HIV-1 infection, response to treatment and establishment of viral latency in a novel humanized T cell-only mouse (TOM) model.

PubMed

Honeycutt, Jenna B; Wahl, Angela; Archin, Nancie; Choudhary, Shailesh; Margolis, David; Garcia, J Victor

2013-10-24

The major targets of HIV infection in humans are CD4⁺ T cells. CD4⁺ T cell depletion is a hallmark of AIDS. Previously, the SCID-hu thy/liv model was used to study the effect of HIV on thymopoeisis in vivo. However, these mice did not develop high levels of peripheral T cell reconstitution and required invasive surgery for infection and analysis. Here, we describe a novel variant of this model in which thy/liv implantation results in systemic reconstitution with human T cells in the absence of any other human hematopoietic lineages. NOD/SCID-hu thy/liv and NSG-hu thy/liv mice were created by implanting human fetal thymus and liver tissues under the kidney capsule of either NOD/SCID or NSG mice. In contrast to NOD/SCID-hu thy/liv mice that show little or no human cells in peripheral blood or tissues, substantial systemic human reconstitution occurs in NSG-hu thy/liv. These mice are exclusively reconstituted with human T cells (i.e. T-cell only mice or TOM). Despite substantial levels of human T cells no signs of graft-versus-host disease (GVHD) were noted in these mice over a period of 14 months. TOM are readily infected after parenteral exposure to HIV-1. HIV replication is sustained in peripheral blood at high levels and results in modest reduction of CD4⁺ T cells. HIV-1 replication in TOM responds to daily administration of combination antiretroviral therapy (ART) resulting in strong suppression of virus replication as determined by undetectable viral load in plasma. Latently HIV infected resting CD4⁺ T cells can be isolated from suppressed mice that can be induced to express HIV ex-vivo upon activation demonstrating the establishment of latency in vivo. NSG-hu thy/liv mice are systemically reconstituted with human T cells. No other human lymphoid lineages are present in these mice (i.e. monocytes/macrophages, B cells and DC are all absent). These T cell only mice do not develop GVHD, are susceptible to HIV-1 infection and can efficiently maintain virus replication. HIV infected TOM undergoing ART harbor latently infected, resting CD4+ T cells.

Mice with Reconstituted Human Immune System Components as a Tool to Study Immune Cell Interactions in EBV Infection.

PubMed

Heuts, Frank; Nagy, Noemi

2017-01-01

Recent developments in mouse models that harbor part of a human immune system have proved extremely valuable to study the in vivo immune response to human specific pathogens such as Epstein-Barr virus. Over the last decades, advances in immunodeficient mouse strains that can be used as recipients for human immune cells have greatly enhanced the use of these models. Here, we describe the generation of mice with reconstituted human immune system (HIS mice) using immunocompromised mice transplanted with human CD34 + hematopoietic stem cells. We will also describe how such mice, in which human immune cells are generated de novo, can be used to study EBV infection.

Mechanism of Actin-Based Motility

NASA Astrophysics Data System (ADS)

Pantaloni, Dominique; Le Clainche, Christophe; Carlier, Marie-France

2001-05-01

Spatially controlled polymerization of actin is at the origin of cell motility and is responsible for the formation of cellular protrusions like lamellipodia. The pathogens Listeria monocytogenes and Shigella flexneri, which undergo actin-based propulsion, are acknowledged models of the leading edge of lamellipodia. Actin-based motility of the bacteria or of functionalized microspheres can be reconstituted in vitro from only five pure proteins. Movement results from the regulated site-directed treadmilling of actin filaments, consistent with observations of actin dynamics in living motile cells and with the biochemical properties of the components of the synthetic motility medium.

Maintenance of Host Leukocytes in Peripheral Immune Compartments Following Lethal Irradiation and Bone Marrow Reconstitution: Implications for Graft Versus Host Disease

PubMed Central

Staley, Elizabeth M.; Tanner, Scott M.; Daft, Joseph G.; Stanus, Andrea L.; Martin, Steven M.; Lorenz, Robin G.

2013-01-01

Bone marrow reconstitution is utilized as a tool for disease treatment and as a research technique to elucidate the function of bone marrow derived cells. Clinically successful engraftment is indicated by the development of a functioning immune repertoire. In research, reconstitution is considered successful if >85% of splenic leukocytes are of donor origins. Previous work suggests that splenic reconstitution may not be indicative of reconstitution in the mucosa. We sought to evaluate mucosal reconstitution in animals following a standard bone marrow eradication and reconstitution technique. Bone marrow was harvested from adult B6.SJL donor mice (CD45.1) and injected via either the retro-orbital or intraperitoneal route into lethally irradiated B6 (CD45.2) adult or neonatal recipients respectively. Expression of CD45 by flow cytometry was used to calculate reconstitution with respect to immune compartment and cell type. In reconstituted adult animals 93.2±1.5% of splenic leukocytes expressed the donor CD45.1 antigen thus meeting the standard definition of reconstitution, however only 58.6±13.6% of intestinal lamina propria lymphocytes and 52.4±16.0% of intestinal intraepithelial lymphocytes were of donor origin, confirming splenic reconstitution fails to represent peripheral immune reconstitution. T-cells in the gastrointestinal tract are the most poorly reconstituted, while B-cells appear to be almost universally replaced by donor cells. The inadequate mucosal reconstitution was not corrected by evaluating later timepoints or by performing the bone marrow transfer during the neonatal period. This demonstration that substantial host T-cells remain in the intestinal mucosa after a “successful” bone marrow transplantation should cause a re-evaluation of data from research bone marrow chimera experiments, as well as the mechanisms for complications after clinical bone marrow transplantation. PMID:23334064

Maintenance of host leukocytes in peripheral immune compartments following lethal irradiation and bone marrow reconstitution: implications for graft versus host disease.

PubMed

Staley, Elizabeth M; Tanner, Scott M; Daft, Joseph G; Stanus, Andrea L; Martin, Steven M; Lorenz, Robin G

2013-03-01

Bone marrow reconstitution is utilized as a tool for disease treatment and as a research technique to elucidate the function of bone marrow derived cells. Clinically successful engraftment is indicated by the development of a functioning immune repertoire. In research, reconstitution is considered successful if >85% of splenic leukocytes are of donor origins. Previous work suggests that splenic reconstitution may not be indicative of reconstitution in the mucosa. We sought to evaluate mucosal reconstitution in animals following a standard bone marrow eradication and reconstitution technique. Bone marrow was harvested from adult B6.SJL donor mice (CD45.1) and injected via either the retro-orbital or intraperitoneal route into lethally irradiated B6 (CD45.2) adult or neonatal recipients respectively. The expression of CD45 by flow cytometry was used to calculate reconstitution with respect to immune compartment and cell type. In reconstituted adult animals 93.2±1.5% of splenic leukocytes expressed the donor CD45.1 antigen thus meeting the standard definition of reconstitution, however only 58.6±13.6% of intestinal lamina propria lymphocytes and 52.4±16.0% of intestinal intraepithelial lymphocytes were of donor origin, confirming splenic reconstitution fails to represent peripheral immune reconstitution. T-cells in the gastrointestinal tract are the most poorly reconstituted, while B-cells appear to be almost universally replaced by donor cells. The inadequate mucosal reconstitution was not corrected by evaluating later time points or by performing the bone marrow transfer during the neonatal period. This demonstration that substantial host T-cells remain in the intestinal mucosa after a "successful" bone marrow transplantation should cause a re-evaluation of data from research bone marrow chimera experiments, as well as the mechanisms for complications after clinical bone marrow transplantation. Copyright © 2013 Elsevier B.V. All rights reserved.

Humoral Immune Reconstitution Kinetics after Allogeneic Hematopoietic Stem Cell Transplantation in Children: A Maturation Block of IgM Memory B Cells May Lead to Impaired Antibody Immune Reconstitution.

PubMed

Abdel-Azim, Hisham; Elshoury, Amro; Mahadeo, Kris M; Parkman, Robertson; Kapoor, Neena

2017-09-01

Although T cell immune reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been well studied, long-term B cell immune reconstitution remains less characterized. We evaluated humoral immune reconstitution among 71 pediatric allo-HSCT recipients. Although tetanus toxoid antibody levels were normal at 1 year after allo-HSCT, antipolysaccharide carbohydrate antibodies remained persistently low for up to 5 years. While naive B cell counts normalized by 6 months, IgM memory B cell deficiency persisted for up to 2 years (P = .01); switched memory B cell deficiency normalized by 1 year after allo-HSCT. CD4 + T cell immune reconstitution correlated with that of switched memory B cells as early as 6 months after allo-HSCT (r = .55, P = .002) but did not correlate with IgM memory B cells at any time point after allo-HSCT. Taken together, this suggests that allo-HSCT recipients have impaired antibody immune reconstitution, mainly due to IgM memory B cell maturation block, compared with more prompt T cell-dependent switched memory cell immune reconstitution. We further explored other factors that might affect humoral immune reconstitution. The use of total body irradiation was associated with lower naive B cells counts at 6 months after HSCT (P = .04) and lower IgM (P = .008) and switched (P = .003) memory B cells up to 2 years. Allo-HSCT recipients with extensive chronic graft-versus-host disease had lower IgM memory B cell counts (P = .03) up to 2 years after allo-HSCT. The use of cord blood was associated with better naive (P = .01), IgM (P = .0005), and switched memory (P = .006) B cells immune reconstitution. These findings may inform future prophylaxis and treatment strategies regarding risk of overwhelming infection, graft-versus-host disease, and post-allogeneic HSCT revaccination. Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Rapid Lymphocyte Reconstitution of Unconditioned Immunodeficient Mice with Non-Self-Renewing Multipotent Hematopoietic Progenitors

PubMed Central

Bhattacharya, Deepta; Bryder, David; Rossi, Derrick J.; Weissman, Irving L.

2015-01-01

The replacement of abnormal hematopoietic stem cells (HSCs) with normal transplanted HSCs can correct a wide range of hematologic disorders. Here, we provide evidence that transplantation of more differentiated progenitor cells can be used to more rapidly correct lymphoid deficiencies in unconditioned immunocompromised mice. Transplantation of flk2+ multipotent progenitors led to robust B and T cell reconstitution that was maintained for at least 16 weeks. Antigenic challenge at 16 weeks post-transplantation revealed that reconstituted lymphocytes maintained a functional repertoire. In contrast to the persistent lymphocytic engraftment, myeloid chimerism was lost by 12 weeks post-transplantation consistent with the fact that flk2+ progenitors are non-self-renewing. Thus, while more differentiated progenitors are capable of rescuing lymphoid deficiencies, transplantation of HSCs must be used for the correction of non-lymphoid disorders, and, we propose, very long-term immune reconstitution. Based on recent evidence, we discuss novel strategies to achieve the replacement of abnormal HSCs without the use of cytotoxic conditioning regimens. PMID:16760650

Immune Reconstitution after Allogeneic Hematopoietic Stem Cell Transplantation

PubMed Central

Ogonek, Justyna; Kralj Juric, Mateja; Ghimire, Sakhila; Varanasi, Pavankumar Reddy; Holler, Ernst; Greinix, Hildegard; Weissinger, Eva

2016-01-01

The timely reconstitution and regain of function of a donor-derived immune system is of utmost importance for the recovery and long-term survival of patients after allogeneic hematopoietic stem cell transplantation (HSCT). Of note, new developments such as umbilical cord blood or haploidentical grafts were associated with prolonged immunodeficiency due to delayed immune reconstitution, raising the need for better understanding and enhancing the process of immune reconstitution and finding strategies to further optimize these transplant procedures. Immune reconstitution post-HSCT occurs in several phases, innate immunity being the first to regain function. The slow T cell reconstitution is regarded as primarily responsible for deleterious infections with latent viruses or fungi, occurrence of graft-versus-host disease, and relapse. Here we aim to summarize the major steps of the adaptive immune reconstitution and will discuss the importance of immune balance in patients after HSCT. PMID:27909435

Factors affecting reconstitution of the T cell compartment in allogeneic haematopoietic cell transplant recipients.

PubMed

Fallen, P R; McGreavey, L; Madrigal, J A; Potter, M; Ethell, M; Prentice, H G; Guimarães, A; Travers, P J

2003-11-01

The factors affecting T cell reconstitution post haematopoietic cell transplantation (HCT) are not well characterised. We carried out a longitudinal analysis of T cell reconstitution in 32 HCT recipients during the first 12 months post transplant. We analysed reconstitution of naïve, memory and effector T cells, their diversity and monitored thymic output using TCR rearrangement excision circles (TRECs). Thymic-independent pathways were responsible for the rapid reconstitution of memory and effector T cells less than 6 months post HCT. Thymic-dependent pathways were activated between 6 and 12 months in the majority of patients with naïve T cell numbers increasing in parallel with TREC levels. Increasing patient age, chronic GVHD and T cell depletion (with or without pretransplant Campath-1H) predicted low TREC levels and slow naïve T cell recovery. Furthermore, increasing patient age also predicted high memory and effector T cell numbers. The effects of post HCT immunosuppression, total body irradiation, donor leucocyte infusions, T cell dose and post HCT infections on T cell recovery were also analysed. However, no effects of these single variables across a variety of different age, GVHD and T cell depletion groups were apparent. This study suggests that future analysis of the factors affecting T cell reconstitution and studies aimed at reactivating the thymus through therapeutic intervention should be analysed in age-, GVHD- and TCD-matched patient groups.

Rapid reconstitution of CMV-specific T-cells after stem-cell transplantation.

PubMed

Widmann, Thomas; Sester, Urban; Schmidt, Tina; Gärtner, Barbara C; Schubert, Jörg; Pfreundschuh, Michael; Sester, Martina

2018-04-13

As reconstitution of virus-specific T-cells is critical to control cytomegalovirus (CMV)-viremia following stem-cell transplantation (SCT), we characterized the dynamics in CMV-specific T-cell reconstitution after SCT. Cytomegalovirus-specific T-cells from 51 SCT-recipients were prospectively quantified and phenotypically characterised by intracellular cytokine-staining after specific stimulation and HLA class-I-specific pentamers using flow cytometry. Cytomegalovirus-specific CD4 T-cells reconstituted after a median of 2.3 (IQR, 2.0-3.0) weeks following autografting, and 4.0 (IQR, 3.0-5.6) weeks after allografting, with CMV-specific T-cells originating from donors and/or recipients. The time for reconstitution of CMV-specific CD4 and CD8 T-cells did not differ (P = .58). Factors delaying the time to initial reconstitution of CMV-specific CD4 T-cells included a negative recipient serostatus (P = .016) and CMV-viremia (P = .026). Percentages of CMV-specific CD4 T-cells significantly increased over time and reached a plateau after 90 days (P = .043). Relative CMV-specific CD4 T-cell levels remained higher in long-term transplant recipients compared with those in controls (P < .0001). However, due to persisting lymphopenia, absolute numbers of CMV-specific T-cells were similar as in controls. Cytomegalovirus-specific T-cells rapidly reconstitute after SCT and their percentages remain high in the long term. In the face of persistent lymphopenia, this results in similar absolute numbers of CMV-specific T-cells as in controls to ensure sufficient pathogen control. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The use of specific antibodies to mediate fusion between Sendai virus envelopes and living cells.

PubMed

Loyter, A; Tomasi, M; Gitman, A G; Etinger, L; Nussbaum, O

1984-01-01

Incubation of Sendai virus particles with non-ionic detergents such as Triton X-100 completely solubilizes the viral envelopes. Removal of the detergent from the supernatant (which contains the two main viral glycoproteins) leads to the formation of fusogenic, reconstituted viral envelopes. Soluble macromolecules such as DNA or proteins can be enclosed within the reconstituted vesicles, while membrane components can be inserted into the viral envelopes. Fusion of such loaded or 'hybrid' reconstituted envelopes with living cells in culture results in either microinjection or transfer of the viral components to the recipient cells. Thus such reconstituted envelopes can serve as efficient carriers for the introduction of macromolecules of biological interest into living cells in culture. A more specific vehicle has been constructed by chemically coupling anti-cell membrane antibodies (anti-human erythrocyte antibody) to the viral envelope. Such antibody-bearing intact virus particles or reconstituted envelopes bound to and fused with virus receptor-depleted cells. In addition, anti-Sendai virus antibodies were coupled to neuraminidase-treated human erythrocytes. Such antibodies mediated the binding and fusion of intact Sendai virus particles and their reconstituted envelopes to virus receptor-depleted cells.

Embryoid body attachment to reconstituted basement membrane induces a genetic program of epithelial differentiation via jun N-terminal kinase signaling.

PubMed

Ho, Hoang-Yen; Moffat, Ryan C; Patel, Rupal V; Awah, Franklin N; Baloue, Kaitrin; Crowe, David L

2010-09-01

Embryonic stem (ES) cells are derived from early stage mammalian embryos and have broad developmental potential. These cells can be manipulated experimentally to generate cells of multiple tissue types which could be important in treating human diseases. The ability to produce relevant amounts of these differentiated cell populations creates the basis for clinical interventions in tissue regeneration and repair. Understanding how embryonic stem cells differentiate also can reveal important insights into cell biology. A previously reported mouse embryonic stem cell model demonstrated that differentiated epithelial cells migrated out of embryoid bodies attached to reconstituted basement membrane. We used genomic technology to profile ES cell populations in order to understand the molecular mechanisms leading to epithelial differentiation. Cells with characteristics of cultured epithelium migrated from embryoid bodies attached to reconstituted basement membrane. However, cells that comprised embryoid bodies also rapidly lost ES cell-specific gene expression and expressed proteins characteristic of stratified epithelia within hours of attachment to basement membrane. Gene expression profiling of sorted cell populations revealed upregulation of the BMP/TGFbeta signaling pathway, which was not sufficient for epithelial differentiation in the absence of basement membrane attachment. Activation of c-jun N-terminal kinase 1 (JNK1) and increased expression of Jun family transcription factors was observed during epithelial differentiation of ES cells. Inhibition of JNK signaling completely blocked epithelial differentiation in this model, revealing a key mechanism by which ES cells adopt epithelial characteristics via basement membrane attachment. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Neonatal bone marrow transplantation of ADA-deficient SCID mice results in immunologic reconstitution despite low levels of engraftment and an absence of selective donor T lymphoid expansion

PubMed Central

Carbonaro, Denise A.; Jin, Xiangyang; Cotoi, Daniel; Mi, Tiejuan; Yu, Xiao-Jin; Skelton, Dianne C.; Dorey, Frederick; Kellems, Rodney E.; Blackburn, Michael R.

2008-01-01

Adenosine deaminase (ADA)–deficient severe combined immune deficiency (SCID) may be treated by allogeneic hematopoietic stem cell transplantation without prior cytoreductive conditioning, although the mechanism of immune reconstitution is unclear. We studied this process in a murine gene knockout model of ADA-deficient SCID. Newborn ADA-deficient pups received transplants of intravenous infusion of normal congenic bone marrow, without prior cytoreductive conditioning, which resulted in long-term survival, multisystem correction, and nearly normal lymphocyte numbers and mitogenic proliferative responses. Only 1% to 3% of lymphocytes and myeloid cells were of donor origin without a selective expansion of donor-derived lymphocytes; immune reconstitution was by endogenous, host-derived ADA-deficient lymphocytes. Preconditioning of neonates with 100 to 400 cGy of total body irradiation before normal donor marrow transplant increased the levels of engrafted donor cells in a radiation dose–dependent manner, but the chimerism levels were similar for lymphoid and myeloid cells. The absence of selective reconstitution by donor T lymphocytes in the ADA-deficient mice indicates that restoration of immune function occurred by rescue of endogenous ADA-deficient lymphocytes through cross-correction from the engrafted ADA-replete donor cells. Thus, ADA-deficient SCID is unique in its responses to nonmyeloablative bone marrow transplantation, which has implications for clinical bone marrow transplantation or gene therapy. PMID:18356486

Neonatal bone marrow transplantation of ADA-deficient SCID mice results in immunologic reconstitution despite low levels of engraftment and an absence of selective donor T lymphoid expansion.

PubMed

Carbonaro, Denise A; Jin, Xiangyang; Cotoi, Daniel; Mi, Tiejuan; Yu, Xiao-Jin; Skelton, Dianne C; Dorey, Frederick; Kellems, Rodney E; Blackburn, Michael R; Kohn, Donald B

2008-06-15

Adenosine deaminase (ADA)-deficient severe combined immune deficiency (SCID) may be treated by allogeneic hematopoietic stem cell transplantation without prior cytoreductive conditioning, although the mechanism of immune reconstitution is unclear. We studied this process in a murine gene knockout model of ADA-deficient SCID. Newborn ADA-deficient pups received transplants of intravenous infusion of normal congenic bone marrow, without prior cytoreductive conditioning, which resulted in long-term survival, multisystem correction, and nearly normal lymphocyte numbers and mitogenic proliferative responses. Only 1% to 3% of lymphocytes and myeloid cells were of donor origin without a selective expansion of donor-derived lymphocytes; immune reconstitution was by endogenous, host-derived ADA-deficient lymphocytes. Preconditioning of neonates with 100 to 400 cGy of total body irradiation before normal donor marrow transplant increased the levels of engrafted donor cells in a radiation dose-dependent manner, but the chimerism levels were similar for lymphoid and myeloid cells. The absence of selective reconstitution by donor T lymphocytes in the ADA-deficient mice indicates that restoration of immune function occurred by rescue of endogenous ADA-deficient lymphocytes through cross-correction from the engrafted ADA-replete donor cells. Thus, ADA-deficient SCID is unique in its responses to nonmyeloablative bone marrow transplantation, which has implications for clinical bone marrow transplantation or gene therapy.

Reconstitution of the NF1 GAP-related domain in NF1-deficient human Schwann cells

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

Thomas, Stacey L.; Neuroscience Program, Loyola University Medical Center, Maywood, IL; Department of Anatomy and Cell Biology, University of Illinois Chicago, Chicago, IL

Schwann cells derived from peripheral nerve sheath tumors from individuals with Neurofibromatosis Type 1 (NF1) are deficient for the protein neurofibromin, which contains a GAP-related domain (NF1-GRD). Neurofibromin-deficient Schwann cells have increased Ras activation, increased proliferation in response to certain growth stimuli, increased angiogenic potential, and altered cell morphology. This study examined whether expression of functional NF1-GRD can reverse the transformed phenotype of neurofibromin-deficient Schwann cells from both benign and malignant peripheral nerve sheath tumors. We reconstituted the NF1-GRD using retroviral transduction and examined the effects on cell morphology, growth potential, and angiogenic potential. NF1-GRD reconstitution resulted in morphologic changes,more » a 16-33% reduction in Ras activation, and a 53% decrease in proliferation in neurofibromin-deficient Schwann cells. However, NF1-GRD reconstitution was not sufficient to decrease the in vitro angiogenic potential of the cells. This study demonstrates that reconstitution of the NF1-GRD can at least partially reverse the transformation of human NF1 tumor-derived Schwann cells.« less

In vitro membrane protein synthesis inside Sec translocon-reconstituted cell-sized liposomes

PubMed Central

Ohta, Naoki; Kato, Yasuhiko; Watanabe, Hajime; Mori, Hirotada; Matsuura, Tomoaki

2016-01-01

Protein synthesis using an in vitro transcription-translation system (IVTT) inside cell-sized liposomes has become a valuable tool to study the properties of biological systems under cell-mimicking conditions. However, previous liposome systems lacked the machinery for membrane protein translocation. Here, we reconstituted the translocon consisting of SecYEG from Escherichia coli inside cell-sized liposomes. The cell-sized liposomes also carry the reconstituted IVTT, thereby providing a cell-mimicking environment for membrane protein synthesis. By using EmrE, a multidrug transporter from E. coli, as a model membrane protein, we found that both the amount and activity of EmrE synthesized inside the liposome is increased approximately three-fold by incorporating the Sec translocon. The topological change of EmrE induced by the translocon was also identified. The membrane integration of 6 out of 9 E. coli inner membrane proteins that was tested was increased by incorporation of the translocon. By introducing the Sec translocon, the membrane integration efficiency of the membrane protein of interest was increased, and enabled the integration of membrane proteins that otherwise cannot be inserted. In addition, this work represents an essential step toward the construction of an artificial cell through a bottom-up approach. PMID:27808179

Partial reconstitution of the CD4+-T-cell compartment in CD4 gene knockout mice restores responses to tuberculosis DNA vaccines.

PubMed

D'Souza, Sushila; Romano, Marta; Korf, Johanna; Wang, Xiao-Ming; Adnet, Pierre-Yves; Huygen, Kris

2006-05-01

Reactivation tuberculosis (TB) is a serious problem in immunocompromised individuals, especially those with human immunodeficiency virus (HIV) coinfection. The adaptive immune response mediated by CD4+ and CD8+ T cells is known to confer protection against TB. Hence, vaccines against TB are designed to activate these two components of the immune system. Anti-TB DNA vaccines encoding the immunodominant proteins Ag85A, Ag85B, and PstS-3 from Mycobacterium tuberculosis are ineffective in mice lacking CD4+ T cells (CD4-/- mice). In this study, we demonstrate that reconstitution of the T-cell compartment in CD4-/- mice restores vaccine-specific antibody and gamma interferon (IFN-gamma) responses to these DNA vaccines. The magnitude of the immune responses correlated with the extent of reconstitution of the CD4+-T-cell compartment. Reconstituted mice vaccinated with DNA encoding PstS-3, known to encode a dominant D(b)-restricted CD8+-T-cell epitope, displayed CD8+-T-cell responses not observed in CD4-/- mice. M. tuberculosis challenge in reconstituted mice led to the extravasation of IFN-gamma-producing CD4+ and CD8+ T cells into lungs, the primary site of bacterial replication. Importantly, a reconstitution of 12 to 15% of the CD4+-T-cell compartment resulted in Ag85B plasmid DNA-mediated protection against a challenge M. tuberculosis infection. Our findings provide evidence that anti-TB DNA vaccines could be effective in immunodeficient individuals after CD4+-T-lymphocyte reconstitution, as may occur following antiretroviral therapy in HIV+ patients.

Epigenetic programming of T cells impacts immune reconstitution in hematopoietic stem cell transplant recipients.

PubMed

Hardy, Kristine; Smith, Corey; Tu, Wen Juan; McCuaig, Robert; Panikkar, Archana; Dasari, Vijayendra; Wu, Fan; Tey, Siok-Keen; Hill, Geoffrey R; Khanna, Rajiv; Rao, Sudha

2018-03-27

Immune reconstitution following hematopoietic stem cell transplantation (HSCT) is critical in preventing harmful sequelae in recipients with cytomegalovirus (CMV) infection. To understand the molecular mechanisms underlying immune reconstitution kinetics, we profiled the transcriptome-chromatin accessibility landscape of CMV-specific CD8 + T cells from HCST recipients with different immune reconstitution efficiencies. CMV-specific T cells from HSCT recipients with stable antiviral immunity expressed higher levels of interferon/defense response and cell cycle genes in an interconnected network involving PI3KCG , STAT5B , NFAT , RBPJ , and lower HDAC6 , increasing chromatin accessibility at the enhancer regions of immune and T-cell receptor signaling pathway genes. By contrast, the transcriptional and epigenomic signatures of CMV-specific T cells from HSCT recipients with unstable immune reconstitution showed commonalities with T-cell responses in other nonresolving chronic infections. These signatures included higher levels of EGR and KLF factors that, along with lower JARID2 expression, maintained higher accessibility at promoter and CpG-rich regions of genes associated with apoptosis. Furthermore, epigenetic targeting via inhibition of HDAC6 or JARID2 enhanced the transcription of genes associated with differential responses, suggesting that drugs targeting epigenomic modifiers may have therapeutic potential for enhancing immune reconstitution in HSCT recipients. Taken together, these analyses demonstrate that transcription factors and chromatin modulators create different chromatin accessibility landscapes in T cells of HSCT recipients that not only affect immediate gene expression but also differentially prime cells for responses to additional signals. Epigenetic therapy may be a promising strategy to promote immune reconstitution in HSCT recipients. © 2018 by The American Society of Hematology.

Long survival and immunologic reconstitution following transplantation with syngeneic or allogeneic fetal liver and neonatal spleen cells. [X radiation, mice

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

Yunis, E.J.; Fernandes, G.; Smith, J.

1976-12-01

Spleen cells from newborn syngeneic and allogeneic mice that lack fully differentiated T lymphocytes can be used as a hematopoietic source to reconstitute both hematopoietic and lymphoid systems of lethally irradiated mice without producing a GVHR. Fetal liver cells from syngeneic and allogeneic mice that lack postthymic T lymphocytes can also be used for hematopoietic and immunologic reconstitution of lethally irradiated mice without producing GVHR. Immunologic deficiency is observed in some experiments in mice given supralethal irradiation (1000 R) and fetal liver as reconstituting hematopoietic tissue. The findings suggest that T cells, at an early stage of differentiation, are moremore » susceptible to tolerance induction than are T lymphocytes at later stages of differentiation and do not, in general, produce GVHR. It is postulated that hematopoietic cells, free of postthymic lymphoid cells, can be used for hematopoietic or immunologic reconstitution and celular engineering without producing GVHD.« less

Modeling Ebola Virus Genome Replication and Transcription with Minigenome Systems.

PubMed

Cressey, Tessa; Brauburger, Kristina; Mühlberger, Elke

2017-01-01

In this chapter, we describe the minigenome system for Ebola virus (EBOV), which reconstitutes EBOV polymerase activity in cells and can be used to model viral genome replication and transcription. This protocol comprises all steps including cell culture, plasmid preparation, transfection, and luciferase reporter assay readout.

Dynamical System Modeling of Immune Reconstitution after Allogeneic Stem Cell Transplantation Identifies Patients at Risk for Adverse Outcomes

PubMed Central

Toor, Amir A.; Sabo, Roy T.; Roberts, Catherine H.; Moore, Bonny L.; Salman, Salman R.; Scalora, Allison F.; Aziz, May T.; Shubar Ali, Ali S.; Hall, Charles E.; Meier, Jeremy; Thorn, Radhika M.; Wang, Elaine; Song, Shiyu; Miller, Kristin; Rizzo, Kathryn; Clark, William B.; McCarty, John M.; Chung, Harold M.; Manjili, Masoud H.; Neale, Michael C.

2016-01-01

Systems that evolve over time and follow mathematical laws as they evolve are called dynamical systems. Lymphocyte recovery and clinical outcomes in 41 allograft recipients conditioned using antithymocyte globulin (ATG) and 4.5-Gy total body irradiation were studied to determine if immune reconstitution could be described as a dynamical system. Survival, relapse, and graft-versus-host disease (GVHD) were not significantly different in 2 cohorts of patients receiving different doses of ATG. However, donor-derived CD3+ cell reconstitution was superior in the lower ATG dose cohort, and there were fewer instances of donor lymphocyte infusion (DLI). Lymphoid recovery was plotted in each individual over time and demonstrated 1 of 3 sigmoid growth patterns: Pattern A (n = 15) had rapid growth with high lymphocyte counts, pattern B (n = 14) had slower growth with intermediate recovery, and pattern C (n = 10) had poor lymphocyte reconstitution. There was a significant association between lymphocyte recovery patterns and both the rate of change of donor-derived CD3+ at day 30 after stem cell transplantation (SCT) and clinical outcomes. GVHD was observed more frequently with pattern A, relapse and DLI more so with pattern C, with a consequent survival advantage in patients with patterns A and B. We conclude that evaluating immune reconstitution after SCT as a dynamical system may differentiate patients at risk of adverse outcomes and allow early intervention to modulate that risk. PMID:25849208

Increasing Stem Cell Dose Promotes Posttransplant Immune Reconstitution.

PubMed

Xu, Ning; Shen, Sylvie; Dolnikov, Alla

2017-04-01

Umbilical cord blood (UCB) transplantation can provide a successful therapeutic option for patients that have no suitable related donor. UCB transplantation is often limited by the relatively small hematopoietic stem cell (HSC) numbers in UCB especially for adult recipients. Early neutrophil and platelet engraftment correlates with the stem cell numbers in UCB transplant. Compared to other HSC sources, immune reconstitution following UCB transplant is slower and complicated by increased frequency of opportunistic infections. The effect of HSC numbers in UCB transplant on immune reconstitution was not thoroughly examined. Using immunocompromised mice transplanted with purified UCB CD34+ stem cells, we have demonstrated that increasing the numbers of CD34+ cells in the transplant promotes hematopoietic and immune reconstitution. At early stages posttransplant, high stem cell dose generated relatively more B cells, while lower dose generated more myeloid and T cells. Thus, the size of the stem cell graft appears to modulate the differentiation potential of infused stem cells. In addition, increasing stem cell dose in the transplant improved CD8+ T cell development and delayed late memory T cell skewing in expense of naive T cells highlighting the importance of HSC dose to maintain the pool of naive T cells able to develop strong immune responses. Transplantation of ex vivo expanded CD34+ cells did not promote, but rather delayed immune reconstitution suggesting the loss of primitive lymphoid precursor cells during ex vivo expansion.

Acetylcholine and acetylcarnitine transport in peritoneum: Role of the SLC22A4 (OCTN1) transporter.

PubMed

Pochini, Lorena; Scalise, Mariafrancesca; Di Silvestre, Sara; Belviso, Stefania; Pandolfi, Assunta; Arduini, Arduino; Bonomini, Mario; Indiveri, Cesare

2016-04-01

A suitable experimental tool based on proteoliposomes for assaying Organic Cation Transporter Novel member 1 (OCTN1) of peritoneum was pointed out. OCTN1, recently acknowledged as acetylcholine transporter, was immunodetected in rat peritoneum. Transport was assayed following flux of radiolabelled TEA, acetylcholine or acetylcarnitine in proteoliposomes reconstituted with peritoneum extract. OCTN1 mediated, besides TEA, also acetylcholine and a slower acetylcarnitine transport. External sodium inhibited acetylcholine uptake but not its release from proteoliposomes. Differently, sodium did not affect acetylcarnitine uptake. These results suggested that physiologically, acetylcholine should be released while acetylcarnitine was taken up by peritoneum cells. Transport was impaired by OCTN1 inhibitors, butyrobetaine, spermine, and choline. Biotin was also found as acetylcholine transport inhibitor. Anti-OCTN1 antibody specifically inhibited acetylcholine transport confirming the involvement of OCTN1. The transporter was also immunodetected in human mesothelial primary cells. Extract from these cells was reconstituted in proteoliposomes. Transport features very similar to those found with rat peritoneum were observed. Validation of the proteoliposome model for peritoneal transport study was then achieved assaying transport in intact mesothelial cells. TEA, butyrobetaine and Na(+) inhibited acetylcholine transport in intact cells while efflux was Na(+) insensitive. Therefore transport features in intact cells overlapped those found in proteoliposomes. Copyright © 2015 Elsevier B.V. All rights reserved.

Identifying risk factors of immune reconstitution inflammatory syndrome in AIDS patients receiving highly active anti-retroviral therapy.

PubMed

He, Bo; Zheng, Yuhuang; Liu, Meng; Zhou, Guoqiang; Chen, Xia; Mamadou, Diallo; He, Yan; Zhou, Huaying; Chen, Zi

2013-01-01

Immune reconstitution inflammation syndrome typically occurs within days after patients undergo highly active anti-retroviral therapy and is a big hurdle for effective treatment of AIDS patients. In this study, we monitored immune reconstitution inflammation syndrome occurrence in 238 AIDS patients treated with highly active anti-retroviral therapy. Among them, immune reconstitution inflammation syndrome occurred in 47 cases (19.7%). Immune reconstitution inflammation syndrome patients had significantly higher rate of opportunistic infection (p<0.001) and persistently lower CD4(+) cell count (p<0.001) compared to the non-immune reconstitution inflammation syndrome patients. In contrast, no significant differences in HIV RNA loads were observed between the immune reconstitution inflammation syndrome group and non-immune reconstitution inflammation syndrome group. These data suggest that a history of opportunistic infection and CD4(+) cell counts at baseline may function as risk factors for immune reconstitution inflammation syndrome occurrence in AIDS patients as well as potential prognostic markers. These findings will improve the management of AIDS with highly active anti-retroviral therapy. Copyright © 2013 Elsevier Editora Ltda. All rights reserved.

Core-shell hydrogel beads with extracellular matrix for tumor spheroid formation.

PubMed

Yu, L; Grist, S M; Nasseri, S S; Cheng, E; Hwang, Y-C E; Ni, C; Cheung, K C

2015-03-01

Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since they may provide a better model of the tumor than conventional monolayer culture. Moreover, tumor cell interaction with the extracellular matrix can determine cell organization and behavior. In this work, a microfluidic system was used to form cell-laden core-shell beads which incorporate elements of the extracellular matrix and support the formation of multicellular spheroids. The bead core (comprising a mixture of alginate, collagen, and reconstituted basement membrane, with gelation by temperature control) and shell (comprising alginate hydrogel, with gelation by ionic crosslinking) were simultaneously formed through flow focusing using a cooled flow path into the microfluidic chip. During droplet gelation, the alginate acts as a fast-gelling shell which aids in preventing droplet coalescence and in maintaining spherical droplet geometry during the slower gelation of the collagen and reconstituted basement membrane components as the beads warm up. After droplet gelation, the encapsulated MCF-7 cells proliferated to form uniform spheroids when the beads contained all three components: alginate, collagen, and reconstituted basement membrane. The dose-dependent response of the MCF-7 cell tumor spheroids to two anticancer drugs, docetaxel and tamoxifen, was compared to conventional monolayer culture.

Stem Cell-Like Differentiation Potentials of Endometrial Side Population Cells as Revealed by a Newly Developed In Vivo Endometrial Stem Cell Assay

PubMed Central

Miyazaki, Kaoru; Maruyama, Tetsuo; Masuda, Hirotaka; Yamasaki, Akiko; Uchida, Sayaka; Oda, Hideyuki; Uchida, Hiroshi; Yoshimura, Yasunori

2012-01-01

Background Endometrial stem/progenitor cells contribute to the cyclical regeneration of human endometrium throughout a woman's reproductive life. Although the candidate cell populations have been extensively studied, no consensus exists regarding which endometrial population represents the stem/progenitor cell fraction in terms of in vivo stem cell activity. We have previously reported that human endometrial side population cells (ESP), but not endometrial main population cells (EMP), exhibit stem cell-like properties, including in vivo reconstitution of endometrium-like tissues when xenotransplanted into immunodeficient mice. The reconstitution efficiency, however, was low presumably because ESP cells alone could not provide a sufficient microenvironment (niche) to support their stem cell activity. The objective of this study was to establish a novel in vivo endometrial stem cell assay employing cell tracking and tissue reconstitution systems and to examine the stem cell properties of ESP through use of this assay. Methodology/Principal Findings ESP and EMP cells isolated from whole endometrial cells were infected with lentivirus to express tandem Tomato (TdTom), a red fluorescent protein. They were mixed with unlabeled whole endometrial cells and then transplanted under the kidney capsule of ovariectomized immunodeficient mice. These mice were treated with estradiol and progesterone for eight weeks and nephrectomized. All of the grafts reconstituted endometrium-like tissues under the kidney capsules. Immunofluorescence revealed that TdTom-positive cells were significantly more abundant in the glandular, stromal, and endothelial cells of the reconstituted endometrium in mice transplanted with TdTom-labeled ESP cells than those with TdTom-labeled EMP cells. Conclusions/Significance We have established a novel in vivo endometrial stem cell assay in which multi-potential differentiation can be identified through cell tracking during in vivo endometrial tissue reconstitution. Using this assay, we demonstrated that ESP cells differentiated into multiple endometrial lineages in the niche provided by whole endometrial cells, indicating that ESP cells are genuine endometrial stem/progenitor cells. PMID:23226538

Multiplex CRISPR/Cas9-Based Genome Editing in Human Hematopoietic Stem Cells Models Clonal Hematopoiesis and Myeloid Neoplasia.

PubMed

Tothova, Zuzana; Krill-Burger, John M; Popova, Katerina D; Landers, Catherine C; Sievers, Quinlan L; Yudovich, David; Belizaire, Roger; Aster, Jon C; Morgan, Elizabeth A; Tsherniak, Aviad; Ebert, Benjamin L

2017-10-05

Hematologic malignancies are driven by combinations of genetic lesions that have been difficult to model in human cells. We used CRISPR/Cas9 genome engineering of primary adult and umbilical cord blood CD34 + human hematopoietic stem and progenitor cells (HSPCs), the cells of origin for myeloid pre-malignant and malignant diseases, followed by transplantation into immunodeficient mice to generate genetic models of clonal hematopoiesis and neoplasia. Human hematopoietic cells bearing mutations in combinations of genes, including cohesin complex genes, observed in myeloid malignancies generated immunophenotypically defined neoplastic clones capable of long-term, multi-lineage reconstitution and serial transplantation. Employing these models to investigate therapeutic efficacy, we found that TET2 and cohesin-mutated hematopoietic cells were sensitive to azacitidine treatment. These findings demonstrate the potential for generating genetically defined models of human myeloid diseases, and they are suitable for examining the biological consequences of somatic mutations and the testing of therapeutic agents. Copyright © 2017 Elsevier Inc. All rights reserved.

Arrestin-3-dependent activation of c-Jun N-terminal kinases (JNKs)

PubMed Central

Kaoud, Tamer S.; Dalby, Kevin N.; Gurevich, Eugenia V.; Gurevich, Vsevolod V.

2015-01-01

Only one out of four arrestin subtypes expressed in mammals, arrestin-3, facilitates the activation of JNK family kinases. Here we describe two different paradigms that allow the elucidation of the mechanisms involved. One is based on reconstitution of signaling modules from purified proteins: arrestin-3, MKK4, MKK7, JNK1, JNK2, and JNK3. The main advantage of this method is that it can unambiguously establish which effects are direct, because only intended purified proteins are present in these assays. The key drawback is that the upstream-most kinases of these cascades, ASK1 or other MAPKKKs, are not available in purified form, limiting reconstitution to incomplete two-kinase modules. The other set of methods analyzes the effects of arrestin-3 on JNK activation in intact cells. In this case, signaling modules include ASK1 and/or other MAPKKKs. However, every cell expresses thousands of different proteins, and their possible effects on the readout cannot be excluded. However, the combination of in vitro reconstitution from purified proteins and cell-based assays enables comprehensive elucidation of the mechanisms of arrestin-3-dependent activation of JNK family kinases. PMID:25737158

Stem cell transplantation as a dynamical system: are clinical outcomes deterministic?

PubMed

Toor, Amir A; Kobulnicky, Jared D; Salman, Salman; Roberts, Catherine H; Jameson-Lee, Max; Meier, Jeremy; Scalora, Allison; Sheth, Nihar; Koparde, Vishal; Serrano, Myrna; Buck, Gregory A; Clark, William B; McCarty, John M; Chung, Harold M; Manjili, Masoud H; Sabo, Roy T; Neale, Michael C

2014-01-01

Outcomes in stem cell transplantation (SCT) are modeled using probability theory. However, the clinical course following SCT appears to demonstrate many characteristics of dynamical systems, especially when outcomes are considered in the context of immune reconstitution. Dynamical systems tend to evolve over time according to mathematically determined rules. Characteristically, the future states of the system are predicated on the states preceding them, and there is sensitivity to initial conditions. In SCT, the interaction between donor T cells and the recipient may be considered as such a system in which, graft source, conditioning, and early immunosuppression profoundly influence immune reconstitution over time. This eventually determines clinical outcomes, either the emergence of tolerance or the development of graft versus host disease. In this paper, parallels between SCT and dynamical systems are explored and a conceptual framework for developing mathematical models to understand disparate transplant outcomes is proposed.

Stem Cell Transplantation as a Dynamical System: Are Clinical Outcomes Deterministic?

PubMed Central

Toor, Amir A.; Kobulnicky, Jared D.; Salman, Salman; Roberts, Catherine H.; Jameson-Lee, Max; Meier, Jeremy; Scalora, Allison; Sheth, Nihar; Koparde, Vishal; Serrano, Myrna; Buck, Gregory A.; Clark, William B.; McCarty, John M.; Chung, Harold M.; Manjili, Masoud H.; Sabo, Roy T.; Neale, Michael C.

2014-01-01

Outcomes in stem cell transplantation (SCT) are modeled using probability theory. However, the clinical course following SCT appears to demonstrate many characteristics of dynamical systems, especially when outcomes are considered in the context of immune reconstitution. Dynamical systems tend to evolve over time according to mathematically determined rules. Characteristically, the future states of the system are predicated on the states preceding them, and there is sensitivity to initial conditions. In SCT, the interaction between donor T cells and the recipient may be considered as such a system in which, graft source, conditioning, and early immunosuppression profoundly influence immune reconstitution over time. This eventually determines clinical outcomes, either the emergence of tolerance or the development of graft versus host disease. In this paper, parallels between SCT and dynamical systems are explored and a conceptual framework for developing mathematical models to understand disparate transplant outcomes is proposed. PMID:25520720

Dynamical System Modeling of Immune Reconstitution after Allogeneic Stem Cell Transplantation Identifies Patients at Risk for Adverse Outcomes.

PubMed

Toor, Amir A; Sabo, Roy T; Roberts, Catherine H; Moore, Bonny L; Salman, Salman R; Scalora, Allison F; Aziz, May T; Shubar Ali, Ali S; Hall, Charles E; Meier, Jeremy; Thorn, Radhika M; Wang, Elaine; Song, Shiyu; Miller, Kristin; Rizzo, Kathryn; Clark, William B; McCarty, John M; Chung, Harold M; Manjili, Masoud H; Neale, Michael C

2015-07-01

Systems that evolve over time and follow mathematical laws as they evolve are called dynamical systems. Lymphocyte recovery and clinical outcomes in 41 allograft recipients conditioned using antithymocyte globulin (ATG) and 4.5-Gy total body irradiation were studied to determine if immune reconstitution could be described as a dynamical system. Survival, relapse, and graft-versus-host disease (GVHD) were not significantly different in 2 cohorts of patients receiving different doses of ATG. However, donor-derived CD3(+) cell reconstitution was superior in the lower ATG dose cohort, and there were fewer instances of donor lymphocyte infusion (DLI). Lymphoid recovery was plotted in each individual over time and demonstrated 1 of 3 sigmoid growth patterns: Pattern A (n = 15) had rapid growth with high lymphocyte counts, pattern B (n = 14) had slower growth with intermediate recovery, and pattern C (n = 10) had poor lymphocyte reconstitution. There was a significant association between lymphocyte recovery patterns and both the rate of change of donor-derived CD3(+) at day 30 after stem cell transplantation (SCT) and clinical outcomes. GVHD was observed more frequently with pattern A, relapse and DLI more so with pattern C, with a consequent survival advantage in patients with patterns A and B. We conclude that evaluating immune reconstitution after SCT as a dynamical system may differentiate patients at risk of adverse outcomes and allow early intervention to modulate that risk. Copyright © 2015 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

7 CFR 718.204 - Reconstitution of allotments, quotas, and bases.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 7 2011-01-01 2011-01-01 false Reconstitution of allotments, quotas, and bases. 718... PROVISIONS APPLICABLE TO MULTIPLE PROGRAMS Reconstitution of Farms, Allotments, Quotas, and Bases § 718.204 Reconstitution of allotments, quotas, and bases. (a) Farms shall be reconstituted in accordance with this subpart...

7 CFR 718.204 - Reconstitution of allotments, quotas, and bases.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 7 2010-01-01 2010-01-01 false Reconstitution of allotments, quotas, and bases. 718... PROVISIONS APPLICABLE TO MULTIPLE PROGRAMS Reconstitution of Farms, Allotments, Quotas, and Bases § 718.204 Reconstitution of allotments, quotas, and bases. (a) Farms shall be reconstituted in accordance with this subpart...

Spontaneous cell sorting of fibroblasts and keratinocytes creates an organotypic human skin equivalent.

PubMed

Wang, C K; Nelson, C F; Brinkman, A M; Miller, A C; Hoeffler, W K

2000-04-01

We show that an inherent ability of two distinct cell types, keratinocytes and fibroblasts, can be relied upon to accurately reconstitute full-thickness human skin including the dermal-epidermal junction by a cell-sorting mechanism. A cell slurry containing both cell types added to silicone chambers implanted on the backs of severe combined immunodeficient mice sorts out to reconstitute a clearly defined dermis and stratified epidermis within 2 wk, forming a cell-sorted skin equivalent. Immunostaining of the cell-sorted skin equivalent with human cell markers showed patterns similar to those of normal full-thickness skin. We compared the cell-sorted skin equivalent model with a composite skin model also made on severe combined immunodeficient mice. The composite grafts were constructed from partially differentiated keratinocyte sheets placed on top of a dermal equivalent constructed of devitalized dermis. Electron microscopy revealed that both models formed ample numbers of normal appearing hemidesmosomes. The cell-sorted skin equivalent model, however, had greater numbers of keratin intermediate filaments within the basal keratinocytes that connected to hemidesmosomes, and on the dermal side both collagen filaments and anchoring fibril connections to the lamina densa were more numerous compared with the composite model. Our results may provide some insight into why, in clinical applications for treating burns and other wounds, composite grafts may exhibit surface instability and blistering for up to a year following grafting, and suggest the possible usefulness of the cell-sorted skin equivalent in future grafting applications.

Ex Vivo Expanded Human Regulatory T Cells Delay Islet Allograft Rejection via Inhibiting Islet-Derived Monocyte Chemoattractant Protein-1 Production in CD34+ Stem Cells-Reconstituted NOD-scid IL2rγnull Mice

PubMed Central

Xiao, Fang; Ma, Liang; Zhao, Min; Huang, Guocai; Mirenda, Vincenzo; Dorling, Anthony

2014-01-01

Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by immune-mediated destruction of insulin-secreting β cells of the pancreas. Near complete dependence on exogenous insulin makes T1DM very difficult to control, with the result that patients are exposed to high blood glucose and risk of diabetic complications and/or intermittent low blood glucose that can cause unconsciousness, fits and even death. Allograft transplantation of pancreatic islets restores normoglycemia with a low risk of surgical complications. However, although successful immediately after transplantation, islets are progressively lost, with most of the patients requiring exogenous insulin within 2 years post-transplant. Therefore, there is an urgent requirement for the development of new strategies to prevent islet rejection. In this study, we explored the importance of human regulatory T cells in the control of islets allograft rejection. We developed a pre-clinical model of human islet transplantation by reconstituting NOD-scid IL2rγnull mice with cord blood-derived human CD34+ stem cells and demonstrated that although the engrafted human immune system mediated the rejection of human islets, their survival was significantly prolonged following adoptive transfer of ex vivo expanded human Tregs. Mechanistically, Tregs inhibited the infiltration of innate immune cells and CD4+ T cells into the graft by down-regulating the islet graft-derived monocyte chemoattractant protein-1. Our findings might contribute to the development of clinical strategies for Treg therapy to control human islet rejection. We also show for the first time that CD34+ cells-reconstituted NOD-scid IL2rγnull mouse model could be beneficial for investigating human innate immunity in vivo. PMID:24594640

Biologically Complex Planar Cell Plasma Membranes Supported on Polyelectrolyte Cushions Enhance Transmembrane Protein Mobility and Retain Native Orientation.

PubMed

Liu, Han-Yuan; Chen, Wei-Liang; Ober, Christopher K; Daniel, Susan

2018-01-23

Reconstituted supported lipid bilayers (SLB) are widely used as in vitro cell-surface models because they are compatible with a variety of surface-based analytical techniques. However, one of the challenges of using SLBs as a model of the cell surface is the limited complexity in membrane composition, including the incorporation of transmembrane proteins and lipid diversity that may impact the activity of those proteins. Additionally, it is challenging to preserve the transmembrane protein native orientation, function, and mobility in SLBs. Here, we leverage the interaction between cell plasma membrane vesicles and polyelectrolyte brushes to create planar bilayers from cell plasma membrane vesicles that have budded from the cell surface. This approach promotes the direct incorporation of membrane proteins and other species into the planar bilayer without using detergent or reconstitution and preserves membrane constituents. Furthermore, the structure of the polyelectrolyte brush serves as a cushion between the planar bilayer and rigid supporting surface, limiting the interaction of the cytosolic domains of membrane proteins with this surface. Single particle tracking was used to analyze the motion of GPI-linked yellow fluorescent proteins (GPI-YFP) and neon-green fused transmembrane P2X2 receptors (P2X2-neon) and shows that this platform retains over 75% mobility of multipass transmembrane proteins in its native membrane environment. An enzyme accessibility assay confirmed that the protein orientation is preserved and results in the extracellular domain facing toward the bulk phase and the cytosolic side facing the support. Because the platform presented here retains the complexity of the cell plasma membrane and preserves protein orientation and mobility, it is a better representative mimic of native cell surfaces, which may find many applications in biological assays aimed at understanding cell membrane phenomena.

Physiologically relevant organs on chips

PubMed Central

Yum, Kyungsuk; Hong, Soon Gweon; Lee, Luke P.

2015-01-01

Recent advances in integrating microengineering and tissue engineering have generated promising microengineered physiological models for experimental medicine and pharmaceutical research. Here we review the recent development of microengineered physiological systems, or organs on chips, that reconstitute the physiologically critical features of specific human tissues and organs and their interactions. This technology uses microengineering approaches to construct organ-specific microenvironments, reconstituting tissue structures, tissue–tissue interactions and interfaces, and dynamic mechanical and biochemical stimuli found in specific organs, to direct cells to assemble into functional tissues. We first discuss microengineering approaches to reproduce the key elements of physiologically important, dynamic mechanical microenvironments, biochemical microenvironments, and microarchitectures of specific tissues and organs in microfluidic cell culture systems. This is followed by examples of microengineered individual organ models that incorporate the key elements of physiological microenvironments into single microfluidic cell culture systems to reproduce organ-level functions. Finally, microengineered multiple organ systems that simulate multiple organ interactions to better represent human physiology, including human responses to drugs, is covered in this review. This emerging organs-on-chips technology has the potential to become an alternative to 2D and 3D cell culture and animal models for experimental medicine, human disease modeling, drug development, and toxicology. PMID:24357624

Assembly kinetics determine the architecture of α-actinin crosslinked F-actin networks.

PubMed

Falzone, Tobias T; Lenz, Martin; Kovar, David R; Gardel, Margaret L

2012-05-29

The actin cytoskeleton is organized into diverse meshworks and bundles that support many aspects of cell physiology. Understanding the self-assembly of these actin-based structures is essential for developing predictive models of cytoskeletal organization. Here we show that the competing kinetics of bundle formation with the onset of dynamic arrest arising from filament entanglements and crosslinking determine the architecture of reconstituted actin networks formed with α-actinin crosslinks. Crosslink-mediated bundle formation only occurs in dilute solutions of highly mobile actin filaments. As actin polymerization proceeds, filament mobility and bundle formation are arrested concomitantly. By controlling the onset of dynamic arrest, perturbations to actin assembly kinetics dramatically alter the architecture of biochemically identical samples. Thus, the morphology of reconstituted F-actin networks is a kinetically determined structure similar to those formed by physical gels and glasses. These results establish mechanisms controlling the structure and mechanics in diverse semiflexible biopolymer networks.

Cognate HLA absence in trans diminishes human NK cell education

PubMed Central

Landtwing, Vanessa; Raykova, Ana; Pezzino, Gaetana; Béziat, Vivien; Graf, Claudine; Moretta, Alessandro; Capaul, Riccarda; Zbinden, Andrea; Malmberg, Karl-Johan; Chijioke, Obinna; Münz, Christian

2016-01-01

NK cells are innate lymphocytes with protective functions against viral infections and tumor formation. Human NK cells carry inhibitory killer cell Ig-like receptors (KIRs), which recognize distinct HLAs. NK cells with KIRs for self-HLA molecules acquire superior cytotoxicity against HLA– tumor cells during education for improved missing-self recognition. Here, we reconstituted mice with human hematopoietic cells from donors with homozygous KIR ligands or with a mix of hematopoietic cells from these homozygous donors, allowing assessment of the resulting KIR repertoire and NK cell education. We found that co-reconstitution with 2 KIR ligand–mismatched compartments did not alter the frequency of KIR-expressing NK cells. However, NK cell education was diminished in mice reconstituted with parallel HLA compartments due to a lack of cognate HLA molecules on leukocytes for the corresponding KIRs. This change in NK cell education in mixed human donor–reconstituted mice improved NK cell–mediated immune control of EBV infection, indicating that mixed hematopoietic cell populations could be exploited to improve NK cell reactivity against leukotropic pathogens. Taken together, these findings indicate that leukocytes lacking cognate HLA ligands can disarm KIR+ NK cells in a manner that may decrease HLA– tumor cell recognition but allows for improved NK cell–mediated immune control of a human γ-herpesvirus. PMID:27571408

Accelerated lymphocyte reconstitution and long-term recovery after transplantation of lentiviral-transduced rhesus CD34+ cells mobilized by G-CSF and plerixafor.

PubMed

Uchida, Naoya; Bonifacino, Aylin; Krouse, Allen E; Metzger, Mark E; Csako, Gyorgy; Lee-Stroka, Agnes; Fasano, Ross M; Leitman, Susan F; Mattapallil, Joseph J; Hsieh, Matthew M; Tisdale, John F; Donahue, Robert E

2011-07-01

Granulocyte colony-stimulating factor (G-CSF) in combination with plerixafor produces significant mobilization of CD34(+) cells in rhesus macaques. We sought to evaluate whether these CD34(+) cells can stably reconstitute blood cells with lentiviral gene marking. We performed hematopoietic stem cell transplantation using G-CSF and plerixafor-mobilized rhesus CD34(+) cells transduced with a lentiviral vector, and these data were compared with those of G-CSF and stem cell factor mobilization. G-CSF and plerixafor mobilization resulted in CD34(+) cell yields that were twofold higher than yields with G-CSF and stem cell factor. CD123 (interleukin-3 receptor) expression was greater in G-CSF and plerixafor-mobilized CD34(+) cells when compared to G-CSF alone. Animals transplanted with G-CSF and plerixafor-mobilized cells showed engraftment of all lineages, similar to animals who received G-CSF and stem cell factor-mobilized grafts. Lymphocyte engraftment was accelerated in animals receiving the G-CSF and plerixafor-mobilized CD34(+) cells. One animal in the G-CSF and plerixafor group developed cold agglutinin-associated skin rash during the first 3 months of rapid lymphocyte recovery. One year after transplantation, all animals had 2% to 10% transgene expression in all blood cell lineages. G-CSF and plerixafor-mobilized CD34(+) cells accelerate lymphocyte engraftment and contain hematopoietic stem cell capable of reconstituting multilineage blood cells. These findings indicate important differences to consider in plerixafor-based hematopoietic stem cell mobilization protocols in rhesus macaques. Published by Elsevier Inc.

LTβR expression on hematopoietic cells regulates acute inflammation and influences maturation of myeloid subpopulations.

PubMed

Wege, Anja K; Huber, Barbara; Wimmer, Nadin; Männel, Daniela N; Hehlgans, Thomas

2014-07-01

Lymphotoxin beta-receptor (LTβR) is involved in the formation and maintenance of secondary lymphoid structures, as well as in the regulation of inflammatory responses. Because LTβR lymphoid structure formation continues to develop in infants, we compared two different chimera models: one using adult mice and the other using a transplantation model of neonatal mice. To elucidate the function of LTβR on lymphoid and non-lymphoid cells, we generated bone marrow chimeras on the wild type C57Bl/6 and the LTβR-deficient (LTβR(-/-)) background, and reconstituted the mice with bone marrow cells reciprocally. These chimeric mice were analyzed in the experimental model of acute dextran sulfate sodium-induced colitis. Interestingly, both models revealed not only equal reconstitution levels but also similar immunological responses: LTβR expression on stromal cells is essential for lymph node formation, whereas LTBR on hematopoietic cells is crucial for a decrease in inflammation. In addition, mice lacking LTβR on hematopoietic cells revealed (a) an increase of immature granulocytic cells in the spleen and (b) a reduced proportion of myeloid cells in peripheral blood and spleen expressing CD11b(+)Ly6C(+)Ly6G(-) (myeloid-derived suppressor cells expression profile). In conclusion, LTβR expression on hematopoietic cells seems to be involved in the down-regulation of acute inflammatory reactions paralleled by the appearance of immature myeloid cells. © The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Reconstitution of adaptive and innate immunity following allogeneic hematopoietic stem cell transplantation in humans.

PubMed

Peggs, K S

2006-01-01

Allogeneic hematopoietic stem cell transplantation is a potentially curative treatment modality for a number of hematologic malignancies, as well as inherited immunodeficiencies and hemoglobinopathies, and may also have a role in selected acquired autoimmune disorders. The complete or near-complete ablation of host immunity and subsequent establishment of donor-derived immunity that is required for successful engraftment and long-term outcomes provide a major obstacle to such transplantation approaches. A delicate balance exists between the need for the reconstituted donor-derived immunity to provide both protection against pathogenic challenges and graft-versus-malignancy activity, and the potentially harmful expansion of alloreactive T-cell clones mediating GvHD. The search for interventions that would allow more rapid and selective reconstitution of beneficial immune specificities continues to be informed by the development of new tools enabling a more precise dissection of the kinetics of reconstituting populations. This review summarizes more recent data on immune reconstitution following allogeneic transplantation in humans.

Hepatic loss of survivin impairs postnatal liver development and promotes expansion of hepatic progenitor cells in mice.

PubMed

Li, Dan; Cen, Jin; Chen, Xiaotao; Conway, Edward M; Ji, Yuan; Hui, Lijian

2013-12-01

Hepatocytes possess a remarkable capacity to regenerate and reconstitute the parenchyma after liver damage. However, in the case of chronic injury, their proliferative potential is impaired and hepatic progenitor cells (HPCs) are activated, resulting in a ductular reaction known as oval cell response. Proapoptotic and survival signals maintain a precise balance to spare hepatocytes and progenitors from hyperplasia and cell death during regeneration. Survivin, a member of the family of inhibitor of apoptosis proteins (IAPs), plays key roles in the proliferation and apoptosis of various cell types. Here, we characterized the in vivo function of Survivin in regulating postnatal liver development and homeostasis using mice carrying conditional Survivin alleles. Hepatic perinatal loss of Survivin causes impaired mitosis, increased genome ploidy, and enlarged cell size in postnatal livers, which eventually leads to hepatocyte apoptosis and triggers tissue damage and inflammation. Subsequently, HPCs that retain genomic Survivin alleles are activated, which finally differentiate into hepatocytes and reconstitute the whole liver. By contrast, inducible ablation of Survivin in adult hepatocytes does not affect HPC activation and liver homeostasis during a long-life period. Perinatal Survivin deletion impairs hepatic mitosis in postnatal liver development, which induces HPC activation and reconstitution in the liver, therefore providing a novel HPC induction model. Copyright © 2013 by the American Association for the Study of Liver Diseases.

Matrigel basement membrane matrix induces eccrine sweat gland cells to reconstitute sweat gland-like structures in nude mice.

PubMed

Li, Haihong; Chen, Lu; Zeng, Shaopeng; Li, Xuexue; Zhang, Xiang; Lin, Changmin; Zhang, Mingjun; Xie, Sitian; He, Yunpu; Shu, Shenyou; Yang, Lvjun; Tang, Shijie; Fu, Xiaobing

2015-03-01

Severe burn results in irreversible damage to eccrine sweat glands, for which no effective treatment is available. Interaction between the extracellular matrix and epithelial cells is critical for proper three-dimensional organization and function of the epithelium. Matrigel-embedded eccrine sweat gland cells were subcutaneously implanted into the inguinal regions of nude mice. Two weeks later, the Matrigel plugs were removed and evaluated for series of detection items. Sweat gland cells developed into sweat gland-like structures in the Matrigel plugs based on: (1) de novo formation of tubular-like structures with one or more hollow lumens, (2) expression of epithelial and sweat gland markers (pancytokeratin, CK5/7/14/19, α-SMA and CEA), (3) basement membrane formation, (4) myoepithelial cells presenting in and encompassing the tubular-like structures, (5) cellular polarization, evident by the expression of tight junction proteins (claudin-1 and ZO-2), anchoring junctions (desmoglein-1 and -2 and E-cadherin) and CEA in the luminal membrane, (6) expression of proteins related to sweat secretion and absorption (Na(+)-K(+)-ATPase α/β, Na(+)-K(+)-2Cl-cotranspoter 1, Na(+)/H(+) exchanger 1, aquaporin-5, epithelial sodium channel, cystic fibrosis transmembrane conductance regulator, potassium channel and vacuolar-type H+-ATPase), and (7) about 20% of the tubular-like structures are de novo coils and 80% are de novo ducts. This study provides not only an excellent model to study eccrine sweat gland development, cytodifferentiation and reconstitution, but also an in vivo model for regeneration of eccrine sweat glands. Copyright © 2015 Elsevier Inc. All rights reserved.

A single-platform approach using flow cytometry and microbeads to evaluate immune reconstitution in mice after bone marrow transplantation.

PubMed

Perruche, Sylvain; Kleinclauss, François; Lienard, Agnès; Robinet, Eric; Tiberghien, Pierre; Saas, Philippe

2004-11-01

The monitoring of immune reconstitution in murine models of HC transplantation, using accurate and automated methods, is necessary in view of the recent developments of hematopoietic cell (HC) transplantation (including reduced intensity conditioning regimens) as well as emerging immunological concepts (such as the involvement of dendritic cells or regulatory T cells). Here, we describe the use of a single-platform approach based on flow cytometry and tubes that contain a defined number of microbeads to evaluate absolute blood cell counts in mice. This method, previously used in humans to quantify CD34+ stem cells or CD4+ T cells in HIV infected patients, was adapted for mouse blood samples. A CD45 gating strategy in this "lyse no wash" protocol makes it possible to discriminate erythroblasts or red blood cell debris from CD45+ leukocytes, thus avoiding cell loss. Tubes contain a lyophilized brightly fluorescent microbead pellet permitting the acquisition of absolute counts of leukocytes after flow cytometric analysis. We compared this method to determine absolute counts of circulating cells with another method combining Unopette reservoir diluted blood samples, hemocytometer, microscopic examination and flow cytometry. The sensitivity of this single-platform approach was evaluated in different situations encountered in allogeneic HC transplantation, including immune cell depletion after different conditioning regimens, activation status of circulating cells after transplantation, evaluation of in vivo cell depletion and hematopoietic progenitor mobilization in the periphery. This single-platform flow cytometric assay can also be proposed to standardize murine (or other mammalian species) leukocyte count determination for physiological, pharmacological/toxicological and diagnostic applications in veterinary practice.

Effect of Low-Intensity Pulsed Ultrasound after Mesenchymal Stromal Cell Injection to Treat Osteochondral Defects: An In Vivo Study.

PubMed

Yamaguchi, Shoki; Aoyama, Tomoki; Ito, Akira; Nagai, Momoko; Iijima, Hirotaka; Tajino, Junichi; Zhang, Xiangkai; Wataru, Kiyan; Kuroki, Hiroshi

2016-12-01

We investigated the effect of low-intensity pulsed ultrasound (LIPUS) treatment combined with mesenchymal stromal cell (MSC) injection for cartilage repair and subchondral bone reconstitution for treatment of osteochondral defects. An osteochondral defect was created on both femur grooves of Wistar rats. Four weeks later, bone marrow MSCs were injected into the right knee joint. The rats were divided into two intervention groups: without or with LIPUS irradiation. Cartilage repair was evaluated histologically based on the Wakitani cartilage repair score. Subchondral bone reconstitution was evaluated as bone volume (BV)/tissue volume (TV) by micro-computed tomography analysis. MSC injection improved the cartilage repair score, and LIPUS irradiation improved BV/TV. Combination treatment promoted both cartilage repair and BV/TV improvement. Thus, MSC injection combined with LIPUS irradiation is more effective than either treatment alone in promoting concurrent cartilage repair and subchondral reconstitution. Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Clearance of CMV viremia and survival after double umbilical cord blood transplantation in adults depends on reconstitution of thymopoiesis.

PubMed

Brown, Julia A; Stevenson, Kristen; Kim, Haesook T; Cutler, Corey; Ballen, Karen; McDonough, Sean; Reynolds, Carol; Herrera, Maria; Liney, Deborah; Ho, Vincent; Kao, Grace; Armand, Philippe; Koreth, John; Alyea, Edwin; McAfee, Steve; Attar, Eyal; Dey, Bimalangshu; Spitzer, Thomas; Soiffer, Robert; Ritz, Jerome; Antin, Joseph H; Boussiotis, Vassiliki A

2010-05-20

Umbilical cord blood grafts are increasingly used as sources of hematopoietic stem cells in adults. Data regarding the outcome of this approach in adults are consistent with delayed and insufficient immune reconstitution resulting in high infection-related morbidity and mortality. Using cytomegalovirus (CMV)-specific immunity as a paradigm, we evaluated the status, mechanism, and clinical implications of immune reconstitution in adults with hematologic malignancies undergoing unrelated double unit cord blood transplantation. Our data indicate that CD8(+) T cells capable of secreting interferon-gamma (IFN-gamma) in a CMV-specific enzyme-linked immunosorbent spot (ELISpot) assay are detectable at 8 weeks after transplantation, before reconstitution of thymopoiesis, but fail to clear CMV viremia. Clearance of CMV viremia occurs later and depends on the recovery of CD4(+)CD45RA(+) T cells, reconstitution of thymopoiesis, and attainment of T-cell receptor rearrangement excision circle (TREC) levels of 2000 or more copies/mug DNA. In addition, overall survival was significantly higher in patients who displayed thymic regeneration and attainment of TREC levels of 2000 or more copies/mug DNA (P = .005). These results indicate that reconstitution of thymopoiesis is critical for long-term clinical outcome in adult recipients of umbilical cord blood transplant. The trial was prospectively registered at http://www.clinicaltrials.gov (NCT00133367).

Identification of GPCR-Interacting Cytosolic Proteins Using HDL Particles and Mass Spectrometry-Based Proteomic Approach

PubMed Central

Chung, Ka Young; Day, Peter W.; Vélez-Ruiz, Gisselle; Sunahara, Roger K.; Kobilka, Brian K.

2013-01-01

G protein-coupled receptors (GPCRs) have critical roles in various physiological and pathophysiological processes, and more than 40% of marketed drugs target GPCRs. Although the canonical downstream target of an agonist-activated GPCR is a G protein heterotrimer; there is a growing body of evidence suggesting that other signaling molecules interact, directly or indirectly, with GPCRs. However, due to the low abundance in the intact cell system and poor solubility of GPCRs, identification of these GPCR-interacting molecules remains challenging. Here, we establish a strategy to overcome these difficulties by using high-density lipoprotein (HDL) particles. We used the β2-adrenergic receptor (β2AR), a GPCR involved in regulating cardiovascular physiology, as a model system. We reconstituted purified β2AR in HDL particles, to mimic the plasma membrane environment, and used the reconstituted receptor as bait to pull-down binding partners from rat heart cytosol. A total of 293 proteins were identified in the full agonist-activated β2AR pull-down, 242 proteins in the inverse agonist-activated β2AR pull-down, and 210 proteins were commonly identified in both pull-downs. A small subset of the β2AR-interacting proteins isolated was confirmed by Western blot; three known β2AR-interacting proteins (Gsα, NHERF-2, and Grb2) and 3 newly identified known β2AR-interacting proteins (AMPKα, acetyl-CoA carboxylase, and UBC-13). Profiling of the identified proteins showed a clear bias toward intracellular signal transduction pathways, which is consistent with the role of β2AR as a cell signaling molecule. This study suggests that HDL particle-reconstituted GPCRs can provide an effective platform method for the identification of GPCR binding partners coupled with a mass spectrometry-based proteomic analysis. PMID:23372797

Intrathymic injection of hematopoietic progenitor cells establishes functional T cell development in a mouse model of severe combined immunodeficiency.

PubMed

Tuckett, Andrea Z; Thornton, Raymond H; O'Reilly, Richard J; van den Brink, Marcel R M; Zakrzewski, Johannes L

2017-05-16

Even though hematopoietic stem cell transplantation can be curative in patients with severe combined immunodeficiency, there is a need for additional strategies boosting T cell immunity in individuals suffering from genetic disorders of lymphoid development. Here we show that image-guided intrathymic injection of hematopoietic stem and progenitor cells in NOD-scid IL2rγ null mice is feasible and facilitates the generation of functional T cells conferring protective immunity. Hematopoietic stem and progenitor cells were isolated from the bone marrow of healthy C57BL/6 mice (wild-type, Luciferase + , CD45.1 + ) and injected intravenously or intrathymically into both male and female, young or aged NOD-scid IL2rγ null recipients. The in vivo fate of injected cells was analyzed by bioluminescence imaging and flow cytometry of thymus- and spleen-derived T cell populations. In addition to T cell reconstitution, we evaluated mice for evidence of immune dysregulation based on diabetes development and graft-versus-host disease. T cell immunity following intrathymic injection of hematopoietic stem and progenitor cells in NOD-scid IL2rγ null mice was assessed in a B cell lymphoma model. Despite the small size of the thymic remnant in NOD-scid IL2rγ null mice, we were able to accomplish precise intrathymic delivery of hematopoietic stem and progenitor cells by ultrasound-guided injection. Thymic reconstitution following intrathymic injection of healthy allogeneic hematopoietic cells was most effective in young male recipients, indicating that even in the setting of severe immunodeficiency, sex and age are important variables for thymic function. Allogeneic T cells generated in intrathymically injected NOD-scid IL2rγ null mice displayed anti-lymphoma activity in vivo, but we found no evidence for severe auto/alloreactivity in T cell-producing NOD-scid IL2rγ null mice, suggesting that immune dysregulation is not a major concern. Our findings suggest that intrathymic injection of donor hematopoietic stem and progenitor cells is a safe and effective strategy to establish protective T cell immunity in a mouse model of severe combined immunodeficiency.

Imaging immune response of skin mast cells in vivo with two-photon microscopy

NASA Astrophysics Data System (ADS)

Li, Chunqiang; Pastila, Riikka K.; Lin, Charles P.

2012-02-01

Intravital multiphoton microscopy has provided insightful information of the dynamic process of immune cells in vivo. However, the use of exogenous labeling agents limits its applications. There is no method to perform functional imaging of mast cells, a population of innate tissue-resident immune cells. Mast cells are widely recognized as the effector cells in allergy. Recently their roles as immunoregulatory cells in certain innate and adaptive immune responses are being actively investigated. Here we report in vivo mouse skin mast cells imaging with two-photon microscopy using endogenous tryptophan as the fluorophore. We studied the following processes. 1) Mast cells degranulation, the first step in the mast cell activation process in which the granules are released into peripheral tissue to trigger downstream reactions. 2) Mast cell reconstitution, a procedure commonly used to study mast cells functioning by comparing the data from wild type mice, mast cell-deficient mice, and mast-cell deficient mice reconstituted with bone marrow-derived mast cells (BMMCs). Imaging the BMMCs engraftment in tissue reveals the mast cells development and the efficiency of BMMCs reconstitution. We observed the reconstitution process for 6 weeks in the ear skin of mast cell-deficient Kit wsh/ w-sh mice by two-photon imaging. Our finding is the first instance of imaging mast cells in vivo with endogenous contrast.

Evidence for naive T-cell repopulation despite thymus irradiation after autologous transplantation in adults with multiple myeloma: role of ex vivo CD34+ selection and age.

PubMed

Malphettes, Marion; Carcelain, Guislaine; Saint-Mezard, Pierre; Leblond, Véronique; Altes, Hester Korthals; Marolleau, Jean-Pierre; Debré, Patrice; Brouet, Jean-Claude; Fermand, Jean-Paul; Autran, Brigitte

2003-03-01

Immunodeficiency following autologous CD34+-purified peripheral blood stem cell (PBSC) transplantation could be related to T-cell depletion of the graft or impaired T-cell reconstitution due to thymus irradiation. Aiming to assess the role of irradiated thymus in T-cell repopulation, we studied 32 adults with multiple myeloma, randomly assigned to receive high-dose therapy including total body irradiation (TBI) followed by autologous transplantation with either unselected or CD34+-selected PBSCs. The median number of reinfused CD3+ cells was lower in the selected group (0.03 versus 14 x 10(6)/kg; P =.002). Lymphocyte subset counts were evaluated from month 3 to 24 after grafting. Naive CD4+ T cells were characterized both by phenotype and by quantification of T-cell receptor rearrangement excision circles (TRECs). The reconstitution of CD3+ and CD4+ T cells was significantly delayed in the CD34+-selected group, but eventually led to counts similar to those found in the unselected group after month 12. Mechanism of reconstitution differed, however, between both groups. Indeed, a marked increase in the naive CD62L+CD45RA+CD4+ subset was observed in the selected group, but not in the unselected group in which half of the CD45RA+CD4+ T cells appear to be CD62L-. Age was identified as an independent adverse factor for CD4+ and CD62L+CD45RA+CD4+ T-cell reconstitution. Our results provide evidence that infusing PBSCs depleted of T cells after TBI in adults delays T-cell reconstitution but accelerates thymic regeneration.

Axi-symmetric patterns of active polar filaments on spherical and composite surfaces

NASA Astrophysics Data System (ADS)

Srivastava, Pragya; Rao, Madan

2014-03-01

Experiments performed on Fission Yeast cells of cylindrical and spherical shapes, rod-shaped bacteria and reconstituted cylindrical liposomes suggest the influence of cell geometry on patterning of cortical actin. A theoretical model based on active hydrodynamic description of cortical actin that includes curvature-orientation coupling predicts spontaneous formation of acto-myosin rings, cables and nodes on cylindrical and spherical geometries [P. Srivastava et al, PRL 110, 168104(2013)]. Stability and dynamics of these patterns is also affected by the cellular shape and has been observed in experiments performed on Fission Yeast cells of spherical shape. Motivated by this, we study the stability and dynamics of axi-symmetric patterns of active polar filaments on the surfaces of spherical, saddle shaped and conical geometry and classify the stable steady state patterns on these surfaces. Based on the analysis of the fluorescence images of Myosin-II during ring slippage we propose a simple mechanical model for ring-sliding based on force balance and make quantitative comparison with the experiments performed on Fission Yeast cells. NSF Grant DMR-1004789 and Syracuse Soft Matter Program.

High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split Renilla luciferase.

PubMed

Kim, Sung Bae; Ozawa, Takeaki; Watanabe, Shigeaki; Umezawa, Yoshio

2004-08-10

Nucleocytoplasmic trafficking of functional proteins plays a key role in regulating gene expressions in response to extracellular signals. We developed a genetically encoded bioluminescent indicator for monitoring the nuclear trafficking of target proteins in vitro and in vivo. The principle is based on reconstitution of split fragments of Renilla reniformis (Rluc) by protein splicing with a DnaE intein (a catalytic subunit of DNA polymerase III). A target cytosolic protein fused to the N-terminal half of Rluc is expressed in mammalian cells. If the protein translocates into the nucleus, the Rluc moiety meets the C-terminal half of Rluc, and full-length Rluc is reconstituted by protein splicing. We demonstrated quantitative cell-based in vitro sensing of ligand-induced translocation of androgen receptor, which allowed high-throughput screening of exo- and endogenous agonists and antagonists. Furthermore, the indicator enabled noninvasive in vivo imaging of the androgen receptor translocation in the brains of living mice with a charge-coupled device imaging system. These rapid and quantitative analyses in vitro and in vivo provide a wide variety of applications for screening pharmacological or toxicological compounds and testing them in living animals.

In vitro reconstitution of interactions in the CARD9 signalosome

PubMed Central

Park, Jin Hee; Choi, Jae Young; Mustafa, Mir Faisal; Park, Hyun Ho

2017-01-01

The caspase-associated recruitment domain (CARD)-containing protein 9 (CARD9) signalosome is composed of CARD9, B-cell CLL/lymphoma 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1). The CARD9 signalosome has been reported to exert critical functions in the immunoreceptor tyrosine-based activation motif-coupled receptor-mediated activation of myeloid cells, through nuclear factor-κB pathways during innate immunity processes. During CARD9 signalosome assembly, BCL10 has been revealed to function as an adaptor protein and to interact with CARD9 via CARD-CARD interactions; BCL10 also interacts with MALT1 via its C-terminal Ser/Thr-rich region and the first immunoglobulin domain of MALT1. The CARD9 signalosome is implicated in critical biological processes; however, its structural and biochemical characteristics have yet to be elucidated. In the present study, CARD9 and BCL10 CARDs were successfully purified and characterized, and their biochemical properties were investigated. In addition, CARD9-BCL10 complexes were reconstituted in vitro under low salt and pH conditions. Furthermore, based on structural modeling data, a scheme was proposed to describe the interactions between CARD9 and BCL10. This provides a further understanding of the mechanism of how the CARD9 signalosome may be assembled. PMID:28765954

Comparative immunohistologic studies in an adoptive transfer model of acute rat cardiac allograft rejection

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

Forbes, R.D.; Lowry, R.P.; Gomersall, M.

1985-07-01

It has been shown that fulminant acute rejection of rat cardiac allografts across a full haplotype disparity may occur as a direct result of adoptive transfer of sensitized W3/25+ MRC OX8- SIg- T helper/DTH syngeneic spleen cells to sublethally irradiated recipients. In order to establish the immunohistologic parameters of this form of rejection, allografts and recipient lymphoid tissue were analyzed using a panel of monoclonal antibodies of known cellular distribution. These data were compared with those obtained following reconstitution of irradiated allograft recipients with unseparated sensitized spleen cells, with unreconstituted irradiated donor recipient pairs, with unmodified first-set rejection, and withmore » induced myocardial infarction of syngeneic heart grafts transplanted to normal and to sublethally irradiated recipients. Rejecting cardiac allografts transplanted to all reconstituted irradiated recipients were characterized by extensive infiltration with MRC OX8+ (T cytotoxic-suppressor, natural killer) cells even when this subset was virtually excluded from the reconstituting inocula. A similar proportional accumulation of MRC OX8+ cells observed at the infarct margins of syngeneic heart grafts transplanted to irradiated unreconstituted recipients greatly exceeded that present in normal nonirradiated controls. These data provide evidence that under conditions of heavy recipient irradiation, MRC OX8+ cells may be sequestered within heart grafts in response to nonspecific injury unrelated to the rejection process.« less

Turnover of bone marrow-derived cells in the irradiated mouse cornea

PubMed Central

Chinnery, Holly R; Humphries, Timothy; Clare, Adam; Dixon, Ariane E; Howes, Kristen; Moran, Caitlin B; Scott, Danielle; Zakrzewski, Marianna; Pearlman, Eric; McMenamin, Paul G

2008-01-01

In light of an increasing awareness of the presence of bone marrow (BM)-derived macrophages in the normal cornea and their uncertain role in corneal diseases, it is important that the turnover rate of these resident immune cells be established. The baseline density and distribution of macrophages in the corneal stroma was investigated in Cx3cr1gfp transgenic mice in which all monocyte-derived cells express enhanced green fluorescent protein (eGFP). To quantify turnover, BM-derived cells from transgenic eGFP mice were transplanted into whole-body irradiated wild-type recipients. Additionally, wild-type BM-derived cells were injected into irradiated Cx3cr1+/gfp recipients, creating reverse chimeras. At 2, 4 and 8 weeks post-reconstitution, the number of eGFP+ cells in each corneal whole mount was calculated using epifluorescence microscopy, immunofluorescence staining and confocal microscopy. The total density of myeloid-derived cells in the normal Cx3cr1+/gfp cornea was 366 cells/mm2. In BM chimeras 2 weeks post-reconstitution, 24% of the myeloid-derived cells had been replenished and were predominantly located in the anterior stroma. By 8 weeks post-reconstitution 75% of the myeloid-derived cells had been replaced and these cells were distributed uniformly throughout the stroma. All donor eGFP+ cells expressed low to moderate levels of CD45 and CD11b, with approximately 25% coexpressing major histocompatibility complex class II, a phenotype characteristic of previous descriptions of corneal stromal macrophages. In conclusion, 75% of the myeloid-derived cells in the mouse corneal stroma are replenished after 8 weeks. These data provide a strong basis for functional investigations of the role of resident stromal macrophages versus non-haematopoietic cells using BM chimeric mice in models of corneal inflammation. PMID:18540963

Determining the structure-mechanics relationships of dense microtubule networks with confocal microscopy and magnetic tweezers-based microrheology.

PubMed

Yang, Yali; Valentine, Megan T

2013-01-01

The microtubule (MT) cytoskeleton is essential in maintaining the shape, strength, and organization of cells. Its spatiotemporal organization is fundamental for numerous dynamic biological processes, and mechanical stress within the MT cytoskeleton provides an important signaling mechanism in mitosis and neural development. This raises important questions about the relationships between structure and mechanics in complex MT structures. In vitro, reconstituted cytoskeletal networks provide a minimal model of cell mechanics while also providing a testing ground for the fundamental polymer physics of stiff polymer gels. Here, we describe our development and implementation of a broad tool kit to study structure-mechanics relationships in reconstituted MT networks, including protocols for the assembly of entangled and cross-linked MT networks, fluorescence imaging, microstructure characterization, construction and calibration of magnetic tweezers devices, and mechanical data collection and analysis. In particular, we present the design and assembly of three neodymium iron boron (NdFeB)-based magnetic tweezers devices optimized for use with MT networks: (1) high-force magnetic tweezers devices that enable the application of nano-Newton forces and possible meso- to macroscale materials characterization; (2) ring-shaped NdFeB-based magnetic tweezers devices that enable oscillatory microrheology measurements; and (3) portable magnetic tweezers devices that enable direct visualization of microscale deformation in soft materials under applied force. Copyright © 2013 Elsevier Inc. All rights reserved.

Physiologically relevant organs on chips.

PubMed

Yum, Kyungsuk; Hong, Soon Gweon; Healy, Kevin E; Lee, Luke P

2014-01-01

Recent advances in integrating microengineering and tissue engineering have generated promising microengineered physiological models for experimental medicine and pharmaceutical research. Here we review the recent development of microengineered physiological systems, or also known as "ogans-on-chips", that reconstitute the physiologically critical features of specific human tissues and organs and their interactions. This technology uses microengineering approaches to construct organ-specific microenvironments, reconstituting tissue structures, tissue-tissue interactions and interfaces, and dynamic mechanical and biochemical stimuli found in specific organs, to direct cells to assemble into functional tissues. We first discuss microengineering approaches to reproduce the key elements of physiologically important, dynamic mechanical microenvironments, biochemical microenvironments, and microarchitectures of specific tissues and organs in microfluidic cell culture systems. This is followed by examples of microengineered individual organ models that incorporate the key elements of physiological microenvironments into single microfluidic cell culture systems to reproduce organ-level functions. Finally, microengineered multiple organ systems that simulate multiple organ interactions to better represent human physiology, including human responses to drugs, is covered in this review. This emerging organs-on-chips technology has the potential to become an alternative to 2D and 3D cell culture and animal models for experimental medicine, human disease modeling, drug development, and toxicology. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inactivation of Nondesiccated and Desiccated Cronobacter sakazakii in Reconstituted Infant Formula by Combination of Citral and Mild Heat.

PubMed

Shi, Chao; Jia, Zhenyu; Sun, Yi; Chen, Yifei; Guo, Du; Liu, Zhiyuan; Wen, Qiwu; Guo, Xiao; Ma, Linlin; Yang, Baowei; Baloch, Allah Bux; Xia, Xiaodong

2017-07-01

The objective of this study was to evaluate the combined effect of citral plus mild heat on nondesiccated and desiccated Cronobacter sakazakii in reconstituted infant formula. Various concentrations of citral (0, 0.3, 0.6, and 0.9%) combined with various temperatures (25, 45, 50, and 55°C) were applied to nondesiccated and desiccated cocktails of three C. sakazakii strains (approximately 6.0 log CFU mL -1 ) in reconstituted infant formula, and the bacterial populations were assayed periodically. The combined treatments had marked antimicrobial effects on C. sakazakii compared with the control. Desiccated cells were more susceptible to citral than were nondesiccated cells in reconstituted infant formula. These findings suggest there is a potential application of citral in combination with mild heat to control C. sakazakii during preparation of reconstituted infant formula.

