Novel mutation patterns in children with steroid-resistant nephrotic syndrome.

Background: Idiopathic nephrotic syndrome (NS) in children poses treatment challenges, with a subset developing steroid-resistant nephrotic syndrome (SRNS). Genetic factors play a role, yet data on paediatric SRNS genetics in India are scarce. We conducted a prospective study using whole-exome sequencing to explore genetic variants and their clinical correlations. Methods: A single-centre prospective study (October 2018-April 2023) enrolled children with SRNS, undergoing renal biopsy and genetic testing per institutional protocol. Clinical, histological, and genetic data were recorded. DNA isolation and next-generation sequencing were conducted for genetic analysis. Data collection included demographics, clinical parameters, and kidney biopsy findings. Syndromic features were evaluated, with second-line immunosuppressive therapy administered. Patient and renal outcomes are presented for patients with and without genetic variants. Results: A total of 680 paediatric NS patients were analysed, with 121 (17.8%) having SRNS and 96 consent to genetic analysis. 69 (71.9%) had early SRNS, 27 (28.1%) late. Among participants, 62 (64.58%) had reportable genetic variants. The most common were in COL4A genes, with 20 (31.7%) positive. Renal biopsy showed focal segmental glomerulosclerosis in 31/42 (74%) with variants, 16/28 (57.1%) without variants. Second-line immunosuppressions varied, with CNIs the most common. Outcomes varied, with partial or complete remission achieved in some while others progressed to ESRD. Conclusion: The study underscores the importance of genetic analysis in paediatric SRNS, revealing variants in 65.7% of cases. COL4A variants were predominant. Variants correlated with varied renal outcomes, highlighting potential prognostic implications. These findings emphasize the value of personalized approaches and further research in managing paediatric SRNS.

Lead and calcium crosstalk tempted acrosome damage and hyperpolarization of spermatozoa: signaling and ultra-structural evidences.

BACKGROUND: Exposure of humans and animals to heavy metals is increasing day-by-day; thus, lead even today remains of significant public health concern. According to CDC, blood lead reference value (BLRV) ranges from 3.5 µg/dl to 5 µg/dl in adults. Recently, almost 2.6% decline in male fertility per year has been reported but the cause is not well established. Lead (Pb2+) affects the size of testis, semen quality, and secretory functions of prostate. But the molecular mechanism(s) of lead toxicity in sperm cells is not clear. Thus, present study was undertaken to evaluate the adverse effects of lead acetate at environmentally relevant exposure levels (0.5, 5, 10 and 20 ppm) on functional and molecular dynamics of spermatozoa of bucks following in vitro exposure for 15 min and 3 h. RESULTS: Lead significantly decreased motility, viable count, and motion kinematic patterns of spermatozoa like curvilinear velocity, straight-line velocity, average path velocity, beat cross frequency and maximum amplitude of head lateral displacement even at 5 ppm concentration. Pb2+ modulated intracellular cAMP and Ca2+ levels in sperm cells through L-type calcium channels and induced spontaneous or premature acrosome reaction (AR) by increasing tyrosine phosphorylation of sperm proteins and downregulated mitochondrial transmembrane potential. Lead significantly increased DNA damage and apoptosis as well. Electron microscopy studies revealed Pb2+ -induced deleterious effects on plasma membrane of head and acrosome including collapsed cristae in mitochondria. CONCLUSIONS: Pb2+ not only mimics Ca2+ but also affects cellular targets involved in generation of cAMP, mitochondrial transmembrane potential, and ionic exchange. Lead seems to interact with Ca2+ channels because of charge similarity and probably enters the sperm cell through these channels and results in hyperpolarization. Our findings also indicate lead-induced TP and intracellular Ca2+ release in spermatozoa which in turn may be responsible for premature acrosome exocytosis which is essential feature of capacitation for fertilization. Thus, lead seems to reduce the fertilizing capacity of spermatozoa even at 0.5 ppm concentrations.

Oro-Respiratory Dysbiosis and Its Modulatory Effect on Lung Mucosal Toxicity during Exposure or Co-Exposure to Carbon Nanotubes and Cigarette Smoke.

