GC-MS analysis and biological activity of hydroalcholic extracts and essential oils of Rhus typhina L. wood (anacardiaceae) in comparison with leaves and fruits.

Hydroalcoholic extracts (HE) and essential oils (EO) of branches, leaves and fruits of Rhus typhina L., were characterized by GC-MS. HE (yealds: branches 68.30 mg/g, leaves 35.82 mg/g and fruits 257.76 mg/g), showed different compositions dominated by gallic acid (33.46%) in branches, its precursor 1-cyclohexane-3,4,5-hydroxy-carboxylic acid (20.55%) in leaves and malic acid (89.15%) in fruits. EO yields were 210 µg/g for branches (main component δ-cadinene, 22.00%), followed by fruits with 132 µg/g (ß-pinene 32.2%) and by leaves with 54 µg/g and phenylacetaldehyde as major component (40.13%). Total phenolic content (TPC) was highest in branches HE (5.87 µg GAE/mL) and in EO leaves (17.71 µg GAE/mL). The highest value of radical scavenging activity (DPPH test) was detected in leaves HE and EO. The branches EO antimicrobial activity was strong against C. albicans and negligible against E. coli. Leaves and fruits EO showed strong activity against C. albicans and intermediate activity against Escherichia coli.

Evaluation of Gut Microbiota in Patients With Vulvovestibular Syndrome.

BACKGROUND: Vulvovestibular syndrome (VVS) or vulvodynia is a chronic, heterogeneous and multifactorial disease that dramatically affects women's health and quality of life. Despite important advancements in understanding VVS etiology have been achieved in the past decades, VVS still remains an elusive and complex condition without identifiable causes and effective treatments. In the present observational, retrospective, case-control study, we sought to investigate whether gut dysbiosis developed in patients with VVS. METHODS: To this aim, we compared both bacterial and fungal composition in VVS patients (n = 74; 34.3 ± 10.9 years old) with those of women without gynecological symptoms (n = 13 healthy control; 38.3 ± 10.4 years old). Furthermore, to assess whether gut ecology may have an impact on gut function, the degree of intestinal inflammation (calprotectin levels) and gut permeability (zonulin levels) were also evaluated. RESULTS: VVS patient developed gut dysbiosis, mainly characterized by a significant increase of Escherichia coli along with increased colonization of mold/yeast compared to healthy controls. Furthermore, fecal levels of zonulin indicated that in VVS patients gut dysbiosis translated into increased gut permeability. CONCLUSION: Our preliminary study, by demonstrating that alterations in gut microbiota and intestinal permeability are present in patients with VVS, highlights the novel notion that gut dysbiosis may be considered an important associated factor for VVS. These findings, if confirmed, may be clinically relevant and may help in choosing further diagnostic methods and more effective therapies for these patients.

Vulvovestibular Syndrome and Vaginal Microbiome: A Simple Evaluation.

BACKGROUND: The vulvovestibular syndrome (VVS) is a chronic, inflammatory, multifactorial, chronic inflammation of the female urogenital access. METHODS: The aim of this anecdotal, observational, retrospective, case-control study was to comparatively evaluate the most common bacterial strains (Lactobacillus spp., Klebsiella spp., Gardnerella spp., and Streptococcus spp.) and fungi (Candida spp., Pennicillum spp., and Aspergillus spp.) in vulvodinic women, and in women without gynecological symptoms (control group). RESULTS: We found that vulvodinic patients had statistically lower Lactobacilli and higher total Fungi concentration. CONCLUSIONS: Our preliminary study is useful to further clarify the etiopathology of vulvodynia and suggest new therapeutic strategies for approaching the VVS.

Pathogenic Gut Flora in Patients With Chronic Heart Failure.

