What Is Hidden in Patients with Unknown Nephropathy? Genetic Screening Could Be the Missing Link in Kidney Transplantation Diagnosis and Management.

Between 15-20% of patients with end stage renal disease (ESRD) do not know the cause of the primary kidney disease and can develop complications after kidney transplantation. We performed a genetic screening in 300 patients with kidney transplantation, or undiagnosed primary renal disease, in order to identify the primary disease cause and discriminate between overlapping phenotypes. We used a custom-made panel for next-generation sequencing (Agilent technology, Santa Clara, CA, USA), including genes associated with Fabry disease, podocytopaties, complement-mediated nephropathies and Alport syndrome-related diseases. We detected candidate diagnostic variants in genes associated with nephrotic syndrome and Focal Segmental Glomerulosclerosis (FSGS) in 29 out of 300 patients, solving about 10% of the probands. We also identified the same genetic cause of the disease (PAX2: c.1266dupC) in three family members with different clinical diagnoses. Interestingly we also found one female patient carrying a novel missense variant, c.1259C>A (p.Thr420Lys), in the GLA gene not previously associated with Fabry disease, which is in silico defined as a likely pathogenic and destabilizing, and associated with a mild alteration in GLA enzymatic activity. The identification of the specific genetic background may provide an opportunity to evaluate the risk of recurrence of the primary disease, especially among patient candidates living with a donor kidney transplant.

The Long Non-coding RNA HOTAIR Controls the Self-renewal, Cell Senescence, and Secretion of Anti-aging Protein α-Klotho in Human Adult Renal Progenitor Cells.

The long non-coding RNAs (lncRNA) play an important role in several biological processes, including some renal diseases. Nevertheless, little is known about lncRNA that are expressed in the healthy kidneys and involved in renal cell homeostasis and development, and even less is known about lncRNA involved in the maintenance of human adult renal stem/progenitor cells (ARPCs) that have been shown to be very important for renal homeostasis and repair processes. Through a whole-genome transcriptome screening, we found that the HOTAIR lncRNA is highly expressed in renal progenitors and potentially involved in cell cycle and senescence biological processes. By CRISPR/Cas9 genome editing, we generated HOTAIR knockout ARPC lines and established a key role of this lncRNA in ARPC self-renewal properties by sustaining their proliferative capacity and limiting the apoptotic process. Intriguingly, the HOTAIR knockout led to the ARPC senescence and to a significant decrease in the CD133 stem cell marker expression which is an inverse marker of ARPC senescence and can regulate renal tubular repair after the damage. Furthermore, we found that ARPCs expressed high levels of the α-Klotho anti-aging protein and especially 2.6-fold higher levels compared to that secreted by renal proximal tubular cells (RPTECs). Finally, we showed that HOTAIR exerts its function through the epigenetic silencing of the cell cycle inhibitor p15 inducing the trimethylation of the histone H3K27. Altogether, these results shed new light on the mechanisms of regulation of these important renal cells and may support the future development of precision therapies for kidney diseases.

High levels of gut-homing immunoglobulin A+ B lymphocytes support the pathogenic role of intestinal mucosal hyperresponsiveness in immunoglobulin A nephropathy patients.

