Mohd Tarique

Design, synthesis, and biological evaluation of pyrimidine derivatives as potential inhibitors of human calcium/calmodulin-dependent protein kinase IV.

Calcium/calmodulin‐dependent protein kinase IV (CAMKIV) is a multifunctional Ser/Thr kinase, associated with cerebral hypoxia, cancer, and neurodegenerative diseases. Here, we report design, synthesis, and biological evaluation of seven pyrimidine‐substituted novel inhibitors of CAMKIV. We successfully synthesized and extensively characterized (ESI‐MS, 1H NMR, and 13C NMR studies) seven compounds that are showing appreciable binding affinity to the CAMKIV. Molecular docking and fluorescence binding studies revealed that compound 1 is showing very high binding free energy (ΔG = −11.52 kcal/mol) and binding affinity (K = 9.2 × 1010 m−1) to the CAMKIV. We further performed MTT assay to check the cytotoxicity and anticancer activity of these compounds. An appreciable IC50 (39 μm) value of compound 1 was observed on human hepatoma cell line and nontoxic till the 400 μm on human embryonic kidney cells. To ensure anticancer activity of all these compounds, we further performed propidium iodide assay to evaluate cell viability and DNA content during the cell cycle. We found that compound 1 is again showing a better anticancer activity on both human hepatoma and human embryonic kidney cell lines.

Association of TNF-α-(308(GG)), IL-10(-819(TT)), IL-10(-1082(GG)) and IL-1R1(+1970(CC)) genotypes with the susceptibility and progression of leprosy in North Indian population.

Leprosy is an infectious disease caused by M. leprae. We analyzed 48 cytokine polymorphisms in 13 (pro as well as anti-inflammatory) cytokine genes using PCR-SSP assay in 102 leprosy patients and 120 healthy controls with intent to find out a link between cytokine polymorphisms and disease susceptibility. TNF-α (-308) GG, IL-10 (-819) TT, IL-10 (-1082) GG and IL1R (+1970) CC genotypes are found to be predominant (p=0.01, p=0.02, p=0.0001 and p=0.001, respectively) in both tuberculoid as well as lepromatous leprosy patients. This observation suggests these genotypes as play the central role(s) in the progression of disease. CBA assay demonstrates the varied serum concentration of these cytokines with respect to their genotypes. The above genotypes appeared as high producer genotypes in our study. Even in presence of high produce genotypes, TNF-α level are found to be affected/masked by the presence of IL-10 in leprosy patients. Expressional masking of TNF-α is associated with the expression of IL-10 in these patients. This is one the negative impact of SNP-SNP interaction in leprosy patients. Therefore, we can conclude that cytokine gene polymorphisms determine the predisposition to the leprosy progression.

IL-12 and IL-23 modulate plasticity of FoxP3+ regulatory T cells in human Leprosy

&NA; Leprosy is a bacterial disease caused by M. leprae. Its clinical spectrum reflects the hosts immune response to the M. leprae and provide an ideal model to investigate the host pathogen interaction and immunological dysregulation. Tregs are high in leprosy patients and responsible for immune suppression of the host by producing IL‐10 and TGF‐&bgr; cytokines. In leprosy, plasticity of Tregs remain unstudied. This is the first study describing the conversion of Tregs into Th1‐like and Th17‐like cells using in vitro cytokine therapy in leprosy patients. Peripheral blood mononuclear cells from leprosy patients were isolated and stimulated with M. leprae antigen (MLCwA), rIL‐12 and rIL‐23 for 48 h. Expression of FoxP3 in CD4+CD25+ Tregs, intracellular cytokines IFN‐&ggr;, TGF‐&bgr;, IL‐10 and IL‐17 in Tregs cells were evaluated by flow cytometry (FACS) after stimulation. rIL‐12 treatment increases the levels of pStat4 in Tregs and IFN‐&ggr; production. In the presence of rIL‐23, pStat3+ and IL‐17A+ cells increase. rIL‐12 and r—IL‐23 treatment downregulated the FoxP3 expression, IL‐10 and TGF‐&bgr; production by Tregs and enhances the expression of co‐stimulatory molecules (CD80, CD86). In conclusion rIL‐12 converts Tregs into IFN‐&ggr; producing cells through STAT‐4 signaling while rIL‐23 converts Tregs into IL‐17 producing cells through STAT‐3 signaling in leprosy patients. This study may helpful to provide a new avenue to overcome the immunosuprression in leprosy patients using in vitro cytokine. Highlightsr‐IL‐12 & r‐IL‐23 downregulate FoxP3 expression inTregs in leprosy patients.r‐IL‐12 converts Tregs into Th1 like cells in leprosy patients.r‐IL‐23 converts Tregs into Th17 like cells in leprosy patients.r‐IL‐12 & r‐IL‐23 downregulate IL‐10, TGF‐&bgr; production by T regs in leprosy.r‐IL‐12 & r‐IL‐23 enhance the expression of co‐stimulatory in leprosy patients.