OBJECTIVES: The goal of this study was to measure the presence of pathogenic gut flora and intestinal permeability (IP) and their correlations with disease severity, venous blood congestion, and inflammation in patients with chronic heart failure (CHF). BACKGROUND: Evidence suggests that translocation of gut flora and/or their toxins from the intestine to the bloodstream is a possible trigger of systemic CHF inflammation. However, the relation between pathogenic gut flora and CHF severity, as well as IP, venous blood congestion as right atrial pressure (RAP), and/or systemic inflammation (C-reactive protein [CRP]), is still unknown. METHODS: This study analyzed 60 well-nourished patients in stable condition with mild CHF (New York Heart Association [NYHA] functional class I to II; n = 30) and moderate to severe CHF (NYHA functional class III to IV; n = 30) and matched healthy control subjects (n = 20). In all subjects, the presence and development in the feces of bacteria and fungi (Candida species) were measured; IP according to cellobiose sugar test results was documented. The study data were then correlated with RAP (echocardiography) and systemic inflammation. RESULTS: Compared with normal control subjects, the entire CHF population had massive quantities of pathogenic bacteria and Candida such as Campylobacter (85.3 ± 3.7 CFU/ml vs. 1.0 ± 0.3 CFU/ml; p < 0.001), Shigella (38.9 ± 12.3 CFU/ml vs. 1.6 ± 0.2 CFU/ml; p < 0.001), Salmonella (31.3 ± 9.1 CFU/ml vs 0 CFU/ml; p < 0.001), Yersinia enterocolitica (22.9 ± 6.3 CFU/ml vs. 0 CFU/ml; p < 0.0001), and Candida species (21.3 ± 1.6 CFU/ml vs. 0.8 ± 0.4 CFU/ml; p < 0.001); altered IP (10.2 ± 1.2 mg vs. 1.5 ± 0.8 mg; p < 0.001); and increased RAP (12.6 ± 0.6 mm Hg) and inflammation (12.5 ± 0.6 mg/dl). These variables were more pronounced in patients with moderate to severe NYHA functional classes than in patients with the mild NYHA functional class. Notably, IP, RAP, and CRP were mutually interrelated (IP vs. RAP, r = 0.55; p < 0.0001; IP vs. CRP, r = 0.78; p < 0.0001; and RAP vs. CRP, r = 0.78; p < 0.0001). CONCLUSIONS: This study showed that patients with CHF may have intestinal overgrowth of pathogenic bacteria and Candida species and increased IP associated with clinical disease severity, venous blood congestion, and inflammation.

Permselective dysfunction of podocyte-podocyte contact upon angiotensin II unravels the molecular target for renoprotective intervention.

Ameliorating the function of the glomerular barrier to circulating proteins by blocking angiotensin II (Ang II) translates into less risk of progression toward end-stage renal failure in diabetic and nondiabetic nephropathies. However, the mechanisms underlying this barrier protection are not clear. Specialized contacts between adjacent podocytes are major candidate targets, and the actin cytoskeleton is emerging as a regulatory element. Here, we present data demonstrating that Ang II induced reorganization of F-actin fibers and redistribution of zonula occludens-1 (ZO-1) that is physically associated with actin in murine podocytes. These effects were paralleled by increased albumin permeability across podocyte monolayers. The F-actin stabilizer jasplakinolide prevented both ZO-1 redistribution and albumin leakage, suggesting that actin cytoskeleton rearrangement is instrumental to podocyte permselective dysfunction induced by Ang II. Changes in both F-actin and ZO-1 patterns were confirmed in glomeruli of rat isolated perfused kidneys on short infusion of Ang II, leading to increased protein excretion. Podocyte dysfunction was mediated by Ang II type 1 receptor and was partly dependent on Src kinase-phospholipase C activation. These data demonstrate that strategies aimed at stabilizing podocyte-podocyte contacts and targeting the relevant intracellular signal transduction are crucial to renoprotection.

Activation of porcine endothelium in response to xenogeneic serum causes thrombosis independently of platelet activation.

