As obesity and Western diet consumption are key factors contributing to gut dysbiosis, we investigated the relationship between intestinal microbiota, obesity, and psoriasis in an imiquimod-based model. C57BL/6 mice were used as follows: psoriasis-induced groups fed continuously with a standard or Western diet, psoriasis-induced group fed with a Western diet and then returned to a standard diet, and controls. For each group, clinicopathological, immune, and metabolic parameters were integrated with microbiome data. The imiquimod-based models displayed human psoriasis features and significant changes in immune parameters. Hence, psoriatic mice on prolonged high-fat intake presented decreased microbial richness and evenness and a gut microbiome composition resembling that of obese mice. Ruminococcus, Clostridium, Desulfovibrio, and Enterorhabdus were the most abundant genera in the obesity-enhanced psoriasis group. Raoultella abundance was linked with psoriasis. Yet, the same pathobionts over-represented in the obese psoriatic mice displayed positive correlations with metabolic stress indicators and proinflammatory factors, indicating potential biomarkers of disease severity. Conversely, Lactobacillus taiwanensis, Alistipes putredinis, and Eubacterium hadrum might be potential taxa for attenuating the metabolic burden in obesity-enhanced psoriasis. Here, we depict the microbial signatures associated with inflammation and metabolic stress in an obesity-aggravated psoriasis mouse model.
Background: SARS-CoV-2 immunity is understudied in cancer patients. Here, we monitored natural/vaccine-induced SARS-CoV-2 immunity in patients with head and neck cancer (HNC) stratified as vaccinated (mRNA/adenovirus-based vaccines), convalescent, and hybrid immunity. Methods: Plasma/PBMC samples were collected from 49 patients with HNC and 14 non-oncologic controls recruited between August 2021 and March 2022. Longitudinal follow-up was performed on 25 HNC patients. Plasma antibodies (Abs) against Spike (S1/S2), receptor-binding domain (RBD), and nucleocapsid (NC) of IgG/IgA isotypes and 25 cytokines/chemokines were quantified using MILLIPLEX® technology. The frequency, phenotype, and isotype of circulating SARS-CoV-2-specific B-cells were studied by flow cytometry using RBD tetramers (Tet++). The proliferation of B-cells and CD4+ and CD8+ T-cells in response to Spike/NC peptides was monitored by a carboxyfluorescein succinimidyl ester (CFSE) assay. Results: Plasma SARS-CoV-2 S1/S2/RBD IgG/IgA Abs were detected in all HNC participants at enrollment median time since immunization (TSI) 117 days at levels similar to controls and were significantly higher in convalescent/hybrid versus vaccinated. NC IgG/IgA Abs were only detected after infection. The frequency of Tet++ B-cells, enriched in the CD27+ memory phenotype and IgG/IgA isotype, positively correlated with plasma levels of RBD IgG/IgA Abs and Spike-specific CD4+ T-cell proliferation, regardless of the immunization status and TSI. Spike/NC-specific B-cell proliferation reached the highest levels in convalescent HNC and was positively correlated with NC IgG Abs, but not with the frequency of Tet++ B-cells. Finally, Tet++ B-cell frequencies remained stable between the two subsequent visits (median TSI: 117 versus 341 days), indicating their ability to persist for a relatively long time. Conclusions: This study monitored SARS-CoV-2 humoral/cellular immunity in an HNC cohort relative to non-oncologic participants and demonstrates that SARS-CoV-2-specific B-cells persist beyond 11 months post-immunization. These findings have implications for the management of HNC in the context of SARS-CoV-2 infection and other viral infections.
