorescence microscopy, RNA extracted for quantitative PCR and protein extracted for Western blotting analysis. F-actin reorganisation was evident in OAF and NP cells subjected to tensile strain; strain induced β-actin at the transcriptional and translational level in OAF cells. β-tubulin mRNA and protein synthesis increased in strained OAF cells, but vimentin expression was significantly inhibited. Cytoskeletal element organisation and expression were less responsive to strain in NP cells. Tensile strain increased type I collagen and differentially regulated extracellular matrix (ECM)-degrading enzymes' mRNA levels in OAF cells. Strain induced type II collagen transcription in NP cells, but had no effect on the transcription of any other genes analysed. Tensile strain induces different mechano-responses in the organisation and/or expression of cytoskeletal elements and on markers of IVD metabolism. Differential mechano-regulation of anabolic and catabolic ECM components in the OAF and NP populations reflects their respective mechanical environments in situ.

Maternal allergic disease does not affect the phenotype of T and B cells or the immune response to allergens in neonates.

PubMed

Rindsjö, E; Joerink, M; Johansson, C; Bremme, K; Malmström, V; Scheynius, A

2010-07-01

It is hypothesized that the in utero environment in allergic mothers can affect the neonatal immune responses. The aim of this study was to analyse the effect of maternal allergic disease on cord blood mononuclear cell (CBMC) phenotype and proliferative responses upon allergen stimulation. Peripheral blood mononuclear cells (PBMC) from 12 allergic and 14 nonallergic mothers and CBMC from their children were analysed. In the mothers, we determined cell proliferation, production of IL-4 and expression of FOXP3 in response to allergen stimulation. In the children, we evaluated cell proliferation and FOXP3 expression following allergen stimulation. Furthermore, expression of different homing markers on T cells and regulatory T cells and maturity of the T cells and B cell subsets were evaluated directly ex vivo. The timothy- and birch-allergic mothers responded with increased proliferation and/or IL-4 production towards timothy and birch extract, respectively, when compared to nonallergic mothers. This could not be explained by impairment of FOXP3(+) regulatory T cells in the allergic mothers. CBMC proliferation and FOXP3 expression in response to allergens were not affected by the allergic status of the mother. Also, phenotype of T cells, FOXP3(+) regulatory T cells and B cells was not affected by the allergic status of the mother. Our results suggest that maternal allergic disease has no effect on the neonatal response to allergens or the phenotype of neonatal lymphocytes. The factors studied here could, however, still affect later development of allergy.

Mesenteric lymph node T cells but not splenic T cells maintain their proliferative response to concanavalin-A following peroral infection with Toxoplasma gondii.

PubMed

Neyer, L E; Kang, H; Remington, J S; Suzuki, Y

1998-12-01

The suppression of T cell responsiveness which occurs after infection with Toxoplasma gondii in mice has been widely studied using spleen cells. Because the natural route of infection with T. gondii is the peroral route, we examined the proliferative responses of mesenteric lymph node (MLN) cells, in addition to spleen cells, to Concanavalin-A (Con-A) in mice perorally infected with T. gondii. Proliferative responses of spleen cells were significantly suppressed seven and ten days after infection when compared with spleen cells from uninfected mice (62% and 91% reduction, respectively). In contrast, proliferative responses of MLN cells from these infected mice did not differ from those of normal MLN cells. Since IFN-gamma-induced reactive nitrogen intermediate (RNI) production has been reported to play a major role in suppression of proliferative responses in spleen cells of infected mice, we compared production of IFN-gamma and RNI by spleen and MLN cells following infection. MLN cells produced as much IFN-gamma as did spleen cells, but produced 70% less nitrite (as a measure of RNI) after Con-A stimulation. Proliferative responses of MLN cells were suppressed when co-cultured with spleen cells from infected mice, and addition of an inhibitor of RNI to these co-culture inhibited this suppression, suggesting that reduced RNI production by MLN cells contributes to their maintenance of higher proliferative responses. These results demonstrated a clear difference in activity of T cells in the MLN and spleen during the acute stage of the infection.

Mitogen-activated protein kinase signal transduction and DNA repair network are involved in aluminum-induced DNA damage and adaptive response in root cells of Allium cepa L.

PubMed Central

Panda, Brahma B.; Achary, V. Mohan M.

2014-01-01

In the current study, we studied the role of signal transduction in aluminum (Al3+)-induced DNA damage and adaptive response in root cells of Allium cepa L. The root cells in planta were treated with Al3+ (800 μM) for 3 h without or with 2 h pre-treatment of inhibitors of mitogen-activated protein kinase (MAPK), and protein phosphatase. Also, root cells in planta were conditioned with Al3+ (10 μM) for 2 h and then subjected to genotoxic challenge of ethyl methane sulfonate (EMS; 5 mM) for 3 h without or with the pre-treatment of the aforementioned inhibitors as well as the inhibitors of translation, transcription, DNA replication and repair. At the end of treatments, roots cells were assayed for cell death and/or DNA damage. The results revealed that Al3+ (800 μM)-induced significant DNA damage and cell death. On the other hand, conditioning with low dose of Al3+ induced adaptive response conferring protection of root cells from genotoxic stress caused by EMS-challenge. Pre-treatment of roots cells with the chosen inhibitors prior to Al3+-conditioning prevented or reduced the adaptive response to EMS genotoxicity. The results of this study suggested the involvement of MAPK and DNA repair network underlying Al-induced DNA damage and adaptive response to genotoxic stress in root cells of A. cepa. PMID:24926302

Impact of mechanical stretch on the cell behaviors of bone and surrounding tissues

PubMed Central

Yu, Hye-Sun; Kim, Jung-Ju; Kim, Hae-Won; Lewis, Mark P; Wall, Ivan

2016-01-01

Mechanical loading is recognized to play an important role in regulating the behaviors of cells in bone and surrounding tissues in vivo. Many in vitro studies have been conducted to determine the effects of mechanical loading on individual cell types of the tissues. In this review, we focus specifically on the use of the Flexercell system as a tool for studying cellular responses to mechanical stretch. We assess the literature describing the impact of mechanical stretch on different cell types from bone, muscle, tendon, ligament, and cartilage, describing individual cell phenotype responses. In addition, we review evidence regarding the mechanotransduction pathways that are activated to potentiate these phenotype responses in different cell populations. PMID:26977284

Classification of human natural killer cells based on migration behavior and cytotoxic response.

PubMed

Vanherberghen, Bruno; Olofsson, Per E; Forslund, Elin; Sternberg-Simon, Michal; Khorshidi, Mohammad Ali; Pacouret, Simon; Guldevall, Karolin; Enqvist, Monika; Malmberg, Karl-Johan; Mehr, Ramit; Önfelt, Björn

2013-02-21

Despite intense scrutiny of the molecular interactions between natural killer (NK) and target cells, few studies have been devoted to dissection of the basic functional heterogeneity in individual NK cell behavior. Using a microchip-based, time-lapse imaging approach allowing the entire contact history of each NK cell to be recorded, in the present study, we were able to quantify how the cytotoxic response varied between individual NK cells. Strikingly, approximately half of the NK cells did not kill any target cells at all, whereas a minority of NK cells was responsible for a majority of the target cell deaths. These dynamic cytotoxicity data allowed categorization of NK cells into 5 distinct classes. A small but particularly active subclass of NK cells killed several target cells in a consecutive fashion. These "serial killers" delivered their lytic hits faster and induced faster target cell death than other NK cells. Fast, necrotic target cell death was correlated with the amount of perforin released by the NK cells. Our data are consistent with a model in which a small fraction of NK cells drives tumor elimination and inflammation.

Radiofrequency currents exert cytotoxic effects in NB69 human neuroblastoma cells but not in peripheral blood mononuclear cells

PubMed Central

HERNÁNDEZ-BULE, MARÍA LUISA; ROLDÁN, ERNESTO; MATILLA, JOAQUÍN; TRILLO, MARÍA ÁNGELES; ÚBEDA, ALEJANDRO

2012-01-01

Recently, a number of electric and electrothermal therapies have been applied to the treatment of specific cancer types. However, the cellular and molecular mechanisms involved in the response to such therapies have not been well characterized yet. Capacitive-resistive electric transfer (CRET) therapy uses electric currents at frequencies within the 0.45–0.6 MHz range to induce hyperthermia in target tissues. Preliminary trials in cancer patients have shown consistent signs that CRET could slow down growth of tumor tissues in brain gliomas, without inducing detectable damage in the surrounding healthy tissue. Previous studies by our group have shown that subthermal treatment with 0.57-MHz electric currents can induce a cytostatic, not cytotoxic response in HepG2 human hepatocarcinoma cells; such effect being mediated by cell cycle alterations. In contrast, the study of the response of NB69 human neuroblastoma cells to the same electric treatment revealed consistent indications of cytotoxic effects. The present study extends the knowledge on the response of NB69 cells to the subthermal stimulus, comparing it to that of primary cultures of human peripheral blood mononuclear cells (PBMC) exposed to the same treatment. The results showed no sensitivity of PBMC to the 0.57 MHz subthermal currents and confirmed that the treatment exerts a cytotoxic action in NB69 cells. The data also revealed a previously undetected cytostatic response of the neuroblastoma cell line. CRET currents affected NB69 cell proliferation by significantly reducing the fraction of cells in the phase G2/M of the cell cycle at 12 h of exposure. These data provide new information on the mechanisms of response to CRET therapy, and are consistent with a cytotoxic and/or cytostatic action of the electric treatment, which would affect human cells of tumor origin but not normal cells with a low proliferation rate. PMID:22843038

Radiofrequency currents exert cytotoxic effects in NB69 human neuroblastoma cells but not in peripheral blood mononuclear cells.

PubMed

Hernández-Bule, María Luisa; Roldán, Ernesto; Matilla, Joaquín; Trillo, María Angeles; Ubeda, Alejandro

2012-10-01

Recently, a number of electric and electrothermal therapies have been applied to the treatment of specific cancer types. However, the cellular and molecular mechanisms involved in the response to such therapies have not been well characterized yet. Capacitive-resistive electric transfer (CRET) therapy uses electric currents at frequencies within the 0.45-0.6 MHz range to induce hyperthermia in target tissues. Preliminary trials in cancer patients have shown consistent signs that CRET could slow down growth of tumor tissues in brain gliomas, without inducing detectable damage in the surrounding healthy tissue. Previous studies by our group have shown that subthermal treatment with 0.57-MHz electric currents can induce a cytostatic, not cytotoxic response in HepG2 human hepatocarcinoma cells; such effect being mediated by cell cycle alterations. In contrast, the study of the response of NB69 human neuroblastoma cells to the same electric treatment revealed consistent indications of cytotoxic effects. The present study extends the knowledge on the response of NB69 cells to the subthermal stimulus, comparing it to that of primary cultures of human peripheral blood mononuclear cells (PBMC) exposed to the same treatment. The results showed no sensitivity of PBMC to the 0.57 MHz subthermal currents and confirmed that the treatment exerts a cytotoxic action in NB69 cells. The data also revealed a previously undetected cytostatic response of the neuroblastoma cell line. CRET currents affected NB69 cell proliferation by significantly reducing the fraction of cells in the phase G2/M of the cell cycle at 12 h of exposure. These data provide new information on the mechanisms of response to CRET therapy, and are consistent with a cytotoxic and/or cytostatic action of the electric treatment, which would affect human cells of tumor origin but not normal cells with a low proliferation rate.

Immunomodulatory Effect of Mesenchymal Stem Cells on B Cells

PubMed Central

Franquesa, Marcella; Hoogduijn, M. J.; Bestard, O.; Grinyó, J. M.

2012-01-01

The research on T cell immunosuppression therapies has attracted most of the attention in clinical transplantation. However, B cells and humoral immune responses are increasingly acknowledged as crucial mediators of chronic allograft rejection. Indeed, humoral immune responses can lead to renal allograft rejection even in patients whose cell-mediated immune responses are well controlled. On the other hand, newly studied B cell subsets with regulatory effects have been linked to tolerance achievement in transplantation. Better understanding of the regulatory and effector B cell responses may therefore lead to new therapeutic approaches. Mesenchymal stem cells (MSC) are arising as a potent therapeutic tool in transplantation due to their regenerative and immunomodulatory properties. The research on MSCs has mainly focused on their effects on T cells and although data regarding the modulatory effects of MSCs on alloantigen-specific humoral response in humans is scarce, it has been demonstrated that MSCs significantly affect B cell functioning. In the present review we will analyze and discuss the results in this field. PMID:22833744

Determinants of public T cell responses.

PubMed

Li, Hanjie; Ye, Congting; Ji, Guoli; Han, Jiahuai

2012-01-01

Historically, sharing T cell receptors (TCRs) between individuals has been speculated to be impossible, considering the dramatic discrepancy between the potential enormity of the TCR repertoire and the limited number of T cells generated in each individual. However, public T cell response, in which multiple individuals share identical TCRs in responding to a same antigenic epitope, has been extensively observed in a variety of immune responses across many species. Public T cell responses enable individuals within a population to generate similar antigen-specific TCRs against certain ubiquitous pathogens, leading to favorable biological outcomes. However, the relatively concentrated feature of TCR repertoire may limit T cell response in a population to some other pathogens. It could be a great benefit for human health if public T cell responses can be manipulated. Therefore, the mechanistic insight of public TCR generation is important to know. Recently, high-throughput DNA sequencing has revolutionized the study of immune receptor repertoires, which allows a much better understanding of the factors that determine the overlap of TCR repertoire among individuals. Here, we summarize the current knowledge on public T-cell response and discuss future challenges in this field.

Gene Expression Profiling and Molecular Signaling of Various Cells in Response to Tricalcium Silicate Cements: A Systematic Review.

PubMed

Rathinam, Elanagai; Rajasekharan, Sivaprakash; Chitturi, Ravi Teja; Declercq, Heidi; Martens, Luc; De Coster, Peter

2016-12-01

The aim of this study was to present a systematic review investigating the gene expression of various cells (other than dental pulp cells) in response to different variants of tricalcium silicate cements (TSCs). A systematic search of the literature was performed by 2 independent reviewers followed by article selection and data extraction. Studies analyzing any cell type except dental pulp stem cells and any variant of tricalcium silicate cement either as the experimental or as the control group were included. A total of 41 relevant articles were included in this review. Among the included studies, ProRoot MTA (Dentsply, Tulsa, OK) was the most commonly studied (69.1%) TSC variant, and 11 cell types were identified, with 13 articles investigating gene expression in osteoblasts. A total of 39 different genes/molecules expressed were found in the selected studies. The experimental group (irrespective of the TSC variant) was identified to express significantly increased gene expression compared with the control group (untreated) in all included studies. Recent studies have provided useful insight into the gene expression and molecular signaling of various cells in response to TSCs, and new elements have been supplied on the pathways activated in this process. TSCs are capable of eliciting a favorable cellular response in periapical regeneration. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Optimization of a cAMP response element signal pathway reporter system.

PubMed

Shan, Qiang; Storm, Daniel R

2010-08-15

A sensitive cAMP response element (CRE) reporter system is essential for studying the cAMP/protein kinase A/cAMP response element binding protein signal pathway. Here we have tested a few CRE promoters and found one with high sensitivity to external stimuli. Using this optimal CRE promoter and the enhanced green fluorescent protein as the reporter, we have established a CRE reporter cell line. This cell line can be used to study the signal pathway by fluorescent microscope, fluorescence-activated cell analysis and luciferase assay. This cell line's sensitivity to forskolin, using the technique of fluorescence-activated cell sorting, was increased to approximately seven times that of its parental HEK 293 cell line, which is currently the most commonly used cell line in the field for the signal pathway study. Therefore, this newly created cell line is potentially useful for studying the signal pathway's modulators, which generally have weaker effect than its mediators. Our research has also established a general procedure for optimizing transcription-based reporter cell lines, which might be useful in performing the same task when studying many other transcription-based signal pathways. (c) 2010 Elsevier B.V. All rights reserved.

Impaired NK Cell Responses to Pertussis and H1N1 Influenza Vaccine Antigens in Human Cytomegalovirus-Infected Individuals

PubMed Central

Nielsen, Carolyn M.; White, Matthew J.; Bottomley, Christian; Lusa, Chiara; Rodríguez-Galán, Ana; Turner, Scarlett E. G.; Goodier, Martin R.

2015-01-01

NK cells contribute to postvaccination immune responses after activation by IL-2 from Ag-specific memory T cells or by cross-linking of the low-affinity IgG receptor, CD16, by Ag–Ab immune complexes. Sensitivity of NK cells to these signals from the adaptive immune system is heterogeneous and influenced by their stage of differentiation. CD56dimCD57+ NK cells are less responsive to IL-2 and produce less IFN-γ in response to T cell–mediated activation than do CD56bright or CD56dimCD57− NK cells. Conversely, NK cell cytotoxicity, as measured by degranulation, is maintained across the CD56dim subsets. Human CMV (HCMV), a highly prevalent herpes virus causing lifelong, usually latent, infections, drives the expansion of the CD56dimCD57+NKG2C+ NK cell population, skewing the NK cell repertoire in favor of cytotoxic responses at the expense of cytokine-driven responses. We hypothesized, therefore, that HCMV seropositivity would be associated with altered NK cell responses to vaccine Ags. In a cross-sectional study of 152 U.K. adults, with HCMV seroprevalence rate of 36%, we find that HCMV seropositivity is associated with lower NK cell IFN-γ production and degranulation after in vitro restimulation with pertussis or H1N1 influenza vaccine Ags. Higher expression of CD57/NKG2C and lower expression of IL-18Rα on NK cells from HCMV seropositive subjects do not fully explain these impaired responses, which are likely the result of multiple receptor–ligand interactions. This study demonstrates for the first time, to our knowledge, that HCMV serostatus influences NK cell contributions to adaptive immunity and raises important questions regarding the impact of HCMV infection on vaccine efficacy. PMID:25855356

Enhanced immune response of MAIT cells in tuberculous pleural effusions depends on cytokine signaling.

PubMed

Jiang, Jing; Chen, Xinchun; An, Hongjuan; Yang, Bingfen; Zhang, Fuping; Cheng, Xiaoxing

2016-09-02

The functions of MAIT cells at the site of Mycobacterium tuberculosis infection in humans are still largely unknown. In this study, the phenotypes and immune response of MAIT cells from tuberculous pleural effusions and peripheral blood were investigated. MAIT cells in tuberculous pleural effusions had greatly enhanced IFN-γ, IL-17F and granzyme B response compared with those in peripheral blood. The level of IFN-γ response in MAIT cells from tuberculous pleural effusions was inversely correlated with the extent of tuberculosis infection (p = 0.0006). To determine whether cytokines drive the immune responses of MAIT cells at the site of tuberculosis infection, the role of IL-1β, IL-2, IL-7, IL-12, IL-15 and IL-18 was investigated. Blockade of IL-2, IL-12 or IL-18 led to significantly reduced production of IFN-γ and/or granzyme B in MAIT cells from tuberculous pleural effusions. Majority of IL-2-producing cells (94.50%) in tuberculous pleural effusions had phenotype of CD3(+)CD4(+), and most IL-12p40-producing cells (91.39%) were CD14(+) cells. MAIT cells had significantly elevated expression of γc receptor which correlated with enhanced immune responses of MAIT cells. It is concluded that MAIT cells from tuberculous pleural effusions exhibited highly elevated immune response to Mtb antigens, which are controlled by cytokines produced by innate/adaptive immune cells.

Immune Impact Induced by PROSTVAC (PSA-TRICOM), a Therapeutic Vaccine for Prostate Cancer

PubMed Central

Gulley, James L.; Madan, Ravi A.; Tsang, Kwong Y.; Jochems, Caroline; Marté, Jennifer L.; Farsaci, Benedetto; Tucker, Jo A.; Hodge, James W.; Liewehr, David J.; Steinberg, Seth M.; Heery, Christopher R.; Schlom, Jeffrey

2013-01-01

PSA-TRICOM (PROSTVAC) is a novel vector-based vaccine designed to generate a robust immune response against prostate-specific antigen (PSA)–expressing tumor cells. The purpose of this report is to present an overview of both published studies and new data in the evaluation of immune responses to the PSA-TRICOM vaccine platform, currently in phase III testing. Of 104 patients tested for T-cell responses, 57% (59/104) demonstrated a ≥ 2-fold increase in PSA-specific T cells 4 weeks after vaccine (median 5-fold increase) compared with pre-vaccine, and 68% (19/28) of patients tested mounted post-vaccine immune responses to tumor-associated antigens not present in the vaccine (antigen-spreading). The PSA-specific immune responses observed 28 days after vaccine (i.e., likely memory cells) are quantitatively similar to the levels of circulating T cells specific for influenza seen in the same patients. Measurements of systemic immune response to PSA may underestimate the true therapeutic immune response (as this does not account for cells that have trafficked to the tumor) and does not include antigen-spreading. Furthermore, while the entire PSA gene is the vaccine, only one epitope of PSA is evaluated in the T-cell responses. Since this therapeutic vaccine is directed at generating a cellular/Th1 immune response (T-cell costimulatory molecules and use of a viral vector), it is not surprising that < 0.6% of patients (2/349) tested have evidence of PSA antibody-induction following vaccine. This suggests that post-vaccine PSA kinetics were not affected by PSA antibodies. An ongoing phase III study will evaluate the systemic immune responses and correlation with clinical outcomes. PMID:24778277

A Systems Vaccinology Approach Reveals Temporal Transcriptomic Changes of Immune Responses to the Yellow Fever 17D Vaccine.

PubMed

Hou, Jue; Wang, Shuhui; Jia, Manxue; Li, Dan; Liu, Ying; Li, Zhengpeng; Zhu, Hong; Xu, Huifang; Sun, Meiping; Lu, Li; Zhou, Zhinan; Peng, Hong; Zhang, Qichen; Fu, Shihong; Liang, Guodong; Yao, Lena; Yu, Xuesong; Carpp, Lindsay N; Huang, Yunda; McElrath, Julie; Self, Steve; Shao, Yiming

2017-08-15

In this study, we used a systems vaccinology approach to identify temporal changes in immune response signatures to the yellow fever (YF)-17D vaccine, with the aim of comprehensively characterizing immune responses associated with protective immunity. We conducted a cohort study in which 21 healthy subjects in China were administered one dose of the YF-17D vaccine; PBMCs were collected at 0 h and then at 4 h and days 1, 2, 3, 5, 7, 14, 28, 84, and 168 postvaccination, and analyzed by transcriptional profiling and immunological assays. At 4 h postvaccination, genes associated with innate cell differentiation and cytokine pathways were dramatically downregulated, whereas receptor genes were upregulated, compared with their baseline levels at 0 h. Immune response pathways were primarily upregulated on days 5 and 7, accompanied by the upregulation of the transcriptional factors JUP, STAT1, and EIF2AK2. We also observed robust activation of innate immunity within 2 d postvaccination and a durable adaptive response, as assessed by transcriptional profiling. Coexpression network analysis indicated that lysosome activity and lymphocyte proliferation were associated with dendritic cell (DC) and CD4 + T cell responses; FGL2, NFAM1, CCR1, and TNFSF13B were involved in these associations. Moreover, individuals who were baseline-seropositive for Abs against another flavivirus exhibited significantly impaired DC, NK cell, and T cell function in response to YF-17D vaccination. Overall, our findings indicate that YF-17D vaccination induces a prompt innate immune response and DC activation, a robust Ag-specific T cell response, and a persistent B cell/memory B cell response. Copyright © 2017 by The American Association of Immunologists, Inc.

Randomized Multicenter Trial of the Effects of Melanoma-Associated Helper Peptides and Cyclophosphamide on the Immunogenicity of a Multipeptide Melanoma Vaccine

PubMed Central

Slingluff, Craig L.; Petroni, Gina R.; Chianese-Bullock, Kimberly A.; Smolkin, Mark E.; Ross, Merrick I.; Haas, Naomi B.; von Mehren, Margaret; Grosh, William W.

2011-01-01

Purpose This multicenter randomized trial was designed to test whether melanoma-associated helper peptides augment CD8+ T-cell responses to a melanoma vaccine and whether cyclophosphamide (CY) pretreatment augments CD4+ or CD8+ T-cell responses to that vaccine. Patients and Methods In all, 167 eligible patients with resected stage IIB to IV melanoma were randomly assigned to four vaccination study arms. Patients were vaccinated with 12 class I major histocompatibility complex–restricted melanoma peptides (12MP) to stimulate CD8+ T cells and were randomly assigned to receive a tetanus helper peptide or a mixture of six melanoma-associated helper peptides (6MHP) to stimulate CD4+ T cells. Before vaccination, patients were also randomly assigned to receive CY pretreatment or not. T-cell responses were assessed by an ex vivo interferon gamma ELISpot assay. Clinical outcomes and toxicities were recorded. Results Vaccination with 12MP plus tetanus induced CD8+ T-cell responses in 78% of patients and CD4+ T-cell responses to tetanus peptide in 93% of patients. Vaccination with 12MP plus 6MHP induced CD8+ responses in 19% of patients and CD4+ responses to 6MHP in 48% of patients. CY had no significant effect on T-cell responses. Overall 3-year survival was 79% (95% CI, 71% to 86%), with no significant differences (at this point) by study arm. Conclusion Melanoma-associated helper peptides paradoxically decreased CD8+ T-cell responses to a melanoma vaccine (P < .001), and CY pretreatment had no immunologic or clinical effect. Prior work showed immunologic and clinical activity of 6MHP alone. Possible explanations for negative effects on CD8 responses include modulation of homing receptor expression or induction of antigen-specific regulatory T cells. PMID:21690475

Red Blood Cell Susceptibility to Pneumolysin

PubMed Central

Bokori-Brown, Monika; Petrov, Peter G.; Khafaji, Mawya A.; Mughal, Muhammad K.; Naylor, Claire E.; Shore, Angela C.; Gooding, Kim M.; Casanova, Francesco; Mitchell, Tim J.; Titball, Richard W.; Winlove, C. Peter

2016-01-01

This study investigated the effect of the biochemical and biophysical properties of the plasma membrane as well as membrane morphology on the susceptibility of human red blood cells to the cholesterol-dependent cytolysin pneumolysin, a key virulence factor of Streptococcus pneumoniae, using single cell studies. We show a correlation between the physical properties of the membrane (bending rigidity and surface and dipole electrostatic potentials) and the susceptibility of red blood cells to pneumolysin-induced hemolysis. We demonstrate that biochemical modifications of the membrane induced by oxidative stress, lipid scrambling, and artificial cell aging modulate the cell response to the toxin. We provide evidence that the diversity of response to pneumolysin in diabetic red blood cells correlates with levels of glycated hemoglobin and that the mechanical properties of the red blood cell plasma membrane are altered in diabetes. Finally, we show that diabetic red blood cells are more resistant to pneumolysin and the related toxin perfringolysin O relative to healthy red blood cells. Taken together, these studies indicate that the diversity of cell response to pneumolysin within a population of human red blood cells is influenced by the biophysical and biochemical status of the plasma membrane and the chemical and/or oxidative stress pre-history of the cell. PMID:26984406

Induction of potent NK cell-dependent anti-myeloma cytotoxic T cells in response to combined mapatumumab and bortezomib.

PubMed

Neeson, Paul J; Hsu, Andy K; Chen, Yin R; Halse, Heloise M; Loh, Joanna; Cordy, Reece; Fielding, Kate; Davis, Joanne; Noske, Josh; Davenport, Alex J; Lindqvist-Gigg, Camilla A; Humphreys, Robin; Tai, Tsin; Prince, H Miles; Trapani, Joseph A; Smyth, Mark J; Ritchie, David S

2015-09-01

There is increasing evidence that some cancer therapies can promote tumor immunogenicity to boost the endogenous antitumor immune response. In this study, we used the novel combination of agonistic anti-TRAIL-R1 antibody (mapatumumab, Mapa) with low dose bortezomib (LDB) for this purpose. The combination induced profound myeloma cell apoptosis, greatly enhanced the uptake of myeloma cell apoptotic bodies by dendritic cell (DC) and induced anti-myeloma cytotoxicity by both CD8 + T cells and NK cells. Cytotoxic lymphocyte expansion was detected within 24 h of commencing therapy and was maximized when myeloma-pulsed DC were co-treated with low dose bortezomib and mapatumumab (LDB+Mapa) in the presence of NK cells. This study shows that Mapa has two distinct but connected modes of action against multiple myeloma (MM). First, when combined with LDB, Mapa produced powerful myeloma cell apoptosis; secondly, it promoted DC priming and an NK cell-mediated expansion of anti-myeloma cytotoxic lymphocyte (CTL). Overall, this study indicates that Mapa can be used to drive potent anti-MM immune responses.

Induction of potent NK cell-dependent anti-myeloma cytotoxic T cells in response to combined mapatumumab and bortezomib

PubMed Central

Neeson, Paul J; Hsu, Andy K; Chen, Yin R; Halse, Heloise M; Loh, Joanna; Cordy, Reece; Fielding, Kate; Davis, Joanne; Noske, Josh; Davenport, Alex J; Lindqvist-Gigg, Camilla A; Humphreys, Robin; Tai, Tsin; Prince, H Miles; Trapani, Joseph A; Smyth, Mark J; Ritchie, David S

2015-01-01

There is increasing evidence that some cancer therapies can promote tumor immunogenicity to boost the endogenous antitumor immune response. In this study, we used the novel combination of agonistic anti-TRAIL-R1 antibody (mapatumumab, Mapa) with low dose bortezomib (LDB) for this purpose. The combination induced profound myeloma cell apoptosis, greatly enhanced the uptake of myeloma cell apoptotic bodies by dendritic cell (DC) and induced anti-myeloma cytotoxicity by both CD8+ T cells and NK cells. Cytotoxic lymphocyte expansion was detected within 24 h of commencing therapy and was maximized when myeloma-pulsed DC were co-treated with low dose bortezomib and mapatumumab (LDB+Mapa) in the presence of NK cells. This study shows that Mapa has two distinct but connected modes of action against multiple myeloma (MM). First, when combined with LDB, Mapa produced powerful myeloma cell apoptosis; secondly, it promoted DC priming and an NK cell-mediated expansion of anti-myeloma cytotoxic lymphocyte (CTL). Overall, this study indicates that Mapa can be used to drive potent anti-MM immune responses. PMID:26405606

Feasibility studies of using the Catfish Immune System to produce monoclonal antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poston, T.M.

1987-03-01

The objective of these studies was to determine the feasibility of using a teleost cell line to produce monoclonal antibodies. Studies were undertaken to demonstrate the production of a polyclonal response of channel catfish (Icatalurus punctatus) challenged with mycotoxins coupled to a protein carrier. Companion studies were also performed to induce a permanent cell line with catfish lymphocytes. Attempts to demonstrate a polyclonal response to haptenized mycotoxins were inconclusive. Tests to induce an immortal, permanent cell line with benzene and x-ray irradiated cells were also inconclusive. 3 refs., 13 tabs.

HIV infection-induced transcriptional program in renal tubular epithelial cells activates a CXCR2-driven CD4+ T-cell chemotactic response.

PubMed

Chen, Ping; Yi, Zhengzi; Zhang, Weijia; Klotman, Mary E; Chen, Benjamin K

2016-07-31

Viral replication and interstitial inflammation play important roles in the pathogenesis of HIV-associated nephropathy. Cell-cell interactions between renal tubule epithelial cells (RTECs) and HIV-infected T cells can trigger efficient virus internalization and viral gene expression by RTEC. To understand how HIV replication initiates HIV-associated nephropathy, we studied the cellular response of RTECs to HIV, examining the transcriptional profiles of primary RTECs exposed to cell-free HIV or HIV-infected T cells. HIV-induced gene expression in hRTECs was examined in vitro by Illumina RNA deep sequencing and revealed an innate response to HIV, which was subclassified by gene ontology biological process terms. Chemokine responses were examined by CD4 T-cell chemotaxis assays. As compared with cell-free virus infection, exposure to HIV-infected T cells elicited a stronger upregulation of inflammatory and immune response genes. A major category of upregulated genes are chemokine/cytokine families involved in inflammation and immune response, including inflammatory cytokines CCL20, IL6 and IL8-related chemokines: IL8, CXCL1, CXCL2, CXCL3, CXCL5 and CXCL6. Supernatants from virus-exposed RTECs contained strong chemoattractant activity on primary CD4 T cells, which was potently blocked by a CXCR2 antagonist that antagonizes IL8-related chemokines. We observed a preferential migration of CXCR2-expressing, central memory CD4 T cells in response to HIV infection of RTECs. Interactions between primary RTECs and HIV-infected T cells result in potent induction of inflammatory response genes and release of cytokines/chemokines from RTECs that can attract additional T cells. Activation of these genes reflects an innate response to HIV by nonimmune cells.

Dual-responsive surfaces modified with phenylboronic acid-containing polymer brush to reversibly capture and release cancer cells.

PubMed

Liu, Hongliang; Li, Yingying; Sun, Kang; Fan, Junbing; Zhang, Pengchao; Meng, Jingxin; Wang, Shutao; Jiang, Lei

2013-05-22

Artificial stimuli-responsive surfaces that can mimic the dynamic function of living systems have attracted much attention. However, there exist few artificial systems capable of responding to dual- or multistimulation as the natural system does. Herein, we synthesize a pH and glucose dual-responsive surface by grafting poly(acrylamidophenylboronic acid) (polyAAPBA) brush from aligned silicon nanowire (SiNW) array. The as-prepared surface can reversibly capture and release targeted cancer cells by precisely controlling pH and glucose concentration, exhibiting dual-responsive AND logic. In the presence of 70 mM glucose, the surface is pH responsive, which can vary from a cell-adhesive state to a cell-repulsive state by changing the pH from 6.8 to 7.8. While keeping the pH at 7.8, the surface becomes glucose responsive--capturing cells in the absence of glucose and releasing cells by adding 70 mM glucose. Through simultaneously changing the pH and glucose concentration from pH 6.8/0 mM glucose to pH 7.8/70 mM glucose, the surface is dual responsive with the capability to switch between cell capture and release for at least 5 cycles. The cell capture and release process on this dual-responsive surface is noninvasive with cell viability higher than 95%. Moreover, topographical interaction between the aligned SiNW array and cell protrusions greatly amplifies the responsiveness and accelerates the response rate of the dual-responsive surface between cell capture and release. The responsive mechanism of the dual-responsive surface is systematically studied using a quartz crystal microbalance, which shows that the competitive binding between polyAAPBA/sialic acid and polyAAPBA/glucose contributes to the dual response. Such dual-responsive surface can significantly impact biomedical and biological applications including cell-based diagnostics, in vivo drug delivery, etc.

Protein nanoparticles are nontoxic, tuneable cell stressors.

PubMed

de Pinho Favaro, Marianna Teixeira; Sánchez-García, Laura; Sánchez-Chardi, Alejandro; Roldán, Mónica; Unzueta, Ugutz; Serna, Naroa; Cano-Garrido, Olivia; Azzoni, Adriano Rodrigues; Ferrer-Miralles, Neus; Villaverde, Antonio; Vázquez, Esther

2018-02-01

Nanoparticle-cell interactions can promote cell toxicity and stimulate particular behavioral patterns, but cell responses to protein nanomaterials have been poorly studied. By repositioning oligomerization domains in a simple, modular self-assembling protein platform, we have generated closely related but distinguishable homomeric nanoparticles. Composed by building blocks with modular domains arranged in different order, they share amino acid composition. These materials, once exposed to cultured cells, are differentially internalized in absence of toxicity and trigger distinctive cell adaptive responses, monitored by the emission of tubular filopodia and enhanced drug sensitivity. The capability to rapidly modulate such cell responses by conventional protein engineering reveals protein nanoparticles as tuneable, versatile and potent cell stressors for cell-targeted conditioning.

LAG-3 confers a competitive disadvantage upon antiviral CD8+ T cell responses1

PubMed Central

Cook, Kevin D.; Whitmire, Jason K.

2016-01-01

Ongoing clinical trials are evaluating the benefits of systemic blockade of lymphocyte activation gene-3 (LAG-3) signals to improve immunity to tumors. Those studies are founded on the well-established inhibitory role of LAG-3 in regulating CD8+ T cells during chronic virus infection and anti-tumor responses. However, the T cell response in LAG-3 deficient mice is similar in size and function to that in wild type animals, suggesting LAG-3 has nuanced immune-regulatory functions. We performed a series of adoptive transfer experiments in mice to better understand the T cell-intrinsic functions of LAG-3 in the regulation of CD8+ T cell responses. Our results indicate that LAG-3 expression by CD8+ T cells inhibits their competitive fitness and results in a slightly reduced rate of cell division in comparison to LAG-3 deficient cells. This cell-intrinsic effect of LAG-3 was consistent across both acute and chronic virus infections. These data show that LAG-3 directly modulates the size of the T cell response and support the use of LAG-3 blockade regimens to enhance CD8+ T cell responses. PMID:27206765

LAG-3 Confers a Competitive Disadvantage upon Antiviral CD8+ T Cell Responses.

PubMed

Cook, Kevin D; Whitmire, Jason K

2016-07-01

Ongoing clinical trials are evaluating the benefits of systemic blockade of lymphocyte activation gene-3 (LAG-3) signals to improve immunity to tumors. Those studies are founded on the well-established inhibitory role of LAG-3 in regulating CD8(+) T cells during chronic virus infection and antitumor responses. However, the T cell response in LAG-3-deficient mice is similar in size and function to that in wild type animals, suggesting LAG-3 has nuanced immune-regulatory functions. We performed a series of adoptive transfer experiments in mice to better understand the T cell-intrinsic functions of LAG-3 in the regulation of CD8(+) T cell responses. Our results indicate that LAG-3 expression by CD8(+) T cells inhibits their competitive fitness and results in a slightly reduced rate of cell division in comparison with LAG-3-deficient cells. This cell-intrinsic effect of LAG-3 was consistent across both acute and chronic virus infections. These data show that LAG-3 directly modulates the size of the T cell response and support the use of LAG-3 blockade regimens to enhance CD8(+) T cell responses. Copyright © 2016 by The American Association of Immunologists, Inc.

Immune activation alters cellular and humoral responses to yellow fever 17D vaccine

PubMed Central

Muyanja, Enoch; Ssemaganda, Aloysius; Ngauv, Pearline; Cubas, Rafael; Perrin, Helene; Srinivasan, Divya; Canderan, Glenda; Lawson, Benton; Kopycinski, Jakub; Graham, Amanda S.; Rowe, Dawne K.; Smith, Michaela J.; Isern, Sharon; Michael, Scott; Silvestri, Guido; Vanderford, Thomas H.; Castro, Erika; Pantaleo, Giuseppe; Singer, Joel; Gillmour, Jill; Kiwanuka, Noah; Nanvubya, Annet; Schmidt, Claudia; Birungi, Josephine; Cox, Josephine; Haddad, Elias K.; Kaleebu, Pontiano; Fast, Patricia; Sekaly, Rafick-Pierre; Trautmann, Lydie

2014-01-01

Background. Defining the parameters that modulate vaccine responses in African populations will be imperative to design effective vaccines for protection against HIV, malaria, tuberculosis, and dengue virus infections. This study aimed to evaluate the contribution of the patient-specific immune microenvironment to the response to the licensed yellow fever vaccine 17D (YF-17D) in an African cohort. Methods. We compared responses to YF-17D in 50 volunteers in Entebbe, Uganda, and 50 volunteers in Lausanne, Switzerland. We measured the CD8+ T cell and B cell responses induced by YF-17D and correlated them with immune parameters analyzed by flow cytometry prior to vaccination. Results. We showed that YF-17D–induced CD8+ T cell and B cell responses were substantially lower in immunized individuals from Entebbe compared with immunized individuals from Lausanne. The impaired vaccine response in the Entebbe cohort associated with reduced YF-17D replication. Prior to vaccination, we observed higher frequencies of exhausted and activated NK cells, differentiated T and B cell subsets and proinflammatory monocytes, suggesting an activated immune microenvironment in the Entebbe volunteers. Interestingly, activation of CD8+ T cells and B cells as well as proinflammatory monocytes at baseline negatively correlated with YF-17D–neutralizing antibody titers after vaccination. Additionally, memory T and B cell responses in preimmunized volunteers exhibited reduced persistence in the Entebbe cohort but were boosted by a second vaccination. Conclusion. Together, these results demonstrate that an activated immune microenvironment prior to vaccination impedes efficacy of the YF-17D vaccine in an African cohort and suggest that vaccine regimens may need to be boosted in African populations to achieve efficient immunity. Trial registration. Registration is not required for observational studies. Funding. This study was funded by Canada’s Global Health Research Initiative, Defense Threat Reduction Agency, National Institute of Allergy and Infectious Diseases, Bill & Melinda Gates Foundation, and United States Agency for International Development. PMID:24911151

Immune activation alters cellular and humoral responses to yellow fever 17D vaccine.

PubMed

Muyanja, Enoch; Ssemaganda, Aloysius; Ngauv, Pearline; Cubas, Rafael; Perrin, Helene; Srinivasan, Divya; Canderan, Glenda; Lawson, Benton; Kopycinski, Jakub; Graham, Amanda S; Rowe, Dawne K; Smith, Michaela J; Isern, Sharon; Michael, Scott; Silvestri, Guido; Vanderford, Thomas H; Castro, Erika; Pantaleo, Giuseppe; Singer, Joel; Gillmour, Jill; Kiwanuka, Noah; Nanvubya, Annet; Schmidt, Claudia; Birungi, Josephine; Cox, Josephine; Haddad, Elias K; Kaleebu, Pontiano; Fast, Patricia; Sekaly, Rafick-Pierre; Trautmann, Lydie; Gaucher, Denis

2014-07-01

Defining the parameters that modulate vaccine responses in African populations will be imperative to design effective vaccines for protection against HIV, malaria, tuberculosis, and dengue virus infections. This study aimed to evaluate the contribution of the patient-specific immune microenvironment to the response to the licensed yellow fever vaccine 17D (YF-17D) in an African cohort. We compared responses to YF-17D in 50 volunteers in Entebbe, Uganda, and 50 volunteers in Lausanne, Switzerland. We measured the CD8+ T cell and B cell responses induced by YF-17D and correlated them with immune parameters analyzed by flow cytometry prior to vaccination. We showed that YF-17D-induced CD8+ T cell and B cell responses were substantially lower in immunized individuals from Entebbe compared with immunized individuals from Lausanne. The impaired vaccine response in the Entebbe cohort associated with reduced YF-17D replication. Prior to vaccination, we observed higher frequencies of exhausted and activated NK cells, differentiated T and B cell subsets and proinflammatory monocytes, suggesting an activated immune microenvironment in the Entebbe volunteers. Interestingly, activation of CD8+ T cells and B cells as well as proinflammatory monocytes at baseline negatively correlated with YF-17D-neutralizing antibody titers after vaccination. Additionally, memory T and B cell responses in preimmunized volunteers exhibited reduced persistence in the Entebbe cohort but were boosted by a second vaccination. Together, these results demonstrate that an activated immune microenvironment prior to vaccination impedes efficacy of the YF-17D vaccine in an African cohort and suggest that vaccine regimens may need to be boosted in African populations to achieve efficient immunity. Registration is not required for observational studies. This study was funded by Canada's Global Health Research Initiative, Defense Threat Reduction Agency, National Institute of Allergy and Infectious Diseases, Bill & Melinda Gates Foundation, and United States Agency for International Development.

Memory T cell proliferative responses and IFN-γ productivity sustain long-lasting efficacy of a Cap-based PCV2 vaccine upon PCV2 natural infection and associated disease.

PubMed

Ferrari, Luca; Borghetti, Paolo; De Angelis, Elena; Martelli, Paolo

2014-04-16

Porcine circovirus type 2 (PCV2) vaccination represents an important measure to cope with PCV2 infection; however, data regarding the modulation of the immune cell compartment are still limited, especially under field conditions. This study is aimed at investigating the features of the cellular immune response in conventional piglets induced by vaccination using a capsid (Cap) protein-based PCV2 vaccine compared to unvaccinated animals when exposed to PCV2 natural infection. Immune reactivity was evaluated by quantifying peripheral cell subsets involved in the anti-viral response and characterizing the interferon-gamma (IFN-γ) secreting cell (SC) responsiveness both in vivo and upon in vitro whole PCV2 recall. The vaccination triggered an early and intense IFN-γ secreting cell response and induced the activation of peripheral lymphocytes. The early increase of IFN-γ SC frequencies resulted in a remarkable and transient tendency to increased IFN-γ productivity in vaccinated pigs. In vaccinated animals, soon before the onset of infection occurred 15-16 weeks post-vaccination, the recalled PCV2-specific immune response was characterized by moderate PCV2-specific IFN-γ secreting cell frequencies and augmented productivity together with reactive CD4+CD8+ memory T cells. Conversely, upon infection, unvaccinated animals showed very high frequencies of IFN-γ secreting cells and a tendency to lower productivity, which paralleled with effector CD4-CD8+ cytotoxic cell responsiveness. The study shows that PCV2 vaccination induces a long-lasting immunity sustained by memory T cells and IFN-γ secreting cells that potentially played a role in preventing the onset of infection; the extent and duration of this reactivity can be an important feature for evaluating the protective immunity induced by vaccination.

A Numerically Subdominant CD8 T Cell Response to Matrix Protein of Respiratory Syncytial Virus Controls Infection with Limited Immunopathology

PubMed Central

Liu, Jie; Haddad, Elias K.; Marceau, Joshua; Morabito, Kaitlyn M.; Rao, Srinivas S.; Filali-Mouhim, Ali; Sekaly, Rafick-Pierre; Graham, Barney S.

2016-01-01

CD8 T cells are involved in pathogen clearance and infection-induced pathology in respiratory syncytial virus (RSV) infection. Studying bulk responses masks the contribution of individual CD8 T cell subsets to protective immunity and immunopathology. In particular, the roles of subdominant responses that are potentially beneficial to the host are rarely appreciated when the focus is on magnitude instead of quality of response. Here, by evaluating CD8 T cell responses in CB6F1 hybrid mice, in which multiple epitopes are recognized, we found that a numerically subdominant CD8 T cell response against DbM187 epitope of the virus matrix protein expressed high avidity TCR and enhanced signaling pathways associated with CD8 T cell effector functions. Each DbM187 T effector cell lysed more infected targets on a per cell basis than the numerically dominant KdM282 T cells, and controlled virus replication more efficiently with less pulmonary inflammation and illness than the previously well-characterized KdM282 T cell response. Our data suggest that the clinical outcome of viral infections is determined by the integrated functional properties of a variety of responding CD8 T cells, and that the highest magnitude response may not necessarily be the best in terms of benefit to the host. Understanding how to induce highly efficient and functional T cells would inform strategies for designing vaccines intended to provide T cell-mediated immunity. PMID:26943673

Long-Term Persistence of Cell-Mediated and Humoral Responses to A(H1N1)pdm09 Influenza Virus Vaccines and the Role of the AS03 Adjuvant System in Adults during Two Randomized Controlled Trials

PubMed Central

Clément, Frédéric; Willekens, Julie; Dewé, Walthère; Walravens, Karl; Vaughn, David W.; Leroux-Roels, Geert

2017-01-01

ABSTRACT We investigated the role of AS03A (here AS03), an α-tocopherol oil-in-water emulsion-based adjuvant system, on the long-term persistence of humoral and cell-mediated immune responses to A(H1N1)pdm09 influenza vaccines. In two studies, a total of 261 healthy adults (≤60 years old) were randomized to receive two doses of AS03-adjuvanted vaccine containing 3.75 μg of hemagglutinin (HA) or nonadjuvanted vaccine containing 15 μg of hemagglutinin (in study A) or 3.75 μg of hemagglutinin (in study B) 21 days apart. Hemagglutination inhibition (HI) antibody, memory B-cell, and CD4+/CD8+ T-cell responses were characterized up to 1 year following dose 1. We also assessed the effects of age and seasonal influenza vaccination history. AS03-adjuvanted (3.75 μg HA) vaccine and nonadjuvanted vaccine at 15 μg but not at 3.75 μg HA elicited HI antibody responses persisting at levels that continued to meet European licensure criteria through month 12. At month 12, the geometric mean titer for AS03-adjuvanted vaccine was similar to that for nonadjuvanted (15-μg) vaccine in study A (1:86 and 1:88, respectively) and higher than that for nonadjuvanted (3.75-μg) vaccine in study B (1:77 and 1:35, respectively). A(H1N1)pdm09-specific CD4+ T-cell and B-cell responses were stronger in AS03-adjuvanted groups and persisted only in these groups for 12 months at levels exceeding prevaccination frequencies. Advancing age and a seasonal vaccination history tended to reduce HI antibody and memory B-cell responses and, albeit less consistently, CD4+ T-cell responses. Thus, AS03 seemed to enhance the persistence of humoral and cell-mediated responses to A(H1N1)pdm09 vaccine, allowing for antigen sparing and mitigating potential negative effects of age and previous seasonal vaccination. (These studies have been registered at ClinicalTrials.gov under registration no. NCT00968539 and NCT00989287.) PMID:28446441

Long-Term Persistence of Cell-Mediated and Humoral Responses to A(H1N1)pdm09 Influenza Virus Vaccines and the Role of the AS03 Adjuvant System in Adults during Two Randomized Controlled Trials.

PubMed

van der Most, Robbert G; Clément, Frédéric; Willekens, Julie; Dewé, Walthère; Walravens, Karl; Vaughn, David W; Leroux-Roels, Geert

2017-06-01

We investigated the role of AS03 A (here AS03), an α-tocopherol oil-in-water emulsion-based adjuvant system, on the long-term persistence of humoral and cell-mediated immune responses to A(H1N1)pdm09 influenza vaccines. In two studies, a total of 261 healthy adults (≤60 years old) were randomized to receive two doses of AS03-adjuvanted vaccine containing 3.75 μg of hemagglutinin (HA) or nonadjuvanted vaccine containing 15 μg of hemagglutinin (in study A) or 3.75 μg of hemagglutinin (in study B) 21 days apart. Hemagglutination inhibition (HI) antibody, memory B-cell, and CD4 + /CD8 + T-cell responses were characterized up to 1 year following dose 1. We also assessed the effects of age and seasonal influenza vaccination history. AS03-adjuvanted (3.75 μg HA) vaccine and nonadjuvanted vaccine at 15 μg but not at 3.75 μg HA elicited HI antibody responses persisting at levels that continued to meet European licensure criteria through month 12. At month 12, the geometric mean titer for AS03-adjuvanted vaccine was similar to that for nonadjuvanted (15-μg) vaccine in study A (1:86 and 1:88, respectively) and higher than that for nonadjuvanted (3.75-μg) vaccine in study B (1:77 and 1:35, respectively). A(H1N1)pdm09-specific CD4 + T-cell and B-cell responses were stronger in AS03-adjuvanted groups and persisted only in these groups for 12 months at levels exceeding prevaccination frequencies. Advancing age and a seasonal vaccination history tended to reduce HI antibody and memory B-cell responses and, albeit less consistently, CD4 + T-cell responses. Thus, AS03 seemed to enhance the persistence of humoral and cell-mediated responses to A(H1N1)pdm09 vaccine, allowing for antigen sparing and mitigating potential negative effects of age and previous seasonal vaccination. (These studies have been registered at ClinicalTrials.gov under registration no. NCT00968539 and NCT00989287.). Copyright © 2017 van der Most et al.

Fish T cells: recent advances through genomics

USGS Publications Warehouse

Laing, Kerry J.; Hansen, John D.

2011-01-01

This brief review is intended to provide a concise overview of the current literature concerning T cells, advances in identifying distinct T cell functional subsets, and in distinguishing effector cells from memory cells. We compare and contrast a wealth of recent progress made in T cell immunology of teleost, elasmobranch, and agnathan fish, to knowledge derived from mammalian T cell studies. From genome studies, fish clearly have most components associated with T cell function and we can speculate on the presence of putative T cell subsets, and the ability to detect their differentiation to form memory cells. Some recombinant proteins for T cell associated cytokines and antibodies for T cell surface receptors have been generated that will facilitate studying the functional roles of teleost T cells during immune responses. Although there is still a long way to go, major advances have occurred in recent years for investigating T cell responses, thus phenotypic and functional characterization is on the near horizon.

Diet-induced obesity does not impact the generation and maintenance of primary memory CD8 T cells.

PubMed

Khan, Shaniya H; Hemann, Emily A; Legge, Kevin L; Norian, Lyse A; Badovinac, Vladimir P

2014-12-15

The extent to which obesity compromises the differentiation and maintenance of protective memory CD8 T cell responses and renders obese individuals susceptible to infection remains unknown. In this study, we show that diet-induced obesity did not impact the maintenance of pre-existing memory CD8 T cells, including acquisition of a long-term memory phenotype (i.e., CD27(hi), CD62L(hi), KLRG1(lo)) and function (i.e., cytokine production, secondary expansion, and memory CD8 T cell-mediated protection). Additionally, obesity did not influence the differentiation and maintenance of newly evoked memory CD8 T cell responses in inbred and outbred hosts generated in response to different types of systemic (LCMV, L. monocytogenes) and/or localized (influenza virus) infections. Interestingly, the rate of naive-to-memory CD8 T cell differentiation after a peptide-coated dendritic cell immunization was similar in lean and obese hosts, suggesting that obesity-associated inflammation, unlike pathogen- or adjuvant-induced inflammation, did not influence the development of endogenous memory CD8 T cell responses. Therefore, our studies reveal that the obese environment does not influence the development or maintenance of memory CD8 T cell responses that are either primed before or after obesity is established, a surprising notion with important implications for future studies aiming to elucidate the role obesity plays in host susceptibility to infections. Copyright © 2014 by The American Association of Immunologists, Inc.

Hit parade for adoptive cell transfer therapy: the best T cells for superior clinical responses.

PubMed

Speiser, Daniel E

2013-04-01

Adoptive cell transfer (ACT) of T cells has great clinical potential, but the numerous variables of this therapy make choices difficult. A new study takes advantage of a novel technology for characterizing the T-cell responses of patients. If applied systematically, this approach may identify biomedical correlates of protection, thereby supporting treatment optimization. ©2013 AACR.

A Simple Proteomics-Based Approach to Identification of Immunodominant Antigens from a Complex Pathogen: Application to the CD4 T Cell Response against Human Herpesvirus 6B

PubMed Central

Becerra-Artiles, Aniuska; Dominguez-Amorocho, Omar; Stern, Lawrence J.; Calvo-Calle, J. Mauricio

2015-01-01

Most of humanity is chronically infected with human herpesvirus 6 (HHV-6), with viral replication controlled at least in part by a poorly characterized CD4 T cell response. Identification of viral epitopes recognized by CD4 T cells is complicated by the large size of the herpesvirus genome and a low frequency of circulating T cells responding to the virus. Here, we present an alternative to classical epitope mapping approaches used to identify major targets of the T cell response to a complex pathogen like HHV-6B. In the approach presented here, extracellular virus preparations or virus-infected cells are fractionated by SDS-PAGE, and eluted fractions are used as source of antigens to study cytokine responses in direct ex vivo T cell activation studies. Fractions inducing significant cytokine responses are analyzed by mass spectrometry to identify viral proteins, and a subset of peptides from these proteins corresponding to predicted HLA-DR binders is tested for IFN-γ production in seropositive donors with diverse HLA haplotypes. Ten HHV-6B viral proteins were identified as immunodominant antigens. The epitope-specific response to HHV-6B virus was complex and variable between individuals. We identified 107 peptides, each recognized by at least one donor, with each donor having a distinctive footprint. Fourteen peptides showed responses in the majority of donors. Responses to these epitopes were validated using in vitro expanded cells and naturally expressed viral proteins. Predicted peptide binding affinities for the eight HLA-DRB1 alleles investigated here correlated only modestly with the observed CD4 T cell responses. Overall, the response to the virus was dominated by peptides from the major capsid protein U57 and major antigenic protein U11, but responses to other proteins including glycoprotein H (U48) and tegument proteins U54 and U14 also were observed. These results provide a means to follow and potentially modulate the CD4 T-cell immune response to HHV-6B. PMID:26599878

Implications of Differential Stress Response Activation Following Non-Frozen Hepatocellular Storage

PubMed Central

Corwin, William L.; Baust, John G.; Van Buskirk, Robert G.

2013-01-01

Hepatocytes are critical for numerous cell therapies and in vitro investigations. A limiting factor for their use in these applications is the ability to process and preserve them without loss of viability or functionality. Normal rat hepatocytes (NHEPs) and human hepatoma (C3A) cells were stored at either 4°C or 37°C to examine post-processing stress responses. Resveratrol and salubrinal were used during storage to determine how targeted molecular stress pathway modulation would affect cell survival. This study revealed that storage outcome is dependent upon numerous factors including: cell type, storage media, storage length, storage temperature, and chemical modulator. These data implicate a molecular-based stress response that is not universal but is specific to the set of conditions under which cells are stored. Further, these findings allude to the potential for targeted protection or destruction of particular cell types for numerous applications, from diagnostic cell selection to cell-based therapy. Ultimately, this study demonstrates the need for further in-depth molecular investigations into the cellular stress response to bioprocessing and preservation. PMID:24845253

Responses to A(H1N1)pdm09 Influenza Vaccines in Participants Previously Vaccinated With Seasonal Influenza Vaccine: A Randomized, Observer-Blind, Controlled Study

PubMed Central

Roy-Ghanta, Sumita; Van der Most, Robbert; Li, Ping; Vaughn, David W.

2014-01-01

Background. Prior receipt of a trivalent seasonal influenza vaccine (TIV) can affect hemagglutination inhibition (HI) antibody responses to pandemic influenza vaccines. We investigated the effect of TIV priming on humoral responses to AS03-adjuvanted and nonadjuvanted A(H1N1)pdm09 vaccines, the role of AS03 on cell-mediated immune (CMI) responses, and vaccine safety. Methods. Healthy adults (aged 19–40 years) were randomized 1:1:1:1 to receive TIV or saline followed 4 months later by 2 doses, 3 weeks apart, of adjuvanted or nonadjuvanted A(H1N1)pdm09 vaccine and followed up to study end (day 507). Pre- and postvaccination responses of HI and neutralizing antibody, CD4+/CD8+ T cells, memory B cells, and plasmablasts were assessed. Results. Ninety-nine of the 133 participants enrolled completed the study. No vaccine-related serious adverse events were recorded. In TIV-primed participants, A(H1N1)pdm09-specific antibody and CD4+ T-cell and memory B-cell responses to the pandemic vaccine tended to be diminished. Vaccine adjuvantation led to increased responses of vaccine-homologous and -heterologous HI and neutralizing antibodies and CD4+ T cells, homologous memory B cells, and plasmablasts. Conclusions. In healthy adults, prior TIV administration decreased humoral and CMI responses to A(H1N1)pdm09 vaccine. Adjuvantation of A(H1N1)pdm09 antigen helped to overcome immune interference between the influenza vaccines. No safety concerns were observed. Registration. Clinical Trials.gov identifier NCT00707967. PMID:24864125

Felix Hoppe-Seyler Lecture 1997. Protective antibody responses against viruses.

PubMed

Zinkernagel, R M

1997-08-01

Neutralizing antibody responses against the acute cytopathic vesicular stomatitis virus (VSV) have been studied in mice to evaluate their general characteristics including specificity, self-/non-self discrimination and memory. IgM responses are generated very early, by day 3 to 4, in a T helper cell-independent fashion and without VSV having polyclonal activating capacities. The order of the glycoprotein tips on the virus envelope (multiple, 8-10 nm distance, paracrystalline) exhibiting the neutralizing determinants are key to this prompt response. These paracrystalline identical multimeric antigens are characteristic of infectious agents and are always reacted against by B cells. Self-antigens that are accessible to B cells in the intact host are either monomeric in serum or mobile multimers on cell surfaces; these configurations need contact dependent or contact independent T help, respectively. Because T help is tolerant against self-antigens, no anti-self B cell responses are usually induced against monomeric self-antigens. If collagen or DNA (rigid multimeric self-antigens) become accessible, however, they may become targets of auto-antibody responses. The antibody repertoire against VSV is partially contained in the germline and partially is generated by somatic mutation; they seem not to undergo affinity-maturation. In any case protection against lethal infection is dependent upon strictly T helper cell dependent IgG generated by day 6 to 7 and reaches a protective level of about 1-10 micrograms/ml. Interesting affinity/avidity and onrate above a minimal threshold are of no apparent advantage for protection in vivo. Maintenance of these antibody levels by antigen depots, and not the presence of memory B cells alone, is key to providing protective immunological memory. Collectively these data suggest that studying biologically important protective antibody responses may modify some of the parameters that have been defined by studying hapten specific antibody responses.

Interferon type I responses to virus infections in carp cells: In vitro studies on Cyprinid herpesvirus 3 and Rhabdovirus carpio infections.

PubMed

Adamek, Mikołaj; Rakus, Krzysztof Ł; Chyb, Jarosław; Brogden, Graham; Huebner, Arne; Irnazarow, Ilgiz; Steinhagen, Dieter

2012-09-01

Interferons (IFNs) are secreted mediators that play a fundamental role in the innate immune response against viruses among all vertebrate classes. Common carp is a host for two highly contagious viruses: spring viraemia of carp virus (Rhabdovirus carpio, SVCV) and the Cyprinid herpesvirus 3 (CyHV-3), which belong to Rhabdoviridae and Alloherpesviridae families, respectively. Both viruses are responsible for significant losses in carp aquaculture. In this paper we studied the mRNA expression profiles of genes encoding for proteins promoting various functions during the interferon pathway, from pattern recognition receptors to antiviral genes, during in vitro viral infection. Furthermore, we investigated the impact of the interferon pathway (stimulated with poly I:C) on CyHV-3 replication and the speed of virus spreading in cell culture. The results showed that two carp viruses, CyHV-3 and SVCV induced fundamentally different type I IFN responses in CCB cells. SVCV induced a high response in all studied genes, whereas CyHV-3 seems to induce no response in CCB cells, but it induces a response in head kidney leukocytes. The lack of an IFN type I response to CyHV-3 could be an indicator of anti-IFN actions of the virus, however the nature of this mechanism has to be evaluated in future studies. Our results also suggest that an activation of type I IFN in CyHV-3 infected cells can limit the spread of the virus in cell culture. This would open the opportunity to treat the disease associated with CyHV-3 by an application of poly I:C in certain cases. Copyright © 2012 Elsevier Ltd. All rights reserved.

HAMLET triggers apoptosis but tumor cell death is independent of caspases, Bcl-2 and p53.

PubMed

Hallgren, O; Gustafsson, L; Irjala, H; Selivanova, G; Orrenius, S; Svanborg, C

2006-02-01

HAMLET (Human alpha-lactalbumin Made Lethal to Tumor cells) triggers selective tumor cell death in vitro and limits tumor progression in vivo. Dying cells show features of apoptosis but it is not clear if the apoptotic response explains tumor cell death. This study examined the contribution of apoptosis to cell death in response to HAMLET. Apoptotic changes like caspase activation, phosphatidyl serine externalization, chromatin condensation were detected in HAMLET-treated tumor cells, but caspase inhibition or Bcl-2 over-expression did not prolong cell survival and the caspase response was Bcl-2 independent. HAMLET translocates to the nuclei and binds directly to chromatin, but the death response was unrelated to the p53 status of the tumor cells. p53 deletions or gain of function mutations did not influence the HAMLET sensitivity of tumor cells. Chromatin condensation was partly caspase dependent, but apoptosis-like marginalization of chromatin was also observed. The results show that tumor cell death in response to HAMLET is independent of caspases, p53 and Bcl-2 even though HAMLET activates an apoptotic response. The use of other cell death pathways allows HAMLET to successfully circumvent fundamental anti-apoptotic strategies that are present in many tumor cells.

Germinal Center B Cell and T Follicular Helper Cell Responses to Viral Vector and Protein-in-Adjuvant Vaccines

PubMed Central

Wang, Chuan; Hart, Matthew; Chui, Cecilia; Ajuogu, Augustine; Brian, Iona J.; de Cassan, Simone C.; Borrow, Persephone; Draper, Simon J.

2016-01-01

There is great interest in the development of Ab-inducing subunit vaccines targeting infections, including HIV, malaria, and Ebola. We previously reported that adenovirus vectored vaccines are potent in priming Ab responses, but uncertainty remains regarding the optimal approach for induction of humoral immune responses. In this study, using OVA as a model Ag, we assessed the magnitude of the primary and anamnestic Ag–specific IgG responses of mice to four clinically relevant vaccine formulations: replication-deficient adenovirus; modified vaccinia Ankara (a poxvirus); protein with alum; and protein in the squalene oil-in-water adjuvant Addavax. We then used flow cytometric assays capable of measuring total and Ag-specific germinal center (GC) B cell and follicular Th cell responses to compare the induction of these responses by the different formulations. We report that adenovirus vectored vaccines induce Ag insert–specific GC B cell and Ab responses of a magnitude comparable to those induced by a potent protein/squalene oil-in-water formulation whereas—despite a robust overall GC response—the insert-specific GC B cell and Ab responses induced by modified vaccinia Ankara were extremely weak. Ag-specific follicular Th cell responses to adenovirus vectored vaccines exceeded those induced by other platforms at day 7 after immunization. We found little evidence that innate immune activation by adenovirus may act as an adjuvant in such a manner that the humoral response to a recombinant protein may be enhanced by coadministering with an adenovirus lacking a transgene of interest. Overall, these studies provide further support for the use of replication-deficient adenoviruses to induce humoral responses. PMID:27412417

PDC-E3BP is not a dominant T-cell autoantigen in primary biliary cirrhosis.

PubMed

McHugh, Anna; Robe, Amanda J; Palmer, Jeremy M; Jones, David E J

2006-05-01

Autoantibody responses reactive with the E2 and E3BP components of pyruvate dehydrogenase complex (PDC), which characterise primary biliary cirrhosis (PBC) crossreact, precluding the identification, from serological studies, of the antigen to which the principal breakdown of tolerance occurs. Although autoreactive T-cell responses to PDC-E2 have been well characterised it is, at present, unclear whether T-cell tolerance breakdown also occurs to PDC-E3BP. The aims of this study were to characterise autoreactive T-cell responses to PDC-E3BP in PBC and potential factors regulating their expression. Peripheral blood T-cell proliferative responses to purified recombinant human PDC-E2 and PDC-E3BP at a range of concentrations were characterised in PBC patients and control subjects. T-cell proliferative responses to both E2 and E3BP were absent from control subjects (median peak stimulation index (SI) to PDC-E2 1.2 [range 0.3-1.9], 0/10 positive (SI>2.32), median peak SI to PDC-E3BP 1.1 [0.7-2.1

Zoledronic Acid-Induced Expansion of γδ T Cells from Early-Stage Breast Cancer Patients: Effect of IL-18 on Helper NK Cells

PubMed Central

Sugie, Tomoharu; Murata-Hirai, Kaoru; Iwasaki, Masashi; Morita, Craig T.; Li, Wen; Okamura, Haruki; Minato, Nagahiro; Toi, Masakazu; Tanaka, Yoshimasa

2013-01-01

Human γδ T cells display potent cytotoxicity against various tumor cells pretreated with zoledronic acid (Zol). Zol has shown benefits when added to adjuvant endocrine therapy for patients with early-stage breast cancer or to standard chemotherapy for patients with multiple myeloma. Although γδ T cells may contribute to this additive effect, the responsiveness of γδ T cells from early-stage breast cancer patients has not been fully investigated. In this study, we determined the number, frequency, and responsiveness of Vγ2Vδ2 T cells from early- and late-stage breast cancer patients and examined the effect of IL-18 on their ex vivo expansion. The responsiveness of Vγ2Vδ2 T cells from patients with low frequencies of Vγ2Vδ2 T cells was significantly diminished. IL-18, however, enhanced ex vivo proliferative responses of Vγ2Vδ2 T cells and helper NK cells from patients with either low or high frequencies of Vγ2Vδ2 T cells. Treatment of breast cancer patients with Zol alone decreased the number of Vγ2Vδ2 T cells and reduced their ex vivo responsiveness. These results demonstrate that Zol can elicit immunological responses by γδ T cells from early-stage breast cancer patients but that frequent in vivo treatment reduces Vγ2Vδ2 T cell numbers and their responsiveness to stimulation. PMID:23151944

Activated leucocyte cell adhesion molecule (ALCAM/CD166) regulates T cell responses in a murine model of food allergy.

PubMed

Kim, Y S; Kim, M N; Lee, K E; Hong, J Y; Oh, M S; Kim, S Y; Kim, K W; Sohn, M H

2018-05-01

Food allergy is a major public health problem. Studies have shown that long-term interactions between activated leucocyte cell adhesion molecule (ALCAM/CD166) on the surface of antigen-presenting cells, and CD6, a co-stimulatory molecule, influence immune responses. However, there are currently no studies on the functions of ALCAM in food allergy. Therefore, we aimed to identify the functions of ALCAM in ovalbumin (OVA)-induced food allergy using ALCAM-deficient mice. Wild-type (WT) and ALCAM-deficient (ALCAM -/- ) mice were sensitized intraperitoneally and with orally fed OVA. The mice were killed, and parameters related to food allergy and T helper type 2 (Th2) immune responses were analysed. ALCAM serum levels increased and mRNA expression decreased in OVA-challenged WT mice. Serum immunoglobulin (Ig)E levels, Th2 cytokine mRNA and histological injuries were higher in OVA-challenged WT mice than in control mice, and these were attenuated in ALCAM -/- mice. T cell proliferation of total cells, CD3 + CD4 + T cells and activated T cells in immune tissues were diminished in OVA-challenged ALCAM -/- mice. Proliferation of co-cultured T cells and dendritic cells (DCs) was decreased by the anti-CD6 antibody. In addition, WT mice sensitized by adoptive transfer of OVA-pulsed ALCAM -/- BM-derived DCs showed reduced immune responses. Lastly, serum ALCAM levels were higher in children with food allergy than in control subjects. In this study, serum levels of ALCAM were elevated in food allergy-induced WT mice and children with food allergy. Moreover, immune responses and T cell activation were attenuated in OVA-challenged ALCAM -/- mice. These results indicate that ALCAM regulates food allergy by affecting T cell activation. © 2018 British Society for Immunology.

Cutting Edge: Eosinophils Undergo Caspase-1-Mediated Pyroptosis in Response to Necrotic Liver Cells.

PubMed

Palacios-Macapagal, Daphne; Connor, Jane; Mustelin, Tomas; Ramalingam, Thirumalai R; Wynn, Thomas A; Davidson, Todd S

2017-08-01

Many chronic liver disorders are characterized by dysregulated immune responses and hepatocyte death. We used an in vivo model to study the immune response to necrotic liver injury and found that necrotic liver cells induced eosinophil recruitment. Necrotic liver induced eosinophil IL-1β and IL-18 secretion, degranulation, and cell death. Caspase-1 inhibitors blocked all of these responses. Caspase-1-mediated cell death with accompanying cytokine release is the hallmark of a novel form of cell death termed pyroptosis. To confirm this response in a disease model, we isolated eosinophils from the livers of Schistosoma mansoni -infected mice. S. mansoni eggs lodge in the hepatic sinusoids of infected mice, resulting in hepatocyte death, inflammation, and progressive liver fibrosis. This response is typified by massive eosinophilia, and we were able to confirm pyroptosis in the infiltrating eosinophils. This demonstrated that pyroptosis is a cellular pathway used by eosinophils in response to large-scale hepatic cell death. Copyright © 2017 by The American Association of Immunologists, Inc.

Human MAIT-cell responses to Escherichia coli: activation, cytokine production, proliferation, and cytotoxicity.

PubMed

Dias, Joana; Sobkowiak, Michał J; Sandberg, Johan K; Leeansyah, Edwin

2016-07-01

Mucosa-associated invariant T cells are a large and relatively recently described innate-like antimicrobial T-cell subset in humans. These cells recognize riboflavin metabolites from a range of microbes presented by evolutionarily conserved major histocompatibility complex, class I-related molecules. Given the innate-like characteristics of mucosa-associated invariant T cells and the novel type of antigens they recognize, new methodology must be developed and existing methods refined to allow comprehensive studies of their role in human immune defense against microbial infection. In this study, we established protocols to examine a range of mucosa-associated invariant T-cell functions as they respond to antigen produced by Escherichia coli These improved and dose- and time-optimized experimental protocols allow detailed studies of MR1-dependent mucosa-associated invariant T-cell responses to Escherichia coli pulsed antigen-presenting cells, as assessed by expression of activation markers and cytokines, by proliferation, and by induction of apoptosis and death in major histocompatibility complex, class I-related-expressing target cells. The novel and optimized protocols establish a framework of methods and open new possibilities to study mucosa-associated invariant T-cell immunobiology, using Escherichia coli as a model antigen. Furthermore, we propose that these robust experimental systems can also be adapted to study mucosa-associated invariant T-cell responses to other microbes and types of antigen-presenting cells. © The Author(s).

Characterization of mTOR-Responsive Truncated mRNAs in Cell Proliferation

DTIC Science & Technology

2017-07-01

AWARD NUMBER: W81XWH-16-1-0135 TITLE: Characterization of mTOR-Responsive Truncated mRNAs in Cell Proliferation PRINCIPAL INVESTIGATOR...TITLE AND SUBTITLE 5a. CONTRACT NUMBER Characterization of mTOR-Responsive Truncated mRNAs in Cell Proliferation 5b. GRANT NUMBER 8W1XWH-16-1...Sclerosis Complex (TSC) 1 or 2 gene leads to deregulated mTOR activation and consequent cell proliferation/growth. Thus, studying the mTOR pathway

Phase I/II evaluation of RV1001, a novel PI3Kδ inhibitor, in spontaneous canine lymphoma.

PubMed

Gardner, Heather L; Rippy, Sarah B; Bear, Misty D; Cronin, Kim L; Heeb, Heather; Burr, Holly; Cannon, Claire M; Penmetsa, Kumar V; Viswanadha, Srikant; Vakkalanka, Swaroop; London, Cheryl A

2018-01-01

RV1001 is a novel, potent, and selective PI3Kδ inhibitor. The purpose of this study was to evaluate the safety and efficacy of RV1001 in canine Non-Hodgkin lymphoma (NHL). Inhibition of endogenous pAKT by RV1001 in primary canine NHL cells was determined by Western blotting. A phase I study of RV1001 was performed in 21 dogs with naïve and drug resistant T and B-cell NHL to assess safety, pharmacokinetic profile, and response to therapy. The objective response rate was 62% (complete response (CR) n = 3; partial response (PR) n = 10), and responses were observed in both naïve and chemotherapy-resistant B and T cell NHL. This study provided the recommended starting dose for a phase II, non-pivotal, exploratory, open label multi-centered clinical trial in 35 dogs with naïve and drug resistant T and B-cell NHL, to further define the efficacy and safety profile of RV1001. The objective response rate in the phase II study was 77% (CR n = 1; PR n = 26). Clinical toxicities were primarily hepatobiliary and gastrointestinal, and were responsive to dose modifications and/or temporary drug discontinuation. Hepatotoxicity was the primary dose limiting toxicity. RV1001 exhibits good oral bioavailability, an acceptable safety profile, and biologic activity with associated inhibition of pAKT in dogs with B and T cell NHL. Data from these studies can be leveraged to help inform the design of future studies involving isoform-selective PI3K inhibitors in humans.

Transcriptomic analysis of rice aleurone cells identified a novel abscisic acid response element.

PubMed

Watanabe, Kenneth A; Homayouni, Arielle; Gu, Lingkun; Huang, Kuan-Ying; Ho, Tuan-Hua David; Shen, Qingxi J

2017-09-01

Seeds serve as a great model to study plant responses to drought stress, which is largely mediated by abscisic acid (ABA). The ABA responsive element (ABRE) is a key cis-regulatory element in ABA signalling. However, its consensus sequence (ACGTG(G/T)C) is present in the promoters of only about 40% of ABA-induced genes in rice aleurone cells, suggesting other ABREs may exist. To identify novel ABREs, RNA sequencing was performed on aleurone cells of rice seeds treated with 20 μM ABA. Gibbs sampling was used to identify enriched elements, and particle bombardment-mediated transient expression studies were performed to verify the function. Gene ontology analysis was performed to predict the roles of genes containing the novel ABREs. This study revealed 2443 ABA-inducible genes and a novel ABRE, designated as ABREN, which was experimentally verified to mediate ABA signalling in rice aleurone cells. Many of the ABREN-containing genes are predicted to be involved in stress responses and transcription. Analysis of other species suggests that the ABREN may be monocot specific. This study also revealed interesting expression patterns of genes involved in ABA metabolism and signalling. Collectively, this study advanced our understanding of diverse cis-regulatory sequences and the transcriptomes underlying ABA responses in rice aleurone cells. © 2017 John Wiley & Sons Ltd.

A Model for Spheroid versus Monolayer Response of SK-N-SH Neuroblastoma Cells to Treatment with 15-Deoxy-PGJ 2

PubMed Central

Dunham, Ann; Chen, Paula X.; Chen, Michelle; Huynh, Milan; Rheingold, Evan; Prosper, Olivia

2016-01-01

Researchers have observed that response of tumor cells to treatment varies depending on whether the cells are grown in monolayer, as in vitro spheroids or in vivo. This study uses data from the literature on monolayer treatment of SK-N-SH neuroblastoma cells with 15-deoxy-PGJ 2 and couples it with data on growth rates for untreated SK-N-SH neuroblastoma cells grown as multicellular spheroids. A linear model is constructed for untreated and treated monolayer data sets, which is tuned to growth, death, and cell cycle data for the monolayer case for both control and treatment with 15-deoxy-PGJ 2. The monolayer model is extended to a five-dimensional nonlinear model of in vitro tumor spheroid growth and treatment that includes compartments of the cell cycle (G 1, S, G 2/M) as well as quiescent (Q) and necrotic (N) cells. Monolayer treatment data for 15-deoxy-PGJ 2 is used to derive a prediction of spheroid response under similar treatments. For short periods of treatment, spheroid response is less pronounced than monolayer response. The simulations suggest that the difference in response to treatment of monolayer versus spheroid cultures observed in laboratory studies is a natural consequence of tumor spheroid physiology rather than any special resistance to treatment. PMID:28044089

Immunomodulatory activity of Zingiber officinale Roscoe, Salvia officinalis L. and Syzygium aromaticum L. essential oils: evidence for humor- and cell-mediated responses.