Increased IL-35 producing Tregs and CD19+IL-35+ cells are associated with disease progression in leprosy patients

BACKGROUND The clinical forms of leprosy consist of a spectrum that reflects the hosts immune response to the M. leprae; it provides an ideal model to study the host pathogen interaction and immunological dysregulation in humans. IL‐10 and TGF‐&bgr; producing Tregs are high in leprosy patients and responsible for immune suppression and M. leprae specific T cells anergy. In leprosy, involvement of IL‐35 producing Tregs and Bregs remain unstudied. OBJECTIVE To study the role of IL‐35 producing Tregs and Bregs in the human leprosy. METHODS Peripheral blood mononuclear cells from leprosy patients were isolated and stimulated with M. leprae antigen (MLCwA) for 48 h. Intracellular cytokine IL‐35 was evaluated in CD4+CD25+ Tregs, CD19+ cells by FACS. Expression of PD‐1 on CD4+CD25+ Tregs, CD19+ cells and its ligand (PD‐L1) on B cells, CD11c cells were evaluated by flow cytometry (FACS). Serum IL‐35 level was estimated by ELISA. RESULTS The frequency of IL‐35 producing Tregs and Bregs cells were found to be high in leprosy patients (p < 0.0001) as compared to healthy controls. These cells produced suppressive cytokine IL‐35 which showed positive correlation with bacteriological index (BI) and TGF‐&bgr; producing Tregs, indicating its suppressive nature. We found higher expression of PD‐1 on Tregs, B cell and its ligand (PD‐L1) on antigen presenting cells in leprosy patients. CONCLUSION This study point out a shift in our understanding of the immunological features that mediate and regulate the immune suppression and the disease progression in leprosy patients with a new paradigm (IL‐35 producing Tregs and Bregs) that is beyond TGF‐&bgr; and IL‐10 producing Treg cells. HIGHLIGHTSIL‐35 producing Treg increases from BT to BL/LL pole of leprosy.CD19+IL‐35+ cell increases from BT to BL/LL pole of leprosy.IL‐35 plays important role in the progression of the disease.IL‐35 shows positive correlation with Bacteriological index (BI).

T helper cells in leprosy: An update

Leprosy is an ancient disease caused by gram positive, rod shaped bacilli called Mycobacterium leprae. Patients present with varied clinico-pathological disease depending on the host immune response to Mycobacterium leprae. Thus tuberculoid (TT) and lepromatous (LL) patients represent two ends of a spectrum where the former shows limited disease, high T cell mediate immune (CMI) response and low antibody (HI) levels in serum. In contrast the latter has low T cell and high humoral immune response i.e antibody levels. The mechanisms underlying these differences have been investigated intensely; however, there is no consensus on the primary immunological basis. Over three decades, Th1 and Th2 paradigm were thought to underling tuberculoid and lepromatous disease respectively. However many patients were shown to have mixed Th1/Th2 pattern of (IFN-γ/IL-4) cytokines. The present review was undertaken with a view to understand the T cells and cytokine dysregulation in leprosy. In recent years the sub classes of T cells that are Regulatory in nature (Treg) have been implicated in immune diseases where they were shown to suppress T cell functions. Additionally Th17 cells secreting IL-17A, IL17F, were implicated in immune inflammation. Taken together these regulatory cells may play a part in influencing immune responses in leprosy.

CD4(+) TCRγδ(+) FoxP3(+) cells: An unidentified population of immunosuppressive cells towards disease progression leprosy patients.

This study, for the first time, reveals the role of M. leprae‐specific CD4+TCRγδ+FoxP3+ cells in the progression and pathogenesis of leprosy. Co‐culture with CD4+CD25− cells suggested the immunosuppressive nature of CD4+TCRγδ+ cells in dose‐dependent manner. Isolation of CD4+TCRγδ+ cells from leprosy patients and then culture in presence of M. leprae cell wall antigens (MLCwA) along with TGF β, IPP and IL‐2 suggested that these cells are M. leprae specific. TGF‐β‐mediated SMAD3 signalling was turned out to be major factor towards the expression of FoxP3 in these cells. SMAD3 silencing during induction of these cells barely showed the induction of FoxP3. High density of SMAD3 binding at TGFβRII in CD4+TCRγδ+FoxP3+ furthermore suggested the TGF‐β‐directed SMAD3 signalling in these cells. Taken together the above data, we can conclude that CD4+TCRγδ+FoxP3+ cells possess the potential to track the severity of the disease in leprosy patients.

