Multi-omics analysis in IgM monoclonal gammopathies reveals epigenetic influence on oncogenesis via DNA methylation.

Currently, the role of DNA methylation in the IgM-monoclonal gammopathy disease spectrum remains poorly understood. In the present study, a multi-omics prospective analysis was conducted integrating DNA methylation, RNA-seq and WES data in 34 subjects [23 WM, 6 IgM-MGUS, 5 normal controls]. Analysis was focused on defining differences between IgM-gammopathies (WM/IgM-MGUS) compared to controls, and specifically between WM and IgM-MGUS. Between groups, genome-wide DNA methylation analysis demonstrated a significant number of differentially methylated regions which were annotated according to genomic region. Next, integration of RNA-seq data was performed to identify potentially epigenetically deregulated pathways. We found that pathways involved in cell cycle, metabolism, cytokine/immune signaling, cytoskeleton, tumor microenvironment, and intracellular signaling were differentially activated and potentially epigenetically regulated. Importantly, there was a positive enrichment of CXCR4 signaling pathway along with several interleukin (IL-6, IL-8, IL15) signaling pathways in WM compared to IgM-MGUS. Further assessment of known tumor suppressor genes and oncogenes uncovered differential promoter methylation of several targets with concordant change in gene expression, including CCND1 and CD79B. Overall, this report defines how aberrant DNA methylation in IgM-gammopathies may play a critical role in the epigenetic control of oncogenesis and key cellular functions.

Monocyte response to SARS-CoV-2 protein ORF8 is associated with severe COVID-19 infection in patients with chronic lymphocytic leukemia.

The open reading frame 8 (ORF8) protein, encoded by the SARS-CoV-2 virus after infection, stimulates monocytes/macrophages to produce pro-inflammatory cytokines. We hypothesized that a positive ex vivo monocyte response to ORF8 protein pre-COVID-19 would be associated with subsequent severe COVID-19. We tested ORF8 ex vivo on peripheral blood mononuclear cells (PBMCs) from 26 anonymous healthy blood donors and measured intracellular cytokine/chemokine levels in monocytes by flow cytometry. The % monocytes staining positive in the sample and change in mean fluorescence intensity (ΔMFI) after ORF8 were used to calculate the adjusted MFI for each cytokine. We then tested pre-COVID-19 PBMC samples from 60 CLL patients who subsequently developed COVID-19 infection. Severe COVID-19 was defined as hospitalization due to COVID-19. In the 26 normal donor samples, the adjusted MFI for interleukin (IL)-1ß, IL-6, IL-8, and CCL-2 were significantly different with ORF8 stimulation vs controls. We next analyzed monocytes from pre-COVID-19 PBMC samples from 60 CLL patients. The adjusted MFI to ORF8 stimulation of monocyte intracellular IL-1ß was associated with severe COVID-19 and a reactive ORF8 monocyte response was defined as an IL- 1ß adjusted MFI ≥ 0.18 (sensitivity 67%, specificity 75%). The median time to hospitalization after infection in CLL patients with a reactive ORF8 response was 12 days versus not reached for patients with a non-reactive ORF8 response with a hazard ratio of 7.7 (95% CI: 2.4-132, p=0.005). These results provide new insight on the monocyte inflammatory response to virus with implications in a broad range of disorders involving monocytes.

Next-Generation Sequencing Reveals the Role of Epigallocatechin-3-Gallate in Regulating Putative Novel and Known microRNAs Which Target the MAPK Pathway in Non-Small-Cell Lung Cancer A549 Cells.

