Science.gov

Sample records for determines cell susceptibility

  1. Endoplasmic reticulum stress-induced autophagy determines the susceptibility of melanoma cells to dabrafenib.

    PubMed

    Ji, Chao; Zhang, Ziping; Chen, Lihong; Zhou, Kunli; Li, Dongjun; Wang, Ping; Huang, Shuying; Gong, Ting; Cheng, Bo

    2016-01-01

    Melanoma is one of the deadliest skin cancers and accounts for most skin-related deaths due to strong resistance to chemotherapy drugs. In the present study, we investigated the mechanisms of dabrafenib-induced drug resistance in human melanoma cell lines A375 and MEL624. Our studies support that both endoplasmic reticulum (ER) stress and autophagy were induced in the melanoma cells after the treatment with dabrafenib. In addition, ER stress-induced autophagy protects melanoma cells from the toxicity of dabrafenib. Moreover, inhibition of both ER stress and autophagy promote the sensitivity of melanoma cells to dabrafenib. Taken together, the data suggest that ER stress-induced autophagy determines the sensitivity of melanoma cells to dabrafenib. These results provide us with promising evidence that the inhibition of autophagy and ER stress could serve a therapeutic effect for the conventional dabrafenib chemotherapy. PMID:27536070

  2. Endoplasmic reticulum stress-induced autophagy determines the susceptibility of melanoma cells to dabrafenib

    PubMed Central

    Ji, Chao; Zhang, Ziping; Chen, Lihong; Zhou, Kunli; Li, Dongjun; Wang, Ping; Huang, Shuying; Gong, Ting; Cheng, Bo

    2016-01-01

    Melanoma is one of the deadliest skin cancers and accounts for most skin-related deaths due to strong resistance to chemotherapy drugs. In the present study, we investigated the mechanisms of dabrafenib-induced drug resistance in human melanoma cell lines A375 and MEL624. Our studies support that both endoplasmic reticulum (ER) stress and autophagy were induced in the melanoma cells after the treatment with dabrafenib. In addition, ER stress-induced autophagy protects melanoma cells from the toxicity of dabrafenib. Moreover, inhibition of both ER stress and autophagy promote the sensitivity of melanoma cells to dabrafenib. Taken together, the data suggest that ER stress-induced autophagy determines the sensitivity of melanoma cells to dabrafenib. These results provide us with promising evidence that the inhibition of autophagy and ER stress could serve a therapeutic effect for the conventional dabrafenib chemotherapy. PMID:27536070

  3. Antimicrobial Susceptibility Patterns of Recent Cuban Mycoplasma genitalium Isolates Determined by a Modified Cell-Culture-Based Method

    PubMed Central

    Mondeja, Brian A.; Rodríguez, Nadia M.; Barroto, Brenda; Blanco, Orestes

    2016-01-01

    Isolation of Mycoplasma genitalium from clinical specimens remains difficult and few strains are available for antimicrobial susceptibility testing. We describe the antimicrobial susceptibility of M. genitalium strains grown in Vero cell culture with first- and second- line antibiotics, using a modified cell-culture-based method. Macrolide- and -fluoroquinolone resistance determinants were detected by sequencing of the 23S and parC genes, respectively. Seven strains were examined, including three new, genetically distinct M. genitalium strains isolated from endocervical and urethral swab specimens from Cuban patients together with four reference strains isolated from specimens collected from men in Denmark, Sweden and Australia. Azithromycin was the most active drug against two of the Cuban M. genitalium strains with MICs values of 0.008 mg/liter, however, one strain was macrolide resistant with an MIC of >8 mg/liter, and the A2059G resistant genotype. Ciprofloxacin was the least active antimicrobial drug and moxifloxacin was the most active fluoroquinolone against the new clinical strains, although an MIC of 1 mg/l was found for two strains. However, no relevant parC mutations were detected. MICs for tetracyclines were 0.5–4 mg/liter. Although the number of Cuban strains was low, the results suggest that a single-dose azithromycin treatment could be ineffective, and that a second-line treatment with moxifloxacin, should become an option in Cuba. To our knowledge, this is the first report of isolation and antibiotic susceptibility testing of M. genitalium strains from the Latin-American region, and the first detection of macrolide resistance in such strains. PMID:27679992

  4. CADM1/TSLC1 Identifies HTLV-1-Infected Cells and Determines Their Susceptibility to CTL-Mediated Lysis

    PubMed Central

    Tanaka, Yuetsu; Taylor, Graham P.; Bangham, Charles R. M.

    2016-01-01

    Human T cell lymphotropic virus-1 (HTLV-1) primarily infects CD4+ T cells, causing inflammatory disorders or a T cell malignancy in 5% to 10% of carriers. The cytotoxic T lymphocyte (CTL) response is a key factor that controls the viral load and thus the risk of disease. The ability to detect the viral protein Tax in primary cells has made it possible to estimate the rate at which Tax-expressing infected cells are eliminated by CTLs in persistently infected people. However, most HTLV-1-infected cells are Tax–at a given time, and their immunophenotype is poorly defined. Here, we aimed to identify a cell-surface molecule expressed by both Tax+ and Tax–HTLV-1-infected cells and use it to analyse the CTL response in fresh peripheral blood mononuclear cells. Cell adhesion molecule 1 (CADM1/TSLC1) was the best single marker of HTLV-1 infection, identifying HTLV-1-infected cells with greater sensitivity and specificity than CD25, CCR4 or ICAM-1. CADM1+CD4+ T cells carried a median of 65% of proviral copies in peripheral blood. In a cohort of 23 individuals, we quantified the rate of CTL-mediated killing of Tax+ and Tax−CADM1+ cells. We show that CADM1 expression is associated with enhanced susceptibility of infected cells to CTL lysis: despite the immunodominance of Tax in the CTL response, Tax+CADM1– cells were inefficiently lysed by CTLs. Upregulation of the CADM1 ligand CRTAM on CD8+ T cells correlated with efficient lysis of infected cells. Tax–CADM1+ cells were lysed at a very low rate by autologous CTLs, however, were efficiently killed when loaded with exogenous peptide antigen. High expression of CADM1 on most HTLV-1-infected cells in the face of enhanced CTL counterselection implies that CADM1 confers a strong benefit on the virus. PMID:27105228

  5. Basal-subtype and MEK-Pl3K feedback signaling determine susceptibility of breast cancer cells to MEK inhibition

    SciTech Connect

    Mirzoeva, Olga K.; Das, Debopriya; Heiser, Laura M.; Bhattacharya, Sanchita; Siwak, Doris; Gendelman, Rina; Bayani, Nora; Wang, Nicholas J.; Neve, Richard M.; Knight, Zachary; Feiler, Heidi S.; Gascard, Philippe; Parvin, Bahram; Spellman, Paul T.; Shokat, Kevan M.; Wyrobek, Andrew J.; Bissell, Mina J.; McCormick, Frank; Kuo, Wen-Lin; Mills, Gordon B.; Gray, Joe W.; Korn, W. Michael

    2009-01-23

    Specific inhibitors of MEK have been developed that efficiently inhibit the oncogenic RAF-MEK-ERK pathway. We employed a systems-based approach to identify breast cancer subtypes particularly susceptible to MEK inhibitors and to understand molecular mechanisms conferring resistance to such compounds. Basal-type breast cancer cells were found to be particularly susceptible to growth-inhibition by small-molecule MEK inhibitors. Activation of the PI3 kinase pathway in response to MEK inhibition through a negative MEK-EGFR-PI3 kinase feedback loop was found to limit efficacy. Interruption of this feedback mechanism by targeting MEK and PI3 kinase produced synergistic effects, including induction of apoptosis and, in some cell lines, cell cycle arrest and protection from apoptosis induced by proapoptotic agents. These findings enhance our understanding of the interconnectivity of oncogenic signal transduction circuits and have implications for the design of future clinical trials of MEK inhibitors in breast cancer by guiding patient selection and suggesting rational combination therapies.

  6. Genetically determined difference in the antiviral action of alpha/beta interferon in cells from mice resistant or susceptible to herpes simplex virus type 2.

    PubMed

    Ellermann-Eriksen, S; Justesen, J; Mogensen, S C

    1986-09-01

    Resistance of mice to infection with herpes simplex virus type 2 (HSV-2) is genetically determined. Embryonic cells from susceptible BALB/c and resistant C57BL/6 mice were equally sensitive to infection with HSV-2 as judged by plaque area, plaquing efficiency, endpoint titration and virus yield. Cells from C57BL/6 mice showed a higher sensitivity than cells from BALB/c mice to the protective action of two preparations of alpha/beta interferon against challenge with HSV-2. This was evident both from c.p.e. inhibition and yield reduction experiments. The difference in sensitivity was dependent on virus dose and was greatest (up to 50-fold) with low virus doses. An analysis of the genetics of the alpha/beta interferon sensitivity in cells from embryos of parental mice and embryos derived from reciprocal matings between HSV-2-resistant and -susceptible mice suggested that interferon sensitivity is inherited as a co-dominant autosomal trait. The induction of the interferon-induced enzyme 2'-5'-oligoadenylate synthetase was also different in cells from the two mouse strains, since significant levels were only detected in cells from C57BL/6 mice. It is suggested that differential interferon sensitivity of cells from HSV-2-resistant and -susceptible mice might be a factor of importance for the course of the infection.

  7. Agreement between the cell culture titrations of canine minute virus determined by two susceptibility-testing methods.

    PubMed

    Pratelli, A; Colao, V

    2013-09-01

    The correct diagnosis of canine minute virus is critical in dog breeding. In this study, the Bland Altman test was used to compare the performance of two susceptibility-testing methods, namely polymerase chain reaction (PCR) and indirect immunofluorescence assay (IFA). The agreement between IFA and PCR in monocytes revealed a mean difference of -1752.16 with 95% confidence and an interval ranging from -3229.80 to -274.53 (SD=2325.62). The agreement between IFA and PCR in Walter Reed canine cells (WRCC) revealed a mean difference of -2396.55 with 95% confidence and an interval ranging from -3774.63 to -1018.48 (SD=2168.93). The Bland Altman test confirmed the overall accuracy of PCR vs IFA and the plot showed that all points were not randomly arranged in the range of average ± 1.96 × SD of the differences.

  8. A plastic SQSTM1/p62-dependent autophagic reserve maintains proteostasis and determines proteasome inhibitor susceptibility in multiple myeloma cells

    PubMed Central

    Milan, Enrico; Perini, Tommaso; Resnati, Massimo; Orfanelli, Ugo; Oliva, Laura; Raimondi, Andrea; Cascio, Paolo; Bachi, Angela; Marcatti, Magda; Ciceri, Fabio; Cenci, Simone

    2015-01-01

    Multiple myeloma (MM) is the paradigmatic proteasome inhibitor (PI) responsive cancer, but many patients fail to respond. An attractive target to enhance sensitivity is (macro)autophagy, recently found essential to bone marrow plasma cells, the normal counterpart of MM. Here, integrating proteomics with hypothesis-driven strategies, we identified the autophagic cargo receptor and adapter protein, SQSTM1/p62 as an essential component of an autophagic reserve that not only synergizes with the proteasome to maintain proteostasis, but also mediates a plastic adaptive response to PIs, and faithfully reports on inherent PI sensitivity. Lentiviral engineering revealed that SQSTM1 is essential for MM cell survival and affords specific PI protection. Under basal conditions, SQSTM1-dependent autophagy alleviates the degradative burden on the proteasome by constitutively disposing of substantial amounts of ubiquitinated proteins. Indeed, its inhibition or stimulation greatly sensitized to, or protected from, PI-induced protein aggregation and cell death. Moreover, under proteasome stress, myeloma cells selectively enhanced SQSTM1 de novo expression and reset its vast endogenous interactome, diverting SQSTM1 from signaling partners to maximize its association with ubiquitinated proteins. Saturation of such autophagic reserve, as indicated by intracellular accumulation of undigested SQSTM1-positive aggregates, specifically discriminated patient-derived myelomas inherently susceptible to PIs from primarily resistant ones. These aggregates correlated with accumulation of the endoplasmic reticulum, which comparative proteomics identified as the main cell compartment targeted by autophagy in MM. Altogether, the data integrate autophagy into our previously established proteasome load-versus-capacity model, and reveal SQSTM1 aggregation as a faithful marker of defective proteostasis, defining a novel prognostic and therapeutic framework for MM. PMID:26043024

  9. VP2 capsid domain of the H-1 parvovirus determines susceptibility of human cancer cells to H-1 viral infection.

    PubMed

    Cho, I-R; Kaowinn, S; Song, J; Kim, S; Koh, S S; Kang, H-Y; Ha, N-C; Lee, K H; Jun, H-S; Chung, Y-H

    2015-05-01

    Although H-1 parvovirus is used as an antitumor agent, not much is known about the relationship between its specific tropism and oncolytic activity. We hypothesize that VP2, a major capsid protein of H-1 virus, determines H-1-specific tropism. To assess this, we constructed chimeric H-1 viruses expressing Kilham rat virus (KRV) capsid proteins, in their complete or partial forms. Chimeric H-1 viruses (CH1, CH2 and CH3) containing the whole KRV VP2 domain could not induce cytolysis in HeLa, A549 and Panc-1 cells. However, the other chimeric H-1 viruses (CH4 and CH5) expressing a partial KRV VP2 domain induced cytolysis. Additionally, the significant cytopathic effect caused by CH4 and CH5 infection in HeLa cells resulted from preferential viral amplification via DNA replication, RNA transcription and protein synthesis. Modeling of VP2 capsid protein showed that two variable regions (VRs) (VR0 and VR2) of H-1 VP2 protein protrude outward, because of the insertion of extra amino-acid residues, as compared with those of KRV VP2 protein. This might explain the precedence of H-1 VP2 protein over KRV in determining oncolytic activity in human cancer cells. Taking these results together, we propose that the VP2 protein of oncolytic H-1 parvovirus determines its specific tropism in human cancer cells.

  10. Determination of anisotropy of diamagnetic susceptibility for nematics and polar anchoring energy coefficient for nematics: substrate systems by wedge cell method

    NASA Astrophysics Data System (ADS)

    Kedzierski, Jerzy; Raszewski, Zbigniew; Kojdecki, Marek A.; Zielinski, Jerzy; Miszczyk, Emilia; Lipinska, Ludwika

    2004-09-01

    A wedge cell of the wedge angle of order of few milliradians was used to measure threshold magnetic fields for the magnetic Freedericksz transition. A nematic liquid crystal filling the cell was of planar orientation enforced by the treatment of the flat boundary plates. A system of interference fringes appeared in the cell placed in normally incident light between analyzer and polarizer crossed. In the vicinity of each fringe the cell could be considered as a flat-parallel one and hence it was equivalent to a system of flat cells of different precisely determined thickness. The threshold magnetic field magnitudes were interpreted as eigenvalues of the boundary eigenvalue problem for the operator of second derivative; the interaction between the nematics and the substrate was described by the Rapini-Papoular formula (i.e. weak coupling was considered). The resulting formulae were used to determine the polar anchoring energy coefficient and the anisotropy of diamagnetic susceptibility after the threshold fields measured. The method was applied to characterize the nematic liquid crystal PCB and the coupling between it and the substrates made of poly(amic acid) MP2. The estimates of material parameters agreed pretty well with those determined by the composite method.

  11. Preoperative determination of susceptibility to malignant hyperthermia.

    PubMed

    Carson, J M; Van Sickels, J E

    1982-07-01

    A careful individual and family history is of utmost importance in evaluating MH susceptibility. Patients having myopathy with or without a significant family history of MH must be suspected of having the condition. The initial evaluation should include routine blood studies and CPK determinations in addition to an electrocardiogram. Full evaluation differentiating rigid from nonrigid types must include a muscle biopsy for microscopic and contraction studies. Platelet ATP depletion with exposure to halothane shows promise as a prescreening test. Susceptible patients can be given both general and local anesthetics if the surgeon and support personnel have a thorough understanding of MH and if proper precautions have been taken and preparation of facilities has been made. PMID:6953194

  12. Developmental Origin of Oligodendrocyte Lineage Cells Determines Response to Demyelination and Susceptibility to Age-Associated Functional Decline

    PubMed Central

    Crawford, Abbe H.; Tripathi, Richa B.; Richardson, William D.; Franklin, Robin J.M.

    2016-01-01

    Summary Oligodendrocyte progenitors (OPs) arise from distinct ventral and dorsal domains within the ventricular germinal zones of the embryonic CNS. The functional significance, if any, of these different populations is not known. Using dual-color reporter mice to distinguish ventrally and dorsally derived OPs, we show that, in response to focal demyelination of the young adult spinal cord or corpus callosum, dorsally derived OPs undergo enhanced proliferation, recruitment, and differentiation as compared with their ventral counterparts, making a proportionally larger contribution to remyelination. However, with increasing age (up to 13 months), the dorsally derived OPs become less able to differentiate into mature oligodendrocytes. Comparison of dorsally and ventrally derived OPs in culture revealed inherent differences in their migration and differentiation capacities. Therefore, the responsiveness of OPs to demyelination, their contribution to remyelination, and their susceptibility to age-associated functional decline are markedly dependent on their developmental site of origin in the developing neural tube. PMID:27149850

  13. Susceptibility of irradiated bovine aortic endothelial cells to injury

    SciTech Connect

    Zhou, M.H.; Dong, Q.; Ts'ao, C.

    1988-11-01

    Using cultured bovine aortic endothelial cells (BAEC), the authors attempted to determine whether prior irradiation would alter the susceptibility of these cells to three known injurious stimuli and, if so, whether the alteration would be related to radiation dose. BAEC were irradiated with 0, 5, or 10 Gy of gamma rays and, on the third postirradiation day, exposed to fibrin, nicotine, or bacterial endotoxin (lipopolysaccharide, LPS). Release of prelabeled 51Cr, representing cell lysis, cell detachment, or a combination of the two, was determined. Significant differences between irradiated and control cells were determined by using paired Student's t-tests. Irradiation did not appear to have altered the sensitivity of BAEC to fibrin-induced injury. Cells irradiated with 10 Gy of gamma rays, but generally not those irradiated with half this dose, showed a heightened susceptibility to nicotine. Contrary to the nicotine results, irradiated cells showed less cell detachment and lysis after exposure to LPS. These results suggest that the susceptibility of irradiated BAEC to harmful stimuli depends largely on the nature of the stimulus as well as the radiation dose.

  14. NOX5-L can stimulate proliferation and apoptosis depending on its levels and cellular context, determining cancer cell susceptibility to cisplatin

    PubMed Central

    Kwon, Eun-Soo; Lim, Jae Cheong; Park, Sung Sup; Kwon, Ki-Sun

    2015-01-01

    The NADPH oxidase, NOX5, is known to stimulate cell proliferation in some cancers by generating reactive oxygen species (ROS). We show here that the long form of NOX5 (NOX5-L) also promotes cell death, and thus determines the balance of proliferation and death, in skin, breast and lung cancer cells. Moderate expression of NOX5-L induced cell proliferation accompanied by AKT and ERK phosphorylation, whereas an increase in NOX5-L above a certain threshold promoted cancer cell death accompanied by caspase-3 activation. Notably, cisplatin treatment increased NOX5-L levels through CREB activation and enhanced NOX5-L activity through augmentation of Ca2+ release and c-Abl expression, ultimately triggering ROS-mediated cancer cell death—a distinct pathway absent in normal cells. These results indicate that NOX5-L determines cellular responses in a concentration- and context-dependent manner. PMID:26513170

  15. Determination of antimicrobial susceptibilities on infected urines without isolation

    NASA Technical Reports Server (NTRS)

    Picciolo, G. L.; Chappelle, E. W.; Deming, J. W.; Shrock, C. G.; Vellend, H.; Barza, M. J.; Weinstein, L. (Inventor)

    1979-01-01

    A method is described for the quick determination of the susceptibilities of various unidentified bacteria contained in an aqueous physiological fluid sample, particularly urine, to one or more antibiotics. A bacterial adenosine triphosphate (ATP) assay is carried out after the elimination of non-bacterial ATP to determine whether an infection exists. If an infection does exist, a portion of the sample is further processed, including subjecting parts of the portion to one or more antibiotics. Growth of the bacteria in the parts are determined, again by an ATP assay, to determine whether the unidentified bacteria in the sample are susceptible to the antibiotic or antibiotics under test.

  16. The large mechanosensitive channel MscL determines bacterial susceptibility to the bacteriocin sublancin 168.

    PubMed

    Kouwen, Thijs R H M; Trip, Erik N; Denham, Emma L; Sibbald, Mark J J B; Dubois, Jean-Yves F; van Dijl, Jan Maarten

    2009-11-01

    Bacillus subtilis strain 168 produces the extremely stable and broad-spectrum lantibiotic sublancin 168. Known sublancin 168-susceptible organisms include important pathogens, such as Staphylococcus aureus. Nevertheless, since its discovery, the mode of action of sublancin 168 has remained elusive. The present studies were, therefore, aimed at the identification of cellular determinants for bacterial susceptibility toward sublancin 168. Growth inhibition and competition assays on plates and in liquid cultures revealed that sublancin 168-mediated growth inhibition of susceptible B. subtilis and S. aureus cells is affected by the NaCl concentration in the growth medium. Added NaCl did not influence the production, activity, or stability of sublancin 168 but, instead, lowered the susceptibility of sensitive cells toward this lantibiotic. Importantly, the susceptibility of B. subtilis and S. aureus cells toward sublancin 168 was shown to depend on the presence of the large mechanosensitive channel of conductance MscL. In contrast, MscL was not involved in susceptibility toward the bacteriocin nisin or Pep5. Taken together, our unprecedented results demonstrate that MscL is a critical and specific determinant in bacterial sublancin 168 susceptibility that may serve either as a direct target for this lantibiotic or as a gate of entry to the cytoplasm. PMID:19738010

  17. Human regulatory T cells control TCR signaling and susceptibility to suppression in CD4+ T cells.

    PubMed

    Chellappa, Stalin; Lieske, Nora V; Hagness, Morten; Line, Pål D; Taskén, Kjetil; Aandahl, Einar M

    2016-07-01

    Human CD4(+)CD25(hi)FOXP3(+) regulatory T cells maintain immunologic tolerance and prevent autoimmune and inflammatory immune responses. Regulatory T cells undergo a similar activation cycle as conventional CD4(+) T cells upon antigen stimulation. Here, we demonstrate that T cell receptors and costimulation are required to activate the regulatory T cell suppressive function. Regulatory T cells suppressed the T cell receptor signaling in effector T cells in a time-dependent manner that corresponded with inhibition of cytokine production and proliferation. Modulation of the activation level and thereby the suppressive capacity of regulatory T cells imposed distinct T cell receptor signaling signatures and hyporesponsiveness in suppressed and proliferating effector T cells and established a threshold for effector T cell proliferation. The immune suppression of effector T cells was completely reversible upon removal of regulatory T cells. However, the strength of prior immune suppression by regulatory T cells and corresponding T cell receptor signaling in effector T cells determined the susceptibility to suppression upon later reexposure to regulatory T cells. These findings demonstrate how the strength of the regulatory T cell suppressive function determines intracellular signaling, immune responsiveness, and the later susceptibility of effector T cells to immune suppression and contribute to unveiling the complex interactions between regulatory T cells and effector T cells. PMID:26715685

  18. New basal cell carcinoma susceptibility loci

    PubMed Central

    Stacey, Simon N.; Helgason, Hannes; Gudjonsson, Sigurjon A.; Thorleifsson, Gudmar; Zink, Florian; Sigurdsson, Asgeir; Kehr, Birte; Gudmundsson, Julius; Sulem, Patrick; Sigurgeirsson, Bardur; Benediktsdottir, Kristrun R.; Thorisdottir, Kristin; Ragnarsson, Rafn; Fuentelsaz, Victoria; Corredera, Cristina; Gilaberte, Yolanda; Grasa, Matilde; Planelles, Dolores; Sanmartin, Onofre; Rudnai, Peter; Gurzau, Eugene; Koppova, Kvetoslava; Nexø, Bjørn A.; Tjønneland, Anne; Overvad, Kim; Jonasson, Jon G.; Tryggvadottir, Laufey; Johannsdottir, Hrefna; Kristinsdottir, Anna M.; Stefansson, Hreinn; Masson, Gisli; Magnusson, Olafur T.; Halldorsson, Bjarni V.; Kong, Augustine; Rafnar, Thorunn; Thorsteinsdottir, Unnur; Vogel, Ulla; Kumar, Rajiv; Nagore, Eduardo; Mayordomo, José I.; Gudbjartsson, Daniel F.; Olafsson, Jon H.; Stefansson, Kari

    2015-01-01

    In an ongoing screen for DNA sequence variants that confer risk of cutaneous basal cell carcinoma (BCC), we conduct a genome-wide association study (GWAS) of 24,988,228 SNPs and small indels detected through whole-genome sequencing of 2,636 Icelanders and imputed into 4,572 BCC patients and 266,358 controls. Here we show the discovery of four new BCC susceptibility loci: 2p24 MYCN (rs57244888[C], OR=0.76, P=4.7 × 10−12), 2q33 CASP8-ALS2CR12 (rs13014235[C], OR=1.15, P=1.5 × 10−9), 8q21 ZFHX4 (rs28727938[G], OR=0.70, P=3.5 × 10−12) and 10p14 GATA3 (rs73635312[A], OR=0.74, P=2.4 × 10−16). Fine mapping reveals that two variants correlated with rs73635312[A] occur in conserved binding sites for the GATA3 transcription factor. In addition, expression microarrays and RNA-seq show that rs13014235[C] and a related SNP rs700635[C] are associated with expression of CASP8 splice variants in which sequences from intron 8 are retained. PMID:25855136

  19. Susceptibility of bovine umbilical cord endothelial cells to bovine herpesviruses and pseudocowpox virus.

    PubMed

    Wellenberg, G J; Verstraten, E R A M; Jongejan, F; Van Oirschot, J T

    2002-07-01

    The purpose of the study was to determine the susceptibility of bovine umbilical cord endothelial (BUE) cells to bovine herpesvirus (BHV) 1, BHV2, BHV4 and BHV5, and to pseudocowpox virus. The detection limits and growth curves of these viruses in BUE cells were compared with those in Vero, Madin-Darby bovine kidney (MDBK). or bovine fetal diploid lung (BFDL) cells. Detection limits were determined by inoculating cell cultures with serial 10-fold dilutions of these viruses, and growth curves by titration of virus, harvested at various times after infecting cells at a multiplicity of infection of 0.1. The detection limits of BHV2 and BHV4 were lower in BUE cells than in Vero or MDBK cells, and cytopathic effects were observed earlier in BUE cells. In addition, BHV2 and BHV4 grew to higher titres in BUE cells than in Vero or MDBK cells. BUE cells appeared to be equally susceptible to BHV5, but less susceptible to BHV1.1 and BHVI.2 than MDBK cells. The study showed that BUE cells are highly susceptible to BHV2 and BHV4. and that the use of BUE cells can improve the laboratory diagnosis of these viruses. The use of BUE cells could also improve the isolation and growth of pseudocowpox virus.

  20. Subtle variations in Pten dose determine cancer susceptibility.

    PubMed

    Alimonti, Andrea; Carracedo, Arkaitz; Clohessy, John G; Trotman, Lloyd C; Nardella, Caterina; Egia, Ainara; Salmena, Leonardo; Sampieri, Katia; Haveman, William J; Brogi, Edi; Richardson, Andrea L; Zhang, Jiangwen; Pandolfi, Pier Paolo

    2010-05-01

    Cancer susceptibility has been attributed to at least one heterozygous genetic alteration in a tumor suppressor gene (TSG). It has been hypothesized that subtle variations in TSG expression can promote cancer development. However, this hypothesis has not yet been definitively supported in vivo. Pten is a TSG frequently lost in human cancer and mutated in inherited cancer-predisposition syndromes. Here we analyze Pten hypermorphic mice (Pten(hy/+)), expressing 80% normal levels of Pten. Pten(hy/+) mice develop a spectrum of tumors, with breast tumors occurring at the highest penetrance. All breast tumors analyzed here retained two intact copies of Pten and maintained Pten levels above heterozygosity. Notably, subtle downregulation of Pten altered the steady-state biology of the mammary tissues and the expression profiles of genes involved in cancer cell proliferation. We present an alterative working model for cancer development in which subtle reductions in the dose of TSGs predispose to tumorigenesis in a tissue-specific manner.

  1. Histamine H2 receptor signaling × environment interactions determine susceptibility to experimental allergic encephalomyelitis

    PubMed Central

    Saligrama, Naresha; Case, Laure K.; Krementsov, Dimitry N.; Teuscher, Cory

    2014-01-01

    Histamine and its receptors are important in both multiple sclerosis and experimental allergic encephalomyelitis (EAE). C57BL/6J (B6) mice deficient for the histamine H2 receptor (H2RKO) are less susceptible to EAE and exhibit blunted Th1 responses. However, whether decreased antigen-specific T-cell effector responses in H2RKO mice were due to a lack of H2R signaling in CD4+ T cells or antigen-presenting cells has remained unclear. We generated transgenic mice expressing H2R specifically in T cells on the H2RKO background, and, using wild-type B6 and H2RKO mice as controls, induced EAE either in the presence or absence of the ancillary adjuvant pertussis toxin (PTX), which models the effects of infectious inflammatory stimuli on autoimmune disease. We monitored the mice for clinical signs of EAE and neuropathology, as well as effector T-cell responses using flow cytometry. EAE severity and neuropathology in H2RKO mice expressing H2R exclusively in T cells become equal to those in wild-type B6 mice only when PTX is used to elicit disease. EAE complementation was associated with frequencies of CD4+IFN-γ+ and CD4+IL-17+ cells that are equal to or greater than those in wild-type B6, respectively. Thus, the regulation of encephalitogenic T-cell responses and EAE susceptibility by H2R signaling in CD4+ T cells is dependent on gene × environment interactions.—Saligrama, N., Case, L. K., Krementsov, D. N., Teuscher, C. Histamine H2 receptor signaling × environment interactions determine susceptibility to experimental allergic encephalomyelitis. PMID:24371118

  2. Subtle variations in Pten dose determine cancer susceptibility

    PubMed Central

    Alimonti, Andrea; Carracedo, Arkaitz; Clohessy, John G; Trotman, Lloyd C; Nardella, Caterina; Egia, Ainara; Salmena, Leonardo; Sampieri, Katia; Haveman, William J; Brogi, Edi; Richardson, Andrea L; Zhang, Jiangwen; Pandolfi, Pier Paolo

    2010-01-01

    Cancer susceptibility has been attributed to at least one heterozygous genetic alteration in a tumor suppressor gene (TSG)1. It has been hypothesized that subtle variations in TSG expression can promote cancer development2,3. However, this hypothesis has not yet been definitively supported in vivo. PTEN is a TSG frequently lost in human cancer and mutated in inherited cancer-predisposition syndromes4. Here, we analyze Pten hypermorphic mice (Ptenhy/+), expressing 80% normal levels of Pten. Ptenhy/+ mice develop a spectrum of tumors, with breast tumors occurring at the highest penetrance. All breast tumors analyzed here retained two intact copies of Pten and maintained Pten levels above heterozygosis. Notably, subtle downregulation of Pten altered the steady-state biology of the mammary tissues and the expression profiles of genes involved in cancer cell proliferation. We present an alterative working model for cancer development in which subtle reductions in the dose of TSGs predispose to tumorigenesis in a tissue-specific manner. PMID:20400965

  3. New common variants affecting susceptibility to basal cell carcinoma.

    PubMed

    Stacey, Simon N; Sulem, Patrick; Masson, Gisli; Gudjonsson, Sigurjon A; Thorleifsson, Gudmar; Jakobsdottir, Margret; Sigurdsson, Asgeir; Gudbjartsson, Daniel F; Sigurgeirsson, Bardur; Benediktsdottir, Kristrun R; Thorisdottir, Kristin; Ragnarsson, Rafn; Scherer, Dominique; Hemminki, Kari; Rudnai, Peter; Gurzau, Eugene; Koppova, Kvetoslava; Botella-Estrada, Rafael; Soriano, Virtudes; Juberias, Pablo; Saez, Berta; Gilaberte, Yolanda; Fuentelsaz, Victoria; Corredera, Cristina; Grasa, Matilde; Höiom, Veronica; Lindblom, Annika; Bonenkamp, Johannes J; van Rossum, Michelle M; Aben, Katja K H; de Vries, Esther; Santinami, Mario; Di Mauro, Maria G; Maurichi, Andrea; Wendt, Judith; Hochleitner, Pia; Pehamberger, Hubert; Gudmundsson, Julius; Magnusdottir, Droplaug N; Gretarsdottir, Solveig; Holm, Hilma; Steinthorsdottir, Valgerdur; Frigge, Michael L; Blondal, Thorarinn; Saemundsdottir, Jona; Bjarnason, Hjördis; Kristjansson, Kristleifur; Bjornsdottir, Gyda; Okamoto, Ichiro; Rivoltini, Licia; Rodolfo, Monica; Kiemeney, Lambertus A; Hansson, Johan; Nagore, Eduardo; Mayordomo, José I; Kumar, Rajiv; Karagas, Margaret R; Nelson, Heather H; Gulcher, Jeffrey R; Rafnar, Thorunn; Thorsteinsdottir, Unnur; Olafsson, Jon H; Kong, Augustine; Stefansson, Kari

    2009-08-01

    In a follow-up to our previously reported genome-wide association study of cutaneous basal cell carcinoma (BCC), we describe here several new susceptibility variants. SNP rs11170164, encoding a G138E substitution in the keratin 5 (KRT5) gene, affects risk of BCC (OR = 1.35, P = 2.1 x 10(-9)). A variant at 9p21 near CDKN2A and CDKN2B also confers susceptibility to BCC (rs2151280[C]; OR = 1.19, P = 6.9 x 10(-9)), as does rs157935[T] at 7q32 near the imprinted gene KLF14 (OR = 1.23, P = 5.7 x 10(-10)). The effect of rs157935[T] is dependent on the parental origin of the risk allele. None of these variants were found to be associated with melanoma or fair-pigmentation traits. A melanoma- and pigmentation-associated variant in the SLC45A2 gene, L374F, is associated with risk of both BCC and squamous cell carcinoma. Finally, we report conclusive evidence that rs401681[C] in the TERT-CLPTM1L locus confers susceptibility to BCC but protects against melanoma. PMID:19578363

  4. Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax

    PubMed Central

    Zimmerman, Peter A.; Ferreira, Marcelo U.; Howes, Rosalind E.; Mercereau-Puijalon, Odile

    2013-01-01

    Resistance to Plasmodium vivax blood-stage infection has been widely recognised to result from absence of the Duffy (Fy) blood group from the surface of red blood cells (RBCs) in individuals of African descent. Interestingly, recent studies from different malaria-endemic regions have begun to reveal new perspectives on the association between Duffy gene polymorphism and P. vivax malaria. In Papua New Guinea and the Americas, heterozygous carriers of a Duffy-negative allele are less susceptible to P. vivax infection than Duffy-positive homozygotes. In Brazil, studies show that the Fya antigen, compared to Fyb, is associated with lower binding to the P. vivax Duffy-binding protein and reduced susceptibility to vivax malaria. Additionally, it is interesting that numerous studies have now shown that P. vivax can infect RBCs and cause clinical disease in Duffy-negative people. This suggests that the relationship between P. vivax and the Duffy antigen is more complex than customarily described. Evidence of P. vivax Duffy-independent red cell invasion indicates that the parasite must be evolving alternative red cell invasion pathways. In this chapter, we review the evidence for P. vivax Duffy-dependent and Duffy-independent red cell invasion. We also consider the influence of further host gene polymorphism associated with malaria endemicity on susceptibility to vivax malaria. The interaction between the parasite and the RBC has significant potential to influence the effectiveness of P. vivax-specific vaccines and drug treatments. Ultimately, the relationships between red cell polymorphisms and P. vivax blood-stage infection will influence our estimates on the population at risk and efforts to eliminate vivax malaria. PMID:23384621

  5. The intersection between cell wall disassembly, ripening, and fruit susceptibility to Botrytis cinerea

    PubMed Central

    Cantu, D.; Vicente, A. R.; Greve, L. C.; Dewey, F. M.; Bennett, A. B.; Labavitch, J. M.; Powell, A. L. T.

    2008-01-01

    Fruit ripening is characterized by processes that modify texture and flavor but also by a dramatic increase in susceptibility to necrotrophic pathogens, such as Botrytis cinerea. Disassembly of the major structural polysaccharides of the cell wall (CW) is a significant process associated with ripening and contributes to fruit softening. In tomato, polygalacturonase (PG) and expansin (Exp) are among the CW proteins that cooperatively participate in ripening-associated CW disassembly. To determine whether endogenous CW disassembly influences the ripening-regulated increase in necrotropic pathogen susceptibility, B. cinerea susceptibility was assessed in transgenic fruit with suppressed polygalacturonase (LePG) and expansin (LeExp1) expression. Suppression of either LePG or LeExp1 alone did not reduce susceptibility but simultaneous suppression of both dramatically reduced the susceptibility of ripening fruit to B. cinerea, as measured by fungal biomass accumulation and by macerating lesion development. These results demonstrate that altering endogenous plant CW disassembly during ripening influences the course of infection by B. cinerea, perhaps by changing the structure or the accessibility of CW substrates to pathogen CW-degrading enzymes. Recognition of the role of ripening-associated CW metabolism in postharvest pathogen susceptibility may be useful in the design and development of strategies to limit pathogen losses during fruit storage, handling, and distribution. PMID:18199833

  6. Genetic Susceptibility to Head and Neck Squamous Cell Carcinoma

    SciTech Connect

    Lacko, Martin; Braakhuis, Boudewijn J.M.; Sturgis, Erich M.; Boedeker, Carsten C.; Suárez, Carlos; Rinaldo, Alessandra; Ferlito, Alfio; Takes, Robert P.

    2014-05-01

    Head-and-neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and its incidence is growing. Although environmental carcinogens and carcinogenic viruses are the main etiologic factors, genetic predisposition obviously plays a risk-modulating role, given that not all individuals exposed to these carcinogens experience the disease. This review highlights some aspects of genetic susceptibility to HNSCC: among others, genetic polymorphisms in biotransformation enzymes, DNA repair pathway, apoptotic pathway, human papillomavirus-related pathways, mitochondrial polymorphisms, and polymorphism related to the bilirubin-metabolized pathway. Furthermore, epigenetic variations, familial forms of HNSCC, functional assays for HNSCC risk assessment, and the implications and perspectives of research on genetic susceptibility in HNSCC are discussed.

  7. Assessment of oxidant susceptibility of red blood cells in various species based on cell deformability.

    PubMed

    Simmonds, Michael J; Meiselman, Herbert J; Marshall-Gradisnik, Sonya M; Pyne, Michael; Kakanis, Michael; Keane, James; Brenu, Ekua; Christy, Rhys; Baskurt, Oguz K

    2011-01-01

    The present study was designed to investigate the oxidant susceptibility of red blood cells (RBC) from four species (echidna, human, koala, Tasmanian devil) based on changes in cellular deformability. These species were specifically chosen based on differences in lifestyle and/or biology associated with varied levels of oxidative stress. The major focus was the influence of superoxide radicals generated within the cell (phenazine methosulfate, PMS, 50 μM) or in the extracellular medium (xanthine oxidase-hypoxanthine, XO-HX, 0.1 U/ml XO) on RBC deformability at various shear stresses (SS). RBC deformability was assessed by laser-diffraction analysis using a "slit-flow ektacytometer". Both superoxide-generating treatments resulted in significant increases of methemoglobin for all species (p < 0.01), with Tasmanian devil RBC demonstrating the most sensitivity to either treatment. PMS caused impaired RBC deformability for all species, but vast interspecies variations were observed: human and koala cells exhibited a similar sigmoid-like response to SS, short-beaked echidna values were markedly lower and only increased slightly with SS, while Tasmanian devil RBC were extremely rigid. The effect of XO-HX on RBC deformability was less when compared with PMS (i.e., smaller increase in rigidity) with the exception of Tasmanian devil RBC which exhibited essentially no deformation even at the highest SS; Tasmanian devil RBC response to XO-HX was thus comparable to that observed with PMS. Our findings indicate that ektacytometry can be used to determine the oxidant susceptibility of RBC from different species which varies significantly among mammals representing diverse lifestyles and evolutionary histories. These differences in susceptibility are consistent with species-specific discrepancies between observed and allometrically-predicted life spans and are compatible with the oxidant theory of aging. PMID:22433570

  8. The importance of growth kinetic analysis in determining bacterial susceptibility against antibiotics and silver nanoparticles.

    PubMed

    Theophel, Karsten; Schacht, Veronika J; Schlüter, Michael; Schnell, Sylvia; Stingu, Catalina-Suzana; Schaumann, Reiner; Bunge, Michael

    2014-01-01

    Routine antibiotics susceptibility testing still relies on standardized cultivation-based analyses, including measurement of inhibition zones in conventional agar diffusion tests and endpoint turbidity-based measurements. Here, we demonstrate that common off-line monitoring and endpoint determination after 18-24 h could be insufficient for reliable growth-dependent evaluation of antibiotic susceptibility. Different minimal inhibitory concentrations were obtained in 20- and 48 h microdilution plate tests using an Enterococcus faecium clinical isolate (strain UKI-MB07) as a model organism. Hence, we used an on-line kinetic assay for simultaneous cultivation and time-resolved growth analysis in a 96-well format instead of off-line susceptibility testing. Growth of the Enterococcus test organism was delayed up to 30 h in the presence of 0.25 μg mL(-1) of vancomycin and 8 μg mL(-1) of fosfomycin, after which pronounced growth was observed. Despite the delayed onset of growth, treatment with fosfomycin, daptomycin, fusidic acid, cefoxitin, or gentamicin resulted in higher maximum growth rates and/or higher final optical density values compared with antibiotic-free controls, indicating that growth stimulation and hormetic effects may occur with extended exposure to sublethal antibiotic concentrations. Whereas neither maximum growth rate nor final cell density correlated with antibiotic concentration, the lag phase duration for some antibiotics was a more meaningful indicator of dose-dependent growth inhibition. Our results also reveal that non-temporal growth profiles are only of limited value for cultivation-based antimicrobial silver nanoparticle susceptibility testing. The exposure to Ag(0) nanoparticles led to plasma membrane damage in a concentration-dependent manner and induced oxidative stress in Enterococcus faecium UKI-MB07, as shown by intracellular ROS accumulation. PMID:25426104

  9. The importance of growth kinetic analysis in determining bacterial susceptibility against antibiotics and silver nanoparticles

    PubMed Central

    Theophel, Karsten; Schacht, Veronika J.; Schlüter, Michael; Schnell, Sylvia; Stingu, Catalina-Suzana; Schaumann, Reiner; Bunge, Michael

    2014-01-01

    Routine antibiotics susceptibility testing still relies on standardized cultivation-based analyses, including measurement of inhibition zones in conventional agar diffusion tests and endpoint turbidity-based measurements. Here, we demonstrate that common off-line monitoring and endpoint determination after 18–24 h could be insufficient for reliable growth-dependent evaluation of antibiotic susceptibility. Different minimal inhibitory concentrations were obtained in 20- and 48 h microdilution plate tests using an Enterococcus faecium clinical isolate (strain UKI-MB07) as a model organism. Hence, we used an on-line kinetic assay for simultaneous cultivation and time-resolved growth analysis in a 96-well format instead of off-line susceptibility testing. Growth of the Enterococcus test organism was delayed up to 30 h in the presence of 0.25 μg mL-1 of vancomycin and 8 μg mL-1 of fosfomycin, after which pronounced growth was observed. Despite the delayed onset of growth, treatment with fosfomycin, daptomycin, fusidic acid, cefoxitin, or gentamicin resulted in higher maximum growth rates and/or higher final optical density values compared with antibiotic-free controls, indicating that growth stimulation and hormetic effects may occur with extended exposure to sublethal antibiotic concentrations. Whereas neither maximum growth rate nor final cell density correlated with antibiotic concentration, the lag phase duration for some antibiotics was a more meaningful indicator of dose-dependent growth inhibition. Our results also reveal that non-temporal growth profiles are only of limited value for cultivation-based antimicrobial silver nanoparticle susceptibility testing. The exposure to Ag(0) nanoparticles led to plasma membrane damage in a concentration-dependent manner and induced oxidative stress in Enterococcus faecium UKI-MB07, as shown by intracellular ROS accumulation. PMID:25426104

  10. The variability of autophagy and cell death susceptibility

    PubMed Central

    Loos, Ben; Engelbrecht, Anna-Mart; Lockshin, Richard A.; Klionsky, Daniel J; Zakeri, Zahra

    2013-01-01

    Impaired autophagic machinery is implicated in a number of diseases such as heart disease, neurodegeneration and cancer. A common denominator in these pathologies is a dysregulation of autophagy that has been linked to a change in susceptibility to cell death. Although we have progressed in understanding the molecular machinery and regulation of the autophagic pathway, many unanswered questions remain. How does the metabolic contribution of autophagy connect with the cell’s history and how does its current autophagic flux affect metabolic status and susceptibility to undergo cell death? How does autophagic flux operate to switch metabolic direction and what are the underlying mechanisms in metabolite and energetic sensing, metabolite substrate provision and metabolic integration during the cellular stress response? In this article we focus on unresolved questions that address issues around the role of autophagy in sensing the energetic environment and its role in actively generating metabolite substrates. We attempt to provide answers by explaining how and when a change in autophagic pathway activity such as primary stress response is able to affect cell viability and when not. By addressing the dynamic metabolic relationship between autophagy, apoptosis and necrosis we provide a new perspective on the parameters that connect autophagic activity, severity of injury and cellular history in a logical manner. Last, by evaluating the cell’s condition and autophagic activity in a clear context of regulatory parameters in the intra- and extracellular environment, this review provides new concepts that set autophagy into an energetic feedback loop, that may assist in our understanding of autophagy in maintaining healthy cells or when it controls the threshold between cell death and cell survival. PMID:23846383

  11. Determinants of in vitro drug susceptibility testing of Plasmodium vivax.

    PubMed

    Russell, B; Chalfein, F; Prasetyorini, B; Kenangalem, E; Piera, K; Suwanarusk, R; Brockman, A; Prayoga, P; Sugiarto, P; Cheng, Q; Tjitra, E; Anstey, N M; Price, R N

    2008-03-01

    In Papua, Indonesia, the antimalarial susceptibility of Plasmodium vivax (n = 216) and P. falciparum (n = 277) was assessed using a modified schizont maturation assay for chloroquine, amodiaquine, artesunate, lumefantrine, mefloquine, and piperaquine. The most effective antimalarial against P. vivax and P. falciparum was artesunate, with geometric mean 50% inhibitory concentrations (IC50s) (95% confidence intervals [CI]) of 1.31 nM (1.07 to 1.59) and 0.64 nM (0.53 to 0.79), respectively. In contrast, the geometric mean chloroquine IC50 for P. vivax was 295 nM (227 to 384) compared to only 47.4 nM (42.2 to 53.3) for P. falciparum. Two factors were found to significantly influence the in vitro drug response of P. vivax: the initial stage of the parasite and the duration of the assay. Isolates of P. vivax initially at the trophozoite stage had significantly higher chloroquine IC50s (478 nM [95% CI, 316 to 722]) than those initially at the ring stage (84.7 nM [95% CI, 45.7 to 157]; P < 0.001). Synchronous isolates of P. vivax and P. falciparum which reached the target of 40% schizonts in the control wells within 30 h had significantly higher geometric mean chloroquine IC50s (435 nM [95% CI, 169 to 1,118] and 55.9 nM [95% CI, 48 to 64.9], respectively) than isolates that took more than 30 h (39.9 nM [14.6 to 110.4] and 36.9 nM [31.2 to 43.7]; P < 0.005). The results demonstrate the marked stage-specific activity of chloroquine with P. vivax and suggest that susceptibility to chloroquine may be associated with variable growth rates. These findings have important implications for the phenotypic and downstream genetic characterization of P. vivax.

  12. Susceptibility to Enzymatic Degradation of Cell Walls From Bean Plants Resistant and Susceptible to Rhizoctonia solani Kuhn.

    PubMed

    Bateman, D F; Van Etten, H D

    1969-05-01

    Enzymes in culture filtrates of Rhizoctonia solani Kuhn grown using 4-day old or 20-day old bean (Phaseolus vulgaris L.) hypocotyl cell walls as a carbon source degraded xylan, galactan, galactomannan, araban, polygalacturonic acid, and carboxymethylcellulose. Extracts of lesions from R. solani infected plants, but not healthy plants, contained similar enzymatic activities. These enzyme sources readily solubilized cell wall constituents containing arabinose, galactose, and glucose from 4-day old, but not from 20-day old, bean cell walls. Analysis of cell walls prepared from infected plants revealed that the alterations in cell wall composition in the diseased host were limited largely to the immediate lesion areas and occurred during the early phases of pathogenesis. The cell walls of young susceptible bean seedlings could be degraded by R. solani enzymes, but the cell walls of older plants which are resistant to this pathogen were not susceptible to enzymatic destruction by the same enzyme preparation.

  13. Genome-wide association study identifies novel susceptibility loci for cutaneous squamous cell carcinoma

    PubMed Central

    Chahal, Harvind S.; Lin, Yuan; Ransohoff, Katherine J.; Hinds, David A.; Wu, Wenting; Dai, Hong-Ji; Qureshi, Abrar A.; Li, Wen-Qing; Kraft, Peter; Tang, Jean Y.; Han, Jiali; Sarin, Kavita Y.

    2016-01-01

    Cutaneous squamous cell carcinoma represents the second most common cutaneous malignancy, affecting 7–11% of Caucasians in the United States. The genetic determinants of susceptibility to cutaneous squamous cell carcinoma remain largely unknown. Here we report the results of a two-stage genome-wide association study of cutaneous squamous cell carcinoma, totalling 7,404 cases and 292,076 controls. Eleven loci reached genome-wide significance (P<5 × 10−8) including seven previously confirmed pigmentation-related loci: MC1R, ASIP, TYR, SLC45A2, OCA2, IRF4 and BNC2. We identify an additional four susceptibility loci: 11q23.3 CADM1, a metastasis suppressor gene involved in modifying tumour interaction with cell-mediated immunity; 2p22.3; 7p21.1 AHR, the dioxin receptor involved in anti-apoptotic pathways and melanoma progression; and 9q34.3 SEC16A, a putative oncogene with roles in secretion and cellular proliferation. These susceptibility loci provide deeper insight into the pathogenesis of squamous cell carcinoma. PMID:27424798

  14. Genome-wide association study identifies novel susceptibility loci for cutaneous squamous cell carcinoma.

    PubMed

    Chahal, Harvind S; Lin, Yuan; Ransohoff, Katherine J; Hinds, David A; Wu, Wenting; Dai, Hong-Ji; Qureshi, Abrar A; Li, Wen-Qing; Kraft, Peter; Tang, Jean Y; Han, Jiali; Sarin, Kavita Y

    2016-01-01

    Cutaneous squamous cell carcinoma represents the second most common cutaneous malignancy, affecting 7-11% of Caucasians in the United States. The genetic determinants of susceptibility to cutaneous squamous cell carcinoma remain largely unknown. Here we report the results of a two-stage genome-wide association study of cutaneous squamous cell carcinoma, totalling 7,404 cases and 292,076 controls. Eleven loci reached genome-wide significance (P<5 × 10(-8)) including seven previously confirmed pigmentation-related loci: MC1R, ASIP, TYR, SLC45A2, OCA2, IRF4 and BNC2. We identify an additional four susceptibility loci: 11q23.3 CADM1, a metastasis suppressor gene involved in modifying tumour interaction with cell-mediated immunity; 2p22.3; 7p21.1 AHR, the dioxin receptor involved in anti-apoptotic pathways and melanoma progression; and 9q34.3 SEC16A, a putative oncogene with roles in secretion and cellular proliferation. These susceptibility loci provide deeper insight into the pathogenesis of squamous cell carcinoma. PMID:27424798

  15. A New Reporter Cell Line to Monitor HIV Infection and Drug Susceptibility in vitro

    NASA Astrophysics Data System (ADS)

    Gervaix, Alain; West, Daniel; Leoni, Lorenzo M.; Richman, Douglas D.; Wong-Staal, Flossie; Corbeil, Jacques

    1997-04-01

    Determination of HIV infectivity in vitro and its inhibition by antiretroviral drugs by monitoring reduction of production of p24 antigen is expensive and time consuming. Such assays also do not allow accurate quantitation of the number of infected cells over time. To develop a simple, rapid, and direct method for monitoring HIV infection, we generated a stable T-cell line (CEM) containing a plasmid encoding the green fluorescent protein (humanized S65T GFP) driven by the HIV-1 long terminal repeat. Clones were selected that displayed low constitutive background fluorescnece, but a high level of GFP expression upon infection with HIV. HIV-1 infection induced a 100- to 1,000-fold increase in relative fluorescence of cells over 2 to 4 days as monitored by fluorescence microscopy, cytofluorimetry, and flow cytometry. Addition of inhibitors of reverse transcriptase, protease, and other targets at different multiplicities of infection permitted the accurate determination of drug susceptibility. This technique also permitted quantitation of infectivity of viral preparations by assessment of number of cells infected in the first round of infection. In conclusion, the CEM-GFP reporter cell line provides a simple, rapid, and direct method for monitoring HIV infectivity titers and antiretroviral drug susceptibility of syncytium-inducing strains.

  16. A rapid antimicrobial susceptibility test based on single-cell morphological analysis.

    PubMed

    Choi, Jungil; Yoo, Jungheon; Lee, Mincheol; Kim, Eun-Geun; Lee, Ji Soo; Lee, Seungok; Joo, Seik; Song, Sang Hoon; Kim, Eui-Chong; Lee, Jung Chan; Kim, Hee Chan; Jung, Yong-Gyun; Kwon, Sunghoon

    2014-12-17

    A rapid antibiotic susceptibility test (AST) is desperately needed in clinical settings for fast and appropriate antibiotic administration. Traditional ASTs, which rely on cell culture, are not suitable for urgent cases of bacterial infection and antibiotic resistance owing to their relatively long test times. We describe a novel AST called single-cell morphological analysis (SCMA) that can determine antimicrobial susceptibility by automatically analyzing and categorizing morphological changes in single bacterial cells under various antimicrobial conditions. The SCMA was tested with four Clinical and Laboratory Standards Institute standard bacterial strains and 189 clinical samples, including extended-spectrum β-lactamase-positive Escherichia coli and Klebsiella pneumoniae, imipenem-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococci from hospitals. The results were compared with the gold standard broth microdilution test. The SCMA results were obtained in less than 4 hours, with 91.5% categorical agreement and 6.51% minor, 2.56% major, and 1.49% very major discrepancies. Thus, SCMA provides rapid and accurate antimicrobial susceptibility data that satisfy the recommended performance of the U.S. Food and Drug Administration. PMID:25520395

  17. Susceptibility of human embryonic stem cell-derived neural cells to Japanese encephalitis virus infection.

    PubMed

    Shen, Shih-Cheng; Shen, Ching-I; Lin, Ho; Chen, Chun-Jung; Chang, Chia-Yu; Chen, Sheng-Mei; Lee, Hsiu-Chin; Lai, Ping-Shan; Su, Hong-Lin

    2014-01-01

    Pluripotent human embryonic stem cells (hESCs) can be efficiently directed to become immature neuroepithelial precursor cells (NPCs) and functional mature neural cells, including neurotransmitter-secreting neurons and glial cells. Investigating the susceptibility of these hESCs-derived neural cells to neurotrophic viruses, such as Japanese encephalitis virus (JEV), provides insight into the viral cell tropism in the infected human brain. We demonstrate that hESC-derived NPCs are highly vulnerable to JEV infection at a low multiplicity of infection (MOI). In addition, glial fibrillary acid protein (GFAP)-expressing glial cells are also susceptible to JEV infection. In contrast, only a few mature neurons were infected at MOI 10 or higher on the third day post-infection. In addition, functional neurotransmitter-secreting neurons are also resistant to JEV infection at high MOI. Moreover, we discover that vimentin intermediate filament, reported as a putative neurovirulent JEV receptor, is highly expressed in NPCs and glial cells, but not mature neurons. These results indicate that the expression of vimentin in neural cells correlates to the cell tropism of JEV. Finally, we further demonstrate that membranous vimentin is necessary for the susceptibility of hESC-derived NPCs to JEV infection.

  18. In vitro antibiotic susceptibilities of Burkholderia mallei (causative agent of glanders) determined by broth microdilution and E-test.

    PubMed

    Heine, H S; England, M J; Waag, D M; Byrne, W R

    2001-07-01

    In vitro susceptibilities to 28 antibiotics were determined for 11 strains of Burkholderia mallei by the broth microdilution method. The B. mallei strains demonstrated susceptibility to aminoglycosides, macrolides, quinolones, doxycycline, piperacillin, ceftazidime, and imipenem. For comparison and evaluation, 17 antibiotic susceptibilities were also determined by the E-test. E-test values were always lower than the broth dilution values. Establishing and comparing antibiotic susceptibilities of specific B. mallei strains will provide reference information for assessing new antibiotic agents.

  19. Degradation/oxidation susceptibility of organic photovoltaic cells in aqueous solutions.

    PubMed

    Habib, K; Husain, A; Al-Hazza, A

    2015-12-01

    A criterion of the degradation/oxidation susceptibility of organic photovoltaic (OPV) cells in aqueous solutions was proposed for the first time. The criterion was derived based on calculating the limit of the ratio value of the polarization resistance of an OPV cell in aqueous solution (Rp(s)) to the polarization resistance of the OPV cell in air (Rp(air)). In other words, the criterion lim(Rp(s)/Rp(air)) = 1 was applied to determine the degradation/oxidation of the OPV cell in the aqueous solution when Rp(air) became equal (increased) to Rp(s) as a function of time of the exposure of the OPV cell to the aqueous solution. This criterion was not only used to determine the degradation/oxidation of different OPV cells in a simulated operational environment but also it was used to determine the electrochemical behavior of OPV cells in deionized water and a polluted water with fine particles of sand. The values of Rp(s) were determined by the electrochemical impedance spectroscopy at low frequency. In addition, the criterion can be applied under diverse test conditions with a predetermined period of OPV operations.

  20. Analysis of target cell susceptibility as a basis for the development of a chemoprotective strategy against benzene-induced hematotoxicities.

    PubMed Central

    Trush, M A; Twerdok, L E; Rembish, S J; Zhu, H; Li, Y

    1996-01-01

    A goal of our research is to identify biochemical factors that underlie the susceptibility of bone marrow cell populations to benzene metabolites so as to develop a mechanistically based chemoprotective strategy that may be used in susceptible humans exposed to benzene. By doing biochemical risk analysis of bone marrow stromal cells from mice and rats and the human myeloid cell lines, HL-60 and ML-1; and by using buthionine sulfoximine and dicumarol we have observed that the susceptibility of these cell populations to hydroquinone (HQ) correlates with their concentration of glutathione (GSH) and activity of quinone reductase (QR). Accordingly, the induction of QR and GSH by 1,2-dithiole-3-thione (D3T) in these cell populations has resulted in a significant protection against the following hydroquinone-mediated toxicities: inhibition of cell proliferation and viability; reduced ability of stromal cells to support myelopoiesis; and altered differentiated of ML-1 cells to monocytes/macrophages. Preliminary in vivo experiments indicate that feeding mice D3T results in an induction of QR in the bone marrow compartment such that stromal cells are more resistant to hydroquinone-induced cytotoxicity in vitro. Overall, these studies suggest that in addition to hepatic cytochrome P4502E1, bone marrow QR and GSH are factors that could determine an individual's relative susceptibility to the toxic effects of benzene. PMID:9118897

  1. Epithelial-macrophage interactions determine pulmonary fibrosis susceptibility in Hermansky-Pudlak syndrome

    PubMed Central

    Young, Lisa R.; Gulleman, Peter M.; Short, Chelsi W.; Tanjore, Harikrishna; Sherrill, Taylor; Qi, Aidong; McBride, Andrew P.; Zaynagetdinov, Rinat; Benjamin, John T.; Lawson, William E.; Novitskiy, Sergey V.; Blackwell, Timothy S.

    2016-01-01

    Alveolar epithelial cell (AEC) dysfunction underlies the pathogenesis of pulmonary fibrosis in Hermansky-Pudlak syndrome (HPS) and other genetic syndromes associated with interstitial lung disease; however, mechanisms linking AEC dysfunction and fibrotic remodeling are incompletely understood. Since increased macrophage recruitment precedes pulmonary fibrosis in HPS, we investigated whether crosstalk between AECs and macrophages determines fibrotic susceptibility. We found that AECs from HPS mice produce excessive MCP-1, which was associated with increased macrophages in the lungs of unchallenged HPS mice. Blocking MCP-1/CCR2 signaling in HPS mice with genetic deficiency of CCR2 or targeted deletion of MCP-1 in AECs normalized macrophage recruitment, decreased AEC apoptosis, and reduced lung fibrosis in these mice following treatment with low-dose bleomycin. We observed increased TGF-β production by HPS macrophages, which was eliminated by CCR2 deletion. Selective deletion of TGF-β in myeloid cells or of TGF-β signaling in AECs through deletion of TGFBR2 protected HPS mice from AEC apoptosis and bleomycin-induced fibrosis. Together, these data reveal a feedback loop in which increased MCP-1 production by dysfunctional AECs results in recruitment and activation of lung macrophages that produce TGF-β, thus amplifying the fibrotic cascade through AEC apoptosis and stimulation of fibrotic remodeling. PMID:27777976

  2. Genetically Determined Susceptibility to Tuberculosis in Mice Causally Involves Accelerated and Enhanced Recruitment of Granulocytes

    PubMed Central

    Keller, Christine; Hoffmann, Reinhard; Lang, Roland; Brandau, Sven; Hermann, Corinna; Ehlers, Stefan

    2006-01-01

    Classical twin studies and recent linkage analyses of African populations have revealed a potential involvement of host genetic factors in susceptibility or resistance to Mycobacterium tuberculosis infection. In order to identify the candidate genes involved and test their causal implication, we capitalized on the mouse model of tuberculosis, since inbred mouse strains also differ substantially in their susceptibility to infection. Two susceptible and two resistant mouse strains were aerogenically infected with 1,000 CFU of M. tuberculosis, and the regulation of gene expression was examined by Affymetrix GeneChip U74A array with total lung RNA 2 and 4 weeks postinfection. Four weeks after infection, 96 genes, many of which are involved in inflammatory cell recruitment and activation, were regulated in common. One hundred seven genes were differentially regulated in susceptible mouse strains, whereas 43 genes were differentially expressed only in resistant mice. Data mining revealed a bias towards the expression of genes involved in granulocyte pathophysiology in susceptible mice, such as an upregulation of those for the neutrophil chemoattractant LIX (CXCL5), interleukin 17 receptor, phosphoinositide kinase 3 delta, or gamma interferon-inducible protein 10. Following M. tuberculosis challenge in both airways or peritoneum, granulocytes were recruited significantly faster and at higher numbers in susceptible than in resistant mice. When granulocytes were efficiently depleted by either of two regimens at the onset of infection, only susceptible mice survived aerosol challenge with M. tuberculosis significantly longer than control mice. We conclude that initially enhanced recruitment of granulocytes contributes to susceptibility to tuberculosis. PMID:16790804

  3. Host genetics determine susceptibility to avian influenza infection and transmission dynamics

    PubMed Central

    Ruiz-Hernandez, Raul; Mwangi, William; Peroval, Marylene; Sadeyen, Jean-Remy; Ascough, Stephanie; Balkissoon, Devanand; Staines, Karen; Boyd, Amy; McCauley, John; Smith, Adrian; Butter, Colin

    2016-01-01

    Host-genetic control of influenza virus infection has been the object of little attention. In this study we determined that two inbred lines of chicken differing in their genetic background , Lines 0 and C-B12, were respectively relatively resistant and susceptible to infection with the low pathogenicity influenza virus A/Turkey/England/647/77 as defined by substantial differences in viral shedding trajectories. Resistant birds, although infected, were unable to transmit virus to contact birds, as ultimately only the presence of a sustained cloacal shedding (and not oropharyngeal shedding) was critical for transmission. Restriction of within-bird transmission of virus occurred in the resistant line, with intra-nares or cloacal infection resulting in only local shedding and failing to transmit fully through the gastro-intestinal-pulmonary tract. Resistance to infection was independent of adaptive immune responses, including the expansion of specific IFNγ secreting cells or production of influenza-specific antibody. Genetic resistance to a novel H9N2 virus was less robust, though significant differences between host genotypes were still clearly evident. The existence of host-genetic determination of the outcome of influenza infection offers tools for the further dissection of this regulation and also for understanding the mechanisms of influenza transmission within and between birds. PMID:27279280

  4. Host genetics determine susceptibility to avian influenza infection and transmission dynamics.

    PubMed

    Ruiz-Hernandez, Raul; Mwangi, William; Peroval, Marylene; Sadeyen, Jean-Remy; Ascough, Stephanie; Balkissoon, Devanand; Staines, Karen; Boyd, Amy; McCauley, John; Smith, Adrian; Butter, Colin

    2016-01-01

    Host-genetic control of influenza virus infection has been the object of little attention. In this study we determined that two inbred lines of chicken differing in their genetic background , Lines 0 and C-B12, were respectively relatively resistant and susceptible to infection with the low pathogenicity influenza virus A/Turkey/England/647/77 as defined by substantial differences in viral shedding trajectories. Resistant birds, although infected, were unable to transmit virus to contact birds, as ultimately only the presence of a sustained cloacal shedding (and not oropharyngeal shedding) was critical for transmission. Restriction of within-bird transmission of virus occurred in the resistant line, with intra-nares or cloacal infection resulting in only local shedding and failing to transmit fully through the gastro-intestinal-pulmonary tract. Resistance to infection was independent of adaptive immune responses, including the expansion of specific IFNγ secreting cells or production of influenza-specific antibody. Genetic resistance to a novel H9N2 virus was less robust, though significant differences between host genotypes were still clearly evident. The existence of host-genetic determination of the outcome of influenza infection offers tools for the further dissection of this regulation and also for understanding the mechanisms of influenza transmission within and between birds.

  5. Host genetics determine susceptibility to avian influenza infection and transmission dynamics.

    PubMed

    Ruiz-Hernandez, Raul; Mwangi, William; Peroval, Marylene; Sadeyen, Jean-Remy; Ascough, Stephanie; Balkissoon, Devanand; Staines, Karen; Boyd, Amy; McCauley, John; Smith, Adrian; Butter, Colin

    2016-01-01

    Host-genetic control of influenza virus infection has been the object of little attention. In this study we determined that two inbred lines of chicken differing in their genetic background , Lines 0 and C-B12, were respectively relatively resistant and susceptible to infection with the low pathogenicity influenza virus A/Turkey/England/647/77 as defined by substantial differences in viral shedding trajectories. Resistant birds, although infected, were unable to transmit virus to contact birds, as ultimately only the presence of a sustained cloacal shedding (and not oropharyngeal shedding) was critical for transmission. Restriction of within-bird transmission of virus occurred in the resistant line, with intra-nares or cloacal infection resulting in only local shedding and failing to transmit fully through the gastro-intestinal-pulmonary tract. Resistance to infection was independent of adaptive immune responses, including the expansion of specific IFNγ secreting cells or production of influenza-specific antibody. Genetic resistance to a novel H9N2 virus was less robust, though significant differences between host genotypes were still clearly evident. The existence of host-genetic determination of the outcome of influenza infection offers tools for the further dissection of this regulation and also for understanding the mechanisms of influenza transmission within and between birds. PMID:27279280

  6. Predominant binding of Theiler's viruses to a 34-kilodalton receptor protein on susceptible cell lines.

    PubMed Central

    Kilpatrick, D R; Lipton, H L

    1991-01-01

    Western immunoblots of BHK-21 cell lysates probed with the highly virulent GDVII and the less virulent BeAn strains of Theiler's murine encephalomyelitis virus (TMEV) revealed predominant binding to a 34-kDa membrane protein and much lower levels of binding to 100- and 18-kDa membrane proteins. Complete inhibition of virus binding to both the 34- and 18-kDa membrane species by excess unlabeled TMEV demonstrated specificity of binding. Virus binding was also blocked by wheat germ agglutinin, which specifically binds to sialic acid residues and blocks TMEV binding to whole BHK-21 cells. Radiolabeled TMEV also bound to 100-, 34-, and 18-kDa membrane proteins expressed on other TMEV permissive cell lines but not on the nonpermissive cell lines tested. These data suggest that a 34-kDa cellular protein may be the primary determinant of susceptibility to TMEV infection by mediating the binding of GDVII and BeAn viruses to susceptible cells. Images PMID:1895381

  7. Decreased Polysaccharide Feruloylation Compromises Plant Cell Wall Integrity and Increases Susceptibility to Necrotrophic Fungal Pathogens

    PubMed Central

    Reem, Nathan T.; Pogorelko, Gennady; Lionetti, Vincenzo; Chambers, Lauran; Held, Michael A.; Bellincampi, Daniela; Zabotina, Olga A.

    2016-01-01

    The complexity of cell wall composition and structure determines the strength, flexibility, and function of the primary cell wall in plants. However, the contribution of the various components to cell wall integrity (CWI) and function remains unclear. Modifications of cell wall composition can induce plant responses known as CWI control. In this study, we used transgenic expression of the fungal feruloyl esterase AnFAE to examine the effect of post-synthetic modification of Arabidopsis and Brachypodium cell walls. Transgenic Arabidopsis plants expressing AnFAE showed a significant reduction of monomeric ferulic acid, decreased amounts of wall-associated extensins, and increased susceptibility to Botrytis cinerea, compared with wild type. Transgenic Brachypodium showed reductions in monomeric and dimeric ferulic acids and increased susceptibility to Bipolaris sorokiniana. Upon infection, transgenic Arabidopsis and Brachypodium plants also showed increased expression of several defense-related genes compared with wild type. These results demonstrate a role, in both monocot and dicot plants, of polysaccharide feruloylation in plant CWI, which contributes to plant resistance to necrotrophic pathogens. PMID:27242834

  8. Decreased Polysaccharide Feruloylation Compromises Plant Cell Wall Integrity and Increases Susceptibility to Necrotrophic Fungal Pathogens.

    PubMed

    Reem, Nathan T; Pogorelko, Gennady; Lionetti, Vincenzo; Chambers, Lauran; Held, Michael A; Bellincampi, Daniela; Zabotina, Olga A

    2016-01-01

    The complexity of cell wall composition and structure determines the strength, flexibility, and function of the primary cell wall in plants. However, the contribution of the various components to cell wall integrity (CWI) and function remains unclear. Modifications of cell wall composition can induce plant responses known as CWI control. In this study, we used transgenic expression of the fungal feruloyl esterase AnFAE to examine the effect of post-synthetic modification of Arabidopsis and Brachypodium cell walls. Transgenic Arabidopsis plants expressing AnFAE showed a significant reduction of monomeric ferulic acid, decreased amounts of wall-associated extensins, and increased susceptibility to Botrytis cinerea, compared with wild type. Transgenic Brachypodium showed reductions in monomeric and dimeric ferulic acids and increased susceptibility to Bipolaris sorokiniana. Upon infection, transgenic Arabidopsis and Brachypodium plants also showed increased expression of several defense-related genes compared with wild type. These results demonstrate a role, in both monocot and dicot plants, of polysaccharide feruloylation in plant CWI, which contributes to plant resistance to necrotrophic pathogens.

  9. Decreased Polysaccharide Feruloylation Compromises Plant Cell Wall Integrity and Increases Susceptibility to Necrotrophic Fungal Pathogens.

    PubMed

    Reem, Nathan T; Pogorelko, Gennady; Lionetti, Vincenzo; Chambers, Lauran; Held, Michael A; Bellincampi, Daniela; Zabotina, Olga A

    2016-01-01

    The complexity of cell wall composition and structure determines the strength, flexibility, and function of the primary cell wall in plants. However, the contribution of the various components to cell wall integrity (CWI) and function remains unclear. Modifications of cell wall composition can induce plant responses known as CWI control. In this study, we used transgenic expression of the fungal feruloyl esterase AnFAE to examine the effect of post-synthetic modification of Arabidopsis and Brachypodium cell walls. Transgenic Arabidopsis plants expressing AnFAE showed a significant reduction of monomeric ferulic acid, decreased amounts of wall-associated extensins, and increased susceptibility to Botrytis cinerea, compared with wild type. Transgenic Brachypodium showed reductions in monomeric and dimeric ferulic acids and increased susceptibility to Bipolaris sorokiniana. Upon infection, transgenic Arabidopsis and Brachypodium plants also showed increased expression of several defense-related genes compared with wild type. These results demonstrate a role, in both monocot and dicot plants, of polysaccharide feruloylation in plant CWI, which contributes to plant resistance to necrotrophic pathogens. PMID:27242834

  10. COMPARISON OF SUSCEPTIBILITY TO INFLUENZA INFECTION IN NASAL EPITHELIAL CELLS OBTAINED FROM SMOKERS AND NON-SMOKERS

    EPA Science Inventory

    Several studies have demonstrated that individuals who smoke have greater susceptibility to influenza infections, as well as other respiratory virus infections, than non-smokers, yet the role of airway epithelial cells in this response is not clear. To determine whether in vivo t...

  11. Quantitative Microplate-Based Growth Assay for Determination of Antifungal Susceptibility of Histoplasma capsulatum Yeasts

    PubMed Central

    Goughenour, Kristie D.; Balada-Llasat, Joan-Miquel

    2015-01-01

    Standardized methodologies for determining the antifungal susceptibility of fungal pathogens is central to the clinical management of invasive fungal disease. Yeast-form fungi can be tested using broth macrodilution and microdilution assays. Reference procedures exist for Candida species and Cryptococcus yeasts; however, no standardized methods have been developed for testing the antifungal susceptibility of yeast forms of the dimorphic systemic fungal pathogens. For the dimorphic fungal pathogen Histoplasma capsulatum, susceptibility to echinocandins differs for the yeast and the filamentous forms, which highlights the need to employ Histoplasma yeasts, not hyphae, in antifungal susceptibility tests. To address this, we developed and optimized methodology for the 96-well microtiter plate-based measurement of Histoplasma yeast growth in vitro. Using optical density, the assay is quantitative for fungal growth with a dynamic range greater than 30-fold. Concentration and assay reaction time parameters were also optimized for colorimetric (MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction) and fluorescent (resazurin reduction) indicators of fungal vitality. We employed this microtiter-based assay to determine the antifungal susceptibility patterns of multiple clinical isolates of Histoplasma representing different phylogenetic groups. This methodology fulfills a critical need for the ability to monitor the effectiveness of antifungals on Histoplasma yeasts, the morphological form present in mammalian hosts and, thus, the form most relevant to disease. PMID:26246483

  12. Bacterial Manipulation of NK Cell Regulatory Activity Increases Susceptibility to Listeria monocytogenes Infection

    PubMed Central

    Guthrie, Brandon S.; Schmidt, Rebecca L.; Jamieson, Amanda; Merkel, Patricia; Knight, Vijaya; Cole, Caroline M.; Raulet, David H.; Lenz, Laurel L.

    2016-01-01

    Natural killer (NK) cells produce interferon (IFN)-γ and thus have been suggested to promote type I immunity during bacterial infections. Yet, Listeria monocytogenes (Lm) and some other pathogens encode proteins that cause increased NK cell activation. Here, we show that stimulation of NK cell activation increases susceptibility during Lm infection despite and independent from robust NK cell production of IFNγ. The increased susceptibility correlated with IL-10 production by responding NK cells. NK cells produced IL-10 as their IFNγ production waned and the Lm virulence protein p60 promoted induction of IL-10 production by mouse and human NK cells. NK cells consequently exerted regulatory effects to suppress accumulation and activation of inflammatory myeloid cells. Our results reveal new dimensions of the role played by NK cells during Lm infection and demonstrate the ability of this bacterial pathogen to exploit the induction of regulatory NK cell activity to increase host susceptibility. PMID:27295349

  13. In Vitro Antibiotic Susceptibilities of Yersinia pestis Determined by Broth Microdilution following CLSI Methods

    PubMed Central

    Hershfield, Jeremy; Marchand, Charles; Miller, Lynda; Halasohoris, Stephanie; Purcell, Bret K.; Worsham, Patricia L.

    2015-01-01

    In vitro susceptibilities to 45 antibiotics were determined for 30 genetically and geographically diverse strains of Yersinia pestis by the broth microdilution method at two temperatures, 28°C and 35°C, following Clinical and Laboratory Standards Institute (CLSI) methods. The Y. pestis strains demonstrated susceptibility to aminoglycosides, quinolones, tetracyclines, β-lactams, cephalosporins, and carbapenems. Only a 1-well shift was observed for the majority of antibiotics between the two temperatures. Establishing and comparing antibiotic susceptibilities of a diverse but specific set of Y. pestis strains by standardized methods and establishing population ranges and MIC50 and MIC90 values provide reference information for assessing new antibiotic agents and also provide a baseline for use in monitoring any future emergence of resistance. PMID:25583720

  14. Rapid determination of antiviral drug susceptibility of herpes simplex virus types 1 and 2 by real-time PCR.

    PubMed

    Thi, Thuong Nguyen; Deback, Claire; Malet, Isabelle; Bonnafous, Pascale; Ait-Arkoub, Zaïna; Agut, Henri

    2006-03-01

    An antiviral drug susceptibility assay of herpes simplex virus (HSV) was developed using real-time PCR quantification of intracellular viral DNA load. The number of HSV DNA copies within Vero cells after 24 h infection was strongly correlated with the number of plaques obtained after 72 h infection. Antiviral drug susceptibility of HSV was determined after virus growth for 24h by measuring the reduction of intracellular HSV DNA in the presence of increasing concentrations of either acyclovir (ACV) or foscarnet (PFA). This assay required neither preliminary titration of infectious stock nor follow-up of cytopathic effect. The 50% inhibitory concentrations (IC50s) obtained with 27 isolates of HSV types 1 and 2 by using this test were significantly correlated with those obtained in parallel with plaque reduction assay taken as the reference method (r=0.91, p<0.0001 and r=0.51, p=0.009 for ACV and PFA, respectively). The threshold real-time PCR IC50s for ACV and PFA resistance did not differ according to HSV type and were determined to be 1.0 and 100 microM, respectively. The real-time PCR susceptibility assay reported here is rapid, reproducible, applicable for HSV-1 as well as HSV-2, and suitable for automation.

  15. Functional BCL-2 regulatory genetic variants contribute to susceptibility of esophageal squamous cell carcinoma.

    PubMed

    Pan, Wenting; Yang, Jinyun; Wei, Jinyu; Chen, Hongwei; Ge, Yunxia; Zhang, Jingfeng; Wang, Zhiqiong; Zhou, Changchun; Yuan, Qipeng; Zhou, Liqing; Yang, Ming

    2015-01-01

    B-cell lymphoma-2 (BCL-2) prevents apoptosis and its overexpression could promote cancer cell survival. Multiple functional BCL-2 genetic polymorphisms, such as rs2279115, rs1801018 and rs1564483, have been identified previously and might be involved in cancer development through deregulating BCL-2 expression. Therefore, we examined associations between these three polymorphisms and esophageal squamous cell carcinoma (ESCC) susceptibility as well as its biological function in vivo. Genotypes were determined in two independent case-control sets consisted of 1588 ESCC patients and 1600 controls from two regions of China. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression. The impact of the rs2279115 polymorphism on BCL-2 expression was detected using esophagus tissues. Our results demonstrated that the BCL-2 rs2279115 AA genotype was significantly associated with decreased ESCC risk compared with the CC genotype (OR = 0.72, 95% CI = 0.57-0.90, P = 0.005), especially in nonsmokers (OR = 0.42, 95% CI = 0.29-0.59, P = 0.001) or nondrinkers (OR = 0.44, 95% CI = 0.32-0.62, P =  .002). Genotype-phenotype correlation studies demonstrated that subjects with the rs2279115 CA and AA genotypes had a statistically significant decrease of BCL-2 mRNA expression compared to the CC genotype in both normal and cancerous esophagus tissues. Our results indicate that the BCL-2 rs2279115 polymorphism contributes to ESCC susceptibility in Chinese populations. PMID:26132559

  16. Susceptibility of Aedes albopictus C6/36 cells to viral infection.

    PubMed

    White, L A

    1987-07-01

    The susceptibility of the C6/36 clone of Aedes albopictus monolayer cell cultures was determined with 46 prototype viruses passed through three subcultures. Viral growth was confirmed by titration of the passage material in other susceptible host systems. Nineteen viruses demonstrated good growth in C6/36 cells: coxsackievirus group A type 10 and group B types 2, 3, 4, and 5; enterovirus 69; mumps virus; poliovirus types 1 to 3; reovirus types 1 to 3; vaccinia virus; dengue virus type 2; eastern equine encephalomyelitis virus; La Crosse virus; Rocio virus; and St. Louis encephalitis virus. Ten viruses did not adapt to growth in the C6/36 cultures. Seventeen other virus strains displayed only limited growth which was primarily restricted to the initial C6/36 passage or was detected by hemagglutinin reactions without observable cell degeneration. Of the 46 viruses, 33 (72%) were capable of initiating infection with a demonstrable cytopathic effect in the initial C6/36 passage. Hemagglutination or complement fixation titers or both were obtained with dengue virus type 2, eastern equine encephalomyelitis virus, La Crosse virus, mumps virus, reovirus types 1 to 3, and Rocio, St. Louis encephalitis, and vaccinia viruses.

  17. Transient Susceptibility of Root Cells in Four Common Legumes to Nodulation by Rhizobia 1

    PubMed Central

    Bhuvaneswari, T. V.; Bhagwat, Arvind A.; Bauer, Wolfgang D.

    1981-01-01

    Root cells of four common legumes were found to remain susceptible to nodulation by rhizobia for only a short period of time. Delayed inoculation experiments conducted with these legume hosts indicated that the initially susceptible region of the root became progressively less susceptible if inoculations were delayed by a few hours. Profiles of the frequency of nodule formation relative to marks indicating the regions of root and root hair development at the time of inoculation indicated that nodulation of Vigna sinensis (L.) Endl. cv California Black Eye and Medicago sativa L. cvs Moapa and Vernal roots was inhibited just below the region that was most susceptible at the time of inoculation. This result suggests the existence of a fast-acting regulatory mechanism in these hosts that prevents overnodulation. Nodulation in white clover may occur in two distinct phases. In addition to the transient susceptibility of preemergent and developing root hair cells, there appeared to be an induced susceptibility of mature clover root hair cells. A cell-free bacterial exudate preparation from Rhizobium trifolii cells was found to render mature root hair cells of white clover more rapidly susceptible to nodulation. PMID:16662065

  18. Memory B cell compartment constitution and susceptibility to recurrent lower respiratory tract infections in young children.

