Shinsmon Jose

Neutrophils are key first responders in the host response to Clostridioides difficile infection (CDI). Although a high number of tissue and blood neutrophils clearly correlates with adverse outcomes in CDI patients, their functional role remains poorly defined. Using murine (CMT-93) and human (Caco-2) intestinal epithelial cell (IEC) lines co-cultured with neutrophils and a pre-clinical mouse model of CDI, we show that activated neutrophils exacerbate C. difficile-induced IEC injury. To identify neutrophil subtypes and biological pathways that contribute to CDI-associated IEC damage, we utilized single-cell RNAseq (scRNAseq) to generate the first transcriptomic atlas of murine bone marrow, blood, and colonic neutrophils in acute CDI. Our analyses identified a novel neutrophil population in the colonic lamina propria, "cNeu3," which exhibited high expression of genes associated with neutrophil activation and degranulation. Informed by cNeu3 signature genes from the scRNAseq dataset, we validated the presence of these neutrophils in mouse colon using flow cytometry (CD11b^hiCD63^hiIL-1β^hiMIP-1α^hiMALT1^hi cells). We found that their abundance correlated with increased epithelial damage. Tumor Necrosis Factor (TNF) was sufficient to polarize mouse bone marrow neutrophils to a cNeu3 phenotype in vitro. Finally, TNF-primed human neutrophils worsened C. difficile toxin-induced IEC damage, which was improved when neutrophil degranulation was blocked. Collectively, our data provide novel insights into neutrophil-mediated pathology during CDI.

Neutrophils are dominant cells during acute immune response to Clostridioides difficile infection (CDI). A higher number of infiltrating colonic neutrophils is clearly linked to greater tissue damage and severe CDI (3, 4). However, the mechanism(s) by which neutrophils exacerbate tissue damage in CDI remain unknown. We investigated the role of a neutrophil subset marked by Olfactomedin-4 expression (OLFM4 ⁺ neutrophils) during CDI. Single-cell transcriptomics reveal that Olfm4 is increased in blood neutrophils of infected mice, and these cells exhibit gene signatures characterized by high expression of degranulation genes. In C. difficile -infected mice, OLFM4 ⁺ neutrophils aggregate to areas of severe intestinal epithelial cell (IEC) damage, and plasma OLFM4 was significantly increased in both C. difficile -infected mice and patients. In vitro , OLFM4 ⁺ neutrophils and recombinant OLFM4 protein exacerbated C. difficile toxin-induced IEC damage. In sum, our studies provide novel insights into neutrophil-mediated pathology and highlight the role of OLFM4 ⁺ neutrophils in worsening CDI-induced IEC damage.

This uniquely original, crowd‑funded laboratory study compared 18 medical‑pharmaceutical and 16 nutraceutical whey protein powders sold in India by analyzing macronutrients, amino‑acid profiles, sugars, heavy metals, mycotoxins, hormones, pesticides and undeclared additives. Nutraceutical products supplied a mean (± SD) 75.6 ± 7.3 g protein/100 g and showed tight agreement with label claims. Ninety‑four per cent met the ≥ 60 g/100 g threshold and exhibited high essential‑amino‑acid densit2y (up to 8.5 g leucine/100 g) with negligible added sugars. By contrast, pharmaceutical powders averaged 29.1 ± 15.4 g protein/100 g; 83% were mis‑labeled beyond ± 5%, and only one product reached the quality benchmark. None exceeded 5 g leucine/100 g, while 44% contained ≥ 2 g sucrose or fructose/100 g, often unlabeled. Heavy‑metal outliers (cadmium, arsenic, copper), aflatoxin, and progesterone appeared more frequently in pharmaceutical brands, and taurine‑based nitrogen spiking was detected in 89% of them versus 12.5% of nutraceuticals. No product contained detectable pesticides or mercury. Overall, nutraceutical powders consistently met compositional expectations, whereas medical‑pharmaceutical formulations delivered lower‑quality protein, higher sugar loads and unreliable labeling, questioning their suitability for therapeutic use.

Homeopathy is a popular alternative medical practice globally. However, homeopathic formulations can cause allergies, anaphylaxis, fatal cardiovascular events, kidney injury, liver failure, and death due to their direct and indirect toxicities. This study examined commonly prescribed homeopathic formulations from 3 categories: classical dilutions, mother tinctures, and proprietary products. This cross-sectional study assessed the quality and safety of popular over-the-counter homeopathic formulations. Each remedy underwent complete organic and inorganic content profiling, including heavy metal detection, alcohol quantification, and steroid detection. We analyzed 134 homeopathic remedies and found high alcohol levels, insect and animal parts, carnivorous plants in alcohol, and toxic heavy metals. Classical dilutions had the highest alcohol content (median: 91.02% v/v). Highly diluted formulations of arsenic and mercury had detectable lead levels. Proprietary medicines also showed various potentially toxic bioactive plant compounds, heavy metal contaminants, industrial-grade solvents, and pharmaceutical intermediates, raising concerns about organ toxicity. Homeopathic formulations revealed high ethanol levels and toxic heavy metals like arsenic, lead, mercury, and cadmium. Tinctures and proprietary formulations contained multiple potentially toxic chemicals. Stricter regulatory oversight is needed, and consumers should be aware that the testimonials and anecdotal claims of homeopathic benefits lack scientific validation and can mask potentially harmful effects.