Lung cancer constitutes 85% of non-small cell lung cancer diagnosed cases. MicroRNAs are novel biomarkers that are capable of modulating multiple oncogenic pathways. Epigallocatechin-3-gallate (EGCG) is a potent chemopreventive and chemotherapeutic agent for cancer. We aimed to identify important known and putative novel microRNAs modulated by EGCG in A549 cells using next-generation sequencing and identify their gene targets. Preliminary analysis revealed an IC50 value of 309 µM with G0/G1 phase arrest at 40 µM EGCG treatment. MicroRNA profiling identified 115 known and 4 putative novel microRNAs in 40 µM and 134 known and 3 putative novel microRNAs in 100 µM EGCG-treated A549 cells. The top 10 up-expressed microRNAs were similar between the untreated control and EGCG-treated A549 cells. An up-expression in oncogenic microRNAs, which belong to broadly conserved seed families, were observed in untreated control and EGCG-treated A549 cells. Kyoto Encyclopedia of Genes and Genomes and Protein Analysis Through Evolutionary Relationships pathway analyses of the validated microRNA targeting genes strengthened the hypothesis that EGCG treatment can modulate microRNAs that play a significant role in the MAPK signaling pathway. Expression profile of microRNAs was validation by quantitative real time PCR of randomly selected microRNAs. This study identified signature microRNAs that can be used as novel biomarkers for lung cancer diagnosis.

Compare the Combined Diagnostic Accuracy of Transient Elastography and Visual Liver Score in Assessing Non-Alcoholic Fatty Liver Disease (NAFLD) and Compare it with Liver Biopsy in Morbidly Obese Patients Undergoing Bariatric Surgery: An Observational Study.

INTRODUCTION: A liver biopsy is the gold standard for the diagnosis of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Here, we combine preoperative transient elastography (TE) and intraoperative standardized visual liver score (VLS) which is compared with intraoperative liver biopsy for prediction of NAFLD and NASH in patients undergoing bariatric surgery. AIM: Evaluate the combined diagnostic accuracy of TE and VLS in assessing NAFLD or NASH and compare it with liver biopsy in patients undergoing bariatric surgery. METHODS: In a prospective cohort of 70 morbidly obese undergoing bariatric surgery, preoperative TE and intraoperative VLS were calculated. Findings of TE and VLS were compared with histology from intraoperative liver biopsy. RESULTS: Histologically, 44 (62.85%) had NAFLD (≥ S1). Significant steatosis was seen in 20 (28.57%) while significant fibrosis was visible in 18 (25.71%). Area Under the Receiver Operating Characteristics (AUROC) TE for diagnosis of NAFLD was excellent (0.844, p = 0.001). At the optimal cutoff of 8.1, the positive predictive value (PPV) was 92.9%, and diagnostic accuracy was 90.6%. VLS had a sensitivity of 90.9% for NAFLD. The combined sensitivity of TE + VLS was 95.5% for ruling out NAFLD. Fourteen (20%) had NASH. VLS had a diagnostic accuracy of 97% in identifying NASH in comparison to TE. AUROC-VLS was 0.987, p ≤ 0.001, and a sensitivity of 100%. The overall sensitivity of combined TE and VLS was 100% with a negative predictive value (NPV) of 100%. CONCLUSION: TE when combined with intraoperative VLS is comparable to liver biopsy and can be used for the diagnosis of NAFLD and NASH in patients undergoing bariatric surgery.

Modulation of T-cell function by myeloid-derived suppressor cells in hematological malignancies.

Myeloid-derived suppressor cells (MDSCs) are pathologically activated neutrophils and monocytes that negatively regulate the immune response to cancer and chronic infections. Abnormal myelopoiesis and pathological activation of myeloid cells generate this heterogeneous population of myeloid-derived suppressor cells. They are characterized by their distinct transcription, phenotypic, biochemical, and functional features. In the tumor microenvironment (TME), myeloid-derived suppressor cells represent an important class of immunosuppressive cells that correlate with tumor burden, stage, and a poor prognosis. Myeloid-derived suppressor cells exert a strong immunosuppressive effect on T-cells (and a broad range of other immune cells), by blocking lymphocyte homing, increasing production of reactive oxygen and nitrogen species, promoting secretion of various cytokines, chemokines, and immune regulatory molecules, stimulation of other immunosuppressive cells, depletion of various metabolites, and upregulation of immune checkpoint molecules. Additionally, the heterogeneity of myeloid-derived suppressor cells in cancer makes their identification challenging. Overall, they serve as a major obstacle for many cancer immunotherapies and targeting them could be a favorable strategy to improve the effectiveness of immunotherapeutic interventions. However, in hematological malignancies, particularly B-cell malignancies, the clinical outcomes of targeting these myeloid-derived suppressor cells is a field that is still to be explored. This review summarizes the complex biology of myeloid-derived suppressor cells with an emphasis on the immunosuppressive pathways used by myeloid-derived suppressor cells to modulate T-cell function in hematological malignancies. In addition, we describe the challenges, therapeutic strategies, and clinical relevance of targeting myeloid-derived suppressor cells in these diseases.

