Probes for INS

Background: Studies using techniques that relied on expression of an X-linked gene suggested predominant clones of smooth muscle cells (SMC) may exist in human atherosclerosis. These studies were limited by spatial resolution and nature of plaque types studied. We investigated whether clones of SMCs exist in unstable human atheroma. Methods and Results: We used a 25 nucleotide deletion in the 3’ UTR of the BGN gene, highly expressed by SMC and prevalent in 30% of females, to study clonal proliferation. Three different types of plaques (erosion, rupture, and adaptive intimal thickening) were selected from females heterozygous for the deletion mutant. Hybridization of target RNA-specific BaseScope probes was conducted to visualize the distribution of mutants and images displayed as a bubble plots. Clonality index was calculated as the percentage of each probe in each ROI. A clonality index equal to or exceeding the three times the standard deviation above the mean of the clonality index of the media in all plaques was considered clonal. In comparing clonality between media and intima, the mean percent ROI with clonality was significantly higher in the intima than in the media (42.3±18.2 vs 18.3±9.6%, P=0.003) and this was consistent for both eroded (27.0±9.8 vs 9.0±3.8%, P=0.04) and ruptured plaques (41.3±10.7 vs 20.0±3.5%, P=0.03). The relationship of dominant clone in the intima and media shows significant concordance in the majority of plaques studied (R=0.72, P

Interferon lambda (IFN-λ) (type III IFN) is constitutively secreted from human placental cells in culture and reduces Zika virus (ZIKV) transplacental transmission in mice. However, the roles of IFN-λ during healthy pregnancy and in restricting congenital infection remain unclear. Here, we used mice lacking the IFN-λ receptor (Ifnlr1-/-) to generate pregnancies lacking either maternal or fetal IFN-λ responsiveness and found that the antiviral effect of IFN-λ resulted from signaling exclusively in maternal tissues. This protective effect depended on gestational stage, as infection earlier in pregnancy (E7 rather than E9) resulted in enhanced transplacental transmission of ZIKV. In Ifnar1-/- dams, which sustain robust ZIKV infection, maternal IFN-λ signaling caused fetal resorption and intrauterine growth restriction. Pregnancy pathology elicited by poly(I·C) treatment also was mediated by maternal IFN-λ signaling, specifically in maternal leukocytes, and also occurred in a gestational stage-dependent manner. These findings identify an unexpected effect of IFN-λ signaling, specifically in maternal (rather than placental or fetal) tissues, which is distinct from the pathogenic effects of IFN-αβ (type I IFN) during pregnancy. These results highlight the complexity of immune signaling at the maternal-fetal interface, where disparate outcomes can result from signaling at different gestational stages. IMPORTANCE Pregnancy is an immunologically complex situation, which must balance protecting the fetus from maternal pathogens with preventing maternal immune rejection of non-self fetal and placental tissue. Cytokines, such as interferon lambda (IFN-λ), contribute to antiviral immunity at the maternal-fetal interface. We found in a mouse model of congenital Zika virus infection that IFN-λ can have either a protective antiviral effect or cause immune-mediated pathology, depending on the stage of gestation when IFN-λ signaling occurs. Remarkably, both the protective and pathogenic effects of IFN-λ occurred through signaling exclusively in maternal immune cells rather than in fetal or placental tissues or in other maternal cell types, identifying a new role for IFN-λ at the maternal-fetal interface.

Background Patients with advanced squamous-cell lung cancer (SQCLC) frequently (46%) exhibit tumor overexpression of fibroblast growth factor receptor (FGFR) messenger ribonucleic acid (mRNA). Rogaratinib is a novel oral pan-FGFR inhibitor with a good safety profile and anti-tumor activity in early clinical trials as a single agent in FGFR pathway-addicted tumors. SAKK 19/18 determined clinical activity of rogaratinib in patients with advanced SQCLC overexpressing FGFR1-3 mRNA. Methods Patients with advanced SQCLC failing standard systemic treatment and with FGFR1-3 mRNA tumor overexpression as defined in the protocol received rogaratinib 600 mg BID until disease progression or intolerable toxicity. A 6-months progression-free survival rate (6mPFS) ≤15% was considered uninteresting (H0), whereas a 6mPFS ≥38% was considered promising (H1). According to a Simon 2-stage design, 2 out of 10 patients of the first stage were required to be progression-free at 6 months. Comprehensive Genomic Profiling was performedusing the Oncomine Comprehensive Assay Plus (Thermo Fisher Scientific). Results Between July 2019 and November 2020, 49 patients were screened and 20 were classified FGFR-positive. Among a total of 15 patients, 6mPFS was reached in 1 patient (6.7%), resulting in trial closure for futility after the first stage. There were 7 (46.7%) patients with stable disease and 5 (33.3%) patients with progressive disease. Median PFS was 1.6 (95% CI 0.9-3.5) months and median overall survival (OS) 3.5 (95% CI 1.0-5.9) months. Most frequent treatment-related adverse events (TRAEs) included hyperphosphatemia in 8 (53%), diarrhea in 5 (33%), stomatitis in 3 (20%) and nail changes in 3 (20%) patients. Grade ≥3 TRAEs occurred in 6 (40%) patients. No associations between mutational profile and treatment outcome were observed. Conclusion Despite preliminary signals of activity, rogaratinib failed to improve PFS in patients with advanced SQCLC overexpressing FGFR mRNA. FGFR inhibitors in SQCLC remain a challenging field, and more in-depth understanding of pathway crosstalks may lead to the development of drug combinations with FGFR inhibitors resulting in improved outcomes.