The oro-respiratory microbiome is impacted by inhalable exposures such as smoking and has been associated with respiratory health conditions. However, the effect of emerging toxicants, particularly engineered nanoparticles, alone or in co-exposure with smoking, is poorly understood. Here, we investigated the impact of sub-chronic exposure to carbon nanotube (CNT) particles, cigarette smoke extract (CSE), and their combination. The oral, nasal, and lung microbiomes were characterized using 16S rRNA-based metagenomics. The exposures caused the following shifts in lung microbiota: CNT led to a change from Proteobacteria and Bacteroidetes to Firmicutes and Tenericutes; CSE caused a shift from Proteobacteria to Bacteroidetes; and co-exposure (CNT+CSE) had a mixed effect, maintaining higher numbers of Bacteroidetes (due to the CNT effect) and Tenericutes (due to the CSE effect) compared to the control group. Oral microbiome analysis revealed an abundance of the following genera: Acinetobacter (CNT), Staphylococcus, Aggregatibacter, Allobaculum, and Streptococcus (CSE), and Alkalibacterium (CNT+CSE). These proinflammatory microbial shifts correlated with changes in the relative expression of lung mucosal homeostasis/defense proteins, viz., aquaporin 1 (AQP-1), surfactant protein A (SP-A), mucin 5b (MUC5B), and IgA. Microbiota depletion reversed these perturbations, albeit to a varying extent, confirming the modulatory role of oro-respiratory dysbiosis in lung mucosal toxicity. This is the first demonstration of specific oro-respiratory microbiome constituents as potential modifiers of toxicant effects in exposed lungs.

Does the peroral chromium administration in young Hariana calves reduce the risk of calf diarrhea by ameliorating insulin response, lactose intolerance, antioxidant status, and immune response?

BACKGROUND: The reduction in insulin sensitivity during rumen development may predispose dairy calves towards lactose intolerance, which could be the reason behind neonatal calf diarrhea (NCD). Chromium (Cr) results in a range of effects when fed to ruminants, but most studies have shown improved insulin sensitivity. The aim of this study was to determine the effect of Cr supplementation on insulin sensitivity, lactose intolerance, diarrhoea and antioxidant, and immune response in young Hariana calves. METHODS: A total of 20 milk-fed Hariana calves were randomly assigned to 1 of 2 treatments, each consisted of 10 calves: (1) a control group without supplemental Cr and (2) a 0.15 mg Cr as Cr-picolinate (CrPic)/kg BW0.75 supplemented group (Cr0.15). RESULTS: A more rapid glucose disappearance with unaltered insulin kinetics during intravenous glucose tolerance test (IVGTT) and oral lactose tolerance test (OLTT) indicates greater insulin sensitivity in Cr supplemented calves. Better insulin sensitivity in Cr supplemented calves was further confirmed by higher values of the quantitative insulin sensitivity check index (QUICKI), revised quantitative insulin sensitivity check index (RQUICKI) and insulin receptor substrate-1 (IRS-1) and lower (P < 0.05) values of homeostasis model assessment-insulin resistance (HOMA-IR) and glucose-to-insulin ratio in Cr supplemented calves during IVGTT. Cr supplementation resulted in a lower (P < 0.05) serum cortisol concentration, whereas serum non-esterified fatty acid (NEFA) concentrations during IVGTT did not differ among the groups. The rise in serum glucose concentrations within 2 h post lactose infusion during OLTT peaked at more than twice the basal glucose concentration, therefore calves were not considered as lactose intolerant. Within monthly blood samples, concentrations of serum insulin were similar among treatments, whereas the Cr supplemented group had lower (P < 0.05) serum glucose concentration and glucose-to-insulin ratio compared with the control group. No treatment differences were detected in the biomarkers of antioxidant status and immunity. Serum Cr concentrations were higher (P < 0.05) in Cr supplemented calves while concentrations of other studied minerals were remained unaltered. The incidence, duration of diarrhea, and faecal score were better (P < 0.05) in calves fed on Cr supplemented diet whereas, no treatment effect was observed on average daily gain (ADG). During the study period, no calves died, and no calves were found to have pneumonia, navel or joint disease. CONCLUSION: Feeding a Cr-supplemented diet improved insulin sensitivity and reduced the risk of diarrhoea in milk-fed young calves, but had no or minimal effects on lactose intolerance, antioxidant status, immune response, and growth performance.

Higher pro-inflammatory cytokines IL-6 and IFN-γ are associated with anti-SARS-CoV-2 spike protein-specific seroconversion in renal allograft recipients.

