COMPUTATIONAL INTERACTION OF ENTOMOPATHOGENIC FUNGAL SECONDARY METABOLITES WITH PROTEINS INVOLVED IN HUMAN XENOBIOTIC DETOXIFICATION

Objective: Entomopathogenic fungi are rich source of secondary metabolites which posses both pharmacological and insecticidal activity. It is essential to assess metabolite toxicity of chemically diverse toxic metabolites of entomopathogenic fungus. Human acetylcholine esterase, cytochrome p450 and glutathione S-transferase are important enzymes involved in human xenobiotic detoxification. Methods: In this study, in silico interaction of 13 selected secondary metabolites of entomopathogenic fungi with the target human proteins were carried out using Molegro Virtual Docker 4.0.2. Results: This study reveals serinocyclin-A, have shown highest binding energy (176.07 KJ mol-1) with glutathione S-transferase followed by helvolic acid, cytochalasin B and beauverolide H have shown considerable inhibition among the metabolites tested. Conclusion: The study concludes that serinocyclin-A, helvonic acid, cytochalasin B and beauverolide among 13 secondary metabolites tested were found to be more toxic and may inhibit the human metabolic pathways

The role of immune responses, focussing on herpes virus specificity and interferon-gamma, in Myocardial Infarction with reperfusion and in Chronic Fatigue Syndrome

Background Immune responses targeting microbes can contribute to chronic inflammation, particularly when the microbes are persistent such as herpes-family Cytomegalovirus (CMV) and EpsteinBarr virus (EBV). Such persistence of antigens can cause T cell exhaustion characterized by loss of appropriate effector functions, expression of inhibitory receptors, as well as failure to return to homeostatic pre-inflammatory conditions. This results in immune senescence and dysregulation which may cause disrupted cell populations, homing and cytokine productions that mediate immunopathology and compromise anti-microbial defences. Aims The aim of the study was to determine whether microbe specific particularly, interferongamma immune responses measured in 2 disease states, where a herpes viral inflammatory aetiology and immune dysregulation are suggested, acute Myocardial Infarction (MI) with reperfusion and Chronic Fatigue Syndrome (CFS), are indicative of disease presence and severity. Patients MI occurs due to blockage of the coronary artery, and treatment involves stent insertion, allowing reperfusion (R), which has associated immunopathology due to T cell homing. A total of 52 MI patients were studied. Blood samples were taken before and after reperfusion to investigate the dynamics of specific T cell homing to the heart, that may contribute to disease severity (reperfusion damage). For 50 CFS patients with measured disease levels, blood samples were taken to examine immune responses including those against microbes implicated in disease (CMV & EBV) compared to healthy controls. Methods T cell immunity was measured by ELIspot and flow cytometry, and cytokine levels in cell culture supernatants were measured using multiplex technology. Statistical analyses were carried out for normality in data sets. The Mann-Whitney test or unpaired t test was used to determine the difference between two unrelated groups. Differences between repeat or paired measurements were determined by Wilcoxon signed rank tests or paired t tests. Associations between measurements were investigated using Spearman correlation. Results and Discussion In MI patients, compared to before reperfusion, levels of the following cell populations fell significantly after reperfusion: terminally-differentiated CD8+ PD-1+ effector memory cells (p=0.016) and CMV-specific IFN-secreting CD4+ T cells (p=0.002) the latter also being associated with injury. This suggests specific pathogenic T cell homing to the heart during reperfusion. In CFS patients, increased disease severity was associated with increased non-specific IFNy production (p=0.008), and reduced percentage of NK cells (p=0.0047). NK cell deficiency may reduce antiviral defences and allow detrimental viral reactivation. Conclusion T cell responses against CMV appeared to have greater involvement in MI + R than CFS. Immune responses involving IFN-γ are demonstrated in both conditions as being associated with disease, and so this cytokine may be considered a disease biomarker and a therapeutic target for both

