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An ideal decellularized allogenic or xenogeneic cardiovascular graft should be capable of preventing thrombus formation after implantation. The antithrombogenicity of the graft is ensured by a confluent endothelial cell layer formed on its surface. Later repopulation and remodeling of the scaffold by the patient’s cells should result in the formation of living autologous tissue. In the work presented here, decellularized porcine pericardium scaffolds were modified by growing a fibrin mesh on the surface and inside the scaffolds, and by attaching heparin and human vascular endothelial growth factor (VEGF) to this mesh. Then the scaffolds were seeded with human adipose tissue-derived stem cells (ASCs). While the ASCs grew only on the surface of the decellularized pericardium, the fibrin-modified scaffolds were entirely repopulated in 28 d, and the scaffolds modified with fibrin, heparin and VEGF were already repopulated within 6 d. Label free mass spectrometry revealed fibronectin, collagens, and other extracellular matrix proteins produced by ASCs during recellularization. Thin layers of human umbilical endothelial cells were formed within 4 d after the cells were seeded on the surfaces of the scaffold, which had previously been seeded with ASCs. The results indicate that an artificial tissue prepared by in vitro recellularization and remodeling of decellularized non-autologous pericardium with autologous ASCs seems to be a promising candidate for cardiovascular grafts capable of accelerating in situ endothelialization. ASCs resemble the valve interstitial cells present in heart valves. An advantage of this approach is that ASCs can easily be collected from the patient by liposuction.

The onset of IgA nephropathy (IgAN), characterized by glomerular deposition of IgA-containing immune complexes, is often associated with synpharyngitic hematuria. Innate immune responses mediated by Toll-like receptors (TLR) may play a role in IgAN onset and/or progression. Here, we assessed the expression of TLR 4, 7, 8, and 9 in renal-biopsy specimens from patients with IgAN, with different degree of proteinuria and eGFR, compared with normal-kidney and disease-control tissues (ANCA-associated vasculitis). Renal-biopsy specimens from 34 patients with IgAN and 7 patients with ANCA-associated vasculitis were used. In addition, we used 15 healthy portions of renal-tissue specimens from kidneys after nephrectomy for cancer as control specimens. Expression of TLR 4, 7, 8, and 9 was assessed using immunohistochemical staining of paraffin-embedded renal-biopsy tissue specimens with specific antibodies and evaluated semiquantitatively by light microscopy. Linear discriminant analysis (LDA) was used to test whether intrarenal staining of TLR 4, 7, 8, and 9 distinguished patients with IgAN from controls or correlated with eGFR and/or proteinuria. eGFR was calculated using the creatinine-based formula. Moreover, the biopsies from patients with IgAN were scored according to the Oxford Classification. LDA showed that staining for TLR 4, 7, 8, and 9 was more intense in specimens from IgAN patients compared to normal kidney tissues. The intensity of intrarenal staining of TLRs discriminated four groups of IgAN patients with different eGFR and proteinuria and MEST scoring.

Butyrate produced by the intestinal microbiota is essential for proper functioning of the intestinal immune system. Total dependence on parenteral nutrition (PN) is associated with numerous adverse effects, including severe microbial dysbiosis and loss of important butyrate producers. We hypothesised that a lack of butyrate produced by the gut microbiota may be compensated by its supplementation in PN mixtures. We tested whetheri.v.butyrate administration would (a) positively modulate intestinal defence mechanisms and (b) counteract PN-induced dysbiosis. Male Wistar rats were randomised to chow, PN, and PN supplemented with 9 mM butyrate (PN+But) for 12 days. Antimicrobial peptides, mucins, tight junction proteins, and cytokine expression were assessed by RT-qPCR. T-cell subpopulations in mesenteric lymph nodes (MLN) were analysed by flow cytometry. Microbiota composition was assessed in caecum content. Butyrate supplementation resulted in increased expression of tight junction proteins (ZO-1, claudin-7, E-cadherin), antimicrobial peptides (Defa 8, Rd5, RegIIIγ), and lysozyme in the ileal mucosa. Butyrate partially alleviated PN-induced intestinal barrier impairment and normalised IL-4, IL-10, and IgA mRNA expression. PN administration was associated with an increase in Tregs in MLN, which was normalised by butyrate. Butyrate increased the total number of CD4+ and decreased a relative amount of CD8+ memory T cells in MLN. Lack of enteral nutrition and PN administration led to a shift in caecal microbiota composition. Butyrate did not reverse the altered expression of most taxa but did influence the abundance of some potentially beneficial/pathogenic genera, which might contribute to its overall beneficial effect.