BACKGROUND: Maintenance immunosuppressive regimens are speculated to hamper immunogenic response against severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) in renal transplant recipients (RTRs) compared to the healthy population. Healthy people with SARS-CoV-2 infection often develop neutralizing antibodies and secret copious quantities of cytokines, leading to virus clearance and sometimes more severe immune-related complications. METHODS: RTRs, either acquired SARS-CoV-2 infection (infection group, n = 132) or were vaccinated with two vaccine doses (vaccination group, n = 78) against SARS-CoV-2, were recruited in the study. Thirty-five unvaccinated RTRs, without anti-SARS-CoV-2 spike protein-specific antibodies, were also included as control. Cytokines interleukine-6 (IL-6), interferon-γ (IFN-γ), TGF-ß, and IL-10 were measured using ELISA. The SARS-CoV-2 spike protein-specific IgG-titer was measured by chemiluminescent microparticle immunoassay methods. RESULTS: The seroconversion rate in the infection group was 115/132 (87.12%), with a median antibody titer 706.40 au/mL (IQR, 215.45-1844.42), and in the vaccination group was 63/78 (80.76%) with antibody titer 1454.20 au/mL (IQR, 80.52-3838.75). The IL-6, IFN-γ, TGF-ß, and IL-10 levels were significantly higher in both the infection and vaccination group compared to healthy control. In the infection group, pro-inflammatory cytokines IL-6 (55.41 ± 24.30 vs. 31.64 ± 16.98 pg/mL, p < .001) and IFN-γ (91.21 ± 33.09 vs. 61.69 ± 33.28 pg/mL, p = .001) were significantly higher in the seroconverter group as compared to non-seroconverter. Similarly, in the vaccination group, pro-inflammatory cytokines IL-6 (50.31 ± 25.67 vs. 30.00 ± 11.19 pg/mL; p = .002) and IFN-γ (65.70 ± 39.78 vs. 32.14 ± 17.48 pg/mL; p = .001) were significantly higher in the seroconverter group compared to non-seroconverter. In contrast, TGF-ß (820.96 ± 415.78 vs. 1045.57 ± 204.66; p = .046) was higher in non-seroconverter. CONCLUSIONS: Pro-inflammatory cytokines IL-6 and IFN-γ were significantly associated with seroconversion after SARS-CoV-2 infection and vaccination in RTRs.

Renal injury, biomarkers, and myositis, an understudied aspect of disease: prospective study in the MyoCite cohort.

Introduction: The mechanisms leading to chronic kidney disease (CKD) in patients with idiopathic inflammatory myopathies (IIMs) are poorly understood. We assessed the prevalence of subclinical renal injury in patients with IIMs, through elevation in biomarker levels of tubular injury and fibrosis (NGAL, KIM1, Activin A, CD163, and Cys-c), and assessed differences between subtypes of IIMs, and the effect of disease activity and duration. Materials and methods: Clinical data, core set measures, sera and urine were prospectively collected from all patients enrolled in the MyoCite cohort from 2017 to 2021. Twenty healthy subjects (HC) and 16 patients with acute kidney injury (AKI) were included as controls. Baseline and follow up data for IIMs were included. Enzyme-linked immunosorbent assay (ELISA) was used to measure urine NGAL (Human Lipocalin-2/NGAL Duoset ELISA, Cat no: DY1757), KIM1 (Human TIM-1/KIM 1/HAVCR Duoset ELISA, Cat.no: DY1750B), Activin A (Human Activin A Duoset ELISA, Cat no: DY338), CD163 (Human CD163 Duoset ELISA,Cat no: DY1607-05), and Cys-c (Human Cystatin C Duoset ELISA, Cat. no.: DY1196) levels, while eGFR (unit mL/min/1.73 m2) was calculated by the Cockcroft-Gault formula and CKD-EPI formula. Results: Analysis of 201 visits of 110 adult patients with IIMs indicated higher normalized biomarker levels compared to HCs, and comparable to patients with AKI, with the exception of NGAL, which was higher in the AKI group. Notably 72 (49%) patients with IIMs had eGFR<90; the levels of the 5 biomarkers were comparable between active and inactive IIMs, and different subtypes of IIMs. Similarly, a poor correlation between urine biomarker levels and core set measures of activity and damage was found. Changes in biomarker levels on follow-up did not correlate with eGFR changes. Discussion: This exploratory analysis of urinary biomarkers identified low eGFR and elevated biomarkers of CKD in nearly half of the patients with IIMs, comparable to patients with AKI and higher than HCs, indicative of potential renal damage in IIMs that may have a lead to complications in other systems.

Lower Circulating Cytotoxic T-Cell Frequency and Higher Intragraft Granzyme-B Expression Are Associated with Inflammatory Interstitial Fibrosis and Tubular Atrophy in Renal Allograft Recipients.

