Background: ANCA-associated vasculitis (AAV) with proteinase 3 (PR3) ANCA is genetically associated with HLA-DP [1], is often relapsing in nature, and has a predisposition for kidneys, lungs and ear-nose-throat involvement [2]. Despite the presence of PR3+ANCA, indicating CD4+T-cell help in the disease, the knowledge about autoreactive CD4+T cells is scarce. Activated T cells have been shown at site of inflammation [3] and involvement of proinflammatory cytokines in circulation is also reported [4, 5]. Objectives: Identification of autoreactive T cells may help to identify the drivers of the immune responses and chronicity. We therefore aimed to investigate PR3-specific CD4+T-cell responses in peripheral blood of AAV patients with a focus on both phenotype and T-cell receptor (TCR) repertoires. Methods: The study included sixty-six patients: 26 with active PR3 autoantibody+ AAV, 21 with inactive but PR3+ AAV and 19 with inactive PR3- AAV. In-vitro cultures with PR3 protein were established to assess antigen-specific cytokine responses in a 3-color fluorospot assay. Deep immunophenotyping was performed by flow cytometry. Antigen-responsive CD4+ T cells were isolated and single cell TCRαβ sequences were generated and analyzed from PR3+ AAV patients (n=5) using a previously published protocol [6]. Results: PBMCs from AAV patients demonstrated an HLA-DP associated cytokine responses to PR3 stimulation including IFN-γ and IL-10, but not IL-17A. This T-cell autoreactivity was found to be confined to a highly differentiated CD4+ T cell population characterized by perforin and GPR56 expression, implicating a cytotoxic feature of the response. Active disease involved a reduction in expression of several markers associated with cytotoxicity amongst the CD4+GPR56+ T cells. Their frequency was also negatively associated with the doses of prednisolone. A similar phenotype was shared with T cells activated by human cytomegalovirus (HCMV) peptides in the same patient cohort. Single cell sequencing of paired alpha beta T-cell receptors (TCRs) revealed different patterns of gene usage between PR3 and HCMV reactive T cells. Moreover, we could identify shared (public) PR3-reactive T-cell clones between different HLA-DPB1*04:01+ patients. Conclusion: PR3 is an autoantigen which provokes ANCA responses in AAV patients. Our study identified PR3-reactive CD4+ T cells at the level of their phenotype and TCR repertoire. The autoreactive CD4+ T cells, present in both active and inactive disease, implicate chronic antigen exposure and the persistence of long-lived T-cell clones. The presence of public autoreactive clones between HLA-DPB1*04:01+ patients suggests an active role for these cells in pathogenesis of AAV and validates the link with predisposed genotype. References: [1]Lyons PA, Rayner TF, Trivedi S, Holle JU, Watts RA, Jayne DR, et al. Genetically distinct subsets within ANCA-associated vasculitis. New England Journal of Medicine. 2012; 367(3):214-223. [2]Kumar Sharma R, Lövström B, Gunnarsson I, Malmström V. Proteinase 3 autoreactivity in Anti-Neutrophil Cytoplasmic Antibody-associated vasculitis–immunological versus clinical features. Scandinavian Journal of Immunology. 2020:e12958. [3]Wilde B, Thewissen M, Damoiseaux J, van Paassen P, Witzke O, Tervaert JWCJAr, et al. T cells in ANCA-associated vasculitis: what can we learn from lesional versus circulating T cells? 2010; 12(1):204. [4]Hoffmann JC, Patschan D, Dihazi H, Müller C, Schwarze K, Henze E, et al. Cytokine profiling in anti neutrophil cytoplasmic antibody-associated vasculitis: a cross-sectional cohort study. Rheumatology international. 2019; 39(11):1907-1917. [5]Berti A, Warner R, Johnson K, Cornec D, Schroeder D, Kabat B, et al. Circulating Cytokine Profiles and ANCA Specificity in Patients with ANCA-Associated Vasculitis. Arthritis & rheumatology (Hoboken, NJ). 2018; 70(7):1114. [6]Han A, Glanville J, Hansmann L, Davis MM. Linking T-cell receptor sequence to functional phenotype at the single-cell level. Nature biotechnology. 2014; 32(7):684-692. Disclosure of Interests: None declared