    PubMed

    Siebert, Johan N; L'huillier, Arnaud G; Grillet, Stéphane; Delhumeau, Cécile; Siegrist, Claire-Anne; Posfay-Barbe, Klara M

    2013-06-01

    A proportion of children have recurrent LRTIs, mostly as a result of Spn, which persist after 2 years of age. Here, we investigate, by flow cytofluorometry, the constitution of the memory B cell compartment in 90 healthy children and 49 children with recurrent LRTIs to determine if an increased susceptibility to recurrent LRTIs results from a delayed or abnormal ontogeny with poor antibody-mediated protection. Total IgA, IgM, IgG, and IgG subclasses were measured by nephelometry, as well as antipneumococcal antibodies by ELISA. Pneumococcal vaccination status was obtained. We show that the memory B cells increase between birth and 2 years of age (1.6% vs. 21.1%, P<0.001) without further significant increase noted per additional years (3-4 years old: 23.3%; 4-5 years old: 22.2%, P>0.40) to reach adult-like values (31.8±11.8%, P=0.08). Proportions of switched and IgM memory B cells were similar in children and adults. Comparatively, LRTI children had no delay in the constitution of their memory B cell compartment (2-3 years old: 26.9%; 3-4 years old: 18.2%; 4-5 years old: 26.8%, P>0.05). Their switched and IgM memory B cells were similar among age categories, and the distribution was overall similar to that of healthy controls. LRTI children had normal total and pneumococcal serotype-specific antibody values but showed a rapid waning of antipneumococcal antibody levels after vaccination. In summary, our results show that the memory B cell compartment is already similarly constituted at 2 years of age in healthy and LRTI children and thus, cannot explain the increased susceptibility to bacterial pneumonia. However, the waning of antibodies might predispose children to recurrent infections in the absence of revaccination.

  19. Morphological characteristics of motor neurons do not determine their relative susceptibility to degeneration in a mouse model of severe spinal muscular atrophy.

    PubMed

    Thomson, Sophie R; Nahon, Joya E; Mutsaers, Chantal A; Thomson, Derek; Hamilton, Gillian; Parson, Simon H; Gillingwater, Thomas H

    2012-01-01

    Spinal muscular atrophy (SMA) is a leading genetic cause of infant mortality, resulting primarily from the degeneration and loss of lower motor neurons. Studies using mouse models of SMA have revealed widespread heterogeneity in the susceptibility of individual motor neurons to neurodegeneration, but the underlying reasons remain unclear. Data from related motor neuron diseases, such as amyotrophic lateral sclerosis (ALS), suggest that morphological properties of motor neurons may regulate susceptibility: in ALS larger motor units innervating fast-twitch muscles degenerate first. We therefore set out to determine whether intrinsic morphological characteristics of motor neurons influenced their relative vulnerability to SMA. Motor neuron vulnerability was mapped across 10 muscle groups in SMA mice. Neither the position of the muscle in the body, nor the fibre type of the muscle innervated, influenced susceptibility. Morphological properties of vulnerable and disease-resistant motor neurons were then determined from single motor units reconstructed in Thy.1-YFP-H mice. None of the parameters we investigated in healthy young adult mice - including motor unit size, motor unit arbor length, branching patterns, motor endplate size, developmental pruning and numbers of terminal Schwann cells at neuromuscular junctions - correlated with vulnerability. We conclude that morphological characteristics of motor neurons are not a major determinant of disease-susceptibility in SMA, in stark contrast to related forms of motor neuron disease such as ALS. This suggests that subtle molecular differences between motor neurons, or extrinsic factors arising from other cell types, are more likely to determine relative susceptibility in SMA.

  20. Determination of Receiver Susceptibility to Radio Frequency Interference from Portable Electronic Devices

    NASA Technical Reports Server (NTRS)

    Nguyen, Truong X.; Ely, Jay J.

    2002-01-01

    With the increasing pressures to allow wireless devices on aircraft, the susceptibility of aircraft receivers to interference from Portable Electronic Devices (PEDs) becomes an increasing concern. Many investigations were conducted in the past, with limited success, to quantify device emissions, path loss, and receiver interference susceptibility thresholds. This paper outlines the recent effort in determining the receiver susceptibility thresholds for ILS, VOR and GPS systems. The effort primarily consists of analysis of data available openly as reported in many RTCA and ICAO documents as well as manufacturers data on receiver sensitivity. Shortcomings with the susceptibility threshold data reported in the RTCA documents are presented, and an approach for an in-depth study is suggested. In addition, intermodulation products were observed and demonstrated in a laboratory experiment when multiple PEDs were in the proximity of each other. These intermodulation effects generate spurious frequencies that may fall within aircraft communication or navigation bands causing undesirable effects. Results from a preliminary analysis are presented that show possible harmful combinations of PEDs and the potentially affected aircraft bands.

  1. Genetic determinants of susceptibility to Mycobacterial infections: IRF8, a new kid on the block.

    PubMed

    Salem, S; Gros, P

    2013-01-01

    Genetic and population studies suggest that onset, progression and ultimate outcome of infection with Mycobacteria, including the agent of tuberculosis Mycobacterium tuberculosis, are strongly influenced by genetic factors. Family-based and case-control linkage and association studies have suggested a complex genetic component for susceptibility to tuberculosis. On the other hand, patients with inborn errors in the IL12/IFNγ circuit may develop disseminated mycobacterial infections following perinatal BCG vaccination. The study of such MSMD (Mendelian Susceptibility to Mycobacterial Diseases) patients has provided much insight into innate and acquired immune defenses against mycobacteria. Parallel genetic analyses in mouse models of mycobacterial infections have also indicated complex genetic control, and have provided candidate genes for parallel testing in humans. Recently, mutations in human IRF8 were discovered and shown to cause two distinct forms of a novel primary immunodeficiency and associated susceptibility to mycobacteria. Autosomal recessive IRF8 deficiency is caused by mutation K108E and associated with severe disease with complete depletion of monocytes and dendritic cells. Mutation T80A causes autosomal dominant IRF8 deficiency and a milder form of the disease with selective loss of a subset of dendritic cells. These findings have established that IRF8 is required for ontogeny of the myeloid lineage and for host response to mycobacteria. The ongoing study of the IRF8 transcriptome has shown promise for the identification of IRF8 dependent pathways that play a critical role in host defense against mycobacteria in particular, and against intracellular pathogens in general.

  2. Endogenous miRNA and target concentrations determine susceptibility to potential ceRNA competition.

    PubMed

    Bosson, Andrew D; Zamudio, Jesse R; Sharp, Phillip A

    2014-11-01

    Target competition (ceRNA crosstalk) within miRNA-regulated gene networks has been proposed to influence biological systems. To assess target competition, we characterize and quantitate miRNA networks in two cell types. Argonaute iCLIP reveals that hierarchical binding of high- to low-affinity miRNA targets is a key characteristic of in vivo activity. Quantification of cellular miRNA and mRNA/ncRNA target pool levels indicates that miRNA:target pool ratios and an affinity partitioned target pool accurately predict in vivo Ago binding profiles and miRNA susceptibility to target competition. Using single-cell reporters, we directly test predictions and estimate that ?3,000 additional high-affinity target sites can affect active miRNA families with low endogenous miRNA:target ratios, such as miR-92/25. In contrast, the highly expressed miR-294 and let-7 families are not susceptible to increases of nearly 10,000 sites. These results show differential susceptibility based on endogenous miRNA:target pool ratios and provide a physiological context for ceRNA competition in vivo.

  3. Endogenous miRNA and Target Concentrations Determine Susceptibility to Potential ceRNA Competition

    PubMed Central

    Bosson, Andrew D.; Zamudio, Jesse R.; Sharp, Phillip A.

    2016-01-01

    SUMMARY Target competition (ceRNA crosstalk) within miRNA-regulated gene networks has been proposed to influence biological systems. To assess target competition, we characterize and quantitate miRNA networks in two cell types. Argonaute iCLIP reveals that hierarchical binding of high- to low-affinity miRNA targets is a key characteristic of in vivo activity. Quantification of cellular miRNA and mRNA/ncRNA target pool levels indicates that miRNA:target pool ratios and an affinity partitioned target pool accurately predict in vivo Ago binding profiles and miRNA susceptibility to target competition. Using single-cell reporters, we directly test predictions and estimate that ~3,000 additional high-affinity target sites can affect active miRNA families with low endogenous miRNA:target ratios, such as miR-92/25. In contrast, the highly expressed miR-294 and let-7 families are not susceptible to increases of nearly 10,000 sites. These results show differential susceptibility based on endogenous miRNA:target pool ratios and provide a physiological context for ceRNA competition in vivo. PMID:25449132

  4. [Susceptibility of induced sickle in samples of heterozygous hemoglobin S patients (sickle cell trait) suffering diabetes mellitus type 2].

    PubMed

    Díaz-Piedra, Pablo; Cervantes-Villagrana, Alberto Rafael; Ramos-Jiménez, Raúl; Presno-Bernal, José Miguel; Cervantes-Villagrana, Rodolfo Daniel

    2015-01-01

    Hemoglobin S is an abnormal protein that induces morphological changes in erythrocyte in low-oxygen conditions. In Mexico, it is reported that up to 13.7% of the population with mutation in one allele are considered asymptomatic (sickle cell trait). The sickle cell trait and diabetes mellitus are conditions that occur together in more than one million patients worldwide. Both diseases possibly produce microvascular changes in retinopathy and acute chest syndrome. The aim of this study was to evaluate the induction of sickle cells in samples of diabetic patients with sickle cell trait to identify altered red cell parameters. We obtained samples of diabetic patients to determine hemoglobin A1c and S; furthermore, red blood cell biometrics data were analyzed. We found that older men with diabetes were susceptible to generate sickle cells and this correlated with reduced red blood cell count and an increase in media cell volume. In samples of women diabetes, there were no differences. We conclude that samples from patients with sickle cell trait and diabetes can cause sickle cells with high frequency in men, with lower red blood cells count and increased mean corpuscular volume as susceptibility parameters.

  5. [Cancer pharmacogenetics: study of genetically determined variations on cancer susceptibility due to xenobiotic exposure].

    PubMed

    Quiñones, Luis; Lee, Kuen; Varela F, Nelson; Escala, Mario; García, Karen; Godoy, Loreto; Castro, Andrés; Soto, Jorge; Saavedra, Iván; Cáceres, Dante

    2006-04-01

    Pharmacogenetics is the study of genetically determined variations in the response to drugs and toxic agents, and their implications on disease. Recently, the discipline has acquired great relevancy due to the development of non-invasive molecular techniques that identify genetic variants in human beings. There is also a need to explain the individual differences in susceptibility to drug actions and disease risk. Genetic variants can modify the magnitude of a pharmacologic effect, toxicity threshold, secondary effects and drug interactions. There are approximately thirty families of drug-metabolizing enzymes with genetic variants that cause functional alterations and variations in pharmacologic activity. We summarize the general knowledge about genetic variants of biotransformation enzymes, their relationship with cancer risk and the role of ethnicity. Cancer pharmacogenetics is another promising and exciting research area that will explain why people with an almost identical group of genes, have a different susceptibility to cancer, whose etiology has genetic and environmental components.

  6. Apparatus and process for determining the susceptibility of microorganisms to antibiotics

    NASA Technical Reports Server (NTRS)

    Gibson, Sandra F. (Inventor); Fadler, Norman L. (Inventor)

    1976-01-01

    A process for determining the susceptibility of microorganisms to antibiotics involves introducing a diluted specimen into discrete quantities of a selective culture medium which favors a specific microorganism in that the microorganism is sustained by the medium and when so sustained will change the optical characteristics of the medium. Only the specific microorganism will alter the optical characteristics. Some of the discrete quantities are blended with known antibiotics, while at least one is not. If the specimen contains the microorganisms favored by the selective medium, the optical characteristics of the discrete quantity of pure selective medium, that is the one without antibiotics, will change. If the antibiotics in any of the other discrete quantities are ineffective against the favored microorganisms, the optical characteristics of those quantities will likewise change. No change in the optical characteristics of a discrete quantity indicates that the favored microorganism is susceptible to the antibiotic in the quantity.

  7. Development of an Accelerated Test Method for the Determination of Susceptibility to Atmospheric Corrosion

    NASA Technical Reports Server (NTRS)

    Ambrose, John R.

    1991-01-01

    The theoretical rationale is presented for use of a repetitive cyclic current reversal voltammetric technique for characterization of localized corrosion processes, including atmospheric corrosion. Applicability of this proposed experimental protocol is applied to characterization of susceptibility to crevice and pitting corrosion, atmospheric corrosion and stress corrosion cracking. Criteria upon which relative susceptibility is based were determined and tested using two iron based alloys commonly in use at NASA-Kennedy; A36 (a low carbon steel) and 4130 (a low alloy steel). Practicality of the procedure was demonstrated by measuring changes in anodic polarization behavior during high frequency current reversal cycles of 25 cycles per second with 1 mA/sq cm current density amplitude in solutions containing Cl anions. The results demonstrated that, due to excessive polarization which affects conductivity of barrier corrosion product layers, A36 was less resistant to atmospheric corrosion than its 4130 counterpart; behavior which was also demonstrated during exposure tests.

  8. Microfluidic devices modulate tumor cell line susceptibility to NK cell recognition.

    PubMed

    Perozziello, Gerardo; La Rocca, Rosanna; Cojoc, Gheorghe; Liberale, Carlo; Malara, Natalia; Simone, Giuseppina; Candeloro, Patrizio; Anichini, Andrea; Tirinato, Luca; Gentile, Francesco; Coluccio, Maria Laura; Carbone, Ennio; Di Fabrizio, Enzo

    2012-09-24

    This study aims to adoptively reduce the major histocompatibility complex class I (MHC-I) molecule surface expression of cancer cells by exposure to microfluid shear stress and a monoclonal antibody. A microfluidic system is developed and tumor cells are injected at different flow rates. The bottom surface of the microfluidic system is biofunctionalized with antibodies (W6/32) specific for the MHC-I molecules with a simple method based on microfluidic protocols. The antibodies promote binding between the bottom surface and the MHC-I molecules on the tumor cell membrane. The cells are injected at an optimized flow rate, then roll on the bottom surface and are subjected to shear stress. The stress is localized and enhanced on the part of the membrane where MHC-I proteins are expressed, since they stick to the antibodies of the system. The localized stress allows a stripping effect and consequent reduction of the MHC-I expression. It is shown that it is possible to specifically treat and recover eukaryotic cells without damaging the biological samples. MHC-I molecule expression on treated and control cell surfaces is measured on tumor and healthy cells. After the cell rolling treatment a clear reduction of MHC-I levels on the tumor cell membrane is observed, whereas no changes are observed on healthy cells (monocytes). The MHC-I reduction is investigated and the possibility that the developed system could induce a loss of these molecules from the tumor cell surface is addressed. The percentage of living tumor cells (viability) that remain after the treatment is measured. The changes induced by the microfluidic system are analyzed by fluorescence-activated cell sorting and confocal microscopy. Cytotoxicity tests show a relevant increased susceptibility of natural killer (NK) cells on microchip-treated tumor cells.

  9. Quantifying susceptibility of CD4+ stem memory T-cells to infection by laboratory adapted and clinical HIV-1 strains.

    PubMed

    Flynn, Jacqueline K; Paukovics, Geza; Cashin, Kieran; Borm, Katharina; Ellett, Anne; Roche, Michael; Jakobsen, Martin R; Churchill, Melissa J; Gorry, Paul R

    2014-02-10

    CD4+ T cells are principal targets for human immunodeficiency virus type 1 (HIV-1) infection. CD4+ T cell subsets are heterogeneous cell populations, divided by functional and phenotypic differences into naïve and memory T cells. The memory CD4+ T cells are further segregated into central, effector and transitional memory cell subsets by functional, phenotypic and homeostatic characteristics. Defining the distribution of HIV-1 infection in different T cell subsets is important, as this can play a role in determining the size and composition of the viral reservoir. Both central memory and transitional memory CD4+ T cells have been described as long-lived viral reservoirs for HIV. Recently, the newly described stem memory T cell subset has also been implicated as a long-lived HIV reservoir. Using green fluorescent protein (GFP) reporter strains of HIV-1 and multi parameter flow cytometry, we developed an assay to simultaneously quantify the susceptibility of stem memory (TSCM), central memory, effector memory, transitional memory and naïve CD4+ T cell subsets, to HIV-1 infection in vitro. We show that TSCM are susceptible to infection with laboratory adapted and clinical HIV-1 strains. Our system facilitates the quantitation of HIV-1 infection in alternative T cell subsets by CCR5- and CXCR4-using viruses across different HIV-1 subtypes, and will be useful for studies of HIV-1 pathogenesis and viral reservoirs.

  10. Quantifying Susceptibility of CD4+ Stem Memory T-Cells to Infection by Laboratory Adapted and Clinical HIV-1 Strains

    PubMed Central

    Flynn, Jacqueline K.; Paukovics, Geza; Cashin, Kieran; Borm, Katharina; Ellett, Anne; Roche, Michael; Jakobsen, Martin R.; Churchill, Melissa J.; Gorry, Paul R.

    2014-01-01

    CD4+ T cells are principal targets for human immunodeficiency virus type 1 (HIV-1) infection. CD4+ T cell subsets are heterogeneous cell populations, divided by functional and phenotypic differences into naïve and memory T cells. The memory CD4+ T cells are further segregated into central, effector and transitional memory cell subsets by functional, phenotypic and homeostatic characteristics. Defining the distribution of HIV-1 infection in different T cell subsets is important, as this can play a role in determining the size and composition of the viral reservoir. Both central memory and transitional memory CD4+ T cells have been described as long-lived viral reservoirs for HIV. Recently, the newly described stem memory T cell subset has also been implicated as a long-lived HIV reservoir. Using green fluorescent protein (GFP) reporter strains of HIV-1 and multi parameter flow cytometry, we developed an assay to simultaneously quantify the susceptibility of stem memory (TSCM), central memory, effector memory, transitional memory and naïve CD4+ T cell subsets, to HIV-1 infection in vitro. We show that TSCM are susceptible to infection with laboratory adapted and clinical HIV-1 strains. Our system facilitates the quantitation of HIV-1 infection in alternative T cell subsets by CCR5- and CXCR4-using viruses across different HIV-1 subtypes, and will be useful for studies of HIV-1 pathogenesis and viral reservoirs. PMID:24517971

  11. Combination treatment with decitabine and ionizing radiation enhances tumor cells susceptibility of T cells

    PubMed Central

    Son, Cheol-Hun; Lee, Hong-Rae; Koh, Eun-Kyoung; Shin, Dong-Yeok; Bae, Jae-Ho; Yang, Kwangmo; Park, You-Soo

    2016-01-01

    Decitabine has been found to have anti-metabolic and anti-tumor activities in various tumor cells. Recently, the use of decitabine in combination with other conventional therapies reportedly resulted in improved anti-tumor activity against various tumors. Ionizing radiation (IR) is widely used as a cancer treatment. Decitabine and IR improve immunogenicity and susceptibility of tumor cells to immune cells by up-regulating the expression of various molecules such as major histocompatibility complex (MHC) class I; natural-killer group 2, member D (NKG2D) ligands; and co-stimulatory molecules. However, the effects of combining decitabine and IR therapies are largely unknown. Our results indicate that decitabine or IR treatment upregulates MHC class I, along with various co-stimulatory molecules in target tumor cells. Furthermore, decitabine and IR combination treatment further upregulates MHC class I, along with the co-stimulatory molecules, when compared to the effect of each treatment alone. Importantly, decitabine treatment further enhanced T cell-mediated cytotoxicity and release of IFN- γ against target tumor cells which is induced by IR. Interestingly, decitabine did not affect NKG2D ligand expression or NK cell-mediated cytotoxicity in target tumor cells. These observations suggest that decitabine may be used as a useful immunomodulator to sensitize tumor cells in combination with other tumor therapies. PMID:27671170

  12. Establishment of Stable, Cell-Mediated Immunity that Makes "Susceptible" Mice Resistant to Leishmania major

    NASA Astrophysics Data System (ADS)

    Bretscher, Peter A.; Wei, Guojian; Menon, Juthika N.; Bielefeldt-Ohmann, Helle

    1992-07-01

    Cell-mediated, but not antibody-mediated, immune responses protect humans against certain pathogens that produce chronic diseases such as leishmaniasis. Effective vaccination against such pathogens must therefore produce an immunological "imprint" so that stable, cell-mediated immunity is induced in all individuals after natural infection. BALB/c mice "innately susceptible" to Leishmania major produce antibodies after substantial infection. In the present study, "susceptible" mice injected with a small number of parasites mounted a cell-mediated response and acquired resistance to a larger, normally pathogenic, challenge. This vaccination strategy may be applicable in diseases in which protection is dependent on cell-mediated immunity.

  13. Genetic and environmental interactions determine seizure susceptibility in epileptic EL mice.

    PubMed

    Todorova, M T; Mantis, J G; Le, M; Kim, C Y; Seyfried, T N

    2006-10-01

    Gene identification has progressed rapidly for monogenic epilepsies, but complex gene-environmental interactions have hindered progress in gene identification for multifactorial epilepsies. We analyzed the role of environmental risk factors in the inheritance of multifactorial idiopathic generalized epilepsy in the EL mouse. Seizure susceptibility was evaluated in the EL (E) and seizure-resistant ABP/LeJ (A) parental mouse strains and in their AEF1 and AEF2 hybrid offspring using a handling-induced seizure test. The seizure test was administered in three environments (environments I, II and III) that differed with respect to the number of seizure tests administered (one test or four tests) and the age of the mice when tested (young or old). The inheritance of seizure susceptibility appeared dominant after repetitive seizure testing in young or old mice, but recessive after a single test in old mice. Heritability was high (0.67-0.77) in each environment. Significant quantitative trait loci (QTL) that were associated with environments I and III (repetitive testing) were found on chromosomes 2 and 9 and colocalized with previously mapped El2 and El4, respectively. The El2 QTL found in environment I associated only with female susceptibility. A novel QTL, El-N, for age-dependent predisposition to seizures was found on proximal chromosome 9 only in environment II. The findings indicate that environmental risk factors determine the genetic architecture of seizure susceptibility in EL mice and suggest that QTL for complex epilepsies should be defined in terms of the environment in which they are expressed.

  14. Uptake and metabolism of clomazone in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures.

    PubMed

    Norman, M A; Liebl, R A; Widholm, J M

    1990-03-01

    Studies were conducted to determine the uptake and metabolism of the pigment synthesis inhibiting herbicide clomazone in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) photomixotrophic cell suspensions. Soybean and cotton on a whole plant level are tolerant and susceptible to clomazone, respectively. Preliminary studies indicated that I(50) values for growth, chlorophyll (Chl), beta-carotene, and lutein were, respectively, >22, 14, 19, and 23 times greater for the soybean cell line (SB-M) 8 days after treatment (DAT) compared to the cotton cell line (COT-M) 16 DAT. Differences in [(14)C]clomazone uptake cannot account for selectivity since there were significantly greater levels of clomazone absorbed by the SB-M cells compared to the COT-M cells for each treatment. The percentage of absorbed clomazone converted to more polar metabolite(s) was significantly greater by the SB-M cells relative to COT-M cells at 6 and 24 hours after treatment, however, only small differences existed between the cell lines by 48 hours after treatment. Nearly identical levels of parental clomazone was recovered from both cell lines for all treatments. A pooled metabolite fraction isolated from SB-M cells had no effect on the leaf pigment content of susceptible velvetleaf (Abutilon theophrasti Medic.) or soybean seedlings. Conversely, a pooled metabolite fraction from COT-M cells reduced the leaf Chl content of velvetleaf. Soybean tolerance to clomazone appears to be due to differential metabolism (bioactivation) and/or differences at the site of action.

  15. Uptake and Metabolism of Clomazone in Tolerant-Soybean and Susceptible-Cotton Photomixotrophic Cell Suspension Cultures 1

    PubMed Central

    Norman, Michael A.; Liebl, Rex A.; Widholm, Jack M.

    1990-01-01

    Studies were conducted to determine the uptake and metabolism of the pigment synthesis inhibiting herbicide clomazone in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) photomixotrophic cell suspensions. Soybean and cotton on a whole plant level are tolerant and susceptible to clomazone, respectively. Preliminary studies indicated that I50 values for growth, chlorophyll (Chl), β-carotene, and lutein were, respectively, >22, 14, 19, and 23 times greater for the soybean cell line (SB-M) 8 days after treatment (DAT) compared to the cotton cell line (COT-M) 16 DAT. Differences in [14C]clomazone uptake cannot account for selectivity since there were significantly greater levels of clomazone absorbed by the SB-M cells compared to the COT-M cells for each treatment. The percentage of absorbed clomazone converted to more polar metabolite(s) was significantly greater by the SB-M cells relative to COT-M cells at 6 and 24 hours after treatment, however, only small differences existed between the cell lines by 48 hours after treatment. Nearly identical levels of parental clomazone was recovered from both cell lines for all treatments. A pooled metabolite fraction isolated from SB-M cells had no effect on the leaf pigment content of susceptible velvetleaf (Abutilon theophrasti Medic.) or soybean seedlings. Conversely, a pooled metabolite fraction from COT-M cells reduced the leaf Chl content of velvetleaf. Soybean tolerance to clomazone appears to be due to differential metabolism (bioactivation) and/or differences at the site of action. PMID:16667349

  16. Uptake and metabolism of clomazone in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures

    SciTech Connect

    Norman, M.A.; Liebl, R.A.; Widholm, J.M. )

    1990-03-01

    Studies were conducted to determine the uptake and metabolism of the pigment synthesis inhibiting herbicide clomazone in tolerant-soybean (Glycine max (L.) Merr. cv Corsoy) and susceptible-cotton (Gossypium hirsutum (L.) cv Stoneville 825) photomixotrophic cell suspensions. Soybean and cotton on a whole plant level are tolerant and susceptible to clomazone, respectively. Preliminary studies indicated that I{sub 50} values for growth, chlorophyll (Chl), {beta}-carotene, and lutein were, respectively, >22, 14, 19, and 23 times greater for the soybean cell line (SB-M) 8 days after treatment (DAT) compared to the cotton cell line (COT-M) 16 DAT. Differences in ({sup 14}C)clomazone uptake cannot account for selectivity since there were significantly greater levels of domazone absorbed by the SB-M cells compared to the COT-M cells for each treatment. The percentage of absorbed clomazone converted to more polar metabolite(s) was significantly greater by the SB-M cells relative to COT-M cells at 6 and 24 hours after treatment, however, only small differences existed between the cell lines by 48 hours after treatment. Nearly identical levels of parental clomazone was recovered from both cell lines for all treatments. A pooled metabolite fraction isolated from SB-M cells had no effect on the leaf pigment content of susceptible velvetleaf or soybean seedlings. Conversely, a pooled metabolite fraction from COT-M cells reduced the leaf Chl content of velvetleaf. Soybean tolerance to clomazone appears to be due to differential metabolism (bioactivation) and/or differences at the site of action.

  17. Technique for magnetic susceptibility determination in the highly doped semiconductors by electron spin resonance

    SciTech Connect

    Veinger, A. I.; Zabrodskii, A. G.; Tisnek, T. V.; Goloshchapov, S. I.; Semenikhin, P. V.

    2014-08-20

    A method for determining the magnetic susceptibility in the highly doped semiconductors is considered. It is suitable for the semiconductors near the metal - insulator transition when the conductivity changes very quickly with the temperature and the resonance line form distorts. A procedure that is based on double integration of the positive part of the derivative of the absorption line having a Dyson shape and takes into account the depth of the skin layer is described. Analysis is made for the example of arsenic-doped germanium samples at a rather high concentration corresponding to the insulator-metal phase transition.

  18. [Testing the susceptibility of cultured cells to infection with bovine leukemia virus].

    PubMed

    Bobáková, M; Lesník, F; Vrtiak, O J

    1985-05-01

    Different cell cultures were studied for their susceptibility to bovine leucosis virus infection. Syncytial assay was used for this study. The FLS/BLV+ cell line served as virus source. Cell lines BHK-21 and ZP-1/58 were found to be susceptible to syncytium formation. Large cells with one to three large nuclei, and loose nuclei reaching the size of syncytium were observed to occur in the BHK-21 and ZP-1/58 cell lines, apart from the syncytial formations. The virus specificity of the syncytia arising in these two cell lines was confirmed by the immunofluorescence assay. In the case of the immunoperoxidase assay, a positive result was obtained only in the BHK-21 cell line. The occurrence of syncytia and large nuclei was observed even in the cases when the BHK-21 cells were infected with the lymphocytes of leucotic cows. PMID:2992148

  19. Protein carbonylation after traumatic brain injury: cell specificity, regional susceptibility, and gender differences.

    PubMed

    Lazarus, Rachel C; Buonora, John E; Jacobowitz, David M; Mueller, Gregory P

    2015-01-01

    Protein carbonylation is a well-documented and quantifiable consequence of oxidative stress in several neuropathologies, including multiple sclerosis, Alzheimer׳s disease, and Parkinson׳s disease. Although oxidative stress is a hallmark of traumatic brain injury (TBI), little work has explored the specific neural regions and cell types in which protein carbonylation occurs. Furthermore, the effect of gender on protein carbonylation after TBI has not been studied. The present investigation was designed to determine the regional and cell specificity of TBI-induced protein carbonylation and how this response to injury is affected by gender. Immunohistochemistry was used to visualize protein carbonylation in the brains of adult male and female Sprague-Dawley rats subjected to controlled cortical impact (CCI) as an injury model of TBI. Cell-specific markers were used to colocalize the presence of carbonylated proteins in specific cell types, including astrocytes, neurons, microglia, and oligodendrocytes. Results also indicated that the injury lesion site, ventral portion of the dorsal third ventricle, and ventricular lining above the median eminence showed dramatic increases in protein carbonylation after injury. Specifically, astrocytes and limited regions of ependymal cells adjacent to the dorsal third ventricle and the median eminence were most susceptible to postinjury protein carbonylation. However, these patterns of differential susceptibility to protein carbonylation were gender dependent, with males showing significantly greater protein carbonylation at sites distant from the lesion. Proteomic analyses were also conducted and determined that the proteins most affected by carbonylation in response to TBI include glial fibrillary acidic protein, dihydropyrimidase-related protein 2, fructose-bisphosphate aldolase C, and fructose-bisphosphate aldolase A. Many other proteins, however, were not carbonylated by CCI. These findings indicate that there is both regional

  20. Protein carbonylation after traumatic brain injury: cell specificity, regional susceptibility, and gender differences.

    PubMed

    Lazarus, Rachel C; Buonora, John E; Jacobowitz, David M; Mueller, Gregory P

    2015-01-01

    Protein carbonylation is a well-documented and quantifiable consequence of oxidative stress in several neuropathologies, including multiple sclerosis, Alzheimer׳s disease, and Parkinson׳s disease. Although oxidative stress is a hallmark of traumatic brain injury (TBI), little work has explored the specific neural regions and cell types in which protein carbonylation occurs. Furthermore, the effect of gender on protein carbonylation after TBI has not been studied. The present investigation was designed to determine the regional and cell specificity of TBI-induced protein carbonylation and how this response to injury is affected by gender. Immunohistochemistry was used to visualize protein carbonylation in the brains of adult male and female Sprague-Dawley rats subjected to controlled cortical impact (CCI) as an injury model of TBI. Cell-specific markers were used to colocalize the presence of carbonylated proteins in specific cell types, including astrocytes, neurons, microglia, and oligodendrocytes. Results also indicated that the injury lesion site, ventral portion of the dorsal third ventricle, and ventricular lining above the median eminence showed dramatic increases in protein carbonylation after injury. Specifically, astrocytes and limited regions of ependymal cells adjacent to the dorsal third ventricle and the median eminence were most susceptible to postinjury protein carbonylation. However, these patterns of differential susceptibility to protein carbonylation were gender dependent, with males showing significantly greater protein carbonylation at sites distant from the lesion. Proteomic analyses were also conducted and determined that the proteins most affected by carbonylation in response to TBI include glial fibrillary acidic protein, dihydropyrimidase-related protein 2, fructose-bisphosphate aldolase C, and fructose-bisphosphate aldolase A. Many other proteins, however, were not carbonylated by CCI. These findings indicate that there is both regional

  1. Site of clomazone action in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures.

    PubMed

    Norman, M A; Liebl, R A; Widholm, J M

    1990-10-01

    Studies were conducted to determine the herbicidal site of clomazone action in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) (SB-M) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) (COT-M) photomixotrophic cell suspension cultures. Although a 10 micromolar clomazone treatment did not significantly reduce the terpene or mixed terpenoid content (microgram per gram fresh weight) of the SB-M cell line, there was over a 70% reduction in the chlorophyll (Chl), carotenoid (CAR), and plastoquinone (PQ) content of the COT-M cell line. The tocopherol (TOC) content was reduced only 35.6%. Reductions in the levels of Chl, CAR, TOC, and PQ indicate that the site of clomazone action in COT-M cells is prior to geranylgeranyl pyrophosphate (GGPP). The clomazone treatment did not significantly reduce the flow of [(14)C]mevalonate ([(14)C]MEV) (nanocuries per gram fresh weight) into CAR and the three mixed terpenoid compounds of SB-M cells. Conversely, [(14)C]MEV incorporation into CAR and the terpene moieties of Chl, PQ, and TOC in COT-M cells was reduced at least 73%, indicating that the site of clomazone action must be after MEV. Sequestration of clomazone away from the chloroplast cannot account for soybean tolerance to clomazone since chloroplasts isolated from both cell lines incubated with [(14)C]clomazone contained a similar amount of radioactivity (disintegrations per minute per microgram of Chl). The possible site(s) of clomazone inhibition include mevalonate kinase, phosphomevalonate kinase, pyrophosphomevalonate decarboxylase, isopentenyl pyrophosphate isomerase, and/or a prenyl transferase.

  2. Genetic background affects susceptibility to tumoral stem cell reprogramming

    PubMed Central

    García-Ramírez, Idoia; Ruiz-Roca, Lucía; Martín-Lorenzo, Alberto; Blanco, Óscar; García-Cenador, María Begoña; García-Criado, Francisco Javier; Vicente-Dueñas, Carolina; Sánchez-García, Isidro

    2013-01-01

    The latest studies of the interactions between oncogenes and its target cell have shown that certain oncogenes may act as passengers to reprogram tissue-specific stem/progenitor cell into a malignant cancer stem cell state. In this study, we show that the genetic background influences this tumoral stem cell reprogramming capacity of the oncogenes using as a model the Sca1-BCRABLp210 mice, where the type of tumor they develop, chronic myeloid leukemia (CML), is a function of tumoral stem cell reprogramming. Sca1-BCRABLp210 mice containing FVB genetic components were significantly more resistant to CML. However, pure Sca1-BCRABLp210 FVB mice developed thymomas that were not seen in the Sca1-BCRABLp210 mice into the B6 background. Collectively, our results demonstrate for the first time that tumoral stem cell reprogramming fate is subject to polymorphic genetic control. PMID:23839033

  3. Antibiotic Susceptibility of Biofilm Cells and Molecular Characterisation of Staphylococcus hominis Isolates from Blood

    PubMed Central

    Mendoza-Olazarán, Soraya; Morfín-Otero, Rayo; Villarreal-Treviño, Licet; Rodríguez-Noriega, Eduardo; Llaca-Díaz, Jorge; Camacho-Ortiz, Adrián; González, Gloria M.; Casillas-Vega, Néstor; Garza-González, Elvira

    2015-01-01

    Objectives We aimed to characterise the staphylococcal cassette chromosome mec (SCCmec) type, genetic relatedness, biofilm formation and composition, icaADBC genes detection, icaD expression, and antibiotic susceptibility of planktonic and biofilm cells of Staphylococcus hominis isolates from blood. Methods The study included 67 S. hominis blood isolates. Methicillin resistance was evaluated with the cefoxitin disk test. mecA gene and SCCmec were detected by multiplex PCR. Genetic relatedness was determined by pulsed-field gel electrophoresis. Biofilm formation and composition were evaluated by staining with crystal violet and by detachment assay, respectively; and the biofilm index (BI) was determined. Detection and expression of icaADBC genes were performed by multiplex PCR and real-time PCR, respectively. Antibiotic susceptibilities of planktonic cells (minimum inhibitory concentration, MIC) and biofilm cells (minimum biofilm eradication concentration, MBEC) were determined by the broth dilution method. Results Eighty-five percent (57/67) of isolates were methicillin resistant and mecA positive. Of the mecA-positive isolates, 66.7% (38/57) carried a new putative SCCmec type. Four clones were detected, with two to five isolates each. Among all isolates, 91% (61/67) were categorised as strong biofilm producers. Biofilm biomass composition was heterogeneous (polysaccharides, proteins and DNA). All isolates presented the icaD gene, and 6.66% (1/15) isolates expressed icaD. This isolate presented the five genes of ica operon. Higher BI and MBEC values than the MIC values were observed for amikacin, vancomycin, linezolid, oxacillin, ciprofloxacin, and chloramphenicol. Conclusions S. hominis isolates were highly resistant to methicillin and other antimicrobials. Most of the detected SCCmec types were different than those described for S. aureus. Isolates indicated low clonality. The results indicate that S. hominis is a strong biofilm producer with an extracellular

  4. The Y chromosome as a regulatory element shaping immune cell transcriptomes and susceptibility to autoimmune disease.

    PubMed

    Case, Laure K; Wall, Emma H; Dragon, Julie A; Saligrama, Naresha; Krementsov, Dimitry N; Moussawi, Mohamad; Zachary, James F; Huber, Sally A; Blankenhorn, Elizabeth P; Teuscher, Cory

    2013-09-01

    Understanding the DNA elements that constitute and control the regulatory genome is critical for the appropriate therapeutic management of complex diseases. Here, using chromosome Y (ChrY) consomic mouse strains on the C57BL/6J (B6) background, we show that susceptibility to two diverse animal models of autoimmune disease, experimental allergic encephalomyelitis (EAE) and experimental myocarditis, correlates with the natural variation in copy number of Sly and Rbmy multicopy ChrY genes. On the B6 background, ChrY possesses gene regulatory properties that impact genome-wide gene expression in pathogenic CD4(+) T cells. Using a ChrY consomic strain on the SJL background, we discovered a preference for ChrY-mediated gene regulation in macrophages, the immune cell subset underlying the EAE sexual dimorphism in SJL mice, rather than CD4(+) T cells. Importantly, in both genetic backgrounds, an inverse correlation exists between the number of Sly and Rbmy ChrY gene copies and the number of significantly up-regulated genes in immune cells, thereby supporting a link between copy number variation of Sly and Rbmy with the ChrY genetic element exerting regulatory properties. Additionally, we show that ChrY polymorphism can determine the sexual dimorphism in EAE and myocarditis. In humans, an analysis of the CD4(+) T cell transcriptome from male multiple sclerosis patients versus healthy controls provides further evidence for an evolutionarily conserved mechanism of gene regulation by ChrY. Thus, as in Drosophila, these data establish the mammalian ChrY as a member of the regulatory genome due to its ability to epigenetically regulate genome-wide gene expression in immune cells. PMID:23800453

  5. The Y chromosome as a regulatory element shaping immune cell transcriptomes and susceptibility to autoimmune disease.

    PubMed

    Case, Laure K; Wall, Emma H; Dragon, Julie A; Saligrama, Naresha; Krementsov, Dimitry N; Moussawi, Mohamad; Zachary, James F; Huber, Sally A; Blankenhorn, Elizabeth P; Teuscher, Cory

    2013-09-01

    Understanding the DNA elements that constitute and control the regulatory genome is critical for the appropriate therapeutic management of complex diseases. Here, using chromosome Y (ChrY) consomic mouse strains on the C57BL/6J (B6) background, we show that susceptibility to two diverse animal models of autoimmune disease, experimental allergic encephalomyelitis (EAE) and experimental myocarditis, correlates with the natural variation in copy number of Sly and Rbmy multicopy ChrY genes. On the B6 background, ChrY possesses gene regulatory properties that impact genome-wide gene expression in pathogenic CD4(+) T cells. Using a ChrY consomic strain on the SJL background, we discovered a preference for ChrY-mediated gene regulation in macrophages, the immune cell subset underlying the EAE sexual dimorphism in SJL mice, rather than CD4(+) T cells. Importantly, in both genetic backgrounds, an inverse correlation exists between the number of Sly and Rbmy ChrY gene copies and the number of significantly up-regulated genes in immune cells, thereby supporting a link between copy number variation of Sly and Rbmy with the ChrY genetic element exerting regulatory properties. Additionally, we show that ChrY polymorphism can determine the sexual dimorphism in EAE and myocarditis. In humans, an analysis of the CD4(+) T cell transcriptome from male multiple sclerosis patients versus healthy controls provides further evidence for an evolutionarily conserved mechanism of gene regulation by ChrY. Thus, as in Drosophila, these data establish the mammalian ChrY as a member of the regulatory genome due to its ability to epigenetically regulate genome-wide gene expression in immune cells.

  6. A Rapid Molecular Test for Determining Yersinia pestis Susceptibility to Ciprofloxacin by the Quantification of Differentially Expressed Marker Genes

    PubMed Central

    Steinberger-Levy, Ida; Shifman, Ohad; Zvi, Anat; Ariel, Naomi; Beth-Din, Adi; Israeli, Ofir; Gur, David; Aftalion, Moshe; Maoz, Sharon; Ber, Raphael

    2016-01-01

    Standard antimicrobial susceptibility tests used to determine bacterial susceptibility to antibiotics are growth dependent and time consuming. The long incubation time required for standard tests may render susceptibility results irrelevant, particularly for patients infected with lethal bacteria that are slow growing on agar but progress rapidly in vivo, such as Yersinia pestis. Here, we present an alternative approach for the rapid determination of antimicrobial susceptibility, based on the quantification of the changes in the expression levels of specific marker genes following exposure to growth-inhibiting concentrations of the antibiotic, using Y. pestis and ciprofloxacin as a model. The marker genes were identified by transcriptomic DNA microarray analysis of the virulent Y. pestis Kimberley53 strain after exposure to specific concentrations of ciprofloxacin for various time periods. We identified several marker genes that were induced following exposure to growth-inhibitory concentrations of ciprofloxacin, and we confirmed the marker expression profiles at additional ciprofloxacin concentrations using quantitative RT-PCR. Eleven candidate marker transcripts were identified, of which four mRNA markers were selected for a rapid quantitative RT-PCR susceptibility test that correctly determined the Minimal Inhibitory Concentration (MIC) values and the categories of susceptibility of several Y. pestis strains and isolates harboring various ciprofloxacin MIC values. The novel molecular susceptibility test requires just 2 h of antibiotic exposure in a 7-h overall test time, in contrast to the 24 h of antibiotic exposure required for a standard microdilution test. PMID:27242774

  7. A Rapid Molecular Test for Determining Yersinia pestis Susceptibility to Ciprofloxacin by the Quantification of Differentially Expressed Marker Genes.

    PubMed

    Steinberger-Levy, Ida; Shifman, Ohad; Zvi, Anat; Ariel, Naomi; Beth-Din, Adi; Israeli, Ofir; Gur, David; Aftalion, Moshe; Maoz, Sharon; Ber, Raphael

    2016-01-01

    Standard antimicrobial susceptibility tests used to determine bacterial susceptibility to antibiotics are growth dependent and time consuming. The long incubation time required for standard tests may render susceptibility results irrelevant, particularly for patients infected with lethal bacteria that are slow growing on agar but progress rapidly in vivo, such as Yersinia pestis. Here, we present an alternative approach for the rapid determination of antimicrobial susceptibility, based on the quantification of the changes in the expression levels of specific marker genes following exposure to growth-inhibiting concentrations of the antibiotic, using Y. pestis and ciprofloxacin as a model. The marker genes were identified by transcriptomic DNA microarray analysis of the virulent Y. pestis Kimberley53 strain after exposure to specific concentrations of ciprofloxacin for various time periods. We identified several marker genes that were induced following exposure to growth-inhibitory concentrations of ciprofloxacin, and we confirmed the marker expression profiles at additional ciprofloxacin concentrations using quantitative RT-PCR. Eleven candidate marker transcripts were identified, of which four mRNA markers were selected for a rapid quantitative RT-PCR susceptibility test that correctly determined the Minimal Inhibitory Concentration (MIC) values and the categories of susceptibility of several Y. pestis strains and isolates harboring various ciprofloxacin MIC values. The novel molecular susceptibility test requires just 2 h of antibiotic exposure in a 7-h overall test time, in contrast to the 24 h of antibiotic exposure required for a standard microdilution test. PMID:27242774

  8. Cell cycle constraints on capsulation and bacteriophage susceptibility

    PubMed Central

    Ardissone, Silvia; Fumeaux, Coralie; Bergé, Matthieu; Beaussart, Audrey; Théraulaz, Laurence; Radhakrishnan, Sunish Kumar; Dufrêne, Yves F; Viollier, Patrick H

    2014-01-01

    Despite the crucial role of bacterial capsules in pathogenesis, it is still unknown if systemic cues such as the cell cycle can control capsule biogenesis. In this study, we show that the capsule of the synchronizable model bacterium Caulobacter crescentus is cell cycle regulated and we unearth a bacterial transglutaminase homolog, HvyA, as restriction factor that prevents capsulation in G1-phase cells. This capsule protects cells from infection by a generalized transducing Caulobacter phage (φCr30), and the loss of HvyA confers insensitivity towards φCr30. Control of capsulation during the cell cycle could serve as a simple means to prevent steric hindrance of flagellar motility or to ensure that phage-mediated genetic exchange happens before the onset of DNA replication. Moreover, the multi-layered regulatory circuitry directing HvyA expression to G1-phase is conserved during evolution, and HvyA orthologues from related Sinorhizobia can prevent capsulation in Caulobacter, indicating that alpha-proteobacteria have retained HvyA activity. DOI: http://dx.doi.org/10.7554/eLife.03587.001 PMID:25421297

  9. Rotenone Susceptibility Phenotype in Olfactory Derived Patient Cells as a Model of Idiopathic Parkinson’s Disease

    PubMed Central

    Matigian, N.; Todorovic, M.; Cook, A. L.; Ravishankar, S.; Dong, L. F.; Neuzil, J.; Silburn, P.; Mackay-Sim, A.; Mellick, G. D.; Wood, S. A.

    2016-01-01

    Parkinson’s disease is a complex age-related neurodegenerative disorder. Approximately 90% of Parkinson’s disease cases are idiopathic, of unknown origin. The aetiology of Parkinson’s disease is not fully understood but increasing evidence implies a failure in fundamental cellular processes including mitochondrial dysfunction and increased oxidative stress. To dissect the cellular events underlying idiopathic Parkinson’s disease, we use primary cell lines established from the olfactory mucosa of Parkinson’s disease patients. Previous metabolic and transcriptomic analyses identified deficiencies in stress response pathways in patient-derived cell lines. The aim of this study was to investigate whether these deficiencies manifested as increased susceptibility, as measured by cell viability, to a range of extrinsic stressors. We identified that patient-derived cells are more sensitive to mitochondrial complex I inhibition and hydrogen peroxide induced oxidative stress, than controls. Exposure to low levels (50 nM) of rotenone led to increased apoptosis in patient-derived cells. We identified an endogenous deficit in mitochondrial complex I in patient-derived cells, but this did not directly correlate with rotenone-sensitivity. We further characterized the sensitivity to rotenone and identified that it was partly associated with heat shock protein 27 levels. Finally, transcriptomic analysis following rotenone exposure revealed that patient-derived cells express a diminished response to rotenone-induced stress compared with cells from healthy controls. Our cellular model of idiopathic Parkinson’s disease displays a clear susceptibility phenotype to mitochondrial stress. The determination of molecular mechanisms underpinning this susceptibility may lead to the identification of biomarkers for either disease onset or progression. PMID:27123847

  10. Gold nanoparticles do not induce myotube cytotoxicity but increase the susceptibility to cell death.

    PubMed

    Leite, Paulo Emílio Corrêa; Pereira, Mariana Rodrigues; do Nascimento Santos, Carlos Antonio; Campos, Andrea Porto Carreiro; Esteves, Ticiana Mota; Granjeiro, José Mauro

    2015-08-01

    Gold nanoparticles (AuNP) have been widely used for many applications, including as biological carriers. A better understanding concerning AuNP safety on muscle cells is crucial, since it could be a potential tool in the nanomedicine field. Here, we describe the impact of polyethylene glycol-coated gold nanoparticles (PEG-AuNP) interaction with differentiated skeletal muscle C2C12 cells on cell viability, mitochondria function, cell signaling related to survival, cytokine levels and susceptibility to apoptosis. Intracellular localization of 4.5 nm PEG-AuNP diameter size was evidenced by STEM-in-SEM in myotube cells. Methods for cytotoxicity analysis showed that PEG-AuNP did not affect cell viability, but intracellular ATP levels and mitochondrial membrane potential increased. Phosphorylation of ERK was not altered but p-AKT levels reduced (p<0.01). Pre-treatment of cells with PEG-AuNP followed by staurosporine induction increased the caspases-3/7 activity. Indeed, cytokines analysis revealed a sharp increase of IFN-γ and TGF-β1 levels after PEG-AuNP treatment, suggesting that inflammatory and fibrotic phenotypes process were activated. These data demonstrate that PEG-AuNP affect the myotube physiology leading these cells to be more susceptible to death stimuli in the presence of staurosporine. Altogether, these results present evidence that PEG-AuNP affect the susceptibility to apoptosis of muscle cells, contributing to development of safer strategies for intramuscular delivery. PMID:25790728

  11. Determination of host genetic susceptibility to genotoxic chemicals in hepatocyte cultures.

    PubMed

    McQueen, C A; Williams, G M

    1982-01-01

    The relationship between acetylator phenotype and genotoxicity, i.e., damage to DNA, has been investigated using freshly isolated hepatocytes, an in-vitro system representing the major organ of N-acetyltransferase (NAT) activity. Hepatocytes were isolated from rapid and slow acetylator rabbits by enzymatic perfusion of the liver. Unscheduled DNA synthesis (UDS), determined by autoradiography, was used as an indicator of DNA damage. Hydralazine (HDZ) elicited UDS in hepatocytes from slow acetylators but little or none in hepatocytes from rapid acetylators. However, hepatocytes from rapid acetylators were more sensitive to the effects of 2-aminofluorene (2-AF). A concentration of 10(-3) M 2-AF was toxic, and 10(-4) M elicited UDS in hepatocytes from rapid acetylators. Hepatocytes from slow acetylators showed UDS when exposed to 10(-3) M. No differences were observed between the two phenotypes in the amount of UDS elicited by 2-acetylaminofluorene. These results demonstrate a correlation between acetylator phenotype and genotoxicity of substrates of NAT. Moreover, as shown by the different responses to HDZ and 2-AF, the sensitive phenotype varies with chemical structure. Thus, the identification of phenotype-dependent differences in the amount of DNA damage in rabbit hepatocytes offers evidence for genetic susceptibility to genotoxic chemicals and indicates that a similar susceptibility would be displayed by humans who express the same polymorphism in NAT activity.

  12. Genetic and environmental determinants of the susceptibility of Amerindian derived populations for having hypertriglyceridemia

    PubMed Central

    Aguilar-Salinas, Carlos A.; Tusie-Luna, Teresa; Pajukanta, Päivi

    2014-01-01

    Here, we discuss potential explanations for the higher prevalence of hypertriglyceridemia in populations with an Amerindian background. Although environmental factors are the triggers, the search for the ethnic related factors that explains the increased susceptibility of the Amerindians is a promising area for research. The study of the genetics of hypertriglyceridemia in Hispanic populations faces several challenges. Ethnicity could be a major confounding variable to prove genetic associations. Despite that, the study of hypertriglyceridemia in Hispanics has resulted in significant contributions. Two GWAS reports have exclusively included Mexican mestizos. Fifty percent of the associations reported in Caucasians could be generalized to the Mexicans, but in many cases the Mexican lead SNP was different than that reported in Europeans. Both reports included new associations with apo B or triglycerides concentrations. The frequency of susceptibility alleles in Mexicans is higher than that found in Europeans for several of the genes with the greatest effect on triglycerides levels. An example is the SNP rs964184 in APOA5. The same trend was observed for ANGPTL3 and TIMD4 variants. In summary, we postulate that the study of the genetic determinants of hypertriglyceridemia in Amerindian populations which have major changes in their lifestyle, may prove to be a great resource to identify new genes and pathways associated with hypertriglyceridemia. PMID:24768220

  13. Enhancement of tumor cell susceptibility to lymphokine-activated killer cells by treatment with the streptococcal preparation OK432.

    PubMed

    Yamaue, H; Tanimura, H; Tsunoda, T; Iwahashi, M; Tani, M; Tamai, M; Noguchi, K; Hotta, T; Arii, K

    1992-01-01

    We investigated whether tumor cell lysis by LAK cells was augmented by treatment with OK432 in vitro. NK and LAK activity against K562 cells was not enhanced by their treatment with OK432. In contrast, the susceptibility of OK432-treated Daudi and KATO-III cells to lysis by LAK cells was enhanced. Succinate dehydrogenase activity and RNA synthesis were impaired in Daudi and KATO-III cells by treatment with OK432, and moreover the expression of HLA Class I antigen and beta 2-microglobulin was inhibited in OK432-treated KATO-III cells. Thus, it is suggested that the enhancement of the susceptibility of OK432-treated tumor cells with regard to succinate dehydrogenase activity, RNA synthesis, and HLA Class I antigen expression.

  14. Viral susceptibility of newly established cell lines from the Hawaiian monk seal Monachus schauinslandi.

    PubMed

    Lu, Yuanan; Aguirre, A Alonso; Wang, Yun; Zeng, Lingbing; Loh, Philip C; Yanagihara, Richard

    2003-12-29

    Ten of 11 cell lines, recently established from the snout (MS-SN), periorbital soft tissue (MS-EY), liver (MS-LV), kidney (MS-KD), lung (MS-LG), spleen (MS-SP), heart (MS-HT), thyroid (MS-TY), brain (MS-BR) and urinary bladder (MS-UB) of a juvenile Hawaiian monk seal Monachus schauinslandi, were evaluated in vitro for their susceptibility to 5 mammalian viruses: herpes simplex virus type 1 (HSV-1), vesicular stomatitis virus (VSV), reovirus type 3 (Reo-3), poliovirus type 1 (Polio-1) and vaccinia virus (Vac); 5 fish viruses: channel catfish herpesvirus (CCV), infectious hematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), fish rhabdovirus carpio (RC) and viral hemorrhagic septicemia virus (VHSV); and 2 marine mammal morbilliviruses: phocine distemper virus (PDV) and dolphin distemper virus (DMV). Four well-established continuous cell-lines of nonhuman primate (Vero) and fish (EPC, CHSE-214 and BB) origin served as controls to standardize the virus infectivity assays. Virus yields were quantified as 50% tissue culture infectious dose (TCID50) ml(-1) on Day 7 post-inoculation. Results of the viral challenge assays revealed that the monk seal cell lines shared a similar pattern of susceptibility to the mammalian viruses. Despite their different tissue origins, all monk seal cells were sensitive to HSV-1, Vac, VSV and Reo-3, but were refractory to Polio-1. A characteristic viral-induced cytopathic effect was noted with VSV and Reo-3, and distinct plaques were observed for HSV-1 and Vac. Monk seal cell lines were also susceptible to PDV and DMV, 2 morbilliviruses isolated from seals and dolphins, respectively. By contrast, these cell lines were generally resistant to VHSV, IHNV and IPNV, with varying susceptibility to RC and CCV. The wide range of viral susceptibility of these monk seal cell lines suggests their potential value in studying viruses of monk seals and other marine mammals. PMID:14960030

  15. IgG subclass responses to Theiler's murine encephalomyelitis virus infection and immunization suggest a dominant role for Th1 cells in susceptible mouse strains.

    PubMed Central

    Peterson, J D; Waltenbaugh, C; Miller, S D

    1992-01-01

    Inbred mouse strains differ in susceptibility to Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease. A strong correlation between disease susceptibility and delayed-type hypersensitivity (DTH) has been previously demonstrated, but no strong correlation between disease susceptibility and total anti-TMEV ELISA titres was shown. Since both DTH and IgG2a antibody production are regulated by CD4+ Th1 cells, we investigated three strains of mice to determine whether antivirus IgG2a antibody levels, like DTH in previous studies, correlated with disease susceptibility. Susceptible SJL/J, intermediately susceptible C3H/HeJ, and resistant C57BL/6 mice were infected intracerebrally (i.c.) with the BeAn strain of TMEV and monitored for clinical signs of demyelination and for levels of TMEV-specific antibody of different IgG subclasses using a particle concentration fluorescence immunoassay (PCFIA). Resistant C57BL/6 mice were found to have significantly lower concentrations of total anti-TMEV antibody than susceptible SJL/J mice and intermediately susceptible C3H/HeJ mice show variable antibody responses. A predominance of anti-TMEV IgG2a (Th1 regulated) antibody was seen in susceptible and intermediately susceptible mice, whereas resistant mice displayed a predominant anti-TMEV IgG1 (Th2 regulated) response accompanied by a marked deficiency of IgG2a. In contrast, immunization of C57BL/6 mice with UV-inactivated TMEV in adjuvant revealed that this strain was not defective either in its ability to generate high levels of anti-TMEV antibody or in its ability to produce IgG2a antibody. These results suggest that the antivirus IgG subclass profile is dependent upon the immunization route, virus viability and/or the use of adjuvant and that the levels of antivirus subclasses may be predictive of disease susceptibility. PMID:1350571

  16. Determination of Second-Order Nonlinear Optical Susceptibility of GaN Films on Sapphire

    NASA Astrophysics Data System (ADS)

    Fujita, Takashi; Hasegawa, Tatsuo; Haraguchi, Masanobu; Okamoto, Toshihiro; Fukui, Masuo; Nakamura, Syuji

    2000-05-01

    The second-order nonlinear susceptibilities of GaN films on sapphire were determined by the Maker fringe technique. In deriving the second-harmonic intensity, the bound wave propagating from the GaN-air interface to the GaN-sapphire interface and that propagating in the opposite direction were taken into account. We obtained |χ(2)zxx|=14.7±0.2 pm/V, |χ(2)xzx|=14.4±0.2 pm/V and |χ(2)zzz|=29.7±0.7 pm/V for the GaN film with a thickness of 2.55 μm using fundamental light with a wavelength of 1.064 μm.

  17. Streptococcus agalactiae in Brazil: serotype distribution, virulence determinants and antimicrobial susceptibility

    PubMed Central

    2014-01-01

    Background Group B Streptococcus (GBS) remains a major cause of neonatal sepsis and is also associated with invasive and noninvasive infections in pregnant women and non-pregnant adults, elderly and patients with underlying medical conditions. Ten capsular serotypes have been recognized, and determination of their distribution within a specific population or geographical region is important as they are major targets for the development of vaccine strategies. We have evaluated the characteristics of GBS isolates recovered from individuals with infections or colonization by this microorganism, living in different geographic regions of Brazil. Methods A total of 434 isolates were identified and serotyped by conventional phenotypic tests. The determination of antimicrobial susceptibility was performed by the disk diffusion method. Genes associated with resistance to erythromycin (ermA, ermB, mefA) and tetracycline (tetK, tetL, tetM, tetO) as well as virulence-associated genes (bac, bca, lmb, scpB) were investigated using PCR. Pulsed-field gel electrophoresis (PFGE) was used to examine the genetic diversity of macrolide-resistant and of a number of selected macrolide-susceptible isolates. Results Overall, serotypes Ia (27.6%), II (19.1%), Ib (18.7%) and V (13.6%) were the most predominant, followed by serotypes IV (8.1%) and III (6.7%). All the isolates were susceptible to the beta-lactam antimicrobials tested and 97% were resistant to tetracycline. Resistance to erythromycin and clindamycin were found in 4.1% and 3% of the isolates, respectively. Among the resistance genes investigated, tetM (99.3%) and tetO (1.8%) were detected among tetracycline-resistant isolates and ermA (39%) and ermB (27.6%) were found among macrolide-resistant isolates. The lmb and scpB virulence genes were detected in all isolates, while bac and bca were detected in 57 (13.1%) and 237 (54.6%) isolates, respectively. Molecular typing by PFGE showed that resistance to erythromycin was associated

  18. Asymmetry and aging of mycobacterial cells lead to variable growth and antibiotic susceptibility.

    PubMed

    Aldridge, Bree B; Fernandez-Suarez, Marta; Heller, Danielle; Ambravaneswaran, Vijay; Irimia, Daniel; Toner, Mehmet; Fortune, Sarah M

    2012-01-01

    Cells use both deterministic and stochastic mechanisms to generate cell-to-cell heterogeneity, which enables the population to better withstand environmental stress. Here we show that, within a clonal population of mycobacteria, there is deterministic heterogeneity in elongation rate that arises because mycobacteria grow in an unusual, unipolar fashion. Division of the asymmetrically growing mother cell gives rise to daughter cells that differ in elongation rate and size. Because the mycobacterial cell division cycle is governed by time, not cell size, rapidly elongating cells do not divide more frequently than slowly elongating cells. The physiologically distinct subpopulations of cells that arise through asymmetric growth and division are differentially susceptible to clinically important classes of antibiotics. PMID:22174129

  19. [Detection of cell death markers as a tool for bacterial antimicrobial susceptibility testing].

    PubMed

    Mlynárčik, P; Kolář, M

    2016-01-01

    Antimicrobial resistance among nosocomial pathogens has emerged as one of the most important health care problems in the new millennium. In this review, we present new methods for bacterial antimicrobial susceptibility testing, based on the detection of antibiotic-mediated cell death markers that could provide valuable alternatives to existing phenotypic approaches in the very near future. PMID:27467325

  20. Differential induction of cytolytic susceptibility by E1A, myc, and ras oncogenes in immortalized cells.

    PubMed Central

    Cook, J L; May, D L; Wilson, B A; Walker, T A

    1989-01-01

    The E1A oncogene of adenovirus serotypes 2 and 5 induces susceptibility to the cytolytic effects of natural killer lymphocytes and activated macrophages when expressed in infected and transformed mammalian cells (cytolysis-susceptible phenotype). E1A and the oncogenes v-myc, long-terminal-repeat-promoted c-myc, and activated c-ras share the ability to immortalize transfected low-passage rodent cells. The cytolytic phenotypes of well-characterized rodent cell lines immortalized by these three oncogenes were defined. In contrast to target cells expressing the intact E1A gene, myc- and ras-expressing, immortalized primary transfectants were resistant to lysis by both types of killer cell populations. The same patterns of susceptibility (E1A) and resistance (myc and ras) to cytolysis were observed in oncogene-transfected continuous rat (REF52) and mouse (NIH 3T3) cell lines, indicating that differences in the cytolytic phenotypes associated with expression of these oncogenes are not due to cell selection during immortalization. The results suggest that the E1A oncogene may possess a functional domain that is different from those of other oncogenes, such as myc and ras, and that the activity linked to this postulated domain is dissociable from the process of immortalization. Images PMID:2526229

  1. Determination of the slow crack growth susceptibility coefficient of dental ceramics using different methods.

    PubMed

    Gonzaga, Carla Castiglia; Cesar, Paulo Francisco; Miranda, Walter Gomes; Yoshimura, Humberto Naoyuki

    2011-11-01

    This study compared three methods for the determination of the slow crack growth susceptibility coefficient (n) of two veneering ceramics (VM7 and d.Sign), two glass-ceramics (Empress and Empress 2) and a glass-infiltrated alumina composite (In-Ceram Alumina). Discs (n = 10) were prepared according to manufacturers' recommendations and polished. The constant stress-rate test was performed at five constant stress rates to calculate n(d) . For the indentation fracture test to determine n(IF) , Vickers indentations were performed and the crack lengths were measured under an optical microscope. For the constant stress test (performed only for d.Sign for the determination of n(s) ) four constant stresses were applied and held constant until the specimens' fracture and the time to failure was recorded. All tests were performed in artificial saliva at 37°C. The n(d) values were 17.2 for Empress 2, followed by d.Sign (20.5), VM7 (26.5), Empress (30.2), and In-Ceram Alumina (31.1). In-Ceram Alumina and Empress 2 showed the highest n(IF) values, 66.0 and 40.2, respectively. The n(IF) values determined for Empress (25.2), d.Sign (25.6), and VM7 (20.1) were similar. The n(s) value determined for d.Sign was 31.4. It can be concluded that the n values determined for the dental ceramics evaluated were significantly influenced by the test method used.

  2. Cell-type-specific enrichment of risk-associated regulatory elements at ovarian cancer susceptibility loci

    PubMed Central

    Coetzee, Simon G.; Shen, Howard C.; Hazelett, Dennis J.; Lawrenson, Kate; Kuchenbaecker, Karoline; Tyrer, Jonathan; Rhie, Suhn K.; Levanon, Keren; Karst, Alison; Drapkin, Ronny; Ramus, Susan J.; Couch, Fergus J.; Offit, Kenneth; Chenevix-Trench, Georgia; Monteiro, Alvaro N.A.; Antoniou, Antonis; Freedman, Matthew; Coetzee, Gerhard A.; Pharoah, Paul D.P.; Noushmehr, Houtan; Gayther, Simon A.

    2015-01-01

    Understanding the regulatory landscape of the human genome is a central question in complex trait genetics. Most single-nucleotide polymorphisms (SNPs) associated with cancer risk lie in non-protein-coding regions, implicating regulatory DNA elements as functional targets of susceptibility variants. Here, we describe genome-wide annotation of regions of open chromatin and histone modification in fallopian tube and ovarian surface epithelial cells (FTSECs, OSECs), the debated cellular origins of high-grade serous ovarian cancers (HGSOCs) and in endometriosis epithelial cells (EECs), the likely precursor of clear cell ovarian carcinomas (CCOCs). The regulatory architecture of these cell types was compared with normal human mammary epithelial cells and LNCaP prostate cancer cells. We observed similar positional patterns of global enhancer signatures across the three different ovarian cancer precursor cell types, and evidence of tissue-specific regulatory signatures compared to non-gynecological cell types. We found significant enrichment for risk-associated SNPs intersecting regulatory biofeatures at 17 known HGSOC susceptibility loci in FTSECs (P = 3.8 × 10−30), OSECs (P = 2.4 × 10−23) and HMECs (P = 6.7 × 10−15) but not for EECs (P = 0.45) or LNCaP cells (P = 0.88). Hierarchical clustering of risk SNPs conditioned on the six different cell types indicates FTSECs and OSECs are highly related (96% of samples using multi-scale bootstrapping) suggesting both cell types may be precursors of HGSOC. These data represent the first description of regulatory catalogues of normal precursor cells for different ovarian cancer subtypes, and provide unique insights into the tissue specific regulatory variation with respect to the likely functional targets of germline genetic susceptibility variants for ovarian cancer. PMID:25804953

  3. Cell wall alterations in the leaves of fusariosis-resistant and susceptible pineapple cultivars.

    PubMed

    de Farias Viégas Aquije, Glória Maria; Zorzal, Poliana Belisário; Buss, David Shaun; Ventura, José Aires; Fernandes, Patricia Machado Bueno; Fernandes, Antonio Alberto Ribeiro

    2010-10-01

    Fusariosis, caused by the fungus Fusarium subglutinans f. sp. ananas (Syn. F. guttiforme), is one of the main phytosanitary threats to pineapple (Ananas comosus var. comosus). Identification of plant cell responses to pathogens is important in understanding the plant-pathogen relationship and establishing strategies to improve and select resistant cultivars. Studies of the structural properties and phenolic content of cell walls in resistant (Vitoria) and susceptible (Perola) pineapple cultivars, related to resistance to the fungus, were performed. The non-chlorophyll base of physiologically mature leaves was inoculated with a conidia suspension. Analyses were performed post-inoculation by light, atomic force, scanning and transmission electron microscopy, and measurement of cell wall-bound phenolic compounds. Non-inoculated leaves were used as controls to define the constitutive tissue characteristics. Analyses indicated that morphological differences, such as cell wall thickness, cicatrization process and lignification, were related to resistance to the pathogen. Atomic force microscopy indicated a considerable difference in the mechanical properties of the resistant and susceptible cultivars, with more structural integrity, associated with higher levels of cell wall-bound phenolics, found in the resistant cultivar. p-Coumaric and ferulic acids were shown to be the major phenolics bound to the cell walls and were found in higher amounts in the resistant cultivar. Leaves of the resistant cultivar had reduced fungal penetration and a faster and more effective cicatrization response compared to the susceptible cultivar.

  4. Cell wall alterations in the leaves of fusariosis-resistant and susceptible pineapple cultivars.

    PubMed

    de Farias Viégas Aquije, Glória Maria; Zorzal, Poliana Belisário; Buss, David Shaun; Ventura, José Aires; Fernandes, Patricia Machado Bueno; Fernandes, Antonio Alberto Ribeiro

    2010-10-01

    Fusariosis, caused by the fungus Fusarium subglutinans f. sp. ananas (Syn. F. guttiforme), is one of the main phytosanitary threats to pineapple (Ananas comosus var. comosus). Identification of plant cell responses to pathogens is important in understanding the plant-pathogen relationship and establishing strategies to improve and select resistant cultivars. Studies of the structural properties and phenolic content of cell walls in resistant (Vitoria) and susceptible (Perola) pineapple cultivars, related to resistance to the fungus, were performed. The non-chlorophyll base of physiologically mature leaves was inoculated with a conidia suspension. Analyses were performed post-inoculation by light, atomic force, scanning and transmission electron microscopy, and measurement of cell wall-bound phenolic compounds. Non-inoculated leaves were used as controls to define the constitutive tissue characteristics. Analyses indicated that morphological differences, such as cell wall thickness, cicatrization process and lignification, were related to resistance to the pathogen. Atomic force microscopy indicated a considerable difference in the mechanical properties of the resistant and susceptible cultivars, with more structural integrity, associated with higher levels of cell wall-bound phenolics, found in the resistant cultivar. p-Coumaric and ferulic acids were shown to be the major phenolics bound to the cell walls and were found in higher amounts in the resistant cultivar. Leaves of the resistant cultivar had reduced fungal penetration and a faster and more effective cicatrization response compared to the susceptible cultivar. PMID:20607243

  5. Retroviral expression of the hepatitis B virus x gene promotes liver cell susceptibility to carcinogen-induced site specific mutagenesis.

    PubMed

    Sohn, S; Jaitovitch-Groisman, I; Benlimame, N; Galipeau, J; Batist, G; Alaoui-Jamali, M A

    2000-06-30

    Mutational inactivation of the tumor suppressor gene p53 is common in hepatocellular carcinomas (HCC). AGG to AGT transversion in codon 249 of exon 7 of the p53 gene occurs in over 50% of HCC from endemic regions, where both chronic infection with the hepatitis B virus (HBV) and exposure to carcinogens such as aflatoxin B1 (AFB1) prevail. In this study, we report the effect of the HBV x protein (HBx) on carcinogen-induced cytotoxicity and AGG to AGT mutation in codon 249 of the p53 gene in the human liver cell line CCL13. Expression of HBx, as revealed by its transactivation function, results in enhanced cell susceptibility to cytotoxicity induced by the AFB1 active metabolite, AFB1-8,9-epoxide, and benzo(a)pyrene diol-epoxide. Under similar conditions, expression of HBx promotes apoptosis in a subset of cell population. Exposure to AFB1-8, 9-epoxide alone induces a low frequency of AGG to AGT mutation in codon 249 of the p53 gene, as determined by an allele-specific polymerase chain reaction (AS-PCR) assay. However, expression of HBx enhances the frequency of AFB1-epoxide-induced AGG to AGT mutation compared to control cells. In summary, this study demonstrates that expression of HBx enhances liver cell susceptibility to carcinogen-induced mutagenesis, possibly through alteration of the balance between DNA repair and apoptosis, two cellular defense mechanisms against genotoxic stress. PMID:10856831

  6. Diabetes increases susceptibility of primary cultures of rat proximal tubular cells to chemically induced injury

    SciTech Connect

    Zhong Qing; Terlecky, Stanley R.; Lash, Lawrence H.

    2009-11-15

    Diabetic nephropathy is characterized by increased oxidative stress and mitochondrial dysfunction. In the present study, we prepared primary cultures of proximal tubular (PT) cells from diabetic rats 30 days after an ip injection of streptozotocin and compared their susceptibility to oxidants (tert-butyl hydroperoxide, methyl vinyl ketone) and a mitochondrial toxicant (antimycin A) with that of PT cells isolated from age-matched control rats, to test the hypothesis that PT cells from diabetic rats exhibit more cellular and mitochondrial injury than those from control rats when exposed to these toxicants. PT cells from diabetic rats exhibited higher basal levels of reactive oxygen species (ROS) and higher mitochondrial membrane potential, demonstrating that the PT cells maintain the diabetic phenotype in primary culture. Incubation with either the oxidants or mitochondrial toxicant resulted in greater necrotic and apoptotic cell death, greater evidence of morphological damage, greater increases in ROS, and greater decreases in mitochondrial membrane potential in PT cells from diabetic rats than in those from control rats. Pretreatment with either the antioxidant N-acetyl-L-cysteine or a catalase mimetic provided equivalent protection of PT cells from both diabetic and control rats. Despite the greater susceptibility to oxidative and mitochondrial injury, both cytoplasmic and mitochondrial glutathione concentrations were markedly higher in PT cells from diabetic rats, suggesting an upregulation of antioxidant processes in diabetic kidney. These results support the hypothesis that primary cultures of PT cells from diabetic rats are a valid model in which to study renal cellular function in the diabetic state.

  7. Analysis of Spleen Cells in Susceptible and Resistant Mice with Experimental Lagochilascariosis

    PubMed Central

    Lara, Priscila Guirão; Prudente, Mariana Felix de Souza; Dias, Neusa Mariana Costa; Tambourgi, Denise Vilarinho; Lino-Junior, Ruy de Souza; Spadafora-Ferreira, Mônica; Carvalhaes, Mara Silvia

    2013-01-01

    Lagochilascariosis is an emerging parasitic disease caused by the helminth Lagochilascaris minor. The experimental mouse model has been used to study the immune response against L. minor infection. In the present work, immunohistochemistry analysis of spleen cells populations was evaluated in susceptible (C57BL/6) and resistant (BALB/c) mice experimentally infected with L. minor. The BALB/c mice exhibited increased spleen cell indexes as follows: F4/80+ at 100 days after infection (DPI), CD4+ at 100 and 250 DPI, CD8+ at 35 and 100 DPI, and CD19+ at 100, 150, and 250 DPI. In the spleens of the infected C57BL/6 mice, increased indexes of the following spleen cells were observed: F4/80+ cells at 250 DPI, CD4+ cells at 150 DPI, CD8+ cells at 35, 150, and 250 DPI, and CD19+ cells at 150 to 250 DPI. The index of spleen cells confirmed the differences between the control and infected groups at several time points following the infection. These data demonstrate an association between a preferential increase in the number of CD4+ and CD19+ spleen cells and resistance to experimental lagochilascariosis in BALB/c mice and between a preferential increase in the number of CD8+ spleen cells and susceptibility in C57BL/6 mice. PMID:27335846

  8. Transgenerational epigenetic effects of the Apobec1 cytidine deaminase deficiency on testicular germ cell tumor susceptibility and embryonic viability.

    PubMed

    Nelson, Vicki R; Heaney, Jason D; Tesar, Paul J; Davidson, Nicholas O; Nadeau, Joseph H

    2012-10-01

    Environmental agents and genetic variants can induce heritable epigenetic changes that affect phenotypic variation and disease risk in many species. These transgenerational effects challenge conventional understanding about the modes and mechanisms of inheritance, but their molecular basis is poorly understood. The Deadend1 (Dnd1) gene enhances susceptibility to testicular germ cell tumors (TGCTs) in mice, in part by interacting epigenetically with other TGCT modifier genes in previous generations. Sequence homology to A1cf, the RNA-binding subunit of the ApoB editing complex, raises the possibility that the function of Dnd1 is related to Apobec1 activity as a cytidine deaminase. We conducted a series of experiments with a genetically engineered deficiency of Apobec1 on the TGCT-susceptible 129/Sv inbred background to determine whether dosage of Apobec1 modifies susceptibility, either alone or in combination with Dnd1, and either in a conventional or a transgenerational manner. In the paternal germ-lineage, Apobec1 deficiency significantly increased susceptibility among heterozygous but not wild-type male offspring, without subsequent transgenerational effects, showing that increased TGCT risk resulting from partial loss of Apobec1 function is inherited in a conventional manner. By contrast, partial deficiency in the maternal germ-lineage led to suppression of TGCTs in both partially and fully deficient males and significantly reduced TGCT risk in a transgenerational manner among wild-type offspring. These heritable epigenetic changes persisted for multiple generations and were fully reversed after consecutive crosses through the alternative germ-lineage. These results suggest that Apobec1 plays a central role in controlling TGCT susceptibility in both a conventional and a transgenerational manner.

  9. In vitro susceptibilities of suspected periodontopathic anaerobes as determined by membrane transfer assay.

    PubMed Central

    Caufield, P W; Allen, D N; Childers, N K

    1987-01-01

    Attempts to devise an antimicrobial approach to combating dentomicrobial infections such as periodontal diseases continue to be hampered by the lack of a relevant in vitro method for determining the susceptibility of suspected periodontopathogens to topically applied antimicrobial agents. Proposed here is a novel in vitro method called the membrane transfer technique, which acknowledges those aspects unique to localized pathogenic infections, particularly those associated with anaerobic bacteria. Bacterial lawns representing six suspected periodontopathic bacteria were prepared on membranes and then placed in contact with different concentrations of antimicrobial agents for 5 min. After incubation for 12 to 24 h, MBCs were determined with the aid of a tetrazolium chloride indicator. Four antimicrobial agents (chlorhexidine, iodine, stannous fluoride, and sodium fluoride) were used to test the applicability of the proposed in vitro method. MBCs were derived for each agent except sodium fluoride against all or most of the six bacterial strains tested. The proposed method may also be useful for examining the bactericidal action of topically applied antimicrobial agents against nonoral infections. Images PMID:3439806

  10. The susceptibility of Aire(-/-) mice to experimental myasthenia gravis involves alterations in regulatory T cells.

    PubMed

    Aricha, Revital; Feferman, Tali; Scott, Hamish S; Souroujon, Miriam C; Berrih-Aknin, Sonia; Fuchs, Sara

    2011-02-01

    The autoimmune regulator (Aire) is involved in the prevention of autoimmunity by promoting thymic expression of tissue restricted antigens which leads to elimination of self-reactive T cells. We found that Aire knockout (KO) mice as well as mouse strains that are susceptible to experimental autoimmune myasthenia gravis (EAMG) have lower thymic expression of acetylcholine receptor (AChR- the main autoantigen in MG), compared to wild type (WT) mice and EAMG-resistant mouse strains, respectively. We demonstrated that Aire KO mice have a significant and reproducible lower frequency of CD4+Foxp3+ cells and a higher expression of Th17 markers in their thymus, compared to wild type (WT) mice. These findings led us to expect that Aire KO mice would display increased susceptibility to EAMG. Surprisingly, when EAMG was induced in young (2 month-old) mice, EAMG was milder in Aire KO than in WT mice for several weeks until the age of about 5 months. However, when EAMG was induced in relatively aged (6 month-old) mice, Aire KO mice presented higher disease severity than WT controls. This age-related change in susceptibility to EAMG correlated with an elevated proportion of Treg cells in the spleens of young but not old KO, compared to WT mice, suggesting a role for peripheral Treg cells in the course of disease. Our observations point to a possible link between Aire and Treg cells and suggest an involvement for both in the pathogenesis of myasthenia.

  11. Interferon-gamma is a strong modulator of NK susceptibility and expression of beta 2-microglobulin but not of transferrin receptors of K562 cells.

    PubMed

    Grönberg, A; Kiessling, R; Fiers, W

    1985-10-01

    The human cell line K562 was treated with human natural leukocyte interferon (IFN-alpha) and recombinant immune interferon (IFN-gamma). Cell cultures exposed to both types of IFNs displayed a reduced susceptibility to the cytotoxic activity of human PBL (NK activity). While this effect occurred preferentially at high doses of IFN-alpha, as little as 10 U/ml of IFN-gamma caused a marked decrease in susceptibility to NK-cell-mediated lysis. Using a monoclonal antibody against human beta2-microglobulin (beta2M) a low level of specific binding to K562 cells was detected. The binding increased after treatment with IFN-alpha (1.4-fold) and IFN-gamma (1.7-fold). The expression of transferrin receptors (TR) was not changed significantly. A hybrid cell line between K562 and a Burkitt's lymphoma-derived cell line displayed a similar pattern of response to IFN-alpha and IFN-gamma as did K562, when effects on NK susceptibility, beta2M expression, and TR expression were studied. The Burkitt's lymphoma line PUT showed no consistent changes in expression of beta2M and TR. These results demonstrate that IFN-gamma is highly efficient in modulating the NK susceptibility, and the expression of beta2M on K562. The presented data do not support a role for expression of TR as the only property that determines the degree of NK susceptibility, since there was no correlation between NK susceptibility and TR expression among the cell lines tested or when IFN-treated and untreated cells were compared.

  12. Miniaturized Antimicrobial Susceptibility Test by Combining Concentration Gradient Generation and Rapid Cell Culturing

    PubMed Central

    Kim, Samuel C.; Cestellos-Blanco, Stefano; Inoue, Keisuke; Zare, Richard N.