Fate of T cells and their secretary proteins during the progression of leprosy

Leprosy is an infectious disease caused by non-cultivable bacteria Mycobacterium leprae. Ridley and Jopling classified the disease into five polar forms, Tuberculoid (TT) and Lepromatous (LL), in between two forms of the disease Borderline tuberculoid (BT), Borderline (BB) and Borderline lepromatous (BL) are laid. The tuberculoid type (BT/TT) leprosy patients show good recall of cellmediated immune (CMI) response and Th1 type of immune response, while lepromatous leprosy (LL) patients show defect in cell-mediated immunity to the causative agent and Th2 type of immune response. Due to distinct clinical and immunological spectra of the disease, leprosy attracted immunologists to consider an ideal model for the study of deregulations of various immune reactions. Recent studies show that Tregs, Th3 (TGF-β, IL-10), IL-35 producing Treg immune response associated with the immune suppressive environment, survival of bugs. IL-17 producing Th17 immune response associated with tuberculoid leprosy and play protective role. γδ T cells also increased from tuberculoid to lepromatous pole of leprosy. In this review, we will discuss the role of various subtypes of T-cell and their cytokines in the pathogenesis of leprosy.

γ δ T cells are associated with inflammation and immunopathogenesis of leprosy reactions

BACKGROUND Leprosy reactions appear episodically in leprosy patients, which lead to high inflammation, morbidity and peripheral nerve damage. The role of Th17 cell has been well studied in leprosy reactions but the role of γδ or unconventional T cells which is an other major source of IL-17 in many diseases, not studied in leprosy reactional episodes. OBJECTIVE The aim of the present study to elucidate the role of γδ T cells in leprosy reactions. METHODOLOGY A total of 40 untreated non-reaction and reactions patients were recruited. PBMCs were isolated and stimulated with M. leprae sonicated antigen (MLSA) for 48 h and immuno-phenotyping was done using flow cytometry. Moreover, γδ T cells were isolated by Magnetic beads technology and mRNA expression of IL-17, IFN-γ, TGF-β and FOXP3 were analyzed by real-time PCR (qPCR) and cytokine was estimated in the culture supernatant by ELISA. RESULTS γδ T cells were significantly increased in both Reversal reaction (RR) and Erythema nodosum leprosum (ENL) reaction patients. These cells produced significant amount of IL-17 and IFN-γ. Furthermore, CD3+TCRγδ+ T cells expressed transient FOXP3 with a low amount of TGF-β in both reactions as compared to stable patients. Moreover, low TGF-β producing TCR-γδ cells were associated with low phosphorylation of STAT5A. CONCLUSION This study will add to our understanding of the immunological features that mediate and regulate the pathogenesis of leprosy and may helpful to reduce the immuno-pathogenesis of leprosy reaction by targeting these cells.

Interleukin-10 Producing Regulatory B Cells Transformed CD4+CD25− Into Tregs and Enhanced Regulatory T Cells Function in Human Leprosy

Regulatory B cells (Bregs) are known to exhibit their regulatory functions through interleukin-10 (IL-10) cytokine which suppress inflammation. There are only a few studies explaining the phenotype and functioning of these cells in contribution to host immunity in leprosy. Here, we evaluated the role of IL-10 producing Bregs in the pathogenesis of leprosy and assessed their immunoregulatory effects on Tregs and effector T cells. We found an increased frequency of Bregs and increased expression of their immune modulatory molecules (IL-10, FoxP3, and PDL-1) in leprosy patients. The potential immunoregulatory mechanism of Bregs was also investigated using MACS sorted Teff (CD4+CD25−) and Treg (CD4+CD25+) cells were cocultured with Bregs to elucidate the effects of Bregs on effector T and regulatory T cells. Cell coculture results showed that purified Bregs cells from leprosy patients convert CD4+CD25− cells into CD4+CD25+ cells. Cell coculture experiments also demonstrated that leprosy derived IL-10 producing Bregs enhance FoxP3 and PD-1 expression in Tregs and enhanced Tregs activity. Blocking of IL-10 receptor confirmed that IL-10 producing Breg has immunomodulatory effect on Tregs and effector T cells as effector T cells are not converted into Tregs and enhanced expression of FoxP3 and PD-1 was not observed on Tregs. Collectively, these findings demonstrate that IL-10 producing Breg cells play an important mechanism in controlling the immunopathogenesis of leprosy and have an immunomodulatory effect on Tregs and effector T cells. Our findings may pave way for novel targets of IL-10 producing Bregs for immunotherapy in leprosy patients.