A field experiment was conducted during rainy season of 2015 and 2016 at Chandra Shekhar Azad University of Agriculture and Technology, Kanpur with the objective to evaluate the effects of different tillage and integrated nutrient management (INM) practices on performance of greengram [Vigna radiata (L.) Wilczek] under greengram + sesame intercropping system. The experiment was laid out in a split-plot design with four tillage practices, viz. conventional tillage (CT), deep tillage (DT), CT + vegetative mulch and DT + vegetative mulch as main plots, and six INM practices, viz. 20-60-40 N-P2O5-K2O kg/ha, 30-60-40 N-P2O5-K2O kg/ha, 20-60-40 N-P2O5-K2O kg/ha + Rhizobium (soil inoculation), 20-60-40 N-P2O5-K2O kg/ha + Rhizobium + phosphorus solubilizing bacteria (PSB), 30-60-40 N-P2O5-K2O kg/ha + Rhizobium and 30-60-40 N-P2O5-K2O kg/ha + Rhizobium + PSB as sub plots with three replications. The DT + vegetative mulch resulted in significantly higher leaf area index (3.10), crop dry matter (10.10 g/plant), seed yield (9.02 q/ha) and net returns (37314.5 INR/ha) with 4.03%, 3.17%, 24.24% and 43% increment over CT, respectively. Among the INM treatments, the 30-60-40 N-P2O5-K2O kg/ha and Rhizobium + PSB resulted in highest leaf area index (3.09), crop dry matter (10.09 g/plant), seed yield (9.14 q/ha), net returns (33346.5 INR/ha), and nitrogen (17.67 kg/ha) and phosphorus (9.31 kg/ha) uptake by seed with mean 3.18%, 2.91%, 21.32%, 43.70%, 27.68% and 34.65% increase over sole application of chemical fertilizers.

Background: Autoimmunity to citrullinated autoantigens forms a critical component of disease pathogenesis in rheumatoid arthritis (RA). Presence of anti-citrullinated protein antibodies (ACPAs) in patients has high diagnostic value. Recently, several citrullinated antigen specific CD4+T cells have been described. However, detailed studies of their T-cell receptor usage and in-vivo profile suffer from the disadvantage that these cells are present at very low frequencies. In this context, we here present a pipeline for TCR repertoire analysis of antigen-specific CD4+T cells from RA patients, including both citrulline and influenza (control) specificities using in-vitro peptide challenge induced-cell expansion. Objectives: To enable studies of the T cell repertoire of citrullinated antigen-specific CD4+T cells in rheumatoid arthritis Methods: Peripheral blood mononuclear cells (PBMCs) (n=7) and synovial fluid mononuclear cells (SFMCs) (n=5) from HLA-DR*0401-postive RA patients were cultured in the presence of citrullinated Tenascin C peptide cocktails or influenza peptides (positive control). Citrulline reactive cells were further supplemented with recombinant human IL-15 and IL-7 on day 2. All cultures were replenished with fresh medium on day 6 and rIL-2 was added every 2 days from then. Assessment of proportion of peptide-HLA-tetramer positive cells was performed using flow cytometry whereby individual antigen-specific CD4+T cells were sorted into 96-well plates containing cell lysis buffer, followed by PCR-based alpha/beta TCR sequencing. TCR sequencing data was demultiplexed and aligned for TCR gene usage using MiXCR. Some tetramer positive cells were sorted into complete medium containing human IL-2 and PHA for expansion of antigen-specific cells. Cells were supplemented with irradiated allogenic PBMCs (30 times number of antigen specific cells). Clones of antigen specific CD4+T cells were further subjected to tetramer staining to confirm expansion of cells. Results: As evidenced by increase in frequency of tetramer positive CD4+T cells, in vitro peptide stimulation resulted in expansion of both influenza specific (Fig. 1a) and citrullinated antigen specific (Fig. 1b) CD4+T cells. Polyclonal in-vitro expansion of tenascin C tetramer positive sorted cells followed by tetramer staining further confirmed antigen specificity and enrichment for antigen specific CD4+T cells after polyclonal stimulation (Fig.1c). TCR repertoire analysis in PB and SF dataset from the first patient showed clonal expansion of influenza specific cells in both sites. Synovial fluid had more diversity of expanding clones as compared to paired PB, with few expanded clones being shared among SF and PB. We observed a more diverse TCR repertoire in citrulline specific CD4+T cells. We also observed sharing of TCR alpha chains among different citrulline specific CD4+T cell clones. Fig. 1 In-vitro expansion of antigen specific CD4+T cells: Conclusion: This method provides a highly suitable approach for investigating TCR specificities of antigen specific CD4+T cells under conditions of low cell yields. Building on this dataset will allow us to assess specific features of TCR usage of autoreactive T cells in RA. PBMCs were cultured in presence of (a) influenza (HA, MP54) and (b) citrullinated tenascin peptides. The proportion of antigen specific CD4+T cells was assessed using HLA-class II tetramer staining. We observed an increase in frequency of (a) Infleunza specific cells (red dots in upper left and lower right quadrants) and (b) citrullinated tenascin C specific cells (red dots in lower right quadrant), at day 13 post culture as compared to day 3. (c) Sorting of citrullinated tenascin specific CD4+T cells, followed by PHA expansion resulted in visible increase in proportion of citrullinated tenascin specific CD4+T cells. Disclosure of Interests: Ravi kumar: None declared, Niyaz Yoosuf: None declared, Christina Gerstner: None declared, Sara Turcinov: None declared, Karine Chemin: None declared, Vivianne Malmström Grant/research support from: VM has had research grants from Janssen Pharmaceutica

