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Sample records for mucin-domain-containing molecule-3 tim-3

  1. Upregulation of T-cell Immunoglobulin and Mucin-Domain Containing-3 (Tim-3) in Monocytes/Macrophages Associates with Gastric Cancer Progression.

    PubMed

    Wang, Zhenxin; Yin, Ni; Zhang, Zixiang; Zhang, Yi; Zhang, Guangbo; Chen, Weichang

    2017-02-01

    T-cell immunoglobulin and mucin-domain containing-3 (Tim-3) is an important immune regulatory molecule in cancer immune system. However, expression and function of Tim-3 in monocytes/macrophages in cancer progression mainly remain unclear. In this study, we analyzed Tim-3 levels in peripheral blood mononuclear cells (PBMCs) from 62 gastric cancer patients and 45 healthy controls using flow cytometry and then associated Tim-3 levels with clinical pathological data from patients. We found Tim-3 level was significantly upregulated in monocytes from gastric cancer patients compared with those from healthy controls, and that upregulated Tim-3 levels associated with depth of tumor invasion and tumor lymph node metastasis and advanced clinical stages of gastric cancer patients. Furthermore, tumor-bearing mouse experiments revealed that Tim-3 level on monocytes/macrophages associated with xenograft formation and growth. In addition, culture of monocytes from healthy controls with gastric cancer cell-conditioned medium upregulated Tim-3 expression, but IL-10, TNF-α, IFN-γ, or GM-CSF treatment or T-bet, Eomes, and T-bet/Eomes double gene knockout did not affect Tim-3 levels in blood monocytes/macrophages from human or mouse, respectively. Gal-9/Tim-3 signal was able to significantly stimulate monocyte to secrete IL-6, IL-8, and IL-10, but not IL-1β, IL-12p70, or TNF-α in presence of LPS. In conclusion, our study demonstrated that Tim-3 expressed by monocyte/macrophages might be an important mechanism in gastric cancer progression.

  2. Expansion of dysfunctional Tim-3-expressing effector memory CD8+ T cells during simian immunodeficiency virus infection in rhesus macaques.

    PubMed

    Fujita, Tsuyoshi; Burwitz, Benjamin J; Chew, Glen M; Reed, Jason S; Pathak, Reesab; Seger, Elizabeth; Clayton, Kiera L; Rini, James M; Ostrowski, Mario A; Ishii, Naoto; Kuroda, Marcelo J; Hansen, Scott G; Sacha, Jonah B; Ndhlovu, Lishomwa C

    2014-12-01

    The T cell Ig- and mucin domain-containing molecule-3 (Tim-3) negative immune checkpoint receptor demarcates functionally exhausted CD8(+) T cells arising from chronic stimulation in viral infections like HIV. Tim-3 blockade leads to improved antiviral CD8(+) T cell responses in vitro and, therefore, represents a novel intervention strategy to restore T cell function in vivo and protect from disease progression. However, the Tim-3 pathway in the physiologically relevant rhesus macaque SIV model of AIDS remains uncharacterized. We report that Tim-3(+)CD8(+) T cell frequencies are significantly increased in lymph nodes, but not in peripheral blood, in SIV-infected animals. Tim-3(+)PD-1(+)CD8(+) T cells are similarly increased during SIV infection and positively correlate with SIV plasma viremia. Tim-3 expression was found primarily on effector memory CD8(+) T cells in all tissues examined. Tim-3(+)CD8(+) T cells have lower Ki-67 content and minimal cytokine responses to SIV compared with Tim-3(-)CD8(+) T cells. During acute-phase SIV replication, Tim-3 expression peaked on SIV-specific CD8(+) T cells by 2 wk postinfection and then rapidly diminished, irrespective of mutational escape of cognate Ag, suggesting non-TCR-driven mechanisms for Tim-3 expression. Thus, rhesus Tim-3 in SIV infection partially mimics human Tim-3 in HIV infection and may serve as a novel model for targeted studies focused on rejuvenating HIV-specific CD8(+) T cell responses.

  3. Increased Tim-3 expression in peripheral NK cells predicts a poorer prognosis and Tim-3 blockade improves NK cell-mediated cytotoxicity in human lung adenocarcinoma.

    PubMed

    Xu, Liyun; Huang, Yanyan; Tan, Linlin; Yu, Wei; Chen, Dongdong; Lu, ChangChang; He, Jianying; Wu, Guoqing; Liu, Xiaoguang; Zhang, Yongkui

    2015-12-01

    T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) has been shown to play an important role in mediating NK-cell function in human diseases. However, the relationship between Tim-3 expression in natural killer (NK) cells and human lung adenocarcinoma remains unclear. We therefore investigated the expression of Tim-3 in NK cells and explored the effect of Tim-3 blockade on NK cell-mediated activity in human lung adenocarcinoma. Upregulated expression of Tim-3 on CD3-CD56+ cells (P<0.05) and CD3-CD56(dim) cells (P<0.05) of patients with lung adenocarcinoma was detected by flow cytometry. Moreover, Tim-3 expression in CD3-CD56+ NK cells was higher in patients with lung adenocarcinoma with lymph node metastasis (LNM) (P<0.05) or with tumor stage T3-T4 (P<0.05). Tim-3 expression in CD56(dim) NK-cell subset was higher in patients with tumor size ≥3cm (P<0.05), or LNM (P<0.05) or with tumor stage T3-T4 (P<0.05). Further analysis showed that higher expressions of Tim-3 on both CD3-CD56+ NK cells and CD56(dim) NK-cell subset were independently correlated with shorter overall survival of patients with lung adenocarcinoma (log-rank test, P=0.0418, 0.0406, respectively). Importantly, blockade of Tim-3 signaling with anti-Tim-3 antibodies resulted in the increased cytotoxicity and IFN-γ production of peripheral NK cells from patients with lung adenocarcinoma. Our data indicate that Tim-3 expression in NK cells can function as a prognostic biomarker in human lung adenocarcinoma and support that Tim-3 could be a new target for an immunotherapeutic strategy.

  4. Tim-3 induces Th2-biased immunity and alternative macrophage activation during Schistosoma japonicum infection.

    PubMed

    Hou, Nan; Piao, Xianyu; Liu, Shuai; Wu, Chuang; Chen, Qijun

    2015-08-01

    T cell immunoglobulin- and mucin-domain-containing molecule 3 (Tim-3) has been regarded as an important regulatory factor in both adaptive and innate immunity. Recently, Tim-3 was reported to be involved in Th2-biased immune responses in mice infected with Schistosoma japonicum, but the exact mechanism behind the involvement of Tim-3 remains unknown. The present study aims to understand the role of Tim-3 in the immune response against S. japonicum infection. Tim-3 expression was determined by flow cytometry, and increased Tim-3 expression was observed on CD4(+) and CD8(+) T cells, NK1.1(+) cells, and CD11b(+) cells from the livers of S. japonicum-infected mice. However, the increased level of Tim-3 was lower in the spleen than in the liver, and no increase in Tim-3 expression was observed on splenic CD8(+) T cells or CD11b(+) cells. The schistosome-induced upregulation of Tim-3 on natural killer (NK) cells was accompanied by reduced NK cell numbers in vitro and in vivo. Tim-3 antibody blockade led to upregulation of inducible nitric oxide synthase and interleukin-12 (IL-12) mRNA in CD11b(+) cells cocultured with soluble egg antigen and downregulation of Arg1 and IL-10, which are markers of M2 macrophages. In summary, we observed schistosome-induced expression of Tim-3 on critical immune cell populations, which may be involved in the Th2-biased immune response and alternative activation of macrophages during infection.

  5. Tim-3 Induces Th2-Biased Immunity and Alternative Macrophage Activation during Schistosoma japonicum Infection

    PubMed Central

    Hou, Nan; Piao, Xianyu; Liu, Shuai; Wu, Chuang

    2015-01-01

    T cell immunoglobulin- and mucin-domain-containing molecule 3 (Tim-3) has been regarded as an important regulatory factor in both adaptive and innate immunity. Recently, Tim-3 was reported to be involved in Th2-biased immune responses in mice infected with Schistosoma japonicum, but the exact mechanism behind the involvement of Tim-3 remains unknown. The present study aims to understand the role of Tim-3 in the immune response against S. japonicum infection. Tim-3 expression was determined by flow cytometry, and increased Tim-3 expression was observed on CD4+ and CD8+ T cells, NK1.1+ cells, and CD11b+ cells from the livers of S. japonicum-infected mice. However, the increased level of Tim-3 was lower in the spleen than in the liver, and no increase in Tim-3 expression was observed on splenic CD8+ T cells or CD11b+ cells. The schistosome-induced upregulation of Tim-3 on natural killer (NK) cells was accompanied by reduced NK cell numbers in vitro and in vivo. Tim-3 antibody blockade led to upregulation of inducible nitric oxide synthase and interleukin-12 (IL-12) mRNA in CD11b+ cells cocultured with soluble egg antigen and downregulation of Arg1 and IL-10, which are markers of M2 macrophages. In summary, we observed schistosome-induced expression of Tim-3 on critical immune cell populations, which may be involved in the Th2-biased immune response and alternative activation of macrophages during infection. PMID:25987707

  6. T cell Ig and mucin domain-containing protein 3 is recruited to the immune synapse, disrupts stable synapse formation, and associates with receptor phosphatases.

    PubMed

    Clayton, Kiera L; Haaland, Matthew S; Douglas-Vail, Matthew B; Mujib, Shariq; Chew, Glen M; Ndhlovu, Lishomwa C; Ostrowski, Mario A

    2014-01-15

    CD8(+) CTLs are adept at killing virally infected cells and cancer cells and releasing cytokines (e.g., IFN-γ) to aid this response. However, during cancer and chronic viral infections, such as with HIV, this CTL response is progressively impaired due to a process called T cell exhaustion. Previous work has shown that the glycoprotein T cell Ig and mucin domain-containing protein 3 (Tim-3) plays a functional role in establishing T cell exhaustion. Tim-3 is highly upregulated on virus and tumor Ag-specific CD8(+) T cells, and antagonizing Tim-3 helps restore function of CD8(+) T cells. However, very little is known of how Tim-3 signals in CTLs. In this study, we assessed the role of Tim-3 at the immunological synapse as well as its interaction with proximal TCR signaling molecules in primary human CD8(+) T cells. Tim-3 was found within CD8(+) T cell lipid rafts at the immunological synapse. Blocking Tim-3 resulted in a significantly greater number of stable synapses being formed between Tim-3(hi)CD8(+) T cells and target cells, suggesting that Tim-3 plays a functional role in synapse formation. Further, we confirmed that Tim-3 interacts with Lck, but not the phospho-active form of Lck. Finally, Tim-3 colocalizes with receptor phosphatases CD45 and CD148, an interaction that is enhanced in the presence of the Tim-3 ligand, galectin-9. Thus, Tim-3 interacts with multiple signaling molecules at the immunological synapse, and characterizing these interactions could aid in the development of therapeutics to restore Tim-3-mediated immune dysfunction.

  7. Combined blockade of Tim-3 and MEK inhibitor enhances the efficacy against melanoma.

    PubMed

    Liu, Yang; Cai, Pengcheng; Wang, Ning; Zhang, Qianwen; Chen, Fenghua; Shi, Liang; Zhang, Yang; Wang, Lin; Hu, Lihua

    2017-03-04

    Insights into the role of the mitogen-activated protein kinase (MAPK) pathway and immune checkpoints have led combined targeted therapy and immunotherapy to be a promising regimen. Trametinib, as a mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor, has demonstrated effectiveness in patients with advanced melanoma. T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3), an immune checkpoint molecule, participates in multiple negative regulation of antitumor immunity. We for the first time to our knowledge reported the combination of trametinib and anti-Tim-3 monoclonal antibody (mAb) in treating B16-F10 melanoma mice. We discovered that trametinib remarkably promoted apoptosis and inhibited cell proliferation while inhibition of MEK improved the expression of Tim-3 and caused the decrease of CD8(+) T cells; to the contrary, anti-Tim-3 mAb enhanced antitumor immunity by stimulating CD8(+) T cells, thus the combined therapy produced potent antitumor effect cooperatively. Taken together, our study provides compelling evidence for combining trametinib and anti-Tim-3 mAb as a potential valuable regimen in treating melanoma.

  8. The Galectin-9/Tim-3 pathway is involved in the regulation of NK cell function at the maternal-fetal interface in early pregnancy.

    PubMed

    Li, Yan-Hong; Zhou, Wen-Hui; Tao, Yu; Wang, Song-Cun; Jiang, Yun-Lan; Zhang, Di; Piao, Hai-Lan; Fu, Qiang; Li, Da-Jin; Du, Mei-Rong

    2016-01-01

    Decidual natural killer (dNK) cells actively participate in the establishment and maintenance of maternal-fetal immune tolerance and act as local guardians against infection. However, how dNK cells maintain the immune balance between tolerance and anti-infection immune responses during pregnancy remains unknown. Here, we demonstrated that the inhibitory molecule T-cell immunoglobulin domain and mucin domain-containing molecule-3 (Tim-3) are expressed on over 60% of dNK cells. Tim-3(+) dNK cells display higher interleukin (IL)-4 and lower tumor necrosis factor (TNF)-α and perforin production. Human trophoblast cells can induce the transformation of peripheral NK cells into a dNK-like phenotype via the secretion of galectin-9 (Gal-9) and the interaction between Gal-9 and Tim-3. In addition, trophoblasts inhibit lipopolysaccharide (LPS)-induced pro-inflammatory cytokine and perforin production by dNK cells, which can be attenuated by Tim-3 neutralizing antibodies. Interestingly, a decreased percentage of Tim-3-expressing dNK cells were observed in human miscarriages and murine abortion-prone models. Moreover, T helper (Th)2-type cytokines were decreased and Th1-type cytokines were increased in Tim-3(+) but not Tim-3(-) dNK cells from human and mouse miscarriages. Therefore, our results suggest that the Gal-9/Tim-3 signal is important for the regulation of dNK cell function, which is beneficial for the maintenance of a normal pregnancy.

  9. Blockade of Tim-3 Pathway Ameliorates Interferon-γ Production from Hepatic CD8+ T Cells in a Mouse Model of Hepatitis B Virus Infection

    PubMed Central

    Ju, Ying; Hou, Nan; Zhang, Xiaoning; Zhao, Di; Liu, Ying; Wang, Jinjin; Luan, Fang; Shi, Wei; Zhu, Faliang; Sun, Wensheng; Zhang, Lining; Gao, Chengjiang; Gao, Lifen; Liang, Xiaohong; Ma, Chunhong

    2009-01-01

    T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) has been reported to participate in the pathogenesis of inflammatory diseases. However, whether Tim-3 is involved in hepatitis B virus (HBV) infection remains unknown. Here, we studied the expression and function of Tim-3 in a hydrodynamics-based mouse model of HBV infection. A significant increase of Tim-3 expression on hepatic T lymphocytes, especially on CD8+ T cells, was demonstrated in HBV model mice from day 7 to day 18. After Tim-3 knockdown by specific shRNAs, significantly increased IFN-γ production from hepatic CD8+ T cells in HBV model mice was observed. Very interestingly, we found Tim-3 expression on CD8+ T cells was higher in HBV model mice with higher serum anti-HBs production. Moreover, Tim-3 knockdown influenced anti-HBs production in vivo. Collectively, our data suggested that Tim-3 might act as a potent regulator of antiviral T-cell responses in HBV infection. PMID:19254478

  10. TIM-3 expression in lymphoma cells predicts chemoresistance in patients with adult T-cell leukemia/lymphoma.

    PubMed

    Horlad, Hasita; Ohnishi, Koji; Ma, Chaoya; Fujiwara, Yukio; Niino, Daisuke; Ohshima, Koichi; Jinushi, Masahisa; Matsuoka, Masao; Takeya, Motohiro; Komohara, Yoshihiro

    2016-08-01

    Adult T-cell leukemia/lymphoma (ATLL), an aggressive type of malignant lymphoma, is highly resistant to chemotherapy. However, the detailed mechanisms of the chemoresistance of ATLL have never been elucidated. We previously demonstrated that direct cell-cell interaction between macrophages and lymphoma cells was significantly associated with lymphoma progression in patients with ATLL. The present study aimed to further analyze the effects of cell-cell interaction between macrophages and ATLL cells by means of cell culture studies and immunohistochemical analysis using human ATLL samples. It was found that direct co-culture with macrophages induced chemoresistance in the ATLL ATN-1 cell line, but not in other cell lines, including TL-Mor, ED and ATL-2S. It was also found that expression of the T cell Ig and mucin domain-containing molecule-3 (TIM-3) was induced in ATN-1 cells by their long-term co-culture with macrophages. TIM-3 gene transfection induced chemoresistance in the ATN-1 cells. Immunostaining of ATLL tissues showed TIM-3 expression in 25 out of 58 ATLL cases. Although TIM-3 expression was not associated with overall survival or T classification, it was associated with resistance to chemotherapy. TIM-3 expression is therefore considered to be a marker for predicting the efficacy of chemotherapy, and TIM-3-associated signals may be a therapeutic target for patients with ATLL.

  11. TIM-3 expression in lymphoma cells predicts chemoresistance in patients with adult T-cell leukemia/lymphoma

    PubMed Central

    Horlad, Hasita; Ohnishi, Koji; Ma, Chaoya; Fujiwara, Yukio; Niino, Daisuke; Ohshima, Koichi; Jinushi, Masahisa; Matsuoka, Masao; Takeya, Motohiro; Komohara, Yoshihiro

    2016-01-01

    Adult T-cell leukemia/lymphoma (ATLL), an aggressive type of malignant lymphoma, is highly resistant to chemotherapy. However, the detailed mechanisms of the chemoresistance of ATLL have never been elucidated. We previously demonstrated that direct cell-cell interaction between macrophages and lymphoma cells was significantly associated with lymphoma progression in patients with ATLL. The present study aimed to further analyze the effects of cell-cell interaction between macrophages and ATLL cells by means of cell culture studies and immunohistochemical analysis using human ATLL samples. It was found that direct co-culture with macrophages induced chemoresistance in the ATLL ATN-1 cell line, but not in other cell lines, including TL-Mor, ED and ATL-2S. It was also found that expression of the T cell Ig and mucin domain-containing molecule-3 (TIM-3) was induced in ATN-1 cells by their long-term co-culture with macrophages. TIM-3 gene transfection induced chemoresistance in the ATN-1 cells. Immunostaining of ATLL tissues showed TIM-3 expression in 25 out of 58 ATLL cases. Although TIM-3 expression was not associated with overall survival or T classification, it was associated with resistance to chemotherapy. TIM-3 expression is therefore considered to be a marker for predicting the efficacy of chemotherapy, and TIM-3-associated signals may be a therapeutic target for patients with ATLL. PMID:27446463

  12. Immune checkpoint proteins PD-1 and TIM-3 are both highly expressed in liver tissues and correlate with their gene polymorphisms in patients with HBV-related hepatocellular carcinoma

    PubMed Central

    Li, Zhu; Li, Na; Li, Fang; Zhou, Zhihua; Sang, Jiao; Chen, Yanping; Han, Qunying; Lv, Yi; Liu, Zhengwen

    2016-01-01

    Abstract Immune checkpoint proteins programmed death-1 (PD-1) and T-cell immunoglobulin domain and mucin domain containing molecule-3 (TIM-3) expression and their gene polymorphisms have separately been shown to be associated with hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC). This study simultaneously examined PD-1 and TIM-3 expression in liver tissues and PD1 and TIM3 polymorphisms and analyzed their correlations in 171 patients with HBV-related HCC and 34 patients with HBV-related cirrhosis. PD-1 and TIM-3 expression in liver tissues were examined by immunohistochemistry and the genotypes of PD1 rs10204525 and TIM3 rs10053538 polymorphisms were determined using genomic DNA extracted from peripheral blood as template. Both PD-1 and TIM-3 expressions in liver infiltrating lymphocytes of HCC tumor tissues were significantly higher than those in tumor adjacent tissues or cirrhotic tissues. The elevated PD-1 and TIM-3 expressions were significantly associated with higher tumor grades. The levels between PD-1 and TIM-3 expression in tumor tissues and tumor adjacent tissues had a significant positive intercorrelation. The expressions of PD-1 and TIM-3 in tumor tissues, tumor adjacent tissues, and cirrhotic tissues were significantly associated with PD1 and TIM3 polymorphisms, with genotype AA of PD1 rs10204525 and genotypes GT+TT of TIM3 rs10053538 being associated with significantly increased PD-1 and TIM-3 expression, respectively. These findings support the potential to improve the efficiency of immune checkpoint-targeted therapy and reduce resistance to the therapy by blocking both PD-1 and TIM-3 and suggest the potential to apply the genotype determination of PD1 rs10204525 and TIM3 rs10053538 as biomarkers of immune checkpoint-directed therapies. PMID:28033288

  13. Immune checkpoint proteins PD-1 and TIM-3 are both highly expressed in liver tissues and correlate with their gene polymorphisms in patients with HBV-related hepatocellular carcinoma.

    PubMed

    Li, Zhu; Li, Na; Li, Fang; Zhou, Zhihua; Sang, Jiao; Chen, Yanping; Han, Qunying; Lv, Yi; Liu, Zhengwen

    2016-12-01

    Immune checkpoint proteins programmed death-1 (PD-1) and T-cell immunoglobulin domain and mucin domain containing molecule-3 (TIM-3) expression and their gene polymorphisms have separately been shown to be associated with hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC). This study simultaneously examined PD-1 and TIM-3 expression in liver tissues and PD1 and TIM3 polymorphisms and analyzed their correlations in 171 patients with HBV-related HCC and 34 patients with HBV-related cirrhosis.PD-1 and TIM-3 expression in liver tissues were examined by immunohistochemistry and the genotypes of PD1 rs10204525 and TIM3 rs10053538 polymorphisms were determined using genomic DNA extracted from peripheral blood as template.Both PD-1 and TIM-3 expressions in liver infiltrating lymphocytes of HCC tumor tissues were significantly higher than those in tumor adjacent tissues or cirrhotic tissues. The elevated PD-1 and TIM-3 expressions were significantly associated with higher tumor grades. The levels between PD-1 and TIM-3 expression in tumor tissues and tumor adjacent tissues had a significant positive intercorrelation. The expressions of PD-1 and TIM-3 in tumor tissues, tumor adjacent tissues, and cirrhotic tissues were significantly associated with PD1 and TIM3 polymorphisms, with genotype AA of PD1 rs10204525 and genotypes GT+TT of TIM3 rs10053538 being associated with significantly increased PD-1 and TIM-3 expression, respectively.These findings support the potential to improve the efficiency of immune checkpoint-targeted therapy and reduce resistance to the therapy by blocking both PD-1 and TIM-3 and suggest the potential to apply the genotype determination of PD1 rs10204525 and TIM3 rs10053538 as biomarkers of immune checkpoint-directed therapies.

  14. The Coordinated Actions of TIM-3 on Cancer and Myeloid Cells in the Regulation of Tumorigenicity and Clinical Prognosis in Clear Cell Renal Cell Carcinomas.

    PubMed

    Komohara, Yoshihiro; Morita, Tomoko; Annan, Dorcas A; Horlad, Hasita; Ohnishi, Koji; Yamada, Sohsuke; Nakayama, Toshiyuki; Kitada, Shohei; Suzu, Shinya; Kinoshita, Ichiro; Dosaka-Akita, Hirotoshi; Akashi, Koichi; Takeya, Motohiro; Jinushi, Masahisa

    2015-09-01

    Clear cell renal cell carcinoma (ccRCC) is one of most common cancers in urogenital organs. Although recent experimental and clinical studies have shown the immunogenic properties of ccRCC as illustrated by the clinical sensitivities to various immunotherapies, the detailed immunoregulatory machineries governing the tumorigenicity of human ccRCC remain largely obscure. In this study, we demonstrated the clinical significance and functional relevance of T-cell immunoglobulin and mucin domain-containing molecule-3 (TIM-3) expressed on tumor cells and myeloid cells in patients with ccRCC. TIM-3 expression was detected on cancer cells and CD204(+) tumor-associated macrophages (TAM), and higher expression level of TIM-3 was positively correlated with shorter progression-free survival (PFS) in patients with ccRCC. We found that TIM-3 expression was detected on a large number of tumors, and there was significant correlation between an increased number of TAMs and high expression level of TIM-3 in patients with ccRCC. Furthermore, TIM-3 rendered RCC cells with the ability to induce resistance to sunitinib and mTOR inhibitors, the standard regimen for patients with ccRCC, as well as stem cell activities. TIM-3 expression was induced on CD14(+) monocytes upon long-term stimulation with RCC cells, and TIM-3-expressing myeloid cells play a critical role in augmenting tumorigenic activities of TIM-3-negative RCC cells. More importantly, treatment with anti-TIM-3 mAb suppressed its tumorigenic effects in in vitro and in vivo settings. These findings indicate the coordinated action of TIM-3 in cancer cells and in myeloid cells regulates the tumorigenicity of human RCC.

  15. Tim-3 is a Marker of Plasmacytoid Dendritic Cell Dysfunction during HIV Infection and Is Associated with the Recruitment of IRF7 and p85 into Lysosomes and with the Submembrane Displacement of TLR9.

    PubMed

    Schwartz, Jordan Ari; Clayton, Kiera L; Mujib, Shariq; Zhang, Hongliang; Rahman, A K M Nur-Ur; Liu, Jun; Yue, Feng Yun; Benko, Erika; Kovacs, Colin; Ostrowski, Mario A

    2017-03-06

    In chronic diseases, such as HIV infection, plasmacytoid dendritic cells (pDCs) are rendered dysfunctional, as measured by their decreased capacity to produce IFN-α. In this study, we identified elevated levels of T cell Ig and mucin-domain containing molecule-3 (Tim-3)-expressing pDCs in the blood of HIV-infected donors. The frequency of Tim-3-expressing pDCs correlated inversely with CD4 T cell counts and positively with HIV viral loads. A lower frequency of pDCs expressing Tim-3 produced IFN-α or TNF-α in response to the TLR7 agonists imiquimod and Sendai virus and to the TLR9 agonist CpG. Thus, Tim-3 may serve as a biomarker of pDC dysfunction in HIV infection. The source and function of Tim-3 was investigated on enriched pDC populations from donors not infected with HIV. Tim-3 induction was achieved in response to viral and artificial stimuli, as well as exogenous IFN-α, and was PI3K dependent. Potent pDC-activating stimuli, such as CpG, imiquimod, and Sendai virus, induced the most Tim-3 expression and subsequent dysfunction. Small interfering RNA knockdown of Tim-3 increased IFN-α secretion in response to activation. Intracellular Tim-3, as measured by confocal microscopy, was dispersed throughout the cytoplasm prior to activation. Postactivation, Tim-3 accumulated at the plasma membrane and associated with disrupted TLR9 at the submembrane. Tim-3-expressing pDCs had reduced IRF7 levels. Furthermore, intracellular Tim-3 colocalized with p85 and IRF7 within LAMP1(+) lysosomes, suggestive of a role in degradation. We conclude that Tim-3 is a biomarker of dysfunctional pDCs and may negatively regulate IFN-α, possibly through interference with TLR signaling and recruitment of IRF7 and p85 into lysosomes, enhancing their degradation.

  16. Modulation of Tim-3 Expression by Antigen-Dependent and -Independent Factors on T Cells from Patients with Chronic Hepatitis B Virus Infection

    PubMed Central

    Dong, Jie; Yang, Xiao-Fei; Wang, Lin-Xu; Wei, Xin; Wang, An-Hui; Hao, Chun-Qiu; Shen, Huan-Jun; Huang, Chang-Xing; Zhang, Ye; Lian, Jian-Qi

    2017-01-01

    T-cell immunoglobulin domain and mucin domain-containing molecule-3 (Tim-3) was up-regulated on viral specific T cells and contributed to T cells exhaustion during chronic hepatitis B virus (HBV) infection. However, modulation of Tim-3 expression was still not fully elucidated. To evaluate the potential viral and inflammatory factors involved in the inductor of Tim-3 expression on T cells, 76 patients with chronic HBV infection (including 40 chronic hepatitis B [CHB] and 36 asymptomatic HBV carriers [AsC]) and 40 of normal controls (NCs) were enrolled in this study. Tim-3 expressions on CD4+ and CD8+ T cells were assessed in response to HBV-encoding antigens, HBV peptide pools, and common γ-chain (γc) cytokines stimulation by flow cytometry. HBV peptides and anti-CD3/CD28 directly induced Tim-3 expression on T cells. γc cytokines also drive Tim-3 up-regulations on both CD4+ and CD8+ T cells in patients with chronic HBV infection. However, γc cytokines did not enhance the Tim-3 inductions by either anti-CD3/CD28 or HBV peptides stimulation. Furthermore, γc cytokines-mediated Tim-3 induction could not be abrogated by γc cytokine receptor-neutralizing antibodies. The current results suggested that elevation of Tim-3 expression on T cells could be regulated by both antigen-dependent and -independent manner in patients with chronic HBV infection. The role of γc cytokines in modulation of inhibitory pathway might be evaluated as immunotherapies in humans.

  17. Genetic variations of PD1 and TIM3 are differentially and interactively associated with the development of cirrhosis and HCC in patients with chronic HBV infection.

    PubMed

    Li, Zhu; Li, Na; Zhu, Qianqian; Zhang, Guoyu; Han, Qunying; Zhang, Pingping; Xun, Meng; Wang, Yawen; Zeng, Xiaoyan; Yang, Cuiling; Liu, Zhengwen

    2013-03-01

    Cooperation or interaction of programmed cell death-1 (PD-1) and T cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3) molecules is more relevant than either molecule alone to immune dysfunction in chronic viral infection and cancers. This study simultaneously investigated polymorphisms at PD1 +8669 and TIM3 -1516 loci in 845 hepatitis B virus (HBV) chronically infected patients [151 asymptomatic carriers, 202 chronic hepatitis, 221 cirrhosis and 271 hepatocellular carcinoma (HCC)], 141 HBV infection resolvers and 318 healthy controls. Multivariate analysis showed that, in addition to gender, age, ALT, albumin and HBV DNA, PD1 +8669 genotype AA was associated with cirrhosis compared with patients without cirrhosis (OR, 2.410; P=0.001). TIM3 -1516 genotypes GT+TT, together with gender, age, ALT, AST, direct bilirubin, albumin and HBeAg status, were associated with HCC compared with cirrhosis patients without HCC (OR, 2.142; P=0.011). The combined carriage of PD1 +8669 AA/TIM3 -1516 GT or TT was higher in cirrhosis and HCC pooled patients than in patients without cirrhosis (OR, 2.326; P=0.020) and in HCC patients than in cirrhosis patients (OR, 2.232; P=0.013). These data suggest that PD1 and TIM3 polymorphisms may differentially and interactively predispose cirrhosis and HCC in chronic HBV infection.

  18. Upregulation of Tim-3 on CD4(+) T cells is associated with Th1/Th2 imbalance in patients with allergic asthma.

    PubMed

    Tang, Fei; Wang, Fukun; An, Liyun; Wang, Xianling

    2015-01-01

    T cell Ig and mucin domain-containing molecule-3 (Tim-3) is a negative regulator preferentially expressed on Th1 cells. Allergic asthma is a clinical syndrome well characterized by Th1/Th2 imbalance. To investigate the role of Tim-3 in the pathogenesis of asthma and its relationship with Th1/Th2 imbalance, a total of 40 patients with allergic asthma and 40 healthy controls were enrolled. Expression of Tim-3 and Th1/Th2 imbalance as well as the relationship between them was analyzed by flow cytometry and real-time PCR. Peripheral blood mononuclear cells (PBMCs) were cultured in vitro and anti-Tim-3 was used to block Tim-3 signaling; Th1/Th2 cytokines in the culture supernatant were detected by enzyme linked immunosorbent assay (ELISA). CD4(+) T cells and B cells were sorted and co-cultured in vitro, and anti-Tim-3 was used to block Tim-3 signaling; Total IgG/IgE in the culture supernatant was detected by ELISA. The mRNA level of T-bet and IFN-γ were significantly decreased in allergic asthma patients, while GATA-3 and IL-4 were significantly increased. Expression of Tim-3 on CD4(+) T cells was much higher in allergic asthma patients and it was negatively correlated with T-bet/GATA-3 ratio or IFN-γ/IL-4 ratio. Blocking of Tim-3 significantly increased Th1 cytokines (TNF-α and IFN-γ) and decreased Th2 cytokines (IL-4, IL-5, IL-13) in the culture supernatant of PBMCs. Blocking of Tim-3 dramatically reduced the production of IgG and IgE in the co-culture supernatant of CD4(+) T cells and B cells. In conclusion, Tim-3 was up-regulated in allergic asthma patients and related with the Th1/Th2 imbalance. Blocking of Tim-3 may be of therapeutic benefit by enhancing the Th1 cytokines response, down-regulating the Th2 cytokines response, and reducing IgG/IgE production.

  19. DNA demethylation of the TIM-3 promoter is critical for its stable expression on T cells.

    PubMed

    Chou, F-C; Kuo, C-C; Chen, H-Y; Chen, H-H; Sytwu, H-K

    2016-04-01

    The T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) is selectively expressed on terminally differentiated T helper 1 (Th1) cells and acts as a negative regulator that terminates Th1 responses. The dysregulation of TIM-3 expression on T cells is associated with several autoimmune phenotypes and with chronic viral infections; however, the mechanism of this regulation is unclear. In this study, we investigated the effect of DNA methylation on the expression of TIM-3. By analyzing the sequences of TIM-3 promoter regions in human and mouse, we identified a CpG island within the TIM-3 promoter and demonstrated that the promoter activity was controlled by DNA methylation. Furthermore, treatment with 5-aza-2'-deoxycytidine enhanced TIM-3 expression on mouse primary CD4(+) T cells under Th0-, Th1- or Th2-polarizing conditions. Finally, pyrosequencing analysis revealed that the methylation level of the TIM-3 promoter gradually decreased after each round of T-cell polarization, and this decrease was inversely correlated with TIM-3 expression. These data suggest that the DNA methylation of the TIM-3 promoter cooperates with lineage-specific transcription factors in the control of Th-cell development. In conclusion, DNA methylation-based regulation of TIM-3 may provide novel insights into understanding the dysregulation of TIM-3 expression under pathogenic conditions.

  20. HCV-specific T cells in HCV/HIV co-infection show elevated frequencies of dual Tim-3/PD-1 expression that correlate with liver disease progression.

    PubMed

    Vali, Bahareh; Jones, R Brad; Sakhdari, Ali; Sheth, Prameet M; Clayton, Kiera; Yue, Feng-Yun; Gyenes, Gabor; Wong, David; Klein, Marina B; Saeed, Sahar; Benko, Erika; Kovacs, Colin; Kaul, Rupert; Ostrowski, Mario A

    2010-09-01

    Co-infection of HCV with HIV has been associated with more rapid progression of HCV-related disease. HCV-specific T-cell immune responses, which are essential for disease control, are attenuated in co-infection with HIV. T-cell exhaustion has recently been implicated in the deficient control of chronic viral infections. In the current study, we investigated the role of programmed death-1 (PD-1) and T-cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3) expression in T-cell exhaustion during HCV/HIV co-infection. We show that in HCV/HIV co-infection, both total and HCV-specific T cells co-express Tim-3 and PD-1 in significantly higher frequencies, compared with HCV mono-infection. Co-expression of these two markers on HCV-specific CD8(+) T cells positively correlated with a clinical parameter of liver disease progression. HCV-specific CD8(+) T cells showed greater frequencies of Tim-3/PD-1 co-expression than HIV-specific CD8(+) T cells, which may indicate a greater degree of exhaustion in the former. Blocking Tim-3 or PD-1 pathways restored both HIV- and HCV-specific CD8(+) T-cell expansion in the blood of co-infected individuals. These data demonstrate that co-expression of Tim-3 and PD-1 may play a significant role in HCV-specific T-cell dysfunction, especially in the setting of HIV co-infection.

  1. TIM3 Mediates T Cell Exhaustion during Mycobacterium tuberculosis Infection.

    PubMed

    Jayaraman, Pushpa; Jacques, Miye K; Zhu, Chen; Steblenko, Katherine M; Stowell, Britni L; Madi, Asaf; Anderson, Ana C; Kuchroo, Vijay K; Behar, Samuel M

    2016-03-01

    While T cell immunity initially limits Mycobacterium tuberculosis infection, why T cell immunity fails to sterilize the infection and allows recrudescence is not clear. One hypothesis is that T cell exhaustion impairs immunity and is detrimental to the outcome of M. tuberculosis infection. Here we provide functional evidence for the development T cell exhaustion during chronic TB. Second, we evaluate the role of the inhibitory receptor T cell immunoglobulin and mucin domain-containing-3 (TIM3) during chronic M. tuberculosis infection. We find that TIM3 expressing T cells accumulate during chronic infection, co-express other inhibitory receptors including PD1, produce less IL-2 and TNF but more IL-10, and are functionally exhausted. Finally, we show that TIM3 blockade restores T cell function and improves bacterial control, particularly in chronically infected susceptible mice. These data show that T cell immunity is suboptimal during chronic M. tuberculosis infection due to T cell exhaustion. Moreover, in chronically infected mice, treatment with anti-TIM3 mAb is an effective therapeutic strategy against tuberculosis.

  2. Overexpression of Tim-3 reduces Helicobacter pylori-associated inflammation through TLR4/NFκB signaling in vitro.

    PubMed

    Wang, Fucai; Mao, Zhirong; Liu, Dongsheng; Yu, Jing; Wang, Youhua; Ye, Wen; Lin, Dongjia; Zhou, Nanjin; Xie, Yong

    2017-03-21

    The present study aimed to investigate the interaction between T-cell immunoglobulin and mucin-domain-containing molecule-3 (Tim-3) and Toll-like receptor 4 (TLR4)/nuclear factor κB (NF‑κB) signaling in Helicobacter pylori-infected RAW264.7 macrophage cells. RAW264.7 cells were co‑cultured with H. pylori SS1 at different bacteria/cell ratios, and subsequently the mRNA expression of Tim‑3, TLR4, and myeloid differentiation factor 88 (MyD88) was measured by reverse transcription-quantitative polymerase chain reaction (RT‑qPCR). Furthermore, the effect of Tim‑3 overexpression was examined by transfection of RAW264.7 with pLVX-IRES-ZsGreen-Tim-3 and co‑culturing with H. pylori. mRNA and protein expression levels were then analyzed for Tim‑3, TLR4, MyD88, and phosphorylated (p‑) NF‑κB by RT‑qPCR and western blot analysis respectively. The concentrations of pro‑inflammatory cytokines [tumor necrosis factor‑α (TNF‑α), interleukin 6 (IL-6), interferon‑γ (IFN‑γ) and interleukin 10 (IL‑10)] released in the culture supernatants were measured by ELISA. H. pylori stimulation resulted in a significant increase of Tim‑3, TLR4, and MyD88 mRNA expression in RAW264.7 cells. H. pylori stimulation upregulated Tim‑3 expression even in the Tim‑3‑overexpressing RAW264.7 cells compared with unstimulated cells. TLR4, MyD88, and pNF‑κB protein expression and pro‑inflammatory cytokines (TNF‑α, IL‑6, and IFN‑γ) release levels were increased in the control RAW264.7 cells following H. pylori infection, but not in the Tim-3-overexpressing RAW264.7 cells. By contrast, IL‑10 levels were decreased following H. pylori infection in both control and Tim‑3‑overexpressing RAW264.7 cells. Overexpression of Tim-3 reduced H. pylori-associated inflammation in RAW264.7 macrophages, by downregulating expression of proteins in the TLR4 pathway and release of pro‑inflammatory cytokines. These findings suggest that Tim‑3 serves a crucial role

  3. Tim-3 Is Upregulated in NK Cells during Early Pregnancy and Inhibits NK Cytotoxicity toward Trophoblast in Galectin-9 Dependent Pathway

    PubMed Central

    Sun, Jintang; Yang, Meixiang; Ban, Yanli; Gao, Wenjuan; Song, Bingfeng; Wang, Yang; Zhang, Yun; Shao, Qianqian; Kong, Beihua; Qu, Xun

    2016-01-01

    NK cells accumulate at the maternal-fetal interface (MFI) and play essential roles in maintaining immune tolerance during pregnancy. The mechanisms that facilitate NK cells tolerance to fetal tissue are largely unknown. T cell Ig and mucin domain-containing protein 3 (Tim-3) is a newly defined molecule with essential immunological function in many physiological and pathological processes. Recent study showed that Tim-3 was involved in the regulation of immune tolerance at MFI. However, whether Tim-3 regulates NK cells cytotoxicity toward trophoblasts is unclear. Here, we showed Tim-3 was mainly expressed by decidual NK cells (dNK) and Tim-3 level in dNK was higher than peripheral NK cells (pNK). Tim-3+ dNK expressed more levels of mature markers CD94 and CD69 than Tim-3- dNK cells and blocking Tim-3 significantly inhibited dNK IFN-γ and TNF-α secretion. Furthermore, we found TGF-β1 may contribute to such up-regulation of Tim-3 in NK cells. Interestingly, blocking Tim-3 enhanced NK cytotoxicity toward trophoblast cell line HTR-8 but not K562. We found HTR-8 expressed Tim-3 ligand Galectin-9, in contrast K562 did not. Small interfering RNA-mediated silencing of Galectin-9 expression enhanced NK cytotoxicity toward HTR-8. We further showed Tim-3/Galecin-9 inhibited NK cytotoxicity toward trophoblast partially via impairing the degranulation process. In addition, clinical data showed that abnormal Tim-3 level on pNK might be associated with recurrent spontaneous abortion (RSA). Thus, our data demonstrate Tim-3/Galectin-9 pathway maintains local tolerance by suppressing NK cytotoxicity toward trophoblasts which may represent a new immunologic tolerance mechanism at MFI. PMID:26789128

  4. Tim-3 Is Upregulated in NK Cells during Early Pregnancy and Inhibits NK Cytotoxicity toward Trophoblast in Galectin-9 Dependent Pathway.

    PubMed

    Sun, Jintang; Yang, Meixiang; Ban, Yanli; Gao, Wenjuan; Song, Bingfeng; Wang, Yang; Zhang, Yun; Shao, Qianqian; Kong, Beihua; Qu, Xun

    2016-01-01

    NK cells accumulate at the maternal-fetal interface (MFI) and play essential roles in maintaining immune tolerance during pregnancy. The mechanisms that facilitate NK cells tolerance to fetal tissue are largely unknown. T cell Ig and mucin domain-containing protein 3 (Tim-3) is a newly defined molecule with essential immunological function in many physiological and pathological processes. Recent study showed that Tim-3 was involved in the regulation of immune tolerance at MFI. However, whether Tim-3 regulates NK cells cytotoxicity toward trophoblasts is unclear. Here, we showed Tim-3 was mainly expressed by decidual NK cells (dNK) and Tim-3 level in dNK was higher than peripheral NK cells (pNK). Tim-3(+) dNK expressed more levels of mature markers CD94 and CD69 than Tim-3- dNK cells and blocking Tim-3 significantly inhibited dNK IFN-γ and TNF-α secretion. Furthermore, we found TGF-β1 may contribute to such up-regulation of Tim-3 in NK cells. Interestingly, blocking Tim-3 enhanced NK cytotoxicity toward trophoblast cell line HTR-8 but not K562. We found HTR-8 expressed Tim-3 ligand Galectin-9, in contrast K562 did not. Small interfering RNA-mediated silencing of Galectin-9 expression enhanced NK cytotoxicity toward HTR-8. We further showed Tim-3/Galecin-9 inhibited NK cytotoxicity toward trophoblast partially via impairing the degranulation process. In addition, clinical data showed that abnormal Tim-3 level on pNK might be associated with recurrent spontaneous abortion (RSA). Thus, our data demonstrate Tim-3/Galectin-9 pathway maintains local tolerance by suppressing NK cytotoxicity toward trophoblasts which may represent a new immunologic tolerance mechanism at MFI.

  5. TIM-3 Suppresses Anti-CD3/CD28-Induced TCR Activation and IL-2 Expression through the NFAT Signaling Pathway.

    PubMed

    Tomkowicz, Brian; Walsh, Eileen; Cotty, Adam; Verona, Raluca; Sabins, Nina; Kaplan, Fred; Santulli-Marotto, Sandy; Chin, Chen-Ni; Mooney, Jill; Lingham, Russell B; Naso, Michael; McCabe, Timothy

    2015-01-01

    TIM-3 (T cell immunoglobulin and mucin-domain containing protein 3) is a member of the TIM family of proteins that is preferentially expressed on Th1 polarized CD4+ and CD8+ T cells. Recent studies indicate that TIM-3 serves as a negative regulator of T cell function (i.e. T cell dependent immune responses, proliferation, tolerance, and exhaustion). Despite having no recognizable inhibitory signaling motifs, the intracellular tail of TIM-3 is apparently indispensable for function. Specifically, the conserved residues Y265/Y272 and surrounding amino acids appear to be critical for function. Mechanistically, several studies suggest that TIM-3 can associate with interleukin inducible T cell kinase (ITK), the Src kinases Fyn and Lck, and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor protein to positively or negatively regulate IL-2 production via NF-κB/NFAT signaling pathways. To begin to address this discrepancy, we examined the effect of TIM-3 in two model systems. First, we generated several Jurkat T cell lines stably expressing human TIM-3 or murine CD28-ECD/human TIM-3 intracellular tail chimeras and examined the effects that TIM-3 exerts on T cell Receptor (TCR)-mediated activation, cytokine secretion, promoter activity, and protein kinase association. In this model, our results demonstrate that TIM-3 inhibits several TCR-mediated phenotypes: i) NF-kB/NFAT activation, ii) CD69 expression, and iii) suppression of IL-2 secretion. To confirm our Jurkat cell observations we developed a primary human CD8+ cell system that expresses endogenous levels of TIM-3. Upon TCR ligation, we observed the loss of NFAT reporter activity and IL-2 secretion, and identified the association of Src kinase Lck, and PLC-γ with TIM-3. Taken together, our results support the conclusion that TIM-3 is a negative regulator of TCR-function by attenuating activation signals mediated by CD3/CD28 co-stimulation.

  6. TIM-3 expression in human osteosarcoma: Correlation with the expression of epithelial-mesenchymal transition-specific biomarkers

    PubMed Central

    SHANG, YONGJUN; LI, ZHANYONG; LI, HONG; XIA, HAIBO; LIN, ZHENHUA

    2013-01-01

    Signals from the T cell Ig- and mucin-domain-containing molecules (TIMs) have been demonstrated to be actively involved in regulating the progression of carcinomas. However, the expression and distribution of these molecules in osteosarcoma, the most common primary bone malignancy with poor prognosis, have not been investigated. In this study, the expression of TIMs was examined in nine invasive human osteosarcomas using immunohistochemistry, and the phenotypes were detected by dual immunofluorescence staining. Using immunohistochemistry, it was observed that only TIM-3, rather than TIM-1 or TIM-4, was expressed in these tumor specimens, where it was localized in the cytoplasm and plasma membrane of tumor cells. Dual immunofluorescence staining revealed that the expression of TIM-3 was observed in all cell types investigated, including CD68+ macrophages, CD31+ endothelial cells, CK-18+ epithelial cells and PCNA+ tumor cells. Notably, in sarcoma cells, TIM-3 was co-expressed with certain biomarkers of epithelial-mesenchymal transition (EMT), including vimentin, Slug, Snail and Smad. These combined results suggest that TIM-3 triggers tumor cells to acquire features of aggressive EMT and may be involved in the pathogenesis of this malignancy. PMID:24137353

  7. Abnormal expression of Tim-3 antigen on peripheral blood T cells is associated with progressive disease in osteosarcoma patients.

    PubMed

    Liu, Hongliang; Zhi, Liqiang; Duan, Ning; Su, Pengxiao

    2016-08-01

    T-cell immunoglobulin and mucin-domain-3-containing molecule 3 (TIM-3) plays a pivotal role in immune regulation and has been found in various tumors. However, the prevalence and distribution of Tim-3 in osteosarcoma (OS) is still unclear. The aim of this study was to investigate the prevalence and distribution of Tim-3 in OS. Tim-3 on peripheral T cells from 82 OS patients and 60 healthy controls were examined by flow cytometry. Plasma levels of IL-2, IFN-γ, and TNF-α were measured by ELSIA. Tim-3 on both CD4(+) T and CD8(+) T cells were significantly upregulated in OS patients compared with healthy controls, Tim-3(+) CD4(+) T, and Tim-3(+) CD8(+) T cells were both negatively associated with serum levels of IL-2 and IFN-γ and TNF-α. In addition, Tim-3 showed similar levels in patients with different tumor sites. Nevertheless, patients with advanced tumor stage, metastasis, and pathological tumor fracture displayed significantly higher Tim-3 on both CD4(+) T cells and CD8(+) T cells than those with early tumor stage, without metastasis and pathological tumor fracture. Moreover, high Tim-3 on peripheral CD4(+) T cells or CD8(+) T were significantly related to poor overall survival (P = 0.014, P = 0.035, respectively). In conclusion, Tim-3 may be a potential diagnostic and prognostic biomarker for OS progression.

  8. TIM-3 Regulates Distinct Functions in Macrophages

    PubMed Central

    Ocaña-Guzman, Ranferi; Torre-Bouscoulet, Luis; Sada-Ovalle, Isabel

    2016-01-01

    The transmembrane protein TIM-3 is a type I protein expressed by sub-types of lymphoid cells, such as lymphocytes Th1, Th17, Tc1, NK, as well as in myeloid cells. Scientific evidence indicates that this molecule acts as a negative regulator of T lymphocyte activation and that its expression is modified in viral infections or autoimmune diseases. In addition to evidence from lymphoid cells, the function of TIM-3 has been investigated in myeloid cells, such as monocytes, macrophages, and dendritic cells (DC), where studies have demonstrated that it can regulate cytokine production, cell activation, and the capture of apoptotic bodies. Despite these advances, the function of TIM-3 in myeloid cells and the molecular mechanisms that this protein regulates are not yet fully understood. This review examines the most recent evidence concerning the function of TIM-3 when expressed in myeloid cells, primarily macrophages, and the potential impact of that function on the field of basic immunology. PMID:27379093

  9. Plasma soluble Tim-3 emerges as an inhibitor in sepsis: sepsis contrary to membrane Tim-3 on monocytes.

    PubMed

    Ren, F; Li, J; Jiang, X; Xiao, K; Zhang, D; Zhao, Z; Ai, J; Hou, C; Jia, Y; Han, G; Xie, L

    2015-11-01

    Immune dysfunction is the main characteristic of sepsis. T cell Ig and mucin domain protein 3 (Tim-3) on the monocytes has been reported to promote immune homeostasis during sepsis, but the influences of plasm soluble Tim-3 (sTim-3) on the immune system during sepsis remain unknown. Here, 100 patients with different severities of sepsis (40 sepsis, 42 severe sepsis, and 18 septic shock) were enrolled in this study. The Tim-3 and human leukocyte antigen-DR (HLA-DR) on the circulating monocytes were detected using flow cytometry. Plasma sTim-3 was detected by enzyme-linked immunosorbent assay. Inflammatory factors and two kinds of A disintegrin and metalloprotease (ADAM) - ADAM10 and ADAM17 were assessed. The Tim-3 and HLA-DR on the monocytes decreased with increasing sepsis severity. The sTim-3 was reduced in the sepsis and severe sepsis patients but was elevated in the septic shock patients who exhibited significant immunosuppression as predicted by HLA-DR. sTim-3 levels were negatively correlated with IL-12 and TNF-α. ADAM10 and ADAM17, sheddases of Tim-3, exhibited trends toward elevations in the septic shock group. In conclusion, sTim-3 was involved in the development of sepsis. The homeostasis-promoting role of the Tim-3 on the monocytes was disrupted, while the inhibitory role of sTim-3 emerged during sepsis-induced immunosuppression.

  10. Lymphoma endothelium preferentially expresses Tim-3 and facilitates the progression of lymphoma by mediating immune evasion

    PubMed Central

    Huang, Xiaoyuan; Bai, Xiangyang; Cao, Yang; Wu, Jingyi; Huang, Mei; Tang, Duozhuang; Tao, Si; Zhu, Tao; Liu, Yanling; Yang, Yang; Zhou, Xiaoxi; Zhao, Yanxia; Wu, Mingfu; Wei, Juncheng; Wang, Daowen; Xu, Gang; Wang, Shixuan

    2010-01-01

    Angiogenesis is increasingly recognized as an important prognosticator associated with the progression of lymphoma and as an attractive target for novel modalities. We report a previously unrecognized mechanism by which lymphoma endothelium facilitates the growth and dissemination of lymphoma by interacting with circulated T cells and suppresses the activation of CD4+ T cells. Global gene expression profiles of microdissected endothelium from lymphoma and reactive lymph nodes revealed that T cell immunoglobulin and mucin domain–containing molecule 3 (Tim-3) was preferentially expressed in lymphoma-derived endothelial cells (ECs). Clinically, the level of Tim-3 in B cell lymphoma endothelium was closely correlated to both dissemination and poor prognosis. In vitro, Tim-3+ ECs modulated T cell response to lymphoma surrogate antigens by suppressing activation of CD4+ T lymphocytes through the activation of the interleukin-6–STAT3 pathway, inhibiting Th1 polarization, and providing protective immunity. In a lymphoma mouse model, Tim-3–expressing ECs promoted the onset, growth, and dissemination of lymphoma by inhibiting activation of CD4+ T cells and Th1 polarization. Our findings strongly argue that the lymphoma endothelium is not only a vessel system but also a functional barrier facilitating the establishment of lymphoma immune tolerance. These findings highlight a novel molecular mechanism that is a potential target for enhancing the efficacy of tumor immunotherapy and controlling metastatic diseases. PMID:20176801

  11. Tim-3 facilitates osteosarcoma proliferation and metastasis through the NF-κB pathway and epithelial-mesenchymal transition.

    PubMed

    Feng, Z M; Guo, S M

    2016-09-02

    The aim of this study was to investigate the expression of T-cell immunoglobulin mucin domain molecule-3 (Tim-3) in osteosarcoma tissues, and analyze its effect on cell proliferation and metastasis in an osteosarcoma cell line. Tim-3 mRNA and protein expression in osteosarcoma tissue was detected by reverse transcriptase-polymerase chain reaction and immunohistochemistry, respectively. Additionally, the cell viability, apoptosis rate, and invasive ability of the osteosarcoma cell line MG-63 were tested using the methyl thiazolyl tetrazolium assay, Annexin V-propidium iodide flow cytometry, and a Transwell assay, respectively, following Tim-3 interference using small interfering RNA (siRNA). We also analyzed the expression of Snail, E-cadherin, vimentin, and nuclear factor (NF)-kB in the cells by western blot. We observed that Tim-3 mRNA and protein was significantly overexpressed in osteosarcoma tissues, compared to the adjacent normal tissue (P < 0.01). Moreover, MG-63 cells transfected with the Tim-3 siRNA presented lower cell viability, a greater number of apoptotic cells, and decreased invasive ability (P < 0.01), compared to control cells. Additionally, we observed a decrease in Snail and vimentin expression, an increase in the E-cadherin level, and an increase in NF-kB p65 phosphorylation (P < 0.01) in Tim-3 siRNA-transfected MG-63 cells. Based on these results, we concluded that Tim-3 is highly expressed in osteosarcoma tissue. Moreover, we speculated that interfering in Tim-3 expression could significantly suppress osteosarcoma cell (MG-63) proliferation and metastasis via the NF-kB/Snail signaling pathway and epithelial-mesenchymal transition.

  12. The regulation of TIM-3 transcription in T cells involves c-Jun binding but not CpG methylation at the TIM-3 promoter.

    PubMed

    Yun, Su Jin; Jun, Ka-Jung; Komori, Kuniharu; Lee, Mi Jin; Kwon, Myung-Hee; Chwae, Yong-Joon; Kim, Kyongmin; Shin, Ho-Joon; Park, Sun

    2016-07-01

    Tim-3 is an immunomodulatory protein that is expressed constitutively on monocytes but is induced in activated T cells. The mechanisms involved in the regulation of TIM-3 transcription are poorly understood. In the present study, we investigated whether methylation of the TIM-3 promoter is involved in regulatingTIM-3 transcription in T cells, and identified a transcription factor that regulates TIM-3 transcription by associating with the TIM-3 minimal promoter region. Pyrosequencing of the TIM-3 promoter up to -1440bp revealed 11 hypermethylated CpG sites and 4 hypomethylated CpG sites in human CD4(+) T cells as well as in CD11b(+) cells. Dimethylation of histone H3 lysine 4 (H3K4), a mark of transcriptional activation, was predominantly found in the proximal TIM-3 promoter -954 to -34bp region, whereas trimethylation of H3K9 and H3K27, which are markers of transcriptional suppression, were mostly observed in the distal promoter -1549 to -1048bp region in human CD4(+) T cells and CD11b(+) cells. However, no change in the methylation status of CpG sites and the histone H3 in the TIM-3 promoter was found during induction of TIM-3 transcription in T cells. Finally, AP-1 involvement in TIM-3 transcription was shown in relation with the TIM-3 minimal promoter -146 to +144bp region. The present study defines the minimal TIM-3 promoter region and demonstrates its interaction with c-Jun during TIM-3 transcription in CD4(+) T cells.

  13. Apoptosis of tumor infiltrating effector TIM-3+CD8+ T cells in colon cancer.

    PubMed

    Kang, Chiao-Wen; Dutta, Avijit; Chang, Li-Yuan; Mahalingam, Jayashri; Lin, Yung-Chang; Chiang, Jy-Ming; Hsu, Chen-Yu; Huang, Ching-Tai; Su, Wan-Ting; Chu, Yu-Yi; Lin, Chun-Yen

    2015-10-23

    TIM-3 functions to enforce CD8+ T cell exhaustion, a dysfunctional state associated with the tolerization of tumor microenvironment. Here we report apoptosis of IFN-γ competent TIM-3+ population of tumor-infiltrating CD8+ T cells in colon cancer. In humans suffering from colorectal cancer, TIM-3+ population is higher in cancer tissue-resident relative to peripheral blood CD8+ T cells. Both the TIM-3+ and TIM-3- cancer tissue-resident CD8+ T cells secrete IFN-γ of comparable levels, although apoptotic cells are more in TIM-3+ compared to TIM-3- population. In mouse CT26 colon tumor model, majority of tumor-infiltrating CD8+ T cells express TIM-3 and execute cytolysis function with higher effector cytokine secretion and apoptosis in TIM-3+ compared to TIM-3- population. The tumor cells secrete galectin-9, which increases apoptosis of tumor-infiltrating CD8+ T cells. Galectin-9/TIM-3 signaling blockade with anti-TIM-3 antibody reduces the apoptosis and in addition, inhibits tumor growth in mice. The blockade increases therapeutic efficacy of cyclophosphamide to treat tumor in mice as well. These results reveal a previously unexplored role of TIM-3 on tumor-infiltrating CD8+ T cells in vivo.

  14. Up-regulation of Tim-3 is associated with poor prognosis of patients with colon cancer.

    PubMed

    Zhou, Encheng; Huang, Qing; Wang, Ji; Fang, Chengfeng; Yang, Leilei; Zhu, Min; Chen, Jianhui; Chen, Lihua; Dong, Milian

    2015-01-01

    Tim-3 (T cell immunoglobulin and mucin domain 3), belonging to the member of the novel Tim family, has been confirmed that it plays a critical negative role in regulating the immune responses against viral infection and carcinoma. Recently, it has also been reported that the over-expression of Tim-3 is associated with poor prognosis in solid tumors. However, the role of Tim-3 in colorectal cancer remains largely unknown. In the current study, we aim to investigate the expression of Tim-3 in colorectal carcinoma and discuss the relationship between Tim-3 expression and colon cancer prognosis, thus speculating the possible role of Tim-3 in colon cancer progression. Colon cancer tissues and paired normal tissue were obtained from 201 patients with colon cancer for preparation of tissue microarray. Tim-3 expression was evaluated by immunohistochemical staining. The Tim-3 expression level was evaluated by q-RT-PCR, western blot and immunocytochemistry in four colon cancer cell lines (HT-29, HCT116, LoVo, SW620). Tim-3 was expressed in 92.5% tumor tissue samples and 86.5% corresponding normal tissue samples. Expression of Tim-3 was significantly higher in tumor tissues than in normal tissues (P < 0.0001). Tim-3 expression in colon cancer tissues is in correlation with colon cancer lymphatic metastasis and TNM (P < 0.0001). Multivariate analysis demonstrated that Tim-3 expression could be a potential independent prognostic factor for colon cancer patients (P < 0.0001). Kaplan-Meier survival analysis result showed that patients with higher Tim-3 expression had a significantly shorter survival time than those with lower Tim-3 expression patients. Our results indicated that Tim-3 might participate in the tumorgenesis of colon cancer and Tim-3 expression might be a potential independent prognostic factor for patients with colorectal cancer.

  15. The immune receptor Tim-3 acts as a trafficker in a Tim-3/galectin-9 autocrine loop in human myeloid leukemia cells

    PubMed Central

    Gonçalves Silva, Isabel; Rüegg, Laura; Gibbs, Bernhard F.; Bardelli, Marco; Fruehwirth, Alexander; Varani, Luca; Berger, Steffen M.; Fasler-Kan, Elizaveta; Sumbayev, Vadim V.

    2016-01-01

    ABSTRACT The immune receptor Tim-3 is often highly expressed in human acute myeloid leukemia (AML) cells where it acts as a growth factor and inflammatory receptor. Recently, it has been demonstrated that Tim-3 forms an autocrine loop with its natural ligand galectin-9 in human AML cells. However, the pathophysiological functions of Tim-3 in human AML cells remain unclear. Here, we report for the first time that Tim-3 is required for galectin-9 secretion in human AML cells. However, this effect is cell-type specific and was found so far to be applicable only to myeloid (and not, for example, lymphoid) leukemia cells. We concluded that AML cells might use Tim-3 as a trafficker for the secretion of galectin-9 which can then be possibly used to impair the anticancer activities of cytotoxic T cells and natural killer (NK) cells. PMID:27622049

  16. The immune receptor Tim-3 acts as a trafficker in a Tim-3/galectin-9 autocrine loop in human myeloid leukemia cells.

    PubMed

    Gonçalves Silva, Isabel; Rüegg, Laura; Gibbs, Bernhard F; Bardelli, Marco; Fruehwirth, Alexander; Varani, Luca; Berger, Steffen M; Fasler-Kan, Elizaveta; Sumbayev, Vadim V

    2016-07-01

    The immune receptor Tim-3 is often highly expressed in human acute myeloid leukemia (AML) cells where it acts as a growth factor and inflammatory receptor. Recently, it has been demonstrated that Tim-3 forms an autocrine loop with its natural ligand galectin-9 in human AML cells. However, the pathophysiological functions of Tim-3 in human AML cells remain unclear. Here, we report for the first time that Tim-3 is required for galectin-9 secretion in human AML cells. However, this effect is cell-type specific and was found so far to be applicable only to myeloid (and not, for example, lymphoid) leukemia cells. We concluded that AML cells might use Tim-3 as a trafficker for the secretion of galectin-9 which can then be possibly used to impair the anticancer activities of cytotoxic T cells and natural killer (NK) cells.

  17. Tim-3 and Tim-4 as the potential targets for antitumor therapy.

    PubMed

    Cheng, Lin; Ruan, Zhihua

    2015-01-01

    Both Tim-3 and Tim-4 belong to the T-cell immunoglobulin and mucin domain (Tim) gene family, which plays a critical role in immunoregulation. Tim-3 has been suggested as a negative regulator of anti-tumor immunity due to its function on inducing T cells exhaustion in cancer. In addition to its expression on exhausted T cells, Tim-3 also has been reported to up-regulate on nature killer (NK) cells and promote NK cells functionally exhausted in cancer. While Tim-3 selectively expression on most types of leukemia stem cells, it promotes the progression of acute myeloid leukemia. Recently, data from experimental models of tumor discovered that Tim-3 and Tim-4 up-regulation on tumor associated dendritic cells and macrophages attenuated the anti-tumor effects of cancer vaccines and chemotherapy. Moreover, co-blockage of Tim-3 and PD-1, Tim-3 and CD137, Tim-3 and carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) could enhance cell-mediated immunity in advanced tumor, and combined treatment with anti-Tim-3 and anti-Tim-4 mAbs further increase the efficacy of cancer vaccines. The therapeutic manipulation of TIM-3 and TIM-4 may provide a novel strategy to improve the clinical efficacy of cancer immunotherapy.

  18. Tim-3 identifies exhausted follicular helper T cells in breast cancer patients.

    PubMed

    Zhu, Shiguang; Lin, Jun; Qiao, Guangdong; Wang, Xingmiao; Xu, Yanping

    2016-09-01

    Breast cancer is the most common cancer diagnosed in women worldwide. Although a series of treatment options have improved the overall 5-year survival rate to 90%, individual responses still vary from patient to patient. New evidence suggested that the infiltration of CXCL13-expressing CD4(+) follicular helper cells (Tfh) in breast tumor predicted better survival. Here, we examined the regulation of Tfh function in breast cancer patients in depth. We found that the frequencies of circulating Tfh cells were not altered in breast cancer patients compared to healthy controls. However, the expression of PD-1 and Tim-3 in Tfh cells was significantly elevated in breast cancer patients. Interestingly, we observed a preferential upregulation of PD-1 in Tim-3(+) Tfh cells compared to Tim-3(-) Tfh cells. Coexpression of PD-1 and Tim-3 is typically a hallmark of functional exhaustion in chronic virus infections and tumor. To examine whether Tim-3(+) identifies exhausted Tfh cells, we stimulated Tfh cells with anti-CD3/CD28, and found that Tim-3(+) T cells expressed reduced frequencies of chemokine CXCL13 and cytokine interleukin 21 (IL-21), and contained fewer proliferating cells, than Tim-3(-) Tfh cells. Compared to those cocultured with Tim-3(-) Tfh cells, naive B cells cocultured with Tim-3(+) Tfh cells resulted in significantly less IgM, IgG and IgA production after 12 day incubation, demonstrating a reduction in Tim-3(+) Tfh-mediated B cell help. Moreover, the frequencies of Tim-3(+) Tfh cells in resected breast tumor were further upregulated than autologous blood, suggesting a participation of Tim-3(+) Tfh cells in tumor physiology. Overall, the data presented here provided new insight in the regulation of Tfh cells in breast cancer patients.

  19. Tim-3 directly enhances CD8 T cell responses to acute Listeria monocytogenes infection

    PubMed Central

    Gorman, Jacob V.; Starbeck-Miller, Gabriel; Pham, Nhat-Long L.; Traver, Geri L.; Rothman, Paul B.; Harty, John T.; Colgan, John D.

    2014-01-01

    Tim-3 is a surface molecule expressed throughout the immune system that can mediate both stimulatory and inhibitory effects. Previous studies have provided evidence that Tim-3 functions to enforce CD8 T cell exhaustion, a dysfunctional state associated with chronic stimulation. In contrast, the role of Tim-3 in the regulation of CD8 T cell responses to acute and transient stimulation remains undefined. To address this knowledge gap, we examined how Tim-3 affects CD8 T cell responses to acute Listeria monocytogenes (LM) infection. Analysis of wild-type (WT) mice infected with LM revealed that Tim-3 was transiently expressed by activated CD8 T cells and was associated primarily with acquisition of an effector phenotype. Comparison of responses to LM by WT and Tim-3 KO mice showed that the absence of Tim-3 significantly reduced the magnitudes of both primary and secondary CD8 T cell responses, which correlated with decreased IFN-γ production and degranulation by Tim-3 KO cells stimulated with peptide antigen ex vivo. To address the T cell-intrinsic role of Tim-3, we analyzed responses to LM infection by WT and Tim-3 KO TCR-transgenic CD8 T cells following adoptive transfer into a shared WT host. In this setting, the accumulation of CD8 T cells and the generation of cytokine-producing cells were significantly reduced by the lack of Tim-3, demonstrating that this molecule has a direct effect on CD8 T cell function. Combined, our results suggest that Tim-3 can mediate a stimulatory effect on CD8 T cell responses to an acute infection. PMID:24567532

  20. Tim-3 enhances FcεRI-proximal signaling to modulate mast cell activation.

    PubMed

    Phong, Binh L; Avery, Lyndsay; Sumpter, Tina L; Gorman, Jacob V; Watkins, Simon C; Colgan, John D; Kane, Lawrence P

    2015-12-14

    T cell (or transmembrane) immunoglobulin and mucin domain protein 3 (Tim-3) has attracted significant attention as a novel immune checkpoint receptor (ICR) on chronically stimulated, often dysfunctional, T cells. Antibodies to Tim-3 can enhance antiviral and antitumor immune responses. Tim-3 is also constitutively expressed by mast cells, NK cells and specific subsets of macrophages and dendritic cells. There is ample evidence for a positive role for Tim-3 in these latter cell types, which is at odds with the model of Tim-3 as an inhibitory molecule on T cells. At this point, little is known about the molecular mechanisms by which Tim-3 regulates the function of T cells or other cell types. We have focused on defining the effects of Tim-3 ligation on mast cell activation, as these cells constitutively express Tim-3 and are activated through an ITAM-containing receptor for IgE (FcεRI), using signaling pathways analogous to those in T cells. Using a variety of gain- and loss-of-function approaches, we find that Tim-3 acts at a receptor-proximal point to enhance Lyn kinase-dependent signaling pathways that modulate both immediate-phase degranulation and late-phase cytokine production downstream of FcεRI ligation.

  1. Enhanced suppressor function of TIM-3+ FoxP3+ regulatory T cells.

    PubMed

    Gautron, Anne-Sophie; Dominguez-Villar, Margarita; de Marcken, Marine; Hafler, David A

    2014-09-01

    T-cell immunoglobulin and mucin domain 3 (TIM-3) is an Ig-superfamily member expressed on IFN-γ-secreting Th1 and Tc1 cells and was identified as a negative regulator of immune tolerance. TIM-3 is expressed by a subset of activated CD4(+) T cells, and anti-CD3/anti-CD28 stimulation increases both the level of expression and the number of TIM-3(+) T cells. In mice, TIM-3 is constitutively expressed on natural regulatory T (Treg) cells and has been identified as a regulatory molecule of alloimmunity through its ability to modulate CD4(+) T-cell differentiation. Here, we examined TIM-3 expression on human Treg cells to determine its role in T-cell suppression. In contrast to mice, TIM-3 is not expressed on Treg cells ex vivo but is upregulated after activation. While TIM-3(+) Treg cells with increased gene expression of LAG3, CTLA4, and FOXP3 are highly efficient suppressors of effector T (Teff) cells, TIM-3(-) Treg cells poorly suppressed Th17 cells as compared with their suppression of Th1 cells; this decreased suppression ability was associated with decreased STAT-3 expression and phosphorylation and reduced gene expression of IL10, EBI3, GZMB, PRF1, IL1Rα, and CCR6. Thus, our results suggest that TIM-3 expression on Treg cells identifies a population highly effective in inhibiting pathogenic Th1- and Th17-cell responses.

  2. Tim-3 promotes intestinal homeostasis in DSS colitis by inhibiting M1 polarization of macrophages.

    PubMed

    Jiang, Xingwei; Yu, Jiahui; Shi, Qingzhu; Xiao, Yan; Wang, Wei; Chen, Guojiang; Zhao, Zhi; Wang, Renxi; Xiao, He; Hou, Chunmei; Feng, Jiannan; Ma, Yuanfang; Shen, Beifen; Wang, Lili; Li, Yan; Han, Gencheng

    2015-10-01

    Tim-3 is involved in the physiopathology of inflammatory bowel disease (IBD), but the underlying mechanism is unknown. Here, we demonstrated that, in mouse with DSS colitis, Tim-3 inhibited the polarization of pathogenic pro-inflammatory M1 macrophages, while Tim-3 downregulation or blockade resulted in an increased M1 response. Adoptive transfer of Tim-3-silenced macrophages worsened DSS colitis and enhanced inflammation, while Tim-3 overexpression attenuated DSS colitis by decreasing the M1 macrophage response. Co-culture of Tim-3-overexpressing macrophages with intestinal lymphocytes decreased the pro-inflammatory response. Tim-3 shaped intestinal macrophage polarization may be TLR-4 dependent since Tim-3 blockade failed to exacerbate colitis or increase M1 macrophage response in the TLR-4 KO model. Finally, Tim-3 signaling inhibited phosphorylation of IRF3, a TLR-4 downstream transcriptional factor regulating macrophage polarization. A better understanding of this pathway may shed new light on colitis pathogenesis and result in a new therapeutic strategy.

  3. Tim-3 enhances FcεRI-proximal signaling to modulate mast cell activation

    PubMed Central

    Phong, Binh L.; Avery, Lyndsay; Sumpter, Tina L.; Gorman, Jacob V.; Watkins, Simon C.; Colgan, John D.

    2015-01-01

    T cell (or transmembrane) immunoglobulin and mucin domain protein 3 (Tim-3) has attracted significant attention as a novel immune checkpoint receptor (ICR) on chronically stimulated, often dysfunctional, T cells. Antibodies to Tim-3 can enhance antiviral and antitumor immune responses. Tim-3 is also constitutively expressed by mast cells, NK cells and specific subsets of macrophages and dendritic cells. There is ample evidence for a positive role for Tim-3 in these latter cell types, which is at odds with the model of Tim-3 as an inhibitory molecule on T cells. At this point, little is known about the molecular mechanisms by which Tim-3 regulates the function of T cells or other cell types. We have focused on defining the effects of Tim-3 ligation on mast cell activation, as these cells constitutively express Tim-3 and are activated through an ITAM-containing receptor for IgE (FcεRI), using signaling pathways analogous to those in T cells. Using a variety of gain- and loss-of-function approaches, we find that Tim-3 acts at a receptor-proximal point to enhance Lyn kinase-dependent signaling pathways that modulate both immediate-phase degranulation and late-phase cytokine production downstream of FcεRI ligation. PMID:26598760

  4. Distinct role of Tim-3 in systemic lupus erythematosus and clear cell renal cell carcinoma.

    PubMed

    Zheng, Hongying; Guo, Xingqing; Tian, Qingwu; Li, Hui; Zhu, Yuanqi

    2015-01-01

    Tim-3 is considered as one of the T-cell immunoglobulin mucin (TIM) gene family members, which contributes to the activating or silencing genes, but the mechanism of Tim-3 function in mediating SLE or tumor metastasis has not been well explored. Here, we reported Tim-3 was high expressed in the peripheral blood mononuclear cells (PBMCs) of patients with SLE, detected by RT-PCR, significantly, GATA-3 mRNA expression also increased in patients with SLE, compared with the healthy control groups. The bioinformatics used to detect the TCGA database indicated the abnormal expression of Tim-3 was involved in several different cancer types. Further, the higher expression of Tim-3 in kidney renal clear cell carcinoma TCGA database indicated it was a marker for worse 5-year survival. The high expression of Tim-3 in different ccRCC cell lines was detected in both RNA level and protein level. Further, two kinds of relative Tim-3 siRNAs in ccRCC cell lines inhibit cell migration and invasion in vitro, However, the inhibition could be partially rescued by the additional GATA3 knockdown. Further, the down regulation in the RNA and protein levels of GATA3, and the negative correlation between Tim-3 and GATA3 implied that suppression of downstream GATA3 was an important mechanism by which Tim-3 triggered metastasis in ccRCC cell lines. Together, our experiments reveal the role for Tim-3 in facilitating SLE or invasive potential of ccRCC cells by either activating GATA3 or inhibiting GATA3, suggesting that Tim-3 might be a potential therapeutic target for treating SLE or clear cell renal cell carcinoma.

  5. Down-regulated expression of Tim-3 promotes invasion and metastasis of colorectal cancer cells.

    PubMed

    Sun, Q Y; Qu, C H; Liu, J Q; Zhang, P; Yao, J

    2017-01-01

    To explore how Tim-3 is expressed and how its expression influences invasion and metastasis of colorectal cancer (CRC) cells. A total of 188 CRC patients were prospectively collected for this study. Meanwhile, 135 normal controls were incorporated during the same period. Intestinal samples of the CRC radical cancerous tissues, paracancerous tissues ( 5.0 cm beyond the cancer tissue) were collected for the following experiment. Furthermore, peripheral venous blood samples (10 ml) were collected from each subject. Immunohistochemical analysis, quantitative real-time polymerase chain reaction (RT-qPCR) and western blot were performed for the detection of Tim-3 in different tissues. The immunohistochemical staining results showed that a positive Tim-3 signal was localized in the cytoplasm and nucleus, observed as yellow or brown granules. Tim-3 was largely expressed in colon carcinoma tissues and normal colon mucosa tissues but was rarely expressed in the cell membrane. RT-qPCR results indicated that Tim-3 mRNA levels were significantly lower in CRC tissues than in paracancerous tissues and normal colon mucosa tissues. A trend of decreased Tim-3 mRNA levels was also found in the paracancerous tissues compared with the normal colon mucosa tissues (all P < 0.05). Western blot results revealed reduced Tim-3 protein expression in CRC tissues compared with normal colon mucosa tissues and paracancerous tissues, and Tim-3 protein expression was much lower in the paracancerous tissues than in the normal colon mucosa tissues (all P < 0.05). Furthermore, obviously lower Tim-3 mRNA levels were found in the poorly differentiated CRC patients and in those with lymph node metastasis and distant metastasis (all P < 0.05). Collectively, Tim-3 expression was mainly located in the cytoplasm and nucleus, showing down-regulated expression in colon carcinoma tissues compared with normal and paracancerous tissues. Reduced Tim-3 expression may promote CRC invasion and metastasis providing a

  6. Tim-3 inhibits macrophage control of Listeria monocytogenes by inhibiting Nrf2.

    PubMed

    Wang, Zhiding; Sun, Dejun; Chen, Guojiang; Li, Ge; Dou, Shuaijie; Wang, Renxi; Xiao, He; Hou, Chunmei; Li, Yan; Feng, Jiannan; Shen, Beifen; Han, Gencheng

    2017-02-16

    T cell immunoglobulin mucin-3 (Tim-3) is an immune checkpoint inhibitor and its dysregulation has been related to T cell tolerance and many immune disorders, such as tumors and infection tolerance. However, the physiopathology roles of Tim-3 in innate immunity remain elusive. Here, we demonstrate that Tim-3 inhibits macrophage phagocytosis of L. monocytogenes by inhibiting the nuclear erythroid 2-related factor 2 (Nrf2) signaling pathway and increases bacterial burden. Tim-3 signaling promotes Nrf2 degradation by increasing its ubiquitination and, as a result, decreasing its nuclear translocation. CD36 and heme oxygenase-1 (HO-1), two downstream molecules in the Tim-3-Nrf2 signaling axis, are involved in the Tim-3- mediated immune evasion of L. monocytogenes both in vitro and in vivo. We here identified new mechanisms by which Tim-3 induces infection tolerance. By modulating the Tim-3 pathway, we demonstrate the feasibility of manipulating macrophage function as a potent tool for treating infectious diseases, such as Listeria infection.

  7. Tim-3: Expression on immune cells and roles at the maternal-fetal interface.

    PubMed

    Hu, Xiao-Hui; Tang, Mao-Xing; Mor, Gil; Liao, Ai-Hua

    2016-11-01

    Successful pregnancy relies on the accurate regulation of the maternal-fetal immune system. Without enough tolerance in the uterine microenvironment, the mother and the hemiallogeneic fetus could not peacefully coexist. T cell immunoglobulin and mucin domain (Tim)-3 is a molecule originally regarded as to be expressed on terminally differentiated IFN-γ expressing CD4(+) T cells (Th1). The engagement of Tim-3 with its ligand, galectin-9 (Gal-9) could induce the exhaustion or apoptosis of effector T cells, and thus might regulate the tolerance. Tim-3 pathway also participates in regulating the activities of CD4(+) regulatory T cells, monocyte-macrophages, dendritic cells and natural killer cells. Dysregulation of Tim-3 expression can elicit excessive or inhibited inflammatory responses and ultimately result in autoimmune diseases, viral or tumor evasion and pregnancy complications. In this review, we will mainly focus on the expression of Tim-3 on local immune cells and its function in pregnancy. In addition, meaningful questions that need further investigation and the potential roles of Tim-3 in fetal tolerance will be discussed. Deeper understanding of the immune checkpoint receptor Tim-3 will shed new light on exploring the pathogenesis of some pregnancy complications, including pre-eclampsia, intrauterine growth restriction, recurrent spontaneous abortion and preterm birth. Tim-3 pathway might be a new target of immune therapy for pregnancy complications in the future.

