Presence of RD149 Deletions in M. tuberculosis Central Asian Strain1 Isolates Affect Growth and TNFα Induction in THP-1 Monocytes

Central Asian Strain 1 (CAS1) is the prevalent Mycobacterium tuberculosis genogroup in South Asia. CAS1 strains carry deletions in RD149 and RD152 regions. Significance of these deletions is as yet unknown. We compared CAS1 strains with RD149 and concurrent RD149-RD152 deletions with CAS1 strains without deletions and with the laboratory reference strain, M. tuberculosis H37Rv for growth and for induction of TNFα, IL6, CCL2 and IL10 in THP-1 cells. Growth of CAS1 strains with deletions was slower in broth (RD149; p = 0.024 and RD149-RD152; p = 0.025) than that of strains without deletions. CAS1 strains with RD149 deletion strains further showed reduced intracellular growth (p = 0.013) in THP-1 cells as compared with strains without deletions, and also as compared with H37Rv (p = 0.007) and with CAS1 RD149-RD152 deletion strains (p = 0.029). All CAS1 strains induced higher levels of TNFα and IL10 secretion in THP-1 cells than H37Rv. Additionally, CAS1 strains with RD149 deletions induced more TNFα secretion than those without deletions (p = 0.013). CAS1 RD149 deletion strains from extrapulmonary sources showed more rapid growth and induced lower levels of TNFα and IL6 secretion in THP-1 cells than isolates from pulmonary sources. This data suggests that presence of RD149 reduces growth and increases the induction of TNFα in host cells by CAS1 strains. Differences observed for extrapulmonary strains may indicate an adaptation which increases potential for dissemination and tropism outside the lung. Overall, we hypothesise that RD149 deletions generate genetic diversity within strains and impact interactions of CAS1 strains with host cells with important clinical consequences

Progressive Visceral Leishmaniasis Is Driven by Dominant Parasite-induced STAT6 Activation and STAT6-dependent Host Arginase 1 Expression

The clinicopathological features of the hamster model of visceral leishmaniasis (VL) closely mimic active human disease. Studies in humans and hamsters indicate that the inability to control parasite replication in VL could be related to ineffective classical macrophage activation. Therefore, we hypothesized that the pathogenesis of VL might be driven by a program of alternative macrophage activation. Indeed, the infected hamster spleen showed low NOS2 but high arg1 enzyme activity and protein and mRNA expression (p<0.001) and increased polyamine synthesis (p<0.05). Increased arginase activity was also evident in macrophages isolated from the spleens of infected hamsters (p<0.05), and arg1 expression was induced by L. donovani in primary hamster peritoneal macrophages (p<0.001) and fibroblasts (p<0.01), and in a hamster fibroblast cell line (p<0.05), without synthesis of endogenous IL-4 or IL-13 or exposure to exogenous cytokines. miRNAi-mediated selective knockdown of hamster arginase 1 (arg1) in BHK cells led to increased generation of nitric oxide and reduced parasite burden (p<0.005). Since many of the genes involved in alternative macrophage activation are regulated by Signal Transducer and Activator of Transcription-6 (STAT6), and because the parasite-induced expression of arg1 occurred in the absence of exogenous IL-4, we considered the possibility that L. donovani was directly activating STAT6. Indeed, exposure of hamster fibroblasts or macrophages to L. donovani resulted in dose-dependent STAT6 activation, even without the addition of exogenous cytokines. Knockdown of hamster STAT6 in BHK cells with miRNAi resulted in reduced arg1 mRNA expression and enhanced control of parasite replication (p<0.0001). Collectively these data indicate that L. donovani infection induces macrophage STAT6 activation and STAT6-dependent arg1 expression, which do not require but are amplified by type 2 cytokines, and which contribute to impaired control of infection

Evaluating the impact of COVID-19 on the adoption of virtual care in general practice in 20 countries (inSIGHT): rationale and study protocol

