The molecular events associated with acute and chronic exposure of mesangial cells (MC) to hyperglycemia were evaluated. We found that, unlike high glucose (HG) and Amadori adducts, advanced glycation end products (AGE) and transforming growth factor-beta (TGF-beta) induced p21waf expression and accumulation of MC in G0/G1. TGF-beta1 blockade inhibited AGE-mediated collagen production but only partially affected AGE-induced p21waf expression and cell-cycle events, indicating that AGE by binding to AGE receptor (RAGE) per se could control MC growth. Moreover, AGE and TGF-beta treatment led to the activation of the signal transduction and activators of transcription (STAT)5 and the formation of a STAT5/p21SIE2 complex. The role of STAT5 in AGE- and TGF-beta-mediated p21waf expression and growth arrest, but not collagen production, was confirmed by the expression of the dominant negative STAT5 (DeltaSTAT5) or the constitutively activated STAT5 (1*6-STAT5) constructs. Finally, in p21waf-/- fibroblasts both AGE and TGF-beta failed to inhibit cell-cycle progression. A potential in vivo role of these mechanisms was sustained by the increasing immunoreactivity for the activated STAT5 and p21(waf) in kidney biopsies from early to advanced stage of diabetic nephropathy. Our data indicate that AGE- and TGF-beta-mediated signals, by converging on STAT5 activation and p21waf expression, may regulate MC growth.

Interleukin-3 (IL-3) expression by tumor-infiltrating lymphocytes (TILs) and its effects on vessel assembly were evaluated. TILs from 'in situ' human breast cancers expressed CD4/CD25 antigens and IL-3. An injection of Matrigel containing SMC and IL-3 or basic-fibroblast growth factor (bFGF) into SCID mice confirmed the neoangiogenetic effect of both factors. However, in response to IL-3, but not to bFGF, only few SMC became incorporated into the nascent vessels. To evaluate the possibility that signals emanated by the nascent vasculature in the presence of IL-3 may negatively regulate SMC recruitment, conditioned media (CM) from IL-3-treated endothelial cells (EC) or SMC were tested for their biological effects on SMC and EC. CM from IL-3-treated SMC stimulated the migration of EC. In contrast, the migration of SMC was not affected by CM from IL-3-stimulated EC; however, it was greatly enhanced by blocking transforming growth factor b (TGFb) activity. TGFb immunoenzymatic assay demonstrated the following: (i) the absence of TGFb activity in CM from IL-3-stimulated EC; (ii) a barely detectable TGFb activity in CM from IL-3-stimulated SMC; and (iii) the presence of TGFb activity in the supernatants of SMC stimulated with CM from IL-3-, but not from bFGF-stimulated EC. Increased TGFb mRNA expression was only detected in SMC stimulated with CM from IL-3-treated EC. Finally, the inhibitory signals induced by IL-3 in vivo were abrogated by the addition of the neutralizing TGFb antibody. Thus, the positive immunostaining for IL-3 by TILs in 'in situ' breast cancers sustains the possibility that early in tumor development, IL-3 can contribute to the chronic immaturity of these vessels.