  8. Microglia activity modulated by T cell Ig and mucin domain protein 3 (Tim-3).

    PubMed

    Wang, Hong-wei; Zhu, Xin-li; Qin, Li-ming; Qian, Hai-jun; Wang, Yiner

    2015-01-01

    Microglia are the main innate immune cells in the central nervous system that are actively involved in maintaining brain homeostasis and diseases. T cell Ig and mucin domain protein 3 (Tim-3) plays critical roles in both the adaptive and the innate immune system and is an emerging therapeutic target for treatment of various disorders. In the brain Tim-3 is specifically expressed on microglia but its functional role is unclear. Here, we showed that Tim-3 was up-regulated on microglia by ATP or LPS stimulation. Tim-3 activation with antibodies increased microglia expression of TGF-β, TNF-α and IL-1β. Blocking of Tim-3 with antibodies decreased the microglial phagocytosis of apoptotic neurons. Tim-3 blocking alleviated the detrimental effect of microglia on neurons and promoted NG2 cell differentiation in co-cultures. Finally, MAPKs namely ERK1/2 and JNK proteins were phosphorylated upon Tim-3 activation in microglia. Data indicated that Tim-3 modulates microglia activity and regulates the interaction of microglia-neural cells.

  9. Tim-3 inhibits macrophage control of Listeria monocytogenes by inhibiting Nrf2

    PubMed Central

    Wang, Zhiding; Sun, Dejun; Chen, Guojiang; Li, Ge; Dou, Shuaijie; Wang, Renxi; Xiao, He; Hou, Chunmei; Li, Yan; Feng, Jiannan; Shen, Beifen; Han, Gencheng

    2017-01-01

    T cell immunoglobulin mucin-3 (Tim-3) is an immune checkpoint inhibitor and its dysregulation has been related to T cell tolerance and many immune disorders, such as tumors and infection tolerance. However, the physiopathology roles of Tim-3 in innate immunity remain elusive. Here, we demonstrate that Tim-3 inhibits macrophage phagocytosis of L. monocytogenes by inhibiting the nuclear erythroid 2-related factor 2 (Nrf2) signaling pathway and increases bacterial burden. Tim-3 signaling promotes Nrf2 degradation by increasing its ubiquitination and, as a result, decreasing its nuclear translocation. CD36 and heme oxygenase-1 (HO-1), two downstream molecules in the Tim-3-Nrf2 signaling axis, are involved in the Tim-3- mediated immune evasion of L. monocytogenes both in vitro and in vivo. We here identified new mechanisms by which Tim-3 induces infection tolerance. By modulating the Tim-3 pathway, we demonstrate the feasibility of manipulating macrophage function as a potent tool for treating infectious diseases, such as Listeria infection. PMID:28205579

  10. Identification of TIM3 2'-fluoro oligonucleotide aptamer by HT-SELEX for cancer immunotherapy.

    PubMed

    Hervas-Stubbs, Sandra; Soldevilla, Mario M; Villanueva, Helena; Mancheño, Uxua; Bendandi, Maurizio; Pastor, Fernando

    2016-01-26

    TIM3 belongs to a family of receptors that are involved in T-cell exhaustion and Treg functions. The development of new therapeutic agents to block this type of receptors is opening a new avenue in cancer immunotherapy. There are currently several clinical trials ongoing to combine different immune-checkpoint blockades to improve the outcome of cancer patients. Among these combinations we should underline PD1:PDL1 axis and TIM3 blockade, which have shown very promising results in preclinical settings. Most of these types of therapeutic agents are protein cell-derived products, which, although broadly used in clinical settings, are still subject to important limitations. In this work we identify by HT-SELEX TIM3 non-antigenic oligonucleotide aptamers (TIM3Apt) that bind with high affinity and specificity to the extracellular motives of TIM3 on the cell surface. The TIM3Apt1 in its monomeric form displays a potent antagonist capacity on TIM3-expressing lymphocytes, determining the increase of IFN-γ secretion. In colon carcinoma tumor-bearing mice, the combinatorial treatment of TIM3Apt1 and PDL1-antibody blockade is synergistic with a remarkable antitumor effect. Immunotherapeutic aptamers could represent an attractive alternative to monoclonal antibodies, as they exhibit important advantages; namely, lower antigenicity, being chemically synthesized agents with a lower price of manufacture, providing higher malleability, and antidote availability.

  11. Impaired functional responses in follicular lymphoma CD8(+)TIM-3(+) T lymphocytes following TCR engagement.

    PubMed

    Gravelle, Pauline; Do, Catherine; Franchet, Camille; Mueller, Sabina; Oberic, Lucie; Ysebaert, Loïc; Larocca, Luigi Maria; Hohaus, Stefan; Calmels, Marie-Noëlle; Frenois, François-Xavier; Kridel, Robert; Gascoyne, Randy D; Laurent, Guy; Brousset, Pierre; Valitutti, Salvatore; Laurent, Camille

    2016-01-01

    Upregulation of T cell immunoglobulin-3 (TIM-3) has been associated with negative regulation of the immune response in chronic infection and cancer, including lymphoma. Here, we investigated the possible correlation between TIM-3 expression by ex vivo cytotoxic T cells (CTL) from follicular lymphoma (FL) biopsies and their functional unresponsiveness that could limit the favorable impact of CTL on disease progression. We report a high percentage of CD8(+)TIM-3(+)T cells in lymph nodes of FL patients. When compared to their CD8(+)TIM-3(-) counterparts, CD8(+)TIM-3(+) T cells exhibited defective cytokine production following TCR engagement. Furthermore, CD8(+)TIM-3(+) T cells display ex vivo markers of lytic granule release and remain unresponsive to further TCR-induced activation of the lytic machinery. Although confocal microscopy showed that TIM-3 expression on CD8(+) T cells correlated with minor alterations of immunological synapse, a selective reduction of ERK signaling in CD8(+)TIM-3(+)T cells was observed by phospho-flow analysis. Finally, short relapse-free survival despite rituximab(R)-chemotherapy was observed in patients with high content of TIM-3(+) cells and a poor infiltrate of granzyme B(+) T cells in FL lymph nodes. Together, our data indicate that, besides selective TCR early signaling defects, TIM-3 expression correlates with unresponsiveness of ex vivo CD8(+) T cells in FL. They show that scores based on the combination of exhaustion and cytolytic markers in FL microenvironment might be instrumental to identify patients at early risk of relapses following R-chemotherapy.

  12. Molecular cloning, characterization and expression analysis of Tim-3 and Galectin-9 in the woodchuck model.

    PubMed

    Liu, Yanan; Wang, Junzhong; Wang, Lu; Wang, Baoju; Yang, Shangqing; Wang, Qin; Luo, Jinzhuo; Feng, Xuemei; Yang, Xuecheng; Lu, Yinping; Roggendorf, Michael; Lu, Mengji; Yang, Dongliang; Liu, Jia

    2017-03-01

    In recent years, a critical role for T cell immunoglobulin mucin domain 3 (Tim-3) and its ligand Galectin-9 (Gal-9) has emerged in infectious disease, autoimmunity and cancer. Manipulating this immune checkpoint may have immunotherapeutic potential and could represent an alternative approach for improving immune responses to viral infections and cancer. The woodchuck (Marmot monax) infected by woodchuck hepatitis virus (WHV) represents an informative animal model to study HBV infection and HCC. In the current study, the cDNA sequences of woodchuck Tim-3 and Gal-9 were cloned, sequenced and characterized. The extracellular domain of Tim-3 cDNA sequence consisted of 576bp coding sequence (CDS) that encoded 192 amino acids. The 1076bp full-length Gal-9 cDNA sequence consisted of 1059bp coding sequence (CDS) that encoded 352 amino acids with a molecular weight of 39.7kDa. The phylogenetic tree analysis revealed that the woodchuck Tim-3 and Gal-9 had the closest genetic relationship with Ictidomys tridecemlineatus. The result of quantification PCR analysis showed that ubiquitous expression of Gal-9 but not Tim-3 in different tissues of naive woodchucks. Elevated liver Gal-9 expression was observed in woodchucks with chronic WHV infection. Moreover, a polyclonal antibody against the extracellular domain of woodchuck Tim-3 were generated and identified by flow cytometry. Our results serve as a foundation for further insight into the role of Tim-3/Galectin-9 signaling pathway in viral hepatitis and HCC in the woodchuck model.

  13. The TIM-3 pathway ameliorates Theiler's murine encephalomyelitis virus-induced demyelinating disease.

    PubMed

    Kaneyama, Tomoki; Tomiki, Hiroki; Tsugane, Sayaka; Inaba, Yuji; Ichikawa, Motoki; Akiba, Hisaya; Yagita, Hideo; Kim, Byung S; Koh, Chang-Sung

    2014-07-01

    Infection by Theiler's murine encephalomyelitis virus (TMEV) in the central nervous system (CNS) induces an immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infection model for human multiple sclerosis. T-cell immunoglobulin and mucin domain-3 (TIM-3) has been demonstrated to play a crucial role in the maintenance of peripheral tolerance. In this study, we examined the regulatory role of the TIM-3 pathway in the development of TMEV-induced demyelinating disease (TMEV-IDD). The expression of TIM-3 was increased at both protein and mRNA levels in the spinal cords of mice with TMEV-IDD compared with naive controls. In addition, by utilizing a blocking mAb, we demonstrate that TIM-3 negatively regulates TMEV-specific ex vivo production of IFN-γ and IL-10 by CD4(+) T cells and IFN-γ by CD8(+) T cells from the CNS of mice with TMEV-IDD at 36 days post-infection (dpi). In vivo blockade of TIM-3 by using the anti-TIM-3 mAb resulted in significant exacerbation of the development of TMEV-IDD both clinically and histologically. The number of infiltrating mononuclear cells in the CNS was also increased in mice administered with anti-TIM-3 mAb both at the induction phase (10 dpi) and at the effector phase (36 dpi). Flow cytometric analysis of intracellular cytokines revealed that the number of CD4(+) T cells producing TNF, IL-4, IL-10 and IL-17 was significantly increased at the effector phase in the CNS of anti-TIM-3 mAb-treated mice. These results suggest that the TIM-3 pathway plays a critical role in the regulation of TMEV-IDD.

  14. A highly conserved tyrosine of Tim-3 is phosphorylated upon stimulation by its ligand galectin-9

    SciTech Connect

    Weyer, Philipp S. van de; Muehlfeit, Michael; Klose, Christoph; Bonventre, Joseph V.; Walz, Gerd; Kuehn, E. Wolfgang . E-mail: wolfgang.kuehn@uniklinik-freiburg.de

    2006-12-15

    Tim-3 is a member of the TIM family of proteins (T-cell immunoglobulin mucin) involved in the regulation of CD4+ T-cells. Tim-3 is a T{sub H}1-specific type 1 membrane protein and regulates T{sub H}1 proliferation and the development of tolerance. Binding of galectin-9 to the extracellular domain of Tim-3 results in apoptosis of T{sub H}1 cells, but the intracellular pathways involved in the regulatory function of Tim-3 are unknown. Unlike Tim-1, which is expressed in renal epithelia and cancer, Tim-3 has not been described in cells other than neuronal or T-cells. Using RT-PCR we demonstrate that Tim-3 is expressed in malignant and non-malignant epithelial tissues. We have cloned Tim-3 from an immortalized liver cell carcinoma line and identified a highly conserved tyrosine in the intracellular tail of Tim-3 (Y265). We demonstrate that Y265 is specifically phosphorylated in vivo by the interleukin inducible T cell kinase (ITK), a kinase which is located in close proximity of the TIM genes on the allergy susceptibility locus 5q33.3. Stimulation of Tim-3 by its ligand galectin-9 results in increased phosphorylation of Y265, suggesting that this tyrosine residue plays an important role in downstream signalling events regulating T-cell fate. Given the role of TIM proteins in autoimmunity and cancer, the conserved SH2 binding domain surrounding Y265 could represent a possible target site for pharmacological intervention.

  15. Elevated TIM3+ hematopoietic stem cells in untreated myelodysplastic syndrome displayed aberrant differentiation, overproliferation and decreased apoptosis.

    PubMed

    Tao, Jing-lian; Li, Li-juan; Fu, Rong; Wang, Hua-quan; Jiang, Hui-juan; Yue, Lan-zhu; Zhang, Wei; Liu, Hui; Ruan, Er-bao; Qu, Wen; Wang, Guo-jin; Wang, Xiao-ming; Wu, Yu-hong; Liu, Hong; Song, Jia; Guan, Jing; Xing, Li-min; Shao, Zong-hong

    2014-06-01

    TIM3, as a negative regulator of anti-tumor immunity, is highly expressed on LSCs, but not on normal HSCs. TIM3 on HSCs in MDS patients has not been clarified. Here, both the percentage of TIM3 on HSCs and the MFI of TIM3+ HSCs were higher in untreated MDS than control and were closed to AML, and excessive TIM3+ HSCs was closely related to clinical parameters: WPSS score, karyotype analysis, morphologic blasts, the number of cytopenia involving hematopoietic lineages, anemia and granulocytopenia. TIM3+ HSCs expressed lower CD11b, TpoR, EpoR, G-CSFR and Annexin V, and higher CD71 and GATA2. TIM3+ HSCs displayed aberrant differentiation, overproliferation and decreased apoptosis. TIM3 might be a promising marker for identifying malignant clone cells in MDS and a candidate for targeted therapy.

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

    PubMed

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

    2015-08-21

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

  17. Expression of human T cell immunoglobulin domain and mucin-3 (TIM-3) and TIM-3 ligands in peripheral blood from patients with systemic lupus erythematosus.

    PubMed

    Jiao, Qingqing; Qian, Qihong; Zhao, Zuotao; Fang, Fumin; Hu, Xiaohan; An, Jingnan; Wu, Jian; Liu, Cuiping

    2016-10-01

    Systemic lupus erythematosus (SLE) is a prototypic systemic autoimmune disease. The T cell immunoglobulin and mucin domain (TIM) family is associated with autoimmune diseases, but its level of expression in the immune cells of patients with SLE is still uncertain. The aim of this study was to examine whether TIM-3 and Galectin-9 (Gal-9) contribute to the pathogenesis of SLE. In total, 30 patients with SLE and 30 healthy controls were recruited, and their levels of TIM-3 expression in peripheral blood mononuclear cells (PBMCs) were examined via flow cytometry. Meanwhile, the levels of Gal-9 expression in serum and in PBMCs were measured via an enzyme-linked immunosorbent assay (ELISA) kit and immunofluorescence staining, respectively. The relation between the level of TIM-3 or Gal-9 expression and the SLE disease activity index (SLEDAI) was also studied. Finally, the function of the TIM-3 and Gal-9 pathway in the pathogenesis of SLE was explored. Our results showed that the levels of expression of TIM-3 and Gal-9 on CD4(+) T cells, CD8(+) T cells, CD56(+) T cells and in serum in patients with SLE were significantly higher than those of healthy controls. We found that the level of Gal-9 expression was significantly higher in both serum and PMBCs of patients with SLE than in healthy controls. The up-regulation of TIM-3 and Gal-9 expression in patients with SLE was closely related to the SLEDAI scores. In addition, Gal-9 blocking antibody significantly inhibited CD3-stimulated PBMC proliferation and Th1-derived cytokines (IL-2, IFN-γ, and TNF-α), Th2-derived cytokines (IL-4, IL-10), a Th17-derived cytokine (IL-17A), and release of a pro-inflammatory factor (IL-6) in patients with SLE. The results suggest that increased expression of TIM-3 and Gal-9 may be a biomarker for SLE diagnosis and that the TIM-3 pathway may be a target for SLE treatment.

  18. Tim-3 blocking rescue macrophage and T cell function against Mycobacterium tuberculosis infection in HIV+ patients

    PubMed Central

    Sada-Ovalle, Isabel; Ocaña-Guzman, Ranferi; Pérez-Patrigeón, Santiago; Chávez-Galán, Leslie; Sierra-Madero, Juan; Torre-Bouscoulet, Luis; Addo, Marylyn M.

    2015-01-01

    Introduction T cell immunoglobulin and mucin domain (Tim) 3 and programmed death 1 (PD-1) are co-inhibitory receptors involved in the so-called T cell exhaustion, and in vivo blockade of these molecules restores T cell dysfunction. High expression of Tim-3 and PD-1 is induced after chronic antigen-specific stimulation of T cells during HIV infection. We have previously demonstrated that the interaction of Tim-3 with its ligand galectin-9 induces macrophage activation and killing of Mycobacterium tuberculosis. Our aim in this study was to analyze the Tim-3 expression profile before and after six months of antiretroviral therapy and the impact of Tim-3 and PD-1 blocking on immunity against M. tuberculosis. Materials and methods HIV+ patients naïve to anti-retroviral therapy (ART) were followed up for six months. Peripheral immune-cell phenotype (CD38/HLA-DR/galectin-9/Tim-3 and PD-1) was assessed by flow cytometry. Supernatants were analyzed with a multiplex cytokine detection system (human Th1/Th2 cytokine Cytometric Bead Array) by flow cytometry. Control of bacterial growth was evaluated by using an in vitro experimental model in which virulent M. tuberculosis-infected macrophages were cultured with T cells in the presence or absence of Tim-3 and PD-1 blocking antibodies. Interleukin-1 beta treatment of infected macrophages was evaluated by enumerating colony-forming units. Results We showed that HIV+ patients had an increased expression of Tim-3 in T cells and were able to control bacterial growth before ART administration. By blocking Tim-3 and PD-1, macrophages and T cells recovered their functionality and had a higher ability to control bacterial growth; this result was partially dependent on the restitution of cytokine production. Conclusions In this study, we demonstrated that increased Tim-3 expression can limit the ability of the immune system to control the infection of intracellular bacteria such as M. tuberculosis. The use of ART and the in vitro

  19. Tim-3 pathway controls regulatory and effector T cell balance during hepatitis C virus infection.

    PubMed

    Moorman, Jonathan P; Wang, Jia M; Zhang, Ying; Ji, Xiao J; Ma, Cheng J; Wu, Xiao Y; Jia, Zhan S; Wang, Ke S; Yao, Zhi Q

    2012-07-15

    Hepatitis C virus (HCV) is remarkable at disrupting human immunity to establish chronic infection. Upregulation of inhibitory signaling pathways (such as T cell Ig and mucin domain protein-3 [Tim-3]) and accumulation of regulatory T cells (Tregs) play pivotal roles in suppressing antiviral effector T cell (Teff) responses that are essential for viral clearance. Although the Tim-3 pathway has been shown to negatively regulate Teffs, its role in regulating Foxp3(+) Tregs is poorly explored. In this study, we investigated whether and how the Tim-3 pathway alters Foxp3(+) Treg development and function in patients with chronic HCV infection. We found that Tim-3 was upregulated, not only on IL-2-producing CD4(+)CD25(+)Foxp3(-) Teffs, but also on CD4(+)CD25(+)Foxp3(+) Tregs, which accumulate in the peripheral blood of chronically HCV-infected individuals when compared with healthy subjects. Tim-3 expression on Foxp3(+) Tregs positively correlated with expression of the proliferation marker Ki67 on Tregs, but it was inversely associated with proliferation of IL-2-producing Teffs. Moreover, Foxp3(+) Tregs were found to be more resistant to, and Foxp3(-) Teffs more sensitive to, TCR activation-induced cell apoptosis, which was reversible by blocking Tim-3 signaling. Consistent with its role in T cell proliferation and apoptosis, blockade of Tim-3 on CD4(+)CD25(+) T cells promoted expansion of Teffs more substantially than Tregs through improving STAT-5 signaling, thus correcting the imbalance of Foxp3(+) Tregs/Foxp3(-) Teffs that was induced by HCV infection. Taken together, the Tim-3 pathway appears to control Treg and Teff balance through altering cell proliferation and apoptosis during HCV infection.

  20. Tim-3 pathway affects NK cell impairment in patients with active tuberculosis.

    PubMed

    Wang, Feng; Hou, Hongyan; Wu, Shiji; Tang, Qing; Huang, Min; Yin, Botao; Huang, Jing; Liu, Weiyong; Mao, Lie; Lu, Yanfang; Sun, Ziyong

    2015-12-01

    Active tuberculosis (TB) patients show impaired NK cell function, and the underlying mechanism remains largely unknown. In this study, we confirmed the decrease in activation, cytokine secretion, and degranulation potential of NK cells in active TB patients. We further investigated whether coinhibitory receptor Tim-3 was involved with impairment of NK cells. Our results revealed that the expression of Tim-3 on NK cells was increased in active TB patients. Tim-3 expression was inversely correlated with IL-12-stimualted IFN-γ production. Moreover, blocking the Tim-3 pathway restored IFN-γ secretion and degranulation of NK cells. Blocking this pathway also increased NK cell cytotoxicity against K562 target cells, and improved the ability of NK cells to control Mtb growth in monocyte-derived macrophages. The Tim-3 expression on NK cells was also observed to be significantly decreased in TB patients post-treatment. In this study, we have identified that Tim-3 is involved with NK cell impairment in TB patients.

  1. Quantitative assessment of TIM-3 polymorphisms and cancer risk in Chinese Han population.

    PubMed

    Gao, Xueren; Yang, Jiaojiao; He, Youji; Zhang, Jianqiong

    2016-06-14

    Previous studies have investigated the associations of TIM-3 polymorphisms (-1516G/T, -574G/T, and +4259T/G) with cancer risk in Chinese Han population, but the results remain conflicting. Therefore, we conducted a meta-analysis to derive a more precise estimation of the associations. The pooled data showed that TIM-3 polymorphisms (-1516G/T, -574G/T, and +4259T/G) were significantly associated with an increased risk of overall cancer in Chinese Han population. Subgroup analyses based on cancer system showed that TIM-3 -1516G/T polymorphism was only associated with an increased risk of digestive system cancer in Chinese Han population. TIM-3 -574G/T polymorphism was associated with an increased risk of digestive system cancer and other cancer in Chinese Han population. TIM-3 +4259T/G polymorphism was only associated with an increased risk of other cancer in Chinese Han population. In summary, our results indicated that TIM-3 polymorphisms (-1516G/T, -574G/T, and +4259T/G) were associated with the increased risk of cancer in Chinese Han population.

  2. Expression of Tim-3 in gastric cancer tissue and its relationship with prognosis.

    PubMed

    Cheng, Gui; Li, Min; Wu, Jun; Ji, Mei; Fang, Cheng; Shi, Hongbing; Zhu, Danxia; Chen, Lujun; Zhao, Jiemin; Shi, Liangrong; Xu, Bin; Zheng, Xiao; Wu, Changping; Jiang, Jingting

    2015-01-01

    As a negative regulatory molecule, T-cell immunoglobulin-and mucin domain-3 (Tim-3) plays a crucial role in the tumor immunological tolerance. In the present study, we aimed to determine the Tim-3 expression in gastric cancer tissue and its relationship with clinicopathological parameters and prognosis. The Tim-3 expression was assessed in 52 gastric cancer specimens and 15 gastritis tissues by flow cytometry, and gastritis tissues served as the control. As a result, we found that the Tim-3 expressions on CD4(+)T cells and CD8(+)T cells in gastric cancer tissue was significantly higher than those in gastritis tissue (P=0.022, P=0.047, respectively). The median expression level of Tim-3 on CD4(+)T cells were significantly correlated with clinicopathological parameters, such as tumor size, lymph node metastasis, the depth of tumor invasion and TNM staging (P=0.042, P=0.026, P=0.001, P=0.003, respectively), while it was not correlated with sex, age and histological subtype (all P>0.05). In CD8(+)T cells, the Tim-3 expression was relevant to tumor invasion and TNM staging (P=0.035, P=0.017, respectively), while it was irrelevant to other clinicopathological parameters (all P>0.05). Additionally, Kaplan-Meier survival curves showed that the median overall survival time of patients with lower Tim-3 expression was greater than that of patients with higher Tim-3 expression in CD4(+)T cells and CD8(+)T cells (χ(2)=18.036, P<0.001 and χ(2)=18.036, P<0.001, respectively). Moreover, the multivariate analysis revealed that the Tim-3 expression and TNM stage were independent prognostic factors for gastric cancer patients (P=0.029, P=0.043 and P=0.003, respectively). These results suggest that Tim-3 played an important role in the development and progression of gastric cancer, and it could be used as an independent prognostic factor for gastric cancer patients.

  3. Negative CD4+TIM-3 Signaling Confers Resistance Against Cold Preservation Damage in Mouse Liver Transplantation

    PubMed Central

    Zhang, Yu; Shen, Xiu-da; Gao, Feng; Nguyen, Terry T.; Shang, Xuanming; Lee, Nayun; Busuttil, Ronald W.; Kupiec-Weglinski, Jerzy W.

    2016-01-01

    Ischemia-reperfusion injury (IRI), an innate immunity-driven local inflammation, remains the major problem in clinical organ transplantation. T cell immunoglobulin and mucin domain (TIM-3) – Galectin-9 (Gal-9) signaling regulates CD4+ Th1 immune responses. Here, we explored TIM-3 – Gal-9 function in a clinically relevant murine model of hepatic cold storage and orthotopic liver transplantation (OLT). C57BL/6 livers, preserved for 20h at 4°C in UW solution, were transplanted to syngeneic mouse recipients. Up-regulation of TIM-3 on OLT-infiltrating activated CD4+ T cells was observed in the early IRI phase (1h). By 6h of reperfusion, OLTs in recipients treated with a blocking anti-TIM-3 Ab were characterized by: 1/ enhanced hepatocellular damage (sALT levels, liver Suzuki's histological score); 2/ polarized cell infiltrate towards Th1/Th17-type phenotype; 3/ depressed T cell exhaustion markers (PD-1, LAG3); and 4/ elevated neutrophil and macrophage infiltration/activation. In parallel studies, adoptive transfer of CD4+ T cells from naïve WT, but not from TIM-3 Tg donors, readily recreated OLT damage in otherwise IR-resistant RAG−/− test recipients. Furthermore, pre-treatment of mice with rGal-9 promoted hepatoprotection against preservation-association liver damage, accompanied by enhanced TIM-3 expression in OLTs. Thus, CD4+ T cell-dependent “negative” TIM-3 costimulation is essential for hepatic homeostasis and resistance against IR stress in OLTs. PMID:25676534

  4. TIM-3 as a Target for Cancer Immunotherapy and Mechanisms of Action.

    PubMed

    Du, Wenwen; Yang, Min; Turner, Abbey; Xu, Chunling; Ferris, Robert L; Huang, Jianan; Kane, Lawrence P; Lu, Binfeng

    2017-03-16

    Cancer immunotherapy has produced impressive clinical results in recent years. Despite the success of the checkpoint blockade strategies targeting cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death receptor 1 (PD-1), a large portion of cancer patients have not yet benefited from this novel therapy. T cell immunoglobulin and mucin domain 3 (TIM-3) has been shown to mediate immune tolerance in mouse models of infectious diseases, alloimmunity, autoimmunity, and tumor Immunity. Thus, targeting TIM-3 emerges as a promising approach for further improvement of current immunotherapy. Despite a large amount of experimental data showing an immune suppressive function of TIM-3 in vivo, the exact mechanisms are not well understood. To enable effective targeting of TIM-3 for tumor immunotherapy, further in-depth mechanistic studies are warranted. These studies will also provide much-needed insight for the rational design of novel combination therapy with other checkpoint blockers. In this review, we summarize key evidence supporting an immune regulatory role of TIM-3 and discuss possible mechanisms of action.

  5. Tim-3 and its role in regulating anti-tumor immunity.

    PubMed

    Das, Madhumita; Zhu, Chen; Kuchroo, Vijay K

    2017-03-01

    Immunotherapy is being increasingly recognized as a key therapeutic modality to treat cancer and represents one of the most exciting treatments for the disease. Fighting cancer with immunotherapy has revolutionized treatment for some patients and therapies targeting the immune checkpoint molecules such as CTLA-4 and PD-1 have achieved durable responses in melanoma, renal cancer, Hodgkin's diseases and lung cancer. However, the success rate of these treatments has been low and a large number of cancers, including colorectal cancer remain largely refractory to CTLA-4 and PD-1 blockade. This has provided impetus to identify other co-inhibitory receptors that could be exploited to enhance response rates of current immunotherapeutic agents and achieve responses to the cancers that are refectory to immunotherapy. Tim-3 is a co-inhibitory receptor that is expressed on IFN-g-producing T cells, FoxP3+ Treg cells and innate immune cells (macrophages and dendritic cells) where it has been shown to suppress their responses upon interaction with their ligand(s). Tim-3 has gained prominence as a potential candidate for cancer immunotherapy, where it has been shown that in vivo blockade of Tim-3 with other check-point inhibitors enhances anti-tumor immunity and suppresses tumor growth in several preclinical tumor models. This review discusses the recent findings on Tim-3, the role it plays in regulating immune responses in different cell types and the rationale for targeting Tim-3 for effective cancer immunotherapy.

  6. The HIF-1/glial TIM-3 axis controls inflammation-associated brain damage under hypoxia

    PubMed Central

    Koh, Han Seok; Chang, Chi Young; Jeon, Sae-Bom; Yoon, Hee Jung; Ahn, Ye-Hyeon; Kim, Hyung-Seok; Kim, In-Hoo; Jeon, Sung Ho; Johnson, Randall S.; Park, Eun Jung

    2015-01-01

    Inflammation is closely related to the extent of damage following cerebral ischaemia, and the targeting of this inflammation has emerged as a promising therapeutic strategy. Here, we present that hypoxia-induced glial T-cell immunoglobulin and mucin domain protein (TIM)-3 can function as a modulator that links inflammation and subsequent brain damage after ischaemia. We find that TIM-3 is highly expressed in hypoxic brain regions of a mouse cerebral hypoxia-ischaemia (H/I) model. TIM-3 is distinctively upregulated in activated microglia and astrocytes, brain resident immune cells, in a hypoxia-inducible factor (HIF)-1-dependent manner. Notably, blockade of TIM-3 markedly reduces infarct size, neuronal cell death, oedema formation and neutrophil infiltration in H/I mice. Hypoxia-triggered neutrophil migration and infarction are also decreased in HIF-1α-deficient mice. Moreover, functional neurological deficits after H/I are significantly improved in both anti-TIM-3-treated mice and myeloid-specific HIF-1α-deficient mice. Further understanding of these insights could serve as the basis for broadening the therapeutic scope against hypoxia-associated brain diseases. PMID:25790768

  7. The HIF-1/glial TIM-3 axis controls inflammation-associated brain damage under hypoxia.

    PubMed

    Koh, Han Seok; Chang, Chi Young; Jeon, Sae-Bom; Yoon, Hee Jung; Ahn, Ye-Hyeon; Kim, Hyung-Seok; Kim, In-Hoo; Jeon, Sung Ho; Johnson, Randall S; Park, Eun Jung

    2015-03-20

    Inflammation is closely related to the extent of damage following cerebral ischaemia, and the targeting of this inflammation has emerged as a promising therapeutic strategy. Here, we present that hypoxia-induced glial T-cell immunoglobulin and mucin domain protein (TIM)-3 can function as a modulator that links inflammation and subsequent brain damage after ischaemia. We find that TIM-3 is highly expressed in hypoxic brain regions of a mouse cerebral hypoxia-ischaemia (H/I) model. TIM-3 is distinctively upregulated in activated microglia and astrocytes, brain resident immune cells, in a hypoxia-inducible factor (HIF)-1-dependent manner. Notably, blockade of TIM-3 markedly reduces infarct size, neuronal cell death, oedema formation and neutrophil infiltration in H/I mice. Hypoxia-triggered neutrophil migration and infarction are also decreased in HIF-1α-deficient mice. Moreover, functional neurological deficits after H/I are significantly improved in both anti-TIM-3-treated mice and myeloid-specific HIF-1α-deficient mice. Further understanding of these insights could serve as the basis for broadening the therapeutic scope against hypoxia-associated brain diseases.

  8. Role of Tim-3 in hepatitis B virus infection: An overview.

    PubMed

    Liu, Yuan; Gao, Li-Fen; Liang, Xiao-Hong; Ma, Chun-Hong

    2016-02-21

    Hepatitis B virus (HBV) infection has received increasing public attention. HBV is the prototypical member of hepadnaviruses, which naturally infect only humans and great apes and induce the acute and persistent chronic infection of hepatocytes. A large body of evidence has demonstrated that dysfunction of the host anti-viral immune response is responsible for persistent HBV replication, unresolved inflammation and disease progression. Many regulatory factors are involved in immune dysfunction. Among these, T cell immunoglobulin domain and mucin domain-3 (Tim-3), one of the immune checkpoint proteins, has attracted increasing attention due to its critical role in regulating both adaptive and innate immune cells. In chronic HBV infection, Tim-3 expression is elevated in many types of immune cells, such as T helper cells, cytotoxic T lymphocytes, dendritic cells, macrophages and natural killer cells. Tim-3 over-expression is often accompanied by impaired function of the above-mentioned immunocytes, and Tim-3 inhibition can at least partially rescue impaired immune function and thus promote viral clearance. A better understanding of the regulatory role of Tim-3 in host immunity during HBV infection will shed new light on the mechanisms of HBV-related liver disease and suggest new therapeutic methods for intervention.

  9. TIM-3 as a Target for Cancer Immunotherapy and Mechanisms of Action

    PubMed Central

    Du, Wenwen; Yang, Min; Turner, Abbey; Xu, Chunling; Ferris, Robert L.; Huang, Jianan; Kane, Lawrence P.; Lu, Binfeng

    2017-01-01

    Cancer immunotherapy has produced impressive clinical results in recent years. Despite the success of the checkpoint blockade strategies targeting cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death receptor 1 (PD-1), a large portion of cancer patients have not yet benefited from this novel therapy. T cell immunoglobulin and mucin domain 3 (TIM-3) has been shown to mediate immune tolerance in mouse models of infectious diseases, alloimmunity, autoimmunity, and tumor Immunity. Thus, targeting TIM-3 emerges as a promising approach for further improvement of current immunotherapy. Despite a large amount of experimental data showing an immune suppressive function of TIM-3 in vivo, the exact mechanisms are not well understood. To enable effective targeting of TIM-3 for tumor immunotherapy, further in-depth mechanistic studies are warranted. These studies will also provide much-needed insight for the rational design of novel combination therapy with other checkpoint blockers. In this review, we summarize key evidence supporting an immune regulatory role of TIM-3 and discuss possible mechanisms of action. PMID:28300768

  10. T-bet-mediated Tim-3 expression dampens monocyte function during chronic hepatitis C virus infection.

    PubMed

    Yi, Wenjing; Zhang, Peixin; Liang, Yan; Zhou, Yun; Shen, Huanjun; Fan, Chao; Moorman, Jonathan P; Yao, Zhi Q; Jia, Zhansheng; Zhang, Ying

    2017-03-01

    Hepatitis C virus (HCV) induces a high rate of chronic infection via dysregulation of host immunity. We have previously shown that T-cell immunoglobulin and mucin domain protein-3 (Tim-3) is up-regulated on monocyte/macrophages (M/Mφ) during chronic HCV infection; little is known, however, about the transcription factor that controls its expression in these cells. In this study, we investigated the role of transcription factor, T-box expressed in T cells (T-bet), in Tim-3 expression in M/Mφ in the setting of HCV infection. We demonstrate that T-bet is constitutively expressed in resting CD14(+) M/Mφ in the peripheral blood. M/Mφ from chronically HCV-infected individuals exhibit a significant increase in T-bet expression that positively correlates with an increased level of Tim-3 expression. Up-regulation of T-bet is also observed in CD14(+) M/Mφ incubated with HCV(+) Huh7.5 cells, as well as in primary M/Mφ or monocytic THP-1 cells exposed to HCV core protein in vitro, which is reversible by blocking HCV core/gC1qR interactions. Moreover, the HCV core-induced up-regulation of T-bet and Tim-3 expression in M/Mφ can be abrogated by incubating the cells with SP600125 - an inhibitor for the c-Jun N-terminal kinase (JNK) signalling pathway. Importantly, silencing T-bet gene expression decreases Tim-3 expression and enhances interleukin-12 secretion as well as signal transducer and activator of transcription 1 phosphorylation. These data suggest that T-bet, induced by the HCV core/gC1qR interaction, enhances Tim-3 expression via the JNK pathway, leading to dampened M/Mφ function during HCV infection. These findings reveal a novel mechanism for Tim-3 regulation via T-bet during HCV infection, providing new targets to combat this global epidemic viral disease.

  11. Identification of TIM3 2′-fluoro oligonucleotide aptamer by HT-SELEX for cancer immunotherapy

    PubMed Central

    Soldevilla, Mario M.; Villanueva, Helena; Mancheño, Uxua; Bendandi, Maurizio

    2016-01-01

    TIM3 belongs to a family of receptors that are involved in T-cell exhaustion and Treg functions. The development of new therapeutic agents to block this type of receptors is opening a new avenue in cancer immunotherapy. There are currently several clinical trials ongoing to combine different immune-checkpoint blockades to improve the outcome of cancer patients. Among these combinations we should underline PD1:PDL1 axis and TIM3 blockade, which have shown very promising results in preclinical settings. Most of these types of therapeutic agents are protein cell-derived products, which, although broadly used in clinical settings, are still subject to important limitations. In this work we identify by HT-SELEX TIM3 non-antigenic oligonucleotide aptamers (TIM3Apt) that bind with high affinity and specificity to the extracellular motives of TIM3 on the cell surface. The TIM3Apt1 in its monomeric form displays a potent antagonist capacity on TIM3-expressing lymphocytes, determining the increase of IFN-γ secretion. In colon carcinoma tumor-bearing mice, the combinatorial treatment of TIM3Apt1 and PDL1-antibody blockade is synergistic with a remarkable antitumor effect. Immunotherapeutic aptamers could represent an attractive alternative to monoclonal antibodies, as they exhibit important advantages; namely, lower antigenicity, being chemically synthesized agents with a lower price of manufacture, providing higher malleability, and antidote availability. PMID:26683225

  12. Differential expression and biochemical activity of the immune receptor Tim-3 in healthy and malignant human myeloid cells.

    PubMed

    Gonçalves Silva, Isabel; Gibbs, Bernhard F; Bardelli, Marco; Varani, Luca; Sumbayev, Vadim V

    2015-10-20

    The T cell immunoglobulin and mucin domain 3 (Tim-3) is a plasma membrane-associated receptor which is involved in a variety of biological responses in human immune cells. It is highly expressed in most acute myeloid leukaemia (AML) cells and therefore may serve as a possible target for AML therapy. However, its biochemical activities in primary human AML cells remain unclear. We therefore analysed the total expression and surface presence of the Tim-3 receptor in primary human AML blasts and healthy primary human leukocytes isolated from human blood. We found that Tim-3 expression was significantly higher in primary AML cells compared to primary healthy leukocytes. Tim-3 receptor molecules were distributed largely on the surface of primary AML cells, whereas in healthy leukocytes Tim-3 protein was mainly expressed intracellularly. In primary human AML blasts, both Tim-3 agonistic antibody and galectin-9 (a Tim-3 natural ligand) significantly upregulated mTOR pathway activity. This was in line with increased accumulation of hypoxia-inducible factor 1 alpha (HIF-1α) and secretion of VEGF and TNF-α. Similar results were obtained in primary human healthy leukocytes. Importantly, in both types of primary cells, Tim-3-mediated effects were compared with those induced by lipopolysaccharide (LPS) and stem cell factor (SCF). Tim-3 induced comparatively moderate responses in both AML cells and healthy leukocytes. However, Tim-3, like LPS, mediated the release of both TNF-α and VEGF, while SCF induced mostly VEGF secretion and did not upregulate TNF-α release.

  13. Expression of the galectin-9-Tim-3 pathway in glioma tissues is associated with the clinical manifestations of glioma.

    PubMed

    Liu, Zengjin; Han, Huamin; He, Xin; Li, Shouwei; Wu, Chenxing; Yu, Chunjiang; Wang, Shengdian

    2016-03-01

    Glioma is known to induce local and systemic immunosuppression, which inhibits antitumor T cell responses. The galectin-9-Tim-3-pathway negatively regulates T cell pathways in the tumor immunosuppressive environment. The present study assessed the expression of Tim-3 and galectin-9 in glioma patients, and evaluated the association between the expression of Tim-3 and galectin-9 with clinical characteristics. The present study identified that Tim-3 expression was significantly increased in peripheral blood T cells of glioma patients compared with those of healthy controls, and was additionally increased on tumor-infiltrating T cells. The expression of Tim-3 on tumor-infiltrating T cells was associated with the World Health Organization (WHO) grade of glioma, but negatively correlated with the Karnofsky Performance Status score of the glioma patients. Immunohistochemical analysis revealed that the expression of galectin-9 in tumor tissues was associated with Tim-3 expression on tumor-infiltrating T cells and the WHO grade of glioma. These findings suggest that the galectin-9-Tim-3 pathway may be critical in the immunoevasion of glioma and may be a potent target for immunotherapy in glioma patients.

  14. Tim-3 is highly expressed in T cells in acute myeloid leukemia and associated with clinicopathological prognostic stratification.

    PubMed

    Li, Caixia; Chen, Xiaochen; Yu, Xiao; Zhu, Yibei; Ma, Chao; Xia, Rui; Ma, Jinfeng; Gu, Caihong; Ye, Lu; Wu, Depei

    2014-01-01

    T cells immunoglobulin mucin 3 (Tim-3) is an important inhibitory stimulatory molecule, which has been reported to play a vital role in the tumor immune escape and be correlated with clinicopathological prognostic stratification in solid tumor. However, the related research is rare of Tim-3 in non-solid tumor, such as acute myeloid leukemia (AML). In this study, we investigated the expression characteristics of Tim-3 on the peripheral blood T cells of newly diagnosed AML patients and its clinical significance. Peripheral blood was obtained from 36 patients with newly diagnosed AML before intervention, with peripheral blood from 20 cases of healthy volunteers collected as normal control. Expression levels of Tim-3 on the peripheral blood T cells were assayed with flow cytometry. We found that Tim-3 expression on the peripheral blood CD4+ T cells and CD8+ T cells in newly diagnosed AML patients were significantly increased compared with that of normal control. CD4+ T cells/CD8+ T cell ratio (CD4/CD8) of peripheral blood in AML patients was significantly correlated with NCCN high risk group. The higher expression level of Tim-3 on CD4+ T cells in the peripheral blood of AML patients had significant correlation with FLT3-ITD mutation, the higher expression level of Tim-3 on CD8+ T cells in AML patients was significantly correlated with NCCN high risk group. To conclude, our results support the concept that Tim-3 is highly expressed on the peripheral blood T cells of AML patients, and Tim-3 expression significantly correlates with clinicopathological prognostic stratification in AMLTim-3, T cell, acute myeloid leukemia, tumor immune escape, clinicopathological prognostic stratification.

  15. Preparation and characterization of a novel nanobody against T-cell immunoglobulin and mucin-3 (TIM-3)

    PubMed Central

    Homayouni, Vida; Ganjalikhani-hakemi, Mazdak; Rezaei, Abbas; Khanahmad, Hossein; Behdani, Mahdi; Lomedasht, Fatemeh Kazemi

    2016-01-01

    Objective(s): As T-cell immunoglobulin and mucin domain 3 (TIM-3) is an immune regulatory molecule; its blocking or stimulating could alter the pattern of immune response towards a desired condition. Based on the unique features of nanobodies, we aimed to construct an anti-TIM-3 nanobody as an appropriate tool for manipulating immune responses for future therapeutic purposes. Materials and Methods: We immunized a camel with TIM-3 antigen and then, synthesized a VHH phage: mid library from its B cell’s transcriptome using nested PCR. Library selection against TIM-3antigen was performed in three rounds of panning. Using phage-ELISA, the most reactive colonies were selected for sub-cloning in soluble protein expression vectors. The Nanobody was purified and confirmed with a nickel-nitrilotriacetic acid (Ni-NTA) column, SDS-PAGE and Western blotting. A flowcytometric analysis was performed to analyze the binding and biologic activities of theTIM-3 specific nanobody with TIM-3 expressing HL-60 and HEK cell lines. Results: Specific 15kD band representing for nanobody was observed on the gel and confirmed with Western blotting. The nanobody showed significant specific immune-reactivity against TIM-3 with a relatively high binding affinity. The nanobody significantly suppressed the proliferation of TIM-3 expressing HL-60 cell line. Conclusion: Finally, we successfully prepared a functional anti-humanTIM-3 specific nanobody with a high affinity and an anti-proliferative activity on an AML cell line in vitro. PMID:27917276

  16. TIM3+FOXP3+ regulatory T cells are tissue-specific promoters of T-cell dysfunction in cancer

    PubMed Central

    Sakuishi, Kaori; Ngiow, Shin Foong; Sullivan, Jenna M.; Teng, Michele W. L.; Kuchroo, Vijay K.; Smyth, Mark J.; Anderson, Ana C.

    2013-01-01

    T-cell immunoglobulin mucin 3 (TIM3) is an inhibitory molecule that has emerged as a key regulator of dysfunctional or exhausted CD8+ T cells arising in chronic diseases such as cancer. In addition to exhausted CD8+ T cells, highly suppressive regulatory T cells (Tregs) represent a significant barrier against the induction of antitumor immunity. We have found that the majority of intratumoral FOXP3+ Tregs express TIM3. TIM3+ Tregs co-express PD-1, are highly suppressive and comprise a specialized subset of tissue Tregs that are rarely observed in the peripheral tissues or blood of tumor-bearing mice. The co-blockade of the TIM3 and PD-1 signaling pathways in vivo results in the downregulation of molecules associated with TIM3+ Treg suppressor functions. This suggests that the potent clinical efficacy of co-blocking TIM3 and PD-1 signal transduction cascades likely stems from the reversal of T-cell exhaustion combined with the inhibition of regulatory T-cell function in tumor tissues. Interestingly, we find that TIM3+ Tregs accumulate in the tumor tissue prior to the appearance of exhausted CD8+ T cells, and that the depletion of Tregs at this stage interferes with the development of the exhausted phenotype by CD8+ T cells. Collectively, our data indicate that TIM3 marks highly suppressive tissue-resident Tregs that play an important role in shaping the antitumor immune response in situ, increasing the value of TIM3-targeting therapeutic strategies against cancer. PMID:23734331

  17. 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.

  18. Co-expression of TIM-3 and CEACAM1 promotes T cell exhaustion in colorectal cancer patients.

    PubMed

    Zhang, Yang; Cai, Pengcheng; Li, Lei; Shi, Liang; Chang, Panpan; Liang, Tao; Yang, Qianqian; Liu, Yang; Wang, Lin; Hu, Lihua

    2017-02-01

    T-cell immunoglobulin domain and mucin domain-3(TIM-3) is an activation induced inhibitory molecule involved in immune tolerance and is recently reported to induce T cell exhaustion which is mediated by carcinoembryonic antigen cell adhesion molecule 1(CEACAM1), another well-known molecule expressed on activated T cells and involved in T cell inhibition. To investigate the expression of TIM-3 and CEACAM1 on circulating CD8(+) T cells and tumor infiltrating lymphocytes (TILs), 65 diagnosed colorectal cancer (CRC) patients and 38 healthy controls were enrolled in this study and the results showed that TIM-3 and CEACAM1 were both highly expressed on circulating CD8(+) T cells in CRC patients and elevated on TILs compared with paraneoplastic T cells. Furthermore, TIM-3(+)CEACAM1(+) CD8(+) T cells represented the most dysfunctional population with the least IFN-γ production. In addition, the expressions of TIM-3 and CEACAM1 were correlated with advanced stage and could be independent risk factors for CRC. We for the first time to our knowledge suggested that co-expression of TIM-3 and CEACAM1 can mediate T cell exhaustion and may be potential biomarkers for CRC prediction, highlighting the possibility of being immunotherapy targets.

  19. Recipient T-Cell TIM-3 and Hepatocyte Galectin-9 Signaling Protects Mouse Liver Transplants Against Ischemia-Reperfusion Injury

    PubMed Central

    Liu, Yuanxing; Ji, Haofeng; Zhang, Yu; Shen, Xiuda; Gao, Feng; He, Xiangyi; Li, Gabriella A.; Busuttil, Ronald W.; Kuchroo, Vijay K.; Kupiec-Weglinski, Jerzy W.

    2015-01-01

    Background & Aims By binding to T-cell immunoglobulin mucin-3 (TIM-3) on activated Th1 cells, Galectin-9 (Gal-9) negatively regulates Th1-type alloimmunity. Although T cells contribute to hepatic ischemia-reperfusion injury (IRI), it is unknown whether negative T cell-dependent TIM-3 costimulation may rescue IR-stressed orthotopic liver transplants (OLT) from innate immunity-driven inflammation. Methods We used WT and TIM-3Tg mice (C57BL6) as liver donors and recipients in a clinically-relevant model of hepatic cold storage (20h at 4°C in UW solution) and syngeneic OLT. Results OLTs in WT or TIM-3Tg->TIM-3Tg groups were resistant against IR- stress, evidenced by preserved hepatocellular function (sALT levels) and liver architecture (Suzuki’s score). In contrast, OLTs in WT or TIM-3Tg->WT groups were susceptible to IRI. TIM-3 induction in recipient circulating CD4+ T cells: 1/ depressed Tbet/IFN-γ, while amplifying GATA3 and IL-4/IL-10 expression in OLTs; 2/ promoted T cell exhaustion (PD-1, LAG-3) phenotype; and 3/ depressed neutrophil and macrophage infiltration/function in OLTs. In parallel studies, we have documented, for the first time that Gal-9, a natural TIM-3 ligand, was produced primarily by and released from IR-stressed hepatocytes, both in-vivo and in-vitro. Moreover, exogenous rGal-9 potentiated liver resistance against IRI by depressing T cell activation and promoting apoptosis of CD4+ T cells. Conclusion Harnessing TIM-3–Gal-9 signaling at T cell–hepatocyte interface facilitates homeostasis in IR-stressed OLTs. Enhancing anti-oxidant hepatocyte Gal-9 potentiates liver IR-resistance. Negative regulation by recipient TIM-3+CD4+ cells provides evidence for cytoprotective functions of a discrete T cell subset, which should be spared when applying T cell-targeted immunosuppression in transplant recipients. PMID:25450716

  20. Interference with Tim-3 protein expression attenuates the invasion of clear cell renal cell carcinoma and aggravates anoikis

    PubMed Central

    Yu, Muming; Lu, Bin; Liu, Yancun; Me, Ying; Wang, Lijun; Li, Hui

    2017-01-01

    Tumor cells resistant to anoikis are considered to be candidates for metastasis. In the present study, the role of Tim-3 in anoikis and its influence on the invasion of clear cell renal cell carcinoma (ccRCC) was investigated. Here, polyhydroxylethylmethacrylate (poly-HEMA) was applied to two ccRCC cell lines, 786-O and Caki-2, to induce detachment from the extracellular matrix (ECM). Tim-3 mRNA and protein expression levels were assayed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot, respectively. Anoikis was measured by Ho33342/PI double staining, acridine orange staining, and further determined using the CytoSelect™ 24-well Anoikis Assay kit. Apoptosis was measured using flow cytometry, E-cadherin and N-cadherin protein expression were determined using western blotting and a Chemicon cell invasion assay kit was used to quantify the invasive capacity of 786-O and Caki-2 cells. It was demonstrated that detachment from the ECM decreases transcription and the protein expression level of Tim-3 in 786-O and Caki-2 cells compared with control cells. Interference with Tim-3 expression using small interfering RNA exacerbated anoikis in 786-O and Caki-2 cells induced by poly-HEMA treatment. E-cadherin upregulation, N-cadherin downregulation, and ECM detachment-induced reduction in invasion ability were all exacerbated by knockdown of Tim-3. In conclusion, interference with Tim-3 expression may attenuate the invasion of renal cell carcinoma by aggravating anoikis, indicating Tim-3 as a potential therapeutic target for treating ccRCC. PMID:28112366

  1. Involvement of Galectin-9/TIM-3 Pathway in the Systemic Inflammatory Response in Early-Onset Preeclampsia

    PubMed Central

    Miko, Eva; Meggyes, Matyas; Bogar, Barbara; Schmitz, Nora; Barakonyi, Aliz; Varnagy, Akos; Farkas, Balint; Tamas, Peter; Bodis, Jozsef; Szekeres-Bartho, Julia; Illes, Zsolt; Szereday, Laszlo

    2013-01-01

    Background Preeclampsia is a common obstetrical disease affecting 3-5% of pregnancies and representing one of the leading causes of both maternal and fetal mortality. Maternal symptoms occur as an excessive systemic inflammatory reaction in response to the placental factors released by the oxidatively stressed and functional impaired placenta. The T-cell immunoglobulin domain and mucin domain (TIM) family is a relatively newly described group of molecules with a conserved structure and important immunological functions. Identification of Galectin-9 as a ligand for TIM-3 has established the Galectin-9/TIM-3 pathway as an important regulator of Th1 immunity and tolerance induction. Methods The aim of our study was to investigate the expression and function of Galectin-9 and TIM-3 molecules by peripheral blood mononuclear cells and the possible role of Galectin-9/TIM-3 pathway in the immunoregulation of healthy pregnancy and early-onset preeclampsia. We determined TIM-3 and Gal-9 expression and cytotoxicicty of peripheral lymphocytes of early-onset preeclamptic women and healthy pregnant woman using flow cytometry. Results Investigating peripheral lymphocytes of women with early-onset preeclampsia, our results showed a decreased TIM-3 expression by T cells, cytotoxic T cells, NK cells and CD56dim NK cells compared to healthy pregnant women. Interestingly, we found a notably increased frequency of Galectin-9 positive cells in each investigated lymphocyte population in the case of early-onset preeclamptic patients. We further demonstrated increased cytotoxic activity by cytotoxic T and CD56dim NK cells in women with early-onset preeclampsia. Our findings showed that the strongest cellular cytotoxic response of lymphocytes occurred in the TIM-3 positive subpopulations of different lymphocytes subsets in early-onset preeclampsia. Conclusion These data suggest that Gal-9/TIM-3 pathway could play an important role in the immune regulation during pregnancy and the altered

  2. Involvement of T cell Ig Mucin-3 (Tim-3) in the negative regulation of inflammatory bowel disease.

    PubMed

    Li, Xia; Chen, Guojiang; Li, Yurong; Wang, Renxi; Wang, Liyan; Lin, Zhou; Gao, Xudong; Feng, Jiannan; Ma, Yuanfang; Shen, Beifen; Li, Yan; Han, Gencheng

    2010-02-01

    Augmented intestinal T cells, especially CD4(+)T cells, are involved in the pathogenesis of inflammatory bowel disease (IBD). We used a murine 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model to investigate whether Tim-3, a negative regulator of CD4(+)T cells, is involved in the suppression of IBD. We found that blocking the Tim-3 signal pathway exacerbated TNBS-induced colitis, as shown by increased weight loss and aggravated tissue injury. Blockade of the Tim-3 pathway resulted in an increase in Tim-3(+)CD4T cells, a biased T effector cell response, and a decrease in Treg cells. It also resulted in an altered profile of co-stimulatory molecules expressed on lymphocytes, which partially explained the biased polarization of different T cell subsets. Our data suggest that the Tim-3 pathway is highly involved in the negative regulation of IBD. A better understanding of this pathway may shed new light on the pathogenesis of this disease.

  3. TIM-3 Rs10515746 (A/C) and Rs10053538 (C/A) Gene Polymorphisms and Risk of Multiple Sclerosis

    PubMed Central

    YAGHOOBI, Esmat; ABEDIAN, Saeed; BABANI, Omid; IZAD, Maryam

    2016-01-01

    Background: Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by auto-reactive T cells against myelin antigens. T-cell immunoglobulin mucin -3 (TIM-3) is a negative regulator glycoprotein expressed by a range of immune cells, including, Th1 cells, activated CD8+ T cells and in a lower level on Th17 cells. A defect in TIM-3 regulation has been shown in multiple sclerosis patients. In humans, several single nucleotide polymorphisms (SNPs) have been identified in the TIM-3 gene and are associated with inflammatory diseases. The aim of this study was to analyze the association between TIM-3 -574A>C and -1516 C>A SNPs in the promoter region, and susceptibility to MS. Methods: DNA samples from 102 patients and 102 healthy controls were genotyped using RFLP-PCR method. Results: In this case-control study, analysis of the alleles and genotypes revealed a significant higher frequency of C/C and lower frequency of A/C genotypes for -574 locus of TIM-3 gene in MS patients (P=0.0002). We also found that C/C genotype for locus of -1516 increased in MS patients, while A/C genotype decreased (P=0.012). Allele C of -574C/C and -1516 C>A SNPs were also more frequent in MS patients (P=0.036 and 0.0027 respectively). Conclusion: -574 A>C and -1516 C>A SNPs in the promoter region of TIM3 gene may affect the disease susceptibility. PMID:27398337

  4. The Influence of Tim-3 Signaling on Central Nervous System Autoimmune Disease is Determined by the Effector Function of the Pathogenic T Cells

    PubMed Central

    Lee, Sarah Y.; Goverman, Joan M.

    2013-01-01

    Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS) mediated by self-reactive, myelin-specific T cells. Both CD4+ and CD8+ T cells play important roles in the pathogenesis of MS. MS is studied using experimental autoimmune encephalomyelitis (EAE), an animal model mediated by myelin-specific T cells. Tim-3 is a cell-surface receptor expressed on CD4+ IFN-γ-secreting Th1 cells, and triggering Tim-3 signaling ameliorated EAE by inducing death in pathogenic Th1 cells in vivo. This suggested that enhancing Tim-3 signaling might be beneficial in patients with MS. However, Tim-3 is also expressed on activated CD8+ T cells, microglia, and dendritic cells (DCs), and the combined effect of manipulating Tim-3 signaling on these cell types during CNS autoimmunity is unknown. Furthermore, CD4+ IL-17-secreting Th17 cells also play a rolein MS but do not express high levels of Tim-3. We investigated Tim-3 signaling in EAE models that include myelin-specific Th17, Th1 and CD8+ T cells. We found that preventing Tim-3 signaling in CD4+ T cells altered the inflammatory pattern in the CNS due to differential effects on Th1 versus Th17 cells. In contrast, preventing Tim-3 signaling during CD8+ T cell-mediated EAE exacerbated disease. We also analyzed the importance of Tim-3 signaling in EAE in innate immune cells. Tim-3 signaling in DCs and microglia did not affect the manifestation of EAE in these models. These results indicate that the therapeutic efficacy of targeting Tim-3 in EAE is dependent on the nature of the effector T cells contributing to the disease. PMID:23562810

  5. A Disintegrin and Metalloprotease (ADAM) 10 and ADAM17 Are Major Sheddases of T Cell Immunoglobulin and Mucin Domain 3 (Tim-3)*

    PubMed Central

    Möller-Hackbarth, Katja; Dewitz, Christin; Schweigert, Olga; Trad, Ahmad; Garbers, Christoph; Rose-John, Stefan; Scheller, Jürgen

    2013-01-01

    T cell immunoglobulin and mucin domain 3 (Tim-3) dampens the response of CD4+ and CD8+ effector T cells via induction of cell death and/or T cell exhaustion and enhances the ability of macrophages to clear pathogens via binding to galectin 9. Here we provide evidence that human Tim-3 is a target of A disintegrin and metalloprotease (ADAM)-mediated ectodomain shedding resulting in a soluble form of Tim-3. We identified ADAM10 and ADAM17 as major sheddases of Tim-3 as shown by ADAM-specific inhibitors and the ADAM10 pro-domain in HEK293 cells and ADAM10/ADAM17-deficient murine embryonic fibroblasts. PMA-induced shedding of Tim-3 was abrogated by deletion of amino acids Glu181–Asp190 of the stalk region and Tim-3 lacking the intracellular domain was not efficiently cleaved after PMA stimulation. Surprisingly, a single lysine residue within the intracellular domain rescues shedding of Tim-3. Shedding of endogenous Tim-3 was found in primary human CD14+ monocytes after PMA and ionomycin stimulation. Importantly, the recently described down-regulation of Tim-3 from Toll-like receptor-activated CD14+ monocytes was caused by ADAM10- and ADAM17-mediated shedding. Inhibition of Tim-3 shedding from lipopolysaccharide-induced monocytes did not influence lipopolysaccharide-induced TNFα and IL-6 but increases IL-12 expression. In summary, we describe Tim-3 as novel target for ADAM-mediated ectodomain shedding and suggest a role of Tim-3 shedding in TLR-mediated immune responses of CD14+ monocytes. PMID:24121505

  6. Tumor-infiltrating Tim-3(+) T cells proliferate avidly except when PD-1 is co-expressed: Evidence for intracellular cross talk.

    PubMed

    Li, Jing; Shayan, Gulidanna; Avery, Lyndsay; Jie, Hyun-Bae; Gildener-Leapman, Neil; Schmitt, Nicole; Lu, Bin Feng; Kane, Lawrence P; Ferris, Robert L

    2016-01-01

    Programmed Death 1 (PD-1) and T cell Ig and mucin domain-3 protein (Tim-3) are immune checkpoint receptors highly expressed on tumor infiltrating T lymphocytes (TIL). PD-1 inhibits T cell activation and type-1 T cell responses, while Tim-3 is proposed to mark more extensively exhausted cells, although the mechanisms underlying Tim-3 function are not clear. Trials of anti-PD-1 therapy have identified a large subset of non-responder patients, likely due to expression of alternative checkpoint molecules like Tim-3. We investigated the phenotypic and functional characteristics of T cells with differential expression of PD-1 (high/low) and Tim-3 (positive/negative), using TIL directly isolated from head and neck squamous cell carcinomas (HNSCC). Unexpectedly, we found that expression of Tim-3 alone does not necessarily mark TIL as dysfunctional/exhausted. In Tim-3-TIL, PD-1 levels correlate with T cell dysfunction, with a PD-1(low/intermed) phenotype identifying recently activated and still functional cells, whereas PD-1(hi)Tim-3(-) T cells are actually exhausted. Nonetheless, PD-1(intermed) cells are still potently suppressed by PD-L1. PD-1 expression was associated with reduced phosphorylation of ribosomal protein S6 (pS6), whereas Tim-3 expression was associated with increased pS6. Using a novel mouse model for inducible Tim-3 expression, we confirmed that expression of Tim-3 does not necessarily render T cells refractory to further activation. These results suggest the existence of PD-1 and Tim-3 crosstalk in regulating antitumor T cell responses, with important implications for anti-PD-1 immunotherapy.

  7. Tim-3 promotes tumor-promoting M2 macrophage polarization by binding to STAT1 and suppressing the STAT1-miR-155 signaling axis.

    PubMed

    Jiang, Xingwei; Zhou, Tingting; Xiao, Yan; Yu, Jiahui; Dou, Shuaijie; Chen, Guojiang; Wang, Renxi; Xiao, He; Hou, Chunmei; Wang, Wei; Shi, Qingzhu; Feng, Jiannan; Ma, Yuanfang; Shen, Beifen; Li, Yan; Han, Gencheng

    2016-01-01

    T cell Ig mucin-3 (Tim-3), an immune checkpoint inhibitor, shows therapeutic potential. However, the molecular mechanism by which Tim-3 regulates immune responses remains to be determined. In particular, very little is known about how Tim-3 works in innate immune cells. Here, we demonstrated that Tim-3 is involved in the development of tumor-promoting M2 macrophages in colon cancer. Manipulation of the Tim-3 pathway significantly affected the polarization status of intestinal macrophages and the progression of colon cancer. The Tim-3 signaling pathway in macrophages was explored using microarray, co-immunoprecipitation, gene mutation, and high-content analysis. For the first time, we demonstrated that Tim-3 polarizes macrophages by directly binding to STAT1 via residue Y256 and Y263 in its intracellular tail and inhibiting the STAT1-miR-155-SOCS1 signaling axis. We also identified a new signaling adaptor of Tim-3 in macrophages, and, by modulating the Tim-3 pathway, demonstrated the feasibility of altering macrophage polarization as a potential tool for treating this kind of disease.

  8. Expression of TIM-3 on CD4+ and CD8+ T cells in the peripheral blood and synovial fluid of rheumatoid arthritis.

    PubMed

    Li, Shufeng; Peng, Dayong; He, Yeteng; Zhang, Hu; Sun, Huaqiang; Shan, Shiying; Song, Yuanlin; Zhang, Shuzhen; Xiao, Hong; Song, Haihan; Zhang, Ming

    2014-10-01

    Rheumatoid arthritis (RA) is characterized by a chronic inflammatory process that targets the synovial lining of diarthrodial joints. TIM-3 plays a key role in the negative regulation of the immune response. In this study, we investigated the expression of TIM-3 on CD4+ and CD8+ T cells from systemic (peripheral blood) and local (synovial fluid) perspectives of RA. Level of TIM-3+ cells from peripheral blood and synovial fluid of patients as well as peripheral blood of healthy controls was measured by flow cytometry. Results showed that TIM-3 expression was significantly increased in both CD4+ and CD8+ T cells in the peripheral blood of RA (p < 0.001 and p < 0.001, respectively). Furthermore, patients revealed even higher expression of TIM-3 in CD4+ and CD8+ T cells in synovial fluid than in peripheral blood. When comparing TIM-3 level with the severity of RA, we identified that the percentage of TIM-3 on both peripheral CD4+ and peripheral CD8+ T cells was negatively correlated with disease activity score 28 (DAS28) of the patients. Similarly, TIM-3 on synovial fluid CD4+ and CD8+ T cells also revealed inverse correlation with DAS28 of the cases. Our data demonstrate a negative correlation between TIM-3 and the disease progression of RA.

  9. Adaptive resistance to anti-PD1 therapy by Tim-3 upregulation is mediated by the PI3K-Akt pathway in head and neck cancer.

    PubMed

    Shayan, Gulidanna; Srivastava, Raghvendra; Li, Jing; Schmitt, Nicole; Kane, Lawrence P; Ferris, Robert L

    2017-01-01

    Programmed Death 1 (PD-1) and T cell Ig and mucin domain-3 protein (Tim-3) are immune checkpoint receptors that are expressed on tumor-infiltrating lymphocytes (TIL) in tumor-bearing mice and humans. As anti-PD-1 single agent response rates are only <20% in head and neck squamous cell carcinoma (HNSCC) patients, it is important to understand how multiple inhibitory checkpoint receptors maintain suppressed cellular immunity. One such receptor, Tim-3, activates downstream proliferative pathways through Akt/S6, and is highly expressed in dysfunctional TIL. We observed that PD-1 and Tim-3 co-expression was associated with a more exhausted phenotype, with the highest PD-1 levels on TIL co-expressing Tim-3. Dampened Akt/S6 phosphorylation in these PD-1(+)Tim-3(+) TIL, when the PD-1 pathway was ligated, suggested that signaling cross-talk could lead to escape through Tim-3 expression. Indeed, PD-1 blockade of human HNSCC TIL led to further Tim-3 upregulation, supporting a circuit of compensatory signaling and potentially permitting escape from anti-PD-1 blockade in the tumor microenvironment. Also, in a murine HNC tumor model that is partially responsive to anti-PD-1 therapy, Tim-3 was upregulated in TIL from persistently growing tumors. Significant antitumor activity was observed after sequential addition of anti-Tim-3 mAb to overcome adaptive resistance to anti-PD-1 mAb. This increased Tim-3-mediated escape of exhausted TIL from PD-1 inhibition that was mediated by phospho-inositol-3 kinase (PI3K)/Akt complex downstream of TCR signaling but not cytokine-mediated pathways. Taken together, we conclude that during PD-1 blockade, TIL upregulate Tim-3 in a PI3K/Akt-dependent manner, providing further support for dual targeting of these molecules for more effective cancer immunotherapy.

  10. The amelioration of composite tissue allograft rejection by TIM-3-modified dendritic cell: Regulation of the balance of regulatory and effector T cells.

    PubMed

    Wang, Yaojun; Zheng, Zhao; Zhu, Xiongxiang; Han, Juntao; Dong, Maolong; Tao, Ke; Wang, Hongtao; Wang, Yunchuan; Hu, Dahai

    2016-01-01

    T cell-dependent immune responses play a central role in allograft rejection. Exploring ways to disarm alloreactive T cells represents a potential strategy to promote long-term allograft acceptance and survival. T cell Ig domain and mucin domain 3 (TIM-3) has previously been demonstrated as a central regulator of T helper 1 (Th1) responses and immune tolerance. Hence, TIM-3 may be an important molecule for decreasing immunological rejection during composite tissue allotransplantation (CTA). In this study, BALB/c and C57BL/6 mice were chosen as the experimental animals. The effects of TIM-3 on allograft rejection were explored using TIM-3-modified mature dendritic cells (TIM-3 mDCs). A laser speckle blood flow (LSBF) imager was used to evaluate blood distribution of the BALB/c mice. ELISA, MTT, ELISPOT assays and flow cytometry analysis were carried out for further researches. We found that TIM-3 could obviously prolong the survival time of the transplanted limbs. And TIM-3 could mitigate the immune response and thus enhance immune tolerance after CTA. Also, TIM-3 can induce lymphocyte hyporesponsiveness, including facilitating lymphocyte apoptosis, decreasing lymphocyte proliferation, and influencing the secretion of inflammatory cytokines by CD4(+) T cells. Furthermore, TIM-3 overexpression could induce CD4(+) T cells to differentiate into regulatory T cells (Tregs), which recalibrate the effector and regulatory arms of the alloimmune response. In summary, we concluded that TIM-3 can mitigate allograft rejection and thus enhance immune tolerance by inducing lymphocyte hyporesponsiveness and increasing the number of Tregs of the alloimmune response. TIM-3 may be a potential therapeutic molecule for allograft rejection in CTA.

  11. [A TIM-3/galectin-9 autocrine stimulatory loop drives self-renewal of human myeloid leukemia stem cells and leukemia progression].

    PubMed

    Kikushige, Yoshikane

    2016-04-01

    Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. We previously reported that the T-cell immunoglobulin mucin-3 (TIM-3) is expressed on the LCS surface in most types of AML. Since only the TIM-3(+), i.e. not the TIM-3(-), fraction of human AML cells can reconstitute human AML in immunodeficient mice, we hypothesized that the TIM-3 has an essential function in maintaining AML LSCs. Herein, we show that TIM-3 and its ligand, galectin-9 (Gal-9), constitute an autocrine loop critical for human AML LSC development. Serum Gal-9 was significantly elevated in primary AML patients and in mice xenografted with human AML. Neutralization of Gal-9 inhibited xenogeneic reconstitution of human AML, as well as Gal-9 ligation of TIM-3 co-activated NF-κB and β-catenin signaling, suggesting that TIM-3 signaling is necessary for LSC self-renewal. Interestingly, identical changes were found to be involved in the progressive transformation of a variety of pre-leukemic disorders into myeloid leukemia. Thus, molecules constituting the TIM-3/Gal-9 autocrine loop are potential therapeutic targets applicable to most types of myeloid leukemia.

  12. PD-1 and Tim-3 pathways are associated with regulatory CD8+ T-cell function in decidua and maintenance of normal pregnancy.

    PubMed

    Wang, S-C; Li, Y-H; Piao, H-L; Hong, X-W; Zhang, D; Xu, Y-Y; Tao, Y; Wang, Y; Yuan, M-M; Li, D-J; Du, M-R

    2015-05-07

    CD8+ T cells are critical in the balance between fetal tolerance and antiviral immunity. T-cell immunoglobulin mucin-3 (Tim-3) and programmed cell death-1 (PD-1) are important negative immune regulatory molecules involved in viral persistence and tumor metastasis. Here, we demonstrate that Tim-3+PD-1+CD8+ T cells from decidua greatly outnumbered those from peripheral blood during human early pregnancy. Co-culture of trophoblasts with CD8+ T cells upregulated PD-1+ and/or Tim-3+ immune cells. Furthermore, the population of CD8+ T cells co-expressing PD-1 and Tim-3 was enriched within the intermediate memory subset in decidua. This population exhibited high proliferative activity and Th2-type cytokine producing capacity. Blockade of Tim-3 and PD-1 resulted in decreased in vitro proliferation and Th2-type cytokine production while increased trophoblast killing and IFN-γ producing capacities of CD8+ T cells. Pregnant CBA/J females challenged with Tim-3 and/or PD-1 blocking antibodies were more susceptible to fetal loss, which was associated with CD8+ T-cell dysfunction. Importantly, the number and function of Tim-3+PD-1+CD8+ T cells in decidua were significantly impaired in miscarriage. These findings underline the important roles of Tim-3 and PD-1 pathways in regulating decidual CD8+ T-cell function and maintaining normal pregnancy.

  13. Preferential Tim-3 expression on Treg and CD8(+) T cells, supported by tumor-associated macrophages, is associated with worse prognosis in gastric cancer.

    PubMed

    Shen, Pinying; Yue, Rongxi; Tang, Jiahong; Si, Haige; Shen, Liqun; Guo, Changsheng; Zhang, Lixin; Han, Huaizhong; Song, Haihan K; Zhao, Pengfei; Wang, Ning; Song, Zongchang; Guo, Chunliang

    2016-01-01

    While infection with H. pylori is a strong risk factor for gastric cancer, most H. pylori-colonized individuals, even those with the high-risk CagA(+)VacA(+) strain, remain asymptomatic over their lifetime. We hypothesized that the discordant outcomes were due to differences in the host immune responses. Previously, Tim-3-mediated immune modulation was observed in H. pylori-challenged mice. In this study, we compared Tim-3-related responses in CagA(+)VacA(+) H. pylori-infected asymptomatic individuals and H. pylori-associated gastric adenocarcinoma patients. We showed that compared to H. pylori-uninfected individuals, both H. pylori-infected asymptomatic and gastric cancer patients upregulated Tim-3 overall. However, the Tim-3 upregulation was enriched on Th1 cells in asymptomatic patients and on Treg and CD8(+) T cells in gastric cancer patients, with respective differences in T cell subset functions. In gastric cancer patients, high Tim-3 expression on Treg and CD8(+) T cells, but not on Th1 cells, was associated with worse prognosis. H. pylori-antigen presentation by tumor-associated macrophages upregulated Tim-3 expression more effectively than by blood monocyte-derived macrophages in vitro. The upregulation of Tim-3 in vitro depended on the concentration of H. pylori antigen but not on whether the cells were from asymptomatic or cancer patients. These data suggest that the discrepancy in Tim-3 upregulation in asymptomatic and cancer subjects is induced by cancer but not the other way around. Once gastric cancer is developed, Tim-3 expression is associated with worse prognosis.

  14. Coexpression of Tim-3 and PD-1 identifies a CD8+ T-cell exhaustion phenotype in mice with disseminated acute myelogenous leukemia.

    PubMed

    Zhou, Qing; Munger, Meghan E; Veenstra, Rachelle G; Weigel, Brenda J; Hirashima, Mitsuomi; Munn, David H; Murphy, William J; Azuma, Miyuki; Anderson, Ana C; Kuchroo, Vijay K; Blazar, Bruce R

    2011-04-28

    Tumor-associated immune suppression can lead to defective T cell-mediated antitumor immunity. Here, we identified a unique phenotype of exhausted T cells in mice with advanced acute myelogenous leukemia (AML). This phenotype is characterized by the coexpression of Tim-3 and PD-1 on CD8(+) T cells in the liver, the major first site of AML metastases. PD-1 and Tim-3 coexpression increased during AML progression. PD-1(+)Tim-3(+) CD8(+) T cells were deficient in their ability to produce IFN-γ, TNF-α, and IL-2 in response to PD-1 ligand (PDL1) and Tim-3 ligand (galectin-9) expressing AML cells. PD-1 knockout (KO), which were partially resistant to AML challenge, up-regulated Tim-3 during AML progression and such Tim-3(+)PD-1- KO CD8(+) T cells had reduced cytokine production. Galectin-9 KO mice were more resistant to AML, which was associated with reduced T-regulatory cell accumulation and a modest induction of PD-1 and Tim-3 expression on CD8(+) T cells. Whereas blocking the PD-1/PDL1 or Tim-3/galectin-9 pathway alone was insufficient to rescue mice from AML lethality, an additive effect was seen in reducing-albeit not eliminating-both tumor burden and lethality when both pathways were blocked. Therefore, combined PD-1/PDL1 and Tim-3/galectin-9 blockade may be beneficial in preventing CD8(+) T-cell exhaustion in patients with hematologic malignancies such as advanced AML.

  15. Characterization of age-associated exhausted CD8⁺ T cells defined by increased expression of Tim-3 and PD-1.

    PubMed

    Lee, Kyoo-A; Shin, Kwang-Soo; Kim, Ga-Young; Song, You Chan; Bae, Eun-Ah; Kim, Il-Kyu; Koh, Choong-Hyun; Kang, Chang-Yuil

    2016-04-01

    Aging is accompanied by altered T-cell responses that result in susceptibility to various diseases. Previous findings on the increased expression of inhibitory receptors, such as programmed cell death protein 1 (PD-1), in the T cells of aged mice emphasize the importance of investigations into the relationship between T-cell exhaustion and aging-associated immune dysfunction. In this study, we demonstrate that T-cell immunoglobulin mucin domain-3 (Tim-3), another exhaustion marker, is up-regulated on aged T cells, especially CD8(+) T cells. Tim-3-expressing cells also produced PD-1, but Tim-3(+) PD-1(+) CD8(+) T cells had a distinct phenotype that included the expression of CD44 and CD62L, from Tim-3(-) PD-1(+) cells. Tim-3(+) PD-1(+) CD8(+) T cells showed more evident properties associated with exhaustion than Tim-3(-) PD-1(+) CD8(+) T cells: an exhaustion-related marker expression profile, proliferative defects following homeostatic or TCR stimulation, and altered production of cytokines. Interestingly, these cells produced a high level of IL-10 and induced normal CD8(+) T cells to produce IL-10, which might contribute to immune dysregulation in aged mice. The generation of Tim-3-expressing CD8(+) T cells in aged mice seems to be mediated by encounters with antigens but not by specific infection, based on their high expression of CD49d and their unbiased TCR Vβ usage. In conclusion, we found that a CD8(+) T-cell population with age-associated exhaustion was distinguishable by its expression of Tim-3. These results provide clues for understanding the alterations that occur in T-cell populations with age and for improving dysfunctions related to the aging of the immune system.

  16. MicroRNA-155 regulates interferon-γ production in natural killer cells via Tim-3 signalling in chronic hepatitis C virus infection.

    PubMed

    Cheng, Yong Q; Ren, Jun P; Zhao, Juan; Wang, Jia M; Zhou, Yun; Li, Guang Y; Moorman, Jonathan P; Yao, Zhi Q

    2015-08-01

    Host immune responses must be tightly regulated by an intricate balance between positive and negative signals while fighting pathogens; persistent pathogens may usurp these regulatory mechanisms to dampen host immunity to facilitate survival in vivo. Here we report that Tim-3, a negative signalling molecule expressed on monocytes and T cells, is up-regulated on natural killer (NK) cells in individuals chronically infected with hepatitis C virus (HCV). Additionally, the transcription factor T-bet was also found to be up-regulated and associated with Tim-3 expression in NK cells during chronic HCV infection. MicroRNA-155 (miR-155), an miRNA that inhibits signalling proteins involved in immune responses, was down-regulated in NK cells by HCV infection. This Tim-3/T-bet over-expression and miR-155 inhibition were recapitulated in vitro by incubating primary NK cells or NK92 cell line with Huh-7 hepatocytes expressing HCV. Reconstitution of miR-155 in NK cells from HCV-infected patients led to a decrease in T-bet/Tim-3 expression and an increase in interferon-γ production. Blocking Tim-3 signalling also enhanced interferon-γ production in NK cells by improving signal transducer and activator of transcription-5 phosphorylation. These data indicate that HCV-induced, miR-155-regulated Tim-3 expression regulates NK cell function, suggesting a novel mechanism for balancing immune clearance and immune injury during chronic viral infection.