PubMed

Carrasco, Fábio Ricardo; Schmidt, Gustavo; Romero, Adriano Lopez; Sartoretto, Juliano Luiz; Caparroz-Assef, Silvana Martins; Bersani-Amado, Ciomar Aparecida; Cuman, Roberto Kenji Nakamura

2009-07-01

The immunomodulatory effect of ginger, Zingiber officinale (Zingiberaceae), sage, Salvia officinalis (Lamiaceae) and clove, Syzygium aromaticum (Myrtaceae), essential oils were evaluated by studying humor- and cell-mediated immune responses. Essential oils were administered to mice (once a day, orally, for a week) previously immunized with sheep red blood cells (SRBCs). Clove essential oil increased the total white blood cell (WBC) count and enhanced the delayed-type hypersensitivity (DTH) response in mice. Moreover, it restored cellular and humoral immune responses in cyclophosphamide-immunosuppressed mice in a dose-dependent manner. Ginger essential oil recovered the humoral immune response in immunosuppressed mice. Contrary to the ginger essential oil response, sage essential oil did not show any immunomodulatory activity. Our findings establish that the immunostimulatory activity found in mice treated with clove essential oil is due to improvement in humor- and cell-mediated immune response mechanisms.

CD4+/CD25+ regulatory cells inhibit activation of tumor-primed CD4+ T cells with IFN-gamma-dependent antiangiogenic activity, as well as long-lasting tumor immunity elicited by peptide vaccination.

PubMed

Casares, Noelia; Arribillaga, Laura; Sarobe, Pablo; Dotor, Javier; Lopez-Diaz de Cerio, Ascensión; Melero, Ignacio; Prieto, Jesús; Borrás-Cuesta, Francisco; Lasarte, Juan J

2003-12-01

CD25(+) regulatory T (T reg) cells suppress the activation/proliferation of other CD4(+) or CD8(+) T cells in vitro. Also, down-regulation of CD25(+) T reg cells enhance antitumor immune responses. In this study, we show that depletion of CD25(+) T reg cells allows the host to induce both CD4(+) and CD8(+) antitumoral responses following tumor challenge. Simultaneous depletion of CD25(+) and CD8(+) cells, as well as adoptive transfer experiments, revealed that tumor-specific CD4(+) T cells, which emerged in the absence of CD25(+) T reg cells, were able to reject CT26 colon cancer cells, a MHC class II-negative tumor. The antitumoral effect mediated by CD4(+) T cells was dependent on IFN-gamma production, which exerted a potent antiangiogenic activity. The capacity of the host to mount this antitumor response is lost once the number of CD25(+) T reg cells is restored over time. However, CD25(+) T reg cell depletion before immunization with AH1 (a cytotoxic T cell determinant from CT26 tumor cells) permits the induction of a long-lasting antitumoral immune response, not observed if immunization is conducted in the presence of regulatory cells. A study of the effect of different levels of depletion of CD25(+) T reg cells before immunization with the peptide AH1 alone, or in combination with a Th determinant, unraveled that Th cells play an important role in overcoming the suppressive effect of CD25(+) T reg on the induction of long-lasting cellular immune responses.

Thermal impulse response and the temperature preference of Escherichia coli

NASA Astrophysics Data System (ADS)

Ryu, William

2010-03-01

From a broad perspective, exposure to environmental temperature changes is a universal condition of living organisms. Escherichia coli is a powerful model system to study how a biochemical network measures and processes thermal information to produce adaptive changes in behavior. E. coli performs thermotaxis, directing its movements to a preferred temperature in spatial thermal gradients. How does the system perform thermotaxis? Where biologically is this analog value of thermal preference stored? Previous studies using populations of cells have shown that E.coli accumulate in spatial thermal gradients, but these experiments did not cleanly separate thermal responses from chemotactic responses. Here we have isolated the thermal behavior by studying the thermal impulse response of single, tethered cells. The motor output of cells was measured in response to small, impulsive increases in temperature, delivered by an infrared laser, over a range of ambient temperature (23 to 43 degrees C). The thermal impulse response at temperatures < 31 degrees C is similar to the chemotactic impulse response: both follow a similar time course, share the same directionality, and show biphasic characteristics. At temperatures > 31 degrees C, some cells show an inverted response, switching from warm- to cold-seeking behavior. The fraction of inverted responses increases nonlinearly with temperature, switching steeply at the preferred temperature of 37 degrees C.

Cholesterol negatively regulates IL-9-producing CD8+ T cell differentiation and antitumor activity.

PubMed

Ma, Xingzhe; Bi, Enguang; Huang, Chunjian; Lu, Yong; Xue, Gang; Guo, Xing; Wang, Aibo; Yang, Maojie; Qian, Jianfei; Dong, Chen; Yi, Qing

2018-05-09

CD8 + T cells can be polarized into IL-9-secreting (Tc9) cells. We previously showed that adoptive therapy using tumor-specific Tc9 cells generated stronger antitumor responses in mouse melanoma than classical Tc1 cells. To understand why Tc9 cells exert stronger antitumor responses, we used gene profiling to compare Tc9 and Tc1 cells. Tc9 cells expressed different levels of cholesterol synthesis and efflux genes and possessed significantly lower cholesterol content than Tc1 cells. Unique to Tc9, but not other CD8 + or CD4 + T cell subsets, manipulating cholesterol content in polarizing Tc9 cells significantly affected IL-9 expression and Tc9 differentiation and antitumor response in vivo. Mechanistic studies showed that IL-9 was indispensable for Tc9 cell persistence and antitumor effects, and cholesterol or its derivatives inhibited IL-9 expression by activating liver X receptors (LXRs), leading to LXR Sumoylation and reduced p65 binding to Il9 promoter. Our study identifies cholesterol as a critical regulator of Tc9 cell differentiation and function. © 2018 Ma et al.

Light responses and morphology of bNOS-immunoreactive neurons in the mouse retina

PubMed Central

Pang, Ji-Jie; Gao, Fan; Wu, Samuel M.

2010-01-01

Nitric oxide (NO), produced by NO synthase (NOS), modulates the function of all retinal neurons and ocular blood vessels and participates in the pathogenesis of ocular diseases. To further understand the regulation of ocular NO release, we systematically studied the morphology, topography and light responses of NOS-containing amacrine cells (NOACs) in dark-adapted mouse retina. Immunohistological staining for neuronal NOS (bNOS), combined with retrograde labeling of ganglion cells (GCs) with Neurobiotin (NB, a gap junction permeable dye) and Lucifer yellow (LY, a less permeable dye), was used to identify NOACs. The light responses of ACs were recorded under whole-cell voltage clamp conditions and cell morphology was examined with a confocal microscope. We found that in dark-adapted conditions bNOS-immunoreactivity (IR) was present primarily in the inner nuclear layer and the ganglion cell layer. bNOS-IR somas were negative for LY, thus they were identified as ACs; nearly 6 % of the cells were labeled by NB but not by LY, indicating that they were dye-coupled with GCs. Three morphological subtypes of NOACs (NI, NII and displaced) were identified. The cell density, inter-cellular distance and the distribution of NOACs were studied in whole retinas. Light evoked depolarizing highly sensitive ON-OFF responses in NI cells and less sensitive OFF responses in NII cells. Frequent (1 to 2 Hz) or abrupt change of light-intensity evoked larger peak responses. The possibility for light to modify NO release from NOACs is discussed. PMID:20503422

Induction of appropriate Th-cell phenotypes: cellular decision-making in heterogeneous environments.

PubMed

van den Ham, H-J; Andeweg, A C; de Boer, R J

2013-11-01

Helper T (Th)-cell differentiation is a key event in the development of the adaptive immune response. By the production of a range of cytokines, Th cells determine the type of immune response that is raised against an invading pathogen. Th cells can adopt many different phenotypes, and Th-cell phenotype decision-making is crucial in mounting effective host responses. This review discusses the different Th-cell phenotypes that have been identified and how Th cells adopt a particular phenotype. The regulation of Th-cell phenotypes has been studied extensively using mathematical models, which have explored the role of regulatory mechanisms such as autocrine cytokine signalling and cross-inhibition between self-activating transcription factors. At the single cell level, Th responses tend to be heterogeneous, but corrections can be made soon after T-cell activation. Although pathogens and the innate immune system provide signals that direct the induction of Th-cell phenotypes, these instructive mechanisms could be easily subverted by pathogens. We discuss that a model of success-driven feedback would select the most appropriate phenotype for clearing a pathogen. Given the heterogeneity in the induction phase of the Th response, such a success-driven feedback loop would allow the selection of effective Th-cell phenotypes while terminating incorrect responses. © 2013 John Wiley & Sons Ltd.

Enhanced immune response of MAIT cells in tuberculous pleural effusions depends on cytokine signaling

PubMed Central

Jiang, Jing; Chen, Xinchun; An, Hongjuan; Yang, Bingfen; Zhang, Fuping; Cheng, Xiaoxing

2016-01-01

The functions of MAIT cells at the site of Mycobacterium tuberculosis infection in humans are still largely unknown. In this study, the phenotypes and immune response of MAIT cells from tuberculous pleural effusions and peripheral blood were investigated. MAIT cells in tuberculous pleural effusions had greatly enhanced IFN-γ, IL-17F and granzyme B response compared with those in peripheral blood. The level of IFN-γ response in MAIT cells from tuberculous pleural effusions was inversely correlated with the extent of tuberculosis infection (p = 0.0006). To determine whether cytokines drive the immune responses of MAIT cells at the site of tuberculosis infection, the role of IL-1β, IL-2, IL-7, IL-12, IL-15 and IL-18 was investigated. Blockade of IL-2, IL-12 or IL-18 led to significantly reduced production of IFN-γ and/or granzyme B in MAIT cells from tuberculous pleural effusions. Majority of IL-2-producing cells (94.50%) in tuberculous pleural effusions had phenotype of CD3+CD4+, and most IL-12p40-producing cells (91.39%) were CD14+ cells. MAIT cells had significantly elevated expression of γc receptor which correlated with enhanced immune responses of MAIT cells. It is concluded that MAIT cells from tuberculous pleural effusions exhibited highly elevated immune response to Mtb antigens, which are controlled by cytokines produced by innate/adaptive immune cells. PMID:27586092

Two alternate strategies for innate immunity to Epstein-Barr virus: One using NK cells and the other NK cells and γδ T cells

PubMed Central

Horowitz, Amir; Nemat-Gorgani, Neda; Münz, Christian

2017-01-01

Most humans become infected with Epstein–Barr virus (EBV), which then persists for life. Infrequently, EBV infection causes infectious mononucleosis (IM) or Burkitt lymphoma (BL). Type I EBV infection, particularly type I BL, stimulates strong responses of innate immune cells. Humans respond to EBV in two alternative ways. Of 24 individuals studied, 13 made strong NK and γδ T cell responses, whereas 11 made feeble γδ T cell responses but stronger NK cell responses. The difference does not correlate with sex, HLA type, or previous exposure to EBV or cytomegalovirus. Cohorts of EBV+ children and pediatric IM patients include both group 1 individuals, with high numbers of γδ T cells, and group 2 individuals, with low numbers. The even balance of groups 1 and 2 in the human population points to both forms of innate immune response to EBV having benefit for human survival. Correlating these distinctive responses with the progress of EBV infection might facilitate the management of EBV-mediated disease. PMID:28468758

Mycobacterium tuberculosis specific CD8(+) T cells rapidly decline with antituberculosis treatment.

PubMed

Nyendak, Melissa R; Park, Byung; Null, Megan D; Baseke, Joy; Swarbrick, Gwendolyn; Mayanja-Kizza, Harriet; Nsereko, Mary; Johnson, Denise F; Gitta, Phineas; Okwera, Alphonse; Goldberg, Stefan; Bozeman, Lorna; Johnson, John L; Boom, W Henry; Lewinsohn, Deborah A; Lewinsohn, David M

2013-01-01

Biomarkers associated with response to therapy in tuberculosis could have broad clinical utility. We postulated that the frequency of Mycobacterium tuberculosis (Mtb) specific CD8(+) T cells, by virtue of detecting intracellular infection, could be a surrogate marker of response to therapy and would decrease during effective antituberculosis treatment. We sought to determine the relationship of Mtb specific CD4(+) T cells and CD8(+) T cells with duration of antituberculosis treatment. We performed a prospective cohort study, enrolling between June 2008 and August 2010, of HIV-uninfected Ugandan adults (n = 50) with acid-fast bacillus smear-positive, culture confirmed pulmonary TB at the onset of antituberculosis treatment and the Mtb specific CD4(+) and CD8(+) T cell responses to ESAT-6 and CFP-10 were measured by IFN-γ ELISPOT at enrollment, week 8 and 24. There was a significant difference in the Mtb specific CD8(+) T response, but not the CD4(+) T cell response, over 24 weeks of antituberculosis treatment (p<0.0001), with an early difference observed at 8 weeks of therapy (p = 0.023). At 24 weeks, the estimated Mtb specific CD8(+) T cell response decreased by 58%. In contrast, there was no significant difference in the Mtb specific CD4(+) T cell during the treatment. The Mtb specific CD4(+) T cell response, but not the CD8(+) response, was negatively impacted by the body mass index. Our data provide evidence that the Mtb specific CD8(+) T cell response declines with antituberculosis treatment and could be a surrogate marker of response to therapy. Additional research is needed to determine if the Mtb specific CD8(+) T cell response can detect early treatment failure, relapse, or to predict disease progression.

A mathematical and experimental simulation of the hematological response to weightlessness

NASA Technical Reports Server (NTRS)

Kimzey, S. L.; Leonard, J. I.; Johnson, P. C.

1979-01-01

A mathematical model of erythropoiesis control was used to simulate the effects of bedrest and zero-g on the circulating red cell mass. The model incorporates the best current understanding of the dynamics of red cell production and destruction and the associated feedback regulation. Specifically studied were the hemodynamic responses of a 28-day bedrest study devised to simulate Skylab experience. The results support the hypothesis that red cell loss during supine bedrest is a normal physiological feedback process in response to hemoconcentration enhanced tissue oxygenation and suppression of red cell production. Model simulation suggested the possibilities that this period was marked by some combination of increased oxygen-hemoglobin affinity, small reduction in mean red cell life span, ineffective erythropoiesis, or abnormal reticulocytosis.

Defective B cell response to T-dependent immunization in lupus-prone mice

PubMed Central

Niu, Haitao; Sobel, Eric S.; Morel, Laurence

2009-01-01

Lupus anti-nuclear Abs show the characteristics of Ag-driven T cell-dependent (TD) humoral responses. If autoAgs elicit the same response as exogenous Ags, lupus should enhance humoral responses to immunization. Blunted responses to various immunizations have, however, been reported in a significant portion of lupus patients. In this study, we show that lupus-prone B6.Sle1.Sle2.Sle3 (B6.TC) mice produce significantly less Ab in response to TD immunization than congenic controls, while producing significantly more total Ig. This blunted Ab response to TD Ag could be reconstituted with B6.TC B and CD4+ T cells. Multiple defects were found in the B6.TC response to NP-KLH as compared to total Ig, including a smaller percentage of B cells participating to the NP-response, a reduced entry into germinal centers, and highly defective production of NP-specific long-lived plasma cells in the bone marrow. B6.TC plasma cells expressed reduced levels of FcγRIIb, which suggests that reduced apoptosis in resident plasma cells prevents the establishment of newly-formed NP-specific plasma cells in bone marrow niches. Overall, these results show that lupus-prone mice responded differently to auto- and exogenous antigens and suggest that low FcγRIIb, hypergammaglobulinemia and high autoantibody production would be predictive of a poor response to immunization in lupus patients. PMID:18924209

Characterization of Effector and Memory T Cell Subsets in the Immune Response to Bovine Tuberculosis in Cattle

PubMed Central

Maggioli, Mayara F.; Palmer, Mitchell V.; Thacker, Tyler C.; Vordermeier, H. Martin; Waters, W. Ray

2015-01-01

Cultured IFN-γ ELISPOT assays are primarily a measure of central memory T cell (Tcm) responses with humans; however, this important subset of lymphocytes is poorly characterized in cattle. Vaccine-elicited cultured IFN-γ ELISPOT responses correlate with protection against bovine tuberculosis in cattle. However, whether this assay measures cattle Tcm responses or not is uncertain. The objective of the present study was to characterize the relative contribution of Tcm (CCR7+, CD62Lhi, CD45RO+), T effector memory (Tem, defined as: CCR7-, CD62Llow/int, CD45RO+), and T effector cells (CCR7-, CD62L-/low, CD45RO-), in the immune response to Mycobacterium bovis. Peripheral blood mononuclear cells (PBMC) from infected cattle were stimulated with a cocktail of M. bovis purified protein derivative, rTb10.4 and rAg85A for 13 days with periodic addition of fresh media and rIL-2. On day 13, cultured PBMC were re-stimulated with medium alone, rESAT-6:CFP10 or PPDb with fresh autologous adherent cells for antigen presentation. Cultured cells (13 days) or fresh PBMCs (ex vivo response) from the same calves were analyzed for IFN-γ production, proliferation, and CD4, CD45RO, CD62L, CD44, and CCR7 expression via flow cytometry after overnight stimulation. In response to mycobacterial antigens, ~75% of CD4+ IFN-γ+ cells in long-term cultures expressed a Tcm phenotype while less than 10% of the ex vivo response consisted of Tcm cells. Upon re-exposure to antigen, long-term cultured cells were highly proliferative, a distinctive characteristic of Tcm, and the predominant phenotype within the long-term cultures switched from Tcm to Tem. These findings suggest that proliferative responses of Tcm cells to some extent occurs simultaneously with reversion to effector phenotypes (mostly Tem). The present study characterizes Tcm cells of cattle and their participation in the response to M. bovis infection. PMID:25879774

Thiol dependent NF-κB suppression and inhibition of T-cell mediated adaptive immune responses by a naturally occurring steroidal lactone Withaferin A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gambhir, Lokesh; Checker, Rahul; Sharma, Deepak

Withaferin A (WA), a steroidal lactone isolated from ayurvedic medicinal plant Withania somnifera, was shown to inhibit tumor growth by inducing oxidative stress and suppressing NF-κB pathway. However, its effect on T-cell mediated adaptive immune responses and the underlying mechanism has not been investigated. Since both T-cell responses and NF-κB pathway are known to be redox sensitive, the present study was undertaken to elucidate the effect of WA on adaptive immune responses in vitro and in vivo. WA inhibited mitogen induced T-cell and B-cell proliferation in vitro without inducing any cell death. It inhibited upregulation of T-cell (CD25, CD69, CD71more » and CD54) and B-cell (CD80, CD86 and MHC-II) activation markers and secretion of Th1 and Th2 cytokines. WA induced oxidative stress by increasing the basal ROS levels and the immunosuppressive effects of WA were abrogated only by thiol anti-oxidants. The redox modulatory effects of WA in T-cells were attributed to its ability to directly interact with free thiols. WA inhibited NF-κB nuclear translocation in lymphocytes and prevented the direct binding of nuclear NF-κB to its consensus sequence. MALDI-TOF analysis using a synthetic NF-κB-p50 peptide containing Cys-62 residue suggested that WA can modify the cysteine residue of NF-κB. The pharmacokinetic studies for WA were also carried out and in vivo efficacy of WA was studied using mouse model of Graft-versus-host disease. In conclusion, WA is a potent inhibitor of T-cell responses and acts via a novel thiol dependent mechanism and inhibition of NF-κB pathway. - Highlights:: • Withaferin A (WA) inhibited T-cell and B-cell mediated immune responses. • WA increased basal ROS levels in lymphocytes. • WA directly interacted with GSH as studied using spectrophotometry and HPLC. • WA inhibited NF-κB nuclear translocation and binding of nuclear NF-κB to DNA. • WA inhibited induction of the graft-versus-host disease in mice.« less

Effects of acidic amino acid antagonists upon the spectral properties of carp horizontal cells: circuitry of the outer retina.

PubMed

Mangel, S C; Ariel, M; Dowling, J E

1985-11-01

The acidic amino acid receptor antagonists, alpha-methylglutamate and alpha-aminoadipate, were applied to the carp retina to study their effects upon the spectral properties of horizontal cells and to elucidate the synaptic connections between horizontal cells and cones. Application of these antagonists strongly hyperpolarized the L-type cone horizontal cells and reduced the responses of these horizontal cells to red light more than to blue light. Application of Co2+ ions to the retina, a procedure which decreases transmitter release, also hyperpolarized the L-type cone horizontal cells but reduced the response of these horizontal cells to red and blue lights equally. These results suggest that red- or long wavelength-sensitive cones release a different transmitter onto L-type cone horizontal cells than do short wavelength-sensitive cones. Application of the acidic amino acid antagonists also revealed details of the feedback pathway from L-type cone horizontal cells to cones. Previous studies have shown that feedback varies directly with stimulus size and that the effects of feedback on the responses of cones are observed as a transient waveform at response onset (a large, hyperpolarizing potential that is quickly followed by a smaller plateau potential). Application of the acidic amino acid antagonists at a dose which partially hyperpolarized the horizontal cells selectively enhanced the response of the cells to blue lights, when full field, and not spot, stimuli were used. The antagonists also eliminated the transient at response onset. These findings are consistent with the presence of a feedback pathway from L-type cone horizontal cells to short wavelength cones but not to long (red-sensitive) cones.

Monitoring the Single-Cell Stress Response of the Diatom Thalassiosira pseudonana by Quantitative Real-Time Reverse Transcription-PCR

PubMed Central

Shi, Xu; Gao, Weimin; Chao, Shih-hui

2013-01-01

Directly monitoring the stress response of microbes to their environments could be one way to inspect the health of microorganisms themselves, as well as the environments in which the microorganisms live. The ultimate resolution for such an endeavor could be down to a single-cell level. In this study, using the diatom Thalassiosira pseudonana as a model species, we aimed to measure gene expression responses of this organism to various stresses at a single-cell level. We developed a single-cell quantitative real-time reverse transcription-PCR (RT-qPCR) protocol and applied it to determine the expression levels of multiple selected genes under nitrogen, phosphate, and iron depletion stress conditions. The results, for the first time, provided a quantitative measurement of gene expression at single-cell levels in T. pseudonana and demonstrated that significant gene expression heterogeneity was present within the cell population. In addition, different expression patterns between single-cell- and bulk-cell-based analyses were also observed for all genes assayed in this study, suggesting that cell response heterogeneity needs to be taken into consideration in order to obtain accurate information that indicates the environmental stress condition. PMID:23315741

Monitoring the single-cell stress response of the diatom Thalassiosira pseudonana by quantitative real-time reverse transcription-PCR.

PubMed

Shi, Xu; Gao, Weimin; Chao, Shih-hui; Zhang, Weiwen; Meldrum, Deirdre R

2013-03-01

Directly monitoring the stress response of microbes to their environments could be one way to inspect the health of microorganisms themselves, as well as the environments in which the microorganisms live. The ultimate resolution for such an endeavor could be down to a single-cell level. In this study, using the diatom Thalassiosira pseudonana as a model species, we aimed to measure gene expression responses of this organism to various stresses at a single-cell level. We developed a single-cell quantitative real-time reverse transcription-PCR (RT-qPCR) protocol and applied it to determine the expression levels of multiple selected genes under nitrogen, phosphate, and iron depletion stress conditions. The results, for the first time, provided a quantitative measurement of gene expression at single-cell levels in T. pseudonana and demonstrated that significant gene expression heterogeneity was present within the cell population. In addition, different expression patterns between single-cell- and bulk-cell-based analyses were also observed for all genes assayed in this study, suggesting that cell response heterogeneity needs to be taken into consideration in order to obtain accurate information that indicates the environmental stress condition.

The response of neurons in areas V1 and MT of the alert rhesus monkey to moving random dot patterns.

PubMed

Snowden, R J; Treue, S; Andersen, R A

1992-01-01

We studied the response of single units to moving random dot patterns in areas V1 and MT of the alert macaque monkey. Most cells could be driven by such patterns; however, many cells in V1 did not give a consistent response but fired at a particular point during stimulus presentation. Thus different dot patterns can produce a markedly different response at any particular time, though the time averaged response is similar. A comparison of the directionality of cells in both V1 and MT using random dot patterns shows the cells of MT to be far more directional. In addition our estimates of the percentage of directional cells in both areas are consistent with previous reports using other stimuli. However, we failed to find a bimodality of directionality in V1 which has been reported in some other studies. The variance associated with response was determined for individual cells. In both areas the variance was found to be approximately equal to the mean response, indicating little difference between extrastriate and striate cortex. These estimates are in broad agreement (though the variance appears a little lower) with those of V1 cells of the anesthetized cat. The response of MT cells was simulated on a computer from the estimates derived from the single unit recordings. While the direction tuning of MT cells is quite wide (mean half-width at half-height approximately 50 degrees) it is shown that the cells can reliably discriminate much smaller changes in direction, and the performance of the cells with the smallest discriminanda were comparable to thresholds measured with human subjects using the same stimuli (approximately 1.1 degrees). Minimum discriminanda for individual cells occurred not at the preferred direction, that is, the peak of their tuning curves, but rather on the steep flanks of their tuning curves. This result suggests that the cells which may mediate the discrimination of motion direction may not be the cells most sensitive to that direction.

B cells in chronic obstructive pulmonary disease: moving to center stage

PubMed Central

Polverino, Francesca; Seys, Leen J. M.; Bracke, Ken R.

2016-01-01

Chronic inflammatory responses in the lungs contribute to the development and progression of chronic obstructive pulmonary disease (COPD). Although research studies focused initially on the contributions of the innate immune system to the pathogenesis of COPD, more recent studies have implicated adaptive immune responses in COPD. In particular, studies have demonstrated increases in B cell counts and increases in the number and size of B cell-rich lymphoid follicles in COPD lungs that correlate directly with COPD severity. There are also increases in lung levels of mediators that promote B cell maturation, activation, and survival in COPD patients. B cell products such as autoantibodies directed against lung cells, components of cells, and extracellular matrix proteins are also present in COPD lungs. These autoantibodies may contribute to lung inflammation and injury in COPD patients, in part, by forming immune complexes that activate complement components. Studies of B cell-deficient mice and human COPD patients have linked B cells most strongly to the emphysema phenotype. However, B cells have protective activities during acute exacerbations of COPD by promoting adaptive immune responses that contribute to host defense against pathogens. This review outlines the evidence that links B cells and B cell-rich lymphoid follicles to the pathogenesis of COPD and the mechanisms involved. It also reviews the potential and limitations of B cells as therapeutic targets to slow the progression of human COPD. PMID:27542809

Lymphocyte responses to phytohaemagglutinin: age-related effects.

PubMed Central

Fernandez, L A; MacSween, J M; Langley, G R

1976-01-01

Cell-mediated immunity is depressed in elderly individuals compared to young individuals, and lymphocytes from elderly individuals have been reported to have impaired lymphocyte responsiveness to stimulation by PHA after 4 days of culture. We have confirmed this observation. However, after 8 days of culture, the lymphocyte responses were greater in elderly normal individuals than in young normal individuals. Responses of lymphocytes from young individuals decreased with time from 4 to 8 days in culture, while there were increased responses with time when lymphocytes from elderly individuals were studied. When adherent cells from lymphocytes of young individuals were removed by passage through protein-coated Degalan-bead columns, the lymphocyte responses to PHA were significantly increased at 8 days. Passage of lymphocytes from elderly individuals through coated Degalan bead columns did not alter the lymphocyte responses. Removal of macrophages from the mononuclear cells obtained from young individuals did not result in increased lymphocyte responses to PHA after 8 days in culture. Removal of adherent cells appeared to have the same effect regardless of the efficiency of removing B cells. The adherent cells removed by the protein coated columns, therefore, appear to be nonphagocytic mononuclear cells which are not B lymphocytes. PMID:1086285

Circulating CXCR5+PD-1+ response predicts influenza vaccine antibody responses in young adults but not elderly adults.

PubMed

Herati, Ramin Sedaghat; Reuter, Morgan A; Dolfi, Douglas V; Mansfield, Kathleen D; Aung, Htin; Badwan, Osama Z; Kurupati, Raj K; Kannan, Senthil; Ertl, Hildegund; Schmader, Kenneth E; Betts, Michael R; Canaday, David H; Wherry, E John

2014-10-01

Although influenza vaccination is recommended for all adults annually, the incidence of vaccine failure, defined as weak or absent increase in neutralizing Ab titers, is increased in the elderly compared with young adults. The T follicular helper cell (Tfh) subset of CD4 T cells provides B cell help in germinal centers and is necessary for class-switched Ab responses. Previous studies suggested a role for circulating Tfh cells (cTfh) following influenza vaccination in adults, but cTfh have not been studied in elderly adults in whom weak vaccine responses are often observed. In this study, we studied cTfh expressing CXCR5 and programmed death-1 (PD-1). cTfh from elderly adults were present at reduced frequency, had decreased in vitro B cell help ability, and had greater expression of ICOS compared with young adults. At 7 d after inactivated influenza vaccination, cTfh correlated with influenza vaccine-specific IgM and IgG responses in young adults but not in elderly adults. In sum, we have identified aging-related changes in cTfh that correlated with reduced influenza vaccine responses. Future rational vaccine design efforts should incorporate Tfh measurement as an immune correlate of protection, particularly in the setting of aging. Copyright © 2014 by The American Association of Immunologists, Inc.

Effect of PDT-treated apoptotic cells on macrophages

NASA Astrophysics Data System (ADS)

Song, Sheng; Xing, Da; Zhou, Fei-fan; Chen, Wei R.

2009-02-01

Recently, the long-term immunological effects of photodynamic therapy have attracted much attention. PDT induced immune response was mainly initiated through necrotic cells and apoptotic cells, as well as immune cells such as macrophages. Nitric oxide (NO) as an important regulatory factor in signal transfer between cells has been wildly studied for generation, development, and metastasis of tumors. NO synthase is a key enzyme in nitric oxide synthesis. However, inducible nitric oxide synthase (iNOS) is usually activated under pathological conditions, such as stress and cancer, which can produce high levels of nitric oxide and contribute to tumor cytotoxicity. In addition, increased NO production by iNOS has been associated with the host immune response and cell apoptosis, which play an important role in many carcinogenesis and anti-carcinoma mechanisms. This study focuses on the NO production in macrophages, induced by mouse breast carcinoma apoptotic cells treated by PDT in vitro, and on the effects of immune response induced by apoptotic cells in tumor cells growth.

Dynamics of the CD8 T-cell response following yellow fever virus 17D immunization

PubMed Central

Co, Mary Dawn T; Kilpatrick, Elizabeth D; Rothman, Alan L

2009-01-01

Management of yellow fever is focused on the prevention of illness by the use of the yellow fever virus (YFV) 17D vaccine. The role of neutralizing antibodies in protection is generally accepted with YFV-specific T cells likely contributing to the control of viral replication. We studied CD8+ T-cell responses to four defined human leucocyte antigen-B35-restricted epitopes in YFV vaccine recipients as a model of the kinetics of cytotoxic T-lymphocyte responses to an acute human viral infection. Multiple features of these epitope-specific responses were analysed after vaccination including magnitude, cytokine production, phenotype and T-cell receptor repertoire. Peak peptide-specific interferon-γ (IFN-γ) responses of almost 1% of CD8+ T cells were seen as early as 2 weeks post-vaccination; however, dominant responses varied between donors. Peptide-specific responses were still detectable at 54 months post-vaccination. Tetramer-positive cells, at high frequencies, were detected as early as 7–9 days, before detectable IFN-γ-producing cells, suggesting a defect in the functional capacity of some antigen-specific cells early post-vaccination. The predominant memory phenotype of the tetramer-positive population was a differentiated effector (CD45RA+ CCR7− CD62L−) phenotype. The T-cell receptor Vβ analysis revealed a diverse oligoclonal repertoire in tetramer-positive T-cell populations in two individuals. These characteristics of the YFV-specific T-cell response could contribute to vaccine effectiveness. PMID:19740333

Dynamics of the CD8 T-cell response following yellow fever virus 17D immunization.

PubMed

Co, Mary Dawn T; Kilpatrick, Elizabeth D; Rothman, Alan L

2009-09-01

Management of yellow fever is focused on the prevention of illness by the use of the yellow fever virus (YFV) 17D vaccine. The role of neutralizing antibodies in protection is generally accepted with YFV-specific T cells likely contributing to the control of viral replication. We studied CD8(+) T-cell responses to four defined human leucocyte antigen-B35-restricted epitopes in YFV vaccine recipients as a model of the kinetics of cytotoxic T-lymphocyte responses to an acute human viral infection. Multiple features of these epitope-specific responses were analysed after vaccination including magnitude, cytokine production, phenotype and T-cell receptor repertoire. Peak peptide-specific interferon-gamma (IFN-gamma) responses of almost 1% of CD8(+) T cells were seen as early as 2 weeks post-vaccination; however, dominant responses varied between donors. Peptide-specific responses were still detectable at 54 months post-vaccination. Tetramer-positive cells, at high frequencies, were detected as early as 7-9 days, before detectable IFN-gamma-producing cells, suggesting a defect in the functional capacity of some antigen-specific cells early post-vaccination. The predominant memory phenotype of the tetramer-positive population was a differentiated effector (CD45RA(+) CCR7(-) CD62L(-)) phenotype. The T-cell receptor Vbeta analysis revealed a diverse oligoclonal repertoire in tetramer-positive T-cell populations in two individuals. These characteristics of the YFV-specific T-cell response could contribute to vaccine effectiveness.

Yersinia vs. host Immunity: how a pathogen evades or triggers a protective response

PubMed Central

Chung, Lawton K.; Bliska, James B.

2015-01-01

The human pathogenic Yersinia species cause diseases that represent a significant source of morbidity and mortality. Despite this, specific mechanisms underlying Yersinia pathogenesis and protective host responses remain poorly understood. Recent studies have shown that Yersinia disrupt cell death pathways, perturb inflammatory processes and exploit immune cells to promote disease. The ensuing host responses following Yersinia infection include coordination of innate and adaptive immune responses in an attempt to control bacterial replication. Here, we highlight current advances in our understanding of the interactions between the pathogenic yersiniae and host cells, as well as the protective host responses mobilized to counteract these pathogens. Together, these studies enhance our understanding of Yersinia pathogenesis and highlight the ongoing battle between host and microbe. PMID:26638030

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peeters, S.H.; Carter, B.G.

An adoptive cell transfer system was defined for studying the long-term IgE anti-ovalbumin (OA) response of B6D2F/sub 1/ mice. The primary IgE anti-OA response given by this strain persisted for more than 8 months and the half-life of the IgE anti-OA in circulation was 10.5 hr as measured by residual passive cutaneous anaphylaxis (PCA) in rat skin. The system described for studying cell recruitment patterns in this response involves the transfer of primed spleen cells to irradiated recipients and the production in these recipients of IgE and hemagglutinating antibody without further overt antigenic challenge. The capacity of primed spleen cellsmore » to transfer the response was detectable 10 days after immunization and increased for the next 6 weeks. After this time the ability of primed cells to transfer the response declined steadily, but was still evident 8 months after immunization of the donor animals. The transferred response itself persisted in the recipients for many months.« less

Differential Response of Bovine Mature Nucleus Pulposus and Notochordal Cells to Hydrostatic Pressure and Glucose Restriction.

PubMed

Saggese, Taryn; Thambyah, Ashvin; Wade, Kelly; McGlashan, Susan Read

2018-05-01

Objective The nucleus pulposus of the human intervertebral disc contains 2 cell types: notochordal (NC) and mature nucleus pulposus (MNP) cells. NC cell loss is associated with disc degeneration and this process may be initiated by mechanical stress and/or nutrient deprivation. This study aimed to investigate the functional responses of NC and MNP cells to hydrostatic pressures and glucose restriction. Design Bovine MNP and NC cells were cultured in 3-dimensional alginate beads under low (0.4-0.8 MPa) and high (1.6-2.4 MPa) dynamic pressure for 24 hours. Cells were cultured in either physiological (5.5 mM) glucose media or glucose-restriction (0.55 mM) media. Finally, the combined effect of glucose restriction and high pressure was examined. Results Cell viability and notochordal phenotypic markers were not significantly altered in response to pressure or glucose restriction. MNP cells responded to low pressure with an increase in glycosaminoglycan (GAG) production while high pressure significantly decreased ACAN gene expression compared with atmospheric controls. NC cells showed no response in matrix gene expression or GAG production with either loading regime. Glucose restriction decreased NC cell TIMP-1 expression but had no effect on MNP cells. The combination of glucose restriction and high pressure only affected MNP cell gene expression, with decreased ACAN, Col2α1, and ADAMTS-5 expression. Conclusion This study shows that NC cells are more resistant to acute mechanical stresses than MNP cells and provides a strong rationale for future studies to further our understanding the role of NC cells within the disc, and the effects of long-term exposure to physical stresses.

Reversible Reprogramming of Circulating Memory T Follicular Helper Cell Function during Chronic HIV Infection

PubMed Central

Cubas, Rafael; van Grevenynghe, Julien; Wills, Saintedym; Kardava, Lela; Santich, Brian H.; Buckner, Clarisa M.; Muir, Roshell; Tardif, Virginie; Nichols, Carmen; Procopio, Francesco; He, Zhong; Metcalf, Talibah; Ghneim, Khader; Locci, Michela; Ancuta, Petronella; Routy, Jean-Pierre; Trautmann, Lydie; Li, Yuxing; McDermott, Adrian B.; Koup, Rick A.; Petrovas, Constantinos; Migueles, Steven A.; Connors, Mark; Tomaras, Georgia D.; Moir, Susan; Crotty, Shane

2015-01-01

Despite the overwhelming benefits of antiretroviral therapy (ART) in curtailing viral load in HIV-infected individuals, ART does not fully restore cellular and humoral immunity. HIV-infected individuals under ART show reduced responses to vaccination and infections and are unable to mount an effective antiviral immune response upon ART cessation. Many factors contribute to these defects, including persistent inflammation, especially in lymphoid tissues, where T follicular helper (Tfh) cells instruct and help B cells launch an effective humoral immune response. In this study we investigated the phenotype and function of circulating memory Tfh cells as a surrogate of Tfh cells in lymph nodes and found significant impairment of this cell population in chronically HIV-infected individuals, leading to reduced B cell responses. We further show that these aberrant memory Tfh cells exhibit an IL-2–responsive gene signature and are more polarized toward a Th1 phenotype. Treatment of functional memory Tfh cells with IL-2 was able to recapitulate the detrimental reprogramming. Importantly, this defect was reversible, as interfering with the IL-2 signaling pathway helped reverse the abnormal differentiation and improved Ab responses. Thus, reversible reprogramming of memory Tfh cells in HIV-infected individuals could be used to enhance Ab responses. Altered microenvironmental conditions in lymphoid tissues leading to altered Tfh cell differentiation could provide one explanation for the poor responsiveness of HIV-infected individuals to new Ags. This explanation has important implications for the development of therapeutic interventions to enhance HIV- and vaccine-mediated Ab responses in patients under ART. PMID:26546609

Evidence for Leydig cell dysfunction in rats with seminiferous tubule damage.

PubMed

Rich, K A; Kerr, J B; de Kretser, D M

1979-02-01

To study the effects of seminiferous tubule damage on Leydig cell function and morphology, rats were treated by fetal irradiation (to induce Sertoli cell-only syndrome, SCO), 3 months administration of hydroxyurea (HU), or chronic feeding of a vitamin A-deficient diet (VAD). Leydig cell function was assessed by the measurement of serum LH and testosterone and the response of serum testosterone to hCG stimulation, while morphology was studied by electron microscopy after perfusion fixation. Serum LH was significantly elevated in each experimental group, while basal serum testosterone was significantly lower only in SCO rats. In all treatment groups, the serum testosterone response to hCG was significantly decreased when measureed as the area under the response curve. Despite a decreased response to hCG, the Leydig cells were larger than normal and showed striking increases in quantities of smooth endoplasmic reticulum, mitochondria and Golgi complex. Leydig cell dysfunction has been demonstrated in animals with varying degrees of seminiferous tubule damage, but paradoxically the cytological features of the Leydig cells were indicative of hypertrophy.

Human Organ Culture: Updating the Approach to Bridge the Gap from In Vitro to In Vivo in Inflammation, Cancer, and Stem Cell Biology.

PubMed

Al-Lamki, Rafia S; Bradley, John R; Pober, Jordan S

2017-01-01

Human studies, critical for developing new diagnostics and therapeutics, are limited by ethical and logistical issues, and preclinical animal studies are often poor predictors of human responses. Standard human cell cultures can address some of these concerns but the absence of the normal tissue microenvironment can alter cellular responses. Three-dimensional cultures that position cells on synthetic matrices, or organoid or organ-on-a-chip cultures, in which different cell spontaneously organize contacts with other cells and natural matrix only partly overcome this limitation. Here, we review how human organ cultures (HOCs) can more faithfully preserve in vivo tissue architecture and can better represent disease-associated changes. We will specifically describe how HOCs can be combined with both traditional and more modern morphological techniques to reveal how anatomic location can alter cellular responses at a molecular level and permit comparisons among different cells and different cell types within the same tissue. Examples are provided involving use of HOCs to study inflammation, cancer, and stem cell biology.

Human Organ Culture: Updating the Approach to Bridge the Gap from In Vitro to In Vivo in Inflammation, Cancer, and Stem Cell Biology

PubMed Central

Al-Lamki, Rafia S.; Bradley, John R.; Pober, Jordan S.

2017-01-01

Human studies, critical for developing new diagnostics and therapeutics, are limited by ethical and logistical issues, and preclinical animal studies are often poor predictors of human responses. Standard human cell cultures can address some of these concerns but the absence of the normal tissue microenvironment can alter cellular responses. Three-dimensional cultures that position cells on synthetic matrices, or organoid or organ-on-a-chip cultures, in which different cell spontaneously organize contacts with other cells and natural matrix only partly overcome this limitation. Here, we review how human organ cultures (HOCs) can more faithfully preserve in vivo tissue architecture and can better represent disease-associated changes. We will specifically describe how HOCs can be combined with both traditional and more modern morphological techniques to reveal how anatomic location can alter cellular responses at a molecular level and permit comparisons among different cells and different cell types within the same tissue. Examples are provided involving use of HOCs to study inflammation, cancer, and stem cell biology. PMID:28955710

Thermal injury-plus-sepsis contributes to a substantial deletion of intestinal mesenteric lymph node CD4 T cell via apoptosis.

PubMed

Fazal, Nadeem; Al-Ghoul, Walid M

2007-09-12

Thermal injury (TI) with septic complications continues to be a serious clinical problem. One of the main concerns in such patients is immunosuppression related to functional derangements in intestinal CD4+ T lymphocytes. Extensive previous studies in thermal injury/septic patients and animal models of thermal injury/sepsis have shown decreased responsiveness of intestinal CD4+ T cells to antigen/mitogen. This hyporesponsiveness could significantly contribute to increase injured host susceptibility to pathogens including those translocating from host's gut lumen. Our previous studies indicated that while thermal injury or sepsis alone lead to suppressed proliferation and IL-2 production of intestinal CD4+ T cells, this study showed a substantial deletion via apoptosis of the Mesenteric Lymph Nodes (MLN) CD4+ T cells. Hence, thermal injury-plus-sepsis contributes not only to suppressed CD4+ T proliferation/IL-2 production but also to a substantial modulation of CD4+ T cell survivability. These findings allow us to conclude that while thermal injury alone can produce attenuated cell mediated responses without an overt change in CD4+ T cell survival, thermal injury with septic complications causes CD4+ T cell death and an irreversible loss of cell-mediated responses. The latter happening could be responsible for high morbidity and mortality in the injured host afflicted with thermal injury plus a critical infection.

Thermal injury-plus-sepsis contributes to a substantial deletion of intestinal mesenteric lymph node CD4+ T cell via apoptosis

PubMed Central

Fazal, Nadeem; Al-Ghoul, Walid M

2007-01-01

Thermal injury (TI) with septic complications continues to be a serious clinical problem. One of the main concerns in such patients is immunosuppression related to functional derangements in intestinal CD4+ T lymphocytes. Extensive previous studies in thermal injury/septic patients and animal models of thermal injury/sepsis have shown decreased responsiveness of intestinal CD4+ T cells to antigen/mitogen. This hyporesponsiveness could significantly contribute to increase injured host susceptibility to pathogens including those translocating from host's gut lumen. Our previous studies indicated that while thermal injury or sepsis alone lead to suppressed proliferation and IL-2 production of intestinal CD4+ T cells, this study showed a substantial deletion via apoptosis of the Mesenteric Lymph Nodes (MLN) CD4+ T cells. Hence, thermal injury-plus-sepsis contributes not only to suppressed CD4+ T proliferation/IL-2 production but also to a substantial modulation of CD4+ T cell survivability. These findings allow us to conclude that while thermal injury alone can produce attenuated cell mediated responses without an overt change in CD4+ T cell survival, thermal injury with septic complications causes CD4+ T cell death and an irreversible loss of cell-mediated responses. The latter happening could be responsible for high morbidity and mortality in the injured host afflicted with thermal injury plus a critical infection. PMID:17895960

Kinetics of ROS generation induced by polycyclic aromatic hydrocarbons and organic extracts from ambient air particulate matter in model human lung cell lines.

PubMed

Libalova, Helena; Milcova, Alena; Cervena, Tereza; Vrbova, Kristyna; Rossnerova, Andrea; Novakova, Zuzana; Topinka, Jan; Rossner, Pavel

2018-03-01

Polycyclic aromatic hydrocarbons (PAHs) associated with particulate matter (PM) may induce oxidative damage via reactive oxygen species (ROS) generation. However, the kinetics of ROS production and the link with antioxidant response induction has not been well studied. To elucidate the differences in oxidative potential of individual PAH compounds and extractable organic matter (EOM) from PM containing various PAH mixtures, we studied ROS formation and antioxidant response [total antioxidant capacity (TAC) and expression of HMOX1 and TXNRD1] in human alveolar basal epithelial cells (A549 cells) and human embryonic lung fibroblasts (HEL12469 cells). We treated the cells with three concentrations of model PAHs (benzo[a]pyrene, B[a]P; 3-nitrobenzanthrone, 3-NBA) and EOM from PM <2.5 μm (PM2.5). ROS levels were evaluated at 8 time intervals (30 min-24 h). In both cell lines, B[a]P treatment was associated with a time-dependent decrease of ROS levels. This trend was more pronounced in HEL12469 cells and was accompanied by increased TAC. A similar response was observed upon 3-NBA treatment in HEL12469 cells. In A549 cells, however, this compound significantly increased superoxide levels. This response was accompanied by the decrease of TAC as well as HMOX1 and TXNRD1 expression. In both cell lines, a short-time exposure to EOMs tended to increase ROS levels, while a marked decrease was observed after longer treatment periods. This was accompanied by the induction of HMOX1 and TXNRD1 expression in HEL12469 cells and increased TAC in A549 cells. In summary, our data indicate that in the studied cell lines B[a]P and EOMs caused a time-dependent decrease of intracellular ROS levels, probably due to the activation of the antioxidant response. This response was not detected in A549 cells following 3-NBA treatment, which acted as a strong superoxide inducer. Pro-oxidant properties of EOMs are limited to short-time exposure periods. Copyright © 2018 Elsevier B.V. All rights reserved.

Inflammatory Response and Barrier Dysfunction by Different e-Cigarette Flavoring Chemicals Identified by Gas Chromatography-Mass Spectrometry in e-Liquids and e-Vapors on Human Lung Epithelial Cells and Fibroblasts.

PubMed

Gerloff, Janice; Sundar, Isaac K; Freter, Robert; Sekera, Emily R; Friedman, Alan E; Robinson, Risa; Pagano, Todd; Rahman, Irfan

2017-03-01

Recent studies suggest that electronic cigarette (e-cig) flavors can be harmful to lung tissue by imposing oxidative stress and inflammatory responses. The potential inflammatory response by lung epithelial cells and fibroblasts exposed to e-cig flavoring chemicals in addition to other risk-anticipated flavor enhancers inhaled by e-cig users is not known. The goal of this study was to evaluate the release of the proinflammatory cytokine (interleukin-8 [IL-8]) and epithelial barrier function in response to different e-cig flavoring chemicals identified in various e-cig e-liquid flavorings and vapors by chemical characterization using gas chromatography-mass spectrometry analysis. Flavorings, such as acetoin (butter), diacetyl, pentanedione, maltol (malt), ortho-vanillin (vanilla), coumarin, and cinnamaldehyde in comparison with tumor necrosis factor alpha (TNFα), were used in this study. Human bronchial epithelial cells (Beas2B), human mucoepidermoid carcinoma epithelial cells (H292), and human lung fibroblasts (HFL-1) were treated with each flavoring chemical for 24 hours. The cells and conditioned media were then collected and analyzed for toxicity (viability %), lung epithelial barrier function, and proinflammatory cytokine IL-8 release. Cell viability was not significantly affected by any of the flavoring chemicals tested at a concentration of 10 μM to 1 mM. Acetoin and diacetyl treatment induced IL-8 release in Beas2B cells. Acetoin- and pentanedione-treated HFL-1 cells produced a differential, but significant response for IL-8 release compared to controls and TNFα. Flavorings, such as ortho-vanillin and maltol, induced IL-8 release in Beas2B cells, but not in H292 cells. Of all the flavoring chemicals tested, acetoin and maltol were more potent inducers of IL-8 release than TNFα in Beas2B and HFL-1 cells. Flavoring chemicals rapidly impaired epithelial barrier function in human bronchial epithelial cells (16-HBE) as measured by electric cell surface impedance sensing. Our findings suggest that some of the e-cig liquids/aerosols containing flavoring chemicals can cause significant loss of epithelial barrier function and proinflammatory response in lung cells.

Characterization of endogenous calcium responses in neuronal cell lines.

PubMed

Vetter, Irina; Lewis, Richard J

2010-03-15

An increasing number of putative therapeutic targets have been identified in recent years for the treatment of neuronal pathophysiologies including pain, epilepsy, stroke and schizophrenia. Many of these targets signal through calcium (Ca(2+)), either by directly facilitating Ca(2+) influx through an ion channel, or through activation of G proteins that couple to intracellular Ca(2+) stores or voltage-gated Ca(2+) channels. Immortalized neuronal cell lines are widely used models to study neuropharmacology. However, systematic pharmacological characterization of the receptors and ion channels expressed in these cell lines is lacking. In this study, we systematically assessed endogenous Ca(2+) signaling in response to addition of agonists at potential therapeutic targets in a range of cell lines of neuronal origin (ND7/23, SH-SY5Y, 50B11, F11 and Neuro2A cells) as well as HEK293 cells, a cell line commonly used for over-expression of receptors and ion channels. This study revealed a remarkable diversity of endogenous Ca(2+) responses in these cell lines, with one or more cell lines responding to addition of trypsin, bradykinin, ATP, nicotine, acetylcholine, histamine and neurotensin. Subtype specificity of these responses was inferred from agonist potency and the effect of receptor subtype specific antagonist. Surprisingly, HEK293 and SH-SY5Y cells responded to the largest number of agonists with potential roles in neuronal signaling. These findings have implications for the heterologous expression of neuronal receptors and ion channels in these cell lines, and highlight the potential of neuron-derived cell lines for the study of a range of endogenously expressed receptors and ion channels that signal through Ca(2+). Crown Copyright 2009. Published by Elsevier Inc. All rights reserved.

Is There Natural Killer Cell Memory and Can It Be Harnessed by Vaccination? Vaccination Strategies Based on NK Cell and ILC Memory.

PubMed

Cooper, Megan A; Fehniger, Todd A; Colonna, Marco

2017-12-18

Studies over the last decade have decisively shown that innate immune natural killer (NK) cells exhibit enhanced long-lasting functional responses following a single activation event. With the increased recognition of memory and memory-like properties of NK cells, questions have arisen with regard to their ability to effectively mediate vaccination responses in humans. Moreover, recently discovered innate lymphoid cells (ILCs) could also potentially exhibit memory-like functions. Here, we review different forms of NK cell memory, and speculate about the ability of these cells and ILCs to meaningfully contribute to vaccination responses. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

High-dose bee venom exposure induces similar tolerogenic B-cell responses in allergic patients and healthy beekeepers.

PubMed

Boonpiyathad, T; Meyer, N; Moniuszko, M; Sokolowska, M; Eljaszewicz, A; Wirz, O F; Tomasiak-Lozowska, M M; Bodzenta-Lukaszyk, A; Ruxrungtham, K; van de Veen, W

2017-03-01

The involvement of B cells in allergen tolerance induction remains largely unexplored. This study investigates the role of B cells in this process, by comparing B-cell responses in allergic patients before and during allergen immunotherapy (AIT) and naturally exposed healthy beekeepers before and during the beekeeping season. Circulating B cells were characterized by flow cytometry. Phospholipase A2 (PLA)-specific B cells were identified using dual-color staining with fluorescently labeled PLA. Expression of regulatory B-cell-associated surface markers, interleukin-10, chemokine receptors, and immunoglobulin heavy-chain isotypes, was measured. Specific and total IgG1, IgG4, IgA, and IgE from plasma as well as culture supernatants of PLA-specific cells were measured by ELISA. Strikingly, similar responses were observed in allergic patients and beekeepers after venom exposure. Both groups showed increased frequencies of plasmablasts, PLA-specific memory B cells, and IL-10-secreting CD73 - CD25 + CD71 + B R 1 cells. Phospholipase A2-specific IgG4-switched memory B cells expanded after bee venom exposure. Interestingly, PLA-specific B cells showed increased CCR5 expression after high-dose allergen exposure while CXCR4, CXCR5, CCR6, and CCR7 expression remained unaffected. This study provides the first detailed characterization of allergen-specific B cells before and after bee venom tolerance induction. The observed B-cell responses in both venom immunotherapy-treated patients and naturally exposed beekeepers suggest a similar functional immunoregulatory role for B cells in allergen tolerance in both groups. These findings can be investigated in other AIT models to determine their potential as biomarkers of early and successful AIT responses. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Effects of spaceflight on levels and activity of immune cells

NASA Technical Reports Server (NTRS)

Sonnenfeld, Gerald; Berry, Wallace D.; Mandel, Adrian D.; Konstantinova, Irena V.; Taylor, Gerald R.

1990-01-01

Experiments were carried out on cells from rats that had been flown on Soviet Biosputnik Cosmos 1887 to explore the effects of speceflight on immune responses. Rat bone marrow cells were examined for their response to colony stimulating factor-M. Rat spleen and bone marrow cells were stained with antibodies directed against cell surface antigenic markers. The results of the studies indicate that bone marrow cells from flown rats showed a decreased response to colony stimulating factor. There was a higher percentage of spleen cells from flown rats staining positively for pan-T-cell, suppressor-T-cell, and interleukin-2 receptor cell surface antigens. A small increase in the percentage of cells staining positively for helper-T-cell antigens was also noted. In addition, a higher percentage of cells that appeared to be part of the myelogenous population of bone marrow cells from flown rats stained positively for surface immunoglobulin.

The therapeutic response of CDDO-Me in the esophageal squamous cell carcinoma (ESCC) cells is mediated by CaMKIIα.

PubMed

Wang, Yan-Yang; Zhou, Shun; Zhao, Ren; Hai, Ping; Zhe, Hong

2016-01-01

CDDO-Me has exhibited a potent anticancer effect in human esophageal squamous cell carcinoma (ESCC) cells in our previous study, but the molecular interactome remains elusive. We applied the approach of stable-isotope labeling by amino acids in cell culture (SILAC) to assess the proteomic responses of CDDO-Me treatment in human ESCC Ec109 cells. The data were subsequently validated using Western blot assay. The results of our study revealed that CDDO-Me increased the expression level of 543 protein molecules, but decreased the expression level of 709 protein molecules in Ec109 cells. Among these modulated protein molecules, calcium/calmodulin-dependent protein kinase type II subunit α (CaMKIIα) was highly expressed in all tested ESCC cell lines, whereas its expression levels were substantially lower in normal control cell line. Its silencing by small interfering RNA inhibited CDDO-Me induced apoptosis and autophagy in ESCC cells. Collectively, these data demonstrate that the therapeutic response of CDDO-Me in the human ESCC cells is mediated by CaMKIIα.

Mast Cell Interactions and Crosstalk in Regulating Allergic Inflammation.

PubMed

Velez, Tania E; Bryce, Paul J; Hulse, Kathryn E

2018-04-17

This review summarizes recent findings on mast cell biology with a focus on IgE-independent roles of mast cells in regulating allergic responses. Recent studies have described novel mast cell-derived molecules, both secreted and membrane-bound, that facilitate cross-talk with a variety of immune effector cells to mediate type 2 inflammatory responses. Mast cells are complex and dynamic cells that are persistent in allergy and are capable of providing signals that lead to the initiation and persistence of allergic mechanisms.

Response to Rhinovirus Infection by Human Airway Epithelial Cells and Peripheral Blood Mononuclear Cells in an In Vitro Two-Chamber Tissue Culture System

PubMed Central

Rajan, Devi; Gaston, Kelsey A.; McCracken, Courtney E.; Erdman, Dean D.; Anderson, Larry J.

2013-01-01

Human rhinovirus (HRV) infections are associated with the common cold, occasionally with more serious lower respiratory tract illnesses, and frequently with asthma exacerbations. The clinical features of HRV infection and its association with asthma exacerbation suggest that some HRV disease results from virus-induced host immune responses to infection. To study the HRV-infection-induced host responses and the contribution of these responses to disease, we have developed an in vitro model of HRV infection of human airway epithelial cells (Calu-3 cells) and subsequent exposure of human peripheral blood mononuclear cells (PBMCs) to these infected cells in a two-chamber trans-well tissue culture system. Using this model, we studied HRV 14 (species B) and HRV 16 (species A) induced cytokine and chemokine responses with PBMCs from four healthy adults. Infection of Calu-3 cells with either virus induced HRV-associated increases in FGF-Basic, IL-15, IL-6, IL-28A, ENA-78 and IP-10. The addition of PBMCs to HRV 14-infected cells gave significant increases in MIP-1β, IL-28A, MCP-2, and IFN-α as compared with mock-infected cells. Interestingly, ENA-78 levels were reduced in HRV 14 infected cells that were exposed to PBMCs. Addition of PBMCs to HRV 16-infected cells did not induce MIP-1β, IL-28A and IFN-α efficiently nor did it decrease ENA-78 levels. Our results demonstrate a clear difference between HRV 14 and HRV 16 and the source of PBMCs, in up or down regulation of several cytokines including those that are linked to airway inflammation. Such differences might be one of the reasons for variation in disease associated with different HRV species including variation in their link to asthma exacerbations as suggested by other studies. Further study of immune responses associated with different HRVs and PBMCs from different patient groups, and the mechanisms leading to these differences, should help characterize pathogenesis of HRV disease and generate novel approaches to its treatment. PMID:23799120

Architecture of a minimal signaling pathway explains the T-cell response to a 1 million-fold variation in antigen affinity and dose

PubMed Central

Lever, Melissa; Lim, Hong-Sheng; Kruger, Philipp; Nguyen, John; Trendel, Nicola; Abu-Shah, Enas; Maini, Philip Kumar; van der Merwe, Philip Anton

2016-01-01

T cells must respond differently to antigens of varying affinity presented at different doses. Previous attempts to map peptide MHC (pMHC) affinity onto T-cell responses have produced inconsistent patterns of responses, preventing formulations of canonical models of T-cell signaling. Here, a systematic analysis of T-cell responses to 1 million-fold variations in both pMHC affinity and dose produced bell-shaped dose–response curves and different optimal pMHC affinities at different pMHC doses. Using sequential model rejection/identification algorithms, we identified a unique, minimal model of cellular signaling incorporating kinetic proofreading with limited signaling coupled to an incoherent feed-forward loop (KPL-IFF) that reproduces these observations. We show that the KPL-IFF model correctly predicts the T-cell response to antigen copresentation. Our work offers a general approach for studying cellular signaling that does not require full details of biochemical pathways. PMID:27702900

Architecture of a minimal signaling pathway explains the T-cell response to a 1 million-fold variation in antigen affinity and dose.

PubMed

Lever, Melissa; Lim, Hong-Sheng; Kruger, Philipp; Nguyen, John; Trendel, Nicola; Abu-Shah, Enas; Maini, Philip Kumar; van der Merwe, Philip Anton; Dushek, Omer

2016-10-25

T cells must respond differently to antigens of varying affinity presented at different doses. Previous attempts to map peptide MHC (pMHC) affinity onto T-cell responses have produced inconsistent patterns of responses, preventing formulations of canonical models of T-cell signaling. Here, a systematic analysis of T-cell responses to 1 million-fold variations in both pMHC affinity and dose produced bell-shaped dose-response curves and different optimal pMHC affinities at different pMHC doses. Using sequential model rejection/identification algorithms, we identified a unique, minimal model of cellular signaling incorporating kinetic proofreading with limited signaling coupled to an incoherent feed-forward loop (KPL-IFF) that reproduces these observations. We show that the KPL-IFF model correctly predicts the T-cell response to antigen copresentation. Our work offers a general approach for studying cellular signaling that does not require full details of biochemical pathways.

Autofluorescence imaging captures heterogeneous drug response differences between 2D and 3D breast cancer cultures

PubMed Central

Cannon, T. M.; Shah, A. T.; Skala, M. C.

2017-01-01

Two-photon microscopy of cellular autofluorescence intensity and lifetime (optical metabolic imaging, or OMI) is a promising tool for preclinical drug development. OMI, which exploits the endogenous fluorescence from the metabolic coenzymes NAD(P)H and FAD, is sensitive to changes in cell metabolism produced by drug treatment. Previous studies have shown that drug response, genetic expression, cell-cell communication, and cell signaling in 3D culture match those of the original in vivo tumor, but not those of 2D culture. The goal of this study is to use OMI to quantify dynamic cell-level metabolic differences in drug response in 2D cell lines vs. 3D organoids generated from xenograft tumors of the same cell origin. BT474 cells and Herceptin-resistant BT474 (HR6) cells were tested. Cells were treated with vehicle control, Herceptin, XL147 (PI3K inhibitor), and the combination. The OMI index was used to quantify response, and is a linear combination of the redox ratio (intensity of NAD(P)H divided by FAD), mean NADH lifetime, and mean FAD lifetime. The results confirm that the OMI index resolves significant differences (p<0.05) in drug response for 2D vs. 3D cultures, specifically for BT474 cells 24 hours after Herceptin treatment, for HR6 cells 24 and 72 hours after combination treatment, and for HR6 cells 72 hours after XL147 treatment. Cell-level analysis of the OMI index also reveals differences in the number of cell sub-populations in 2D vs. 3D culture at 24, 48, and 72 hours post-treatment in control and treated groups. Finally, significant increases (p<0.05) in the mean lifetime of NADH and FAD were measured in 2D vs. 3D for both cell lines at 72 hours post-treatment in control and all treatment groups. These whole-population differences in the mean NADH and FAD lifetimes are supported by differences in the number of cell sub-populations in 2D vs. 3D. Overall, these studies confirm that OMI is sensitive to differences in drug response in 2D vs. 3D, and provides further information on dynamic changes in the relative abundance of metabolic cell sub-populations that contribute to this difference. PMID:28663873

Heterogeneity of Human CD4(+) T Cells Against Microbes.

PubMed

Sallusto, Federica

2016-05-20

CD4(+) T helper (Th) cells play a central role in the adaptive immune response by providing help to B cells and cytotoxic T cells and by releasing different types of cytokines in tissues to mediate protection against a wide range of pathogenic microorganisms. These functions are performed by different types of Th cells endowed with distinct migratory capacities and effector functions. Here we discuss how studies of the human T cell response to microbes have advanced our understanding of Th cell functional heterogeneity, in particular with the discovery of a distinct Th1 subset involved in the response to Mycobacteria and the characterization of two types of Th17 cells specific for extracellular bacteria or fungi. We also review new approaches to dissect at the clonal level the human CD4(+) T cell response induced by pathogens or vaccines that have revealed an unexpected degree of intraclonal diversification and propose a progressive and selective model of CD4(+) T cell differentiation.

Use of a novel antigen expressing system to study the Salmonella enterica serovar Typhi protein recognition by T cells

PubMed Central

Lou, David; Steiner, Stephanie; Rezwanul, Tasmia; Guo, Qin; Picking, William D.; Nene, Vishvanath; Sztein, Marcelo B.

2017-01-01

Salmonella enterica serovar Typhi (S. Typhi), the causative agent of the typhoid fever, is a pathogen of great public health importance. Typhoid vaccines have the potential to be cost-effective measures towards combating this disease, yet the antigens triggering host protective immune responses are largely unknown. Given the key role of cellular-mediated immunity in S. Typhi protection, it is crucial to identify S. Typhi proteins involved in T-cell responses. Here, cells from individuals immunized with Ty21a typhoid vaccine were collected before and after immunization and used as effectors. We also used an innovative antigen expressing system based on the infection of B-cells with recombinant Escherichia coli (E. coli) expressing one of four S. Typhi gene products (i.e., SifA, OmpC, FliC, GroEL) as targets. Using flow cytometry, we found that the pattern of response to specific S. Typhi proteins was variable. Some individuals responded to all four proteins while others responded to only one or two proteins. We next evaluated whether T-cells responding to recombinant E. coli also possess the ability to respond to purified proteins. We observed that CD4+ cell responses, but not CD8+ cell responses, to recombinant E. coli were significantly associated with the responses to purified proteins. Thus, our results demonstrate the feasibility of using an E. coli expressing system to uncover the antigen specificity of T-cells and highlight its applicability to vaccine studies. These results also emphasize the importance of selecting the stimuli appropriately when evaluating CD4+ and CD8+ cell responses. PMID:28873442

Role of IL-4 receptor α-positive CD4(+) T cells in chronic airway hyperresponsiveness.

PubMed

Kirstein, Frank; Nieuwenhuizen, Natalie E; Jayakumar, Jaisubash; Horsnell, William G C; Brombacher, Frank

2016-06-01

TH2 cells and their cytokines are associated with allergic asthma in human subjects and with mouse models of allergic airway disease. IL-4 signaling through the IL-4 receptor α (IL-4Rα) chain on CD4(+) T cells leads to TH2 cell differentiation in vitro, implying that IL-4Rα-responsive CD4(+) T cells are critical for the induction of allergic asthma. However, mechanisms regulating acute and chronic allergen-specific TH2 responses in vivo remain incompletely understood. This study defines the requirements for IL-4Rα-responsive CD4(+) T cells and the IL-4Rα ligands IL-4 and IL-13 in the development of allergen-specific TH2 responses during the onset and chronic phase of experimental allergic airway disease. Development of acute and chronic ovalbumin (OVA)-induced allergic asthma was assessed weekly in CD4(+) T cell-specific IL-4Rα-deficient BALB/c mice (Lck(cre)IL-4Rα(-/lox)) and respective control mice in the presence or absence of IL-4 or IL-13. During acute allergic airway disease, IL-4 deficiency did not prevent the onset of TH2 immune responses and OVA-induced airway hyperresponsiveness or goblet cell hyperplasia, irrespective of the presence or absence of IL-4Rα-responsive CD4(+) T cells. In contrast, deficiency of IL-13 prevented allergic asthma, irrespective of the presence or absence of IL-4Rα-responsive CD4(+) T cells. Importantly, chronic allergic inflammation and airway hyperresponsiveness were dependent on IL-4Rα-responsive CD4(+) T cells. Deficiency in IL-4Rα-responsive CD4(+) T cells resulted in increased numbers of IL-17-producing T cells and, consequently, increased airway neutrophilia. IL-4-responsive T helper cells are dispensable for acute OVA-induced airway disease but crucial in maintaining chronic asthmatic pathology. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Cell-to-cell contact dependence and junctional protein content are correlated with in vivo maturation of pancreatic beta cells.

PubMed

Santos-Silva, Junia Carolina; Carvalho, Carolina Prado de França; de Oliveira, Ricardo Beltrame; Boschero, Antonio Carlos; Collares-Buzato, Carla Beatriz

2012-07-01

In this study, we investigated the cellular distribution of junctional proteins and the dependence on cell-cell contacts of pancreatic beta cells during animal development. Fetus and newborn rat islets, which display a relatively poor insulin secretory response to glucose, present an immature morphology and cytoarchitecture when compared with young and adult islets that are responsive to glucose. At the perinatal stage, beta cells display a low junctional content of neural cell adhesion molecule (N-CAM), α- and β-catenins, ZO-1, and F-actin, while a differential distribution of N-CAM and Pan-cadherin was seen in beta cells and nonbeta cells only from young and adult islets. In the absence of intercellular contacts, the glucose-stimulated insulin secretion was completely blocked in adult beta cells, but after reaggregation they partially reestablished the secretory response to glucose. By contrast, neonatal beta cells were poorly responsive to sugar, regardless of whether they were arranged as intact islets or as isolated cells. Interestingly, after 10 days of culturing, neonatal beta cells, known to display increased junctional protein content in vitro, became responsive to glucose and concomitantly dependent on cell-cell contacts. Therefore, our data suggest that the developmental acquisition of an adult-like insulin secretory pattern is paralleled by a dependence on direct cell-cell interactions.

9-AAA inhibits growth and induces apoptosis in human melanoma A375 and rat prostate adenocarcinoma AT-2 and Mat-LyLu cell lines but does not affect the growth and viability of normal fibroblasts.

PubMed

Korohoda, Włodzimierz; Hapek, Anna; Pietrzak, Monika; Ryszawy, Damian; Madeja, Zbigniew

2016-11-01

The present study found that, similarly to 5-fluorouracil, low concentrations (1-10 µM) of 9-aminoacridine (9-AAA) inhibited the growth of the two rat prostate cancer AT-2 and Mat-LyLu cell lines and the human melanoma A375 cell line. However, at the same concentrations, 9-AAA had no effect on the growth and apoptosis of normal human skin fibroblasts (HSFs). The differences between the cellular responses of the AT-2 and Mat-LyLu cell lines, which differ in malignancy, were found to be relatively small compared with the differences between normal HSFs and the cancer cell lines. Visible effects on the cell growth and survival of tumor cell lines were observed after 24-48 h of treatment with 9-AAA, and increased over time. The inhibition of cancer cell growth was found to be due to the gradually increasing number of cells dying by apoptosis, which was observed using two methods, direct counting and FlowSight analysis. Simultaneously, cell motile activity decreased to the same degree in cancer and normal cells within the first 8 h of incubation in the presence of 9-AAA. The results presented in the current study suggest that short-lasting tests for potential anticancer substances can be insufficient; which may result in cell type-dependent differences in the responses of cells to tested compounds that act with a delay being overlooked. The observed differences in responses between normal human fibroblasts and cancer cells to 9-AAA show the requirement for additional studies to be performed simultaneously on differently reacting cancer and normal cells, to determine the molecular mechanisms responsible for these differences.

Antibody response against Betaferon® in immune tolerant mice: involvement of marginal zone B-cells and CD4+ T-cells and apparent lack of immunological memory.

PubMed

Sauerborn, Melody; van Beers, Miranda M C; Jiskoot, Wim; Kijanka, Grzegorz M; Boon, Louis; Schellekens, Huub; Brinks, Vera

2013-01-01

The immunological processes underlying immunogenicity of recombinant human therapeutics are poorly understood. Using an immune tolerant mouse model we previously demonstrated that aggregates are a major trigger of the antidrug antibody (ADA) response against recombinant human interferon beta (rhIFNβ) products including Betaferon®, and that immunological memory seems to be lacking after a rechallenge with non-aggregated rhIFNβ. The apparent absence of immunological memory indicates a CD4+ T-cell independent (Tind) immune response underlying ADA formation against Betaferon®. This hypothesis was tested. Using the immune tolerant mouse model we first validated that rechallenge with highly aggregated rhIFNβ (Betaferon®) does not lead to a subsequent fast increase in ADA titers, suggesting a lack of immunological memory. Next we assessed whether Betaferon® could act as Tind antigen by inactivation of marginal zone (MZ) B-cells during treatment. MZ B-cells are major effector cells involved in a Tind immune response. In a following experiment we depleted the mice from CD4+ T-cells to test their involvement in the ADA response against Betaferon®. Inactivation of MZ B-cells at the start of Betaferon® treatment drastically lowered ADA levels, suggesting a Tind immune response. However, persistent depletion of CD4+ T-cells before and during Betaferon® treatment abolished the ADA response in almost all mice. The immune response against rhIFNβ in immune tolerant mice is neither a T-cell independent nor a classical T-cell dependent immune response. Further studies are needed to confirm absence of immunological memory (cells).

Regeneration of Xenopus laevis spinal cord requires Sox2/3 expressing cells

PubMed Central

Muñoz, Rosana; Edwards-Faret, Gabriela; Moreno, Mauricio; Zuñiga, Nikole; Cline, Hollis; Larraín, Juan

2016-01-01

Spinal cord regeneration is very inefficient in humans, causing paraplegia and quadriplegia. Studying model organisms that can regenerate the spinal cord in response to injury could be useful for understanding the cellular and molecular mechanisms that explain why this process fails in humans. Here, we use Xenopus laevis as a model organism to study spinal cord repair. Histological and functional analyses showed that larvae at pre-metamorphic stages restore anatomical continuity of the spinal cord and recover swimming after complete spinal cord transection. These regenerative capabilities decrease with onset of metamorphosis. The ability to study regenerative and non-regenerative stages in Xenopus laevis makes it a unique model system to study regeneration. We studied the response of Sox2/3 expressing cells to spinal cord injury and their function in the regenerative process. We found that cells expressing Sox2 and/or Sox3 are present in the ventricular zone of regenerative animals and decrease in non-regenerative froglets. Bromodeoxyuridine (BrdU) experiments and in vivo time-lapse imaging studies using green fluorescent protein (GFP) expression driven by the Sox3 promoter showed a rapid, transient and massive proliferation of Sox2/3+ cells in response to injury in the regenerative stages. The in vivo imaging also demonstrated that Sox2/3+ neural progenitor cells generate neurons in response to injury. In contrast, these cells showed a delayed and very limited response in non-regenerative froglets. Sox2 knockdown and overexpression of a dominant negative form of Sox2 disrupts locomotor and anatomical-histological recovery. We also found that neurogenesis markers increase in response to injury in regenerative but not in non-regenerative animals. We conclude that Sox2 is necessary for spinal cord regeneration and suggest a model whereby spinal cord injury activates proliferation of Sox2/3 expressing cells and their differentiation into neurons, a mechanism that is lost in non-regenerative froglets. PMID:25797152

Rapid flow-induced responses in endothelial cells

NASA Technical Reports Server (NTRS)

Stamatas, G. N.; McIntire, L. V.

2001-01-01

Endothelial cells alter their morphology, growth rate, and metabolism in response to fluid shear stress. To study rapid flow-induced responses in the 3D endothelial cell morphology and calcium distribution, coupled fluorescence microscopy with optical sectioning, digital imaging, and numerical deconvolution techniques have been utilized. Results demonstrate that within the first minutes of flow application nuclear calcium is increasing. In the same time frame whole cell height and nuclear height are reduced by about 1 microm. Whole cell height changes may facilitate reduction of shear stress gradients on the luminal surface, whereas nuclear structural changes may be important for modulating endothelial growth rate and metabolism. To study the role of the cytoskeleton in these responses, endothelial cells have been treated with specific disrupters (acrylamide, cytochalasin D, and colchicine) of each of the cytoskeleton elements (intermediate filaments, microfilaments, and microtubules, respectively). None of these compounds had any effect on the shear-induced calcium response. Cytochalasin D and acrylamide did not affect the shear-induced nuclear morphology changes. Colchicine, however, completely abrogated the response, indicating that microtubules may be implicated in force transmission from the plasma membrane to the nucleus. A pedagogical model based on tensegrity theory principles is presented that is consistent with the results on the 3D endothelial morphology.

Adjuvant-enhanced CD4 T Cell Responses are Critical to Durable Vaccine Immunity.

PubMed

Martins, Karen A O; Cooper, Christopher L; Stronsky, Sabrina M; Norris, Sarah L W; Kwilas, Steven A; Steffens, Jesse T; Benko, Jacqueline G; van Tongeren, Sean A; Bavari, Sina

2016-01-01

Protein-based vaccines offer a safer alternative to live-attenuated or inactivated vaccines but have limited immunogenicity. The identification of adjuvants that augment immunogenicity, specifically in a manner that is durable and antigen-specific, is therefore critical for advanced development. In this study, we use the filovirus virus-like particle (VLP) as a model protein-based vaccine in order to evaluate the impact of four candidate vaccine adjuvants on enhancing long term protection from Ebola virus challenge. Adjuvants tested include poly-ICLC (Hiltonol), MPLA, CpG 2395, and alhydrogel. We compared and contrasted antibody responses, neutralizing antibody responses, effector T cell responses, and T follicular helper (Tfh) cell frequencies with each adjuvant's impact on durable protection. We demonstrate that in this system, the most effective adjuvant elicits a Th1-skewed antibody response and strong CD4 T cell responses, including an increase in Tfh frequency. Using immune-deficient animals and adoptive transfer of serum and cells from vaccinated animals into naïve animals, we further demonstrate that serum and CD4 T cells play a critical role in conferring protection within effective vaccination regimens. These studies inform on the requirements of long term immune protection, which can potentially be used to guide screening of clinical-grade adjuvants for vaccine clinical development.

TACI is required for efficient plasma cell differentiation in response to T-independent type 2 antigens.