Early recovery of T-cell function predicts improved survival after T-cell depleted allogeneic transplant.

PubMed

Goldberg, Jenna D; Zheng, Junting; Ratan, Ravin; Small, Trudy N; Lai, Kuan-Chi; Boulad, Farid; Castro-Malaspina, Hugo; Giralt, Sergio A; Jakubowski, Ann A; Kernan, Nancy A; O'Reilly, Richard J; Papadopoulos, Esperanza B; Young, James W; van den Brink, Marcel R M; Heller, Glenn; Perales, Miguel-Angel

2017-08-01

Infection, relapse, and GVHD can complicate allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although the effect of poor immune recovery on infection risk is well-established, there are limited data on the effect of immune reconstitution on relapse and survival, especially following T-cell depletion (TCD). To characterize the pattern of immune reconstitution in the first year after transplant and its effects on survival and relapse, we performed a retrospective study in 375 recipients of a myeloablative TCD allo-HSCT for hematologic malignancies. We noted that different subsets recover sequentially, CD8 + T cells first, followed by total CD4 + and naïve CD4 + T cells, indicating thymic recovery during the first year after HSCT. In the multivariate model, a fully HLA-matched donor and recovery of T-cell function, assessed by PHA response at 6 months, were the only factors independently associated with OS and EFS. In conclusion, T-cell recovery is an important predictor of outcome after TCD allo-HSCT.

Comparison of outcomes of hematopoietic stem cell transplantation without chemotherapy conditioning by using matched sibling and unrelated donors for treatment of severe combined immunodeficiency.

PubMed

Dvorak, Christopher C; Hassan, Amel; Slatter, Mary A; Hönig, Manfred; Lankester, Arjan C; Buckley, Rebecca H; Pulsipher, Michael A; Davis, Jeffrey H; Güngör, Tayfun; Gabriel, Melissa; Bleesing, Jacob H; Bunin, Nancy; Sedlacek, Petr; Connelly, James A; Crawford, David F; Notarangelo, Luigi D; Pai, Sung-Yun; Hassid, Jake; Veys, Paul; Gennery, Andrew R; Cowan, Morton J

2014-10-01

Patients with severe combined immunodeficiency disease who have matched sibling donors (MSDs) can proceed to hematopoietic cell transplantation (HCT) without conditioning chemotherapy. We sought to determine whether the results of HCT without chemotherapy-based conditioning from matched unrelated donors (URDs), either from volunteer adults or umbilical cord blood, are comparable with those from MSDs. We performed a multicenter survey of severe combined immunodeficiency transplantation centers in North America, Europe, and Australia to compile retrospective data on patients who have undergone unconditioned HCT from either URDs (n = 37) or MSDs (n = 66). Most patients undergoing URD HCT (92%) achieved donor T-cell engraftment compared with 97% for those with MSDs; however, estimated 5-year overall and event-free survival were worse for URD recipients (71% and 60%, respectively) compared with MSD recipients (92% and 89%, respectively; P < .01 for both). URD recipients who received pre-HCT serotherapy had similar 5-year overall survival (100%) to MSD recipients. The incidences of grade II to IV acute and chronic graft-versus-host disease were higher in URD (50% and 39%, respectively) compared with MSD (22% and 5%, respectively) recipients (P < .01 for both). In the surviving patients there was no difference in T-cell reconstitution at the last follow-up between the URD and MSD recipients; however, MSD recipients were more likely to achieve B-cell reconstitution (72% vs 17%, P < .001). Unconditioned URD HCT achieves excellent rates of donor T-cell engraftment similar to that seen in MSD recipients, and reconstitution rates are adequate. However, only a minority will have myeloid and B-cell reconstitution, and attention must be paid to graft-versus-host disease prophylaxis. This approach might be safer in children ineligible for intense regimens to spare the potential complications of chemotherapy. Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells

PubMed Central

Hess, David A.; Wirthlin, Louisa; Craft, Timothy P.; Herrbrich, Phillip E.; Hohm, Sarah A.; Lahey, Ryan; Eades, William C.; Creer, Michael H.; Nolta, Jan A.

2006-01-01

The development of novel cell-based therapies requires understanding of distinct human hematopoietic stem and progenitor cell populations. We recently isolated reconstituting hematopoietic stem cells (HSCs) by lineage depletion and purification based on high aldehyde dehydrogenase activity (ALDHhiLin- cells). Here, we further dissected the ALDHhi-Lin- population by selection for CD133, a surface molecule expressed on progenitors from hematopoietic, endothelial, and neural lineages. ALDHhiCD133+Lin- cells were primarily CD34+, but also included CD34-CD38-CD133+ cells, a phenotype previously associated with repopulating function. Both ALDHhiCD133-Lin- and ALDHhiCD133+Lin- cells demonstrated distinct clonogenic progenitor function in vitro, whereas only the ALDHhiCD133+Lin- population seeded the murine bone marrow 48 hours after transplantation. Significant human cell repopulation was observed only in NOD/SCID and NOD/SCID β2M-null mice that received transplants of ALDHhiCD133+Lin- cells. Limiting dilution analysis demonstrated a 10-fold increase in the frequency of NOD/SCID repopulating cells compared with CD133+Lin- cells, suggesting that high ALDH activity further purified cells with repopulating function. Transplanted ALDHhiCD133+Lin- cells also maintained primitive hematopoietic phenotypes (CD34+CD38-) and demonstrated enhanced repopulating function in recipients of serial, secondary transplants. Cell selection based on ALDH activity and CD133 expression provides a novel purification of HSCs with long-term repopulating function and may be considered an alternative to CD34 cell selection for stem cell therapies. PMID:16269619

Development of 3D imaging technique of reconstructed human epidermis with immortalized human epidermal cell line.

PubMed

Inoue, Yu; Hasegawa, Seiji; Miyachi, Katsuma; Yamada, Takaaki; Nakata, Satoru; Ipponjima, Sari; Hibi, Terumasa; Nemoto, Tomomi; Tanaka, Masahiko; Suzuki, Ryo; Hirashima, Naohide

2018-05-01

The epidermis, the outermost layer of the skin, retains moisture and functions as a physical barrier against the external environment. Epidermal cells are continuously replaced by turnover, and thus to understand in detail the dynamic cellular events in the epidermis, techniques to observe live tissues in 3D are required. Here, we established a live 3D imaging technique for epidermis models. We first obtained immortalized human epidermal cell lines which have a normal differentiation capacity and fluorescence-labelled cytoplasm or nuclei. The reconstituted 3D epidermis was prepared with these lines. Using this culture system, we were able to observe the structure of the reconstituted epidermis live in 3D, which was similar to an in vivo epidermis, and evaluate the effect of a skin irritant. This technique may be useful for dermatological science and drug development. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Reconstitution of Intestinal CD4 and Th17 T Cells in Antiretroviral Therapy Suppressed HIV-Infected Subjects: Implication for Residual Immune Activation from the Results of a Clinical Trial

PubMed Central

d'Ettorre, Gabriella; Baroncelli, Silvia; Micci, Luca; Ceccarelli, Giancarlo; Andreotti, Mauro; Sharma, Prachi; Fanello, Gianfranco; Fiocca, Fausto; Cavallari, Eugenio Nelson; Giustini, Noemi; Mallano, Alessandra; Galluzzo, Clementina M.; Vella, Stefano; Mastroianni, Claudio M.; Silvestri, Guido; Paiardini, Mirko; Vullo, Vincenzo

2014-01-01

Introduction During HIV infection the severe depletion of intestinal CD4+ T-cells is associated with microbial translocation, systemic immune activation, and disease progression. This study examined intestinal and peripheral CD4+ T-cell subsets reconstitution under combined antiretroviral therapy (cART), and systemic immune activation markers. Methods This longitudinal single-arm pilot study evaluates CD4+ T cells, including Th1 and Th17, in gut and blood and soluble markers for inflammation in HIV-infected individuals before (M0) and after eight (M8) months of cART. From January 2010 to December 2011, 10 HIV-1 naïve patients were screened and 9 enrolled. Blood and gut CD4+ T-cells subsets and cellular immune activation were determined by flow-cytometry and plasma soluble CD14 by ELISA. CD4+ Th17 cells were detected in gut biopsies by immunohistochemistry. Microbial translocation was measured by limulus-amebocyte-lysate assay to detect bacterial lipopolysaccharide (LPS) and PCR Real Time to detect plasma bacterial 16S rDNA. Results Eight months of cART increased intestinal CD4+ and Th17 cells and reduced levels of T-cell activation and proliferation. The magnitude of intestinal CD4+ T-cell reconstitution correlated with the reduction of plasma LPS. Importantly, the magnitude of Th17 cells reconstitution correlated directly with blood CD4+ T-cell recovery. Conclusion Short-term antiretroviral therapy resulted in a significant increase in the levels of total and Th17 CD4+ T-cells in the gut mucosa and in decline of T-cell activation. The observation that pre-treatment levels of CD4+ and of CD8+ T-cell activation are predictors of the magnitude of Th17 cell reconstitution following cART provides further rationale for an early initiation of cART in HIV-infected individuals. Trial Registration ClinicalTrials.gov NCT02097381 PMID:25340778

In vitro studies of actin filament and network dynamics

PubMed Central

Mullins, R Dyche; Hansen, Scott D

2013-01-01

Now that many genomes have been sequenced, a central concern of cell biology is to understand how the proteins they encode work together to create living matter. In vitro studies form an essential part of this program because understanding cellular functions of biological molecules often requires isolating them and reconstituting their activities. In particular, many elements of the actin cytoskeleton were first discovered by biochemical methods and their cellular functions deduced from in vitro experiments. We highlight recent advances that have come from in vitro studies, beginning with studies of actin filaments, and ending with multi-component reconstitutions of complex actin-based processes, including force-generation and cell spreading. We describe both scientific results and the technical innovations that made them possible. PMID:23267766

Immunological Outcome in Haploidentical-HSC Transplanted Patients Treated with IL-10-Anergized Donor T Cells

PubMed Central

Bacchetta, Rosa; Lucarelli, Barbarella; Sartirana, Claudia; Gregori, Silvia; Lupo Stanghellini, Maria T.; Miqueu, Patrick; Tomiuk, Stefan; Hernandez-Fuentes, Maria; Gianolini, Monica E.; Greco, Raffaella; Bernardi, Massimo; Zappone, Elisabetta; Rossini, Silvano; Janssen, Uwe; Ambrosi, Alessandro; Salomoni, Monica; Peccatori, Jacopo; Ciceri, Fabio; Roncarolo, Maria-Grazia

2013-01-01

T-cell therapy after hematopoietic stem cell transplantation (HSCT) has been used alone or in combination with immunosuppression to cure hematologic malignancies and to prevent disease recurrence. Here, we describe the outcome of patients with high-risk/advanced stage hematologic malignancies, who received T-cell depleted (TCD) haploidentical-HSCT (haplo-HSCT) combined with donor T lymphocytes pretreated with IL-10 (ALT-TEN trial). IL-10-anergized donor T cells (IL-10-DLI) contained T regulatory type 1 (Tr1) cells specific for the host alloantigens, limiting donor-vs.-host-reactivity, and memory T cells able to respond to pathogens. IL-10-DLI were infused in 12 patients with the goal of improving immune reconstitution after haplo-HSCT without increasing the risk of graft-versus-host-disease (GvHD). IL-10-DLI led to fast immune reconstitution in five patients. In four out of the five patients, total T-cell counts, TCR-Vβ repertoire and T-cell functions progressively normalized after IL-10-DLI. These four patients are alive, in complete disease remission and immunosuppression-free at 7.2 years (median follow-up) after haplo-HSCT. Transient GvHD was observed in the immune reconstituted (IR) patients, despite persistent host-specific hypo-responsiveness of donor T cells in vitro and enrichment of cells with Tr1-specific biomarkers in vivo. Gene-expression profiles of IR patients showed a common signature of tolerance. This study provides the first indication of the feasibility of Tr1 cell-based therapy and paves way for the use of these Tr1 cells as adjuvant treatment for malignancies and immune-mediated disorders. PMID:24550909

Inorganic Kernel-Reconstituted Lipoprotein Biomimetic Nanovehicles Enable Efficient Targeting “Trojan Horse” Delivery of STAT3-Decoy Oligonucleotide for Overcoming TRAIL Resistance

PubMed Central

Shi, Kai; Xue, Jianxiu; Fang, Yan; Bi, Hongshu; Gao, Shan; Yang, Dongjuan; Lu, Anqi; Li, Yuai; Chen, Yao; Ke, Liyuan

2017-01-01

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis in a variety of tumor cells, but not most normal cells. Nevertheless, its therapeutic potential is limited due to the frequent occurrence of resistance in tumor cells, especially hepatocellular carcinoma cell lines. Therefore, we investigated the reversal effect of STAT3-decoy oligonucleotides (ODNs) on TRAIL resistance. Methods. Considering that the drawback of poor cellular permeability and rapid degradation in vivo limited ODNs' further clinical applications, we developed a biomimetic calcium phosphate-reconstituted low density lipoprotein nanovehicle (CaP@LDL) that would serve as a “Trojan horse” to carry STAT3-decoy ODNs into tumor cells and then regulate TRAIL-induced apoptosis. Results. In comparison with native ODNs, the reconstituted CaP@LDL packaged ODNs showed significantly increased serum stability, cellular transfection, in vitro synergistic cytotoxicity and apoptosis in hepatoma cells, while there was no cytotoxicity to normal cells. The improved TRAIL sensitization is attributed to blocking of STAT3 signaling and consequent expression of the downstream target antiapoptotic gene. Following systemic administration, CaP@LDL displayed LDL-mimicking pharmacokinetic behavior such as attenuated blood clearance as well as enhanced accumulation in tumor and hepatorenal sites. With the synergistic combination of decoyODN/CaP@LDL, TRAIL dramatically inhibited hepatic tumor growth in a xenograft model and induced significant tumor apoptosis in vivo. Conclusion. These results suggested that CaP@LDL-mediated STAT3-decoy ODN delivery might be a promising new strategy for reversing TRAIL resistance in hepatocellular carcinoma therapy. PMID:29158840

Inorganic Kernel-Reconstituted Lipoprotein Biomimetic Nanovehicles Enable Efficient Targeting "Trojan Horse" Delivery of STAT3-Decoy Oligonucleotide for Overcoming TRAIL Resistance.

PubMed

Shi, Kai; Xue, Jianxiu; Fang, Yan; Bi, Hongshu; Gao, Shan; Yang, Dongjuan; Lu, Anqi; Li, Yuai; Chen, Yao; Ke, Liyuan

2017-01-01

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis in a variety of tumor cells, but not most normal cells. Nevertheless, its therapeutic potential is limited due to the frequent occurrence of resistance in tumor cells, especially hepatocellular carcinoma cell lines. Therefore, we investigated the reversal effect of STAT3-decoy oligonucleotides (ODNs) on TRAIL resistance. Methods . Considering that the drawback of poor cellular permeability and rapid degradation in vivo limited ODNs' further clinical applications, we developed a biomimetic calcium phosphate-reconstituted low density lipoprotein nanovehicle (CaP@LDL) that would serve as a "Trojan horse" to carry STAT3-decoy ODNs into tumor cells and then regulate TRAIL-induced apoptosis. Results . In comparison with native ODNs, the reconstituted CaP@LDL packaged ODNs showed significantly increased serum stability, cellular transfection, in vitro synergistic cytotoxicity and apoptosis in hepatoma cells, while there was no cytotoxicity to normal cells. The improved TRAIL sensitization is attributed to blocking of STAT3 signaling and consequent expression of the downstream target antiapoptotic gene. Following systemic administration, CaP@LDL displayed LDL-mimicking pharmacokinetic behavior such as attenuated blood clearance as well as enhanced accumulation in tumor and hepatorenal sites. With the synergistic combination of decoyODN/CaP@LDL, TRAIL dramatically inhibited hepatic tumor growth in a xenograft model and induced significant tumor apoptosis in vivo . Conclusion. These results suggested that CaP@LDL-mediated STAT3-decoy ODN delivery might be a promising new strategy for reversing TRAIL resistance in hepatocellular carcinoma therapy.

Low-density lipoprotein reconstituted by pyropheophorbide cholesteryl oleate as target-specific photosensitizer.

PubMed

Zheng, Gang; Li, Hui; Zhang, Min; Lund-Katz, Sissel; Chance, Britton; Glickson, Jerry D

2002-01-01

To target tumors overexpressing low-density lipoprotein receptors (LDLr), a pyropheophorbide cholesterol oleate conjugate was synthesized and successfully reconstituted into the low-density lipoprotein (LDL) lipid core. Laser scanning confocal microscopy studies demonstrated that this photosensitizer-reconstituted LDL can be internalized via LDLr by human hepatoblastoma G(2) (HepG(2)) tumor cells.

Multipotent Adult Progenitor Cells Suppress T Cell Activation in In Vivo Models of Homeostatic Proliferation in a Prostaglandin E2-Dependent Manner

PubMed Central

Carty, Fiona; Corbett, Jennifer M.; Cunha, João Paulo M. C. M.; Reading, James L.; Tree, Timothy I. M.; Ting, Anthony E.; Stubblefield, Samantha R.; English, Karen

2018-01-01

Lymphodepletion strategies are used in the setting of transplantation (including bone marrow, hematopoietic cell, and solid organ) to create space or to prevent allograft rejection and graft versus host disease. Following lymphodepletion, there is an excess of IL-7 available, and T cells that escape depletion respond to this cytokine undergoing accelerated proliferation. Moreover, this environment promotes the skew of T cells to a Th1 pro-inflammatory phenotype. Existing immunosuppressive regimens fail to control this homeostatic proliferative (HP) response, and thus the development of strategies to successfully control HP while sparing T cell reconstitution (providing a functioning immune system) represents a significant unmet need in patients requiring lymphodepletion. Multipotent adult progenitor cells (MAPC®) have the capacity to control T cell proliferation and Th1 cytokine production. Herein, this study shows that MAPC cells suppressed anti-thymocyte globulin-induced cytokine production but spared T cell reconstitution in a pre-clinical model of lymphodepletion. Importantly, MAPC cells administered intraperitoneally were efficacious in suppressing interferon-γ production and in promoting the expansion of regulatory T cells in the lymph nodes. MAPC cells administered intraperitoneally accumulated in the omentum but were not present in the spleen suggesting a role for soluble factors. MAPC cells suppressed lymphopenia-induced cytokine production in a prostaglandin E2-dependent manner. This study suggests that MAPC cell therapy may be useful as a novel strategy to target lymphopenia-induced pathogenic T cell responses in lymphodepleted patients. PMID:29740426

Assembly Kinetics Determine the Architecture of α-actinin Crosslinked F-actin Networks

PubMed Central

Falzone, Tobias T.; Lenz, Martin; Kovar, David R.; Gardel, Margaret L.

2013-01-01

The actin cytoskeleton is organized into diverse meshworks and bundles that support many aspects of cell physiology. Understanding the self-assembly of these actin-based structures is essential for developing predictive models of cytoskeletal organization. Here we show that the competing kinetics of bundle formation with the onset of dynamic arrest arising from filament entanglements and cross-linking determine the architecture of reconstituted actin networks formed with α-actinin cross-links. Cross-link mediated bundle formation only occurs in dilute solutions of highly mobile actin filaments. As actin polymerization proceeds, filament mobility and bundle formation are arrested concomitantly. By controlling the onset of dynamic arrest, perturbations to actin assembly kinetics dramatically alter the architecture of biochemically identical samples. Thus, the morphology of reconstituted F-actin networks is a kinetically determined structure similar to those formed by physical gels and glasses. These results establish mechanisms controlling the structure and mechanics in diverse semi-flexible biopolymer networks. PMID:22643888

Elucidation of the biosynthesis of carnosic acid and its reconstitution in yeast

NASA Astrophysics Data System (ADS)

Scheler, Ulschan; Brandt, Wolfgang; Porzel, Andrea; Rothe, Kathleen; Manzano, David; Božić, Dragana; Papaefthimiou, Dimitra; Balcke, Gerd Ulrich; Henning, Anja; Lohse, Swanhild; Marillonnet, Sylvestre; Kanellis, Angelos K.; Ferrer, Albert; Tissier, Alain

2016-10-01

Rosemary extracts containing the phenolic diterpenes carnosic acid and its derivative carnosol are approved food additives used in an increasingly wide range of products to enhance shelf-life, thanks to their high anti-oxidant activity. We describe here the elucidation of the complete biosynthetic pathway of carnosic acid and its reconstitution in yeast cells. Cytochrome P450 oxygenases (CYP76AH22-24) from Rosmarinus officinalis and Salvia fruticosa already characterized as ferruginol synthases are also able to produce 11-hydroxyferruginol. Modelling-based mutagenesis of three amino acids in the related ferruginol synthase (CYP76AH1) from S. miltiorrhiza is sufficient to convert it to a 11-hydroxyferruginol synthase (HFS). The three sequential C20 oxidations for the conversion of 11-hydroxyferruginol to carnosic acid are catalysed by the related CYP76AK6-8. The availability of the genes for the biosynthesis of carnosic acid opens opportunities for the metabolic engineering of phenolic diterpenes, a class of compounds with potent anti-oxidant, anti-inflammatory and anti-tumour activities.

Keratinocytes propagated in serum-free, feeder-free culture conditions fail to form stratified epidermis in a reconstituted skin model.

PubMed

Lamb, Rebecca; Ambler, Carrie A

2013-01-01

Primary human epidermal stem cells isolated from skin tissues and subsequently expanded in tissue culture are used for human therapeutic use to reconstitute skin on patients and to generate artificial skin in culture for academic and commercial research. Classically, epidermal cells, known as keratinocytes, required fibroblast feeder support and serum-containing media for serial propagation. In alignment with global efforts to remove potential animal contaminants, many serum-free, feeder-free culture methods have been developed that support derivation and growth of these cells in 2-dimensional culture. Here we show that keratinocytes grown continually in serum-free and feeder-free conditions were unable to form into a stratified, mature epidermis in a skin equivalent model. This is not due to loss of cell potential as keratinocytes propagated in serum-free, feeder-free conditions retain their ability to form stratified epidermis when re-introduced to classic serum-containing media. Extracellular calcium supplementation failed to improve epidermis development. In contrast, the addition of serum to commercial, growth media developed for serum-free expansion of keratinocytes facilitated 3-dimensional stratification in our skin equivalent model. Moreover, the addition of heat-inactivated serum improved the epidermis structure and thickness, suggesting that serum contains factors that both aid and inhibit stratification.

Keratinocytes Propagated in Serum-Free, Feeder-Free Culture Conditions Fail to Form Stratified Epidermis in a Reconstituted Skin Model

PubMed Central

Lamb, Rebecca; Ambler, Carrie A.

2013-01-01

Primary human epidermal stem cells isolated from skin tissues and subsequently expanded in tissue culture are used for human therapeutic use to reconstitute skin on patients and to generate artificial skin in culture for academic and commercial research. Classically, epidermal cells, known as keratinocytes, required fibroblast feeder support and serum-containing media for serial propagation. In alignment with global efforts to remove potential animal contaminants, many serum-free, feeder-free culture methods have been developed that support derivation and growth of these cells in 2-dimensional culture. Here we show that keratinocytes grown continually in serum-free and feeder-free conditions were unable to form into a stratified, mature epidermis in a skin equivalent model. This is not due to loss of cell potential as keratinocytes propagated in serum-free, feeder-free conditions retain their ability to form stratified epidermis when re-introduced to classic serum-containing media. Extracellular calcium supplementation failed to improve epidermis development. In contrast, the addition of serum to commercial, growth media developed for serum-free expansion of keratinocytes facilitated 3-dimensional stratification in our skin equivalent model. Moreover, the addition of heat-inactivated serum improved the epidermis structure and thickness, suggesting that serum contains factors that both aid and inhibit stratification. PMID:23326335

Accelerating immune reconstitution after hematopoietic stem cell transplantation

PubMed Central

Tzannou, Ifigeneia; Leen, Ann M

2014-01-01

Viral infections remain a significant cause of morbidity and mortality after hematopoietic stem cell transplantation. Pharmacologic agents are effective against some pathogens, but they are costly and can be associated with significant toxicities. Thus, many groups have investigated adoptive T-cell transfer as a means of hastening immune reconstitution and preventing and treating viral infections. This review discusses the immunotherapeutic strategies that have been explored. PMID:25505959

Generation of 3D Skin Equivalents Fully Reconstituted from Human Induced Pluripotent Stem Cells (iPSCs)

PubMed Central

Guo, Zongyou; Liu, Liang; Higgins, Claire A.; Christiano, Angela M.

2013-01-01

Recent generation of patient-specific induced pluripotent stem cells (PS-iPSCs) provides significant advantages for cell- and gene-based therapy. Establishment of iPSC-based therapy for skin diseases requires efficient methodology for differentiating iPSCs into both keratinocytes and fibroblasts, the major cellular components of the skin, as well as the reconstruction of skin structures using these iPSC-derived skin components. We previously reported generation of keratinocytes from human iPSCs for use in the treatment of recessive dystrophic epidermolysis bullosa (RDEB) caused by mutations in the COL7A1 gene. Here, we developed a protocol for differentiating iPSCs into dermal fibroblasts, which also produce type VII collagen and therefore also have the potential to treat RDEB. Moreover, we generated in vitro 3D skin equivalents composed exclusively human iPSC-derived keratinocytes and fibroblasts for disease models and regenerative therapies for skin diseases, first demonstrating that iPSCs can provide the basis for modeling a human organ derived entirely from two different types of iPSC-derived cells. PMID:24147053

Electrochemically Controlled Reconstitution of Immobilized Ferritins for Bioelectronic Applications

NASA Technical Reports Server (NTRS)

Kim, Jae-Woo; Choi, Sang H.; Lillehei, Peter T.; Chu, Sang-Hong; King, Glen C.; Watt, Gerald D.

2007-01-01

Site-specific reconstituted nanoparticles were fabricated via electrochemically-controlled biomineralization through the immobilization of biomolecules. The work reported herein includes the immobilization of ferritin with various surface modifications, the electrochemical biomineralization of ferritins with different inorganic cores, and the electrocatalytic reduction of oxygen on the reconstituted Pt-cored ferritins. Protein immobilization on the substrate is achieved by anchoring ferritins with dithiobis-N-succinimidyl propionate (DTSP). A reconstitution process of site-specific electrochemical biomineralization with a protein cage loads ferritins with different core materials. The ferritin acts as a nano-scale template, a biocompatible cage, and a separator between the nanoparticles. This first demonstration of electrochemically controlled site-specific reconstitution of biomolecules provides a new tool for biomineralization and opens the way to produce the bio-templated nanoparticles by electrochemical control. The nanosized platinum-cored ferritins on gold displayed good catalytic activity for the electrochemical reduction of oxygen, which is applicable to biofuel cell applications. This results in a smaller catalyst loading on the electrodes for fuel cells or other bioelectronic devices.

Establishment of 3D Co-Culture Models from Different Stages of Human Tongue Tumorigenesis: Utility in Understanding Neoplastic Progression.

PubMed

Sawant, Sharada; Dongre, Harsh; Singh, Archana Kumari; Joshi, Shriya; Costea, Daniela Elena; Mahadik, Snehal; Ahire, Chetan; Makani, Vidhi; Dange, Prerana; Sharma, Shilpi; Chaukar, Devendra; Vaidya, Milind

2016-01-01

To study multistep tumorigenesis process, there is a need of in-vitro 3D model simulating in-vivo tissue. Present study aimed to reconstitute in-vitro tissue models comprising various stages of neoplastic progression of tongue tumorigenesis and to evaluate the utility of these models to investigate the role of stromal fibroblasts in maintenance of desmosomal anchoring junctions using transmission electron microscopy. We reconstituted in-vitro models representing normal, dysplastic, and malignant tissues by seeding primary keratinocytes on either fibroblast embedded in collagen matrix or plain collagen matrix in growth factor-free medium. The findings of histomorphometry, immunohistochemistry, and electron microscopy analyses of the three types of 3D cultures showed that the stratified growth, cell proliferation, and differentiation were comparable between co-cultures and their respective native tissues; however, they largely differed in cultures grown without fibroblasts. The immunostaining intensity of proteins, viz., desmoplakin, desmoglein, and plakoglobin, was reduced as the disease stage increased in all co-cultures as observed in respective native tissues. Desmosome-like structures were identified using immunogold labeling in these cultures. Moreover, electron microscopic observations revealed that the desmosome number and their length were significantly reduced and intercellular spaces were increased in cultures grown without fibroblasts when compared with their co-culture counterparts. Our results showed that the major steps of tongue tumorigenesis can be reproduced in-vitro. Stromal fibroblasts play a role in regulation of epithelial thickness, cell proliferation, differentiation, and maintenance of desmosomalanchoring junctions in in-vitro grown tissues. The reconstituted co-culture models could help to answer various biological questions especially related to tongue tumorigenesis.

Establishment of 3D Co-Culture Models from Different Stages of Human Tongue Tumorigenesis: Utility in Understanding Neoplastic Progression

PubMed Central

Sawant, Sharada; Dongre, Harsh; Singh, Archana Kumari; Joshi, Shriya; Costea, Daniela Elena; Mahadik, Snehal; Ahire, Chetan; Makani, Vidhi; Dange, Prerana; Sharma, Shilpi; Chaukar, Devendra; Vaidya, Milind

2016-01-01

To study multistep tumorigenesis process, there is a need of in-vitro 3D model simulating in-vivo tissue. Present study aimed to reconstitute in-vitro tissue models comprising various stages of neoplastic progression of tongue tumorigenesis and to evaluate the utility of these models to investigate the role of stromal fibroblasts in maintenance of desmosomal anchoring junctions using transmission electron microscopy. We reconstituted in-vitro models representing normal, dysplastic, and malignant tissues by seeding primary keratinocytes on either fibroblast embedded in collagen matrix or plain collagen matrix in growth factor-free medium. The findings of histomorphometry, immunohistochemistry, and electron microscopy analyses of the three types of 3D cultures showed that the stratified growth, cell proliferation, and differentiation were comparable between co-cultures and their respective native tissues; however, they largely differed in cultures grown without fibroblasts. The immunostaining intensity of proteins, viz., desmoplakin, desmoglein, and plakoglobin, was reduced as the disease stage increased in all co-cultures as observed in respective native tissues. Desmosome-like structures were identified using immunogold labeling in these cultures. Moreover, electron microscopic observations revealed that the desmosome number and their length were significantly reduced and intercellular spaces were increased in cultures grown without fibroblasts when compared with their co-culture counterparts. Our results showed that the major steps of tongue tumorigenesis can be reproduced in-vitro. Stromal fibroblasts play a role in regulation of epithelial thickness, cell proliferation, differentiation, and maintenance of desmosomalanchoring junctions in in-vitro grown tissues. The reconstituted co-culture models could help to answer various biological questions especially related to tongue tumorigenesis. PMID:27501241

Antifibrotic Therapy in Simian Immunodeficiency Virus Infection Preserves CD4+ T-Cell Populations and Improves Immune Reconstitution With Antiretroviral Therapy

PubMed Central

Estes, Jacob D.; Reilly, Cavan; Trubey, Charles M.; Fletcher, Courtney V.; Cory, Theodore J.; Piatak, Michael; Russ, Samuel; Anderson, Jodi; Reimann, Thomas G.; Star, Robert; Smith, Anthony; Tracy, Russell P.; Berglund, Anna; Schmidt, Thomas; Coalter, Vicky; Chertova, Elena; Smedley, Jeremy; Haase, Ashley T.; Lifson, Jeffrey D.; Schacker, Timothy W.

2015-01-01

Even with prolonged antiretroviral therapy (ART), many human immunodeficiency virus-infected individuals have <500 CD4+ T cells/µL, and CD4+ T cells in lymphoid tissues remain severely depleted, due in part to fibrosis of the paracortical T-cell zone (TZ) that impairs homeostatic mechanisms required for T-cell survival. We therefore used antifibrotic therapy in simian immunodeficiency virus-infected rhesus macaques to determine whether decreased TZ fibrosis would improve reconstitution of peripheral and lymphoid CD4+ T cells. Treatment with the antifibrotic drug pirfenidone preserved TZ architecture and was associated with significantly larger populations of CD4+ T cells in peripheral blood and lymphoid tissues. Combining pirfenidone with an ART regimen was associated with greater preservation of CD4+ T cells than ART alone and was also associated with higher pirfenidone concentrations. These data support a potential role for antifibrotic drug treatment as adjunctive therapy with ART to improve immune reconstitution. PMID:25246534

Prediction of equibiaxial loading stress in collagen-based extracellular matrix using a three-dimensional unit cell model.

PubMed

Susilo, Monica E; Bell, Brett J; Roeder, Blayne A; Voytik-Harbin, Sherry L; Kokini, Klod; Nauman, Eric A

2013-03-01

Mechanical signals are important factors in determining cell fate. Therefore, insights as to how mechanical signals are transferred between the cell and its surrounding three-dimensional collagen fibril network will provide a basis for designing the optimum extracellular matrix (ECM) microenvironment for tissue regeneration. Previously we described a cellular solid model to predict fibril microstructure-mechanical relationships of reconstituted collagen matrices due to unidirectional loads (Acta Biomater 2010;6:1471-86). The model consisted of representative volume elements made up of an interconnected network of flexible struts. The present study extends this work by adapting the model to account for microstructural anisotropy of the collagen fibrils and a biaxial loading environment. The model was calibrated based on uniaxial tensile data and used to predict the equibiaxial tensile stress-stretch relationship. Modifications to the model significantly improved its predictive capacity for equibiaxial loading data. With a comparable fibril length (model 5.9-8μm, measured 7.5μm) and appropriate fibril anisotropy the anisotropic model provides a better representation of the collagen fibril microstructure. Such models are important tools for tissue engineering because they facilitate prediction of microstructure-mechanical relationships for collagen matrices over a wide range of microstructures and provide a framework for predicting cell-ECM interactions. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A Retrospective Cohort Study of Lesion Distribution of HIV-1 Infection Patients With Cryptococcal Meningoencephalitis on MRI: Correlation With Immunity and Immune Reconstitution.

PubMed

Xia, Shuang; Li, Xueqin; Shi, Yanbin; Liu, Jinxin; Zhang, Mengjie; Gu, Tenghui; Pan, Shinong; Song, Liucun; Xu, Jinsheng; Sun, Yan; Zhao, Qingxia; Lu, Zhiyan; Lu, Puxuan; Li, Hongjun

2016-02-01

The objective of this paper is to correlate the MRI distribution of cryptococcal meningoencephalitis in HIV-1 infection patients with CD4 T cell count and immune reconstitution effect.A large retrospective cohort study of HIV patients from multi-HIV centers in China was studied to demonstrate the MRI distribution of cryptococcal meningoencephalitis and its correlation with the different immune status.The consecutive clinical and neuroimaging data of 55 HIV-1-infected patients with cryptococcal meningoencephalitis collected at multi-HIV centers in China during the years of 2011 to 2014 was retrospectively analyzed. The enrolled patients were divided into 2 groups based on the distribution of lesions. One group of patients had their lesions at the central brain (group 1, n = 34) and the other group of patients had their lesions at the superficial brain (group 2, n = 21). We explored their MRI characterization of brain. In addition, we also compared their CD4 T cell counts and immune reconstitution effects between the 2 groups based on the imaging findings.No statistical difference was found in terms of age and gender between the 2 groups. The medians of CD4 T cell counts were 11.67 cells/mm (3.00-52.00 cells/mm) in group 1 and 42.00 cells/mm (10.00-252.00 cells/mm) in group 2. Statistical difference of CD4 T cell count was found between the 2 groups (P = 0.023). Thirteen patients in group 1 (13/34) and 12 patients in group 2 (12/21) received highly active antiretroviral treatment (HAART). Patients of group 2 received HAART therapy more frequently than patients of group 1 (P = 0.021).Central and superficial brain lesions detected by MR imaging in HIV-1-infected patients with cryptococcal meningoencephalitis are in correlation with the host immunity and HAART therapy.

Reconstitution of SNARE proteins into solid-supported lipid bilayer stacks and X-ray structure analysis.

PubMed

Xu, Yihui; Kuhlmann, Jan; Brennich, Martha; Komorowski, Karlo; Jahn, Reinhard; Steinem, Claudia; Salditt, Tim

2018-02-01

SNAREs are known as an important family of proteins mediating vesicle fusion. For various biophysical studies, they have been reconstituted into supported single bilayers via proteoliposome adsorption and rupture. In this study we extended this method to the reconstitution of SNAREs into supported multilamellar lipid membranes, i.e. oriented multibilayer stacks, as an ideal model system for X-ray structure analysis (X-ray reflectivity and diffraction). The reconstitution was implemented through a pathway of proteomicelle, proteoliposome and multibilayer. To monitor the structural evolution in each step, we used small-angle X-ray scattering for the proteomicelles and proteoliposomes, followed by X-ray reflectivity and grazing-incidence small-angle scattering for the multibilayers. Results show that SNAREs can be successfully reconstituted into supported multibilayers, with high enough orientational alignment for the application of surface sensitive X-ray characterizations. Based on this protocol, we then investigated the effect of SNAREs on the structure and phase diagram of the lipid membranes. Beyond this application, this reconstitution protocol could also be useful for X-ray analysis of many further membrane proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Immunopathology of immune reconstitution inflammatory syndrome in Whipple's disease.

PubMed

Moos, Verena; Feurle, Gerhard E; Schinnerling, Katina; Geelhaar, Anika; Friebel, Julian; Allers, Kristina; Moter, Annette; Kikhney, Judith; Loddenkemper, Christoph; Kühl, Anja A; Erben, Ulrike; Fenollar, Florence; Raoult, Didier; Schneider, Thomas

2013-03-01

During antimicrobial treatment of classic Whipple's disease (CWD), the chronic systemic infection with Tropheryma whipplei, immune reconstitution inflammatory syndrome (IRIS), is a serious complication. The aim of our study was to characterize the immunological processes underlying IRIS in CWD. Following the definition of IRIS, we describe histological features of IRIS and immunological parameters of 24 CWD IRIS patients, 189 CWD patients without IRIS, and 89 healthy individuals. T cell reconstitution, Th1 reactivity, and the phenotype of T cells were described in the peripheral blood, and infiltration of CD4(+) T cells and regulatory T cells in the duodenal mucosa was determined. During IRIS, tissues were heavily infiltrated by CD3(+), predominantly CD45RO(+)CD4(+) T cells. In the periphery, initial reduction of CD4(+) cell counts and their reconstitution on treatment was more pronounced in CWD patients with IRIS than in those without IRIS. The ratio of activated and regulatory CD4(+) T cells, nonspecific Th1 reactivity, and the proportion of naive among CD4(+) T cells was high, whereas serum IL-10 was low during IRIS. T. whipplei-specific Th1 reactivity remained suppressed before and after emergence of IRIS. The findings that IRIS in CWD mainly are mediated by nonspecific activation of CD4(+) T cells and that it is not sufficiently counterbalanced by regulatory T cells indicate that flare-up of pathogen-specific immunoreactivity is not instrumental in the pathogenesis of IRIS in CWD.

Monitoring of Immune and Microbial Reconstitution in (HCT) and Novel Immunotherapies

ClinicalTrials.gov

2018-06-25

Immune and Microbial Reconstitution; Systemic Viral Infection; Acute-graft-versus-host Disease; Chronic Graft-versus-host-disease; Recurrent Malignancy; Cytokine Release Syndrome; Allogenic Related Donors; Cell Therapy/Immunotherapy Patients

Autologous transplantation of blood stem cells mobilized with filgrastim alone in 93 patients with malignancies: the number of CD34+ cells reinfused is the only factor predicting both granulocyte and platelet recovery.

PubMed

Faucher, C; Le Corroller, A G; Chabannon, C; Viens, P; Stoppa, A M; Bouabdallah, R; Camerlo, J; Vey, N; Gravis, G; Gastaut, J A; Novakovitch, G; Mannoni, P; Bardou, V J; Moatti, J P; Maraninchi, D; Blaise, D

1996-12-01

High-dose chemotherapy (HDC) supported by autologous transplantation of blood stem cells (BSC) is used increasingly for patients with poor-risk malignancies. We report our experience with 93 consecutive patients who were mobilized with recombinant human granulocyte colony-stimulating factor (rhG-CSF) alone. They received a fixed dose of G-CSF for 5 or 6 days, and BSC were collected by leukapheresis. Aphereses were evaluated for MNC, CD34+ cells, and CFU-GM counts and cryopreserved. All patients received a conditioning regimen without TBI. Engraftment was assessed as the first of 2 consecutive days on which patients achieved 0.5 and 1 x 10(9)/L neutrophils and an unsupported platelet count of 25 x 10(9)/L. Multivariate analysis was performed to study patients and graft characteristics that could influence reconstitution. The G-CSF priming regimen was well tolerated and allowed collection of BSC for all patients, 66% of them achieving >3 x 10(6)/kg CD34+ cells, and 86% achieving >10 x 10(4) CFU-GM/kg. The numbers of collected CD34 and CFU-GM cells were highly correlated. The number of courses of chemotherapy prior to collection, a diagnosis of breast cancer, the use of rhG-CSF posttransplant, and the numbers of CFU-GM and CD34+ cells reinfused were correlated with hematologic recovery. In a multivariate analysis, however, the number of CD34+ cells was the only factor independently influencing both granulocyte and platelet recovery. Patients who received at least 3 x 10(6)/kg CD34+ cells achieved granulocyte reconstitution on day 11 after reinfusion (range 8-15) and an unsupported platelet count of 25 x 10(9)/l on day 14 (range 12-180), significantly earlier than patients who received fewer cells (p < 0.001). In addition, G-CSF administration postreinfusion independently enhanced granulocyte reconstitution but not platelet recovery. In conclusion, CD34+ cell number appears to be the only factor predicting both granulocyte and platelet reconstitution. Based on this study, the collection of a minimal number of 3 x 10(6)/kg CD34+ cells appears desirable.

Reverse and forward engineering of protein pattern formation.

PubMed

Kretschmer, Simon; Harrington, Leon; Schwille, Petra

2018-05-26

Living systems employ protein pattern formation to regulate important life processes in space and time. Although pattern-forming protein networks have been identified in various prokaryotes and eukaryotes, their systematic experimental characterization is challenging owing to the complex environment of living cells. In turn, cell-free systems are ideally suited for this goal, as they offer defined molecular environments that can be precisely controlled and manipulated. Towards revealing the molecular basis of protein pattern formation, we outline two complementary approaches: the biochemical reverse engineering of reconstituted networks and the de novo design, or forward engineering, of artificial self-organizing systems. We first illustrate the reverse engineering approach by the example of the Escherichia coli Min system, a model system for protein self-organization based on the reversible and energy-dependent interaction of the ATPase MinD and its activating protein MinE with a lipid membrane. By reconstituting MinE mutants impaired in ATPase stimulation, we demonstrate how large-scale Min protein patterns are modulated by MinE activity and concentration. We then provide a perspective on the de novo design of self-organizing protein networks. Tightly integrated reverse and forward engineering approaches will be key to understanding and engineering the intriguing phenomenon of protein pattern formation.This article is part of the theme issue 'Self-organization in cell biology'. © 2018 The Author(s).

Development of an animal model of progressive vaccinia in nu/nu mice and the use of bioluminescence imaging for assessment of the efficacy of monoclonal antibodies against vaccinial B5 and L1 proteins.

PubMed

Zaitseva, Marina; Thomas, Antonia; Meseda, Clement A; Cheung, Charles Y K; Diaz, Claudia G; Xiang, Yan; Crotty, Shane; Golding, Hana

2017-08-01

Bioluminescence imaging (BLI) was used to follow dissemination of recombinant vaccinia virus (VACV) expressing luciferase (IHD-J-Luc) in BALB/c nu/nu mice treated post-challenge with monoclonal antibodies (MAbs) against L1 and B5 VACV proteins in a model of Progressive Vaccinia (PV). Areas Under the flux Curve (AUC) were calculated for viral loads in multiple organs in individual mice. Following scarification with 10 5  pfu, IHD-J-Luc VACV undergoes fast replication at the injection site and disseminates rapidly to the inguinal lymph nodes followed by spleen, liver, and axillary lymph nodes within 2-3 days and before primary lesions are visible at the site of scarification. Extension of survival in nude mice treated with a combination of anti-B5 and anti-L1 MAbs 24 h post challenge correlated with a significant reduction in viral load at the site of scarification and delayed systemic dissemination. Nude mice reconstituted with 10 4  T cells prior to challenge with IHD-J-Luc, and treated with MAbs post-challenge, survived infection, cleared the virus from all organs and scarification site, and developed anti-VACV IgG and VACV-specific polyfunctional CD8 + T cells that co-expressed the degranulation marker CD107a, and IFNγ and TNFα cytokines. All T cell reconstituted mice survived intranasal re-challenge with IHD-J-Luc (10 4  pfu) two months after the primary infection. Thus, using BLI to monitor VACV replication in a PV model, we showed that anti-VACV MAbs administered post challenge extended survival of nude mice and protected T cell reconstituted nude mice from lethality by reducing replication at the site of scarification and systemic dissemination of VACV. Published by Elsevier B.V.

Avian germplasm preservation: embryonic stem cells or primordial germ cells?

PubMed

Petitte, J N

2006-02-01

Presently, avian genetic resources are best maintained as living collections of birds. Unfortunately, these stocks have been under constant pressure to be destroyed because of the decline in the number of Poultry Science Departments and pressures to cut costs at land grant institutions. Cryopreservation of semen is often suggested as a means to bank avian germplasm. However, this is only applicable for single-gene traits and does not allow for full reconstitution of the genetics of the original line. Over the last 15 yr, advances in the manipulation of the early chick embryo, manipulation of primordial germ cells (PGC), and the culture of embryonic stem cells (ESC) suggests that cryopreservation of blastodermal cells, ESC, or PGC might offer a means to preserve the entire genome of highly selected, specialized stocks of poultry. Freezing each of these cell types is possible with varying degrees of efficiency. Similarly, the effectiveness of generating germ line chimeras using blastodermal cells, ESC, or PGC also varies greatly. Other factors that must be considered include the choice of the recipient lines to develop the germ line chimeras and the number of individuals needed to reconstitute the line. Finally, the low efficiency rate of reconstitution and the high cost associated with current technologies makes these approaches prohibitive. Significant challenges remain to be overcome before the entire genome of poultry stocks can be routinely cryoperserved and reconstituted.

Understanding force-generating microtubule systems through in vitro reconstitution

PubMed Central

Kok, Maurits; Dogterom, Marileen

2016-01-01

ABSTRACT Microtubules switch between growing and shrinking states, a feature known as dynamic instability. The biochemical parameters underlying dynamic instability are modulated by a wide variety of microtubule-associated proteins that enable the strict control of microtubule dynamics in cells. The forces generated by controlled growth and shrinkage of microtubules drive a large range of processes, including organelle positioning, mitotic spindle assembly, and chromosome segregation. In the past decade, our understanding of microtubule dynamics and microtubule force generation has progressed significantly. Here, we review the microtubule-intrinsic process of dynamic instability, the effect of external factors on this process, and how the resulting forces act on various biological systems. Recently, reconstitution-based approaches have strongly benefited from extensive biochemical and biophysical characterization of individual components that are involved in regulating or transmitting microtubule-driven forces. We will focus on the current state of reconstituting increasingly complex biological systems and provide new directions for future developments. PMID:27715396

Reconstitution of a Patterned Neural Tube from Single Mouse Embryonic Stem Cells.

PubMed

Ishihara, Keisuke; Ranga, Adrian; Lutolf, Matthias P; Tanaka, Elly M; Meinhardt, Andrea

2017-01-01

The recapitulation of tissue development and patterning in three-dimensional (3D) culture is an important dimension of stem cell research. Here, we describe a 3D culture protocol in which single mouse ES cells embedded in Matrigel under neural induction conditions clonally form a lumen containing, oval-shaped epithelial structure within 3 days. By Day 7 an apicobasally polarized neuroepithelium with uniformly dorsal cell identity forms. Treatment with retinoic acid at Day 2 results in posteriorization and self-organization of dorsal-ventral neural tube patterning. Neural tube organoid growth is also supported by pure laminin gels as well as poly(ethylene glycol) (PEG)-based artificial extracellular matrix hydrogels, which can be fine-tuned for key microenvironment characteristics. The rapid generation of a simple, patterned tissue in well-defined culture conditions makes the neural tube organoid a tractable model for studying neural stem cell self-organization.

The cooked meat-derived mammary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine promotes invasive behaviour of breast cancer cells.

PubMed

Lauber, Sandra N; Gooderham, Nigel J

2011-01-11

The cooked meat derived genotoxic carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces cancer of the colon, prostate and mammary gland when fed to rats. Epidemiology studies link these tumours to a Western diet and exposure to heterocyclic amines such as PhIP. We have shown that PhIP is also potently estrogenic and have proposed that this hormonal activity contributes to its target site carcinogenicity. We now postulate that the estrogenic properties of PhIP influence metastatic potential. We have used an in vitro assay for cell invasion based upon digestion and migration through a reconstituted basement membrane model. Zymography and immunoblotting were used to confirm PhIP-mediated changes associated with induction of the invasive phenotype. Treatment of the mammary cancer cell lines MCF-7 and T47D with PhIP induces cells to digest and migrate through a reconstituted basement membrane. The response was dose dependent, observed at sub-nanomolar concentrations of PhIP and was inhibited by the antiestrogen ICI 182,780. The PhIP-induced invasive phenotype was associated with expression of cathepsin D, cyclooxygenase-2 and matrix metalloproteinase activity. These findings emphasise the range and potency of the biological activities associated with this cooked meat product and mechanistically support the tissue-specific carcinogenicity of the chemical. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Reconstitution of hepatic tissue architectures from fetal liver cells obtained from a three-dimensional culture with a rotating wall vessel bioreactor.

PubMed

Ishikawa, Momotaro; Sekine, Keisuke; Okamura, Ai; Zheng, Yun-wen; Ueno, Yasuharu; Koike, Naoto; Tanaka, Junzo; Taniguchi, Hideki

2011-06-01

Reconstitution of tissue architecture in vitro is important because it enables researchers to investigate the interactions and mutual relationships between cells and cellular signals involved in the three-dimensional (3D) construction of tissues. To date, in vitro methods for producing tissues with highly ordered structure and high levels of function have met with limited success although a variety of 3D culture systems have been investigated. In this study, we reconstituted functional hepatic tissue including mature hepatocyte and blood vessel-like structures accompanied with bile duct-like structures from E15.5 fetal liver cells, which contained more hepatic stem/progenitor cells comparing with neonatal liver cells. The culture was performed in a simulated microgravity environment produced by a rotating wall vessel (RWV) bioreactor. The hepatocytes in the reconstituted 3D tissue were found to be capable of producing albumin and storing glycogen. Additionally, bile canaliculi between hepatocytes, characteristics of adult hepatocyte in vivo were also formed. Apart from this, bile duct structure secreting mucin was shown to form complicated tubular branches. Furthermore, gene expression analysis by semi-quantitative RT-PCR revealed the elevated levels of mature hepatocyte markers as well as genes with the hepatic function. With RWV culture system, we could produce functionally reconstituted liver tissue and this might be useful in pharmaceutical industry including drug screening and testing and other applications such as an alternative approach to experimental animals. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Reconstitution of actin-based motility of Listeria and Shigella using pure proteins

NASA Astrophysics Data System (ADS)

Loisel, Thomas P.; Boujemaa, Rajaa; Pantaloni, Dominique; Carlier, Marie-France

1999-10-01

Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, α-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.

Age and CD4 count at initiation of antiretroviral therapy in HIV-infected children: effects on long-term T-cell reconstitution.

PubMed

Lewis, Joanna; Walker, A Sarah; Castro, Hannah; De Rossi, Anita; Gibb, Diana M; Giaquinto, Carlo; Klein, Nigel; Callard, Robin

2012-02-15

Effective therapies and reduced AIDS-related morbidity and mortality have shifted the focus in pediatric human immunodeficiency virus (HIV) from minimizing short-term disease progression to maintaining optimal long-term health. We describe the effects of children's age and pre-antiretroviral therapy (ART) CD4 count on long-term CD4 T-cell reconstitution. CD4 counts in perinatally HIV-infected, therapy-naive children in the Paediatric European Network for the Treatment of AIDS 5 trial were monitored following initiation of ART for a median 5.7 years. In a substudy, naive and memory CD4 counts were recorded. Age-standardized measurements were analyzed using monophasic, asymptotic nonlinear mixed-effects models. One hundred twenty-seven children were studied. Older children had lower age-adjusted CD4 counts in the long term and at treatment initiation (P < .001). At all ages, lower counts before treatment were associated with impaired recovery (P < .001). Age-adjusted naive CD4 counts increased on a timescale comparable to overall CD4 T-cell reconstitution, whereas age-adjusted memory CD4 counts increased less, albeit on a faster timescale. It appears the immature immune system can recover well from HIV infection via the naive pool. However, this potential is progressively damaged with age and/or duration of infection. Current guidelines may therefore not optimize long-term immunological health.

CCL3L1-CCR5 genotype influences durability of immune recovery during antiretroviral therapy of HIV-1–infected individuals

PubMed Central

Ahuja, Sunil K; Kulkarni, Hemant; Catano, Gabriel; Agan, Brian K; Camargo, Jose F; He, Weijing; O'Connell, Robert J; Marconi, Vincent C; Delmar, Judith; Eron, Joseph; Clark, Robert A; Frost, Simon; Martin, Jeffrey; Ahuja, Seema S; Deeks, Steven G; Little, Susan; Richman, Douglas; Hecht, Frederick M; Dolan, Matthew J

2008-01-01

The basis for the extensive variability seen in the reconstitution of CD4+ T cell counts in HIV-infected individuals receiving highly active antiretroviral therapy (HAART) is not fully known. Here, we show that variations in CCL3L1 gene dose and CCR5 genotype, but not major histocompatibility complex HLA alleles, influence immune reconstitution, especially when HAART is initiated at <350 CD4+ T cells/mm3. The CCL3L1-CCR5 genotypes favoring CD4+ T cell recovery are similar to those that blunted CD4+ T cell depletion during the time before HAART became available (pre-HAART era), suggesting that a common CCL3L1-CCR5 genetic pathway regulates the balance between pathogenic and reparative processes from early in the disease course. Hence, CCL3L1-CCR5 variations influence HIV pathogenesis even in the presence of HAART and, therefore, may prospectively identify subjects in whom earlier initiation of therapy is more likely to mitigate immunologic failure despite viral suppression by HAART. Furthermore, as reconstitution of CD4+ cells during HAART is more sensitive to CCL3L1 dose than to CCR5 genotypes, CCL3L1 analogs might be efficacious in supporting immunological reconstitution. PMID:18376407

CD62L− memory T cells enhance T-cell regeneration after allogeneic stem cell transplantation by eliminating host resistance in mice

PubMed Central

Zhang, Jifeng; Barefoot, Brice E.; Mo, Wenjian; Deoliveira, Divino; Son, Jessica; Cui, Xiuyu; Ramsburg, Elizabeth

2012-01-01

A major challenge in allogeneic hematopoietic cell transplantation is how to transfer T-cell immunity without causing graft-versus-host disease (GVHD). Effector memory T cells (CD62L−) are a cell subset that can potentially address this challenge because they do not induce GVHD. Here, we investigated how CD62L− T cells contributed to phenotypic and functional T-cell reconstitution after transplantation. On transfer into allogeneic recipients, CD62L− T cells were activated and expressed multiple cytokines and cytotoxic molecules. CD62L− T cells were able to deplete host radioresistant T cells and facilitate hematopoietic engraftment, resulting in enhanced de novo T-cell regeneration. Enhanced functional immune reconstitution was demonstrated in CD62L− T-cell recipients using a tumor and an influenza virus challenge model. Even though CD62L− T cells are able to respond to alloantigens and deplete host radioresistant immune cells in GVHD recipients, alloreactive CD62L− T cells lost the reactivity over time and were eventually tolerant to alloantigens as a result of prolonged antigen exposure, suggesting a mechanism by which CD62L− T cells were able to eliminate host resistance without causing GVHD. These data further highlight the unique characteristics of CD62L− T cells and their potential applications in clinical hematopoietic cell transplantation. PMID:22596261

Infiltration of CD4+ lymphocytes into the brain contributes to neurodegeneration in a mouse model of Parkinson disease

PubMed Central

Brochard, Vanessa; Combadière, Béhazine; Prigent, Annick; Laouar, Yasmina; Perrin, Aline; Beray-Berthat, Virginie; Bonduelle, Olivia; Alvarez-Fischer, Daniel; Callebert, Jacques; Launay, Jean-Marie; Duyckaerts, Charles; Flavell, Richard A.; Hirsch, Etienne C.; Hunot, Stéphane

2008-01-01

Parkinson disease (PD) is a neurodegenerative disorder characterized by a loss of dopamine-containing neurons. Mounting evidence suggests that dopaminergic cell death is influenced by the innate immune system. However, the pathogenic role of the adaptive immune system in PD remains enigmatic. Here we showed that CD8+ and CD4+ T cells but not B cells had invaded the brain in both postmortem human PD specimens and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD during the course of neuronal degeneration. We further demonstrated that MPTP-induced dopaminergic cell death was markedly attenuated in the absence of mature T lymphocytes in 2 different immunodeficient mouse strains (Rag1–/– and Tcrb–/– mice). Importantly, similar attenuation of MPTP-induced dopaminergic cell death was seen in mice lacking CD4 as well as in Rag1–/– mice reconstituted with FasL-deficient splenocytes. However, mice lacking CD8 and Rag1–/– mice reconstituted with IFN-γ–deficient splenocytes were not protected. These data indicate that T cell–mediated dopaminergic toxicity is almost exclusively arbitrated by CD4+ T cells and requires the expression of FasL but not IFNγ. Further, our data may provide a rationale for targeting the adaptive arm of the immune system as a therapeutic strategy in PD. PMID:19104149

Reconstitution radicicol containing apolipoprotein B lipoparticle and tracing its cell uptake process by super resolution fluorescent microscopy.

NASA Astrophysics Data System (ADS)

Lin, Chung Ching; Lin, Po-Yen; Chang, Chia-Ching

Apolipoprotein B (apoB) is the only protein of LDL. LDL delivers cholesterol, triacylglycerides and lipids to the target cells. Reconstitute apoB lipoparticle (rABL) will be an idea drug delivery vehicle for hydrophobic and amphiphilic materials delivery. It is challenged to renature ApoB into its functional state from denatured state. By using modified bile salt and radicicol (Rad) added over-critical refolding process, apoB can be restored into its native like state. The intrinsic fluorescence of apoB increased during the refolding process. Moreover, radicicol (Rad) molecules have been encapsulated into reconstitute rABL (Rad@rABL). To investigate the cell uptake mechanism of Rad@rABL, a super resolution ground state depletion (GSD) microscopy is used in this research. Fluorescence labeled Rad@rABL can be traced within the tumor cell. Key words: LDL, radicicol, protein refolding, super resolution microscopy.

Engraftment and reconstitution of hematopoiesis is dependent on VEGFR2 mediated regeneration of sinusoidal endothelial cells

PubMed Central

Hooper, Andrea T.; Butler, Jason M.; Nolan, Daniel J; Kranz, Andrea; Iida, Kaoruko; Kobayashi, Mariko; Kopp, Hans-Georg; Shido, Koji; Petit, Isabelle; Yanger, Kilangsungla; James, Daylon; Witte, Larry; Zhu, Zhenping; Wu, Yan; Pytowski, Bronislaw; Rosenwaks, Zev; Mittal, Vivek; Sato, Thomas N.; Rafii, Shahin

2011-01-01

SUMMARY The phenotypic attributes and molecular determinants for the regeneration of bone marrow (BM) sinusoidal endothelial cells (SECs) and their contribution to hematopoiesis are unknown. We show that after myelosuppression VEGFR2 activation promotes reassembly of regressed SECs, reconstituting hematopoietic stem and progenitor cells (HSPCs). VEGFR2 and VEGFR3 expression are restricted to BM vasculature, demarcating a continuous network of VEGFR2+VEGFR3+Sca1− SECs and VEGFR2+VEGFR3−Sca1+ arterioles. While chemotherapy (5FU) and sublethal irradiation (650 rad) induce minor SEC regression, lethal irradiation (950 rad) induces severe regression of SECs requiring BM transplantation (BMT) for regeneration. Conditional deletion of VEGFR2 in adult mice blocks regeneration of SECs in sublethally irradiated animals, preventing hematopoietic reconstitution. Inhibition of VEGFR2 signaling in lethally irradiated wild type mice rescued with BMT severely impairs SEC reconstruction, preventing engraftment and reconstitution of HSPCs. Therefore, activation of VEGFR2 is critical for regeneration of VEGFR3+Sca1− SECs that are essential for engraftment and restoration of HSPCs and hematopoiesis. PMID:19265665

Analyzing structure-function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells.

PubMed

Rank, Lisa; Veith, Sebastian; Gwosch, Eva C; Demgenski, Janine; Ganz, Magdalena; Jongmans, Marjolijn C; Vogel, Christopher; Fischbach, Arthur; Buerger, Stefanie; Fischer, Jan M F; Zubel, Tabea; Stier, Anna; Renner, Christina; Schmalz, Michael; Beneke, Sascha; Groettrup, Marcus; Kuiper, Roland P; Bürkle, Alexander; Ferrando-May, Elisa; Mangerich, Aswin

2016-12-01

Genotoxic stress activates PARP1, resulting in the post-translational modification of proteins with poly(ADP-ribose) (PAR). We genetically deleted PARP1 in one of the most widely used human cell systems, i.e. HeLa cells, via TALEN-mediated gene targeting. After comprehensive characterization of these cells during genotoxic stress, we analyzed structure-function relationships of PARP1 by reconstituting PARP1 KO cells with a series of PARP1 variants. Firstly, we verified that the PARP1\\E988K mutant exhibits mono-ADP-ribosylation activity and we demonstrate that the PARP1\\L713F mutant is constitutively active in cells. Secondly, both mutants exhibit distinct recruitment kinetics to sites of laser-induced DNA damage, which can potentially be attributed to non-covalent PARP1-PAR interaction via several PAR binding motifs. Thirdly, both mutants had distinct functional consequences in cellular patho-physiology, i.e. PARP1\\L713F expression triggered apoptosis, whereas PARP1\\E988K reconstitution caused a DNA-damage-induced G2 arrest. Importantly, both effects could be rescued by PARP inhibitor treatment, indicating distinct cellular consequences of constitutive PARylation and mono(ADP-ribosyl)ation. Finally, we demonstrate that the cancer-associated PARP1 SNP variant (V762A) as well as a newly identified inherited PARP1 mutation (F304L\\V762A) present in a patient with pediatric colorectal carcinoma exhibit altered biochemical and cellular properties, thereby potentially supporting human carcinogenesis. Together, we establish a novel cellular model for PARylation research, by revealing strong structure-function relationships of natural and artificial PARP1 variants. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Reconstruction of Multiple Facial Nerve Branches Using Skeletal Muscle-Derived Multipotent Stem Cell Sheet-Pellet Transplantation.

PubMed

Saito, Kosuke; Tamaki, Tetsuro; Hirata, Maki; Hashimoto, Hiroyuki; Nakazato, Kenei; Nakajima, Nobuyuki; Kazuno, Akihito; Sakai, Akihiro; Iida, Masahiro; Okami, Kenji

2015-01-01

Head and neck cancer is often diagnosed at advanced stages, and surgical resection with wide margins is generally indicated, despite this treatment being associated with poor postoperative quality of life (QOL). We have previously reported on the therapeutic effects of skeletal muscle-derived multipotent stem cells (Sk-MSCs), which exert reconstitution capacity for muscle-nerve-blood vessel units. Recently, we further developed a 3D patch-transplantation system using Sk-MSC sheet-pellets. The aim of this study is the application of the 3D Sk-MSC transplantation system to the reconstitution of facial complex nerve-vascular networks after severe damage. Mouse experiments were performed for histological analysis and rats were used for functional examinations. The Sk-MSC sheet-pellets were prepared from GFP-Tg mice and SD rats, and were transplanted into the facial resection model (ST). Culture medium was transplanted as a control (NT). In the mouse experiment, facial-nerve-palsy (FNP) scoring was performed weekly during the recovery period, and immunohistochemistry was used for the evaluation of histological recovery after 8 weeks. In rats, contractility of facial muscles was measured via electrical stimulation of facial nerves root, as the marker of total functional recovery at 8 weeks after transplantation. The ST-group showed significantly higher FNP (about three fold) scores when compared to the NT-group after 2-8 weeks. Similarly, significant functional recovery of whisker movement muscles was confirmed in the ST-group at 8 weeks after transplantation. In addition, engrafted GFP+ cells formed complex branches of nerve-vascular networks, with differentiation into Schwann cells and perineurial/endoneurial cells, as well as vascular endothelial and smooth muscle cells. Thus, Sk-MSC sheet-pellet transplantation is potentially useful for functional reconstitution therapy of large defects in facial nerve-vascular networks.

Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

PubMed Central

Rank, Lisa; Veith, Sebastian; Gwosch, Eva C.; Demgenski, Janine; Ganz, Magdalena; Jongmans, Marjolijn C.; Vogel, Christopher; Fischbach, Arthur; Buerger, Stefanie; Fischer, Jan M.F.; Zubel, Tabea; Stier, Anna; Renner, Christina; Schmalz, Michael; Beneke, Sascha; Groettrup, Marcus; Kuiper, Roland P.; Bürkle, Alexander; Ferrando-May, Elisa; Mangerich, Aswin

2016-01-01

Genotoxic stress activates PARP1, resulting in the post-translational modification of proteins with poly(ADP-ribose) (PAR). We genetically deleted PARP1 in one of the most widely used human cell systems, i.e. HeLa cells, via TALEN-mediated gene targeting. After comprehensive characterization of these cells during genotoxic stress, we analyzed structure–function relationships of PARP1 by reconstituting PARP1 KO cells with a series of PARP1 variants. Firstly, we verified that the PARP1\\E988K mutant exhibits mono-ADP-ribosylation activity and we demonstrate that the PARP1\\L713F mutant is constitutively active in cells. Secondly, both mutants exhibit distinct recruitment kinetics to sites of laser-induced DNA damage, which can potentially be attributed to non-covalent PARP1–PAR interaction via several PAR binding motifs. Thirdly, both mutants had distinct functional consequences in cellular patho-physiology, i.e. PARP1\\L713F expression triggered apoptosis, whereas PARP1\\E988K reconstitution caused a DNA-damage-induced G2 arrest. Importantly, both effects could be rescued by PARP inhibitor treatment, indicating distinct cellular consequences of constitutive PARylation and mono(ADP-ribosyl)ation. Finally, we demonstrate that the cancer-associated PARP1 SNP variant (V762A) as well as a newly identified inherited PARP1 mutation (F304L\\V762A) present in a patient with pediatric colorectal carcinoma exhibit altered biochemical and cellular properties, thereby potentially supporting human carcinogenesis. Together, we establish a novel cellular model for PARylation research, by revealing strong structure–function relationships of natural and artificial PARP1 variants. PMID:27694308

GVHD prevents NK-cell-dependent leukemia and virus-specific innate immunity.

PubMed

Bunting, Mark D; Varelias, Antiopi; Souza-Fonseca-Guimaraes, Fernando; Schuster, Iona S; Lineburg, Katie E; Kuns, Rachel D; Fleming, Peter; Locke, Kelly R; Huntington, Nicholas D; Blazar, Bruce R; Lane, Steven W; Tey, Siok-Keen; MacDonald, Kelli P A; Smyth, Mark J; Degli-Esposti, Mariapia A; Hill, Geoffrey R

2017-02-02

Allogeneic bone marrow transplantation (allo-BMT) is a curative therapy for hematological malignancies, but is associated with significant complications, principally graft-versus-host disease (GVHD) and opportunistic infections. Natural killer (NK) cells mediate important innate immunity that provides a temporal bridge until the reconstruction of adaptive immunity. Here, we show that the development of GVHD after allo-BMT prevented NK-cell reconstitution, particularly within the maturing M1 and M2 NK-cell subsets in association with exaggerated activation, apoptosis, and autophagy. Donor T cells were critical in this process by limiting the availability of interleukin 15 (IL-15), and administration of IL-15/IL-15Rα or immune suppression with rapamycin could restore NK-cell reconstitution. Importantly, the NK-cell defect induced by GVHD resulted in the failure of NK-cell-dependent in vivo cytotoxicity and graft-versus-leukemia effects. Control of cytomegalovirus infection after allo-BMT was also impaired during GVHD. Thus, during GVHD, donor T cells compete with NK cells for IL-15 thereby inducing profound defects in NK-cell reconstitution that compromise both leukemia and pathogen-specific immunity. © 2017 by The American Society of Hematology.

CD56-enriched donor cell infusion after post-transplantation cyclophosphamide for haploidentical transplantation of advanced myeloid malignancies is associated with prompt reconstitution of mature natural killer cells and regulatory T cells with reduced incidence of acute graft versus host disease: A pilot study.

PubMed

Jaiswal, Sarita Rani; Zaman, Shamsur; Nedunchezhian, Murugaiyan; Chakrabarti, Aditi; Bhakuni, Prakash; Ahmed, Margoob; Sharma, Kanika; Rawat, Sheh; O'donnell, Paul; Chakrabarti, Suparno

2017-04-01

We conducted a pilot study on the feasibility of CD56-enriched donor cell infusion after post-transplantation cyclophosphamide (PTCy) for 10 patients with advanced myeloid malignancies undergoing haploidentical peripheral blood stem cell transplantation with cyclosporine alone as graft-versus-host disease (GVHD) prophylaxis and compared the outcome and immune reconstitution with a control group of 20 patients undergoing the same without CD56-enriched donor cell infusion. An early and rapid surge of mature NK cells as well as CD4 + T cells and regulatory T cells (Tregs) was noted compared with the control group. KIR of donor phenotype reconstituted as early as day 30 with expression of CD56 dim CD16 + NKG2A - KIR + phenotype. None experienced viral or fungal infections, and non-relapse mortality was 10% only. The incidence of grade 2-4 acute GVHD was 50% in the control group with none in the CD56 group (P = 0.01). Only two had de novo chronic GVHD in each group. Relapse occurred in five patients in CD56 group with a median follow-up of 12 months, similar to the control group. Our preliminary data show that CD56 + donor cell infusion after PTCy and short-course cyclosporine is feasible with prompt engraftment, rapid reconstitution of CD4 + T cells, Tregs and NK cells and reduced incidence of acute GVHD. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Cryopreservation and in vitro culture of primary cell types from lung tissue of a stranded pygmy sperm whale (Kogia breviceps).

PubMed

Annalaura Mancia; Spyropoulos, Demetri D; McFee, Wayne E; Newton, Danforth A; Baatz, John E

2012-01-01

Current models for in vitro studies of tissue function and physiology, including responses to hypoxia or environmental toxins, are limited and rely heavily on standard 2-dimensional (2-D) cultures with immortalized murine or human cell lines. To develop a new more powerful model system, we have pursued methods to establish and expand cultures of primary lung cell types and reconstituted tissues from marine mammals. What little is known about the physiology of the deep-sea diving pygmy sperm whale (PSW), Kogia breviceps, comes primarily from stranding events that occur along the coast of the southeastern United States. Thus, development of a method for preserving live tissues and retrieving live cells from deceased stranded individuals was initiated. This report documents successful cryopreservation of PSW lung tissue. We established in vitro cultures of primary lung cell types from tissue fragments that had been cryopreserved several months earlier at the stranding event. Dissociation of cryopreserved lung tissues readily provides a variety of primary cell types that, to varying degrees, can be expanded and further studied/manipulated in cell culture. In addition, PSW-specific molecular markers have been developed that permitted the monitoring of fibroblast, alveolar type II, and vascular endothelial cell types. Reconstitution of 3-D cultures of lung tissues with these cell types is now underway. This novel system may facilitate the development of rare or disease-specific lung tissue models (e.g., to test causes of PSW stranding events and lead to improved treatments for pulmonary hypertension or reperfusion injury in humans). Also, the establishment of a "living" tissue bank biorepository for rare/endangered species could serve multiple purposes as surrogates for freshly isolated samples. Copyright © 2011 Elsevier Inc. All rights reserved.

Isolation of Mouse Hair Follicle Bulge Stem Cells and Their Functional Analysis in a Reconstitution Assay.

PubMed

Zheng, Ying; Hsieh, Jen-Chih; Escandon, Julia; Cotsarelis, George

2016-01-01

The hair follicle (HF) is a dynamic structure readily accessible within the skin, and contains various pools of stem cells that have a broad regenerative potential during normal homeostasis and in response to injury. Recent discoveries demonstrating the multipotent capabilities of hair follicle stem cells and the easy access to skin tissue make the HF an attractive source for isolating stem cells and their subsequent application in tissue engineering and regenerative medicine. Here, we describe the isolation and purification of hair follicle bulge stem cells from mouse skin, and hair reconstitution assays that allows the functional analysis of multipotent stem cells.

Dynamic equilibrium of reconstituting hematopoietic stem cell populations.

PubMed

O'Quigley, John

2010-12-01

Clonal dominance in hematopoietic stem cell populations is an important question of interest but not one we can directly answer. Any estimates are based on indirect measurement. For marked populations, we can equate empirical and theoretical moments for binomial sampling, in particular we can use the well-known formula for the sampling variation of a binomial proportion. The empirical variance itself cannot always be reliably estimated and some caution is needed. We describe the difficulties here and identify ready solutions which only require appropriate use of variance-stabilizing transformations. From these we obtain estimators for the steady state, or dynamic equilibrium, of the number of hematopoietic stem cells involved in repopulating the marrow. The calculations themselves are not too involved. We give the distribution theory for the estimator as well as simple approximations for practical application. As an illustration, we rework on data recently gathered to address the question as to whether or not reconstitution of marrow grafts in the clinical setting might be considered to be oligoclonal.

Expansion of brain T cells in homeostatic conditions in lymphopenic Rag2(-/-) mice.

PubMed

Song, Chang; Nicholson, James D; Clark, Sarah M; Li, Xin; Keegan, Achsah D; Tonelli, Leonardo H

2016-10-01

The concept of the brain as an immune privileged organ is rapidly evolving in light of new findings outlining the sophisticated relationship between the central nervous and the immune systems. The role of T cells in brain development and function, as well as modulation of behavior has been demonstrated by an increasing number of studies. Moreover, recent studies have redefined the existence of a brain lymphatic system and the presence of T cells in specific brain structures, such as the meninges and choroid plexus. Nevertheless, much information is needed to further the understanding of brain T cells and their relationship with the central nervous system under non-inflammatory conditions. In the present study we employed the Rag2(-/-) mouse model of lymphocyte deficiency and reconstitution by adoptive transfer to study the temporal and anatomical expansion of T cells in the brain under homeostatic conditions. Lymphopenic Rag2(-/-) mice were reconstituted with 10 million lymphoid cells and studied at one, two and four weeks after transfer. Moreover, lymphoid cells and purified CD4(+) and CD8(+) T cells from transgenic GFP expressing mice were used to define the neuroanatomical localization of transferred cells. T cell numbers were very low in the brain of reconstituted mice up to one week after transfer and significantly increased by 2weeks, reaching wild type values at 4weeks after transfer. CD4(+) T cells were the most abundant lymphocyte subtype found in the brain followed by CD8(+) T cells and lastly B cells. Furthermore, proliferation studies showed that CD4(+) T cells expand more rapidly than CD8(+) T cells. Lymphoid cells localize abundantly in meningeal structures, choroid plexus, and circumventricular organs. Lymphocytes were also found in vascular and perivascular spaces and in the brain parenchyma across several regions of the brain, in particular in structures rich in white matter content. These results provide proof of concept that the brain meningeal system, as well as vascular and perivascular spaces, are homing sites of lymphocytes and suggest the possibility of a brain specific T cell subtype. Published by Elsevier Inc.

A mouse model for ulcerative colitis based on NOD-scid IL2R γnull mice reconstituted with peripheral blood mononuclear cells from affected individuals.

PubMed

Palamides, Pia; Jodeleit, Henrika; Föhlinger, Michael; Beigel, Florian; Herbach, Nadja; Mueller, Thomas; Wolf, Eckhard; Siebeck, Matthias; Gropp, Roswitha

2016-09-01

Animal models reflective of ulcerative colitis (UC) remain a major challenge, and yet are crucial to understand mechanisms underlying the onset of disease and inflammatory characteristics of relapses and remission. Mouse models in which colitis-like symptoms are induced through challenge with toxins such as oxazolone, dextran sodium sulfate (DSS) or 2,4,6-trinitrobenzenesulfonic acid (TNBS) have been instrumental in understanding the inflammatory processes of UC. However, these neither reflect the heterogeneous symptoms observed in the UC-affected population nor can they be used to test the efficacy of inhibitors developed against human targets where high sequence and structural similarity of the respective ligands is lacking. In an attempt to overcome these problems, we have developed a mouse model that relies on NOD-scid IL2R γ(null) mice reconstituted with peripheral blood mononuclear cells derived from UC-affected individuals. Upon challenge with ethanol, mice developed colitis-like symptoms and changes in the colon architecture, characterized by influx of inflammatory cells, edema, crypt loss, crypt abscesses and epithelial hyperplasia, as previously observed in immune-competent mice. TARC, TGFβ1 and HGF expression increased in distal parts of the colon. Analysis of human leucocytes isolated from mouse spleen revealed an increase in frequencies of CD1a+, CD64+, CD163+ and TSLPR+ CD14+ monocytes, and antigen-experienced CD44+ CD4+ and CD8+ T-cells in response to ethanol. Analysis of human leucocytes from the colon of challenged mice identified CD14+ monocytes and CD11b+ monocytes as the predominant populations. Quantitative real-time PCR (RT-PCR) analysis from distal parts of the colon indicated that IFNγ might be one of the cytokines driving inflammation. Treatment with infliximab ameliorated symptoms and pathological manifestations, whereas pitrakinra had no therapeutic benefit. Thus, this model is partially reflective of the human disease and might help to increase the translation of animal and clinical studies. © 2016. Published by The Company of Biologists Ltd.

Hair Follicle Generation by Injections of Adult Human Follicular Epithelial and Dermal Papilla Cells into Nude Mice

PubMed Central

Nilforoushzadeh, Mohammadali; Rahimi Jameh, Elham; Jaffary, Fariba; Abolhasani, Ehsan; Keshtmand, Gelavizh; Zarkob, Hajar; Mohammadi, Parvaneh; Aghdami, Nasser

2017-01-01

Objective Dermal papilla and hair epithelial stem cells regulate hair formation and the growth cycle. Damage to or loss of these cells can cause hair loss. Although several studies claim to reconstitute hairs using rodent cells in an animal model, additional research is needed to develop a stable human hair follicle reconstitution protocol. In this study, we have evaluated hair induction by injecting adult cultured human dermal papilla cells and a mixture of hair epithelial and dermal papilla cells in a mouse model. Materials and Methods In this experimental study, discarded human scalp skins were used to obtain dermal papilla and hair epithelial cells. After separation, cells were cultured and assessed for their characteristics. We randomly allocated 15 C57BL/6 nude mice into three groups that received injections in their dorsal skin. The first group received cultured dermal papilla cells, the second group received a mixture of cultured epithelial and dermal papilla cells, and the third group (control) received a placebo [phosphate-buffered saline (PBS-)]. Results Histopathologic examination of the injection sites showed evidence of hair growth in samples that received cells compared with the control group. However, the group that received epithelial and dermal papilla cells had visible evidence of hair growth. PKH tracing confirmed the presence of transplanted cells in the new hair. Conclusion Our data showed that injection of a combination of adult human cultured dermal papilla and epithelial cells could induce hair growth in nude mice. This study emphasized that the combination of human adult cultured dermal papilla and epithelial cells could induce new hair in nude mice. PMID:28670518

Critical role of γ4 chain in the expression of functional Vγ4Vδ1 T cell receptor of gastric tumour-infiltrating γδT lymphocytes.

PubMed

Jiang, Y; Tang, F; Li, Z; Cui, L; He, W

2012-01-01

Vγ4Vδ1 T cell receptor (TCRγ4δ1)-expressing γδT cells were the most dominant subset in gastric tumour-infiltrating γδT cells (γδTIL) we recently analyzed. To study the essential roles of γ and δ chains in assembly and function of TCRγ4δ1, we sequenced and constructed them into lentiviral vectors for the reconstitution of TCRγ4δ1 using different modalities of transduction. We were able to efficiently reconstitute TCRγ4δ1 with functional activities when both γ4 and δ1 chains are coexpressed in TCR-negative J.RT3-T3.5 cells. However, the expression of δ1 chain is greatly diminished when γ4 expression is absent, suggesting that the coexpressing γ4 is critical in maintaining the folding and stability of δ1 product. To functionally study the reconstituted TCRγ4δ1, we examined the cytolytic activity of TCRγ4δ1-reconstituted J.RT3-T3.5 cells and cytokine secretion and found the receptors are fully functional, but their functionality also requires the presence of γ4. Our results demonstrated that γ4 is critical for the stability of δ1 and the function of TCRγ4δ1. © 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.

Lipoprotein-biomimetic nanostructure enables efficient targeting delivery of siRNA to Ras-activated glioblastoma cells via macropinocytosis

NASA Astrophysics Data System (ADS)

Huang, Jia-Lin; Jiang, Gan; Song, Qing-Xiang; Gu, Xiao; Hu, Meng; Wang, Xiao-Lin; Song, Hua-Hua; Chen, Le-Pei; Lin, Ying-Ying; Jiang, Di; Chen, Jun; Feng, Jun-Feng; Qiu, Yong-Ming; Jiang, Ji-Yao; Jiang, Xin-Guo; Chen, Hong-Zhuan; Gao, Xiao-Ling

2017-05-01

Hyperactivated Ras regulates many oncogenic pathways in several malignant human cancers including glioblastoma and it is an attractive target for cancer therapies. Ras activation in cancer cells drives protein internalization via macropinocytosis as a key nutrient-gaining process. By utilizing this unique endocytosis pathway, here we create a biologically inspired nanostructure that can induce cancer cells to `drink drugs' for targeting activating transcription factor-5 (ATF5), an overexpressed anti-apoptotic transcription factor in glioblastoma. Apolipoprotein E3-reconstituted high-density lipoprotein is used to encapsulate the siRNA-loaded calcium phosphate core and facilitate it to penetrate the blood-brain barrier, thus targeting the glioblastoma cells in a macropinocytosis-dependent manner. The nanostructure carrying ATF5 siRNA exerts remarkable RNA-interfering efficiency, increases glioblastoma cell apoptosis and inhibits tumour cell growth both in vitro and in xenograft tumour models. This strategy of targeting the macropinocytosis caused by Ras activation provides a nanoparticle-based approach for precision therapy in glioblastoma and other Ras-activated cancers.

Adoptive immunotherapy with allodepleted donor T-cells improves immune reconstitution after haploidentical stem cell transplantation

PubMed Central

Amrolia, Persis J.; Muccioli-Casadei, Giada; Huls, Helen; Adams, Stuart; Durett, April; Gee, Adrian; Yvon, Eric; Weiss, Heidi; Cobbold, Mark; Gaspar, H. Bobby; Rooney, Cliona; Kuehnle, Ingrid; Ghetie, Victor; Schindler, John; Krance, Robert; Heslop, Helen E.; Veys, Paul; Vitetta, Ellen; Brenner, Malcolm K.

2006-01-01

Poor T lymphocyte reconstitution limits the use of haploidentical stem cell transplantation (SCT) because it results in a high mortality from viral infections. One approach to overcome this problem is to infuse donor T cells from which alloreactive lymphocytes have been selectively depleted, but the immunologic benefit of this approach is unknown. We have used an anti-CD25 immunotoxin to deplete alloreactive lymphocytes and have compared immune reconstitution after allodepleted donor T cells were infused at 2 dose levels into recipients of T-cell–depleted haploidentical SCT. Eight patients were treated at 104 cells/kg/dose, and 8 patients received 105 cells/kg/dose. Patients receiving 105 cells/kg/dose showed significantly improved T-cell recovery at 3, 4, and 5 months after SCT compared with those receiving 104 cells/kg/dose (P < .05). Accelerated T-cell recovery occurred as a result of expansion of the effector memory (CD45RA–CCR-7-) population (P < .05), suggesting that protective T-cell responses are likely to be long lived. T-cell–receptor signal joint excision circles (TRECs) were not detected in reconstituting T cells in dose-level 2 patients, indicating they are likely to be derived from the infused allodepleted cells. Spectratyping of the T cells at 4 months demonstrated a polyclonal Vβ repertoire. Using tetramer and enzyme-linked immunospot (ELISPOT) assays, we have observed cytomegalovirus (CMV)– and Epstein-Barr virus (EBV)–specific responses in 4 of 6 evaluable patients at dose level 2 as early as 2 to 4 months after transplantation, whereas such responses were not observed until 6 to 12 months in dose-level 1 patients. The incidence of significant acute (2 of 16) and chronic graft-versus-host disease (GVHD; 2 of 15) was low. These data demonstrate that allodepleted donor T cells can be safely used to improve T-cell recovery after haploidentical SCT and may broaden the applicability of this approach. PMID:16741253

Integument pattern formation involves genetic and epigenetic controls: feather arrays simulated by digital hormone models

PubMed Central

Jiang, Ting-Xin; Widelitz, Randall B.; Shen, Wei-Min; Will, Peter; Wu, Da-Yu; Lin, Chih-Min; Jung, Han-Sung; Chuong, Cheng-Ming

2015-01-01

Pattern formation is a fundamental morphogenetic process. Models based on genetic and epigenetic control have been proposed but remain controversial. Here we use feather morphogenesis for further evaluation. Adhesion molecules and/or signaling molecules were first expressed homogenously in feather tracts (restrictive mode, appear earlier) or directly in bud or inter-bud regions (de novo mode, appear later). They either activate or inhibit bud formation, but paradoxically co-localize in the bud. Using feather bud reconstitution, we showed that completely dissociated cells can reform periodic patterns without reference to previous positional codes. The patterning process has the characteristics of being self-organizing, dynamic and plastic. The final pattern is an equilibrium state reached by competition, and the number and size of buds can be altered based on cell number and activator/inhibitor ratio, respectively. We developed a Digital Hormone Model which consists of (1) competent cells without identity that move randomly in a space, (2) extracellular signaling hormones which diffuse by a reaction-diffusion mechanism and activate or inhibit cell adhesion, and (3) cells which respond with topological stochastic actions manifested as changes in cell adhesion. Based on probability, the results are cell clusters arranged in dots or stripes. Thus genetic control provides combinational molecular information which defines the properties of the cells but not the final pattern. Epigenetic control governs interactions among cells and their environment based on physical-chemical rules (such as those described in the Digital Hormone Model). Complex integument patterning is the sum of these two components of control and that is why integument patterns are usually similar but non-identical. These principles may be shared by other pattern formation processes such as barb ridge formation, fingerprints, pigmentation patterning, etc. The Digital Hormone Model can also be applied to swarming robot navigation, reaching intelligent automata and representing a self-re-configurable type of control rather than a follow-the-instruction type of control. PMID:15272377

Antigen presentation under the influence of 'immune evasion' proteins and its modulation by interferon-gamma: implications for immunotherapy of cytomegalovirus infection with antiviral CD8 T cells.

