Decoding the role of aldosterone in glycation-induced diabetic complications.

Diabetes-mediated development of micro and macro-vascular complications is a global concern. One of the factors is hyperglycemia induced the non-enzymatic formation of advanced glycation end products (AGEs). Accumulated AGEs bind with receptor of AGEs (RAGE) causing inflammation, oxidative stress and extracellular matrix proteins (ECM) modifications responsible for fibrosis, cell damage and tissue remodeling. Moreover, during hyperglycemia, aldosterone (Aldo) secretion increases, and its interaction with mineralocorticoid receptor (MR) through genomic and non-genomic pathways leads to inflammation and fibrosis. Extensive research on individual involvement of AGEs-RAGE and Aldo-MR pathways in the development of diabetic nephropathy (DN), cardiovascular diseases (CVDs), and impaired immune system has led to the discovery of therapeutic drugs. Despite mutual repercussions, the cross-talk between AGEs-RAGE and Aldo-MR pathways remains unresolved. Hence, this review focuses on the possible interaction of Aldo and glycation in DN and CVDs, considering the clinical significance of mutual molecular targets.

Immunogenicity of SARS-CoV-2 vaccines BBV152 (COVAXIN®) and ChAdOx1 nCoV-19 (COVISHIELD™) in seronegative and seropositive individuals in India: a multicentre, nonrandomised observational study.

Background: There are limited global data on head-to-head comparisons of vaccine platforms assessing both humoral and cellular immune responses, stratified by pre-vaccination serostatus. The COVID-19 vaccination drive for the Indian population in the age group 18-45 years began in April 2021 when seropositivity rates in the general population were rising due to the delta wave of COVID-19 pandemic during April-May 2021. Methods: Between June 30, 2021, and Jan 28, 2022, we enrolled 691 participants in the age group 18-45 years across four clinical sites in India. In this non-randomised and laboratory blinded study, participants received either two doses of Covaxin® (4 weeks apart) or two doses of Covishield™ (12 weeks apart) as per the national vaccination policy. The primary outcome was the seroconversion rate and the geometric mean titre (GMT) of antibodies against the SARS-CoV-2 spike and nucleocapsid proteins post two doses. The secondary outcome was the frequency of cellular immune responses pre- and post-vaccination. Findings: When compared to pre-vaccination baseline, both vaccines elicited statistically significant seroconversion and binding antibody levels in both seronegative and seropositive individuals. In the per-protocol cohort, Covishield™ elicited higher antibody responses than Covaxin® as measured by seroconversion rate (98.3% vs 74.4%, p < 0.0001 in seronegative individuals; 91.7% vs 66.9%, p < 0.0001 in seropositive individuals) as well as by anti-spike antibody levels against the ancestral strain (GMT 1272.1 vs 75.4 binding antibody units/ml [BAU/ml], p < 0.0001 in seronegative individuals; 2089.07 vs 585.7 BAU/ml, p < 0.0001 in seropositive individuals). As participants at all clinical sites were not recruited at the same time, site-specific immunogenicity was impacted by the timing of vaccination relative to the delta and omicron waves. Surrogate neutralising antibody responses against variants-of-concern including delta and omicron was higher in Covishield™ recipients than in Covaxin® recipients; and in seropositive than in seronegative individuals after both vaccination and asymptomatic infection (omicron variant). T cell responses are reported from only one of the four site cohorts where the vaccination schedule preceded the omicron wave. In seronegative individuals, Covishield™ elicited both CD4+ and CD8+ spike-specific cytokine-producing T cells whereas Covaxin® elicited mainly CD4+ spike-specific T cells. Neither vaccine showed significant post-vaccination expansion of spike-specific T cells in seropositive individuals. Interpretation: Covishield™ elicited immune responses of higher magnitude and breadth than Covaxin® in both seronegative individuals and seropositive individuals, across cohorts representing the pre-vaccination immune history of most of the vaccinated Indian population. Funding: Corporate social responsibility (CSR) funding from Hindustan Unilever Limited (HUL) and Unilever India Pvt. Ltd. (UIPL).