Background & Aims The prevalence of non-alcoholic fatty liver disease (NAFLD) and its severe form, non-alcoholic steatohepatitis (NASH), is increasing. Subjects with NASH often develop liver fibrosis and advanced liver fibrosis is the main determinant of mortality in NASH patients. We and others have reported that STAT3 contributes to liver fibrosis and hepatocellular carcinoma in mice. Methods Here, we explored whether STAT3 activation in hepatocytes and in non-hepatocytes areas, measured by phospho-STAT3 (pSTAT3), is associated with liver fibrosis progression in 133 patients with NAFLD. We further characterized the molecular and cellular determinants of STAT3 activation by integrating spatial distribution and transcriptomic changes in fibrotic NAFLD liver. Results pSTAT3 scores in non-hepatocytes areas progressively increased with fibrosis severity (r=0.53, p

Gastric cancer (GC) is one of the most common cancer types in the world with a high mortality rate. Hereditary predisposition for GC is not fully elucidated so far. The aim of this study was identification of possible new candidate genes, associated with the increased risk of gastric cancer development. Whole exome sequencing (WES) was performed on 18 DNA samples from adenocarcinoma specimens and non-tumor-bearing healthy stomach tissue from the same patient. Three pathogenic variants were identified: c.1320+1G>A in the CDH1 gene and c.27_28insCCCAGCCCCAGCTACCA (p.Ala9fs) of the VEGFA gene were found only in the tumor tissue, whereas c.G1874C (p.Cys625Ser) in the FANCA gene was found in both the tumor and normal tissue. These changes were found only in patients with diffuse gastric cancer and were absent in the DNA of healthy donors.

Antisense oligonucleotides (AONs) are short, synthetic nucleic acid sequences that work by modulating exon incorporation at the level of pre-mRNA. In Duchenne muscular dystrophy (DMD), a fatal muscle degenerative disorder caused by mutations in the DMD gene, AONs skip specific exons to correct the reading frame, producing an internally shortened but partly functional dystrophin protein. Golodirsen is an approved AON phosphorodiamidate morpholino oligomer (PMO) that specifically targets DMD exon 53. In the clinical study 4053-101, we demonstrated that intravenous golodirsen administration induces an unequivocal exon skipping and protein restoration in all the treated patients, but with inter-patient variability. We used fibroblasts isolated from the patients in this clinical trial, that were induced to undergo myogenic differentiation in vitro by expression of MyoD, to better understand the reasons behind the observed variability. We evaluated the amount and the molecular weight of dystrophin protein in treated and non-treated patient cells, by an automated capillary-based immunoassay (WES) system. In these in-vitro studies we demonstrated that the amount of protein was comparable to the previous in-vivo study and that the size of the restored protein was compatible with the different genomic deletions carried by patients. Next, we used an in-situ RNA hybridization technique, BaseScope, to investigate the sub-cellular localization of the DMD transcript in treated and non-treated differentiated patient-derived myogenic cells in vitro, which allowed us to assess the ratio of skipped and unskipped products. Our study provides additional information on the dynamics of DMD mRNA in patients and may help to better understand the biological reasons underpinning variability in dystrophin restoration that can be seen in AON clinical trials.