Comparison of Fecal Calprotectin with Different Endoscopic Scores in the Assessment of Ulcerative Colitis (UC) Activity and Its Utility in Differentiating IBS from IBD.

Background: Ulcerative colitis (UC), a chronic inflammatory disease of gastrointestinal tract, can have initial presentation which is clinically difficult to differentiate from functional bowel disorders [irritable bowel syndrome (IBS) and irritable bowel disease (IBD)]. Conventional laboratory tests, such as erythrocyte sedimentation rate (ESR), C-reactive protein, and albumin express systemic patient responses instead of intestinal inflammation. In the last decade, fecal calprotectin, a calcium-binding protein, has been suggested as a sensitive marker of intestinal inflammation. However, only few studies have investigated its role in relation with the extent of the disease. Aim: To evaluate the usefulness of fecal calprotectin as a biomarker for disease activity in UC, its correlation with disease extent and its utility in differentiating IBS from IBD. Methods: A total of 75 patients (50 cases with colonoscopic evidence of inflammation and 25 cases with normal colonoscopic examination) were included in the study. Fecal calprotectin test was done on the day of colonoscopy. Severity of the disease was assessed by modified Mayo's endoscopy score (MMES). Results: Age and baseline parameters were comparable in both the groups (UC and IBS). Patients in the ulcerative group had tachycardia (95 vs 74), high ESR (26 vs 20), high leukocytes count (9198 vs 8852), high fecal calprotectin (594 vs 29), low albumin (3.00 vs 3.80) and low hemoglobin (11 vs 13.40). Minimum and maximum MMES were 2 and 13.2. A significant correlation was observed between fecal calprotectin and MMES (p-value < 0.001). Conclusion: Fecal calprotectin is a simple, noninvasive, cost-effective marker that is strongly associated with colorectal inflammation; moreover, it has better role in the differentiation of IBD (UC) from IBS. How to cite this article: Acharya K, Bhardwaj V, Chuahan I, et al. Comparison of Fecal Calprotectin with Different Endoscopic Scores in the Assessment of Ulcerative Colitis (UC) Activity and Its Utility in Differentiating IBS from IBD. Euroasian J Hepato-Gastroenterol 2023;13(2):120-123.

Real-world data on renal transplantations from a tertiary-care hospital in North India, in context of Indian regulatory act-transplantation of human organs and tissues act (THOTA): A retrospective analysis.