Butyrate produced by the intestinal microbiota is essential for proper functioning of the intestinal immune system. Total dependence on parenteral nutrition (PN) is associated with numerous adverse effects, including severe microbial dysbiosis and loss of important butyrate producers. We hypothesised that a lack of butyrate produced by the gut microbiota may be compensated by its supplementation in PN mixtures. We tested whetheri.v.butyrate administration would (a) positively modulate intestinal defence mechanisms and (b) counteract PN-induced dysbiosis. Male Wistar rats were randomised to chow, PN, and PN supplemented with 9 mM butyrate (PN+But) for 12 days. Antimicrobial peptides, mucins, tight junction proteins and cytokine expression were assessed by RT-qPCR. T-cell subpopulations in mesenteric lymph nodes (MLN) were analysed by flow cytometry. Microbiota composition was assessed in caecum content. Butyrate supplementation resulted in increased expression of tight junction proteins (ZO-1, claudin-7, E-cadherin), antimicrobial peptides (Defa 8, Rd5, RegIIIγ) and lysozyme in the ileal mucosa. Butyrate partially alleviated PN-induced intestinal barrier impairment and normalised IL-4, IL-10 and IgA mRNA expression. PN administration was associated with an increase in Tregs in MLN, which was normalised by butyrate. Butyrate increased the total number of CD4+ and decreased a relative amount of CD8+ memory T cells in MLN. Lack of enteral nutrition and PN administration led to a shift in caecal microbiota composition. Butyrate did not reverse the altered expression of most taxa but did influence the abundance of some potentially beneficial/ pathogenic genera, which might contribute to its overall beneficial effect.

Acetaminophen or paracetamol (APAP) overdose is a common cause of liver injury. Silymarin (SLM) is a hepatoprotective agent widely used for treating liver injury of different origin. In order to evaluate the possible beneficial effects of SLM, Balb/c mice were pretreated with SLM (100 mg/kg b.wt. per os) once daily for three days. Two hours after the last SLM dose, the mice were administered APAP (300 mg/kg b.wt. i.p.) and killed 6 (T6), 12 (T12) and 24 (T24) hours later. SLM-treated mice exhibited a significant reduction in APAP-induced liver injury, assessed according to AST and ALT release and histological examination. SLM treatment significantly reduced superoxide production, as indicated by lower GSSG content, lower HO-1 induction, alleviated nitrosative stress, decreased p-JNK activation and direct measurement of mitochondrial superoxide production in vitro. SLM did not affect the APAP-induced decrease in CYP2E1 activity and expression during the first 12 hrs. Neutrophil infiltration and enhanced expression of inflammatory markers were first detected at T12 in both groups. Inflammation progressed in the APAP group at T24 but became attenuated in SLM-treated animals. Histological examination suggests that necrosis the dominant cell death pathway in APAP intoxication, which is partially preventable by SLM pretreatment. We demonstrate that SLM significantly protects against APAP-induced liver damage through the scavenger activity of SLM and the reduction of superoxide and peroxynitrite content. Neutrophil-induced damage is probably secondary to necrosis development.

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, pConclusion: Subclinical acute kidney inflammation has transcriptional profile of immune injury of lower extend compared to clinical acute inflammation.

BACKGROUND: Delayed graft function (DGF) caused by ischemia/reperfusion injury (I/RI) negatively influences the outcome of kidney transplantation. This prospective single-center study characterized the intrarenal transcriptome during I/RI as a means of identifying genes associated with DGF development. METHODS: Characterization of the intrarenal transcription profile associated with I/RI was carried out on three sequential graft biopsies from respective allografts before and during transplantation. The intragraft expression of 92 candidate genes was measured using quantitative real-time reverse transcriptase polymerase chain reaction (2) in delayed (n=9) and primary function allografts (n=26). RESULTS: Cold storage was not associated with significant changes to the expression profile of the target gene transcripts; however, up-regulation of 16 genes associated with enhanced activation of innate and adaptive immune responses and apoptosis was observed after reperfusion. Multivariate logistic regression analysis revealed that higher tubular atrophy scores (ct) together with a lower expression of Netrin-1 might predict DGF development (training area under the receiver operating curve=0.89, cross-validated area under the receiver operating curve=0.81). CONCLUSIONS: Poor baseline tubular cell quality (defined by a higher rate of tubular atrophy) combined with the reduced potential of apoptotic survival factors represented by decreased Netrin-1 gene expression were associated with delayed kidney graft function.