  17. Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer

    PubMed Central

    2013-01-01

    Background T-cell immunoglobulin and mucin domain 3 (TIM-3) is known as a negative immune regulator and emerging data have implicated TIM-3 a pivotal role in suppressing antitumor immunity. The co-stimulatory receptor CD137 is transiently upregulated on T-cells following activation and increases their proliferation and survival when engaged. Although antagonistic anti-TIM-3 or agonistic anti-CD137 antibodies can promote the rejection of several murine tumors, some poorly immunogenic tumors were refractory to this treatment. In this study, we sought to evaluate whether combined TIM-3 blockade and CD137 activation would significantly improve the immunotherapy in the murine ID8 ovarian cancer model. Methods Mice with established ID8 tumor were intraperitoneally injected with single or combined anti-TIM-3/CD137 monoclonal antibody (mAb); mice survival was recorded, the composition and gene expression of tumor-infiltrating immune cells in these mice was analyzed by flow cytometry and quantitative RT-PCR respectively, and the function of CD8+ cells was evaluated by ELISA and cytotoxicity assay. Results Either anti-TIM-3 or CD137 mAb alone, although effective in 3 days established tumor, was unable to prevent tumor progression in mice bearing 10 days established tumor, however, combined anti-TIM-3/CD137 mAb significantly inhibited the growth of these tumors with 60% of mice tumor free 90 days after tumor inoculation. Therapeutic efficacy was associated with a systemic immune response with memory and antigen specificity, required CD4+ cells and CD8+ cells. The 2 mAb combination increased CD4+ and CD8+ cells and decreased immunosuppressive CD4+FoxP3+ regulatory T (Treg) cells and CD11b+Gr-1+ myeloid suppressor cells (MDSC) at tumor sites, giving rise to significantly elevated ratios of CD4+ and CD8+ cells to Treg and MDSC; This is consistent with biasing local immune response towards an immunostimulatory Th1 type and is further supported by quantitative RT-PCR data

  18. Association of TIM-1 5383-5397ins/del and TIM-3 -1541C>T polymorphisms with multiple sclerosis in Isfahan population.

    PubMed

    Mazrouei, F; Ganjalikhani-Hakemi, M; Salehi, R; Alesahebfosoul, F; Etemadifar, M; Pouladian, M; Meshkat, R; Nekoueian, Sh; Zarkesh-Esfahani, H; Ziyaee-Ghahnaviyeh, M

    2016-06-01

    Multiple sclerosis (MS) is an organ-specific autoimmune disease in central nervous system, affecting about 2.5 million people around the world. Probable involvement of two newly identified immunoregulator molecules, TIM-1 and TIM-3, has been reported in autoimmune diseases. In this study, for the first time, the association of TIM-1 5383-5397ins/del and TIM-3 -1541C>T polymorphisms with MS in an Iranian population was considered. The results of our study showed that there is no significant association between TIM-1 5383-5397ins/del and MS (P = 0.38); however, the frequency of CT genotype of TIM-3 -1541C>T in patient group was significantly higher than the control group, and there was a significant association between CT genotype and MS (P = 0.009, OR = 4.08).

  19. Tumor antigen-specific CD8(+) T cells are negatively regulated by PD-1 and Tim-3 in human gastric cancer.

    PubMed

    Lu, Xu; Yang, Lin; Yao, Daxing; Wu, Xuan; Li, Jingpo; Liu, Xuesong; Deng, Lijuan; Huang, Caiting; Wang, Yue; Li, Dan; Liu, Jingwei

    2017-03-01

    Cytotoxic CD8 T lymphocytes that are present in tumors and capable of recognizing tumor epitopes are nevertheless generally important in eliciting tumor rejection. NY-ESO-1 is a major target of CD8(+) T cell recognition in gastric cancer (GC) and is among the most immunogenic tumor antigens defined to date. Thus, identifying the immune escape mechanisms responsible for inducing tumor-specific CD8(+) T cell dysfunction may reveal effective strategies for immunotherapy. In an effort to understand in vivo tolerance mechanisms, we assessed the phenotype and function of NY-ESO-1-specific CD8(+) T cells derived from peripheral blood lymphocytes (PBLs) and tumor-associated lymphocytes (TALs) of GC patients. Here, we report that Tim-3 expression defines a subpopulation of PD-1(+) exhausted NY-ESO-1-specific CD8(+) T cell and PD-1(+)Tim-3(+) CD8(+) T cells represented the largest subset of NY-ESO-1-specific CD8(+) T cells in GC patients. Functionally, CD8(+)PD-1(+)Tim-3(+) T cells were more impaired in IFN-γ, TNF-α and IL-2 production compared with PD-1(+)Tim-3(-) or PD-1(-)Tim-3(-) subsets. Dual blockade of Tim-3 and PD-1 during T-cell priming efficiently augmented proliferation and cytokine production by NY-ESO-1-specific CD8(+) T cells could potentially be improved by therapeutic targeting of these inhibitory receptors, indicating that antitumor function of NY-ESO-1-specific CD8(+) T cells could potentially be improved by therapeutic targeting of these inhibitory receptors.

  20. Tim-3 alters the balance of IL-12/IL-23 and drives TH17 cells: role in hepatitis B vaccine failure during hepatitis C infection

    PubMed Central

    Wang, Jia M.; Ma, Cheng J.; Li, Guang Y.; Wu, Xiao Y.; Thayer, Penny; Greer, Pamela; Smith, Ashley M.; High, Kevin P.; Moorman, Jonathan P; Yao, Zhi Q.

    2013-01-01

    Hepatitis B virus (HBV) vaccination is recommended for individuals with hepatitis C virus (HCV) infection given their shared risk factors and increased liver-related morbidity and mortality upon super-infection. Vaccine responses in this setting are often blunted, with poor response rates to HBV vaccinations in chronically HCV-infected individuals compared to healthy subjects. In this study, we investigated the role of T cell immunoglobulin mucin domain-3 (Tim-3)-mediated immune regulation in HBV vaccine responses during HCV infection. We found that Tim-3, a marker for T cell exhaustion, was over-expressed on monocytes, leading to a differential regulation of IL-12/IL-23 production with in turn TH17 cell accumulation, in HCV-infected HBV vaccine non-responders compared to HCV-infected HBV vaccine responders or healthy subjects (HS). Importantly, ex vivo blockade of Tim-3 signaling corrected the imbalance of IL-12/IL-23 as well as the IL-17 bias observed in HBV vaccine non-responders during HCV infection. These results suggest that Tim-3-mediated dysregulation of innate to adaptive immune responses is involved in HBV vaccine failure in individuals with chronic HCV infection, raising the possibility that blocking this negative signaling pathway might improve the success rate of HBV immunization in the setting of chronic viral infection. PMID:23499521

  1. IL-15 induces strong but short-lived tumor-infiltrating CD8 T cell responses through the regulation of Tim-3 in breast cancer

    SciTech Connect

    Heon, Elise K.; Wulan, Hasi; Macdonald, Loch P.; Malek, Adel O.; Braunstein, Glenn H.; Eaves, Connie G.; Schattner, Mark D.; Allen, Peter M.; Alexander, Michael O.; Hawkins, Cynthia A.; McGovern, Dermot W.; Freeman, Richard L.; Amir, Eitan P.; Huse, Jason D.; Zaltzman, Jeffrey S.; Kauff, Noah P.; Meyers, Paul G.; Gleason, Michelle H.; Overholtzer, Michael G.; Wiseman, Sam S.; and others

    2015-08-14

    IL-15 has pivotal roles in the control of CD8{sup +} memory T cells and has been investigated as a therapeutic option in cancer therapy. Although IL-15 and IL-2 share many functions together, including the stimulation of CD8 T cell proliferation and IFN-γ production, the different in vivo roles of IL-15 and IL-2 have been increasingly recognized. Here, we explored the different effects of IL-15 and IL-2 on tumor-infiltrating (TI) T cells from resected breast tumors. We found that neither IL-2 nor IL-15 induced intratumoral CD8 T cell proliferation by itself, but after CD3/CD28-stimulation, IL-15 induced significantly higher proliferation than IL-2 during early time points, at day 2, day 3 and day 6. However, the IL-15-induced proliferation leveled off at day 9 and day 12, whereas IL-2 induced lower but progressive proliferation at each time point. Furthermore, IL-15 caused an early and robust increase of IFN-γ in the supernatant of TI cell cultures, which diminished at later time points, while the IL-2-induced IFN-γ production remained constant over time. In addition, the IL-15-costimulated CD8 T cells presented higher frequencies of apoptotic cells. The diminishing IL-15-induced response was possibly due to regulatory and/or exhaustion mechanisms. We did not observe increased IL-10 or PD-1 upregulation, but we have found an increase of Tim-3 upregulation on IL-15-, but not IL-2-stimulated cells. Blocking Tim-3 function using anti-Tim-3 antibodies resulted in increased IL-15-induced proliferation and IFN-γ production for a prolonged period of time, whereas adding Tim-3 ligand galectin 9 led to reduced proliferation and IFN-γ production. Our results suggest that IL-15 in combination of Tim-3 blocking antibodies could potentially act as an IL-2 alternative in tumor CD8 T cell expansion in vitro, a crucial step in adoptive T cell therapy. - Highlights: • We explored the effects of IL-15 and IL-2 on tumor-infiltrating (TI) T cells of breast cancer. • IL-15

  2. PD-1 and Tim-3 regulate the expansion of tumor antigen-specific CD8+ T cells induced by melanoma vaccines

    PubMed Central

    Fourcade, Julien; Sun, Zhaojun; Pagliano, Ornella; Chauvin, Joe-Marc; Sander, Cindy; Janjic, Bratislav; Tarhini, Ahmad A.; Tawbi, Hussein A.; Kirkwood, John M.; Moschos, Stergios; Wang, Hong; Guillaume, Philippe; Luescher, Immanuel F.; Krieg, Arthur; Anderson, Ana C.; Kuchroo, Vijay K.; Zarour, Hassane M.

    2014-01-01

    Although melanoma vaccines stimulate tumor antigen (TA)-specific CD8+ T cells, objective clinical responses are rarely observed. To investigate this discrepancy, we evaluated the character of vaccine-induced CD8+ T cells with regard to the inhibitory T cell co-receptors PD-1 and Tim-3 in metastatic melanoma patients who were administered tumor vaccines. The vaccines included incomplete Freund's adjuvant (IFA), CpG oligodeoxynucleotide (CpG) and the HLA-A2-restricted analog peptide NY-ESO-1 157-165V, either by itself or in combination with the pan-DR epitope NY-ESO-1 119-143. Both vaccines stimulated rapid TA-specific CD8+ T-cell responses detected ex vivo, however, TA-specific CD8+ T cells produced more IFN-γ and exhibited higher lytic function upon immunization with MHC class I and class II epitopes. Notably, the vast majority of vaccine-induced CD8+ T cells upregulated PD-1 and a minority also upregulated Tim-3. Levels of PD-1 and Tim-3 expression by vaccine-induced CD8+ T cells at the time of vaccine administration correlated inversely with their expansion in vivo. Dual blockade of PD-1 and Tim-3 enhanced the expansion and cytokine production of vaccine-induced CD8+ T cells in vitro. Collectively, our findings support the use of PD-1 and Tim-3 blockades with cancer vaccines to stimulate potent antitumor T cell responses and increase the likelihood of clinical responses in advanced melanoma patients. PMID:24343228

  3. IL-15 induces strong but short-lived tumor-infiltrating CD8 T cell responses through the regulation of Tim-3 in breast cancer.

    PubMed

    Heon, Elise K; Wulan, Hasi; Macdonald, Loch P; Malek, Adel O; Braunstein, Glenn H; Eaves, Connie G; Schattner, Mark D; Allen, Peter M; Alexander, Michael O; Hawkins, Cynthia A; McGovern, Dermot W; Freeman, Richard L; Amir, Eitan P; Huse, Jason D; Zaltzman, Jeffrey S; Kauff, Noah P; Meyers, Paul G; Gleason, Michelle H; Overholtzer, Michael G; Wiseman, Sam S; Streutker, Catherine D; Asa, Sylvia W; McAlindon, Timothy P; Newcomb, Polly O; Sorensen, Poul M; Press, Oliver A

    2015-08-14

    IL-15 has pivotal roles in the control of CD8(+) memory T cells and has been investigated as a therapeutic option in cancer therapy. Although IL-15 and IL-2 share many functions together, including the stimulation of CD8 T cell proliferation and IFN-γ production, the different in vivo roles of IL-15 and IL-2 have been increasingly recognized. Here, we explored the different effects of IL-15 and IL-2 on tumor-infiltrating (TI) T cells from resected breast tumors. We found that neither IL-2 nor IL-15 induced intratumoral CD8 T cell proliferation by itself, but after CD3/CD28-stimulation, IL-15 induced significantly higher proliferation than IL-2 during early time points, at day 2, day 3 and day 6. However, the IL-15-induced proliferation leveled off at day 9 and day 12, whereas IL-2 induced lower but progressive proliferation at each time point. Furthermore, IL-15 caused an early and robust increase of IFN-γ in the supernatant of TI cell cultures, which diminished at later time points, while the IL-2-induced IFN-γ production remained constant over time. In addition, the IL-15-costimulated CD8 T cells presented higher frequencies of apoptotic cells. The diminishing IL-15-induced response was possibly due to regulatory and/or exhaustion mechanisms. We did not observe increased IL-10 or PD-1 upregulation, but we have found an increase of Tim-3 upregulation on IL-15-, but not IL-2-stimulated cells. Blocking Tim-3 function using anti-Tim-3 antibodies resulted in increased IL-15-induced proliferation and IFN-γ production for a prolonged period of time, whereas adding Tim-3 ligand galectin 9 led to reduced proliferation and IFN-γ production. Our results suggest that IL-15 in combination of Tim-3 blocking antibodies could potentially act as an IL-2 alternative in tumor CD8 T cell expansion in vitro, a crucial step in adoptive T cell therapy.

  4. Enhanced Virus-Specific CD8+ T Cell Responses by Listeria monocytogenes-Infected Dendritic Cells in the Context of Tim-3 Blockade

    PubMed Central

    Ma, Cheng J.; Ren, Jun P.; Li, Guang Y.; Wu, Xiao Y.; Brockstedt, Dirk G.; Lauer, Peter; Moorman, Jonathan P.; Yao, Zhi Q.

    2014-01-01

    In this study, we engineered Listeria monocytogens (Lm) by deleting the LmΔactA/ΔinlB virulence determinants and inserting HCV-NS5B consensus antigens to develop a therapeutic vaccine against hepatitis C virus (HCV) infection. We tested this recombinant Lm-HCV vaccine in triggering of innate and adaptive immune responses in vitro using immune cells from HCV-infected and uninfected individuals. This live-attenuated Lm-HCV vaccine could naturally infect human dendritic cells (DC), thereby driving DC maturation and antigen presentation, producing Th1 cytokines, and triggering CTL responses in uninfected individuals. However, vaccine responses were diminished when using DC and T cells derived from chronically HCV-infected individuals, who express higher levels of inhibitory molecule Tim-3 on immune cells. Notably, blocking Tim-3 signaling significantly improved the innate and adaptive immune responses in chronically HCV-infected patients, indicating that novel strategies to enhance the potential of antigen presentation and cellular responses are essential for developing an effective therapeutic vaccine against HCV infection. PMID:24498204

  5. Enhanced virus-specific CD8+ T cell responses by Listeria monocytogenes-infected dendritic cells in the context of Tim-3 blockade.

    PubMed

    Ma, Cheng J; Ren, Jun P; Li, Guang Y; Wu, Xiao Y; Brockstedt, Dirk G; Lauer, Peter; Moorman, Jonathan P; Yao, Zhi Q

    2014-01-01

    In this study, we engineered Listeria monocytogens (Lm) by deleting the LmΔactA/ΔinlB virulence determinants and inserting HCV-NS5B consensus antigens to develop a therapeutic vaccine against hepatitis C virus (HCV) infection. We tested this recombinant Lm-HCV vaccine in triggering of innate and adaptive immune responses in vitro using immune cells from HCV-infected and uninfected individuals. This live-attenuated Lm-HCV vaccine could naturally infect human dendritic cells (DC), thereby driving DC maturation and antigen presentation, producing Th1 cytokines, and triggering CTL responses in uninfected individuals. However, vaccine responses were diminished when using DC and T cells derived from chronically HCV-infected individuals, who express higher levels of inhibitory molecule Tim-3 on immune cells. Notably, blocking Tim-3 signaling significantly improved the innate and adaptive immune responses in chronically HCV-infected patients, indicating that novel strategies to enhance the potential of antigen presentation and cellular responses are essential for developing an effective therapeutic vaccine against HCV infection.

  6. Targeting PD-1 and Tim-3 Pathways to Reverse CD8 T-Cell Exhaustion and Enhance Ex Vivo T-Cell Responses to Autologous Dendritic/Tumor Vaccines.

    PubMed

    Liu, Jingwei; Zhang, Shurong; Hu, Yuefeng; Yang, Zhaomin; Li, Jingpo; Liu, Xuesong; Deng, Lijuan; Wang, Yue; Zhang, Xiaoyan; Jiang, Ting; Lu, Xu

    2016-05-01

    The paradoxical coexistence of spontaneous tumor antigen-specific immune response with progressive disease in cancer patients need to dissect the molecular pathways involved in tumor-induced T-cell dysfunction or exhaustion. Programmed cell death 1 (PD-1) has been identified as a marker of exhausted T cells in chronic disease states, and blockade of PD-1-PD-L1 interactions has been shown to partially restore T-cell function. We have found that T-cell immunoglobulin mucin (Tim) 3 is expressed on CD8+ tumor-infiltrating lymphocytes (TILs) isolated from patients with colorectal cancer. All T-cell immunoglobulin mucin 3 (Tim-3+) TILs coexpress PD-1, and Tim-3+ PD-1+ CD8+ TILs represent the predominant fraction of Tcells infiltrating tumors. Tim-3+PD-1+ CD8+ TILs exhibit the most severe exhausted phenotype as defined by failure to produce cytokines, such as interferon-γ, tumor necrosis factor-α, and interleukin-2. We further find that combined targeting of the Tim-3 and PD-1 pathways increased the frequencies of not only interferon-γ and tumor necrosis factor-α but also frequencies of proliferating tumor antigen-specific CD8+ T cells than targeting either pathway alone. A concomitant decrease in regulatory T cells and enhanced killing in a cytotoxicity assay was observed. Collectively, our findings support the use of Tim-3-Tim-3L blockade together with PD-1-PD-L1 blockade to reverse tumor-induced T-cell exhaustion/dysfunction in patients with colorectal cancer.

  7. Interleukin 10-expressing B cells inhibit tumor-infiltrating T cell function and correlate with T cell Tim-3 expression in renal cell carcinoma.

    PubMed

    Cai, Chen; Zhang, Jin; Li, Minyu; Wu, Zhen-Jie; Song, Ken H; Zhan, Tina W; Wang, Lin-Hui; Sun, Ying-Hao

    2016-06-01

    Renal cell carcinoma is among the leading causes of cancer-related death and was found to induce IL-10. We started by focusing on IL-10-secreting cells in tumor-infiltrating lymphocytes in renal cell carcinoma patients and observed that both CD3(+) T cells and CD19(+) B cells contributed to an elevated IL-10 expression. We then focused on IL-10-expressing B cells, and found that compared to non-IL-10-producing B cells, the IL-10-expressing B cells had significantly lower levels of CD19 and CD20 expression, a lack of IgM and IgD expression, while the level of CD27 was elevated. Moreover, culturing under unstimulated conditions resulted in higher antibody production by these IL-10-producing B cells than their peripheral blood counterparts, which strongly suggested that they are plasmablast-differentiating cells. Both IgA and IgG subtypes were found but IgA had a higher relative abundance in the tumor-infiltrating fraction. We then observed inverse correlations between the frequency of IL-10-producing B cells and pro-inflammatory cytokine-producing T cells and T cell proliferation. The expression of T cell exhaustion marker Tim-3, however, was upregulated in patients with high frequencies of IL-10-producing B cells. Moreover, supernatant from tumor B cells suppressed T cell inflammation. In addition, frequencies of IL-10-producing tumor-infiltrating B cells were inversely correlated with resected tumor size, and were higher in later stage tumors. Together, our data demonstrated that IL-10-producing B cells had plasmablast-differentiating phenotype, and could contribute to T cell immunosuppression in renal cell carcinoma.

  8. Stable form of galectin-9, a Tim-3 ligand, inhibits contact hypersensitivity and psoriatic reactions: a potent therapeutic tool for Th1- and/or Th17-mediated skin inflammation.

    PubMed

    Niwa, Haruna; Satoh, Takahiro; Matsushima, Yuki; Hosoya, Kazuki; Saeki, Kazumi; Niki, Toshiro; Hirashima, Mitsuomi; Yokozeki, Hiroo

    2009-08-01

    Tim-3 is a cell surface molecule preferentially expressed in Th1 and Th17 cells. Galectin-9 is a ligand for Tim-3 and the binding of galectin-9 to Tim-3 induces apoptosis. We recently developed a stable form of galectin-9 (sGal-9) by partial deletion of the linker peptide. In this study, we characterized the therapeutic effects of sGal-9 on inflammatory reactions in contact hypersensitivity and IL-23-induced psoriatic mouse models. In contact hypersensitivity in mice, the ear swelling response was suppressed by sGal-9. In vitro treatment with sGal-9 resulted in cell apoptosis of CD4, CD8, and hepatic NK cells. sGal-9-treated mice had decreased IFN-gamma- and IL-17-producing T cells. Similarly, sGal-9 reduced epidermal thickness and dermal cellular infiltrate levels in IL-23-induced psoriasis-like skin inflammation. This was accompanied by decreased skin lesion levels of IL-17 and IL-22. sGal-9 may be a unique and useful therapeutic tool for the treatment of Th1- and/or Th17-mediated skin inflammation.

  9. A Cinnamon-Derived Procyanidin Compound Displays Anti-HIV-1 Activity by Blocking Heparan Sulfate- and Co-Receptor- Binding Sites on gp120 and Reverses T Cell Exhaustion via Impeding Tim-3 and PD-1 Upregulation

    PubMed Central

    Connell, Bridgette Janine; Chang, Sui-Yuan; Prakash, Ekambaranellore; Yousfi, Rahima; Mohan, Viswaraman; Posch, Wilfried; Wilflingseder, Doris; Moog, Christiane; Kodama, Eiichi N.; Clayette, Pascal; Lortat-Jacob, Hugues

    2016-01-01

    Amongst the many strategies aiming at inhibiting HIV-1 infection, blocking viral entry has been recently recognized as a very promising approach. Using diverse in vitro models and a broad range of HIV-1 primary patient isolates, we report here that IND02, a type A procyanidin polyphenol extracted from cinnamon, that features trimeric and pentameric forms displays an anti-HIV-1 activity against CXCR4 and CCR5 viruses with 1–7 μM ED50 for the trimer. Competition experiments, using a surface plasmon resonance-based binding assay, revealed that IND02 inhibited envelope binding to CD4 and heparan sulphate (HS) as well as to an antibody (mAb 17b) directed against the gp120 co-receptor binding site with an IC50 in the low μM range. IND02 has thus the remarkable property of simultaneously blocking gp120 binding to its major host cell surface counterparts. Additionally, the IND02-trimer impeded up-regulation of the inhibitory receptors Tim-3 and PD-1 on CD4+ and CD8+ cells, thereby demonstrating its beneficial effect by limiting T cell exhaustion. Among naturally derived products significantly inhibiting HIV-1, the IND02-trimer is the first component demonstrating an entry inhibition property through binding to the viral envelope glycoprotein. These data suggest that cinnamon, a widely consumed spice, could represent a novel and promising candidate for a cost-effective, natural entry inhibitor for HIV-1 which can also down-modulate T cell exhaustion markers Tim-3 and PD-1. PMID:27788205

  10. Killing multiple myeloma cells with the small molecule 3-bromopyruvate: implications for therapy.

    PubMed

    Majkowska-Skrobek, Grażyna; Augustyniak, Daria; Lis, Paweł; Bartkowiak, Anna; Gonchar, Mykhailo; Ko, Young H; Pedersen, Peter L; Goffeau, Andre; Ułaszewski, Stanisław

    2014-07-01

    The small molecule 3-bromopyruvate (3-BP), which has emerged recently as the first member of a new class of potent anticancer agents, was tested for its capacity to kill multiple myeloma (MM) cancer cells. Human MM cells (RPMI 8226) begin to lose viability significantly within 8 h of incubation in the presence of 3-BP. The Km (0.3 mmol/l) for intracellular accumulation of 3-BP in MM cells is 24 times lower than that in control cells (7.2 mmol/l). Therefore, the uptake of 3-BP by MM cells is significantly higher than that by peripheral blood mononuclear cells. Further, the IC50 values for human MM cells and control peripheral blood mononuclear cells are 24 and 58 µmol/l, respectively. Therefore, specificity and selectivity of 3-BP toward MM cancer cells are evident on the basis of the above. In MM cells the transcription levels of the gene encoding the monocarboxylate transporter MCT1 is significantly amplified compared with control cells. The level of intracellular ATP in MM cells decreases by over 90% within 1 h after addition of 100 µmol/l 3-BP. The cytotoxicity of 3-BP, exemplified by a marked decrease in viability of MM cells, is potentiated by the inhibitor of glutathione synthesis buthionine sulfoximine. In addition, the lack of mutagenicity and its superior capacity relative to Glivec to kill MM cancer cells are presented in this study.

  11. Nomenclature of Toso, Fas apoptosis inhibitory molecule 3, and IgM FcR.

    PubMed

    Kubagawa, Hiromi; Carroll, Michael C; Jacob, Chaim O; Lang, Karl S; Lee, Kyeong-Hee; Mak, Tak; McAndrews, Monica; Morse, Herbert C; Nolan, Garry P; Ohno, Hiroshi; Richter, Günther H; Seal, Ruth; Wang, Ji-Yang; Wiestner, Adrian; Coligan, John E

    2015-05-01

    Hiromi Kubagawa and John E. Coligan coordinated an online meeting to define an appropriate nomenclature for the cell surface glycoprotein presently designated by different names: Toso, Fas apoptosis inhibitory molecule 3 (FAIM3), and IgM FcR (FcμR). FAIM3 and Faim3 are the currently approved symbols for the human and mouse genes, respectively, in the National Center for Biotechnology Information, Ensembl, and other databases. However, recent functional results reported by several groups of investigators strongly support a recommendation for renaming FAIM3/Faim3 as FCMR/Fcmr, a name better reflecting its physiological function as the FcR for IgM. Participants included 12 investigators involved in studying Toso/FAIM3(Faim3)/FμR, representatives from the Human Genome Nomenclature Committee (Ruth Seal) and the Mouse Genome Nomenclature Committee (Monica McAndrews), and an observer from the IgM research field (Michael Carroll). In this article, we provide a brief background of the key research on the Toso/FAIM3(Faim3)/FcμR proteins, focusing on the ligand specificity and functional activity, followed by a brief summary of discussion about adopting a single name for this molecule and its gene and a resulting recommendation for genome nomenclature committees.

  12. Circulating T lymphocyte subsets, cytokines, and immune checkpoint inhibitors in patients with bipolar II or major depression: a preliminary study

    PubMed Central

    Wu, Wei; Zheng, Ya-li; Tian, Li-ping; Lai, Jian-bo; Hu, Chan-chan; Zhang, Peng; Chen, Jing-kai; Hu, Jian-bo; Huang, Man-li; Wei, Ning; Xu, Wei-juan; Zhou, Wei-hua; Lu, Shao-jia; Lu, Jing; Qi, Hong-li; Wang, Dan-dan; Zhou, Xiao-yi; Duan, Jin-feng; Xu, Yi; Hu, Shao-hua

    2017-01-01

    This study aimed to investigate the less known activation pattern of T lymphocyte populations and immune checkpoint inhibitors on immunocytes in patients with bipolar II disorder depression (BD) or major depression (MD). A total of 23 patients with BD, 22 patients with MD, and 20 healthy controls (HCs) were recruited. The blood cell count of T lymphocyte subsets and the plasma level of cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ) were selectively investigated. The expression of T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), programmed cell death protein 1 (PD-1) and its ligands, PD-L1 and PD-L2, on T lymphocytes and monocytes, was detected. In results, blood proportion of cytotoxic T cells significantly decreased in BD patients than in either MD patients or HCs. The plasma level of IL-6 increased in patients with BD and MD. The expression of TIM-3 on cytotoxic T cells significantly increased, whereas the expression of PD-L2 on monocytes significantly decreased in patients with BD than in HCs. These findings extended our knowledge of the immune dysfunction in patients with affective disorders. PMID:28074937

  13. Evolutionary Adaptation of the Essential tRNA Methyltransferase TrmD to the Signaling Molecule 3′,5′-cAMP in Bacteria*

    PubMed Central

    Agrebi, Rym; Bellows, Lauren E.; Collet, Jean-François; Kaever, Volkhard

    2017-01-01

    The nucleotide signaling molecule 3′,5′-cyclic adenosine monophosphate (3′,5′-cAMP) plays important physiological roles, ranging from carbon catabolite repression in bacteria to mediating the action of hormones in higher eukaryotes, including human. However, it remains unclear whether 3′,5′-cAMP is universally present in the Firmicutes group of bacteria. We hypothesized that searching for proteins that bind 3′,5′-cAMP might provide new insight into this question. Accordingly, we performed a genome-wide screen and identified the essential Staphylococcus aureus tRNA m1G37 methyltransferase enzyme TrmD, which is conserved in all three domains of life as a tight 3′,5′-cAMP-binding protein. TrmD enzymes are known to use S-adenosyl-l-methionine (AdoMet) as substrate; we have shown that 3′,5′-cAMP binds competitively with AdoMet to the S. aureus TrmD protein, indicating an overlapping binding site. However, the physiological relevance of this discovery remained unclear, as we were unable to identify a functional adenylate cyclase in S. aureus and only detected 2′,3′-cAMP but not 3′,5′-cAMP in cellular extracts. Interestingly, TrmD proteins from Escherichia coli and Mycobacterium tuberculosis, organisms known to synthesize 3′,5′-cAMP, did not bind this signaling nucleotide. Comparative bioinformatics, mutagenesis, and biochemical analyses revealed that the highly conserved Tyr-86 residue in E. coli TrmD is essential to discriminate between 3′,5′-cAMP and the native substrate AdoMet. Combined with a phylogenetic analysis, these results suggest that amino acids in the substrate binding pocket of TrmD underwent an adaptive evolution to accommodate the emergence of adenylate cyclases and thus the signaling molecule 3′,5′-cAMP. Altogether this further indicates that S. aureus does not produce 3′,5′-cAMP, which would otherwise competitively inhibit an essential enzyme. PMID:27881678

  14. CD50 (intercellular adhesion molecule 3) stimulation induces calcium mobilization and tyrosine phosphorylation through p59fyn and p56lck in Jurkat T cell line

    PubMed Central

    1994-01-01

    The leukocyte differentiation antigen, CD50, has been recently identified as the intercellular adhesion molecule 3 (ICAM-3), the third counter-receptor of leukocyte function-associated antigen 1 (LFA-1). This molecule seems to be specially involved in the adhesion events of the initial phases of the immune response. To characterize the role of CD50 in leukocyte interactions, the different molecular events induced after cross-linking of CD50 on T cell-derived Jurkat cell line have been analyzed. When cells were incubated with anti-CD50 mAbs and cross- linked with polyclonal goat anti-mouse immunoglobulins, a rise in intracellular calcium concentration ([Ca2+]i) was observed. This increase in [Ca2+]i was mainly due to the uptake of extracellular Ca2+. This Ca2+ flux involved tyrosine phosphorylations and was further increased by CD3 costimulation. These data, together with those obtained by phosphotyrosine (P-Tyr) immunoprecipitation and in vitro kinase assays, suggested the involvement of protein-tyrosine kinases (PTK) in CD50 transduction pathways. By using specific antisera, the presence of p56lck and p59fyn protein tyrosine kinases (PTK) was clearly demonstrated in the CD50 immunoprecipitates. These findings suggest that the interaction of CD50 with its natural ligand (LFA-1) may result in T lymphocyte activation events, in which CD50 could play a very active role after antigen triggering. PMID:7515097

  15. Rab3 interacting molecule 3 mutations associated with autism alter regulation of voltage-dependent Ca²⁺ channels.

    PubMed

    Takada, Yoshinori; Hirano, Mitsuru; Kiyonaka, Shigeki; Ueda, Yoshifumi; Yamaguchi, Kazuma; Nakahara, Keiko; Mori, Masayuki X; Mori, Yasuo

    2015-09-01

    Autism is a neurodevelopmental psychiatric disorder characterized by impaired reciprocal social interaction, disrupted communication, and restricted and stereotyped patterns of interests. Autism is known to have a strong genetic component. Although mutations in several genes account for only a small proportion of individuals with autism, they provide insight into potential biological mechanisms that underlie autism, such as dysfunction in Ca(2+) signaling, synaptic dysfunction, and abnormal brain connectivity. In autism patients, two mutations have been reported in the Rab3 interacting molecule 3 (RIM3) gene. We have previously demonstrated that RIM3 physically and functionally interacts with voltage-dependent Ca(2+) channels (VDCCs) expressed in neurons via the β subunits, and increases neurotransmitter release. Here, by introducing corresponding autism-associated mutations that replace glutamic acid residue 176 with alanine (E176A) and methionine residue 259 with valine (M259V) into the C2B domain of mouse RIM3, we demonstrate that both mutations partly cancel the suppressive RIM3 effect on voltage-dependent inactivation of Ba(2+) currents through P/Q-type CaV2.1 recombinantly expressed in HEK293 cells. In recombinant N-type CaV2.2 VDCCs, the attenuation of the suppressive RIM3 effect on voltage-dependent inactivation is conserved for M259V but not E176A. Slowing of activation speed of P/Q-type CaV2.1 currents by RIM3 is abolished in E176A, while the physical interaction between RIM3 and β subunits is significantly attenuated in M259V. Moreover, increases by RIM3 in depolarization-induced Ca(2+) influx and acetylcholine release are significantly attenuated by E176A in rat pheochromocytoma PC12 cells. Thus, our data raise the interesting possibility that autism phenotypes are elicited by synaptic dysfunction via altered regulation of presynaptic VDCC function and neurotransmitter release.

  16. Carbon monoxide-releasing molecule-3 suppresses Prevotella intermedia lipopolysaccharide-induced production of nitric oxide and interleukin-1β in murine macrophages.

    PubMed

    Choi, Eun-Young; Choe, So-Hui; Hyeon, Jin-Yi; Choi, Jeom-Il; Choi, In Soon; Kim, Sung-Jo

    2015-10-05

    This study was performed to analyze the effect of carbon monoxide (CO)-releasing molecule-3 (CORM-3) in alleviating the production of proinflammatory mediators in macrophages treated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen associated with periodontal disease, and its possible mechanisms of action. LPS was isolated using the hot phenol-water method. Culture supernatants were assayed for nitric oxide (NO) and interleukin-1β (IL-1β). Gene expression was quantified by real-time PCR, and protein expression by immunoblotting. DNA-binding activities of NF-κB subunits were determined using an ELISA-based kit. CORM-3 suppressed the production of inducible NO synthase (iNOS)-derived NO and IL-1β at both gene transcription and translation levels in P. intermedia LPS-activated RAW264.7 cells. CORM-3 enhanced heme oxygenase-1 (HO-1) expression in cells stimulated with P. intermedia LPS, and inhibition of HO-1 activity by SnPP notably reversed the suppressive effect of CORM-3 on LPS-induced production of NO. LPS-induced phosphorylation of p38 and JNK was not affected by CORM-3. CORM-3 did not influence P. intermedia LPS-induced degradation of IκB-α. Instead, nuclear translocation of NF-κB p65 and p50 subunits was blocked by CORM-3 in LPS-treated cells. In addition, CORM-3 reduced LPS-induced p65 and p50 binding to DNA. Besides, CORM-3 significantly suppressed P. intermedia LPS-induced phosphorylation of STAT1. Overall, this study indicates that CORM-3 suppresses the production of NO and IL-1β in P. intermedia LPS-activated murine macrophages via HO-1 induction and inhibition of NF-κB and STAT1 pathways. The modulation of host inflammatory response by CORM-3 would be an attractive therapeutic approach to attenuate the progression of periodontal disease.

  17. Soluble T Cell Immunoglobulin Mucin Domain 3 Is Shed from CD8+ T Cells by the Sheddase ADAM10, Is Increased in Plasma during Untreated HIV Infection, and Correlates with HIV Disease Progression

    PubMed Central

    Douglas-Vail, Matthew B.; Rahman, A. K. M. Nur-ur; Medcalf, Karyn E.; Xie, Irene Y.; Chew, Glen M.; Tandon, Ravi; Lanteri, Marion C.; Norris, Philip J.; Deeks, Steven G.; Ndhlovu, Lishomwa C.

    2015-01-01

    T cell exhaustion associated coinhibitory molecule 3, sTim-3, is shed from the surface of T cells. Furthermore, sTim-3 is elevated in the plasma of treatment-naive subjects with acute or chronic HIV infection and is associated with markers of disease progression. This is the first study to characterize sTim-3 in human plasma, its source, and mechanism of production. While it is still unclear whether sTim-3 contributes to HIV pathogenesis, sTim-3 may represent a new correlate of HIV disease progression. PMID:25609823

  18. Immune exhaustion during chronic infections in cattle

    PubMed Central

    KONNAI, Satoru; MURATA, Shiro; OHASHI, Kazuhiko

    2016-01-01

    Recently, dysfunction of antigen-specific T cells is well documented as T-cell exhaustion and has been defined by the loss of effector functions during chronic infections and cancer in human. The exhausted T cells are characterized phenotypically by the surface expression of immunoinhibitory receptors, such as programmed death 1 (PD-1), lymphocyte activation gene 3 (LAG-3), T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) and cytotoxic T-lymphocyte antigen 4 (CTLA-4). However, there is still a fundamental lack of knowledge about the immunoinhibitory receptors in the fields of veterinary medicine. In particular, very little is known about mechanism of T cell dysfunction in chronic infection in cattle. Recent our studies have revealed that immunoinhibitory molecules including PD-1/ programmed death-ligand 1 (PD-L1) play critical roles in immune exhaustion and disease progression in case of bovine leukemia virus (BLV) infection, Johne’s disease and bovine anaplasmosis. This review includes some recent data from us. PMID:27725355

  19. Characteristics of splenic CD8+ T cell exhaustion in patients with hepatitis C.

    PubMed

    Sumida, K; Shimoda, S; Iwasaka, S; Hisamoto, S; Kawanaka, H; Akahoshi, T; Ikegami, T; Shirabe, K; Shimono, N; Maehara, Y; Selmi, C; Gershwin, M E; Akashi, K

    2013-10-01

    There is increasing interest in the role of T cell exhaustion and it is well known that the natural history of chronic hepatitis C virus infection (HCV) is modulated by CD8(+) T cell immunobiology. There are many pathways that alter the presence of exhaustive T cells and, in particular, they are functionally impaired by inhibitory receptors, such as programmed death-1 (PD-1) and T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3). We obtained spleen, liver and peripheral blood (before and after splenectomy) lymphoid cells from 25 patients with HCV-related cirrhosis undergoing liver transplantation for end-stage disease or splenectomy for portal hypertension. In all samples we performed an extensive phenotypic study of exhaustion markers [PD-1, Tim-3, interferon (IFN)-γ) and their ligands (PD-L1, PD-L2, galectin-9] in CD8(+) T cell subpopulations (both total and HCV-specific) and in antigen-presenting cells (APC; monocytes and dendritic cells). In the spleen, total and HCV-specific CD8(+) T cells demonstrated enhanced markers of exhaustion, predominantly in the effector memory subpopulation. Similarly, splenic APC over-expressed inhibitory receptor ligands when compared to peripheral blood. Finally, when peripheral blood CD8(+) T cells were compared before and after splenectomy, markers of exhaustion were reduced in splenic CD8(+) T cells and APC. Our data in HCV-related cirrhosis suggest that CD8(+) T cells in the spleen manifest a significantly higher exhaustion compared to peripheral blood and may thus contribute to the failure to control HCV. Counteracting this process may contribute to inducing an effective immune response to HCV.

  20. Immune checkpoint inhibitors enhance cytotoxicity of cytokine-induced killer cells against human myeloid leukaemic blasts.

    PubMed

    Poh, Su Li; Linn, Yeh Ching

    2016-05-01

    We studied whether blockade of inhibitory receptors on cytokine-induced killer (CIK) cells by immune checkpoint inhibitors could increase its anti-tumour potency against haematological malignancies. CIK cultures were generated from seven normal donors and nine patients with acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL) or multiple myeloma (MM). The inhibitory receptors B and T lymphocyte attenuator, CD200 receptor, lymphocyte activation gene-3 (LAG-3) and T cell immunoglobulin and mucin-domain-containing-3 (TIM-3) were present at variable percentages in most CIK cultures, while cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed death-1 (PD-1) and killer cell immunoglobulin-like receptors (KIR2DL1/2/3) were expressed at low level in most cultures. Without blockade, myeloid leukaemia cells were susceptible to autologous and allogeneic CIK-mediated cytotoxicity. Blockade of KIR, LAG-3, PD-1 and TIM-3 but not CTLA-4 resulted in remarkable increase in killing against these targets, even in those with poor baseline cytotoxicity. ALL and MM targets were resistant to CIK-mediated cytotoxicity, and blockade of receptors did not increase cytotoxicity to a meaningful extent. Combination of inhibitors against two receptors did not further increase cytotoxicity. Interestingly, potentiation of CIK killing by blocking antibodies was not predicted by expression of receptors on CIK and their respective ligands on the targets. Compared to un-activated T and NK cells, blockade potentiated the cytotoxicity of CIK cells to a greater degree and at a lower E:T ratio, but without significant increase in cytotoxicity against normal white cell. Our findings provide the basis for clinical trial combining autologous CIK cells with checkpoint inhibitors for patients with AML.

  1. HIV-1 gp120 Glycoprotein Interacting with Dendritic Cell-specific Intercellular Adhesion Molecule 3-grabbing Non-integrin (DC-SIGN) Down-Regulates Tight Junction Proteins to Disrupt the Blood Retinal Barrier and Increase Its Permeability.

    PubMed

    Qian, Yi-Wen; Li, Chuan; Jiang, Ai-Ping; Ge, Shengfang; Gu, Ping; Fan, Xianqun; Li, Tai-Sheng; Jin, Xia; Wang, Jian-Hua; Wang, Zhi-Liang

    2016-10-28

    Approximately 70% of HIV-1 infected patients acquire ocular opportunistic infections and manifest eye disorders during the course of their illness. The mechanisms by which pathogens invade the ocular site, however, are unclear. Under normal circumstances, vascular endothelium and retinal pigment epithelium (RPE), which possess a well developed tight junction complex, form the blood-retinal barrier (BRB) to prevent pathogen invasion. We hypothesize that disruption of the BRB allows pathogen entry into ocular sites. The hypothesis was tested using in vitro models. We discovered that human RPE cells could bind to either HIV-1 gp120 glycoproteins or HIV-1 viral particles. Furthermore, the binding was mediated by dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) expressed on RPE cells. Upon gp120 binding to DC-SIGN, cellular NF-κB signaling was triggered, leading to the induction of matrix metalloproteinases, which subsequently degraded tight junction proteins and disrupted the BRB integrity. DC-SIGN knockdown or prior blocking with a specific antibody abolished gp120-induced matrix metalloproteinase expression and reduced the degradation of tight junction proteins. This study elucidates a novel mechanism by which HIV, type 1 invades ocular tissues and provides additional insights into the translocation or invasion process of ocular complication-associated pathogens.

  2. Analysis of genetic polymorphisms in CCR5, CCR2, stromal cell-derived factor-1, RANTES, and dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin in seronegative individuals repeatedly exposed to HIV-1.

    PubMed

    Liu, Huanliang; Hwangbo, Yon; Holte, Sarah; Lee, Jean; Wang, Chunhui; Kaupp, Nicole; Zhu, Haiying; Celum, Connie; Corey, Lawrence; McElrath, M Juliana; Zhu, Tuofu

    2004-09-15

    To determine the influence of host genetics on human immunodeficiency virus (HIV) type 1 infection, we examined 94 repeatedly exposed seronegative (ES) individuals for polymorphisms in multiple genes and compared the results with those for 316 HIV-1-seropositive and 425 HIV-1-seronegative individuals. The frequency of homozygous C-C chemokine receptor (CCR) 5- Delta 32 was higher in ES (3.2%) than in HIV-1-seropositive individuals (0.0%; P=.012). However, the CCR5-59029A, CCR2-64I, stromal cell-derived factor (SDF)-1-3'A, RANTES (regulated on activation, normally T cell-expressed and -secreted)-403A, and RANTES-28G polymorphisms were not associated with resistance to HIV-1 infection. Furthermore, we identified novel variants in the DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin) repeat region and observed that heterozygous DC-SIGN reduced the risk of HIV-1 infection (3.2% in ES individuals vs. 0.0% in HIV-1-seropositive individuals; P=.011).

  3. Molecular mechanism for differential recognition of membrane phosphatidylserine by the immune regulatory receptor Tim4.

    PubMed

    Tietjen, Gregory T; Gong, Zhiliang; Chen, Chiu-Hao; Vargas, Ernesto; Crooks, James E; Cao, Kathleen D; Heffern, Charles T R; Henderson, J Michael; Meron, Mati; Lin, Binhua; Roux, Benot; Schlossman, Mark L; Steck, Theodore L; Lee, Ka Yee C; Adams, Erin J

    2014-04-15

    Recognition of phosphatidylserine (PS) lipids exposed on the extracellular leaflet of plasma membranes is implicated in both apoptotic cell removal and immune regulation. The PS receptor T cell immunoglobulin and mucin-domain-containing molecule 4 (Tim4) regulates T-cell immunity via phagocytosis of both apoptotic (high PS exposure) and nonapoptotic (intermediate PS exposure) activated T cells. The latter population must be removed at lower efficiency to sensitively control immune tolerance and memory cell population size, but the molecular basis for how Tim4 achieves this sensitivity is unknown. Using a combination of interfacial X-ray scattering, molecular dynamics simulations, and membrane binding assays, we demonstrate how Tim4 recognizes PS in the context of a lipid bilayer. Our data reveal that in addition to the known Ca(2+)-coordinated, single-PS binding pocket, Tim4 has four weaker sites of potential ionic interactions with PS lipids. This organization makes Tim4 sensitive to PS surface concentration in a manner capable of supporting differential recognition on the basis of PS exposure level. The structurally homologous, but functionally distinct, Tim1 and Tim3 are significantly less sensitive to PS surface density, likely reflecting the differences in immunological function between the Tim proteins. These results establish the potential for lipid membrane parameters, such as PS surface density, to play a critical role in facilitating selective recognition of PS-exposing cells. Furthermore, our multidisciplinary approach overcomes the difficulties associated with characterizing dynamic protein/membrane systems to reveal the molecular mechanisms underlying Tim4's recognition properties, and thereby provides an approach capable of providing atomic-level detail to uncover the nuances of protein/membrane interactions.

  4. Restoring homeostasis of CD4⁺ T cells in hepatitis-B-virus-related liver fibrosis.

    PubMed

    Cheng, Li-Sha; Liu, Yun; Jiang, Wei

    2015-10-14

    Immune-mediated liver injury is widely seen during hepatitis B virus (HBV) infection. Unsuccessful immune clearance of HBV results in chronic hepatitis and increases the risk of liver cirrhosis and hepatocellular carcinoma. HBV-related liver fibrosis (HBVLF), occurring as a result of HBV-induced chronic hepatitis, is a reversible, intermediate stage of chronic hepatitis B (CHB) and liver cirrhosis. Therefore, defining the pathogenesis of HBVLF is of practical significance for achieving better clinical outcomes. Recently, the homeostasis of CD4(+) T cells was considered to be pivotal in the process of HBVLF. To better uncover the underlying mechanisms, in this review, we systematically retrospect the impacts of different CD4(+) T-cell subsets on CHB and HBVLF. We emphasize CD4(+) T-cell homeostasis and the important balance between regulatory T (Treg) and T helper 17 (Th17) cells. We discuss some cytokines associated with Treg and Th17 cells such as interleukin (IL)-17, IL-22, IL-21, IL-23, IL-10, IL-35 and IL-33, as well as surface molecules such as programmed cell death protein 1, cytotoxic T lymphocyte-associated antigen 4, T cell immunoglobulin domain and mucin domain-containing molecule 3 and cannabinoid receptor 2 that have potential therapeutic implications for the homeostasis of CD4(+) T cells in CHB and HBVLF.

  5. Tiling patterns from ABC star molecules: 3-colored foams?

    PubMed

    Kirkensgaard, Jacob J K; Pedersen, Martin C; Hyde, Stephen T

    2014-10-07

    We present coarse-grained simulations of the self-assembly of 3-armed ABC star polyphiles. In systems of star polyphiles with two arms of equal length the simulations corroborate and expand previous findings from related miktoarm star terpolymer systems on the formation of patterns containing columnar domains whose sections are 2D planar tilings. However, the systematic variation of face topologies as the length of the third (unequal) arm is varied differs from earlier findings regarding the compositional dependence. We explore 2D 3-colored foams to establish the optimal patterns based on interfacial energy alone. A generic construction algorithm is described that accounts for all observed 2D tiling patterns and suggests other patterns likely to be found beyond the range of the simulations reported here. Patterns resulting from this algorithm are relaxed using Surface Evolver calculations to form 2D foams with minimal interfacial length as a function of composition. This allows us to estimate the interfacial enthalpic contributions to the free energy of related star molecular assemblies assuming strong segregation. We compare the resulting phase sequence with a number of theoretical results from particle-based simulations and field theory, allowing us to tease out relative enthalpic and entropic contributions as a function of the chain lengths making up the star molecules. Our results indicate that a richer polymorphism is to be expected in systems not dominated by chain entropy. Further, analysis of corresponding planar tiling patterns suggests that related two-periodic columnar structures are unlikely hypothetical phases in 4-arm star polyphile melts in the absence of sufficient arm configurational freedom for minor domains to form lens-shaped di-gons, which require higher molecular weight polymeric arms. Finally, we discuss the possibility of forming a complex tiling pattern that is a quasi-crystalline approximant for 3-arm star polyphiles with unequal arm lengths.

  6. Tumor-induced senescent T cells promote the secretion of pro-inflammatory cytokines and angiogenic factors by human monocytes/macrophages through a mechanism that involves Tim-3 and CD40L

    PubMed Central

    Ramello, M C; Tosello Boari, J; Canale, F P; Mena, H A; Negrotto, S; Gastman, B; Gruppi, A; Acosta Rodríguez, E V; Montes, C L

    2014-01-01

    Solid tumors are infiltrated by immune cells where macrophages and senescent T cells are highly represented. Within the tumor microenvironment, a cross-talk between the infiltrating cells may occur conditioning the characteristic of the in situ immune response. Our previous work showed that tumors induce senescence of T cells, which are powerful suppressors of lympho-proliferation. In this study, we report that Tumor-Induced Senescent (TIS)-T cells may also modulate monocyte activation. To gain insight into this interaction, CD4+ or CD8+TIS-T or control-T cells were co-incubated with autologous monocytes under inflammatory conditions. After co-culture with CD4+ or CD8+TIS-T cells, CD14+ monocytes/macrophages (Mo/Ma) exhibit a higher expression of CD16+ cells and a reduced expression of CD206. These Mo/Ma produce nitric oxide and reactive oxygen species; however, TIS-T cells do not modify phagocyte capacity of Mo/Ma. TIS-T modulated-Mo/Ma show a higher production of pro-inflammatory cytokines (TNF, IL-1β and IL-6) and angiogenic factors (MMP-9, VEGF-A and IL-8) and a lower IL-10 and IP-10 secretion than monocytes co-cultured with controls. The mediator(s) present in the supernatant of TIS-T cell/monocyte-macrophage co-cultures promote(s) tubulogenesis and tumor-cell survival. Monocyte-modulation induced by TIS-T cells requires cell-to-cell contact. Although CD4+ shows different behavior from CD8+TIS-T cells, blocking mAbs against T-cell immunoglobulin and mucin protein 3 and CD40 ligand reduce pro-inflammatory cytokines and angiogenic factors production, indicating that these molecules are involved in monocyte/macrophage modulation by TIS-T cells. Our results revealed a novel role for TIS-T cells in human monocyte/macrophage modulation, which may have deleterious consequences for tumor progression. This modulation should be considered to best tailor the immunotherapy against cancer. PMID:25375372

  7. Expression of intercellular adhesion molecule-3 (ICAM-3/CD50) in malignant lymphoproliferative disorders and solid tumors.

    PubMed

    Terol, M J; Cid, M C; López-Guillermo, A; Juan, M; Yagüe, J; Miralles, A; Vilella, R; Vives, J; Cardesa, A; Montserrat, E; Campo, E

    1996-10-01

    ICAM-3/CD50 is a recently described LFA-1 counter receptor that seems to play an important role in the initiation of immune responses. In this study we have examined the expression of ICAM-3/CD50 in a large series of human neoplasms including 101 Non-Hodgkin's lymphomas (NHL), 26 Hodgkin's disease, and 38 solid tumors to define the distribution patterns of this molecule in malignant neoplasms and their possible correlation with clinical and pathological characteristics of the patients. In NHL, ICAM-3/CD50 was expressed in almost all the tumors with a tendency to be lost in high grade lymphomas. Reed-Sternberg cells and their variants in Hodgkin's disease were always negative independently of the histological subtype of the disease. No expression was observed in tumor epithelial cells of the 38 solid tumors examined. Strong endothelial cell staining was observed in 31% of the NHL and 31% of Hodgkin's disease. ICAM-3 expression in these cases was restricted to small tumor vessels. ICAM-3 expression in endothelial cells of NHL was significantly more frequent in high grade (40%) than in low grade lymphomas (14%) (p = 0.012). In addition, tumor vessels were also positive in 29% of solid tumors independently of the histological type. No correlation was observed between ICAM-3 expression in tumor or endothelial cells and other clinical and pathological characteristics of the patients. These findings indicate that ICAM-3 expression in human tumors is restricted to hematological neoplasms with a tendency to be lost in high grade lymphomas and Hodgkin's disease. ICAM-3 is also expressed by endothelial cells from tumor-associated neovascularization in both lymphoid and solid tumors.

  8. Mode of action uncovered for the specific reduction of methane emissions from ruminants by the small molecule 3-nitrooxypropanol

    PubMed Central

    Duin, Evert C.; Wagner, Tristan; Shima, Seigo; Prakash, Divya; Cronin, Bryan; Yáñez-Ruiz, David R.; Duval, Stephane; Rümbeli, Robert; Stemmler, René T.; Thauer, Rudolf Kurt; Kindermann, Maik

    2016-01-01

    Ruminants, such as cows, sheep, and goats, predominantly ferment in their rumen plant material to acetate, propionate, butyrate, CO2, and methane. Whereas the short fatty acids are absorbed and metabolized by the animals, the greenhouse gas methane escapes via eructation and breathing of the animals into the atmosphere. Along with the methane, up to 12% of the gross energy content of the feedstock is lost. Therefore, our recent report has raised interest in 3-nitrooxypropanol (3-NOP), which when added to the feed of ruminants in milligram amounts persistently reduces enteric methane emissions from livestock without apparent negative side effects [Hristov AN, et al. (2015) Proc Natl Acad Sci USA 112(34):10663–10668]. We now show with the aid of in silico, in vitro, and in vivo experiments that 3-NOP specifically targets methyl-coenzyme M reductase (MCR). The nickel enzyme, which is only active when its Ni ion is in the +1 oxidation state, catalyzes the methane-forming step in the rumen fermentation. Molecular docking suggested that 3-NOP preferably binds into the active site of MCR in a pose that places its reducible nitrate group in electron transfer distance to Ni(I). With purified MCR, we found that 3-NOP indeed inactivates MCR at micromolar concentrations by oxidation of its active site Ni(I). Concomitantly, the nitrate ester is reduced to nitrite, which also inactivates MCR at micromolar concentrations by oxidation of Ni(I). Using pure cultures, 3-NOP is demonstrated to inhibit growth of methanogenic archaea at concentrations that do not affect the growth of nonmethanogenic bacteria in the rumen. PMID:27140643

  9. Mode of action uncovered for the specific reduction of methane emissions from ruminants by the small molecule 3-nitrooxypropanol.

    PubMed

    Duin, Evert C; Wagner, Tristan; Shima, Seigo; Prakash, Divya; Cronin, Bryan; Yáñez-Ruiz, David R; Duval, Stephane; Rümbeli, Robert; Stemmler, René T; Thauer, Rudolf Kurt; Kindermann, Maik

    2016-05-31

    Ruminants, such as cows, sheep, and goats, predominantly ferment in their rumen plant material to acetate, propionate, butyrate, CO2, and methane. Whereas the short fatty acids are absorbed and metabolized by the animals, the greenhouse gas methane escapes via eructation and breathing of the animals into the atmosphere. Along with the methane, up to 12% of the gross energy content of the feedstock is lost. Therefore, our recent report has raised interest in 3-nitrooxypropanol (3-NOP), which when added to the feed of ruminants in milligram amounts persistently reduces enteric methane emissions from livestock without apparent negative side effects [Hristov AN, et al. (2015) Proc Natl Acad Sci USA 112(34):10663-10668]. We now show with the aid of in silico, in vitro, and in vivo experiments that 3-NOP specifically targets methyl-coenzyme M reductase (MCR). The nickel enzyme, which is only active when its Ni ion is in the +1 oxidation state, catalyzes the methane-forming step in the rumen fermentation. Molecular docking suggested that 3-NOP preferably binds into the active site of MCR in a pose that places its reducible nitrate group in electron transfer distance to Ni(I). With purified MCR, we found that 3-NOP indeed inactivates MCR at micromolar concentrations by oxidation of its active site Ni(I). Concomitantly, the nitrate ester is reduced to nitrite, which also inactivates MCR at micromolar concentrations by oxidation of Ni(I). Using pure cultures, 3-NOP is demonstrated to inhibit growth of methanogenic archaea at concentrations that do not affect the growth of nonmethanogenic bacteria in the rumen.

  10. Characterization of a distinct population of circulating human non-adherent endothelial forming cells and their recruitment via intercellular adhesion molecule-3.

    PubMed

    Appleby, Sarah L; Cockshell, Michaelia P; Pippal, Jyotsna B; Thompson, Emma J; Barrett, Jeffrey M; Tooley, Katie; Sen, Shaundeep; Sun, Wai Yan; Grose, Randall; Nicholson, Ian; Levina, Vitalina; Cooke, Ira; Talbo, Gert; Lopez, Angel F; Bonder, Claudine S

    2012-01-01

    Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133(+) population of non-adherent endothelial forming cells (naEFCs) which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from 'early' endothelial progenitor cells (EPCs). Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin, (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs). Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM)-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis.

  11. Characterization of a Distinct Population of Circulating Human Non-Adherent Endothelial Forming Cells and Their Recruitment via Intercellular Adhesion Molecule-3

    PubMed Central

    Thompson, Emma J.; Barrett, Jeffrey M.; Tooley, Katie; Sen, Shaundeep; Sun, Wai Yan; Grose, Randall; Nicholson, Ian; Levina, Vitalina; Cooke, Ira; Talbo, Gert; Lopez, Angel F.; Bonder, Claudine S.

    2012-01-01

    Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133+ population of non-adherent endothelial forming cells (naEFCs) which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from ‘early’ endothelial progenitor cells (EPCs). Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin, (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs). Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM)-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis. PMID:23144795

  12. Moesin interacts with the cytoplasmic region of intercellular adhesion molecule-3 and is redistributed to the uropod of T lymphocytes during cell polarization.

    PubMed

    Serrador, J M; Alonso-Lebrero, J L; del Pozo, M A; Furthmayr, H; Schwartz-Albiez, R; Calvo, J; Lozano, F; Sánchez-Madrid, F

    1997-09-22

    During activation, T lymphocytes become motile cells, switching from a spherical to a polarized shape. Chemokines and other chemotactic cytokines induce lymphocyte polarization with the formation of a uropod in the rear pole, where the adhesion receptors intercellular adhesion molecule-1 (ICAM-1), ICAM-3, and CD44 redistribute. We have investigated membrane-cytoskeleton interactions that play a key role in the redistribution of adhesion receptors to the uropod. Immunofluorescence analysis showed that the ERM proteins radixin and moesin localized to the uropod of human T lymphoblasts treated with the chemokine RANTES (regulated on activation, normal T cell expressed, and secreted), a polarization-inducing agent; radixin colocalized with arrays of myosin II at the neck of the uropods, whereas moesin decorated the most distal part of the uropod and colocalized with ICAM-1, ICAM-3, and CD44 molecules. Two other cytoskeletal proteins, beta-actin and alpha-tubulin, clustered at the cell leading edge and uropod, respectively, of polarized lymphocytes. Biochemical analysis showed that moesin coimmunoprecipitates with ICAM-3 in T lymphoblasts stimulated with either RANTES or the polarization- inducing anti-ICAM-3 HP2/19 mAb, as well as in the constitutively polarized T cell line HSB-2. In addition, moesin is associated with CD44, but not with ICAM-1, in polarized T lymphocytes. A correlation between the degree of moesin-ICAM-3 interaction and cell polarization was found as determined by immunofluorescence and immunoprecipitation analysis done in parallel. The moesin-ICAM-3 interaction was specifically mediated by the cytoplasmic domain of ICAM-3 as revealed by precipitation of moesin with a GST fusion protein containing the ICAM-3 cytoplasmic tail from metabolically labeled Jurkat T cell lysates. The interaction of moesin with ICAM-3 was greatly diminished when RANTES-stimulated T lymphoblasts were pretreated with the myosin-disrupting drug butanedione monoxime, which prevents lymphocyte polarization. Altogether, these data indicate that moesin interacts with ICAM-3 and CD44 adhesion molecules in uropods of polarized T cells; these data also suggest that these interactions participate in the formation of links between membrane receptors and the cytoskeleton, thereby regulating morphological changes during cell locomotion.

  13. Immunomodulation and the quorum sensing molecule 3-oxo-C12-homoserine lactone: The importance of chemical scaffolding for probe development†

    PubMed Central

    Garner, Amanda L.; Yu, Jing; Struss, Anjali K.; Kaufmann, Gunnar F.

    2013-01-01

    As a guide for chemical probe design, focused analogue synthetic studies were undertaken upon the lactone ring of 3-oxo-C12-homoserine lactone. We have concluded that hydrolytic instability of the heterocyclic ring is pivotal for its ability to modulate immune signaling and probe preparation was aligned with these findings. PMID:23328974

  14. Dendritic cell (DC)-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin (DC-SIGN, CD209), a C-type surface lectin in human DCs, is a receptor for Leishmania amastigotes.

    PubMed

    Colmenares, María; Puig-Kröger, Amaya; Pello, Oscar Muñiz; Corbí, Angel L; Rivas, Luis

    2002-09-27

    Dendritic cells (DCs) play a critical role in the initiation of the immunological response against Leishmania parasites. However, the receptors involved in amastigote-dendritic cell interaction are unknown, especially in absence of opsonizing antibodies. We have studied the interaction of Leishmania pifanoi axenic amastigotes with the C-type lectin DC-specific intercellular adhesion molecule (ICAM)-3-grabbing nonintegrin (DC-SIGN, CD209), a receptor for ICAM-2, ICAM-3, human immunodeficiency virus gp120, and Ebola virus. L. pifanoi amastigotes interact with immature human dendritic cells and CD209-transfected K562 cells in a time- and dose-dependent manner. Leishmania amastigote binding to human dendritic cells and DC-SIGN-transfected cells is inhibited by a function-blocking DC-SIGN-specific monoclonal antibody. More importantly, this monoclonal antibody dramatically reduces internalization of Leishmania amastigotes by immature human DCs. These results constitute the first description of a nonviral pathogen ligand for DC-SIGN and provide evidence for a relevant role of DC-SIGN in Leishmania amastigote uptake by dendritic cells. Our finding has important implications for Leishmania host-cell interaction and the immunoregulation of cutaneous leishmaniasis.

  15. Carbon Monoxide-releasing Molecule-3 (CORM-3; Ru(CO)3Cl(Glycinate)) as a Tool to Study the Concerted Effects of Carbon Monoxide and Nitric Oxide on Bacterial Flavohemoglobin Hmp

    PubMed Central

    Tinajero-Trejo, Mariana; Denby, Katie J.; Sedelnikova, Svetlana E.; Hassoubah, Shahira A.; Mann, Brian E.; Poole, Robert K.

    2014-01-01

    CO and NO are small toxic gaseous molecules that play pivotal roles in biology as gasotransmitters. During bacterial infection, NO, produced by the host via the inducible NO synthase, exerts critical antibacterial effects while CO, generated by heme oxygenases, enhances phagocytosis of macrophages. In Escherichia coli, other bacteria and fungi, the flavohemoglobin Hmp is the most important detoxification mechanism converting NO and O2 to the ion nitrate (NO3−). The protoheme of Hmp binds not only O2 and NO, but also CO so that this ligand is expected to be an inhibitor of NO detoxification in vivo and in vitro. CORM-3 (Ru(CO)3Cl(glycinate)) is a metal carbonyl compound extensively used and recently shown to have potent antibacterial properties. In this study, attenuation of the NO resistance of E. coli by CORM-3 is demonstrated in vivo. However, polarographic measurements showed that CO gas, but not CORM-3, produced inhibition of the NO detoxification activity of Hmp in vitro. Nevertheless, CO release from CORM-3 in the presence of soluble cellular compounds is demonstrated by formation of carboxy-Hmp. We show that the inability of CORM-3 to inhibit the activity of purified Hmp is due to slow release of CO in protein solutions alone i.e. when sodium dithionite, widely used in previous studies of CO release from CORM-3, is excluded. Finally, we measure intracellular CO released from CORM-3 by following the formation of carboxy-Hmp in respiring cells. CORM-3 is a tool to explore the concerted effects of CO and NO in vivo. PMID:25193663

  16. Expression of immune checkpoints in T cells of esophageal cancer patients

    PubMed Central

    Xie, Jinhua; Wang, Ji; Cheng, Shouliang; Zheng, Liangfeng; Ji, Feiyue; Yang, Lin; Zhang, Yan; Ji, Haoming

    2016-01-01

    Inhibition of immune checkpoint proteins (checkpoints) has become a promising anti-esophageal cancer strategy. We here tested expressions of immune checkpoints in human esophageal cancers. Our results showed the expressions of many immune checkpoints, including CD28, CD27, CD137L, programmed death 1 (PD-1), T cell immunoglobulin mucin-3 (TIM-3), T cell Ig and ITIM domain (TIGIT), CD160, cytotoxic T lymphocyte antigen 4 (CTLA-4), CD200, CD137 and CD158, were dysregulated in peripheral T cells of esophageal cancer patients. Further, the expressions of PD-1, TIM-3 and TIGIT were upregulated in tumor infiltrating lymphocytes (TILs), which might be associated with TILs exhaustion. Meanwhile, the expressions of PD-1 and TIM-3 on CD4+ T cells were closely associated with clinic pathological features of esophageal cancer patients. These results indicate that co-inhibitory receptors PD-1, TIM-3 and TIGIT may be potential therapeutic oncotargets for esophageal cancer. PMID:27577071

  17. Tim-4 protects mice against lipopolysaccharide-induced endotoxic shock by suppressing the NF-κB signaling pathway.

    PubMed

    Xu, Liyun; Zhao, Peiqing; Xu, Yong; Gao, Lishuang; Wang, Hongxing; Jia, Xiaoxia; Ma, Hongxin; Liang, Xiaoxong; Ma, Chunxong; Gao, Lifen

    2016-11-01

    Endotoxic shock is the primary cause of morbidity and mortality in hospital patients, creating an urgent need to explore the mechanisms involved in sepsis. Our previous studies showed that T-cell immunoglobulin- and mucin-domain-containing molecule-4 (Tim-4) attenuated the inflammatory response through regulating the functions of macrophages. However, the mechanism by which Tim-4 does this has not been fully elucidated. In this study, we found that Tim-4 expression was increased in lipopolysaccharide (LPS)-induced endotoxic shock. Interestingly, the survival rate of mice in the Tim-4 overexpression group was higher than that of the control group after LPS administration. To investigate the function of Tim-4 in LPS-induced inflammation, we further demonstrated that Tim-4 attenuated LPS-induced endotoxic shock by inhibiting cytokine production by macrophages. Blocking expression of Tim-4 and nuclear factor-kappa B (NF-κB) signal inhibition showed that Tim-4 inhibited cytokine production via NF-κB signaling pathway. This study indicates that Tim-4 may exert its immune modulation by regulating inflammatory factor secretion and might act as a novel potential target for inflammatory diseases, especially endotoxic shock.

  18. Molecular characterization of T-cell immunoglobulin mucin domain-3 and Galectin-9 genes of swamp- and riverine-type water buffaloes.

    PubMed

    Duran, P L H; Padiernos, R B C; Abella, E A; Konnai, S; Mingala, C N

    2015-12-01

    Molecular characterization of T-cell immunoglobulin mucin domain-3 (TIM-3) and Galectin-9 (GAL-9) genes of swamp- and riverine-type water buffaloes was conducted to compare these genes with other species; determine the unique characteristic specific in water buffalo; and provide baseline information for the assessment of disease progression in buffalo species. TIM-3 and GAL-9 genes were amplified, purified, sequenced and characterized. The sequence result of TIM-3 in both types of water buffaloes contained 843 nucleotides encoding to 280 amino acids while GAL-9 of swamp-type and riverine-type water buffaloes contained 1023 and 972 nucleotides encoding to 340 and 323 amino acids, respectively. Meanwhile, the nucleotide and amino sequence of TIM-3 in water buffalo were 83-98% and 94-97% identical with other artiodactyl species, respectively. On the other hand, GAL-9 nucleotide and amino acid sequence in water buffalo were 85-98% and 76-96% identical with other artiodactyl species. The tyrosine-kinase phosphorylation motif and potential glycosylation sites were conserved within the tribe Bovinae. It is imperative to have further studies in the assessment of the role of these genes in disease progression in water buffalo during chronic infection. The study is the first report that describes the genetic characteristic of TIM-3 and GAL-9 genes in water buffalo.

  19. Replacement of Oxygen by Sulfur in Small Organic Molecules. 3. Theoretical Studies on the Tautomeric Equilibria of the 2OH and 4OH-Substituted Oxazole and Thiazole and the 3OH and 4OH-Substituted Isoxazole and Isothiazole in the Isolated State and in Solution

    PubMed Central

    Nagy, Peter I.

    2016-01-01

    This follow-up paper completes the author’s investigations to explore the in-solution structural preferences and relative free energies of all OH-substituted oxazole, thiazole, isoxazole, and isothiazole systems. The polarizable continuum dielectric solvent method calculations in the integral-equation formalism (IEF-PCM) were performed at the DFT/B97D/aug-cc-pv(q+(d))z level for the stable neutral tautomers with geometries optimized in dichloromethane and aqueous solution. With the exception of the predictions for the predominant tautomers of the 3OH isoxazole and isothiazole, the results of the IEF-PCM calculations for identifying the most stable tautomer of the given species in the two selected solvents agreed with those from experimental investigations. The calculations predict that the hydroxy proton, with the exception for the 4OH isoxazole and 4OH isothiazole, moves preferentially to the ring nitrogen or to a ring carbon atom in parallel with the development of a C=O group. The remaining, low-fraction OH tautomers will not be observable in the equilibrium compositions. Relative solvation free energies obtained by the free energy perturbation method implemented in Monte Carlo simulations are in moderate accord with the IEF-PCM results, but consideration of the ΔGsolv/MC values in calculating ΔGstot maintains the tautomeric preferences. It was revealed from the Monte Carlo solution structure analyses that the S atom is not a hydrogen-bond acceptor in any OH-substituted thiazole or isothiazole, and the OH-substituted isoxazole and oxazole ring oxygens may act as a weak hydrogen-bond acceptor at most. The molecules form 1.0−3.4 solute−water hydrogen bonds in generally unexplored numbers at some specific solute sites. Nonetheless, hydrogen-bond formation is favorable with the NH, C=O and OH groups. PMID:27409605

  20. A bioinformatics approach identifies signal transducer and activator of transcription-3 and checkpoint kinase 1 as upstream regulators of kidney injury molecule-1 after kidney injury.

    PubMed

    Ajay, Amrendra Kumar; Kim, Tae-Min; Ramirez-Gonzalez, Victoria; Park, Peter J; Frank, David A; Vaidya, Vishal S

    2014-01-01

    Kidney injury molecule-1 (KIM-1)/T cell Ig and mucin domain-containing protein-1 (TIM-1) is upregulated more than other proteins after AKI, and it is highly expressed in renal damage of various etiologies. In this capacity, KIM-1/TIM-1 acts as a phosphatidylserine receptor on the surface of injured proximal tubular epithelial cells, mediating phagocytosis of apoptotic cells, and it may also act as a costimulatory molecule for immune cells. Despite recognition of KIM-1 as an important therapeutic target for kidney disease, the regulators of KIM-1 transcription in the kidney remain unknown. Using a bioinformatics approach, we identified upstream regulators of KIM-1 after AKI. In response to tubular injury in rat and human kidneys or oxidant stress in human proximal tubular epithelial cells (HPTECs), KIM-1 expression increased significantly in a manner that corresponded temporally and regionally with increased phosphorylation of checkpoint kinase 1 (Chk1) and STAT3. Both ischemic and oxidant stress resulted in a dramatic increase in reactive oxygen species that phosphorylated and activated Chk1, which subsequently bound to STAT3, phosphorylating it at S727. Furthermore, STAT3 bound to the KIM-1 promoter after ischemic and oxidant stress, and pharmacological or genetic induction of STAT3 in HPTECs increased KIM-1 mRNA and protein levels. Conversely, inhibition of STAT3 using siRNAs or dominant negative mutants reduced KIM-1 expression in a kidney cancer cell line (769-P) that expresses high basal levels of KIM-1. These observations highlight Chk1 and STAT3 as critical upstream regulators of KIM-1 expression after AKI and may suggest novel approaches for therapeutic intervention.

  1. T Cell Immunoglobulin Mucin-3 Crystal Structure Reveals a Galectin-9-Independent Ligand-Binding Surface

    SciTech Connect

    Cao,E.; Zang, X.; Ramagopal, U.; Mukhopadhaya, A.; Fedorov, A.; Fedorov, E.; Zencheck, W.; Lary, J.; Cole, J.; et al.

    2007-01-01

    The T cell immunoglobulin mucin (Tim) family of receptors regulates effector CD4+ T cell functions and is implicated in autoimmune and allergic diseases. Tim-3 induces immunological tolerance, and engagement of the Tim-3 immunoglobulin variable (IgV) domain by galectin-9 is important for appropriate termination of T helper 1-immune responses. The 2 {angstrom} crystal structure of the Tim-3 IgV domain demonstrated that four cysteines, which are invariant within the Tim family, form two noncanonical disulfide bonds, resulting in a surface not present in other immunoglobulin superfamily members. Biochemical and biophysical studies demonstrated that this unique structural feature mediates a previously unidentified galectin-9-independent binding process and suggested that this structural feature is conserved within the entire Tim family. The current work provided a graphic example of the relationship between sequence, structure, and function and suggested that the interplay between multiple Tim-3-binding activities contributes to the regulated assembly of signaling complexes required for effective Th1-mediated immunity.

  2. Prognostic and predictive aspects of the tumor immune microenvironment and immune checkpoints in malignant pleural mesothelioma.

    PubMed

    Marcq, Elly; Siozopoulou, Vasiliki; De Waele, Jorrit; van Audenaerde, Jonas; Zwaenepoel, Karen; Santermans, Eva; Hens, Niel; Pauwels, Patrick; van Meerbeeck, Jan P; Smits, Evelien L J

    2017-01-01

    Malignant pleural mesothelioma (MPM) is an aggressive cancer with a poor prognosis and an increasing incidence, for which novel therapeutic strategies are urgently required. Since the immune system has been described to play a presumed role in the protection against MPM, characterization of its tumor immune microenvironment (TME) and immune checkpoints can identify new immunotherapeutic targets and their predictive and/or prognostic value. To characterize the TME and the immune checkpoint expression profile, we performed immunohistochemistry (IHC) on formalin-fixed paraffin embedded (FFPE) tissue sections from 54 MPM patients (40 at time of diagnosis; 14 treated with chemotherapy). We stained for PD-1, PD-L1, TIM-3, LAG-3, CD4, CD8, CD45RO, granzyme B, FoxP3 and CD68. Furthermore, we analyzed the relationship between the immunological parameters and survival, as well as response to chemotherapy. We found that TIM-3, PD-1 and PD-L1 were expressed on both immune and tumor cells. Strikingly, PD-1 and PD-L1 expression on tumor cells was only seen in unpretreated samples. No LAG-3 expression was observed. CD45RO expression in the stroma was an independent negative predictive factor for response on chemotherapy, while CD4 and TIM-3 expression in lymphoid aggregates were independent prognostic factors for better outcome. Our data propose TIM-3 as a promising new target in mesothelioma. Chemotherapy influences the expression of immune checkpoints and therefore further research on the best combination treatment schedule is required.

  3. Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints.

    PubMed

    Koyama, Shohei; Akbay, Esra A; Li, Yvonne Y; Herter-Sprie, Grit S; Buczkowski, Kevin A; Richards, William G; Gandhi, Leena; Redig, Amanda J; Rodig, Scott J; Asahina, Hajime; Jones, Robert E; Kulkarni, Meghana M; Kuraguchi, Mari; Palakurthi, Sangeetha; Fecci, Peter E; Johnson, Bruce E; Janne, Pasi A; Engelman, Jeffrey A; Gangadharan, Sidharta P; Costa, Daniel B; Freeman, Gordon J; Bueno, Raphael; Hodi, F Stephen; Dranoff, Glenn; Wong, Kwok-Kin; Hammerman, Peter S

    2016-02-17

    Despite compelling antitumour activity of antibodies targeting the programmed death 1 (PD-1): programmed death ligand 1 (PD-L1) immune checkpoint in lung cancer, resistance to these therapies has increasingly been observed. In this study, to elucidate mechanisms of adaptive resistance, we analyse the tumour immune microenvironment in the context of anti-PD-1 therapy in two fully immunocompetent mouse models of lung adenocarcinoma. In tumours progressing following response to anti-PD-1 therapy, we observe upregulation of alternative immune checkpoints, notably T-cell immunoglobulin mucin-3 (TIM-3), in PD-1 antibody bound T cells and demonstrate a survival advantage with addition of a TIM-3 blocking antibody following failure of PD-1 blockade. Two patients who developed adaptive resistance to anti-PD-1 treatment also show a similar TIM-3 upregulation in blocking antibody-bound T cells at treatment failure. These data suggest that upregulation of TIM-3 and other immune checkpoints may be targetable biomarkers associated with adaptive resistance to PD-1 blockade.

  4. The role of LAT in increased CD8+ T cell exhaustion in trigeminal ganglia of mice latently infected with herpes simplex virus 1.

    PubMed

    Allen, Sariah J; Hamrah, Pedram; Gate, David; Mott, Kevin R; Mantopoulos, Dimosthenis; Zheng, Lixin; Town, Terrence; Jones, Clinton; von Andrian, Ulrich H; Freeman, Gordon J; Sharpe, Arlene H; BenMohamed, Lbachir; Ahmed, Rafi; Wechsler, Steven L; Ghiasi, Homayon

    2011-05-01

    Herpes simplex virus (HSV) infection is a classic example of latent viral infection in humans and experimental animal models. The HSV-1 latency-associated transcript (LAT) plays a major role in the HSV-1 latency reactivation cycle and thus in recurrent disease. Whether the presence of LAT leads to generation of dysfunctional T cell responses in the trigeminal ganglia (TG) of latently infected mice is not known. To address this issue, we used LAT-positive [LAT(+)] and LAT-deficient [LAT(-)] viruses to evaluate the effect of LAT on CD8 T cell exhaustion in TG of latently infected mice. The amount of latency as determined by quantitative reverse transcription-PCR (qRT-PCR) of viral DNA in total TG extracts was 3-fold higher with LAT(+) than with LAT(-) virus. LAT expression and increased latency correlated with increased mRNA levels of CD8, PD-1, and Tim-3. PD-1 is both a marker for exhaustion and a primary factor leading to exhaustion, and Tim-3 can also contribute to exhaustion. These results suggested that LAT(+) TG contain both more CD8(+) T cells and more CD8(+) T cells expressing the exhaustion markers PD-1 and Tim-3. This was confirmed by flow cytometry analyses of expression of CD3/CD8/PD-1/Tim-3, HSV-1, CD8(+) T cell pentamer (specific for a peptide derived from residues 498 to 505 of glycoprotein B [gB(498-505)]), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-α). The functional significance of PD-1 and its ligands in HSV-1 latency was demonstrated by the significantly reduced amount of HSV-1 latency in PD-1- and PD-L1-deficient mice. Together, these results may suggest that both PD-1 and Tim-3 are mediators of CD8(+) T cell exhaustion and latency in HSV-1 infection.