Aldosterone, Methylglyoxal, and Glycated Albumin Interaction with Macrophage Cells Affects Their Viability, Activation, and Differentiation.

BACKGROUND: The inflammatory response in diabetes is strongly correlated with increasing amounts of advanced glycation end products (AGEs), methylglyoxal (MGO), aldosterone (Aldo), and activation of macrophages. Aldo is known to be associated with increased pro-inflammatory responses in general, but its significance in inflammatory responses under glycated circumstances has yet to be understood. In the current work, the aim of our study was to study the macrophage immune response in the presence of AGEs, MGO, and Aldo to comprehend their combined impact on diabetes-associated complications. METHODS AND RESULTS: The viability of macrophages upon treatment with glycated HSA (Gly-HSA) promoted cell growth as the concentration increased from 100 to 500 µg/mL, whereas MGO at a high concentration (≥300 µM) significantly hampered cell growth. At lower concentrations (0.5-5 nM), Aldo strongly promoted cell growth, whereas at higher concentrations (50 nM), it was seen to inhibit growth when used for cell treatment for 24 h. Aldo had no effect on MGO-induced cell growth inhibition after 24 h of treatment. However, compared to MGO or Aldo treatment alone, an additional decrease in viability could be seen after 48 h of treatment with a combination of MGO and Aldo. Treatment with Aldo and MGO induced expression of TNF-α independently and when combined. However, when combined, Aldo and MGO significantly suppressed the expression of TGF-ß. Aldo, Gly-HSA, and MGO strongly induced the transcription of NF-κB and RAGE mRNA and, as expected, also promoted the formation of reactive oxygen species. Also, by inducing iNOS and MHC-II and suppressing CD206 transcript expression, Gly-HSA strongly favored the differentiation of macrophages into M1 type (pro-inflammatory). On the other hand, the combination of Aldo and MGO strongly induced the expression of MHC-II, CD206, and ARG1 (M2 macrophage marker). These findings suggest that Gly-HSA, MGO, and Aldo differently influence macrophage survival, activation, and differentiation. CONCLUSIONS: Overall, this study gives an insight into the effects of glycated protein and MGO in the presence of Aldo on macrophage survival, activation, differentiation, and inflammatory response.

Liquid Biopsies: Emerging role and clinical applications in solid tumours.

Late detection and lack of precision diagnostics are the major challenges in cancer prevention and management. Biomarker discovery in specific cancers, especially at the pre-invasive stage, is vital for early diagnosis, positive treatment response, and good disease prognosis. Traditional diagnostic measures require invasive procedures such as tissue excision using a needle, an endoscope, and/or surgical resection which can be unsafe, expensive, and painful. Additionally, the presence of comorbid conditions in individuals might render them ineligible for undertaking a tissue biopsy, and in some cases, it is difficult to access tumours depending on the site of occurrence. In this context, liquid biopsies are being explored for their clinical significance in solid malignancies management. These non-invasive or minimally invasive methods are being developed primarily for identification of biomarkers for early diagnosis and targeted therapeutics. In this review, we have summarised the use and importance of liquid biopsy as significant tool in diagnosis, prognosis prediction, and therapeutic development. We have also discussed the challenges that are encountered and future perspective.

Efficient T cell-B cell collaboration guides autoantibody epitope bias and onset of celiac disease.

