The Chemical Composition of Achillea wilhelmsii C. Koch and Its Desirable Effects on Hyperglycemia, Inflammatory Mediators and Hypercholesterolemia as Risk Factors for Cardiometabolic Disease.

This study was done to identify the content compounds of Achillea wilhelmsii (A. wilhelmsii) and to evaluate its hypoglycemic and anti-hypercholesterolemic activity and effect on inflammatory mediators. The extracts and fractions of A. wilhelmsii were thoroughly analyzed using high performance liquid chromatography (HPLC), and the total content of phenols and flavonoids was determined. The hypoglycemic activity was evaluated in vivo using alloxan-induced diabetic mice. The effect upon inflammatory mediators was evaluated in vitro using the human monocytic leukemia cell line (THP-1). The anti-hypercholesterolemic activity was evaluated in vitro using the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase assay kit. The water extract (WE)-treated group showed the highest reduction in the fasting blood glucose levels (FBGL). The chloroform fraction (CF) and ethyl acetate fraction (EAF) both showed a significant ability to reduce the secretion of tumor necrosis factor alpha (TNF-α). The EAF, however, also attenuated the levels of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). The CF showed the most significant 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) inhibition activity. The five main compounds in the CF were isolated and identified. Out of the five compounds in the CF, 1ß,10ß-epoxydesacetoxymatricarin (CP1) and leucodin (CP2) showed the highest anti-hypercholesterolemic potential. A molecular docking study provided corresponding results.

Antiurease activity of plants growing in the Czech Republic.

The antiurease activity of the aqueous extracts of 42 plants growing in the Czech Republic was investigated. A phenol-hypochlorite reaction was used for the determination of ammonia produced by urease. The inhibitory activity of the extracts at a concentration of 0.2 mg/mL varied from 17.8% to 80.0%. Extracts from six Potentilla species expressed inhibitory activity against jack bean urease. They were further investigated for their phenolic constituents and the major compounds were subjected to molecular docking. The results revealed that both jack bean urease and Helicobacter pylori urease were inhibited by quercetin-3-O-ß-D-galactopyranoside-6″-gallate (1), myricetin-3-O-ß-D-glucuronide (2), tiliroside (3) and B-type procyanidin (4). The antiurease activity of the investigated Potentilla species is probably due to the presence of complex phenolic constituents such as flavonoid glycosides and catechin dimers.

An overview of urease and its relation to the ureolytic bacteria and the search for new urease inhibitors.

UNLABELLED: In this review, an overview of the available literature on urease is presented. Urease is an enzyme which catalyzes the hydrolysis of urea. The occurrence of ureases and their functions are discussed thoroughly. The relationship of urease to ureolytic bacteria is examined, and the currently available urease inhibitors, both inorganic and natural, are presented. Finally, the importance of urease and current and future applications of new inhibitors and explored. KEYWORDS: bacteria inhibitor urease.

Molecular markers of rejection and tolerance: lessons from clinical research.

In terms of finding specific molecular markers associated with graft outcome, attempts have been made to study whole genome transcripts using microarray assays or to study the effect of number of genes of interest using quantitative real-time polymerase chain reaction. Using these techniques, molecular phenotypes of rejection have been characterized, and the variability of the clinical outcome besides similar morphology explained in part. Recently, several specific transcripts including naïve B cell regulation have been identified in the peripheral blood of operationally tolerant kidney transplant recipients. The decrease in immature B cell-related transcripts in the peripheral blood in patients with immunosuppression was shown to be associated with acute rejection. Similarly, tolerance-associated antigen 1 transcripts were identified in biopsies and regulatory T cell transcripts in urine and biopsies in patients without rejection. Better understanding of molecular processes associated with allograft rejection or alloantigen hyporesponsiveness/tolerance may help to improve our knowledge about graft pathology and identify novel markers suitable for future monitoring and guided therapy and finally improve the outcome of kidney transplantation.