Background and Objectives: Inflammatory interstitial fibrosis and tubular atrophy (i-IFTA) is an inflammation in the area of tubular atrophy and fibrosis. i-IFTA is poorly associated with graft outcome and associated with infiltration of inflammatory mononuclear cells. A cytotoxic T cell is a granzyme B+CD8+CD3+ T cell, mainly secret granzyme B. Granzyme B is a serine protease that may mediate allograft injury and inflammatory interstitial fibrosis and tubular atrophy (i-IFTA). However, there is no report identifying the association of granzyme B with i-IFTA after a long post-transplant interval. Material and Methods: In this study, we have measured the cytotoxic T-cell frequency with flow cytometry, serum and PBMCs culture supernatants granzyme-B levels with ELISA and intragraft granzyme-B mRNA transcript expression with the RT-PCR in RTRs in 30 patients with biopsy-proven i-IFTA and 10 patients with stable graft function. Result: The frequency of cytotoxic T cells (CD3+CD8+ granzyme B+) in SGF vs. i-IFTA was (27.96 ± 4.86 vs. 23.19 ± 3.85%, p = 0.011), the serum granzyme-B level was (100.82 ± 22.41 vs. 130.32 ± 46.60, p = 0.038 pg/mL) and the intragraft granzyme-B mRNA transcript expression was (1.01 ± 0.048 vs. 2.10 ± 1.02, p < 0.001 fold). The frequency of CD3+ T cells in SGF vs. i-IFTA was (66.08 ± 6.8 vs. 65.18 ± 9.35%; p = 0.68) and that of CD3+CD8+ T cells was (37.29 ± 4.11 vs. 34.68 ± 5.43%; p = 0.28), which were similar between the 2 groups. CTLc frequency was negatively correlated with urine proteinuria (r = -0.51, p < 0.001), serum creatinine (r = -0.28, p = 0.007) and eGFR (r = -0.28, p = 0.037). Similarly, the PBMC culture supernatants granzyme-B level was negatively correlated with urine proteinuria (r = -0.37, p < 0.001) and serum creatinine (r = -0.31, p = 0.002), while the serum granzyme-B level (r = 0.343, p = 0.001) and intragraft granzyme-B mRNA transcript expression (r = 0.38, p < 0.001) were positively correlated with proteinuria. Conclusions: A decrease in the CTLc frequency in circulation and an increased serum granzyme-B level and intragraft granzyme-B mRNA expression shows that cytotoxic T cells may mediate the allograft injury in RTRs with i-IFTA by releasing granzyme B in serum and intragraft tissue.

Antibody response to ChAdOx1 nCoV-19 (Covishield®) vaccine in people on maintenance hemodialysis.

INTRODUCTION: People on renal replacement therapy (RRT) have a high risk of COVID-19 infection and subsequent death. COVID-19 vaccination is strongly recommended for those on RRT. Data are limited on the immune response of the ChAdOx1 nCoV-19/AZD1222 (Covishield®) vaccine in patients on RRT. METHODS: A prospective cohort of adult (age > 18 years), on RRT in the form of hemodialysis were included and received two intramuscular doses of Covishield®. A blood specimen of 5.0 mL was collected at two time points, within a few days before administering the first dose of the vaccine and at 4-16 weeks after the second dose. According to their prior COVID-19 infection status, the participants were grouped as (i) prior symptomatic COVID-19 infection, (ii) prior asymptomatic COVID-19 infection, and (iii) no prior COVID-19 infection. RESULTS: A large proportion (81%) of participants had anti-spike antibodies (ASAb) before vaccination, and a reasonable proportion (30%) also had neutralizing antibodies (NAb). The titer of ASAb was relatively low (207 U/mL) before vaccination. The ASAb titer (9405 [1635-25,000] U/mL) and percentage of NAb (96.4% [59.6-98.1%]) were markedly increased following the administration of two doses of the vaccine. The participants' prior COVID-19 exposure status did not influence the rise in ASAb titer and NAb percentage. Further, administering two doses of the Covishield vaccine helps them achieve a high ASAb titer. CONCLUSION: Two doses of ChAdOx1 nCoV-19/AZD1222 (Covishield®) vaccine, given 12 weeks apart, achieve a high titer of ASAb and a high percentage of NAb in people on hemodialysis.

Differential modulation of lung aquaporins among other pathophysiological markers in acute (Cl2 gas) and chronic (carbon nanoparticles, cigarette smoke) respiratory toxicity mouse models.