    2015-01-01

    Effective treatment of bacterial infection relies on timely diagnosis and proper prescription of antibiotic drugs. The antimicrobial susceptibility test (AST) is one of the most crucial experimental procedures, providing the baseline information for choosing effective antibiotic agents and their dosages. Conventional methods, however, require long incubation times or significant instrumentation costs to obtain test results. We propose a lab-on-a-chip approach to perform AST in a simple, economic, and rapid manner. Our assay platform miniaturizes the standard broth microdilution method on a microfluidic device (20 × 20 mm) that generates an antibiotic concentration gradient and delivers antibiotic-containing culture media to eight 30-nL chambers for cell culture. When tested with 20 μL samples of a model bacterial strain (E. coli ATCC 25922) treated with ampicillin or streptomycin, our method allows for the determination of minimum inhibitory concentrations consistent with the microdilution test in three hours, which is almost a factor of ten more rapid than the standard method. PMID:27025635

  13. Insect Gut Symbiont Susceptibility to Host Antimicrobial Peptides Caused by Alteration of the Bacterial Cell Envelope*

    PubMed Central

    Kim, Jiyeun Kate; Son, Dae Woo; Kim, Chan-Hee; Cho, Jae Hyun; Marchetti, Roberta; Silipo, Alba; Sturiale, Luisa; Park, Ha Young; Huh, Ye Rang; Nakayama, Hiroshi; Fukatsu, Takema; Molinaro, Antonio; Lee, Bok Luel

    2015-01-01

    The molecular characterization of symbionts is pivotal for understanding the cross-talk between symbionts and hosts. In addition to valuable knowledge obtained from symbiont genomic studies, the biochemical characterization of symbionts is important to fully understand symbiotic interactions. The bean bug (Riptortus pedestris) has been recognized as a useful experimental insect gut symbiosis model system because of its cultivatable Burkholderia symbionts. This system is greatly advantageous because it allows the acquisition of a large quantity of homogeneous symbionts from the host midgut. Using these naïve gut symbionts, it is possible to directly compare in vivo symbiotic cells with in vitro cultured cells using biochemical approaches. With the goal of understanding molecular changes that occur in Burkholderia cells as they adapt to the Riptortus gut environment, we first elucidated that symbiotic Burkholderia cells are highly susceptible to purified Riptortus antimicrobial peptides. In search of the mechanisms of the increased immunosusceptibility of symbionts, we found striking differences in cell envelope structures between cultured and symbiotic Burkholderia cells. The bacterial lipopolysaccharide O antigen was absent from symbiotic cells examined by gel electrophoretic and mass spectrometric analyses, and their membranes were more sensitive to detergent lysis. These changes in the cell envelope were responsible for the increased susceptibility of the Burkholderia symbionts to host innate immunity. Our results suggest that the symbiotic interactions between the Riptortus host and Burkholderia gut symbionts induce bacterial cell envelope changes to achieve successful gut symbiosis. PMID:26116716

  14. Changes in the biocide susceptibility of Staphylococcus epidermidis and Escherichia coli cells associated with rapid attachment to plastic surfaces.

    PubMed

    Das, J R; Bhakoo, M; Jones, M V; Gilbert, P

    1998-05-01

    Differences in opacity between wells of a microtitre plate containing different volumes of inoculated growth medium reflected planktonic growth without any contribution from cells attached at the well surface. Simple algebra and a knowledge of the dependence of optical density upon sample path length (volume) for suspensions of differing cell density enables the generation of growth curves for attached populations (biofilms). In this manner, minimum inhibitory concentrations (MICs) were determined at various stages of growth (0-20 h), both for cells growing attached to the bases of the plate wells and, simultaneously, for cells growing in suspension above them. Biocides included cetrimide, polyhexamethylene biguanide, peracetic acid, phenoxyethanol and chloroxylenol. Results, expressed as planktonic:biofilm MIC ratios, showed susceptibility to change, not only as a function of attachment and biofilm formation, but also with respect to the nature of the chemical agent. In some instances, changes in susceptibility greater than twofold occurred immediately on attachment and could occur in the presence of biocide concentrations which exceeded the MIC.

  15. Phenotypic characterization in mice of thymus target cells susceptible to productive infection by the radiation leukemia virus

    SciTech Connect

    Boniver, J.; Decleve, A.; Honsik, C.; Libermann, M.; Kaplan, H.S.

    1981-11-01

    The spread of virus repliction was studied by electron microscopy in the thymuses of inbred C57BL/Ka mice after intrathymic inoculation of the radiation leukemia virus (RadLV). The first type C-budding virus particles appeared in scarce blast cells of the subcapsular zone. Most of these blast cells were ''X-cells,'' i.e., the thymus lymphoid cells most actively engaged in DNA synthesis. Virus replication spread to the entire cortical blast cell population and, from day 7 on, to the small cortical lymphocytes. The first virus-producing cells were derived from a very few target cells (approx. =0.001-0.003% of thymocytes) susceptible to RadLV infection. For determination of the phenotypes of these target cells, various thymocyte subpopulations obtained through a battery of cell separation methods were tested for their ability to support the replication of RadLV/VL/sub 3/ virus in short-term culture. Most of these target cells were sensitive to the lytic effect of hydrocortisone and migrated in the fastest fraction of a 1Xg sedimentation gradient, together with the majority of (/sup 3/H)thymidine-incorporating blast cells. They exhibited an intermediate density and expressed H-2 and Thy 1.2 cell surface antigens, although they were not found preferentially among the high Thy 1.2 population to which most of the cortical blast cells belonged. The spread of RadLV within the thymus and the surface phenotype characteristics of target cells indicate that these cells correspond to a thymocyte subset at the earliest stage of thymic lymphopoiesis and may be transitional between the prothymocytes and the subcapsular blast cell population.

  16. Detecting bacteria and Determining Their Susceptibility to Antibiotics by Stochastic Confinement in Nanoliter Droplets using Plug-Based Microfluidics

    SciTech Connect

    Boedicker, J.; Li, L; Kline, T; Ismagilov, R

    2008-01-01

    This article describes plug-based microfluidic technology that enables rapid detection and drug susceptibility screening of bacteria in samples, including complex biological matrices, without pre-incubation. Unlike conventional bacterial culture and detection methods, which rely on incubation of a sample to increase the concentration of bacteria to detectable levels, this method confines individual bacteria into droplets nanoliters in volume. When single cells are confined into plugs of small volume such that the loading is less than one bacterium per plug, the detection time is proportional to plug volume. Confinement increases cell density and allows released molecules to accumulate around the cell, eliminating the pre-incubation step and reducing the time required to detect the bacteria. We refer to this approach as stochastic confinement. Using the microfluidic hybrid method, this technology was used to determine the antibiogram - or chart of antibiotic sensitivity - of methicillin-resistant Staphylococcus aureus (MRSA) to many antibiotics in a single experiment and to measure the minimal inhibitory concentration (MIC) of the drug cefoxitin (CFX) against this strain. In addition, this technology was used to distinguish between sensitive and resistant strains of S. aureus in samples of human blood plasma. High-throughput microfluidic techniques combined with single-cell measurements also enable multiple tests to be performed simultaneously on a single sample containing bacteria. This technology may provide a method of rapid and effective patient-specific treatment of bacterial infections and could be extended to a variety of applications that require multiple functional tests of bacterial samples on reduced timescales.

  17. Barley disease susceptibility factor RACB acts in epidermal cell polarity and positioning of the nucleus

    PubMed Central

    Scheler, Björn; Schnepf, Vera; Galgenmüller, Carolina; Ranf, Stefanie; Hückelhoven, Ralph

    2016-01-01

    RHO GTPases are regulators of cell polarity and immunity in eukaryotes. In plants, RHO-like RAC/ROP GTPases are regulators of cell shaping, hormone responses, and responses to microbial pathogens. The barley (Hordeum vulgare L.) RAC/ROP protein RACB is required for full susceptibility to penetration by Blumeria graminis f.sp. hordei (Bgh), the barley powdery mildew fungus. Disease susceptibility factors often control host immune responses. Here we show that RACB does not interfere with early microbe-associated molecular pattern-triggered immune responses such as the oxidative burst or activation of mitogen-activated protein kinases. RACB also supports rather than restricts expression of defence-related genes in barley. Instead, silencing of RACB expression by RNAi leads to defects in cell polarity. In particular, initiation and maintenance of root hair growth and development of stomatal subsidiary cells by asymmetric cell division is affected by silencing expression of RACB. Nucleus migration is a common factor of developmental cell polarity and cell-autonomous interaction with Bgh. RACB is required for positioning of the nucleus near the site of attack from Bgh. We therefore suggest that Bgh profits from RACB’s function in cell polarity rather than from immunity-regulating functions of RACB. PMID:27056842

  18. Barley disease susceptibility factor RACB acts in epidermal cell polarity and positioning of the nucleus.

    PubMed

    Scheler, Björn; Schnepf, Vera; Galgenmüller, Carolina; Ranf, Stefanie; Hückelhoven, Ralph

    2016-05-01

    RHO GTPases are regulators of cell polarity and immunity in eukaryotes. In plants, RHO-like RAC/ROP GTPases are regulators of cell shaping, hormone responses, and responses to microbial pathogens. The barley (Hordeum vulgare L.) RAC/ROP protein RACB is required for full susceptibility to penetration by Blumeria graminis f.sp. hordei (Bgh), the barley powdery mildew fungus. Disease susceptibility factors often control host immune responses. Here we show that RACB does not interfere with early microbe-associated molecular pattern-triggered immune responses such as the oxidative burst or activation of mitogen-activated protein kinases. RACB also supports rather than restricts expression of defence-related genes in barley. Instead, silencing of RACB expression by RNAi leads to defects in cell polarity. In particular, initiation and maintenance of root hair growth and development of stomatal subsidiary cells by asymmetric cell division is affected by silencing expression of RACB. Nucleus migration is a common factor of developmental cell polarity and cell-autonomous interaction with Bgh RACB is required for positioning of the nucleus near the site of attack from Bgh We therefore suggest that Bgh profits from RACB's function in cell polarity rather than from immunity-regulating functions of RACB.

  19. Replication of Legionella Pneumophila in Human Cells: Why are We Susceptible?

    PubMed Central

    Khweek, Arwa Abu; Amer, Amal

    2010-01-01

    Legionella pneumophila is the causative agent of Legionnaires’ disease, a serious and often fatal form of pneumonia. The susceptibility to L. pneumophila arises from the ability of this intracellular pathogen to multiply in human alveolar macrophages and monocytes. L. pneumophila also replicates in several professional and non-professional phagocytic human-derived cell lines. With the exception of the A/J mouse strain, most mice strains are restrictive, thus they do not support L. pneumophila replication. Mice lacking the NOD-like receptor Nlrc4 or caspase-1 are also susceptible to L. pneumophila. On the other hand, in the susceptible human hosts, L. pneumophila utilizes several strategies to ensure intracellular replication and protect itself against the host immune system. Most of these strategies converge to prevent the fusion of the L. pneumophila phagosome with the lysosome, inhibiting host cell apoptosis, activating survival pathways, and sequestering essential nutrients for replication and pathogenesis. In this review, we summarize survival mechanisms employed by L. pneumophila to maintain its replication in human cells. In addition, we highlight different human-derived cell lines that support the multiplication of this intracellular bacterium. Therefore, these in vitro models can be applicable and are reproducible when investigating L. pneumophila/phagocyte interactions at the molecular and cellular levels in the human host. PMID:21687775

  20. FT-IR spectrometry utilization for determining changes in erythrocyte susceptibility to oxidative stress

    NASA Astrophysics Data System (ADS)

    Petibois, Cyril; Deleris, Gdrard Y. R.

    2004-07-01

    We tested the hypothesis that FT-IR spectrometry was useful for determining oxidative stress damage on erythrocytes. Endurance-trained subjects performed a standardized endurance-training session at 75% of maximal oxygen consumption each week over 19 consecutive weeks. Capillary blood samples were taken before and after test-sessions and plasma and erythrocytes were separately analyzed using Fourier-transform infrared spectrometry. Exercise-induced change in plasma concentrations and erythrocyte IR absorptivities (vC-Hn of fatty acyl moieties, vC=O and δN-H of proteins, vP=O of phospholipids, vCOO- of amino-acids, and vC-O of lactate) were monitored and compared to training level. First training weeks induced normalization of plasma concentration changes during exercise (unchanged for glucose, moderately increased for lactate, high increases for triglycerides, glycerol, and fatty acids) while erythrocyte phospholipids alteration remained elevated (P < 0.05). Further, training reduced the exercise-induced erythrocyte lactate content increase (vC-O; P < 0.05) and phospholipids alteration (vC-Hn and vP=O; P < 0.05) during exercise. These changes paralleled the lowering of exercise-induced hemoconcentration (P < 0.05) and plasma lactate concentration increase during exercise (P < 0.05). These correlated changes between plasma and erythrocyte parameters suggest that hemoconcentration and lactate acidosis (plasmatic and intracellular) are important factors contributing to reduce erythrocyte susceptibility to oxidative stress during chronic endurance training.

  1. Determination of arterial input function in dynamic susceptibility contrast MRI using group independent component analysis technique

    NASA Astrophysics Data System (ADS)

    Chen, Sharon; Liu, Ho-Ling; Yang, Yihong; Hsu, Yuan-Yu; Chuang, Keh-Shih

    2006-12-01

    Quantification of cerebral blood flow (CBF) with dynamic susceptibility contrast (DSC) magnetic resonance imaging (MRI) requires the determination of the arterial input function (AIF). The segmentation of surrounding tissue by manual selection is error-prone due to the partial volume artifacts. Independent component analysis (ICA) has the advantage in automatically decomposing the signals into interpretable components. Recently group ICA technique has been applied to fMRI study and showed reduced variance caused by motion artifact and noise. In this work, we investigated the feasibility and efficacy of the use of group ICA technique to extract the AIF. Both simulated and in vivo data were analyzed in this study. The simulation data of eight phantoms were generated using randomized lesion locations and time activity curves. The clinical data were obtained from spin-echo EPI MR scans performed in seven normal subjects. Group ICA technique was applied to analyze data through concatenating across seven subjects. The AIFs were calculated from the weighted average of the signals in the region selected by ICA. Preliminary results of this study showed that group ICA technique could not extract accurate AIF information from regions around the vessel. The mismatched location of vessels within the group reduced the benefits of group study.

  2. Properties of food folates determined by stability and susceptibility to intestinal pteroylpolyglutamate hydrolase action.

    PubMed

    Seyoum, E; Selhub, J

    1998-11-01

    The intestinal absorption of folate occurs at the monoglutamyl level, and an important measure of food folate bioavailability is how much folate from the food reaches the intestinal sites in forms that can readily be absorbed. In the absence of protecting agents, e.g., vitamin C and reduced thiols, many labile folates may be lost during cooking and during residence in the acid-peptic milieu of the stomach. On the other hand, the presence of polyglutamyl folate necessitates the action of intestinal hydrolases, which could be affected by food constituents. In this study, we developed an in vitro assay for the determination of an index of food folate availability. The index of folate availability in this study was defined as that proportion of folate that has been identified as monoglutamyl derivatives after tests for stability and susceptibility to an enzymatic hydrolysis. The index of folate availability varied widely among foods. The highest index was for egg yolk (72.2%), followed by cow's livers (55.7%), orange juice (21. 3%), cabbage (6.0%), lima beans (4.5%) and lettuce (2.9%). Yeast folate had the lowest index (0.3%). The availability indices generated by this study correlate with the indices of the bioavailability of the corresponding food folate observed in earlier studies, R2 = 0.529 (P = 0.068). Additional information is required on the bioavailability of other food products to test the usefulness of this in vitro approach for assessing food folate availability.

  3. Susceptibility to ulcerative colitis in Hungarian patients determined by gene-gene interactions

    PubMed Central

    Sarlos, Patricia; Varszegi, Dalma; Csongei, Veronika; Magyari, Lili; Jaromi, Luca; Nagy, Lajos; Melegh, Bela

    2014-01-01

    AIM: To study the inflammatory bowel disease-5 locus (IBD5) and interleukin-23 receptor (IL23R) gene variants in UC patients and test for gene-gene interaction. METHODS: The study population (n = 625) was comprised of 320 unrelated ulcerative colitis (UC) patients with Caucasian origin and 316 age- and gender-matched, healthy controls. Five variants in the IBD5 locus (IGR2198a_1 rs11739135, IGR2096a_1 rs12521868, IGR2230a_1 rs17622208, SLC22A4 rs1050152 and SLC22A5 rs2631367) and two of the IL23R gene (rs1004819, rs2201841) were analysed. PCR and restriction fragment length polymorphism methods were used for genotyping, the SLC22A4 rs1050152 genotypes were determined by direct sequencing. Interactions and specific genotype combinations of the seven variants were tested by binary logistic regression analysis. The IL23R genotypes were stratified by IBD5 genotypes for further interaction analyses. RESULTS: For the IL23R rs1004819 A allele we found significantly higher allele frequency (P = 0.032) in UC patients compared to control subjects. The SNP rs1004819 showed significant association with UC risk for carriers (P = 0.004, OR = 1.606; 95%CI: 1.160-2.223) and the SNP rs2201841 for homozygotes (P = 0.030, OR = 1.983; 95%CI: 1.069-3.678). Individually none of the IBD5 markers conferred risk to UC development. There was no evidence for statistical interaction either between IBD5 loci and IL23R genes using logistic regression analysis. After genotype stratification, we could detect a positive association on the background of rs1004819 A allele for SLC22A4 T, SLC22A5 C, IGR2198a_1 C or IGR2096a_1 T allele, the highest OR was calculated in the presence of SLC22A4 T allele (P = 0.005, OR = 2.015; 95%CI: 1.230-3.300). There was no association with UC for any combinations of rs1004819 and IGR2230a_1. The IL23R rs2201841 homozygous genotype and IBD5 carrier status together did not confer susceptibility for UC. CONCLUSION: The present study has shown that UC susceptibility

  4. Susceptibility of chicken lymphoid cells to infectious bursal disease virus does not correlate with the presence of specific binding sites.

    PubMed

    Nieper, H; Müller, H

    1996-06-01

    Pathogenic serotype 1 strains of infectious bursal disease virus (IBDV) replicate efficiently in lymphoid cells of the bursa of Fabricius of chicken. Lymphoid cells in other organs are not susceptible. Apathogenic serotype 2 strains do not replicate in lymphoid bursa cells or in other lymphoid cells. Chicken embryo fibroblasts (CEF), however, efficiently replicate strains of either serotype. Binding studies showed that strains of both IBDV serotypes bind to lymphoid cells isolated from the bursa, thymus or spleen, indicating that restriction of IBDV replication to lymphoid B cells is not determined by the presence of specific receptor sites. The specificity of binding was demonstrated by saturation and competition experiments. These revealed the presence of different receptors: CEF had receptors common to both serotypes and specific ones for each serotype. Receptor sites common to both serotypes were also present on lymphoid cells; however, additional serotype-specific sites were only demonstrated for the apathogenic serotype 2 strain. Strains of both serotypes specifically bound to proteins with molecular masses of 40 kDa and 46 kDa, exposed on the surface of CEF and lymphoid cells. Competition experiments indicated that these proteins might represent the common receptor sites of IBDV. PMID:8683211

  5. In Situ Microscopy Analysis Reveals Local Innate Immune Response Developed around Brucella Infected Cells in Resistant and Susceptible Mice

    PubMed Central

    Copin, Richard; Vitry, Marie-Alice; Hanot Mambres, Delphine; Machelart, Arnaud; De Trez, Carl; Vanderwinden, Jean-Marie; Magez, Stefan; Akira, Shizuo; Ryffel, Bernhard; Carlier, Yves; Letesson, Jean-Jacques; Muraille, Eric

    2012-01-01

    Brucella are facultative intracellular bacteria that chronically infect humans and animals causing brucellosis. Brucella are able to invade and replicate in a broad range of cell lines in vitro, however the cells supporting bacterial growth in vivo are largely unknown. In order to identify these, we used a Brucella melitensis strain stably expressing mCherry fluorescent protein to determine the phenotype of infected cells in spleen and liver, two major sites of B. melitensis growth in mice. In both tissues, the majority of primary infected cells expressed the F4/80 myeloid marker. The peak of infection correlated with granuloma development. These structures were mainly composed of CD11b+ F4/80+ MHC-II+ cells expressing iNOS/NOS2 enzyme. A fraction of these cells also expressed CD11c marker and appeared similar to inflammatory dendritic cells (DCs). Analysis of genetically deficient mice revealed that differentiation of iNOS+ inflammatory DC, granuloma formation and control of bacterial growth were deeply affected by the absence of MyD88, IL-12p35 and IFN-γ molecules. During chronic phase of infection in susceptible mice, we identified a particular subset of DC expressing both CD11c and CD205, serving as a reservoir for the bacteria. Taken together, our results describe the cellular nature of immune effectors involved during Brucella infection and reveal a previously unappreciated role for DC subsets, both as effectors and reservoir cells, in the pathogenesis of brucellosis. PMID:22479178

  6. A new approach to determine the susceptibility of bacteria to antibiotics directly from positive blood culture bottles in two hours.

    PubMed

    March, Gabriel A; García-Loygorri, María C; Simarro, María; Gutiérrez, María P; Orduña, Antonio; Bratos, Miguel A

    2015-02-01

    The rapid identification and antibiotic susceptibility test of bacteria causing bloodstream infections are given a very high priority by clinical laboratories. In an effort to reduce the time required for performing antibiotic susceptibility test (AST), we have developed a new method to be applied from positive blood culture bottles. The design of method was performed using blood culture bottles prepared artificially with five strains which have a known susceptibility. An aliquot of the blood culture was subcultured in the presence of specific antibiotics and bacterial counts were monitored using the Sysmex UF-1000i flow cytometer at different times up to 180min. Receiver operating curve (ROC) analysis allowed us to find out the cut-off point for differentiating between sensitive and resistant strains to the tested antibiotic. This procedure was then validated against standard commercial methods on a total of 100 positive blood culture bottles from patients. First, bacterial identification was performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) directly from positive blood culture bottles as we have previously reported. Secondly, antibiotic susceptibility test was performed in the same way that was carried out in artificially prepared blood culture bottles. Our results indicate that antibiotic susceptibility test can be determined as early as 120min since a blood culture bottle is flagged as positive. The essential agreement between our susceptibility test and commercial methods (E-test, MicroScan and Vitek) was 99%. In summary, we conclude that reliable results on bacterial identification and antibiotic susceptibility test performed directly from positive blood culture bottles can be obtained within 3h.

  7. A novel function of the human oncogene Stil: Regulation of PC12 cell toxic susceptibility through the Shh pathway

    PubMed Central

    Li, Lei; Carr, Aprell L.; Sun, Lei; Drewing, Audrey; Lee, Jessica; Rao, Zihe

    2015-01-01

    The human oncogene SCL/TAL1 interrupting locus (Stil) is highly conserved in vertebrate species. Here, we report new findings of Stil in the regulation of toxic susceptibility in mammalian dopaminergic (DA)-like PC12 cells. RNAi-mediated knockdown of Stil expression did not affect the survival of proliferating PC12 cells but caused a significant amount of cell death in differentiated neurons after toxic drug treatment. In contrast, overexpression of Stil increased toxic susceptibility only in proliferating cells but produced no effect in mature neurons. Exogenetic inactivation or activation of the Sonic hedgehog (Shh) signaling transduction mimicked the effect of Stil knockdown or overexpression in regulation of PC12 cell toxic susceptibility, suggesting that Stil exerts its role through the Shh pathway. Together, the data provide evidence for novel functions of the human oncogene Stil in neural toxic susceptibility. PMID:26549353

  8. Phase Determination of Second-Order Surface Susceptibility Tensor of Liquid Crystal Monolayer Using Ultra-Thin Film Local Oscillator

    NASA Astrophysics Data System (ADS)

    Sei, Masaki; Nagayama, Kohei; Kajikawa, Kotaro; Ishii, Hisao; Seki, Kazuhiko; Kondo, Katsumi; Matsumoto, Yoshiyasu; Ouchi, Yukio

    1998-04-01

    We demonstrated full determination of second-order nonlinear susceptibility of a 4‧-n-octyl-4-cyanobiphenyl (8CB) liquid crystal (LC) monolayer adsorbed on a second-harmonic (SH) active polyimide (PI) substrate. In order to separate the SH signal of the LC film from that of the PI film, we adopted an interferometry technique of second-harmonic generation (SHG) using an ultra-thin film local oscillator. We have found a variety of phases in the components of susceptibility: those of χzii and χizi are almost the same but the phase of χzzz differs by 80° from the other two. The phases of the components of the surface susceptibility tensor are not always identical. This fact indicates that the surface SH response is more complicated than what we expected.

  9. B7-deficient autoreactive T cells are highly susceptible to suppression by CD4(+)CD25(+) regulatory T cells.

    PubMed

    May, Kenneth F; Chang, Xing; Zhang, Huiming; Lute, Kenneth D; Zhou, Penghui; Kocak, Ergun; Zheng, Pan; Liu, Yang

    2007-02-01

    CD4(+)CD25(+) regulatory T cells (Tregs) suppress immunity to infections and tumors as well as autoimmunity and graft-vs-host disease. Since Tregs constitutively express CTLA-4 and activated T cells express B7-1 and B7-2, it has been suggested that the interaction between CTLA-4 on Tregs and B7-1/2 on the effector T cells may be required for immune suppression. In this study, we report that autopathogenic T cells from B7-deficient mice cause multiorgan inflammation when adoptively transferred into syngeneic RAG-1-deficient hosts. More importantly, this inflammation is suppressed by adoptive transfer of purified wild-type (WT) CD4(+)CD25(+) T cells. WT Tregs also inhibited lymphoproliferation and acquisition of activation markers by the B7-deficient T cells. An in vitro suppressor assay revealed that WT and B7-deficient T cells are equally susceptible to WT Treg regulation. These results demonstrate that B7-deficient T cells are highly susceptible to immune suppression by WT Tregs and refute the hypothesis that B7-CTLA-4 interaction between effector T cells and Tregs plays an essential role in Treg function.

  10. Testosterone Increases Susceptibility to Amebic Liver Abscess in Mice and Mediates Inhibition of IFNγ Secretion in Natural Killer T Cells

    PubMed Central

    Lotter, Hannelore; Helk, Elena; Bernin, Hannah; Jacobs, Thomas; Prehn, Cornelia; Adamski, Jerzy; González-Roldán, Nestor; Holst, Otto; Tannich, Egbert

    2013-01-01

    Amebic liver abscess (ALA), a parasitic disease due to infection with the protozoan Entamoeba histolytica, occurs age and gender dependent with strong preferences for adult males. Using a mouse model for ALA with a similar male bias for the disease, we have investigated the role of female and male sexual hormones and provide evidence for a strong contribution of testosterone. Removal of testosterone by orchiectomy significantly reduced sizes of abscesses in male mice, while substitution of testosterone increased development of ALA in female mice. Activation of natural killer T (NKT) cells, which are known to be important for the control of ALA, is influenced by testosterone. Specifically activated NKT cells isolated from female mice produce more IFNγ compared to NKT cells derived from male mice. This high level production of IFNγ in female derived NKT cells was inhibited by testosterone substitution, while the IFNγ production in male derived NKT cells was increased by orchiectomy. Gender dependent differences were not a result of differences in the total number of NKT cells, but a result of a higher activation potential for the CD4− NKT cell subpopulation in female mice. Taken together, we conclude that the hormone status of the host, in particular the testosterone level, determines susceptibility to ALA at least in a mouse model of the disease. PMID:23424637

  11. Pancreatic beta cells are highly susceptible to oxidative and ER stresses during the development of diabetes.

    PubMed

    Gorasia, Dhana G; Dudek, Nadine L; Veith, Paul D; Shankar, Renu; Safavi-Hemami, Helena; Williamson, Nicholas A; Reynolds, Eric C; Hubbard, Michael J; Purcell, Anthony W

    2015-02-01

    The complex interplay of many cell types and the temporal heterogeneity of pancreatic islet composition obscure the direct role of resident alpha and beta cells in the development of Type 1 diabetes. Therefore, in addition to studying islets isolated from non-obese diabetic mice, we analyzed homogeneous cell populations of murine alpha (αTC-1) and beta (NIT-1) cell lines to understand the role and differential survival of these two predominant islet cell populations. A total of 56 proteins in NIT-1 cells and 50 in αTC-1 cells were differentially expressed when exposed to proinflammatory cytokines. The major difference in the protein expression between cytokine-treated NIT-1 and αTC-1 cells was free radical scavenging enzymes. A similar observation was made in cytokine-treated whole islets, where a comprehensive analysis of subcellular fractions revealed that 438 unique proteins were differentially expressed under inflammatory conditions. Our data indicate that beta cells are relatively susceptible to ER and oxidative stress and reveal key pathways that are dysregulated in beta cells during cytokine exposure. Additionally, in the islets, inflammation also leads to enhanced antigen presentation, which completes a three-way insult on beta cells, rendering them targets of infiltrating T lymphocytes.

  12. Shape determination in motile cells

    NASA Astrophysics Data System (ADS)

    Mogilner, Alex

    2010-03-01

    Flat, simple shaped, rapidly gliding fish keratocyte cell is the model system of choice to study cell motility. The cell motile appendage, lamellipod, has a characteristic bent-rectangular shape. Recent experiments showed that the lamellipodial geometry is tightly correlated with cell speed and with actin dynamics. These quantitative data combined with computational modeling suggest that a model for robust actin treadmill inside the 'unstretchable membrane bag'. According to this model, a force balance between membrane tension and growing and pushing actin network distributed unevenly along the cell periphery can explain the cell shape and motility. However, when adhesion of the cell to the surface weakens, the actin dynamics become less regular, and myosin-powered contraction starts playing crucial role in stabilizing the cell shape. I will illustrate how the combination of theoretical and experimental approaches helped to unravel the keratocyte motile behavior.

  13. Food makes you a target: disentangling genetic, physiological, and behavioral effects determining susceptibility to infection.

    PubMed

    Seppälä, Otto; Karvonen, Anssi; Haataja, Maarit; Kuosa, Marja; Jokela, Jukka

    2011-05-01

    Genetics, physiology, and behavior are all expected to influence the susceptibility of hosts to parasites. Furthermore, interactions between genetic and other factors are suggested to contribute to the maintenance of genetic polymorphism in resistance when the relative susceptibility of host genotypes is context dependent. We used a maternal sibship design and long- and short-term food deprivation treatments to test the role of family-level genetic variation, body condition, physiological state, and foraging behavior on the susceptibility of Lymnaea stagnalis snails to infection by a trematode parasite that uses chemical cues to locate its hosts. In experimental exposures, we found that snails in the long-term food deprivation treatment contracted fewer parasites than snails that were continuously well-fed, possibly because well-fed snails grew larger and attracted more transmission stages. When we kept the long-term feeding rates the same, but manipulated the physiological state and foraging behavior of the snails with short-term food deprivation treatment, we found that snails that were fed before the exposure contracted more parasites than snails that were fed during the exposure. This suggests that direct physiological effects of food processing, but not foraging behavior, predisposed snails to infection. Feeding treatments also affected the family-level variation in snail susceptibility, suggesting that the relative susceptibility of host genotypes was context dependent.

  14. Reduced expression IRF7 in nasal epithelial cells from smokers as a potential mechanism mediating enhanced susceptibility to influenza

    EPA Science Inventory

    Rationale: Smokers are more susceptible to viral infections, including influenza virus, yet the mechanisms mediating this effect are not known. Methods: We have established an in vitro model of differentiated nasal epithelial cells from smokers, which maintain enhanced levels...

  15. [Comparative analysis of the susceptibility and productivity of respiratory tract target cells of mice and rats exposed to inflienza virus in vitro].

    PubMed

    Zhukov, V A; Shishkina, L N; Sergeev, A A; Malkova, E M; Riabchikova, E I; Petrishchenko, V A; Sergeev, A N; Ustiuzhanina, N V; Nesvizhskiĭ, Iu V; Vorob'ev, A A

    2008-01-01

    The levels of susceptibility to influenza virus A/Aichi/2/68 H3N2 and the virus yield were determined using primary cells of the trachea and lungs of CD-1 mice and Wistar rats, and for 3 sets of cells obtained from primary lung cells of the both species by centrifugation in the gradient of density and by sedimentation on a surface. The values of ID50 virus dose for 10(6) cells and virus yield per 1 infected cell determined for primary mice cells were 4.0+/-0.47 and 3.2+/-0.27 IgEID50 (lung cells), 3.8+/-0.17 and 3.3+/-0.20 IgEID50 (tracheal cells), and those determined for primary rat cells were 4.0+/-0.35 and 2.1+/-0.24 IgEID50 (lung cells), 3.7+/-0.27 and 2.2+/-0.46 IgEID50 (tracheal cells). The values of ID50 and yield measured for mixtures of cells obtained from primary lung cells by centrifugation in gradient of density and by sedimentation on a surface differed insignificantly (p = 0.05) from the values of the corresponding parameters measured for lung and tracheal cells for both rats and mice. The analysis of data on the variation of the concentrations of different cell types in the experimental cell mixtures shows that type 1 and 2 alveolocytes possess significantly lower (p = 0.05) susceptibility and productivity vs. ciliated cells of the both species. The investigation was conducted within the frame of the ISTC/DARPA#450p project.

  16. Association of single nucleotide polymorphisms in cell cycle regulatory genes with oral cancer susceptibility.

    PubMed

    Murali, Abitha; Nalinakumari, K R; Thomas, Shaji; Kannan, S

    2014-09-01

    Alterations in the regulation of the cell cycle are strongly linked to tumorigenesis, so genetic variants in genes critical to control of the cycle are good candidates to have their association with susceptibility to oral cancer assessed. In this hospital-based, case-control study of 445 patients who had been newly-diagnosed with oral cancer and 449 unaffected controls, we used a multigenic approach to examine the associations among a panel of 10 selected polymorphisms in the pathway of the cell cycle that were possibly susceptible to oral cancer. Six of 9 single nucleotide polymorphisms in the cell cycle showed significant risks for oral cancer, the highest risk being evident for p27 (rs34329; Odds ratio 3.05, 95% CI 2.12 to 4.40). A significant risk of oral cancer was also evident for individual polymorphisms of cyclin E (rs1406), cyclin H (rs3093816), cyclin D1-1 (rs647451), cyclin D2 (rs3217901) and Rb1-2 (rs3092904). The risk of oral cancer increased significantly as the number of unfavourable genotypes in the pathway increased, and so the results point to a stronger combined effect of polymorphisms in important cell cycle regulatory genes on predisposition to oral cancer. PMID:24947332

  17. Susceptibility of human tonsillar epithelial cells to enterovirus 71 with normal cytokine response.

    PubMed

    Xie, Guang-Cheng; Guo, Ni-Jun; Grénman, Reidar; Wang, Hong; Wang, Ying; Vuorenmma, Minna; Zhang, Qing; Zhang, Shuang; Li, Hui-Ying; Pang, Li-Li; Li, Dan-Di; Jin, Miao; Sun, Xiao-Man; Kong, Xiang-Yu; Duan, Zhao-Jun

    2016-07-01

    A recent histopathologic study implicated human tonsillar crypt epithelium as an important site for EV71 replication in EV71-caused fatal cases. This study aimed to confirm the susceptibility of human tonsillar epithelium to EV71. Two human tonsillar epithelial cell lines (UT-SCC-60A and UT-SCC-60B) were susceptive to EV71, and PI3K/AKT, p38, ERK1/2, and JNK1/2 signal pathways were activated. Interferon-α, IL-8, IL-1β, IL-6 and IL-12p40 were induced and regulated by PI3K/AKT, p38, ERK1/2, and JNK1/2 signal pathways. PI3K/AKT pathway activation appeared to suppress the induction of TNF-α, which induced cell survival by inhibiting GSK-3β. The activation of NF-κB was observed but inhibited by these pathways in EV71 infection. Furthermore, ERK1/2 and JNK1/2 were essential for efficient EV71 replication. Human tonsillar epithelial cells support EV71 replication and display innate antiviral immunity in vitro, indicating that human tonsillar epithelial cells may be novel targets for EV71 infection and replication in vivo. PMID:27107253

  18. Susceptibility of human tonsillar epithelial cells to enterovirus 71 with normal cytokine response.

    PubMed

    Xie, Guang-Cheng; Guo, Ni-Jun; Grénman, Reidar; Wang, Hong; Wang, Ying; Vuorenmma, Minna; Zhang, Qing; Zhang, Shuang; Li, Hui-Ying; Pang, Li-Li; Li, Dan-Di; Jin, Miao; Sun, Xiao-Man; Kong, Xiang-Yu; Duan, Zhao-Jun

    2016-07-01

    A recent histopathologic study implicated human tonsillar crypt epithelium as an important site for EV71 replication in EV71-caused fatal cases. This study aimed to confirm the susceptibility of human tonsillar epithelium to EV71. Two human tonsillar epithelial cell lines (UT-SCC-60A and UT-SCC-60B) were susceptive to EV71, and PI3K/AKT, p38, ERK1/2, and JNK1/2 signal pathways were activated. Interferon-α, IL-8, IL-1β, IL-6 and IL-12p40 were induced and regulated by PI3K/AKT, p38, ERK1/2, and JNK1/2 signal pathways. PI3K/AKT pathway activation appeared to suppress the induction of TNF-α, which induced cell survival by inhibiting GSK-3β. The activation of NF-κB was observed but inhibited by these pathways in EV71 infection. Furthermore, ERK1/2 and JNK1/2 were essential for efficient EV71 replication. Human tonsillar epithelial cells support EV71 replication and display innate antiviral immunity in vitro, indicating that human tonsillar epithelial cells may be novel targets for EV71 infection and replication in vivo.

  19. Dendritic cells play a role in host susceptibility to Cryptosporidium parvum infection.

    PubMed

    Bedi, Brahmchetna; McNair, Nina N; Mead, Jan R

    2014-01-01

    Our previous studies have described dendritic cells (DCs) to be important sources of Th1 cytokines such as IL-12 and IL-2 in vitro, following stimulation with Cryptosporidium parvum antigens. We further established the role of DCs during cryptosporidiosis using a diphtheria toxin promoter regulated transgenic CD11c-DTR/EGFP mouse model. In vivo depletion of CD11c(+) cells in CD11c-DTR-Tg mice significantly increased susceptibility to C. parvum infection. Adoptive transfer of unstimulated or antigen stimulated DCs into CD11c(+) depleted CD11c-DTR-Tg mice resulted in an early decrease in parasite load at 4 days post infection. However, this response was transient since parasite load increased in mice engrafted with either unstimulated DCs or DCs stimulated with solubilized antigen by 6 days post infection. In contrast, in mice engrafted with DCs stimulated with live sporozoites, parasite load remained low during the entire period, suggesting the development of a more effective and sustained response. A corresponding increase in IFN-γ expression in T cells from spleen and mesenteric lymph nodes was also noted. Consistent with the in vivo engraftment study, DCs that are pulsed with live sporozoites in vitro and co-cultured with CD4(+) and CD8(+) T cells produced higher IFN-γ levels. Our study establishes the importance of DCs in susceptibility to infection by C. parvum and as important mediators of immune responses.

  20. Towards in vitro DT/DNT testing: Assaying chemical susceptibility in early differentiating NT2 cells.

    PubMed

    Menzner, Ann-Katrin; Abolpour Mofrad, Sepideh; Friedrich, Oliver; Gilbert, Daniel F

    2015-12-01

    Human pluripotent embryonal carcinoma (NT2) cells are increasingly considered as a suitable model for in vitro toxicity testing, e.g. developmental toxicity and neurotoxicity (DT/DNT) studies, as they undergo neuronal differentiation upon stimulation with retinoic acid (RA) and permit toxicity testing at different stages of maturation. NT2 cells have recently been reported to show specific changes in dielectric resistance profiles during differentiation which can be observed as early as 24h upon RA-stimulation. These observations suggest altered susceptibility to chemicals at an early stage of differentiation. However, chemical susceptibility of early differentiating NT cells has not yet been studied. To address this question, we have established a cell fitness screening assay based on the analysis of intracellular ATP levels and we applied the assay in a large-scale drug screening experiment in NT2 stem cells and early differentiating NT2 cells. Subsequent analysis of ranked fitness phenotypes revealed 19 chemicals with differential toxicity profile in early differentiating NT2 cells. To evaluate whether any of the identified drugs have previously been associated with DT/DNT, we conducted a literature search on the identified molecules and quantified the fraction of chemicals assigned to the FDA (Food and Drug Administration) pregnancy risk categories (PRC) N, A, B, C, D, and X in the hit list and the small molecule library. While the fractions of the categories N and B were decreased (0.81 and 0.35-fold), the classes C, D and X were increased (1.35, 1.47 and 3.27-fold) in the hit list compared to the chemical library. From these data as well as from the literature review, identifying large fractions of chemicals being directly (∼42%) and indirectly associated with DT/DNT (∼32%), we conclude that our method may be beneficial to systematic in vitro-based primary screening for developmental toxicants and neurotoxicants and we propose cell fitness screening in

  1. Polymorphisms of the cell surface receptor control mouse susceptibilities to xenotropic and polytropic leukemia viruses.

    PubMed

    Marin, M; Tailor, C S; Nouri, A; Kozak, S L; Kabat, D

    1999-11-01

    The differential susceptibilities of mouse strains to xenotropic and polytropic murine leukemia viruses (X-MLVs and P-MLVs, respectively) are poorly understood but may involve multiple mechanisms. Recent evidence has demonstrated that these viruses use a common cell surface receptor (the X-receptor) for infection of human cells. We describe the properties of X-receptor cDNAs with distinct sequences cloned from five laboratory and wild strains of mice and from hamsters and minks. Expression of these cDNAs in resistant cells conferred susceptibilities to the same viruses that naturally infect the animals from which the cDNAs were derived. Thus, a laboratory mouse (NIH Swiss) X-receptor conferred susceptibility to P-MLVs but not to X-MLVs, whereas those from humans, minks, and several wild mice (Mus dunni, SC-1 cells, and Mus spretus) mediated infections by both X-MLVs and P-MLVs. In contrast, X-receptors from the resistant mouse strain Mus castaneus and from hamsters were inactive as viral receptors. These results suggest that X-receptor polymorphisms are a primary cause of resistances of mice to members of the X-MLV/P-MLV family of retroviruses and are responsible for the xenotropism of X-MLVs in laboratory mice. By site-directed mutagenesis, we substituted sequences between the X-receptors of M. dunni and NIH Swiss mice. The NIH Swiss protein contains two key differences (K500E in presumptive extracellular loop 3 [ECL 3] and a T582 deletion in ECL 4) that are both required to block X-MLV infections. Accordingly, a single inverse mutation in the NIH Swiss protein conferred X-MLV susceptibility. Furthermore, expression of an X-MLV envelope glycoprotein in Chinese hamster ovary cells interfered efficiently with X-MLV and P-MLV infections mediated by X-receptors that contained K500 and/or T582 but had no effect on P-MLV infections mediated by X-receptors that lacked these amino acids. In contrast, moderate expression of a P-MLV (MCF247) envelope glycoprotein did not

  2. CD26/DPP4 Cell-Surface Expression in Bat Cells Correlates with Bat Cell Susceptibility to Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Infection and Evolution of Persistent Infection

    PubMed Central

    Caì, Yíngyún; Yú, Shuǐqìng; Postnikova, Elena N.; Mazur, Steven; Bernbaum, John G.; Burk, Robin; Zhāng, Téngfēi; Radoshitzky, Sheli R.; Müller, Marcel A.; Jordan, Ingo; Bollinger, Laura; Hensley, Lisa E.; Jahrling, Peter B.; Kuhn, Jens H.

    2014-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) is a recently isolated betacoronavirus identified as the etiologic agent of a frequently fatal disease in Western Asia, Middle East respiratory syndrome. Attempts to identify the natural reservoirs of MERS-CoV have focused in part on dromedaries. Bats are also suspected to be reservoirs based on frequent detection of other betacoronaviruses in these mammals. For this study, ten distinct cell lines derived from bats of divergent species were exposed to MERS-CoV. Plaque assays, immunofluorescence assays, and transmission electron microscopy confirmed that six bat cell lines can be productively infected. We found that the susceptibility or resistance of these bat cell lines directly correlates with the presence or absence of cell surface-expressed CD26/DPP4, the functional human receptor for MERS-CoV. Human anti-CD26/DPP4 antibodies inhibited infection of susceptible bat cells in a dose-dependent manner. Overexpression of human CD26/DPP4 receptor conferred MERS-CoV susceptibility to resistant bat cell lines. Finally, sequential passage of MERS-CoV in permissive bat cells established persistent infection with concomitant downregulation of CD26/DPP4 surface expression. Together, these results imply that bats indeed could be among the MERS-CoV host spectrum, and that cellular restriction of MERS-CoV is determined by CD26/DPP4 expression rather than by downstream restriction factors. PMID:25409519

  3. Methodology for determining susceptibility of rough rice to Rhyzopertha dominica (L.) and Sitotroga cerealella (Olivier)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Varieties of rough rice, Oryzae sativa (L)., were obtained from different sources in the south-central United States and evaluated for susceptibility to the lesser grain borer, Rhyzopertha dominica (Fab.), and the Angoumois grain moth, Sitotroga cerealella (Olivier), in laboratory studies. Adult R. ...

  4. Carbapenem Susceptibility Patterns for Clinical Isolates of Mycobacterium abscessus Determined by the Etest Method▿

    PubMed Central

    Chihara, Shingo; Smith, Geremy; Petti, Cathy A.

    2010-01-01

    Mycobacterium abscessus is resistant to multiple antibiotics, creating treatment challenges. Carbapenems are tested to increase therapeutic alternatives. We performed in vitro susceptibility testing by Etest of four carbapenems for M. abscessus isolates. Imipenem demonstrated the most in vitro activity, and testing of other carbapenems provided no additional value. PMID:20018813

  5. Depressed T-cell proliferation associated with susceptibility to experimental Taenia crassiceps infection.

    PubMed Central

    Sciutto, E; Fragoso, G; Baca, M; De la Cruz, V; Lemus, L; Lamoyi, E

    1995-01-01

    Peritoneal infection with Taenia crassiceps cysticerci of naturally resistant (C57BL/10J and C57BL/6J) and susceptible (BALB/cAnN) mice induces a cellular immune depression. T-cell proliferation in response to concanavalin A (ConA) or anti-CD3 was significantly depressed in infected mice of all strains tested. However, in resistant mice, the diminished response to ConA was transient and animals recovered normal responsiveness at day 40, whereas susceptible mice remained suppressed throughout the 40 days of the experiment. In contrast, the proliferative response to anti-CD3 was lower in infected mice than in noninfected controls regardless of differences in natural susceptibility of the strains. Intraperitoneal injection of mice with a parasite extract also induced a depression of the response to ConA, although not as strong as that produced by the parasite itself. This depression is not due to direct effects by parasite antigens over host lymphocytes, as proliferation is not affected by the presence of cysticercal antigens added in vitro. Diminished interleukin-2 production during the parasitosis accounts at least in part for the diminished responses to ConA. A primary infection favors parasite establishment after a second challenge, pointing to the relevance of the immunodepression in generating a host environment favorable to the parasite. PMID:7768609

  6. Space Systems - Safety and Compatibility of Materials - Method to Determine the Ignition Susceptibility of Materials or Components to Particle Impact

    NASA Technical Reports Server (NTRS)

    Hirsch, David B.

    2011-01-01

    The scope of this International Technical Specification is to provide a method to determine the ignition susceptibility of materials and components to particle impact. The method can be used to determine the conditions at which ignition and consumption of a specimen material occurs when impacted by single or multiple particles entrained in a flow of gaseous oxygen (GOX). Alternatively, the method can be used to determine if a specific material or component is subject to ignition and sustained combustion in a given flow environment when impacted by single or multiple particles entrained in a flow of GOX.

  7. Hypergravity Alters the Susceptibility of Cells to Anoxia-Reoxygenation Injury

    NASA Technical Reports Server (NTRS)

    McCloud, Henry; Pink, Yulondo; Harris-Hooker, Sandra A.; Melhado, Caroline D.; Sanford, Gary L.

    1997-01-01

    Gravity is a physical force, much like shear stress or mechanical stretch, and should affect organ and cellular function. Researchers have shown that gravity plays a role in ventilation and blood flow distribution, gas exchange, alveolar size and mechanical stresses within the lung. Short exposure to microgravity produced marked alterations in lung blood flow and ventilation distribution while hypergravity exaggerated the regional differences in lung structure and function resulting in reduced ventilation at the base and no ventilation of the upper half of the lung. Microgravity also decreased metabolic activity in cardiac cells, WI-38 embryonic lung cells, and human lymphocytes. Rats, in the tail-suspended head-down tilt model, experienced transient loss of lung water, contrary to an expected increase due to pooling of blood in the pulmonary vasculature. Hypergravity has also been found to increase the proliferation of several different cell lines (e.g., chick embryo fibroblasts) while decreasing cell motility and slowing liver regeneration following partial hepatectomy. These studies show that changes in the gravity environment will affect several aspects of organ and cellular function and produce major change in blood flow and tissue/organ perfusion. However, these past studies have not addressed whether ischemia-reperfusion injury will be exacerbated or ameliorated by changes in the gravity environment, e.g., space flight. Currently, nothing is known about how gravity will affect the susceptibility of different lung and vascular cells to this type of injury. We conducted studies that addressed the following question: Does the susceptibility of lung fibroblasts, vascular smooth muscle, and endothelial cells to anoxia/reoxygenation injury change following exposure to hypergravity conditions?

  8. Susceptibility of colorectal-carcinoma cells to natural-killer-mediated lysis: relationship to CEA expression and degree of differentiation.

    PubMed

    Prado, I B; Laudanna, A A; Carneiro, C R

    1995-06-01

    This study addresses the relevance of colorectal-carcinoma-cell (CRC) CEA expression and degree of differentiation in natural-killer(NK)-mediated lysis susceptibility. A 51Cr-release cytotoxicity assay performed with 5 human CRC lines demonstrated that CRC CEA expression was related to resistance to NK lysis. Moreover, the addition of anti-CEA Fab fragments to the assay led to a significant increase of lysability of high-CEA-producing and NK-resistant cells (LS 174-T), whereas purified CEA drastically reduced lysis of low-CEA-producing and NK-susceptible cells (LISP-I) in a dose-dependent manner. These results strongly suggest that CEA plays a causal role in CRC resistance to NK lysis. Nevertheless, our data did not demonstrate CEA binding to effector cell surface, suggesting that CEA expression can protect CRC, possibly by preventing NK-tumor-cell adhesion to occur. Our results also show that CRC susceptibility to NK lysis was related to a less differentiated phenotype. HCT-8, which are poorly differentiated and low-CEA-producing cells, were cultured in vitro in the presence of the differentiation agent sodium butyrate. Treated cells became less susceptible to NK lysis as they progressed towards a more differentiated phenotype. However, CEA production was not altered upon differentiation. Our study thus demonstrates that both features, CEA expression and degree of cellular differentiation, may individually influence CRC susceptibility to NK lysis. PMID:7790122

  9. Determination of antifungal susceptibility patterns among the environmental isolates of Aspergillus fumigatus in Iran

    PubMed Central

    Mohammadi, Faezeh; Dehghan, Parvin; Nekoeian, Shahram; Hashemi, Seyed Jamal

    2016-01-01

    Background: In recent years, triazole-resistant environmental isolates of Aspergillus fumigatus have emerged in Europe and Asia. Azole resistance has been reported in patients who are treated with long-term azole therapy or exposure of the fungus spores to the azole fungicides used in agriculture. To date, a wide range of mutations in A. fumigatus have been described conferring azole-resistance, which commonly involves modifications in the cyp51A gene. We investigated antifungal susceptibility pattern of environmental isolates of A. fumigatus. Materials and Methods: In this study, 170 environmental samples collected from indoors surfaces of three hospitals in Iran. It was used β-tubulin gene to confirm the all of A. fumigatus isolates, which was identified by conventional methods. Furthermore, the antifungal susceptibility of itraconazole, voriconazole, and posaconazole was investigated using broth microdilution test, according to European Committee on Antimicrobial Susceptibility testing reference method. Results: From a total of 158 environmental molds fungi obtained from the hospitals, 58 isolates were identified as A. fumigatus by amplification of expected size of β-tubulin gene (~500 bp). In this study, in vitro antifungal susceptibility testing has shown that there were not high minimum inhibitory concentration values of triazole antifungals in all of the 58 environmental isolates of A. fumigatus. Conclusion: Our findings demonstrated that there was not azole-resistant among environmental isolates of A. fumigatus. Medical triazoles compounds have structural similarity with triazole fungicide compounds in agriculture, therefore, resistance development through exposure to triazole fungicide compounds in the environment is important but it sounds there is not a serious health problem in drug resistance in environmental isolates in Iran.

  10. Determination of antifungal susceptibility patterns among the environmental isolates of Aspergillus fumigatus in Iran

    PubMed Central

    Mohammadi, Faezeh; Dehghan, Parvin; Nekoeian, Shahram; Hashemi, Seyed Jamal

    2016-01-01

    Background: In recent years, triazole-resistant environmental isolates of Aspergillus fumigatus have emerged in Europe and Asia. Azole resistance has been reported in patients who are treated with long-term azole therapy or exposure of the fungus spores to the azole fungicides used in agriculture. To date, a wide range of mutations in A. fumigatus have been described conferring azole-resistance, which commonly involves modifications in the cyp51A gene. We investigated antifungal susceptibility pattern of environmental isolates of A. fumigatus. Materials and Methods: In this study, 170 environmental samples collected from indoors surfaces of three hospitals in Iran. It was used β-tubulin gene to confirm the all of A. fumigatus isolates, which was identified by conventional methods. Furthermore, the antifungal susceptibility of itraconazole, voriconazole, and posaconazole was investigated using broth microdilution test, according to European Committee on Antimicrobial Susceptibility testing reference method. Results: From a total of 158 environmental molds fungi obtained from the hospitals, 58 isolates were identified as A. fumigatus by amplification of expected size of β-tubulin gene (~500 bp). In this study, in vitro antifungal susceptibility testing has shown that there were not high minimum inhibitory concentration values of triazole antifungals in all of the 58 environmental isolates of A. fumigatus. Conclusion: Our findings demonstrated that there was not azole-resistant among environmental isolates of A. fumigatus. Medical triazoles compounds have structural similarity with triazole fungicide compounds in agriculture, therefore, resistance development through exposure to triazole fungicide compounds in the environment is important but it sounds there is not a serious health problem in drug resistance in environmental isolates in Iran. PMID:27656605

  11. Determination of homozygous susceptible strain in Culex quinquefasciatus (Say), using single raft sib-selection method.

    PubMed

    Hidayati, H; Nazni, W A; Mohd, S A

    2008-04-01

    The standard laboratory strain was found to be heterozygous for susceptibility. Hence, an attempt was made to obtain a homozygous susceptible strain in Culex quinquefasciatus (Say) using single raft sib-selection method. Lab-bred females of Cx. quinquefasciatus from insectariums, Unit of Medical Entomology were used in the experiment. After blood feeding Cx. quinquefasciatus mosquitoes laid eggs in raft form, ten rafts selected randomly for the test. Each egg raft was introduced into a plastic tray from number one to number ten. Twenty-five third stage larvae from each tray were exposed to 17.5 microl from 500mg/l malathion in a paper cup label number 1 to number ten. In the bioassay, which had 100% mortality, the respective larva in that particular tray was bred to adult stage for the following generation. Less than 7days old female mosquitoes that emerged from F(0) were used in the test. The F(0) and the subsequent adult and larval stage generations were subjected to adult and larval bioassay. After selection for about 10 generations, a homozygous susceptible strain in Cx. quinquefasciatus was obtained.

  12. Cryopreserved Mesenchymal Stromal Cells Are Susceptible to T-Cell Mediated Apoptosis Which Is Partly Rescued by IFNγ Licensing.

    PubMed

    Chinnadurai, Raghavan; Copland, Ian B; Garcia, Marco A; Petersen, Christopher T; Lewis, Christopher N; Waller, Edmund K; Kirk, Allan D; Galipeau, Jacques

    2016-09-01

    We have previously demonstrated that cryopreservation and thawing lead to altered Mesenchymal stromal cells (MSC) functionalities. Here, we further analyzed MSC's fitness post freeze-thaw. We have observed that thawed MSC can suppress T-cell proliferation when separated from them by transwell membrane and the effect is lost in a MSC:T-cell coculture system. Unlike actively growing MSCs, thawed MSCs were lysed upon coculture with activated autologous Peripheral Blood Mononuclear Cells (PBMCs) and the lysing effect was further enhanced with allogeneic PBMCs. The use of DMSO-free cryoprotectants or substitution of Human Serum Albumin (HSA) with human platelet lysate in freezing media and use of autophagy or caspase inhibitors did not prevent thaw defects. We tested the hypothesis that IFNγ prelicensing before cryobanking can enhance MSC fitness post thaw. Post thawing, IFNγ licensed MSCs inhibit T cell proliferation as well as fresh MSCs and this effect can be blocked by 1-methyl Tryptophan, an Indoleamine 2,3-dioxygenase (IDO) inhibitor. In addition, IFNγ prelicensed thawed MSCs inhibit the degranulation of cytotoxic T cells while IFNγ unlicensed thawed MSCs failed to do so. However, IFNγ prelicensed thawed MSCs do not deploy lung tropism in vivo following intravenous injection as well as fresh MSCs suggesting that IFNγ prelicensing does not fully rescue thaw-induced lung homing defect. We identified reversible and irreversible cryoinjury mechanisms that result in susceptibility to host T-cell cytolysis and affect MSC's cell survival and tissue distribution. The susceptibility of MSC to negative effects of cryopreservation and the potential to mitigate the effects with IFNγ prelicensing may inform strategies to enhance the therapeutic efficacy of MSC in clinical use. Stem Cells 2016;34:2429-2442. PMID:27299362

  13. Cells susceptible to epithelial-mesenchymal transition are enriched in stem-like side population cells from prostate cancer.

    PubMed

    Luo, Yong; Cui, Xinhao; Zhao, Jiahui; Han, Yili; Li, Mingchuan; Lin, Yunhua; Jiang, Yongguang; Lan, Ling

    2014-02-01

    Accumulating evidence suggests that epithelial-mesenchymal transition (EMT) acts as an important factor for the promotion of tumor progression. Strategies for suppressing EMT remain the subject of ongoing research. In the present study, fluorescence-activated cell sorting (FACS) was used to isolate side population (SP) cells from human prostate cancer (PCa) cell lines and xenograft tissues. After identifying their molecular and functional stem-like characteristics, stem-like SP cells from a cell line and from xenograft tissue were transfected with hypoxia inducible factor-1α (HIF-1α). The potential of the prostate stem-like SP cells to undergo EMT was compared with that in their bulk counterparts after HIF-1α introduction. Stem-like SP cells acquired more complete EMT molecular features and exhibited stronger aggressive capability than the homologous bulk population cells both in vitro (proliferation and invasion) and in vivo (tumorigenesis and metastasis formation). We, therefore, concluded that EMT is closely associated with tumor heterogeneity, and that PCa cells susceptible to EMT are enriched in stem-like SP cells. These findings disclose a new approach, targeting the cellular basis of the EMT process that may help to identify effective and accurate methods for suppressing tumor growth and preventing distant dissemination.

  14. Cell-specific RNA aptamer against human CCR5 specifically targets HIV-1 susceptible cells and inhibits HIV-1 infectivity.

    PubMed

    Zhou, Jiehua; Satheesan, Sangeetha; Li, Haitang; Weinberg, Marc S; Morris, Kevin V; Burnett, John C; Rossi, John J

    2015-03-19

    The C-C chemokine receptor type 5 (CCR5) is a receptor expressed by T cells and macrophages that serves as a coreceptor for macrophage-tropic HIV-1. Loss of CCR5 is associated with resistance to HIV-1. Here, we combine the live-cell-based SELEX with high-throughput sequencing technology to generate CCR5 RNA aptamers capable of specifically targeting HIV-1 susceptible cells (as small interfering RNA [siRNA] delivery agent) and inhibiting HIV-1 infectivity (as antiviral agent) via block of the CCR5 required for HIV-1 to enter cells. One of the best candidates, G-3, efficiently bound and was internalized into human CCR5-expressing cells. The G-3 specifically neutralized R5 virus infection in primary peripheral blood mononuclear cells, and in vivo generated human CD4(+) T cells with a nanomolar inhibitory concentration 50%. G-3 was also capable of transferring functional siRNAs to CCR5-expressing cells. Collectively, the cell-specific, internalizing, CCR5-targeted aptamers and aptamer-siRNA conjugates offer promise for overcoming some of the current challenges of drug resistance in HIV-1 by providing cell-type- or tissue-specific delivery of various therapeutic moieties.

  15. Relative susceptibilities of male germ cells to genetic defects induced by cancer chemotherapies

    SciTech Connect

    Wyrobek, A J; Schmid, T E; Marchetti, F

    2004-06-15

    Some chemotherapy regimens include agents that are mutagenic or clastogenic in model systems. This raises concerns that cancer survivors, who were treated before or during their reproductive years, may be at increased risks for abnormal reproductive outcomes. However, the available data from offspring of cancer survivors are limited, representing diverse cancers, therapies, time-to-pregnancies, and reproductive outcomes. Rodent breeding data after paternal exposures to individual chemotherapeutic agents illustrate the complexity of factors that influence the risk for transmitted genetic damage including agent, dose, endpoint, and the germ-cell susceptibility profiles that vary across agents. Direct measurements of chromosomal abnormalities in sperm of mice and humans by sperm FISH have corroborated the differences in germ-cell susceptibilities. The available evidence suggests that the risk of producing chromosomally defective sperm is highest during the first few weeks after the end of chemotherapy, and decays with time. Thus, sperm samples provided immediately after the initiation of cancer therapies may contain treatment-induced genetic defects that will jeopardize the genetic health of offspring.

  16. Simultaneous Identification and Susceptibility Determination to Multiple Antibiotics of Staphylococcus aureus by Bacteriophage Amplification Detection Combined with Mass Spectrometry.

    PubMed

    Rees, Jon C; Pierce, Carrie L; Schieltz, David M; Barr, John R

    2015-07-01

    The continued advance of antibiotic resistance in clinically relevant bacterial strains necessitates the development and refinement of assays that can rapidly and cost-effectively identify bacteria and determine their susceptibility to a panel of antibiotics. A methodology is described herein that exploits the specificity and physiology of the Staphylococci bacteriophage K to identify Staphylococcus aureus (S. aureus) and determine its susceptibility to clindamycin and cefoxitin. The method uses liquid chromatography-mass spectrometry to monitor the replication of bacteriophage after it is used to infect samples thought to contain S. aureus. Amplification of bacteriophage K indicates the sample contains S. aureus, for it is only in the presence of a suitable host that bacteriophage K can amplify. If bacteriophage amplification is detected in samples containing the antibiotics clindamycin or cefoxitin, the sample is deemed to be resistant to these antibiotics, respectively, for bacteriophage can only amplify in a viable host. Thus, with a single work flow, S. aureus can be detected in an unknown sample and susceptibility to clindamycin and cefoxitin can be ascertained. This Article discusses implications for the use of bacteriophage amplification in the clinical laboratory.

  17. Herpes simplex virus type 2 modulates the susceptibility of human bladder cells to uropathogenic bacteria.

    PubMed

    Superti, F; Longhi, C; Di Biase, A M; Tinari, A; Marchetti, M; Pisani, S; Gallinelli, C; Chiarini, F; Seganti, L

    2001-09-01

    The present study analyses the susceptibility of human bladder-derived cells (HT-1376) to the infection by herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis, as well as to the adhesiveness of uropathogenic bacteria. HT-1376 cells were efficiently infected by HSV-2 strain 333, as demonstrated by immunofluorescence staining of viral antigens, titration of cytopathic effect, and visualisation by transmission electron microscopy. This cell model was also prone to C. trachomatis (serovar E, Bour strain) replication and to the adherence of clinical uropathogenic isolates of Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris and Enterococcus faecalis. The pre-infection of HT-1376 cells with HSV-2 caused a tenfold increased adherence of an E. coli strain (U1), isolated from a patient affected by severe haemorrhagic cystitis, whereas in HSV-2 pre-infected cells the number of C. trachomatis inclusion bodies was significantly reduced. Our findings indicate that these cells are a suitable in vitro model for studying infection and super-infection of the lower urinary tract by viruses and bacteria.

  18. Valproic acid cooperates with hydralazine to augment the susceptibility of human osteosarcoma cells to Fas- and NK cell-mediated cell death.

    PubMed

    Yamanegi, Koji; Yamane, Junko; Kobayashi, Kenta; Kato-Kogoe, Nahoko; Ohyama, Hideki; Nakasho, Keiji; Yamada, Naoko; Hata, Masaki; Fukunaga, Satoru; Futani, Hiroyuki; Okamura, Haruki; Terada, Nobuyuki

    2012-07-01

    We investigated the effects of valproic acid (VPA), a histone deacetylase inhibitor, in combination with hydralazine, a DNA methylation inhibitor, on the expression of cell-surface Fas and MHC-class I-related chain molecules A and B (MICA and B), the ligands of NKG2D which is an activating receptor of NK cells, and on production of their soluble forms in HOS, U-2 OS and SaOS-2 human osteosarcoma cell lines. We also examined the susceptibility of these cells to Fas- and NK cell-mediated cell death. VPA did not increase the expression of Fas on the surface of osteosarcoma cells, while hydralazine did, and the combination of VPA with hydralazine increased the expression of cell-surface Fas. In contrast, the combination of VPA with hydralazine did not increase the production of soluble Fas by osteosarcoma cells. Both VPA and hydralazine increased the expression of cell-surface MICA and B in osteosarcoma cells, and their combination induced a greater increase in their expression. VPA inhibited the production of both soluble MICA and MICB by osteosarcoma cells while hydralazine produced no effect. Both VPA and hydralazine enhanced the susceptibility of osteosarcoma cells to Fas- and NK cell-mediated cell death and the combination of VPA with hydralazine further enhanced the effects. The present results suggest that combined administration of VPA and hydrazine is valuable for enhancing the therapeutic effects of immunotherapy for osteosarcomas.

  19. Sendai virus utilizes specific sialyloligosaccharides as host cell receptor determinants.

    PubMed Central

    Markwell, M A; Paulson, J C

    1980-01-01

    Purified sialyltransferases (CMP-N-acetyl-neuraminate:D-galactosyl-glycoprotein N-acetylneuraminyl-transferase, EC 2.4.99.1) in conjunction with neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) were used to produce cell surface sialyloligosaccharides of defined sequence to investigate their role in paramyxovirus infection of host cells. Infection of Madin-Darby bovine kidney cells by Sendai virus was monitored by hemagglutination titer of the virus produced and by changes in morphological characteristics. By either criterion, treatment of the cells with Vibrio cholerae neuraminidase to remove cell surface sialic acids rendered them resistant to infection by Sendai virus. Endogenous replacement of receptors by the cell occurred slowly but supported maximal levels of infection within 6 hr. In contrast, sialylation during a 20-min incubation with CMP-sialic acid and beta-galactoside alpha 2,3-sialytransferase restored full susceptibility to infection. This enzyme elaborates the NeuAc alpha 2,3Gal beta 1,3GalNAc (NeuAc, N-acetylneuraminic acid) sequence on glycoproteins and glycolipids. No restoration of infectivity was observed when neuraminidase-treated cells were sialylated by using beta-galactoside alpha 2,6-sialytransferase, which elaborates the NeuAc-alpha 2,6Gal beta 1,4GlcNAc sequence. These results suggest that sialyloligosaccharide receptor determinants of defined sequence are required for Sendai virus infection of host cells. Images PMID:6255459

  20. Susceptibility of Chinese Perch Brain (CPB) Cell and Mandarin Fish to Red-Spotted Grouper Nervous Necrosis Virus (RGNNV) Infection

    PubMed Central

    Tu, Jiagang; Chen, Wenjie; Fu, Xiaozhe; Lin, Qiang; Chang, Ouqin; Zhao, Lijuan; Lan, Jiangfeng; Li, Ningqiu; Lin, Li

    2016-01-01

    Nervous necrosis virus (NNV) is the causative agent of viral encephalopathy and retinopathy (VER), a neurological disease responsible for high mortality of fish species worldwide. Taking advantage of our established Chinese perch brain (CPB) cell line derived from brain tissues of Mandarin fish (Siniperca chuatsi), the susceptibility of CPB cell to Red-Spotted Grouper nervous necrosis virus (RGNNV) was evaluated. The results showed that RGNNV replicated well in CPB cells, resulting in cellular apoptosis. Moreover, the susceptibility of Mandarin fish to RGNNV was also evaluated. Abnormal swimming was observed in RGNNV-infected Mandarin fish. In addition, the cellular vacuolation and viral particles were also observed in brain tissues of RGNNV-infected Mandarin fish by Hematoxylin-eosin staining or electronic microscopy. The established RGNNV susceptible brain cell line from freshwater fish will pave a new way for the study of the pathogenicity and replication of NNV in the future. PMID:27213348

  1. Susceptibility of Chinese Perch Brain (CPB) Cell and Mandarin Fish to Red-Spotted Grouper Nervous Necrosis Virus (RGNNV) Infection.

    PubMed

    Tu, Jiagang; Chen, Wenjie; Fu, Xiaozhe; Lin, Qiang; Chang, Ouqin; Zhao, Lijuan; Lan, Jiangfeng; Li, Ningqiu; Lin, Li

    2016-01-01

    Nervous necrosis virus (NNV) is the causative agent of viral encephalopathy and retinopathy (VER), a neurological disease responsible for high mortality of fish species worldwide. Taking advantage of our established Chinese perch brain (CPB) cell line derived from brain tissues of Mandarin fish (Siniperca chuatsi), the susceptibility of CPB cell to Red-Spotted Grouper nervous necrosis virus (RGNNV) was evaluated. The results showed that RGNNV replicated well in CPB cells, resulting in cellular apoptosis. Moreover, the susceptibility of Mandarin fish to RGNNV was also evaluated. Abnormal swimming was observed in RGNNV-infected Mandarin fish. In addition, the cellular vacuolation and viral particles were also observed in brain tissues of RGNNV-infected Mandarin fish by Hematoxylin-eosin staining or electronic microscopy. The established RGNNV susceptible brain cell line from freshwater fish will pave a new way for the study of the pathogenicity and replication of NNV in the future. PMID:27213348

  2. Automated determination of chemical functionalisation addition routes based on magnetic susceptibility and nucleus independent chemical shifts

    NASA Astrophysics Data System (ADS)

    Van Lier, G.; Ewels, C. P.; Geerlings, P.

    2008-07-01

    We present a modified version of our previously reported meta-code SACHA, for systematic analysis of chemical addition. The code automates the generation of structures, running of quantum chemical codes, and selection of preferential isomers based on chosen selection rules. While the selection rules for the previous version were based on the total system energy, predicting purely thermodynamic addition patterns, we examine here the possibility of using other system parameters, notably magnetic susceptibility as a descriptor of global aromaticity, and nucleus independent chemical shifts (NICS) as local aromaticity descriptor.

  3. Differential susceptibility of macrophage cell lines to Bacillus anthracis-Vollum 1B.

    PubMed

    Gutting, B W; Gaske, K S; Schilling, A S; Slaterbeck, A F; Sobota, L; Mackie, R S; Buhr, T L

    2005-03-01

    Bacillus anthracis (BA) is a spore forming bacterium and the causative agent of anthrax disease. Macrophages (Mphis) play a central role in anthrax disease. An important step in disease progression is the ability of BA to secrete lethal toxin (LeTx) that kills Mphis. LeTx is a heterodimer composed of protective antigen (PA) and lethal factor (LF). Researchers have shown that Mphi cell lines demonstrate differential susceptibility to purified LeTx; for example RAW264.7 and J774A.1 Mphis are sensitive to LeTx whereas IC-21 Mphis are resistant. Research has also suggested that exogenous factors, including other BA proteins, can influence the activity of LeTx. For this reason, the objective of the current work was to examine if RAW264.7, J774A.1, and IC-21 Mphis demonstrated differential susceptibility when cultured with a LeTx-producing strain of BA. Here, we co-cultured Mphis with LeTx+ Vollum 1B (V1B) spores for >15 h and assayed for Mphi cell death by morphology, trypan blue (TB) staining, neutral red (NR) activity, and lactate dehydrogenase (LDH) activity in the culture media. Following the addition of V1B spores, necrosis (approximately 50% mortality) was observed in RAW264.7 and J774A.1 Mphis at 7.5 and 10 h, respectively. By 15 h, both RAW264.7 and J774A.1 Mphis demonstrated 100% mortality. In contrast, IC-21 Mphis, under identical culture conditions, remained viable (98%) and activated throughout the course of the experiment (>24 h). The mechanism of RAW264.7 cell death appeared to involve LeTx because the V1B-induced cytotoxicity was dose-dependently reversed by the addition of anti-PA antibody to the culture media. These observations suggest there is differential susceptibility of Mphi cell lines to the LeTx+ V1B strain of BA. Further development of this in vitro model may be useful to further characterize the interactions between Mphis and BA spores. PMID:15649636

  4. CD52-Negative NK Cells Are Abundant in the Liver and Less Susceptible to Alemtuzumab Treatment

    PubMed Central

    Matsuura, Toshiharu; Muraoka, Izumi; Tryphonopoulos, Panagiotis; Fan, Ji; Tekin, Akin; Selvaggi, Gennaro; Levi, David; Ruiz, Phillip; Ricordi, Camillo; Vianna, Rodrigo; Ohdan, Hideki; Waldmann, Herman; Tzakis, Andreas G.; Nishida, Seigo

    2016-01-01

    Background T-cell depleting strategies have become an integral part of immunosuppressive regimens in organ transplantation. Alemtuzumab is a humanized monoclonal antibody against CD52, a cell-surface antigen on several immune cells. It has been suggested that lymphocyte depletion increases the risk of serious infections. However, this has not been observed with short-term alemtuzumab treatment in an organ transplant setting. For induction therapy using alemtuzumab following liver transplantation, we found that T- and B-cell numbers declined rapidly after alemtuzumab therapy; however, the natural killer (NK) cell number was sustained. NK cells are important effectors of innate immunity. Since the effects of alemtuzumab on NK cell functions, especially those of liver NK cells, are unknown, this study aimed to investigate this in detail. Methods To assess the effect of alemtuzumab on NK cells, samples were obtained from 7 organ donors and examined by flow cytometry using Annexin V and propidium iodide. Phenotypical and functional differences within subsets of NK cells with different levels of CD52 expression were determined by flow cytometry and in vitro cytotoxicity assays. Results CD52 expression on NK cells was lower than that on other lymphocyte subsets. The liver contained a large number of CD52− NK cells compared with the peripheral blood. In vitro treatment of liver-derived NK cells with alemtuzumab did not result in cell death. In contrast, co-incubation with alemtuzumab induced cell death in peripheral blood mononuclear cells and non-NK cells in the liver. Furthermore, CD52− liver NK cells were more cytotoxic and produced more IFN-γ than CD52+ NK cells after cytokine activation. Conclusion The liver contains a large number of CD52− NK cells. These cells are refractory to alemtuzumab and have robust activity. These findings indicate that CD52− NK cells persist and could protect against infection after alemtuzumab-based lymphocyte depletion. PMID

  5. Low in situ expression of antioxidative enzymes in rat cerebellar granular cells susceptible to methylmercury.

    PubMed

    Fujimura, M; Usuki, F

    2014-01-01

    Methylmercury (MeHg), an environmental neurotoxicant, induces site-specific toxicity in the brain. Although oxidative stress has been demonstrated with MeHg toxicity, the site-specific toxicity is not completely understood. Among the cerebellar neurons, cerebellar granule cells (CGCs) appear vulnerable to MeHg, whereas Purkinje cells and molecular layer neurons are resistant. Here, we use a MeHg-intoxicated rat model to investigate these cerebellar neurons for the different causes of susceptibility to MeHg. Rats were exposed to 20 ppm MeHg for 4 weeks and subsequently exhibited neuropathological changes in the cerebellum that were similar to those observed in humans. We first isolated the three cerebellar neuron types using a microdissection system and then performed real-time PCR analyses for antioxidative enzymes. We observed that expression of manganese-superoxide dismutase (Mn-SOD), glutathione peroxidase 1 (GPx1), and thioredoxin reductase 1 (TRxR1) was significantly higher in Purkinje cells and molecular layer neurons than in CGCs. Finally, we performed immunohistochemical analyses on the cerebellum. Immunohistochemistry showed increased expression of Mn-SOD, GPx1, and TRxR1 in Purkinje cells and molecular layer neurons, which was coincident with the mRNA expression patterns. Considering Mn-SOD, GPx1, and TRxR1 are critical for protecting cells against MeHg intoxication, the results indicate that low expression of these antioxidative enzymes increases CGCs vulnerability to MeHg toxicity.

  6. Stem Cells as Hormone Targets That Lead to Increased Cancer Susceptibility

    PubMed Central

    Calderon-Gierszal, Esther L.; Hu, Wen-Yang

    2015-01-01

    Major advances during the past decade have permitted a clearer understanding of processes that regulate stem cell self-renewal and lineage commitment toward differentiated progeny that populate all tissues. Considerable evidence has also accumulated to indicate that aberrations in the stem and progenitor cell populations can lead to increased cancer risk in specific organs systems. It is long recognized that environmental factors play a major role in cancer etiology, and emerging data suggest that endocrine-disrupting chemicals (EDCs) may contribute to an increased cancer risk. Using the prostate gland as a model system, the present review highlights recent data that find that estrogens and EDCs can reprogram prostate stem and progenitor cell populations, leading to increased cancer susceptibility. We propose that stem cell programming during early development in hormone-regulated tissues may lead to heightened sensitivity to early-life EDC exposures and that aberrant stem cell reprogramming by EDCs may contribute to the developmental basis of adult cancer risk. PMID:26241068

  7. Susceptibility of mouse minute virus to inactivation by heat in two cell culture media types.

    PubMed

    Schleh, Marc; Romanowski, Peter; Bhebe, Prince; Zhang, Li; Chinniah, Shivanthi; Lawrence, Bill; Bashiri, Houman; Gaduh, Asri; Rajurs, Viveka; Rasmussen, Brian; Chuck, Alice; Dehghani, Houman

    2009-01-01

    Viral contaminations of biopharmaceutical manufacturing cell culture facilities are a significant threat and one for which having a risk mitigation strategy is highly desirable. High temperature, short time (HTST) mammalian cell media treatment may potentially safeguard manufacturing facilities from such contaminations. HTST is thought to inactivate virions by denaturing proteins of the viral capsid, and there is evidence that HTST provides ample virucidal efficacy against nonenveloped or naked viruses such as mouse minute virus (MMV), a parvovirus. The aim of the studies presented herein was to further delineate the susceptibility of MMV, known to have contaminated mammalian cell manufacturing facilities, to heat by exposing virus-spiked cell culture media to a broad range of temperatures and for various times of exposure. The results of these studies show that HTST is capable of inactivating MMV by three orders of magnitude or more. Thus, we believe that HTST is a useful technology for the purposes of providing a barrier to adventitious contamination of mammalian cell culture processes in the biopharmaceutical industry.

  8. Stem Cells as Hormone Targets That Lead to Increased Cancer Susceptibility.

    PubMed

    Prins, Gail S; Calderon-Gierszal, Esther L; Hu, Wen-Yang

    2015-10-01

    Major advances during the past decade have permitted a clearer understanding of processes that regulate stem cell self-renewal and lineage commitment toward differentiated progeny that populate all tissues. Considerable evidence has also accumulated to indicate that aberrations in the stem and progenitor cell populations can lead to increased cancer risk in specific organs systems. It is long recognized that environmental factors play a major role in cancer etiology, and emerging data suggest that endocrine-disrupting chemicals (EDCs) may contribute to an increased cancer risk. Using the prostate gland as a model system, the present review highlights recent data that find that estrogens and EDCs can reprogram prostate stem and progenitor cell populations, leading to increased cancer susceptibility. We propose that stem cell programming during early development in hormone-regulated tissues may lead to heightened sensitivity to early-life EDC exposures and that aberrant stem cell reprogramming by EDCs may contribute to the developmental basis of adult cancer risk. PMID:26241068

  9. Determining Outdoor CPV Cell Temperature (Presentation)

    SciTech Connect

    Muller, M.

    2011-04-01

    An accurate method is needed for determining cell temperature when measuring CPV modules outdoors. It has been suggested that cell temperature can be calculated though a procedure that shutters sunlight to the cells while measuring the transients in open-circuit voltage (Voc) and heat sink temperature. This presentation documents application of this shutter procedure to multiple CPV modules at NREL. The challenges and limitations are presented along with an alternate approach to measuring CPV cell operating temperature.

  10. Determining Outdoor CPV Cell Temperature: Preprint

    SciTech Connect

    Muller, M.; Deline, C.; Marion, B.; Kurtz, S.; Bosco, N.

    2011-07-01

    An accurate method is needed for determining cell temperature when measuring CPV modules outdoors. It has been suggested that cell temperature can be calculated though a procedure that shutters sunlight to the cells while measuring the transients in open-circuit voltage (Voc) and heat sink temperature. This paper documents application of this shutter procedure to multiple CPV modules at NREL. The challenges and limitations are presented along with an alternate approach to measuring CPV cell operating temperature.

  11. Site of Clomazone Action in Tolerant-Soybean and Susceptible-Cotton Photomixotrophic Cell Suspension Cultures 1

    PubMed Central

    Norman, Michael A.; Liebl, Rex A.; Widholm, Jack M.