Topological insulator thin films of Bi2Se3 have been deposited on SrTiO3 (111) substrate by dc magnetron sputtering and annealed in Se environment. X-ray Diffraction (XRD) measurement shows (00l) (l = 3n, where n is an integer) type of reflections over 2 theta range of 10-80 degrees without any other peaks indicating that the films are of single crystalline quality along the [001] growth direction confirming that highly c-axis-preferred orientated Bi2Se3 thin films can be obtained by relatively simpler and easily scalable dc magnetron sputtering technique. The results are also confirmed by Field Emission Scanning Electron Microscopy (FESEM), Energy Dispersive X-ray Spectroscopy (EDXS), Photo-electron Spectroscopy (PES) and X-ray absorption spectroscopy (XAS) measurements.

ABSTRACT Congenital heart defects affect ∼2% of live births and often involve malformations of the semilunar (aortic and pulmonic) valves. We previously reported a highly penetrant GATA4 p.Gly296Ser mutation in familial, congenital atrial septal defects and pulmonic valve stenosis and showed that mice harboring the orthologous G295S disease-causing mutation display not only atrial septal defects, but also semilunar valve stenosis. Here, we aimed to characterize the role of Gata4 in semilunar valve development and stenosis using the Gata4G295Ski/wt mouse model. GATA4 is highly expressed in developing valve endothelial and interstitial cells. Echocardiographic examination of Gata4G295Ski/wt mice at 2 months and 1 year of age identified functional semilunar valve stenosis predominantly affecting the aortic valve with distal leaflet thickening and severe extracellular matrix (ECM) disorganization. Examination of the aortic valve at earlier postnatal timepoints demonstrated similar ECM abnormalities consistent with congenital disease. Analysis at embryonic timepoints showed a reduction in aortic valve cushion volume at embryonic day (E)13.5, predominantly affecting the non-coronary cusp (NCC). Although total cusp volume recovered by E15.5, the NCC cusp remained statistically smaller. As endothelial to mesenchymal transition (EMT)-derived cells contribute significantly to the NCC, we performed proximal outflow tract cushion explant assays and found EMT deficits in Gata4G295Ski/wt embryos along with deficits in cell proliferation. RNA-seq analysis of E15.5 outflow tracts of mutant embryos suggested a disease state and identified changes in genes involved in ECM and cell migration as well as dysregulation of Wnt signaling. By utilizing a mouse model harboring a human disease-causing mutation, we demonstrate a novel role for GATA4 in congenital semilunar valve stenosis. This article has an associated First Person interview with the joint first authors of the paper.

The present study was conducted to evaluate the toxic effects of cadmium and chlorpyrifos and their interaction on liver and kidney of broiler chicken. Twenty four birds were divided into four groups. Group-I was kept as control. Group-II, III and IV birds were administered with cadmium (50 mg/L in drinking water), chlorpyrifos (4 mg/kg body weight orally) and cadmium (50 mg/L in drinking water) + chlorpyrifos (4 mg/kg body weight orally), respectively and observed for 45 days. Blood samples were collected on day 15, 30 and 45 for biochemical parameters, which showed significant (P<0.01) increase in ALT, AST, ALP and creatinine. Significant (P<0.05) increase in the glucose, urea, uric acid and significant (P<0.05) decrease in total protein, albuminand globulin were observed. Histopathologically, liver revealed necrosis of hepatocytes, haemorrhages, infiltration of heterophils and mononuclear cells. Kidneys showed cortical tubular degeneration and desquamation, glomerular atrophy and hyalinization. Histopathological andbiochemical changes were more marked in group IV birds, which indicated the synergistic effects of these environmental toxicantsin producing pronounced hepatotoxic and nephrotoxic alterations.