PubMed

Mantchev, George T; Cortesão, Catarina S; Rebrovich, Michelle; Cascalho, Marilia; Bram, Richard J

2007-08-15

The control of systemic infection by encapsulated microorganisms requires T-independent type II (TI-2) Ab responses to bacterial polysaccharides. To understand how such responses evolve, we explored the function of transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI), a member of the TNFR family, required for TI-2 Ab production. Quasimonoclonal (QM) mice produce robust TI-2 responses to 4-hydroxy-3-nitrophenylacetate (NP)-Ficoll, owing to the high precursor frequency of NP-specific B cells in the marginal zone of the spleen. QM mice that lack TACI produce decreased numbers of IgM (2-fold) and IgG (1.6-fold) NP-specific ASCs, compared with TACI-positive QM mice in response to immunization with NP-Ficoll. Our studies indicate that TACI acts at a remote time from activation because TACI is not necessary for activation and proliferation of B cells both in vitro and in vivo. Instead, TACI-deficient QM B cells remained in the cell cycle longer than TACI-proficient QM cells and had impaired plasma cell differentiation in response to NP-Ficoll. We conclude that TACI has dual B cell-autonomous functions, inhibiting prolonged B cell proliferation and stimulating plasma cell differentiation, thus resolving the longstanding paradox that TACI may have both B cell-inhibitory and -stimulatory functions. By promoting plasma cell differentiation earlier during clonal expansion, TACI may decrease the chances of autoantibody production by somatic hypermutation of Ig genes in response to T-independent Ags.

Mesenchymal stem cells induce dermal fibroblast responses to injury

PubMed Central

Smith, Andria N.; Willis, Elise; Chan, Vincent T.; Muffley, Lara A.; Isik, F. Frank; Gibran, Nicole S.; Hocking, Anne M.

2009-01-01

Although bone marrow-derived mesenchymal stem cells have been shown to promote repair when applied to cutaneous wounds, the mechanism for this response remains to be determined. The aim of this study was to determine the effects of paracrine signaling from mesenchymal stem cells on dermal fibroblast responses to injury including proliferation, migration and expression of genes important in wound repair. Dermal fibroblasts were co-cultured with bone marrow-derived mesenchymal stem cells grown in inserts, which allowed for paracrine interactions without direct cell contact. In this co-culture model, bone marrow-derived mesenchymal stem cells regulate dermal fibroblast proliferation, migration and gene expression. When co-cultured with mesenchymal stem cells, dermal fibroblasts show increased proliferation and accelerated migration in a scratch assay. A chemotaxis assay also demonstrated that dermal fibroblasts migrate towards bone marrow-derived mesenchymal stem cells. A PCR array was used to analyze the effect of mesenchymal stem cells on dermal fibroblast gene expression. In response to mesenchymal stem cells, dermal fibroblasts up-regulate integrin alpha 7 expression and down-regulate expression of ICAM1, VCAM1 and MMP11. These observations suggest that mesenchymal stem cells may provide an important early signal for dermal fibroblast responses to cutaneous injury. PMID:19666021

Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

PubMed Central

Anthony, Donald D.; Milkovich, Kimberly A.; Zhang, Wenji; Rodriguez, Benigno; Yonkers, Nicole L.; Tary-Lehmann, Magdalena; Lehmann, Paul V.

2012-01-01

Chronic allograft rejection is in part mediated by host T cells that recognize allogeneic antigens on transplanted tissue. One factor that determines the outcome of a T cell response is clonal size, while another is the effector quality. Studies of alloimmune predictors of transplant graft survival have most commonly focused on only one measure of the alloimmune response. Because differing qualities and frequencies of the allospecific T cell response may provide distinctly different information we analyzed the relationship between frequency of soluble antigen and allo-antigen specific memory IFN-γ secreting CD4 and CD8 T cells, their ability to secrete IL-2, and their proliferative capacity, while accounting for cognate and bystander proliferation. The results show proliferative responses primarily reflect on IL-2 production by antigen-specific T cells, and that proliferating cells in such assays entail a considerable fraction of bystander cells. On the other hand, proliferation (and IL-2 production) did not reflect on the frequency of IFN-γ producing memory cells, a finding particularly accentuated in the CD8 T cell compartment. These data provide rationale for considering both frequency and effector function of pre-transplant T cell reactivity when analyzing immune predictors of graft rejection. PMID:24710419

Effect of filgrastim (recombinant human granulocyte colony stimulating factor) on IgE responses in human asthma: a case study.

PubMed

Smith-Norowitz, Tamar A; Joks, Rauno; Norowitz, Kevin B; Chice, Seto; Durkin, Helen G; Bluth, Martin H

2013-10-01

The role of peripheral blood progenitor cell mobilization on Immunoglobulin E (IgE) responses has not been studied. Distributions of blood lymphocytes (CD4+, CD8+, CD8+CD60+, CD19+, CD23+, CD16/56+, CD25, CD45RA+, CD45RO+, CD34+), and levels of serum immunoglobulins (IgM, IgG, IgA, IgE) were studied in an allergic asthmatic serum IgE+ (181IU/mL) adult (m/45 y/o) donor undergoing routine stem cell mobilization protocol (American Society of Hematology) before (day-30), during (day 4), and after (1 wk post last dose) filgrastim (subcutaneous, 480 mcg, 2qd) treatment (flow cytometry, nephelometry, UniCAP Total IgE Fluoro enzyme immunoassay). On day 4 of filgrastim treatment, numbers of CD8+CD60+T cells and CD23+ blood cells dramatically increased (98% and 240% respectively) compared with pre treatment. In contrast on day 4 of treatment, serum IgE levels decreased (>50%) compared with pre treatment. CD8+CD60+T cells and CD23+ blood cells and serum IgE levels approached pre-treatment levels at 1 week post treatment. Filgrastim treatment transiently increases numbers of CD8+CD60+T and CD23+ expressing cells, which are known to regulate human IgE responses, while also transiently suppressing ongoing IgE responses. These results suggest that filgrastim affects IgE related responses, and may be useful in modulating allergic responses. Copyright © 2013 Elsevier Ltd. All rights reserved.

The suppression of mitogen responses associated with resistance to experimental autoimmune encephalomyelitis requires adherent and T cells.

PubMed

Lyman, W D; Brosnan, C F; Kadish, A S; Raine, C S

1984-05-01

Resistance to experimental autoimmune encephalomyelitis (EAE) in Hartley guinea pigs has previously been reported to be associated with disease-specific antigen-induced suppression of mitogen responses in vitro. The present studies were initiated to investigate the requirement for different cell populations in this suppression. Intact and adherent-cell-depleted cultures of spleen cells from experimental and control animals were incubated with myelin basic protein (MBP), the major antigen of EAE, with the T-cell mitogen concanavalin A (Con A) alone or with Con A in the presence of MBP. In agreement with previous studies, MBP-induced suppression of the Con A response was observed only in cultures derived from resistant animals. In addition, it was observed that this suppression was abrogated by depletion of adherent cells. When cells from resistant and susceptible animals were mixed, suppression occurred only in the presence of nonadherent cells from resistant guinea pigs. Adherent cells from either resistant or susceptible animals functioned equally well. Cultures of purified E-rosette-forming cells (E+) from resistant animals (i.e., T cells) showed no suppression. Similarly, cells from these same animals which were depleted of E+ cells (i.e., non-T cells) did not demonstrate suppression in vitro. Upon reconstitution of spleen cell populations from resistant guinea pigs by mixing E+ and E- cells, suppression was restored. These experiments show that this model of suppression in vitro requires adherent cells as well as T cells and suggests that antigen-induced suppression of mitogen responses is dependent upon a cell-mediated immunologic mechanism.

Grapevine cell early activation of specific responses to DIMEB, a resveratrol elicitor

PubMed Central

Zamboni, Anita; Gatto, Pamela; Cestaro, Alessandro; Pilati, Stefania; Viola, Roberto; Mattivi, Fulvio; Moser, Claudio; Velasco, Riccardo

2009-01-01

Background In response to pathogen attack, grapevine synthesizes phytoalexins belonging to the family of stilbenes. Grapevine cell cultures represent a good model system for studying the basic mechanisms of plant response to biotic and abiotic elicitors. Among these, modified β-cyclodextrins seem to act as true elicitors inducing strong production of the stilbene resveratrol. Results The transcriptome changes of Vitis riparia × Vitis berlandieri grapevine cells in response to the modified β-cyclodextrin, DIMEB, were analyzed 2 and 6 h after treatment using a suppression subtractive hybridization experiment and a microarray analysis respectively. At both time points, we identified a specific set of induced genes belonging to the general phenylpropanoid metabolism, including stilbenes and hydroxycinnamates, and to defence proteins such as PR proteins and chitinases. At 6 h we also observed a down-regulation of the genes involved in cell division and cell-wall loosening. Conclusions We report the first large-scale study of the molecular effects of DIMEB, a resveratrol inducer, on grapevine cell cultures. This molecule seems to mimic a defence elicitor which enhances the physical barriers of the cell, stops cell division and induces phytoalexin synthesis. PMID:19660119

Cellular Immune Responses to Live Attenuated Japanese Encephalitis (JE) Vaccine SA14-14-2 in Adults in a JE/Dengue Co-Endemic Area.

PubMed

Turtle, Lance; Tatullo, Filippo; Bali, Tanushka; Ravi, Vasanthapuram; Soni, Mohammed; Chan, Sajesh; Chib, Savita; Venkataswamy, Manjunatha M; Fadnis, Prachi; Yaïch, Mansour; Fernandez, Stefan; Klenerman, Paul; Satchidanandam, Vijaya; Solomon, Tom

2017-01-01

Japanese encephalitis (JE) virus (JEV) causes severe epidemic encephalitis across Asia, for which the live attenuated vaccine SA14-14-2 is being used increasingly. JEV is a flavivirus, and is closely related to dengue virus (DENV), which is co-endemic in many parts of Asia, with clinically relevant interactions. There is no information on the human T cell response to SA14-14-2, or whether responses to SA14-14-2 cross-react with DENV. We used live attenuated JE vaccine SA14-14-2 as a model for studying T cell responses to JEV infection in adults, and to determine whether these T cell responses are cross-reactive with DENV, and other flaviviruses. We conducted a single arm, open label clinical trial (registration: clinicaltrials.gov NCT01656200) to study T cell responses to SA14-14-2 in adults in South India, an area endemic for JE and dengue. Ten out of 16 (62.5%) participants seroconverted to JEV SA14-14-2, and geometric mean neutralising antibody (NAb) titre was 18.5. Proliferation responses were commonly present before vaccination in the absence of NAb, indicating a likely high degree of previous flavivirus exposure. Thirteen of 15 (87%) participants made T cell interferon-gamma (IFNγ) responses against JEV proteins. In four subjects tested, at least some T cell epitopes mapped cross-reacted with DENV and other flaviviruses. JEV SA14-14-2 was more immunogenic for T cell IFNγ than for NAb in adults in this JE/DENV co-endemic area. The proliferation positive, NAb negative combination may represent a new marker of long term immunity/exposure to JE. T cell responses can cross-react between JE vaccine and DENV in a co-endemic area, illustrating a need for greater knowledge on such responses to inform the development of next-generation vaccines effective against both diseases. clinicaltrials.gov (NCT01656200).

Effect of age and latent CMV infection on CD8+ CD56+ T cells (NKT-like) frequency and functionality.

PubMed

Hassouneh, Fakhri; Campos, Carmen; López-Sejas, Nelson; Alonso, Corona; Tarazona, Raquel; Solana, Rafael; Pera, Alejandra

2016-09-01

Changes in the T cell pool caused by CMV infection have been proposed to contribute to immunosenescence, but it has been postulated that CMV can also have some beneficial effects in young individuals improving the immune response to other pathogens. T cells expressing CD56 (NKT-like cells) are cytotoxic effector cells with a significant role in the immune response against cancer. We have studied how age and latent CMV infection affect the frequency of NKT-like cells (CD8+ CD56+ T cells) and their response to Staphylococcal Enterotoxin B (SEB) in the context of CMV and ageing. NKT-like cell percentage increases with the combination of both CMV and age. The response to SEB and the polyfunctional index of NKT-like cells also increase with age in CMV-seropositive individuals. In young individuals, CMV infection induces a shift on the polyfunctional profile of CD8+ CD56- T cells not observed on the NKT-like cells response. NKT-like cells expressing CD57 are expanded in CMV-seropositive individuals and are more polyfunctional than their CD57-  counterpart. In addition CD57- NKT-like cells are more polyfunctional than CD8+ CD56- CD57- T cells. The results support that the expansion of polyfunctional NKT-cells may have a beneficial effect on the immune response against pathogens. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Tumour response endpoints in the BA1112 rat sarcoma.

PubMed Central

Martin, D. F.; Moulder, J. E.; Fischer, J. J.

1980-01-01

The rat rhabdomyosarcoma BA1112 has a number of features which make it a useful model for the study of tumour response to radiation therapy. It is a transplantable tumour, isologous to an inbred line of WAG/Rij rats and it elicits no demonstrable host immune response. The tumour grows locally at the implantation site and rarely metastasizes. It is known to contain hypoxic cells which reoxygenate during a prolonged course of fractionated radiation therapy. The growth and radiation response characteristics of the tumour have remained stable for over 15 years. A newly developed in vitro assay for tumour cell clonogenicity greatly facilitates the measurement of radiation dose-response curves and the monitoring of cell response following in vivo treatment. The in vivo response of the tumour to fractionated radiation therapy is analysed in terms of cellular response, reoxygenation and cell proliferation. PMID:6932936

T cell cytokine responses to stimulation with Ureaplasma parvum in pregnancy.

PubMed

Friedland, Yael D; Lee-Pullen, Tracey F; Nathan, Elizabeth A; Watts, Rory; Keelan, Jeffrey A; Payne, Matthew S; Ireland, Demelza J

2016-08-01

Ureaplasma spp. are a common vaginal microorganism causally linked to inflammation-driven preterm birth (PTB). The nature of the immune response to Ureaplasma spp. may influence PTB risk. This study sought to define maternal T cell cytokine responses to in vitro stimulation with Ureaplasma parvum serovar 3 (UpSV3) in vaginally colonised (UP+) and non-colonised (UP-) pregnant women. Whole blood flow cytometry demonstrated an increase (p=0.027) in the baseline frequency of IFNγ-positive CD3(+)CD4(-)(CD8(+)) T cells in UP+ women. UpSV3 stimulation resulted in a significant and specific increase (p=0.001) in the frequency of IFNγ-positive CD3(+)CD4(-)(CD8(+)) T cells, regardless of vaginal colonisation status. UpSV3 stimulation also increased the frequency of IFNγ-positive CD3(+)CD4(+) T cells, particularly in the UP+ group (p=0.003). This is the first published study to examine T cell responses to Ureaplasma spp. Future appropriately-powered studies are needed to assess whether insufficient priming or a loss of tolerance to Ureaplasma spp. is occurring in UP+ women at risk of PTB. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Vascular endothelial growth factor-A enhances indoleamine 2,3-dioxygenase expression by dendritic cells and subsequently impacts lymphocyte proliferation

PubMed Central

Marti, Luciana Cavalheiro; Pavon, Lorena; Severino, Patricia; Sibov, Tatiana; Guilhen, Daiane; Moreira-Filho, Carlos Alberto

2013-01-01

Dendritic cells (DCs) are antigen (Ag)-presenting cells that activate and stimulate effective immune responses by T cells, but can also act as negative regulators of these responses and thus play important roles in immune regulation. Pro-angiogenic vascular endothelial growth factor (VEGF) has been shown to cause defective DC differentiation and maturation. Previous studies have demonstrated that the addition of VEGF to DC cultures renders these cells weak stimulators of Ag-specific T cells due to the inhibitory effects mediated by VEGF receptor 1 (VEGFR1) and/or VEGFR2 signalling. As the enzyme indoleamine 2,3-dioxygenase (IDO) is recognised as an important negative regulator of immune responses, this study aimed to investigate whether VEGF affects the expression of IDO by DCs and whether VEGF-matured DCs acquire a suppressor phenotype. Our results are the first to demonstrate that VEGF increases the expression and activity of IDO in DCs, which has a suppressive effect on Ag-specific and mitogen-stimulated lymphocyte proliferation. These mechanisms have broad implications for the study of immunological responses and tolerance under conditions as diverse as cancer, graft rejection and autoimmunity. PMID:24141959

Intralesional corticosteroid injections in the treatment of central giant cell lesions of the jaws: A meta-analytic study

PubMed Central

Araújo, Phelype M.; de Souza-Carvalho, Abrahao C.; Cavalcante, Roberta B.; Sant’Ana, Eduardo; Nongueira, Renato L.

2013-01-01

Objective: The aim of this study was to evaluate the response of treatment of central giant cell lesion to intralesional corticosteroid injections. Study Design: Review of articles indexed in PubMed on the topic between the years 1988 and 2011, and development of a descriptive meta-analysis of the results. Results: Sample of 41 patients primarily treated with intralesional corticosteroid injections was obtained, with a male female ratio of 1:0.95, being 23 aggressive and 18 non-aggressive central giant cell lesions. Triamcinolone acetonide and triamcinolone hexacetonide were the drugs used, and 78.0% cases were considered as good result, 14.6% were considered as moderate response and 7.3% were considered as negative result to treatment. Considering the aggressiveness, 88.9% of non-aggressive lesions presented a good response to treatment, in aggressive central giant cell lesions, 69.6% presented a good response to intralesional corticosteroid injections. Conclusion: In view of the results analyzed, intralesional corticosteroid injections could be considered as first treatment option for central giant cell lesion. Key words:Central giant cell lesion, corticosteroids injections, triamcinolone hexacetonide, triamcinolone acetonide. PMID:23385503

A randomized phase II trial comparing two different sequence combinations of autologous vaccine and human recombinant interferon gamma and human recombinant interferon alpha2B therapy in patients with metastatic renal cell carcinoma: clinical outcome and analysis of immunological parameters.

PubMed

Schwaab, T; Heaney, J A; Schned, A R; Harris, R D; Cole, B F; Noelle, R J; Phillips, D M; Stempkowski, L; Ernstoff, M S

2000-04-01

The clinical observation of spontaneous regression in patients with renal cell carcinoma (RCC) and the response to various immunotherapeutic therapies strongly suggest a role for the host immune system in this disease. Prior studies showed that sequential administration of interferon (IFN) gamma and IFN alpha to RCC patients was safe. Clinical responses as well as immune changes in the peripheral blood mononuclear cell compartment were observed. Autologous tumor cell vaccines (AV) have also demonstrated activity in renal cell carcinoma. We hypothesize that the addition of AV to sequential IFN gamma and a therapy might improve the tumor-specific immune response by providing an appropriate source of antigen in the appropriate cytokine environment. To our knowledge, this is the first trial using AV combined with IFN alpha and IFN gamma. The purpose of this study was to evaluate the feasibility of manufacturing and administering (AV) from resected tumor samples, and administration of AV with combination IFN gamma and IFN alpha therapy. Finally, the impact on immunological parameters of these treatment options was assessed. Patients with metastatic RCC were randomly assigned to receive AV plus bCG along with a sequential administration of IFN gamma and a either together or after initiation of vaccine. Toxicity and clinical responses were evaluated. Modulations of the immune system were investigated by analyzing phenotype, cytokine mRNA expression, T cell proliferation and cytotoxicity in the peripheral blood mononuclear cell compartment. Fourteen patients with metastatic renal cell carcinoma were enrolled in this study; 9 were available for response evaluation. In a 70 day period, 3 (33%) showed mixed responses, 5 (56%) stable disease and 1 (11%) progression of disease. Toxicities were consistent with previous clinical reports. In the flow-cytometry phenotype analysis, stimulation of distinct subsets of circulating T-lymphocytes and a decrease of CD8+ T cell subsets was demonstrated. T-cell proliferation to allogeneic tumor cell stimulation improved following treatment. IL-4 and IL-5 mRNA levels were reduced in all patients after treatment. Patients who responded to treatment did not produce any IL-4 mRNA at all, before or after treatment. AV with IFNgamma and IFNalpha therapy might induce a MHC class-mediated cytotoxic T lymphocyte (CTL) response. We suggest that adequate therapy might direct T cell response toward a Th1 type response. We hypothesize a state of improved immune readiness in patients who might eventually respond to immunotherapy.

Spontaneous T-cell responses against the immune check point programmed-death-ligand 1 (PD-L1) in patients with chronic myeloproliferative neoplasms correlate with disease stage and clinical response.

PubMed

Holmström, Morten Orebo; Riley, Caroline Hasselbalch; Skov, Vibe; Svane, Inge Marie; Hasselbalch, Hans Carl; Andersen, Mads Hald

2018-01-01

The Chronic Myeloproliferative Neoplasms (MPN) are cancers characterized by hyperinflammation and immune deregulation. Concurrently, the expression of the immune check point programmed death ligand 1 (PD-L1) is induced by inflammation. In this study we report on the occurrence of spontaneous T cell responses against a PD-L1 derived epitope in patients with MPN. We show that 71% of patients display a significant immune response against PD-L1, and patients with advanced MPN have significantly fewer and weaker PD-L1 specific immune responses compared to patients with non-advanced MPN. The PD-L1 specific T cell responses are CD4 + T cell responses, and by gene expression analysis we show that expression of PD-L1 is enhanced in patients with MPN. This could imply that the tumor specific immune response in MPN could be enhanced by vaccination with PD-L1 derived epitopes by boosting the anti-regulatory immune response hereby allowing tumor specific T cell to exert anti-tumor immunity.

Impaired Cytogenetic Damage Repair and Cell Cycle Regulation in Response to Ionizing Radiation in Human Fibroblast Cells with Individual Knock-down of 25 Genes

NASA Technical Reports Server (NTRS)

Zhang, Ye; Rohde, Larry; Emami, Kamal; Hammond, Dianne; Casey, Rachael; Mehta, Satish; Jeevarajan, Antony; Pierson, Duane; Wu, Honglu

2008-01-01

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have demonstrated that genes with upregulated expression induced by IR may play important roles in DNA damage sensing, cell cycle checkpoint and chromosomal repair, the relationship between the regulation of gene expression by IR and its impact on cytogenetic responses to ionizing radiation has not been systematically studied. In our present study, the expression of 25 genes selected based on their transcriptional changes in response to IR or from their known DNA repair roles were individually knocked down by siRNA transfection in human fibroblast cells. Chromosome aberrations (CA) and micronuclei (MN) formation were measured as the cytogenetic endpoints. Our results showed that the yield of MN and/or CA formation were significantly increased by suppressed expression of 5 genes that included Ku70 in the DSB repair pathway; XPA in the NER pathway; RPA1 in the MMR pathway; RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes including MRE11A, RAD51 in the DSB pathway, and SESN1 and SUMO1 showed significant inhibition of cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, p21 and MLH1 expression resulted in both enhanced cell cycle progression and significantly higher yield of cytogenetic damage, indicating the involvement of these gene products in both cell cycle control and DNA damage repair. Of these 11 genes that affected the cytogenetic response, 9 were up-regulated in the cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulating the biological consequences after IR. Failure to express these IR-responsive genes, such as by gene mutation, could seriously change the outcome of the post IR scenario and lead to carcinogenesis.

TOPK modulates tumour-specific radiosensitivity and correlates with recurrence after prostate radiotherapy

PubMed Central

Pirovano, Giacomo; Ashton, Thomas M; Herbert, Katharine J; Bryant, Richard J; Verrill, Clare L; Cerundolo, Lucia; Buffa, Francesca M; Prevo, Remko; Harrap, Iona; Ryan, Anderson J; Macaulay, Valentine; McKenna, William G; Higgins, Geoff S

2017-01-01

Background: Tumour-specific radiosensitising treatments may enhance the efficacy of radiotherapy without exacerbating side effects. In this study we determined the radiation response following depletion or inhibition of TOPK, a mitogen-activated protein kinase kinase family Ser/Thr protein kinase that is upregulated in many cancers. Methods: Radiation response was studied in a wide range of cancer cell lines and normal cells using colony formation assays. The effect on cell cycle progression was assessed and the relationship between TOPK expression and therapeutic efficacy was studied in a cohort of 128 prostate cancer patients treated with radical radiotherapy. Results: TOPK knockdown did not alter radiation response in normal tissues, but significantly enhanced radiosensitivity in cancer cells. This result was recapitulated in TOPK knockout cells and with the TOPK inhibitor, OTS964. TOPK depletion altered the G1/S transition and G2/M arrest in response to radiation. Furthermore, TOPK depletion increased chromosomal aberrations, multinucleation and apoptotic cell death after irradiation. These results suggest a possible role for TOPK in the radiation-induced DNA damage checkpoints. These findings have clinical relevance, as elevated TOPK protein expression was associated with poorer clinical outcomes in prostate cancer patients treated with radical radiotherapy. Conclusions: This study demonstrates that TOPK disruption may cause tumour-specific radiosensitisation in multiple different tumour types. PMID:28677687

TOPK modulates tumour-specific radiosensitivity and correlates with recurrence after prostate radiotherapy.

PubMed

Pirovano, Giacomo; Ashton, Thomas M; Herbert, Katharine J; Bryant, Richard J; Verrill, Clare L; Cerundolo, Lucia; Buffa, Francesca M; Prevo, Remko; Harrap, Iona; Ryan, Anderson J; Macaulay, Valentine; McKenna, William G; Higgins, Geoff S

2017-08-08

Tumour-specific radiosensitising treatments may enhance the efficacy of radiotherapy without exacerbating side effects. In this study we determined the radiation response following depletion or inhibition of TOPK, a mitogen-activated protein kinase kinase family Ser/Thr protein kinase that is upregulated in many cancers. Radiation response was studied in a wide range of cancer cell lines and normal cells using colony formation assays. The effect on cell cycle progression was assessed and the relationship between TOPK expression and therapeutic efficacy was studied in a cohort of 128 prostate cancer patients treated with radical radiotherapy. TOPK knockdown did not alter radiation response in normal tissues, but significantly enhanced radiosensitivity in cancer cells. This result was recapitulated in TOPK knockout cells and with the TOPK inhibitor, OTS964. TOPK depletion altered the G 1 /S transition and G 2 /M arrest in response to radiation. Furthermore, TOPK depletion increased chromosomal aberrations, multinucleation and apoptotic cell death after irradiation. These results suggest a possible role for TOPK in the radiation-induced DNA damage checkpoints. These findings have clinical relevance, as elevated TOPK protein expression was associated with poorer clinical outcomes in prostate cancer patients treated with radical radiotherapy. This study demonstrates that TOPK disruption may cause tumour-specific radiosensitisation in multiple different tumour types.

The role of the cytoskeleton in sensing changes in gravity by nonspecialized cells.

PubMed

Vorselen, Daan; Roos, Wouter H; MacKintosh, Fred C; Wuite, Gijs J L; van Loon, Jack J W A

2014-02-01

A large body of evidence indicates that single cells in vitro respond to changes in gravity, and that this response might play an important role for physiological changes at the organism level during spaceflight. Gravity can lead to changes in cell proliferation, differentiation, signaling, and gene expression. At first glance, gravitational forces seem too small to affect bodies with the size of a cell. Thus, the initial response to gravity is both puzzling and important for understanding physiological changes in space. This also offers a unique environment to study the mechanical response of cells. In the past 2 decades, important steps have been made in the field of mechanobiology, and we use these advances to reevaluate the response of single cells to changes in gravity. Recent studies have focused on the cytoskeleton as initial gravity sensor. Thus, we review the observed changes in the cytoskeleton in a microgravity environment, both during spaceflight and in ground-based simulation techniques. We also evaluate to what degree the current experimental evidence supports the cytoskeleton as primary gravity sensor. Finally, we consider how the cytoskeleton itself could be affected by changed gravity. To make the next step toward understanding the response of cells to altered gravity, the challenge will be to track changes quantitatively and on short timescales.

Quantitative analysis of circadian single cell oscillations in response to temperature

PubMed Central

Kramer, Achim; Herzel, Hanspeter

2018-01-01

Body temperature rhythms synchronize circadian oscillations in different tissues, depending on the degree of cellular coupling: the responsiveness to temperature is higher when single circadian oscillators are uncoupled. So far, the role of coupling in temperature responsiveness has only been studied in organotypic tissue slices of the central circadian pacemaker, because it has been assumed that peripheral target organs behave like uncoupled multicellular oscillators. Since recent studies indicate that some peripheral tissues may exhibit cellular coupling as well, we asked whether peripheral network dynamics also influence temperature responsiveness. Using a novel technique for long-term, high-resolution bioluminescence imaging of primary cultured cells, exposed to repeated temperature cycles, we were able to quantitatively measure period, phase, and amplitude of central (suprachiasmatic nuclei neuron dispersals) and peripheral (mouse ear fibroblasts) single cell oscillations in response to temperature. Employing temperature cycles of different lengths, and different cell densities, we found that some circadian characteristics appear cell-autonomous, e.g. period responses, while others seem to depend on the quality/degree of cellular communication, e.g. phase relationships, robustness of the oscillation, and amplitude. Overall, our findings indicate a strong dependence on the cell’s ability for intercellular communication, which is not only true for neuronal pacemakers, but, importantly, also for cells in peripheral tissues. Hence, they stress the importance of comparative studies that evaluate the degree of coupling in a given tissue, before it may be used effectively as a target for meaningful circadian manipulation. PMID:29293562

Circulatory shear flow alters the viability and proliferation of circulating colon cancer cells

NASA Astrophysics Data System (ADS)

Fan, Rong; Emery, Travis; Zhang, Yongguo; Xia, Yuxuan; Sun, Jun; Wan, Jiandi

2016-06-01

During cancer metastasis, circulating tumor cells constantly experience hemodynamic shear stress in the circulation. Cellular responses to shear stress including cell viability and proliferation thus play critical roles in cancer metastasis. Here, we developed a microfluidic approach to establish a circulatory microenvironment and studied circulating human colon cancer HCT116 cells in response to a variety of magnitude of shear stress and circulating time. Our results showed that cell viability decreased with the increase of circulating time, but increased with the magnitude of wall shear stress. Proliferation of cells survived from circulation could be maintained when physiologically relevant wall shear stresses were applied. High wall shear stress (60.5 dyne/cm2), however, led to decreased cell proliferation at long circulating time (1 h). We further showed that the expression levels of β-catenin and c-myc, proliferation regulators, were significantly enhanced by increasing wall shear stress. The presented study provides a new insight to the roles of circulatory shear stress in cellular responses of circulating tumor cells in a physiologically relevant model, and thus will be of interest for the study of cancer cell mechanosensing and cancer metastasis.

Methods for Studying Ciliary-Mediated Chemoresponse in Paramecium.

PubMed

Valentine, Megan Smith; Van Houten, Judith L

2016-01-01

Paramecium is a useful model organism for the study of ciliary-mediated chemical sensing and response. Here we describe ways to take advantage of Paramecium to study chemoresponse.Unicellular organisms like the ciliated protozoan Paramecium sense and respond to chemicals in their environment (Van Houten, Ann Rev Physiol 54:639-663, 1992; Van Houten, Trends Neurosci 17:62-71, 1994). A thousand or more cilia that cover Paramecium cells serve as antennae for chemical signals, similar to ciliary function in a large variety of metazoan cell types that have primary or motile cilia (Berbari et al., Curr Biol 19(13):R526-R535, 2009; Singla V, Reiter J, Science 313:629-633, 2006). The Paramecium cilia also produce the motor output of the detection of chemical cues by controlling swimming behavior. Therefore, in Paramecium the cilia serve multiple roles of detection and response.We present this chapter in three sections to describe the methods for (1) assaying populations of cells for their behavioral responses to chemicals (attraction and repulsion), (2) characterization of the chemoreceptors and associated channels of the cilia using proteomics and binding assays, and (3) electrophysiological analysis of individual cells' responses to chemicals. These methods are applied to wild type cells, mutants, transformed cells that express tagged proteins, and cells depleted of gene products by RNA Interference (RNAi).

Differential CD4+ versus CD8+ T-cell responses elicited by different poxvirus-based human immunodeficiency virus type 1 vaccine candidates provide comparable efficacies in primates.

PubMed

Mooij, Petra; Balla-Jhagjhoorsingh, Sunita S; Koopman, Gerrit; Beenhakker, Niels; van Haaften, Patricia; Baak, Ilona; Nieuwenhuis, Ivonne G; Kondova, Ivanela; Wagner, Ralf; Wolf, Hans; Gómez, Carmen E; Nájera, José L; Jiménez, Victoria; Esteban, Mariano; Heeney, Jonathan L

2008-03-01

Poxvirus vectors have proven to be highly effective for boosting immune responses in diverse vaccine settings. Recent reports reveal marked differences in the gene expression of human dendritic cells infected with two leading poxvirus-based human immunodeficiency virus (HIV) vaccine candidates, New York vaccinia virus (NYVAC) and modified vaccinia virus Ankara (MVA). To understand how complex genomic changes in these two vaccine vectors translate into antigen-specific systemic immune responses, we undertook a head-to-head vaccine immunogenicity and efficacy study in the pathogenic HIV type 1 (HIV-1) model of AIDS in Indian rhesus macaques. Differences in the immune responses in outbred animals were not distinguished by enzyme-linked immunospot assays, but differences were distinguished by multiparameter fluorescence-activated cell sorter analysis, revealing a difference between the number of animals with both CD4(+) and CD8(+) T-cell responses to vaccine inserts (MVA) and those that elicit a dominant CD4(+) T-cell response (NYVAC). Remarkably, vector-induced differences in CD4(+)/CD8(+) T-cell immune responses persisted for more than a year after challenge and even accompanied antigenic modulation throughout the control of chronic infection. Importantly, strong preexposure HIV-1/simian immunodeficiency virus-specific CD4(+) T-cell responses did not prove deleterious with respect to accelerated disease progression. In contrast, in this setting, animals with strong vaccine-induced polyfunctional CD4(+) T-cell responses showed efficacies similar to those with stronger CD8(+) T-cell responses.

Evidence that shock-induced immune suppression is mediated by adrenal hormones and peripheral beta-adrenergic receptors.

PubMed

Cunnick, J E; Lysle, D T; Kucinski, B J; Rabin, B S

1990-07-01

Our previous work has demonstrated that presentations of mild foot-shock to Lewis rats induces a suppression of splenic and peripheral blood lymphocyte responses to nonspecific T-cell mitogens. The present study demonstrated that adrenalectomy prevented the shock-induced suppression of the mitogenic response of peripheral blood T-cells but did not attenuate the suppression of splenic T-cells. Conversely, the beta-adrenergic receptor antagonists, propranolol and nadolol, attenuated the shock-induced suppression of splenic T-cells in a dose-dependent manner but did not attenuate suppression of the blood mitogen response. These data indicate that distinct mechanisms mediate the shock-induced suppression of T-cell responsiveness to mitogens in the spleen and the peripheral blood. The results indicate that the peripheral release of catecholamines is responsible for splenic immune suppression and that adrenal hormones, which do not interact with beta-adrenergic receptors, are responsible for shock-induced suppression of blood mitogenic responses.

Quantitative Analyses of Synergistic Responses between Cannabidiol and DNA-Damaging Agents on the Proliferation and Viability of Glioblastoma and Neural Progenitor Cells in Culture

PubMed Central

Deng, Liting; Ng, Lindsay; Ozawa, Tatsuya

2017-01-01

Evidence suggests that the nonpsychotropic cannabis-derived compound, cannabidiol (CBD), has antineoplastic activity in multiple types of cancers, including glioblastoma multiforme (GBM). DNA-damaging agents remain the main standard of care treatment available for patients diagnosed with GBM. Here we studied the antiproliferative and cell-killing activity of CBD alone and in combination with DNA-damaging agents (temozolomide, carmustine, or cisplatin) in several human GBM cell lines and in mouse primary GBM cells in cultures. This activity was also studied in mouse neural progenitor cells (NPCs) in culture to assess for potential central nervous system toxicity. We found that CBD induced a dose-dependent reduction of both proliferation and viability of all cells with similar potencies, suggesting no preferential activity for cancer cells. Hill plot analysis indicates an allosteric mechanism of action triggered by CBD in all cells. Cotreatment regimens combining CBD and DNA-damaging agents produced synergistic antiproliferating and cell-killing responses over a limited range of concentrations in all human GBM cell lines and mouse GBM cells as well as in mouse NPCs. Remarkably, antagonistic responses occurred at low concentrations in select human GBM cell lines and in mouse GBM cells. Our study suggests limited synergistic activity when combining CBD and DNA-damaging agents in treating GBM cells, along with little to no therapeutic window when considering NPCs. PMID:27821713

Radiation-induced bystander effect and adaptive response in mammalian cells

NASA Technical Reports Server (NTRS)

Zhou, H.; Randers-Pehrson, G.; Waldren, C. A.; Hei, T. K.

2004-01-01

Two conflicting phenomena, bystander effect and adaptive response, are important in determining the biological responses at low doses of radiation and have the potential to impact the shape of the dose-response relationship. Using the Columbia University charged-particle microbeam and the highly sensitive AL cell mutagenic assay, we show here that non-irradiated cells acquire mutagenesis through direct contact with cells whose nuclei have been traversed with a single alpha particle each. Pretreatment of cells with a low dose of X-rays four hours before alpha particle irradiation significantly decreased this bystander mutagenic response. Results from the present study address some of the fundamental issues regarding both the actual target and radiation dose effect and can contribute to our current understanding in radiation risk assessment. c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.

Memory T cell proliferative responses and IFN-γ productivity sustain long-lasting efficacy of a Cap-based PCV2 vaccine upon PCV2 natural infection and associated disease

PubMed Central

2014-01-01

Porcine circovirus type 2 (PCV2) vaccination represents an important measure to cope with PCV2 infection; however, data regarding the modulation of the immune cell compartment are still limited, especially under field conditions. This study is aimed at investigating the features of the cellular immune response in conventional piglets induced by vaccination using a capsid (Cap) protein-based PCV2 vaccine compared to unvaccinated animals when exposed to PCV2 natural infection. Immune reactivity was evaluated by quantifying peripheral cell subsets involved in the anti-viral response and characterizing the interferon-gamma (IFN-γ) secreting cell (SC) responsiveness both in vivo and upon in vitro whole PCV2 recall. The vaccination triggered an early and intense IFN-γ secreting cell response and induced the activation of peripheral lymphocytes. The early increase of IFN-γ SC frequencies resulted in a remarkable and transient tendency to increased IFN-γ productivity in vaccinated pigs. In vaccinated animals, soon before the onset of infection occurred 15-16 weeks post-vaccination, the recalled PCV2-specific immune response was characterized by moderate PCV2-specific IFN-γ secreting cell frequencies and augmented productivity together with reactive CD4+CD8+ memory T cells. Conversely, upon infection, unvaccinated animals showed very high frequencies of IFN-γ secreting cells and a tendency to lower productivity, which paralleled with effector CD4–CD8+ cytotoxic cell responsiveness. The study shows that PCV2 vaccination induces a long-lasting immunity sustained by memory T cells and IFN-γ secreting cells that potentially played a role in preventing the onset of infection; the extent and duration of this reactivity can be an important feature for evaluating the protective immunity induced by vaccination. PMID:24735253

B-cell activation with CD40L or CpG measures the function of B-cell subsets and identifies specific defects in immunodeficient patients.

PubMed

Marasco, Emiliano; Farroni, Chiara; Cascioli, Simona; Marcellini, Valentina; Scarsella, Marco; Giorda, Ezio; Piano Mortari, Eva; Leonardi, Lucia; Scarselli, Alessia; Valentini, Diletta; Cancrini, Caterina; Duse, Marzia; Grimsholm, Ola; Carsetti, Rita

2017-01-01

Around 65% of primary immunodeficiencies are antibody deficiencies. Functional tests are useful tools to study B-cell functions in vitro. However, no accepted guidelines for performing and evaluating functional tests have been issued yet. Here, we report our experience on the study of B-cell functions in infancy and throughout childhood. We show that T-independent stimulation with CpG measures proliferation and differentiation potential of memory B cells. Switched memory B cells respond better than IgM memory B cells. On the other hand, CD40L, a T-dependent stimulus, does not induce plasma cell differentiation, but causes proliferation of naïve and memory B cells. During childhood, the production of plasmablasts in response to CpG increases with age mirroring the development of memory B cells. The response to CD40L does not change with age. In patients with selective IgA deficiency (SIgAD), we observed that switched memory B cells are reduced due to the absence of IgA memory B cells. In agreement, IgA plasma cells are not generated in response to CpG. Unexpectedly, B cells from SIgAD patients show a reduced proliferative response to CD40L. Our results demonstrate that functional tests are an important tool to assess the functions of the humoral immune system. © 2016 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Generation of protective T cell-independent antiviral antibody responses in SCID mice reconstituted with follicular or marginal zone B cells.

PubMed

Guay, Heath M; Mishra, Rabinarayan; Garcea, Robert L; Welsh, Raymond M; Szomolanyi-Tsuda, Eva

2009-07-01

B cells generated in the bone marrow of adult mice enter the periphery as transitional B cells and subsequently differentiate into one of two phenotypically and functionally distinct subsets, marginal zone (MZ) or follicular (Fo) B cells. Recent reports indicate, however, that in response to environmental cues, such as lymphopenia, mature Fo B cells can change to display phenotypic markers characteristic of MZ B cells. Previously, we found that splenic B cells transferred to SCID mice responded to polyoma virus (PyV) infection with T cell-independent (TI) IgM and IgG secretion, reducing the viral load and protecting mice from the lethal effect of the infection. The contribution of MZ and Fo B cell subsets to this antiviral TI-2 response, however, has not been addressed. In this study, we show that both sort-purified MZ and Fo B cells generate protective TI Ab responses to PyV infection when transferred into SCID mice. Moreover, the transferred Fo B cells in the spleens of the PyV-infected SCID mice change phenotype, with many of them displaying MZ B cell characteristics. These findings demonstrate the plasticity of the B cell subsets in virus-infected hosts and show for the first time that B cells derived exclusively from Fo B cells can effectively function in antiviral TI-2 responses.

T Lymphocytes Do Not Directly Mediate the Protective Effect of Estrogen on Experimental Autoimmune Encephalomyelitis

PubMed Central

Polanczyk, Magdalena J.; Jones, Richard E.; Subramanian, Sandhya; Afentoulis, Michael; Rich, Cathleen; Zakroczymski, Melissa; Cooke, Paul; Vandenbark, Arthur A.; Offner, Halina

2004-01-01

Gender influences mediated by 17β-estradiol (E2) have been associated with susceptibility to and severity of autoimmune diseases such as diabetes, arthritis, and multiple sclerosis. In this regard, we have shown that estrogen receptor-α (Esr1) is crucial for the protective effect of 17β-estradiol (E2) in murine experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis. The expression of estrogen receptors among various immune cells (eg, T and B lymphocytes, antigen-presenting cells) suggests that the therapeutic effect of E2 is likely mediated directly through specific receptor binding. However, the target immune cell populations responsive to E2 treatment have not been identified. In the current study, we induced EAE in T-cell-deficient, severe combined immunodeficient mice or in immunocompetent mice with encephalitogenic T cells from wild-type Esr1+/+ or Esr1 knockout (Esr1−/−) donors and compared the protective E2 responses. The results showed that E2-responsive, Esr1+/+ disease-inducing encephalitogenic T cells were neither necessary nor sufficient for E2-mediated protection from EAE. Instead, the therapeutic response appeared to be mediated through direct effects on nonlymphocytic, E2-responsive cells and down-regulation of the inflammatory response in the central nervous system. These results provide the first demonstration that the protective effect of E2 on EAE is not mediated directly through E2-responsive T cells and raise the alternative possibility that nonlymphocytic cells such as macrophages, dendritic cells, or other nonlymphocytic cells are primarily responsive to E2 treatment in EAE. PMID:15579449

Mucorales spores induce a proinflammatory cytokine response in human mononuclear phagocytes and harbor no rodlet hydrophobins.

PubMed

Wurster, Sebastian; Thielen, Vanessa; Weis, Philipp; Walther, Paul; Elias, Johannes; Waaga-Gasser, Ana Maria; Dragan, Mariola; Dandekar, Thomas; Einsele, Hermann; Löffler, Jürgen; Ullmann, Andrew J

2017-11-17

Mucormycoses are life-threatening infections in immunocompromised patients. This study characterizes the response of human mononuclear cells to different Mucorales and Ascomycota. PBMC, monocytes, and monocyte derived dendritic cells (moDCs) from healthy donors were stimulated with resting and germinated stages of Mucorales and Ascomycota. Cytokine response and expression of activation markers were studied. Both inactivated germ tubes and resting spores of Rhizopus arrhizus and other human pathogenic Mucorales species significantly stimulated mRNA synthesis and secretion of proinflammatory cytokines. Moreover, R. arrhizus spores induced the upregulation of co-stimulatory molecules on moDCs and a specific T-helper cell response. Removal of rodlet hydrophobins by hydrofluoric acid treatment of A. fumigatus conidia resulted in enhanced immunogenicity, whereas the cytokine response of PBMCs to dormant R. arrhizus spores was not influenced by hydrofluoric acid. Scanning electron micrographs of Mucorales spores did not exhibit any morphological correlates of rodlet hydrophobins. Taken together, this study revealed striking differences in the response of human mononuclear cells to resting stages of Ascomycota and Mucorales, which may be explained by absence of an immunoprotective hydrophobin layer in Mucorales spores.

Contribution of engineered nanomaterials physicochemical properties to mast cell degranulation

NASA Astrophysics Data System (ADS)

Johnson, Monica M.; Mendoza, Ryan; Raghavendra, Achyut J.; Podila, Ramakrishna; Brown, Jared M.

2017-03-01

The rapid development of engineered nanomaterials (ENMs) has grown dramatically in the last decade, with increased use in consumer products, industrial materials, and nanomedicines. However, due to increased manufacturing, there is concern that human and environmental exposures may lead to adverse immune outcomes. Mast cells, central to the innate immune response, are one of the earliest sensors of environmental insult and have been shown to play a role in ENM-mediated immune responses. Our laboratory previously determined that mast cells are activated via a non-FcɛRI mediated response following silver nanoparticle (Ag NP) exposure, which was dependent upon key physicochemical properties. Using bone marrow-derived mast cells (BMMCs), we tested the hypothesis that ENM physicochemical properties influence mast cell degranulation. Exposure to 13 physicochemically distinct ENMs caused a range of mast degranulation responses, with smaller sized Ag NPs (5 nm and 20 nm) causing the most dramatic response. Mast cell responses were dependent on ENMs physicochemical properties such as size, apparent surface area, and zeta potential. Surprisingly, minimal ENM cellular association by mast cells was not correlated with mast cell degranulation. This study suggests that a subset of ENMs may elicit an allergic response and contribute to the exacerbation of allergic diseases.

Metabolomic Responses of Guard Cells and Mesophyll Cells to Bicarbonate

PubMed Central

Misra, Biswapriya B.; de Armas, Evaldo; Tong, Zhaohui; Chen, Sixue

2015-01-01

Anthropogenic CO2 presently at 400 ppm is expected to reach 550 ppm in 2050, an increment expected to affect plant growth and productivity. Paired stomatal guard cells (GCs) are the gate-way for water, CO2, and pathogen, while mesophyll cells (MCs) represent the bulk cell-type of green leaves mainly for photosynthesis. We used the two different cell types, i.e., GCs and MCs from canola (Brassica napus) to profile metabolomic changes upon increased CO2 through supplementation with bicarbonate (HCO3 -). Two metabolomics platforms enabled quantification of 268 metabolites in a time-course study to reveal short-term responses. The HCO3 - responsive metabolomes of the cell types differed in their responsiveness. The MCs demonstrated increased amino acids, phenylpropanoids, redox metabolites, auxins and cytokinins, all of which were decreased in GCs in response to HCO3 -. In addition, the GCs showed differential increases of primary C-metabolites, N-metabolites (e.g., purines and amino acids), and defense-responsive pathways (e.g., alkaloids, phenolics, and flavonoids) as compared to the MCs, indicating differential C/N homeostasis in the cell-types. The metabolomics results provide insights into plant responses and crop productivity under future climatic changes where elevated CO2 conditions are to take center-stage. PMID:26641455

Blocking Glycolytic Metabolism Increases Memory T Cells and Antitumor Function | Center for Cancer Research

Cancer.gov

CD8+ T cells are a major component of the cellular immune response, which is necessary to control a variety of bacterial and viral infections. CD8+ T cells also play a major role in the cell-mediated antitumor immune response. After encountering antigen, naïve CD8+ T cells undergo an extensive period of proliferation and expansion, and differentiate into effector cells and distinct memory T cell subsets. Preclinical studies using adoptive transfer of purified CD8+ T cells have shown that the ability of T cells to proliferate and survive for a long time after transfer is associated with effective antitumor and antiviral responses. Understanding how the formation of long-lived memory T cell subsets is controlled may enable development of more potent immunotherapies against cancer and infectious diseases.

Yersinia versus host immunity: how a pathogen evades or triggers a protective response.

PubMed

Chung, Lawton K; Bliska, James B

2016-02-01

The human pathogenic Yersinia species cause diseases that represent a significant source of morbidity and mortality. Despite this, specific mechanisms underlying Yersinia pathogenesis and protective host responses remain poorly understood. Recent studies have shown that Yersinia disrupt cell death pathways, perturb inflammatory processes and exploit immune cells to promote disease. The ensuing host responses following Yersinia infection include coordination of innate and adaptive immune responses in an attempt to control bacterial replication. Here, we highlight current advances in our understanding of the interactions between the pathogenic yersiniae and host cells, as well as the protective host responses mobilized to counteract these pathogens. Together, these studies enhance our understanding of Yersinia pathogenesis and highlight the ongoing battle between host and microbe. Copyright © 2015 Elsevier Ltd. All rights reserved.

Individual human cell responses to low doses of chemicals studied by synchrotron infrared spectromicroscopy

NASA Astrophysics Data System (ADS)

Holman, Hoi-Ying N.; Goth-Goldstein, Regine; Blakely, Elanor A.; Bjornstad, Kathy; Martin, Michael C.; McKinney, Wayne R.

2000-05-01

Vibrational spectroscopy, when combined with synchrotron radiation-based (SR) microscopy, is a powerful new analytical tool with high spatial resolution for detecting biochemical changes in the individual living cells. In contrast to other microscopy methods that require fixing, drying, staining or labeling, SR-FTIR microscopy probes intact living cells providing a composite view of all of the molecular response and the ability to monitor the response over time in the same cell. Observed spectral changes include all types of lesions induced in that cell as well as cellular responses to external and internal stresses. These spectral changes combined with other analytical tools may provide a fundamental understanding of the key molecular mechanisms induced in response to stresses created by low- doses of chemicals. In this study we used the high spatial - resolution SR-FTIR vibrational spectromicroscopy as a sensitive analytical tool to detect chemical- and radiation- induced changes in individual human cells. Our preliminary spectral measurements indicate that this technique is sensitive enough to detect changes in nucleic acids and proteins of cells treated with environmentally relevant concentrations of dioxin. This technique has the potential to distinguish changes from exogenous or endogenous oxidative processes. Future development of this technique will allow rapid monitoring of cellular processes such as drug metabolism, early detection of disease, bio- compatibility of implant materials, cellular repair mechanisms, self assembly of cellular apparatus, cell differentiation and fetal development.

Enhanced Vaccine-Induced CD8+ T Cell Responses to Malaria Antigen ME-TRAP by Fusion to MHC Class II Invariant Chain

PubMed Central

Spencer, Alexandra J.; Cottingham, Matthew G.; Jenks, Jennifer A.; Longley, Rhea J.; Capone, Stefania; Colloca, Stefano; Folgori, Antonella; Cortese, Riccardo; Nicosia, Alfredo; Bregu, Migena; Hill, Adrian V. S.

2014-01-01

The orthodox role of the invariant chain (CD74; Ii) is in antigen presentation to CD4+ T cells, but enhanced CD8+ T cells responses have been reported after vaccination with vectored viral vaccines encoding a fusion of Ii to the antigen of interest. In this study we assessed whether fusion of the malarial antigen, ME-TRAP, to Ii could increase the vaccine-induced CD8+ T cell response. Following single or heterologous prime-boost vaccination of mice with a recombinant chimpanzee adenovirus vector, ChAd63, or recombinant modified vaccinia virus Ankara (MVA), higher frequencies of antigen-specific CD4+ and CD8+ T cells were observed, with the largest increases observed following a ChAd63-MVA heterologous prime-boost regimen. Studies in non-human primates confirmed the ability of Ii-fusion to augment the T cell response, where a 4-fold increase was maintained up to 11 weeks after the MVA boost. Of the numerous different approaches explored to increase vectored vaccine induced immunogenicity over the years, fusion to the invariant chain showed a consistent enhancement in CD8+ T cell responses across different animal species and may therefore find application in the development of vaccines against human malaria and other diseases where high levels of cell-mediated immunity are required. PMID:24945248

In vitro suppression of spontaneous erythrocyte autoimmune responses with lymphocytes activated with concanavalin A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramshaw, I.A.; Woodsworth, M.; Eidinger, D.

1979-01-01

When normal mouse spleen cells are cultured in vitro, large numbers of cells develop that produce antibody toward antigens found on bromelain-treated mouse erythrocytes (BrMRBC). The in vitro culture also generates T cells that mediate DTH toward these antigens. We have suggested that under in vivo conditions, suppressor T cells maintain these immune responses at a low level but that this suppression wanes when the cells are cultured in vitro. The present study examines the effect of concanavalin A (Con A) on the in vitro development of humoral and cell-mediated immunity to BrMRBC. Mitogenic concentrations of Con A prevented themore » development of both the PFC and T/sub DTH/ responses toward BrMRBC. The Con A-induced suppression was due to the induction of suppressor T cells; thus the addition of Con A-activated cells to fresh spleen cell cultures prevented the development of both the PFC and T/sub DTH/ response against BrMRBC.« less

Can dendritic cells see light?

NASA Astrophysics Data System (ADS)

Chen, Aaron C.-H.; Huang, Ying-Ying; Sharma, Sulbha K.; Hamblin, Michael R.

2010-02-01

There are many reports showing that low-level light/laser therapy (LLLT) can enhance wound healing, upregulate cell proliferation and has anti-apoptotic effects by activating intracellular protective genes. In the field of immune response study, it is not known with any certainty whether light/laser is proinflammatory or anti-inflammatory. Increasingly in recent times dendritic cells have been found to play an important role in inflammation and the immunological response. In this study, we try to look at the impact of low level near infrared light (810-nm) on murine bone-marrow derived dendritic cells. Changes in surface markers, including MHC II, CD80 and CD11c and the secretion of interleukins induced by light may provide additional evidence to reveal the mystery of how light affects the maturation of dendritic cells as well how these light-induced mature dendritic cells would affect the activation of adaptive immune response.

Suppression of Antigen-Specific T Cell Responses by the Kaposi's Sarcoma-Associated Herpesvirus Viral OX2 Protein and Its Cellular Orthologue, CD200

PubMed Central

Misstear, Karen; Chanas, Simon A.; Rezaee, S. A. Rahim; Colman, Rachel; Quinn, Laura L.; Long, Heather M.; Goodyear, Oliver; Lord, Janet M.; Hislop, Andrew D.

2012-01-01

Regulating appropriate activation of the immune response in the healthy host despite continual immune surveillance dictates that immune responses must be either self-limiting and therefore negatively regulated following their activation or prevented from developing inappropriately. In the case of antigen-specific T cells, their response is attenuated by several mechanisms, including ligation of CTLA-4 and PD-1. Through the study of the viral OX2 (vOX2) immunoregulator encoded by Kaposi's sarcoma-associated herpesvirus (KSHV), we have identified a T cell-attenuating role both for this protein and for CD200, a cellular orthologue of the viral vOX2 protein. In vitro, antigen-presenting cells (APC) expressing either native vOX2 or CD200 suppressed two functions of cognate antigen-specific T cell clones: gamma interferon (IFN-γ) production and mobilization of CD107a, a cytolytic granule component and measure of target cell killing ability. Mechanistically, vOX2 and CD200 expression on APC suppressed the phosphorylation of ERK1/2 mitogen-activated protein kinase in responding T cells. These data provide the first evidence for a role of both KSHV vOX2 and cellular CD200 in the negative regulation of antigen-specific T cell responses. They suggest that KSHV has evolved to harness the host CD200-based mechanism of attenuation of T cell responses to facilitate virus persistence and dissemination within the infected individual. Moreover, our studies define a new paradigm in immune modulation by viruses: the provision of a negative costimulatory signal to T cells by a virus-encoded orthologue of CD200. PMID:22491458

Circulating and tumor-infiltrating Tim-3 in patients with colorectal cancer

PubMed Central

Gao, Quanli; Yuan, Peng; Zhao, Peng; Yuan, Huijuan; Fan, Huijie; Li, Tiepeng; Qin, Peng; Han, Lu; Fang, Weijia; Suo, Zhenhe

2015-01-01

T-cell exhaustion represents a progressive loss of T-cell function. The inhibitory receptor PD-1 is known to negatively regulate CD8+ T cell responses directed against tumor antigen, but the blockades of PD-1 pathway didn't show the objective responses in patients with colorectal cancer (CRC). Thus, further exploring the molecular mechanism responsible for inducing T-cell dysfunction in CRC patients may reveal effective strategies for immune therapy. This study aims to characterize co-inhibitory receptors on T cells in CRC patients to identify novel targets for immunotherapy. In this study, peripheral blood samples from 20 healthy controls and 54 consented CRC patients, and tumor and matched paraneoplastic tissues from 7 patients with advanced CRC, subjected to multicolor flow cytometric analysis of the expression of PD-1 and Tim-3 receptors on CD8+ T cells. It was found that CRC patients presented with significantly higher levels of circulating Tim-3+PD-1+CD8+ T cells compared to the healthy controls (medians of 3.12% and 1.99%, respectively, p = 0.0403). A similar increase of Tim-3+PD-1+CD8+ T cells was also observed in the tumor tissues compared to paraneoplastic tussues. Tim-3+PD-1+CD8+ T cells in tumor tissues produced even less cytokine than that in paraneoplastic tissues. Functional ex vivo experiments showed that Tim-3+PD-1+CD8+ T cells produced significantly less IFN-γ than Tim-3−PD-1−CD8+ T cells, followed by Tim-3+PD-1−CD8+ T cells, and Tim-3−PD-1+CD8+ T cells, indicating a stronger inhibition of IFN-γ production of Tim-3+CD8+ T cells. It is also found in this study that Tim-3+PD-1+CD8+ T cell increase in circulation was correlated with clinical cancer stage but not histologic grade and serum concentrations of cancer biomarker CEA. Our results indicate that upregulation of the inhibitory receptor Tim-3 may restrict T cell responses in CRC patients, and therefore blockage of Tim-3 and thus restoring T cell responses may be a potential therapeutic approach for CRC patients. PMID:26008981

Fas/FasL interaction mediates imbalanced cytokine/cytotoxicity responses of iNKT cells against Jurkat cells.

PubMed

Dou, Rui; Hong, Zhenya; Tan, Xiaosheng; Hu, Fenfen; Ding, Yajie; Wang, Wei; Liang, Zhihui; Zhong, Rongrong; Wu, Xiongwen; Weng, Xiufang

2018-07-01

The rapid antitumor cytokine production and direct cytotoxicity confer invariant NKT (iNKT) cells ideal candidates for cancer therapy. However, the therapeutic potential of iNKT cells in T-cell malignant diseases remains elusive, as antigen presentation by T cells (T-T presentation) has been suggested to induce hyporesponsiveness of iNKT cells. In this study, we found discrepancies in iNKT cell responses against two T cell-origin cell lines (Jurkat and Molt-4). Human iNKT cells exhibited more intensive cytotoxicity and less efficient cytokine production in response to Fas-bearing Jurkat cells than those to the Fas-negative tumor cells (Molt-4 and myeloid-derived K562). The imbalanced cytokine/cytotoxicity responses of iNKT cells against Jurkat cells were CD1d-dependent and relied mostly on Fas/FasL interaction. The impairment in cytokine production could be overcome by Fas/FasL blocking antibodies and exogenous IL-2. Elevated CD1d levels as well as CD1d and Fas co-localization were found in T-cell lymphomas. However, defects in frequency and function of circulating iNKT cells were observed in the patients, which could be partly rescued by exogenous IL-2. Collectively, the Fas/FasL-dependent aberrant iNKT cell responses and the reversibility of the defects suggest the distinct iNKT cell manipulation in CD1d- and Fas-bearing T cell malignancies. Copyright © 2018. Published by Elsevier Ltd.

Dynamic quantitative photothermal monitoring of cell death of individual human red blood cells upon glucose depletion

NASA Astrophysics Data System (ADS)

Vasudevan, Srivathsan; Chen, George Chung Kit; Andika, Marta; Agarwal, Shuchi; Chen, Peng; Olivo, Malini

2010-09-01

Red blood cells (RBCs) have been found to undergo ``programmed cell death,'' or eryptosis, and understanding this process can provide more information about apoptosis of nucleated cells. Photothermal (PT) response, a label-free photothermal noninvasive technique, is proposed as a tool to monitor the cell death process of living human RBCs upon glucose depletion. Since the physiological status of the dying cells is highly sensitive to photothermal parameters (e.g., thermal diffusivity, absorption, etc.), we applied linear PT response to continuously monitor the death mechanism of RBC when depleted of glucose. The kinetics of the assay where the cell's PT response transforms from linear to nonlinear regime is reported. In addition, quantitative monitoring was performed by extracting the relevant photothermal parameters from the PT response. Twofold increases in thermal diffusivity and size reduction were found in the linear PT response during cell death. Our results reveal that photothermal parameters change earlier than phosphatidylserine externalization (used for fluorescent studies), allowing us to detect the initial stage of eryptosis in a quantitative manner. Hence, the proposed tool, in addition to detection of eryptosis earlier than fluorescence, could also reveal physiological status of the cells through quantitative photothermal parameter extraction.

Ex Vivo Induction of Multiple Myeloma-specific Immune Responses by Monocyte-derived Dendritic Cells Following Stimulation by Whole-tumor Antigen of Autologous Myeloma Cells.

PubMed

Vasileiou, Spyridoula; Baltadakis, Ioannis; Delimpasi, Sosanna; Karatza, Maria-Helena; Liapis, Konstantinos; Garofalaki, Maria; Tziotziou, Eirini; Poulopoulou, Zoe; Karakasis, Dimitri; Harhalakis, Nicholas

2017-09-01

The introduction of novel agents has significantly expanded treatment options for multiple myeloma (MM), albeit long-term disease control cannot be achieved in the majority of patients. Vaccination with MM antigen-loaded dendritic cells (DCs) represents an alternative strategy that is currently being explored. The aim of this study was to assess the immunogenic potential of ex vivo-generated monocyte-derived DCs (moDCs), following stimulation with the whole-antigen array of autologous myeloma cells (AMC). MoDCs were loaded with antigens of myeloma cells by 2 different methods: phagocytosis of apoptotic bodies from γ-irradiated AMC, or transfection with AMC total RNA by square-wave electroporation. Twenty patients with MM were enrolled in the study. Following stimulation and maturation, moDCs were tested for their capacity to induce T-helper 1 and cytotoxic T lymphocyte responses in vitro. Both strategies were effective in the induction of myeloma-specific cytotoxic T lymphocyte and T-helper 1 cells, as demonstrated by cytotoxicity and ELISpot assays. On the whole, T-cell responses were observed in 18 cases by either method of DC pulsing. We conclude that both whole-tumor antigen approaches are efficient in priming autologous antimyeloma T-cell responses and warrant further study aiming at the development of individualized DC vaccines for MM patients.

Responses of indigenous microorganisms to soil incubation as viewed by transmission electron microscopy of cell thin sections.

NASA Technical Reports Server (NTRS)

Bae, h. C.; Casida, L. E., Jr.

1973-01-01

Indigenous soil microorganisms were cultivated in their soil habitat with 50% moisture capacity at 30 C for two weeks. Changes in microorganism cells were studied by electron microscopy during incubation, with particular attention to the dormant cell growth and to the ability of cystlike cells to germinate and reencyst. The responses of various cell species to incubation conditions are described and illustrated by photomicrographs.

Variability in Cell Response of Cronobacter sakazakii after Mild-Heat Treatments and Its Impact on Food Safety

PubMed Central

Parra-Flores, Julio; Juneja, Vijay; Garcia de Fernando, Gonzalo; Aguirre, Juan

2016-01-01

Cronobacter spp. have been responsible for severe infections in infants associated with consumption of powdered infant formula and follow-up formulae. Despite several risk assessments described in published studies, few approaches have considered the tremendous variability in cell response that small micropopulations or single cells can have in infant formula during storage, preparation or post process/preparation before the feeding of infants. Stochastic approaches can better describe microbial single cell response than deterministic models as we prove in this study. A large variability of lag phase was observed in single cell and micropopulations of ≤50 cells. This variability increased as the heat shock increased and growth temperature decreased. Obviously, variability of growth of individual Cronobacter sakazakii cell is affected by inoculum size, growth temperature and the probability of cells able to grow at the conditions imposed by the experimental conditions should be taken into account, especially when errors in bottle-preparation practices, such as improper holding temperatures, or manipulation, may lead to growth of the pathogen to a critical cell level. The mean probability of illness from initial inoculum size of 1 cell was below 0.2 in all the cases and for inoculum size of 50 cells the mean probability of illness, in most of the cases, was above 0.7. PMID:27148223

Characterisation of the immune response to type I collagen in scleroderma

PubMed Central

Warrington, Kenneth J; Nair, Usha; Carbone, Laura D; Kang, Andrew H; Postlethwaite, Arnold E

2006-01-01

This study was conducted to examine the frequency, phenotype, and functional profile of T lymphocytes that proliferate in response to type I collagen (CI) in patients with scleroderma (SSc). Peripheral blood mononuclear cells (PBMCs) from SSc patients, healthy controls, and rheumatoid arthritis disease controls were labeled with carboxy-fluorescein diacetate, succinimidyl ester (CFSE), cultured with or without antigen (bovine CI) for 14 days, and analysed by flow cytometry. Surface markers of proliferating cells were identified by multi-color flow cytometry. T-cell lines were derived after sorting for proliferating T cells (CFSElow). Cytokine expression in CI-responsive T cells was detected by intracellular staining/flow cytometry and by multiplex cytokine bead assay (Bio-Plex). A T-cell proliferative response to CI was detected in 8 of 25 (32%) SSc patients, but was infrequent in healthy or disease controls (3.6%; p = 0.009). The proliferating T cells expressed a CD4+, activated (CD25+), memory (CD45RO+) phenotype. Proliferation to CI did not correlate with disease duration or extent of skin involvement. T-cell lines were generated using in vitro CI stimulation to study the functional profile of these cells. Following activation of CI-reactive T cells, we detected intracellular interferon (IFN)-γ but not interleukin (IL)-4 by flow cytometry. Supernatants from the T-cell lines generated in vitro contained IL-2, IFN-γ, GM-CSF (granulocyte macrophage-colony-stimulating factor), and tumour necrosis factor-α, but little or no IL-4 and IL-10, suggesting that CI-responsive T cells express a predominantly Th1 cytokine pattern. In conclusion, circulating memory CD4 T cells that proliferate to CI are present in a subset of patients with SSc, but are infrequent in healthy or disease controls. PMID:16879746

gp49B-mediated negative regulation of antibody production by memory and marginal zone B cells.

PubMed

Fukao, Saori; Haniuda, Kei; Nojima, Takuya; Takai, Toshiyuki; Kitamura, Daisuke

2014-07-15

The rapid Ab responses observed after primary and secondary immunizations are mainly derived from marginal zone (MZ) and memory B cells, respectively, but it is largely unknown how these responses are negatively regulated. Several inhibitory receptors have been identified and their roles have been studied, but mainly on follicular B cells and much less so on MZ B, and never on memory B cells. gp49B is an Ig superfamily member that contains two ITIMs in its cytoplasmic tail, and it has been shown to negatively regulate mast cell, macrophage, and NK cell responses. In this study, we demonstrate that gp49B is preferentially expressed on memory and MZ B cells. We show that gp49B(-/-) mice produce more IgM after a primary immunization and more IgM and IgG1 after a secondary immunization than gp49B(+/+) mice in T cell-dependent immune responses. Memory and MZ B cells from gp49B(-/-) mice also produce more Abs upon in vitro stimulation with CD40 than those from gp49B(+/+) mice. The in vitro IgM production by MZ B cells from gp49B(+/+), but not gp49B(-/-), mice is suppressed by interaction with a putative gp49B ligand, the integrin αvβ3 heterodimer. In addition, gp49B(-/-) mice exhibited exaggerated IgE production in the memory recall response. These results suggest that plasma cell development from memory and MZ B cells, as well as subsequent Ab production, are suppressed via gp49B. In memory B cells, this suppression also prevents excessive IgE production, thus curtailing allergic diseases. Copyright © 2014 by The American Association of Immunologists, Inc.

Antibody production studied by means of the LHG assay*

PubMed Central

Wortis, H. H.; Taylor, R. B.; Dresser, D. W.

1966-01-01

By means of a modified plaque assay the numbers of cells producing 19S antibody (direct PFC) and 7S antibody (developed PFC) have been studied separately. Significant and somewhat surprising differences in the responses of the two kinds of antibody-producing cell have been noted. It is clear that the route of injection of the antigen is of importance in determining the response in the spleen. The response of both types of PFC was found to be biphasic. The total increase in spleen cell number after immunization was not readily accounted for by the increase in specific PFC. PMID:5954775

Nanoscale definition of substrate materials to direct human adult stem cells towards tissue specific populations.

PubMed

Curran, Judith M; Chen, Rui; Stokes, Robert; Irvine, Eleanor; Graham, Duncan; Gubbins, Earl; Delaney, Deany; Amro, Nabil; Sanedrin, Raymond; Jamil, Haris; Hunt, John A

2010-03-01

The development of homogenously nano-patterned chemically modified surfaces that can be used to initiate a cellular response, particularly stem cell differentiation, in a highly controlled manner without the need for exogenous biological factors has never been reported, due to that fact that precisely defined and reproducible systems have not been available that can be used to study cell/material interactions and unlock the potential of a material driven cell response. Until now material driven stem cell (furthermore any cell) responses have been variable due to the limitations in definition and reproducibility of the underlying substrate and the lack of true homogeneity of modifications that can dictate a cellular response at a sub-micron level that can effectively control initial cell interactions of all cells that contact the surface. Here we report the successful design and use of homogenously molecularly nanopatterned surfaces to control initial stem cell adhesion and hence function. The highly specified nano-patterned arrays were compared directly to silane modified bulk coated substrates that have previously been proven to initiate mesenchymal stem cell (MSC) differentiation in a heterogenous manner, the aim of this study was to prove the efficiency of these previously observed cell responses could be enhanced by the incorporation of nano-patterns. Nano-patterned surfaces were prepared by Dip Pen Nanolithography (DPN) to produce arrays of 70 nm sized dots separated by defined spacings of 140, 280 and 1000 nm with terminal functionalities of carboxyl, amino, methyl and hydroxyl and used to control cell growth. These nanopatterned surfaces exhibited unprecedented control of initial cell interactions and will change the capabilities for stem cell definition in vitro and then cell based medical therapies. In addition to highlighting the ability of the materials to control stem cell functionality on an unprecedented scale this research also introduces the successful scale-up of DPN and the novel chemistries and systems to facilitate the production of homogeneously patterned substrates (5 mm2) that are applicable for use in in vitro cell conditions over prolonged periods for complete control of material driven cell responses.

Rabbit spleen B lymphocytes as helper cells in lymphocyte activation by concanavalin A and phytohaemagglutinin.

PubMed

Cavaillon, J M; Udupa, T N; Chou, C T; Cinader, B; Dubiski, S

1982-01-01

Using rosetting methods, we have purified rabbit B cells and studied their interactions with T cells purified by passage over an anti-immunoglobulin-coated Degalan beads column. B cells enhance the response of T cells to concanavalin A (Con A) and phytohaemagglutinin. In regulation of the response to Con A, an adherent cell is a third participating cell. B-cell preparation contain a minority of cells that can respond to T mitogens with the help of non-proliferating T cells, but the proportion of these responding cells is small, and the involvement of the T-cell impurity cannot be excluded.

Molecular pathways mediating differential responses to lipopolysaccharide between human and baboon arterial endothelial cells.

PubMed

Shi, Qiang; Cox, Laura A; Glenn, Jeremy; Tejero, Maria E; Hondara, Vida; Vandeberg, John L; Wang, Xing Li

2010-02-01

1. Vascular inflammation plays a critical role in atherogenesis. Previously, we showed that baboon arterial endothelial cells (BAEC) were hyporesponsive to lipopolysaccharide (LPS) compared with human arterial endothelial cells (HAEC). 2. In the present study, we investigated mechanisms underlying differential responses between HAEC and BAEC to tumour necrosis factor (TNF)-alpha and LPS. 3. Both HAEC and BAEC responded similarly to TNF-alpha. However, BAEC showed retarded responses to LPS in expression of E-selectin, intercellular adhesion molecule-1, monocyte chemotactic protein-1 and interleukin-8 (P < 0.05). These changes were confirmed at the mRNA level. Tumour necrosis factor-alpha activated nuclear factor-kappaB members such as p50, p52, p65, c-rel and RelB in both HAEC and BAEC. In contrast, LPS activated p50 and p65 only in HAEC. Using microarray assays, we found that TNF receptor-associated factor 2 (TRAF-2), TNF receptor superfamily, member 1A-associated via death domain (TRADD) and nuclear factors such as nuclear factor of kappa in B-cells inhibitor, alpha (NFKBIA) and nuclear factor of kappa in B-cells inhibitor, beta (NFKBIB) were upregulated by LPS only in HAEC. Although the baseline expression of Toll-like receptor (TLR) 4 was low in both HAEC and BAEC, TNF-alpha activated TLR4 expression in both cell types. Although LPS increased TLR4 expression only in HAEC, human and baboon peripheral blood mononuclear cells exhibited similar TLR4 expression and response to LPS. Transfecting BAEC with TLR4/myeloid differentiation protein-2 overexpression vector conferred BAEC responsiveness to LPS. 4. The findings of the present study indicate that an altered TLR4 system may be responsible for the resistance of baboon endothelial cells to LPS. Given the importance of TLR4 in human immune responses and vascular diseases, the natural resistance of baboons to LPS/TLR4-initiated inflammation could make the baboon a valuable animal model in which to study how inflammation affects atherogenesis.

Ki-67 expression reveals strong, transient influenza specific CD4 T cell responses after adult vaccination.

PubMed

Li, Xi; Miao, Hongyu; Henn, Alicia; Topham, David J; Wu, Hulin; Zand, Martin S; Mosmann, Tim R

2012-06-29

Although previous studies have found minimal changes in CD4 T cell responses after vaccination of adults with trivalent inactivated influenza vaccine, daily sampling and monitoring of the proliferation marker Ki-67 have now been used to reveal that a substantial fraction of influenza-specific CD4 T cells respond to vaccination. At 4-6 days after vaccination, there is a sharp rise in the numbers of Ki-67-expressing PBMC that produce IFNγ, IL-2 and/or TNFα in vitro in response to influenza vaccine or peptide. Ki-67(+) cell numbers then decline rapidly, and 10 days after vaccination, both Ki-67(+) and overall influenza-specific cell numbers are similar to pre-vaccination levels. These results provide a tool for assessing the quality and quantity of CD4 T cell responses to different influenza vaccines, and raise the possibility that the anti-influenza T cell memory response may be qualitatively altered by vaccination, even if the overall memory cell numbers do not change significantly. Copyright © 2012. Published by Elsevier Ltd.

Bone marrow support of the heart in pressure overload is lost with aging.

PubMed

Sopko, Nikolai A; Turturice, Benjamin A; Becker, Mitchell E; Brown, Chase R; Dong, Feng; Popović, Zoran B; Penn, Marc S

2010-12-21

Exogenous stem cell delivery is under investigation to prevent and treat cardiac dysfunction. It is less studied as to the extent endogenous bone marrow derived stem cells contribute to cardiac homeostais in response to stress and the affects of aging on this stress response. To determine the role of bone marrow (BM) derived stem cells on cardiac homeostasis in response to pressure overload (PO) and how this response is altered by aging. Young (8 weeks) and old (>40 weeks) C57/b6 mice underwent homo- and heterochronic BM transplantation prior to transverse aortic constriction (TAC). We found that older BM is associated with decreased cardiac function following TAC. This decreased function is associated with decrease in BM cell engraftment, increased myocyte apoptosis, decreased myocyte hypertrophy, increased myocardial fibrosis and decreased cardiac function. Additionally, there is a decrease in activation of resident cells within the heart in response to PO in old mice. Interestingly, these effects are not due to alterations in vascular density or inflammation in response to PO or differences in ex vivo stem cell migration between young and old mice. BM derived stem cells are activated in response to cardiac PO, and the recruitment of BM derived cells are involved in cardiac myocyte hypertrophy and maintenance of function in response to PO which is lost with aging.

Vaccine-specific antibody secreting cells are a robust early marker of LAIV-induced B-cell response in ferrets.

PubMed

Cherukuri, Anu; Servat, Esteban; Woo, Jennifer

2012-01-05

Currently, a robust set of immune correlates for live attenuated influenza vaccine (LAIV) efficacy in humans has not been fully elucidated. The serum hemagglutination inhibition (HAI) assay has been historically used to measure humoral immune responses to injectable inactivated influenza vaccination. However, serum antibody titers do not reliably reflect the complete mechanism of action of LAIV, which is an intranasally delivered vaccine and is expected to induce local mucosal and cellular immune responses in addition to humoral immune responses. Therefore, we designed a study to evaluate potential immune correlates of LAIV vaccination in the ferret animal model of influenza infection. Ferrets were vaccinated with increasing doses of LAIV and four weeks later challenged with a homologous wild-type (wt) H1N1 strain. Humoral immune responses measured following LAIV vaccination included HAI, serum antibodies and antibody secreting cells (ASC); and the responses were found to correlate with the dose level of LAIV administered in this model. Protection from wt virus challenge was determined by measuring inhibition of wt viral replication in nasal washes and in lung tissue. Results demonstrated that LAIV doses ≥ 5.0 log(10) Plaque Forming Units (PFU) elicited vaccine-specific IgG and IgA ASC frequencies and induced complete protection in the lungs. Further, we developed a novel model utilizing seropositive older ferrets to demonstrate that in the background of previous wt influenza infection LAIV induces a robust vaccine-specific B-cell response even in the absence of serum antibody response, a result that suggests that effector B-cell responses generated by LAIV are not inhibited by prior viral exposure. Finally, we demonstrated that LAIV elicits strain-specific memory B-cell responses that are measurable in a background of wt influenza infections. Taken together, results from these studies identified the antigen-specific ASC frequency as a useful early biomarker of LAIV-induced B-cell immune response. Copyright © 2011 Elsevier Ltd. All rights reserved.

