BACKGROUND: Steroid avoidance in immunosuppression in kidney transplantation offers several metabolic advantages, however it is associated with higher early acute rejection rate. Cellular and molecular mechanisms of this phenomenon remain poorly understood. METHODS: In this single center observational study, low-risk kidney transplant recipients randomized into large multicenter prospective ADVANCE trial with steroid avoidance/early withdrawal and center standard of care treated patients were monitored for 12months. The expressions of 28 transcripts, associated with alloimmune response and operational tolerance, were evaluated in the peripheral blood using RT-qPCR at 0, 7, 14, 90 and 365 postoperative days (POD) and in the protocol graft biopsy at 3months while lymphocyte subpopulations were analyzed by flow-cytometry within the follow-up. RESULTS: Both steroid avoidance and withdrawal regimens were associated with significantly higher granzyme B (GZMB) transcript at POD 14 and perforin 1 (PRF1) transcript at POD 7. The higher interleukin 2 (IL-2) expression at POD 7 was detected only in the steroid avoidance group. Initial steroids decreased the expression SH2D1B transcript at POD14 and there were no further differences in other operational tolerance transcripts among groups. The statistically significant decrease in absolute numbers of peripheral NK cells in the first 14days was observed in the standard of care group only. There were no differences in analyzed intrarenal transcripts in 3-month biopsies among groups. CONCLUSIONS: The enhanced expression of some of Th1 associated transcripts and limited effects on NK cells of steroid avoidance immunosuppression suggest higher susceptibility for early acute rejection.

• Klíčová slova
• ADVANCE trial, Kidney transplantation, Rejection, Steroid elimination, Tolerance,
• MeSH
• buňky NK imunologie   metabolismus   MeSH
• dospělí MeSH
• imunosupresivní léčba metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• prospektivní studie MeSH
• regulace genové exprese * MeSH
• senioři MeSH
• Th1 buňky imunologie   metabolismus   MeSH
• transplantace ledvin * MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• pozorovací studie MeSH
• randomizované kontrolované studie MeSH

BACKGROUND: There is high medical need for safe long-term immunosuppression monotherapy in kidney transplantation. Selective targeting of post-transplant alloantigen-(re)activated effector-T cells by anti-TNF antibodies after global T cell depletion may allow safe drug minimization, however, it is unsolved what might be the best maintenance monotherapy. METHODS: In this open, prospective observational single-centre trial, 20 primary deceased donor kidney transplant recipients received 2x20 mg Alemtuzumab (d0/d1) followed by 5 mg/kg Infliximab (d2). For 14 days all patients received only tacrolimus, then they were allocated to either receive tacrolimus (TAC, n = 13) or sirolimus (SIR, n = 7) monotherapy, respectively. Protocol biopsies and extensive immune monitoring were performed and patients were followed-up for 60 months. RESULTS: TAC-monotherapy resulted in excellent graft survival (5yr 92%, 95%CI: 56.6-98.9) and function, normal histology, and no proteinuria. Immune monitoring revealed low intragraft inflammation (urinary IP-10) and hints for the development of operational tolerance signature in the TAC- but not SIR-group. Remarkably, the TAC-monotherapy was successful in all five presensitized (ELISPOT+) patients. However, recruitment into SIR-arm was stopped (after n = 7) because of high incidence of proteinuria and acute/chronic rejection in biopsies. No opportunistic infections occurred during follow-up. CONCLUSIONS: In conclusion, our novel fast-track TAC-monotherapy protocol is likely to be safe and preliminary results indicated an excellent 5-year outcome, however, a full-scale study will be needed to confirm our findings. TRIAL REGISTRATION: EudraCT Number: 2006-003110-18.