BACKGROUND: Endothelial cell activation and microvascular thrombosis are hallmarks of hyperacute xenograft rejection. However, the molecular determinants of platelet-endothelial interaction and thrombus formation are poorly understood. This study investigated whether: (i) xenogeneic human serum (HS), as a source of xenoreactive antibodies and complement, activates porcine aortic endothelial cells (PAEC) to promote thrombus formation under high shear stress; (ii) the endothelial adhesive proteins vitronectin receptor and P-selectin are involved in the von Willebrand factor (VWF)-platelet interaction during the thrombotic process under flow; (iii) reactive oxygen species (ROS) are activated by complement and served as intracellular signals for adhesive protein up-regulation. METHODS: The PAEC were pre-exposed for 90 min in static conditions to medium plus 10, 20, and 50% HS or 20% porcine serum (PS), as control, then cells were perfused at 50 dynes/cm2 in a parallel plate flow chamber with human blood and area occupied by thrombi was measured. The role of complement in HS-induced thrombus formation was assessed by incubating PAEC with 20% HS in the presence of soluble complement receptor type 1 (sCR1) before blood perfusion. The effect of platelet activation was assessed using human blood treated or not with ADP and then flowed over PAEC pre-exposed to 20% HS or 20% PS as control. To identify the endothelial adhesive proteins involved in thrombus formation PAEC treated with 20% HS were then incubated with anti-vitronectin receptor antibody, anti-P-selectin antibody or P-selectin glycoprotein ligand-1 (PSGL-1), the soluble ligand of P-selectin, before the adhesion assay. Confocal microscopy was used to detect changes in endothelial adhesive protein expression. VWF interaction with platelet receptors GPIb and alphaIIbbeta3 was assessed adding aurin tricarboxylic acid (ATA) and anti-alphaIIbbeta3 antibody to blood before perfusion. The ROS involvement in xenogeneic serum-induced thrombus formation was determined studying the intracellular production of hydrogen peroxide (H2O2). The effect of antioxidants and metal chelators on HS-induced thrombus formation was evaluated treating PAEC with pyrrolidine dithiocarbamate (PDTC) or 1,3-dimethyl-2-thiourea (DMTU) before and during incubation with 20% HS followed by blood perfusion. The effect of antioxidants and sCR1 on ROS generation was investigated treating PAEC with PDTC or DMTU before and during incubation with 20% HS. Intracellular ROS generation was measured by fluorescence spectroscopy using the probe dihydrorhodamine 123 (DHR-123). RESULTS: Human serum but not PS caused thrombus formation on PAEC under high shear stress. Blockade of complement activation by sCR1 prevented xenogeneic serum-induced thrombus formation. Activated platelets did not promote thrombus formation on resting endothelium, and did not further increase platelet deposition on xenogeneic serum-treated PAEC. Vitronectin receptor and P-selectin were up-regulated on the endothelial surface by HS. Their functional blockade by specific antibodies prevented platelet deposition and thrombus formation. H2O2 production significantly increased when PAEC were exposed to the xenogeneic condition. Antioxidants and sCR1 completely prevented thrombus formation by reducing excessive ROS production and the expression of vitronectin receptor and P-selectin. CONCLUSIONS: Xenogeneic complement induces endothelial cell activation and thrombosis which is independent of platelet activation. Complement deposition elicits a rapid generation of ROS that lead to overexpression of endothelial adhesive molecules instrumental for platelet deposition.

In response to protein load podocytes reorganize cytoskeleton and modulate endothelin-1 gene: implication for permselective dysfunction of chronic nephropathies.

Effacement of podocyte foot processes occurs in many proteinuric nephropathies and is accompanied by rearrangement of the actin cytoskeleton. Here, we studied whether protein overload affects intracellular pathways, leading to cytoskeletal architecture changes and ultimately to podocyte dysfunction. Mouse podocytes bound and endocytosed both albumin and IgG via receptor-specific mechanisms. Protein overload caused redistribution of F-actin fibers instrumental to up-regulation of the prepro-endothelin (ET)-1 gene and production of the corresponding peptide. Increased DNA-binding activity for nuclear factor (NF)-kappaB and Ap-1 nuclear proteins was measured in nuclear extracts of podocytes exposed to excess proteins. Both Y27632, which inhibits Rho kinase-dependent stress fiber formation, and jasplakinolide, an F-actin stabilizer, decreased NF-kappaB and Ap-1 activity and reduced ET-1 expression. This suggested a role for the cytoskeleton, through activated Rho, in the regulation of the ET-1 peptide. Focal adhesion kinase (FAK), an integrin-associated nonreceptor tyrosine kinase, was phosphorylated by albumin treatment via Rho kinase-triggered actin reorganization. FAK activation led to NF-kappaB- and Ap-1-dependent ET-1 expression. These data suggest that reorganization of the actin cytoskeletal network in response to protein load is implicated in modulation of the ET-1 gene via Rho kinase-dependent FAK activation of NF-kappaB and Ap-1 in differentiated podocytes. Increased ET-1 generation might alter glomerular permselectivity and amplify the noxious effect of protein overload on dysfunctional podocytes.

Mesenchymal stem cells are renotropic, helping to repair the kidney and improve function in acute renal failure.

Injury to a target organ can be sensed by bone marrow stem cells that migrate to the site of damage, undergo differentiation, and promote structural and functional repair. This remarkable stem cell capacity prompted an investigation of the potential of mesenchymal and hematopoietic stem cells to cure acute renal failure. The model of renal injury induced in mice by the anticancer agent cisplatin was chosen. Injection of mesenchymal stem cells of male bone marrow origin remarkably protected cisplatin-treated syngeneic female mice from renal function impairment and severe tubular injury. Y chromosome-containing cells localized in the context of the tubular epithelial lining and displayed binding sites for Lens culinaris lectin, indicating that mesenchymal stem cells engraft the damaged kidney and differentiate into tubular epithelial cells, thereby restoring renal structure and function. Mesenchymal stem cells markedly accelerated tubular proliferation in response to cisplatin-induced damage, as revealed by higher numbers of Ki-67-positive cells within the tubuli with respect to cisplatin-treated mice that were given saline. Hematopoietic stem cells failed to exert beneficial effects. These results offer a strong case for exploring the possibility that mesenchymal stem cells by virtue of their renotropic property and tubular regenerative potential may have a role in the treatment of acute renal failure in humans.