OBJECTIVE:Our understanding of the pathophysiology of pancreatic neuroendocrine tumors (PanNETs) remains incomplete, largely due to their historically underestimated incidence and the perception of these tumors as rare and slow-growing cancers. Additionally, conventional reliance on histological examination alone is gradually being supplemented by the exploration and introduction of molecular biomarkers, such as microRNAs (miRNAs). As miRNAs modulate the expression of multiple genes and pathways involved in the tumorigenesis of PanNETs, these biomarkers hold considerable promise for diagnosis and prognosis applications. In this study, we aimed to identify miRNAs as tissue markers associated with the diagnosis of PanNETs. METHODS:We conducted a case-control study including: 7 PanNETs and 19 nontumoral pancreatic tissues obtained from Romanian patients. The samples underwent miRNA profiling via quantitative RT-PCR to assess the expression of 84 miRNAs. Our results were compared with those obtained by reanalyzing a public dataset. Furthermore, we structured our miRNA expression data according to their targeted mRNAs and their roles in signaling pathways. RESULTS:Fourteen miRNAs (miR-1, miR-133a-3p, miR-210-3p, miR-7-5p, miR-10a-5p, miR-92b-3p, miR-132-3p, miR-221-3p, miR-29b-3p, miR-107, miR-103a-3p, let-7b-5p, miR-148a-3p, and miR-202-3p) were identified as differentially expressed by comparing PanNETs with pancreatic nontumoral tissues, with six miRNAs (miR-7-5p, miR-92b-3p, miR-29b-3p, miR-107, miR-103a-3p, and miR-148a-3p) also found in the public dataset analyzed. Bioinformatic analysis revealed that the 14 identified miRNAs target 17 genes. Reanalyzing two public gene expression datasets, five of these genes have been found differentially expressed in PanNET compared to controls. CONCLUSIONS:Our preliminary results, albeit limited by a small sample size, highlighted a specific miRNA expression pattern able to distinguish tumoral from normal pancreatic tissue. The diagnostic performance of these miRNAs, matching with circulating miRNAs and validated in more homogeneous and large cohorts, could represent a starting point for improving the diagnostic accuracy of PanNETs.
MiR-210 is widely recognized as the quintessential hypoxia-responsive miRNA and is thought to fine-tune various facets of cellular homeostasis. We hereby present an integrative appraisal of the phenotypic and molecular repercussions of disrupting the corresponding locus in human and mouse cells using multiple genetic strategies. In brief, MIR210 deletion led to decreased cellular fitness and suboptimal responses to several stress types. Transcriptomic comparisons via different profiling platforms, performed independently by members of this collaboration, revealed consistent deregulation of neighboring genes, in locus-disrupted cells. Interestingly, the anticipated enrichment of miR-210 targets failed to materialize in unbiased analyses. Our results point to the biological significance of unrecognized regulatory elements that overlap miRNA genes and should serve as a note of caution for studies based on the genetic disruption of such loci.
Background: Cutaneous melanoma (CM) is a heterogeneous and highly metastatic disease with unpredictable clinical behaviour, for which the most effective pharmacological strategies are still being sought. In the present study, we describe the workflow for RUNX2 KO generation in B16 melanoma cells, an approach that according to preliminary data from international databases may contribute to defusing CM resistance to targeted and immunotherapies. Methods: The role of the RUNX2 gene in CM was investigated using the TIMER2.0 (http://timer.cistrome.org/) and TISIDB (http://cis.hku.hk/TISIDB/) databases. Two gRNAs targeting exon 4 of RUNX2 were designed and cloned into the px459v2.0 plasmid, as previously described1. The B16 melanoma cells were transfected with 2.5 μg of plasmid DNA using Lipofectamine 3000 in DMEM (without antibiotics). After one day, cells were selected with complete DMEM containing 1 μg/ml puromycin for six days. Cells were further subjected to monoclonal cell isolation by seeding 0.3 cells/well in 96-well plates. Genomic DNA was extracted from the transfected bulk cell population and isolated clones and PCR-amplified with primers spanning the gRNA-target region. RUNX2 KO was confirmed by Western blot. Results: According to the TIMER2.0 and TISIDB databases, RUNX2 shows positive correlations with the most notorious immune inhibitors within the CM tumour microenvironment and is overexpressed in CM patients unresponsive to immune and targeted therapies. Therefore, we hypothesize that orchestrating RUNX2 KO in melanoma may be a promising strategy to improve tumour response to therapeutic approaches. Five B16 clones were subjected to Sanger sequencing and all showed editing events. In particular, they showed an in-frame deletion within the Runt domain of the RUNX2 gene. In addition, these five clones showed no RUNX2 protein expression by Western blot. Conclusions: Our study presents the workflow for obtaining genetically engineered melanoma cells that can be further exploited to dissect the biological roles of RUNX2 in syngeneic B16 melanoma mouse models.
