Soluble ACE2 correlates with severe COVID-19 and can impair antibody responses.

Identifying immune modulators that impact neutralizing antibody responses against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is of great relevance. We postulated that high serum concentrations of soluble angiotensin-converting enzyme 2 (sACE2) might mask the spike and interfere with antibody maturation toward the SARS-CoV-2-receptor-binding motif (RBM). We tested 717 longitudinal samples from 295 COVID-19 patients and showed a 2- to 10-fold increase of enzymatically active sACE2 (a-sACE2), with up to 1 µg/mL total sACE2 in moderate and severe patients. Fifty percent of COVID-19 sera inhibited ACE2 activity, in contrast to 1.3% of healthy donors and 4% of non-COVID-19 pneumonia patients. A mild inverse correlation of a-sACE2 with RBM-directed serum antibodies was observed. In silico, we show that sACE2 concentrations measured in COVID-19 sera can disrupt germinal center formation and inhibit timely production of high-affinity antibodies. We suggest that sACE2 is a biomarker for COVID-19 and that soluble receptors may contribute to immune suppression informing vaccine design.

Chronicity of high and low level mupirocin resistance in Staphylococcus aureus from 30 Indian hospitals.

Mupirocin is one of the most effective topically used antibiotic for the treatment of dermatitis, nasal carriage, decolonization of methicillin susceptible Staphylococcus aureus and eradication of methicillin resistant Staphylococcus aureus. Extensive use of this antibiotic has resulted in mupirocin resistance in Staphylococcus aureus which is a matter of concern. This study was conducted to evaluate the high and low level of mupirocin resistance in Staphylococcus aureus collected from various Indian hospitals. A total of 600 samples, of which 436 were pus specimens and 164 wound site swabs were collected from 30 Indian hospitals. Disc diffusion and agar dilution methods were used to test mupirocin susceptibility in methicillin resistant Staphylococcus aureus. Out of 600 Staphylococcus aureus isolates, 176 isolates (29.33%) were found to be methicillin resistant Staphylococcus aureus (MRSA). Out of 176 non-duplicate MRSA strains, 138 isolates were found to be mupirocin sensitive, 21 isolates had high level resistance whereas 17 isolates had low level resistance to mupirocin, which contributed 78.41%, 11.93% and 9.66% respectively. Multidrug resistant susceptibility was tested for all the MRSA with Cefuroxime, Cotrimoxazole and Vancomycin antibiotics. All the high and low level resistant strain were subjected to genome screening for mupA ileS gene respectively. mupA gene was found positive in all the high level resistant strain and out of 17 low level resistant strain, 16 strain were found point mutation in V588F of ileS gene. Overall, high rate of mupirocin resistance was found in the studied samples which might be a result of indiscriminate use of mupirocin in the population of studied region. This data emphasizes the urgent need for formulation of a well-defined and regulated guidelines for mupirocin use. Moreover, continuous surveillance is needed for the use of mupirocin and routine test should be performed to detect MRSA in patients and health care personnel to prevent MRSA infections.

Amount of antigen, T follicular helper cells and affinity of founder cells shape the diversity of germinal center B cells: A computational study.

A variety of B cell clones seed the germinal centers, where a selection stringency expands the fitter clones to generate higher affinity antibodies. However, recent experiments suggest that germinal centers often retain a diverse set of B cell clones with a range of affinities and concurrently carry out affinity maturation. Amid a tendency to flourish germinal centers with fitter clones, how several B cell clones with differing affinities can be concurrently selected remains poorly understood. Such a permissive selection may allow non-immunodominant clones, which are often rare and of low-affinity, to somatically hypermutate and result in a broad and diverse B cell response. How the constituent elements of germinal centers, their quantity and kinetics may modulate diversity of B cells, has not been addressed well. By implementing a state-of-the-art agent-based model of germinal center, here, we study how these factors impact temporal evolution of B cell clonal diversity and its underlying balance with affinity maturation. While we find that the extent of selection stringency dictates clonal dominance, limited antigen availability on follicular dendritic cells is shown to expedite the loss of diversity of B cells as germinal centers mature. Intriguingly, the emergence of a diverse set of germinal center B cells depends on high affinity founder cells. Our analysis also reveals a substantial number of T follicular helper cells to be essential in balancing affinity maturation with clonal diversity, as a low number of T follicular helper cells impedes affinity maturation and also contracts the scope for a diverse B cell response. Our results have implications for eliciting antibody responses to non-immunodominant specificities of the pathogens by controlling the regulators of the germinal center reaction, thereby pivoting a way for vaccine development to generate broadly protective antibodies.

