Expression of FBXW11 in normal and disease-associated osteogenic cells.

The ubiquitin-proteasome system (UPS) plays an important role in maintaining cellular homeostasis by degrading a multitude of key regulatory proteins. FBXW11, also known as b-TrCP2, belongs to the F-box family, which targets the proteins to be degraded by UPS. Transcription factors or proteins associated with cell cycle can be modulated by FBXW11, which may stimulate or inhibit cellular proliferation. Although FBXW11 has been investigated in embryogenesis and cancer, its expression has not been evaluated in osteogenic cells. With the aim to explore FBXW11gene expression modulation in the osteogenic lineage we performed molecular investigations in mesenchymal stem cells (MSCs) and osteogenic cells in normal and pathological conditions. In vitro experiments as well as ex vivo investigations have been performed. In particular, we explored the FBXW11 expression in normal osteogenic cells as well as in cells of cleidocranial dysplasia (CCD) patients or osteosarcoma cells. Our data showed that FBXW11 expression is modulated during osteogenesis and overexpressed in circulating MSCs and in osteogenically stimulated cells of CCD patients. In addition, FBXW11 is post-transcriptionally regulated in osteosarcoma cells leading to increased levels of beta-catenin. In conclusion, our findings show the modulation of FBXW11 in osteogenic lineage and its dysregulation in impaired osteogenic cells.

Human Melanoma Cells Differentially Express RNASEL/RNase-L and miR-146a-5p under Sex Hormonal Stimulation.

Polymorphisms in the ribonuclease L (RNASEL) coding gene and hsa-miR-146a-5p (miR-146a) have been associated with melanoma in a sex-specific manner. We hypothesized that RNASEL and miR-146a expression could be influenced by sex hormones playing a role in the female advantages observed in melanoma incidence and survival. Thus, we explored the effects of testosterone and 17ß-estradiol on RNASEL and miR-146a expression in LM-20 and A375 melanoma cell lines. Direct targeting of miR-146a to the 3' untranslated region (3'UTR) of RNASEL was examined using a luciferase reporter system. Our results indicate that RNASEL is a direct target of miR-146a in both melanoma cell lines. Trough qPCR and western blot analyses, we explored the effect of miR-146a mimic transfection in the presence of each hormone either on RNASEL mRNA level or on protein expression of RNase-L, the enzyme codified by RNASEL gene. In the presence of testosterone or 17ß-estradiol, miR-146a overexpression did not influence RNASEL transcript level in LM-20 cell line, but it slightly induced RNASEL mRNA level in A375 cells. Remarkably, miR-146a overexpression was able to repress the protein level of RNase-L in both LM-20 and A375 cells in the presence of each hormone, as well as to elicit high expression levels of the activated form of the extracellular signal-regulated kinases (ERK)1/2, hence confirming the pro-tumorigenic role of miR-146a overexpression in melanoma. Thereafter, we assessed if the administration of each hormone could affect the endogenous expression of RNASEL and miR-146a genes in LM-20 and A375 cell lines. Testosterone exerted no significant effect on RNASEL gene expression in both cell lines, while 17ß-estradiol enhanced RNASEL transcript level at least in LM-20 melanoma cells. Conversely, miR-146a transcript augmented only in the presence of testosterone in either melanoma cell line. Importantly, each hormone acted quite the opposite regarding the RNase-L protein expression, i.e., testosterone significantly decreased RNase-L expression, whereas 17ß-estradiol increased it. Overall, the data show that, in melanoma cells treated with 17ß-estradiol, RNase-L expression increased likely by transcriptional induction of its gene. Testosterone, instead, decreased RNase-L expression in melanoma cell lines with a post-transcriptional mechanism in which miR-146a could play a role. In conclusion, the pro-tumor activity of androgen hormone in melanoma cells could be exacerbated by both miR-146a increase and RNase-L downregulation. These events may contribute to the worse outcome in male melanoma patients.

Upregulation of miR-34a-5p, miR-20a-3p and miR-29a-3p by Onconase in A375 Melanoma Cells Correlates with the Downregulation of Specific Onco-Proteins.