Objective The degree to which sarcoidosis patients are affected by autoimmune diseases is poorly understood. Prior studies of autoimmune co-morbidities in sarcoidosis have focused on populations outside the USA or have been impeded by small sample sizes and limited scope. This case-control study evaluated the association between sarcoidosis and autoimmune diseases in a large, diverse cohort based in the USA. Methods We used data from the All of Us research programme to conduct a case-control study involving patients ≥18 years old, from 2018 to the present, diagnosed with sarcoidosis. Sarcoidosis cases and age-, sex- and race-matched controls were identified in a 1:4 ratio. Autoimmune co-morbidities were compared between sarcoidosis patients and controls in univariable and multivariable analyses using logistic regression. The degree of association was measured using the odds ratio (OR). Results A total of 1408 sarcoidosis cases and 5632 controls were included in this study. Seven of 24 examined autoimmune diseases were significantly associated with sarcoidosis in our multivariable analysis (P < 0.05). The composite variable of any autoimmune disease was also significantly associated with sarcoidosis (OR = 2.29, P < 0.001). Conclusion We demonstrate an association between sarcoidosis and multiple autoimmune diseases in a large and diverse cohort based in the USA. These results underscore the need for careful screening of sarcoidosis patients for concomitant autoimmune disease.

RESULTS: We identified highly abundant miR-BARTs in the 4 cell lines and the EBV-infected T- or NK-cells from 12 sCAEBV patient's PBMCs (age of 17 to 47 y.o). The expression of miR-BHRFs was not detected in these cells. miR-BART7-3p, miR-BART6-3p, and miR-BART5-5p were the top three expressed among the EBV-derived miRNAs. The highest miR-BART expression among all samples was miR-BART7-3p. We also confirmed the expression of miR-BART7-3p and miR-BART5-5p by _in situ_ hybridization in histological specimens of 3 patients which were observable. Two reports have demonstrated deletion in part of BART region in approximately 30% of sCAEBV cases (_Okuno et al, Nature Microbiology, 2018. Wongwiwat et al, J. Virology, 2022)_. In contrast, no deletion was detected in the region encoding miR-BARTs of EBV obtained from 10 sCAEBV patients. Finally, we examined the role of miR-BART7-3p in sCAEBV. Inhibition of miR-BART7-3p by the inhibitor did not show significant effects on cell proliferation in SNT16 and SNK10 cells. However, GO analysis showed upregulation of immune activation-related genes after miR-BART7-3p inhibition. These results suggest that miR-BART7-3p may function as an immunosuppressor in sCAEBV.

Inflammatory bowel disease (IBD) is a significant global health problem that involves chronic intestinal inflammation and can involve severe comorbidities, including intestinal fibrosis and inflammation-associated colorectal cancer (CRC). Disease-associated alterations to the intestinal microbiota often include fecal enrichment of Enterobacteriaceae, which are strongly implicated in IBD development. This dysbiosis of intestinal flora accompanies changes in microbial metabolites, shaping host:microbe interactions and disease risk. While there have been numerous studies linking specific bacterial taxa with IBD development, our understanding of microbial function in the context of IBD is limited. Several classes of microbial metabolites have been directly implicated in IBD disease progression, including bacterial siderophores and genotoxins. Yet, our microbiota still harbors thousands of uncharacterized microbial products. In-depth discovery and characterization of disease-associated microbial metabolites is necessary to target these products in IBD treatment strategies. Towards improving our understanding of microbiota metabolites in IBD, it is important to recognize how host relevant factors influence microbiota function. For example, changes in host inflammation status, metal availability, interbacterial community structure, and xenobiotics all play an important role in shaping gut microbial ecology. In this minireview, we outline how each of these factors influences gut microbial function, with a specific focus on IBD-associated Enterobacteriaceae metabolites. Importantly, we discuss how altering the intestinal microenvironment could improve the treatment of intestinal inflammation and associated disorders, like intestinal fibrosis and CRC.

The connexin gene family is the most prevalent gene that contributes to hearing loss. Connexins 26 and 30, encoded by GJB2 and GJB6, respectively, are the most abundantly expressed connexins in the inner ear. Connexin 43, which is encoded by GJA1, appears to be widely expressed in various organs, including the heart, skin, the brain, and the inner ear. The mutations that arise in GJB2, GJB6, and GJA1 can all result in comprehensive or non-comprehensive genetic deafness in newborns. As it is predicted that connexins include at least 20 isoforms in humans, the biosynthesis, structural composition, and degradation of connexins must be precisely regulated so that the gap junctions can properly operate. Certain mutations result in connexins possessing a faulty subcellular localization, failing to transport to the cell membrane and preventing gap junction formation, ultimately leading to connexin dysfunction and hearing loss. In this review, we provide a discussion of the transport models for connexin 43, connexins 30 and 26, mutations affecting trafficking pathways of these connexins, the existing controversies in the trafficking pathways of connexins, and the molecules involved in connexin trafficking and their functions. This review can contribute to a new way of understanding the etiological principles of connexin mutations and finding therapeutic strategies for hereditary deafness.