Comparative transduction mechanisms of hair cells in the bullfrog uticulus. 2: Sensitivity and response dynamics to hair bundle displacement

NASA Technical Reports Server (NTRS)

Baird, Richard A.

1994-01-01

The present study was motivated by an interest in seeing whether hair cell types in the bullfrog utriculus might differ in their voltage responses to hair bundle displacement. Particular interest was in assessing the contributions of two factors to the responses of utricular hair cells. First, interest in examining the effect of hair bundle morphology on the sensitivity of hair cells to natural stimulation was motivated by the observation that vestibular hair cells, unlike many auditory hair cells, are not free-standing but rather linked to an accessory cupular or otolithic membrane via the tip of their kinocilium. Interest also laid in examining the contribution, if any, of adaptation to the response properties of utricular hair cells. Hair cells in auditory and vibratory inner ear endorgans adapt to maintained displacements of their hair bundles, sharply limiting their low frequency sensitivity. This adaptation is mediated by a shift in the displacement-response curve (DRC) of the hair cell along the displacement axis. Observations suggest that the adaptation process occurs within the hair bundle and precedes mechanoelectric transduction. Recent observations of time-dependent changes in hair bundle stiffness are consistent with this conclusion. Adaptation would be expected to be most useful in inner ear endorgans in which hair cells are subject to large static displacements that could potentially saturate their instantaneous response and compromise their sensitivity to high frequency stimulation. The adaptation process also permits hair cells to maintain their sensory hair bundle in the most sensitive portion of their DRC. In vestibular otolith organs in which static sensitivity is desirable, any adaptation process in the hair cells may be undesirable. The rate and extent of the decline of the voltage responses was measured of utricular hair cells to step and sinusoidal hair bundle displacements. Then for similar resting potentials and response amplitudes, the voltage responses of individual hair cells were compared to both hair bundle displacement and intracellular current.

Clinical implications of basic science discoveries: janus resurrected--two faces of B cell and plasma cell biology.

PubMed

Woodle, E S; Rothstein, D M

2015-01-01

B cells play a complex role in the immune response. In addition to giving rise to plasma cells (PCs) and promoting T cell responses via antigen presentation, they perform immunoregulatory functions. This knowledge has created concerns regarding nonspecific B cell depletional therapy because of the potential to paradoxically augment immune responses. Recent studies now indicate that PCs have immune functions beyond immunoglobulin synthesis. Evidence for a new role for PCs as potent regulatory cells (via IL-10 and IL-35 production) is discussed including the implications for PC-targeted therapies currently being developed for clinical transplantation. © Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.

Controlled Breast Cancer Microarrays for the Deconvolution of Cellular Multilayering and Density Effects upon Drug Responses

PubMed Central

Håkanson, Maria; Kobel, Stefan; Lutolf, Matthias P.; Textor, Marcus; Cukierman, Edna; Charnley, Mirren

2012-01-01

Background Increasing evidence shows that the cancer microenvironment affects both tumorigenesis and the response of cancer to drug treatment. Therefore in vitro models that selectively reflect characteristics of the in vivo environment are greatly needed. Current methods allow us to screen the effect of extrinsic parameters such as matrix composition and to model the complex and three-dimensional (3D) cancer environment. However, 3D models that reflect characteristics of the in vivo environment are typically too complex and do not allow the separation of discrete extrinsic parameters. Methodology/Principal Findings In this study we used a poly(ethylene glycol) (PEG) hydrogel-based microwell array to model breast cancer cell behavior in multilayer cell clusters that allows a rigorous control of the environment. The innovative array fabrication enables different matrix proteins to be integrated into the bottom surface of microwells. Thereby, extrinsic parameters including dimensionality, type of matrix coating and the extent of cell-cell adhesion could be independently studied. Our results suggest that cell to matrix interactions and increased cell-cell adhesion, at high cell density, induce independent effects on the response to Taxol in multilayer breast cancer cell clusters. In addition, comparing the levels of apoptosis and proliferation revealed that drug resistance mediated by cell-cell adhesion can be related to altered cell cycle regulation. Conversely, the matrix-dependent response to Taxol did not correlate with proliferation changes suggesting that cell death inhibition may be responsible for this effect. Conclusions/Significance The application of the PEG hydrogel platform provided novel insight into the independent role of extrinsic parameters controlling drug response. The presented platform may not only become a useful tool for basic research related to the role of the cancer microenvironment but could also serve as a complementary platform for in vitro drug development. PMID:22792141

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

PubMed Central

Santana, Vinicius Canato; Almeida, Rafael Ribeiro; Ribeiro, Susan Pereira; Ferreira, Luís Carlos de Souza; Kalil, Jorge; Rosa, Daniela Santoro; Cunha-Neto, Edecio

2015-01-01

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity. PMID:26602876

Ca2+ and MgATP2- dependence of shortening in skinned single smooth muscle cells.

PubMed

Warshaw, D M; McBride, W J; Hubbard, M S

1987-04-01

Most studies of skinned smooth muscle have been performed in whole tissue preparations. In this study, we report the development of a chemically skinned single smooth muscle cell preparation from the toad, Bufo marinus, stomach. Isolated smooth muscle cells were skinned using saponin. The effect of various ionic environments (i.e., changing free Ca2+ and MgATP2-) on skinned cell contractile response was assessed by measuring cell lengths from populations of cells using a computer-assisted length-measuring system. Comparison of cell length histograms were used to determine the extent of cell shortening in response to a given ionic perturbation. Once skinned, the single cells shortened with a sensitivity to free calcium (ED50 = 1.5 microM Ca2+) that was three orders of magnitude lower than potassium depolarized cells (ED50 = 1.5 mM Ca2+). In addition to the calcium sensitivity, the effect of free MgATP2- on the extent of cell shortening was investigated. The extent of cell shortening was dependent on free MgATP2- with the maximum shortening response occurring at MgATP2- greater than 1 mM.

Targeting of non-dominant antigens as a vaccine strategy to broaden T-cell responses during chronic viral infection.

PubMed

Holst, Peter J; Jensen, Benjamin A H; Ragonnaud, Emeline; Thomsen, Allan R; Christensen, Jan P

2015-01-01

In this study, we compared adenoviral vaccine vectors with the capacity to induce equally potent immune responses against non-dominant and immunodominant epitopes of murine lymphocytic choriomeningitis virus (LCMV). Our results demonstrate that vaccination targeting non-dominant epitopes facilitates potent virus-induced T-cell responses against immunodominant epitopes during subsequent challenge with highly invasive virus. In contrast, when an immunodominant epitope was included in the vaccine, the T-cell response associated with viral challenge remained focussed on that epitope. Early after challenge with live virus, the CD8+ T cells specific for vaccine-encoded epitopes, displayed a phenotype typically associated with prolonged/persistent antigenic stimulation marked by high levels of KLRG-1, as compared to T cells reacting to epitopes not included in the vaccine. Notably, this association was lost over time in T cells specific for the dominant T cell epitopes, and these cells were fully capable of expanding in response to a new viral challenge. Overall, our data suggests a potential for broadening of the antiviral CD8+ T-cell response by selecting non-dominant antigens to be targeted by vaccination. In addition, our findings suggest that prior adenoviral vaccination is not likely to negatively impact the long-term and protective immune response induced and maintained by a vaccine-attenuated chronic viral infection.

A Novel 3 Dimensional Stromal-based Model for In Vitro Chemotherapy Sensitivity Testing of Leukemia Cells

PubMed Central

Aljitawi, Omar S.; Li, Dandan; Xiao, Yinghua; Zhang, Da; Ramachandran, Karthik; Stehno-Bittel, Lisa; Van Veldhuizen, Peter; Lin, Tara L.; Kambhampati, Suman; Garimella, Rama

2014-01-01

The disparate responses of leukemia cells to chemotherapy in vivo, compared to in vitro, is partly related to the interactions of leukemic cells and the 3 dimensional (3D) bone marrow stromal microenvironment. We investigated the effects of chemotherapy agents on leukemic cell lines co-cultured with human bone marrow mesenchymal stem cell (hu-BM-MSC) in 3D. Comparison was made to leukemic cells treated in suspension, or grown on a hu-BM-MSC monolayer (2D conditions). We demonstrated that leukemic cells cultured in 3D were more resistant to drug-induced apoptosis compared to cells cultured in 2D or in suspension. We also demonstrated significant differences in leukemic cell response to chemotherapy using different leukemic cell lines cultured in 3D. We suggest that the differential responses to chemotherapy in 3D may be related to the expression of N-cadherin in the co-culture system. This unique model provides an opportunity to study leukemic cell responses to chemotherapy in 3D. PMID:23566162

Effects of chemotherapy on immune responses in dogs with cancer.

PubMed

Walter, Claudia U; Biller, Barbara J; Lana, Susan E; Bachand, Annette M; Dow, Steven W

2006-01-01

Chemotherapy is assumed to be immunosuppressive; yet to the authors' knowledge, the effects of common chemotherapy protocols on adaptive immune responses in dogs with cancer have not been fully evaluated. Therefore, a study was conducted to evaluate the effects of 2 common chemotherapy protocols on T- and B-cell numbers and humoral immune responses to de novo vaccination in dogs with cancer. Twenty-one dogs with cancer (12 with lymphoma, 9 with osteosarcoma) were enrolled in a prospective study to assess effects of doxorubicin versus multi-drug chemotherapy on adaptive immunity. Numbers of circulating T and B cells were assessed by flow cytometry, and antibody responses to de novo vaccination were assessed before, during, and after chemotherapy. The T- and B-cell numbers before treatment also were compared with those of healthy, age-matched, control dogs. Prior to treatment, dogs with cancer had significantly fewer (P < .05) CD4+ T cells and CD8+ T cells than did healthy dogs. Doxorubicin treatment did not cause a significant decrease in T- or B-cell numbers, whereas treatment with combination chemotherapy caused a significant and persistent decrease in B-cell numbers. Antibody titers after vaccination were not significantly different between control and chemotherapy-treated dogs. These findings suggest that chemotherapy may have less impact on T-cell numbers and ability to mount antibody responses in dogs with cancer than was previously anticipated, though dogs with lymphoma or osteosarcoma appear to be relatively T-cell deficient before initiation of chemotherapy.

Jasmonic Acid Signaling Modulates Ozone-Induced Hypersensitive Cell Death

PubMed Central

Rao, Mulpuri V.; Lee, Hyung-il; Creelman, Robert A.; Mullet, John E.; Davis, Keith R.

2000-01-01

Recent studies suggest that cross-talk between salicylic acid (SA)–, jasmonic acid (JA)–, and ethylene-dependent signaling pathways regulates plant responses to both abiotic and biotic stress factors. Earlier studies demonstrated that ozone (O3) exposure activates a hypersensitive response (HR)–like cell death pathway in the Arabidopsis ecotype Cvi-0. We now have confirmed the role of SA and JA signaling in influencing O3-induced cell death. Expression of salicylate hydroxylase (NahG) in Cvi-0 reduced O3-induced cell death. Methyl jasmonate (Me-JA) pretreatment of Cvi-0 decreased O3-induced H2O2 content and SA concentrations and completely abolished O3-induced cell death. Cvi-0 synthesized as much JA as did Col-0 in response to O3 exposure but exhibited much less sensitivity to exogenous Me-JA. Analyses of the responses to O3 of the JA-signaling mutants jar1 and fad3/7/8 also demonstrated an antagonistic relationship between JA- and SA-signaling pathways in controlling the magnitude of O3-induced HR-like cell death. PMID:11006337

Jasmonic acid signaling modulates ozone-induced hypersensitive cell death.

PubMed

Rao, M V; Lee, H; Creelman, R A; Mullet, J E; Davis, K R

2000-09-01

Recent studies suggest that cross-talk between salicylic acid (SA)-, jasmonic acid (JA)-, and ethylene-dependent signaling pathways regulates plant responses to both abiotic and biotic stress factors. Earlier studies demonstrated that ozone (O(3)) exposure activates a hypersensitive response (HR)-like cell death pathway in the Arabidopsis ecotype Cvi-0. We now have confirmed the role of SA and JA signaling in influencing O(3)-induced cell death. Expression of salicylate hydroxylase (NahG) in Cvi-0 reduced O(3)-induced cell death. Methyl jasmonate (Me-JA) pretreatment of Cvi-0 decreased O(3)-induced H(2)O(2) content and SA concentrations and completely abolished O(3)-induced cell death. Cvi-0 synthesized as much JA as did Col-0 in response to O(3) exposure but exhibited much less sensitivity to exogenous Me-JA. Analyses of the responses to O(3) of the JA-signaling mutants jar1 and fad3/7/8 also demonstrated an antagonistic relationship between JA- and SA-signaling pathways in controlling the magnitude of O(3)-induced HR-like cell death.

A photoelectrochemical platform for the capture and release of rare single cells.

PubMed

Parker, Stephen G; Yang, Ying; Ciampi, Simone; Gupta, Bakul; Kimpton, Kathleen; Mansfeld, Friederike M; Kavallaris, Maria; Gaus, Katharina; Gooding, J Justin

2018-06-12

For many normal and aberrant cell behaviours, it is important to understand the origin of cellular heterogeneity. Although powerful methods for studying cell heterogeneity have emerged, they are more suitable for common rather than rare cells. Exploring the heterogeneity of rare single cells is challenging because these rare cells must be first pre-concentrated and undergo analysis prior to classification and expansion. Here, a versatile capture & release platform consisting of an antibody-modified and electrochemically cleavable semiconducting silicon surface for release of individual cells of interest is presented. The captured cells can be interrogated microscopically and tested for drug responsiveness prior to release and recovery. The capture & release strategy was applied to identify rare tumour cells from whole blood, monitor the uptake of, and response to, doxorubicin and subsequently select cells for single-cell gene expression based on their response to the doxorubicin.

Comparison of the canine corneal epithelial cell sheets cultivated from limbal stem cells on canine amniotic membrane, atelocollagen gel, and temperature-responsive culture dish.

PubMed

Nam, Eunryel; Fujita, Naoki; Morita, Maresuke; Tsuzuki, Keiko; Lin, Hsing Yi; Chung, Cheng Shu; Nakagawa, Takayuki; Nishimura, Ryohei

2015-07-01

The current study compared canine corneal epithelial cell sheets cultivated from limbal stem cells on amniotic membrane, atelocollagen gel, and temperature-responsive culture dish. We collected limbal epithelial cells from the intact eyes of beagles and cultivated the cells on denuded canine amniotic membranes, temperature-responsive cell culture labware, and collagen gel with 3T3 feeder cells. Immunofluorescence staining for Ki-67 was used to analyze the capacity of cell proliferation in the sheets. Immunofluorescence staining was also performed for the corneal epithelium-specific marker cytokeratin 3 and putative stem cell markers ABCG2 and p63. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to detect ABCG2 and p63. The growth rates of the cultivated cells, or the times it took them to reach confluency, were different for the three scaffolds. The cultivated sheet on the temperature-responsive dish consisted of 2-3 layers, while those on the collagen gel and on the amniotic membrane consisted of 5-8 layers. The basal layer cells grown on all three scaffolds expressed putative stem cell markers. In real-time RT-PCR analysis, the highest level of p63 was observed in the sheets grown on collagen gel. In this study, the cells cultured on the collagen gel demonstrated a capacity for cell proliferation, and the expressions of stem cells in the sheets suggested that collagen gel is the most suitable carrier for clinical use. © 2014 American College of Veterinary Ophthalmologists.

ESAT-6 Targeting to DEC205+ Antigen Presenting Cells Induces Specific-T Cell Responses against ESAT-6 and Reduces Pulmonary Infection with Virulent Mycobacterium tuberculosis.

PubMed

Silva-Sánchez, Aarón; Meza-Pérez, Selene; Flores-Langarica, Adriana; Donis-Maturano, Luis; Estrada-García, Iris; Calderón-Amador, Juana; Hernández-Pando, Rogelio; Idoyaga, Juliana; Steinman, Ralph M; Flores-Romo, Leopoldo

2015-01-01

Airways infection with Mycobacterium tuberculosis (Mtb) is contained mostly by T cell responses, however, Mtb has developed evasion mechanisms which affect antigen presenting cell (APC) maturation/recruitment delaying the onset of Ag-specific T cell responses. Hypothetically, bypassing the natural infection routes by delivering antigens directly to APCs may overcome the pathogen's naturally evolved evasion mechanisms, thus facilitating the induction of protective immune responses. We generated a murine monoclonal fusion antibody (α-DEC-ESAT) to deliver Early Secretory Antigen Target (ESAT)-6 directly to DEC205+ APCs and to assess its in vivo effects on protection associated responses (IFN-γ production, in vivo CTL killing, and pulmonary mycobacterial load). Treatment with α-DEC-ESAT alone induced ESAT-6-specific IFN-γ producing CD4+ T cells and prime-boost immunization prior to Mtb infection resulted in early influx (d14 post-infection) and increased IFN-γ+ production by specific T cells in the lungs, compared to scarce IFN-γ production in control mice. In vivo CTL killing was quantified in relevant tissues upon transferring target cells loaded with mycobacterial antigens. During infection, α-DEC-ESAT-treated mice showed increased target cell killing in the lungs, where histology revealed cellular infiltrate and considerably reduced bacterial burden. Targeting the mycobacterial antigen ESAT-6 to DEC205+ APCs before infection expands specific T cell clones responsible for early T cell responses (IFN-γ production and CTL activity) and substantially reduces lung bacterial burden. Delivering mycobacterial antigens directly to APCs provides a unique approach to study in vivo the role of APCs and specific T cell responses to assess their potential anti-mycobacterial functions.

ESAT-6 Targeting to DEC205+ Antigen Presenting Cells Induces Specific-T Cell Responses against ESAT-6 and Reduces Pulmonary Infection with Virulent Mycobacterium tuberculosis

PubMed Central

Silva-Sánchez, Aarón; Meza-Pérez, Selene; Flores-Langarica, Adriana; Donis-Maturano, Luis; Estrada-García, Iris; Calderón-Amador, Juana; Hernández-Pando, Rogelio; Idoyaga, Juliana; Flores-Romo, Leopoldo

2015-01-01

Airways infection with Mycobacterium tuberculosis (Mtb) is contained mostly by T cell responses, however, Mtb has developed evasion mechanisms which affect antigen presenting cell (APC) maturation/recruitment delaying the onset of Ag-specific T cell responses. Hypothetically, bypassing the natural infection routes by delivering antigens directly to APCs may overcome the pathogen’s naturally evolved evasion mechanisms, thus facilitating the induction of protective immune responses. We generated a murine monoclonal fusion antibody (α-DEC-ESAT) to deliver Early Secretory Antigen Target (ESAT)-6 directly to DEC205+ APCs and to assess its in vivo effects on protection associated responses (IFN-γ production, in vivo CTL killing, and pulmonary mycobacterial load). Treatment with α-DEC-ESAT alone induced ESAT-6-specific IFN-γ producing CD4+ T cells and prime-boost immunization prior to Mtb infection resulted in early influx (d14 post-infection) and increased IFN-γ+ production by specific T cells in the lungs, compared to scarce IFN-γ production in control mice. In vivo CTL killing was quantified in relevant tissues upon transferring target cells loaded with mycobacterial antigens. During infection, α-DEC-ESAT-treated mice showed increased target cell killing in the lungs, where histology revealed cellular infiltrate and considerably reduced bacterial burden. Targeting the mycobacterial antigen ESAT-6 to DEC205+ APCs before infection expands specific T cell clones responsible for early T cell responses (IFN-γ production and CTL activity) and substantially reduces lung bacterial burden. Delivering mycobacterial antigens directly to APCs provides a unique approach to study in vivo the role of APCs and specific T cell responses to assess their potential anti-mycobacterial functions. PMID:25915045

Mycobacterium tuberculosis Specific CD8+ T Cells Rapidly Decline with Antituberculosis Treatment

PubMed Central

Nyendak, Melissa R.; Park, Byung; Null, Megan D.; Baseke, Joy; Swarbrick, Gwendolyn; Mayanja-Kizza, Harriet; Nsereko, Mary; Johnson, Denise F.; Gitta, Phineas; Okwera, Alphonse; Goldberg, Stefan; Bozeman, Lorna; Johnson, John L.; Boom, W. Henry; Lewinsohn, Deborah A.; Lewinsohn, David M.

2013-01-01

Rationale Biomarkers associated with response to therapy in tuberculosis could have broad clinical utility. We postulated that the frequency of Mycobacterium tuberculosis (Mtb) specific CD8+ T cells, by virtue of detecting intracellular infection, could be a surrogate marker of response to therapy and would decrease during effective antituberculosis treatment. Objectives: We sought to determine the relationship of Mtb specific CD4+ T cells and CD8+ T cells with duration of antituberculosis treatment. Materials and Methods We performed a prospective cohort study, enrolling between June 2008 and August 2010, of HIV-uninfected Ugandan adults (n = 50) with acid-fast bacillus smear-positive, culture confirmed pulmonary TB at the onset of antituberculosis treatment and the Mtb specific CD4+ and CD8+ T cell responses to ESAT-6 and CFP-10 were measured by IFN-γ ELISPOT at enrollment, week 8 and 24. Results There was a significant difference in the Mtb specific CD8+ T response, but not the CD4+ T cell response, over 24 weeks of antituberculosis treatment (p<0.0001), with an early difference observed at 8 weeks of therapy (p = 0.023). At 24 weeks, the estimated Mtb specific CD8+ T cell response decreased by 58%. In contrast, there was no significant difference in the Mtb specific CD4+ T cell during the treatment. The Mtb specific CD4+ T cell response, but not the CD8+ response, was negatively impacted by the body mass index. Conclusions Our data provide evidence that the Mtb specific CD8+ T cell response declines with antituberculosis treatment and could be a surrogate marker of response to therapy. Additional research is needed to determine if the Mtb specific CD8+ T cell response can detect early treatment failure, relapse, or to predict disease progression. PMID:24324704

Reduced immune responses in chimeric mice engrafted with bone marrow cells from mice with airways inflammation.

PubMed

Scott, Naomi M; Ng, Royce L X; McGonigle, Terence A; Gorman, Shelley; Hart, Prue H

2015-11-01

During respiratory inflammation, it is generally assumed that dendritic cells differentiating from the bone marrow are immunogenic rather than immunoregulatory. Using chimeric mice, the outcomes of airways inflammation on bone marrow progenitor cells were studied. Immune responses were analyzed in chimeric mice engrafted for >16 weeks with bone marrow cells from mice with experimental allergic airways disease (EAAD). Responses to sensitization and challenge with the allergen causing inflammation in the bone marrow-donor mice were significantly reduced in the chimeric mice engrafted with bone marrow cells from mice with EAAD (EAAD-chimeric). Responses to intranasal LPS and topical fluorescein isothiocyanate (non-specific challenges) were significantly attenuated. Fewer activated dendritic cells from the airways and skin of the EAAD-chimeric mice could be tracked to the draining lymph nodes, and may contribute to the significantly reduced antigen/chemical-induced hypertrophy in the draining nodes, and the reduced immune responses to sensitizing allergens. Dendritic cells differentiating in vitro from the bone marrow of >16 weeks reconstituted EAAD-chimeric mice retained an ability to poorly prime immune responses when transferred into naïve mice. Dendritic cells developing from bone marrow progenitors during airways inflammation are altered such that daughter cells have reduced antigen priming capabilities.

Cell response to long term mechanical interaction with nanopipettes

NASA Astrophysics Data System (ADS)

Orynbayeva, Zulfiya; Singhal, Riju; Vitol, Elina; Bouchard, Michael; Azizkhan-Clifford, Jane; Layton, Bradley; Friedman, Gary; Gogotsi, Yury

2009-03-01

Traditional microinjection into cells is performed over a relatively short term. Pipettes are typically withdrawn following any kind of injection. On the other hand, there is growing interest in using nanopipettes for cellular and subcellular probing. This interest is partly due to new developments in nanopipette technology which employ carbon nanotubes and provide robustness, flexibility, and biocompatibility. However, as far as we know, no systematic study of physiological, biochemical, and biophysical processes associated with cell response to lengthy mechanical stimulations by nanopipette probing have been performed so far. We present a detailed investigation of a wide range of effects of long term pipette insertion into a cell. Both traditional glass micropipettes and the novel carbon nanotube-tipped probes were involved in this study. The mechanism of Ca2+ response to the mechanical stimuli introduced by the nanopipette, and the role of different organelles in this mechanism were studied. We hypothesize that the calcium response is a function of cytoskeleton integrity and the mode of coupling between the cytoskeleton and the plasma membrane domains.

CXCL13-producing TFH cells link immune suppression and adaptive memory in human breast cancer

PubMed Central

Gu-Trantien, Chunyan; Migliori, Edoardo; de Wind, Alexandre; Brohée, Sylvain; Garaud, Soizic; Noël, Grégory; Dang Chi, Vu Luan; Lodewyckx, Jean-Nicolas; Naveaux, Céline; Duvillier, Hugues; Larsimont, Denis

2017-01-01

T follicular helper cells (TFH cells) are important regulators of antigen-specific B cell responses. The B cell chemoattractant CXCL13 has recently been linked with TFH cell infiltration and improved survival in human cancer. Although human TFH cells can produce CXCL13, their immune functions are currently unknown. This study presents data from human breast cancer, advocating a role for tumor-infiltrating CXCL13-producing (CXCR5–) TFH cells, here named TFHX13 cells, in promoting local memory B cell differentiation. TFHX13 cells potentially trigger tertiary lymphoid structure formation and thereby generate germinal center B cell responses at the tumor site. Follicular DCs are not potent CXCL13 producers in breast tumor tissues. We used the TFH cell markers PD-1 and ICOS to identify distinct effector and regulatory CD4+ T cell subpopulations in breast tumors. TFHX13 cells are an important component of the PD-1hiICOSint effector subpopulation and coexpanded with PD-1intICOShiFOXP3hi Tregs. IL2 deprivation induces CXCL13 expression in vitro with a synergistic effect from TGFβ1, providing insight into TFHX13 cell differentiation in response to Treg accumulation, similar to conventional TFH cell responses. Our data suggest that human TFHX13 cell differentiation may be a key factor in converting Treg-mediated immune suppression to de novo activation of adaptive antitumor humoral responses in the chronic inflammatory breast cancer microenvironment. PMID:28570278

Intrinsic attenuation of post-irradiation calcium and ER stress imparts significant radioprotection to lepidopteran insect cells.

PubMed

Guleria, Ayushi; Thukral, Neha; Chandna, Sudhir

2018-04-15

Sf9 lepidopteran insect cells are 100-200 times more radioresistant than mammalian cells. This distinctive feature thus makes them suitable for studies exploring radioprotective molecular mechanisms. It has been established from previous studies of our group that downstream mitochondrial apoptotic signaling pathways in Sf9 cells are quite similar to mammalian cells, implicating the upstream signaling pathways in their extensive radioresistance. In the present study, intracellular and mitochondrial calcium levels remained unaltered in Sf9 cells in response to radiation, in sharp contrast to human (HEK293T) cells. The isolated mitochondria from Sf9 cells exhibited nearly 1.5 times greater calcium retention capacity than mammalian cells, highlighting their inherent stress resilience. Importantly, UPR/ER stress marker proteins (p-eIF2α, GRP4 and SERCA) remained unaltered by radiation and suggested highly attenuated ER and calcium stress. Lack of SERCA induction further corroborates the lack of radiation-induced calcium mobilization in these cells. The expression of CaMKII, an important effector molecule of calcium signaling, did not alter in response to radiation. Inhibiting CaMKII by KN-93 or suppressing CaM by siRNA failed to alter Sf9 cells response to radiation and suggests CaM-CaMKII independent radiation signaling. Therefore, this study suggests that attenuated calcium signaling/ER stress is an important determinant of lepidopteran cell radioresistance. Copyright © 2018 Elsevier Inc. All rights reserved.

Human CD4+ T-cell response to hepatitis delta virus: identification of multiple epitopes and characterization of T-helper cytokine profiles.

PubMed Central

Nisini, R; Paroli, M; Accapezzato, D; Bonino, F; Rosina, F; Santantonio, T; Sallusto, F; Amoroso, A; Houghton, M; Barnaba, V

1997-01-01

The T-cell-mediated immune response plays a crucial role in defense against hepatotropic viruses as well as in the pathogenesis of viral chronic hepatitides. However, very little is known about the role of specific T cells during hepatitis delta virus (HDV) infection in humans. In this study, the T-cell response to HDV in chronic hepatitis B virus (HBV) carriers with HDV superinfection was investigated at different levels. Analysis of peripheral blood mononuclear cell (PBMC) proliferation in response to a recombinant form of large hepatitis delta antigen (HDAg) revealed that 8 of 30 patients studied (27%) specifically responded to HDAg. By employing synthetic peptides spanning the entire HDAg sequence, we found that T-cell recognition was directed against different antigenic determinants, with patient-to-patient variation in the pattern of response to peptides. Interestingly, all responders had signs of inactive HDV-induced disease, while none of the patients with active disease and none of the control subjects showed any significant proliferation. More accurate information about the specific T-cell response was obtained at the clonal level. A panel of HDAg-specific CD4+ T-cell clones from three HDV-infected individuals and fine-specificity analysis revealed that the clones tested individually recognized four epitopes corresponding to amino acids (aa) 26 to 41, 50 to 65, 66 to 81, or 106 to 121 of HDAg sequence. The study of human leukocyte antigen (HLA) restriction revealed that peptides 50 to 65 and 106 to 121 were presented to specific T cells in association with multiple class II molecules. In addition, peptide 26 to 41 was efficiently generated after processing of HDAg through the endogenous processing pathway. Cytokine secretion analysis showed that all the CD4+ T-cell clones assayed were able to produce high levels of gamma interferon (IFN-gamma), belonging either to T helper-1 (Th1) or Th0 subsets and that some of them were cytotoxic in a specific assay. This study provides the first evidence that detection of a specific T-cell response to HDAg in the peripheral blood of individuals with hepatitis delta is related to the decrease of HDV-induced disease activity. The HDAg epitopes identified here and particularly those recognized by CD4+ T cells in association with multiple major histocompatibility complex class II molecules may be potentially exploited for the preparation of a vaccine for prophylaxis and therapy of HDV infection. PMID:9032359

Effect of human umbilical cord blood stem cell transplantation on oval cell response in 2-AAF/CCL4 liver injury model: experimental immunohistochemical study.

PubMed

Abdellatif, Hussein; Shiha, Gamal; Saleh, Dalia M; Eltahry, Huda; Botros, Kamal G

2017-01-01

Oval cells, specific liver progenitors, are activated in response to injury. The human umbilical cord blood (hUCB) is a possible source of transplantable hepatic progenitors and can be used in cases of severe liver injury. We detected the effect of hUCB stem cell transplantation on natural response of oval cells to injury. Twenty-four female albino rats were randomly divided into three groups: (A) control, (B) liver injury with hepatocyte block, and (C) hUCB transplanted group. Hepatocyte block was performed by administration of 2-acetylaminofluorene (2-AAF) for 12 days. CCL4 was administrated at day 5 from experiment start. Animals were sacrificed at 9 days post CCL4 administration, and samples were collected for biochemical and histopathological analysis. Oval cell response to injury was evaluated by the percentage of oval cells in the liver tissue and frequency of cells incorporated into new ducts. Immunohistochemical analysis of oval cell response to injury was performed. There was significant deviation in the hUCB-transplanted (4.9 ± 1.4) and liver injury groups (2.4 ± 0.9) as compared to control (0.89 ± 0.4) 9 days post injury. Detection of oval cell response was dependant on OV-6 immunoreactivity. For mere localization of cells with human origin, CD34 antihuman immunoreactivity was performed. There was no significant difference in endogenous OV-6 immunoreactivity following stem cell transplantation as compared to the liver injury group. In vivo transplantation of cord blood stem cells (hUCB) does not interfere with natural oval cell response to liver injury.

Aryl hydrocarbon receptor activation impairs the priming but not the recall of influenza virus-specific CD8+ T cells in the lung.

PubMed

Lawrence, B Paige; Roberts, Alan D; Neumiller, Joshua J; Cundiff, Jennifer A; Woodland, David L

2006-11-01

The response of CD8+ T cells to influenza virus is very sensitive to modulation by aryl hydrocarbon receptor (AhR) agonists; however, the mechanism underlying AhR-mediated alterations in CD8+ T cell function remains unclear. Moreover, very little is known regarding how AhR activation affects anamnestic CD8+ T cell responses. In this study, we analyzed how AhR activation by the pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters the in vivo distribution and frequency of CD8+ T cells specific for three different influenza A virus epitopes during and after the resolution of a primary infection. We then determined the effects of TCDD on the expansion of virus-specific memory CD8+ T cells during recall challenge. Adoptive transfer of AhR-null CD8+ T cells into congenic AhR(+/+) recipients, and the generation of CD45.2AhR(-/-)-->CD45.1AhR(+/+) chimeric mice demonstrate that AhR-regulated events within hemopoietic cells, but not directly within CD8+ T cells, underlie suppressed expansion of virus-specific CD8+ T cells during primary infection. Using a dual-adoptive transfer approach, we directly compared the responsiveness of virus-specific memory CD8+ T cells created in the presence or absence of TCDD, which revealed that despite profound suppression of the primary response to influenza virus, the recall response of virus-specific CD8+ T cells that form in the presence of TCDD is only mildly impaired. Thus, the delayed kinetics of the recall response in TCDD-treated mice reflects the fact that there are fewer memory cells at the time of reinfection rather than an inherent defect in the responsive capacity of virus-specific memory CD8+ cells.

NaCl responsive taste cells in the mouse fungiform taste buds.

PubMed

Yoshida, R; Horio, N; Murata, Y; Yasumatsu, K; Shigemura, N; Ninomiya, Y

2009-03-17

Previous studies have demonstrated that rodents' chorda tympani (CT) nerve fibers responding to NaCl can be classified according to their sensitivities to the epithelial sodium channel (ENaC) blocker amiloride into two groups: amiloride-sensitive (AS) and -insensitive (AI). The AS fibers were shown to respond specifically to NaCl, whereas AI fibers broadly respond to various electrolytes, including NaCl. These data suggest that salt taste transduction in taste cells may be composed of at least two different systems; AS and AI ones. To further address this issue, we investigated the responses to NaCl, KCl and HCl and the amiloride sensitivity of mouse fungiform papilla taste bud cells which are innervated by the CT nerve. Comparable with the CT data, the results indicated that 56 NaCl-responsive cells tested were classified into two groups; 25 cells ( approximately 44%) narrowly responded to NaCl and their NaCl response were inhibited by amiloride (AS cells), whereas the remaining 31 cells ( approximately 56%) responded not only to NaCl, but to KCl and/or HCl and showed no amiloride inhibition of NaCl responses (AI cells). Amiloride applied to the basolateral side of taste cells had no effect on NaCl responses in the AS and AI cells. Single cell reverse transcription-polymerase chain reaction (RT-PCR) experiments indicated that ENaC subunit mRNA was expressed in a subset of AS cells. These findings suggest that the mouse fungiform taste bud is composed of AS and AI cells that can transmit taste information differently to their corresponding types of CT fibers, and apical ENaCs may be involved in the NaCl responses of AS cells.

Studies on the immunopotentiating effects of a streptococcal preparation, OK-432. I. Enhancement of T cell-mediated immune responses of mice.

PubMed Central

Kai, S; Tanaka, J; Nomoto, K; Torisu, M

1979-01-01

The effects of the anti-tumour agent OK-432 on the immune response to hamster erythrocytes (HRBC) and nucleated chicken erythrocytes (CRBC) were studied in inbred SL mice. Mice were treated repeatedly with OK-432 before immunization with erythrocytes in saline. The cytotoxicity of CRBC-primed spleen cells, as demonstrated by 51Cr release from labelled CRBC, was markedly increased by treatment with OD-432. The delayed footpad reaction to CRBC was significantly augmented by treatment with OK-432. These results in mice indicate that OK-432 can enhance the cellular immune responses which require the contribution of T cells. Such an activation of T cells by OK-432 was observed in the humoral immune response to a trinitrophenyl group. Augmentation of anti-hapten antibody production, suggesting the enhancement of helper T cell activity by OK-432, was noticed after immunization with trinitrophenyl conjugated to erythrocytes. Furthermore, this enhancement of helper T cell activity by OK-432 was confirmed by utilizing an adoptive transfer system. These results support the possibility that T cell activation may be one of the important effects of OK-432 as an immunopotentiator. PMID:314874

Thermo-responsive cell culture carrier: Effects on macrophage functionality and detachment efficiency.

PubMed

Rennert, Knut; Nitschke, Mirko; Wallert, Maria; Keune, Natalie; Raasch, Martin; Lorkowski, Stefan; Mosig, Alexander S

2017-01-01

Harvesting cultivated macrophages for tissue engineering purposes by enzymatic digestion of cell adhesion molecules can potentially result in unintended activation, altered function, or behavior of these cells. Thermo-responsive polymer is a promising tool that allows for gentle macrophage detachment without artificial activation prior to subculture within engineered tissue constructs. We therefore characterized different species of thermo-responsive polymers for their suitability as cell substrate and to mediate gentle macrophage detachment by temperature shift. Primary human monocyte- and THP-1-derived macrophages were cultured on thermo-responsive polymers and characterized for phagocytosis and cytokine secretion in response to lipopolysaccharide stimulation. We found that both cell types differentially respond in dependence of culture and stimulation on thermo-responsive polymers. In contrast to THP-1 macrophages, primary monocyte-derived macrophages showed no signs of impaired viability, artificial activation, or altered functionality due to culture on thermo-responsive polymers compared to conventional cell culture. Our study demonstrates that along with commercially available UpCell carriers, two other thermo-responsive polymers based on poly(vinyl methyl ether) blends are attractive candidates for differentiation and gentle detachment of primary monocyte-derived macrophages. In summary, we observed similar functionality and viability of primary monocyte-derived macrophages cultured on thermo-responsive polymers compared to standard cell culture surfaces. While this first generation of custom-made thermo-responsive polymers does not yet outperform standard culture approaches, our results are very promising and provide the basis for exploiting the unique advantages offered by custom-made thermo-responsive polymers to further improve macrophage culture and recovery in the future, including the covalent binding of signaling molecules and the reduction of centrifugation and washing steps. Optimizing these and other benefits of thermo-responsive polymers could greatly improve the culture of macrophages for tissue engineering applications.

The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq.

PubMed

Wills, Quin F; Mellado-Gomez, Esther; Nolan, Rory; Warner, Damien; Sharma, Eshita; Broxholme, John; Wright, Benjamin; Lockstone, Helen; James, William; Lynch, Mark; Gonzales, Michael; West, Jay; Leyrat, Anne; Padilla-Parra, Sergi; Filippi, Sarah; Holmes, Chris; Moore, Michael D; Bowden, Rory

2017-01-07

Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity, despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages. We introduce the programmable Polaris™ microfluidic lab-on-chip for single-cell sequencing, which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1, such as an altered oxidative stress response, have a large paracrine signaling component. Furthermore, we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation, both associated with the oxidative stress response and altered proteostasis. Interestingly, SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'), with no known co-regulation. As single-cell methods continue to mature, so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture, live-cell imaging, and single-cell sequencing, we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages, for example, react to inflammation and form treatment resistant HIV reservoirs.

Cell viability study of thermo-responsive core-shell superparamagnetic nanoparticles for multimodal cancer therapy

NASA Astrophysics Data System (ADS)

Shah, Saqlain A.; Majeed, A.; Shafique, M. A.; Rashid, K.; Awan, Saif-Ullah

2014-02-01

This is a vital extension of our previously published work. Thermo-responsive copolymer coated superparamagnetic MnFe2O4 nanoparticles are tested for cell viability and affinity on HeLa carcinoma cells under different conditions. Nanoparticles were loaded with anticancer drug doxorubicin. Composite nanoparticles of average diameter 45 nm were of core-shell structure having magnetic core of about 18 nm. Magnetic hyperthermia effects on cell viability and drug delivery were studied by exposing the cell suspension to high frequency magnetic field, and living cells were quantified using MTT method. There was almost absence of drug release at 37 °C. Drug was released at temperatures above lower critical solution temperature (LCST) by magnetic heating. LCST of the thermo-responsive copolymer was observed to be around 39 °C. Below this temperature, copolymer was hydrophilic and swelled. But above LCST, copolymer could become hydrophobic, expel water and drug and shrink in volume. Combination of hyperthermia and drug delivery effectively treated cancer cells.

Viability and DNA damage responses of TPPII-deficient Myc- and Ras-transformed fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsurumi, Chizuko; Firat, Elke; Gaedicke, Simone

2009-09-04

Tripeptidyl peptidase II (TPPII) is a giant cytosolic protease. Previous protease inhibitor, overexpression and siRNA studies suggested that TPPII is important for viability and proliferation of tumor cells, and for their ionizing radiation-induced DNA damage response. The possibility that TPPII could be targeted for tumor therapy prompted us to study its role in transformed cells following genetic TPPII deletion. We generated cell lines from primary fibroblasts having conditional (floxed) TPPII alleles, transformed them with both the c-myc and H-ras oncogenes, and deleted TPPII using retroviral self-deleting Cre recombinase. Clonally derived TPPIIflox/flox and TPPII-/- transformed fibroblasts showed no influences of TPPIImore » expression on basal cell survival and proliferation, nor on radiation-induced p53 activation, p21 induction, cell cycle arrest, apoptosis, or clonogenic cell death. Thus, our results do not support a generally important role of TPPII for viability and proliferation of transformed cells or their p53-mediated DNA damage response.« less

Viability and DNA damage responses of TPPII-deficient Myc- and Ras-transformed fibroblasts.

PubMed

Tsurumi, Chizuko; Firat, Elke; Gaedicke, Simone; Huai, Jisen; Mandal, Pankaj Kumar; Niedermann, Gabriele

2009-09-04

Tripeptidyl peptidase II (TPPII) is a giant cytosolic protease. Previous protease inhibitor, overexpression and siRNA studies suggested that TPPII is important for viability and proliferation of tumor cells, and for their ionizing radiation-induced DNA damage response. The possibility that TPPII could be targeted for tumor therapy prompted us to study its role in transformed cells following genetic TPPII deletion. We generated cell lines from primary fibroblasts having conditional (floxed) TPPII alleles, transformed them with both the c-myc and H-ras oncogenes, and deleted TPPII using retroviral self-deleting Cre recombinase. Clonally derived TPPIIflox/flox and TPPII-/- transformed fibroblasts showed no influences of TPPII expression on basal cell survival and proliferation, nor on radiation-induced p53 activation, p21 induction, cell cycle arrest, apoptosis, or clonogenic cell death. Thus, our results do not support a generally important role of TPPII for viability and proliferation of transformed cells or their p53-mediated DNA damage response.

Cellulose conjugated FITC-labelled mesoporous silica nanoparticles: intracellular accumulation and stimuli responsive doxorubicin release

NASA Astrophysics Data System (ADS)

Hakeem, Abdul; Zahid, Fouzia; Duan, Ruixue; Asif, Muhammad; Zhang, Tianchi; Zhang, Zhenyu; Cheng, Yong; Lou, Xiaoding; Xia, Fan

2016-02-01

Herein, we design novel cellulose conjugated mesoporous silica nanoparticle (CLS-MSP) based nanotherapeutics for stimuli responsive intracellular doxorubicin (DOX) delivery. DOX molecules are entrapped in pores of the fabricated mesoporous silica nanoparticles (MSPs) while cellulose is used as an encapsulating material through esterification on the outlet of the pores of the MSPs to avoid premature DOX release under physiological conditions. In in vitro studies, stimuli responsive DOX release is successfully achieved from DOX loaded cellulose conjugated mesoporous silica nanoparticles (DOX/CLS-MSPs) by pH and cellulase triggers. Intracellular accumulation of DOX/CLS-MSPs in human liver cancer cells (HepG2 cells) is investigated through confocal microscope magnification. Cell viability of HepG2 cells is determined as the percentage of the cells incubated with DOX/CLS-MSPs compared with that of non-incubated cells through an MTT assay.Herein, we design novel cellulose conjugated mesoporous silica nanoparticle (CLS-MSP) based nanotherapeutics for stimuli responsive intracellular doxorubicin (DOX) delivery. DOX molecules are entrapped in pores of the fabricated mesoporous silica nanoparticles (MSPs) while cellulose is used as an encapsulating material through esterification on the outlet of the pores of the MSPs to avoid premature DOX release under physiological conditions. In in vitro studies, stimuli responsive DOX release is successfully achieved from DOX loaded cellulose conjugated mesoporous silica nanoparticles (DOX/CLS-MSPs) by pH and cellulase triggers. Intracellular accumulation of DOX/CLS-MSPs in human liver cancer cells (HepG2 cells) is investigated through confocal microscope magnification. Cell viability of HepG2 cells is determined as the percentage of the cells incubated with DOX/CLS-MSPs compared with that of non-incubated cells through an MTT assay. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr08753h

Flow cells for bioanalytical and bioprocess applications with optimized dynamic response and flow characteristics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lancaster, V.R.; Modlin, D.N.

1994-12-31

In this study, the authors present a method for design and characterization of flow cells developed for minimum flow volume and optimal dynamic response with a given central observation area. The dynamic response of a circular shaped dual ported flow cell was compared to that obtained from a flow cell whose optimized shape was determined using this method. In the optimized flow cell design, the flow rate at the nominal operating pressure increased by 50% whereas the flow cell volume was reduced by 70%. In addition, the dynamic response of the new flow cell was found to be 200% fastermore » than the circular flow cell. The fluid dynamic analysis included simple graphical techniques utilizing free stream vorticity functions and Hagen-Poiseuille relationships. The flow cell dynamic response was measured using a fluorescence detection system. The fluoresce in emission from a 400{micro}m spot located at the exit port was measured as a function of time after switching the input to the flow cell between fluorescent and non-fluorescent solutions. Analysis of results revealed the system could be reasonably characterized as a first order dynamic system. Although some evidence of second order behavior was also observed, it is reasonable to assume that a first order model will provide adequate predictive capability for many real world applications. Given a set of flow cell requirements, the methods presented in this study can be used to design and characterize flow cells with lower reagent consumption and reduced purging times. These improvements can be readily translated into reduced process times and/or lower usage of high cost reagents.« less

Induction of CD8 T-cell responses restricted to multiple HLA class I alleles in a cancer patient by immunization with a 20-mer NY-ESO-1f (NY-ESO-1 91-110) peptide.

PubMed

Eikawa, Shingo; Kakimi, Kazuhiro; Isobe, Midori; Kuzushima, Kiyotaka; Luescher, Immanuel; Ohue, Yoshihiro; Ikeuchi, Kazuhiro; Uenaka, Akiko; Nishikawa, Hiroyoshi; Udono, Heiichiro; Oka, Mikio; Nakayama, Eiichi

2013-01-15

Immunogenicity of a long 20-mer NY-ESO-1f peptide vaccine was evaluated in a lung cancer patient TK-f01, immunized with the peptide with Picibanil OK-432 and Montanide ISA-51. We showed that internalization of the peptide was necessary to present CD8 T-cell epitopes on APC, contrasting with the direct presentation of the short epitope. CD8 T-cell responses restricted to all five HLA class I alleles were induced in the patient after the peptide vaccination. Clonal analysis showed that B*35:01 and B*52:01-restricted CD8 T-cell responses were the two dominant responses. The minimal epitopes recognized by A*24:02, B*35:01, B*52:01 and C*12:02-restricted CD8 T-cell clones were defined and peptide/HLA tetramers were produced. NY-ESO-1 91-101 on A*24:02, NY-ESO-1 92-102 on B*35:01, NY-ESO-1 96-104 on B*52:01 and NY-ESO-1 96-104 on C*12:02 were new epitopes first defined in this study. Identification of the A*24:02 epitope is highly relevant for studying the Japanese population because of its high expression frequency (60%). High affinity CD8 T-cells recognizing tumor cells naturally expressing the epitopes and matched HLA were induced at a significant level. The findings suggest the usefulness of a long 20-mer NY-ESO-1f peptide harboring multiple CD8 T-cell epitopes as an NY-ESO-1 vaccine. Characterization of CD8 T-cell responses in immunomonitoring using peptide/HLA tetramers revealed that multiple CD8 T-cell responses comprised the dominant response. Copyright © 2012 UICC.

Comprehensive transcriptome analyses correlated with untargeted metabolome reveal differentially expressed pathways in response to cell wall alterations.

PubMed

Reem, Nathan T; Chen, Han-Yi; Hur, Manhoi; Zhao, Xuefeng; Wurtele, Eve Syrkin; Li, Xu; Li, Ling; Zabotina, Olga

2018-03-01

This research provides new insights into plant response to cell wall perturbations through correlation of transcriptome and metabolome datasets obtained from transgenic plants expressing cell wall-modifying enzymes. Plants respond to changes in their cell walls in order to protect themselves from pathogens and other stresses. Cell wall modifications in Arabidopsis thaliana have profound effects on gene expression and defense response, but the cell signaling mechanisms underlying these responses are not well understood. Three transgenic Arabidopsis lines, two with reduced cell wall acetylation (AnAXE and AnRAE) and one with reduced feruloylation (AnFAE), were used in this study to investigate the plant responses to cell wall modifications. RNA-Seq in combination with untargeted metabolome was employed to assess differential gene expression and metabolite abundance. RNA-Seq results were correlated with metabolite abundances to determine the pathways involved in response to cell wall modifications introduced in each line. The resulting pathway enrichments revealed the deacetylation events in AnAXE and AnRAE plants induced similar responses, notably, upregulation of aromatic amino acid biosynthesis and changes in regulation of primary metabolic pathways that supply substrates to specialized metabolism, particularly those related to defense responses. In contrast, genes and metabolites of lipid biosynthetic pathways and peroxidases involved in lignin polymerization were downregulated in AnFAE plants. These results elucidate how primary metabolism responds to extracellular stimuli. Combining the transcriptomics and metabolomics datasets increased the power of pathway prediction, and demonstrated the complexity of pathways involved in cell wall-mediated signaling.

Cell wall pectic arabinans influence the mechanical properties of Arabidopsis thaliana inflorescence stems and their response to mechanical stress.

PubMed

Verhertbruggen, Yves; Marcus, Susan E; Chen, Jianshe; Knox, J Paul

2013-08-01

Little is known of the dynamics of plant cell wall matrix polysaccharides in response to the impact of mechanical stress on plant organs. The capacity of the imposition of a mechanical stress (periodic brushing) to reduce the height of the inflorescence stem of Arabidopsis thaliana seedlings has been used to study the role of pectic arabinans in the mechanical properties and stress responsiveness of a plant organ. The arabinan-deficient-1 (arad1) mutation that affects arabinan structures in epidermal cell walls of inflorescence stems is demonstrated to reduce the impact on inflorescence stem heights caused by mechanical stress. The arabinan-deficient-2 (arad2) mutation, that does not have detectable impact on arabinan structures, is also shown to reduce the impact on stem heights caused by mechanical stress. The LM13 linear arabinan epitope is specifically detected in epidermal cell walls of the younger, flexible regions of inflorescence stems and increases in abundance at the base of inflorescence stems in response to an imposed mechanical stress. The strain (percentage deformation) of stem epidermal cells in the double mutant arad1 × arad2 is lower in unbrushed plants than in wild-type plants, but rises to wild-type levels in response to brushing. The study demonstrates the complexity of arabinan structures within plant cell walls and also that their contribution to cell wall mechanical properties is a factor influencing responsiveness to mechanical stress.

Effects of Frequency and Acceleration Amplitude on Osteoblast Mechanical Vibration Responses: A Finite Element Study

PubMed Central

Hsu, Hung-Yao

2016-01-01

Bone cells are deformed according to mechanical stimulation they receive and their mechanical characteristics. However, how osteoblasts are affected by mechanical vibration frequency and acceleration amplitude remains unclear. By developing 3D osteoblast finite element (FE) models, this study investigated the effect of cell shapes on vibration characteristics and effect of acceleration (vibration intensity) on vibrational responses of cultured osteoblasts. Firstly, the developed FE models predicted natural frequencies of osteoblasts within 6.85–48.69 Hz. Then, three different levels of acceleration of base excitation were selected (0.5, 1, and 2 g) to simulate vibrational responses, and acceleration of base excitation was found to have no influence on natural frequencies of osteoblasts. However, vibration response values of displacement, stress, and strain increased with the increase of acceleration. Finally, stress and stress distributions of osteoblast models under 0.5 g acceleration in Z-direction were investigated further. It was revealed that resonance frequencies can be a monotonic function of cell height or bottom area when cell volumes and material properties were assumed as constants. These findings will be useful in understanding how forces are transferred and influence osteoblast mechanical responses during vibrations and in providing guidance for cell culture and external vibration loading in experimental and clinical osteogenesis studies. PMID:28074178

Effects of pilocarpine and cevimeline on Ca2+ mobilization in rat parotid acini and ducts.

PubMed

Ono, Kentaro; Inagaki, Tomohiro; Iida, Taichi; Hosokawa, Ryuji; Inenaga, Kiyotoshi

2009-01-01

Previous reports suggested that there is no significant difference in the binding affinity of pilocarpine and cevimeline on muscarinic receptors (1). However, in vivo studies from our laboratory suggested that pilocarpine-induced salivation from the parotid gland is greater than that induced by cevimeline in rats. Therefore, in the present study, sialogogue-induced intracellular Ca(2+) mobilization was investigated in isolated parotid gland cells in vitro. Pilocarpine and cevimeline increased the intracellular Ca(2+) concentration of parotid gland acinar and duct cells over 1 microM in a dose-dependent manner. Pilocarpine-induced responses were higher than cevimeline-induced responses. Ca(2+) responses to both agents were completely blocked by 1 microM 4-DAMP, an M3 muscarinic receptor antagonist. In the absence of extracellular Ca(2+), both sialogogues induced transient Ca(2+) increase in parotid gland acinar cells. These results suggest that the sialogogues stimulate some common routes via the Ca(2+) signaling in parotid gland acinar cells. We also report a significant difference of Ca(2+) responses in concentration between pilocarpine and cevimeline in parotid gland acinar cells. The different Ca(2+) responses between the sialogogues would explain the different saliva volumes from the parotid gland between them that we have observed in previous in vivo studies.

Pneumococcal surface protein A (PspA) is effective at eliciting T cell-mediated responses during invasive pneumococcal disease in adults

PubMed Central

Baril, L; Dietemann, J; Essevaz-Roulet, M; Béniguel, L; Coan, P; Briles, D E; Guy, B; Cozon, G

2006-01-01

Humoral immune response is essential for protection against invasive pneumococcal disease and this property is the basis of the polysaccharide-based anti-pneumococcal vaccines. Pneumococcal surface protein A (PspA), a cell-wall-associated surface protein, is a promising component for the next generation of pneumococcal vaccines. This PspA antigen has been shown to stimulate an antibody-based immunity. In the present study, we evaluated the capacity of PspA to stimulate CD4+ T cells which are needed for the correct development of a B cell based immune response in humans. Cellular immunity to PspA was evaluated by whole-blood culture with different pneumococcal antigens, followed by flow cytometric detection of activated CD4+CD25+ T cells. T cell-mediated immune responses to recombinant PspA proteins were assessed in acute-phase and convalescent blood from adults with invasive pneumococcal disease and in blood from healthy subjects. All cases had detectable antibodies against PspA on admission. We found that invasive pneumococcal disease induced transient T cell depletion but adaptive immune responses strengthened markedly during convalescence. The increased production of both interleukin (IL)-10 and interferon (IFN)-γ during convalescence suggests that these cytokines may be involved in modulating antibody-based immunity to pneumococcal disease. We demonstrated that PspA is efficient at eliciting T cell immune responses and antibodies to PspA. This study broadens the applicability of recombinant PspA as potent pneumococcal antigen for vaccination against S. pneumoniae. PMID:16879247

Periostin inhibits mechanical stretch-induced apoptosis in osteoblast-like MG-63 cells.

PubMed

Yu, Kai-Wen; Yao, Chung-Chen; Jeng, Jiiang-Huei; Shieh, Hao-Ying; Chen, Yi-Jane

2018-04-01

Appropriate mechanical stress plays an important role in regulating the proliferation and differentiation of osteoblasts, whereas high-level mechanical stress may be harmful and compromise cell survival. Periostin, a matricellular protein, is essential in maintaining functional integrity of bone and collagen-rich connective tissue in response to mechanical stress. This study investigated whether or not high-level mechanical stretch induces cell apoptosis and the regulatory role of periostin in mechanical stretch-induced apoptosis in osteoblastic cells. Osteoblast-like MG-63 cells were seeded onto Bio-Flex I culture plates and subjected to cyclic mechanical stretching (15% elongation, 0.1 Hz) in a Flexercell tension plus system-5000. The same process was applied to cells pre-treated with exogenous human recombinant periostin before mechanical stretching. We used a chromatin condensation and membrane permeability dead cell apoptosis kit to evaluate the stretch-induced cell responses. Expression of caspase-3 and cPARP was examined by immunofluorescent stain and flow cytometry. The expression of periostin in MG-63 cells is involved in the TGF-β signaling pathway. High-level cyclic mechanical stretch induced apoptotic responses in MG-63 osteoblastic cells. The percentages of apoptotic cells and cells expressing cPARP protein increased in the groups of cells subjected to mechanical stretch, but these responses were absent in the presence of exogenous periostin. Our study revealed that high-level mechanical stretch induces apoptotic cell death, and that periostin plays a protective role against mechanical stretch-induced apoptosis in osteoblastic cells. Copyright © 2017. Published by Elsevier B.V.

Effect of chemical mutagens and carcinogens on gene expression profiles in human TK6 cells.

PubMed

Godderis, Lode; Thomas, Reuben; Hubbard, Alan E; Tabish, Ali M; Hoet, Peter; Zhang, Luoping; Smith, Martyn T; Veulemans, Hendrik; McHale, Cliona M

2012-01-01

Characterization of toxicogenomic signatures of carcinogen exposure holds significant promise for mechanistic and predictive toxicology. In vitro transcriptomic studies allow the comparison of the response to chemicals with diverse mode of actions under controlled experimental conditions. We conducted an in vitro study in TK6 cells to characterize gene expression signatures of exposure to 15 genotoxic carcinogens frequently used in European industries. We also examined the dose-responsive changes in gene expression, and perturbation of biochemical pathways in response to these carcinogens. TK6 cells were exposed at 3 dose levels for 24 h with and without S9 human metabolic mix. Since S9 had an impact on gene expression (885 genes), we analyzed the gene expression data from cells cultures incubated with S9 and without S9 independently. The ribosome pathway was affected by all chemical-dose combinations. However in general, no similar gene expression was observed among carcinogens. Further, pathways, i.e. cell cycle, DNA repair mechanisms, RNA degradation, that were common within sets of chemical-dose combination were suggested by clustergram. Linear trends in dose-response of gene expression were observed for Trichloroethylene, Benz[a]anthracene, Epichlorohydrin, Benzene, and Hydroquinone. The significantly altered genes were involved in the regulation of (anti-) apoptosis, maintenance of cell survival, tumor necrosis factor-related pathways and immune response, in agreement with several other studies. Similarly in S9+ cultures, Benz[a]pyrene, Styrene and Trichloroethylene each modified over 1000 genes at high concentrations. Our findings expand our understanding of the transcriptomic response to genotoxic carcinogens, revealing the alteration of diverse sets of genes and pathways involved in cellular homeostasis and cell cycle control.

Effect of Chemical Mutagens and Carcinogens on Gene Expression Profiles in Human TK6 Cells

PubMed Central

Godderis, Lode; Thomas, Reuben; Hubbard, Alan E.; Tabish, Ali M.; Hoet, Peter; Zhang, Luoping; Smith, Martyn T.; Veulemans, Hendrik; McHale, Cliona M.

2012-01-01

Characterization of toxicogenomic signatures of carcinogen exposure holds significant promise for mechanistic and predictive toxicology. In vitro transcriptomic studies allow the comparison of the response to chemicals with diverse mode of actions under controlled experimental conditions. We conducted an in vitro study in TK6 cells to characterize gene expression signatures of exposure to 15 genotoxic carcinogens frequently used in European industries. We also examined the dose-responsive changes in gene expression, and perturbation of biochemical pathways in response to these carcinogens. TK6 cells were exposed at 3 dose levels for 24 h with and without S9 human metabolic mix. Since S9 had an impact on gene expression (885 genes), we analyzed the gene expression data from cells cultures incubated with S9 and without S9 independently. The ribosome pathway was affected by all chemical-dose combinations. However in general, no similar gene expression was observed among carcinogens. Further, pathways, i.e. cell cycle, DNA repair mechanisms, RNA degradation, that were common within sets of chemical-dose combination were suggested by clustergram. Linear trends in dose–response of gene expression were observed for Trichloroethylene, Benz[a]anthracene, Epichlorohydrin, Benzene, and Hydroquinone. The significantly altered genes were involved in the regulation of (anti-) apoptosis, maintenance of cell survival, tumor necrosis factor-related pathways and immune response, in agreement with several other studies. Similarly in S9+ cultures, Benz[a]pyrene, Styrene and Trichloroethylene each modified over 1000 genes at high concentrations. Our findings expand our understanding of the transcriptomic response to genotoxic carcinogens, revealing the alteration of diverse sets of genes and pathways involved in cellular homeostasis and cell cycle control. PMID:22723965

Fluorescence-Activated Cell Sorting-Based Analysis Reveals an Asymmetric Induction of Interferon-Stimulated Genes in Response to Seasonal Influenza A Virus

PubMed Central

von Recum-Knepper, Jessica; Sadewasser, Anne; Weinheimer, Viola K.

2015-01-01

ABSTRACT Influenza A virus (IAV) infection provokes an antiviral response involving the expression of type I and III interferons (IFN) and IFN-stimulated genes (ISGs) in infected cell cultures. However, the spatiotemporal dynamics of the IFN reaction are incompletely understood, as previous studies investigated mainly the population responses of virus-infected cultures, although substantial cell-to-cell variability has been documented. We devised a fluorescence-activated cell sorting-based assay to simultaneously quantify expression of viral antigens and ISGs, such as ISG15, MxA, and IFIT1, in IAV-infected cell cultures at the single-cell level. This approach revealed that seasonal IAV triggers an unexpected asymmetric response, as the major cell populations expressed either viral antigen or ISG, but rarely both. Further investigations identified a role of the viral NS1 protein in blocking ISG expression in infected cells, which surprisingly did not reduce paracrine IFN signaling to noninfected cells. Interestingly, viral ISG control was impaired in cultures infected with avian-origin IAV, including the H7N9 virus from eastern China. This phenotype was traced back to polymorphic NS1 amino acids known to be important for stable binding of the polyadenylation factor CPSF30 and concomitant suppression of host cell gene expression. Most significantly, mutation of two amino acids within the CPSF30 attachment site of NS1 from seasonal IAV diminished the strict control of ISG expression in infected cells and substantially attenuated virus replication. In conclusion, our approach revealed an asymmetric, NS1-dependent ISG induction in cultures infected with seasonal IAV, which appears to be essential for efficient virus propagation. IMPORTANCE Interferons are expressed by infected cells in response to IAV infection and play important roles in the antiviral immune response by inducing hundreds of interferon-stimulated genes (ISGs). Unlike many previous studies, we investigated the ISG response at the single-cell level, enabling novel insights into this virus-host interaction. Hence, cell cultures infected with seasonal IAV displayed an asymmetric ISG induction that was confined almost exclusively to noninfected cells. In comparison, ISG expression was observed in larger cell populations infected with avian-origin IAV, suggesting a more resolute antiviral response to these strains. Strict control of ISG expression by seasonal IAV was explained by the binding of the viral NS1 protein to the polyadenylation factor CPSF30, which reduces host cell gene expression. Mutational disruption of CPSF30 binding within NS1 concomitantly attenuated ISG control and replication of seasonal IAV, illustrating the importance of maintaining an asymmetric ISG response for efficient virus propagation. PMID:25903337

Role of inhibition in the specification of orientation selectivity of cells in the cat striate cortex.

PubMed

Bonds, A B

1989-01-01

Mechanisms supporting orientation selectivity of cat striate cortical cells were studied by stimulation with two superimposed sine-wave gratings of different orientations. One grating (base) generated a discharge of known amplitude which could be modified by the second grating (mask). Masks presented at nonoptimal orientations usually reduced the base-generated response, but the degree of reduction varied widely between cells. Cells with narrow orientation tuning tended to be more susceptible to mask presence than broadly tuned cells; similarly, simple cells generally showed more response reduction than did complex cells. The base and mask stimuli were drifted at different temporal frequencies which, in simple cells, permitted the identification of individual response components from each stimulus. This revealed that the reduction of the base response by the mask usually did not vary regularly with mask orientation, although response facilitation from the mask was orientation selective. In some sharply tuned simple cells, response reduction had clear local maxima near the limits of the cell's orientation-tuning function. Response reduction resulted from a nearly pure rightward shift of the response versus log contrast function. The lowest mask contrast yielding reduction was within 0.1-0.3 log unit of the lowest contrast effective for excitation. The temporal-frequency bandpass of the response-reduction mechanism resembled that of most cortical cells. The spatial-frequency bandpass was much broader than is typical for single cortical cells, spanning essentially the entire visual range of the cat. These findings are compatible with a model in which weak intrinsic orientation-selective excitation is enhanced in two stages: (1) control of threshold by nonorientation-selective inhibition that is continuously dependent on stimulus contrast; and (2) in the more narrowly tuned cells, orientation-selective inhibition that has local maxima serving to increase the slope of the orientation-tuning function.

Regulatory T cells in cattle and their potential role in bovine paratuberculosis.

PubMed

Coussens, Paul M; Sipkovsky, Sue; Murphy, Brooke; Roussey, Jon; Colvin, Christopher J

2012-05-01

The intracellular bacterium Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in wild and domestic ruminants. Johne's disease presents as a chronic enteritis with severe inflammation of intestinal tissues, characterized by widespread infiltration of macrophages, the target cell of MAP. Clinical signs of Johne's disease are typically accompanied by a loss of peripheral CD4+ T cell responses to MAP antigens and an increase in anti-MAP serum IgG levels. Recently, it was proposed that regulatory T cells might develop over the lengthy course of subclinical MAP infection. In the past five years, significant progress in defining bovine regulatory T cells has been made. These studies grew out of observations that IL-10 is produced by PBMCs in response to MAP antigen stimulation and that neutralization of this IL-10 could enhance IFN-γ production from MAP-antigen reactive effector T cells. Depletion studies revealed that MAP responsive cell populations producing IL-10 were largely CD4+ and CD25+, although monocytes have also been shown to produce IL-10 in response to MAP. In addition, evidence for a regulatory population of γδ T cells has also begun to accumulate. We summarize current thinking regarding regulatory T cells in MAP infection and provide data suggesting a potential link between regulatory T cells, bovine leukemia virus, and MAP. Copyright © 2012 Elsevier Ltd. All rights reserved.

Immunostimulatory Activity of the Cytokine-Based Biologic, IRX-2, on Human Papillomavirus-Exposed Langerhans Cells

PubMed Central

Da Silva, Diane M.; Woodham, Andrew W.; Naylor, Paul H.; Egan, James E.; Berinstein, Neil L.

2016-01-01

Langerhans cells (LCs) are the antigen-presenting cells of the epithelial layer and are responsible for initiating immune responses against skin and mucosa-invading viruses. Human papillomavirus (HPV)-mediated suppression of LC function is a crucial mechanism of HPV immune evasion, which can lead to persistent infection and development of several human cancers, including cervical, anal, and head and neck cancers. The cell-derived cytokine-based biologic, IRX-2, consists of multiple well-defined cytokines and is broadly active on various immune cell subsets. In this study, we investigated primary human LC activation after exposure to HPV16, followed by treatment with IRX-2 in vitro, and evaluated their subsequent ability to induce HPV16-specific T cells. In contrast to its activity on dendritic cells, HPV16 alone is not sufficient to induce phenotypic and functional activation of LCs. However, IRX-2 induces a significant upregulation of antigen presentation and costimulatory molecules, T helper 1 (Th1)-associated cytokine release, and chemokine-directed migration of LCs pre-exposed to HPV16. Furthermore, LCs treated with IRX-2 after HPV16 exposure induced CD8+ T-cell responses against specific HLA-A*0201-binding HPV16 T-cell epitopes. The present study suggests that IRX-2 is an attractive immunomodulator for assisting the immune response in eradication of HPV-infected cells, thereby potentially preventing HPV-induced cancers. PMID:26653678

Immunostimulatory Activity of the Cytokine-Based Biologic, IRX-2, on Human Papillomavirus-Exposed Langerhans Cells.

PubMed

Da Silva, Diane M; Woodham, Andrew W; Naylor, Paul H; Egan, James E; Berinstein, Neil L; Kast, W Martin

2016-05-01

Langerhans cells (LCs) are the antigen-presenting cells of the epithelial layer and are responsible for initiating immune responses against skin and mucosa-invading viruses. Human papillomavirus (HPV)-mediated suppression of LC function is a crucial mechanism of HPV immune evasion, which can lead to persistent infection and development of several human cancers, including cervical, anal, and head and neck cancers. The cell-derived cytokine-based biologic, IRX-2, consists of multiple well-defined cytokines and is broadly active on various immune cell subsets. In this study, we investigated primary human LC activation after exposure to HPV16, followed by treatment with IRX-2 in vitro, and evaluated their subsequent ability to induce HPV16-specific T cells. In contrast to its activity on dendritic cells, HPV16 alone is not sufficient to induce phenotypic and functional activation of LCs. However, IRX-2 induces a significant upregulation of antigen presentation and costimulatory molecules, T helper 1 (Th1)-associated cytokine release, and chemokine-directed migration of LCs pre-exposed to HPV16. Furthermore, LCs treated with IRX-2 after HPV16 exposure induced CD8(+) T-cell responses against specific HLA-A*0201-binding HPV16 T-cell epitopes. The present study suggests that IRX-2 is an attractive immunomodulator for assisting the immune response in eradication of HPV-infected cells, thereby potentially preventing HPV-induced cancers.

Intravenous infusion of apoptotic cells simultaneously with allogeneic hematopoietic grafts alters anti-donor humoral immune responses.

PubMed

Perruche, Sylvain; Kleinclauss, François; Bittencourt, Marcelo de Carvalho; Paris, Dominique; Tiberghien, Pierre; Saas, Philippe

2004-08-01

Intravenous infusion of apoptotic donor or third-party leukocytes simultaneously with an allogeneic donor bone marrow (BM) graft favors engraftment across major histocompatibility barriers. While verifying that such apoptotic cell infusion might not also be associated with antibody (Ab)-mediated allo-immune responses, we found, rather strikingly, that apoptotic cell infusion could in fact successfully prevent a humoral allo-immunization against a BM graft in mice. Indeed, among recipients having rejected their BM graft, prior apoptotic cell infusion was associated with a near absence of Ab-mediated allo-responses, while such an immunization was frequently observed in the absence of apoptotic cell infusion. This was also observed when infusing host apoptotic cells, thus showing that the prevention of immunization was linked to the apoptotic state of the cells rather than mediated by residual anti-recipient activity. In vivo anti-transforming growth factor-beta (TGF-beta) treatment resulted in the loss of this apoptotic cell infusion-associated protective effect on humoral allo-responses. Further studies will determine whether apoptotic cell infusion, in addition to hematopoietic graft facilitation might also contribute to preventing deleterious Ab-mediated allo-responses in various transplantation settings.

Dendritic Cell Immune Responses in HIV-1 Controllers.

PubMed

Martin-Gayo, Enrique; Yu, Xu G

2017-02-01

Robust HIV-1-specific CD8 T cell responses are currently regarded as the main correlate of immune defense in rare individuals who achieve natural, drug-free control of HIV-1; however, the mechanisms that support evolution of such powerful immune responses are not well understood. Dendritic cells (DCs) are specialized innate immune cells critical for immune recognition, immune regulation, and immune induction, but their possible contribution to HIV-1 immune defense in controllers remains ill-defined. Recent studies suggest that myeloid DCs from controllers have improved abilities to recognize HIV-1 through cytoplasmic immune sensors, resulting in more potent, cell-intrinsic type I interferon secretion in response to viral infection. This innate immune response may facilitate DC-mediated induction of highly potent antiviral HIV-1-specific T cells. Moreover, protective HLA class I isotypes restricting HIV-1-specific CD8 T cells may influence DC function through specific interactions with innate myelomonocytic MHC class I receptors from the leukocyte immunoglobulin-like receptor family. Bi-directional interactions between dendritic cells and HIV-1-specific T cells may contribute to natural HIV-1 immune control, highlighting the importance of a fine-tuned interplay between innate and adaptive immune activities for effective antiviral immune defense.

Preserved immune functionality and high CMV-specific T-cell responses in HIV-infected individuals with poor CD4+ T-cell immune recovery.

PubMed

Gómez-Mora, Elisabet; García, Elisabet; Urrea, Victor; Massanella, Marta; Puig, Jordi; Negredo, Eugenia; Clotet, Bonaventura; Blanco, Julià; Cabrera, Cecilia

2017-09-15

Poor CD4 + T-cell recovery after cART has been associated with skewed T-cell maturation, inflammation and immunosenescence; however, T-cell functionality in those individuals has not been fully characterized. In the present study, we assessed T-cell function by assessing cytokine production after polyclonal, CMV and HIV stimulations of T-cells from ART-suppressed HIV-infected individuals with CD4 + T-cell counts >350 cells/μL (immunoconcordants) or <350 cells/μL (immunodiscordants). A group of HIV-uninfected individuals were also included as controls. Since CMV co-infection significantly affected T-cell maturation and polyfunctionality, only CMV + individuals were analyzed. Despite their reduced and skewed CD4 + T-cell compartment, immunodiscordant individuals showed preserved polyclonal and HIV-specific responses. However, CMV response in immunodiscordant participants was significantly different from immunoconcordant or HIV-seronegative individuals. In immunodiscordant subjects, the magnitude of IFN-γ + CD8 + and IL-2 + CD4 + T-cells in response to CMV was higher and differently associated with the CD4 + T-cell maturation profile., showing an increased frequency of naïve, central memory and EMRA CMV-specific CD4 + T-cells. In conclusion, CD4 + and CD8 + T-cell polyfunctionality was not reduced in immunodiscordant individuals, although heightened CMV-specific immune responses, likely related to subclinical CMV reactivations, may be contributing to the skewed T-cell maturation and the higher risk of clinical progression observed in those individuals.

Mesenchymal stem cells induce dermal fibroblast responses to injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Andria N., E-mail: snosmith@u.washington.edu; Willis, Elise, E-mail: elise.willis@gmail.com; Chan, Vincent T.

2010-01-01

Although bone marrow-derived mesenchymal stem cells have been shown to promote repair when applied to cutaneous wounds, the mechanism for this response remains to be determined. The aim of this study was to determine the effects of paracrine signaling from mesenchymal stem cells on dermal fibroblast responses to injury including proliferation, migration and expression of genes important in wound repair. Dermal fibroblasts were co-cultured with bone marrow-derived mesenchymal stem cells grown in inserts, which allowed for paracrine interactions without direct cell contact. In this co-culture model, bone marrow-derived mesenchymal stem cells regulate dermal fibroblast proliferation, migration and gene expression. Whenmore » co-cultured with mesenchymal stem cells, dermal fibroblasts show increased proliferation and accelerated migration in a scratch assay. A chemotaxis assay also demonstrated that dermal fibroblasts migrate towards bone marrow-derived mesenchymal stem cells. A PCR array was used to analyze the effect of mesenchymal stem cells on dermal fibroblast gene expression. In response to mesenchymal stem cells, dermal fibroblasts up-regulate integrin alpha 7 expression and down-regulate expression of ICAM1, VCAM1 and MMP11. These observations suggest that mesenchymal stem cells may provide an important early signal for dermal fibroblast responses to cutaneous injury.« less

Long-term antibody memory induced by synthetic peptide vaccination is protective against Streptococcus pyogenes infection and is independent of memory T-cell help

PubMed Central

Pandey, Manisha; Wykes, Michelle N; Hartas, Jon; Good, Michael F; Batzloff, Michael R

2013-01-01

Streptococcus pyogenes (group A streptococcus; GAS) is a leading human pathogen associated with a diverse array of mucosal and systemic infections. Vaccination with J8, a conserved region synthetic peptide derived from the M-protein of GAS and containing only 12 amino acids from GAS, when conjugated to DT, has been shown to protect mice against a lethal GAS challenge. Protection has been previously shown to be antibody-mediated. J8 does not contain a dominant GAS-specific T-cell epitope. The current study examined long-term antibody memory and dissected the role of B and T-cells. Our results demonstrated that vaccination generates specific memory B-cells and long-lasting antibody responses. The memory B-cell response can be activated following boost with antigen or limiting numbers of whole bacteria. We further show that these memory responses protect against systemic infection with GAS. T-cell help is required for activation of memory B-cells but can be provided by naïve T-cells responding directly to GAS at the time of infection. Thus, individuals whose T-cells do not recognize the short synthetic peptide in the vaccine will be able to generate a protective and rapid memory antibody response at the time of infection. These studies significantly strengthen previous findings, which showed that protection by the J8-DT vaccine is antibody-mediated and suggest that in vaccine design for other organisms the source of T-cell help for antibody responses need not be limited to sequences from the organism itself. PMID:23401589

A Pharmacogenetic Approach to Identify Mutant Forms of α-Galactosidase A that Respond to a Pharmacological Chaperone for Fabry Disease

PubMed Central

Wu, Xiaoyang; Katz, Evan; Valle, Maria Cecilia Della; Mascioli, Kirsten; Flanagan, John J; Castelli, Jeffrey P; Schiffmann, Raphael; Boudes, Pol; Lockhart, David J; Valenzano, Kenneth J; Benjamin, Elfrida R

2011-01-01

Fabry disease is caused by mutations in the gene (GLA) that encodes α-galactosidase A (α-Gal A). The iminosugar AT1001 (GR181413A, migalastat hydrochloride, 1-deoxygalactonojirimycin) is a pharmacological chaperone that selectively binds and stabilizes α-Gal A, increasing total cellular levels and activity for some mutant forms (defined as “responsive”). In this study, we developed a cell-based assay in cultured HEK-293 cells to identify mutant forms of α-Gal A that are responsive to AT1001. Concentration-dependent increases in α-Gal A activity in response to AT1001 were shown for 49 (60%) of 81 mutant forms. The responses of α-Gal A mutant forms were generally consistent with the responses observed in male Fabry patient-derived lymphoblasts. Importantly, the HEK-293 cell responses of 19 α-Gal A mutant forms to a clinically achievable concentration of AT1001 (10 µM) were generally consistent with observed increases in α-Gal A activity in peripheral blood mononuclear cells from male Fabry patients orally administered AT1001 during Phase 2 clinical studies. This indicates that the cell-based responses can identify mutant forms of α-Gal A that are likely to respond to AT1001 in vivo. Thus, the HEK-293 cell-based assay may be a useful aid in the identification of Fabry patients with AT1001-responsive mutant forms. Hum Mutat 32:1–13, 2011. © 2011 Wiley-Liss, Inc. PMID:21598360

Characterization of guinea pig T cell responses elicited after EP-assisted delivery of DNA vaccines to the skin

PubMed Central

Schultheis, Katherine; Schaefer, Hubert; Yung, Bryan S.; Oh, Janet; Muthumani, Karuppiah; Humeau, Laurent; Broderick, Kate E.