Onconase (ONC) is an amphibian secretory ribonuclease displaying cytostatic and cytotoxic activities against many mammalian tumors, including melanoma. ONC principally damages tRNA species, but also other non-coding RNAs, although its precise targets are not known. We investigated the ONC ability to modulate the expression of 16 onco-suppressor microRNAs (miRNAs) in the A375 BRAF-mutated melanoma cell line. RT-PCR and immunoblots were used to measure the expression levels of miRNAs and their regulated proteins, respectively. In silico study was carried out to verify the relations between miRNAs and their mRNA targets. A375 cell transfection with miR-20a-3p and miR-34a-5p mimics or inhibitors was performed. The onco-suppressors miR-20a-3p, miR-29a-3p and miR-34a-5p were highly expressed in 48-h ONC-treated A375 cells. The cytostatic effect of ONC in A375 cells was mechanistically explained by the sharp inhibition of cyclins D1 and A2 expression level, as well as by downregulation of retinoblastoma protein and cyclin-dependent-kinase-2 activities. Remarkably, the expression of kinases ERK1/2 and Akt, as well as of the hypoxia inducible factor-1α, was inhibited by ONC. All these proteins control pro-survival pathways. Finally, many crucial proteins involved in migration, invasion and metastatic potential were downregulated by ONC. Results obtained from transfection of miR-20a-3p and miR-34a-5p inhibitors in the presence of ONC show that these miRNAs may participate in the antitumor effects of ONC in the A375 cell line. In conclusion, we identified many intracellular downregulated proteins involved in melanoma cell proliferation, metabolism and progression. All mRNAs coding these proteins may be targets of miR-20a-3p, miR-29a-3p and/or miR-34a-5p, which are in turn upregulated by ONC. Data suggest that several known ONC anti-proliferative and anti-metastatic activities in A375 melanoma cells might depend on the upregulation of onco-suppressor miRNAs. Notably, miRNAs stability depends on the upstream regulation by long-non-coding-RNAs or circular-RNAs that can, in turn, be damaged by ONC ribonucleolytic activity.

3,5-Dicaffeoylquinic Acid Lowers 3T3-L1 Mitotic Clonal Expansion and Adipocyte Differentiation by Enhancing Heme Oxygenase-1 Expression.

Adipogenesis is a complex process in which cell commitment and mitotic clonal expansion (MCE) are in-sequence crucial events leading to terminal adipocyte differentiation. The molecules able to block some key signals in this cascade can hamper adipogenesis becoming promising agents to counteract hyperplasia and hypertrophy of adipose tissue. Mono- and di-caffeoylquinic acid isomers are biologically active polyphenols, displaying in vitro and in vivo antioxidant, hepatoprotective, anti-diabetic and anti-obesity properties. Among these isomers, 3,5-dicaffeoylquinic acid (DCQA) has been reported to inhibit lipid accumulation in adipose cells more successfully than others. Thus, we investigated DCQA effects and molecular mechanisms on 3T3-L1 pre-adipocytes induced to differentiate with a hormonal cocktail (MDI). Oil Red O incorporation assessed that DCQA pre-treatment inhibited lipid accumulation in 3T3-L1 cells induced to differentiate for 10 days. At this time, an increased phosphorylation of both AMP-activated kinase and acetyl-CoA carboxylase, as well as a strong decrease in fatty acid synthase protein level, were registered by immunoblotting, thereby suggesting that DCQA treatment can reduce fatty acid anabolism in 3T3-L1 adipocytes. Furthermore, BrdU incorporation assay, performed 48 h after hormonal stimulation, revealed that DCQA treatment was also able to hinder the 3T3-L1 cell proliferation during the MCE, which is an essential step in the adipogenic process. Thus, we focused our attention on early signals triggered by the differentiation stimuli. In the first hours after hormonal cocktail administration, the activation of ERK1/2 and Akt kinases, or CREB and STAT3 transcription factors, was not affected by DCQA pre-treatment. Whereas 24 h after MDI induction, DCQA pre-treated cells showed increased level of the transcription factor Nrf2, that induced the expression of the antioxidant enzyme heme oxygenase 1 (HO-1). In control samples, the expression level of HO-1 was reduced 24 h after MDI induction in comparison with the higher amount of HO-1 protein found at 2 h. The HO-1 decrease was functional by allowing reactive oxygen species to boost and allowing cell proliferation induction at the beginning of MCE phase. Instead, in DCQA-treated cells the HO-1 expression was maintained at high levels for a further 24 h; in fact, its expression decreased only 48 h after MDI stimulation. The longer period in which HO-1 expression remained high led to a delay of the MCE phase, with a subsequent inhibition of both C/EBP-α expression and adipocyte terminal differentiation. In conclusion, DCQA counteracting an excessive adipose tissue expansion may become an attractive option in obesity treatment.