A Retrospective Community Perspective: Bridging the Health Disparity Gap with Obesity as the Leading Comorbidity in Disparities in Health Outcomes in the COVID-19 Pandemic.

COVID-19 is a viral infection that resulted in a global pandemic. In the United States, COVID-19 caused incommensurate deaths, especially among members of minority groups. Previous literature shows comorbidities such as hypertension (HTN), diabetes mellitus (DM) and obesity (OBS) have been implicated in the severity of COVID-19 cases regardless of racial or ethnic group classification. However, minority populations, particularly people of African descent experienced higher mortality as they carry a disproportionately heavier burden in comorbidities cases. In this study we first confirm current literature on COVID-19 incidence and its correlation with the prevalence of comorbidities in various racial and ethnic populations, using anonymous and aggregated data from the Nashville General Hospital at Meharry, an Institute for the Study of Minority Health. We also evaluated the prevalence of comorbidities in minority patients and computed the correlation between the COVID-19 incidence and a permuted prevalence of comorbidities. A total of 959 patients were reviewed and our study indicates COVID-19 patients classified as Non-Hispanic Blacks (NHB) were approximately 3 times more likely to have an HTN or DM or both HTN and DM diagnosis. The chances double to be approximately six times higher when an OBS diagnosis is included singularly or in conjunction with either HTN or DM or both HTN and DM.

Application of C-Terminal Clostridium Perfringens Enterotoxin in Treatment of Brain Metastasis from Breast Cancer.

Claudin-4 is part of the Claudin family of transmembrane tight junction (TJ) proteins found in almost all tissues and, together with adherens junctions and desmosomes, forms epithelial and endothelial junctional complexes. Although the distribution of Claudin-4 occurs in many cell types, the level of expression is cell-specific. Claudin proteins regulate cell proliferation and differentiation by binding cell-signaling ligands, and its expression is upregulated in several cancers. As a result, alterations in Claudin expression patterns or distribution are vital in the pathology of cancer. Profiling the genetic expression of Claudin-4 showed that Claudin-4 is also a receptor for the clostridium perfringens enterotoxin (CPE) and that Claudin-4 has a high sequence similarity with CPE's high-affinity receptor. CPE is cytolytic due to its ability to form pores in cellular membranes, and CPE treatment in breast cancer cells have shown promising results due to the high expression of Claudin-4. The C-terminal fragment of CPE (c-CPE) provides a less toxic alternative for drug delivery into breast cancer cells, particularly metastatic tumors in the brain, especially as Claudin-4 expression in the central nervous system (CNS) is low. Therefore, c-CPE provides a unique avenue for the treatment of breast-brain metastatic tumors.

Increased B Cell Selection Stringency In Germinal Centers Can Explain Improved COVID-19 Vaccine Efficacies With Low Dose Prime or Delayed Boost.

The efficacy of COVID-19 vaccines appears to depend in complex ways on the vaccine dosage and the interval between the prime and boost doses. Unexpectedly, lower dose prime and longer prime-boost intervals have yielded higher efficacies in clinical trials. To elucidate the origins of these effects, we developed a stochastic simulation model of the germinal center (GC) reaction and predicted the antibody responses elicited by different vaccination protocols. The simulations predicted that a lower dose prime could increase the selection stringency in GCs due to reduced antigen availability, resulting in the selection of GC B cells with higher affinities for the target antigen. The boost could relax this selection stringency and allow the expansion of the higher affinity GC B cells selected, improving the overall response. With a longer dosing interval, the decay in the antigen with time following the prime could further increase the selection stringency, amplifying this effect. The effect remained in our simulations even when new GCs following the boost had to be seeded by memory B cells formed following the prime. These predictions offer a plausible explanation of the observed paradoxical effects of dosage and dosing interval on vaccine efficacy. Tuning the selection stringency in the GCs using prime-boost dosages and dosing intervals as handles may help improve vaccine efficacies.