2016-01-01

The skin is an ideal target tissue for vaccine delivery for a number of reasons. It is highly accessible, and most importantly, enriched in professional antigen presenting cells. Possessing strong similarities to human skin physiology and displaying a defined epidermis, the guinea pig is an appropriate model to study epidermal delivery of vaccine. However, whilst we have characterized the humoral responses in the guinea pig associated with skin vaccine protocols we have yet to investigate the T cell responses. In response to this inadequacy, we developed an IFN-γ ELISpot assay to characterize the cellular immune response in the peripheral blood of guinea pigs. Using a nucleoprotein (NP) influenza pDNA vaccination regimen, we characterized host T cell responses. After delivery of the DNA vaccine to the guinea pig epidermis we detected robust and rapid T cell responses. The levels of IFN-γ spot-forming units averaged approximately 5000 per million cells after two immunizations. These responses were broad in that multiple regions across the NP antigen elicited a T cell response. Interestingly, we identified a number of NP immunodominant T cell epitopes to be conserved across an outbred guinea pig population, a phenomenon which was also observed after immunization with a RSV DNA vaccine. We believe this data enhances our understanding of the cellular immune response elicited to a vaccine in guinea pigs, and globally, will advance the use of this model for vaccine development, especially those targeting skin as a delivery site. PMID:27894716

Characterization of guinea pig T cell responses elicited after EP-assisted delivery of DNA vaccines to the skin.

PubMed

Schultheis, Katherine; Schaefer, Hubert; Yung, Bryan S; Oh, Janet; Muthumani, Karuppiah; Humeau, Laurent; Broderick, Kate E; Smith, Trevor R F

2017-01-03

The skin is an ideal target tissue for vaccine delivery for a number of reasons. It is highly accessible, and most importantly, enriched in professional antigen presenting cells. Possessing strong similarities to human skin physiology and displaying a defined epidermis, the guinea pig is an appropriate model to study epidermal delivery of vaccine. However, whilst we have characterized the humoral responses in the guinea pig associated with skin vaccine protocols we have yet to investigate the T cell responses. In response to this inadequacy, we developed an IFN-γ ELISpot assay to characterize the cellular immune response in the peripheral blood of guinea pigs. Using a nucleoprotein (NP) influenza pDNA vaccination regimen, we characterized host T cell responses. After delivery of the DNA vaccine to the guinea pig epidermis we detected robust and rapid T cell responses. The levels of IFN-γ spot-forming units averaged approximately 5000 per million cells after two immunizations. These responses were broad in that multiple regions across the NP antigen elicited a T cell response. Interestingly, we identified a number of NP immunodominant T cell epitopes to be conserved across an outbred guinea pig population, a phenomenon which was also observed after immunization with a RSV DNA vaccine. We believe this data enhances our understanding of the cellular immune response elicited to a vaccine in guinea pigs, and globally, will advance the use of this model for vaccine development, especially those targeting skin as a delivery site. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Motility of vestibular hair cells in the chick.

PubMed

Ogata, Y; Sekitani, T

1993-01-01

Recent studies of the outer hair cells in cochlea have demonstrated active motilities. However, very little study has been done on the vestibular hair cells (VHCs). The present study shows the motile response of the VHCs induced by application of Ca2+/ATP promoting contraction. Reversible cell shape changes could be shown in 10 of 16 isolated type I hair cells and 9 of 15 isolated type II hair cells by applying the contraction solution. Furthermore, the sensory hair bundles in the utricular epithelium pivoted around the base and stood perpendicularly to the apical borderline of the epithelium in response to the application of the same solution. It is suggested that the contraction of the isolated VHCs may be transferred to tension which causes the sensory hair bundles to restrict their motion in normal tissue, instead of changing the cell shape.

Loss of the ex vivo but not the reinducible CD8+ T-cell response to Tax in human T-cell leukemia virus type 1-infected patients with adult T-cell leukemia/lymphoma.

PubMed

Arnulf, B; Thorel, M; Poirot, Y; Tamouza, R; Boulanger, E; Jaccard, A; Oksenhendler, E; Hermine, O; Pique, C

2004-01-01

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy (HAM). In asymptomatic carriers and HAM patients, HTLV-1 infection leads to a vigorous cytotoxic T-cell (CTL) response mainly directed to the regulatory Tax protein. In contrast, initial studies showed that anti-HTLV-1 CTL activities were not reproductively detected in ATLL patients, neither ex vivo, nor after in vitro restimulation. To better understand this discrepancy, we explored the anti-HTLV-1 CD8+ T-cell response of eight ATLL patients by using in vitro restimulated or freshly isolated CD8+ T cells. In all the ATLL patients, we found that mitogenic activation allowed the induction of CD8+ T cells able to lyse autologous HTLV-1-infected cells and/or to produce IFNgamma in response to Tax peptides. In contrast, only a minority of the patients possessed CD8+ cells able to respond ex vivo to the same epitopes. These findings indicate that although a restimulatable anti-HTLV-1 CTL activity persists during ATLL, the specific ex vivo response is not constantly maintained. This provides definitive evidence that the CD8+ T-cell response to HTLV-1 is affected by ATLL development and reveals that a major defect concerns the generation and/or the functionality of CD8+ effectors.

Microengineering as a tool to study substratum modulation and cell behaviour.

PubMed

Keatch, R P; Armoogum, K; Schor, S L; Pridham, M S; Banks, K; Khor, T Y; Matthew, C

2002-01-01

This research is an investigation of the means by which geometrical parameters (e.g. area and shape) and various surface attributes (materials and surface finish) of microengineered structures can modulate cellular response. This is based on biological observations indicating that: (i) the response of tissue cells to injury is determined by the net signal transduction response elicited by soluble regulatory molecules (e.g. cytokines), (ii) common matrix constituents (e.g. collagen) directly affect cell behaviour by the same signal transduction mechanisms mediating cytokine bioactivity, (iii) cellular response to cytokines is modulated by the precise nature of the extracellular matrix to which the target cells are adherent, including its biochemical composition and physical structure.

Phenytoin promotes Th2 type immune response in mice

PubMed Central

Okada, K; Sugiura, T; Kuroda, E; Tsuji, S; Yamashita, U

2001-01-01

The effects of chronic administration of phenytoin, a common anticonvulsive drug, on immune responses were studied in mice. Anti-keyhole limpet haemocyanin (KLH) IgE antibody response after KLH-immunization was enhanced in phenytoin-treated mice. Proliferative responses of spleen cells induced with KLH, concanavalin A (ConA), lipopolysaccharide and anti-CD3 antibody were reduced in phenytoin-treated mice. Accessory function of spleen adherent cells on ConA-induced T cell proliferative response was reduced in phenytoin-treated mice. KLH-induced IL-4 production of spleen cells was enhanced, while IFN-γ production was reduced in phenytoin-treated mice. In addition, production of IL-1α, but not IL-6 and IL-12 by spleen adherent cells from phenytoin-treated mice was reduced. Natural killer cell activity was reduced in phenytoin-treated mice. These results suggest that phenytoin treatment preferentially induces a Th2 type response. We also observed that plasma ACTH and corticosterone levels were increased in phenytoin-treated mice, and speculated that phenytoin might act directly and indirectly, through HPA axis activation, on the immune system to modulate Th1/Th2 balance. PMID:11472401

Human Memory CD4+ T Cell Immune Responses against Giardia lamblia

PubMed Central

Sørnes, Steinar; Peirasmaki, Dimitra; Svärd, Staffan; Langeland, Nina

2015-01-01

The intestinal protozoan parasite Giardia lamblia may cause severe prolonged diarrheal disease or pass unnoticed as an asymptomatic infection. T cells seem to play an important role in the immune response to Giardia infection, and memory responses may last years. Recently, TH17 responses have been found in three animal studies of Giardia infection. The aim of this study was to characterize the human CD4+ T cell responses to Giardia. Peripheral blood mononuclear cells (PBMCs) were obtained from 21 returning travelers with recent or ongoing giardiasis and 12 low-risk healthy controls and stimulated in vitro with Giardia lamblia proteins. Production of tumor necrosis factor alpha (TNF-α), gamma interferon, interleukin-17A (IL-17A), IL-10, and IL-4 was measured in CD4+ effector memory (EM) T cells after 24 h by flow cytometry. After 6 days of culture, activation and proliferation were measured by flow cytometry, while an array of inflammatory cytokine levels in supernatants were measured with multiplex assays. We found the number of IL-17A-producing CD4+ EM T cells, as well as that of cells simultaneously producing both IL-17A and TNF-α, to be significantly elevated in the Giardia-exposed individuals after 24 h of antigen stimulation. In supernatants of PBMCs stimulated with Giardia antigens for 6 days, we found inflammation-associated cytokines, including 1L-17A, as well as CD4+ T cell activation and proliferation, to be significantly elevated in the Giardia-exposed individuals. We conclude that symptomatic Giardia infection in humans induces a CD4+ EM T cell response of which IL-17A production seems to be an important component. PMID:26376930

Pharmacological characterization of extracellular acidification rate responses in human D2(long), D3 and D4.4 receptors expressed in Chinese hamster ovary cells

PubMed Central

Coldwell, M C; Boyfield, I; Brown, A M; Stemp, G; Middlemiss, D N

1999-01-01

This study characterized pharmacologically the functional responses to agonists at human dopamine D2(long) (hD2), D3 (hD3) and D4.4 (hD4) zreceptors separately expressed in cloned cells using the cytosensor microphysiometer. Dopaminergic receptor agonists caused increases in extracellular acidification rate in adherent Chinese hamster ovary (CHO) clones expressing hD2, hD3 or hD4 receptors. Acidification rate responses to agonists in other cell lines expressing these receptors were smaller than those in adherent CHO cells. The time courses and maximum increases in acidification rate of the agonist responses in adherent CHO cells were different between the three dopamine receptor clones. Responses were blocked by pretreatment of cells with pertussis toxin or amiloride analogues. Most agonists had full intrinsic activity at each of the dopamine receptor subtypes, as compared to quinpirole, however both enantiomers of UH-232 and (−)3-PPP were partial agonists in this assay system. The functional potency of full agonists at each of the three receptors expressed in CHO cells was either higher than, or similar to, the apparent inhibition constants (Ki) determined in [125I]-iodosulpride competition binding studies. Functional selectivities of the agonists were less than radioligand binding selectivities. The rank orders of agonist potencies and selectivities were similar, but not identical, to the rank orders of radioligand binding affinities and selectivities. The dopamine receptor antagonists, iodosulpride and clozapine, had no effect on basal acidification rates but inhibited acidification responses in CHO cells to quinpirole in an apparently competitive manner. Antagonist potencies closely matched their radioligand binding affinities in these cells. PMID:10455259

The use of flow cytometry to examine calcium signalling by TRPV1 in mixed cell populations.

PubMed

Assas, Bakri M; Abdulaal, Wesam H; Wakid, Majed H; Zakai, Haytham A; Miyan, J; Pennock, J L

2017-06-15

Flow cytometric analysis of calcium mobilisation has been in use for many years in the study of specific receptor engagement or isolated cell:cell communication. However, calcium mobilisation/signaling is key to many cell functions including apoptosis, mobility and immune responses. Here we combine multiplex surface staining of whole spleen with Indo-1 AM to visualise calcium mobilisation and examine calcium signaling in a mixed immune cell culture over time. We demonstrate responses to a TRPV1 agonist in distinct cell subtypes without the need for cell separation. Multi parameter staining alongside Indo-1 AM to demonstrate calcium mobilization allows the study of real time calcium signaling in a complex environment. Copyright © 2017. Published by Elsevier Inc.

Trichomonas vaginalis α-Actinin 2 Modulates Host Immune Responses by Inducing Tolerogenic Dendritic Cells via IL-10 Production from Regulatory T Cells.

PubMed

Lee, Hye-Yeon; Kim, Juri; Ryu, Jae-Sook; Park, Soon-Jung

2017-08-01

Trichomonas vaginalis is a pathogen that triggers severe immune responses in hosts. T. vaginalis α-actinin 2, Tvα-actinin 2, has been used to diagnose trichomoniasis. This study was undertaken to examine the role of Tvα-actinin 2 as an antigenic molecule to induce immune responses from humans. Western blot analysis using anti-Tvα-actinin 2 antibodies indicated its presence in the secreted proteins of T. vaginalis. ELISA was employed to measure cytokine production by vaginal epithelial cells, prostate cells, mouse dendritic cells (DCs), or T cells stimulated with T. vaginalis or Tvα-actinin 2 protein. Both T. vaginalis and rTvα-actinin 2 induced cytokine production from epithelial cell lines, including IL-10. Moreover, CD4+CD25- regulatory T cells (Treg cells) incubated with rTvα-actinin 2-treated DCs produced high levels of IL-10. These data indicate that Tvα-actinin 2 modulates immune responses via IL-10 production by Treg cells.

Monitoring the Systemic Human Memory B Cell Compartment of Melanoma Patients for Anti-Tumor IgG Antibodies

PubMed Central

Gilbert, Amy E.; Karagiannis, Panagiotis; Dodev, Tihomir; Koers, Alexander; Lacy, Katie; Josephs, Debra H.; Takhar, Pooja; Geh, Jenny L. C.; Healy, Ciaran; Harries, Mark; Acland, Katharine M.; Rudman, Sarah M.; Beavil, Rebecca L.; Blower, Philip J.; Beavil, Andrew J.; Gould, Hannah J.; Spicer, James; Nestle, Frank O.; Karagiannis, Sophia N.

2011-01-01

Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells. We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (P<0.0001). Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21) (P<0.0001). Overall, 28% of melanoma patient-derived B cell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells. Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer. PMID:21559411

Verification of immune response optimality through cybernetic modeling.

PubMed

Batt, B C; Kompala, D S

1990-02-09

An immune response cascade that is T cell independent begins with the stimulation of virgin lymphocytes by antigen to differentiate into large lymphocytes. These immune cells can either replicate themselves or differentiate into plasma cells or memory cells. Plasma cells produce antibody at a specific rate up to two orders of magnitude greater than large lymphocytes. However, plasma cells have short life-spans and cannot replicate. Memory cells produce only surface antibody, but in the event of a subsequent infection by the same antigen, memory cells revert rapidly to large lymphocytes. Immunologic memory is maintained throughout the organism's lifetime. Many immunologists believe that the optimal response strategy calls for large lymphocytes to replicate first, then differentiate into plasma cells and when the antigen has been nearly eliminated, they form memory cells. A mathematical model incorporating the concept of cybernetics has been developed to study the optimality of the immune response. Derived from the matching law of microeconomics, cybernetic variables control the allocation of large lymphocytes to maximize the instantaneous antibody production rate at any time during the response in order to most efficiently inactivate the antigen. A mouse is selected as the model organism and bacteria as the replicating antigen. In addition to verifying the optimal switching strategy, results showing how the immune response is affected by antigen growth rate, initial antigen concentration, and the number of antibodies required to eliminate an antigen are included.

Natural killer cells regulate T cell immune responses in primary biliary cirrhosis.

PubMed

Shimoda, Shinji; Hisamoto, Satomi; Harada, Kenichi; Iwasaka, Sho; Chong, Yong; Nakamura, Minoru; Bekki, Yuki; Yoshizumi, Tomoharu; Shirabe, Ken; Ikegami, Toru; Maehara, Yoshihiko; He, Xiao-Song; Gershwin, M Eric; Akashi, Koichi

2015-12-01

The hallmark of primary biliary cirrhosis (PBC) is the presence of autoreactive T- and B-cell responses that target biliary epithelial cells (BECs). Biliary cell cytotoxicity is dependent upon initiation of innate immune responses followed by chronic adaptive, as well as bystander, mechanisms. Critical to these mechanisms are interactions between natural killer (NK) cells and BECs. We have taken advantage of the ability to isolate relatively pure viable preparations of liver-derived NK cells, BECs, and endothelial cells, and studied interactions between NK cells and BECs and focused on the mechanisms that activate autoreactive T cells, their dependence on interferon (IFN)-γ, and expression of BEC major histocompatibility complex (MHC) class I and II molecules. Here we show that at a high NK/BEC ratio, NK cells are cytotoxic for autologous BECs, but are not dependent on autoantigen, yet still activate autoreactive CD4(+) T cells in the presence of antigen presenting cells. In contrast, at a low NK/BEC ratio, BECs are not lysed, but IFN-γ production is induced, which facilitates expression of MHC class I and II molecules on BEC and protects them from lysis upon subsequent exposure to autoreactive NK cells. Furthermore, IFN-γ secreted from NK cells after exposure to autologous BECs is essential for this protective function and enables autoreactive CD4(+) T cells to become cytopathic. NK cell-mediated innate immune responses are likely critical at the initial stage of PBC, but also facilitate and maintain the chronic cytopathic effect of autoantigen-specific T cells, essential for progression of disease. © 2015 by the American Association for the Study of Liver Diseases.

Nasal Epithelial Cells as Surrogates for Bronchial Epithelial Cells in Airway Inflammation Studies

PubMed Central

McDougall, Catherine M.; Blaylock, Morgan G.; Douglas, J. Graham; Brooker, Richard J.; Helms, Peter J.; Walsh, Garry M.

2008-01-01

The nose is an attractive source of airway epithelial cells, particularly in populations in which bronchoscopy may not be possible. However, substituting nasal cells for bronchial epithelial cells in the study of airway inflammation depends upon comparability of responses, and evidence for this is lacking. Our objective was to determine whether nasal epithelial cell inflammatory mediator release and receptor expression reflect those of bronchial epithelial cells. Paired cultures of undifferentiated nasal and bronchial epithelial cells were obtained from brushings from 35 subjects, including 5 children. Cells were subject to morphologic and immunocytochemical assessment. Mediator release from resting and cytokine-stimulated cell monolayers was determined, as was cell surface receptor expression. Nasal and bronchial cells had identical epithelial morphology and uniform expression of cytokeratin 19. There were no differences in constitutive expression of CD44, intercellular adhesion molecule-1, αvβ3, and αvβ5. Despite significantly higher constitutive release of IL-8, IL-6, RANTES (regulated on activation, normal T cell expressed and secreted), and matrix metalloproteinase (MMP)-9 from nasal compared with bronchial cells, the increments in release of all studied mediators in response to stimulation with IL-1β and TNF-α were similar, and there were significant positive correlations between nasal and bronchial cell secretion of IL-6, RANTES, vascular endothelial growth factor, monocyte chemoattractant protein-1, MMP-9, and tissue inhibitor of metalloproteinase-1. Despite differences in absolute mediator levels, the responses of nasal and bronchial epithelial cells to cytokine stimulation were similar, expression of relevant surface receptors was comparable, and there were significant correlations between nasal and bronchial cell mediator release. Therefore, nasal epithelial cultures constitute an accessible surrogate for studying lower airway inflammation. PMID:18483420

Nasal epithelial cells as surrogates for bronchial epithelial cells in airway inflammation studies.

PubMed

McDougall, Catherine M; Blaylock, Morgan G; Douglas, J Graham; Brooker, Richard J; Helms, Peter J; Walsh, Garry M

2008-11-01

The nose is an attractive source of airway epithelial cells, particularly in populations in which bronchoscopy may not be possible. However, substituting nasal cells for bronchial epithelial cells in the study of airway inflammation depends upon comparability of responses, and evidence for this is lacking. Our objective was to determine whether nasal epithelial cell inflammatory mediator release and receptor expression reflect those of bronchial epithelial cells. Paired cultures of undifferentiated nasal and bronchial epithelial cells were obtained from brushings from 35 subjects, including 5 children. Cells were subject to morphologic and immunocytochemical assessment. Mediator release from resting and cytokine-stimulated cell monolayers was determined, as was cell surface receptor expression. Nasal and bronchial cells had identical epithelial morphology and uniform expression of cytokeratin 19. There were no differences in constitutive expression of CD44, intercellular adhesion molecule-1, alphavbeta3, and alphavbeta5. Despite significantly higher constitutive release of IL-8, IL-6, RANTES (regulated on activation, normal T cell expressed and secreted), and matrix metalloproteinase (MMP)-9 from nasal compared with bronchial cells, the increments in release of all studied mediators in response to stimulation with IL-1beta and TNF-alpha were similar, and there were significant positive correlations between nasal and bronchial cell secretion of IL-6, RANTES, vascular endothelial growth factor, monocyte chemoattractant protein-1, MMP-9, and tissue inhibitor of metalloproteinase-1. Despite differences in absolute mediator levels, the responses of nasal and bronchial epithelial cells to cytokine stimulation were similar, expression of relevant surface receptors was comparable, and there were significant correlations between nasal and bronchial cell mediator release. Therefore, nasal epithelial cultures constitute an accessible surrogate for studying lower airway inflammation.

Response of Human Prostate Cancer Cells to Mitoxantrone Treatment in Simulated Microgravity Environment

NASA Astrophysics Data System (ADS)

Zhang, Ye; Wu, Honglu

2012-07-01

RESPONSE OF HUMAN PROSTATE CANCER CELLS TO MITOXANTRONE TREATMENT IN SIMULATED MICROGRAVITY ENVIRONMENT Ye Zhang1,2, Christopher Edwards3, and Honglu Wu1 1 NASA-Johnson Space Center, Houston, TX 2 Wyle Integrated Science and Engineering Group, Houston, TX 3 Oregon State University, Corvallis, OR This study explores the changes in growth of human prostate cancer cells (LNCaP) and their response to the treatment of an antineoplastic agent, mitoxantrone, under the simulated microgravity condition. In comparison to static 1g, microgravity and simulated microgravity have been shown to alter global gene expression patterns and protein levels in various cultured cell models or animals. However, very little is known about the effect of altered gravity on the responses of cells to the treatment of drugs, especially chemotherapy drugs. To test the hypothesis that zero gravity would result in altered regulations of cells in response to antineoplastic agents, we cultured LNCaP cells in either a High Aspect Ratio Vessel (HARV) bioreactor at the rotating condition to model microgravity in space or in the static condition as control, and treated the cells with mitoxantrone. Cell growth, as well as expressions of oxidative stress related genes, were analyzed after the drug treatment. Compared to static 1g controls, the cells cultured in the simulated microgravity environment did not present significant differences in cell viability, growth rate, or cell cycle distribution. However, after mitoxantrone treatment, a significant proportion of bioreactor cultured cells became apoptotic or was arrested in G2. Several oxidative stress related genes also showed a higher expression level post mitoxantrone treatment. Our results indicate that simulated microgravity may alter the response of LNCaP cells to mitoxantrone treatment. Understanding the mechanisms by which cells respond to drugs differently in an altered gravity environment will be useful for the improvement of cancer treatment on the ground. This study explores the changes in growth of human prostate cancer cells (LNCaP) and their response to the treatment of an antineoplastic agent, mitoxantrone, under the simulated microgravity condition. In comparison to static 1g, microgravity and simulated microgravity have been shown to alter global gene expression patterns and protein levels in various cultured cell models or animals. However, very little is known about the effect of altered gravity on the responses of cells to the treatment of drugs, especially chemotherapy drugs. To test the hypothesis that zero gravity would result in altered regulations of cells in response to antineoplastic agents, we cultured LNCaP cells in either a High Aspect Ratio Vessel (HARV) bioreactor at the rotating condition to model microgravity in space or in the static condition as control, and treated the cells with mitoxantrone. Cell growth, as well as expressions of oxidative stress related genes, were analyzed after the drug treatment. Compared to static 1g controls, the cells cultured in the simulated microgravity environment did not present significant differences in cell viability, growth rate, or cell cycle distribution. However, after mitoxantrone treatment, a significant proportion of bioreactor cultured cells became apoptotic or was arrested in G2. Several oxidative stress related genes also showed a higher expression level post mitoxantrone treatment. Our results indicate that simulated microgravity may alter the response of LNCaP cells to mitoxantrone treatment. Understanding the mechanisms by which cells respond to drugs differently in an altered gravity environment will be useful for the improvement of cancer treatment on the ground.

Cell-like features imprinted in the physical nano- and micro-topography of the environment modify the responses to anti-cancer drugs of endometrial cancer cells.

PubMed

Tan, Li Hui; Sykes, Peter H; Alkaisi, Maan M; Evans, John J

2017-02-14

Topographical features of cells at nanometre resolution were fabricated in polystyrene. The study investigated the effect of physical topography on the response of cancer cells to the common anticancer drugs, paclitaxel and doxorubicin. Human endometrial cancer cells (Ishikawa) were incubated on substrates containing cell-like features that had been fabricated using our bioimprint methodology to create moulds of cells with positive (convex) and negative (concave) topography. Control cultures were performed on flat substrates. Effects of the drugs on caspase-3 expression, proliferating nuclear antigen (PCNA) expression, cell number and vascular endothelial growth factor (VEGF) secretion were determined. Results revealed that the topography influenced the cell responses in a drug-dependent manner i.e. paclitaxel effects were sensitive to topography differently to those of doxorubicin. In addition, function signalling pathways were sensitive to the detailed topography i.e. positive imprint and negative imprint induced distinct response patterns. The results in this study show for the first time that a culture surface with cell-like topography, that has both nano- and micro-resolution, influences endometrial cancer cell responses to chemotherapy drugs. The effects are dependent on the topography and also on the chemotherapy drug. In particular, the platforms described have potential to provide substrates with high physical relevancy on which to undertake preclinical testing of new drugs. The method also allows for use of different cell types to provide cell-specific topography. The results imply that physical architecture of the cancer cell environment may be a suitable prospective target to enhance clinical activity of traditional drugs. Additionally or alternatively we provide compelling support for the notion that understanding the physical component of the nano- and micro-environment may encourage a redirection of drug development. Further, our observation that the cells distinguish between the different cell-like topographies (positive and negative bioimprints) indicates that a realistic topography is advantageous as growth platforms in experiment design.

Identical Strength of the T Cell Responses against E2, nsP1 and Capsid CHIKV Proteins in Recovered and Chronic Patients after the Epidemics of 2005-2006 in La Reunion Island

PubMed Central

Pellé, Olivier; Samri, Assia; Jaffar-Bandjee, Marie-Christine; Gasque, Philippe; Autran, Brigitte

2013-01-01

To characterize the immunity developed by patients infected by chikungunya virus (CHIKV), we studied the intensity and specificity of CHIKV-specific T cells mediated responses in chronic and recovered patients at 12 to 24 months post-infection. T cells were challenged in vitro against CHIKV synthetic peptides covering the length of three viral proteins, capsid, E2 and nsP1 proteins as well as all inactivated virus particles. Cytokine production was assessed by ELISPOT and intracellular labeling. T cells producing IFN-γ were detected against CHIKV in 85% patient’s cells either by direct ELISPOT assay (69% of patients) or after expansion of memory T cells allowing the detection of both CD4 and CD8 specific-T cells in 16% additional cases. The IFN-γ response was mainly engaged in response to nsP1 or E2 (52% and 46% cases, respectively) but in only 27% cases against the capsid. The anti-E2 response represented half the magnitude of the total CHIKV IFN-γ production and was mainly directed against the C-terminal half part of the protein. Almost all patients had conserved a T cell specific response against CHIKV with a clear hierarchy of T cell responses (CD8 > CD4) engaged against E2 > nsP1 > capsid. More importantly, the intensity of responses was not significantly different between recovered and chronic patients. These findings constitute key elements to a better understanding of patient T cell immunoreactivity against CHIKV and argue against a possible defect of T cell immunoresponse in the chronicity post-CHIKV infection. PMID:24376836

Zoledronic acid induces dose-dependent increase of antigen-specific CD8 T-cell responses in combination with peptide/poly-IC vaccine.

PubMed

Park, Hye-Mi; Cho, Hyun-Il; Shin, Chang-Ae; Shon, Hyun-Jung; Kim, Tai-Gyu

2016-03-04

Zoledronic acid (ZA) is used for treating osteoporosis and for preventing skeletal fractures in cancer patients suffering from myeloma and prostate cancer. It is also reported to directly induce cancer cell apoptosis and indirectly modulate T-cell immune response as an antitumor agent. In this study, the effect of ZA following peptide/polyinosinic-polycytidylic acid (poly-IC) vaccination was investigated in a murine tumor model. The combination of ZA with peptide/poly-IC vaccine showed a synergistic effect on the induction of antigen-specific CD8 T-cell response. Three consecutive intravenous administrations of ZA was defined to induce the highest CD8 T-cell response. Further, total splenocyte counts and antigen-specific CD8 T-cell response gradually increased depending on the dose of ZA. In tumor-bearing mice, ZA showed a dose-dependent decrease of growth and prolonged survival. Treatment with ZA only decreased the number of CD11b(+)Gr1(+) myeloid cells in blood. Our results demonstrate that the use of ZA could improve antitumor immune responses induced by the peptide/poly-IC vaccine. Copyright © 2016 Elsevier Ltd. All rights reserved.

An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi

PubMed Central

Jin, Chenggang; Roen, Diana R.; Lehmann, Paul V.; Kellermann, Gottfried H.

2013-01-01

Lyme Borreliosis is an infectious disease caused by the spirochete Borrelia burgdorferi that is transmitted through the bite of infected ticks. Both B cell-mediated humoral immunity and T cell immunity develop during natural Borrelia infection. However, compared with humoral immunity, the T cell response to Borrelia infection has not been well elucidated. In this study, a novel T cell-based assay was developed and validated for the sensitive detection of antigen-specific T cell response to B. burgdorferi. Using interferon-γ as a biomarker, we developed a new enzyme-linked immunospot method (iSpot LymeTM) to detect Borrelia antigen-specific effector/memory T cells that were activated in vivo by exposing them to recombinant Borrelia antigens ex vivo. To test this new method as a potential laboratory diagnostic tool, we performed a clinical study with a cohort of Borrelia positive patients and healthy controls. We demonstrated that the iSpot Lyme assay has a significantly higher specificity and sensitivity compared with the Western Blot assay that is currently used as a diagnostic measure. A comprehensive evaluation of the T cell response to Borrelia infection should, therefore, provide new insights into the pathogenesis, diagnosis, treatment and monitoring of Lyme disease. PMID:24709800

Inflammatory Response and Barrier Dysfunction by Different e-Cigarette Flavoring Chemicals Identified by Gas Chromatography–Mass Spectrometry in e-Liquids and e-Vapors on Human Lung Epithelial Cells and Fibroblasts

PubMed Central

Gerloff, Janice; Sundar, Isaac K.; Freter, Robert; Sekera, Emily R.; Friedman, Alan E.; Robinson, Risa; Pagano, Todd

2017-01-01

Abstract Recent studies suggest that electronic cigarette (e-cig) flavors can be harmful to lung tissue by imposing oxidative stress and inflammatory responses. The potential inflammatory response by lung epithelial cells and fibroblasts exposed to e-cig flavoring chemicals in addition to other risk-anticipated flavor enhancers inhaled by e-cig users is not known. The goal of this study was to evaluate the release of the proinflammatory cytokine (interleukin-8 [IL-8]) and epithelial barrier function in response to different e-cig flavoring chemicals identified in various e-cig e-liquid flavorings and vapors by chemical characterization using gas chromatography–mass spectrometry analysis. Flavorings, such as acetoin (butter), diacetyl, pentanedione, maltol (malt), ortho-vanillin (vanilla), coumarin, and cinnamaldehyde in comparison with tumor necrosis factor alpha (TNFα), were used in this study. Human bronchial epithelial cells (Beas2B), human mucoepidermoid carcinoma epithelial cells (H292), and human lung fibroblasts (HFL-1) were treated with each flavoring chemical for 24 hours. The cells and conditioned media were then collected and analyzed for toxicity (viability %), lung epithelial barrier function, and proinflammatory cytokine IL-8 release. Cell viability was not significantly affected by any of the flavoring chemicals tested at a concentration of 10 μM to 1 mM. Acetoin and diacetyl treatment induced IL-8 release in Beas2B cells. Acetoin- and pentanedione-treated HFL-1 cells produced a differential, but significant response for IL-8 release compared to controls and TNFα. Flavorings, such as ortho-vanillin and maltol, induced IL-8 release in Beas2B cells, but not in H292 cells. Of all the flavoring chemicals tested, acetoin and maltol were more potent inducers of IL-8 release than TNFα in Beas2B and HFL-1 cells. Flavoring chemicals rapidly impaired epithelial barrier function in human bronchial epithelial cells (16-HBE) as measured by electric cell surface impedance sensing. Our findings suggest that some of the e-cig liquids/aerosols containing flavoring chemicals can cause significant loss of epithelial barrier function and proinflammatory response in lung cells. PMID:28337465

Different subsets of natural killer T cells may vary in their roles in health and disease

PubMed Central

Kumar, Vipin; Delovitch, Terry L

2014-01-01

Natural killer T cells (NKT) can regulate innate and adaptive immune responses. Type I and type II NKT cell subsets recognize different lipid antigens presented by CD1d, an MHC class-I-like molecule. Most type I NKT cells express a semi-invariant T-cell receptor (TCR), but a major subset of type II NKT cells reactive to a self antigen sulphatide use an oligoclonal TCR. Whereas TCR-α dominates CD1d-lipid recognition by type I NKT cells, TCR-α and TCR-β contribute equally to CD1d-lipid recognition by type II NKT cells. These variable modes of NKT cell recognition of lipid–CD1d complexes activate a host of cytokine-dependent responses that can either exacerbate or protect from disease. Recent studies of chronic inflammatory and autoimmune diseases have led to a hypothesis that: (i) although type I NKT cells can promote pathogenic and regulatory responses, they are more frequently pathogenic, and (ii) type II NKT cells are predominantly inhibitory and protective from such responses and diseases. This review focuses on a further test of this hypothesis by the use of recently developed techniques, intravital imaging and mass cytometry, to analyse the molecular and cellular dynamics of type I and type II NKT cell antigen-presenting cell motility, interaction, activation and immunoregulation that promote immune responses leading to health versus disease outcomes. PMID:24428389

Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

NASA Technical Reports Server (NTRS)

Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Casey, Rachael; Wu, Honglu

2008-01-01

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in DSB repair, and its impact on cytogenetic responses has not been systematically studied. In the present study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by transfection with small interfering RNA in human fibroblast cells. The purpose of this study is to identify new roles of these selected genes on regulating DSB repair and cell cycle progression , as measured in the micronuclei formation and chromosome aberration. In response to IR, the formation of MN was significantly increased by suppressed expression of 5 genes: Ku70 in the DSB repair pathway, XPA in the NER pathway, RPA1 in the MMR pathway, and RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, P21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Most of the 11 genes that affected cytogenetic responses are not known to have clear roles influencing DBS repair. Nine of these 11 genes were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate the biological consequences after IR.

CD8+ T Cells Provide an Immunologic Signature of Tuberculosis in Young Children

PubMed Central

Nyendak, Melissa; Kiguli, Sarah; Zalwango, Sarah; Mori, Tomi; Mayanja-Kizza, Harriet; Balyejusa, Stephen; Null, Megan; Baseke, Joy; Mulindwa, Deo; Byrd, Laura; Swarbrick, Gwendolyn; Scott, Christine; Johnson, Denise F.; Malone, LaShaunda; Mudido-Musoke, Philipa; Boom, W. Henry; Lewinsohn, David M.; Lewinsohn, Deborah A.

2012-01-01

Rationale: The immunologic events surrounding primary Mycobacterium tuberculosis infection and development of tuberculosis remain controversial. Young children who develop tuberculosis do so quickly after first exposure, thus permitting study of immune response to primary infection and disease. We hypothesized that M. tuberculosis–specific CD8+ T cells are generated in response to high bacillary loads occurring during tuberculosis. Objectives: To determine if M. tuberculosis–specific T cells are generated among healthy children exposed to M. tuberculosis and children with tuberculosis. Methods: Enzyme-linked immunosorbent spot assays were used to measure IFN-γ production in response to M. tuberculosis–specific proteins ESAT-6/CFP-10 by peripheral blood mononuclear cells and CD8+ T cells isolated from Ugandan children hospitalized with tuberculosis (n = 96) or healthy tuberculosis contacts (n = 62). Measurements and Main Results: The proportion of positive CD8+ T-cell assays and magnitude of CD8+ T-cell responses were significantly greater among young (<5 yr) tuberculosis cases compared with young contacts (P = 0.02, Fisher exact test, P = 0.01, Wilcoxon rank-sum, respectively). M. tuberculosis–specific T-cell responses measured in peripheral blood mononuclear cells were equivalent between groups. Conclusions: Among young children, M. tuberculosis–specific CD8+ T cells develop in response to high bacillary loads, as occurs during tuberculosis, and are unlikely to be found after M. tuberculosis exposure. T-cell responses measured in peripheral blood mononuclear cells are generated after M. tuberculosis exposure alone, and thus cannot distinguish exposure from disease. In young children, IFN-γ–producing M. tuberculosis–specific CD8+ T cells provide an immunologic signature of primary M. tuberculosis infection resulting in disease. PMID:22071329

Tim-3 directly enhances CD8 T cell responses to acute Listeria monocytogenes infection

PubMed Central

Gorman, Jacob V.; Starbeck-Miller, Gabriel; Pham, Nhat-Long L.; Traver, Geri L.; Rothman, Paul B.; Harty, John T.; Colgan, John D.

2014-01-01

Tim-3 is a surface molecule expressed throughout the immune system that can mediate both stimulatory and inhibitory effects. Previous studies have provided evidence that Tim-3 functions to enforce CD8 T cell exhaustion, a dysfunctional state associated with chronic stimulation. In contrast, the role of Tim-3 in the regulation of CD8 T cell responses to acute and transient stimulation remains undefined. To address this knowledge gap, we examined how Tim-3 affects CD8 T cell responses to acute Listeria monocytogenes (LM) infection. Analysis of wild-type (WT) mice infected with LM revealed that Tim-3 was transiently expressed by activated CD8 T cells and was associated primarily with acquisition of an effector phenotype. Comparison of responses to LM by WT and Tim-3 KO mice showed that the absence of Tim-3 significantly reduced the magnitudes of both primary and secondary CD8 T cell responses, which correlated with decreased IFN-γ production and degranulation by Tim-3 KO cells stimulated with peptide antigen ex vivo. To address the T cell-intrinsic role of Tim-3, we analyzed responses to LM infection by WT and Tim-3 KO TCR-transgenic CD8 T cells following adoptive transfer into a shared WT host. In this setting, the accumulation of CD8 T cells and the generation of cytokine-producing cells were significantly reduced by the lack of Tim-3, demonstrating that this molecule has a direct effect on CD8 T cell function. Combined, our results suggest that Tim-3 can mediate a stimulatory effect on CD8 T cell responses to an acute infection. PMID:24567532

Fabrication of thermo-responsive PNIPAAm-g-ETFE for cell culture dishes by pre-irradiation grafting

NASA Astrophysics Data System (ADS)

Yamahara, Yumi; Nagasawa, Naotsugu; Taguchi, Mitsumasa; Oshima, Akihiro; Washio, Masakazu

2018-01-01

Thermo-responsive templates for the cell cultivation based on Poly(tetrafluoroethylene-co-ethylene) (ETFE) were fabricated by pre-irradiation grafting of N-isoproplyacrylamide (NIPAAm) monomer by electron beam (EB) irradiation under nitrogen gas atmosphere at room temperature, and their characteristic properties were studied. The detachment of cultured HeLa cells from fabricated thermo-responsive templates were attempted. Furthermore, the reaction mechanism is proposed using ESR spectroscopy and FT-IR spectroscopy. It is confirmed that the cultured HeLa cells were detached from fabricated thermo-responsive templates at 20 °C. Water contact angle analysis indicated that obtained templates had thermo-response around 30 °C. It is suggested that the grafted polymer chains would mainly react with peroxy radicals (-CF2-CF(OO･)-) on tetrafluoroethylene unit in ETFE.

ONC201 selectively induces apoptosis in cutaneous T-cell lymphoma cells via activating pro-apoptotic integrated stress response and inactivating JAK/STAT and NF-κB pathways

PubMed Central

Ni, Xiao; Zhang, Xiang; Hu, Cheng-Hui; Langridge, Timothy; Tarapore, Rohinton S.; Allen, Joshua E.; Oster, Wolfgang; Duvic, Madeleine

2017-01-01

Cutaneous T-cell lymphomas (CTCLs) are extremely symptomatic and still incurable, and more effective and less toxic therapies are urgently needed. ONC201, an imipridone compound, has shown efficacy in pre-clinical studies in multiple advanced cancers. This study was to evaluate the anti-tumor activity of ONC201 on CTCL cells. The effect of ONC201 on the cell growth and apoptosis were evaluated in CTCL cell lines (n=8) and primary CD4+ malignant T cells isolated from CTCL patients (n=5). ONC201 showed a time-dependent cell growth inhibition in all treated cell lines with a concentration range of 1.25-10.0 μM. ONC201 also induced apoptosis in tested cells with a narrow concentration range of 2.5-10.0 μM, evidenced by increased Annexin V+ cells, accompanied by accumulated sub-G1 portions. ONC201 only induced apoptosis in CD4+ malignant T cells, not in normal CD4+ T cells. The activating transcription factor 4 (ATF4), a hallmark of integrated stress response, was upregulated in response to ONC201 whereas Akt was downregulated. In addition, molecules in JAK/STAT and NF-κB pathways, as well as IL-32β, were downregulated following ONC201 treatment. Thus, ONC201 exerts a potent and selective anti-tumor effect on CTCL cells. Its efficacy may involve activating integrated stress response through ATF4 and inactivating JAK/STAT and NF-κB pathways. PMID:28977902

ONC201 selectively induces apoptosis in cutaneous T-cell lymphoma cells via activating pro-apoptotic integrated stress response and inactivating JAK/STAT and NF-κB pathways.

PubMed

Ni, Xiao; Zhang, Xiang; Hu, Cheng-Hui; Langridge, Timothy; Tarapore, Rohinton S; Allen, Joshua E; Oster, Wolfgang; Duvic, Madeleine

2017-09-22

Cutaneous T-cell lymphomas (CTCLs) are extremely symptomatic and still incurable, and more effective and less toxic therapies are urgently needed. ONC201, an imipridone compound, has shown efficacy in pre-clinical studies in multiple advanced cancers. This study was to evaluate the anti-tumor activity of ONC201 on CTCL cells. The effect of ONC201 on the cell growth and apoptosis were evaluated in CTCL cell lines (n=8) and primary CD4 + malignant T cells isolated from CTCL patients (n=5). ONC201 showed a time-dependent cell growth inhibition in all treated cell lines with a concentration range of 1.25-10.0 μM. ONC201 also induced apoptosis in tested cells with a narrow concentration range of 2.5-10.0 μM, evidenced by increased Annexin V + cells, accompanied by accumulated sub-G1 portions. ONC201 only induced apoptosis in CD4 + malignant T cells, not in normal CD4 + T cells. The activating transcription factor 4 (ATF4), a hallmark of integrated stress response, was upregulated in response to ONC201 whereas Akt was downregulated. In addition, molecules in JAK/STAT and NF-κB pathways, as well as IL-32β, were downregulated following ONC201 treatment. Thus, ONC201 exerts a potent and selective anti-tumor effect on CTCL cells. Its efficacy may involve activating integrated stress response through ATF4 and inactivating JAK/STAT and NF-κB pathways.

Counterbalancing Regulation in Response Memory of a Positively Autoregulated Two-Component System.

PubMed

Gao, Rong; Godfrey, Katherine A; Sufian, Mahir A; Stock, Ann M

2017-09-15

Fluctuations in nutrient availability often result in recurrent exposures to the same stimulus conditions. The ability to memorize the past event and use the "memory" to make adjustments to current behaviors can lead to a more efficient adaptation to the recurring stimulus. A short-term phenotypic memory can be conferred via carryover of the response proteins to facilitate the recurrent response, but the additional accumulation of response proteins can lead to a deviation from response homeostasis. We used the Escherichia coli PhoB/PhoR two-component system (TCS) as a model system to study how cells cope with the recurrence of environmental phosphate (Pi) starvation conditions. We discovered that "memory" of prior Pi starvation can exert distinct effects through two regulatory pathways, the TCS signaling pathway and the stress response pathway. Although carryover of TCS proteins can lead to higher initial levels of transcription factor PhoB and a faster initial response in prestarved cells than in cells not starved, the response enhancement can be overcome by an earlier and greater repression of promoter activity in prestarved cells due to the memory of the stress response. The repression counterbalances the carryover of the response proteins, leading to a homeostatic response whether or not cells are prestimulated. A computational model based on sigma factor competition was developed to understand the memory of stress response and to predict the homeostasis of other PhoB-regulated response proteins. Our insight into the history-dependent PhoBR response may provide a general understanding of how TCSs respond to recurring stimuli and adapt to fluctuating environmental conditions. IMPORTANCE Bacterial cells in their natural environments experience scenarios that are far more complex than are typically replicated in laboratory experiments. The architectures of signaling systems and the integration of multiple adaptive pathways have evolved to deal with such complexity. In this study, we examined the molecular "memory" that is generated by previous exposure to stimulus. Under our experimental conditions, activating effects of autoregulated two-component signaling and inhibitory effects of the stress response counterbalanced the transcriptional output to approach response homeostasis whether or not cells had been preexposed to stimulus. Modeling allows prediction of response behavior in different scenarios and demonstrates both the robustness of the system output and its sensitivity to historical parameters such as timing and levels of exposure to stimuli. Copyright © 2017 American Society for Microbiology.

Counterbalancing Regulation in Response Memory of a Positively Autoregulated Two-Component System

PubMed Central

Gao, Rong; Godfrey, Katherine A.; Sufian, Mahir A.

2017-01-01

ABSTRACT Fluctuations in nutrient availability often result in recurrent exposures to the same stimulus conditions. The ability to memorize the past event and use the “memory” to make adjustments to current behaviors can lead to a more efficient adaptation to the recurring stimulus. A short-term phenotypic memory can be conferred via carryover of the response proteins to facilitate the recurrent response, but the additional accumulation of response proteins can lead to a deviation from response homeostasis. We used the Escherichia coli PhoB/PhoR two-component system (TCS) as a model system to study how cells cope with the recurrence of environmental phosphate (Pi) starvation conditions. We discovered that “memory” of prior Pi starvation can exert distinct effects through two regulatory pathways, the TCS signaling pathway and the stress response pathway. Although carryover of TCS proteins can lead to higher initial levels of transcription factor PhoB and a faster initial response in prestarved cells than in cells not starved, the response enhancement can be overcome by an earlier and greater repression of promoter activity in prestarved cells due to the memory of the stress response. The repression counterbalances the carryover of the response proteins, leading to a homeostatic response whether or not cells are prestimulated. A computational model based on sigma factor competition was developed to understand the memory of stress response and to predict the homeostasis of other PhoB-regulated response proteins. Our insight into the history-dependent PhoBR response may provide a general understanding of how TCSs respond to recurring stimuli and adapt to fluctuating environmental conditions. IMPORTANCE Bacterial cells in their natural environments experience scenarios that are far more complex than are typically replicated in laboratory experiments. The architectures of signaling systems and the integration of multiple adaptive pathways have evolved to deal with such complexity. In this study, we examined the molecular “memory” that is generated by previous exposure to stimulus. Under our experimental conditions, activating effects of autoregulated two-component signaling and inhibitory effects of the stress response counterbalanced the transcriptional output to approach response homeostasis whether or not cells had been preexposed to stimulus. Modeling allows prediction of response behavior in different scenarios and demonstrates both the robustness of the system output and its sensitivity to historical parameters such as timing and levels of exposure to stimuli. PMID:28674072

Quantitative Analyses of Synergistic Responses between Cannabidiol and DNA-Damaging Agents on the Proliferation and Viability of Glioblastoma and Neural Progenitor Cells in Culture.

PubMed

Deng, Liting; Ng, Lindsay; Ozawa, Tatsuya; Stella, Nephi

2017-01-01

Evidence suggests that the nonpsychotropic cannabis-derived compound, cannabidiol (CBD), has antineoplastic activity in multiple types of cancers, including glioblastoma multiforme (GBM). DNA-damaging agents remain the main standard of care treatment available for patients diagnosed with GBM. Here we studied the antiproliferative and cell-killing activity of CBD alone and in combination with DNA-damaging agents (temozolomide, carmustine, or cisplatin) in several human GBM cell lines and in mouse primary GBM cells in cultures. This activity was also studied in mouse neural progenitor cells (NPCs) in culture to assess for potential central nervous system toxicity. We found that CBD induced a dose-dependent reduction of both proliferation and viability of all cells with similar potencies, suggesting no preferential activity for cancer cells. Hill plot analysis indicates an allosteric mechanism of action triggered by CBD in all cells. Cotreatment regimens combining CBD and DNA-damaging agents produced synergistic antiproliferating and cell-killing responses over a limited range of concentrations in all human GBM cell lines and mouse GBM cells as well as in mouse NPCs. Remarkably, antagonistic responses occurred at low concentrations in select human GBM cell lines and in mouse GBM cells. Our study suggests limited synergistic activity when combining CBD and DNA-damaging agents in treating GBM cells, along with little to no therapeutic window when considering NPCs. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Invariant NKT Cells Regulate the CD8 T Cell Response during Theiler's Virus Infection

PubMed Central

Mars, Lennart T.; Mas, Magali; Beaudoin, Lucie; Bauer, Jan; Leite-de-Moraes, Maria; Lehuen, Agnès; Bureau, Jean-Francois; Liblau, Roland S.

2014-01-01

Invariant NKT cells are innate lymphocytes with a broad tissue distribution. Here we demonstrate that iNKT cells reside in the central nervous system (CNS) in the absence of inflammation. Their presence in the CNS dramatically augments following inoculation of C57Bl/6 mice with the neurotropic Theiler's murine encephalomyelitis virus (TMEV). At the peak of inflammation the cellular infiltrate comprises 45 000 iNKT cells for 1 250 CD8 T cells specific for the immunodominant TMEV epitope. To study the interaction between these two T cell subsets, we infected both iNKT cell deficient Jα18-/- mice and iNKT cell enriched Vα14 transgenic mice with TMEV. The CD8 T cell response readily cleared TMEV infection in the iNKT cell deficient mice. However, in the iNKT cell enriched mice TMEV infection persisted and was associated with significant mortality. This was caused by the inhibition of the CD8 T cell response in the cervical lymph nodes and spleen after T cell priming. Taken together we demonstrate that iNKT cells reside in the CNS in the absence of inflammation and that their enrichment is associated with the inhibition of the anti-viral CD8 T cell response and an augmented mortality during acute encephalomyelitis. PMID:24498175

Relationship between Functional Profile of HIV-1 Specific CD8 T Cells and Epitope Variability with the Selection of Escape Mutants in Acute HIV-1 Infection

PubMed Central

Goonetilleke, Nilu; Liu, Michael K. P.; Turnbull, Emma L.; Salazar-Gonzalez, Jesus F.; Hawkins, Natalie; Self, Steve; Watson, Sydeaka; Betts, Michael R.; Gay, Cynthia; McGhee, Kara; Pellegrino, Pierre; Williams, Ian; Tomaras, Georgia D.; Haynes, Barton F.; Gray, Clive M.; Borrow, Persephone; Roederer, Mario; McMichael, Andrew J.; Weinhold, Kent J.

2011-01-01

In the present study, we analyzed the functional profile of CD8+ T-cell responses directed against autologous transmitted/founder HIV-1 isolates during acute and early infection, and examined whether multifunctionality is required for selection of virus escape mutations. Seven anti-retroviral therapy-naïve subjects were studied in detail between 1 and 87 weeks following onset of symptoms of acute HIV-1 infection. Synthetic peptides representing the autologous transmitted/founder HIV-1 sequences were used in multiparameter flow cytometry assays to determine the functionality of HIV-1-specific CD8+ T memory cells. In all seven patients, the earliest T cell responses were predominantly oligofunctional, although the relative contribution of multifunctional cell responses increased significantly with time from infection. Interestingly, only the magnitude of the total and not of the poly-functional T-cell responses was significantly associated with the selection of escape mutants. However, the high contribution of MIP-1β-producing CD8+ T-cells to the total response suggests that mechanisms not limited to cytotoxicity could be exerting immune pressure during acute infection. Lastly, we show that epitope entropy, reflecting the capacity of the epitope to tolerate mutational change and defined as the diversity of epitope sequences at the population level, was also correlated with rate of emergence of escape mutants. PMID:21347345

DNA sequence responsible for the amplification of adjacent genes.

PubMed

Pasion, S G; Hartigan, J A; Kumar, V; Biswas, D K

1987-10-01

A 10.3-kb DNA fragment in the 5'-flanking region of the rat prolactin (rPRL) gene was isolated from F1BGH(1)2C1, a strain of rat pituitary tumor cells (GH cells) that produces prolactin in response to 5-bromodeoxyuridine (BrdU). Following transfection and integration into genomic DNA of recipient mouse L cells, this DNA induced amplification of the adjacent thymidine kinase gene from Herpes simplex virus type 1 (HSV1TK). We confirmed the ability of this "Amplicon" sequence to induce amplification of other linked or unlinked genes in DNA-mediated gene transfer studies. When transferred into the mouse L cells with the 10.3-5'rPRL gene sequence of BrdU-responsive cells, both the human growth hormone and the HSV1TK genes are amplified in response to 5-bromodeoxyuridine. This observation is substantiated by BrdU-induced amplification of the cotransferred bacterial Neo gene. Cotransfection studies reveal that the BrdU-induced amplification capability is associated with a 4-kb DNA sequence in the 5'-flanking region of the rPRL gene of BrdU-responsive cells. These results demonstrate that genes of heterologous origin, linked or unlinked, and selected or unselected, can be coamplified when located within the amplification boundary of the Amplicon sequence.

Development of New Photovoltaic Devices Based on Multi Wall Carbon Nanotubes and Nanoparticles

DTIC Science & Technology

2013-03-01

response is registered in all the photon spectral range studied. The new kind of Graetzel-like solar cell device was built without dye and TiO2 , showing...response is registered in all the photon spectral range studied. - The new kind of Graetzel (DSSC, Dye Synthesized Solar Cell ) built without Dye and TiO2 ...an IPCE up to 20%. 15. SUBJECT TERMS EOARD, organic solar cells , photovoltaics, carbon nanotubes 16. SECURITY CLASSIFICATION

Clonal expansion under the microscope: studying lymphocyte activation and differentiation using live-cell imaging.

PubMed

Polonsky, Michal; Chain, Benjamin; Friedman, Nir

2016-03-01

Clonal expansion of lymphocytes is a hallmark of vertebrate adaptive immunity. A small number of precursor cells that recognize a specific antigen proliferate into expanded clones, differentiate and acquire various effector and memory phenotypes, which promote effective immune responses. Recent studies establish a large degree of heterogeneity in the level of expansion and in cell state between and within expanding clones. Studying these processes in vivo, while providing insightful information on the level of heterogeneity, is challenging due to the complex microenvironment and the inability to continuously track individual cells over extended periods of time. Live cell imaging of ex vivo cultures within micro fabricated arrays provides an attractive methodology for studying clonal expansion. These experiments facilitate continuous acquisition of a large number of parameters on cell number, proliferation, death and differentiation state, with single-cell resolution on thousands of expanding clones that grow within controlled environments. Such data can reveal stochastic and instructive mechanisms that contribute to observed heterogeneity and elucidate the sequential order of differentiation events. Intercellular interactions can also be studied within these arrays by following responses of a controlled number of interacting cells, all trapped within the same microwell. Here we describe implementations of live-cell imaging within microwell arrays for studies of lymphocyte clonal expansion, portray insights already gained from these experiments and outline directions for future research. These tools, together with in vivo experiments tracking single-cell responses, will expand our understanding of adaptive immunity and the ways by which it can be manipulated.

Antigen-specific IgA B memory cell responses to Shigella antigens elicited in volunteers immunized with live attenuated Shigella flexneri 2a oral vaccine candidates

PubMed Central

Simon, J. K.; Maciel, M.; Weld, E.D.; Wahid, R.; Pasetti, M.F.; Picking, W.L.; Kotloff, K. L.; Levine, M. M.; Sztein, M. B.

2011-01-01

We studied the induction of antigen-specific IgA memory B cells (BM) in volunteers who received live attenuated Shigella flexneri 2a vaccines. Subjects ingested a single oral dose of 107, 108 or 109 CFU of S. flexneri 2a with deletions in guaBA (CVD 1204) or in guaBA, set and sen (CVD 1208). Antigen-specific serum and stool antibody responses to LPS and Ipa B were measured on days 0, 7, 14, 28 and 42. IgA BM cells specific to LPS, Ipa B and total IgA were assessed on days 0 and 28. We show the induction of significant LPS-specific IgA BM cells in anti-LPS IgA seroresponders. Positive correlations were found between anti-LPS IgA BM cells and anti-LPS IgA in serum and stool; IgA BM cell responses to IpaB were also observed. These BM cell responses are likely play an important role in modulating the magnitude and longevity of the humoral response. PMID:21388888

Vaccine-elicited SIV and HIV envelope-specific IgA and IgG memory B cells in rhesus macaque peripheral blood correlate with functional antibody responses and reduced viremia

PubMed Central

Brocca-Cofano, Egidio; McKinnon, Katherine; Demberg, Thorsten; Venzon, David; Hidajat, Rachmat; Xiao, Peng; Daltabuit-Test, Mara; Patterson, L. Jean; Robert-Guroff, Marjorie

2011-01-01

An effective HIV vaccine requires strong systemic and mucosal, cellular and humoral immunity. Numerous non-human primate studies have investigated memory T cells, but not memory B cells. Humoral immunologic memory is mediated by long-lived antibody-secreting plasma cells and differentiation of memory B cells into short-lived plasma blasts following re-exposure to immunizing antigen. Here we studied memory B cells in vaccinated rhesus macaques. PBMC were stimulated polyclonally using CD40 Ligand, IL-21 and CpG to induce B cell proliferation and differentiation into antibody secreting cells (ASC). Flow cytometry was used for phenotyping and evaluating proliferation by CFSE dilution. B cell responses were quantified by ELISPOT. Methodology was established using PBMC of vaccinated elite-controller macaques that exhibited strong, multi-functional antibody activities. Subsequently, memory B cells elicited by two replicating Ad-recombinant prime/envelope boost regimens were retrospectively evaluated pre- and post- SIV and SHIV challenges. The vaccine regimens induced SIV and HIV Env-specific IgG and IgA memory B cells. Prior to challenge, IgA memory B cells were more numerous than IgG memory B cells, reflecting the mucosal priming immunizations. Pre- and post-challenge memory B cells were correlated with functional antibody responses including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated viral inhibition (ADCVI) and transcytosis inhibition. Post-challenge, Env-specific IgG and IgA memory B cells were correlated with reduced chronic viremia. We conclude that functional antibody responses elicited by our prime/boost regimen were effectively incorporated into the memory B cell pool where they contributed to control of viremia following re-exposure to the immunizing antigen. PMID:21382487

Immune Responses to HIV and SIV in Mucosal Tissues: “Location, Location, Location”

PubMed Central

Shacklett, Barbara L.

2010-01-01

Purpose of review This review summarizes research literature regarding mucosal immunity to HIV and SIV, with an emphasis on work published within the past 18 months. Recent findings Notable recent studies have focused on the pivotal events occurring within mucosal tissues during acute HIV/SIV infection that serve to establish a balance between detrimental immune activation and beneficial adaptive responses. In cervicovaginal mucosa, an early inflammatory response leads to recruitment of susceptible target cells. At this acute stage, the in vivo ratio between CD8+ effector cells and infected CD4+ T-cells may be critical for limiting viral dissemination. Acute infection is also accompanied by loss of germinal center architecture and T/B cell apoptosis in Peyer’s patches of the gastrointestinal tract. During chronic infection, mucosal CD8+ T-cells may play a role in immune control, as suggested by studies of elite controllers. Summary Mucosal tissues serve as the major portal of entry for HIV, and house a majority of the body’s lymphocytes, including CD4+ T-cells that are targets for infection. Recent studies have focused renewed attention on events occurring immediately after transmission, and underscore the concept that the balance between inflammation and protective immunity is established by host responses in mucosal tissues. PMID:20543589

Synthesis of Nanoscale TiO2 and Study of the Effect of Their Crystal Structure on Single Cell Response

PubMed Central

Ismagilov, Z. R.; Shikina, N. V.; Mazurkova, N. A.; Tsikoza, L. T.; Tuzikov, F. V.; Ushakov, V. A.; Ishchenko, A. V.; Rudina, N. A.; Korneev, D. V.; Ryabchikova, E. I.

2012-01-01

To study the effect of nanoscale titanium dioxide (TiO2) on cell responses, we synthesized four modifications of the TiO2 (amorphous, anatase, brookite, and rutile) capable of keeping their physicochemical characteristics in a cell culture medium. The modifications of nanoscale TiO2 were obtained by hydrolysis of TiCl4 and Ti(i-OC3H7)4 (TIP) upon variation of the synthesis conditions; their textural, morphological, structural, and dispersion characteristics were examined by a set of physicochemical methods: XRD, BET, SAXS, DLS, AFM, SEM, and HR-TEM. The effect of synthesis conditions (nature of precursor, pH, temperature, and addition of a complexing agent) on the structural-dispersion properties of TiO2 nanoparticles was studied. The hydrolysis methods providing the preparation of amorphous, anatase, brookite, and rutile modifications of TiO2 nanoparticles 3–5 nm in size were selected. Examination of different forms of TiO2 nanoparticles interaction with MDCK cells by transmission electron microscopy of ultrathin sections revealed different cell responses after treatment with different crystalline modifications and amorphous form of TiO2. The obtained results allowed us to conclude that direct contact of the nanoparticles with cell plasma membrane is the primary and critical step of their interaction and defines a subsequent response of the cell. PMID:22623903

The cell-mediated immune response to ectromelia virus infection. Secondary response in vitro: specificity, nature of effector and responder cells and requirements for induction of antigenic changes in stimulator cells.

PubMed

Pang, T; Blanden, R V

1976-06-01

An in vitro culture method was used to study secondary cell-mediated responses to ectromelia virus infection in mice. Infected, syngeneic spleen cells or peritoneal cells were efficient "stimulator" cells when cultured with "responder" cells obtained from mice infected with ectromelia 4-6 weeks previously. The kinetics of generation of cytotoxic cells in cultures were determined; a peak occurred on days 4-5. A separation procedure performed on the cytotoxic cells showed that activity was associated mainly with the Ig-negative subpopulation (T cell-rich) and that H-2 compatibility between cytotoxic cells and target cells was required. The secondary response was virus-specific, at the level of both induction and target cell lysis, at least so far as ectromelia and lymphocytic choriomeningitis (LCM) viruses are concerned. Seperation of responder cells prior to culture showed that a potent secondary response was generated with the Ig-negative (T cell-rich) subpopulation and only a weak response was observed when the responder cells were Ig-positive (rich in B cells). Infected stimulator cells did not appear to secrete significant amounts of soluble antigen into the medium over 4 days of culture. Thus, antigenic patterns effective in memory T cell stimulation may be largely associated with the surfaces of infected cells.Pretreatment of ectromelia virus with UV- or gamma-irradiation did not impair its ability to induce antigenic changes in stimulator cells. Stimulator cells treated with UV-or gamma-irradiated virus for 1 h and then immediately with pactamycin to inhibit further viral protein synthesis and replication were efficient stimulators, thus indicating that antigenic changes are induced very rapidly on the surface of stimulator cells after uptake of virus. These treatments are being used to further characterize the cellular requirements in the stimulator population.

Bovine apolipoprotein B-100 is a dominant immunogen in therapeutic cell populations cultured in fetal calf serum in mice and humans

PubMed Central

Sakamoto, Norihisa; Tsuji, Kazuhide; Muul, Linda M.; Lawler, Ann M.; Petricoin, Emanuel F.; Candotti, Fabio; Metcalf, Julia A.; Tavel, Jorge A.; Lane, H. Clifford; Urba, Walter J.; Fox, Bernard A.; Varki, Ajit; Lunney, Joan K.

2007-01-01

Recent studies have demonstrated that cell populations intended for therapeutic purposes that are cultured in heterologous animal products can acquire xenoantigens, potentially limiting their utility. In investigations of the immune response to murine embryonic stem cells, we found that a strong antibody response was generated after the second infusion. Both polyclonal and monoclonal antibody responses, derived from immunized mice, were found to be specific for bovine apolipoprotein B-100, which binds to abundant low-density lipoprotein receptors on the cell surface and is internalized. Here we show that in the majority of patients administered 3 different types of cell-based therapies using cells grown in fetal calf serum-containing media, an antibody response to bovine apolipoprotein B-100 develops after the second infusion and is the dominant specificity. The known and potential clinical effects of such antibodies are discussed. PMID:17395779

Anti-HBV response to toll-like receptor 7 agonist GS-9620 is associated with intrahepatic aggregates of T cells and B cells.

PubMed

Li, Li; Barry, Vivian; Daffis, Stephane; Niu, Congrong; Huntzicker, Erik; French, Dorothy M; Mikaelian, Igor; Lanford, Robert E; Delaney, William E; Fletcher, Simon P

2018-05-01

GS-9620, an oral agonist of toll-like receptor 7, is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the chimpanzee and woodchuck models of CHB. Herein, we investigated the immunomodulatory mechanisms underlying these antiviral effects. Archived liver biopsies and paired peripheral blood mononuclear cell samples from a previous chimpanzee study were analyzed by RNA sequencing, quantitative reverse transcription PCR, immunohistochemistry (IHC) and in situ hybridization (ISH). GS-9620 treatment of CHB chimpanzees induced an intrahepatic transcriptional profile significantly enriched with genes associated with hepatitis B virus (HBV) clearance in acutely infected chimpanzees. Type I and II interferon, CD8 + T cell and B cell transcriptional signatures were associated with treatment response, together with evidence of hepatocyte death and liver regeneration. IHC and ISH confirmed an increase in intrahepatic CD8 + T cell and B cell numbers during treatment, and revealed that GS-9620 transiently induced aggregates predominantly comprised of CD8 + T cells and B cells in portal regions. There were no follicular dendritic cells or IgG-positive cells in these lymphoid aggregates and very few CD11b + myeloid cells. There was no change in intrahepatic natural killer cell number during GS-9620 treatment. The antiviral response to GS-9620 treatment in CHB chimpanzees was associated with an intrahepatic interferon response and formation of lymphoid aggregates in the liver. Our data indicate these intrahepatic structures are not fully differentiated follicles containing germinal center reactions. However, the temporal correlation between development of these T and B cell aggregates and the antiviral response to treatment suggests they play a role in promoting an effective immune response against HBV. New therapies to treat chronic hepatitis B (CHB) are urgently needed. In this study we performed a retrospective analysis of liver and blood samples from a chimpanzee model of CHB to help understand how GS-9620, a drug in clinical trials, suppressed hepatitis B virus (HBV). We found that the antiviral response to GS-9620 was associated with accumulation of immune cells in the liver that can either kill cells infected with HBV or can produce antibodies that may prevent HBV from infecting new liver cells. These findings have important implications for how GS-9620 may be used in patients and may also help guide the development of new therapies to treat chronic HBV infection. Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Blocking Junctional Adhesion Molecule C Enhances Dendritic Cell Migration and Boosts the Immune Responses against Leishmania major

PubMed Central

Ballet, Romain; Emre, Yalin; Jemelin, Stéphane; Charmoy, Mélanie; Tacchini-Cottier, Fabienne; Imhof, Beat A.

2014-01-01

The recruitment of dendritic cells to sites of infections and their migration to lymph nodes is fundamental for antigen processing and presentation to T cells. In the present study, we showed that antibody blockade of junctional adhesion molecule C (JAM-C) on endothelial cells removed JAM-C away from junctions and increased vascular permeability after L. major infection. This has multiple consequences on the output of the immune response. In resistant C57BL/6 and susceptible BALB/c mice, we found higher numbers of innate immune cells migrating from blood to the site of infection. The subsequent migration of dendritic cells (DCs) from the skin to the draining lymph node was also improved, thereby boosting the induction of the adaptive immune response. In C57BL/6 mice, JAM-C blockade after L. major injection led to an enhanced IFN-γ dominated T helper 1 (Th1) response with reduced skin lesions and parasite burden. Conversely, anti JAM-C treatment increased the IL-4-driven T helper 2 (Th2) response in BALB/c mice with disease exacerbation. Overall, our results show that JAM-C blockade can finely-tune the innate cell migration and accelerate the consequent immune response to L. major without changing the type of the T helper cell response. PMID:25474593

Genes involved in immortalization of human mammary cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stampfer, Martha R.; Yaswen, Paul

2001-09-27

Breast cancer progression is characterized by inappropriate cell growth. Normal cells cease growth after a limited number of cell divisions--a process called cellular senescence-while tumor cells may acquire the ability to proliferate indefinitely (immortality). Inappropriate expression of specific oncogenes in a key cellular signaling pathway (Ras, Raf) can promote tumorigenicity in immortal cells, while causing finite lifespan cells to undergo a rapid senescence-like arrest. We have studied when in the course of transformation of cultured human mammary epithelial cells (HMEC), the response to overexpressed oncogenic Raf changes from being tumor-suppressive to tumor enhancing, and what are the molecular underpinnings ofmore » this response. Our data indicate: (1) HMEC acquire the ability to maintain growth in the presence of oncogenic Raf not simply as a consequence of overcoming senescence, but as a result of a newly discovered step in the process of immortal transformation uncovered by our lab, termed conversion. Immortal cells that have not undergone conversion (e.g., cells immortalized by exogenous introduction of the immortalizing enzyme, telomerase) remain growth inhibited. (2) Finite lifespan HMEC growth arrest in response to oncogenic Raf using mediators of growth inhibition that are very different from those used in response to oncogenic Raf by rodent cells and certain other human cell types, including the connective tissue cells from the same breast tissue. While many diverse cell types appear to have in common a tumor-suppressive response to this oncogenic signal, they also have developed multiple mechanisms to elicit this response. Understanding how cancer cells acquire the crucial capacity to be immortal and to abrogate normal tumor-suppressive mechanisms may serve both to increase our understanding of breast cancer progression, and to provide new targets for therapeutic intervention. Our results indicate that normal HMEC have novel means of enforcing a Raf-induced growth arrest and that this tumor suppressive function is lost at a specific stage in malignant transformation. Further studies to elucidate the ways by which immortal, converted HMEC escape this arrest may provide a more complete model of breast carcinogenesis as well as ways to intervene in that process.« less

Immunotoxicity and biodistribution analysis of arsenic trioxide in C57Bl/6 mice following a 2-week inhalation exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burchiel, Scott W., E-mail: Sburchiel@salud.unm.ed; Mitchell, Leah A.; Lauer, Fredine T.

2009-12-15

In these studies the immunotoxicity of arsenic trioxide (ATO, As{sub 2}O{sub 3}) was evaluated in mice following 14 days of inhalation exposures (nose only, 3 h per day) at concentrations of 50 mug/m{sup 3} and 1 mg/m{sup 3}. A biodistribution analysis performed immediately after inhalation exposures revealed highest levels of arsenic in the kidneys, bladder, liver, and lung. Spleen cell levels were comparable to those found in the blood, with the highest concentration of arsenic detected in the spleen being 150 mug/g tissue following the 1 mg/m{sup 3} exposures. No spleen cell cytotoxicity was observed at either of the twomore » exposure levels. There were no changes in spleen cell surface marker expression for B cells, T cells, macrophages, and natural killer (NK) cells. There were also no changes detected in the B cell (LPS-stimulated) and T cell (Con A-stimulated) proliferative responses of spleen cells, and no changes were found in the NK-mediated lysis of Yac-1 target cells. The primary T-dependent antibody response was, however, found to be highly susceptible to ATO suppression. Both the 50 mug/m{sup 3} and 1 mg/m{sup 3} exposures produced greater than 70% suppression of the humoral immune response to sheep red blood cells. Thus, the primary finding of this study is that the T-dependent humoral immune response is extremely sensitive to suppression by ATO and assessment of humoral immune responses should be considered in evaluating the health effects of arsenic containing agents.« less

Natural Killer Dendritic Cells Enhance Immune Responses Elicited by α -Galactosylceramide-Stimulated Natural Killer T Cells.

PubMed

Lee, Sung Won; Park, Hyun Jung; Kim, Nayoung; Hong, Seokmann

2013-01-01

Natural killer dendritic cells (NKDCs) possess potent anti-tumor activity, but the cellular effect of NKDC interactions with other innate immune cells is unclear. In this study, we demonstrate that the interaction of NKDCs and natural killer T (NKT) cells is required for the anti-tumor immune responses that are elicited by α -galactosylceramide ( α -GC) in mice. The rapid and strong expression of interferon- γ by NKDCs after α -GC stimulation was dependent on NKT cells. Various NK and DC molecular markers and cytotoxic molecules were up-regulated following α -GC administration. This up-regulation could improve NKDC presentation of tumor antigens and increase cytotoxicity against tumor cells. NKDCs were required for the stimulation of DCs, NK cells, and NKT cells. The strong anti-tumor immune responses elicited by α -GC may be due to the down-regulation of regulatory T cells. Furthermore, the depletion of NKDCs dampened the tumor clearance mediated by α -GC-stimulated NKT cells in vivo. Taken together, these results indicate that complex interactions of innate immune cells might be required to achieve optimal anti-tumor immune responses during the early stages of tumorigenesis.

The Subcellular Location of Ovalbumin in Plasmodium berghei Blood Stages Influences the Magnitude of T-Cell Responses

PubMed Central

Lin, Jing-Wen; Shaw, Tovah N.; Annoura, Takeshi; Fougère, Aurélie; Bouchier, Pascale; Chevalley-Maurel, Séverine; Kroeze, Hans; Franke-Fayard, Blandine; Janse, Chris J.; Couper, Kevin N.

2014-01-01

Model antigens are frequently introduced into pathogens to study determinants that influence T-cell responses to infections. To address whether an antigen's subcellular location influences the nature and magnitude of antigen-specific T-cell responses, we generated Plasmodium berghei parasites expressing the model antigen ovalbumin (OVA) either in the parasite cytoplasm or on the parasitophorous vacuole membrane (PVM). For cytosolic expression, OVA alone or conjugated to mCherry was expressed from a strong constitutive promoter (OVAhsp70 or OVA::mCherryhsp70); for PVM expression, OVA was fused to HEP17/EXP1 (OVA::Hep17hep17). Unexpectedly, OVA expression in OVAhsp70 parasites was very low, but when OVA was fused to mCherry (OVA::mCherryhsp70), it was highly expressed. OVA expression in OVA::Hep17hep17 parasites was strong but significantly less than that in OVA::mCherryhsp70 parasites. These transgenic parasites were used to examine the effects of antigen subcellular location and expression level on the development of T-cell responses during blood-stage infections. While all OVA-expressing parasites induced activation and proliferation of OVA-specific CD8+ T cells (OT-I) and CD4+ T cells (OT-II), the level of activation varied: OVA::Hep17hep17 parasites induced significantly stronger splenic and intracerebral OT-I and OT-II responses than those of OVA::mCherryhsp70 parasites, but OVA::mCherryhsp70 parasites promoted stronger OT-I and OT-II responses than those of OVAhsp70 parasites. Despite lower OVA expression levels, OVA::Hep17hep17 parasites induced stronger T-cell responses than those of OVA::mCherryhsp70 parasites. These results indicate that unconjugated cytosolic OVA is not stably expressed in Plasmodium parasites and, importantly, that its cellular location and expression level influence both the induction and magnitude of parasite-specific T-cell responses. These parasites represent useful tools for studying the development and function of antigen-specific T-cell responses during malaria infection. PMID:25156724

T cell chronic lymphocytic leukaemia with suppressor phenotype.