    1990-01-01

    Studies were conducted to determine the herbicidal site of clomazone action in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) (SB-M) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) (COT-M) photomixotrophic cell suspension cultures. Although a 10 micromolar clomazone treatment did not significantly reduce the terpene or mixed terpenoid content (microgram per gram fresh weight) of the SB-M cell line, there was over a 70% reduction in the chlorophyll (Chl), carotenoid (CAR), and plastoquinone (PQ) content of the COT-M cell line. The tocopherol (TOC) content was reduced only 35.6%. Reductions in the levels of Chl, CAR, TOC, and PQ indicate that the site of clomazone action in COT-M cells is prior to geranylgeranyl pyrophosphate (GGPP). The clomazone treatment did not significantly reduce the flow of [14C]mevalonate ([14C]MEV) (nanocuries per gram fresh weight) into CAR and the three mixed terpenoid compounds of SB-M cells. Conversely, [14C]MEV incorporation into CAR and the terpene moieties of Chl, PQ, and TOC in COT-M cells was reduced at least 73%, indicating that the site of clomazone action must be after MEV. Sequestration of clomazone away from the chloroplast cannot account for soybean tolerance to clomazone since chloroplasts isolated from both cell lines incubated with [14C]clomazone contained a similar amount of radioactivity (disintegrations per minute per microgram of Chl). The possible site(s) of clomazone inhibition include mevalonate kinase, phosphomevalonate kinase, pyrophosphomevalonate decarboxylase, isopentenyl pyrophosphate isomerase, and/or a prenyl transferase. PMID:16667768

  12. Transformation of adrenal medullary chromaffin cells increases asthmatic susceptibility in pups from allergen-sensitized rats

    PubMed Central

    2012-01-01

    Background Studies have shown that epinephrine release is impaired in patients with asthma. The pregnancy of female rats (dams) with asthma promotes in their pups the differentiation of adrenal medulla chromaffin cells (AMCCs) into sympathetic neurons, mediated by nerve growth factor, which leads to a reduction in epinephrine secretion. However, the relatedness between the alteration of AMCCs and increased asthma susceptibility in such offspring has not been established. Methods In this study, we observed the effects of allergization via ovalbumin on rat pups born of asthmatic dams. Results Compared to the offspring of untreated controls, bronchial hyperreactivity and airway inflammation were more severe in the pups from sensitized (asthmatic) dams. In pups exposed to nerve growth factor (NGF) in utero these effects were aggravated further, but the effects were blocked in pups whose dams had been treated with anti-NGF. Furthermore, alterations in AMCC phenotype corresponded to the degree of bronchial hyperreactivity and lung lesions of the different treatment groups. Such AMCC alterations included degranulation of chromaffin granules, reduction of epinephrine and phenylethanolamine-n-methyl transferase, and elevation of NGF and peripherin levels. Conclusions Our results present evidence that asthma during the pregnancy of rat dams promotes asthma susceptibility in their offspring, and that the transformation of AMCCs to neurons induced by NGF plays an important role in this process. PMID:23137120

  13. Neural stem cell-encoded temporal patterning delineates an early window of malignant susceptibility in Drosophila

    PubMed Central

    Narbonne-Reveau, Karine; Lanet, Elodie; Dillard, Caroline; Foppolo, Sophie; Chen, Ching-Huan; Parrinello, Hugues; Rialle, Stéphanie; Sokol, Nicholas S; Maurange, Cédric

    2016-01-01

    Pediatric neural tumors are often initiated during early development and can undergo very rapid transformation. However, the molecular basis of this early malignant susceptibility remains unknown. During Drosophila development, neural stem cells (NSCs) divide asymmetrically and generate intermediate progenitors that rapidly differentiate in neurons. Upon gene inactivation, these progeny can dedifferentiate and generate malignant tumors. Here, we find that intermediate progenitors are prone to malignancy only when born during an early window of development while expressing the transcription factor Chinmo, and the mRNA-binding proteins Imp/IGF2BP and Lin-28. These genes compose an oncogenic module that is coopted upon dedifferentiation of early-born intermediate progenitors to drive unlimited tumor growth. In late larvae, temporal transcription factor progression in NSCs silences the module, thereby limiting mitotic potential and terminating the window of malignant susceptibility. Thus, this study identifies the gene regulatory network that confers malignant potential to neural tumors with early developmental origins. DOI: http://dx.doi.org/10.7554/eLife.13463.001 PMID:27296804

  14. Development of Similar Broth Microdilution Methods to Determine the Antimicrobial Susceptibility of Flavobacterium columnare and F. psychrophilum.

    PubMed

    Gieseker, Charles M; Crosby, Tina C; Mayer, Tamara D; Bodeis, Sonya M; Stine, Cynthia B

    2016-03-01

    Flavobacterium columnare and F. psychrophilum are major fish pathogens that cause diseases that may require antimicrobial therapy. Choice of appropriate treatment is dependent upon determining the antimicrobial susceptibility of isolates. Therefore we optimized methods for broth microdilution testing of F. columnare and F. psychrophilum to facilitate standardizing an antimicrobial susceptibility test. We developed adaptations to make reproducible broth inoculums and confirmed the proper incubation time and media composition. We tested the stability of potential quality-control bacteria and compared test results between different operators. Log phase occurred at 48 h for F. columnare and 72-96 h for F. psychrophilum, confirming the test should be incubated at 28°C for approximately 48 h and at 18°C for approximately 96 h, respectively. The most consistent susceptibility results were achieved with plain, 4-g/L, dilute Mueller-Hinton broth supplemented with dilute calcium and magnesium. Supplementing the broth with horse serum did not improve growth. The quality-control strains, Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658, yielded stable minimal inhibitory concentrations (MIC) against all seven antimicrobials tested after 30 passes at 28°C and 15 passes at 18°C. In comparison tests, most MICs of the isolates agreed 100% within one drug dilution for ampicillin, florfenicol, and oxytetracycline. The agreement was lower with the ormetoprim-sulfdimethoxine combination, but there was at least 75% agreement for all but one isolate. These experiments have provided methods to help standardize antimicrobial susceptibility testing of these nutritionally fastidious aquatic bacteria. Received June 24, 2015; accepted October 2, 2015.

  15. Species-specific PCR for the Diagnosis and Determination of Antibiotic Susceptibilities of Brucella Strains Isolated from Tehran, Iran

    PubMed Central

    Irajian, Gholam Reza; Masjedian Jazi, Faramarz; Mirnejad, Reza; Piranfar, Vahhab; Zahraei salehi, Taghi; Amir Mozafari, Noor; Ghaznavi-rad, Ehsanollah; Khormali, Mahmoud

    2016-01-01

    Background: Brucellosis is an endemic zoonotic disease in the Middle East. This study intended to design a uniplex PCR assay for the detection and differentiation of Brucella at the species level and determining the antibiotic susceptibility pattern of Brucella in Iran. Methods: Sixty-eight Brucella specimens (38 animal and 30 human specimens) were analyzed using PCR (using one pair of primers). Antibiotic susceptibility patterns were evaluated and compared using the E-Test and disk diffusion susceptibility test. Tigecycline susceptibility pattern was compared with other antibiotics. Results: Thirty six isolates of B. melitensis, 2 isolates of B. abortus and 1 isolate of B. suis from the 38 animal specimens, 24 isolates of B. melitensis and 6 isolates of B. abortus from the 30 human specimens were differentiated. The MIC50 values of doxycycline for human and animal specimens were 125 and 10 μg/ml, respectively, tigecycline 0.064 μg/ml for human specimens and 0.125μg/ml for animal specimens, and trimethoprim/ sulfamethoxazole and ciprofloxacin 0.065 and 0.125μg/ml, respectively, for both human and animal specimens. The highest MIC50 value of streptomycin in the human specimens was 0.5μg/ml and 1μg/ml for the animal specimens. The greatest resistance shown was to tetracycline and gentamicin, respectively. Conclusion: Uniplex PCR for the detection and differentiation of Brucella at the strain level is faster and less expensive than multiplex PCR, and the antibiotics doxycycline, rifampin, trimethoprim-sulfamethoxazole, ciprofloxacin, and ofloxacin are the most effective antibiotics for treating brucellosis. Resistance to tigecycline is increasing, and we recommend that it be used in a combination regimen. PMID:27799972

  16. Selective Susceptibility of Human Skin Antigen Presenting Cells to Productive Dengue Virus Infection

    PubMed Central

    Cerny, Daniela; Haniffa, Muzlifah; Shin, Amanda; Bigliardi, Paul; Tan, Bien Keem; Lee, Bernett; Poidinger, Michael; Tan, Ern Yu; Ginhoux, Florent; Fink, Katja

    2014-01-01

    Dengue is a growing global concern with 390 million people infected each year. Dengue virus (DENV) is transmitted by mosquitoes, thus host cells in the skin are the first point of contact with the virus. Human skin contains several populations of antigen-presenting cells which could drive the immune response to DENV in vivo: epidermal Langerhans cells (LCs), three populations of dermal dendritic cells (DCs), and macrophages. Using samples of normal human skin we detected productive infection of CD14+ and CD1c+ DCs, LCs and dermal macrophages, which was independent of DC-SIGN expression. LCs produced the highest viral titers and were less sensitive to IFN-β. Nanostring gene expression data showed significant up-regulation of IFN-β, STAT-1 and CCL5 upon viral exposure in susceptible DC populations. In mice infected intra-dermally with DENV we detected parallel populations of infected DCs originating from the dermis and migrating to the skin-draining lymph nodes. Therefore dermal DCs may simultaneously facilitate systemic spread of DENV and initiate the adaptive anti-viral immune response. PMID:25474532

  17. Reducing the Level of Undecaprenyl Pyrophosphate Synthase Has Complex Effects on Susceptibility to Cell Wall Antibiotics.

    PubMed

    Lee, Yong Heon; Helmann, John D

    2013-06-24

    Undecaprenyl pyrophosphate synthase (UppS) catalyzes the formation of the C55 lipid carrier (UPP) that is essential for bacterial peptidoglycan biosynthesis. Here we selected a vancomycin (VAN)-resistant derivative of Bacillus subtilis W168 which contains a single-point mutation in the ribosome-binding site (RBS) of the uppS gene designated uppS1. Genetic reconstruction experiments demonstrate that the uppS1 allele is sufficient to confer low-level VAN resistance and causes reduced UppS translation. The decreased level of UppS renders B. subtilis slightly more susceptible to many late-acting cell wall antibiotics including β-lactams, but significantly more resistant to fosfomycin and D-cycloserine, antibiotics that interfere with the very early steps of cell wall synthesis. We further show that the uppS1 allele leads to slightly elevated expression of the σ(M) regulon, possibly helping to compensate for the stress caused by a decrease in UPP levels. Notably, the uppS1 mutation increases resistance to VAN, fosfomycin, and D-cycloserine in wild-type cells, but this effect is greatly reduced or eliminated in a sigM mutant background. Our findings suggest that, although UppS is an attractive antibacterial target, incomplete inhibition of UppS function may lead to increased resistance to some cell wall-active antibiotics. PMID:23796923

  18. Virulence determinants, antimicrobial susceptibility, and molecular profiles of Erysipelothrix rhusiopathiae strains isolated from China.

    PubMed

    Ding, Yi; Zhu, Dongmei; Zhang, Jianmin; Yang, Longsheng; Wang, Xiangru; Chen, Huanchun; Tan, Chen

    2015-11-01

    The aim of this study was to understand the epidemiology, serotype, antibiotic sensitivity, and clonal structure of Erysipelothrix rhusiopathiae strains in China. Forty-eight strains were collected from seven provinces during the period from 2012 to 2013. Pulse-field electrophoresis identified 32 different patterns which were classified into clonal groups A–D. Most pulsed-field gel electrophoresis (PFGE) patterns were observed in clonal complex A and B, suggesting high diversity of genetic characterization in these two predominant clonal complexes. Antibiotic sensitivity test shows that all the stains were susceptible to ampicillin, erythromycin, and cefotaxime, and resistant to kanamycin, cefazolin, sulfadiazine, and amikacin. Erythromycin and ampicillin are recommended as first-line antibiotics for treatment of E. rhusiopathiae in China. The high variation in PFGE pattern among the main clonal groups shows that the E. rhusiopathiae in China may originate from different lineages and sources instead of from expansion of a single clonal lineage across different regions. PMID:26975059

  19. Virulence determinants, antimicrobial susceptibility, and molecular profiles of Erysipelothrix rhusiopathiae strains isolated from China

    PubMed Central

    Ding, Yi; Zhu, Dongmei; Zhang, Jianmin; Yang, Longsheng; Wang, Xiangru; Chen, Huanchun; Tan, Chen

    2015-01-01

    The aim of this study was to understand the epidemiology, serotype, antibiotic sensitivity, and clonal structure of Erysipelothrix rhusiopathiae strains in China. Forty-eight strains were collected from seven provinces during the period from 2012 to 2013. Pulse-field electrophoresis identified 32 different patterns which were classified into clonal groups A–D. Most pulsed-field gel electrophoresis (PFGE) patterns were observed in clonal complex A and B, suggesting high diversity of genetic characterization in these two predominant clonal complexes. Antibiotic sensitivity test shows that all the stains were susceptible to ampicillin, erythromycin, and cefotaxime, and resistant to kanamycin, cefazolin, sulfadiazine, and amikacin. Erythromycin and ampicillin are recommended as first-line antibiotics for treatment of E. rhusiopathiae in China. The high variation in PFGE pattern among the main clonal groups shows that the E. rhusiopathiae in China may originate from different lineages and sources instead of from expansion of a single clonal lineage across different regions. PMID:26975059

  20. Increased NHC Cells in the Peritoneal Cavity of Plasmacytoma Susceptible BALB/c Mouse

    PubMed Central

    Sánchez-González, Berenice; García-Vázquez, Francisco Javier; Farfán-Morales, José Eduardo; Jiménez-Zamudio, Luis Antonio

    2015-01-01

    BALB/c strain mice are unique in that they develop murine plasmacytoma (MPC) as a consequence of the inflammation induced by pristane oil injection in the peritoneal cavity. In this work the Treg, Th17, B1, B2, and NHC lymphocyte populations from the peritoneal environment of BALB/c, the susceptible strain, and C57BL/6 mice, which do not develop MPC after oil treatment, were studied. Both oil-treated strains showed decreased levels of Th17 lymphocytes, no significant variation in Treg lymphocytes, and a drastic decrease of all B lymphocyte populations. However, only oil-induced BALB/c showed increased levels of natural helper cells (NHC) which could be important in the myeloma induction. PMID:26504358

  1. Cancer therapy. Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility.

    PubMed

    Yu, Min; Bardia, Aditya; Aceto, Nicola; Bersani, Francesca; Madden, Marissa W; Donaldson, Maria C; Desai, Rushil; Zhu, Huili; Comaills, Valentine; Zheng, Zongli; Wittner, Ben S; Stojanov, Petar; Brachtel, Elena; Sgroi, Dennis; Kapur, Ravi; Shioda, Toshihiro; Ting, David T; Ramaswamy, Sridhar; Getz, Gad; Iafrate, A John; Benes, Cyril; Toner, Mehmet; Maheswaran, Shyamala; Haber, Daniel A

    2014-07-11

    Circulating tumor cells (CTCs) are present at low concentrations in the peripheral blood of patients with solid tumors. It has been proposed that the isolation, ex vivo culture, and characterization of CTCs may provide an opportunity to noninvasively monitor the changing patterns of drug susceptibility in individual patients as their tumors acquire new mutations. In a proof-of-concept study, we established CTC cultures from six patients with estrogen receptor-positive breast cancer. Three of five CTC lines tested were tumorigenic in mice. Genome sequencing of the CTC lines revealed preexisting mutations in the PIK3CA gene and newly acquired mutations in the estrogen receptor gene (ESR1), PIK3CA gene, and fibroblast growth factor receptor gene (FGFR2), among others. Drug sensitivity testing of CTC lines with multiple mutations revealed potential new therapeutic targets. With optimization of CTC culture conditions, this strategy may help identify the best therapies for individual cancer patients over the course of their disease.

  2. The influence of T cell receptor and cytokine genes on sarcoidosis susceptibility in African Americans.

    PubMed

    Rybicki, B A; Maliarik, M J; Malvitz, E; Sheffer, R G; Major, M; Popovich, J; Iannuzzi, M C

    1999-09-01

    The pathogenesis of sarcoidosis, a multisystem granulomatous disorder, is mediated through immunoregulatory pathways. While sarcoidosis clusters in families, inherited risk factors remain undefined. In search of possible sarcoidosis susceptibility genes, we examined anonymous polymorphic genetic markers tightly linked to six different candidate gene regions on chromosomes 2q13, 5q31, 6p23-25, 7p14-15, 14q11 and 22q11. These candidate regions contain T cell receptor, interleukin (IL) and interferon regulatory factor (IRF) genes. Our study population consisted of 105 African-American sarcoidosis cases and 95 unrelated healthy controls. The allelic frequency distribution of two out of the six markers, IL-1 alpha marker (p = 0.010) on 2q13 and the F13A marker (p = 0.0006) on 6p23-25, was statistically significantly different in cases compared with controls. The two alleles most strongly associated with sarcoidosis were IL-1 alpha*137 (Odds Ratio (OR) = 2.60; 95% confidence interval (CI) = 1.36-4.98) and F13A*188 (OR = 2.42; 95% CI = 1.37-4.30). Individuals that had both of these alleles were at a six-fold increased risk for sarcoidosis (OR = 6.19; 95% CI = 2.54-15.10). Restricting the analysis to cases with at least one first or second-degree relative affected with sarcoidosis increased the OR to 15.38. IL-1 levels are elevated in sarcoidosis and the F13A marker is tightly linked to a gene that codes for a newly identified interferon regulatory factor protein (IRF-4), which is thought to play a role in T cell effector functions. Our results suggest genetic susceptibility to sarcoidosis may be conferred by more than one immune-related gene that act synergistically on disease risk.

  3. Myeloid Dendritic Cells (DCs) of Mice Susceptible to Paracoccidioidomycosis Suppress T Cell Responses whereas Myeloid and Plasmacytoid DCs from Resistant Mice Induce Effector and Regulatory T Cells

    PubMed Central

    Pina, Adriana; Frank de Araujo, Eliseu; Felonato, Maíra; Loures, Flávio V.; Feriotti, Claudia; Bernardino, Simone; Barbuto, José Alexandre M.

    2013-01-01

    The protective adaptive immune response in paracoccidioidomycosis, a mycosis endemic among humans, is mediated by T cell immunity, whereas impaired T cell responses are associated with severe, progressive disease. The early host response to Paracoccidioides brasiliensis infection is not known since the disease is diagnosed at later phases of infection. Our laboratory established a murine model of infection where susceptible mice reproduce the severe disease, while resistant mice develop a mild infection. This work aimed to characterize the influence of dendritic cells in the innate and adaptive immunity of susceptible and resistant mice. We verified that P. brasiliensis infection induced in bone marrow-derived dendritic cells (DCs) of susceptible mice a prevalent proinflammatory myeloid phenotype that secreted high levels of interleukin-12 (IL-12), tumor necrosis factor alpha, and IL-β, whereas in resistant mice, a mixed population of myeloid and plasmacytoid DCs secreting proinflammatory cytokines and expressing elevated levels of secreted and membrane-bound transforming growth factor β was observed. In proliferation assays, the proinflammatory DCs from B10.A mice induced anergy of naïve T cells, whereas the mixed DC subsets from resistant mice induced the concomitant proliferation of effector and regulatory T cells (Tregs). Equivalent results were observed during pulmonary infection. The susceptible mice displayed preferential expansion of proinflammatory myeloid DCs, resulting in impaired proliferation of effector T cells. Conversely, the resistant mice developed myeloid and plasmacytoid DCs that efficiently expanded gamma interferon-, IL-4-, and IL-17-positive effector T cells associated with increased development of Tregs. Our work highlights the deleterious effect of excessive innate proinflammatory reactions and provides new evidence for the importance of immunomodulation during pulmonary paracoccidioidomycosis. PMID:23340311

  4. Cell fate determination in the vertebrate retina.

    PubMed Central

    Cepko, C L; Austin, C P; Yang, X; Alexiades, M; Ezzeddine, D

    1996-01-01

    In the vertebrate central nervous system, the retina has been a useful model for studies of cell fate determination. Recent results from studies conducted in vitro and in vivo suggest a model of retinal development in which both the progenitor cells and the environment change over time. The model is based upon the notion that the mitotic cells within the retina change in their response properties, or "competence", during development. These changes presage the ordered appearance of distinct cell types during development and appear to be necessary for the production of the distinct cell types. As the response properties of the cells change, so too do the environmental signals that the cells encounter. Together, intrinsic properties and extrinsic cues direct the choice of cell fate. Images Fig. 2 Fig. 5 PMID:8570600

  5. Sex-determined susceptibility and differential MUC2 mRNA expression during the course of murine intestinal eimeriosis.

    PubMed

    Dkhil, Mohamed A

    2015-01-01

    Parasitic diseases differ in prevalence, course, and severity between males and females. The study was designed to compare males with females for the susceptibility to Eimeria papillata infection as well as the expression of the mucin gene, MUC2. Oocysts output was detected to be more in the feces of male mice (3.5 × 10(4) ± 4000 oocysts/g feces) than in females (2 × 10(4) ± 2000 oocysts/g feces). In addition, infected males showed a significant higher number of meronts, gamonts, and developing oocysts compared to infected female mice. Moreover, E. papillata induced a marked goblet cell hypoplasia where the jejuna of infected male mice contained lower numbers of goblet cells per ten villus-crypt units compared to infected females. Also, the expression of MUC2 mRNA is found to be more expressed in infected females than males. In addition, testosterone, nitric oxide, and inducible nitric oxide synthase activities were found to be higher in infected male mice than in infected females. In general, male Swiss albino mice have been shown to be relatively more susceptible to infection with E. papilaata when compared with female mice.

  6. Effect on quarter milk somatic cell count and antimicrobial susceptibility of Staphylococcus rostri causing intramammary infection in dairy water buffaloes.

    PubMed

    Locatelli, C; Piepers, S; De Vliegher, S; Barberio, A; Supré, K; Scaccabarozzi, L; Pisoni, G; Bronzo, V; Haesebrouck, F; Moroni, P

    2013-06-01

    In many parts of the world, coagulase-negative staphylococci (CNS) are the predominant cause of intramammary infections (IMI) in dairy cows and in water buffaloes, as well. A longitudinal field study was carried out on one well-managed dairy water buffalo herd to determine the prevalence and distribution of CNS and a recently described CNS-species, Staphylococcus rostri, in milk samples to explore its relevance for buffaloes' udder health throughout lactation, and to gain insight into the susceptibility of the latter species toward commonly used antimicrobials. Twice weekly quarter milk samples from a cohort of 11 lactating water buffaloes were collected over an 8-mo period. The CNS (n=109; 76.2% of all culture-positive samples) were the predominant pathogens causing IMI, followed by Corynebacterium bovis (n=11; 7.6%) and Streptococcus spp. (n=9; 6.2%) other than Stretococcus uberis (n=2; 1.4%). Thirty-seven hemolytic staphylococci suspected to be Staphylococcus aureus were further differentiated using transfer DNA-intergenic spacer-PCR and rpoB-gene sequencing because they were coagulase-negative. Thirty-three of those isolates were identified as Staph. rostri, whereas 2 others were identified as Staphylococcus epidermidis. None of the Staph. rostri isolates displayed resistance to the antimicrobial agents tested. Mean quarter milk somatic cell count (qSCC) of all samples collected throughout lactation was 20,970 cells/mL. The qSCC at sampling of quarters infected with Staph. rostri (34,466 cells/mL) and CNS other than Staph. rostri (34,813 cells/mL) were significantly higher than the qSCC of noninfected quarters (20,287 cells/mL), yet not significantly different from each other. These findings provide novel insight into the prevalence and distribution, antimicrobial susceptibility, and relevance of Staph. rostri compared with other CNS species causing IMI in water buffaloes. Further studies are needed to pinpoint the relevance, niches, and transmission routes of

  7. MHC-I expression renders catecholaminergic neurons susceptible to T-cell-mediated degeneration

    PubMed Central

    Cebrián, Carolina; Zucca, Fabio A.; Mauri, Pierluigi; Steinbeck, Julius A.; Studer, Lorenz; Scherzer, Clemens R.; Kanter, Ellen; Budhu, Sadna; Mandelbaum, Jonathan; Vonsattel, Jean P.; Zecca, Luigi; Loike, John D.; Sulzer, David

    2014-01-01

    Subsets of rodent neurons are reported to express major histocompatibilty complex class I (MHC-I), but such expression has not been reported in normal adult human neurons. Here we provide evidence from immunolabel, RNA expression, and mass spectrometry analysis of postmortem samples that human catecholaminergic substantia nigra and locus coeruleus neurons express MHC-I, and that this molecule is inducible in human stem cell derived dopamine (DA) neurons. Catecholamine murine cultured neurons are more responsive to induction of MHC-I by gamma-interferon than other neuronal populations. Neuronal MHC-I is also induced by factors released from microglia activated by neuromelanin or alpha-synuclein, or high cytosolic DA and/or oxidative stress. DA neurons internalize foreign ovalbumin and display antigen derived from this protein by MHC-I, which triggers DA neuronal death in the presence of appropriate cytotoxic T-cells. Thus, neuronal MHC-I can trigger antigenic response, and catecholamine neurons may be particularly susceptible to T cell-mediated cytotoxic attack. PMID:24736453

  8. Evidence for strain-specific differences in benzene toxicity as a function of host target cell susceptibility.

    PubMed

    Neun, D J; Penn, A; Snyder, C A

    1992-01-01

    It has long been recognized that benzene exposure produces disparate toxic responses among different species or even among different strains within the same species. There is ample evidence that species- or strain-dependent differences in metabolic activity correlate with the disparate responses to benzene. However, bone marrow cells (the putative targets of benzene toxicity) may also exhibit species- or strain-dependent differences in susceptibility to the toxic effects of benzene. To investigate this hypothesis, two sets of companion experiments were performed. First, two strains of mice, Swiss Webster (SW) and C57B1/6J (C57), were exposed to 300 ppm benzene via inhalation and the effects of the exposures were determined on bone marrow cellularity and the development of bone marrow CFU-e (Colony Forming Unit-erythroid, an early red cell progenitor). Second, bone marrow cells from the same strains were exposed in vitro to five known benzene metabolites (1,4 benzoquinone, catechol, hydroquinone, muconic acid, and phenol) individually and in binary combinations. Benzene exposure, in vivo, reduced bone marrow cellularity and the development of CFU-e in both strains; however, reductions in both these endpoints were more severe in the SW strain. When bone marrow cells from the two strains were exposed in vitro to the five benzene metabolites individually, benzoquinone, hydroquinone, and catechol reduced the numbers of CFU-e in both strains in dose-dependent responses, phenol weakly reduced the numbers of the C57 CFU-e only and in a non-dose-dependent manner, and muconic acid was without effect on cells from either strain.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Adiponectin is critical in determining susceptibility to depressive behaviors and has antidepressant-like activity.

    PubMed

    Liu, Jing; Guo, Ming; Zhang, Di; Cheng, Shao-Ying; Liu, Meilian; Ding, Jun; Scherer, Philipp E; Liu, Feng; Lu, Xin-Yun

    2012-07-24

    Depression is a debilitating mental illness and is often comorbid with metabolic disorders such as type 2 diabetes. Adiponectin is an adipocyte-derived hormone with antidiabetic and insulin-sensitizing properties. Here we show that adiponectin levels in plasma are reduced in a chronic social-defeat stress model of depression, which correlates with decreased social interaction time. A reduction in adiponectin levels caused by haploinsufficiency results in increased susceptibility to social aversion, "anhedonia," and learned helplessness and causes impaired glucocorticoid-mediated negative feedback on the hypothalamic-pituitary-adrenal (HPA) axis. Intracerebroventricular (i.c.v.) injection of an adiponectin neutralizing antibody precipitates stress-induced depressive-like behavior. Conversely, i.c.v. administration of exogenous adiponectin produces antidepressant-like behavioral effects in normal-weight mice and in diet-induced obese diabetic mice. Taken together, these results suggest a critical role of adiponectin in depressive-like behaviors and point to a potential innovative therapeutic approach for depressive disorders.

  10. Adiponectin is critical in determining susceptibility to depressive behaviors and has antidepressant-like activity

    PubMed Central

    Liu, Jing; Guo, Ming; Zhang, Di; Cheng, Shao-Ying; Liu, Meilian; Ding, Jun; Scherer, Philipp E.; Liu, Feng; Lu, Xin-Yun

    2012-01-01

    Depression is a debilitating mental illness and is often comorbid with metabolic disorders such as type 2 diabetes. Adiponectin is an adipocyte–derived hormone with antidiabetic and insulin-sensitizing properties. Here we show that adiponectin levels in plasma are reduced in a chronic social-defeat stress model of depression, which correlates with decreased social interaction time. A reduction in adiponectin levels caused by haploinsufficiency results in increased susceptibility to social aversion, “anhedonia,” and learned helplessness and causes impaired glucocorticoid-mediated negative feedback on the hypothalamic–pituitary–adrenal (HPA) axis. Intracerebroventricular (i.c.v.) injection of an adiponectin neutralizing antibody precipitates stress-induced depressive-like behavior. Conversely, i.c.v. administration of exogenous adiponectin produces antidepressant-like behavioral effects in normal-weight mice and in diet-induced obese diabetic mice. Taken together, these results suggest a critical role of adiponectin in depressive-like behaviors and point to a potential innovative therapeutic approach for depressive disorders. PMID:22778410

  11. Method of determining the number of cells in cell culture

    SciTech Connect

    Connolly, D.T.

    1990-06-12

    This patent describes a color-sensitivity method for determining the number of cells in in vitro cell culture at a sensitivity as low as about 100 or about 500 cells. It comprises lysing the cells and incubating the lysate with p-nitrophenyl phosphate at acid pH for a predetermined period of time at a temperature of from about 35{degrees} to about 38{degrees}C. and then measuring the color development at 400 to 420 nanometers and correlating the color development with cell number by comparing with a control standard of known cell number.

  12. Influence of MDM2 polymorphisms on squamous cell carcinoma susceptibility: a meta-analysis

    PubMed Central

    Yu, Huanxin; Li, Haiyan; Zhang, Jinling; Liu, Gang

    2016-01-01

    Purpose Controversial associations between single-nucleotide polymorphisms (rs2279744, rs937283, rs3730485) of the MDM2 gene and the etiology of squamous cell carcinomas (SCCs) have been reported. This merits further comprehensive assessment. Materials and methods We systematically reviewed the available data and conducted an updated meta-analysis to evaluate the genetic effect of MDM2 polymorphisms in SCC susceptibility, using Stata/SE 12.0 software. Results After screening, 7,987 SCC cases and 12,954 controls from 26 eligible case–control studies were enrolled. Overall, compared with the control group, a significantly increased SCC risk was observed for the MDM2 rs2279744 polymorphism in the Asian population (test of association: odds ratio [OR] 1.12, P=0.027 for G vs T; OR 1.26, P=0.016 for GG vs TT; OR 1.25, P<0.001 for GG vs TT + TG; and OR 1.08, P=0.023 for carrier G vs T). In subgroup analysis by SCC type, a similarly increased esophageal SCC risk was detected (OR 1.19, P<0.001 for G vs T; OR 1.46, P<0.001 for GG vs TT; and OR 1.48, P=0.005 for GG vs TT + TG). Furthermore, MDM2–TP53 double mutation was statistically associated with increased SCC susceptibility overall (OR 1.52, P=0.001), especially in the Asian population (OR 1.49, P=0.022). However, no significant difference between the control and case groups was obtained for MDM2 rs937283 or rs3730485 under any genetic model (all P>0.05). Conclusion Our results highlight a positive association between the GG genotype of MDM2 rs2279744 polymorphism and an increased risk of esophageal SCC in the Asian population, which needs to be clarified by more large-scale studies. PMID:27785069

  13. A novel method to measure HLA-DM-susceptibility of peptides bound to MHC class II molecules based on peptide binding competition assay and differential IC(50) determination.

    PubMed

    Yin, Liusong; Stern, Lawrence J

    2014-04-01

    HLA-DM (DM) functions as a peptide editor that mediates the exchange of peptides loaded onto MHCII molecules by accelerating peptide dissociation and association kinetics. The relative DM-susceptibility of peptides bound to MHCII molecules correlates with antigen presentation and immunodominance hierarchy, and measurement of DM-susceptibility has been a key effort in this field. Current assays of DM-susceptibility, based on differential peptide dissociation rates measured for individually labeled peptides over a long time base, are difficult and cumbersome. Here, we present a novel method to measure DM-susceptibility based on peptide binding competition assays performed in the presence and absence of DM, reported as a delta-IC(50) (change in 50% inhibition concentration) value. We simulated binding competition reactions of peptides with various intrinsic and DM-catalyzed kinetic parameters and found that under a wide range of conditions the delta-IC(50) value is highly correlated with DM-susceptibility as measured in off-rate assay. We confirmed experimentally that DM-susceptibility measured by delta-IC(50) is comparable to that measured by traditional off-rate assay for peptides with known DM-susceptibility hierarchy. The major advantage of this method is that it allows simple, fast and high throughput measurement of DM-susceptibility for a large set of unlabeled peptides in studies of the mechanism of DM action and for identification of CD4+ T cell epitopes.

  14. Antifungal Susceptibility in Serum and Virulence Determinants of Candida Bloodstream Isolates from Hong Kong

    PubMed Central

    Seneviratne, Chaminda J.; Rajan, Suhasini; Wong, Sarah S. W.; Tsang, Dominic N. C.; Lai, Christopher K. C.; Samaranayake, Lakshman P.; Jin, Lijian

    2016-01-01

    Candida bloodstream infections (CBI) are one of the most common nosocomial infections globally, and they account for a high mortality rate. The increasing global prevalence of drug-resistant Candida strains has also been posing a challenge to clinicians. In this study, we comprehensively evaluated the biofilm formation and production of hemolysin and proteinase of 63 CBI isolates derived from a hospital setting in Hong Kong as well as their antifungal susceptibility both in the presence and in the absence of human serum, using standard methodology. Candida albicans was the predominant species among the 63 CBI isolates collected, and non-albicans Candida species accounted for approximately one third of the isolates (36.5%). Of them, Candida tropicalis was the most common non-albicans Candida species. A high proportion (31.7%) of the CBI isolates (40% of C. albicans isolates, 10% of C. tropicalis isolates, 11% of C. parapsilosis isolates, and 100% of C. glabrata isolates) were found to be resistant to fluconazole. One of the isolates (C. tropicalis) was resistant to amphotericin B. A rising prevalence of drug-resistance CBI isolates in Hong Kong was observed with reference to a previous study. Notably, all non-albicans Candida species, showed increased hemolytic activity relative to C. albicans, whilst C. albicans, C. tropicalis, and C. parapsilosis exhibited proteinase activities. Majority of the isolates were capable of forming mature biofilms. Interestingly, the presence of serum distorted the yeast sensitivity to fluconazole, but not amphotericin B. Taken together, our findings demonstrate that CBI isolates of Candida have the potential to express to varying extent their virulence attributes (e.g., biofilm formation, hemolysin production, and proteinase activity) and these, together with perturbations in their antifungal sensitivity in the presence of serum, may contribute to treatment complication in candidemia. The effect of serum on antifungal activity

  15. Up-regulation of TRPV1 in mononuclear cells of end-stage kidney disease patients increases susceptibility to N-arachidonoyl-dopamine (NADA)-induced cell death.

    PubMed

    Saunders, Cassandra I; Fassett, Robert G; Geraghty, Dominic P

    2009-10-01

    Transient receptor potential vanilloid (TRPV) 1 channels function as sensors for a variety of noxious and inflammatory signals, including capsaicin, heat and protons, and are up-regulated under inflammatory conditions. As end-stage kidney disease (ESKD) is associated with chronic inflammation, impaired immunity and depressed lymphocyte numbers, we sought to determine whether altered TRPV1 (and related TRPV2) expression in immune cells might be a contributing factor. TRPV1 and TRPV2 mRNA expression in peripheral blood mononuclear cells (PBMC) was similar in controls and ESKD patients by quantitative real-time RT-PCR. However, using immunocytochemistry, TRPV1-immunoreactivity was significantly higher and TRPV2-immunoreactivity was significantly lower in PBMC from ESKD patients compared to controls. The plant-derived TRPV1 agonists, capsaicin and resiniferatoxin (RTX) and the putative endovanilloid/endocannabinoids, N-arachidonoyl-dopamine (NADA) and N-oleoyl-dopamine (OLDA), induced concentration-dependent death of PBMC from healthy donors with a rank order of potency of RTX>NADA>OLDA>capsaicin. TRPV1 (5'-iodoresiniferatoxin) and cannabinoid (CB2; AM630) receptor antagonists blocked the cytotoxic effect of NADA. In subsequent experiments, PBMC from ESKD patients exhibited significantly increased susceptibility to NADA-induced death compared to PBMC from controls. The apparent up-regulation of TRPV1 may be a response to the inflammatory milieu in which PBMC exist in ESKD and may be responsible for the increased susceptibility of these cells to NADA-induced death, providing a possible explanation as to why ESKD patients have reduced lymphocyte counts and impaired immune function. Thus, TRPV1 (and possibly CB2) antagonists may have potential for the treatment of immune dysfunction in ESKD.

  16. Childhood acute lymphoblastic leukemia: genetic determinants of susceptibility and disease outcome.

    PubMed

    Krajinovic, M; Labuda, D; Sinnett, D

    2001-01-01

    The origin of acute lymphoblastic leukemia (ALL), the most common pediatric cancer, can be explained by a combination of genetic factors and environmental exposure. The environmental toxicants to which an individual is exposed are biotransformed and eliminated from the body after metabolic conversion mediated by Phase I and Phase II xenobiotic-metabolizing enzymes. Phase I enzymes catalyze hydroxylation, reduction and oxidation reactions of xenobiotics (carcinogens/drugs), often converting them into more active or toxic compounds. Phase II enzymes catalyze conjugation reactions (glucuronidation, acetylation, methylation), thereby converting the metabolites into non-reactive, water-soluble products that are eliminated from the organism. The genetic polymorphism underlying the variation in enzyme activity can modify susceptibility to diverse adult cancers, probably by influencing the activation and removal of toxicants or drugs. Here we present an overview of the role of genetic variants of certain Phase I and Phase II enzymes in the development of childhood ALL, a good model for such studies because of its short latency period. The genetic contribution to the development of ALL is examined by association studies that analyze the loci of Phase I enzymes (cytochrome P-450, myeloperoxidase) and Phase II enzymes (quinone-oxidoreductase, glutathione-S-transferase, N-acetyltransferase). The loci of the enzyme variants CYPlA1, CYP2E1, NQO1, GSTM1, GSTP1, NAT2 are associated with disease development, and evidence of gene-gene interactions has emerged as well. Despite the improvements in treatment, resistant cases of ALL remain a leading cause of cancer-related death in children. Although the underlying mechanism of drug resistance is not well understood, differences in the capacity of ALL patients to process drugs and environmental carcinogens could play a role by modifying the risk of recurrent malignancy, as well as the response to therapy. Therefore, polymorphic genes

  17. Plant cell wall dynamics and wall-related susceptibility in plant-pathogen interactions.

    PubMed

    Bellincampi, Daniela; Cervone, Felice; Lionetti, Vincenzo

    2014-01-01

    The cell wall is a dynamic structure that often determines the outcome of the interactions between plants and pathogens. It is a barrier that pathogens need to breach to colonize the plant tissue. While fungal necrotrophs extensively destroy the integrity of the cell wall through the combined action of degrading enzymes, biotrophic fungi require a more localized and controlled degradation of the cell wall in order to keep the host cells alive and utilize their feeding structures. Also bacteria and nematodes need to degrade the plant cell wall at a certain stage of their infection process, to obtain nutrients for their growth. Plants have developed a system for sensing pathogens and monitoring the cell wall integrity, upon which they activate defense responses that lead to a dynamic cell wall remodeling required to prevent the disease. Pathogens, on the other hand, may exploit the host cell wall metabolism to support the infection. We review here the strategies utilized by both plants and pathogens to prevail in the cell wall battleground.

  18. Plant cell wall dynamics and wall-related susceptibility in plant–pathogen interactions

    PubMed Central

    Bellincampi, Daniela; Cervone, Felice; Lionetti, Vincenzo

    2014-01-01

    The cell wall is a dynamic structure that often determines the outcome of the interactions between plants and pathogens. It is a barrier that pathogens need to breach to colonize the plant tissue. While fungal necrotrophs extensively destroy the integrity of the cell wall through the combined action of degrading enzymes, biotrophic fungi require a more localized and controlled degradation of the cell wall in order to keep the host cells alive and utilize their feeding structures. Also bacteria and nematodes need to degrade the plant cell wall at a certain stage of their infection process, to obtain nutrients for their growth. Plants have developed a system for sensing pathogens and monitoring the cell wall integrity, upon which they activate defense responses that lead to a dynamic cell wall remodeling required to prevent the disease. Pathogens, on the other hand, may exploit the host cell wall metabolism to support the infection. We review here the strategies utilized by both plants and pathogens to prevail in the cell wall battleground. PMID:24904623

  19. Conjugation between quinolone-susceptible bacteria can generate mutations in the quinolone resistance-determining region, inducing quinolone resistance.

    PubMed

    Pitondo-Silva, André; Martins, Vinicius Vicente; Silva, Carolina Fávero da; Stehling, Eliana Guedes

    2015-02-01

    Quinolones are an important group of antibacterial agents that can inhibit DNA gyrase and topoisomerase IV activity. DNA gyrase is responsible for maintaining bacteria in a negatively supercoiled state, being composed of subunits A and B. Topoisomerase IV is a homologue of DNA gyrase and consists of two subunits codified by the parC and parE genes. Mutations in gyrA and gyrB of DNA gyrase may confer resistance to quinolones, and the majority of resistant strains show mutations between positions 67 and 106 of gyrA, a region denoted the quinolone resistance-determining region (QRDR). The most frequent substitutions occur at positions 83 and 87, but little is known about the mechanisms promoting appearance of mutations in the QRDR. The present study proposes that some mutations in the QRDR could be generated as a result of the natural mechanism of conjugation between bacteria in their natural habitat. This event was observed following conjugation in vitro of two different isolates of quinolone-susceptible Pseudomonas aeruginosa, which transferred plasmids of different molecular weights to a recipient strain of Escherichia coli (HB101), also quinolone-susceptible, generating two different transconjugants that presented mutations in DNA gyrase and acquisition of resistance to all quinolones tested. PMID:25262036

  20. Cytokine production in arthritis susceptible and resistant rats: a study with arthritogenic and non-arthritogenic Lactobacillus cell walls.

    PubMed

    Simelyte, E; Isomäki, P; Rimpiläinen, M; Zhang, X; Toivanen, P

    2001-02-01

    The basis of the different susceptibility to bacterial cell wall-induced arthritis between Lewis and Fischer rats is unclear. Likewise, it is not known why cell walls of some species of Lactobacillus are arthritogenic and those of others are not. With these two questions in mind, we investigated the role of anti-inflammatory (interleukin (IL)-10, IL-4) and proinflammatory (tumour necrosis factor (TNF)-alpha, IL-1 beta) cytokines in Lewis and Fischer rats injected intraperitoneally with cell walls from arthritogenic or nonarthritogenic species of Lactobacillus. Cytokine levels in the serum and in vitro production by peritoneal macrophages and splenocytes were studied. The results obtained indicate that the differences in the production of IL-10, IL-4, TNF-alpha or IL-1 beta do not explain the difference in the arthritis susceptibility between Lewis and Fischer rats. Likewise, the arthritogenicity of different Lactobacillus cell walls appears not to be dependent on their capacity to stimulate cytokine production. PMID:11169216

  1. Respiratory protease/antiprotease balance determines susceptibility to viral infection and can be modified by nutritional antioxidants

    PubMed Central

    Meyer, Megan

    2015-01-01

    The respiratory epithelium functions as a central orchestrator to initiate and organize responses to inhaled stimuli. Proteases and antiproteases are secreted from the respiratory epithelium and are involved in respiratory homeostasis. Modifications to the protease/antiprotease balance can lead to the development of lung diseases such as emphysema or chronic obstructive pulmonary disease. Furthermore, altered protease/antiprotease balance, in favor for increased protease activity, is associated with increased susceptibility to respiratory viral infections such as influenza virus. However, nutritional antioxidants induce antiprotease expression/secretion and decrease protease expression/activity, to protect against viral infection. As such, this review will elucidate the impact of this balance in the context of respiratory viral infection and lung disease, to further highlight the role epithelial cell-derived proteases and antiproteases contribute to respiratory immune function. Furthermore, this review will offer the use of nutritional antioxidants as possible therapeutics to boost respiratory mucosal responses and/or protect against infection. PMID:25888573

  2. Respiratory protease/antiprotease balance determines susceptibility to viral infection and can be modified by nutritional antioxidants.

    PubMed

    Meyer, Megan; Jaspers, Ilona

    2015-06-15

    The respiratory epithelium functions as a central orchestrator to initiate and organize responses to inhaled stimuli. Proteases and antiproteases are secreted from the respiratory epithelium and are involved in respiratory homeostasis. Modifications to the protease/antiprotease balance can lead to the development of lung diseases such as emphysema or chronic obstructive pulmonary disease. Furthermore, altered protease/antiprotease balance, in favor for increased protease activity, is associated with increased susceptibility to respiratory viral infections such as influenza virus. However, nutritional antioxidants induce antiprotease expression/secretion and decrease protease expression/activity, to protect against viral infection. As such, this review will elucidate the impact of this balance in the context of respiratory viral infection and lung disease, to further highlight the role epithelial cell-derived proteases and antiproteases contribute to respiratory immune function. Furthermore, this review will offer the use of nutritional antioxidants as possible therapeutics to boost respiratory mucosal responses and/or protect against infection.

  3. Determination of hot-spot susceptibility of multistring photovoltaic modules in a central-station application

    NASA Technical Reports Server (NTRS)

    Gonzalez, C. C.; Weaver, R. W.; Ross, R. G., Jr.; Spencer, R.; Arnett, J. C.

    1984-01-01

    Part of the effort of the Jet Propulsion Laboratory (JPL) Flat-Plate Solar Array Project (FSA) includes a program to improve module and array reliability. A collaborative activity with industry dealing with the problem of hot-spot heating due to the shadowing of photovoltaic cells in modules and arrays containing several paralleled cell strings is described. The use of multiparallel strings in large central-station arrays introduces the likelihood of unequal current sharing and increased heating levels. Test results that relate power dissipated, current imbalance, cross-strapping frequency, and shadow configuration to hot-spot heating levels are presented. Recommendations for circuit design configurations appropriate to central-station applications that reduce the risk of hot-spot problems are offered. Guidelines are provided for developing hot-spot tests for arrays when current imbalance is a threat.

  4. Investigation of the influence of different physico-chemical parameters upon the susceptibility of planktonic and adherent Escherichia coli cells to beta-lactams and quinolones.

    PubMed

    Drăcea, O; Iordache, C; Bucur, M; Bleotu, C; Banu, O; Ungureanu, C; Cristea, D; Lixandru, M S; Larion, Cristina; Necula, G; Lazăr, V; Chifiriuc, M C

    2009-01-01

    The purpose of this study was to evaluate the influence of different physico-chemical parameters on Escherichia coli susceptibility to ceftriaxone (CRO), cefotaxime (CTX), imipenem (IMP), and nalidixic acid (as marker for resistance by impermeability). The influence of chemical composition of culture medium was evaluated by the comparative assessment of inhibition growth diameters on different solid media: Mueller Hinton Medium (MH), Plate Count Agar Medium (PCA), MacConkey Medium (MC) and Eosin Methylen Blue Medium (EMB). In order to evaluate the differences in antibiotic susceptibility between the biofilm embedded and planktonic cells, an original, simple experimental model was used, by including the bacterial cells in an agar layer, mimicking the biofilm matrix. Our results demonstrated that the inhibition diameter zone was much larger on PCA, EMB and MC than on MH, considered as general standard medium for the antibiosusceptibility testings (CLSI). When bacterial cells were included in the agar matrix, the growth inhibition diameters obtained for different beta-lactams proved to be different of planktonic cells, i.e.: for CTX, a narrow inhibition diameter was obtained, demonstrating the low efficiency of this antibiotic in the treatment of biofilm associated infections, whereas the CRO proved the same efficiency against planktonic as well as to agar embedded bacteria. The different susceptibility results obtained for the cells embedded in the agar matrix by an adapted disk diffusion method are pleading for the necessity to assess new adapted standard methods and specific parameters in the purpose to determine the antibiotic resistance of bacterial cells isolated from biofilm associated infections.

  5. Determination of methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteria in blood by capillary zone electrophoresis.

    PubMed

    Horká, Marie; Tesařová, Marie; Karásek, Pavel; Růžička, Filip; Holá, Veronika; Sittová, Martina; Roth, Michal

    2015-04-01

    Serious bloodstream infections are a significant complication in critically ill patients. The treatment of these infections has become more difficult because of the increasing prevalence of multiresistant strains, especially methicillin-resistant Staphylococcus aureus (MRSA). Rapid differentiation of low number of MRSA from methicillin-susceptible S. aureus (MSSA) cells (10(1)-10(2) cells mL(-1)) in blood is necessary for fast effective antibiotic therapy. Currently, three groups of techniques, phenotyping, genotyping, and mass spectrometry, are used for MRSA and MSSA strains differentiation. Most of these techniques are time-consuming. PCR and other molecular techniques allow the detection and differentiation between MSSA and MRSA directly from blood cultures. These methods alone are rapid and they have good reproducibility and repeatability. Potential disadvantages of the genotyping methods include their discrimination ability, technical complexity, financial costs, and difficult interpretation of the results. Recently, capillary electrophoresis (CZE) was successfully used to differentiate between the agar-cultivated MRSA and MSSA strains in fused silica capillaries etched with supercritical water and modified with (3-glycidyloxypropyl)trimethoxysilane. The possible use of CZE as a fast and low-cost method for distinguishing between the blood-incubated MRSA or MSSA cells has been tested in this manuscript. Our goal was to test low amounts of bacteria (∼10(2) cell mL(-1)) similar to those in clinical samples. The migration times of the purified blood-incubated cells and the agar-cultivated cells were different from each other. However, their isoelectric point was the same for all strains.

  6. [The refractory susceptibility in determination of sulfur in organic drugs using the Schoniger method].

    PubMed

    Listov, S A; Arsamastsev, A P; Gamanina, G J

    1988-10-01

    Conventional methods of the determination of sulphur in organic drugs were studied (Schöniger method) and new methods developed. Emphasis was put on the disturbance effect of 5 elements, which often occur in the structure of organic drug compounds. It could be shown, that the disturbance effect of nitrogen and chlorine was overcome by the use of carbamide and ammonium carbonate in the absorption solution. The effect of fluorine was abolished by the use of boric acid and a special two-spiral technique of the analysis. Based on these studies concrete recommendations for the Schöniger method of sulphur determination in drugs of various composition of elements were given. PMID:3212013

  7. [The refractory susceptibility in determination of sulfur in organic drugs using the Schoniger method].

    PubMed

    Listov, S A; Arsamastsev, A P; Gamanina, G J

    1988-10-01

    Conventional methods of the determination of sulphur in organic drugs were studied (Schöniger method) and new methods developed. Emphasis was put on the disturbance effect of 5 elements, which often occur in the structure of organic drug compounds. It could be shown, that the disturbance effect of nitrogen and chlorine was overcome by the use of carbamide and ammonium carbonate in the absorption solution. The effect of fluorine was abolished by the use of boric acid and a special two-spiral technique of the analysis. Based on these studies concrete recommendations for the Schöniger method of sulphur determination in drugs of various composition of elements were given.

  8. High vancomycin MICs within the susceptible range in Staphylococcus aureus bacteraemia isolates are associated with increased cell wall thickness and reduced intracellular killing by human phagocytes.

    PubMed

    Falcón, Rocío; Martínez, Alba; Albert, Eliseo; Madrid, Silvia; Oltra, Rosa; Giménez, Estela; Soriano, Mario; Vinuesa, Víctor; Gozalbo, Daniel; Gil, María Luisa; Navarro, David

    2016-05-01

    Vancomycin minimum inhibitory concentrations (MICs) at the upper end of the susceptible range for Staphylococcus aureus have been associated with poor clinical outcomes of bloodstream infections. We tested the hypothesis that high vancomycin MICs in S. aureus bacteraemia isolates are associated with increased cell wall thickness and suboptimal bacterial internalisation or lysis by human phagocytes. In total, 95 isolates were evaluated. Original vancomycin MICs were determined by Etest. The susceptibility of S. aureus isolates to killing by phagocytes was assessed in a human whole blood assay. Internalisation of bacterial cells by phagocytes was investigated by flow cytometry. Cell wall thickness was evaluated by transmission electron microscopy. Genotypic analysis of S. aureus isolates was performed using a DNA microarray system. Vancomycin MICs were significantly higher (P=0.006) in isolates that were killed suboptimally (killing index <60%) compared with those killed efficiently (killing index >70%) and tended to correlate inversely (P=0.08) with the killing indices. Isolates in both killing groups were internalised by human neutrophils and monocytes with comparable efficiency. The cell wall was significantly thicker (P=0.03) in isolates in the low killing group. No genotypic differences were found between the isolates in both killing groups. In summary, high vancomycin MICs in S. aureus bacteraemia isolates were associated with increased cell wall thickness and reduced intracellular killing by phagocytes. PMID:27056298

  9. In Vitro Amphotericin B Susceptibility of Malassezia pachydermatis Determined by the CLSI Broth Microdilution Method and Etest Using Lipid-Enriched Media

    PubMed Central

    Álvarez-Pérez, Sergio; Peláez, Teresa; Cutuli, Maite; García, Marta E.

    2014-01-01

    We determined the in vitro amphotericin B susceptibility of 60 Malassezia pachydermatis isolates by the CLSI broth microdilution method and the Etest using lipid-enriched media. All isolates were susceptible at MICs of ≤1 μg/ml, confirming the high activity of amphotericin B against this yeast species. Overall, the essential agreement between the tested methods was high (80% and 96.7% after 48 h and 72 h, respectively), and all discrepancies were regarded as nonsubstantial. PMID:24752258

  10. Determination of surface complex nonlinear optical susceptibilities and molecular orientational distribution functions using resonant surface second-harmonic generation

    NASA Astrophysics Data System (ADS)

    Park, Byoungchoo; Yoo, Jeong-Geun; Sakai, Takahiro; Hoshi, Hajime; Ishikawa, Ken; Takezoe, Hideo

    1998-10-01

    Using the resonant optical surface second-harmonic generation (SHG), we have determined the relative values of the complex nonlinear optical (NLO) components (χzzz, χzxx, and χxxz) at isotropic interfaces (C∞v) of a polymer with SHG active side chains. The introduced configuration of the SHG experiment was a polarizer-rotating quarter wave plate-sample-analyzer. It was shown that this configuration gives information on complex NLO coefficients without using the Kleinmann symmetry. For the experiments, we measured resonant surface SHG from the air-polymer and the substrate-polymer interfaces of a thick polymer film. By theoretically fitting the SHG data, we unambiguously determined the nonlinear susceptibility components at the both interfaces of the polymer film. Moreover, unbiased molecular orientational distribution functions (ODFs) at both interfaces were also determined using the modified maximum entropy method. The obtained ODFs were found to be quite different from the previous ones obtained by assuming the Kleinmann symmetry, indicating the important role of the imaginary part of χ's played when determining ODFs.

  11. Identification of four new susceptibility loci for testicular germ cell tumour

    PubMed Central

    Litchfield, Kevin; Holroyd, Amy; Lloyd, Amy; Broderick, Peter; Nsengimana, Jérémie; Eeles, Rosalind; Easton, Douglas F; Dudakia, Darshna; Bishop, D. Timothy; Reid, Alison; Huddart, Robert A.; Grotmol, Tom; Wiklund, Fredrik; Shipley, Janet; Houlston, Richard S.; Turnbull, Clare

    2015-01-01

    Genome-wide association studies (GWAS) have identified multiple risk loci for testicular germ cell tumour (TGCT), revealing a polygenic model of disease susceptibility strongly influenced by common variation. To identify additional single-nucleotide polymorphisms (SNPs) associated with TGCT, we conducted a multistage GWAS with a combined data set of >25,000 individuals (6,059 cases and 19,094 controls). We identified new risk loci for TGCT at 3q23 (rs11705932, TFDP2, P=1.5 × 10−9), 11q14.1 (rs7107174, GAB2, P=9.7 × 10−11), 16p13.13 (rs4561483, GSPT1, P=1.6 × 10−8) and 16q24.2 (rs55637647, ZFPM1, P=3.4 × 10−9). We additionally present detailed functional analysis of these loci, identifying a statistically significant relationship between rs4561483 risk genotype and increased GSPT1 expression in TGCT patient samples. These findings provide additional support for a polygenic model of TGCT risk and further insight into the biological basis of disease development. PMID:26503584

  12. Testicular germ cell tumor susceptibility associated with the UCK2 locus on chromosome 1q23.

    PubMed

    Schumacher, Fredrick R; Wang, Zhaoming; Skotheim, Rolf I; Koster, Roelof; Chung, Charles C; Hildebrandt, Michelle A T; Kratz, Christian P; Bakken, Anne C; Bishop, D Timothy; Cook, Michael B; Erickson, R Loren; Fosså, Sophie D; Greene, Mark H; Jacobs, Kevin B; Kanetsky, Peter A; Kolonel, Laurence N; Loud, Jennifer T; Korde, Larissa A; Le Marchand, Loic; Lewinger, Juan Pablo; Lothe, Ragnhild A; Pike, Malcolm C; Rahman, Nazneen; Rubertone, Mark V; Schwartz, Stephen M; Siegmund, Kimberly D; Skinner, Eila C; Turnbull, Clare; Van Den Berg, David J; Wu, Xifeng; Yeager, Meredith; Nathanson, Katherine L; Chanock, Stephen J; Cortessis, Victoria K; McGlynn, Katherine A

    2013-07-01

    Genome-wide association studies (GWASs) have identified multiple common genetic variants associated with an increased risk of testicular germ cell tumors (TGCTs). A previous GWAS reported a possible TGCT susceptibility locus on chromosome 1q23 in the UCK2 gene, but failed to reach genome-wide significance following replication. We interrogated this region by conducting a meta-analysis of two independent GWASs including a total of 940 TGCT cases and 1559 controls for 122 single-nucleotide polymorphisms (SNPs) on chromosome 1q23 and followed up the most significant SNPs in an additional 2202 TGCT cases and 2386 controls from four case-control studies. We observed genome-wide significant associations for several UCK2 markers, the most significant of which was for rs3790665 (PCombined = 6.0 × 10(-9)). Additional support is provided from an independent familial study of TGCT where a significant over-transmission for rs3790665 with TGCT risk was observed (PFBAT = 2.3 × 10(-3)). Here, we provide substantial evidence for the association between UCK2 genetic variation and TGCT risk. PMID:23462292

  13. Lgn1, a gene that determines susceptibility to Legionella pneumophila, maps to mouse chromosome 13

    SciTech Connect

    Dietrich, W.F.; Damron, D.M.; Lander, E.S.

    1995-04-10

    The intracellular pathogen Legionella pneumophila is unable to replicate in macrophages derived from most inbred mouse strains. Here, we report the mapping of a gene, called Lgn1, that determines whether mouse macrophages are permissive for the intracellular replication of L. pneumophila. Although Lgn1 has been previously reported to map to mouse chromosome 15, we show here that it actually maps to chromosome 13, between D13Mit128 and D13Mit70. In the absence of any regional candidates for Lgn1, this map position will facilitate positional cloning attempts directed at this gene. 22 refs., 2 figs., 2 tabs.

  14. Use of new T-cell-based cell lines expressing two luciferase reporters for accurately evaluating susceptibility to anti-human immunodeficiency virus type 1 drugs.

    PubMed

    Chiba-Mizutani, Tomoko; Miura, Hideka; Matsuda, Masakazu; Matsuda, Zene; Yokomaku, Yoshiyuki; Miyauchi, Kosuke; Nishizawa, Masako; Yamamoto, Naoki; Sugiura, Wataru

    2007-02-01

    Two new T-cell-based reporter cell lines were established to measure human immunodeficiency virus type 1 (HIV-1) infectivity. One cell line naturally expresses CD4 and CXCR4, making it susceptible to X4-tropic viruses, and the other cell line, in which a CCR5 expression vector was introduced, is susceptible to both X4- and R5-tropic viruses. Reporter cells were constructed by transfecting the human T-cell line HPB-Ma, which demonstrates high susceptibility to HIV-1, with genomes expressing two different luciferase reporters, HIV-1 long terminal repeat-driven firefly luciferase and cytomegalovirus promoter-driven renilla luciferase. Upon HIV infection, the cells expressed firefly luciferase at levels that were highly correlated (r2=0.91 to 0.98) with the production of the capsid antigen p24. The cells also constitutively expressed renilla luciferase, which was used to monitor cell numbers and viability. The reliability of the cell lines for two in vitro applications, drug resistance phenotyping and drug screening, was confirmed. As HIV-1 efficiently replicated in these cells, they could be used for multiple-round replication assays as an alternative method to a single-cycle replication protocol. Coefficients of variation for drug susceptibility evaluated with the cell lines ranged from 17 to 41%. The new cell lines were beneficial for evaluating antiretroviral drug resistance. Firefly luciferase gave a wider dynamic range for evaluating virus infectivity, and the introduction of renilla luciferase improved assay reproducibility. The cell lines were also beneficial for screening new antiretroviral agents, as false inhibition caused by the cytotoxicity of test compounds was easily detected by monitoring renilla luciferase activity.

  15. Arsenic Mediated Disruption of Promyelocytic Leukemia Protein Nuclear Bodies Induces Ganciclovir Susceptibility in Epstein-Barr Positive Epithelial Cells

    PubMed Central

    Sides, Mark D.; Block, Gregory J.; Shan, Bin; Esteves, Kyle C.; Lin, Zhen; Flemington, Erik K.; Lasky, Joseph A.

    2011-01-01

    Promyelocytic leukemia protein nuclear bodies (PML NBs) have been implicated in host immune response to viral infection. PML NBs are targeted for degradation during reactivation of herpes viruses, suggesting that disruption of PML NB function supports this aspect of the viral life cycle. The Epstein-Barr virus (EBV) Latent Membrane Protein 1 (LMP1) has been shown to suppress EBV reactivation. Our finding that LMP1 induces PML NB immunofluorescence intensity led to the hypothesis that LMP1 may modulate PML NBs as a means of maintaining EBV latency. Increased PML protein and morphometric changes in PML NBs were observed in EBV infected alveolar epithelial cells and nasopharyngeal carcinoma cells. Treatment with low dose arsenic trioxide disrupted PML NBs, induced expression of EBV lytic proteins, and conferred ganciclovir susceptibility. This study introduces an effective modality to induce susceptibility to ganciclovir in epithelial cells with implications for the treatment of EBV associated pathologies. PMID:21605886

  16. Arsenic mediated disruption of promyelocytic leukemia protein nuclear bodies induces ganciclovir susceptibility in Epstein-Barr positive epithelial cells.

    PubMed

    Sides, Mark D; Block, Gregory J; Shan, Bin; Esteves, Kyle C; Lin, Zhen; Flemington, Erik K; Lasky, Joseph A

    2011-07-20

    Promyelocytic leukemia protein nuclear bodies (PML NBs) have been implicated in host immune response to viral infection. PML NBs are targeted for degradation during reactivation of herpes viruses, suggesting that disruption of PML NB function supports this aspect of the viral life cycle. The Epstein-Barr virus (EBV) Latent Membrane Protein 1 (LMP1) has been shown to suppress EBV reactivation. Our finding that LMP1 induces PML NB immunofluorescence intensity led to the hypothesis that LMP1 may modulate PML NBs as a means of maintaining EBV latency. Increased PML protein and morphometric changes in PML NBs were observed in EBV infected alveolar epithelial cells and nasopharyngeal carcinoma cells. Treatment with low dose arsenic trioxide disrupted PML NBs, induced expression of EBV lytic proteins, and conferred ganciclovir susceptibility. This study introduces an effective modality to induce susceptibility to ganciclovir in epithelial cells with implications for the treatment of EBV associated pathologies. PMID:21605886

  17. Arsenic mediated disruption of promyelocytic leukemia protein nuclear bodies induces ganciclovir susceptibility in Epstein-Barr positive epithelial cells

    SciTech Connect

    Sides, Mark D.; Block, Gregory J.; Shan, Bin; Esteves, Kyle C.; Lin, Zhen; Flemington, Erik K.; Lasky, Joseph A.

    2011-06-20

    Promyelocytic leukemia protein nuclear bodies (PML NBs) have been implicated in host immune response to viral infection. PML NBs are targeted for degradation during reactivation of herpes viruses, suggesting that disruption of PML NB function supports this aspect of the viral life cycle. The Epstein-Barr virus (EBV) Latent Membrane Protein 1 (LMP1) has been shown to suppress EBV reactivation. Our finding that LMP1 induces PML NB immunofluorescence intensity led to the hypothesis that LMP1 may modulate PML NBs as a means of maintaining EBV latency. Increased PML protein and morphometric changes in PML NBs were observed in EBV infected alveolar epithelial cells and nasopharyngeal carcinoma cells. Treatment with low dose arsenic trioxide disrupted PML NBs, induced expression of EBV lytic proteins, and conferred ganciclovir susceptibility. This study introduces an effective modality to induce susceptibility to ganciclovir in epithelial cells with implications for the treatment of EBV associated pathologies.

  18. Observation of reversible, rapid changes in drug susceptibility of hypoxic tumor cells in a microfluidic device.

    PubMed

    Germain, Todd; Ansari, Megan; Pappas, Dimitri

    2016-09-14

    Hypoxia is a major stimulus for increased drug resistance and for survival of tumor cells. Work from our group and others has shown that hypoxia increases resistance to anti-cancer compounds, radiation, and other damage-pathway cytotoxic agents. In this work we utilize a microfluidic culture system capable of rapid switching of local oxygen concentrations to determine changes in drug resistance in prostate cancer cells. We observed rapid adaptation to hypoxia, with drug resistance to 2 μM staurosporine established within 30 min of hypoxia. Annexin-V/Sytox Green apoptosis assays over 9 h showed 78.0% viability, compared to 84.5% viability in control cells (normoxic cells with no staurosporine). Normoxic cells exposed to the same staurosporine concentration had a viability of 48.6% after 9 h. Hypoxia adaptation was rapid and reversible, with Hypoxic cells treated with 20% oxygen for 30 min responding to staurosporine with 51.6% viability after drug treatment for 9 h. Induction of apoptosis through the receptor-mediated pathway, which bypasses anti-apoptosis mechanisms induced by hypoxia, resulted in 39.4 ± 7% cell viability. The rapid reversibility indicates co-treatment of oxygen with anti-cancer compounds may be a potential therapeutic target. PMID:27566353

  19. A cell line (NTU-MV) established from Maruca vitrata (Lepidoptera: Pyralidae): Characterization, viral susceptibility, and polyhedra production.

    PubMed

    Yeh, Shih-Chia; Lee, Song-Tay; Wu, Chih-Yu; Wang, Chung-Hsiung

    2007-10-01

    Here we describe the establishment of a new cell line, NTU-MV, derived from pupal tissues of an economically important pest, the legume pod borer Maruca vitrata. This cell line contained four major cell types: polymorphic cells, round cells, spindle-shaped cells, and comma cells. The doubling time of MV cells in TNM-FH medium supplemented with 8% FBS at 28 degrees C was 27h. The chromosome numbers of MV cells varied widely from 16 to 268. Compared to other insect cell lines, the MV cell line produced distinct isozyme patterns with esterase, malate dehydrogenase (MDH), and lactate dehydrogenase (LDH). Confirmation that NTU-MV was derived from M. vitrata was demonstrated by showing that the sequence of the internal transcribed spacer regions (ITS) of the MV cells was 98% identical to that of M. vitrata larvae. Two NTU-MV cell strains, NTU-MV1 and NTU-MV56, were selected based on susceptibility to MaviMNPV (M. vitrata multiple nucleopolyhedrovirus). NTU-MV, MV1, and MV56 cells showed a high susceptibility to MaviMNPV and produced high yields of polyhedra (47-50OBs/cell, 4x10(7)-5.96x10(7)OBs/ml) after 2 weeks of MaviMNPV infection. We conclude that the NTU-MV cell line will be a useful tool for studying MaviMNPV as well as for the mass production of MaviMNPV polyhedra for the biocontrol of M. vitrata.

  20. Novel Method Based on Real-Time Cell Analysis for Drug Susceptibility Testing of Herpes Simplex Virus and Human Cytomegalovirus.

    PubMed

    Piret, Jocelyne; Goyette, Nathalie; Boivin, Guy

    2016-08-01

    The plaque reduction assay (PRA) is the gold standard phenotypic method to determine herpes simplex virus (HSV) and human cytomegalovirus (HCMV) susceptibilities to antiviral drugs. However, this assay is subjective and labor intensive. Here, we describe a novel antiviral phenotypic method based on real-time cell analysis (RTCA) that measures electronic impedance over time. The effective drug concentrations that reduced by 50% (EC50s) the cytopathic effects induced by HSV-1 and HCMV were evaluated by both methods. The EC50s of acyclovir and foscarnet against a reference wild-type (WT) HSV-1 strain in Vero cells were, respectively, 0.5 μM and 32.6 μM by PRA and 0.8 μM and 93.6 μM by RTCA. The EC50 ratios for acyclovir against several HSV-1 thymidine kinase (TK) mutants were 101.8×, 73.4×, 28.8×, and 35.4× (PRA) and 18.0×, 52.0×, 5.5×, and 87.8× (RTCA) compared to those for the WT. The EC50 ratios for acyclovir and foscarnet against the HSV-1 TK/DNA polymerase mutant were 182.8× and 9.7× (PRA) and >125.0× and 10.8× (RTCA) compared to the WT. The EC50s of ganciclovir and foscarnet against WT HCMV strain AD169 in fibroblasts were, respectively, 1.6 μM and 27.8 μM by PRA and 5.0 μM and 111.4 μM by RTCA. The EC50 ratios of ganciclovir against the HCMV UL97 mutant were 3.8× (PRA) and 8.2× (RTCA) compared to those for the WT. The EC50 ratios of ganciclovir and foscarnet against the HCMV UL97/DNA polymerase mutant were 17.1× and 12.1× (PRA) and 14.7× and 4.6× (RTCA) compared to those for the WT. RTCA allows objective drug susceptibility testing of HSV and HCMV and could permit high-throughput screening of new antivirals. PMID:27252463

  1. Surface-attached cells, biofilms and biocide susceptibility: implications for hospital cleaning and disinfection.

    PubMed

    Otter, J A; Vickery, K; Walker, J T; deLancey Pulcini, E; Stoodley, P; Goldenberg, S D; Salkeld, J A G; Chewins, J; Yezli, S; Edgeworth, J D

    2015-01-01

    Microbes tend to attach to available surfaces and readily form biofilms, which is problematic in healthcare settings. Biofilms are traditionally associated with wet or damp surfaces such as indwelling medical devices and tubing on medical equipment. However, microbes can survive for extended periods in a desiccated state on dry hospital surfaces, and biofilms have recently been discovered on dry hospital surfaces. Microbes attached to surfaces and in biofilms are less susceptible to biocides, antibiotics and physical stress. Thus, surface attachment and/or biofilm formation may explain how vegetative bacteria can survive on surfaces for weeks to months (or more), interfere with attempts to recover microbes through environmental sampling, and provide a mixed bacterial population for the horizontal transfer of resistance genes. The capacity of existing detergent formulations and disinfectants to disrupt biofilms may have an important and previously unrecognized role in determining their effectiveness in the field, which should be reflected in testing standards. There is a need for further research to elucidate the nature and physiology of microbes on dry hospital surfaces, specifically the prevalence and composition of biofilms. This will inform new approaches to hospital cleaning and disinfection, including novel surfaces that reduce microbial attachment and improve microbial detachment, and methods to augment the activity of biocides against surface-attached microbes such as bacteriophages and antimicrobial peptides. Future strategies to address environmental contamination on hospital surfaces should consider the presence of microbes attached to surfaces, including biofilms.

  2. Differential Susceptibilities to BmNPV Infection of Two Cell Lines Derived from the Same Silkworm Ovarian Tissues

    PubMed Central

    Zhang, Chun-Dong; He, Qian; Dong, Zhan-Qi; Cao, Ming-Ya; Dong, Xiao-Long; Pan, Cai-Xia; Lu, Cheng; Pan, Min-Hui

    2014-01-01

    We previously established and characterized two insect cell lines (BmN-SWU1 and BmN-SWU2) from Bombyx mori ovaries. Here, we examined their differential susceptibilities to Bombyx mori nucleopolyhedrovirus (BmNPV) despite having originated from the same tissue source. BmN-SWU1 cells were susceptible and supported high titers of BmNPV replication, while BmN-SWU2 cells were resistant to BmNPV infection. Subcellular localization analysis demonstrated that very few BmNPV particles could be imported into BmN-SWU2 cells. However, initiation of BmNPV DNA replication but not amplification was detected in BmN-SWU2 cells after transfection with vA4prm-VP39-EGFP bacmid DNA. BmNPV transcription assays showed that late and very late but not early viral genes apparently were blocked in BmNSWU2 cells by unknown mechanisms. Further syncytium formation assays demonstrated that the BmNPV envelope fusion protein GP64 could not mediate BmN-SWU2 host cell-cell membrane fusion. Taken together, these results indicate that these two cell lines represent optimal tools for investigating host-virus interactions and insect antiviral mechanisms. PMID:25221982

  3. Interferon-γ-induced activation of JAK1 and JAK2 suppresses tumor cell susceptibility to NK cells through upregulation of PD-L1 expression

    PubMed Central

    Bellucci, Roberto; Martin, Allison; Bommarito, Davide; Wang, Kathy; Hansen, Steen H; Freeman, Gordon J; Ritz, Jerome

    2015-01-01

    Inhibition of JAK1 or JAK2 in human tumor cells was previously shown to increase susceptibility of these cells to NK cell lysis. In the present study, we examined the cellular mechanisms that mediate this effect in hematopoietic tumor cell lines and primary tumor cells. Incubation of tumor cells with supernatant from activated NK cells or interferon-gamma (IFNγ)-induced activation of pSTAT1 and increased expression of PD-L1 without altering expression of other activating or inhibitory NK cell ligands. These functional effects were blocked by chemical JAK inhibition or shRNAs targeting JAK1, JAK2 or STAT1. Inhibition of IFNγ signaling also prevented the upregulation of PD-L1 and blocking PD-L1 resulted in increased tumor lysis by NK cells. These results show that NK cell activation and secretion of IFNγ results in activation of JAK1, JAK2 and STAT1 in tumor cells, resulting in rapid up-regulation of PD-L1 expression. Increased expression of PD-L1 results in increased resistance to NK cell lysis. Blockade of JAK pathway activation prevents increased PD-L1 expression resulting in increased susceptibility of tumor cells to NK cell activity. These observations suggest that JAK pathway inhibitors as well as PD-1 and PD-L1 antibodies may work synergistically with other immune therapies by preventing IFN-induced inhibition of NK cell-mediated tumor cell lysis. PMID:26155422

  4. Determination of Morphological, Biometric and Biochemical Susceptibilities in Healthy Eurasier Dogs with Suspected Inherited Glaucoma

    PubMed Central

    Goulle, Frédéric; Thomas, Philippe; Isard, Pierre-François; Azoulay, Thierry; Lafarge-Beurlet, Stéphanie; Woods, Mike; Lavillegrand, Sylvie; Ivkovic, Ivana; Neveux, Nathalie; Sahel, José-Alain; Picaud, Serge; Froger, Nicolas

    2014-01-01

    In both humans and dogs, the primary risk factor for glaucoma is high intraocular pressure (IOP), which may be caused by iridocorneal angle (ICA) abnormalities. Oxidative stress has also been implicated in retinal ganglion cell damage associated with glaucoma. A suspected inherited form of glaucoma was recently identified in Eurasier dogs (EDs), a breed for which pedigrees are readily available. Because of difficulties in assessing ICA morphology in dogs with advanced glaucoma, we selected a cohort of apparently healthy dogsfor the investigation of ICA morphological status, IOP and plasma concentrations of oxidative stress biomarkers. We aimed to establish correlations between these factors, to identify predictive markers of glaucoma in this dog breed. A cohort of 28 subjects, volunteered for inclusion by their owners, was selected by veterinary surgeons. These dogs were assigned to four groups: young males, young females (1–3 years old), adult males and adult females (4–8 years old). Ocular examination included ophthalmoscopy, tonometry, gonioscopy, biometry and ultrasound biomicroscopy (UBM), and the evaluation of oxidative stress biomarkers consisting of measurements of plasma glutathione peroxidase (GP) activity and taurine and metabolic precursor (methionine and cysteine) concentrations in plasma. The prevalence of pectinate ligament abnormalities was significantly higher in adult EDs than in young dogs. Moreover, in adult females, high IOP was significantly correlated with a short axial globe length, and a particularly large distance between Schwalbe's line and the anterior lens capsule. GP activity levels were significantly lower in EDs than in a randomized control group of dogs, and plasma taurine concentrations were higher. Hence, ICA abnormalities were associated with weaker antioxidant defenses in EDs, potentially counteracted by higher plasma taurine concentrations. This study suggests that EDs may constitute an appropriate canine model for the

  5. Determination of morphological, biometric and biochemical susceptibilities in healthy Eurasier dogs with suspected inherited glaucoma.

    PubMed

    Boillot, Thomas; Rosolen, Serge G; Dulaurent, Thomas; Goulle, Frédéric; Thomas, Philippe; Isard, Pierre-François; Azoulay, Thierry; Lafarge-Beurlet, Stéphanie; Woods, Mike; Lavillegrand, Sylvie; Ivkovic, Ivana; Neveux, Nathalie; Sahel, José-Alain; Picaud, Serge; Froger, Nicolas

    2014-01-01

    In both humans and dogs, the primary risk factor for glaucoma is high intraocular pressure (IOP), which may be caused by iridocorneal angle (ICA) abnormalities. Oxidative stress has also been implicated in retinal ganglion cell damage associated with glaucoma. A suspected inherited form of glaucoma was recently identified in Eurasier dogs (EDs), a breed for which pedigrees are readily available. Because of difficulties in assessing ICA morphology in dogs with advanced glaucoma, we selected a cohort of apparently healthy dogsfor the investigation of ICA morphological status, IOP and plasma concentrations of oxidative stress biomarkers. We aimed to establish correlations between these factors, to identify predictive markers of glaucoma in this dog breed. A cohort of 28 subjects, volunteered for inclusion by their owners, was selected by veterinary surgeons. These dogs were assigned to four groups: young males, young females (1-3 years old), adult males and adult females (4-8 years old). Ocular examination included ophthalmoscopy, tonometry, gonioscopy, biometry and ultrasound biomicroscopy (UBM), and the evaluation of oxidative stress biomarkers consisting of measurements of plasma glutathione peroxidase (GP) activity and taurine and metabolic precursor (methionine and cysteine) concentrations in plasma. The prevalence of pectinate ligament abnormalities was significantly higher in adult EDs than in young dogs. Moreover, in adult females, high IOP was significantly correlated with a short axial globe length, and a particularly large distance between Schwalbe's line and the anterior lens capsule. GP activity levels were significantly lower in EDs than in a randomized control group of dogs, and plasma taurine concentrations were higher. Hence, ICA abnormalities were associated with weaker antioxidant defenses in EDs, potentially counteracted by higher plasma taurine concentrations. This study suggests that EDs may constitute an appropriate canine model for the

  6. Evaluation of agar dilution and broth microdilution methods to determine the disinfectant susceptibility.

    PubMed

    Wu, Guoyan; Yang, Qianru; Long, Mei; Guo, Lijuan; Li, Bei; Meng, Yue; Zhang, Anyun; Wang, Hongning; Liu, Shuliang; Zou, Likou

    2015-11-01

    A variety of disinfectants have been widely used in veterinary hygiene, food industries and environments, which could induce the development of bacterial resistance to disinfectants. The methods used to investigate antimicrobial effects of disinfectant vary considerably among studies, making comparisons difficult. In this study, agar dilution and broth microdilution methods were used to compare the antimicrobial activities of four quaternary ammonium compounds (QACs) against foodborne and zoonotic pathogens. The potential relationship between the presence of QACs resistance genes and phenotypic resistance to QACs was also investigated. Our results indicated that the minimum inhibitory concentrations (MICs) determined by two methods might be different depended upon different QACs and bacteria applied. Regardless of the testing methods, Klebsiella pneumoniae was more tolerant among Gram-negative strains to four QACs, followed by Salmonella and Escherichia coli. The agreement between MICs obtained by the two methods was good, for benzalkonium chloride (78.15%), didecyldimethylammonium chloride (DDAC) (82.35%), cetylpyridinium chloride (CTPC) (97.48%) and cetyltrimethylammonium bromide (CTAB) (99.16%), respectively. Among all Gram-negative bacteria, 94.55% (n=52) of qacEΔ1-positive strains showed higher MICs (512 mg l(-1)) to CTAB. The qacEΔ1 gene was highly associated (P<0.05) with the high MICs of QACs (⩾512 mg l(-1)). In addition, DDAC remained as the most effective disinfectant against both Gram-positive and Gram-negative bacteria. This is the first study that compared the agar dilution and broth microdilution methods to assess the antimicrobial activity of QACs. The study demonstrated the need to standardize method that would be used in evaluating QACs antimicrobial properties in the future.

  7. T cell and non-T cell compartments can independently determine resistance to Leishmania major

    PubMed Central

    1995-01-01

    In experimental murine cutaneous leishmaniasis caused by Leishmania major (Lm), the cellular determinants governing development of protective or exacerbative T cells are not well understood. We, therefore, attempted to determine the influence of T cell and non-T cell compartments on disease outcome. To this end, T cell chimeric mice were constructed using adult thymectomized lethally irradiated, bone marrow-reconstituted (ATXBM) animals of genetically resistant, C57BL/6, or susceptible, BALB/c, backgrounds. These hosts were engrafted with naive T cell populations from H-2-congenic susceptible, BALB.B6-H-2b, or resistant, C57BL/6.C-H-2d, animals, respectively. Chimeric mice were then infected with Lm, and disease outcome was monitored. BALB/c T cell chimeric mice, BALB/c ATXBM hosts given naive C57BL/6.C-H-2d T cells, resolved their infections as indicated by reductions in both lesion size and parasite numbers. Furthermore, the mice developed typical Th1 (interferon[IFN]-gamma hiinterleukin[IL]-4lo) cytokine patterns. In contrast, both sham chimeric, BALB/c ATXBM hosts given naive BALB/c T cells, and control irradiated euthymic mice succumbed to infection, producing Th2 profiles (IFN-gamma loIL-4hiIL-10hi). C57BL/6 T cell chimeras, C57BL/6 ATXBM hosts given naive BALB.B6-H-2b T cells, resolved their infections as did C57BL/6 sham chimeras and euthymic controls. Interestingly, whereas C57BL/6 control animals produced Th1 cytokines, chimeric animals progressed from Th0 (IFN-gamma hiIL-4hiIL- 10hi) to Th2 (IFN-gamma loIL-4hiIL-10hi) cytokine profiles as cure ensued. Both reconstitution and chimeric status of all mice were confirmed by flow cytometry. In addition, T cell receptor V beta usage of Lm-specific blasts was determined. In all cases, V beta use was multiclonal, involving primarily V beta 2, 4, 6, 8.1, 8.2, 8.3, 10, and 14, with relative V beta frequencies differing between H-2b and H-2d animals. Most importantly, however, these differences did not segregate

  8. Preliminary AMS Study in Cretaceous Igneous Rocks of Valle Chico Complex, Uruguay: Statistical Determination of Magnetic Susceptibility

    NASA Astrophysics Data System (ADS)

    Barcelona, H.; Mena, M.; Sanchez-Bettucci, L.