Immunomodulatory Effects of a Concoction of Natural Bioactive Compounds-Mechanistic Insights.

Natural bioactive compounds derived from plant-based products are known for their biological immunomodulatory activities. They possess systemic pleiotropic effects, minimal side effects, and very low toxicities. Plant-based bioactive compounds have tremendous potential as natural therapeutic entities against various disease conditions and act as anti-inflammatory, antioxidant, anti-mutagenic, anti-microbial, anti-viral, anti-tumour, anti-allergic, neuroprotective, and cardioprotective agents. A herbal formulation extract including five biologically active compounds: Apigenin, Quercetin, Betulinic acid, Oleanolic acid, and ß-Sitosterol can impart several immunomodulatory effects. In this review, we systematically present the impact of these compounds on important molecular signaling pathways, including inflammation, immunity, redox metabolism, neuroinflammation, neutropenia, cell growth, apoptosis, and cell cycle. The review corroborates the beneficial effect of these compounds and shows considerable potential to be used as a safer, more cost-effective treatment for several diseases by affecting the major nodal points of various stimulatory pathways.

Preferential Presentation of High-Affinity Immune Complexes in Germinal Centers Can Explain How Passive Immunization Improves the Humoral Response.

Passive immunization (PI) with external antibodies has been used classically for rapid but temporary alleviation of disease. Transcending this role, recent studies have shown PI to induce lasting improvements in natural antibody production, suggesting that PI could become a powerful tool to engineer humoral responses. We propose a mechanism with which PI can alter the humoral response. Antigen-specific B cells evolve and get selected in germinal centers (GCs) on the basis of their ability to acquire antigen from antibody-antigen complexes presented in GCs. When external antibodies of high affinity for antigen are used, they form the majority of the complexes in GCs, letting only B cells with even higher affinities be selected. Using an in silico GC reaction model, we show that this mechanism explains the improved humoral responses following PI. The model also synthesizes several independent experimental observations, indicating the robustness of the mechanism, and proposes tunable handles to optimize PI.

Cloning and sequence analysis of the gene encoding LipL32 of Leptospira interrogans serovar Sejroe.

Leptospira, a member of the order Spirochaetales, is the causative agent of leptospirosis, an important zoonosis encountered worldwide. The Leptospira interrogans serovar Sejroe was grown in EMJH medium and its DNA was isolated using standard techniques. The LipL32 gene was amplified using the reported primer of Kirschneri of LipL32. The amplified product was found to comprise 756 base pairs. This amplified gene fragment of LipL32 lipoprotein was cloned in E. coli (DH5 alpha) cells using pDrive plasmid as a vector. The recombinant cells were selected on LB agar medium containing ampicillin, X-gal and isopropyl-beta-D-thiogalactopyranoside. Plasmid was extracted from the recombinant white colonies, and restriction endonuclease (RE) analysis was carried out using PstI and SalI. On partial sequence analysis, the product exhibited 756 base pairs, corresponding to 251 amino acids. The cloned gene could be further used for expression of recombinant protein for serodiagnosis of leptospirosis.

Immunoreactive outer membrane proteins of Leptospira interrogans serovar Canicola strain Hond Utrecht IV.