B-cell-related biomarkers of tolerance are up-regulated in rejection-free kidney transplant recipients.

BACKGROUND: Molecular signatures have recently been identified in operationally tolerant long-term kidney transplant patients; however, their expression in patients on immunosuppression remains unclear. METHODS: In this prospective study, the gene expression profiles of eight selected tolerance-associated genes (MS4A1, CD79B, TCL1A, TMEM176B, FOXP3, TOAG-1, MAN1A1, and TLR5) in the peripheral blood of 67 kidney transplant recipients at days 0, 7, 14, 21, 28, 60, 90, and at 6 and 12 months, and in graft biopsies were measured. Similarly, using flow cytometry, CD45CD19CD3 B-cell counts were evaluated in the follow-up. Expression patterns were compared among patients with biopsy-proven acute rejection, borderline changes, and in rejection-free patients. A generalized linear mixed model with gamma distribution for repeated measures adjusted for induction therapy was used for statistical analysis of longitudinal data and Kruskal-Wallis test for case biopsy data. RESULTS: Compared to patients with rejection, a significantly higher number of peripheral B cells were observed during follow-up in rejection-free patients and in patients with borderline changes. Gene expression patterns of MS4A1 (CD20), TCL1A, CD79B, TOAG-1, and FOXP3 genes were significantly higher in rejection-free patients as compared to rejection group with the highest differences during the first 3 months. In contrast, TMEM176B (TORID) was up-regulated in the rejection group. Similar trends were also observed between patients with borderline changes and acute rejection. Higher intragraft expression of TOAG-1 was observed in rejection-free patients. CONCLUSIONS: These observations suggest an association of B-cell signatures, seen also in drug-free tolerant patients, with controlled alloimmune response.

Molecular networks involved in the immune control of BK polyomavirus.

BK polyomavirus infection is the important cause of virus-related nephropathy following kidney transplantation. BK virus reactivates in 30%-80% of kidney transplant recipients resulting in BK virus-related nephropathy in 1%-10% of cases. Currently, the molecular processes associated with asymptomatic infections in transplant patients infected with BK virus remain unclear. In this study we evaluate intrarenal molecular processes during different stages of BKV infection. The gene expression profiles of 90 target genes known to be associated with immune response were evaluated in kidney graft biopsy material using TaqMan low density array. Three patient groups were examined: control patients with no evidence of BK virus reactivation (n = 11), infected asymptomatic patients (n = 9), and patients with BK virus nephropathy (n = 10). Analysis of biopsies from asymptomatic viruria patients resulted in the identification of 5 differentially expressed genes (CD3E, CD68, CCR2, ICAM-1, and SKI) (P < 0.05), and functional analysis showed a significantly heightened presence of costimulatory signals (e.g., CD40/CD40L; P < 0.05). Gene ontology analysis revealed several biological networks associated with BKV immune control in comparison to the control group. This study demonstrated that asymptomatic BK viruria is associated with a different intrarenal regulation of several genes implicating in antiviral immune response.

Long-term outcomes of pre-emptive valganciclovir compared with valacyclovir prophylaxis for prevention of cytomegalovirus in renal transplantation.

Prevention of cytomegalovirus (CMV) is essential in organ transplantation. The two main strategies are pre-emptive therapy, in which one screens for and treats asymptomatic CMV viremia, and universal antiviral prophylaxis. We compared these strategies and examined long-term outcomes in a randomized, open-label, single-center trial. We randomly assigned 70 renal transplant recipients (CMV-seropositive recipient or donor) to 3-month prophylaxis with valacyclovir (n=34) or pre-emptive valganciclovir for significant CMV viremia detected at predefined assessments through month 12 (n=36). Among the 55 patients who had a protocol biopsy specimen available at 3 years to allow assessment of the primary outcome, 9 (38%) of 24 patients in the prophylaxis group and 6 (19%) of 31 patients in the pre-emptive therapy group had moderate to severe interstitial fibrosis and tubular atrophy (odds ratio, 2.50; 95% confidence interval, 0.74-8.43; P=0.22). The prophylaxis group had significantly higher intrarenal mRNA expression of genes involved in fibrogenesis. The occurrence of CMV disease was similar in both groups, but pre-emptive therapy improved 4-year graft survival (92% versus 74%; P=0.049) as a result of worse outcomes in patients with late-onset CMV viremia. In conclusion, compared with valacyclovir prophylaxis, pre-emptive valganciclovir therapy may lead to less severe interstitial fibrosis and tubular atrophy and to significantly better graft survival.