BACKGROUND: Immunoglobulin A nephropathy (IgAN) is the most frequent primary glomerulonephritis. The role of the microbiota and mucosal immunity in the pathogenesis of IgAN remains a key element. To date, the hypothetical relationship between commensal bacteria, elevated tumour necrosis factor (TNF) superfamily member 13 [also known as B-cell activating factor (BAFF)] levels, perturbed homoeostasis of intestinal-activated B cells and intestinal IgA class switch has not been clearly shown in IgAN patients. METHODS: We studied the intestinal-renal axis connections, analysing levels of BAFF, TNF ligand superfamily member 13 (APRIL) and intestinal-activated B cells in IgAN patients, healthy subjects (HSs) and patients with non-IgA glomerulonephritides. RESULTS: IgAN patients had increased serum levels of BAFF cytokine, correlating with higher amounts of five specific microbiota metabolites, and high APRIL cytokine serum levels. We also found that subjects with IgAN have a higher level of circulating gut-homing (CCR9+ ß7 integrin+) regultory B cells, memory B cells and IgA+ memory B cells compared with HSs. Finally, we found that IgAN patients had high levels of both total plasmablasts (PBs) and intestinal-homing PBs. Interestingly, PBs significantly increased in IgAN but not in patients with other glomerulonephritides. CONCLUSIONS: Our results demonstrate a significant difference in the amount of intestinal-activated B lymphocytes between IgAN patients and HSs, confirming the hypothesis of the pathogenic role of intestinal mucosal hyperresponsiveness in IgAN. The intestinal-renal axis plays a crucial role in IgAN and several factors may contribute to its complex pathogenesis and provide an important area of research for novel targeted therapies to modulate progression of the disease.

Inhibition of Lysine 63 Ubiquitination Prevents the Progression of Renal Fibrosis in Diabetic DBA/2J Mice.

Diabetic nephropathy (DN) is the most frequent cause of end-stage renal disease. Tubulointerstitial accumulation of lysine 63 (K63)-ubiquitinated (Ub) proteins is involved in the progression of DN fibrosis and correlates with urinary miR-27b-3p downregulation. We explored the renoprotective effect of an inhibitor of K63-Ub (NSC697923), alone or in combination with the ACE-inhibitor ramipril, in vitro and in vivo. Proximal tubular epithelial cells and diabetic DBA/2J mice were treated with NSC697923 and/or ramipril. K63-Ub protein accumulation along with α-SMA, collagen I and III, FSP-1, vimentin, p16INK4A expression, SA-α Gal staining, Sirius Red, and PAS staining were measured. Finally, we measured the urinary albumin to creatinine ratio (uACR), and urinary miR-27b-3p expression in mice. NSC697923, both alone and in association with ramipril, in vitro and in vivo inhibited hyperglycemia-induced epithelial to mesenchymal transition by significantly reducing K63-Ub proteins, α-SMA, collagen I, vimentin, FSP-1 expression, and collagen III along with tubulointerstitial and glomerular fibrosis. Treated mice also showed recovery of urinary miR-27b-3p and restored expression of p16INK4A. Moreover, NSC697923 in combination with ramipril demonstrated a trend in the reduction of uACR. In conclusion, we suggest that selective inhibition of K63-Ub, when combined with the conventional treatment with ACE inhibitors, might represent a novel treatment strategy to prevent the progression of fibrosis and proteinuria in diabetic nephropathy and we propose miR-27b-3p as a biomarker of treatment efficacy.

Renal progenitor cells revert LPS-induced endothelial-to-mesenchymal transition by secreting CXCL6, SAA4, and BPIFA2 antiseptic peptides.

Endothelial dysfunction is a hallmark of LPS-induced acute kidney injury (AKI). Endothelial cells (ECs) acquired a fibroblast-like phenotype and contributed to myofibroblast generation through the endothelial-to-mesenchymal transition (EndMT) process. Of note, human adult renal stem/progenitor cells (ARPCs) enhance the tubular regenerative mechanism during AKI but little is known about their effects on ECs. Following LPS exposure, ECs proliferated, decreased EC markers CD31 and vascular endothelial cadherin, and up-regulated myofibroblast markers, collagen I, and vimentin. The coculture with ARPCs normalized the EC proliferation rate and abrogated the LPS-induced EndMT. The gene expression analysis showed that most of the genes modulated in LPS-stimulated ARPCs belong to cell activation and defense response pathways. We showed that the ARPC-specific antifibrotic effect is exerted by the secretion of CXCL6, SAA4, and BPIFA2 produced after the anaphylatoxin stimulation. Next, we investigated the molecular signaling that underlies the ARPC protective mechanism and found that renal progenitors diverge from differentiated tubular cells and ECs in myeloid differentiation primary response 88-independent pathway activation. Finally, in a swine model of LPS-induced AKI, we observed that activated ARPCs secreted CXCL6, SAA4, and BPIFA2 as a defense response. These data open new perspectives on the treatment of both sepsis- and endotoxemia-induced AKI, suggesting an underestimated role of ARPCs in preventing endothelial dysfunction and novel strategies to protect the endothelial compartment and promote kidney repair.-Sallustio, F., Stasi, A., Curci, C., Divella, C., Picerno, A., Franzin, R., De Palma, G., Rutigliano, M., Lucarelli, G., Battaglia, M., Staffieri, F., Crovace, A., Pertosa, G. B., Castellano, G., Gallone, A., Gesualdo, L. Renal progenitor cells revert LPS-induced endothelial-to-mesenchymal transition by secreting CXCL6, SAA4, and BPIFA2 antiseptic peptides.