PubMed

Fink, Annette; Lemmermann, Niels A W; Gillert-Marien, Dorothea; Thomas, Doris; Freitag, Kirsten; Böhm, Verena; Wilhelmi, Vanessa; Reifenberg, Kurt; Reddehase, Matthias J; Holtappels, Rafaela

2012-11-01

Cytomegalovirus (CMV) disease with multiple organ manifestations is the most feared viral complication limiting the success of hematopoietic cell transplantation as a therapy of hematopoietic malignancies. A timely endogenous reconstitution of CD8 T cells controls CMV infection, and adoptive transfer of antiviral CD8 T cells is a therapeutic option to prevent CMV disease by bridging the gap between an early CMV reactivation and delayed endogenous reconstitution of protective immunity. Preclinical research in murine models has provided 'proof of concept' for CD8 T-cell therapy of CMV disease. Protection by CD8 T cells appears to be in conflict with the finding that CMVs encode proteins that inhibit antigen presentation to CD8 T cells by interfering with the constitutive trafficking of peptide-loaded MHC class I molecules (pMHC-I complexes) to the cell surface. Here, we have systematically explored antigen presentation in the presence of the three currently noted immune evasion proteins of murine CMV in all possible combinations and its modulation by pre-treatment of cells with interferon-gamma (IFN-γ). The data reveal improvement in antigen processing by pre-treatment with IFN-γ can almost overrule the inhibitory function of immune evasion molecules in terms of pMHC-I expression levels capable of triggering most of the specific CD8 T cells, though the intensity of stimulation did not retrieve their full functional capacity. Notably, an in vivo conditioning of host tissue cells with IFN-γ in adoptive cell transfer recipients constitutively overexpressing IFN-γ (B6-SAP-IFN-γ mice) enhanced the antiviral efficiency of CD8 T cells in this transgenic cytoimmunotherapy model.

Probing the luminal microenvironment of reconstituted epithelial microtissues

PubMed Central

Cerchiari, Alec E.; Samy, Karen E.; Todhunter, Michael E.; Schlesinger, Erica; Henise, Jeff; Rieken, Christopher; Gartner, Zev J.; Desai, Tejal A.

2016-01-01

Polymeric microparticles can serve as carriers or sensors to instruct or characterize tissue biology. However, incorporating microparticles into tissues for in vitro assays remains a challenge. We exploit three-dimensional cell-patterning technologies and directed epithelial self-organization to deliver microparticles to the lumen of reconstituted human intestinal microtissues. We also develop a novel pH-sensitive microsensor that can measure the luminal pH of reconstituted epithelial microtissues. These studies offer a novel approach for investigating luminal microenvironments and drug-delivery across epithelial barriers. PMID:27619235

Critical role of CD4 T cells in maintaining lymphoid tissue structure for immune cell homeostasis and reconstitution.

PubMed

Zeng, Ming; Paiardini, Mirko; Engram, Jessica C; Beilman, Greg J; Chipman, Jeffrey G; Schacker, Timothy W; Silvestri, Guido; Haase, Ashley T

2012-08-30

Loss of the fibroblastic reticular cell (FRC) network in lymphoid tissues during HIV-1 infection has been shown to impair the survival of naive T cells and limit immune reconstitution after antiretroviral therapy. What causes this FRC loss is unknown. Because FRC loss correlates with loss of both naive CD4 and CD8 T-cell subsets and decreased lymphotoxin-β, a key factor for maintenance of FRC network, we hypothesized that loss of naive T cells is responsible for loss of the FRC network. To test this hypothesis, we assessed the consequences of antibody-mediated depletion of CD4 and CD8 T cells in rhesus macaques and sooty mangabeys. We found that only CD4 T-cell depletion resulted in FRC loss in both species and that this loss was caused by decreased lymphotoxin-β mainly produced by the CD4 T cells. We further found the same dependence of the FRC network on CD4 T cells in HIV-1-infected patients before and after antiretroviral therapy and in other immunodeficiency conditions, such as CD4 depletion in cancer patients induced by chemotherapy and irradiation. CD4 T cells thus play a central role in the maintenance of lymphoid tissue structure necessary for their own homeostasis and reconstitution.

Glucose 6-phosphate dehydrogenase deficient subjects may be better "storers" than donors of red blood cells.

PubMed

Tzounakas, Vassilis L; Kriebardis, Anastasios G; Georgatzakou, Hara T; Foudoulaki-Paparizos, Leontini E; Dzieciatkowska, Monika; Wither, Matthew J; Nemkov, Travis; Hansen, Kirk C; Papassideri, Issidora S; D'Alessandro, Angelo; Antonelou, Marianna H

2016-07-01

Storage of packed red blood cells (RBCs) is associated with progressive accumulation of lesions, mostly triggered by energy and oxidative stresses, which potentially compromise the effectiveness of the transfusion therapy. Concerns arise as to whether glucose 6-phosphate dehydrogenase deficient subjects (G6PD(-)), ~5% of the population in the Mediterranean area, should be accepted as routine donors in the light of the increased oxidative stress their RBCs suffer from. To address this question, we first performed morphology (scanning electron microscopy), physiology and omics (proteomics and metabolomics) analyses on stored RBCs from healthy or G6PD(-) donors. We then used an in vitro model of transfusion to simulate transfusion outcomes involving G6PD(-) donors or recipients, by reconstituting G6PD(-) stored or fresh blood with fresh or stored blood from healthy volunteers, respectively, at body temperature. We found that G6PD(-) cells store well in relation to energy, calcium and morphology related parameters, though at the expenses of a compromised anti-oxidant system. Additional stimuli, mimicking post-transfusion conditions (37°C, reconstitution with fresh healthy blood, incubation with oxidants) promoted hemolysis and oxidative lesions in stored G6PD(-) cells in comparison to controls. On the other hand, stored healthy RBC units showed better oxidative parameters and lower removal signaling when reconstituted with G6PD(-) fresh blood compared to control. Although the measured parameters of stored RBCs from the G6PD deficient donors appeared to be acceptable, the results from the in vitro model of transfusion suggest that G6PD(-) RBCs could be more susceptible to hemolysis and oxidative stresses post-transfusion. On the other hand, their chronic exposure to oxidative stress might make them good recipients, as they better tolerate exposure to oxidatively damaged long stored healthy RBCs. Copyright © 2016 Elsevier Inc. All rights reserved.

Brief report: Circulating markers of fibrosis are associated with immune reconstitution status in HIV-infected men.

PubMed

Tobolowsky, F A; Wada, N; Martinez-Maza, O; Magpantay, L; Koletar, S L; Palella, F J; Brown, T T; Lake, J E

2018-01-01

Lymphoid tissue fibrosis may contribute to incomplete immune reconstitution on antiretroviral therapy (ART) via local CD4+ T lymphocyte (CD4) depletion. Hyaluronic acid (HA) increases with fibrotic burden. CXCL4 concentrations increase in response to pro-fibrotic stimuli, but lower CXCL4 concentrations in HIV-infected individuals may reflect successful immune evasion by HIV. We investigated relationships between circulating HA and CXCL4 concentrations and immune reconstitution on ART in HIV-infected Multicenter AIDS Cohort Study participants. HIV-infected men on ART for >1 year with cryopreserved plasma samples and suppressed post-ART HIV-1 RNA were included. Men with post-ART CD4 <200 cells/mm3 were defined as immunologic non-responders (n = 25). Age-/race-matched men with post-ART CD4 >500 cells/mm3 served as controls (n = 49). HA and CXCL4 concentrations were measured via ELISA. Median pre-ART CD4 was 297 cells/mm3 for non-responders vs 386 cells/mm3 for controls. Median post-ART CD4 was 141 cells/mm3 for non-responders and 815 cells/mm3 for controls. HIV infection duration was 23 years, with median time on ART 13 years for non-responders vs 11 years for controls. Pre-ART HA and CXCL4 concentrations did not vary by eventual immune reconstitution status. Post-ART HA concentrations tended to be higher (85 vs 36 ng/mL, p = 0.07) and CXCL4 concentrations were lower (563 vs 1459 ng/mL, p = 0.01) among non-responders. Among men with paired pre-/post-ART samples, non-responders had greater HA increases and CXCL4 decreases than controls (HA: 50 vs 12 ng/mL, p = 0.04; CXCL4: -1258 vs -405 ng/mL, p = 0.01). Higher circulating concentrations of HA and lower concentrations of CXCL4 are associated with failure of immune reconstitution on ART.

Brief report: Circulating markers of fibrosis are associated with immune reconstitution status in HIV-infected men

PubMed Central

Wada, N.; Martinez-Maza, O.; Magpantay, L.; Koletar, S. L.; Palella, F. J.; Brown, T. T.; Lake, J. E.

2018-01-01

Introduction Lymphoid tissue fibrosis may contribute to incomplete immune reconstitution on antiretroviral therapy (ART) via local CD4+ T lymphocyte (CD4) depletion. Hyaluronic acid (HA) increases with fibrotic burden. CXCL4 concentrations increase in response to pro-fibrotic stimuli, but lower CXCL4 concentrations in HIV-infected individuals may reflect successful immune evasion by HIV. We investigated relationships between circulating HA and CXCL4 concentrations and immune reconstitution on ART in HIV-infected Multicenter AIDS Cohort Study participants. Methods HIV-infected men on ART for >1 year with cryopreserved plasma samples and suppressed post-ART HIV-1 RNA were included. Men with post-ART CD4 <200 cells/mm3 were defined as immunologic non-responders (n = 25). Age-/race-matched men with post-ART CD4 >500 cells/mm3 served as controls (n = 49). HA and CXCL4 concentrations were measured via ELISA. Results Median pre-ART CD4 was 297 cells/mm3 for non-responders vs 386 cells/mm3 for controls. Median post-ART CD4 was 141 cells/mm3 for non-responders and 815 cells/mm3 for controls. HIV infection duration was 23 years, with median time on ART 13 years for non-responders vs 11 years for controls. Pre-ART HA and CXCL4 concentrations did not vary by eventual immune reconstitution status. Post-ART HA concentrations tended to be higher (85 vs 36 ng/mL, p = 0.07) and CXCL4 concentrations were lower (563 vs 1459 ng/mL, p = 0.01) among non-responders. Among men with paired pre-/post-ART samples, non-responders had greater HA increases and CXCL4 decreases than controls (HA: 50 vs 12 ng/mL, p = 0.04; CXCL4: -1258 vs -405 ng/mL, p = 0.01). Conclusions Higher circulating concentrations of HA and lower concentrations of CXCL4 are associated with failure of immune reconstitution on ART. PMID:29381717

Measurement of the intracellular ph in human stomach cells: a novel approach to evaluate the gastric acid secretory potential of coffee beverages.

PubMed

Weiss, Carola; Rubach, Malte; Lang, Roman; Seebach, Elisabeth; Blumberg, Simone; Frank, Oliver; Hofmann, Thomas; Somoza, Veronika

2010-02-10

As the consumption of coffee beverages sometimes is reported to cause gastric irritation, for which an increased stomach acid secretion is one of the promoting factors, different processing technologies such as steam-treatment have been developed to reduce putative stomach irritating compounds. There is evidence-based data neither on the effect of detailed processing variations nor on individual coffee components affecting the proton secretory activity (PSA). This work aimed at developing a screening model suitable for investigating the effects of commercial coffee beverages and components thereof on human parietal cells. Human gastric cancer cells (HGT-1) were treated with reconstituted freeze-dried coffee beverages prepared from customary coffee products such as regular coffee (RC, n = 4), mild bean coffee (MBC, n = 5), stomach friendly coffee (SFC, n = 4), and SFC decaffeinated (SFCD, n = 3). PSA was analyzed by flow cytometry using the pH-sensitive dye SNARF-AM. Treatment of the cells with MBC did not result in a PSA different from RC treatment (p

Mechano-genetic DNA hydrogels as a simple, reconstituted model to probe the effect of active fluctuations on gene transcription

NASA Astrophysics Data System (ADS)

Nguyen, Dan; Saleh, Omar

Active fluctuations - non-directed fluctuations attributable, not to thermal energy, but to non-equilibrium processes - are thought to influence biology by increasing the diffusive motion of biomolecules. Dense DNA regions within cells (i.e. chromatin) are expected to exhibit such phenomena, as they are cross-linked networks that continually experience propagating forces arising from dynamic cellular activity. Additional agitation within these gene-encoding DNA networks could have potential genetic consequences. By changing the local mobility of transcriptional machinery and regulatory proteins towards/from their binding sites, and thereby influencing transcription rates, active fluctuations could prove to be a physical means of modulating gene expression. To begin probing this effect, we construct genetic DNA hydrogels, as a simple, reconstituted model of chromatin, and quantify transcriptional output from these hydrogels in the presence/absence of active fluctuations.

A propagating ATPase gradient drives transport of surface-confined cellular cargo

NASA Astrophysics Data System (ADS)

Vecchiarelli, Anthony; Neuman, Keir; Mizuuchi, Kiyoshi

2014-03-01

The process of DNA segregation is of central importance for all organisms. Although eukaryotic mitosis is relatively well established, the most common mechanism employed for bacterial DNA segregation has been unclear. ParA ATPases form dynamic patterns on the bacterial nucleoid, to spatially organize plasmids, chromosomes and other large cellular cargo, but the force generating mechanism has been a source of controversy and debate. A dominant view proposes that ParA-mediated transport and cargo positioning occurs via a filament-based mechanism that resembles eukaryotic mitosis. Here we present direct evidence against such models. Our cell-free reconstitution supports a non-filament-based mode of transport that may be as widely found in nature as actin filaments and microtubules.

Establishment of an in vitro transcription system for Peste des petits ruminant virus.

PubMed

Yunus, Mohammad; Shaila, Melkote S

2012-12-05

Peste-des-petits ruminants virus (PPRV) is a non segmented negative strand RNA virus of the genus Morbillivirus within Paramyxoviridae family. Negative strand RNA viruses are known to carry nucleocapsid (N) protein, phospho (P) protein and RNA polymerase (L protein) packaged within the virion which possess all activities required for transcription, post-transcriptional modification of mRNA and replication. In order to understand the mechanism of transcription and replication of the virus, an in vitro transcription reconstitution system is required. In the present work, an in vitro transcription system has been developed with ribonucleoprotein (RNP) complex purified from virus infected cells as well as partially purified recombinant polymerase (L-P) complex from insect cells along with N-RNA (genomic RNA encapsidated by N protein) template isolated from virus infected cells. RNP complex isolated from virus infected cells and recombinant L-P complex purified from insect cells was used to reconstitute transcription on N-RNA template. The requirement for this transcription reconstitution has been defined. Transcription of viral genes in the in vitro system was confirmed by PCR amplification of cDNAs corresponding to individual transcripts using gene specific primers. In order to measure the relative expression level of viral transcripts, real time PCR analysis was carried out. qPCR analysis of the transcription products made in vitro showed a gradient of polarity of transcription from 3' end to 5' end of the genome similar to that exhibited by the virus in infected cells. This report describes for the first time, the development of an in vitro transcription reconstitution system for PPRV with RNP complex purified from infected cells and recombinant L-P complex expressed in insect cells. Both the complexes were able to synthesize all the mRNA species in vitro, exhibiting a gradient of polarity in transcription.

A stromal cell free culture system generates mouse pro-T cells that can reconstitute T-cell compartments in vivo.

PubMed

Gehre, Nadine; Nusser, Anja; von Muenchow, Lilly; Tussiwand, Roxane; Engdahl, Corinne; Capoferri, Giuseppina; Bosco, Nabil; Ceredig, Rhodri; Rolink, Antonius G

2015-03-01

T-cell lymphopenia following BM transplantation or diseases such as AIDS result in immunodeficiency. Novel approaches to ameliorate this situation are urgently required. Herein, we describe a novel stromal cell free culture system in which Lineage(-) Sca1(+)c-kit(+) BM hematopoietic progenitors very efficiently differentiate into pro-T cells. This culture system consists of plate-bound Delta-like 4 Notch ligand and the cytokines SCF and IL-7. The pro-T cells developing in these cultures express CD25, CD117, and partially CD44; express cytoplasmic CD3ε; and have their TCRβ locus partially D-J rearranged. They could be expanded for over 3 months and used to reconstitute the T-cell compartments of sublethally irradiated T-cell-deficient CD3ε(-/-) mice or lethally irradiated WT mice. Pro-T cells generated in this system could partially correct the T-cell lymphopenia of pre-Tα(-/-) mice. However, reconstituted CD3ε(-/-) mice suffered from a wasting disease that was prevented by co-injection of purified CD4(+) CD25(high) WT Treg cells. In a T-cell-sufficient or T-lymphopenic setting, the development of disease was not observed. Thus, this in vitro culture system represents a powerful tool to generate large numbers of pro-T cells for transplantation and possibly with clinical applications. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Three-dimensional cultures modeling premalignant progression of human breast epithelial cells: role of cysteine cathepsins.

PubMed

Mullins, Stefanie R; Sameni, Mansoureth; Blum, Galia; Bogyo, Matthew; Sloane, Bonnie F; Moin, Kamiar

2012-12-01

The expression of the cysteine protease cathepsin B is increased in early stages of human breast cancer.To assess the potential role of cathepsin B in premalignant progression of breast epithelial cells, we employed a 3D reconstituted basement membrane overlay culture model of MCF10A human breast epithelial cells and isogenic variants that replicate the in vivo phenotypes of hyper plasia(MCF10AneoT) and atypical hyperplasia (MCF10AT1). MCF10A cells developed into polarized acinar structures with central lumens. In contrast, MCF10AneoT and MCF10AT1 cells form larger structures in which the lumens are filled with cells. CA074Me, a cell-permeable inhibitor selective for the cysteine cathepsins B and L,reduced proliferation and increased apoptosis of MCF10A, MCF10AneoT and MCF10AT1 cells in 3D culture. We detected active cysteine cathepsins in the isogenic MCF10 variants in 3D culture with GB111, a cell-permeable activity based probe, and established differential inhibition of cathepsin B in our 3D cultures. We conclude that cathepsin B promotes proliferation and premalignant progression of breast epithelial cells. These findings are consistent with studies by others showing that deletion of cathepsin B in the transgenic MMTV-PyMT mice, a murine model that is predisposed to development of mammary cancer, reduces malignant progression.

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.

Evaluation of the severe combined immunodeficient (SCID) mouse as an animal model for dengue viral infection.

PubMed

Wu, S J; Hayes, C G; Dubois, D R; Windheuser, M G; Kang, Y H; Watts, D M; Sieckmann, D G

1995-05-01

Severe combined immunodeficient (SCID) mice reconstituted with human peripheral blood lymphocytes (hu-PBL) were evaluated as an animal model for demonstrating dengue (DEN) viral infection. Reconstituted mice (hu-PBL-SCID) that demonstrated successful engraftment by the presence of serum titers of human immunoglobulin (Ig) were inoculated intraperitoneally with DEN virus serotype 1 (DEN-1). Serial blood samples were taken postinoculation and assayed for virus in C6/36 cells. The identity of all viral isolates was confirmed by an immunofluorescence antibody assay using DEN-1 monoclonal antibody. A total of six experiments were performed using different procedures of reconstitution and infection, and in three of these experiments, DEN-1 virus was recovered from the hu-PBL-SCID mice. In the first successful experiment, DEN-1 virus was recovered on postinoculation day (PID) 24 from blood, spleen, thymus, and lung tissues of one of eight hu-PBL-SCID mice. A second group of eight hu-PBL-SCID mice were inoculated with human monocytes infected in vitro with DEN-1 virus. Virus was recovered from the blood of mice between PID 15 and 23, and from lung tissue of one of these mice. In a third experiment, seven SCID mice were treated initially with anti-asialo GM1 antibody to eliminate natural killer cells, and then were injected simultaneously with a mixture of hu-PBL and DEN-1 virus. Virus was demonstrated in the blood of one mouse on PID 38, and in another mouse on PID 8, 12, 20, 24, and 36.(ABSTRACT TRUNCATED AT 250 WORDS)

Proximal location of mouse prostate epithelial stem cells

PubMed Central

Tsujimura, Akira; Koikawa, Yasuhiro; Salm, Sarah; Takao, Tetsuya; Coetzee, Sandra; Moscatelli, David; Shapiro, Ellen; Lepor, Herbert; Sun, Tung-Tien; Wilson, E. Lynette

2002-01-01

Stem cells are believed to regulate normal prostatic homeostasis and to play a role in the etiology of prostate cancer and benign prostatic hyperplasia. We show here that the proximal region of mouse prostatic ducts is enriched in a subpopulation of epithelial cells that exhibit three important attributes of epithelial stem cells: they are slow cycling, possess a high in vitro proliferative potential, and can reconstitute highly branched glandular ductal structures in collagen gels. We propose a model of prostatic homeostasis in which mouse prostatic epithelial stem cells are concentrated in the proximal region of prostatic ducts while the transit-amplifying cells occupy the distal region of the ducts. This model can account for many biological differences between cells of the proximal and distal regions, and has implications for prostatic disease formation. PMID:12082083

Imaging CXCL12-CXCR4 Signaling and Inhibition in Ovarian Cancer

DTIC Science & Technology

2013-10-01

arrestin 2 and reconstitution of luciferase enzymes to produce light (see attached manuscript from PLoS One). Fig 2. CXCL12-dependent activation...With the luciferase complementa- tion system, interactions between CXCR4 and b-arrestin 2 reconstitute active click beetle luciferase to produce light ...dimensional cell culture experiments We plated 1.56104 HeyA8-CXCR4-CBRN/Ar-CBC cells per well in black wall 96 well plates one day before assays. We

Stem cell therapies in cardiovascular disease A "realistic" appraisal.

PubMed

Partovian, Chohreh; Simons, Michael

2008-01-01

The possibility of reconstituting the damaged heart has introduced a new paradigm in cardiovascular biology and created the potential for a new therapeutic approach in the cardiovascular field, where there is a compelling need for innovative treatments. While the results of animal and early clinical studies are encouraging, the more direct use of cell-based therapies in patients is still long-reached. Gaps in our basic understanding of mechanisms, lack of important randomized, double blind, and controlled clinical trials, as well as technology development for cell production are among challenges to be overcome before full translation of cell based therapies in clinical arena. This review focuses on summarizing the latest knowledge in stem cell therapy for cardiovascular diseases.

Central nervous system demyelinating disease protection by the human commensal Bacteroides fragilis depends on polysaccharide A expression.

PubMed

Ochoa-Repáraz, Javier; Mielcarz, Daniel W; Ditrio, Lauren E; Burroughs, Ashley R; Begum-Haque, Sakhina; Dasgupta, Suryasarathi; Kasper, Dennis L; Kasper, Lloyd H

2010-10-01

The importance of gut commensal bacteria in maintaining immune homeostasis is increasingly understood. We recently described that alteration of the gut microflora can affect a population of Foxp3(+)T(reg) cells that regulate demyelination in experimental autoimmune encephalomyelitis (EAE), the experimental model of human multiple sclerosis. We now extend our previous observations on the role of commensal bacteria in CNS demyelination, and we demonstrate that Bacteroides fragilis producing a bacterial capsular polysaccharide Ag can protect against EAE. Recolonization with wild type B. fragilis maintained resistance to EAE, whereas reconstitution with polysaccharide A-deficient B. fragilis restored EAE susceptibility. Enhanced numbers of Foxp3(+)T(reg) cells in the cervical lymph nodes were observed after intestinal recolonization with either strain of B. fragilis. Ex vivo, CD4(+)T cells obtained from mice reconstituted with wild type B. fragilis had significantly enhanced rates of conversion into IL-10-producing Foxp3(+)T(reg) cells and offered greater protection against disease. Our results suggest an important role for commensal bacterial Ags, in particular B. fragilis expressing polysaccharide A, in protecting against CNS demyelination in EAE and perhaps human multiple sclerosis.

A plasmid-based reverse genetics system for influenza A virus.

PubMed Central

Pleschka, S; Jaskunas, R; Engelhardt, O G; Zürcher, T; Palese, P; García-Sastre, A

1996-01-01

A reverse genetics system for negative-strand RNA viruses was first successfully developed for influenza viruses. This technology involved the transfection of in vitro-reconstituted ribonucleoprotein (RNP) complexes into influenza virus-infected cells. We have now developed a method that allows intracellular reconstitution of RNP complexes from plasmid-based expression vectors. Expression of a viral RNA-like transcript is achieved from a plasmid containing a truncated human polymerase I (polI) promoter and a ribozyme sequence that generates the desired 3' end by autocatalytic cleavage. The polI-driven plasmid is cotransfected into human 293 cells with polII-responsive plasmids that express the viral PB1, PB2, PA, and NP proteins. This exclusively plasmid-driven system results in the efficient transcription and replication of the viral RNA-like reporter and allows the study of cis- and trans-acting signals involved in the transcription and replication of influenza virus RNAs. Using this system, we have also been able to rescue a synthetic neuraminidase gene into a recombinant influenza virus. This method represents a convenient alternative to the previously established RNP transfection system. PMID:8648766

Protective Immunity in Mice Achieved with Dry Powder Formulation and Alternative Delivery of Plague F1-V Vaccine▿

PubMed Central

Huang, Joanne; D'Souza, Ajit J.; Alarcon, Jason B.; Mikszta, John A.; Ford, Brandi M.; Ferriter, Matthew S.; Evans, Michelle; Stewart, Todd; Amemiya, Kei; Ulrich, Robert G.; Sullivan, Vincent J.

2009-01-01

The potential use of Yersinia pestis as a bioterror agent is a great concern. Development of a stable powder vaccine against Y. pestis and administration of the vaccine by minimally invasive methods could provide an alternative to the traditional liquid formulation and intramuscular injection. We evaluated a spray-freeze-dried powder vaccine containing a recombinant F1-V fusion protein of Y. pestis for vaccination against plaque in a mouse model. Mice were immunized with reconstituted spray-freeze-dried F1-V powder via intramuscular injection, microneedle-based intradermal delivery, or noninvasive intranasal administration. By intramuscular injection, the reconstituted powder induced serum antibody responses and provided protection against lethal subcutaneous challenge with 1,000 50% lethal doses of Y. pestis at levels equivalent to those elicited by unprocessed liquid formulations (70 to 90% protection). The feasibility of intradermal and intranasal delivery of reconstituted powder F1-V vaccine was also demonstrated. Overall, microneedle-based intradermal delivery was shown to be similar in efficacy to intramuscular injection, while intranasal administration required an extra dose of vaccine to achieve similar protection. In addition, the results suggest that seroconversion against F1 may be a better predictor of protection against Y. pestis challenge than seroconversion against either F1-V or V. In summary, we demonstrate the preclinical feasibility of using a reconstituted powder F1-V formulation and microneedle-based intradermal delivery to provide protective immunity against plague in a mouse model. Intranasal delivery, while feasible, was less effective than injection in this study. The potential use of these alternative delivery methods and a powder vaccine formulation may result in substantial health and economic benefits. PMID:19261773

Protective immunity in mice achieved with dry powder formulation and alternative delivery of plague F1-V vaccine.

PubMed

Huang, Joanne; D'Souza, Ajit J; Alarcon, Jason B; Mikszta, John A; Ford, Brandi M; Ferriter, Matthew S; Evans, Michelle; Stewart, Todd; Amemiya, Kei; Ulrich, Robert G; Sullivan, Vincent J

2009-05-01

The potential use of Yersinia pestis as a bioterror agent is a great concern. Development of a stable powder vaccine against Y. pestis and administration of the vaccine by minimally invasive methods could provide an alternative to the traditional liquid formulation and intramuscular injection. We evaluated a spray-freeze-dried powder vaccine containing a recombinant F1-V fusion protein of Y. pestis for vaccination against plaque in a mouse model. Mice were immunized with reconstituted spray-freeze-dried F1-V powder via intramuscular injection, microneedle-based intradermal delivery, or noninvasive intranasal administration. By intramuscular injection, the reconstituted powder induced serum antibody responses and provided protection against lethal subcutaneous challenge with 1,000 50% lethal doses of Y. pestis at levels equivalent to those elicited by unprocessed liquid formulations (70 to 90% protection). The feasibility of intradermal and intranasal delivery of reconstituted powder F1-V vaccine was also demonstrated. Overall, microneedle-based intradermal delivery was shown to be similar in efficacy to intramuscular injection, while intranasal administration required an extra dose of vaccine to achieve similar protection. In addition, the results suggest that seroconversion against F1 may be a better predictor of protection against Y. pestis challenge than seroconversion against either F1-V or V. In summary, we demonstrate the preclinical feasibility of using a reconstituted powder F1-V formulation and microneedle-based intradermal delivery to provide protective immunity against plague in a mouse model. Intranasal delivery, while feasible, was less effective than injection in this study. The potential use of these alternative delivery methods and a powder vaccine formulation may result in substantial health and economic benefits.

In vitro reconstitution of T cell receptor-mediated segregation of the CD45 phosphatase

PubMed Central

Carbone, Catherine B.; Fernandes, Ricardo A.; Hui, Enfu; Su, Xiaolei; Garcia, K. Christopher; Vale, Ronald D.

2017-01-01

T cell signaling initiates upon the binding of peptide-loaded MHC (pMHC) on an antigen-presenting cell to the T cell receptor (TCR) on a T cell. TCR phosphorylation in response to pMHC binding is accompanied by segregation of the transmembrane phosphatase CD45 away from TCR–pMHC complexes. The kinetic segregation hypothesis proposes that CD45 exclusion shifts the local kinase–phosphatase balance to favor TCR phosphorylation. Spatial partitioning may arise from the size difference between the large CD45 extracellular domain and the smaller TCR–pMHC complex, although parsing potential contributions of extracellular protein size, actin activity, and lipid domains is difficult in living cells. Here, we reconstitute segregation of CD45 from bound receptor–ligand pairs using purified proteins on model membranes. Using a model receptor–ligand pair (FRB–FKBP), we first test physical and computational predictions for protein organization at membrane interfaces. We then show that the TCR–pMHC interaction causes partial exclusion of CD45. Comparing two developmentally regulated isoforms of CD45, the larger RABC variant is excluded more rapidly and efficiently (∼50%) than the smaller R0 isoform (∼20%), suggesting that CD45 isotypes could regulate signaling thresholds in different T cell subtypes. Similar to the sensitivity of T cell signaling, TCR–pMHC interactions with Kds of ≤15 µM were needed to exclude CD45. We further show that the coreceptor PD-1 with its ligand PD-L1, immunotherapy targets that inhibit T cell signaling, also exclude CD45. These results demonstrate that the binding energies of physiological receptor–ligand pairs on the T cell are sufficient to create spatial organization at membrane–membrane interfaces. PMID:29042512

Effect of high moisture storage of pearl millet (Pennisetum typhoides) with or without feed enzymes on growth and nutrient utilization in broiler chickens.

PubMed

Manwar, Satish Jagannath; Mandal, Asit Baran

2009-08-01

Effect of reconstitution of pearl millet with or without enzymes on its utilization in broiler chickens was studied. The pearl millet grains were reconstituted by adding water to raise the moisture level to 30%, followed by storage in sealed plastic buckets with or without feed enzymes (0.5 g/kg) for 21 days at room temperature (25 degrees C). Subsequently, the grains were sun-dried to reduce the moisture content up to 10% to avoid mould growth. Nine dietary treatments were formulated incorporating pearl millet either raw with or without enzymes or reconstituted with or without enzymes in maize-soya based control diet replacing maize at 50 and 75% levels. The birds fed on diets containing enzyme reconstituted pearl millet accrued higher body weight than maize based control diet. Addition of enzymes to raw pearl-millet based diet improved the body weight gain significantly. The reconstitution of pearl millet with or without enzymes increased (P < 0.01) the dietary nitrogen corrected apparent metabolizable energy (AMEn) values and the highest improvement (6.11%) was recorded in diets containing pearl millet reconstituted with enzymes at 75% level of maize replacement. The percent nitrogen retention in pearl millet based diets was comparable to maize based control diet. It may be concluded that the supplementation of the feed enzymes or reconstitution of pearl millet may improve the utilization of pearl millet in broiler chickens.

IL-9-Producing Mast Cell Precursors and Food Allergy

DTIC Science & Technology

2017-10-01

established genetically modified murine strains, a new reconstitution model of experimental food allergy, and the system to acquire duodenal biopsy...development in vivo using murine model of food allergy. Other Subtasks which are designed to study the molecular mechanisms underlying the FcεR signaling...for effective MMC9 expansion using FcεR deficient mice (Fig. 6). The cellular and molecular mechanisms underlining the FcεR signaling pathway will

Identification and Characterization of a Dendritic Cell Precursor in Parenchymal Lung Tissue.

PubMed

von Garnier, Christophe; Blank, Fabian; Rothen-Rutishauser, Barbara; Goethert, Joachim R; Holt, Patrick G; Stumbles, Philip A; Strickland, Deborah H

2017-03-01

The pulmonary parenchymal and mucosal microenvironments are constantly exposed to the external environment and thus require continuous surveillance to maintain steady-state immunological homeostasis. This is achieved by a mobile network of pulmonary dendritic cells (DC) and macrophages (mø) that constantly sample and process microenvironmental antigens into signals that can initiate or dampen inflammation, either locally or after onward migration to draining lymph nodes. The constant steady-state turnover of pulmonary DC and mø requires replenishment from bone marrow precursors; however, the nature of the pulmonary precursor cell (PC) remains unclear, although recent studies suggest that subsets of pulmonary DC may derive from circulating monocytic precursors. In the current study, we describe a population of cells in steady-state mouse lung tissue that has the surface phenotypic and ultrastructural characteristics of a common DC progenitor. Irradiation and reconstitution studies confirmed the bone marrow origins of this PC and showed that it had rapid depletion and reconstitution kinetics that were similar to those of DC, with a 50% repopulation by donor-derived cells by Days 7-9 after reconstitution. This was significantly faster than the rates observed for mø, which showed 50% repopulation by donor-derived cells beyond Days 16-21 after reconstitution. Purified PC gained antigen-presenting function and a cell surface phenotype similar to that of pulmonary DC after maturation in vitro, with light and electron microscopy confirming a myeloid DC morphology. To the best of our knowledge, this is the first study to describe a PC for DC in lung tissue; the findings have implications for the restoration of pulmonary immunological homeostasis after bone marrow transplant.

Wrinkling of a spherical lipid interface induced by actomyosin cortex

NASA Astrophysics Data System (ADS)

Ito, Hiroaki; Nishigami, Yukinori; Sonobe, Seiji; Ichikawa, Masatoshi

2015-12-01

Actomyosin actively generates contractile forces that provide the plasma membrane with the deformation stresses essential to carry out biological processes. Although the contractile property of purified actomyosin has been extensively studied, to understand the physical contribution of the actomyosin contractile force on a deformable membrane is still a challenging problem and of great interest in the field of biophysics. Here, we reconstitute a model system with a cell-sized deformable interface that exhibits anomalous curvature-dependent wrinkling caused by the actomyosin cortex underneath the spherical closed interface. Through a shape analysis of the wrinkling deformation, we find that the dominant contributor to the wrinkled shape changes from bending elasticity to stretching elasticity of the reconstituted cortex upon increasing the droplet curvature radius of the order of the cell size, i.e., tens of micrometers. The observed curvature dependence is explained by the theoretical description of the cortex elasticity and contractility. Our present results provide a fundamental insight into the deformation of a curved membrane induced by the actomyosin cortex.

Mismatch repair deficient hematopoietic stem cells are preleukemic stem cells

PubMed Central

Gerson, Stanton L.

2017-01-01

Whereas transformation events in hematopoietic malignancies may occur at different developmental stages, the initial mutation originates in hematopoietic stem cells (HSCs), creating a preleukemic stem cell (PLSC). Subsequent mutations at either stem cell or progenitor cell levels transform the PLSC into lymphoma/leukemia initiating cells (LIC). Thymic lymphomas have been thought to develop from developing thymocytes. T cell progenitors are generated from HSCs in the bone marrow (BM), but maturation and proliferation of T cells as well as T-lymphomagenesis depends on both regulatory mechanisms and microenvironment within the thymus. We studied PLSC linked to thymic lymphomas. In this study, we use MSH2-/- mice as a model to investigate the existence of PLSC and the evolution of PLSC to LIC. Following BM transplantation, we found that MSH2-/- BM cells from young mice are able to fully reconstitute multiple hematopoietic lineages of lethally irradiated wild-type recipients. However, all recipients developed thymic lymphomas within three and four months post transplantation. Transplantation of different fractions of BM cells or thymocytes from young health MSH2-/- mice showed that an HSC enriched fraction always reconstituted hematopoiesis followed by lymphoma development. In addition, lymphomas did not occur in thymectomized recipients of MSH2-/- BM. These results suggest that HSCs with DNA repair defects such as MSH2-/- are PLSCs because they retain hematopoietic function, but also carry an obligate lymphomagenic potential within their T-cell progeny that is dependent on the thymic microenvironment. PMID:28767666

Bat-mouse bone marrow chimera: a novel animal model for dissecting the uniqueness of the bat immune system.

PubMed

Yong, Kylie Su Mei; Ng, Justin Han Jia; Her, Zhisheng; Hey, Ying Ying; Tan, Sue Yee; Tan, Wilson Wei Sheng; Irac, Sergio Erdal; Liu, Min; Chan, Xue Ying; Gunawan, Merry; Foo, Randy Jee Hiang; Low, Dolyce Hong Wen; Mendenhall, Ian Hewitt; Chionh, Yok Teng; Dutertre, Charles-Antoine; Chen, Qingfeng; Wang, Lin-Fa

2018-03-16

Bats are an important animal model with long lifespans, low incidences of tumorigenesis and an ability to asymptomatically harbour pathogens. Currently, in vivo studies of bats are hampered due to their low reproduction rates. To overcome this, we transplanted bat cells from bone marrow (BM) and spleen into an immunodeficient mouse strain NOD-scid IL-2R -/- (NSG), and have successfully established stable, long-term reconstitution of bat immune cells in mice (bat-mice). Immune functionality of our bat-mouse model was demonstrated through generation of antigen-specific antibody response by bat cells following immunization. Post-engraftment of total bat BM cells and splenocytes, bat immune cells survived, expanded and repopulated the mouse without any observable clinical abnormalities. Utilizing bat's remarkable immunological functions, this novel model has a potential to be transformed into a powerful platform for basic and translational research.

Critical role of CD4 T cells in maintaining lymphoid tissue structure for immune cell homeostasis and reconstitution

PubMed Central

Zeng, Ming; Paiardini, Mirko; Engram, Jessica C.; Beilman, Greg J.; Chipman, Jeffrey G.; Schacker, Timothy W.; Silvestri, Guido

2012-01-01

Loss of the fibroblastic reticular cell (FRC) network in lymphoid tissues during HIV-1 infection has been shown to impair the survival of naive T cells and limit immune reconstitution after antiretroviral therapy. What causes this FRC loss is unknown. Because FRC loss correlates with loss of both naive CD4 and CD8 T-cell subsets and decreased lymphotoxin-β, a key factor for maintenance of FRC network, we hypothesized that loss of naive T cells is responsible for loss of the FRC network. To test this hypothesis, we assessed the consequences of antibody-mediated depletion of CD4 and CD8 T cells in rhesus macaques and sooty mangabeys. We found that only CD4 T-cell depletion resulted in FRC loss in both species and that this loss was caused by decreased lymphotoxin-β mainly produced by the CD4 T cells. We further found the same dependence of the FRC network on CD4 T cells in HIV-1–infected patients before and after antiretroviral therapy and in other immunodeficiency conditions, such as CD4 depletion in cancer patients induced by chemotherapy and irradiation. CD4 T cells thus play a central role in the maintenance of lymphoid tissue structure necessary for their own homeostasis and reconstitution. PMID:22613799

Non-animal sensitization testing: state-of-the-art.

PubMed

Vandebriel, Rob J; van Loveren, Henk

2010-05-01

Predictive tests to identify the sensitizing properties of chemicals are carried out using animals. In the European Union timelines for phasing out many standard animal tests were established for cosmetics. Following this policy, the new European Chemicals Legislation (REACH) favors alternative methods, if validated and appropriate. In this review the authors aim to provide a state-of-the art overview of alternative methods (in silico, in chemico, and in vitro) to identify contact and respiratory sensitizing capacity and in some occasions give a measure of potency. The past few years have seen major advances in QSAR (quantitative structure-activity relationship) models where especially mechanism-based models have great potential, peptide reactivity assays where multiple parameters can be measured simultaneously, providing a more complete reactivity profile, and cell-based assays. Several cell-based assays are in development, not only using different cell types, but also several specifically developed assays such as three-dimenionally (3D)-reconstituted skin models, an antioxidant response reporter assay, determination of signaling pathways, and gene profiling. Some of these assays show relatively high sensitivity and specificity for a large number of sensitizers and should enter validation (or are indeed entering this process). Integrating multiple assays in a decision tree or integrated testing system is a next step, but has yet to be developed. Adequate risk assessment, however, is likely to require significantly more time and efforts.

A microengineered pathophysiological model of early-stage breast cancer.

PubMed

Choi, Yoonseok; Hyun, Eunjeh; Seo, Jeongyun; Blundell, Cassidy; Kim, Hee Chan; Lee, Eunhee; Lee, Su Hyun; Moon, Aree; Moon, Woo Kyung; Huh, Dongeun

2015-08-21

A mounting body of evidence in cancer research suggests that the local microenvironment of tumor cells has a profound influence on cancer progression and metastasis. In vitro studies on the tumor microenvironment and its pharmacological modulation, however, are often hampered by the technical challenges associated with creating physiological cell culture environments that integrate cancer cells with the key components of their native niche such as neighboring cells and extracellular matrix (ECM) to mimic complex microarchitecture of cancerous tissue. Using early-stage breast cancer as a model disease, here we describe a biomimetic microengineering strategy to reconstitute three-dimensional (3D) structural organization and microenvironment of breast tumors in human cell-based in vitro models. Specifically, we developed a microsystem that enabled co-culture of breast tumor spheroids with human mammary ductal epithelial cells and mammary fibroblasts in a compartmentalized 3D microfluidic device to replicate microarchitecture of breast ductal carcinoma in situ (DCIS). We also explored the potential of this breast cancer-on-a-chip system as a drug screening platform by evaluating the efficacy and toxicity of an anticancer drug (paclitaxel). Our microengineered disease model represents the first critical step towards recapitulating pathophysiological complexity of breast cancer, and may serve as an enabling tool to systematically examine the contribution of the breast cancer microenvironment to the progression of DCIS to an invasive form of the disease.

Putative Virulence Factor Expression by Clinical and Food Isolates of Bacillus spp. after Growth in Reconstituted Infant Milk Formulae

PubMed Central

Rowan, Neil J.; Deans, Karen; Anderson, John G.; Gemmell, Curtis G.; Hunter, Iain S.; Chaithong, Thararat

2001-01-01

Forty-seven strains representing 14 different Bacillus species isolated from clinical and food samples were grown in reconstituted infant milk formulae (IMF) and subsequently assessed for adherence to, invasion of, and cytotoxicity toward HEp-2 and Caco-2 cells. Cell-free supernatant fluids from 38 strains (81%) were shown to be cytotoxic, 43 strains (91%) adhered to the test cell lines, and 23 strains (49%) demonstrated various levels of invasion. Of the 21 Bacillus cereus strains examined, 5 (24%) were invasive. A larger percentage of clinically derived Bacillus species (20%) than of similar species tested from the food environment were invasive. Increased invasion occurred after growth of selected Bacillus species in reconstituted IMF containing glucose. While PCR primer studies revealed that many different Bacillus species contained DNA sequences encoding the hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T, not all of these isolates expressed these diarrheagenic genes after growth in reconstituted IMF. Of the 47 Bacillus isolates examined, 3 isolates of B. cereus and 1 isolate of B. subtilis produced the HBL enterotoxin after 18 h of growth in brain heart infusion broth. However, eight isolates belonging to the species B. cereus, B. licheniformis, B. circulans, and B. megaterium were found to produce this enterotoxin after growth in reconstituted IMF when assessed with the B. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (RPLA) kit. It is concluded that several Bacillus species occurring occasionally in clinical specimens and food samples are of potential medical significance due to the expression of putative virulence factors. PMID:11525980

Reconstruction of Multiple Facial Nerve Branches Using Skeletal Muscle-Derived Multipotent Stem Cell Sheet-Pellet Transplantation

PubMed Central

Saito, Kosuke; Tamaki, Tetsuro; Hirata, Maki; Hashimoto, Hiroyuki; Nakazato, Kenei; Nakajima, Nobuyuki; Kazuno, Akihito; Sakai, Akihiro; Iida, Masahiro; Okami, Kenji

2015-01-01

Head and neck cancer is often diagnosed at advanced stages, and surgical resection with wide margins is generally indicated, despite this treatment being associated with poor postoperative quality of life (QOL). We have previously reported on the therapeutic effects of skeletal muscle-derived multipotent stem cells (Sk-MSCs), which exert reconstitution capacity for muscle-nerve-blood vessel units. Recently, we further developed a 3D patch-transplantation system using Sk-MSC sheet-pellets. The aim of this study is the application of the 3D Sk-MSC transplantation system to the reconstitution of facial complex nerve-vascular networks after severe damage. Mouse experiments were performed for histological analysis and rats were used for functional examinations. The Sk-MSC sheet-pellets were prepared from GFP-Tg mice and SD rats, and were transplanted into the facial resection model (ST). Culture medium was transplanted as a control (NT). In the mouse experiment, facial-nerve-palsy (FNP) scoring was performed weekly during the recovery period, and immunohistochemistry was used for the evaluation of histological recovery after 8 weeks. In rats, contractility of facial muscles was measured via electrical stimulation of facial nerves root, as the marker of total functional recovery at 8 weeks after transplantation. The ST-group showed significantly higher FNP (about three fold) scores when compared to the NT-group after 2–8 weeks. Similarly, significant functional recovery of whisker movement muscles was confirmed in the ST-group at 8 weeks after transplantation. In addition, engrafted GFP+ cells formed complex branches of nerve-vascular networks, with differentiation into Schwann cells and perineurial/endoneurial cells, as well as vascular endothelial and smooth muscle cells. Thus, Sk-MSC sheet-pellet transplantation is potentially useful for functional reconstitution therapy of large defects in facial nerve-vascular networks. PMID:26372044

Modified methods for growing 3-D skin equivalents: an update.

PubMed

Lamb, Rebecca; Ambler, Carrie A

2014-01-01

Artificial epidermis can be reconstituted in vitro by seeding primary epidermal cells (keratinocytes) onto a supportive substrate and then growing the developing skin equivalent at the air-liquid interface. In vitro skin models are widely used to study skin biology and for industrial drug and cosmetic testing. Here, we describe updated methods for growing 3-dimensional skin equivalents using de-vitalized, de-epidermalized dermis (DED) substrates including methods for DED substrate preparation, cell seeding, growth conditions, and fixation procedures.

Use of microgravity bioreactors for development of an in vitro rat salivary gland cell culture model

NASA Technical Reports Server (NTRS)

Lewis, M. L.; Moriarity, D. M.; Campbell, P. S.

1993-01-01

During development, salivary gland (SG) cells both secrete factors which modulate cellular behavior and express specific hormone receptors. Whether SG cell growth is modulated by an autocrine epidermal growth factor (EGF) receptor-mediated signal transduction pathway is not clearly understood. SG tissue is the synthesis site for functionally distinct products including growth factors, digestive enzymes, and homeostasis maintaining factors. Historically, SG cells have proven difficult to grow and may be only maintained as limited three-dimensional ductal-type structures in collagen gels or on reconstituted basement membrane gels. A novel approach to establishing primary rat SG cultures is use of microgravity bioreactors originally designed by NASA as low-shear culture systems for predicting cell growth and differentiation in the microgravity environment of space. These completely fluid-filled bioreactors, which are oriented horizontally and rotate, have proven advantageous for Earth-based culture of three-dimensional cell assemblies, tissue-like aggregates, and glandular structures. Use of microgravity bioreactors for establishing in vitro models to investigate steroid-mediated secretion of EGF by normal SG cells may also prove useful for the investigation of cancer and other salivary gland disorders. These microgravity bioreactors promise challenging opportunities for future applications in basic and applied cell research.

A stapled BIM peptide overcomes apoptotic resistance in hematologic cancers

PubMed Central

LaBelle, James L.; Katz, Samuel G.; Bird, Gregory H.; Gavathiotis, Evripidis; Stewart, Michelle L.; Lawrence, Chelsea; Fisher, Jill K.; Godes, Marina; Pitter, Kenneth; Kung, Andrew L.; Walensky, Loren D.

2012-01-01

Cancer cells subvert the natural balance between cellular life and death, achieving immortality through pathologic enforcement of survival pathways and blockade of cell death mechanisms. Pro-apoptotic BCL-2 family proteins are frequently disarmed in relapsed and refractory cancer through genetic deletion or interaction-based neutralization by overexpressed antiapoptotic proteins, resulting in resistance to chemotherapy and radiation treatments. New pharmacologic strategies are urgently needed to overcome these formidable apoptotic blockades. We harnessed the natural killing activity of BCL-2–interacting mediator of cell death (BIM), which contains one of the most potent BH3 death domains of the BCL-2 protein family, to restore BH3-dependent cell death in resistant hematologic cancers. A hydrocarbon-stapled peptide modeled after the BIM BH3 helix broadly targeted BCL-2 family proteins with high affinity, blocked inhibitory antiapoptotic interactions, directly triggered proapoptotic activity, and induced dose-responsive and BH3 sequence–specific cell death of hematologic cancer cells. The therapeutic potential of stapled BIM BH3 was highlighted by the selective activation of cell death in the aberrant lymphoid infiltrates of mice reconstituted with BIM-deficient bone marrow and in a human AML xenograft model. Thus, we found that broad and multimodal targeting of the BCL-2 family pathway can overcome pathologic barriers to cell death. PMID:22622039

Apolipoprotein E3 Mediated Targeted Brain Delivery of Reconstituted High Density Lipoprotein Bearing 3, 10, And 17 Nm Hydrophobic Core Gold Nanoparticles

NASA Astrophysics Data System (ADS)

Chuang, Skylar T.

We have developed a high density lipoprotein (HDL)-based platform for transport and delivery of hydrophobic gold nanoparticles (AuNP). The ability of apolipoprotein E3 (apoE3) to act as a ligand for the low-density lipoprotein receptor (LDLr) was exploited to gain entry of HDL with AuNP into glioblastoma cells. AuNP of 3, 10 and 17 nm diameter, the latter two synthesized by phase transfer process, were solubilized by integration into reconstituted HDL (rHDL). Absorption spectroscopy indicated the presence of stable particles with signature surface plasmon bands, while electron microscopy revealed AuNP embedded in rHDL core. The rHDL-AuNP complexes displayed robust binding to the LDLr, were internalized by the glioblastoma cells, and appeared as aggregated AuNP in the endosomal-lysosomal compartments. The rHDL-AuNP generated little cytotoxicity and were able to cross the blood brain barrier. The findings bear significance since they offer an effective means of delivering AuNP across tumor cell membrane.

Storage Duration of Autologous Stem Cell Preparations Has No Impact on Hematopoietic Recovery after Transplantation.

PubMed

Lisenko, Katharina; Pavel, Petra; Kriegsmann, Mark; Bruckner, Thomas; Hillengass, Jens; Goldschmidt, Hartmut; Witzens-Harig, Mathias; Ho, Anthony D; Wuchter, Patrick

2017-04-01

Peripheral blood stem cells (PBSCs) are widely used for autologous blood stem cell transplantation (ABSCT). These cells must be stored for months or even years, usually at temperatures ≤-140°C, until their use. Although several in vitro studies on CD34 + viability and clonogenic assays of PBSCs after long-term storage have been reported, only a few publications have investigated the influence of long-term storage on in vivo hematopoietic reconstitution. In this study, we retrospectively analyzed hematopoietic recovery after storage of PBSCs via controlled-rate freezing (CRF) and cryostorage in 10% DMSO at ≤-140°C in 105 patients with multiple myeloma who received high-dose melphalan before ABSCT. Three groups of PBSC transplantation (n = 247) were delineated based on the storage period: short-term (≤12 months, n = 143), medium-term (>12 and ≤60 months, n = 75), and long-term storage (>60 months, n = 29). A neutrophil increase of ≥.5 × 10 9 /L in medium-term or long-term PBSC cryopreservation groups was observed at day 14 after ABSCT; this increase was comparable to patients who received briefly stored PBSCs (day 15). No negative effect of PBSC storage duration was observed on leucocyte or neutrophil reconstitution. Platelet reconstitutions of ≥20  × 10 9 /L and 50 × 10 9 /L were observed after median times of 10 to 11 and 13 to 14 days after ABSCT, respectively. No influence of PBSC storage duration on platelet recovery of ≥20  × 10 9 /L and ≥50 × 10 9 /L was observed in the 3 storage groups (P = .07, P = .32). The number of previous ABSCTs also had no significant impact upon hematopoietic reconstitution. In conclusion, these results indicate that long-term cryopreservation of PBSC products at vapor nitrogen temperature after CRF does not have a negative effect on hematopoietic recovery even after prolonged storage. Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Immune reconstitution after haematopoietic cell transplantation in children: immunophenotype analysis with regard to factors affecting the speed of recovery.

PubMed

Kalwak, Krzysztof; Gorczyńska, Ewa; Toporski, Jacek; Turkiewicz, Dominik; Slociak, Malgorzata; Ussowicz, Marek; Latos-Grazyńska, Elzbieta; Król, Marzena; Boguslawska-Jaworska, Janina; Chybicka, Alicja

2002-07-01

Immune reconstitution was studied prospectively in 66 children who underwent 77 haematopoietic cell transplantations (HCT): 46 autologous HCTs in 39 patients and 31 allogeneic HCTs in 27 patients. We studied the dynamic analysis of immune recovery with regard to potential factors affecting its speed, including age, type of HCT, diagnosis, graft-versus-host disease (GvHD) and cytomegalovirus (CMV) infection reactivation. Absolute counts of different lymphocyte subsets and immunoglobulin serum levels were determined in peripheral blood of patients on d -7 and +16, and then at various intervals up to 24 months post transplant. Common patterns of immune recovery after both allogeneic and autologous HCT were identified: (i) CD4+CD45RO+ peripheral T-cell expansion on d +16; (ii) inverted CD4+:CD8+ ratio from d +30 onwards; (iii) rapid natural killer (NK) cell (CD16+/-CD56+) count normalization. We observed prolonged T-cell lymphopenia (CD3+, CD3+CD4+, CD4+CD45RA+) until 24 months after autologous HCT, whereas in the allogeneic setting CD3+CD4+ cells, including naive CD45RA+ cells, returned to normal values at 9 months post transplant. Age > 10 years and coexistence of GvHD and CMV reactivation were associated with a substantial delay in T- (CD4+, including CD45RA+) and B-cell recovery after allogeneic HCT. Multidrug GvHD prophylaxis resulted in impaired T- (CD4+, CD4+CD45RA+) and B-cell reconstitution only in the early phase after allogeneic HCT (up to 4 months). Our results demonstrated that T-cell recovery was severely impaired in children after autologous HCT. It should be emphasized that specific approaches to enhance immune reconstitution are necessary to control minimal residual disease and avoid the risk of infectious complications in the autologous setting. Thymic involution after allogeneic HCT seems to be associated with age and coexistence of GvHD and CMV reactivation.

Long-term human immune system reconstitution in non-obese diabetic (NOD)-Rag (-)-γ chain (-) (NRG) mice is similar but not identical to the original stem cell donor.

PubMed

Harris, D T; Badowski, M; Balamurugan, A; Yang, O O

2013-12-01

The murine immune system is not necessarily identical to it human counterpart, which has led to the construction of humanized mice. The current study analysed whether or not a human immune system contained within the non-obese diabetic (NOD)-Rag1(null) -γ chain(null) (NRG) mouse model was an accurate representation of the original stem cell donor and if multiple mice constructed from the same donor were similar to one another. To that end, lightly irradiated NRG mice were injected intrahepatically on day 1 of life with purified cord blood-derived CD34(+) stem and progenitor cells. Multiple mice were constructed from each cord blood donor. Mice were analysed quarterly for changes in the immune system, and followed for periods up to 12 months post-transplant. Mice from the same donor were compared directly with each other as well as with the original donor. Analyses were performed for immune reconstitution, including flow cytometry, T cell receptor (TCR) and B cell receptor (BCR) spectratyping. It was observed that NRG mice could be 'humanized' long-term using cord blood stem cells, and that animals constructed from the same cord blood donor were nearly identical to one another, but quite different from the original stem cell donor immune system. © 2013 British Society for Immunology.

Restoration of Viral Immunity in Immunodeficient Humans by the Adoptive Transfer of T Cell Clones

NASA Astrophysics Data System (ADS)

Riddell, Stanley R.; Watanabe, Kathe S.; Goodrich, James M.; Li, Cheng R.; Agha, Mounzer E.; Greenberg, Philip D.

1992-07-01

The adoptive transfer of antigen-specific T cells to establish immunity is an effective therapy for viral infections and tumors in animal models. The application of this approach to human disease would require the isolation and in vitro expansion of human antigen-specific T cells and evidence that such T cells persist and function in vivo after transfer. Cytomegalovirus-specific CD8^+ cytotoxic T cell (CTL) clones could be isolated from bone marrow donors, propagated in vitro, and adoptively transferred to immunodeficient bone marrow transplant recipients. No toxicity developed and the clones provided persistent reconstitution of CD8^+ cytomegalovirus-specific CTL responses.

A homozygous CARD9 mutation in a family with susceptibility to fungal infections.

PubMed

Glocker, Erik-Oliver; Hennigs, Andre; Nabavi, Mohammad; Schäffer, Alejandro A; Woellner, Cristina; Salzer, Ulrich; Pfeifer, Dietmar; Veelken, Hendrik; Warnatz, Klaus; Tahami, Fariba; Jamal, Sarah; Manguiat, Annabelle; Rezaei, Nima; Amirzargar, Ali Akbar; Plebani, Alessandro; Hannesschläger, Nicole; Gross, Olaf; Ruland, Jürgen; Grimbacher, Bodo

2009-10-29

Chronic mucocutaneous candidiasis may be manifested as a primary immunodeficiency characterized by persistent or recurrent infections of the mucosa or the skin with candida species. Most cases are sporadic, but both autosomal dominant inheritance and autosomal recessive inheritance have been described. We performed genetic studies in 36 members of a large, consanguineous five-generation family, in which 4 members had recurrent fungal infections and an additional 3 members died during adolescence, 2 after invasive infection of the brain with candida species. All 36 family members were enrolled in the study, and 22 had blood samples taken for DNA analysis. Homozygosity mapping was used to locate the mutated gene. In the 4 affected family members (patients) and the 18 unaffected members we sequenced CARD9, the gene encoding the caspase recruitment domain-containing protein 9, carried out T-cell phenotyping, and performed functional studies, with the use of either leukocytes from the patients or a reconstituted murine model of the genetic defect. We found linkage (lod score, 3.6) to a genomic interval on chromosome 9q, including CARD9. All four patients had a homozygous point mutation in CARD9, resulting in a premature termination codon (Q295X). Healthy family members had wild-type expression of the CARD9 protein; the four patients lacked wild-type expression, which was associated with low numbers of Th17 cells (helper T cells producing interleukin-17). Functional studies based on genetic reconstitution of myeloid cells from Card9(-/-) mice showed that the Q295X mutation impairs innate signaling from the antifungal pattern-recognition receptor dectin-1. An autosomal recessive form of susceptibility to chronic mucocutaneous candidiasis is associated with homozygous mutations in CARD9. 2009 Massachusetts Medical Society

Long-term outcome after haploidentical stem cell transplant and infusion of T cells expressing the inducible caspase 9 safety transgene

PubMed Central

Zhou, Xiaoou; Di Stasi, Antonio; Tey, Siok-Keen; Krance, Robert A.; Martinez, Caridad; Leung, Kathryn S.; Durett, April G.; Wu, Meng-Fen; Liu, Hao; Leen, Ann M.; Savoldo, Barbara; Lin, Yu-Feng; Grilley, Bambi J.; Gee, Adrian P.; Spencer, David M.; Rooney, Cliona M.; Heslop, Helen E.; Brenner, Malcolm K.

2014-01-01

Adoptive transfer of donor-derived T lymphocytes expressing a safety switch may promote immune reconstitution in patients undergoing haploidentical hematopoietic stem cell transplant (haplo-HSCT) without the risk for uncontrolled graft versus host disease (GvHD). Thus, patients who develop GvHD after infusion of allodepleted donor-derived T cells expressing an inducible human caspase 9 (iC9) had their disease effectively controlled by a single administration of a small-molecule drug (AP1903) that dimerizes and activates the iC9 transgene. We now report the long-term follow-up of 10 patients infused with such safety switch-modified T cells. We find long-term persistence of iC9-modified (iC9-T) T cells in vivo in the absence of emerging oligoclonality and a robust immunologic benefit, mediated initially by the infused cells themselves and subsequently by an apparently accelerated reconstitution of endogenous naive T lymphocytes. As a consequence, these patients have immediate and sustained protection from major pathogens, including cytomegalovirus, adenovirus, BK virus, and Epstein-Barr virus in the absence of acute or chronic GvHD, supporting the beneficial effects of this approach to immune reconstitution after haplo-HSCT. This study was registered at www.clinicaltrials.gov as #NCT00710892. PMID:24753538

MCF-10A-NeoST: A New Cell System for Studying Cell-ECM and Cell-Cell Interactions in Breast Cancer

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

Zantek, Nicole Dodge; Walker-Daniels, Jennifer; Stewart, Jane

2001-08-22

There is a continuing need for genetically matched cell systems to model cellular behaviors that are frequently observed in aggressive breast cancers. We report here the isolation and initial characterization of a spontaneously arising variant of MCF-10A cells, NeoST, which provides a new model to study cell adhesion and signal transduction in breast cancer. NeoST cells recapitulate important biological and biochemical features of metastatic breast cancer, including anchorage-independent growth, invasiveness in threedimensional reconstituted membranes, loss of E-cadherin expression, and increased tyrosine kinase activity. A comprehensive analysis of tyrosine kinase expression revealed overexpression or functional activation of the Axl, FAK, andmore » EphA2 tyrosine kinases in transformed MCF-10A cells. MCF-10A and these new derivatives provide a genetically matched model to study defects in cell adhesion and signaling that are relevant to cellular behaviors that often typify aggressive breast cancer cells.« less

Proceedings from the National Cancer Institute's Second International Workshop on the Biology, Prevention, and Treatment of Relapse after Hematopoietic Stem Cell Transplantation: Part I. Biology of relapse after transplantation.

PubMed

Gress, Ronald E; Miller, Jeffrey S; Battiwalla, Minoo; Bishop, Michael R; Giralt, Sergio A; Hardy, Nancy M; Kröger, Nicolaus; Wayne, Alan S; Landau, Dan A; Wu, Catherine J

2013-11-01

In the National Cancer Institute's Second Workshop on the Biology, Prevention, and Treatment of Relapse after Hematopoietic Stem Cell Transplantation, the Scientific/Educational Session on the Biology of Relapse discussed recent advances in understanding some of the host-, disease-, and transplantation-related contributions to relapse, emphasizing concepts with potential therapeutic implications. Relapse after hematopoietic stem cell transplantation (HSCT) represents tumor escape, from the cytotoxic effects of the conditioning regimen and from immunologic control mediated by reconstituted lymphocyte populations. Factors influencing the biology of the therapeutic graft-versus-malignancy (GVM) effect-and relapse-include conditioning regimen effects on lymphocyte populations and homeostasis, immunologic niches, and the tumor microenvironment; reconstitution of lymphocyte populations and establishment of functional immune competence; and genetic heterogeneity within the malignancy defining potential for clonal escape. Recent developments in T cell and natural killer cell homeostasis and reconstitution are reviewed, with implications for prevention and treatment of relapse, as is the application of modern genome sequencing to defining the biologic basis of GVM, clonal escape, and relapse after HSCT. Published by Elsevier Inc.

A majority of mice show long-term expression of a human. beta. -globin gene after retrovirus transfer into hematopoietic stem cells

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

Bender, M.A.; Gelinas, R.E.; Miller

1989-04-01

Murine bone marrow was infected with a high-titer retrovirus vector containing the human {beta}-globin and neomycin phosphotransferase genes. Anemic W/W/sup v/ mice were transplanted with infected marrow which in some cases had been exposed to the selective agent G418. Human {beta}-globin expression was monitored in transplanted animals by using a monoclonal antibody specific for human {beta}-globin polypeptide, and hematopoietic reconstitution was monitored by using donor and recipient mice which differed in hemoglobin type. In some experiments all transplanted mice expressed the human {beta}-globin polypeptide for over 4 months, and up to 50% of peripheral erythrocytes contained detectable levels of polypeptide.more » DNA analysis of transplanted animals revealed that virtually every myeloid cell contained a provirus. Integration site analysis and reconstitution of secondary marrow recipients suggested that every mouse was reconstituted with at least one infected stem cell which had extensive repopulation capability. The ability to consistently transfer an active {beta}-globin gene into mouse hematopoietic cells improves the feasibility of using these techniques for somatic cell gene therapy in humans.« less

Human eccrine sweat gland cells reconstitute polarized spheroids when subcutaneously implanted with Matrigel in nude mice.

PubMed

Li, Haihong; Zhang, Mingjun; Chen, Liyun; Li, Xuexue; Zhang, Bingna

2016-10-01

Increasing evidence indicates that maintenance of cell polarity plays a pivotal role in the regulation of glandular homeostasis and function. We examine the markers for polarity at different time points to investigate the formation of cell polarity during 3D reconstitution of eccrine sweat glands. Mixtures of eccrine sweat gland cells and Matrigel were injected subcutaneously into the inguinal regions of nude mice. At 2, 3, 4, 5 and 6 weeks post-implantation, Matrigel plugs were removed and immunostained for basal collagen IV, lateral β-catenin, lateroapical ZO-1 and apical F-actin. The results showed that the cell polarity of the spheroids appeared in sequence. Formation of basal polarity was prior to lateral, apical and lateroapical polarity. Collagen IV was detected basally at 2 weeks, β-catenin laterally and ZO-1 lateroapically at 3 weeks, and F-actin apically at 4 weeks post-implantation. At week 5 and week 6, the localization and the positive percentage of collagen IV, β-catenin, ZO-1 or F-actin in spheroids was similar to that in native eccrine sweat glands. We conclude that the reconstituted 3D eccrine sweat glands are functional or potentially functional.

NK Cells and Other Innate Lymphoid Cells in Hematopoietic Stem Cell Transplantation.

PubMed

Vacca, Paola; Montaldo, Elisa; Croxatto, Daniele; Moretta, Francesca; Bertaina, Alice; Vitale, Chiara; Locatelli, Franco; Mingari, Maria Cristina; Moretta, Lorenzo

2016-01-01

Natural killer (NK) cells play a major role in the T-cell depleted haploidentical hematopoietic stem cell transplantation (haplo-HSCT) to cure high-risk leukemias. NK cells belong to the expanding family of innate lymphoid cells (ILCs). At variance with NK cells, the other ILC populations (ILC1/2/3) are non-cytolytic, while they secrete different patterns of cytokines. ILCs provide host defenses against viruses, bacteria, and parasites, drive lymphoid organogenesis, and contribute to tissue remodeling. In haplo-HSCT patients, the extensive T-cell depletion is required to prevent graft-versus-host disease (GvHD) but increases risks of developing a wide range of life-threatening infections. However, these patients may rely on innate defenses that are reconstituted more rapidly than the adaptive ones. In this context, ILCs may represent important players in the early phases following transplantation. They may contribute to tissue homeostasis/remodeling and lymphoid tissue reconstitution. While the reconstitution of NK cell repertoire and its role in haplo-HSCT have been largely investigated, little information is available on ILCs. Of note, CD34(+) cells isolated from different sources of HSC may differentiate in vitro toward various ILC subsets. Moreover, cytokines released from leukemia blasts (e.g., IL-1β) may alter the proportions of NK cells and ILC3, suggesting the possibility that leukemia may skew the ILC repertoire. Further studies are required to define the timing of ILC development and their potential protective role after HSCT.

Caenorhabditis elegans vulval cell fate patterning

NASA Astrophysics Data System (ADS)

Félix, Marie-Anne

2012-08-01

The spatial patterning of three cell fates in a row of competent cells is exemplified by vulva development in the nematode Caenorhabditis elegans. The intercellular signaling network that underlies fate specification is well understood, yet quantitative aspects remain to be elucidated. Quantitative models of the network allow us to test the effect of parameter variation on the cell fate pattern output. Among the parameter sets that allow us to reach the wild-type pattern, two general developmental patterning mechanisms of the three fates can be found: sequential inductions and morphogen-based induction, the former being more robust to parameter variation. Experimentally, the vulval cell fate pattern is robust to stochastic and environmental challenges, and minor variants can be detected. The exception is the fate of the anterior cell, P3.p, which is sensitive to stochastic variation and spontaneous mutation, and is also evolving the fastest. Other vulval precursor cell fates can be affected by mutation, yet little natural variation can be found, suggesting stabilizing selection. Despite this fate pattern conservation, different Caenorhabditis species respond differently to perturbations of the system. In the quantitative models, different parameter sets can reconstitute their response to perturbation, suggesting that network variation among Caenorhabditis species may be quantitative. Network rewiring likely occurred at longer evolutionary scales.

Combination immunotherapy and active-specific tumor cell vaccination augments anti-cancer immunity in a mouse model of gastric cancer

PubMed Central

2011-01-01

Background Active-specific immunotherapy used as an adjuvant therapeutic strategy is rather unexplored for cancers with poorly characterized tumor antigens like gastric cancer. The aim of this study was to augment a therapeutic immune response to a low immunogenic tumor cell line derived from a spontaneous gastric tumor of a CEA424-SV40 large T antigen (CEA424-SV40 TAg) transgenic mouse. Methods Mice were treated with a lymphodepleting dose of cyclophosphamide prior to reconstitution with syngeneic spleen cells and vaccination with a whole tumor cell vaccine combined with GM-CSF (a treatment strategy abbreviated as LRAST). Anti-tumor activity to subcutaneous tumor challenge was examined in a prophylactic as well as a therapeutic setting and compared to corresponding controls. Results LRAST enhances tumor-specific T cell responses and efficiently inhibits growth of subsequent transplanted tumor cells. In addition, LRAST tended to slow down growth of established tumors. The improved anti-tumor immune response was accompanied by a transient decrease in the frequency and absolute number of CD4+CD25+FoxP3+ T cells (Tregs). Conclusions Our data support the concept that whole tumor cell vaccination in a lymphodepleted and reconstituted host in combination with GM-CSF induces therapeutic tumor-specific T cells. However, the long-term efficacy of the treatment may be dampened by the recurrence of Tregs. Strategies to counteract suppressive immune mechanisms are required to further evaluate this therapeutic vaccination protocol. PMID:21859450

Inflammation Causes Resistance to Anti-CD20-Mediated B Cell Depletion.

PubMed

Laws, L H; Parker, C E; Cherala, G; Koguchi, Y; Waisman, A; Slifka, M K; Oberbarnscheidt, M H; Obhrai, J S; Yeung, M Y; Riella, L V

2016-11-01

B cells play a central role in antibody-mediated rejection and certain autoimmune diseases. However, B cell-targeted therapy such as anti-CD20 B cell-depleting antibody (aCD20) has yielded mixed results in improving outcomes. In this study, we investigated whether an accelerated B cell reconstitution leading to aCD20 depletion resistance could account for these discrepancies. Using a transplantation model, we found that antigen-independent inflammation, likely through toll-like receptor (TLR) signaling, was sufficient to mitigate B cell depletion. Secondary lymphoid organs had a quicker recovery of B cells when compared to peripheral blood. Inflammation altered the pharmacokinetics (PK) and pharmacodynamics (PD) of aCD20 therapy by shortening drug half-life and accelerating the reconstitution of the peripheral B cell pool by bone marrow-derived B cell precursors. IVIG (intravenous immunoglobulin) coadministration also shortened aCD20 drug half-life and led to accelerated B cell recovery. Repeated aCD20 dosing restored B cell depletion and delayed allograft rejection, especially B cell-dependent, antibody-independent allograft rejection. These data demonstrate the importance of further clinical studies of the PK/PD of monoclonal antibody treatment in inflammatory conditions. The data also highlight the disconnect between B cell depletion on peripheral blood compared to secondary lymphoid organs, the deleterious effect of IVIG when given with aCD20 and the relevance of redosing of aCD20 for effective B cell depletion in alloimmunity. © Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

Question 7: Biosynthesis of Phosphatidic Acid in Liposome Compartments Toward the Self-Reproduction of Minimal Cells

NASA Astrophysics Data System (ADS)

Kuruma, Yutetsu

2007-10-01

Self-reproduction is one of main properties that define living cells. In order to explore the self-reproduction process for the study of early cells, and to develop a research line somehow connected to the origin of life, we have built up a constructive ‘synthetic cells (minimal cells)’ approach. The minimal cells approach consists in the investigation of the minimal number of elements to accomplish simple cell-like processes like self-reproduction. Such approach belongs to the field of synthetic biology. The minimal cells are reconstructed from a totally reconstituted cell-free protein synthesis system (PURESYSTEM) and liposome compartments as containers. Based on this approach, we synthesized two membrane proteins (enzymes), GPAT and LPAAT, which are involved in the phosphatidic acid biosynthesis in bacteria. Both membrane proteins were successfully synthesized by PURESYSTEM encapsulated inside POPC liposomes. Additionally, the enzymatic activity of GPAT was restored by mixing the expressed enzyme with lipid and by forming liposomes in situ. Through these experimental evidences, here we present a possible model to achieve self-reproduction in minimal cells. Our results would contribute to the idea that early cells could have been built by an extremely small number of genes.

The Adult Livers of Immunodeficient Mice Support Human Hematopoiesis: Evidence for a Hepatic Mast Cell Population that Develops Early in Human Ontogeny

PubMed Central

Muench, Marcus O.; Beyer, Ashley I.; Fomin, Marina E.; Thakker, Rahul; Mulvaney, Usha S.; Nakamura, Masato; Suemizu, Hiroshi; Bárcena, Alicia

2014-01-01

The liver plays a vital role in hematopoiesis during mammalian prenatal development but its hematopoietic output declines during the perinatal period. Nonetheless, hepatic hematopoiesis is believed to persist into adulthood. We sought to model human adult-liver hematopoiesis by transplantation of fetal and neonatal hematopoietic stem cells (HSCs) into adult immunodeficient mice. Livers were found to be engrafted with human cells consisting primarily of monocytes and B-cells with lesser contributions by erythrocytes, T-cells, NK-cells and mast-cells. A resident population of CD117++CD203c+ mast cells was also documented in human midgestation liver, indicating that these cells comprise part of the liver's resident immune cell repertoire throughout human ontogeny. The murine liver was shown to support human multilineage hematopoiesis up to 321 days after transplant. Evidence of murine hepatic hematopoiesis was also found in common mouse strains as old as 2 years. Human HSC engraftment of the murine liver was demonstrated by detection of high proliferative-potential colony-forming cells in clonal cultures, observation of CD38−CD34++ and CD133+CD34++ cells by flow cytometry, and hematopoietic reconstitution of secondary transplant recipients of chimeric liver cells. Additionally, chimeric mice with both hematopoietic and endothelial reconstitution were generated by intrasplenic injection of immunodeficient mice with liver specific expression of the urokinase-type plasminogen activator (uPA) transgene. In conclusion, the murine liver is shown to be a hematopoietic organ throughout adult life that can also support human hematopoiesis in severely immunodeficient strains. Further humanization of the murine liver can be achieved in mice harboring an uPA transgene, which support engraftment of non-hematopoietic cells types. Thus, offering a model system to study the interaction of diverse human liver cell types that regulate hematopoiesis and immune function in the liver. PMID:24819392

Architecture of the ring formed by the tubulin homologue FtsZ in bacterial cell division

PubMed Central

Szwedziak, Piotr; Wang, Qing; Bharat, Tanmay A M; Tsim, Matthew; Löwe, Jan

2014-01-01

Membrane constriction is a prerequisite for cell division. The most common membrane constriction system in prokaryotes is based on the tubulin homologue FtsZ, whose filaments in E. coli are anchored to the membrane by FtsA and enable the formation of the Z-ring and divisome. The precise architecture of the FtsZ ring has remained enigmatic. In this study, we report three-dimensional arrangements of FtsZ and FtsA filaments in C. crescentus and E. coli cells and inside constricting liposomes by means of electron cryomicroscopy and cryotomography. In vivo and in vitro, the Z-ring is composed of a small, single-layered band of filaments parallel to the membrane, creating a continuous ring through lateral filament contacts. Visualisation of the in vitro reconstituted constrictions as well as a complete tracing of the helical paths of the filaments with a molecular model favour a mechanism of FtsZ-based membrane constriction that is likely to be accompanied by filament sliding. DOI: http://dx.doi.org/10.7554/eLife.04601.001 PMID:25490152

Immunologic reconstitution during PEG-ADA therapy in an unusual mosaic ADA deficient patient.

PubMed

Liu, Ping; Santisteban, Ines; Burroughs, Lauri M; Ochs, Hans D; Torgerson, Troy R; Hershfield, Michael S; Rawlings, David J; Scharenberg, Andrew M

2009-02-01

We report detailed genetic and immunologic studies in a patient diagnosed with adenosine deaminase (ADA) deficiency and combined immune deficiency at age 5 years. At the time of diagnosis, although all other lymphocyte subsets were depleted, circulating CD8(+) T cells with a terminally differentiated phenotype were abundant and expressed normal ADA activity due to a reversion mutation in a CD8(+) T cell or precursor. Over the first 9 months of replacement therapy with PEG-ADA, the patient steadily accumulated mature naïve CD4(+) and CD8(+) T cells, as well as CD4(+)/FOXP3(+) regulatory T cells, consistent with restoration of a functional cellular immune system. While CD19(+) naïve B cells also accumulated in response to PEG-ADA therapy, a high proportion of these B cells exhibited an immature surface marker phenotype even after 9 months, and immunization with neoantigen bacteriophage varphiX174 demonstrated a markedly subnormal humoral immune response. Our observations in this single patient have important implications for gene therapy of human ADA deficiency, as they indicate that ADA expression within even a large circulating lymphocyte population may not be sufficient to support adequate immune reconstitution. They also suggest that an immature surface marker phenotype of the peripheral B cell compartment may be a useful surrogate marker for incomplete humoral immune reconstitution during enzyme replacement, and possibly other forms of hematopoietic cell therapies.

Correction of murine Rag2 severe combined immunodeficiency by lentiviral gene therapy using a codon-optimized RAG2 therapeutic transgene.

PubMed

van Til, Niek P; de Boer, Helen; Mashamba, Nomusa; Wabik, Agnieszka; Huston, Marshall; Visser, Trudi P; Fontana, Elena; Poliani, Pietro Luigi; Cassani, Barbara; Zhang, Fang; Thrasher, Adrian J; Villa, Anna; Wagemaker, Gerard

2012-10-01

Recombination activating gene 2 (RAG2) deficiency results in severe combined immunodeficiency (SCID) with complete lack of T and B lymphocytes. Initial gammaretroviral gene therapy trials for other types of SCID proved effective, but also revealed the necessity of safe vector design. We report the development of lentiviral vectors with the spleen focus forming virus (SF) promoter driving codon-optimized human RAG2 (RAG2co), which improved phenotype amelioration compared to native RAG2 in Rag2(-/-) mice. With the RAG2co therapeutic transgene, T-cell receptor (TCR) and immunoglobulin repertoire, T-cell mitogen responses, plasma immunoglobulin levels and T-cell dependent and independent specific antibody responses were restored. However, the thymus double positive T-cell population remained subnormal, possibly due to the SF virus derived element being sensitive to methylation/silencing in the thymus, which was prevented by replacing the SF promoter by the previously reported silencing resistant element (ubiquitous chromatin opening element (UCOE)), and also improved B-cell reconstitution to eventually near normal levels. Weak cellular promoters were effective in T-cell reconstitution, but deficient in B-cell reconstitution. We conclude that immune functions are corrected in Rag2(-/-) mice by genetic modification of stem cells using the UCOE driven codon-optimized RAG2, providing a valid optional vector for clinical implementation.

Immune Reconstitution After Allogeneic Hematopoietic Stem Cell Transplantation and Association With Occurrence and Outcome of Invasive Aspergillosis.

PubMed

Stuehler, Claudia; Kuenzli, Esther; Jaeger, Veronika K; Baettig, Veronika; Ferracin, Fabrizia; Rajacic, Zarko; Kaiser, Deborah; Bernardini, Claudia; Forrer, Pascal; Weisser, Maja; Elzi, Luigia; Battegay, Manuel; Halter, Joerg; Passweg, Jakob; Khanna, Nina

2015-09-15

Invasive aspergillosis (IA) remains a leading cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplantation (HSCT). To date, no reliable immunological biomarkers for management and outcome of IA exist. Here, we investigated reconstitution of antifungal immunity in patients during the first 12 months after HSCT and correlated it with IA. Fifty-one patients were included, 9 with probable/proven IA. We determined quantitative and qualitative reconstitution of polymorphonuclear (PMN), CD4, CD8, and natural killer (NK) cells against Aspergillus fumigatus over 5 time points and compared the values to healthy donors. Absolute CD4 and CD8 cell counts, antigen-specific T-cell responses, and killing capacity of PMN against A. fumigatus were significantly decreased in all patients over 12 months. In patients with probable/proven IA, reactive oxygen species (ROS) production tended to be lower compared to patients without IA, and absolute NK-cell counts remained below 200 cells/µL. Patients with well-controlled IA showed significantly higher ROS production and NK-cell counts compared to patients with poor outcome. This study highlights the importance of functional PMN, T-cell, and NK-cell immunity for the outcome of IA. Larger multicenter studies should address the potential use of NK-cell counts for the management of antifungal therapy. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Ex Vivo Expanded Human Regulatory T Cells Can Prolong Survival of a Human Islet Allograft in a Humanized Mouse Model

PubMed Central

Wu, Douglas C.; Hester, Joanna; Nadig, Satish N.; Zhang, Wei; Trzonkowski, Piotr; Gray, Derek; Hughes, Stephen; Johnson, Paul; Wood, Kathryn J.

2013-01-01

Background Human regulatory T cells (Treg) offer an attractive adjunctive therapy to reduce current reliance on lifelong, nonspecific immunosuppression after transplantation. Here, we evaluated the ability of ex vivo expanded human Treg to prevent the rejection of islets of Langerhans in a humanized mouse model and examined the mechanisms involved. Methods We engrafted human pancreatic islets of Langerhans into the renal subcapsular space of immunodeficient BALB/c.rag2−/−.cγ−/− mice, previously rendered diabetic via injection of the β-cell toxin streptozocin. After the establishment of stable euglycemia, mice were reconstituted with allogeneic human peripheral blood mononuclear cells (PBMC) and the resultant alloreactive response studied. Ex vivo expanded CD25highCD4+ human Treg, which expressed FoxP3, CTLA-4, and CD62L and remained CD127low, were then cotransferred together with human PBMC and islet allografts and monitored for evidence of rejection. Results Human islets transplanted into diabetic immunodeficient mice reversed diabetes but were rejected rapidly after the mice were reconstituted with allogeneic human PBMC. Cotransfer of purified, ex vivo expanded human Treg prolonged islet allograft survival resulting in the accumulation of Treg in the peripheral lymphoid tissue and suppression of proliferation and interferon-γ production by T cells. In vitro, Treg suppressed activation of signal transducers and activators of transcription and inhibited the effector differentiation of responder T cells. Conclusions Ex vivo expanded Treg retain regulatory activity in vivo, can protect a human islet allograft from rejection by suppressing signal transducers and activators of transcription activation and inhibiting T-cell differentiation, and have clinical potential as an adjunctive cellular therapy. PMID:23917725

Transplantation of motoneurons derived from MASH1-transfected mouse ES cells reconstitutes neural networks and improves motor function in hemiplegic mice.

PubMed

Ikeda, Ritsuko; Kurokawa, Manae S; Chiba, Shunmei; Yoshikawa, Hideshi; Hashimoto, Takuo; Tadokoro, Mamoru; Suzuki, Noboru

2004-10-01

Mouse embryonic stem (ES) cells were transfected with a MASH1 expression vector and G418-resistant cells were selected. The MASH1-transfected cells became neuron-like appearance and expressed betaIIItubulin and panNCAM. Glial fibrillary acidic protein (GFAP) and galactocerebroside (GalC)-expressing cells were rarely detected. Half of the neural cells differentiated into the Islet1+ motoneuron lineage. Thus, we obtained motoneuron lineage-enriched neuronal cells by transfection of ES cells with MASH1. A hemiplegic model of mice was developed by cryogenic injury of the motor cortex, and motoneuron lineage-enriched neuronal cells were transplanted underneath the injured motor cortex neighboring the periventricular region. The motor function of the recipients was assessed by a beam walking and rotarod tests, whereby the results gradually improved, but little improvement was observed in vehicle injected control mice. We found that the grafted cells not only remained close to the implantation site, but also exhibited substantial migration, penetrating into the damaged lesion in a directed manner up to the cortical region. Grafted neuronal cells that had migrated into the cortex were elongated axon-positive for neurofilament middle chain (NFM). Synaptophysin immunostaining showed a positive staining pattern around the graft, suggesting that the transplanted neurons interacted with the recipient neurons to form a neural network. Our study suggests that the motoneuron lineage can be induced from ES cells, and grafted cells adapt to the host environment and can reconstitute a neural network to improve motor function of a paralyzed limb.

A simplified approach to quasi-linear viscoelastic modeling

PubMed Central

Nekouzadeh, Ali; Pryse, Kenneth M.; Elson, Elliot L.; Genin, Guy M.

2007-01-01

The fitting of quasi-linear viscoelastic (QLV) constitutive models to material data often involves somewhat cumbersome numerical convolution. A new approach to treating quasi-linearity in one dimension is described and applied to characterize the behavior of reconstituted collagen. This approach is based on a new principle for including nonlinearity and requires considerably less computation than other comparable models for both model calibration and response prediction, especially for smoothly applied stretching. Additionally, the approach allows relaxation to adapt with the strain history. The modeling approach is demonstrated through tests on pure reconstituted collagen. Sequences of “ramp-and-hold” stretching tests were applied to rectangular collagen specimens. The relaxation force data from the “hold” was used to calibrate a new “adaptive QLV model” and several models from literature, and the force data from the “ramp” was used to check the accuracy of model predictions. Additionally, the ability of the models to predict the force response on a reloading of the specimen was assessed. The “adaptive QLV model” based on this new approach predicts collagen behavior comparably to or better than existing models, with much less computation. PMID:17499254

Long-term monoclonal reconstitution of erythropoiesis in genetically anemic W/Wv mice by injection of 5-fluorouracil-treated bone marrow cells of Pgk-1b/Pgk-1a mice

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

Nakano, T.; Waki, N.; Asai, H.