Molecular phenotypes of kidney graft rejection.

In the last decade, gene expression studies of kidney transplants provided an opportunity to better understand the development and regulation of kidney graft rejection. This review outlines the progress in the definition of biomarkers of rejection and, above all, concentrates on studies of the molecular phenotype of rejection. This phenotype, rather than morphological characterization, may be critical for assessing the ongoing processes in the graft and for the outcome prediction.

A prospective longitudinal study of BK virus infection in 120 Czech renal transplant recipients.

Polyomavirus BK (BKV) is a common human polyomavirus that rarely causes clinical symptoms in immunocompetent individuals. However, BK virus reactivation occurs in 20-40% of kidney transplant patients and 1-10% of cases present with BK virus-associated nephropathy (BKVN) and reduced kidney allograft survival. In this study, 120 consecutive renal allograft recipients were monitored for BK virus replication by real-time PCR (qPCR) in the blood and urine during the first year post-transplantation and risk factors for BK viremia, viruria, and polyoma BKV-associated nephropathy were evaluated. Receiver operating characteristic curve analysis was used to determine the cutoff points for assessing the risk of developing BKVN. In total, 1,243 samples were tested. BK-DNAuria >10(7) copies/ml and BK-DNAemia >10(4) copies/ml were found in 25.8% and 5% of the samples screened, respectively, during the 12 month follow-up period. BKVN was confirmed histologically in 3/120 patients and viremic patients were treated with dialysis for longer time periods and had higher levels of panel [corrected] reactive antibodies. Patients with viruria were also treated longer with dialysis and had impaired graft function 12 months post-transplantation. Patients with sustained viruria exhibited more acute rejection episodes than patients with transient viruria. Using receiver operating characteristic curve analysis, the cutoff point for viremia and viruria was redefined to 10(3) copies/ml serum for BK viremia and a cutoff point of 6.7 × 10(7) copies/ml in urine. In conclusion, polyoma BK viremia and viruria are frequent findings in kidney transplant recipients that warrant intensive monitoring as a means of preventing graft failure [corrected].

Association of advanced vasculopathy and transforming growth factor-beta1 gene expression with immunoglobulin A nephropathy progression.

BACKGROUND: The mechanism of IgA nephropathy (IgAN) progression remains ill-defined. In this prospective study, the prognostic role of clinical, histological and molecular markers over a 2-year follow-up was evaluated. METHODS: Fifty-one patients with biopsy-proven IgAN were followed for 24 months. Besides routine histology, the intrarenal gene expressions of cytokines and chemokines were quantified by reverse transcription quantitative real-time polymerase chain reaction, and the presence of lymphocytes and macrophages were immunohistochemically examined. RESULTS: Higher transforming growth factor-ß1 and severe chronic vasculopathy (but not glomerulosclerosis, interstitial fibrosis or lymphocyte infiltrate) were associated with the IgAN progression 24 months after biopsy. The gene expression of chemokine (C-C motif) ligands 2 and 5, hepatocyte growth factor, bone morphogenic protein-7 and transforming growth factor-ß1 and the interstitial infiltrate of T and B lymphocytes and macrophages were significantly associated with serum creatinine and glomerular filtration rate at the time of biopsy. The intrarenal chemokine (C-C motif) ligand 2 and hepatocyte growth factor gene expression were associated with the proteinuria. CONCLUSIONS: Besides the known risk factors for chronic kidney disease, advanced vasculopathy and molecular signatures of fibrogenesis were associated with the IgAN progression.