Bone marrow fibroblasts overexpress miR-27b and miR-214 in step with multiple myeloma progression, dependent on tumour cell-derived exosomes.

Aberrant microRNA (miR) expression has an important role in tumour progression, but its involvement in bone marrow fibroblasts of multiple myeloma patients remains undefined. We demonstrate that a specific miR profile in bone marrow fibroblasts parallels the transition from monoclonal gammopathy of undetermined significance (MGUS) to myeloma. Overexpression of miR-27b-3p and miR-214-3p triggers proliferation and apoptosis resistance in myeloma fibroblasts via the FBXW7 and PTEN/AKT/GSK3 pathways, respectively. Transient transfection of miR-27b-3p and miR-214-3p inhibitors demonstrates a cooperation between these two miRNAs in the expression of the anti-apoptotic factor MCL1, suggesting that miR-27b-3p and miR-214-3p negatively regulate myeloma fibroblast apoptosis. Furthermore, myeloma cells modulate miR-27b-3p and miR-214-3p expression in fibroblasts through the release of exosomes. Indeed, tumour cell-derived exosomes induce an overexpression of both miRNAs in MGUS fibroblasts not through a simple transfer mechanism but by de novo synthesis triggered by the transfer of exosomal WWC2 protein that regulates the Hippo pathway. Increased levels of miR-27b-3p and miR-214-3p in MGUS fibroblasts co-cultured with myeloma cell-derived exosomes enhance the expression of fibroblast activation markers αSMA and FAP. These data show that the MGUS-to-myeloma transition entails an aberrant miRNA profile in marrow fibroblasts and highlight a key role of myeloma cells in modifying the bone marrow microenvironment by reprogramming the marrow fibroblasts' behaviour. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Altered Phosphorylation of Cytoskeleton Proteins in Peripheral Blood Mononuclear Cells Characterizes Chronic Antibody-Mediated Rejection in Kidney Transplantation.

Chronic antibody-mediated rejection (CAMR) is the major cause of kidney transplant failure. The molecular mechanisms underlying this event are still poorly defined and this lack of knowledge deeply influences the potential therapeutic strategies. The aim of our study was to analyze the phosphoproteome of peripheral blood mononuclear cells (PBMCs), to identify cellular signaling networks differentially activated in CAMR. Phosphoproteins isolated from PBMCs of biopsy proven CAMR, kidney transplant recipients with normal graft function and histology and healthy immunocompetent individuals, have been investigated by proteomic analysis. Phosphoproteomic results were confirmed by Western blot and PBMCs' confocal microscopy analyses. Overall, 38 PBMCs samples were analyzed. A differential analysis of PBMCs' phosphoproteomes revealed an increase of lactotransferrin, actin-related protein 2 (ARPC2) and calgranulin-B in antibody-mediated rejection patients, compared to controls. Increased expression of phosphorylated ARPC2 and its correlation to F-actin filaments were confirmed in CAMR patients. Our results are the first evidence of altered cytoskeleton organization in circulating immune cells of CAMR patients. The increased expression of phosphorylated ARPC2 found in the PBMCs of our patients, and its association with derangement of F-actin filaments, might suggest that proteins regulating actin dynamics in immune cells could be involved in the mechanism of CAMR of kidney grafts.