    2009-05-01

    The Valle Chico Complex, at southeast Uruguay, is related Paraná-Etendeka Province. The study involved basaltic lavas, quarz-syenites, and rhyolitic and trachytic dikes. Samples were taken from 18 sites and the AMS of 250 specimens was analyzed. The AMS is modeled by a second order tensor K and it graphical representation is a symmetric ellipsoid. The axes relations determine parameters which describe different properties like shape, lineation, and foliation, degree of anisotropy and bulk magnetic susceptibility. Under this perspective, one lava, dike, or igneous body can be considered a mosaic of magnetic susceptibility domains (MSD). The DSM is an area with specific degree of homogeneity in the distribution of parameters values and cinematic conditions. An average tensor would weigh only one MSD, but if the site is a mosaic, subsets of specimens with similar parameters can be created. Hypothesis tests can be used to establish parameter similarities. It would be suitable considered as a MSD the subsets with statistically significant differences in at least one of its means parameters, and therefore, be treated independently. Once defined the MSDs the tensor analysis continues. The basalt-andesitic lavas present MSD with an NNW magnetic foliation, dipping 10. The K1 are sub-horizontal, oriented E-W and reprsent the magmatic flow direction. The quartz-syenites show a variable magnetic fabric or prolate ellipsoids mayor axes dispose parallel to the flow direction (10 to the SSE). Deformed syenites show N300/11 magnetic foliation, consistent with the trend of fractures. The K1 is subvertical. The MSD defined in rhyolitic dikes have magnetic foliations consistent with the structural trend. The trachytic dikes show an important indetermination in the magnetic response. However, a 62/N90 magnetic lineation was defined. The MSDs obtained are consistent with the geological structures and contribute to the knowledge of the tectonic, magmatic and kinematic events.

  9. Differential effect of IL10 and TNFα genotypes on determining susceptibility to discoid and systemic lupus erythematosus

    PubMed Central

    Suarez, A; Lopez, P; Mozo, L; Gutierrez, C

    2005-01-01

    Objective: To ascertain the possible involvement of functional interleukin 10 (IL10) and tumour necrosis α (TNFα) cytokine promoter polymorphisms on the susceptibility to discoid and systemic lupus erythematosus (DLE, SLE), and their associations with immunological features. Methods: Single nucleotide polymorphisms of the IL10 (–1082, –819, and –592) and TNFα (–308) genes were determined using allele specific probes in 248 lupus patients and 343 matched controls. To assess functional significance of genotypes, basal mRNA cytokine levels were quantified in 106 genotyped healthy controls by real time RT-PCR. Specific autoantibodies and cutaneous manifestations were analysed in SLE patients and associated with functional genotypes. Results: After analysing the distribution of IL10 and TNFα transcript levels according to promoter genotypes in healthy individuals, patients and controls were classified into functional single and combined genotypes according to the expected high or low constitutive cytokine production. High TNFα genotypes (–308AA or AG) were associated with SLE independently of IL10 alleles, whereas the risk of developing DLE and the prevalence of discoid lesion in SLE were higher in the high IL10/low TNFα producer group (–1082GG/–308GG). Cytokine interaction also influences the appearance of autoantibodies. Antibodies against Sm are prevalent among low producer patients for both cytokines, a genotype not associated with lupus incidence, whereas low IL10/high TNFα patients have the highest frequency of antibodies to SSa and SSb. Conclusions: IL10/TNFα interaction influences susceptibility to DLE and the appearance of specific autoantibodies in SLE patients, whereas high TNFα producer genotypes represent a significant risk factor for SLE. PMID:15800006

  10. Antigen conformation determines processing requirements for T-cell activation.

    PubMed Central

    Streicher, H Z; Berkower, I J; Busch, M; Gurd, F R; Berzofsky, J A

    1984-01-01

    We studied the difference in requirements for processing and presentation to a single T-cell clone of four different forms of the same epitope of sperm whale myoglobin--namely, on the native protein, on two conformationally altered forms of the protein, or as a 22-residue antigenic peptide fragment. The T-cell clone was I-Ed-restricted and specific for an epitope on the CNBr fragment 132-153 involving Lys-140. As inhibitors of macrophage processing of antigen, we used several agents that inhibit lysosomal function: the weak bases chloroquine and NH4Cl, the cationic ionophore monensin, and the competitive protease inhibitor leupeptin. When these agents were used to inhibit processing of antigen by presenting cells and then washed out before T cells were added to culture, they inhibited the presentation of native antigen but not of fragment 132-153. To our surprise, the intact but denatured form, S-methylmyoglobin, behaved like the fragment not like the native protein. Apomyoglobin was intermediate in susceptibility to inhibition. Thus, native myoglobin requires a processing step that appears to involve lysosomal proteolysis, which is not required by fragment 132-153 or the denatured unfolded forms. For an antigen the size of myoglobin (Mr 17,800), it appears that unfolding of the native conformation, rather than further reduction in size, is the critical parameter determining the need for processing. Since a major difference between native myoglobin and the other forms is the greater accessibility in the latter of sites, such as hydrophobic residues, buried in the native protein, we propose that processing may be necessary to expose these sites, perhaps for interaction with the cell membrane or the Ia of the antigen-presenting cell. PMID:6333686

  11. A Multidirectional Non-Cell Autonomous Control and a Genetic Interaction Restricting Tobacco Etch Virus Susceptibility in Arabidopsis

    PubMed Central

    Gopalan, Suresh

    2007-01-01

    Background Viruses constitute a major class of pathogens that infect a variety of hosts. Understanding the intricacies of signaling during host-virus interactions should aid in designing disease prevention strategies and in understanding mechanistic aspects of host and pathogen signaling machinery. Methodology/Principal Findings An Arabidopsis mutant, B149, impaired in susceptibility to Tobacco etch virus (TEV), a positive strand RNA virus of picoRNA family, was identified using a high-throughput genetic screen and a counterselection scheme. The defects include initiation of infection foci, rate of cell-to-cell movement and long distance movement. Conclusions/Significance The defect in infectivity is conferred by a recessive locus. Molecular genetic analysis and complementation analysis with three alleles of a previously published mutant lsp1 (loss of susceptibility to potyviruses) indicate a genetic interaction conferring haploinsufficiency between the B149 locus and certain alleles of lsp1 resulting in impaired host susceptibility. The pattern of restriction of TEV foci on leaves at or near the boundaries of certain cell types and leaf boundaries suggest dysregulation of a multidirectional non-cell autonomous regulatory mechanism. Understanding the nature of this multidirectional signal and the molecular genetic mechanism conferring it should potentially reveal a novel arsenal in the cellular machinery. PMID:17912362

  12. The susceptibility of primary cultured rhesus macaque kidney epithelial cells to rhesus cytomegalovirus strains.

    PubMed

    Yue, Yujuan; Kaur, Amitinder; Lilja, Anders; Diamond, Don J; Walter, Mark R; Barry, Peter A

    2016-06-01

    Kidney epithelial cells are common targets for human and rhesus cytomegalovirus (HCMV and RhCMV) in vivo, and represent an important reservoir for long-term CMV shedding in urine. To better understand the role of kidney epithelial cells in primate CMV natural history, primary cultures of rhesus macaque kidney epithelial cells (MKE) were established and tested for infectivity by five RhCMV strains, including two wild-type strains (UCD52 and UCD59) and three strains containing different coding contents in UL/b'. The latter strains included 180.92 [containing an intact RhUL128-RhUL130-R hUL131 (RhUL128L) locus but deleted for the UL/b' RhUL148-rh167-loci], 68-1 (RhUL128L-defective and fibroblast-tropic) and BRh68-1.2 (the RhUL128L-repaired version of 68-1). As demonstrated by RhCMV cytopathic effect, plaque formation, growth kinetics and early virus entry, we showed that MKE were differentially susceptible to RhCMV infection, related to UL/b' coding contents of the different strains. UCD52 and UCD59 replicated vigorously in MKE, 68-1 replicated poorly, and 180.92 grew with intermediate kinetics. Reconstitution of RhUL128L in 68-1 (BRh68-1.2) restored its replication efficiency in MKE as compared to UCD52 and UCD59, consistent with the essential role of UL128L for HCMV epithelial tropism. Further analysis revealed that the UL/b' UL148-rh167-loci deletion in 180.92 impaired RhUL132 (rh160) expression. Given that 180.92 retains an intact RhUL128L, but genetically or functionally lacks genes from RhUL132 (rh160) to rh167 in UL/b', its attenuated infection efficiency indicated that, along with RhUL128L, an additional protein(s) encoded within the UL/b' RhUL132 (rh160)-rh167 region (potentially, RhUL132 and/or RhUL148) is indispensable for efficient replication in MKE. PMID:26974598

  13. [Tools for determining health of phytoplankton cells

    SciTech Connect

    Not Available

    1992-01-01

    The primary purpose of the proposed research is to develop molecular tools for determining the health of marine phytoplankton on an individual cell basis. Since the definition of healthy in phytoplankton cells is elusive, we propose to develop markers for several different metabolic processes indicative of physiological state: photosynthetic activity, esterase activity, membrane permeability, and mitochondrial activity. One underlying motivation is to develop methods which will allow us to evaluate the hypothesis that, while healthy cells release very little dissolved organic carbon (DOC), many phytoplankton communities are comprised of unhealthy or physiologically stressed cells which release a large proportion of total photosynthate directly into the pool of labile DOC. This is proposed to be especially true in continental shelf and coastal environments where zones of productivity are patchy and phytoplankton populations adapted to one regime can be easily transported into waters which differ in salinity, nutrient supply, and/or turbidity. The significance of the work, however, extends beyond this immediate goal since there are presently relatively few methods which allow us to estimate the physiological state of phytoplankton cells.When we evaluate population sizes of phytoplankton in the water column or examine fecal pellets, particulate aggregates, or other material, we generally work in ignorance of the activity of the cells except as the average cell-specific activity is estimated from bulk measurements. This approach effectively hides any differences in the relative contribution of different taxa or individuals to overall productivity eventhough most flux processes are sensitive to physiological and taxonomically determined differences among members of the community.

  14. [Tools for determining health of phytoplankton cells

    SciTech Connect

    Not Available

    1992-12-31

    The primary purpose of the proposed research is to develop molecular tools for determining the health of marine phytoplankton on an individual cell basis. Since the definition of healthy in phytoplankton cells is elusive, we propose to develop markers for several different metabolic processes indicative of physiological state: photosynthetic activity, esterase activity, membrane permeability, and mitochondrial activity. One underlying motivation is to develop methods which will allow us to evaluate the hypothesis that, while healthy cells release very little dissolved organic carbon (DOC), many phytoplankton communities are comprised of unhealthy or physiologically stressed cells which release a large proportion of total photosynthate directly into the pool of labile DOC. This is proposed to be especially true in continental shelf and coastal environments where zones of productivity are patchy and phytoplankton populations adapted to one regime can be easily transported into waters which differ in salinity, nutrient supply, and/or turbidity. The significance of the work, however, extends beyond this immediate goal since there are presently relatively few methods which allow us to estimate the physiological state of phytoplankton cells.When we evaluate population sizes of phytoplankton in the water column or examine fecal pellets, particulate aggregates, or other material, we generally work in ignorance of the activity of the cells except as the average cell-specific activity is estimated from bulk measurements. This approach effectively hides any differences in the relative contribution of different taxa or individuals to overall productivity eventhough most flux processes are sensitive to physiological and taxonomically determined differences among members of the community.

  15. Susceptibility testing of Actinobacillus pleuropneumoniae in Denmark. Evaluation of three different media of MIC-determinations and tablet diffusion tests.

    PubMed

    Aarestrup, F M; Jensen, N E

    1999-02-12

    This study was conducted to compare the applicability of three different media in sensitivity testing of Actinobacillus pleuropneumoniae by means of MIC and tablet diffusion tests. The media used were: modified PPLO agar, chocolatized Mueller-Hinton-II and Columbia agar supplemented with NAD. Seven antimicrobial agents were tested: ceftiofur, enrofloxacin, penicillin, spectinomycin, tiamulin, trimethoprim + sulfadiazine and tylosin, against 40 randomly selected A. pleuropneumoniae isolates. In general, good agreement was found between results obtained with all combinations of media, most antimicrobials tested and the two-test systems. Some variations between media were observed for spectinomycin, tiamulin and tylosin. For ceftiofur and trimethoprim + sulfadiazine some isolates with low MIC-values were classified as resistant using tablet diffusion, indicating that the break points of resistance for these antimicrobials using the tablet diffusion tests need adjustment. Using current break points for resistance with MIC-determinations, all isolates tested susceptible to ceftiofur, enrofloxacin, penicillin, tiamulin and trimethoprim + sulfadiazine. A larger number of isolates tested resistant to spectinomycin and tylosin on all three media using both MIC determinations and tablet diffusion. PMID:10063535

  16. B1a cells enhance susceptibility to infection with virulent Francisella tularensis via modulation of NK/NKT cell responses*

    PubMed Central

    Crane, Deborah D.; Griffin, Amanda J.; Wehrly, Tara D.; Bosio, Catharine M.

    2013-01-01

    B1a cells are an important source of natural antibodies, antibodies directed against T-independent antigens, and are a primary source of IL-10. Bruton's tyrosine kinase (btk) is a cytoplasmic kinase that is essential for mediating signals from the B cell receptor and is critical for development of B1a cells. Consequentially, animals lacking btk have few B1a cells, minimal antibody responses, and can preferentially generate Th1 type immune responses following infection. B1a cells have been shown to aid in protection against infection with attenuated Francisella tularensis but their role in infection mediated by fully virulent F. tularensis is not known. Therefore, we utilized mice with defective btk (XID mice) to determine the contribution of B1a cells in defense against the virulent, F. tularensis ssp. tularensis strain SchuS4. Surprisingly, XID mice displayed increased resistance to pulmonary infection with F. tularensis. Specifically, XID mice had enhanced clearance of bacteria from the lung and spleen and significantly greater survival of infection compared to wild type controls. We revealed that resistance to infection in XID mice was associated with decreased numbers of IL-10 producing B1a cells and concomitant increased numbers of IL-12 producing macrophages and IFN-γ producing NK/NKT cells. Adoptive transfer of wild type B1a cells into XID mice reversed the control of bacterial replication. Similarly, depletion of NK/NKT cells also increased bacterial burdens in XID mice. Together, our data suggest B cell-NK/NKT cell crosstalk is a critical pivot controlling survival of infection with virulent F. tularensis. PMID:23378429

  17. Susceptibility Testing

    MedlinePlus

    ... page helpful? Also known as: Sensitivity Testing; Drug Resistance Testing; Culture and Sensitivity; C & S; Antimicrobial Susceptibility Formal name: Bacterial and Fungal Susceptibility Testing Related tests: Urine Culture ; ...

  18. Norwalk Virus Does Not Replicate in Human Macrophages or Dendritic Cells Derived from the Peripheral Blood of Susceptible Humans

    PubMed Central

    Lay, Margarita K.; Atmar, Robert L.; Guix, Susana; Bharadwaj, Uddalak; He, Hong; Neill, Frederick H.; Sastry, Jagannadha K.; Yao, Qizhi; Estes, Mary K.

    2010-01-01

    Human noroviruses are difficult to study due to the lack of an efficient in vitro cell culture system or small animal model. Murine norovirus replicates in murine macrophages (MΦ) and dendritic cells (DCs), raising the possibility that human NoVs might replicate in such human cell types. To test this hypothesis, we evaluated DCs and MΦ derived from monocyte subsets and CD11c+ DCs isolated from peripheral blood mononuclear cells of individuals susceptible to Norwalk virus (NV) infection. These cells were exposed to NV and replication was evaluated by immunofluorescence and by quantitative RT-PCR. A few PBMC-derived DCs expressed NV proteins. However, NV RNA did not increase in any of the cells tested. These results demonstrate that NV does not replicate in human CD11c+ DCs, monocyte-derived DCs and MΦ, but abortive infection may occur in a few DCs. These results suggest that NV tropism is distinct from that of murine noroviruses. PMID:20667573

  19. Evaluation of spectrophotometric and HPLC methods for shikimic acid determination in plants: models in glyphosate-resistant and -susceptible crops.

    PubMed

    Zelaya, Ian A; Anderson, Jennifer A H; Owen, Micheal D K; Landes, Reid D

    2011-03-23

    Endogenous shikimic acid determinations are routinely used to assess the efficacy of glyphosate in plants. Numerous analytical methods exist in the public domain for the detection of shikimic acid, yet the most commonly cited comprise spectrophotometric and high-pressure liquid chromatography (HPLC) methods. This paper compares an HPLC and two spectrophotometric methods (Spec 1 and Spec 2) and assesses the effectiveness in the detection of shikimic acid in the tissues of glyphosate-treated plants. Furthermore, the study evaluates the versatility of two acid-based shikimic acid extraction methods and assesses the longevity of plant extract samples under different storage conditions. Finally, Spec 1 and Spec 2 are further characterized with respect to (1) the capacity to discern between shikimic acid and chemically related alicyclic hydroxy acids, (2) the stability of the chromophore (t1/2), (3) the detection limits, and (4) the cost and simplicity of undertaking the analytical procedure. Overall, spectrophotometric methods were more cost-effective and simpler to execute yet provided a narrower detection limit compared to HPLC. All three methods were specific to shikimic acid and detected the compound in the tissues of glyphosate-susceptible crops, increasing exponentially in concentration within 24 h of glyphosate application and plateauing at approximately 72 h. Spec 1 estimated more shikimic acid in identical plant extract samples compared to Spec 2 and, likewise, HPLC detection was more effective than spectrophotometric determinations. Given the unprecedented global adoption of glyphosate-resistant crops and concomitant use of glyphosate, an effective and accurate assessment of glyphosate efficacy is important. Endogenous shikimic acid determinations are instrumental in corroborating the efficacy of glyphosate and therefore have numerous applications in herbicide research and related areas of science as well as resolving many commercial issues as a consequence of

  20. T lymphocytes bearing the gamma delta T cell receptor are susceptible to steroid-induced programmed cell death.

    PubMed

    Spinozzi, F; Agea, E; Bistoni, O; Travetti, A; Migliorati, G; Moraca, R; Nicoletti, I; Riccardi, C; Paoletti, F P; Vaccaro, R

    1995-05-01

    The mechanisms by which glucocorticoids suppress immune responses have not yet been clearly defined. In steroid-sensitive pathological conditions, an increase in gamma delta T cells can occur in certain untreated systemic autoimmune disorders and seems to be a peristent feature in most cases of systemic lupus erythematosus (SLE). Our previously published data demonstrated that immunosuppressive therapy normalized this expanded SLE T cell subset in parallel with clinical remission of the symptoms. To establish how corticosteroid treatment determines the disappearance of peripheral blood gamma delta T lymphocytes, circulating alpha beta and gamma delta T lymphocytes from seven SLE subjects with active disease and seven healthy individuals were cultured in the presence or absence of 10(-7) M Dexamethasone (DEX). Cell suspensions were then analysed for DNA fragmentation, characteristic of apoptotic cell death, by a new cytofluorimetric method. Conventional agarose-gel electrophoresis on the same T cell populations was carried out for comparison. Regular follow-ups for 6 months revealed in vivo steroid treatment determined a dramatic fall in SLE blood gamma delta T cells, and in vitro experiments seem to indicate that DEX-triggered apoptotic signals are confined to the double negative (CD4-CD8-) gamma delta T cell subpopulation which disappears after in vivo immunosuppressive therapy. Clinical and pathological remission of some autoimmune diseases is often obtained by corticosteroids. Our results offer new insights on the mechanisms through these hormones exert their potent inhibitory activities on immune system cells postulated to play a role in the generation of autoimmune responses. PMID:7725070

  1. Establishment of cell line from embryonic tissue of Pipistrellus ceylonicus bat species from India & its susceptibility to different viruses

    PubMed Central

    Mourya, Devendra T.; Lakra, Rajen J.; Yadav, Pragya D.; Tyagi, Preeti; Raut, Chandrashekhar G.; Shete, Anita M.; Singh, Dinesh K.

    2013-01-01

    Background & objectives: Pipistrellus ceylonicus bat species is widely distributed in South Asia, with additional populations recorded in China and Southeast Asia. Bats are the natural reservoir hosts for a number of emerging zoonotic diseases. Attempts to isolate bat-borne viruses in various terrestrial mammalian cell lines have sometimes been unsuccessful. The bat cell lines are useful in isolation and propagation of many of the viruses harboured by bats. New stable bat cell lines are needed to help such investigations and to assist in the study of bat immunology and virus-host interactions. In this study we made an attempt to develop a cell line from P. ceylonicus bats. Methods: An effort was made to establish cell line from embryo of P. ceylonicus species of bat after seeding to Dulbecco's modified eagle medium (DMEM) supplemented with 10 per cent foetal bovine serum; a primary cell line was established and designated as NIV-BtEPC. Mitochondrial DNA profile analysis was done using cyt-b and ND-1 gene sequences from the cell line. Phylogenetic tree was constructed using neighbour-joining algorithm for cyt-b and ND-1 genes with 1000-bootstrap replicates. Results: NIV-BtEPC cell line was susceptible to Chandipura (CHPV) and novel adenovirus (BtAdv-RLM) isolated from Rousettus leschenaulti from India but did not support multiplication of a number of Bunyaviruses, Alphaviruses and Flavivirus. This might be useful for isolation of a range of viruses and investigation of unknown aetiological agents. Interpretation & conclusions: In this study, a new bat cell line was developed from P. ceylonicus. This cell line was successfully tested for the susceptibility to Chandipura and BtAdv-RLM virus isolated from bats. The approach developed and optimised in this study may be applicable to the other species of bats and this established cell line can be used to facilitate virus isolation and basic research into virus-host interaction. PMID:24056599

  2. Extracellular pH Modulates Neuroendocrine Prostate Cancer Cell Metabolism and Susceptibility to the Mitochondrial Inhibitor Niclosamide.

    PubMed

    Ippolito, Joseph E; Brandenburg, Matthew W; Ge, Xia; Crowley, Jan R; Kirmess, Kristopher M; Som, Avik; D'Avignon, D Andre; Arbeit, Jeffrey M; Achilefu, Samuel; Yarasheski, Kevin E; Milbrandt, Jeffrey

    2016-01-01

    Neuroendocrine prostate cancer is a lethal variant of prostate cancer that is associated with castrate-resistant growth, metastasis, and mortality. The tumor environment of neuroendocrine prostate cancer is heterogeneous and characterized by hypoxia, necrosis, and numerous mitoses. Although acidic extracellular pH has been implicated in aggressive cancer features including metastasis and therapeutic resistance, its role in neuroendocrine prostate cancer physiology and metabolism has not yet been explored. We used the well-characterized PNEC cell line as a model to establish the effects of extracellular pH (pH 6.5, 7.4, and 8.5) on neuroendocrine prostate cancer cell metabolism. We discovered that alkalinization of extracellular pH converted cellular metabolism to a nutrient consumption-dependent state that was susceptible to glucose deprivation, glutamine deprivation, and 2-deoxyglucose (2-DG) mediated inhibition of glycolysis. Conversely, acidic pH shifted cellular metabolism toward an oxidative phosphorylation (OXPHOS)-dependent state that was susceptible to OXPHOS inhibition. Based upon this mechanistic knowledge of pH-dependent metabolism, we identified that the FDA-approved anti-helminthic niclosamide depolarized mitochondrial potential and depleted ATP levels in PNEC cells whose effects were enhanced in acidic pH. To further establish relevance of these findings, we tested the effects of extracellular pH on susceptibility to nutrient deprivation and OXPHOS inhibition in a cohort of castrate-resistant prostate cancer cell lines C4-2B, PC-3, and PC-3M. We discovered similar pH-dependent toxicity profiles among all cell lines with these treatments. These findings underscore a potential importance to acidic extracellular pH in the modulation of cell metabolism in tumors and development of an emerging paradigm that exploits the synergy of environment and therapeutic efficacy in cancer. PMID:27438712

  3. Extracellular pH Modulates Neuroendocrine Prostate Cancer Cell Metabolism and Susceptibility to the Mitochondrial Inhibitor Niclosamide

    PubMed Central

    Ippolito, Joseph E.; Brandenburg, Matthew W.; Ge, Xia; Crowley, Jan R.; Kirmess, Kristopher M.; Som, Avik; D’Avignon, D. Andre; Arbeit, Jeffrey M.; Achilefu, Samuel; Yarasheski, Kevin E.; Milbrandt, Jeffrey

    2016-01-01

    Neuroendocrine prostate cancer is a lethal variant of prostate cancer that is associated with castrate-resistant growth, metastasis, and mortality. The tumor environment of neuroendocrine prostate cancer is heterogeneous and characterized by hypoxia, necrosis, and numerous mitoses. Although acidic extracellular pH has been implicated in aggressive cancer features including metastasis and therapeutic resistance, its role in neuroendocrine prostate cancer physiology and metabolism has not yet been explored. We used the well-characterized PNEC cell line as a model to establish the effects of extracellular pH (pH 6.5, 7.4, and 8.5) on neuroendocrine prostate cancer cell metabolism. We discovered that alkalinization of extracellular pH converted cellular metabolism to a nutrient consumption-dependent state that was susceptible to glucose deprivation, glutamine deprivation, and 2-deoxyglucose (2-DG) mediated inhibition of glycolysis. Conversely, acidic pH shifted cellular metabolism toward an oxidative phosphorylation (OXPHOS)-dependent state that was susceptible to OXPHOS inhibition. Based upon this mechanistic knowledge of pH-dependent metabolism, we identified that the FDA-approved anti-helminthic niclosamide depolarized mitochondrial potential and depleted ATP levels in PNEC cells whose effects were enhanced in acidic pH. To further establish relevance of these findings, we tested the effects of extracellular pH on susceptibility to nutrient deprivation and OXPHOS inhibition in a cohort of castrate-resistant prostate cancer cell lines C4-2B, PC-3, and PC-3M. We discovered similar pH-dependent toxicity profiles among all cell lines with these treatments. These findings underscore a potential importance to acidic extracellular pH in the modulation of cell metabolism in tumors and development of an emerging paradigm that exploits the synergy of environment and therapeutic efficacy in cancer. PMID:27438712

  4. Extracellular pH Modulates Neuroendocrine Prostate Cancer Cell Metabolism and Susceptibility to the Mitochondrial Inhibitor Niclosamide.

    PubMed

    Ippolito, Joseph E; Brandenburg, Matthew W; Ge, Xia; Crowley, Jan R; Kirmess, Kristopher M; Som, Avik; D'Avignon, D Andre; Arbeit, Jeffrey M; Achilefu, Samuel; Yarasheski, Kevin E; Milbrandt, Jeffrey

    2016-01-01

    Neuroendocrine prostate cancer is a lethal variant of prostate cancer that is associated with castrate-resistant growth, metastasis, and mortality. The tumor environment of neuroendocrine prostate cancer is heterogeneous and characterized by hypoxia, necrosis, and numerous mitoses. Although acidic extracellular pH has been implicated in aggressive cancer features including metastasis and therapeutic resistance, its role in neuroendocrine prostate cancer physiology and metabolism has not yet been explored. We used the well-characterized PNEC cell line as a model to establish the effects of extracellular pH (pH 6.5, 7.4, and 8.5) on neuroendocrine prostate cancer cell metabolism. We discovered that alkalinization of extracellular pH converted cellular metabolism to a nutrient consumption-dependent state that was susceptible to glucose deprivation, glutamine deprivation, and 2-deoxyglucose (2-DG) mediated inhibition of glycolysis. Conversely, acidic pH shifted cellular metabolism toward an oxidative phosphorylation (OXPHOS)-dependent state that was susceptible to OXPHOS inhibition. Based upon this mechanistic knowledge of pH-dependent metabolism, we identified that the FDA-approved anti-helminthic niclosamide depolarized mitochondrial potential and depleted ATP levels in PNEC cells whose effects were enhanced in acidic pH. To further establish relevance of these findings, we tested the effects of extracellular pH on susceptibility to nutrient deprivation and OXPHOS inhibition in a cohort of castrate-resistant prostate cancer cell lines C4-2B, PC-3, and PC-3M. We discovered similar pH-dependent toxicity profiles among all cell lines with these treatments. These findings underscore a potential importance to acidic extracellular pH in the modulation of cell metabolism in tumors and development of an emerging paradigm that exploits the synergy of environment and therapeutic efficacy in cancer.

  5. Determining Physical Properties of the Cell Cortex.

    PubMed

    Saha, Arnab; Nishikawa, Masatoshi; Behrndt, Martin; Heisenberg, Carl-Philipp; Jülicher, Frank; Grill, Stephan W

    2016-03-29

    Actin and myosin assemble into a thin layer of a highly dynamic network underneath the membrane of eukaryotic cells. This network generates the forces that drive cell- and tissue-scale morphogenetic processes. The effective material properties of this active network determine large-scale deformations and other morphogenetic events. For example, the characteristic time of stress relaxation (the Maxwell time τM) in the actomyosin sets the timescale of large-scale deformation of the cortex. Similarly, the characteristic length of stress propagation (the hydrodynamic length λ) sets the length scale of slow deformations, and a large hydrodynamic length is a prerequisite for long-ranged cortical flows. Here we introduce a method to determine physical parameters of the actomyosin cortical layer in vivo directly from laser ablation experiments. For this we investigate the cortical response to laser ablation in the one-cell-stage Caenorhabditis elegans embryo and in the gastrulating zebrafish embryo. These responses can be interpreted using a coarse-grained physical description of the cortex in terms of a two-dimensional thin film of an active viscoelastic gel. To determine the Maxwell time τM, the hydrodynamic length λ, the ratio of active stress ζΔμ, and per-area friction γ, we evaluated the response to laser ablation in two different ways: by quantifying flow and density fields as a function of space and time, and by determining the time evolution of the shape of the ablated region. Importantly, both methods provide best-fit physical parameters that are in close agreement with each other and that are similar to previous estimates in the two systems. Our method provides an accurate and robust means for measuring physical parameters of the actomyosin cortical layer. It can be useful for investigations of actomyosin mechanics at the cellular-scale, but also for providing insights into the active mechanics processes that govern tissue-scale morphogenesis. PMID

  6. A Bacterial Cell Shape-Determining Inhibitor.

    PubMed

    Liu, Yanjie; Frirdich, Emilisa; Taylor, Jennifer A; Chan, Anson C K; Blair, Kris M; Vermeulen, Jenny; Ha, Reuben; Murphy, Michael E P; Salama, Nina R; Gaynor, Erin C; Tanner, Martin E

    2016-04-15

    Helicobacter pylori and Campylobacter jejuni are human pathogens and causative agents of gastric ulcers/cancer and gastroenteritis, respectively. Recent studies have uncovered a series of proteases that are responsible for maintaining the helical shape of these organisms. The H. pylori metalloprotease Csd4 and its C. jejuni homologue Pgp1 cleave the amide bond between meso-diaminopimelate and iso-d-glutamic acid in truncated peptidoglycan side chains. Deletion of either csd4 or pgp1 results in bacteria with a straight rod phenotype, a reduced ability to move in viscous media, and reduced pathogenicity. In this work, a phosphinic acid-based pseudodipeptide inhibitor was designed to act as a tetrahedral intermediate analog against the Csd4 enzyme. The phosphinic acid was shown to inhibit the cleavage of the alternate substrate, Ac-l-Ala-iso-d-Glu-meso-Dap, with a Ki value of 1.5 μM. Structural analysis of the Csd4-inhibitor complex shows that the phosphinic acid displaces the zinc-bound water and chelates the metal in a bidentate fashion. The phosphinate oxygens also interact with the key acid/base residue, Glu222, and the oxyanion-stabilizing residue, Arg86. The results are consistent with the "promoted-water pathway" mechanism for carboxypeptidase A catalysis. Studies on cultured bacteria showed that the inhibitor causes significant cell straightening when incubated with H. pylori at millimolar concentrations. A diminished, yet observable, effect on the morphology of C. jejuni was also apparent. Cell straightening was more pronounced with an acapsular C. jejuni mutant strain compared to the wild type, suggesting that the capsule impaired inhibitor accessibility. These studies demonstrate that a highly polar compound is capable of crossing the outer membrane and altering cell shape, presumably by inhibiting cell shape determinant proteases. Peptidoglycan proteases acting as cell shape determinants represent novel targets for the development of antimicrobials

  7. Class I (H-2Kb) gene transfection reduces susceptibility of YAC-1 lymphoma targets to natural killer cells.

    PubMed

    Carlow, D A; Payne, U; Hozumi, N; Roder, J C; Czitrom, A A

    1990-04-01

    A "hybrid gene" (MTKb) comprised of the human metallothionein IIA promoter ligated to the genomic sequence of the major histocompatibility complex class I (H-2Kb) gene was subcloned into the expression vector pSV2neo and transfected into the natural killer (NK) cell-sensitive YAC-1 lymphoma. The Kb gene product was readily detectable on the cell surface of G418-resistant transfectants using both Kb-specific monoclonal antibodies and H-2b-specific cytolytic T cells. Unlike control pSV2neo transfectants, MTKb-pSV2neo transfectants were relatively resistant to lysis by NK cells from H-2a, H-2b, H-2k or H-2 (a x b)F1 haplotype mice. These data strongly suggest that the effects of MHC expression on susceptibility to NK cells can be mediated by a single and well-defined class I molecule, Kb.

  8. Laboratory bioassay studies to determine methoprene susceptibility in a natural population of Ochlerotatus taeniorhynchus from the Florida keys.

    PubMed

    Floore, Omas G; Smith, John P; Shaffer, Kenneth R; Schreiber, Eric T

    2002-06-01

    Larvae of Florida Keys Ochlerotatus taeniorhynchus collected from No Name Key were colonized and evaluated against technical S-methoprene in laboratory beaker tests. Glassware was treated with a silanizing reagent before testing to prevent methoprene attachment to the glassware. The No Name Key strain was compared with a susceptible laboratory strain of Oc. taeniorhynchus. Five serial dilutions (0.0100, 0.0050, 0.0010, 0.0005, and 0.0001 microg/ml) and an untreated control were evaluated. Tests were conducted in water baths with a constant water temperature of 27+/-1 degrees C and 250 ml of 3 per thousand salt water. Twenty-five late 3rd-stage larvae were placed in each beaker. Bioassay samples were analyzed by probit analysis and the median lethal concentration (LC50), 90% lethal concentration (LC90), and 95% lethal concentration (LC95) values; confidence limits; chi2 value; slope; and standard error were determined. The Florida Keys No Name Key strain exhibited no significant differences at the LC50, LC90, and LC95 levels from the laboratory strain in these studies.

  9. Candida colonization in urine samples of ICU patients: determination of etiology, antifungal susceptibility testing and evaluation of associated risk factors.

    PubMed

    Singla, Nidhi; Gulati, Neelam; Kaistha, Neelam; Chander, Jagdish

    2012-08-01

    The presence of Candida in urine presents a therapeutic challenge for the physician as it is often asymptomatic, and management guidelines have not been clearly laid down on this issue. The presence of Candida in urine may represent contamination of clinical sample, actual colonization of the lower urinary tract or may be a true indicator of invasive infection of lower and/or upper urinary tract. In a clinical setting like the ICU, multiple risk factors for Candida colonization may be present in the same patient, thereby increasing the chances of candiduria, manifold. In the present study on 80 patients in ICU, high rate of Candida colonization (57.5%) was found in urine samples of ICU patients with C. tropicalis (57.3%) being the predominant species. We also isolated 8 strains of Trichosporon species, all of these presented as a mixed infection along with Candida species. Among the various risk factors studied, urinary catheterization and previous antibiotic therapy were identified as statistically significant (P value <0.05). The minimum inhibitory concentration of the isolates was determined for amphotericin B, fluconazole and itraconazole by E-test. Most of the isolates were susceptible to amphotericin B. The C. parapsilosis strains did not show any drug resistance; however, resistance to fluconazole was observed 18.6, 27.27, 50 and 25% in C. tropicalis, C. albicans, C. glabrata and Trichosporon species, respectively.

  10. Antimicrobial susceptibility, virulence determinant carriage and molecular characteristics of Staphylococcus aureus isolates associated with skin and soft tissue infections.

    PubMed

    Yu, Fangyou; Liu, Yunling; Lv, Jinnan; Qi, Xiuqin; Lu, Chaohui; Ding, Yu; Li, Dan; Liu, Huanle; Wang, Liangxing

    2015-01-01

    A better understanding of the antimicrobial susceptibility, carriage of virulence determinants and molecular characteristics of Staphylococcus aureus isolates associated with skin and soft tissue infections (SSTIs) may provide further insights related to clinical outcomes with these infections. From January 2012 to September 2013, a total of 128 non-duplicate S. aureus isolates were recovered from patients with SSTIs. All 128 S. aureus SSTI isolates carried at least five virulence genes tested. Virulence genes detected among at least 70% of all tested isolates included hld (100%), hla (95.3%), icaA (96.9%), clf (99.2%), sdrC (79.7%), sdrD (70.3%), and sdrE (72.7%). The prevalence of MRSA isolates with 10 virulence genes tested (54.4%, 31/56) was significantly higher than that among MSSA isolates (35.2%, 25/71) (p<0.05). The positive rates of seb, sen, sem, sdrE and pvl among MRSA isolates were significantly higher than among MSSA isolates (p<0.05). ST7 and ST630 accounting for 10.9% were found to be the predominant STs. The most prevalent spa type was t091 (8.6%). MRSA-ST59-SCCmec IV was the most common clone (12.3%) among MRSA isolates whereas among MSSA isolates the dominant clone was MSSA-ST7 (15.5%). Six main clonal complexes (CCs) were found, including CC5 (52.3%), CC7 (11.7%), CC59 (8.6%), CC88 (6.3%), CC398 (4.7%), and CC121 (3.1%). A higher carriage of seb and sec was found among CC59 isolates. In comparison to CC5 and CC7 isolates, those with the highest carriage rates (>80.0%) of sdrC and sdrD, CC59 isolates had lower prevalence of these two virulence genes. All CC59 isolates were susceptible to gentamicin and trimethoprim/sulfamethoxazole, while CC5 and CC7 isolates had resistance rates to these two antimicrobials of 25.4% and 20.9%, and 40.0% and 40.0%, respectively. The resistance rates for tetracycline, clindamycin, and erythromycin among CC5 isolates were lower than among CC7 and CC59 isolates. In conclusion, the molecular typing of S. aureus SSTI

  11. Histone Deacetylase Inhibitors Enhance CD4 T Cell Susceptibility to NK Cell Killing but Reduce NK Cell Function

    PubMed Central

    Pace, Matthew; Williams, James; Kurioka, Ayako; Gerry, Andrew B.; Jakobsen, Bent; Klenerman, Paul; Nwokolo, Nneka; Fox, Julie

    2016-01-01

    In the search for a cure for HIV-1 infection, histone deacetylase inhibitors (HDACi) are being investigated as activators of latently infected CD4 T cells to promote their targeting by cytotoxic T-lymphocytes (CTL). However, HDACi may also inhibit CTL function, suggesting different immunotherapy approaches may need to be explored. Here, we study the impact of different HDACi on both Natural Killer (NK) and CTL targeting of HIV-1 infected cells. We found HDACi down-regulated HLA class I expression independently of HIV-1 Nef which, without significantly compromising CTL function, led to enhanced targeting by NK cells. HDACi-treated HIV-1-infected CD4 T cells were also more effectively cleared than untreated controls during NK co-culture. However, HDACi impaired NK function, reducing degranulation and killing capacity. Depending on the HDACi and dose, this impairment could counteract the benefit gained by treating infected target cells. These data suggest that following HDACi-induced HLA class I down-regulation NK cells kill HIV-1-infected cells, although HDACi-mediated NK cell inhibition may negate this effect. Our data emphasize the importance of studying the effects of potential interventions on both targets and effectors. PMID:27529554

  12. Bovine Mx1 enables resistance against foot-and-mouth disease virus in naturally susceptible cells by inhibiting the replication of viral RNA.

    PubMed

    Wang, H-M; Xia, X-Z; Hu, G-X; Yu, L; He, H-B

    2016-03-01

    Innate immunity, especially the anti-viral genes, exerts an important barrier function in preventing viral infections. Myxovirus-resistant (Mx) gene take an anti-viral role, whereas its effects on foot-and-mouth disease virus (FMDV) in naturally susceptible cells are still unclear. The bovine primary fetal tracheal epithelial cell line BPTE-siMx1, in which bovine Mx1 gene was silenced, was established and treated with IFN alpha for 6 hr before FMDV infection. The copy numbers of the negative and positive strand viral RNA were determined by strand-specific real-time fluorescence quantitative RT-PCR. The TCID50 of BPTE-siMx1 cells increased at least 17-fold as compared to control cells BPTE-LacZ at 8 hr post infection, thus silencing of bovine Mx1 could promote the replication of FMDV. The amount of both the negative and positive strand viral RNA in BPTE-siMx1 cells significantly increased as compared to BPTE-LacZ cells, indicating that the replication levels of viral RNA were promoted by silencing bovine Mx1. The bovine Mx1 gene could provide resistance against FMDV in the bovine primary fetal tracheal epithelial cells via suppressing the replication of viral RNA. PMID:26982472

  13. Bovine Mx1 enables resistance against foot-and-mouth disease virus in naturally susceptible cells by inhibiting the replication of viral RNA.

    PubMed

    Wang, H-M; Xia, X-Z; Hu, G-X; Yu, L; He, H-B

    2016-03-01

    Innate immunity, especially the anti-viral genes, exerts an important barrier function in preventing viral infections. Myxovirus-resistant (Mx) gene take an anti-viral role, whereas its effects on foot-and-mouth disease virus (FMDV) in naturally susceptible cells are still unclear. The bovine primary fetal tracheal epithelial cell line BPTE-siMx1, in which bovine Mx1 gene was silenced, was established and treated with IFN alpha for 6 hr before FMDV infection. The copy numbers of the negative and positive strand viral RNA were determined by strand-specific real-time fluorescence quantitative RT-PCR. The TCID50 of BPTE-siMx1 cells increased at least 17-fold as compared to control cells BPTE-LacZ at 8 hr post infection, thus silencing of bovine Mx1 could promote the replication of FMDV. The amount of both the negative and positive strand viral RNA in BPTE-siMx1 cells significantly increased as compared to BPTE-LacZ cells, indicating that the replication levels of viral RNA were promoted by silencing bovine Mx1. The bovine Mx1 gene could provide resistance against FMDV in the bovine primary fetal tracheal epithelial cells via suppressing the replication of viral RNA.

  14. Human skin neural crest progenitor cells are susceptible to BRAF(V600E)-induced transformation.

    PubMed

    Kumar, S M; Dai, J; Li, S; Yang, R; Yu, H; Nathanson, K L; Liu, S; Zhou, H; Guo, J; Xu, X

    2014-02-13

    Adult stem cells are multipotent and persist in small numbers in adult tissues throughout the lifespan of an organism. Unlike differentiated cells, adult stem cells are intrinsically resistant to senescence. It is unclear how adult stem cells in solid organs respond to oncogenic stimulation and whether these cells have a role in tumor initiation. We report here that expression of BRAF(V600E) in human neural crest progenitor cells (hNCPCs) did not induce growth arrest as seen in human melanocytes, but instead, increased their cell proliferation capacity. These cells (hNCPCs(V600E)) acquired anchorage-independent growth ability and were weakly tumorigenic in vivo. Unlike in human melanocytes, BRAF(V600E) expression in hNCPCs did not induce p16(INK4a) expression. BRAF(V600E) induced elevated expression of CDK2, CDK4, MITF and EST1/2 protein in hNCPCs, and also induced melanocytic differentiation of these cells. Furthermore, overexpression of MITF in hNCPCs(V600E) dramatically increased their tumorigenicity and resulted in fully transformed tumor cells. These findings indicate that hNCPCs are susceptible to BRAF(V600E)-induced transformation, and MITF potentiates the oncogenic effect of BRAF(V600E) in these progenitor cells. These results suggest that the hNCPCs are potential targets for BRAF(V600E)-induced melanocytic tumor formation.

  15. Human skin neural crest progenitor cells are susceptible to BRAF(V600E)-induced transformation.

    PubMed

    Kumar, S M; Dai, J; Li, S; Yang, R; Yu, H; Nathanson, K L; Liu, S; Zhou, H; Guo, J; Xu, X

    2014-02-13

    Adult stem cells are multipotent and persist in small numbers in adult tissues throughout the lifespan of an organism. Unlike differentiated cells, adult stem cells are intrinsically resistant to senescence. It is unclear how adult stem cells in solid organs respond to oncogenic stimulation and whether these cells have a role in tumor initiation. We report here that expression of BRAF(V600E) in human neural crest progenitor cells (hNCPCs) did not induce growth arrest as seen in human melanocytes, but instead, increased their cell proliferation capacity. These cells (hNCPCs(V600E)) acquired anchorage-independent growth ability and were weakly tumorigenic in vivo. Unlike in human melanocytes, BRAF(V600E) expression in hNCPCs did not induce p16(INK4a) expression. BRAF(V600E) induced elevated expression of CDK2, CDK4, MITF and EST1/2 protein in hNCPCs, and also induced melanocytic differentiation of these cells. Furthermore, overexpression of MITF in hNCPCs(V600E) dramatically increased their tumorigenicity and resulted in fully transformed tumor cells. These findings indicate that hNCPCs are susceptible to BRAF(V600E)-induced transformation, and MITF potentiates the oncogenic effect of BRAF(V600E) in these progenitor cells. These results suggest that the hNCPCs are potential targets for BRAF(V600E)-induced melanocytic tumor formation. PMID:23334329

  16. A Natural Variant of the T Cell Receptor-Signaling Molecule Vav1 Reduces Both Effector T Cell Functions and Susceptibility to Neuroinflammation

    PubMed Central

    Kassem, Sahar; Bernard, Isabelle; Dejean, Anne S.; Liblau, Roland; Fournié, Gilbert J.; Colacios, Céline

    2016-01-01

    The guanine nucleotide exchange factor Vav1 is essential for transducing T cell antigen receptor signals and therefore plays an important role in T cell development and activation. Our previous genetic studies identified a locus on rat chromosome 9 that controls the susceptibility to neuroinflammation and contains a non-synonymous polymorphism in the major candidate gene Vav1. To formally demonstrate the causal implication of this polymorphism, we generated a knock-in mouse bearing this polymorphism (Vav1R63W). Using this model, we show that Vav1R63W mice display reduced susceptibility to experimental autoimmune encephalomyelitis (EAE) induced by MOG35-55 peptide immunization. This is associated with a lower production of effector cytokines (IFN-γ, IL-17 and GM-CSF) by autoreactive CD4 T cells. Despite increased proportion of Foxp3+ regulatory T cells in Vav1R63W mice, we show that this lowered cytokine production is intrinsic to effector CD4 T cells and that Treg depletion has no impact on EAE development. Finally, we provide a mechanism for the above phenotype by showing that the Vav1R63W variant has normal enzymatic activity but reduced adaptor functions. Together, these data highlight the importance of Vav1 adaptor functions in the production of inflammatory cytokines by effector T cells and in the susceptibility to neuroinflammation. PMID:27438086

  17. Automated Determination of Arterial Input Function for Dynamic Susceptibility Contrast MRI from Regions around Arteries Using Independent Component Analysis

    PubMed Central

    Lai, Jui-Jen; Chang, Chin-Ching

    2016-01-01

    Purpose. Quantitative cerebral blood flow (CBF) measurement using dynamic susceptibility contrast- (DSC-) MRI requires accurate estimation of the arterial input function (AIF). The present work utilized the independent component analysis (ICA) method to determine the AIF in the regions adjacent to the middle cerebral artery (MCA) by the alleviated confounding of partial volume effect. Materials and Methods. A series of spin-echo EPI MR scans were performed in 10 normal subjects. All subjects received 0.2 mmol/kg Gd-DTPA contrast agent. AIFs were calculated by two methods: (1) the region of interest (ROI) selected manually and (2) weighted average of each component selected by ICA (weighted-ICA). The singular value decomposition (SVD) method was then employed to deconvolve the AIF from the tissue concentration time curve to obtain quantitative CBF values. Results. The CBF values calculated by the weighted-ICA method were 41.1 ± 4.9 and 22.1 ± 2.3 mL/100 g/min for cortical gray matter (GM) and deep white matter (WM) regions, respectively. The CBF values obtained based on the manual ROIs were 53.6 ± 12.0 and 27.9 ± 5.9 mL/100 g/min for the same two regions, respectively. Conclusion. The weighted-ICA method allowed semiautomatic and straightforward extraction of the ROI adjacent to MCA. Through eliminating the partial volume effect to minimum, the CBF thus determined may reflect more accurate physical characteristics of the T2⁎ signal changes induced by the contrast agent. PMID:27547451

  18. 7,12-dimethylbenz(a)anthracene-induced DNA binding and repair synthesis in susceptible and nonsusceptible mammary epithelial cells in culture. [Rats

    SciTech Connect

    Tay, L.K.; Russo, J.

    1981-07-01

    The effect of age and parity on the binding of 7,12-dimethylbenz(a)anthracene (DMBA) to DNA and the repair of DMBA-damaged DNA have been demonstrated in logarithmic phase and confluent mammary epithelial cell cultures from young virgin (YV), old virgin (OV), and parous (P) noninbred and inbred Sprague-Dawley rats. Excision repair was determined by measuring, in the presence of hydroxyurea and 5-bromodeoxyuridine, tritiated thymidine incorporation into DNA during the repair process. These results suggest that age and parity not only lower the binding of DMBA to mammary epithelial cell DNA but also increase the efficiency of DNA repair processes, which may explain the lower susceptibility of OV and P rats to DMBA-induced mammary carcinogenesis.

  19. Elevation of susceptibility to ozone-induced acute tracheobronchial injury in transgenic mice deficient in Clara cell secretory protein

    SciTech Connect

    Plopper, C.G. . E-mail: cgplopper@ucdavis.edu; Mango, G.W.; Hatch, G.E.; Wong, V.J.; Toskala, E.; Reynolds, S.D.; Tarkington, B.K.; Stripp, B.R.

    2006-05-15

    Increases in Clara cell abundance or cellular expression of Clara cell secretory protein (CCSP) may cause increased tolerance of the lung to acute oxidant injury by repeated exposure to ozone (O{sub 3}). This study defines how disruption of the gene for CCSP synthesis affects the susceptibility of tracheobronchial epithelium to acute oxidant injury. Mice homozygous for a null allele of the CCSP gene (CCSP-/-) and wild type (CCSP+/+) littermates were exposed to ozone (0.2 ppm, 8 h; 1 ppm, 8 h) or filtered air. Injury was evaluated by light and scanning electron microscopy, and the abundance of necrotic, ciliated, and nonciliated cells was estimated by morphometry. Proximal and midlevel intrapulmonary airways and terminal bronchioles were evaluated. There was no difference in airway epithelial composition between CCSP+/+ and CCSP-/- mice exposed to filtered air, and exposure to 0.2 ppm ozone caused little injury to the epithelium of both CCSP+/+ and CCSP-/- mice. After exposure to 1.0 ppm ozone, CCSP-/- mice suffered from a greater degree of epithelial injury throughout the airways compared to CCSP+/+ mice. CCSP-/- mice had both ciliated and nonciliated cell injury. Furthermore, lack of CCSP was associated with a shift in airway injury to include proximal airway generations. Therefore, we conclude that CCSP modulates the susceptibility of the epithelium to oxidant-induced injury. Whether this is due to the presence of CCSP on the acellular lining layer surface and/or its intracellular distribution in the secretory cell population needs to be defined.

  20. Elevation of susceptibility to ozone-induced acute tracheobronchial injury in transgenic mice deficient in Clara cell secretory protein.

    PubMed

    Plopper, C G; Mango, G W; Hatch, G E; Wong, V J; Toskala, E; Reynolds, S D; Tarkington, B K; Stripp, B R

    2006-05-15

    Increases in Clara cell abundance or cellular expression of Clara cell secretory protein (CCSP) may cause increased tolerance of the lung to acute oxidant injury by repeated exposure to ozone (O3). This study defines how disruption of the gene for CCSP synthesis affects the susceptibility of tracheobronchial epithelium to acute oxidant injury. Mice homozygous for a null allele of the CCSP gene (CCSP-/-) and wild type (CCSP+/+) littermates were exposed to ozone (0.2 ppm, 8 h; 1 ppm, 8 h) or filtered air. Injury was evaluated by light and scanning electron microscopy, and the abundance of necrotic, ciliated, and nonciliated cells was estimated by morphometry. Proximal and midlevel intrapulmonary airways and terminal bronchioles were evaluated. There was no difference in airway epithelial composition between CCSP+/+ and CCSP-/- mice exposed to filtered air, and exposure to 0.2 ppm ozone caused little injury to the epithelium of both CCSP+/+ and CCSP-/- mice. After exposure to 1.0 ppm ozone, CCSP-/- mice suffered from a greater degree of epithelial injury throughout the airways compared to CCSP+/+ mice. CCSP-/- mice had both ciliated and nonciliated cell injury. Furthermore, lack of CCSP was associated with a shift in airway injury to include proximal airway generations. Therefore, we conclude that CCSP modulates the susceptibility of the epithelium to oxidant-induced injury. Whether this is due to the presence of CCSP on the acellular lining layer surface and/or its intracellular distribution in the secretory cell population needs to be defined.

  1. The frequency of α4β7high memory CD4+ T cells correlates with susceptibility to rectal SIV infection

    PubMed Central

    Martinelli, Elena; Veglia, Filippo; Goode, Diana; Guerra-Perez, Natalia; Aravantinou, Meropi; Arthos, James; Piatak, Michael; Lifson, Jeffrey D.; Blanchard, James; Gettie, Agegnehu; Robbiani, Melissa

    2013-01-01

    Background Integrin α4β7 (α4β7) mediates the homing of CD4+ T cells to gut-associated lymphoid tissues (GALT), which constitute a highly favorable environment for HIV expansion and dissemination. HIV and SIV envelope proteins bind to and signal through α4β7 and during acute infection SIV preferentially infects α4β7high CD4+ T cells. We postulated that the availability of these cells at the time of challenge could influence mucosal SIV transmission and acute viral load (VL). Methods We challenged 17 rhesus macaques with 3000 TCID50 of SIVmac239 rectally and followed the subsets of α4β7+ T and dendritic cells (DCs) by flow cytometry in blood and tissues, before and after challenge. Results We found that the frequency of memory CD4+ T cells that expressed high levels of α4β7 (α4β7high memory CD4+ T cells) in blood before challenge correlated strongly with susceptibility to infection and acute VL. Notably, not only at the time of challenge, but also their frequency 3 weeks before challenge correlated with infection. This association extended to the rectal tissue as we observed a strong direct correlation between the frequency of α4β7high memory CD4+ T cells in blood and rectum before and after challenge. The frequency of α4β7+ myeloid DCs and α4β7high CD80+ DCs also correlated with infection and acute VL, while blood CCR5+ and CD69+ CD4+ T cells could not be associated with infection. Conclusions Our results suggest that animals with higher frequency of α4β7high CD4+ T cells in circulation and in rectal tissue could be more susceptible to SIV rectal transmission. PMID:23797688

  2. Cell-specific RNA aptamer against human CCR5 specifically targets HIV-1 susceptible and inhibits HIV-1 infectivity

    PubMed Central

    Zhou, Jiehua; Satheesan, Sangeetha; Li, Haitang; Weinberg, Marc S.; Morris, Kevin V.; Burnett, John; Rossi, John

    2015-01-01

    SUMMARY The C-C chemokine receptor type 5 (CCR5) is a receptor expressed by T-cells and macrophages that serves as a co-receptor for macrophage-tropic HIV-1. Loss of CCR5 is associated with resistance to HIV-1. Here we combine the live cell-based SELEX with high throughput sequencing technology to generate CCR5 RNA aptamers capable of specifically targeting HIV-1 susceptible cells (as siRNA delivery agent) and inhibiting HIV-1 infectivity (as antiviral agent) via block of the CCR5 required for HIV-1 to enter cells. One of the best candidates, G-3, efficiently bound and was internalized into human CCR5 expressing cells. The G-3 specifically neutralized R5 virus infection in primary peripheral blood mononuclear cells, and in vivo generated human CD4+ T cells with a nanomolar IC50. G-3 was also capable of transferring functional siRNAs to CCR5 expressing cells. Collectively, the cell-specific, internalizing, CCR5-targeted aptamers and aptamer-siRNA conjugates offer promise for overcoming some of the current challenges of drug resistance in HIV-1 by providing cell-type- or tissue-specific delivery of various therapeutic moieties. PMID:25754473

  3. Cell proliferation is a key determinant of the outcome of FOXO3a activation

    SciTech Connect

    Poulsen, Raewyn C. Carr, Andrew J.; Hulley, Philippa A.

    2015-06-19

    The FOXO family of forkhead transcription factors have a pivotal role in determining cell fate in response to oxidative stress. FOXO activity can either promote cell survival or induce cell death. Increased FOXO-mediated cell death has been implicated in the pathogenesis of degenerative diseases affecting musculoskeletal tissues. The aim of this study was to determine the conditions under which one member of the FOXO family, FOXO3a, promotes cell survival as opposed to cell death. Treatment of primary human tenocytes with 1 pM hydrogen peroxide for 18 h resulted in increased protein levels of FOXO3a. In peroxide-treated cells cultured in low serum media, FOXO3a inhibited cell proliferation and protected against apoptosis. However in peroxide treated cells cultured in high serum media, cell proliferation was unchanged but level of apoptosis significantly increased. Similarly, in tenocytes transduced to over-express FOXO3a, cell proliferation was inhibited and level of apoptosis unchanged in cells cultured in low serum. However there was a robust increase in cell death in FOXO3a-expressing cells cultured in high serum. Inhibition of cell proliferation in either peroxide-treated or FOXO3a-expressing cells cultured in high serum protected against apoptosis induction. Conversely, addition of a Chk2 inhibitor to peroxide-treated or FOXO3a-expressing cells overrode the inhibitory effect of FOXO3a on cell proliferation and led to increased apoptosis in cells cultured in low serum. This study demonstrates that proliferating cells may be particularly susceptible to the apoptosis-inducing actions of FOXO3a. Inhibition of cell proliferation by FOXO3a may be a critical event in allowing the pro-survival rather than the pro-apoptotic activity of FOXO3a to prevail. - Highlights: • FOXO3a activity can result in either promotion of cell survival or apoptosis. • The outcome of FOXO3a activation differs in proliferating compared to non-proliferating cells. • Proliferating

  4. Resistance to infection with Eimeria vermiformis in mouse radiation chimeras is determined by donor bone-marrow cells

    SciTech Connect

    Joysey, H.S.; Wakelin, D.; Rose, M.E.

    1988-05-01

    The course of infection with Eimeria vermiformis was determined in BALB/b, BALB/c, and C57BL/10ScSn (B10) mice and in radiation chimeras prepared from the H-2-compatible BALB/b and B10 mice. The BALB strains, irrespective of H-2 haplotype, were resistant, the B10 mice were susceptible, and in the chimeras infection was characterized by the genotype of the donated bone-marrow cells and not by the phenotype of the recipient. Thus, the genetic control of relative resistance or susceptibility to infection with this parasite is expressed through bone-marrow-derived cells.

  5. Murine granulated metrial gland cells are susceptible to Chlamydia psittaci infection in vivo.

    PubMed

    Sánchez, J; Buendía, A J; Salinas, J; Bernabé, A; Rodolakis, A; Stewart, I J

    1996-09-01

    Granulated metrial gland (GMG) cells are the most numerous lymphoid cells in the uteroplacental unit in rodent pregnancy. In an experimental murine model of abortion-causing infection, we have studied the responses of GMG cells to Chlamydia psittaci. Chlamydial inclusions have been found within GMG cells, both in apparently healthy cells and in cells with degenerative changes. Establishing the existence of GMG cells infected by C. psittaci opens a new and interesting chapter in the study of these cells.

  6. Murine granulated metrial gland cells are susceptible to Chlamydia psittaci infection in vivo.

    PubMed Central

    Sánchez, J; Buendía, A J; Salinas, J; Bernabé, A; Rodolakis, A; Stewart, I J

    1996-01-01

    Granulated metrial gland (GMG) cells are the most numerous lymphoid cells in the uteroplacental unit in rodent pregnancy. In an experimental murine model of abortion-causing infection, we have studied the responses of GMG cells to Chlamydia psittaci. Chlamydial inclusions have been found within GMG cells, both in apparently healthy cells and in cells with degenerative changes. Establishing the existence of GMG cells infected by C. psittaci opens a new and interesting chapter in the study of these cells. PMID:8751945

  7. Influence of malignant cell clonogenic capacities and position along the maturation pathway on their susceptibility to lymphokine-activated killer cell cytotoxicity.

    PubMed

    Thomas, X; Anglaret, B; Adeleine, P; Maritaz, O; Bailly, M; Fiere, D; Archimbaud, E

    1998-01-01

    In order to investigate the sensitivity of malignant target cells to lysis by LAK cells according to their clonogenic capacities and their position along the maturation pathway, we compared clonogenic and chromium release cytotoxicity assays performed on human hematopoietic cell lines using Effector: Target ratios of 1:1, 3:1, 6:1, 12:1, 24:1, 48:1 and 96:1, and studied the sensitivity of HL-60 and U937 human cell lines after exposure to different factors including GM-CSF, SCF, IFN, Retinoic acid (RA), DMSO, and TPA which are able to recruit cells into the cell cycle or to induce cell differentiation. There was a good correlation between the lysis of the target cells using 51Cr release and the growth inhibition in semisolid medium. The degree of inhibition was significantly higher using the colony growth assay (p = 0.006). Regarding the effects of culturing cell lines with proliferating and differentiating agents on the sensitivity of these cell lines to LAK cytolysis, a correlation was noted between the proliferative response of the U937 cell line and susceptibility to LAK cell lysis (p = 0.01), while results appeared close to significance with HL-60. The most significant effects were a decreased sensitivity of HL-60 to LAK lysis with RA (p < 0.001) and TPA (p < 0.001), and an increased susceptibility of U937 to LAK lysis with GM-CSF (p < 0.0001). In studies planned to investigate whether susceptibility of treated cells to LAK activity was a consequence of a downregulation of adhesion molecules expressed on target cell surface, the proportion of cells expressing adhesion molecules was not significantly changed, except for CD54 expression on HL-60 cells which showed a higher expression, after cells were treated with RA or DMSO. We conclude that clonogenic cells are more sensitive to LAK cell lysis and that cell line sensitivity to LAK cytolysis can be modulated by a variety of agents of potential therapeutic use. The poor correlation between adhesion molecules

  8. Water chemical shift in 1H NMR of red cells: effects of pH when transmembrane magnetic susceptibility differences are low.

    PubMed

    Larkin, Timothy J; Bubb, William A; Kuchel, Philip W

    2008-04-01

    The (1)H magic angle spinning (MAS) NMR spectrum of water in erythrocyte suspensions shows peaks from each of the intracellular and extracellular water pools. The splitting is a true chemical shift and is brought about by the elimination of water exchange under MAS conditions due to physical separation of the two water populations. The size of the chemical shift difference is determined by the concentration of intracellular protein affecting the average extent of hydrogen bonding of water. We present here a model of the chemical shift behavior for water in erythrocytes under normal high-resolution NMR conditions based on results from MAS experiments on these cells exposed to different pH and osmotic conditions. The model accurately predicts the chemical shift of water for a static sample, and the results demonstrate that in high-resolution NMR experiments the chemical shift of water will appear to be invariant if differences in magnetic susceptibility across the cell membrane are minimal (<10% of the magnetic susceptibility of water). Thus, changes in the shape and chemical shift of the water resonance are not due to pH changes in the physiological range. The findings are fundamental to an interpretation of the mechanism of chemical shift effects on the water resonance that may occur in functional MRI.

  9. Transactivation of the TIEG1 confers growth inhibition of transforming growth factor-β-susceptible hepatocellular carcinoma cells

    PubMed Central

    Jiang, Lei; Lai, Yiu-Kay; Zhang, Jin-Fang; Chan, Chu-Yan; Lu, Gang; Lin, Marie CM; He, Ming-Liang; Li, Ji-Cheng; Kung, Hsiang-Fu

    2012-01-01

    AIM: To investigate the role of transforming growth factor (TGF)-β-inducible early gene 1 (TIEG1) in TGF-β-induced growth inhibition in hepatocellular carcinoma (HCC) cells. METHODS: Human hepatocyte and HCC cell lines with varied susceptibilities to TGF-β1 were tested by methylthiazoletetrazolium (MTT) assay. The expression changes of Smad2, Smad3, Smad4, Smad7, TIEG1 and TIEG2 gene following treatment with TGF-β1 in a TGF-β-sensitive hepatocyte cell line (MIHA), a TGF-β-sensitive hepatoma cell line (Hep3B) and two TGF-β-insensitive hepatoma cell lines (HepG2 and Bel7404) were examined. SiRNA targeting TIEG1 was transfected into Hep3B cells and the sensitivity of cells to TGF-β1 was examined. Overexpression of TIEG1 was induced by lentiviral-mediated transduction in TGF-β1-resistant hepatoma cell lines (Bel7404 and HepG2). MTT assay and 4’,6-Diamidino-2-phenylindole staining were used to identify cell viability and apoptosis, respectively. The expression level of stathmin was measured by reverse transcriptase polymerase chain reaction and Western-blotting analysis, and stathmin promoter activity by TIEG1 was monitored by a luciferase reporter gene system. RESULTS: TIEG1 was significantly upregulated by TGF-β1 in the TGF-β1-sensitive HCC cell line, Hep3B, but not in the resistant cell lines. The suppression of TIEG1 by siRNAs decreased the sensitivity of Hep3B cells to TGF-β1, whereas the overexpression of TIEG1 mediated growth inhibition and apoptosis in TGF-β1-resistant HCC cell lines, which resembled those of TGF-β1-sensitive HCC cells treated with TGF-β1. Our data further suggested that stathmin was a direct target of TIEG1, as stathmin was significantly downregulated by TIEG1 overexpression, and stathmin promoter activity was inhibited by TIEG1 in a dose-dependent manner. CONCLUSION: Our data suggest that transactivation of TIEG1 conferred growth inhibition of TGF-β-susceptible human HCC cells. PMID:22563190

  10. [Comparison of microdilution and disk diffusion methods for the detection of fluconazole and voriconazole susceptibility against clinical Candida glabrata isolates and determination of changing susceptibility with new CLSI breakpoints].

    PubMed

    Hazırolan, Gülşen; Sarıbaş, Zeynep; Arıkan Akdağlı, Sevtap

    2016-07-01

    Candida albicans is the most frequently isolated species as the causative agent of Candida infections. However, in recent years, the isolation rate of non-albicans Candida species have increased. In many centers, Candida glabrata is one of the commonly isolated non-albicans species of C.glabrata infections which are difficult-to-treat due to decreased susceptibility to fluconazole and cross-resistance to other azoles. The aims of this study were to determine the in vitro susceptibility profiles of clinical C.glabrata isolates against fluconazole and voriconazole by microdilution and disk diffusion methods and to evaluate the results with both the previous (CLSI) and current species-specific CLSI (Clinical and Laboratory Standards Institute) clinical breakpoints. A total of 70 C.glabrata strains isolated from clinical samples were included in the study. The identification of the isolates was performed by morphologic examination on cornmeal Tween 80 agar and assimilation profiles obtained by using ID32C (BioMérieux, France). Broth microdilution and disk diffusion methods were performed according to CLSI M27-A3 and CLSI M44-A2 documents, respectively. The results were evaluated according to CLSI M27-A3 and M44-A2 documents and new vs. species-specific CLSI breakpoints. By using both previous and new CLSI breakpoints, broth microdilution test results showed that voriconazole has greater in vitro activity than fluconazole against C.glabrata isolates. For the two drugs tested, very major error was not observed with disk diffusion method when microdilution method was considered as the reference method. Since "susceptible" category no more exists for fluconazole vs. C.glabrata, the isolates that were interpreted as susceptible by previous breakpoints were evaluated as susceptible-dose dependent by current CLSI breakpoints. Since species-specific breakpoints remain yet undetermined for voriconazole, comparative analysis was not possible for this agent. The results obtained

  11. Restoration of susceptibility of intracellular methicillin-resistant Staphylococcus aureus to beta-lactams: comparison of strains, cells, and antibiotics.

    PubMed

    Lemaire, Sandrine; Olivier, Aurélie; Van Bambeke, Françoise; Tulkens, Paul M; Appelbaum, Peter C; Glupczynski, Youri

    2008-08-01

    Staphylococcus aureus invades eukaryotic cells. When methicillin-resistant S. aureus (MRSA) ATCC 33591 is phagocytized by human THP-1 macrophages, complete restoration of susceptibility to cloxacillin and meropenem is shown and the strain becomes indistinguishable from MSSA ATCC 25923 due to the acid pH prevailing in phagolysosomes (S. Lemaire et al., Antimicrob. Agents Chemother. 51:1627-1632, 2007). We examined whether this observation can be extended to (i) strains of current clinical and epidemiological interest (three hospital-acquired MRSA [HA-MRSA] strains, two community-acquired MRSA [CA-MRSA] strains, two HA-MRSA strains with the vancomycin-intermediate phenotype, one HA-MRSA strain with the vancomycin-resistant phenotype, and one animal [porcine] MRSA strain), (ii) activated THP-1 cells and nonprofessional phagocytes (keratinocytes, Calu-3 bronchial epithelial cells), and (iii) other beta-lactams (imipenem, oxacillin, cefuroxime, cefepime). All strains showed (i) a marked reduction in MICs in broth at pH 5.5 compared with the MIC at pH 7.4 and (ii) sigmoidal dose-response curves with cloxacillin (0.01x to 100x MIC, 24 h of incubation) after phagocytosis by THP-1 macrophages that were indistinguishable from each other and from the dose-response curve for methicillin-susceptible S. aureus (MSSA) ATCC 25923 (relative potency [50% effect], 6.09x MIC [95% confidence interval {CI}, 4.50 to 8.25]; relative efficacy [change in bacterial counts over the original inoculum for an infinitely large cloxacillin concentration, or maximal effect], -0.69 log CFU [95% CI, -0.79 to -0.58]). Similar dose-response curves for cloxacillin were also observed with MSSA ATCC 25923 and MRSA ATCC 33591 after phagocytosis by activated THP-1 macrophages, keratinocytes, and Calu-3 cells. By contrast, there was a lower level of restoration of susceptibility of MRSA ATCC 33591 to cefuroxime and cefepime after phagocytosis by THP-1 macrophages, even when the data were normalized for

  12. The genomic landscape of testicular germ cell tumours: from susceptibility to treatment.

    PubMed

    Litchfield, Kevin; Levy, Max; Huddart, Robert A; Shipley, Janet; Turnbull, Clare

    2016-07-01

    The genomic landscape of testicular germ cell tumour (TGCT) can be summarized using four overarching hypotheses. Firstly, TGCT risk is dominated by inherited genetic factors, which determine nearly half of all disease risk and are highly polygenic in nature. Secondly KIT-KITLG signalling is currently the major pathway that is implicated in TGCT formation, both as a predisposition risk factor and a somatic driver event. Results from genome-wide association studies have also consistently suggested that other closely related pathways involved in male germ cell development and sex determination are associated with TGCT risk. Thirdly, the method of disease formation is unique, with tumours universally stemming from a noninvasive precursor lesion, probably of fetal origin, which lies dormant through childhood into adolescence and then eventually begins malignant growth in early adulthood. Formation of a 12p isochromosome, a hallmark of TGCT observed in nearly all tumours, is likely to be a key triggering event for malignant transformation. Finally, TGCT have been shown to have a distinctive somatic mutational profile, with a low rate of point mutations contrasted with frequent large-scale chromosomal gains. These four hypotheses by no means constitute a complete model that explains TGCT tumorigenesis, but advances in genomic technologies have enabled considerable progress in describing and understanding the disease. Further advancing our understanding of the genomic basis of TGCT offers a clear opportunity for clinical benefit in terms of preventing invasive cancer arising in young men, decreasing the burden of chemotherapy-related survivorship issues and reducing mortality in the minority of patients who have treatment-refractory disease. PMID:27296647

  13. Potentiation of a tumor cell susceptibility to autologous CTL killing by restoration of wild-type p53 function.

    PubMed

    Thiery, Jérôme; Dorothée, Guillaume; Haddada, Hedi; Echchakir, Hamid; Richon, Catherine; Stancou, Rodica; Vergnon, Isabelle; Benard, Jean; Mami-Chouaib, Fathia; Chouaib, Salem

    2003-06-15

    Inactivation of p53 has been implicated in many types of tumors particularly in non-small cell lung carcinoma, one of the most common cancers in which p53 mutation has been frequently identified. The aim of this study was to investigate the influence of p53 status on the regulation of tumor susceptibility to specific CTL-mediated cell death. For this purpose, we used a cytotoxic T lymphocyte clone, Heu127, able to lyse the human autologous lung carcinoma cell line, IGR-Heu, in a HLA-A2-restricted manner. Direct genomic DNA sequencing revealed that IGR-Heu expresses a mutated p53 at codon 132 of the exon 5 which results in the loss of p53 capacity to induce the expression of the p53-regulated gene product p21(waf/CIP1). Initial experiments demonstrated that IGR-Heu was resistant to Fas, TNF, and TRAIL apoptotic pathways. This correlated with the lack of p55 TNFRI, Fas, DR4, and DR5 expression. The effect of wild-type (wt) p53 restoration on the sensitization of IGR-Heu to autologous CTL clone lysis was investigated following infection of the tumor cell line with a recombinant adenovirus encoding the wt p53 (Adwtp53). We demonstrate that the restoration of wt p53 expression and function resulted in a significant potentiation of target cell susceptibility to CTL-mediated lysis. The wt p53-induced optimization of tumor cell killing by specific CTL involves at least in part Fas-mediated pathway via induction of CD95 expression by tumor cells but does not appear to interfere with granzyme B cytotoxic pathway. PMID:12794118

  14. Propofol Increases Host Susceptibility to Microbial Infection by Reducing Subpopulations of Mature Immune Effector Cells at Sites of Infection

    PubMed Central

    Visvabharathy, Lavanya; Xayarath, Bobbi; Weinberg, Guy; Shilling, Rebecca A.; Freitag, Nancy E.

    2015-01-01

    Anesthetics are known to modulate host immune responses, but separating the variables of surgery from anesthesia when analyzing hospital acquired infections is often difficult. Here, the bacterial pathogen Listeria monocytogenes (Lm) was used to assess the impact of the common anesthetic propofol on host susceptibility to infection. Brief sedation of mice with physiologically relevant concentrations of propofol increased bacterial burdens in target organs by more than 10,000-fold relative to infected control animals. The adverse effects of propofol sedation on immune clearance of Lm persisted after recovery from sedation, as animals given the drug remained susceptible to infection for days following anesthesia. In contrast to propofol, sedation with alternative anesthetics such as ketamine/xylazine or pentobarbital did not increase susceptibility to systemic Lm infection. Propofol altered systemic cytokine and chemokine expression during infection, and prevented effective bacterial clearance by inhibiting the recruitment and/or activity of immune effector cells at sites of infection. Propofol exposure induced a marked reduction in marginal zone macrophages in the spleens of Lm infected mice, resulting in bacterial dissemination into deep tissue. Propofol also significantly increased mouse kidney abscess formation following infection with the common nosocomial pathogen Staphylococcus aureus. Taken together, these data indicate that even brief exposure to propofol severely compromises host resistance to microbial infection for days after recovery from sedation. PMID:26381144

  15. Establishment, characterization, and virus susceptibility of a new marine cell line from red spotted grouper (Epinephelus akaara).

    PubMed

    Zhou, Guang-Zhou; Li, Zheng-Qiu; Yuan, Xiu-Ping; Zhang, Qi-Ya

    2007-01-01

    A marine fish cell line from the snout of red spotted grouper Epinephelus akaara, a protogynous hermaphrodite, was established, characterized, and subcultured with more than 60 passages. The grouper snout cell line (GSC) cells multiplied well in Dulbecco's modified Eagle's medium (DMEM) medium supplemented with 10% fetal bovine serum. The optimal growth temperature was 25 degrees C, and morphologically the cells were fibroblastic. Chromosome analysis revealed that the GSC cell line has a normal diploid karyotype with 2n = 8st + 40t. A virus titration study indicated that the cells were susceptible to turbot Scophthalmus Maximus rhabdovirus (SMRV) (10(8.5) TCID(50) ml(-1)), while the viral titer of frog Rana grylio virus 9807 (RGV(9807)) reached 10(3.5) TCID(50) ml(-1). The infection was confirmed by cytopathic effect (CPE), immunofluorescence, and electron microscopy experiments, which detected the viral particles in the cytoplasm of virus-infected cells, respectively. Further, significant fluorescent signals were observed when the GSC cells were transfected with pEGFP vector DNA, indicating their potential utility for transgenic and genetic manipulation studies.

  16. T Helper1/T Helper2 Cells and Resistance/Susceptibility to Leishmania Infection: Is This Paradigm Still Relevant?

    PubMed Central

    Alexander, James; Brombacher, Frank

    2012-01-01

    Work in large part on Leishmania major in the 1980s identified two distinct apparently counter-regulatory CD4+ T cell populations, T helper (h)1 and Th2, that controlled resistance/susceptibility to infection respectively. However, the generation of IL-4−/− mice in the 1990s questioned the paramount role of this Th2 archetypal cytokine in the non-healing response to Leishmania infection. The more recent characterization of CD4+ T cell regulatory populations and further effector CD4+ T helper populations, Th17, Th9, and T follicular (f)h cells as well as the acknowledged plasticity in T helper cell function has further added to the complexity of host pathogen interactions. These interactions are complicated by the multiplicity of cells that respond to CD4+ T cell subset signatory cytokines, as well as the diversity of Leishmania species that are often subject to significantly different immune-regulatory controls. In this article we review current knowledge with regard to the role of CD4+ T cells and their products during Leishmania infection. In particular we update on our studies using conditional IL-4Rα gene-deficient mice that have allowed dissection of the cell interplay dictating the disease outcomes of the major Leishmania species infecting humans. PMID:22566961

  17. Determining the Efficacy of Magnetic Susceptibility as an Analytical Tool in the Middle Devonian Gas Bearing Shale of Taylor County, West Virginia

    NASA Astrophysics Data System (ADS)

    Baird, John

    The magnetic susceptibility of two Middle Devonian shale units, the Mahantango Formation and Marcellus Shale, was recorded in order to determine if magnetic susceptibility could be used to predict (1) transgressive and regressive cycles, (2) brittleness, and (3) total organic content (TOC). A core from Taylor County, West Virginia was selected for this purpose. Transgressive and regressive cycles were detected through variations of magnetic susceptibility values with maximum flooding surfaces indicated by troughs in the data and maximum regressive surfaces indicated by peaks. A sequence stratigraphic framework based upon variations in gamma ray and density measurements was used to establish a standard to gauge the accuracy of predictions made through magnetic susceptibility. It was found that the accuracy of the magnetic susceptibility method was similar to the gamma-density method in detecting a large 2nd order cycle, when both shale units were evaluated together. When the units were evaluated separately, it was found that both methods detected the same 3rd order cycles. However, within the Mahantango Formation the magnetic susceptibility method was more accurate recording 4th order cycles that the gamma-density method did not. Conversely, within the Marcellus Shale, the gamma-density method was more accurate recording 4th order cycles that the magnetic susceptibility method did not. It was concluded that the increased accuracy of the gamma-density method in the Marcellus shale was due to an increased sensitivity in the gamma ray and density logs as a response to the large amounts of TOC in the formation This increased sensitivity allowed for smaller variations to be more easily detected. The Mahantango Formation does not have large quantities of TOC. This diminished the sensitivity of the gamma and density logs allowing for the magnetic susceptibility method to be more accurate. It was assumed that variations in brittleness are driven by transgressive and

  18. Tat protein expression in MDBK cells does not confer susceptibility to bovine immunodeficiency virus.

    PubMed

    Kempster, S; Collins, M E; Brownlie, J

    2002-03-01

    The ability of BIV strain R29 to infect bovine cell lines in the presence or absence of a functional lentiviral Tat protein is described. Jembrana disease virus (JDV) Tat protein was stably expressed in MDBK cells. No viral replication could be detected in this cell line after infection with BIV R29. Transfection of MDBK cells and MDBK Tat expressing cells with BIV R29 proviral DNA established that BIV R29 could not replicate in MDBK cells. Whether viral entry into MDBK cells is also a block to BIV R29 infection of MDBK cells has yet to be established.

  19. Determinants of Human Immunodeficiency Virus Type 1 Baseline Susceptibility to the Fusion Inhibitors Enfuvirtide and T-649 Reside outside the Peptide Interaction Site

    PubMed Central

    Heil, Marintha L.; Decker, Julie M.; Sfakianos, Jeffrey N.; Shaw, George M.; Hunter, Eric; Derdeyn, Cynthia A.