INTRODUCTION: Renal transplantation is the treatment of choice for patients suffering from end stage renal disease (ESRD). Indian regulations defined under Transplantation of Human Organs and Tissues Act (THOTA), 2014 restricts organ donations to near-related living donors to curb any malpractices like 'paid donors' in living-donor kidney transplantation (LDKT). The aim of our study was to look at real-world data of donor-recipient pairs and to identify relationship of donors (with their respective patients) and the common (or uncommon) DNA profiling methods used for supporting "claimed relationship" in accordance with the regulations. MATERIAL AND METHODS: The donors were categorized and grouped into near-related donor, donors other than near-related donors, swap donors and deceased donors. Claimed relationship was confirmed, commonly by HLA typing, using SSOP method. In few cases, which were uncommon (and infrequent), autosomal DNA analysis, mitochondrial DNA analysis and Y-STR DNA analysis were performed to support the claimed relationship. Data collected included age, gender, relationship, DNA profiling test method. RESULTS: Among the 514 donor-recipient pairs evaluated, numbers of female donors out-numbered male donors. The decreasing order of relationships in near-related donor group were wife>mother>father>sister>son>brother>husband> daughter>grandmother. 11.9% of donors were in the category of donors other than near-related donors. In 97.86% cases, the claimed relationship was supported by HLA typing and in just 2.1% cases autosomal DNA analysis>mitochondrial DNA analysis> Y-STR DNA analysis, in this order, were performed to establish relationship. CONCLUSION: This study brought out gender disparity with women out-numbering men as donors. Among recipients, access to renal transplant was largely restricted to men. As far as relationship of donors to recipients was concerned, mostly near-related family members, like wife, were donors and claimed relationship was almost always (99%) was corroborated by HLA typing.

Secreted ORF8 induces monocytic pro-inflammatory cytokines through NLRP3 pathways in patients with severe COVID-19.

Despite extensive research, the specific factor associated with SARS-CoV-2 infection that mediates the life-threatening inflammatory cytokine response in patients with severe COVID-19 remains unidentified. Herein we demonstrate that the virus-encoded Open Reading Frame 8 (ORF8) protein is abundantly secreted as a glycoprotein in vitro and in symptomatic patients with COVID-19. ORF8 specifically binds to the NOD-like receptor family pyrin domain-containing 3 (NLRP3) in CD14+ monocytes to induce inflammasomal cytokine/chemokine responses including IL1ß, IL8, and CCL2. Levels of ORF8 protein in the blood correlate with severity and disease-specific mortality in patients with acute SARS-CoV-2 infection. Furthermore, the ORF8-induced inflammasome response was readily inhibited by the NLRP3 inhibitor MCC950 in vitro. Our study identifies a dominant cause of pathogenesis, its underlying mechanism, and a potential new treatment strategy for severe COVID-19.

Exercise-induced myokines downregulates the ACE2 level in bronchial epithelial cells: Implications for SARS-CoV-2 prevention.

BACKGROUND: The Covid-19 pandemic has emerged as the leading public health challenge of our time (20th century). While vaccinations have finally blunted the death rate, concern has remained about more virulent forms highlighting the need for alternative approaches. Epidemiological studies indicate that physical activity has been shown to decrease the risk of infection of some respiratory viruses. Part of the salutary effects of exercise is believed to be through the elaboration of cytokines by contracting skeletal muscles (termed myokines). The objective of this study was to investigate whether exercise-induced myokines would mitigate the SARS-CoV-2 infectivity of the bronchial epithelium through modulating the SARS-CoV-2 Covid-19 receptor (angiotensin-converting enzyme 2 -ACE2) its priming enzyme, transmembrane serine protease 2 (TMPRSS2). METHODS: We utilized a cell culture model of exercise to generate myokines by differentiating C2C12 cells into myotubules and inducing them to contract via low-frequency electric pulse stimulation. Condition media was concentrated via centrifugation and applied to human immortalized human bronchial epithelium cell line (6HBE14o) along with conditioned media from unstimulated myotubules as controls. Following exposure to myokines, the 16HBE14o cells were harvested and subjected to quantitative RT-PCR and Enzyme-Linked Immunosorbent Assay (ELISA) for assessment of mRNA and protein levels of ACE2 and TMPRSS2, respectively. Pilot proteomic data was performed with isotope barcoding and mass spectroscopy. RESULTS: Quantitative Real-Time PCR of 16HBE14o with 48 h treated unstimulated vs. stimulated myokine treatment revealed a reduction of ACE2 and TMPRSS2 mRNA by 32% (p<2.69x10-5) and 41% (p<4.57x10-5), respectively. The high sensitivity of ELISAs showed downregulation of ACE2 and TMPRSS2 protein expression in 16HBE14o cells by 53% (p<0.01) and 32% (p<0.03) respectively with 48 h treated. For rigor, this work was replicated in the human lung cancer cell line A549, which mirrored the downregulation. Proteomic analysis showed dramatic alteration in myokine profile between contracted and uncontracted C2C12 tubules. CONCLUSIONS: The current study explores a novel approach of a modified exercise cell culture system and uses ACE2 and TMPRSS2 as a surrogate marker of SARS-CoV-2 infectivity. In conclusion, we demonstrated biological data supporting exercise's protective effect against Covid-19. These further strengthen myokines' beneficial role as potential therapeutic targets against SARS-CoV-2 and similar viruses albeit these preliminary cell culture studies will require future validation in animal models.