Inhaled toxic chemicals and particulates are known to disrupt lung homeostasis causing pulmonary toxicity and tissue injury. However, biomarkers of such exposures and their underlying mechanisms are poorly understood, especially for emerging toxicants such as engineered nanoparticles and chemical threat agents such as chlorine gas (Cl2). Aquaporins (AQPs), commonly referred to as water channels, are known to play roles in lung homeostasis and pathophysiology. However, little is known on their regulation in toxicant-induced lung injuries. Here, we compared four lung toxicity models namely, acute chemical exposure (Cl2)-, chronic particulate exposure (carbon nanotubes/CNT)-, chronic chemical exposure (cigarette smoke extract/CSE)-, and a chronic co-exposure (CNT + CSE)- model, for modulation of lung aquaporins (AQPs 1, 3, 4, and 5) in relation to other pathophysiological endpoints. These included markers of compromised state of lung mucosal lining [mucin 5b (MUC5B) and surfactant protein A (SP-A)] and lung-blood barrier [protein content in bronchoalveolar lavage (BAL) fluid and, cell tight junction proteins occludin and zona-occludens]. The results showed toxicity model-specific regulation of AQPs measured in terms of mRNA abundance. A differential upregulation was observed for AQP1 in acute Cl2 exposure model (14.71-fold; p = 0.002) and AQP3 in chronic CNT exposure model (3.83-fold; p = 0.044). In contrast, AQP4 was downregulated in chronic CSE model whereas AQP5 showed no significant change in any of the models. SP-A and MUC5B expression showed a decreasing pattern across all toxicity models except the acute Cl2 toxicity model, which showed a highly significant upregulation of MUC5B (25.95-fold; p = 0.003). This was consistent with other significant pathophysiological changes observed in this acute model, particularly a compromised lung epithelial-endothelial barrier indicated by significantly increased protein infiltration and expression of tight junction proteins, and more severe histopathological (structural and immunological) changes. To our knowledge, this is the first report on lung AQPs as molecular targets of the study toxicants. The differentially regulated AQPs, AQP1 in acute Cl2 exposure versus AQP3 in chronic CNT nanoparticle exposure, in conjunction with the corresponding differentially impacted pathophysiological endpoints (particularly MUC5B) could potentially serve as predictive markers of toxicant type-specific pulmonary injury and as candidates for future investigation for clinical intervention.

Crosstalk between gut microbiota and lung inflammation in murine toxicity models of respiratory exposure or co-exposure to carbon nanotube particles and cigarette smoke extract.

Carbon nanotubes (CNTs) are emerging environmental and occupational toxicants known to induce lung immunotoxicity. While the underlying mechanisms are evolving, it is yet unknown whether inhaled CNTs would cause abnormalities in gut microbiota (dysbiosis), and if such microbiota alteration plays a role in the modulation of CNT-induced lung immunotoxicity. It is also unknown whether co-exposure to tobacco smoke will modulate CNT effects. We compared the effects of lung exposure to multi-wall CNT, cigarette smoke extract (CSE), and their combination (CNT + CSE) in a 4-week chronic toxicity mouse model. The exposures induced differential perturbations in gut microbiome as evidenced by altered microbial α- and ß- diversity, indicating a lung-to-gut communication. The gut dysbiosis due to CNTs, unlike CSE, was characterized by an increase in Firmicutes/Bacteroidetes ratio typically associated with proinflammatory condition. Notably, while all three exposures reduced Proteobacteria, the CNT exposure and co-exposure induced appearance of Tenericutes and Cyanobacteria, respectively, implicating them as potential biomarkers of exposure. CNTs differentially induced certain lung proinflammatory mediators (TNF-α, IL-1ß, CCL2, CXCL5) whereas CNTs and CSE commonly induced other mediators (CXCL1 and TGF-ß). The co-exposure showed either a component-dominant effect or a summative effect for both dysbiosis and lung inflammation. Depletion of gut microbiota attenuated both the differentially-induced and commonly-induced (TGF-ß) lung inflammatory mediators as well as granulomas indicating gut-to-lung communication and a modulatory role of gut dysbiosis. Taken together, the results demonstrated gut dysbiosis as a systemic effect of inhaled CNTs and provided the first evidence of a bidirectional gut-lung crosstalk modulating CNT lung immunotoxicity.

Room Temperature Sputtered Aluminum-Doped ZnO Thin Film Transparent Electrode for Application in Solar Cells and for Low-Band-Gap Optoelectronic Devices.

Aluminum-doped zinc oxide (AZO) is a popular, low-cost, nontoxic material that finds application as a transparent conducting electrode in photonic, sensing, and photovoltaic devices. We report the AZO thin films with a high figure of merit on large-area glass substrates by direct current magnetron sputtering without any intentional substrate heating. Furthermore, a simple thermal post-treatment to improve the transmittance of AZO thin film in the infrared region for its application in low-band-gap devices is presented. High optoelectronic properties are obtained by optimizing oxygen content during the sputtering process. The structural, morphological, optoelectrical, and photoluminescence characterization of cold sputtered AZO films is investigated for its latent applications. AZO thin films with an electrical sheet resistance of 8.8 Ω/□ and a visible light transmittance of 78.5% with thickness uniformity above 95% are achieved on 300 mm × 300 mm glass substrate. The AZO film with optimized process conditions is employed as a transparent electrode to fabricate a copper-indium-gallium-selenide-based thin film solar cell, demonstrating 11.8% power conversion efficiency. The AZO film with optimized sputter conditions was post-treated in ambient conditions with an Al blanket to suppress the resistivity by proper organization of the defects due to Al3+ consumption and point defects, resulting in improved transparency (85%) in the infrared region with a sheet resistance of 40 Ω/□. This has great potential for developing scalable and low-cost AZO thin films for transparent electrodes in a wide range of the spectrum.

