UDP-glucose 4, 6-dehydratase Activity Plays an Important Role in Maintaining Cell Wall Integrity and Virulence of Candida albicans

Candida albicans, a human fungal pathogen, undergoes morphogenetic changes that are associated with virulence. We report here that GAL102 in C. albicans encodes a homolog of dTDP-glucose 4,6-dehydratase, an enzyme that affects cell wall properties as well as virulence of many pathogenic bacteria. We found that GAL102 deletion leads to greater sensitivity to antifungal drugs and cell wall destabilizing agents like Calcofluor white and Congo red. The mutant also formed biofilms consisting mainly of hyphal cells that show less turgor. The NMR analysis of cell wall mannans of gal102 deletion strain revealed that a major constituent of mannan is missing and the phosphomannan component known to affect virulence is greatly reduced. We also observed that there was a substantial reduction in the expression of genes involved in biofilm formation but increase in the expression of genes encoding glycosylphosphatidylinositol-anchored proteins in the mutant. These, along with altered mannosylation of cell wall proteins together might be responsible for multiple phenotypes displayed by the mutant. Finally, the mutant was unable to grow in the presence of resident peritoneal macrophages and elicited a weak pro-inflammatory cytokine response in vitro. Similarly, this mutant elicited a poor serum pro-inflammatory cytokine response as judged by IFNγ and TNFα levels and showed reduced virulence in a mouse model of systemic candidiasis. Importantly, an Ala substitution for a conserved Lys residue in the active site motif YXXXK, that abrogates the enzyme activity also showed reduced virulence and increased filamentation similar to the gal102 deletion strain. Since inactivating the enzyme encoded by GAL102 makes the cells sensitive to antifungal drugs and reduces its virulence, it can serve as a potential drug target in combination therapies for C. albicans and related pathogens

Manipulation of Signal Transduction Pathways of Chronic Myelogenous Leukemia Cells That Lead to Apoptosis

Leukemia was first described by European physicians during the mid-19th century. During autopsies, physicians noted cases of profoundly elevated white cell counts—today we know that many of these white cells were nonfunctional leukemic cells—and very low red cell counts. For this reason, the condition was referred to as weisses blut (German for “white blood”). Later, the term leukemia (Greek leukos, “white”; haima, “blood”) was applied to the disease (5). Leukemia or leukaemia starts when an immature blood cell in the bone marrow, known as a progenitor cell, becomes cancerous, dividing uncontrollably and overriding the body’s normal restrictions on cell division. Over time, the marrow becomes crowded with cancerous cells, all of them descendants of the first abnormal cell and devoid of normal healthy blood cells. The malignant cells may also accumulate in a patient’s lymph nodes, spleen, and elsewhere. At the time of diagnosis, up to a trillion leukemic cells may be present in the body. Thus, leukemia is a cancer of the blood or bone marrow characterized by an abnormal proliferation of blood cells, usually white blood cells (leukocytes). It is part of the broad group of diseases called hematological neoplasms.According to the American Cancer Society, leukemia is the sixth leading cause of cancer deaths among men and the seventh leading cause of cancer deaths among women. Each year in the United States about 31,000 new cases of leukemia are diagnosed and the disease causes an estimated 22,000 deaths. It accounts for about onethird of all cancers in children under age 15

IL-4 Alone without the Involvement of GM-CSF Transforms Human Peripheral Blood Monocytes to a CD1adim, CD83+ Myeloid Dendritic Cell Subset

Myeloid dendritic cells (DCs) are conventionally generated by culturing human peripheral blood monocytes in the presence of GM-CSF and IL-4. Here we report that IL-4 alone, in the absence of detectable endogenous GM-CSF, transforms human peripheral blood monocytes to a CD1adim DC subset that could be matured to CD83+ DCs. Absence of endogenous GM-CSF in IL-4-DC was demonstrated by RT-PCR and flow cytometry. With the exception of CD1a expression, surface marker, morphology and phagocytic activity of these DCs (IL-4-DC) were similar to myeloid DCs (GM-IL-4-DC) conventionally generated in the presence of GM-CSF and IL-4. Conventional GM-IL-4-DC produced less IL-12 compared with IL-4-DC after stimulation with anti-CD40 monoclonal antibody, or LPS plus IFN-g, although the difference was more prominent when LPS plus IFN-g was used as the stimulus. The GM-IL-4-DC also induced less frequent IFN- g+ T cells in a mixed leukocyte reaction (MLR) than that of IL-4-DC. Yields of IL-4-DCs were marginally lower than that of GM-IL-4-DCs. Our data indicate that peripheral blood monocytes can be transformed to CD1a-deficient myeloid DCs solely by IL-4, and these IL-4-DCs are likely to induce a stronger Th1 response than conventional GMIL- 4-DC

Innate immune mechanisms to oral pathogens in oral mucosa of HIV-infected individuals

A crucial aspect of mucosal HIV transmission is the interaction between HIV, the local environmental milieu and immune cells. The oral mucosa comprises many host cell types including epithelial cells, CD4 + T cells, dendritic cells and monocytes/macrophages, as well as a diverse microbiome predominantly comprising bacterial species. While the oral epithelium is one of the first sites exposed to HIV through oral-genital contact and nursing infants, it is largely thought to be resistant to HIV transmission via mechanisms that are still unclear. HIV-1 infection is also associated with predisposition to secondary infections, such as tuberculosis, and other diseases including cancer. This review addresses the following questions that were discussed at the 8th World Workshop on Oral Health and Disease in AIDS held in Bali, Indonesia, 13 September –15 September 2019: (a) How does HIV infection affect epithelial cell signalling? (b) How does HIV infection affect the production of cytokines and other innate antimicrobial factors, (c) How is the mucosal distribution and function of immune cells altered in HIV infection? (d) How do T cells affect HIV (oral) pathogenesis and cancer? (e) How does HIV infection lead to susceptibility to TB infections