Enhancer of zeste 2 polycomb repressive complex 2 subunit polymorphisms in melanoma skin cancer risk.

Melanoma is the most deadly skin cancer, and its incidence is growing. EZH2, a member of the Polycomb Group (PcGs) proteins family, plays an important biological role in the occurrence and development of melanoma. EZH2 germline genetic polymorphisms have not been yet evaluated in melanoma predisposition. Three hundred thirty sporadic Italian melanoma patients and 333 healthy volunteers were genotyped to analyse the association between EZH2 variants rs6950683, rs2302427, rs3757441, rs2072408 and melanoma risk. The functionality of rs6950683 alleles was investigated in keratinocytes (HaCat), melanoma cells (A375) and human embryonic kidney cells (HEK293), using promoter-reporter assays. Genotype distribution of SNPs showed that rs6950683T and rs3757441C alleles were positively associated with melanoma risk (P = .003 and .004, respectively). Haplotype analysis revealed that TCCA and CCCG haplotypes were associated with a higher risk of melanoma (P = .02 and .04, respectively). Functional assays demonstrated that allele rs6950683T reduce promoter activity in the three cell lines analysed compared to C allele. rs6950683T and rs3757441C alleles in the EZH2 gene appear positively associated with melanoma risk in the analysed population. In addition, we demonstrated for the first time the functional role of rs6950683 upstream polymorphism on EZH2 gene expression regulation.

miR-146a-5p impairs melanoma resistance to kinase inhibitors by targeting COX2 and regulating NFkB-mediated inflammatory mediators.

BACKGROUND: Targeted therapy with BRAF and MEK inhibitors has improved the survival of patients with BRAF-mutated metastatic melanoma, but most patients relapse upon the onset of drug resistance induced by mechanisms including genetic and epigenetic events. Among the epigenetic alterations, microRNA perturbation is associated with the development of kinase inhibitor resistance. Here, we identified and studied the role of miR-146a-5p dysregulation in melanoma drug resistance. METHODS: The miR-146a-5p-regulated NFkB signaling network was identified in drug-resistant cell lines and melanoma tumor samples by expression profiling and knock-in and knock-out studies. A bioinformatic data analysis identified COX2 as a central gene regulated by miR-146a-5p and NFkB. The effects of miR-146a-5p/COX2 manipulation were studied in vitro in cell lines and with 3D cultures of treatment-resistant tumor explants from patients progressing during therapy. RESULTS: miR-146a-5p expression was inversely correlated with drug sensitivity and COX2 expression and was reduced in BRAF and MEK inhibitor-resistant melanoma cells and tissues. Forced miR-146a-5p expression reduced COX2 activity and significantly increased drug sensitivity by hampering prosurvival NFkB signaling, leading to reduced proliferation and enhanced apoptosis. Similar effects were obtained by inhibiting COX2 by celecoxib, a clinically approved COX2 inhibitor. CONCLUSIONS: Deregulation of the miR-146a-5p/COX2 axis occurs in the development of melanoma resistance to targeted drugs in melanoma patients. This finding reveals novel targets for more effective combination treatment. Video Abstract.

Upregulated serum miR-128-3p in progressive and relapse-free multiple sclerosis patients.