Computational Analysis Reveals Monomethylated Triazolopyrimidine as a Novel Inhibitor of SARS-CoV-2 RNA-Dependent RNA Polymerase (RdRp).

The human population is still facing appalling conditions due to several outbreaks of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) virus. The absence of specific drugs, appropriate vaccines for mutants, and knowledge of potential therapeutic agents makes this situation more difficult. Several 1, 2, 4-triazolo [1, 5-a] pyrimidine (TP)-derivative compounds were comprehensively studied for antiviral activities against RNA polymerase of HIV, HCV, and influenza viruses, and showed immense pharmacological interest. Therefore, TP-derivative compounds can be repurposed against the RNA-dependent RNA polymerase (RdRp) protein of SARS-CoV-2. In this study, a meta-analysis was performed to ensure the genomic variability and stability of the SARS-CoV-2 RdRp protein. The molecular docking of natural and synthetic TP compounds to RdRp and molecular dynamic (MD) simulations were performed to analyse the dynamic behaviour of TP compounds at the active site of the RdRp protein. TP compounds were also docked against other non-structural proteins (NSP1, NSP2, NSP3, NSP5, NSP8, NSP13, and NSP15) of SARS-CoV-2. Furthermore, the inhibition potential of TP compounds was compared with Remdesivir and Favipiravir drugs as a positive control. Additionally, TP compounds were analysed for inhibitory activity against SARS-CoV RdRp protein. This study demonstrates that TP analogues (monomethylated triazolopyrimidine and essramycin) represent potential lead molecules for designing an effective inhibitor to control viral replication. Furthermore, in vitro and in vivo studies will strengthen the use of these inhibitors as suitable drug candidates against SARS-CoV-2.

B-cell receptor signaling and genetic lesions in TP53 and CDKN2A/CDKN2B cooperate in Richter transformation.

B-cell receptor (BCR) signals play a critical role in the pathogenesis of chronic lymphocytic leukemia (CLL), but their role in regulating CLL cell proliferation has still not been firmly established. Unlike normal B cells, CLL cells do not proliferate in vitro upon engagement of the BCR, suggesting that CLL cell proliferation is regulated by other signals from the microenvironment, such as those provided by Toll-like receptors or T cells. Here, we report that BCR engagement of human and murine CLL cells induces several positive regulators of the cell cycle, but simultaneously induces the negative regulators CDKN1A, CDKN2A, and CDKN2B, which block cell-cycle progression. We further show that introduction of genetic lesions that downregulate these cell-cycle inhibitors, such as inactivating lesions in CDKN2A, CDKN2B, and the CDKN1A regulator TP53, leads to more aggressive disease in a murine in vivo CLL model and spontaneous proliferation in vitro that is BCR dependent but independent of costimulatory signals. Importantly, inactivating lesions in CDKN2A, CDKN2B, and TP53 frequently co-occur in Richter syndrome (RS), and BCR stimulation of human RS cells with such lesions is sufficient to induce proliferation. We also show that tumor cells with combined TP53 and CDKN2A/2B abnormalities remain sensitive to BCR-inhibitor treatment and are synergistically sensitive to the combination of a BCR and cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor both in vitro and in vivo. These data provide evidence that BCR signals are directly involved in driving CLL cell proliferation and reveal a novel mechanism of Richter transformation.

Bhendi Yellow Vein Mosaic Virus and Bhendi Yellow Vein Mosaic Betasatellite Cause Enation Leaf Curl Disease and Alter Host Phytochemical Contents in Okra.

Whitefly (Bemisia tabaci)-transmitted begomoviruses cause severe diseases in numerous economically important dicotyledonous plants. Okra enation leaf curl disease (OELCuD) has emerged as a serious threat to okra (Abelmoschus esculentus L. Moench) cultivation in the Indian subcontinent. This study reports the association of a monopartite begomovirus (bhendi yellow vein mosaic virus; BYVMV) and betasatellite (bhendi yellow vein mosaic betasatellite; BYVB) with OELCuD in the Mau region of Uttar Pradesh, India. The BYVMV alone inoculated Nicotiana benthamiana and A. esculentus cv. Pusa Sawani plants developed mild symptoms. Co-inoculation of BYVMV and BYVB resulted in a reduced incubation period, an increased symptom severity, and an enhanced BYVMV accumulation by Southern hybridization and quantitative real-time PCR. This is the first study that satisfies Koch's postulates for OELCuD in its natural host. Activities of various antioxidative enzymes were significantly increased in the virus-inoculated okra plants. Differential responses in various biochemical components (such as photosynthetic pigments, phenol, proline, and sugar) in diseased okra plants were observed. This change in phytochemical responses is significant in understanding its impact on virus pathogenesis and disease development.