BACKGROUND & OBJECTIVES: Leptospirosis is a severe and complex zoonotic disease prevalent in many countries including India. Current leptospiral research is focussed on the identification of the outer membrane proteins (OMPs) of the organism that could be used in developing diagnostic assays for leptospirosis. METHODS: The Leptospira interrogans serovar Canicola was grown in EMJH medium and the cells were subjected to sarcosyl detergent treatment. The sarcosyl soluble (SS) and sarcosyl insoluble (SI) fractions were analyzed by SDS-PAGE and immunoblotting to deduce their protein profile and identifying various immunodominant antigens. RESULTS: The protein profile of SS fractions indicated the presence of three major bands of 41, 32 and 25 kDa and minor bands of 85 and 46 kDa. The SI fraction in serovar Canicola revealed the presence of 112, 93, 77, 43, 36, 29 and 22.5 kDa as major bands and minor bands of 102 and 53 kDa. In immunoblotting, the SS proteins of 41, 32 and 25 kDa and SI proteins of 112, 77, 36 and 22.5 kDa were detected to be major immunogenic proteins. INTERPRETATION & CONCLUSION: In our study immunogenic proteins were extracted from SS and SI fractions and OMPs were similar to those reported in other pathogenic Leptospira strains. These OMPs being unique to all the pathogenic leptospires, can be targeted for diagnostic purpose. Further analysis of the cellular location and expression of leptospiral proteins will be useful in the annotation of genomic sequence data and in providing insight into the biology of Leptospira cells.

Modification of radiation damage to mitochondrial system in vivo by Podophyllum hexandrum: mechanistic aspects.

The present study was undertaken to investigate whether RP-1 treatment protected mitochondrial system against radiation damage and also to unravel the mechanism associated with this process. Radioprotection of mitochondrial system by Podophyllum hexandrum (RP-1) was investigated to understand its mechanism of action. Levels of superoxide anion (O2-), reduced or oxidized glutathione (GSH or GSSG), thiobarbituric acid reactive substance (TBARS), protein carbonyl (PC), ATP, NADH-ubiquinone oxidoreductase (complex-I), NADH-cytochrome c oxidoreductase (complex I/II), succinate-cytochrome c oxidoreductase (complex II/III) and mitochondrial membrane potential (MMP) were studied in mitochondria isolated from liver of mice belonging to various treatment groups. Whole body y-irradiation (10 Gy) significantly (p < 0.01) increased the formation of O2-, PC, and TBARS, upto 24 h as compared to untreated control. RP-1 treatment (200 mg/kg b.w.) to mice 2 h before irradiation reduced the radiation-induced O2- generation within 4 h and formation of TBARS and PC upto 24 h significantly (p < 0.01). Singularly irradiation or RP-1 treatment significantly (p < 0.01) increased the levels of glutathione within an hour, as compared to untreated control. Pre-irradiation administration of RP-1 enhanced levels of GSH induced increase in complex I (upto 16 h), complex I/III (4 h) complex II/III activity (upto 24 h; p < 0.01) and inhibited the radiation-induced decrease in MMP significantly (24 h; p < 0.01). The present study indicates that RP-1 itself modulates several mitichondrial perameters due to its influence on the biochemical milieu within and outside the cells. However, RP-1 treatment before irradiation modulates radiation induced perturbations such as the increase in electron transport chain enzyme activity, formation of O2-, TBARS and PC to offer radioprotection.

Radiation protection of HepG2 cells by Podophyllum hexandrum Royale.

Radioprotection by an aqueous extract of Podophyllum hexandrum (RP-1) was investigated in HepG2 cells by evaluating colony forming efficacy (CFE), redox status of mitochondria, reactive oxygen species (ROS), generation of nitric oxide (NO), peroxidation of lipids and intracellular glutathione. Lower concentrations of RP-1 (0.1 and 1 microg/ml) rendered maximum radioprotection when administered 1 or 2 h before irradiation. Higher concentrations (5 and 10 microg/ml) however were less effective when administered 1 or 2 h before irradiation, but were more effective with increased time intervals (4 or 8 h) between RP-1 administration and irradiation. RP-1 pre-treatment also significantly inhibited radiation-induced MTT reduction in a concentration and time-dependent manner by decreasing gamma radiation-induced leakage of electrons from electron transport chain. Pre-irradiation administration of RP-1 significantly reduced both ROS and NO generation and enhanced glutathione levels, thereby inhibiting lipid peroxidation.