    2004-01-01

    The peptide fusion inhibitor (PFI) enfuvirtide is the first of a new class of entry inhibitors to receive FDA approval. We previously determined the susceptibility of 55 PFI-naïve-patient isolates to enfuvirtide and a second peptide inhibitor, T-649. Seven of the 55 viral isolates were insusceptible to enfuvirtide, T-649, or both inhibitors in the absence of prior exposure. To determine the molecular basis of the insusceptible phenotypes, we PCR amplified and cloned five PFI-insusceptible and one PFI-susceptible, full-length, biologically functional env genes and characterized viruses pseudotyped with the Env proteins in a single-round drug sensitivity assay. Overall, the mean 50% inhibitory concentrations of enfuvirtide and T-649 for the PFI-insusceptible Env pseudotypes were 1.4 to 1.7 log10 and 1.2 to 1.8 log10 greater, respectively, than those for a PFI-susceptible lab strain, NLHX; however, all of the PFI-insusceptible Env proteins conserved the sequence of a critical enfuvirtide interaction site (residues 36 to 38 of gp41, GIV) in HR-1. In contrast, multiple amino acid changes were observed C-terminal to HR-1, many of which were located in regions of HR-2 corresponding to the PFI. Nevertheless, peptides based on patient-derived HR-2 sequences were not more potent inhibitors than enfuvirtide or T-649, arguing that the basis of PFI susceptibility is not a higher-affinity, competitive HR-1/HR-2 interaction. These results demonstrate that regions of Env outside the enfuvirtide interaction site can significantly impact the PFI susceptibility of patient-derived Env, even prior to drug exposure. We hypothesize that both gp120 gene- and gp41 gene-encoded determinants that minimize the window of opportunity for PFI to bind provide a growth advantage and possibly a predisposition to resistance to this new class of drugs in vivo. PMID:15220433

  20. Effects of Female Sex Hormones on Susceptibility to HSV-2 in Vaginal Cells Grown in Air-Liquid Interface.

    PubMed

    Lee, Yung; Dizzell, Sara E; Leung, Vivian; Nazli, Aisha; Zahoor, Muhammad A; Fichorova, Raina N; Kaushic, Charu

    2016-01-01

    The lower female reproductive tract (FRT) is comprised of the cervix and vagina, surfaces that are continuously exposed to a variety of commensal and pathogenic organisms. Sexually transmitted viruses, such as herpes simplex virus type 2 (HSV-2), have to traverse the mucosal epithelial lining of the FRT to establish infection. The majority of current culture systems that model the host-pathogen interactions in the mucosal epithelium have limitations in simulating physiological conditions as they employ a liquid-liquid interface (LLI), in which both apical and basolateral surfaces are submerged in growth medium. We designed the current study to simulate in vivo conditions by growing an immortalized vaginal epithelial cell line (Vk2/E6E7) in culture with an air-liquid interface (ALI) and examined the effects of female sex hormones on their growth, differentiation, and susceptibility to HSV-2 under these conditions, in comparison to LLI cultures. ALI conditions induced Vk2/E6E7 cells to grow into multi-layered cultures compared to the monolayers present in LLI conditions. Vk2 cells in ALI showed higher production of cytokeratin in the presence of estradiol (E2), compared to cells grown in progesterone (P4). Cells grown under ALI conditions were exposed to HSV-2-green fluorescent protein (GFP) and the highest infection and replication was observed in the presence of P4. Altogether, this study suggests that ALI cultures more closely simulate the in vivo conditions of the FRT compared to the conventional LLI cultures. Furthermore, under these conditions P4 was found to confer higher susceptibility to HSV-2 infection in vaginal cells. The vaginal ALI culture system offers a better alternative to study host-pathogen interactions. PMID:27589787

  1. Upregulation of retinoic acid receptor-β reverses drug resistance in cholangiocarcinoma cells by enhancing susceptibility to apoptosis

    PubMed Central

    Ren, Hong-Yue; Chen, Bo; Huang, Gui-Li; Liu, Yu; Shen, Dong-Yan

    2016-01-01

    Retinoic acid receptor β (RARβ), a known tumor suppressor gene, is frequently silenced in numerous malignant types of tumor. Recent reports have demonstrated that loss of RARβ expression may be responsible, in part, for the drug resistance observed in clinical trials. However, little is known about the role of RARβ in regulating drug sensitivity in patients with cholangiocarcinoma (CCA) with a high risk of mortality and poor outcomes. In the present study, low RARβ expression was observed in the majority of CCA tissues investigated (28/33, 84.8%). In addition, the CCA cell line QBC939, which exhibits low RARβ expression, was found to be significantly resistant to chemotherapeutic agents compared with SK-ChA-1, MZ-ChA-1 and Hccc9810 CCA cell lines, which exhibit high RARβ expression. Furthermore, upregulation of RARβ significantly enhanced the sensitivity of QBC939 cells to common chemotherapeutic agents both in vitro and in vivo. Upregulation of RARβ was shown to increase the expression of proapoptotic genes bax, bak and bim, in addition to caspase-3 activity, and decrease the expression of antiapoptotic genes bcl-2, bcl-xL and mcl-1. As a result, CCA cells were more susceptible to caspase-dependent apoptosis. Taken together, these data suggest that RARβ upregulation rendered CCA cells more sensitive to chemotherapeutic agents by increasing the susceptibility of cells to caspase-dependent apoptosis. These results support the hypothesis that RARβ may be an ideal chemosensitization target for the treatment of patients with drug-resistant CCA. PMID:27599527

  2. Upregulation of retinoic acid receptor-β reverses drug resistance in cholangiocarcinoma cells by enhancing susceptibility to apoptosis.

    PubMed

    Ren, Hong-Yue; Chen, Bo; Huang, Gui-Li; Liu, Yu; Shen, Dong-Yan

    2016-10-01

    Retinoic acid receptor β (RARβ), a known tumor suppressor gene, is frequently silenced in numerous malignant types of tumor. Recent reports have demonstrated that loss of RARβ expression may be responsible, in part, for the drug resistance observed in clinical trials. However, little is known about the role of RARβ in regulating drug sensitivity in patients with cholangiocarcinoma (CCA) with a high risk of mortality and poor outcomes. In the present study, low RARβ expression was observed in the majority of CCA tissues investigated (28/33, 84.8%). In addition, the CCA cell line QBC939, which exhibits low RARβ expression, was found to be significantly resistant to chemotherapeutic agents compared with SK‑ChA‑1, MZ‑ChA‑1 and Hccc9810 CCA cell lines, which exhibit high RARβ expression. Furthermore, upregulation of RARβ significantly enhanced the sensitivity of QBC939 cells to common chemotherapeutic agents both in vitro and in vivo. Upregulation of RARβ was shown to increase the expression of proapoptotic genes bax, bak and bim, in addition to caspase‑3 activity, and decrease the expression of antiapoptotic genes bcl‑2, bcl‑xL and mcl‑1. As a result, CCA cells were more susceptible to caspase‑dependent apoptosis. Taken together, these data suggest that RARβ upregulation rendered CCA cells more sensitive to chemotherapeutic agents by increasing the susceptibility of cells to caspase-dependent apoptosis. These results support the hypothesis that RARβ may be an ideal chemosensitization target for the treatment of patients with drug-resistant CCA. PMID:27599527

  3. Effects of Female Sex Hormones on Susceptibility to HSV-2 in Vaginal Cells Grown in Air-Liquid Interface

    PubMed Central

    Lee, Yung; Dizzell, Sara E.; Leung, Vivian; Nazli, Aisha; Zahoor, Muhammad A.; Fichorova, Raina N.; Kaushic, Charu

    2016-01-01

    The lower female reproductive tract (FRT) is comprised of the cervix and vagina, surfaces that are continuously exposed to a variety of commensal and pathogenic organisms. Sexually transmitted viruses, such as herpes simplex virus type 2 (HSV-2), have to traverse the mucosal epithelial lining of the FRT to establish infection. The majority of current culture systems that model the host-pathogen interactions in the mucosal epithelium have limitations in simulating physiological conditions as they employ a liquid-liquid interface (LLI), in which both apical and basolateral surfaces are submerged in growth medium. We designed the current study to simulate in vivo conditions by growing an immortalized vaginal epithelial cell line (Vk2/E6E7) in culture with an air-liquid interface (ALI) and examined the effects of female sex hormones on their growth, differentiation, and susceptibility to HSV-2 under these conditions, in comparison to LLI cultures. ALI conditions induced Vk2/E6E7 cells to grow into multi-layered cultures compared to the monolayers present in LLI conditions. Vk2 cells in ALI showed higher production of cytokeratin in the presence of estradiol (E2), compared to cells grown in progesterone (P4). Cells grown under ALI conditions were exposed to HSV-2-green fluorescent protein (GFP) and the highest infection and replication was observed in the presence of P4. Altogether, this study suggests that ALI cultures more closely simulate the in vivo conditions of the FRT compared to the conventional LLI cultures. Furthermore, under these conditions P4 was found to confer higher susceptibility to HSV-2 infection in vaginal cells. The vaginal ALI culture system offers a better alternative to study host-pathogen interactions. PMID:27589787

  4. Validation of Six Genetic Determinants of Susceptibility to Estrogen-Induced Mammary Cancer in the Rat and Assessment of Their Relevance to Breast Cancer Risk in Humans

    PubMed Central

    Colletti, John A.; Leland-Wavrin, Kristin M.; Kurz, Scott G.; Hickman, Maureen Peters; Seiler, Nicole L.; Samanas, Nyssa Becker; Eckert, Quincy A.; Dennison, Kirsten L.; Ding, Lina; Schaffer, Beverly S.; Shull, James D.

    2014-01-01

    When treated with 17β-estradiol, female ACI rats (Rattus norvegicus) rapidly develop mammary cancers that share multiple phenotypes with luminal breast cancers. Seven distinct quantitative trait loci that harbor genetic determinants of susceptibility to 17β-estradiol−induced mammary cancer have been mapped in reciprocal intercrosses between susceptible ACI rats and resistant Brown Norway (BN) rats. A panel of unique congenic rat strains has now been generated and characterized to confirm the existence of these quantitative trait loci, designated Emca3 through Emca9, and to quantify their individual effects on susceptibility to 17β-estradiol−induced mammary cancer. Each congenic strain carries BN alleles spanning an individual Emca locus, introgressed onto the ACI genetic background. Data presented herein indicate that BN alleles at Emca3, Emca4, Emca5, Emca6, and Emca9 reduce susceptibility to 17β-estradiol−induced mammary cancer, whereas BN alleles at Emca7 increase susceptibility, thereby confirming the previous interval mapping data. All of these Emca loci are orthologous to regions of the human genome that have been demonstrated in genome-wide association studies to harbor genetic variants that influence breast cancer risk. Moreover, four of the Emca loci are orthologous to loci in humans that have been associated with mammographic breast density, a biomarker of breast cancer risk. This study further establishes the relevance of the ACI and derived congenic rat models of 17β-estradiol−induced mammary cancer for defining the genetic bases of breast cancer susceptibility and elucidating the mechanisms through which 17β-estradiol contributes to breast cancer development. PMID:24875630

  5. Ultrastructural and Metabolic Determinants of Resistance to Azo-dye and Susceptibility to Nitrosamine Carcinogenesis of the Guinea-pig

    PubMed Central

    Bryant, G. M.; Sohal, R. S.; Argus, M. F.; Arcos, J. C.

    1977-01-01

    During diethylnitrosamine (DEN) administration, a distinctive difference was observed between rats and guinea-pigs in the sequence of ultrastructural changes in the hepatic endoplasmic reticulum (ER). In DEN-induced hepatic tumour cells in the guinea-pig there was extensive proliferation of the rough ER, while the smooth ER was quite sparse; in the premalignant liver the opposite was noted. This is in contrast to the rat, in which administration of either DEN or 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB) brings about, in both premalignant and malignant hepatic tissue, proliferation of the smooth ER and sparsity of the rough ER. Yet, as in the rat, the number of ribosomes on the outer surface of the guinea-pig liver rough ER is greatly reduced and this is paralleled by a 49% decrease of the RNA/protein ratio as early as 4 weeks of nitrosamine administration. The decrease of RNA/protein ratio and ultrastructurally observed loss of ribosomes from the ER, following nitrosamine administration, correlate with a decrease of photometric response of microsomal suspensions to the sulphydryl probe, p-chloromercuribenzoate. While azo-dye-reductase activity is higher in untreated rats than in untreated guinea-pigs, feeding 3′-Me-DAB for 6 weeks brings about a 76% decrease in the rat, but no significant decrease in the guinea-pig, which is refractory to azo-dye carcinogenesis. Thus, the ability of the liver to inactivate the dye is greatly decreased in the rat, but not in the guinea-pig, as administration progresses toward the threshold dose for tumorigenesis. On the other hand, constitutive levels of nitrosamine dealkylase are identical in the 2 species and remain essentially unchanged following administration of DEN for 10 weeks. Inasmuch as nitrosamine dealkylation represents activating metabolism, this provides a rationale for the comparable susceptibility of the rat and guinea-pig to DEN carcinogenesis. Of the 2 enzymes in the 2 species, it is only azo

  6. An investigation of the genetic basis of increased susceptibility to neutralization by anti-fusion glycoprotein antibody arising on passage of human respiratory syncytial virus in cell culture.

    PubMed

    Hiriote, W; Gias, E L Michael; Welsh, S H; Toms, G L

    2015-01-01

    Human respiratory syncytial virus isolates have previously been shown to exhibit resistance to neutralization by anti-fusion glycoprotein antibodies that is lost on passage in cell culture. Early passage resistant and late passage susceptible stocks of two virus isolates from different epidemics were cloned by plaque purification. Early passage stocks of both isolates yielded predominantly neutralization resistant clones while late passage stocks yielded predominantly susceptible clones. On further characterization of resistant and susceptible clones, resistant virus yields were lower and they were relatively resistant to both neutralization and fusion inhibition by anti-F murine monoclonal antibodies and were also resistant to neutralization by human sera and by Palivizumab. The full genome of resistant and susceptible clones from one of the isolates was sequenced. Four differences, confirmed by sequencing sister clones, were found between resistant and susceptible clones, one in each of the SH, G, F, and L genes.

  7. The efficiency of silencing expression of the gene coding STAT3 transcriptional factor and susceptibility of bladder cancer cells to apoptosis

    PubMed Central

    Bednarek, Ilona; Sypniewski, Daniel; Gawlik, Natalia; Goraus, Karol

    2012-01-01

    Aim of the study Abnormalities in signaling as well as altered gene expression have been identified in numerous diseases, including cancer. The biological functions of signal transducer and activator of transcription 3 (STAT3) are very broad. It is thought that STAT3 can also contribute to oncogenesis. RNA interference (RNAi) is one of the most efficient tools for silencing gene expression within cells. The main goal of the study was to verify the effectiveness of STAT3 gene silencing and its influence on cell proliferation and activation of apoptosis in bladder cancer cells. Material and methods The study was conducted on cellular material, which was the stable human bladder cancer cell line T24. The synthesis of shRNA (short hairpin RNA) interfering with the STAT3 gene was based on pSUPER. neo expression vector. The gene expression at the mRNA level was determined by the real-time PCR method. The influence of STAT3 gene silencing on apoptosis induced in cells with modulated STAT3 expression was evaluated using parallel quantification of mono- and oligonucleosomal DNA degradation of genomic DNA. Results In transfected T24 cells, the STAT3 mRNA expression decreased to the level of 68.3% compared to the scrambled (SCR) control. Silencing the STAT3 gene induced changes in the phenotype of T24 cells. Statistically significant differences in cell proliferation (p = 0.0318) and apoptosis induction (p = 0.0376) were observed. Conclusions Application of the designed shRNA for the STAT3 gene contributed to a decrease of expression of the examined gene. It also decreased the proliferation and increased the susceptibility to apoptosis in T24 bladder cancer cells. PMID:23788901

  8. Oxaliplatin regulates expression of stress ligands in ovarian cancer cells and modulates their susceptibility to natural killer cell-mediated cytotoxicity.

    PubMed

    Siew, Yin-Yin; Neo, Soek-Ying; Yew, Hui-Chuing; Lim, Shun-Wei; Ng, Yi-Cheng; Lew, Si-Min; Seetoh, Wei-Guang; Seow, See-Voon; Koh, Hwee-Ling

    2015-12-01

    Selected cytotoxic chemicals can provoke the immune system to recognize and destroy malignant tumors. Most of the studies on immunogenic cell death are focused on the signals that operate on a series of receptors expressed by dendritic cells to induce tumor antigen-specific T-cell responses. Here, we explored the effects of oxaliplatin, an immunogenic cell death inducer, on the induction of stress ligands and promotion of natural killer (NK) cell-mediated cytotoxicity in human ovarian cancer cells. The results indicated that treatment of tumor cells with oxaliplatin induced the production of type I interferons and chemokines and enhanced the expression of major histocompatibility complex class I-related chains (MIC) A/B, UL16-binding protein (ULBP)-3, CD155 and TNF-related apoptosis-inducing ligand (TRAIL)-R1/R2. Furthermore, oxaliplatin but not cisplatin treatment enhanced susceptibility of ovarian cancer cells to NK cell-mediated cytolysis. In addition, activated NK cells completely abrogated the growth of cancer cells that were pretreated with oxaliplatin. However, cancer cells pretreated with the same concentration of oxaliplatin alone were capable of potentiating regrowth over a period of time. These results suggest an advantage in combining oxaliplatin and NK cell-based therapy in the treatment of ovarian cancer. Further investigation on such potential combination therapy is warranted.

  9. TIM-3 Genetic Variations Affect Susceptibility to Osteoarthritis by Interfering with Interferon Gamma in CD4+ T Cells.

    PubMed

    Li, Shufeng; Ren, Yanjun; Peng, Dayong; Yuan, Zhen; Shan, Shiying; Sun, Huaqiang; Yan, Xinfeng; Xiao, Hong; Li, Guang; Song, Haihan

    2015-10-01

    Osteoarthritis (OA) is the most common type of arthritis, in which T cell responses and cytokines may play critical roles in the development of the disease. TIM-3 may affect immune responses and is correlated with decreased expression of interferon gamma (INF-γ) in CD4+ T cells. In the current study, we investigated the association between polymorphisms in the TIM-3 gene and susceptibility to OA. Two polymorphisms in TIM-3, -574G/T and +4259T/G polymorphisms, were identified in OA cases and healthy donors by polymerase chain reaction-restriction fragment length polymorphism method. Data revealed that the prevalence of TIM-3 +4259T/G genotype was significantly elevated in OA patients than in the healthy donors after adjustment (Odds ratio [OR] = 2.67, 95% confidence interval [CI] 1.32-5.11, P < 0.001). Similarly, the TIM-3 +4259G allele presented a positive association with the risk of OA after adjustment (OR = 2.58, 95% CI 1.29-4.82, P = 0.003). The TIM-3 -574G/T polymorphism did not show any correlation with the disease. We further examined whether the two TIM-3 polymorphisms could affect INF-γ expression in CD4+ T cells. Data revealed that subjects carrying polymorphic +4259TG genotype had significantly higher mRNA and protein levels of INF-γ in CD4+ T cells compared to wild-type GG genotype (P < 0.001 and P < 0.01). These results indicated that TIM-3 polymorphism is associated with increased susceptibility to OA possibly by upregulating INF-γ expression in CD4+ T cells.

  10. Functional deficit of T regulatory cells in Fulani, an ethnic group with low susceptibility to Plasmodium falciparum malaria

    PubMed Central

    Torcia, Maria G.; Santarlasci, Veronica; Cosmi, Lorenzo; Clemente, AnnMaria; Maggi, Laura; Mangano, Valentina D.; Verra, Federica; Bancone, Germana; Nebie, Issa; Sirima, Bienvenu Sodiomon; Liotta, Francesco; Frosali, Francesca; Angeli, Roberta; Severini, Carlo; Sannella, Anna R.; Bonini, Paolo; Lucibello, Maria; Maggi, Enrico; Garaci, Enrico; Coluzzi, Mario; Cozzolino, Federico; Annunziato, Francesco; Romagnani, Sergio; Modiano, David

    2008-01-01

    Previous interethnic comparative studies on the susceptibility to malaria performed in West Africa showed that Fulani are more resistant to Plasmodium falciparum malaria than are sympatric ethnic groups. This lower susceptibility is not associated to classic malaria-resistance genes, and the analysis of the immune response to P. falciparum sporozoite and blood stage antigens, as well as non-malaria antigens, revealed higher immune reactivity in Fulani. In the present study we compared the expression profile of a panel of genes involved in immune response in peripheral blood mononuclear cells (PBMC) from Fulani and sympatric Mossi from Burkina Faso. An increased expression of T helper 1 (TH1)-related genes (IL-18, IFNγ, and TBX21) and TH2-related genes (IL-4 and GATA3) and a reduced expression of genes distinctive of T regulatory activity (CTLA4 and FOXP3) were observed in Fulani. Microarray analysis on RNA from CD4+CD25+ (T regulatory) cells, performed with a panel of cDNA probes specific for 96 genes involved in immune modulation, indicated obvious differences between the two ethnic groups with 23% of genes, including TGFβ, TGFβRs, CTLA4, and FOXP3, less expressed in Fulani compared with Mossi and European donors not exposed to malaria. As further indications of a low T regulatory cell activity, Fulani showed lower serum levels of TGFβ and higher concentrations of the proinflammatory chemokines CXCL10 and CCL22 compared with Mossi; moreover, the proliferative response of Fulani to malaria antigens was not affected by the depletion of CD25+ regulatory cells whereas that of Mossi was significantly increased. The results suggest that the higher resistance to malaria of the Fulani could derive from a functional deficit of T regulatory cells. PMID:18174328

  11. Heat shock enhances the susceptibility of BHK cells to rotavirus infection through the facilitation of entry and post-entry virus replication steps.

    PubMed

    López, Tomás; López, Susana; Arias, Carlos F

    2006-10-01

    Rotavirus infection is known to induce several cellular stress proteins, although their possible involvement in the replication cycle of the virus has not been studied. In addition, the heat shock cognate protein hsc70 has been shown to function as a post-attachment receptor during virus entry. In this work we have studied the effect of heat shock on the susceptibility of cells to rotavirus infection. BHK cells, which are largely refractory to the virus, became about 100-fold more susceptible when heat-treated, while the rotavirus highly susceptible MA104 cells did not significantly modified their susceptibility upon heat stress, suggesting that heat shock induces factors that are rate-limiting the replication of rotaviruses in BHK but not in MA104 cells. The heat treatment was shown to facilitate the rotavirus infection of BHK cells at the penetration and post-penetration levels, and each of these stages seems to contribute comparably to the overall observed 100-fold increase in infectivity. Since the binding of the virus to the cell surface was not affected, the caloric stress probably facilitates the penetration and/or uncoating of the virus. The pathway of virus entry into heat-shocked BHK cells seems to be similar to that used in MA104 cells, since treatments that affect MA104 cell infection also affected rotavirus infectivity in heat-treated BHK cells.

  12. Variable susceptibility of ovarian cancer cells to non-thermal plasma-activated medium

    PubMed Central

    UTSUMI, FUMI; KAJIYAMA, HIROAKI; NAKAMURA, KAE; TANAKA, HIROMASA; MIZUNO, MASAAKI; TOYOKUNI, SHINNYA; HORI, MASARU; KIKKAWA, FUMITAKA

    2016-01-01

    Non-thermal atmospheric pressure plasma has been widely studied in recent years in many fields, including cancer treatment. However, its efficiency for inducing apoptosis sometimes varies depending on the cell species and experimental conditions. The aim of this study was to elucidate what causes these differences in responses to plasma treatment. Using four ovarian cancer cell lines, the cell density had a markedly negative impact on the proliferation inhibition rate (PIR) and it was more obvious in OVCAR-3 and NOS2 cells. Furthermore, TOV21G and ES-2 cells were drastically sensitive to plasma-activated medium (PAM) compared with the other two cell lines. We demonstrated that the proportion of reactive oxygen species and cell number had a marked impact on the effect of PAM against ovarian cancer cells. Additionally it was suggested that the morphological features of cells were also closely related PMID:27035127

  13. Altered mitochondrial dynamics and response to insulin in cybrid cells harboring a diabetes-susceptible mitochondrial DNA haplogroup.

    PubMed

    Kuo, Hsiao-Mei; Weng, Shao-Wen; Chang, Alice Y W; Huang, Hung-Tu; Lin, Hung-Yu; Chuang, Jiin-Haur; Lin, Tsu-Kung; Liou, Chia-Wei; Tai, Ming-Hong; Lin, Ching-Yi; Wang, Pei-Wen

    2016-07-01

    The advantage of using a cytoplasmic hybrid (cybrid) model to study the genetic effects of mitochondria is that the cells have the same nuclear genomic background. We previously demonstrated the independent role of mitochondria in the pathogenesis of insulin resistance (IR) and pro-inflammation in type 2 diabetes. In this study, we compared mitochondrial dynamics and related physiological functions between cybrid cells harboring diabetes-susceptible (B4) and diabetes-protective (D4) mitochondrial haplogroups, especially the responses before and after insulin stimulation. Cybrid B4 showed a more fragmented mitochondrial network, impaired mitochondrial biogenesis and bioenergetics, increased apoptosis and ineffective mitophagy and a low expression of fusion-related molecules. Upon insulin stimulation, increases in network formation, mitochondrial DNA (mtDNA) content, and ATP production were observed only in cybrid D4. Insulin promoted a pro-fusion dynamic status in both cybrids, but the trend was greater in cybrid D4. In cybrid B4, the imbalance of mitochondrial dynamics and impaired biogenesis and bioenergetics, and increased apoptosis were significantly improved in response to antioxidant treatment. We concluded that diabetes-susceptible mtDNA variants are themselves resistant to insulin. PMID:27107769

  14. Human mesothelial cells are unusually susceptible to simian virus 40-mediated transformation and asbestos cocarcinogenicity

    PubMed Central

    Bocchetta, Maurizio; Di Resta, Ilaria; Powers, Amy; Fresco, Raoul; Tosolini, Alessandra; Testa, Joseph R.; Pass, Harvey I.; Rizzo, Paola; Carbone, Michele

    2000-01-01

    Mesothelioma, a malignancy associated with asbestos, has been recently linked to simian virus 40 (SV40). We found that infection of human mesothelial cells by SV40 is very different from the semipermissive infection thought to be characteristic of human cells. Mesothelial cells are uniformly infected but not lysed by SV40, a mechanism related to p53, and undergo cell transformation at an extremely high rate. Exposure of mesothelial cells to asbestos complemented SV40 mutants in transformation. Our data provide a mechanistic explanation for the ability of SV40 to transform mesothelial cells preferentially and indicate that asbestos and SV40 may be cocarcinogens. PMID:10954737

  15. Murine lupus susceptibility locus Sle1a requires the expression of two sub-loci to induce inflammatory T cells.

    PubMed

    Cuda, C M; Zeumer, L; Sobel, E S; Croker, B P; Morel, L

    2010-10-01

    The NZM2410-derived Sle1a lupus susceptibility locus induces activated autoreactive CD4(+) T cells and reduces the number and function of Foxp3(+) regulatory T cells (Tregs). In this study, we first showed that Sle1a contributes to autoimmunity by increasing antinuclear antibody production when expressed on either NZB or NZW heterozygous genomes, and by enhancing the chronic graft versus host disease response indicating an expansion of the autoreactive B-cell pool. Screening two non-overlapping recombinants, the Sle1a.1 and Sle1a.2 intervals that cover the entire Sle1a locus, revealed that both Sle1a.1 and Sle1a.2 were necessary for the full Sle1a phenotype. Sle1a.1, and to a lesser extent Sle1a.2, significantly affected CD4(+) T-cell activation as well as Treg differentiation and function. Sle1a.2 also increased the production of autoreactive B cells. As the Sle1a.1 and Sle1a.2 intervals contain only 1 and 15 known genes, respectively, this study considerably reduces the number of candidate genes responsible for the production of autoreactive T cells. These results also show that the Sle1 locus is an excellent model for the genetic architecture of lupus, in which a major obligate phenotype results from the coexpression of multiple genetic variants with individual weak effects.

  16. Establishment and partial characterization of a cell line from burbot Lota lota maculosa: susceptibility to IHNV, IPNV and VHSV.

    USGS Publications Warehouse

    Batts, William N.; Polinski, Mark P.; Drennan, John D.; Ireland, Susan C.; Cain, Kenneth D.

    2010-01-01

    This study describes the development and partial characterization of a continuous fibroblastic-like cell line (BEF-1) developed from late stage embryos of North American burbot Lota lota maculosa. This cell line has been maintained for over 5 yr and 100 passages in vitro. Cells were cultured using Eagle’s minimum essential medium with Earle’s salts (MEM) supplemented with GlutaMAX™, and 10% fetal bovine serum (FBS), pH 7.4. The addition of penicillin-streptomycin-neomycin (PSN) antibiotic mixture (0.05, 0.05, 0.1 mg ml–1, respectively) did not negatively influence cell replication; however, the antimycotic Fungizone™ (2.5 µg ml–1, amphotericin B) caused cell rounding and resulted in a severe decrease in cell proliferation. Optimal incubation temperature has been observed between 15 and 23°C, and at these temperatures cultures are routinely passed using standard trypsinization methods every 5 to 7 d at a split ratio of 1:3 or 1:4. The cell line was susceptible to isolates of the M and U North American genotypes of infectious hematopoietic necrosis virus (IHNV), and to isolates of genotypes I, IVa, and IVb of viral hemorrhagic septicemia virus (VHSV). In contrast, the cell line was refractory to infection by 2 North American isolates of infectious pancreatic necrosis virus (IPNV) from serotypes A1 and A9. This cell line provides a new laboratory tool, will allow further investigation into viral diseases of burbot and possibly other species, and is the first immortalized cell line reported from a species in the Gadidae (cod) family.

  17. Apoptotic susceptibility to DNA damage of pluripotent stem cells facilitates pharmacologic purging of teratoma risk.

    PubMed

    Smith, Alyson J; Nelson, Natalie G; Oommen, Saji; Hartjes, Katherine A; Folmes, Clifford D; Terzic, Andre; Nelson, Timothy J

    2012-10-01

    Pluripotent stem cells have been the focus of bioengineering efforts designed to generate regenerative products, yet harnessing therapeutic capacity while minimizing risk of dysregulated growth remains a challenge. The risk of residual undifferentiated stem cells within a differentiated progenitor population requires a targeted approach to eliminate contaminating cells prior to delivery. In this study we aimed to validate a toxicity strategy that could selectively purge pluripotent stem cells in response to DNA damage and avoid risk of uncontrolled cell growth upon transplantation. Compared with somatic cell types, embryonic stem cells and induced pluripotent stem cells displayed hypersensitivity to apoptotic induction by genotoxic agents. Notably, hypersensitivity in pluripotent stem cells was stage-specific and consistently lost upon in vitro differentiation, with the mean half-maximal inhibitory concentration increasing nearly 2 orders of magnitude with tissue specification. Quantitative polymerase chain reaction and Western blotting demonstrated that the innate response was mediated through upregulation of the BH3-only protein Puma in both natural and induced pluripotent stem cells. Pretreatment with genotoxic etoposide purged hypersensitive pluripotent stem cells to yield a progenitor population refractory to teratoma formation upon transplantation. Collectively, this study exploits a hypersensitive apoptotic response to DNA damage within pluripotent stem cells to decrease risk of dysregulated growth and augment the safety profile of transplant-ready, bioengineered progenitor cells.

  18. Establishment of an indicator cell line for monitoring bovine immunodeficiency virus infection and inhibitor susceptibility.

    PubMed

    Yao, Xue; Su, Yang; Liu, Chang; Tan, Juan; Liu, Li; Geng, Yun-Qi; Qiao, Wen-Tao

    2010-01-01

    Indicator cell lines are useful biological tools for monitoring virus infection. In order to monitor infection with bovine immunodeficiency virus (BIV) in vitro, an indicator cell line derived from baby hamster kidney cells which contains integrated copies of an enhanced green fluorescent protein gene driven by the BIV long terminal repeat was constructed. The BIV indicator cell line, designated BIVE, can detect BIV infection more easily and effectively than the established method, which involves the observation of cell cytopathic effects. Furthermore, viral titration using an assay based on the indicator cells is 100 times more sensitive than the assay based on cytopathic effect. The finding that BIV can infect the hamster cell line expands the known host range of BIV in vitro. The BIV indicator cell line could also be used for the evaluation of the inhibitory effect of antiviral agents. The fusion inhibition effect of the heptad repeat 2 region of the BIV envelope protein could also be quantified.

  19. Inflammasome priming increases retinal pigment epithelial cell susceptibility to lipofuscin phototoxicity by changing the cell death mechanism from apoptosis to pyroptosis.

    PubMed

    Brandstetter, Carolina; Patt, Joshua; Holz, Frank G; Krohne, Tim U

    2016-08-01

    Progressive death of retinal pigment epithelium (RPE) cells is a hallmark of age-related macular degeneration (AMD), the leading cause of blindness in all developed countries. Photooxidative damage and activation of the NLRP3 inflammasome have been suggested as contributing factors to this process. We investigated the effects of inflammasome activation on oxidative damage-induced RPE cell death. In primary human RPE cells and ARPE-19 cells, lipofuscin accumulated following incubation with oxidatively modified photoreceptor outer segments. Oxidative stress was induced by blue light irradiation (dominant wavelength: 448nm, irradiance: 0.8mW/cm(2), duration: 3 to 6h) of lipofuscin-loaded cells and resulted in cell death by apoptosis. Prior inflammasome priming by IL-1α or complement activation product C5a altered the cell death mechanism to pyroptosis and resulted in a significant increase of the phototoxic effect. Following IL-1α priming, viability 24h after irradiation was reduced in primary RPE cells and ARPE-19 cells from 65.3% and 56.7% to 22.6% (p=0.003) and 5.1% (p=0.0002), respectively. Inflammasome-mediated IL-1β release occurred only in association with pyroptotic cell lysis. Inflammasome priming by conditioned media of pyroptotic cells likewise increased cell death. Suppression of inflammasome activation by inhibition of caspase-1 or cathepsins B and L significantly reduced cell death in primed cells. In summary, inflammasome priming by IL-1α, C5a, or conditioned media of pyroptotic cells increases RPE cell susceptibility to photooxidative damage-mediated cell death and changes the mechanism of induced cell death from apoptosis to pyroptosis. This process may contribute to RPE degeneration in AMD and provide new targets for intervention. PMID:27240191

  20. Erythrocyte enrichment in hematopoietic progenitor cell cultures based on magnetic susceptibility of the hemoglobin.

    PubMed

    Jin, Xiaoxia; Abbot, Stewart; Zhang, Xiaokui; Kang, Lin; Voskinarian-Berse, Vanessa; Zhao, Rui; Kameneva, Marina V; Moore, Lee R; Chalmers, Jeffrey J; Zborowski, Maciej

    2012-01-01

    Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes.

  1. Role of Akata cell membrane fluidity in susceptibility to Epstein-Barr virus infection.

    PubMed

    Pozzi, D; Lisi, A; Grimaldi, S

    1995-01-01

    Infection by Epstein-Barr virus (EBV), a B lymphotropic human herpesvirus, of its target cells is initiated by the binding of the viral envelope glycoprotein gp350/220 to a 145-kDa cell membrane glycoprotein (CD21, CR2) which also serves as the receptor for the complement fragment C3d (Fingeroth et al., 1984; Nemerow et al., 1987). We used the fluorescent probe 1-6-diphenyl-1,3,5-hexatriene (DPH), extremely sensitive to the polar environment, in order to analyse the membrane viscosity distribution in single cells of two lymphoid cell lines, Raji and Akata. Lipid analysis on both cell lines showed a slightly lower cholesterol:phospholipid molar ratio on Akata than on Raji cells. Measurements of cell fluidity by DPH polarization in native cells and after cholesterol enrichment indicated that the apparent Akata membrane viscosity was lower than the viscosity of Raji cells. To examine the possibility that this difference could be correlated to a difference in the behaviour of Akata and Raji cells in expressing EBV early antigens, both lines were superinfected with the EBV non-transforming P3HR1 strain. We report here evidence that lipid composition can regulate EBV entry into cells. PMID:8539494

  2. A rapid method for the determination of microbial susceptibility using the firefly luciferase assay for adenosine triphosphate (ATP)

    NASA Technical Reports Server (NTRS)

    Vellend, H.; Tuttle, S. A.; Barza, M.; Weinstein, L.; Picciolo, G. L.; Chappelle, E. W.

    1975-01-01

    Luciferase assay for adenosine triphosphate (ATP) was optimized for pure bacteria in broth in order to evaluate if changes in bacterial ATP content could be used as a rapid measure of antibiotic effect on microorganisms. Broth cultures of log phase bacteria were incubated at 310 K (37 C) for 2.5 hours at antimicrobial concentrations which resulted in the best discrimination between sensitive and resistant strains. Eighty-seven strains of 11 bacterial species were studied for their susceptibility to 12 commonly used antimicrobial agents: ampicillin, Penicillin G, nafcillin, carbenicillin, cephalothin, tetracycline, erythromycin, clindamycin, gentamicin, nitrofurantoin, colistin, and chloramplenicol. The major advantage of the ATP system over existing methods of rapid microbial susceptibility testing is that the assay can be made specific for bacterial ATP.

  3. A locus on chromosome 7 determines myocardial cell necrosis and calcification (dystrophic cardiac calcinosis) in mice.

    PubMed Central

    Ivandic, B T; Qiao, J H; Machleder, D; Liao, F; Drake, T A; Lusis, A J

    1996-01-01

    Dystrophic cardiac calcinosis, an age-related cardiomyopathy that occurs among certain inbred strains of mice, involves myocardial injury, necrosis, and calcification. Using a complete linkage map approach and quantitative trait locus analysis, we sought to identify genetic loci determining dystrophic cardiac calcinosis in an F2 intercross of resistant C57BL/6J and susceptible C3H/HeJ inbred strains. We identified a single major locus, designated Dyscalc, located on proximal chromosome 7 in a region syntenic with human chromosomes 19q13 and 11p15. The statistical significance of Dyscalc (logarithm of odds score 14.6) was tested by analysis of permuted trait data. Analysis of BxH recombinant inbred strains confirmed the mapping position. The inheritance pattern indicated that this locus influences susceptibility of cells both to enter necrosis and to subsequently undergo calcification. Images Fig. 1 Fig. 3 PMID:8643601

  4. Slow mitochondrial repair of 5'-AMP renders mtDNA susceptible to damage in APTX deficient cells.

    PubMed

    Akbari, Mansour; Sykora, Peter; Bohr, Vilhelm A

    2015-08-10

    Aborted DNA ligation events in eukaryotic cells can generate 5'-adenylated (5'-AMP) DNA termini that can be removed from DNA by aprataxin (APTX). Mutations in APTX cause an inherited human disease syndrome characterized by early-onset progressive ataxia with ocular motor apraxia (AOA1). APTX is found in the nuclei and mitochondria of eukaryotic cells. Depletion of APTX causes mitochondrial dysfunction and renders the mitochondrial genome, but not the nuclear genome susceptible to damage. The biochemical processes that link APTX deficiency to mitochondrial dysfunction have not been well elucidated. Here, we monitored the repair of 5'-AMP DNA damage in nuclear and mitochondrial extracts from human APTX(+/+) and APTX(-/-) cells. The efficiency of repair of 5'-AMP DNA was much lower in mitochondrial than in nuclear protein extracts, and resulted in persistent DNA repair intermediates in APTX deficient cells. Moreover, the removal of 5'-AMP from DNA was significantly slower in the mitochondrial extracts from human cell lines and mouse tissues compared with their corresponding nuclear extracts. These results suggest that, contrary to nuclear DNA repair, mitochondrial DNA repair is not able to compensate for APTX deficiency resulting in the accumulation of mitochondrial DNA damage.

  5. Autophagy-deficiency in hepatic progenitor cells leads to the defects of stemness and enhances susceptibility to neoplastic transformation.

    PubMed

    Xue, Feng; Hu, Lei; Ge, Ruiliang; Yang, Lixue; Liu, Kai; Li, Yunyun; Sun, Yanfu; Wang, Kui

    2016-02-01

    Autophagy is a highly conserved and lysosome-dependent degradation process which assists in cell survival and tissue homeostasis. Although previous reports have shown that deletion of the essential autophagy gene disturbs stem cell maintenance in some cell types such as hematopoietic and neural cells, it remains unclear how autophagy-deficiency influences hepatic progenitor cells (HPCs). Here we report that Atg5-deficiency in HPCs delays HPC-mediated rat liver regeneration in vivo. In vitro researches further demonstrate that loss of autophagy decreases the abilities of colony and spheroid formations, and disrupts the induction of hepatic differentiation in HPCs. Meanwhile, autophagy-deficiency increases the accumulations of damaged mitochondria and mitochondrial reactive oxygen species (mtROS) and suppresses homologous recombination (HR) pathway of DNA damage repair in HPCs. Moreover, in both diethylnitrosamine (DEN) and CCl4 models, autophagy-deficiency accelerates neoplastic transformation of HPCs. In conclusion, these findings demonstrate that autophagy contributes to stemness maintenance and reduces susceptibility to neoplastic transformation in HPCs.

  6. Slow mitochondrial repair of 5'-AMP renders mtDNA susceptible to damage in APTX deficient cells.

    PubMed

    Akbari, Mansour; Sykora, Peter; Bohr, Vilhelm A

    2015-01-01

    Aborted DNA ligation events in eukaryotic cells can generate 5'-adenylated (5'-AMP) DNA termini that can be removed from DNA by aprataxin (APTX). Mutations in APTX cause an inherited human disease syndrome characterized by early-onset progressive ataxia with ocular motor apraxia (AOA1). APTX is found in the nuclei and mitochondria of eukaryotic cells. Depletion of APTX causes mitochondrial dysfunction and renders the mitochondrial genome, but not the nuclear genome susceptible to damage. The biochemical processes that link APTX deficiency to mitochondrial dysfunction have not been well elucidated. Here, we monitored the repair of 5'-AMP DNA damage in nuclear and mitochondrial extracts from human APTX(+/+) and APTX(-/-) cells. The efficiency of repair of 5'-AMP DNA was much lower in mitochondrial than in nuclear protein extracts, and resulted in persistent DNA repair intermediates in APTX deficient cells. Moreover, the removal of 5'-AMP from DNA was significantly slower in the mitochondrial extracts from human cell lines and mouse tissues compared with their corresponding nuclear extracts. These results suggest that, contrary to nuclear DNA repair, mitochondrial DNA repair is not able to compensate for APTX deficiency resulting in the accumulation of mitochondrial DNA damage. PMID:26256098

  7. Site of clomazone action in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures. [Glycine max (L. ); Gossypium hirsutum

    SciTech Connect

    Norman, M.A.; Liebl, R.A.; Widholm, J.M. )

    1990-10-01

    Studies were conducted to determine the herbicidal site of clomazone action in tolerant-soybean (Glycine max (L.) Merr. cv Corsoy) (SB-M) and susceptible-cotton (Gossypium hirsutum (L.) cv Stoneville 825) (COT-M) photomixotrophic cell suspension cultures. Although a 10 micromolar clomazone treatment did not significantly reduce the terpene or mixed terpenoid content (microgram per gram fresh weight) of the SB-M cell line, there was over a 70% reduction in the chlorophyll (Chl), carotenoid (CAR), and plastoquinone (PQ) content of the COT-M cell line. The tocopherol (TOC) content was reduced only 35.6%. Reductions in the levels of Chl, CAR, TOC, and PQ indicate that the site of clomazone action in COT-M cells is prior to geranylgeranyl pyrophosphate (GGPP). The clomazone treatment did not significantly reduce the flow of ({sup 14}C)mevalonate (({sup 14}C)MEV) (nanocuries per gram fresh weight) into CAR and the three mixed terpenoid compounds of SB-M cells. Conversely, ({sup 14}C)MEV incorporation into CAR and the terpene moieties of Chl, PQ, and TOC in COT-M cells was reduced at least 73%, indicating that the site of clomazone action must be after MEV. Sequestration of clomazone away from the chloroplast cannot account for soybean tolerance to clomazone since chloroplasts isolated from both cell lines incubated with ({sup 14}C)clomazone contained a similar amount of radioactivity (disintegrations per minute per microgram of Chl). The possible site(s) of clomazone inhibition include mevalonate kinase, phosphomevalonate kinase, pyrophosphomevalonate decarboxylase, isopentenyl pyrophosphate isomerase, and/or a prenyl transferase.

  8. Susceptibility of a line of dolphin kidney cell culture to several herpesviruses.

    PubMed

    Kadoi, K; Mochizuki, A; Ikeda, T; Kamata, H; Yukawa, M; Inoue, Y

    1992-01-01

    A cell line was established from cell cultures of kidney cortex of a pantropical spotted dolphin, Stenella attenuate. The replication of 6 strains of herpesviruses was studied in the cells. Five strains of them, herpes simplex virus type I and type II, equine rhinopneumonitis virus, infectious bovine rhinotracheitis virus and Aujeszky's disease virus, were grown fairly well in showing clear cytopathic effects and plaques under agar overlay medium. PMID:1460566

  9. Progesterone augments cell susceptibility to HIV-1 and HIV-1/HSV-2 co-infections.

    PubMed

    Ragupathy, Viswanath; Xue, Wang; Tan, Ji; Devadas, Krishnakumar; Gao, Yamei; Hewlett, Indira

    2016-10-01

    In human immunodeficiency virus type 1 (HIV-1)-infected women, oral or injectable progesterone containing contraceptive pills may enhance HIV-1 acquisition in vivo, and the mechanism by which this occurs is not fully understood. In developing countries, Herpes simplex virus type-2 (HSV-2) co-infection has been shown to be a risk for increase of HIV-1 acquisition and, if co-infected women use progesterone pills, infections may increase several fold. In this study, we used an in vitro cell culture system to study the effects of progesterone on HIV-1 replication and to explore the molecular mechanism of progesterone effects on infected cells. In our in vitro model, CEMss cells (lymphoblastoid cell line) were infected with either HIV-1 alone or co-infected with HSV-2. HIV-1 viral load was measured with and without sex hormone treatment. Progesterone-treated cells showed an increase in HIV-1 viral load (1411.2 pg/mL) compared with cells without progesterone treatment (993.1 pg/mL). Increased cell death was noted with HSV-2 co-infection and in progesterone-treated cells. Similar observations were noted in peripheral blood mononuclear cells (PBMC) cells derived from three female donors. Progesterone-treated cells also showed reduced antiviral efficacy. Inflammatory cytokines and associations with biomarkers of disease progression were explored. Progesterone upregulated inflammatory cytokines and chemokines conversely and downregulated anti-apoptotic Bcl-2 expression. Nuclear protein analysis by electrophoretic mobility shift assay showed the association of progesterone with progesterone response element (PRE), which may lead to downregulation of Bcl-2. These data indicate that progesterone treatment enhances HIV-1 replication in infected cells and co-infection with HSV-2 may further fuel this process. PMID:27538988

  10. Establishment, characterization and viral susceptibility of 3 new cell lines from snakehead, Channa striatus (Blooch).

    PubMed

    Zhao, Zhengshan; Montgomery-Brock, Dee; Lee, Cheng-Sheng; Lu, Yuanan

    2003-01-01

    Three cell lines were established from muscle (SHMS), heart (SHHT) and swim bladder (SHSB) of snakehead (Channa striatus). The cells grew initially at 25 degrees C in L15 medium supplemented with 20% fetal bovine serum and have been subcultured 13-18 times since their initiation on June 25, 2002. Growth of the snakehead cells was serum-dependent and plating efficiencies ranged from 22-29%. These snakehead cells grew well in RPMI 1640 and L-15 media, which are commonly used for cultivation of animal and mammalian cells and retained 95.9-96.6% cell viability following storage for 4 months in liquid nitrogen. Karyotyping indicated that these snakehead-derived cell lines remained diploid with a chromosome count of 44 at their early passage (passage 8-14). These cell lines were sensitive to CCV, VHSV, SVCV, IPN and SHRV; they were refractory to IHNV. These newly established cell lines are currently being used for the investigation of snakehead viral diseases in Hawaii and will be available for future isolation and study of snakehead viruses.

  11. Cell density determines epithelial migration in culture.

    PubMed Central

    Rosen, P; Misfeldt, D S

    1980-01-01

    The dog kidney epithelial cell line (MDCK) has been shown to exhibit a density-correlated inhibition of growth at approxmately 6.6 X 10(5) cells per cm2. When a confluent monolayer at its maximal density was wounded by removal of a wide swath of cells, migration of the cell sheet into the denuded area occurred. Precise measurements of the rate of migration for 5 day showed that the cells accelerated at a uniform rate of 0.24 micrometer . hr-2 and, by extrapolation, possessed an apparent initial velocity of 2.8 micrometer . hr-1 at the time of wounding. The apparent initial velocity was considered to be the result of a brief (< 10 hr) and rapid acceleration dependent on cell density. To verify this, wounds were made at different densities below the maximum. In these experiments, the cells did not migrate until a "threshold" density of 2.0 X 10(5) cells per cm2 was reached regardless of the density at the time of wounding. At the threshold density, the cell sheet began to accelerate at the previously measured rate (0.24 micrometer . hr-2). Any increase in density by cell division was balanced by cell migration, so that the same threshold density was maintained by the migrating cells. Each migrating cell sustained the movement of the cell sheet at a constant rate of acceleration. It is proposed that an acceleration is, in general, characteristic of the vectorial movement of an epithelial cell sheet. Images PMID:6933523

  12. Digital Quantification of DNA Replication and Chromosome Segregation Enables Determination of Antimicrobial Susceptibility after only 15 Minutes of Antibiotic Exposure.

    PubMed

    Schoepp, Nathan G; Khorosheva, Eugenia M; Schlappi, Travis S; Curtis, Matthew S; Humphries, Romney M; Hindler, Janet A; Ismagilov, Rustem F

    2016-08-01

    Rapid antimicrobial susceptibility testing (AST) would decrease misuse and overuse of antibiotics. The "holy grail" of AST is a phenotype-based test that can be performed within a doctor visit. Such a test requires the ability to determine a pathogen's susceptibility after only a short antibiotic exposure. Herein, digital PCR (dPCR) was employed to test whether measuring DNA replication of the target pathogen through digital single-molecule counting would shorten the required time of antibiotic exposure. Partitioning bacterial chromosomal DNA into many small volumes during dPCR enabled AST results after short exposure times by 1) precise quantification and 2) a measurement of how antibiotics affect the states of macromolecular assembly of bacterial chromosomes. This digital AST (dAST) determined susceptibility of clinical isolates from urinary tract infections (UTIs) after 15 min of exposure for all four antibiotic classes relevant to UTIs. This work lays the foundation to develop a rapid, point-of-care AST and strengthen global antibiotic stewardship.

  13. Dihydrolipoic but not alpha-lipoic acid affects susceptibility of eukaryotic cells to bacterial invasion.

    PubMed

    Bozhokina, Ekaterina; Khaitlina, Sofia; Gamaley, Irina

    2015-05-01

    Sensitivity of eukaryotic cells to facultative pathogens can depend on physiological state of host cells. Previously we have shown that pretreatment of HeLa cells with N-acetylcysteine (NAC) makes the cells 2-3-fold more sensitive to invasion by the wild-type Serratia grimesii and recombinant Escherichia coli expressing gene of actin-specific metalloprotease grimelysin [1]. To evaluate the impact of chemically different antioxidants, in the present work we studied the effects of α-Lipoic acid (LA) and dihydrolipoic acid (DHLA) on efficiency of S. grimesii and recombinant E. coli expressing grimelysin gene to penetrate into HeLa and CaCo cells. Similarly to the effect of NAC, pretreatment of HeLa and CaCo cells with 0.6 or 1.25 mM DHLA increased the entry of grimelysin producing bacteria by a factor of 2.5 and 3 for the wild-type S. grimesii and recombinant E. coli, respectively. In contrast, pretreatment of the cells with 0.6 or 1.25 mM LA did not affect the bacteria uptake. The increased invasion of HeLa and CaCo cells correlated with the enhanced expression of E-cadherin and β-catenin genes, whereas expression of these genes in the LA-treated cells was not changed. Comparison of these results suggests that it is sulfhydryl group of DHLA that promotes efficient modification of cell properties assisting bacterial uptake. We assume that the NAC- and DHLA-induced stimulation of the E-cadherin-catenin pathway contributes to the increased internalization of the grimelysin producing bacteria within transformed cells.

  14. Association between CYP1A1 Ile462Val Polymorphism and Oral Squamous Cell Carcinoma Susceptibility: Evidence from 13 Investigations

    PubMed Central

    Yang, Xiao-Lei; Xie, Shang; Jiang, Yi-Yan; Shi, Chang; Cai, Zhi-Gang; Chen, Su-Xiu

    2015-01-01

    CYP1A1 Ile462Val polymorphism might play a key role in pathogenesis of oral squamous cell carcinoma (OSCC). Many case-control studies have investigated the association between CYP1A1 Ile462Val polymorphism and OSCC susceptibility. However, the conclusions are inconsistent. To aim a convincible conclusion, we carried out a meta-analysis to systematically evaluate the association of CYP1A1 Ile462Val polymorphism with OSCC susceptibility. We searched Pubmed, Web of Science, Ovid and Embase databases for available publications. The odds ratio (OR) with the corresponding 95% confidence interval (95% CI) was carried out to estimate the association. A total of 13 papers including 1468 cases and 2183 controls were included, a significant increased OSCC risk was observed in recessive model (OR=1.64, 95% CI=1.08-2.49), but not other genetic models. Our results suggest that the homozygous variant of CYP1A1 Ile462Val might be a risk factor of OSCC. PMID:25767599

  15. Aneuploid cells are differentially susceptible to caspase-mediated death during embryonic cerebral cortical development.

    PubMed

    Peterson, Suzanne E; Yang, Amy H; Bushman, Diane M; Westra, Jurjen W; Yung, Yun C; Barral, Serena; Mutoh, Tetsuji; Rehen, Stevens K; Chun, Jerold

    2012-11-14

    Neural progenitor cells, neurons, and glia of the normal vertebrate brain are diversely aneuploid, forming mosaics of intermixed aneuploid and euploid cells. The functional significance of neural mosaic aneuploidy is not known; however, the generation of aneuploidy during embryonic neurogenesis, coincident with caspase-dependent programmed cell death (PCD), suggests that a cell's karyotype could influence its survival within the CNS. To address this hypothesis, PCD in the mouse embryonic cerebral cortex was attenuated by global pharmacological inhibition of caspases or genetic removal of caspase-3 or caspase-9. The chromosomal repertoire of individual brain cells was then assessed by chromosome counting, spectral karyotyping, fluorescence in situ hybridization, and DNA content flow cytometry. Reducing PCD resulted in markedly enhanced mosaicism that was comprised of increased numbers of cells with the following: (1) numerical aneuploidy (chromosome losses or gains); (2) extreme forms of numerical aneuploidy (>5 chromosomes lost or gained); and (3) rare karyotypes, including those with coincident chromosome loss and gain, or absence of both members of a chromosome pair (nullisomy). Interestingly, mildly aneuploid (<5 chromosomes lost or gained) populations remained comparatively unchanged. These data demonstrate functional non-equivalence of distinguishable aneuploidies on neural cell survival, providing evidence that somatically generated, cell-autonomous genomic alterations have consequences for neural development and possibly other brain functions. PMID:23152605

  16. Anisotropy of out-of-phase magnetic susceptibility of rocks as a tool for direct determination of magnetic sub-fabrics of some minerals

    NASA Astrophysics Data System (ADS)

    Hrouda, Frantisek; Chadima, Martin; Jezek, Josef; Pokorny, Jiri

    2015-04-01

    The magnetic susceptibility measured in alternating field can in general be resolved into a component that is in-phase with the applied field and a component that is out-of-phase. While in diamagnetic, paramagnetic and many ferromagnetic materials the phase is effectively zero, in some ferromagnetic minerals, such as pyrrhotite, hematite, titanomagnetite or ultra-fine magnetically viscous grains of magnetite, it is clearly non-zero. The anisotropy of out-of-phase susceptibility (opAMS) offers itself as a tool for the direct determination of the magnetic sub-fabrics of the minerals with non-zero phase. The SAFYR program, originally destined for computing the in-phase AMS (ipAMS) measured with the MFK1-FA Kappabridge, was modified in such a way that it calculates also the opAMS using exactly the same calculus; the ipAMS and opAMS are determined in one measuring process. Five groups of specimens showing clearly non-zero phase were used to investigate the precisions in determining both the ipAMS and opAMS as well as the relationship between the ipAMS and opAMS. In the specimens investigated, the out-of-phase susceptibility is one to two orders of magnitude lower than the in-phase susceptibility. If the mean out-of-phase susceptibility is higher than 5 × 10-5 [SI], the accuracy in determination of the opAMS, expressed in terms of fitting error and confidence angles, is comparable to or only slightly worse than that of the ipAMS. In the specimens whose magnetism is dominantly carried by a mineral with non-zero phase, the ellipsoids of opAMS and ipAMS show similar shapes and orientations of respective principal directions. The degree of anisotropy, however, is significantly higher in opAMS than in ipAMS. The reasons for this phenomenon are not fully known, one of them can be masking effect of the minerals with zero-phase. In the specimens possessing non-coaxial magnetic sub-fabrics of the minerals with non-zero and zero-phase, not only the degrees of AMS, but also

  17. Cell-on-hydrogel platform made of agar and alginate for rapid, low-cost, multidimensional test of antimicrobial susceptibility.

    PubMed

    Sun, Han; Liu, Zhengzhi; Hu, Chong; Ren, Kangning

    2016-08-01

    Antimicrobial resistance (AMR) is a rapidly increasing threat to the effective treatment of infectious diseases worldwide. The two major remedies include: (1) using narrow-spectrum antibiotics based on rapid diagnosis; and (2) developing new antibiotics. A key part of both remedies is the antimicrobial susceptibility test (AST). However, the current standard ASTs that monitor colony formation are costly and time-consuming and the new strategies proposed are not yet practical to be implemented. Herein, we report a strategy to fabricate whole-hydrogel microfluidic chips using alginate-doped agar. This agar-based microfabrication makes it possible to prepare inexpensive hydrogel devices, and allows a seamless link between microfluidics and conventional agar-based cell culture. Different from common microfluidic systems, in our system the cells are cultured on top of the device, similar to normal agar plate culture; on the other hand, the microfluidic channels inside the hydrogel allow precise generation of linear gradient of drugs, thus giving a better performance than the conventional disk diffusion method. Cells in this system are not exposed to any shear flow, which allows the reliable tracking of individual cells and AST results to be obtained within 2-3 hours. Furthermore, our system could test the synergistic effect of drugs through two-dimensional gradient generation. Finally, the platform could be directly implemented to new drug discovery and other applications wherein a fast, cost-efficient method for studying the response of microorganisms upon drug administration is desirable. PMID:27452345

  18. Decreased SIRT3 in aged human mesenchymal stromal/stem cells increases cellular susceptibility to oxidative stress.

    PubMed

    Wang, Xue-Qing; Shao, Yong; Ma, Chong-Yi; Chen, Wei; Sun, Lu; Liu, Wei; Zhang, Dong-Yang; Fu, Bi-Cheng; Liu, Kai-Yu; Jia, Zhi-Bo; Xie, Bao-Dong; Jiang, Shu-Lin; Li, Ren-Ke; Tian, Hai

    2014-11-01

    Sirtuin3 (SIRT3) is an important member of the sirtuin family of protein deacetylases that is localized to mitochondria and linked to lifespan extension in organisms ranging from yeast to humans. As aged cells have less regenerative capacity and are more susceptible to oxidative stress, we investigated the effect of ageing on SIRT3 levels and its correlation with antioxidant enzyme activities. Here, we show that severe oxidative stress reduces SIRT3 levels in young human mesenchymal stromal/stem cells (hMSCs). Overexpression of SIRT3 improved hMSCs resistance to the detrimental effects of oxidative stress. By activating manganese superoxide dismutase (MnSOD) and catalase (CAT), SIRT3 protects hMSCs from apoptosis under stress. SIRT3 expression, levels of MnSOD and CAT, as well as cell survival showed little difference in old versus young hMSCs under normal growth conditions, whereas older cells had a significantly reduced capacity to withstand oxidative stress compared to their younger counterparts. Expression of the short 28 kD SIRT3 isoform was higher, while the long 44 kD isoform expression was lower in young myocardial tissues compared with older ones. These results suggest that the active short isoform of SIRT3 protects hMSCs from oxidative injury by increasing the expression and activity of antioxidant enzymes. The expression of this short isoform decreases in cardiac tissue during ageing, leading to a reduced capacity for the heart to withstand oxidative stress. PMID:25210848

  19. Histone deacetylase inhibitors stimulate the susceptibility of A549 cells to a plasma-activated medium treatment.

    PubMed

    Adachi, Tetsuo; Kano, Ayame; Nonomura, Saho; Kamiya, Tetsuro; Hara, Hirokazu

    2016-09-15

    The number of potential applications of non-thermal atmospheric pressure plasma (NTAPP) discharges in medicine, particularly in cancer therapy, has increased in recent years. NTAPP has been shown to affect cells not only by direct irradiation, but also by an indirect treatment with previously prepared plasma-activated medium (PAM). Histone deacetylase (HDAC) inhibitors have the potential to enhance susceptibility to anticancer drugs and radiation. The aim of the present study was to demonstrate the advantage of the combined application of PAM and HDAC inhibitors on A549 cancer cell survival and elucidate the underlying mechanisms. Cell death with DNA breaks in the nucleus was greater using combined regimens of PAM and HDAC inhibitors such as trichostatin A (TSA) and valproic acid (VPA) than a single PAM treatment and was accompanied by the activation of poly (ADP-ribose) polymerase-1 (PARP-1), depletion of ATP, and elevations in intracellular calcium levels. Moreover, the expression of Rad 51, a DNA repair factor in homologous recombination pathways, was significantly suppressed by the treatment with HDAC inhibitors. These results demonstrate that HDAC inhibitors may synergistically induce the sensitivity of cancer cells to PAM components. PMID:27470189

  20. Differential susceptibility of cells expressing allogeneic MHC or viral antigen to killing by antigen-specific CTL.

    PubMed

    Lee, Koutetsu; Takenaka, Hiroshi; Yoneda, Yukio; Goto, Toshiyuki; Sano, Kouichi; Nakanishi, Mahito; Eguchi, Akiko; Okada, Masashi; Tashiro, Junko; Sakurai, Kanji; Kubota, Takahiro; Yoshida, Ryotaro

    2004-01-01

    CD8(+) cytotoxic T lymphocytes (CTLs) generated by immunization with allogeneic cells or viral infection are able to lyse allogeneic or virally infected in vitro cells (e.g., lymphoma and mastocytoma). In contrast, it is reported that CD8(+) T cells are not essential for allograft rejection (e.g., heart and skin), and that clearance of influenza or the Sendai virus from virus-infected respiratory epithelium is normal or only slightly delayed after a primary viral challenge of CD8-knockout mice. To address this controversy, we generated H-2(d)-specific CD8(+) CTLs by a mixed lymphocyte culture and examined the susceptibility of a panel of H-2(d) cells to CTL lysis. KLN205 squamous cell carcinoma, Meth A fibrosarcoma, and BALB/c skin components were found to be resistant to CTL-mediated lysis. This resistance did not appear to be related to a reduced expression of MHC class I molecules, and all these cells could block the recognition of H-2(d) targets by CTLs in cold target inhibition assays. We extended our observation by persistently infecting the same panel of cell lines with defective-interfering Sendai virus particles. The Meth A and KLN205 lines infected with a variant Sendai virus were resistant to lysis by Sendai virus-specific CTLs. The Sendai virus-infected Meth A and KLN205 lines were able to block the lysis of Sendai virus-infected targets by CTLs in cold target inhibition assays. Taken together, these results suggest that not all in vivo tissues may be sensitive to CTL lysis.

  1. Osmotin, a plant antifungal protein, subverts signal transduction to enhance fungal cell susceptibility.

    PubMed

    Yun, D J; Ibeas, J I; Lee, H; Coca, M A; Narasimhan, M L; Uesono, Y; Hasegawa, P M; Pardo, J M; Bressan, R A

    1998-05-01

    The plant pathogenesis-related protein osmotin is an antifungal cytotoxic agent that causes rapid cell death in the yeast S. cerevisiae. We show here that osmotin uses a signal transduction pathway to weaken defensive cell wall barriers and increase its cytotoxic efficacy. The pathway activated by osmotin includes the regulatory elements of the mating pheromone response STE4, STE18, STE20, STE5, STE11, STE7, FUS3, KSS1, and STE12. Neither the pheromone receptor nor its associated G protein alpha subunit GPA1 are required for osmotin action. However, mutation of SST2, a negative regulator of G alpha proteins, resulted in supersensitivity to osmotin. Phosphorylation of STE7 was rapidly stimulated by osmotin preceding any changes in cell vitality or morphology. These results demonstrate that osmotin subverts target cell signal transduction as part of its mechanism of action. PMID:9660964

  2. Differential Susceptibility of Human Pleural and Peritoneal Mesothelial Cells to Asbestos Exposure.

    PubMed

    Dragon, Julie; Thompson, Joyce; MacPherson, Maximilian; Shukla, Arti

    2015-08-01

    Malignant mesothelioma (MM) is an aggressive cancer of mesothelial cells of pleural and peritoneal cavities. In 85% of cases both pleural and peritoneal MM is caused by asbestos exposure. Although both are asbestos-induced cancers, the incidence of pleural MM is significantly higher (85%) than peritoneal MM (15%). It has been proposed that carcinogenesis is a result of asbestos-induced inflammation but it is not clear what contributes to the differences observed between incidences of these two cancers. We hypothesize that the observed differences in incidences of pleural and peritoneal MM are the result of differences in the direct response of these cell types to asbestos rather than to differences mediated by the in vivo microenvironment. To test this hypothesis we characterized cellular responses to asbestos in a controlled environment. We found significantly greater changes in genome-wide expression in response to asbestos exposure in pleural mesothelial cells as compared to peritoneal mesothelial cells. In particular, a greater response in many common genes (IL-8, ATF3, CXCL2, CXCL3, IL-6, GOS2) was seen in pleural mesothelial cells as compared to peritoneal mesothelial cells. Unique genes expressed in pleural mesothelial cells were mainly pro-inflammatory (G-CSF, IL-1β, IL-1α, GREM1) and have previously been shown to be involved in development of MM. Our results are consistent with the hypothesis that differences in incidences of pleural and peritoneal MM upon exposure to asbestos are the result of differences in mesothelial cell physiology that lead to differences in the inflammatory response, which leads to cancer.

  3. Differential susceptibility of human pleural and peritoneal mesothelial cells to asbestos exposure

    PubMed Central

    Dragon, Julie; Thompson, Joyce; MacPherson, Maximilian; Shukla, Arti

    2015-01-01

    Malignant mesothelioma (MM) is an aggressive cancer of mesothelial cells of pleural and peritoneal cavities. In 85% of cases both pleural and peritoneal MM is caused by asbestos exposure. Although both are asbestos-induced cancers, the incidence of pleural MM is significantly higher (85%) than peritoneal MM (15%). It has been proposed that carcinogenesis is a result of asbestos-induced inflammation but it is not clear what contributes to the differences observed between incidences of these two cancers. We hypothesize that the observed differences in incidences of pleural and peritoneal MM are the result of differences in the direct response of these cell types to asbestos rather than to differences mediated by the in vivo microenvironment. To test this hypothesis we characterized cellular responses to asbestos in a controlled environment. We found significantly greater changes in genome-wide expression in response to asbestos exposure in pleural mesothelial cells as compared to peritoneal mesothelial cells. In particular, a greater response in many common genes (IL-8, ATF3, CXCL2, CXCL3, IL-6, GOS2) was seen in pleural mesothelial cells as compared to peritoneal mesothelial cells. Unique genes expressed in pleural mesothelial cells were mainly pro-inflammatory (G-CSF, IL-1β, IL-1α, GREM1) and have previously been shown to be involved in development of MM. Our results are consistent with the hypothesis that differences in incidences of pleural and peritoneal MM upon exposure to asbestos are the result of differences in mesothelial cell physiology that lead to differences in the inflammatory response, which leads to cancer. PMID:25757056

  4. The surface receptor is a major determinant of the cell tropism of influenza C virus.

    PubMed

    Herrler, G; Klenk, H D

    1987-07-01

    N-Acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) has been shown to be a high-affinity receptor determinant for attachment of influenza C virus to erythrocytes (G. N. Rogers, G. Herrler, J. C. Paulson, and H-D. Klenk, 1986, J. Biol. Chem. 261, 5947-5951). In this report the nature of the cell surface receptor for influenza C virus on tissue culture cells was analyzed. Pretreatment with either neuraminidase or neuraminate 9-O-acetylesterase was found to render LLC-MK2 cells resistant to infection by influenza C virus as evidenced by the failure to detect virus release into the medium by hemagglutination titration. Susceptibility to infection was fully restored after incubation of neuraminidase-treated cells with bovine brain gangliosides known to contain Neu5,9Ac2. These results indicate that (i) Neu5,9Ac2 is the primary receptor determinant required for influenza C virus to attach to tissue culture cells and to initiate infection and (ii) gangliosides containing this type of sialic acid are potential receptors for influenza C virus. Several cell lines which are resistant to infection by this virus were able to release influenza C virus into the medium provided they were incubated with bovine brain gangliosides prior to virus infection. This result indicates that lack of appropriate receptors on the cell surface is a major reason for the restricted cell tropism of influenza C virus.

  5. Sex determination in mammalian germ cells

    PubMed Central

    Spiller, Cassy M; Bowles, Josephine

    2015-01-01

    Germ cells are the precursors of the sperm and oocytes and hence are critical for survival of the species. In mammals, they are specified during fetal life, migrate to the developing gonads and then undergo a critical period during which they are instructed, by the soma, to adopt the appropriate sexual fate. In a fetal ovary, germ cells enter meiosis and commit to oogenesis, whereas in a fetal testis, they avoid entry into meiosis and instead undergo mitotic arrest and mature toward spermatogenesis. Here, we discuss what we know so far about the regulation of sex-specific differentiation of germ cells, considering extrinsic molecular cues produced by somatic cells, as well as critical intrinsic changes within the germ cells. This review focuses almost exclusively on our understanding of these events in the mouse model. PMID:25791730

  6. Antifungal susceptibility of clinical and environmental isolates of Cryptococcus neoformans to four antifungal drugs determined by two techniques.

    PubMed

    Moraes, E M P; Prímola, N S; Hamdan, Júnia Soares

    2003-06-01

    A total of 64 Cryptococcus neoformans strains, including clinical and environmental Brazilian isolates var. neoformans and var. gattii, were tested for susceptibility to amphotericin B, 5-flucytosine, fluconazole and itraconazole. The tests were performed according to the recommendations of National Committee of Clinical Laboratory Standards and the method of macrodilution in liquid medium of Shadomy et al. [Manual de Microbiologia Clínica, 4th ed. Buenos Aires: Editorial Medica Panamericana, 1987: 1229-38]. For most drugs there was a significant difference between the readings taken at 24 and 48 h with both methods. When the minimum inhibitory concentrations obtained by the two techniques were compared, significant differences were observed for amphotericin B and fluconazole. Overall, differences in drug susceptibility with respect to the origin of the isolates or the variety of the fungus were not observed. As an exception, the gattii variety exhibited a high resistance rate to amphotericin B when the technique of Shadomy et al. was applied, a fact possibly related to the greater difficulty for treatment of the disease caused by this fungal variety.

  7. Susceptibility to T cell-mediated liver injury is enhanced in asialoglycoprotein receptor-deficient mice.

    PubMed

    McVicker, Benita L; Thiele, Geoffrey M; Casey, Carol A; Osna, Natalia A; Tuma, Dean J

    2013-05-01

    T cell activation and associated pro-inflammatory cytokine production is a pathological feature of inflammatory liver disease. It is also known that liver injury is associated with marked impairments in the function of many hepatic proteins including a hepatocyte-specific binding protein, the asialoglycoprotein receptor (ASGPR). Recently, it has been suggested that hepatic ASGPRs may play an important role in the physiological regulation of T lymphocytes, leading to our hypothesis that ASGPR defects correlate with inflammatory-mediated events in liver diseases. Therefore, in this study we investigated whether changes in hepatocellular ASGPR expression were related to the dysregulation of intrahepatic T lymphocytes and correlate with the development of T-cell mediated hepatitis. Mice lacking functional ASGPRs (receptor-deficient, RD), and wild-type (WT) controls were intravenously injected with T-cell mitogens, Concanavalin A (Con A) or anti-CD3 antibody. As a result of T cell mitogen treatment, RD mice lacking hepatic ASGPRs displayed enhancements in liver pathology, transaminase activities, proinflammatory cytokine expression, and caspase activation compared to that observed in normal WT mice. Furthermore, FACS analysis demonstrated that T-cell mitogen administration resulted in a significant rise in the percentage of CD8+ lymphocytes present in the livers of RD animals versus WT mice. Since these two mouse strains differ only in whether they express the hepatic ASGPR, it can be concluded that proper ASGPR function exerts a protective effect against T cell mediated hepatitis and that impairments to this hepatic receptor could be related to the accumulation of cytotoxic T cells that are observed in inflammatory liver diseases.

  8. Association of susceptibility to septic shock with platelet endothelial cell adhesion molecule-1 gene Leu125Val polymorphism and serum sPECAM-1 levels in sepsis patients.

    PubMed

    Sun, Wei; Li, Fang-Shun; Zhang, Yuan-Huai; Wang, Xiao-Ping; Wang, Chao-Rong

    2015-01-01

    Sepsis is a systemic inflammatory response to infection and includes severe sepsis, septic shock and death. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is one cell adhesion molecule expressed on platelets and leukocytes. It regulates platelet activation and mediates transendothelial migration of leukocytes, thus maintaining the integrity of the vasculature. There are some animal experiments associated with the protective role of PECAM-1 against septic shock. However few host genetic risk factors have been identified for sepsis severity and susceptibility to septic shock. A case-control study was conducted, which included 217 patients with sepsis and 90 control subjects recruited from our hospital. One single nucleotide polymorphisms (SNP) of PECAM-1 gene Leu125Val (C373G) was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Serum soluble PECAM-1 (sPECAM-1) levels were determined by enzyme-linked immunosorbent assay (ELISA). Our results showed that the CG and GG genotypes of SNP in Leu125Val of PECAM-1 (rs668: C>G) was significantly associated with increased susceptibility to septic shock compared with CC genotype in sepsis patients (CG genotype, OR: 2.493, 95% CI: 1.175~5.287, P = 0.016; GG genotype: OR: 3.328, 95% CI: 1.445~7.666, P = 0.004). The serum levels of sPECAM-1 in the sepsis patients (47.1 ± 17.5 ng/ml) were significantly higher than those in the healthy controls (61.3 ± 20.9 ng/ml, P<0.01). Among sepsis patients, the serum levels of sPECAM-1 were significantly higher in CG and GG genotype than in CC genotype. In septic shock patients, nonsurvivors (83.7 ± 12.6 ng/ml, n = 69) had a significantly higher serum sPECAM-1 level than the survivors (76.9 ± 12.7 ng/ml, n = 53) (P<0.01). In conclusion, PECAM-1 Leu125Val polymorphism and its sPECAM-1 levels are associated with sepsis severity and susceptibility to septic shock. PMID:26884965

  9. Association of susceptibility to septic shock with platelet endothelial cell adhesion molecule-1 gene Leu125Val polymorphism and serum sPECAM-1 levels in sepsis patients

    PubMed Central

    Sun, Wei; Li, Fang-Shun; Zhang, Yuan-Huai; Wang, Xiao-Ping; Wang, Chao-Rong

    2015-01-01

    Sepsis is a systemic inflammatory response to infection and includes severe sepsis, septic shock and death. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is one cell adhesion molecule expressed on platelets and leukocytes. It regulates platelet activation and mediates transendothelial migration of leukocytes, thus maintaining the integrity of the vasculature. There are some animal experiments associated with the protective role of PECAM-1 against septic shock. However few host genetic risk factors have been identified for sepsis severity and susceptibility to septic shock. A case-control study was conducted, which included 217 patients with sepsis and 90 control subjects recruited from our hospital. One single nucleotide polymorphisms (SNP) of PECAM-1 gene Leu125Val (C373G) was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Serum soluble PECAM-1 (sPECAM-1) levels were determined by enzyme-linked immunosorbent assay (ELISA). Our results showed that the CG and GG genotypes of SNP in Leu125Val of PECAM-1 (rs668: C>G) was significantly associated with increased susceptibility to septic shock compared with CC genotype in sepsis patients (CG genotype, OR: 2.493, 95% CI: 1.175~5.287, P = 0.016; GG genotype: OR: 3.328, 95% CI: 1.445~7.666, P = 0.004). The serum levels of sPECAM-1 in the sepsis patients (47.1 ± 17.5 ng/ml) were significantly higher than those in the healthy controls (61.3 ± 20.9 ng/ml, P<0.01). Among sepsis patients, the serum levels of sPECAM-1 were significantly higher in CG and GG genotype than in CC genotype. In septic shock patients, nonsurvivors (83.7 ± 12.6 ng/ml, n = 69) had a significantly higher serum sPECAM-1 level than the survivors (76.9 ± 12.7 ng/ml, n = 53) (P<0.01). In conclusion, PECAM-1 Leu125Val polymorphism and its sPECAM-1 levels are associated with sepsis severity and susceptibility to septic shock. PMID:26884965

  10. Method for determining properties of red blood cells

    DOEpatents

    Gourley, Paul L.

    2001-01-01

    A method for quantifying the concentration of hemoglobin in a cell, and indicia of anemia, comprises determining the wavelength of the longitudinal mode of a liquid in a laser microcavity; determining the wavelength of the fundamental transverse mode of a red blood cell in the liquid in the laser microcavity; and determining if the cell is anemic from the difference between the wavelength of the longitudinal mode and the fundamental transverse mode. In addition to measuring hemoglobin, the invention includes a method using intracavity laser spectroscopy to measure the change in spectra as a function of time for measuring the influx of water into a red blood cell and the cell's subsequent rupture.

  11. Cell Transmembrane Receptors Determine Tissue Pattern Stability

    NASA Astrophysics Data System (ADS)

    Beyer, Tilo; Meyer-Hermann, Michael

    2008-10-01

    The analysis of biological systems requires mathematical tools that represent their complexity from the molecular scale up to the tissue level. The formation of cell aggregates by chemotaxis is investigated using Delaunay object dynamics. It is found that when cells migrate fast such that the chemokine distribution is far from equilibrium, the details of the chemokine receptor dynamics can induce an internalization driven instability of cell aggregates. The instability occurs in a parameter regime relevant for lymphoid tissue and is similar to ectopic lymphoid structures.

  12. Differential type I interferon activation and susceptibility of dendritic cell populations to porcine arterivirus

    PubMed Central

    Loving, Crystal L; Brockmeier, Susan L; Sacco, Randy E

    2007-01-01

    Dendritic cells (DCs) play a role in anti-viral immunity by providing early innate protection against viral replication and by presenting antigen to T cells for initiation of the adaptive immune response. Studies show the adaptive response to porcine reproductive and respiratory syndrome virus (PRRSV) is ineffective for complete viral elimination. Other studies describe the kinetics of the adaptive response to PRRSV, but have not investigated the early response by DCs. We hypothesize that there is an aberrant activation of DCs early in PRRSV infection; consequently, the adaptive response is triggered inadequately. The current study characterized a subtype of porcine lung DCs (L-DCs) and investigated the ability of PRRSV to infect and replicate in L-DCs and monocyte-derived DCs (MDDCs). Furthermore, the type I interferon anti-viral response to PRRSV with and without the addition of recombinant porcine IFN-α (rpIFN-α), an important cytokine that signals for anti-viral mediator activation, was analysed. Results show that PRRSV replicated in MDDCs but not L-DCs, providing evidence that these cells have followed distinct differentiation pathways. Although both cell types responded to PRRSV with an induction of IFN-β mRNA, the magnitude and duration of the response differed between cell types. The addition of rpIFN-α was protective in MDDCs, and mRNA synthesis of Mx (myxovirus resistant) and PKR (double-stranded RNA dependent protein kinase) was observed in both cell types after rpIFN-α addition. Overall, PRRSV replicated in MDDCs but not L-DCs, and rpIFN-α was required for the transcription of protective anti-viral mediators. DC response to PRRSV was limited to IFN-β transcription, which may be inadequate in triggering the adaptive immune response. PMID:17116172

  13. Two monokines, interleukin 1 and tumor necrosis factor, render cultured vascular endothelial cells susceptible to lysis by antibodies circulating during Kawasaki syndrome

    PubMed Central

    1986-01-01

    Kawasaki syndrome (KS) is an acute febrile illness of early childhood characterized by diffuse vasculitis and marked immune activation. The present study was undertaken to determine whether the acute phase of KS is associated with circulating cytotoxic antibodies directed to target antigens induced on vascular endothelium by the monokines, IL-1, or tumor necrosis factor (TNF). Sera from 20 patients with acute KS, 11 patients in the convalescent phase of KS, and 17 age-matched controls were assessed for complement-dependent cytotoxic activity against 111In- labeled human endothelial cells (HEC), dermal fibroblasts, and vascular smooth muscle cells. Sera from patients with acute KS but not the other subject groups caused significant (p less than 0.01) complement- mediated killing of IL-1- or TNF-stimulated HEC. None of the sera tested had cytotoxicity against control HEC cultures or the other target cell types, with or without IL-1 or TNF pretreatment. Expression of the IL-1- or TNF-inducible target antigens on endothelial cells was rapid and transient, peaking at 4 h and disappearing after 24 h despite continued incubation with monokine. In contrast, we have previously shown that IFN-gamma requires 72 h to render HEC susceptible to lysis with acute KS sera. Serum adsorption studies demonstrated that IL-1- and TNF-inducible endothelial target antigens are distinct from IFN- gamma-inducible antigens. These observations suggest that mediator secretion by activated monocyte/macrophages could be a predisposing factor to the development of vascular injury in acute KS. Although our present observations have been restricted to KS, the development of cytotoxic antibodies directed to monokine-inducible endothelial cell antigens may also be found in other vasculitides accompanied by immune activation. PMID:3491174

  14. Human NK cell repertoire diversity reflects immune experience and correlates with viral susceptibility

    PubMed Central

    Strauss-Albee, Dara M.; Fukuyama, Julia; Liang, Emily C.; Yao, Yi; Jarrell, Justin A.; Drake, Alison L.; Kinuthia, John; Montgomery, Ruth R.; John-Stewart, Grace; Holmes, Susan; Blish, Catherine A.

    2015-01-01

    Innate natural killer (NK) cells are diverse at the single-cell level because of variegated expressions of activating and inhibitory receptors, yet the developmental roots and functional consequences of this diversity remain unknown. Because NK cells are critical for antiviral and antitumor responses, a better understanding of their diversity could lead to an improved ability to harness them therapeutically. We found that NK diversity is lower at birth than in adults. During an antiviral response to either HIV-1 or West Nile virus, NK diversity increases, resulting in terminal differentiation and cytokine production at the cost of cell division and degranulation. In African women matched for HIV-1 exposure risk, high NK diversity is associated with increased risk of HIV-1 acquisition. Existing diversity may therefore decrease the flexibility of the antiviral response. Collectively, the data reveal that human NK diversity is a previously undefined metric of immune history and function that may be clinically useful in forecasting the outcomes of infection and malignancy. PMID:26203083

  15. Ceramide and neurodegeneration: Susceptibility of neurons and oligodendrocytes to cell damage and death

    PubMed Central

    Jana, Arundhati; Hogan, Edward L.; Pahan, Kalipada

    2009-01-01

    Neurodegenerative disorders are marked by extensive neuronal apoptosis and gliosis. Although several apoptosis-inducing agents have been described, understanding of the regulatory mechanisms underlying modes of cell death is incomplete. A major breakthrough in delineation of the mechanism of cell death came from elucidation of the sphingomyelin (SM)-ceramide pathway that has received worldwide attention in recent years. The SM pathway induces apoptosis, differentiation, proliferation, and growth arrest depending upon cell and receptor types, and on downstream targets. Sphingomyelin, a plasma membrane constituent, is abundant in mammalian nervous system, and ceramide, its primary catabolic product released by activation of either neutral or acidic sphingomyelinase, serves as a potential lipid second messenger or mediator molecule modulating diverse cellular signaling pathways. Neutral sphingomyelinase (NSMase) is a key enzyme in the regulated activation of the SM cycle and is particularly sensitive to oxidative stress. In a context of increasing clarification of the mechanisms of neurodegeneration, we thought that it would be useful to review details of recent findings that we and others have made concerning different pro-apoptotic neurotoxins including proinflammatory cytokines, hypoxia-induced SM hydrolysis and ceramide production that induce cell death in human primary neurons and primary oligodendrocytes: redox sensitive events. What has and is emerging is a vista of therapeutically important ceramide regulation affecting a variety of different neurodegenerative and neuroinflammatory disorders. PMID:19147160

  16. Predicting drug susceptibility of non–small cell lung cancers based on genetic lesions

    PubMed Central

    Sos, Martin L.; Michel, Kathrin; Zander, Thomas; Weiss, Jonathan; Frommolt, Peter; Peifer, Martin; Li, Danan; Ullrich, Roland; Koker, Mirjam; Fischer, Florian; Shimamura, Takeshi; Rauh, Daniel; Mermel, Craig; Fischer, Stefanie; Stückrath, Isabel; Heynck, Stefanie; Beroukhim, Rameen; Lin, William; Winckler, Wendy; Shah, Kinjal; LaFramboise, Thomas; Moriarty, Whei F.; Hanna, Megan; Tolosi, Laura; Rahnenführer, Jörg; Verhaak, Roel; Chiang, Derek; Getz, Gad; Hellmich, Martin; Wolf, Jürgen; Girard, Luc; Peyton, Michael; Weir, Barbara A.; Chen, Tzu-Hsiu; Greulich, Heidi; Barretina, Jordi; Shapiro, Geoffrey I.; Garraway, Levi A.; Gazdar, Adi F.; Minna, John D.; Meyerson, Matthew; Wong, Kwok-Kin; Thomas, Roman K.