Potential predictive markers in protocol biopsies for premature renal graft loss.

BACKGROUND/AIMS: Protocol biopsies offer new possibilities to predict kidney allograft outcome. The aim of this study was to find clinical, laboratory, morphological and molecular predictors of short-term renal graft survival. METHODS: Three-month protocol kidney graft biopsy was carried out on 257 patients. The real-time RT-PCR was used to identify intragraft mRNA expression of several cytokines and chemokines and predictive statistics was performed to find markers connected with the risk of premature graft failure. RESULTS: Compared to patients with normal morphology at 3 months, patients with subclinical rejection including borderline changes had experienced more frequent (p < 0.001) acute rejections before 3-month biopsy, serum creatinine >or=170 micromol/l (p < 0.01), and higher intrarenal expression of RANTES, IP-10 (p < 0.001), C3, CD3, IgJ (p < 0.01) and CD20 (p < 0.05). There was a significant correlation between subclinical rejection and the occurrence of late acute rejection and graft failure at the first year after transplantation. Moreover, higher RANTES and IP-10 expressions in subclinical rejection predicted graft loss at one year after transplantation in the univariate analysis. CONCLUSIONS: Patients with subclinical rejection including borderline changes in 3-month biopsy and particularly those with higher intrarenal expression of RANTES and IP-10 mRNA were found to be at risk for premature kidney graft loss.

Molecular phenotypes of acute rejection predict kidney graft prognosis.

Earlier detection of antibody-mediated rejection of kidney allografts may improve graft outcomes. Profiling of gene expression holds promise for the diagnosis and prognosis of antibody-mediated rejection. Here, we identified 730 patients who received kidney transplants during 2002-2005, including 21 patients (2.9%) who experienced early acute antibody-mediated rejection. We also identified a matched group of 43 patients with early acute T cell-mediated rejection to serve as controls. Compared with patients with T cell-mediated rejection, those with antibody-mediated rejection had significantly higher intrarenal mRNA expression of the cytoprotective heme oxygenase-1 but had lower expression of the regulatory T cell marker forkhead box P3 (FoxP3), the B cell marker CD20, and the chemokine regulated upon activation, normal T cell expressed and secreted (RANTES). T cell infiltration was similar in both groups of patients. Compared with grafts that had a favorable course, those that failed as a result of antibody-mediated rejection had expression profiles suggesting a lack of regulation (less FoxP3, TGF-beta1, RANTES, and CD20). Grafts that failed as a result of T cell-mediated rejection only revealed lower expression of CD20 mRNA. In summary, these data suggest that severe antibody-mediated rejection and T cell-mediated rejection result in graft loss by distinct mechanisms. Molecular phenotypes of early acute rejection might help to identify grafts with poor prognosis, allowing earlier application of additional therapies.

Haplotypic structure of ABCB1/MDR1 gene modifies the risk of the acute allograft rejection in renal transplant recipients.

BACKGROUND: Bioavailability of tacrolimus (Tac) and cyclosporine is determined by cytochrome P450IIIA and by P-glycoprotein encoded by the CYP3A4/CYP3A5 and ABCB1 genes. Polymorphisms in these genes have been suggested to influence acute rejection and pharmacokinetics in renal transplantation. We aimed to validate these findings in a haplotype analysis. METHODS: A total of 832 renal transplant recipients were genotyped for the CYP3A4 -288A>G, CYP3A5 +6986G>A, ABCB1 +1236C>T, +2677G>T>A, and +3435C>T polymorphisms. Their association with acute rejection and with pharmacokinetic parameters was analyzed in haplotype models. RESULTS: Apart from human leukocyte antigen-DR mismatches, delayed graft function and age at renal transplantation, acute rejection was also predicted by the [ABCB1 +1236C; +2677G; +3435T] haplotype. Allograft survival was determined by donor age, age at renal transplantation, delayed graft function, cold ischemia, and history of more than two acute rejections. Homozygotes for the [CYP3A4 -288A; CYP3A5 +6986G] haplotype achieved earlier therapeutic concentrations of Tac and a higher concentration to dose ratio at week 1. ABCB1 haplotypes did not influence pharmacokinetic parameters. CONCLUSIONS: ABCB1 haplotypes modify the risk of acute rejection, suggesting that ABCB1 allelic arrangement is a stronger regulator of P-glycoprotein activity than single polymorphisms. The risk of acute rejection determined by ABCB1 is independent of pharmacokinetic parameters. CYP3A haplotypes control the bioavailability of Tac, but do not modify the risk of acute rejection.