BACKGROUND: Circulating microRNAs have emerged as novel multiple sclerosis (MS) biomarkers. AIMS: To assess the association between candidate miR expression in serum samples of patients with MS and the disease course. METHODS: Serum levels of ten microRNAs (ie, miR-199, miR-128-3p, miR-20a-5p, miR-27a-3p, miR-15b-5p, miR-325, miR-92a1-5p, miR-223-5p, miR-22-5p, and miR-23a-5p) were measured in 74 MS cases and 17 non-MS controls consecutively enrolled at Verona University Hospital. The association of microRNA expression with patients' clinical and MRI features was analyzed. Candidate microRNAs were detected by real-time PCR and expressed as ratio of each microRNA level to a normalizer. RESULTS: Serum miR-128-3p levels were higher in progressive than relapsing MS (median ratio 2.86 vs 0.73, P = .036). In addition, miR-128-3p was upregulated in patients without relapses after sample collection compared to cases who relapsed (1.64 vs 0.82; P = .014). miR-128-3p levels and relapse rate were inversely correlated (r = -.44, P = .008). CONCLUSIONS: Serum levels of mir-128-3p could be related to biological mechanisms underlying MS activity and progression.

Regulation of microRNAs in Satellite Cell Renewal, Muscle Function, Sarcopenia and the Role of Exercise.

Sarcopenia refers to a condition of progressive loss of skeletal muscle mass and function associated with a higher risk of falls and fractures in older adults. Musculoskeletal aging leads to reduced muscle mass and strength, affecting the quality of life in elderly people. In recent years, several studies contributed to improve the knowledge of the pathophysiological alterations that lead to skeletal muscle dysfunction; however, the molecular mechanisms underlying sarcopenia are still not fully understood. Muscle development and homeostasis require a fine gene expression modulation by mechanisms in which microRNAs (miRNAs) play a crucial role. miRNAs modulate key steps of skeletal myogenesis including satellite cells renewal, skeletal muscle plasticity, and regeneration. Here, we provide an overview of the general aspects of muscle regeneration and miRNAs role in skeletal mass homeostasis and plasticity with a special interest in their expression in sarcopenia and skeletal muscle adaptation to exercise in the elderly.

Correlations between gene expression highlight a different activation of ACE/TLR4/PTGS2 signaling in symptomatic and asymptomatic plaques in atherosclerotic patients.

Inflammation has a key role and translates the effects of many known risk factors for the disease in atherosclerotic vulnerable plaques. Aiming to look into the elements that induce the development of either a vulnerable or stable atherosclerotic plaque, and considering that inflammation has a central role in the progression of lesions, we analyzed the expression of genes involved in the ACE/TLR4/PTGS2 signaling in carotid plaques of symptomatic and asymptomatic patients. Patients with internal carotid artery stenosis undergoing carotid endarterectomy at Verona University Hospital were included in this study. A total of 71 patients was considered for gene expression analysis (29 atherothrombotic stroke patients and 42 asymptomatic patients). Total RNA was extracted from the excised plaques and expression of PTGS2, ACE, TLR4, PTGER4, PTGER3, EPRAP and ACSL4 genes was analyzed by real-time PCR. The correlation between the pair of genes was studied by Spearman coefficient. From the analyzed genes, we did not observe any individual difference in gene expression but the network of co-expressed genes suggests a different activation of pathways in the two groups of plaques.

Melanoma risk alleles are associated with downregulation of the MTAP gene and hypermethylation of a CpG island upstream of the gene in dermal fibroblasts.

Several association studies and GWAS on melanoma skin cancer risk have reported statistically significant signals on 9p21.3 region, where MTAP gene maps. None of the associated SNPs identified in these studies lie in the coding region of the gene and the causative relation of risk alleles with melanoma predisposition has not been elucidated. MTAP has a tumor suppressor activity and epigenetic silencing has been described in melanoma cell lines. In the present study, we show that melanoma risk alleles correlate with a MTAP allele-specific hyper-methylation and down-regulation of gene expression.

HDAC9, TWIST1 and FERD3L gene expression in asymptomatic stable and unstable carotid plaques.

BACKGROUND AND OBJECTIVES: A variant located at the end of HDAC9 gene within clusters of DNAse I sensitivity zones and histone modification hotspots has been associated with large vessel stroke and could be linked to plaque instability. The aim of the study is to define if an altered expression of HDAC9, TWIST1 and FERD3L genes could be involved in plaque vulnerability. METHODS: Histological classification and gene expression analysis were performed in 6 stable and 16 unstable plaques obtained from asymptomatic patients undergoing endarterectomy. Gene expression was analysed by real-time PCR. RESULTS AND CONCLUSIONS: TWIST1 gene expression resulted higher in stable plaques (P < 0.02). HDAC9 gene expression followed a similar trend (P = 0.11). These results highlighting the significant correlation between TWIST and HDAC9 gene expression suggest that both genes may contribute to plaque stability in a coordinated way.