Polymer Properties: Functionalization and Surface Modified Nanoparticles

Herein, the various polymer properties and the underlying mechanism for the functionalization and surface modification of polymer nanoparticles have been discussed. There are numerous polymer particles designed and developed for various applications. The synthesis and characterization of different types of polymers followed by the engineering of nanoparticles and capsules depend on various factors. There are too many polymerization methods approached for the development of nanoparticles with desired surface properties. The ring-opening polymerization (ROP), emulsion polymerization (EP), atom transfer radical polymerization (ATRP), and free radical micro initiation are the significant approaches for the polymerization reactions. The polymer nanoparticle functionalization and modification of their surfaces based on requirements is an essential task. The solvent concentration, pH, temperature, and sonication have played a vital role to tune the morphology of polymer nanoparticles and capsules. Different characterizations such as FTIR, NMR (1H and 13C), HRMS, and MALDI-TOF are used for preliminary structural and confirmations. Further, SEM, FE-SEM, TEM, AFM, BET, XRD, Raman, EDAX, TGA-DSC, DLS, and zeta potential were used for morphological and thermal properties

Novel elvitegravir nanoformulation for drug delivery across the blood-brain barrier to achieve HIV-1 suppression in the CNS macrophages

The use of antiretroviral therapy (ART) has remarkably decreased the morbidity associated with HIV-1 infection, however, the prevalence of HIV-1-associated neurocognitive disorders (HAND) is still increasing. The blood-brain barrier (BBB) is the major impediment for penetration of antiretroviral drugs, causing therapeutics to reach only suboptimal level to the brain. Conventional antiretroviral drug regimens are not sufficient to improve the treatment outcomes of HAND. In our recent report, we have developed a poloxamer-PLGA nanoformulation loaded with elvitegravir (EVG), a commonly used antiretroviral drug. The nanoformulated EVG is capable of elevating intracellular drug uptake and simultaneously enhance viral suppression in HIV-1-infected macrophages. In this work, we identified the clinical parameters including stability, biocompatibility, protein corona, cellular internalization pathway of EVG nanoformulation for its potential clinical translation. We further assessed the ability of this EVG nanoformulation to cross the in vitro BBB model and suppress the HIV-1 in macrophage cells. Compared with EVG native drug, our EVG nanoformulation demonstrated an improved BBB model penetration cross the in vitro BBB model and an enhanced HIV-1 suppression in HIV-1-infected human monocyte-derived macrophages after crossing the BBB model without altering the BBB model integrity. Overall, this is an innovative and optimized treatment strategy that has a potential for therapeutic interventions in reducing HAND

Myocardial Ischemia and Reperfusion Leads to Transient CD8 Immune Deficiency and Accelerated Immunosenescence in CMV-Seropositive Patients

Rationale: There is mounting evidence of a higher incidence of coronary heart disease (CHD) in cytomegalovirus (CMV) seropositive individuals. Objective: The aim of this study was to investigate whether acute MI triggers an inflammatory T-cell response that might lead to accelerated immunosenescence in CMV-seropositive patients. Methods and Results: Thirty-four patients with acute MI undergoing primary PCI (PPCI) were longitudinally studied within 3 months following reperfusion (Cohort A). In addition, 54 patients with acute and chronic MI were analyzed in a cross-sectional study (Cohort B). CMV-seropositive patients demonstrated a greater fall in the concentration of terminally differentiated CD8 effector memory T cells (TEMRA) in peripheral blood during the first 30 min of reperfusion compared with CMV-seronegative patients (-192 vs. -63 cells/µl; p=0.008), correlating with the expression of programmed cell death-1 (PD-1) before PPCI (r=0.8; p=0.0002). A significant proportion of TEMRA cells remained depleted for at least 3 months in CMV-seropositive patients. Using high-throughput 13-parameter flow cytometry and HLA class I CMV-specific dextramers, we confirmed an acute and persistent depletion of terminally differentiated TEMRA and CMV-specific CD8+ cells in CMV-seropositive patients. Long-term reconstitution of the TEMRA pool in chronic CMV-seropositive post-MI patients was associated with signs of terminal differentiation including an increase in KLRG1 and shorter telomere length in CD8+ T cells (2225 bp vs. 3397 bp; p<0.001). Conclusions: Myocardial ischemia and reperfusion in CMV-seropositive patients undergoing PPCI leads to acute loss of antigen-specific, terminally differentiated CD8 T-cells, possibly through PD-1-dependent programmed cell death. Our results suggest that acute MI and reperfusion accelerate immunosenescence in CMV-seropositive patients

Anti-bacterial antibody and T cell responses in Bronchiectasis are differentially associated with lung colonization and disease