Genetic variability of major inflammatory mediators has no impact on the outcome of kidney transplantation.

BACKGROUND: Functionally relevant polymorphisms in genes of the Th1 and Th2-inflammatory pathway influence the susceptibility to acute rejection (AR), chronic allograft nephropathy (CAN), and subclinical rejection (SR) as well as graft survival after renal transplantation. Because these findings have not been validated, we sought confirmatory evidence of these associations in a larger group of renal transplant recipients. METHODS: A total of 436 kidney transplant recipients were genotyped for 9 single nucleotide polymorphisms (TNF-alpha-308G/A, MCP-1-2518A/G, RANTES-403G/A, -109T/C and -28C/G, CCR2+190G/A, IFN-gamma+874A/T, TGF-beta+869T/C and +915G/C) and for the 32-bp indel polymorphism in CCR5. The effects of these polymorphisms on the incidence of AR, SR, CAN and graft survival were analyzed in single locus and haplotype models. RESULTS: Single locus analysis revealed that there was no significant difference in the distribution of the genotype frequencies between patients with and without AR, and between patients with CAN or SR, and individuals without CAN. Furthermore, no influence of any of the polymorphisms on the long-term graft survival was observed. Haplotype [TGF-beta +869G; TGF-beta +915C] seemed to be associated with the presence of SR (odds ratio: 3.45, 95% confidence interval: 1.19 - 9.99, P=0.023), but the association was nonsignificant due to the insufficient power. CONCLUSION: In contrast to previous allelic association studies, neither of the polymorphisms has been associated with the outcome of kidney transplantation in the single locus analysis nor in the haplotype model. Our findings reinforce the need for more rigorous research compliant with the currently accepted standards for polymorphism-disease association studies.

Intrarenal cytokine and chemokine gene expression and kidney graft outcome.

AIMS: Proinflammatory cytokines are thought to play an important role in various kidney graft diseases resulting in interstitial fibrosis and tubular atrophy frequently found in case biopsies. To explore the role of various cytokines and chemokines in the long-term graft outcome, the transcription patterns of their genes in kidney allograft biopsies were evaluated. METHODS: The real-time RT-PCR was used to identify intragraft mRNA expression of cytokines and chemokines in 74 kidney graft recipients and the results were correlated with histological and clinical parameters and long-term graft outcome. RESULTS: We observed up-regulated IL-10 (p < 0.001), TGF-beta1, IL-6, MCP-1, RANTES (p < 0.01) and TNF-alpha (p < 0.05) mRNA expression in patients with chronic allograft nephropathy (CAN) as compared to controls. There were positive correlations between the mRNA expression of IL-6 (p < 0.001), IL-10 (p < 0.01), TNF-alpha, MCP-1 (p < 0.05) and the proteinuria. The up-regulation of intrarenal MCP-1 in patients with CAN increased the risk for the graft failure within the next 42 months (OR 5.1, p < 0.05). Kaplan-Meier survival analysis revealed that proteinuria and higher intragraft expression of TGF-beta1 and MCP-1 predict a poor kidney graft outcome. CONCLUSION: Expression patterns of intrarenal proinflammatory genes might discriminate patients at a higher risk for the earlier allograft failure.