Adult Renal Stem/Progenitor Cells Can Modulate T Regulatory Cells and Double Negative T Cells.

Adult Renal Stem/Progenitor Cells (ARPCs) have been recently identified in the human kidney and several studies show their active role in kidney repair processes during acute or chronic injury. However, little is known about their immunomodulatory properties and their capacity to regulate specific T cell subpopulations. We co-cultured ARPCs activated by triggering Toll-Like Receptor 2 (TLR2) with human peripheral blood mononuclear cells for 5 days and 15 days and studied their immunomodulatory capacity on T cell subpopulations. We found that activated-ARPCs were able to decrease T cell proliferation but did not affect CD8+ and CD4+ T cells. Instead, Tregs and CD3+ CD4- CD8- double-negative (DN) T cells decreased after 5 days and increased after 15 days of co-culture. In addition, we found that PAI1, MCP1, GM-CSF, and CXCL1 were significantly expressed by TLR2-activated ARPCs alone and were up-regulated in T cells co-cultured with activated ARPCs. The exogenous cocktail of cytokines was able to reproduce the immunomodulatory effects of the co-culture with activated ARPCs. These data showed that ARPCs can regulate immune response by inducing Tregs and DN T cells cell modulation, which are involved in the balance between immune tolerance and autoimmunity.

Do thermal treatments influence the ultrafast opto-thermal processes of eumelanin?

After light absorption, melanin converts very rapidly the energy gained into heat. The time scale of this process ranges from tens of femtoseconds to a few nanoseconds. Femtosecond transient absorption allows for exploration of such photo-induced carrier dynamics to observe the de-excitation pathways of the biological complex. Here, we report on the ultrafast relaxation of suspensions of Sepia melanin in DMSO at room temperature using a femtosecond broadband pump and probe technique by photoexciting in the UV and probing in the entire visible range. In particular, we focus on the possible role that different heat treatments, performed in the temperature range 30-80 °C  might have on the relaxation of charge carriers photogenerated by UV radiation in such suspensions. Experimental data indicate that in all the investigated suspensions, photoexcited carriers always follow a tri-exponential route to relaxation. Moreover, we find that the relaxation time constants are essentially the same in all cases, within the experimental error. We take this as evidence that all the investigated suspensions essentially exhibit the same relaxation dynamics, regardless of the temperature at which the heat treatment has been performed and of the heat-induced denaturation of the proteinaceous compounds bound to the photoactive pigment. Our experiments represent a significant step towards the understanding of the stability of melanin with respect to temperature changes.

A transcriptomics study of hereditary angioedema attacks.

BACKGROUND: Hereditary angioedema (HAE) caused by C1-inhibitor deficiency is a lifelong illness characterized by recurrent acute attacks of localized skin or mucosal edema. Activation of the kallikrein/bradykinin pathway at the endothelial cell level has a relevant pathogenetic role in acute HAE attacks. Moreover, other pathways are involved given the variable clinical expression of the disease in different patients. OBJECTIVE: We sought to explore the involvement of other putative genes in edema formation. METHODS: We performed a PBMC microarray gene expression analysis on RNA isolated from patients with HAE during an acute attack and compared them with the transcriptomic profile of the same patients in the remission phase. RESULTS: Gene expression analysis identified 23 genes significantly modulated during acute attacks that are involved primarily in the natural killer cell signaling and leukocyte extravasation signaling pathways. Gene set enrichment analysis showed a significant activation of relevant biological processes, such as response to external stimuli and protein processing (q < 0.05), suggesting involvement of PBMCs during acute HAE attacks. Upregulation of 2 genes, those encoding adrenomedullin and cellular receptor for urokinase plasminogen activator (uPAR), which occurs during an acute attack, was confirmed in PBMCs of 20 additional patients with HAE by using real-time PCR. Finally, in vitro studies demonstrated the involvement of uPAR in the generation of bradykinin and endothelial leakage. CONCLUSIONS: Our study demonstrates the increase in levels of adrenomedullin and uPAR in PBMCs during an acute HAE attack. Activation of these genes usually involved in regulation of vascular tone and in inflammatory response might have a pathogenic role by amplifying bradykinin production and edema formation in patients with HAE.