• MeSH
• antigen CD52 MeSH
• antigeny nádorové MeSH
• biologické markery metabolismus   MeSH
• CD antigeny MeSH
• dospělí MeSH
• glykoproteiny antagonisté a inhibitory   MeSH
• imunosupresiva terapeutické užití   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• monitorování imunologické MeSH
• prospektivní studie MeSH
• rejekce štěpu farmakoterapie   etiologie   MeSH
• sirolimus terapeutické užití   MeSH
• stanovení celkové genové exprese MeSH
• takrolimus terapeutické užití   MeSH
• TNF-alfa antagonisté a inhibitory   MeSH
• transplantace ledvin škodlivé účinky   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• klinické zkoušky MeSH
• pozorovací studie MeSH
• Názvy látek
• antigen CD52 MeSH
• antigeny nádorové MeSH
• biologické markery MeSH
• CD antigeny MeSH
• CD52 protein, human MeSH Prohlížeč
• glykoproteiny MeSH
• imunosupresiva MeSH
• sirolimus MeSH
• takrolimus MeSH
• TNF-alfa MeSH

Secondary hyperparathyroidism is a well-known complication of end-stage renal disease (ESRD). Both nodular and diffuse parathyroid hyperplasia occur in ESRD patients. However, their distinct molecular mechanisms remain poorly understood. Parathyroid tissue obtained from ESRD patients who had undergone parathyroidectomy was used for Illumina transcriptome screening and subsequently for discriminatory gene analysis, pathway mapping, and gene annotation enrichment analysis. Results were further validated using quantitative RT-PCR on the independent larger cohort. Microarray screening proved homogeneity of gene transcripts in hemodialysis patients compared with the transplant cohort and primary hyperparathyroidism; therefore, further experiments were performed in hemodialysis patients only. Enrichment analysis conducted on 485 differentially expressed genes between nodular and diffuse parathyroid hyperplasia revealed highly significant differences in Gene Ontology terms and the Kyoto Encyclopedia of Genes and Genomes database in ribosome structure (P = 3.70 × 10-18). Next, quantitative RT-PCR validation of the top differently expressed genes from microarray analysis proved higher expression of RAN guanine nucleotide release factor (RANGRF; P < 0.001), calcyclin-binding protein (CACYBP; P < 0.05), and exocyst complex component 8 (EXOC8; P < 0.05) and lower expression of peptidylprolyl cis/trans-isomerase and NIMA-interacting 1 (PIN1; P < 0.01) mRNA in nodular hyperplasia. Multivariate analysis revealed higher RANGRF and lower PIN1 expression along with parathyroid weight to be associated with nodular hyperplasia. In conclusion, our study suggests the RANGRF transcript, which controls RNA metabolism, to be likely involved in pathways associated with the switch to nodular parathyroid growth. This transcript, along with PIN1 transcript, which influences parathyroid hormone secretion, may represent new therapeutical targets to cure secondary hyperparathyroidism.

• Klíčová slova
• diffuse parathyroid hyperplasia, microarray, nodular parathyroid hyperplasia,
• MeSH
• chronické selhání ledvin komplikace   terapie   MeSH
• dialýza ledvin * MeSH
• dospělí MeSH
• fokální nodulární hyperplazie etiologie   genetika   terapie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• messenger RNA biosyntéza   genetika   MeSH
• multigenová rodina genetika   MeSH
• parathormon krev   MeSH
• paratyreoidea patologie   MeSH
• paratyreoidektomie MeSH
• primární hyperparatyreóza patologie   MeSH
• regulace genové exprese genetika   MeSH
• sekundární hyperparatyreóza etiologie   genetika   terapie   MeSH
• senioři MeSH
• stanovení celkové genové exprese MeSH
• transkriptom genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• parathormon MeSH

B cells play an important role in the immune responses which affect the outcomes of kidney allografts. Dynamic changes of B-cell compartments in clinical kidney transplantation are still poorly understood. B-cell subsets were prospectively monitored using flow cytometry for 1 year in 98 kidney transplant recipients. Data were correlated with immunosuppression and clinical outcomes. An increase in the total population of B lymphocytes was observed during the first week after transplantation. The level of IgM(high) CD38(high) CD24(high) transitional B cells reduced significantly up until the third month, with partial repopulation in the first year. Lower numbers of transitional B cells in the third month were associated with higher risk of graft rejection. IgM(+) IgD(+) CD27(-) naive B cells did not change within follow-up. IgM(+) CD27(+) nonswitched memory B cells and IgM(-) CD27(+) switched memory B cells increased on post-operative day 7. IgM(-) CD38(high) CD27(high) plasmablasts showed similar kinetics during the first post-transplant year, similar to transitional B cells. In conclusion, sensitized kidney transplant recipients as well as those with either acute or chronic rejection within the first post-transplant year exhibited lower levels of transitional B cells. Therefore, these data further support the hypothesis that transitional B cells have a protective role in kidney transplantation.