Neutrophils are key first responders to Clostridioides difficile infection (CDI). Excessive tissue and blood neutrophils are associated with worse histopathology and adverse outcomes, however their functional role during CDI remains poorly defined. Utilizing intestinal epithelial cell (IEC)-neutrophil co-cultures and a pre-clinical animal model of CDI, we show that neutrophils exacerbate C. difficile -induced IEC injury. We utilized cutting-edge single-cell transcriptomics to illuminate neutrophil subtypes and biological pathways that could exacerbate CDI-associated IEC damage. As such, we have established the first transcriptomics atlas of bone marrow (BM), blood, and colonic neutrophils after CDI. We found that CDI altered the developmental trajectory of BM and blood neutrophils towards populations that exhibit gene signatures associated with pro-inflammatory responses and neutrophil-mediated tissue damage. Similarly, the transcriptomic signature of colonic neutrophils was consistent with hyper-inflammatory and highly differentiated cells that had amplified expression of cytokine-mediated signaling and degranulation priming genes. One of the top 10 variable features in colonic neutrophils was the gene for neutrophil glycoprotein, Olfactomedin 4 (OLFM4). CDI enhanced OLFM4 mRNA and protein expression in neutrophils, and OLFM4 ⁺ cells aggregated to areas of severe IEC damage. Compared to uninfected controls, both humans and mice with CDI had higher concentrations of circulating OLFM4; and in mice, OLFM4 deficiency resulted in faster recovery and better survival after infection. Collectively, these studies provide novel insights into neutrophil-mediated pathology after CDI and highlight the pathogenic role of OLFM4 ⁺ neutrophils in regulating CDI-induced IEC damage. One Sentence Summary: Utilizing single-cell transcriptomics, IEC-epithelial co-cultures, and pre-clinical models of CDI, we have identified a subset of neutrophils that are marked by OLFM4 expression as pathogenic determinants of IEC barrier damage after CDI.

Nitric oxide is a versatile mediator formed by enzymes called nitric oxide synthases. It has numerous homeostatic functions and important roles in inflammation. Within the inflamed brain, microglia and astrocytes produce large amounts of nitric oxide during inflammation. Excessive nitric oxide causes neuronal toxicity and death and mesenchymal stem cells can be used as an approach to limit the neuronal damage caused by neuroinflammation. Mesenchymal stem cell therapy ameliorates inflammation and neuronal damage in disease models of Alzheimer’s disease, Parkinson’s disease, and other neuroinflammatory disorders. Interestingly, we have reported that in vitro, mesenchymal stem cells themselves contribute to a rise in nitric oxide levels through microglial cues. This may be an undesirable effect and highlights a possible need to explore acellular approaches for mesenchymal stem cell therapy in the central nervous system.

Neutrophils are key first-responders in the innate immune response to C. difficile infection (CDI) and play a central role in disease pathogenesis. Studies have clearly shown that tissue neutrophil numbers need to be tightly regulated for optimal CDI outcomes: while excessive colonic neutrophilia is associated with severe CDI, neutrophil depletion also results in worse outcomes. However, the biological mechanisms that control CDI-induced neutrophilia remain poorly defined. C-X-C chemokine receptor 2 (CXCR2) is a chemotactic receptor that is critical in neutrophil mobilization from bone marrow to blood and tissue sites. We have previously reported that a single nucleotide polymorphism (SNP) in leptin receptor (LEPR), present in up to 50% of people, influenced CDI-induced neutrophil CXCR2 expression and tissue neutrophilia. Homozygosity for mutant LEPR (i.e. RR genotype) was associated with higher CXCR2 expression and more tissue neutrophils. Here, we investigated the biological mechanisms that regulate neutrophil CXCR2 expression after CDI, and the influence of host genetics on this process. Our data reveal that: a) CXCR2 plays a key role in CDI-induced neutrophil extravasation from blood to colonic tissue; b) plasma from C. difficile-infected mice upregulated CXCR2 on bone marrow neutrophils; c) plasma from C. difficile-infected RR mice induced a higher magnitude of CXCR2 upregulation and had more IL-1β; and d) IL-1β neutralization reduced CXCR2 expression on bone marrow and blood neutrophils and their subsequent accrual to colonic tissue. In sum, our data indicate that IL-1β is a key molecular mediator that communicates between gastro-intestinal tract (i.e. site of CDI) and bone marrow (i.e. primary neutrophil reservoir) and regulates the intensity of CDI-induced tissue neutrophilia by modulating CXCR2 expression. Further, our studies highlight the importance of host genetics in affecting these innate immune responses and provide novel insights into the mechanisms by which a common SNP influences CDI-induced neutrophilia.