B cells play important roles in autoimmune diseases through autoantibody production, cytokine secretion, or antigen presentation to T cells. In most cases, the contribution of B cells as antigen-presenting cells is not well understood. We have studied the autoantibody response against the enzyme transglutaminase 2 (TG2) in celiac disease patients by generating recombinant antibodies from single gut plasma cells reactive with discrete antigen domains and by undertaking proteomic analysis of anti-TG2 serum antibodies. The majority of the cells recognized epitopes in the N-terminal domain of TG2. Antibodies recognizing C-terminal epitopes interfered with TG2 cross-linking activity, and B cells specific for C-terminal epitopes were inefficient at taking up TG2-gluten complexes for presentation to gluten-specific T cells. The bias toward N-terminal epitopes hence reflects efficient T-B collaboration. Production of antibodies against N-terminal epitopes coincided with clinical onset of disease, suggesting that TG2-reactive B cells with certain epitope specificities could be the main antigen-presenting cells for pathogenic, gluten-specific T cells. The link between B cell epitopes, antigen presentation, and disease onset provides insight into the pathogenic mechanisms of a T cell-mediated autoimmune condition.

miR-191 modulates B-cell development and targets transcription factors E2A, Foxp1, and Egr1.

The interdependence of posttranscriptional gene regulation via miRNA and transcriptional regulatory networks in lymphocyte development is poorly understood. Here, we identified miR-191 as direct upstream modulator of a transcriptional module comprising the transcription factors Foxp1, E2A, and Egr1. Deletion as well as ectopic expression of miR-191 resulted in developmental arrest in B lineage cells, indicating that fine tuning of the combined expression levels of Foxp1, E2A, and Egr1, which in turn control somatic recombination and cytokine-driven expansion, constitutes a prerequisite for efficient B-cell development. In conclusion, we propose that miR-191 acts as a rheostat in B-cell development by fine tuning a key transcriptional program.

Induced B Cell Development in Adult Mice.

We employed the B-Indu-Rag1 model in which the coding exon of recombination-activating gene 1 (Rag1) is inactivated by inversion. It is flanked by inverted loxP sites. Accordingly, B cell development is stopped at the pro/pre B-I cell precursor stage. A B cell-specific Cre recombinase fused to a mutated estrogen receptor allows the induction of RAG1 function and B cell development by application of Tamoxifen. Since Rag1 function is recovered in a non-self-renewing precursor cell, only single waves of development can be induced. Using this system, we could determine that B cells minimally require 5 days to undergo development from pro/preB-I cells to the large and 6 days to the small preB-II cell stage. First immature transitional (T) 1 and T2 B cells could be detected in the bone marrow at day 6 and day 7, respectively, while their appearance in the spleen took one additional day. We also tested a contribution of adult bone marrow to the pool of B-1 cells. Sublethally irradiated syngeneic WT mice were adoptively transferred with bone marrow of B-Indu-Rag1 mice and B cell development was induced after 6 weeks. A significant portion of donor derived B-1 cells could be detected in such adult mice. Finally, early VH gene usage was tested after induction of B cell development. During the earliest time points the VH genes proximal to D/J were found to be predominantly rearranged. At later time points, the large family of the most distal VH prevailed.

High-Throughput Single-Cell Analysis of B Cell Receptor Usage among Autoantigen-Specific Plasma Cells in Celiac Disease.

Characterization of Ag-specific BCR repertoires is essential for understanding disease mechanisms involving humoral immunity. This is optimally done by interrogation of paired H chain V region (VH) and L chain V region (VL) sequences of individual and Ag-specific B cells. By applying single-cell high-throughput sequencing on gut lesion plasma cells (PCs), we have analyzed the transglutaminase 2 (TG2)-specific VH:VL autoantibody repertoire of celiac disease (CD) patients. Autoantibodies against TG2 are a hallmark of CD, and anti-TG2 IgA-producing gut PCs accumulate in patients upon gluten ingestion. Altogether, we analyzed paired VH and VL sequences of 1482 TG2-specific and 1421 non-TG2-specific gut PCs from 10 CD patients. Among TG2-specific PCs, we observed a striking bias in IGHV and IGKV/IGLV gene usage, as well as pairing preferences with a particular presence of the IGHV5-51:IGKV1-5 pair. Selective and biased VH:VL pairing was particularly evident among expanded clones. In general, TG2-specific PCs had lower numbers of mutations both in VH and VL genes than in non-TG2-specific PCs. TG2-specific PCs using IGHV5-51 had particularly few mutations. Importantly, VL segments paired with IGHV5-51 displayed proportionally low mutation numbers, suggesting that the low mutation rate among IGHV5-51 PCs is dictated by the BCR specificity. Finally, we observed selective amino acid changes in VH and VL and striking CDR3 length and J segment selection among TG2-specific IGHV5-51:IGKV1-5 pairs. Hence this study reveals features of a disease- and Ag-specific autoantibody repertoire with preferred VH:VL usage and pairings, limited mutations, clonal dominance, and selection of particular CDR3 sequences.

