Tetrahydropyrazolo[1,5-a]Pyrimidine-3-Carboxamide and N-Benzyl-6′,7′-Dihydrospiro[Piperidine-4,4′-Thieno[3,2-c]Pyran] analogues with bactericidal efficacy against Mycobacterium tuberculosis targeting MmpL3

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. As part of our efforts towards the discovery of new anti-tubercular leads, a number of potent tetrahydropyrazolo[1,5-a]pyrimidine-3-ca​rboxamide(THPP) and N-benzyl-6′,7′-dihydrospiro[piperidine-4,​4′-thieno[3,2-c]pyran](Spiro) analogues were recently identified against Mycobacterium tuberculosis and Mycobacterium bovis BCG through a high-throughput whole-cell screening campaign. Herein, we describe the attractive in vitro and in vivo anti-tubercular profiles of both lead series. The generation of M. tuberculosis spontaneous mutants and subsequent whole genome sequencing of several resistant mutants identified single mutations in the essential mmpL3 gene. This ‘genetic phenotype’ was further confirmed by a ‘chemical phenotype’, whereby M. bovis BCG treated with both the THPP and Spiro series resulted in the accumulation of trehalose monomycolate. In vivo efficacy evaluation of two optimized THPP and Spiro leads showed how the compounds were able to reduce >2 logs bacterial cfu counts in the lungs of infected mice

Interferon-Alpha Administration Enhances CD8+ T Cell Activation in HIV Infection

Type I interferons play important roles in innate immune defense. In HIV infection, type I interferons may delay disease progression by inhibiting viral replication while at the same time accelerating disease progression by contributing to chronic immune activation.To investigate the effects of type I interferons in HIV-infection, we obtained cryopreserved peripheral blood mononuclear cell samples from 10 subjects who participated in AIDS Clinical Trials Group Study 5192, a trial investigating the activity of systemic administration of IFNα for twelve weeks to patients with untreated HIV infection. Using flow cytometry, we examined changes in cell cycle status and expression of activation antigens by circulating T cells and their maturation subsets before, during and after IFNα treatment.The proportion of CD38+HLA-DR+CD8+ T cells increased from a mean of 11.7% at baseline to 24.1% after twelve weeks of interferon treatment (p = 0.006). These frequencies dropped to an average of 20.1% six weeks after the end of treatment. In contrast to CD8+ T cells, the frequencies of activated CD4+ T cells did not change with administration of type I interferon (mean percentage of CD38+DR+ cells = 2.62% at baseline and 2.17% after 12 weeks of interferon therapy). As plasma HIV levels fell with interferon therapy, this was correlated with a "paradoxical" increase in CD8+ T cell activation (p<0.001).Administration of type I interferon increased expression of the activation markers CD38 and HLA DR on CD8+ T cells but not on CD4+ T cells of HIV+ persons. These observations suggest that type I interferons may contribute to the high levels of CD8+ T cell activation that occur during HIV infection

<雜録>歐洲ニ於ケル農業的勞働關係

HIV infection provokes a myriad of pathological effects on the immune system where many markers of CD4+ T cell dysfunction have been identified. However, most studies to date have focused on single/double measurements of immune dysfunction, while the identification of pathological CD4+ T cell clusters that is highly associated to a specific biomarker for HIV disease remain less studied. Here, multi-parametric flow cytometry was used to investigate immune activation, exhaustion, and senescence of diverse maturation phenotypes of CD4+ T cells. The traditional method of manual data analysis was compared to a multidimensional clustering tool, FLOw Clustering with K (FLOCK) in two cohorts of 47 untreated HIV-infected individuals and 21 age and sex matched healthy controls. In order to reduce the subjectivity of FLOCK, we developed an "artificial reference", using 2% of all CD4+ gated T cells from each of the HIV-infected individuals. Principle component analyses demonstrated that using an artificial reference lead to a better separation of the HIV-infected individuals from the healthy controls as compared to using a single HIV-infected subject as a reference or analyzing data manually. Multiple correlation analyses between laboratory parameters and pathological CD4+ clusters revealed that the CD4/CD8 ratio was the preeminent surrogate marker of CD4+ T cells dysfunction using all three methods. Increased frequencies of an early-differentiated CD4+ T cell cluster with high CD38, HLA-DR and PD-1 expression were best correlated (Rho = -0.80, P value = 1.96×10-11) with HIV disease progression as measured by the CD4/CD8 ratio. The novel approach described here can be used to identify cell clusters that distinguish healthy from HIV infected subjects and is biologically relevant for HIV disease progression. These results further emphasize that a simple measurement of the CD4/CD8 ratio is a useful biomarker for assessment of combined CD4+ T cell dysfunction in chronic HIV disease