A New Vision of IgA Nephropathy: The Missing Link.

IgA Nephropathy (IgAN) is a primary glomerulonephritis problem worldwide that develops mainly in the 2nd and 3rd decade of life and reaches end-stage kidney disease after 20 years from the biopsy-proven diagnosis, implying a great socio-economic burden. IgAN may occur in a sporadic or familial form. Studies on familial IgAN have shown that 66% of asymptomatic relatives carry immunological defects such as high IgA serum levels, abnormal spontaneous in vitro production of IgA from peripheral blood mononuclear cells (PBMCs), high serum levels of aberrantly glycosylated IgA1, and an altered PBMC cytokine production profile. Recent findings led us to focus our attention on a new perspective to study the pathogenesis of this disease, and new studies showed the involvement of factors driven by environment, lifestyle or diet that could affect the disease. In this review, we describe the results of studies carried out in IgAN patients derived from genomic and epigenomic studies. Moreover, we discuss the role of the microbiome in the disease. Finally, we suggest a new vision to consider IgA Nephropathy as a disease that is not disconnected from the environment in which we live but influenced, in addition to the genetic background, also by other environmental and behavioral factors that could be useful for developing precision nephrology and personalized therapy.

Administration of a polyphenol-enriched feed to farmed sea bass (Dicentrarchus labrax L.): Kidney melanomacrophages response.

The reinforcement of the defense mechanism of fish, through the administration of immunostimulants, is considered as a promising alternative to vaccines. Natural immunostimulants such as polyphenols, flavanoids, pigments and essential oils can modulate the innate immune response. In lower vertebrates, melano-macrophage centres, i.e. clusters of pigment-containing cells forming the extracutaneous pigment system, are wide-spread in the stroma of the haemopoietic tissue, mainly in kidney and spleen. In fishes, melano-macrophage centres play an important role in the immune response against antigenic stimulants and pathogens. In the present study, we evaluated the effect of a polyphenol-enriched diet on the health status of European sea bass (Dicentrarchus labrax L.). Farmed sea bass were administered a feed containing a phytocomplex, rich in catechins and epigallocatechins, which was obtained from the seeds of Canosina Nero di Troia Vitis vinifera and mixed with conventional feed at two different concentrations. The effects of such a diet were investigated in juvenile and commercial size samples, i.e. undergoing a short- and long-term period of diet, respectively, focusing on their extracutaneous pigmentary system and, in more detail, on the enzymatic activities leading to melanin biosynthesis. Our results show that prolonged dietary treatments with higher concentration of polyphenols might modulate tyrosinase activity and gene expression in commercial size fishes. An increase of melano-macrophage activity is correlated to a stimulation of cytoprotective functions against antigenic stimulants and pathogens, as an expression of a robust and protective adaptive immune response.

A multidisciplinary study of the extracutaneous pigment system of European sea bass (Dicentrarchus labrax L.). A possible relationship between kidney disease and dopa oxidase activity level.

Infectious diseases and breeding conditions can influence fish health status. Furthermore it is well known that human and animal health are strongly correlated. In lower vertebrates melano-macrophage centres, clusters of pigment-containing cells forming the extracutaneous pigment system, are widespread in the stroma of the haemopoietic tissue, mainly in kidney and spleen. In fishes, melano-macrophage centres play an important role in the immune response against antigenic stimulants and pathogens. Hence, they are employed as biomarker of fish health status. We have investigated this cell system in the European sea bass (Dicentrarchus labrax L.) following the enzyme activities involved in melanin biosynthesis. We have found a possible relationship between kidney disease of farmed fishes and dopa oxidase activity level, suggesting it as an indicator of kidney disease. Moreover variations of dopa oxidase activity in extracutaneous pigment system have been observed with respect to environmental temperature. At last, for the first time, using femtosecond transient absorption spectroscopy (Femto-TA), we pointed out that pigment-containing cells of fish kidney tissue present melanin pigments.