Experimental colitis is exacerbated by concomitant infection with Mycobacterium avium ssp. paratuberculosis.

BACKGROUND: Crohn's disease (CD) is a chronic inflammatory disorder of the human gastrointestinal tract. Although genetic, immunological, environmental, and bacterial factors have been implicated, the pathogenesis is incompletely understood. The histopathological appearance of CD strikingly resembles Johne's disease, a ruminant inflammatory bowel disease, caused by Mycobacterium avium ssp. paratuberculosis (MAP), but a causative role of MAP in CD has not been established. In this work, we hypothesized that MAP might exacerbate an already existing intestinal disease. METHODS: We combined dextran sulfate sodium (DSS)-induced colitis with MAP infection in mice and monitored the immune response and bacterial count in different organs. RESULTS: An increased size of liver and spleen was observed in DSS-treated and MAP-infected animals (DSS + MAP) as compared with DSS-treated uninfected (DSS + PBS) mice. Similarly, DSS treatment increased the number and size of MAP-induced liver granulomas and enhanced the MAP counts in enteric tissue. MAP infection in turn delayed the mucosal healing of DSS-induced tissue damage. Finally, high numbers of MAP were found in mesenteric fat tissue causing large granuloma and necrotic regions. CONCLUSIONS: Taken together, we present an in vivo model to study the role of MAP infection in CD. Our results confirm the hypothesis that MAP is able to exacerbate existing intestinal inflammation.

An intrinsic propensity of murine peritoneal B1b cells to switch to IgA in presence of TGF-ß and retinoic acid.

AIMS: In the present study we have investigated the comparative switching propensity of murine peritoneal and splenic B cell subpopulations to IgA in presence of retinoic acid (RA) and TGF-ß. METHODS AND RESULTS: To study the influence of RA and TGF-ß on switching of B cell subpopulations to IgA, peritoneal (B1a, B1b and B2 cells) and splenic (B1a, marginal zone, and B2) B cells from normal BALB/c mice were FACS purified, cultured for 4 days in presence of RA and TGF-ß and the number of IgA producing cells was determined by ELISPOT assay or FACS analysis. In presence of TGF-ß, peritoneal B1b cells switched to IgA more potently than other peritoneal B cell subpopulations. When TGF-ß was combined with retinoic acid (RA), switching to IgA was even more pronounced. Under these conditions, "innate" B cells like peritoneal and splenic B1 cells and MZ B cells produced IgA more readily than B2 cells. Additionally, high frequency of nucleotide exchanges indicating somatic hypermutation in VH regions was observed. Besides IgA induction, RA treatment of sorted PEC and splenic B cells led to expression of gut homing molecules - α4ß7 and CCR9. Intraperitoneal transfer of RA-treated B1 cells into Rag1(-/-) recipients resulted in IgA in serum and gut lavage, most efficiently amongst B1b cell recipients. CONCLUSION: Present study demonstrates the differential and synergistic effect of RA and TGF-ß on switching of different B cell subpopulations to IgA and establishes the prominence of peritoneal B1b cells in switching to IgA under the influence of these two factors. Our study extends our knowledge about the existing differences among B cell subpopulations with regards to IgA production and indicates towards their differential contribution to gut associated humoral immunity.