Two distinct monopartite begomovirus-betasatellite complexes in western India cause tomato leaf curl disease.

In India, begomovirus infection causing tomato leaf curl disease (ToLCD) is a major constraint for tomato productivity. Here, we have identified two distinct monopartite begomovirus and betasatellite complexes causing ToLCD in the western part of India. A new monopartite begomovirus (Tomato leaf curl Mumbai virus, ToLCMumV) and betasatellite (Tomato leaf curl Mumbai betasatellite, ToLCMumB) were isolated from the Mumbai sample. A distinct Tomato leaf curl Gandhinagar virus (ToLCGanV) and Tomato leaf curl Gandhinagar betasatellite (ToLCGanB) were identified from the Gandhinagar sample. Both of the cloned begomoviruses were recombinants. The demonstration of systemic infection caused by begomovirus (ToLCGanV or ToLCMumV) alone in N. benthamiana and tomato (a virus resistant variety) emphasizes that they were monopartite begomoviruses. Co-inoculation of cognate begomovirus and betasatellite reduces the incubation period and increases symptom severity. Thus, Koch's postulates were satisfied for these virus complexes. Further, an enhanced accumulation of ToLCGanV was detected in the presence of cognate ToLCGanB, however ToLCMumB did not influence the level of ToLCMumV in the agro-inoculated tomato plants. Our results indicate that the cloned viruses form potential virus resistance breaking disease complexes in India. This necessitates to investigate the spread of these disease complexes to major tomato growing regions in the country.

MMP-7 derived peptides with MHC class-I binding motifs from canine mammary tumor tissue elicit strong antigen-specific T-cell responses in BALB/c mice.

Matrix Metalloproteinases (MMPs)-induced altered proteolysis of extracellular matrix proteins and basement membrane holds the key for tumor progression and metastasis. Matrix metalloproteinases-7 (Matrilysin), the smallest member of the MMP family also performs quite alike; thus serves as a potential candidate for anti-tumor immunotherapy. Conversely, being an endogenous tumor-associated antigen (TAA), targeting MMP-7 for immunization is challenging. But MMP-7-based xenovaccine can surmount the obstacle of poor immunogenicity and immunological tolerance, often encountered in TAA-based conventional vaccine for anti-tumor immunotherapy. This paves the way for investigating the potential of MMP-7-derived major histocompatibility complex (MHC)-binding peptides to elicit precise epitope-specific T-cell responses towards their possible inclusion in anti-tumor vaccine formulations. Perhaps it also ushers the path of achieving multiple epitope-based broad and universal cellular immunity. In current experiment, an immunoinformatics approach has been employed to identify the putative canine matrix matelloproteinases-7 (cMMP-7)-derived peptides with MHC class-I-binding motifs which can elicit potent antigen-specific immune responses in BALB/c mice. Immunization with the cMMP-7 DNA vaccine induced a strong CD8+ cytotoxic T lymphocytes (CTLs) and Th1- type response, with high level of gamma interferon (IFN-γ) production in BALB/c mice. The two identified putative MHC-I-binding nonameric peptides (Peptide32-40 and Peptide175-183) from cMMP-7 induced significant lymphocyte proliferation along with the production of IFN-γ from CD8+ T-cells in mice immunized with cMMP-7 DNA vaccine. The current observation has depicted the immunogenic potential of the two cMMP-7-derived nonapeptides for their possible exploitation in xenovaccine-mediated anti-tumor immunotherapy in mouse model.

Effects of Injectable Trace Minerals (ITMs) on Th1/Th2 Cytokine Balance of Newborn Calves with Tropical Theileriosis.