Melanogenesis in visceral tissues of Salmo salar. A link between immunity and pigment production?

Melanogenesis is mostly studied in melanocytes and melanoma cells, but much less is known about other pigment cell systems. Liver, spleen, kidney, and other organs of lower vertebrates harbour a visceral pigment cell system with an embryonic origin that differs from that of melanocytes. In teleosts, melanin-containing cells occur in the reticulo-endothelial system and are mainly in the kidney and spleen. The Atlantic salmon (Salmo salar L.) is an ichthyic breeding species of considerable economic importance. The accumulation of pigments in salmon visceral organs and musculature adversely affects the quality of fish products and is a problem for the aquaculture industry. With the aim to reveal novel functions and behaviour of the salmonid extracutaneous pigment system, we investigated aspects of the melanogenic systems in the tissues of Atlantic salmon, as well as in SHK-1 cells, which is a long-term cell line derived from macrophages of the Atlantic salmon head-kidney. We demonstrate that a melanogenic system is present in SHK-1 cells, head-kidney, and spleen tissues. As teleosts lack lymph nodes and Peyer's patches, the head-kidney and spleen are regarded as the most important secondary lymphoid organs. The detection of tyrosinase activity in lymphoid organs indicates that a link exists between the extracutaneous pigmentary system and the immune system in salmon.

Probiotic features of Lactobacillus plantarum mutant strains.

In this study, the probiotic potential of Lactobacillus plantarum wild-type and derivative mutant strains was investigated. Bacterial survival was evaluated in an in vitro system, simulating the transit along the human oro-gastro-intestinal tract. Interaction with human gut epithelial cells was studied by assessing bacterial adhesive ability to Caco-2 cells and induction of genes involved in innate immunity. L. plantarum strains were resistant to the combined stress at the various steps of the simulated gastrointestinal tract. Major decreases in the viability of L. plantarum cells were observed mainly under drastic acidic conditions (pH ≤ 2.0) of the gastric compartment. Abiotic stresses associated to small intestine poorly affected bacterial viability. All the bacterial strains significantly adhered to Caco-2 cells, with the ΔctsR mutant strain exhibiting the highest adhesion. Induction of immune-related genes resulted higher upon incubation with heat-inactivated bacteria rather than with live ones. For specific genes, a differential transcriptional pattern was observed upon stimulation with different L. plantarum strains, evidencing a possible role of the knocked out bacterial genes in the modulation of host cell response. In particular, cells from Δhsp18.55 and ΔftsH mutants strongly triggered immune defence genes. Our study highlights the relevance of microbial genetic background in host-probiotic interaction and might contribute to identify candidate bacterial genes and molecules involved in probiosis.

Pre-Transplant Expression of CCR-2 in Kidney Transplant Recipients Is Associated With the Development of Delayed Graft Function.