Background: In recent decades, virtual care has emerged as a promising option to support primary care delivery. However, despite the potential, adoption rates remained low. With the outbreak of COVID-19, it has suddenly been pushed to the forefront of care delivery. As we progress into the second year of the COVID-19 pandemic, there is a need and opportunity to review the impact remote care had in primary care settings and reassess its potential future role. This study aims to explore the perspectives of General Practitioners (GPs) / Family Doctors on a.) use of virtual care during the COVID-19 pandemic; b.) perceived impact on quality and safety of care; c.) essential factors for high-quality and sustainable use of virtual care in the future. Methods: Online cross-sectional questionnaire of GPs, distributed across 20 countries. The survey was hosted in Qualtrics and distributed using email, social media, and the researchers’ personal contact networks. General Practitioners were eligible for the survey if they were working mainly in primary care during the period of the COVID-19 pandemic. Descriptive statistical analysis will be performed for quantitative variables, and relationships between the use of virtual care and perceptions on impact on quality and safety of care, and participants’ characteristics, may be explored. Qualitative data (free-text responses) will be analysed using framework analysis. Results: Data collection took place from June to September 2020. As of this manuscript’s submission, a total of 1,605 GP respondents participated in the questionnaire. Further data analysis is currently ongoing. Discussion: The study will provide a comprehensive overview of the availability of virtual care technologies, perceived impact on quality and safety of care and essential factors for high-quality future use. In addition, a description of the underlying factors that influence this adoption and perceptions, in both individual GP/FD characteristics, and the context in which they work, will be provided. While the COVID-19 pandemic may prove the first great stress test of the capabilities, capacity, and robustness of digital systems currently in use, remote care will likely remain an increasingly common approach in the future. There is an imperative to identify the main lessons from this unexpected transformation and use them to inform policy decisions and health service design

The immunological footprint of CMV in HIV-1 patients stable on long-term ART

BACKGROUND:Most HIV-infected persons are cytomegalovirus (CMV) seropositive and retain latent virus that can be reactivated by immune activation. Their T cell populations express markers reflecting a late stage of differentiation, but the contributions of HIV and CMV to this profile are unclear. We investigated the immunological "footprint" of CMV in HIV patients who had a history of extreme immunodeficiency but were now stable on antiretroviral therapy (ART).RESULTS:Twenty CMV seropositive HIV patients &gt;50years old with nadir CD4 T-cell counts &lt;200 cells/mul were studied after &gt;12years on ART. 16 CMV seropositive and 9 CMV seronegative healthy controls were included. CMV antibody titres were higher in HIV patients than controls (P&lt;0.001-0.003). Levels of soluble B-cell activating factor (sBAFF) were elevated in patients (P=0.002) and correlated with levels of CMV antibodies (P=0.03-0.002), with no clear relationship in controls. CD8 T-cell IFNgamma responses to the IE1 peptide (VLE) remained elevated in HIV patients (P=0.005). The CD57 + CD45RA + CD27 phenotype of CD8 T-cells correlated with age (r=0.60, P=0.006), antibodies against CMV IE1 protein (r=0.44, P=0.06) and CD4 T-cell IFNgamma response to CMV lysate (r=0.45, P=0.05).CONCLUSIONS:Humoral and T-cell responses to CMV remained elevated in HIV patients after &gt;12years on ART. Age and presence of CMV disease influenced CD8 T-cell phenotypes. Elevated levels of sBAFF may be a consequence of HIV disease and contribute to high titres of CMV antibody

Specific, targetable interactions with the microenvironment influence imatinib-resistant chronic myeloid leukemia

Therapy resistance in leukemia may be due to cancer cell-intrinsic and/or -extrinsic mechanisms. Mutations within BCR-ABL1, the oncogene giving rise to chronic myeloid leukemia (CML), lead to resistance to tyrosine kinase inhibitors (TKI), and some are associated with clinically more aggressive disease and worse outcome. Using the retroviral transduction/transplantation model of CML and human cell lines we faithfully recapitulate accelerated disease course in TKI resistance. We show in various models, that murine and human imatinib-resistant leukemia cells positive for the oncogene BCR-ABL1T315I differ from BCR-ABL1 native (BCR-ABL1) cells with regards to niche location and specific niche interactions. We implicate a pathway via integrin β3, integrin-linked kinase (ILK) and its role in deposition of the extracellular matrix (ECM) protein fibronectin as causative of these differences. We demonstrate a trend towards a reduced BCR-ABL1T315I+ tumor burden and significantly prolonged survival of mice with BCR-ABL1T315I+ CML treated with fibronectin or an ILK inhibitor in xenogeneic and syngeneic murine transplantation models, respectively. These data suggest that interactions with ECM proteins via the integrin β3/ILK-mediated signaling pathway in BCR-ABL1T315I+ cells differentially and specifically influence leukemia progression. Niche targeting via modulation of the ECM may be a feasible therapeutic approach to consider in this setting