Association of promoter polymorphism -765G>C in the PTGS2 gene with malignant melanoma in Italian patients and its correlation to gene expression in dermal fibroblasts.

Prostaglandins, especially prostaglandin E synthetase (PGE2), influence carcinogenesis by promoting cell proliferation, inhibiting apoptosis, stimulating angiogenesis and mediating immune suppression. Cyclooxygenase-2, coded by the PTGS2 gene, is the key enzyme in the production of prostaglandins. In melanoma, Cox-2 is over expressed in primary malignant melanoma (MM) and in their corresponding metastases. Polymorphisms in the promoter region of PTGS2 gene can modulate gene expression and could modify individual susceptibility to MM. Two hundred and forty melanoma patients and 342 controls were genotyped for polymorphisms -765G>C (rs20417) and -1195A>G (rs689466). Allele -765C frequency was significantly higher in melanoma patients. No allele frequency differences for -1195A>G polymorphism were observed. Haplotype analysis revealed that the haplotypes carrying the minor alleles were associated to a higher risk of melanoma (P = 0.02). Expression analysis showed that allele -765C is associated to a higher gene expression and could represent a risk allele by affecting the functionality of the promoter.

Cyclooxygenase 2, toll-like receptor 4 and interleukin 1ß mRNA expression in atherosclerotic plaques of type 2 diabetic patients.

OBJECTIVES AND DESIGN: Inflammation has a prominent role in the development of atherosclerosis. Type 2 diabetes could contribute to atherosclerosis development by promoting inflammation. This status might accelerate changes in intrinsic vascular wall cells and favor plaque formation. Cyclooxygenase 2 (COX-2) is highly expressed in atherosclerotic plaques. COX-2 gene expression is promoted through activation of toll-like receptor 4 (TLR4) and pro-inflammatory cytokine interleukin 1ß (IL1-ß). Aim of this study is to investigate whether expression profiles of pro-inflammatory genes such as COX-2, TLR4 and IL1-ß in atherosclerotic plaques are altered in type 2 diabetes (T2D). METHODS: Total RNA was isolated from plaques of atherosclerotic patients and expression of COX-2, TLR4, IL1-ß analyzed using real-time PCR. Histological analysis was performed on sections of the plaque to establish the degree of instability. RESULTS: Statistically significant differences in mRNA expression of COX-2 and IL1-ß were found in plaques of T2D compared with non-T2D patients. A multi-variable linear regression model suggests that COX-2 mRNA expression is affected by T2D pathology and IL1-ß mRNA expression in atherosclerotic plaques. CONCLUSIONS: Our results support the hypothesis that T2D pathology contributes in vivo to increase the inflammatory process associated with the atherosclerotic plaque formation, as shown by an increment of COX-2 and IL1-ß mRNA expression.

Sex-dependent interaction of PTGS2 with miR-146a as risk factor for melanoma and the impact of sex hormones in gene expression in skin cells.

Gender disparity in melanoma is a complex issue where sex hormones could be engaged. Differences in genetic variations are important in understanding the mechanisms of sex disparity in melanoma. Post-transcriptional regulation of prostaglandin-endoperoxide synthase (PTGS2) mRNA occurs through a complex interplay of specific trans-acting RNA-binding proteins and microRNAs. MiR-146a is a key player in melanoma, modulating immune responses and tumor microenvironment (TME). Polymorphisms in PTGS2 gene rs20415GC have been associated with an increased risk of melanoma. Epistasis between polymorphisms rs20415GC was investigated by genotyping 453 melanoma patients and 382 control individuals. The effects of testosterone and 17ß-estradiol were analyzed in keratinocytes and two melanoma cell lines. The rs2910164GG showed a higher risk in the presence of the genotype rs20417CC in the male population. Testosterone and 17ß-estradiol act differently on PTGS2 and miR-146a expression, depending on the cell type. Testosterone augments PTGS2 gene expression in keratinocytes and miR-146a in melanoma cells. While 17ß-estradiol only increases miR-146a expression in HaCaT cells. The present study indicates a sex-specific relation between miR-146a and PTGS2 polymorphisms with melanoma cancer risk. Testosterone and 17ß-estradiol act differently on the expression of PTGS2 and miR-146a depending on the skin cell type.