  5. Epigallocatechin-3-gallate Prevents Triptolide-Induced Hepatic Injury by Restoring the Th17/Treg Balance in Mice.

    PubMed

    Yu, Shu-Jing; Jiang, Rong; Mazzu, Ying Z; Wei, Cai-Bing; Sun, Zong-Liang; Zhang, Yu-Zhen; Zhou, Lian-Di; Zhang, Qi-Hui

    2016-01-01

    Drug-induced liver injury (DILI) is the most common cause of acute liver failure. Disruption of the Th17/Treg balance can lead to hepatic inflammation, which causes the main symptoms of DILI. Here we investigate the protective mechanisms of (-)-Epigallocatechin-3-gallate (EGCG) on triptolide (TP)-induced DILI that shows the Th17/Treg imbalance. Pretreatment with EGCG (5[Formula: see text]mg/kg) for 10 days before TP (0.5[Formula: see text]mg/kg) administration in mice significantly reduced the increased alanine aminotransferase (ALT) level ([Formula: see text]) induced by TP treatment. The hepatic histology analysis further proved that EGCG protected mice from TP-induced liver injury. The imbalance of Th17/Treg was induced by TP treatment, as shown by the upregulation of TLR4 and downregulation of Tim3 expression. EGCG pretreatment can maintain the expression of TLR4 and Tim3 at normal levels to restore the Th17/Treg imbalance. In addition, EGCG can block the TP-induced expression of the downstream targets of TLR4, including MyD88, NF[Formula: see text]B, and retinoid related orphan receptor (ROR-[Formula: see text]t), while EGCG can restore the TP inhibition of forkhead/winged-helix family transcriptional repressor p3 (FoxP3) that is the downstream target of Tim3. Consequently, EGCG pretreatment can effectively inhibit the Th17-related pro-inflammatory cytokine (e.g. IL-17 and IL-6) upregulation induced by TP treatment. However, TP inhibition of Treg-related anti-inflammatory cytokine IL-10 production was restored by EGCG pretreatment. Taken together, these results suggest that EGCG possesses significant protective properties against TP-induced hepatic inflammatory injury, and that these properties are carried out via the restoration of the Th17/Treg imbalance by the inhibition of the TLR4 signaling pathway and the enhanced activation of the Tim3 signaling pathway.

  6. Study of Increasing Lead Times in Major Weapon Systems Acquisition.

    DTIC Science & Technology

    1982-07-31

    Increasing Lead Tim ........ . 3-36 3.3.5.1.1 Market Factors . . . . . . . . .... ................... 3-36 3.3.5.1.2 Industrial Factors...factors, industry factors, or market factors. (For listings of specific causes identified, see Tables 3-2, 3-5, 3-8, 3-11, 3-13, and 3-16.) Some of...technicians, and other skilled craftsmen. * Market - The significant competition of comercial demands in certain business sectors such as aerospace and

  7. Tr1-Like T Cells – An Enigmatic Regulatory T Cell Lineage

    PubMed Central

    White, Anna Malgorzata; Wraith, David C.

    2016-01-01

    The immune system evolved to respond to foreign invaders and prevent autoimmunity to self-antigens. Several types of regulatory T cells facilitate the latter process. These include a subset of Foxp3− CD4+ T cells able to secrete IL-10 in an antigen-specific manner, type 1 regulatory (Tr1) T cells. Although their suppressive function has been confirmed both in vitro and in vivo, their phenotype remains poorly defined. It has been suggested that the surface markers LAG-3 and CD49b are biomarkers for murine and human Tr1 cells. Here, we discuss these findings in the context of our data regarding the expression pattern of inhibitory receptors (IRs) CD49b, TIM-3, PD-1, TIGIT, LAG-3, and ICOS on Tr1-like human T cells generated in vitro from CD4+ memory T cells stimulated with αCD3 and αCD28 antibodies. We found that there were no differences in IR expression between IL-10+ and IL-10− T cells. However, CD4+IL-10+ T cells isolated ex vivo, following a short stimulation and cytokine secretion assay, contained significantly higher proportions of TIM-3+ and PD-1+ cells. They also expressed significantly higher TIGIT mRNA and showed a trend toward increased TIM-3 mRNA levels. These data led us to conclude that large pools of IRs may be stored intracellularly; hence, they may not represent ideal candidates as cell surface biomarkers for Tr1-like T cells. PMID:27683580

  8. Blockage of Galectin-receptor Interactions by α-lactose Exacerbates Plasmodium berghei-induced Pulmonary Immunopathology

    PubMed Central

    Liu, Jinfeng; Huang, Shiguang; Su, Xin-zhuan; Song, Jianping; Lu, Fangli

    2016-01-01

    Malaria-associated acute lung injury (ALI) is a frequent complication of severe malaria that is often caused by “excessive” immune responses. To better understand the mechanism of ALI in malaria infection, here we investigated the roles of galectin (Gal)-1, 3, 8, 9 and the receptors of Gal-9 (Tim-3, CD44, CD137, and PDI) in malaria-induced ALI. We injected alpha (α)-lactose into mice-infected with Plasmodium berghei ANKA (PbANKA) to block galectins and found significantly elevated total proteins in bronchoalveolar lavage fluid, higher parasitemia and tissue parasite burden, and increased numbers of CD68+ alveolar macrophages as well as apoptotic cells in the lungs after blockage. Additionally, mRNA levels of Gal-9, Tim-3, CD44, CD137, and PDI were significantly increased in the lungs at day 5 after infection, and the levels of CD137, IFN-α, IFN-β, IFN-γ, IL-4, and IL-10 in the lungs were also increased after α-lactose treatment. Similarly, the levels of Gal-9, Tim-3, IFN-α, IFN-β, IFN-γ, and IL-10 were all significantly increased in murine peritoneal macrophages co-cultured with PbANKA-infected red blood cells in vitro; but only IFN-α and IFN-β were significantly increased after α-lactose treatment. Our data indicate that Gal-9 interaction with its multiple receptors play an important role in murine malaria-associated ALI. PMID:27554340

  9. Cytotoxic T Cells in PD-L1-Positive Malignant Pleural Mesotheliomas Are Counterbalanced by Distinct Immunosuppressive Factors.

    PubMed

    Awad, Mark M; Jones, Robert E; Liu, Hongye; Lizotte, Patrick H; Ivanova, Elena V; Kulkarni, Meghana; Herter-Sprie, Grit S; Liao, Xiaoyun; Santos, Abigail A; Bittinger, Mark A; Keogh, Lauren; Koyama, Shohei; Almonte, Christina; English, Jessie M; Barlow, Julianne; Richards, William G; Barbie, David A; Bass, Adam J; Rodig, Scott J; Hodi, F Stephen; Wucherpfennig, Kai W; Jänne, Pasi A; Sholl, Lynette M; Hammerman, Peter S; Wong, Kwok-Kin; Bueno, Raphael

    2016-12-01

    PD-L1 immunohistochemical staining does not always predict whether a cancer will respond to treatment with PD-1 inhibitors. We sought to characterize immune cell infiltrates and the expression of T-cell inhibitor markers in PD-L1-positive and PD-L1-negative malignant pleural mesothelioma samples. We developed a method for immune cell phenotyping using flow cytometry on solid tumors that have been dissociated into single-cell suspensions and applied this technique to analyze 43 resected malignant pleural mesothelioma specimens. Compared with PD-L1-negative tumors, PD-L1-positive tumors had significantly more infiltrating CD45(+) immune cells, a significantly higher proportion of infiltrating CD3(+) T cells, and a significantly higher percentage of CD3(+) cells displaying the activated HLA-DR(+)/CD38(+) phenotype. PD-L1-positive tumors also had a significantly higher proportion of proliferating CD8(+) T cells, a higher fraction of FOXP3(+)/CD4(+) Tregs, and increased expression of PD-1 and TIM-3 on CD4(+) and CD8(+) T cells. Double-positive PD-1(+)/TIM-3(+) CD8(+) T cells were more commonly found on PD-L1-positive tumors. Compared with epithelioid tumors, sarcomatoid and biphasic mesothelioma samples were significantly more likely to be PD-L1 positive and showed more infiltration with CD3(+) T cells and PD-1(+)/TIM-3(+) CD8(+) T cells. Immunologic phenotypes in mesothelioma differ based on PD-L1 status and histologic subtype. Successful incorporation of comprehensive immune profiling by flow cytometry into prospective clinical trials could refine our ability to predict which patients will respond to specific immune checkpoint blockade strategies. Cancer Immunol Res; 4(12); 1038-48. ©2016 AACR.

  10. Structure of full-length Toxascaris leonina galectin with two carbohydrate-recognition domains.

    PubMed

    Jeong, Mi Suk; Hwang, Hyun Gi; Yu, Hak Sun; Jang, Se Bok

    2013-02-01

    The full-length crystal structure of Toxascaris leonine galectin (Tl-gal), a galectin-9 homologue protein, was determined at a resolution of 2.0 Å. Galectin-9 exhibits a variety of biological functions, including cell aggregation, eosinophil chemoattraction, activation and apoptosis of murine thymocytes, T cells and human melanoma cells. Similar to this galectin, Tl-gal may function as a regulatory molecule in the host immune system; however, no molecular or structural information has been reported for Tl-gal. Moreover, until now, there have been no reports of a full-length galectin structure. There are two molecules of Tl-gal per asymmetric unit in space group P2(1)2(1)2(1), and the N-terminal and C-terminal carbohydrate-recognition domains (NCRD and CCRD) of Tl-gal are composed of six-stranded β-sheets and five-stranded β-sheets with a short α-helix. The NCRD of Tl-gal resembles that of human galectin-7 and its CCRD resembles human galectin-9, but the residues in the interface and loop regions of the NCRD and CCRD are flexible and are related to interaction. Engagement of the T-cell immunoglobulin mucin-3 (Tim-3) immunoglobulin variable (IgV) domain by a galectin-9 ligand is known to be important for appropriate termination of T-helper 1 immune responses. To investigate the binding site of Tl-gal, the interaction between Tl-gal and Tim-3 was modelled. Tim-3 is docked into a major groove of the Tl-gal structure, which is larger and deeper than the minor groove. The structural information presented here will provide insight into the development of novel anti-inflammatory agents or selective modulators of immune response.

  11. High Expression of Antiviral and Vitamin D Pathway Genes Are a Natural Characteristic of a Small Cohort of HIV-1-Exposed Seronegative Individuals.

    PubMed

    Aguilar-Jimenez, Wbeimar; Saulle, Irma; Trabattoni, Daria; Vichi, Francesca; Lo Caputo, Sergio; Mazzotta, Francesco; Rugeles, Maria T; Clerici, Mario; Biasin, Mara

    2017-01-01

    Natural resistance to HIV-1 infection is influenced by genetics, viral-exposure, and endogenous immunomodulators such as vitamin D (VitD), being a multifactorial phenomenon that characterizes HIV-1-exposed seronegative individuals (HESNs). We compared mRNA expression of 10 antivirals, 5 immunoregulators, and 3 VitD pathway genes by qRT-PCR in cells of a small cohort of 11 HESNs, 16 healthy-controls (HCs), and 11 seropositives (SPs) at baseline, in response to calcidiol (VitD precursor) and/or aldithriol-2-(AT2)-inactivated HIV-1. In addition, the expression of TIM-3 on T and NK cells of six HCs after calcidiol and calcitriol (active VitD) treatments was evaluated by flow cytometry. Calcidiol increased the mRNA expression of HAVCR2 (TIM-3; Th1-cells inhibitor) in HCs and HESNs. AT2-HIV-1 increased the mRNA expression of the activating VitD enzyme CYP27B1, of the endogenous antiviral proteins MX2, TRIM22, APOBEC3G, and of immunoregulators ERAP2 and HAVCR2, but reduced the mRNA expression of VitD receptor (VDR) and antiviral peptides PI3 and CAMP in all groups. Remarkably, higher mRNA levels of VDR, CYP27B1, PI3, CAMP, SLPI, and of ERAP2 were found in HESNs compared to HCs either at baseline or after stimuli. Furthermore, calcitriol increases the percentage of CD4+ T cells expressing TIM-3 protein compared to EtOH controls. These results suggest that high mRNA expression of antiviral and VitD pathway genes could be genetically determined in HESNs more than viral-induced at least in peripheral blood mononuclear cells. Moreover, the virus could potentiate bio-activation and use of VitD, maintaining the homeostasis of the immune system. Interestingly, VitD-induced TIM-3 on T cells, a T cell inhibitory and anti-HIV-1 molecule, requires further studies to analyze the functional outcomes during HIV-1 infection.

  12. High Expression of Antiviral and Vitamin D Pathway Genes Are a Natural Characteristic of a Small Cohort of HIV-1-Exposed Seronegative Individuals

    PubMed Central

    Aguilar-Jimenez, Wbeimar; Saulle, Irma; Trabattoni, Daria; Vichi, Francesca; Lo Caputo, Sergio; Mazzotta, Francesco; Rugeles, Maria T.; Clerici, Mario; Biasin, Mara

    2017-01-01

    Natural resistance to HIV-1 infection is influenced by genetics, viral-exposure, and endogenous immunomodulators such as vitamin D (VitD), being a multifactorial phenomenon that characterizes HIV-1-exposed seronegative individuals (HESNs). We compared mRNA expression of 10 antivirals, 5 immunoregulators, and 3 VitD pathway genes by qRT-PCR in cells of a small cohort of 11 HESNs, 16 healthy-controls (HCs), and 11 seropositives (SPs) at baseline, in response to calcidiol (VitD precursor) and/or aldithriol-2-(AT2)-inactivated HIV-1. In addition, the expression of TIM-3 on T and NK cells of six HCs after calcidiol and calcitriol (active VitD) treatments was evaluated by flow cytometry. Calcidiol increased the mRNA expression of HAVCR2 (TIM-3; Th1-cells inhibitor) in HCs and HESNs. AT2-HIV-1 increased the mRNA expression of the activating VitD enzyme CYP27B1, of the endogenous antiviral proteins MX2, TRIM22, APOBEC3G, and of immunoregulators ERAP2 and HAVCR2, but reduced the mRNA expression of VitD receptor (VDR) and antiviral peptides PI3 and CAMP in all groups. Remarkably, higher mRNA levels of VDR, CYP27B1, PI3, CAMP, SLPI, and of ERAP2 were found in HESNs compared to HCs either at baseline or after stimuli. Furthermore, calcitriol increases the percentage of CD4+ T cells expressing TIM-3 protein compared to EtOH controls. These results suggest that high mRNA expression of antiviral and VitD pathway genes could be genetically determined in HESNs more than viral-induced at least in peripheral blood mononuclear cells. Moreover, the virus could potentiate bio-activation and use of VitD, maintaining the homeostasis of the immune system. Interestingly, VitD-induced TIM-3 on T cells, a T cell inhibitory and anti-HIV-1 molecule, requires further studies to analyze the functional outcomes during HIV-1 infection. PMID:28243241

  13. Development of Titanium Alloy Casting Technology

    DTIC Science & Technology

    1976-08-01

    q Opp (0~ ~V~w Het221(47) ()Ha 221(45 TIiC-loT-lC-, , P, L.q& r .- 6N.’ ( Heat 24221. ( 9497 ) (1) Heat 24224 (9493) Ti-13Cu-lCON Ti-M3u-Le.5N 1760F...N.D. Ag, Sc, Na, In, Y, V, De, B, Nb , Ge, W, Bi, Cr, Te Hlydrogen M.D. Chlorine 0.07 0.09 -- ( 0.005-Coors) Oxygen (0. 65-Oremet) Nitrogen (0. 61

  14. TIGIT predominantly regulates the immune response via regulatory T cells

    PubMed Central

    Kurtulus, Sema; Sakuishi, Kaori; Ngiow, Shin-Foong; Joller, Nicole; Tan, Dewar J.; Teng, Michele W.L.; Smyth, Mark J.; Kuchroo, Vijay K.; Anderson, Ana C.

    2015-01-01

    Coinhibitory receptors are critical for the maintenance of immune homeostasis. Upregulation of these receptors on effector T cells terminates T cell responses, while their expression on Tregs promotes their suppressor function. Understanding the function of coinhibitory receptors in effector T cells and Tregs is crucial, as therapies that target coinhibitory receptors are currently at the forefront of treatment strategies for cancer and other chronic diseases. T cell Ig and ITIM domain (TIGIT) is a recently identified coinhibitory receptor that is found on the surface of a variety of lymphoid cells, and its role in immune regulation is just beginning to be elucidated. We examined TIGIT-mediated immune regulation in different murine cancer models and determined that TIGIT marks the most dysfunctional subset of CD8+ T cells in tumor tissue as well as tumor-tissue Tregs with a highly active and suppressive phenotype. We demonstrated that TIGIT signaling in Tregs directs their phenotype and that TIGIT primarily suppresses antitumor immunity via Tregs and not CD8+ T cells. Moreover, TIGIT+ Tregs upregulated expression of the coinhibitory receptor TIM-3 in tumor tissue, and TIM-3 and TIGIT synergized to suppress antitumor immune responses. Our findings provide mechanistic insight into how TIGIT regulates immune responses in chronic disease settings. PMID:26413872

  15. Follicular Regulatory CD8 T Cells Impair the Germinal Center Response in SIV and Ex Vivo HIV Infection

    PubMed Central

    Folkvord, Joy M.; Levy, David N.; Rakasz, Eva G.; Connick, Elizabeth

    2016-01-01

    During chronic HIV infection, viral replication is concentrated in secondary lymphoid follicles. Cytotoxic CD8 T cells control HIV replication in extrafollicular regions, but not in the follicle. Here, we show CXCR5hiCD44hiCD8 T cells are a regulatory subset differing from conventional CD8 T cells, and constitute the majority of CD8 T cells in the follicle. This subset, CD8 follicular regulatory T cells (CD8 TFR), expand in chronic SIV infection, exhibit enhanced expression of Tim-3 and IL-10, and express less perforin compared to conventional CD8 T cells. CD8 TFR modestly limit HIV replication in follicular helper T cells (TFH), impair TFH IL-21 production via Tim-3, and inhibit IgG production by B cells during ex vivo HIV infection. CD8 TFR induce TFH apoptosis through HLA-E, but induce less apoptosis than conventional CD8 T cells. These data demonstrate that a unique regulatory CD8 population exists in follicles that impairs GC function in HIV infection. PMID:27716848

  16. CD8+ T cells of chronic HCV-infected patients express multiple negative immune checkpoints following stimulation with HCV peptides.

    PubMed

    Barathan, Muttiah; Mohamed, Rosmawati; Vadivelu, Jamuna; Chang, Li Yen; Vignesh, Ramachandran; Krishnan, Jayalakshmi; Sigamani, Panneer; Saeidi, Alireza; Ram, M Ravishankar; Velu, Vijayakumar; Larsson, Marie; Shankar, Esaki M

    2017-03-01

    Hepatitis C virus (HCV)-specific CD4+ and CD8+ T cells are key to successful viral clearance in HCV disease. Accumulation of exhausted HCV-specific T cells during chronic infection results in considerable loss of protective functional immune responses. The role of T-cell exhaustion in chronic HCV disease remains poorly understood. Here, we studied the frequency of HCV peptide-stimulated T cells expressing negative immune checkpoints (PD-1, CTLA-4, TRAIL, TIM-3 and BTLA) by flow cytometry, and measured the levels of Th1/Th2/Th17 cytokines secreted by T cells by a commercial Multi-Analyte ELISArray™ following in vitro stimulation of T cells using HCV peptides and phytohemagglutinin (PHA). HCV peptide-stimulated CD4+ and CD8+ T cells of chronic HCV (CHC) patients showed significant increase of CTLA-4. Furthermore, HCV peptide-stimulated CD4+ T cells of CHC patients also displayed relatively higher levels of PD-1 and TRAIL, whereas TIM-3 was up-regulated on HCV peptide-stimulated CD8+ T cells. Whereas the levels of IL-10 and TGF-β1 were significantly increased, the levels of pro-inflammatory cytokines IL-2, TNF-α, IL-17A and IL-6 were markedly decreased in the T cell cultures of CHC patients. Chronic HCV infection results in functional exhaustion of CD4+ and CD8+ T cells likely contributing to viral persistence.

  17. TIGIT predominantly regulates the immune response via regulatory T cells.

    PubMed

    Kurtulus, Sema; Sakuishi, Kaori; Ngiow, Shin-Foong; Joller, Nicole; Tan, Dewar J; Teng, Michele W L; Smyth, Mark J; Kuchroo, Vijay K; Anderson, Ana C

    2015-11-02

    Coinhibitory receptors are critical for the maintenance of immune homeostasis. Upregulation of these receptors on effector T cells terminates T cell responses, while their expression on Tregs promotes their suppressor function. Understanding the function of coinhibitory receptors in effector T cells and Tregs is crucial, as therapies that target coinhibitory receptors are currently at the forefront of treatment strategies for cancer and other chronic diseases. T cell Ig and ITIM domain (TIGIT) is a recently identified coinhibitory receptor that is found on the surface of a variety of lymphoid cells, and its role in immune regulation is just beginning to be elucidated. We examined TIGIT-mediated immune regulation in different murine cancer models and determined that TIGIT marks the most dysfunctional subset of CD8+ T cells in tumor tissue as well as tumor-tissue Tregs with a highly active and suppressive phenotype. We demonstrated that TIGIT signaling in Tregs directs their phenotype and that TIGIT primarily suppresses antitumor immunity via Tregs and not CD8+ T cells. Moreover, TIGIT+ Tregs upregulated expression of the coinhibitory receptor TIM-3 in tumor tissue, and TIM-3 and TIGIT synergized to suppress antitumor immune responses. Our findings provide mechanistic insight into how TIGIT regulates immune responses in chronic disease settings.

  18. Interferon response factor 3 is crucial to poly-I:C induced NK cell activity and control of B16 melanoma growth.

    PubMed

    Moore, Tyler C; Kumm, Phyllis M; Brown, Deborah M; Petro, Thomas M

    2014-04-28

    Interferon Response Factor 3 (IRF3) induces several NK-cell activating factors, is activated by poly-I:C, an experimental cancer therapeutic, but is suppressed during many viral infections. IRF3 Knockout (KO) mice exhibited enhanced B16 melanoma growth, impaired intratumoral NK cell infiltration, but not an impaired poly-I:C therapeutic effect due to direct suppression of B16 growth. IRF3 was responsible for poly-I:C decrease in TIM-3 expression by intratumoral dendritic cells, induction of NK-cell Granzyme B and IFN-γ, and induction of macrophage IL-12, IL-15, IL-6, and IRF3-dependent NK-activating molecule (INAM). Thus, IRF3 is a key factor controlling melanoma growth through NK-cell activities, especially during poly-I:C therapy.

  19. Immunological biomarkers predict HIV-1 viral rebound after treatment interruption

    PubMed Central

    Hurst, Jacob; Hoffmann, Matthias; Pace, Matthew; Williams, James P.; Thornhill, John; Hamlyn, Elizabeth; Meyerowitz, Jodi; Willberg, Chris; Koelsch, Kersten K.; Robinson, Nicola; Brown, Helen; Fisher, Martin; Kinloch, Sabine; Cooper, David A.; Schechter, Mauro; Tambussi, Giuseppe; Fidler, Sarah; Babiker, Abdel; Weber, Jonathan; Kelleher, Anthony D.; Phillips, Rodney E.; Frater, John

    2015-01-01

    Treatment of HIV-1 infection with antiretroviral therapy (ART) in the weeks following transmission may induce a state of ‘post-treatment control' (PTC) in some patients, in whom viraemia remains undetectable when ART is stopped. Explaining PTC could help our understanding of the processes that maintain viral persistence. Here we show that immunological biomarkers can predict time to viral rebound after stopping ART by analysing data from a randomized study of primary HIV-1 infection incorporating a treatment interruption (TI) after 48 weeks of ART (the SPARTAC trial). T-cell exhaustion markers PD-1, Tim-3 and Lag-3 measured prior to ART strongly predict time to the return of viraemia. These data indicate that T-cell exhaustion markers may identify those latently infected cells with a higher proclivity to viral transcription. Our results may open new avenues for understanding the mechanisms underlying PTC, and eventually HIV-1 eradication. PMID:26449164

  20. Immunological biomarkers predict HIV-1 viral rebound after treatment interruption.

    PubMed

    Hurst, Jacob; Hoffmann, Matthias; Pace, Matthew; Williams, James P; Thornhill, John; Hamlyn, Elizabeth; Meyerowitz, Jodi; Willberg, Chris; Koelsch, Kersten K; Robinson, Nicola; Brown, Helen; Fisher, Martin; Kinloch, Sabine; Cooper, David A; Schechter, Mauro; Tambussi, Giuseppe; Fidler, Sarah; Babiker, Abdel; Weber, Jonathan; Kelleher, Anthony D; Phillips, Rodney E; Frater, John

    2015-10-09

    Treatment of HIV-1 infection with antiretroviral therapy (ART) in the weeks following transmission may induce a state of 'post-treatment control' (PTC) in some patients, in whom viraemia remains undetectable when ART is stopped. Explaining PTC could help our understanding of the processes that maintain viral persistence. Here we show that immunological biomarkers can predict time to viral rebound after stopping ART by analysing data from a randomized study of primary HIV-1 infection incorporating a treatment interruption (TI) after 48 weeks of ART (the SPARTAC trial). T-cell exhaustion markers PD-1, Tim-3 and Lag-3 measured prior to ART strongly predict time to the return of viraemia. These data indicate that T-cell exhaustion markers may identify those latently infected cells with a higher proclivity to viral transcription. Our results may open new avenues for understanding the mechanisms underlying PTC, and eventually HIV-1 eradication.

  1. Impaired T cell function in malignant pleural effusion is caused by TGF-β derived predominantly from macrophages.

    PubMed

    Li, Lifeng; Yang, Li; Wang, Liping; Wang, Fei; Zhang, Zhen; Li, Jieyao; Yue, Dongli; Chen, Xinfeng; Ping, Yu; Huang, Lan; Zhang, Bin; Zhang, Yi

    2016-11-15

    Malignant pleural effusion (MPE) is an indication of advanced cancer. Immune dysfunction often occurs in MPE. We aimed to identify the reason for impaired T cell activity in MPE from lung cancer patients and to provide clues toward potential immune therapies for MPE. The surface inhibitory molecules and cytotoxic activity of T cells in MPE and peripheral blood (PB) were analyzed using flow cytometry. Levels of inflammatory cytokines in MPE and PB were tested using ELISA. TGF-β expression in tumor-associated macrophages (TAMs) was also analyzed. The effect of TAMs on T cells was verified in vitro. Lastly, changes in T cells were evaluated following treatment with anti-TGF-β antibody. We found that expression levels of Tim-3, PD-1 and CTLA-4 in T cells from MPE were upregulated compared with those from PB, but levels of IFN-γ and Granzyme B were downregulated (p < 0.05). The amount of TGF-β was significantly higher in MPE than in PB (p < 0.05). TGF-β was mainly produced by TAMs in MPE. When T cells were co-cultured with TAMs, expression levels of Tim-3, PD-1 and CTLA-4 were significantly higher than controls, whereas levels of IFN-γ and Granzyme B were significantly decreased, in a dose-dependent manner (p < 0.05). In vitro treatment with anti-TGF-β antibody restored the impaired T cell cytotoxic activity in MPE. Our results indicate that macrophage-derived TGF-β plays an important role in impaired T cell cytotoxicity. It will therefore be valuable to develop therapeutic strategies against TGF-β pathway for MPE therapy of lung cancer.

  2. Plasma biomarkers of risk for death in a multicenter phase 3 trial with uniform transplant characteristics post–allogeneic HCT

    PubMed Central

    Abu Zaid, Mohammad; Wu, Juan; Wu, Cindy; Logan, Brent R.; Yu, Jeffrey; Cutler, Corey; Antin, Joseph H.; Paczesny, Sophie

    2017-01-01

    A phase 3 clinical trial (BMT CTN 0402) comparing tacrolimus/sirolimus (Tac/Sir) vs tacrolimus/methotrexate (Tac/Mtx) as graft-versus-host disease (GVHD) prophylaxis after matched-related allogeneic hematopoietic cell transplantation (HCT) recently showed no difference between study arms in acute GVHD-free survival. Within this setting of a prospective, multicenter study with uniform GVHD prophylaxis, conditioning regimen, and donor source, we explored the correlation of 10 previously identified biomarkers with clinical outcomes after allogeneic HCT. We measured biomarkers from plasma samples collected in 211 patients using enzyme-linked immunosorbent assay (Tac/Sir = 104, Tac/Mtx = 107). High suppression of tumorigenicity-2 (ST2) and T-cell immunoglobulin mucin-3 (TIM3) at day 28 correlated with 2-year nonrelapse mortality in multivariate analysis (P = .0050, P = .0075, respectively) and in a proportional hazards model with time-dependent covariates (adjusted hazard ratio: 2.43 [1.49–3.95], P = .0038 and 4.87 [2.53–9.34], P < .0001, respectively). High ST2 and TIM3 correlated with overall survival. Chemokine (C-X-C motif) ligand 9 (CXCL9) levels above the median were associated with chronic GVHD compared with levels below the median in a time-dependent proportional hazard analysis (P = .0069). Low L-Ficolin was associated with hepatic veno-occlusive disease (P = .0053, AUC = 0.80). We confirmed the correlation of plasma-derived proteins, previously assessed in single-center cohorts, with clinical outcomes after allogeneic HCT within this prospective, multicenter study. PMID:27827824

  3. Intraepithelial macrophage infiltration is related to a high number of regulatory T cells and promotes a progressive course of HPV-induced vulvar neoplasia.

    PubMed

    van Esch, Edith M G; van Poelgeest, Mariette I E; Trimbos, J Baptist M Z; Fleuren, Gert Jan; Jordanova, Ekaterina S; van der Burg, Sjoerd H

    2015-02-15

    Human papilloma virus (HPV)-induced usual-type vulvar intraepithelial neoplasia (uVIN) is infiltrated by myeloid cells but the type and role of these cells is unclear. We used triple immunofluorescent confocal microscopy to locate, identify and quantify myeloid cells based on their staining pattern for CD14, CD33 and CD163 in a cohort of 43 primary and 20 recurrent uVIN lesions, 21 carcinomas and 26 normal vulvar tissues. The progressive course of uVIN is characterized by an increase in both intraepithelial and stromal mature M1 and M2 macrophages. While the M2 macrophages outnumber M1 macrophages in healthy controls and uVIN, they are matched in number by M1 macrophages in cancer. Importantly, uVIN patients with a dense intraepithelial infiltration with mature CD14+ macrophages (irrespective of M1 or M2 type) displayed approximately a six times higher risk to develop a recurrence and a high number of these cells constituted an independent prognostic factor for recurrence. In addition, a dense intraepithelial CD14+ cell infiltration was associated with high numbers of intraepithelial CD4+ Tregs and low numbers of stromal CD8+TIM3+ T cells. Patients with low numbers of intraepithelial CD14+ cells and high numbers of stromal CD8+TIM3+ cells showed the best recurrence-free survival. These data clearly show the importance of the local immune response in HPV-induced vulvar neoplasia and may be of help in predicting the prognosis of patients or their response to immunotherapy.

  4. Galectin-9 is rapidly released during acute HIV-1 infection and remains sustained at high levels despite viral suppression even in elite controllers.

    PubMed

    Tandon, Ravi; Chew, Glen M; Byron, Mary M; Borrow, Persephone; Niki, Toshiro; Hirashima, Mitsuomi; Barbour, Jason D; Norris, Philip J; Lanteri, Marion C; Martin, Jeffrey N; Deeks, Steven G; Ndhlovu, Lishomwa C

    2014-07-01

    Galectin-9 (Gal-9) is a β-galactosidase-binding lectin that promotes apoptosis, tissue inflammation, and T cell immune exhaustion, and alters HIV infection in part through engagement with the T cell immunoglobulin mucin domain-3 (Tim-3) receptor and protein disulfide isomerases (PDI). Gal-9 was initially thought to be an eosinophil attractant, but is now known to mediate multiple complex signaling events that affect T cells in both an immunosuppressive and inflammatory manner. To understand the kinetics of circulating Gal-9 levels during HIV infection we measured Gal-9 in plasma during HIV acquisition, in subjects with chronic HIV infection with differing virus control, and in uninfected individuals. During acute HIV infection, circulating Gal-9 was detected as early as 5 days after quantifiable HIV RNA and tracked plasma levels of interleukin (IL)-10, tumor necrosis factor (TNF)-α, and IL-1β. In chronic HIV infection, Gal-9 levels positively correlated with plasma HIV RNA levels (r=0.29; p=0.023), and remained significantly elevated during suppressive antiretroviral therapy (median: 225.3 pg/ml) and in elite controllers (263.3 pg/ml) compared to age-matched HIV-uninfected controls (54 pg/ml). Our findings identify Gal-9 as a novel component of the first wave of the cytokine storm in acute HIV infection that is sustained at elevated levels in virally suppressed subjects and suggest that Gal-9:Tim-3 crosstalk remains active in elite controllers and antiretroviral (ARV)-suppressed subjects, potentially contributing to ongoing inflammation and persistent T cell dysfunction.

  5. Exhaustion of Activated CD8 T Cells Predicts Disease Progression in Primary HIV-1 Infection.

    PubMed

    Hoffmann, Matthias; Pantazis, Nikos; Martin, Genevieve E; Hickling, Stephen; Hurst, Jacob; Meyerowitz, Jodi; Willberg, Christian B; Robinson, Nicola; Brown, Helen; Fisher, Martin; Kinloch, Sabine; Babiker, Abdel; Weber, Jonathan; Nwokolo, Nneka; Fox, Julie; Fidler, Sarah; Phillips, Rodney; Frater, John

    2016-07-01

    The rate at which HIV-1 infected individuals progress to AIDS is highly variable and impacted by T cell immunity. CD8 T cell inhibitory molecules are up-regulated in HIV-1 infection and associate with immune dysfunction. We evaluated participants (n = 122) recruited to the SPARTAC randomised clinical trial to determine whether CD8 T cell exhaustion markers PD-1, Lag-3 and Tim-3 were associated with immune activation and disease progression. Expression of PD-1, Tim-3, Lag-3 and CD38 on CD8 T cells from the closest pre-therapy time-point to seroconversion was measured by flow cytometry, and correlated with surrogate markers of HIV-1 disease (HIV-1 plasma viral load (pVL) and CD4 T cell count) and the trial endpoint (time to CD4 count <350 cells/μl or initiation of antiretroviral therapy). To explore the functional significance of these markers, co-expression of Eomes, T-bet and CD39 was assessed. Expression of PD-1 on CD8 and CD38 CD8 T cells correlated with pVL and CD4 count at baseline, and predicted time to the trial endpoint. Lag-3 expression was associated with pVL but not CD4 count. For all exhaustion markers, expression of CD38 on CD8 T cells increased the strength of associations. In Cox models, progression to the trial endpoint was most marked for PD-1/CD38 co-expressing cells, with evidence for a stronger effect within 12 weeks from confirmed diagnosis of PHI. The effect of PD-1 and Lag-3 expression on CD8 T cells retained statistical significance in Cox proportional hazards models including antiretroviral therapy and CD4 count, but not pVL as co-variants. Expression of 'exhaustion' or 'immune checkpoint' markers in early HIV-1 infection is associated with clinical progression and is impacted by immune activation and the duration of infection. New markers to identify exhausted T cells and novel interventions to reverse exhaustion may inform the development of novel immunotherapeutic approaches.

  6. Exhaustion of Activated CD8 T Cells Predicts Disease Progression in Primary HIV-1 Infection

    PubMed Central

    Hickling, Stephen; Hurst, Jacob; Meyerowitz, Jodi; Willberg, Christian B.; Robinson, Nicola; Brown, Helen; Kinloch, Sabine; Babiker, Abdel; Nwokolo, Nneka; Fox, Julie; Fidler, Sarah; Phillips, Rodney; Frater, John

    2016-01-01

    The rate at which HIV-1 infected individuals progress to AIDS is highly variable and impacted by T cell immunity. CD8 T cell inhibitory molecules are up-regulated in HIV-1 infection and associate with immune dysfunction. We evaluated participants (n = 122) recruited to the SPARTAC randomised clinical trial to determine whether CD8 T cell exhaustion markers PD-1, Lag-3 and Tim-3 were associated with immune activation and disease progression. Expression of PD-1, Tim-3, Lag-3 and CD38 on CD8 T cells from the closest pre-therapy time-point to seroconversion was measured by flow cytometry, and correlated with surrogate markers of HIV-1 disease (HIV-1 plasma viral load (pVL) and CD4 T cell count) and the trial endpoint (time to CD4 count <350 cells/μl or initiation of antiretroviral therapy). To explore the functional significance of these markers, co-expression of Eomes, T-bet and CD39 was assessed. Expression of PD-1 on CD8 and CD38 CD8 T cells correlated with pVL and CD4 count at baseline, and predicted time to the trial endpoint. Lag-3 expression was associated with pVL but not CD4 count. For all exhaustion markers, expression of CD38 on CD8 T cells increased the strength of associations. In Cox models, progression to the trial endpoint was most marked for PD-1/CD38 co-expressing cells, with evidence for a stronger effect within 12 weeks from confirmed diagnosis of PHI. The effect of PD-1 and Lag-3 expression on CD8 T cells retained statistical significance in Cox proportional hazards models including antiretroviral therapy and CD4 count, but not pVL as co-variants. Expression of ‘exhaustion’ or ‘immune checkpoint’ markers in early HIV-1 infection is associated with clinical progression and is impacted by immune activation and the duration of infection. New markers to identify exhausted T cells and novel interventions to reverse exhaustion may inform the development of novel immunotherapeutic approaches. PMID:27415828

  7. TIM-1 Promotes Hepatitis C Virus Cell Attachment and Infection.

    PubMed

    Wang, Jing; Qiao, Luhua; Hou, Zhouhua; Luo, Guangxiang

    2017-01-15

    Human TIM and TAM family proteins were recently found to serve as phosphatidylserine (PS) receptors which promote infections by many different viruses, including dengue virus, West Nile virus, Ebola virus, Marburg virus, and Zika virus. In the present study, we provide substantial evidence demonstrating that TIM-1 is important for efficient infection by hepatitis C virus (HCV). The knockdown of TIM-1 expression significantly reduced HCV infection but not HCV RNA replication. Likewise, TIM-1 knockout in Huh-7.5 cells remarkably lowered HCV cell attachment and subsequent HCV infection. More significantly, the impairment of HCV infection in the TIM-1 knockout cells could be restored completely by ectopic expression of TIM-1 but not TIM-3 or TIM-4. Additionally, HCV infection and cell attachment were inhibited by PS but not by phosphatidylcholine (PC), demonstrating that TIM-1-mediated enhancement of HCV infection is PS dependent. The exposure of PS on the HCV envelope was confirmed by immunoprecipitation of HCV particles with a PS-specific monoclonal antibody. Collectively, these findings demonstrate that TIM-1 promotes HCV infection by serving as an attachment receptor for binding to PS exposed on the HCV envelope.

  8. Immunomodulatory Drugs: Immune Checkpoint Agents in Acute Leukemia

    PubMed Central

    Knaus, Hanna A.; Kanakry, Christopher G.; Luznik, Leo; Gojo, Ivana

    2016-01-01

    Intrinsic immune responses to acute leukemia are inhibited by a variety of mechanisms, such as aberrant antigen expression by leukemia cells, secretion of immunosuppressive cytokines and expression of inhibitory enzymes in the tumor microenvironment, expansion of immunoregulatory cells, and activation of immune checkpoint pathways, all leading to T cell dysfunction and/or exhaustion. Leukemic cells, similar to other tumor cells, hijack these inhibitory pathways to evade immune recognition and destruction by cytotoxic T lymphocytes. Thus, blockade of immune checkpoints has emerged as a highly promising approach to augment innate anti-tumor immunity in order to treat malignancies. Most evidence for the clinical efficacy of this immunotherapeutic strategy has been seen in patients with metastatic melanoma, where anti-CTLA-4 and anti-PD-1 antibodies have recently revolutionized treatment of this lethal disease with otherwise limited treatment options. To meet the high demand for new treatment strategies in acute leukemia, clinical testing of these promising therapies is commencing. Herein, we review the biology of multiple inhibitory checkpoints (including CTLA-4, PD-1, TIM-3, LAG-3, BTLA, and CD200R) and their contribution to immune evasion by acute leukemias. In addition, we discuss the current state of preclinical and clinical studies of immune checkpoint inhibition in acute leukemia, which seek to harness the body’s own immune system to fight leukemic cells. PMID:25981611

  9. Galectin-9 is Involved in Immunosuppression Mediated by Human Bone Marrow-derived Clonal Mesenchymal Stem Cells.

    PubMed

    Kim, Si-Na; Lee, Hyun-Joo; Jeon, Myung-Shin; Yi, TacGhee; Song, Sun U

    2015-10-01

    Bone marrow-derived mesenchymal stem cells (MSCs) have immunomodulatory properties and can suppress exaggerated pro-inflammatory immune responses. Although the exact mechanisms remain unclear, a variety of soluble factors are known to contribute to MSC-mediated immunosuppression. However, functional redundancy in the immunosuppressive properties of MSCs indicates that other uncharacterized factors could be involved. Galectin-9, a member of the β-galactoside binding galectin family, has emerged as an important regulator of innate and adaptive immunity. We examined whether galectin-9 contributes to MSC-mediated immunosuppression. Galectin-9 was strongly induced and secreted from human MSCs upon stimulation with pro-inflammatory cytokines. An in vitro immunosuppression assay using a knockdown approach revealed that galectin-9-deficient MSCs do not exert immunosuppressive activity. We also provided evidence that galectin-9 may contribute to MSC-mediated immunosuppression by binding to its receptor, TIM-3, expressed on activated lymphocytes, leading to apoptotic cell death of activated lymphocytes. Taken together, our findings demonstrate that galectin-9 is involved in MSC-mediated immunosuppression and represents a potential therapeutic factor for the treatment of inflammatory diseases.

  10. Clinical and pharmacodynamic analysis of pomalidomide dosing strategies in myeloma: impact of immune activation and cereblon targets

    PubMed Central

    Sehgal, Kartik; Das, Rituparna; Zhang, Lin; Verma, Rakesh; Deng, Yanhong; Kocoglu, Mehmet; Vasquez, Juan; Koduru, Srinivas; Ren, Yan; Wang, Maria; Couto, Suzana; Breider, Mike; Hansel, Donna; Seropian, Stuart; Cooper, Dennis; Thakurta, Anjan; Yao, Xiaopan; Dhodapkar, Kavita M.

    2015-01-01

    In preclinical studies, pomalidomide mediated both direct antitumor effects and immune activation by binding cereblon. However, the impact of drug-induced immune activation and cereblon/ikaros in antitumor effects of pomalidomide in vivo is unknown. Here we evaluated the clinical and pharmacodynamic effects of continuous or intermittent dosing strategies of pomalidomide/dexamethasone in lenalidomide-refractory myeloma in a randomized trial. Intermittent dosing led to greater tumor reduction at the cost of more frequent adverse events. Both cohorts experienced similar event-free and overall survival. Both regimens led to a distinct pattern but similar degree of mid-cycle immune activation, manifested as increased expression of cytokines and lytic genes in T and natural killer (NK) cells. Pomalidomide induced poly-functional T-cell activation, with increased proportion of coinhibitory receptor BTLA+ T cells and Tim-3+ NK cells. Baseline levels of ikaros and aiolos protein in tumor cells did not correlate with response or survival. Pomalidomide led to rapid decline in Ikaros in T and NK cells in vivo, and therapy-induced activation of CD8+ T cells correlated with clinical response. These data demonstrate that pomalidomide leads to strong and rapid immunomodulatory effects involving both innate and adaptive immunity, even in heavily pretreated multiple myeloma, which correlates with clinical antitumor effects. This trial was registered at www.clinicaltrials.gov as #NCT01319422. PMID:25869284

  11. Control of Immune Response to Allogeneic Embryonic Stem Cells by CD3 Antibody-Mediated Operational Tolerance Induction.

    PubMed

    Calderon, D; Prot, M; You, S; Marquet, C; Bellamy, V; Bruneval, P; Valette, F; de Almeida, P; Wu, J C; Pucéat, M; Menasché, P; Chatenoud, L

    2016-02-01

    Implantation of embryonic stem cells (ESCs) and their differentiated derivatives into allogeneic hosts triggers an immune response that represents a hurdle to clinical application. We established in autoimmunity and in transplantation that CD3 antibody therapy induces a state of immune tolerance. Promising results have been obtained with CD3 antibodies in the clinic. In this study, we tested whether this strategy can prolong the survival of undifferentiated ESCs and their differentiated derivatives in histoincompatible hosts. Recipients of either mouse ESC-derived embryoid bodies (EBs) or cardiac progenitors received a single short tolerogenic regimen of CD3 antibody. In immunocompetent mice, allogeneic EBs and cardiac progenitors were rejected within 20-25 days. Recipients treated with CD3 antibody showed long-term survival of implanted cardiac progenitors or EBs. In due course, EBs became teratomas, the growth of which was self-limited. Regulatory CD4(+)FoxP3(+) T cells and signaling through the PD1/PDL1 pathway played key roles in the CD3 antibody therapeutic effect. Gene profiling emphasized the importance of TGF-β and the inhibitory T cell coreceptor Tim3 to the observed effect. These results demonstrate that CD3 antibody administered alone promotes prolonged survival of allogeneic ESC derivatives and thus could prove useful for enhancing cell engraftment in the absence of chronic immunosuppression.

  12. Sequential transcriptional changes dictate safe and effective antigen-specific immunotherapy.

    PubMed

    Burton, Bronwen R; Britton, Graham J; Fang, Hai; Verhagen, Johan; Smithers, Ben; Sabatos-Peyton, Catherine A; Carney, Laura J; Gough, Julian; Strobel, Stephan; Wraith, David C

    2014-09-03

    Antigen-specific immunotherapy combats autoimmunity or allergy by reinstating immunological tolerance to target antigens without compromising immune function. Optimization of dosing strategy is critical for effective modulation of pathogenic CD4(+) T-cell activity. Here we report that dose escalation is imperative for safe, subcutaneous delivery of the high self-antigen doses required for effective tolerance induction and elicits anergic, interleukin (IL)-10-secreting regulatory CD4(+) T cells. Analysis of the CD4(+) T-cell transcriptome, at consecutive stages of escalating dose immunotherapy, reveals progressive suppression of transcripts positively regulating inflammatory effector function and repression of cell cycle pathways. We identify transcription factors, c-Maf and NFIL3, and negative co-stimulatory molecules, LAG-3, TIGIT, PD-1 and TIM-3, which characterize this regulatory CD4(+) T-cell population and whose expression correlates with the immunoregulatory cytokine IL-10. These results provide a rationale for dose escalation in T-cell-directed immunotherapy and reveal novel immunological and transcriptional signatures as surrogate markers of successful immunotherapy.

  13. Sequential transcriptional changes dictate safe and effective antigen-specific immunotherapy

    PubMed Central

    Burton, Bronwen R.; Britton, Graham J.; Fang, Hai; Verhagen, Johan; Smithers, Ben; Sabatos-Peyton, Catherine A.; Carney, Laura J.; Gough, Julian; Strobel, Stephan; Wraith, David C.

    2014-01-01

    Antigen-specific immunotherapy combats autoimmunity or allergy by reinstating immunological tolerance to target antigens without compromising immune function. Optimization of dosing strategy is critical for effective modulation of pathogenic CD4+ T-cell activity. Here we report that dose escalation is imperative for safe, subcutaneous delivery of the high self-antigen doses required for effective tolerance induction and elicits anergic, interleukin (IL)-10-secreting regulatory CD4+ T cells. Analysis of the CD4+ T-cell transcriptome, at consecutive stages of escalating dose immunotherapy, reveals progressive suppression of transcripts positively regulating inflammatory effector function and repression of cell cycle pathways. We identify transcription factors, c-Maf and NFIL3, and negative co-stimulatory molecules, LAG-3, TIGIT, PD-1 and TIM-3, which characterize this regulatory CD4+ T-cell population and whose expression correlates with the immunoregulatory cytokine IL-10. These results provide a rationale for dose escalation in T-cell-directed immunotherapy and reveal novel immunological and transcriptional signatures as surrogate markers of successful immunotherapy. PMID:25182274

  14. IL-21 signaling is essential for optimal host resistance against Mycobacterium tuberculosis infection

    PubMed Central

    Booty, Matthew G.; Barreira-Silva, Palmira; Carpenter, Stephen M.; Nunes-Alves, Cláudio; Jacques, Miye K.; Stowell, Britni L.; Jayaraman, Pushpa; Beamer, Gillian; Behar, Samuel M.

    2016-01-01

    IL-21 is produced predominantly by activated CD4+ T cells and has pleiotropic effects on immunity via the IL-21 receptor (IL-21R), a member of the common gamma chain (γc) cytokine receptor family. We show that IL-21 signaling plays a crucial role in T cell responses during Mycobacterium tuberculosis infection by augmenting CD8+ T cell priming, promoting T cell accumulation in the lungs, and enhancing T cell cytokine production. In the absence of IL-21 signaling, more CD4+ and CD8+ T cells in chronically infected mice express the T cell inhibitory molecules PD-1 and TIM-3. We correlate these immune alterations with increased susceptibility of IL-21R−/− mice, which have increased lung bacterial burden and earlier mortality compared to WT mice. Finally, to causally link the immune defects with host susceptibility, we use an adoptive transfer model to show that IL-21R−/− T cells transfer less protection than WT T cells. These results prove that IL-21 signaling has an intrinsic role in promoting the protective capacity of T cells. Thus, the net effect of IL-21 signaling is to enhance host resistance to M. tuberculosis. These data position IL-21 as a candidate biomarker of resistance to tuberculosis. PMID:27819295

  15. The Role of Coinhibitory Signaling Pathways in Transplantation and Tolerance

    PubMed Central

    McGrath, Martina M.; Najafian, Nader

    2012-01-01

    Negative costimulatory molecules, acting through so-called inhibitory pathways, play a crucial role in the control of T cell responses. This negative “second signal” opposes T cell receptor activation and leads to downregulation of T cell proliferation and promotes antigen specific tolerance. Much interest has focused upon these pathways in recent years as a method to control detrimental alloresponses and promote allograft tolerance. However, recent experimental data highlights the complexity of negative costimulatory pathways in alloimmunity. Varying effects are observed from molecules expressed on donor and recipient tissues and also depending upon the activation status of immune cells involved. There appears to be significant overlap and redundancy within these systems, rendering this a challenging area to understand and exploit therapeutically. In this article, we will review the literature at the current time regarding the major negative costimulation pathways including CTLA-4:B7, PD-1:PD-L1/PD-L2 and PD-L1:B7-1, B7-H3, B7-H4, HVEM:BTLA/CD160, and TIM-3:Galectin-9. We aim to outline the role of these pathways in alloimmunity and discuss their potential applications for tolerance induction in transplantation. PMID:22566929

  16. Specific CD8+ T cell response immunotherapy for hepatocellular carcinoma and viral hepatitis

    PubMed Central

    Moreno-Cubero, Elia; Larrubia, Juan-Ramón

    2016-01-01

    Hepatocellular carcinoma (HCC), chronic hepatitis B (CHB) and chronic hepatitis C (CHC) are characterized by exhaustion of the specific CD8+ T cell response. This process involves enhancement of negative co-stimulatory molecules, such as programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte antigen-4 (CTLA-4), 2B4, Tim-3, CD160 and LAG-3, which is linked to intrahepatic overexpression of some of the cognate ligands, such as PD-L1, on antigen presenting cells and thereby favouring a tolerogenic environment. Therapies that disrupt these negative signalling mechanisms represent promising therapeutic tools with the potential to restore reactivity of the specific CD8+ T cell response. In this review we discuss the impressive in vitro and in vivo results that have been recently achieved in HCC, CHB and CHC by blocking these negative receptors with monoclonal antibodies against these immune checkpoint modulators. The article mainly focuses on the role of CTLA-4 and PD-1 blocking monoclonal antibodies, the first ones to have reached clinical practice. The humanized monoclonal antibodies against CTLA-4 (tremelimumab and ipilimumab) and PD-1 (nivolumab and pembrolizumab) have yielded good results in testing of HCC and chronic viral hepatitis patients. Trelimumab, in particular, has shown a significant increase in the time to progression in HCC, while nivolumab has shown a remarkable effect on hepatitis C viral load reduction. The research on the role of ipilimumab, nivolumab and pembrolizumab on HCC is currently underway. PMID:27605882

  17. CEACAM1 mediates B cell aggregation in central nervous system autoimmunity

    PubMed Central

    Rovituso, Damiano M.; Scheffler, Laura; Wunsch, Marie; Dörck, Sebastian; Ulzheimer, Jochen; Bayas, Antonios; Steinman, Lawrence; Ergün, Süleyman; Kuerten, Stefanie

    2016-01-01

    B cell aggregates in the central nervous system (CNS) have been associated with rapid disease progression in patients with multiple sclerosis (MS). Here we demonstrate a key role of carcinoembryogenic antigen-related cell adhesion molecule1 (CEACAM1) in B cell aggregate formation in MS patients and a B cell-dependent mouse model of MS. CEACAM1 expression was increased on peripheral blood B cells and CEACAM1+ B cells were present in brain infiltrates of MS patients. Administration of the anti-CEACAM1 antibody T84.1 was efficient in blocking aggregation of B cells derived from MS patients. Along these lines, application of the monoclonal anti-CEACAM1 antibody mCC1 was able to inhibit CNS B cell aggregate formation and significantly attenuated established MS-like disease in mice in the absence of any adverse effects. CEACAM1 was co-expressed with the regulator molecule T cell immunoglobulin and mucin domain −3 (TIM-3) on B cells, a novel molecule that has recently been described to induce anergy in T cells. Interestingly, elevated coexpression on B cells coincided with an autoreactive T helper cell phenotype in MS patients. Overall, these data identify CEACAM1 as a clinically highly interesting target in MS pathogenesis and open new therapeutic avenues for the treatment of the disease. PMID:27435215

  18. The use of PCR to detect Neospora caninum DNA in the blood of naturally infected cows.

    PubMed

    Okeoma, C M; Williamson, N B; Pomroy, W E; Stowell, K M; Gillespie, L

    2004-08-06

    Twelve 2-year old heifers in their fifth month of gestation when pregnancy tested were used in this study. Six heifers aborted at approximately 4 months of gestation and had blood samples drawn less than 6 weeks after the abortions were identified. Blood samples were also drawn from three sero-positive pregnant and three sero-negative pregnant heifers. DNA was isolated from the samples and a 350 bp fragment of the Nc-5 gene was PCR amplified using primer pair Np21+ and Np6+. Also, the Internal Transcribed Spacer 1 (ITS1) was PCR amplified using Tim 3 and Tim 11 primer pair. The Nc-5 gene fragment was cloned, sequenced and the sequence BLAST-tested. Similarly, the ITS1 product was sequenced and BLAST-tested. The BLAST test results revealed that Neospora caninum DNA was present in these blood samples indicating that polymerase chain reaction can be used in the detection of N. caninum DNA in the blood of sero-positive cows.

  19. Interleukin-35 limits anti-tumor immunity

    PubMed Central

    Turnis, Meghan E.; Sawant, Deepali V.; Szymczak-Workman, Andrea L.; Andrews, Lawrence P.; Delgoffe, Greg M.; Yano, Hiroshi; Beres, Amy J.; Vogel, Peter; Workman, Creg J.; Vignali, Dario A. A.

    2016-01-01

    Summary Regulatory T (Treg) cells pose a major barrier to effective anti-tumor immunity. Although Treg cell depletion enhances tumor rejection, the ensuing autoimmune sequelae limits its utility in the clinic and highlights the need for limiting Treg cell activity within the tumor microenvironment. Interleukin-35 (IL-35) is a Treg cell-secreted cytokine that inhibits T cell proliferation and function. Using an IL-35 reporter mouse, we observed substantial enrichment of IL-35+ Treg cells in tumors. Neutralization with an IL-35-specific antibody or Treg cell-restricted deletion of IL-35 production limited tumor growth in multiple murine models of human cancer. Limiting intratumoral IL-35 enhanced T cell proliferation, effector function, antigen-specific responses, and long-term T cell memory. Treg cell-derived IL-35 promoted the expression of multiple inhibitory receptors (PD1, TIM3, LAG3), thereby facilitating intratumoral T cell exhaustion. These findings reveal previously unappreciated roles for IL-35 in limiting anti-tumor immunity and contributing to T cell dysfunction in the tumor microenvironment. PMID:26872697

  20. Chronic hepatitis C virus infection triggers spontaneous differential expression of biosignatures associated with T cell exhaustion and apoptosis signaling in peripheral blood mononucleocytes.

    PubMed

    Barathan, Muttiah; Gopal, Kaliappan; Mohamed, Rosmawati; Ellegård, Rada; Saeidi, Alireza; Vadivelu, Jamuna; Ansari, Abdul W; Rothan, Hussin A; Ravishankar Ram, M; Zandi, Keivan; Chang, Li Y; Vignesh, Ramachandran; Che, Karlhans F; Kamarulzaman, Adeeba; Velu, Vijayakumar; Larsson, Marie; Kamarul, Tunku; Shankar, Esaki M

    2015-04-01

    Persistent hepatitis C virus (HCV) infection appears to trigger the onset of immune exhaustion to potentially assist viral persistence in the host, eventually leading to hepatocellular carcinoma. The role of HCV on the spontaneous expression of markers suggestive of immune exhaustion and spontaneous apoptosis in immune cells of chronic HCV (CHC) disease largely remain elusive. We investigated the peripheral blood mononuclear cells of CHC patients to determine the spontaneous recruitment of cellular reactive oxygen species (cROS), immunoregulatory and exhaustion markers relative to healthy controls. Using a commercial QuantiGenePlex(®) 2.0 assay, we determined the spontaneous expression profile of 80 different pro- and anti-apoptotic genes in persistent HCV disease. Onset of spontaneous apoptosis significantly correlated with the up-regulation of cROS, indoleamine 2,3-dioxygenase (IDO), cyclooxygenase-2/prostaglandin H synthase (COX-2/PGHS), Foxp3, Dtx1, Blimp1, Lag3 and Cd160. Besides, spontaneous differential surface protein expression suggestive of T cell inhibition viz., TRAIL, TIM-3, PD-1 and BTLA on CD4+ and CD8+ T cells, and CTLA-4 on CD4+ T cells was also evident. Increased up-regulation of Tnf, Tp73, Casp14, Tnfrsf11b, Bik and Birc8 was observed, whereas FasLG, Fas, Ripk2, Casp3, Dapk1, Tnfrsf21, and Cflar were moderately up-regulated in HCV-infected subjects. Our observation suggests the spontaneous onset of apoptosis signaling and T cell exhaustion in chronic HCV disease.

  1. Interleukin-35 Limits Anti-Tumor Immunity.

    PubMed

    Turnis, Meghan E; Sawant, Deepali V; Szymczak-Workman, Andrea L; Andrews, Lawrence P; Delgoffe, Greg M; Yano, Hiroshi; Beres, Amy J; Vogel, Peter; Workman, Creg J; Vignali, Dario A A

    2016-02-16

    Regulatory T (Treg) cells pose a major barrier to effective anti-tumor immunity. Although Treg cell depletion enhances tumor rejection, the ensuing autoimmune sequelae limits its utility in the clinic and highlights the need for limiting Treg cell activity within the tumor microenvironment. Interleukin-35 (IL-35) is a Treg cell-secreted cytokine that inhibits T cell proliferation and function. Using an IL-35 reporter mouse, we observed substantial enrichment of IL-35(+) Treg cells in tumors. Neutralization with an IL-35-specific antibody or Treg cell-restricted deletion of IL-35 production limited tumor growth in multiple murine models of human cancer. Limiting intratumoral IL-35 enhanced T cell proliferation, effector function, antigen-specific responses, and long-term T cell memory. Treg cell-derived IL-35 promoted the expression of multiple inhibitory receptors (PD1, TIM3, LAG3), thereby facilitating intratumoral T cell exhaustion. These findings reveal previously unappreciated roles for IL-35 in limiting anti-tumor immunity and contributing to T cell dysfunction in the tumor microenvironment.

  2. Immunomodulatory Drugs: Immune Checkpoint Agents in Acute Leukemia.

    PubMed

    Knaus, Hanna A; Kanakry, Christopher G; Luznik, Leo; Gojo, Ivana

    2017-01-01

    Intrinsic immune responses to acute leukemia are inhibited by a variety of mechanisms, such as aberrant antigen expression by leukemia cells, secretion of immunosuppressive cytokines and expression of inhibitory enzymes in the tumor microenvironment, expansion of immunoregulatory cells, and activation of immune checkpoint pathways, all leading to T cell dysfunction and/or exhaustion. Leukemic cells, similar to other tumor cells, hijack these inhibitory pathways to evade immune recognition and destruction by cytotoxic T lymphocytes. Thus, blockade of immune checkpoints has emerged as a highly promising approach to augment innate anti-tumor immunity in order to treat malignancies. Most evidence for the clinical efficacy of this immunotherapeutic strategy has been seen in patients with metastatic melanoma, where anti-CTLA-4 and anti-PD-1 antibodies have recently revolutionized treatment of this lethal disease with otherwise limited treatment options. To meet the high demand for new treatment strategies in acute leukemia, clinical testing of these promising therapies is commencing. Herein, we review the biology of multiple inhibitory checkpoints (including CTLA-4, PD-1, TIM-3, LAG-3, BTLA, and CD200R) and their contribution to immune evasion by acute leukemias. In addition, we discuss the current state of preclinical and clinical studies of immune checkpoint inhibition in acute leukemia, which seek to harness the body's own immune system to fight leukemic cells.

  3. Galectins expressed differently in genetically susceptible C57BL/6 and resistant BALB/c mice during acute ocular Toxoplasma gondii infection.

    PubMed

    Chen, S-J; Zhang, Y-X; Huang, S-G; Lu, F-L

    2017-03-09

    Ocular toxoplasmosis (OT) caused by Toxoplasma gondii is a major cause of infectious uveitis, however little is known about its immunopathological mechanism. Susceptible C57BL/6 (B6) and resistant BALB/c mice were intravitreally infected with 500 tachyzoites of the RH strain of T. gondii. B6 mice showed more severe ocular pathology and higher parasite loads in the eyes. The levels of galectin (Gal)-9 and its receptors (Tim-3 and CD137), interferon (IFN)-γ, IL-6 and IL-10 were significantly higher in the eyes of B6 mice than those of BALB/c mice; however, the levels of IFN-α and -β were significantly decreased in the eyes and CLNs of B6 mice but significantly increased in BALB/c mice after infection. After blockage of galectin-receptor interactions by α-lactose, neither ocular immunopathology nor parasite loads were different from those of infected BALB/c mice without α-lactose treatment. Although the expressions of Gal-9/receptor were significantly increased in B6 mice and Gal-1 and -3 were upregulated in both strains of mice upon ocular T. gondii infection, blockage of galectins did not change the ocular pathogenesis of genetic resistant BALB/c mice. However, IFN-α and -β were differently expressed in B6 and BALB/c mice, suggesting that type I IFNs may play a protective role in experimental OT.

  4. Immune-mediated antitumor effect by type 2 diabetes drug, metformin

    PubMed Central

    Eikawa, Shingo; Nishida, Mikako; Mizukami, Shusaku; Yamazaki, Chihiro; Nakayama, Eiichi; Udono, Heiichiro

    2015-01-01

    Metformin, a prescribed drug for type 2 diabetes, has been reported to have anti-cancer effects; however, the underlying mechanism is poorly understood. Here we show that this mechanism may be immune-mediated. Metformin enabled normal but not T-cell–deficient SCID mice to reject solid tumors. In addition, it increased the number of CD8+ tumor-infiltrating lymphocytes (TILs) and protected them from apoptosis and exhaustion characterized by decreased production of IL-2, TNFα, and IFNγ. CD8+ TILs capable of producing multiple cytokines were mainly PD-1−Tim-3+, an effector memory subset responsible for tumor rejection. Combined use of metformin and cancer vaccine improved CD8+ TIL multifunctionality. The adoptive transfer of antigen-specific CD8+ T cells treated with metformin concentrations as low as 10 μM showed efficient migration into tumors while maintaining multifunctionality in a manner sensitive to the AMP-activated protein kinase (AMPK) inhibitor compound C. Therefore, a direct effect of metformin on CD8+ T cells is critical for protection against the inevitable functional exhaustion in the tumor microenvironment. PMID:25624476

  5. Progression of Lung Cancer Is Associated with Increased Dysfunction of T Cells Defined by Coexpression of Multiple Inhibitory Receptors.

    PubMed

    Thommen, Daniela S; Schreiner, Jens; Müller, Philipp; Herzig, Petra; Roller, Andreas; Belousov, Anton; Umana, Pablo; Pisa, Pavel; Klein, Christian; Bacac, Marina; Fischer, Ozana S; Moersig, Wolfgang; Savic Prince, Spasenija; Levitsky, Victor; Karanikas, Vaios; Lardinois, Didier; Zippelius, Alfred

    2015-12-01

    Dysfunctional T cells present in malignant lesions are characterized by a sustained and highly diverse expression of inhibitory receptors, also referred to as immune checkpoints. Yet, their relative functional significance in different cancer types remains incompletely understood. In this study, we provide a comprehensive characterization of the diversity and expression patterns of inhibitory receptors on tumor-infiltrating T cells from patients with non-small cell lung cancer. In spite of the large heterogeneity observed in the amount of PD-1, Tim-3, CTLA-4, LAG-3, and BTLA expressed on intratumoral CD8(+) T cells from 32 patients, a clear correlation was established between increased expression of these inhibitory coreceptors and progression of the disease. Notably, the latter was accompanied by a progressively impaired capacity of T cells to respond to polyclonal activation. Coexpression of several inhibitory receptors was gradually acquired, with early PD-1 and late LAG-3/BTLA expression. PD-1 blockade was able to restore T-cell function only in a subset of patients. A high percentage of PD-1(hi) T cells was correlated with poor restoration of T-cell function upon PD-1 blockade. Of note, PD-1(hi) expression marked a particularly dysfunctional T-cell subset characterized by coexpression of multiple inhibitory receptors and thus may assist in identifying patients likely to respond to inhibitory receptor-specific antibodies. Overall, these data may provide a framework for future personalized T-cell-based therapies aiming at restoration of tumor-infiltrating lymphocyte effector functions.

  6. IL2Rβ-dependent signals drive terminal exhaustion and suppress memory development during chronic viral infection.

    PubMed

    Beltra, Jean-Christophe; Bourbonnais, Sara; Bédard, Nathalie; Charpentier, Tania; Boulangé, Moana; Michaud, Eva; Boufaied, Ines; Bruneau, Julie; Shoukry, Naglaa H; Lamarre, Alain; Decaluwe, Hélène

    2016-09-13

    Exhaustion of CD8(+) T cells severely impedes the adaptive immune response to chronic viral infections. Despite major advances in our understanding of the molecular regulation of exhaustion, the cytokines that directly control this process during chronicity remain unknown. We demonstrate a direct impact of IL-2 and IL-15, two common gamma-chain-dependent cytokines, on CD8(+) T-cell exhaustion. Common to both cytokine receptors, the IL-2 receptor β (IL2Rβ) chain is selectively maintained on CD8(+) T cells during chronic lymphocytic choriomeningitis virus and hepatitis C virus infections. Its expression correlates with exhaustion severity and identifies terminally exhausted CD8(+) T cells both in mice and humans. Genetic ablation of the IL2Rβ chain on CD8(+) T cells restrains inhibitory receptor induction, in particular 2B4 and Tim-3; precludes terminal differentiation of highly defective PD-1(hi) effectors; and rescues memory T-cell development and responsiveness to IL-7-dependent signals. Together, we ascribe a previously unexpected role to IL-2 and IL-15 as instigators of CD8(+) T-cell exhaustion during chronic viral infection.

  7. Chronic Activation of Innate Immunity Correlates With Poor Prognosis in Cancer Patients Treated With Oncolytic Adenovirus.

    PubMed

    Taipale, Kristian; Liikanen, Ilkka; Juhila, Juuso; Turkki, Riku; Tähtinen, Siri; Kankainen, Matti; Vassilev, Lotta; Ristimäki, Ari; Koski, Anniina; Kanerva, Anna; Diaconu, Iulia; Cerullo, Vincenzo; Vähä-Koskela, Markus; Oksanen, Minna; Linder, Nina; Joensuu, Timo; Lundin, Johan; Hemminki, Akseli

    2016-02-01

    Despite many clinical trials conducted with oncolytic viruses, the exact tumor-level mechanisms affecting therapeutic efficacy have not been established. Currently there are no biomarkers available that would predict the clinical outcome to any oncolytic virus. To assess the baseline immunological phenotype and find potential prognostic biomarkers, we monitored mRNA expression levels in 31 tumor biopsy or fluid samples from 27 patients treated with oncolytic adenovirus. Additionally, protein expression was studied from 19 biopsies using immunohistochemical staining. We found highly significant changes in several signaling pathways and genes associated with immune responses, such as B-cell receptor signaling (P < 0.001), granulocyte macrophage colony-stimulating factor (GM-CSF) signaling (P < 0.001), and leukocyte extravasation signaling (P < 0.001), in patients surviving a shorter time than their controls. In immunohistochemical analysis, markers CD4 and CD163 were significantly elevated (P = 0.020 and P = 0.016 respectively), in patients with shorter than expected survival. Interestingly, T-cell exhaustion marker TIM-3 was also found to be significantly upregulated (P = 0.006) in patients with poor prognosis. Collectively, these data suggest that activation of several functions of the innate immunity before treatment is associated with inferior survival in patients treated with oncolytic adenovirus. Conversely, lack of chronic innate inflammation at baseline may predict improved treatment outcome, as suggested by good overall prognosis.

  8. An adaptive immune response driven by mature, antigen-experienced T and B cells within the microenvironment of oral squamous cell carcinoma.

    PubMed

    Quan, Hongzhi; Fang, Liangjuan; Pan, Hao; Deng, Zhiyuan; Gao, Shan; Liu, Ousheng; Wang, Yuehong; Hu, Yanjia; Fang, Xiaodan; Yao, Zhigang; Guo, Feng; Lu, Ruohuang; Xia, Kun; Tang, Zhangui

    2016-06-15

    Lymphocyte infiltrates have been observed in the microenvironment of oral cancer; however, little is known about whether the immune response of the lymphocyte infiltrate affects tumor biology. For a deeper understanding of the role of the infiltrating-lymphocytes in oral squamous cell carcinoma (OSCC), we characterized the lymphocyte infiltrate repertoires and defined their features. Immunohistochemistry revealed considerable T and B cell infiltrates and lymphoid follicles with germinal center-like structures within the tumor microenvironment. Flow cytometry demonstrated that populations of antigen-experienced CD4+ and CD8+ cells were present, as well as an enrichment of regulatory T cells; and T cells expressing programmed death-1 (PD-1) and T cell Ig and mucin protein-3 (Tim-3), indicative of exhaustion, within the tumor microenvironment. Characterization of tumor-infiltrating B cells revealed clear evidence of antigen exposure, in that the cardinal features of an antigen-driven B cell response were present, including somatic mutation, clonal expansion, intraclonal variation and isotype switching. Collectively, our results point to an adaptive immune response occurring within the OSCC microenvironment, which may be sustained by the expression of specific antigens in the tumor.

  9. Implication of combined PD-L1/PD-1 blockade with cytokine-induced killer cells as a synergistic immunotherapy for gastrointestinal cancer

    PubMed Central

    Geng, Ruixuan; Ge, Xiaoxiao; Tang, Wenbo; Chang, Jinjia; Wu, Zheng; Liu, Xinyang; Lin, Ying; Zhang, Zhe; Li, Jin

    2016-01-01

    Cytokine-induced killer (CIK) cells represent a realistic approach in cancer immunotherapy with confirmed survival benefits in the context of metastatic solid tumors. However, therapeutic effects are limited to a fraction of patients. In this study, immune-resistance elements and ideal combination therapies were explored. Initially, phenotypic analysis was performed to document CD3, CD56, NKG2D, DNAM-1, PD-L1, PD-1, CTLA-4, TIM-3, 2B4, and LAG-3 on CIK cells. Upon engagement of CIK cells with the tumor cells, expression of PD-1 on CIK cells and PD-L1 on both cells were up-regulated. Over-expression of PD-L1 levels on tumor cells via lentiviral transduction inhibited tumoricidal activity of CIK cells, and neutralizing of PD-L1/PD-1 signaling axis could enhance their tumor-killing effect. Conversely, blockade of NKG2D, a major activating receptor of CIK cells, largely caused dysfunction of CIK cells. Functional study showed an increase of NKG2D levels along with PD-L1/PD-1 blockade in the presence of other immune effector molecule secretion. Additionally, combined therapy of CIK infusion and PD-L1/PD-1 blockade caused a delay of in vivo tumor growth and exhibited a survival advantage over untreated mice. These results provide a preclinical proof-of-concept for simultaneous PD-L1/PD-1 pathways blockade along with CIK infusion as a novel immunotherapy for unresectable cancers. PMID:26871284

  10. HTLV-1 Infection and Neuropathogenesis in the Context of Rag1(-/-)γc(-/-) (RAG1-Hu) and BLT Mice.

    PubMed

    Ginwala, Rashida; Caruso, Breanna; Khan, Zafar K; Pattekar, Ajinkya; Chew, Glen M; Corley, Michael J; Loonawat, Ronak; Jacobson, Steven; Sreedhar, Sreesha; Ndhlovu, Lishomwa C; Jain, Pooja

    2017-04-04

    To date, the lack of a suitable small animal model has hindered our understanding of Human T-cell lymphotropic virus (HTLV)-1 chronic infection and associated neuropathogenesis defined as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The host immune response plays a critical role in the outcome of HTLV-1 infection, which could be better tested in the context of humanized (hu) mice. Thus, we employ here the Balb/c-Rag1(-/-)γc(-/-) or Rag1 as well as Bone marrow-Liver-Thymic (BLT) mouse models for engraftment of human CD34(+) hematopoietic stem cells. Flow cytometry and histological analyses confirmed reconstitution of Rag1 and BLT mice with human immune cells. Following HTLV-1 infection, proviral load (PVL) was detected in the blood of Rag-1 and BLT hu-mice as early as 2 weeks post-infection (wpi) with sustained elevation in the subsequent weeks followed by Tax expression. Additionally, infection was compared between adult and neonatal Rag1 mice with both PVL and Tax expression considerably higher in the adult Rag1 mice as compared to the neonates. Establishment of peripheral infection led to lymphocytic infiltration with concomitant Tax expression and resulting myelin disruption within the central nervous system of infected mice. In addition, up-regulation in the expression of several immune checkpoint mediators such as programmed cell death-1 (PD-1), T-cell Ig and ITIM domain (TIGIT), and T cell Ig and mucin domain-3 protein (Tim-3) were observed on CD8(+) T cells in various organs including the CNS of infected hu-mice. Collectively, these studies represent the first attempt to establish HTLV-1 neuropathogenesis in the context of Rag-1 and BLT hu-mice as potential novel tools for understanding HTLV-1 neuropathogenesis and testing of novel therapies such as immune checkpoint blockade in the amelioration of chronic HTLV-1 infection.

  11. Impaired NK Cell Activation and Chemotaxis toward Dendritic Cells Exposed to Complement-Opsonized HIV-1.

    PubMed

    Ellegård, Rada; Crisci, Elisa; Andersson, Jonas; Shankar, Esaki M; Nyström, Sofia; Hinkula, Jorma; Larsson, Marie

    2015-08-15

    Mucosa resident dendritic cells (DCs) may represent one of the first immune cells that HIV-1 encounters during sexual transmission. The virions in body fluids can be opsonized with complement factors because of HIV-mediated triggering of the complement cascade, and this appears to influence numerous aspects of the immune defense targeting the virus. One key attribute of host defense is the ability to attract immune cells to the site of infection. In this study, we investigated whether the opsonization of HIV with complement (C-HIV) or a mixture of complement and Abs (CI-HIV) affected the cytokine and chemokine responses generated by DCs, as well as their ability to attract other immune cells. We found that the expression levels of CXCL8, CXCL10, CCL3, and CCL17 were lowered after exposure to either C-HIV or CI-HIV relative to free HIV (F-HIV). DCs exposed to F-HIV induced higher cell migration, consisting mainly of NK cells, compared with opsonized virus, and the chemotaxis of NK cells was dependent on CCL3 and CXCL10. NK cell exposure to supernatants derived from HIV-exposed DCs showed that F-HIV induced phenotypic activation (e.g., increased levels of TIM3, CD69, and CD25) and effector function (e.g., production of IFNγ and killing of target cells) in NK cells, whereas C-HIV and CI-HIV did not. The impairment of NK cell recruitment by DCs exposed to complement-opsonized HIV and the lack of NK activation may contribute to the failure of innate immune responses to control HIV at the site of initial mucosa infection.

  12. The PD-L1/PD-1 pathway promotes dysfunction, but not "exhaustion", in tumor-responding T cells from pleural effusions in lung cancer patients.

    PubMed

    Prado-Garcia, Heriberto; Romero-Garcia, Susana; Puerto-Aquino, Alejandra; Rumbo-Nava, Uriel

    2017-03-13

    Malignant pleural effusions are frequent in patients with advanced stages of lung cancer and are commonly infiltrated by lymphocytes and tumor cells. CD8+ T cells from these effusions have reduced effector functions. The programmed death receptor 1(PD-1)/programmed death ligand 1 (PD-L1) pathway is involved in T-cell exhaustion, and it might be responsible for T-cell dysfunction in lung cancer patients. Here, we show that PD-L1 is expressed on tumor cell samples from malignant effusions, on lung cancer cell lines, and, interestingly, on MRC-5 lung fibroblasts. PD-L1 was up-regulated in lung cancer cell lines upon treatment with IFN-gamma, but not under hypoxic conditions, as detected by RT-qPCR and flow cytometry. Blockade of PD-L1 on tumor cells restored granzyme-B expression in allogenic CD8+ T cells in vitro. Remarkably, pleural effusion CD8+ T cells that responded to the tumor antigens MAGE-3A and WT-1 (identified as CD137+ cells) were lower in frequency than CMV pp65-responding CD8+ T cells and did not have an exhausted phenotype (PD-1+ TIM-3+). Nonetheless, tumor-responding CD8+ T cells had a memory phenotype and expressed higher levels of PD-1. A PD-L1 blocking antibody increased the expression of granzyme-B and perforin on polyclonal- and tumor-stimulated CD8+ T cells. Taken together, our data show that rather than being exhausted, tumor-responding CD8+ T cells are not completely differentiated into effector cells and are prone to negative regulation by PD-L1. Hence, our study provides evidence that lung cancer patients respond to immunotherapy due to blockade of the PD-L1/PD-1 pathway.

  13. Protein kinase C theta is required for efficient induction of IL-10-secreting T cells

    PubMed Central

    Burton, Bronwen R.