Background: Delayed graft function (DGF) leads to a reduced graft survival. Donors' features have been always considered as key pathogenic factors in this setting. The aim of our study was to evaluate the recipients' characteristics in the development of DGF. Methods: We enrolled 932 kidney graft recipients from 466 donors; 226 recipients experienced DGF. In 290 donors, both recipients presented with early graft function (EGF, group A), in 50 both recipients experienced DGF (group B), and in 126 one recipient presented with DGF and the other with EGF (group C). In group C, we selected 7 couples of DGF/EGF recipients and we evaluated the transcriptomic profile by microarray on circulating mononuclear cells harvested before transplantation. Results were validated by qPCR in an independent group of 25 EGF/DGF couples. Findings: In the whole study group, DGF was associated with clinical characteristics related to both donors and recipient. In group C, DGF was significantly associated with body mass index, hemodialysis, and number of mismatches. In the same group, we identified 411 genes differently expressed before transplantation between recipients discordant for the transplant outcome. Those genes were involved in immune dysfunction and inflammation. In particular, we observed a significant increase in DGF patients in the expression of C-C chemokine receptor type 2 (CCR2), the monocyte chemoattractant protein-1 (MCP-1) receptor. CCR-2 upregulation was confirmed in an independent cohort of patients. Conclusions: Our results suggest that recipients' clinical/immunological features, potentially modulated by dialysis, are associated with the development of DGF independently of donors' features.

Identification and monitoring of Copy Number Variants (CNV) in monoclonal gammopathy.

Monoclonal gammopathy of undetermined significance (MGUS) represents the pre-clinical stage of Multiple Myeloma (MM) with the 5% of MGUS progresses to MM. Although the progression from MGUS to MM has not been completely characterized, it is possible to monitor the DNA modifications of patients diagnosed with MGUS to detect early specific genomic abnormalities, including copy number variations (CNV). The CNVs of chromosome 1q and chromosome 13q are associated with a worse prognosis in MM.In the present study, we showed that it is possible to monitor the 1q21 gain and 13q deletion frequencies in gDNA using digital PCR. The CNV analysis of three cell lines with a well-characterized cytogenetic profile were compared with measures performed by a real-time PCR approach and with a digital PCR approach. Then, we analyzed CNVs in CD138+ plasma cells isolated from bone marrow of MGUS and MM patients.Our results show that digital PCR and targeted DNA monitoring represent a specific and accurate technique for the early detection of specific genomic abnormalities both in MM and in MGUS patients.Our results could represent a remarkable advancement in MM and MGUS diagnosis and in CNV analysis for the evaluation of the risk of progression from MGUS to MM.

Analysis of the Physico-Chemical, Mechanical and Biological Properties of Crosslinked Type-I Collagen from Horse Tendon: Towards the Development of Ideal Scaffolding Material for Urethral Regeneration.

Urethral stenosis is a pathological condition that consists in the narrowing of the urethral lumen because of the formation of scar tissue. Unfortunately, none of the current surgical approaches represent an optimal solution because of the high stricture recurrence rate. In this context, we preliminarily explored the potential of an insoluble type-I collagen from horse tendon as scaffolding material for the development of innovative devices for the regeneration of injured urethral tracts. Non-porous collagen-based substrates were produced and optimized, in terms of crosslinking density of the macromolecular structure, to either provide mechanical properties compliant with the urinary tract physiological stress and better sustain tissue regeneration. The effect of the adopted crosslinking strategy on the protein integrity and on the substrate physical-chemical, mechanical and biological properties was investigated in comparison with a decellularized matrix from porcine small intestinal submucosa (SIS patch), an extensively used xenograft licensed for clinical use in urology. The optimized production protocols allowed the preservation of the type I collagen native structure and the realization of a substrate with appealing end-use properties. The biological response, preliminarily investigated by immunofluorescence experiments on human adult renal stem/progenitor cells until 28 days, showed the formation of a stem-cell monolayer within 14 days and the onset of spheroids within 28 days. These results suggested the great potential of the collagen-based material for the development of scaffolds for urethral plate regeneration and for in vitro cellular studies.

Characterization of the CtsR stress response regulon in Lactobacillus plantarum.

Lactobacillus plantarum ctsR was characterized. ctsR was found to be cotranscribed with clpC and induced in response to various abiotic stresses. ctsR deletion conferred a heat-sensitive phenotype with peculiar cell morphological features. The transcriptional pattern of putative CtsR regulon genes was examined in the Delta ctsR mutant. Direct CtsR-dependent regulation was demonstrated by DNA-binding assays using recombinant CtsR and the promoters of the ctsR-clpC operon and hsp1.