The spleen colony-forming assay does not represent the number of hematopoietic stem cells with extensive self-maintaining capacity because five to 50 spleen colony-forming units (CFU-S) are necessary to rescue a genetically anemic (WB X C57BL/6)F1-W/Wv(WBB6F1-W/Wv) mouse. We investigated which is more important for the reconstitution of erythropoiesis, the transplantation of multiple CFU-S or that of a single stem cell with extensive self-maintaining potential. The electrophoretic pattern of hemoglobin was used as a marker of reconstitution and that of phosphoglycerate kinase (PGK), an X chromosome-linked enzyme, as a tool for estimating the number of stem cells. For this purpose, we developedmore » the C57BL/6 congeneic strain with the Pgk-1a gene. Bone marrow cells were harvested after injection of 5-fluorouracil from C57BL/6-Pgk-1b/Pgk-1a female mice in which each stem cell had either A-type PGK or B-type PGK due to the random inactivation of one or two X chromosomes. When a relatively small number of bone marrow cells (ie, 10(3) or 3 X 10(3) were injected into 200-rad-irradiated WBB6F1-W/Wv mice, the hemoglobin pattern changed from the recipient type (Hbbd/Hbbs) to the donor type (Hbbs/Hbbs) in seven of 150 mice for at least 8 weeks. Erythrocytes of all these WBB6F1-W/Wv mice showed either A-type PGK alone or B-type PGK alone during the time of reconstitution, which suggests that a single stem cell with extensive self-maintaining potential may sustain the whole erythropoiesis of a mouse for at least 8 weeks.« less

Functional Reconstitution of Staphylococcus aureus Truncated AgrC Histidine Kinase in a Model Membrane System

PubMed Central

Liu, Baoquan; Wang, Jianfeng; Xiong, Wen; Zhao, Pengchao; Fan, Shengdi

2013-01-01

The integral membrane protein AgrC is a histidine kinase whose sensor domains interact with an autoinducing peptide, resulting in a series of downstream responses. In this study, truncated AgrCTM5-6C and AgrCTM5-6C-GFP with GFP as a reporter gene were produced using a bacterial system. Purified AgrCTM5-6C and AgrCTM5-6C-GFP were reconstituted into liposomes by a detergent-mediated method. To achieve high-yield protein incorporation, we investigated the effect of different detergents on protein reconstitution efficiency. The highest incorporation was found with N,N-dimethyldode-cylamine N-oxide during complete liposome solubilization, which resulted in a yield of 85±5%. The COOH-terminus of the protein AgrCTM5-6C was almost exclusively oriented towards the inside of the vesicles. AgrCTM5-6C in proteoliposomes exhibited approximately a 6-fold increase in constitutive activity compared with AgrCTM5-6C in detergent micelles. The reconstitution of AgrCTM5-6C or AgrCTM5-6C-GFP was characterized using dynamic light scattering, fluorescence microscopy, and transmission electron microscopy. Based on the results, the optimal conditions for protein incorporation were defined. These findings contribute to the study of membrane protein structure and function in vitro using a reconstitution system. PMID:24303011

Non-CD34+ cells, especially CD8+ cytotoxic T cells and CD56+ natural killer cells, rather than CD34 cells, predict early engraftment and better transplantation outcomes in patients with hematologic malignancies after allogeneic peripheral stem cell transplantation.

PubMed

Kim, Dong Hwan; Won, Dong Il; Lee, Nan Young; Sohn, Sang Kyun; Suh, Jang Soo; Lee, Kyu Bo

2006-07-01

The effect of the transplant dose of each cell subset on engraftment kinetics and transplantation outcomes was evaluated in HLA-identical allogeneic peripheral blood stem cell transplantation (PBSCT). Sixty-nine patients were included in this retrospective study. Engraftment kinetics, transplantation outcomes, and immune reconstitution up to 1 year after transplantation were analyzed according to the transplant dose of CD34+ and non-CD34+ cells, including natural killer (NK) cells and CD8+ cytotoxic T (Tc) cells. An accelerated neutrophil engraftment was strongly associated with a higher transplant dose of NK cells (12 versus 16 days, P < .001) and Tc cells (13 versus 16 days, P < .001) but not CD34+ cells (P = .442). Survival analyses revealed a favorable prognosis for patients who received a higher dose of non-CD34+ cell subsets, rather than CD34+ cells, in terms of overall survival (OS; P = .024 for NK cells and .050 for Tc cells) and nonrelapse mortality (NRM; P = .005 for NK cells, .060 for Tc cells). In addition, a higher transplant dose of NK and Tc cells was correlated with a faster lymphoid reconstitution. In multivariate analyses, rapid neutrophil engraftment was correlated with a higher transplant dose of NK cells (P = .001) and Tc cells (P = .004). Moreover, an increased OS was associated with the NK cell dose (P = .007) and chronic graft-versus-host disease (P = .009), whereas a decreased NRM was associated with the NK dose (P = .024). In conclusion, in a PBSCT setting, a higher transplant dose of NK and Tc cells accelerated neutrophil engraftment, improved the immune reconstitution, and decreased NRM, thereby increasing OS after allogeneic PBSCT.

Poised Regeneration of Zebrafish Melanocytes Involves Direct Differentiation and Concurrent Replenishment of Tissue-Resident Progenitor Cells.

PubMed

Iyengar, Sharanya; Kasheta, Melissa; Ceol, Craig J

2015-06-22

Efficient regeneration following injury is critical for maintaining tissue function and enabling organismal survival. Cells reconstituting damaged tissue are often generated from resident stem or progenitor cells or from cells that have dedifferentiated and become proliferative. While lineage-tracing studies have defined cellular sources of regeneration in many tissues, the process by which these cells execute the regenerative process is largely obscure. Here, we have identified tissue-resident progenitor cells that mediate regeneration of zebrafish stripe melanocytes and defined how these cells reconstitute pigmentation. Nearly all regeneration melanocytes arise through direct differentiation of progenitor cells. Wnt signaling is activated prior to differentiation, and inhibition of Wnt signaling impairs regeneration. Additional progenitors divide symmetrically to sustain the pool of progenitor cells. Combining direct differentiation with symmetric progenitor divisions may serve as a means to rapidly repair injured tissue while preserving the capacity to regenerate. Copyright © 2015 Elsevier Inc. All rights reserved.

Intensive fibrosarcoma-binding capability of the reconstituted analog and its antitumor activity.

PubMed

Xu, Jian; Du, Yue; Liu, Wen-Juan; Li, Liang; Li, Yi; Wang, Xiao-Fei; Yi, Hong-Fei; Shan, Chuan-Kun; Xia, Gui-Min; Liu, Xiu-Jun; Zhen, Yong-Su

2018-11-01

Fibrosarcomas are highly aggressive malignant tumors. It is urgently needed to explore targeted drugs and modalities for more effective therapy. Matrix metalloproteinases (MMPs) play important roles in tumor progression and metastasis, while several MMPs are highly expressed in fibrosarcomas. In addition, tissue inhibitor of metalloproteinase 2 (TIMP2) displays specific interaction with MMPs. Therefore, TIMP2 may play an active role in the development of fibrosarcoma-targeting agents. In the current study, a TIMP2-based recombinant protein LT and its enediyne-integrated analog LTE were prepared; furthermore, the fibrosarcoma-binding intensity and antitumor activity were investigated. As shown, intense and selective binding capability of the protein LT to human fibrosarcoma specimens was confirmed by tissue microarray. Moreover, LTE, the enediyne-integrated analog of LT, exerted highly potent cytotoxicity to fibrosarcoma HT1080 cells, induced apoptosis, and caused G2/M arrest. LTE at 0.1 nM markedly suppressed the migration and invasion of HT1080 cells. LTE at tolerated dose of 0.6 mg/kg inhibited the tumor growth of fibrosarcoma xenograft in athymic mice. The study provides evidence that the TIMP2-based reconstituted analog LTE may be useful as a targeted drug for fibrosarcome therapy.

C/EBPβ in bone marrow is essential for diet induced inflammation, cholesterol balance, and atherosclerosis.

PubMed

Rahman, Shaikh M; Baquero, Karalee C; Choudhury, Mahua; Janssen, Rachel C; de la Houssaye, Becky A; Sun, Ming; Miyazaki-Anzai, Shinobu; Wang, Shu; Moustaid-Moussa, Naima; Miyazaki, Makoto; Friedman, Jacob E

2016-07-01

Atherosclerosis is both a chronic inflammatory disease and a lipid metabolism disorder. C/EBPβ is well documented for its role in the development of hematopoietic cells and integration of lipid metabolism. However, C/EBPβ's role in atherosclerotic progression has not been examined. We assessed the impact of hematopoietic CEBPβ deletion in ApoE(-/-) mice on hyperlipidemia, inflammatory responses and lesion formation in the aorta. ApoE(-/-) mice were reconstituted with bone marrow cells derived from either WT or C/EBPβ(-/-) mice and placed on low fat or high fat/high cholesterol diet for 11 weeks. Hematopoietic C/EBPβ deletion in ApoE(-/-) mice reduced blood and hepatic lipids and gene expression of hepatic stearoyl CoA desaturase 1 and fatty acid synthase while expression of ATP binding cassette transporter G1, cholesterol 7-alpha-hydroxylase, and liver X receptor alpha genes were significantly increased. ApoE(-/-) mice reconstituted with C/EBPβ(-/-) bone marrow cells also significantly reduced blood cytokine levels and reduced lesion area in aortic sinuses compared with ApoE(-/-) mice reconstituted with WT bone marrow cells. Silencing of C/EBPβ in RAW264.7 macrophage cells prevented oxLDL-mediated foam cell formation and inflammatory cytokine secretion in conditioned medium. C/EBPβ in hematopoietic cells is crucial to regulate diet-induced inflammation, hyperlipidemia and atherosclerosis development. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Long-term outcome after haploidentical stem cell transplant and infusion of T cells expressing the inducible caspase 9 safety transgene.

PubMed

Zhou, Xiaoou; Di Stasi, Antonio; Tey, Siok-Keen; Krance, Robert A; Martinez, Caridad; Leung, Kathryn S; Durett, April G; Wu, Meng-Fen; Liu, Hao; Leen, Ann M; Savoldo, Barbara; Lin, Yu-Feng; Grilley, Bambi J; Gee, Adrian P; Spencer, David M; Rooney, Cliona M; Heslop, Helen E; Brenner, Malcolm K; Dotti, Gianpietro

2014-06-19

Adoptive transfer of donor-derived T lymphocytes expressing a safety switch may promote immune reconstitution in patients undergoing haploidentical hematopoietic stem cell transplant (haplo-HSCT) without the risk for uncontrolled graft versus host disease (GvHD). Thus, patients who develop GvHD after infusion of allodepleted donor-derived T cells expressing an inducible human caspase 9 (iC9) had their disease effectively controlled by a single administration of a small-molecule drug (AP1903) that dimerizes and activates the iC9 transgene. We now report the long-term follow-up of 10 patients infused with such safety switch-modified T cells. We find long-term persistence of iC9-modified (iC9-T) T cells in vivo in the absence of emerging oligoclonality and a robust immunologic benefit, mediated initially by the infused cells themselves and subsequently by an apparently accelerated reconstitution of endogenous naive T lymphocytes. As a consequence, these patients have immediate and sustained protection from major pathogens, including cytomegalovirus, adenovirus, BK virus, and Epstein-Barr virus in the absence of acute or chronic GvHD, supporting the beneficial effects of this approach to immune reconstitution after haplo-HSCT. This study was registered at www.clinicaltrials.gov as #NCT00710892. © 2014 by The American Society of Hematology.

Optochemical Control of Protein Localization and Activity within Cell-like Compartments.

PubMed

Caldwell, Reese M; Bermudez, Jessica G; Thai, David; Aonbangkhen, Chanat; Schuster, Benjamin S; Courtney, Taylor; Deiters, Alexander; Hammer, Daniel A; Chenoweth, David M; Good, Matthew C

2018-05-08

We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.

Synthesis and characterization of tethered lipid assemblies for membrane protein reconstitution (Review).

PubMed

Veneziano, Rémi; Rossi, Claire; Chenal, Alexandre; Brenner, Catherine; Ladant, Daniel; Chopineau, Joël

2017-09-28

Biological membranes and their related molecular mechanisms are essential for all living organisms. Membranes host numerous proteins and are responsible for the exchange of molecules and ions, cell signaling, and cell compartmentation. Indeed, the plasma membrane delimits the intracellular compartment from the extracellular environment and intracellular membranes. Biological membranes also play a major role in metabolism regulation and cellular physiology (e.g., mitochondrial membranes). The elaboration of membrane based biomimetic systems allows us to reconstitute and investigate, in controlled conditions, biological events occurring at the membrane interface. A whole variety of model membrane systems have been developed in the last few decades. Among these models, supported membranes were developed on various hydrophilic supports. The use of solid supports enables the direct use of surface sensitive techniques (e.g., surface plasmon resonance, quartz crystal microbalance, and atomic force microscopy) to monitor and quantify events occurring at the membrane surface. Tethered bilayer membranes (tBLMs) could be considered as an achievement of the first solid supported membranes described by the McConnell group. Tethered bilayers on solid supports were designed to delimit an inside compartment from an outside one. They were used for measuring interactions with ligands or incorporating large membrane proteins or complexes without interference with the support. In this context, the authors developed an easy concept of versatile tBLMs assembled on amino coated substrates that are formed upon the vesicle fusion rupture process applicable to protein-free vesicles as well as proteoliposomes. The phospholipid bilayer (natural or synthetic lipids) incorporated 5% of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly ethylene glycol-N-hydroxy succinimide to ensure the anchorage of the bilayer to the amino coated surface. The conditions for the formation of tBLMs on amino-coated gold and glass were optimized for protein-free vesicles. This biomimetic membrane delimits an inside "trans" compartment separated from an outside reservoir "cis." Using this tBLM construction, the authors were interested in deciphering two complex molecular mechanisms involving membrane-associated proteins. The first one concerns two mitochondrial proteins, i.e., the porin voltage dependent anion channel (VDAC) embedded in the outer membrane and the nucleotide transporter (adenine nucleotide translocase) that interacts dynamically during mitochondrial pathophysiology. The purified VDAC porin was first reconstituted in proteoliposomes that were subsequently assembled on an amino coated support to form a biomimetic membrane. As a major result, VDAC was reconstituted in this tBLM and calcium channeling was demonstrated across the lipid bilayer. The same two-compartment biomimetic membrane design was further engineered to study the translocation mechanism of a bacterial toxin, the adenylate cyclase toxin, CyaA, from Bordetella pertussis. As a result, the authors developed an elegant in vitro translocation toolkit applicable to potentially a large panel of proteins transported across membranes.

Intracellular production of hydrogels and synthetic RNA granules by multivalent molecular interactions

NASA Astrophysics Data System (ADS)

Nakamura, Hideki; Lee, Albert A.; Afshar, Ali Sobhi; Watanabe, Shigeki; Rho, Elmer; Razavi, Shiva; Suarez, Allister; Lin, Yu-Chun; Tanigawa, Makoto; Huang, Brian; Derose, Robert; Bobb, Diana; Hong, William; Gabelli, Sandra B.; Goutsias, John; Inoue, Takanari

2018-01-01

Some protein components of intracellular non-membrane-bound entities, such as RNA granules, are known to form hydrogels in vitro. The physico-chemical properties and functional role of these intracellular hydrogels are difficult to study, primarily due to technical challenges in probing these materials in situ. Here, we present iPOLYMER, a strategy for a rapid induction of protein-based hydrogels inside living cells that explores the chemically inducible dimerization paradigm. Biochemical and biophysical characterizations aided by computational modelling show that the polymer network formed in the cytosol resembles a physiological hydrogel-like entity that acts as a size-dependent molecular sieve. We functionalize these polymers with RNA-binding motifs that sequester polyadenine-containing nucleotides to synthetically mimic RNA granules. These results show that iPOLYMER can be used to synthetically reconstitute the nucleation of biologically functional entities, including RNA granules in intact cells.

Expression of HLA Class II Molecules in Humanized NOD.Rag1KO.IL2RgcKO Mice is Critical for Development and Function of Human T and B Cells

DTIC Science & Technology

2011-05-17

HSC-infused DRAG and control mice were immunized with 1 flocculation unit of TT vaccine ( Sanofi Pasteur) by the intramuscular route, and the titers... Vaccine Program, Naval Medical Research Center/Walter Reed Army Institute of Research, Silver Spring, Maryland, United States of America, 2 Department...human vaccines prior to clinical trials. However, current humanized mouse models show sub-optimal human T cell reconstitution and limited ability to

Haematopoietic development and immunological function in the absence of cathepsin D

PubMed Central

Tulone, Calogero; Uchiyama, Yasuo; Novelli, Marco; Grosvenor, Nicholas; Saftig, Paul; Chain, Benjamin M

2007-01-01

Background Cathepsin D is a well-characterized aspartic protease expressed ubiquitously in lysosomes. Cathepsin D deficiency is associated with a spectrum of pathologies leading ultimately to death. Cathepsin D is expressed at high levels in many cells of the immune system, but its role in immune function is not well understood. This study examines the reconstitution and function of the immune system in the absence of cathepsin D, using bone marrow radiation chimaeras in which all haematopoietic cells are derived from cathepsin D deficient mice. Results Cathepsin D deficient bone marrow cells fully reconstitute the major cellular components of both the adaptive and innate immune systems. Spleen cells from cathepsin D deficient chimaeric mice contained an increased number of autofluorescent granules characteristic of lipofuscin positive lysosomal storage diseases. Biochemical and ultrastructural changes in cathepsin D deficient spleen are consistent with increased autolysosomal activity. Chimaeric mice were immunised with either soluble (dinitrophenylated bovine gamma globulin) or particulate (sheep red blood cells) antigens. Both antigens induced equivalent immune responses in wild type or cathepsin D deficient chimaeras. Conclusion All the parameters of haematopoietic reconstitution and adaptive immunity which were measured in this study were found to be normal in the absence of cathepsin D, even though cathepsin D deficiency leads to dysregulation of lysosomal function. PMID:17897442

Interleukin-6, Produced by Resident Cells of the Central Nervous System and Infiltrating Cells, Contributes to the Development of Seizures following Viral Infection▿

PubMed Central

Libbey, Jane E.; Kennett, Nikki J.; Wilcox, Karen S.; White, H. Steve; Fujinami, Robert S.

2011-01-01

Cells that can participate in an innate immune response within the central nervous system (CNS) include infiltrating cells (polymorphonuclear leukocytes [PMNs], macrophages, and natural killer [NK] cells) and resident cells (microglia and sometimes astrocytes). The proinflammatory cytokine interleukin-6 (IL-6) is produced by all of these cells and has been implicated in the development of behavioral seizures in the Theiler's murine encephalomyelitis virus (TMEV)-induced seizure model. The assessment, via PCR arrays, of the mRNA expression levels of a large number of chemokines (ligands and receptors) in TMEV-infected and mock-infected C57BL/6 mice both with and without seizures did not clearly demonstrate the involvement of PMNs, monocytes/macrophages, or NK cells in the development of seizures, possibly due to overlapping function of the chemokines. Additionally, C57BL/6 mice unable to recruit or depleted of infiltrating PMNs and NK cells had seizure rates comparable to those of controls following TMEV infection, and therefore PMNs and NK cells do not significantly contribute to seizure development. In contrast, C57BL/6 mice treated with minocycline, which affects monocytes/macrophages, microglial cells, and PMNs, had significantly fewer seizures than controls following TMEV infection, indicating monocytes/macrophages and resident microglial cells are important in seizure development. Irradiated bone marrow chimeric mice that were either IL-6-deficient mice reconstituted with wild-type bone marrow cells or wild-type mice reconstituted with IL-6-deficient bone marrow cells developed significantly fewer behavioral seizures following TMEV infection. Therefore, both resident CNS cells and infiltrating cells are necessary for seizure development. PMID:21543484

Radioprotection by polysaccharides alone and in combination with aminothiols

NASA Astrophysics Data System (ADS)

Patchen, Myra L.; Macvittie, Thomas J.; Solberg, Brian D.; D'Alesandro, Michele M.; Brook, Itzhak

We demonstrated that glucan, a beta-1,3 polysaccharide immunomodulator, enhances survival of mice when administered before radiation exposure. Glucan's prophylactic survival-enhancing effects are mediated by several mechanisms including (1) increasing macrophage-mediated resistance to potentially lethal postirradiation opportunistic infections, (2) increasing the Do of hematopoietic progenitor cells, and (3) accelerating hematopoietic reconstitution. In addition, even when administered shortly after some otherwise lethal doses of radiation, glucan increases survival. Glucan's therapeutic survival-enhancing effects are also mediated through its ability to enhance macrophage function and to accelerate hematopoietic reconstitution; glucan's therapeutic potential, however, is ultimately dependent on the survival of a critical number of hematopoietic stem cells capable of responding to glucan's stimulatory effects. Preirradiation administration of the traditional aminothiol radioprotectants WR-2721 and WR-3689 has been previously demonstrated to be an extremely effective means to increase hematopoietic stem cell survival. Therapeutic glucan treatment administered in combination with preirradiation WR-2721 or WR-3689 treatment synergistically increases both hematopoietic reconstitution and survival. Such combined modality treatments offer new promise in treating acute radiation injury.

Promotion of hair follicle development and trichogenesis by Wnt-10b in cultured embryonic skin and in reconstituted skin

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

Ouji, Yukiteru; Yoshikawa, Masahide; Shiroi, Akira

2006-06-30

We previously showed that Wnt-10b promoted the differentiation of primary skin epithelial cells (MPSEC) toward hair shaft and inner root sheath of the hair follicle (IRS) cells in vitro. In the present study, we found that Wnt-10b promotes the development of hair follicles using a culture of mouse embryonic skin tissue and trichogenesis using a reconstitution experiment with nude mice. Hair follicle development was observed in skin taken from mouse embryos on embryonic day 10.5 following a 2-day culture with recombinant Wnt-10b (rWnt-10b), however, not without rWnt-10b. Brown hair growth was observed at the site of reconstituted skin in Balb/cmore » nude mice where dermal fibroblasts and keratinocytes, derived from C3H/HeN new born mice, were transplanted with Wnt-10b-producing COS cells (Wnt-COS). Without the co-transplantation of Wnt-COS, no hair growth was observed. Our results suggest an important role of Wnt-10b in the initiation of hair follicle development and following trichogenesis.« less

A new protein-protein interaction sensor based on tripartite split-GFP association.

PubMed

Cabantous, Stéphanie; Nguyen, Hau B; Pedelacq, Jean-Denis; Koraïchi, Faten; Chaudhary, Anu; Ganguly, Kumkum; Lockard, Meghan A; Favre, Gilles; Terwilliger, Thomas C; Waldo, Geoffrey S

2013-10-04

Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.

A New Protein-Protein Interaction Sensor Based on Tripartite Split-GFP Association

PubMed Central

Cabantous, Stéphanie; Nguyen, Hau B.; Pedelacq, Jean-Denis; Koraïchi, Faten; Chaudhary, Anu; Ganguly, Kumkum; Lockard, Meghan A.; Favre, Gilles; Terwilliger, Thomas C.; Waldo, Geoffrey S.

2013-01-01

Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence. PMID:24092409

Clinicopathological correlates in HIV seropositive tuberculosis cases presenting with jaundice after initiating antiretroviral therapy with a structured review of the literature.

PubMed

Barr, David A; Ramdial, Pravistadevi K

2012-10-14

The development of jaundice after initiation of HAART in HIV-TB co-infected patients is a challenging presentation in resource constrained settings, and is often attributed to drug induced liver injury (DILI).Some investigators have described hepatic tuberculosis Immune Reconstitution Inflammatory Syndrome (TB-IRIS) as a cause of liver disease in patients initiating HAART, which could also cause jaundice. We report the clinical and histopathological features of five HIV-TB co-infected patients presenting with a syndrome of jaundice, tender hepatomegaly, bile canalicular enzyme rise and return of constitutional symptoms within 8 weeks of initiation of highly active antiretroviral therapy (HAART) for advanced HIV infection at a rural clinic in KwaZulu Natal, South Africa.All five patients had been diagnosed with tuberculosis infection prior to HAART initiation and were on antituberculous medication at time of developing jaundice. There was evidence of multiple aetiologies of liver injury in all patients. However, based on clinical course and pathological findings, predominant hepatic injury was thought to be drug induced in one case and hepatic tuberculosis associated immune reconstitution inflammatory syndrome (TB-IRIS) in the other four.In these later 4 patients, liver biopsy findings included necrotising and non-necrotising granulomatous inflammation in the lobules and portal tracts. The granulomas demonstrated - in addition to epithelioid histiocytes and Langhans giant cells - neutrophils, plasma cells and large numbers of lymphocytes, which are not features of a conventional untreated tuberculous response. In this high TB prevalent, low resource setting, TB-IRIS may be an important cause of jaundice post-HAART initiation. Clinicopathological correlation is essential for optimal diagnosis. Further multi-organ based histopathological studies in the context of immune reconstitution would be useful to clinicians in low resource settings dealing with this challenging presentation.

Cell-free protein synthesis: the state of the art.

PubMed

Whittaker, James W

2013-02-01

Cell-free protein synthesis harnesses the synthetic power of biology, programming the ribosomal translational machinery of the cell to create macromolecular products. Like PCR, which uses cellular replication machinery to create a DNA amplifier, cell-free protein synthesis is emerging as a transformative technology with broad applications in protein engineering, biopharmaceutical development, and post-genomic research. By breaking free from the constraints of cell-based systems, it takes the next step towards synthetic biology. Recent advances in reconstituted cell-free protein synthesis (Protein synthesis Using Recombinant Elements expression systems) are creating new opportunities to tailor the reactions for specialized applications including in vitro protein evolution, printing protein microarrays, isotopic labeling, and incorporating nonnatural amino acids.

Chronic nerve compression alters Schwann cell myelin architecture in a murine model

PubMed Central

Gupta, Ranjan; Nassiri, Nima; Hazel, Antony; Bathen, Mary; Mozaffar, Tahseen

2011-01-01

Introduction Myelinating Schwann cells compartmentalize their outermost layer to form actin-rich channels known as Cajal bands. Here, we investigate changes in Schwann cell architecture and cytoplasmic morphology in a novel mouse model of carpal tunnel syndrome. Methods Chronic nerve compression (CNC) injury was created in wild-type and slow-Wallerian degeneration (WldS) mice. Over 12 weeks, nerves were electrodiagnostically assessed, and Schwann cell morphology was thoroughly evaluated. Results A decline in nerve conduction velocity and increase in g-ratio is observed without early axonal damage. Schwann cells display shortened internodal lengths and severely disrupted Cajal bands. Quite surprisingly, the latter is reconstituted without improvements to nerve conduction velocity. Discussion Chronic entrapment injuries like carpal tunnel syndrome are primarily mediated by the Schwann cell response, wherein decreases in internodal length and myelin thickness disrupt the efficiency of impulse propagation. Restitution of Cajal bands is not sufficient for remyelination post-CNC injury. PMID:22246880

Acute immunosuppression and syngeneic bone marrow transplantation in ocular autoimmunity abort disease, but do not result in induction of long-term protection.

PubMed

Savion, S; Silver, P B; Chan, C C; Caspi, R R

1998-09-01

Acute immunosuppression induced by total body irradiation (TBI) or cyclophosphamide (Cy) treatment, followed by syngeneic bone marrow transplantation (SBMT), was reported to be effective in inducing long-term tolerance in some autoimmune disease models. We examined the efficacy of this approach in the mouse model of experimental autoimmune uveoretinitis (EAU). Development of EAU induced by the interphotoreceptor retinoid binding protein (IRBP) was abolished almost completely by either TBI or Cy treatment, followed by SBMT, instituted one week after priming. In parallel, IRBP-specific delayed-type hypersensitivity (DTH) responses and lymph node cell proliferation were strongly suppressed or abolished. However, when these IRBP-immunized, lymphoablated and BM reconstituted mice were rechallenged with the immunizing antigen seven weeks after the primary immunization, they were not protected from developing disease, despite the fact that DTH and lymph node cell proliferation to the antigen were suppressed relative to controls. TBI treatment appeared somewhat more effective than Cy treatment as judged by its more profound effect on immunological responses. These results demonstrate the ability of acute immunosuppression followed by reconstitution of the immune system to inhibit the development of EAU, although long-term protection from disease was not achieved.

Pathogen inactivation treatment of plasma and platelet concentrates and their predicted functionality in massive transfusion protocols.

PubMed

Arbaeen, Ahmad F; Schubert, Peter; Serrano, Katherine; Carter, Cedric J; Culibrk, Brankica; Devine, Dana V

2017-05-01

Trauma transfusion packages for hemorrhage control consist of red blood cells, plasma, and platelets at a set ratio. Although pathogen reduction improves the transfusion safety of platelet and plasma units, there is an associated reduction in quality. This study aimed to investigate the impact of riboflavin/ultraviolet light-treated plasma or platelets in transfusion trauma packages composed of red blood cell, plasma, and platelet units in a ratio of 1:1:1 in vitro by modeling transfusion scenarios for trauma patients and assessing function by rotational thromboelastometry. Pathogen-reduced or untreated plasma and buffy coat platelet concentrate units produced in plasma were used in different combinations with red blood cells in trauma transfusion packages. After reconstitution of these packages with hemodiluted blood, the hemostatic functionality was analyzed by rotational thromboelastometry. Hemostatic profiles of pathogen-inactivated buffy coat platelet concentrate and plasma indicated decreased activity compared with their respective controls. Reconstitution of hemodiluted blood (hematocrit = 20%) with packages that contained treated or nontreated components resulted in increased alpha and maximum clot firmness and enhanced clot-formation time. Simulating transfusion scenarios based on 30% blood replacement with a transfusion trauma package resulted in a nonsignificant difference in rotational thromboelastometry parameters between packages containing treated and nontreated blood components (p ≥ 0.05). Effects of pathogen inactivation treatment were evident when the trauma package percentage was 50% or greater and contained both pathogen inactivation-treated plasma and buffy coat platelet concentrate. Rotational thromboelastometry investigations suggest that there is relatively little impact of pathogen inactivation treatment on whole blood clot formation unless large amounts of treated components are used. © 2017 AABB.

Immunologic reconstitution during PEG-ADA therapy in an unusual mosaic ADA-deficient patient

PubMed Central

Liu, Ping; Santisteban, Ines; Burroughs, Laurie M.; Ochs, Hans D.; Torgerson, Troy R.; Hershfield, Michael S.; Rawlings, David J.; Scharenberg, Andrew M.

2009-01-01

We report detailed genetic and immunologic studies in a patient diagnosed with adenosine deaminase (ADA) deficiency and combined immune deficiency at age 5 years. At the time of diagnosis, although all other lymphocyte subsets were depleted, circulating CD8+ T cells with a terminally differentiated phenotype were abundant and expressed normal ADA activity due to a reversion mutation in a CD8+ T cell or precursor. Over the first 9 months of replacement therapy with PEG-ADA, the patient steadily accumulated mature naïve CD4+ and CD8+ T cells, as well as CD4+/FOXP3+ regulatory T cells, consistent with restoration of a functional cellular immune system. While CD19+ naïve B cells also accumulated in response to PEG-ADA therapy, a high proportion of these B cells exhibited an immature surface marker phenotype even after 9 months, and immunization with neoantigen bacteriophage φX174 demonstrated a markedly subnormal humoral immune response. Our observations in this single patient have important implications for gene therapy of human ADA deficiency, as they indicate that ADA expression within even a large circulating lymphocyte population may not be sufficient to support adequate immune reconstitution. They also suggest that an immature surface marker phenotype of the peripheral B cell compartment may be a useful surrogate marker for incomplete humoral immune reconstitution during enzyme replacement, and possibly other forms of hematopoietic cell therapies. PMID:18952502

The role of poverty in explaining health variations in 7-year-old children from different family structures: findings from the UK Millennium Cohort Study.

PubMed

Pearce, Anna; Lewis, Hannah; Law, Catherine

2013-02-01

Despite rises in reconstituted and lone-parent families, relatively little is known about how the health of children in different family types varies, and the extent to which any differences might be explained by poverty. The authors examined this using cross-sectional data on 13 681 seven-year-olds from the Millennium Cohort Study. The authors estimated RRs and 95% CIs for having poor physical (general health, long-standing illness, injury, overweight, asthma, fits) and mental health (using strengths and difficulties scores) according to family structure using Poisson regression. The authors adjusted for confounders (aRR) and then investigated the role of poverty as a mediator by entering a poverty score (based on income, receipt of benefits, subjective poverty and material deprivation) into the main model. Children living in reconstituted and lone-parent families were at a slight increased risk of poor health compared with those living with two natural parents. Adjusting for poverty tended to remove the elevated risk of poor physical health in children living in lone-parent and reconstituted families. However, for the mental health outcomes, poverty tended to remove the elevated risk for lone parents but not for reconstituted families. For example, the aRR for borderline-abnormal total difficulties fell from 1.45 (1.22 to 1.72) to 1.34 (1.13 to 1.59) in children living in reconstituted families and from 1.29 (1.14 to 1.45) to 1.05 (0.92 to 1.19) in those living with lone parents. Poor physical and mental health was slightly more prevalent in children living in lone-parent or reconstituted families. Poverty reduction may help to reduce these differences, especially for children living with lone parents; however, alternative mechanisms should be also explored, particularly for children living in reconstituted families.

The glutathione redox system is essential to prevent ferroptosis caused by impaired lipid metabolism in clear cell renal cell carcinoma.

PubMed

Miess, Heike; Dankworth, Beatrice; Gouw, Arvin M; Rosenfeldt, Mathias; Schmitz, Werner; Jiang, Ming; Saunders, Becky; Howell, Michael; Downward, Julian; Felsher, Dean W; Peck, Barrie; Schulze, Almut

2018-06-05

Metabolic reprogramming is a prominent feature of clear cell renal cell carcinoma (ccRCC). Here we investigated metabolic dependencies in a panel of ccRCC cell lines using nutrient depletion, functional RNAi screening and inhibitor treatment. We found that ccRCC cells are highly sensitive to the depletion of glutamine or cystine, two amino acids required for glutathione (GSH) synthesis. Moreover, silencing of enzymes of the GSH biosynthesis pathway or glutathione peroxidases, which depend on GSH for the removal of cellular hydroperoxides, selectively reduced viability of ccRCC cells but did not affect the growth of non-malignant renal epithelial cells. Inhibition of GSH synthesis triggered ferroptosis, an iron-dependent form of cell death associated with enhanced lipid peroxidation. VHL is a major tumour suppressor in ccRCC and loss of VHL leads to stabilisation of hypoxia inducible factors HIF-1α and HIF-2α. Restoration of functional VHL via exogenous expression of pVHL reverted ccRCC cells to an oxidative metabolism and rendered them insensitive to the induction of ferroptosis. VHL reconstituted cells also exhibited reduced lipid storage and higher expression of genes associated with oxidiative phosphorylation and fatty acid metabolism. Importantly, inhibition of β-oxidation or mitochondrial ATP-synthesis restored ferroptosis sensitivity in VHL reconstituted cells. We also found that inhibition of GSH synthesis blocked tumour growth in a MYC-dependent mouse model of renal cancer. Together, our data suggest that reduced fatty acid metabolism due to inhibition of β-oxidation renders renal cancer cells highly dependent on the GSH/GPX pathway to prevent lipid peroxidation and ferroptotic cell death.

Programmed cell death 1 inhibits inflammatory helper T-cell development through controlling the innate immune response

PubMed Central

Rui, Yuxiang; Honjo, Tasuku; Chikuma, Shunsuke

2013-01-01

Programmed cell death 1 (PD-1) is an inhibitory coreceptor on immune cells and is essential for self-tolerance because mice genetically lacking PD-1 (PD-1−/−) develop spontaneous autoimmune diseases. PD-1−/− mice are also susceptible to severe experimental autoimmune encephalomyelitis (EAE), characterized by a massive production of effector/memory T cells against myelin autoantigen, the mechanism of which is not fully understood. We found that an increased primary response of PD-1−/− mice to heat-killed mycobacteria (HKMTB), an adjuvant for EAE, contributed to the enhanced production of T-helper 17 (Th17) cells. Splenocytes from HKMTB-immunized, lymphocyte-deficient PD-1−/− recombination activating gene (RAG)2−/− mice were found to drive antigen-specific Th17 cell differentiation more efficiently than splenocytes from HKMTB-immunized PD-1+/+ RAG2−/− mice. This result suggested PD-1’s involvement in the regulation of innate immune responses. Mice reconstituted with PD-1−/− RAG2−/− bone marrow and PD-1+/+ CD4+ T cells developed more severe EAE compared with the ones reconstituted with PD-1+/+ RAG2−/− bone marrow and PD-1+/+ CD4+ T cells. We found that upon recognition of HKMTB, CD11b+ macrophages from PD-1−/− mice produced very high levels of IL-6, which helped promote naive CD4+ T-cell differentiation into IL-17–producing cells. We propose a model in which PD-1 negatively regulates antimycobacterial responses by suppressing innate immune cells, which in turn prevents autoreactive T-cell priming and differentiation to inflammatory effector T cells. PMID:24043779

A novel microfluidic model can mimic organ-specific metastasis of circulating tumor cells.

PubMed

Kong, Jing; Luo, Yong; Jin, Dong; An, Fan; Zhang, Wenyuan; Liu, Lilu; Li, Jiao; Fang, Shimeng; Li, Xiaojie; Yang, Xuesong; Lin, Bingcheng; Liu, Tingjiao

2016-11-29

A biomimetic microsystem might compensate costly and time-consuming animal metastatic models. Herein we developed a biomimetic microfluidic model to study cancer metastasis. Primary cells isolated from different organs were cultured on the microlfuidic model to represent individual organs. Breast and salivary gland cancer cells were driven to flow over primary cell culture chambers, mimicking dynamic adhesion of circulating tumor cells (CTCs) to endothelium in vivo. These flowing artificial CTCs showed different metastatic potentials to lung on the microfluidic model. The traditional nude mouse model of lung metastasis was performed to investigate the physiological similarity of the microfluidic model to animal models. It was found that the metastatic potential of different cancer cells assessed by the microfluidic model was in agreement with that assessed by the nude mouse model. Furthermore, it was demonstrated that the metastatic inhibitor AMD3100 inhibited lung metastasis effectively in both the microfluidic model and the nude mouse model. Then the microfluidic model was used to mimick liver and bone metastasis of CTCs and confirm the potential for research of multiple-organ metastasis. Thus, the metastasis of CTCs to different organs was reconstituted on the microfluidic model. It may expand the capabilities of traditional cell culture models, providing a low-cost, time-saving, and rapid alternative to animal models.

A strategy for tissue self-organization that is robust to cellular heterogeneity and plasticity.

PubMed

Cerchiari, Alec E; Garbe, James C; Jee, Noel Y; Todhunter, Michael E; Broaders, Kyle E; Peehl, Donna M; Desai, Tejal A; LaBarge, Mark A; Thomson, Matthew; Gartner, Zev J

2015-02-17

Developing tissues contain motile populations of cells that can self-organize into spatially ordered tissues based on differences in their interfacial surface energies. However, it is unclear how self-organization by this mechanism remains robust when interfacial energies become heterogeneous in either time or space. The ducts and acini of the human mammary gland are prototypical heterogeneous and dynamic tissues comprising two concentrically arranged cell types. To investigate the consequences of cellular heterogeneity and plasticity on cell positioning in the mammary gland, we reconstituted its self-organization from aggregates of primary cells in vitro. We find that self-organization is dominated by the interfacial energy of the tissue-ECM boundary, rather than by differential homo- and heterotypic energies of cell-cell interaction. Surprisingly, interactions with the tissue-ECM boundary are binary, in that only one cell type interacts appreciably with the boundary. Using mathematical modeling and cell-type-specific knockdown of key regulators of cell-cell cohesion, we show that this strategy of self-organization is robust to severe perturbations affecting cell-cell contact formation. We also find that this mechanism of self-organization is conserved in the human prostate. Therefore, a binary interfacial interaction with the tissue boundary provides a flexible and generalizable strategy for forming and maintaining the structure of two-component tissues that exhibit abundant heterogeneity and plasticity. Our model also predicts that mutations affecting binary cell-ECM interactions are catastrophic and could contribute to loss of tissue architecture in diseases such as breast cancer.

Proflavine sensitivity of RNA processing in isolated nuclei.

PubMed Central

Yannarell, A; Niemann, M; Schumm, D E; Webb, T E

1977-01-01

The intercalating agent proflavine inhibits the processing and subsequent release of preformed messenger RNA and ribosomal RNA from isolated liver nuclei to surrogate cytoplasm. The direct effect of proflavine on these processes, as monitored in a reconstituted cell-free system, supports the theory that base-paired segments (i.e. hairpin loops) in the precursor RNA's are involved as recognition sites in nuclear RNA processing. PMID:866181

Secreted protein acidic and rich in cysteine functions in colitis via IL17A regulation in mucosal CD4+ T cells.

PubMed

Tanaka, Makoto; Takagi, Tomohisa; Naito, Yuji; Uchiyama, Kazuhiko; Hotta, Yuma; Toyokawa, Yuki; Ushiroda, Chihiro; Hirai, Yasuko; Aoi, Wataru; Higashimura, Yasuki; Mizushima, Katsura; Okayama, Tetsuya; Katada, Kazuhiro; Kamada, Kazuhiro; Ishikawa, Takeshi; Handa, Osamu; Itoh, Yoshito

2018-03-01

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycol that regulates cell proliferation, tissue repair, and tumorigenesis. Despite evidence linking SPARC to inflammation, the mechanisms are unclear. Accordingly, the role of SPARC in intestinal inflammation was investigated. Colitis was induced in wild-type (WT) and SPARC knockout (KO) mice using trinitrobenzene sulfonic acid (TNBS). Colons were assessed for damage; leukocyte infiltration; Tnf, Ifng, Il17a, and Il10 mRNA expression; and histology. Cytokine profiling of colonic lamina propria mononuclear cells (LPMCs) was performed by flow cytometry. Naïve CD4 + T cells were isolated from WT and SPARC KO mouse spleens, and the effect of SPARC on Th17 cell differentiation was examined. Recombination activating gene 1 knockout (RAG1 KO) mice reconstituted with T cells from either WT or SPARC KO mice were investigated. Trinitrobenzene sulfonic acid exposure significantly reduced bodyweight and increased mucosal inflammation, leukocyte infiltration, and Il17a mRNA expression in WT relative to SPARC KO mice. The percentage of IL17A-producing CD4 + T cells among LPMCs from KO mice was lower than that in WT mice when both groups were exposed to TNBS. Th17 cell differentiation was suppressed in cells from SPARC KO mice. In the T cell transfer colitis model, RAG1 KO mice receiving T cells from WT mice were more severely affected than those reconstituted with cells from SPARC KO mice. Secreted protein acidic and rich in cysteine accelerates colonic mucosal inflammation via modulation of IL17A-producing CD4 + T cells. SPARC is a potential therapeutic target for conditions involving intestinal inflammation. © 2017 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

Reconstitution of a secondary cell wall in a secondary cell wall-deficient Arabidopsis mutant.

PubMed

Sakamoto, Shingo; Mitsuda, Nobutaka

2015-02-01

The secondary cell wall constitutes a rigid frame of cells in plant tissues where rigidity is required. Deposition of the secondary cell wall in fiber cells contributes to the production of wood in woody plants. The secondary cell wall is assembled through co-operative activities of many enzymes, and their gene expression is precisely regulated by a pyramidal cascade of transcription factors. Deposition of a transmuted secondary cell wall in empty fiber cells by expressing selected gene(s) in this cascade has not been attempted previously. In this proof-of-concept study, we expressed chimeric activators of 24 transcription factors that are preferentially expressed in the stem, in empty fiber cells of the Arabidopsis nst1-1 nst3-1 double mutant, which lacks a secondary cell wall in fiber cells, under the control of the NST3 promoter. The chimeric activators of MYB46, SND2 and ANAC075, as well as NST3, reconstituted a secondary cell wall with different characteristics from those of the wild type in terms of its composition. The transgenic lines expressing the SND2 or ANAC075 chimeric activator showed increased glucose and xylose, and lower lignin content, whereas the transgenic line expressing the MYB46 chimeric activator showed increased mannose content. The expression profile of downstream genes in each transgenic line was also different from that of the wild type. This study proposed a new screening strategy to identify factors of secondary wall formation and also suggested the potential of the artificially reconstituted secondary cell walls as a novel raw material for production of bioethanol and other chemicals. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

Naphthalocyanine-reconstituted LDL nanoparticles for in vivo cancer imaging and treatment

PubMed Central

Song, Liping; Li, Hui; Sunar, Ulas; Chen, Juan; Corbin, Ian; Yodh, Arjun G; Zheng, Gang

2007-01-01

Low density lipoproteins (LDLs) are naturally occurring nanoparticles that are biocompatible, biodegradable and non-immunogenic. Moreover, the size of LDL particle is precisely controlled (~22 nm) by its apoB-100 component, setting them apart from liposomes and lipid micelles. LDL particles have long been proposed as a nanocarrier for targeted delivery of diagnostics and therapeutics to LDL receptor (LDLR)-positive cancers. Here, we report the design and synthesis of a novel naphthalocyanine (Nc)-based photodynamic therapy (PDT) agent, SiNcBOA, and describe its efficient reconstitution into LDL core (100:1 payload). Possessing a near-infrared (NIR) absorption wavelength (>800 nm) and extremely high extinction coefficient (>105 M–1cm–1), SiNcBOA holds the promise of treating deeply seated tumors. Reconstituted LDL particles (r-Nc-LDL) maintain the size and shape of native LDL as determined by transmission electron microscopy, and also retain their LDLR-mediated uptake by cancer cells as demonstrated by confocal microscopy. Its preferential uptake by tumor vs normal tissue was confirmed in vivo by noninvasive optical imaging technique, demonstrating the feasibility of using this nanoparticle for NIR imaging-guided PDT of cancer. PMID:18203443

Micro- and nanofabrication methods for ion channel reconstitution in bilayer lipid membranes

NASA Astrophysics Data System (ADS)

Tadaki, Daisuke; Yamaura, Daichi; Arata, Kohei; Ohori, Takeshi; Ma, Teng; Yamamoto, Hideaki; Niwano, Michio; Hirano-Iwata, Ayumi

2018-03-01

The self-assembled bilayer lipid membrane (BLM) forms the basic structure of the cell membrane and serves as a major barrier against ion movement. Ion channel proteins function as gated pores that permit ion permeation across the BLM. The reconstitution of ion channel proteins in artificially formed BLMs represents a well-defined system for investigating channel functions and screening drug effects on ion channels. In this review, we will discuss our recent microfabrication approaches to the formation of stable BLMs containing ion channel proteins as a potential platform for next-generation drug screening systems. BLMs formed in a microaperture having a tapered edge exhibited highly stable properties, such as a lifetime of ∼65 h and tolerance to solution changes even after the incorporation of the human ether-a-go-go-related gene (hERG) channel. We also explore a new method of efficiently incorporating human ion channels into BLMs by centrifugation. Our approaches to the formation of stable BLMs and efficient channel incorporation markedly improve the experimental efficiency of BLM reconstitution systems, leading to the realization of a BLM-based high-throughput platform for functional assays of various ion channels.

[Clinical and experimental study of treating aplastic anemia with fetal liver cell suspension and fetal liver cell-free suspension].

PubMed

Han, J R; Yuan, S W; Ren, Q F

1990-06-01

Fresh fetal liver obtained from 3- to 6-month fetus was prepared. Fetal liver cell suspension (FLC) or fetal liver cell-free suspension (FLCF) were then transfused into two groups of patient of aplastic anemia. 15 of 21 patients of aplastic anemia treated with FLC showed reconstitution of haemopoietic function or improvement of peripheral blood pictures, while 27 of 30 patients treated with FLCF showed reconstitution or improvement. It is verified that there is a stimulating factor for CFU-CM, BFU-E, and CFU-E and also a immunologic stimulant for improving the nonspecific immunologic function of the organism as shown by clinical analysis and experimental study. It is obvious that the therapeutic effect of FLCF is much better than that of the FLC.

A disassembly-driven mechanism explains F-actin-mediated chromosome transport in starfish oocytes

PubMed Central

Bun, Philippe; Dmitrieff, Serge; Belmonte, Julio M

2018-01-01

While contraction of sarcomeric actomyosin assemblies is well understood, this is not the case for disordered networks of actin filaments (F-actin) driving diverse essential processes in animal cells. For example, at the onset of meiosis in starfish oocytes a contractile F-actin network forms in the nuclear region transporting embedded chromosomes to the assembling microtubule spindle. Here, we addressed the mechanism driving contraction of this 3D disordered F-actin network by comparing quantitative observations to computational models. We analyzed 3D chromosome trajectories and imaged filament dynamics to monitor network behavior under various physical and chemical perturbations. We found no evidence of myosin activity driving network contractility. Instead, our observations are well explained by models based on a disassembly-driven contractile mechanism. We reconstitute this disassembly-based contractile system in silico revealing a simple architecture that robustly drives chromosome transport to prevent aneuploidy in the large oocyte, a prerequisite for normal embryonic development. PMID:29350616

W' expenditure and reconstitution during severe intensity constant power exercise: mechanistic insight into the determinants of W'.

PubMed

Broxterman, Ryan M; Skiba, Phillip F; Craig, Jesse C; Wilcox, Samuel L; Ade, Carl J; Barstow, Thomas J

2016-10-01

The sustainable duration of severe intensity exercise is well-predicted by critical power (CP) and the curvature constant (W'). The development of the W'BAL model allows for the pattern of W' expenditure and reconstitution to be characterized and this model has been applied to intermittent exercise protocols. The purpose of this investigation was to assess the influence of relaxation phase duration and exercise intensity on W' reconstitution during dynamic constant power severe intensity exercise. Six men (24.6 ± 0.9 years, height: 173.5 ± 1.9 cm, body mass: 78.9 ± 5.6 kg) performed severe intensity dynamic handgrip exercise to task failure using 50% and 20% duty cycles. The W'BAL model was fit to each exercise test and the time constant for W' reconstitution (τW') was determined. The τW' was significantly longer for the 50% duty cycle (1640 ± 262 sec) than the 20% duty cycle (863 ± 84 sec, P = 0.02). Additionally, the relationship between τW' and CP was well described as an exponential decay (r(2) = 0.90, P < 0.0001). In conclusion, the W'BAL model is able to characterize the expenditure and reconstitution of W' across the contraction-relaxation cycles comprising severe intensity constant power handgrip exercise. Moreover, the reconstitution of W' during constant power severe intensity exercise is influenced by the relative exercise intensity, the duration of relaxation between contractions, and CP. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Widespread Non-Hematopoietic Tissue Distribution by Transplanted Human Progenitor Cells with High Aldehyde Dehydrogenase Activity

PubMed Central

Hess, David A.; Craft, Timothy P.; Wirthlin, Louisa; Hohm, Sarah; Zhou, Ping; Eades, William C.; Creer, Michael H.; Sands, Mark S.; Nolta, Jan A.

2011-01-01

Transplanted adult progenitor cells distribute to peripheral organs and can promote endogenous cellular repair in damaged tissues. However, development of cell-based regenerative therapies has been hindered by the lack of pre-clinical models to efficiently assess multiple organ distribution and difficulty defining human cells with regenerative function. After transplantation into beta-glucuronidase (GUSB)-deficient NOD/SCID/MPSVII mice, we characterized the distribution of lineage depleted human umbilical cord blood-derived cells purified by selection using high aldehyde dehydrogenase activity (ALDH) with CD133 co-expression. ALDHhi or ALDHhiCD133+ cells produced robust hematopoietic reconstitution, and variable levels of tissue distribution in multiple organs. GUSB+ donor cells that co-expressed human (HLA-A,B,C) and hematopoietic (CD45+) cell surface markers were the primary cell phenotype found adjacent to the vascular beds of several tissues, including islet and ductal regions of mouse pancreata. In contrast, variable phenotypes were detected in the chimeric liver, with HLA+/CD45+ cells demonstrating robust GUSB expression adjacent to blood vessels, and CD45−/HLA− cells with diluted GUSB expression predominant in the liver parenchyma. However, true non-hematopoietic human (HLA+/CD45−) cells were rarely detected in other peripheral tissues, suggesting that these GUSB+/HLA−/CD45− cells in the liver were a result of downregulated human surface marker expression in vivo, not widespread seeding of non-hematopoietic cells. However, relying solely on continued expression of cell surface markers, as employed in traditional xenotransplantation models, may underestimate true tissue distribution. ALDH-expressing progenitor cells demonstrated widespread and tissue-specific distribution of variable cellular phenotypes, indicating that these adult progenitor cells should be explored in transplantation models of tissue damage. PMID:18055447

Repopulating hematopoietic stem cells from steady-state blood before and after ex vivo culture are enriched in CD34+CD133+CXCR4low fraction.

PubMed

Lapostolle, Véronique; Chevaleyre, Jean; Duchez, Pascale; Rodriguez, Laura; Vlaski-Lafarge, Marija; Sandvig, Ioanna; Brunet de la Grange, Philippe; Ivanovic, Zoran

2018-06-01

Feasibility of ex vivo expansion allows us to consider the steady-state peripheral blood as an alternative source of hematopoietic stem progenitor cells for transplantation when growth factor-induced cell mobilization is contraindicated or inapplicable. Ex vivo expansion dramatically enhances the in vivo reconstituting cell population from steady-state blood. In order to investigate phenotype and the expression of homing molecules, CD34, CD133, CD90, CD45RA, CD26 and CD9 expression was determined on sorted CD34+ cells according to CXCR4 (neg, low, bright) and CD133 expression before and after ex vivo expansion. Hematopoietic stem cell activity was determined in vivo on the basis of hematopoietic repopulation of primary and secondary recipients - NSG immuno-deficient mice. In vivo reconstituting cells in steady-state blood CD34+ cell fraction before expansion belong to the CD133+ population and are CXCR4low or, to a lesser extent, CXCR4neg, while after ex vivo expansion they are contained in only the CD133+CXCR4low cells. The failure of CXCR4bright population to engraft is probably due to the exclusive expression of CD26 by these cells. The limiting-dilution analysis showed that both repopulating cell number and individual proliferative capacity were enhanced by ex vivo expansion. Thus, steady-state peripheral blood cells exhibit a different phenotype compared to mobilized and cord blood ones, as well as to those issued from the bone marrow. This data represent the first phenotypic characterization of steady-state blood cells exhibiting short and long term hematopoietic reconstituting potential, which can be expanded ex vivo, a sine qua non for their subsequent use for transplantation. Copyright © 2018, Ferrata Storti Foundation.

Reconstitution of in vivo macrophage-tumor cell pairing and streaming motility on one-dimensional micro-patterned substrates

PubMed Central

Sharma, Ved P.; Beaty, Brian T.; Patsialou, Antonia; Liu, Huiping; Clarke, Michael; Cox, Dianne; Condeelis, John S.; Eddy, Robert J.

2014-01-01

In mammary tumors, intravital imaging techniques have uncovered an essential role for macrophages during tumor cell invasion and metastasis mediated by an epidermal growth factor (EGF)/colony stimulating factor-1 (CSF-1) paracrine loop. It was previously demonstrated that mammary tumors in mice derived from rat carcinoma cells (MTLn3) exhibited high velocity migration on extracellular matrix (ECM) fibers. These cells form paracrine loop-dependent linear assemblies of alternating host macrophages and tumor cells known as “streams.” Here, we confirm by intravital imaging that similar streams form in close association with ECM fibers in a highly metastatic patient-derived orthotopic mammary tumor (TN1). To understand the in vivo cell motility behaviors observed in streams, an in vitro model of fibrillar tumor ECM utilizing adhesive 1D micropatterned substrates was developed. MTLn3 cells on 1D fibronectin or type I collagen substrates migrated with higher velocity than on 2D substrates and displayed enhanced lamellipodial protrusion and increased motility upon local interaction and pairing with bone marrow-derived macrophages (BMMs). Inhibitors of EGF or CSF-1 signaling disrupted this interaction and reduced tumor cell velocity and protrusion, validating the requirement for an intact paracrine loop. Both TN1 and MTLn3 cells in the presence of BMMs were capable of co-assembling into linear arrays of alternating tumor cells and BMMs that resembled streams in vivo, suggesting the stream assembly is cell autonomous and can be reconstituted on 1D substrates. Our results validate the use of 1D micropatterned substrates as a simple and defined approach to study fibrillar ECM-dependent cell pairing, migration and relay chemotaxis as a complementary tool to intravital imaging. PMID:24634804

Coupling of g proteins to reconstituted monomers and tetramers of the M2 muscarinic receptor.

PubMed

Redka, Dar'ya S; Morizumi, Takefumi; Elmslie, Gwendolynne; Paranthaman, Pranavan; Shivnaraine, Rabindra V; Ellis, John; Ernst, Oliver P; Wells, James W

2014-08-29

G protein-coupled receptors can be reconstituted as monomers in nanodiscs and as tetramers in liposomes. When reconstituted with G proteins, both forms enable an allosteric interaction between agonists and guanylyl nucleotides. Both forms, therefore, are candidates for the complex that controls signaling at the level of the receptor. To identify the biologically relevant form, reconstituted monomers and tetramers of the purified M2 muscarinic receptor were compared with muscarinic receptors in sarcolemmal membranes for the effect of guanosine 5'-[β,γ-imido]triphosphate (GMP-PNP) on the inhibition of N-[(3)H]methylscopolamine by the agonist oxotremorine-M. With monomers, a stepwise increase in the concentration of GMP-PNP effected a lateral, rightward shift in the semilogarithmic binding profile (i.e. a progressive decrease in the apparent affinity of oxotremorine-M). With tetramers and receptors in sarcolemmal membranes, GMP-PNP effected a vertical, upward shift (i.e. an apparent redistribution of sites from a state of high affinity to one of low affinity with no change in affinity per se). The data were analyzed in terms of a mechanistic scheme based on a ligand-regulated equilibrium between uncoupled and G protein-coupled receptors (the "ternary complex model"). The model predicts a rightward shift in the presence of GMP-PNP and could not account for the effects at tetramers in vesicles or receptors in sarcolemmal membranes. Monomers present a special case of the model in which agonists and guanylyl nucleotides interact within a complex that is both constitutive and stable. The results favor oligomers of the M2 receptor over monomers as the biologically relevant state for coupling to G proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Construction of fusogenic vesicles bearing specific antibodies. Targeting of reconstituted Sendai virus envelopes towards neuraminidase-treated human erythrocytes.

PubMed

Gitman, A G; Loyter, A

1984-08-10

The cross-linking reagents succinimidyl-4-(p-maleimidophenyl)-butyrate and N-succinimidyl-3-(2-pyridyldithio)-propionate were used to covalently attach antibodies against human erythrocytes to the thiol-containing paraffin, dodecanethiol. The complex formed, dodecanethiol-maleimidophenylbutyrate (or pyridyldithiopropionate)-antibody was inserted into the membranes of reconstituted Sendai virus envelopes. This was achieved by addition of the dodecanethiol-maleimidophenylbutyrate-antibody to a detergent solution (Triton X-100) containing the viral envelope phospholipids and glycoproteins. Removal of the detergent led to the formation of vesicles containing the viral glycoprotein and the dodecanethiol-maleimidophenylbutyrate (or pyridyldithiopropionate)-antibody complexes within the same membrane. Reconstituted Sendai virus envelope-bearing antibodies against human erythrocytes were able to fuse with human erythrocytes (as was reflected by reconstituted Sendai virus envelope-induced hemolysis) from which the natural virus receptors were removed by treatment with neuraminidase. Thus, it appears that anti-human erythrocyte antibodies could substitute for the viral binding protein (hemagglutinin/neuraminidase glycoprotein) in mediating functional binding of the virus particles to the cell plasma membranes. Furthermore, from the results of the present work, it may be inferred that in addition to being the viral-binding protein, hemagglutinin/neuraminidase glycoprotein actively participates in the process of virus-cell fusion.

An Improved Protocol for Efficient Engraftment in NOD/LTSZ-SCIDIL-2RγNULL Mice Allows HIV Replication and Development of Anti-HIV Immune Responses

PubMed Central

Singh, Maneesh; Singh, Pratibha; Gaudray, Gilles; Musumeci, Lucia; Thielen, Caroline; Vaira, Dolores; Vandergeeten, Claire; Delacroix, Laurence; Van Gulck, Ellen; Vanham, Guido; de Leval, Laurence; Rahmouni, Souad; Moutschen, Michel

2012-01-01

Cord blood hematopoietic progenitor cells (CB-HPCs) transplanted immunodeficient NOD/LtsZ-scidIL2Rγnull (NSG) and NOD/SCID/IL2Rγnull (NOG) mice need efficient human cell engraftment for long-term HIV-1 replication studies. Total body irradiation (TBI) is a classical myeloablation regimen used to improve engraftment levels of human cells in these humanized mice. Some recent reports suggest the use of busulfan as a myeloablation regimen to transplant HPCs in neonatal and adult NSG mice. In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3–4 week old NSG mice. In this CB-CD34+transplanted NSG mice engraftment efficiency of human CD45+cell is over 90% in peripheral blood. Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days. These humanized NSG mice have shown long-lasting viremia after HIV-1JRCSF and HIV-1Bal inoculation through intravenous and rectal routes. We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection. Humanized NSG mice reconstituted according to our new protocol produced, moderate cellular and humoral immune responses to HIV-1 postinfection. We believe that NSG mice reconstituted according to our easy to use protocol will provide a better in vivo model for HIV-1 replication and anti-HIV-1 therapy trials. PMID:22675567

Immune mechanisms in organ allograft rejection. V. Pivotal role of the cytotoxic-suppressor T cell subset in the rejection of heart grafts bearing isolated class I disparities in the inbred rat

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

Lowry, R.P.; Forbes, R.D.; Blackburn, J.H.

1985-11-01

The cellular requirements for rejection of heart grafts bearing isolated major histocompatibility complex (MHC) subregion RT1A-encoded class I disparities was assessed by adoptive transfer. Sublethally irradiated (780 rads) (PVG X WF)F1 recipients of irradiated PVG-RT1r1 heart grafts were selectively reconstituted with spleen cells from syngeneic donors previously sensitized with two sequential PVG-RT1r1 skin grafts. PVG-RT1r1 heart grafts were rejected acutely in recipients reconstituted with 10 X 10(6) unfractionated immune spleen cells or inocula (5 X 10(6) cells) depleted of SIg+ cells, but additional depletion of cytotoxic T cells and their precursors resulted in marked prolongation of graft survival. Reducing themore » reconstituting inocula from 4 X 10(6) to 2.5 X 10(6) spleen cells prolonged graft survival to that observed in unreconstituted recipients. Additional studies were performed to define the immunologic basis for prolonged survival of PVG-RT1r1 heart grafts in homozygous PVG recipients. Although lymphoid cells of naive PVG failed to proliferate on coculture with irradiated PVG-RT1r1, bulk cultures yielding but weak and variable CTL generation, lymphoid cells from specifically sensitized PVG proliferated and generated greater cytotoxic T lymphocyte (CTL) activity under identical conditions, strongly suggesting, therefore, that prolonged heart graft survival in this strain combination is related to low CTL precursor frequency.« less

Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance

PubMed Central

Romano Ibarra, Guillermo S; Paul, Biswajit; Sather, Blythe D; Younan, Patrick M; Sommer, Karen; Kowalski, John P; Hale, Malika; Stoddard, Barry; Jarjour, Jordan; Astrakhan, Alexander; Kiem, Hans-Peter; Rawlings, David J

2016-01-01

A naturally occurring 32-base pair deletion of the HIV-1 co-receptor CCR5 has demonstrated protection against HIV infection of human CD4+ T cells. Recent genetic engineering approaches using engineered nucleases to disrupt the gene and mimic this mutation show promise for HIV therapy. We developed a megaTAL nuclease targeting the third extracellular loop of CCR5 that we delivered to primary human T cells by mRNA transfection. The CCR5 megaTAL nuclease established resistance to HIV in cell lines and disrupted the expression of CCR5 on primary human CD4+ T cells with a high efficiency, achieving up to 80% modification of the locus in primary cells as measured by molecular analysis. Gene-modified cells engrafted at levels equivalent to unmodified cells when transplanted into immunodeficient mice. Furthermore, genetically modified CD4+ cells were preferentially expanded during HIV-1 infection in vivo in an immunodeficient mouse model. Our results demonstrate the feasibility of targeting CCR5 in primary T cells using an engineered megaTAL nuclease, and the potential to use gene-modified cells to reconstitute a patient's immune system and provide protection from HIV infection. PMID:27741222

Prolonged CD4 T Cell Lymphopenia Increases Morbidity and Mortality after Renal Transplantation

PubMed Central

Courivaud, Cécile; Bamoulid, Jamal; Vivet, Bérengère; Chabroux, Aline; Deschamps, Marina; Rebibou, Jean-Michel; Ferrand, Christophe; Chalopin, Jean-Marc; Tiberghien, Pierre; Saas, Philippe

2010-01-01

Prolonged CD4 T cell lymphopenia after administration of polyclonal anti-thymocyte globulins increases the rate of posttransplantation morbidity, but whether impaired immune reconstitution affects survival is unknown. We studied the effect of CD4 T cell lymphopenia on survival in 302 consecutive prevalent renal transplant recipients and the role of thymic function in CD4 T cell reconstitution and posttransplantation outcomes in 100 consecutive incident renal transplant recipients. We followed the prevalent cohort for a mean duration of 92 months. Of these 302 patients, 81 (27%) had persistent CD4 T cell counts <300/mm3 and 36 (12%) died during follow-up. We observed a higher death rate in patients with CD4 T cell lymphopenia persisting for >1 year (24.1 versus 7.6%; P < 0.001). Furthermore, in Cox regression analysis, CD4 T cell lymphopenia associated with a nearly five-fold risk for death (adjusted hazard ratio [HR] 4.63; 95% confidence interval [CI] 1.91 to 10.65; P = 0.001). In the incident cohort, we estimated thymic function by T cell receptor excision circles (TRECs) per 150,000 CD3+ cells, which predicted efficient CD4 T cell reconstitution. Higher pretransplantation TREC values associated with lower risks for cancer (adjusted HR 0.39; 95% CI 0.15 to 0.97; P = 0.046) and infection (HR 0.29; 95% CI 0.11 to 0.78; P = 0.013). In summary, prolonged polyclonal anti-thymocyte globulin–induced CD4 T cell lymphopenia is an independent risk factor for death. Determination of pretransplantation thymic function may identify patients at higher risk for CD4 T cell lymphopenia and posttransplantation morbidity, including cancer and infections. PMID:20203160

Prolonged CD4 T cell lymphopenia increases morbidity and mortality after renal transplantation.

PubMed

Ducloux, Didier; Courivaud, Cécile; Bamoulid, Jamal; Vivet, Bérengère; Chabroux, Aline; Deschamps, Marina; Rebibou, Jean-Michel; Ferrand, Christophe; Chalopin, Jean-Marc; Tiberghien, Pierre; Saas, Philippe

2010-05-01

Prolonged CD4 T cell lymphopenia after administration of polyclonal anti-thymocyte globulins increases the rate of posttransplantation morbidity, but whether impaired immune reconstitution affects survival is unknown. We studied the effect of CD4 T cell lymphopenia on survival in 302 consecutive prevalent renal transplant recipients and the role of thymic function in CD4 T cell reconstitution and posttransplantation outcomes in 100 consecutive incident renal transplant recipients. We followed the prevalent cohort for a mean duration of 92 months. Of these 302 patients, 81 (27%) had persistent CD4 T cell counts <300/mm3 and 36 (12%) died during follow-up. We observed a higher death rate in patients with CD4 T cell lymphopenia persisting for >1 year (24.1 versus 7.6%; P < 0.001). Furthermore, in Cox regression analysis, CD4 T cell lymphopenia associated with a nearly five-fold risk for death (adjusted hazard ratio [HR] 4.63; 95% confidence interval [CI] 1.91 to 10.65; P = 0.001). In the incident cohort, we estimated thymic function by T cell receptor excision circles (TRECs) per 150,000 CD3+ cells, which predicted efficient CD4 T cell reconstitution. Higher pretransplantation TREC values associated with lower risks for cancer (adjusted HR 0.39; 95% CI 0.15 to 0.97; P = 0.046) and infection (HR 0.29; 95% CI 0.11 to 0.78; P = 0.013). In summary, prolonged polyclonal anti-thymocyte globulin-induced CD4 T cell lymphopenia is an independent risk factor for death. Determination of pretransplantation thymic function may identify patients at higher risk for CD4 T cell lymphopenia and posttransplantation morbidity, including cancer and infections.

Childhood Hematopoietic Cell Transplantation (PDQ®)—Health Professional Version

Cancer.gov

Childhood hematopoietic cell transplantation involves the infusion of blood stem cells into a patient to reconstitute the blood system. Get detailed information about autologous and allogeneic transplant, HLA matching, preparative regimens, and complications in this summary for clinicians.

Adenosine deaminase deficiency: a review.

PubMed

Flinn, Aisling M; Gennery, Andrew R

2018-04-24

Adenosine deaminase (ADA) deficiency leads to an accumulation of toxic purine degradation by-products, most potently affecting lymphocytes, leading to adenosine deaminase-deficient severe combined immunodeficiency. Whilst most notable affects are on lymphocytes, other manifestations include skeletal abnormalities, neurodevelopmental affects and pulmonary manifestations associated with pulmonary-alveolar proteinosis. Affected patients present in early infancy, usually with persistent infection, or with pulmonary insufficiency. Three treatment options are currently available. Initial treatment with enzyme replacement therapy may alleviate acute symptoms and enable partial immunological reconstitution, but treatment is life-long, immune reconstitution is incomplete, and the reconstituted immune system may nullify the effects of the enzyme replacement. Hematopoietic stem cell transplant has long been established as the treatment of choice, particularly where a matched sibling or well matched unrelated donor is available. More recently, the use of gene addition techniques to correct the genetic defect in autologous haematopoietic stem cells treatment has demonstrated immunological and clinical efficacy. This article reviews the biology, clinical presentation, diagnosis and treatment of ADA-deficiency.

Cell-cycle regulation of formin-mediated actin cable assembly

PubMed Central

Miao, Yansong; Wong, Catherine C. L.; Mennella, Vito; Michelot, Alphée; Agard, David A.; Holt, Liam J.; Yates, John R.; Drubin, David G.

2013-01-01

Assembly of appropriately oriented actin cables nucleated by formin proteins is necessary for many biological processes in diverse eukaryotes. However, compared with knowledge of how nucleation of dendritic actin filament arrays by the actin-related protein-2/3 complex is regulated, the in vivo regulatory mechanisms for actin cable formation are less clear. To gain insights into mechanisms for regulating actin cable assembly, we reconstituted the assembly process in vitro by introducing microspheres functionalized with the C terminus of the budding yeast formin Bni1 into extracts prepared from yeast cells at different cell-cycle stages. EM studies showed that unbranched actin filament bundles were reconstituted successfully in the yeast extracts. Only extracts enriched in the mitotic cyclin Clb2 were competent for actin cable assembly, and cyclin-dependent kinase 1 activity was indispensible. Cyclin-dependent kinase 1 activity also was found to regulate cable assembly in vivo. Here we present evidence that formin cell-cycle regulation is conserved in vertebrates. The use of the cable-reconstitution system to test roles for the key actin-binding proteins tropomyosin, capping protein, and cofilin provided important insights into assembly regulation. Furthermore, using mass spectrometry, we identified components of the actin cables formed in yeast extracts, providing the basis for comprehensive understanding of cable assembly and regulation. PMID:24133141

Effect of Vibrio cholerae neuraminidase on the mitogen response of T lymphocytes. I. Enhancement of macrophage T-lymphocyte cooperation in concanavalin-A-induced lymphocyte activation.

PubMed

Knop, J

1980-12-01

Vibrio cholerae neuraminidase (VCN) enhances the immune response of lymphocytes in various systems, such as antigen- and mitogen-induced blastogenesis, mixed lymphocyte culture (MLC) and tumor-cell response. We used macrophage-depleted and reconstituted murine lymph-node T-cells to investigate the effect of VCN on macrophage-T-lymphocyte co-operation in Con-A-induced lymphocyte activation. In unfractionated lymph-node cells VCN enhanced the Con-A-induced lymphocyte activation as measured by 3H-thymidine (3H-dThd) incorporation. Removing macrophages from the cells resulted in a significantly diminished response. In addition the enhancing effect of VCN was greatly reduced. Reconstitution of the lymphocyte cultures with macrophages in increasing numbers and from various sources rstored the lymphocyte response and the enhancing effect of VCN. VCN proved to be most efficient in cultures reconstituted with normal peritoneal macrophages. Some effect was also observed using bone-marrow-derived (BM) macrophages. However, higher numbers of normal PE macrophages in the presence of VCN inhibited lymphocyte activation, and inhibition by thioglycollate-broth-induced macrophages was considerably increased by VCN. These results suggest that VCN acts by increasing the efficiency of macrophage-T lymphocyte interaction.

Cytomegalovirus shapes long-term immune reconstitution after allogeneic stem cell transplantation

PubMed Central

Itzykson, Raphael; Robin, Marie; Moins-Teisserenc, Helene; Delord, Marc; Busson, Marc; Xhaard, Aliénor; de Fontebrune, Flore Sicre; de Latour, Régis Peffault; Toubert, Antoine; Socié, Gérard

2015-01-01

Immune reconstitution after allogeneic stem cell transplantation is a dynamic and complex process depending on the recipient and donor characteristics, on the modalities of transplantation, and on the occurrence of graft-versus-host disease. Multivariate methods widely used for gene expression profiling can simultaneously analyze the patterns of a great number of biological variables on a heterogeneous set of patients. Here we use these methods on flow cytometry assessment of up to 25 lymphocyte populations to analyze the global pattern of long-term immune reconstitution after transplantation. Immune patterns were most distinct from healthy controls at six months, and had not yet fully recovered as long as two years after transplant. The two principal determinants of variability were linked to the balance of B and CD8+ T cells and of natural killer and B cells, respectively. Recipient’s cytomegalovirus serostatus, cytomegalovirus replication, and chronic graft-versus-host disease were the main factors shaping the immune pattern one year after transplant. We identified a complex signature of under- and over-representation of immune populations dictated by recipient’s cytomegalovirus seropositivity. Finally, we identified dimensions of variance in immune patterns as significant predictors of long-term non-relapse mortality, independently of chronic graft-versus-host disease. PMID:25261095

Reconstitution of full‐thickness skin by microcolumn grafting

PubMed Central

Wang, Ying; Vuong, Linh N.; Fisher, Jeremy M.; Farinelli, William A.; Anderson, R. Rox

2016-01-01

Abstract In addition to providing a physical barrier, skin also serves a diverse range of physiological functions through different specialized resident cell types/structures, including melanocytes (pigmentation and protection against ultraviolet radiation), Langerhans cells (adaptive immunity), fibroblasts (maintaining extracellular matrix, paracrine regulation of keratinocytes), sweat glands (thermoregulation) and hair follicles (hair growth, sensation and a stem cell reservoir). Restoration of these functional elements has been a long‐standing challenge in efforts to engineer skin tissue, while autologous skin grafting is limited by the scarcity of donor site skin and morbidity caused by skin harvesting. We demonstrate an alternative approach of harvesting and then implanting μm‐scale, full‐thickness columns of human skin tissue, which can be removed from a donor site with minimal morbidity and no scarring. Fresh human skin microcolumns were used to reconstitute skin in wounds on immunodeficient mice. The restored skin recapitulated many key features of normal human skin tissue, including epidermal architecture, diverse skin cell populations, adnexal structures and sweat production in response to cholinergic stimulation. These promising preclinical results suggest that harvesting and grafting of microcolumns may be useful for reconstituting fully functional skin in human wounds, without donor site morbidity. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd. PMID:27296503

Inhibiting host-pathogen interactions using membrane-based nanostructures.

PubMed

Bricarello, Daniel A; Patel, Mira A; Parikh, Atul N

2012-06-01

Virulent strains of bacteria and viruses recognize host cells by their plasma membrane receptors and often exploit the native translocation machinery to invade the cell. A promising therapeutic concept for early interruption of pathogen infection is to subvert this pathogenic trickery using exogenously introduced decoys that present high-affinity mimics of cellular receptors. This review highlights emerging applications of molecularly engineered lipid-bilayer-based nanostructures, namely (i) functionalized liposomes, (ii) supported colloidal bilayers or protocells and (iii) reconstituted lipoproteins, which display functional cellular receptors in optimized conformational and aggregative states. These decoys outcompete host cell receptors by preferentially binding to and neutralizing virulence factors of both bacteria and viruses, thereby promising a new approach to antipathogenic therapy. Copyright © 2012 Elsevier Ltd. All rights reserved.

Inflammation induced by mast cell deficiency rather than the loss of interstitial cells of Cajal causes smooth muscle dysfunction in W/Wv mice

PubMed Central

Winston, John H.; Chen, Jinghong; Shi, Xuan-Zheng; Sarna, Sushil K.

2014-01-01

The initial hypothesis suggested that the interstitial cells of Cajal (ICC) played an essential role in mediating enteric neuronal input to smooth muscle cells. Much information for this hypothesis came from studies in W/Wv mice lacking ICC. However, mast cells, which play critical roles in regulating inflammation in their microenvironment, are also absent in W/Wv mice. We tested the hypothesis that the depletion of mast cells in W/Wv mice generates inflammation in fundus muscularis externa (ME) that impairs smooth muscle reactivity to Ach, independent of the depletion of ICC. We performed experiments on the fundus ME from wild type (WT) and W/Wv mice before and after reconstitution of mast cells by bone marrow transplant. We found that mast cell deficiency in W/Wv mice significantly increased COX-2 and iNOS expression and decreased smooth muscle reactivity to Ach. Mast cell reconstitution or concurrent blockade of COX-2 and iNOS restored smooth muscle contractility without affecting the suppression of c-kit in W/Wv mice. The expression of nNOS and ChAT were suppressed in W/Wv mice; mast cell reconstitution did not restore them. We conclude that innate inflammation induced by mast cell deficiency in W/Wv mice impairs smooth muscle contractility independent of ICC deficiency. The impairment of smooth muscle contractility and the suppression of the enzymes regulating the synthesis of Ach and NO in W/Wv mice need to be considered in evaluating the role of ICC in regulating smooth muscle and enteric neuronal function in W/Wv mice. PMID:24550836

Lower Pre-Treatment T Cell Activation in Early- and Late-Onset Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome

PubMed Central

Goovaerts, Odin; Jennes, Wim; Massinga-Loembé, Marguerite; Ondoa, Pascale; Ceulemans, Ann; Vereecken, Chris; Worodria, William; Mayanja-Kizza, Harriet; Colebunders, Robert; Kestens, Luc

2015-01-01

Background Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an inflammatory complication in HIV-TB co-infected patients receiving antiretroviral therapy (ART). The role of disturbed T cell reconstitution in TB-IRIS is not well understood. We investigated T cell activation and maturation profiles in patients who developed TB-IRIS at different intervals during ART. Methods Twenty-two HIV-TB patients who developed early-onset TB-IRIS and 10 who developed late-onset TB-IRIS were matched for age, sex and CD4 count to equal numbers of HIV-TB patients who did not develop TB-IRIS. Flow cytometry analysis was performed on fresh blood, drawn before and after ART initiation and during TB-IRIS events. T cell activation and maturation was measured on CD4+ and CD8+ T cells using CD45RO, CD38, HLA-DR, CCR7 and CD27 antibodies. Results CD8+ T cell activation before ART was decreased in both early-onset (77% vs. 82%, p = 0.014) and late-onset (71% vs. 83%, p = 0.012) TB-IRIS patients compared to non-IRIS controls. After ART initiation, the observed differences in T cell activation disappeared. During late-onset, but not early-onset TB-IRIS, we observed a skewing from memory to terminal effector CD4+ and CD8+ T cell populations (p≤0.028). Conclusion Our data provide evidence of reduced CD8+ T cell activation before ART as a common predisposing factor of early- and late-onset TB-IRIS. The occurrence of TB-IRIS itself was not marked by an over-activated CD8+ T cell compartment. Late- but not early-onset TB-IRIS was characterized by a more terminally differentiated T cell phenotype. PMID:26208109

Evaluation of an intravenous preparation information system for improving the reconstitution and dilution process.

PubMed

Jo, Yun Hee; Shin, Wan Gyoon; Lee, Ju-Yeun; Yang, Bo Ram; Yu, Yun Mi; Jung, Sun Hoi; Kim, Hyang Sook

2016-10-01

There are very few studies reporting the impact of providing intravenous (IV) preparation information on quality use of antimicrobials, particularly regarding their reconstitution and dilution. Therefore, to improve these processes in IV antimicrobial administration, an IV preparation information system (IPIS) was implemented in a hospital. We aimed to evaluate the effect of improving reconstitution and dilution by implementing an IPIS in the electronic medical record (EMR) system. Prescriptions and activity records of nurses for injectable antimicrobials that required reconstitution and dilution for IV preparation from January 2008 to December 2013 were retrieved from EMR, and assessed based on packaging label information for reconstituting and diluting solutions. We defined proper reconstitution and dilution as occurring when the reconstitution and dilution solutions prescribed were consistent with the nurses' acting records. The types of intervention in the IPIS were as follows: a pop-up alert for proper reconstitution and passive guidance for proper dilution. We calculated the monthly proper reconstitution rate (PRR) and proper dilution rate (PDR) and evaluated the changes in these rates and trends using interrupted time series analyses. Prior to the initiation of the reconstitution alert and dilution information, the PRR and PDR were 12.7 and 46.1%, respectively. The reconstitution alert of the IPIS rapidly increased the PRR by 41% (p<0.001), after which the PRR decreased by 0.9% (p=0.013) per month after several months. However, there was no significant change in the rate or trend of the PDR during the study period. This study demonstrated that the provision of reconstitution alerts by the IPIS contributed to improving the reconstitution process of IV antimicrobial injection administration. However, providing passive information on dilution solutions was ineffective. Furthermore, solutions to ensure the continuous effectiveness of alert systems are warranted and should be actively sought. Copyright © 2016. Published by Elsevier Ireland Ltd.

The potential association of CMV-specific CD8+ T lymphocyte reconstitution with the risk of CMV reactivation and persistency in post allogeneic stem cell transplant patients.

PubMed

Shams El-Din, Ahmed Ali; El-Desoukey, Nermeen Ahmed; Amin Tawadrous, Dalia Gamil; Fouad, Neveen Mohammed Baha El-Din; Abdel-Mooti, Mohammed; Hotar, Said Fathy

2018-01-09

development of cytomegalovirus (CMV)-specific CD8+ T cell response is crucial in preventing symptomatic CMV infection specially, in stem cell transplant (SCT) patients. The aim of this study was to evaluate CMV-specific CD8+ T cell reconstitution in allogeneic SCT recipients and to study the possible association between CMV-specific CD8+ T cell recovery with protection from CMV reactivation and persistency. Human leuKocyte antigen (HLA)-tetramers were used for CMV-specific CD8+ cell quantitation by Flow cytometry in twenty post-allogeneic SCT patients. Nine patients (45%) developed rapid recovery of CMV-specific CD8+ cells, among them; 7 patients (78%) had no CMV reactivation in the first 95 days post-transplant. Five patients had developed persistent CMV viremia; all of them had not developed CMV-specific CD8+ recovery till day 95 post-transplant. Patients with persistent CMV viremia had a statistically significant lower means of CMV-specific CD8+ percent and absolute count compared to those without persistent viremia (p = .001, .015), respectively. The incidence of CMV reactivation and persistency was higher among patients with delayed CMV-specific CD8+ reconstitution in the first 95 days post-transplant. CMV-specific CD8+ cells can help in categorizing patients into risk groups: (early recovery/low risk) and (delayed recovery/increased risk), this tool may guide clinicians in the selection of patients who may profit from prophylactic antiviral therapy and frequent viral monitoring.

Monocyte-derived dendritic cells induce a house dust mite-specific Th2 allergic inflammation in the lung of humanized SCID mice: involvement of CCR7.

PubMed

Hammad, Hamida; Lambrecht, Bart N; Pochard, Pierre; Gosset, Philippe; Marquillies, Philippe; Tonnel, André-Bernard; Pestel, Joël

2002-08-01

In rodents, airway dendritic cells (DCs) capture inhaled Ag, undergo maturation, and migrate to the draining mediastinal lymph nodes (MLN) to initiate the Ag-specific T cell response. However, the role of human DCs in the pathogenesis of the Th2 cell-mediated disease asthma remains to be clarified. Here, by using SCID mice engrafted with T cells from either house dust mite (HDM)-allergic patients or healthy donors, we show that DCs pulsed with Der p 1, one of the major allergens of HDM, and injected intratracheally into naive animals migrated into the MLN. In the MLN, Der p 1-pulsed DCs from allergic patients induced the proliferation of IL-4-producing CD4(+) T cells, whereas those from healthy donors induced IFN-gamma-secreting cells. In reconstituted human PBMC-reconstituted SCID mice primed with pulsed DCs from allergic patients, repeated exposure to aerosols of HDM induced 1) a strong pulmonary inflammatory reaction rich in T cells and eosinophils, 2) an increase in IL-4 and IL-5 production in the lung lavage fluid, and 3) increased IgE production compared with that in mice primed with unpulsed DCs. All these effects were reduced following in vivo neutralization of the CCR7 ligand secondary lymphoid tissue chemokine. These data in human PBMC-reconstituted SCID mice show that monocyte-derived DCs might play a key role in the pathogenesis of the pulmonary allergic response by inducing Th2 effector function following migration to the MLN.

The phenotype of cancer cell invasion controlled by fibril diameter and pore size of 3D collagen networks.

PubMed

Sapudom, Jiranuwat; Rubner, Stefan; Martin, Steve; Kurth, Tony; Riedel, Stefanie; Mierke, Claudia T; Pompe, Tilo

2015-06-01

The behavior of cancer cells is strongly influenced by the properties of extracellular microenvironments, including topology, mechanics and composition. As topological and mechanical properties of the extracellular matrix are hard to access and control for in-depth studies of underlying mechanisms in vivo, defined biomimetic in vitro models are needed. Herein we show, how pore size and fibril diameter of collagen I networks distinctively regulate cancer cell morphology and invasion. Three-dimensional collagen I matrices with a tight control of pore size, fibril diameter and stiffness were reconstituted by adjustment of concentration and pH value during matrix reconstitution. At first, a detailed analysis of topology and mechanics of matrices using confocal laser scanning microscopy, image analysis tools and force spectroscopy indicate pore size and not fibril diameter as the major determinant of matrix elasticity. Secondly, by using two different breast cancer cell lines (MDA-MB-231 and MCF-7), we demonstrate collagen fibril diameter--and not pore size--to primarily regulate cell morphology, cluster formation and invasion. Invasiveness increased and clustering decreased with increasing fibril diameter for both, the highly invasive MDA-MB-231 cells with mesenchymal migratory phenotype and the MCF-7 cells with amoeboid migratory phenotype. As this behavior was independent of overall pore size, matrix elasticity is shown to be not the major determinant of the cell characteristics. Our work emphasizes the complex relationship between structural-mechanical properties of the extracellular matrix and invasive behavior of cancer cells. It suggests a correlation of migratory and invasive phenotype of cancer cells in dependence on topological and mechanical features of the length scale of single fibrils and not on coarse-grained network properties. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cre-mediated cell ablation contests mast cell contribution in models of antibody- and T cell-mediated autoimmunity.