    2017-01-01

    Secretion of interleukin-10 (IL-10) by CD4+ T cells is an essential immunoregulatory mechanism. The work presented here assesses the role of the signaling molecule protein kinase C theta (PKCθ) in the induction of IL-10 expression in CD4+ T cells. Using wildtype and PKCθ-deficient Tg4 T cell receptor transgenic mice, we implemented a well-described protocol of repeated doses of myelin basic protein (MBP)Ac1-9[4Y] antigen to induce Tr1-like IL-10+ T cells. We find that PKCθ is required for the efficient induction of IL-10 following antigen administration. Both serum concentrations of IL-10 and the proportion of IL-10+ T cells were reduced in PKCθ-deficient mice relative to wildtype mice following [4Y] treatment. We further characterized the T cells of [4Y] treated PKCθ-deficient Tg4 mice and found reduced expression of the transcription factors cMaf, Nfil3 and FoxP3 and the surface receptors PD-1 and Tim3, all of which have been associated with the differentiation or function of IL-10+ T cells. Finally, we demonstrated that, unlike [4Y] treated wildtype Tg4 T cells, cells from PKCθ-deficient mice were unable to suppress the priming of naïve T cells in vitro and in vivo. In summary, we present data demonstrating a role for PKCθ in the induction of suppressive, IL-10-secreting T cells induced in TCR-transgenic mice following chronic antigen administration. This should be considered when contemplating PKCθ as a suitable drug target for inducing immune suppression and graft tolerance. PMID:28158245

  14. Graves’ Disease Is Associated with a Defective Expression of the Immune Regulatory Molecule Galectin-9 in Antigen-Presenting Dendritic Cells

    PubMed Central

    de la Fuente, Hortensia; Rodríguez-Muñoz, Ana; Ramos-Levi, Ana; Sampedro-Nuñez, Miguel; Sánchez-Madrid, Francisco; González-Amaro, Roberto; Marazuela, Mónica

    2015-01-01

    Introduction Patients with autoimmune thyroid disease (AITD) show defects in their immune-regulatory mechanisms. Herein we assessed the expression and function of galectin-1 and galectin-9 (Gal-1, Gal-9) in dendritic cells (DCs) from patients with AITD. Materials and Methods Peripheral blood samples from 25 patients with Graves’ disease (GD), 11 Hashimoto’s thyroiditis (HT), and 24 healthy subjects were studied. Thyroid tissue samples from 44 patients with AITD and 22 patients with goiter were also analyzed. Expression and function of Gal-1 and Gal-9 was assessed by quantitative RT-PCR, immunofluorescence and flow cytometry. Results A diminished expression of Gal-9, but not of Gal-1, by peripheral blood DCs was observed in GD patients, mainly in those with Graves´ ophthalmopathy, and a significant negative association between disease severity and Gal-9 expression was detected. In addition, the mRNA levels of Gal-9 and its ligand TIM-3 were increased in thyroid tissue from AITD patients and its expression was associated with the levels of Th1/Th12/Th17 cytokines. Immunofluorescence studies proved that intrathyroidal Gal-9 expression was confined to DCs and macrophages. Finally, in vitro functional assays showed that exogenous Gal-9 had a suppressive effect on the release of Th1/Th2/Th17 cytokines by DC/lymphocyte autologous co-cultures from both AITD patients and healthy controls. Conclusions The altered pattern of expression of Gal-9 in peripheral blood DCs from GD patients, its correlation with disease severity as well as its ability to suppress cytokine release suggest that Gal-9 could be involved in the pathogenesis of AITD. PMID:25880730

  15. TIGIT and PD-1 impair tumor antigen–specific CD8+ T cells in melanoma patients

    PubMed Central

    Chauvin, Joe-Marc; Pagliano, Ornella; Fourcade, Julien; Sun, Zhaojun; Wang, Hong; Sander, Cindy; Kirkwood, John M.; Chen, Tseng-hui Timothy; Maurer, Mark; Korman, Alan J.; Zarour, Hassane M.

    2015-01-01

    T cell Ig and ITIM domain (TIGIT) is an inhibitory receptor expressed by activated T cells, Tregs, and NK cells. Here, we determined that TIGIT is upregulated on tumor antigen–specific (TA-specific) CD8+ T cells and CD8+ tumor-infiltrating lymphocytes (TILs) from patients with melanoma, and these TIGIT-expressing CD8+ T cells often coexpress the inhibitory receptor PD-1. Moreover, CD8+ TILs from patients exhibited downregulation of the costimulatory molecule CD226, which competes with TIGIT for the same ligand, supporting a TIGIT/CD226 imbalance in metastatic melanoma. TIGIT marked early T cell activation and was further upregulated by T cells upon PD-1 blockade and in dysfunctional PD-1+TIM-3+ TA-specific CD8+ T cells. PD-1+TIGIT+, PD-1–TIGIT+, and PD-1+TIGIT– CD8+ TILs had similar functional capacities ex vivo, suggesting that TIGIT alone, or together with PD-1, is not indicative of T cell dysfunction. However, in the presence of TIGIT ligand–expressing cells, TIGIT and PD-1 blockade additively increased proliferation, cytokine production, and degranulation of both TA-specific CD8+ T cells and CD8+ TILs. Collectively, our results show that TIGIT and PD-1 regulate the expansion and function of TA-specific CD8+ T cells and CD8+ TILs in melanoma patients and suggest that dual TIGIT and PD-1 blockade should be further explored to elicit potent antitumor CD8+ T cell responses in patients with advanced melanoma. PMID:25866972

  16. CD4+ primary T cells expressing HCV-core protein upregulate Foxp3 and IL-10, suppressing CD4 and CD8 T cells.

    PubMed

    Fernandez-Ponce, Cecilia; Dominguez-Villar, Margarita; Aguado, Enrique; Garcia-Cozar, Francisco

    2014-01-01

    Adaptive T cell responses are critical for controlling HCV infection. While there is clinical evidence of a relevant role for regulatory T cells in chronic HCV-infected patients, based on their increased number and function; mechanisms underlying such a phenomena are still poorly understood. Accumulating evidence suggests that proteins from Hepatitis C virus can suppress host immune responses. We and others have shown that HCV is present in CD4+ lymphocytes from chronically infected patients and that HCV-core protein induces a state of unresponsiveness in the CD4+ tumor cell line Jurkat. Here we show that CD4+ primary T cells lentivirally transduced with HCV-core, not only acquire an anergic phenotype but also inhibit IL-2 production and proliferation of bystander CD4+ or CD8+ T cells in response to anti-CD3 plus anti-CD28 stimulation. Core-transduced CD4+ T cells show a phenotype characterized by an increased basal secretion of the regulatory cytokine IL-10, a decreased IFN-γ production upon stimulation, as well as expression of regulatory T cell markers, CTLA-4, and Foxp3. A significant induction of CD4+CD25+CD127(low)PD-1(high)TIM-3(high) regulatory T cells with an exhausted phenotype was also observed. Moreover, CCR7 expression decreased in HCV-core expressing CD4+ T cells explaining their sequestration in inflamed tissues such as the infected liver. This work provides a new perspective on de novo generation of regulatory CD4+ T cells in the periphery, induced by the expression of a single viral protein.

  17. Polyfunctional Melan-A-specific tumor-reactive CD8+ T cells elicited by dacarbazine treatment before peptide-vaccination depends on AKT activation sustained by ICOS

    PubMed Central

    Franzese, Ornella; Palermo, Belinda; Di Donna, Cosmo; Sperduti, Isabella; Ferraresi, Virginia; Stabile, Helena; Gismondi, Angela; Santoni, Angela; Nisticò, Paola

    2016-01-01

    ABSTRACT The identification of activation pathways linked to antitumor T-cell polyfunctionality in long surviving patients is of great relevance in the new era of immunotherapy. We have recently reported that dacarbazine (DTIC) injected one day before peptide-vaccination plus IFN-α improves the antitumor lytic activity and enlarges the repertoire of Melan-A-specific T-cell clones, as compared with vaccination alone, impacting the overall survival of melanoma patients. To identify the mechanisms responsible for this improvement of the immune response, we have analyzed the endogenous and treatment-induced antigen (Ag)-specific response in a panel of Melan-A-specific CD8+ T-cell clones in terms of differentiation phenotype, inhibitory receptor profile, polyfunctionality and AKT activation. Here, we show that Melan-A-specific CD8+ T cells isolated from patients treated with chemoimmunotherapy possess a late differentiated phenotype as defined by the absence of CD28 and CD27 co-stimulatory molecules and high levels of LAG-3, TIM-3 and PD-1 inhibitory receptors. Nevertheless, they show higher proliferative potential and an improved antitumor polyfunctional effector profile in terms of co-production of TNF-α, IFNγ and Granzyme-B (GrB) compared with cells derived from patients treated with vaccination alone. Polyfunctionality is dependent on an active AKT signaling related to the engagement of the co-stimulatory molecule ICOS. We suggest that this phenotypic and functional signature is dictated by a fine-tuned balance between TCR triggering, AKT activation, co-stimulatory and inhibitory signals induced by chemoimmunotherapy and may be associated with antitumor T cells able to protect patients from tumor recurrence. PMID:27467927

  18. Multiparametric profiling of non–small-cell lung cancers reveals distinct immunophenotypes

    PubMed Central

    Lizotte, Patrick H.; Ivanova, Elena V.; Awad, Mark M.; Jones, Robert E.; Keogh, Lauren; Liu, Hongye; Dries, Ruben; Herter-Sprie, Grit S.; Santos, Abigail; Feeney, Nora B.; Paweletz, Cloud P.; Kulkarni, Meghana M.; Bass, Adam J.; Rustgi, Anil K.; Yuan, Guo-Cheng; Kufe, Donald W.; Jänne, Pasi A.; Hammerman, Peter S.; Sholl, Lynette M.; Hodi, F. Stephen; Richards, William G.; Bueno, Raphael; English, Jessie M.; Bittinger, Mark A.

    2016-01-01

    BACKGROUND. Immune checkpoint blockade improves survival in a subset of patients with non–small-cell lung cancer (NSCLC), but robust biomarkers that predict response to PD-1 pathway inhibitors are lacking. Furthermore, our understanding of the diversity of the NSCLC tumor immune microenvironment remains limited. METHODS. We performed comprehensive flow cytometric immunoprofiling on both tumor and immune cells from 51 NSCLCs and integrated this analysis with clinical and histopathologic characteristics, next-generation sequencing, mRNA expression, and PD-L1 immunohistochemistry (IHC). RESULTS. Cytometric profiling identified an immunologically “hot” cluster with abundant CD8+ T cells expressing high levels of PD-1 and TIM-3 and an immunologically “cold” cluster with lower relative abundance of CD8+ T cells and expression of inhibitory markers. The “hot” cluster was highly enriched for expression of genes associated with T cell trafficking and cytotoxic function and high PD-L1 expression by IHC. There was no correlation between immunophenotype and KRAS or EGFR mutation, or patient smoking history, but we did observe an enrichment of squamous subtype and tumors with higher mutation burden in the “hot” cluster. Additionally, approximately 20% of cases had high B cell infiltrates with a subset producing IL-10. CONCLUSIONS. Our results support the use of immune-based metrics to study response and resistance to immunotherapy in lung cancer. FUNDING. The Robert A. and Renée E. Belfer Family Foundation, Expect Miracles Foundation, Starr Cancer Consortium, Stand Up to Cancer Foundation, Conquer Cancer Foundation, International Association for the Study of Lung Cancer, National Cancer Institute (R01 CA205150), and the Damon Runyon Cancer Research Foundation. PMID:27699239

  19. Genetic architecture of local adaptation in lunar and diurnal emergence times of the marine midge Clunio marinus (Chironomidae, Diptera).

    PubMed

    Kaiser, Tobias S; Heckel, David G

    2012-01-01

    Circadian rhythms pre-adapt the physiology of most organisms to predictable daily changes in the environment. Some marine organisms also show endogenous circalunar rhythms. The genetic basis of the circalunar clock and its interaction with the circadian clock is unknown. Both clocks can be studied in the marine midge Clunio marinus (Chironomidae, Diptera), as different populations have different local adaptations in their lunar and diurnal rhythms of adult emergence, which can be analyzed by crossing experiments. We investigated the genetic basis of population variation in clock properties by constructing the first genetic linkage map for this species, and performing quantitative trait locus (QTL) analysis on variation in both lunar and diurnal timing. The genome has a genetic length of 167-193 centimorgans based on a linkage map using 344 markers, and a physical size of 95-140 megabases estimated by flow cytometry. Mapping the sex determining locus shows that females are the heterogametic sex, unlike most other Chironomidae. We identified two QTL each for lunar emergence time and diurnal emergence time. The distribution of QTL confirms a previously hypothesized genetic basis to a correlation of lunar and diurnal emergence times in natural populations. Mapping of clock genes and light receptors identified ciliary opsin 2 (cOps2) as a candidate to be involved in both lunar and diurnal timing; cryptochrome 1 (cry1) as a candidate gene for lunar timing; and two timeless (tim2, tim3) genes as candidate genes for diurnal timing. This QTL analysis of lunar rhythmicity, the first in any species, provides a unique entree into the molecular analysis of the lunar clock.

  20. The IDO1 selective inhibitor epacadostat enhances dendritic cell immunogenicity and lytic ability of tumor antigen-specific T cells

    PubMed Central

    Jochems, Caroline; Fantini, Massimo; Fernando, Romaine I.; Kwilas, Anna R.; Donahue, Renee N.; Lepone, Lauren M.; Grenga, Italia; Kim, Young-Seung; Brechbiel, Martin W.; Gulley, James L.; Madan, Ravi A.; Heery, Christopher R.; Hodge, James W.; Newton, Robert

    2016-01-01

    Epacadostat is a novel inhibitor of indoleamine-2,3-dioxygenase-1 (IDO1) that suppresses systemic tryptophan catabolism and is currently being evaluated in ongoing clinical trials. We investigated the effects of epacadostat on (a) human dendritic cells (DCs) with respect to maturation and ability to activate human tumor antigen-specific cytotoxic T-cell (CTL) lines, and subsequent T-cell lysis of tumor cells, (b) human regulatory T cells (Tregs), and (c) human peripheral blood mononuclear cells (PBMCs) in vitro. Simultaneous treatment with epacadostat and IFN-γ plus lipopolysaccharide (LPS) did not change the phenotype of matured human DCs, and as expected decreased the tryptophan breakdown and kynurenine production. Peptide-specific T-cell lines stimulated with DCs pulsed with peptide produced significantly more IFN-γ, TNFα, GM-CSF and IL-8 if the DCs were treated with epacadostat. These T cells also displayed higher levels of tumor cell lysis on a per cell basis. Epacadostat also significantly decreased Treg proliferation induced by IDO production from IFN-γ plus LPS matured human DCs, although the Treg phenotype did not change. Multicolor flow cytometry was performed on human PBMCs treated with epacadostat; analysis of 123 discrete immune cell subsets revealed no changes in major immune cell types, an increase in activated CD83+ conventional DCs, and a decrease in immature activated Tim3+ NK cells. These studies show for the first time several effects of epacadostat on human DCs, and subsequent effects on CTL and Tregs, and provide a rationale as to how epacadostat could potentially increase the efficacy of immunotherapeutics, including cancer vaccines. PMID:27192116

  1. Multiparametric profiling of non-small-cell lung cancers reveals distinct immunophenotypes.

    PubMed

    Lizotte, Patrick H; Ivanova, Elena V; Awad, Mark M; Jones, Robert E; Keogh, Lauren; Liu, Hongye; Dries, Ruben; Almonte, Christina; Herter-Sprie, Grit S; Santos, Abigail; Feeney, Nora B; Paweletz, Cloud P; Kulkarni, Meghana M; Bass, Adam J; Rustgi, Anil K; Yuan, Guo-Cheng; Kufe, Donald W; Jänne, Pasi A; Hammerman, Peter S; Sholl, Lynette M; Hodi, F Stephen; Richards, William G; Bueno, Raphael; English, Jessie M; Bittinger, Mark A; Wong, Kwok-Kin

    2016-09-08

    BACKGROUND. Immune checkpoint blockade improves survival in a subset of patients with non-small-cell lung cancer (NSCLC), but robust biomarkers that predict response to PD-1 pathway inhibitors are lacking. Furthermore, our understanding of the diversity of the NSCLC tumor immune microenvironment remains limited. METHODS. We performed comprehensive flow cytometric immunoprofiling on both tumor and immune cells from 51 NSCLCs and integrated this analysis with clinical and histopathologic characteristics, next-generation sequencing, mRNA expression, and PD-L1 immunohistochemistry (IHC). RESULTS. Cytometric profiling identified an immunologically "hot" cluster with abundant CD8(+) T cells expressing high levels of PD-1 and TIM-3 and an immunologically "cold" cluster with lower relative abundance of CD8(+) T cells and expression of inhibitory markers. The "hot" cluster was highly enriched for expression of genes associated with T cell trafficking and cytotoxic function and high PD-L1 expression by IHC. There was no correlation between immunophenotype and KRAS or EGFR mutation, or patient smoking history, but we did observe an enrichment of squamous subtype and tumors with higher mutation burden in the "hot" cluster. Additionally, approximately 20% of cases had high B cell infiltrates with a subset producing IL-10. CONCLUSIONS. Our results support the use of immune-based metrics to study response and resistance to immunotherapy in lung cancer. FUNDING. The Robert A. and Renée E. Belfer Family Foundation, Expect Miracles Foundation, Starr Cancer Consortium, Stand Up to Cancer Foundation, Conquer Cancer Foundation, International Association for the Study of Lung Cancer, National Cancer Institute (R01 CA205150), and the Damon Runyon Cancer Research Foundation.

  2. T-cell responses against CD19+ pediatric acute lymphoblastic leukemia mediated by bispecific T-cell engager (BiTE) are regulated contrarily by PD-L1 and CD80/CD86 on leukemic blasts

    PubMed Central

    Feucht, Judith; Kayser, Simone; Gorodezki, David; Hamieh, Mohamad; Döring, Michaela; Blaeschke, Franziska; Schlegel, Patrick; Bösmüller, Hans; Quintanilla-Fend, Leticia; Ebinger, Martin; Lang, Peter; Handgretinger, Rupert; Feuchtinger, Tobias

    2016-01-01

    T-cell immunotherapies are promising options in relapsed/refractory B-precursor acute lymphoblastic leukemia (ALL). We investigated the effect of co-signaling molecules on T-cell attack against leukemia mediated by CD19/CD3-bispecific T-cell engager. Primary CD19+ ALL blasts (n≥10) and physiologic CD19+CD10+ bone marrow precursors were screened for 20 co-signaling molecules. PD-L1, PD-1, LAG-3, CD40, CD86, CD27, CD70 and HVEM revealed different stimulatory and inhibitory profiles of pediatric ALL compared to physiologic cells, with PD-L1 and CD86 as most prominent inhibitory and stimulatory markers. PD-L1 was increased in relapsed ALL patients (n=11) and in ALLs refractory to Blinatumomab (n=5). Exhaustion markers (PD-1, TIM-3) were significantly higher on patients' T cells compared to physiologic controls. T-cell proliferation and effector function was target-cell dependent and correlated to expression of co-signaling molecules. Blockade of inhibitory PD-1-PD-L and CTLA-4-CD80/86 pathways enhanced T-cell function whereas blockade of co-stimulatory CD28-CD80/86 interaction significantly reduced T-cell function. Combination of Blinatumomab and anti-PD-1 antibody was feasible and induced an anti-leukemic in vivo response in a 12-year-old patient with refractory ALL. In conclusion, ALL cells actively regulate T-cell function by expression of co-signaling molecules and modify efficacy of therapeutic T-cell attack against ALL. Inhibitory interactions of leukemia-induced checkpoint molecules can guide future T-cell therapies. PMID:27708227

  3. Genetic Architecture of Local Adaptation in Lunar and Diurnal Emergence Times of the Marine Midge Clunio marinus (Chironomidae, Diptera)

    PubMed Central

    Kaiser, Tobias S.; Heckel, David G.

    2012-01-01

    Circadian rhythms pre-adapt the physiology of most organisms to predictable daily changes in the environment. Some marine organisms also show endogenous circalunar rhythms. The genetic basis of the circalunar clock and its interaction with the circadian clock is unknown. Both clocks can be studied in the marine midge Clunio marinus (Chironomidae, Diptera), as different populations have different local adaptations in their lunar and diurnal rhythms of adult emergence, which can be analyzed by crossing experiments. We investigated the genetic basis of population variation in clock properties by constructing the first genetic linkage map for this species, and performing quantitative trait locus (QTL) analysis on variation in both lunar and diurnal timing. The genome has a genetic length of 167–193 centimorgans based on a linkage map using 344 markers, and a physical size of 95–140 megabases estimated by flow cytometry. Mapping the sex determining locus shows that females are the heterogametic sex, unlike most other Chironomidae. We identified two QTL each for lunar emergence time and diurnal emergence time. The distribution of QTL confirms a previously hypothesized genetic basis to a correlation of lunar and diurnal emergence times in natural populations. Mapping of clock genes and light receptors identified ciliary opsin 2 (cOps2) as a candidate to be involved in both lunar and diurnal timing; cryptochrome 1 (cry1) as a candidate gene for lunar timing; and two timeless (tim2, tim3) genes as candidate genes for diurnal timing. This QTL analysis of lunar rhythmicity, the first in any species, provides a unique entree into the molecular analysis of the lunar clock. PMID:22384150

  4. PD-1 identifies the patient-specific CD8⁺ tumor-reactive repertoire infiltrating human tumors.

    PubMed

    Gros, Alena; Robbins, Paul F; Yao, Xin; Li, Yong F; Turcotte, Simon; Tran, Eric; Wunderlich, John R; Mixon, Arnold; Farid, Shawn; Dudley, Mark E; Hanada, Ken-Ichi; Almeida, Jorge R; Darko, Sam; Douek, Daniel C; Yang, James C; Rosenberg, Steven A

    2014-05-01

    Adoptive transfer of tumor-infiltrating lymphocytes (TILs) can mediate regression of metastatic melanoma; however, TILs are a heterogeneous population, and there are no effective markers to specifically identify and select the repertoire of tumor-reactive and mutation-specific CD8⁺ lymphocytes. The lack of biomarkers limits the ability to study these cells and develop strategies to enhance clinical efficacy and extend this therapy to other malignancies. Here, we evaluated unique phenotypic traits of CD8⁺ TILs and TCR β chain (TCRβ) clonotypic frequency in melanoma tumors to identify patient-specific repertoires of tumor-reactive CD8⁺ lymphocytes. In all 6 tumors studied, expression of the inhibitory receptors programmed cell death 1 (PD-1; also known as CD279), lymphocyte-activation gene 3 (LAG-3; also known as CD223), and T cell immunoglobulin and mucin domain 3 (TIM-3) on CD8⁺ TILs identified the autologous tumor-reactive repertoire, including mutated neoantigen-specific CD8⁺ lymphocytes, whereas only a fraction of the tumor-reactive population expressed the costimulatory receptor 4-1BB (also known as CD137). TCRβ deep sequencing revealed oligoclonal expansion of specific TCRβ clonotypes in CD8⁺PD-1⁺ compared with CD8⁺PD-1- TIL populations. Furthermore, the most highly expanded TCRβ clonotypes in the CD8⁺ and the CD8⁺PD-1⁺ populations recognized the autologous tumor and included clonotypes targeting mutated antigens. Thus, in addition to the well-documented negative regulatory role of PD-1 in T cells, our findings demonstrate that PD-1 expression on CD8⁺ TILs also accurately identifies the repertoire of clonally expanded tumor-reactive cells and reveal a dual importance of PD-1 expression in the tumor microenvironment.

  5. TIGIT and PD-1 impair tumor antigen-specific CD8⁺ T cells in melanoma patients.

    PubMed

    Chauvin, Joe-Marc; Pagliano, Ornella; Fourcade, Julien; Sun, Zhaojun; Wang, Hong; Sander, Cindy; Kirkwood, John M; Chen, Tseng-hui Timothy; Maurer, Mark; Korman, Alan J; Zarour, Hassane M

    2015-05-01

    T cell Ig and ITIM domain (TIGIT) is an inhibitory receptor expressed by activated T cells, Tregs, and NK cells. Here, we determined that TIGIT is upregulated on tumor antigen-specific (TA-specific) CD8⁺ T cells and CD8⁺ tumor-infiltrating lymphocytes (TILs) from patients with melanoma, and these TIGIT-expressing CD8⁺ T cells often coexpress the inhibitory receptor PD-1. Moreover, CD8⁺ TILs from patients exhibited downregulation of the costimulatory molecule CD226, which competes with TIGIT for the same ligand, supporting a TIGIT/CD226 imbalance in metastatic melanoma. TIGIT marked early T cell activation and was further upregulated by T cells upon PD-1 blockade and in dysfunctional PD-1⁺TIM-3⁺ TA-specific CD8⁺ T cells. PD-1⁺TIGIT⁺, PD-1⁻TIGIT⁺, and PD-1⁺TIGIT⁻ CD8⁺ TILs had similar functional capacities ex vivo, suggesting that TIGIT alone, or together with PD-1, is not indicative of T cell dysfunction. However, in the presence of TIGIT ligand-expressing cells, TIGIT and PD-1 blockade additively increased proliferation, cytokine production, and degranulation of both TA-specific CD8⁺ T cells and CD8⁺ TILs. Collectively, our results show that TIGIT and PD-1 regulate the expansion and function of TA-specific CD8⁺ T cells and CD8⁺ TILs in melanoma patients and suggest that dual TIGIT and PD-1 blockade should be further explored to elicit potent antitumor CD8⁺ T cell responses in patients with advanced melanoma.

  6. Biology Labs That Work: The Best of How-To-Do-Its. Volume II.

    ERIC Educational Resources Information Center

    Black, Suzanne, Ed.; Moore, Randy, Ed.; Haugen, Heidi, Ed.

    This selected collection of How-To-Do-It articles published in the American Biology Teacher during the past six years presents experiments that can be conducted safely under properly trained and responsible teacher supervision. Contents include: (1) "General Biology and the Nature of Science"; (2) "Cells and Molecules"; (3) "Microbes and Fungi";…

  7. Theoretical and experimental investigations on molecular structure of 7-Chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one with cytotoxic studies

    NASA Astrophysics Data System (ADS)

    Satheeshkumar, Rajendran; Shankar, Ramasamy; Kaminsky, Werner; Kalaiselvi, Sivalingam; Padma, Viswanadha Vijaya; Rajendra Prasad, Karnam Jayarampillai

    2016-04-01

    7-Chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one (3) is synthesized from 2-amino-5-chlorobenzophenone (1) and 1,2-cyclohexanedione (2) in the presence of catalyst InCl3. FT-IR, FT-Raman and FT-NMR spectra of molecule 3 have been recorded and the structure was confirmed by single crystal X-ray diffraction. CDCl3 and DMSO-d6 FT-NMR spectra and 1H and 13C NMR chemical shifts have been measured in molecule 3 and calculated at the B3LYP/6-311G (d,p) and MO6-2x/6-311G (d,p) levels of theory. Similarly calculated vibrational frequencies were found in good agreement with experimental findings. The optimized geometry of molecule 3 was compared with experimental XRD values. DFT calculations of the molecular electrostatic potential (MEP) and HOMO - LUMO frontier orbitals identified chemically active sites of molecule 3 responsible for its bioactivity. The title compound, 3 exhibits higher cytotoxicity in Human breast cancer cells (MCF-7) compared to human lung adenocarcinoma cells (A549).

  8. Electronic and Nuclear Factors in Charge and Excitation Transfer

    SciTech Connect

    Piotr Piotrowiak

    2004-09-28

    We report the and/or state of several subprojects of our DOE sponsored research on Electronic and Nuclear Factors in Electron and Excitation Transfer: (1) Construction of an ultrafast Ti:sapphire amplifier. (2) Mediation of electronic interactions in host-guest molecules. (3) Theoretical models of electrolytes in weakly polar media. (4) Symmetry effects in intramolecular excitation transfer.

  9. The Herpes Simplex Virus Latency-Associated Transcript Gene Is Associated with a Broader Repertoire of Virus-Specific Exhausted CD8+ T Cells Retained within the Trigeminal Ganglia of Latently Infected HLA Transgenic Rabbits

    PubMed Central

    Srivastava, Ruchi; Dervillez, Xavier; Khan, Arif A.; Chentoufi, Aziz A.; Chilukuri, Sravya; Shukr, Nora; Fazli, Yasmin; Ong, Nicolas N.; Afifi, Rasha E.; Osorio, Nelson; Geertsema, Roger; Nesburn, Anthony B.

    2016-01-01

    ABSTRACT Persistent pathogens, such as herpes simplex virus 1 (HSV-1), have evolved a variety of immune evasion strategies to avoid being detected and destroyed by the host's immune system. A dynamic cross talk appears to occur between the HSV-1 latency-associated transcript (LAT), the only viral gene that is abundantly transcribed during latency, and the CD8+ T cells that reside in HSV-1 latently infected human and rabbit trigeminal ganglia (TG). The reactivation phenotype of TG that are latently infected with wild-type HSV-1 or with LAT-rescued mutant (i.e., LAT+ TG) is significantly higher than TG latently infected with LAT-null mutant (i.e., LAT− TG). Whether LAT promotes virus reactivation by selectively shaping a unique repertoire of HSV-specific CD8+ T cells in LAT+ TG is unknown. In the present study, we assessed the frequency, function, and exhaustion status of TG-resident CD8+ T cells specific to 40 epitopes derived from HSV-1 gB, gD, VP11/12, and VP13/14 proteins, in human leukocyte antigen (HLA-A*0201) transgenic rabbits infected ocularly with LAT+ versus LAT– virus. Compared to CD8+ T cells from LAT– TG, CD8+ T cells from LAT+ TG (i) recognized a broader selection of nonoverlapping HSV-1 epitopes, (ii) expressed higher levels of PD-1, TIM-3, and CTLA-4 markers of exhaustion, and (iii) produced less tumor necrosis factor alpha, gamma interferon, and granzyme B. These results suggest a novel immune evasion mechanism by which the HSV-1 LAT may contribute to the shaping of a broader repertoire of exhausted HSV-specific CD8+ T cells in latently infected TG, thus allowing for increased viral reactivation. IMPORTANCE A significantly larger repertoire of dysfunctional (exhausted) HSV-specific CD8+ T cells were found in the TG of HLA transgenic rabbits latently infected with wild-type HSV-1 or with LAT-rescued mutant (i.e., LAT+ TG) than in a more restricted repertoire of functional HSV-specific CD8+ T cells in the TG of HLA transgenic rabbits latently

  10. Tumor-associated macrophages subvert T-cell function and correlate with reduced survival in clear cell renal cell carcinoma

    PubMed Central

    Dannenmann, Stefanie Regine; Thielicke, Julia; Stöckli, Martina; Matter, Claudia; von Boehmer, Lotta; Cecconi, Virginia; Hermanns, Thomas; Hefermehl, Lukas; Schraml, Peter; Moch, Holger; Knuth, Alexander; van den Broek, Maries

    2013-01-01

    Although malignant cells can be recognized and controlled by the immune system, in patients with clinically apparent cancer immunosurveillance has failed. To better understand local immunoregulatory processes that impact on cancer progression, we correlated intratumoral immunological profiles with the survival of patients affected by primary clear cell renal cell carcinoma (ccRCC). A retrospective analysis of 54 primary ccRCC samples for 31 different immune response-related transcripts, revealed a negative correlation of CD68 (a marker of tumor-associated macrophages, TAMs) and FOXP3 (a marker of regulatory T cells, Tregs) with survival. The subsequent analysis of 12 TAM-related transcripts revealed an association between the genes coding for CD163, interferon regulatory factor 4 (IRF4) and fibronectin 1 (FN1), all of which have been linked to the M2 TAM phenotype, with reduced survival and increased tumor stage, whereas the opposite was the case for the M1-associated gene coding for inducible nitric oxide synthetase (iNOS). The M2 signature of (CD68+) TAMs was found to correlate with CD163 expression, as determined in prospectively collected fresh ccRCC tissue samples. Upon co-culture with autologous tumor cells, CD11b+ cells isolated from paired blood samples expressed CD163 and other M2-associated proteins, suggesting that the malignant cells promote the accumulation of M2 TAMs. Furthermore, the tumor-associated milieu as well as isolated TAMs induced the skewing of autologous, blood-derived CD4+ T cells toward a more immunosuppressive phenotype, as shown by decreased production of effector cytokines, increased production of interleukin-10 (IL-10) and enhanced expression of the co-inhibitory molecules programmed death 1 (PD-1) and T-cell immunoglobulin mucin 3 (TIM-3). Taken together, our data suggest that ccRCC progressively attracts macrophages and induces their skewing into M2 TAMs, in turn subverting tumor-infiltrating T cells such that immunoregulatory

  11. Tumor-associated macrophages subvert T-cell function and correlate with reduced survival in clear cell renal cell carcinoma.

    PubMed

    Dannenmann, Stefanie Regine; Thielicke, Julia; Stöckli, Martina; Matter, Claudia; von Boehmer, Lotta; Cecconi, Virginia; Hermanns, Thomas; Hefermehl, Lukas; Schraml, Peter; Moch, Holger; Knuth, Alexander; van den Broek, Maries

    2013-03-01

    Although malignant cells can be recognized and controlled by the immune system, in patients with clinically apparent cancer immunosurveillance has failed. To better understand local immunoregulatory processes that impact on cancer progression, we correlated intratumoral immunological profiles with the survival of patients affected by primary clear cell renal cell carcinoma (ccRCC). A retrospective analysis of 54 primary ccRCC samples for 31 different immune response-related transcripts, revealed a negative correlation of CD68 (a marker of tumor-associated macrophages, TAMs) and FOXP3 (a marker of regulatory T cells, Tregs) with survival. The subsequent analysis of 12 TAM-related transcripts revealed an association between the genes coding for CD163, interferon regulatory factor 4 (IRF4) and fibronectin 1 (FN1), all of which have been linked to the M2 TAM phenotype, with reduced survival and increased tumor stage, whereas the opposite was the case for the M1-associated gene coding for inducible nitric oxide synthetase (iNOS). The M2 signature of (CD68(+)) TAMs was found to correlate with CD163 expression, as determined in prospectively collected fresh ccRCC tissue samples. Upon co-culture with autologous tumor cells, CD11b(+) cells isolated from paired blood samples expressed CD163 and other M2-associated proteins, suggesting that the malignant cells promote the accumulation of M2 TAMs. Furthermore, the tumor-associated milieu as well as isolated TAMs induced the skewing of autologous, blood-derived CD4(+) T cells toward a more immunosuppressive phenotype, as shown by decreased production of effector cytokines, increased production of interleukin-10 (IL-10) and enhanced expression of the co-inhibitory molecules programmed death 1 (PD-1) and T-cell immunoglobulin mucin 3 (TIM-3). Taken together, our data suggest that ccRCC progressively attracts macrophages and induces their skewing into M2 TAMs, in turn subverting tumor-infiltrating T cells such that

  12. CD127 Expression, Exhaustion Status and Antigen Specific Proliferation Predict Sustained Virologic Response to IFN in HCV/HIV Co-Infected Individuals

    PubMed Central

    Kared, Hassen; Saeed, Sahar; Klein, Marina B.; Shoukry, Naglaa H.

    2014-01-01

    Hepatitis C virus (HCV) infection is a major cause of morbidity and mortality in the HIV co-infected population. Interferon-alpha (IFN-α) remains a major component of anti-HCV therapy despite its deleterious effects on the immune system. Furthermore, IFN-α was recently shown to diminish the size of the latent HIV reservoir. The objectives of this study were to monitor the impact of IFN-α on T cell phenotype and proliferation of HIV and HCV-specific T cells during IFN therapy, and to identify immune markers that can predict the response to IFN in HICV/HIV co-infected patients. We performed longitudinal analyses of T cell numbers, phenotype and function in co-infected patients undergoing IFN-α therapy with different outcomes including IFN-α non-responders (NR) (n = 9) and patients who achieved sustained virologic response (SVR) (n = 19). We examined the expression of activation (CD38, HLA-DR), functional (CD127) and exhaustion markers (PD1, Tim-3, CD160 and CD244) on total CD4 and CD8 T cells before, during and after therapy. In addition, we examined the HIV- and HCV-specific proliferative responses against HIV-p24 and HCV-NS3 proteins. Frequencies of CD127+ CD4 T cells were higher in SVR than in NR patients at baseline. An increase in CD127 expression on CD8 T cells was observed after IFN-α therapy in all patients. In addition, CD8 T cells from NR patients expressed a higher exhaustion status at baseline. Finally, SVR patients exhibited higher proliferative response against both HIV and HCV antigens at baseline. Altogether, SVR correlated with higher expression of CD127, lower T cell exhaustion status and better HIV and HCV proliferative responses at baseline. Such factors might be used as non-invasive methods to predict the success of IFN–based therapies in co-infected individuals. PMID:25007250

  13. HCV coinfection contributes to HIV pathogenesis by increasing immune exhaustion in CD8 T-cells

    PubMed Central

    Rallón, Norma; García, Marcial; García-Samaniego, Javier; Rodríguez, Noelia; Cabello, Alfonso; Restrepo, Clara; Álvarez, Beatriz; García, Rosa; Górgolas, Miguel; Benito, José M.

    2017-01-01

    Background There are several contributors to HIV-pathogenesis or insufficient control of the infection. However, whether HIV/HCV-coinfected population exhibits worst evolution of HIV-pathogenesis remains unclear. Recently, some markers of immune exhaustion have been proposed as preferentially upregulated on T-cells during HIV-infection. Herein, we have analyzed T-cell exhaustion together with several other contributors to HIV-pathogenesis that could be affected by HCV-coinfection. Patients and methods Ninety-six patients with chronic HIV-infection (60 HIV-monoinfected and 36 HIV/HCV-coinfected), and 20 healthy controls were included in the study. All patients were untreated for both infections. Several CD4 and CD8 T-cell subsets involved in HIV-pathogenesis were investigated. Non-parametric tests were used to establish differences between groups and associations between variables. Multivariate linear regression was used to ascertain the variables independently associated with CD4 counts. Results HIV-patients presented significant differences compared to healthy controls in most of the parameters analyzed. Both HIV and HIV/HCV groups were comparable in terms of age, CD4 counts and HIV-viremia. Compared to HIV group, HIV/HCV group presented significantly higher levels of exhaustion (Tim3+PD1- subset) in total CD8+ T-cells (p = 0.003), and higher levels of exhaustion in CD8+HLADR+CD38+ (p = 0.04), CD8+HLADR-CD38+ (p = 0.009) and CD8+HLADR-CD38- (p = 0.006) subsets of CD8+ T-cells. Interestingly these differences were maintained after adjusting by CD4 counts and HIV-viremia. Conclusions We show a significant impact of HCV-coinfection on CD8 T-cells exhaustion, an important parameter associated with CD8 T-cell dysfunction in the setting of chronic HIV-infection. The relevance of this phenomenon on immunological and/or clinical HIV progression prompts HCV treatment to improve management of coinfected patients. PMID:28323897

  14. Inhibitory Receptor Expression Depends More Dominantly on Differentiation and Activation than “Exhaustion” of Human CD8 T Cells

    PubMed Central

    Legat, Amandine; Speiser, Daniel E.; Pircher, Hanspeter; Zehn, Dietmar; Fuertes Marraco, Silvia A.

    2013-01-01

    Under conditions of chronic antigen stimulation, such as persistent viral infection and cancer, CD8 T cells may diminish effector function, which has been termed “exhaustion.” Expression of inhibitory Receptors (iRs) is often regarded as a hallmark of “exhaustion.” Here we studied the expression of eight different iRs by CD8 T cells of healthy humans, including CTLA-4, PD1, TIM3, LAG3, 2B4, BTLA, CD160, and KLRG1. We show that many iRs are expressed upon activation, and with progressive differentiation to effector cells, even in absence of long-term (“chronic”) antigenic stimulation. In particular, we evaluated the direct relationship between iR expression and functionality in CD8 T cells by using anti-CD3 and anti-CD28 stimulation to stimulate all cells and differentiation subsets. We observed a striking up-regulation of certain iRs following the cytokine production wave, in agreement with the notion that iRs function as a negative feedback mechanism. Intriguingly, we found no major impairment of cytokine production in cells positive for a broad array of iRs, as previously shown for PD1 in healthy donors. Rather, the expression of the various iRs strongly correlated with T cell differentiation or activation states, or both. Furthermore, we analyzed CD8 T cells from lymph nodes (LNs) of melanoma patients. Interestingly, we found altered iR expression and lower cytokine production by T cells from metastatic LNs, but also from non-metastatic LNs, likely due to mechanisms which are not related to exhaustion. Together, our data shows that expression of iRs per se does not mark dysfunctional cells, but is rather tightly linked to activation and differentiation. This study highlights the importance of considering the status of activation and differentiation for the study and the clinical monitoring of CD8 T cells. PMID:24391639

  15. Modulation of Innate Immune Mechanisms to Enhance Leishmania Vaccine-Induced Immunity: Role of Coinhibitory Molecules

    PubMed Central

    Gannavaram, Sreenivas; Bhattacharya, Parna; Ismail, Nevien; Kaul, Amit; Singh, Rakesh; Nakhasi, Hira L.

    2016-01-01

    No licensed human vaccines are currently available against any parasitic disease including leishmaniasis. Several antileishmanial vaccine formulations have been tested in various animal models, including genetically modified live-attenuated parasite vaccines. Experimental infection studies have shown that Leishmania parasites utilize a broad range of strategies to undermine effector properties of host phagocytic cells, i.e., dendritic cells (DCs) and macrophages (MΦ). Furthermore, Leishmania parasites have evolved strategies to actively inhibit TH1 polarizing functions of DCs and to condition the infected MΦ toward anti-inflammatory/alternative/M2 phenotype. The altered phenotype of phagocytic cells is characterized by decreased production of antimicrobial reactive oxygen, nitrogen molecules, and pro-inflammatory cytokines, such as IFN-γ, IL-12, and TNF-α. These early events limit the activation of TH1-effector cells and set the stage for pathogenesis. Furthermore, this early control of innate immunity by the virulent parasites results in substantial alteration in the adaptive immunity characterized by reduced proliferation of CD4+ and CD8+ T cells and TH2-biased immunity that results in production of anti-inflammatory cytokines, such as TGF-β, and IL-10. More recent studies have also documented the induction of coinhibitory ligands, such as CTLA-4, PD-L1, CD200, and Tim-3, that induce exhaustion and/or non-proliferation in antigen-experienced T cells. Most of these studies focus on viral infections in chronic phase, thus limiting the direct application of these results to parasitic infections and much less to parasitic vaccines. However, these studies suggest that vaccine-induced protective immunity can be modulated using strategies that enhance the costimulation that might reduce the threshold necessary for T cell activation and conversely by strategies that reduce or block inhibitory molecules, such as PD-L1 and CD200. In this review, we will focus on the

  16. A Hybrid Computational-Experimental Framework for Microbial Chemical Synthesis via Enzyme Channeling

    DTIC Science & Technology

    2007-12-05

    its natural ligand, the freely diffusible quorum signaling molecule 3-oxooctanyl-l-homoserine lactone (OHHL), the TraR protein is a monomer that is...conformational switch for sensing molecules other than OHHL. We expect that a bevy of small molecule switches can created using the GFP-TraR backbone...AUTHORS 7. PERFORMING ORGANIZATION NAMES AND ADDRESSES U.S. Army Research Office P.O. Box 12211 Research Triangle Park, NC 27709-2211 15. SUBJECT

  17. Mannosyl Glycodendritic Structure Inhibits DC-SIGN-Mediated Ebola Virus Infection in cis and in trans

    PubMed Central

    Lasala, Fátima; Arce, Eva; Otero, Joaquín R.; Rojo, Javier; Delgado, Rafael

    2003-01-01

    We have designed a glycodendritic structure, BH30sucMan, that blocks the interaction between dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and Ebola virus (EBOV) envelope. BH30sucMan inhibits DC-SIGN-mediated EBOV infection at nanomolar concentrations. BH30sucMan may counteract important steps of the infective process of EBOV and, potentially, of microorganisms shown to exploit DC-SIGN for cell entry and infection. PMID:14638512

  18. Magnetic Trapping of Atomic Nitrogen (14N) and Cotrapping of NH (X3sigma)

    DTIC Science & Technology

    2008-11-12

    model using cold molecules 3. Recently, several interacting systems have been studied: a Bose - Einstein condensate with dipolar inter- actions 4...coefficient with 3He of kin1.8 10−14 cm3 s−1 due to the electronic interaction anisotropy induced by spin - orbit coupling 44. The small inelastic rate... spin is weakly coupled to the internuclear axis. NH, a molecule studied by many groups 26–28, is of this type. Second, the atomic partner should have

  19. Programmed Pathogen Sense and Destroy Circuits

    DTIC Science & Technology

    2009-02-18

    evolve the Pseudomonas aeruginosa quorum sensing transcription factor LasR to respond to the signal molecule 3OC12HSL with higher sensitivity and...sentinel circuits in recombinant E. coli cells with components of canonical quorum sensing (QS) signaling pathways. These pathways are normally used by...Pathogen Detection Expanded Accomplishments a) Accomplishments In the canonical gram-negative Quorum Sensing system, an I-protein synthase produces

  20. 2008 Annual Report

    DTIC Science & Technology

    2008-01-01

    intracellular sensor of small molecules . Since there are currently no generic reporters for intracellular metabolites, we sought to develop a tool for sensing ...freely diffusible quorum signaling molecule 3-oxooctanyl-l-homoserine lactone (OHHL), the TraR protein is a monomer that is highly unstable in the...for sensing molecules other than OHHL. We expect that a collection of small molecule switches can be created using the GFP-TraR backbone, simply by

  1. Synthetic Metabolic Channels for Improving Microbial Production of 1,2,4-butanetriol

    DTIC Science & Technology

    2009-01-01

    In the absence of its natural ligand, the freely diffusible quorum signaling molecule 3- oxooctanyl-l-homoserine lactone (OHHL), the TraR protein...replicate experiments. 4 During the last year, we have begun engineering the GFP-TraR conformational switch for sensing molecules other than...that the TraR-GFP fusion has an exquisite ability to be stabilized by the binding of a small molecule ligand and thus “ sense ” the presence of

  2. Blue-green variable light-emitting diode based on organic-molecule-doped polymer

    NASA Astrophysics Data System (ADS)

    Xu, Chunxiang; Cui, Yiping; Shen, Yingzhong; Gu, Hongwei; Pan, Yi; Li, Yinkui

    1999-09-01

    Monolayer organic light-emitting diodes based on the organic molecule [(3,4-dimethoxybenzyldehycle-2'-hydroxy naphthylimine)dimethyl gallium]-doped [poly(2-mehtyoxy-5-ethyloxy)-4-di-(2-methyoxy-5'-octaoxy)phenylene vinylene] have been fabricated by a spin-coating method. Color variation from green to blue has been observed. The results have been attributed to the variation of the recombination zone and the charge transfer between the materials.

  3. Synthesis of a Precursor to Sacubitril Using Enabling Technologies.

    PubMed

    Lau, Shing-Hing; Bourne, Samuel L; Martin, Benjamin; Schenkel, Berthold; Penn, Gerhard; Ley, Steven V

    2015-11-06

    An efficient preparation of a precursor to the neprilysin inhibitor sacubitril is described. The convergent synthesis features a diastereoselective Reformatsky-type carbethoxyallylation and a rhodium-catalyzed stereoselective hydrogenation for installation of the two key stereocenters. Moreover, by integrating machine-assisted methods with batch processes, this procedure allows a safe and rapid production of the key intermediates which are promptly transformed to the target molecule (3·HCl) over 7 steps in 54% overall yield.

  4. United States Air Force Summer Research Program -- 1992 High School Apprenticeship Program (HSAP) Reports. Volume 16. Arnold Engineering Development Center Civil Engineering Laboratory

    DTIC Science & Technology

    1992-01-01

    the FORTRAN and IDL codes are portable to workstations across the network and may be easily modified for the analysis of different molecules. 3-2...codes are portable on workstations across the network . Observations Through creating a program to visually analyze the electron probability density...calculations of set-up time for the test and other calculations such as mega -watt hours, air-on hours, and user-occupancy hours are contained on the lengthy

  5. Maintenance of Paraoxonase 2 Activity as a Strategy to Attenuate P. Aeruginosa Virulence

    DTIC Science & Technology

    2013-10-01

    Bacterial Pathogenesis, Host Defense, Host-Pathogen Interactions, Innate Immunity, Paraoxonase, Pseudomonas aeruginosa, Quorum Sensing 16. SECURITY...esterase that has been shown to efficiently hydrolyze, and thereby inactivate, the P. aeruginosa quorum sensing molecule 3OC12(1). This suggests that...PON2 may be an important component of the innate defense which can disrupt bacterial quorum sensing , limiting the pathogenicity of the bacteria. We

  6. The Role of MUC1 Cytoplasmic Domain in Tumorigenesis

    DTIC Science & Technology

    2005-05-01

    the extracellular matrix via integrin receptors. FAK plays an important role in integrin-mediated signaling and modulating processes such as cell...acts as a proadhesive molecule, 3-kinase and P-catenin. These findings may provide a facilitating the metastatic process by allowing migrating... processes such as cell growth, 18) from Santa Cruz was used for immunoprecipitation and differentiation, survival, and migration (Hanks et al

  7. Direct Simulation Monte Carlo for Atmospheric Entry. 1. Theoretical Basis and Physical Models

    DTIC Science & Technology

    2009-09-01

    rotational degrees of freedom (=2 for a diatomic molecule; =3 for a polyatomic molecule), k is Boltzmann’s constant, and T is the temperature. When...in [42] and [43]. 3.5 Charged Species Ions and electrons are formed in sufficiently energetic hypersonic flows first through associative ionization...would be required otherwise. However, electrostatic attraction means that electrons and ions interact with one another such that electron diffusion is

  8. Computational chemistry and aeroassisted orbital transfer vehicles

    NASA Technical Reports Server (NTRS)

    Cooper, D. M.; Jaffe, R. L.; Arnold, J. O.

    1985-01-01

    An analysis of the radiative heating phenomena encountered during a typical aeroassisted orbital transfer vehicle (AOTV) trajectory was made to determine the potential impact of computational chemistry on AOTV design technology. Both equilibrium and nonequilibrium radiation mechanisms were considered. This analysis showed that computational chemistry can be used to predict (1) radiative intensity factors and spectroscopic data; (2) the excitation rates of both atoms and molecules; (3) high-temperature reaction rate constants for metathesis and charge exchange reactions; (4) particle ionization and neutralization rates and cross sections; and (5) spectral line widths.

  9. A Ferrocene-Quinoxaline Derivative as a Highly Selective Probe for Colorimetric and Redox Sensing of Toxic Mercury(II) Cations

    PubMed Central

    Zapata, Fabiola; Caballero, Antonio; Molina, Pedro; Tarraga, Alberto

    2010-01-01

    A new chemosensor molecule 3 based on a ferrocene-quinoxaline dyad recognizes mercury (II) cations in acetonitrile solution. Upon recognition, an anodic shift of the ferrocene/ferrocenium oxidation peaks and a progressive red-shift (Δλ = 140 nm) of the low-energy band, are observed in its absorption spectrum. This change in the absorption spectrum is accompanied by a colour change from orange to deep green, which can be used for a “naked-eye” detection of this metal cation. PMID:22163528

  10. Skeletal hybridization and PfRIO-2 kinase modeling for synthesis of α-pyrone analogs as anti-malarial agent.

    PubMed

    Parveen, Afsana; Chakraborty, Arnish; Konreddy, Ananda Kumar; Chakravarty, Harapriya; Sharon, Ashoke; Trivedi, Vishal; Bal, Chandralata

    2013-01-01

    The pharmacophoric hybridization and computational design approach were applied to generate a novel series of α-pyrone analogs as plausible anti-malarial lead candidate. A putative active site in flexible loop close to wing-helix domain of PfRIO2 kinase was explored computationally to understand the molecular basis of ligand binding. All the synthesized molecules (3a-g) exhibited in vitro antimalarial activity. Oxidative stress induced by 3a-d were calculated and found to be significantly higher in case of 3b. Therefore, 3b, which shown most significant result was identified as promising lead for further SAR study to develop potent anti-malarials.

  11. Free Radical-Surface Interactions Using Multiphoton Ionization of Free Radicals

    DTIC Science & Technology

    1989-01-01

    Atoms, Rgf4PI 9 t Free Radl!cals)aj"i Atoms, Cross Section -’ r RE)*I of Free Radicals arid Atonn. 𔄃 43S’RACT (Conti n reverse if necessary Ind identi...Phy’s 71,2682f 19’q molecules,3 whereas the etch probability on a fluorinated ’M. T . Duignan, J. W. Hudgens, and J. R Wyatt, 1. Phys. Chem 86, 4156 (1982...80. (15) Kerr, J. A.; Wright. J. P. J Chem. Soc., Faraday Trans. 1 1915. 81. (17) Adams. T . E.; Morrtson. R J S.; Grant, E R Rev Sci Instrum 1471. 1980

  12. Life Sciences Issues for a Mission to Mars

    NASA Technical Reports Server (NTRS)

    1997-01-01

    Session MP5 includes short reports on: (1) Cardiovascular Concerns for a Mars Mission: Autonomic and Biomechanical Effects; (2) Reducing the Risk of Space Radiation Induced Bioeffects: Vehicle Design and Protectant Molecules; (3) Musculoskeletal Issues for Long Duration Mission: Muscle Mass Preservation, Renal Stone Risk Factors, Countermeasures, and Contingency Treatment Planning; (4) Psychological Issues and Crew Selection for a Mars Mission: Maximizing the Mix for the Long Haul; and (5) Issues in Crew Health, Medical Selection and Medical Officer (CMO) Training for a Mission to Mars.

  13. Conformational evaluation and detailed 1H and 13C NMR assignments of eremophilanolides.

    PubMed

    Burgueño-Tapia, Eleuterio; Hernández, Luis R; Reséndiz-Villalobos, Adriana Y; Joseph-Nathan, Pedro

    2004-10-01

    Extensive application of 1D and 2D NMR methodology, combined with molecular modeling, allowed the complete 1H and 13C NMR assignments of eremophilanolides from Senecio toluccanus. Comparison of the experimental 1H, 1H coupling constant values with those generated employing a generalized Karplus-type relationship, using dihedral angles extracted from MMX and DFT calculations, revealed that the epoxidized eremophilanolides 1 and 2 show conformational rigidity at room temperature, whereas molecules 3-6, containing an isolated double bond, are conformationally mobile.

  14. Highly-Immunogenic Virally-Vectored T-cell Vaccines Cannot Overcome Subversion of the T-cell Response by HCV during Chronic Infection

    PubMed Central

    Swadling, Leo; Halliday, John; Kelly, Christabel; Brown, Anthony; Capone, Stefania; Ansari, M. Azim; Bonsall, David; Richardson, Rachel; Hartnell, Felicity; Collier, Jane; Ammendola, Virginia; Del Sorbo, Mariarosaria; Von Delft, Annette; Traboni, Cinzia; Hill, Adrian V. S.; Colloca, Stefano; Nicosia, Alfredo; Cortese, Riccardo; Klenerman, Paul; Folgori, Antonella; Barnes, Eleanor

    2016-01-01

    An effective therapeutic vaccine for the treatment of chronic hepatitis C virus (HCV) infection, as an adjunct to newly developed directly-acting antivirals (DAA), or for the prevention of reinfection, would significantly reduce the global burden of disease associated with chronic HCV infection. A recombinant chimpanzee adenoviral (ChAd3) vector and a modified vaccinia Ankara (MVA), encoding the non-structural proteins of HCV (NSmut), used in a heterologous prime/boost regimen induced multi-specific, high-magnitude, durable HCV-specific CD4+ and CD8+ T-cell responses in healthy volunteers, and was more immunogenic than a heterologous Ad regimen. We now assess the immunogenicity of this vaccine regimen in HCV infected patients (including patients with a low viral load suppressed with interferon/ribavirin therapy), determine T-cell cross-reactivity to endogenous virus, and compare immunogenicity with that observed previously in both healthy volunteers and in HCV infected patients vaccinated with the heterologous Ad regimen. Vaccination of HCV infected patients with ChAd3-NSmut/MVA-NSmut was well tolerated. Vaccine-induced HCV-specific T-cell responses were detected in 8/12 patients; however, CD4+ T-cell responses were rarely detected, and the overall magnitude of HCV-specific T-cell responses was markedly reduced when compared to vaccinated healthy volunteers. Furthermore, HCV-specific cells had a distinct partially-functional phenotype (lower expression of activation markers, granzyme B, and TNFα production, weaker in vitro proliferation, and higher Tim3 expression, with comparable Tbet and Eomes expression) compared to healthy volunteers. Robust anti-vector T-cells and antibodies were induced, showing that there is no global defect in immunity. The level of viremia at the time of vaccination did not correlate with the magnitude of the vaccine-induced T-cell response. Full-length, next-generation sequencing of the circulating virus demonstrated that T-cells were

  15. Highly-Immunogenic Virally-Vectored T-cell Vaccines Cannot Overcome Subversion of the T-cell Response by HCV during Chronic Infection.

    PubMed

    Swadling, Leo; Halliday, John; Kelly, Christabel; Brown, Anthony; Capone, Stefania; Ansari, M Azim; Bonsall, David; Richardson, Rachel; Hartnell, Felicity; Collier, Jane; Ammendola, Virginia; Del Sorbo, Mariarosaria; Von Delft, Annette; Traboni, Cinzia; Hill, Adrian V S; Colloca, Stefano; Nicosia, Alfredo; Cortese, Riccardo; Klenerman, Paul; Folgori, Antonella; Barnes, Eleanor

    2016-08-02

    An effective therapeutic vaccine for the treatment of chronic hepatitis C virus (HCV) infection, as an adjunct to newly developed directly-acting antivirals (DAA), or for the prevention of reinfection, would significantly reduce the global burden of disease associated with chronic HCV infection. A recombinant chimpanzee adenoviral (ChAd3) vector and a modified vaccinia Ankara (MVA), encoding the non-structural proteins of HCV (NSmut), used in a heterologous prime/boost regimen induced multi-specific, high-magnitude, durable HCV-specific CD4+ and CD8+ T-cell responses in healthy volunteers, and was more immunogenic than a heterologous Ad regimen. We now assess the immunogenicity of this vaccine regimen in HCV infected patients (including patients with a low viral load suppressed with interferon/ribavirin therapy), determine T-cell cross-reactivity to endogenous virus, and compare immunogenicity with that observed previously in both healthy volunteers and in HCV infected patients vaccinated with the heterologous Ad regimen. Vaccination of HCV infected patients with ChAd3-NSmut/MVA-NSmut was well tolerated. Vaccine-induced HCV-specific T-cell responses were detected in 8/12 patients; however, CD4+ T-cell responses were rarely detected, and the overall magnitude of HCV-specific T-cell responses was markedly reduced when compared to vaccinated healthy volunteers. Furthermore, HCV-specific cells had a distinct partially-functional phenotype (lower expression of activation markers, granzyme B, and TNFα production, weaker in vitro proliferation, and higher Tim3 expression, with comparable Tbet and Eomes expression) compared to healthy volunteers. Robust anti-vector T-cells and antibodies were induced, showing that there is no global defect in immunity. The level of viremia at the time of vaccination did not correlate with the magnitude of the vaccine-induced T-cell response. Full-length, next-generation sequencing of the circulating virus demonstrated that T-cells were

  16. Identification of cell surface molecules involved in dystroglycan-independent Lassa virus cell entry.

    PubMed

    Shimojima, Masayuki; Ströher, Ute; Ebihara, Hideki; Feldmann, Heinz; Kawaoka, Yoshihiro

    2012-02-01

    Although O-mannosylated dystroglycan is a receptor for Lassa virus, a causative agent of Lassa fever, recent findings suggest the existence of an alternative receptor(s). Here we identified four molecules as receptors for Lassa virus: Axl and Tyro3, from the TAM family, and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and liver and lymph node sinusoidal endothelial calcium-dependent lectin (LSECtin), from the C-type lectin family. These molecules enhanced the binding of Lassa virus to cells and mediated infection independently of dystroglycan. Axl- or Tyro3-mediated infection required intracellular signaling via the tyrosine kinase activity of Axl or Tyro3, whereas DC-SIGN- or LSECtin-mediated infection and binding were dependent on a specific carbohydrate and on ions. The identification of these four molecules as Lassa virus receptors advances our understanding of Lassa virus cell entry.

  17. Spin control in ladderlike hexanuclear copper(II) complexes with metallacyclophane cores.

    PubMed

    Pardo, Emilio; Bernot, Kevin; Julve, Miguel; Lloret, Francesc; Cano, Joan; Ruiz-García, Rafael; Delgado, Fernando S; Ruiz-Pérez, Catalina; Ottenwaelder, Xavier; Journaux, Yves

    2004-05-03

    Two new hexanuclear oxamatocopper(II) complexes 3 and 4 have been synthesized from the binuclear copper(II) complexes of the meta- and para-phenylenebis(oxamate) ligands, respectively. Complexes 3 and 4 possess an overall ladderlike structure made up of two oxamate-bridged linear trinuclear units ("rails") connected through two phenylenediamidate bridges ("rungs") between the central copper atoms to give metallacyclic cores of the meta- and para-cyclophane type, respectively. They show different ground spin states, S = 1 (3) or S = 0 (4), depending on the substitution pattern in the aromatic spacers. The triplet state molecule 3 containing two spin doublet Cu(II)3 units connected by two m-phenylenediamidate bridges represents a successful extension of the concept of "ferromagnetic coupling units" to metal complexes, which is a well-known approach toward high spin organic radicals.

  18. The Fe(III) and Ga(III) coordination chemistry of 3-(1-hydroxymethylidene) and 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione: novel tetramic acid degradation products of homoserine lactone bacterial quorum sensing molecules.

    PubMed

    Romano, Ariel A; Hahn, Tobias; Davis, Nicole; Lowery, Colin A; Struss, Anjali K; Janda, Kim D; Böttger, Lars H; Matzanke, Berthold F; Carrano, Carl J

    2012-02-01

    Bacteria use small diffusible molecules to exchange information in a process called quorum sensing (QS). An important class of quorum sensing molecules used by Gram-negative bacteria is the family of N-acylhomoserine lactones (HSL). It was recently discovered that a degradation product of the QS molecule 3-oxo-C(12)-homoserine lactone, the tetramic acid 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione, is a potent antibacterial agent, thus implying roles for QS outside of simply communication. Because these tetramic acids also appear to bind iron with appreciable affinity it was suggested that metal binding might contribute to their biological activity. Here, using a variety of spectroscopic tools, we describe the coordination chemistry of both the methylidene and decylidene tetramic acid derivatives with Fe(III) and Ga(III) and discuss the potential biological significance of such metal binding.

  19. In Silico Molecular Docking and In Vitro Antidiabetic Studies of Dihydropyrimido[4,5-a]acridin-2-amines

    PubMed Central

    Bharathi, A.; Roopan, Selvaraj Mohana; Vasavi, C. S.; Munusami, Punnagai; Gayathri, G. A.; Gayathri, M.

    2014-01-01

    An in vitro antidiabetic activity on α-amylase and α–glucosidase activity of novel 10-chloro-4-(2-chlorophenyl)-12-phenyl-5,6-dihydropyrimido[4,5-a]acridin-2-amines (3a–3f) were evaluated. Structures of the synthesized molecules were studied by FT-IR, 1H NMR, 13C NMR, EI-MS, and single crystal X-ray structural analysis data. An in silico molecular docking was performed on synthesized molecules (3a–3f). Overall studies indicate that compound 3e is a promising compound leading to the development of selective inhibition of α-amylase and α-glucosidase. PMID:24991576

  20. Transition-moment directions of selected carbocyanines from emission anisotropy and linear dichroism measurements in uniaxially stretched polymer films

    NASA Astrophysics Data System (ADS)

    Synak, Anna; Bojarski, Piotr

    2005-12-01

    Based on the Kawski-Gryczyński method the values of angle between absorption and fluorescence transition moments of carbocyanines are given. This method is applied to the linear molecules: 3,3'-diethylthiacyanine iodide (DTTHCI), diethyloxacarbocyanine iodide (DOCI), 3,3'-diethyl-9-methylthiacarbocyanine iodide (MDTCI), diethylthiacarbocyanine iodide (DTCI) and 3,3'-diethyloxadicarbocyanine iodide (DODCI). Similarly located transition moments polarized approximately along the long axis of DTTHCI, DOCI, MDTCI, DTCI and DODCI are responsible for absorption and fluorescence ( β ⩽ 10°), when exciting in the long wavelength absorption band. The results are compared with relevant data obtained from linear dichroism measurements, energy migration data in partly ordered films and general Perrin formula.

  1. A new alligator-clip compound for molecular electronics

    NASA Astrophysics Data System (ADS)

    Jacob, Timo; Blanco, Mario; Goddard, William A.

    2004-06-01

    We used the B3LYP flavor of density functional calculations to study new alligator-clip compounds for molecular electronics with platinum electrodes. First, with commonly used S-based linkage molecule 3-methyl-1,2-dithiolane (MDTL) we find that after chemisorption on Pt(1 1 1) the most stable structure is ring-opened with a binding energy of 32.44 kcal/mol. Among several alternative alligator-clip compounds we find that P-based molecules lead to much higher binding energies. For the ring-closed structure of 3-methyl-1,2-diphospholane (MDPL) a binding energy of 47.72 kcal/mol was calculated and even 54.88 kcal/mol for the ring-opened molecule. Thus, MDPL provides a more stable link to the metal surface and might increase the conductance.

  2. The Fe(III) and Ga(III) coordination chemistry of 3-(1-hydroxymethylidene) and 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione: Novel tetramic acid degradation products of homoserine lactone bacterial quorum sensing molecules

    PubMed Central

    Romano, Ariel A.; Hahn, Tobias; Davis, Nicole; Lowery, Colin A.; Struss, Anjali K.; Janda, Kim D.; Böttger, Lars H.; Matzanke, Berthold F.; Carrano, Carl J.

    2011-01-01

    Bacteria use small diffusible molecules to exchange information in a process called quorum sensing (QS). An important class of quorum sensing molecules used by Gram-negative bacteria is the family of N-acylhomoserine lactones (HSL). It was recently discovered that a degradation product of the QS molecule 3-oxo-C12-homoserine lactone, the tetramic acid 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione, is a potent antibacterial agent, thus implying roles for QS outside of simply communication. Because these tetramic acids also appear to bind iron with appreciable affinity it was suggested that metal binding might contribute to their biological activity. Here, using a variety of spectroscopic tools, we describe the coordination chemistry of both the methylidene and decylidene tetramic acid derivatives with Fe(III) and Ga(III) and discuss the potential biological significance of such metal binding. PMID:22178671

  3. Simulation of Initial Stages of the Methanol to Gasoline Process in Acidic Zeolites

    NASA Astrophysics Data System (ADS)

    Stich, I.; Hytha, M.; Gale, J. D.; Terakura, K.; Payne, M. C.

    2000-03-01

    Methanol to gasoline process is one of the most studied applications of zeolites in current commercial production. This complex process involves: (1) initial adsorption of methanol in the zeolite, (2) activation of the adsorbed methanol molecules, (3) formation of the first intermediates (dimethyl ether (DME)), (4) formation of the first -C-C- bonds. Extensive studies of the stages (1)-(3) have been performed using the method of ab initio molecular dynamics (within DFT in the GGA approximation) and the method of thermodynamic integration to compute the free energy profile for formation of the first intermediate. We find that the initial adsorption (physisorption vs. chemisorption) depends on the adsorption conditions such as zeolite framework and methanol loading. Under certain combination of these conditions the chemisorbed species undergo activation. The activated species are very susceptible to nucleophilic attack to form DME. The computed free energy profile shows that this reaction is entropically controlled with significant differences between the total and free energy profiles.

  4. Construction of hybrid material with double chemical bond from functional bridge ligand: Molecular modification, lotus root-like micromorphology and strong luminescence

    NASA Astrophysics Data System (ADS)

    Yan, Bing; Sui, Yu-Long

    2006-07-01

    Modifying benzoic acid with a cross-linking molecule (3-aminopropyl)triethoxysilane (abbreviated as APES), a fictional molecular bridge with double reactivity was achieved by the amidation reaction between them. Then the modified functional molecule, which behaving as a bridge, both coordinate with terbium ion through amide's oxygen atom and form the Si-O chemical bond in an in situ sol-gel process with matrix precursor (tetraethoxysilane, TEOS). As a result, a novel molecular hybrid material (Tb-BA-APES) with double chemical bond (Tb-O coordination bond and Si-O covalent bond) was constructed. The strong luminescence of Tb 3+ substantiates optimum energy couple and effective intramolecular energy transfer between the triplet state energy of modified ligand bridge and emissive energy level of Tb 3+. Especially SEM of the molecular hybrid material exhibits unexpected microlotus root-like pore morphology.

  5. Synthesis and characterization of novel benzohydrazide as potential antibacterial agents from natural product vanillin and wintergreen oil

    NASA Astrophysics Data System (ADS)

    Setyawati, Amri; Wahyuningsih, Tutik Dwi; Purwono, Bambang

    2017-03-01

    A chalcone-like benzohydrazide derivatives (3) has been synthesis from natural resources vanillin (1a) and wintergreen oil (1b). This compound was synthesis as modified natural resource antibacterial agent. Some modification was done to increase the biological activity. Bromide was introduced to the vanillin structure to increase the activity (2a), whereas Hydrazine monohydrate was reacted with wintergreen oil to make new nucleophile (2b). Furthermore, chalcone like benzohydrazide compound was synthesized by stirring 5-bromovanillin (2a) with salicyl hydrazine (2b) at room temperature for 2-3 hours. The product was analyzed by FTIR, GCMS, 1H- and 13C-NMR to confirm its structure. The result showed that 5-bromovanillin, salicyl hydrazine, and benzohydrazide were successfully synthesized with 98, 78, and 33% of yield respectively. The target molecule 3 achieved with yellowish color with m.p. 106-111 °C, 97% purity.

  6. The physiological role of DC-SIGN: a tale of mice and men.

    PubMed

    Garcia-Vallejo, Juan J; van Kooyk, Yvette

    2013-10-01

    The innate immune receptor DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin) was discovered over a decade ago and was initially identified as a pattern recognition receptor. In addition to its ability to recognize a broad range of pathogen-derived ligands and self-glycoproteins, DC-SIGN also mediates intercellular adhesion, as well as antigen uptake and signaling, which is a functional hallmark of dendritic cells (DCs). Most research on DC-SIGN has relied on in vitro studies. The in vivo function of DC-SIGN is difficult to address, in part because there are eight genetic homologs in mice with no clear DC-SIGN ortholog. Here, we summarize the functions attributed to DC-SIGN based on in vitro data and discuss the limitations of available mouse models to uncover the physiological role of this receptor in vivo.

  7. Unexpected interplay of bonding height and energy level alignment at heteromolecular hybrid interfaces.

    PubMed

    Stadtmüller, Benjamin; Lüftner, Daniel; Willenbockel, Martin; Reinisch, Eva M; Sueyoshi, Tomoki; Koller, Georg; Soubatch, Serguei; Ramsey, Michael G; Puschnig, Peter; Tautz, F Stefan; Kumpf, Christian

    2014-04-16

    Although geometric and electronic properties of any physical or chemical system are always mutually coupled by the rules of quantum mechanics, counterintuitive coincidences between the two are sometimes observed. The coadsorption of the organic molecules 3,4,9,10-perylene tetracarboxylic dianhydride and copper-II-phthalocyanine on Ag(111) represents such a case, since geometric and electronic structures appear to be decoupled: one molecule moves away from the substrate while its electronic structure indicates a stronger chemical interaction, and vice versa for the other. Our comprehensive experimental and ab-initio theoretical study reveals that, mediated by the metal surface, both species mutually amplify their charge-donating and -accepting characters, respectively. This resolves the apparent paradox, and demonstrates with exceptional clarity how geometric and electronic bonding parameters are intertwined at metal-organic interfaces.

  8. Ionic model for highly compressed solid hydrogen

    NASA Astrophysics Data System (ADS)

    Yakub, E. S.

    2013-05-01

    We propose a simple ionic model for high-pressure conducting phase IV of solid hydrogen observed recently at room temperature. It is based on an assumption of dissociative ionization of hydrogen molecules 3H2=2H2(+)+2H(-) induced by high compression. The proposed model predicts the first order transition of molecular hydrogen solid into partly ionic conducting phase at megabar pressures and describes the temperature dependence of resistivity at room temperature. Its predictions are consistent with high temperature shock-compression experiments which exhibit conductivity of multiply shocked hydrogen. The location of phase transition line, the volume change, and the ionization degree in solid phase IV are estimated.

  9. Future perspectives in melanoma research : Meeting report from the "Melanoma Bridge". Napoli, December 1st-4th 2015.

    PubMed

    Ascierto, Paolo A; Agarwala, Sanjiv; Botti, Gerardo; Cesano, Alessandra; Ciliberto, Gennaro; Davies, Michael A; Demaria, Sandra; Dummer, Reinhard; Eggermont, Alexander M; Ferrone, Soldano; Fu, Yang Xin; Gajewski, Thomas F; Garbe, Claus; Huber, Veronica; Khleif, Samir; Krauthammer, Michael; Lo, Roger S; Masucci, Giuseppe; Palmieri, Giuseppe; Postow, Michael; Puzanov, Igor; Silk, Ann; Spranger, Stefani; Stroncek, David F; Tarhini, Ahmad; Taube, Janis M; Testori, Alessandro; Wang, Ena; Wargo, Jennifer A; Yee, Cassian; Zarour, Hassane; Zitvogel, Laurence; Fox, Bernard A; Mozzillo, Nicola; Marincola, Francesco M; Thurin, Magdalena

    2016-11-15

    T cell receptor (TCR) modified T cells; (ii) tumor heterogeneity including changes in antigenic profiles over time and location in individual patient; and (iii) a variety of immune-suppressive mechanisms in the tumor microenvironment (TME) including T regulatory cells (Treg), myeloid derived suppressor cells (MDSC) and immunosuppressive cytokines. In addition, complex interaction of tumor-immune system further increases the level of difficulties in the process of biomarkers development and their validation for clinical use. Recent clinical trial results have highlighted the potential for combination therapies that include immunomodulating agents such as anti-PD-1 and anti-CTLA-4. Agents targeting other immune inhibitory (e.g., Tim-3) or immune stimulating (e.g., CD137) receptors on T cells and other approaches such as adoptive cell transfer are tested for clinical efficacy in melanoma as well. These agents are also being tested in combination with targeted therapies to improve upon shorter-term responses thus far seen with targeted therapy. Various locoregional interventions that demonstrate promising results in treatment of advanced melanoma are also integrated with immunotherapy agents and the combinations with cytotoxic chemotherapy and inhibitors of angiogenesis are changing the evolving landscape of therapeutic options and are being evaluated to prevent or delay resistance and to further improve survival rates for melanoma patients' population. This meeting's specific focus was on advances in immunotherapy and combination therapy for melanoma. The importance of understanding of melanoma genomic background for development of novel therapies and biomarkers for clinical application to predict the treatment response was an integral part of the meeting. The overall emphasis on biomarkers supports novel concepts toward integrating biomarkers into personalized-medicine approach for treatment of patients with melanoma across the entire spectrum of disease stage

  10. Theoretical characterization on photoelectric properties of benzothiadiazole- and fluorene-based small molecule acceptor materials for the organic photovoltaics.

    PubMed

    Sui, Mingyue; Li, Shuangbao; Pan, Qingqing; Sun, Guangyan; Geng, Yun

    2017-01-01

    The upper efficiency of heterojunction organic photovoltaics depends on the increased open-circuit voltage (V oc) and short-circuit current (J sc). So, a higher lowest unoccupied molecular orbital (LUMO) level is necessary for organic acceptor material to possess higher V oc and more photons absorbsorption in the solar spectrum is needed for larger J sc. In this article, we theoretically designed some small molecule acceptors (2∼5) based on fluorene (F), benzothiadiazole, and cyano group (CN) referring to the reported acceptor material 2-[{7-(9,9-di-n-propyl-9H-fluoren-2-yl)benzo[c][1,2,5]thiadiazol-4-yl}methylene]malononitrile (1), the crucial parameters affecting photoelectrical properties of compounds 2∼5 were evaluated by the density functional theory (DFT) and time dependent density functional theory (TDDFT) methods. The results reveal that compared with 1, 3 and 4 could have the better complementary absorption spectra with P3HT, the increased LUMO level, the improved V oc, and the decreased electronic organization energy (λ e). From the simulation of transition density matrix, it is very clear that the excitons of molecules 3 and 4 are easier to separate in the material surface. Therefore, 3 and 4 may become potential acceptor candidates for organic photovoltaic cells. In addition, with the increased number of CN, the optoelectronic properties of the molecules show a regular change, mainly improve the LUMO level, energy gap, V oc, and absorption intensity. In summary, reasonably adjusting CN can effectively improve the photovoltaic properties of small molecule acceptors. Graphical Abstract Structure-property relationship of small molecule acceptors could be rationally evaluated in the article. The changes of conjugate length and CN are important strategies to alter the photovoltaic properties of small molecule acceptors. Therefore, taking the K12/1 as a reference, we have theoretically designed a series of small molecule acceptors (2-4). The calculated

  11. Structure and energetic characteristics of methane hydrates. From single cage to triple cage: A DFT-D study

    NASA Astrophysics Data System (ADS)

    Giricheva, N. I.; Ischenko, A. A.; Yusupov, V. I.; Bagratashvili, V. N.; Girichev, G. V.

    2017-03-01

    Electronic, geometrical, vibrational and energetic characteristics of the ice I TDT fragment consisted of dodecahedron H2O[512] (D) fused with two tetrakaidecahedrons H2O[51262] (T) and of the TDT cluster with three encapsulated CH4 molecules (3CH4·TDT) were calculated using a DFT/B97-D/6-311++G(2d,2p) approach. Binding energies, hydrogen bonding energies, energies of encapsulation of methane molecules into small D- and large T-cages of the TDT fragment, energies of frontier orbitals, the translational and librational frequencies, as well as the intramolecular vibrations of methane within the cages of different sizes were studied. Similar characteristics of isolated D- and T-cages and clathrates CH4·D and CH4·T were studied as function of compression/expansion of their oxygen skeletons using DFT/B97-D, LC-B3LYP, B3LYP-D2 methods.

  12. Metal Ion Enhanced Charge Transfer in a Terpyridine-bis-Pyrene System

    PubMed Central

    D'Aléo, Anthony; Cecchetto, Elio; De Cola, Luisa; Williams, René M.

    2009-01-01

    The synthesis, electrochemical and photophysical properties of a branched molecule 3,5-bis(pyrene-1-yl)-4′-phenyl-2,2′:6′,2″-terpyridine are reported. Spectroscopy in different solvents reveals that an optical electron transfer from the pyrene donor to the terpyridyl electron acceptor can occur in polar media, as the system displays both charge transfer (CT) absorption and CT emission. Furthermore, the study of the zinc complex as well as the bis-protonated form shows an enhancement of the electron transfer character of the system, by an increase of the acceptor strength. This is accompanied by a large increase of the non-radiative processes. With sub-nanosecond transient absorption spectroscopy, the CT state, consisting of the pyrene radical cation and the terpyridine radical anion, has been detected. At room temperature, the study of the nanosecond transient absorption spectra reveals the formation of a low-lying triplet excited state that we attribute to the pyrene moiety through which the CT state decays. At 77K, the absence of the terpyridine triplet emission also suggests the population of a low-lying triplet state of the pyrene unit. PMID:22412328

  13. MXene: a new family of promising hydrogen storage medium.

    PubMed

    Hu, Qianku; Sun, Dandan; Wu, Qinghua; Wang, Haiyan; Wang, Libo; Liu, Baozhong; Zhou, Aiguo; He, Julong

    2013-12-27

    Searching for reversible hydrogen storage materials operated under ambient conditions is a big challenge for material scientists and chemists. In this work, using density functional calculations, we systematically investigated the hydrogen storage properties of the two-dimensional (2D) Ti2C phase, which is a representative of the recently synthesized MXene materials ( ACS Nano 2012 , 6 , 1322 ). As a constituent element of 2D Ti2C phase, the Ti atoms are fastened tightly by the strong Ti-C covalent bonds, and thus the long-standing clustering problem of transition metal does not exist. Combining with the calculated binding energy of 0.272 eV, ab initio molecular dynamic simulations confirmed the hydrogen molecules (3.4 wt % hydrogen storage capacity) bound by Kubas-type interaction can be adsorbed and released reversibly under ambient conditions. Meanwhile, the hydrogen storage properties of the other two MXene phases (Sc2C and V2C) were also evaluated, and the results were similar to those of Ti2C. Therefore, the MXene family including more than 20 members was expected to be a good candidate for reversible hydrogen storage materials under ambient conditions.