In this paper, PANI–WO3 composite was synthesized via chemical polymerization method. The results were well supported by scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR), UV–visible spectrophotometer and sensing measurements. The formation of PANI–WO3 was confirmed by XRD analysis. The average crystallite size was evaluated using Debye-Scherer’s formula. The functional group present in the PANI–WO3 was confirmed using FTIR spectrometer. UV–visible spectra showed the presence of different rings and energy band gaps of various samples were calculated. Further, the thin films of samples have been fabricated using spin coater. The sensing studies at 10, 20, 30 and 40 % of WO3 in PANI were carried out at room temperature. Sensing results demonstrate the fabricated porous PANI–WO3 composite thin films which is a challenging material for the development of the humidity sensors.

Environmental toxicity due to pesticides, metals and non metals is producing havoc to public health. Toxic effects of sub chronic exposure of cypermethrin, a synthetic pyrethroidandsodium fluoride, the most electronegative non-metalwas studied in Wistar rats, with respect to oxidative damage, biochemical and histopathological alterations in liver. Rats were divided into four groups of six each: group I was used as control. Group II, III and IV were orally treated with cypermethrin (25 mg/kg body weight), Sodium fluoride (8mg/kgbody weight) and cypermethrin plus sodium fluoride, respectively for 45 days. Cypermethrin and sodium fluoride in combination (group IV) produced a marked decrease in haematological parameters including haemoglobin, PCV, TEC, MCV, MCH, TLC and lymphocytes as well as significant increase in neutrophils compared to other grioups. Biochemical estimation showed significant increase in enzymes (ALT, AST and ALP), glucose and urea, and significant decrease in total protein and albumin in rats with combined toxicity. The mean values of SOD, GSH and catalase revealed significant decrease while there was significant increase in LPO in liver homogenate in toxicity groups. Grossly, liver was pale with occasional presence of pinpoint haemorrhages and mottling on the dorsal surface. Microscopically, liver revealed congested and distended sinusoids with hyperplasia of Kupffer cells, sub-acute hepatitis characterized by area of necrosis of hepatocytes along with infiltration of mononuclear cells and degenerative changes. These changes were more marked in combined toxicity group.

In the present work nanostructured ferric oxide has been synthesized using sol–gel method. Thin films of ferric oxide were fabricated via spin coating process. The surface of the thin film was scanned by scanning electron microscope that exhibited the surface morphology of ferric oxide nanostructures. The material was also characterized by XRD, Acoustic particle sizer and FTIR. All the particles distributed on the surface have some spaces among them known as pores. These pores serve as adsorption sites for moisture and other gases. EDX confirmed the elements forming the ferric oxide in pure form. The particle size of the ferric oxide was estimated as ~12.2 nm. The pore size of the film was ~50 nm i.e., nature is mesoporous. Annealing effect on the surface morphology was also observed. Humidity sensing, electrical as well as optical of the prepared film was carried out. The results showed the suitability of material for the development of humidity sensors. Variations in resistance with the exposure of LPG were recorded and found that resistance of film increases with the increasing exposure time and concentration of gas. The maximum sensing response of the sensor was recorded as 3.26 for 1000 ppm at room temperature. The response and recovery times of the sensor were found to be ~12 and 9 min, respectively.

Primary amoebic meningoencephalitis (PAM) due to Naegleria fowleri was detected in a 36-year-old, Indian countryman who had a history of taking bath in the village pond. He was admitted in a semi comatosed condition with severe frontal headache, neck stiffness, intermittent fever, nausea, vomiting, left hemiparesis and seizures. Computerized tomography (CT) scan of brain showed a soft tissue non-enhancing mass with erosion of sphenoid sinus. However CSF findings showed no fungal or bacterial pathogen. Trophozoites of Naegleria fowleri were detected in the direct microscopic examination of CSF and these were grown in culture on non-nutrient agar. The patient was put on amphotericin-B, rifampicin and ceftazidime but his condition deteriorated and was taken home by his relatives in a moribund condition against medical advice and subsequently died. A literature review of 7 previous reports of PAM in India is also presented. Four of theses eight cases were non lethal. The mean age was 13.06 years with male: female ratio of 7:1. History of contact with water was present in four cases. Trophozoites could be identified in all 8 cases in this series.