PubMed Central

Hofman, F M; Smith, D; Hocking, W

1982-01-01

The peripheral blood cells from a patient with T cell chronic lymphocytic leukaemia were examined for surface marker and functional characteristics. Eighty-91% of the peripheral blood cells formed SRBC rosettes and 22-49% possessed Fc receptors; 73% of the peripheral blood cells were reactive with the OKT8 antiserum and 61% expressed DR antigens. Response to PHA stimulation was markedly reduced, whereas allogeneic responsiveness in mixed leucocyte culture was intact. The ability of Con A-stimulated peripheral blood cells to generate suppressor activity in a mixed leucocyte reaction was deficient, whereas suppression of in vitro immunoglobulin synthesis was greater than normal. The leukaemic peripheral blood cell population expressed a T suppressor phenotype. Functional studies suggest that these cells were derived from the subset of T lymphocytes with regulatory activity for immunoglobulin synthesis as opposed to mitogenic responsiveness. PMID:6215199

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wyrobek, A. J.; Manohar, C. F.; Nelson, D. O.

We investigated the low dose dependency of the transcriptional response of human cells to characterize the shape and biological functions associated with the dose response curve and to identify common and conserved functions of low dose expressed genes across cells and tissues. Human lymphoblastoid (HL) cells from two unrelated individuals were exposed to graded doses of radiation spanning the range of 1-10 cGy were analyzed by transcriptome profiling, qPCR and bioinformatics, in comparison to sham irradiated samples. A set of {approx}80 genes showed consistent responses in both cell lines; these genes were associated with homeostasis mechanisms (e.g., membrane signaling, moleculemore » transport), subcellular locations (e.g., Golgi, and endoplasmic reticulum), and involved diverse signal transduction pathways. The majority of radiation-modulated genes had plateau-like responses across 1-10 cGy, some with suggestive evidence that transcription was modulated at doses below 1 cGy. MYC, FOS and TP53 were the major network nodes of the low-dose response in HL cells. Comparison our low dose expression findings in HL cells with those of prior studies in mouse brain after whole body exposure, in human keratinocyte cultures, and in endothelial cells cultures, indicates that certain components of the low dose radiation response are broadly conserved across cell types and tissues, independent of proliferation status.« less

Effector cell signature in peripheral blood following nasal allergen challenge in grass pollen allergic individuals.

PubMed

Shamji, M H; Bellido, V; Scadding, G W; Layhadi, J A; Cheung, D K M; Calderon, M A; Asare, A; Gao, Z; Turka, L A; Tchao, N; Togias, A; Phippard, D; Durham, S R

2015-02-01

Several studies have demonstrated the time course of inflammatory mediators in nasal fluids following nasal allergen challenge (NAC), whereas the effects of NAC on cells in the periphery are unknown. We examined the time course of effector cell markers (for basophils, dendritic cells and T cells) in peripheral blood after nasal grass pollen allergen challenge. Twelve participants with seasonal allergic rhinitis underwent a control (diluent) challenge followed by NAC after an interval of 14 days. Nasal symptoms and peak nasal inspiratory flow (PNIF) were recorded along with peripheral basophil, T-cell and dendritic cell responses (flow cytometry), T-cell proliferative responses (thymidine incorporation), and cytokine expression (FluoroSpot assay). Robust increases in nasal symptoms and decreases in PNIF were observed during the early (0-1 h) response and modest significant changes during the late (1-24 h) response. Sequential peaks in peripheral blood basophil activation markers were observed (CD107a at 3 h, CD63 at 6 h, and CD203c(bright) at 24 h). T effector/memory cells (CD4(+) CD25(lo) ) were increased at 6 h and accompanied by increases in CD80(+) and CD86(+) plasmacytoid dendritic cells (pDCs). Ex vivo grass antigen-driven T-cell proliferative responses and the frequency of IL-4(+) CD4(+) T cells were significantly increased at 6 h after NAC when compared to the control day. Basophil, T-cell, and dendritic cell activation increased the frequency of allergen-driven IL-4(+) CD4(+) T cells, and T-cell proliferative responses are detectable in the periphery after NAC. These data confirm systemic cellular activation following a local nasal provocation. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Modelling and Simulation of the Dynamics of the Antigen-Specific T Cell Response Using Variable Structure Control Theory.

PubMed

Anelone, Anet J N; Spurgeon, Sarah K

2016-01-01

Experimental and mathematical studies in immunology have revealed that the dynamics of the programmed T cell response to vigorous infection can be conveniently modelled using a sigmoidal or a discontinuous immune response function. This paper hypothesizes strong synergies between this existing work and the dynamical behaviour of engineering systems with a variable structure control (VSC) law. These findings motivate the interpretation of the immune system as a variable structure control system. It is shown that dynamical properties as well as conditions to analytically assess the transition from health to disease can be developed for the specific T cell response from the theory of variable structure control. In particular, it is shown that the robustness properties of the specific T cell response as observed in experiments can be explained analytically using a VSC perspective. Further, the predictive capacity of the VSC framework to determine the T cell help required to overcome chronic Lymphocytic Choriomeningitis Virus (LCMV) infection is demonstrated. The findings demonstrate that studying the immune system using variable structure control theory provides a new framework for evaluating immunological dynamics and experimental observations. A modelling and simulation tool results with predictive capacity to determine how to modify the immune response to achieve healthy outcomes which may have application in drug development and vaccine design.

Endothelium as a transducing surface.

PubMed

Ryan, U S

1989-02-01

Endothelial cells responses to a variety of agonists include release of endothelium dependent vasodilators, such as endothelium dependent relaxing factor (EDRF) and prostacyclin (PGI2). These substances act on vascular smooth muscle to cause relaxation and also have potent anti-aggregatory effects on platelets. A study of the mechanisms of signal transduction involved in these processes was undertaken. An investigation of intracellular calcium using FURA-2 and INDO-1 loaded endothelial cells shows transient elevation in response to vasodilator agonists. The calcium content of endothelial cells calculated using 45Ca flux techniques is increased in response to bradykinin and thrombin. Receptor activation leads to increased phosphoinositide turnover in endothelial cells and activates protein kinase C, the latter may be involved in feedback regulation. Patch clamp studies have demonstrated receptor-operated ionic channels in the endothelial cell membrane. Thus, intracellular calcium concentration is elevated in response to receptor activation, both as a result of liberation of calcium from intracellular stores and calcium entry from extracellular sources. Endothelial cells also respond to particulate stimuli. They can selectively bind and phagocytize bacteria. Phagocytosis leads to generation of superoxide aionin, a process which also seems to be controlled by elevation of intracellular calcium and activation of protein kinase C. In addition phagocytosis activates endothelial cells resulting in increased migration, division and further phagocytosis. All in all, the plethora of different endothelial responses to a variety of stimuli suggests a complex and multipotent cell type.(ABSTRACT TRUNCATED AT 250 WORDS)

Mathematical modeling provides kinetic details of the human immune response to vaccination

PubMed Central

Le, Dustin; Miller, Joseph D.; Ganusov, Vitaly V.

2015-01-01

With major advances in experimental techniques to track antigen-specific immune responses many basic questions on the kinetics of virus-specific immunity in humans remain unanswered. To gain insights into kinetics of T and B cell responses in human volunteers we combined mathematical models and experimental data from recent studies employing vaccines against yellow fever and smallpox. Yellow fever virus-specific CD8 T cell population expanded slowly with the average doubling time of 2 days peaking 2.5 weeks post immunization. Interestingly, we found that the peak of the yellow fever-specific CD8 T cell response was determined by the rate of T cell proliferation and not by the precursor frequency of antigen-specific cells as has been suggested in several studies in mice. We also found that while the frequency of virus-specific T cells increased slowly, the slow increase could still accurately explain clearance of yellow fever virus in the blood. Our additional mathematical model described well the kinetics of virus-specific antibody-secreting cell and antibody response to vaccinia virus in vaccinated individuals suggesting that most of antibodies in 3 months post immunization were derived from the population of circulating antibody-secreting cells. Taken together, our analysis provided novel insights into mechanisms by which live vaccines induce immunity to viral infections and highlighted challenges of applying methods of mathematical modeling to the current, state-of-the-art yet limited immunological data. PMID:25621280

Mathematical modeling provides kinetic details of the human immune response to vaccination.

PubMed

Le, Dustin; Miller, Joseph D; Ganusov, Vitaly V

2014-01-01

With major advances in experimental techniques to track antigen-specific immune responses many basic questions on the kinetics of virus-specific immunity in humans remain unanswered. To gain insights into kinetics of T and B cell responses in human volunteers we combined mathematical models and experimental data from recent studies employing vaccines against yellow fever and smallpox. Yellow fever virus-specific CD8 T cell population expanded slowly with the average doubling time of 2 days peaking 2.5 weeks post immunization. Interestingly, we found that the peak of the yellow fever-specific CD8 T cell response was determined by the rate of T cell proliferation and not by the precursor frequency of antigen-specific cells as has been suggested in several studies in mice. We also found that while the frequency of virus-specific T cells increased slowly, the slow increase could still accurately explain clearance of yellow fever virus in the blood. Our additional mathematical model described well the kinetics of virus-specific antibody-secreting cell and antibody response to vaccinia virus in vaccinated individuals suggesting that most of antibodies in 3 months post immunization were derived from the population of circulating antibody-secreting cells. Taken together, our analysis provided novel insights into mechanisms by which live vaccines induce immunity to viral infections and highlighted challenges of applying methods of mathematical modeling to the current, state-of-the-art yet limited immunological data.

TLR4 signaling shapes B cell dynamics via MyD88-dependent pathways and Rac GTPases.

PubMed

Barrio, Laura; Saez de Guinoa, Julia; Carrasco, Yolanda R

2013-10-01

B cells use a plethora of TLR to recognize pathogen-derived ligands. These innate signals have an important function in the B cell adaptive immune response and modify their trafficking and tissue location. The direct role of TLR signaling on B cell dynamics nonetheless remains almost entirely unknown. In this study, we used a state-of-the-art two-dimensional model combined with real-time microscopy to study the effect of TLR4 stimulation on mouse B cell motility in response to chemokines. We show that a minimum stimulation period is necessary for TLR4 modification of B cell behavior. TLR4 stimulation increased B cell polarization, migration, and directionality; these increases were dependent on the MyD88 signaling pathway and did not require ERK or p38 MAPK activity downstream of TLR4. In addition, TLR4 stimulation enhanced Rac GTPase activity and promoted sustained Rac activation in response to chemokines. These results increase our understanding of the regulation of B cell dynamics by innate signals and the underlying molecular mechanisms.

Immune-responsiveness of CD4+ T cells during Streptococcus suis serotype 2 infection

PubMed Central

Lecours, Marie-Pier; Letendre, Corinne; Clarke, Damian; Lemire, Paul; Galbas, Tristan; Benoit-Biancamano, Marie-Odile; Thibodeau, Jacques; Gottschalk, Marcelo; Segura, Mariela

2016-01-01

The pathogenesis of Streptococcus suis infection, a major swine and human pathogen, is only partially understood and knowledge on the host adaptive immune response is critically scarce. Yet, S. suis virulence factors, particularly its capsular polysaccharide (CPS), enable this bacterium to modulate dendritic cell (DC) functions and potentially impair the immune response. This study aimed to evaluate modulation of T cell activation during S. suis infection and the role of DCs in this response. S. suis-stimulated total mouse splenocytes readily produced TNF-α, IL-6, IFN-γ, CCL3, CXCL9, and IL-10. Ex vivo and in vivo analyses revealed the involvement of CD4+ T cells and a Th1 response. Nevertheless, during S. suis infection, levels of the Th1-derived cytokines TNF-α and IFN-γ were very low. A transient splenic depletion of CD4+ T cells and a poor memory response were also observed. Moreover, CD4+ T cells secreted IL-10 and failed to up-regulate optimal levels of CD40L and CD69 in coculture with DCs. The CPS hampered release of several T cell-derived cytokines in vitro. Finally, a correlation was established between severe clinical signs of S. suis disease and impaired antibody responses. Altogether, these results suggest S. suis interferes with the adaptive immune response. PMID:27905502

New generation humanized mice for virus research: Comparative aspects and future prospects

PubMed Central

Akkina, Ramesh

2014-01-01

Work with human specific viruses will greatly benefit from the use of an in vivo system that provides human target cells and tissues in a physiological setting. In this regard humanized mice (hu-Mice) have played an important role in our understanding of viral pathogenesis and testing of therapeutic strategies. Limitations with earlier versions of hu-Mice that lacked a functioning human immune system are currently being overcome. The new generation hu-Mouse models are capable of multilineage human hematopoiesis and generate T cells, B cells, macrophages and dendritic cells required for an adaptive human immune response. Now any human specific pathogen that can infect humanized mice can be studied in the context of ongoing infection and immune responses. Two leading humanized mouse models are currently employed: the hu-HSC model is created by transplantation of human hematopoietic stem cells (HSC), whereas the BLT mouse model is prepared by transplantation of human fetal liver, thymus and HSC. A number of human specific viruses such as HIV-1, dengue, EBV and HCV are being studied intensively in these systems. Both models permit infection by mucosal routes with viruses such as HIV-1 thus allowing transmission prevention studies. Cellular and humoral immune responses are seen in both the models. While there is efficient antigen specific IgM production, IgG responses are suboptimal due to inefficient immunoglobulin class switching. With the maturation of T cells occurring in the autologous human thymus, BLT mice permit human HLA restricted T cell responses in contrast to hu-HSC mice. However, the strength of the immune responses needs further improvement in both models to reach the levels seen in humans. The scope of hu-Mice use is further broadened by transplantation of additional tissues like human liver thus permitting immunopathogenesis studies on hepatotropic viruses such as HCV. Numerous studies that encompass antivirals, gene therapy, viral evolution, and the generation of human monoclonal antibodies have been conducted with promising results in these mice. For further improvement of the new hu-Mouse models, ongoing work is focused on generating new strains of immunodeficient mice transgenic for human HLA molecules to strengthen immune responses and human cytokines and growth factors to improve human cell reconstitution and their homeostatic maintenance. PMID:23217612

Bim is required for T-cell allogeneic responses and graft-versus-host disease in vivo

PubMed Central

Yu, Yu; Yu, Jing; Iclozan, Cristina; Kaosaard, Kane; Anasetti, Claudio; Yu, Xue-Zhong

2012-01-01

Bim, a BH3-only Bcl-2-family protein, is essential for T-cell negative selection in the thymus as well as for the death of activated T cells in the periphery. The role of Bim has been extensively studied in T-cell responses to self-antigens and viral infections. Recent findings on Bim in autoimmunity triggered our interest in investigating whether Bim may play a role in another disease with inflammatory symptoms as graft-versus-host disease (GVHD). Here we report that Bim is required for optimal T-cell responses to alloantigens in vivo and for the development of GVHD. Using murine models of allogeneic bone marrow transplantation (BMT), we found that donor T cells deficient for Bim are impaired in the induction of GVHD primarily due to a significant defect in T cell activation and expansion in vivo. Upon TCR engagement, Bim-/- T cells exhibited selective defects in CD69 expression and phosphorylation of PLCγ1. Our studies uncover a novel aspect of Bim function in T-cell activation with important implications in understanding the mechanisms of T-cell activation and tolerance under allogeneic transplantation. PMID:22432091

Cytokine activation induces human memory-like NK cells.

PubMed

Romee, Rizwan; Schneider, Stephanie E; Leong, Jeffrey W; Chase, Julie M; Keppel, Catherine R; Sullivan, Ryan P; Cooper, Megan A; Fehniger, Todd A

2012-12-06

Natural killer (NK) cells are lymphocytes that play an important role in the immune response to infection and malignancy. Recent studies in mice have shown that stimulation of NK cells with cytokines or in the context of a viral infection results in memory-like properties. We hypothesized that human NK cells exhibit such memory-like properties with an enhanced recall response after cytokine preactivation. In the present study, we show that human NK cells preactivated briefly with cytokine combinations including IL-12, IL-15, and IL-18 followed by a 7- to 21-day rest have enhanced IFN-γ production after restimulation with IL-12 + IL-15, IL-12 + IL-18, or K562 leukemia cells. This memory-like phenotype was retained in proliferating NK cells. In CD56(dim) NK cells, the memory-like IFN-γ response was correlated with the expression of CD94, NKG2A, NKG2C, and CD69 and a lack of CD57 and KIR. Therefore, human NK cells have functional memory-like properties after cytokine activation, which provides a novel rationale for integrating preactivation with combinations of IL-12, IL-15, and IL-18 into NK cell immunotherapy strategies.

The Plasma Concentration of the B Cell Activating Factor Is Increased in Children With Acute Malaria

PubMed Central

Nduati, Eunice; Gwela, Agnes; Karanja, Henry; Mugyenyi, Cleopatra; Langhorne, Jean; Marsh, Kevin

2011-01-01

Malaria-specific antibody responses in children often appear to be short-lived but the mechanisms underlying this phenomenon are not well understood. In this study, we investigated the relationship between the B-cell activating factor (BAFF) and its receptors expressed on B cells with antibody responses during and after acute malaria in children. Our results demonstrate that BAFF plasma levels increased during acute malarial disease and reflected disease severity. The expression profiles for BAFF receptors on B cells agreed with rapid activation and differentiation of a proportion of B cells to plasma cells. However, BAFF receptor (BAFF-R) expression was reduced on all peripheral blood B cells during acute infection, but those children with the highest level of BAFF-R expression on B cells maintained schizont-specific immunoglobin G (IgG) over a period of 4 months, indicating that dysregulation of BAFF-R expression on B cells may contribute to short-lived antibody responses to malarial antigens in children. In summary, this study suggests a potential role for BAFF during malaria disease, both as a marker for disease severity and in shaping the differentiation pattern of antigen-specific B cells. PMID:21849293

A characterization of the ZFL cell line and primary hepatocytes as in vitro liver cell models for the zebrafish (Danio rerio).

PubMed

Eide, Marta; Rusten, Marte; Male, Rune; Jensen, Knut Helge Midtbø; Goksøyr, Anders

2014-02-01

The zebrafish (Danio rerio) is a widely used model species in biomedical research. The ZFL cell line, established from zebrafish liver, and freshly isolated primary hepatocytes from zebrafish have been used in several toxicological studies. However, no previous report has compared and characterized these two systems at the level of gene expression. The aim of this study was to evaluate the ZFL cell line in comparison to primary hepatocytes as in vitro models for studying effects of environmental contaminants in zebrafish liver. Using quantitative real-time PCR, the basal level and transcriptional induction potential of key genes involved in toxic responses in the ZFL cell line, primary hepatocytes and whole liver from zebrafish were compared. The study showed that the ZFL cells have lower levels of mRNA of most selected genes compared to zebrafish liver. The induced gene transcription following exposure to ligand was much lower in ZFL cells compared to zebrafish primary hepatocytes at the doses tested. Importantly, oestrogen receptor and vitellogenin genes showed low basal transcription and no induction response in the ZFL cell line. In conclusion, it appears that primary hepatocytes are well suited for studying environmental contaminants including xenoestrogens, but may show large sex-dependent differences in gene transcription. The ZFL cell line shows potential in toxicological studies involving the aryl hydrocarbon receptor pathway. However, low potential for transcriptional induction of genes in general should be expected, especially notable when studying estrogenic responses. Copyright © 2013 Elsevier B.V. All rights reserved.

Dual function of CD70 in viral infection: modulator of early cytokine responses and activator of adaptive responses1

PubMed Central

Allam, Atef; Swiecki, Melissa; Vermi, William; Ashwell, Jonathan D.; Colonna, Marco

2014-01-01

The role of the tumor necrosis factor family member CD70 in adaptive T cell responses has been intensively studied but its function in innate responses is still under investigation. Here we show that CD70 inhibits the early innate response to murine cytomegalovirus (MCMV) but is essential for the optimal generation of virus-specific CD8 T cells. CD70-/- mice reacted to MCMV infection with a robust type I interferon and proinflammatory cytokine response. This response was sufficient for initial control of MCMV, although at later time points, CD70-/- mice became more susceptible to MCMV infection. The heightened cytokine response during the early phase of MCMV infection in CD70-/- mice was paralleled by a reduction in regulatory T cells (Treg). Treg from naïve CD70-/- mice were not as efficient at suppressing T cell proliferation compared to Treg from naïve WT mice and depletion of Treg during MCMV infection in Foxp3-DTR mice or in WT mice recapitulated the phenotype observed in CD70-/- mice. Our study demonstrates that while CD70 is required for the activation of the antiviral adaptive response, it has a regulatory role in early cytokine responses to viruses such as MCMV, possibly through maintenance of Treg survival and function. PMID:24913981

The effect of strychnine, bicuculline, and picrotoxin on X and Y cells in the cat retina

PubMed Central

1979-01-01

The effect of intravenous strychnine and the GABA antagonists picrotoxin and bicuculline upon the discharge pattern of center- surround-organized cat retinal ganglion cells of X and Y type were studied. Stimuli (mostly scotopic, and some photopic) were selected such that responses from both on and off-center cells were either due to the center, due to the surround, or clearly mixed. Pre-drug control responses were obtained, and their behavior following administration of the antagonists was observed for periods up to several hours. X-cell responses were affected in a consistent manner by strychnine while being unaffected by GABA antagonists. All observed changes following strychnine were consistent with a shift in center-surround balance of X cells in favor of the center. For Y-cell responses to flashing annuli following strychnine, there was either no shift or a relatively small shift in center-surround balance. Compared to X-cell responses to flashing lights, those of Y cells were very little affected by strychnine and in most cases were unaffected. It thus appears that glycine plays a similar role in receptive field organization of X cells as does GABA in Y cells (Kirby and Enroth-Cugell, 1976. J. Gen. Physiol. 68:465-484). PMID:479822

Comparison of cytotoxic T lymphocyte responses against pancreatic cancer induced by dendritic cells transfected with total tumor RNA and fusion hybrided with tumor cell

PubMed Central

Chen, Jiang; Li, Hong-Yu; Wang, Di; Shao, Xiao-Dong

2015-01-01

Pancreatic cancer (PC) is a deadly human malignancy. Dendritic cell (DC)-based immunotherapy with whole tumor antigens demonstrates potential efficiency in cancer treatment. Tumor RNA and tumor fusion hybrid cells are sources of whole tumor antigens for preparing DC tumor vaccines. However, the efficacy of these sources in eliciting immune responses against PC has not yet to be directly compared. In the present study, patient-derived PC cells and DCs were fused (DC–tumor hybrids) and primary cultured PC cell-derived total RNA was electroporated into autologous DCs (DC–tumor RNA). The antitumor immune responses induced by DC–tumor hybrids and DC–tumor RNA were compared directly. The results showed that both RNA and hybrid methodologies could induce tumor-specific cytotoxic T lymphocyte (CTL) responses, but pulsing DCs with total tumor RNA could induce a higher frequency of activated CTLs and T-helper cells than fusing DCs with autologous tumor cells. In addition, DC–tumor RNA triggered stronger autologous tumor cell lysis than DC–tumor hybrids. It could be concluded that DCs pulsed with whole tumor RNA are superior to those fused with tumor cells in priming anti-PC CTL responses. Electroporation with total tumor RNA may be more suitable for DC-based PC vaccination. PMID:25736302

Visible red and infrared light alters gene expression in human marrow stromal fibroblast cells.

PubMed

Guo, J; Wang, Q; Wai, D; Zhang, Q Z; Shi, S H; Le, A D; Shi, S T; Yen, S L-K

2015-04-01

This study tested whether or not gene expression in human marrow stromal fibroblast (MSF) cells depends on light wavelength and energy density. Primary cultures of isolated human bone marrow stem cells (hBMSC) were exposed to visible red (VR, 633 nm) and infrared (IR, 830 nm) radiation wavelengths from a light emitting diode (LED) over a range of energy densities (0.5, 1.0, 1.5, and 2.0 Joules/cm2) Cultured cells were assayed for cell proliferation, osteogenic potential, adipogenesis, mRNA and protein content. mRNA was analyzed by microarray and compared among different wavelengths and energy densities. Mesenchymal and epithelial cell responses were compared to determine whether responses were cell type specific. Protein array analysis was used to further analyze key pathways identified by microarrays. Different wavelengths and energy densities produced unique sets of genes identified by microarray analysis. Pathway analysis pointed to TGF-beta 1 in the visible red and Akt 1 in the infrared wavelengths as key pathways to study. TGF-beta protein arrays suggested switching from canonical to non-canonical TGF-beta pathways with increases to longer IR wavelengths. Microarrays suggest RANKL and MMP 10 followed IR energy density dose-response curves. Epithelial and mesenchymal cells respond differently to stimulation by light suggesting cell type-specific response is possible. These studies demonstrate differential gene expression with different wavelengths, energy densities and cell types. These differences in gene expression have the potential to be exploited for therapeutic purposes and can help explain contradictory results in the literature when wavelengths, energy densities and cell types differ. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Generation of mice deficient in RNA-binding motif protein 3 (RBM3) and characterization of its role in innate immune responses and cell growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsuda, Atsushi; Core Research for Evolution Science and Technology, Japan Science and Technology Agency, Chiyoda-ku, Tokyo 102-0075; Ogawa, Masahiro

Highlights: {yields} We identified RNA-binding motif protein 3 (RBM3) as CpG-B DNA-binding protein. {yields} RBM3 translocates from the nucleus to the cytoplasm and co-localized with CpG-B DNA. {yields} We newly generated Rbm3-deficient (Rbm3{sup -/-}) mice. {yields} DNA-mediated cytokine gene induction was normally occured in Rbm3{sup -/-} cells. {yields}Rbm3{sup -/-} MEFs showed poorer proliferation rate and increased number of G2-phase cells. -- Abstract: The activation of innate immune responses is critical to host defense against microbial infections, wherein nucleic acid-sensing pattern recognition receptors recognize DNA or RNA from viruses or bacteria and activate downstream signaling pathways. In a search for newmore » DNA-sensing molecules that regulate innate immune responses, we identified RNA-binding motif protein 3 (RBM3), whose role has been implicated in the regulation of cell growth. In this study, we generated Rbm3-deficient (Rbm3{sup -/-}) mice to study the role of RBM3 in immune responses and cell growth. Despite evidence for its interaction with immunogenic DNA in a cell, no overt phenotypic abnormalities were found in cells from Rbm3{sup -/-} mice for the DNA-mediated induction of cytokine genes. Interestingly, however, Rbm3{sup -/-} mouse embryonic fibroblasts (MEFs) showed poorer proliferation rates as compared to control MEFs. Further cell cycle analysis revealed that Rbm3{sup -/-} MEFs have markedly increased number of G2-phase cells, suggesting a hitherto unknown role of RBM3 in the G2-phase control. Thus, these mutant mice and cells may provide new tools with which to study the mechanisms underlying the regulation of cell cycle and oncogenesis.« less

A critical role of T follicular helper cells in human mucosal anti-influenza response that can be enhanced by immunological adjuvant CpG-DNA.

PubMed

Aljurayyan, A N; Sharma, R; Upile, N; Beer, H; Vaughan, C; Xie, C; Achar, P; Ahmed, M S; McNamara, P S; Gordon, S B; Zhang, Q

2016-08-01

T Follicular helper cells (TFH) are considered critical for B cell antibody response, and recent efforts have focused on promoting TFH in order to enhance vaccine efficacy. We studied the frequency and function of TFH in nasopharynx-associated lymphoid tissues (NALT) from children and adults, and its role in anti-influenza antibody response following stimulation by a live-attenuated influenza vaccine (LAIV) or an inactivated seasonal virus antigen (sH1N1). We further studied whether CpG-DNA promotes TFH and by which enhances anti-influenza response. We showed NALT from children aged 1.5-10 years contained abundant TFH, suggesting efficient priming of TFH during early childhood. Stimulation by LAIV induced a marked increase in TFH that correlated with a strong production of anti-hemagglutinin (HA) IgA/IgG/IgM antibodies in tonsillar cells. Stimulation by the inactivated sH1N1 antigen induced a small increase in TFH which was markedly enhanced by CpG-DNA, accompanied by enhanced anti-HA antibody responses. In B cell co-culture experiment, anti-HA responses were only seen in the presence of TFH, and addition of plasmacytoid dendritic cell to TFH-B cell co-culture enhanced the TFH-mediated antibody production following CpG-DNA and sH1N1 antigen stimulation. Induction of TFH differentiation from naïve T cells was also shown following the stimulation. Our results support a critical role of TFH in human mucosal anti-influenza antibody response. Use of an adjuvant such as CpG-DNA that has the capacity to promote TFH by which to enhance antigen-induced antibody responses in NALT tissue may have important implications for future vaccination strategies against respiratory pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

Interleukin (IL)-1 Receptor Signaling on Graft Parenchymal Cells Regulates Memory and De Novo Donor-Reactive CD8 T Cell Responses to Cardiac Allografts1

PubMed Central

Iida, Shoichi; Tsuda, Hidetoshi; Tanaka, Toshiaki; Kish, Danielle D.; Abe, Toyofumi; Su, Charles A.; Abe, Ryo; Tanabe, Kazunari; Valujskikh, Anna; Baldwin, William M.; Fairchild, Robert L.

2016-01-01

Reperfusion of organ allografts induces a potent inflammatory response that directs rapid memory T cell, neutrophil and macrophage graft infiltration and their activation to express functions mediating graft tissue injury. The role of cardiac allograft IL-1 receptor signaling in this early inflammation and the downstream primary alloimmune response was investigated. When compared to complete MHC-mismatched wild type cardiac allografts, IL-1R−/− allografts had marked decreases in endogenous memory CD8 T cell and neutrophil infiltration and expression of proinflammatory mediators at early times after transplant whereas endogenous memory CD4 T cell and macrophage infiltration was not decreased. IL-1R−/− allograft recipients also had marked decreases in de novo donor-reactive CD8, but not CD4, T cell development to IFN-γ-producing cells. CD8 T cell-mediated rejection of IL-1R−/− cardiac allografts took 3 weeks longer than wild type allografts. Cardiac allografts from reciprocal bone marrow reconstituted IL-1R−/−/wild type chimeric donors indicated that IL-1R signaling on graft non-hematopoietic-derived, but not bone marrow-derived, cells is required for the potent donor-reactive memory and primary CD8 T cell alloimmune responses observed in response to wild type allografts. These studies implicate IL-1R-mediated signals by allograft parenchymal cells in generating the stimuli provoking development and elicitation of optimal alloimmune responses to the grafts. PMID:26856697

Persistence of memory B-cell and T-cell responses to the quadrivalent HPV vaccine in HIV-infected children.

PubMed

Weinberg, Adriana; Huang, Sharon; Moscicki, Anna-Barbara; Saah, Afred; Levin, Myron J

2018-04-24

To determine the magnitude and persistence of quadrivalent human papillomavirus (HPV)16 and HPV18 B-cell and T-cell memory after three or four doses of quadrivalent HPV vaccine (QHPV) in HIV-infected children. Seventy-four HIV-infected children immunized with four doses and 23 with three doses of QHPV had HPV16 and HPV18 IgG B-cell and IFNγ and IL2 T-cell ELISPOT performed at 2, 3.5 and 4-5 years after the last dose. HPV16 and HPV18 T-cell responses were similar in both treatment groups, with higher responses to HPV16 vs. HPV18. These HPV T-cell responses correlated with HIV disease characteristics at the study visits. Global T-cell function declined over time as measured by nonspecific mitogenic stimulation. B-cell memory was similar across treatment groups and HPV genotypes. There was a decline in HPV-specific B-cell memory over time that reached statistical significance for HPV16 in the four-dose group. B-cell and T-cell memory did not significantly differ after either three or four doses of QHPV in HIV-infected children. The clinical consequences of decreasing global T-cell function and HPV B-cell memory over time in HIV-infected children requires further investigation.

Understanding the T cell immune response in SARS coronavirus infection

PubMed Central

Janice Oh, Hsueh-Ling; Ken-En Gan, Samuel; Bertoletti, Antonio; Tan, Yee-Joo

2012-01-01

The severe acute respiratory syndrome (SARS) epidemic started in late 2002 and swiftly spread across 5 continents with a mortality rate of around 10%. Although the epidemic was eventually controlled through the implementation of strict quarantine measures, there continues a need to investigate the SARS coronavirus (SARS-CoV) and develop interventions should it re-emerge. Numerous studies have shown that neutralizing antibodies against the virus can be found in patients infected with SARS-CoV within days upon the onset of illness and lasting up to several months. In contrast, there is little data on the kinetics of T cell responses during SARS-CoV infection and little is known about their role in the recovery process. However, recent studies in mice suggest the importance of T cells in viral clearance during SARS-CoV infection. Moreover, a growing number of studies have investigated the memory T cell responses in recovered SARS patients. This review covers the available literature on the emerging importance of T cell responses in SARS-CoV infection, particularly on the mapping of cytotoxic T lymphocyte (CTL) epitopes, longevity, polyfunctionality and human leukocyte antigen (HLA) association as well as their potential implications on treatment and vaccine development. PMID:26038429

Understanding the T cell immune response in SARS coronavirus infection.

PubMed

Janice Oh, Hsueh-Ling; Ken-En Gan, Samuel; Bertoletti, Antonio; Tan, Yee-Joo

2012-09-01

The severe acute respiratory syndrome (SARS) epidemic started in late 2002 and swiftly spread across 5 continents with a mortality rate of around 10%. Although the epidemic was eventually controlled through the implementation of strict quarantine measures, there continues a need to investigate the SARS coronavirus (SARS-CoV) and develop interventions should it re-emerge. Numerous studies have shown that neutralizing antibodies against the virus can be found in patients infected with SARS-CoV within days upon the onset of illness and lasting up to several months. In contrast, there is little data on the kinetics of T cell responses during SARS-CoV infection and little is known about their role in the recovery process. However, recent studies in mice suggest the importance of T cells in viral clearance during SARS-CoV infection. Moreover, a growing number of studies have investigated the memory T cell responses in recovered SARS patients. This review covers the available literature on the emerging importance of T cell responses in SARS-CoV infection, particularly on the mapping of cytotoxic T lymphocyte (CTL) epitopes, longevity, polyfunctionality and human leukocyte antigen (HLA) association as well as their potential implications on treatment and vaccine development.

ANALYSIS OF RISK FROM EXPOSURE TO ALDICARB USING IMMUNE RESPONSE OF NONUNIFORM POPULATIONS OF MICE

EPA Science Inventory

The immunomodulation response of mice to low levels of aldicarb in drinking water was investigated in four series of studies. he splenic plaque forming cell (PFC) response to red sheep cells were measured for treatment levels of 0.01 to 1000 ppb (ug/kg). ased on their beginning a...

Increased T cell proliferative responses to islet antigens identify clinical responders to anti-CD20 monoclonal antibody (rituximab) therapy in type 1 diabetes.

PubMed

Herold, Kevan C; Pescovitz, Mark D; McGee, Paula; Krause-Steinrauf, Heidi; Spain, Lisa M; Bourcier, Kasia; Asare, Adam; Liu, Zhugong; Lachin, John M; Dosch, H Michael

2011-08-15

Type 1 diabetes mellitus is believed to be due to the autoimmune destruction of β-cells by T lymphocytes, but a single course of rituximab, a monoclonal anti-CD20 B lymphocyte Ab, can attenuate C-peptide loss over the first year of disease. The effects of B cell depletion on disease-associated T cell responses have not been studied. We compare changes in lymphocyte subsets, T cell proliferative responses to disease-associated target Ags, and C-peptide levels of participants who did (responders) or did not (nonresponders) show signs of β-cell preservation 1 y after rituximab therapy in a placebo-controlled TrialNet trial. Rituximab decreased B lymphocyte levels after four weekly doses of mAb. T cell proliferative responses to diabetes-associated Ags were present at baseline in 75% of anti-CD20- and 82% of placebo-treated subjects and were not different over time. However, in rituximab-treated subjects with significant C-peptide preservation at 6 mo (58%), the proliferative responses to diabetes-associated total (p = 0.032), islet-specific (p = 0.048), and neuronal autoantigens (p = 0.005) increased over the 12-mo observation period. This relationship was not seen in placebo-treated patients. We conclude that in patients with type 1 diabetes mellitus, anti-B cell mAb causes increased proliferative responses to diabetes Ags and attenuated β-cell loss. The way in which these responses affect the disease course remains unknown.

Metabolomics Analysis of Hormone-Responsive and Triple-Negative Breast Cancer Cell Responses to Paclitaxel Identify Key Metabolic Differences.

PubMed

Stewart, Delisha A; Winnike, Jason H; McRitchie, Susan L; Clark, Robert F; Pathmasiri, Wimal W; Sumner, Susan J

2016-09-02

To date, no targeted therapies are available to treat triple negative breast cancer (TNBC), while other breast cancer subtypes are responsive to current therapeutic treatment. Metabolomics was conducted to reveal differences in two hormone receptor-negative TNBC cell lines and two hormone receptor-positive Luminal A cell lines. Studies were conducted in the presence and absence of paclitaxel (Taxol). TNBC cell lines had higher levels of amino acids, branched-chain amino acids, nucleotides, and nucleotide sugars and lower levels of proliferation-related metabolites like choline compared with Luminal A cell lines. In the presence of paclitaxel, each cell line showed unique metabolic responses, with some similarities by type. For example, in the Luminal A cell lines, levels of lactate and creatine decreased while certain choline metabolites and myo-inositol increased with paclitaxel. In the TNBC cell lines levels of glutamine, glutamate, and glutathione increased, whereas lysine, proline, and valine decreased in the presence of drug. Profiling secreted inflammatory cytokines in the conditioned media demonstrated a greater response to paclitaxel in the hormone-positive Luminal cells compared with a secretion profile that suggested greater drug resistance in the TNBC cells. The most significant differences distinguishing the cell types based on pathway enrichment analyses were related to amino acid, lipid and carbohydrate metabolism pathways, whereas several biological pathways were differentiated between the cell lines following treatment.

A Rapid-Response Humoral Vaccine Platform Exploiting Pre-Existing Non-Cognate Populations of Anti-Vaccine or Anti-Viral CD4+ T Helper Cells to Confirm B Cell Activation.

PubMed

Hills, Thomas; Jakeman, Phillip G; Carlisle, Robert C; Klenerman, Paul; Seymour, Leonard W; Cawood, Ryan

2016-01-01

The need for CD4+ T cell responses to arise de novo following vaccination can limit the speed of B cell responses. Populations of pre-existing vaccine-induced or anti-viral CD4+ T cells recognising distinct antigens could be exploited to overcome this limitation. We hypothesise that liposomal vaccine particles encapsulating epitopes that are recognised, after processing and B cell MHCII presentation, by pre-existing CD4+ T cells will exploit this pre-existing T cell help and result in improved antibody responses to distinct target antigens displayed on the particle surface. Liposomal vaccine particles were engineered to display the malaria circumsporozoite (CSP) antigen on their surface, with helper CD4+ epitopes from distinct vaccine or viral antigens contained within the particle core, ensuring the B cell response is raised but focused against CSP. In vivo vaccination studies were then conducted in C57Bl/6 mice as models of either vaccine-induced pre-existing CD4+ T cell immunity (using ovalbumin-OVA) or virus-induced pre-existing CD4+ T cell immunity (murine cytomegalovirus-MCMV). Following the establishment of pre-existing by vaccination (OVA in the adjuvant TiterMax® Gold) or infection with MCMV, mice were administered CSP-coated liposomal vaccines containing the relevant OVA or MCMV core CD4+ T cell epitopes. In mice with pre-existing anti-OVA CD4+ T cell immunity, these vaccine particles elicited rapid, high-titre, isotype-switched CSP-specific antibody responses-consistent with the involvement of anti-OVA T helper cells in confirming activation of anti-CSP B cells. Responses were further improved by entrapping TLR9 agonists, combining humoral vaccination signals 'one', 'two' and 'three' within one particle. Herpes viruses can establish chronic infection and elicit significant, persistent cellular immune responses. We then demonstrate that this principle can be extended to re-purpose pre-existing anti-MCMV immunity to enhance anti-CSP vaccine responses-the first description of a strategy to specifically exploit anti-cytomegalovirus immunity to augment vaccination against a target antigen.

Multifactorial analysis of human blood cell responses to clinical total body irradiation

NASA Technical Reports Server (NTRS)

Yuhas, J. M.; Stokes, T. R.; Lushbaugh, C. C.

1972-01-01

Multiple regression analysis techniques are used to study the effects of therapeutic radiation exposure, number of fractions, and time on such quantal responses as tumor control and skin injury. The potential of these methods for the analysis of human blood cell responses is demonstrated and estimates are given of the effects of total amount of exposure and time of protraction in determining the minimum white blood cell concentration observed after exposure of patients from four disease groups.

Histone deacetylase inhibitors prevent activation-induced cell death and promote anti-tumor immunity

PubMed Central

Cao, K; Wang, G; Li, W; Zhang, L; Wang, R; Huang, Y; Du, L; Jiang, J; Wu, C; He, X; Roberts, A I; Li, F; Rabson, A B; Wang, Y; Shi, Y

2015-01-01

The poor efficacy of the in vivo anti-tumor immune response has been partially attributed to ineffective T-cell responses mounted against the tumor. Fas-FasL-dependent activation-induced cell death (AICD) of T cells is believed to be a major contributor to compromised anti-tumor immunity. The molecular mechanisms of AICD are well-investigated, yet the possibility of regulating AICD for cancer therapy remains to be explored. In this study, we show that histone deacetylase inhibitors (HDACIs) can inhibit apoptosis of CD4+ T cells within the tumor, thereby enhancing anti-tumor immune responses and suppressing melanoma growth. This inhibitory effect is specific for AICD through suppressing NFAT1-regulated FasL expression on activated CD4+ T cells. In gld/gld mice with mutation in FasL, the beneficial effect of HDACIs on AICD of infiltrating CD4+ T cells is not seen, confirming the critical role of FasL regulation in the anti-tumor effect of HDACIs. Importantly, we found that the co-administration of HDACIs and anti-CTLA4 could further enhance the infiltration of CD4+ T cells and achieve a synergistic therapeutic effect on tumor. Therefore, our study demonstrates that the modulation of AICD of tumor-infiltrating CD4+ T cells using HDACIs can enhance anti-tumor immune responses, uncovering a novel mechanism underlying the anti-tumor effect of HDACIs. PMID:25745993

Dextromethorphan mediated bitter taste receptor activation in the pulmonary circuit causes vasoconstriction.

PubMed

Upadhyaya, Jasbir D; Singh, Nisha; Sikarwar, Anurag S; Chakraborty, Raja; Pydi, Sai P; Bhullar, Rajinder P; Dakshinamurti, Shyamala; Chelikani, Prashen

2014-01-01

Activation of bitter taste receptors (T2Rs) in human airway smooth muscle cells leads to muscle relaxation and bronchodilation. This finding led to our hypothesis that T2Rs are expressed in human pulmonary artery smooth muscle cells and might be involved in regulating the vascular tone. RT-PCR was performed to reveal the expression of T2Rs in human pulmonary artery smooth muscle cells. Of the 25 T2Rs, 21 were expressed in these cells. Functional characterization was done by calcium imaging after stimulating the cells with different bitter agonists. Increased calcium responses were observed with most of the agonists, the largest increase seen for dextromethorphan. Previously in site-directed mutational studies, we have characterized the response of T2R1 to dextromethorphan, therefore, T2R1 was selected for further analysis in this study. Knockdown with T2R1 specific shRNA decreased mRNA levels, protein levels and dextromethorphan-induced calcium responses in pulmonary artery smooth muscle cells by up to 50%. To analyze if T2Rs are involved in regulating the pulmonary vascular tone, ex vivo studies using pulmonary arterial and airway rings were pursued. Myographic studies using porcine pulmonary arterial and airway rings showed that stimulation with dextromethorphan led to contraction of the pulmonary arterial and relaxation of the airway rings. This study shows that dextromethorphan, acting through T2R1, causes vasoconstrictor responses in the pulmonary circuit and relaxation in the airways.

Male Rat Germ Cells Display Age-Dependent and Cell-Specific Susceptibility in Response to Oxidative Stress Challenges1

PubMed Central

Selvaratnam, Johanna; Paul, Catriona; Robaire, Bernard

2015-01-01

For decades male germ cells were considered unaffected by aging, due to the fact that males continue to generate sperm into old age; however, evidence indicates that germ cells from aged males are of lower quality than those of young males. The current study examines the effects of aging on pachytene spermatocytes and round spermatids, and is the first study to culture these cells in isolation for an extended period. Our objective is to determine the cell-specific responses germ cells have to aging and oxidative insult. Culturing isolated germ cells from young and aged Brown Norway rats revealed that germ cells from aged males displayed an earlier decline in viability, elevated levels of reactive oxygen species (ROS), and increased spermatocyte DNA damage, compared to young males. Furthermore, oxidative insult by prooxidant 3-morpholinosydnonimine provides insight into how spermatocytes and spermatids manage excess ROS. Genome-wide microarray analyses revealed that several transcripts for antioxidants, Sod1, Cat, and Prdxs, were up-regulated in response to ROS in germ cells from young males while being expressed at lower levels in the aged. In contrast, the expression of DNA damage repair genes Rad50 and Atm were increased in the germ cells from aged animals. Our data indicate that as germ cells undergo spermatogenesis, they adapt and respond to oxidative stress differently, depending on their phase of development, and the process of aging results in redox dysfunction. Thus, even at early stages of spermatogenesis, germ cells from aged males are unable to mount an appropriate response to manage oxidative stress. PMID:26224006

Cerebral regulatory T cells restrain microglia/macrophage-mediated inflammatory responses via IL-10.

PubMed

Xie, Luokun; Choudhury, Gourav Roy; Winters, Ali; Yang, Shao-Hua; Jin, Kunlin

2015-01-01

Forkhead box P3 (Foxp3)(+) regulatory T (Treg) cells maintain the immune tolerance and prevent inflammatory responses in the periphery. However, the presence of Treg cells in the CNS under steady state has not been studied. Here, for the first time, we show a substantial TCRαβ (+) CD4(+) Foxp3(+) T-cell population (cerebral Treg cells) in the rat cerebrum, constituting more than 15% of the cerebral CD4(+) T-cell compartment. Cerebral Treg cells showed an activated/memory phenotype and expressed many Treg-cell signature genes at higher levels than peripheral Treg cells. Consistent with their activated/memory phenotype, cerebral Treg cells robustly restrained the LPS-induced inflammatory responses of brain microglia/macrophages, suggesting a role in maintaining the cerebral homeostasis by inhibiting the neuroinflammation. In addition, brain astrocytes were the helper cells that sustained Foxp3 expression in Treg cells through IL-2/STAT5 signaling, showing that the interaction between astrocytes and Treg cells contributes to the maintenance of Treg-cell identity in the brain. Taken together, our work represents the first study to characterize the phenotypic and functional features of Treg cells in the rat cerebrum. Our data have provided a novel insight for the contribution of Treg cells to the immunosurveillance and immunomodulation in the cerebrum under steady state. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cerebral regulatory T cells restrain microglia/macrophage-mediated inflammatory responses via IL-10

PubMed Central

Xie, Luokun; Choudhury, Gourav Roy; Winters, Ali; Yang, Shao-Hua; Jin, Kunlin

2014-01-01

Forkhead box P3 (Foxp3)+ regulatory T (Treg) cells maintain the immune tolerance and prevent inflammatory responses in the periphery. However, the presence of Treg cells in the central nervous system under steady state has not been studied. Here, for the first time, we show a substantial TCRαβ+CD4+Foxp3+ T-cell population (cerebral Treg cells) in the normal rat cerebrum, constituting more than 15% of the cerebral CD4+ T-cell compartment. Cerebral Treg cells showed an activated/memory phenotype and expressed many Treg-cell signature genes at higher levels than peripheral Treg cells. Consistent with their activated/memory phenotype, cerebral Treg cells robustly restrained the LPS-induced inflammatory responses of brain microglia/macrophages, suggesting a role in maintaining the cerebral homeostasis by inhibiting the neuroinflammation. In addition, brain astrocytes were the helper cells that sustained Foxp3 expression in Treg cells through IL-2/STAT5 signaling, showing that the interaction between astrocytes and Treg cells contributes to the maintenance of Treg-cell identity in the brain. Taken together, our work represents the first study to characterize the phenotypic and functional features of Treg cells in the normal rat cerebrum. Our data have provided a novel insight for the contribution of Treg cells to the immunosurveillance and immunomodulation in the cerebrum under steady state. PMID:25329858

A Tunable Diffusion-Consumption Mechanism of Cytokine Propagation Enables Plasticity in Cell-to-Cell Communication in the Immune System.

PubMed

Oyler-Yaniv, Alon; Oyler-Yaniv, Jennifer; Whitlock, Benjamin M; Liu, Zhiduo; Germain, Ronald N; Huse, Morgan; Altan-Bonnet, Grégoire; Krichevsky, Oleg

2017-04-18

Immune cells communicate by exchanging cytokines to achieve a context-appropriate response, but the distances over which such communication happens are not known. Here, we used theoretical considerations and experimental models of immune responses in vitro and in vivo to quantify the spatial extent of cytokine communications in dense tissues. We established that competition between cytokine diffusion and consumption generated spatial niches of high cytokine concentrations with sharp boundaries. The size of these self-assembled niches scaled with the density of cytokine-consuming cells, a parameter that gets tuned during immune responses. In vivo, we measured interactions on length scales of 80-120 μm, which resulted in a high degree of cell-to-cell variance in cytokine exposure. Such heterogeneous distributions of cytokines were a source of non-genetic cell-to-cell variability that is often overlooked in single-cell studies. Our findings thus provide a basis for understanding variability in the patterning of immune responses by diffusible factors. Published by Elsevier Inc.

X-ray-induced bystander responses reduce spontaneous mutations in V79 cells

PubMed Central

Maeda, Munetoshi; Kobayashi, Katsumi; Matsumoto, Hideki; Usami, Noriko; Tomita, Masanori

2013-01-01

The potential for carcinogenic risks is increased by radiation-induced bystander responses; these responses are the biological effects in unirradiated cells that receive signals from the neighboring irradiated cells. Bystander responses have attracted attention in modern radiobiology because they are characterized by non-linear responses to low-dose radiation. We used a synchrotron X-ray microbeam irradiation system developed at the Photon Factory, High Energy Accelerator Research Organization, KEK, and showed that nitric oxide (NO)-mediated bystander cell death increased biphasically in a dose-dependent manner. Here, we irradiated five cell nuclei using 10 × 10 µm2 5.35 keV X-ray beams and then measured the mutation frequency at the hypoxanthine-guanosine phosphoribosyl transferase (HPRT) locus in bystander cells. The mutation frequency with the null radiation dose was 2.6 × 10–5 (background level), and the frequency decreased to 5.3 × 10–6 with a dose of approximately 1 Gy (absorbed dose in the nucleus of irradiated cells). At high doses, the mutation frequency returned to the background level. A similar biphasic dose-response effect was observed for bystander cell death. Furthermore, we found that incubation with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), a specific scavenger of NO, suppressed not only the biphasic increase in bystander cell death but also the biphasic reduction in mutation frequency of bystander cells. These results indicate that the increase in bystander cell death involves mechanisms that suppress mutagenesis. This study has thus shown that radiation-induced bystander responses could affect processes that protect the cell against naturally occurring alterations such as mutations. PMID:23660275

Alkaline ceramidase 2 and its bioactive product sphingosine are novel regulators of the DNA damage response

PubMed Central

Xu, Ruijuan; Wang, Kai; Mileva, Izolda; Hannun, Yusuf A.; Obeid, Lina M.; Mao, Cungui

2016-01-01

Human cells respond to DNA damage by elevating sphingosine, a bioactive sphingolipid that induces programmed cell death (PCD) in response to various forms of stress, but its regulation and role in the DNA damage response remain obscure. Herein we demonstrate that DNA damage increases sphingosine levels in tumor cells by upregulating alkaline ceramidase 2 (ACER2) and that the upregulation of the ACER2/sphingosine pathway induces PCD in response to DNA damage by increasing the production of reactive oxygen species (ROS). Treatment with the DNA damaging agent doxorubicin increased both ACER2 expression and sphingosine levels in HCT116 cells in a dose-dependent manner. ACER2 overexpression increased sphingosine in HeLa cells whereas knocking down ACER2 inhibited the doxorubicin-induced increase in sphingosine in HCT116 cells, suggesting that DNA damage elevates sphingosine by upregulating ACER2. Knocking down ACER2 inhibited an increase in the apoptotic and necrotic cell population and the cleavage of poly ADP ribose polymerase (PARP) in HCT116 cells in response to doxorubicin as well as doxorubicin-induced release of lactate dehydrogenase (LDH) from these cells. Similar to treatment with doxorubicin, ACER2 overexpression induced an increase in the apoptotic and necrotic cell population and PARP cleavage in HeLa cells and LDH release from cells, suggesting that ACER2 upregulation mediates PCD in response to DNA damage through sphingosine. Mechanistic studies demonstrated that the upregulation of the ACER2/sphingosine pathway induces PCD by increasing ROS levels. Taken together, these results suggest that the ACER2/sphingosine pathway mediates PCD in response to DNA damage through ROS production. PMID:26943039

Heterogeneity of T Cell Responses to Pandemic pH1N1 Monovalent Vaccine in HIV-Infected Pregnant Women.

PubMed

Weinberg, Adriana; Muresan, Petronella; Richardson, Kelly; Fenton, Terence; Dominguez, Teresa; Bloom, Anthony; Watts, D Heather; Abzug, Mark J; Nachman, Sharon A; Levin, Myron J

2015-11-01

We investigated the Th1 protective and regulatory T and B cell (Treg and Breg) responses to pH1N1 monovalent influenza vaccine (IIV1) in HIV-infected pregnant women on combination antiretroviral therapy (cART). Peripheral blood mononuclear cells (PBMCs) from 52 study participants were cryopreserved before and after vaccination and analyzed by flow cytometry. pH1N1-specific Th1, Treg, and Breg responses were measured in PBMCs after in vitro stimulation with pH1N1 and control antigen. The cohort analysis did not detect changes in pH1N1-Th1, Treg, or Breg subsets postvaccination. However, individual analyses distinguished subjects who mounted vigorous Th1 responses postvaccination from others who did not. Postvaccination, high pH1N1-Th1 correlated with high pH1N1-Treg and Breg responses, suggesting that low influenza effector responses did not result from excessive vaccine-induced immune regulation. High postvaccination pH1N1-Th1 responses correlated with baseline high PHA- and pH1N1-IFN-γ ELISpot and circulating CD4(+)CD39(+)% and CD8(+)CD39(+)% Treg, with low CD8(+) cell numbers and CD19(+)FOXP3(+)% Breg, but not with CD4(+) cell numbers or HIV viral load. These data highlight the heterogeneity of T cell responses to vaccines in HIV-infected individuals on cART. Predictors of robust Th1 responses to IIV include CD8(+) cell numbers, T cell functionality, and circulating Breg and Treg.

CD70-deficiency impairs effector CD8 T cell generation and viral clearance but is dispensable for the recall response to LCMV

PubMed Central

Munitic, Ivana; Kuka, Mirela; Allam, Atef; Scoville, Jonathan P.; Ashwell, Jonathan D.

2012-01-01

CD27 interactions with its ligand, CD70, are thought to be necessary for optimal primary and memory adaptive immune responses to a variety of pathogens. Thus far all studies addressing the function of the CD27-CD70 axis have been performed either in mice lacking CD27, overexpressing CD70, or in which these receptors were blocked or mimicked by antibodies or recombinant soluble CD70. Because these methods have in some cases led to divergent results, we generated CD70-deficient mice to directly assess its role in vivo. We find that lack of CD70-mediated stimulation during primary responses to LCMV lowered the magnitude of CD8 antigen-specific T cell response, resulting in impaired viral clearance, without affecting CD4 T cell responses. Unexpectedly, CD70-CD27 costimulation was not needed for memory CD8 T cell generation or the ability to mount a recall response to LCMV. Adoptive transfers of wild type (WT) memory T cells into CD70−/− or WT hosts also showed no need for CD70-mediated stimulation during the course of the recall response. Moreover, CD70-expression by CD8 T cells could not rescue endogenous CD70−/− cells from defective expansion, arguing against a role for CD70-mediated T:T help in this model. Therefore, CD70 appears to be an important factor in the initiation of a robust and effective primary response but dispensable for CD8 T cell memory responses. PMID:23269247

Definition of a pool of epitopes that recapitulates the T cell reactivity against major house dust mite allergens.

PubMed

Hinz, D; Oseroff, C; Pham, J; Sidney, J; Peters, B; Sette, A

2015-10-01

Allergens from house dust mites (HDM) are a common cause of asthma. Der p and Der f from Dermatophagoides sp. are strong immunogens in humans. Allergen extracts are used to study T helper (Th2) cell responses to HDM, which are implicated in the development and regulation of allergic disease. To define an epitope mixture that recapitulates, and might substitute for, HDM extract in terms of detecting and characterizing Th2 cell responses. Peripheral blood mononuclear cells (PBMC) from 52 HDM allergic and 10 non-allergic individuals were stimulated with HDM extracts and assayed with a set of 178 peptides spanning mite allergens group Der p 1, 2, 23 and Der f group 1 and 2 allergens. A pool of the most dominant T cell epitopes identified in the present study and from published literature was assembled and tested for ex vivo T cell responses. Correlation with HDM-specific IgE titres was examined. Patterns of T cell reactivity to Der p and Der f - derived peptides revealed a large number of epitopes. Clear patterns of immunodominance were apparent, with HDM allergen group 1 and 2 dominant over group 23. Furthermore, within a given antigen, 6-11 epitopes accounted for the vast majority of responses. Based on these results and published data, a comprehensive dust mite pool (DMP) of epitopes was designed and found to allow detection of ex vivo T cell responses. DMP ex vivo reactivity correlated with HDM-specific IgE titres and was similar to that detected with commonly used HDM extracts. Ex vivo DMP stimulation was associated with a predominant Th2 response in allergic donors, and minor reactivity of T cells producing IFNγ, IL17 and IL10. A detailed map of Der p and Der f antigens defined a pool of epitopes that can be used to detect ex vivo HDM responses. © 2015 John Wiley & Sons Ltd.

Kidney dendritic cells in acute and chronic renal disease.

PubMed

Hochheiser, Katharina; Tittel, André; Kurts, Christian

2011-06-01

Dendritic cells are not only the master regulators of adaptive immunity, but also participate profoundly in innate immune responses. Much has been learned about their basic immunological functions and their roles in various diseases. Comparatively little is still known about their role in renal disease, despite their obvious potential to affect immune responses in the kidney, and immune responses that are directed against renal components. Kidney dendritic cells form an abundant network in the renal tubulointerstitium and constantly survey the environment for signs of injury or infection, in order to alert the immune system to the need to initiate defensive action. Recent studies have identified a role for dendritic cells in several murine models of acute renal injury and chronic nephritis. Here we summarize the current knowledge on the role of kidney dendritic cells that has been obtained from the study of murine models of renal disease. © 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.

Peripheral T cell lymphomas: an immunological study of seven unusual cases.

PubMed

Raziuddin, S; Latif, A B; Arif, S; Ahad, A; Zaidi, A Z

1988-05-01

A multiparameter study of malignant lymph node cells and peripheral blood lymphocytes of seven patients with peripheral T cell lymphoma is presented. The results of monoclonal marker studies showed three cases of helper-suppressor T cell lymphoma (OKT4+, OKT8+), one case of suppressor T cell lymphoma (OKT8+), and three cases of helper T cell lymphoma (OKT4+). Immunophenotypic heterogeneity of neoplastic T cells with expression of pan-T antigens, OKT3+, and OKT11+ (erythrocyte rosetting+) was observed in most patients. Six of the seven cases tested showed Ia and DR antigens. No relationship was detected between patterns of reactivity with T cell reagents and histological types. When tested, the in-vitro malignant T cells of five patients proliferated in response to concanavalin A (Con A), but had poor response to phytohaemagglutinin. The interleukin 2 receptors showed maximum expression on Con A-activated T cells of five patients, and phytohaemagglutinin-activated T cells of one patient. The neoplastic T cells (OKT4+, OKT8+) of one patient studied had suppressor activity for IgG and IgA, and helper activity for IgM synthesis on pokeweed mitogen-induced normal B cell differentiations.

Tumoral expression of IL-33 inhibits tumor growth and modifies the tumor microenvironment through CD8+ T and NK cells.

PubMed

Gao, Xin; Wang, Xuefeng; Yang, Qianting; Zhao, Xin; Wen, Wen; Li, Gang; Lu, Junfeng; Qin, Wenxin; Qi, Yuan; Xie, Fang; Jiang, Jingting; Wu, Changping; Zhang, Xueguang; Chen, Xinchun; Turnquist, Heth; Zhu, Yibei; Lu, Binfeng

2015-01-01

Cancer immunotherapy has shown great promise as a new standard cancer therapeutic modality. However, the response rates are limited for current approach that depends on enhancing spontaneous antitumor immune responses. Therefore, increasing tumor immunogenicity by expressing appropriate cytokines should further improve the current immunotherapy. IL-33 is a member of the IL-1 family of cytokines and is released by necrotic epithelial cells or activated innate immune cells and is thus considered a "danger" signal. The role of IL-33 in promoting type 2 immune responses and tissue inflammation has been well established. However, whether IL-33 drives antitumor immune responses is controversial. Our previous work established that IL-33 promoted the function of CD8(+) T cells. In this study, we showed that the expression of IL-33 in two types of cancer cells potently inhibited tumor growth and metastasis. Mechanistically, IL-33 increased numbers and IFN-γ production by CD8(+) T and NK cells in tumor tissues, thereby inducing a tumor microenvironment favoring tumor eradication. Importantly, IL-33 greatly increased tumor Ag-specific CD8(+) T cells. Furthermore, both NK and CD8(+) T cells were required for the antitumor effect of IL-33. Moreover, depletion of regulatory T cells worked synergistically with IL-33 expression for tumor elimination. Our studies established "alarmin" IL-33 as a promising new cytokine for tumor immunotherapy through promoting cancer-eradicating type 1 immune responses. Copyright © 2014 by The American Association of Immunologists, Inc.

The biological and practical significance of antigenic variability in protective T cell responses against Theileria parva.

PubMed

Morrison, W I

2007-08-19

The evolution of antigenically distinct pathogen strains that fail to cross-protect is well documented for pathogens controlled primarily by humoral immune responses. Unlike antibodies, which recognise native proteins, protective T cells can potentially recognise epitopes in a variety of proteins that are not necessarily displayed on the pathogen surface. Moreover, individual hosts of different MHC genotypes generally respond to different sets of epitopes. It is therefore less easy to envisage how strain restricted immunity can arise for pathogens controlled by T cell responses, particularly in antigenically complex parasites. Nevertheless, strain restricted immunity is clearly a feature of a number of parasitic infections, where immunity is known to be mediated by T cell responses. One such parasite is Theileria parva which induces potent CD8 T cell responses that play an important role in immunity. CD8 T cells specific for parasitized lymphoblasts exhibit strain specificity, which appears to correlate with the ability of parasite strains to cross-protect. Studies using recently identified T. parva antigens recognised by CD8 T cells have shown that the strain restricted nature of immunity is a consequence of the CD8 T cell response in individual animals being focused on a limited number of dominant polymorphic antigenic determinants. Responses in animals of different MHC genotypes are often directed to different parasite antigens, indicating that, at the host population level, a larger number of parasite proteins can serve as targets for the protective T cell response. Nevertheless, the finding that parasite strains show overlapping antigenic profiles, probably as a consequence of sexual recombination, suggests that induction of responses to an extended but limited set of antigens in individual animals may overcome the strain restricted nature of immunity.

Quantitative regulation of B cell division destiny by signal strength.

PubMed

Turner, Marian L; Hawkins, Edwin D; Hodgkin, Philip D

2008-07-01

Differentiation to Ab secreting and isotype-switched effector cells is tightly linked to cell division and therefore the degree of proliferation strongly influences the nature of the immune response. The maximum number of divisions reached, termed the population division destiny, is stochastically distributed in the population and is an important parameter in the quantitative outcome of lymphocyte responses. In this study, we further assessed the variables that regulate B cell division destiny in vitro in response to T cell- and TLR-dependent stimuli. Both the concentration and duration of stimulation were able to regulate the average maximum number of divisions undergone for each stimulus. Notably, a maximum division destiny was reached during provision of repeated saturating stimulation, revealing that an intrinsic limit to proliferation exists even under these conditions. This limit was linked directly to division number rather than time of exposure to stimulation and operated independently of the survival regulation of the cells. These results demonstrate that a B cell population's division destiny is regulable by the stimulatory conditions up to an inherent maximum value. Division destiny is a crucial parameter in regulating the extent of B cell responses and thereby also the nature of the immune response mounted.

Hsp70 enhances presentation of FMDV antigen to bovine CD4+ T cells in vitro

PubMed Central

McLaughlin, Kerry; Seago, Julian; Robinson, Lucy; Kelly, Charles; Charleston, Bryan

2010-01-01

Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious acute vesicular disease affecting cloven-hoofed animals, including cattle, sheep and pigs. The current vaccine induces a rapid humoral response, but the duration of the protective antibody response is variable, possibly associated with a variable specific CD4+ T cell response. We investigated the use of heat shock protein 70 (Hsp70) as a molecular chaperone to target viral antigen to the Major Histocompatibility Complex (MHC) class II pathway of antigen presenting cells and generate enhanced MHC II-restricted CD4+ T cell responses in cattle. Monocytes and CD4+ T cells from FMDV vaccinated cattle were stimulated in vitro with complexes of Hsp70 and FMDV peptide, or peptide alone. Hsp70 was found to consistently improve the presentation of a 25-mer FMDV peptide to CD4+ T cells, as measured by T cell proliferation. Complex formation was required for the enhanced effects and Hsp70 alone did not stimulate proliferation. This study provides further evidence that Hsp70:peptide complexes can enhance antigen-specific CD4+ T cell responses in vitro for an important pathogen of livestock. PMID:20167197

Hepatocyte-derived exosomes promote T follicular regulatory cell expansion during hepatitis C virus infection.

PubMed

Cobb, Dustin A; Kim, Ok-Kyung; Golden-Mason, Lucy; Rosen, Hugo R; Hahn, Young S

2018-01-01

Hepatitis C virus (HCV) is a global health concern that can cause severe liver disease, such as cirrhosis and hepatocellular carcinoma. Control of HCV requires vigorous T-cell responses, yet CD4 + T cells in chronic HCV patients are dysfunctional. T follicular regulatory (Tfr) cells are a subset of regulatory T cells that suppress T follicular helper (Tfh) cells and the generation of high affinity antibody-producing B cells. In this study, we examined the accumulation of Tfr cells in the liver compartment during chronic HCV infection and defined the cellular and molecular mechanisms underlying their expansion. Our analysis revealed a substantial population of Tfr cells in livers of chronic HCV patients that is absent in liver tissues from nonviral hepatitis or healthy subjects. Coculture of PBMCs from healthy subjects with HCV-infected hepatoma cells resulted in preferential expansion of circulating Tfr cells, leading to suppression of Tfh cells. Additionally, coculture of tonsillar cells with infected hepatoma cells lead to an expansion of germinal center Tfr. Notably, expansion was mediated by transforming growth factor beta (TGF-β)-containing exosomes released from HCV-infected hepatocytes given that blockade of exosome-associated TGF-β or inhibition of exosome release abrogated Tfr expansion. These results show that liver-derived exosomes play a pivotal role in the accumulation of Tfr cells, likely leading to suppression of Tfh responses in HCV-infected patients. Our study identifies a novel pathway in which HCV infection in hepatocytes exacerbates Tfr cell responses to subvert antiviral immunity. (Hepatology 2018;67:71-85). © 2017 by the American Association for the Study of Liver Diseases.

High-throughput autofluorescence flow cytometry of breast cancer metabolism (Conference Presentation)

NASA Astrophysics Data System (ADS)

Shah, Amy T.; Cannon, Taylor M.; Higginbotham, Jim N.; Skala, Melissa C.

2016-02-01

Tumor heterogeneity poses challenges for devising optimal treatment regimens for cancer patients. In particular, subpopulations of cells can escape treatment and cause relapse. There is a need for methods to characterize tumor heterogeneity of treatment response. Cell metabolism is altered in cancer (Warburg effect), and cells use the autofluorescent cofactor NADH in numerous metabolic reactions. Previous studies have shown that microscopy measurements of NADH autofluorescence are sensitive to treatment response in breast cancer, and these techniques typically assess hundreds of cells per group. An alternative approach is flow cytometry, which measures fluorescence on a single-cell level and is attractive for characterizing tumor heterogeneity because it achieves high-throughput analysis and cell sorting in millions of cells per group. Current applications for flow cytometry rely on staining with fluorophores. This study characterizes flow cytometry measurements of NADH autofluorescence in breast cancer cells. Preliminary results indicate flow cytometry of NADH is sensitive to cyanide perturbation, which inhibits oxidative phosphorylation, in nonmalignant MCF10A cells. Additionally, flow cytometry is sensitive to higher NADH intensity for HER2-positive SKBr3 cells compared with triple-negative MDA-MB-231 cells. These results agree with previous microscopy studies. Finally, a mixture of SKBr3 and MDA-MB-231 cells were sorted into each cell type using NADH intensity. Sorted cells were cultured, and microscopy validation showed the expected morphology for each cell type. Ultimately, flow cytometry could be applied to characterize tumor heterogeneity based on treatment response and sort cell subpopulations based on metabolic profile. These achievements could enable individualized treatment strategies and improved patient outcomes.

Multi-scale modeling of the CD8 immune response

NASA Astrophysics Data System (ADS)

Barbarroux, Loic; Michel, Philippe; Adimy, Mostafa; Crauste, Fabien

2016-06-01

During the primary CD8 T-Cell immune response to an intracellular pathogen, CD8 T-Cells undergo exponential proliferation and continuous differentiation, acquiring cytotoxic capabilities to address the infection and memorize the corresponding antigen. After cleaning the organism, the only CD8 T-Cells left are antigen-specific memory cells whose role is to respond stronger and faster in case they are presented this very same antigen again. That is how vaccines work: a small quantity of a weakened pathogen is introduced in the organism to trigger the primary response, generating corresponding memory cells in the process, giving the organism a way to defend himself in case it encounters the same pathogen again. To investigate this process, we propose a non linear, multi-scale mathematical model of the CD8 T-Cells immune response due to vaccination using a maturity structured partial differential equation. At the intracellular scale, the level of expression of key proteins is modeled by a delay differential equation system, which gives the speeds of maturation for each cell. The population of cells is modeled by a maturity structured equation whose speeds are given by the intracellular model. We focus here on building the model, as well as its asymptotic study. Finally, we display numerical simulations showing the model can reproduce the biological dynamics of the cell population for both the primary response and the secondary responses.

Multi-scale modeling of the CD8 immune response

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barbarroux, Loic, E-mail: loic.barbarroux@doctorant.ec-lyon.fr; Ecole Centrale de Lyon, 36 avenue Guy de Collongue, 69134 Ecully; Michel, Philippe, E-mail: philippe.michel@ec-lyon.fr

During the primary CD8 T-Cell immune response to an intracellular pathogen, CD8 T-Cells undergo exponential proliferation and continuous differentiation, acquiring cytotoxic capabilities to address the infection and memorize the corresponding antigen. After cleaning the organism, the only CD8 T-Cells left are antigen-specific memory cells whose role is to respond stronger and faster in case they are presented this very same antigen again. That is how vaccines work: a small quantity of a weakened pathogen is introduced in the organism to trigger the primary response, generating corresponding memory cells in the process, giving the organism a way to defend himself inmore » case it encounters the same pathogen again. To investigate this process, we propose a non linear, multi-scale mathematical model of the CD8 T-Cells immune response due to vaccination using a maturity structured partial differential equation. At the intracellular scale, the level of expression of key proteins is modeled by a delay differential equation system, which gives the speeds of maturation for each cell. The population of cells is modeled by a maturity structured equation whose speeds are given by the intracellular model. We focus here on building the model, as well as its asymptotic study. Finally, we display numerical simulations showing the model can reproduce the biological dynamics of the cell population for both the primary response and the secondary responses.« less

Investigating cell mechanics with atomic force microscopy

PubMed Central

Haase, Kristina; Pelling, Andrew E.

2015-01-01

Transmission of mechanical force is crucial for normal cell development and functioning. However, the process of mechanotransduction cannot be studied in isolation from cell mechanics. Thus, in order to understand how cells ‘feel’, we must first understand how they deform and recover from physical perturbations. Owing to its versatility, atomic force microscopy (AFM) has become a popular tool to study intrinsic cellular mechanical properties. Used to directly manipulate and examine whole and subcellular reactions, AFM allows for top-down and reconstitutive approaches to mechanical characterization. These studies show that the responses of cells and their components are complex, and largely depend on the magnitude and time scale of loading. In this review, we generally describe the mechanotransductive process through discussion of well-known mechanosensors. We then focus on discussion of recent examples where AFM is used to specifically probe the elastic and inelastic responses of single cells undergoing deformation. We present a brief overview of classical and current models often used to characterize observed cellular phenomena in response to force. Both simple mechanistic models and complex nonlinear models have been used to describe the observed cellular behaviours, however a unifying description of cell mechanics has not yet been resolved. PMID:25589563

Using Brillouin microspectroscopy to characterize adipocytes' response to lipid droplet accumulation

NASA Astrophysics Data System (ADS)

Troyanova-Wood, Maria; Coker, Zachary; Traverso, Andrew; Yakovlev, Vladislav V.

2017-02-01

Obesity and overweight are accompanied by an enlargement of adipocytes, which is commonly related to the increasing number or size of lipid droplets within the cells. Some studies have shown that the accumulation of lipid droplets within adipocytes results in their increased stiffness. Recently, Brillouin microspectroscopy has been introduced as a nondestructive method of imaging the elasticity of cells. Unlike other imaging modalities, it is capable of assessing the elastic properties on both tissue- and cell levels. In this study, Brillouin spectroscopy was used to measure the elasticity changes in response to accumulation of lipid droplets within adipocyte during adipogenesis. The cell line used in the study is 3T3-L1, with chemically-induced differentiation from pre-adipocytes to mature adipocytes. The Brillouin shift measurements of the cells before and after differentiation indicate that the stiffness of adipocytes increases due to accumulation of lipid droplets. The results are in agreement with previous atomic force microscopy (AFM) nanoindentation studies. Brillouin microspectroscopy is a technique suitable for measuring the changes of elasticity of adipocytes in response to lipid droplet accumulation.

The effect of veiled cells on lymphocyte function.

PubMed

Balfour, B; O'Brien, J A; Perera, M; Clarke, J; Sumerska, T; Knight, S C

1982-01-01

The response of lymphocytes to stimulation with Con A has been studied in the presence of veiled cells collected from the afferent lymph. In these enriched cultures the response occurred earlier with smaller numbers of lymphocytes and at lower concentrations of Con A. Veiled cells also caused clumping of lymphocytes in unstimulated cultures. In stimulated cultures small cells with veil-like projections appeared after 48 hr, but were not seen in unstimulated cultures.

A stochastic chemical dynamic approach to correlate autoimmunity and optimal vitamin-D range.

PubMed

Roy, Susmita; Shrinivas, Krishna; Bagchi, Biman

2014-01-01

Motivated by several recent experimental observations that vitamin-D could interact with antigen presenting cells (APCs) and T-lymphocyte cells (T-cells) to promote and to regulate different stages of immune response, we developed a coarse grained but general kinetic model in an attempt to capture the role of vitamin-D in immunomodulatory responses. Our kinetic model, developed using the ideas of chemical network theory, leads to a system of nine coupled equations that we solve both by direct and by stochastic (Gillespie) methods. Both the analyses consistently provide detail information on the dependence of immune response to the variation of critical rate parameters. We find that although vitamin-D plays a negligible role in the initial immune response, it exerts a profound influence in the long term, especially in helping the system to achieve a new, stable steady state. The study explores the role of vitamin-D in preserving an observed bistability in the phase diagram (spanned by system parameters) of immune regulation, thus allowing the response to tolerate a wide range of pathogenic stimulation which could help in resisting autoimmune diseases. We also study how vitamin-D affects the time dependent population of dendritic cells that connect between innate and adaptive immune responses. Variations in dose dependent response of anti-inflammatory and pro-inflammatory T-cell populations to vitamin-D correlate well with recent experimental results. Our kinetic model allows for an estimation of the range of optimum level of vitamin-D required for smooth functioning of the immune system and for control of both hyper-regulation and inflammation. Most importantly, the present study reveals that an overdose or toxic level of vitamin-D or any steroid analogue could give rise to too large a tolerant response, leading to an inefficacy in adaptive immune function.

Systemic Analysis of Heat Shock Response Induced by Heat Shock and a Proteasome Inhibitor MG132

PubMed Central

Kim, Hee-Jung; Joo, Hye Joon; Kim, Yung Hee; Ahn, Soyeon; Chang, Jun; Hwang, Kyu-Baek; Lee, Dong-Hee; Lee, Kong-Joo

2011-01-01

The molecular basis of heat shock response (HSR), a cellular defense mechanism against various stresses, is not well understood. In this, the first comprehensive analysis of gene expression changes in response to heat shock and MG132 (a proteasome inhibitor), both of which are known to induce heat shock proteins (Hsps), we compared the responses of normal mouse fibrosarcoma cell line, RIF- 1, and its thermotolerant variant cell line, TR-RIF-1 (TR), to the two stresses. The cellular responses we examined included Hsp expressions, cell viability, total protein synthesis patterns, and accumulation of poly-ubiquitinated proteins. We also compared the mRNA expression profiles and kinetics, in the two cell lines exposed to the two stresses, using microarray analysis. In contrast to RIF-1 cells, TR cells resist heat shock caused changes in cell viability and whole-cell protein synthesis. The patterns of total cellular protein synthesis and accumulation of poly-ubiquitinated proteins in the two cell lines were distinct, depending on the stress and the cell line. Microarray analysis revealed that the gene expression pattern of TR cells was faster and more transient than that of RIF-1 cells, in response to heat shock, while both RIF-1 and TR cells showed similar kinetics of mRNA expression in response to MG132. We also found that 2,208 genes were up-regulated more than 2 fold and could sort them into three groups: 1) genes regulated by both heat shock and MG132, (e.g. chaperones); 2) those regulated only by heat shock (e.g. DNA binding proteins including histones); and 3) those regulated only by MG132 (e.g. innate immunity and defense related molecules). This study shows that heat shock and MG132 share some aspects of HSR signaling pathway, at the same time, inducing distinct stress response signaling pathways, triggered by distinct abnormal proteins. PMID:21738571

Induction of IL21 in Peripheral T Follicular Helper Cells Is an Indicator of Influenza Vaccine Response in a Previously Vaccinated HIV-Infected Pediatric Cohort.