PubMed

Feyerabend, Thorsten B; Weiser, Anne; Tietz, Annette; Stassen, Michael; Harris, Nicola; Kopf, Manfred; Radermacher, Peter; Möller, Peter; Benoist, Christophe; Mathis, Diane; Fehling, Hans Jörg; Rodewald, Hans-Reimer

2011-11-23

Immunological functions of mast cells remain poorly understood. Studies in Kit mutant mice suggest key roles for mast cells in certain antibody- and T cell-mediated autoimmune diseases. However, Kit mutations affect multiple cell types of both immune and nonimmune origin. Here, we show that targeted insertion of Cre-recombinase into the mast cell carboxypeptidase A3 locus deleted mast cells in connective and mucosal tissues by a genotoxic Trp53-dependent mechanism. Cre-mediated mast cell eradication (Cre-Master) mice had, with the exception of a lack of mast cells and reduced basophils, a normal immune system. Cre-Master mice were refractory to IgE-mediated anaphylaxis, and this defect was rescued by mast cell reconstitution. This mast cell-deficient strain was fully susceptible to antibody-induced autoimmune arthritis and to experimental autoimmune encephalomyelitis. Differences comparing Kit mutant mast cell deficiency models to selectively mast cell-deficient mice call for a systematic re-evaluation of immunological functions of mast cells beyond allergy. Copyright © 2011 Elsevier Inc. All rights reserved.

Determining P-glycoprotein-drug interactions: evaluation of reconstituted P-glycoprotein in a liposomal system and LLC-MDR1 polarized cell monolayers

PubMed Central

Melchior, Donald L.; Sharom, Frances J.; Evers, Raymond; Wright, George E.; Chu, Joseph W.K.; Wright, Stephen E.; Chu, Xiaoyan; Yabut, Jocelyn

2012-01-01

Introduction P-Glycoprotein (ABCB1, MDR1) is a multidrug efflux pump that is a member of the ATP-binding cassette (ABC) superfamily. Many drugs in common clinical use are either substrates or inhibitors of this transporter. Quantitative details of P-glycoprotein inhibition by pharmaceutical agents are essential for assessment of their pharmacokinetic behavior and prevention of negative patient reactions. Cell-based systems have been widely used for determination of drug interactions with P-glycoprotein, but they suffer from several disadvantages, and results are often widely variable between laboratories. We aimed to demonstrate that a novel liposomal system employing contemporary biochemical methodologies could measure the ability of clinically used drugs to inhibit the P-glycoprotein pump. To accomplish this we compared results with those of cell-based approaches. Methods Purified transport-competent hamster Abcb1a P-glycoprotein was reconstituted into a unilamellar liposomal system, Fluorosome-trans-pgp, whose aqueous interior contains fluorescent drug sensors. This provides a well-defined system for measuring P-glycoprotein transport inhibition by test drugs in real time using rapid fluorescence-based technology. Results Inhibition of ATP-driven transport by Fluorosome-trans-pgp employed a panel of 46 representative drugs. Resulting IC50 values correlated well (r2 = 0.80) with Kd values for drug binding to purified P-glycoprotein. They also showed a similar trend to transport inhibition data obtained using LLC-MDR1 cell monolayers. Fluorosome-trans-pgp IC50 values were in agreement with published results of digoxin drug-drug interaction studies in humans. Discussion This novel approach using a liposomal system and fluorescence-based technology is shown to be suitable to study whether marketed drugs and drug candidates are P-glycoprotein inhibitors. The assay is rapid, allowing a 7-point IC50 determination in <6 minutes, and requires minimal quantities of test drug. The method is amenable to robotics and offers a cost advantage relative to conventional cell-based assays. The well-defined nature of this assay also obviates many of the inherent complications and ambiguities of cell-based systems. PMID:22394995

Determining P-glycoprotein-drug interactions: evaluation of reconstituted P-glycoprotein in a liposomal system and LLC-MDR1 polarized cell monolayers.

PubMed

Melchior, Donald L; Sharom, Frances J; Evers, Raymond; Wright, George E; Chu, Joseph W K; Wright, Stephen E; Chu, Xiaoyan; Yabut, Jocelyn

2012-03-01

P-Glycoprotein (ABCB1, MDR1) is a multidrug efflux pump that is a member of the ATP-binding cassette (ABC) superfamily. Many drugs in common clinical use are either substrates or inhibitors of this transporter. Quantitative details of P-glycoprotein inhibition by pharmaceutical agents are essential for assessment of their pharmacokinetic behavior and prevention of negative patient reactions. Cell-based systems have been widely used for determination of drug interactions with P-glycoprotein, but they suffer from several disadvantages, and results are often widely variable between laboratories. We aimed to demonstrate that a novel liposomal system employing contemporary biochemical methodologies could measure the ability of clinically used drugs to inhibit the P-glycoprotein pump. To accomplish this we compared results with those of cell-based approaches. Purified transport-competent hamster Abcb1a P-glycoprotein was reconstituted into a unilamellar liposomal system, Fluorosome-trans-pgp, whose aqueous interior contains fluorescent drug sensors. This provides a well-defined system for measuring P-glycoprotein transport inhibition by test drugs in real time using rapid fluorescence-based technology. Inhibition of ATP-driven transport by Fluorosome-trans-pgp employed a panel of 46 representative drugs. Resulting IC50 values correlated well (r2=0.80) with Kd values for drug binding to purified P-glycoprotein. They also showed a similar trend to transport inhibition data obtained using LLC-MDR1 cell monolayers. Fluorosome-trans-pgp IC50 values were in agreement with published results of digoxin drug-drug interaction studies in humans. This novel approach using a liposomal system and fluorescence-based technology is shown to be suitable to study whether marketed drugs and drug candidates are P-glycoprotein inhibitors. The assay is rapid, allowing a 7-point IC50 determination in <6 min, and requires minimal quantities of test drug. The method is amenable to robotics and offers a cost advantage relative to conventional cell-based assays. The well-defined nature of this assay also obviates many of the inherent complications and ambiguities of cell-based systems. Copyright © 2012 Elsevier Inc. All rights reserved.

Expression, purification and functional reconstitution of slack sodium-activated potassium channels.

PubMed

Yan, Yangyang; Yang, Youshan; Bian, Shumin; Sigworth, Fred J

2012-11-01

The slack (slo2.2) gene codes for a potassium-channel α-subunit of the 6TM voltage-gated channel family. Expression of slack results in Na(+)-activated potassium channel activity in various cell types. We describe the purification and reconstitution of Slack protein and show that the Slack α-subunit alone is sufficient for potassium channel activity activated by sodium ions as assayed in planar bilayer membranes and in membrane vesicles.

In silico reconstitution of Listeria propulsion exhibits nano-saltation.

PubMed

Alberts, Jonathan B; Odell, Garrett M

2004-12-01

To understand how the actin-polymerization-mediated movements in cells emerge from myriad individual protein-protein interactions, we developed a computational model of Listeria monocytogenes propulsion that explicitly simulates a large number of monomer-scale biochemical and mechanical interactions. The literature on actin networks and L. monocytogenes motility provides the foundation for a realistic mathematical/computer simulation, because most of the key rate constants governing actin network dynamics have been measured. We use a cluster of 80 Linux processors and our own suite of simulation and analysis software to characterize salient features of bacterial motion. Our "in silico reconstitution" produces qualitatively realistic bacterial motion with regard to speed and persistence of motion and actin tail morphology. The model also produces smaller scale emergent behavior; we demonstrate how the observed nano-saltatory motion of L. monocytogenes,in which runs punctuate pauses, can emerge from a cooperative binding and breaking of attachments between actin filaments and the bacterium. We describe our modeling methodology in detail, as it is likely to be useful for understanding any subcellular system in which the dynamics of many simple interactions lead to complex emergent behavior, e.g., lamellipodia and filopodia extension, cellular organization, and cytokinesis.

IL-2 receptor γ-chain molecule is critical for intestinal T-cell reconstitution in humanized mice.

PubMed

Denton, P W; Nochi, T; Lim, A; Krisko, J F; Martinez-Torres, F; Choudhary, S K; Wahl, A; Olesen, R; Zou, W; Di Santo, J P; Margolis, D M; Garcia, J V

2012-09-01

Intestinal immune cells are important in host defense, yet the determinants for human lymphoid homeostasis in the intestines are poorly understood. In contrast, lymphoid homeostasis has been studied extensively in mice, where the requirement for a functional common γ-chain molecule has been established. We hypothesized that humanized mice could offer insights into human intestinal lymphoid homeostasis if generated in a strain with an intact mouse common γ-chain molecule. To address this hypothesis, we used three mouse strains (non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) (N/S); NOD/SCID γ-chain(-/-) (NSG); and Rag2(-/-) γ-chain(-/-) (DKO)) and two humanization techniques (bone marrow liver thymus (BLT) and human CD34(+) cell bone marrow transplant of newborn mice (hu)) to generate four common types of humanized mice: N/S-BLT, NSG-BLT, NSG-hu, and DKO-hu mice. The highest levels of intestinal human T cells throughout the small and large intestines were observed in N/S-BLT mice, which have an intact common γ-chain molecule. Furthermore, the small intestine lamina propria T-cell populations of N/S-BLT mice exhibit a human intestine-specific surface phenotype. Thus, the extensive intestinal immune reconstitution of N/S-BLT mice was both quantitatively and qualitatively better when compared with the other models tested such that N/S-BLT mice are well suited for the analysis of human intestinal lymphocyte trafficking and human-specific diseases affecting the intestines.

Chromosome Instability Underlies Hematopoietic Stem Cell Dysfunction and Lymphoid Neoplasia Associated with Impaired Fbw7-Mediated Cyclin E Regulation

PubMed Central

Siu, Ka Tat; Xu, Yanfei; Swartz, Kelsey L.; Bhattacharyya, Mitra; Gurbuxani, Sandeep; Hua, Youjia

2014-01-01

The Fbw7 ubiquitin ligase critically regulates hematopoietic stem cell (HSC) function, though the precise contribution of individual substrate ubiquitination pathways to HSC homeostasis is unknown. In the work reported here, we used a mouse model in which we introduced two knock-in mutations (T74A and T393A [changes of T to A at positions 74 and 393]) to disrupt Fbw7-dependent regulation of cyclin E, its prototypic substrate, and to examine the consequences of cyclin E dysregulation for HSC function. Serial transplantation revealed that cyclin ET74A T393A HSCs self-renewed normally; however, we identified defects in their multilineage reconstituting capacity. By inducing hematologic stress, we exposed an impaired self-renewal phenotype in cyclin E knock-in HSCs that was associated with defective cell cycle exit and the emergence of chromosome instability (CIN). Importantly, p53 deletion induced both defects in self-renewal and multilineage reconstitution in cyclin E knock-in HSCs with serial transplantation and CIN in hematopoietic stem and progenitor cells. Moreover, CIN was a feature of fatal T-cell malignancies that ultimately developed in recipients of cyclin ET74A T393A; p53-null HSCs. Together, our findings demonstrate the importance of Fbw7-dependent cyclin E control to the hematopoietic system and highlight CIN as a characteristic feature of HSC dysfunction and malignancy induced by deregulated cyclin E. PMID:24958101

Construction of membrane-bound artificial cells using microfluidics: a new frontier in bottom-up synthetic biology.

PubMed

Elani, Yuval

2016-06-15

The quest to construct artificial cells from the bottom-up using simple building blocks has received much attention over recent decades and is one of the grand challenges in synthetic biology. Cell mimics that are encapsulated by lipid membranes are a particularly powerful class of artificial cells due to their biocompatibility and the ability to reconstitute biological machinery within them. One of the key obstacles in the field centres on the following: how can membrane-based artificial cells be generated in a controlled way and in high-throughput? In particular, how can they be constructed to have precisely defined parameters including size, biomolecular composition and spatial organization? Microfluidic generation strategies have proved instrumental in addressing these questions. This article will outline some of the major principles underpinning membrane-based artificial cells and their construction using microfluidics, and will detail some recent landmarks that have been achieved. © 2016 The Author(s).

Humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice sustain the complex vertebrate life cycle of Plasmodium falciparum malaria.

PubMed

Wijayalath, Wathsala; Majji, Sai; Villasante, Eileen F; Brumeanu, Teodor D; Richie, Thomas L; Casares, Sofia

2014-09-30

Malaria is a deadly infectious disease affecting millions of people in tropical and sub-tropical countries. Among the five species of Plasmodium parasites that infect humans, Plasmodium falciparum accounts for the highest morbidity and mortality associated with malaria. Since humans are the only natural hosts for P. falciparum, the lack of convenient animal models has hindered the understanding of disease pathogenesis and prompted the need of testing anti-malarial drugs and vaccines directly in human trials. Humanized mice hosting human cells represent new pre-clinical models for infectious diseases that affect only humans. In this study, the ability of human-immune-system humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice to sustain infection with P. falciparum was explored. Four week-old DRAG mice were infused with HLA-matched human haematopoietic stem cells (HSC) and examined for reconstitution of human liver cells and erythrocytes. Upon challenge with infectious P. falciparum sporozoites (NF54 strain) humanized DRAG mice were examined for liver stage infection, blood stage infection, and transmission to Anopheles stephensi mosquitoes. Humanized DRAG mice reconstituted human hepatocytes, Kupffer cells, liver endothelial cells, and erythrocytes. Upon intravenous challenge with P. falciparum sporozoites, DRAG mice sustained liver to blood stage infection (average 3-5 parasites/microlitre blood) and allowed transmission to An. stephensi mosquitoes. Infected DRAG mice elicited antibody and cellular responses to the blood stage parasites and self-cured the infection by day 45 post-challenge. DRAG mice represent the first human-immune-system humanized mouse model that sustains the complex vertebrate life cycle of P. falciparum without the need of exogenous injection of human hepatocytes/erythrocytes or P. falciparum parasite adaptation. The ability of DRAG mice to elicit specific human immune responses to P. falciparum parasites may help deciphering immune correlates of protection and to identify protective malaria antigens.

Interleukin-4-dependent innate collaboration between iNKT cells and B-1 B cells controls adaptative contact sensitivity

PubMed Central

Campos, Regis A; Szczepanik, Marian; Itakura, Atsuko; Lisbonne, Mariette; Dey, Neelendu; Leite-de-Moraes, Maria C; Askenase, Philip W

2006-01-01

We showed that hepatic Vα14+ invariant natural killer T (iNKT) cells, via their rapid interleukin (IL)-4 production, activate B-1 cells to initiate contact sensitivity (CS). This innate collaboration was absent in IL-4–/– and signal transducer and activator of transcription (STAT)-6–/– mice and was inhibited by anti-IL-4 treatment. These mice have defective CS because they fail to locally recruit the sensitized effector T cells of acquired immunity. Their CS is reconstituted by transfer of downstream-acting 1-day immune B-1 cells from wild-type mice. Responses were not reconstituted with B-1 cells from IL-4 receptor-α–/– or STAT-6–/– mice, nor by IL-4 treatment of B cell-deficient mice at immunization. Finally, IL-4 was preferentially and transiently produced by hepatic iNKT cells within 7 min after sensitization to mediate collaboration between innate-like iNKT cells and the B-1 B cells that participate in the recruitment of effector T cells in vivo. PMID:16556268

Reconstituted fresh whole blood improves clinical outcomes compared with stored component blood therapy for neonates undergoing cardiopulmonary bypass for cardiac surgery: a randomized controlled trial.

PubMed

Gruenwald, Colleen E; McCrindle, Brian W; Crawford-Lean, Lynn; Holtby, Helen; Parshuram, Christopher; Massicotte, Patricia; Van Arsdell, Glen

2008-12-01

This study compared the effects of reconstituted fresh whole blood against standard blood component therapy in neonates undergoing cardiac surgery. Patients less than 1 month of age were randomized to receive either reconstituted fresh whole blood (n = 31) or standard blood component therapy (n = 33) to prime the bypass circuit and for transfusion during the 24 hours after cardiopulmonary bypass. Primary outcome was chest tube drainage; secondary outcomes included transfusion needs, inotrope score, ventilation time, and hospital length of stay. Patients who received reconstituted fresh whole blood had significantly less postoperative chest tube volume loss per kilogram of body weight (7.7 mL/kg vs 11.8 mL/kg; P = .03). Standard blood component therapy was associated with higher inotropic score (6.6 vs 3.3; P = .002), longer ventilation times (164 hours vs 119 hours; P = .04), as well as longer hospital stays (18 days vs 12 days; P = .006) than patients receiving reconstituted fresh whole blood. Of the different factors associated with the use of reconstituted fresh whole blood, lower platelet counts at 10 minutes and at the end of cardiopulmonary bypass, older age of cells used in the prime and throughout bypass, and exposures to higher number of allogeneic donors were found to be independent predictors of poor clinical outcomes. Reconstituted fresh whole blood used for the prime, throughout cardiopulmonary bypass, and for all transfusion requirements within the first 24 hours postoperatively results in reduced chest tube volume loss and improved clinical outcomes in neonatal patients undergoing cardiac surgery.

Microengineered physiological biomimicry: organs-on-chips.

PubMed

Huh, Dongeun; Torisawa, Yu-suke; Hamilton, Geraldine A; Kim, Hyun Jung; Ingber, Donald E

2012-06-21

Microscale engineering technologies provide unprecedented opportunities to create cell culture microenvironments that go beyond current three-dimensional in vitro models by recapitulating the critical tissue-tissue interfaces, spatiotemporal chemical gradients, and dynamic mechanical microenvironments of living organs. Here we review recent advances in this field made over the past two years that are focused on the development of 'Organs-on-Chips' in which living cells are cultured within microfluidic devices that have been microengineered to reconstitute tissue arrangements observed in living organs in order to study physiology in an organ-specific context and to develop specialized in vitro disease models. We discuss the potential of organs-on-chips as alternatives to conventional cell culture models and animal testing for pharmaceutical and toxicology applications. We also explore challenges that lie ahead if this field is to fulfil its promise to transform the future of drug development and chemical safety testing.

Antimicrobial efficacy of phytochemicals against Bacillus cereus in reconstituted infant rice cereal.

PubMed

Cetin-Karaca, Hayriye; Newman, Melissa C

2018-02-01

The objective of this study was to determine the potential use of Trans-cinnamaldehyde (TC), (-)-Epigallocatechin gallate (EGCG) and [10]-Gingerol (GI) to inhibit the growth of B. cereus in infant rice cereal reconstituted with infant formula. Samples were inoculated with either vegetative cells or spores of B. cereus (ATCC 14579), and they were treated with 500 ppm (mg/L) TC, EGCG and GI. They were stored at 7 °C, 23 °C, or 37 °C for 0, 4, 8 and 24 h to simulate advance preparation, handling and temperature abuse. At 23 °C no growth was observed with TC over 24 h. TC also showed the highest antimicrobial activity 37 °C by inhibiting the growth of B. cereus vegetative cells by 0.83 log CFU/g and B. cereus spores by 2.0 log CFU/g after 24 h. B. cereus (ATCC 14579) did not grow at 7 °C over 24 h and TC had no effect on its survival. Significant differences (P < 0.05) were found in color and aroma of rice cereal samples containing EGCG and TC, respectively. Additionally, TC exhibited a cinnamon taste, while EGCG gave a purple color to the reconstituted rice cereal. These results indicate that TC may serve as a potential natural antimicrobial in reconstituted infant rice cereal even when utilized at low concentrations, inhibiting both vegetative cells and spores of B. cereus. Copyright © 2017. Published by Elsevier Ltd.

[Hematopoietic Reconstitution and Prognosis of HLA Matched and Haploidentical Hematopoietic Stem Cell Transplantation Using Modified FC/ATG Conditioning for Treatment of Severe Aplastic Anemia].

PubMed

Wang, Li; Wu, Ya-Mei; Cao, Yong-Bin; Li, Xiao-Hong; Xu, Li-Xin; Wang, Hai-Tao; Gao, Ya-Hui; Wu, Xiao-Xiong

2016-12-01

To analyse the feasibility and compare differences between hematopoietic reconstitution and prognosis of patients with severe aplastic anemia(SAA) after matched sibling donor (MSD) or haploidentical family donor (HFD) hematopoietic stem cell transplantation (HSCT) using the modified FC/ATG conditioning. The clinical data of 56 patients with SAA who received HSCT in First Affiliated Hospital of Chinese PLA General Hospital from January 2011 to June 2016 were analyzed retrospectively. The hematopoietic reconstitution, graft verus host disease (GVHD), transplantation related toxicity (TRT) and prognosis after transplantation were compared. Furthermore, the modifed conditioning FC/ATG included low-dose cyclophosphamide (total dose 100 mg/kg), infustion of third-party donor-derived mesenchymal stem cells. All 56 patients with MSD-HSCT or HFD-HSCT achieved hematopoietic reconstitution. Among them, not only the recovery of neutrophils and platelets, but also the incidences of III-IV aGVHD, extensive cGVHD and TRT were not significantly different (the P value were 0.58, 0.61, 0.73, 0.73 and 0.67, respectively). After following-up for 32(2-66) months, 48 patients alive well, the 1-year overall survival rates were 86% in HFD-HSCT group and 89% in MSD-HSCT group, respectively (P=0.58). After HSCT using the modifed FC/ATG conditioning, patients with SAA achieved stable engraftment, low toxicity, mild GVHD and excellent outcomes. Furthermore, the HFD-HSCT achieved comparable outcomes to MSD-HSCT and may be served as an alternate therapy for patients with SAA.

Analysis of the `Biarez Favre' and `Burland' models for the compressibility of remoulded clays

NASA Astrophysics Data System (ADS)

Favre, Jean-Louis; Hattab, Mahdia

2008-01-01

This study aims at comparing the prediction by the Biarez and Favre model as well as by the more recent Burland one, established for reconstituted normally consolidated clays submitted to oedometric loading. The former, proposed in the 1970s, uses the liquidity index IL, and while the latter introduces a parameter, Iv, which is a normalised void index based on two characteristic void ratios ( e100* and e1000*) corresponding to the oedometric curve of σv=100 kPa and σv=1000 kPa. The aim of these models is to predict the compressibility parameters based on the identification of parameters represented by the Atterberg limits ( wL, wP, Ip) as well as of other physical parameters such as the void ratio e or the natural water content wnat, taking into account the effective overburden pressure σv. These models, which represent the intrinsic properties of clays under compression, are compared with two experimental curves, the first one representing remoulded and reconstituted clay, and the other one a deepwater clay sediment taken from the Gulf of Guinea at a depth of 700 m.

Stem/progenitor cell-like properties of desmoglein 3dim cells in primary and immortalized keratinocyte lines.

PubMed

Wan, Hong; Yuan, Ming; Simpson, Cathy; Allen, Kirsty; Gavins, Felicity N E; Ikram, Mohammed S; Basu, Subham; Baksh, Nuzhat; O'Toole, Edel A; Hart, Ian R

2007-05-01

We showed previously that primary keratinocytes selected for low desmoglein 3 (Dsg3) expression levels exhibited increased colony-forming efficiency and heightened proliferative potential relative to cells with higher Dsg3 expression levels, characteristics consistent with a more "stem/progenitor cell-like" phenotype. Here, we have confirmed that Dsg3(dim) cells derived from cultured primary human adult keratinocytes have comparability with alpha(6)(bri)/CD71(dim) stem cells in terms of colony-forming efficiency. Moreover, these Dsg3(dim) cells exhibit increased reconstituting ability in in vitro organotypic culture on de-epidermalized dermis (DED); they are small, actively cycling cells, and they express elevated levels of various p63 isoforms. In parallel, using the two immortalized keratinocyte cell lines HaCaT and NTERT, we obtained essentially similar though occasionally different findings. Thus, reduced colony-forming efficiency by Dsg3(bri) cells consistently was observed in both cell lines even though the cell cycle profile and levels of p63 isoforms in the bri and dim populations differed between these two cell lines. Dsg3(dim) cells from both immortalized lines produced thicker and better ordered hierarchical structural organization of reconstituted epidermis relative to Dsg3(bri) and sorted control cells. Dsg3(dim) HaCaT cells also show sebocyte-like differentiation in the basal compartment of skin reconstituted after a 4-week organotypic culture. No differences in percentages of side population cells (also a putative marker of stem cells) were detected between Dsg3(dim) and Dsg3(bri) populations. Taken together our data indicate that Dsg3(dim) populations from primary human adult keratinocytes and long-term established keratinocyte lines possess certain stem/progenitor cell-like properties, although the side population characteristic is not one of these features. Disclosure of potential conflicts of interest is found at the end of this article.

Hematopoietic Stem Cells Are the Major Source of Multilineage Hematopoiesis in Adult Animals.

PubMed

Sawai, Catherine M; Babovic, Sonja; Upadhaya, Samik; Knapp, David J H F; Lavin, Yonit; Lau, Colleen M; Goloborodko, Anton; Feng, Jue; Fujisaki, Joji; Ding, Lei; Mirny, Leonid A; Merad, Miriam; Eaves, Connie J; Reizis, Boris

2016-09-20

Hematopoietic stem cells (HSCs) sustain long-term reconstitution of hematopoiesis in transplantation recipients, yet their role in the endogenous steady-state hematopoiesis remains unclear. In particular, recent studies suggested that HSCs provide a relatively minor contribution to immune cell development in adults. We directed transgene expression in a fraction of HSCs that maintained reconstituting activity during serial transplantations. Inducible genetic labeling showed that transgene-expressing HSCs gave rise to other phenotypic HSCs, confirming their top position in the differentiation hierarchy. The labeled HSCs rapidly contributed to committed progenitors of all lineages and to mature myeloid cells and lymphocytes, but not to B-1a cells or tissue macrophages. Importantly, labeled HSCs gave rise to more than two-thirds of all myeloid cells and platelets in adult mice, and this contribution could be accelerated by an induced interferon response. Thus, classically defined HSCs maintain immune cell development in the steady state and during systemic cytokine responses. Copyright © 2016 Elsevier Inc. All rights reserved.

Module Based Complexity Formation: Periodic Patterning in Feathers and Hairs

PubMed Central

Chuong, Cheng-Ming; Yeh, Chao-Yuan; Jiang, Ting-Xin; Widelitz, Randall

2012-01-01

Patterns describe order which emerges from homogeneity. Complex patterns on the integument are striking because of their visibility throughout an organism's lifespan. Periodic patterning is an effective design because the ensemble of hair or feather follicles (modules) allows the generation of complexity, including regional variations and cyclic regeneration, giving the skin appendages a new lease on life. Spatial patterns include the arrangements of feathers and hairs in specified number, size, and spacing. We explore how a field of equivalent progenitor cells can generate periodically arranged modules based on genetic information, physical-chemical rules and developmental timing. Reconstitution experiments suggest a competitive equilibrium regulated by activators / inhibitors involving Turing reaction-diffusion. Temporal patterns result from oscillating stem cell activities within each module (micro-environment regulation), reflected as growth (anagen) and resting (telogen) phases during the cycling of feather and hair follicles. Stimulating modules with activators initiates the spread of regenerative hair waves, while global inhibitors outside each module (macro-environment) prevent this. Different wave patterns can be simulated by Cellular Automata principles. Hormonal status and seasonal changes can modulate appendage phenotypes, leading to “organ metamorphosis”, with multiple ectodermal organ phenotypes generated from the same precursors. We discuss potential evolutionary novel steps using this module based complexity in several amniote integument organs, exemplified by the spectacular peacock feather pattern. We thus explore the application of the acquired knowledge of patterning in tissue engineering. New hair follicles can be generated after wounding. Hairs and feathers can be reconstituted through self-organization of dissociated progenitor cells. PMID:23539312

Module-based complexity formation: periodic patterning in feathers and hairs.

PubMed

Chuong, Cheng-Ming; Yeh, Chao-Yuan; Jiang, Ting-Xin; Widelitz, Randall

2013-01-01

Patterns describe order which emerges from homogeneity. Complex patterns on the integument are striking because of their visibility throughout an organism’s lifespan. Periodic patterning is an effective design because the ensemble of hair or feather follicles (modules) allows the generation of complexity, including regional variations and cyclic regeneration, giving the skin appendages a new lease on life. Spatial patterns include the arrangements of feathers and hairs in specific number, size, and spacing.We explorehowa field of equivalent progenitor cells can generate periodically arranged modules based on genetic information, physical–chemical rules and developmental timing. Reconstitution experiments suggest a competitive equilibrium regulated by activators/inhibitors involving Turing reaction-diffusion. Temporal patterns result from oscillating stem cell activities within each module (microenvironment regulation), reflected as growth (anagen) and resting (telogen) phases during the cycling of feather and hair follicles. Stimulating modules with activators initiates the spread of regenerative hair waves, while global inhibitors outside each module (macroenvironment) prevent this. Different wave patterns can be simulated by cellular automata principles. Hormonal status and seasonal changes can modulate appendage phenotypes, leading to ‘organ metamorphosis’, with multiple ectodermal organ phenotypes generated from the same precursors. We discuss potential novel evolutionary steps using this module-based complexity in several amniote integument organs, exemplified by the spectacular peacock feather pattern. We thus explore the application of the acquired knowledge of patterning in tissue engineering. New hair follicles can be generated after wounding. Hairs and feathers can be reconstituted through self-organization of dissociated progenitor cells. © 2012 Wiley Periodicals, Inc.

Quantitative, non-invasive imaging of radiation-induced DNA double strand breaks in vivo

PubMed Central

Li, Wenrong; Li, Fang; Huang, Qian; Shen, Jingping; Wolf, Frank; He, Yujun; Liu, Xinjian; Hu, Y. Angela; Bedford, Joel. S.; Li, Chuan-Yuan

2011-01-01

DNA double strand breaks is a major form of DNA damage and a key mechanism through which radiotherapy and some chemotherapeutic agents kill cancer cells. Despite its importance, measuring DNA double strand breaks is still a tedious task that is normally carried out by gel electrophoresis or immunofluorescence staining. Here we report a novel approach to image and quantify DNA double strand breaks in live mammalian cells through bi-fragment luciferase reconstitution. N- and C- terminal fragments of firefly luciferase gene were fused with H2AX and MDC1 genes, respectively. Our strategy was based on the established fact that at the sites of DNA double strand breaks, H2AX protein is phosphoryated and physically associates with the MDC1 protein, thus bringing together N- and C- luciferase fragments and reconstituting luciferase activity. Our strategy allowed serial, non-invasive quantification of DNA double strand breaks in cells irradiated with x-rays and 56Fe ions. Furthermore, it allowed for the evaluation of DNA double strand breaks (DSBs) non-invasively in vivo in irradiated tumors over two weeks. Surprisingly, we detected a second wave of DSB induction in irradiated tumor cells days after radiation exposure in addition to the initial rapid induction of DSBs. We conclude that our new split-luciferase based method for imaging γ-H2AX-MDC1 interaction is a powerful new tool to study DNA double strand break repair kinetics in vivo with considerable advantage for experiments requiring observations over an extended period of time. PMID:21527553

Modeling the dynamics of shape generation and sensing by proteins on lipid membranes

NASA Astrophysics Data System (ADS)

Walani, Nikhil; Arroyo, Marino

Lipid membranes are fluid surfaces with flexural resistance that interact with proteins to perform their function in a biological context. A set of these proteins are responsible for shaping the lipid membranes, or of sensing curvature. A large body of work has examined the curvature sensing and generation properties of these proteins. Even though such processes are fundamentally dynamical in cells and in in vitro reconstituted systems, theoretical and computational studies have largely focussed on equilibrium thermodynamics. In this work, we propose a theoretical framework based on Onsager's variational principle of irreversible thermodynamics that captures the dynamics of adsorption, diffusion, and shape generation or sensing of proteins on lipid surfaces. We acknowledge the funds from European Research Council CoG- 681434 to support this research.

Adherence to stainless steel by foodborne microorganisms during growth in model food systems.

PubMed

Hood, S K; Zottola, E A

1997-07-22

Biofilm formation on stainless steel by Salmonella typhimurium, Listeria monocytogenes, Escherichia coli O157:H7, Pseudomonas fragi and Pseudomonas fluorescens during growth in model food systems was studied. Test growth media included tryptic soy broth (TSB), diluted TSB (dTSB), 1% reconstituted skim milk (RSM) and diluted meat juice (DMJ). Adherent cells were stained with acridine orange and enumerated using epifluorescent microscopy and computerized image analysis. Cells were observed on the stainless steel surface after 1 h in all of the media. However, the increases in the number of adherent cells over time was seen only with S. typhimurium in DMJ, E. coli O157:H7 in TSB, dTSB and DMJ, P. fragi in RSM and P. fluorescens in RSM. The medium which produced the highest observed level of adherent cells was different for each microorganism.

In vitro models of tail contraction and cytoplasmic streaming in amoeboid cells.

PubMed

Janson, L W; Taylor, D L

1993-10-01

We have developed a reconstituted gel-sol and contractile model system that mimics the structure and dynamics found at the ectoplasm/endoplasm interface in the tails of many amoeboid cells. We tested the role of gel-sol transformations of the actin-based cytoskeleton in the regulation of contraction and in the generation of endoplasm from ectoplasm. In a model system with fully phosphorylated myosin II, we demonstrated that either decreasing the actin filament length distribution or decreasing the extent of actin filament cross-linking initiated both a weakening of the gel strength and contraction. However, streaming of the solated gel components occurred only under conditions where the length distribution of actin was decreased, causing a self-destruct process of continued solation and contraction of the gel. These results offer significant support that gel strength plays an important role in the regulation of actin/myosin II-based contractions of the tail cortex in many amoeboid cells as defined by the solation-contraction coupling hypothesis (Taylor, D. L., and M. Fechheimer. 1982. Phil. Trans. Soc. Lond. B. 299:185-197). The competing processes of solation and contraction of the gel would appear to be mutually exclusive. However, it is the temporal-spatial balance of the rate and extent of two stages of solation, coupled to contraction, that can explain the conversion of gelled ectoplasm in the tail to a solated endoplasm within the same small volume, generation of a force for the retraction of tails, maintenance of cell polarity, and creation of a positive hydrostatic pressure to push against the newly formed endoplasm. The mechanism of solation-contraction of cortical cytoplasm may be a general component of the normal movement of a variety of amoeboid cells and may also be a component of other contractile events such as cytokinesis.

Thrombopoietin treatment of one graft in a double cord blood transplant provides early platelet recovery while contributing to long-term engraftment in NSG mice.

PubMed

van der Garde, Mark; van Hensbergen, Yvette; Brand, Anneke; Slot, Manon C; de Graaf-Dijkstra, Alice; Mulder, Arend; Watt, Suzanne M; Zwaginga, Jaap Jan

2015-01-01

Human cord blood (CB) hematopoietic stem cell (HSC) transplants demonstrate delayed early neutrophil and platelet recovery and delayed longer term immune reconstitution compared to bone marrow and mobilized peripheral blood transplants. Despite advances in enhancing early neutrophil engraftment, platelet recovery after CB transplantation is not significantly altered when compared to contemporaneous controls. Recent studies have identified a platelet-biased murine HSC subset, maintained by thrombopoietin (TPO), which has enhanced capacity for short- and long-term platelet reconstitution, can self-renew, and can give rise to myeloid- and lymphoid-biased HSCs. In previous studies, we have shown that transplantation of human CB CD34(+) cells precultured in TPO as a single graft accelerates early platelet recovery as well as yielding long-term repopulation in immune-deficient mice. In this study, using a double CB murine transplant model, we investigated whether TPO cultured human CB CD34(+) cells have a competitive advantage or disadvantage over untreated human CB CD34(+) cells in terms of (1) short-term and longer term platelet recovery and (2) longer term hematological recovery. Our studies demonstrate that the TPO treated graft shows accelerated early platelet recovery without impairing the platelet engraftment of untreated CD34(+) cells. Notably, this was followed by a dominant contribution to platelet production through the untreated CD34(+) cell graft over the intermediate to longer term. Furthermore, although the contribution of the TPO treated graft to long-term hematological engraftment was reduced, the TPO treated and untreated grafts both contributed significantly to long-term chimerism in vivo.

Generation of folliculogenic human epithelial stem cells from induced pluripotent stem cells

NASA Astrophysics Data System (ADS)

Yang, Ruifeng; Zheng, Ying; Burrows, Michelle; Liu, Shujing; Wei, Zhi; Nace, Arben; Guo, Wei; Kumar, Suresh; Cotsarelis, George; Xu, Xiaowei

2014-01-01

Epithelial stem cells (EpSCs) in the hair follicle bulge are required for hair follicle growth and cycling. The isolation and propagation of human EpSCs for tissue engineering purposes remains a challenge. Here we develop a strategy to differentiate human iPSCs (hiPSCs) into CD200+/ITGA6+ EpSCs that can reconstitute the epithelial components of the hair follicle and interfollicular epidermis. The hiPSC-derived CD200+/ITGA6+ cells show a similar gene expression signature as EpSCs directly isolated from human hair follicles. Human iPSC-derived CD200+/ITGA6+ cells are capable of generating all hair follicle lineages including the hair shaft, and the inner and outer root sheaths in skin reconstitution assays. The regenerated hair follicles possess a KRT15+ stem cell population and produce hair shafts expressing hair-specific keratins. These results suggest an approach for generating large numbers of human EpSCs for tissue engineering and new treatments for hair loss, wound healing and other degenerative skin disorders.

Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice.

PubMed

Fuster, José J; MacLauchlan, Susan; Zuriaga, María A; Polackal, Maya N; Ostriker, Allison C; Chakraborty, Raja; Wu, Chia-Ling; Sano, Soichi; Muralidharan, Sujatha; Rius, Cristina; Vuong, Jacqueline; Jacob, Sophia; Muralidhar, Varsha; Robertson, Avril A B; Cooper, Matthew A; Andrés, Vicente; Hirschi, Karen K; Martin, Kathleen A; Walsh, Kenneth

2017-02-24

Human aging is associated with an increased frequency of somatic mutations in hematopoietic cells. Several of these recurrent mutations, including those in the gene encoding the epigenetic modifier enzyme TET2, promote expansion of the mutant blood cells. This clonal hematopoiesis correlates with an increased risk of atherosclerotic cardiovascular disease. We studied the effects of the expansion of Tet2 -mutant cells in atherosclerosis-prone, low-density lipoprotein receptor-deficient ( Ldlr -/- ) mice. We found that partial bone marrow reconstitution with TET2-deficient cells was sufficient for their clonal expansion and led to a marked increase in atherosclerotic plaque size. TET2-deficient macrophages exhibited an increase in NLRP3 inflammasome-mediated interleukin-1β secretion. An NLRP3 inhibitor showed greater atheroprotective activity in chimeric mice reconstituted with TET2-deficient cells than in nonchimeric mice. These results support the hypothesis that somatic TET2 mutations in blood cells play a causal role in atherosclerosis. Copyright © 2017, American Association for the Advancement of Science.

Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice

PubMed Central

Fuster, José J.; MacLauchlan, Susan; Zuriaga, María A.; Polackal, Maya N.; Ostriker, Allison C.; Chakraborty, Raja; Wu, Chia-Ling; Sano, Soichi; Muralidharan, Sujatha; Rius, Cristina; Vuong, Jacqueline; Jacob, Sophia; Muralidhar, Varsha; Robertson, Avril A. B.; Cooper, Matthew A.; Andrés, Vicente; Hirschi, Karen K.; Martin, Kathleen A.; Walsh, Kenneth

2017-01-01

Human aging is associated with an increased frequency of somatic mutations in hematopoietic cells. Several of these recurrent mutations, including those in the gene encoding the epigenetic modifier enzyme TET2, promote expansion of the mutant blood cells. This clonal hematopoiesis correlates with an increased risk of atherosclerotic cardiovascular disease. We studied the effects of the expansion of Tet2-mutant cells in atherosclerosis-prone, low-density lipoprotein receptor–deficient (Ldlr−/−) mice. We found that partial bone marrow reconstitution with TET2-deficient cells was sufficient for their clonal expansion and led to a marked increase in atherosclerotic plaque size. TET2-deficient macrophages exhibited an increase in NLRP3 inflammasome–mediated interleukin-1β secretion. An NLRP3 inhibitor showed greater atheroprotective activity in chimeric mice reconstituted with TET2-deficient cells than in nonchimeric mice. These results support the hypothesis that somatic TET2 mutations in blood cells play a causal role in atherosclerosis. PMID:28104796

Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

PubMed Central

Fujii, Hodaka

2007-01-01

Binding of IL-2 to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2R-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. However, Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling. PMID:17266928

Cells in 3D-reconstitutued eccrine sweat gland cell spheroids differentiate into gross cystic disease fluid protein 15-expressing dark secretory cells and carbonic anhydrase II-expressing clear secretory cells.

PubMed

Li, Haihong; Chen, Liyun; Zhang, Mingjun; Zhang, Bingna

2017-07-01

Secretory coils of eccrine sweat glands are composed of myoepithelial cells, dark secretory cells and clear secretory cells. The two types of cells play important roles in sweat secretion. In our previous study, we demonstrated that the 3D-reconstituted eccrine sweat gland cell spheroids differentiate into secretory coil-like structures. However, whether the secretory coil-like structures further differentiate into dark secretory cells and clear secretory cells were is still unknown. In this study, we detected the differentiation of clear and dark secretory cells in the 3D-reconstituted eccrine sweat gland cell spheroids using the dark secretory cell-specific marker, GCDFP-15, and clear secretory cell-specific marker, CAII by immunofluorescence staining. Results showed that there were both GCDFP-15- and CAII-expressing cells in 12-week-old 3D spheroids, similar to native eccrine sweat glands, indicating that the spheroids possess a cellular structure capable of sweat secretion. We conclude that the 12-week 3D spheroids may have secretory capability. Copyright © 2017 Elsevier GmbH. All rights reserved.

Reconstitution of full-thickness skin by microcolumn grafting.

PubMed

Tam, Joshua; Wang, Ying; Vuong, Linh N; Fisher, Jeremy M; Farinelli, William A; Anderson, R Rox

2017-10-01

In addition to providing a physical barrier, skin also serves a diverse range of physiological functions through different specialized resident cell types/structures, including melanocytes (pigmentation and protection against ultraviolet radiation), Langerhans cells (adaptive immunity), fibroblasts (maintaining extracellular matrix, paracrine regulation of keratinocytes), sweat glands (thermoregulation) and hair follicles (hair growth, sensation and a stem cell reservoir). Restoration of these functional elements has been a long-standing challenge in efforts to engineer skin tissue, while autologous skin grafting is limited by the scarcity of donor site skin and morbidity caused by skin harvesting. We demonstrate an alternative approach of harvesting and then implanting μm-scale, full-thickness columns of human skin tissue, which can be removed from a donor site with minimal morbidity and no scarring. Fresh human skin microcolumns were used to reconstitute skin in wounds on immunodeficient mice. The restored skin recapitulated many key features of normal human skin tissue, including epidermal architecture, diverse skin cell populations, adnexal structures and sweat production in response to cholinergic stimulation. These promising preclinical results suggest that harvesting and grafting of microcolumns may be useful for reconstituting fully functional skin in human wounds, without donor site morbidity. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

MaxSynBio - Avenues towards creating cells from the bottom up.

PubMed

Schwille, Petra; Spatz, Joachim; Landfester, Katharina; Bodenschatz, Eberhard; Herminghaus, Stephan; Sourjik, Victor; Erb, Tobias; Bastiaens, Philippe; Lipowsky, Reinhard; Hyman, Anthony; Dabrock, Peter; Baret, Jean-Christophe; Vidakovic-Koch, Tanja; Bieling, Peter; Dimova, Rumiana; Mutschler, Hannes; Robinson, Tom; Tang, Dora; Wegner, Seraphine; Sundmacher, Kai

2018-05-11

A large Max Planck-based German research consortium ('MaxSynBio') was formed to investigate living systems from a fundamental perspective. The research program of MaxSynBio relies solely on the bottom-up approach to Synthetic Biology. MaxSynBio focuses on the detailed analysis and understanding of essential processes of life, via their modular reconstitution in minimal synthetic systems. The ultimate goal is to construct a basic living unit entirely from non-living components. The fundamental insights gained from the activities in MaxSynBio can eventually be utilized for establishing a new generation of biotechnological processes, which would be based on synthetic cell constructs that replace natural cells currently used in conventional biotechnology. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Alpha-tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II.

PubMed

Dong, L-F; Low, P; Dyason, J C; Wang, X-F; Prochazka, L; Witting, P K; Freeman, R; Swettenham, E; Valis, K; Liu, J; Zobalova, R; Turanek, J; Spitz, D R; Domann, F E; Scheffler, I E; Ralph, S J; Neuzil, J

2008-07-17

Alpha-tocopheryl succinate (alpha-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of alpha-TOS has not been identified. Here, we show that alpha-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ)-binding site (Q(P) and Q(D), respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of alpha-TOS compared to that of UbQ for the Q(P) and Q(D) sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and underwent apoptosis in the presence of alpha-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to alpha-TOS. We propose that alpha-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy.

α-Tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II

PubMed Central

Dong, Lan-Feng; Low, Pauline; Dyason, Jeffrey C.; Wang, Xiu-Fang; Prochazka, Lubomir; Witting, Paul K.; Freeman, Ruth; Swettenham, Emma; Valis, Karel; Liu, Ji; Zobalova, Renata; Turanek, Jaroslav; Spitz, Doug R.; Domann, Frederick E.; Scheffler, Immo E.; Ralph, Stephen J.; Neuzil, Jiri

2009-01-01

α-Tocopheryl succinate (α-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of α-TOS has not been identified. Here we show that α-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ) binding site (QP and QD, respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of α-TOS compared to that of UbQ for the QP and QD sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and undergo apoptosis in the presence of α-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to α-TOS. We propose that α-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy. PMID:18372923

Weakly self-reactive T-cell clones can homeostatically expand when present at low numbers.

PubMed

Vrisekoop, Nienke; Artusa, Patricio; Monteiro, Joao P; Mandl, Judith N

2017-01-01

T-cell division is central to maintaining a stable T-cell pool in adults. It also enables T-cell expansion in neonates, and after depletion by chemotherapy, bone marrow transplantation, or infection. The same signals required for T-cell survival in lymphoreplete settings, IL-7 and T-cell receptor (TCR) interactions with self-peptide MHC (pMHC), induce division when T-cell numbers are low. The strength of reactivity for self-pMHC has been shown to correlate with the capacity of T cells to undergo lymphopenia-induced proliferation (LIP), in that weakly self-reactive T cells are unable to divide, implying that T-cell reconstitution would significantly skew the TCR repertoire toward TCRs with greater self-reactivity and thus compromise T-cell diversity. Here, we show that while CD4 + T cells with low self-pMHC reactivity experience more intense competition, they are able to divide when present at low enough cell numbers. Thus, at physiological precursor frequencies CD4 + T cells with low self-pMHC reactivity are able to contribute to the reconstitution of the T-cell pool. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Immunological effects of nilotinib prophylaxis after allogeneic stem cell transplantation in patients with advanced chronic myeloid leukemia or philadelphia chromosome-positive acute lymphoblastic leukemia

PubMed Central

Shouval, Roni; Eldror, Shiran; Lev, Atar; Davidson, Jacqueline; Rosenthal, Esther; Volchek, Yulia; Shem-Tov, Noga; Yerushalmi, Ronit; Shimoni, Avichai; Somech, Raz; Nagler, Arnon

2017-01-01

Allogeneic stem cell transplantation remains the standard treatment for resistant advanced chronic myeloid leukemia and Philadelphia chromosome–positive acute lymphoblastic leukemia. Relapse is the major cause of treatment failure in both diseases. Post-allo-SCT administration of TKIs could potentially reduce relapse rates, but concerns regarding their effect on immune reconstitution have been raised. We aimed to assess immune functions of 12 advanced CML and Ph+ ALL patients who received post-allo-SCT nilotinib. Lymphocyte subpopulations and their functional activities including T-cell response to mitogens, NK cytotoxic activity and thymic function, determined by quantification of the T cell receptor (TCR) excision circles (TREC) and TCR repertoire, were evaluated at several time points, including pre-nilotib-post-allo-SCT, and up to 365 days on nilotinib treatment. NK cells were the first to recover post allo-SCT. Concomitant to nilotinib administration, total lymphocyte counts and subpopulations gradually increased. CD8 T cells were rapidly reconstituted and continued to increase until day 180 post SCT, while CD4 T cells counts were low until 180−270 days post nilotinib treatment. T-cell response to mitogenic stimulation was not inhibited by nilotinib administration. Thymic activity, measured by TREC copies and surface membrane expression of 24 different TCR Vβ families, was evident in all patients at the end of follow-up after allo-SCT and nilotinib treatment. Finally, nilotinib did not inhibit NK cytotoxic activity. In conclusion, administration of nilotinib post allo-SCT, in attempt to reduce relapse rates or progression of Ph+ ALL and CML, did not jeopardize immune reconstitution or function following transplantation. PMID:27880933

Sensitivity of small myosin II ensembles from different isoforms to mechanical load and ATP concentration.

PubMed

Erdmann, Thorsten; Bartelheimer, Kathrin; Schwarz, Ulrich S

2016-11-01

Based on a detailed crossbridge model for individual myosin II motors, we systematically study the influence of mechanical load and adenosine triphosphate (ATP) concentration on small myosin II ensembles made from different isoforms. For skeletal and smooth muscle myosin II, which are often used in actomyosin gels that reconstitute cell contractility, fast forward movement is restricted to a small region of phase space with low mechanical load and high ATP concentration, which is also characterized by frequent ensemble detachment. At high load, these ensembles are stalled or move backwards, but forward motion can be restored by decreasing ATP concentration. In contrast, small ensembles of nonmuscle myosin II isoforms, which are found in the cytoskeleton of nonmuscle cells, are hardly affected by ATP concentration due to the slow kinetics of the bound states. For all isoforms, the thermodynamic efficiency of ensemble movement increases with decreasing ATP concentration, but this effect is weaker for the nonmuscle myosin II isoforms.

Intracellular production of hydrogels and synthetic RNA granules by multivalent enhancers

PubMed Central

Nakamura, Hideki; Lee, Albert A.; Afshar, Ali Sobhi; Watanabe, Shigeki; Rho, Elmer; Razavi, Shiva; Suarez, Allison; Lin, Yu-Chun; Tanigawa, Makoto; Huang, Brian; DeRose, Robert; Bobb, Diana; Hong, William; Gabelli, Sandra B.; Goutsias, John; Inoue, Takanari

2018-01-01

Non-membrane bound, hydrogel-like entities, such as RNA granules, nucleate essential cellular functions through their unique physico-chemical properties. However, these intracellular hydrogels have not been as extensively studied as their extracellular counterparts, primarily due to technical challenges in probing these materials in situ. Here, by taking advantage of a chemically inducible dimerization paradigm, we developed iPOLYMER, a strategy for rapid induction of protein-based hydrogels inside living cells. A series of biochemical and biophysical characterizations, in conjunction with computational modeling, revealed that the polymer network formed in the cytosol resembles a physiological hydrogel-like entity that behaves as a size-dependent molecular sieve. We studied several properties of the gel and functionalized it with RNA binding motifs that sequester polyadenine-containing nucleotides to synthetically mimic RNA granules. Therefore, we here demonstrate that iPOLYMER presents a unique and powerful approach to synthetically reconstitute hydrogel-like structures including RNA granules in intact cells. PMID:29115293

Patterns of hemopoietic reconstitution in nonobese diabetic mice: dichotomy of allogeneic resistance versus competitive advantage of disease-resistant marrow.

PubMed

Kaufman, C L; Li, H; Ildstad, S T

1997-03-01

Complete replacement of the immune system via allogeneic bone marrow transplantation is sufficient to prevent diabetes in the nonobese diabetic (NOD) mouse model. In the present study we examined whether mixed allogeneic reconstitution would be sufficient to interrupt the autoimmune process with respect to occurrence of overt diabetes, as well as preexisting autoimmune insulitis. NOD mice were lethally irradiated and reconstituted with a mixture of NOD and B10.BR marrow. A relative resistance to allogeneic bone marrow engraftment was noted in NOD recipients of the mixed bone marrow inoculum, compared with disease-resistant controls. Moreover, unlike disease-resistant controls, all animals that initially repopulated as mixed donor/host chimeras became predominantly allogeneic by 4 mo, suggesting a competitive advantage for long term engraftment for disease-resistant marrow. All but one mouse in the group that engrafted with allogeneic marrow remained free of diabetes for the entire follow-up period (n = 22). Moreover, in all animals examined, virtually all islets were free of insulitis. In contrast, 74% of NOD mice that received similar conditioning and failed to engraft with donor marrow developed acute diabetes and intra-islet insulitis was present in all animals examined. These data suggest that NOD mice exhibit a relative resistance to engraftment compared with disease-resistant recipients. Conversely, animals that initially repopulated as a mixture of syngeneic and donor marrow become converted to virtually all donor by 4 mo. These data provide additional support that a defective stem cell is responsible for autoimmune diabetes in this experimental model.

Improving reconstituted HDL composition for efficient post-ischemic reduction of ischemia reperfusion injury.

PubMed

Brulhart-Meynet, Marie-Claude; Braunersreuther, Vincent; Brinck, Jonas; Montecucco, Fabrizio; Prost, Jean-Christophe; Thomas, Aurelien; Galan, Katia; Pelli, Graziano; Pedretti, Sarah; Vuilleumier, Nicolas; Mach, François; Lecour, Sandrine; James, Richard W; Frias, Miguel A

2015-01-01

New evidence shows that high density lipoproteins (HDL) have protective effects beyond their role in reverse cholesterol transport. Reconstituted HDL (rHDL) offer an attractive means of clinically exploiting these novel effects including cardioprotection against ischemia reperfusion injury (IRI). However, basic rHDL composition is limited to apolipoprotein AI (apoAI) and phospholipids; addition of bioactive compound may enhance its beneficial effects. The aim of this study was to investigate the role of rHDL in post-ischemic model, and to analyze the potential impact of sphingosine-1-phosphate (S1P) in rHDL formulations. The impact of HDL on IRI was investigated using complementary in vivo, ex vivo and in vitro IRI models. Acute post-ischemic treatment with native HDL significantly reduced infarct size and cell death in the ex vivo, isolated heart (Langendorff) model and the in vivo model (-48%, p<0.01). Treatment with rHDL of basic formulation (apoAI + phospholipids) had a non-significant impact on cell death in vitro and on the infarct size ex vivo and in vivo. In contrast, rHDL containing S1P had a highly significant, protective influence ex vivo, and in vivo (-50%, p<0.01). This impact was comparable with the effects observed with native HDL. Pro-survival signaling proteins, Akt, STAT3 and ERK1/2 were similarly activated by HDL and rHDL containing S1P both in vitro (isolated cardiomyocytes) and in vivo. HDL afford protection against IRI in a clinically relevant model (post-ischemia). rHDL is significantly protective if supplemented with S1P. The protective impact of HDL appears to target directly the cardiomyocyte.

Phthalocyanine-labeled LDL for tumor imaging and photodynamic therapy

NASA Astrophysics Data System (ADS)

Li, Hui; Marotta, Diane; Kim, Soungkyoo; Chance, Britton; Glickson, Jerry D.; Busch, Theresa M.; Zheng, Gang

2005-01-01

Current limitation of both near-infrared (NIR) tumor imaging and photodynamic therapy (PDT) is their lack of sufficient tumor-to-tissue contrast due to the relatively non-specific nature of delivering dye to the tumor, which has led to false negatives for NIR imaging and inadequate therapeutic ratio for PDT. Hence, agents targeting "cancer signatures", i.e. molecules that accumulate selectively in cancer cells, are particular attractive. One of these signatures is low-density-lipoprotein receptor (LDLR), which is overexpressed in many tumors. We have developed pyropheophorbide cholesterol oleate reconstituted LDL as a LDLR-targeting photosensitizer (PS) and demonstrated its LDLR-mediated uptake in vitro and in vivo. To improve the labeling efficiency for achieving high probe/protein ratio, tetra-t-butyl silicon phthalocyanine bearing two oleate moieties at its axial positions, (tBu)4SiPcBOA, was designed and synthesized. This compound was designed to 1) prevent the PS aggregation; 2) improve the PS solubility in non-polar solvent; and 3) maximize the PS binding to LDL phospholipid monolayer. Using this novel strategy, (tBu)4SiPcBOA was reconstituted into LDL (r-SiPcBOA-LDL) with a very high payload (500:1 molar ratio). In addition, (tBu)4SiPcBOA reconstituted acetylated LDL (r-SiPcBOA)-AcLDL with similar payload was also prepared. Since Ac-LDL cannot bind to LDLR, (r-SiPcBOA)-AcLDL can serve as the negative control to evaluate LDLR targeting specificity. For biological evaluation of these new agents, confocal microscopy and in vitro PDT protocols were performed using LDLR-overexpressing human hepatoblastoma G2 (HepG2) tumor model. These studies suggest that LDL serves as a delivery vehicle to bring large amount of the NIR/PDT agents selectively to tumor cells overexpressing LDLR.

Nanoporous silica-based protocells at multiple scales for designs of life and nanomedicine

DOE PAGES

Sun, Jie; Jakobsson, Eric; Wang, Yingxiao; ...

2015-01-19

In this study, various protocell models have been constructed de novo with the bottom-up approach. Here we describe a silica-based protocell composed of a nanoporous amorphous silica core encapsulated within a lipid bilayer built by self-assembly that provides for independent definition of cell interior and the surface membrane. In this review, we will first describe the essential features of this architecture and then summarize the current development of silica-based protocells at both micro- and nanoscale with diverse functionalities. As the structure of the silica is relatively static, silica-core protocells do not have the ability to change shape, but their interiormore » structure provides a highly crowded and, in some cases, authentic scaffold upon which biomolecular components and systems could be reconstituted. In basic research, the larger protocells based on precise silica replicas of cells could be developed into geometrically realistic bioreactor platforms to enable cellular functions like coupled biochemical reactions, while in translational research smaller protocells based on mesoporous silica nanoparticles are being developed for targeted nanomedicine. Ultimately we see two different motivations for protocell research and development: (1) to emulate life in order to understand it; and (2) to use biomimicry to engineer desired cellular interactions.« less

Nanoporous silica-based protocells at multiple scales for designs of life and nanomedicine

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

Sun, Jie; Jakobsson, Eric; Wang, Yingxiao

In this study, various protocell models have been constructed de novo with the bottom-up approach. Here we describe a silica-based protocell composed of a nanoporous amorphous silica core encapsulated within a lipid bilayer built by self-assembly that provides for independent definition of cell interior and the surface membrane. In this review, we will first describe the essential features of this architecture and then summarize the current development of silica-based protocells at both micro- and nanoscale with diverse functionalities. As the structure of the silica is relatively static, silica-core protocells do not have the ability to change shape, but their interiormore » structure provides a highly crowded and, in some cases, authentic scaffold upon which biomolecular components and systems could be reconstituted. In basic research, the larger protocells based on precise silica replicas of cells could be developed into geometrically realistic bioreactor platforms to enable cellular functions like coupled biochemical reactions, while in translational research smaller protocells based on mesoporous silica nanoparticles are being developed for targeted nanomedicine. Ultimately we see two different motivations for protocell research and development: (1) to emulate life in order to understand it; and (2) to use biomimicry to engineer desired cellular interactions.« less

5′CAG and 5′CTG Repeats Create Differential Impediment to the Progression of a Minimal Reconstituted T4 Replisome Depending on the Concentration of dNTPs

PubMed Central

Delagoutte, Emmanuelle; Baldacci, Giuseppe

2011-01-01

Instability of repetitive sequences originates from strand misalignment during repair or replicative DNA synthesis. To investigate the activity of reconstituted T4 replisomes across trinucleotide repeats (TNRs) during leading strand DNA synthesis, we developed a method to build replication miniforks containing a TNR unit of defined sequence and length. Each minifork consists of three strands, primer, leading strand template, and lagging strand template with a 5′ single-stranded (ss) tail. Each strand is prepared independently, and the minifork is assembled by hybridization of the three strands. Using these miniforks and a minimal reconstituted T4 replisome, we show that during leading strand DNA synthesis, the dNTP concentration dictates which strand of the structure-forming 5′CAG/5′CTG repeat creates the strongest impediment to the minimal replication complex. We discuss this result in the light of the known fluctuation of dNTP concentration during the cell cycle and cell growth and the known concentration balance among individual dNTPs. PMID:22096622

Dissection of Signaling Events Downstream of the c-Mpl Receptor in Murine Hematopoietic Stem Cells Via Motif-Engineered Chimeric Receptors.

PubMed

Saka, Koichiro; Lai, Chen-Yi; Nojima, Masanori; Kawahara, Masahiro; Otsu, Makoto; Nakauchi, Hiromitsu; Nagamune, Teruyuki

2018-02-01

Hematopoietic stem cells (HSCs) are a valuable resource in transplantation medicine. Cytokines are often used to culture HSCs aiming at better clinical outcomes through enhancement of HSC reconstitution capability. Roles for each signal molecule downstream of receptors in HSCs, however, remain puzzling due to complexity of the cytokine-signaling network. Engineered receptors that are non-responsive to endogenous cytokines represent an attractive tool for dissection of signaling events. We here tested a previously developed chimeric receptor (CR) system in primary murine HSCs, target cells that are indispensable for analysis of stem cell activity. Each CR contains tyrosine motifs that enable selective activation of signal molecules located downstream of the c-Mpl receptor upon stimulation by an artificial ligand. Signaling through a control CR with a wild-type c-Mpl cytoplasmic tail sufficed to enhance HSC proliferation and colony formation in cooperation with stem cell factor (SCF). Among a series of CRs, only one compatible with selective Stat5 activation showed similar positive effects. The HSCs maintained ex vivo in these environments retained long-term reconstitution ability following transplantation. This ability was also demonstrated in secondary recipients, indicating effective transmission of stem cell-supportive signals into HSCs via these artificial CRs during culture. Selective activation of Stat5 through CR ex vivo favored preservation of lymphoid potential in long-term reconstituting HSCs, but not of myeloid potential, exemplifying possible dissection of signals downstream of c-Mpl. These CR systems therefore offer a useful tool to scrutinize complex signaling pathways in HSCs.

Recombination-activating gene 1 (Rag1)-deficient mice with severe combined immunodeficiency treated with lentiviral gene therapy demonstrate autoimmune Omenn-like syndrome.

PubMed

van Til, Niek P; Sarwari, Roya; Visser, Trudi P; Hauer, Julia; Lagresle-Peyrou, Chantal; van der Velden, Guus; Malshetty, Vidyasagar; Cortes, Patricia; Jollet, Arnaud; Danos, Olivier; Cassani, Barbara; Zhang, Fang; Thrasher, Adrian J; Fontana, Elena; Poliani, Pietro L; Cavazzana, Marina; Verstegen, Monique M A; Villa, Anna; Wagemaker, Gerard

2014-04-01

Recombination-activating gene 1 (RAG1) deficiency results in severe combined immunodeficiency (SCID) caused by a complete lack of T and B lymphocytes. If untreated, patients succumb to recurrent infections. We sought to develop lentiviral gene therapy for RAG1-induced SCID and to test its safety. Constructs containing the viral spleen-focus-forming virus (SF), ubiquitous promoters, or cell type-restricted promoters driving sequence-optimized RAG1 were compared for efficacy and safety in sublethally preconditioned Rag1(-/-) mice undergoing transplantation with transduced bone marrow progenitors. Peripheral blood CD3(+) T-cell reconstitution was achieved with SF, ubiquitous promoters, and cell type-restricted promoters but 3- to 18-fold lower than that seen in wild-type mice, and with a compromised CD4(+)/CD8(+) ratio. Mitogen-mediated T-cell responses and T cell-dependent and T cell-independent B-cell responses were not restored, and T-cell receptor patterns were skewed. Reconstitution of mature peripheral blood B cells was approximately 20-fold less for the SF vector than in wild-type mice and often not detectable with the other promoters, and plasma immunoglobulin levels were abnormal. Two months after transplantation, gene therapy-treated mice had rashes with cellular tissue infiltrates, activated peripheral blood CD44(+)CD69(+) T cells, high plasma IgE levels, antibodies against double-stranded DNA, and increased B cell-activating factor levels. Only rather high SF vector copy numbers could boost T- and B-cell reconstitution, but mRNA expression levels during T- and B-cell progenitor stages consistently remained less than wild-type levels. These results underline that further development is required for improved expression to successfully treat patients with RAG1-induced SCID while maintaining low vector copy numbers and minimizing potential risks, including autoimmune reactions resembling Omenn syndrome. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Childhood Hematopoietic Cell Transplantation (PDQ®)—Health Professional Version

Cancer.gov

Hematopoietic cell transplantation involves the infusion of blood stem cells (peripheral/umbilical cord blood, bone marrow) into a patient to reconstitute the blood system. Get detailed information about autologous and allogeneic transplant, including cell selection, HLA matching, and preparative regimens, and the acute complications and late effects of treatment in this summary for clinicians.

A 10-aa-long sequence in SLP-76 upstream of the Gads binding site is essential for T cell development and function.

PubMed

Kumar, Lalit; Feske, Stefan; Rao, Anjana; Geha, Raif S

2005-12-27

The adapter SLP-76 is essential for T cell development and function. SLP-76 binds to the src homology 3 domain of Lck in vitro. This interaction depends on amino acids 185-194 of SLP-76. To examine the role of the Lck-binding region of SLP-76 in T cell development and function, SLP-76(-/-) mice were reconstituted with an SLP-76 mutant that lacks amino acids 185-194. Double and single positive thymocytes from reconstituted mice were severely reduced in numbers and exhibited impaired positive selection and increased apoptosis. Peripheral T cells were also reduced in numbers, exhibited impaired phospholipase C-gamma1 and Erk phosphorylation, and failed to flux calcium, secrete IL-2, and proliferate in response to T cell antigen receptor ligation. Delayed cutaneous hypersensitivity responses and Ab responses to T cell-dependent antigen were severely impaired. These results indicate that the Lck binding region of SLP-76 is essential for T cell antigen receptor signaling and normal T cell development and function.

T Cell Receptor Excision Circle (TREC) Monitoring after Allogeneic Stem Cell Transplantation; a Predictive Marker for Complications and Clinical Outcome

PubMed Central

Gaballa, Ahmed; Sundin, Mikael; Stikvoort, Arwen; Abumaree, Muhamed; Uzunel, Mehmet; Sairafi, Darius; Uhlin, Michael

2016-01-01

Allogeneic hematopoietic stem cell transplantation (HSCT) is a well-established treatment modality for a variety of malignant diseases as well as for inborn errors of the metabolism or immune system. Regardless of disease origin, good clinical effects are dependent on proper immune reconstitution. T cells are responsible for both the beneficial graft-versus-leukemia (GVL) effect against malignant cells and protection against infections. The immune recovery of T cells relies initially on peripheral expansion of mature cells from the graft and later on the differentiation and maturation from donor-derived hematopoietic stem cells. The formation of new T cells occurs in the thymus and as a byproduct, T cell receptor excision circles (TRECs) are released upon rearrangement of the T cell receptor. Detection of TRECs by PCR is a reliable method for estimating the amount of newly formed T cells in the circulation and, indirectly, for estimating thymic function. Here, we discuss the role of TREC analysis in the prediction of clinical outcome after allogeneic HSCT. Due to the pivotal role of T cell reconstitution we propose that TREC analysis should be included as a key indicator in the post-HSCT follow-up. PMID:27727179

Emergent Vortex Patterns in Systems of Self-Propelled, Chiral Particles

NASA Astrophysics Data System (ADS)

Huber, Lorenz; Denk, Jonas; Reithmann, Emanuel; Frey, Erwin

Self-organization of FtsZ polymers is vital for Z-ring assembly during bacterial cell division, and has been studied using reconstituted in vitro model systems. Employing Brownian dynamics simulations and a Boltzmann approach, we model FtsZ polymers as active particles moving along chiral circular paths. With both theoretical approaches we find self-organization into vortex structures and characterize different states in parameter states. Our work demonstrates that these patterns are robust and are generic for active chiral matter. Moreover, we show that the dynamics at the onset of pattern formation is described by a generalized complex Ginzburg-Landau equation.

Risk factors for mortality in a south Indian population on generic antiretroviral therapy.

PubMed

Rupali, Priscilla; Mannam, Sam; Bella, Annie; John, Lydia; Rajkumar, S; Clarence, Peace; Pulimood, Susanne A; Samuel, Prasanna; Karthik, Rajiv; Abraham, Ooriapadickal Cherian; Mathai, Dilip

2012-12-01

Antiretroviral treatment (ART) programs from low-income countries utilizing standardized ART regimens, simplified approaches to clinical decision making and basic lab monitoring have reported high mortality rates. We determined the risk factors for mortality among HIV-infected adults following the initiation of ART from a single center in south India. ART-naive HIV-infected south Indian adults attending the Infectious Diseases clinic in a 2000-bed academic medical center in south India who were initiated on ART (generic, fixed-dose combinations) as per the national guidelines were followed up. Cases (32 patients who died) were compared with age and sex matched controls. Eight-hundred and twenty-two patients were started on ART from January 1, 2000 to December 31, 2008. The cumulative mortality was 6.8% (56/822). Among the cases mean age was 44 years, 18% were women and mean CD4 counts was 107 cells/microl. Among the controls mean age was 41 years, 18% were women and mean CD4 counts were 113 cells/microl. Stavudine based ART was predominant 62.5% in the cases vs 37.5% in the controls, followed by zidovudine based therapy in 31.2% of cases and 43.7% in the controls. Tenofovir based therapy was used in 6.2% of cases vs 18.7% in the controls. The commonest causes of death were drug toxicity 19%, advanced Acquired Immunodeficiency Syndrome (AIDS) in 37%, Immune Reconstitution Inflammatory Syndrome (IRIS) in 16%, non AIDS related deaths in 22% and malignancies 6%. In a univariate analysis, absolute lymphocyte count <1200 cells/cmm (p=0.03), development of immune reconstitution inflammatory syndrome (IRIS) (p=0.000) and mean CD4 cell count increase <75 cells/microl after 1 year of ART (p=0.001) were significantly associated with mortality. The mortality among our patients was comparable to that reported from other low-income countries. Earlier initiation of ART may reduce the high mortality rates observed.

Quantitative stability of hematopoietic stem and progenitor cell clonal output in rhesus macaques receiving transplants

PubMed Central

Koelle, Samson J.

2017-01-01

Autologous transplantation of hematopoietic stem and progenitor cells lentivirally labeled with unique oligonucleotide barcodes flanked by sequencing primer targets enables quantitative assessment of the self-renewal and differentiation patterns of these cells in a myeloablative rhesus macaque model. Compared with other approaches to clonal tracking, this approach is highly quantitative and reproducible. We documented stable multipotent long-term hematopoietic clonal output of monocytes, granulocytes, B cells, and T cells from a polyclonal pool of hematopoietic stem and progenitor cells in 4 macaques observed for up to 49 months posttransplantation. A broad range of clonal behaviors characterized by contribution level and biases toward certain cell types were extremely stable over time. Correlations between granulocyte and monocyte clonalities were greatest, followed by correlations between these cell types and B cells. We also detected quantitative expansion of T cell–biased clones consistent with an adaptive immune response. In contrast to recent data from a nonquantitative murine model, there was little evidence for clonal succession after initial hematopoietic reconstitution. These findings have important implications for human hematopoiesis, given the similarities between macaque and human physiologies. PMID:28087539

T-cell phenotype and function following a first cART regimen containing either a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor in HIV-infected late presenters: results from a retrospective, ex vivo study.

PubMed

Tincati, Camilla; Savoldi, Alessia; Cannizzo, E Stefania; Bellistrì, Giusi M; Termini, Roberta; Garau, Marzia; Mancusi, Daniela; d'Arminio Monforte, Antonella; Marchetti, Giulia

2016-01-01

We aimed to comparatively assess darunavir/ritonavir (DRV/r) and efavirenz (EFV)-based first-line cART regimens in the reconstitution of T-cell phenotype and function in HIV-infected, late presenter subjects. Retrospective, ex vivo study on stored peripheral blood mononuclear cell samples of cART-naive, HIV-infected individuals with CD4(+) T-cell counts <50>250/µl upon cART initiation with either DRV/r or EFV as third drugs of standard antiretroviral regimens. CD4(+) and CD8(+) T-cell maturation (CCR7/CD45RA) and proliferation (Ki67), CD8(+) T-cell activation (CD38/HLA-DR) as well as HIV- and cytomegalovirus (CMV)-specific responses (CD4/CD8/IL-2/IFN-γ) were studied by flow cytometry at baseline (T0), T3, T6 and T12 months. Soluble inflammatory markers (IL-6 and sCD14) were measured in plasma at T0 and T12. Wilcoxon and Mann-Whitney tests were used for statistics. A total of 19 patients started DRV/r and 15 EFV. Both regimens accounted for suppression of the HIV RNA load (<40 copies/ml), reconstitution of absolute CD4(+) T-cells and CD4(+)/CD8(+) T-cell ratio. All study participants displayed a significant decrease of activated HLA-DR(+)CD38(+) CD8(+) T-cells at all study time points, yet no differences were found between study groups in T-cell activation and maturation phenotype. From a functional standpoint, only individuals receiving DRV/r displayed transitory recovery of HIV-specific IL-2(+)IFN-γ(-) CD4(+) T-cells (T3: P=0.006) and IL-2(-)IFN-γ(+) CD8(+) T-cells (T3: P=0.032). DRV/r- and EFV-based regimens have an equal effect on T-cell phenotype and function in HIV late presenters. A temporary restoration of HIV-specific T-cell immunity early in the course of therapy with DRV/r possibly implies a more effective control over HIV in the first months following a PI/r-based regimen, even at late stage of disease.

Clinicopathological correlates in HIV seropositive tuberculosis cases presenting with jaundice after initiating antiretroviral therapy with a structured review of the literature

PubMed Central

2012-01-01

Background The development of jaundice after initiation of HAART in HIV-TB co-infected patients is a challenging presentation in resource constrained settings, and is often attributed to drug induced liver injury (DILI).Some investigators have described hepatic tuberculosis Immune Reconstitution Inflammatory Syndrome (TB-IRIS) as a cause of liver disease in patients initiating HAART, which could also cause jaundice. Case presentations We report the clinical and histopathological features of five HIV-TB co-infected patients presenting with a syndrome of jaundice, tender hepatomegaly, bile canalicular enzyme rise and return of constitutional symptoms within 8 weeks of initiation of highly active antiretroviral therapy (HAART) for advanced HIV infection at a rural clinic in KwaZulu Natal, South Africa. All five patients had been diagnosed with tuberculosis infection prior to HAART initiation and were on antituberculous medication at time of developing jaundice. There was evidence of multiple aetiologies of liver injury in all patients. However, based on clinical course and pathological findings, predominant hepatic injury was thought to be drug induced in one case and hepatic tuberculosis associated immune reconstitution inflammatory syndrome (TB-IRIS) in the other four. In these later 4 patients, liver biopsy findings included necrotising and non-necrotising granulomatous inflammation in the lobules and portal tracts. The granulomas demonstrated – in addition to epithelioid histiocytes and Langhans giant cells – neutrophils, plasma cells and large numbers of lymphocytes, which are not features of a conventional untreated tuberculous response. Conclusion In this high TB prevalent, low resource setting, TB-IRIS may be an important cause of jaundice post-HAART initiation. Clinicopathological correlation is essential for optimal diagnosis. Further multi-organ based histopathological studies in the context of immune reconstitution would be useful to clinicians in low resource settings dealing with this challenging presentation. PMID:23061403

A Novel Function for the nm23-Hl Gene: Overexpression in Human Breast Carcinoma Cells Leads to the Formation of Basement Membrane and Growth Arrest

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

Howlett, Anthony R; Petersen, Ole W; Steeg, Patricia S

1994-01-01

We have developed a culture system using reconstituted basement membrane components in which normal human mammary epithelial cells exhibit several aspects of the development and differentiation process, including formation of acinar-like structures, production and basal deposition of basement membrane components, and production and apical secretion of sialomucins. Cell lines and cultures from human breast carcinomas failed to recapitulate this process. The data indicate the importance of cellular interactions with the basement membrane in the regulation of normal breast differentiation and, potentially, its loss in neoplasia. Our purpose was to use this assay to investigate the role of the putative metastasismore » suppressor gene nm23-H1 in mammary development and differentiation. The metastatic human breast carcinoma cell line MDA-MB-435, clones transfected with a control pCMVBamneo vector, and clones transfected with pCMVBamneo vector containing nm23-H1 complementary DNA (the latter of which exhibited a substantial reduction in spontaneous metastatic potential in vivo) were cultured within a reconstituted basement membrane. Clones were examined for formation of acinus-like spheres, deposition of basement membrane components, production of sialomucin, polarization, and growth arrest. In contrast to the parental cell line and control transfectants, MDA-MB-435 breast carcinoma cells overexpressing Nm23-H1 protein regained several aspects of the normal phenotype within reconstituted basement membrane. Nm23-H1 protein-positive cells formed organized acinus-like spheres, deposited the basement membrane components type IV collagen and, to some extent, laminin to the outside of the spheres, expressed sialomucin, and growth arrested. Growth arrest of Nm23-H1 protein-positive cells was preceded by and correlated with formation of a basement membrane, suggesting a causal relationship. The data indicate a previously unidentified cause-and-effect relationship between nm23-H1 gene expression and morphological-biosynthetic-growth aspects of breast differentiation in this model system. While the basement membrane microenvironment is capable of directing the differentiation of normal human breast cells, neoplastic transformation abrogates this relationship, suggesting that intrinsic cellular events are also critical to this process. The data identify nm23-H1 gene expression as one of these events, suggesting an important role in the modulation of cellular responsiveness to the microenvironment. The data also identify previously unknown growth inhibitory effects of nm23-H1 gene overexpression.« less

Coupling of G Proteins to Reconstituted Monomers and Tetramers of the M2 Muscarinic Receptor*

PubMed Central

Redka, Dar'ya S.; Morizumi, Takefumi; Elmslie, Gwendolynne; Paranthaman, Pranavan; Shivnaraine, Rabindra V.; Ellis, John; Ernst, Oliver P.; Wells, James W.

2014-01-01

G protein-coupled receptors can be reconstituted as monomers in nanodiscs and as tetramers in liposomes. When reconstituted with G proteins, both forms enable an allosteric interaction between agonists and guanylyl nucleotides. Both forms, therefore, are candidates for the complex that controls signaling at the level of the receptor. To identify the biologically relevant form, reconstituted monomers and tetramers of the purified M2 muscarinic receptor were compared with muscarinic receptors in sarcolemmal membranes for the effect of guanosine 5′-[β,γ-imido]triphosphate (GMP-PNP) on the inhibition of N-[3H]methylscopolamine by the agonist oxotremorine-M. With monomers, a stepwise increase in the concentration of GMP-PNP effected a lateral, rightward shift in the semilogarithmic binding profile (i.e. a progressive decrease in the apparent affinity of oxotremorine-M). With tetramers and receptors in sarcolemmal membranes, GMP-PNP effected a vertical, upward shift (i.e. an apparent redistribution of sites from a state of high affinity to one of low affinity with no change in affinity per se). The data were analyzed in terms of a mechanistic scheme based on a ligand-regulated equilibrium between uncoupled and G protein-coupled receptors (the “ternary complex model”). The model predicts a rightward shift in the presence of GMP-PNP and could not account for the effects at tetramers in vesicles or receptors in sarcolemmal membranes. Monomers present a special case of the model in which agonists and guanylyl nucleotides interact within a complex that is both constitutive and stable. The results favor oligomers of the M2 receptor over monomers as the biologically relevant state for coupling to G proteins. PMID:25023280

B1 Cell IgE Impedes Mast Cell-Mediated Enhancement of Parasite Expulsion through B2 IgE Blockade.

PubMed

Martin, Rebecca K; Damle, Sheela R; Valentine, Yolander A; Zellner, Matthew P; James, Briana N; Lownik, Joseph C; Luker, Andrea J; Davis, Elijah H; DeMeules, Martha M; Khandjian, Laura M; Finkelman, Fred D; Urban, Joseph F; Conrad, Daniel H

2018-02-13

Helminth infection is known for generating large amounts of poly-specific IgE. Here we demonstrate that innate-like B1 cells are responsible for this IgE production during infection with the nematode parasites Nippostrongylus brasiliensis and Heligmosomoides polygyrus bakeri. In vitro analysis of B1 cell immunoglobulin class switch recombination to IgE demonstrated a requirement for anti-CD40 and IL-4 that was further enhanced when IL-5 was added or when the B1 source was helminth infected mice. An IL-25-induced upregulation of IgE in B1 cells was also demonstrated. In T cell-reconstituted RAG1 -/- mice, N. brasiliensis clearance was enhanced with the addition of B2 cells in an IgE-dependent manner. This enhanced clearance was impeded by reconstitution with IgE sufficient B1 cells. Mucosal mast cells mediated the B2 cell enhancement of clearance in the absence of B1 cells. The data support B1 cell IgE secretion as a regulatory response exploited by the helminth. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Diffusion of naltrexone across reconstituted human oral epithelium and histomorphological features.

PubMed

Giannola, Libero Italo; De Caro, Viviana; Giandalia, Giulia; Siragusa, Maria Gabriella; Campisi, Giuseppina; Florena, Ada Maria; Ciach, Tomasz

2007-02-01

In transbuccal absorption a major limitation could be the low permeability of the mucosa which implies low drug bioavailability. The ability of naltrexone hydrochloride (NLX) to penetrate a resembling histologically human buccal mucosa was assessed and the occurrence of any histomorphological changes observed. We used reconstituted human oral (RHO) non-keratinised epithelium as mucosal section and a Transwell diffusion cells system as bicompartmental model. Buccal permeation was expressed in terms of drug flux (J(s)) and permeability coefficients (K(p)). Data were collected using both artificial and natural human saliva. The main finding was that RHO does not restrain NLX permeation. Drug transport across the epithelium was observed also in presence of various concentrations of penetration enhancers, without any significant differences. On the contrary, the flux throughout the mucosa was extensively affected by iontophoresis. Histologically, no sign of flogosis was observed in any specimen under experiment without iontophoresis, whereas cytoarchitectural changes, up to nuclear pycnosis or cellular swelling, were determined as a consequence of the application of electric fields.

Role of the 2B4 Receptor in CD8+ T-Cell-Dependent Immune Control of Epstein-Barr Virus Infection in Mice With Reconstituted Human Immune System Components.

PubMed

Chijioke, Obinna; Marcenaro, Emanuela; Moretta, Alessandro; Capaul, Riccarda; Münz, Christian

2015-09-01

Patients with X-linked lymphoproliferative (XLP) disease due to deficiency in the adaptor molecule signaling lymphocytic activation molecule-associated protein (SAP) are highly susceptible to one specific viral pathogen, the Epstein-Barr virus (EBV). This susceptibility might result from impaired CD8(+) T-cell and natural killer cell responses to EBV infection in these patients. We demonstrate that antibody blocking of the SAP-dependent 2B4 receptor is sufficient to induce XLP-like aggravation of EBV disease in mice with reconstituted human immune system components. CD8(+) T cells require 2B4 for EBV-specific immune control, because 2B4 blockade after CD8(+) T-cell depletion did not further aggravate symptoms of EBV infection. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Notch-dependent T-lineage commitment occurs at extrathymic sites following bone marrow transplantation

PubMed Central

Maillard, Ivan; Schwarz, Benjamin A.; Sambandam, Arivazhagan; Fang, Terry; Shestova, Olga; Xu, Lanwei; Bhandoola, Avinash; Pear, Warren S.

2006-01-01

Early T-lineage progenitors (ETPs) arise after colonization of the thymus by multipotent bone marrow progenitors. ETPs likely serve as physiologic progenitors of T-cell development in adult mice, although alternative T-cell differentiation pathways may exist. While we were investigating mechanisms of T-cell reconstitution after bone marrow transplantation (BMT), we found that efficient donor-derived thymopoiesis occurred before the pool of ETPs had been replenished. Simultaneously, T lineage–restricted progenitors were generated at extrathymic sites, both in the spleen and in peripheral lymph nodes, but not in the bone marrow or liver. The generation of these T lineage–committed cells occurred through a Notch-dependent differentiation process. Multipotent bone marrow progenitors efficiently gave rise to extrathymic T lineage–committed cells, whereas common lymphoid progenitors did not. Our data show plasticity of T-lineage commitment sites in the post-BMT environment and indicate that Notch-driven extrathymic Tlineage commitment from multipotent progenitors may contribute to early T-lineage reconstitution after BMT. PMID:16397133

Long-term engraftment, graft-vs.-host disease, and immunologic reconstitution after experimental transplantation of allogeneic peripheral blood cells from G-CSF-treated donors.

PubMed

Pan, L; Bressler, S; Cooke, K R; Krenger, W; Karandikar, M; Ferrara, J L

1996-10-01

Peripheral blood cells (PBPC) are an alternative source of bone marrow for allogeneic transplantation. Reports from recent clinical trials granulocyte colony-stimulating factor (G-CSF)-mobilized PBPC for allogeneic transplantation show incidence and severity of graft-vs.-host disease (GVHD) similar to those observed in conventional bone marrow transplantation (BMT), despite the presence of 10- to 20-fold more T cell in the PBPC inoculum. In the present study, we examined the effects of pretreatment of donors with G-CSF on GVHD, long-term engraftment, and lymphocyte reconstitution in a murine parent-->F1 model (B6.Ly-5a-->B6d2F1) using splenocytes as a source of peripheral progenitor cells. Recipients of splenocytes from G-CSF-treated donors experienced less mortality from acute GVHD and showed sustained weight gain by day 100 after transplantation. At that time, there was no histological evidence od GVHD in either liver or gut. Recipients of splenocytes from G-CSF-treated donors showed complete donor engraftment within 1 month, which was sustained until the end of the observation period. In contrast, recipients of T cell-depleted splenocytes showed slower donor engraftment and persistent donor/host chimerism. In addition, lymphocyte phenotype and function in mice receiving splenocytes from G-CSF-treated donors was significantly restored by day 100 after transplantation. Thus, the use of G-CSF-mobilized PBPC may provide significant advantages to conventional BMT by reducing GVHD without impairing long-term engraftment and immunologic reconstruction.

Amelioration of murine beta-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both gamma-globin and the MGMT drug-resistance gene.

PubMed

Zhao, Huifen; Pestina, Tamara I; Nasimuzzaman, Md; Mehta, Perdeep; Hargrove, Phillip W; Persons, Derek A

2009-06-04

Correction of murine models of beta-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However, transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human gamma-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT), driven by a constitutive cellular promoter. MGMT expression provides cellular resistance to alkylator drugs, which can be administered to kill residual untransduced, diseased HSCs, whereas transduced cells are protected. Mice transplanted with beta-thalassemic HSCs transduced with a gamma-globin/MGMT vector initially had subtherapeutic levels of red cells expressing gamma-globin. To enrich gamma-globin-expressing cells, transplanted mice were treated with the alkylator agent 1,3-bis-chloroethyl-1-nitrosourea. This resulted in significant increases in the number of gamma-globin-expressing red cells and the amount of fetal hemoglobin, leading to resolution of anemia. Selection of transduced HSCs was also obtained when cells were drug-treated before transplantation. Mice that received these cells demonstrated reconstitution with therapeutic levels of gamma-globin-expressing cells. These data suggest that MGMT-based drug selection holds promise as a modality to improve gene therapy for beta-thalassemia.

Reconstruction of the immune system after unrelated or partially matched T-cell-depleted bone marrow transplantation in children: immunophenotypic analysis and factors affecting the speed of recovery.