Interferon-gamma induced cell death: Regulation and contributions of nitric oxide, cJun N-terminal kinase, reactive oxygen species and peroxynitrite

Interferon-gamma (Ifn gamma), a known immunomodulatory cytokine, regulates cell proliferation and survival. In this study, the mechanisms leading to the selective susceptibility of some tumor cells to Ifn gamma were deciphered. Seven different mouse tumor cell lines tested demonstrated upregulation of MHC class I to variable extents with Ifn gamma; however, only the cell lines, H6 hepatoma and L929 fibrosarcoma, that produce higher amounts of nitric oxide (NO) and reactive oxygen species (ROS) are sensitive to Ifn gamma-induced cell death. NO inhibitors greatly reduce Ifn gamma-induced ROS; however, ROS inhibitors did not affect the levels of Ifn gamma-induced NO, demonstrating that NO regulates ROS. Consequently, NO inhibitors are more effective, compared to ROS inhibitors, in reducing Ifn gamma-induced cell death. Further analysis revealed that Ifn gamma induces peroxynitrite and 3-nitrotyrosine amounts and a peroxynitrite scavenger, FeTPPS, reduces cell death. Ifn gamma treatment induces the phosphorylation of c-jun N-terminal kinase (Jnk) in H6 and L929 but not CT26, a colon carcinoma cell line, which is resistant to Ifn gamma-mediated death. Jnk activation downstream to NO leads to induction of ROS, peroxynitrite and cell death in response to Ifn gamma. Importantly, three cell lines tested, i.e. CT26, EL4 and Neuro2a, that are resistant to cell death with Ifn gamma alone become sensitive to the combination of Ifn gamma and NO donor or ROS inducer in a peroxynitrite-dependent manner. Overall, this study delineates the key roles of NO as the initiator and Jnk, ROS, and peroxynitrite as the effectors during Ifn gamma-mediated cell death. The implications of these findings in the Ifn gamma-mediated treatment of malignancies are discussed. (C) 2014 Elsevier B.V. All rights reserved

Leishmania donovani Infection of Human Myeloid Dendritic Cells Leads to a Th1 Response in CD4+ T Cells from Healthy Donors and Patients with Kala-Azar.

The role played by dendritic cells (DCs) in Leishmania donovani infection is poorly understood. Here, we report that L. donovani amastigotes efficiently infect human peripheral-blood monocyte–derived DCs. Opsonization with normal human serum enhanced the infectivity of amastigotes and promastigotes only marginally. Surface attachment versus internalization was distinguished by incubation of DCs with live, fluorescein isothiocyanate–labeled parasites, followed by quenching with crystal violet. Infection with amastigotes was accompanied by DC maturation, as was evident from the up-regulation of maturation-associated cell-surface markers, the nuclear translocation of RelB, and the release of cytokines. Amastigote-primed DCs produced inflammatory cytokines in response to subsequent treatment with interferon-g or anti-CD40 monoclonal antibody. When cocultured, amastigote-infected DCs induced T helper cell type 1 (Th1) responses both in naive allogeneic CD4+ T cells and in autologous CD4+ T cells from patients with kala-azar and up-regulated the expression of T-bet. Our data reveal that infection with L. donovani amastigotes induces a Th1 cytokine milieu in both DCs and T cells

N-acetyl cysteine enhances imatinib-induced apoptosis of Bcr-Abl+ cells by endothelial nitric oxide synthase-mediated production of nitric oxide

Introduction Imatinib, a small-molecule inhibitor of the Bcr-Abl kinase, is a successful drug for treating chronic myeloid leukemia (CML). Bcr-Abl kinase stimulates the production of H2O2, which in turn activates Abl kinase. We therefore evaluated whether N-acetyl cysteine (NAC), a ROS scavenger improves imatinib efficacy. Materials and methods Effects of imatinib and NAC either alone or in combination were assessed on Bcr-Abl? cells to measure apoptosis. Role of nitric oxide (NO) in NAC-induced enhanced cytotoxicity was assessed using pharmacological inhibitors and siRNAs of nitric oxide synthase isoforms. We report that imatinib-induced apoptosis of imatinib-resistant and imatinib-sensitive Bcr-Abl? CML cell lines and primary cells from CML patients significantly enhanced by co-treatment with NAC compared to imatinib treatment alone. In contrast, another ROS scavenger glutathione reversed imatinib-mediated killing. NAC-mediated enhanced killing correlated with cleavage of caspases, PARP and up-regulation and down regulation of pro- and anti-apoptotic family of proteins, respectively. Co-treatment with NAC leads to enhanced production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). Involvement of eNOS dependent NO in NACmediated enhancement of imatinib-induced cell death was confirmed by nitric oxide synthase (NOS) specific pharmacological inhibitors and siRNAs. Indeed, NO donor sodium nitroprusside (SNP) also enhanced imatinib-mediated apoptosis of Bcr-Abl? cells. Conclusion NAC enhances imatinib-induced apoptosis of Bcr-Abl? cells by endothelial nitric oxide synthasemediated production of nitric oxide