B-1-cell subpopulations contribute differently to gut immunity.

In mice, B-1 (B1a/B1b) cells are mainly located in the peritoneal cavity. B-1 cells are well known for their role in the early stages of Ab-mediated immune responses against pathogenic invasion as well as for the production of natural IgM antibodies. Although such B cells have been claimed to give rise to intestinal plasma cells producing IgA, a clear role of B-1 cells in IgA production in the gut-associated tissues is still not defined. Here, we employed the transgenic L2 mouse model characterized by the lack of B-2 cells and presence of B-1 cells as major B-cell subpopulation. The oligoclonality of the Ab repertoire in this mouse allowed us to take typical B1a cell VH sequences as indicators of the presence of IgM-producing B-1a cells in Peyer's patches as well as in lamina propria. However, amongst the IgAVH sequences recovered from the same tissues, none of the sequences showed B1a-cell specificity. Interestingly, all IgAVH sequences derived from the lamina propria of L2 mice displayed extensive numbers of nucleotide exchanges, indicating somatic hypermutation, and affinity maturation. This suggests that the contribution of natural unmutated IgA by B-1a cells to intestinal immunity is negligible.

Induction of B-cell development in adult mice reveals the ability of bone marrow to produce B-1a cells.

To study B-cell development from bone marrow (BM), we generated recombination-activating gene 1 (Rag1)-targeted mice lacking mature lymphocytes. B-cell development can be induced in such mice by B cell-specific restoration of a functional Rag1 transcription unit. Follicular and marginal zone B cells populated the spleen when Rag1 expression was permitted. Notably, the peritoneal cavity was dominated by bona fide B-1a cells, as judged by surface markers and functional properties. These BM-derived B-1a cells exhibited a polyclonal VDJ repertoire with substantial N nucleotide insertions. Nevertheless, physiologic frequencies of phosphatidylcholine-specific B cells were detected. Importantly, the BM of young and 5-month-old mice was indistinguishable with regard to the potential to generate B-1a cells.

Somatic hypermutation in peritoneal B1b cells.

Murine B1 cells have been shown to be able to switch to IgA in vitro. In agreement, we could demonstrate in the peritoneum of mice the presence of IgA producing B1 cells. Interestingly, enzyme-linked immunospot assays of lipopolysaccharide stimulated cultures revealed that only the B1b cell subpopulation contained high numbers of such cells while IgA producing B cells were rare amongst the B2 and B1a cell populations. This was confirmed by RT-PCR on sorted peritoneal B cell subpopulations. In addition, the variable regions associated with IgA of peritoneal B1b cells displayed extensive variation due to somatic hypermutation. In contrast, mutations were found only at low frequencies in VH regions associated with IgM of both B1 cell populations. Thus, peritoneal B1b cells display many similarities to B2 cells. This finding is consistent with the idea of a layered immune system in which peritoneal B1a and splenic follicular B2 cells appear at the two extremes and peritoneal B1b and B2 cells represent intermediates.

Loss of lambda2(315) transgene copy numbers influences the development of B1 cells.

Transgenic L2 mice contain high numbers of the lambda2(315) immunoglobulin L chain gene in their germ line. They are characterized by an almost complete block in B2 cell development and dominance of B1 cells in their periphery. This was attributed to high transgene expression. Here, we describe a variant of such mice (L2V), which has lost half of the transgene copies. This results in decreased transgene expression. Consequently, such mice display less severe isotype exclusion and an increase in B cells expressing endogenous kappa light chains. In addition, the B2 cell compartment is enlarged. Nevertheless, L2V mice exhibit phosphatidylcholine (PtC) binding B cells expressing lambda L chains as well as an unaltered number of B1a cells expressing the dominating specificity usually encountered in L2 mice. Since in L2V mice transgene integration and regulation is identical to L2 mice, the correlation of decreased transgene expression and increased presence of B2 cells strongly suggests that high transgene expression is decisive for development of B1 cells in L2 mice.