PubMed

Kook, H; Goldman, F; Padley, D; Giller, R; Rumelhart, S; Holida, M; Lee, N; Peters, C; Comito, M; Huling, D; Trigg, M

1996-08-01

We prospectively studied immune reconstitution in 102 children who underwent T-lymphocyte depleted bone marrow transplants using either closely matched unrelated donors or partially matched familial donors by assaying total lymphocyte counts (TLC), T-cell subsets, B cells, and natural killer cells. TLC, CD3+, and CD4+ T-cell counts remained depressed until 2 to 3 years posttransplant, whereas CD8+ T-cell counts normalized by 18 months, resulting in an inverted CD4:CD8 ratio until 12 months posttransplant. Although the percentage of NK cells was elevated early posttransplant, their absolute numbers remained normal. CD20+ B cells were depressed until 12 to 18 months posttransplant. Factors affecting immunophenotypic recovery were analyzed by nonparametric statistics. Younger patients tended to have higher TLC posttransplant. Higher marrow cell doses were not associated with hastened immunophenotypic recovery. Graft-versus-host disease (GVHD) and/or its treatment significantly delayed the immune reconstitution of CD3+, CD4+, and CD20+ cells. The presence of cytomegalovirus was associated with increased CD8+ counts and a decrease in the percentages of CD4+ and CD20+ cells.

T Lymphocytes Influence the Mineralization Process of Bone

PubMed Central

El Khassawna, Thaqif; Serra, Alessandro; Bucher, Christian H.; Petersen, Ansgar; Schlundt, Claudia; Könnecke, Ireen; Malhan, Deeksha; Wendler, Sebastian; Schell, Hanna; Volk, Hans-Dieter; Schmidt-Bleek, Katharina; Duda, Georg N.

2017-01-01

Bone is a unique organ able to regenerate itself after injuries. This regeneration requires the local interplay between different biological systems such as inflammation and matrix formation. Structural reconstitution is initiated by an inflammatory response orchestrated by the host immune system. However, the individual role of T cells and B cells in regeneration and their relationship to bone tissue reconstitution remain unknown. Comparing bone and fracture healing in animals with and without mature T and B cells revealed the essential role of these immune cells in determining the tissue mineralization and thus the bone quality. Bone without mature T and B cells is stiffer when compared to wild-type bone thus lacking the elasticity that helps to absorb forces, thus preventing fractures. In-depth analysis showed dysregulations in collagen deposition and osteoblast distribution upon lack of mature T and B cells. These changes in matrix deposition have been correlated with T cells rather than B cells within this study. This work presents, for the first time, a direct link between immune cells and matrix formation during bone healing after fracture. It illustrates specifically the role of T cells in the collagen organization process and the lack thereof in the absence of T cells. PMID:28596766

A strategy for tissue self-organization that is robust to cellular heterogeneity and plasticity

PubMed Central

Cerchiari, Alec E.; Garbe, James C.; Jee, Noel Y.; Todhunter, Michael E.; Broaders, Kyle E.; Peehl, Donna M.; Desai, Tejal A.; LaBarge, Mark A.; Thomson, Matthew; Gartner, Zev J.

2015-01-01

Developing tissues contain motile populations of cells that can self-organize into spatially ordered tissues based on differences in their interfacial surface energies. However, it is unclear how self-organization by this mechanism remains robust when interfacial energies become heterogeneous in either time or space. The ducts and acini of the human mammary gland are prototypical heterogeneous and dynamic tissues comprising two concentrically arranged cell types. To investigate the consequences of cellular heterogeneity and plasticity on cell positioning in the mammary gland, we reconstituted its self-organization from aggregates of primary cells in vitro. We find that self-organization is dominated by the interfacial energy of the tissue–ECM boundary, rather than by differential homo- and heterotypic energies of cell–cell interaction. Surprisingly, interactions with the tissue–ECM boundary are binary, in that only one cell type interacts appreciably with the boundary. Using mathematical modeling and cell-type-specific knockdown of key regulators of cell–cell cohesion, we show that this strategy of self-organization is robust to severe perturbations affecting cell–cell contact formation. We also find that this mechanism of self-organization is conserved in the human prostate. Therefore, a binary interfacial interaction with the tissue boundary provides a flexible and generalizable strategy for forming and maintaining the structure of two-component tissues that exhibit abundant heterogeneity and plasticity. Our model also predicts that mutations affecting binary cell–ECM interactions are catastrophic and could contribute to loss of tissue architecture in diseases such as breast cancer. PMID:25633040

Caveolins/caveolae protect adipocytes from fatty acid-mediated lipotoxicity.

PubMed

Meshulam, Tova; Breen, Michael R; Liu, Libin; Parton, Robert G; Pilch, Paul F

2011-08-01

Mice and humans lacking functional caveolae are dyslipidemic and have reduced fat stores and smaller fat cells. To test the role of caveolins/caveolae in maintaining lipid stores and adipocyte integrity, we compared lipolysis in caveolin-1 (Cav1)-null fat cells to that in cells reconstituted for caveolae by caveolin-1 re-expression. We find that the Cav1-null cells have a modestly enhanced rate of lipolysis and reduced cellular integrity compared with reconstituted cells as determined by the release of lipid metabolites and lactic dehydrogenase, respectively, into the media. There are no apparent differences in the levels of lipolytic enzymes or hormonally stimulated phosphorylation events in the two cell lines. In addition, acute fasting, which dramatically raises circulating fatty acid levels in vivo, causes a significant upregulation of caveolar protein constituents. These results are consistent with the hypothesis that caveolae protect fat cells from the lipotoxic effects of elevated levels fatty acids, which are weak detergents at physiological pH, by virtue of the property of caveolae to form detergent-resistant membrane domains.

Caveolins/caveolae protect adipocytes from fatty acid-mediated lipotoxicity

PubMed Central

Meshulam, Tova; Breen, Michael R.; Liu, Libin; Parton, Robert G.; Pilch, Paul F.

2011-01-01

Mice and humans lacking functional caveolae are dyslipidemic and have reduced fat stores and smaller fat cells. To test the role of caveolins/caveolae in maintaining lipid stores and adipocyte integrity, we compared lipolysis in caveolin-1 (Cav1)-null fat cells to that in cells reconstituted for caveolae by caveolin-1 re-expression. We find that the Cav1-null cells have a modestly enhanced rate of lipolysis and reduced cellular integrity compared with reconstituted cells as determined by the release of lipid metabolites and lactic dehydrogenase, respectively, into the media. There are no apparent differences in the levels of lipolytic enzymes or hormonally stimulated phosphorylation events in the two cell lines. In addition, acute fasting, which dramatically raises circulating fatty acid levels in vivo, causes a significant upregulation of caveolar protein constituents. These results are consistent with the hypothesis that caveolae protect fat cells from the lipotoxic effects of elevated levels fatty acids, which are weak detergents at physiological pH, by virtue of the property of caveolae to form detergent-resistant membrane domains. PMID:21652731

Engineering artificial cells by combining HeLa-based cell-free expression and ultra-thin double emulsion template

PubMed Central

Ho, Kwun Yin; Murray, Victoria L.; Liu, Allen P.

2015-01-01

Generation of artificial cells provides the bridge needed to cover the gap between studying the complexity of biological processes in whole cells and studying these same processes in an in vitro reconstituted system. Artificial cells are defined as the encapsulation of biologically active material in a biological or synthetic membrane. Here, we describe a robust and general method to produce artificial cells for the purpose of mimicking one or more behaviors of a cell. A microfluidic double emulsion system is used to encapsulate a mammalian cell free expression system that is able to express membrane proteins into the bilayer or soluble proteins inside the vesicles. The development of a robust platform that allows the assembly of artificial cells is valuable in understanding subcellular functions and emergent behaviors in a more cell-like environment as well as for creating novel signaling pathways to achieve specific cellular behaviors. PMID:25997354

Estrogen Regulates Bone Turnover by Targeting RANKL Expression in Bone Lining Cells.

PubMed

Streicher, Carmen; Heyny, Alexandra; Andrukhova, Olena; Haigl, Barbara; Slavic, Svetlana; Schüler, Christiane; Kollmann, Karoline; Kantner, Ingrid; Sexl, Veronika; Kleiter, Miriam; Hofbauer, Lorenz C; Kostenuik, Paul J; Erben, Reinhold G

2017-07-25

Estrogen is critical for skeletal homeostasis and regulates bone remodeling, in part, by modulating the expression of receptor activator of NF-κB ligand (RANKL), an essential cytokine for bone resorption by osteoclasts. RANKL can be produced by a variety of hematopoietic (e.g. T and B-cell) and mesenchymal (osteoblast lineage, chondrocyte) cell types. The cellular mechanisms by which estrogen acts on bone are still a matter of controversy. By using murine reconstitution models that allow for selective deletion of estrogen receptor-alpha (ERα) or selective inhibition of RANKL in hematopoietic vs. mesenchymal cells, in conjunction with in situ expression profiling in bone cells, we identified bone lining cells as important gatekeepers of estrogen-controlled bone resorption. Our data indicate that the increase in bone resorption observed in states of estrogen deficiency in mice is mainly caused by lack of ERα-mediated suppression of RANKL expression in bone lining cells.

Fibril growth kinetics link buffer conditions and topology of 3D collagen I networks.

PubMed

Kalbitzer, Liv; Pompe, Tilo

2018-02-01

Three-dimensional fibrillar networks reconstituted from collagen I are widely used as biomimetic scaffolds for in vitro and in vivo cell studies. Various physicochemical parameters of buffer conditions for in vitro fibril formation are well known, including pH-value, ion concentrations and temperature. However, there is a lack of a detailed understanding of reconstituting well-defined 3D network topologies, which is required to mimic specific properties of the native extracellular matrix. We screened a wide range of relevant physicochemical buffer conditions and characterized the topology of the reconstituted 3D networks in terms of mean pore size and fibril diameter. A congruent analysis of fibril formation kinetics by turbidimetry revealed the adjustment of the lateral growth phase of fibrils by buffer conditions to be key in the determination of pore size and fibril diameter of the networks. Although the kinetics of nucleation and linear growth phase were affected by buffer conditions as well, network topology was independent of those two growth phases. Overall, the results of our study provide necessary insights into how to engineer 3D collagen matrices with an independent control over topology parameters, in order to mimic in vivo tissues in in vitro experiments and tissue engineering applications. The study reports a comprehensive analysis of physicochemical conditions of buffer solutions to reconstitute defined 3D collagen I matrices. By a combined analysis of network topology, i.e., pore size and fibril diameter, and the kinetics of fibril formation we can reveal the dependence of 3D network topology on buffer conditions, such as pH-value, phosphate concentration and sodium chloride content. With those results we are now able to provide engineering strategies to independently tune the topology parameters of widely used 3D collagen scaffolds based on the buffer conditions. By that, we enable the straightforward mimicking of extracellular matrices of in vivo tissues for in vitro cell culture experiments and tissue engineering applications. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Solution stability of Captisol-stabilized melphalan (Evomela) versus Propylene glycol-based melphalan hydrochloride injection.

PubMed

Singh, Ramsharan; Chen, Jin; Miller, Teresa; Bergren, Michael; Mallik, Rangan

2016-12-14

The objective of this study was to compare the stability of recently approved Captisol-stabilized propylene glycol-free melphalan injection (Evomela™) against currently marketed propylene glycol-based melphalan injection. The products were compared as reconstituted solutions in vials as well as admixture solutions prepared from normal saline in infusion bags. Evomela and propylene glycol-based melphalan injection were reconstituted in normal saline and organic custom diluent, respectively, according to their package insert instructions. The reconstituted solutions were diluted in normal saline to obtain drug admixture solutions at specific drug concentrations. Stability of the solutions was studied at room temperature by assay of melphalan and determination of melphalan-related impurities. Results show that based on the increase in total impurities in propylene glycol-based melphalan injection at 0.45 mg/mL, Evomela admixture solutions are about 5, 9, 15 and 29 times more stable at concentrations of 0.45, 1.0, 2.0 and 5.0 mg/mL, respectively. Results confirmed that reconstituted Evomela solution can be stored in the vial for up to 1 h at RT or for up to 24 h at refrigerated temperature (2-8 °C) with no significant degradation. After storage in the vial, it remains stable for an additional 3-29 h after preparation of admixture solution in infusion bags at concentrations of 0.25-5.0 mg/mL, respectively. In addition, Evomela solution in saline, at concentration of 5.0 mg/mL melphalan was bacteriostatic through 72 h storage at 2-8 °C. Formulation of melphalan with Captisol technology significantly improved stability compared to melphalan hydrochloride reconstituted with propylene-glycol based diluents.

Candida Drug Resistance Protein 1, a Major Multidrug ATP Binding Cassette Transporter of Candida albicans, Translocates Fluorescent Phospholipids in a Reconstituted System†

PubMed Central

Shukla, Sudhanshu; Rai, Versha; Saini, Preeti; Banerjee, Dibyendu; Menon, Anant K.; Prasad, Rajendra

2008-01-01

Candida albicans drug resistance protein 1 (Cdr1p), an ATP-dependent drug efflux pump, contributes to multidrug resistance in Candida-infected immunocompromised patients. Previous cell-based assays suggested that Cdr1p also acts as a phospholipid translocator. To investigate this, we reconstituted purified Cdr1p into sealed membrane vesicles. Comparison of the ATPase activities of sealed and permeabilized proteoliposomes indicated that Cdr1p was asymmetrically reconstituted such that ~70% of the molecules had their ATP binding sites accessible to the extravesicular space. Fluorescent glycerophospholipids were incorporated into the outer leaflet of the proteoliposomes, and their transport into the inner leaflet was tracked with a quenching assay using membrane-impermeant dithionite. We observed ATP-dependent transport of the fluorescent lipids into the inner leaflet of the vesicles. With ~6 molecules of Cdr1p per vesicle on average, the half-time to reach the maximal extent of transport was ~15 min. Transport was reduced in vesicles reconstituted with Cdr1p variants with impaired ATPase activity and could be competed out to different levels by a molar excess of drugs such as fluconazole and miconazole that are known to be effluxed by Cdr1p. Transport was not affected by ampicillin, a compound that is not effluxed by Cdr1p. Our results suggest a direct link between the ability of Cdr1p to translocate fluorescent phospholipids and efflux drugs. We note that only a few members of the ABC superfamily of Candida have a well-defined role as drug exporters; thus, lipid translocation mediated by Cdr1p could reflect its cellular function. PMID:17924650

CD45{sup low}c-Kit{sup high} cells have hematopoietic properties in the mouse aorta-gonad-mesonephros region

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

Nobuhisa, Ikuo, E-mail: nobuhisa.scr@mri.tmd.ac.jp; Department of Cell Fate Modulation, Institute of Molecular Embryology and Genetics/Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, 860-0811; Yamasaki, Shoutarou

Long-term reconstituting hematopoietic stem cells first arise from the aorta of the aorta-gonad-mesonephros (AGM) region in a mouse embryo. We have previously reported that in cultures of the dispersed AGM region, CD45{sup low}c-Kit{sup +} cells possess the ability to reconstitute multilineage hematopoietic cells, but investigations are needed to show that this is not a cultured artifact and to clarify when and how this population is present. Based on the expression profile of CD45 and c-Kit in freshly dissociated AGM cells from embryonic day 9.5 (E9.5) to E12.5 and aorta cells in the AGM from E13.5 to E15.5, we defined sixmore » cell populations (CD45{sup -}c-Kit{sup -}, CD45{sup -}c-Kit{sup low}, CD45{sup -}c-Kit{sup high}, CD45{sup low}c-Kit{sup high}, CD45{sup high}c-Kit{sup high}, and CD45{sup high}c-Kit{sup very} {sup low}). Among these six populations, CD45{sup low}c-Kit{sup high} cells were most able to form hematopoietic cell colonies, but their ability decreased after E11.5 and was undetectable at E13.5 and later. The CD45{sup low}c-Kit{sup high} cells showed multipotency in vitro. We demonstrated further enrichment of hematopoietic activity in the Hoechst dye-effluxing side population among the CD45{sup low}c-Kit{sup high} cells. Here, we determined that CD45{sup low}c-Kit{sup high} cells arise from the lateral plate mesoderm using embryonic stem cell-derived differentiation system. In conclusion, CD45{sup low}c-Kit{sup high} cells are the major hematopoietic cells of mouse AGM.« less

Experimental Human Cell and Tissue Models of Pemphigus

PubMed Central

van der Wier, Gerda; Pas, Hendri H.; Jonkman, Marcel F.

2010-01-01

Pemphigus is a chronic mucocutaneous autoimmune bullous disease that is characterized by loss of cell-cell contact in skin and/or mucous membranes. Past research has successfully identified desmosomes as immunological targets and has demonstrated that acantholysis is initiated through direct binding of IgG. The exact mechanisms of acantholysis, however, are still missing. Experimental model systems have contributed considerably to today's knowledge and are still a favourite tool of research. In this paper we will describe to what extent human cell and tissue models represent the in vivo situation, for example, organ cultures of human skin, keratinocyte cultures, and human skin grafted on mice and, furthermore, how suitable they are to study the pathogenesis of pemphigus. Organ cultures closely mimic the architecture of the epidermis but are less suitable to answer posed biochemical questions. Cultured keratinocyte monolayers are convenient in this respect, but their desmosomal make-up in terms of adhesion molecules does not exactly reflect the in vivo situation. Reconstituted skin is a relatively new model that approaches organ culture. In models of human skin grafted on mice, acantholysis can be studied in actual human skin but now with all the advantages of an animal model. PMID:20585596

Family Therapy with Reconstituted Families: A Crisis-Induction Approach.

ERIC Educational Resources Information Center

Baptiste, David A.

1983-01-01

Describes a crisis-based therapeutic approach for overcoming resistance in reconstituted families. Presents therapeutically induced crisis as a means through which therapists might purposefully disequilibrate families in which resistance is high and subsequently redirect them to meaningful change. Reviews implications and contraindications for the…

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

Murine Visceral Leishmaniasis: IgM and Polyclonal B-Cell Activation Lead to Disease Exacerbation

PubMed Central

Deak, Eszter; Jayakumar, Asha; Wing Cho, Ka; Goldsmith-Pestana, Karen; Dondji, Blaise; Lambris, John D.; McMahon-Pratt, Diane

2010-01-01

In visceral leishmaniasis, the draining lymph node (DLN) is the initial site for colonization and establishment of infection after intradermal transmission by the sand fly vector; however, little is known about the developing immune response within this site. Using an intradermal infection model, which allows for parasite visceralization, we have examined the ongoing immune responses in the DLN of BALB/c mice infected with L. infantum. Although not unexpected, at early times post-infection there is a marked B cell expansion in the DLN, which persists throughout infection. However, the characteristics of this response were of interest; as early as day 7 post-infection, polyclonal antibodies (TNP, OVA, chromatin) were observed and the levels appeared comparable to the specific anti-leishmania response. Although B-cell-deficient JHD BALB/c mice are relatively resistant to infection, neither B-cell-derived IL-10 nor B-cell antigen presentation appear to be primarily responsible for the elevated parasitemia. However, passive transfer and reconstitution of JHD BALB/c with secretory immunoglobulins, (IgM or IgG; specific or non-specific immune complexes) results in increased susceptibility to L. infantum infection. Further, JHD BALB/c mice transgenetically reconstituted to secrete IgM demonstrated exacerbated disease in comparison to wild type BALB/c mice as early as 2 days post-infection. Evidence suggests that complement activation (generation of C5a) and signaling via the C5aR (CD88) is related to the disease exacerbation caused by IgM rather than cytokine levels (IL-10 or IFN-γ). Overall these studies indicate that polyclonal B cell activation, which is known to be associated with human visceral leishmaniasis, is an early and intrinsic characteristic of disease and may represent a target for therapeutic intervention. PMID:20213734

Smooth muscle cells healing atherosclerotic plaque disruptions are of local, not blood, origin in apolipoprotein E knockout mice.

PubMed

Bentzon, Jacob F; Sondergaard, Claus S; Kassem, Moustapha; Falk, Erling

2007-10-30

Signs of preceding episodes of plaque rupture and smooth muscle cell (SMC)-mediated healing are common in atherosclerotic plaques, but the source of the healing SMCs is unknown. Recent studies suggest that activated platelets adhering to sites of injury recruit neointimal SMCs from circulating bone marrow-derived progenitor cells. Here, we analyzed the contribution of this mechanism to plaque healing after spontaneous and mechanical plaque disruption in apolipoprotein E knockout (apoE-/-) mice. To determine the origin of SMCs after spontaneous plaque disruption, irradiated 18-month-old apoE-/- mice were reconstituted with bone marrow cells from enhanced green fluorescent protein (eGFP) transgenic apoE-/- mice and examined when they died up to 9 months later. Plaque hemorrhage, indicating previous plaque disruption, was widely present, but no bone marrow-derived eGFP+ SMCs were detected. To examine the origin of healing SMCs in a model that recapitulates more features of human plaque rupture and healing, we developed a mechanical technique that produced consistent plaque disruption, superimposed thrombosis, and SMC-mediated plaque healing in apoE-/- mice. Mechanical plaque disruption was produced in irradiated apoE-/- mice reconstituted with eGFP+ apoE-/- bone marrow cells and in carotid bifurcations cross-grafted between apoE-/- and eGFP+ apoE-/- mice. Apart from few non-graft-derived SMCs near the anastomosis site in 1 transplanted carotid bifurcation, no SMCs originating from outside the local arterial segment were detected in healed plaques. Healing SMCs after atherosclerotic plaque disruption are derived entirely from the local arterial wall and not circulating progenitor cells in apoE-/- mice.

Noninvasive Quantification of Retinal Microglia Using Widefield Autofluorescence Imaging.

PubMed

Kokona, Despina; Schneider, Nadia; Giannakaki-Zimmermann, Helena; Jovanovic, Joel; Ebneter, Andreas; Zinkernagel, Martin

2017-04-01

To validate widefield autofluorescence (AF) in vivo imaging of the retina in mice expressing green fluorescent protein (gfp) in microglia, and to monitor retinal microglia reconstitution in vivo after lethal irradiation and bone marrow transplantation. Transgenic Cx3cr1gfp/gfp and wildtype Balb/c mice were used in this study. A confocal scanning laser ophthalmoscope was used for AF imaging with a 55° and a widefield 102° lens. Intrasession reproducibility was assessed for each lens. To investigate reconstitution in vivo, bone marrow from Cx3cr1gfp/gfp mice was used to rescue lethally irradiated wildtype mice. Data were compared to confocal microscopy of retinal flat mounts. Both the 55° and the 102° lens produced high resolution images of retinal microglia with similar microglia density. However, compared to the 55° lens, the widefield 102° lens captured approximately 3.6 times more microglia cells (1515 ± 123 cells versus 445 ± 76 cells [mean ± SD], for 102° and 55°, respectively, P < 0.001). No statistical difference in the number of gfp positive cells within corresponding areas was observed within the same imaging session. Imaging of microglia reconstitution showed a similar time course compared to flat mount preparations with an excellent correlation between microglia cell numbers in AF and gfp-stained flat mounts (R = 0.92, P < 0.0001). Widefield AF imaging of mice with gfp expressing microglia can be used to quantify retinal microglia. In vivo microglia counts corresponded very well with ex vivo counts on retinal flat mounts. As such, AF imaging can largely replace ex vivo quantification.

The mechanisms of the protective effects of reconstituted skim milk during convective droplet drying of lactic acid bacteria.

PubMed

Zheng, Xufeng; Fu, Nan; Duan, Manlei; Woo, Meng Wai; Selomulya, Cordelia; Chen, Xiao Dong

2015-10-01

Reconstituted skim milk (RSM) is a reputed protective carrier for improving the survival ratio of lactic acid bacteria (LAB) after spray drying; however the underlying mechanisms of the prominent protection remains unclear. In this study, the inactivation histories of two LAB strains during droplet drying with four carriers were experimentally determined, and the effects of droplet drying parameters on LAB inactivation were investigated. For the first time, the possible contribution of each RSM components to the maintenance of LAB viability during drying was discussed. Rapid inactivation of LAB cells only started at the later stage of drying, where RSM could maintain viability better upon both high droplet temperature and low moisture content than the other three carriers tested. Such protective effects was attributed to calcium and milk proteins rather than lactose. Upon the rapidly increasing droplet temperature at the later stage, calcium might enhance the heat resistance of LAB cells, whereas proteins might lead to a mild temperature variation rate which was beneficial to cell survival. LAB cells dried in the reconstituted whole milk showed the most advanced transition of rapid viability loss, with transition temperature at around 60°C, in contrast to 65-70°C in lactose and MRS carriers and 75°C in the RSM carrier. The detrimental effects could be due to the high level of milk fat content. The proposed effects of each RSM components on LAB viability would be useful for constructing more powerful protectants for production of active dry LAB cells via spray drying. Copyright © 2015 Elsevier Ltd. All rights reserved.

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro

PubMed Central

Purushotham, Pallinti; Cho, Sung Hyun; Díaz-Moreno, Sara M.; Kumar, Manish; Nixon, B. Tracy; Bulone, Vincent; Zimmer, Jochen

2016-01-01

Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme’s N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils. PMID:27647898

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro.

PubMed

Purushotham, Pallinti; Cho, Sung Hyun; Díaz-Moreno, Sara M; Kumar, Manish; Nixon, B Tracy; Bulone, Vincent; Zimmer, Jochen

2016-10-04

Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme's N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils.

In vitro reconstitution of the active T. castaneum telomerase.

PubMed

Schuller, Anthony P; Harkisheimer, Michael J; Skordalakes, Emmanuel

2011-07-14

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro. Telomerase is a specialized reverse transcriptase that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit. Additionally, telomerase is dormant in most somatic cells in adults, but is active in cancer cells where it promotes cell immortality. The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres. Prior to 2008, only structures for individual telomerase domains had been solved. A major breakthrough in this field came from the determination of the crystal structure of the active, catalytic subunit of T. castaneum telomerase, TcTERT. Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

Protein-based microhydraulic transport for controllable actuation

NASA Astrophysics Data System (ADS)

Sundaresan, Vishnu Baba; Leo, Donald J.

2006-03-01

Plants have the ability to develop large mechanical force from chemical energy available with bio-fuels. The energy released by the cleavage of a terminal phosphate ion during the hydrolysis of a bio- fuel assists the transport of ions and fluids in cellular homeostasis. Materials that develop pressure and hence strain similar to the response of plants to an external stimuli are classified as nastic materials. Calculations for controlled actuation of an active material inspired by biological transport mechanism demonstrated the feasibility of developing such a material with actuation energy densities on the order of 100 kJ/m 3. The mathematical model for a simplified proof of concept actuator referred to as micro hydraulic actuator uses ion transporters extracted from plants reconstituted on a synthetic bilayer lipid membrane (BLM). Thermodynamic model of the concept actuator predicted the ability to develop 5 percent normalized deformation in thickness of the micro- hydraulic actuator. Controlled fluid transport through AtSUT4 (Proton-sucrose co-transporter from Arabidopsis thaliana) reconstituted on a 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-L- Serine] (Sodium Salt) (POPS), 1-Palmitoyl-2-Oleoyl-sn-Glycero-3- Phosphoethanolamine (POPE) BLM on a porous lead silicate glass plate (50μm with 61μm pitch) was driven by proton gradient. Bulk fluid flux of 1.2 μl/min was observed for each microliter of AtSUT4 transporter suspension (16.6 mg/ml in pH7.0 medium) reconstituted on the BLM. The flux rate is observed to be dependent on the concentration of sucrose present in pH4 buffer. Flux rate of 10 μl/min is observed for 5 mM sucrose in the first 10 minutes. The observed flux scales linearly with BLM area and the amount of proteins reconstituted on the lipid membrane. This article details the next step in the development of the micro hydraulic actuator - fluid transport driven by exergonic Adenosine triphosphate (ATP) hydrolysis reaction in the presence of ATP-phosphohydrolase (red beet ATP-ase) enzyme in the reconstituted bilayer.

Reconstituted asbestos matrix for fuel cells

NASA Technical Reports Server (NTRS)

Mcbryar, H.

1975-01-01

Method is described for reprocessing commercially available asbestos matrix stock to yield greater porosity and bubble pressure (due to increased surface tension), improved homogeneity, and greater uniformity.

Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells.

PubMed

Hayashi, Katsuhiko; Saitou, Mitinori

2013-08-01

Oogenesis is an integrated process through which an egg acquires the potential for totipotency, a fundamental condition for creating new individuals. Reconstitution of oogenesis in a culture that generates eggs with proper function from pluripotent stem cells (PSCs) is therefore one of the key goals in basic biology as well as in reproductive medicine. Here we describe a stepwise protocol for the generation of eggs from mouse PSCs, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs and iPSCs are first induced into primordial germ cell-like cells (PGCLCs) that are in turn aggregated with somatic cells of female embryonic gonads, the precursors for adult ovaries. Induction of PGCLCs followed by aggregation with the somatic cells takes up to 8 d. The aggregations are then transplanted under the ovarian bursa, in which PGCLCs grow into germinal vesicle (GV) oocytes in ∼1 month. The PGCLC-derived GV oocytes can be matured into eggs in 1 d by in vitro maturation (IVM), and they can be fertilized with spermatozoa by in vitro fertilization (IVF) to obtain healthy and fertile offspring. This method provides an initial step toward reconstitution of the entire process of oogenesis in vitro.

Two endogenous proteins that induce cell wall extension in plants

NASA Technical Reports Server (NTRS)

McQueen-Mason, S.; Durachko, D. M.; Cosgrove, D. J.

1992-01-01

Plant cell enlargement is regulated by wall relaxation and yielding, which is thought to be catalyzed by elusive "wall-loosening" enzymes. By employing a reconstitution approach, we found that a crude protein extract from the cell walls of growing cucumber seedlings possessed the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from various dicot stems and the leaves of amaryllidaceous monocots, but was less effective on grass coleoptile walls. Endogenous and reconstituted wall extension activities showed similar sensitivities to pH, metal ions, thiol reducing agents, proteases, and boiling in methanol or water. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD associated with the activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall. These proteins appear to mediate "acid growth" responses of isolated walls and may catalyze plant cell wall extension by a novel biochemical mechanism.

Hematopoietic Stem Cells in Regenerative Medicine: Astray or on the Path?

PubMed Central

Müller, Albrecht M.; Huppertz, Sascha; Henschler, Reinhard

2016-01-01

Hematopoietic stem cells (HSCs) are the best characterized adult stem cells and the only stem cell type in routine clinical use. The concept of stem cell transplantation laid the foundations for the development of novel cell therapies within, and even outside, the hematopoietic system. Here, we report on the history of hematopoietic cell transplantation (HCT) and of HSC isolation, we briefly summarize the capabilities of HSCs to reconstitute the entire hemato/lymphoid cell system, and we assess current indications for HCT. We aim to draw the lines between areas where HCT has been firmly established, areas where HCT can in the future be expected to be of clinical benefit using their regenerative functions, and areas where doubts persist. We further review clinical trials for diverse approaches that are based on HCT. Finally, we highlight the advent of genome editing in HSCs and critically view the use of HSCs in non-hematopoietic tissue regeneration. PMID:27721700

T-cell suicide gene therapy prompts thymic renewal in adults after hematopoietic stem cell transplantation.

PubMed

Vago, Luca; Oliveira, Giacomo; Bondanza, Attilio; Noviello, Maddalena; Soldati, Corrado; Ghio, Domenico; Brigida, Immacolata; Greco, Raffaella; Lupo Stanghellini, Maria Teresa; Peccatori, Jacopo; Fracchia, Sergio; Del Fiacco, Matteo; Traversari, Catia; Aiuti, Alessandro; Del Maschio, Alessandro; Bordignon, Claudio; Ciceri, Fabio; Bonini, Chiara

2012-08-30

The genetic modification of T cells with a suicide gene grants a mechanism of control of adverse reactions, allowing safe infusion after partially incompatible hematopoietic stem cell transplantation (HSCT). In the TK007 clinical trial, 22 adults with hematologic malignancies experienced a rapid and sustained immune recovery after T cell-depleted HSCT and serial infusions of purified donor T cells expressing the HSV thymidine kinase suicide gene (TK+ cells). After a first wave of circulating TK+ cells, the majority of T cells supporting long-term immune reconstitution did not carry the suicide gene and displayed high numbers of naive lymphocytes, suggesting the thymus-dependent development of T cells, occurring only upon TK+ -cell engraftment. Accordingly, after the infusions, we documented an increase in circulating TCR excision circles and CD31+ recent thymic emigrants and a substantial expansion of the active thymic tissue as shown by chest tomography scans. Interestingly, a peak in the serum level of IL-7 was observed after each infusion of TK+ cells, anticipating the appearance of newly generated T cells. The results of the present study show that the infusion of genetically modified donor T cells after HSCT can drive the recovery of thymic activity in adults, leading to immune reconstitution.

Leprosy reversal reaction as immune reconstitution inflammatory syndrome in patients with AIDS.

PubMed

Batista, Mariana D; Porro, Adriana M; Maeda, Solange M; Gomes, Elimar E; Yoshioka, Márcia C N; Enokihara, Mílvia M S S; Tomimori, Jane

2008-03-15

We report 2 instances in which reactional borderline leprosy manifested itself as an immune reconstitution phenomenon in patients with acquired immunodeficiency syndrome. We discuss the clinical, laboratory-based, histopathologic, and immunohistochemical characteristics of both patients. Furthermore, we review similar reports from the literature.

Cutting the brakes on hematopoietic regeneration by blocking TGFβ to limit chemotherapy-induced myelosuppression

PubMed Central

Brenet, Fabienne; Scandura, Joseph M

2015-01-01

Hematopoietic stressors such as infection, bleeding, or toxic injury trigger a hematopoietic adaptation that sacrifices hematopoietic stem and progenitor cell (HSPC) quiescence to meet an urgent need for new blood cell production. Once the hematopoietic demands are adequately met, homeostasis must be restored. Transforming growth factor β (TGFβ) signaling is a central mediator mandating the return of HSPCs to quiescence after stress. Blockade of TGFβ signaling after hematopoietic stress delays the return of cycling HSPCs to quiescence and in so doing promotes hematopoietic stem cell (HSC) self-renewal and accelerates hematopoietic reconstitution. These findings open the door to new therapeutics that modulate the hematopoietic adaptation to stress. In this review, we will discuss the complex context-dependent activities of TGFβ in hematopoiesis and the potential benefits and limitations of using TGFβ pathway inhibitors to promote multilineage hematopoietic reconstitution after myelosuppressive chemotherapy. PMID:27308454

In vitro-reconstituted nucleoids can block mitochondrial DNA replication and transcription.

PubMed

Farge, Géraldine; Mehmedovic, Majda; Baclayon, Marian; van den Wildenberg, Siet M J L; Roos, Wouter H; Gustafsson, Claes M; Wuite, Gijs J L; Falkenberg, Maria

2014-07-10

The mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000-10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A (TFAM). Here, we reconstitute nucleoid-like particles in vitro and demonstrate that small changes in TFAM levels dramatically impact the fraction of DNA molecules available for transcription and DNA replication. Compaction by TFAM is highly cooperative, and at physiological ratios of TFAM to DNA, there are large variations in compaction, from fully compacted nucleoids to naked DNA. In compacted nucleoids, TFAM forms stable protein filaments on DNA that block melting and prevent progression of the replication and transcription machineries. Based on our observations, we suggest that small variations in the TFAM-to-mtDNA ratio may be used to regulate mitochondrial gene transcription and DNA replication. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Postchallenge Administration of Brincidofovir Protects Healthy and Immune-Deficient Mice Reconstituted with Limited Numbers of T Cells from Lethal Challenge with IHD-J-Luc Vaccinia Virus

PubMed Central

McCullough, Kevin Tyler; Cruz, Stephanie; Thomas, Antonia; Diaz, Claudia G.; Keilholz, Laurie; Grossi, Irma M.; Trost, Lawrence C.; Golding, Hana

2015-01-01

ABSTRACT Protection from lethality by postchallenge administration of brincidofovir (BCV, CMX001) was studied in normal and immune-deficient (nude, nu/nu) BALB/c mice infected with vaccinia virus (VACV). Whole-body bioluminescence imaging was used to record total fluxes in the nasal cavity, lungs, spleen, and liver and to enumerate pox lesions on tails of mice infected via the intranasal route with 105 PFU of recombinant IHD-J-Luc VACV expressing luciferase. Areas under the flux curve (AUCs) were calculated for individual mice to assess viral loads. A three-dose regimen of 20 mg/kg BCV administered every 48 h starting either on day 1 or day 2 postchallenge protected 100% of mice. Initiating BCV treatment earlier was more efficient in reducing viral loads and in providing protection from pox lesion development. All BCV-treated mice that survived challenge were also protected from rechallenge with IHD-J-Luc or WRvFire VACV without additional treatment. In immune-deficient mice, BCV protected animals from lethality and reduced viral loads while animals were on the drug. Viral recrudescence occurred within 4 to 9 days, and mice succumbed ∼10 to 20 days after treatment termination. Nude mice reconstituted with 105 T cells prior to challenge with 104 PFU of IHD-J-Luc and treated with BCV postchallenge survived the infection, cleared the virus from all organs, and survived rechallenge with 105 PFU of IHD-J-Luc VACV without additional BCV treatment. Together, these data suggest that BCV protects immunocompetent and partially T cell-reconstituted immune-deficient mice from lethality, reduces viral dissemination in organs, prevents pox lesion development, and permits generation of VACV-specific memory. IMPORTANCE Mass vaccination is the primary element of the public health response to a smallpox outbreak. In addition to vaccination, however, antiviral drugs are required for individuals with uncertain exposure status to smallpox or for whom vaccination is contraindicated. Whole-body bioluminescence imaging was used to study the effect of brincidofovir (BCV) in normal and immune-deficient (nu/nu) mice infected with vaccinia virus, a model of smallpox. Postchallenge administration of 20 mg/kg BCV rescued normal and immune-deficient mice partially reconstituted with T cells from lethality and significantly reduced viral loads in organs. All BCV-treated mice that survived infection were protected from rechallenge without additional treatment. In immune-deficient mice, BCV extended survival. The data show that BCV controls viral replication at the site of challenge and reduces viral dissemination to internal organs, thus providing a shield for the developing adaptive immunity that clears the host of virus and builds virus-specific immunological memory. PMID:25589648

The aerosol rabbit model of TB latency, reactivation and immune reconstitution inflammatory syndrome

PubMed Central

Manabe, Yukari C.; Kesavan, Anup K.; Lopez-Molina, Javier; Hatem, Christine L.; Brooks, Megan; Fujiwara, Ricardo; Hochstein, Karl; Pitt, M. Louise M.; Tufariello, JoAnn; Chan, John; McMurray, David N.; Bishai, William R.; Dannenberg, Arthur M.; Mendez, Susana

2015-01-01

The large reservoir of human latent tuberculosis (TB) contributes to the global success of the pathogen, Mycobacterium tuberculosis (Mtb). We sought to test whether aerosol infection of rabbits with Mtb H37Rv could model paucibacillary human latent TB. The lung burden of infection peaked at 5 weeks after aerosol infection followed by host containment of infection that was achieved in all rabbits. One-third of rabbits had at least one caseous granuloma with culturable bacilli at 36 weeks after infection suggesting persistent paucibacillary infection. Corticosteroid-induced immunosuppression initiated after disease containment resulted in reactivation of disease. Seventy-two percent of rabbits had culturable bacilli in the right upper lung lobe homogenates compared to none of the untreated controls. Discontinuation of dexamethasone led to predictable lymphoid recovery, with a proportion of rabbits developing multicentric large caseous granuloma. The development and severity of the immune reconstitution inflammatory syndrome (IRIS) was dependent on the antigen load at the time of immunosuppression and subsequent bacillary replication during corticosteroid-induced immunosuppression. Clinically, many aspects were similar to IRIS in severely immunosuppressed HIV-infected patients who have functional restoration of T cells in response to effective (highly active) antiretroviral therapy. This corticosteroid model is the only animal model of the IRIS. Further study of the rabbit model of TB latency, reactivation and IRIS may be important in understanding the immunopathogenesis of these poorly modeled states as well as for improved diagnostics for specific stages of disease. PMID:18068491

Ciliate telomerase RNA loop IV nucleotides promote hierarchical RNP assembly and holoenzyme stability.

PubMed

Robart, Aaron R; O'Connor, Catherine M; Collins, Kathleen

2010-03-01

Telomerase adds simple-sequence repeats to chromosome 3' ends to compensate for the loss of repeats with each round of genome replication. To accomplish this de novo DNA synthesis, telomerase uses a template within its integral RNA component. In addition to providing the template, the telomerase RNA subunit (TER) also harbors nontemplate motifs that contribute to the specialized telomerase catalytic cycle of reiterative repeat synthesis. Most nontemplate TER motifs function through linkage with the template, but in ciliate and vertebrate telomerases, a stem-loop motif binds telomerase reverse transcriptase (TERT) and reconstitutes full activity of the minimal recombinant TERT+TER RNP, even when physically separated from the template. Here, we resolve the functional requirements for this motif of ciliate TER in physiological RNP context using the Tetrahymena thermophila p65-TER-TERT core RNP reconstituted in vitro and the holoenzyme reconstituted in vivo. Contrary to expectation based on assays of the minimal recombinant RNP, we find that none of a panel of individual loop IV nucleotide substitutions impacts the profile of telomerase product synthesis when reconstituted as physiological core RNP or holoenzyme RNP. However, loop IV nucleotide substitutions do variably reduce assembly of TERT with the p65-TER complex in vitro and reduce the accumulation and stability of telomerase RNP in endogenous holoenzyme context. Our results point to a unifying model of a conformational activation role for this TER motif in the telomerase RNP enzyme.

Elucidating the molecular mechanisms underlying cellular response to biophysical cues using synthetic biology approaches

PubMed Central

Denning, Denise; Roos, Wouter H.

2016-01-01

ABSTRACT The use of synthetic surfaces and materials to influence and study cell behavior has vastly progressed our understanding of the underlying molecular mechanisms involved in cellular response to physicochemical and biophysical cues. Reconstituting cytoskeletal proteins and interfacing them with a defined microenvironment has also garnered deep insight into the engineering mechanisms existing within the cell. This review presents recent experimental findings on the influence of several parameters of the extracellular environment on cell behavior and fate, such as substrate topography, stiffness, chemistry and charge. In addition, the use of synthetic environments to measure physical properties of the reconstituted cytoskeleton and their interaction with intracellular proteins such as molecular motors is discussed, which is relevant for understanding cell migration, division and structural integrity, as well as intracellular transport. Insight is provided regarding the next steps to be taken in this interdisciplinary field, in order to achieve the global aim of artificially directing cellular response. PMID:27266767

Rapid Reconstitution Packages (RRPs) implemented by integration of computational fluid dynamics (CFD) and 3D printed microfluidics.

PubMed

Chi, Albert; Curi, Sebastian; Clayton, Kevin; Luciano, David; Klauber, Kameron; Alexander-Katz, Alfredo; D'hers, Sebastian; Elman, Noel M

2014-08-01

Rapid Reconstitution Packages (RRPs) are portable platforms that integrate microfluidics for rapid reconstitution of lyophilized drugs. Rapid reconstitution of lyophilized drugs using standard vials and syringes is an error-prone process. RRPs were designed using computational fluid dynamics (CFD) techniques to optimize fluidic structures for rapid mixing and integrating physical properties of targeted drugs and diluents. Devices were manufactured using stereo lithography 3D printing for micrometer structural precision and rapid prototyping. Tissue plasminogen activator (tPA) was selected as the initial model drug to test the RRPs as it is unstable in solution. tPA is a thrombolytic drug, stored in lyophilized form, required in emergency settings for which rapid reconstitution is of critical importance. RRP performance and drug stability were evaluated by high-performance liquid chromatography (HPLC) to characterize release kinetics. In addition, enzyme-linked immunosorbent assays (ELISAs) were performed to test for drug activity after the RRPs were exposed to various controlled temperature conditions. Experimental results showed that RRPs provided effective reconstitution of tPA that strongly correlated with CFD results. Simulation and experimental results show that release kinetics can be adjusted by tuning the device structural dimensions and diluent drug physical parameters. The design of RRPs can be tailored for a number of applications by taking into account physical parameters of the active pharmaceutical ingredients (APIs), excipients, and diluents. RRPs are portable platforms that can be utilized for reconstitution of emergency drugs in time-critical therapies.

Restricted intra-embryonic origin of bona fide hematopoietic stem cells in the chicken

PubMed Central

Yvernogeau, Laurent

2017-01-01

Hematopoietic stem cells (HSCs), which are responsible for blood cell production, are generated during embryonic development. Human and chicken embryos share features that position the chicken as a reliable and accessible alternative model to study developmental hematopoiesis. However, the existence of HSCs has never been formally proven in chicken embryos. Here, we have established a complete cartography and quantification of hematopoietic cells in the aorta during development. We demonstrate the existence of bona fide HSCs, originating from the chicken embryo aorta (and not the yolk sac, allantois or head), through an in vivo transplantation assay. Embryos transplanted in ovo with GFP embryonic tissues on the chorio-allantoic membrane provided multilineage reconstitution in adulthood. Historically, most breakthrough discoveries in the field of developmental hematopoiesis were first made in birds and later extended to mammals. Our study sheds new light on the avian model as a valuable system to study HSC production and regulation in vivo. PMID:28526756

Cell-Free Reconstitution of Multivesicular Body Formation and Receptor Sorting

PubMed Central

Sun, Wei; Vida, Thomas A.; Sirisaengtaksin, Natalie; Merrill, Samuel A.; Hanson, Phyllis I.; Bean, Andrew J.

2010-01-01

The number of surface membrane proteins and their residence time on the plasma membrane are critical determinants of cellular responses to cues that can control plasticity, growth and differentiation. After internalization, the ultimate fate of many plasma membrane proteins is dependent on whether they are sorted for internalization into the lumenal vesicles of multivesicular bodies (MVBs), an obligate step prior to lysosomal degradation. To help to elucidate the mechanisms underlying MVB sorting, we have developed a novel cell-free assay that reconstitutes the sorting of a prototypical membrane protein, the epidermal growth factor receptor, with which we have probed some of its molecular requirements. The sorting event measured is dependent on cytosol, ATP, time, temperature and an intact proton gradient. Depletion of Hrs inhibited biochemical and morphological measures of sorting that were rescued by inclusion of recombinant Hrs in the assay. Moreover, depletion of signal-transducing adaptor molecule (STAM), or addition of mutated ATPase-deficient Vps4, also inhibited sorting. This assay reconstitutes the maturation of late endosomes, including the formation of internal vesicles and the sorting of a membrane protein, and allows biochemical investigation of this process. PMID:20214752

Immune Reconstitution Reactions in Human Immunodeficiency Virus–Negative Patients

PubMed Central

Scharschmidt, Tiffany C.; Amerson, Erin H.; Rosenberg, Oren S.; Jacobs, Richard A.; McCalmont, Timothy H.; Shinkai, Kanade

2013-01-01

Background Immune reconstitution inflammatory syndrome (IRIS) is a phenomenon initially described in patients with human immunodeficiency virus. Upon initiation of combination antiretroviral therapy, recovery of cellular immunity triggers inflammation to a preexisting infection or antigen that causes paradoxical worsening of clinical disease. A similar phenomenon can occur in human immunodeficiency virus–negative patients, including pregnant women, neutropenic hosts, solidorgan or stem cell transplant recipients, and patients receiving tumor necrosis factor inhibitors. Observations We report a case of leprosy unmasking and downgrading reaction after stem cell transplantation that highlights some of the challenges inherent to the diagnosis of IRIS, especially in patients without human immunodeficiency virus infection, as well as review the spectrum of previously reported cases of IRIS reactions in this population. Conclusions The mechanism of immune reconstitution reactions is complex and variable, depending on the underlying antigen and the mechanism of immunosuppression or shift in immune status. Use of the term IRIS can aid our recognition of an important phenomenon that occurs in the setting of immunosuppression or shifts in immunity but should not deter us from thinking critically about the distinct processes that underlie this heterogeneous group of conditions. PMID:23324760

[Hematopoietic reconstitution after transplantation of uncontrolled-rate cryopreservation autologous peripheral blood hematopoietic stem cells using -80 °C mechanical freezer].

PubMed

Liu, Mo; Zhao, Yu; Sun, Jing-Fen; Zhao, Wei; Wang, Li-Li; Yu, Li

2015-02-01

This study was to identify the efficacy of -80°C cryopreservated peripheral blood hemato-poietic stem cell (PBHSC) transplantation for hematopoietic reanstitution in patients. The efficacy of 104 patients underwent autologous peripheral blood hematopoietic stem cell transplantation using uncontrolled-rate freezing and storage at -80°C was evaluated. This cryopreservation method could effectively cryopreserve peripheral blood stem cells. Out of 104 patients only 2 patients died, other patients got hematologic reconstition satisfactorily, the median engrafement times of neutrophils and platelet were 12 and 14 days respectively, the activity of cells after rehabilitation was 94%, the mean recovery rates of CD34(+) cells and mononuclear cells (MNC) were 86% and 80.3% respectively. There were no significant influences on engrafement time in sex, chemotherapy circles and radiotherapy. The engrafement of leukocytes associated with amount of CD34(+) cells. This simple uncontrolled-rate freezing PBHSC at -80°C is safe, effective and economic, and can meet clinical needs. As compared with the classical cryopreservation, there were no significant differences in hematopoietic reconstitution. Therefore, this method worth to popularize and apply in clinic.

Immune Reconstitution Inflammatory Syndrome (IRIS): What pathologists should know.

PubMed

Nelson, Ann Marie; Manabe, Yukari C; Lucas, Sebastian B

2017-07-01

Antiretroviral therapy has significantly improved the quality and length of life for those patients able to access effective and sustained treatment. The resulting restoration of the immune response is associated with a change in the clinical presentation of opportunistic infections, and the histologic reaction to pathogens. A complex combination of alterations in host response across the stages of HIV infection has been documented over the past 3 decades. The defects are seen in both acute and chronic phases of inflammation and involve innate and adaptive immunity. In advanced stages of HIV infection, the marked disruption of lymphoid tissue and loss of follicular dendritic cells limits the host's ability to process antigen and mount specific responses to pathogens. There are qualitative and quantitative defects in CD4 T cells due to HIV infection. The resulting indirect effects include loss of cytokine production, dysregulation of B-cell function, loss of cellular mediated immunity and "holes" in the immunologic repertoire that may not be restored with the use of antiretroviral therapy. Immune reconstitution allows the host to respond to and control infection, but a significant number of patients will have atypical inflammatory syndromes during the recovery period. We briefly discuss the impact of HIV infection on the immune system and give an overview of the spectrum of conditions attributed to the Immune Reconstitution Inflammatory syndrome (IRIS). Copyright © 2017 Elsevier Inc. All rights reserved.

[Effect of Spatholobus suberctus on adhesion, invasion, migration and metastasis of melanoma cells].

PubMed

Xu, Jian-Ya; Gu, Qin; Xia, Wei-Jun

2010-10-01

To study the effect of Spatholobus suberctus, a kind of Chinese Traditional Medicine which can dissolve the stasis by activating the blood circulation, on invasion, adhesion, migration and metastasis of B16-BL6 metastatic mouse melanoma cells and its mechanism. The proliferation, adhesion, invasion and migration capacity of B16-BL6 metastatic cells was evaluated by MTP assay, adhesion assay and reconstituted basement membrane invasion and migration assay in vitro respectively. Mouse spontaneous motility melanoma model was used to study the effect of Spatholobus suberctus on metastasis in vivo. At the highest innoxious concentration, the extracts of Spatholobus suberctus inhibited the adhesion and invasion capacity of B16-BL6 metastatic cells significantly. In the mouse spontaneous melanoma model, the lung metastatic nodes number and its volume were significantly decreased after continuously treated with the extracts of Spatholobus suberctu. The extracts of Spatholobus suberctu can inhibit the metastasis of of B16-BI6 metastatic mouse melanoma cells and its mechanism may be inhibiting the capability of B16-BL6 cells in adhering to the ECM and invading the basement membrane.

Role of structural barriers for carotenoid bioaccessibility upon high pressure homogenization.

PubMed

Palmero, Paola; Panozzo, Agnese; Colle, Ines; Chigwedere, Claire; Hendrickx, Marc; Van Loey, Ann

2016-05-15

A specific approach to investigate the effect of high pressure homogenization on the carotenoid bioaccessibility in tomato-based products was developed. Six different tomato-based model systems were reconstituted in order to target the specific role of the natural structural barriers (chromoplast substructure/cell wall) and of the phases (soluble/insoluble) in determining the carotenoid bioaccessibility and viscosity changes upon high pressure homogenization. Results indicated that in the absence of natural structural barriers (carotenoid enriched oil), the soluble and insoluble phases determined the carotenoid bioaccessibility upon processing whereas, in their presence, these barriers governed the bioaccessibility. Furthermore, it was shown that the increment of the viscosity upon high pressure homogenization is determined by the presence of insoluble phase, however, this result was related to the initial ratio of the soluble:insoluble phases in the system. In addition, no relationship between the changes in viscosity and carotenoid bioaccessibility upon high pressure homogenization was found. Copyright © 2015 Elsevier Ltd. All rights reserved.

Micron-scale plasma membrane curvature is recognized by the septin cytoskeleton

PubMed Central

Bridges, Andrew A.; Jentzsch, Maximilian S.; Oakes, Patrick W.; Occhipinti, Patricia

2016-01-01

Cells change shape in response to diverse environmental and developmental conditions, creating topologies with micron-scale features. Although individual proteins can sense nanometer-scale membrane curvature, it is unclear if a cell could also use nanometer-scale components to sense micron-scale contours, such as the cytokinetic furrow and base of neuronal branches. Septins are filament-forming proteins that serve as signaling platforms and are frequently associated with areas of the plasma membrane where there is micron-scale curvature, including the cytokinetic furrow and the base of cell protrusions. We report here that fungal and human septins are able to distinguish between different degrees of micron-scale curvature in cells. By preparing supported lipid bilayers on beads of different curvature, we reconstitute and measure the intrinsic septin curvature preference. We conclude that micron-scale curvature recognition is a fundamental property of the septin cytoskeleton that provides the cell with a mechanism to know its local shape. PMID:27044896

Tunability of collagen matrix mechanical properties via multiple modes of mineralization

PubMed Central

Smith, Lester J.; Deymier, Alix C.; Boyle, John J.; Li, Zhen; Linderman, Stephen W.; Pasteris, Jill D.; Xia, Younan; Genin, Guy M.; Thomopoulos, Stavros

2016-01-01

Functionally graded, mineralized collagen tissues exist at soft-to-hard material attachments throughout the body. However, the details of how collagen and hydroxyapatite mineral (HA) interact are not fully understood, hampering efforts to develop tissue-engineered constructs that can assist with repair of injuries at the attachments of soft tissues to bone. In this study, spatial control of mineralization was achieved in collagen matrices using simulated body fluids (SBFs). Based upon previous observations of poor bonding between reconstituted collagen and HA deposited using SBF, we hypothesized that mineralizing collagen in the presence of fetuin (which inhibits surface mineralization) would lead to more mineral deposition within the scaffold and therefore a greater increase in stiffness and toughness compared with collagen mineralized without fetuin. We tested this hypothesis through integrated synthesis, mechanical testing and modelling of graded, mineralized reconstituted collagen constructs. Results supported the hypothesis, and further suggested that mineralization on the interior of reconstituted collagen constructs, as promoted by fetuin, led to superior bonding between HA and collagen. The results provide us guidance for the development of mineralized collagen scaffolds, with implications for bone and tendon-to-bone tissue engineering. PMID:26855755

Chronic active Epstein-Barr virus infection (CAEBV) successfully treated with allogeneic peripheral blood stem cell transplantation.

PubMed

Taketani, T; Kikuchi, A; Inatomi, J; Hanada, R; Kawaguchi, H; Ida, K; Oh-Ishi, T; Arai, T; Kishimoto, H; Yamamoto, K

2002-03-01

We report a pediatric case of CAEBV and T cell-based Hodgkin's-like disease successfully treated with allo PBSCT from an HLA-matched sibling. The diagnosis of CAEBV was made from clinical signs and the presence of the EBV genome in PBMC and tumor cells. Conditioning with busulfan (BU) + etoposide (VP16) + cyclophosphamide (CY) was effective and well tolerated. EBV was totally eradicated by 3 months after allo PBSCT. Although she suffered from chronic GVHD of the liver, she has been well and free of disease for 47 months since PBSCT. We suggest allo PBSCT for CAEBV as a potent therapeutic strategy for eradication of the EBV genome and allowing immunological reconstitution.

Somatic mutations and affinity maturation are impaired by excessive numbers of T follicular helper cells and restored by Treg cells or memory T cells.

PubMed

Preite, Silvia; Baumjohann, Dirk; Foglierini, Mathilde; Basso, Camilla; Ronchi, Francesca; Fernandez Rodriguez, Blanca M; Corti, Davide; Lanzavecchia, Antonio; Sallusto, Federica

2015-11-01

We previously reported that Cd3e-deficient mice adoptively transferred with CD4(+) T cells generate high numbers of T follicular helper (Tfh) cells, which go on to induce a strong B-cell and germinal center (GC) reaction. Here, we show that in this system, GC B cells display an altered distribution between the dark and light zones, and express low levels of activation-induced cytidine deaminase. Furthermore, GC B cells from Cd3e(-/-) mice accumulate fewer somatic mutations as compared with GC B cells from wild-type mice, and exhibit impaired affinity maturation and reduced differentiation into long-lived plasma cells. Reconstitution of Cd3e(-/-) mice with regulatory T (Treg) cells restored Tfh-cell numbers, GC B-cell numbers and B-cell distribution within dark and light zones, and the rate of antibody somatic mutations. Tfh-cell numbers and GC B-cell numbers and dynamics were also restored by pre-reconstitution of Cd3e(-/-) mice with Cxcr5(-/-) Treg cells or non-regulatory, memory CD4(+) T cells. Taken together, these findings underline the importance of a quantitatively regulated Tfh-cell response for an efficient and long-lasting serological response. © 2015 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pharmacokinetic and pharmacodynamic studies of two different rabbit antithymocyte globulin dosing regimens: results of a randomized trial.

PubMed

Büchler, Matthias; Longuet, Hélène; Lemoine, Roxane; Herr, Florence; Gatault, Philippe; Thibault, Gilles; Ternant, David; Foulon, Christine; Pilorge, Bernadette; Lemay, Djamila; Sung, Crystal; Halimi, Jean-Michel; Baron, Christophe; Lebranchu, Yvon

2013-03-01

Rabbit antithymocyte globulin (rATG; Thymoglobulin(®)) is currently used to prevent acute rejection in kidney transplantation. The dose and regimen of rATG have not been optimized. Moreover, the impact of different treatment regimens on T-cell phenotype reconstitution remains unknown. We conducted a prospective randomized study of 17 renal transplant patients to determine the pharmacokinetics of total and active (bound to human cells) rATG and T-cell phenotype reconstitution after rATG administration. Patients received rATG at a total dose of 6mg/kg, administered either as 1.5mg/kg/day on days 0-3 (Group 1, n=8) or 3mg/kg on days 0 and 3 (Group 2, n=9). All patients received tacrolimus, mycophenolate mofetil and steroids. Blood samples were assayed for total rATG by enzyme linked immunosorbent assay and active rATG by flow cytometry. Maximum concentrations and terminal half-lives were similar between the two groups but at month 3 Group 1 had significantly lower values for total rATG (concentration was 6.2±1.1μg/mL versus 10.2±2.9μg/mL in Group 2, p=0.027) and total rATG dose-normalized AUC (374±83dayg/mL versus 508±149dayg/mL in Group 2, p=0.046). Time to sub-therapeutic levels (<1μg/mL) of active rATG was significantly shorter in Group 1 (18.75±6.9days versus 20±7.5days in Group 2, p<0.001). rATG induced significant depletion followed by slow reconstitution of CD3(+), CD4(+) and CD8(+) cells, with no marked differences between groups. B-cell count was unaffected, whereas CD3(-)CD56(+) NK-cell depletion was observed in both groups. rATG induced a significant decrease in the proportion of naïve CD4(+) T-cells, which plateaued after month 1 in Group 1 and after month 6 in Group 2. The proportion of central memory CD4(+) T-cells increased to a similar extent in both groups (Group 1: 38±18% at baseline, 74±23% at one year, p=0.009; Group 2: 32±14% at baseline, 65±14% at one year, p=0.001). In conclusion, our results suggest that the dosing regimen for rATG induction influences pharmacokinetic parameters without affecting the quality of immune reconstitution. Copyright © 2013 Elsevier B.V. All rights reserved.

Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells

PubMed Central

Kobayashi, Hideki; Butler, Jason M.; O'Donnell, Rebekah; Kobayashi, Mariko; Ding, Bi-Sen; Bonner, Bryant; Chiu, Vi K.; Nolan, Daniel J.; Shido, Koji; Benjamin, Laura; Rafii, Shahin

2010-01-01

Endothelial cells establish an instructive vascular niche that reconstitutes haematopoietic stem and progenitor cells (HSPCs) through release of specific paracrine growth factors, known as angiocrine factors. However, the mechanism by which endothelial cells balance the rate of proliferation and lineage-specific differentiation of HSPCs is unknown. Here, we demonstrate that Akt activation in endothelial cells, through recruitment of mTOR, but not the FoxO pathway, upregulates specific angiocrine factors that support expansion of CD34−Flt3− KLS HSPCs with long-term haematopoietic stem cell (LT-HSC) repopulation capacity. Conversely, co-activation of Akt-stimulated endothelial cells with p42/44 MAPK shifts the balance towards maintenance and differentiation of the HSPCs. Selective activation of Akt1 in the endothelial cells of adult mice increased the number of colony forming units in the spleen and CD34−Flt3− KLS HSPCs with LT-HSC activity in the bone marrow, accelerating haematopoietic recovery. Therefore, the activation state of endothelial cells modulates reconstitution of HSPCs through the upregulation of angiocrine factors, with Akt–mTOR-activated endothelial cells supporting the self-renewal of LT-HSCs and expansion of HSPCs, whereas MAPK co-activation favours maintenance and lineage-specific differentiation of HSPCs. PMID:20972423

Molecular Memory of Morphologies by Septins during Neuron Generation Allows Early Polarity Inheritance.

PubMed

Boubakar, Leila; Falk, Julien; Ducuing, Hugo; Thoinet, Karine; Reynaud, Florie; Derrington, Edmund; Castellani, Valérie

2017-08-16

Transmission of polarity established early during cell lineage history is emerging as a key process guiding cell differentiation. Highly polarized neurons provide a fascinating model to study inheritance of polarity over cell generations and across morphological transitions. Neural crest cells (NCCs) migrate to the dorsal root ganglia to generate neurons directly or after cell divisions in situ. Using live imaging of vertebrate embryo slices, we found that bipolar NCC progenitors lose their polarity, retracting their processes to round for division, but generate neurons with bipolar morphology by emitting processes from the same locations as the progenitor. Monitoring the dynamics of Septins, which play key roles in yeast polarity, indicates that Septin 7 tags process sites for re-initiation of process growth following mitosis. Interfering with Septins blocks this mechanism. Thus, Septins store polarity features during mitotic rounding so that daughters can reconstitute the initial progenitor polarity. Copyright © 2017 Elsevier Inc. All rights reserved.

Reconstitution of high affinity. cap alpha. /sub 2/ adrenergic agonist binding by fusion with a pertussis toxin substrate

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

Kim, M.H.; Neubig, R.R.

1986-03-05

High affinity ..cap alpha../sub 2/ adrenergic agonist binding is thought to occur via a coupling of the ..cap alpha../sub 2/ receptor with N/sub i/, the inhibitory guanyl nucleotide binding protein. Human platelet membranes pretreated at pH 11.5 exhibit a selective inactivation of agonist binding and N/sub i/. To further study the mechanism of agonist binding, alkali treated membranes (ATM) were mixed with membranes pretreated with 10 ..mu..M phenoxybenzamine to block ..cap alpha../sub 2/ receptors (POB-M). The combined membrane pellet was incubated in 50% polyethylene glycol (PEG) to promote membrane-membrane fusion and assayed for binding to the ..cap alpha../sub 2/ agonistmore » (/sup 3/H)UK 14,304 (UK) and the antagonist (/sup 3/H) yohimbine. PEG treatment resulted in a 2-4 fold enhancement of UK binding whereas yohimbine binding was unchanged. No enhancement of UK binding was observed in the absence of PEG treatment. The reconstitution was dependent on the addition of POB-M. They found that a 1:1 ratio of POB-M:ATM was optimal. Reconstituted binding was inhibited by GppNHp. Fusion of rat C6 glioma cell membranes, which do not contain ..cap alpha../sub 2/ receptors, also enhanced agonist binding to ATM. Fusion of C6 membranes from cells treated with pertussis toxin did not enhance (/sup 3/H) UK binding. These data show that a pertussis toxin sensitive membrane component, possibly N/sub i/, can reconstitute high affinity ..cap alpha../sub 2/ agonist binding.« less

Comparison of the fluorescence kinetics of detergent-solubilized and membrane-reconstituted LH2 complexes from Rps. acidophila and Rb. sphaeroides.

PubMed

Pflock, Tobias; Dezi, Manuela; Venturoli, Giovanni; Cogdell, Richard J; Köhler, Jürgen; Oellerich, Silke

2008-01-01

Picosecond time-resolved fluorescence spectroscopy has been used in order to compare the fluorescence kinetics of detergent-solubilized and membrane-reconstituted light-harvesting 2 (LH2) complexes from the purple bacteria Rhodopseudomonas (Rps.) acidophila and Rhodobacter (Rb.) sphaeroides. LH2 complexes were reconstituted in phospholipid model membranes at different lipid:protein-ratios and all samples were studied exciting with a wide range of excitation densities. While the detergent-solubilized LH2 complexes from Rps. acidophila showed monoexponential decay kinetics (tau(f )= 980 ps) for excitation densities of up to 3.10(13) photons/(pulse.cm(2)), the membrane-reconstituted LH2 complexes showed multiexponential kinetics even at low excitation densities and high lipid:protein-ratios. The latter finding indicates an efficient clustering of LH2 complexes in the phospholipid membranes. Similar results were obtained for the LH2 complexes from Rb. sphaeroides.

Candida albicans ISW2 Regulates Chlamydospore Suspensor Cell Formation and Virulence In Vivo in a Mouse Model of Disseminated Candidiasis

PubMed Central

Lionakis, Michail S.; Nickerson, Kenneth W.

2016-01-01

Formation of chlamydospores by Candida albicans was an established medical diagnostic test to confirm candidiasis before the molecular era. However, the functional role and pathological relevance of this in vitro morphological transition to pathogenesis in vivo remain unclear. We compared the physical properties of in vitro-induced chlamydospores with those of large C. albicans cells purified by density gradient centrifugation from Candida-infected mouse kidneys. The morphological and physical properties of these cells in kidneys of mice infected intravenously with wild type C. albicans confirmed that chlamydospores can form in infected kidneys. A previously reported chlamydospore-null Δisw2/Δisw2 mutant was used to investigate its role in virulence and chlamydospore induction. Virulence of the Δisw2/Δisw2 mutant strain was reduced 3.4-fold compared to wild type C. albicans or the ISW2 reconstituted strain. Altered host inflammatory reactions to the null mutant further indicate that ISW2 is a virulence factor in C. albicans. ISW2 deletion abolished chlamydospore formation within infected mouse kidneys, whereas the reconstituted strain restored chlamydospore formation in kidneys. Under chlamydospore inducing conditions in vitro, deletion of ISW2 significantly delayed chlamydospore formation, and those late induced chlamydospores lacked associated suspensor cells while attaching laterally to hyphae via novel spore-hypha septa. Our findings establish the induction of chlamydospores by C. albicans during mouse kidney colonization. Our results indicate that ISW2 is not strictly required for chlamydospores formation but is necessary for suspensor cell formation. The importance of ISW2 in chlamydospore morphogenesis and virulence may lead to additional insights into morphological differentiation and pathogenesis of C. albicans in the host microenvironment. PMID:27727302

The Use of Novel Therapies to Reconstitute Blood Cell Production Production and Promote Organ Performance, Using Bone Marrow Failure as Modela Model

DTIC Science & Technology

2015-10-01

reported to be on Zenpep for gastro intestinal absorption issues and there are no data on the absorption of Leucine in this setting) . There was one...gastrointestinal absorption issues. As there are no data on the absorption of Leucine in this setting , the subject was withdrawn. 19DEC2014 Subject 12-002... Leucine is one of the branched chain amino acids and has been shown to upregulate protein translation . This is a pilot study to test the feasibility of

Direct Reprogramming of Murine Fibroblasts to Hematopoietic Progenitor Cells

PubMed Central

Batta, Kiran; Florkowska, Magdalena; Kouskoff, Valerie; Lacaud, Georges

2014-01-01

Summary Recent reports have shown that somatic cells, under appropriate culture conditions, could be directly reprogrammed to cardiac, hepatic, or neuronal phenotype by lineage-specific transcription factors. In this study, we demonstrate that both embryonic and adult somatic fibroblasts can be efficiently reprogrammed to clonal multilineage hematopoietic progenitors by the ectopic expression of the transcription factors ERG, GATA2, LMO2, RUNX1c, and SCL. These reprogrammed cells were stably expanded on stromal cells and possessed short-term reconstitution ability in vivo. Loss of p53 function facilitated reprogramming to blood, and p53−/− reprogrammed cells efficiently generated erythroid, megakaryocytic, myeloid, and lymphoid lineages. Genome-wide analyses revealed that generation of hematopoietic progenitors was preceded by the appearance of hemogenic endothelial cells expressing endothelial and hematopoietic genes. Altogether, our findings suggest that direct reprogramming could represent a valid alternative approach to the differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) for disease modeling and autologous blood cell therapies. PMID:25466247

Combining G-CSF with a blockade of adhesion strongly improves the reconstitutive capacity of mobilized hematopoietic progenitor cells.

PubMed

Christ, O; Kronenwett, R; Haas, R; Zöller, M

2001-03-01

Mobilization of hematopoietic progenitor cells is achieved mainly by application of growth factors and, more recently, by blockade of adhesion. In this report, we describe the advantages of a combined treatment with granulocyte colony-stimulating factor (G-CSF) and anti-VLA4 (CD49d)/anti-CD44 as compared to treatment with the individual components. Mobilization by intravenous injection of anti-CD44, anti-VLA4, or G-CSF was controlled in spleen and bone marrow with regard to frequencies of multipotential colony-forming unit (C-CFU), marrow repopulating ability, long-term reconstitution, recovery of myelopoiesis, and regain of immunocompetence. Mobilization by anti-CD44 had a strong effect on expansion of early progenitor cells in the bone marrow, while the recovery in the spleen was poor. In anti-CD49d-mobilized noncommitted and committed progenitors, progenitor expansion was less pronounced, but settlement in the spleen was quite efficient. Thus, anti-CD44 and anti-CD49d differently influenced mobilization. Accordingly, mobilization and recovery after transfer were improved by combining anti-CD44 with anti-CD49d treatment. Mobilization by G-CSF was most efficient with respect to recovery of progenitor cells in the spleen. However, when transferring G-CSF-mobilized cells, regain of immunocompetence was strongly delayed. This disadvantage could be overridden when progenitor cells were mobilized via blockade of adhesion and when expansion of these mobilized progenitor cells was supported by low-dose G-CSF only during the last 24 hours before transfer. Mobilization of pluripotent progenitor cells via antibody blockade of CD44 or CD49d or via G-CSF relies on distinct mechanisms. Therefore, the reconstitutive capacity of a transplant can be significantly improved by mobilization regimens combining antibody with low-dose G-CSF treatment.

FLT3-ITD cooperates with inv(16) to promote progression to acute myeloid leukemia

PubMed Central

Kim, Hyung-Gyoon; Kojima, Kyoko; Swindle, C. Scott; Cotta, Claudiu V.; Huo, Yongliang; Reddy, Vishnu

2008-01-01

The inversion of chromosome 16 in the inv(16)(p13q22) is one of the most frequent cytogenetic abnormalities observed in acute myeloid leukemia (AML). The inv(16) fuses the core binding factor (CBF) beta subunit with the coiled-coil rod domain of smooth muscle myosin heavy chain (SMMHC). Expression of CBFβ-SMMHC in mice does not promote AML in the absence of secondary mutations. Patient samples with the inv(16) also possess mutually exclusive activating mutations in either N-RAS, K-RAS, or the receptor tyrosine kinases, c-KIT and FLT3, in almost 70% of cases. To test whether an activating mutation of FLT3 (FLT3-ITD) would cooperate with CBFβ-SMMHC to promote AML, we coexpressed both mutations in hematopoietic progenitor cells used to reconstitute lethally irradiated mice. Analysis of transplanted animals showed strong selection for CBFβ-SMMHC/FLT3-ITD–expressing cells in bone marrow and peripheral blood. Compared with animals transplanted with only CBFβ-SMMHC–expressing cells, FLT3-ITD further restricted early myeloid differentiation and promoted peripheralization of primitive myeloblasts as early as 2.5 weeks after transplantation. FLT3-ITD also accelerated disease progression in all CBFβ-SMMHC/FLT3-ITD–reconstituted animals, which died of a highly aggressive and transplantable AML within 3 to 5 months. These results indicate that FLT3-activating mutations can cooperate with CBFβ-SMMHC in an animal model of inv(16)-associated AML. PMID:17967943

Human natural killer cells prevent infectious mononucleosis features by targeting lytic Epstein-Barr virus infection

PubMed Central

Chijioke, Obinna; Müller, Anne; Feederle, Regina; Barros, Mario Henrique M.; Krieg, Carsten; Emmel, Vanessa; Marcenaro, Emanuela; Leung, Carol S.; Antsiferova, Olga; Landtwing, Vanessa; Bossart, Walter; Moretta, Alessandro; Hassan, Rocio; Boyman, Onur; Niedobitek, Gerald; Delecluse, Henri-Jacques; Capaul, Riccarda; Münz, Christian

2014-01-01

SUMMARY Primary infection with the human oncogenic Epstein Barr virus (EBV) can result in infectious mononucleosis (IM), a self-limiting disease caused by massive lymphocyte expansion, which predisposes for the development of distinct EBV-associated lymphomas. It remains unclear why some individuals experience this symptomatic primary EBV infection, while the majority acquires the virus asymptomatically. Using a mouse model with reconstituted human immune system components, we show here that depletion of human natural killer (NK) cells enhances IM symptoms and promotes EBV-associated tumorigenesis, mainly due to loss of immune control over lytic EBV infection. These data suggest that failure of innate immune control by human NK cells augments symptomatic lytic EBV infection, which drives lymphocyte expansion and predisposes for EBV-associated malignancies. PMID:24360958

Shaping up synthetic cells

NASA Astrophysics Data System (ADS)

Mulla, Yuval; Aufderhorst-Roberts, Anders; Koenderink, Gijsje H.

2018-07-01

How do the cells in our body reconfigure their shape to achieve complex tasks like migration and mitosis, yet maintain their shape in response to forces exerted by, for instance, blood flow and muscle action? Cell shape control is defined by a delicate mechanical balance between active force generation and passive material properties of the plasma membrane and the cytoskeleton. The cytoskeleton forms a space-spanning fibrous network comprising three subsystems: actin, microtubules and intermediate filaments. Bottom-up reconstitution of minimal synthetic cells where these cytoskeletal subsystems are encapsulated inside a lipid vesicle provides a powerful avenue to dissect the force balance that governs cell shape control. Although encapsulation is technically demanding, a steady stream of advances in this technique has made the reconstitution of shape-changing minimal cells increasingly feasible. In this topical review we provide a route-map of the recent advances in cytoskeletal encapsulation techniques and outline recent reports that demonstrate shape change phenomena in simple biomimetic vesicle systems. We end with an outlook toward the next steps required to achieve more complex shape changes with the ultimate aim of building a fully functional synthetic cell with the capability to autonomously grow, divide and move.

Biomimetics: reconstitution of low-density lipoprotein for targeted drug delivery and related theranostic applications.

PubMed

Zhu, Chunlei; Xia, Younan

2017-12-11

Low-density lipoprotein (LDL), one of the four major groups of lipoproteins for lipid transport in vivo, is emerging as an attractive carrier for the targeted delivery of theranostic agents. In contrast to the synthetic systems, LDL particles are intrinsically biocompatible and biodegradable, together with reduced immunogenicity and natural capabilities to target cancerous cells and to escape from the recognition and elimination by the reticuloendothelial system. Enticed by these attributes, a number of strategies have been developed for reconstituting LDL particles, including conjugation to the apolipoprotein, insertion into the phospholipid layer, and loading into the core. Here we present a tutorial review on the development of reconstituted LDL (rLDL) particles for theranostic applications. We start with a brief introduction to LDL and LDL receptor, as well as the advantages of using rLDL particles as a natural and versatile platform for the targeted delivery of theranostic agents. After a discussion of commonly used strategies for the reconstitution of LDL, we highlight the applications of rLDL particles in the staging of disease progression, treatment of lesioned tissues, and delivery of photosensitizers for photodynamic cancer therapy. We finish this review with a perspective on the remaining challenges and future directions.

Concurrent CMV and EBV DNAemia is significantly correlated with a delay in the response to HAART in treatment-naive HIV type 1-positive patients.

PubMed

Panagiotakis, Simeon H; Soufla, Giannoula; Baritaki, Stavroula; Sourvinos, George; Passam, Andreas; Zagoreos, Ioannis; Stavrianeas, Nikolaos; Spandidos, Demetrios A

2007-01-01

The purpose of this study was to assess the qualitative single and multiple herpes virus DNAemia in the peripheral blood leukocytes (PBLs) of HIV-1-positive patients and its impact on the response to highly active antiretroviral therapy (HAART) and immune reconstitution. All (163) HIV-1-positive patients attending "Syngros AIDS Referral Center" from November 2000 to February 2001 were recruited. CMV, HSV-1, HSV-2, EBV, and HHV-8 DNA were detected in PBLs by polymerase chain reaction (PCR). Patients' follow-up comprised regular measurements of CD4(+) T cell count and HIV-1 viral load (VL) for an average period of 21 months. Immune reconstitution was defined as an increase in the CD4 T cell count by above 200 cells/micro l, while response to HAART was defined as a decrease in HIV-1 VL to undetectable levels. Single and multiple herpetic DNAemia in PBLs was found to be significantly higher in HIV-1-positive patients compared to healthy controls (p < 0.02) for all the viruses detected apart from HSV-2, which was not detected in the PBLs of either population. Concurrent CMV and EBV DNAemia significantly correlates with a delay in the response to HAART (p = 0.033) in treatment-naive patients. Untreated patients with a CD4(+) T cell count <200 cells/micro l, and with either CMV or EBV DNAemia, presented a delayed increase in the CD4 count after initiation of HAART (p = 0.035 and p = 0.037 respectively), while multiple herpetic DNAemia in the above patients was borderline associated with immune reconstitution (p = 0.068). Conclusively, CMV and EBV DNAemia may be poor prognostic factors for the response to HAART in treatment-naive HIV-1 patients.

Regulation by CD45 of the tyrosine phosphorylation of high affinity IgE receptor beta- and gamma-chains.

PubMed

Adamczewski, M; Numerof, R P; Koretzky, G A; Kinet, J P

1995-04-01

Previous studies using tyrosine phosphatase inhibitors have implicated tyrosine phosphatases in the signal transduction pathway initiated by aggregation of Fc epsilon RI, the high affinity receptor for IgE. To define more precisely a role for the tyrosine phosphatase CD45 in Fc epsilon RI-mediated signaling, we have transfected the three subunits of Fc epsilon RI into wild-type Jurkat and a CD45-deficient Jurkat derivative. Here we demonstrate that CD45 is necessary for the initiation of calcium flux through the transfected Fc epsilon RI. In contrast to the effect of phosphatase inhibitors, the tyrosine phosphorylation levels of beta and gamma after aggregation of Fc epsilon RI are surprisingly reduced, relative to wild-type Jurkat, in the CD45-deficient cells. After reconstitution of the CD45-deficient cells with a chimeric molecule containing the cytoplasmic phosphatase domains of CD45, both the base line and activation-induced tyrosine phosphorylation levels are increased. By examining Lck autophosphorylation, we find that Fc epsilon RI aggregation induces an increase in Lck enzymatic activity only in wild-type Jurkat and the CD45-deficient Jurkat reconstituted with chimeric CD45. This regulation of src-family tyrosine kinase activity may be the means by which CD45 controls aggregation-induced receptor phosphorylation.

Reactive Oxygen Species Produced by Prostate Cancer Cells Cause Castrate-Resistant Cell Growth by Inducing B-cell Lymphotoxin Release

DTIC Science & Technology

2013-06-01

data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this...Gaussia luciferase reconstitution, high throughput screen, small molecule inhibitors, human prostate carcinoma cells, pharmacokinetcs, prostate cancer...cells. The increase in ROS levels is probably due to an induction of a polyamine oxidation pathway and specific small molecule inhibitors of this

Cytoprotection: Immune and Matrix Modulation of Tissue Repair

DTIC Science & Technology

2011-04-14

the damaged external urethral sphincter and significantly improved VLPP. Poster #BS39 THE EFFECT OF DONOR AGE ON INDUCED PLURIPOTENT STEM CELLS FROM...is an extracellular matrix hydrogel that contains cross-linked HMW-HA, which has been used to advantage in the growth of other stem cell types, but...that, after treatment with regenerating or reconstituted cells or stem cells , the viability of those therapeutic cells is often threatened by the

Natural and adoptive T-cell immunity against herpes family viruses after allogeneic hematopoietic stem cell transplantation.

PubMed

Thomas, Simone; Herr, Wolfgang

2011-06-01

Reactivated infections with herpes family-related cytomegalovirus, Epstein-Barr virus and varicella zoster virus are serious and sometimes life-threatening complications for patients undergoing allogeneic hematopoietic stem cell transplantation. The pathogenesis of these infections critically involves the slow and inefficient recovery of antiviral T-cell immunity after transplantation. Although efficient drugs to decrease viral load during this vulnerable period have been developed, long-term control of herpes viruses and protection from associated diseases require the sufficient reconstitution of virus-specific memory T cells. To heal the deficiency by immunotherapeutic means, numerous research groups have developed antiviral vaccines and strategies based on the adoptive transfer of virus-specific T cells. This article summarizes the substantial progress made in this field during the past two decades and gives future perspectives about challenges that need to be addressed before antigen-specific immunotherapy against herpes family viruses can be implemented in general clinical practice.

7 CFR 718.206 - Determining farms, tracts, allotments, quotas, and bases when reconstitution is made by division.

Code of Federal Regulations, 2011 CFR

2011-01-01

..., ACREAGE ALLOTMENTS, AND PRODUCTION ADJUSTMENT PROVISIONS APPLICABLE TO MULTIPLE PROGRAMS Reconstitution of... available land, cultural operations, and changes in type of farming. (e) The cropland method is the pro-rata... the method used did not provide an equitable distribution considering available land, cultural...

Multidrug Transport Protein NorM from Vibrio cholerae Simultaneously Couples to Sodium- and Proton-Motive Force*

PubMed Central

Jin, Yoonhee; Nair, Asha; van Veen, Hendrik W.

2014-01-01

Membrane transporters belonging to the multidrug and toxic compound extrusion family mediate the efflux of unrelated pharmaceuticals from the interior of the cell in organisms ranging from bacteria to human. These proteins are thought to fall into two classes that couple substrate efflux to the influx of either Na+ or H+. We studied the energetics of drug extrusion by NorM from Vibrio cholerae in proteoliposomes in which purified NorM protein was functionally reconstituted in an inside-out orientation. We establish that NorM simultaneously couples to the sodium-motive force and proton-motive force, and biochemically identify protein regions and residues that play important roles in Na+ or H+ binding. As the positions of protons are not available in current medium and high-resolution crystal structures of multidrug and toxic compound extrusion transporters, our findings add a previously unrecognized parameter to mechanistic models based of these structures. PMID:24711447

Artificial Lipid Membranes: Past, Present, and Future

PubMed Central

Siontorou, Christina G.; Nikoleli, Georgia-Paraskevi; Nikolelis, Dimitrios P.

2017-01-01

The multifaceted role of biological membranes prompted early the development of artificial lipid-based models with a primary view of reconstituting the natural functions in vitro so as to study and exploit chemoreception for sensor engineering. Over the years, a fair amount of knowledge on the artificial lipid membranes, as both, suspended or supported lipid films and liposomes, has been disseminated and has helped to diversify and expand initial scopes. Artificial lipid membranes can be constructed by several methods, stabilized by various means, functionalized in a variety of ways, experimented upon intensively, and broadly utilized in sensor development, drug testing, drug discovery or as molecular tools and research probes for elucidating the mechanics and the mechanisms of biological membranes. This paper reviews the state-of-the-art, discusses the diversity of applications, and presents future perspectives. The newly-introduced field of artificial cells further broadens the applicability of artificial membranes in studying the evolution of life. PMID:28933723

Assessing the risk of CMV reactivation and reconstitution of antiviral immune response post bone marrow transplantation by the QuantiFERON-CMV-assay and real time PCR.

PubMed

Krawczyk, Adalbert; Ackermann, Jessica; Goitowski, Birgit; Trenschel, Rudolf; Ditschkowski, Markus; Timm, Jörg; Ottinger, Hellmut; Beelen, Dietrich W; Grüner, Nico; Fiedler, Melanie

CMV reactivation is a major cause of severe complications in allogeneic hematopoietic stem cell transplant (HSCT) recipients. The risk of CMV reactivation depends on the serostatus (+/-) of the donor (D) and recipient (R). The reconstitution of CMV-specific T-cell responses after transplantation is crucial for the control of CMV reactivation. The study aimed to determine the cellular immune status correlating with protection from high-level CMV viremia (>5000 copies/ml) and disease. We monitored CMV-specific cellular immune responses in 9 high-risk (D-/R+), 14 intermediate risk (D+/R+) and 3 low risk individuals (D+/R-), and 8 CMV negative controls (D-/R-). Interferon- γ (IFN-γ) levels as a marker for the CD8+ T-cell response were determined by the QuantiFERON-CMV-assay and compared to viral loads determined by PCR. Early CMV reactivation was detected in all high-risk and 13/14 intermediate risk individuals. High-level viremia was detected in 5/7 high and 7/14 intermediate risk patients. Reconstitution of the CMV-specific cellular immune response started from 3 months after transplantation and resulted in protection against CMV reactivation. Re-establishing of CMV-specific T-cell immune responses with IFN- γ levels >8.9 IU/ml is crucial for protection from high-level CMV viremia. Monitoring of HSCT-recipients with the QuantiFERON-CMV-assay might be of great benefit to optimize antiviral treatment. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

A very low geno2pheno false positive rate is associated with poor viro-immunological response in drug-naïve patients starting a first-line HAART.