  14. Small Molecule AKAP-Protein Kinase A (PKA) Interaction Disruptors That Activate PKA Interfere with Compartmentalized cAMP Signaling in Cardiac Myocytes*

    PubMed Central

    Christian, Frank; Szaszák, Márta; Friedl, Sabine; Drewianka, Stephan; Lorenz, Dorothea; Goncalves, Andrey; Furkert, Jens; Vargas, Carolyn; Schmieder, Peter; Götz, Frank; Zühlke, Kerstin; Moutty, Marie; Göttert, Hendrikje; Joshi, Mangesh; Reif, Bernd; Haase, Hannelore; Morano, Ingo; Grossmann, Solveig; Klukovits, Anna; Verli, Judit; Gáspár, Róbert; Noack, Claudia; Bergmann, Martin; Kass, Robert; Hampel, Kornelia; Kashin, Dmitry; Genieser, Hans-Gottfried; Herberg, Friedrich W.; Willoughby, Debbie; Cooper, Dermot M. F.; Baillie, George S.; Houslay, Miles D.; von Kries, Jens Peter; Zimmermann, Bastian; Rosenthal, Walter; Klussmann, Enno

    2011-01-01

    A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3′-diamino-4,4′-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating β-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure. PMID:21177871

  15. Synthesis, spectral analysis and quantum chemical studies on molecular geometry, chemical reactivity of 7-chloro-9-(2‧-chlorophenyl)-2,3-dihydroacridin-4(1H)-one and 7-chloro-9-(2‧-fluorophenyl)-2,3-dihydroacridin-4(1H)-one

    NASA Astrophysics Data System (ADS)

    Satheeshkumar, Rajendran; Sayin, Koray; Kaminsky, Werner; Rajendra Prasad, Karnam Jayarampillai

    2017-01-01

    7-Chloro-9-(2'-chlorophenyl)-2,3-dihydroacridin-4(1H)-one (3a) and 7-chloro-9-(2'-fluorophenyl)-2,3-dihydroacridin-4-(1H)-one (3b) were synthesized from 2-amino-2‧,5-dichlorobenzophenone (1a) and 2-amino-5-chloro-2'-fluorobenzophenone (1b) respectively with 1,2-cyclohexanedione (2) in the presence of 1-butyl-3-methylimidazolium tetrafluoroborate and InCl3 condition. The synthesized compounds have been recorded of FT-IR, NMR spectra and the structure was further confirmed by using single crystal X-ray diffraction. The synthesized compounds have been further checked the photo physical properties like UV, emission and fluorescent quantum yields were calculated. FT-NMR spectra and 1H and 13C NMR chemical shifts have been measured and computational calculations of compounds 3 are done by using B3LYP method with 6-311G basis set in gas phase. Similarly calculated vibrational frequencies were found in good agreement with experimental findings. The optimized geometry of molecules 3 was compared with experimental XRD values. DFT calculations of the molecular electrostatic potential (MEP) and HOMO - LUMO frontier orbitals identified chemically active sites of compounds 3 responsible for its chemical reactivity.

  16. Entangled states decoherence in coupled molecular spin clusters

    NASA Astrophysics Data System (ADS)

    Troiani, Filippo; Szallas, Attila; Bellini, Valerio; Affronte, Marco

    2010-03-01

    Localized electron spins in solid-state systems are widely investigated as potential building blocks of quantum devices and computers. While most efforts in the field have been focused on semiconductor low-dimensional structures, molecular antiferromagnets were recently recognized as alternative implementations of effective few-level spin systems. Heterometallic, Cr-based spin rings behave as effective spin-1/2 systems at low temperature and show long decoherence times [1]; besides, they can be chemically linked and magnetically coupled in a controllable fascion [2]. Here, we theoretically investigate the decoherence of the Bell states in such ring dimers, resulting from hyperfine interactions with nuclear spins. Based on a microscopic description of the molecules [3], we simulate the effect of inhomogeneous broadening, spectral diffusion and electron-nuclear entanglement on the electron-spin coherence, estimating the role of the different nuclei (and of possible chemical substitutions), as well as the effect of simple spin-echo sequences. References: [1] F. Troiani, et al., Phys. Rev. Lett. 94, 207208 (2005). [2] G. A. Timco, S: Carretta, F. Troiani et al., Nature Nanotech. 4, 173 (2009). [3] F. Troiani, V. Bellini, and M. Affronte, Phys. Rev. B 77, 054428 (2008).

  17. Noninvasive detection of weapons of mass destruction using terahertz radiation

    NASA Astrophysics Data System (ADS)

    Campbell, Matthew B.; Heilweil, Edwin J.

    2003-08-01

    The growing and immediate threat of biological and chemical weapons has placed urgency on the development of chemical and biological warfare agent (CWA/BWA) screening devices. Specifically, the ability to detect CWA/BWA prior to deployment is paramount to mitigating the threat without exposing individuals to its effects. SPARTA, Inc. and NIST are currently investigating the feasibility of using far-infrared radiation, or terahertz (THz, 1 THz = 1012 Hz) radiation, to non-invasively detect biological and chemical agents, explosives and drugs/narcotics inside sealed containers. Small-to-medium sized molecules (3-100 atoms) in gas, liquid and solid phases consistently exhibit identifiable spectral features in the far-IR portion of the spectrum. Many compounds associated with weapons of mass destruction are made up of molecules of this size. The THz portion of the spectrum lies between visible light and radio waves, allowing for partial transmission of 0.3-10.0 THz (30-1000 μm, 10-330 cm-1) light through most common materials. Therefore, transmission measurements of THz light can potentially be used to non-invasively detect the presence of CWA/BWA, explosives and drugs in the pathway of a THz radiation beam.

  18. Interaction of L-SIGN with hepatitis C virus envelope protein E2 up-regulates Raf-MEK-ERK pathway.

    PubMed

    Zhao, Lan-Juan; Wang, Wen; Ren, Hao; Qi, Zhong-Tian

    2013-07-01

    Liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin (L-SIGN) facilitates hepatitis C virus (HCV) infection through interaction with HCV envelope protein E2. Signaling events triggered by the E2 via L-SIGN are poorly understood. Here, kinase cascades of Raf-MEK-ERK pathway were defined upon the E2 treatment in NIH3T3 cells with stable expression of L-SIGN. The E2 bound to the cells through interaction with L-SIGN and such binding subsequently resulted in phosphorylation and activation of Raf, MEK, and ERK. Blockage of L-SIGN with antibody against L-SIGN reduced the E2-induced phosphorylation of Raf, MEK, and ERK. In the cells infected with cell culture-derived HCV, phosphorylation of these kinases was enhanced by the E2. Up-regulation of Raf-MEK-ERK pathway by HCV E2 via L-SIGN provides new insights into signaling cascade of L-SIGN, and might be a potential target for control and prevention of HCV infection.

  19. Binding of DC-SIGN to glycoproteins expressed in glycoengineered Pichia pastoris.

    PubMed

    Cukan, Michael C; Hopkins, Daniel; Burnina, Irina; Button, Michelle; Giaccone, Erin; Houston-Cummings, Nga Rewa; Jiang, Youwei; Li, Fang; Mallem, Muralidhar; Mitchell, Teresa; Moore, Renée; Nylen, Adam; Prinz, Bianka; Rios, Sandra; Sharkey, Nathan; Zha, Dongxing; Hamilton, Stephen; Li, Huijuan; Stadheim, Terrance A

    2012-12-14

    Previous studies have shown that glycoproteins expressed in wild-type Pichia pastoris bind to Dendritic cell-SIGN (DC-Specific Intercellular adhesion molecule-3 Grabbing Nonintegrin), a mannose-binding receptor found on dendritic cells in peripheral tissues which is involved in antigen presentation and the initiation of an immune response. However, the binding of DC-SIGN to glycoproteins purified from P. pastoris strains engineered to express humanized N- and O-linked glycans has not been tested to date. In this study, the binding of glycoproteins with specific high-mannose or human N- and O-linked glycan structures to DC-SIGN was tested. Proteins with humanized N-glycans including Man5 structures and O-glycans (up to as many as 24) with single mannose chain length showed DC-SIGN binding that was comparable to that measured for a CHO-produced IgG1 which lacks O-linked mannose. Glycoproteins with wild-type N-glycans and mannotriose and higher O-glycans bound to DC-SIGN in a manner that was strongly inhibited by either the use of enzymatic N-deglycosylation or sodium meta-periodate oxidation. Mannan purified from humanized P. pastoris also showed lower ability to inhibit DC-SIGN binding to glycoproteins with wild type fungal glycosylation than mannan purified from wild type strains. This study shows that humanized P. pastoris can produce glycoproteins that do not bind to DC-SIGN.

  20. Epoxidation of plasmalogens: source for long-chain alpha-hydroxyaldehydes in subcellular fractions of bovine liver.

    PubMed Central

    Loidl-Stahlhofen, A; Hannemann, K; Felde, R; Spiteller, G

    1995-01-01

    1. Masked long-chain alpha-hydroxyaldehydes were trapped in all subcellular fractions of bovine liver by application of pentafluorbenzyloxime derivatization [van Kuijk, Thomas, Stephens and Dratz (1986) Biochem. Biophys. Res. Commun. 139, 144-149] and quantified via GLC/MS using characteristic ion traces. 2. The chain-length profile of long-chain 2-hydroxyalkanales clearly indicates their relationship to plasmalogens as precursor molecules. 3. The previously postulated existence of alpha-acyloxyplasmalogens as precursor molecules of masked long-chain alpha-hydroxyaldehydes in bovine tissue lipids [Lutz and Spiteller (1991) Liebigs Ann. Chem. 1991, 563-567] was excluded. 4. The constant oxidation rate of plasmalogens in all subcellular fractions provides conclusive evidence for a non-enzymic plasmalogen epoxidation process (probably via hydroperoxy radicals). 5. The high reactivity of alpha-hydroxyaldehydes sheds some doubt on the postulation that plasmalogens protect mammalian cells against oxidative stress as postulated previously [Morand, Zoeller and Raetz (1988) J. Biol. Chem. 263, 11590-11596; Morand, Zoeller and Raetz (1988) J. Biol. Chem. 263, 11597-11606]. Images Figure 4 PMID:7639697

  1. Combination of an aromatic core and aromatic side chains which constitutes discotic liquid crystal and organogel supramolecular assemblies.

    PubMed

    Ishi-i, Tsutomu; Hirayama, Tomoyuki; Murakami, Ko-ichi; Tashiro, Hiroshi; Thiemann, Thies; Kubo, Kanji; Mori, Akira; Yamasaki, Sumio; Akao, Tetsuyuki; Tsuboyama, Akira; Mukaide, Taihei; Ueno, Kazunori; Mataka, Shuntaro

    2005-02-15

    This paper reports unique and unusual formations of columnar liquid crystals and organogels by self-assembling discotic molecules, which are composed of an aromatic hexaazatriphenylene (HAT) core and six flexible aromatic side chains. In HAT derivatives 3a, with 4'-(N,N-diphenylamino)biphenyl-4-yl chains, 3b, with 4'-[N-(2-naphthyl)-N-phenylamino]biphenyl-4-yl chains, and 3c, with 4'-phenoxybiphenyl-4-yl chains, the two-dimensional hexagonal packings can be created by their self-assembling in the liquid crystalline phase, which were characterized by polarizing optical microscopy, differential scanning calorimetry, and X-ray diffraction analysis. In certain solvents, HAT molecules 3a-c can form the viscoelastic fluid organogels, in which one-dimensional aggregates composed of the HAT molecules are self-assembled and entangled into three-dimensional network structures. The organogel structures were analyzed by scanning electron microscopy observation, (1)H NMR, UV-vis, and circular dichroism spectroscopy. In contrast to 3a-c, none of the liquid crystalline and organogel phases could be formed from 3d and 3e with short aromatic side chains including a phenylene spacer, and 3f (except a few specific solutions) and 3g without terminal diarylamino and phenoxy groups. In 3a-c, the aromatic side chains with terminal flexible groups make up soft regions that cooperatively stabilize the liquid crystalline and organogel supramolecular structures together with the hard regions of the hexaazatriphenylene core.

  2. Magnetic moment enhancement and spin polarization switch of the manganese phthalocyanine molecule on an IrMn(100) surface.

    PubMed

    Sun, X; Wang, B; Pratt, A; Yamauchi, Y

    2014-07-21

    The geometric, electronic, and magnetic structures of a manganese phthalocyanine (MnPc) molecule on an antiferromagnetic IrMn(100) surface are studied by density functional theory calculations. Two kinds of orientation of the adsorbed MnPc molecule are predicted to coexist due to molecular self-assembly on the surface-a top-site geometry with the Mn-N bonds aligned along the ⟨100⟩ direction, and a hollow-site orientation in which the Mn-N bonds are parallel to the ⟨110⟩ direction. The MnPc molecule is antiferromagnetically coupled to the substrate at the top site with a slight reduction in the magnetic moment of the Mn atom of the MnPc molecule (Mnmol). In contrast, the magnetic moment of the Mnmol is enhanced to 4.28 μB at the hollow site, a value larger than that in the free MnPc molecule (3.51 μB). Molecular distortion induced by adsorption is revealed to be responsible for the enhancement of the magnetic moment. Furthermore, the spin polarization of the Mnmol atom at around the Fermi level is found to change from negative to positive through an elongation of the Mn-N bonds of the MnPc. We propose that a reversible switch of the low/high magnetic moment and negative/positive spin polarization might be realized through some mechanical engineering methods.

  3. EPR oxygen imaging and hyperpolarized 13C MRI of pyruvate metabolism as noninvasive biomarkers of tumor treatment response to a glycolysis inhibitor 3-bromopyruvate.

    PubMed

    Matsumoto, Shingo; Saito, Keita; Yasui, Hironobu; Morris, H Douglas; Munasinghe, Jeeva P; Lizak, Martin; Merkle, Hellmut; Ardenkjaer-Larsen, Jan Henrik; Choudhuri, Rajani; Devasahayam, Nallathamby; Subramanian, Sankaran; Koretsky, Alan P; Mitchell, James B; Krishna, Murali C

    2013-05-01

    The hypoxic nature of tumors results in treatment resistance and poor prognosis. To spare limited oxygen for more crucial pathways, hypoxic cancerous cells suppress mitochondrial oxidative phosphorylation and promote glycolysis for energy production. Thereby, inhibition of glycolysis has the potential to overcome treatment resistance of hypoxic tumors. Here, EPR imaging was used to evaluate oxygen dependent efficacy on hypoxia-sensitive drug. The small molecule 3-bromopyruvate blocks glycolysis pathway by inhibiting hypoxia inducible enzymes and enhanced cytotoxicity of 3-bromopyruvate under hypoxic conditions has been reported in vitro. However, the efficacy of 3-bromopyruvate was substantially attenuated in hypoxic tumor regions (pO2<10 mmHg) in vivo using squamous cell carcinoma (SCCVII)-bearing mouse model. Metabolic MRI studies using hyperpolarized 13C-labeled pyruvate showed that monocarboxylate transporter-1 is the major transporter for pyruvate and the analog 3-bromopyruvate in SCCVII tumor. The discrepant results between in vitro and in vivo data were attributed to biphasic oxygen dependent expression of monocarboxylate transporter-1 in vivo. Expression of monocarboxylate transporter-1 was enhanced in moderately hypoxic (8-15 mmHg) tumor regions but down regulated in severely hypoxic (<5 mmHg) tumor regions. These results emphasize the importance of noninvasive imaging biomarkers to confirm the action of hypoxia-activated drugs.

  4. CNS myelin induces regulatory functions of DC-SIGN–expressing, antigen-presenting cells via cognate interaction with MOG

    PubMed Central

    García-Vallejo, J.J.; Ilarregui, J.M.; Kalay, H.; Chamorro, S.; Koning, N.; Unger, W.W.; Ambrosini, M.; Montserrat, V.; Fernandes, R.J.; Bruijns, S.C.M.; van Weering, J.R.T.; Paauw, N.J.; O’Toole, T.; van Horssen, J.; van der Valk, P.; Nazmi, K.; Bolscher, J.G.M.; Bajramovic, J.; Dijkstra, C.D.; ’t Hart, B.A.

    2014-01-01

    Myelin oligodendrocyte glycoprotein (MOG), a constituent of central nervous system myelin, is an important autoantigen in the neuroinflammatory disease multiple sclerosis (MS). However, its function remains unknown. Here, we show that, in healthy human myelin, MOG is decorated with fucosylated N-glycans that support recognition by the C-type lectin receptor (CLR) DC-specific intercellular adhesion molecule-3–grabbing nonintegrin (DC-SIGN) on microglia and DCs. The interaction of MOG with DC-SIGN in the context of simultaneous TLR4 activation resulted in enhanced IL-10 secretion and decreased T cell proliferation in a DC-SIGN-, glycosylation-, and Raf1-dependent manner. Exposure of oligodendrocytes to proinflammatory factors resulted in the down-regulation of fucosyltransferase expression, reflected by altered glycosylation at the MS lesion site. Indeed, removal of fucose on myelin reduced DC-SIGN–dependent homeostatic control, and resulted in inflammasome activation, increased T cell proliferation, and differentiation toward a Th17-prone phenotype. These data demonstrate a new role for myelin glycosylation in the control of immune homeostasis in the healthy human brain through the MOG–DC-SIGN homeostatic regulatory axis, which is comprised by inflammatory insults that affect glycosylation. This phenomenon should be considered as a basis to restore immune tolerance in MS. PMID:24935259

  5. Glycolipid-based TLR4 Modulators and Fluorescent Probes: Rational Design, Synthesis, and Biological Properties.

    PubMed

    Ciaramelli, Carlotta; Calabrese, Valentina; Sestito, Stefania E; Pérez-Regidor, Lucia; Klett, Javier; Oblak, Alja; Jerala, Roman; Piazza, Matteo; Martín-Santamaría, Sonsoles; Peri, Francesco

    2016-08-01

    The cationic glycolipid IAXO-102, a potent TLR4 antagonist targeting both MD-2 and CD14 co-receptors, has been used as scaffold to design new potential TLR4 modulators and fluorescent labels for the TLR4 receptor complex (membrane TLR4.MD-2 dimer and CD14). The primary amino group of IAXO-102, not involved in direct interaction with MD-2 and CD14 receptors, has been exploited to covalently attach a fluorescein (molecules 1 and 2) or to link two molecules of IAXO-102 through diamine and diammonium spacers, obtaining 'dimeric' molecules 3 and 4. The structure-based rational design of compounds 1-4 was guided by the optimization of MD-2 and CD14 binding. Compounds 1 and 2 inhibited TLR4 activation, in a concentration-dependent manner, and signaling in HEK-Blue TLR4 cells. The fluorescent labeling of murine macrophages by molecule 1 was inhibited by LPS and was also abrogated when cell surface proteins were digested by trypsin, thus suggesting an interaction of fluorescent probe 1 with membrane proteins of the TLR4 receptor system.

  6. Dual impact of chronic liver disease and amaebiasis on immunopathogenesis of primary osteoarthritis in Egyptians.

    PubMed

    El-Dardiry, Samia A; Shafik, Sherine R; Wagih, Ayman; Amir, El-Amir M; El-Yamany, Sahar; Selim, Sahar; Amr, Yaser; Hawas, Samir El-Sayed

    2007-08-01

    Sixty cases with primary knee OA were equally categorized into six groups with EHI (Gs 1, 2, 3) or without (Gs 4, 5, 6). GI included cases with HCV, GII cases with RHS & HCV and GIII cases with a history of non-active schistosomiasis whereas Gs 4, 5 & 6 included cases without EHI. Clinical examination with inclusion criteria of pathological manifestations w\\as associated with biochemical evaluation of adhesion molecules (E-selectin, P-selectin, intracellular adhesion molecule-3 "ICAM-3") in plasma and synovial fluid. Synovial fluid indices (IgG, IgA, IgM, & C3) were evaluated as well as indices of inflammation and oxidative stress (Beta 2 microglobulin, Haptaglobulin, fibronectin, total thiol, superoxide dismutase, thiobarbituric acid reactive substance & hyaluronan) in synovial fluid and indices activating fibrogenesis in serum and plasma (procollagen III, plasma prolidase, Interleukin-1 beta, Interleukin-6 & TNF alpha). The results showed a positive relationship between indices activating vascular damage, fibrogenesis and immuno-inflammatory response with higher change magnitude in EHI cases particularly with combined HCV & RHS. This implement the dual role of hepatic insult and intestinal amoebiasis on immune mediated mechanisms activating inflammatory response in OA cases reflecting common signaling pathways associated with pathogenesis of multifaceted origin.

  7. Extraction of trivalent lanthanides and actinides by a synergistic mixture of thenoyltrifluoroacetone and a linear polymer

    SciTech Connect

    Ensor, D.D.; Shah, A.H.

    1984-01-01

    Mixtures of a linear polyether, K-5, and thenoyltrifluoroacetone, HTTA, have been shown to exhibit synergistic character in the extraction of trivalent lanthanides and actinides. The effect of the addition of K-5 to the organic phase on the extractions of Ce(III), Eu(III), Tm(III), Am(III), Cm(III), Bk(III), and Cf(III) by HTTA in chloroform from 0.5M NaNO/sub 3/ at 25/sup 0/C has been measured. These results indicate the extraction is enhanced by the formation of an adduct containing one metal molecule, 3 TTA molecules, and one K-5 molecule in the organic phase. The organic phase stability constants for the formation of these synergistic species have been calculated for all the metals studied. The magnitude of these organic phase stability constants for K-5 are similar to other common neutral donors. The order of stability does not follow the normal trend based on charge-to-radius ratio, but follows a pattern based on size, with Am(III) being the most stable.

  8. Galleria mellonella as a model system to study Acinetobacter baumannii pathogenesis and therapeutics.

    PubMed

    Peleg, Anton Y; Jara, Sebastian; Monga, Divya; Eliopoulos, George M; Moellering, Robert C; Mylonakis, Eleftherios

    2009-06-01

    Nonmammalian model systems of infection such as Galleria mellonella (caterpillars of the greater wax moth) have significant logistical and ethical advantages over mammalian models. In this study, we utilize G. mellonella caterpillars to study host-pathogen interactions with the gram-negative organism Acinetobacter baumannii and determine the utility of this infection model to study antibacterial efficacy. After infecting G. mellonella caterpillars with a reference A. baumannii strain, we observed that the rate of G. mellonella killing was dependent on the infection inoculum and the incubation temperature postinfection, with greater killing at 37 degrees C than at 30 degrees C (P = 0.01). A. baumannii strains caused greater killing than the less-pathogenic species Acinetobacter baylyi and Acinetobacter lwoffii (P < 0.001). Community-acquired A. baumannii caused greater killing than a reference hospital-acquired strain (P < 0.01). Reduced levels of production of the quorum-sensing molecule 3-hydroxy-C(12)-homoserine lactone caused no change in A. baumannii virulence against G. mellonella. Treatment of a lethal A. baumannii infection with antibiotics that had in vitro activity against the infecting A. baumannii strain significantly prolonged the survival of G. mellonella caterpillars compared with treatment with antibiotics to which the bacteria were resistant. G. mellonella is a relatively simple, nonmammalian model system that can be used to facilitate the in vivo study of host-pathogen interactions in A. baumannii and the efficacy of antibacterial agents.

  9. Quantitative analysis of 3-OH oxylipins in fermentation yeast.

    PubMed

    Potter, Greg; Xia, Wei; Budge, Suzanne M; Speers, R Alex

    2017-02-01

    Despite the ubiquitous distribution of oxylipins in plants, animals, and microbes, and the application of numerous analytical techniques to study these molecules, 3-OH oxylipins have never been quantitatively assayed in yeasts. The formation of heptafluorobutyrate methyl ester derivatives and subsequent analysis with gas chromatography - negative chemical ionization - mass spectrometry allowed for the first determination of yeast 3-OH oxylipins. The concentration of 3-OH 10:0 (0.68-4.82 ng/mg dry cell mass) in the SMA strain of Saccharomyces pastorianus grown in laboratory-scale beverage fermentations was elevated relative to oxylipin concentrations in plant tissues and macroalgae. In fermenting yeasts, the onset of 3-OH oxylipin formation has been related to fermentation progression and flocculation initiation. When the SMA strain was grown in laboratory-scale fermentations, the maximal sugar consumption rate preceded the lowest concentration of 3-OH 10:0 by ∼4.5 h and a distinct increase in 3-OH 10:0 concentration by ∼16.5 h.

  10. Zero-Mode Waveguide detection of biomolecules transport through artificial nanopores and nuclear pore complexes

    NASA Astrophysics Data System (ADS)

    Auger, Thomas; Auvray, Loic; Montel, Fabien

    We have developed a novel single molecule optical observation method using a custom Zero-Mode Waveguide setup to study the translocation of biopolymers through artificial and biological nanopores. Our work focuses on two aspects. First we monitored the flow driven injection of DNA molecules through solid state nanopores and showed that DNA starts translocating over a flow threshold independent of the pore radius, the DNA concentration and length. We demonstrate that the translocation is controlled by an energy barrier as proposed by the de Gennes - Brochard suction model. The height of the energy barrier can be modulated by functionalizing the nanopores with PEG-Thiols. More recently we adapted our setup to the study of transport through the nuclear pore complex (NPC) using extracted nuclear membranes from Xenopus Laevis oocytes. We aim at probing the conformation of unstructured proteins - the FG-Nucleoporins - crowding the central channel of the NPC by monitoring the free diffusion of small Dextran molecules (3kDa). We have been able to estimate the radius of the central pore of the NPC. We want to study the effects of transporter molecules, which have a high affinity for the FG-Nups, on the central pore size and correlate it to the conformation of FG-Nups.

  11. Femtosecond study on the isomerization dynamics of NK88. II. Excited-state dynamics

    NASA Astrophysics Data System (ADS)

    Vogt, Gerhard; Nuernberger, Patrick; Gerber, Gustav; Improta, Roberto; Santoro, Fabrizio

    2006-07-01

    The molecule 3,3'-diethyl-2,2'-thiacyanine isomerizes after irradiation with light of the proper wavelength. After excitation, it undergoes a transition, in which one or more conical intersections are involved, back to the ground state to form different product photoisomers. The dynamics before and directly after the transition back to the ground state is investigated by transient absorption spectroscopy in a wavelength region of 360-950nm, as well as by fluorescence upconversion. It is shown that the excited-state dynamics are governed by two time scales: a short one with a decay time of less than 2ps and a long one with about 9ps. A thorough comparison of the experimental results with those of configuration interaction singles and time-dependent density functional theory calculations suggests that these dynamics are related to two competing pathways differing in the molecular twisting on the excited surface after photoexcitation. From the experimental point of view this picture arises taking into account the time scales for ground-state bleach, excited-state absorption, stimulated emission, fluorescence, and assumed hot ground-state absorption both in the solvent methanol and ethylene glycol.

  12. Virulence in Pectobacterium atrosepticum is regulated by a coincidence circuit involving quorum sensing and the stress alarmone, (p)ppGpp.

    PubMed

    Bowden, Steven D; Eyres, Alison; Chung, Jade C S; Monson, Rita E; Thompson, Arthur; Salmond, George P C; Spring, David R; Welch, Martin

    2013-11-01

    Pectobacterium atrosepticum (Pca) is a Gram-negative phytopathogen which causes disease by secreting plant cell wall degrading exoenzymes (PCWDEs). Previous studies have shown that PCWDE production is regulated by (i) the intercellular quorum sensing (QS) signal molecule, 3-oxo-hexanoyl-l-homoserine lactone (OHHL), and (ii) the intracellular 'alarmone', (p)ppGpp, which reports on nutrient limitation. Here we show that these two signals form an integrated coincidence circuit which ensures that metabolically costly PCWDE synthesis does not occur unless the population is simultaneously quorate and nutrient limited. A (p)ppGpp null ΔrelAΔspoT mutant was defective in both OHHL and PCWDE production, and nutritional supplementation of wild type cultures (which suppresses (p)ppGpp production) also suppressed OHHL and PCWDE production. There was a substantial overlap in the transcriptome of a (p)ppGpp deficient relA mutant and of a QS defective expI (OHHL synthase) mutant, especially with regards to virulence-associated genes. Random transposon mutagenesis revealed that disruption of rsmA was sufficient to restore PCWDE production in the (p)ppGpp null strain. We found that the ratio of RsmA protein to its RNA antagonist, rsmB, was modulated independently by (p)ppGpp and QS. While QS predominantly controlled virulence by modulating RsmA levels, (p)ppGpp exerted regulation through the modulation of the RsmA antagonist, rsmB.

  13. Fragment-based strategy for structural optimization in combination with 3D-QSAR.

    PubMed

    Yuan, Haoliang; Tai, Wenting; Hu, Shihe; Liu, Haichun; Zhang, Yanmin; Yao, Sihui; Ran, Ting; Lu, Shuai; Ke, Zhipeng; Xiong, Xiao; Xu, Jinxing; Chen, Yadong; Lu, Tao

    2013-10-01

    Fragment-based drug design has emerged as an important methodology for lead discovery and drug design. Different with other studies focused on fragment library design and active fragment identification, a fragment-based strategy was developed in combination with three-dimensional quantitative structure-activity relationship (3D-QSAR) for structural optimization in this study. Based on a validated scaffold or fragment hit, a series of structural optimization was conducted to convert it to lead compounds, including 3D-QSAR modelling, active site analysis, fragment-based structural optimization and evaluation of new molecules. 3D-QSAR models and active site analysis provided sufficient information for confirming the SAR and pharmacophoric features for fragments. This strategy was evaluated through the structural optimization on a c-Met inhibitor scaffold 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one, which resulted in an c-Met inhibitor with high inhibitory activity. Our study suggested the effectiveness of this fragment-based strategy and the druggability of our newly explored active region. The reliability of this strategy indicated it could also be applied to facilitate lead optimization of other targets.

  14. Label-Free LC-MS/MS Proteomic Analysis of Cerebrospinal Fluid Identifies Protein/Pathway Alterations and Candidate Biomarkers for Amyotrophic Lateral Sclerosis.

    PubMed

    Collins, Mahlon A; An, Jiyan; Hood, Brian L; Conrads, Thomas P; Bowser, Robert P

    2015-11-06

    Analysis of the cerebrospinal fluid (CSF) proteome has proven valuable to the study of neurodegenerative disorders. To identify new protein/pathway alterations and candidate biomarkers for amyotrophic lateral sclerosis (ALS), we performed comparative proteomic profiling of CSF from sporadic ALS (sALS), healthy control (HC), and other neurological disease (OND) subjects using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1712 CSF proteins were detected and relatively quantified by spectral counting. Levels of several proteins with diverse biological functions were significantly altered in sALS samples. Enrichment analysis was used to link these alterations to biological pathways, which were predominantly related to inflammation, neuronal activity, and extracellular matrix regulation. We then used our CSF proteomic profiles to create a support vector machines classifier capable of discriminating training set ALS from non-ALS (HC and OND) samples. Four classifier proteins, WD repeat-containing protein 63, amyloid-like protein 1, SPARC-like protein 1, and cell adhesion molecule 3, were identified by feature selection and externally validated. The resultant classifier distinguished ALS from non-ALS samples with 83% sensitivity and 100% specificity in an independent test set. Collectively, our results illustrate the utility of CSF proteomic profiling for identifying ALS protein/pathway alterations and candidate disease biomarkers.

  15. Application of polymer based stationary phases in high performance liquid chromatography and capillary high performance liquid chromatography hyphenated to microcoil 1H nuclear magnetic resonance spectroscopy.

    PubMed

    Grynbaum, Marc David; Meyer, Christoph; Putzbach, Karsten; Rehbein, Jens; Albert, Klaus

    2007-07-13

    The increased demand for chromatographic materials that are able to achieve a fast separation of large quantities of structure analogues is a great challenge. It is known that polymer based chromatographic materials have a higher loadability, compared to silica based sorbents. Unfortunately these polymer materials cannot be used under high pressure which is necessary in order to obtain high flow rates, and hence long times are needed to perform a separation. However, by immobilizing a polymer on a mechanically stable porous silica core, this problem can be circumvented and higher flows become feasible on these materials. Especially for capillary liquid chromatography hyphenated with nuclear magnetic resonance a high loadability is of great importance in order to obtain sharp, resolved, and concentrated peaks thus resulting in a good signal to noise ratio in the NMR experiment. Therefore, a highly shape selective chromatographic sorbent was developed by covalently immobilizing a poly(ethylene-co-acrylic) acid copolymer (-CH(2)CH(2)-)(x)[CH(2)CH(CO(2)H)-](y) (x=119, y=2.4) with a mass fraction of acrylic acid of 5% as stationary phase on silica via a spacer molecule (3-glycidoxypropyltrimethoxysilane). First, the loadability of this sorbent compared to C(30) is demonstrated by the HPLC separation of two xanthophyll isomers. Subsequently, it has been successfully employed in the hyphenation of capillary HPLC with microcoil (1)H NMR spectroscopy by separating and identifying a highly concentrated solution of the tocopherol homologues.

  16. Effects of Almond- and Olive Oil-Based Docosahexaenoic- and Vitamin E-Enriched Beverage Dietary Supplementation on Inflammation Associated to Exercise and Age.

    PubMed

    Capó, Xavier; Martorell, Miquel; Sureda, Antoni; Riera, Joan; Drobnic, Franchek; Tur, Josep Antoni; Pons, Antoni

    2016-10-09

    n-3-polyunsaturated fatty acids and polyphenols are potential key factors for the treatment and prevention of chronic inflammation associated to ageing and non-communicable diseases. The aim was to analyse effects of an almond and olive oil beverage enriched with α-tocopherol and docosahexaenoic, exercise and age on inflammatory plasma markers, and immune gene expression in peripheral blood mononuclear cells (PBMCs). Five young and five senior athletes who were supplemented for five weeks with a functional beverage performed a stress test under controlled conditions before and after beverage supplementation. Blood samples were taken immediately before and 1 h after each test. Plasma, erythrocytes and PBMCs were isolated. Beverage supplementation increased plasmatic Tumour Necrosis Factor α (TNFα) levels depending on age and exercise. Exercise increased plasma non esterified fatty acids (NEFAs), soluble Intercellular adhesion molecule 3 (sICAM3) and soluble L-selectin (sL-Selectin), and this increase was attenuated by the supplementation. Exercise increased PGE2 plasma levels in supplemented young and in senior placebo athletes. Exercise increased NFkβ-activated levels in PBMCs, which are primed to a pro-inflammatory response increasing pro-inflammatory genes expression after the exercise mainly in the young group after the supplementation. The functional beverage supplementation to young athletes enhances a pro-inflammatory circulating environment in response to the exercise that was less evident in the senior group.

  17. Modulation of CD14 and TLR4.MD-2 activities by a synthetic lipid A mimetic

    PubMed Central

    Cighetti, Roberto; Ciaramelli, Carlotta; Sestito, Stefania Enza; Zanoni, Ivan; Kubik, Łukasz; Ardá-Freire, Ana; Calabrese, Valentina; Granucci, Francesca; Jerala, Roman; Martín-Santamaría, Sonsoles; Jiménez-Barbero, Jesus

    2014-01-01

    Monosaccharide lipid A mimetics composed by a glucosamine core linked to two fatty acid chains and bearing one or two phosphates have been synthesized. While compounds 1 and 2, with one phosphate group, were practically inactive in inhibiting LPS-induced TLR4 signaling and cytokine production in HEK-blue™ cells and murine macrophages, compound 3 with two phosphates was found to be active in efficiently inhibiting TLR4 signal in both cell types. The direct interaction of molecule 3 with MD-2 co-receptor has been investigated by means of NMR and molecular modeling/docking analysis. This compound also interacts directly with CD14 receptor, stimulating its internalization by endocytosis. Experiments on macrophages show that the effect on CD14 reinforces the activity on MD-2.TLR4, because compound 3 activity is higher when CD14 is important for TLR4 signaling i,e, at low LPS concentration. The dual MD-2 and CD14 targeting, accompanied by good solubility in water and lack of toxicity, suggests the use of monosaccharide 3 as a lead compound to develop drugs directed against TLR4-related syndromes. PMID:24339336

  18. Understanding the movements of metal whiskers

    NASA Astrophysics Data System (ADS)

    Karpov, V. G.

    2015-06-01

    Metal whiskers often grow across leads of electric equipment causing short circuits and raising significant reliability issues. Their nature remains a mystery after several decades of research. It was observed that metal whiskers exhibit large amplitude movements under gentle air flow or, according to some testimonies, without obvious stimuli. Understanding the physics behind that movements would give additional insights into the nature of metal whiskers. Here, we quantitatively analyze possible mechanisms of the observed movements: (1) minute air currents; (2) Brownian motion due to random bombardments with the air molecules; (3) mechanically caused movements, such as (a) transmitted external vibrations, and (b) torque exerted due to material propagation along curved whiskers (the garden hose instability); (4) time dependent electric fields due to diffusion of ions; and (5) non-equilibrium electric fields making it possible for some whiskers to move. For all these mechanisms, we provide numerical estimates. Our conclusion is that the observed movements are likely due to the air currents or electric recharging caused by external light or similar factors.

  19. Fundamental frequency from classical molecular dynamics.

    PubMed

    Yamada, Tomonori; Aida, Misako

    2015-02-07

    We give a theoretical validation for calculating fundamental frequencies of a molecule from classical molecular dynamics (MD) when its anharmonicity is small enough to be treated by perturbation theory. We specifically give concrete answers to the following questions: (1) What is the appropriate initial condition of classical MD to calculate the fundamental frequency? (2) From that condition, how accurately can we extract fundamental frequencies of a molecule? (3) What is the benefit of using ab initio MD for frequency calculations? Our analytical approaches to those questions are classical and quantum normal form theories. As numerical examples we perform two types of MD to calculate fundamental frequencies of H2O with MP2/aug-cc-pVTZ: one is based on the quartic force field and the other one is direct ab initio MD, where the potential energies and the gradients are calculated on the fly. From those calculations, we show comparisons of the frequencies from MD with the post vibrational self-consistent field calculations, second- and fourth-order perturbation theories, and experiments. We also apply direct ab initio MD to frequency calculations of C-H vibrational modes of tetracene and naphthalene. We conclude that MD can give the same accuracy in fundamental frequency calculation as second-order perturbation theory but the computational cost is lower for large molecules.

  20. A Close Look at the Structure of the TiO2-APTES Interface in Hybrid Nanomaterials and Its Degradation Pathway: An Experimental and Theoretical Study

    PubMed Central

    2016-01-01

    The surface functionalization of TiO2-based materials with alkylsilanes is attractive in several cutting-edge applications, such as photovoltaics, sensors, and nanocarriers for the controlled release of bioactive molecules. (3-Aminopropyl)triethoxysilane (APTES) is able to self-assemble to form monolayers on TiO2 surfaces, but its adsorption geometry and solar-induced photodegradation pathways are not well understood. We here employ advanced experimental (XPS, NEXAFS, AFM, HR-TEM, and FT-IR) and theoretical (plane-wave DFT) tools to investigate the preferential interaction mode of APTES on anatase TiO2. We demonstrate that monomeric APTES chemisorption should proceed through covalent Si–O–Ti bonds. Although dimerization of the silane through Si–O–Si bonds is possible, further polymerization on the surface is scarcely probable. Terminal amino groups are expected to be partially involved in strong charge-assisted hydrogen bonds with surface hydroxyl groups of TiO2, resulting in a reduced propensity to react with other species. Solar-induced mineralization proceeds through preferential cleavage of the alkyl groups, leading to the rapid loss of the terminal NH2 moieties, whereas the Si-bearing head of APTES undergoes slower oxidation and remains bound to the surface. The suitability of employing the silane as a linker with other chemical species is discussed in the context of controlled degradation of APTES monolayers for drug release and surface patterning. PMID:28191270

  1. Size dependent transition to solid hydrogen and argon clusters probed via spectroscopy of PTCDA embedded in helium nanodroplets

    NASA Astrophysics Data System (ADS)

    Dvorak, Matthieu; Müller, Markus; Bünermann, Oliver; Stienkemeier, Frank

    2014-04-01

    Complexes made of either ArN or (H2)N clusters (N = 1-170) and a single PTCDA molecule (3,4,9,10-perylene-tetracarboxylic-dianhydride) are assembled inside helium droplets and spectroscopically studied via laser-induced fluorescence spectroscopy. The frequency shift and line-broadening are analyzed as a function of N and of the pick-up order of the PTCDA and cluster material in order to track liquid or solid properties of the clusters. For argon, the solid phase is observed for N > 10 above which the pick-up order dramatically influences the localization of the chromophore with respect to the Ar cluster. If the droplets are doped first with Ar, the chromophore remains on the surface of a solid cluster whereas for the reversed pick-up order the molecule is surrounded by an argon shell. At N < 10 wetting and the formation of the first solvation shell are observed. For para-hydrogen, a transition to the solid is observed at N ˜ 20-25, confirming previous theoretical predictions on the existence of a liquid-like phase at such small sizes, even below the bulk hydrogen freezing temperature.

  2. Small molecule AKAP-protein kinase A (PKA) interaction disruptors that activate PKA interfere with compartmentalized cAMP signaling in cardiac myocytes.

    PubMed

    Christian, Frank; Szaszák, Márta; Friedl, Sabine; Drewianka, Stephan; Lorenz, Dorothea; Goncalves, Andrey; Furkert, Jens; Vargas, Carolyn; Schmieder, Peter; Götz, Frank; Zühlke, Kerstin; Moutty, Marie; Göttert, Hendrikje; Joshi, Mangesh; Reif, Bernd; Haase, Hannelore; Morano, Ingo; Grossmann, Solveig; Klukovits, Anna; Verli, Judit; Gáspár, Róbert; Noack, Claudia; Bergmann, Martin; Kass, Robert; Hampel, Kornelia; Kashin, Dmitry; Genieser, Hans-Gottfried; Herberg, Friedrich W; Willoughby, Debbie; Cooper, Dermot M F; Baillie, George S; Houslay, Miles D; von Kries, Jens Peter; Zimmermann, Bastian; Rosenthal, Walter; Klussmann, Enno

    2011-03-18

    A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating β-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.

  3. Host Langerin (CD207) is a receptor for Yersinia pestis phagocytosis and promotes dissemination

    PubMed Central

    Yang, Kun; Park, Chae G; Cheong, Cheolho; Bulgheresi, Silvia; Zhang, Shusheng; Zhang, Pei; He, Yingxia; Jiang, Lingyu; Huang, Hongping; Ding, Honghui; Wu, Yiping; Wang, Shaogang; Zhang, Lin; Li, Anyi; Xia, Lianxu; Bartra, Sara S; Plano, Gregory V; Skurnik, Mikael; Klena, John D; Chen, Tie

    2015-01-01

    Yersinia pestis is a Gram-negative bacterium that causes plague. After Y. pestis overcomes the skin barrier, it encounters antigen-presenting cells (APCs), such as Langerhans and dendritic cells. They transport the bacteria from the skin to the lymph nodes. However, the molecular mechanisms involved in bacterial transmission are unclear. Langerhans cells (LCs) express Langerin (CD207), a calcium-dependent (C-type) lectin. Furthermore, Y. pestis possesses exposed core oligosaccharides. In this study, we show that Y. pestis invades LCs and Langerin-expressing transfectants. However, when the bacterial core oligosaccharides are shielded or truncated, Y. pestis propensity to invade Langerhans and Langerin-expressing cells decreases. Moreover, the interaction of Y. pestis with Langerin-expressing transfectants is inhibited by purified Langerin, a DC-SIGN (DC-specific intercellular adhesion molecule 3 grabbing nonintegrin)-like molecule, an anti-CD207 antibody, purified core oligosaccharides and several oligosaccharides. Furthermore, covering core oligosaccharides reduces the mortality associated with murine infection by adversely affecting the transmission of Y. pestis to lymph nodes. These results demonstrate that direct interaction of core oligosaccharides with Langerin facilitates the invasion of LCs by Y. pestis. Therefore, Langerin-mediated binding of Y. pestis to APCs may promote its dissemination and infection. PMID:25829141

  4. On-tissue chemical derivatization of 3-methoxysalicylamine for MALDI-imaging mass spectrometry

    PubMed Central

    Chacon, Almary; Zagol-Ikapitte, Irene; Amarnath, Venkataraman; Reyzer, Michelle L.; Oates, John A.; Caprioli, Richard M.; Boutaud, Olivier

    2011-01-01

    MALDI-imaging mass spectrometry (IMS) has been shown to be a powerful tool to study drug distributions in organ tissue as well as whole animal bodies. Nevertheless, not all drugs are amenable to MALDI while others may be limited by poor sensitivity poor sensitivity. The use of chemical derivatization to improve detection of small molecules by mass spectrometry techniques is well documented. To our knowledge, however, this approach has not been applied to direct tissue analysis of small organic molecules. In this manuscript, we demonstrate the use of on-tissue chemical derivatization of a small organic molecule, 3-methoxysalicylamine (3-MoSA) a scavenger of γ -ketoaldehydes. Derivatization of 3-MoSA with 1,1′-thiocarbonyldiimidazole (TCDI) results in an oxothiazolidine derivative which is detected with much greater sensitivity by MALDI than 3-MoSA itself. TCDI treatment of tissue from mice dosed with 3-MoSA allowed images to be obtained showing its spatial distribution as well as its pharmacokinetic profile in different organs. These images correlated well with results obtained from HPLC-MS/MS analyses of the same tissues. These results provide proof-of-concept that on-tissue chemical derivatization can be used to improve detection of a small organic molecule by MALDI-IMS. PMID:21834023

  5. Host-guest interaction of 3-hydroxyflavone and 7-hydroxyflavone with cucurbit [7]uril: A spectroscopic and calorimetric approach.

    PubMed

    Ahmed, Sayeed Ashique; Maity, Banibrata; Duley, Soma Seth; Seth, Debabrata

    2017-03-01

    The modulation of photophysical behaviour of small organic molecules in the presence of macrocycles is one of the most interesting areas of research. In this work we reported the interaction of two biologically active molecules 3-hydroxyflavone and 7-hydroxyflavone with macrocyclic host cucurbit [7]uril in aqueous medium. To investigate the change of photophysical properties of these two flavones, we have used steady state absorption, fluorescence, time resolved fluorescence emission spectroscopy and isothermal titration calorimetric technique. It is observed that on complexation with cucurbit [7]uril, the excited state proton transfer processes in both flavones have been facilitated. Isothermal titration calorimetric method was used in order to investigate the involvement of thermodynamic parameters in complexation between flavone with cucurbit [7]uril. The changes in thermodynamic properties due to the complexation of the flavones molecules with cucurbit [7]urils help to understand about the governing parameters involved in this complexation. The inclusion of flavone molecules inside the cavity of cucurbit [7]uril molecules was studied theoretically to decipher the molecular orientation of flavones in the presence of cucurbit [7]uril. The structure of HOMO and LUMO of the complexes between cucurbit [7]uril with flavones was reported. This study will be helpful to get the knowledge about the modulation of photophysical properties of the flavones molecules on addition of macrocyclic host cucurbit [7]uril. This study will be helpful for the use of cucurbit [7]uril as a potential drug delivery system.

  6. A comparison of force fields and calculation methods for vibration intervals of isotopic H3(+) molecules

    NASA Astrophysics Data System (ADS)

    Carney, G. D.; Adler-Golden, S. M.; Lesseski, D. C.

    1986-04-01

    This paper reports (1) improved values for low-lying vibration intervals of H3(+), H2D(+), D2H(+), and D3(+) calculated using the variational method and Simons-Parr-Finlan (1973) representations of the Carney-Porter (1976) and Dykstra-Swope (1979) ab initio H3(+) potential energy surfaces, (2) quartic normal coordinate force fields for isotopic H3(+) molecules, (3) comparisons of variational and second-order perturbation theory, and (4) convergence properties of the Lai-Hagstrom internal coordinate vibrational Hamiltonian. Standard deviations between experimental and ab initio fundamental vibration intervals of H3(+), H2D(+), D2H(+), and D3(+) for these potential surfaces are 6.9 (Carney-Porter) and 1.2/cm (Dykstra-Swope). The standard deviations between perturbation theory and exact variational fundamentals are 5 and 10/cm for the respective surfaces. The internal coordinate Hamiltonian is found to be less efficient than the previously employed 't' coordinate Hamiltonian for these molecules, except in the case of H2D(+).

  7. Structure-property relationship of 3-(N-phthalimidomethyl)-4-amino-1,2,4-triazole-5-thione: A structural, spectroscopic and DFT study

    NASA Astrophysics Data System (ADS)

    Tamer, Ömer; Bhatti, Moazzam H.; Yunus, Uzma; Nadeem, Muhammad; Avcı, Davut; Atalay, Yusuf; Yaqub, Azra; Quershi, Rumana

    2017-04-01

    The title molecule, 3-(N-phthalimidomethyl)-4-amino-1,2,4-triazole-5-thione (C11H9N5O2S), was synthesized by the fusion of N-Phthaloylglycine and thiocarbohydrazide at 145 °C. In this study, we have investigated the crystal structure, photophysical properties as well as the relation between the molecular structure and nonlinear optical properties of 2-(4-Amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-ylmethyl)isoindoline-1,3-dione. For this purpose, the molecular structure, vibration spectrum, electronic absorption spectrum, 1H and 13C NMR spectra have been evaluated by both of the experimental techniques and density functional theory method. A detailed assignment of vibrational bands has been performed on the basis of potential energy distribution analysis. Additionally, UV-Vis spectrum was recorded in different solvents in order to examine the solvent effect on the electronic absorption spectrum. NBO analysis has been carried out to investigate intra-molecular charge transfer interactions. Finally, nonlinear optical properties of the title compound have been investigated by using M062X level of density functional theory.

  8. Synthesis, photophysical and electrochemical properties of two novel carbazole-based dye molecules

    NASA Astrophysics Data System (ADS)

    Zhang, Qing; Zhu, Weiju; Fang, Min; Yin, Fangfang; Li, Cun

    2015-01-01

    Two carbazole-based dye molecules: 3-(6-benzothiazol-2-yl-9H-hexylcarbazole-3-yl)-2-cyano-acylic acid (D3) and 3-[5-(6-benzothiazol-2-yl-9H-hexylcarbazole-3-yl)-thiophen-2-yl]-2-cyan-acylic acid (D4) were synthesized by an approach from carbazole derivate using Vilsmeier-Haack, Suzuki cross-coupling and Knoevenagel reactions. Their physical and electrochemical properties were investigated. D3 and D4 exhibit different optical properties, such as UV absorption, photoluminescence, fluorescence quantum yield and fluorescence lifetime in different solvents. Compared with D3 without a thiophene unit, the maximum absorption wavelength of D4 red-shift obviously and its fluorescence intensity is also enhanced. A shift of the EHOMO and ELUMO is observed for D3 (EHOMO = 2.06 V, ELUMO = -1.39 V vs. NHE) and D4 (EHOMO = 1.73 V, ELUMO = -1.33 V vs. NHE). D3 and D4 can be used as dyes for dye-sensitized solar cells (DSSCs) with TiO2 nanomaterial because their EHOMO are lower than the conduction band edge of TiO2 [-0.5 V (vs. NHE)] and their ELUMO are higher than the I3-/I- redox potential [0.42 V (vs. NHE)].

  9. Histology-directed and imaging mass spectrometry: an emerging technology in ectopic calcification

    PubMed Central

    De Santis, Giorgio; Caprioli, Richard M; Quaglino, Daniela

    2015-01-01

    The present study was designed to demonstrate the potential of an optimized histology directed protein identification combined with imaging mass spectrometry technology to reveal and identify molecules associated to ectopic calcification in human tissue. As a proof of concept, mineralized and non-mineralized areas were compared within the same dermal tissue obtained from a patient affected by Pseudoxanthoma elasticum, a genetic disorder characterized by calcification only at specific sites of soft connective tissues. Data have been technically validated on a contralateral dermal tissue from the same subject and compared with those from control healthy skin. Results demonstrate that this approach 1) significantly reduces the effects generated by techniques that, disrupting tissue organization, blend data from affected and unaffected areas; 2) demonstrates that, abolishing differences due to inter-individual variability, mineralized and non-mineralized areas within the same sample have a specific protein profile and have a different distribution of molecules; 3) avoiding the bias of focusing on already known molecules, reveals a number of proteins that have been never related to the disease nor to the calcification process, thus paving the way for the selection of new molecules to be validated as pathogenic or as potential pharmacological targets. PMID:25595835

  10. S-layer proteins from Lactobacillus sp. inhibit bacterial infection by blockage of DC-SIGN cell receptor.

    PubMed

    Prado Acosta, Mariano; Ruzal, Sandra M; Cordo, Sandra M

    2016-11-01

    Many species of Lactobacillus sp. possess Surface(s) layer proteins in their envelope. Among other important characteristics S-layer from Lactobacillus acidophilus binds to the cellular receptor DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin; CD209), which is involved in adhesion and infection of several families of bacteria. In this report we investigate the activity of new S-layer proteins from the Lactobacillus family (Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus helveticus and Lactobacillus kefiri) over the infection of representative microorganisms important to human health. After the treatment of DC-SIGN expressing cells with these proteins, we were able to diminish bacterial infection by up to 79% in both gram negative and mycobacterial models. We discovered that pre-treatment of the bacteria with S-layers from Lactobacillus acidophilus and Lactobacillus brevis reduced bacteria viability but also prevent infection by the pathogenic bacteria. We also proved the importance of the glycosylation of the S-layer from Lactobacillus kefiri in the binding to the receptor and thus inhibition of infection. This novel characteristic of the S-layers proteins may contribute to the already reported pathogen exclusion activity for these Lactobacillus probiotic strains; and might be also considered as a novel enzymatic antimicrobial agents to inhibit bacterial infection and entry to host cells.

  11. 3-O-Hydroxytyrosol glucuronide and 4-O-hydroxytyrosol glucuronide reduce endoplasmic reticulum stress in vitro.

    PubMed

    Giordano, Elena; Dangles, Olivier; Rakotomanomana, Njara; Baracchini, Silvia; Visioli, Francesco

    2015-10-01

    Endoplasmic reticulum (ER) stress is important for atherosclerosis development and is mediated by the unfolded protein response (UPR). In this work, we synthesized two among the most physiologically-prominent hydroxytyrosol HT hepatic metabolites, i.e. 3-O-HT glucuronide and 4-O-HT glucuronide and we tested their activities on ER stress (in human hepatocarcinoma HepG2 cells), to gain further insight into the cardiopreventive properties of HT, extra virgin olive oil, and the Mediterranean diet. We report that 3-O-HT glucuronide and 4-O-HT glucuronide inhibit tunicamycin-induced ER stress. As compared with the effects of the parent molecule, 3-O-HT glucuronide and 4-O-HT glucuronide at 10 μM and 25 μM alone induced a milder change in mRNA expression levels of both CCAAT-enhancer-binding protein homologous protein (CHOP) and glucose regulated protein GRP78 immunoglobulin heavy chain binding protein (BiP). In conclusion, we add further evidence to the hypothesis that the HT intake might be atheroprotective and reiterate the usefulness to preferably use high-quality, high-(poly)phenol extra virgin olive oil as a prominent condiment.

  12. Exploring the possibility to store the mixed oxygen-hydrogen cluster in clathrate hydrate in molar ratio 1:2 (O2+2H2).

    PubMed

    Qin, Yan; Du, Qi-Shi; Xie, Neng-Zhong; Li, Jian-Xiu; Huang, Ri-Bo

    2017-02-01

    An interesting possibility is explored: storing the mixture of oxygen and hydrogen in clathrate hydrate in molar ratio 1:2. The interaction energies between oxygen, hydrogen, and clathrate hydrate are calculated using high level quantum chemical methods. The useful conclusion points from this study are summarized as follows. (1) The interaction energies of oxygen-hydrogen mixed cluster are larger than the energies of pure hydrogen molecular cluster. (2) The affinity of oxygen molecules with water molecules is larger than that of the hydrogen molecules with water molecules. (3) The dimension of O2-2H2 interaction structure is smaller than the dimension of CO2-2H2 interaction structure. (4) The escaping energy of oxygen molecules from the hydrate cell is larger than that of the hydrogen molecules. (5) The high affinity of the oxygen molecules with both the water molecules and the hydrogen molecules may promote the stability of oxygen-hydrogen mixture in the clathrate hydrate. Therefore it is possible to store the mixed (O2+2H2) cluster in clathrate hydrate.

  13. Molecular orbital theory of ballistic electron transport through molecules

    NASA Astrophysics Data System (ADS)

    Ernzerhof, Matthias; Rocheleau, Philippe; Goyer, Francois

    2009-03-01

    Electron transport through molecules occurs, for instance, in STM imaging and in conductance measurements on molecular electronic devices (MEDs). To model these phenomena, we use a non-Hermitian model Hamiltonian [1] for the description of open systems that exchange current density with their environment. We derive qualitative, molecular-orbital-based rules relating molecular structure and conductance. We show how side groups attached to molecular conductors [2] can completely suppress the conductance. We discuss interference effects in aromatic molecules [3] that can also inhibit electron transport. Rules are developed [1] for the prediction of Fano resonances. All these phenomena are explained with a molecular orbital theory [1,4] for molecules attached to macroscopic reservoirs. [1] F. Goyer, M. Ernzerhof, and M. Zhuang, JCP 126, 144104 (2007); M. Ernzerhof, JCP 127, 204709 (2007). [2] M. Ernzerhof, M. Zhuang, and P. Rocheleau, JCP 123, 134704 (2005); G. C. Solomon, D Q. Andrews, R P. Van Duyne, and M A. Ratner, JACS 130, 7788 (2008). [3] M. Ernzerhof, H. Bahmann, F. Goyer, M. Zhuang, and P. Rocheleau, JCTC 2, 1291 (2006); G. C. Solomon, D. Q. Andrews, R. P. Van Duyne, and M. A. Ratner, JCP 129, 054701 (2008). [4] B.T. Pickup, P.W. Fowler, CPL 459, 198 (2008); P. Rocheleau and M. Ernzerhof, JCP, submitted.

  14. Molecular structure, vibrational spectra, natural bond orbital and thermodynamic analysis of 3,6-dichloro-4-methylpyridazine and 3,6-dichloropyridazine-4-carboxylic acid by dft approach.

    PubMed

    Prabavathi, N; Senthil Nayaki, N; Venkatram Reddy, B

    2015-02-05

    Vibrational spectral analysis of the molecules 3,6-dichloro-4-methylpyridazine (DMP) and 3,6-dichloropyridazine-4-carboxylic acid (DPC) was carried out using FT-IR and FT-Raman spectroscopic techniques. The molecular structure and vibrational spectra of DMP and DPC were obtained by the density functional theory (DFT) method, using B3LYP functional, with 6-311++G(d,p) basis set. A detailed interpretation of the Infrared and Raman spectra of the two molecules were reported based on potential energy distribution (PED). The theoretically predicted FTIR and FT-Raman spectra of the titled molecules have been simulated and were compared with the experimental spectra. Determination of electric dipole moment (μ) and hyperpolarizability β0 helps to study the non-linear optical (NLO) behavior of DMP and DPC. Stability of the molecules arising from hyperconjugative interactions, charge delocalization have been analyzed using natural bond orbital (NBO) analysis. (13)C and (1)H NMR spectra were recorded and (13)C and (1)H NMR chemical shifts of the molecules were calculated using the gauge independent atomic orbital (GIAO) method. UV-visible spectrum of the compounds was also recorded in the region 200-1100 nm and electronic properties, HOMO (Highest Occupied Molecular Orbitals) and LUMO (Lowest Unoccupied Molecular Orbitals) energies were measured by time-dependent TD-DFT approach. Charge density distribution and site of chemical reactivity of the molecule have been studied by mapping electron density isosurface with molecular electrostatic potential (MESP).

  15. Comprehensive Map of Molecules Implicated in Obesity

    PubMed Central

    Agrawal, Stuti

    2016-01-01

    Obesity is a global epidemic affecting over 1.5 billion people and is one of the risk factors for several diseases such as type 2 diabetes mellitus and hypertension. We have constructed a comprehensive map of the molecules reported to be implicated in obesity. A deep curation strategy was complemented by a novel semi-automated text mining system in order to screen 1,000 full-length research articles and over 90,000 abstracts that are relevant to obesity. We obtain a scale free network of 804 nodes and 971 edges, composed of 510 proteins, 115 genes, 62 complexes, 23 RNA molecules, 83 simple molecules, 3 phenotype and 3 drugs in “bow-tie” architecture. We classify this network into 5 modules and identify new links between the recently discovered fat mass and obesity associated FTO gene with well studied examples such as insulin and leptin. We further built an automated docking pipeline to dock orlistat as well as other drugs against the 24,000 proteins in the human structural proteome to explain the therapeutics and side effects at a network level. Based upon our experiments, we propose that therapeutic effect comes through the binding of one drug with several molecules in target network, and the binding propensity is both statistically significant and different in comparison with any other part of human structural proteome. PMID:26886906

  16. Hydroxy Cinnamic Acid Derivatives as Partial PPARγ agonists: In Silico Studies, Synthesis and Biological Characterization Against Chronic Myeloid Leukemia Cell Line (K562).

    PubMed

    Joshi, Hardik; Marulkar, Kavita; Gota, Vikram; Ramaa, C S

    2016-06-06

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor that regulates the expression of many genes relevant to carcinogenesis. By analogy to selective estrogen receptor modulator for treatment of cancer, selective or partial PPARγ agonists are considered clinically important for chemotherapy of cancer. A series of p-coumaric (3a-3y) and ferulic acid (4a-4y) derivatives were designed and docked and virtually studied for their molecular properties. Synthesized derivatives were assessed to check their effect on non-transformed hepatocytes and further evaluated for their anti-proliferative potential on K562. Molecules 3c, 3m, 4c and 4m were found to have GI50 value less than 50μM. These molecules were found to block G0/G1 phase of cell cycle in dose dependent manner. Western blot analysis revealed that these molecules inhibit proliferating cell nuclear antigen (PCNA) and cyclin D1 expression. Collectively, these results suggest that these molecules could play a role as a novel therapeutic strategy for chronic myeloid leukemia.

  17. Chiral Sensitivity in the Dissociative Electron Attachment of Halocamphor Molecules

    NASA Astrophysics Data System (ADS)

    Dreiling, Joan

    2016-05-01

    We have demonstrated chirally-dependent molecular destruction when incident longitudinally-spin-polarized (chiral) electrons break bonds in chiral molecules. This chiral sensitivity was observed through an asymmetry in the dissociative electron attachment (DEA) reaction rate with chiral 3-bromocamphor (C10 H15 BrO). Such an observation provides an unambiguous demonstration of the idea underlying the Vester-Ulbricht hypothesis, which attempts to explain the origins of the homochirality that is observed in many biological systems. While the lack of inversion symmetry in these reactions allows the effects we observe to occur, their dynamic causes are poorly understood. We have further studied the asymmetries in the DEA rates for two additional halocamphor molecules, 3-iodocamphor (C10 H15 IO) and 10-iodocamphor, in a systematic effort to illuminate the mechanisms responsible for the chiral sensitivity. The DEA signal depends on the sign of the incident electron helicity for a given target handedness in all molecules, and it varies with both the atomic number and the location of the heaviest atom in the molecule. Surprisingly, the DEA asymmetries for 10-iodocamphor, in which the heaviest atom is farther from a chiral center than for the other molecules, produced the largest asymmetries. This work was performed at the University of Nebraska-Lincoln. This project was funded by NSF Grant PHY-1206067.

  18. Glycoprotein B7-H3 overexpression and aberrant glycosylation in oral cancer and immune response

    PubMed Central

    Chen, Jung-Tsu; Chen, Chein-Hung; Ku, Ko-Li; Hsiao, Michael; Chiang, Chun-Pin; Hsu, Tsui-Ling; Chen, Min-Huey; Wong, Chi-Huey

    2015-01-01

    The incidence and mortality rate of oral cancer continue to rise, partly due to the lack of effective early diagnosis and increasing environmental exposure to cancer-causing agents. To identify new markers for oral cancer, we used a sialylation probe to investigate the glycoproteins differentially expressed on oral cancer cells. Of the glycoproteins identified, B7 Homolog 3 (B7-H3) was significantly overexpressed in oral squamous cell carcinoma (OSCC), and its overexpression correlated with larger tumor size, advanced clinical stage, and low survival rate in OSCC patients. In addition, knockdown of B7-H3 suppressed tumor cell proliferation, and restoration of B7-H3 expression enhanced tumor growth. It was also found that the N-glycans of B7-H3 from Ca9-22 oral cancer cells contain the terminal α-galactose and are more diverse with higher fucosylation and better interaction with DC-SIGN [DC-specific intercellular adhesion molecule-3 (ICAM-3)–grabbing nonintegrin] and Langerin on immune cells than that from normal cells, suggesting that the glycans on B7-H3 may also play an important role in the disease. PMID:26438868

  19. Electrochemical serotonin monitoring of poly(ethylenedioxythiophene):poly(sodium 4-styrenesulfonate)-modified fluorine-doped tin oxide by predeposition of self-assembled 4-pyridylporphyrin.

    PubMed

    Song, Min-Jung; Kim, Sangsig; Ki Min, Nam; Jin, Joon-Hyung

    2014-02-15

    A 5,10,15,20-tetrakis(4-pyridyl)-21H,23H-porphyrin (TPyP)-modified self-assembled functional layer was prepared on a fluorine-doped tin oxide (FTO) substrate. We employed a bifunctional molecule, 3-iodopropionate (3IP), to covalently bind TPyP to the FTO substrate. The 3IP-monolayered FTO and the TPyP-3IP-bilayered FTO electrodes were characterized by cyclic voltammetry, electrochemical impedance spectroscopy, and Fourier transform-infrared spectroscopy. Compared to conventional electropolymerized poly(ethylenedioxythiophene):poly(sodium 4-styrenesulfonate) (PEDOT:PSS) film on bare FTO, the PEDOT:PSS film on the TPyP-3IP-bilayered FTO showed better sensitivity and selectivity in monitoring serotonin in the presence of high concentrations of interfering agents such as ascorbic acid, urea, D-(+)-glucose, epinephrine, and L-3,4-dihydroxyphenylalanine. Both PEDOT:PSS films on the bare FTO and the TPyP-3IP-bilayered FTO showed electrocatalytic effects in serotonin detection, and only the TPyP-3IP-based PEDOT:PSS film acted as a pH resistant buffer layer in the selective detection of serotonin.

  20. Dendritic cells respond to nasopharygeal carcinoma cells through annexin A2-recognizing DC-SIGN.

    PubMed

    Chao, Pin-Zhir; Hsieh, Ming-Shium; Cheng, Chao-Wen; Hsu, Tin-Jui; Lin, Yun-Tien; Lai, Chang-Hao; Liao, Chen-Chung; Chen, Wei-Yu; Leung, Ting-Kai; Lee, Fei-Peng; Lin, Yung-Feng; Chen, Chien-Ho

    2015-01-01

    Dendritic cells (DCs) play an essential role in immunity and are used in cancer immunotherapy. However, these cells can be tuned by tumors with immunosuppressive responses. DC-specific intercellular adhesion molecule 3-Grabbing Nonintegrin (DC-SIGN), a C-type lectin expressed on DCs, recognizes certain carbohydrate structures which can be found on cancer cells. Nasopharyngeal carcinoma (NPC) is an epithelial cell-derived malignant tumor, in which immune response remains unclear. This research is to reveal the molecular link on NPC cells that induces the immunosuppressive responses in DCs. In this article, we report identification of annexin A2 (ANXA2) on NPC cells as a ligand for DC-SIGN on DCs. N-linked mannose-rich glycan on ANXA2 may mediate the interaction. ANXA2 was abundantly expressed in NPC, and knockdown of ANXA2 suppressed NPC xenograft in mice, suggesting a crucial role of ANXA2 in NPC growth. Interaction with NPC cells caused DC-SIGN activation in DCs. Consequently DC maturation and the proinflammatory interleukin (IL)-12 production were inhibited, and the immunosuppressive IL-10 production was promoted. Blockage of either DC-SIGN or ANXA2 eliminated the production of IL-10 from DCs. This report suggests that suppression of ANXA2 at its expression or glycosylation on NPC may improve DC-mediated immunotherapy for the tumor.

  1. Monovalent mannose-based DC-SIGN antagonists: targeting the hydrophobic groove of the receptor.

    PubMed

    Tomašić, Tihomir; Hajšek, David; Švajger, Urban; Luzar, Jernej; Obermajer, Nataša; Petit-Haertlein, Isabelle; Fieschi, Franck; Anderluh, Marko

    2014-03-21

    Dendritic cell-specific, intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) is a C-type lectin expressed specifically on dendritic cells. It is a primary site for recognition and binding of various pathogens and thus a promising therapeutic target for inhibition of pathogen entry and subsequent prevention of immune defense cell infection. We report the design and synthesis of d-mannose-based DC-SIGN antagonists bearing diaryl substituted 1,3-diaminopropanol or glycerol moieties incorporated to target the hydrophobic groove of the receptor. The designed glycomimetics were evaluated by in vitro assay of the isolated DC-SIGN extracellular domain for their ability to compete with HIV-1 gp120 for binding to the DC-SIGN carbohydrate recognition domain. Compounds 14d and 14e, that display IC50 values of 40 μM and 50 μM, are among the most potent monovalent DC-SIGN antagonists reported. The antagonistic effect of all the synthesized compounds was further evaluated by a one-point in vitro assay that measures DC adhesion. Compounds 14d, 14e, 18d and 18e were shown to act as functional antagonists of DC-SIGN-mediated DC adhesion. The binding mode of 14d was also studied by molecular docking and molecular dynamics simulation, which revealed flexibility of 14d in the binding site and provides a basis for further optimization.

  2. Beyond attachment: Roles of DC-SIGN in dengue virus infection.

    PubMed

    Liu, Ping; Ridilla, Marc; Patel, Pratik; Betts, Laurie; Gallichotte, Emily; Shahidi, Lidea; Thompson, Nancy L; Jacobson, Ken

    2017-04-01

    Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), a C-type lectin expressed on the plasma membrane by human immature dendritic cells, is a receptor for numerous viruses including Ebola, SARS and dengue. A controversial question has been whether DC-SIGN functions as a complete receptor for both binding and internalization of dengue virus (DENV) or whether it is solely a cell surface attachment factor, requiring either hand-off to another receptor or a co-receptor for internalization. To examine this question, we used 4 cell types: human immature dendritic cells and NIH3T3 cells expressing either wild-type DC-SIGN or 2 internalization-deficient DC-SIGN mutants, in which either the 3 cytoplasmic internalization motifs are silenced by alanine substitutions or the cytoplasmic region is truncated. Using confocal and super-resolution imaging and high content single particle tracking, we investigated DENV binding, DC-SIGN surface transport, endocytosis, as well as cell infectivity. DC-SIGN was found colocalized with DENV inside cells suggesting hand-off at the plasma membrane to another receptor did not occur. Moreover, all 3 DC-SIGN molecules on NIH3T3 cells supported cell infection. These results imply the involvement of a co-receptor because cells expressing the internalization-deficient mutants could still be infected.

  3. Distinct usage of three C-type lectins by Japanese encephalitis virus: DC-SIGN, DC-SIGNR, and LSECtin.

    PubMed

    Shimojima, Masayuki; Takenouchi, Atsushi; Shimoda, Hiroshi; Kimura, Naho; Maeda, Ken

    2014-08-01

    Infection with West Nile virus and dengue virus, two mosquito-borne flaviviruses, is enhanced by two calcium-dependent lectins: dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), and its related molecule (DC-SIGNR). The present study examined the relationship between Japanese encephalitis virus (JEV) infection and three lectins: DC-SIGN, DC-SIGNR, and liver sinusoidal endothelial cell lectin (LSECtin). Expression of DC-SIGNR resulted in robust JEV proliferation in a lymphoid cell line, Daudi cells, which was otherwise non-permissive to infection. DC-SIGN expression caused moderate JEV proliferation, with effects that varied according to the cells in which JEV was prepared. LSECtin expression had comparatively minor, but consistent, effects, in all cell types used in JEV preparation. While DC-SIGN/DC-SIGNR-mediated JEV infection was inhibited by yeast mannan, LSECtin-mediated infection was inhibited by N-acetylglucosamine β1-2 mannose. Although involvement of DC-SIGN/DC-SIGNR in infection seems to be a common characteristic, this is the first report on usage of LSECtin in mosquito-borne flavivirus infection.

  4. Synthesis and microarray-assisted binding studies of core xylose and fucose containing N-glycans.

    PubMed

    Brzezicka, Katarzyna; Echeverria, Begoña; Serna, Sonia; van Diepen, Angela; Hokke, Cornelis H; Reichardt, Niels-Christian

    2015-05-15

    The synthesis of a collection of 33 xylosylated and core-fucosylated N-glycans found only in nonmammalian organisms such as plants and parasitic helminths has been achieved by employing a highly convergent chemo-enzymatic approach. The influence of these core modifications on the interaction with plant lectins, with the human lectin DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Nonintegrin), and with serum antibodies from schistosome-infected individuals was studied. Core xylosylation markedly reduced or completely abolished binding to several mannose-binding plant lectins and to DC-SIGN, a C-type lectin receptor present on antigen presenting cells. Employing the synthetic collection of core-fucosylated and core-xylosylated N-glycans in the context of a larger glycan array including structures lacking these core modifications, we were able to dissect core xylose and core fucose specific antiglycan antibody responses in S. mansoni infection sera, and we observed clear and immunologically relevant differences between children and adult groups infected with this parasite. The work presented here suggests that, quite similar to bisecting N-acetylglucosamine, core xylose distorts the conformation of the unsubstituted glycan, with important implications for the immunogenicity and protein binding properties of complex N-glycans.

  5. High-Performance Silver Window Electrodes for Top-Illuminated Organic Photovoltaics Using an Organo-molybdenum Oxide Bronze Interlayer.

    PubMed

    Tyler, Martin S; Walker, Marc; Hatton, Ross A

    2016-05-18

    We report an organo-molybdenumn oxide bronze that enables the fabrication of high-performance silver window electrodes for top-illuminated solution processed organic photovoltaics without complicating the process of device fabrication. This hybrid material combines the function of wide-band-gap interlayer for efficient hole extraction with the role of metal electrode seed layer, enabling the fabrication of highly transparent, low-sheet-resistance silver window electrodes. Additionally it is also processed from ethanol, which ensures orthogonality with a large range of solution processed organic semiconductors. The key organic component is the low cost small molecule 3-mercaptopropionic acid, which (i) promotes metal film formation and imparts robustness at low metal thickness, (ii) reduces the contact resistance at the Ag/molybdenumn oxide bronze interface, (iii) and greatly improves the film forming properties. Silver electrodes with a thickness of 8 nm deposited by simple vacuum evaporation onto this hybrid interlayer have a sheet resistance as low as 9.7 Ohms per square and mean transparency ∼80% over the wavelength range 400-900 nm without the aid of an antireflecting layer, which makes them well-matched to the needs of organic photovoltaics and applicable to perovskite photovoltaics. The application of this hybrid material is demonstrated in two types of top-illuminated organic photovoltaic devices.

  6. Effects of Almond- and Olive Oil-Based Docosahexaenoic- and Vitamin E-Enriched Beverage Dietary Supplementation on Inflammation Associated to Exercise and Age

    PubMed Central

    Capó, Xavier; Martorell, Miquel; Sureda, Antoni; Riera, Joan; Drobnic, Franchek; Tur, Josep Antoni; Pons, Antoni

    2016-01-01

    n-3-polyunsaturated fatty acids and polyphenols are potential key factors for the treatment and prevention of chronic inflammation associated to ageing and non-communicable diseases. The aim was to analyse effects of an almond and olive oil beverage enriched with α-tocopherol and docosahexaenoic, exercise and age on inflammatory plasma markers, and immune gene expression in peripheral blood mononuclear cells (PBMCs). Five young and five senior athletes who were supplemented for five weeks with a functional beverage performed a stress test under controlled conditions before and after beverage supplementation. Blood samples were taken immediately before and 1 h after each test. Plasma, erythrocytes and PBMCs were isolated. Beverage supplementation increased plasmatic Tumour Necrosis Factor α (TNFα) levels depending on age and exercise. Exercise increased plasma non esterified fatty acids (NEFAs), soluble Intercellular adhesion molecule 3 (sICAM3) and soluble L-selectin (sL-Selectin), and this increase was attenuated by the supplementation. Exercise increased PGE2 plasma levels in supplemented young and in senior placebo athletes. Exercise increased NFkβ-activated levels in PBMCs, which are primed to a pro-inflammatory response increasing pro-inflammatory genes expression after the exercise mainly in the young group after the supplementation. The functional beverage supplementation to young athletes enhances a pro-inflammatory circulating environment in response to the exercise that was less evident in the senior group. PMID:27735833

  7. Near-Field CARS with Micro- and Nano-Particle

    NASA Astrophysics Data System (ADS)

    Ooi, C. H. Raymond

    2010-08-01

    Spatial dependence of coherent anti-Stokes Raman scattering (CARS) intensity and spectra for a spherical particle are studied for different sizes, ranging from micrometers to nanometers. Effects of near field on the spectra are analyzed, showing potential application as nano-sensor in microscopy and imaging. The results can be extended to an array of nanospheres. The CARS process has been developed into a versatile real-time detection technique in spectroscopy and microscopy [1]. In particularly, backscattered ultra-violet CARS implemented on LIDAR system [2] is promising for remote detection of molecular species present in hazardous biological aerosols with microscale dimension. In practice, the aerosols could be in any dimension. Thus, we need to know study a modified the setup of the CARS technique for reliable detection of chemicals in micro- and nano-particles using near-field effects. We have developed a nonlinear semiclassical microscopic theory to describe the CARS spectra for a particle composed of a collection of arbitrarily complex molecules [3] as well as simple few levels quantum systems [2]. The theory provides useful results on the CARS spectra for any observation angle and for any form of laser pulses [3]. Here, we focus on the spectra in the near field. We wish to study how the spectra vary with the near field distance with focused laser pulses. We also analyze to what extend the dimension of the particle and the focusing laser affect the lensing effect which could enhance the backscattered light.

  8. Two types of photoluminescence blinking revealed by single quantum dot spectroelectrochemistry

    PubMed Central

    Galland, Christophe; Ghosh, Yagnaseni; Steinbrück, Andrea; Sykora, Milan; Hollingsworth, Jennifer A.; Klimov, Victor I.; Htoon, Han

    2012-01-01

    Photoluminescence (PL) intermittency (blinking), or random switching between states of high- (ON) and low (OFF) emissivities, is a universal property of molecular emitters exhibited by dyes1, polymers2, biological molecules3 and artificial nanostructures such as nanocrystal quantum dots, carbon nanotubes, and nanowires4,5,6. For the past fifteen years, colloidal nanocrystals have been used as a model system for studies of this phenomenon.5,6 The occurrence of OFF periods in nanocrystal emission has been commonly attributed to the presence of an additional charge7, which leads to PL quenching by nonradiative Auger recombination.8 However, the “charging” model was recently challenged in several reports.9,10 Here, to clarify the role of charging in PL intermittency, we perform time-resolved PL studies of individual nanocrystals while controlling electrochemically the degree of their charging. We find that two distinct mechanisms can lead to PL intermittency. We identify conventional blinking (A-type) due to charging/discharging of the nanocrystal core when lower PL intensities correlate with shorter PL lifetimes. Importantly, we observe a different blinking (B-type), when large changes in the PL intensity are not accompanied by significant changes in PL dynamics. We attribute this blinking behavior to charge fluctuations in the electron-accepting surface sites. When unoccupied, these sites intercept hot electrons before they relax into emitting core states. Both blinking mechanisms can be controlled electrochemically and under appropriate potential blinking can be completely suppressed. PMID:22071764

  9. Structure of dimethylphenyl betaine hydrochloride studied by X-ray diffraction, DFT calculation, NMR and FTIR spectra

    NASA Astrophysics Data System (ADS)

    Szafran, M.; Katrusiak, A.; Dega-Szafran, Z.; Kowalczyk, I.

    2013-01-01

    The structure of dimethylphenyl betaine hydrochloride (1) has been studied by X-ray diffraction, DFT calculations, NMR and FTIR spectra. The crystals are monoclinic, space group P21/c. In the crystal, the Cl- anion is connected with protonated betaine through the O-H⋯Cl- hydrogen bond of 2.943(2) Å. The structures in the gas phase (2) and water solution (3) have been optimized by the B3LYP/6-311++G(d,p) approach and the geometrical results have been compared with the X-ray data of 1. The FTIR spectrum of the solid compound is consistent with the X-ray results. The probable assignments of the anharmonic experimental vibrational frequencies of the investigated chloride (1) based on the calculated harmonic frequencies in water solution (3) are proposed. The correlations between the experimental 1H and 13C NMR chemical shifts (δexp) of 1 in D2O and the magnetic isotropic shielding constants (σcalc) calculated by the GIAO/B3LYP/6-311G++(d,p) approach, using the screening solvation model (COSMO), δexp = a + b σcalc, for optimized molecule 3 in water solution are linear and correctly reproduce the experimental chemical shifts.

  10. Reactive oxygen species production by human dendritic cells involves TLR2 and dectin-1 and is essential for efficient immune response against Mycobacteria.