PubMed

de Armas, Lesley R; Cotugno, Nicola; Pallikkuth, Suresh; Pan, Li; Rinaldi, Stefano; Sanchez, M Celeste; Gonzalez, Louis; Cagigi, Alberto; Rossi, Paolo; Palma, Paolo; Pahwa, Savita

2017-03-01

HIV-infected patients of all ages frequently underperform in response to seasonal influenza vaccination, despite virologic control of HIV. The molecular mechanisms governing this impairment, as well as predictive biomarkers for responsiveness, remain unknown. This study was performed in samples obtained prevaccination (T0) from HIV-infected children who received the 2012-2013 seasonal influenza vaccine. Response status was determined based on established criterion for hemagglutination inhibition titer; participants with a hemagglutination titer ≥1:40 plus a ≥4-fold increase over T0 at 3 wk postvaccination were designated as responders. All children had a history of prior influenza vaccinations. At T0, the frequencies of CD4 T cell subsets, including peripheral T follicular helper (pTfh) cells, which provide help to B cells for developing into Ab-secreting cells, were similar between responders and nonresponders. However, in response to in vitro stimulation with influenza A/California/7/2009 (H1N1) Ag, differential gene expression related to pTfh cell function was observed by Fluidigm high-density RT-PCR between responders and nonresponders. In responders, H1N1 stimulation at T0 also resulted in CXCR5 induction (mRNA and protein) in CD4 T cells and IL21 gene induction in pTfh cells that were strongly associated with H1N1-specific B cell responses postvaccination. In contrast, CD4 T cells of nonresponders exhibited increased expression of IL2 and STAT5 genes, which are known to antagonize peripheral Tfh cell function. These results suggest that the quality of pTfh cells at the time of immunization is important for influenza vaccine responses and provide a rationale for targeted, ex vivo Ag-driven molecular profiling of purified immune cells to detect predictive biomarkers of the vaccine response. Copyright © 2017 by The American Association of Immunologists, Inc.

Human and Murine Innate Immune Cell Populations Display Common and Distinct Response Patterns during Their In Vitro Interaction with the Pathogenic Mold Aspergillus fumigatus.

PubMed

Hellmann, Anna-Maria; Lother, Jasmin; Wurster, Sebastian; Lutz, Manfred B; Schmitt, Anna Lena; Morton, Charles Oliver; Eyrich, Matthias; Czakai, Kristin; Einsele, Hermann; Loeffler, Juergen

2017-01-01

Aspergillus fumigatus is the main cause of invasive fungal infections occurring almost exclusively in immunocompromised patients. An improved understanding of the initial innate immune response is key to the development of better diagnostic tools and new treatment options. Mice are commonly used to study immune defense mechanisms during the infection of the mammalian host with A. fumigatus . However, little is known about functional differences between the human and murine immune response against this fungal pathogen. Thus, we performed a comparative functional analysis of human and murine dendritic cells (DCs), macrophages, and polymorphonuclear cells (PMNs) using standardized and reproducible working conditions, laboratory protocols, and readout assays. A. fumigatus did not provoke identical responses in murine and human immune cells but rather initiated relatively specific responses. While human DCs showed a significantly stronger upregulation of their maturation markers and major histocompatibility complex molecules and phagocytosed A. fumigatus more efficiently compared to their murine counterparts, murine PMNs and macrophages exhibited a significantly stronger release of reactive oxygen species after exposure to A. fumigatus . For all studied cell types, human and murine samples differed in their cytokine response to conidia or germ tubes of A. fumigatus . Furthermore, Dectin-1 showed inverse expression patterns on human and murine DCs after fungal stimulation. These specific differences should be carefully considered and highlight potential limitations in the transferability of murine host-pathogen interaction studies.

Human and Murine Innate Immune Cell Populations Display Common and Distinct Response Patterns during Their In Vitro Interaction with the Pathogenic Mold Aspergillus fumigatus

PubMed Central

Hellmann, Anna-Maria; Lother, Jasmin; Wurster, Sebastian; Lutz, Manfred B.; Schmitt, Anna Lena; Morton, Charles Oliver; Eyrich, Matthias; Czakai, Kristin; Einsele, Hermann; Loeffler, Juergen

2017-01-01

Aspergillus fumigatus is the main cause of invasive fungal infections occurring almost exclusively in immunocompromised patients. An improved understanding of the initial innate immune response is key to the development of better diagnostic tools and new treatment options. Mice are commonly used to study immune defense mechanisms during the infection of the mammalian host with A. fumigatus. However, little is known about functional differences between the human and murine immune response against this fungal pathogen. Thus, we performed a comparative functional analysis of human and murine dendritic cells (DCs), macrophages, and polymorphonuclear cells (PMNs) using standardized and reproducible working conditions, laboratory protocols, and readout assays. A. fumigatus did not provoke identical responses in murine and human immune cells but rather initiated relatively specific responses. While human DCs showed a significantly stronger upregulation of their maturation markers and major histocompatibility complex molecules and phagocytosed A. fumigatus more efficiently compared to their murine counterparts, murine PMNs and macrophages exhibited a significantly stronger release of reactive oxygen species after exposure to A. fumigatus. For all studied cell types, human and murine samples differed in their cytokine response to conidia or germ tubes of A. fumigatus. Furthermore, Dectin-1 showed inverse expression patterns on human and murine DCs after fungal stimulation. These specific differences should be carefully considered and highlight potential limitations in the transferability of murine host–pathogen interaction studies. PMID:29270175

Single-Cell Analysis Reveals Early Manifestation of Cancerous Phenotype in Pre-Malignant Esophageal Cells

PubMed Central

Wang, Jiangxin; Shi, Xu; Johnson, Roger H.; Kelbauskas, Laimonas; Zhang, Weiwen; Meldrum, Deirdre R.

2013-01-01

Cellular heterogeneity plays a pivotal role in a variety of functional processes in vivo including carcinogenesis. However, our knowledge about cell-to-cell diversity and how differences in individual cells manifest in alterations at the population level remains very limited mainly due to the lack of appropriate tools enabling studies at the single-cell level. We present a study on changes in cellular heterogeneity in the context of pre-malignant progression in response to hypoxic stress. Utilizing pre-malignant progression of Barrett’s esophagus (BE) as a disease model system we studied molecular mechanisms underlying the progression from metaplastic to dysplastic (pre-cancerous) stage. We used newly developed methods enabling measurements of cell-to-cell differences in copy numbers of mitochondrial DNA, expression levels of a set of mitochondrial and nuclear genes involved in hypoxia response pathways, and mitochondrial membrane potential. In contrast to bulk cell studies reported earlier, our study shows significant differences between metaplastic and dysplastic BE cells in both average values and single-cell parameter distributions of mtDNA copy numbers, mitochondrial function, and mRNA expression levels of studied genes. Based on single-cell data analysis, we propose that mitochondria may be one of the key factors in pre-malignant progression in BE. PMID:24116039

Characteristics of the early immune response following transplantation of mouse ES cell derived insulin-producing cell clusters.

PubMed

Boyd, Ashleigh S; Wood, Kathryn J

2010-06-04

The fully differentiated progeny of ES cells (ESC) may eventually be used for cell replacement therapy (CRT). However, elements of the innate immune system may contribute to damage or destruction of these tissues when transplanted. Herein, we assessed the hitherto ill-defined contribution of the early innate immune response in CRT after transplantation of either ESC derived insulin producing cell clusters (IPCCs) or adult pancreatic islets. Ingress of neutrophil or macrophage cells was noted immediately at the site of IPCC transplantation, but this infiltration was attenuated by day three. Gene profiling identified specific inflammatory cytokines and chemokines that were either absent or sharply reduced by three days after IPCC transplantation. Thus, IPCC transplantation provoked less of an early immune response than pancreatic islet transplantation. Our study offers insights into the characteristics of the immune response of an ESC derived tissue in the incipient stages following transplantation and suggests potential strategies to inhibit cell damage to ensure their long-term perpetuation and functionality in CRT.

Arctigenin from Fructus Arctii is a novel suppressor of heat shock response in mammalian cells

PubMed Central

Ishihara, Keiichi; Yamagishi, Nobuyuki; Saito, Youhei; Takasaki, Midori; Konoshima, Takao; Hatayama, Takumi

2006-01-01

Because heat shock proteins (Hsps) are involved in protecting cells and in the pathophysiology of diseases such as inflammation, cancer, and neurodegenerative disorders, the use of regulators of the expression of Hsps in mammalian cells seems to be useful as a potential therapeutic modality. To identify compounds that modulate the response to heat shock, we analyzed several natural products using a mammalian cell line containing an hsp promoter-regulated reporter gene. In this study, we found that an extract from Fructus Arctii markedly suppressed the expression of Hsp induced by heat shock. A component of the extract arctigenin, but not the component arctiin, suppressed the response at the level of the activation of heat shock transcription factor, the induction of mRNA, and the synthesis and accumulation of Hsp. Furthermore, arctigenin inhibited the acquisition of thermotolerance in mammalian cells, including cancer cells. Thus, arctigenin seemed to be a new suppressive regulator of heat shock response in mammalian cells, and may be useful for hyperthermia cancer therapy. PMID:16817321

Comparison of human and rhesus macaque T-cell responses elicited by boosting with NYVAC encoding human immunodeficiency virus type 1 clade C immunogens.

PubMed

Mooij, Petra; Balla-Jhagjhoorsingh, Sunita S; Beenhakker, Niels; van Haaften, Patricia; Baak, Ilona; Nieuwenhuis, Ivonne G; Heidari, Shirin; Wolf, Hans; Frachette, Marie-Joelle; Bieler, Kurt; Sheppard, Neil; Harari, Alexandre; Bart, Pierre-Alexandre; Liljeström, Peter; Wagner, Ralf; Pantaleo, Giuseppe; Heeney, Jonathan L

2009-06-01

Rhesus macaques (Macaca mulatta) have played a valuable role in the development of human immunodeficiency virus (HIV) vaccine candidates prior to human clinical trials. However, changes and/or improvements in immunogen quality in the good manufacturing practice (GMP) process or changes in adjuvants, schedule, route, dose, or readouts have compromised the direct comparison of T-cell responses between species. Here we report a comparative study in which T-cell responses from humans and macaques to HIV type 1 antigens (Gag, Pol, Nef, and Env) were induced by the same vaccine batches prepared under GMP and administered according to the same schedules in the absence and presence of priming. Priming with DNA (humans and macaques) or alphavirus (macaques) and boosting with NYVAC induced robust and broad antigen-specific responses, with highly similar Env-specific gamma interferon (IFN-gamma) enzyme-linked immunospot assay responses in rhesus monkeys and human volunteers. Persistent cytokine responses of antigen-specific CD4(+) and CD8(+) T cells of the central memory as well as the effector memory phenotype, capable of simultaneously eliciting multiple cytokines (IFN-gamma, interleukin 2, and tumor necrosis factor alpha), were induced. Responses were highly similar in humans and primates, confirming earlier data indicating that priming is essential for inducing robust NYVAC-boosted IFN-gamma T-cell responses. While significant similarities were observed in Env-specific responses in both species, differences were also observed with respect to responses to other HIV antigens. Future studies with other vaccines using identical lots, immunization schedules, and readouts will establish a broader data set of species similarities and differences with which increased confidence in predicting human responses may be achieved.

Comparison of Human and Rhesus Macaque T-Cell Responses Elicited by Boosting with NYVAC Encoding Human Immunodeficiency Virus Type 1 Clade C Immunogens▿

PubMed Central

Mooij, Petra; Balla-Jhagjhoorsingh, Sunita S.; Beenhakker, Niels; van Haaften, Patricia; Baak, Ilona; Nieuwenhuis, Ivonne G.; Heidari, Shirin; Wolf, Hans; Frachette, Marie-Joelle; Bieler, Kurt; Sheppard, Neil; Harari, Alexandre; Bart, Pierre-Alexandre; Liljeström, Peter; Wagner, Ralf; Pantaleo, Giuseppe; Heeney, Jonathan L.

2009-01-01

Rhesus macaques (Macaca mulatta) have played a valuable role in the development of human immunodeficiency virus (HIV) vaccine candidates prior to human clinical trials. However, changes and/or improvements in immunogen quality in the good manufacturing practice (GMP) process or changes in adjuvants, schedule, route, dose, or readouts have compromised the direct comparison of T-cell responses between species. Here we report a comparative study in which T-cell responses from humans and macaques to HIV type 1 antigens (Gag, Pol, Nef, and Env) were induced by the same vaccine batches prepared under GMP and administered according to the same schedules in the absence and presence of priming. Priming with DNA (humans and macaques) or alphavirus (macaques) and boosting with NYVAC induced robust and broad antigen-specific responses, with highly similar Env-specific gamma interferon (IFN-γ) enzyme-linked immunospot assay responses in rhesus monkeys and human volunteers. Persistent cytokine responses of antigen-specific CD4+ and CD8+ T cells of the central memory as well as the effector memory phenotype, capable of simultaneously eliciting multiple cytokines (IFN-γ, interleukin 2, and tumor necrosis factor alpha), were induced. Responses were highly similar in humans and primates, confirming earlier data indicating that priming is essential for inducing robust NYVAC-boosted IFN-γ T-cell responses. While significant similarities were observed in Env-specific responses in both species, differences were also observed with respect to responses to other HIV antigens. Future studies with other vaccines using identical lots, immunization schedules, and readouts will establish a broader data set of species similarities and differences with which increased confidence in predicting human responses may be achieved. PMID:19321612

Radiotherapy as an immunological booster in patients with metastatic melanoma or renal cell carcinoma treated with high-dose Interleukin-2: evaluation of biomarkers of immunologic and therapeutic response.

PubMed

Ridolfi, Laura; de Rosa, Francesco; Ridolfi, Ruggero; Gentili, Giorgia; Valmorri, Linda; Scarpi, Emanuela; Parisi, Elisabetta; Romeo, Antonino; Guidoboni, Massimo

2014-09-23

Tumor cells killed by radiation therapy (RT) are a potentially good source of antigens for dendritic cell (DC) uptake and presentation to T-cells. RT upregulates cell death receptors such as Fas/CD95 and MHC-I, induces the expression of co-stimulatory molecules on tumor cells, and promotes production of pro-inflammatory cytokines. High-dose interleukin-2 (HD-IL-2) bolus has been shown to obtain objective response rates ranging from 15% to 17% in patients with metastatic melanoma or renal cell carcinoma (RCC), with 6% to 8% of cases experiencing a durable complete response. However, HD-IL-2 is also associated with severe side-effects; if it is to remain a component of the curative treatment strategy in patients with metastatic melanoma or RCC, its therapeutic efficacy must be improved and patients who are most likely to benefit from treatment must be identified a priori. We designed a clinical study combining immunomodulating RT and HD-IL-2 to evaluate their clinical and immunological efficacy and to explore the predictive and prognostic value of 1) tumor-specific immune response and 2) serum levels of proangiogenic cytokines. The primary endpoint of this proof-of-principle phase II study is immune response. Secondary endpoints are the identification of biomarkers potentially predictive of response, toxicity, response rate and overall survival. Three daily doses of booster radiotherapy (XRT) at 6-12 Gy will be administered to at least one metastatic field on days -3 to -1 before the first and third cycle. Treatment with IL-2 (dose 18 MIU/m2/day by continuous IV infusion for 72 hours) will start on day +1 and will be repeated every 3 weeks for up to 4 cycles and then every 4 weeks for a further 2 cycles. Immune response against tumor antigens expressed by melanoma and/or RCC will be evaluated during treatment. Circulating immune effectors and regulators, e.g. cytotoxic T lymphocytes and regulatory T cells, as well as serum levels of proangiogenic/proinflammatory cytokines will also be quantified. This study aims to evaluate the potential immunological synergism between HD-IL-2 and XRT, and to identify biomarkers that are predictive of response to IL-2 in order to spare potentially non responding patients from toxicity. EudraCT no. 2012-001786-32.

Immunologic hierarchy, class II MHC promiscuity, and epitope spreading of a melanoma helper peptide vaccine.

PubMed

Hu, Yinin; Petroni, Gina R; Olson, Walter C; Czarkowski, Andrea; Smolkin, Mark E; Grosh, William W; Chianese-Bullock, Kimberly A; Slingluff, Craig L

2014-08-01

Immunization with a combination melanoma helper peptide (6MHP) vaccine has been shown to induce CD4(+) T cell responses, which are associated with patient survival. In the present study, we define the relative immunogenicity and HLA allele promiscuity of individual helper peptides and identify helper peptide-mediated augmentation of specific CD8(+) T cell responses. Thirty-seven participants with stage IIIB-IV melanoma were vaccinated with 6MHP in incomplete Freund's adjuvant. The 6MHP vaccine is comprised of 6 peptides representing melanocytic differentiation proteins gp100, tyrosinase, Melan-A/MART-1, and cancer testis antigens from the MAGE family. CD4(+) and CD8(+) T cell responses were assessed in peripheral blood and in sentinel immunized nodes (SIN) by thymidine uptake after exposure to helper peptides and by direct interferon-γ ELIspot assay against 14 MHC class I-restricted peptides. Vaccine-induced CD4(+) T cell responses to individual epitopes were detected in the SIN of 63 % (22/35) and in the peripheral blood of 38 % (14/37) of participants for an overall response rate of 65 % (24/37). The most frequently immunogenic peptides were MAGE-A3281-295 (49 %) and tyrosinase386-406 (32 %). Responses were not limited to HLA restrictions originally described. Vaccine-associated CD8(+) T cell responses against class I-restricted peptides were observed in 45 % (5/11) of evaluable participants. The 6MHP vaccine induces both CD4(+) and CD8(+) T cell responses against melanoma antigens. CD4(+) T cell responses were detected beyond reported HLA-DR restrictions. Induction of CD8(+) T cell responses suggests epitope spreading and systemic activity mediated at the tumor site.

Different Regions of the Malaria Merozoite Surface Protein 1 of Plasmodium chabaudi Elicit Distinct T-Cell and Antibody Isotype Responses

PubMed Central

Quin, Stuart J.; Langhorne, Jean

2001-01-01

In this study we have investigated the antibody and CD4 T-cell responses to the well-characterized malaria vaccine candidate MSP-1 during the course of a primary Plasmodium chabaudi chabaudi (AS) infection. Specific antibody responses can be detected within the first week of infection, and CD4 T cells can be detected after 3 weeks of infection. The magnitude of the CD4 T-cell response elicited during a primary infection depended upon the region of MSP-1. In general, the highest precursor frequencies were obtained when a recombinant MSP-1 fragment corresponding to amino acids 900 to 1507 was used as the antigen in vitro. By contrast, proliferative and cytokine responses against amino acids 1508 to 1766 containing the C-terminal 21-kDa region of the molecule were low. The characteristic interleukin 4 (IL-4) switch that occurs in the CD4 T-cell population after an acute blood stage P. c. chabaudi infection was only consistently observed in the response to the amino acid 900 to 1507 MSP1 fragment. A lower frequency of IL-4-producing cells was seen in response to other regions. Although the magnitudes of the immunoglobulin G antibody responses to the different regions of MSP-1 were similar, the isotype composition of each response was distinct, and there was no obvious relationship with the type of T helper cells generated. Interestingly, a relatively high antibody response to the C-terminal region of MSP-1 was observed, suggesting that T-cell epitopes outside of this region may provide the necessary cognate help for specific antibody production. PMID:11254580

Mechanistic studies of systemic immune responses induced by laser-nanotechnology

NASA Astrophysics Data System (ADS)

Chen, Wei R.; Zhou, Feifan; Henderson, Brock; Vasquez, Bailey; Liu, Hong; Hode, Tomas; Nordquist, Robert E.

2014-02-01

With the help of the specific absorption spectrum of carbon nanotubes, we achieved selective photothermal tumor cell destruction, particularly using a near-infrared laser to reduce potential damage to untargeted tissues. Combined with immunological stimulation, using a novel adjuvant, we also observed the anti-tumor immune responses when treating animal tumors using the laser-nano treatment. In fact, the local application of laser-nano-immunotherapy appeared to result in a systemic curative effect. In our mechanistic study, we found that the laser-nano-immuno treatment can activate antigen-presenting cells, such as dendritic cells (DCs). More importantly, the uptake and presentation of antigens by these antigen presenting cells were significantly enhanced, as shown by the strong binding of tumor cells and DCs as well as the proliferation of T cells caused by the DCs after the DCs had been incubated with laser-nano-immuno treated tumors. These cellular observations provide evidence that a systemic anti-tumor immune response was induced by the combination of laser and nanotechnology.

Center/surround organization of retinal bipolar cells: High correlation of fundamental responses of center and surround to sinusoidal contrasts

PubMed Central

Burkhardt, Dwight A.; Bartoletti, Theodore M.; Thoreson, Wallace B.

2012-01-01

Receptive field organization of cone-driven bipolar cells was investigated by intracellular recording in the intact light-adapted retina of the tiger salamander (Ambystoma tigrinum). Centered spots and concentric annuli of optimum dimensions were used to selectively stimulate the receptive field center and surround with sinusoidal modulations of contrast at 3 Hz. At low contrasts, responses of both the center and surround of both ON and OFF bipolar cells were linear, showing high gain and thus contrast enhancement relative to cones. The contrast/response curves for the fundamental response, measured by a Fast Fourier Transform, reached half maximum amplitude quickly at 13% contrast followed by saturation at high contrasts. The variation of the normalized amplitude of the center and surround responses was remarkably similar, showing linear regression over the entire response range with very high correlations, r2 = 0.97 for both ON and OFF cells. The contrast/response curves of both center and surround for both ON and OFF cells were well fit (r2 = 0.98) by an equation for single-site binding. In about half the cells studied, the nonlinear waveforms of center and surround could be brought into coincidence by scaling and shifting the surround response in time. This implies that a nonlinearity, common to both center and surround, occurs after polarity inversion at the cone feedback synapse. Evidence from paired whole-cell recordings between single cones and OFF bipolar cells suggests that substantial nonlinearity is not due to transmission at the cone synapse but instead arises from intrinsic bipolar cell and network mechanisms. When sinusoidal contrast modulations were applied to the center and surround simultaneously, clear additivity was observed for small responses in both ON and OFF cells, whereas the interaction was strikingly nonadditive for large responses. The contribution of the surround was then greatly reduced, suggesting attenuation at the cone feedback synapse. PMID:21439110

Mechanisms underlying cellular responses of cells from haemopoietic tissue to low dose/low LET radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Munira A Kadhim

2010-03-05

To accurately define the risks associated with human exposure to relevant environmental doses of low LET ionizing radiation, it is necessary to completely understand the biological effects at very low doses (i.e., less than 0.1 Gy), including the lowest possible dose, that of a single electron track traversal. At such low doses, a range of studies have shown responses in biological systems which are not related to the direct interaction of radiation tracks with DNA. The role of these “non-targeted” responses in critical tissues is poorly understood and little is known regarding the underlying mechanisms. Although critical for dosimetry andmore » risk assessment, the role of individual genetic susceptibility in radiation risk is not satisfactorily defined at present. The aim of the proposed grant is to critically evaluate radiation-induced genomic instability and bystander responses in key stem cell populations from haemopoietic tissue. Using stem cells from two mouse strains (CBA/H and C57BL/6J) known to differ in their susceptibility to radiation effects, we plan to carefully dissect the role of genetic predisposition on two non-targeted radiation responses in these models; the bystander effect and genomic instability, which we believe are closely related. We will specifically focus on the effects of low doses of low LET radiation, down to doses approaching a single electron traversal. Using conventional X-ray and γ-ray sources, novel dish separation and targeted irradiation approaches, we will be able to assess the role of genetic variation under various bystander conditions at doses down to a few electron tracks. Irradiations will be carried out using facilities in routine operation for bystander targeted studies. Mechanistic studies of instability and the bystander response in different cell lineages will focus initially on the role of cytokines which have been shown to be involved in bystander signaling and the initiation of instability. These studies also aim to uncover protein mediators of the bystander responses using advanced proteomic screening of factors released from irradiated, bystander and unstable cells. Integral to these studies will be an assessment of the role of genetic susceptibility in these responses, using CBA/H and C57BL/6J mice. The relevance of in vivo interactions between stem cells and the stem cell niche will be explored in the future by re-implantation techniques of previously irradiated cells. The above studies will provide fundamental mechanistic information relating genetic predisposition to important low dose phenomena, and will aid in the development of Department of Energy policy, as well as radiation risk policy for the public and the workplace. We believe the proposed studies accurately reflect the goals of the DOE low dose program.« less

Invariant NKT cells inhibit development of the Th17 lineage

PubMed Central

Mars, Lennart T.; Araujo, Luiza; Kerschen, Philippe; Diem, Séverine; Bourgeois, Elvire; Van, Linh Pham; Carrié, Nadège; Dy, Michel; Liblau, Roland S.; Herbelin, André

2009-01-01

T cells differentiate into functionally distinct effector subsets in response to pathogen encounter. Cells of the innate immune system direct this process; CD1d-restricted invariant natural killer T (iNKT) cells, for example, can either promote or inhibit Th1 and Th2 responses. Recently, a new subset of CD4+ T helper cells, called Th17, was identified that is implicated in mucosal immunity and autoimmune disorders. To investigate the influence of iNKT cells on the differentiation of naïve T cells we used an adoptive transfer model of traceable antigen-specific CD4+ T cells. Transferred naïve CD25−CD62L+ CD4+ T cells were primed by antigen immunization of the recipient mice, permitting their expansion and Th17 differentiation. This study establishes that in vivo activation of iNKT cells during T-cell priming impedes the commitment of naïve T cells to the Th17 lineage. In vivo cytokine neutralization experiments revealed a role for IL-4, IL-10, and IFN-γ in the iNKT-cell-mediated regulation of T-cell lineage development. Moreover, by comparing IL-17 production by antigen-experienced T cells from unmanipulated wild-type mice and iNKT-cell-deficient mice, we demonstrate an enhanced Th17 response in mice lacking iNKT cells. This invigorated Th17 response reverts to physiological levels when iNKT cells are introduced into Jα18−/− mice by adoptive transfer, indicating that iNKT cells control the Th17 compartment at steady state. We conclude that iNKT cells play an important role in limiting development of the Th17 lineage and suggest that iNKT cells provide a natural barrier against Th17 responses. PMID:19325124

Multiplexed Nanoplasmonic Temporal Profiling of T-Cell Response under Immunomodulatory Agent Exposure

PubMed Central

2016-01-01

Immunomodulatory drugs—agents regulating the immune response—are commonly used for treating immune system disorders and minimizing graft versus host disease in persons receiving organ transplants. At the cellular level, immunosuppressant drugs are used to inhibit pro-inflammatory or tissue-damaging responses of cells. However, few studies have so far precisely characterized the cellular-level effect of immunomodulatory treatment. The primary challenge arises due to the rapid and transient nature of T-cell immune responses to such treatment. T-cell responses involve a highly interactive network of different types of cytokines, which makes precise monitoring of drug-modulated T-cell response difficult. Here, we present a nanoplasmonic biosensing approach to quantitatively characterize cytokine secretion behaviors of T cells with a fine time-resolution (every 10 min) that are altered by an immunosuppressive drug used in the treatment of T-cell-mediated diseases. With a microfluidic platform integrating antibody-conjugated gold nanorod (AuNR) arrays, the technique enables simultaneous multi-time-point measurements of pro-inflammatory (IL-2, IFN-γ, and TNF-α) and anti-inflammatory (IL-10) cytokines secreted by T cells. The integrated nanoplasmonic biosensors achieve precise measurements with low operating sample volume (1 μL), short assay time (∼30 min), heightened sensitivity (∼20–30 pg/mL), and negligible sensor crosstalk. Data obtained from the multicytokine secretion profiles with high practicality resulting from all of these sensing capabilities provide a comprehensive picture of the time-varying cellular functional state during pharmacologic immunosuppression. The capability to monitor cellular functional response demonstrated in this study has great potential to ultimately permit personalized immunomodulatory treatment. PMID:27478873

Immunological characterization of the aryl hydrocarbon receptor (AHR) knockout rat in the presence and absence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

PubMed

Phadnis-Moghe, Ashwini S; Chen, Weimin; Li, Jinpeng; Crawford, Robert B; Bach, Anthony; D'Ingillo, Shawna; Kovalova, Natalia; Suarez-Martinez, Jose E; Kaplan, Barbara L F; Harrill, Joshua A; Budinsky, Robert; Rowlands, J Craig; Thomas, Russell S; Kaminski, Norbert E

2016-08-10

The aryl hydrocarbon receptor (AHR) has been extensively characterized for the essential role it plays in mediating the toxic responses elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Despite similarities across animal species, species-specific differences exist in the profile of toxicity and sensitivity to TCDD owing, in part, to differences in the AHR. Newer reports have implicated the importance of AHR in the development and regulation of the immune system. Our present studies seek to further explore the essential role of AHR in lymphoid tissue composition, B cell function and the immunological responses after TCDD administration using the recently established AHR KO rats. Comprehensive immune cell phenotyping showed a decrease in the CD8 + T cell, CD11c + populations and an increase in NKT cells in 3-week-old AHR KO rats compared to the WT controls. The lipopolysaccharide-induced IgM response and proliferation was markedly suppressed in the WT but not in the AHR KO B cells in the presence of TCDD. However, the percentage of LPS-activated IgM + B cells was significantly higher in the AHR KO B cells as compared to that of WT suggesting the role of AHR in regulating the IgM response. The use of an AHR antagonist further alluded to the endogenous role of AHR in regulating B cell responses in the rat. Overall, the studies report for the first time, comprehensive immune cell phenotyping of the AHR KO rat and the endogenous role of AHR in the regulation of B cell function in the rat. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Effects of wear particles of polyether-ether-ketone and cobalt-chromium-molybdenum on CD4- and CD8-T-cell responses

PubMed Central

Du, Zhe; Wang, Shujun; Yue, Bing; Wang, Ying; Wang, You

2018-01-01

T-cells, second only to macrophages, are often considered as the potential cells involved in debris-related failure of arthroplasty. Here, we assessed the effects of particulate wear debris on T-cells and inflammatory reactions. Blood samples from 25 donors were incubated with polyether-ether-ketone (PEEK) and cobalt-chromium-molybdenum (CoCrMo) particles generated by custom cryo-milling and pulverization. The T-cell phenotypes were assessed using immunostaining and flow cytometry. For the in vivo study, 0.1 mL of each particle suspension (approximately 1.0 × 108 wear particles) was injected into murine knee joints; the synovium and spleen were collected one week after the operation for histological examination and immunofluorescence staining. The T-cell responses observed included low-level activation of Th1, Th2, Th17, and CD8+ pathways after 72 h of co-culture of the particles with peripheral blood mononuclear cells. Obvious CD8+ T-cell responses were observed in local synovium and peripheral spleen, with higher inflammatory cytokine expression in the CoCrMo group. Relatively minor cytotoxic and immunological reactions were observed in vitro, with PEEK and CoCrMo particle-induced immune responses being primarily mediated by CD8+ T-cells, rather than CD4+ T-cells, in vivo. Overall, PEEK wear particles induced fewer inflammatory reactions than CoCrMo particles. This study verified that PEEK was suitable as a potential alternative for metals in total knee replacements in terms of the immunological reaction to PEEK particles, and shed light on the effects of wear particles from polymer and metal-based implants on immune responses. PMID:29541407

Effects of wear particles of polyether-ether-ketone and cobalt-chromium-molybdenum on CD4- and CD8-T-cell responses.

PubMed

Du, Zhe; Wang, Shujun; Yue, Bing; Wang, Ying; Wang, You

2018-02-16

T-cells, second only to macrophages, are often considered as the potential cells involved in debris-related failure of arthroplasty. Here, we assessed the effects of particulate wear debris on T-cells and inflammatory reactions. Blood samples from 25 donors were incubated with polyether-ether-ketone (PEEK) and cobalt-chromium-molybdenum (CoCrMo) particles generated by custom cryo-milling and pulverization. The T-cell phenotypes were assessed using immunostaining and flow cytometry. For the in vivo study, 0.1 mL of each particle suspension (approximately 1.0 × 10 8 wear particles) was injected into murine knee joints; the synovium and spleen were collected one week after the operation for histological examination and immunofluorescence staining. The T-cell responses observed included low-level activation of Th1, Th2, Th17, and CD8+ pathways after 72 h of co-culture of the particles with peripheral blood mononuclear cells. Obvious CD8+ T-cell responses were observed in local synovium and peripheral spleen, with higher inflammatory cytokine expression in the CoCrMo group. Relatively minor cytotoxic and immunological reactions were observed in vitro , with PEEK and CoCrMo particle-induced immune responses being primarily mediated by CD8+ T-cells, rather than CD4+ T-cells, in vivo . Overall, PEEK wear particles induced fewer inflammatory reactions than CoCrMo particles. This study verified that PEEK was suitable as a potential alternative for metals in total knee replacements in terms of the immunological reaction to PEEK particles, and shed light on the effects of wear particles from polymer and metal-based implants on immune responses.

Transcriptional activation of rat creatine kinase B by 17beta-estradiol in MCF-7 cells involves an estrogen responsive element and GC-rich sites.

PubMed

Wang, F; Samudio, I; Safe, S

2001-01-01

The rat creatine kinase B (CKB) gene is induced by estrogen in the uterus, and constructs containing rat CKB gene promoter inserts are highly estrogen-responsive in cell culture. Analysis of the upstream -568 to -523 region of the promoter in HeLa cells has identified an imperfect palindromic estrogen response element (ERE) that is required for hormone inducibility. Analysis of the CKB gene promoter in MCF-7 breast cancer cells confirmed that pCKB7 (containing the -568 to -523 promoter insert) was estrogen-responsive in transient transfection studies. However, mutation and deletion analysis of this region of the promoter showed that two GC-rich sites and the concensus ERE were functional cis-elements that bound estrogen receptor alpha (ERalpha)/Sp1 and ERalpha proteins, respectively. The role of these elements was confirmed in gel mobility shift and chromatin immunoprecipitation assays and transfection studies in MDA-MB-231 and Schneider Drosophila SL-2 cells. These results show that transcriptional activation of CKB by estrogen is dependent, in part, on ERalpha/Sp1 action which is cell context-dependent. Copyright 2001 Wiley-Liss, Inc.

Epidermal Viral Immunity Induced by CD8α+ Dendritic Cells But Not by Langerhans Cells

NASA Astrophysics Data System (ADS)

Allan, Rhys S.; Smith, Chris M.; Belz, Gabrielle T.; van Lint, Allison L.; Wakim, Linda M.; Heath, William R.; Carbone, Francis R.

2003-09-01

The classical paradigm for dendritic cell function derives from the study of Langerhans cells, which predominate within skin epidermis. After an encounter with foreign agents, Langerhans cells are thought to migrate to draining lymph nodes, where they initiate T cell priming. Contrary to this, we show here that infection of murine epidermis by herpes simplex virus did not result in the priming of virus-specific cytotoxic T lymphocytes by Langerhans cells. Rather, the priming response required a distinct CD8α+ dendritic cell subset. Thus, the traditional view of Langerhans cells in epidermal immunity needs to be revisited to accommodate a requirement for other dendritic cells in this response.

TP53 status and response to chemotherapy in breast cancer.

PubMed

Bertheau, Philippe; Espié, Marc; Turpin, Elisabeth; Lehmann, Jacqueline; Plassa, Louis-François; Varna, Mariana; Janin, Anne; de Thé, Hugues

2008-01-01

Despite its central role in the control of apoptosis, senescence and cell cycle arrest, the tumor suppressor protein p53 remains an enigma for its possible role in predicting response to chemotherapy in cancer patients. Many studies remained inconclusive, others showed a better response for tumors with normal p53, and some recent studies showed adverse effects of normal p53 for response to treatment. p53 is not only a powerful pro-apoptotic factor in response to drug-induced DNA damages but also a potential inducer of cell cycle arrest, protecting tumor cells from further cytotoxic damages. Our review describes the classical as well as the more recent concepts. In order to draw definite conclusions, future works should use more reliable methods to assess the TP53 status and should address more homogeneous tumor subpopulations treated with homogeneous chemotherapy regimens. Copyright 2008 S. Karger AG, Basel.

Photovoltaic cells for laser power beaming

NASA Technical Reports Server (NTRS)

Landis, Geoffrey A.; Jain, Raj K.

1992-01-01

To better understand cell response to pulsed illumination at high intensity, the PC-1DC finite-element computer model was used to analyze the response of solar cells to pulsed laser illumination. Over 50% efficiency was calculated for both InP and GaAs cells under steady-state illumination near the optimum wavelength. The time-dependent response of a high-efficiency GaAs concentrator cell to a laser pulse was modelled, and the effect of laser intensity, wavelength, and bias point was studied. Designing a cell to accommodate pulsed input can be done either by accepting the pulsed output and designing a cell to minimize adverse effects due to series resistance and inductance, or to design a cell with a long enough minority carrier lifetime, so that the output of the cell will not follow the pulse shape. Two such design possibilities are a monolithic, low-inductance voltage-adding GaAs cell, or a high-efficiency, light-trapping silicon cell. The advantages of each design will be discussed.

Evidence of B cell immune responses to acute lymphoblastic leukemia in murine allogeneic hematopoietic stem cell transplantation recipients treated with donor lymphocyte infusion and/or vaccination.

PubMed

Mullen, Craig A; Campbell, Andrew; Tkachenko, Olena; Jansson, Johan; Hsu, Yu-Chiao

2011-02-01

These experiments explored mechanisms of control of acute lymphoblastic leukemia (ALL) following allogeneic hematopoietic stem cell transplantation using a murine model of MHC-matched, minor histocompatibility antigen-mismatched transplantation. The central hypothesis examined was that addition of active vaccination against leukemia cells would substantially increase the effectiveness of allogeneic donor lymphocyte infusion (DLI) against ALL present in the host after transplantation. Although vaccination did increase the magnitude of type I T cell responses against leukemia cells associated with DLI, it did not lead to substantial improvement in long-term survival. Analysis of immunologic mechanisms of leukemia progression demonstrated that the failure of vaccination was not because of antigen loss in leukemia cells. However, analysis of survival provided surprising findings that, in addition to very modest type I T cell responses, a B cell response that produced antibodies that bind leukemia cells was found in long-term survivors. The risk of death from leukemia was significantly lower in recipients that had higher levels of such antibodies. These studies raise the hypothesis that stimulation of B cell responses after transplantation may provide a novel way to enhance allogeneic graft-versus-leukemia effects associated with transplantation. Copyright © 2011 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

CD24(hi)CD27(+) B cells from patients with allergic asthma have impaired regulatory activity in response to lipopolysaccharide.

PubMed

van der Vlugt, L E P M; Mlejnek, E; Ozir-Fazalalikhan, A; Janssen Bonas, M; Dijksman, T R; Labuda, L A; Schot, R; Guigas, B; Möller, G M; Hiemstra, P S; Yazdanbakhsh, M; Smits, H H

2014-04-01

Regulatory B cells have been identified that strongly reduce allergic and auto-immune inflammation in experimental models by producing IL-10. Recently, several human regulatory B-cell subsets with an impaired function in auto-immunity have been described, but there is no information on regulatory B cells in allergic asthma. In this study, the frequency and function of IL-10 producing B-cell subsets in allergic asthma were investigated. Isolated peripheral blood B cells from 13 patients with allergic asthma and matched healthy controls were analyzed for the expression of different regulatory B-cell markers. Next, the B cells were activated by lipopolysaccharide (LPS), CpG or through the B-cell receptor, followed by co-culture with endogenous memory CD4(+) T cells and house dust mite allergen DerP1. Lower number of IL-10 producing B cells were found in patients in response to LPS, however, this was not the case when B cells were activated through the B-cell receptor or by CpG. Further dissection showed that only the CD24(hi)CD27(+) B-cell subset was reduced in number and IL-10 production to LPS. In response to DerP1, CD4(+) T cells from patients co-cultured with LPS-primed total B cells produced less IL-10 compared to similar cultures from controls. These results are in line with the finding that sorted CD24(hi)CD27(+) B cells are responsible for the induction of IL-10(+) CD4(+) T cells. Taken together, these data indicate that CD24(hi)CD27(+) B cells from allergic asthma patients produce less IL-10 in response to LPS leading to a weaker IL-10 induction in T cells in response to DerP1, which may play a role in allergic asthma. © 2013 John Wiley & Sons Ltd.

Mechanobiology of the Meniscus

PubMed Central

McNulty, Amy L.; Guilak, Farshid

2015-01-01

The meniscus plays a critical biomechanical role in the knee, providing load support, joint stability, and congruity. Importantly, growing evidence indicates that the mechanobiologic response of meniscal cells plays a critical role in the physiologic, pathologic, and repair responses of the meniscus. Here we review experimental and theoretical studies that have begun to directly measure the biomechanical effects of joint loading on the meniscus under physiologic and pathologic conditions, showing that the menisci are exposed to high contact stresses, resulting in a complex and nonuniform stress-strain environment within the tissue. By combining microscale measurements of the mechanical properties of meniscal cells and their pericellular and extracellular matrix regions, theoretical and experimental models indicate that the cells in the meniscus are exposed to a complex and inhomogeneous environment of stress, strain, fluid pressure, fluid flow, and a variety of physicochemical factors. Studies across a range of culture systems from isolated cells to tissues have revealed that the biological response of meniscal cells is directly influenced by physical factors, such as tension, compression, and hydrostatic pressure. In addition, these studies have provided new insights into the mechanotransduction mechanisms by which physical signals are converted into metabolic or pro/anti-inflammatory responses. Taken together, these in vivo and in vitro studies show that mechanical factors play an important role in the health, degeneration, and regeneration of the meniscus. A more thorough understanding of the mechanobiologic responses of the meniscus will hopefully lead to therapeutic approaches to prevent degeneration and enhance repair of the meniscus. PMID:25731738

Validity and reproducibility of measurement of islet autoreactivity by T-cell assays in subjects with early type 1 diabetes.

PubMed

Herold, Kevan C; Brooks-Worrell, Barbara; Palmer, Jerry; Dosch, H Michael; Peakman, Mark; Gottlieb, Peter; Reijonen, Helena; Arif, Sefina; Spain, Lisa M; Thompson, Clinton; Lachin, John M

2009-11-01

Type 1 diabetes results from an immunemediated destruction of beta-cells, likely to be mediated by T lymphocytes, but the sensitivity, specificity, and other measures of validity of existing assays for islet autoreactive T-cells are not well established. Such assays are vital for monitoring responses to interventions that may modulate disease progression. We studied the ability of cellular assays to discriminate responses in patients with type 1 diabetes and normal control subjects in a randomized blinded study in the U.S. and U.K. We evaluated the reproducibility of these measurements overall and to individual analytes from repeat collections. Responses in the cellular immunoblot, U.K.-ELISPOT, and T-cell proliferation assays could differentiate patients from control subjects with odds ratios of 21.7, 3.44, and 3.36, respectively, with sensitivity and specificity as high as 74 and 88%. The class II tetramer and U.S. ELISPOT assays performed less well. Despite the significant association of the responses with type 1 diabetes, the reproducibility of the measured responses, both overall and individual analytes, was relatively low. Positive samples from normal control subjects (i.e., false positives) were generally isolated to single assays. The cellular immunoblot, U.K.-ELISPOT, and T-cell proliferation assays can distinguish responses from patients with type 1 diabetes and healthy control subjects. The limited reproducibility of the measurements overall and of responses to individual analytes may reflect the difficulty in detection of low frequency of antigen-specific T-cells or variability in their appearance in peripheral blood.

Depressed immune surveillance against cancer: role of deficient T cell: extracellular matrix interactions.

PubMed

Górski, A; Castronovo, V; Stepień-Sopniewska, B; Grieb, P; Ryba, M; Mrowiec, T; Korczak-Kowalska, G; Wierzbicki, P; Matysiak, W; Dybowska, B

1994-07-01

Although T cells infiltrate malignant tumors, the local immune response is usually inefficient and tumors escape destruction. While extracellular matrix proteins strongly costimulate T cell responses in normal individuals, our studies indicate that peripheral blood T cells from cancer patients and tumor infiltrating cells respond poorly or are resistant to stimulative signals mediated by collagen I and IV and fibronectin. Moreover, the adhesive properties of cancer T cells are markedly depressed. Those functional deficiencies are paralleled by variable deficits in integrin and non-integrin T cell receptors for extracellular matrix. Immunotherapy with BCG causes a dramatic but transient increase in T cell: ECM interactions.

Commensal oral bacteria antigens prime human dendritic cells to induce Th1, Th2 or Treg differentiation.

PubMed

Kopitar, A N; Ihan Hren, N; Ihan, A

2006-02-01

In various immunopathologic conditions, bacterial flora induce an immune response which results in inflammatory manifestations, e.g. periapical granuloma. Dendritic cells provide the main orchestration of specific immune responses. The aim of our study was to test the capacity of distinct oral bacterial antigens (prepared from Streptococcus mitis, Propionibacterium acnes, and Bacteroides spp.) to prime human dendritic cells for stimulation of the T-lymphocyte response. To assess the T-lymphocyte response, the expression of CD25, CD69, intracellular interferon gamma (cIFN-gamma), and intracellular interleukin 4 (cIL-4) was determined. Dendritic cells were prepared from leukocyte buffy coat from healthy blood donors. Monocytes were stimulated with IL-4 and GM-CSF and dendritic cells activated with bacterial lysates. Cell suspensions contained up to 90% dendritic cells, which represented 2-12% of the initial number of mononuclear cells. Lymphocyte subsets that developed in lymphocyte cultures after 1 week of stimulation were analyzed by flow cytometry. Dendritic cells, primed with antigens of Bacteroides fragilis have shown significantly higher activation and expression of intercellular IFN-gamma by T lymphocytes compared to negative controls. The dendritic cells primed with antigens of P. acnes had no effect on T-lymphocyte activation or cytokine production; instead they induced differentiation of T lymphocytes into CD25bright cells (regulatory T cells) with a potentially inhibitory effect on immune response. Dendritic cells primed with antigens of S. mitis induced increased expression of cIL-4. We conclude that commensal oral bacteria antigens prepared from B. fragilis, S. mitis, and P. acnes prime human dendritic cells to induce Th1, Th2, and T(reg) differentiation, respectively. This may advance our understanding of immunopathologic manifestations in the oral cavity and offer new possibilities for redirecting immune responses in mucosal vaccination.

High-resolution Identification and Separation of Living Cell Types by Multiple microRNA-responsive Synthetic mRNAs.

PubMed

Endo, Kei; Hayashi, Karin; Saito, Hirohide

2016-02-23

The precise identification and separation of living cell types is critical to both study cell function and prepare cells for medical applications. However, intracellular information to distinguish live cells remains largely inaccessible. Here, we develop a method for high-resolution identification and separation of cell types by quantifying multiple microRNA (miRNA) activities in live cell populations. We found that a set of miRNA-responsive, in vitro synthesized mRNAs identify a specific cell population as a sharp peak and clearly separate different cell types based on less than two-fold differences in miRNA activities. Increasing the number of miRNA-responsive mRNAs enhanced the capability for cell identification and separation, as we precisely and simultaneously distinguished different cell types with similar miRNA profiles. In addition, the set of synthetic mRNAs separated HeLa cells into subgroups, uncovering heterogeneity of the cells and the level of resolution achievable. Our method could identify target live cells and improve the efficiency of cell purification from heterogeneous populations.

In situ observation of single cell response to acoustic droplet vaporization: Membrane deformation, permeabilization, and blebbing.

PubMed

Qin, Dui; Zhang, Lei; Chang, Nan; Ni, Pengying; Zong, Yujin; Bouakaz, Ayache; Wan, Mingxi; Feng, Yi

2018-02-06

In this study, the bioeffects of acoustic droplet vaporization (ADV) on adjacent cells were investigated by evaluating the real-time cell response at the single-cell level in situ, using a combined ultrasound-exposure and optical imaging system. Two imaging modalities, high-speed and fluorescence imaging, were used to observe ADV bubble dynamics and to evaluate the impact on cell membrane permeabilization (i.e., sonoporation) using propidium iodide (PI) uptake as an indicator. The results indicated that ADV mainly led to irreversible rather than reversible sonoporation. Further, the rate of irreversible sonoporation significantly increased with increasing nanodroplet concentration, ultrasound amplitude, and pulse duration. The results suggested that sonoporation is correlated to the rapid formation, expansion, and contraction of ADV bubbles near cells, and strongly depends on ADV bubble size and bubble-to-cell distance when subjected to short ultrasound pulses (1 μs). Moreover, the displacement of ADV bubbles was larger when using a long ultrasound pulse (20 μs), resulting in considerable cell membrane deformation and a more irreversible sonoporation rate. During sonoporation, cell membrane blebbing as a recovery manoeuvre was also investigated, indicating the essential role of Ca 2+ influx in the membrane blebbing response. This study has helped us gain further insights into the dynamic behavior of ADV bubbles near cells, ADV bubble-cell interactions, and real-time cell response, which are invaluable in the development of optimal approaches for ADV-associated theranostic applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Modifications in astrocyte morphology and calcium signaling induced by a brain capillary endothelial cell line.

PubMed

Yoder, Elizabeth J

2002-04-15

Astrocytes extend specialized endfoot processes to perisynaptic and perivascular regions, and thus are positioned to mediate the bidirectional flow of metabolic, ionic, and other transmissive substances between neurons and the blood stream. While mutual structural and functional interactions between neurons and astrocytes have been documented, less is known about the interactions between astrocytes and cerebrovascular cells. For example, although the ability of astrocytes to induce structural and functional changes in endothelial cells is established, the reciprocity of brain endothelial cells to induce changes in astrocytes is undetermined. This issue is addressed in the present study. Changes in primary cultures of neonatal mouse cortical astrocytes were investigated following their coculture with mouse brain capillary endothelial (bEnd3) cells. The presence of bEnd3 cells altered the morphology of astrocytes by transforming them from confluent monolayers into networks of elongated multicellular columns. These columns did not occur when either bEnd3 cells or astrocytes were cocultured with other cell types, suggesting that astrocytes undergo specific morphological consequences when placed in close proximity to brain endothelial cells. In addition to these structural changes, the pharmacological profile of astrocytes was modified by coculture with bEnd3 cells. Astrocytes in the cocultures showed an increased Ca2+ responsiveness to bradykinin and glutamate, but no change in responsiveness to ATP, as compared to controls. Coculturing the astrocytes with a neuronal cell line resulted in increased responsiveness of the glial responses to glutamate but not to bradykinin. These studies indicate that brain endothelial cells induce changes in astrocyte morphology and pharmacology. Copyright 2002 Wiley-Liss, Inc.

Sympathetic neural signaling via the β2-adrenergic receptor suppresses T-cell receptor-mediated human and mouse CD8(+) T-cell effector function.

PubMed

Estrada, Leonardo D; Ağaç, Didem; Farrar, J David

2016-08-01

Postganglionic sympathetic neurons innervate secondary lymphoid organs and secrete norepinephrine (NE) as the primary neurotransmitter. NE binds and signals through five distinct members of the adrenergic receptor family. In this study, we show elevated expression of the β2-adrenergic receptor (ADRB2) on primary human CD8(+) effector memory T cells. Treatment of both human and murine CD8(+) T cells with NE decreased IFN-γ and TNF-α secretion and suppressed their cytolytic capacity in response to T-cell receptor (TCR) activation. The effects of NE were specifically reversed by β2-specific antagonists. Adrb2(-/-) CD8(+) T cells were completely resistant to the effects of NE. Further, the ADRB2-specific pharmacological ligand, albuterol, significantly suppressed effector functions in both human and mouse CD8(+) T cells. While both TCR activation and stimulation with IL-12 + IL-18 were able to induce inflammatory cytokine secretion, NE failed to suppress IFN-γ secretion in response to IL-12 + IL18. Finally, the long-acting ADRB2-specific agonist, salmeterol, markedly reduced the cytokine secretion capacity of CD8(+) T cells in response to infection with vesicular stomatitis virus. This study reveals a novel intrinsic role for ADRB2 signaling in CD8(+) T-cell function and underscores the novel role this pathway plays in adaptive T-cell responses to infection. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The G-protein coupled receptor, GPR84 regulates IL-4 production by T lymphocytes in response to CD3 crosslinking.

PubMed

Venkataraman, Chandrasekar; Kuo, Frederick

2005-11-15

The orphan G-protein coupled receptor, GPR84 is highly expressed in the bone marrow, and in splenic T cells and B cells. In this study, GPR84-deficient mice were generated to understand the biological function of this orphan receptor. The proliferation of T and B cells in response to various mitogens was normal in GPR84-deficient mice. Interestingly, primary stimulation of T cells with anti-CD3 resulted in increased IL-4 but not IL-2 or IFN-gamma production in GPR84(-/-) mice compared to wild-type mice. Augmented IL-4 production in GPR84-deficient T cells was not related to increased frequency of IL-4-secreting cells in response to anti-CD3 stimulation. In fact, stimulation with anti-CD3 and anti-CD28 resulted in increased levels of IL-4 but not IFN-gamma steady-state mRNA in GPR84(-/-) T cells. In addition, Th2 effector cells generated in vitro from GPR84(-/-) mice produced higher levels of IL-4, IL-5 and IL-13 compared to wild-type mice. However, there was no detectable difference in the extent of IL-4 and IL-5 production between the two groups of mice in response to antigen stimulation of spleen cells, isolated from mice previously immunized with OVA in alum. These studies reveal a novel role for GPR84 in regulating early IL-4 gene expression in activated T cells.

Biology of the cell cycle inhibitor p21(CDKN1A): molecular mechanisms and relevance in chemical toxicology.

PubMed

Dutto, Ilaria; Tillhon, Micol; Cazzalini, Ornella; Stivala, Lucia A; Prosperi, Ennio

2015-02-01

The cell cycle inhibitor p21(CDKN1A) is a protein playing multiple roles not only in the DNA damage response, but also in many cellular processes during unperturbed cell growth. The main, well-known function of p21 is to arrest cell cycle progression by inhibiting the activity of cyclin-dependent kinases. In addition, p21 is involved in the regulation of transcription, apoptosis, DNA repair, as well as cell motility. However, p21 appears to a have a dual-face behavior because, in addition to its tumor suppressor functions, it may act as an oncogene, depending on the cell type and on the cellular localization. As a biomarker of the cell response to different toxic stimuli, p21 expression and functions have been analyzed in an impressive number of studies investigating the activity of several types of chemicals, in order to determine their possible harmful effects on human cells. Here, we review these studies in order to highlight the different roles p21 may play in the cell response to chemical exposure and to better evaluate the information provided by this biomarker.

An in vitro study of functional maturation of murine thymus cells.

PubMed

Chakravarty, A K

1977-05-26

Critical time of onset of thymus cell functions in ontogeny was studied in vitro. Collaborative function in an antibody response and ability to induce a graft-versus-host (GvH) response by murine thymocytes from different stages of ontogeny were investigated. Thymocytes from as early as 16-day mouse embryos were capable of collaborating in the antibody response to sheep-erythrocyte-antigen in vitro following 24 h of pretreatment with concanavalin A (con A). By contrast, maturation of thymus cell function as measured by competence to induce a graft-versus-host reaction, was first manifested by newborn thymus cells, and pretreatment with con A did not facilitate the maturation of this thymus cell function. Experiments to understand the effect of con A on the expression of cell surface antigens have also been reported. Con A-treated thymus cells of different ontogenic stages tested were less susceptible to killing by anti-theta serum than nontreated thymus cells; reverse was true with anti-H-2 serum. The significance of the differential susceptibility of con A-treated thymus cells to anti-sera treatment and the finding that mouse thymocytes can provide helper function as early as the 16th day of gestation have been discussed.

Bee venom processes human skin lipids for presentation by CD1a

PubMed Central

Bourgeois, Elvire A.; Subramaniam, Sumithra; Cheng, Tan-Yun; De Jong, Annemieke; Layre, Emilie; Ly, Dalam; Salimi, Maryam; Legaspi, Annaliza; Modlin, Robert L.; Salio, Mariolina; Cerundolo, Vincenzo

2015-01-01

Venoms frequently co-opt host immune responses, so study of their mode of action can provide insight into novel inflammatory pathways. Using bee and wasp venom responses as a model system, we investigated whether venoms contain CD1-presented antigens. Here, we show that venoms activate human T cells via CD1a proteins. Whereas CD1 proteins typically present lipids, chromatographic separation of venoms unexpectedly showed that stimulatory factors partition into protein-containing fractions. This finding was explained by demonstrating that bee venom–derived phospholipase A2 (PLA2) activates T cells through generation of small neoantigens, such as free fatty acids and lysophospholipids, from common phosphodiacylglycerides. Patient studies showed that injected PLA2 generates lysophospholipids within human skin in vivo, and polyclonal T cell responses are dependent on CD1a protein and PLA2. These findings support a previously unknown skin immune response based on T cell recognition of CD1a proteins and lipid neoantigen generated in vivo by phospholipases. The findings have implications for skin barrier sensing by T cells and mechanisms underlying phospholipase-dependent inflammatory skin disease. PMID:25584012

Development of an efficient, non-viral transfection method for studying gene function and bone growth in human primary cranial suture mesenchymal cells reveals that the cells respond to BMP2 and BMP3.

PubMed

Dwivedi, Prem P; Anderson, Peter J; Powell, Barry C

2012-08-03

Achieving efficient introduction of plasmid DNA into primary cultures of mammalian cells is a common problem in biomedical research. Human primary cranial suture cells are derived from the connective mesenchymal tissue between the bone forming regions at the edges of the calvarial plates of the skull. Typically they are referred to as suture mesenchymal cells and are a heterogeneous population responsible for driving the rapid skull growth that occurs in utero and postnatally. To better understand the molecular mechanisms involved in skull growth, and in abnormal growth conditions, such as craniosynostosis, caused by premature bony fusion, it is essential to be able to easily introduce genes into primary bone forming cells to study their function. A comparison of several lipid-based techniques with two electroporation-based techniques demonstrated that the electroporation method known as nucleofection produced the best transfection efficiency. The parameters of nucleofection, including cell number, amount of DNA and nucleofection program, were optimized for transfection efficiency and cell survival. Two different genes and two promoter reporter vectors were used to validate the nucleofection method and the responses of human primary suture mesenchymal cells by fluorescence microscopy, RT-PCR and the dual luciferase assay. Quantification of bone morphogenetic protein (BMP) signalling using luciferase reporters demonstrated robust responses of the cells to both osteogenic BMP2 and to the anti-osteogenic BMP3. A nucleofection protocol has been developed that provides a simple and efficient, non-viral alternative method for in vitro studies of gene and protein function in human skull growth. Human primary suture mesenchymal cells exhibit robust responses to BMP2 and BMP3, and thus nucleofection can be a valuable method for studying the potential competing action of these two bone growth factors in a model system of cranial bone growth.

Cytomegalovirus-Responsive CD8+ T Cells Expand After Solid Organ Transplantation in the Absence of CMV Disease.

PubMed

Higdon, L E; Trofe-Clark, J; Liu, S; Margulies, K B; Sahoo, M K; Blumberg, E; Pinsky, B A; Maltzman, J S

2017-08-01

Cytomegalovirus (CMV) is a major cause of morbidity and mortality in solid organ transplant recipients. Approximately 60% of adults are CMV seropositive, indicating previous exposure. Following resolution of the primary infection, CMV remains in a latent state. Reactivation is controlled by memory T cells in healthy individuals; transplant recipients have reduced memory T cell function due to chronic immunosuppressive therapies. In this study, CD8 + T cell responses to CMV polypeptides immediate-early-1 and pp65 were analyzed in 16 CMV-seropositive kidney and heart transplant recipients longitudinally pretransplantation and posttransplantation. All patients received standard of care maintenance immunosuppression, antiviral prophylaxis, and CMV viral load monitoring, with approximately half receiving T cell-depleting induction therapy. The frequency of CMV-responsive CD8 + T cells, defined by the production of effector molecules in response to CMV peptides, increased during the course of 1 year posttransplantation. The increase commenced after the completion of antiviral prophylaxis, and these T cells tended to be terminally differentiated effector cells. Based on this small cohort, these data suggest that even in the absence of disease, antigenic exposure may continually shape the CMV-responsive T cell population posttransplantation. © 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.

Epithelium-Innate Immune Cell Axis in Mucosal Responses to SIV

PubMed Central

Shang, L.; Duan, L.; Perkey, K. E.; Wietgrefe, S.; Zupancic, M.; Smith, A. J.; Southern, P. J.; Johnson, R. P.; Haase, A. T.

2016-01-01

In the SIV-rhesus macaque model of HIV-1 transmission to women, one hallmark of the mucosal response to exposure to high doses of SIV is CD4 T cell recruitment that fuels local virus expansion in early infection. In this study, we systematically analyzed the cellular events and chemoattractant profiles in cervical tissues that precede CD4 T cell recruitment. We show that vaginal exposure to the SIV inoculum rapidly induces chemokine expression in cervical epithelium including CCL3, CCL20, and CXCL8. The chemokine expression is associated with early recruitment of macrophages and plasmacytoid dendritic cells that are co-clustered underneath the cervical epithelium. Production of chemokines CCL3 and CXCL8 by these cells in turn generates a chemokine gradient that is spatially correlated with the recruitment of CD4 T cells. We further show that the protection of SIVmac239Δnef vaccination against vaginal challenge is correlated with the absence of this epithelium-innate immune cell-CD4 T cell axis response in the cervical mucosa. Our results reveal a critical role for cervical epithelium in initiating early mucosal responses to vaginal infection, highlight an important role for macrophages in target cell recruitment and provide further evidence of a paradoxical dampening effect of a protective vaccine on these early mucosal responses. PMID:27435105

The utility of the new generation of humanized mice to study HIV-1 infection: transmission, prevention, pathogenesis, and treatment

PubMed Central

2011-01-01

Substantial improvements have been made in recent years in the ability to engraft human cells and tissues into immunodeficient mice. The use of human hematopoietic stem cells (HSCs) leads to multi-lineage human hematopoiesis accompanied by production of a variety of human immune cell types. Population of murine primary and secondary lymphoid organs with human cells occurs, and long-term engraftment has been achieved. Engrafted cells are capable of producing human innate and adaptive immune responses, making these models the most physiologically relevant humanized animal models to date. New models have been successfully infected by a variety of strains of Human Immunodeficiency Virus Type 1 (HIV-1), accompanied by virus replication in lymphoid and non-lymphoid organs, including the gut-associated lymphoid tissue, the male and female reproductive tracts, and the brain. Multiple forms of virus-induced pathogenesis are present, and human T cell and antibody responses to HIV-1 are detected. These humanized mice are susceptible to a high rate of rectal and vaginal transmission of HIV-1 across an intact epithelium, indicating the potential to study vaccines and microbicides. Antiviral drugs, siRNAs, and hematopoietic stem cell gene therapy strategies have all been shown to be effective at reducing viral load and preventing or reversing helper T cell loss in humanized mice, indicating that they will serve as an important preclinical model to study new therapeutic modalities. HIV-1 has also been shown to evolve in response to selective pressures in humanized mice, thus showing that the model will be useful to study and/or predict viral evolution in response to drug or immune pressures. The purpose of this review is to summarize the findings reported to date on all new humanized mouse models (those transplanted with human HSCs) in regards to HIV-1 sexual transmission, pathogenesis, anti-HIV-1 immune responses, viral evolution, pre- and post-exposure prophylaxis, and gene therapeutic strategies. PMID:21835012

The rat suprachiasmatic nucleus: the master clock ticks at 30 Hz

PubMed Central

Tsuji, Takahiro; Tsuji, Chiharu; Ludwig, Mike

2016-01-01

Key points Light‐responsive neurones in the rat suprachiasmatic nucleus discharge with a harmonic distribution of interspike intervals, whereas unresponsive neurones seldom do.This harmonic patterning has a fundamental frequency of close to 30 Hz, and is the same in light‐on cells as in light‐off cells, and is unaffected by exposure to light.Light‐on cells are more active than light‐off cells in both subjective day and subjective night, and both light‐on cells and light‐off cells respond more strongly to changes in light intensity during the subjective night than during the subjective day.Paired recordings indicate that the discharge of adjacent light‐responsive cells is very tightly synchronized.The gap junction inhibitor carbenoxolone increases the spontaneous activity of suprachiasmatic nucleus neurones but does not block the harmonic discharge patterning. Abstract The suprachiasmatic nucleus (SCN) of the hypothalamus has an essential role in orchestrating circadian rhythms of behaviour and physiology. In the present study, we recorded from single SCN neurons in urethane‐anaesthetized rats, categorized them by the statistical features of their electrical activity and by their responses to light, and examined how activity in the light phase differs from activity in the dark phase. We classified cells as light‐on cells or light‐off cells according to how their firing rate changed in acute response to light, or as non‐responsive cells. In both sets of light‐responsive neurons, responses to light were stronger at subjective night than in subjective day. Neuronal firing patterns were analysed by constructing hazard functions from interspike interval data. For most light‐responsive cells, the hazard functions showed a multimodal distribution, with a harmonic sequence of modes, indicating that spike activity was driven by an oscillatory input with a fundamental frequency of close to 30 Hz; this harmonic pattern was rarely seen in non‐responsive SCN cells. The frequency of the rhythm was the same in light‐on cells as in light‐off cells, was the same in subjective day as at subjective night, and was unaffected by exposure to light. Paired recordings indicated that the discharge of adjacent light‐responsive neurons was very tightly synchronized, consistent with electrical coupling. PMID:27061101

Multiple host defense defects in failure of C57BL/6 ep/ep (pale ear) mice to resolve visceral Leishmania donovani infection.

PubMed Central

Murray, H W; Hariprashad, J; McDermott, D F; Stoeckle, M Y

1996-01-01

Euthymic C57BL/L ep/ep (pale ear [PE]) mice halt the visceral replication of intracellular Leishmania donovani but fail to properly resolve infection. A previous study identified an isolated defect in tissue granuloma formation in these mice; CD4+ and CD8+ cell number, gamma interferon (IFN-gamma) production, and macrophage antimicrobial activity in vitro were all intact. New in vivo results reported here suggest a considerably more complex immune defect, with evidence indicating (i) enhanced control over L. donovani after transfer of normal C57BL/6 spleen cells, (ii) a partially suppressive Th2 cell-associated response mediated by interleukin-4 (IL-4) but not reversed by CD4+ cell depletion, (iii) absent responses to endogenous Th1 cell lymphokines (IFN-gamma and IL-2) but preserved responsiveness to endogenous tumor necrosis factor alpha, (iv) absent responses to exogenous treatment with recognized antileishmanial cytokines (IFN-gamma, IL-2, IL-12, and granulocyte-macrophage colony-stimulating factor [GM-CSF]) not corrected by transfer of C57BL/6 spleen cells, and (v) a deficient response to antimony chemotherapy. Defective hepatic granuloma formation was not corrected by transfer of C57BL/6 spleen cells or by anti-IL-4 administration. While treatment with IL-2 and GM-CSF modified the tissue reaction and induced selected effector cells to encase tissue macrophages, no antileishmanial activity resulted. Together, these observations suggest that the failure of PE mice to resolve visceral L. donovani infection likely represents expression of multiple suboptimal immune responses and/or partial defects, probably involving a combination of T-cell dysfunction, a Th2 cell response, and target cell (macrophage) hyporesponsiveness. PMID:8557335

B cell immunopoiesis: visualizing the impact of CD40 engagement on the course of T cell-independent immune responses in an Ig transgenic system.

PubMed

Erickson, L D; Vogel, L A; Cascalho, M; Wong, J; Wabl, M; Durell, B G; Noelle, R J

2000-11-01

This study tracks the fate of antigen-reactive B cells through follicular and extrafollicular responses and addresses the function of CD40 in these processes. The unique feature of this system is the use of transgenic B cells in which the heavy chain locus has been altered by site-directed insertion of a rearranged V(H) DJ(H) exon such that they are able to clonally expand, isotype-switch and follow a normal course of differentiation upon immunization. These Ig transgenic B cells when adoptively transferred into non-transgenic (Tg) mice in measured amounts expanded and differentiated distinctively in response to T cell-independent (TI) or T cell-dependent (TD) antigens. The capacity of these Tg B cells to faithfully recapitulate the humoral immune response to TI and TD antigens provides the means to track clonal B cell behavior in vivo. Challenge with TI antigen in the presence of agonistic anti-CD40 mAb resulted in well-defined alterations of the TI response. In vivo triggering of Tg B cells with TI antigen and CD40 caused an increase in the levels IgG produced and a broadening of the Ig isotype profile, characteristics which partially mimic TD responses. Although some TD characteristics were induced by TI antigen and CD40 triggering, the Tg B cells failed to acquire a germinal center phenotype and failed to generate a memory response. Therefore, TD-like immunity can be only partially reconstituted with CD40 agonists and TI antigens, suggesting that there are additional signals required for germinal center formation and development of memory.

A single dose of inactivated hepatitis A vaccine promotes HAV-specific memory cellular response similar to that induced by a natural infection.

PubMed

Melgaço, Juliana Gil; Morgado, Lucas Nóbrega; Santiago, Marta Almeida; Oliveira, Jaqueline Mendes de; Lewis-Ximenez, Lia Laura; Hasselmann, Bárbara; Cruz, Oswaldo Gonçalves; Pinto, Marcelo Alves; Vitral, Claudia Lamarca

2015-07-31

Based on current studies on the effects of single dose vaccines on antibody production, Latin American countries have adopted a single dose vaccine program. However, no data are available on the activation of cellular response to a single dose of hepatitis A. Our study investigated the functional reactivity of the memory cell phenotype after hepatitis A virus (HAV) stimulation through administration of the first or second dose of HAV vaccine and compared the response to that of a baseline group to an initial natural infection. Proliferation assays showed that the first vaccine dose induced HAV-specific cellular response; this response was similar to that induced by a second dose or an initial natural infection. Thus, from the first dose to the second dose, increase in the frequencies of classical memory B cells, TCD8 cells, and central memory TCD4 and TCD8 cells were observed. Regarding cytokine production, increased IL-6, IL-10, TNF, and IFNγ levels were observed after vaccination. Our findings suggest that a single dose of HAV vaccine promotes HAV-specific memory cell response similar to that induced by a natural infection. The HAV-specific T cell immunity induced by primary vaccination persisted independently of the protective plasma antibody level. In addition, our results suggest that a single dose immunization system could serve as an alternative strategy for the prevention of hepatitis A in developing countries. Copyright © 2015 Elsevier Ltd. All rights reserved.

Alloimmune Responses of Humanized Mice to Human Pluripotent Stem Cell Therapeutics.

PubMed

Kooreman, Nigel G; de Almeida, Patricia E; Stack, Jonathan P; Nelakanti, Raman V; Diecke, Sebastian; Shao, Ning-Yi; Swijnenburg, Rutger-Jan; Sanchez-Freire, Veronica; Matsa, Elena; Liu, Chun; Connolly, Andrew J; Hamming, Jaap F; Quax, Paul H A; Brehm, Michael A; Greiner, Dale L; Shultz, Leonard D; Wu, Joseph C

2017-08-22

There is growing interest in using embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) derivatives for tissue regeneration. However, an increased understanding of human immune responses to stem cell-derived allografts is necessary for maintaining long-term graft persistence. To model this alloimmunity, humanized mice engrafted with human hematopoietic and immune cells could prove to be useful. In this study, an in-depth analysis of graft-infiltrating human lymphocytes and splenocytes revealed that humanized mice incompletely model human immune responses toward allogeneic stem cells and their derivatives. Furthermore, using an "allogenized" mouse model, we show the feasibility of reconstituting immunodeficient mice with a functional mouse immune system and describe a key role of innate immune cells in the rejection of mouse stem cell allografts. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Response of the oligodendrocyte progenitor cell population (defined by NG2 labelling) to demyelination of the adult spinal cord.

PubMed

Keirstead, H S; Levine, J M; Blakemore, W F

1998-02-01

Elucidation of the response of oligodendrocyte progenitor cell populations to demyelination in the adult central nervous system (CNS) is critical to understanding why remyelination fails in multiple sclerosis. Using the anti-NG2 monoclonal antibody to identify oligodendrocyte progenitor cells, we have documented their response to antibody-induced demyelination in the dorsal column of the adult rat spinal cord. The number of NG2+ cells in the vicinity of demyelinated lesions increased by 72% over the course of 3 days following the onset of demyelination. This increase in NG2+ cell numbers did not reflect a nonspecific staining of reactive cells, as GFAP, OX-42, and Rip antibodies did not co-localise with NG2 + cells in double immunostained tissue sections. NG2 + cells incorporated BrdU 48-72 h following the onset of demyelination. After the onset of remyelination (10-14 days), the number of NG2+ cells decreased to 46% of control levels and remained consistently low for 2 months. When spinal cords were exposed to 40 Grays of x-irradiation prior to demyelination, the number of NG2+ cells decreased to 48% of control levels by 3 days following the onset of demyelination and remained unchanged at 3 weeks. Since 40 Grays of x-irradiation kills dividing cells, these studies illustrate a responsive and nonresponsive NG2+ cell population following demyelination in the adult spinal cord and suggest that the responsive NG2+ cell population does not renew itself.

Different subsets of natural killer T cells may vary in their roles in health and disease.

PubMed

Kumar, Vipin; Delovitch, Terry L

2014-07-01

Natural killer T cells (NKT) can regulate innate and adaptive immune responses. Type I and type II NKT cell subsets recognize different lipid antigens presented by CD1d, an MHC class-I-like molecule. Most type I NKT cells express a semi-invariant T-cell receptor (TCR), but a major subset of type II NKT cells reactive to a self antigen sulphatide use an oligoclonal TCR. Whereas TCR-α dominates CD1d-lipid recognition by type I NKT cells, TCR-α and TCR-β contribute equally to CD1d-lipid recognition by type II NKT cells. These variable modes of NKT cell recognition of lipid-CD1d complexes activate a host of cytokine-dependent responses that can either exacerbate or protect from disease. Recent studies of chronic inflammatory and autoimmune diseases have led to a hypothesis that: (i) although type I NKT cells can promote pathogenic and regulatory responses, they are more frequently pathogenic, and (ii) type II NKT cells are predominantly inhibitory and protective from such responses and diseases. This review focuses on a further test of this hypothesis by the use of recently developed techniques, intravital imaging and mass cytometry, to analyse the molecular and cellular dynamics of type I and type II NKT cell antigen-presenting cell motility, interaction, activation and immunoregulation that promote immune responses leading to health versus disease outcomes. © 2014 John Wiley & Sons Ltd.

A single-cell correlative nanoelectromechanosensing approach to detect cancerous transformation: monitoring the function of F-actin microfilaments in the modulation of the ion channel activity

NASA Astrophysics Data System (ADS)

AbdolahadThe Authors With Same Contributions., Mohammad; Saeidi, Ali; Janmaleki, Mohsen; Mashinchian, Omid; Taghinejad, Mohammad; Taghinejad, Hossein; Azimi, Soheil; Mahmoudi, Morteza; Mohajerzadeh, Shams

2015-01-01

Cancerous transformation may be dependent on correlation between electrical disruptions in the cell membrane and mechanical disruptions of cytoskeleton structures. Silicon nanotube (SiNT)-based electrical probes, as ultra-accurate signal recorders with subcellular resolution, may create many opportunities for fundamental biological research and biomedical applications. Here, we used this technology to electrically monitor cellular mechanosensing. The SiNT probe was combined with an electrically activated glass micropipette aspiration system to achieve a new cancer diagnostic technique that is based on real-time correlation between mechanical and electrical behaviour of single cells. Our studies demonstrated marked changes in the electrical response following increases in the mechanical aspiration force in healthy cells. In contrast, such responses were extremely weak for malignant cells. Confocal microscopy results showed the impact of actin microfilament remodelling on the reduction of the electrical response for aspirated cancer cells due to the significant role of actin in modulating the ion channel activity in the cell membrane.Cancerous transformation may be dependent on correlation between electrical disruptions in the cell membrane and mechanical disruptions of cytoskeleton structures. Silicon nanotube (SiNT)-based electrical probes, as ultra-accurate signal recorders with subcellular resolution, may create many opportunities for fundamental biological research and biomedical applications. Here, we used this technology to electrically monitor cellular mechanosensing. The SiNT probe was combined with an electrically activated glass micropipette aspiration system to achieve a new cancer diagnostic technique that is based on real-time correlation between mechanical and electrical behaviour of single cells. Our studies demonstrated marked changes in the electrical response following increases in the mechanical aspiration force in healthy cells. In contrast, such responses were extremely weak for malignant cells. Confocal microscopy results showed the impact of actin microfilament remodelling on the reduction of the electrical response for aspirated cancer cells due to the significant role of actin in modulating the ion channel activity in the cell membrane. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr06102k

Immune selection of tumor cells in TCR β-chain transgenic mice.