Children with the new coronavirus disease 2019 (COVID-19) have milder symptoms and a better prognosis than adult patients. Several investigations assessed type I, II, and III interferon (IFN) signatures in SARS-CoV-2 infected adults, however no data are available for pediatric patients. RIM28 and SETDB1 regulate the transcription of multiple genes involved in the immune response as well as of human endogenous retroviruses (HERVs). Exogenous viral infections can trigger the activation of HERVs, which in turn can induce inflammatory and immune reactions. Despite the potential cross-talks between SARS-CoV-2 infection and TRIM28, SETDB1, and HERVs, information on their expressions in COVID-19 patients is lacking. We assessed, through a PCR real time Taqman amplification assay, the transcription levels of six IFN-I stimulated genes, IFN-II and three of its sensitive genes, three IFN-lIIs, as well as of TRIM28, SETDB1, pol genes of HERV-H, -K, and -W families, and of env genes of Syncytin (SYN)1, SYN2, and multiple sclerosis-associated retrovirus (MRSV) in peripheral blood from COVID-19 children nd in control uninfected subjects. Higher expression levels of IFN-I and IFN-II inducible genes were observed in 36 COVID-19-infected children with mild or moderate disease as compared to uninfected controls, whereas their concentrations decreased in 17 children with severe disease and in 11 with multisystem inflammatory syndrome (MIS-C). Similar findings were found for the expression of TRIM-28, SETDB1, and every HERV gene. Positive correlations emerged between the transcriptional levels of type I and II IFNs, TRIM28, SETDB1, and HERVs in COVID-19 patients. IFN-III expressions were comparable in each group of subjects. This preserved induction of IFN-λs could contribute to the better control of the infection in children as compared to adults, in whom IFN-III deficiency has been reported. The upregulation of IFN-I, IFN-II, TRIM28, SETDB1, and HERVs in children with mild symptoms, their declines in severe cases or with MIS-C, and the positive correlations of their transcription in SARS-CoV-2-infected children suggest that they may play important roles in conditioning the evolution of the infection.

We previously demonstrated that integrin-dependent adhesion activates STAT5A, a well known target of IL-3–mediated signaling. Here, we show that in endothelial cells the active β1 integrin constitutively associates with the unphosphorylated IL-3 receptor (IL-3R) β common subunit. This association is not sufficient for activating downstream signals. Indeed, only upon fibronectin adhesion is Janus Kinase 2 (JAK2) recruited to the β1 integrin–IL-3R complex and triggers IL-3R β common phosphorylation, leading to the formation of docking sites for activated STAT5A. These events are IL-3 independent but require the integrity of the IL-3R β common. IL-3 treatment increases JAK2 activation and STAT5A and STAT5B tyrosine and serine phosphorylation and leads to cell cycle progression in adherent cells. Expression of an inactive STAT5A inhibits cell cycle progression upon IL-3 treatment, identifying integrin-dependent STAT5A activation as a priming event for IL-3–mediated S phase entry. Consistently, overexpression of a constitutive active STAT5A leads to anchorage-independent cell cycle progression. Therefore, these data provide strong evidence that integrin-dependent STAT5A activation controls IL-3–mediated proliferation.

Chronic hepatitis C virus (HCV) infection is associated with several hepatic and extrahepatic complications, including cancers and autoimmune disorders, whose frequency is reduced but not abolished after drug-induced viral clearance. The causes of these complications and of their persistence are ill-defined. Human endogenous retroviruses (HERVs) are remnants of ancestral infections and constitute 8% of the human genome. Most HERV elements are inactive, but some are transcribed. HERV overexpression is associated with many cancers and autoimmune diseases with a putative pathogenetic role. Several viral infections trigger HERV activation, but there are no studies on HCV-infected subjects. We assessed, through a PCR real-time amplification assay, the transcription levels of the pol genes of HERV-H, -K, and -W, and of their repressor TRIM28 in white blood cells (WBCs) of vertically infected children, both before and after therapy with direct-acting antivirals (DAAs). The results documented significantly higher expressions of HERV-H-pol and HERV-K-pol, not of HERV-W-pol, in HCV-infected subjects as compared to age-matched controls. HERV RNA levels remained unchanged after DAA-driven viral clearance. No significant variations in transcription levels of TRIM28 were observed in infected subjects. Our findings demonstrate HERV-H-pol and HERV-K-pol overexpression in subjects with chronic HCV infection, without variations after a positive response to DAAs; this might justify their predisposition to cancers and autoimmune disorders that persist after a DAA-induced resolution of viremia.