Colonic Epithelial Angiotensin-Converting Enzyme 2 (ACE2) Expression in Blacks and Whites: Potential Implications for Pathogenesis Covid-19 Racial Disparities.

BACKGROUND: Covid-19 toll is disproportionate in Blacks although the mechanisms remain incompletely understood. From a biological perspective, several host proteins have received most attention as logical susceptibility targets. Specifically, angiotensin-converting enzyme 2 (ACE2) serves as the epithelial cell receptor and acts in concert with transmembrane protease serine 2 (TMPRSS2). Intriguingly, ACE2 can also suppress the inflammatory response and therefore may impact the severity of Covid-19 infections (from the exuberant immune response a.k.a. "cytokine storm"). We, therefore, assessed expression of ACE2 and TMPRSS2 in Blacks versus Whites. METHODS: Archived mucosal biopsies from colonoscopic biopsies of visually normal rectal mucosa without concurrent neoplasia or inflammation were used for this study. Total mRNA was isolated and subjected to real-time polymerase chain reaction for ACE2, and TMPRSS2 was assessed from non-Hispanic Blacks (n = 45) and non-Hispanic Whites (n = 38). GAPDH and beta-actin were used for normalization. Multivariable analysis was performed using Analyse-IT software. RESULTS: ACE2 and TMPRSS2 levels were not altered by gender, BMI, or age. ACE2 levels were lower in Blacks than Whites achieving statistical significance in multivariable (0.51-fold, p = 0.03) but not quite in univariable (p = 0.07) analysis. This downregulation was mirrored in TMRPSS2 in both univariable (p = 0.03) and multivariable analyses (0.41-fold, p = 0.02). Moreover, there was a strong correlation between ACE2 and TMPRSS2 levels (r-squared = 0.78). CONCLUSIONS: To our knowledge, this is the first report on racial differences inACE2 and TMPRSS2 mucosal expression. This may provide potential biological underpinnings for the disproportionately higher mortality of Covid-19 in Blacks and should spur future studies.

Phenotype, Function, and Clinical Significance of CD26+ and CD161+Tregs in Splenic Marginal Zone Lymphoma.

PURPOSE: Regulatory T-cells (Treg) are essential to Tregs homeostasis and modulate the antitumor immune response in patients with lymphoma. However, the biology and prognostic impact of Tregs in splenic marginal zone lymphoma (SMZL) have not been studied. EXPERIMENTAL DESIGN: Biopsy specimens from 24 patients with SMZL and 12 reactive spleens (rSP) from individuals without lymphoma were analyzed by using CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), CyTOF (mass cytometry) analysis, and flow cytometry to explore the phenotype, transcriptomic profile, and clinical significance of intratumoral Tregs and their subsets. The biological characteristics and cell signaling pathways of intratumoral Treg subsets were confirmed by in vitro functional assays. RESULTS: We found that Tregs are more abundant in SMZL patients' spleens than rSP, and Tregs from patients with SMZL and rSP can be separated into CD161+Treg and CD26+Treg subsets. CD161+Tregs are increased in SMZL but have dysregulated immune function. We found that CD161+Treg and CD26+Tregs have unique gene expression and phenotypic profiles and are differentially correlated with patient outcomes. Specifically, increased CD161+Tregs are significantly associated with a favorable prognosis in patients with SMZL, whereas CD26+Tregs are associated with a poor prognosis. Furthermore, activation of the IL2/STAT5 pathway contributes to the induction of CD26+Tregs and can be reversed by STAT5 inhibition. CONCLUSIONS: IL2/STAT5-mediated expansion of CD26+Tregs contributes to a poor clinical outcome in SMZL and may represent a therapeutic opportunity in this disease.