PubMed

Armenia, Daniele; Soulie, Cathia; Di Carlo, Domenico; Fabeni, Lavinia; Gori, Caterina; Forbici, Federica; Svicher, Valentina; Bertoli, Ada; Sarmati, Loredana; Giuliani, Massimo; Latini, Alessandra; Boumis, Evangelo; Zaccarelli, Mauro; Bellagamba, Rita; Andreoni, Massimo; Marcelin, Anne-Geneviève; Calvez, Vincent; Antinori, Andrea; Ceccherini-Silberstein, Francesca; Perno, Carlo-Federico; Santoro, Maria Mercedes

2014-01-01

We previously found that a very low geno2pheno false positive rate (FPR ≤ 2%) defines a viral population associated with low CD4 cell count and the highest amount of X4-quasispecies. In this study, we aimed at evaluating whether FPR ≤ 2% might impact on the viro-immunological response in HIV-1 infected patients starting a first-line HAART. The analysis was performed on 305 HIV-1 B subtype infected drug-naïve patients who started their first-line HAART. Baseline FPR (%) values were stratified according to the following ranges: ≤ 2; 2-5; 5-10; 10-20; 20-60; >60. The impact of genotypically-inferred tropism on the time to achieve immunological reconstitution (a CD4 cell count gain from HAART initiation ≥ 150 cells/mm(3)) and on the time to achieve virological success (the first HIV-RNA measurement <50 copies/mL from HAART initiation) was evaluated by survival analyses. Overall, at therapy start, 27% of patients had FPR ≤ 10 (6%, FPR ≤ 2; 7%, FPR 2-5; 14%, FPR 5-10). By 12 months of therapy the rate of immunological reconstitution was overall 75.5%, and it was significantly lower for FPR ≤ 2 (54.1%) in comparison to other FPR ranks (78.8%, FPR 2-5; 77.5%, FPR 5-10; 71.7%, FPR 10-20; 81.8%, FPR 20-60; 75.1%, FPR >60; p = 0.008). The overall proportion of patients achieving virological success was 95.5% by 12 months of therapy. Multivariable Cox analyses showed that patients having pre-HAART FPR ≤ 2% had a significant lower relative adjusted hazard [95% C.I.] both to achieve immunological reconstitution (0.37 [0.20-0.71], p = 0.003) and to achieve virological success (0.50 [0.26-0.94], p = 0.031) than those with pre-HAART FPR >60%. Beyond the genotypically-inferred tropism determination, FPR ≤ 2% predicts both a poor immunological reconstitution and a lower virological response in drug-naïve patients who started their first-line therapy. This parameter could be useful to identify patients potentially with less chance of achieving adequate immunological reconstitution and virological undetectability.

The NLRP3 Inflammasome Promotes Age-related Thymic Demise and Immunosenescence

PubMed Central

Youm, Yun-Hee; Kanneganti, Thirumala-Devi; Vandanmagsar, Bolormaa; Zhu, Xuewei; Ravussin, Anthony; Adijiang, Ayinuer; Owen, John S.; Thomas, Michael J.; Francis, Joseph; Parks, John S.; Dixit, Vishwa Deep

2013-01-01

The collapse of thymic stromal cell microenvironment with age and resultant inability of the thymus to produce naïve T cells contributes to lower immune-surveillance in the elderly. Here we show that age-related increase in ‘lipotoxic danger signals’ such as free cholesterol (FC) and ceramides, leads to thymic caspase-1 activation via the Nlrp3 inflammasome. Elimination of Nlrp3 and Asc, a critical adaptor required for inflammasome assembly, reduces age-related thymic atrophy and results in an increase in cortical thymic epithelial cells, T cell progenitors and maintenance of T cell repertoire diversity. Using a mouse model of irradiation and hematopoietic stem cell transplantation (HSCT), we show that deletion of the Nlrp3 inflammasome accelerates T cell reconstitution and immune recovery in middle-aged animals. Collectively, these data demonstrate that lowering inflammasome-dependent caspase-1 activation increases thymic lymphopoiesis and suggest that Nlrp3 inflammasome inhibitors may aid the reestablishment of a diverse T cell repertoire in middle-aged or elderly patients undergoing HSCT. PMID:22832107

In Vitro Microfluidic Models for Neurodegenerative Disorders.

PubMed

Osaki, Tatsuya; Shin, Yoojin; Sivathanu, Vivek; Campisi, Marco; Kamm, Roger D

2018-01-01

Microfluidic devices enable novel means of emulating neurodegenerative disease pathophysiology in vitro. These organ-on-a-chip systems can potentially reduce animal testing and substitute (or augment) simple 2D culture systems. Reconstituting critical features of neurodegenerative diseases in a biomimetic system using microfluidics can thereby accelerate drug discovery and improve our understanding of the mechanisms of several currently incurable diseases. This review describes latest advances in modeling neurodegenerative diseases in the central nervous system and the peripheral nervous system. First, this study summarizes fundamental advantages of microfluidic devices in the creation of compartmentalized cell culture microenvironments for the co-culture of neurons, glial cells, endothelial cells, and skeletal muscle cells and in their recapitulation of spatiotemporal chemical gradients and mechanical microenvironments. Then, this reviews neurodegenerative-disease-on-a-chip models focusing on Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Finally, this study discusses about current drawbacks of these models and strategies that may overcome them. These organ-on-chip technologies can be useful to be the first line of testing line in drug development and toxicology studies, which can contribute significantly to minimize the phase of animal testing steps. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Meningeal mast cell-T cell crosstalk regulates T cell encephalitogenicity.

PubMed

Russi, Abigail E; Walker-Caulfield, Margaret E; Guo, Yong; Lucchinetti, Claudia F; Brown, Melissa A

2016-09-01

GM-CSF is a cytokine produced by T helper (Th) cells that plays an essential role in orchestrating neuroinflammation in experimental autoimmune encephalomyelitis, a rodent model of multiple sclerosis. Yet where and how Th cells acquire GM-CSF expression is unknown. In this study we identify mast cells in the meninges, tripartite tissues surrounding the brain and spinal cord, as important contributors to antigen-specific Th cell accumulation and GM-CSF expression. In the absence of mast cells, Th cells do not accumulate in the meninges nor produce GM-CSF. Mast cell-T cell co-culture experiments and selective mast cell reconstitution of the meninges of mast cell-deficient mice reveal that resident meningeal mast cells are an early source of caspase-1-dependent IL-1β that licenses Th cells to produce GM-CSF and become encephalitogenic. We also provide evidence of mast cell-T cell co-localization in the meninges and CNS of recently diagnosed acute MS patients indicating similar interactions may occur in human demyelinating disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Reconstituted yogurt from yogurt cultured milk powder mix has better overall characteristics than reconstituted yogurt from commercial yogurt powder.

PubMed

Song, Lijie; Aryana, Kayanush J

2014-10-01

For manufacture of commercial yogurt powder, yogurt has to go through a drying process, which substantially lowers the yogurt culture counts, so the potential health benefits of the yogurt culture bacteria are reduced. Also, upon reconstitution, commercial yogurt powder does not taste like yogurt and has an off-flavor. The objective was to study the microbial, physicochemical, and sensory characteristics of reconstituted yogurt from yogurt cultured milk powder (YCMP) mix and reconstituted yogurt from commercial yogurt powder (CYP). The CYP reconstituted yogurt was the control and YCMP mix reconstituted yogurt was the treatment. Microbial and physicochemical characteristics of the CYP reconstituted yogurt and YCMP mix reconstituted yogurt were analyzed daily for the first week and then weekly for a period of 8 wk. Sensory consumer testing of CYP reconstituted yogurt and YCMP mix reconstituted yogurt was conducted with 100 consumers. At 56 d, YCMP mix reconstituted yogurt had 5 log cfu/mL higher counts of Streptococcus thermophilus than the control (CYP reconstituted yogurt). Also, Lactobacillus bulgaricus counts of YCMP mix reconstituted yogurt were 6.55 log cfu/mL at 28 d and were 5.35 log cfu/mL at 56 d, whereas the CYP reconstituted yogurt from 28 d onwards had a count of <10 cfu/mL. The YCMP mix reconstituted yogurt also had significantly higher apparent viscosity and sensory scores for appearance, color, aroma, taste, thickness, overall liking, consumer acceptability, and purchase intent than CYP reconstituted yogurt. Overall, YCMP mix reconstituted yogurt had more desirable characteristics than CYP reconstituted yogurt. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Amperometric cholesterol biosensor based on in situ reconstituted cholesterol oxidase on an immobilized monolayer of flavin adenine dinucleotide cofactor.

PubMed

Vidal, Juan-C; Espuelas, Javier; Castillo, Juan-R

2004-10-01

A new amperometric biosensor for determining cholesterol based on deflavination of the enzyme cholesterol oxidase (ChOx) and subsequent reconstitution of the apo-protein with a complexed flavin adenine dinucleotide (FAD) monolayer is described. The charge transfer mediator pyrroquinoline quinone (PQQ) was covalently bound to a cystamine self-assembled monolayer (SAM) on an Au electrode. Boronic acid (BA) was then bound to PQQ using the carbodiimide procedure, and the BA ligand was complexed to the FAD molecules on which the apo-ChOx was subsequently reconstituted. The effective release of the FAD from the enzyme and the successful reconstitution were verified using molecular fluorescence and cyclic voltammetry. The optimal orientation of FAD toward the PQQ mediator and the distances between FAD and PQQ and between PQQ and electrode enhance the charge transfer, very high sensitivity (about 2,500 nAmM(-1)cm(-2)) being obtained for cholesterol determination. The biosensor is selective toward electroactive interferents (ascorbic acid and uric acid) and was tested in reference serum samples, demonstrating excellent accuracy (relative errors below 3% in all cases). The biosensor activity can be successfully regenerated in a simple process by successive reconstitution with batches of recently prepared apo-ChOx on the same immobilized Au/SAM-PQQ-BA-FAD monolayer (it was tested five times); the lifetime of the biosensor is about 45-60 days.

Alterations of idiotypic profiles: The cellular basis of T15 dominance in BALB/c mice

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

Wemhoff, G.A.; Quintans, J.

1987-01-01

Phosphorylcholine (PC) is a component of cell walls and membranes from a variety of widely distributed microorganisms. It is highly immunogenic in mice and most murine strains have circulating anti-PC antibodies which are known to confer protection against certain bacterial infections. BALB/c mice offer a striking example of a high responsiveness to PC, a propensity to generate PC-binding myelomas, and a great restriction of idiotype expression in anti-PC antibodies; in fact, most BALB/c anti-PC IgM antibodies express the T15 idiotype marker. Although it has been suspected that T15 dominance is somewhat related to the continuous antigenic load presented by microorganismalmore » flora found in conventional mice, a complete experimental account of how antigenic selection brings about such extreme idiotypic dominance is not yet available. In the studies presented below, we investigated the role played by the host environment, T cells, and antigen in affecting the generation of the anti-PC T15 idiotype profile in lethally irradiated adoptive hosts reconstituted with syngeneic neonatal liver cells. The results presented herein indicate that the transfer of mature carrier-primed T cells with neonatal liver cells does not influence the generation of the T15 idiotype profile. We also demonstrated that anti-T15 idiotype suppressed mice, used as lethally irradiated hosts of immature immunocompetent cells, allow an increased rate of reconstitution of the anti-PC response when compared to nonsuppressed hosts. Since the administration of a T15+ anti-PC antibody inhibits both reconstitution and idiotype expansion, we conclude that T15+ B cells do not self-promote themselves. In contrast, we observed that exposure of adoptive hosts to PC antigens can enhance the anti-PC response and alter the idiotypic profile in favor of T15-bearing clones.« less

Heterogeneous and monoclonal helper T cells induce similar anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody populations in the primary adoptive response. II. Lambda light chain dominance and idiotope expression.

PubMed

Smith, F I; Tesch, H; Rajewsky, K

1984-02-01

When the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) is presented on different carrier molecules, different anti-NP antibody responses are stimulated. On stimulation with NP-lipopolysaccharide (LPS) [T-independent type 1 (TI-1) antigen] kappa + antibodies are the major population, whereas on stimulation with NP-Ficoll [T-independent type 2 (TI-2) antigen], NP-keyhole limpet hemocyanin (KLH) or NP-chicken gamma globulin (CG) [T-dependent (TD) antigens], lambda 1+ antibodies dominate. The relative contribution of idiotopes Ac38 or Ac146 to the lambda 1+ anti-NP response was also different on comparison of TI-1 with TI-2 or TD anti-NP responses. We investigated whether light chain- or idiotype-specific T cells are responsible for these differences. Analysis of the anti-NP response of nude mice after immunization with NP-Ficoll showed lambda 1 dominance. Likewise primary adoptive transfer experiments using carrier-specific T cell lines to reconstitute the TD anti-NP response to NP-KLH or NP-CG, showed that help from carrier-specific T cells alone is capable of stimulating the characteristic lambda 1 dominant response. No significant difference could be found in the levels of Ac38 and Ac146 idiotope expression between mice reconstituted with splenic T cells and those reconstituted with T cell lines. These results suggest that light chain- or idiotype-specific T cells are required neither for the production of lambda 1 light chain dominance, nor for the appearance of idiotopes characteristic of the primary anti-NP response. The possible reasons for differences seen in both light chain and idiotope expression between primary anti-NP responses to the TI-1 antigen NP-LPS and those to TD or TI-2 antigens are discussed.

Fabrication of 3D Reconstituted Organoid Arrays by DNA-programmed Assembly of Cells (DPAC)

PubMed Central

Todhunter, Michael E; Weber, Robert J; Farlow, Justin; Jee, Noel Y; Cerchiari, Alec E; Gartner, Zev J

2016-01-01

Tissues are the organizational units of function in metazoan organisms. Tissues comprise an assortment of cellular building blocks, soluble factors, and extracellular matrix (ECM) that are composed into specific three dimensional (3D) structures. The capacity to reconstitute tissues in vitro with the structural complexity observed in vivo is key to understanding processes such as morphogenesis, homeostasis, and disease. In this unit, we describe DNA-programmed Assembly of Cells (DPAC), a method to fabricate viable, functional arrays of organoid-like tissues within 3D ECM gels. In DPAC, dissociated cells are chemically functionalized with degradable oligonucleotide “velcro,” allowing rapid, specific, and reversible cell adhesion to a two-dimensional (2D) template patterned with complementary DNA. An iterative assembly process builds up organoids, layer-by-layer, from this initial 2D template and into the third dimension. Cleavage of the DNA releases the completed array of tissues that are captured and fully embedded in ECM gels for culture and observation. DPAC controls the size, shape, composition, and spatial heterogeneity of organoids, and permits positioning constituent cells with single-cell resolution even within cultures several centimeters long. PMID:27622567

Enhanced Expansion and Sustained Inductive Function of Skin‐Derived Precursor Cells in Computer‐Controlled Stirred Suspension Bioreactors

PubMed Central

Agabalyan, Natacha A.; Borys, Breanna S.; Sparks, Holly D.; Boon, Kathryn; Raharjo, Eko W.; Abbasi, Sepideh; Kallos, Michael S.

2016-01-01

Abstract Endogenous dermal stem cells (DSCs) reside in the adult hair follicle mesenchyme and can be isolated and grown in vitro as self‐renewing colonies called skin‐derived precursors (SKPs). Following transplantation into skin, SKPs can generate new dermis and reconstitute the dermal papilla and connective tissue sheath, suggesting they could have important therapeutic value for the treatment of skin disease (alopecia) or injury. Controlled cell culture processes must be developed to efficiently and safely generate sufficient stem cell numbers for clinical use. Compared with static culture, stirred‐suspension bioreactors generated fivefold greater expansion of viable SKPs. SKPs from each condition were able to repopulate the dermal stem cell niche within established hair follicles. Both conditions were also capable of inducing de novo hair follicle formation and exhibited bipotency, reconstituting the dermal papilla and connective tissue sheath, although the efficiency was significantly reduced in bioreactor‐expanded SKPs compared with static conditions. We conclude that automated bioreactor processing could be used to efficiently generate large numbers of autologous DSCs while maintaining their inherent regenerative function. Stem Cells Translational Medicine 2017;6:434–443 PMID:28191777

Effects of Combined CCR5/Integrase Inhibitors-Based Regimen on Mucosal Immunity in HIV-Infected Patients Naïve to Antiretroviral Therapy: A Pilot Randomized Trial.

PubMed

Serrano-Villar, Sergio; Sainz, Talia; Ma, Zhong-Min; Utay, Netanya S; Chun, Tae-Wook; Wook-Chun, Tae; Mann, Surinder; Kashuba, Angela D; Siewe, Basile; Albanese, Anthony; Troia-Cancio, Paolo; Sinclair, Elizabeth; Somasunderam, Anoma; Yotter, Tammy; Deeks, Steven G; Landay, Alan; Pollard, Richard B; Miller, Christopher J; Moreno, Santiago; Asmuth, David M

2016-01-01

Whether initiation of antiretroviral therapy (ART) regimens aimed at achieving greater concentrations within gut associated lymphoid tissue (GALT) impacts the level of mucosal immune reconstitution, inflammatory markers and the viral reservoir remains unknown. We included 12 HIV- controls and 32 ART-naïve HIV patients who were randomized to efavirenz, maraviroc or maraviroc+raltegravir, each with fixed-dose tenofovir disoproxil fumarate/emtricitabine. Rectal and duodenal biopsies were obtained at baseline and at 9 months of ART. We performed a comprehensive assay of T-cell subsets by flow cytometry, T-cell density in intestinal biopsies, plasma and tissue concentrations of antiretroviral drugs by high-performance liquid chromatography/mass spectroscopy, and plasma interleukin-6 (IL-6), lipoteichoic acid (LTA), soluble CD14 (sCD14) and zonulin-1 each measured by ELISA. Total cell-associated HIV DNA was measured in PBMC and rectal and duodenal mononuclear cells. Twenty-six HIV-infected patients completed the follow-up. In the duodenum, the quadruple regimen resulted in greater CD8+ T-cell density decline, greater normalization of mucosal CCR5+CD4+ T-cells and increase of the naïve/memory CD8+ T-cell ratio, and a greater decline of sCD14 levels and duodenal HIV DNA levels (P = 0.004 and P = 0.067, respectively), with no changes in HIV RNA in plasma or tissue. Maraviroc showed the highest drug distribution to the gut tissue, and duodenal concentrations correlated well with other T-cell markers in duodenum, i.e., the CD4/CD8 ratio, %CD4+ and %CD8+ HLA-DR+CD38+ T-cells. Maraviroc use elicited greater activation of the mucosal naïve CD8+ T-cell subset, ameliorated the distribution of the CD8+ T-cell maturational subsets and induced higher improvement of zonulin-1 levels. These data suggest that combined CCR5 and integrase inhibitor based combination therapy in ART treatment naïve patients might more effectively reconstitute duodenal immunity, decrease inflammatory markers and impact on HIV persistence by cell-dependent mechanisms, and show unique effects of MVC in duodenal immunity driven by higher drug tissue penetration and possibly by class-dependent effects.

Mitochondrial Ca2+ influx targets cardiolipin to disintegrate respiratory chain complex II for cell death induction

PubMed Central

Hwang, M-S; Schwall, C T; Pazarentzos, E; Datler, C; Alder, N N; Grimm, S

2014-01-01

Massive Ca2+ influx into mitochondria is critically involved in cell death induction but it is unknown how this activates the organelle for cell destruction. Using multiple approaches including subcellular fractionation, FRET in intact cells, and in vitro reconstitutions, we show that mitochondrial Ca2+ influx prompts complex II of the respiratory chain to disintegrate, thereby releasing an enzymatically competent sub-complex that generates excessive reactive oxygen species (ROS) for cell death induction. This Ca2+-dependent dissociation of complex II is also observed in model membrane systems, but not when cardiolipin is replaced with a lipid devoid of Ca2+ binding. Cardiolipin is known to associate with complex II and upon Ca2+ binding coalesces into separate homotypic clusters. When complex II is deprived of this lipid, it disintegrates for ROS formation and cell death. Our results reveal Ca2+ binding to cardiolipin for complex II disintegration as a pivotal step for oxidative stress and cell death induction. PMID:24948011

Immune reconstitution inflammatory syndrome in HIV and sporotrichosis coinfection: report of two cases and review of the literature.

PubMed

Lyra, Marcelo Rosandiski; Nascimento, Maria Letícia Fernandes Oliveira; Varon, Andréa Gina; Pimentel, Maria Inês Fernandes; Antonio, Liliane de Fátima; Saheki, Maurício Naoto; Bedoya-Pacheco, Sandro Javier; Valle, Antonio Carlos Francesconi do

2014-01-01

We report 2 cases of patients with immune reconstitution inflammatory syndrome (IRIS) associated with cutaneous disseminated sporotrichosis and human immunodeficiency virus (HIV) coinfection. The patients received specific treatment for sporotrichosis. However, after 4 and 5 weeks from the beginning of antiretroviral therapy, both patients experienced clinical exacerbation of skin lesions despite increased T CD4+ cells (T cells cluster of differentiation 4 positive) count and decreased viral load. Despite this exacerbation, subsequent mycological examination after systemic corticosteroid administration did not reveal fungal growth. Accordingly, they were diagnosed with IRIS. However, the sudden withdrawal of the corticosteroids resulted in the recurrence of IRIS symptoms. No serious adverse effects could be attributed to prednisone. We recommend corticosteroid treatment for mild-to-moderate cases of IRIS in sporotrichosis and HIV coinfection with close follow-up.

Reconstituted AIM2 inflammasome in cell-free system.

PubMed

Kaneko, Naoe; Ito, Yuki; Iwasaki, Tomoyuki; Takeda, Hiroyuki; Sawasaki, Tatsuya; Migita, Kiyoshi; Agematsu, Kazunaga; Kawakami, Atsushi; Morikawa, Shinnosuke; Mokuda, Sho; Kurata, Mie; Masumoto, Junya

2015-11-01

Absent in melanoma 2 (AIM2) is an intracellular pattern-recognition receptor, which is a member of the PYHIN protein family, consisting of a PYD domain and an IFN-inducible nuclear localization (HIN) domain. AIM2 is reported to oligomerize with adaptor protein ASC upon sensing bacterial and viral cytosolic DNA in order to form the AIM2 inflammasome, which activates caspase-1 leading to IL-1β secretion. Dysregulation of AIM2 inflammasome is supposed to result in autoinflammatory and autoimmune diseases. Thus, the development of new targeted drugs against AIM2 inflammasome would be important for the treatment of these diseases. However, since AIM2 inflammasome is an intracellular receptor, enforced internalization of both ligands and candidate molecules is necessary for the screening of AIM2-inflammasome-targeted molecules. We developed a reconstituted AIM2 inflammasome in a cell-free system with amplified luminescent proximity homogeneous assay (Alpha). Strong Alpha signal was detected upon incubation with poly-deoxyadenylic-deoxythymidylic acid, poly(dA:dT), whereas no Alpha signal was detected upon incubation with muramyl dipeptide, one of the NLR ligands of Nod2 ligand. The interaction between AIM2 and ASC was disrupted by an anti-human ASC monoclonal antibody, CRID3, a class of diarylsulfonylurea-containing compounds, and glycyrrhizin, a substance found in liquorice root. Thus, the reconstituted AIM2 inflammasome in a cell-free system is useful for screening AIM2-inflammasome-targeted therapeutic molecules. Copyright © 2015 Elsevier B.V. All rights reserved.

Oncogenic Nras has bimodal effects on stem cells that sustainably increase competitiveness.

PubMed

Li, Qing; Bohin, Natacha; Wen, Tiffany; Ng, Victor; Magee, Jeffrey; Chen, Shann-Ching; Shannon, Kevin; Morrison, Sean J

2013-12-05

'Pre-leukaemic' mutations are thought to promote clonal expansion of haematopoietic stem cells (HSCs) by increasing self-renewal and competitiveness; however, mutations that increase HSC proliferation tend to reduce competitiveness and self-renewal potential, raising the question of how a mutant HSC can sustainably outcompete wild-type HSCs. Activating mutations in NRAS are prevalent in human myeloproliferative neoplasms and leukaemia. Here we show that a single allele of oncogenic Nras(G12D) increases HSC proliferation but also increases reconstituting and self-renewal potential upon serial transplantation in irradiated mice, all prior to leukaemia initiation. Nras(G12D) also confers long-term self-renewal potential to multipotent progenitors. To explore the mechanism by which Nras(G12D) promotes HSC proliferation and self-renewal, we assessed cell-cycle kinetics using H2B-GFP label retention and 5-bromodeoxyuridine (BrdU) incorporation. Nras(G12D) had a bimodal effect on HSCs, increasing the frequency with which some HSCs divide and reducing the frequency with which others divide. This mirrored bimodal effects on reconstituting potential, as rarely dividing Nras(G12D) HSCs outcompeted wild-type HSCs, whereas frequently dividing Nras(G12D) HSCs did not. Nras(G12D) caused these effects by promoting STAT5 signalling, inducing different transcriptional responses in different subsets of HSCs. One signal can therefore increase HSC proliferation, competitiveness and self-renewal through bimodal effects on HSC gene expression, cycling and reconstituting potential.

Bone marrow-derived SP cells can contribute to the respiratory tract of mice in vivo.

PubMed

Macpherson, Heather; Keir, Pamela; Webb, Sheila; Samuel, Kay; Boyle, Shelagh; Bickmore, Wendy; Forrester, Lesley; Dorin, Julia

2005-06-01

Recent work has indicated that adult bone marrow-derived cells have the ability to contribute to both the haematopoietic system and other organs. Haematopoietic reconstitution by whole bone marrow and selected but not fully characterised cell populations have resulted in reports indicating high-level repopulation of lung epithelia. The well-characterised cells from the side population have a robust ability for haematopoietic reconstitution. We have used freshly isolated side population cells derived from ROSA26 adult bone marrow and demonstrate that despite being unable to contribute to embryos following blastocyst injection, or air liquid interface cultures or denuded tracheal xenografts, they could contribute to the tracheal epithelium in vivo. Epithelial damage is reported to be important in encouraging the recruitment of marrow-derived stem cells into non-haematopoietic organs. Here we demonstrate that mice engrafted with side population cells have donor-derived cells present in the epithelial lining of the trachea following damage and repair. Donor-derived cells were found at a frequency of 0.83%. Widefield and confocal microscopy revealed donor cells that expressed cytokeratins, indicative of cells of an epithelial nature. These results imply that SP haematopoietic stem cells from the bone marrow do not have the ability to contribute to airway epithelia themselves but require factors present in vivo to allow them to acquire characteristics of this tissue.

Secretion of Hepatitis C Virus Replication Intermediates Reduces Activation of Toll-Like Receptor 3 in Hepatocytes.

PubMed

Grünvogel, Oliver; Colasanti, Ombretta; Lee, Ji-Young; Klöss, Volker; Belouzard, Sandrine; Reustle, Anna; Esser-Nobis, Katharina; Hesebeck-Brinckmann, Jasper; Mutz, Pascal; Hoffmann, Katrin; Mehrabi, Arianeb; Koschny, Ronald; Vondran, Florian W R; Gotthardt, Daniel; Schnitzler, Paul; Neumann-Haefelin, Christoph; Thimme, Robert; Binder, Marco; Bartenschlager, Ralf; Dubuisson, Jean; Dalpke, Alexander H; Lohmann, Volker

2018-06-01

Hepatitis C virus (HCV) infections most often result in chronic outcomes, although the virus constantly produces replication intermediates, in particular double-stranded RNA (dsRNA), representing potent inducers of innate immunity. We aimed to characterize the fate of HCV dsRNA in hepatocyte cultures to identify mechanisms contributing to viral persistence in presence of an active innate immune response. We analyzed hepatocyte-based culture models for HCV for induction of innate immunity, secretion of virus positive- or negative-strand RNA, and viral replication using different quantification methods and microscopy techniques. Expression of pattern recognition receptors was reconstituted in hepatoma cells by lentiviral transduction. HCV-infected cells secrete substantial amounts of virus positive- and negative-strand RNAs in extracellular vesicles (EVs), toward the apical and basolateral domain of hepatocytes. Secretion of negative-strand RNA was independent from virus production, and viral RNA secreted in EVs contained higher relative amounts of negative-strands, indicating that mostly virus dsRNA is released. A substantial part of viral replication complexes and dsRNA was found in the endosomal compartment and multivesicular bodies, indicating that secretion of HCV replication intermediates is mediated by the exosomal pathway. Block of vesicle release in HCV-positive cells increased intracellular dsRNA levels and increased activation of toll-like receptor 3, inhibiting HCV replication. Using hepatocyte-based culture models for HCV, we found a portion of HCV dsRNA intermediates to be released from infected cells in EVs, which reduces activation of toll-like receptor 3. This represents a novel mechanism how HCV evades host immune responses, potentially contributing to viral persistence. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Plasmacytoid predendritic cells initiate psoriasis through interferon-α production

PubMed Central

Nestle, Frank O.; Conrad, Curdin; Tun-Kyi, Adrian; Homey, Bernhard; Gombert, Michael; Boyman, Onur; Burg, Günter; Liu, Yong-Jun; Gilliet, Michel

2005-01-01

Psoriasis is one of the most common T cell–mediated autoimmune diseases in humans. Although a role for the innate immune system in driving the autoimmune T cell cascade has been proposed, its nature remains elusive. We show that plasmacytoid predendritic cells (PDCs), the natural interferon (IFN)-α–producing cells, infiltrate the skin of psoriatic patients and become activated to produce IFN-α early during disease formation. In a xenograft model of human psoriasis, we demonstrate that blocking IFN-α signaling or inhibiting the ability of PDCs to produce IFN-α prevented the T cell–dependent development of psoriasis. Furthermore, IFN-α reconstitution experiments demonstrated that PDC-derived IFN-α is essential to drive the development of psoriasis in vivo. These findings uncover a novel innate immune pathway for triggering a common human autoimmune disease and suggest that PDCs and PDC-derived IFN-α represent potential early targets for the treatment of psoriasis. PMID:15998792

Respiratory syncytial virus subunit vaccine based on a recombinant fusion protein expressed transiently in mammalian cells.

PubMed

Nallet, Sophie; Amacker, Mario; Westerfeld, Nicole; Baldi, Lucia; König, Iwo; Hacker, David L; Zaborosch, Christiane; Zurbriggen, Rinaldo; Wurm, Florian M

2009-10-30

Although respiratory syncytial virus (RSV) causes severe lower respiratory tract infection in infants and adults at risk, no RSV vaccine is currently available. In this report, efforts toward the generation of an RSV subunit vaccine using recombinant RSV fusion protein (rRSV-F) are described. The recombinant protein was produced by transient gene expression (TGE) in suspension-adapted human embryonic kidney cells (HEK-293E) in 4 L orbitally shaken bioreactors. It was then purified and formulated in immunostimulating reconstituted influenza virosomes (IRIVs). The candidate vaccine induced anti-RSV-F neutralizing antibodies in mice, and challenge studies in cotton rats are ongoing. If successful in preclinical and clinical trials, this will be the first recombinant subunit vaccine produced by large-scale TGE in mammalian cells.

WHEAT GERM CELL-FREE TRANSLATION, PURIFICATION, AND ASSEMBLY OF A FUNCTIONAL HUMAN STEAROYL-COA DESATURASE COMPLEX

PubMed Central

Goren, Michael A.; Fox, Brian G.

2008-01-01

A wheat germ cell-free extract was used to perform in vitro translation of human stearoyl-CoA desaturase in the presence of unilamelar liposomes, and near complete transfer of the expressed integral membrane protein into the liposome was observed. Moreover, co-translation of the desaturase along with human cytochrome b5 led to transfer of both membrane proteins into the liposomes. A simple, single step purification via centrifugation in a density gradient yielded proteoliposomes with the desaturase in high purity as judged by capillary electrophoresis. After in vitro reconstitution of the non-heme iron and heme active sites, the function of the reconstituted enzyme complex was demonstrated by conversion of stearoyl-CoA to oleoyl-CoA. This simple translation approach obviates the use of detergents or other lipids to stabilize and isolate a catalytically active integral membrane enzyme. The applicability of cell-free translation to the assembly and purification of other integral membrane protein complexes is discussed. PMID:18765284

Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex.

PubMed

Goren, Michael A; Fox, Brian G

2008-12-01

A wheat germ cell-free extract was used to perform in vitro translation of human stearoyl-CoA desaturase in the presence of unilamelar liposomes, and near complete transfer of the expressed integral membrane protein into the liposome was observed. Moreover, co-translation of the desaturase along with human cytochrome b(5) led to transfer of both membrane proteins into the liposomes. A simple, single step purification via centrifugation in a density gradient yielded proteoliposomes with the desaturase in high purity as judged by capillary electrophoresis. After in vitro reconstitution of the non-heme iron and heme active sites, the function of the reconstituted enzyme complex was demonstrated by conversion of stearoyl-CoA to oleoyl-CoA. This simple translation approach obviates the use of detergents or other lipids to stabilize and isolate a catalytically active integral membrane enzyme. The applicability of cell-free translation to the assembly and purification of other integral membrane protein complexes is discussed.

One-way membrane trafficking of SOS in receptor-triggered Ras activation.

PubMed

Christensen, Sune M; Tu, Hsiung-Lin; Jun, Jesse E; Alvarez, Steven; Triplet, Meredith G; Iwig, Jeffrey S; Yadav, Kamlesh K; Bar-Sagi, Dafna; Roose, Jeroen P; Groves, Jay T

2016-09-01

SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2-SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted-membrane experiments, these Grb2-independent interactions were sufficient to retain human SOS on the membrane for many minutes, during which a single SOS molecule could processively activate thousands of Ras molecules. These observations raised questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative assays of reconstituted SOS-deficient chicken B-cell signaling systems combined with single-molecule measurements in supported membranes. These studies revealed an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until being actively removed via endocytosis.

One-way membrane trafficking of SOS in receptor-triggered Ras activation

PubMed Central

Christensen, Sune M.; Tu, Hsiung-Lin; Jun, Jesse E.; Alvarez, Steven; Triplet, Meredith G.; Iwig, Jeffrey S.; Yadav, Kamlesh K.; Bar-Sagi, Dafna; Roose, Jeroen P.; Groves, Jay T.

2016-01-01

SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane-recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2:SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted membrane experiments, these Grb2-independent interactions are sufficient to retain SOS on the membrane for many minutes, during which a single SOS molecule can processively activate thousands of Ras molecules. These observations raise questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative reconstituted SOS-deficient chicken B cell signaling systems combined with single molecule measurements in supported membranes. These studies reveal an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until it is actively removed via endocytosis. PMID:27501536

MiR-29b antagonizes the pro-inflammatory tumor-promoting activity of multiple myeloma-educated dendritic cells

PubMed Central

Botta, C; Cucè, M; Pitari, M R; Caracciolo, D; Gullà, A; Morelli, E; Riillo, C; Biamonte, L; Gallo Cantafio, M E; Prabhala, R; Mignogna, C; Di Vito, A; Altomare, E; Amodio, N; Di Martino, M T; Correale, P; Rossi, M; Giordano, A; Munshi, N C; Tagliaferri, P; Tassone, P

2018-01-01

Dendritic cells (DCs) have a key role in regulating tumor immunity, tumor cell growth and drug resistance. We hypothesized that multiple myeloma (MM) cells might recruit and reprogram DCs to a tumor-permissive phenotype by changes within their microRNA (miRNA) network. By analyzing six different miRNA-profiling data sets, miR-29b was identified as the only miRNA upregulated in normal mature DCs and significantly downregulated in tumor-associated DCs. This finding was validated in primary DCs co-cultured in vitro with MM cell lines and in primary bone marrow DCs from MM patients. In DCs co-cultured with MM cells, enforced expression of miR-29b counteracted pro-inflammatory pathways, including signal transducer and activator of transcription 3 and nuclear factor-κB, and cytokine/chemokine signaling networks, which correlated with patients’ adverse prognosis and development of bone disease. Moreover, miR-29b downregulated interleukin-23 in vitro and in the SCID-synth-hu in vivo model, and antagonized a Th17 inflammatory response. All together, these effects translated into strong anti-proliferative activity and reduction of genomic instability of MM cells. Our study demonstrates that MM reprograms the DCs functional phenotype by downregulating miR-29b whose reconstitution impairs DCs ability to sustain MM cell growth and survival. These results underscore miR-29b as an innovative and attractive candidate for miRNA-based immune therapy of MM. PMID:29158557

Over-expression in Escherichia coli, purification and reconstitution in liposomes of the third member of the OCTN sub-family: The mouse carnitine transporter OCTN3

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

Scalise, Mariafrancesca; Galluccio, Michele; Pochini, Lorena

2012-05-25

Highlights: Black-Right-Pointing-Pointer mOCTN3 transport protein has been cloned in pET-21a(+) and over-expressed in Escherichia coli. Black-Right-Pointing-Pointer The expressed mOCTN3 has been purified to homogeneity by Ni-chelating chromatography. Black-Right-Pointing-Pointer The protein solubilised in Triton X-100 has been reconstituted in liposomes. Black-Right-Pointing-Pointer Recombinant mOCTN3 catalyses transport of carnitine by a uniport mode. -- Abstract: pET-21a(+)-mOCTN3-6His was constructed and used for over-expression in Escherichia coli Rosetta(DE3)pLysS. After IPTG induction a protein with apparent molecular mass of 53 kDa was collected in the insoluble fraction of the cell lysate and purified by Ni{sup 2+}-chelating chromatography with a yield of 2 mg/l of cell culture.more » The over-expressed protein was identified with mOCTN3 by anti-His antibody and reconstitution in liposomes. mOCTN3 required peculiar conditions for optimal expression and reconstitution in liposomes. The protein catalyzed a time dependent [{sup 3}H]carnitine uptake which was stimulated by intraliposomal ATP and nearly independent of the pH. The K{sub m} for carnitine was 36 {mu}M. [{sup 3}H]carnitine transport was inhibited by carnitine analogues and some Cys and NH{sub 2} reagents. This paper represents the first outcome in over-expressing, in active form, the third member of the OCTN sub-family, mOCTN3, in E. coli.« less

Successful control of Epstein-Barr virus (EBV)-infected cells by allogeneic nonmyeloablative stem cell transplantation in a patient with the lethal form of chronic active EBV infection.

PubMed

Uehara, Taeko; Nakaseko, Chiaki; Hara, Satoru; Harima, Akane; Ejiri, Megumi; Yokota, Akira; Saito, Yasushi; Nishimura, Miki

2004-08-01

Chronic active Epstein-Barr virus infection (CAEBV) is a heterogeneous EBV-related disorder, ranging from mild/moderate forms to rapidly lethal disorders. The lethal form of CAEBV is characterized by multiple organ failure, hemophagocytic syndrome, and development of lymphomas. Allogeneic stem cell transplantation is considered as the only potentially curative treatment for the lethal form of CAEBV, but it is not always desirable because of the high incidence of regimen-related toxicities. A 17-year-old female with CAEBV, who was refractory to conventional therapies and considered to be unable to receive a myeloablative regimen because of multiple organ dysfunction, underwent allogeneic nonmyeloablative stem cell transplantation (allo-NST) before developing a hematological malignancy. She has been well without any signs of CAEBV for 27 months after allo-NST, and we confirmed that specific cytotoxic T lymphocyte activity against EBV was reconstituted. This outcome suggests that allo-NST can control CAEBV by reconstituting the host immunity against EBV. Copyright 2004 Wiley-Liss, Inc.

Schinus terebinthifolius Raddi extract and linoleic acid from Passiflora edulis synergistically decrease melanin synthesis in B16 cells and reconstituted epidermis.

PubMed

Jorge, A T S; Arroteia, K F; Santos, I A; Andres, E; Medina, S P H; Ferrari, C R; Lourenço, C B; Biaggio, R M T T; Moreira, P L

2012-10-01

Several treatments for skin whitening are available today, but few of them are completely adequate, especially owing to the carcinogenic potential attributed to classical drugs like hydroquinone, arbutin and kojic acid. To provide an alternative and safer technology for whitening, we developed two botanical compounds originated from Brazilian biodiversity, an extract of Schinus terebinthifolius Raddi and a linoleic acid fraction isolated from Passiflora edulis oil. The whitening effect of these compounds was assessed using biochemical assays and in vitro models including cellular assays and equivalent skin. The results showed that S. terebinthifolius Raddi extract is able to reduce the tyrosinase activity in vitro, and the combination of this extract with linoleic acid is able to decrease the level of melanin produced by B16 cells cultured with melanocyte-stimulating hormone. Furthermore, melanin was also reduced in human reconstituted epidermis (containing melanocytes) treated with the compounds. The combination of the compounds may provide a synergistic positive whitening effect rather than their isolated use. Finally, we demonstrated that the performance of these mixed compounds is comparable to classical molecules used for skin whitening, as kojic acid. This new natural mixture could be considered an alternative therapeutic agent for treating hyperpigmentation and an effective component in whitening cosmetics. © 2012 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

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

Defresne, M.P.; Greimers, R.; Lenaerts, P.

A split-dose regimen of whole-body irradiation (4 X 175 rad at weekly intervals) induced thymic lymphomas in C57BL/Ka mice after a latent period of 3-9 months. Meanwhile, preleukemia cells arose in the thymus and bone marrow and persisted until the onset of lymphomas. Simultaneously, thymic lymphopoiesis was impaired; thymocyte numbers were subnormal and thymic nurse cells disappeared in a progressive but irreversible fashion. The depletion of these lymphoepithelial complexes, which are normally involved in the early steps of thymic lymphopoiesis, was related to altered prothymocyte activity in bone marrow and to damaged thymic microenvironment, perhaps as a consequence of themore » presence of preleukemia cells. The grafting of normal bone marrow cells after irradiation prevented the development of lymphomas. However, marrow reconstitution did not inhibit the induction of preleukemia cells. They disappeared from the thymus during the second part of the latent period. At the same time, thymic lymphopoiesis was restored; thymocytes and nurse cell numbers returned to normal as a consequence of the proliferation of grafted marrow-derived cells within the thymus. The results thus demonstrated an intimate relationship between preleukemia cells and an alteration of thymic lymphopoiesis, which particularly involved the nurse cell microenvironment. Some preleukemia cells in marrow-reconstituted, irradiated mice derived from the unirradiated marrow inoculate. Thus these cells acquired neoplastic potential through a factor present in the irradiated tissues. The nature of this indirect mechanism was briefly discussed.« less

Reconstitution of lymphocyte subpopulations after hematopoietic stem cell transplantation: comparison of hematologic malignancies and donor types in event-free patients.

PubMed

Park, Borae G; Park, Chan-Jeoung; Jang, Seongsoo; Chi, Hyun-Sook; Kim, Dae-Young; Lee, Jung-Hee; Lee, Je-Hwan; Lee, Kyoo-Hyung

2015-12-01

The reconstitution of different immunocyte subsets after hematopoietic stem cell transplantation (HSCT), follows different timelines. We prospectively investigated changes in lymphocyte subsets after HSCT and their associations with primary diagnosis, conditioning regimen, and HSCT type in event-free patients. A total of 95 patients (48 with acute myeloid leukemia, 22 with acute lymphoid leukemia, and 25 with myelodysplastic syndrome) who underwent allogeneic HSCT (34 sibling matched, 37 unrelated matched, and 24 haploidentical HSCT) but did not experience any events such as relapse or death were enrolled in this study. Lymphocyte subpopulations (T cells, helper/inducer T cells, cytotoxic/suppressor T cells, memory T cells, regulatory T cells, natural killer (NK) cells, NK-T cells, and B cells) were quantified by flow cytometry of peripheral blood from recipients 7 days before and 1, 2, 3, 6, and 12 months after HSCT. Leukocyte counts recovered within 1 month after HSCT. However, the number of T and B lymphocytes recovered at 2 months after HSCT. NK cell counts recovered shortly after haploidentical HSCT. However, T lymphocytes and their subpopulations showed delayed recovery after haploidentical HSCT. Lymphocyte subsets showed different sequential patterns according to HSCT type but no differences were seen according to primary diagnosis or conditioning regimen. Copyright © 2015 Elsevier Ltd. All rights reserved.

Human natural killer cells prevent infectious mononucleosis features by targeting lytic Epstein-Barr virus infection.

PubMed

Chijioke, Obinna; Müller, Anne; Feederle, Regina; Barros, Mario Henrique M; Krieg, Carsten; Emmel, Vanessa; Marcenaro, Emanuela; Leung, Carol S; Antsiferova, Olga; Landtwing, Vanessa; Bossart, Walter; Moretta, Alessandro; Hassan, Rocio; Boyman, Onur; Niedobitek, Gerald; Delecluse, Henri-Jacques; Capaul, Riccarda; Münz, Christian

2013-12-26

Primary infection with the human oncogenic Epstein-Barr virus (EBV) can result in infectious mononucleosis (IM), a self-limiting disease caused by massive lymphocyte expansion that predisposes for the development of distinct EBV-associated lymphomas. Why some individuals experience this symptomatic primary EBV infection, whereas the majority acquires the virus asymptomatically, remains unclear. Using a mouse model with reconstituted human immune system components, we show that depletion of human natural killer (NK) cells enhances IM symptoms and promotes EBV-associated tumorigenesis mainly because of a loss of immune control over lytic EBV infection. These data suggest that failure of innate immune control by human NK cells augments symptomatic lytic EBV infection, which drives lymphocyte expansion and predisposes for EBV-associated malignancies. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

In Vivo RNA Interference Screening Identifies a Leukemia-Specific Dependence on Integrin Beta 3 Signaling

PubMed Central

Miller, Peter G.; Al-Shahrour, Fatima; Hartwell, Kimberly A.; Chu, Lisa P.; Järås, Marcus; Puram, Rishi V.; Puissant, Alexandre; Callahan, Kevin P.; Ashton, John; McConkey, Marie E.; Poveromo, Luke P.; Cowley, Glenn S.; Kharas, Michael G.; Labelle, Myriam; Shterental, Sebastian; Fujisaki, Joji; Silberstein, Lev; Alexe, Gabriela; Al-Hajj, Muhammad A.; Shelton, Christopher A.; Armstrong, Scott A.; Root, David E.; Scadden, David T.; Hynes, Richard O.; Mukherjee, Siddhartha; Stegmaier, Kimberly; Jordan, Craig T.; Ebert, Benjamin L.

2013-01-01

SUMMARY We used an in vivo short hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo, and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase, Syk. In contrast, loss of Itgb3 in normal HSPCs did not affect engraftment, reconstitution, or differentiation. Finally, we confirmed that Itgb3 is dispensable for normal hematopoiesis and required for leukemogenesis using an Itgb3 knockout mouse model. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML. PMID:23770013

Understanding the reconstitution of the B-cell compartment in bone marrow and blood after treatment for B-cell precursor acute lymphoblastic leukaemia.

PubMed

Theunissen, Prisca M J; van den Branden, Anouk; Van Der Sluijs-Gelling, Alita; De Haas, Valerie; Beishuizen, Auke; van Dongen, Jacques J M; Van Der Velden, Vincent H J

2017-07-01

A better understanding of the reconstitution of the B-cell compartment during and after treatment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) will help to assess the immunological status and needs of post-treatment BCP-ALL patients. Using 8-colour flow cytometry and proliferation-assays, we studied the composition and proliferation of both the B-cell precursor (BCP) population in the bone marrow (BM) and mature B-cell population in peripheral blood (PB) during and after BCP-ALL therapy. We found a normal BCP differentiation pattern and a delayed formation of classical CD38 dim -naive mature B-cells, natural effector B-cells and memory B-cells in patients after chemotherapy. This B-cell differentiation/maturation pattern was strikingly similar to that during initial B-cell development in healthy infants. Tissue-resident plasma cells appeared to be partly protected from chemotherapy. Also, we found that the fast recovery of naive mature B-cell numbers after chemotherapy was the result of increased de novo BCP generation, rather than enhanced B-cell proliferation in BM or PB. These results indicate that post-treatment BCP-ALL patients will eventually re-establish a B-cell compartment with a composition and B-cell receptor repertoire similar to that in healthy children. Additionally, the formation of a new memory B-cell compartment suggests that revaccination might be beneficial after BCP-ALL therapy. © 2017 John Wiley & Sons Ltd.

Isolation, Characterization, and Transplantation of Bone Marrow-Derived Cell Components with Hematopoietic Stem Cell Niche Properties

PubMed Central

Ahmadbeigi, Naser; Vasei, Mohammad; Gheisari, Yousof; Mortazavi, Yousef; Azadmanesh, Kayhan; Omidkhoda, Azadeh; Janzamin, Ehsan; Nardi, Nance Beyer

2013-01-01

Although the unique role of hematopoietic stem cell (HSC) niche in hematopoiesis has long been recognized, unsuccessful isolation of intact niche units limited their in vitro study, manipulation, and therapeutic application. Here, we isolated cell complexes based on size fractionation from mouse bone marrow (BM), characterized the derived cells, and transplanted them to irradiated mice. These cell complexes were the origin of both BM mesenchymal stem cells and various hematopoietic lineages when kept in appropriate culture conditions. They also had the potential of recruiting circulating HSC. Intraperitoneal transplantation of these structures into irradiated mice not only showed long-lasting hematopoietic multilineage reconstitution, but also could recover the stromal cells of BM. In conclusion, this study for the first time provides evidences on the feasibility and efficacy of transplantation of HSC in association with their native specialized microenvironment. As the molecular cross-talk between HSC and niche is crucial for their proper function, the proposed method could be considered as a novel hematopoietic transplantation strategy. PMID:23879861

Reconstituted human gingival epithelium: nonsubmerged in vitro model.

PubMed

Delcourt-Huard, A; Corlu, A; Joffre, A; Magloire, H; Bonnaure-Mallet, M

1997-01-01

Many studies have shown that human gingival keratinocytes grown in submerged culture fail to attain optimal differentiation. This study reports an in vitro culture system for oral gingival epithelial cells, in which they are grown at the air-liquid interface, on polycarbonate inserts, in the presence of an NIH-3T3 feeder layer. This model was compared with two submerged culture methods for gingival keratinocytes, on type 1 collagen gel and on an NIH-3T3 feeder layer. Transmission electron microscopy showed an advanced level of stratification (over six layers of cells) for cultures grown at the air-liquid interface. Immunofluorescence and electrophoretic patterns showed the presence of cytokeratins 10 and 11 in cytoskeletal protein extracts of these cultured keratinocytes. In this air-liquid interface culture model, in the presence of NIH-3T3 feeder cells, keratinocytes can achieve an advanced level of stratification and differentiation and a resemblance to in vivo gingiva. The obtention of a highly differentiated epithelium will permit in vitro pharmacological studies and studies on the biocompatability of certain alloys with the superficial periodontium; it will also provide grafts for patients undergoing periodontal surgery.

Macrophages sustain HIV replication in vivo independently of T cells.

PubMed

Honeycutt, Jenna B; Wahl, Angela; Baker, Caroline; Spagnuolo, Rae Ann; Foster, John; Zakharova, Oksana; Wietgrefe, Stephen; Caro-Vegas, Carolina; Madden, Victoria; Sharpe, Garrett; Haase, Ashley T; Eron, Joseph J; Garcia, J Victor

2016-04-01

Macrophages have long been considered to contribute to HIV infection of the CNS; however, a recent study has contradicted this early work and suggests that myeloid cells are not an in vivo source of virus production. Here, we addressed the role of macrophages in HIV infection by first analyzing monocytes isolated from viremic patients and patients undergoing antiretroviral treatment. We were unable to find viral DNA or viral outgrowth in monocytes isolated from peripheral blood. To determine whether tissue macrophages are productively infected, we used 3 different but complementary humanized mouse models. Two of these models (bone marrow/liver/thymus [BLT] mice and T cell-only mice [ToM]) have been previously described, and the third model was generated by reconstituting immunodeficient mice with human CD34+ hematopoietic stem cells that were devoid of human T cells (myeloid-only mice [MoM]) to specifically evaluate HIV replication in this population. Using MoM, we demonstrated that macrophages can sustain HIV replication in the absence of T cells; HIV-infected macrophages are distributed in various tissues including the brain; replication-competent virus can be rescued ex vivo from infected macrophages; and infected macrophages can establish de novo infection. Together, these results demonstrate that macrophages represent a genuine target for HIV infection in vivo that can sustain and transmit infection.

Functional reconstitution of the voltage-regulated sodium channel purified from electroplax of Electrophorus electricus

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

Rosenberg, R.L.

1985-01-01

The voltage-regulated NA channel is responsible for the depolarization of the excitable cell membrane during the normal action potential. This research has focused on the functional properties of the Na channel, purified from detergent extracts of electroplax membranes of the electric eel, and reconstituted into vesicles of defined phospholipid. These properties were assessed by measuring neurotoxin-modulated ion flux into the reconstituted membrane vesicles and by recording the single-channel currents of the purified channel by the patch-clamp method. The binding of tritiated tetrodotoxin (TTX) was employed as a marker for the purification of the channel. Two high-resolution fractionation steps, based onmore » molecular charge and protein size, were used to obtain a preparation that is 80% homogeneous for a large peptide of 270,000 daltons. Radiotracer /sup 22/Na/sup +/ influx into the vesicles was stimulated by veratridine and by batrachotoxin (BTX) at concentrations of 100 ..mu..M and 5 ..mu..M, respectively. The stimulation by BTX was greater than that by veratridine, and can be as much as 16-fold over control influx levels. The stimulated influx is blocked by TTX with a K/sub i/ of 35 nM, and by local anesthetics in the normal pharmacological range. Large multilamellar vesicles prepared with a freeze-thaw step are suitable for single-channel recording techniques. When excised patches of the reconstituted membranes were voltage-clamped in the absence of activating neurotoxins, voltage-dependent single-channel currents were recorded. These displayed properties similar to those from native membranes of nerve and muscle. These results indicate that the protein purified on the basis of TTX binding is a functional Na channel possessing these functional domains: the ion-selective channel, the voltage sensors controlling activation and inactivation, and the sites of action of TTX, alkaloid neurotoxins, and local anesthetics.« less

Defective homing is associated with altered Cdc42 activity in cells from patients with Fanconi anemia group A

PubMed Central

Zhang, Xiaoling; Shang, Xun; Guo, Fukun; Murphy, Kim; Kirby, Michelle; Kelly, Patrick; Reeves, Lilith; Smith, Franklin O.; Williams, David A.

2008-01-01

Previous studies showed that Fanconi anemia (FA) murine stem cells have defective reconstitution after bone marrow (BM) transplantation. The mechanism underlying this defect is not known. Here, we report defective homing of FA patient BM progenitors transplanted into mouse models. Using cells from patients carrying mutations in FA complementation group A (FA-A), we show that when transplanted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) recipient mice, FA-A BM cells exhibited impaired homing activity. FA-A cells also showed defects in both cell-cell and cell-matrix adhesion. Complementation of FA-A deficiency by reexpression of FANCA readily restored adhesion of FA-A cells. A significant decrease in the activity of the Rho GTPase Cdc42 was found associated with these defective functions in patient-derived cells, and expression of a constitutively active Cdc42 mutant was able to rescue the adhesion defect of FA-A cells. These results provide the first evidence that FA proteins influence human BM progenitor homing and adhesion via the small GTPase Cdc42-regulated signaling pathway. PMID:18565850

Size-dependent protein segregation at membrane interfaces

NASA Astrophysics Data System (ADS)

Schmid, Eva M.; Bakalar, Matthew H.; Choudhuri, Kaushik; Weichsel, Julian; Ann, Hyoung Sook; Geissler, Phillip L.; Dustin, Michael L.; Fletcher, Daniel A.

2016-07-01

Membrane interfaces formed at cell-cell junctions are associated with characteristic patterns of membrane proteins whose organization is critical for intracellular signalling. To isolate the role of membrane protein size in pattern formation, we reconstituted model membrane interfaces in vitro using giant unilamellar vesicles decorated with synthetic binding and non-binding proteins. We show that size differences between membrane proteins can drastically alter their organization at membrane interfaces, with as little as a ~5 nm increase in non-binding protein size driving its exclusion from the interface. Combining in vitro measurements with Monte Carlo simulations, we find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and thermally driven membrane height fluctuations that transiently limit access to the interface. This sensitive and highly effective means of physically segregating proteins has implications for cell-cell contacts such as T-cell immunological synapses (for example, CD45 exclusion) and epithelial cell junctions (for example, E-cadherin enrichment), as well as for protein sorting at intracellular contact points between membrane-bound organelles.

IL-7 receptor blockade following T cell depletion promotes long-term allograft survival

PubMed Central

Mai, Hoa-Le; Boeffard, Françoise; Longis, Julie; Danger, Richard; Martinet, Bernard; Haspot, Fabienne; Vanhove, Bernard; Brouard, Sophie; Soulillou, Jean-Paul

2014-01-01

T cell depletion is commonly used in organ transplantation for immunosuppression; however, a restoration of T cell homeostasis following depletion leads to increased memory T cells, which may promote transplant rejection. The cytokine IL-7 is important for controlling lymphopoiesis under both normal and lymphopenic conditions. Here, we investigated whether blocking IL-7 signaling with a mAb that targets IL-7 receptor α (IL-7Rα) alone or following T cell depletion confers an advantage for allograft survival in murine transplant models. We found that IL-7R blockade alone induced indefinite pancreatic islet allograft survival if anti–IL-7R treatment was started 3 weeks before graft. IL-7R blockade following anti-CD4– and anti-CD8–mediated T cell depletion markedly prolonged skin allograft survival. Furthermore, IL-7 inhibition in combination with T cell depletion synergized with either CTLA-4Ig administration or suboptimal doses of tacrolimus to induce long-term skin graft acceptance in this stringent transplant model. Together, these therapies inhibited T cell reconstitution, decreased memory T cell numbers, increased the relative frequency of Tregs, and abrogated both cellular and humoral alloimmune responses. Our data suggest that IL-7R blockade following T cell depletion has potential as a robust, immunosuppressive therapy in transplantation. PMID:24569454

Secreted aspartic proteases are not required for invasion of reconstituted human epithelia by Candida albicans.

PubMed

Lermann, Ulrich; Morschhäuser, Joachim

2008-11-01

A well-known virulence attribute of the human-pathogenic yeast Candida albicans is the secretion of aspartic proteases (Saps), which may contribute to colonization and infection of different host niches by degrading tissue barriers, destroying host defence molecules, or digesting proteins for nutrient supply. The role of individual Sap isoenzymes, which are encoded by a large gene family, for the pathogenicity of C. albicans has been investigated by assessing the virulence of mutants lacking specific SAP genes and by studying the expression pattern of the SAP genes in various models of superficial and systemic infections. We used a recombination-based genetic reporter system to detect the induction of the SAP1-SAP6 genes during infection of reconstituted human vaginal epithelium. Only SAP5, but none of the other tested SAP genes, was detectably activated in this in vitro infection model. To directly address the importance of the SAP1-SAP6 genes for invasion of reconstituted human epithelia (RHE), we constructed a set of mutants of the wild-type C. albicans model strain SC5314 in which either single or multiple SAP genes were specifically deleted. Even mutants lacking all of the SAP1-SAP3 or the SAP4-SAP6 genes displayed the same capacity to invade and damage both oral and vaginal RHE as their wild-type parental strain, in contrast to a nonfilamentous efg1Delta mutant that was avirulent under these conditions. We therefore conclude from these results that the secreted aspartic proteases Sap1p-Sap6p are not required for invasion of RHE by C. albicans.

Current Knowledge and Priorities for Future Research in Late Effects after Hematopoietic Stem Cell Transplantation (HCT) for Severe Combined Immunodeficiency (SCID) Patients: a Consensus Statement from the Second Pediatric Blood and Marrow Transplant Consortium International Conference on Late Effects after Pediatric HCT

PubMed Central

Heimall, J; Puck, J; Buckley, R H; Fleisher, T A; Gennery, A R; Neven, B; Slatter, M; Haddad, E; Notarangelo, L; Baker, KS; Dietz, A C; Duncan, C; Pulsipher, M A; Cowan, MJ

2017-01-01

Severe Combined Immunodeficiency (SCID) is one of the most common indications for pediatric hematopoietic cell transplantation (HCT) in patients with primary immunodeficiency (PID). Historically, SCID was diagnosed in infants who presented with opportunistic infections within the first year of life. With newborn screening (NBS) for SCID in most of the U.S., the majority of infants with SCID are now diagnosed and treated in the first 3.5 months of life, although in the rest of the world, the lack of NBS means that most infants with SCID still present with infections. The average survival for transplanted SCID patients currently is >70% at 3 years post-transplant, although this can vary significantly based on multiple factors including age and infection status at the time of transplantation, type of donor source utilized, manipulation of graft prior to transplant, GVHD prophylaxis, type of conditioning (if any) utilized and underlying genotype of SCID. In at least one study of SCID patients who received no conditioning, long-term survival was 77% at 8.7 years (range out to 26 years) post-transplantation. While a majority of patients with SCID will engraft T cells without any conditioning therapy, depending on genotype, donor source, HLA match and presence of circulating maternal cells a sizable percentage of these will fail to achieve full immune reconstitution. Without conditioning, T cell reconstitution typically occurs, although not always fully, while B cell engraftment does not—leaving some molecular types of SCID patients with intrinsically defective B cells in most cases dependent on regular infusions of immunoglobulin. Because of this, many centers have used conditioning with alkylating agents including busulfan or melphalan known to open marrow niches in attempts to achieve B cell reconstitution. Thus, it is imperative that we understand the potential late effects of these agents in this patient population. There are also non-immunologic risks associated with HCT for SCID that appear to be dependent upon the genotype of the patient. In this report we have evaluated the published data on late effects and attempted to summarize the known risks associated with conditioning and alternative donor sources. These data, while informative, are also a clear demonstration that there is still much to be learned from the SCID population in terms of their post-HCT outcomes. This paper will summarize current findings and recommend further research in areas considered high priority. Specific guidelines regarding a recommended approach to long-term follow up, including laboratory and clinical monitoring will be forthcoming in a subsequent paper. PMID:28068510

Epstein-Barr virus lymphoproliferative disease after hematopoietic stem cell transplant.

PubMed

Rouce, Rayne H; Louis, Chrystal U; Heslop, Helen E

2014-11-01

Epstein-Barr virus (EBV) reactivation can cause significant morbidity and mortality after allogeneic hematopoietic stem cell transplant. Delays in reconstitution of EBV-specific T lymphocyte activity can lead to life-threatening EBV lymphoproliferative disease (EBV-PTLD). This review highlights recent advances in the understanding of pathophysiology, risk factors, diagnosis, and management of EBV viremia and PTLD. During the past decade, early detection strategies, such as serial measurement of EBV-DNA load, have helped identify high-risk patients and diagnose early lymphoproliferation. The most significant advances have come in the form of innovative treatment options, including manipulation of the balance between outgrowing EBV-infected B cells and the EBV cytotoxic T lymphocyte response, and targeting infected B cells with monoclonal antibodies, chemotherapy, unmanipulated donor lymphocytes, and donor or more recently third-party EBV cytotoxic T lymphocytes. Defining criteria for preemptive therapy remains a challenge. EBV reactivation is a significant complication after stem cell transplant. Continued improvements in risk stratification and treatment options are required to improve the morbidity and mortality caused by EBV-associated diseases. Current approaches use rituximab to deplete B cells or adoptive transfer of EBV cytotoxic T lymphocyte to reconstitute immunity. The availability of rapid EBV-specific T cell products offers the possibility of improved outcomes.

Role of claudin species-specific dynamics in reconstitution and remodeling of the zonula occludens.

PubMed

Yamazaki, Yuji; Tokumasu, Reitaro; Kimura, Hiroshi; Tsukita, Sachiko

2011-05-01

Tight-junction strands, which are organized into the beltlike cell-cell adhesive structure called the zonula occludens (TJ), create the paracellular permselective barrier in epithelial cells. The TJ is constructed on the basis of the zonula adherens (AJ) by polymerized claudins in a process mediated by ZO-1/2, but whether the 24 individual claudin family members play different roles at the TJ is unclear. Here we established a cell system for examining the polymerization of individual claudins in the presence of ZO-1/2 using an epithelial-like cell line, SF7, which lacked endogenous TJs and expressed no claudin but claudin-12 in immunofluorescence and real-time PCR assays. In stable SF7-derived lines, exogenous claudin-7, -14, or -19, but no other claudins, individually reconstituted TJs, each with a distinct TJ-strand pattern, as revealed by freeze-fracture analyses. Fluorescence recovery after photobleaching (FRAP) analyses of the claudin dynamics in these and other epithelial cells suggested that slow FRAP-recovery dynamics of claudins play a critical role in regulating their polymerization around AJs, which are loosely coupled with ZO-1/2, to form TJs. Furthermore, the distinct claudin stabilities in different cell types may help to understand how TJs regulate paracellular permeability by altering the paracellular flux and the paracellular ion permeability.

Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module

PubMed Central

Roostalu, Johanna; Cade, Nicholas I.; Surrey, Thomas

2016-01-01

Spindle assembly and function require precise control of microtubule nucleation and dynamics. The chromatin-driven spindle assembly pathway exerts such control locally in the vicinity of chromosomes. One of the key targets of this pathway is TPX2. The molecular mechanism of how TPX2 stimulates microtubule nucleation is not understood. Using microscopy-based dynamic in vitro reconstitution assays with purified proteins, we find that human TPX2 directly stabilises growing microtubule ends and stimulates microtubule nucleation by stabilising early microtubule nucleation intermediates. Human microtubule polymerase chTOG (XMAP215/Msps/Stu2p/Dis1/Alp14 homolog) only weakly promotes nucleation, but acts synergistically with TPX2. Hence, a combination of distinct and complementary activities is sufficient for efficient microtubule formation in vitro. Importins control the efficiency of the microtubule nucleation by selectively blocking TPX2’s interaction with microtubule nucleation intermediates. This in vitro reconstitution reveals the molecular mechanism of regulated microtubule formation by a minimal nucleation module essential for chromatin-dependent microtubule nucleation in cells. PMID:26414402

Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module.

PubMed

Roostalu, Johanna; Cade, Nicholas I; Surrey, Thomas

2015-11-01

Spindle assembly and function require precise control of microtubule nucleation and dynamics. The chromatin-driven spindle assembly pathway exerts such control locally in the vicinity of chromosomes. One of the key targets of this pathway is TPX2. The molecular mechanism of how TPX2 stimulates microtubule nucleation is not understood. Using microscopy-based dynamic in vitro reconstitution assays with purified proteins, we find that human TPX2 directly stabilizes growing microtubule ends and stimulates microtubule nucleation by stabilizing early microtubule nucleation intermediates. Human microtubule polymerase chTOG (XMAP215/Msps/Stu2p/Dis1/Alp14 homologue) only weakly promotes nucleation, but acts synergistically with TPX2. Hence, a combination of distinct and complementary activities is sufficient for efficient microtubule formation in vitro. Importins control the efficiency of the microtubule nucleation by selectively blocking the interaction of TPX2 with microtubule nucleation intermediates. This in vitro reconstitution reveals the molecular mechanism of regulated microtubule formation by a minimal nucleation module essential for chromatin-dependent microtubule nucleation in cells.

Control of Cytomegalovirus Viremia after Allogeneic Stem Cell Transplantation: A Review on CMV-Specific T Cell Reconstitution.

PubMed

van der Heiden, Pim; Marijt, Erik; Falkenburg, Fred; Jedema, Inge

2018-04-04

Recipients of allogeneic stem cell transplantation (alloSCT) are at risk for reactivation of endogenous herpesviruses due to profound and prolonged T cell deficiency following conditions such as graft-versus-host disease, immunosuppression, and/or T cell depletion. Reactivation of endogenous cytomegalovirus (CMV) is the most frequently occurring herpesvirus reactivation following alloSCT. Antiviral medication is often used in pre-emptive treatment strategies initiated when increases in CMV viral loads are detected as a result of active reactivation of the virus. Despite pre-emptive antiviral treatment, the incidence of CMV disease in CMV-seropositive alloSCT patients is still 10% at 1 year following alloSCT. This illustrates the necessity for adequate CMV-specific T cell immunity for long-term control of CMV and prevention of CMV disease. In this review, we analyzed the available studies on the influence of donor CMV status on CMV-specific T cell reconstitution and CMV disease. Furthermore, we reviewed the available studies on the safety and efficacy of adoptive transfer of donor CMV-specific T cells for the prevention and treatment of CMV disease following alloSCT, including studies on adoptive transfer of third-party CMV-specific T cells as a possible alternative when donor T cells are not available. Copyright © 2018 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Immune Reconstitution After Autologous Hematopoietic Stem Cell Transplantation in Crohn’s Disease: Current Status and Future Directions. A Review on Behalf of the EBMT Autoimmune Diseases Working Party and the Autologous Stem Cell Transplantation In Refractory CD—Low Intensity Therapy Evaluation Study Investigators

PubMed Central

Pockley, Alan Graham; Lindsay, James O.; Foulds, Gemma A.; Rutella, Sergio; Gribben, John G.; Alexander, Tobias; Snowden, John A.

2018-01-01

Patients with treatment refractory Crohn’s disease (CD) suffer debilitating symptoms, poor quality of life, and reduced work productivity. Surgery to resect inflamed and fibrotic intestine may mandate creation of a stoma and is often declined by patients. Such patients continue to be exposed to medical therapy that is ineffective, often expensive and still associated with a burden of adverse effects. Over the last two decades, autologous hematopoietic stem cell transplantation (auto-HSCT) has emerged as a promising treatment option for patients with severe autoimmune diseases (ADs). Mechanistic studies have provided proof of concept that auto-HSCT can restore immunological tolerance in chronic autoimmunity via the eradication of pathological immune responses and a profound reconfiguration of the immune system. Herein, we review current experience of auto-HSCT for the treatment of CD as well as approaches that have been used to monitor immune reconstitution following auto-HSCT in patients with ADs, including CD. We also detail immune reconstitution studies that have been integrated into the randomized controlled Autologous Stem cell Transplantation In refractory CD—Low Intensity Therapy Evaluation trial, which is designed to test the hypothesis that auto-HSCT using reduced intensity mobilization and conditioning regimens will be a safe and effective means of inducing sustained control in refractory CD compared to standard of care. Immunological profiling will generate insight into the pathogenesis of the disease, restoration of responsiveness to anti-TNF therapy in patients with recurrence of endoscopic disease and immunological events that precede the onset of disease in patients that relapse after auto-HSCT. PMID:29670622

Synthesis and Self-Assembly of Cellulose Microfibrils from Reconstituted Cellulose Synthase.

PubMed

Cho, Sung Hyun; Purushotham, Pallinti; Fang, Chao; Maranas, Cassandra; Díaz-Moreno, Sara M; Bulone, Vincent; Zimmer, Jochen; Kumar, Manish; Nixon, B Tracy

2017-09-01

Cellulose, the major component of plant cell walls, can be converted to bioethanol and is thus highly studied. In plants, cellulose is produced by cellulose synthase, a processive family-2 glycosyltransferase. In plant cell walls, individual β-1,4-glucan chains polymerized by CesA are assembled into microfibrils that are frequently bundled into macrofibrils. An in vitro system in which cellulose is synthesized and assembled into fibrils would facilitate detailed study of this process. Here, we report the heterologous expression and partial purification of His-tagged CesA5 from Physcomitrella patens Immunoblot analysis and mass spectrometry confirmed enrichment of PpCesA5. The recombinant protein was functional when reconstituted into liposomes made from yeast total lipid extract. The functional studies included incorporation of radiolabeled Glc, linkage analysis, and imaging of cellulose microfibril formation using transmission electron microscopy. Several microfibrils were observed either inside or on the outer surface of proteoliposomes, and strikingly, several thinner fibrils formed ordered bundles that either covered the surfaces of proteoliposomes or were spawned from liposome surfaces. We also report this arrangement of fibrils made by proteoliposomes bearing CesA8 from hybrid aspen. These observations describe minimal systems of membrane-reconstituted CesAs that polymerize β-1,4-glucan chains that coalesce to form microfibrils and higher-ordered macrofibrils. How these micro- and macrofibrils relate to those found in primary and secondary plant cell walls is uncertain, but their presence enables further study of the mechanisms that govern the formation and assembly of fibrillar cellulosic structures and cell wall composites during or after the polymerization process controlled by CesA proteins. © 2017 American Society of Plant Biologists. All Rights Reserved.

Synthesis and Self-Assembly of Cellulose Microfibrils from Reconstituted Cellulose Synthase1[OPEN

PubMed Central

Purushotham, Pallinti; Fang, Chao; Maranas, Cassandra; Bulone, Vincent

2017-01-01

Cellulose, the major component of plant cell walls, can be converted to bioethanol and is thus highly studied. In plants, cellulose is produced by cellulose synthase, a processive family-2 glycosyltransferase. In plant cell walls, individual β-1,4-glucan chains polymerized by CesA are assembled into microfibrils that are frequently bundled into macrofibrils. An in vitro system in which cellulose is synthesized and assembled into fibrils would facilitate detailed study of this process. Here, we report the heterologous expression and partial purification of His-tagged CesA5 from Physcomitrella patens. Immunoblot analysis and mass spectrometry confirmed enrichment of PpCesA5. The recombinant protein was functional when reconstituted into liposomes made from yeast total lipid extract. The functional studies included incorporation of radiolabeled Glc, linkage analysis, and imaging of cellulose microfibril formation using transmission electron microscopy. Several microfibrils were observed either inside or on the outer surface of proteoliposomes, and strikingly, several thinner fibrils formed ordered bundles that either covered the surfaces of proteoliposomes or were spawned from liposome surfaces. We also report this arrangement of fibrils made by proteoliposomes bearing CesA8 from hybrid aspen. These observations describe minimal systems of membrane-reconstituted CesAs that polymerize β-1,4-glucan chains that coalesce to form microfibrils and higher-ordered macrofibrils. How these micro- and macrofibrils relate to those found in primary and secondary plant cell walls is uncertain, but their presence enables further study of the mechanisms that govern the formation and assembly of fibrillar cellulosic structures and cell wall composites during or after the polymerization process controlled by CesA proteins. PMID:28768815

Frequent downregulation of miR-34 family in human ovarian cancers.

PubMed

Corney, David C; Hwang, Chang-Il; Matoso, Andres; Vogt, Markus; Flesken-Nikitin, Andrea; Godwin, Andrew K; Kamat, Aparna A; Sood, Anil K; Ellenson, Lora H; Hermeking, Heiko; Nikitin, Alexander Yu

2010-02-15

The miR-34 family is directly transactivated by tumor suppressor p53, which is frequently mutated in human epithelial ovarian cancer (EOC). We hypothesized that miR-34 expression would be decreased in EOC and that reconstituted miR-34 expression might reduce cell proliferation and invasion of EOC cells. miR-34 expression was determined by quantitative reverse transcription-PCR and in situ hybridization in a panel of 83 human EOC samples. Functional characterization of miR-34 was accomplished by reconstitution of miR-34 expression in EOC cells with synthetic pre-miR molecules followed by determining changes in proliferation, apoptosis, and invasion. miR-34a expression is decreased in 100%, and miR-34b*/c in 72%, of EOC with p53 mutation, whereas miR-34a is also downregulated in 93% of tumors with wild-type p53. Furthermore, expression of miR-34b*/c is significantly reduced in stage IV tumors compared with stage III (P = 0.0171 and P = 0.0029, respectively). Additionally, we observed promoter methylation and copy number variations at mir-34. In situ hybridization showed that miR-34a expression is inversely correlated with MET immunohistochemical staining, consistent with translational inhibition by miR-34a. Finally, miR-34 reconstitution experiments in p53 mutant EOC cells resulted in reduced proliferation, motility, and invasion, the latter of which was dependent on MET expression. Our work suggests that miR-34 family plays an important role in EOC pathogenesis and reduced expression of miR-34b*/c may be particularly important for progression to the most advanced stages. Part of miR-34 effects on motility and invasion may be explained by regulation of MET, which is frequently overexpressed in EOC.

Studying Mechanosensitivity of Two-Pore Domain K+ Channels in Cellular and Reconstituted Proteoliposome Membranes.

PubMed

Del Mármol, Josefina; Rietmeijer, Robert A; Brohawn, Stephen G

2018-01-01

Mechanical force sensation is fundamental to a wide breadth of biology from the classic senses of touch, pain, hearing, and balance to less conspicuous sensations of proprioception, blood pressure, and osmolarity and basic aspects of cell growth, differentiation, and development. These diverse and essential systems use force-gated (or mechanosensitive) ion channels that convert mechanical stimuli into cellular electrical signals. TRAAK, TREK1, and TREK2 are K + -selective ion channels of the two-pore domain K + (K2P) family that are mechanosensitive: they are gated open by increasing membrane tension. TRAAK and TREK channels are thought to play roles in somatosensory and other mechanosensory processes in neuronal and non-neuronal tissues. Here, we present protocols for three assays to study mechanical activation of these channels in cell membranes: (1) cell swelling, (2) cell poking, and (3) patched membrane stretching. Patched membrane stretching is also applicable to the study of mechanosensitive K2P channel activity in a cell-free system and a procedure for proteoliposome reconstitution and patching is also presented. These approaches are also readily applicable to the study of other mechanosensitive ion channels.

Collection and use of circulating hematopoietic progenitor cells.

PubMed

Lee, J H; Klein, H G

1995-02-01

Although lymphocytes and monocytes are becoming increasingly important in transfusion therapy, peripheral stem cells have been responsible for the recent explosive interest in harvesting mononuclear cells from the peripheral circulation. Despite their low concentration in peripheral blood and the consequent difficulty in cell collection, circulating hematopoietic progenitor cells are collected and used almost routinely. These mononuclear cells, possessing the capacity for hematopoietic reconstitution and the potential for definitive therapy of a variety of disorders, have been the focus of recent intense interest in transfusion medicine.

[Development of the next generation humanized mouse for drug discovery].

PubMed

Ito, Ryoji

A humanized mouse, which is efficiently engrafted human cells and tissues, is an important tool to mimic human physiology for biomedical researches. Since 2000s, severe combined immunodeficient mouse strains such as NOG, BRG, and NSG mice have been generated. They are great recipients to create humanized mouse models compared to previous other immunodeficient strains due to their multiple dysfunctions of innate and acquired immunity. Especially, the transfer of human hematopoietic stem cells into these immunodeficient mice has been enabled to reconstitute human immune systems, because the mice show high engraftment level of human leukocyte in peripheral blood (～50%), spleen and bone marrow (60～90%) and generate well-differentiated multilineage human immune cells including lymphoid and myeloid lineage cells. Using these mice, several human disease models such as cancer, allergy, graft-versus-host disease (GVHD), and etc. have been established to understand the pathogenic mechanisms of the diseases and to evaluate the efficacy and safety of novel drugs. In this review, I provide an overview of recent advances in the humanized mouse technology, including generation of novel platforms of genetically modified NOG (next generation NOG) mice and some applications of them to create human disease models for drug discovery in preclinical researches.

Monoclonal antibody disulfide reduction during manufacturing

PubMed Central

Hutterer, Katariina M.; Hong, Robert W.; Lull, Jonathon; Zhao, Xiaoyang; Wang, Tian; Pei, Rex; Le, M. Eleanor; Borisov, Oleg; Piper, Rob; Liu, Yaoqing Diana; Petty, Krista; Apostol, Izydor; Flynn, Gregory C.

2013-01-01

Manufacturing-induced disulfide reduction has recently been reported for monoclonal human immunoglobulin gamma (IgG) antibodies, a widely used modality in the biopharmaceutical industry. This effect has been tied to components of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the effect of process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the end of production displayed large variations in the extent of antibody reduction between different products, including no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted model in which process variables could be isolated from product properties, we found that antibody reduction was dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity following the order IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production. PMID:23751615

Streptococcal cell wall-induced arthritis and adjuvant arthritis in F344----Lewis and in Lewis----F344 bone marrow chimeras

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

van Bruggen, M.C.; van den Broek, M.F.; van den Berg, W.B.

1991-09-01

Streptococcal cell wall (SCW)-induced arthritis and adjuvant arthritis (AA) are rat models for chronic, erosive polyarthritis. Both models can be induced in susceptible Lewis rats, whereas F344 rats are resistant. In AA as well as in SCW arthritis, antigen-specific T lymphocytes have been demonstrated to be crucial for chronic disease. In this communication the authors describe their studies to probe the cellular mechanism responsible for the difference in susceptibility of Lewis and F344, using bone marrow chimeras. By transplanting bone marrow cells from F344 into lethally irradiated Lewis recipients, Lewis rats were rendered resistant to SCW arthritis induction. F344 ratsmore » reconstituted with Lewis bone marrow, i.e., Lewis----F344 chimeras, develop an arthritis upon SCW injection. For AA comparable results were obtained. These data suggest that both resistance and susceptibility to bacterium-induced chronic arthritis are mediated by hemopoietic/immune cells and that the recipiental environment does not influence the susceptibility to chronic joint inflammation.« less

Identification of the yeast mitochondrial transporter for oxaloacetate and sulfate.

PubMed

Palmieri, L; Vozza, A; Agrimi, G; De Marco, V; Runswick, M J; Palmieri, F; Walker, J E

1999-08-06

Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family, including the OAC protein. The transport specificities of some family members are known, but most are not. The function of the OAC has been revealed by overproduction in Escherichia coli, reconstitution into liposomes, and demonstration that the proteoliposomes transport malonate, oxaloacetate, sulfate, and thiosulfate. Reconstituted OAC catalyzes both unidirectional transport and exchange of substrates. In S. cerevisiae, OAC is in inner mitochondrial membranes, and deletion of its gene greatly reduces transport of oxaloacetate sulfate, thiosulfate, and malonate. Mitochondria from wild-type cells swelled in isoosmotic solutions of ammonium salts of oxaloacetate, sulfate, thiosulfate, and malonate, indicating that these anions are cotransported with protons. Overexpression of OAC in the deletion strain increased greatly the [(35)S]sulfate/sulfate and [(35)S]sulfate/oxaloacetate exchanges in proteoliposomes reconstituted with digitonin extracts of mitochondria. The main physiological role of OAC appears to be to use the proton-motive force to take up into mitochondria oxaloacetate produced from pyruvate by cytoplasmic pyruvate carboxylase.

Biologically Derived Nanoparticle Arrays via a Site-Specific Reconstitution of Ferritin and their Electrochemistry

NASA Technical Reports Server (NTRS)

Kim, Jae-Woo; Choi, Sang H.; Lillehei, Peter T.; King, Glen C.; Elliott, James R.; Chu, Sang-Hyon; Park, Yeonjoon; Watt, Gerald D.

2004-01-01

Nanoparticle arrays biologically derived from an electrochemically-controlled site-specific biomineralization were fabricated on a gold substrate through the immobilization process of biomolecules. The work reported herein includes the immobilization of ferritin with various surface modifications, the electrochemical biomineralization of ferritins with different inorganic cores, the fabrication of self-assembled arrays with the immobilized ferritin, and the electrochemical characterization of various core materials. Protein immobilization on the substrate is achieved by anchoring ferritins with dithiobis-N-succinimidyl propionate (DTSP). A reconstitution process of electrochemical site-specific biomineralization with a protein cage loads ferritins with different core materials such as Pt, Co, Mn, and Ni. The ferritin acts as a nano-scale template, a biocompatible cage, and a separator between the nanoparticles. The nano-sized metalcored ferritins on a gold substrate displayed a good electrochemical activity for the electron transport and storage, which is suitable for bioelectronics applications such as biofuel cell, bionanobattery, biosensors, etc. Keywords: Ferritin, immobilization, site-specific reconstitution, biomineralization, and bioelectronics

Channel function reconstitution and re-animation: a single-channel strategy in the postcrystal age

PubMed Central

Oiki, Shigetoshi

2015-01-01

The most essential properties of ion channels for their physiologically relevant functions are ion-selective permeation and gating. Among the channel species, the potassium channel is primordial and the most ubiquitous in the biological world, and knowledge of this channel underlies the understanding of features of other ion channels. The strategy applied to studying channels changed dramatically after the crystal structure of the potassium channel was resolved. Given the abundant structural information available, we exploited the bacterial KcsA potassium channel as a simple model channel. In the postcrystal age, there are two effective frameworks with which to decipher the functional codes present in the channel structure, namely reconstitution and re-animation. Complex channel proteins are decomposed into essential functional components, and well-examined parts are rebuilt for integrating channel function in the membrane (reconstitution). Permeation and gating are dynamic operations, and one imagines the active channel by breathing life into the ‘frozen’ crystal (re-animation). Capturing the motion of channels at the single-molecule level is necessary to characterize the behaviour of functioning channels. Advanced techniques, including diffracted X-ray tracking, lipid bilayer methods and high-speed atomic force microscopy, have been used. Here, I present dynamic pictures of the KcsA potassium channel from the submolecular conformational changes to the supramolecular collective behaviour of channels in the membrane. These results form an integrated picture of the active channel and offer insights into the processes underlying the physiological function of the channel in the cell membrane. PMID:25833254

Channel function reconstitution and re-animation: a single-channel strategy in the postcrystal age.

PubMed

Oiki, Shigetoshi

2015-06-15

The most essential properties of ion channels for their physiologically relevant functions are ion-selective permeation and gating. Among the channel species, the potassium channel is primordial and the most ubiquitous in the biological world, and knowledge of this channel underlies the understanding of features of other ion channels. The strategy applied to studying channels changed dramatically after the crystal structure of the potassium channel was resolved. Given the abundant structural information available, we exploited the bacterial KcsA potassium channel as a simple model channel. In the postcrystal age, there are two effective frameworks with which to decipher the functional codes present in the channel structure, namely reconstitution and re-animation. Complex channel proteins are decomposed into essential functional components, and well-examined parts are rebuilt for integrating channel function in the membrane (reconstitution). Permeation and gating are dynamic operations, and one imagines the active channel by breathing life into the 'frozen' crystal (re-animation). Capturing the motion of channels at the single-molecule level is necessary to characterize the behaviour of functioning channels. Advanced techniques, including diffracted X-ray tracking, lipid bilayer methods and high-speed atomic force microscopy, have been used. Here, I present dynamic pictures of the KcsA potassium channel from the submolecular conformational changes to the supramolecular collective behaviour of channels in the membrane. These results form an integrated picture of the active channel and offer insights into the processes underlying the physiological function of the channel in the cell membrane. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Immunological and physical evaluation of the multistage tuberculosis subunit vaccine candidate H56/CAF01 formulated as a spray-dried powder.

PubMed

Thakur, Aneesh; Ingvarsson, Pall Thor; Schmidt, Signe Tandrup; Rose, Fabrice; Andersen, Peter; Christensen, Dennis; Foged, Camilla

2018-05-31

Liquid vaccine dosage forms have limited stability and require refrigeration during their manufacture, distribution and storage. In contrast, solid vaccine dosage forms, produced by for example spray drying, offer improved storage stability and reduced dependence on cold-chain facilities. This is advantageous for mass immunization campaigns for global public health threats, e.g., tuberculosis (TB), and offers cheaper vaccine distribution. The multistage subunit vaccine antigen H56, which is a fusion protein of the Mycobacterium tuberculosis (Mtb) antigens Ag85B, ESAT-6, and Rv2660, has been shown to confer protective efficacy against active TB before and after Mtb exposure in preclinical models, and it is currently undergoing clinical phase 2a testing. In several studies, including a recent study comparing multiple clinically relevant vaccine adjuvants, the T helper type 1 (Th1)/Th17-inducing adjuvant CAF01 was the most efficacious adjuvant for H56 to stimulate protective immunity against Mtb. With the long-term goal of designing a thermostable and self-administrable dry powder vaccine based on H56 and CAF01 for inhalation, we compared H56 spray-dried with CAF01 with the non-spray-dried H56/CAF01 vaccine with respect to their ability to induce systemic Th1, Th17 and humoral responses after subcutaneous immunization. Here we show that spray drying of the H56/CAF01 vaccine results in preserved antigenic epitope recognition and adjuvant activity of CAF01, and the spray-dried, reconstituted vaccine induces antigen-specific Th1, Th17 and humoral immune responses, which are comparable to those stimulated by the non-spray-dried H56/CAF01 vaccine. In addition, the spray-dried and reconstituted H56/CAF01 vaccine promotes similar polyfunctional CD4 + T-cell responses as the non-spray-dried vaccine. Thus, our study provides proof-of-concept that spray drying of the subunit vaccine H56/CAF01 preserves vaccine-induced humoral and cell-mediated immune responses. These results support our ongoing efforts to develop a thermostable, dry powder-based TB vaccine. Copyright © 2018 Elsevier Ltd. All rights reserved.

Site-specific differences in T cell frequencies and phenotypes in the blood and gut of HIV-uninfected and ART-treated HIV+ adults.