    2009-01-01

    Somatic genetic alterations in cancers have been linked with response to targeted therapeutics by creation of specific dependency on activated oncogenic signaling pathways. However, no tools currently exist to systematically connect such genetic lesions to therapeutic vulnerability. We have therefore developed a genomics approach to identify lesions associated with therapeutically relevant oncogene dependency. Using integrated genomic profiling, we have demonstrated that the genomes of a large panel of human non–small cell lung cancer (NSCLC) cell lines are highly representative of those of primary NSCLC tumors. Using cell-based compound screening coupled with diverse computational approaches to integrate orthogonal genomic and biochemical data sets, we identified molecular and genomic predictors of therapeutic response to clinically relevant compounds. Using this approach, we showed that v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations confer enhanced Hsp90 dependency and validated this finding in mice with KRAS-driven lung adenocarcinoma, as these mice exhibited dramatic tumor regression when treated with an Hsp90 inhibitor. In addition, we found that cells with copy number enhancement of v-abl Abelson murine leukemia viral oncogene homolog 2 (ABL2) and ephrin receptor kinase and v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) (SRC) kinase family genes were exquisitely sensitive to treatment with the SRC/ABL inhibitor dasatinib, both in vitro and when it xenografted into mice. Thus, genomically annotated cell-line collections may help translate cancer genomics information into clinical practice by defining critical pathway dependencies amenable to therapeutic inhibition. PMID:19451690

  17. Human embryonic stem cells and derived contractile embryoid bodies are susceptible to Coxsakievirus B infection and respond to interferon Iβ treatment.

    PubMed

    Scassa, María E; Jaquenod de Giusti, Carolina; Questa, María; Pretre, Gabriela; Richardson, Guillermo A Videla; Bluguermann, Carolina; Romorini, Leonardo; Ferrer, María F; Sevlever, Gustavo E; Miriuka, Santiago G; Gómez, Ricardo M

    2011-01-01

    We studied the susceptibility of human embryonic stem cells and derived contractile embryoid bodies from WAO9, HUES-5 and HUES-16 cell lines to Coxsackievirus B infection. After validating stem cell-like properties and cardiac phenotype, Coxsackievirus B receptors CAR and DAF, as well as type I interferon receptors were detected in all cell lines and differentiation stages studied. Real-time PCR analysis showed that CAR mRNA levels were 3.4-fold higher in undifferentiated cells, while DAF transcript levels were 2.78-fold more abundant in differentiated cultures (P<0.05). All cell lines were susceptible to Coxsackievirus serotypes B1-5 infection as shown by RT-PCR detection of viral RNA, immunofluorescence detection of viral protein and infectivity titration of cell culture supernatants resulting in cell death. Supernatants infectivity titers 24-48 h post-infection ranged from 10⁵-10⁶ plaque forming units (PFU)/ml, the highest titers were detected in undifferentiated cells. Cell viability detected by a colorimetric assay, showed inverse correlation with infectivity titers of cell culture supernatants. Treatment with 100 U of interferon Iβ significantly reduced viral replication and associated cell death during a 24-48 h observation period, as detected by reduced infectivity titers in the supernatants and increased cell viability by a colorimetric assay, respectively. We propose human embryonic stem cell and derived contractile embryoid bodies as a valid model to study cardiac Coxsackievirus B infection.

  18. Contact inhibition of locomotion determines cell-cell and cell-substrate forces in tissues.

    PubMed

    Zimmermann, Juliane; Camley, Brian A; Rappel, Wouter-Jan; Levine, Herbert

    2016-03-01

    Cells organized in tissues exert forces on their neighbors and their environment. Those cellular forces determine tissue homeostasis as well as reorganization during embryonic development and wound healing. To understand how cellular forces are generated and how they can influence the tissue state, we develop a particle-based simulation model for adhesive cell clusters and monolayers. Cells are contractile, exert forces on their substrate and on each other, and interact through contact inhibition of locomotion (CIL), meaning that cell-cell contacts suppress force transduction to the substrate and propulsion forces align away from neighbors. Our model captures the traction force patterns of small clusters of nonmotile cells and larger sheets of motile Madin-Darby canine kidney (MDCK) cells. In agreement with observations in a spreading MDCK colony, the cell density in the center increases as cells divide and the tissue grows. A feedback between cell density, CIL, and cell-cell adhesion gives rise to a linear relationship between cell density and intercellular tensile stress and forces the tissue into a nonmotile state characterized by a broad distribution of traction forces. Our model also captures the experimentally observed tissue flow around circular obstacles, and CIL accounts for traction forces at the edge. PMID:26903658

  19. Role of skin immune cells on the host susceptibility to mosquito-borne viruses.

    PubMed

    Briant, Laurence; Desprès, Philippe; Choumet, Valérie; Missé, Dorothée

    2014-09-01

    Due to climate change and the propagation of competent arthropods worldwide, arboviruses have become pathogens of major medical importance. Early transmission to vertebrates is initiated by skin puncture and deposition of virus together with arthropod saliva in the epidermis and dermis. Saliva components have the capacity to modulate skin cell responses by enhancing and/or counteracting initial replication and establishment of systemic viral infection. Here, we review the nature of the cells targeted by arboviruses at the skin level and discuss the type of cellular responses elicited by these pathogens in light of the immunomodulatory properties of arthropod vector-derived salivary factors injected at the inoculation site. Understanding cutaneous arbovirus-host interactions may provide new clues for the design of future therapeutics. PMID:25043586

  20. Deletion of aquaporin-4 renders retinal glial cells more susceptible to osmotic stress.

    PubMed

    Pannicke, Thomas; Wurm, Antje; Iandiev, Ianors; Hollborn, Margrit; Linnertz, Regina; Binder, Devin K; Kohen, Leon; Wiedemann, Peter; Steinhäuser, Christian; Reichenbach, Andreas; Bringmann, Andreas

    2010-10-01

    The glial water channel aquaporin-4 (AQP4) is implicated in the control of ion and osmohomeostasis in the sensory retina. Using retinal slices from AQP4-deficient and wild-type mice, we investigated whether AQP4 is involved in the regulation of glial cell volume under altered osmotic conditions. Superfusion of retinal slices with a hypoosmolar solution induced a rapid swelling of glial somata in tissues from AQP4 null mice but not from wild-type mice. The swelling was mediated by oxidative stress, inflammatory lipid mediators, and sodium influx into the cells and was prevented by activation of glutamatergic and purinergic receptors. Distinct inflammatory proteins, including interleukin-1 beta, interleukin-6, and inducible nitric oxide synthase, were up-regulated in the retina of AQP4 null mice compared with control, whereas cyclooxygenase-2 was down-regulated. The data suggest that water flux through AQP4 is involved in the rapid volume regulation of retinal glial (Müller) cells in response to osmotic stress and that deletion of AQP4 results in an inflammatory response of the retinal tissue. Possible implications of the data for understanding the pathophysiology of neuromyelitis optica, a human disease that has been suggested to involve serum antibodies to AQP4, are discussed. PMID:20544823

  1. Promoter methylation and expression of CDH1 and susceptibility and prognosis of eyelid squamous cell carcinoma.

    PubMed

    Wang, Yong-Qiang; Yuan, Ye; Jiang, Shan; Jiang, Hua

    2016-07-01

    Eyelid skin tumors are the most frequent type of cancer in ophthalmology. And, eyelid squamous cell carcinoma (SCC) accounts for a large part of it. CDH1 encodes E-cadherin, a glycoprotein that plays an important part in cell-cell interaction. Loss of CDH1 function was suspected to be associated with tumorigenesis. Methylation of CDH1 promotors can alter the expression of its protein and is also considered as a contributor to various cancers. In this study, CDH1 methylation and expression profile as well as prognosis of 38 cases of eyelid SCC and the corresponding adjacent tissues were analyzed to clarify the role of CDH1 methylation in SCC carcinogenesis and prognosis. Methylation was detected by PCR, and CDH1 expression was evaluated by immunohistochemistry. We observed that CDH1 methylation is significantly correlated with decreased CDH1 protein expression in eyelid SCC patients. Patients with methylation and low expression of CDH1 are significantly associated with advanced and aggressive phenotypes. Therefore, CDH1 methylation and CDH1 expression are both independent prognostic factors for prognosis of eyelid SCC patients.

  2. Enhanced susceptibility of B lymphoma cells to measles virus by Epstein-Barr virus type III latency that upregulates CD150/signaling lymphocytic activation molecule.

    PubMed

    Takeda, Satoshi; Kanbayashi, Daiki; Kurata, Takako; Yoshiyama, Hironori; Komano, Jun

    2014-02-01

    Measles virus (MV) is one of the candidates for the application of oncolytic virotherapy (OVT). Although an advanced clinical study has been reported on a T-cell lymphoma, the potential of MV OVT against B-cell lymphomas remains to be clarified. We found that an EBV-transformed B lymphoblastoid cell line, a model for diffuse large B-cell lymphoma, and EBV-positive Burkitt's lymphoma cells bearing type III latency were highly susceptible to the cytolysis induced by an MV vaccine strain CAM-70. As analyzed by EBV-positive and -negative counterparts of the same cytogenetic background, type III EBV latency, not type I, was shown to augment the susceptibility of B lymphoma cells to MV-induced cytolysis. Cell surface levels of CD150/signaling lymphocytic activation molecule, a receptor of MV, were upregulated in B lymphoma cell lines with type III EBV latency by 3.8-fold, on average. The cytolytic activity of CD150-tropic WT MV was akin to that of CD46- and CD150-tropic CAM-70, suggesting that CD150 is critical for the susceptibility to MV-induced cytolysis. Among EBV-encoded genes, latent membrane protein 1 was responsible for the CD150 upregulation. It was notable that the majority of B lymphoma cell lines of type III EBV latency showed higher susceptibility to the non-Edmonston-derived CAM-70 than to the Edmonston-derived Schwarz strain. This is the first report indicating the potential of non-Edmonston MV strain for the application of OVT. Furthermore, a cellular regulator of MV replication was implicated that functions in a vaccine strain-specific fashion. Altogether, the MV OVT should serve as an alternative therapy against EBV-positive diffuse large B-cell lymphoma with type III EBV latency.

  3. [Morphology and cytochemistry of Aedes aegypti's cell cultures (Diptera: Culicidae) and susceptibility to Leishmania panamensis (Kinetoplastida: Trypanosomatidae)].

    PubMed

    Miranda, Alfonso Arturo; Sarmiento, Ladys; Caldas, María Leonor; Zapata, Cristina; Bello, Felio Jesús

    2008-06-01

    Morphology and cytochemistry of Aedes aegypti's cell cultures (Diptera: Culicidae) and susceptibility to Leishmania panamensis (Kinetoplastida: Trypanosomatidae). The first cellular line of Aedes aegypti was developed by Grace in 1966; afterwards, other cellular lines of this species have been generated. These have been used for the study of pathogenic organisms like viruses, bacteria and parasites, which demonstrates their importance in biomedical applications. This research describes, for the first time, some cytochemical characteristics of A. aegypti cell cultures, that were infected with (MHOM/CO/87CL412) strain of Leishmania panamensis. A morphological study of the cell culture was also carried out. Maintenance of the cell culture, parasites and infection in vitro were carried out in the Laboratory of Entomology, Cell Biology and Genetics of the Universidad de La Salle. The cell cultures infected with the parasite were maintained in a mixture of mediums Grace/L15, supplemented with 10 % fetal bovine serum (FBS) at pH 6.8 and a temperature of 26 degrees C, during 3, 6 and 9 post-infection days. After this, these cell cultures were processed through High Resolution Light Microscopy (HRLM) and Transmission Electron Microscopy (TEM) based on standard protocols defined by the Group of Microscopy and Image Analyses of the Instituto Nacional de Salud. Semi-fine slices of 1 microm colored with toluidine blue were used for the morphological analysis of the culture, and ultra fine cuts of 60 to 90 nm stained with uranyl acetate and lead citrate where used for the ultrastructural study. In addition, PAS and peroxidase staining was carried out in cells fixed with methanol. The morphometric study was analyzed with software ImageJ (NIH). In the semi-fine slices, small cells were observed showing fibroblastic appearance 10.84 +/- 2.54 microm in length and 5.31 +/- 1.26 microm wide; other cells had epithelial appearance with a great peripheral nucleus, voluminous and

  4. The 12p13.33/RAD52 locus and genetic susceptibility to squamous cell cancers of upper aerodigestive tract.

    PubMed

    Delahaye-Sourdeix, Manon; Oliver, Javier; Timofeeva, Maria N; Gaborieau, Valérie; Johansson, Mattias; Chabrier, Amélie; Wozniak, Magdalena B; Brenner, Darren R; Vallée, Maxime P; Anantharaman, Devasena; Lagiou, Pagona; Holcátová, Ivana; Richiardi, Lorenzo; Kjaerheim, Kristina; Agudo, Antonio; Castellsagué, Xavier; Macfarlane, Tatiana V; Barzan, Luigi; Canova, Cristina; Thakker, Nalin S; Conway, David I; Znaor, Ariana; Healy, Claire M; Ahrens, Wolfgang; Zaridze, David; Szeszenia-Dabrowska, Neonilia; Lissowska, Jolanta; Fabianova, Eleonora; Mates, Ioan Nicolae; Bencko, Vladimir; Foretova, Lenka; Janout, Vladimir; Curado, Maria Paula; Koifman, Sergio; Menezes, Ana; Wünsch-Filho, Victor; Eluf-Neto, José; Boffetta, Paolo; Garrote, Leticia Fernández; Serraino, Diego; Lener, Marcin; Jaworowska, Ewa; Lubiński, Jan; Boccia, Stefania; Rajkumar, Thangarajan; Samant, Tanuja A; Mahimkar, Manoj B; Matsuo, Keitaro; Franceschi, Silvia; Byrnes, Graham; Brennan, Paul; McKay, James D

    2015-01-01

    Genetic variants located within the 12p13.33/RAD52 locus have been associated with lung squamous cell carcinoma (LUSC). Here, within 5,947 UADT cancers and 7,789 controls from 9 different studies, we found rs10849605, a common intronic variant in RAD52, to be also associated with upper aerodigestive tract (UADT) squamous cell carcinoma cases (OR = 1.09, 95% CI: 1.04-1.15, p = 6x10(-4)). We additionally identified rs10849605 as a RAD52 cis-eQTL inUADT(p = 1x10(-3)) and LUSC (p = 9x10(-4)) tumours, with the UADT/LUSC risk allele correlated with increased RAD52 expression levels. The 12p13.33 locus, encompassing rs10849605/RAD52, was identified as a significant somatic focal copy number amplification in UADT(n = 374, q-value = 0.075) and LUSC (n = 464, q-value = 0.007) tumors and correlated with higher RAD52 tumor expression levels (p = 6x10(-48) and p = 3x10(-29) in UADT and LUSC, respectively). In combination, these results implicate increased RAD52 expression in both genetic susceptibility and tumorigenesis of UADT and LUSC tumors.

  5. The hydration state of human red blood cells and their susceptibility to invasion by Plasmodium falciparum.

    PubMed

    Tiffert, Teresa; Lew, Virgilio L; Ginsburg, Hagai; Krugliak, Miriam; Croisille, Laure; Mohandas, Narla

    2005-06-15

    In most inherited red blood cell (RBC) disorders with high gene frequencies in malaria-endemic regions, the distribution of RBC hydration states is much wider than normal. The relationship between the hydration state of circulating RBCs and protection against severe falciparum malaria remains unexplored. The present investigation was prompted by a casual observation suggesting that falciparum merozoites were unable to invade isotonically dehydrated normal RBCs. We designed an experimental model to induce uniform and stable isotonic volume changes in RBC populations from healthy donors by increasing or decreasing their KCl contents through a reversible K(+) permeabilization pulse. Swollen and mildly dehydrated RBCs were able to sustain Plasmodium falciparum cultures with similar efficiency to untreated RBCs. However, parasite invasion and growth were progressively reduced in dehydrated RBCs. In a parallel study, P falciparum invasion was investigated in density-fractionated RBCs from healthy subjects and from individuals with inherited RBC abnormalities affecting primarily hemoglobin (Hb) or the RBC membrane (thalassemias, hereditary ovalocytosis, xerocytosis, Hb CC, and Hb CS). Invasion was invariably reduced in the dense cell fractions in all conditions. These results suggest that the presence of dense RBCs is a protective factor, additional to any other protection mechanism prevailing in each of the different pathologies. PMID:15728121

  6. The hydration state of human red blood cells and their susceptibility to invasion by Plasmodium falciparum

    PubMed Central

    Tiffert, Teresa; Lew, Virgilio L.; Ginsburg, Hagai; Krugliak, Miriam; Croisille, Laure; Mohandas, Narla

    2005-01-01

    In most inherited red blood cell (RBC) disorders with high gene frequencies in malaria-endemic regions, the distribution of RBC hydration states is much wider than normal. The relationship between the hydration state of circulating RBCs and protection against severe falciparum malaria remains unexplored. The present investigation was prompted by a casual observation suggesting that falciparum merozoites were unable to invade isotonically dehydrated normal RBCs. We designed an experimental model to induce uniform and stable isotonic volume changes in RBC populations from healthy donors by increasing or decreasing their KCl contents through a reversible K+ permeabilization pulse. Swollen and mildly dehydrated RBCs were able to sustain Plasmodium falciparum cultures with similar efficiency to untreated RBCs. However, parasite invasion and growth were progressively reduced in dehydrated RBCs. In a parallel study, P falciparum invasion was investigated in density-fractionated RBCs from healthy subjects and from individuals with inherited RBC abnormalities affecting primarily hemoglobin (Hb) or the RBC membrane (thalassemias, hereditary ovalocytosis, xerocytosis, Hb CC, and Hb CS). Invasion was invariably reduced in the dense cell fractions in all conditions. These results suggest that the presence of dense RBCs is a protective factor, additional to any other protection mechanism prevailing in each of the different pathologies. (Blood. 2005; 105:4853-4860) PMID:15728121

  7. A human astrocytoma cell line is highly susceptible to infection with Trypanosoma cruzi.

    PubMed

    Vargas-Zambrano, Juan Camilo; Lasso, Paola; Cuellar, Adriana; Puerta, Concepción Judith; González, John Mario

    2013-04-01

    Astrocytes play a vital role in neuronal protection, homeostasis, vascular interchange and the local immune response. Some viruses and parasites can cross the blood-brain barrier and infect glia. Trypanosoma cruzi, the aetiological agent of Chagas disease, can seriously compromise the central nervous system, mainly in immune-suppressed individuals, but also during the acute phase of the infection. In this report, the infective capacity of T. cruzi in a human astrocyte tumour-derived cell line was studied. Astrocytes exposed to trypomastigotes (1:10 ratio) produced intracellular amastigotes and new trypomastigotes emerged by day 4 post-infection (p.i.). At day 6 p.i., 93% of the cells were infected. Using flow cytometry, changes were observed in both the expression of major histocompatibility complex class I and II molecules and the chemokine secretion pattern of astrocytes exposed to the parasite. Blocking the low-density lipoprotein receptor on astrocytes did not reduce parasite intracellular infection. Thus, T. cruzi can infect astrocytes and modulate the immune response during central nervous system infection.

  8. ERK2 mediates inner hair cell survival and decreases susceptibility to noise-induced hearing loss

    PubMed Central

    Kurioka, Takaomi; Matsunobu, Takeshi; Satoh, Yasushi; Niwa, Katsuki; Endo, Shogo; Fujioka, Masato; Shiotani, Akihiro

    2015-01-01

    Extracellular signal-regulated kinase (ERK) is a member of the family of mitogen-activated protein kinases (MAPKs) and coordinately regulates a multitude of cellular processes. In response to a variety of extracellular stimuli, phosphorylation of both threonine and tyrosine residues activates ERK. Recent evidence indicates that ERK is activated in response to cellular stress such as acoustic trauma. However, the specific role of ERK isoforms in auditory function is not fully understood. Here, we show that the isoform ERK2 plays an important role in regulating hair cell (HC) survival and noise-induced hearing loss (NIHL) in mice (C57BL/6J). We found that conditional knockout mice deficient for Erk2 in the inner ear HCs had hearing comparable to control mice and exhibited no HC loss under normal conditions. However, we found that these knockout mice were more vulnerable to noise and had blunted recovery from NIHL compared to control mice. Furthermore, we observed a significantly lower survival rate of inner hair cells in these mice compared to control mice. Our results indicate that ERK2 plays important roles in the survival of HC in NIHL. PMID:26577290

  9. C60(Nd) nanoparticles enhance chemotherapeutic susceptibility of cancer cells by modulation of autophagy

    NASA Astrophysics Data System (ADS)

    Wei, Pengfei; Zhang, Li; Lu, Yang; Man, Na; Wen, Longping

    2010-12-01

    Autophagy, an evolutionally conserved intracellular process degrading cytoplasmic proteins and organelles for recycling, has become one of the most remarkable strategies applied in cancer research. The fullerene C60 nanoparticle (nC60) has been shown to induce autophagy and sensitize chemotherapeutic killing of cancer cells, but the details still remain unknown. Here we show that a water-dispersed nanoparticle solution of derivatized fullerene C60, C60(Nd) nanoparticles (nC60(Nd)), has greater potential in inducing autophagy and sensitizing chemotherapeutic killing of both normal and drug-resistant cancer cells than nC60 does in an autophagy-dependent fashion. Additionally we further demonstrated that autophagy induced by nC60/C60(Nd) and Rapamycin had completely different roles in cancer chemotherapy. Our results, for the first time, revealed a novel and more potent derivative of the C60 nanoparticle in enhancing the cytotoxicity of chemotherapeutic agents and reducing drug resistance through autophagy modulation, which may ultimately lead to novel therapeutic strategies in cancer therapy.

  10. Nitrite Modulates Bacterial Antibiotic Susceptibility and Biofilm Formation in Association with Airway Epithelial Cells

    PubMed Central

    Zemke, Anna C; Shiva, Sruti; Burn, Jane L.; Moskowitz, Samuel M.; Pilewski, Joseph M.; Gladwin, Mark T.; Bomberger, Jennifer M.

    2014-01-01

    Pseudomonas aeruginosa is the major pathogenic bacteria in cystic fibrosis and other forms of bronchiectasis. Growth in antibiotic resistant biofilms contributes to the virulence of this organism. Sodium nitrite has antimicrobial properties and has been tolerated as a nebulized compound at high concentrations in human subjects with pulmonary hypertension; however, its effects have not been evaluated on biotic biofilms or in combination with other clinically useful antibiotics. We grew P. aeruginosa on the apical surface of primary human airway epithelial cells to test the efficacy of sodium nitrite against biotic biofilms. Nitrite alone prevented 99% of biofilm growth. We then identified significant cooperative interactions between nitrite and polymyxins. For P. aeruginosa growing on primary CF airway cells, combining nitrite and colistimethate resulted in an additional log of bacterial inhibition compared to treating with either agent alone. Nitrite and colistimethate additively inhibited oxygen consumption by P. aeruginosa. Surprisingly, while the antimicrobial effects of nitrite in planktonic, aerated cultures are nitric oxide (NO) dependent, antimicrobial effects in other growth conditions are not. The inhibitory effect of nitrite on bacterial oxygen consumption and biofilm growth did not require NO as an intermediate as chemically scavenging NO did not block growth inhibition. These data suggest an NO-radical independent nitrosative or oxidative inhibition of respiration. The combination of nebulized sodium nitrite and colistimethate may provide a novel therapy for chronic P. aeruginosa airway infections, because sodium nitrite, unlike other antibiotic respiratory chain ‘poisons’, can be safely nebulized at high concentration in humans. PMID:25229185

  11. ERK2 mediates inner hair cell survival and decreases susceptibility to noise-induced hearing loss.

    PubMed

    Kurioka, Takaomi; Matsunobu, Takeshi; Satoh, Yasushi; Niwa, Katsuki; Endo, Shogo; Fujioka, Masato; Shiotani, Akihiro

    2015-01-01

    Extracellular signal-regulated kinase (ERK) is a member of the family of mitogen-activated protein kinases (MAPKs) and coordinately regulates a multitude of cellular processes. In response to a variety of extracellular stimuli, phosphorylation of both threonine and tyrosine residues activates ERK. Recent evidence indicates that ERK is activated in response to cellular stress such as acoustic trauma. However, the specific role of ERK isoforms in auditory function is not fully understood. Here, we show that the isoform ERK2 plays an important role in regulating hair cell (HC) survival and noise-induced hearing loss (NIHL) in mice (C57BL/6J). We found that conditional knockout mice deficient for Erk2 in the inner ear HCs had hearing comparable to control mice and exhibited no HC loss under normal conditions. However, we found that these knockout mice were more vulnerable to noise and had blunted recovery from NIHL compared to control mice. Furthermore, we observed a significantly lower survival rate of inner hair cells in these mice compared to control mice. Our results indicate that ERK2 plays important roles in the survival of HC in NIHL.

  12. Alcohol metabolism in human cells causes DNA damage and activates the Fanconi anemia – breast cancer susceptibility (FA-BRCA) DNA damage response network

    PubMed Central

    Abraham, Jessy; Balbo, Silvia; Crabb, David; Brooks, P.J.

    2011-01-01

    Background We recently reported that exposure of human cells in vitro to acetaldehyde resulted in activation of the Fanconi anemia-breast cancer associated (FA-BRCA) DNA damage response network. Methods To determine whether intracellular generation of acetaldehyde from ethanol metabolism can cause DNA damage and activate the FA-BRCA network, we engineered HeLa cells to metabolize alcohol by expression of human alcohol dehydrogenase 1B. Results Incubation of HeLa-ADH1B cells with ethanol (20 mM) resulted in acetaldehyde accumulation in the media which was prevented by co-incubation with 4-methyl pyrazole (4-MP), a specific inhibitor of ADH. Ethanol treatment of HeLa-ADH1B cells produced a 4-fold increase in the acetaldehyde-DNA adduct, N2-ethylidene-dGuo, and also resulted in activation of the Fanconi anemia -breast cancer susceptibility (FA-BRCA) DNA damage response network, as indicated by a monoubiquitination of FANCD2, and phosphorylation of BRCA1. Ser 1524 was identified as one site of BRCA1 phosphorylation. The increased levels of DNA adducts, FANCD2 monoubiquitination, and BRCA1 phosphorylation were all blocked by 4-MP, indicating that acetaldehyde, rather than ethanol itself, was responsible for all three responses. Importantly, the ethanol concentration we used is within the range that can be attained in the human body during social drinking. Conclusions Our results indicate that intracellular metabolism of ethanol to acetaldehyde results in DNA damage which activates the FA-BRCA DNA damage response network. PMID:21919919

  13. [Rapid antibiotic susceptibility test in Clinical Microbiology].

    PubMed

    March Rosselló, Gabriel Alberto; Bratos Pérez, Miguel Ángel

    2016-01-01

    The most widely used antibiotic susceptibility testing methods in Clinical Microbiology are based on the phenotypic detection of antibiotic resistance by measuring bacterial growth in the presence of the antibiotic being tested. These conventional methods take typically 24hours to obtain results. A review is presented here of recently developed techniques for the rapid determination of antibiotic susceptibility. Data obtained with different methods such as molecular techniques, flow cytometry, chemiluminescence, mass spectrometry, commercial methods used in routine work, colorimetric methods, nephelometry, microarrays, microfluids, and methods based on cell disruption and sequencing, are analyzed and discussed in detail.

  14. Different surface charge of colistin-susceptible and -resistant Acinetobacter baumannii cells measured with zeta potential as a function of growth phase and colistin treatment

    PubMed Central

    Soon, Rachel L.; Nation, Roger L.; Cockram, Stewart; Moffatt, Jennifer H.; Harper, Marina; Adler, Ben; Boyce, John D.; Larson, Ian; Li, Jian

    2011-01-01

    Objectives Electrostatic forces mediate the initial interaction between cationic colistin and Gram-negative bacterial cells. Lipopolysaccharide (LPS) loss mediates colistin resistance in some A. baumannii strains. Our aim was to determine the surface charge of colistin-susceptible and –resistant A. baumannii as a function of growth phase and in response to polymyxin treatment. Methods The zeta potential of A. baumannii ATCC 19606 and 10 clinical multidrug-resistant strains (MICs 0.5–2 mg/L) was assessed. Colistin-resistant derivatives (MIC >128 mg/L) of wild-type strains were selected in the presence of 10 mg/L colistin, including the LPS-deficient lpxA mutant, ATCC 19606R. To determine the contribution of LPS to surface charge, two complemented ATCC 19606R derivatives were examined, namely ATCC 19606R + lpxA (containing an intact lpxA gene) and ATCC 19606R + V (containing empty vector). Investigations were conducted as a function of growth phase and polymyxin treatment (1, 4 and 8 mg/L). Results Wild-type cells exhibited a greater negative charge (−60.5 ± 2.36 to −26.2 ± 2.56 mV) thancolistin-resistant cells (−49.2 ± 3.09 to −19.1 ± 2.80 mV) at mid-log phase (ANOVA, P < 0.05). Opposing growth-phase trends were observed for both phenotypes: wild-type cells displayed reduced negative charge and colistin-resistant cells displayed increased negative charge at stationary compared with mid-logarithmic phase. Polymyxin exposure resulted in a concentration-dependent increase in zeta potential. Examination of ATCC 19606R and complemented strains supported the importance of LPS in determining surface charge, suggesting a potential mechanism of colistin resistance. Conclusions Zeta potential differences between A. baumannii phenotypes probably reflect compositional outer-membrane variations that impact the electrostatic component of colistin activity. PMID:21081544

  15. Presence of plasmid-mediated quinolone resistance determinants and mutations in gyrase and topoisomerase in Salmonella enterica isolates with resistance and reduced susceptibility to ciprofloxacin.

    PubMed

    Casas, Monique Ribeiro Tiba; Camargo, Carlos Henrique; Soares, Flávia Barrosa; da Silveira, Wanderley Dias; Fernandes, Sueli Aparecida

    2016-05-01

    In recent decades, the emergence and spread of resistance to nalidixic acid are usually associated with reduced susceptibility to ciprofloxacin among Salmonella serotypes. The aims of this study were to investigate the mechanisms associated with resistance to fluoroquinolone and the clonal relatedness of Salmonella strains isolated from human and nonhuman origins, in a 5-year period in São Paulo, Brazil. Antimicrobial susceptibility testing for Salmonella isolates was performed. PCR and DNA sequencing were accomplished to identify mutations in the quinolone resistance-determining regions of the topoisomerase genes and to determine the fluoroquinolone determinants. The strains presented MIC to ciprofloxacin ranging from 0.125 to 8.0 mg/L (all nonsusceptible). From these, 16 strains (17.5%) were resistant to ciprofloxacin (MIC ≥1 mg/L) and belonging to serotypes Typhimurium, I. 4,5,12:i:-, Enteritidis, and Heidelberg. Amplification and DNA sequencing of topoisomerases genes identified multiple amino acid substitutions in GyrA and ParC. No mutations were identified in GyrB, and 1 amino acid substitution was identified in ParE. Among the 16 Salmonella strains resistant to ciprofloxacin, 8 S. I. 4,5,12:i:- presenting mutations in gyrA and parE genes were grouped into the same pulsotype. Plasmid-mediated quinolone resistance (PMQR) determinants: qnrB, aac(6')-lb-cr, and oqxA/B were detected among 13 strains. To the best of our knowledge, this is the first work to report Salmonella isolates resistant to ciprofloxacin in Brazil. Indeed, this is the first detection of PMQR determinants in Salmonella strains from Sao Paulo State. These findings alert for the potential spread of quinolone resistance of Salmonella strains, particularly in S. I. 4,5,12:i:-, a prevalent serotype implicated in human disease and foodborne outbreaks.

  16. Properties of magnetoelectric susceptibility

    NASA Astrophysics Data System (ADS)

    Zhai, Junyi; Li, Jiefang; Viehland, Dwight; Bichurin, M. I.

    2006-03-01

    The magnetoelectric (ME) susceptibility is the principle property of ME materials, determining the connection between polarization (or electric induction) and an external magnetic field. Since measurement of the ME susceptibility over a wide frequency range [1] and the design of new ME devices require more information about the ME susceptibility, the present work has focused on this property in detail. First, we consider the ME susceptibility as a complex parameter with both real and the imaginary parts, advancing a methodology for measurement of these values. Second, we have analyzed the ME susceptibility, for example a trilayer laminate composite of Terfenol-D/PZT, Terfenol-D, and found a maximum value of ˜3.5x10-7s/m in the electromechanical resonance range. In addition, we have studied the internal structure of the ME susceptibility: i.e., its dependencies on phase volume fractions, layer thickness, and choice of materials couple. Our results for the ME susceptibility will allow it to be more correctly used both as a fundamental materials property and also in potential ME device applications. [1] M.I. Bichurin, V.M. Petrov,Yu.V. Kiliba, and G. Srinivasan. Phys. Rev. B 66, 134404 (2002).

  17. Epistatic interactions between Tgfb1 and genetic loci, Tgfbm2 and Tgfbm3, determine susceptibility to an asthmatic stimulus.

    PubMed

    Freimuth, Julia; Clermont, Frederic F; Huang, Xiaozhu; DeSapio, Angela; Tokuyasu, Taku A; Sheppard, Dean; Akhurst, Rosemary J

    2012-10-30

    TGFβ activation and signaling have been extensively studied in experimental models of allergen-induced asthma as potential therapeutic targets during chronic or acute phases of the disease. Outcomes of experimental manipulation of TGFβ activity have been variable, in part due to use of different model systems. Using an ovalbumin (OVA)-induced mouse model of asthma, we here show that innate variation within TGFβ1 genetic modifier loci, Tgfbm2 and Tgfbm3, alters disease susceptibility. Specifically, Tgfbm2(129) and Tgfbm3(C57) synergize to reverse accentuated airway hyperresponsiveness (AHR) caused by low TGFβ1 levels in Tgfb1(+/-) mice of the NIH/OlaHsd strain. Moreover, epistatic interaction between Tgfbm2(129) and Tgfbm3(C57) uncouples the inflammatory response to ovalbumin from those of airway remodeling and airway hyperresponsiveness, illustrating independent genetic control of these responses. We conclude that differential inheritance of genetic variants of Tgfbm genes alters biological responses to reduced TGFβ1 signaling in an experimental asthma model. TGFβ antagonists for treatment of lung diseases might therefore give diverse outcomes, dependent on genetic variation. PMID:23064636

  18. Is correction necessary when clinically determining quantitative cerebral perfusion parameters from multi-slice dynamic susceptibility contrast MR studies?

    NASA Astrophysics Data System (ADS)

    Salluzzi, M.; Frayne, R.; Smith, M. R.

    2006-01-01

    Several groups have modified the standard singular value decomposition (SVD) algorithm to produce delay-insensitive cerebral blood flow (CBF) estimates from dynamic susceptibility contrast (DSC) perfusion studies. However, new dependences of CBF estimates on bolus arrival times and slice position in multi-slice studies have been recently recognized. These conflicting findings can be reconciled by accounting for several experimental and algorithmic factors. Using simulation and clinical studies, the non-simultaneous measurement of arterial and tissue concentration curves (relative slice position) in a multi-slice study is shown to affect time-related perfusion parameters, e.g. arterial-tissue-delay measurements. However, the current clinical impact of relative slice position on amplitude-related perfusion parameters, e.g. CBF, can be expected to be small unless any of the following conditions are present individually or in combination: (a) high concentration curve signal-to-noise ratios, (b) small tissue mean transit times, (c) narrow arterial input functions or (d) low temporal resolution of the DSC image sequence. Recent improvements in magnetic resonance (MR) technology can easily be expected to lead to scenarios where these effects become increasingly important sources of inaccuracy for all perfusion parameter estimates. We show that using Fourier interpolated (high temporal resolution) residue functions reduces the systematic error of the perfusion parameters obtained from multi-slice studies. Preliminary results associated with this paper were presented at ISMRM 12th Scientific Meeting and Exhibition, Kyoto, Japan, 2004.

  19. Genetic susceptibility to anthracycline-related congestive heart failure in survivors of haematopoietic cell transplantation.

    PubMed

    Armenian, Saro H; Ding, Yan; Mills, George; Sun, Canlan; Venkataraman, Kalyanasundaram; Wong, Florence Lennie; Neuhausen, Susan L; Senitzer, David; Wang, Shirong; Forman, Stephen J; Bhatia, Smita

    2013-10-01

    Haematopoietic cell transplantation (HCT) survivors are at increased risk for developing congestive heart failure (CHF), primarily due to pre-HCT exposure to anthracyclines. We examined the association between the development of CHF after HCT and polymorphisms in 16 candidate genes involved in anthracycline metabolism, iron homeostasis, anti-oxidant defence, and myocardial remodelling. A nested case-control study design was used. Cases (post-HCT CHF) were identified from 2950 patients who underwent HCT between 1988 and 2007 at City of Hope and had survived ≥1 year. This cohort formed the sampling frame for selecting controls (without CHF) matched on: age, race/ethnicity, cumulative anthracycline exposure, stem cell source (allogeneic, autologous), and length of follow-up. Seventy-seven cases with pre-HCT germline DNA and 178 controls were genotyped. Multivariate analysis revealed that the odds of CHF was higher in females [Odds Ratio (OR) = 2·9, P < 0·01], individuals with pre-HCT chest radiation (OR = 4·7, P = 0·05), hypertension (OR = 2·9, P = 0·01), and with variants of genes coding for the NAD(P)H-oxidase subunit RAC2 (rs13058338, 7508T→A; OR = 2·8, P < 0·01), HFE (rs1799945, 63C→G; OR = 2·5, P = 0·05) or the doxorubicin efflux transporter ABCC2 (rs8187710, 1515G→A; OR = 4·3, P < 0·01). A combined (clinical and genetic) CHF predictive model performed better [area under the curve (AUC), 0·79] than the genetic (AUC = 0·67) or the clinical (AUC = 0·69) models alone.

  20. Oocyte-specific differences in cell-cycle control create an innate susceptibility to meiotic errors.

    PubMed

    Nagaoka, So Iha; Hodges, Craig A; Albertini, David F; Hunt, Patricia Ann

    2011-04-26

    Segregation of homologs at the first meiotic division (MI) is facilitated by crossovers and by a physical constraint imposed on sister kinetochores that facilitates monopolar attachment to the MI spindle. Recombination failure or premature separation of homologs results in univalent chromosomes at MI, and univalents constrained to form monopolar attachments should be inherently unstable and trigger the spindle assembly checkpoint (SAC). Although univalents trigger cell-cycle arrest in the male, this is not the case in mammalian oocytes. Because the spindle assembly portion of the SAC appears to function normally, two hypotheses have been proposed to explain the lack of response to univalents: (1) reduced stringency of the oocyte SAC to aberrant chromosome behavior, and (2) the ability of univalents to satisfy the SAC by forming bipolar attachments. The present study of Mlh1 mutant mice demonstrates that metaphase alignment is not a prerequisite for anaphase onset and provides strong evidence that MI spindle stabilization and anaphase onset require stable bipolar attachment of a critical mass--but not all--of chromosomes. We postulate that subtle differences in SAC-mediated control make the human oocyte inherently error prone and contribute to the age-related increase in aneuploidy.

  1. Increased susceptibility to ethylmercury-induced mitochondrial dysfunction in a subset of autism lymphoblastoid cell lines.

    PubMed

    Rose, Shannon; Wynne, Rebecca; Frye, Richard E; Melnyk, Stepan; James, S Jill

    2015-01-01

    The association of autism spectrum disorders with oxidative stress, redox imbalance, and mitochondrial dysfunction has become increasingly recognized. In this study, extracellular flux analysis was used to compare mitochondrial respiration in lymphoblastoid cell lines (LCLs) from individuals with autism and unaffected controls exposed to ethylmercury, an environmental toxin known to deplete glutathione and induce oxidative stress and mitochondrial dysfunction. We also tested whether pretreating the autism LCLs with N-acetyl cysteine (NAC) to increase glutathione concentrations conferred protection from ethylmercury. Examination of 16 autism/control LCL pairs revealed that a subgroup (31%) of autism LCLs exhibited a greater reduction in ATP-linked respiration, maximal respiratory capacity, and reserve capacity when exposed to ethylmercury, compared to control LCLs. These respiratory parameters were significantly elevated at baseline in the ethylmercury-sensitive autism subgroup as compared to control LCLs. NAC pretreatment of the sensitive subgroup reduced (normalized) baseline respiratory parameters and blunted the exaggerated ethylmercury-induced reserve capacity depletion. These findings suggest that the epidemiological link between environmental mercury exposure and an increased risk of developing autism may be mediated through mitochondrial dysfunction and support the notion that a subset of individuals with autism may be vulnerable to environmental influences with detrimental effects on development through mitochondrial dysfunction.

  2. Increased Susceptibility to Ethylmercury-Induced Mitochondrial Dysfunction in a Subset of Autism Lymphoblastoid Cell Lines

    PubMed Central

    Wynne, Rebecca; Frye, Richard E.; Melnyk, Stepan; James, S. Jill

    2015-01-01

    The association of autism spectrum disorders with oxidative stress, redox imbalance, and mitochondrial dysfunction has become increasingly recognized. In this study, extracellular flux analysis was used to compare mitochondrial respiration in lymphoblastoid cell lines (LCLs) from individuals with autism and unaffected controls exposed to ethylmercury, an environmental toxin known to deplete glutathione and induce oxidative stress and mitochondrial dysfunction. We also tested whether pretreating the autism LCLs with N-acetyl cysteine (NAC) to increase glutathione concentrations conferred protection from ethylmercury. Examination of 16 autism/control LCL pairs revealed that a subgroup (31%) of autism LCLs exhibited a greater reduction in ATP-linked respiration, maximal respiratory capacity, and reserve capacity when exposed to ethylmercury, compared to control LCLs. These respiratory parameters were significantly elevated at baseline in the ethylmercury-sensitive autism subgroup as compared to control LCLs. NAC pretreatment of the sensitive subgroup reduced (normalized) baseline respiratory parameters and blunted the exaggerated ethylmercury-induced reserve capacity depletion. These findings suggest that the epidemiological link between environmental mercury exposure and an increased risk of developing autism may be mediated through mitochondrial dysfunction and support the notion that a subset of individuals with autism may be vulnerable to environmental influences with detrimental effects on development through mitochondrial dysfunction. PMID:25688267

  3. The lupus susceptibility locus Sle1 facilitates the peripheral development and selection of anti-DNA B cells through impaired receptor editing.

    PubMed

    Chang, Soog-Hee; Kim, Tae-Joo; Kim, Young-Joo; Liu, Yang; Min, So-Youn; Park, Min-Jung; Park, Hyun-Sil; Lee, Sun-Kyung; Nam, Ki-Hoan; Kim, Ho-Youn; Mohan, Chandra; Kim, Hang-Rae

    2014-06-15

    Systemic lupus erythematosus is characterized by the spontaneous production of IgG autoantibodies in patients and lupus-prone mice. In this study, we investigated the effect of the Sle1 lupus susceptibility locus on the peripheral development of 56R(+) anti-DNA transgenic B cells by tracking 56R(+) B cells in mice without (B6.56R) or with (B6.Sle1.56R) the Sle1 locus. Compared with B6.56R mice, B6.Sle1.56R mice exhibited increased class-switched IgG2a anti-DNA Abs in their serum, encoded by the transgene. Interestingly, within the spleen, Sle1 facilitated the development of these cells into clusters of IgG2a class-switched B cells juxtaposed to CD4(+) T cells within extrafollicular sites. Through sequence analysis of B cell hybridomas, we also found that B cells from B6.Sle1.56R mice are inefficient at Ig H and L chain editing. Thus, the Ig H chains in Sle1.56R(+) B cells are partnered more often with cationic L chains that facilitate DNA binding. Taken together, these findings indicate that the Sle1 lupus-susceptibility locus may facilitate the emergence of anti-DNA B cells by subduing BCR revision and possibly by shaping the extrafollicular development of effector B cells, although the precise molecular mechanisms await further study.

  4. Optical determination of intracellular water in apoptotic cells.

    PubMed

    Model, Michael A; Schonbrun, Ethan

    2013-12-01

    Intracellular water plays a critical role in apoptotic and necrotic cell death. We describe a method for quantifying cell water by application of two previously described variants of transmission microscopy. By taking two axially displaced brightfield images, the phase shift of the transmitted wave was computed using the transport-of-intensity equation. At the same time, cell thickness was determined by transmission through an externally applied dye ('transmission-through-dye' microscopy); switching between these two imaging modalities was accomplished by simply changing the illumination wavelength. The sets of data thus obtained allow computation of the refractive index and cell water content within individual cells. The method was illustrated using cells treated with apoptotic agents staurosporine and actinomycin D and with necrosis inducer ionomycin. Water imaging allows discrimination between apoptotic volume decrease due to dehydration from that due to detachment of apoptotic bodies and can be used on samples where cell volume determination alone would be difficult or insufficient.

  5. Optical system for determining physical characteristics of a solar cell

    DOEpatents

    Sopori, Bhushan L.

    2001-01-01

    The invention provides an improved optical system for determining the physical characteristics of a solar cell. The system comprises a lamp means for projecting light in a wide solid-angle onto the surface of the cell; a chamber for receiving the light through an entrance port, the chamber having an interior light absorbing spherical surface, an exit port for receiving a beam of light reflected substantially normal to the cell, a cell support, and an lower aperture for releasing light into a light absorbing baffle; a means for dispersing the reflection into monochromatic components; a means for detecting an intensity of the components; and a means for reporting the determination.

  6. Molecular and bioenergetic differences between cells with African versus European inherited mitochondrial DNA haplogroups: implications for population susceptibility to diseases.

    PubMed

    Kenney, M Cristina; Chwa, Marilyn; Atilano, Shari R; Falatoonzadeh, Payam; Ramirez, Claudio; Malik, Deepika; Tarek, Mohamed; Del Carpio, Javier Cáceres; Nesburn, Anthony B; Boyer, David S; Kuppermann, Baruch D; Vawter, Marquis P; Jazwinski, S Michal; Miceli, Michael V; Wallace, Douglas C; Udar, Nitin

    2014-02-01

    The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP (adenosine triphosphate) turnover rates and lower levels of reactive oxygen species production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 (chemokine, CC motif, receptor 3) signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases. PMID:24200652

  7. Association of genetic variants of cancer stem cell gene CD44 haplotypes with gallbladder cancer susceptibility in North Indian population.

    PubMed

    Sharma, Kiran Lata; Yadav, Anu; Gupta, Annapurna; Tulsayan, Sonam; Kumar, Vijay; Misra, Sanjeev; Kumar, Ashok; Mittal, Balraj

    2014-03-01

    CD44 is an important marker for cancer stem cells. Germline variants in CD44 gene have been associated with susceptibility to breast and nasopharyngeal carcinomas but no study in gallbladder cancer (GBC) has been done yet. The present study included 405 GBC patients and 200 healthy controls from North India. Tagger SNPs for CD44 were selected from the GIH population data. Genotyping was carried out by PCR-RFLP and Taqman probes. Statistical analysis was done by SPSS. Bonferroni correction was applied in subgroup analysis. Logistic regression analysis showed no individual association of CD44 polymorphisms with GBC risk. However, [CCAT] haplotype was associated with overall reduced risk of GBC [P = 0.04, odds ratios (OR) = 0.47]. Gender stratification revealed that [CCAT] and [TAGT] haplotypes were significantly associated with decreased risk in female GBC patients [P = 0.022, OR = 0.38; P = 0.011, OR = 0.17, respectively]. The CAAT haplotype was marginally associated with low GBC risk in patients with co-existing gallstones [P = 0.026, OR = 0.53]. The cancer risk was not further modified with tobacco usage or age of onset. In silico analysis showed change in transcriptional regulation of selected SNPs. This study reports an important role of CD44 haplotypes with reduced risk of GBC.

  8. Lack of IL-1 Receptor-Associated Kinase-4 Leads to Defective Th1 Cell Responses and Renders Mice Susceptible to Mycobacterial Infection.

    PubMed

    Marinho, Fábio V; Fahel, Júlia S; Scanga, Charles A; Gomes, Marco Tulio R; Guimarães, Gabriela; Carvalho, Gabrielle R M; Morales, Stefanny V; Báfica, André; Oliveira, Sergio Costa

    2016-09-01

    The Toll-like and IL-1 family receptors play critical roles in innate and adaptive immunity against intracellular pathogens. Although previous data demonstrated the importance of TLRs and IL-1R signaling events for the establishment of an effective immune response to mycobacteria, the possible function of the adaptor molecule IL-1R-associated kinase (IRAK)-4 against this pathogen has not been addressed. In this study, we determined the role of IRAK-4 in signaling pathways responsible for controlling mycobacterial infections. This kinase is important for the production of IL-12 and TNF-α by macrophages and dendritic cells exposed to mycobacteria. Moreover, Mycobacterium bovis-infected IRAK-4-knockout macrophages displayed impaired MAPK and NF-κB activation. IL-1β secretion and caspase-1 activation were also dependent on IRAK-4 signaling. Mice lacking IRAK-4 showed increased M. bovis burden in spleen, liver, and lungs and smaller liver granulomas during 60 d of infection compared with wild-type mice. Furthermore, 80% of IRAK-4(-/-) mice succumbed to virulent M. tuberculosis within 100 d following low-dose infection. This increased susceptibility to mycobacteria correlated with reduced IFN-γ/TNF-α recall responses by splenocytes, as well as fewer IL-12p70-producing APCs. Additionally, we observed that IRAK-4 is also important for the production of IFN-γ by CD4(+) T cells from infected mice. Finally, THP-1 cells treated with an IRAK-4 inhibitor and exposed to M. bovis showed reduced TNF-α and IL-12, suggesting that the results found in mice can be extended to humans. In summary, these data demonstrate that IRAK-4 is essential for innate and adaptive immunity and necessary for efficient control of mycobacterial infections.

  9. p53-regulated autophagy is controlled by glycolysis and determines cell fate

    PubMed Central

    Duan, Lei; Perez, Ricardo E.; Davaadelger, Batzaya; Dedkova, Elena N.; Blatter, Lothar A.; Maki, Carl G.

    2015-01-01

    The tumor suppressor p53 regulates downstream targets that determine cell fate. Canonical p53 functions include inducing apoptosis, growth arrest, and senescence. Non-canonical p53 functions include its ability to promote or inhibit autophagy and its ability to regulate metabolism. The extent to which autophagy and/or metabolic regulation determines cell fate by p53 is unclear. To address this, we compared cells resistant or sensitive to apoptosis by the p53 activator Nutlin-3a. In resistant cells, glycolysis was maintained upon Nutlin-3a treatment, and activated p53 promoted prosurvival autophagy. In contrast, in apoptosis sensitive cells activated p53 increased superoxide levels and inhibited glycolysis through repression of glycolytic pathway genes. Glycolysis inhibition and increased superoxide inhibited autophagy by repressing ATG genes essential for autophagic vesicle maturation. Inhibiting glycolysis increased superoxide and blocked autophagy in apoptosis-resistant cells, causing p62-dependent caspase-8 activation. Finally, treatment with 2-DG or the autophagy inhibitors chloroquine or bafilomycin A1 sensitized resistant cells to Nutlin-3a-induced apoptosis. Together, these findings reveal novel links between glycolysis and autophagy that determine apoptosis-sensitivity in response to p53. Specifically, the findings indicate 1) that glycolysis plays an essential role in autophagy by limiting superoxide levels and maintaining expression of ATG genes required for autophagic vesicle maturation, 2) that p53 can promote or inhibit autophagy depending on the status of glycolysis, and 3) that inhibiting protective autophagy can expand the breadth of cells susceptible to Nutlin-3a induced apoptosis. PMID:26337205

  10. Determining surface magnetic susceptibility of loess-paleosol sections based on spectral features: Application to a UHD 185 hyperspectral image

    NASA Astrophysics Data System (ADS)

    Cui, Jing; Zhang, Shimin; Zhang, Jingfa; Liu, Xudong; Ding, Rui; Liu, Hanyong

    2016-08-01

    Magnetic susceptibility (MS) records of loess-paleosol sequences have been considered a measure of the degree of pedogenic activity and are considered to be excellent proxies for terrestrial climatic fluctuations. However, the MS of single (vertical) path variations occasionally represents site-specific influences rather than monsoonal changes (depending on the position of the path). Few studies have used remote sensing techniques to map the surface MS information of loess-paleosol sections. Hyperspectral techniques provide an efficient, economical and quantitative alternative. In this study, stepwise regression was used to build MS estimation models based on spectral features. Six MS models based on spectral features were established. Test datasets indicated that our models are very successful, all resulting in R2 > 0.92 and RMSEs ranging from 4.5736 to 6.80475. The slope change between 810 nm and 880 nm (b880/b810) observed in all models played an important role in MS estimation. Models 5 and 6 have higher RMSEs and relatively lower SAM values, although the R2 values are both above 0.95. The RMSEs of the first four models are similar. Therefore, the first four models were thought to be more stable and useful. UHD 185, a new generation of commercial hyperspectral imaging sensor, was used for surface MS mapping of a loess-paleosol section by model 1 and model 2. The MS map corresponded well to the loess sequences. The MS values obtained from the UHD 185 data are convincing and consistent with the measured data (R2 > 0.85). The trend in changing MS values is clear, suggesting that model 1 and model 2 could produce reasonable loess-paleosol section surface maps from the UHD 185 image, although there is a linear offset between the estimated MS and the measured MS. The methodology proposed here can be used to map MS on a much larger scale. Because of the limit of the spectral range, the performances of model 3 and model 4 with the image were not discussed. However

  11. Determinants of Default from Tuberculosis Treatment among Patients with Drug-Susceptible Tuberculosis in Karachi, Pakistan: A Mixed Methods Study

    PubMed Central

    Chida, Natasha; Ansari, Zara; Hussain, Hamidah; Jaswal, Maria; Symes, Stephen; Khan, Aamir J.; Mohammed, Shama

    2015-01-01

    Purpose Non-adherence to tuberculosis therapy can lead to drug resistance, prolonged infectiousness, and death; therefore, understanding what causes treatment default is important. Pakistan has one of the highest burdens of tuberculosis in the world, yet there have been no qualitative studies in Pakistan that have specifically examined why default occurs. We conducted a mixed methods study at a tuberculosis clinic in Karachi to understand why patients with drug-susceptible tuberculosis default from treatment, and to identify factors associated with default. Patients attending this clinic pick up medications weekly and undergo family-supported directly observed therapy. Methods In-depth interviews were administered to 21 patients who had defaulted. We also compared patients who defaulted with those who were cured, had completed, or had failed treatment in 2013. Results Qualitative analyses showed the most common reasons for default were the financial burden of treatment, and medication side effects and beliefs. The influence of finances on other causes of default was also prominent, as was concern about the effect of treatment on family members. In quantitative analysis, of 2120 patients, 301 (14.2%) defaulted. Univariate analysis found that male gender (OR: 1.34, 95% CI: 1.04–1.71), being 35–59 years of age (OR: 1.54, 95% CI: 1.14–2.08), or being 60 years of age or older (OR: 1.84, 95% CI: 1.17–2.88) were associated with default. After adjusting for gender, disease site, and patient category, being 35–59 years of age (aOR: 1.49, 95% CI: 1.10–2.03) or 60 years of age or older (aOR: 1.76, 95% CI: 1.12–2.77) were associated with default. Conclusions In multivariate analysis age was the only variable associated with default. This lack of identifiable risk factors and our qualitative findings imply that default is complex and often due to extrinsic and medication-related factors. More tolerable medications, improved side effect management, and innovative

  12. Genetic Variants of Retinoic Acid Receptor-Related Orphan Receptor Alpha Determine Susceptibility to Type 2 Diabetes Mellitus in Han Chinese

    PubMed Central

    Zhang, Yuwei; Liu, Yulan; Liu, Yin; Zhang, Yanjie; Su, Zhiguang

    2016-01-01

    Retinoic acid receptor-related orphan receptor alpha (RORA) plays a key role in the regulation of lipid and glucose metabolism and insulin expression that are implicated in the development of type 2 diabetes mellitus (T2DM). However, the effects of genetic variants in the RORA gene on the susceptibility to T2DM remain unknown. Nine tagging single-nucleotide polymorphisms (SNPs) were screened by using the SNaPshot method in 427 patients with T2DM and 408 normal controls. Association between genotypes and haplotypes derived from these SNPs with T2DM was analyzed using different genetic models. Allele and genotype frequencies at rs10851685 were significantly different between T2DM patients and control subjects (allele: p = 0.009, Odds ratios (OR) = 1.36 [95% Confidence intervals (CI) = 1.08–1.72]; genotype: p = 0.029). The minor allele T, at rs10851685, was potentially associated with an increased risk of T2DM in the dominant model, displaying OR of 1.38 (95% CI: 1.04–1.82, p = 0.025) in subjects with genotypes TA+TT vs. AA. In haplotype analysis, we observed that haplotypes GGTGTAACT, GGTGTAACC, and GATATAACT were significantly associated with increased risk of T2DM, while haplotypes GATGAAGTT, AGTGAAGTT, and AATGAAATT were protective against T2DM. These data suggest that the genetic variation in RORA might determine a Chinese Han individual’s susceptibility to T2DM. PMID:27556492

  13. Genetic Variants of Retinoic Acid Receptor-Related Orphan Receptor Alpha Determine Susceptibility to Type 2 Diabetes Mellitus in Han Chinese.

    PubMed

    Zhang, Yuwei; Liu, Yulan; Liu, Yin; Zhang, Yanjie; Su, Zhiguang

    2016-01-01

    Retinoic acid receptor-related orphan receptor alpha (RORA) plays a key role in the regulation of lipid and glucose metabolism and insulin expression that are implicated in the development of type 2 diabetes mellitus (T2DM). However, the effects of genetic variants in the RORA gene on the susceptibility to T2DM remain unknown. Nine tagging single-nucleotide polymorphisms (SNPs) were screened by using the SNaPshot method in 427 patients with T2DM and 408 normal controls. Association between genotypes and haplotypes derived from these SNPs with T2DM was analyzed using different genetic models. Allele and genotype frequencies at rs10851685 were significantly different between T2DM patients and control subjects (allele: p = 0.009, Odds ratios (OR) = 1.36 [95% Confidence intervals (CI) = 1.08-1.72]; genotype: p = 0.029). The minor allele T, at rs10851685, was potentially associated with an increased risk of T2DM in the dominant model, displaying OR of 1.38 (95% CI: 1.04-1.82, p = 0.025) in subjects with genotypes TA+TT vs. AA. In haplotype analysis, we observed that haplotypes GGTGTAACT, GGTGTAACC, and GATATAACT were significantly associated with increased risk of T2DM, while haplotypes GATGAAGTT, AGTGAAGTT, and AATGAAATT were protective against T2DM. These data suggest that the genetic variation in RORA might determine a Chinese Han individual's susceptibility to T2DM. PMID:27556492

  14. Identity-by-descent approaches identify regions of importance for genetic susceptibility to hereditary esophageal squamous cell carcinoma.

    PubMed

    Ko, Josephine My; Zhang, Peng; Law, Simon; Fan, Yanhui; Song, You-Qiang; Zhao, Xue Ke; Wong, Elibe H W; Tang, Sa; Song, Xin; Lung, Maria Li; Wang, Li Dong

    2014-08-01

    Worldwide, the highest prevalence of esophageal cancer (EC) occurs in Northern China. High-density SNP arrays allow identification of identity-by-descent (IBD) segments in genomic DNAs representative of shared common ancestral regions. We utilized IBD approaches to map susceptibility loci associated with low-penetrance SNPs in high-risk Henan hereditary esophageal squamous cell carcinoma (ESCC) patients. Affymetrix GeneChip Human mapping SNP array IBD analysis was performed in 32 Henan family history-positive (FH+) ESCC patients, 18 Henan healthy unrelated individuals, and 45 Chinese individuals from a CHB HapMap dataset using PLink (scoring IBD segments individually) and Beagle (scoring of shared IBD segments among case/case vs. control/control pairs) software. Both analyses identified longer IBD segment lengths associated with FH+ ESCC compared to controls. However, there was no strong evidence for a genetic founder effect. Pairing IBD analysis with BEAGLE identified 8 critical IBD segments residing at 2q32.1-q32.2, 3p22.3-p22.2, 4q21.1-q21.21, 7p22.2, 8q23.2-q23.3, 10q23.33-q24.1, 14q24.3 and 16q11.2-q12.1, which were more significantly shared among case/case compared to control/control. The shared IBD segments in FH+ ESCC samples with no overlap with control/CHB Hapmap may encompass potential cancer susceptibility loci. Selected targeted genes, PLCE1, GPT2, SIAH1 and CYP2C-18, residing within the IBD segments at 10q23.33-q24.1 and 16q11.2-q12.1, had statistically significant differential expression in primary ESCC tissues and are likely involved in ESCC carcinogenesis. The importance of these IBD segments to the etiology and development of ESCC in high-risk areas requires further study with expanded sample sizes. This is the first report employing the pairing IBD approach for elucidation of the genetic basis of hereditary ESCC in Henan by applying high throughput SNP array analysis.

  15. Degradation of endogenous bacterial cell wall polymers by the muralytic enzyme mutanolysin prevents hepatobiliary injury in genetically susceptible rats with experimental intestinal bacterial overgrowth.

    PubMed Central

    Lichtman, S N; Okoruwa, E E; Keku, J; Schwab, J H; Sartor, R B

    1992-01-01

    Jejunal self-filling blind loops with subsequent small bowel bacterial overgrowth (SBBO) induce hepatobiliary injury in genetically susceptible Lewis rats. Lesions consist of portal tract inflammation, bile duct proliferation, and destruction. To determine the pathogenesis of SBBO-induced hepatobiliary injury, we treated Lewis rats with SBBO by using several agents with different mechanisms of activity. Buffer treatment, ursodeoxycholic acid, prednisone, methotrexate, and cyclosporin A failed to prevent SBBO-induced injury as demonstrated by increased plasma aspartate aminotransferase (AST) and elevated histology scores. However, hepatic injury was prevented by mutanolysin, a muralytic enzyme whose only known activity is to split the beta 1-4 N-acetylmuramyl-N-acetylglucosamine linkage of peptidoglycan-polysaccharide (PG-PS), a bacterial cell wall polymer with potent inflammatory and immunoregulatory properties. Mutanolysin therapy started on the day blind loops were surgically created and continued for 8 wk significantly diminished AST (101 +/- 37 U/liter) and liver histology scores (2.2 +/- 2.7) compared to buffer-treated rats (228 +/- 146 U/liter, P < 0.05, 8.2 +/- 1.9, P < 0.001 respectively). Mutanolysin treatment started during the early phase of hepatic injury, 16-21 d after surgery, decreased AST in 7 of 11 rats from 142 +/- 80 to 103 +/- 24 U/liter contrasted to increased AST in 9 of 11 buffer-treated rats from 108 +/- 52 to 247 +/- 142 U/liter, P < 0.05. Mutanolysin did not change total bacterial numbers within the loop, eliminate Bacteroides sp., have in vitro antibiotic effects, or diminish mucosal PG-PS transport. However, mutanolysin treatment prevented elevation of plasma anti-PG antibodies and tumor necrosis factor-alpha (TNF alpha) levels which occurred in buffer treated rats with SBBO and decreased TNF alpha production in isolated Kupffer cells stimulated in vitro with PG-PS. Based on the preventive and therapeutic activity of this highly specific

  16. Factors Determining Staphylococcus aureus Susceptibility to Photoantimicrobial Chemotherapy: RsbU Activity, Staphyloxanthin Level, and Membrane Fluidity.

    PubMed

    Kossakowska-Zwierucho, Monika; Kaźmierkiewicz, Rajmund; Bielawski, Krzysztof P; Nakonieczna, Joanna

    2016-01-01

    Photoantimicrobial chemotherapy (PACT) constitutes a particular type of stress condition, in which bacterial cells induce a pleiotropic and as yet unexplored effect. In light of this, the key master regulators are of putative significance to the overall phototoxic outcome. In Staphylococcus aureus, the alternative sigma factor σ(B) controls the expression of genes involved in the response to environmental stress. We show that aberration of any sigB operon genes in S. aureus USA300 isogenic mutants causes a pronounced sensitization (>5 log10 reduction in CFU drop) to PACT with selected photosensitizers, namely protoporphyrin diarginate, zinc phthalocyanine and rose bengal. This effect is partly due to aberration-coupled staphyloxanthin synthesis inhibition. We identified frequent mutations in RsbU, a σ(B) activator, in PACT-vulnerable clinical isolates of S. aureus, resulting in σ(B) activity impairment. Locations of significant changes in protein structure (IS256 insertion, early STOP codon occurrence, substitutions A230T and A276D) were shown in a theoretical model of S. aureus RsbU. As a phenotypic hallmark of PACT-vulnerable S. aureus strains, we observed an increased fluidity of bacterial cell membrane, which is a result of staphyloxanthin content and other yet unidentified factors. Our research indicates σ(B) as a promising target of adjunctive antimicrobial therapy and suggests that enhanced cell membrane fluidity may be an adjuvant strategy in PACT. PMID:27486456

  17. Factors Determining Staphylococcus aureus Susceptibility to Photoantimicrobial Chemotherapy: RsbU Activity, Staphyloxanthin Level, and Membrane Fluidity

    PubMed Central

    Kossakowska-Zwierucho, Monika; Kaźmierkiewicz, Rajmund; Bielawski, Krzysztof P.; Nakonieczna, Joanna

    2016-01-01

    Photoantimicrobial chemotherapy (PACT) constitutes a particular type of stress condition, in which bacterial cells induce a pleiotropic and as yet unexplored effect. In light of this, the key master regulators are of putative significance to the overall phototoxic outcome. In Staphylococcus aureus, the alternative sigma factor σB controls the expression of genes involved in the response to environmental stress. We show that aberration of any sigB operon genes in S. aureus USA300 isogenic mutants causes a pronounced sensitization (>5 log10 reduction in CFU drop) to PACT with selected photosensitizers, namely protoporphyrin diarginate, zinc phthalocyanine and rose bengal. This effect is partly due to aberration-coupled staphyloxanthin synthesis inhibition. We identified frequent mutations in RsbU, a σB activator, in PACT-vulnerable clinical isolates of S. aureus, resulting in σB activity impairment. Locations of significant changes in protein structure (IS256 insertion, early STOP codon occurrence, substitutions A230T and A276D) were shown in a theoretical model of S. aureus RsbU. As a phenotypic hallmark of PACT-vulnerable S. aureus strains, we observed an increased fluidity of bacterial cell membrane, which is a result of staphyloxanthin content and other yet unidentified factors. Our research indicates σB as a promising target of adjunctive antimicrobial therapy and suggests that enhanced cell membrane fluidity may be an adjuvant strategy in PACT. PMID:27486456

  18. Human intrahepatic regulatory T cells are functional, require IL‐2 from effector cells for survival, and are susceptible to Fas ligand‐mediated apoptosis

    PubMed Central

    Chen, Yung‐Yi; Jeffery, Hannah C.; Hunter, Stuart; Bhogal, Ricky; Birtwistle, Jane; Braitch, Manjit Kaur; Roberts, Sheree; Ming, Mikaela; Hannah, Jack; Thomas, Clare; Adali, Gupse; Hübscher, Stefan G.; Syn, Wing‐Kin; Afford, Simon; Lalor, Patricia F.; Adams, David H.

    2016-01-01

    Regulatory T cells (Treg) suppress T effector cell proliferation and maintain immune homeostasis. Autoimmune liver diseases persist despite high frequencies of Treg in the liver, suggesting that the local hepatic microenvironment might affect Treg stability, survival, and function. We hypothesized that interactions between Treg and endothelial cells during recruitment and then with epithelial cells within the liver affect Treg stability, survival, and function. To model this, we explored the function of Treg after migration through human hepatic sinusoidal‐endothelium (postendothelial migrated Treg [PEM Treg]) and the effect of subsequent interactions with cholangiocytes and local proinflammatory cytokines on survival and stability of Treg. Our findings suggest that the intrahepatic microenvironment is highly enriched with proinflammatory cytokines but deficient in the Treg survival cytokine interleukin (IL)‐2. Migration through endothelium into a model mimicking the inflamed liver microenvironment did not affect Treg stability; however, functional capacity was reduced. Furthermore, the addition of exogenous IL‐2 enhanced PEM Treg phosphorylated STAT5 signaling compared with PEMCD8. CD4 and CD8 T cells are the main source of IL‐2 in the inflamed liver. Liver‐infiltrating Treg reside close to bile ducts and coculture with cholangiocytes or their supernatants induced preferential apoptosis of Treg compared with CD8 effector cells. Treg from diseased livers expressed high levels of CD95, and their apoptosis was inhibited by IL‐2 or blockade of CD95. Conclusion: Recruitment through endothelium does not impair Treg stability, but a proinflammatory microenvironment deficient in IL‐2 leads to impaired function and increased susceptibility of Treg to epithelial cell‐induced Fas‐mediated apoptosis. These results provide a mechanism to explain Treg dysfunction in inflamed tissues and suggest that IL‐2 supplementation, particularly if used in conjunction

  19. Lack of the programmed death-1 receptor renders host susceptible to enteric microbial infection through impairing the production of the mucosal natural killer cell effector molecules.

    PubMed

    Solaymani-Mohammadi, Shahram; Lakhdari, Omar; Minev, Ivelina; Shenouda, Steve; Frey, Blake F; Billeskov, Rolf; Singer, Steven M; Berzofsky, Jay A; Eckmann, Lars; Kagnoff, Martin F

    2016-03-01

    The programmed death-1 receptor is expressed on a wide range of immune effector cells, including T cells, natural killer T cells, dendritic cells, macrophages, and natural killer cells. In malignancies and chronic viral infections, increased expression of programmed death-1 by T cells is generally associated with a poor prognosis. However, its role in early host microbial defense at the intestinal mucosa is not well understood. We report that programmed death-1 expression is increased on conventional natural killer cells but not on CD4(+), CD8(+) or natural killer T cells, or CD11b(+) or CD11c(+) macrophages or dendritic cells after infection with the mouse pathogen Citrobacter rodentium. Mice genetically deficient in programmed death-1 or treated with anti-programmed death-1 antibody were more susceptible to acute enteric and systemic infection with Citrobacter rodentium. Wild-type but not programmed death-1-deficient mice infected with Citrobacter rodentium showed significantly increased expression of the conventional mucosal NK cell effector molecules granzyme B and perforin. In contrast, natural killer cells from programmed death-1-deficient mice had impaired expression of those mediators. Consistent with programmed death-1 being important for intracellular expression of natural killer cell effector molecules, mice depleted of natural killer cells and perforin-deficient mice manifested increased susceptibility to acute enteric infection with Citrobacter rodentium. Our findings suggest that increased programmed death-1 signaling pathway expression by conventional natural killer cells promotes host protection at the intestinal mucosa during acute infection with a bacterial gut pathogen by enhancing the expression and production of important effectors of natural killer cell function.

  20. Cell polarity determinants establish asymmetry in MEN signaling

    PubMed Central

    Monje-Casas, Fernando; Amon, Angelika

    2009-01-01

    Summary Components of the Mitotic Exit Network (MEN), a signaling pathway that triggers exit from mitosis, localize to the spindle pole body (SPB) that migrates into the daughter cell during anaphase but are largely absent from the SPB that remains in the mother cell. Through the analysis of one of the determinants of this asymmetry, Bfa1, we find that the machinery responsible for establishing cell polarity and cytoplasmic microtubules collaborate to establish MEN asymmetry. In cells defective in the Cdc42 signaling pathway or the formin Bni1, Bfa1 localizes to both SPBs. The quantitative analysis of Bfa1 localization further shows that Bfa1 can associate with both SPBs in a transient and highly dynamic fashion, but the protein is stabilized on the SPB that migrates into the daughter cell during anaphase through microtubule – bud cortex interactions. Our results indicate that mother – daughter cell asymmetry determinants establish MEN signaling asymmetry through microtubule – bud cortex interactions. PMID:19154724

  1. Biologic effects of microwave exposure. II. Studies on the mechanisms controlling susceptibility to microwave-induced increases in complement receptor-positive spleen cells

    SciTech Connect

    Schlagel, C.J.; Sulek, K.; Ho, H.S.; Leach, W.M.; Ahmed, A.; Woody, J.N.

    1980-01-01

    In attempting to evaluate the mechanisms responsible for susceptibility to the inductive increase in splenic complement receptor-positive (CR+) cells following exposure to 2450-MHz microwaves, it was found that sensitivity to microwave-induced CR+ cell increases was under genetic control. In particular, evidence was accumulated suggesting that regulation was under the control of a gene or genes closely associated with but outside of the mouse major histocompatibility complex (H-2). All responsive strains of mice tested were of the H-2k haplotype, while mice of the H-2a, H-2b, H-2d and H-1i5 haplotypes were refractory to the microwave-induced increases in CR+ cells. By utilizing certain H-2k strains of mice that were genetically unable to respond to endotoxin, we were able to show that these strains of mice responded to microwaves, but not to endotoxin, by increasing CR+ cells. Microwave-induced increases in CR+ cells were not mimicked by the intraperitoneal injection of hydrocortisone. Athymic mice responded to microwave exposure, indicating that this event was not regulated by the T-cell population. Mice less than eight weeks old were found not to be susceptible to exposure to 2450-MHz microwaves. These studies indicate that microwaves do induce changes in the population of cells with specific cell-surface receptors, that susceptibility to these changes is under genetic control, and that it is unlikely that endotoxin, corticosteroids, or regulatory T cells play a significant role in the mechanisms regulating these increases.

  2. Characterization, anticancer drug susceptibility and atRA-induced growth inhibition of a novel cell line (HUMEMS) established from pleural effusion of alveolar rhabdomyosarcoma of breast tissue.

    PubMed

    Ohi, Satoshi

    2007-05-01

    We recently established a cell line derived from pleural effusion from a 13-year-old girl with primary alveolar rhabdomyosarcoma (RMS with a chromosomal translocation t[2;13]) in the breast tissue. The cell line was designated as HUMEMS. Cases of primary alveolar RMS swelling in the breast are extremely rare (about 0.2% of all RMSs). Therefore, the HUMEMS cell line is an important material for studying therapeutics for malignant tumors in children. The HUMEMS cell line we isolated consisted of two morphological subtypes. One type (SSN cells) is small in size and has a single nucleus. Another (LMN cells) is large in size and has two or more nuclei. Both SSN cells and LMN cells were immunohistochemically positive for desmin and slightly positive for myoglobin. Our data suggested LMN cells are well-differentiated SSN cells. Moreover, in some of the LMN cells, rapid cell contractions (1-5 times/10 sec) were observed. We investigated the anticancer drug susceptibility of the HUMEMS cell line with an oxygen electrode apparatus (Daikin, DOX-10, JPN) and effect of all-trans-retinoic acid (atRA) to the cell line. The atRA-treatment inhibited proliferation of the HUMEMS cells.

  3. Formative cell divisions: principal determinants of plant morphogenesis.

    PubMed

    Smolarkiewicz, Michalina; Dhonukshe, Pankaj

    2013-03-01

    Formative cell divisions utilizing precise rotations of cell division planes generate and spatially place asymmetric daughters to produce different cell layers. Therefore, by shaping tissues and organs, formative cell divisions dictate multicellular morphogenesis. In animal formative cell divisions, the orientation of the mitotic spindle and cell division planes relies on intrinsic and extrinsic cortical polarity cues. Plants lack known key players from animals, and cell division planes are determined prior to the mitotic spindle stage. Therefore, it appears that plants have evolved specialized mechanisms to execute formative cell divisions. Despite their profound influence on plant architecture, molecular players and cellular mechanisms regulating formative divisions in plants are not well understood. This is because formative cell divisions in plants have been difficult to track owing to their submerged positions and imprecise timings of occurrence. However, by identifying a spatiotemporally inducible cell division plane switch system applicable for advanced microscopy techniques, recent studies have begun to uncover molecular modules and mechanisms for formative cell divisions. The identified molecular modules comprise developmentally triggered transcriptional cascades feeding onto microtubule regulators that now allow dissection of the hierarchy of the events at better spatiotemporal resolutions. Here, we survey the current advances in understanding of formative cell divisions in plants in the context of embryogenesis, stem cell functionality and post-embryonic organ formation.

  4. Formative cell divisions: principal determinants of plant morphogenesis.

    PubMed

    Smolarkiewicz, Michalina; Dhonukshe, Pankaj

    2013-03-01

    Formative cell divisions utilizing precise rotations of cell division planes generate and spatially place asymmetric daughters to produce different cell layers. Therefore, by shaping tissues and organs, formative cell divisions dictate multicellular morphogenesis. In animal formative cell divisions, the orientation of the mitotic spindle and cell division planes relies on intrinsic and extrinsic cortical polarity cues. Plants lack known key players from animals, and cell division planes are determined prior to the mitotic spindle stage. Therefore, it appears that plants have evolved specialized mechanisms to execute formative cell divisions. Despite their profound influence on plant architecture, molecular players and cellular mechanisms regulating formative divisions in plants are not well understood. This is because formative cell divisions in plants have been difficult to track owing to their submerged positions and imprecise timings of occurrence. However, by identifying a spatiotemporally inducible cell division plane switch system applicable for advanced microscopy techniques, recent studies have begun to uncover molecular modules and mechanisms for formative cell divisions. The identified molecular modules comprise developmentally triggered transcriptional cascades feeding onto microtubule regulators that now allow dissection of the hierarchy of the events at better spatiotemporal resolutions. Here, we survey the current advances in understanding of formative cell divisions in plants in the context of embryogenesis, stem cell functionality and post-embryonic organ formation. PMID:23248201

  5. [Validation of the modified algorithm for predicting host susceptibility to viruses taking into account susceptibility parameters of primary target cell cultures and natural immunity factors].

    PubMed

    Zhukov, V A; Shishkina, L N; Safatov, A S; Sergeev, A A; P'iankov, O V; Petrishchenko, V A; Zaĭtsev, B N; Toporkov, V S; Sergeev, A N; Nesvizhskiĭ, Iu V; Vorob'ev, A A

    2010-01-01

    The paper presents results of testing a modified algorithm for predicting virus ID50 values in a host of interest by extrapolation from a model host taking into account immune neutralizing factors and thermal inactivation of the virus. The method was tested for A/Aichi/2/68 influenza virus in SPF Wistar rats, SPF CD-1 mice and conventional ICR mice. Each species was used as a host of interest while the other two served as model hosts. Primary lung and trachea cells and secretory factors of the rats' airway epithelium were used to measure parameters needed for the purpose of prediction. Predicted ID50 values were not significantly different (p = 0.05) from those experimentally measured in vivo. The study was supported by ISTC/DARPA Agreement 450p.

  6. Class I-deficient resistant mice intracerebrally inoculated with Theiler's virus show an increased T cell response to viral antigens and susceptibility to demyelination.

    PubMed

    Pullen, L C; Miller, S D; Dal Canto, M C; Kim, B S

    1993-09-01

    Intracerebral inoculation of Theiler's murine encephalomyelitis virus (TMEV) results in immune-mediated demyelination in susceptible mouse strains. The histology of TMEV-induced demyelination is similar to that seen in patients suffering from multiple sclerosis. It was previously shown that the susceptibility of mice to TMEV-induced demyelination in certain strain combinations is closely associated with the major histocompatibility complex (MHC) class I locus. Here we examine disease susceptibility of beta 2-microglobulin (beta 2M)-deficient transgenic mice lacking class I expression and functional CD8+ T cells. In contrast to TMEV-infected parental C57BL/6 mice, the transgenics develop high levels of virus-specific DTH and T cell proliferation accompanied by an increased frequency of central nervous system (CNS) demyelinating lesions. However, clinical signs of demyelination were not noted. Neither antibody titer nor viral persistence were significantly affected in the beta 2M-deficient mice. These results suggest that in the absence of functional class I/CD8+ cells, the class II-restricted T cell response to TMEV is enhanced and CNS pathogenesis is heightened, although the level is not severe enough to result in clinical disease. When the TMEV-infected mice were subcutaneously immunized with virus, however, the beta 2M-deficient mice displayed clinical symptoms. Therefore, our results strongly suggest that CD8+ T cells do not directly contribute to CNS demyelination. In contrast, such T cells appear to be primarily involved in down-regulation of a potentially damaging CD4+ T cell response in resistant animals, although some of the T cells may play a role in clearing viral persistence in the CNS, resulting in the protection of the host from viral demyelination.

  7. Expression of T cell receptor V beta transcripts in central nervous system of mice susceptible and resistant to Theiler's virus-induced demyelination.