EGCG Nanoparticles Attenuate Aluminum Chloride Induced Neurobehavioral Deficits, Beta Amyloid and Tau Pathology in a Rat Model of Alzheimer's Disease.

Rational: Alzheimer's disease (AD) is a neurodegenerative pathology characterized by the presence of neuritic plaques and neurofibrillary tangles. Aluminum has been reported to play an important role in the etiology and pathogenesis of this disease. Hence, the present study aimed to evaluate the neuroprotective role of epigallocatechin-gallate (EGCG) loaded nanoparticles (nanoEGCG) against aluminum chloride (AlCl3) induced neurobehavioral and pathological changes in AD induced rats. Method: 100 mg/kg body weight AlCl3 was administered orally for 60 days, which was followed by 10 mg/kg body weight free EGCG and nanoEGCG treatment for 30 days. Morris water maze, open field and novel object recognition tests were employed for neurobehavioral assessment of the rats. This was followed by histopathological assessment of the cortex and the hippocampus in the rat brain. For further validation biochemical, immunohistochemistry and western blot assays were carried out. Result: Aluminum exposure reduced the exploratory and locomotor activities in open field and significantly reduced the memory and learning curve of rats in Morris water maze and novel object recognition tests. These neurobehavioral impairments were significantly attenuated in nanoEGCG treated rats. Histopathological assessment of the cortex and hippocampus of AlCl3 induced rat brains showed the presence of both neuritic plaques and neurofibrillary tangles. In nanoEGCG treated rats this pathology was absent. Significant increase in biochemical, immunohistochemical and protein levels was noted in AlCl3 induced rats. While these levels were greatly reduced in nanoEGCG treated rats. Conclusion: In conclusion, this study strengthens the hypothesis that EGCG nanoparticles can reverse memory loss, neuritic plaque and neurofibrillary tangles formation.

Biologics for the treatment of pyoderma gangrenosum in ulcerative colitis.

Pyoderma gangrenosum (PG) is an uncommon extra-intestinal manifestation of inflammatory bowel disease (IBD). Despite limited published literature, biologics have caused a paradigm shift in the management of this difficult-to-treat skin condition. The clinical data and outcomes of three patients with active ulcerative colitis and concurrent PG treated with biologics (infliximab two and adalimumab one) are reviewed in this report. Biologics were added because of the sub-optimal response of the colonic symptoms and skin lesions to parenteral hydrocortisone therapy. All three patients showed a dramatic response to the addition of the biologics. In view of the rapid healing of the skin lesions, superior response rate, and the additional benefit of improvement in the underlying colonic disease following treatment, anti-tumor necrosis factor blockers should be considered as a first line therapy in the management of PG with underlying IBD.

T-cell lymphoma with raised serum glycoprotein-125(CA-125) and raised ascitic fluid adenosinedeaminase levels initially presenting as ascites: a case report and review of literature.

We report a case of T-cell lymphoma presenting as ascites, omental thickening, and raised serum glycoprotein-125 (CA-125) and high-ascitic fluid adenosinedeaminase levels. This case was clinically suspected to be peritoneal carcinomatosis or peritoneal tuberculosis based on clinical, biochemical, and radiological features. However, effusion cytology, cell block, and immunohistochemistry confirmed this case as T-cell lymphoma. This revealed the role of effusion cytology and cell-block preparation with imunohistochemistry in the diagnosis.