• Klíčová slova
• B cells, kidney transplantation, rejection, transitional B cells,
• MeSH
• alografty MeSH
• antigen CD24 metabolismus   MeSH
• antigeny CD27 metabolismus   MeSH
• antigeny CD38 metabolismus   MeSH
• časové faktory MeSH
• dítě MeSH
• dospělí MeSH
• imunologická paměť imunologie   MeSH
• imunosupresivní léčba MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• plazmatické buňky imunologie   MeSH
• podskupiny B-lymfocytů imunologie   MeSH
• předškolní dítě MeSH
• prekurzorové B-lymfoidní buňky imunologie   MeSH
• příjemce transplantátu MeSH
• prospektivní studie MeSH
• rejekce štěpu imunologie   MeSH
• senioři MeSH
• senzitivita a specificita MeSH
• transplantace ledvin * MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigen CD24 MeSH
• antigeny CD27 MeSH
• antigeny CD38 MeSH

The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3) in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information from 48 hrs onwards.

• MeSH
• inzulin metabolismus   MeSH
• krysa rodu Rattus MeSH
• Langerhansovy ostrůvky metabolismus   MeSH
• sekrece inzulinu MeSH
• stanovení celkové genové exprese * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inzulin MeSH

BACKGROUND: Induction therapy can improve kidney transplantation (KTx) outcomes, but little is known about the mechanisms underlying its effects. METHODS: The mRNA levels of T cell-related genes associated with tolerance or rejection (CD247, GZMB, PRF1, FOXP3, MAN1A1, TCAIM, and TLR5) and lymphocyte subpopulations were monitored prospectively in the peripheral blood of 60 kidney transplant recipients before and 7, 14, 21, 28, 60, 90 days, 6 months, and 12 months after KTx. Patients were treated with calcineurin inhibitor-based triple immunosuppression and induction with rabbit anti-thymocyte globulin (rATG, n = 24), basiliximab (n = 17), or without induction (no-induction, n = 19). A generalized linear mixed model with gamma distribution for repeated measures, adjusted for rejection, recipient/donor age and delayed graft function, was used for statistical analysis. RESULTS: rATG treatment caused an intense reduction in all T cell type population and natural killer (NK) cells within 7 days, then a slow increase and repopulation was observed. This was also noticed in the expression levels of CD247, FOXP3, GZMB, and PRF1. The basiliximab group exhibited higher CD247, GZMB, FOXP3 and TCAIM mRNA levels and regulatory T cell (Treg) counts than the no-induction group. The levels of MAN1A1 and TLR5 mRNA expressions were increased, whereas TCAIM decreased in the rATG group as compared with those in the no-induction group. CONCLUSION: The rATG induction therapy was associated with decreased T and NK cell-related transcript levels and with upregulation of two rejection-associated transcripts (MAN1A1 and TLR5) shortly after KTx. Basiliximab treatment was associated with increased absolute number of Treg cells, and increased level of FOXP3 and TCAIM expression.