Analysis of apoptosis in lymph nodes of HIV-infected persons. Intensity of apoptosis correlates with the general state of activation of the lymphoid tissue and not with stage of disease or viral burden

The occurrence of in vivo apoptosis was investigated in lymph node sections obtained from HIV-infected persons at different stages of disease. The degree of apoptosis in lymph nodes from HIV-infected individuals was compared with that observed in lymph nodes obtained from HIV-negative individuals. Apoptosis was readily detected in lymph nodes obtained from both HIV-negative and HIV-positive persons; however, the degree of apoptosis in lymph nodes obtained from HIV-positive persons was three to four times higher than that observed in the lymph nodes obtained from HIV-negative persons. In contrast to HIV-negative lymph nodes in which apoptosis was confined largely to germinal centers, in HIV-positive lymph nodes all functional compartments of the lymph node (i.e., cortex, paracortex, and sinuses) were extensively involved by this phenomenon. Furthermore, a significant correlation was observed between intensity of apoptosis and degree of activation of the lymphoid tissue associated with HIV infection. In contrast, intensity of apoptosis correlated neither with the clinical stage of HIV disease nor with the viral burden in the lymph node. Finally, apoptosis was not restricted only to CD4+ T cells; both B cells and CD8+ T cells were found to undergo apoptosis. Taken together, these results indicate that the increased intensity of the apoptotic phenomenon in HIV infection is caused by the general state of immune activation, and is independent of the progression of HIV disease and of the levels of viral load

The effect of the PI3K/AKT pathway inhibition on leiomyosarcoma cells

9002 Background: Leiomyosarcomas (LMS) are one of the most common histological types of soft tissue sarcomas and are usually resistant to standard chemotherapy. Novel agents that can cause apoptosi..

The effect of the proteosome inhibitor, PS 341 on leiomyosarcoma cells

9040 Background: Leiomyosarcomas (LMS) are one of the most common histologic types of soft tissue sarcoma and are usually resistant to standard chemotherapy. Bortezomib (PS 341) a reversible inhibi..

Relationship between the frequency of HIV-specific CD8(+) T cells and the level of CD38(+)CD8(+) T cells in untreated HIV-infected individuals

CD8(+) T cells are critical for effective host defenses against viral infections. Studies addressing HIV-induced immune responses in infected individuals have suggested that CD8(+) T cells play an important role in controlling viral replication. However, despite an abundance of HIV-specific CD8(+) T cells, HIV is not contained in many untreated patients. Active HIV replication is associated with numerous immunologic changes, the most notable and consistent of which is an increase in CD8(+) T cells expressing CD38. Previous studies have demonstrated that the expression of CD38 on CD8(+) T cells is associated with poor prognostic outcome in infected individuals with detectable plasma viremia; however, the relationship between the expression of CD38 and the frequency of HIV-specific CD8(+) T cells is unclear. We demonstrate a correlation between levels of HIV-specific CD8(+) T cells and levels of CD8(+) T cells expressing CD38 in untreated patients. The distribution of HIV-specific CD8(+) T cells was heavily skewed toward CD38(+)CD8(+) T cells in patients with a high percentage of CD38(+)CD8(+) T cells. Spontaneous/Fas-mediated apoptosis in CD38(+)CD8(+) T cells was significantly higher in patients with high percentages of CD38(+)CD8(+) T cells. Our data suggest that a substantial proportion of the HIV-specific CD8(+) T cells present in CD38(+)CD8(+) T cells in patients with active viral replication arise by HIV-driven aberrant immune activation and may not manifest effective cytolytic activity against infected targets due to a high degree of susceptibility to apoptosis, thus providing an explanation of why HIV is not successfully contained by CD8(+) T cells in such individuals