    PubMed

    Romero, María Mercedes; Basile, Juan Ignacio; Corra Feo, Laura; López, Beatriz; Ritacco, Viviana; Alemán, Mercedes

    2016-06-01

    Tuberculosis remains the single largest infectious disease with 10 million new cases and two million deaths that are estimated to occur yearly, more than any time in history. The intracellular replication of Mycobacterium tuberculosis (Mtb) and its spread from the lungs to other sites occur before the development of adaptive immune responses. Dendritic cells (DC) are professional antigen-presenting cells whose maturation is critical for the onset of the protective immune response against tuberculosis disease and may vary depending on the nature of the cell wall of Mtb strain. Here, we describe the role of the endogenous production of reactive oxygen species (ROS) on DC maturation and expansion of Mtb-specific lymphocytes. Here, we show that Mtb induces DC maturation through TLR2/dectin-1 by generating of ROS and through Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) in a ROS independently manner. Based on the differences observed in the ability to induce DC maturation, ROS production and lymphocyte proliferation by those Mtb families widespread in South America, i.e., Haarlem and Latin American Mediterranean and the reference strain H37Rv, we propose that variance in ROS production might contribute to immune evasion affecting DC maturation and antigen presentation.

  11. Crystal structures and redox responses coupled with ion recognition of p-benzoquinone- and hydroquinone-fused [18]crown-6.

    PubMed

    Kobayashi, Takayuki; Nakane, Yuta; Takeda, Takashi; Hoshino, Norihisa; Kawai, Hidetoshi; Akutagawa, Tomoyuki

    2015-02-01

    The crystal structures and redox properties of p-benzoquinone (BQ)-fused [18]crown-6 1 and bis-BQ-fused [18]crown-6 2 were examined. The anion radicals of these BQ molecules were stabilized by addition of metal ions, through effective electrostatic interactions between the negatively charged BQ moiety and positively charged ion-capturing [18]crown-6 unit. The electrostatic interactions and solvation energy played important roles in determining the magnitudes of anodic redox shifts in the reduction potentials. Regular π-stacking of BQ units and regular arrays of [18]crown-6 units were observed in crystal 2, in which one-dimensional π-electron columns were separated by ionic channels. The hydroquinone-fused [18]crown-6 molecule 3 and a new BQ- and phenol-fused [18]crown-6 derivative 4 were obtained as single crystals. The molecular conformations of [18]crown-6 in crystal 3 and hydrated crystal 3⋅H2 O were different from each other.

  12. Effect of 3-hydroxyflavone on pig embryos produced by parthenogenesis or somatic cell nuclear transfer.

    PubMed

    Uhm, Sang Jun; Gupta, Mukesh Kumar; Das, Ziban Chandra; Lim, Kyoung Tae; Yang, Ji Hoon; Lee, Hoon Taek

    2011-02-01

    This study evaluated the effect of 3-hydroxyflavone (a flavonoid having hydroxyl group at 3 carbon position) on embryos using parthenogenetic activation (PA) and cloned pig embryos as model system. There was no evidence for embryo toxicity of 3-hydroxyflavone in a wide concentration range of 1-100 μM. On the contrary, 3-hydroxyflavone significantly improved the in vitro development and quality of PA embryos that was associated with the activation of ERK signaling molecules and reduction in Caspase 3 expression. Furthermore, 3-hydroxyflavone rescued the in vitro development and embryo quality of in vitro aged oocytes by inhibiting ROS activity and activating ERK signaling. The beneficial effects of 3-hydroxyflavone on PA embryos were consistent both in PVA- and BSA-containing embryo culture medium and in cloned embryos. These results suggest that, contrary to those of other structurally related flavonoid molecules, 3-hydroxyflavone may be useful as a therapeutic drug for improving the developmental potential of aged oocytes in assisted reproductive technologies.

  13. Viral piracy: HIV-1 targets dendritic cells for transmission.

    PubMed

    Lekkerkerker, Annemarie N; van Kooyk, Yvette; Geijtenbeek, Teunis B H

    2006-04-01

    Dendritic cells (DCs), the professional antigen presenting cells, are critical for host immunity by inducing specific immune responses against a broad variety of pathogens. Remarkably the human immunodeficiency virus-1 (HIV-1) subverts DC function leading to spread of the virus. At an early phase of HIV-1 transmission, DCs capture HIV-1 at mucosal surfaces and transmit the virus to T cells in secondary lymphoid tissues. Capture of the virus on DCs takes place via C-type lectins of which the dendritic cell-specific intercellular adhesion molecule-3 (ICAM-3) grabbing nonintegrin (DC-SIGN) is the best studied. DC-SIGN-captured HIV-1 particles accumulate in CD81(+) multivesicular bodies (MVBs) in DCs and are subsequently transmitted to CD4+ T cells resulting in infection of T cells. The viral cell-to-cell transmission takes place at the DC-T cell interface termed the infectious synapse. Recent studies demonstrate that direct infection of DCs contributes to the transmission to T cells at a later phase. Moreover, the infected DCs may function as cellular reservoirs for HIV-1. This review discusses the different processes that govern viral piracy of DCs by HIV-1, emphasizing the intracellular routing of the virus from capture on the cell surface to egress in the infectious synapse.

  14. Fragment-based strategy for structural optimization in combination with 3D-QSAR

    NASA Astrophysics Data System (ADS)

    Yuan, Haoliang; Tai, Wenting; Hu, Shihe; Liu, Haichun; Zhang, Yanmin; Yao, Sihui; Ran, Ting; Lu, Shuai; Ke, Zhipeng; Xiong, Xiao; Xu, Jinxing; Chen, Yadong; Lu, Tao

    2013-10-01

    Fragment-based drug design has emerged as an important methodology for lead discovery and drug design. Different with other studies focused on fragment library design and active fragment identification, a fragment-based strategy was developed in combination with three-dimensional quantitative structure-activity relationship (3D-QSAR) for structural optimization in this study. Based on a validated scaffold or fragment hit, a series of structural optimization was conducted to convert it to lead compounds, including 3D-QSAR modelling, active site analysis, fragment-based structural optimization and evaluation of new molecules. 3D-QSAR models and active site analysis provided sufficient information for confirming the SAR and pharmacophoric features for fragments. This strategy was evaluated through the structural optimization on a c-Met inhibitor scaffold 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one, which resulted in an c-Met inhibitor with high inhibitory activity. Our study suggested the effectiveness of this fragment-based strategy and the druggability of our newly explored active region. The reliability of this strategy indicated it could also be applied to facilitate lead optimization of other targets.

  15. Effects of dopaminergic compounds on carbonic anhydrase isozymes I, II, and VI.

    PubMed

    Sentürk, Murat; Ekinci, Deniz; Göksu, Süleyman; Supuran, Claudiu T

    2012-06-01

    Studies on carbonic anhydrase (CA, EC 4.2.1.1) inhibitors have increased due to several therapeutic applications while there are few investigations on activators. Here we investigated CA inhibitory and activatory capacities of a series of dopaminergic compounds on human carbonic anhydrase (hCA) isozymes I, II, and VI. 2-Amino-1,2,3,4-tetrahydronaphthalene-6,7-diol hydrobromide and 2-amino-1,2,3,4-tetrahydronaphthalene-5,6-diol hydrobromide were found to show effective inhibitory action on hCA I and II whereas 2-amino-5,6-dibromoindan hydrobromide and 2-amino-5-bromoindan hydrobromide exhibited only moderate inhibition against both isoforms, being more effective inhibitors of hCA VI. K(i) values of the molecules 3-6 were in the range of 41.12-363 μM against hCA I, of 0.381-470 μM against hCA II and of 0.578-1.152 μM against hCA VI, respectively. Compound 7 behaved as a CA activator with K(A) values of 27.3 μM against hCA I, of 18.4 μM against hCA II and of 8.73 μM against hCA VI, respectively.

  16. Electrolyte layering at the calcite(104)-water interface indicated by Rb+- and Se(VI) K-edge resonant interface diffraction

    SciTech Connect

    Heberling, F.; Eng, P.; Denecke, M. A.; Lützenkirchen, J.; Geckeis, H.

    2014-09-22

    Calcite–water interface reactions are of major importance in various environmental settings as well as in industrial applications. Here we present resonant interface diffraction results on the calcite(104)–aqueous solution interface, measured in solutions containing either 10 mmol L-1 RbCl or 0.5 mmol L-1 Se(VI). Results indicate that Rb+ ions enter the surface adsorbed water layers and adsorb at the calcite(104)–water interface in an inner-sphere fashion. A detailed analysis based on specular and off-specular resonant interface diffraction data reveals three distinct Rb+ adsorption species: one 1.2 Å above the surface, the second associated with surface adsorbed water molecules 3.2 Å above the surface, and the third adsorbed in an outer-sphere fashion 5.6 Å above the surface. A peak in resonant amplitude between L = 1.5 and L = 3.0 is interpreted as signal from a layered electrolyte structure. The presence of a layered electrolyte structure seems to be confirmed by data measured in the presence of Se(VI).

  17. Highly selective mercury(II) cations detection in mixed-aqueous media by a ferrocene-based fluorescent receptor.

    PubMed

    Alfonso, María; Contreras-García, Julia; Espinosa, Arturo; Tárraga, Alberto; Molina, Pedro

    2012-04-21

    A new chemosensor molecule 3 based on a ferrocene-imidazophenanthrophenazine dyad effectively recognizes Hg(2+) in an aqueous environment through three different channels. Upon recognition, an anodic shift of the ferrocene-ferrocenium oxidation potential (ΔE(1/2) = 240 mV) and a progressive red shift (Δλ = 17 nm) of the low energy band in its absorption spectrum is produced. The emission spectrum of 3 in an aqueous environment, CH(3)CN-EtOH-H(2)O (65:25:10), and conducted at pH = 7.4 (20 × 10(-3) M HEPES) (Φ = 0.003), is perturbed after addition of Hg(2+) cations and an intense and structureless red shift emission band at 494 nm (Δλ = 92 nm) appeared along with an increase of the intensity of the emission band (CHEF = 77), the quantum yield (Φ = 0.054) resulted in a 18-fold increase. The combined (1)H NMR data of the complex and the theoretical calculations suggest the proposed bridging coordination mode.

  18. Shape-Engineering of Self-Assembled Organic Single Microcrystal as Optical Microresonator for laser Applications

    NASA Astrophysics Data System (ADS)

    Wang, Xuedong; Liao, Qing; Lu, Xiaomei; Li, Hui; Xu, Zhenzhen; Fu, Hongbing

    2014-11-01

    Single micro/nanocrystals based on π-conjugated organic molecules have caused tremendous interests in the optoelectronic applications in laser, optical waveguide, nonlinear optics, and field effect transistors. However, the controlled synthesis of these organic micro/nanocrystals with regular shapes is very difficult to achieve, because the weak interaction (van der Waals' force, ca. 5 kJ/mol) between organic molecules could not dominate the kinetic process of crystal growth. Herein, we develop an elaborate strategy, selective adhesion to organic crystal plane by the hydrogen-bonding interaction (ca. 40 kJ/mol), for modulating the kinetic process of the formation of microcrystal, which leads to the self-assembly of one organic molecule 3-[4-(dimethylamino)phenyl]-1-(2-hy-droxyphenyl)prop-2-en-1-on (HDMAC) into one-dimensional (1D) microwires and 2D microdisks respectively. Furthermore, these as-prepared microcrystals demonstrate shape-dependent microresonator properties that 1D microwires act as Fabry-Pérot (FP) mode lasing resonator and 2D microdisks provide the whispering-gallery-mode (WGM) resonator for lasing oscillator. More significantly, through the investigation of the size-effect on the laser performance, single-mode lasing at red wavelength was successfully achieved in the self-assembled 2D organic microdisk at room temperature. These easily fabricated organic single-crystalline microcrystals with controlled shapes are the natural laser sources, which offer considerable promise for the multi-functionalities of coherent light devices integrated on the optics microchip.

  19. Transcriptome of the NTS in exercise-trained spontaneously hypertensive rats: implications for NTS function and plasticity in regulating blood pressure.

    PubMed

    Waki, Hidefumi; Gouraud, Sabine S; Bhuiyan, Mohammad E R; Takagishi, Miwa; Yamazaki, Toshiya; Kohsaka, Akira; Maeda, Masanobu

    2013-01-07

    The nucleus tractus solitarii (NTS) controls the cardiovascular system during exercise, and alteration of its function may underlie exercise-induced cardiovascular adaptation. To understand the molecular basis of the NTS's plasticity in regulating blood pressure (BP) and its potential contribution to the antihypertensive effects, we characterized the gene expression profiles at the level of the NTS after long-term daily wheel running in spontaneously hypertensive rats (SHRs). Genome-wide microarray analysis was performed to screen for differentially expressed genes in the NTS between exercise-trained (12 wk) and control SHRs. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes database revealed that daily exercise altered the expression levels of NTS genes that are functionally associated with metabolic pathways (5 genes), neuroactive ligand-receptor interactions (4 genes), cell adhesion molecules (3 genes), and cytokine-cytokine receptor interactions (3 genes). One of the genes that belonged to the neuroactive ligand-receptor interactions category was histamine receptor H(1). Since we confirmed that the pressor response induced by activation of this receptor is increased after long-term daily exercise, it is suggested that functional plasticity in the histaminergic system may mediate the facilitation of blood pressure control in response to exercise but may not be involved in the lowered basal BP level found in exercise-trained SHRs. Since abnormal inflammatory states in the NTS are known to be prohypertensive in SHRs, altered gene expression of the inflammatory molecules identified in this study may be related to the antihypertensive effects in exercise-trained SHRs, although such speculation awaits functional validation.

  20. Lactobacillus reuteri Surface Mucus Adhesins Upregulate Inflammatory Responses Through Interactions With Innate C-Type Lectin Receptors

    PubMed Central

    Bene, Krisztián P.; Kavanaugh, Devon W.; Leclaire, Charlotte; Gunning, Allan P.; MacKenzie, Donald A.; Wittmann, Alexandra; Young, Ian D.; Kawasaki, Norihito; Rajnavolgyi, Eva; Juge, Nathalie

    2017-01-01

    The vertebrate gut symbiont Lactobacillus reuteri exhibits strain-specific adhesion and health-promoting properties. Here, we investigated the role of the mucus adhesins, CmbA and MUB, upon interaction of L. reuteri ATCC PTA 6475 and ATCC 53608 strains with human monocyte-derived dendritic cells (moDCs). We showed that mucus adhesins increased the capacity of L. reuteri strains to interact with moDCs and promoted phagocytosis. Our data also indicated that mucus adhesins mediate anti- and pro-inflammatory effects by the induction of interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-α), IL-1β, IL-6, and IL-12 cytokines. L. reuteri ATCC PTA 6475 and ATCC 53608 were exclusively able to induce moDC-mediated Th1 and Th17 immune responses. We further showed that purified MUB activates moDCs and induces Th1 polarized immune responses associated with increased IFNγ production. MUB appeared to mediate these effects via binding to C-type lectin receptors (CLRs), as shown using cell reporter assays. Blocking moDCs with antibodies against DC-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) or Dectin-2 did not affect the uptake of the MUB-expressing strain, but reduced the production of TNF-α and IL-6 by moDCs significantly, in line with the Th1 polarizing capacity of moDCs. The direct interaction between MUB and CLRs was further confirmed by atomic force spectroscopy. Taken together these data suggest that mucus adhesins expressed at the cell surface of L. reuteri strains may exert immunoregulatory effects in the gut through modulating the Th1-promoting capacity of DCs upon interaction with C-type lectins. PMID:28326063

  1. Spectroscopic investigation of photoinduced charge-transfer processes in FTO/TiO2/N719 photoanodes with and without covalent attachment through silane-based linkers.

    PubMed

    Pandit, Bill; Luitel, Tulashi; Cummins, Dustin R; Thapa, Arjun K; Druffel, Thad; Zamborini, Frank; Liu, Jinjun

    2013-12-19

    Understanding electron-transfer (ET) processes in dye-sensitized solar cells (DSSCs) is crucial to improving their device performance. Recently, covalent attachment of dye molecules to mesoporous semiconductor nanoparticle films via molecular linkers has been employed to increase the stability of DSSC photoanodes. The power conversion efficiency (PCE) of these DSSCs, however, is lower than DSSCs with conventional unmodified photoanodes in this study. Ultrafast transient absorption pump-probe spectroscopy (TAPPS) has been used to study the electron injection process from N719 dye molecules to TiO2 nanoparticles (NPs) in DSSC photoanodes with and without the presence of two silane-based linker molecules: 3-aminopropyltriethoxysilane (APTES) and p-aminophenyltrimethoxysilane (APhS). Ultrafast biphasic electron injection kinetics were observed in all three photoanodes using a 530 nm pump wavelength and 860 nm probe wavelength. Both the slow and fast decay components, attributed to electron injection from singlet and triplet excited states, respectively, of the N719 dye to the TiO2 conduction band, are hindered by the molecular linkers. The hindering effect is less significant with the APhS linker than the APTES linker and is more significant for the singlet-state channel than the triplet-state one. Electron injection from the vibrationally excited states is less affected by the linkers. The spectroscopic results are interpreted on the basis of the standard ET theory and can be used to guide selection of molecular linkers for DSSCs with better device performance. Other factors that affect the efficiency and stability of the DSSCs are also discussed. The relatively lower PCE of the covalently attached photoanodes is attributed to the multilayer and aggregation of the dye molecules as well as the linkers.

  2. The Porphobilinogen Conundrum in Prebiotic Routes to Tetrapyrrole Macrocycles

    NASA Astrophysics Data System (ADS)

    Taniguchi, Masahiko; Ptaszek, Marcin; Chandrashaker, Vanampally; Lindsey, Jonathan S.

    2017-03-01

    Attempts to develop a credible prebiotic route to tetrapyrroles have relied on enzyme-free recapitulation of the extant biosynthesis, but this process has foundered from the inability to form the pyrrole porphobilinogen ( PBG) in good yield by self-condensation of the precursor δ-aminolevulinic acid ( ALA). PBG undergoes robust oligomerization in aqueous solution to give uroporphyrinogen (4 isomers) in good yield. ALA, PBG, and uroporphyrinogen III are universal precursors to all known tetrapyrrole macrocycles. The enzymic formation of PBG entails carbon-carbon bond formation between the less stable enolate/enamine of one ALA molecule (3-position) and the carbonyl/imine (4-position) of the second ALA molecule; without enzymes, the first ALA reacts at the more stable enolate/enamine (5-position) and gives the pyrrole pseudo-PBG. pseudo-PBG cannot self-condense, yet has one open α-pyrrole position and is proposed to be a terminator of oligopyrromethane chain-growth from PBG. Here, 23 analogues of ALA have been subjected to density functional theoretical (DFT) calculations, but no motif has been identified that directs reaction at the 3-position. Deuteriation experiments suggested 5-(phosphonooxy)levulinic acid would react preferentially at the 3- versus 5-position, but a hybrid condensation with ALA gave no observable uroporphyrin. The results suggest efforts toward a biomimetic, enzyme-free route to tetrapyrroles from ALA should turn away from structure-directed reactions and focus on catalysts that orient the two aminoketones to form PBG in a kinetically controlled process, thereby avoiding formation of pseudo-PBG.

  3. Phase-Variable Control of Multiple Phenotypes in Acinetobacter baumannii Strain AB5075

    PubMed Central

    Tipton, Kyle A.; Dimitrova, Daniela

    2015-01-01

    ABSTRACT Acinetobacter baumannii strain AB5075 produces colonies with two opacity phenotypes, designated opaque and translucent. These phenotypes were unstable and opaque and translucent colony variants were observed to interconvert at high frequency, suggesting that a phase-variable mechanism was responsible. The frequency of phase variation both within colonies and in broth cultures increased in a cell density-dependent manner and was mediated by the accumulation of an extracellular factor. This factor was distinct from the known A. baumannii signaling molecule 3-OH C12-homoserine lactone. Opaque and translucent colony variants exhibited a number of phenotypic differences, including cell morphology, surface motility, biofilm formation, antibiotic resistance, and virulence in a Galleria mellonella model. Additional clinical isolates exhibited a similar phase-variable control of colony opacity, suggesting that this may be a common feature of A. baumannii. IMPORTANCE A novel phase-variable mechanism has been identified in Acinetobacter baumannii that results in an interconversion between opaque and translucent colony phenotypes. This phase variation also coordinately regulates motility, cell shape, biofilm formation, antibiotic resistance, and virulence. The frequency of phase variation is increased at high cell density via a diffusible extracellular signal. To our knowledge, this report presents the first example of phase variation in A. baumannii and also the first example of quorum sensing-mediated control of phase variation in a bacterium. The findings are important, as this phase-variable mechanism can be identified only via changes in colony opacity using oblique light; therefore, many researchers studying A. baumannii may unknowingly be working with different colony variants. PMID:26013481

  4. A small molecule modulator of prion protein increases human mesenchymal stem cell lifespan, ex vivo expansion, and engraftment to bone marrow in NOD/SCID mice.

    PubMed

    Mohanty, Sindhu T; Cairney, Claire J; Chantry, Andrew D; Madan, Sanjeev; Fernandes, James A; Howe, Steven J; Moore, Harry D; Thompson, Mark J; Chen, Beining; Thrasher, Adrian; Keith, W Nicol; Bellantuono, Ilaria

    2012-06-01

    Human mesenchymal stem cells (hMSCs) have been shown to have potential in regenerative approaches in bone and blood. Most protocols rely on their in vitro expansion prior to clinical use. However, several groups including our own have shown that hMSCs lose proliferation and differentiation ability with serial passage in culture, limiting their clinical applications. Cellular prion protein (PrP) has been shown to enhance proliferation and promote self-renewal of hematopoietic, mammary gland, and neural stem cells. Here we show, for the first time, that expression of PrP decreased in hMSC following ex vivo expansion. When PrP expression was knocked down, hMSC showed significant reduction in proliferation and differentiation. In contrast, hMSC expanded in the presence of small molecule 3/689, a modulator of PrP expression, showed retention of PrP expression with ex vivo expansion and extended lifespan up to 10 population doublings. Moreover, cultures produced a 300-fold increase in the number of cells generated. These cells showed a 10-fold increase in engraftment levels in bone marrow 5 weeks post-transplant. hMSC treated with 3/689 showed enhanced protection from DNA damage and enhanced cell cycle progression, in line with data obtained by gene expression profiling. Moreover, upregulation of superoxide dismutase-2 (SOD2) was also observed in hMSC expanded in the presence of 3/689. The increase in SOD2 was dependent on PrP expression and suggests increased scavenging of reactive oxygen species as mechanism of action. These data point to PrP as a good target for chemical intervention in stem cell regenerative medicine.

  5. Spectral and structural studies of dimethylphenyl betaine hydrate

    NASA Astrophysics Data System (ADS)

    Szafran, M.; Ostrowska, K.; Katrusiak, A.; Dega-Szafran, Z.

    2014-07-01

    Hydrates of betaines can be divided into four groups depending on the interactions of their water molecules with the carboxylate group. Dimethylphenyl betaine crystallizes as monohydrate (1), in which water molecules mediate in hydrogen bonds between the carboxylate groups. The water molecules are H-bonded only to one oxygen atom of the dimethylphenyl betaine molecules and link them into a chain via two O(1 W)sbnd H⋯O hydrogen bonds of the lengths 2.779(2) and 2.846(2) Å. The structures of monomer (2) and dimer (4) hydrates in vacuum, and the structure of monomer (3) in an aqueous environment have been optimized by the B3LYP/6-311++G(d,p) approach and the geometrical results have been compared with the X-ray diffraction data of 1. The calculated IR frequencies for the optimized structure have been used for the assignments of FTIR bands, the broad absorption band in the range 3415-3230 cm-1 has been assigned to the O(1w)sbnd H⋯O hydrogen bonds. The correlations between the experimental 1H and 13C NMR chemical shifts (δexp) of 1 in D2O and the magnetic isotropic shielding constants (σcalc) calculated by the GIAO/B3LYP/6-311G++(d,p) approach, using the screening solvation model (COSMO), δexp = a + b σcalc, for optimized molecule 3 in water solution are linear and well reproduce the experimental chemical shifts.

  6. Core-to-valence spectroscopic detection of the CH{sub 2}Br radical and element-specific femtosecond photodissociation dynamics of CH{sub 2}IBr

    SciTech Connect

    Attar, Andrew R.; Piticco, Lorena; Leone, Stephen R.

    2014-10-28

    Element-specific single photon photodissociation dynamics of CH{sub 2}IBr and core-to-valence absorption spectroscopy of CH{sub 2}Br radicals are investigated using femtosecond high-harmonic extreme ultraviolet (XUV) transient absorption spectroscopy. Photodissociation of CH{sub 2}IBr along both the C–I or C–Br reaction coordinates is observed in real-time following excitation at 266 nm. At this wavelength, C–I dissociation is the dominant reaction channel and C–Br dissociation is observed as a minor pathway. Both photodissociation pathways are probed simultaneously through individual 4d(I) N{sub 4/5} and 3d(Br) M{sub 4/5} core-to-valence transitions. The 3d(Br) M{sub 4/5} pre-edge absorption spectrum of the CH{sub 2}Br radical photoproduct corresponding to the C–I dissociation channel is characterized for the first time. Although the radical's singly occupied molecular orbital (SOMO) is mostly localized on the central carbon atom, the 3d(Br) → π{sup *}(SOMO) resonances at 68.5 eV and 69.5 eV are detected 2 eV below the parent molecule 3d(Br) → σ{sup *}(LUMO) transitions. Core-to-valence XUV absorption spectroscopy provides a unique probe of the local electronic structure of the radical species in reference to the Br reporter atom. The measured times for C–I dissociation leading to I and I{sup *} atomic products are 48 ± 12 fs and 44 ± 4 fs, respectively, while the measured C–Br dissociation time leading to atomic Br is 114 ± 17 fs. The investigation performed here demonstrates the capability of femtosecond time-resolved core-level spectroscopy utilizing multiple reporter atoms simultaneously.

  7. HLA-A2-restricted cytotoxic T lymphocyte epitopes from human hepsin as novel targets for prostate cancer immunotherapy.

    PubMed

    Guo, J; Li, G; Tang, J; Cao, X-B; Zhou, Q-Y; Fan, Z-J; Zhu, B; Pan, X-H

    2013-09-01

    Hepsin is a type II transmembrane serine protease that is overexpressed in prostate cancer, and it is associated with prostate cancer cellular migration and invasion. Therefore, HPN is a biomarker for prostate cancer. CD8(+) T cells play an important role in tumour immunity. This study predicted and identified HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitopes in human hepsin protein. HLA-A2-restricted CTL epitopes were identified using the following four-step procedure: (1) a computer program generated predicted epitopes from the amino acid sequence of human hepsin; (2) an HLA-A2-binding assay detected the affinity of the predicted epitopes to the HLA-A2 molecule; (3) the primary T cell response against the predicted epitopes was stimulated in vitro; and (4) the induced CTLs towards different types of hepsin- or HLA-A2-expressing prostate cancer cells were detected. Five candidate peptides were identified. The effectors that were induced by human hepsin epitopes containing residues 229 to 237 (Hpn229; GLQLGVQAV), 268 to 276 (Hpn268; PLTEYIQPV) and 191 to 199 (Hpn199; SLLSGDWVL) effectively lysed LNCaP prostate cancer cells that were hepsin-positive and HLA-A2 matched. These peptide-specific CTLs did not lyse normal liver cells with low hepsin levels. Hpn229, Hpn268 and Hpn199 increased the frequency of IFN-γ-producing T cells compared with the negative peptide. These results suggest that the Hpn229, Hpn268 and Hpn199 epitopes are novel HLA-A2-restricted CTL epitopes that are capable of inducing hepsin-specific CTLs in vitro. Hpn229, Hpn268 and Hpn199 peptide-based vaccines may be useful for immunotherapy in patients with prostate cancer.

  8. Development and evaluation of a double antibody sandwich ELISA for the detection of human sDC-SIGN.

    PubMed

    Chen, Shang-Liang; Li, Yan-Li; Tang, Yuan; Chen, Zhi-Cheng; Zhou, Jing; Zhou, Jia; Lu, Xiao; Zhao, Na; Chen, Zheng-Liang; Zuo, Daming

    2016-09-01

    sDC-SIGN is the soluble form of dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN, CD209), which is a molecule involved with pathogen recognition and immune regulation. However, there is no commercially available ELISA kit for detecting human sDC-SIGN, and the normal range of this molecule is unknown. Here, we describe an ELISA for detecting human sDC-SIGN with high specificity. First, sDC-SIGN protein was expressed and purified. Monoclonal and polyclonal antibodies were then raised against the purified protein and subsequently characterized. A sandwich ELISA was developed using polyclonal antibodies specific for sDC-SIGN for capture and a biotin-labeled monoclonal antibody specific for sDC-SIGN for detection of protein. This method has sensitivity up to 0.2 ng/ml. Using this ELISA, we found that the concentration of sDC-SIGN in sera of healthy volunteers ranges from 0-319 ng/ml with a mean concentration of 27.14 ng/ml. Interestingly, the concentration of sDC-SIGN in sera from patients with cancer or chronic hepatitis B virus (CHB) infection was lower than that of health controls. The mean concentrations of sDC-SIGN in cancer patients and chronic hepatitis B virus infection patients were 3.2 ng/ml and 3.8 ng/ml, respectively. We developed a sandwich ELISA for detecting human sDC-SIGN and demonstrated its use by assessing sera concentrations of sDC-SIGN in patients with cancer and chronic CHB infection compared to that of healthy controls.

  9. N-Glycans on the Rift Valley Fever Virus Envelope Glycoproteins Gn and Gc Redundantly Support Viral Infection via DC-SIGN

    PubMed Central

    Phoenix, Inaia; Nishiyama, Shoko; Lokugamage, Nandadeva; Hill, Terence E.; Huante, Matthew B.; Slack, Olga A.L.; Carpio, Victor H.; Freiberg, Alexander N.; Ikegami, Tetsuro

    2016-01-01

    Rift Valley fever is a mosquito-transmitted, zoonotic disease that infects humans and ruminants. Dendritic cell specific intercellular adhesion molecule 3 (ICAM-3) grabbing non-integrin (DC-SIGN) acts as a receptor for members of the phlebovirus genus. The Rift Valley fever virus (RVFV) glycoproteins (Gn/Gc) encode five putative N-glycan sequons (asparagine (N)–any amino acid (X)–serine (S)/threonine (T)) at positions: N438 (Gn), and N794, N829, N1035, and N1077 (Gc). The N-glycosylation profile and significance in viral infection via DC-SIGN have not been elucidated. Gc N-glycosylation was first evaluated by using Gc asparagine (N) to glutamine (Q) mutants. Subsequently, we generated a series of recombinant RVFV MP-12 strain mutants, which encode N-to-Q mutations, and the infectivity of each mutant in Jurkat cells stably expressing DC-SIGN was evaluated. Results showed that Gc N794, N1035, and N1077 were N-glycosylated but N829 was not. Gc N1077 was heterogeneously N-glycosylated. RVFV Gc made two distinct N-glycoforms: “Gc-large” and “Gc-small”, and N1077 was responsible for “Gc-large” band. RVFV showed increased infection of cells expressing DC-SIGN compared to cells lacking DC-SIGN. Infection via DC-SIGN was increased in the presence of either Gn N438 or Gc N1077. Our study showed that N-glycans on the Gc and Gn surface glycoproteins redundantly support RVFV infection via DC-SIGN. PMID:27223297

  10. Striatal Signaling in L-DOPA-Induced Dyskinesia: Common Mechanisms with Drug Abuse and Long Term Memory Involving D1 Dopamine Receptor Stimulation

    PubMed Central

    Murer, Mario Gustavo; Moratalla, Rosario

    2011-01-01

    Parkinson’s disease is a common neurodegenerative disorder caused by the degeneration of midbrain substantia nigra dopaminergic neurons that project to the striatum. Despite extensive investigation aimed at finding new therapeutic approaches, the dopamine precursor molecule, 3,4-dihydroxyphenyl-l-alanine (l-DOPA), remains the most effective and commonly used treatment. However, chronic treatment and disease progression lead to changes in the brain’s response to l-DOPA, resulting in decreased therapeutic effect and the appearance of dyskinesias. l-DOPA-induced dyskinesia (LID) interferes significantly with normal motor activity and persists unless l-DOPA dosages are reduced to below therapeutic levels. Thus, controlling LID is one of the major challenges in Parkinson’s disease therapy. LID is the result of intermittent stimulation of supersensitive D1 dopamine receptors located in the very severely denervated striatal neurons. Through increased coupling to Gαolf, resulting in greater stimulation of adenylyl-cyclase, D1 receptors phosphorylate DARPP-32, and other protein kinase A targets. Moreover, D1 receptor stimulation activates extracellular signal-regulated kinase and triggers a signaling pathway involving mammalian target for rapamycin and modifications of histones that results in changes in translation, chromatin modification, and gene transcription. In turn, sensitization of D1 receptor signaling causes a widespread increase in the metabolic response to D1 agonists and changes in the activity of basal ganglia neurons that correlate with the severity of LID. Importantly, different studies suggest that dyskinesias may share mechanisms with drug abuse and long term memory involving D1 receptor activation. Here we review evidence implicating D1 receptor signaling in the genesis of LID, analyze mechanisms that may translate enhanced D1 signaling into dyskinetic movements, and discuss the possibility that the mechanisms underlying LID are not unique to the

  11. A translational study "case report" on the small molecule "energy blocker" 3-bromopyruvate (3BP) as a potent anticancer agent: from bench side to bedside.

    PubMed

    Ko, Y H; Verhoeven, H A; Lee, M J; Corbin, D J; Vogl, T J; Pedersen, P L

    2012-02-01

    The small alkylating molecule, 3-bromopyruvate (3BP), is a potent and specific anticancer agent. 3BP is different in its action from most currently available chemo-drugs. Thus, 3BP targets cancer cells' energy metabolism, both its high glycolysis ("Warburg Effect") and mitochondrial oxidative phosphorylation. This inhibits/ blocks total energy production leading to a depletion of energy reserves. Moreover, 3BP as an "Energy Blocker", is very rapid in killing such cells. This is in sharp contrast to most commonly used anticancer agents that usually take longer to show a noticeable effect. In addition, 3BP at its effective concentrations that kill cancer cells has little or no effect on normal cells. Therefore, 3BP can be considered a member, perhaps one of the first, of a new class of anticancer agents. Following 3BP's discovery as a novel anticancer agent in vitro in the Year 2000 (Published in Ko et al. Can Lett 173:83-91, 2001), and also as a highly effective and rapid anticancer agent in vivo shortly thereafter (Ko et al. Biochem Biophys Res Commun 324:269-275, 2004), its efficacy as a potent anticancer agent in humans was demonstrated. Here, based on translational research, we report results of a case study in a young adult cancer patient with fibrolamellar hepatocellular carcinoma. Thus, a bench side discovery in the Department of Biological Chemistry at Johns Hopkins University, School of Medicine was taken effectively to bedside treatment at Johann Wolfgang Goethe University Frankfurt/Main Hospital, Germany. The results obtained hold promise for 3BP as a future cancer therapeutic without apparent cyto-toxicity when formulated properly.

  12. The molecule of DC-SIGN captures enterovirus 71 and confers dendritic cell-mediated viral trans-infection

    PubMed Central

    2014-01-01

    Background Enterovirus 71 (EV71) is the main causative agent of hand, foot and mouth disease that occurs in young children. Neither antiviral agents nor vaccines are available for efficiently combating viral infection. Study of EV71–host interplay is important for understanding viral infection and developing strategies for prevention and therapy. Here the interactions of EV71 with human dendritic cells were analyzed. Methods EV71 capture, endocytosis, infection, and degradation in monocyte-derived dendritic cells (MDDCs) were detected by Flow cytometry or real-time (RT-) PCR, and MDDCs-mediated EV71 trans-infection of RD cells was determined via coculture system. Cell morphology or viability was monitored with microscopy or flow cytometry. SiRNA interference was used to knock down gene expression. Results MDDCs can bind EV71, but these loaded-EV71 particles in MDDCs underwent a rapid degradation in the absence of efficient replication; once the captured EV71 encountered susceptible cells, MDDCs efficiently transferred surface-bound viruses to target cells. The molecule of DC-SIGN (DC-specific intercellular adhesion molecule-3 grabbing nonintegrin) mediated viral binding and transfer, because interference of DC-SIGN expression with specific siRNAs reduced EV71 binding and impaired MDDC-mediated viral trans-infection, and exogenous expression of DC-SIGN molecule on Raji cell initiated viral binding and subsequent transmission. Conclusion MDDCs could bind efficiently EV71 viruses through viral binding to DC-SIGN molecule, and these captured-viruses could be transferred to susceptible cells for robust infection. The novel finding of DC-mediated EV71 dissemination might facilitate elucidation of EV71 primary infection and benefit searching for new clues for preventing viruses from initial infection. PMID:24620896

  13. AM3 modulates dendritic cell pathogen recognition capabilities by targeting DC-SIGN.

    PubMed

    Serrano-Gómez, Diego; Martínez-Nuñez, Rocío T; Sierra-Filardi, Elena; Izquierdo, Nuria; Colmenares, María; Pla, Jesús; Rivas, Luis; Martinez-Picado, Javier; Jimenez-Barbero, Jesús; Alonso-Lebrero, José Luis; González, Salvador; Corbí, Angel L

    2007-07-01

    AM3 (Inmunoferon) is an orally effective immunomodulator that influences the regulatory and effector functions of the immune system whose molecular mechanisms of action are mostly unknown. We hypothesized that the polysaccharide moiety of AM3 (IF-S) might affect immune responses by modulating the lectin-dependent pathogen recognition abilities of human dendritic cells. IF-S inhibited binding of viral, fungal, and parasite pathogens by human monocyte-derived dendritic cells in a dose-dependent manner. IF-S specifically impaired the pathogen recognition capabilities of DC-SIGN, as it reduced the attachment of Candida, Aspergillus, and Leishmania to DC-SIGN transfectants. IF-S also inhibited the interaction of DC-SIGN with both its cellular counterreceptor (intercellular adhesion molecule 3) and the human immunodeficiency virus (HIV) type 1 gp120 protein and blocked the DC-SIGN-dependent capture of HIV virions and the HIV trans-infection capability of DC-SIGN transfectants. IF-S promoted DC-SIGN internalization in DCs without affecting mannose receptor expression, and (1)D saturation transfer difference nuclear magnetic resonance demonstrated that IF-S directly interacts with DC-SIGN on the cell surface. Therefore, the polysaccharide moiety of AM3 directly influences pathogen recognition by dendritic cells by interacting with DC-SIGN. Our results indicate that DC-SIGN is the target for an immunomodulator and imply that the adjuvant and immunomodulatory actions of AM3 are mediated, at least in part, by alteration of the DC-SIGN functional activities.

  14. Matrix molecularly imprinted mesoporous sol-gel sorbent for efficient solid-phase extraction of chloramphenicol from milk.

    PubMed

    Samanidou, Victoria; Kehagia, Maria; Kabir, Abuzar; Furton, Kenneth G

    2016-03-31

    Highly selective and efficient chloramphenicol imprinted sol-gel silica based inorganic polymeric sorbent (sol-gel MIP) was synthesized via matrix imprinting approach for the extraction of chloramphenicol in milk. Chloramphenicol was used as the template molecule, 3-aminopropyltriethoxysilane (3-APTES) and triethoxyphenylsilane (TEPS) as the functional precursors, tetramethyl orthosilicate (TMOS) as the cross-linker, isopropanol as the solvent/porogen, and HCl as the sol-gel catalyst. Non-imprinted sol-gel polymer (sol-gel NIP) was synthesized under identical conditions in absence of template molecules for comparison purpose. Both synthesized materials were characterized by Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FT-IR) and nitrogen adsorption porosimetry, which unambiguously confirmed their significant structural and morphological differences. The synthesized MIP and NIP materials were evaluated as sorbents for molecularly imprinted solid phase extraction (MISPE) of chloramphenicol in milk. The effect of critical extraction parameters (flow rate, elution solvent, sample and eluent volume, selectivity coefficient, retention capacity) was studied in terms of retention and desorption of chloramphenicol. Competition and cross reactivity tests have proved that sol-gel MIP sorbent possesses significantly higher specific retention and enrichment capacity for chloramphenicol compared to its non-imprinted analogue. The maximum imprinting factor (IF) was found as 9.7, whereas the highest adsorption capacity of chloramphenicol by sol-gel MIP was 23 mg/g. The sol-gel MIP was found to be adequately selective towards chloramphenicol to provide the necessary minimum required performance limit (MRPL) of 0.3 μg/kg set forth by European Commission after analysis by LC-MS even without requiring time consuming solvent evaporation and sample reconstitution step, often considered as an integral part in solid phase extraction work-flow. Intra and

  15. A tandem mass spectrometric study of bile acids: interpretation of fragmentation pathways and differentiation of steroid isomers.

    PubMed

    Qiao, Xue; Ye, Min; Liu, Chun-fang; Yang, Wen-zhi; Miao, Wen-juan; Dong, Jing; Guo, De-an

    2012-02-01

    Bile acids are steroids with a pentanoic acid substituent at C-17. They are the terminal products of cholesterol excretion, and play critical physiological roles in human and animals. Bile acids are easy to detect but difficult to identify by using mass spectrometry due to their poly-ring structure and various hydroxylation patterns. In this study, fragmentation pathways of 18 free and conjugated bile acids were interpreted by using tandem mass spectrometry. The analyses were conducted on ion trap and triple quadrupole mass spectrometers. Upon collision-induced dissociation, the conjugated bile acids could cleave into glycine or taurine related fragments, together with the steroid skeleton. Fragmentations of free bile acids were further elucidated, especially by atmospheric pressure chemical ionization mass spectrometry in positive ion mode. Aside from universally observed neutral losses, eliminations occurred on bile acid carbon rings were proposed for the first time. Moreover, four isomeric 5β-cholanic acid hydroxyl derivatives (3α,6α-, 3α,7β-, 3α,7α-, and 3α,12α-) were differentiated using electrospray ionization in negative ion mode: 3α,7β-OH substituent inclined to eliminate H(2)O and CH(2)O(2) groups; 3α,6α-OH substituent preferred neutral loss of two H(2)O molecules; 3α,12α-OH substituent apt to lose the carboxyl in the form of CO(2) molecule; and 3α,7α-OH substituent exhibited no further fragmentation after dehydration. This study provided specific interpretation for mass spectra of bile acids. The results could contribute to bile acid analyses, especially in clinical assays and metabonomic studies.

  16. Synthesis and characterization of a new fluorogenic active-site titrant of serine proteases.

    PubMed

    Livingston, D C; Brocklehurst, J R; Cannon, J F; Leytus, S P; Wehrly, J A; Peltz, S W; Peltz, G A; Mangel, W F

    1981-07-21

    The molecule 3',6'-bis(4-guanidinobenzoyloxy)-5-[N'-(4-carboxyphenyl)thioureido[spirop]isobenzofuran-1-(3H),9'-[9H]xanthen]-3-one, abbreviated FDE, was designed and synthesized as a fluorogenic active-site titrant for serine proteases. It is an analogue of p-nitrophenyl p-guanidino-benzoate (NPGB) in which a fluorescein derivative is substituted for p-nitrophenol. FDE and NPGB exhibit similar kinetic characteristics in an active-site titration of trypsin in phosphate-buffered saline, pH 7.2. The rate of acylation with FDE is extremely fast (k2 = 1.05 s-1) and the rate of deacylation extremely slow (k3 = 1.66 X 10(-5) s-1). The Ks is 3.06 X 10(-6) M, and the Km(app) is 4.85 X 10(-11) M. With two of the serine proteases involved in fibrinolysis, the rate of acylation with FDE is also fast, K2 = 0.112 s-1 for urokinase and 0.799 s-1 for plasmin, and the rate of deacylation is slow, k3 = 3.64 X 10(-4) s-1 for urokinase and 6.27 X 10(-6) s-1 for plasmin. The solubility limit of FDE in phosphate-buffered saline is 1.3 X 10(-5) M, and the first-order rate constant for spontaneous hydrolysis is 5.1 X 10(-6) s-1. The major difference between FDE and NPGB is the detectability of the product in an active-site titration. p-Nitrophenol can be detected at concentrations no lower than 10(-6) M whereas fluorescein can be detected at concentrations as low as 10(-12) M. Thus, FDE should be useful in quantitatively assaying serine proteases as very low concentrations.

  17. Engineering Graphene Conductivity for Flexible and High-Frequency Applications.

    PubMed

    Samuels, Alexander J; Carey, J David

    2015-10-14

    Advances in lightweight, flexible, and conformal electronic devices depend on materials that exhibit high electrical conductivity coupled with high mechanical strength. Defect-free graphene is one such material that satisfies both these requirements and which offers a range of attractive and tunable electrical, optoelectronic, and plasmonic characteristics for devices that operate at microwave, terahertz, infrared, or optical frequencies. Essential to the future success of such devices is therefore the ability to control the frequency-dependent conductivity of graphene. Looking to accelerate the development of high-frequency applications of graphene, here we demonstrate how readily accessible and processable organic and organometallic molecules can efficiently dope graphene to carrier densities in excess of 10(13) cm(-2) with conductivities at gigahertz frequencies in excess of 60 mS. In using the molecule 3,6-difluoro-2,5,7,7,8,8-hexacyanoquinodimethane (F2-HCNQ), a high charge transfer (CT) of 0.5 electrons per adsorbed molecule is calculated, resulting in p-type doping of graphene. n-Type doping is achieved using cobaltocene and the sulfur-containing molecule tetrathiafulvalene (TTF) with a CT of 0.41 and 0.24 electrons donated per adsorbed molecule, respectively. Efficient CT is associated with the interaction between the π electrons present in the molecule and in graphene. Calculation of the high-frequency conductivity shows dispersion-less behavior of the real component of the conductivity over a wide range of gigahertz frequencies. Potential high-frequency applications in graphene antennas and communications that can exploit these properties and the broader impacts of using molecular doping to modify functional materials that possess a low-energy Dirac cone are also discussed.

  18. Lentivirus-mediated RNA interference of DC-SIGN expression inhibits human immunodeficiency virus transmission from dendritic cells to T cells.

    PubMed

    Arrighi, Jean-François; Pion, Marjorie; Wiznerowicz, Maciej; Geijtenbeek, Teunis B; Garcia, Eduardo; Abraham, Shahnaz; Leuba, Florence; Dutoit, Valérie; Ducrey-Rundquist, Odile; van Kooyk, Yvette; Trono, Didier; Piguet, Vincent

    2004-10-01

    In the early events of human immunodeficiency virus type 1 (HIV-1) infection, immature dendritic cells (DCs) expressing the DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) receptor capture small amounts of HIV-1 on mucosal surfaces and spread viral infection to CD4(+) T cells in lymph nodes (22, 34, 45). RNA interference has emerged as a powerful tool to gain insight into gene function. For this purpose, lentiviral vectors that express short hairpin RNA (shRNA) for the delivery of small interfering RNA (siRNA) into mammalian cells represent a powerful tool to achieve stable gene silencing. In order to interfere with DC-SIGN function, we developed shRNA-expressing lentiviral vectors capable of conditionally suppressing DC-SIGN expression. Selectivity of inhibition of human DC-SIGN and L-SIGN and chimpanzee and rhesus macaque DC-SIGN was obtained by using distinct siRNAs. Suppression of DC-SIGN expression inhibited the attachment of the gp120 envelope glycoprotein of HIV-1 to DC-SIGN transfectants, as well as transfer of HIV-1 to target cells in trans. Furthermore, shRNA-expressing lentiviral vectors were capable of efficiently suppressing DC-SIGN expression in primary human DCs. DC-SIGN-negative DCs were unable to enhance transfer of HIV-1 infectivity to T cells in trans, demonstrating an essential role for the DC-SIGN receptor in transferring infectious viral particles from DCs to T cells. The present system should have broad applications for studying the function of DC-SIGN in the pathogenesis of HIV as well as other pathogens also recognized by this receptor.

  19. Identification and in vitro evaluation of new leads as selective and competitive glycogen synthase kinase-3β inhibitors through ligand and structure based drug design.

    PubMed

    Darshit, B S; Balaji, B; Rani, P; Ramanathan, M

    2014-09-01

    Glycogen synthase kinase-3β elicits multi-functional effects on intracellular signaling pathways, thereby making the kinase a therapeutic target in multiple pathologies. Hence, it is important to selectively inhibit GSK-3β over structurally and biologically similar targets, such as CDK5. The current study was designed to identify and evaluate novel ATP-competitive GSK-3β inhibitors. The study was designed to identify new leads by ligand based drug design, structure based drug design and in vitro evaluation. The best validated pharmacophore model (AADRRR) identified using LBDD was derived from a dataset of 135 molecules. There were 357 primary hits within the SPECS database using this pharmacophore model. A SBDD approach to the GSK-3β and CDK5 proteins was applied to all primary hits, and 5 selective inhibitors were identified for GSK-3β. GSK-3β and CDK5 in vitro kinase inhibition assays were performed with these molecules to confirm their selectivity for GSK-3β. The molecules showed IC50 values ranging from 0.825μM to 1.116μM and were 23- to 57-fold selective for GSK-3β. Of all the molecules, molecule 3 had the lowest IC50 value of 0.825μM. Our research identified molecules possessing benzothiophene, isoquinoline, thiazolidinedione imidazo-isoquinoline and quinazolinone scaffolds. Potency of these molecules may be due to H-bond interaction with backbone residues of Val135, Asp133 and side chain interaction with Tyr134. Selectivity over CDK5 may be due to side chain interactions with Asp200, backbone of Val61, ionic interaction with Lys60 and π-cationic interaction with Arg141. These selective molecules were also exhibited small atom hydrophobicity and H-bond interaction with water molecule.

  20. Danshensu, a major water-soluble component of Salvia miltiorrhiza, enhances the radioresponse for Lewis Lung Carcinoma xenografts in mice

    PubMed Central

    Cao, Hong-Ying; Ding, Rui-Lin; Li, Meng; Yang, Mao-Nan; Yang, Ling-Lin; Wu, Jing-Bo; Yang, Bo; Wang, Jing; Luo, Cui-Lian; Wen, Qing-Lian

    2017-01-01

    The molecule 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid (danshensu), a herbal preparation used in traditional Chinese medicine, has been found to possess potential antitumor and anti-angiogenesis effects. The aim of the present study was to investigate the efficacy of the combination of radiation therapy (RT) with danshensu in the treatment of Lewis lung carcinoma (LLC) xenografts, whilst exploring and evaluating the mechanism involved. In total, 8-week old female C57BL/6J mice were randomly assigned into 3 groups to receive: RT, RT + cisplatin and RT + danshensu, respectively, when LLC reached 100–150 mm3. Each group was divided into 7 subgroups according to the different irradiation doses that were administered. Tumor growth curves were created and the sensitization enhancement ratios of the drugs were calculated. The experiment was then repeated, and the 4 groups of tumor-bearing mice were treated with natural saline, danshensu, RT + danshensu and RT, respectively. The mice were sacrificed on day 7, and tumor tissue and blood were collected to determine microvessel density, the expression of proangiogenic factors, and the levels of blood thromboxane B2 and 6-keto-prostaglandin-F1α. Tumor hypoxia was also detected using in vivo fluorescence imaging. With respect to LLC xenografts, treatment with danshensu + RT significantly enhanced the effects of tumor growth inhibition (P<0.05). Furthermore, tumor vasculature was remodeled and microcirculation was improved, which significantly reduced tumor hypoxia (P<0.05). The present study demonstrated that danshensu significantly enhanced the radioresponse of LLC xenografts in mice. The mechanism involved may be associated with the alleviation of tumor cell hypoxia following treatment with danshensu + RT, caused by the improvement of tumor microcirculation and the remodeling of tumor vasculature. PMID:28356936

  1. Adhesion molecules in vernal keratoconjunctivitis

    PubMed Central

    El-Asrar, A.; Geboes, K.; Al-Kharashi, S.; Tabbara, K.; Missotten, L.; Desmet, V.

    1997-01-01

    AIMS/BACKGROUND—Adhesion molecules play a key role in the selective recruitment of different leucocyte population to inflammatory sites. The purpose of the present study was to investigate the presence and distribution of adhesion molecules in the conjunctiva of patients with vernal keratoconjunctivitis (VKC).
METHODS—The presence and distribution of adhesion molecules were studied in 14 conjunctival biopsy specimens from seven patients with active VKC and in four normal conjunctival biopsy specimens. We used a panel of specific monoclonal antibodies (mAbs) directed against intercellular adhesion molecule-1 (ICAM-1), intercellular adhesion molecule-3 (ICAM-3), lymphocyte function associated antigen-1 (LFA-1), very late activation antigen-4 (VLA-4), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leucocyte adhesion molecule-1 (ELAM-1). In addition, a panel of mAbs were used to characterise the composition of the inflammatory infiltrate.
RESULTS—In the normal conjunctiva, ICAM-1 was expressed on the vascular endothelium only, LFA-1 and ICAM-3 on epithelial and stromal mononuclear cells , and VLA-4 on stromal mononuclear cells. The expression of VCAM-1 and ELAM-1 was absent. The number of cells expressing adhesion molecules was found to be markedly increased in all VKC specimens. This was concurrent with a heavy inflammatory infiltrate. Strong ICAM-1 expression was induced on the basal epithelial cells, and vascular endothelial cells. Furthermore, ICAM-1 was expressed on stromal mononuclear cells. LFA-1 and ICAM-3 were expressed on the majority of epithelial and stromal infiltrating mononuclear cells. VLA-4 expression was noted on stromal mononuclear cells. Compared with controls, VKC specimens showed significantly more ICAM-3+, LFA-1+, and VLA-4+ cells. VCAM-1 and ELAM-1 were induced on the vascular endothelial cells.
CONCLUSIONS—Increased expression of adhesion molecules may play an important role in the pathogenesis of VKC.

 PMID

  2. Exposure of airway epithelial cells to Pseudomonas aeruginosa biofilm-derived quorum sensing molecules decrease the activity of the anti-oxidant response element bound by NRF2.

    PubMed

    Roussel, Lucie; Rousseau, Simon

    2017-02-05

    Chronic bacterial infections in cystic fibrosis lung disease are often characterized by Pseudomonas aeruginosa biofilms that are regulated by bacterial intercellular signals termed quorum sensing (QS), such as N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL). This study reports that biofilm-derived exoproducts decrease the transcriptional activity of the anti-oxidant response element in bronchial epithelial cells. In a live co-culture assay of BEAS-2B cells and P. aeruginosa biofilm, the QS molecule 3OC12-HSL was an important but not sole contributor to the inhibition of basal NRF2 luciferase reporter activity. Moreover, biofilm-derived exoproducts and 3OC12-HSL decrease the expression of endogenous antioxidant response element-regulated genes hemeoxygenase-1 (HO-1) and NAD(P)H Quinone Dehydrogenase-1 (NQO-1) while they increase IL-8 expression. As previously reported, IL-8 expression is partially dependent on p38 MAPK activity, but the inhibitory effect of biofilm QS molecules on HO-1 and NQO-1 expression occurs independently of this protein kinase. Finally, the transfection of CFTRdelF508 but not its wild type counterpart decreases basal, planktonic PsaDM and sulforaphane-driven NRF2 luciferase reporter activity in BEAS-2B cells. Therefore, the presence of quorum sensing molecules derived from bacterial biofilms lowers the transcriptional activity of the anti-oxidant response element, which may contribute to the establishment of chronic bacterial infections, especially in the presence of mutated CFTR. Increasing NRF2 activity may thus be a promising strategy to potentiate anti-biofilm activity in cystic fibrosis lung disease.

  3. Differential Immune Modulatory Activity of Pseudomonas aeruginosa Quorum-Sensing Signal Molecules

    PubMed Central

    Hooi, Doreen S. W.; Bycroft, Barrie W.; Chhabra, Siri Ram; Williams, Paul; Pritchard, David I.

    2004-01-01

    Pseudomonas aeruginosa releases a spectrum of well-regulated virulence factors, controlled by intercellular communication (quorum sensing) and mediated through the production of small diffusible quorum-sensing signal molecules (QSSM). We hypothesize that QSSM may in fact serve a dual purpose, also allowing bacterial colonization via their intrinsic immune-modulatory capacity. One class of signal molecule, the N-acylhomoserine lactones, has pleiotropic effects on eukaryotic cells, particularly those involved in host immunity. In the present study, we have determined the comparative effects of two chemically distinct and endobronchially detectable QSSM, N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) and 2-heptyl-3-hydroxy-4 (1H)-quinolone or the Pseudomonas quinolone signal (PQS), on human leukocytes exposed to a series of stimuli designed to detect differential immunological activity in vitro. 3-Oxo-C12-HSL and PQS displayed differential effects on the release of interleukin-2 (IL-2) when human T cells were activated via the T-cell receptor and CD28 (a costimulatory molecule). 3-Oxo-C12-HSL inhibited cell proliferation and IL-2 release; PQS inhibited cell proliferation without affecting IL-2 release. Both molecules inhibited cell proliferation and the release of IL-2 following mitogen stimulation. Furthermore, in the presence of Escherichia coli lipopolysaccharide, 3-oxo-C12-HSL inhibited tumor necrosis factor alpha release from human monocytes, as reported previously (K. Tateda et al., Infect. Immun. 64:37-43, 1996), whereas PQS did not inhibit in this assay. These data highlight the presence of two differentially active immune modulatory QSSM from P. aeruginosa, which are detectable endobronchially and may be active at the host/pathogen interface during infection with P. aeruginosa, should the bronchial airway lymphoid tissues prove to be accessible to QSSM. PMID:15501777

  4. Differential immune modulatory activity of Pseudomonas aeruginosa quorum-sensing signal molecules.

    PubMed

    Hooi, Doreen S W; Bycroft, Barrie W; Chhabra, Siri Ram; Williams, Paul; Pritchard, David I

    2004-11-01

    Pseudomonas aeruginosa releases a spectrum of well-regulated virulence factors, controlled by intercellular communication (quorum sensing) and mediated through the production of small diffusible quorum-sensing signal molecules (QSSM). We hypothesize that QSSM may in fact serve a dual purpose, also allowing bacterial colonization via their intrinsic immune-modulatory capacity. One class of signal molecule, the N-acylhomoserine lactones, has pleiotropic effects on eukaryotic cells, particularly those involved in host immunity. In the present study, we have determined the comparative effects of two chemically distinct and endobronchially detectable QSSM, N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and 2-heptyl-3-hydroxy-4 (1H)-quinolone or the Pseudomonas quinolone signal (PQS), on human leukocytes exposed to a series of stimuli designed to detect differential immunological activity in vitro. 3-Oxo-C12-HSL and PQS displayed differential effects on the release of interleukin-2 (IL-2) when human T cells were activated via the T-cell receptor and CD28 (a costimulatory molecule). 3-Oxo-C12-HSL inhibited cell proliferation and IL-2 release; PQS inhibited cell proliferation without affecting IL-2 release. Both molecules inhibited cell proliferation and the release of IL-2 following mitogen stimulation. Furthermore, in the presence of Escherichia coli lipopolysaccharide, 3-oxo-C12-HSL inhibited tumor necrosis factor alpha release from human monocytes, as reported previously (K. Tateda et al., Infect. Immun. 64:37-43, 1996), whereas PQS did not inhibit in this assay. These data highlight the presence of two differentially active immune modulatory QSSM from P. aeruginosa, which are detectable endobronchially and may be active at the host/pathogen interface during infection with P. aeruginosa, should the bronchial airway lymphoid tissues prove to be accessible to QSSM.

  5. Analysis of the magnetically induced current density of molecules consisting of annelated aromatic and antiaromatic hydrocarbon rings.

    PubMed

    Sundholm, Dage; Berger, Raphael J F; Fliegl, Heike

    2016-06-21

    Magnetically induced current susceptibilities and current pathways have been calculated for molecules consisting of two pentalene groups annelated with a benzene (1) or naphthalene (2) moiety. Current strength susceptibilities have been obtained by numerically integrating separately the diatropic and paratropic contributions to the current flow passing planes through chosen bonds of the molecules. The current density calculations provide novel and unambiguous current pathways for the unusual molecules with annelated aromatic and antiaromatic hydrocarbon moieties. The calculations show that the benzene and naphthalene moieties annelated with two pentalene units as in molecules 1 and 2, respectively, are unexpectedly antiaromatic sustaining only a local paratropic ring current around the ring, whereas a weak diatropic current flows around the C-H moiety of the benzene ring. For 1 and 2, the individual five-membered rings of the pentalenes are antiaromatic and a slightly weaker semilocal paratropic current flows around the two pentalene rings. Molecules 1 and 2 do not sustain any net global ring current. The naphthalene moiety of the molecule consisting of a naphthalene annelated with two pentalene units (3) does not sustain any strong ring current that is typical for naphthalene. Instead, half of the diatropic current passing the naphthalene moiety forms a zig-zag pattern along the C-C bonds of the naphthalene moiety that are not shared with the pentalene moieties and one third of the current continues around the whole molecule partially cancelling the very strong paratropic semilocal ring current of the pentalenes. For molecule 3, the pentalene moieties and the individual five-membered rings of the pentalenes are more antiaromatic than for 1 and 2. The calculated current patterns elucidate why the compounds with formally [4n + 2] π-electrons have unusual aromatic properties violating the Hückel π-electron count rule. The current density calculations also provide

  6. Ferroptosis: process and function.

    PubMed

    Xie, Y; Hou, W; Song, X; Yu, Y; Huang, J; Sun, X; Kang, R; Tang, D

    2016-03-01

    Ferroptosis is a recently recognized form of regulated cell death. It is characterized morphologically by the presence of smaller than normal mitochondria with condensed mitochondrial membrane densities, reduction or vanishing of mitochondria crista, and outer mitochondrial membrane rupture. It can be induced by experimental compounds (e.g., erastin, Ras-selective lethal small molecule 3, and buthionine sulfoximine) or clinical drugs (e.g., sulfasalazine, sorafenib, and artesunate) in cancer cells and certain normal cells (e.g., kidney tubule cells, neurons, fibroblasts, and T cells). Activation of mitochondrial voltage-dependent anion channels and mitogen-activated protein kinases, upregulation of endoplasmic reticulum stress, and inhibition of cystine/glutamate antiporter is involved in the induction of ferroptosis. This process is characterized by the accumulation of lipid peroxidation products and lethal reactive oxygen species (ROS) derived from iron metabolism and can be pharmacologically inhibited by iron chelators (e.g., deferoxamine and desferrioxamine mesylate) and lipid peroxidation inhibitors (e.g., ferrostatin, liproxstatin, and zileuton). Glutathione peroxidase 4, heat shock protein beta-1, and nuclear factor erythroid 2-related factor 2 function as negative regulators of ferroptosis by limiting ROS production and reducing cellular iron uptake, respectively. In contrast, NADPH oxidase and p53 (especially acetylation-defective mutant p53) act as positive regulators of ferroptosis by promotion of ROS production and inhibition of expression of SLC7A11 (a specific light-chain subunit of the cystine/glutamate antiporter), respectively. Misregulated ferroptosis has been implicated in multiple physiological and pathological processes, including cancer cell death, neurotoxicity, neurodegenerative diseases, acute renal failure, drug-induced hepatotoxicity, hepatic and heart ischemia/reperfusion injury, and T-cell immunity. In this review, we summarize the

  7. A novel ICK peptide from the Loxosceles intermedia (brown spider) venom gland: cloning, heterologous expression and immunological cross-reactivity approaches.

    PubMed

    Matsubara, Fernando Hitomi; Gremski, Luiza Helena; Meissner, Gabriel Otto; Constantino Lopes, Eduardo Soares; Gremski, Waldemiro; Senff-Ribeiro, Andrea; Chaim, Olga Meiri; Veiga, Silvio Sanches

    2013-09-01

    The venom of a Loxosceles spider is composed of a complex mixture of biologically active components, consisting predominantly of low molecular mass molecules (3-45 kDa). Transcriptome analysis of the Loxosceles intermedia venom gland revealed ESTs with similarity to the previously described LiTx peptides. Sequences similar to the LiTx3 isoform were the most abundant, representing approximately 13.9% of all ESTs and 32% of the toxin-encoding messengers. These peptides are grouped in the ICK (Inhibitor Cystine Knot) family, which contains single chain molecules with low molecular mass (3-10 kDa). Due to their high number of cysteine residues, ICK peptides form intramolecular disulfide bridges. The aims of this study were to clone and express a novel ICK peptide isoform, as well as produce specific hyperimmune serum for immunoassays. The corresponding cDNA was amplified by PCR using specific primers containing restriction sites for the XhoI and BamHI enzymes; this PCR product was then ligated in the pET-14b vector and transformed into E. coli AD494 (DE3) cells. The peptide was expressed by IPTG induction for 4 h at 30 °C and purified by affinity chromatography with Ni-NTA resin. Hyperimmune serum to the recombinant peptide was produced in rabbits and was able to specifically recognize both the purified recombinant peptide and the native form present in the venom. Furthermore, the recombinant peptide was recognized by antisera raised against L. intermedia, L. gaucho and L. laeta whole venoms. The recombinant peptide obtained will enable future studies to characterize its biological activity, as well as investigations regarding possible biotechnological applications.

  8. Tissue-Specific Inactivation of Type 2 Deiodinase Reveals Multilevel Control of Fatty Acid Oxidation by Thyroid Hormone in the Mouse

    PubMed Central

    Fonseca, Tatiana L.; Werneck-De-Castro, Joao Pedro; Castillo, Melany; Bocco, Barbara M.L.C.; Fernandes, Gustavo W.; McAninch, Elizabeth A.; Ignacio, Daniele L.; Moises, Caio C.S.; Ferreira, Alexandre; Gereben, Balázs

    2014-01-01

    Type 2 deiodinase (D2) converts the prohormone thyroxine (T4) to the metabolically active molecule 3,5,3′-triiodothyronine (T3), but its global inactivation unexpectedly lowers the respiratory exchange rate (respiratory quotient [RQ]) and decreases food intake. Here we used FloxD2 mice to generate systemically euthyroid fat-specific (FAT), astrocyte-specific (ASTRO), or skeletal-muscle-specific (SKM) D2 knockout (D2KO) mice that were monitored continuously. The ASTRO-D2KO mice also exhibited lower diurnal RQ and greater contribution of fatty acid oxidation to energy expenditure, but no differences in food intake were observed. In contrast, the FAT-D2KO mouse exhibited sustained (24 h) increase in RQ values, increased food intake, tolerance to glucose, and sensitivity to insulin, all supporting greater contribution of carbohydrate oxidation to energy expenditure. Furthermore, FAT-D2KO animals that were kept on a high-fat diet for 8 weeks gained more body weight and fat, indicating impaired brown adipose tissue (BAT) thermogenesis and/or inability to oxidize the fat excess. Acclimatization of FAT-D2KO mice at thermoneutrality dissipated both features of this phenotype. Muscle D2 does not seem to play a significant metabolic role given that SKM-D2KO animals exhibited no phenotype. The present findings are unique in that they were obtained in systemically euthyroid animals, revealing that brain D2 plays a dominant albeit indirect role in fatty acid oxidation via its sympathetic control of BAT activity. D2-generated T3 in BAT accelerates fatty acid oxidation and protects against diet-induced obesity. PMID:24487027

  9. Understanding the complexation of Eu3 + with potential ligands used for preferential separation of lanthanides and actinides in various stages of nuclear fuel cycle: A luminescence investigation

    NASA Astrophysics Data System (ADS)

    Sengupta, Arijit; Kadam, R. M.

    2017-02-01

    A systematic photoluminescence based investigation was carried out to understand the complexation of Eu3 + with different ligands (TBP: tri-n-butyl phosphate, DHOA: di-n-hexyl octanamide, Cyanex 923: tri-n-alkyl phosphine oxide and Cyanex 272: Bis (2,4,4 trimethyl) pentyl phosphinic acid) used for preferential separation of lanthanides and actinides in various stages of nuclear fuel cycle. In case of TBP and DHOA complexes, 3 ligand molecules coordinated in monodentate fashion and 3 nitrate ion in bidentate fashion to Eu3 + to satisfy the 9 coordination of Eu. In case of Cyanex 923 and Cyanex 272 complexes, 3 ligand molecules, 3 nitrate ion and 3 water molecules coordinated to Eu3 + in monodentate fashion. The Eu complexes of TBP and DHOA were found to have D3h local symmetry while that for Cyanex 923 and Cyanex 272 were C3h. Judd-Ofelt analysis of these systems revealed that the covalency of Eusbnd O bond followed the trend DHOA > TBP > Cyanex 272 > Cyanex 923. Different photophysical properties like radiative and non-radiative life time, branching ratio for different transitions, magnetic and electric dipole moment transition probabilities and quantum efficiency were also evaluated and compared for these systems. The magnetic dipole transition probability was found to be almost independent of ligand field perturbation while electric dipole transition probability for 5D0-7F2 transition was found to be hypersensitive with ligand field with a trend DHOA > TBP > Cyanex 272 > Cyanex 923. Supplementary Table 2: Determination of inner sphere water molecules from the different empirical formulae reported in the literature.

  10. Weak Ergodicity Breaking of Receptor Motion in Living Cells Stemming from Random Diffusivity

    NASA Astrophysics Data System (ADS)

    Manzo, Carlo; Torreno-Pina, Juan A.; Massignan, Pietro; Lapeyre, Gerald J.; Lewenstein, Maciej; Garcia Parajo, Maria F.

    2015-01-01

    Molecular transport in living systems regulates numerous processes underlying biological function. Although many cellular components exhibit anomalous diffusion, only recently has the subdiffusive motion been associated with nonergodic behavior. These findings have stimulated new questions for their implications in statistical mechanics and cell biology. Is nonergodicity a common strategy shared by living systems? Which physical mechanisms generate it? What are its implications for biological function? Here, we use single-particle tracking to demonstrate that the motion of dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), a receptor with unique pathogen-recognition capabilities, reveals nonergodic subdiffusion on living-cell membranes In contrast to previous studies, this behavior is incompatible with transient immobilization, and, therefore, it cannot be interpreted according to continuous-time random-walk theory. We show that the receptor undergoes changes of diffusivity, consistent with the current view of the cell membrane as a highly dynamic and diverse environment. Simulations based on a model of an ordinary random walk in complex media quantitatively reproduce all our observations, pointing toward diffusion heterogeneity as the cause of DC-SIGN behavior. By studying different receptor mutants, we further correlate receptor motion to its molecular structure, thus establishing a strong link between nonergodicity and biological function. These results underscore the role of disorder in cell membranes and its connection with function regulation. Because of its generality, our approach offers a framework to interpret anomalous transport in other complex media where dynamic heterogeneity might play a major role, such as those found, e.g., in soft condensed matter, geology, and ecology.

  11. A Atomic-Scale View of Motion and Interactions on Surfaces

    NASA Astrophysics Data System (ADS)

    Stranick, Stephan Jeffrey

    The scanning tunneling microscope (STM) affords an atomic-scale view of the motions and the interactions of adsorbates on surfaces. One of the aspects of STM that makes it ideal for the study of surface motions is the ability to probe the dynamics of the system as well as the resulting structures with atomic resolution. Motion on surfaces is critical in surface processes where adsorbates must reach special sites to undergo reaction; in film growth to allow epitaxial or other special growth modes; and in other surface phenomena such as etching, corrosion, and wetting. Rates of adsorbate motion are often dominated by the effects of interactions with sites such as steps, other defects, and coadsorbates. The interactions of adsorbates on surfaces continue to be of fundamental interest and of technological importance. Adsorbate-adsorbate interactions are generally of three types: dipole-dipole, orbital overlap, and indirect or substrate-mediated. These interactions determine the energetics, structure, and dynamics of the surface-adsorbate system. In this thesis, the advances in our fundamental understanding of surface dynamics and interactions as well as the advances in our technological capabilities to study these processes are outlined and described: We have (1) elucidated the interactions and dynamics of benzene molecules on a Cu{111 } surface on the atomic scale, (2) provided "real space" evidence for the nanometer-scale phase separation of two similar molecules, (3) demonstrated a new mechanism for surface diffusion of an adsorbate-substrate atom complex, (4) developed a new technique for the topographic and spectroscopic characterization of insulating films and solids, and (5) demonstrated that short-range interactions can result in long-range order.

  12. DNA binding by a new metallointercalator that contains a proflavine group bearing a hanging chelating unit.

    PubMed

    Bazzicalupi, Carla; Bencini, Andrea; Bianchi, Antonio; Biver, Tarita; Boggioni, Alessia; Bonacchi, Sara; Danesi, Andrea; Giorgi, Claudia; Gratteri, Paola; Ingraín, Antonio Marchal; Secco, Fernando; Sissi, Claudia; Valtancoli, Barbara; Venturini, Marcella

    2008-01-01

    The new bifunctional molecule 3,6-diamine-9-[6,6-bis(2-aminoethyl)-1,6-diaminohexyl]acridine (D), which is characterised by both an aromatic moiety and a separate metal-complexing polyamine centre, has been synthesised. The characteristics of D and its ZnII complex ([ZnD]) (protonation and metal-complexing constants, optical properties and self-aggregation phenomena) have been analysed by means of NMR spectroscopy, potentiometric, spectrophotometric and spectrofluorimetric techniques. The equilibria and kinetics of the binding process of D and [ZnD] to calf thymus DNA have been investigated at I=0.11 M (NaCl) and 298.1 K by using spectroscopic methods and the stopped-flow technique. Static measurements show biphasic behaviour for both D-DNA and [ZnD]-DNA systems; this reveals the occurrence of two different binding processes depending on the polymer-to-dye molar ratio (P/D). The binding mode that occurs at low P/D values is interpreted in terms of external binding with a notable contribution from the polyamine residue. The binding mode at high P/D values corresponds to intercalation of the proflavine residue. Stopped-flow, circular dichroism and supercoiled-DNA unwinding experiments corroborate the proposed mechanism. Molecular-modelling studies support the intercalative process and evidence the influence of NH+...O interactions between the protonated acridine nitrogen atom and the oxygen atoms of the polyanion; these interactions play a key role in determining the conformation of DNA adducts.

  13. SKLB70326, a novel small-molecule inhibitor of cell-cycle progression, induces G{sub 0}/G{sub 1} phase arrest and apoptosis in human hepatic carcinoma cells

    SciTech Connect

    Han, Yuanyuan; He, Haiyun; Peng, Feng; Liu, Jiyan; Dai, Xiaoyun; Lin, Hongjun; Xu, Youzhi; Zhou, Tian; Mao, Yongqiu; Xie, Gang; Yang, Shengyong; Yu, Luoting; Yang, Li; Zhao, Yinglan

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer SKLB70326 is a novel compound and has activity of anti-HCC. Black-Right-Pointing-Pointer SKLB70326 induces cell cycle arrest and apoptosis in HepG2 cells. Black-Right-Pointing-Pointer SKLB70326 induces G{sub 0}/G{sub 1} phase arrest via inhibiting the activity of CDK2, CDK4 and CDK6. Black-Right-Pointing-Pointer SKLB70326 induces apoptosis through the intrinsic pathway. -- Abstract: We previously reported the potential of a novel small molecule 3-amino-6-(3-methoxyphenyl)thieno[2.3-b]pyridine-2-carboxamide (SKLB70326) as an anticancer agent. In the present study, we investigated the anticancer effects and possible mechanisms of SKLB70326 in vitro. We found that SKLB70326 treatment significantly inhibited human hepatic carcinoma cell proliferation in vitro, and the HepG2 cell line was the most sensitive to its treatment. The inhibition of cell proliferation correlated with G{sub 0}/G{sub 1} phase arrest, which was followed by apoptotic cell death. The SKLB70326-mediated cell-cycle arrest was associated with the downregulation of cyclin-dependent kinase (CDK) 2, CDK4 and CDK6 but not cyclin D1 or cyclin E. The phosphorylation of the retinoblastoma protein (Rb) was also observed. SKLB70326 treatment induced apoptotic cell death via the activation of PARP, caspase-3, caspase-9 and Bax as well as the downregulation of Bcl-2. The expression levels of p53 and p21 were also induced by SKLB70326 treatment. Moreover, SKLB70326 treatment was well tolerated. In conclusion, SKLB70326, a novel cell-cycle inhibitor, notably inhibits HepG2 cell proliferation through the induction of G{sub 0}/G{sub 1} phase arrest and subsequent apoptosis. Its potential as a candidate anticancer agent warrants further investigation.

  14. Synthesis, molecular structure, hydrogen-bonding, NBO and chemical reactivity analysis of a novel 1,9-bis(2-cyano-2-ethoxycarbonylvinyl)-5-(4-hydroxyphenyl)-dipyrromethane: a combined experimental and theoretical (DFT and QTAIM) approach.

    PubMed

    Singh, R N; Kumar, Amit; Tiwari, R K; Rawat, Poonam

    2013-09-01

    The spectroscopic analysis of a newly synthesized 1,9-bis(2-cyano-2-ethoxycarbonylvinyl)-5-(4-hydroxyphenyl)-dipyrromethane (3) has been carried out using (1)H NMR, UV-Visible, FT-IR and Mass spectroscopic techniques. All the quantum chemical calculations have been carried out using DFT level of theory, B3LYP functional and 6-31G(d,p) as basis set. Thermodynamic parameters (H, G, S) of all the reactants and products have been used to determine the nature of the chemical reaction. The chemical shift of pyrrolic NH in (1)H NMR spectrum appears at 9.4 ppm due to intramolecular hydrogen bonding. TD-DFT calculation shows the nature of electronic transitions as π→π(*) within the molecule. A combined experimental and theoretical vibrational analysis designates the existence of H-bonding between pyrrole N-H as proton donor and nitrogen of cyanide as proton acceptor, therefore, lowering in stretching vibration of NH and CN. To investigate the strength and nature of H-bonding, topological parameters at bond critical points (BCPs) are analyzed by 'Quantum theory of Atoms in molecules' (QTAIMs). Natural bond orbitals (NBOs) analysis has been carried out to investigate the intramolecular conjugative and hyperconjugative interactions within molecule and their second order stabilization energy (E((2))). Global electrophilicity index (ω=4.528 eV) shows that title molecule (3) is a strong electrophile. The maximum values of local electrophilic reactivity descriptors (fk(+),sk(+),ωk(+)) at vinyl carbon (C6/C22) of (3) indicate that these sites are more prone to nucleophilic attacks.

  15. Synthesis, superoxide dismutase, nuclease, and anticancer activities of copper(II) complexes incorporating bis(2-picolyl)amine with different counter anions

    NASA Astrophysics Data System (ADS)

    Ibrahim, Mohamed M.; Ramadan, Abdel-Motaleb M.; Mersal, Gaber A. M.; El-Shazly, Samir A.

    2011-07-01

    Interaction of the tridentate ligand bis(2-picolyl)amine L with copper(II) salts gave a series of copper(II) complexes with the formula types: [ LCu(X) 2] (X = Cl -1, = Br -2), [( LCu (H 2O)(μ-SO 4)( LCu(H 2O)]SO 43, [ LCu(OAc)](OAc )H 2O 4, [ LCu(H 2O) 2](Y) 2 (Y = NO3-5, = ClO4-6). Their structures and properties were characterized by elemental analysis, thermal analysis (TGA), IR, UV-vis and ESR spectroscopy, electrochemical measurements including cyclic voltammetry and electrical molar conductivity, and magnetic moment measurements. A square pyramidal geometry is proposed for the halogeno complexes 1 and 2 in monomeric structures. For sulfate complex, the sulfate group bridged two copper(II) ions of the two [N 3O] donor units to give the dimeric complex molecule 3 in square pyramidal environment around the copper(II) ions. In the case of complexes 4- 6, square planar stereochemistries in monomeric structures are suggested. The SOD biomimetic catalytic activity of the obtained complexes was assessed for their ability to inhibit the reduction of nitroblue tetrazolium (NBT). The catalytic efficiency of O2- scavenging by complexes depends on the nature of the particular acidic anion radical incorporated in the complex molecule and follows the order: NO3- > ClO4- > Br - ⩾ Cl - > SO4- > AcO -. A probable mechanistic implications for the catalytic dismutation of O2- by copper(II) complexes are proposed. Furthermore, complex 1 exhibits significant hydrolytic cleavage of the genomic DNA in the absence of any external additives. In addition, the in vitro study of cytotoxicity of complex 1 on colon cancer cell line (Caco-2) indicates that the complex has the potential to act as an effective anticancer drug with IC 50 value of 156 ± 0.35 μM.