Background: As a way to determine markers of infection or disease informing disease management, and to reveal disease-associated immune mechanisms, this study sought to measure antibody and T cell responses against key lung pathogens and to relate these to patients’ microbial colonization status, exacerbation history and lung function, in Bronchiectasis (BR) and Chronic Obstructive Pulmonary Disease (COPD). Methods: 119 patients with stable BR, 58 with COPD and 28 healthy volunteers were recruited and spirometry was performed. Bacterial lysates were used to measure specific antibody responses by ELISA and T cells by ELIspot. Cytokine secretion by lysate-stimulated T cells was measured by multiplex cytokine assay whilst activation phenotype was measured by flow cytometry. Results: Typical colonization profiles were observed in BR and COPD, dominated by P.aeruginosa, H.influenzae, S.pneumoniae and M.catarrhalis. Colonization frequency was greater in BR, showing association with increased antibody responses against P.aeruginosa compared to COPD and HV, and with sensitivity of 73% and specificity of 95%. Interferon-gamma T cell responses against P.aeruginosa and S.pneumoniae were reduced in BR and COPD, whilst reactive T cells in BR had similar markers of homing and senescence compared to healthy volunteers. Exacerbation frequency in BR was associated with increased antibodies against P. aeruginosa, M.catarrhalis and S.maltophilia. T cell responses against H.influenzae showed positive correlation with FEV1% (r=0.201, p=0.033) and negative correlation with Bronchiectasis Severity Index (r=-0.287, p=0.0035). Conclusion: Our findings suggest a difference in antibody and T cell immunity in BR, with antibody being a marker of exposure and disease in BR for P.aeruginosa, M.catarrhalis and H.influenzae, and T cells a marker of reduced disease for H.influenzae

An Elvitegravir Nanoformulation Crosses the Blood–Brain Barrier and Suppresses HIV-1 Replication in Microglia

Even with an efficient combination of antiretroviral therapy (ART), which significantly decreases viral load in human immunodeficiency virus type 1 (HIV-1)-positive individuals, the occurrence of HIV-1-associated neurocognitive disorders (HAND) still exists. Microglia have been shown to have a significant role in HIV-1 replication in the brain and in subsequent HAND pathogenesis. However, due to the limited ability of ART drugs to cross the blood–brain barrier (BBB) after systemic administration, in addition to efflux transporter expression on microglia, the efficacy of ART drugs for viral suppression in microglia is suboptimal. Previously, we developed novel poly (lactic-co-glycolic acid) (PLGA)-based elvitegravir nanoparticles (PLGA-EVG NPs), which showed improved BBB penetration in vitro and improved viral suppression in HIV-1-infected primary macrophages, after crossing an in vitro BBB model. Our objective in the current study was to evaluate the efficacy of our PLGA-EVG NPs in an important central nervous system (CNS) HIV-1 reservoir, i.e., microglia. In this study, we evaluated the cyto-compatibility of the PLGA-EVG NPs in microglia, using an XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay and cellular morphology observation. We also studied the endocytosis pathway and the subcellular localization of PLGA NPs in microglia, using various endocytosis inhibitors and subcellular localization markers. We determined the ability of PLGA-EVG NPs to suppress HIV-1 replication in microglia, after crossing an in vitro BBB model. We also studied the drug levels in mouse plasma and brain tissue, using immunodeficient NOD scid gamma (NSG) mice, and performed a pilot study, to evaluate the efficacy of PLGA-EVG NPs on viral suppression in the CNS, using an HIV-1 encephalitic (HIVE) mouse model. From our results, the PLGA-EVG NPs showed ~100% biocompatibility with microglia, as compared to control cells. The internalization of PLGA NPs in microglia occurred through caveolae-/clathrin-mediated endocytosis. PLGA NPs can also escape from endo-lysosomal compartments and deliver the therapeutics to cells efficiently. More importantly, the PLGA-EVG NPs were able to show ~25% more viral suppression in HIV-1-infected human-monocyte-derived microglia-like cells after crossing the in vitro BBB compared to the EVG native drug, without altering BBB integrity. PLGA-EVG NPs also showed a ~two-fold higher level in mouse brain and a trend of decreasing CNS HIV-1 viral load in HIV-1-infected mice. Overall, these results help us to create a safe and efficient drug delivery method to target HIV-1 reservoirs in the CNS, for potential clinical use