PubMed

Silaeva, Yulia Yu; Grinenko, Tatyana S; Vagida, Murad S; Kalinina, Anastasia A; Khromykh, Ludmila M; Kazansky, Dmitry B

2014-10-01

The concept of immunological surveillance implies that immunogenic variants of tumor cells arising in the organism can be recognized by the immune system. Tumor progression is provided by somatic evolution of tumor cells under the pressure of the immune system. The loss of MHC Class I molecules on the surface of tumor cells is one of the most known outcomes of immune selection. This study developed a model of immune selection based on the immune response of TCR 1d1 single β-chain transgenic B10.D2(R101) (K(d)I(d)D(b)) mice to allogeneic EL4 (H-2(b)) thymoma cells. In wild-type B10.D2(R101) mice, immunization with EL4 cells induced a vigorous CTL response targeted to the H-2K(b) molecule and results in full rejection of the tumor cells. In contrast, transgenic mice developed a compromised proliferative response in mixed-lymphocyte response assays and were unable to reject transplanted allogeneic EL4 cells. During the immune response to EL4 cells, CD8(+) T-lymphocytes with endogenous β-chains accumulated predominantly in the spleen of transgenic mice and only a small part of the T-lymphocytes expressing transgenic β-chains became CD8(+)CD44(+)CD62L(-) effectors. Then, instead of a full elimination of tumor cells as in wild-type mice, a reproducible prolonged equilibrium phase and subsequent escape was observed in transgenic mice that resulted in death of 90% of the mice in 40-60 days after grafting. Prolonged exposure of tumor cells to the pressure of the immune system in transgenic mice in vivo resulted in a stable loss of H-2K(b) molecules on the EL4 cell surface. Genetic manipulation of the T-lymphocyte repertoire was sufficient to reproduce the classic pattern of interactions between tumor cells and the immune system, usually observed in reliable syngeneic models of anti-tumor immunity. This newly-developed model could be used in further studies of immunoregulatory circuits common for transplantational and anti-tumor immune responses.

Characterization of Specific Immune Responses to Different Aspergillus Antigens during the Course of Invasive Aspergillosis in Hematologic Patients

PubMed Central

Beauvais, Anne; Beau, Remi; Candoni, Anna; Maertens, Johan; Rossi, Giulio; Morselli, Monica; Zanetti, Eleonora; Quadrelli, Chiara; Codeluppi, Mauro; Guaraldi, Giovanni; Pagano, Livio; Caira, Morena; Giovane, Cinzia Del; Maccaferri, Monica; Stefani, Alessandro; Morandi, Uliano; Tazzioli, Giovanni; Girardis, Massimo; Delia, Mario; Specchia, Giorgina; Longo, Giuseppe; Marasca, Roberto; Narni, Franco; Merli, Francesco; Imovilli, Annalisa; Apolone, Giovanni; Carvalho, Agostinho; Comoli, Patrizia; Romani, Luigina; Latgè, Jean Paul; Luppi, Mario

2013-01-01

Several studies in mouse model of invasive aspergillosis (IA) and in healthy donors have shown that different Aspergillus antigens may stimulate different adaptive immune responses. However, the occurrence of Aspergillus-specific T cells have not yet been reported in patients with the disease. In patients with IA, we have investigated during the infection: a) whether and how specific T-cell responses to different Aspergillus antigens occur and develop; b) which antigens elicit the highest frequencies of protective immune responses and, c) whether such protective T cells could be expanded ex-vivo. Forty hematologic patients have been studied, including 22 patients with IA and 18 controls. Specific T cells producing IL-10, IFN-γ, IL-4 and IL-17A have been characterized through enzyme linked immunospot and cytokine secretion assays on 88 peripheral blood (PB) samples, by using the following recombinant antigens: GEL1p, CRF1p, PEP1p, SOD1p, α1–3glucan, β1–3glucan, galactomannan. Specific T cells were expanded through short term culture. Aspergillus-specific T cells producing non-protective interleukin-10 (IL-10) and protective interferon-gamma (IFN-γ) have been detected to all the antigens only in IA patients. Lower numbers of specific T cells producing IL-4 and IL-17A have also been shown. Protective T cells targeted predominantly Aspergillus cell wall antigens, tended to increase during the IA course and to be associated with a better clinical outcome. Aspergillus-specific T cells could be successfully generated from the PB of 8 out of 8 patients with IA and included cytotoxic subsets able to lyse Aspergillus hyphae. Aspergillus specific T-cell responses contribute to the clearance of the pathogen in immunosuppressed patients with IA and Aspergillus cell wall antigens are those mainly targeted by protective immune responses. Cytotoxic specific T cells can be expanded from immunosuppressed patients even during the infection by using the above mentioned antigens. These findings may be exploited for immunotherapeutic purposes in patients with IA. PMID:24023936

Increased CD8+ T Cell Response to Epstein-Barr Virus Lytic Antigens in the Active Phase of Multiple Sclerosis

PubMed Central

Angelini, Daniela F.; Serafini, Barbara; Piras, Eleonora; Severa, Martina; Coccia, Eliana M.; Rosicarelli, Barbara; Ruggieri, Serena; Gasperini, Claudio; Buttari, Fabio; Centonze, Diego; Mechelli, Rosella; Salvetti, Marco; Borsellino, Giovanna; Aloisi, Francesca; Battistini, Luca

2013-01-01

It has long been known that multiple sclerosis (MS) is associated with an increased Epstein-Barr virus (EBV) seroprevalence and high immune reactivity to EBV and that infectious mononucleosis increases MS risk. This evidence led to postulate that EBV infection plays a role in MS etiopathogenesis, although the mechanisms are debated. This study was designed to assess the prevalence and magnitude of CD8+ T-cell responses to EBV latent (EBNA-3A, LMP-2A) and lytic (BZLF-1, BMLF-1) antigens in relapsing-remitting MS patients (n = 113) and healthy donors (HD) (n = 43) and to investigate whether the EBV-specific CD8+ T cell response correlates with disease activity, as defined by clinical evaluation and gadolinium-enhanced magnetic resonance imaging. Using HLA class I pentamers, lytic antigen-specific CD8+ T cell responses were detected in fewer untreated inactive MS patients than in active MS patients and HD while the frequency of CD8+ T cells specific for EBV lytic and latent antigens was higher in active and inactive MS patients, respectively. In contrast, the CD8+ T cell response to cytomegalovirus did not differ between HD and MS patients, irrespective of the disease phase. Marked differences in the prevalence of EBV-specific CD8+ T cell responses were observed in patients treated with interferon-β and natalizumab, two licensed drugs for relapsing-remitting MS. Longitudinal studies revealed expansion of CD8+ T cells specific for EBV lytic antigens during active disease in untreated MS patients but not in relapse-free, natalizumab-treated patients. Analysis of post-mortem MS brain samples showed expression of the EBV lytic protein BZLF-1 and interactions between cytotoxic CD8+ T cells and EBV lytically infected plasma cells in inflammatory white matter lesions and meninges. We therefore propose that inability to control EBV infection during inactive MS could set the stage for intracerebral viral reactivation and disease relapse. PMID:23592979

Towards Predicting the Response of a Solid Tumour to Chemotherapy and Radiotherapy Treatments: Clinical Insights from a Computational Model

PubMed Central

Powathil, Gibin G.; Adamson, Douglas J. A.; Chaplain, Mark A. J.

2013-01-01

In this paper we use a hybrid multiscale mathematical model that incorporates both individual cell behaviour through the cell-cycle and the effects of the changing microenvironment through oxygen dynamics to study the multiple effects of radiation therapy. The oxygenation status of the cells is considered as one of the important prognostic markers for determining radiation therapy, as hypoxic cells are less radiosensitive. Another factor that critically affects radiation sensitivity is cell-cycle regulation. The effects of radiation therapy are included in the model using a modified linear quadratic model for the radiation damage, incorporating the effects of hypoxia and cell-cycle in determining the cell-cycle phase-specific radiosensitivity. Furthermore, after irradiation, an individual cell's cell-cycle dynamics are intrinsically modified through the activation of pathways responsible for repair mechanisms, often resulting in a delay/arrest in the cell-cycle. The model is then used to study various combinations of multiple doses of cell-cycle dependent chemotherapies and radiation therapy, as radiation may work better by the partial synchronisation of cells in the most radiosensitive phase of the cell-cycle. Moreover, using this multi-scale model, we investigate the optimum sequencing and scheduling of these multi-modality treatments, and the impact of internal and external heterogeneity on the spatio-temporal patterning of the distribution of tumour cells and their response to different treatment schedules. PMID:23874170

The regrowth kinetic of the surviving population is independent of acute and chronic responses to temozolomide in glioblastoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Silva, Andrew Oliveira, E-mail: andrewbiomed@gmail.com; Dalsin, Eloisa, E-mail: dalsineloisa@gmail.com; Onzi, Giovana Ravizzoni, E-mail: gioonzi@gmail.com

Chemotherapy acts on cancer cells by producing multiple effects on a cell population including cell cycle arrest, necrosis, apoptosis and senescence. However, often a subpopulation of cells survives and the behavior of this subpopulation, which is responsible for cancer recurrence, remains obscure. Here we investigated the in vitro short- and long-term responses of six glioblastoma cell lines to clinically relevant doses of temozolomide for 5 days followed by 23 days of recovery, mimicking the standard schedule used in glioblastoma patient for this drug. These cells presented different profiles of sensitivity to temozolomide with varying levels of cell cycle arrest, autophagymore » and senescence, followed by a regrowth of the surviving cells. The initial reduction in cell number and the subsequent regrowth was analyzed with four new parameters applied to Cumulative Population Doubling (CPD) curves that describe the overall sensitivity of the population and the characteristic of the regrowth: the relative end point CPD (RendCPD); the relative Area Under Curve (rAUC); the Relative Time to Cross a Threshold (RTCT); and the Relative Proliferation Rate (RPR). Surprisingly, the kinetics of regrowth were not predicted by the mechanisms activated after treatment nor by the acute or overall sensitivity. With this study we added new parameters that describe key responses of glioblastoma cell populations to temozolomide treatment. These parameters can also be applied to other cell types and treatments and will help to understand the behavior of the surviving cancer cells after treatment and shed light on studies of cancer resistance and recurrence. - Highlights: • Little is known about the behavior of the glioma cells surviving to TMZ. • The short- and long-term response of six glioma cells lines to TMZ varies considerably. • These glioma cells lines recovered proliferation after therapeutic levels of TMZ. • The growth velocity of the surviving cells was different from the untreated cells. • The kinetic of regrowth was not predicted by any TMZ-triggered mechanism.« less

Neuron-derived orphan receptor 1 transduces survival signals in neuronal cells in response to hypoxia-induced apoptotic insults.

PubMed

Chio, Chung-Ching; Wei, Li; Chen, Tyng Guey; Lin, Chien-Min; Shieh, Ja-Ping; Yeh, Poh-Shiow; Chen, Ruei-Ming

2016-06-01

OBJECT Hypoxia can induce cell death or trigger adaptive mechanisms to guarantee cell survival. Neuron-derived orphan receptor 1 (NOR-1) works as an early-response protein in response to a variety of environmental stresses. In this study, the authors evaluated the roles of NOR-1 in hypoxia-induced neuronal insults. METHODS Neuro-2a cells were exposed to oxygen/glucose deprivation (OGD). Cell viability, cell morphology, cas-pase-3 activity, DNA fragmentation, and cell apoptosis were assayed to determine the mechanisms of OGD-induced neuronal insults. RNA and protein analyses were carried out to evaluate the effects of OGD on expressions of NOR-1, cAMP response element-binding (CREB), and cellular inhibitor of apoptosis protein 2 (cIAP2) genes. Translations of these gene expressions were knocked down using RNA interference. Mice subjected to traumatic brain injury (TBI) and NOR-1 was immunodetected. RESULTS Exposure of neuro-2a cells to OGD decreased cell viability in a time-dependent manner. Additionally, OGD led to cell shrinkage, DNA fragmentation, and cell apoptosis. In parallel, treatment of neuro-2a cells with OGD time dependently increased cellular NOR-1 mRNA and protein expressions. Interestingly, administration of TBI also augmented NOR-1 levels in the impacted regions of mice. As to the mechanism, exposure to OGD increased nuclear levels of the transcription factor CREB protein. Downregulating CREB expression using RNA interference simultaneously inhibited OGD-induced NOR-1 mRNA expression. Also, levels of cIAP2 mRNA and protein in neuro-2a cells were augmented by OGD. After reducing cIAP2 translation, OGD-induced cell death was reduced. Sequentially, application of NOR-1 small interfering RNA to neuro-2a cells significantly inhibited OGD-induced cIAP2 mRNA expression and concurrently alleviated hypoxia-induced alterations in cell viability, caspase-3 activation, DNA damage, and cell apoptosis. CONCLUSIONS This study shows that NOR-1 can transduce survival signals in neuronal cells responsible for hypoxiainduced apoptotic insults through activation of a CREB/cIAP2-dependent mechanism.

1,25-Dihydroxyvitamin D3 and its analogues increase catalase at the mRNA, protein and activity level in a canine transitional carcinoma cell line.

PubMed

Middleton, R P; Nelson, R; Li, Q; Blanton, A; Labuda, J A; Vitt, J; Inpanbutr, N

2015-12-01

Antioxidant enzymes, such as catalase, superoxide dismutases (SOD), MnSOD and Cu/ZnSOD, protect cells by scavenging reactive oxygen species (ROS). Numerous studies have reported the anti-cancer effects of 1,25-dihydroxyvitamin D3 (calcitriol) and its related analogues, seocalcitol and analogue V. In this study, canine bladder transitional cell carcinoma (cbTCC) cells were used to determine effects of calcitriol and its related analogues on antioxidant enzyme gene expression, protein expression and activity. Catalase mRNA was increased in response to calcitriol (10(-7) M), and seocalcitol (10(-7) and 10(-9) M). MnSOD mRNA was decreased in response to calcitriol at 10(-7) M. Catalase was significantly increased in response to calcitriol (10(-7) and 10(-9) M), and seocalcitol (10(-9) M). Catalase enzymatic activity increased in response to calcitriol, seocalcitol and analogue V (10(-9) M). In addition, global gene expression analysis identified the involvement of mitogen-activated protein kinase (MAPK) signalling in cbTCC's response to calcitriol and seocalcitol treatment.

Stimuli-responsive protamine-based biodegradable nanocapsules for enhanced bioavailability and intracellular delivery of anticancer agents

NASA Astrophysics Data System (ADS)

Radhakrishnan, Krishna; Thomas, Midhun B.; Pulakkat, Sreeranjini; Gnanadhas, Divya P.; Chakravortty, Dipshikha; Raichur, Ashok M.

2015-08-01

Enzyme- and pH-responsive polyelectrolyte nanocapsules having diameters in the range of 200 ± 20 nm were fabricated by means of Layer-by-Layer assembly of biopolymers, protamine, and heparin, and then loaded with anticancer drug doxorubicin. The incorporation of the FDA-approved peptide drug protamine as a wall component rendered the capsules responsive to enzyme stimuli. The stimuli-responsive drug release from these nanocapsules was evaluated, and further modulation of capsule permeability to avoid premature release was demonstrated by crosslinking the wall components. The interaction of the nanocapsules with cancer cells was studied using MCF-7 breast cancer cells. These capsules were readily internalized and disintegrated inside the cells, culminating in the release of the loaded doxorubicin and subsequent cell death as observed by confocal microscopy and MTT Assay. The bioavailability studies performed using BALB/c mice revealed that the encapsulated doxorubicin exhibited enhanced bioavailability compared to free doxorubicin. Our results indicate that this stimuli-responsive system fabricated from clinically used FDA-approved molecules and exhibiting minimal premature release has great potential for drug-delivery applications.

Collective cell migration during inflammatory response

NASA Astrophysics Data System (ADS)

Wu, Di; Stroka, Kimberly; Aranda-Espinoza, Helim

2012-02-01

Wound scratch healing assays of endothelial cell monolayers is a simple model to study collective cell migration as a function of biological signals. A signal of particular interest is the immune response, which after initial wounding in vivo causes the release of various inflammatory factors such as tumor necrosis alpha (TNF-α). TNF-α is an innate inflammatory cytokine that can induce cell growth, cell necrosis, and change cell morphology. We studied the effects of TNF-α on collective cell migration using the wound healing assays and measured several migration metrics, such as rate of scratch closure, velocities of leading edge and bulk cells, closure index, and velocity correlation functions between migrating cells. We observed that TNF-α alters all migratory metrics as a function of the size of the scratch and TNF-α content. The changes observed in migration correlate with actin reorganization upon TNF-α exposure.

EdU induces DNA damage response and cell death in mESC in culture.

PubMed

Kohlmeier, Fanni; Maya-Mendoza, Apolinar; Jackson, Dean A

2013-03-01

Recently, a novel DNA replication precursor analogue called 5-ethynyl-2'-deoxyuridine (EdU) has been widely used to monitor DNA synthesis as an alternative to bromodeoxyuridine. Use of EdU benefits from simplicity and reproducibility and the simple chemical detection systems allows excellent preservation of nuclear structure. However, the alkyne moiety is highly reactive, raising the possibility that incorporation might compromise genome stability. To assess the extent of possible DNA damage, we have analysed the effect of EdU incorporation into DNA during short- and long-term cell culture using a variety of cell lines. We show that EdU incorporation has no measurable impact on the rate of elongation of replication forks during synthesis. However, using different cell lines we find that during long-term cell culture variable responses to EdU incorporation are seen, which range from delayed cell cycle progression to complete cell cycle arrest. The most profound phenotypes were seen in mouse embryonic stem cells, which following incorporation of EdU accumulated in the G2/M-phase of the cell cycle before undergoing apoptosis. In long-term cell culture, EdU incorporation also triggered a DNA damage response in all cell types analysed. Our study shows that while EdU is extremely useful to tag sites of on-going replication, for long-term studies (i.e. beyond the cell cycle in which labelling is performed), a careful analysis of cell cycle perturbations must be performed in order to ensure that any conclusions made after EdU treatment are not a direct consequence of EdU-dependent activation of cell stress responses.

Radiation-induced genomic instability and bystander effects: related inflammatory-type responses to radiation-induced stress and injury? A review.

PubMed

Lorimore, S A; Wright, E G

2003-01-01

To review studies of radiation responses in the haemopoietic system in the context of radiation-induced genomic instability, bystander effects and inflammatory-type processes. There is considerable evidence that cells that themselves are not exposed to ionizing radiation but are the progeny of cells irradiated many cell divisions previously may express a high frequency of gene mutations, chromosomal aberrations and cell death. These effects are collectively known as radiation-induced genomic instability. A second untargeted effect results in non-irradiated cells exhibiting responses typically associated with direct radiation exposure but occurs as a consequence of contact with irradiated cells or by receiving soluble signals from irradiated cells. These effects are collectively known as radiation-induced bystander effects. Reported effects include increases or decreases in damage-inducible and stress-related proteins; increases or decreases in reactive oxygen species, cell death or cell proliferation, and induction of mutations and chromosome aberrations. This array of responses is reminiscent of effects mediated by cytokines and other similar regulatory factors that may involve, but do not necessarily require, gap junction-mediated transfer, have multiple inducers and a variety of context-dependent consequences in different cell systems. That chromosomal instability in haemopoietic cells can be induced by an indirect bystander-type mechanism both in vitro and in vivo provides a potential link between these two untargeted effects and there are radiation responses in vivo consistent with the microenvironment contributing secondary cell damage as a consequence of an inflammatory-type response to radiation-induced injury. Intercellular signalling, production of cytokines and free radicals are features of inflammatory responses that have the potential for both bystander-mediated and persisting damage as well as for conferring a predisposition to malignancy. The induction of bystander effects and instabilities may reflect interrelated aspects of a non-specific inflammatory-type response to radiation-induced stress and injury and be involved in a variety of the pathological consequences of radiation exposures.

Immediate and long-term galvanotactic responses of Amoeba proteus to dc electric fields.

PubMed

Korohoda, W; Mycielska, M; Janda, E; Madeja, Z

2000-01-01

The long-term and immediate galvanotactic responses of Amoeba proteus to the direct current electric fields (dcEFs) were studied with the methods of computer-aided image analysis. It was found that in contrast to earlier reports, amoebae continued locomotion towards cathode (the negative pole) for hours and the increase in the field strength in the range 300-600 mV/mm caused the straightening of cell trajectories accompanied by the decreased frequency of the lateral pseudopods formation and lesser change in the speed of cell movement. In the cell regions pointing to the anode, the formation of new pseudopodia was prevented and the higher cEFs strength the more extended were the regions in which formation of new pseudopods was inhibited. Replacement of calcium with magnesium in the extracellular medium reduced the galvanotactic cell responses. Research on the localisation and kinetics of the primary cell responses to the dcEF or to change in its direction revealed that the primary cell responses occurred at the anode oriented cell regions. The cell response to the field reversal appeared to be localised and to take place in less than 1 sec. First the retraction and withdrawal of the anode-directed pseudopodium was observed whereas the uroid (cell tail) moved for 10-40 sec in the original direction before it begun to react to the field reversal. The exposure of amoebae to the dcEFs sensitised them to the reversion in the field direction and induced an acceleration of cell responses. The results presented are difficult to reconcile with the attempt to explain the cell galvanotaxis as a consequence of the membrane protein lateral electrophoresis or electroosmosis. It is suggested that the lateral electrophoresis of ions and the modification of ionic conditions at the vicinity of ion channels may be involved in the induction of fast responses of cells to external dcEFs. Copyright 2000 Wiley-Liss, Inc.

Surveillance Study of Influenza Occurrence and Immunity in a Wisconsin Cohort During the 2009 Pandemic

PubMed Central

Lo, Chia-Yun; Strobl, Susan L.; Dunham, Kimberly; Wang, Wei; Stewart, Lucy; Misplon, Julia A.; Garcia, Mayra; Gao, Jin; Ozawa, Tatsuhiko; Price, Graeme E.; Navidad, Jose; Gradus, Steve; Bhattacharyya, Sanjib; Viboud, Cecile; Eichelberger, Maryna C.; Weiss, Carol D.; Gorski, Jack

2017-01-01

Abstract Background. Antibody and T-cell immunity to conserved influenza virus antigens can protect animals against infection with diverse influenza strains. Although immunity against conserved antigens occurs in humans, whether such responses provide cross-protection in humans and could be harnessed as the basis for universal influenza vaccines is controversial. The 2009 pandemic provided an opportunity to investigate whether pre-existing cross-reactive immunity affected susceptibility to infection. Methods. In 2009, we banked sera and peripheral blood mononuclear cells (PBMC) from blood donors, then monitored them for pandemic influenza infection (pH1N1) by polymerase chain reaction or seroconversion. Antibodies to hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix 2 (M2), and HA-pseudotypes were measured in sera. T-cell inteferon-γ enzyme-linked immunospot responses were measured in PBMC. Results. There were 13 infections in 117 evaluable donors. Pre-existing T-cell reactivity to pH1N1 was substantial (of 153 donors tested, 146 had >100 spot-forming cells/106 cells). Antibodies reactive with pH1N1 were common: anti-NP (all donors) and anti-M2 (44% of donors). Pseudotype-neutralizing antibodies to H1 were detected, but not to highly conserved HA epitopes. Unexpectedly, donors with symptomatic pH1N1 infection had sharp rises in HA pseudotype-neutralizing antibodies, not only pH1N1 but also against multiple seasonal H1s. In addition, an exploratory study of a T-cell marker (response to NP418-426) identified probable infection missed by standard criteria. Conclusions. Although the number of infections was inadequate for conclusions about mechanisms of protection, this study documents the wide variety of pre-existing, cross-reactive, humoral and cellular immune responses to pandemic influenza virus antigens in humans. These responses can be compared with results of other studies and explored in universal influenza vaccine studies. PMID:28730155

SILAC-Based Quantitative Proteomic Analysis of Human Lung Cell Response to Copper Oxide Nanoparticles

PubMed Central

Edelmann, Mariola J.; Shack, Leslie A.; Naske, Caitlin D.; Walters, Keisha B.; Nanduri, Bindu

2014-01-01

Copper (II) oxide (CuO) nanoparticles (NP) are widely used in industry and medicine. In our study we evaluated the response of BEAS-2B human lung cells to CuO NP, using Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics and phosphoproteomics. Pathway modeling of the protein differential expression showed that CuO NP affect proteins relevant in cellular function and maintenance, protein synthesis, cell death and survival, cell cycle and cell morphology. Some of the signaling pathways represented by BEAS-2B proteins responsive to the NP included mTOR signaling, protein ubiquitination pathway, actin cytoskeleton signaling and epithelial adherens junction signaling. Follow-up experiments showed that CuO NP altered actin cytoskeleton, protein phosphorylation and protein ubiquitination level. PMID:25470785

Inhibition of lymphocyte proliferation and antibody production in vitro by silica, talc, bentonite or Corynebacterium parvum: involvement of peroxidative processes.

PubMed

Hoffeld, J T

1983-05-01

This study was undertaken to determine whether and by what means particles which induce granulomata in vivo can affect murine spleen lymphoproliferative and antibody responses in vitro. Particles of silica, talc, Bentonite or C. parvum cells inhibited lipopolysaccharide- or concanavalin A-stimulated proliferation and sheep red blood cell-induced antibody response in vitro. The inhibition required at least 48 hours exposure of the cells to the particles. The late onset of inhibition and its reproducibility at different cell or mitogen concentrations implicated particle-induced injury to both phagocytes and lymphocytes. Either alpha-tocopherol or 2-mercaptoethanol prevented the particle-induced inhibition of spleen cell responses. alpha-Tocopherol and 2-mercaptoethanol have in common the capacity to protect cells against membrane lipid peroxidation. The inhibitory peroxidative process(es) implicated by these studies are most likely attributable to: (a) stimulation of oxidative metabolism of phagocytic cells by particles; and (b) iron-catalyzed peroxidation directly by the particles. These data may be relevant in understanding the pathogenesis of and devising therapeutic approaches toward various granulomatous conditions.

Olfactory Nerve–Evoked, Metabotropic Glutamate Receptor–Mediated Synaptic Responses in Rat Olfactory Bulb Mitral Cells

PubMed Central

Ennis, Matthew; Zhu, Mingyan; Heinbockel, Thomas; Hayar, Abdallah

2008-01-01

The group I metabotropic glutamate receptor (mGluR) subtype, mGluR1, is highly expressed on the apical dendrites of olfactory bulb mitral cells and thus may be activated by glutamate released from olfactory nerve (ON) terminals. Previous studies have shown that mGluR1 agonists directly excite mitral cells. In the present study, we investigated the involvement of mGluR1 in ON-evoked responses in mitral cells in rat olfactory bulb slices using patch-clamp electrophysiology. In voltage-clamp recordings, the average EPSC evoked by single ON shocks or brief trains of ON stimulation (six pulses at 50 Hz) in normal physiological conditions were not significantly affected by the nonselective mGluR antagonist LY341495 (50–100 μM) or the mGluR1-specific antagonist LY367385 (100 μM); ON-evoked responses were attenuated, however, in a subset (36%) of cells. In the presence of blockers of ionotropic glutamate and GABA receptors, application of the glutamate uptake inhibitors THA (300 μM) and TBOA (100 μM) revealed large-amplitude, long-duration responses to ON stimulation, whereas responses elicited by antidromic activation of mitral/tufted cells were unaffected. Magnitudes of the ON-evoked responses elicited in the presence of THA–TBOA were dependent on stimulation intensity and frequency, and were maximal during high-frequency (50-Hz) bursts of ON spikes, which occur during odor stimulation. ON-evoked responses elicited in the presence of THA–TBOA were significantly reduced or completely blocked by LY341495 or LY367385 (100 μM). These results demonstrate that glutamate transporters tightly regulate access of synaptically evoked glutamate from ON terminals to postsynaptic mGluR1s on mitral cell apical dendrites. Taken together with other findings, the present results suggest that mGluR1s may not play a major role in phasic responses to ON input, but instead may play an important role in shaping slow oscillatory activity in mitral cells and/or activity-dependent regulation of plasticity at ON–mitral cell synapses. PMID:16394070

Cellular Interactions and Immune Response of Spherical Nucleic Acid (SNA) Nanoconjugates

NASA Astrophysics Data System (ADS)

Massich, Matthew David

Spherical nucleic acid (SNA) nanoconjugates consist of a densely packed monolayer shell of highly-oriented oligonucleotides covalently bound to a gold nanoparticle core. The nanoconjugates exhibit several important qualities, which make them useful for various biological applications, such as antisense gene regulation strategies and the intracellular detection of biomolecules. The focus of this thesis was to characterize the nanoconjugates interaction with cultured cells and specifically the immune response to their intracellular presence. The immune response of macrophage cells to internalized nanoconjugates was studied, and due to the dense functionalization of oligonucleotides on the surface of the nanoparticle and the resulting high localized salt concentration the innate immune response to the nanoconjugates is ˜25-fold less when compared to a lipoplex carrying the same sequence. Additionally, genome-wide expression profiling was used to study the biological response of cultured cells to the nanoconjugates. The biological response of HeLa cells to gold nanoparticles stabilized by weakly bound ligands was significant, yet when these same nanoparticles were stably functionalized with covalently attached oligonucleotides the cells showed no measurable response. In human keratinocytes, the oligonucleotide sequences caused 427 genes to be differentially expressed when complexed with Dharmafect, but when the oligonucleotides were conjugated to nanoparticles only 7 genes were differentially expressed. Beyond characterizing the cellular interactions and immune response of the nanoconjugates, the optimal length of siRNA (from 19--34 base pairs) that induces the most gene knockdown while maintaining limited immune activation was determined to be 24 base pairs. Further, the SNAs were shown to be useful as a potential antiviral gene therapy by demonstrating approximately 50% knockdown of the Ebola VP35 gene. Lastly, a scanning probe-enabled method was used to rapidly create nanoscale fibronectin patterns over large areas with a range of feature sizes, thereby opening the field of nanocombinatorics. This allowed the investigation of the relationship between fibronectin feature size and stem cell fate. MSCs cultured on nanoscale fibronectin features directed differentiation toward osteogenesis to a greater extent than cells grown on both microscale features and cells grown on non-patterned fibronectin substrates with osteogenic inducing media, demonstrating a new method for controlling stem cell fate.

Mechanics regulates ATP-stimulated collective calcium response in fibroblast cells

PubMed Central

Lembong, Josephine; Sabass, Benedikt; Sun, Bo; Rogers, Matthew E.; Stone, Howard A.

2015-01-01

Cells constantly sense their chemical and mechanical environments. We study the effect of mechanics on the ATP-induced collective calcium response of fibroblast cells in experiments that mimic various tissue environments. We find that closely packed two-dimensional cell cultures on a soft polyacrylamide gel (Young's modulus E = 690 Pa) contain more cells exhibiting calcium oscillations than cultures on a rigid substrate (E = 36 000 Pa). Calcium responses of cells on soft substrates show a slower decay of calcium level relative to those on rigid substrates. Actin enhancement and disruption experiments for the cell cultures allow us to conclude that actin filaments determine the collective Ca2+ oscillatory behaviour in the culture. Inhibition of gap junctions results in a decrease of the oscillation period and reduced correlation of calcium responses, which suggests additional complexity of signalling upon cell–cell contact. Moreover, the frequency of calcium oscillations is independent of the rigidity of the substrate but depends on ATP concentration. We compare our results with those from similar experiments on individual cells. Overall, our observations show that collective chemical signalling in cell cultures via calcium depends critically on the mechanical environment. PMID:26063818

Antigen presenting cells (APCs) from thermally injured and/or septic rats modulate CD4+ T cell responses of naive rat.

PubMed

Fazal, Nadeem; Raziuddin, Syed; Khan, Mehdi; Al-Ghoul, Walid M

2006-01-01

Regulation of immune response is marked by complex interactions among the cells that recognize and present antigens. Antigen presenting cells (APCs), the antigen presenting cell component of the innate immune response plays an important role in effector CD4+ T cell response. Thermal injury and/or superimposed sepsis in rats' leads to suppressed CD4+ T cell functions. We investigated modulations of CD4+ T cell function by APCs (purified non-T cells) from thermally injured and/or septic rats. Rats were subjected to 30% total body surface area scald burn or exposed to 37 degrees C water (Sham burn) and sepsis was induced by cecal-ligation and puncture (CLP) method. At day 3 post-injury animals were sacrificed and CD4+ T cells and APCs from mesenteric lymph nodes (MLN) were obtained using magnetic microbead isolation procedure. APCs from injured rats were co-cultured with sham rat MLN CD4+ T cells and proliferative responses (thymidine incorporation), phenotypic changes (Flow cytometry), IL-2 production (ELISA) and CTLA-4 mRNA (RT-PCR) were determined in naive rat CD4+ T cells. The data indicate that APCs from thermally injured and/or septic rats when co-cultured with CD4+ T cells suppressed CD4+ T cell effector functions. This lack of CD4+ T cell activation was accompanied with altered co-stimulatory molecules, i.e., CD28 and/or CTLA-4 (CD152). In conclusion, our studies indicated that defective APCs from thermally injured and/or septic rats modulate CD4+ T cell functions via changes in co-stimulatory molecules expressed on naive CD4+ T cells. This altered APC: CD4+ T cell interaction leads to suppressed CD4+ T cell activation of healthy animals.

The plant cell wall integrity maintenance mechanism--a case study of a cell wall plasma membrane signaling network.

PubMed

Hamann, Thorsten

2015-04-01

Some of the most important functions of plant cell walls are protection against biotic/abiotic stress and structural support during growth and development. A prerequisite for plant cell walls to perform these functions is the ability to perceive different types of stimuli in both qualitative and quantitative manners and initiate appropriate responses. The responses in turn involve adaptive changes in cellular and cell wall metabolism leading to modifications in the structures originally required for perception. While our knowledge about the underlying plant mechanisms is limited, results from Saccharomyces cerevisiae suggest the cell wall integrity maintenance mechanism represents an excellent example to illustrate how the molecular mechanisms responsible for stimulus perception, signal transduction and integration can function. Here I will review the available knowledge about the yeast cell wall integrity maintenance system for illustration purposes, summarize the limited knowledge available about the corresponding plant mechanism and discuss the relevance of the plant cell wall integrity maintenance mechanism in biotic stress responses. Copyright © 2014 Elsevier Ltd. All rights reserved.

Neutral Polymer Micelle Carriers with pH-Responsive, Endosome-Releasing Activity Modulate Antigen Trafficking to Enhance CD8 T-Cell Responses

PubMed Central

Keller, Salka; Wilson, John T; Patilea, Gabriela I; Kern, Hanna B; Convertine, Anthony J; Stayton, Patrick S

2014-01-01

Synthetic subunit vaccines need to induce CD8+ cytotoxic T-cell (CTL) responses for effective vaccination against intracellular pathogens. Most subunit vaccines primarily generate humoral immune responses, with a weaker than desired CD8+ cytotoxic T-cell response. Here, a neutral, pH-responsive polymer micelle carrier that alters intracellular antigen trafficking was shown to enhance CD8+ T-cell responses with a correlated increase in cytosolic delivery and a decrease in exocytosis. Polymer diblock carriers consisted of a N-(2-hydroxypropyl) methacrylamide corona block with pendant pyridyl disulfide groups for reversible conjugation of thiolated ovalbumin, and a tercopolymer ampholytic core-forming block composed of propylacrylic acid (PAA), dimethylaminoethyl methacrylate (DMAEMA), and butyl methacrylate (BMA). The diblock copolymers self-assembled into 25–30 nm diameter micellar nanoparticles. Conjugation of ovalbumin to the micelles significantly enhanced antigen cross-presentation in vitro relative to free ovalbumin, an unconjugated physical mixture of ovalbumin and polymer, and a non pH-responsive micelle-ovalbumin control. Mechanistic studies in a murine dendritic cell line (DC2.4) demonstrated micelle-mediated enhancements in intracellular antigen retention and cytosolic antigen accumulation. Approximately 90% of initially internalized ovalbumin-conjugated micelles were retained in cells after 1.5 h, compared to only ~40% for controls. Furthermore, cells dosed with conjugates displayed 67-fold higher cytosolic antigen levels relative to soluble ovalbumin 4 h post uptake. Subcutaneous immunization of mice with ovalbumin-polymer conjugates significantly enhanced antigen-specific CD8+ T cell responses (0.4 % IFN-γ+ of CD8+) compared to immunization with soluble protein, ovalbumin and polymer mixture, and the control micelle without endosome-releasing activity. Additionally, pH-responsive carrier facilitated antigen delivery to antigen presenting cells in the draining lymph nodes. As early as 90 min post injection ova-micelle conjugates were associated with 28% and 55% of dendritic cells and macrophages, respectively. After 24 h, conjugates preferentially associated with dendritic cells, affording 30-, 3-, and 3-fold enhancements in uptake relative to free protein, physical mixture, and the non pH-responsive conjugate controls, respectively. These results demonstrate the potential of pH-responsive polymeric micelles for use in vaccine applications that rely on CD8+ T cell activation. PMID:24698946

Neutral polymer micelle carriers with pH-responsive, endosome-releasing activity modulate antigen trafficking to enhance CD8(+) T cell responses.

PubMed

Keller, Salka; Wilson, John T; Patilea, Gabriela I; Kern, Hanna B; Convertine, Anthony J; Stayton, Patrick S

2014-10-10

Synthetic subunit vaccines need to induce CD8(+) cytotoxic T cell (CTL) responses for effective vaccination against intracellular pathogens. Most subunit vaccines primarily generate humoral immune responses, with a weaker than desired CD8(+) cytotoxic T cell response. Here, a neutral, pH-responsive polymer micelle carrier that alters intracellular antigen trafficking was shown to enhance CD8(+) T cell responses with a correlated increase in cytosolic delivery and a decrease in exocytosis. Polymer diblock carriers consisted of a N-(2-hydroxypropyl) methacrylamide corona block with pendent pyridyl disulfide groups for reversible conjugation of thiolated ovalbumin, and a tercopolymer ampholytic core-forming block composed of propylacrylic acid (PAA), dimethylaminoethyl methacrylate (DMAEMA), and butyl methacrylate (BMA). The diblock copolymers self-assembled into 25-30nm diameter micellar nanoparticles. Conjugation of ovalbumin to the micelles significantly enhanced antigen cross-presentation in vitro relative to free ovalbumin, an unconjugated physical mixture of ovalbumin and polymer, and a non-pH-responsive micelle-ovalbumin control. Mechanistic studies in a murine dendritic cell line (DC 2.4) demonstrated micelle-mediated enhancements in intracellular antigen retention and cytosolic antigen accumulation. Approximately 90% of initially internalized ovalbumin-conjugated micelles were retained in cells after 1.5h, compared to only ~40% for controls. Furthermore, cells dosed with conjugates displayed 67-fold higher cytosolic antigen levels relative to soluble ovalbumin 4h post uptake. Subcutaneous immunization of mice with ovalbumin-polymer conjugates significantly enhanced antigen-specific CD8(+) T cell responses (0.4% IFN-γ(+) of CD8(+)) compared to immunization with soluble protein, ovalbumin and polymer mixture, and the control micelle without endosome-releasing activity. Additionally, pH-responsive carrier facilitated antigen delivery to antigen presenting cells in the draining lymph nodes. As early as 90min post injection, ova-micelle conjugates were associated with 28% and 55% of dendritic cells and macrophages, respectively. After 24h, conjugates preferentially associated with dendritic cells, affording 30-, 3-, and 3-fold enhancements in uptake relative to free protein, physical mixture, and the non-pH-responsive conjugate controls, respectively. These results demonstrate the potential of pH-responsive polymeric micelles for use in vaccine applications that rely on CD8(+) T cell activation. Copyright © 2014 Elsevier B.V. All rights reserved.

Ionic mechanisms underlying the responses of off-center bipolar cells in the carp retina. II. Studies on responses evoked by transretinal current stimulation.

PubMed

Kaneko, A; Saito, T

1983-04-01

Transretinal current pulses flowing from the receptor side to the vitreous side of the retina cause transient release of transmitter from the photoreceptor terminals, and in off-center bipolar cells they evoke transient depolarizations with a brief (less than 1 ms) synaptic delay. Since it is known that the presence of Na+ in the external medium is not essential for this type of transmitter release, we used this procedure to examine the role of [Na+]o in the generation of light-evoked responses (hyperpolarizing to spot illumination in the receptive field center and depolarizing to an annulus in the surround) of this type of bipolar cell. When the cell membrane was steadily depolarized by current injection through the recording microelectrode, the depolarizing response evoked by the transretinal current pulses decreased in amplitude and reversed its polarity at above +45 mV. Conversely, the response amplitude increased when the cell was steadily hyperpolarized. The reversal potential seems to be lowered in low [Na+]o (28 mM). Removal of Na+ from the superfusate hyperpolarized the cell and both the light-evoked and current-evoked responses disappeared. From these observations, it is hypothesized that the hyperpolarizing center response of the off-center bipolar cells is a result of removal of sustained depolarization produced by sodium permeability increase.

Merck Ad5/HIV induces broad innate immune activation that predicts CD8⁺ T-cell responses but is attenuated by preexisting Ad5 immunity.

PubMed

Zak, Daniel E; Andersen-Nissen, Erica; Peterson, Eric R; Sato, Alicia; Hamilton, M Kristina; Borgerding, Joleen; Krishnamurty, Akshay T; Chang, Joanne T; Adams, Devin J; Hensley, Tiffany R; Salter, Alexander I; Morgan, Cecilia A; Duerr, Ann C; De Rosa, Stephen C; Aderem, Alan; McElrath, M Juliana

2012-12-11

To better understand how innate immune responses to vaccination can lead to lasting protective immunity, we used a systems approach to define immune signatures in humans over 1 wk following MRKAd5/HIV vaccination that predicted subsequent HIV-specific T-cell responses. Within 24 h, striking increases in peripheral blood mononuclear cell gene expression associated with inflammation, IFN response, and myeloid cell trafficking occurred, and lymphocyte-specific transcripts decreased. These alterations were corroborated by marked serum inflammatory cytokine elevations and egress of circulating lymphocytes. Responses of vaccinees with preexisting adenovirus serotype 5 (Ad5) neutralizing antibodies were strongly attenuated, suggesting that enhanced HIV acquisition in Ad5-seropositive subgroups in the Step Study may relate to the lack of appropriate innate activation rather than to increased systemic immune activation. Importantly, patterns of chemoattractant cytokine responses at 24 h and alterations in 209 peripheral blood mononuclear cell transcripts at 72 h were predictive of subsequent induction and magnitude of HIV-specific CD8(+) T-cell responses. This systems approach provides a framework to compare innate responses induced by vectors, as shown here by contrasting the more rapid, robust response to MRKAd5/HIV with that to yellow fever vaccine. When applied iteratively, the findings may permit selection of HIV vaccine candidates eliciting innate immune response profiles more likely to drive HIV protective immunity.

Relationship of ovarian stimulation response with vascular endothelial growth factor and degree of granulosa cell apoptosis.

PubMed

Quintana, R; Kopcow, L; Marconi, G; Sueldo, C; Speranza, G; Barañao, R I

2001-09-01

The aim of this study was to evaluate the concentration of vascular endothelial growth factor (VEGF) in follicular fluid and in granulosa cell cultures in relation to the degree of apoptosis in granulosa cells from patients with different types of ovarian response to controlled ovarian hyperstimulation. We studied 30 women who underwent controlled ovarian hyperstimulation and oocyte retrieval. Group A comprised patients with 1-4 follicles (n = 10), group B patients with 5-14 follicles (n = 10) and group C patients with >15 follicles (n = 10). Mean (+/-SD) VEGF concentrations in follicular fluid were 1232 +/- 209, 813 +/- 198 and 396 +/- 103 pg/ml for groups A, B and C respectively (P > 0.01). Concentrations of VEGF in granulosa cell supernatant were 684 +/- 316, 1101 +/- 295 and 1596 +/- 227 pg/ml respectively (P < 0.05). Percentages of apoptotic cells in granulosa cells culture was 55.02 +/- 7.5, 23.98 +/- 4.4 and 14.2 +/- 2.3% respectively (A versus B, P < 0.01, A versus C, P < 0.006, B versus C, NS). Our findings showed that in patients with decreased ovarian response to controlled ovarian hyperstimulation, follicular fluid VEGF concentration is elevated, the concentration from granulosa cells culture supernatant is decreased and the percentage of apoptotic granulosa cells is increased, while opposite findings occurred in patients with normal or hyper-responses.

Oligoclonal CD8+ T-cell expansion in patients with chronic hepatitis C is associated with liver pathology and poor response to interferon-alpha therapy.

PubMed

Manfras, Burkhard J; Weidenbach, Hans; Beckh, Karl-Heinz; Kern, Peter; Möller, Peter; Adler, Guido; Mertens, Thomas; Boehm, Bernhard O

2004-05-01

The role of CD8(+) T lymphocytes in chronic hepatitis C virus (HCV) infection and in liver injury with subsequent development of fibrosis and cirrhosis is poorly understood. To address this question, we performed a follow-up study including 27 chronically HCV-infected individuals. We determined clonality and phenotypes of circulating CD8(+) T cells employing TCRBV spectratyping. Antigen specificity was tested by rMHC-peptide tetramer staining and stimulation with recombinant HCV antigens. In addition, T-cell clonality and phenotypes were followed during the variable clinical response of interferon- (IFN) alpha treatment. We could demonstrate that CD8(+) T-cell expansions were significantly associated with liver fibrosis and cirrhosis. Likewise, increased oligoclonality of circulating CD8(+) T cells in chronic HCV infection was identified as an indicator for poor clinical response to IFN-alpha therapy. Moreover, we also found that IFN-alpha therapy enhanced the differentiation of CD8(+) T cells towards a late differentiation phenotype (CD28(-) CD57(+)). In cases of virus elimination the disappearance of expanded terminally differentiated CD8(+) cells was observed. Thus, this study identifies an association of clonal expansions of circulating CD8(+) T cells with liver pathology and provides a possible explanation for the fact that response to IFN-alpha therapy diminishes with the duration of infection.

A CD133-expressing murine liver oval cell population with bilineage potential.

PubMed

Rountree, C Bart; Barsky, Lora; Ge, Shundi; Zhu, Judy; Senadheera, Shantha; Crooks, Gay M

2007-10-01

Although oval cells are postulated to be adult liver stem cells, a well-defined phenotype of a bipotent liver stem cell remains elusive. The heterogeneity of cells within the oval cell fraction has hindered lineage potential studies. Our goal was to identify an enriched population of bipotent oval cells using a combination of flow cytometry and single cell gene expression in conjunction with lineage-specific liver injury models. Expression of cell surface markers on nonparenchymal, nonhematopoietic (CD45-) cells were characterized. Cell populations were isolated by flow cytometry for gene expression studies. 3,5-Diethoxycarbonyl-1,4-dihydrocollidine toxic injury induced cell cycling and expansion specifically in the subpopulation of oval cells in the periportal zone that express CD133. CD133+CD45- cells expressed hepatoblast and stem cell-associated genes, and single cells coexpressed both hepatocyte and cholangiocyte-associated genes, indicating bilineage potential. CD133+CD45- cells proliferated in response to liver injury. Following toxic hepatocyte damage, CD133+CD45- cells demonstrated upregulated expression of the hepatocyte gene Albumin. In contrast, toxic cholangiocyte injury resulted in upregulation of the cholangiocyte gene Ck19. After 21-28 days in culture, CD133+CD45- cells continued to generate cells of both hepatocyte and cholangiocyte lineages. Thus, CD133 expression identifies a population of oval cells in adult murine liver with the gene expression profile and function of primitive, bipotent liver stem cells. In response to lineage-specific injury, these cells demonstrate a lineage-appropriate genetic response. Disclosure of potential conflicts of interest is found at the end of this article.

Host responses in human skin after conventional intradermal injection or microneedle administration of virus-like-particle influenza vaccine.

PubMed

Pearton, Marc; Pirri, Daniela; Kang, Sang-Moo; Compans, Richard W; Birchall, James C

2013-10-01

Miniaturized microneedle devices are being developed for painlessly targeting vaccines to the immune cell populations in skin. As skin immunization studies are generally restricted to animal models however, where skin architecture and immunity is greatly different to human, surprisingly little is known about the local human response to intradermal (ID) vaccines. Here surgically excised human skin is used to explore for the first time the complex molecular and cellular host responses to a candidate influenza vaccine comprising nanoparticulate virus-like-particles (VLPs), administered via conventional hypodermic injection or reduced scale microneedles. Responses at the molecular level are determined by microarray analysis (47,296 discrete transcripts) and validated by quantitative PCR (96 genes). Cellular response is probed through monitoring migration of dendritic cells in viable skin tissue. Gene expression mapping, ontological analysis, and qPCR reveal up-regulation of a host of genes responsible for key immunomodulatory processes and host viral response, including cell recruitment, activation, migration, and T cell interaction following both ID and microneedle injection of VLPs; the response from the microneedles being more subtle. Significant morphological and migratory changes to skin dendritic cells are also apparent following microneedle VLP delivery. This is the first study displaying the global, multifaceted immunological events that occur at the site of vaccine deposition in human skin and will subsequently influence the degree and nature of innate and adaptive immune responses. An increased understanding of the detailed similarities and differences in response against antigen administered via different delivery modalities will inform the development of improved vaccines and vaccine delivery systems. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

EphA2 is an epithelial cell pattern recognition receptor for fungal β-glucans

PubMed Central

Swidergall, Marc; Solis, Norma V.; Lionakis, Michail S.; Filler, Scott G.

2017-01-01

Oral epithelial cells discriminate between pathogenic and non-pathogenic stimuli, and only induce an inflammatory response when they are exposed to high levels of a potentially harmful microorganism. The pattern recognition receptors (PRRs) in epithelial cells that mediate this differential response are poorly understood. Here, we demonstrate that the ephrin type-A receptor 2 (EphA2) is an oral epithelial cell PRR that binds to exposed β-glucans on the surface of the fungal pathogen Candida albicans. Binding of C. albicans to EphA2 on oral epithelial cells activates signal transducer and activator of transcription 3 (Stat3) and mitogen-activated protein kinase signaling in an inoculum-dependent manner, and is required for induction of a pro-inflammatory and antifungal response. EphA2−/− mice have impaired inflammatory responses and reduced IL-17 signaling during oropharyngeal candidiasis, resulting in more severe disease. Our study reveals that EphA2 functions as PRR for β-glucans that senses epithelial cell fungal burden and is required for the maximal mucosal inflammatory response to C. albicans. PMID:29133884

Odontoblast-Like Cells Differentiated from Dental Pulp Stem Cells Retain Their Phenotype after Subcultivation

PubMed Central

Baldión, Paula A.; Velandia-Romero, Myriam L.

2018-01-01

Odontoblasts, the main cell type in teeth pulp tissue, are not cultivable and they are responsible for the first line of response after dental restauration. Studies on dental materials cytotoxicity and odontoblast cells physiology require large quantity of homogenous cells retaining most of the phenotype characteristics. Odontoblast-like cells (OLC) were differentiated from human dental pulp stem cells using differentiation medium (containing TGF-β1), and OLC expanded after trypsinization (EXP-21) were evaluated and compared. Despite a slower cell growth curve, EXP-21 cells express similarly the odontoblast markers dentinal sialophosphoprotein and dentin matrix protein-1 concomitantly with RUNX2 transcripts and low alkaline phosphatase activity as expected. Both OLC and EXP-21 cells showed similar mineral deposition activity evidenced by alizarin red and von Kossa staining. These results pointed out minor changes in phenotype of subcultured EXP-21 regarding the primarily differentiated OLC, making the subcultivation of these cells a useful strategy to obtain odontoblasts for biocompatibility or cell physiology studies in dentistry. PMID:29670655

Correlation between thyroidal and peripheral blood total T cells, CD8+ T cells, and CD8+ T- regulatory cells and T-cell reactivity to calsequestrin and collagen XIII in patients with Graves' ophthalmopathy.

PubMed

Al-Ansari, Farah; Lahooti, Hooshang; Stokes, Leanne; Edirimanne, Senarath; Wall, Jack

2018-05-22

Purpose/aim of the study: Graves' ophthalmopathy (GO) is closely related to the thyroid autoimmune disorder Graves' disease. Previous studies have suggested roles for thyroidal CD8 +  T cells and autoimmunity against calsequestrin-1 (CASQ)-1 in the link between thyroidal and orbital autoimmune reactions in GO. A role for autoimmunity against CollXIII has also been suggested. In this study, we aimed to investigate correlations between some thyroidal and peripheral blood T-cell subsets and thyroidal T-cell reactivity against CASQ1 and CollXIII in patients with GO. Fresh thyroid tissues were processed by enzyme digestion and density gradient to isolate mononuclear cells (MNCs). Peripheral blood MNCs were also isolated using density gradient. Flow-cytometric analysis was used to identify the various T-cell subsets. T -cell reactivity to CASQ1 and CollXIII was measured by a 5-day culture of the MNCs and BrdU uptake method. We found a positive correlation between thyroidal CD8 +  T cells and CD8 +  T-regulatory (T-reg) cells in patients with GO. Thyroidal T cells from two out of the three patients with GO tested (66.7%) showed a positive response to CASQ1, while thyroidal T cells from none of the six Graves' Disease patients without ophthalmopathy (GD) tested showed a positive response to this antigen. Thyroidal T cells from these patient groups however, showed no significant differences in their response to CollXIII. Our observations provide further evidence for a possible role of thyroidal CD8 +  T cells, CD8 +  T-reg cells and the autoantigen CASQ1 in the link between thyroidal and orbital autoimmune reactions of GO.

The Value of Analysis of Standardized Placement Exams: A Case Study of Cell Structure.

ERIC Educational Resources Information Center

Blystone, Robert V.

This study focused on potential pedagological uses of standardized placement exams. A sample of 250 exams of the May 1984 Biology Advanced Placement (AP) exam was obtained and student responses to the question on cell structure were analyzed. The frequency of particular responses to the question is listed and trends and patterns in the responses…

Plasma and memory B cell responses targeting O-specific polysaccharide (OSP) are associated with protection against Vibrio cholerae O1 infection among household contacts of cholera patients in Bangladesh.

PubMed

Aktar, Amena; Rahman, M Arifur; Afrin, Sadia; Akter, Aklima; Uddin, Taher; Yasmin, Tahirah; Sami, Md Israk Nur; Dash, Pinki; Jahan, Sultana Rownok; Chowdhury, Fahima; Khan, Ashraful I; LaRocque, Regina C; Charles, Richelle C; Bhuiyan, Taufiqur Rahman; Mandlik, Anjali; Kelly, Meagan; Kováč, Pavol; Xu, Peng; Calderwood, Stephen B; Harris, Jason B; Qadri, Firdausi; Ryan, Edward T

2018-04-01

The mediators of protection against cholera, a severe dehydrating illness of humans caused by Vibrio cholerae, are unknown. We have previously shown that plasma IgA as well as memory B IgG cells targeting lipopolysaccharide (LPS) of Vibrio cholerae O1 correlate with protection against V. cholerae O1 infection among household contacts of cholera patients. Protection against cholera is serogroup specific, and serogroup specificity is defined by the O-specific polysaccharide (OSP) component of LPS. Therefore, we prospectively followed household contacts of cholera patients to determine whether OSP-specific immune responses present at the time of enrollment are associated with protection against V. cholerae infection. In this study, we enrolled two hundred forty two household contacts of one hundred fifty index patients who were infected with Vibrio cholerae. We determined OSP-specific memory B cells and plasma IgA, IgG and IgM antibody responses on study entry (day 2). The presence of OSP-specific plasma IgA, IgM, and IgG antibody responses on study entry were associated with a decrease in the risk of infection in household contacts (IgA, p = 0.015; IgM, p = 0.01, and IgG, p = 0.024). In addition, the presence of OSP-specific IgG memory B cell responses in peripheral blood on study entry was also associated with a decreased risk of infection (44% reduction; 95% CI: 31.1 to 99.8) in contacts. No protection was associated with cholera toxin B subunit (CtxB)-specific memory B cell responses. These results suggest that immune responses that target OSP, both in plasma and memory responses, may be important in mediating protection against infection with V. cholerae O1.

Plasma and memory B cell responses targeting O-specific polysaccharide (OSP) are associated with protection against Vibrio cholerae O1 infection among household contacts of cholera patients in Bangladesh

PubMed Central

Aktar, Amena; Rahman, M. Arifur; Afrin, Sadia; Akter, Aklima; Uddin, Taher; Yasmin, Tahirah; Sami, Md. Israk Nur; Dash, Pinki; Jahan, Sultana Rownok; Chowdhury, Fahima; Khan, Ashraful I.; LaRocque, Regina C.; Charles, Richelle C.; Bhuiyan, Taufiqur Rahman; Mandlik, Anjali; Kelly, Meagan; Kováč, Pavol; Xu, Peng; Calderwood, Stephen B.; Harris, Jason B.; Ryan, Edward T.

2018-01-01

Background The mediators of protection against cholera, a severe dehydrating illness of humans caused by Vibrio cholerae, are unknown. We have previously shown that plasma IgA as well as memory B IgG cells targeting lipopolysaccharide (LPS) of Vibrio cholerae O1 correlate with protection against V. cholerae O1 infection among household contacts of cholera patients. Protection against cholera is serogroup specific, and serogroup specificity is defined by the O-specific polysaccharide (OSP) component of LPS. Therefore, we prospectively followed household contacts of cholera patients to determine whether OSP-specific immune responses present at the time of enrollment are associated with protection against V. cholerae infection. Methodology In this study, we enrolled two hundred forty two household contacts of one hundred fifty index patients who were infected with Vibrio cholerae. We determined OSP-specific memory B cells and plasma IgA, IgG and IgM antibody responses on study entry (day 2). Principle findings The presence of OSP-specific plasma IgA, IgM, and IgG antibody responses on study entry were associated with a decrease in the risk of infection in household contacts (IgA, p = 0.015; IgM, p = 0.01, and IgG, p = 0.024). In addition, the presence of OSP-specific IgG memory B cell responses in peripheral blood on study entry was also associated with a decreased risk of infection (44% reduction; 95% CI: 31.1 to 99.8) in contacts. No protection was associated with cholera toxin B subunit (CtxB)-specific memory B cell responses. Conclusion These results suggest that immune responses that target OSP, both in plasma and memory responses, may be important in mediating protection against infection with V. cholerae O1. PMID:29684006

Effect of interleukins on the proliferation and survival of B cell chronic lymphocytic leukaemia cells.

PubMed Central

Mainou-Fowler, T; Copplestone, J A; Prentice, A G

1995-01-01

AIMS--To investigate the effects of interleukin (IL) 1, 2, 4, and 5 on the proliferation and survival of peripheral blood B cells from patients with B chronic lymphocytic leukaemia (B-CLL) and compare them with the effects on normal peripheral blood B cells. METHODS--The proliferation and survival of pokeweed mitogen (PWM) activated B cells from B-CLL (n = 12) and normal peripheral blood (n = 5) were studied in vitro in response to IL-1, IL-2 IL-4, and IL-5. Survival of cells in cultures with or without added interleukins was studied by microscopic examination of cells and DNA agarose gel electrophoresis. RESULTS--Proliferation was observed in both B-CLL and normal peripheral blood cells on culture with IL-2 alone and also in some, but not all, B-CLL and normal peripheral blood cells with IL-1 and IL-4. However, there was greater variability in B-CLL cell responses than in normal peripheral blood cells. Il-5 did not affect normal peripheral blood cell proliferation but it increased proliferation in two B-CLL cases. Synergistic effects of these cytokines were not detected. IL-4 inhibited normal peripheral blood and B-CLL cell proliferation after the addition of IL-2. Inhibition of B-CLL cell responses to IL-2 was also observed with IL-5 and Il-1. Survival of B-CLL cells in cultures was enhanced with IL-4 not by an increase in proliferation but by reduced apoptosis. No such effect was seen in normal peripheral blood cells. IL-2 had a less noticeable antiapoptotic effect; IL-5 enhanced apoptosis in B-CLL cells. CONCLUSIONS--B-CLL and normal peripheral blood cells proliferated equally well in response to IL-2. IL-4 had a much lower effect on B-CLL cell proliferation, but had noticeable antiapoptotic activity. IL-5 enhanced cell death by apoptosis. Images PMID:7629299

Herbal preparation (HemoHIM) enhanced functional maturation of bone marrow-derived dendritic cells mediated toll-like receptor 4.

PubMed

Lee, Sung-Ju; Kim, Jong-Jin; Kang, Kyung-Yun; Hwang, Yun-Ho; Jeong, Gil-Yeon; Jo, Sung-kee; Jung, Uhee; Park, Hae-Ran; Yee, Sung-Tae

2016-02-19

HemoHIM, which is an herbal preparation of three edible herbs (Angelicam gigas Nakai, Cnidium offinale Makino, and Peaonia japonica Miyabe), is known to have various biological and immunological activities, but the modulatory effects of this preparation on dendritic cells (DCs)-mediated immune responses have not been examined previously. DCs are a unique group of white blood cells that initiate primary immune responses by capturing, processing, and presenting antigens to T cells. In the present study, we investigated the effect of HemoHIM on the functional and phenotypic maturation of murine bone marrow-derived dendritic cells (BMDCs) both in vitro and in vivo. The expression of co-stimulatory molecules (CD40, CD80, CD86, MHC I, and MHC II) and the production of cytokines (IL-1β, IL-6, IL-12p70, and TNF-α) were increased by HemoHIM in BMDCs. Furthermore, the antigen-uptake ability of BMDCs was decreased by HemoHIM, and the antigen-presenting ability of HemoHIM-treated mature BMDCs increased TLR4-dependent CD4(+) and CD8(+) T cell responses. Our findings demonstrated that HemoHIM induces TLR4-mediated BMDCs functional and phenotypic maturation through in vivo and in vitro. And our study showed the antigen-presenting ability that HemoHIM-treated mature BMDCs increase CD4(+) and CD8(+) T cell responses by in vitro. These results suggest that HemoHIM has the potential to mediate DC immune responses.

Factor X/Xa elicits protective signaling responses in endothelial cells directly via PAR-2 and indirectly via endothelial protein C receptor-dependent recruitment of PAR-1.

PubMed

Bae, Jong-Sup; Yang, Likui; Rezaie, Alireza R

2010-11-05

We recently demonstrated that the Gla domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier-protective response if endothelial cells were preincubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a cross-talk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells.

An integrated approach to characterize transcription factor and microRNA regulatory networks involved in Schwann cell response to peripheral nerve injury

PubMed Central

2013-01-01

Background The regenerative response of Schwann cells after peripheral nerve injury is a critical process directly related to the pathophysiology of a number of neurodegenerative diseases. This SC injury response is dependent on an intricate gene regulatory program coordinated by a number of transcription factors and microRNAs, but the interactions among them remain largely unknown. Uncovering the transcriptional and post-transcriptional regulatory networks governing the Schwann cell injury response is a key step towards a better understanding of Schwann cell biology and may help develop novel therapies for related diseases. Performing such comprehensive network analysis requires systematic bioinformatics methods to integrate multiple genomic datasets. Results In this study we present a computational pipeline to infer transcription factor and microRNA regulatory networks. Our approach combined mRNA and microRNA expression profiling data, ChIP-Seq data of transcription factors, and computational transcription factor and microRNA target prediction. Using mRNA and microRNA expression data collected in a Schwann cell injury model, we constructed a regulatory network and studied regulatory pathways involved in Schwann cell response to injury. Furthermore, we analyzed network motifs and obtained insights on cooperative regulation of transcription factors and microRNAs in Schwann cell injury recovery. Conclusions This work demonstrates a systematic method for gene regulatory network inference that may be used to gain new information on gene regulation by transcription factors and microRNAs. PMID:23387820

Parasite Fate and Involvement of Infected Cells in the Induction of CD4+ and CD8+ T Cell Responses to Toxoplasma gondii

PubMed Central

Dupont, Christopher D.; Christian, David A.; Selleck, Elizabeth M.; Pepper, Marion; Leney-Greene, Michael; Harms Pritchard, Gretchen; Koshy, Anita A.; Wagage, Sagie; Reuter, Morgan A.; Sibley, L. David; Betts, Michael R.; Hunter, Christopher A.

2014-01-01

During infection with the intracellular parasite Toxoplasma gondii, the presentation of parasite-derived antigens to CD4+ and CD8+ T cells is essential for long-term resistance to this pathogen. Fundamental questions remain regarding the roles of phagocytosis and active invasion in the events that lead to the processing and presentation of parasite antigens. To understand the most proximal events in this process, an attenuated non-replicating strain of T. gondii (the cpsII strain) was combined with a cytometry-based approach to distinguish active invasion from phagocytic uptake. In vivo studies revealed that T. gondii disproportionately infected dendritic cells and macrophages, and that infected dendritic cells and macrophages displayed an activated phenotype characterized by enhanced levels of CD86 compared to cells that had phagocytosed the parasite, thus suggesting a role for these cells in priming naïve T cells. Indeed, dendritic cells were required for optimal CD4+ and CD8+ T cell responses, and the phagocytosis of heat-killed or invasion-blocked parasites was not sufficient to induce T cell responses. Rather, the selective transfer of cpsII-infected dendritic cells or macrophages (but not those that had phagocytosed the parasite) to naïve mice potently induced CD4+ and CD8+ T cell responses, and conferred protection against challenge with virulent T. gondii. Collectively, these results point toward a critical role for actively infected host cells in initiating T. gondii-specific CD4+ and CD8+ T cell responses. PMID:24722202

NKp46+ Innate Lymphoid Cells Dampen Vaginal CD8 T Cell Responses following Local Immunization with a Cholera Toxin-Based Vaccine

PubMed Central

Luci, Carmelo; Bekri, Selma; Bihl, Franck; Pini, Jonathan; Bourdely, Pierre; Nouhen, Kelly; Malgogne, Angélique; Walzer, Thierry; Braud, Véronique M.; Anjuère, Fabienne

2015-01-01

Innate and adaptive immune cells work in concert to generate efficient protection at mucosal surface. Vaginal mucosa is an epithelial tissue that contains innate and adaptive immune effector cells. Our previous studies demonstrated that vaginal administration of Cholera toxin -based vaccines generate antigen-specific CD8 T cells through the stimulation of local dendritic cells (DC). Innate lymphoid cells (ILC) are a group of lymphocytes localized in epithelial tissues that have important immune functions against pathogens and in tissue homeostasis. Their contribution to vaccine-induced mucosal T cell responses is an important issue for the design of protective vaccines. We report here that the vaginal mucosa contains a heterogeneous population of NKp46+ ILC that includes conventional NK cells and ILC1-like cells. We show that vaginal NKp46+ ILC dampen vaccine-induced CD8 T cell responses generated after local immunization. Indeed, in vivo depletion of NKp46+ ILC with anti-NK1.1 antibody or NKG2D blockade increases the magnitude of vaginal OVA-specific CD8 T cells. Furthermore, such treatments also increase the number of DC in the vagina. NKG2D ligands being expressed by vaginal DC but not by CD8 T cells, these results support that NKp46+ ILC limit mucosal CD8 T cell responses indirectly through the NKG2D-dependent elimination of vaginal DC. Our data reveal an unappreciated role of NKp46+ ILC in the regulation of mucosal CD8 T cell responses. PMID:26630176

IL-1 Receptor Signaling on Graft Parenchymal Cells Regulates Memory and De Novo Donor-Reactive CD8 T Cell Responses to Cardiac Allografts.

PubMed

Iida, Shoichi; Tsuda, Hidetoshi; Tanaka, Toshiaki; Kish, Danielle D; Abe, Toyofumi; Su, Charles A; Abe, Ryo; Tanabe, Kazunari; Valujskikh, Anna; Baldwin, William M; Fairchild, Robert L

2016-03-15

Reperfusion of organ allografts induces a potent inflammatory response that directs rapid memory T cell, neutrophil, and macrophage graft infiltration and their activation to express functions mediating graft tissue injury. The role of cardiac allograft IL-1 receptor (IL-1R) signaling in this early inflammation and the downstream primary alloimmune response was investigated. When compared with complete MHC-mismatched wild-type cardiac allografts, IL-1R(-/-) allografts had marked decreases in endogenous memory CD8 T cell and neutrophil infiltration and expression of proinflammatory mediators at early times after transplant, whereas endogenous memory CD4 T cell and macrophage infiltration was not decreased. IL-1R(-/-) allograft recipients also had marked decreases in de novo donor-reactive CD8, but not CD4, T cell development to IFN-γ-producing cells. CD8 T cell-mediated rejection of IL-1R(-/-) cardiac allografts took 3 wk longer than wild-type allografts. Cardiac allografts from reciprocal bone marrow reconstituted IL-1R(-/-)/wild-type chimeric donors indicated that IL-1R signaling on graft nonhematopoietic-derived, but not bone marrow-derived, cells is required for the potent donor-reactive memory and primary CD8 T cell alloimmune responses observed in response to wild-type allografts. These studies implicate IL-1R-mediated signals by allograft parenchymal cells in generating the stimuli-provoking development and elicitation of optimal alloimmune responses to the grafts. Copyright © 2016 by The American Association of Immunologists, Inc.

Separability of stimulus parameter encoding by on-off directionally selective rabbit retinal ganglion cells

PubMed Central

Nowak, Przemyslaw; Dobbins, Allan C.; Gawne, Timothy J.; Grzywacz, Norberto M.

2011-01-01

The ganglion cell output of the retina constitutes a bottleneck in sensory processing in that ganglion cells must encode multiple stimulus parameters in their responses. Here we investigate encoding strategies of On-Off directionally selective retinal ganglion cells (On-Off DS RGCs) in rabbits, a class of cells dedicated to representing motion. The exquisite axial discrimination of these cells to preferred vs. null direction motion is well documented: it is invariant with respect to speed, contrast, spatial configuration, spatial frequency, and motion extent. However, these cells have broad direction tuning curves and their responses also vary as a function of other parameters such as speed and contrast. In this study, we examined whether the variation in responses across multiple stimulus parameters is systematic, that is the same for all cells, and separable, such that the response to a stimulus is a product of the effects of each stimulus parameter alone. We extracellularly recorded single On-Off DS RGCs in a superfused eyecup preparation while stimulating them with moving bars. We found that spike count responses of these cells scaled as independent functions of direction, speed, and luminance. Moreover, the speed and luminance functions were common across the whole sample of cells. Based on these findings, we developed a model that accurately predicted responses of On-Off DS RGCs as products of separable functions of direction, speed, and luminance (r = 0.98; P < 0.0001). Such a multiplicatively separable encoding strategy may simplify the decoding of these cells' outputs by the higher visual centers. PMID:21325684

Assessing Estrogen-Induced Proliferative Response in an Endometrial Cancer Cell Line Using a Universally Applicable Methodological Guide.

PubMed

Parkes, Christina; Kamal, Areege; Valentijn, Anthony J; Alnafakh, Rafah; Gross, Stephane R; Barraclough, Roger; Moss, Diana; Kirwan, John; Hapangama, Dharani K

2018-01-01

Translational endometrial cancer (EC) research benefits from an in vitro experimental approach using EC cell lines. We demonstrated the steps that are required to examine estrogen-induced proliferative response, a simple yet important research question pertinent to EC, and devised a pragmatic methodological workflow for using EC cell lines in experimental models. Comprehensive review of all commercially available EC cell lines was carried out, and Ishikawa cell line was selected to study the estrogen responsiveness with HEC1A, RL95-2, and MFE280 cell lines as comparators where appropriate, examining relevant differential molecular (steroid receptors) and functional (phenotype, anchorage-independent growth, hormone responsiveness, migration, invasion, and chemosensitivity) characteristics in 2-dimensional and 3-dimensional cultures in vitro using immunocytochemistry, immunofluorescence, quantitative polymerase chain reaction, and Western blotting. In vivo tumor, formation, and chemosensitivity were also assessed in a chick chorioallantoic membrane model. Short tandem repeat analysis authenticated the purchased cell lines, whereas gifted cells deviated significantly from the published profile. We demonstrate the importance of prior assessment of the suitability of each cell line for the chosen in vitro experimental technique. Prior establishment of baseline, nonenriched conditions was required to induce a proliferative response to estrogen. The chorioallantoic membrane model was a suitable in vivo multicellular animal model for EC for producing rapid and reproducible data. We have developed a methodological guide for EC researchers when using endometrial cell lines to answer important translational research questions (exemplified by estrogen-responsive cell proliferation) to facilitate robust data, while saving time and resources.

T cell epitope immunotherapy ameliorates allergic responses in a murine model of shrimp allergy.

PubMed

Wai, C Y Y; Leung, N Y H; Leung, P S C; Chu, K H

2016-03-01

Shellfish allergy is one of the most common food hypersensitivities worldwide but allergen-specific immunotherapy for shellfish allergy is not yet available. We believe that T cell peptide-based immunotherapy holds the potential for modulating allergic responses without IgE cross-linking. We sought to identify the immunodominant T cell epitopes of tropomyosin, the major shrimp allergen of Metapenaeus ensis (Met e 1), and to evaluate their therapeutic effects in a Balb/c mouse model of Met e 1 hypersensitivity. T cell epitopes of Met e 1 were first identified based on the proliferation and cytokine responses of splenocytes isolated from Met e 1-sensitized Balb/c mice upon stimulation by 18 synthetic peptides that span the full-length Met e 1. The immunodominant T cell peptides identified were then fed orally to Met e 1-sensitized Balb/c mice twice a week for four weeks. Allergic responses, serological antibody levels, intestinal histology and systemic and local cytokine profiles were compared between the treated and the untreated groups. Six major Met e 1 T cell epitopes were identified. Mice treated with the T cell epitope peptide mixture demonstrated an amelioration of systemic allergic symptoms and a significant reduction in Th2-associated antibody and cytokine responses. These benefits were accompanied by a shift to a balanced Th1/Th2 response, induction of IgG2a antibodies possessing in vitro and in vivo blocking activities and the induction of regulatory T cell responses. T cell epitope-based oral immunotherapy is effective in reducing allergic responses towards shrimp tropomyosin. This is a novel strategy for clinical management of shellfish allergy and is a model for mechanistic studies of oral immunotherapy. © 2015 John Wiley & Sons Ltd.

Viral Infection of Tumors Overcomes Resistance to PD-1-immunotherapy by Broadening Neoantigenome-directed T-cell Responses.

PubMed

Woller, Norman; Gürlevik, Engin; Fleischmann-Mundt, Bettina; Schumacher, Anja; Knocke, Sarah; Kloos, Arnold M; Saborowski, Michael; Geffers, Robert; Manns, Michael P; Wirth, Thomas C; Kubicka, Stefan; Kühnel, Florian

2015-10-01

There is evidence that viral oncolysis is synergistic with immune checkpoint inhibition in cancer therapy but the underlying mechanisms are unclear. Here, we investigated whether local viral infection of malignant tumors is capable of overcoming systemic resistance to PD-1-immunotherapy by modulating the spectrum of tumor-directed CD8 T-cells. To focus on neoantigen-specific CD8 T-cell responses, we performed transcriptomic sequencing of PD-1-resistant CMT64 lung adenocarcinoma cells followed by algorithm-based neoepitope prediction. Investigations on neoepitope-specific T-cell responses in tumor-bearing mice demonstrated that PD-1 immunotherapy was insufficient whereas viral oncolysis elicited cytotoxic T-cell responses to a conserved panel of neoepitopes. After combined treatment, we observed that PD-1-blockade did not affect the magnitude of oncolysis-mediated antitumoral immune responses but a broader spectrum of T-cell responses including additional neoepitopes was observed. Oncolysis of the primary tumor significantly abrogated systemic resistance to PD-1-immunotherapy leading to improved elimination of disseminated lung tumors. Our observations were confirmed in a transgenic murine model of liver cancer where viral oncolysis strongly induced PD-L1 expression in primary liver tumors and lung metastasis. Furthermore, we demonstrated that combined treatment completely inhibited dissemination in a CD8 T-cell-dependent manner. Therefore, our results strongly recommend further evaluation of virotherapy and concomitant PD-1 immunotherapy in clinical studies.

Plasmacytoid dendritic cells control dengue and Chikungunya virus infections via IRF7-regulated interferon responses

PubMed Central

Zafirova, Biljana; This, Sébastien; Coléon, Séverin; Décembre, Elodie; Paidassi, Helena; Bouvier, Isabelle; Joubert, Pierre-Emmanuel; Duffy, Darragh; Walzer, Thierry

2018-01-01

Type I interferon (IFN-I) responses are critical for the control of RNA virus infections, however, many viruses, including Dengue (DENV) and Chikungunya (CHIKV) virus, do not directly activate plasmacytoid dendritic cells (pDCs), robust IFN-I producing cells. Herein, we demonstrated that DENV and CHIKV infected cells are sensed by pDCs, indirectly, resulting in selective IRF7 activation and IFN-I production, in the absence of other inflammatory cytokine responses. To elucidate pDC immunomodulatory functions, we developed a mouse model in which IRF7 signaling is restricted to pDC. Despite undetectable levels of IFN-I protein, pDC-restricted IRF7 signaling controlled both viruses and was sufficient to protect mice from lethal CHIKV infection. Early pDC IRF7-signaling resulted in amplification of downstream antiviral responses, including an accelerated natural killer (NK) cell-mediated type II IFN response. These studies revealed the dominant, yet indirect role of pDC IRF7-signaling in directing both type I and II IFN responses during arbovirus infections. PMID:29914621

Calcium ion propagation in cultured keratinocytes and other cells in skin in response to hydraulic pressure stimulation.

PubMed

Goto, Makiko; Ikeyama, Kazuyuki; Tsutsumi, Moe; Denda, Sumiko; Denda, Mitsuhiro

2010-07-01

We have previously suggested that a variety of environmental factors might be first sensed by epidermal keratinocytes, which represent the frontier of the body. To further examine this idea, in the present study, we examined the intracellular calcium responses of cultured keratinocytes to external hydraulic pressure. First, we compared the responses of undifferentiated and differentiated keratinocytes with those of fibroblasts, vascular endothelial cells (VEC), and lymphatic endothelial cells. Elevation of intracellular calcium was observed after application of pressure to keratinocytes, fibroblasts, and VEC. The calcium propagation extended over a larger area and continued for a longer period of time in differentiated keratinocytes, as compared with the other cells. The response of the keratinocytes was dramatically reduced when the cells were incubated in medium without calcium. Application of a non-selective transient receptor potential (TRP) channel blocker also attenuated the calcium response. These results suggest that differentiated keratinocytes are sensitive to external pressure and that TRP might be involved in the mechanism of their response. (c) 2010 Wiley-Liss, Inc.