Injectable trace minerals (ITMs) could provide a potential alternative way of trace mineral delivery for sick animals. Therefore, evaluation of ameliorative potentials of ITMs (copper, manganese, selenium, and zinc) on the circulating Th1/Th2 cytokine misbalance in Theileria annulata-infected calves was aimed. Forty-three T. annulata-infected newborn calves were randomly allocated into four groups: buparvaquone alone-treated group (BUPA), buparvaquone + oxytetracycline (BUPA + OXY)-treated group, buparvaquone + injectable trace minerals (BUPA + ITMs)-treated group, and BUPA + OXY + ITM-treated group. Blood samples were collected from each of the calves before the start of therapy (day 0) and on day 14 post-therapy. Serum contents of pro- and anti-inflammatory cytokines were estimated by bovine specific ELISA kits. On day 14 post-therapy, significant amelioration in the circulating levels of the studied cytokines was not observed in the calves treated with BUPA, while the calves treated with BUPA + OXY revealed significant (P ≤ 0.04) amelioration in the circulating tumour necrosis factor-α (TNF-α) level. The calves treated with BUPA + ITMs revealed significant (P ≤ 0.041) elevation in the circulating interferon-γ (IFN-γ) and significant (P ≤ 0.011) reduction in the interleukin-10 (IL-10) levels. Moreover, the calves treated with BUPA + OXY + ITMs revealed significant reduction in TNF-α (P ≤ 0.0001) and IL-10 (P ≤ 0.012) contents, and significant elevation in IFN-γ (P ≤ 0.0002) content on day 14 post-therapy. None of the treated calve group revealed significant alteration in the circulating level of transforming growth factor-ß (TGF-ß) on day 14 post-therapy. In conclusion, administration of ITMs to the therapeutic regimen of newborn calves with tropical theileriosis could be a promising therapeutic strategy. ITMs can be recommended for the amelioration of immunological misbalance due to tropical theileriosis in newborn calves.

Hidden Granzyme B-Mediated Injury in Chronic Active Antibody-Mediated Rejection.

OBJECTIVES: Antibody-mediated injury in chronic active antibody-mediated rejection, possibly with other effector T cells, may play a role in graft injury. The role of inflammatory cells in the inflammation and fibrosis and tubular atrophy region has been recently advocated in the progression of injury. Cytotoxic T cells play a prominent role in T-cell-mediated rejection; however, the possible role of cytotoxic T cells in circulation and the intragraft compartment in chronic active antibody-mediated rejection, a common immunological cause of long-term graft failure, has not been well-studied. MATERIALS AND METHODS: We measured the frequency of circulating cytotoxic T cells with flow cytometry, serum granzyme B level by enzyme-linked immunosorbent assay and intragraft granzyme B+ cell, and mRNA by immunohistochemistry and real-time polymerase chain reaction in biopsy tissue from living donor renal allograft recipients with stable graft function and chronic active antibody-mediated rejection. RESULTS: The frequency of CD3+ and CD3+CD8+ T cells was similar in both stable graft function patients and chronic active antibody-mediated rejection patients. The frequency of CD3+CD8+granzyme B+ cytotoxic T cells was significantly lower in peripheral blood. Serum granzyme B level and intragraft number of granzyme B+ cells (counts/mm²) were also significantly higher in the chronic active antibody-mediated rejection group compared with that of patients with stable graft function. The intragraft granzyme B+ T cell count was positively correlated with serum creatinine and 24-hour urine proteinuria but negatively correlated with estimated glomerular filtration rate. CONCLUSIONS: Granzyme B mediates covert graft injury in patients with chronic active antibody-mediated rejection in addition to antibody-mediated injury.

Freezing-thawing induces deprotamination, cryocapacitation-associated changes; DNA fragmentation; and reduced progesterone sensitivity in buck spermatozoa.

In the present study, there was evaluation of cryocapacitation-associated changes, apoptotic-like changes, deprotamination, total antioxidant capacity (TAC), and in vitro sperm functional attributes in Barbari bucks after freezing-thawing. The correlation between deprotamination and sperm functional characteristics was established. Using immunoblotting procedures, there was detection of the presence of a single 28-kDa protein band corresponding to protamine-1. The localization in the head region of the spermatozoa was further validated by an immunofluorescence test. Capacitated (B-) and acrosome-reacted (AR-) pattern spermatozoa, spermatozoa with the externalization of phosphatidylserine and a relatively lesser mitochondrial transmembrane potential, and deprotamination and DNA fragmentation was greater (P < 0.05) after freezing-thawing and indicated there were cryocapacitation- and apoptotic-like changes, respectively. Furthermore, the detection of phosphorylation of tyrosine-containing proteins with use of immunoblotting and immunofluorescence procedures confirmed there were cryocapacitation-like changes in the buck spermatozoa after freezing-thawing. Total antioxidant capacity (TAC), in vitro thermal resistance response, Vanguard distance, progesterone sensitivity, and in vitro capacitation response were less (P < 0.05) in the spermatozoa after freezing-thawing compared with spermatozoa after initial dilution and equilibration. Deprotamination (chromomycin A3-positive cells, CMA3+) and DNA fragmentation (TUNEL+ve) were positively correlated with B- and AR-pattern spermatozoa, while other values for other variables were negatively correlated. In conclusion, the results of this study indicated there was protamine-1 in buck spermatozoa and after freezing-thawing there was a loss of protamine-1 combined with cryocapacitation-associated changes and apoptotic-like changes in buck spermatozoa. Spermatozoa deprotamination might be attributed to increased DNA fragmentation, resulting in compromised fertilizing capacity of buck spermatozoa.