    PubMed

    Rodriguez, M; Prayoonwiwat, N; Zhou, P; David, C

    1993-08-01

    We utilized the polymerase chain reaction (PCR) to examine for preferential expression of T cell receptor (TcR) V beta s in T cells infiltrating the central nervous system (CNS) of mice infected with Theiler's virus. Infection of susceptible strains of mice with Theiler's virus results in demyelinating disease similar to multiple sclerosis. At 7 days following infection, no difference was observed in TcR V beta usage in lymphocytes infiltrating the CNS between resistant (B10.K) and susceptible (B10.Q or SJL/J) mice. In susceptible mice with prominent demyelination (day 45 and 238 following infection), no preferential expression of TcR V beta s was observed in the CNS. There is strong evidence that T cells play a major role in pathogenesis of TMEV-induced demyelination. There is increasing evidence using recombinant inbred strain mice with TcR V beta deletions that T cells expressing certain TcR V beta genes may be critical in the disease process. Yet, analysis of TcR V beta expression on T cells in CNS using PCR technology did not provide a way to dissect which antigen-specific T cells play a role in disease. These results confirm that during active demyelination specific as well as non-specific T cells are recruited to the CNS. Even though the pathogenesis of TMEV-induced disease may not be identical to that in multiple sclerosis, it is unlikely that similar approaches utilizing polymerase chain reaction will provide insights to the role of T cell receptors in the pathogenesis of multiple sclerosis.

  8. Cell fate determination in cisplatin resistance and chemosensitization.

    PubMed

    Luong, Khanh V; Wang, Ling; Roberts, Brett J; Wahl, James K; Peng, Aimin

    2016-04-26

    Understanding the determination of cell fate choices after cancer treatment will shed new light on cancer resistance. In this study, we quantitatively analyzed the individual cell fate choice in resistant UM-SCC-38 head and neck cancer cells exposed to cisplatin. Our study revealed a highly heterogeneous pattern of cell fate choices in UM-SCC-38 cells, in comparison to that of the control, non-tumorigenic keratinocyte HaCaT cells. In both UM-SCC-38 and HaCaT cell lines, the majority of cell death occurred during the immediate interphase without mitotic entry, whereas significant portions of UM-SCC-38 cells survived the treatment via either checkpoint arrest or checkpoint slippage. Interestingly, checkpoint slippage occurred predominantly in cells treated in late S and G2 phases, and cells in M-phase were hypersensitive to cisplatin. Moreover, although the cisplatin-resistant progression of mitosis exhibited no delay in general, prolonged mitosis was correlated with the induction of cell death in mitosis. The finding thus suggested a combinatorial treatment using cisplatin and an agent that blocks mitotic exit. Consistently, we showed a strong synergy between cisplatin and the proteasome inhibitor Mg132. Finally, targeting the DNA damage checkpoint using inhibitors of ATR, but not ATM, effectively sensitized UM-SCC-38 to cisplatin treatment. Surprisingly, checkpoint targeting eliminated both checkpoint arrest and checkpoint slippage, and augmented the induction of cell death in interphase without mitotic entry. Taken together, our study, by profiling cell fate determination after cisplatin treatment, reveals new insights into chemoresistance and suggests combinatorial strategies that potentially overcome cancer resistance. PMID:26993599

  9. Cell fate determination in cisplatin resistance and chemosensitization

    PubMed Central

    Luong, Khanh V.; Wang, Ling; Roberts, Brett J.; Wahl, James K.; Peng, Aimin

    2016-01-01

    Understanding the determination of cell fate choices after cancer treatment will shed new light on cancer resistance. In this study, we quantitatively analyzed the individual cell fate choice in resistant UM-SCC-38 head and neck cancer cells exposed to cisplatin. Our study revealed a highly heterogeneous pattern of cell fate choices in UM-SCC-38 cells, in comparison to that of the control, non-tumorigenic keratinocyte HaCaT cells. In both UM-SCC-38 and HaCaT cell lines, the majority of cell death occurred during the immediate interphase without mitotic entry, whereas significant portions of UM-SCC-38 cells survived the treatment via either checkpoint arrest or checkpoint slippage. Interestingly, checkpoint slippage occurred predominantly in cells treated in late S and G2 phases, and cells in M-phase were hypersensitive to cisplatin. Moreover, although the cisplatin-resistant progression of mitosis exhibited no delay in general, prolonged mitosis was correlated with the induction of cell death in mitosis. The finding thus suggested a combinatorial treatment using cisplatin and an agent that blocks mitotic exit. Consistently, we showed a strong synergy between cisplatin and the proteasome inhibitor Mg132. Finally, targeting the DNA damage checkpoint using inhibitors of ATR, but not ATM, effectively sensitized UM-SCC-38 to cisplatin treatment. Surprisingly, checkpoint targeting eliminated both checkpoint arrest and checkpoint slippage, and augmented the induction of cell death in interphase without mitotic entry. Taken together, our study, by profiling cell fate determination after cisplatin treatment, reveals new insights into chemoresistance and suggests combinatorial strategies that potentially overcome cancer resistance. PMID:26993599

  10. Experimental infection of Haemonchus contortus strains resistant and susceptible to benzimidazoles and the effect on mast cells distribution in the stomach of Mongolian gerbils (Meriones unguiculatus).

    PubMed

    Königová, Alzbeta; Hrckova, Gabriela; Velebný, Samuel; Corba, Július; Várady, Marián

    2008-03-01

    Establishment rate of Haemonchus contortus in non-suppressed and immunosuppressed gerbils within 14 days post-infection was compared after inoculation with 1,000 third-stage larvae (L3), exsheathed BZ-susceptible larvae. Based on significantly higher number of larvae in gerbils receiving low doses of immunosuppressant agent hydrocortisone, development of benzimidazole (BZ)-susceptible and BZ-resistant strain of nematode in the stomach was studied on days 4, 7, 10, and 14 p.i. Sections of stomach from both groups of animals were examined for overall histopathological response and dynamics of mucosal mast cells (MMC) and connective tissue mast cells (CTMC). In the immunosuppressed gerbils, H. contortus L3 stage larvae developed to the L4 stage on days 10 and 14 p.i., and their sex ratio was higher toward female worms. Significantly higher ratios of establishment rate were recorded for BZ-susceptible than BZ-resistant strain. Infection elicited strong inflammation mainly in the lamina propria mucosae, where MMC numbers peaked on day 7 p.i., being present in a significantly higher numbers in gerbils infected with BZ-susceptible strain. Infection with BZ-susceptible strain of nematode also resulted in a higher number of CTMC in comparison with the effect of BZ-resistant strain, which were observed in the tela submucosa only. Thus, H. contortus infection in gerbils seems to be a suitable model to study host-parasite interactions. Our results indicate that BZ-resistant strain of H. contortus have a decreased capacity to establish infection in direct relation with lower mucosal and connective tissue MCs counts in the stomach.

  11. NMR determination of the orientation of the magnetic susceptibility tensor in cyanometmyoglobin: A new probe of steric tilt of bound ligand

    SciTech Connect

    Emerson, S.D.; La Mar, G.N. )

    1990-02-13

    The experimentally determined paramagnetic dipolar shifts for noncoordinated amino acid side-chain protons in the heme pocket of sperm whale cyanometmyoglobin were used to determine in solution the orientation of the principal axes for the paramagnetic susceptibility tensor relative to the heme iron molecular coordinates. The determination was made by a least-squares search for the unique Euler rotation angles which convert the geometric factors in the molecular (crystal) coordinates to ones that correctly predict each of 41 known dipolar shifts by using the magnetic anisotropies computed previously. An excellent fit to experimental shifts was obtained, which also provided predictions that allowed subsequent new assignments to be made. The magnetic axes are oriented so that the z axis is tipped {approximately}15{degree} from the heme normal toward the heme {delta}-meso-H and coincides approximately with the characterized FeCO tilt axis in the isostructural MbCO complex. Since the FeCO and FeCN units are isostructural, the authors propose that the dominant protein constraint that tips the magnetic z axis from the heme normal is the tilt of the FeCN by steric interactions with the distal residues. It is shown that the proximal His ring nonlabile proton hyperfine shifts provide direct and exquisitely sensitive indicators of the degree of the z axis tilt that may serve as a valuable probe for characterizing variable steric interactions in the distal pocket of both point mutants and natural genetic variants of myoglobin.

  12. Cryptococcus neoformans biofilm formation depends on surface support and carbon source and reduces fungal cell susceptibility to heat, cold, and UV light.

    PubMed

    Martinez, Luis R; Casadevall, Arturo

    2007-07-01

    The fungus Cryptococcus neoformans possesses a polysaccharide capsule and can form biofilms on medical devices. We describe the characteristics of C. neoformans biofilm development using a microtiter plate model, microscopic examinations, and a colorimetric 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay to observe the metabolic activity of cryptococci within a biofilm. A strong correlation between XTT and CFU assays was demonstrated. Chemical analysis of the exopolymeric material revealed sugar composition consisting predominantly of xylose, mannose, and glucose, indicating the presence of other polysaccharides in addition to glucurunoxylomannan. Biofilm formation was affected by surface support differences, conditioning films on the surface, characteristics of the medium, and properties of the microbial cell. A specific antibody to the capsular polysaccharide of this fungus was used to stain the extracellular polysaccharide matrix of the fungal biofilms using light and confocal microscopy. Additionally, the susceptibility of C. neoformans biofilms and planktonic cells to environmental stress was investigated using XTT reduction and CFU assays. Biofilms were less susceptible to heat, cold, and UV light exposition than their planktonic counterparts. Our findings demonstrate that fungal biofilm formation is dependent on support surface characteristics and that growth in the biofilm state makes fungal cells less susceptible to potential environmental stresses. PMID:17513597

  13. Mycobacterium tuberculosis Genotypes Determined by Spoligotyping to Be Circulating in Colombia between 1999 and 2012 and Their Possible Associations with Transmission and Susceptibility to First-Line Drugs

    PubMed Central

    Puerto, Gloria; Erazo, Lina; Wintaco, Maira; Castro, Claudia; Ribón, Wellman; Guerrero, Martha Inírida

    2015-01-01

    Introduction Tuberculosis (TB) remains a primary public health problem worldwide. The number of multidrug-resistant tuberculosis (MDR TB) cases has increased in recent years in Colombia. Knowledge of M. tuberculosis genotypes defined by spoligotyping can help determine the circulation of genotypes that must be controlled to prevent the spread of TB. Objective To describe the genotypes of M. tuberculosis using spoligotyping in resistant and drug-sensitive isolates and their possible associations with susceptibility to first-line drugs. Methods An analytical observational study was conducted that included 741 isolates of M. tuberculosis from patients. The isolates originated from 31 departments and were obtained by systematic surveillance between 1999 and 2012. Results In total 61.94% of the isolates were resistant to 1 or more drugs, and 147 isolates were MDR. In total, 170 genotypes were found in the population structure of Colombian M. tuberculosis isolates. The isolates were mainly represented by four families: LAM (39.9%), Haarlem (19%), Orphan (17%) and T (9%). The SIT42 (LAM 9) was the most common genotype and contained 24.7% of the isolates, followed by the genotypes SIT62 (Haarlem1), SIT53 (T1), and SIT50 (H3). A high clustering of isolates was evident with 79.8% of the isolates classified into 32 groups. The Beijing family was associated with resistant isolates, whereas the Haarlem and T families were associated with sensitive isolates. The Haarlem family was also associated with grouped isolates (p = 0.031). Conclusions A high proportion (approximately 80%) of isolates was found in clusters; these clusters were not associated with resistance to first-line drugs. The Beijing family was associated with drug resistance, whereas the T and Haarlem families were associated with susceptibility in the Colombian isolates studied. PMID:26066494

  14. Topoisomerase II and IV quinolone resistance-determining regions in Stenotrophomonas maltophilia clinical isolates with different levels of quinolone susceptibility.

    PubMed

    Valdezate, Sylvia; Vindel, Ana; Echeita, Aurora; Baquero, Fernando; Cantó, Rafael

    2002-03-01

    The quinolone resistance-determining regions (QRDRs) of topoisomerase II and IV genes from Stenotrophomonas maltophilia ATCC 13637 were sequenced and compared with the corresponding regions of 32 unrelated S. maltophilia clinical strains for which ciprofloxacin MICs ranged from 0.1 to 64 microg/ml. GyrA (Leu-55 to Gln-155, Escherichia coli numbering), GyrB (Met-391 to Phe-513), ParC (Ile-34 to Arg-124), and ParE (Leu-396 to Leu-567) fragments from strain ATCC 13637 showed high degrees of identity to the corresponding regions from the phytopathogen Xylella fastidiosa, with the degrees of identity ranging from 85.0 to 93.5%. Lower degrees of identity to the corresponding regions from Pseudomonas aeruginosa (70.9 to 88.6%) and E. coli (73.0 to 88.6%) were observed. Amino acid changes were present in GyrA fragments from 9 of the 32 strains at positions 70, 85, 90, 103, 112, 113, 119, and 124; but there was no consistent relation to higher ciprofloxacin MICs. The absence of changes at positions 83 and 87, commonly involved in quinolone resistance in gram-negative bacteria, was unexpected. The GyrB sequences were identical in all strains, and only one strain (ciprofloxacin MIC, 16 microg/ml) showed a ParC amino acid change (Ser-80-->Arg). In contrast, a high frequency (16 of 32 strains) of amino acid replacements was present in ParE. The frequencies of alterations at positions 437, 465, 477, and 485 were higher (P < 0.05) in strains from cystic fibrosis patients, but these changes were not linked with high ciprofloxacin MICs. An efflux phenotype, screened by the detection of decreases of at least twofold doubling dilutions of the ciprofloxacin MIC in the presence of carbonyl cyanide m-chlorophenylhydrazone (0.5 microg/ml) or reserpine (10 microg/ml), was suspected in seven strains. These results suggest that topoisomerases II and IV may not be the primary targets involved in quinolone resistance in S. maltophilia.

  15. Topoisomerase II and IV Quinolone Resistance-Determining Regions in Stenotrophomonas maltophilia Clinical Isolates with Different Levels of Quinolone Susceptibility

    PubMed Central

    Valdezate, Sylvia; Vindel, Ana; Echeita, Aurora; Baquero, Fernando; Cantó, Rafael

    2002-01-01

    The quinolone resistance-determining regions (QRDRs) of topoisomerase II and IV genes from Stenotrophomonas maltophilia ATCC 13637 were sequenced and compared with the corresponding regions of 32 unrelated S. maltophilia clinical strains for which ciprofloxacin MICs ranged from 0.1 to 64 μg/ml. GyrA (Leu-55 to Gln-155, Escherichia coli numbering), GyrB (Met-391 to Phe-513), ParC (Ile-34 to Arg-124), and ParE (Leu-396 to Leu-567) fragments from strain ATCC 13637 showed high degrees of identity to the corresponding regions from the phytopathogen Xylella fastidiosa, with the degrees of identity ranging from 85.0 to 93.5%. Lower degrees of identity to the corresponding regions from Pseudomonas aeruginosa (70.9 to 88.6%) and E. coli (73.0 to 88.6%) were observed. Amino acid changes were present in GyrA fragments from 9 of the 32 strains at positions 70, 85, 90, 103, 112, 113, 119, and 124; but there was no consistent relation to higher ciprofloxacin MICs. The absence of changes at positions 83 and 87, commonly involved in quinolone resistance in gram-negative bacteria, was unexpected. The GyrB sequences were identical in all strains, and only one strain (ciprofloxacin MIC, 16 μg/ml) showed a ParC amino acid change (Ser-80→Arg). In contrast, a high frequency (16 of 32 strains) of amino acid replacements was present in ParE. The frequencies of alterations at positions 437, 465, 477, and 485 were higher (P < 0.05) in strains from cystic fibrosis patients, but these changes were not linked with high ciprofloxacin MICs. An efflux phenotype, screened by the detection of decreases of at least twofold doubling dilutions of the ciprofloxacin MIC in the presence of carbonyl cyanide m-chlorophenylhydrazone (0.5 μg/ml) or reserpine (10 μg/ml), was suspected in seven strains. These results suggest that topoisomerases II and IV may not be the primary targets involved in quinolone resistance in S. maltophilia. PMID:11850246

  16. Reduced glycosylation of human cell lines increases susceptibility to CD4-independent infection by human immunodeficiency virus type 2 (LAV-2/B).

    PubMed Central

    Talbot, S J; Weiss, R A; Schulz, T F

    1995-01-01

    The human immunodeficiency virus type 2 (HIV-2) strain LAV-2/B is able to infect a variety of human cell lines via a CD4-independent pathway. We have used the glycosylation inhibitors tunicamycin, swainsonine, and deoxymannojirimycin to further characterize this putative alternative receptor for HIV-2 (LAV-2/B). These antibiotics resulted in an increase (5- to 30-fold) in the susceptibility of a variety of CD4- human cell lines to infection by LAV-2/B (RD, HeLa, HT29, Rsb, Heb7a, Hos, and Daudi). Several nonprimate cell lines (mink Mv-1-lu, rabbit SIRC, hamster a23, mouse NIH 3T3, cat CCC, and rat HSN) remained resistant to infection by LAV-2/B after treatment with glycosylation inhibitors, suggesting that they do not express the HIV-2 CD4-independent receptor. Two of these nonprimate cell lines are readily infected by HIV-2 when they express CD4 (Mv-1-lu and CCC). Treatment of human cells with neuraminidase had no effect on subsequent infection by LAV-2/B, suggesting that the increase in susceptibility to infection of deglycosylated cells is not due to a change in the electrostatic charge of the cell surface. Treatment of RD CD4- cells and HeLa CD4+ cells with a variety of proteases resulted in a 75 to 90% decrease in infection by LAV-2/B when compared with untreated cells. Taken together, all these data suggest that HIV-2 can utilize a membrane glycoprotein other than CD4 to attach and fuse with a variety of human cells. PMID:7745686

  17. Cell age, suspending medium and metal ion influence the susceptibility of Escherichia coli O157:H7 to water-soluble maltose chitosan derivative.

    PubMed

    Yang, Tsui-Chu; Li, Chin-Fung; Chou, Cheng-Chun

    2007-02-15

    The effects of cell age, suspending medium and metal ions on the susceptibility of Escherichia coli O157:H7 to the water-soluble maltose chitosan derivative were investigated. In addition, the leakage of glucose, protein (absorbance at 280 nm) and lactate dehydrogenase (LDH) induced by maltose chitosan derivative in saline solution and deionized water, was examined. Cells of E. coli O157:H7 in the mid-exponential phase (6 h) were most susceptible to the chitosan derivative followed by cells in the late-exponential phase (12 h) and stationary phase (24 h). In addition, it was found that the susceptibility of the test organism to the maltose chitosan derivative was less in saline solution than in deionized water. The viable population of E. coli O157:H7 in deionized water containing the maltose chitosan derivative (500 ppm), was reduced from ca 7.6 log cfu/ml to a non-detectable level after 10 h of incubation at 37 degrees C compared to a viable population of ca 6.2 log cfu/ml noted in the chitosan derivative-containing-saline solution. After cells of E. coli O157:H7 were exposed to the chitosan derivative in deionized water, a marked increase in the levels of glucose, protein and LDH activity was observed in the supernatant of cell suspension compared to cells of test organism exposed to the saline solution containing chitosan derivative. Metal ions were also found to reduce the antibacterial activity of chitosan derivative. Their effectiveness increased at greater concentrations and varied with the kinds of metal ions with Ba(2+) the most effective and Mg(2+) the least effective.

  18. Glycolysis determines dichotomous regulation of T cell subsets in hypoxia

    PubMed Central

    Xu, Yang; Zhang, Ming; Savoldo, Barbara; Metelitsa, Leonid S.; Rodgers, John; Yustein, Jason T.; Neilson, Joel R.

    2016-01-01

    Hypoxia occurs in many pathological conditions, including chronic inflammation and tumors, and is considered to be an inhibitor of T cell function. However, robust T cell responses occur at many hypoxic inflammatory sites, suggesting that functions of some subsets are stimulated under low oxygen conditions. Here, we investigated how hypoxic conditions influence human T cell functions and found that, in contrast to naive and central memory T cells (TN and TCM), hypoxia enhances the proliferation, viability, and cytotoxic action of effector memory T cells (TEM). Enhanced TEM expansion in hypoxia corresponded to high hypoxia-inducible factor 1α (HIF1α) expression and glycolytic activity compared with that observed in TN and TCM. We determined that the glycolytic enzyme GAPDH negatively regulates HIF1A expression by binding to adenylate-uridylate–rich elements in the 3′-UTR region of HIF1A mRNA in glycolytically inactive TN and TCM. Conversely, active glycolysis with decreased GAPDH availability in TEM resulted in elevated HIF1α expression. Furthermore, GAPDH overexpression reduced HIF1α expression and impaired proliferation and survival of T cells in hypoxia, indicating that high glycolytic metabolism drives increases in HIF1α to enhance TEM function during hypoxia. This work demonstrates that glycolytic metabolism regulates the translation of HIF1A to determine T cell responses to hypoxia and implicates GAPDH as a potential mechanism for controlling T cell function in peripheral tissue. PMID:27294526

  19. Cell fate determination by ubiquitin-dependent regulation of translation

    PubMed Central

    Werner, Achim; Iwasaki, Shintaro; McGourty, Colleen; Medina-Ruiz, Sofia; Teerikorpi, Nia; Fedrigo, Indro; Ingolia, Nicholas T.; Rape, Michael

    2015-01-01

    Metazoan development depends on accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates 1. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell fate determination is less well understood. Here, we have identified the vertebrate-specific ubiquitin ligase CUL3KBTBD8 as an essential regulator of neural crest specification. CUL3KBTBD8 monoubiquitylates NOLC1 and its paralog TCOF1, whose mutation underlies the neurocristopathy Treacher Collins Syndrome 2,3. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favor of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell fate determination. PMID:26399832

  20. An autologous T cell clone overcomes intra-melanoma heterogeneity for susceptibility to cell-mediated lysis by using multiple lytic mechanisms: in vitro and in vivo analysis.

    PubMed

    Mazzocchi, A; Rodolfo, M; Parmiani, G; Anichini, A

    1991-01-01

    An HLA-A2-specific cytotoxic T lymphocyte (CTL) clone (CTL49), capable of killing the HLA-A2-negative autologous melanoma (Me665/2) in a T cell receptor and MHC-independent fashion, lysed six of 16 Me665/2 tumour clones in short-term (4 and 18 hour) 51Cr-release assays. In long-term (96 hour) lytic assays, CTL49 could lyse all the 16 tumour clones. The lysis observed in the 96 hour assay could be enhanced by stimulating CTL49 with anti-CD3 monoclonal antibodies (MAb) and interleukin-2 (IL-2). Supernatants of anti-CD3- or antigen-stimulated CTL49, known to contain tumour necrosis factor (TNF) alpha and interferon (IFN)gamma, were also able to lyse all but one (665/2/51) of the tumour clones in 96 hour assays. Absence of lysis of tumour clone 2/51 by supernatants correlated with resistance of the same tumour clone to lysis by recombinant IFN-gamma plus TNF-alpha. Antibodies to TNF-alpha and, to a lesser extent, to IFN-gamma, reduced significantly the 96 hour lysis of Me2/9 and Me2/10, two of the tumour clones killed in long term but not in short term assays. Winn assays in nude mice revealed that CTL49, stimulated with anti-CD3-MAb plus IL-2, could abolish tumour cell growth when injected together with clones 2/9 or 2/10. These results indicate that intra-tumour heterogeneity for susceptibility to lysis can be overcome even by a single CTL clone providing that appropriate signals (i.e. anti-CD3-MAb and IL-2) are supplied to an effector able to mediate tumour cell lysis by multiple mechanisms. PMID:1841713

  1. Determination of cell electroporation in small-volume samples.

    PubMed

    Saulis, Gintautas; Praneviciŭte, Rita

    2007-01-01

    Expose of cells to electric field pulses increases the cell membrane permeability. Intracellular potassium ions leak out of the cells through aqueous pores created in the membrane. This release is used here for the determination of the fraction of electroporated cells. To determine cell membrane electroporation in small-volume samples (40-50 miacrol), mini both potassium ion-selective and reference electrodes, with tip diameters of 1-1.5 mm and minimum immersion depths of 1 mm, were utilized. The obtained calibration graph was linear within the concentration range 0.2-100 mM. The slope was 50-51 and 53-56 mV per concentration order at 10-11 and 19-21 degrees C, respectively. Detection limit of the electrode was determined to be 0.05-0.08 mM, however, it was possible to work down to concentrations in the range of 0.01 mM. Experiments have been carried out on human erythrocytes exposed to a square-wave electric pulse with the duration of 0.1-2 ms. The extracellular potassium concentrations were in the range between 0.04-0.08 mM (intact cells) and 3-5 mM (100% electroporation). The obtained dependences of the fraction of electroporated cells on the pulse intensity were of a sigmoid shape. The dependence of the pulse amplitude required to electroporate 50% of cells on the pulse duration, obtained from the release of intracellular potassium ions, coincided with the one determined from the extent of hemolysis after 24 h-incubation at low temperature.

  2. Specificity of regulatory CD4+CD25+ T cells for self-T cell receptor determinants.

    PubMed

    Buenafe, Abigail C; Tsaknaridis, Laura; Spencer, Leslie; Hicks, Kevin S; McMahan, Rachel H; Watson, Lisa; Culbertson, Nicole E; Latocha, Dorian; Wegmann, Keith; Finn, Tom; Bartholomew, Richard; Burrows, Gregory G; Whitham, Ruth; Bourdette, Dennis N; Jones, Richard E; Offner, Halina; Chou, Yuan K; Vandenbark, Arthur A

    2004-04-01

    Although the phenotypic and regulatory properties of the CD4(+)CD25(+) T cell lineage (Treg cells) have been well described, the specificities remain largely unknown. We demonstrate here that the CD4(+)CD25(+) Treg population includes the recognition of a broad spectrum of human TCR CDR2 determinants found in the germline V gene repertoire as well as that of a clonotypic nongermline-encoded CDR3beta sequence present in a recombinant soluble T cell receptor (TCR) protein. Regulatory activity was demonstrated in T cell lines responsive to TCR but not in T cell lines responsive to control antigens. Inhibitory activity of TCR-reactive T cells required cell-cell contact and involved CTLA-4, GITR, IL-10, and IL-17. Thus, the T-T regulatory network includes Treg cells with specificity directed toward self-TCR determinants.

  3. Establishment and evaluation of a murine αvβ3-integrin-expressing cell line with increased susceptibility to Foot-and-mouth disease virus

    PubMed Central

    Zhang, Wei; Lian, Kaiqi; Yang, Fan; Yang, Yang; Zhu, Zhijian; Zhu, Zixiang; Cao, Weijun; Mao, Ruoqing; Jin, Ye; He, Jijun; Guo, Jianhong; Liu, Xiangtao

    2015-01-01

    Integrin αvβ3 plays a major role in various signaling pathways, cell apoptosis, and tumor angiogenesis. To examine the functions and roles of αvβ3 integrin, a stable CHO-677 cell line expressing the murine αvβ3 heterodimer (designated as "CHO-677-mαvβ3" cells) was established using a highly efficient lentiviral-mediated gene transfer technique. Integrin subunits αv and β3 were detected at the gene and protein levels by polymerase chain reaction (PCR) and indirect immunofluorescent assay (IFA), respectively, in the CHO-677-mαvβ3 cell line at the 20th passage, implying that these genes were successfully introduced into the CHO-677 cells and expressed stably. A plaque-forming assay, 50% tissue culture infective dose (TCID50), real-time quantitative reverse transcription-PCR, and IFA were used to detect the replication levels of Foot-and-mouth disease virus (FMDV) in the CHO-677-mαvβ3 cell line. After infection with FMDV/O/ZK/93, the cell line showed a significant increase in viral RNA and protein compared with CHO-677 cells. These findings suggest that we successfully established a stable αvβ3-receptor-expressing cell line with increased susceptibility to FMDV. This cell line will be very useful for further investigation of αvβ3 integrin, and as a cell model for FMDV research. PMID:25643796

  4. Autophagy variation within a cell population determines cell fate through selective degradation of Fap-1.

    PubMed

    Gump, Jacob M; Staskiewicz, Leah; Morgan, Michael J; Bamberg, Alison; Riches, David W H; Thorburn, Andrew

    2014-01-01

    Autophagy regulates cell death both positively and negatively, but the molecular basis for this paradox remains inadequately characterized. We demonstrate here that transient cell-to-cell variations in autophagy can promote either cell death or survival depending on the stimulus and cell type. By separating cells with high and low basal autophagy using flow cytometry, we demonstrate that autophagy determines which cells live or die in response to death receptor activation. We have determined that selective autophagic degradation of the phosphatase Fap-1 promotes Fas apoptosis in Type I cells, which do not require mitochondrial permeabilization for efficient apoptosis. Conversely, autophagy inhibits apoptosis in Type II cells (which require mitochondrial involvement) or on treatment with TRAIL in either Type I or II cells. These data illustrate that differences in autophagy in a cell population determine cell fate in a stimulus- and cell-type-specific manner. This example of selective autophagy of an apoptosis regulator may represent a general mechanism for context-specific regulation of cell fate by autophagy. PMID:24316673

  5. Two subpopulations of human triple-negative thymic cells are susceptible to infection by human immunodeficiency virus type 1 in vitro.

    PubMed Central

    Valentin, H; Nugeyre, M T; Vuillier, F; Boumsell, L; Schmid, M; Barré-Sinoussi, F; Pereira, R A

    1994-01-01

    Some infants infected with human immunodeficiency virus type 1 (HIV-1) rapidly develop a fatal disease characterized by a severe lymphopenia. To explain the immune dysfunction, we proposed a mechanism by which a nongeneration of CD4+ T cells is caused by HIV-1 infection of thymic cells. To examine this hypothesis, we infected primary triple-negative (TN; phenotypically CD3- CD4- CD8-), CD1a- TN, or CD1a+ TN thymic cell subsets. Our data indicate that by flow cytometry, TN, CD1a- TN, and CD1a+ TN cells remain CD4 negative throughout the culture period. We demonstrated that TN and CD1a+ TN thymic cell subsets are susceptible to HIV-1 as is the entire thymic cell population, whereas CD1a- TN cells are not. A limited number of infected TN cells are expressing HIV-1 but the level of transcription is very high in permissive cells, as detected by in situ hybridization. We then performed blocking experiments on TN cells to examine the mechanism of HIV-1 entry into these cells. CD4 (OKT4a) monoclonal antibody blocks their infection. Finally, infection experiments on two subpopulations of TN cells (CD2+ CD7+ and CD2- CD7-) indicate that infected TN cells may correspond to both immature thymocytes and thymic dendritic cells. These data are of particular interest since infection of thymic stromal cells might result in an impairment of T-cell differentiation, which may explain a nongeneration of functional CD4+ T-cell population in the thymus. This phenomenon may play a role in AIDS pathogenesis, in particular in infants born from seropositive mothers. Images PMID:7512158

  6. Integrin α4β7 expression increases HIV susceptibility in activated cervical CD4+ T cells via an HIV attachment-independent mechanism

    PubMed Central

    Ding, Jian; Tasker, Carley; Lespinasse, Pierre; Dai, Jihong; Fitzgerald-Bocarsly, Patricia; Lu, Wuyuan; Heller, Debra; Chang, Theresa L.

    2015-01-01

    Background CD4+ T cells, the principal target in acute SIV and HIV infection, are crucial for the establishment and dissemination of HIV infection in mucosal tissues. Studies indicate that α4β7 CD4+ T cells are preferentially infected by HIV in vitro and during acute SIV infection. The integrin α4β7 is thought to promote HIV capture by target cells; however, the role of integrin α4β7 in HIV transmission remains controversial. In this study, we characterized immune phenotypes of human cervical T cells and examined HIV preference in integrin α4β7+ CD4+ T cells. In vitro all-trans retinoic acid differentiated peripheral CD4+ T cells (at-RA differentiated cells) were included as a comparison. Results In both peripheral and cervical cells, the majority of HIV p24+ cells were activated CD4+ T cells expressing integrin α4β7. Among infected at-RA differentiated cells, the frequency of CCR5 expression was higher in HIV p24+ cells than in HIV p24- cells; no such difference was observed in cervical cells. Neither the cyclic hexapeptide CWLDVC nor a monoclonal antibody against integrin α4β7 blocked HIV attachment or gp120 binding to target cells regardless of the presence of CD4, indicating that integrin α4β7 did not facilitate HIV capture by target cells. Conclusion Integrin α4β7 expression increases HIV susceptibility, but the mechanism is not through promoting HIV binding to target cells. PMID:26167616

  7. Comparative analysis of virulence determinants, antibiotic susceptibility patterns and serogrouping of atypical enteropathogenic Escherichia coli versus typical enteropathogenic E. coli in India.

    PubMed

    Malvi, Supriya; Appannanavar, Suma; Mohan, Balvinder; Kaur, Harsimran; Gautam, Neha; Bharti, Bhavneet; Kumar, Yashwant; Taneja, Neelam

    2015-10-01

    The epidemiology of enteropathogenic Escherichia coli (EPEC) and the significance of isolation of atypical EPEC (aEPEC) in childhood diarrhoea have not been well studied in an Indian context. A comparative study was undertaken to investigate virulence determinants, antibiotic susceptibility patterns and serogrouping of typical EPEC (tEPEC) versus aEPEC causing diarrhoea in children. A total of 400 prospective and 500 retrospective E. coli isolates were included. PCR was performed for eae, bfpA, efa, nleB, nleE, cdt, ehxA and paa genes. The Clinical and Laboratory Standards Institute's disc diffusion test was used to determine the antimicrobial susceptibility. Phenotypic screening of extended spectrum β-lactamases (ESBLs), AmpC and Klebsiella pneumoniae carbapenemase (KPC) production, and molecular detection of bla(NDM-1), bla(VIM), bla(CTX-M-15), bla(IMP) and bla(KPC) were performed. aEPEC (57.6 %) were more common as compared with tEPEC (42.3 %). The occurrence of virulence genes was observed to be three times higher in aEPEC as compared with tEPEC, efa1 (14.7 % of aEPEC, 4 % of tEPEC) being the most common. Most of the isolates did not belong to the classical EPEC O-serogroups. The highest resistance was observed against amoxicillin (93.22 %) followed by quinolones (83 %), cephalosporins (37.28 %), cotrimoxazole (35.59 %) and carbapenems (30.5 %). Overall equal numbers of aEPEC (41.17 %) and tEPEC (40 %) were observed to be multidrug-resistant. Fifteen EPEC strains demonstrated presence of ESBLs, five produced AmpC and four each produced metallo-β-lactamases and KPC-type carbapenemases; eight, seven and one isolate(s) each were positive for bla(VIM), bla(CTX-M-15) and bla(NDM-1), respectively. Here, to the best of our knowledge, we report for the first time on carbapenem resistance and the presence of bla(NDM-1) and bla(CTX-M-15) in EPEC isolates from India. PMID:26233663

  8. Comparative analysis of virulence determinants, antibiotic susceptibility patterns and serogrouping of atypical enteropathogenic Escherichia coli versus typical enteropathogenic E. coli in India.

    PubMed

    Malvi, Supriya; Appannanavar, Suma; Mohan, Balvinder; Kaur, Harsimran; Gautam, Neha; Bharti, Bhavneet; Kumar, Yashwant; Taneja, Neelam

    2015-10-01

    The epidemiology of enteropathogenic Escherichia coli (EPEC) and the significance of isolation of atypical EPEC (aEPEC) in childhood diarrhoea have not been well studied in an Indian context. A comparative study was undertaken to investigate virulence determinants, antibiotic susceptibility patterns and serogrouping of typical EPEC (tEPEC) versus aEPEC causing diarrhoea in children. A total of 400 prospective and 500 retrospective E. coli isolates were included. PCR was performed for eae, bfpA, efa, nleB, nleE, cdt, ehxA and paa genes. The Clinical and Laboratory Standards Institute's disc diffusion test was used to determine the antimicrobial susceptibility. Phenotypic screening of extended spectrum β-lactamases (ESBLs), AmpC and Klebsiella pneumoniae carbapenemase (KPC) production, and molecular detection of bla(NDM-1), bla(VIM), bla(CTX-M-15), bla(IMP) and bla(KPC) were performed. aEPEC (57.6 %) were more common as compared with tEPEC (42.3 %). The occurrence of virulence genes was observed to be three times higher in aEPEC as compared with tEPEC, efa1 (14.7 % of aEPEC, 4 % of tEPEC) being the most common. Most of the isolates did not belong to the classical EPEC O-serogroups. The highest resistance was observed against amoxicillin (93.22 %) followed by quinolones (83 %), cephalosporins (37.28 %), cotrimoxazole (35.59 %) and carbapenems (30.5 %). Overall equal numbers of aEPEC (41.17 %) and tEPEC (40 %) were observed to be multidrug-resistant. Fifteen EPEC strains demonstrated presence of ESBLs, five produced AmpC and four each produced metallo-β-lactamases and KPC-type carbapenemases; eight, seven and one isolate(s) each were positive for bla(VIM), bla(CTX-M-15) and bla(NDM-1), respectively. Here, to the best of our knowledge, we report for the first time on carbapenem resistance and the presence of bla(NDM-1) and bla(CTX-M-15) in EPEC isolates from India.

  9. Histone Deacetylation Critically Determines T-cell Subset Radiosensitivity1

    PubMed Central

    Pugh, Jason L.; Sukhina, Alona S.; Seed, Thomas M.; Manley, Nancy R.; Sempowski, Gregory A.; van den Brink, Marcel R.M.; Smithey, Megan J.; Nikolich-Zugich, Janko

    2014-01-01

    Lymphocytes are sensitive to ionizing radiation and naïve lymphocytes are more radiosensitive than their memory counterparts. Less is known about radiosensitivity of memory cell subsets. We examined the radiosensitivity of naïve (TN), effector memory (TEM), and central memory (TCM) T cell subsets in C57BL/6 mice, and found TEM to be more resistant to radiation-induced apoptosis than either TN or TCM. Surprisingly, we found no correlation between the extent of radiation-induced apoptosis in T cell subsets and : (i) levels of pro- and anti-apoptotic Bcl-2 family members; or (ii) the H2-AX content and maximal γH2-AX fold change. Rather, TEM cell survival correlated with higher levels of immediate γH2-AX marking, immediate break binding and genome-wide open chromatin structure. T cells were able to mark DNA damage seemingly instantly (30 s), even if kept on ice. Relaxing chromatin with the histone deacetylase inhibitor valproic acid following radiation or etoposide treatment, improved the survival of TCM and TN cells up to levels seen in the resistant TEM cells, but did not improve survival from caspase-mediated apoptosis. We conclude that an open genome-wide chromatin state is the key determinant of efficient immediate repair of DNA damage in T cells, explaining the observed T cell subset radiosensitivity differences. PMID:24990082

  10. Single-nucleotide polymorphisms and haplotypes of membrane type 1-matrix metalloproteinase in susceptibility and clinical significance of squamous cell neoplasia of uterine cervix in Taiwan women.

    PubMed

    Tee, Yi-Torng; Liu, Yu-Fan; Chang, Jinghua Tsai; Yang, Shun-Fa; Chen, Shiuan-Chih; Han, Chih-Ping; Wang, Po-Hui; Liao, Chiung-Ling

    2012-09-01

    Membrane type 1-matrix metalloproteinase (MT1-MMP) participates in the activity of MMP-2, which correlates with cancer of uterine cervix. Single-nucleotide polymorphisms (SNPs) in promoter and exon of MT1-MMP may influence their binding with transcription factors and gene transcription. To date, no study reports the association of the MT1-MMP polymorphisms with cervical neoplasia. Therefore, we investigated the influence of the MT1-MMP gene polymorphisms on the susceptibility and clinicopathological variables of cervical neoplasia for women in Taiwan. We recruited 72 patients with cervical squamous cell carcinoma and 63 with high-grade dysplasia as 1 subgroup. Meanwhile, 280 control women were included as another subgroup. The SNPs rs1003349 (site -165), rs2236307 (+7096), and rs3751489 (+8153) as well as rs2236302 (site +6727) of MT1-MMP gene were determined by polymerase chain reaction (PCR)-restriction fragment length polymorphism and real-time PCR genotyping, respectively. Then, we correlated these SNPs and haplotypes with the development of cervical neoplasia and cancer clinicopathological variables. We found that women with CC genotype in rs2236307 SNP exhibited a more risk to develop cervical neoplasia as compared with those with wild genotype TT. Haplotypes -165 T, +6727 C, +7096 C, +8153 G or -165 G, +6727 G, +7096 T, and +8153 G and diplotypes including at least 1 type of these haplotypes of MT1-MMP gene showed a higher risk of cervical neoplasia. However, both haplotypes were not significantly correlated with the clinicopathological characteristics of cervical cancer. In conclusion, Taiwan women with variant homozygote CC (+7096) and haplotypes, TCCG and GGTG, of MT1-MMP exhibit more risk in developing cervical neoplasia.

  11. Determinism and probability in the development of the cell theory.

    PubMed

    Duchesneau, François

    2012-09-01

    A return to Claude Bernard's original use of the concept of 'determinism' displays the fact that natural laws were presumed to rule over all natural processes. In a more restricted sense, the term boiled down to a mere presupposition of constant determinant causes for those processes, leaving aside any particular ontological principle, even stochastic. The history of the cell theory until around 1900 was dominated by a twofold conception of determinant causes. Along a reductionist trend, cells' structures and processes were supposed to be accounted for through their analysis into detailed partial mechanisms. But a more holistic approach tended to subsume those analytic means and the mechanism involved under a program of global functional determinations. When mitotic and meiotic sequences in nuclear replication were being unveiled and that neo-Mendelian genetics was being grafted onto cytology and embryology, a conception of strict determinism at the nuclear level, principally represented by Wilhelm Roux and August Weismann, would seem to rule unilaterally over the mosaic interpretation of the cleavage of blastomeres. But, as shown by E.B. Wilson, in developmental processes there occur contingent outcomes of cell division which observations and experiments reveal. This induces the need to admit 'epigenetic' determinants and relativize the presumed 'preformation' of thedevelopmental phases by making room for an emergent order which the accidental circumstances of gene replication would trigger on. PMID:22542690

  12. Both CD133+ and CD133- medulloblastoma cell lines express ligands for triggering NK receptors and are susceptible to NK-mediated cytotoxicity.

    PubMed

    Castriconi, Roberta; Dondero, Alessandra; Negri, Francesca; Bellora, Francesca; Nozza, Paolo; Carnemolla, Barbara; Raso, Alessandro; Moretta, Lorenzo; Moretta, Alessandro; Bottino, Cristina

    2007-11-01

    Adoptive cellular immunotherapy has been proposed as an additional treatment of medulloblastoma, an intracranial tumor characterized by a particularly poor prognosis. However, little is known on the ability of the immune system to effectively attack this tumor. In this study, we show that activated human NK cells efficiently kill medulloblastoma cell lines in vitro. NK-mediated killing involved different activating receptors (including NKp46, NKp30, DNAM-1 and NKG2D) and correlated with the presence of their specific ligands on tumor cells. In contrast, the absence of major adhesion interactions, such as LFA-1/ICAM did not impair the NK-mediated cytotoxicity. Medulloblastoma expressed a number of tumor-associated molecules including CD146 and CD133, considered a marker for cancer stem cells. Remarkably, both CD133-positive and CD133-negative cell lines were susceptible to lysis. Tumor cells also expressed molecules that are currently used as diagnostic tools for neuroblastoma cell identification. In particular, B7 homolog 3 (B7-H3) was expressed by all the medulloblastoma cell lines analyzed, while the presence of GD(2) and NB84 was restricted to given cell lines and/or marked a defined tumor cell subset.

  13. Viral susceptibility, transfection and growth of SPB--a fish neural progenitor cell line from the brain of snubnose pompano, Trachinotus blochii (Lacépède).

    PubMed

    Wen, C-M; Ku, C-C; Wang, C-S

    2013-07-01

    This study investigates the susceptibilities of the SPB cell line to fish viruses including giant seaperch iridovirus (GSIV-K1), red sea bream iridovirus (RSIV-Ku), grouper nervous necrosis virus (GNNV-K1), chum salmon reovirus (CSV) and eel herpesvirus (HVA). GSIV-K1, RSIV-Ku and CSV replicated well in SPB cells, with a significant cytopathic effect and virus production. However, the cells were HVA and GNNV refractory. To examine the ability of SPB cells to stably express foreign protein, expression vectors encoding GNNV B1 and B2 fused to enhanced green fluorescent protein (EGFP) and GSIV ORF35L fused to DsRed were constructed and introduced by transfection into SPB cells. Stable transfectants displayed different morphologies compared with SPB and with each other. EGFP-B1 was predominantly localized in the nuclei, EFPF-B2 was distributed throughout the cytoplasm and nucleus, and granular 35L-DsRed was localized with secreted vesicles. The expression of EFPF-B2 in SPB cells produced blebs on the surface, but the cells showing stable expression of EGFP, EGFP-B1 or 35L-DsRed showed normal morphologies. Results show the SPB cells and the transfected cells grow well at temperatures between 20 and 35 °C and with serum-dependent growth. SPB cells are suitable for studies on foreign protein expression and virology.

  14. Establishment of a new cell line from the snout tissue of golden pompano Trachinotus ovatus, and its application in virus susceptibility.

    PubMed

    Yu, Y; Wei, S; Wang, Z; Huang, X; Huang, Y; Cai, J; Li, C; Qin, Q

    2016-06-01

    A new marine-fish cell line, designated GPS, was established from the snout tissue of golden pompano Trachinotus ovatus. GPS cells multiplied well in Leibovitz's L-15 containing 10% foetal bovine serum (FBS) at 28° C and the cells have been subcultured for >60 passages. Polymerase chain reaction (PCR) amplification of 16S ribosomal (r)RNA confirmed the origin of this cell line from T. ovatus. Chromosome analysis showed that GPS cells exhibited chromosomal aneuploidy with a modal chromosome number of 54. Bright green fluorescence signal was observed in enhanced green fluorescent protein (EGFP)-N3 transfected cells, indicating that GPS cells could be used to investigate gene functions in vitro. The GPS cells were highly susceptible to Singapore grouper iridovirus (SGIV), which was demonstrated by the presence of severe cytopathic effect (CPE) and increased viral titres. Real-time quantitative PCR and Western blot analysis showed that the viral gene transcription and protein synthesis occurred during SGIV infection in GPS cells. Thus, this study described the characteristic of a new cell line from the snout tissue of T. ovatus that could be used as a tool for propagation of iridovirus and genetic manipulation to investigate host-pathogen interactions.

  15. Gag-Positive Reservoir Cells Are Susceptible to HIV-Specific Cytotoxic T Lymphocyte Mediated Clearance In Vitro and Can Be Detected In Vivo

    PubMed Central

    Graf, Erin H.; Pace, Matthew J.; Peterson, Bennett A.; Lynch, Lindsay J.; Chukwulebe, Steve B.; Mexas, Angela M.; Shaheen, Farida; Martin, Jeffrey N.; Deeks, Steven G.; Connors, Mark; Migueles, Stephen A.; O’Doherty, Una

    2013-01-01

    Resting CD4+ T cells infected with HIV persist in the presence of suppressive anti-viral therapy (ART) and are barriers to a cure. One potential curative approach, therapeutic vaccination, is fueled by recognition of the ability of a subset of elite controllers (EC) to control virus without therapy due to robust anti-HIV immune responses. Controllers have low levels of integrated HIV DNA and low levels of replication competent virus, suggesting a small reservoir. As our recent data indicates some reservoir cells can produce HIV proteins (termed GPR cells for Gag-positive reservoir cells), we hypothesized that a fraction of HIV-expressing resting CD4+ T cells could be efficiently targeted and cleared in individuals who control HIV via anti-HIV cytotoxic T lymphocytes (CTL). To test this we examined if superinfected resting CD4+ T cells from EC express HIV Gag without producing infectious virus and the susceptibility of these cells to CTL. We found that resting CD4+ T cells expressed HIV Gag and were cleared by autologous CD8+ T cells from EC. Importantly, we found the extent of CTL clearance in our in vitro assay correlates with in vivo reservoir size and that a population of Gag expressing resting CD4+ T cells exists in vivo in patients well controlled on therapy. PMID:23951263

  16. MHC-matched induced pluripotent stem cells can attenuate cellular and humoral immune responses but are still susceptible to innate immunity in pigs.

    PubMed

    Mizukami, Yoshihisa; Abe, Tomoyuki; Shibata, Hiroaki; Makimura, Yukitoshi; Fujishiro, Shuh-hei; Yanase, Kimihide; Hishikawa, Shuji; Kobayashi, Eiji; Hanazono, Yutaka

    2014-01-01

    Recent studies have revealed negligible immunogenicity of induced pluripotent stem (iPS) cells in syngeneic mice and in autologous monkeys. Therefore, human iPS cells would not elicit immune responses in the autologous setting. However, given that human leukocyte antigen (HLA)-matched allogeneic iPS cells would likely be used for medical applications, a more faithful model system is needed to reflect HLA-matched allogeneic settings. Here we examined whether iPS cells induce immune responses in the swine leukocyte antigen (SLA)-matched setting. iPS cells were generated from the SLA-defined C1 strain of Clawn miniature swine, which were confirmed to develop teratomas in mice, and transplanted into the testes (n = 4) and ovary (n = 1) of C1 pigs. No teratomas were found in pigs on 47 to 125 days after transplantation. A Mixed lymphocyte reaction revealed that T-cell responses to the transplanted MHC-matched (C1) iPS cells were significantly lower compared to allogeneic cells. The humoral immune responses were also attenuated in the C1-to-C1 setting. More importantly, even MHC-matched iPS cells were susceptible to innate immunity, NK cells and serum complement. iPS cells lacked the expression of SLA class I and sialic acids. The in vitro cytotoxic assay showed that C1 iPS cells were targeted by NK cells and serum complement of C1. In vivo, the C1 iPS cells developed larger teratomas in NK-deficient NOG (T-B-NK-) mice (n = 10) than in NK-competent NOD/SCID (T-B-NK+) mice (n = 8) (p<0.01). In addition, C1 iPS cell failed to form teratomas after incubation with the porcine complement-active serum. Taken together, MHC-matched iPS cells can attenuate cellular and humoral immune responses, but still susceptible to innate immunity in pigs.

  17. Galvanic Cells and the Determination of Equilibrium Constants

    ERIC Educational Resources Information Center

    Brosmer, Jonathan L.; Peters, Dennis G.

    2012-01-01

    Readily assembled mini-galvanic cells can be employed to compare their observed voltages with those predicted from the Nernst equation and to determine solubility products for silver halides and overall formation constants for metal-ammonia complexes. Results obtained by students in both an honors-level first-year course in general chemistry and…

  18. Nodavirus infection induces a great innate cell-mediated cytotoxic activity in resistant, gilthead seabream, and susceptible, European sea bass, teleost fish.

    PubMed

    Chaves-Pozo, Elena; Guardiola, Francisco A; Meseguer, José; Esteban, María A; Cuesta, Alberto

    2012-11-01

    Viral nervous necrosis (VNN) virus produces great mortalities in fish having susceptible and reservoir species between the most important marine aquaculture species. Cell-mediated cytotoxicity (CMC) is considered, towards the interferon (IFN), the most important mechanism of the immune response to fight against viral infections but it has been very scarcely evaluated. We aimed to evaluate the effects of VNNV infection in the reservoir gilthead seabream (Sparus aurata) and susceptible European sea bass (Dicentrarchus labrax). Firstly, after experimental infection we found mortalities in the sea bass (55%) but no in the seabream. Moreover, VNN virus replicates in the brain of both species as it was reflected by the high up-regulation of the Mx gene expression. Interestingly, the head-kidney leucocyte cell-mediated cytotoxic activity was significantly increased in both species reaching highest activity at 7 days: 3.65- and 2.7-fold increase in seabream and sea bass, respectively. This is supported by the significant up-regulation of the non-specific cytotoxic cell receptor (NCCRP-1) in the two fish species. By contrast, phagocytosis was unaffected in both species. The respiratory burst was increased in seabream 7 days post-infection whilst in sea bass this activity was significantly decreased at days 7 and 15. Our results demonstrate the significance of the CMC activity in both gilthead seabream and European sea bass against nodavirus infections but further studies are still needed to understand the role of cytotoxic cells in the antiviral immune response and the mechanisms involved in either reservoir or susceptible fish species. PMID:22981914

  19. The Fatty Acid Signaling Molecule cis-2-Decenoic Acid Increases Metabolic Activity and Reverts Persister Cells to an Antimicrobial-Susceptible State

    PubMed Central

    Morozov, Aleksey; Planzos, Penny; Zelaya, Hector M.

    2014-01-01

    Persister cells, which are tolerant to antimicrobials, contribute to biofilm recalcitrance to therapeutic agents. In turn, the ability to kill persister cells is believed to significantly improve efforts in eradicating biofilm-related, chronic infections. While much research has focused on elucidating the mechanism(s) by which persister cells form, little is known about the mechanism or factors that enable persister cells to revert to an active and susceptible state. Here, we demonstrate that cis-2-decenoic acid (cis-DA), a fatty acid signaling molecule, is able to change the status of Pseudomonas aeruginosa and Escherichia coli persister cells from a dormant to a metabolically active state without an increase in cell number. This cell awakening is supported by an increase of the persister cells' respiratory activity together with changes in protein abundance and increases of the transcript expression levels of several metabolic markers, including acpP, 16S rRNA, atpH, and ppx. Given that most antimicrobials target actively growing cells, we also explored the effect of cis-DA on enhancing antibiotic efficacy in killing persister cells due to their inability to keep a persister cell state. Compared to antimicrobial treatment alone, combinational treatments of persister cell subpopulations with antimicrobials and cis-DA resulted in a significantly greater decrease in cell viability. In addition, the presence of cis-DA led to a decrease in the number of persister cells isolated. We thus demonstrate the ability of a fatty acid signaling molecule to revert bacterial cells from a tolerant phenotype to a metabolically active, antimicrobial-sensitive state. PMID:25192989

  20. Direct determination of phosphatase activity from physiological substrates in cells.

    PubMed

    Ren, Zhongyuan; Do, Le Duy; Bechkoff, Géraldine; Mebarek, Saida; Keloglu, Nermin; Ahamada, Saandia; Meena, Saurabh; Magne, David; Pikula, Slawomir; Wu, Yuqing; Buchet, René

    2015-01-01

    A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min(-1) mg(-1) for PPi, to 56 ± 11 nmol min(-1) mg(-1) for AMP, to 79 ± 23 nmol min(-1) mg(-1) for beta-glycerophosphate and to 73 ± 15 nmol min(-1) mg(-1) for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole--a TNAP inhibitor--served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC50 value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes. PMID:25785438

  1. Direct determination of phosphatase activity from physiological substrates in cells.

    PubMed

    Ren, Zhongyuan; Do, Le Duy; Bechkoff, Géraldine; Mebarek, Saida; Keloglu, Nermin; Ahamada, Saandia; Meena, Saurabh; Magne, David; Pikula, Slawomir; Wu, Yuqing; Buchet, René

    2015-01-01

    A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min(-1) mg(-1) for PPi, to 56 ± 11 nmol min(-1) mg(-1) for AMP, to 79 ± 23 nmol min(-1) mg(-1) for beta-glycerophosphate and to 73 ± 15 nmol min(-1) mg(-1) for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole--a TNAP inhibitor--served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC50 value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.

  2. Increased cellular apoptosis susceptibility (CSE1L/CAS) protein expression promotes protrusion extension and enhances migration of MCF-7 breast cancer cells

    SciTech Connect

    Tai, Cheng-Jeng; Shen, Shing-Chuan; Lee, Woan-Ruoh; Liao, Ching-Fong; Deng, Win-Ping; Chiou, Hung-Yi; Hsieh, Cheng-I; Tung, Jai-Nien; Chen, Ching-Shyang; Chiou, Jeng-Fong; Li, Li-Tzu; Lin, Chuang-Yu; Hsu, Chung-Huei; Jiang, Ming-Chung

    2010-10-15

    Microtubules are part of cell structures that play a role in regulating the migration of cancer cells. The cellular apoptosis susceptibility (CSE1L/CAS) protein is a microtubule-associated protein that is highly expressed in cancer. We report here that CSE1L regulates the association of {alpha}-tubulin with {beta}-tubulin and promotes the migration of MCF-7 breast cancer cells. CSE1L was associated with {alpha}-tubulin and {beta}-tubulin in GST (glutathione S-transferase) pull-down and immunoprecipitation assays. CSE1L-GFP (green fluorescence protein) fusion protein experiments showed that the N-terminal of CSE1L interacted with microtubules. Increased CSE1L expression resulted in decreased tyrosine phosphorylation of {alpha}-tubulin and {beta}-tubulin, increased {alpha}-tubulin and {beta}-tubulin association, and enhanced assembly of microtubules. Cell protrusions or pseudopodia are temporary extensions of the plasma membrane and are implicated in cancer cell migration and invasion. Increased CSE1L expression increased the extension of MCF-7 cell protrusions. In vitro migration assay showed that enhanced CSE1L expression increased the migration of MCF-7 cells. Our results indicate that CSE1L plays a role in regulating the extension of cell protrusions and promotes the migration of cancer cells.

  3. Restoration of Susceptibility of Intracellular Methicillin-Resistant Staphylococcus aureus to β-Lactams: Comparison of Strains, Cells, and Antibiotics▿ †

    PubMed Central

    Lemaire, Sandrine; Olivier, Aurélie; Van Bambeke, Françoise; Tulkens, Paul M.; Appelbaum, Peter C.; Glupczynski, Youri

    2008-01-01

    Staphylococcus aureus invades eukaryotic cells. When methicillin-resistant S. aureus (MRSA) ATCC 33591 is phagocytized by human THP-1 macrophages, complete restoration of susceptibility to cloxacillin and meropenem is shown and the strain becomes indistinguishable from MSSA ATCC 25923 due to the acid pH prevailing in phagolysosomes (S. Lemaire et al., Antimicrob. Agents Chemother. 51:1627-1632, 2007). We examined whether this observation can be extended to (i) strains of current clinical and epidemiological interest (three hospital-acquired MRSA [HA-MRSA] strains, two community-acquired MRSA [CA-MRSA] strains, two HA-MRSA strains with the vancomycin-intermediate phenotype, one HA-MRSA strain with the vancomycin-resistant phenotype, and one animal [porcine] MRSA strain), (ii) activated THP-1 cells and nonprofessional phagocytes (keratinocytes, Calu-3 bronchial epithelial cells), and (iii) other β-lactams (imipenem, oxacillin, cefuroxime, cefepime). All strains showed (i) a marked reduction in MICs in broth at pH 5.5 compared with the MIC at pH 7.4 and (ii) sigmoidal dose-response curves with cloxacillin (0.01× to 100× MIC, 24 h of incubation) after phagocytosis by THP-1 macrophages that were indistinguishable from each other and from the dose-response curve for methicillin-susceptible S. aureus (MSSA) ATCC 25923 (relative potency [50% effect], 6.09× MIC [95% confidence interval {CI}, 4.50 to 8.25]; relative efficacy [change in bacterial counts over the original inoculum for an infinitely large cloxacillin concentration, or maximal effect], −0.69 log CFU [95% CI, −0.79 to −0.58]). Similar dose-response curves for cloxacillin were also observed with MSSA ATCC 25923 and MRSA ATCC 33591 after phagocytosis by activated THP-1 macrophages, keratinocytes, and Calu-3 cells. By contrast, there was a lower level of restoration of susceptibility of MRSA ATCC 33591 to cefuroxime and cefepime after phagocytosis by THP-1 macrophages, even when the data were normalized

  4. Disparate effects of depletion of CD1d-reactive T cells during early versus late stages of disease in a genetically susceptible model of lupus.

    PubMed

    Jacinto, J; Kim, P J; Singh, R R

    2012-04-01

    Some T cells react with lipid antigens bound to antigen-presenting molecule CD1d. Numbers and functions of a subset of such lipid-reactive T cells are reduced in patients with systemic lupus erythematosus (SLE) and their relatives, as well as in genetically susceptible and chemically induced animal models of lupus-like disease. We have reported that the germline deletion of CD1d exacerbates lupus, suggesting a protective role of these cells in the development of lupus. The use of a knockout mouse model in this study, however, did not allow examination of the role of these cells at different stages of disease. Here, we describe an approach to deplete CD1d-dependent T cells, which allowed us to investigate the role of these cells at different stages of disease in genetically lupus-prone NZB/NZW F1 (BWF1) mice. Repeated intravenous injections of large numbers of CD1d-transfected cells resulted in ∼50-75% reduction in these cells, as defined by the expression of CD4, NK1.1 and CD122, and lack of expression of CD62 ligand. TCR γδ (+)NK1.1(+) cells were also reduced in the recipients of CD1d-transfected cells as compared with control recipients. Such depletion of CD1d-reactive T cells in preclinical BWF1 mice resulted in disease acceleration with a significant increase in proteinuria and mortality. In older BWF1 mice having advanced nephritis, however, such depletion of CD1d-reactive T cells resulted in some disease improvement. Taken together, these data as well as our published studies suggest that CD1d-reactive T cells protect against the development of lupus in animal models. However, these cells appear to be unable to suppress established lupus nephritis in these animals, and might even play a disease aggravating role in late stages of disease.

  5. Cell-Type Specific Determinants of NRAMP1 Expression in Professional Phagocytes

    PubMed Central

    Cellier, Mathieu F. M.

    2013-01-01

    The Natural resistance-associated macrophage protein 1 (Nramp1 or Solute carrier 11 member 1, Slc11a1) transports divalent metals across the membrane of late endosomes and lysosomes in professional phagocytes. Nramp1 represents an ancient eukaryotic cell-autonomous defense whereas the gene duplication that yielded Nramp1 and Nramp2 predated the origin of Sarcopterygians (lobe-finned fishes and tetrapods). SLC11A1 genetic polymorphisms associated with human resistance to tuberculosis consist of potential regulatory variants. Herein, current knowledge of the regulation of SLC11A1 gene expression is reviewed and comprehensive analysis of ENCODE data available for hematopoietic cell-types suggests a hypothesis for the regulation of SLC11A1 expression during myeloid development and phagocyte functional polarization. SLC11A1 is part of a 34.6 kb CTCF-insulated locus scattered with predicted regulatory elements: a 3' enhancer, a large 5' enhancer domain and four elements spread around the transcription start site (TSS), including several C/EBP and PU.1 sites. SLC11A1 locus ends appear mobilized by ETS-related factors early during myelopoiesis; activation of both 5' and 3' enhancers in myelo-monocytic cells correlate with transcription factor binding at the TSS. Characterizing the corresponding cis/trans determinants functionally will establish the mechanisms involved and possibly reveal genetic variation that impacts susceptibility to infectious or immune diseases. PMID:24832660

  6. Growth maintenance of the maize primary root at low water potentials involves increases in cell-wall extension properties, expansin activity, and wall susceptibility to expansins.

    PubMed Central

    Wu, Y; Sharp, R E; Durachko, D M; Cosgrove, D J

    1996-01-01

    Previous work on the growth biophysics of maize (Zea mays L.) primary roots suggested that cell walls in the apical 5 mm of the elongation zone increased their yielding ability as an adaptive response to low turgor and water potential (psi w). To test this hypothesis more directly, we measured the acid-induced extension of isolated walls from roots grown at high (-0.03 MPa) or low (-1.6 MPa) psi w using an extensometer. Acid-induced extension was greatly increased in the apical 5 mm and was largely eliminated in the 5- to 10-mm region of roots grown at low psi w. This pattern is consistent with the maintenance of elongation toward the apex and the shortening of the elongation zone in these roots. Wall proteins extracted from the elongation zone possessed expansin activity, which increased substantially in roots grown at low psi w. Western blots likewise indicated higher expansin abundance in the roots at low psi w. Additionally, the susceptibility of walls to expansin action was higher in the apical 5 mm of roots at low psi w than in roots at high psi w. The basal region of the elongation zone (5-10 mm) did not extend in response to expansins, indicating that loss of susceptibility to expansins was associated with growth cessation in this region. Our results indicate that both the increase in expansin activity and the increase in cell-wall susceptibility to expansins play a role in enhancing cell-wall yielding and, therefore, in maintaining elongation in the apical region of maize primary roots at low psi w. PMID:11536740

  7. Dendritic Cells from HIV Controllers Have Low Susceptibility to HIV-1 Infection In Vitro but High Capacity to Capture HIV-1 Particles.

    PubMed

    Hamimi, Chiraz; David, Annie; Versmisse, Pierre; Weiss, Laurence; Bruel, Timothée; Zucman, David; Appay, Victor; Moris, Arnaud; Ungeheuer, Marie-Noëlle; Lascoux-Combe, Caroline; Barré-Sinoussi, Françoise; Muller-Trutwin, Michaela; Boufassa, Faroudy; Lambotte, Olivier; Pancino, Gianfranco; Sáez-Cirión, Asier

    2016-01-01

    HIV controllers (HICs), rare HIV-1 infected individuals able to control viral replication without antiretroviral therapy, are characterized by an efficient polyfunctional and cytolytic HIV-specific CD8+ T cell response. The mechanisms underlying the induction and maintenance of such response in many HICs despite controlled viremia are not clear. Dendritic cells play a crucial role in the generation and reactivation of T cell responses but scarce information is available on those cells in HICs. We found that monocyte derived dendritic cells (MDDCs) from HICs are less permissive to HIV-1 infection than cells from healthy donors. In contrast MDDCs from HICs are particularly efficient at capturing HIV-1 particles when compared to cells from healthy donors or HIV-1 patients with suppressed viral load on antiretroviral treatment. MDDCs from HICs expressed on their surface high levels of syndecan-3, DC-SIGN and MMR, which could cooperate to facilitate HIV-1 capture. The combination of low susceptibility to HIV-1 infection but enhanced capacity to capture particles might allow MDDCs from HICs to preserve their function from the deleterious effect of infection while facilitating induction of HIV-specific CD8+ T cells by cross-presentation in a context of low viremia. PMID:27505169

  8. Dendritic Cells from HIV Controllers Have Low Susceptibility to HIV-1 Infection In Vitro but High Capacity to Capture HIV-1 Particles

    PubMed Central

    Hamimi, Chiraz; David, Annie; Versmisse, Pierre; Weiss, Laurence; Bruel, Timothée; Zucman, David; Appay, Victor; Moris, Arnaud; Ungeheuer, Marie-Noëlle; Lascoux-Combe, Caroline; Barré-Sinoussi, Françoise; Muller-Trutwin, Michaela; Boufassa, Faroudy; Lambotte, Olivier; Pancino, Gianfranco; Sáez-Cirión, Asier

    2016-01-01

    HIV controllers (HICs), rare HIV-1 infected individuals able to control viral replication without antiretroviral therapy, are characterized by an efficient polyfunctional and cytolytic HIV-specific CD8+ T cell response. The mechanisms underlying the induction and maintenance of such response in many HICs despite controlled viremia are not clear. Dendritic cells play a crucial role in the generation and reactivation of T cell responses but scarce information is available on those cells in HICs. We found that monocyte derived dendritic cells (MDDCs) from HICs are less permissive to HIV-1 infection than cells from healthy donors. In contrast MDDCs from HICs are particularly efficient at capturing HIV-1 particles when compared to cells from healthy donors or HIV-1 patients with suppressed viral load on antiretroviral treatment. MDDCs from HICs expressed on their surface high levels of syndecan-3, DC-SIGN and MMR, which could cooperate to facilitate HIV-1 capture. The combination of low susceptibility to HIV-1 infection but enhanced capacity to capture particles might allow MDDCs from HICs to preserve their function from the deleterious effect of infection while facilitating induction of HIV-specific CD8+ T cells by cross-presentation in a context of low viremia. PMID:27505169

  9. Establishment of a new cell line from the heart of giant grouper, Epinephelus lanceolatus (Bloch), and its application in toxicology and virus susceptibility.

    PubMed

    Guo, C Y; Huang, Y H; Wei, S N; Ouyang, Z L; Yan, Y; Huang, X H; Qin, Q W

    2015-02-01

    A new marine fish cell line, derived from the heart of giant grouper, Epinephelus lanceolatus (Bloch), was established and characterized. The cell line was designated as ELGH and subcultured with more than 60 passages. The ELGH cells were mainly composed of fibroblast-like cells and multiplied well in Leibovitz's L-15 medium supplemented with 10% foetal bovine serum (FBS) at 28 °C. Chromosome analysis indicated that the modal chromosome number was 48. The fluorescent signals were detected in ELGH when transfected with green fluorescent protein reporter plasmids. The 50% cytotoxic concentration (CC50 ) of the extracellular products (ECPs) from Streptococcus iniae and Vibrio alginolyticus E333 on ELGH cells was 60.02 and 12.49 μg mL(-1), respectively. Moreover, the ELGH cells showed susceptibility to Singapore grouper iridovirus (SGIV), but not to soft-shelled turtle iridovirus (STIV), red-spotted grouper nervous necrosis virus (RGNNV) and spring viremia of carp virus (SVCV), which was demonstrated by the presence of a severe cytopathic effect (CPE) and increased viral titres. In addition, electron microscopy observation showed that abundant virus particles were present in the infected cells. Taken together, our data above provided the potential utility of ELGH cells for transgenic and genetic manipulation, as well as cytotoxicity testing and virus pathogenesis.

  10. RB-resistant Abl kinase induces delayed cell cycle progression and increases susceptibility to apoptosis upon cellular stresses through interaction with p53.

    PubMed

    Cho, Jae-We; Chung, Junah; Baek, Won-Ki; Suh, Seong-Il; Kwon, Taeg Kyu; Park, Jong-Wook; Suh, Min-Ho

    2003-06-01

    c-Abl, a non-receptor tyrosine kinase, is found in both nucleus and cytoplasm of proliferating fibroblasts. RB negatively regulates the kinase activity of c-Abl. Overexpression of kinase active c-Abl can overcome RB-induced growth arrest in Saos-2 cells. However, we previously reported that disruption of the RB matchmaker function leads to delayed cell cycle progression in the presence of p53. In this study, we investigated whether overexpression of mutant c-Abl (AS2, RB-resistant Abl kinase) not only lead to delayed cell cycle progression but also make cells susceptible to apoptosis under coexpression with a fragment of RB C pocket in human skin fibroblast. AS2 expressing cells showed delayed cell growth rate in normal growth condition. After genotoxic stress such as etoposide treatment, AS2 expressing cells readily progressed into apoptosis through p53 and caspase-3 activations. Our results suggest that expression of AS2 not only induces delayed cell cycle progression but also results in increased sensitivity to apoptosis in the presence of p53. PMID:12738983

  11. Polymorphisms of cell cycle regulator genes CCND1 G870A and TP53 C215G: Association with colorectal cancer susceptibility risk in a Malaysian population

    PubMed Central

    ZAHARY, MOHD NIZAM; AHMAD AIZAT, ABDUL AZIZ; KAUR, GURJEET; YEONG YEH, LEE; MAZUWIN, MAYA; ANKATHIL, RAVINDRAN

    2015-01-01

    Colorectal cancer (CRC) occurs as a more common sporadic form and a less common familial form. Our earlier analysis of germline mutations of mismatch repair genes confirmed only 32% of familial CRC cases as Lynch syndrome cases. It was hypothesized that the remaining familial aggregation may be ‘polygenic’ due to single nucleotide polymorphisms (SNPs) of low penetrance genes involved in cancer predisposition pathways, such as cell cycle regulation and apoptosis pathways. The current case-control study involving 104 CRC patients (52 sporadic and 52 familial) and 104 normal healthy controls investigated the contribution of the SNPs cyclin D1 (CCND1) G870A and tumor protein p53 (TP53) C215G in modulating familial and sporadic CRC susceptibility risk. DNA was extracted from peripheral blood and the polymorphisms were genotyped by employing a polymerase chain reaction-restriction fragment length polymorphism method. The association between these polymorphisms and CRC susceptibility risk was calculated using a binary logistic regression analysis and deriving odds ratios (ORs). The A/A variant genotype of CCND1 and G/G variant genotype of TP53 exhibited a significantly greater association with the risk of sporadic CRC [CCND1: OR, 3.471; 95% confidence interval (CI), 1.443–8.350; P=0.005. TP53: OR, 2.829; CI, 1.119–7.152; P=0.026] as well as familial CRC susceptibility (CCND1: OR, 3.086; CI, 1.270–7.497; P=0.019. TP53: OR, 3.048; CI, 1.147–8.097; P=0.030). The results suggest a potential role of the SNPs CCND1 G870A and TP53 C215G in the modulation of sporadic and familial CRC susceptibility risk. PMID:26722315

  12. Determination of optimum electrolyte composition for molten carbonate fuel cells

    SciTech Connect

    Yuh, C.Y.; Pigeaud, A.

    1987-01-01

    The objective of this study is to determine the optimum electrolyte composition for molten carbonate fuel cells. To accomplish this, the contractor will provide: (1) Comprehensive reports of on-going efforts to optimize carbonate composition. (2) A list of characteristics affected by electrolyte composition variations (e.g. ionic conductivity, vapor pressure, melting range, gas solubility, exchange current densities on NiO, corrosion and cathode dissolution effects). (3) Assessment of the overall effects that these characteristics have on state-of-the-art cell voltage and lifetime.

  13. Determination of optimum electrolyte composition for molten carbonate fuel cells

    SciTech Connect

    Yuh, C.Y.; Pigeaud, A.

    1988-03-01

    The objective of this study is to determine the optimum electrolyte composition for molten carbonate fuel cells. To accomplish this, the contractor will provide: (1) Comprehensive reports of on-going efforts to optimize carbonate composition. (2) A list of characteristics affected by electrolyte composition variations (e.g. ionic conductivity, vapor pressure, melting range, gas solubility, exchange current densities on NiO, corrosion and cathode dissolution effects). (3) Assessment of the overall effects that these characteristics have on state-of-the-art cell voltage and lifetime.

  14. Determination of optimum electrolyte composition for molten carbonate fuel cells

    SciTech Connect

    Yuh, C.Y.; Pigeaud, A.

    1987-01-01

    The objective of this study is to determine the optimum electrolyte composition for molten carbonate fuel cells. To accomplish this, the contractor will provide: (1) Comprehensive reports of on-going efforts to optimize carbonate composition. (2) A list of characteristics affected by electrolyte composition variations (e.g. ionic conductivity, vapor pressure, melting range, gas solubility, exchange current densities on NiO, corrosion and cathode dissolution effects). (3) Assessment of the overall effects that these characteristics have state-of-the-art cell voltage and lifetime.

  15. Determination of optimum electrolyte composition for molten carbonate fuel cells

    SciTech Connect

    Yuh, C.Y.; Pigeaud, A.

    1988-06-01

    The objective of this study is to determine the optimum electrolyte composition for molten carbonate fuel cells. To accomplish this, the contractor will provide: (1) Comprehensive reports of on-going efforts to optimize carbonate composition. (2) A list of characteristics affected by electrolyte composition variations (e.g. ionic conductivity, vapor pressure, melting range, gas solubility, exchange current densities on NiO, corrosion and cathode dissolution effects). (3) Assessment of the overall effects that these characteristics have on state-of-the-art cell voltage and lifetime.

  16. Determination of nematics material parameters by measurements of Fredericksz transitions in crossed electric and magnetic fields in wedge cell

    NASA Astrophysics Data System (ADS)

    Kedzierski, Jerzy; Raszewski, Zbigniew; Kojdecki, Marek A.; Miszczyk, Emilia; Perkowski, Pawel; Piecek, Wiktor; Lipinska, Ludwika

    2004-09-01

    Wedge cells of the wedge angle of order of few milliradians were used to measure the threshold voltages for the Freedericksz transition in electric and magnetic fields crossed. A nematic liquid crystal, PCB, filling cells was of planar orientation enforced by the treatment of the flat boundary plates, covered with poly(amic acid) PM9. A system of interference fringes appeared in each cell placed in normally incident light between analyzer and polarizer crossed. In the vicinity of each fringe a cell could be considered as a flat-parallel one and hence it was equivalent to the system of flat cells of different precisely determined thickness. Threshold voltages, parameterized by magnetic field magnitudes were interpreted as the eigenvalues of the boundary eigenvalue problem for the operator of the second derivative; the interaction between the nematics and the substrates was assumed very strong with the anchoring direction parallel to the cell boundaries. The resulting formulae were used to determine the anisotropy of diamagnetic susceptibility and the splay elastic constant of PCB after the threshold magnitudes measured. The estimates of material parameters agreed well with those determined by the composite method.

  17. Receptor-specific regulation of B-cell susceptibility to Fas-mediated apoptosis and a novel Fas apoptosis inhibitory molecule.

    PubMed

    Rothstein, T L; Zhong, X; Schram, B R; Negm, R S; Donohoe, T J; Cabral, D S; Foote, L C; Schneider, T J

    2000-08-01

    The susceptibility of primary B cells to Fas (APO-1, CD95)-mediated apoptosis is modulated by signals derived from additional surface receptors: CD40 engagement produces upregulation of Fas expression and marked sensitivity to Fas-induced cell death, whereas antigen receptor engagement, or interleukin-4 receptor (IL-4R) engagement, inhibits Fas killing and thereby produces Fas resistance, even in otherwise susceptible, CD40-stimulated targets. Surface immunoglobulin (sIg) and IL-4R utilize distinct signaling pathways to produce Fas resistance that rely on protein kinase C and signal transducer and activator of transcription 6, respectively sIg signaling for inducible Fas resistance requires nuclear factor-kappaB and depends on new macromolecular synthesis. Proximate mediators for Fas resistance include the known anti-apoptotic gene products Bcl-xL and FLIP (but not Btk), and a novel anti-apoptotic gene that encodes Fas apoptosis inhibitory molecule (FAIM). FAIM was identified by differential display and was cloned as two alternatively spliced forms: FAIM-S is broadly expressed, whereas faim-L expression is tissue specific. faim is highly evolutionarily conserved, suggesting an important function throughout phylogeny. Inducible resistance to Fas-mediated apoptosis is speculated to protect antigen-specific B cells during potentially dangerous interactions with FasL-bearing T cells; the elevated sIg-signaling threshold for inducible Fas resistance in autoreactive, tolerant B cells would insure against autoimmunity. However, aberrant acquisition of Fas resistance may allow autoreactive B cells to escape Fas deletion and malignant lymphocytes to thwart antitumor immunity.

  18. Multi-walled carbon nanotube length as a critical determinant of bioreactivity with primary human pulmonary alveolar cells

    PubMed Central

    Sweeney, Sinbad; Berhanu, Deborah; Misra, Superb K.; Thorley, Andrew J.; Valsami-Jones, Eugenia; Tetley, Teresa D.

    2015-01-01

    Multiwalled carbon nanotube (MWCNT) length is suggested to critically determine their pulmonary toxicity. This stems from in vitro and in vivo rodent studies and in vitro human studies using cell lines (typically cancerous). There is little data using primary human lung cells. We addressed this knowledge gap, using highly relevant, primary human alveolar cell models exposed to precisely synthesized and thoroughly characterized MWCNTs. In this work, transformed human alveolar type-I-like epithelial cells (TT1), primary human alveolar type-II epithelial cells (ATII) and alveolar macrophages (AM) were treated with increasing concentrations of MWCNTs before measuring cytotoxicity, inflammatory mediator release and MAP kinase signalling. Strikingly, we observed that short MWCNTs (~0.6 µm in length) induced significantly greater responses from the epithelial cells, whilst AM were particularly susceptible to long MWCNTs (~20 µm). These differences in the pattern of mediator release were associated with alternative profiles of JNK, p38 and ERK1/2 MAP kinase signal transduction within each cell type. This study, using highly relevant target human alveolar cells and well defined and characterized MWCNTs, shows marked cellular responses to the MWCNTs that vary according to the target cell type, as well as the aspect ratio of the MWCNT. PMID:25780270

  19. Susceptibility of pathogenic and nonpathogenic Naegleria ssp

    SciTech Connect

    Whiteman, L.Y.

    1988-01-01

    The susceptibility of four species of Naegleria amoebae to complement-mediated lysis was determined. The amoebicidal activity of normal human serum (NHS) and normal guinea pig serum (NGPS) for Naegleria amoebae was measured by an in vitro cytotoxicity assay. Release of radioactivity from amoebae labeled with {sup 3}H-uridine and visual observation with a compound microscope were used as indices of lysis. Susceptibility or resistance to complement-mediated lysis in vitro correlated with the in vivo pathogenic potential. Nonpathogenic Naegleria amoebae were lysed at a faster rate and at higher cell concentrations than were pathogenic amoebae. Electrophoretic analysis of NHS incubated with pathogenic or nonpathogenic Naegleria spp. demonstrated that amoebae activate the complement cascade resulting in the production of C3 and C5 complement cleavage products. Treatment with papain or trypsin for 1 h, but not with sialidase, increase the susceptibility of highly pathogenic, mouse-passaged N. fowleri to lysis. Treatment with actinomycin D, cycloheximide or various protease inhibitors for 4 h did not increase susceptibility to lysis. Neither a repair process involving de novo protein synthesis nor a complement-inactivating protease appear to account for the increase resistance of N. fowleri amoebae to complement-mediated lysis. A binding study with {sup 125}I radiolabeled C9 indicated that the terminal complement component does not remain stably bound to the membrane of pathogenic amoebae.

  20. Lysosome biogenesis/scattering increases host cell susceptibility to invasion by Trypanosoma cruzi metacyclic forms and resistance to tissue culture trypomastigotes

    PubMed Central

    Cortez, Cristian; Real, Fernando

    2015-01-01

    Summary A fundamental question to be clarified concerning the host cell invasion by Trypanosoma cruzi is whether the insect‐borne and mammalian‐stage parasites use similar mechanisms for invasion. To address that question, we analysed the cell invasion capacity of metacyclic trypomastigotes (MT) and tissue culture trypomastigotes (TCT) under diverse conditions. Incubation of parasites for 1 h with HeLa cells in nutrient‐deprived medium, a condition that triggered lysosome biogenesis and scattering, increased MT invasion and reduced TCT entry into cells. Sucrose‐induced lysosome biogenesis increased HeLa cell susceptibility to MT and resistance to TCT. Treatment of cells with rapamycin, which inhibits mammalian target of rapamycin (mTOR), induced perinuclear lysosome accumulation and reduced MT invasion while augmenting TCT invasion. Metacylic trypomastigotes, but not TCT, induced mTOR dephosphorylation and the nuclear translocation of transcription factor EB (TFEB), a mTOR‐associated lysosome biogenesis regulator. Lysosome biogenesis/scattering was stimulated upon HeLa cell interaction with MT but not with TCT. Recently, internalized MT, but not TCT, were surrounded by colocalized lysosome marker LAMP2 and mTOR. The recombinant gp82 protein, the MT‐specific surface molecule that mediates invasion, induced mTOR dephosphorylation, nuclear TFEB translocation and lysosome biogenesis/scattering. Taken together, our data clearly indicate that MT invasion is mainly lysosome‐dependent, whereas TCT entry is predominantly lysosome‐independent. PMID:26572924