PubMed

Yukl, Steven A; Shergill, Amandeep K; Girling, Valerie; Li, Qingsheng; Killian, Maudi; Epling, Lorrie; Li, Peilin; Kaiser, Philipp; Haase, Ashley; Havlir, Diane V; McQuaid, Kenneth; Sinclair, Elizabeth; Wong, Joseph K

2015-01-01

Gastrointestinal T lymphocytes are critical for mucosal immunity and HIV pathogenesis, yet little is known about normal T cell numbers and phenotypes in different regions of the gut, or the degree to which ART can restore levels to those of HIV-uninfected individuals. To investigate these questions, we measured T cell frequencies and markers of memory, activation, anergy, and homing in the blood, ileum, and rectum of HIV- and ART-suppressed HIV+ adults. In HIV- individuals, T cell frequencies and phenotypes differed significantly between sites. Compared to HIV- adults, HIV+ adults had lower absolute CD4+T cell counts in the ileal lamina propria and lower relative CD4+T cell counts in the blood and ileum. In the gut, HIV+ adults had a higher proportion of CD38+ CD4+T cells, a lower proportion of terminally-differentiated effector cells, and, in the rectum, a higher proportion of CTLA-4+ CD4+T cells. In HIV+ individuals, relative CD4+T cell numbers in the ileum correlated with the proportion of CTLA-4+ CD4+T cells, whereas in the rectum, they tended to correlate with the proportion of circulating CD4+T cells expressing α4β7 or CCR6. Mechanisms of T cell reconstitution may differ throughout the gut, with homing contributing more in the rectum while ileal reconstitution is associated with mucosal CD4+T cell anergy.

A mouse air pouch model for evaluating the immune response to Taenia crassiceps infection.

PubMed

Gaspar, Emanuelle B; Sakai, Yuriko I; Gaspari, Elizabeth De

2014-02-01

The experimental system of Taenia crassiceps cysticerci infection in BALB/c mice is considered to be the most representative model of cysticercosis. In our work, mice were sacrificed 7 and 30days after infection, and pouch fluid was collected to determine the number of accumulated cells and the concentrations of IFNγ, IL-2, IL-4, IL-6, IL-10 and nitric oxide. The injection of 50 nonbudding cysticerci into normal mouse dorsal air pouches induced a high level of IFNγ and nitric oxide production relative to the parasite load. The air pouch provides a convenient cavity that allows studying the cellular immunological aspects of the T. crassiceps parasite. The nonbudding cysticerci recovered from the air pouches contained cells that can reconstitute complete cysts in the peritoneal cavity of mice. In conclusion, these results demonstrate that the air pouch model is an alternative tool for the evaluation of the immune characteristics of T. crassiceps infection. Copyright © 2013 Elsevier Inc. All rights reserved.

A New Extensively Characterised Conditionally Immortal Muscle Cell-Line for Investigating Therapeutic Strategies in Muscular Dystrophies

PubMed Central

Muses, Sofia; Morgan, Jennifer E.; Wells, Dominic J.

2011-01-01

A new conditionally immortal satellite cell-derived cell-line, H2K 2B4, was generated from the H2Kb-tsA58 immortomouse. Under permissive conditions H2K 2B4 cells terminally differentiate in vitro to form uniform myotubes with a myogenic protein profile comparable with freshly isolated satellite cells. Following engraftment into immunodeficient dystrophin-deficient mice, H2K 2B4 cells regenerated host muscle with donor derived myofibres that persisted for at least 24 weeks, without forming tumours. These cells were readily transfectable using both retrovirus and the non-viral transfection methods and importantly upon transplantation, were able to reconstitute the satellite cell niche with functional donor derived satellite cells. Finally using the Class II DNA transposon, Sleeping Beauty, we successfully integrated a reporter plasmid into the genome of H2K 2B4 cells without hindering the myogenic differentiation. Overall, these data suggest that H2K 2B4 cells represent a readily transfectable stable cell-line in which to investigate future stem cell based therapies for muscle disease. PMID:21935475

A new extensively characterised conditionally immortal muscle cell-line for investigating therapeutic strategies in muscular dystrophies.

PubMed

Muses, Sofia; Morgan, Jennifer E; Wells, Dominic J

2011-01-01

A new conditionally immortal satellite cell-derived cell-line, H2K 2B4, was generated from the H2K(b)-tsA58 immortomouse. Under permissive conditions H2K 2B4 cells terminally differentiate in vitro to form uniform myotubes with a myogenic protein profile comparable with freshly isolated satellite cells. Following engraftment into immunodeficient dystrophin-deficient mice, H2K 2B4 cells regenerated host muscle with donor derived myofibres that persisted for at least 24 weeks, without forming tumours. These cells were readily transfectable using both retrovirus and the non-viral transfection methods and importantly upon transplantation, were able to reconstitute the satellite cell niche with functional donor derived satellite cells. Finally using the Class II DNA transposon, Sleeping Beauty, we successfully integrated a reporter plasmid into the genome of H2K 2B4 cells without hindering the myogenic differentiation. Overall, these data suggest that H2K 2B4 cells represent a readily transfectable stable cell-line in which to investigate future stem cell based therapies for muscle disease.

Different competitive capacities of Stat4- and Stat6-deficient CD4+ T cells during lymphophenia-driven proliferation.

PubMed

Sanchez-Guajardo, Vanesa; Borghans, José A M; Marquez, Maria-Elena; Garcia, Sylvie; Freitas, Antonio A

2005-02-01

The outcome of an immune response relies on the competitive capacities acquired through differentiation of CD4(+) T cells into Th1 or Th2 effector cells. Because Stat4 and Stat6 proteins are implicated in the Th1 vs Th2 generation and maintenance, respectively, we compare in this study the kinetics of Stat4(-/-) and Stat6(-/-) CD4(+) T cells during competitive bone marrow reconstitution and lymphopenia-driven proliferation. After bone marrow transplantation, both populations reconstitute the peripheral T cell pools equally well. After transfer into lymphopenic hosts, wild-type and Stat6(-/-) CD4(+) T cells show a proliferation advantage, which is early associated with the expression of an active phospho-Stat4 and the down-regulation of Stat6. Despite these differences, Stat4- and Stat6-deficient T cells reach similar steady state numbers. However, when both Stat4(-/-) and Stat6(-/-) CD4(+) T cells are coinjected into the same hosts, the Stat6(-/-) cells become dominant and out-compete Stat4(-/-) cells. These findings suggest that cell activation, through the Stat4 pathway and the down-regulation of Stat6, confers to pro-Th1 T cells a slight proliferation advantage that in a competitive situation has major late repercussions, because it modifies the final homeostatic equilibrium of the populations and favors the establishment of Th1 CD4(+) T cell dominance.

Lineage-specific T-cell reconstitution following in vivo CD4+ and CD8+ lymphocyte depletion in nonhuman primates.

PubMed

Engram, Jessica C; Cervasi, Barbara; Borghans, Jose A M; Klatt, Nichole R; Gordon, Shari N; Chahroudi, Ann; Else, James G; Mittler, Robert S; Sodora, Donald L; de Boer, Rob J; Brenchley, Jason M; Silvestri, Guido; Paiardini, Mirko

2010-08-05

Many features of T-cell homeostasis in primates are still unclear, thus limiting our understanding of AIDS pathogenesis, in which T-cell homeostasis is lost. Here, we performed experiments of in vivo CD4(+) or CD8(+) lymphocyte depletion in 2 nonhuman primate species, rhesus macaques (RMs) and sooty mangabeys (SMs). Whereas RMs develop AIDS after infection with simian immunodeficiency virus (SIV), SIV-infected SMs are typically AIDS-resistant. We found that, in both species, most CD4(+) or CD8(+) T cells in blood and lymph nodes were depleted after treatment with their respective antibodies. These CD4(+) and CD8(+) lymphocyte depletions were followed by a largely lineage-specific CD4(+) and CD8(+) T-cell proliferation, involving mainly memory T cells, which correlated with interleukin-7 plasma levels. Interestingly, SMs showed a faster repopulation of naive CD4(+) T cells than RMs. In addition, in both species CD8(+) T-cell repopulation was faster than that of CD4(+) T cells, with CD8(+) T cells reconstituting a normal pool within 60 days and CD4(+) T cells remaining below baseline levels up to day 180 after depletion. While this study revealed subtle differences in CD4(+) T-cell repopulation in an AIDS-sensitive versus an AIDS-resistant species, such differences may have particular relevance in the presence of active SIV repli cation, where CD4(+) T-cell destruction is chronic.

Lineage-specific T-cell reconstitution following in vivo CD4+ and CD8+ lymphocyte depletion in nonhuman primates

PubMed Central

Engram, Jessica C.; Cervasi, Barbara; Borghans, Jose A. M.; Klatt, Nichole R.; Gordon, Shari N.; Chahroudi, Ann; Else, James G.; Mittler, Robert S.; Sodora, Donald L.; de Boer, Rob J.; Brenchley, Jason M.; Silvestri, Guido

2010-01-01

Many features of T-cell homeostasis in primates are still unclear, thus limiting our understanding of AIDS pathogenesis, in which T-cell homeostasis is lost. Here, we performed experiments of in vivo CD4+ or CD8+ lymphocyte depletion in 2 nonhuman primate species, rhesus macaques (RMs) and sooty mangabeys (SMs). Whereas RMs develop AIDS after infection with simian immunodeficiency virus (SIV), SIV-infected SMs are typically AIDS-resistant. We found that, in both species, most CD4+ or CD8+ T cells in blood and lymph nodes were depleted after treatment with their respective antibodies. These CD4+ and CD8+ lymphocyte depletions were followed by a largely lineage-specific CD4+ and CD8+ T-cell proliferation, involving mainly memory T cells, which correlated with interleukin-7 plasma levels. Interestingly, SMs showed a faster repopulation of naive CD4+ T cells than RMs. In addition, in both species CD8+ T-cell repopulation was faster than that of CD4+ T cells, with CD8+ T cells reconstituting a normal pool within 60 days and CD4+ T cells remaining below baseline levels up to day 180 after depletion. While this study revealed subtle differences in CD4+ T-cell repopulation in an AIDS-sensitive versus an AIDS-resistant species, such differences may have particular relevance in the presence of active SIV repli cation, where CD4+ T-cell destruction is chronic. PMID:20484087

Method for simultaneous use of a single additive for coal flotation, dewatering, and reconstitution

DOEpatents

Wen, Wu-Wey; Gray, McMahan L.; Champagne, Kenneth J.

1995-01-01

A single dose of additive contributes to three consecutive fine coal unit operations, i.e., flotation, dewatering and reconstitution, whereby the fine coal is first combined with water in a predetermined proportion so as to formulate a slurry. The slurry is then mixed with a heavy hydrocarbon-based emulsion in a second predetermined proportion and at a first predetermined mixing speed and for a predetermined period of time. The conditioned slurry is then cleaned by a froth flotation method to form a clean coal froth and then the froth is dewatered by vacuum filtration or a centrifugation process to form reconstituted products that are dried to dust-less clumps prior to combustion.

Model for the architecture of caveolae based on a flexible, net-like assembly of Cavin1 and Caveolin discs

PubMed Central

Stoeber, Miriam; Schellenberger, Pascale; Siebert, C. Alistair; Leyrat, Cedric; Helenius, Ari

2016-01-01

Caveolae are invaginated plasma membrane domains involved in mechanosensing, signaling, endocytosis, and membrane homeostasis. Oligomers of membrane-embedded caveolins and peripherally attached cavins form the caveolar coat whose structure has remained elusive. Here, purified Cavin1 60S complexes were analyzed structurally in solution and after liposome reconstitution by electron cryotomography. Cavin1 adopted a flexible, net-like protein mesh able to form polyhedral lattices on phosphatidylserine-containing vesicles. Mutating the two coiled-coil domains in Cavin1 revealed that they mediate distinct assembly steps during 60S complex formation. The organization of the cavin coat corresponded to a polyhedral nano-net held together by coiled-coil segments. Positive residues around the C-terminal coiled-coil domain were required for membrane binding. Purified caveolin 8S oligomers assumed disc-shaped arrangements of sizes that are consistent with the discs occupying the faces in the caveolar polyhedra. Polygonal caveolar membrane profiles were revealed in tomograms of native caveolae inside cells. We propose a model with a regular dodecahedron as structural basis for the caveolae architecture. PMID:27834731

Characterization of Particle Translocation through Mucin Hydrogels

PubMed Central

Lieleg, Oliver; Vladescu, Ioana; Ribbeck, Katharina

2010-01-01

Abstract Biological functional entities surround themselves with selective barriers that control the passage of certain classes of macromolecules while rejecting others. A prominent example of such a selective permeability barrier is given by mucus. Mucus is a biopolymer-based hydrogel that lines all wet epithelial surfaces of the human body. It regulates the uptake of nutrients from our gastrointestinal system, adjusts itself with the menstrual cycle to control the passage of sperm, and shields the underlying cells from pathogens such as bacteria and viruses. In the case of drug delivery, the mucus barrier needs to be overcome for successful medical treatment. Despite its importance for both physiology and medical applications, the underlying principles which regulate the permeability of mucus remain enigmatic. Here, we analyze the mobility of microscopic particles in reconstituted mucin hydrogels. We show that electrostatic interactions between diffusing particles and mucin polymers regulate the permeability properties of reconstituted mucin hydrogels. As a consequence, various parameters such as particle surface charge and mucin density, and buffer conditions such as pH and ionic strength, can modulate the microscopic barrier function of the mucin hydrogel. Our findings suggest that the permeability of a biopolymer-based hydrogel such as native mucus can be tuned to a wide range of settings in different compartments of our bodies. PMID:20441741

Perceptual Characterization and Analysis of Aroma Mixtures Using Gas Chromatography Recomposition-Olfactometry

PubMed Central

Johnson, Arielle J.; Hirson, Gregory D.; Ebeler, Susan E.

2012-01-01

This paper describes the design of a new instrumental technique, Gas Chromatography Recomposition-Olfactometry (GC-R), that adapts the reconstitution technique used in flavor chemistry studies by extracting volatiles from a sample by headspace solid-phase microextraction (SPME), separating the extract on a capillary GC column, and recombining individual compounds selectively as they elute off of the column into a mixture for sensory analysis (Figure 1). Using the chromatogram of a mixture as a map, the GC-R instrument allows the operator to “cut apart" and recombine the components of the mixture at will, selecting compounds, peaks, or sections based on retention time to include or exclude in a reconstitution for sensory analysis. Selective recombination is accomplished with the installation of a Deans Switch directly in-line with the column, which directs compounds either to waste or to a cryotrap at the operator's discretion. This enables the creation of, for example, aroma reconstitutions incorporating all of the volatiles in a sample, including instrumentally undetectable compounds as well those present at concentrations below sensory thresholds, thus correcting for the “reconstitution discrepancy" sometimes noted in flavor chemistry studies. Using only flowering lavender (Lavandula angustifola ‘Hidcote Blue’) as a source for volatiles, we used the instrument to build mixtures of subsets of lavender volatiles in-instrument and characterized their aroma qualities with a sensory panel. We showed evidence of additive, masking, and synergistic effects in these mixtures and of “lavender' aroma character as an emergent property of specific mixtures. This was accomplished without the need for chemical standards, reductive aroma models, or calculation of Odor Activity Values, and is broadly applicable to any aroma or flavor. PMID:22912722

Perceptual characterization and analysis of aroma mixtures using gas chromatography recomposition-olfactometry.

PubMed

Johnson, Arielle J; Hirson, Gregory D; Ebeler, Susan E

2012-01-01

This paper describes the design of a new instrumental technique, Gas Chromatography Recomposition-Olfactometry (GC-R), that adapts the reconstitution technique used in flavor chemistry studies by extracting volatiles from a sample by headspace solid-phase microextraction (SPME), separating the extract on a capillary GC column, and recombining individual compounds selectively as they elute off of the column into a mixture for sensory analysis (Figure 1). Using the chromatogram of a mixture as a map, the GC-R instrument allows the operator to "cut apart" and recombine the components of the mixture at will, selecting compounds, peaks, or sections based on retention time to include or exclude in a reconstitution for sensory analysis. Selective recombination is accomplished with the installation of a Deans Switch directly in-line with the column, which directs compounds either to waste or to a cryotrap at the operator's discretion. This enables the creation of, for example, aroma reconstitutions incorporating all of the volatiles in a sample, including instrumentally undetectable compounds as well those present at concentrations below sensory thresholds, thus correcting for the "reconstitution discrepancy" sometimes noted in flavor chemistry studies. Using only flowering lavender (Lavandula angustifola 'Hidcote Blue') as a source for volatiles, we used the instrument to build mixtures of subsets of lavender volatiles in-instrument and characterized their aroma qualities with a sensory panel. We showed evidence of additive, masking, and synergistic effects in these mixtures and of "lavender' aroma character as an emergent property of specific mixtures. This was accomplished without the need for chemical standards, reductive aroma models, or calculation of Odor Activity Values, and is broadly applicable to any aroma or flavor.

Tethered bilayer lipid membranes (tBLMs): interest and applications for biological membrane investigations.

PubMed

Rebaud, Samuel; Maniti, Ofelia; Girard-Egrot, Agnès P

2014-12-01

Biological membranes play a central role in the biology of the cell. They are not only the hydrophobic barrier allowing separation between two water soluble compartments but also a supra-molecular entity that has vital structural functions. Notably, they are involved in many exchange processes between the outside and inside cellular spaces. Accounting for the complexity of cell membranes, reliable models are needed to acquire current knowledge of the molecular processes occurring in membranes. To simplify the investigation of lipid/protein interactions, the use of biomimetic membranes is an approach that allows manipulation of the lipid composition of specific domains and/or the protein composition, and the evaluation of the reciprocal effects. Since the middle of the 80's, lipid bilayer membranes have been constantly developed as models of biological membranes with the ultimate goal to reincorporate membrane proteins for their functional investigation. In this review, after a brief description of the planar lipid bilayers as biomimetic membrane models, we will focus on the construction of the tethered Bilayer Lipid Membranes, the most promising model for efficient membrane protein reconstitution and investigation of molecular processes occurring in cell membranes. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

A Very Low Geno2pheno False Positive Rate Is Associated with Poor Viro-Immunological Response in Drug-Naïve Patients Starting a First-Line HAART

PubMed Central

Armenia, Daniele; Soulie, Cathia; Di Carlo, Domenico; Fabeni, Lavinia; Gori, Caterina; Forbici, Federica; Svicher, Valentina; Bertoli, Ada; Sarmati, Loredana; Giuliani, Massimo; Latini, Alessandra; Boumis, Evangelo; Zaccarelli, Mauro; Bellagamba, Rita; Andreoni, Massimo; Marcelin, Anne-Geneviève; Calvez, Vincent; Antinori, Andrea; Ceccherini-Silberstein, Francesca; Perno, Carlo-Federico; Santoro, Maria Mercedes

2014-01-01

Background We previously found that a very low geno2pheno false positive rate (FPR ≤2%) defines a viral population associated with low CD4 cell count and the highest amount of X4-quasispecies. In this study, we aimed at evaluating whether FPR ≤2% might impact on the viro-immunological response in HIV-1 infected patients starting a first-line HAART. Methods The analysis was performed on 305 HIV-1 B subtype infected drug-naïve patients who started their first-line HAART. Baseline FPR (%) values were stratified according to the following ranges: ≤2; 2–5; 5–10; 10–20; 20–60; >60. The impact of genotypically-inferred tropism on the time to achieve immunological reconstitution (a CD4 cell count gain from HAART initiation ≥150 cells/mm3) and on the time to achieve virological success (the first HIV-RNA measurement <50 copies/mL from HAART initiation) was evaluated by survival analyses. Results Overall, at therapy start, 27% of patients had FPR ≤10 (6%, FPR ≤2; 7%, FPR 2–5; 14%, FPR 5–10). By 12 months of therapy the rate of immunological reconstitution was overall 75.5%, and it was significantly lower for FPR ≤2 (54.1%) in comparison to other FPR ranks (78.8%, FPR 2–5; 77.5%, FPR 5–10; 71.7%, FPR 10–20; 81.8%, FPR 20–60; 75.1%, FPR >60; p = 0.008). The overall proportion of patients achieving virological success was 95.5% by 12 months of therapy. Multivariable Cox analyses showed that patients having pre-HAART FPR ≤2% had a significant lower relative adjusted hazard [95% C.I.] both to achieve immunological reconstitution (0.37 [0.20–0.71], p = 0.003) and to achieve virological success (0.50 [0.26–0.94], p = 0.031) than those with pre-HAART FPR >60%. Conclusions Beyond the genotypically-inferred tropism determination, FPR ≤2% predicts both a poor immunological reconstitution and a lower virological response in drug-naïve patients who started their first-line therapy. This parameter could be useful to identify patients potentially with less chance of achieving adequate immunological reconstitution and virological undetectability. PMID:25153969

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

Mavrakis, K. J.; McDonald, E. R.; Schlabach, M. R.

5-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway. The MTAP gene is frequently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. By interrogating data from a large-scale short hairpin RNA–mediated screen across 390 cancer cell line models, we found that the viability of MTAP-deficient cancer cells is impaired by depletion of the protein arginine methyltransferase PRMT5. MTAP-deleted cells accumulate the metabolite methylthioadenosine (MTA), which we found to inhibit PRMT5 methyltransferase activity. Deletion of MTAP in MTAP-proficient cells rendered them sensitive to PRMT5 depletion. Conversely, reconstitution of MTAP in anmore » MTAP-deficient cell line rescued PRMT5 dependence. Thus, MTA accumulation in MTAP–deleted cancers creates a hypomorphic PRMT5 state that is selectively sensitized toward further PRMT5 inhibition. Inhibitors of PRMT5 that leverage this dysregulated metabolic state merit further investigation as a potential therapy for MTAP/CDKN2A-deleted tumors.« less

Inhibitory effect of magnolol on tumour metastasis in mice.

PubMed

Ikeda, Koji; Sakai, Yoshimichi; Nagase, Hisamitsu

2003-09-01

It has previously been reported that magnolol, a phenolic compound isolated from Magnolia obovata, inhibited tumour cell invasion in vitro. The purpose of this study was to investigate the antimetastatic effect of magnolol on tumour metastasis in vivo with experimental and spontaneous metastasis models and to clarify the mechanism. The antimetastatic effects of magnolol were evaluated by an experimental liver and spleen metastasis model using L5178Y-ML25 lymphoma, or an experimental and spontaneous lung metastasis model using B16-BL6 melanoma. Intraperitoneal (i.p.) administration of 2 or 10 mg/kg of magnolol significantly suppressed liver and spleen metastasis or lung metastasis. As for the spontaneous lung metastasis model using B16-BL6 melanoma, multiple i.p. administrations of 10 mg/kg of magnolol after and before tumour inoculation significantly suppressed lung metastasis and primary tumour growth. In addition, magnolol significantly inhibited B16-BL6 cell invasion of the reconstituted basement membrane (Matrigel, MG) without affecting cell growth. These data from the in vivo experiments suggest that magnolol possesses strong antimetastatic ability and that it may be a lead compound for drug development. The antimetastatic action of magnolol is considered to be due to its ability to inhibit tumour cell invasion. Copyright 2003 John Wiley & Sons, Ltd.

Essential role of caspase-8 in p53/p73-dependent apoptosis induced by etoposide in head and neck carcinoma cells

PubMed Central

2011-01-01

Background Caspase-8 is a key upstream mediator in death receptor-mediated apoptosis and also participates in mitochondria-mediated apoptosis via cleavage of proapoptotic Bid. However, the role of caspase-8 in p53- and p73-dependent apoptosis induced by genotoxic drugs remains unclear. We recently reported that the reconstitution of procaspase-8 is sufficient for sensitizing cisplatin- but not etoposide-induced apoptosis, in chemoresistant and caspase-8 deficient HOC313 head and neck squamous cell carcinoma (HNSCC) cells. Results We show that p53/p73-dependent caspase-8 activation is required for sensitizing etoposide-induced apoptosis by utilizing HOC313 cells carrying a temperature-sensitive p53G285K mutant. Restoration of wild-type p53 function under the permissive conditions, together with etoposide treatment, led to substantial transcriptional activation of proapoptotic Noxa and PUMA, but failed to induce apoptosis. In addition to p53 restoration, caspase-8 reconstitution was needed for sensitization to etoposide-induced apoptosis, mitochondria depolarization, and cleavage of the procaspases-3, and -9. In etoposide-sensitive Ca9-22 cells carrying a temperature-insensitive mutant p53, siRNA-based p73 knockdown blocked etoposide-induced apoptosis and procaspase-8 cleavage. However, induction of p73 protein and up-regulation of Noxa and PUMA, although observed in Ca9-22 cells, were hardly detected in etoposide-treated HOC313 cells under non-permissive conditions, suggesting a contribution of p73 reduction to etoposide resistance in HOC313 cells. Finally, the caspase-9 inhibitor Ac-LEHD-CHO or caspase-9 siRNA blocked etoposide-induced caspase-8 activation, Bid cleavage, and apoptosis in both cell lines, indicating that p53/p73-dependent caspase-8 activation lies downstream of mitochondria. Conclusions we conclude that p53 and p73 can act as upstream regulators of caspase-8, and that caspase-8 is an essential mediator of the p53/p73-dependent apoptosis induced by etoposide in HNSCC cells. Our data suggest the importance of caspase-8-mediated positive feedback amplification in the p53/p73-dependent apoptosis induced by etoposide in HNSCC cells. PMID:21801448

Essential role of caspase-8 in p53/p73-dependent apoptosis induced by etoposide in head and neck carcinoma cells.

PubMed

Liu, Juan; Uematsu, Hiroshi; Tsuchida, Nobuo; Ikeda, Masa-Aki

2011-07-31

Caspase-8 is a key upstream mediator in death receptor-mediated apoptosis and also participates in mitochondria-mediated apoptosis via cleavage of proapoptotic Bid. However, the role of caspase-8 in p53- and p73-dependent apoptosis induced by genotoxic drugs remains unclear. We recently reported that the reconstitution of procaspase-8 is sufficient for sensitizing cisplatin- but not etoposide-induced apoptosis, in chemoresistant and caspase-8 deficient HOC313 head and neck squamous cell carcinoma (HNSCC) cells. We show that p53/p73-dependent caspase-8 activation is required for sensitizing etoposide-induced apoptosis by utilizing HOC313 cells carrying a temperature-sensitive p53G285K mutant. Restoration of wild-type p53 function under the permissive conditions, together with etoposide treatment, led to substantial transcriptional activation of proapoptotic Noxa and PUMA, but failed to induce apoptosis. In addition to p53 restoration, caspase-8 reconstitution was needed for sensitization to etoposide-induced apoptosis, mitochondria depolarization, and cleavage of the procaspases-3, and -9. In etoposide-sensitive Ca9-22 cells carrying a temperature-insensitive mutant p53, siRNA-based p73 knockdown blocked etoposide-induced apoptosis and procaspase-8 cleavage. However, induction of p73 protein and up-regulation of Noxa and PUMA, although observed in Ca9-22 cells, were hardly detected in etoposide-treated HOC313 cells under non-permissive conditions, suggesting a contribution of p73 reduction to etoposide resistance in HOC313 cells. Finally, the caspase-9 inhibitor Ac-LEHD-CHO or caspase-9 siRNA blocked etoposide-induced caspase-8 activation, Bid cleavage, and apoptosis in both cell lines, indicating that p53/p73-dependent caspase-8 activation lies downstream of mitochondria. we conclude that p53 and p73 can act as upstream regulators of caspase-8, and that caspase-8 is an essential mediator of the p53/p73-dependent apoptosis induced by etoposide in HNSCC cells. Our data suggest the importance of caspase-8-mediated positive feedback amplification in the p53/p73-dependent apoptosis induced by etoposide in HNSCC cells.

HSC extrinsic sex-related and intrinsic autoimmune disease-related human B-cell variation is recapitulated in humanized mice.

PubMed

Borsotti, Chiara; Danzl, Nichole M; Nauman, Grace; Hölzl, Markus A; French, Clare; Chavez, Estefania; Khosravi-Maharlooei, Mohsen; Glauzy, Salome; Delmotte, Fabien R; Meffre, Eric; Savage, David G; Campbell, Sean R; Goland, Robin; Greenberg, Ellen; Bi, Jing; Satwani, Prakash; Yang, Suxiao; Bathon, Joan; Winchester, Robert; Sykes, Megan

2017-10-24

B cells play a major role in antigen presentation and antibody production in the development of autoimmune diseases, and some of these diseases disproportionally occur in females. Moreover, immune responses tend to be stronger in female vs male humans and mice. Because it is challenging to distinguish intrinsic from extrinsic influences on human immune responses, we used a personalized immune (PI) humanized mouse model, in which immune systems were generated de novo from adult human hematopoietic stem cells (HSCs) in immunodeficient mice. We assessed the effect of recipient sex and of donor autoimmune diseases (type 1 diabetes [T1D] and rheumatoid arthritis [RA]) on human B-cell development in PI mice. We observed that human B-cell levels were increased in female recipients regardless of the source of human HSCs or the strain of immunodeficient recipient mice. Moreover, mice injected with T1D- or RA-derived HSCs displayed B-cell abnormalities compared with healthy control HSC-derived mice, including altered B-cell levels, increased proportions of mature B cells and reduced CD19 expression. Our study revealed an HSC-extrinsic effect of recipient sex on human B-cell reconstitution. Moreover, the PI humanized mouse model revealed HSC-intrinsic defects in central B-cell tolerance that recapitulated those in patients with autoimmune diseases. These results demonstrate the utility of humanized mouse models as a tool to better understand human immune cell development and regulation.

Evaluation of accessory cell heterogeneity. I. Differential accessory cell requirement for T helper cell activation and for T-B cooperation.

PubMed

Ramila, G; Studer, S; Kennedy, M; Sklenar, I; Erb, P

1985-01-01

Several Ia+ tumor cell lines and peritoneal exudate macrophages were tested as accessory cells (AC) for the activation of antigen-specific T cells and for T-B cooperation. The macrophages and all the Ia+ tumor lines tested induced the release of lymphokines from T cells in a major histocompatibility complex (MHC)-restricted fashion and reconstituted the antibody responses of AC-depleted spleen cells or of purified T and B cells. However, only the normal macrophages but none of the tumor lines induced carrier-specific T helper (Th) cells which help B cells for specific antihapten antibody responses by linked recognition. For T-B cooperation accessory cells were also required, but in contrast to Th cell activation any type of Ia+ AC (e.g. macrophage or tumor line) was effective. Strong MHC-restriction between the lymphocytes and the AC was seen if antigen-pulsed AC were added into the AC-depleted T-B cooperation cultures. If the AC and antigen were concomitantly added to the AC-depleted T-B cultures, MHC-restriction was less obvious. Concanavalin A supernatant reconstituted the response of AC-depleted T-B cultures provided antigen-specific Th cells and the hapten-carrier conjugate were present. If, however, tumor line-activated T cells were added instead of macrophage-induced Th cells, no cooperation with B cells took place even in the presence of Con A supernatant. The results obtained demonstrate a differential AC requirement for the induction of Th cells depending on the differentiation stage of the Th cells.

[The effect of Angelica sinensis on adhesion, invasion, migration and metastasis of melanoma cells].

PubMed

Gu, Qin; Xu, Jian-ya; Cheng, Luo-gen; Xia, Wei-jun

2007-03-01

To study the effect of Angelica sinensis on invasion, adhesion, migration and metastasis of B16-BL6 metastatic mouse melanoma cells and discuss its functional mechanism. The proliferation, adhesion, invasion and migration capacity of B16-BL6 metastatic cells was evaluated by MTT assay, adhesion assay and reconstituted basement membrane invasion and migration assay in vitro respectively. Mouse spontaneous melanoma model was used to study the effect of Angelica sinensis on metastasis in vivo. The extract of Angelica sinensis inhibited the proliferation of B16-BL6 metastatic cells and its migration capacity significantly. It regulated bidirectionally the adhesion of B16-BL6 metastatic cells to the basement component laminin while it had no effect on the invasion capacity. In the mouse spotaneous melanoma model, the lung metastatic nodes number and its volume were significantly decreased after continuously treated with the extract of Angelica sinensis at the concentration of 3.67 mg/kg. The extract of Angelica sinensis can inhibit the metastasis of of B16-BL6 metastatic mouse melanoma cells and its mechanism is maybe that Angelica sinensis can inhibit the B16-BL6 cells adhering to the ECM and reduce the migration of B16-BL6 cells.

Hematologic, immunologic reconstitution, and outcome of 342 autologous peripheral blood stem cell transplantations after cryopreservation in a -80°C mechanical freezer and preserved less than 6 months.

PubMed

Calvet, Laure; Cabrespine, Aurélie; Boiret-Dupré, Nathalie; Merlin, Etienne; Paillard, Catherine; Berger, Marc; Bay, Jacques-Olivier; Tournilhac, Olivier; Halle, Pascale

2013-03-01

Controlled-rate freezing and storage in nitrogen is the standard technique for cryopreservation of peripheral hematopoietic progenitor cells (PHPCs) but presents high cost and dimethyl sulfoxide (DMSO) toxicity. Cryopreservation at -80°C, by uncontrolled rate freezing with only 3.5% DMSO, preserves the functional capacities of PHPCs, produces successful engraftment, and reduces toxicity during infusion. Long-term hematopoietic and immunologic reconstitution for 342 autografts (311 adults, 31 children) after PHPCs were cryopreserved at -80°C was studied at 3, 6, and 12 months. The median (range) storage time of PHPCs cryopreserved was 1.7 (0.1-5.99) months. Hemoglobin (Hb), white blood cells, and platelets (PLTs) reach normal values to trilineage at 12 months for 39% patients. Multivariate analysis shows a significant impact on CD34+ infused and on conditioning regimen for PLTs. Hb was influenced by growth factor administration at 3 months. Long-term recovery is also highly dependent on blood counts (Hb, PLT, and neutrophil) at start of high-dose chemotherapy. Only 43% of patients had reached normal lymphocyte values at 12 months after transplant, and a profound CD4+ T-lymphocyte deficit remained, as others reported. Transplantation with PHPCs cryopreserved at -80°C for no more than 6 months is satisfactory for long-term hematopoietic and immunologic reconstitution, even if a profound CD4+ T lymphocyte deficit persists at 1 year. This easier and cheaper cryopreservation method also leads to successful engraftment. © 2012 American Association of Blood Banks.

Mature induced-pluripotent-stem-cell-derived human podocytes reconstitute kidney glomerular-capillary-wall function on a chip

PubMed Central

Musah, Samira; Mammoto, Akiko; Ferrante, Thomas C.; Jeanty, Sauveur S. F.; Hirano-Kobayashi, Mariko; Mammoto, Tadanori; Roberts, Kristen; Chung, Seyoon; Novak, Richard; Ingram, Miles; Fatanat-Didar, Tohid; Koshy, Sandeep; Weaver, James C.; Church, George M.; Ingber, Donald E.

2017-01-01

An in vitro model of the human kidney glomerulus — the major site of blood filtration — could facilitate drug discovery and illuminate kidney-disease mechanisms. Microfluidic organ-on-a-chip technology has been used to model the human proximal tubule, yet a kidney-glomerulus-on-a-chip has not been possible because of the lack of functional human podocytes — the cells that regulate selective permeability in the glomerulus. Here, we demonstrate an efficient (> 90%) and chemically defined method for directing the differentiation of human induced pluripotent stem (hiPS) cells into podocytes that express markers of the mature phenotype (nephrin+, WT1+, podocin+, Pax2−) and that exhibit primary and secondary foot processes. We also show that the hiPS-cell-derived podocytes produce glomerular basement-membrane collagen and recapitulate the natural tissue/tissue interface of the glomerulus, as well as the differential clearance of albumin and inulin, when co-cultured with human glomerular endothelial cells in an organ-on-a-chip microfluidic device. The glomerulus-on-a-chip also mimics adriamycin-induced albuminuria and podocyte injury. This in vitro model of human glomerular function with mature human podocytes may facilitate drug development and personalized-medicine applications. PMID:29038743

Macrophages sustain HIV replication in vivo independently of T cells

PubMed Central

Wahl, Angela; Baker, Caroline; Spagnuolo, Rae Ann; Foster, John; Zakharova, Oksana; Wietgrefe, Stephen; Caro-Vegas, Carolina; Sharpe, Garrett; Haase, Ashley T.; Eron, Joseph J.; Garcia, J. Victor

2016-01-01

Macrophages have long been considered to contribute to HIV infection of the CNS; however, a recent study has contradicted this early work and suggests that myeloid cells are not an in vivo source of virus production. Here, we addressed the role of macrophages in HIV infection by first analyzing monocytes isolated from viremic patients and patients undergoing antiretroviral treatment. We were unable to find viral DNA or viral outgrowth in monocytes isolated from peripheral blood. To determine whether tissue macrophages are productively infected, we used 3 different but complementary humanized mouse models. Two of these models (bone marrow/liver/thymus [BLT] mice and T cell–only mice [ToM]) have been previously described, and the third model was generated by reconstituting immunodeficient mice with human CD34+ hematopoietic stem cells that were devoid of human T cells (myeloid-only mice [MoM]) to specifically evaluate HIV replication in this population. Using MoM, we demonstrated that macrophages can sustain HIV replication in the absence of T cells; HIV-infected macrophages are distributed in various tissues including the brain; replication-competent virus can be rescued ex vivo from infected macrophages; and infected macrophages can establish de novo infection. Together, these results demonstrate that macrophages represent a genuine target for HIV infection in vivo that can sustain and transmit infection. PMID:26950420

Donor Satellite Cell Engraftment is Significantly Augmented When the Host Niche is Preserved and Endogenous Satellite Cells are Incapacitated

PubMed Central

Boldrin, Luisa; Neal, Alice; Zammit, Peter S; Muntoni, Francesco; Morgan, Jennifer E

2012-01-01

Stem cell transplantation is already in clinical practice for certain genetic diseases and is a promising therapy for dystrophic muscle. We used the mdx mouse model of Duchenne muscular dystrophy to investigate the effect of the host satellite cell niche on the contribution of donor muscle stem cells (satellite cells) to muscle regeneration. We found that incapacitation of the host satellite cells and preservation of the muscle niche promote donor satellite cell contribution to muscle regeneration and functional reconstitution of the satellite cell compartment. But, if the host niche is not promptly refilled, or is filled by competent host satellite cells, it becomes nonfunctional and donor engraftment is negligible. Application of this regimen to aged host muscles also promotes efficient regeneration from aged donor satellite cells. In contrast, if the niche is destroyed, yet host satellite cells remain proliferation-competent, donor-derived engraftment is trivial. Thus preservation of the satellite cell niche, concomitant with functional impairment of the majority of satellite cells within dystrophic human muscles, may improve the efficiency of stem cell therapy. Stem Cells2012;30:1971–1984 PMID:22730231

The Effects of Low Dose Irradiation on Inflammatory Response Proteins in a 3D Reconstituted Human Skin Tissue Model

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

Varnum, Susan M.; Springer, David L.; Chaffee, Mary E.

Skin responses to moderate and high doses of ionizing radiation include the induction of DNA repair, apoptosis, and stress response pathways. Additionally, numerous studies indicate that radiation exposure leads to inflammatory responses in skin cells and tissue. However, the inflammatory response of skin tissue to low dose radiation (<10 cGy) is poorly understood. In order to address this, we have utilized a reconstituted human skin tissue model (MatTek EpiDerm FT) and assessed changes in 23 cytokines twenty-four and forty eight hours following treatment of skin with either 3 or 10 cGy low-dose of radiation. Three cytokines, IFN-γ, IL-2, MIP-1α, weremore » significantly altered in response to low dose radiation. In contrast, seven cytokines were significantly altered in response to a high radiation dose of 200 cGy (IL-2, IL-10, IL-13, IFN-γ, MIP-1α, TNF α, and VEGF) or the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (G-CSF, GM-CSF, IL-1α, IL-8, MIP-1α, MIP-1β, RANTES). Additionally, radiation induced inflammation appears to have a distinct cytokine response relative to the non-radiation induced stressor, TPA. Overall, these results indicate that there are subtle changes in the inflammatory protein levels following exposure to low dose radiation and this response is a sub-set of what is seen following a high dose in a human skin tissue model.« less

Combined effects of interleukin-7 and stem cell factor administration on lymphopoiesis after murine bone marrow transplantation.

PubMed

Chung, Brile; Min, Dullei; Joo, Lukas W; Krampf, Mark R; Huang, Jing; Yang, Yujun; Shashidhar, Sumana; Brown, Janice; Dudl, Eric P; Weinberg, Kenneth I

2011-01-01

The decreased ability of the thymus to generate T cells after bone marrow transplantation (BMT) is a clinically significant problem. Interleukin (IL)-7 and stem cell factor (SCF) induce proliferation, differentiation, and survival of thymocytes. Although previous studies have shown that administration of recombinant human IL-7 (rhIL-7) after murine and human BMT improves thymopoiesis and immune function, whether administration of SCF exerts similar effects is unclear. To evaluate independent or combinatorial effects of IL-7 and SCF in post-BMT thymopoiesis, bone marrow (BM)-derived mesenchymal stem cells transduced ex vivo with the rhIL-7 or murine SCF (mSCF) genes were cotransplanted with T cell-depleted BM cells into lethally irradiated mice. Although rhIL-7 and mSCF each improved immune reconstitution, the combination treatment had a significantly greater effect than either cytokine alone. Moreover, the combination treatment significantly increased donor-derived common lymphoid progenitors (CLPs) in BM, suggesting that transplanted CLPs expand more rapidly in response to IL-7 and SCF and may promote immune reconstitution. Our findings demonstrate that IL-7 and SCF might be therapeutically useful for enhancing de novo T cell development. Furthermore, combination therapy may allow the administration of lower doses of IL-7, thereby decreasing the likelihood of IL-7-mediated expansion of mature T cells. 2011. Published by Elsevier Inc.

Assembly of multiple cell gradients directed by three-dimensional microfluidic channels.

PubMed

Li, Yiwei; Feng, Xiaojun; Wang, Yachao; Du, Wei; Chen, Peng; Liu, Chao; Liu, Bi-Feng

2015-08-07

Active control over the cell gradient is essential for understanding biological systems and the reconstitution of the functionality of many types of tissues, particularly for organ-on-a-chip. Here, we propose a three-dimensional (3D) microfluidic strategy for generating controllable cell gradients. In this approach, a homogeneous cell suspension is loaded into a 3D stair-shaped PDMS microchannel to generate a cell gradient within 10 min by sedimentation. We demonstrate that cell gradients of various profiles (exponential and piecewise linear) can be achieved by precisely controlling the height of each layer during the fabrication. With sequential seeding, we further demonstrate the generation of two overlapping cell gradients on the same glass substrate with pre-defined designs. The cell gradient-based QD cytotoxicity assay also demonstrated that cell behaviors and resistances were regulated by the changes in cell density. These results reveal that the proposed 3D microfluidic strategy provides a simple and versatile means for establishing controllable gradients in cell density, opening up a new avenue for reconstructing functional tissues.

The Nlrp3 inflammasome promotes age-related thymic demise and immunosenescence.

PubMed

Youm, Yun-Hee; Kanneganti, Thirumala-Devi; Vandanmagsar, Bolormaa; Zhu, Xuewei; Ravussin, Anthony; Adijiang, Ayinuer; Owen, John S; Thomas, Michael J; Francis, Joseph; Parks, John S; Dixit, Vishwa Deep

2012-01-26

The collapse of thymic stromal cell microenvironment with age and resultant inability of the thymus to produce naive T cells contributes to lower immune-surveillance in the elderly. Here we show that age-related increase in 'lipotoxic danger signals' such as free cholesterol (FC) and ceramides, leads to thymic caspase-1 activation via the Nlrp3 inflammasome. Elimination of Nlrp3 and Asc, a critical adaptor required for inflammasome assembly, reduces age-related thymic atrophy and results in an increase in cortical thymic epithelial cells, T cell progenitors and maintenance of T cell repertoire diversity. Using a mouse model of irradiation and hematopoietic stem cell transplantation (HSCT), we show that deletion of the Nlrp3 inflammasome accelerates T cell reconstitution and immune recovery in middle-aged animals. Collectively, these data demonstrate that lowering inflammasome-dependent caspase-1 activation increases thymic lymphopoiesis and suggest that Nlrp3 inflammasome inhibitors may aid the re-establishment of a diverse T cell repertoire in middle-aged or elderly patients undergoing HSCT. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

Growth and characterization of different human rhinovirus C types in three-dimensional human airway epithelia reconstituted in vitro

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

Tapparel, Caroline, E-mail: Caroline.Tapparel@hcuge.ch; Sobo, Komla; Constant, Samuel

New molecular diagnostic tools have recently allowed the discovery of human rhinovirus species C (HRV-C) that may be overrepresented in children with lower respiratory tract complications. Unlike HRV-A and HRV-B, HRV-C cannot be propagated in conventional immortalized cell lines and their biological properties have been difficult to study. Recent studies have described the successful amplification of HRV-C15, HRV-C11, and HRV-C41 in sinus mucosal organ cultures and in fully differentiated human airway epithelial cells. Consistent with these studies, we report that a panel of clinical HRV-C specimens including HRV-C2, HRV-C7, HRV-C12, HRV-C15, and HRV-C29 types were all capable of mediating productivemore » infection in reconstituted 3D human primary upper airway epithelial tissues and that the virions enter and exit preferentially through the apical surface. Similar to HRV-A and HRV-B, our data support the acid sensitivity of HRV-C. We observed also that the optimum temperature requirement during HRV-C growth may be type-dependent. - Highlights: • A 3D human upper airway epithelia reconstituted in vitro supports HRV-C growth. • HRV-Cs enter and exit preferentially at the apical side of this ALI culture system. • HRV-Cs are acid sensitive. • Temperature sensitivity may be type-dependent for HRV-Cs.« less

CMV reactivation drives posttransplant T-cell reconstitution and results in defects in the underlying TCRβ repertoire

PubMed Central

Suessmuth, Yvonne; Mukherjee, Rithun; Watkins, Benjamin; Koura, Divya T.; Finstermeier, Knut; Desmarais, Cindy; Stempora, Linda; Horan, John T.; Langston, Amelia; Qayed, Muna; Khoury, Hanna J.; Grizzle, Audrey; Cheeseman, Jennifer A.; Conger, Jason A.; Robertson, Jennifer; Garrett, Aneesah; Kirk, Allan D.; Waller, Edmund K.; Blazar, Bruce R.; Mehta, Aneesh K.; Robins, Harlan S.

2015-01-01

Although cytomegalovirus (CMV) reactivation has long been implicated in posttransplant immune dysfunction, the molecular mechanisms that drive this phenomenon remain undetermined. To address this, we combined multiparameter flow cytometric analysis and T-cell subpopulation sorting with high-throughput sequencing of the T-cell repertoire, to produce a thorough evaluation of the impact of CMV reactivation on T-cell reconstitution after unrelated-donor hematopoietic stem cell transplant. We observed that CMV reactivation drove a >50-fold specific expansion of Granzyme Bhigh/CD28low/CD57high/CD8+ effector memory T cells (Tem) and resulted in a linked contraction of all naive T cells, including CD31+/CD4+ putative thymic emigrants. T-cell receptor β (TCRβ) deep sequencing revealed a striking contraction of CD8+ Tem diversity due to CMV-specific clonal expansions in reactivating patients. In addition to querying the topography of the expanding CMV-specific T-cell clones, deep sequencing allowed us, for the first time, to exhaustively evaluate the underlying TCR repertoire. Our results reveal new evidence for significant defects in the underlying CD8 Tem TCR repertoire in patients who reactivate CMV, providing the first molecular evidence that, in addition to driving expansion of virus-specific cells, CMV reactivation has a detrimental impact on the integrity and heterogeneity of the rest of the T-cell repertoire. This trial was registered at www.clinicaltrials.gov as #NCT01012492. PMID:25852054

RT-PCR detection of Candida albicans ALS gene expression in the reconstituted human epithelium (RHE) model of oral candidiasis and in model biofilms.

PubMed

Green, Clayton B; Cheng, Georgina; Chandra, Jyotsna; Mukherjee, Pranab; Ghannoum, Mahmoud A; Hoyer, Lois L

2004-02-01

An RT-PCR assay was developed to analyse expression patterns of genes in the Candida albicans ALS (agglutinin-like sequence) family. Inoculation of a reconstituted human buccal epithelium (RHE) model of mucocutaneous candidiasis with strain SC5314 showed destruction of the epithelial layer by C. albicans and also formation of an upper fungal layer that had characteristics similar to a biofilm. RT-PCR analysis of total RNA samples extracted from C. albicans-inoculated buccal RHE showed that ALS1, ALS2, ALS3, ALS4, ALS5 and ALS9 were consistently detected over time as destruction of the RHE progressed. Detection of transcripts from ALS7, and particularly from ALS6, was more sporadic, but not associated with a strictly temporal pattern. The expression pattern of ALS genes in C. albicans cultures used to inoculate the RHE was similar to that observed in the RHE model, suggesting that contact of C. albicans with buccal RHE does little to alter ALS gene expression. RT-PCR analysis of RNA samples extracted from model denture and catheter biofilms showed similar gene expression patterns to the buccal RHE specimens. Results from the RT-PCR analysis of biofilm RNA specimens were consistent between various C. albicans strains during biofilm development and were comparable to gene expression patterns in planktonic cells. The RT-PCR assay described here will be useful for analysis of human clinical specimens and samples from other disease models. The method will provide further insight into the role of ALS genes and their encoded proteins in the diverse interactions between C. albicans and its host.

Expanding xylose metabolism in yeast for plant cell wall conversion to biofuels.

PubMed

Li, Xin; Yu, Vivian Yaci; Lin, Yuping; Chomvong, Kulika; Estrela, Raíssa; Park, Annsea; Liang, Julie M; Znameroski, Elizabeth A; Feehan, Joanna; Kim, Soo Rin; Jin, Yong-Su; Glass, N Louise; Cate, Jamie H D

2015-02-03

Sustainable biofuel production from renewable biomass will require the efficient and complete use of all abundant sugars in the plant cell wall. Using the cellulolytic fungus Neurospora crassa as a model, we identified a xylodextrin transport and consumption pathway required for its growth on hemicellulose. Reconstitution of this xylodextrin utilization pathway in Saccharomyces cerevisiae revealed that fungal xylose reductases act as xylodextrin reductases, producing xylosyl-xylitol oligomers as metabolic intermediates. These xylosyl-xylitol intermediates are generated by diverse fungi and bacteria, indicating that xylodextrin reduction is widespread in nature. Xylodextrins and xylosyl-xylitol oligomers are then hydrolyzed by two hydrolases to generate intracellular xylose and xylitol. Xylodextrin consumption using a xylodextrin transporter, xylodextrin reductases and tandem intracellular hydrolases in cofermentations with sucrose and glucose greatly expands the capacity of yeast to use plant cell wall-derived sugars and has the potential to increase the efficiency of both first-generation and next-generation biofuel production.

Hepatic progenitor cells of biliary origin with liver repopulation capacity

PubMed Central

Boulter, Luke; Tsuchiya, Atsunori; Cole, Alicia M; Hay, Trevor; Guest, Rachel V; Wojtacha, Davina; Man, Tak Yung; Mackinnon, Alison; Ridgway, Rachel A; Kendall, Timothy; Williams, Michael J; Jamieson, Thomas; Raven, Alex; Hay, David C; Iredale, John P; Clarke, Alan R; Sansom, Owen J; Forbes, Stuart J

2015-01-01

Summary Hepatocytes and cholangiocytes self renew following liver injury. Following severe injury hepatocytes are increasingly senescent, whether Hepatic Progenitor Cells (HPCs) then contribute to liver regeneration is unclear. Here, we describe a mouse model where Mdm2 is inducibly deleted in over 98% of hepatocytes, causing apoptosis, necrosis and senescence with nearly all hepatocytes expressing p21. This results in florid HPC activation, which is necessary for survival, followed by complete, functional liver reconstitution. HPCs isolated from genetically normal mice, using cell surface markers, were highly expandable and phenotypically stable in vitro. These HPCs were transplanted into adult mouse livers where hepatocyte Mdm2 was repeatedly deleted, creating a non-competitive repopulation assay. Transplanted HPCs contributed significantly to restoration of liver parenchyma, regenerating hepatocytes and biliary epithelia, highlighting their in vivo lineage potency. HPCs are therefore a potential future alternative to hepatocyte or liver transplantation for liver disease. PMID:26192438

Bioprinting of 3D Tissue Models Using Decellularized Extracellular Matrix Bioink.

PubMed

Pati, Falguni; Cho, Dong-Woo

2017-01-01

Bioprinting provides an exciting opportunity to print and pattern all the components that make up a tissue-cells and extracellular matrix (ECM) material-in three dimensions (3D) to generate tissue analogues. A large number of materials have been used for making bioinks; however, majority of them cannot represent the complexity of natural ECM and thus are unable to reconstitute the intrinsic cellular morphologies and functions. We present here a method for making of bioink from decellularized extracellular matrices (dECMs) and a protocol for bioprinting of cell-laden constructs with this novel bioink. The dECM bioink is capable of providing an optimized microenvironment that is conducive to the growth of 3D structured tissue. We have prepared bioinks from different tissues, including adipose, cartilage and heart tissues and achieved high cell viability and functionality of the bioprinted tissue structures using our novel bioink.

Targeted Delivery of Small Interfering RNA Using Reconstituted High-Density Lipoprotein Nanoparticles12

PubMed Central

Shahzad, Mian MK; Mangala, Lingegowda S; Han, Hee Dong; Lu, Chunhua; Bottsford-Miller, Justin; Nishimura, Masato; Mora, Edna M; Lee, Jeong-Won; Stone, Rebecca L; Pecot, Chad V; Thanapprapasr, Duangmani; Roh, Ju-Won; Gaur, Puja; Nair, Maya P; Park, Yun-Yong; Sabnis, Nirupama; Deavers, Michael T; Lee, Ju-Seog; Ellis, Lee M; Lopez-Berestein, Gabriel; McConathy, Walter J; Prokai, Laszlo; Lacko, Andras G; Sood, Anil K

2011-01-01

RNA interference holds tremendous potential as a therapeutic approach, especially in the treatment of malignant tumors. However, efficient and biocompatible delivery methods are needed for systemic delivery of small interfering RNA (siRNA). To maintain a high level of growth, tumor cells scavenge high-density lipoprotein (HDL) particles by overexpressing its receptor: scavenger receptor type B1 (SR-B1). In this study, we exploited this cellular characteristic to achieve efficient siRNA delivery and established a novel formulation of siRNA by incorporating it into reconstituted HDL (rHDL) nanoparticles. Here, we demonstrate that rHDL nanoparticles facilitate highly efficient systemic delivery of siRNA in vivo, mediated by the SR-B1. Moreover, in therapeutic proof-of-concept studies, these nanoparticles were effective in silencing the expression of two proteins that are key to cancer growth and metastasis (signal transducer and activator of transcription 3 and focal adhesion kinase) in orthotopic mouse models of ovarian and colorectal cancer. These data indicate that an rHDL nanoparticle is a novel and highly efficient siRNA carrier, and therefore, this novel technology could serve as the foundation for new cancer therapeutic approaches. PMID:21472135

Adiponectin Deficiency Impairs Maternal Metabolic Adaptation to Pregnancy in Mice.

PubMed

Qiao, Liping; Wattez, Jean-Sebastien; Lee, Samuel; Nguyen, Amanda; Schaack, Jerome; Hay, William W; Shao, Jianhua

2017-05-01

Hypoadiponectinemia has been widely observed in patients with gestational diabetes mellitus (GDM). To investigate the causal role of hypoadiponectinemia in GDM, adiponectin gene knockout ( Adipoq -/- ) and wild-type (WT) mice were crossed to produce pregnant mouse models with or without adiponectin deficiency. Adenoviral vector-mediated in vivo transduction was used to reconstitute adiponectin during late pregnancy. Results showed that Adipoq -/- dams developed glucose intolerance and hyperlipidemia in late pregnancy. Increased fetal body weight was detected in Adipoq -/- dams. Adiponectin reconstitution abolished these metabolic defects in Adipoq -/- dams. Hepatic glucose and triglyceride production rates of Adipoq -/- dams were significantly higher than those of WT dams. Robustly enhanced lipolysis was found in gonadal fat of Adipoq -/- dams. Interestingly, similar levels of insulin-induced glucose disposal and insulin signaling in metabolically active tissues in Adipoq -/- and WT dams indicated that maternal adiponectin deficiency does not reduce insulin sensitivity. However, remarkably decreased serum insulin concentrations were observed in Adipoq -/- dams. Furthermore, β-cell mass, but not glucose-stimulated insulin release, in Adipoq -/- dams was significantly reduced compared with WT dams. Together, these results demonstrate that adiponectin plays an important role in controlling maternal metabolic adaptation to pregnancy. © 2017 by the American Diabetes Association.

Adiponectin Deficiency Impairs Maternal Metabolic Adaptation to Pregnancy in Mice

PubMed Central

Qiao, Liping; Wattez, Jean-Sebastien; Lee, Samuel; Nguyen, Amanda; Schaack, Jerome; Hay, William W.

2017-01-01

Hypoadiponectinemia has been widely observed in patients with gestational diabetes mellitus (GDM). To investigate the causal role of hypoadiponectinemia in GDM, adiponectin gene knockout (Adipoq−/−) and wild-type (WT) mice were crossed to produce pregnant mouse models with or without adiponectin deficiency. Adenoviral vector–mediated in vivo transduction was used to reconstitute adiponectin during late pregnancy. Results showed that Adipoq−/− dams developed glucose intolerance and hyperlipidemia in late pregnancy. Increased fetal body weight was detected in Adipoq−/− dams. Adiponectin reconstitution abolished these metabolic defects in Adipoq−/− dams. Hepatic glucose and triglyceride production rates of Adipoq−/− dams were significantly higher than those of WT dams. Robustly enhanced lipolysis was found in gonadal fat of Adipoq−/− dams. Interestingly, similar levels of insulin-induced glucose disposal and insulin signaling in metabolically active tissues in Adipoq−/− and WT dams indicated that maternal adiponectin deficiency does not reduce insulin sensitivity. However, remarkably decreased serum insulin concentrations were observed in Adipoq−/− dams. Furthermore, β-cell mass, but not glucose-stimulated insulin release, in Adipoq−/− dams was significantly reduced compared with WT dams. Together, these results demonstrate that adiponectin plays an important role in controlling maternal metabolic adaptation to pregnancy. PMID:28073830

Risk factors for increased immune reconstitution in response to Mycobacterium tuberculosis antigens in tuberculosis HIV-infected, antiretroviral-naïve patients.

PubMed

da Silva, Tatiana Pereira; Giacoia-Gripp, Carmem Beatriz Wagner; Schmaltz, Carolina A; Sant'Anna, Flavia Marinho; Saad, Maria Helena; Matos, Juliana Arruda de; de Lima E Silva, Julio Castro Alves; Rolla, Valeria Cavalcanti; Morgado, Mariza Gonçalves

2017-09-06

Little is known regarding the restoration of the specific immune response after combined antiretroviral therapy (cART) and anti-tuberculosis (TB) therapy introduction among TB-HIV patients. In this study, we examined the immune response of TB-HIV patients to Mycobacterium tuberculosis (Mtb) antigens to evaluate the response dynamics to different antigens over time. Moreover, we also evaluated the influence of two different doses of efavirenz and the factors associated with immune reconstitution. This is a longitudinal study nested in a clinical trial, where cART was initiated during the baseline visit (D0), which occurred 30 ± 10 days after the introduction of anti-TB therapy. Follow-up visits were performed at 30, 60, 90 and 180 days after cART initiation. The production of IFN-γ upon in vitro stimulation with Mtb antigens purified protein derivative (PPD), ESAT-6 and 38 kDa/CFP-10 using ELISpot was examined at baseline and follow-up visits. Sixty-one patients, all ART-naïve, were selected and included in the immune reconstitution analysis; seven (11.5%) developed Immune Reconstitution Inflammatory Syndrome (IRIS). The Mtb specific immune response was higher for the PPD antigen followed by 38 kDa/CFP-10 and increased in the first 60 days after cART initiation. In multivariate analysis, the variables independently associated with increased IFN-γ production in response to PPD antigen were CD4 + T cell counts <200 cells/mm 3 at baseline, age, site of tuberculosis, 800 mg efavirenz dose and follow-up CD4 + T cell counts. Moreover, the factors associated with the production of IFN-γ in response to 38 kDa/CFP-10 were detectable HIV viral load (VL) and CD4 + T cell counts at follow-up visits of ≥200 cells/mm 3 . These findings highlight the differences in immune response according to the specificity of the Mtb antigen, which contributes to a better understanding of TB-HIV immunopathogenesis. IFN-γ production elicited by PPD and 38 kDa/CFP-10 antigens have a greater magnitude compared to ESAT-6 and are associated with different factors. The low response to ESAT-6, even during immune restoration, suggests that this antigen is not adequate to assess the immune response of immunosuppressed TB-HIV patients.

Activation of Saccharomyces cerevisiae Mlh1-Pms1 Endonuclease in a Reconstituted Mismatch Repair System.

PubMed

Smith, Catherine E; Bowen, Nikki; Graham, William J; Goellner, Eva M; Srivatsan, Anjana; Kolodner, Richard D

2015-08-28

Previous studies reported the reconstitution of an Mlh1-Pms1-independent 5' nick-directed mismatch repair (MMR) reaction using Saccharomyces cerevisiae proteins. Here we describe the reconstitution of a mispair-dependent Mlh1-Pms1 endonuclease activation reaction requiring Msh2-Msh6 (or Msh2-Msh3), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) and a reconstituted Mlh1-Pms1-dependent 3' nick-directed MMR reaction requiring Msh2-Msh6 (or Msh2-Msh3), exonuclease 1 (Exo1), replication protein A (RPA), RFC, PCNA, and DNA polymerase δ. Both reactions required Mg(2+) and Mn(2+) for optimal activity. The MMR reaction also required two reaction stages in which the first stage required incubation of Mlh1-Pms1 with substrate DNA, with or without Msh2-Msh6 (or Msh2-Msh3), PCNA, and RFC but did not require nicking of the substrate, followed by a second stage in which other proteins were added. Analysis of different mutant proteins demonstrated that both reactions required a functional Mlh1-Pms1 endonuclease active site, as well as mispair recognition and Mlh1-Pms1 recruitment by Msh2-Msh6 but not sliding clamp formation. Mutant Mlh1-Pms1 and PCNA proteins that were defective for Exo1-independent but not Exo1-dependent MMR in vivo were partially defective in the Mlh1-Pms1 endonuclease and MMR reactions, suggesting that both reactions reflect the activation of Mlh1-Pms1 seen in Exo1-independent MMR in vivo. The availability of this reconstituted MMR reaction should now make it possible to better study both Exo1-independent and Exo1-dependent MMR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Activation of Saccharomyces cerevisiae Mlh1-Pms1 Endonuclease in a Reconstituted Mismatch Repair System*

PubMed Central

Smith, Catherine E.; Bowen, Nikki; Graham, William J.; Goellner, Eva M.; Srivatsan, Anjana; Kolodner, Richard D.

2015-01-01

Previous studies reported the reconstitution of an Mlh1-Pms1-independent 5′ nick-directed mismatch repair (MMR) reaction using Saccharomyces cerevisiae proteins. Here we describe the reconstitution of a mispair-dependent Mlh1-Pms1 endonuclease activation reaction requiring Msh2-Msh6 (or Msh2-Msh3), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) and a reconstituted Mlh1-Pms1-dependent 3′ nick-directed MMR reaction requiring Msh2-Msh6 (or Msh2-Msh3), exonuclease 1 (Exo1), replication protein A (RPA), RFC, PCNA, and DNA polymerase δ. Both reactions required Mg2+ and Mn2+ for optimal activity. The MMR reaction also required two reaction stages in which the first stage required incubation of Mlh1-Pms1 with substrate DNA, with or without Msh2-Msh6 (or Msh2-Msh3), PCNA, and RFC but did not require nicking of the substrate, followed by a second stage in which other proteins were added. Analysis of different mutant proteins demonstrated that both reactions required a functional Mlh1-Pms1 endonuclease active site, as well as mispair recognition and Mlh1-Pms1 recruitment by Msh2-Msh6 but not sliding clamp formation. Mutant Mlh1-Pms1 and PCNA proteins that were defective for Exo1-independent but not Exo1-dependent MMR in vivo were partially defective in the Mlh1-Pms1 endonuclease and MMR reactions, suggesting that both reactions reflect the activation of Mlh1-Pms1 seen in Exo1-independent MMR in vivo. The availability of this reconstituted MMR reaction should now make it possible to better study both Exo1-independent and Exo1-dependent MMR. PMID:26170454

Low levels of aflatoxin B1, ricin and milk enhance recombinant protein production in mammalian cells

USDA-ARS?s Scientific Manuscript database

Changing the optimal tissue culture medium by adding low levels of environmental stress such as 1 µM of the fungal toxin aflatoxin B1 (AFB1), 1 ng of the castor bean protein toxin ricin in transduced mammalian cells or 1% reconstituted milk enhances transcription and increases production of the foll...

Cellular Sites of Immunologic Unresponsiveness*

PubMed Central

Chiller, Jacques M.; Habicht, Gail S.; Weigle, William O.

1970-01-01

The reconstitution of the immune response of lethally irradiated mice to human γ-globulin is dependent on the synergistic action of bone marrow with thymus cells. Immunologic unresponsiveness appears to involve a functional defect at each of these cellular levels, inasmuch as neither bone marrow nor thymus cells from unresponsive donors are capable of demonstrating synergism in combination with their normal counterpart. PMID:4192271

Lentiviral gene transfer regenerates hematopoietic stem cells in a mouse model for Mpl-deficient aplastic anemia.

PubMed

Heckl, Dirk; Wicke, Daniel C; Brugman, Martijn H; Meyer, Johann; Schambach, Axel; Büsche, Guntram; Ballmaier, Matthias; Baum, Christopher; Modlich, Ute

2011-04-07

Thpo/Mpl signaling plays an important role in the maintenance of hematopoietic stem cells (HSCs) in addition to its role in megakaryopoiesis. Patients with inactivating mutations in Mpl develop thrombocytopenia and aplastic anemia because of progressive loss of HSCs. Yet, it is unknown whether this loss of HSCs is an irreversible process. In this study, we used the Mpl knockout (Mpl(-/-)) mouse model and expressed Mpl from newly developed lentiviral vectors specifically in the physiologic Mpl target populations, namely, HSCs and megakaryocytes. After validating lineage-specific expression in vivo using lentiviral eGFP reporter vectors, we performed bone marrow transplantation of transduced Mpl(-/-) bone marrow cells into Mpl(-/-) mice. We show that restoration of Mpl expression from transcriptionally targeted vectors prevents lethal adverse reactions of ectopic Mpl expression, replenishes the HSC pool, restores stem cell properties, and corrects platelet production. In some mice, megakaryocyte counts were atypically high, accompanied by bone neo-formation and marrow fibrosis. Gene-corrected Mpl(-/-) cells had increased long-term repopulating potential, with a marked increase in lineage(-)Sca1(+)cKit(+) cells and early progenitor populations in reconstituted mice. Transcriptome analysis of lineage(-)Sca1(+)cKit(+) cells in Mpl-corrected mice showed functional adjustment of genes involved in HSC self-renewal.

Effects of the lipid environment, cholesterol and bile acids on the function of the purified and reconstituted human ABCG2 protein.

PubMed

Telbisz, Ágnes; Özvegy-Laczka, Csilla; Hegedűs, Tamás; Váradi, András; Sarkadi, Balázs

2013-03-01

The human ABCG2 multidrug transporter actively extrudes a wide range of hydrophobic drugs and xenobiotics recognized by the transporter in the membrane phase. In order to examine the molecular nature of the transporter and its effects on the lipid environment, we have established an efficient protocol for the purification and reconstitution of the functional protein. We found that the drug-stimulated ATPase and the transport activity of ABCG2 are fully preserved by applying excess lipids and mild detergents during solubilization, whereas a detergent-induced dissociation of the ABCG2 dimer causes an irreversible inactivation. By using the purified and reconstituted protein we demonstrate that cholesterol is an essential activator, whereas bile acids are important modulators of ABCG2 activity. Both wild-type ABCG2 and its R482G mutant variant require cholesterol for full activity, although they exhibit different cholesterol sensitivities. Bile acids strongly decrease the basal ABCG2-ATPase activity both in the wild-type ABCG2 and in the mutant variant. These data reinforce the results for the modulatory effects of cholesterol and bile acids of ABCG2 investigated in a complex cell membrane environment. Moreover, these experiments open the possibility to perform functional and structural studies with a purified, reconstituted and highly active ABCG2 multidrug transporter.

An essential role for platelet-activating factor in activating mast cell migration following ultraviolet irradiation

PubMed Central

Chacón-Salinas, Rommel; Chen, Limo; Chávez-Blanco, Alma D.; Limón-Flores, Alberto Y.; Ma, Ying; Ullrich, Stephen E.

2014-01-01

The UVB (290–320 nm) radiation in sunlight is responsible for inducing skin cancer. Exposure to UV radiation is also immunosuppressive, and the systemic immune suppression induced by UV is a well-recognized risk factor for cancer induction. As UVB radiation is absorbed within the upper layers of the skin, indirect mechanisms must play a role in activating systemic immune suppression. One prominent example is mast cell migration, which from the skin to the draining LN is an essential step in the cascade of events leading to immune suppression. What triggers mast cell migration is not entirely clear. Here, we tested the hypothesis that PAF, a lipid mediator of inflammation produced by the skin in response to UV exposure, is involved. Mast cell-deficient mice (KitW-sh/W-sh) are resistant to the suppressive effect of UV radiation, and reconstituting mast cell-deficient mice with normal bone marrow-derived mast cells restores susceptibility to immunosuppression. However, when mast cells from PAFR−/− mice were used, the reconstituted mice were not susceptible to the suppressive effects of UV. Furthermore, PAFR−/− mice showed impaired UV-induced mast cell migration when compared with WT mice. Finally, injecting PAF into WT mice mimicked the effect of UV irradiation and induced mast cell migration but not in PAFR−/− mice. Our findings indicate that PAFR binding induces mast cells to migrate from the skin to the LNs, where they mediate immune suppression. PMID:24009177

Fifty Years of Research in ARDS. Cell-based Therapy for Acute Respiratory Distress Syndrome. Biology and Potential Therapeutic Value.

PubMed

Laffey, John G; Matthay, Michael A

2017-08-01

On the basis of several preclinical studies, cell-based therapy has emerged as a potential new therapeutic for acute respiratory distress syndrome (ARDS). Of the various cell-based therapy options, mesenchymal stem/stromal cells (MSCs) from bone marrow, adipose tissue, and umbilical cord have the most experimental data to support their potential efficacy for lung injury from both infectious and noninfectious causes. Mechanistically, MSCs exert their beneficial effects by release of paracrine factors, microvesicles, and transfer of mitochondria, all of which have antiinflammatory and pro-resolving effects on injured lung endothelium and alveolar epithelium, including enhancing the resolution of pulmonary edema by up-regulating sodium-dependent alveolar fluid clearance. MSCs also have antimicrobial effects mediated by release of antimicrobial factors and by up-regulating monocyte/macrophage phagocytosis. Phase 2a clinical trials to establish safety in ARDS are in progress, and two phase 1 trials did not report any serious adverse events. Several issues need further study, including: determining the optimal methods for large-scale production, reconstitution of cryopreserved cells for clinical use, defining cell potency assays, and determining the therapeutic potential of conditioned media derived from MSCs. Because ARDS is a heterogeneous syndrome, targeting MSCs to patients with ARDS with a more hyperinflammatory endotype may further enhance their potential for efficacy.

Future Perspectives: Therapeutic Targeting of Notch Signalling May Become a Strategy in Patients Receiving Stem Cell Transplantation for Hematologic Malignancies

PubMed Central

Ersvaer, Elisabeth; Hatfield, Kimberley J.; Reikvam, Håkon; Bruserud, Øystein

2011-01-01

The human Notch system consists of 5 ligands and 4 membrane receptors with promiscuous ligand binding, and Notch-initiated signalling interacts with a wide range of other intracellular pathways. The receptor signalling seems important for regulation of normal and malignant hematopoiesis, development of the cellular immune system, and regulation of immune responses. Several Notch-targeting agents are now being developed, including natural receptor ligands, agonistic and antagonistic antibodies, and inhibitors of intracellular Notch-initiated signalling. Some of these agents are in clinical trials, and several therapeutic strategies seem possible in stem cell recipients: (i) agonists may be used for stem cell expansion and possibly to enhance posttransplant lymphoid reconstitution; (ii) receptor-specific agonists or antagonists can be used for immunomodulation; (iii) Notch targeting may have direct anticancer effects. Although the effects of therapeutic targeting are difficult to predict due to promiscuous ligand binding, targeting of this system may represent an opportunity to achieve combined effects with earlier posttransplant reconstitution, immunomodulation, or direct anticancer effects. PMID:22046566

Bone marrow transplantation reveals an essential synergy between neuronal and hemopoietic cell neurokinin production in pulmonary inflammation.

PubMed

Chavolla-Calderón, Mara; Bayer, Meggan K; Fontán, J Julio Pérez

2003-04-01

Neurogenic inflammation is believed to originate with the antidromic release of substance P, and of other neurokinins encoded by the preprotachykinin A (PPT-A) gene, from unmyelinated nerve fibers (C-fibers) following noxious stimuli. Consistent with this concept, we show here that selective sensory-fiber denervation with capsaicin and targeted deletion of the PPT-A gene protect murine lungs against both immune complex-mediated and stretch-mediated injuries. Reconstitution of PPT-A gene-deleted mice with WT bone marrow does not abrogate this protection, demonstrating a critical role for PPT-A gene expression by sensory neurons in pulmonary inflammation. Surprisingly, reconstitution of WT mice with PPT-A gene-deficient bone marrow also confers protection against pulmonary injury, revealing that PPT-A gene expression in hemopoietic cells has a previously unanticipated essential role in tissue injury. Taken together, these findings demonstrate a critical synergy between capsaicin-sensitive sensory fibers and hemopoietic cells in neurokinin-mediated inflammation and suggest that such synergy may be the basis for a stereotypical mechanism of response to injury in the respiratory tract.

Bone marrow transplantation reveals an essential synergy between neuronal and hemopoietic cell neurokinin production in pulmonary inflammation

PubMed Central

Chavolla-Calderón, Mara; Bayer, Meggan K.; Fontán, J. Julio Pérez

2003-01-01

Neurogenic inflammation is believed to originate with the antidromic release of substance P, and of other neurokinins encoded by the preprotachykinin A (PPT-A) gene, from unmyelinated nerve fibers (C-fibers) following noxious stimuli. Consistent with this concept, we show here that selective sensory-fiber denervation with capsaicin and targeted deletion of the PPT-A gene protect murine lungs against both immune complex–mediated and stretch-mediated injuries. Reconstitution of PPT-A gene–deleted mice with WT bone marrow does not abrogate this protection, demonstrating a critical role for PPT-A gene expression by sensory neurons in pulmonary inflammation. Surprisingly, reconstitution of WT mice with PPT-A gene–deficient bone marrow also confers protection against pulmonary injury, revealing that PPT-A gene expression in hemopoietic cells has a previously unanticipated essential role in tissue injury. Taken together, these findings demonstrate a critical synergy between capsaicin-sensitive sensory fibers and hemopoietic cells in neurokinin-mediated inflammation and suggest that such synergy may be the basis for a stereotypical mechanism of response to injury in the respiratory tract. PMID:12671046

Control of temporal activation of hepatitis C virus-induced interferon response by domain 2 of nonstructural protein 5A.

PubMed

Hiet, Marie-Sophie; Bauhofer, Oliver; Zayas, Margarita; Roth, Hanna; Tanaka, Yasuhito; Schirmacher, Peter; Willemsen, Joschka; Grünvogel, Oliver; Bender, Silke; Binder, Marco; Lohmann, Volker; Lotteau, Vincent; Ruggieri, Alessia; Bartenschlager, Ralf

2015-10-01

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a multifunctional protein playing a crucial role in diverse steps of the viral replication cycle and perturbing multiple host cell pathways. We showed previously that removal of a region in domain 2 (D2) of NS5A (mutant NS5A(D2Δ)) is dispensable for viral replication in hepatoma cell lines. By using a mouse model and immune-competent cell systems, we studied the role of D2 in controlling the innate immune response. In vivo replication competence of NS5A(D2Δ) was studied in transgenic mice with human liver xenografts. Results were validated using primary human hepatocytes (PHHs) and mechanistic analyses were conducted in engineered Huh7 hepatoma cells with reconstituted innate signaling pathways. Although the deletion in NS5A removed most of the interferon (IFN) sensitivity determining-region, mutant NS5A(D2Δ) was as sensitive as the wild type to IFN-α and IFN-λ in vitro, but severely attenuated in vivo. This attenuation could be recapitulated in PHHs and was linked to higher activation of the IFN response, concomitant with reduced viral replication and virus production. Importantly, immune-reconstituted Huh7-derived cell lines revealed a sequential activation of the IFN-response via RIG-I (retinoic acid-inducible gene I) and MDA5 (Myeloma differentiation associated factor 5), respectively, that was significantly higher in the case of the mutant lacking most of NS5A D2. Our study reveals an important role of NS5A D2 for suppression of the IFN response that is activated by HCV via RIG-I and MDA5 in a sequential manner. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Monitoring developmental force distributions in reconstituted embryonic epithelia.

PubMed

Przybyla, L; Lakins, J N; Sunyer, R; Trepat, X; Weaver, V M

2016-02-01

The way cells are organized within a tissue dictates how they sense and respond to extracellular signals, as cues are received and interpreted based on expression and organization of receptors, downstream signaling proteins, and transcription factors. Part of this microenvironmental context is the result of forces acting on the cell, including forces from other cells or from the cellular substrate or basement membrane. However, measuring forces exerted on and by cells is difficult, particularly in an in vivo context, and interpreting how forces affect downstream cellular processes poses an even greater challenge. Here, we present a simple method for monitoring and analyzing forces generated from cell collectives. We demonstrate the ability to generate traction force data from human embryonic stem cells grown in large organized epithelial sheets to determine the magnitude and organization of cell-ECM and cell-cell forces within a self-renewing colony. We show that this method can be used to measure forces in a dynamic hESC system and demonstrate the ability to map intracolony protein localization to force organization. Copyright © 2015 Elsevier Inc. All rights reserved.

Consequences of Molecular-Scale Non-Equilibrium Activity on the Dynamics and Mechanics of Self-Assembled Actin-Based Structures and Materials

NASA Astrophysics Data System (ADS)

Marshall Mccall, Patrick

Living cells are hierarchically self-organized forms of active soft matter: molecules on the nanometer scale form functional structures and organelles on the micron scale, which then compose cells on the scale of 10s of microns. While the biological functions of intracellular organelles are defined by the composition and properties of the structures themselves, how those bulk properties emerge from the properties and interactions of individual molecules remains poorly understood. Actin, a globular protein which self-assembles into dynamic semi-flexible polymers, is the basic structural material of cells and the major component of many functional organelles. In this thesis, I have used purified actin as a model system to explore the interplay between molecular-scale dynamics and organelle-scale functionality, with particular focus on the role of molecular-scale non-equilibrium activity. One of the most canonical forms of molecular-scale non-equilibrium activity is that of mechanoenzymes, also called motor proteins. These proteins utilized the free energy liberated by hydrolysis of ATP to perform mechanical work, thereby introducing non-equilibrium "active" stresses on the molecular scale. Combining experiments with mathematical modeling, we demonstrate in this thesis that non-equilibrium motor activity is sufficient to drive self-organization and pattern formation of the multimeric actin-binding motor protein Myosin II on 1D reconstituted actomyosin bundles. Like myosin, actin is itself an ATPase. However, nono-equilibrium ATP hydrolysis on actin is known to regulate the stability and assembly kinetics of actin filaments rather than generate active stresses per se. At the level of single actin filaments, the inhomogeneous nucleotide composition generated along the filament length by hydrolysis directs binding of regulatory proteins like cofilin, which mediate filament disassembly and thereby accelerate actin filament turnover. The concequences of this non-equilibrium turnover on the steady-state properties of collections of filaments remained unclear. Here, I reconstituted tunable, non-equilibrium actin turnover dynamics in entangled solutions of actin filaments as a model of the actin cortex of living cells. We found that this non-equilibrium turnover decouples solution mechanics from microstructure, enabling structurally indistinguishable materials to behave effectively as either viscous fluids or elastic gels. Additionally, we employed computer simulations to identify the dynamical regime in which actin turnover controls the effective viscosity of 2D cross-linked actin networks in the presence of motors. Additionally, I examine in this thesis the localization and self-assembly of actin filaments in condensed liquid phases called polyelectrolyte coacervates as a model membrane-less organelle. We find that concentration of actin through spontaneous partitioning preferentially to the coacervate phase accelerates the assembly of filaments. These filaments then localize to the coacervate-bulk interface, generating particles with visco-elastic shells surrounding liquid cores. In this case, the properties of the condensed phase enable regulation of actin assembly dynamics.

Molecular requirements for actin-based lamella formation in Drosophila S2 cells

PubMed Central

Rogers, Stephen L.; Wiedemann, Ursula; Stuurman, Nico; Vale, Ronald D.

2003-01-01

Cell migration occurs through the protrusion of the actin-enriched lamella. Here, we investigated the effects of RNAi depletion of ∼90 proteins implicated in actin function on lamella formation in Drosophila S2 cells. Similar to in vitro reconstitution studies of actin-based Listeria movement, we find that lamellae formation requires a relatively small set of proteins that participate in actin nucleation (Arp2/3 and SCAR), barbed end capping (capping protein), filament depolymerization (cofilin and Aip1), and actin monomer binding (profilin and cyclase-associated protein). Lamellae are initiated by parallel and partially redundant signaling pathways involving Rac GTPases and the adaptor protein Nck, which stimulate SCAR, an Arp2/3 activator. We also show that RNAi of three proteins (kette, Abi, and Sra-1) known to copurify with and inhibit SCAR in vitro leads to SCAR degradation, revealing a novel function of this protein complex in SCAR stability. Our results have identified an essential set of proteins involved in actin dynamics during lamella formation in Drosophila S2 cells. PMID:12975351

Evidence that electrostatic interactions between vesicle-associated membrane protein 2 and acidic phospholipids may modulate the fusion of transport vesicles with the plasma membrane.

PubMed

Williams, Dumaine; Vicôgne, Jérome; Zaitseva, Irina; McLaughlin, Stuart; Pessin, Jeffrey E

2009-12-01

The juxtamembrane domain of vesicle-associated membrane protein (VAMP) 2 (also known as synaptobrevin2) contains a conserved cluster of basic/hydrophobic residues that may play an important role in membrane fusion. Our measurements on peptides corresponding to this domain determine the electrostatic and hydrophobic energies by which this domain of VAMP2 could bind to the adjacent lipid bilayer in an insulin granule or other transport vesicle. Mutation of residues within the juxtamembrane domain that reduce the VAMP2 net positive charge, and thus its interaction with membranes, inhibits secretion of insulin granules in beta cells. Increasing salt concentration in permeabilized cells, which reduces electrostatic interactions, also results in an inhibition of insulin secretion. Similarly, amphipathic weak bases (e.g., sphingosine) that reverse the negative electrostatic surface potential of a bilayer reverse membrane binding of the positively charged juxtamembrane domain of a reconstituted VAMP2 protein and inhibit membrane fusion. We propose a model in which the positively charged VAMP and syntaxin juxtamembrane regions facilitate fusion by bridging the negatively charged vesicle and plasma membrane leaflets.

Characteristics of autoimmune thyroid disease occurring as a late complication of immune reconstitution in patients with advanced human immunodeficiency virus (HIV) disease.

PubMed

Chen, Fabian; Day, Sara L; Metcalfe, Russell A; Sethi, Gulshan; Kapembwa, Moses S; Brook, M Gary; Churchill, Duncan; de Ruiter, Annemiek; Robinson, Stephen; Lacey, Charles J; Weetman, Anthony P

2005-03-01

Experimental evidence from animal models has provided a framework for our current understanding of autoimmune disease pathogenesis and supports the importance of genetic predisposition, molecular mimicry, and immune dysregulation. However, only recently has evidence emerged to support the role of immune dysregulation in human organ-specific autoimmune disease. In the current study of the "late" manifestation of autoimmune thyroid disease (AITD) in a cohort of human immunodeficiency virus (HIV)-positive patients following highly active antiretroviral therapy (HAART), we discuss how immune dysregulation and factors associated with the immunopathology of HIV infection fit the current understanding of autoimmunity and provide a plausible basis for our clinical observations. De novo diagnoses of thyroid disease were identified between 1996 and 2002 in 7 HIV treatment centers (5/7 centers completed the study). Patients were diagnosed as clinical case entities and not discovered through thyroid function test screening. Paired plasma specimens were used to demonstrate sequential rise in thyroid antibodies. Seventeen patients were diagnosed with AITD (median age, 38 yr; 65% were of black African or black Caribbean ethnicity; and 82% were female). The median duration of immune reconstitution was 17 months. Graves disease (GD) was diagnosed in 15 of 17 patients. One patient developed hashithyrotoxicosis with atypically raised C-reactive protein, and another developed hypothyroidism. One GD patient had associated secondary hypoadrenalism. The estimated combined prevalence of GD for 4 treatment centers for female patients was 7/234 and for males was 2/1289. The denominator numbers were matched controls, from 4 centers able to provide data, who commenced HAART during the same time (January 1996 to July 2002) and who did not develop clinical AITD. The mean baseline pre-HAART CD4 count was 67 cells/mL, and the mean increase from nadir to AITD presentation was 355 cells/mL. AITD patients were more likely than controls (95% confidence interval, chi-square test) to be severely compromised at baseline (as defined by a CD4 count < 200 cells/mL or the presence of an acquired immunodeficiency syndrome [AIDS]-defining diagnosis), and to experience greater CD4 increments following HAART. AITD may be a late manifestation of immune reconstitution in HIV-positive patients taking HAART, and immune dysregulation may be an important factor.

Induction of Skin-Derived Precursor Cells from Human Induced Pluripotent Stem Cells.

PubMed

Sugiyama-Nakagiri, Yoriko; Fujimura, Tsutomu; Moriwaki, Shigeru

2016-01-01

The generation of full thickness human skin from dissociated cells is an attractive approach not only for treating skin diseases, but also for treating many systemic disorders. However, it is currently not possible to obtain an unlimited number of skin dermal cells. The goal of this study was to develop a procedure to produce skin dermal stem cells from induced pluripotent stem cells (iPSCs). Skin-derived precursor cells (SKPs) were isolated as adult dermal precursors that could differentiate into both neural and mesodermal progenies and could reconstitute the dermis. Thus, we attempted to generate SKPs from iPSCs that could reconstitute the skin dermis. Human iPSCs were initially cultured with recombinant noggin and SB431542, an inhibitor of activin/nodal and TGFβ signaling, to induce neural crest progenitor cells. Those cells were then treated with SKP medium that included CHIR99021, a WNT signal activator. The induction efficacy from neural crest progenitor cells to SKPs was more than 97%. No other modifiers tested were able to induce those cells. Those human iPSC-derived SKPs (hiPSC-SKPs) showed a similar gene expression signature to SKPs isolated from human skin dermis. Human iPSC-SKPs differentiated into neural and mesodermal progenies, including adipocytes, skeletogenic cell types and Schwann cells. Moreover, they could be induced to follicular type keratinization when co-cultured with human epidermal keratinocytes. We here provide a new efficient protocol to create human skin dermal stem cells from hiPSCs that could contribute to the treatment of various skin disorders.