Polymorphism -2604G>A variants in TLR4 promoter are associated with different gene expression level in peripheral blood of atherosclerotic patients.

Toll-like receptor-4 (TLR4) is a primary receptor of the innate immune reaction and compelling evidence demonstrates its involvement in the pathogenesis of atherosclerosis and stroke. TLR4 is constitutively expressed on monocytes and endothelial cells; it is highly expressed in atherosclerotic plaques and in peripheral blood of patients after ischemic stroke. Polymorphisms in the promoter region that alter the transcriptional regulation of this gene may represent genetic risk factors involved in the predisposition to atherosclerotic disease. In this study we investigated the effect on TLR4 gene expression of three polymorphisms in the upstream regulatory region at positions -1607T>C/rs10759932, -2026A>G/rs1927914 and -2604G>A/rs10759931 in peripheral blood of atherosclerotic patients. RNA from individuals homozygous for the -2604A allele showed a lower expression of the gene when compared to patients carrying the counterparts GG+GA. Electrophoretic mobility shift assays showed differences in the electrophoretic mobility of the DNA-nuclear protein complexes formed by the G>A variants, suggesting that the two alleles differ in their binding affinity to transcriptional factors.

Common CFTR haplotypes and susceptibility to chronic pancreatitis and congenital bilateral absence of the vas deferens.

CFTR mutations enhance susceptibility for idiopathic chronic pancreatitis (ICP) and congenital bilateral absence of the vas deferens (CBAVD); however, it is unknown why CFTR heterozygotes are at increased disease risk. We recently showed that common CFTR variants are associated with aberrantly spliced transcripts. Here, we genotyped for common CFTR variants and tested for associations in two ICP (ICP-A: 126 patients, 319 controls; ICP-B: 666 patients, 1,181 controls) and a CBAVD population (305 patients, 319 controls). Haplotype H10 (TG11-T7-470V) conferred protection (ICP-A: OR 0.19, P<0.0001; ICP-B: OR 0.78, P = 0.06; CBAVD OR 0.08, P<0.001), whereas haplotype H3 (TG10-T7-470M) increased disease risk (ICP-A: OR 8.34, P = 0.003; ICP-B: OR 1.88, P = 0.007; CBAVD: OR 5.67, P = 0.01). The risk of heterozygous CFTR mutations carriers for ICP (OR 2.44, P<0.001) and CBAVD (OR 14.73, P<0.001) was fully abrogated by the H10/H10 genotype. Similarly, ICP risk of heterozygous p.Asn34Ser SPINK1 mutation carriers (OR 10.34, P<0.001) was compensated by H10/H10. Thus, common CFTR haplotypes modulate ICP and CBAVD susceptibility alone and in heterozygous CFTR and p.Asn34Ser mutation carriers. Determination of these haplotypes helps to stratify carriers into high- and low-risk subjects, providing helpful information for genetic counseling.

Analysis of the 3'UTR of the prostaglandin synthetase-2 (PTGS-2/COX-2) gene in non-melanoma skin cancer after organ transplantation.

To define the potential involvement of polymorphisms in the 3'untranslated region (3'UTR) of the prostaglandin synthetase-2 (PTGS-2) gene to non-melanoma skin cancer (NMSC) predisposition after transplantation, we screened for genetic variant, relevant parts of this region. It contains binding sites for trans-acting factors, an alternative polyadenylation site and putative target sequences for miRNAs. Variant +8473T>C did not appear to play a functional role in the regulation of gene expression in human keratinocyte-transfected cells. In addition to the well-known +8473T>C, we identified four polymorphisms: +8293G>C, +10259T>G, +10267G>A and +10335G>A. No allele frequency differences were observed between cases and controls neither for +8473T>C nor for any of the identified polymorphisms, suggesting that polymorphisms in the 3'UTR of the PTGS2 gene are rare and unlikely to represent risk factor for NMSC after transplantation.