• MeSH
• antigeny CD3 genetika   MeSH
• antilymfocytární sérum terapeutické užití   MeSH
• basiliximab MeSH
• dospělí MeSH
• exprese genu účinky léků   MeSH
• forkhead transkripční faktory genetika   MeSH
• granzymy genetika   MeSH
• imunologická tolerance účinky léků   genetika   MeSH
• imunosupresiva terapeutické užití   MeSH
• imunosupresivní léčba metody   MeSH
• indukční chemoterapie metody   MeSH
• inhibitory kalcineurinu terapeutické užití   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mannosidasy genetika   MeSH
• messenger RNA krev   MeSH
• mladý dospělý MeSH
• monoklonální protilátky terapeutické užití   MeSH
• NKT buňky účinky léků   imunologie   MeSH
• perforin genetika   MeSH
• počet lymfocytů MeSH
• prospektivní studie MeSH
• regulační T-lymfocyty účinky léků   imunologie   MeSH
• rejekce štěpu genetika   imunologie   prevence a kontrola   MeSH
• rekombinantní fúzní proteiny terapeutické užití   MeSH
• senioři MeSH
• transplantace ledvin metody   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD3 MeSH
• antilymfocytární sérum MeSH
• basiliximab MeSH
• CD3 antigen, zeta chain MeSH Prohlížeč
• forkhead transkripční faktory MeSH
• FOXP3 protein, human MeSH Prohlížeč
• granzymy MeSH
• GZMB protein, human MeSH Prohlížeč
• imunosupresiva MeSH
• inhibitory kalcineurinu MeSH
• mannosidasy MeSH
• mannosyl-oligosaccharide 1,2-alpha-mannosidase MeSH Prohlížeč
• messenger RNA MeSH
• monoklonální protilátky MeSH
• perforin MeSH
• PRF1 protein, human MeSH Prohlížeč
• rekombinantní fúzní proteiny MeSH

BACKGROUND/AIMS: Subclinical rejection diagnosed from protocol biopsies is thought to be a risk factor of long- term allograft dysfunction. The reason why in some patients subclinical rejection does not represent risk for progression is not fully understood. METHODS: The intragraft expression of 376 target genes involved in chemokine defense, apoptosis, inflammation, tolerance and TGF-β signalling pathways was measured using quantitative real-time RT-PCR (2(-)∆∆(Ct)) method in subclinical inflammation (SCI, n=10), clinical inflammation in acute T-cell mediated rejection (CI, n=10) and no rejection samples (n=9). RESULTS: Clinical inflammation group showed a increased expression of genes for chemotaxis mediating cytokines (CCL1, CCL17, CCL24, CCL25, CCL26), cytokine receptors (CCR1, CCRL2, IL1RAPL2, CXCR5), proinflammatory cytokines (IL12A, LTA), inflammatory mediator (PTAFR), complement protein C3, executioner protein of apoptosis (CASP7), growth factor (TGFA), colony stimulating factor (CSF-2), proteins involved in dendritic cells differentiation and interaction (CD209, LAMP3), regulation of immune response (LILRB2, LILBRB4). The quantitative difference in transcripts signature between SCI and CI is consistent with stronger proinflammatory setting of CI. Prostaglandin E2 receptor gene expression was independently associated with lower risk of further graft function deterioration (OR 0.11, CI 0.01-0.78, p<0.0001). CONCLUSION: Subclinical acute kidney inflammation has transcriptional profile of immune injury of lower extend compared to clinical acute inflammation.

• MeSH
• apoptóza MeSH
• biopsie MeSH
• chemokiny metabolismus   MeSH
• cytokiny metabolismus   MeSH
• dospělí MeSH
• exprese genu genetika   MeSH
• imunosupresiva terapeutické užití   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mediátory zánětu metabolismus   MeSH
• přežívání štěpu MeSH
• progrese nemoci MeSH
• receptory cytokinové metabolismus   MeSH
• rejekce štěpu genetika   metabolismus   MeSH
• riziko MeSH
• RNA analýza   biosyntéza   MeSH
• senioři MeSH
• signální transdukce MeSH
• transformující růstový faktor beta metabolismus   MeSH
• transplantace ledvin * MeSH
• výsledek terapie MeSH
• zánět patologie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chemokiny MeSH
• cytokiny MeSH
• imunosupresiva MeSH
• mediátory zánětu MeSH
• receptory cytokinové MeSH
• RNA MeSH
• transformující růstový faktor beta MeSH