Maturation of the definitive adrenal cortex occurs between 3 and 6 weeks post-partum and involves onset of CYP11B2 expression and establishment of the laminin-encased 3D structure of glomeruli that contain rosettes of 10 to 15 zona glomerulosa (zG) cells that work in coordination to produce optimal amounts of aldosterone. Although this process is dependent on canonical WNT/b-catenin signaling, cellular sources of WNT ligands remain elusive and the mechanisms involved in the extensive extra-cellular matrix remodeling associated with rosette/glomeruli morphogenesis are unknown. Beyond their role in innate immunity, macrophages are involved in extra-cellular matrix remodeling under a wide variety of pathophysiological conditions and have the capacity to produce WNT ligands. This, together with the presence of macrophages within the zG cells, strongly suggest that macrophages may play a role in zG morphogenesis and differentiation. Supporting this idea, a recent publication has shown that intra-tissular aldosterone concentration was reduced in the absence of macrophages under stress conditions. However, whether macrophages play a direct role in controlling aldosterone secretion or an indirect role by remodelling the postnatal zG is unknown. The presence of tissue resident macrophages in a specific zone is dependent on the production of trophic factors such as IL34, CSF1, CSF2 or CX3CL1 by nearby, tissue resident ‘niche’ cells. In return, macrophages are thought to provide positive ‘feedback’ signals to their niche, generating mutually beneficial circuits between the niche and its macrophages. To gain insight into the role of macrophages in zG morphogenesis and homeostasis, we used single cell sequencing and RNAscope analyses to show expression of CX3CL1 in the zG and of CX3CR1 in macrophages. Interestingly, CX3CL1 expression in the zG was downstream of WNT signalling, suggesting existence of a bi-directional interaction between macrophages and zG. To further study the role of macrophages during the maturation of the zG, we pharmacologically depleted macrophages by the small molecule inhibitor Pexidartinib at different time points between 3- and 12-weeks post-partum, when maturation of the zG occurs. Short-term depletion of macrophages resulted in a more disorganized and elongated zG, suggesting a delay in maturation. However, long-term depletion of macrophages resulted in exacerbated maturation of the rosettes, suggesting that the short-term zG defect was followed by establishment of a compensatory mechanism to allow formation of rosettes even in the absence of macrophages. Whether these perturbations of the zG are correlated with an altered production of aldosterone is still under study.

GPR37 is an orphan GPCR and expressed in different brain regions. However, its biological function in pain regulation remains poorly understood. Recently, we identified Neuroprotectin D1 (NPD1) as a novel ligand of GPR37. NPD1 is a specialized pro-resolving mediator (SPM) and bio-synthesized from fish oil DHA (docosahexaenoic acid) . Here we reported a protective role of GRP37/NPD1 signaling in traumatic brain injury (TBI)-induced neuropathic pain. Mild TBI was induced by closed-head impact and the neuropathic pain was assessed by periorbital and cutaneous mechanical allodynia. In contrast to DHA (300 μg) showing no effects, peri-surgical treatment of NPD1 via intravenous injection (300 ng) effectively prevented TBI-induced locomotor deficiency and mechanical hypersensitivity in mice. Intraperitoneal post-treatment of NPD1 also significantly reduced established neuropathic pain in TBI mice. We also found that NPD1 treatment could inhibit TBI-induced neuroinflammation, characterized by microglia and astrocyte activation in the cortex and hippocampus. Furthermore, demyelination occurred after TBI, which was prevented by peri-surgical treatment of NPD1. RNAscope in situ hybridization revealed high Gpr37 mRNA expression in oligodendrocytes of the motor cortex and hippocampus . TBI resulted in a significant decrease in GPR37 expression, which could be restored by NPD1 treatment. GPR37 is protective and mice lacking Gpr37 (Gpr37-/-) exhibited prolonged pain symptoms after TBI. Furthermore, the protective effect of NPD1 was abolished in Gpr37-/- mice. All these findings suggest that activation of NPD1/GPR37 signaling pathway is a promising therapeutic strategy for preventing and treating neuropathic pain and its comorbidities after TBI. Funding: DoD grant W81XWH2110885.