  16. Shape-Engineering of Self-Assembled Organic Single Microcrystal as Optical Microresonator for laser Applications

    PubMed Central

    Wang, Xuedong; Liao, Qing; Lu, Xiaomei; Li, Hui; Xu, Zhenzhen; Fu, Hongbing

    2014-01-01

    Single micro/nanocrystals based on π-conjugated organic molecules have caused tremendous interests in the optoelectronic applications in laser, optical waveguide, nonlinear optics, and field effect transistors. However, the controlled synthesis of these organic micro/nanocrystals with regular shapes is very difficult to achieve, because the weak interaction (van der Waals' force, ca. 5 kJ/mol) between organic molecules could not dominate the kinetic process of crystal growth. Herein, we develop an elaborate strategy, selective adhesion to organic crystal plane by the hydrogen-bonding interaction (ca. 40 kJ/mol), for modulating the kinetic process of the formation of microcrystal, which leads to the self-assembly of one organic molecule 3-[4-(dimethylamino)phenyl]-1-(2-hy-droxyphenyl)prop-2-en-1-on (HDMAC) into one-dimensional (1D) microwires and 2D microdisks respectively. Furthermore, these as-prepared microcrystals demonstrate shape-dependent microresonator properties that 1D microwires act as Fabry-Pérot (FP) mode lasing resonator and 2D microdisks provide the whispering-gallery-mode (WGM) resonator for lasing oscillator. More significantly, through the investigation of the size-effect on the laser performance, single-mode lasing at red wavelength was successfully achieved in the self-assembled 2D organic microdisk at room temperature. These easily fabricated organic single-crystalline microcrystals with controlled shapes are the natural laser sources, which offer considerable promise for the multi-functionalities of coherent light devices integrated on the optics microchip. PMID:25388213

  17. Production of sialylated O-linked glycans in Pichia pastoris.

    PubMed

    Hamilton, Stephen R; Cook, W James; Gomathinayagam, Sujatha; Burnina, Irina; Bukowski, John; Hopkins, Daniel; Schwartz, Shaina; Du, Min; Sharkey, Nathan J; Bobrowicz, Piotr; Wildt, Stefan; Li, Huijuan; Stadheim, Terrance A; Nett, Juergen H

    2013-10-01

    The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. Previously, we have reported the glycoengineering of this organism to produce human-like N-linked glycans but up to now no one has addressed engineering the O-linked glycosylation pathway. Typically, O-linked glycans produced by wild-type P. pastoris are linear chains of four to five α-linked mannose residues, which may be capped with β- or phospho-mannose. Previous genetic engineering of the N-linked glycosylation pathway of P. pastoris has eliminated both of these two latter modifications, resulting in O-linked glycans which are linear α-linked mannose structures. Here, we describe a method for the co-expression of an α-1,2-mannosidase, which reduces these glycans to primarily a single O-linked mannose residue. In doing so, we have reduced the potential of these glycans to interact with carbohydrate-binding proteins, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin. Furthermore, the introduction of the enzyme protein-O-linked-mannose β-1,2-N-acetylglucosaminyltransferase 1, resulted in the capping of the single O-linked mannose residues with N-acetylglucosamine. Subsequently, this glycoform was extended into human-like sialylated glycans, similar in structure to α-dystroglycan-type glycoforms. As such, this represents the first example of sialylated O-linked glycans being produced in yeast and extends the utility of the P. pastoris production platform beyond N-linked glycosylated biotherapeutics to include molecules possessing O-linked glycans.

  18. Association of DC-SIGNR Expression in Peripheral Blood Mononuclear Cells with DC-SIGNR Genotypes in HIV-1 Infection.

    PubMed

    Chaudhary, Omkar; Kumar, Sanjeev; Bala, Manju; Singh, Jasbir; Hazarika, Anjali; Luthra, Kalpana

    2015-10-01

    Dendritic cell-specific intracellular adhesion molecule 3 grabbing nonintegrin related molecule (DC-SIGNR) is a C-type lectin, calcium-dependent carbohydrate-binding protein, which can act as a cell-adhesion and pathogen recognition receptor. DC-SIGNR is known to be highly expressed on liver sinusoidal cells and in the lymph nodes. However, its expression in peripheral blood mononuclear cells (PBMCs) in HIV-1 infection has not been addressed. Therefore, this study determined the expression of DC-SIGNR in PBMCs of HIV-1-infected patients and healthy seronegative individuals by real-time polymerase chain reaction and assessed its correlation with CD4+ T cell counts and DC-SIGNR genotypes. A significantly higher expression of DC-SIGNR was observed in the PBMCs of HIV-1-infected patients compared with healthy seronegative individuals. Further, there was a negative correlation between DC-SIGNR expression and CD4+ T cell counts and positive with viral load, with higher DC-SIGNR expression in the PBMCs of HIV-1-infected patients with a CD4+ T cell count <200 cells/μL than those with >200 cells/μL. This is the first study to report the expression of DC-SIGNR in PBMCs of HIV-1-infected patients. A salient finding of this study is that the DC-SIGNR expression was higher in HIV-1-infected patients, and its positive correlation with viral load and negative with CD4+ T cells counts suggesting a potential role of DC-SIGNR in HIV-1 infection.

  19. Gp120 binding with DC-SIGN induces reactivation of HIV-1 provirus via the NF-κB signaling pathway.

    PubMed

    Jin, Changzhong; Li, Jie; Cheng, Linfang; Liu, Fumin; Wu, Nanping

    2016-03-01

    The reactivation mechanism of latent human immunodeficiency virus type 1 (HIV-1) infection is unclear, especially in dendritic cells (DC). DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) binds with HIV-1 and other pathogens to activate the extracellular regulated protein kinase (ERK) and nuclear factor-kappa B (NF-κB) pathways and regulate cytokine expression. We hypothesized that DC-SIGN-induced signaling pathways may activate HIV-1 provirus. To investigate this hypothesis, we generated a model by transfecting 293T cells with a DC-SIGN expression plasmid and an HIV-1 5' long terminal repeat (LTR) reporter plasmid, and then stimulated the 293T cells with HIV-1 gp120 protein, wild-type HIV-1 or VSV-G-pNL4.3 pseudotype virus (without gp120 protein). It was found that the HIV-1 5'LTR was reactivated by HIV-1 gp120 in DC-SIGN-expressing 293T cells. Then the HIV-1 chronically infected CEM-Bru cells were transfected with DC-SIGN expression plasmid and stimulated by HIV-1 gp120 protein. It was found that early and late HIV-1 provirus replication was reactivated by the HIV-1 gp120/DC-SIGN stimulation. We then investigated the involvement of the ERK, p38 mitogen-activated protein kinases and NF-κB signaling pathways in HIV-1 gp120/DC-SIGN-induced activation of HIV-1 provirus by inhibiting the pathways specifically. Our results indicated that HIV-1 gp120/DC-SIGN stimulation reactivates latent HIV-1 provirus via the NF-κB signal pathway.

  20. N-Glycans on the Rift Valley Fever Virus Envelope Glycoproteins Gn and Gc Redundantly Support Viral Infection via DC-SIGN.

    PubMed

    Phoenix, Inaia; Nishiyama, Shoko; Lokugamage, Nandadeva; Hill, Terence E; Huante, Matthew B; Slack, Olga A L; Carpio, Victor H; Freiberg, Alexander N; Ikegami, Tetsuro

    2016-05-23

    Rift Valley fever is a mosquito-transmitted, zoonotic disease that infects humans and ruminants. Dendritic cell specific intercellular adhesion molecule 3 (ICAM-3) grabbing non-integrin (DC-SIGN) acts as a receptor for members of the phlebovirus genus. The Rift Valley fever virus (RVFV) glycoproteins (Gn/Gc) encode five putative N-glycan sequons (asparagine (N)-any amino acid (X)-serine (S)/threonine (T)) at positions: N438 (Gn), and N794, N829, N1035, and N1077 (Gc). The N-glycosylation profile and significance in viral infection via DC-SIGN have not been elucidated. Gc N-glycosylation was first evaluated by using Gc asparagine (N) to glutamine (Q) mutants. Subsequently, we generated a series of recombinant RVFV MP-12 strain mutants, which encode N-to-Q mutations, and the infectivity of each mutant in Jurkat cells stably expressing DC-SIGN was evaluated. Results showed that Gc N794, N1035, and N1077 were N-glycosylated but N829 was not. Gc N1077 was heterogeneously N-glycosylated. RVFV Gc made two distinct N-glycoforms: "Gc-large" and "Gc-small", and N1077 was responsible for "Gc-large" band. RVFV showed increased infection of cells expressing DC-SIGN compared to cells lacking DC-SIGN. Infection via DC-SIGN was increased in the presence of either Gn N438 or Gc N1077. Our study showed that N-glycans on the Gc and Gn surface glycoproteins redundantly support RVFV infection via DC-SIGN.

  1. Structural characterization of the DC-SIGN-Lewis(X) complex.

    PubMed

    Pederson, Kari; Mitchell, Daniel A; Prestegard, James H

    2014-09-09

    Dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is a C-type lectin highly expressed on the surface of antigen-presenting dendritic cells. DC-SIGN mediates interactions among dendritic cells, pathogens, and a variety of epithelia, myeloid cells, and endothelia by binding to high mannose residues on pathogenic invaders or fucosylated residues on the membranes of other immune cells. Although these interactions are normally beneficial, they can also contribute to disease. The structural characterization of binding geometries is therefore of interest as a basis for the construction of mimetics that can mediate the effects of abnormal immune response. Here, we report the structural characteristics of the interaction of the DC-SIGN carbohydrate recognition domain (CRD) with a common fucosylated entity, the Lewis(X) trisaccharide (Le(X)), using NMR methods. Titration of the monomeric DC-SIGN CRD with Le(X) monitored by 2D NMR revealed significant perturbations of DC-SIGN cross-peak positions in (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra and identified residues near the binding site. Additionally, saturation transfer difference (STD) and transferred nuclear Overhauser effect (trNOE) NMR experiments, using a tetrameric form of DC-SIGN, identified binding epitopes and bound conformations of the Le(X) ligand. The restraints derived from these multiple experiments were used to generate models for the binding of Le(X) to the DC-SIGN CRD. Ranking of the models based on the fit of model-based simulations of the trNOE data and STD buildup curves suggested conformations distinct from those seen in previous crystal structures. The new conformations offer insight into how differences between binding of Lewis(X) and mannose-terminated saccharides may be propagated.

  2. DC-SIGN expression on podocytes and its role in inflammatory immune response of lupus nephritis.

    PubMed

    Cai, Minchao; Zhou, Tong; Wang, Xuan; Shang, Minghua; Zhang, Yueyue; Luo, Maocai; Xu, Chundi; Yuan, Weijie

    2016-03-01

    Podocytes, the main target of immune complex, participate actively in the development of glomerular injury as immune cells. Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) is an innate immune molecular that has an immune recognition function, and is involved in mediation of cell adhesion and immunoregulation. Here we explored the expression of DC-SIGN on podocytes and its role in immune and inflammatory responses in lupus nephritis (LN). Expression of DC-SIGN and immunoglobulin (Ig)G1 was observed in glomeruli of LN patients. DC-SIGN was co-expressed with nephrin on podocytes. Accompanied by increased proteinuria of LN mice, DC-SIGN and IgG1 expressions were observed in the glomeruli from 20 weeks, and the renal function deteriorated up to 24 weeks. Mice with anti-DC-SIGN antibody showed reduced proteinuria and remission of renal function. After the podocytes were stimulated by serum of LN mice in vitro, the expression of DC-SIGN, major histocompatibility complex (MHC) class II and CD80 was up-regulated, stimulation of T cell proliferation was enhanced and the interferon (IFN)-γ/interleukin (IL)-4 ratio increased. However, anti-DC-SIGN antibody treatment reversed these events. These results suggested that podocytes in LN can exert DC-like function through their expression of DC-SIGN, which may be involved in immune and inflammatory responses of renal tissues. However, blockage of DC-SIGN can inhibit immune functions of podocytes, which may have preventive and therapeutic effects.

  3. AFM force spectroscopy reveals how subtle structural differences affect the interaction strength between Candida albicans and DC-SIGN.

    PubMed

    te Riet, Joost; Reinieren-Beeren, Inge; Figdor, Carl G; Cambi, Alessandra

    2015-11-01

    The fungus Candida albicans is the most common cause of mycotic infections in immunocompromised hosts. Little is known about the initial interactions between Candida and immune cell receptors, such as the C-type lectin dendritic cell-specific intracellular cell adhesion molecule-3 (ICAM-3)-grabbing non-integrin (DC-SIGN), because a detailed characterization at the structural level is lacking. DC-SIGN recognizes specific Candida-associated molecular patterns, that is, mannan structures present in the cell wall of Candida. The molecular recognition mechanism is however poorly understood. We postulated that small differences in mannan-branching may result in considerable differences in the binding affinity. Here, we exploit atomic force microscope-based dynamic force spectroscopy with single Candida cells to gain better insight in the carbohydrate recognition capacity of DC-SIGN. We demonstrate that slight differences in the N-mannan structure of Candida, that is, the absence or presence of a phosphomannan side chain, results in differences in the recognition by DC-SIGN as follows: (i) it contributes to the compliance of the outer cell wall of Candida, and (ii) its presence results in a higher binding energy of 1.6 kB T. The single-bond affinity of tetrameric DC-SIGN for wild-type C. albicans is ~10.7 kB T and a dissociation constant kD of 23 μM, which is relatively strong compared with other carbohydrate-protein interactions described in the literature. In conclusion, this study shows that DC-SIGN specifically recognizes mannan patterns on C. albicans with high affinity. Knowledge on the binding pocket of DC-SIGN and its pathogenic ligands will lead to a better understanding of how fungal-associated carbohydrate structures are recognized by receptors of the immune system and can ultimately contribute to the development of new anti-fungal drugs.

  4. Human DC-SIGN binds specific human milk glycans.

    PubMed

    Noll, Alexander J; Yu, Ying; Lasanajak, Yi; Duska-McEwen, Geralyn; Buck, Rachael H; Smith, David F; Cummings, Richard D

    2016-05-15

    Human milk glycans (HMGs) are prebiotics, pathogen receptor decoys and regulators of host physiology and immune responses. Mechanistically, human lectins (glycan-binding proteins, hGBP) expressed by dendritic cells (DCs) are of major interest, as these cells directly contact HMGs. To explore such interactions, we screened many C-type lectins and sialic acid-binding immunoglobulin-like lectins (Siglecs) expressed by DCs for glycan binding on microarrays presenting over 200 HMGs. Unexpectedly, DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) showed robust binding to many HMGs, whereas other C-type lectins failed to bind, and Siglec-5 and Siglec-9 showed weak binding to a few glycans. By contrast, most hGBP bound to multiple glycans on other microarrays lacking HMGs. An α-linked fucose residue was characteristic of HMGs bound by DC-SIGN. Binding of DC-SIGN to the simple HMGs 2'-fucosyl-lactose (2'-FL) and 3-fucosyl-lactose (3-FL) was confirmed by flow cytometry to beads conjugated with 2'-FL or 3-FL, as well as the ability of the free glycans to inhibit DC-SIGN binding. 2'-FL had an IC50 of ∼1 mM for DC-SIGN, which is within the physiological concentration of 2'-FL in human milk. These results demonstrate that DC-SIGN among the many hGBP expressed by DCs binds to α-fucosylated HMGs, and suggest that such interactions may be important in influencing immune responses in the developing infant.

  5. Human Milk Blocks DC-SIGN-Pathogen Interaction via MUC1.

    PubMed

    Koning, Nathalie; Kessen, Sabine F M; Van Der Voorn, J Patrick; Appelmelk, Ben J; Jeurink, Prescilla V; Knippels, Leon M J; Garssen, Johan; Van Kooyk, Yvette

    2015-01-01

    Beneficial effects of breastfeeding are well-recognized and include both immediate neonatal protection against pathogens and long-term protection against allergies and autoimmune diseases. Although several proteins have been identified to have anti-viral or anti-bacterial effects like secretory IgA or lactoferrin, the mechanisms of immune modulation are not fully understood. Recent studies identified important beneficial effects of glycans in human milk, such as those expressed in oligosaccharides or on glycoproteins. Glycans are recognized by the carbohydrate receptors C-type lectins on dendritic cell (DC) and specific tissue macrophages, which exert important functions in immune modulation and immune homeostasis. A well-characterized C-type lectin is dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), which binds terminal fucose. The present study shows that in human milk, MUC1 is the major milk glycoprotein that binds to the lectin domain of DC-SIGN and prevents pathogen interaction through the presence of Lewis x-type oligosaccharides. Surprisingly, this was specific for human milk, as formula, bovine or camel milk did not show any presence of proteins that interacted with DC-SIGN. The expression of DC-SIGN is found in young infants along the entire gastrointestinal tract. Our data thus suggest the importance of human milk glycoproteins for blocking pathogen interaction to DC in young children. Moreover, a potential benefit of human milk later in life in shaping the infants immune system through DC-SIGN cannot be ruled out.

  6. Defining the Mode of Action of Tetramic Acid Antibacterials Derived from Pseudomonas aeruginosa Quorum Sensing Signals

    PubMed Central

    Lowery, Colin A.; Park, Junguk; Gloeckner, Christian; Meijler, Michael M.; Mueller, Ryan S.; Boshoff, Helena I.; Ulrich, Ricky L.; Barry, Clifton E.; Bartlett, Douglas H.; Kravchenko, Vladimir V.; Kaufmann, Gunnar F.; Janda, Kim D.

    2009-01-01

    In Nature, bacteria rarely exist as single, isolated entities, but rather as communities comprised of many other species including higher host organisms. To survive in these competitive environments, microorganisms have developed elaborate tactics such as the formation of biofilms and the production of antimicrobial toxins. Recently, it was discovered that the Gram-negative bacterium Pseudomonas aeruginosa, an opportunistic human pathogen, produces an antibiotic, 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione (C12-TA), derived from one of its quorum sensing molecules. Here, we present a comprehensive study of the expanded spectrum of C12-TA antibacterial activity against microbial competitors encountered by P. aeruginosa in Nature as well as significant human pathogens. The mechanism of action of C12-TA was also elucidated and C12-TA was found to dissipate both the membrane potential and pH gradient of Gram-positive bacteria, correlating well with cell death. Notably, in stark contrast to its parent molecule 3-oxo-dodecanoyl homoserine lactone (3-oxo-C12-HSL), neither activation of cellular stress pathways nor cytotoxicity was observed in human cells treated with C12-TA. Our results suggest that the QS machinery of P. aeruginosa has evolved for a dual-function, both to signal others of the same species, and also to defend against both host immunity and competing bacteria. Because of the broad-spectrum antibacterial activity, established mode of action, lack of rapid resistance development, and tolerance by human cells, the C12-TA scaffold may also serve as a new lead compound for the development of antimicrobial therapeutics. PMID:19807189

  7. Magnetic moment enhancement and spin polarization switch of the manganese phthalocyanine molecule on an IrMn(100) surface

    SciTech Connect

    Sun, X.; Wang, B.; Pratt, A.; Yamauchi, Y.

    2014-07-21

    The geometric, electronic, and magnetic structures of a manganese phthalocyanine (MnPc) molecule on an antiferromagnetic IrMn(100) surface are studied by density functional theory calculations. Two kinds of orientation of the adsorbed MnPc molecule are predicted to coexist due to molecular self-assembly on the surface—a top-site geometry with the Mn–N bonds aligned along the 〈100〉 direction, and a hollow-site orientation in which the Mn–N bonds are parallel to the 〈110〉 direction. The MnPc molecule is antiferromagnetically coupled to the substrate at the top site with a slight reduction in the magnetic moment of the Mn atom of the MnPc molecule (Mn{sub mol}). In contrast, the magnetic moment of the Mn{sub mol} is enhanced to 4.28 μB at the hollow site, a value larger than that in the free MnPc molecule (3.51 μB). Molecular distortion induced by adsorption is revealed to be responsible for the enhancement of the magnetic moment. Furthermore, the spin polarization of the Mn{sub mol} atom at around the Fermi level is found to change from negative to positive through an elongation of the Mn–N bonds of the MnPc. We propose that a reversible switch of the low/high magnetic moment and negative/positive spin polarization might be realized through some mechanical engineering methods.

  8. Synthesis of low color, atomic oxygen resistant polyimides

    NASA Technical Reports Server (NTRS)

    MacInnes, Dave

    1995-01-01

    The purpose of this project was to develop low color, atomic oxygen resistant polyimides for potential applications on spacecraft in low earth orbit. The material is needed in order to protect satellites and spacecraft from the gases and radiation found at those altitudes. Phosphorous containing polyimides have been shown to be especially resistant to corrosion and weight loss under oxygen plasma. Unfortunately the color of these phosphorous containing polyimides is still too high for them to be good heat insulators. While they are not as effective as teflon, the current material of choice. polyimides are much less dense than teflon and would be especially valuable if they could be made with low color. The approach taken was to synthesize a monomer which would contain the elements needed for giving the final polyimide its desired properties. In particular the monomer should incorporate phosphine or phosphine oxides and have bulky side groups to block any color forming charge transfer structures. The target molecule, 3,5-di-(trifluoromethylphenyl)-bis(3-aminophenyl) phosphine oxide, (containing both a phosphine oxide group and a bulky ditrifluoromethylphenyl group) was synthesized via three reactions in overall yield of 21 percent. In addition, a model compound, bis(3-phenylamine) phenyl phosphine oxide, was synthesized two different ways in order to establish the conditions for the nitration of phosphine oxides and their reduction to the amine. Finally, a trisubstituted phosphine oxide was synthesized. In all, seven phosphorus containing organic compounds were synthesized, purified and characterized. The model compound was reacted with oxydiphthalic anhydride to form a polyamic acid with inherent viscosity of 0.34. This material was cast into a film and heated, forming a normally colored fairly strong polyimide with a Tg of 240 C. The target compound was reacted with 6-fluorodiphthalic anhydride to give a polyamic acid with inherent viscosity of 0.19 and cast to

  9. An IL-27/Stat3 axis induces expression of programmed cell death 1 ligands (PD-L1/2) on infiltrating macrophages in lymphoma.

    PubMed

    Horlad, Hasita; Ma, Chaoya; Yano, Hiromu; Pan, Cheng; Ohnishi, Koji; Fujiwara, Yukio; Endo, Shinya; Kikukawa, Yoshitaka; Okuno, Yutaka; Matsuoka, Masao; Takeya, Motohiro; Komohara, Yoshihiro

    2016-11-01

    Immune escape and tolerance in the tumor microenvironment are closely involved in tumor progression, and are caused by T-cell exhaustion and mediated by the inhibitory signaling of immune checkpoint molecules including programmed death-1 (PD-1), cytotoxic T-lymphocyte associated protein 4, and T-cell immunoglobulin and mucin domaincontaining molecule-3. In the present study, we investigated the expression of the PD-1 ligand 1 (PD-L1) in a lymphoma microenvironment using paraffin-embedded tissue samples, and subsequently studied the detailed mechanism of upregulation of PD-L1 on macrophages using cultured human macrophages and lymphoma cell lines. We found that macrophages in lymphoma tissues of almost all cases of adult T-cell leukemia/lymphoma (ATLL), follicular lymphoma and diffuse large B-cell lymphoma expressed PD-L1. Cell culture studies showed that the conditioned medium of ATL-T and SLVL cell lines induced increased expression of PD-L1/2 on macrophages, and that this PD-L1/2 overexpression was dependent on activation of signal transducer and activator of transcription 3 (Stat3). In vitro studies including cytokine array analysis showed that IL-27 (heterodimer of p28 and EBI3) induced overexpression of PD-L1/2 on macrophages via Stat3 activation. Because lymphoma cell lines produced IL-27B (EBI3) but not IL-27p28, it was proposed that the IL-27p28 derived from macrophages and the IL-27B (EBI3) derived from lymphoma cells formed an IL-27 (heterodimer) that induced PD-L1/2 overexpression. Although the significance of PD-L1/2 expressions on macrophages in lymphoma progression has never been clarified, an IL-27-Stat3 axis might be a target for immunotherapy for lymphoma patients.

  10. Upregulation of miR-150* and miR-630 Induces Apoptosis in Pancreatic Cancer Cells by Targeting IGF-1R

    PubMed Central

    Farhana, Lulu; Dawson, Marcia I.; Murshed, Farhan; Das, Jayanta K.; Rishi, Arun K.; Fontana, Joseph A.

    2013-01-01

    MicroRNAs have been implicated in many critical cellular processes including apoptosis. We have previously found that apoptosis in pancreatic cancer cells was induced by adamantyl retinoid-related (ARR) molecule 3-Cl-AHPC. Here we report that 3-Cl-AHPC-dependent apoptosis involves regulating a number of microRNAs including miR-150* and miR-630. 3-Cl-AHPC stimulated miR-150* expression and caused decreased expression of c-Myb and IGF-1R in the pancreatic cancer cells. 3-Cl-AHPC-mediated reduction of c-Myb resulted in diminished binding of c-Myb with IGF-1R and Bcl-2 promoters, thereby causing repression of their transcription and protein expression. Over-expression of miR-150* also resulted in diminished levels of c-Myb and Bcl-2 proteins. Furthermore, the addition of the miRNA inhibitor 2′-O-methylated miR-150 blocked 3-Cl-AHPC-mediated increase in miR-150* levels and abrogated loss of c-Myb protein. Knockdown of c-Myb in PANC-1 cells resulted in enhanced apoptosis both in the presence or absence of 3-Cl-AHPC confirming the anti-apoptotic property of c-Myb. Overexpression of miR-630 also induced apoptosis in the pancreatic cancer cells and inhibited target protein IGF-1R mRNA and protein expression. Together these results implicate key roles for miR-150* and miR-630 and their targeting of IGF-1R to promote apoptosis in pancreatic cancer cells. PMID:23675407

  11. Atomistic Pseudopotential Calculations of the Electronic and Optical Properties of Self-Assembled Quantum Dots

    NASA Astrophysics Data System (ADS)

    Bester, Gabriel

    2006-03-01

    The optical spectrum and the charging energies of semiconductor quantum dots have been recently measured with high accuracy. Both of these experimental techniques probe many-body states that are not directly described by independent particle theories such as the density functional theory. On the other hand, quasi- particle theories that can in principle address the problem, such as GW, are computationally too demanding for the study of nanostructures (as opposed to clusters) where many thousands of atoms are involved. One way to approach this problem is to use the effective mass approximation or the k.p method and choose a confinement potential that reproduces a few known experimental facts (e.g. the splitting between confined levels). These methods can provide a good initial guess but were shown to be too crude to enable a quantitative comparision with recent experiments. We therefore adopt a bottom-up atomistic approach where instead of starting from a simplified approach, such as effective mass, and progressively increase the complexity by adding parameters, we start from the accurate atomistic description (LDA or GW) and work ourselves up using a few well controlled approximations.I will first present the method, namely (i) the scheme that is used to derive the empirical pseudopotentials including the piezoelectric effect, (ii) the choices that have to be made for the basis used to expand the wave functions, (iii) the inclusion of corelations through Bethe-Salpeter-like treatment. I will then present recent applications of the theory to calculate the fine-structure [1] of excitons and charged excitons, the charging spectra of holes [2] and the degree or entanglement stored in a quantum dot molecule [3].[1] G. Bester, S.V. Nair, A. Zunger, prb 67, 161306 (2003). [2] L. He, G. Bester, A. Zunger, PRL (in press). [3] G. Bester, J. Shumway, A. Zunger, PRL 93, 047401 (2004)

  12. High taurine levels in the Solemya velum symbiosis.

    PubMed

    Conway, N M; McDowell Capuzzo, J E

    1992-05-01

    1. To compare biochemical differences between bivalves with and without endosymbiotic chemoautotrophic bacteria, specimens of Solemya velum, a bivalve species known to contain bacterial endosymbionts, and the symbiont-free soft-shelled clam Mya arenaria, were collected from the same subtidal reducing sediments during October and November 1988. 2. Total and free amino acid compositions were determined for both species. Protein-bound amino acids were calculated as the difference between total and free amino acids. In addition, stable isotope ratios of the total and free amino acids of each species were measured to determine potential sources for these molecules. 3. Both species had similar total hydrolyzable- and protein-bound amino acid compositions; approximately 50% of the protein-bound amino acids were essential amino acids. In S. velum, the small size of the digestive system suggests that these amino acids are probably synthesized by the endosymbiotic bacteria and translocated to the animal tissue. The delta 13C and delta 15N ratios of the amino acids are very similar to the isotope ratios previously found in both the endosymbionts and whole tissues of S. velum. The relative and absolute amounts of free amino acids are very different in the two species. In S. velum, the absolute concentrations of taurine, a sulfur-containing amino acid, were greater than the total free amino acid concentrations found in other bivalves. 4. The delta 34S ratios of the free amino acids of S. velum, which were predominantly composed of taurine, were extremely negative (-17.2/1000) suggesting that taurine is synthesized using sulfur originally derived from external reduced sulfur sources, such as pore water sulfides. The possible roles for taurine in this animal-bacteria symbiosis are discussed.

  13. Ferroptosis: process and function

    PubMed Central

    Xie, Y; Hou, W; Song, X; Yu, Y; Huang, J; Sun, X; Kang, R; Tang, D

    2016-01-01

    Ferroptosis is a recently recognized form of regulated cell death. It is characterized morphologically by the presence of smaller than normal mitochondria with condensed mitochondrial membrane densities, reduction or vanishing of mitochondria crista, and outer mitochondrial membrane rupture. It can be induced by experimental compounds (e.g., erastin, Ras-selective lethal small molecule 3, and buthionine sulfoximine) or clinical drugs (e.g., sulfasalazine, sorafenib, and artesunate) in cancer cells and certain normal cells (e.g., kidney tubule cells, neurons, fibroblasts, and T cells). Activation of mitochondrial voltage-dependent anion channels and mitogen-activated protein kinases, upregulation of endoplasmic reticulum stress, and inhibition of cystine/glutamate antiporter is involved in the induction of ferroptosis. This process is characterized by the accumulation of lipid peroxidation products and lethal reactive oxygen species (ROS) derived from iron metabolism and can be pharmacologically inhibited by iron chelators (e.g., deferoxamine and desferrioxamine mesylate) and lipid peroxidation inhibitors (e.g., ferrostatin, liproxstatin, and zileuton). Glutathione peroxidase 4, heat shock protein beta-1, and nuclear factor erythroid 2-related factor 2 function as negative regulators of ferroptosis by limiting ROS production and reducing cellular iron uptake, respectively. In contrast, NADPH oxidase and p53 (especially acetylation-defective mutant p53) act as positive regulators of ferroptosis by promotion of ROS production and inhibition of expression of SLC7A11 (a specific light-chain subunit of the cystine/glutamate antiporter), respectively. Misregulated ferroptosis has been implicated in multiple physiological and pathological processes, including cancer cell death, neurotoxicity, neurodegenerative diseases, acute renal failure, drug-induced hepatotoxicity, hepatic and heart ischemia/reperfusion injury, and T-cell immunity. In this review, we summarize the

  14. Microglia Transcriptome Changes in a Model of Depressive Behavior after Immune Challenge

    PubMed Central

    Gonzalez-Pena, Dianelys; Nixon, Scott E.; O’Connor, Jason C.; Southey, Bruce R.; Lawson, Marcus A.; McCusker, Robert H.; Borras, Tania; Machuca, Debbie; Hernandez, Alvaro G.; Dantzer, Robert; Kelley, Keith W.; Rodriguez-Zas, Sandra L.

    2016-01-01

    Depression symptoms following immune response to a challenge have been reported after the recovery from sickness. A RNA-Seq study of the dysregulation of the microglia transcriptome in a model of inflammation-associated depressive behavior was undertaken. The transcriptome of microglia from mice at day 7 after Bacille Calmette Guérin (BCG) challenge was compared to that from unchallenged Control mice and to the transcriptome from peripheral macrophages from the same mice. Among the 562 and 3,851 genes differentially expressed between BCG-challenged and Control mice in microglia and macrophages respectively, 353 genes overlapped between these cells types. Among the most differentially expressed genes in the microglia, serum amyloid A3 (Saa3) and cell adhesion molecule 3 (Cadm3) were over-expressed and coiled-coil domain containing 162 (Ccdc162) and titin-cap (Tcap) were under-expressed in BCG-challenged relative to Control. Many of the differentially expressed genes between BCG-challenged and Control mice were associated with neurological disorders encompassing depression symptoms. Across cell types, S100 calcium binding protein A9 (S100A9), interleukin 1 beta (Il1b) and kynurenine 3-monooxygenase (Kmo) were differentially expressed between challenged and control mice. Immune response, chemotaxis, and chemokine activity were among the functional categories enriched by the differentially expressed genes. Functional categories enriched among the 9,117 genes differentially expressed between cell types included leukocyte regulation and activation, chemokine and cytokine activities, MAP kinase activity, and apoptosis. More than 200 genes exhibited alternative splicing events between cell types including WNK lysine deficient protein kinase 1 (Wnk1) and microtubule-actin crosslinking factor 1(Macf1). Network visualization revealed the capability of microglia to exhibit transcriptome dysregulation in response to immune challenge still after resolution of sickness symptoms

  15. Analyses at High Spatial Resolution of Organic Molecules in Extraterrestrial Samples: Two-Step Laser Mass Spectrometry: Search for Polycyclic Aromatic Hydrocarbons in Antarctic Meteorite and Micrometeorite Samples

    NASA Technical Reports Server (NTRS)

    Zare, Richard N.

    1998-01-01

    Perhaps the best way to summarize the past three-year grant period is to cite the publications and present a brief synopsis of each: 1. "Indigenous Polycyclic Aromatic Hydrocarbon Molecules in Circumstellar Graphite Grains." Bulk C-12/C-13 isotope ratios observed in some graphite grains extracted from primitive meteorites point strongly to a circumstellar origin. By applying our technique of microprobe two-step laser desorption laser ionization mass spectrometry ((mu)L(sup 2)MS) to individual circumstellar graphite grains we have measured the C-12/C-13 isotope ratio of various polycyclic aromatic hydrocarbons (PAHS) found in these grains. 2. "Deuterium Enrichments in Cluster IDPS," Large enrichments in the D/H isotope ratios in IDPs likely arise from the preservation of presolar molecules. 3. "Evidence for thermalization of surface-disorder molecules at heating rates of 10(exp 8) K/s". A careful study of the ((mu)L(sup 2)MS) of aniline-d(sub 7) from a single-crystal surface (0001) of sapphire (al2O3) shows that all measured properties are consistent with a thermal mechanism for desorption. 4. "Search for past life on Mars; possible relic biogenic activity in Martian meteorite ALH 84001. The authors examined the Martian meteorite ALH 84001 and found several lines of evidence compatible with existence of past primitive (single-cell) life on early Mars. 5. "Microprobe two-step laser mass spectrometry as an analytical tool for meteorite samples". THis paper presents a comprehensive review of (mu)L(sup 2)MS and how this technique can be applied to meteoritic samples. 6. "Indigenous polycyclic aromatic hydrocarbons in circumstellar graphite grains from primitive meteorites". The C-12/C-13 isotope ratios were measured for PAHs in a total of 89 spherical graphite grains. 7. "Observation of indigenous polycyclic aromatic hydrocarbons in "Giant" carbonaceous antarctic micrometeorites." The (mu)L(sup 2)MS method was used to establish the nature and distribution of PAHs in

  16. A new microRNA signal pathway regulated by long noncoding RNA TGFB2-OT1 in autophagy and inflammation of vascular endothelial cells

    PubMed Central

    Huang, ShuYa; Lu, Wei; Ge, Di; Meng, Ning; Li, Ying; Su, Le; Zhang, ShangLi; Zhang, Yun; Zhao, BaoXiang; Miao, JunYing

    2015-01-01

    TGFB2-OT1 (TGFB2 overlapping transcript 1) is a newly discovered long noncoding RNA (lncRNA) derived from the 3′UTR of TGFB2. It can regulate autophagy in vascular endothelial cells (VECs). However, the mechanisms of TGFB2-OT1 action are unclear, and whether it is involved in VECs dysfunction needs investigation. Here, the level of TGFB2-OT1 was markedly increased by lipopolysaccharide and oxidized low-density lipoprotein, 2 VECs inflammation triggers. A chemical small molecule, 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO) significantly decreased TGFB2-OT1 levels and inhibited the effect of LPS and oxLDL. The NUPR1 level was upregulated by the 2 inflammation inducers and modulated the TGFB2-OT1 level by promoting the expression of TIA1, responsible for TGFB2-OT1 processing. We focused on how TGFB2-OT1 regulated autophagy and inflammation. Use of miRNA chip assay, TGFB2-OT1 overexpression technology and 3BDO revealed that TGFB2-OT1 regulated the levels of 3 microRNAs, MIR3960, MIR4488 and MIR4459. Further studies confirmed that TGFB2-OT1 acted as a competing endogenous RNA, bound to MIR3960, MIR4488 and MIR4459, then regulated the expression of the miRNA targets CERS1 (ceramide synthase 1), NAT8L (N-acetyltransferase 8-like [GCN5-related, putative]), and LARP1 (La ribonucleoprotein domain family, member 1). CERS1 and NAT8L participate in autophagy by affecting mitochondrial function. TGFB2-OT1 increased the LARP1 level, which promoted SQSTM1 (sequestosome 1) expression, NFKB RELA and CASP1 activation, and then production of IL6, IL8 and IL1B in VECs. Thus, NUPR1 and TIA1 may control the level of TGFB2-OT1, and TGFB2-OT1 bound to MIR3960, MIR4488 and MIR4459, which targeted CERS1, NAT8L, and LARP1, respectively, the key proteins involved in autophagy and inflammation. PMID:26565952

  17. Initial mechanisms for the unimolecular decomposition of electronically excited bisfuroxan based energetic materials.

    PubMed

    Yuan, Bing; Bernstein, Elliot R

    2017-01-07

    Unimolecular decomposition of energetic molecules, 3,3'-diamino-4,4'-bisfuroxan (labeled as A) and 4,4'-diamino-3,3'-bisfuroxan (labeled as B), has been explored via 226/236 nm single photon laser excitation/decomposition. These two energetic molecules, subsequent to UV excitation, create NO as an initial decomposition product at the nanosecond excitation energies (5.0-5.5 eV) with warm vibrational temperature (1170 ± 50 K for A, 1400 ± 50 K for B) and cold rotational temperature (<55 K). Initial decomposition mechanisms for these two electronically excited, isolated molecules are explored at the complete active space self-consistent field (CASSCF(12,12)/6-31G(d)) level with and without MP2 correction. Potential energy surface calculations illustrate that conical intersections play an essential role in the calculated decomposition mechanisms. Based on experimental observations and theoretical calculations, NO product is released through opening of the furoxan ring: ring opening can occur either on the S1 excited or S0 ground electronic state. The reaction path with the lowest energetic barrier is that for which the furoxan ring opens on the S1 state via the breaking of the N1-O1 bond. Subsequently, the molecule moves to the ground S0 state through related ring-opening conical intersections, and an NO product is formed on the ground state surface with little rotational excitation at the last NO dissociation step. For the ground state ring opening decomposition mechanism, the N-O bond and C-N bond break together in order to generate dissociated NO. With the MP2 correction for the CASSCF(12,12) surface, the potential energies of molecules with dissociated NO product are in the range from 2.04 to 3.14 eV, close to the theoretical result for the density functional theory (B3LYP) and MP2 methods. The CASMP2(12,12) corrected approach is essential in order to obtain a reasonable potential energy surface that corresponds to the observed decomposition behavior of these

  18. Inhibition of Predator Attraction to Kairomones by Non-Host Plant Volatiles for Herbivores: A Bypass-Trophic Signal

    PubMed Central

    Zhang, Qing-He; Schlyter, Fredrik

    2010-01-01

    Background Insect predators and parasitoids exploit attractive chemical signals from lower trophic levels as kairomones to locate their herbivore prey and hosts. We hypothesized that specific chemical cues from prey non-hosts and non-habitats, which are not part of the trophic chain, are also recognized by predators and would inhibit attraction to the host/prey kairomone signals. To test our hypothesis, we studied the olfactory physiology and behavior of a predaceous beetle, Thanasimus formicarius (L.) (Coleoptera: Cleridae), in relation to specific angiosperm plant volatiles, which are non-host volatiles (NHV) for its conifer-feeding bark beetle prey. Methodology/Principal Findings Olfactory detection in the clerid was confirmed by gas chromatography coupled to electroantennographic detection (GC-EAD) for a subset of NHV components. Among NHV, we identified two strongly antennally active molecules, 3-octanol and 1-octen-3-ol. We tested the potential inhibition of the combination of these two NHV on the walking and flight responses of the clerid to known kairomonal attractants such as synthetic mixtures of bark beetle (Ips spp.) aggregation pheromone components (cis-verbenol, ipsdienol, and E-myrcenol) combined with conifer (Picea and Pinus spp.) monoterpenes (α-pinene, terpinolene, and Δ3-carene). There was a strong inhibitory effect, both in the laboratory (effect size d = −3.2, walking bioassay) and in the field (d = −1.0, flight trapping). This is the first report of combining antennal detection (GC-EAD) and behavioral responses to identify semiochemical molecules that bypass the trophic system, signaling habitat information rather than food related information. Conclusions/Significance Our results, along with recent reports on hymenopteran parasitoids and coleopteran predators, suggest that some NHV chemicals for herbivores are part of specific behavioral signals for the higher trophic level and not part of a background noise. Such bypass

  19. A Systematic Approach for Computing Zero-Point Energy, Quantum Partition Function, and Tunneling Effect Based on Kleinert's Variational Perturbation Theory.

    PubMed

    Wong, Kin-Yiu; Gao, Jiali

    2008-09-09

    In this paper, we describe an automated integration-free path-integral (AIF-PI) method, based on Kleinert's variational perturbation (KP) theory, to treat internuclear quantum-statistical effects in molecular systems. We have developed an analytical method to obtain the centroid potential as a function of the variational parameter in the KP theory, which avoids numerical difficulties in path-integral Monte Carlo or molecular dynamics simulations, especially at the limit of zero-temperature. Consequently, the variational calculations using the KP theory can be efficiently carried out beyond the first order, i.e., the Giachetti-Tognetti-Feynman-Kleinert variational approach, for realistic chemical applications. By making use of the approximation of independent instantaneous normal modes (INM), the AIF-PI method can readily be applied to many-body systems. Previously, we have shown that in the INM approximation, the AIF-PI method is accurate for computing the quantum partition function of a water molecule (3 degrees of freedom) and the quantum correction factor for the collinear H(3) reaction rate (2 degrees of freedom). In this work, the accuracy and properties of the KP theory are further investigated by using the first three order perturbations on an asymmetric double-well potential, the bond vibrations of H(2), HF, and HCl represented by the Morse potential, and a proton-transfer barrier modeled by the Eckart potential. The zero-point energy, quantum partition function, and tunneling factor for these systems have been determined and are found to be in excellent agreement with the exact quantum results. Using our new analytical results at the zero-temperature limit, we show that the minimum value of the computed centroid potential in the KP theory is in excellent agreement with the ground state energy (zero-point energy) and the position of the centroid potential minimum is the expectation value of particle position in wave mechanics. The fast convergent property

  20. Garlic as an inhibitor of Pseudomonas aeruginosa quorum sensing in cystic fibrosis--a pilot randomized controlled trial.

    PubMed

    Smyth, Alan R; Cifelli, Paramita M; Ortori, Catharine A; Righetti, Karima; Lewis, Sarah; Erskine, Penny; Holland, Elaine D; Givskov, Michael; Williams, Paul; Cámara, Miguel; Barrett, David A; Knox, Alan

    2010-04-01

    Pseudomonas aeruginosa forms biofilms in the cystic fibrosis lung. Quorum sensing (QS) controls biofilm maturation, immune evasion, antibiotic tolerance and virulence factor production. Garlic shows QS inhibitory activity in vitro and in animal models. We report the first clinical trial in man of a QS inhibitor.We randomized 34 patients to garlic or olive oil capsules (both 656 mg daily). Clinical outcomes and safety bloods were measured at baseline and after 8 weeks treatment. In this exploratory study, analysis was per protocol.Eight patients withdrew, leaving 26 for analysis (13 garlic). With placebo, there was a greater decline in mean (SD) percentage change from baseline FEV(1) [-3.6% (11.3)] than with garlic [-2.0% (12.3)]. This was not significant (mean difference = 1.6, 95% CI -12.7 to 15.9, P = 0.8). The mean (SD) increase in weight was greater with garlic [1.0% (2.0)] than with placebo [0.6% (2.0)]--non-significant (mean difference = 0.4%, 95% CI -1.3 to 2.0, P = 0.6). The median (range) change in clinical score with garlic was -1 (-3 to 5) and 1 (-1 to 4) with placebo (negative score means improvement). This was non-significant [median difference = -1 (-3 to 0), P = 0.16]. In the garlic group, seven patients had IV antibiotics versus five placebo. There was a highly significant correlation between plasma and sputum measurements of the QS molecule 3-oxo-C12-HSL (Pearson correlation coefficient = 0.914, P = 0.004). At the end of treatment five patients in each group had abnormal liver function or triglycerides and five garlic patients (one placebo) reported minor adverse effects.Garlic capsules were well tolerated. Although there was no significant effect of garlic compared to placebo in this pilot study, there was a suggestion of improvement with garlic which should be investigated in a larger trial.

  1. Docking, molecular dynamics and quantitative structure-activity relationship studies for HEPTs and DABOs as HIV-1 reverse transcriptase inhibitors.

    PubMed

    Mao, Yating; Li, Yan; Hao, Ming; Zhang, Shuwei; Ai, Chunzhi

    2012-05-01

    As a key component in combination therapy for acquired immunodeficiency syndrome (AIDS), non-nucleoside reverse transcriptase inhibitors (NNRTIs) have been proven to be an essential way in stopping HIV-1 replication. In the present work, in silico studies were conducted on a series of 119 NNRTIs, including 1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT) and dihydroalkoxybenzyloxopyrimidine (DABO) derivatives by using the comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), docking simulations and molecular dynamics (MD). The statistical results of the optimal model, the ligand-based CoMSIA one (Q(2) = 0.48, R(ncv)(2) =0.847, R(pre)(2) = 0.745) validates its satisfactory predictive capacity both internally and externally. The contour maps, docking and MD results correlate well with each other, drawing conclusions as follows: 1) Compounds with bulky substituents in position-6 of ring A, hydrophobic groups around position- 1, 2, 6 are preferable to the biological activities; 2) Two hydrogen bonds between RT inhibitor and the Tyr 318, Lys 101 residues, respectively, and a π-π bond between the inhibitor and Trp 188 are formed and crucial to the orientation of the active conformation of the molecules; 3) The binding pocket is essentially hydrophobic, which are determined by residues such as Trp 229, Tyr 318, Val 179, Tyr 188 and Val 108, and hydrophobic substituents may bring an improvement to the biological activity; 4) DABO and HEPT derivatives have different structures but take a similar mechanism to inhibit RT. The potency difference between two isomers in HEPTs can be explained by the distinct locations of the 6-naphthylmethyl substituent and the reasons are explained in details. All these results could be employed to alter the structural scaffold in order to develop new HIV-1 RT inhibitors that have an improved biological property. To the best of our knowledge, this is the first report on 3D

  2. A Hybrid Chalcone Combining the Trimethoxyphenyl and Isatinyl Groups Targets Multiple Oncogenic Proteins and Pathways in Hepatocellular Carcinoma Cells

    PubMed Central

    Cao, Lili; Zhang, Lijun; Zhao, Xiang; Zhang, Ye

    2016-01-01

    Small molecule inhibitors that can simultaneously inhibit multiple oncogenic proteins in essential pathways are promising therapeutic chemicals for hepatocellular carcinoma (HCC). To combine the anticancer effects of combretastatins, chalcones and isatins, we synthesized a novel hybrid molecule 3’,4’,5’-trimethoxy-5-chloro-isatinylchalcone (3MCIC). 3MCIC inhibited proliferation of cultured HepG2 cells, causing rounding-up of the cells and massive vacuole accumulation in the cytoplasm. Paxillin and focal adhesion plaques were downregulated by 3MCIC. Surprisingly, unlike the microtubule (MT)-targeting agent CA-4 that inhibits tubulin polymerization, 3MCIC stabilized tubulin polymers both in living cells and in cell lysates. 3MCIC treatment reduced cyclin B1, CDK1, p-CDK1/2, and Rb, but increased p53 and p21. Moreover, 3MCIC caused GSK3β degradation by promoting GSK3β-Ser9 phosphorylation. Nevertheless, 3MCIC inhibited the Wnt/β-catenin pathway by downregulating β-catenin, c-Myc, cyclin D1 and E2F1. 3MCIC treatment not only activated the caspase-3-dependent apoptotic pathway, but also caused massive autophagy evidenced by rapid and drastic changes of LC3 and p62. 3MCIC also promoted cleavage and maturation of the lysosomal protease cathepsin D. Using ligand-affinity chromatography (LAC), target proteins captured onto the Sephacryl S1000-C12-3MCIC resins were isolated and analyzed by mass spectrometry (MS). Some of the LAC-MS identified targets, i.e., septin-2, vimentin, pan-cytokeratin, nucleolin, EF1α1/2, EBP1 (PA2G4), cyclin B1 and GSK3β, were further detected by Western blotting. Moreover, both septin-2 and HIF-1α decreased drastically in 3MCIC-treated HepG2 cells. Our data suggest that 3MCIC is a promising anticancer lead compound with novel targeting mechanisms, and also demonstrate the efficiency of LAC-MS based target identification in anticancer drug development. PMID:27525972

  3. The Legionella pneumophila orphan sensor kinase LqsT regulates competence and pathogen-host interactions as a component of the LAI-1 circuit.

    PubMed

    Kessler, Aline; Schell, Ursula; Sahr, Tobias; Tiaden, André; Harrison, Christopher; Buchrieser, Carmen; Hilbi, Hubert

    2013-02-01

    Legionella pneumophila is an amoeba-resistant opportunistic pathogen that performs cell-cell communication through the signalling molecule 3-hydroxypentadecane-4-one (LAI-1, Legionella autoinducer-1). The lqs (Legionella quorum sensing) gene cluster encodes the LAI-1 autoinducer synthase LqsA, the cognate sensor kinase LqsS and the response regulator LqsR. Here we show that the Lqs system includes an 'orphan' homologue of LqsS termed LqsT. Compared with wild-type L. pneumophila, strains lacking lqsT or both lqsS and lqsT show increased salt resistance, greatly enhanced natural competence for DNA acquisition and impaired uptake by phagocytes. Sensitive novel single round growth assays and competition experiments using Acanthamoeba castellanii revealed that ΔlqsT and ΔlqsS-ΔlqsT, as well as ΔlqsA and other lqs mutant strains are impaired for intracellular growth and cannot compete against wild-type bacteria upon co-infection. In contrast to the ΔlqsS strain, ΔlqsT does not produce extracellular filaments. The phenotypes of the ΔlqsS-ΔlqsT strain are partially complemented by either lqsT or lqsS, but are not reversed by overexpression of lqsA, suggesting that LqsT and LqsS are the sole LAI-1-responsive sensor kinases in L. pneumophila. In agreement with the different phenotypes of the ΔlqsT and ΔlqsS strains, lqsT and lqsS are differentially expressed in the post-exponential growth phase, and transcriptome studies indicated that 90% of the genes, which are downregulated in absence of lqsT, are upregulated in absence of lqsS. Reciprocally regulated genes encode components of a 133 kb genomic 'fitness island' or translocated effector proteins implicated in virulence. Together, these results reveal a unique organization of the L. pneumophila Lqs system comprising two partially antagonistic LAI-1-responsive sensor kinases, LqsT and LqsS, which regulate distinct pools of genes implicated in pathogen-host cell interactions, competence, expression of a

  4. Effect of Culture Supernatant Derived from Trichophyton Rubrum Grown in the Nail Medium on the Innate Immunity-related Molecules of HaCaT

    PubMed Central

    Huang, Xin-Zhu; Liang, Pan-Pan; Ma, Han; Yi, Jin-Ling; Yin, Song-Chao; Chen, Zhi-Rui; Li, Mei-Rong; Lai, Wei; Chen, Jian

    2015-01-01

    Background: Trichophyton rubrum is superficial fungi characteristically confined to dead keratinized tissues. These observations suggest that the soluble components released by the fungus could influence the host immune response in a cell in contact-free manner. Therefore, this research aimed to analyze whether the culture supernatant derived from T. rubrum grown in the nail medium could elicit the immune response of keratinocyte effectively. Methods: The culture supernatants of two strains (T1a, TXHB) were compared for the β-glucan concentrations and their capacity to impact the innate immunity of keratinocytes. The β-glucan concentrations in the supernatants were determined with the fungal G-test kit and protein concentrations with bicinchoninic acid protein quantitative method, then HaCaT was stimulated with different concentrations of culture supernatants by adopting morphological method to select a suitable dosage. Expressions of host defense genes were assessed by quantitative polymerase chain reaction after the HaCaT was stimulated with the culture supernatants. Data were analyzed with one-way analysis of variance, followed by the least significant difference test. Results: The T. rubrum strains (T1a and TXHB) released β-glucan of 87.530 ± 37.581 pg/ml and 15.747 ± 6.453 pg/ml, respectively into the media. The messenger RNA (mRNA) expressions of toll-like receptor-2 (TLR2), TLR4, and CARD9 were moderately up-regulated in HaCaT within 6-h applications of both supernatants. HaCaT cells were more responsive to T1a than TXHB. The slight increase of dendritic cells-specific intercellular adhesion molecule 3-grabbing nonintegrin expression was faster and stronger, induced by T1a supernatant than TXHB. The moderate decreases of RNase 7, the slight up-regulations of Dectin-1 and interleukin-8 at the mRNA level were detected only in response to T1a rather than TXHB. After a long-time contact, all the elevated defense genes decreased after 24 h. Conclusion: The

  5. Recombinant human serum amyloid A (apoSAAp) binds cholesterol and modulates cholesterol flux.

    PubMed

    Liang, J S; Sipe, J D

    1995-01-01

    During acute inflammation, the serum amyloid A (apoSAA) proteins apoSAA1 and apoSAA2 are transiently associated with high density lipoproteins (HDL) in concentrations of as much as 1000-fold more than their concentrations during homeostasis; however, their effect on HDL function is unclear. Recombinant apoSAAp, a hybrid of the closely related human apoSAA1 and apoSAA2 isoforms, was found to exhibit a high affinity for cholesterol. The adsorption of apoSAAp to polystyrene microtiter wells at physiological pH, temperature, and salt concentration was inhibited and reversed by cholesterol. ApoSAAp, to a greater extent than apoA-I, albumin, or fetal bovine serum, enhanced diffusion of cholesterol from HDL across a membrane that retained molecules > 3.5 kDa. Cholesterol from 25 nM to 125 microM inhibited binding of [3H]cholesterol to 167 nM apoSAAp. A cholesterol binding assay was developed to determine the dissociation constant for binding of [3H]cholesterol to apoSAAp; Kd = 1.7 +/- 0.3 x 10(-7) M and the maximum binding capacity (Bmax) is 1.1 +/- 0.1 mol/mol. After binding cholesterol, the apparent size of apoSAAp as determined by gel filtration on Sephacryl S-100 was increased from 12 to 23 kDa. ApoSAAp enhanced free [14C]cholesterol uptake from tissue culture medium by HepG2 cells, an effect that was dose dependent and blocked by polyclonal antibodies to human apoSAA1 and apoSAA2. ApoSAAp, unlike apoA-I, was taken up from serum-free medium by HepG2 cells and appeared to be degraded by cell-associated enzymes. Unlike peritoneal exudate cells, human HepG2 hepatoma cells do not secrete an enzyme that degrades apoSAAp. These results suggest that apoSAA can potentially serve as a transient cholesterol-binding protein.

  6. Endogenous ligands of imidazoline receptors: classic and immunoreactive clonidine-displacing substance and agmatine.

    PubMed

    Reis, D J; Li, G; Regunathan, S

    1995-07-12

    1. There are several endogenous ligands that bind to I-receptors of both the I1 and I2 subclass. These include: (a) classic CDS, a partially purified entity isolated by the criteria that it displaces binding ligands to alpha 2- and I-receptors; (b) immunoreactive (ir)-CDS, a moiety that binds to antibodies raised against clonidine, para-amino-clonidine, or idazoxan; and (c) agmatine. 2. Classic-CDS, not yet defined structurally, binds to I1, I2, and alpha 2-adrenergic receptors, is neither a peptide nor a catecholamine, and has purportedly a molecular weight of 588 Da. By ligand binding assays, it was found in brain, serum, CSF, and placenta and in a neural-glial cell line. Partially purified classic CDS is bioactive. Like clonidine, it contracts aorta and vas deferens and inhibits platelet aggregation, effects largely attributable to agonism at alpha 2-adrenergic receptors. Unlike clonidine, it contracts rat gastric fundus and releases catecholamines from chromaffin cells, effects attributable to actions at I-receptors. Injected into the RVL, classic CDS alters arterial pressure, but the direction of change of pressure has differed between groups of investigators. However, in the absence of structure, it is possible that ligand binding and bioactivity may be attributable to different molecules. 3. Ir-CDS, also of unknown structure, is a material(s) that binds to antibodies raised against clonidine, PAC, or idazoxan. Ir-CDS, measured by radioimmunoassay, is unevenly distributed in brain with highest concentrations in the hypothalamus, midbrain, and dorsal medulla. It is contained in the gastric fundus, adrenal gland, heart, kidney, and serum in amounts substantially higher than found in brain. Ir-CDS may be elevated in the serum of some patients with hypertension and in the CSF of patients with structural brain disease. The concentration of ir-CDS and bioactivity on gastric fundus directly correlates, suggesting that it may share similarities with classic

  7. Inhibition of HIV-1 transmission in trans from dendritic cells to CD4+ T lymphocytes by natural antibodies to the CRD domain of DC-SIGN purified from breast milk and intravenous immunoglobulins

    PubMed Central

    Requena, Mary; Bouhlal, Hicham; Nasreddine, Nadine; Saidi, Hela; Gody, Jean-Chrysostome; Aubry, Sylvie; Grésenguet, Gérard; Kazatchkine, Michel D; Sekaly, Rafick-Pierre; Bélec, Laurent; Hocini, Hakim

    2008-01-01

    The present study demonstrates that human breast milk and normal human polyclonal immunoglobulins purified from plasma [intravenous immunoglobulins (IVIg)] contain functional natural immunoglobulin A (IgA) and IgG antibodies directed against the carbohydrate recognition domain (CRD) domain of the dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) molecule, which is involved in the binding of human immunodeficiency virus (HIV)-1 to dendritic cells (DCs). Antibodies to DC-SIGN CRD were affinity-purified on a matrix to which a synthetic peptide corresponding to the N-terminal CRD domain (amino-acid 342–amino-acid 371) had been coupled. The affinity-purified antibodies bound to the DC-SIGN peptide and to the native DC-SIGN molecule expressed by HeLa DC-SIGN+ cells and immature monocyte-derived dendritic cells (iMDDCs), in a specific and dose-dependent manner. At an optimal dose of 200 µg/ml, natural antibodies to DC-SIGN CRD peptide purified from breast milk and IVIg stained 25 and 20% of HeLa DC-SIGN+ cells and 32 and 12% of iMDDCs, respectively. Anti-DC-SIGN CRD peptide antibodies inhibited the attachment of virus to HeLa DC-SIGN by up to 78% and the attachment to iMDDCs by only 20%. Both breast milk- and IVIg-derived natural antibodies to the CRD peptide inhibited 60% of the transmission in trans of HIV-1JRCSF, an R5-tropic strain, from iMDDCs to CD4+ T lymphocytes. Taken together, these observations suggest that the attachment of HIV to DCs and transmission in trans to autologous CD4+ T lymphocytes occur through two independent mechanisms. Our data support a role of natural antibodies to DC-SIGN in the modulation of postnatal HIV transmission through breast-feeding and in the natural host defence against HIV-1 in infected individuals. PMID:17999675

  8. Modeling Electrical Transport through Nucleic Acids

    NASA Astrophysics Data System (ADS)

    Qi, Jianqing

    Nucleic acids play a vital role in many biological systems and activities. In recent years, engineers and scientists have been interested in studying their electrical properties. The motivation for these studies stems from the following facts: (1) the bases, which form the building blocks of nucleic acids, have unique ionization potentials. Further, nucleic acids are one of the few nanomaterials that can be reproducibly manufactured with a high degree of accuracy (though admittedly their placement at desired locations remains a challenge). As a result, designed strands with specific sequences may offer unique device properties; (2) electrical methods offer potential for sequencing nucleic acids based on a single molecule; (3) electrical methods for disease detection based on the current flowing through nucleic acids are beginning to be demonstrated. While experiments in the above mentioned areas is promising, a deeper understanding of the electrical current flow through the nucleic acids needs to be developed. The modeling of current flowing in these molecules is complex because: (1) they are based on atomic scale contacts between nucleic acids and metal, which cannot be reproducibly built; (2) the conductivity of nucleic acids is easily influenced by the environment, which is constantly changing; and (3) the nucleic acids by themselves are floppy. This thesis focuses on the modeling of electrical transport through nucleic acids that are connected to two metal electrodes at nanoscale. We first develop a decoherent transport model for the double-stranded helix based on the Landauer-Buttiker framework. This model is rationalized by comparison with an experiment that measured the conductance of four different DNA strands. The developed model is then used to study the: (1) potential to make barriers and wells for quantum transport using specifically engineered sequences; (2) change in the electrical properties of a specific DNA strand with and without methylation; (3

  9. Characterization of Glycoprotein-Mediated Entry of Severe Fever with Thrombocytopenia Syndrome Virus

    PubMed Central

    Tani, Hideki; Shimojima, Masayuki; Fukushi, Shuetsu; Yoshikawa, Tomoki; Fukuma, Aiko; Taniguchi, Satoshi; Morikawa, Shigeru

    2016-01-01

    ABSTRACT Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever with a high case fatality rate caused by SFTS virus (SFTSV). Effective vaccines and specific therapies for SFTS are urgently sought, and investigation into virus-host cell interactions is expected to contribute to the development of antiviral strategies. In this study, we have developed a pseudotype vesicular stomatitis virus (VSV) bearing the unmodified Gn/Gc glycoproteins (GPs) of SFTSV (SFTSVpv). We have analyzed the host cell entry of this pseudotype virus and native SFTSV. Both SFTSVpv and SFTSV exhibited high infectivity in various mammalian cell lines. The use of lysosomotropic agents indicated that virus entry occurred via pH-dependent endocytosis. SFTSVpv and SFTSV infectivity was neutralized by serial dilutions of convalescent-phase patient sera. Entry of SFTSVpv and growth of SFTSV were increased in Raji cells expressing not only the C-type lectin dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) but also DC-SIGN-related (DC-SIGNR) and liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin). 25-Hydroxycholesterol (25HC), a soluble oxysterol metabolite, inhibited the cell entry of SFTSVpv and the membrane fusion of SFTSV. These results indicate that pH-dependent endocytosis of SFTSVpv and SFTSV is enhanced by attachment to certain C-type lectins. SFTSVpv is an appropriate model for the investigation of SFTSV-GP-mediated cell entry and virus neutralization at lower biosafety levels. Furthermore, 25HC may represent a potential antiviral agent against SFTS. IMPORTANCE SFTSV is a recently discovered bunyavirus associated with SFTS, a viral hemorrhagic fever with a high case fatality rate endemic to China, South Korea, and Japan. Because little is known about the characteristics of the envelope protein and entry mechanisms of SFTSV, further studies will be required for the development of a vaccine or effective

  10. Brugia malayi Antigen (BmA) Inhibits HIV-1 Trans-Infection but Neither BmA nor ES-62 Alter HIV-1 Infectivity of DC Induced CD4+ Th-Cells

    PubMed Central

    Mouser, Emily E. I. M.; Pollakis, Georgios; Yazdanbakhsh, Maria; Harnett, William

    2016-01-01

    One of the hallmarks of HIV-1 disease is the association of heightened CD4+ T-cell activation with HIV-1 replication. Parasitic helminths including filarial nematodes have evolved numerous and complex mechanisms to skew, dampen and evade human immune responses suggesting that HIV-1 infection may be modulated in co-infected individuals. Here we studied the effects of two filarial nematode products, adult worm antigen from Brugia malayi (BmA) and excretory-secretory product 62 (ES-62) from Acanthocheilonema viteae on HIV-1 infection in vitro. Neither BmA nor ES-62 influenced HIV-1 replication in CD4+ enriched T-cells, with either a CCR5- or CXCR4-using virus. BmA, but not ES-62, had the capacity to bind the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) thereby inhibiting HIV-1 trans-infection of CD4+ enriched T-cells. As for their effect on DCs, neither BmA nor ES-62 could enhance or inhibit DC maturation as determined by CD83, CD86 and HLA-DR expression, or the production of IL-6, IL-10, IL-12 and TNF-α. As expected, due to the unaltered DC phenotype, no differences were found in CD4+ T helper (Th) cell phenotypes induced by DCs treated with either BmA or ES-62. Moreover, the HIV-1 susceptibility of the Th-cell populations induced by BmA or ES-62 exposed DCs was unaffected for both CCR5- and CXCR4-using HIV-1 viruses. In conclusion, although BmA has the potential capacity to interfere with HIV-1 transmission or initial viral dissemination through preventing the virus from interacting with DCs, no differences in the Th-cell polarizing capacity of DCs exposed to BmA or ES-62 were observed. Neither antigenic source demonstrated beneficial or detrimental effects on the HIV-1 susceptibility of CD4+ Th-cells induced by exposed DCs. PMID:26808476

  11. A Systematic Approach for Computing Zero-Point Energy, Quantum Partition Function, and Tunneling Effect Based on Kleinert’s Variational Perturbation Theory

    PubMed Central

    Wong, Kin-Yiu; Gao, Jiali

    2009-01-01

    In this paper, we describe an automated integration-free path-integral (AIF-PI) method, based on Kleinert’s variational perturbation (KP) theory, to treat internuclear quantum-statistical effects in molecular systems. We have developed an analytical method to obtain the centroid potential as a function of the variational parameter in the KP theory, which avoids numerical difficulties in path-integral Monte Carlo or molecular dynamics simulations, especially at the limit of zero-temperature. Consequently, the variational calculations using the KP theory can be efficiently carried out beyond the first order, i.e., the Giachetti-Tognetti-Feynman-Kleinert variational approach, for realistic chemical applications. By making use of the approximation of independent instantaneous normal modes (INM), the AIF-PI method can readily be applied to many-body systems. Previously, we have shown that in the INM approximation, the AIF-PI method is accurate for computing the quantum partition function of a water molecule (3 degrees of freedom) and the quantum correction factor for the collinear H3 reaction rate (2 degrees of freedom). In this work, the accuracy and properties of the KP theory are further investigated by using the first three order perturbations on an asymmetric double-well potential, the bond vibrations of H2, HF, and HCl represented by the Morse potential, and a proton-transfer barrier modeled by the Eckart potential. The zero-point energy, quantum partition function, and tunneling factor for these systems have been determined and are found to be in excellent agreement with the exact quantum results. Using our new analytical results at the zero-temperature limit, we show that the minimum value of the computed centroid potential in the KP theory is in excellent agreement with the ground state energy (zero-point energy) and the position of the centroid potential minimum is the expectation value of particle position in wave mechanics. The fast convergent property of

  12. The molecular mechanism of fullerene-inhibited aggregation of Alzheimer's β-amyloid peptide fragment

    NASA Astrophysics Data System (ADS)

    Xie, Luogang; Luo, Yin; Lin, Dongdong; Xi, Wenhui; Yang, Xinju; Wei, Guanghong

    2014-07-01

    Amyloid deposits are implicated in the pathogenesis of many neurodegenerative diseases such as Alzheimer's disease (AD). The inhibition of β-sheet formation has been considered as the primary therapeutic strategy for AD. Increasing data show that nanoparticles can retard or promote the fibrillation of amyloid-β (Aβ) peptides depending on the physicochemical properties of nanoparticles, however, the underlying molecular mechanism remains elusive. In this study, our replica exchange molecular dynamics (REMD) simulations show that fullerene nanoparticle - C60 (with a fullerene : peptide molar ratio greater than 1 : 8) can dramatically prevent β-sheet formation of Aβ(16-22) peptides. Atomic force microscopy (AFM) experiments further confirm the inhibitory effect of C60 on Aβ(16-22) fibrillation, in support of our REMD simulations. An important finding from our REMD simulations is that fullerene C180, albeit with the same number of carbon atoms as three C60 molecules (3C60) and smaller surface area than 3C60, displays an unexpected stronger inhibitory effect on the β-sheet formation of Aβ(16-22) peptides. A detailed analysis of the fullerene-peptide interaction reveals that the stronger inhibition of β-sheet formation by C180 results from the strong hydrophobic and aromatic-stacking interactions of the fullerene hexagonal rings with the Phe rings relative to the pentagonal rings. The strong interactions between the fullerene nanoparticles and Aβ(16-22) peptides significantly weaken the peptide-peptide interaction that is important for β-sheet formation, thus retarding Aβ(16-22) fibrillation. Overall, our studies reveal the significant role of fullerene hexagonal rings in the inhibition of Aβ(16-22) fibrillation and provide novel insight into the development of drug candidates against Alzheimer's disease.Amyloid deposits are implicated in the pathogenesis of many neurodegenerative diseases such as Alzheimer's disease (AD). The inhibition of

  13. 3-Bromopyruvate (3BP) a fast acting, promising, powerful, specific, and effective "small molecule" anti-cancer agent taken from labside to bedside: introduction to a special issue.

    PubMed

    Pedersen, Peter L

    2012-02-01

    Although the "Warburg effect", i.e., elevated glucose metabolism to lactic acid (glycolysis) even in the presence of oxygen, has been recognized as the most common biochemical phenotype of cancer for over 80 years, its biochemical and genetic basis remained unknown for over 50 years. Work focused on elucidating the underlying mechanism(s) of the "Warburg effect" commenced in the author's laboratory in 1969. By 1985 among the novel findings made two related most directly to the basis of the "Warburg effect", the first that the mitochondrial content of tumors exhibiting this phenotype is markedly decreased relative to the tissue of origin, and the second that such mitochondria have markedly elevated amounts of the enzyme hexokinase-2 (HK2) bound to their outer membrane. HK2 is the first of a number of enzymes in cancer cells involved in metabolizing the sugar glucose to lactic acid. At its mitochondrial location HK2 binds at/near the protein VDAC (voltage dependent anion channel), escapes inhibition by its product glucose-6-phosphate, and gains access to mitochondrial produced ATP. As shown by others, it also helps immortalize cancer cells, i.e., prevents cell death. Based on these studies, the author's laboratory commenced experiments to elucidate the gene basis for the overexpression of HK2 in cancer. These studies led to both the discovery of a unique HK2 promoter region markedly activated by both hypoxic conditions and moderately activated by several metabolites (e.g., glucose), Also discovered was the promoter's regulation by epigenetic events (i.e., methylation, demethylation). Finally, the author's laboratory turned to the most important objective. Could they selectively and completely destroy cancerous tumors in animals? This led to the discovery in an experiment conceived, designed, and conducted by Young Ko that the small molecule 3-bromopyruvate (3BP), the subject of this mini-review series, is an incredibly powerful and swift acting anticancer agent

  14. Use of a chemical trigger for electron transfer to characterize a precursor to cluster X in assembly of the iron-radical cofactor of Escherichia coli ribonucleotide reductase.

    PubMed

    Saleh, Lana; Krebs, Carsten; Ley, Brenda A; Naik, Sunail; Huynh, Boi Hanh; Bollinger, J Martin

    2004-05-25

    A key step in generation of the catalytically essential tyrosyl radical (Y122(*)) in protein R2 of Escherichia coli ribonucleotide reductase is electron transfer (ET) from the near-surface residue, tryptophan 48 (W48), to a (Fe(2)O(2))(4+) complex formed by addition of O(2) to the carboxylate-bridged diiron(II) cluster. Because this step is rapid, the (Fe(2)O(2))(4+) complex does not accumulate and, therefore, has not been characterized. The product of the ET step is a "diradical" intermediate state containing the well-characterized Fe(IV)Fe(III) cluster, X, and a W48 cation radical (W48(+)(*)). The latter may be reduced from solution to complete the two-step transfer of an electron to the buried diiron site. In this study, a (Fe(2)O(2))(4+) state that is probably the precursor to the X-W48(+)(*) diradical state in the reaction of the wild-type protein (R2-wt) has been characterized by exploitation of the observation that in R2 variants with W48 replaced with alanine (A), the otherwise disabled ET step can be mediated by indole compounds. Mixing of the Fe(II) complex of R2-W48A/Y122F with O(2) results in accumulation of an intermediate state that rapidly converts to X upon mixing with 3-methylindole (3-MI). The state comprises at least two species, of which each exhibits an apparent Mössbauer quadrupole doublet with parameters characteristic of high-spin Fe(III) ions. The isomer shifts of these complexes and absence of magnetic hyperfine coupling in their Mössbauer spectra suggest that both are antiferromagnetically coupled diiron(III) clusters. The fact that both rapidly convert to X upon treatment with a molecule (3-MI) shown in the preceding paper to mediate ET in W48A R2 variants indicates that they are more oxidized than X by one electron, which suggests that they have a bound peroxide equivalent. Their failure to exhibit either the long-wavelength absorption (at 650-750 nm) or Mössbauer doublet with high isomer shift (>0.6 mm/s) that are characteristic of

  15. Carbon monoxide contributes to the constipating effects of granisetron in rat colon

    PubMed Central

    Nacci, Carmela; Fanelli, Margherita; Potenza, Maria Assunta; Leo, Valentina; Montagnani, Monica; De Salvia, Maria Antonietta

    2016-01-01

    AIM To investigate the mechanisms underlying the potential contribution of the heme oxygenase/carbon monoxide (HO/CO) pathway in the constipating effects of granisetron. METHODS For in vivo studies, gastrointestinal motility was evaluated in male rats acutely treated with granisetron [25, 50, 75 μg/kg/subcutaneous (sc)], zinc protoporphyrin IX [ZnPPIX, 50 μg/kg/intraperitoneal (ip)] and hemin (50 μmol/L/kg/ip), alone or in combination. For in vitro studies, the contractile neurogenic response to electrical field stimulation (EFS, 3, 5, 10 Hz, 14 V, 1 ms, pulse trains lasting 10 s), as well as the contractile myogenic response to acetylcholine (ACh, 0.1-100 μmol/L) were evaluated on colon specimens incubated with granisetron (3 μmol/L, 15 min), ZnPPIX (10 μmol/L, 60 min) or CO-releasing molecule-3 (CORM-3, 100, 200, 400 μmol/L) alone or in combination. These experiments were performed under co-treatment with or without atropine (3 μmol/L, a muscarinic receptor antagonist) or NG-nitro-L-Arginine (L-NNA, 100 μmol/L, a nitric oxide synthase inhibitor). RESULTS Administration of granisetron (50, 75 μg/kg) in vivo significantly increased the time to first defecation (P = 0.045 vs vehicle-treated rats), clearly suggesting a constipating effect of this drug. Although administration of ZnPPIX or hemin alone had no effect on this gastrointestinal motility parameter, ZnPPIX co-administered with granisetron abolished the granisetron-induced constipation. On the other hand, co-administration of hemin and granisetron did not modify the increased constipation observed under granisetron alone. When administered in vitro, granisetron alone (3 μmol/L) did not significantly modify the colon’s contractile response to either EFS or ACh. Incubation with ZnPPIX alone (10 μmol/L) significantly reduced the colon’s contractile response to EFS (P = 0.016) but had no effect on contractile response to ACh. Co-administration of ZnPPIX and atropine (3 μmol/L) abolished the Zn

  16. PREFACE: 14th International Conference on the Physics of Highly Charged Ions (HCI 2008)

    NASA Astrophysics Data System (ADS)

    Azuma, Toshiyuki; Nakamura, Nobuyuki; Yamada, Chikashi

    2009-07-01

    the morning of 1 September. In the evening we invited the foreign delegates to the Tokyo-EBIT laboratory and entertained them with sushi and YEBISU beer at the lab (YEBISU is a popular brand of beer in Japan and also the nickname of the Tokyo-EBIT project, Young Electron Beam Ion Source Unit). Cans of YEBISU beer also helped us to make the poster sessions lively, on the evenings of 2 and 4 September. The best poster of each session was selected by the vote of International Advisory Board, and two young scientists were awarded the prizes. On the afternoon of 3 September, we took an excursion to Lake Yamanaka near Mount Fuji for a conference outing. The conference dinner was then held at a hotel alongside Lake Yamanaka from where we really enjoyed the view of Mount Fuji. In the summer it is quite rare to have clear weather, but a teru-teru bozu (a Japanese paper doll with a wish for good weather) placed on the top page of the conference WEB site brought us clear skies for the dinner. About 200 scientists from 22 countries registered for the HCI2008. The number of invited talks was 18 and the contributed papers 206. These proceedings contain 9 invited talks and 107 contributed articles. Following the previous conference, they are grouped into the five categories of: (1) Fundamental aspects, structure and spectroscopy; (2) Collisions with electrons, ions, atoms and molecules; (3) Interactions with clusters, surfaces and solids; (4) Interactions with photons, plasmas and strong field processes, and (5) Production, experimental developments and applications. We are happy that the conference was completely successful, and this success owes much to the sponsors. We acknowledge the support from the Japan Society for the Promotion of Science (JSPS), The International Union of Pure and Applied Physics (IUPAP), Matsuo Foundation, Iwatani Naoji Foundation, CASIO Science Promotion Foundation, and The Society for Atomic Collision Research. The next International Conference on the

  17. 16th international conference on the physics of highly charged ions

    NASA Astrophysics Data System (ADS)

    Fritzsche, Stephan; Stöhlker, Thomas; Surzhykov, Andrey

    2013-09-01

    physicists. The conference was held in the Physics Lecture Hall at the New Campus of Heidelberg University. On the evening of 2 September, the day before the opening of HCI 2012, all participants were welcomed warmly at the foyer of this lecture hall, whose decorative glass front provides a view upon artificial ponds and water lilies at this time of the year. For many colleagues and delegates, this evening offered a hearty re-encounter with each other, along with wine and other beverages. The conference then opened on the morning of 3 September, and an exciting program was organized by the local committee with the help of the International Advisory Board, including 5 invited talks, 10 progress reports as well as 26 selected talks. In addition, more than 230 posters were presented in two sessions, with beer and brezels aside. On Tuesday evening, an exciting public lecture on Heavy ions in therapy and space was given by Marco Durante from the GSI Helmholtz Center and Technical University in Darmstadt. Moreover, many of the participants joined the guided tour through the old city of Heidelberg with its famous (ruins of the) castle, and several the Solar Boat Trip. On Thursday night, we all enjoyed the conference dinner with home-brewed beer and regional specialties at the 'Kulturbrauerei' in the historic center of Heidelberg. Finally, scientific tours were also organized to GSI Darmstadt and the Max-Planck Institute for Nuclear Physics in Heidelberg on the last day of the conference, and attracted much consideration. We here present the proceedings of this conference that contain a total of 104 contributions, including invited papers, progress reports and contributed papers. As previously, these papers are grouped into five categories. (1) Fundamental aspects, structure and spectroscopy. (2) Collisions with electrons, ions, atoms and molecules. (3) Interactions with clusters, surfaces and solids. (4) Interactions with photons, plasmas and strong field processes. (5) Production

  18. Optical Detection of Anomalous Nitrogen in Comets

    NASA Astrophysics Data System (ADS)

    2003-12-01

    studies will provide crucial information about the detailed composition of a much larger number of comets than hitherto possible and hence, more information about the primordial matter from which the solar system formed. A better understanding of the origins of the cometary material (in particular the HCN and CN molecules [3]) and the connection with heavier organic molecules is highly desirable. This is especially so in view of the probable rôle of comets in bringing to the young Earth materials essential for the subsequent formation of life on our planet . PR Photo 28a/03 : Comet LINEAR (C/2000 WM1) - direct image and UVES slit position. PR Photo 28b/03 : Part of the UVES spectrum of Comet LINEAR (C/2000 WM1) with CN-band. PR Photo 28c/03 : Identification of nitrogen-15 in the spectrum. Cometary material Knowledge of the abundance of the stable isotopes [2] of the light elements in different solar system objects provides critical clues to the origin and early evolution of these objects and of the system as a whole. In order to gain the best possible insight into the origins and formation of the niche in which we live, it is therefore important to determine such isotopic abundance ratios in as many members of the solar family as possible. This is particularly true for comets, believed to be carriers of well-preserved specimens of the pristine material from which the solar system was made, some 4,600 million years ago. However, the detailed study of cometary material is a difficult task. Measurements of isotopic ratios is an especially daunting undertaking, mainly because of the extreme weakness of the spectral signatures (emissions) of the less abundant species like carbon-13, nitrogen-15, etc.. Measurements of microwave emission from those atoms suffer from additional, inherent uncertainties connected to the much stronger emission of the more abundant species. Measurements in the optical spectral region thus take on particular importance in this context. However, it is