Rehabilitation and Biomarkers of Stroke Recovery: Study Protocol for a Randomized Controlled Trial.

Background: Stroke is a leading cause of disability. Nonetheless, the care pathway for stroke rehabilitation takes partially into account the needs of chronic patients. This is due in part to the lack of evidence about the mechanisms of recovery after stroke, together with the poor knowledge of related and influencing factors. Here we report on the study protocol "Rehabilitation and Biomarkers of Stroke Recovery," which consists of 7 work-packages and mainly aim to investigate the effects of long-term neurorehabilitation on stroke patients and to define a related profile of (clinical-biological, imaging, neurophysiological, and genetic-molecular) biomarkers of long-term recovery after stroke. The work-package 1 will represent the main part of this protocol and aims to compare the long-term effects of intensive self-rehabilitation vs. usual (rehabilitation) care for stroke. Methods: We planned to include a total of 134 adult subacute stroke patients (no more than 3 months since onset) suffering from multidomain disability as a consequence of first-ever unilateral ischemic stroke. Eligible participants will be randomly assigned to one of the following groups: intensive self-rehabilitation (based on the principles of "Guided Self-Rehabilitation Contract") vs. usual care (routine practice). Treatment will last 1 year, and patients will be evaluated every 3 months according to their clinical presentation. The following outcomes will be considered in the main work-package: Fugl-Meyer assessment, Cognitive Oxford Screen Barthel Index, structural and functional neuroimaging, cortical excitability, and motor and somatosensory evoked potentials. Discussion: This trial will deal with the effects of an intensive self-management rehabilitation protocol and a related set of biomarkers. It will also investigate the role of training intensity on long-term recovery after stroke. In addition, it will define a set of biomarkers related to post-stroke recovery and neurorehabilitation outcome in order to detect patients with greater potential and define long-term individualized rehabilitation programs. Clinical Trial Registration: www.ClinicalTrials.gov, identifier: NCT04323501.

Primary IgA nephropathy is more severe in TGF-beta1 high secretor patients.

BACKGROUND: IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide and is characterized by extremely variable clinical and morphological features and outcome. TGF-beta1 has a key role in fibrogenesis and the progression of renal damage. Its production is under genetic control. METHODS: We recruited 105 Italian biopsy-proven IgAN patients for genotyping for the TGF-beta1 C-509T, T869C (COD 10) and G915C (COD 25) polymorphisms; 200 healthy blood donors were used as normal controls. Glomerular and interstitial mRNA levels of TGF-beta1 were assessed by real-time PCR in 34 patients to seek relationships with clinical, renal histopathological features and outcome. RESULTS: The genotype distributions in the IgAN population were not statistically different from the controls. The COD 10 TT genotype was associated with more severe histological damage as assessed by Lee's classification (CC 50%, CT 39.6% and TT 17.2% were graded as mild; CC 35.7%, CT 43.7% and TT 44.8% as moderate, and CC 14.3%, CT 16.7% and TT 37.9% as severe [p=0.0049]) and with severe interstitial infiltrates (CC 10.4%, CT 35.2% and TT 54.2% [p=0.03]). A higher interstitial immunodeposition was observed for TGF-beta1, collagen IV and alpha-SMA in patients with the COD 10 T allele (p=0.045, p=0.049, p=0.032, respectively). The T allele was associated with significantly higher TGF-beta1 mRNA levels in the interstitium (TT+CT vs. CC: 0.52 +/- 0.16 vs. 0.18 +/- 0.10 copies/mL, respectively; p=0.000). The T allele was also associated with higher mRNA levels in glomeruli, though the difference was not statistically significant. Finally, the T allele was significantly associated with a worse prognosis, the end points being reached by 40% of TT+CT and 32% of CC patients (p=0.009). CONCLUSIONS: In primary IgA nephropathy, the T allele of the TGF-beta1 COD 10 C/T polymorphism seems to be associated with more severe histological lesions, higher renal TGF-beta1 mRNA levels and a worse prognosis. This polymorphism seems to be functionally relevant and to have a prognostic impact.