Aim: Studies have shown that Lactobacilli reute ri probiotics can affect cells that play a key role in the immune system. This in vivo Italian study investigated how Lactobacillus reuteri DSM 17938 influenced CC-chemokine receptor 7 (CCR7) and interleukin 10 (IL-10) in breastfed colicky infants. Methods: Our University hospital in Turin recruited 50 healthy outpatients, at a median age of approximately 1 month, from September 2017 to August 2018. They were randomized to daily Lactobacillus reuteri DSM17938 (1 × 10 8 cfu) or a placebo for 28 days from recruitment. We collected peripheral blood and evaluated the expression of CCR7 messenger ribonucleic acid using the real-time TaqMan reverse transcription polymerase chain reaction method at baseline and after the study period. Results: We found increased expression of CC-chemokine receptor 7 in infants treated with the probiotic, but not the controls ( p < 0.0026). No differences were observed for interleukin 10 after the study period in either group. At baseline, daily crying time was comparable in the probiotic and control groups: 341 (25) vs. 337 (29) min., respectively ( p = 0.450). After 28 days, daily mean crying time decrease statistically in the probiotic group: 78 (23) vs. 232 (31), respectively ( p < 0.001). Conclusion: The increase in CC-chemokine receptor 7 might have been a response to probiotic treatment. As a relatively small sample was used to conduct this study, our research needs to be replicated in different settings, and over time, to produce comparable findings.

No abstract

Objective-To characterize the molecules and the mechanisms regulating the neoangiogenetic process in advanced atherosclerotic plaques. Methods and Results-Western blot and immunofluorescence analysis of atherosclerotic specimens demonstrated that unlike neovessels from early lesions that expressed vascular endothelial growth factor (VEGF) and angiopoietin1 (Angio1), vessels from advanced lesions expressed VEGF and angiopoietin 2 (Angio2). Moreover, only few neovessels from advanced lesions showed a positive immunostaining for proliferating cell nuclear antigen. Angio1-elicited and Angio2-elicited intracellular events in endothelial cells (EC) demonstrated that while Angio1 triggered Erk1/Erk2 mitogen activated protein kinases (MAPK) and Akt activation, Angio2 (50 ng/mL) induced STAT5 activation and p21 waf expression and increased the fraction of cells in G1. Both Angio2-mediated events were abrogated by expressing a dominant negative STAT5 construct (⌬STAT5). Consistent with the expression of Angio2 in neovessels of advanced lesions a transcriptionally active STAT5 was detected. Moreover, co-immunoprecipitation experiments revealed the presence of a STAT5/Tie2 molecular complex in neointima vessels from advanced, but not from early, lesions. 3 Consistently, in vivo developing primary lesions might be considered as underperfused and dependent on neovessel formation for continued growth. 4 Angiogenesis in mature tissues is composed of a series of cellular responses including activation of EC proliferation by angiogenic factors, such as VEGF, fibroblast growth factor (FGF), and angiopoietins (Angio) and formation of tube-like structures and vessel stabilization. 5 Compared with the developmental angiogenesis, the role of angiopoietins in pathological angiogenesis is less understood. The angiopoietin family consists of four members, Angio1 to 4. 6 -8 Angio1 and 4 stimulate their receptor tyrosine kinase Tie2, whereas Angio2 and 3 by binding to Tie2 antagonize Angio1 signaling. 6 -8 Signal transduction pathways via Tie2 and VEGF receptors (VEGFR) have been extensively examined. 9,10 In particular, Korpelainem et al showed that Tie2 and VEGFR1 overexpression triggers the phosphorylation of the signal transducer and activator of transcription (STAT) 3 and STAT5. 11 STAT5 belongs to the family of transcriptional factors, which, on activation, binds to the receptor via their src homology 2 domain (SH2). 12 Recruited STATs, dimerize, translocate to the nucleus, and activate transcription of target genes mainly involved in regulation of cell cycle progression. 12,13 Cell cycle phases are coordinated by regulatory proteins denoted as cyclins and cyclin-dependent kinases (CDKs). 14 Phase-specific cyclin/CDK complexes confer specificity and orderly progression through the cell cycle. Initially, cyclin D/CDK4 or cyclin E/CDK2 complexes, in cooperation with proliferating cell nuclear antigen (PCNA), coordinate DNA duplication by regulating the transition through the G1 and S phase. 15 Furthermore, cell cycle progression is ...