Shiga toxin-2 triggers endothelial leukocyte adhesion and transmigration via NF-kappaB dependent up-regulation of IL-8 and MCP-1.

BACKGROUND: Shiga toxin (Stx)-producing E. coli is a causative agent of the epidemic form of hemolytic uremic syndrome (HUS), the most common cause of acute renal failure in children. Endothelial injury and leukocyte activation are instrumental to the development of microangiopathic lesions. To obtain more insight into the mechanisms favoring endothelium-leukocyte interaction, we studied (1) the effect of Stx-2 on leukocyte adhesion and transmigration in human endothelial cells under flow; (2) the effect of Stx-2 on endothelial expression of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) and their functional role in the adhesive phenomena; and (3) the role of nuclear factor-kappaB (NF-kappaB) in endothelial chemokine expression. METHODS: For adhesion experiments, human umbilical vein endothelial cells (HUVEC) and human glomerular endothelial cells (GEC) were incubated for 24 hours with Stx-2 (25 pmol/L), with or without anti-IL-8 or MCP-1 antibodies, and then exposed to leukocyte suspension under flow (1.5 dynes/cm2). IL-8 and MCP-1 expression was evaluated in Stx-2 treated endothelial cells (6 hours) by Northern blot. NF-kappaB activity was assessed by electrophoretic mobility shift assay. The role of NF-kappaB in Stx-induced chemokines was evaluated by transfecting HUVEC with an adenovirus coding for IkappaBalpha. RESULTS: Stx-2 significantly enhanced the number of leukocytes that adhered and then migrated across the endothelium. Stx-2 increased the expression of IL-8 and MCP-1, which was preceded by NF-kappaB activation. Blocking of endothelial IL-8 and MCP-1 with corresponding antibodies significantly inhibited Stx-induced leukocyte adhesion and migration either in HUVEC or GEC. Adenovirus-mediated gene transfer of IkappaBalpha down-regulated IL-8 and MCP-1 mRNA and also inhibited the adhesion and transmigration of leukocytes in Stx-treated HUVEC. CONCLUSIONS: Stx-2 via a transcriptional activation mechanism specifically mediated by NF-kappaB up-regulates endothelial MCP-1 and IL-8. Both chemokines are important modulators of leukocyte adhesion and transmigration under flow. These findings might be relevant to understand the nature of microvascular lesions in HUS and open future perspectives for better treatment of microvascular thrombosis.

Transforming growth factor-beta1 is up-regulated by podocytes in response to excess intraglomerular passage of proteins: a central pathway in progressive glomerulosclerosis.

Chronic diseases of the kidney have a progressive course toward organ failure. Common pathway mechanisms of progressive injury, irrespectively of the etiology of the underlying diseases, include glomerular capillary hypertension and enhanced passage of plasma proteins across the glomerular capillary barrier because of impaired permselective function. These changes are associated with podocyte injury and glomerular sclerosis. Direct evidence for causal roles is lacking, particularly for the link between intraglomerular protein deposition and sclerosing reaction. Because transforming growth factor-beta1 (TGF-beta1) is the putative central mediator of scarring, we hypothesized that TGF-beta1 can be up-regulated by protein overload of podocytes thereby contributing to sclerosis. In rats with renal mass reduction, protein accumulation in podocytes as a consequence of enhanced transcapillary passage preceded podocyte dedifferentiation and injury, increase in TGF-beta1 expression in podocytes, and TGF-beta1-dependent activation of mesangial cells. Angiotensin-converting enzyme inhibitor prevented both accumulation of plasma proteins and TGF-beta1 overexpression in podocytes and sclerosis. Albumin load on podocytes in vitro caused loss of the synaptopodin differentiation marker and enhanced TGF-beta1 mRNA and protein. Conditioned medium of albumin-stimulated podocytes induced a sclerosing phenotype in mesangial cells, an effect mimicked by TGF-beta1 and blocked by anti-TGF-beta1 antibodies. Thus, the passage of excess plasma proteins across the glomerular capillary wall is the trigger of podocyte dysfunction and of a TGF-beta1-mediated mechanism underlying sclerosis. Agents to reduce TGF-beta1, possibly combined with angiotensin blockade, should have priority in novel approaches to treatment of progressive nephropathies.