Aims Recent studies in patients with dilated cardiomyopathy (DCM) have detected the genome of Borrelia burgdorferi sensu lato (BBSL) in endomyocardial biopsy (EMB) specimens using a qualitative polymerase chain reaction (PCR), suggesting a causal link between Lyme disease and DCM in areas in which Lyme disease is endemic. We aimed to study this relationship using a comprehensive molecular analysis detecting BBSL in EMB samples. Methods and results We performed a comprehensive histopathological, immunohistochemical, ultrastructural, and molecular analysis targeting cardiotropic viruses and BBSL in EMB specimens of 41 individuals with recent-onset DCM and 15 controls with end-stage coronary artery disease. Specifically, quantitative PCR and electron microscopy of EMB specimens were employed. In addition, autoantibodies and manifestation of autoimmune diseases were evaluated in both groups. Individuals with recent-onset DCM presented more frequently with myocardial BBSL persistence as compared with the control group (24% vs. 0%, P = 0.035). In contrast, the prevalence of parvovirus B19 and cytomegalovirus was similar in both groups. Sequence analysis of borrelial DNA revealed the following genospecies: Borrelia burgdorferi sensu stricto in three patients (30%), Borrelia afzelii in two patients (20%), and Borrelia garinii in four patients (40%), the results being inconclusive in one case. BBSL-positive DCM patients had a higher prevalence of organ-specific autoimmune diseases in comparison with the remaining DCM patients (50% vs. 16%, P = 0.030). Conclusion Myocardial persistence of BBSL may be involved in the pathophysiology of DCM in individuals living in areas in which Lyme disease is endemic.

In vitro labeling of pancreatic islets with iron nanoparticles enables their direct posttransplant visualization by magnetic resonance; however, there is still a discrepancy in the fate of iron nanoparticles. This study was performed to detail the labeling process, consequently to improve the labeling efficacy and to confirm safety for islet cells. The islets were visible on T 2 *-weighted magnetic resonance images as hypointense spots immediately after 1-hr cultivation. Although at this time already the sufficient superparamagnetic effect was achieved, most of the particles were deposed in islet macrophages and only later were they found in endosomes of endocrine islet cells. The iron content depended on length of culture period. The labeled islets showed an intact ultrastructure, responded normally to glucose stimulation in vitro, and were able to treat experimental diabetes. For purpose of subsequent magnetic resonance imaging, a 24-hr culture with ferucarbotran leads to sufficient labeling with no apparent adverse effect on beta cell morphology or function.

Aim: Organic anion-transporting polypeptides OATP1B1 and OATP1B3 are sinusoidal membrane transporters mediating liver uptake of a wide range of substrates including conjugated and unconjugated bilirubin, xenobiotics and drugs. Absence of OATP1Bs in the liver causes Rotor syndrome. Our aim was to correlate OATP1B expression with hyperbilirubinemia in common liver diseases. Methods: Immunoreactivity of five antibodies against human OATP1Bs was tested on frozen and formalin-fixed paraffin-embedded liver tissue of mouse strains transgenic for SLCO1B1 or SLCO1B3 and on human specimens. The proportion of hepatocytes expressing OATP1Bs was then assessed immunohistologically in formalin-fixed paraffin-embedded liver samples obtained from patients with hepatocellular and primary biliary liver diseases. UGT1A1 promoter TATA-box and SLCO1B1 rs4149056 genotyping was performed to rule out individuals predisposed to hyperbilirubinemia. Results: The most specific detection of OATP1B3 was achieved with the H-52 (sc-98981) antibody. OATP1B1 was specifically recognized with the ESL (ab15441) anti-OATP1B1 antibody, but only in frozen sections. The MDQ (ab15442) anti-OATP1B1 antibody cross-reacted with both OATP1B proteins in liver tissue of the transgenic mouse strains. Expression of the OATP1B proteins was decreased in advanced liver diseases and inversely correlated with serum bilirubin levels. The reduction was more pronounced in advanced primary biliary diseases (1.9±1.1 vs. 2.7±0.6; P=0.009). Conclusions: Down-regulation of OATP1B proteins may contribute to pathogenesis of jaundice accompanying advanced cholestatic liver diseases.

Background: Renal ischemia and hypertension have been suggested to be involved in the progression of renal diseases. Recently, we developed a model of accelerated major histocompatibility complex-independent renal injury, where high-renin hypertension aggravates functional and morphological changes induced by ischemia/reperfusion (I/R). In this model, we evaluated the effect of immunosuppressant mycophenolate mofetil (MMF) to test its capability to slow the progression of accelerated nephropathy. Methods: 34 anesthetized uninephrectomized hypertensive transgenic (mREN2)27 rats (TGR) received a clamp on the renal pedicle for 45 min. Animals were treated with MMF 10 mg/kg/day (n = 10), 20 mg/kg/day (n = 10) or placebo (n = 10) orally via gavage for 12 weeks. Four animals were sham operated and not treated. Proteinuria and blood pressure were evaluated throughout the experiment. At the end of the experiment, kidney function was evaluated and kidneys harvested for morphological analysis and immunohistochemistry (CD4+, CD8+ lymphocytes and specific rat monocyte/macrophage marker ED-1+ cells). Results: At week 12, both MMF-treated groups had lower proteinuria as compared to the placebo group (MMF 10: 22.4 ± 9.8, MMF 20: 20.9 ± 5.6 vs. 126.7 ± 35.8; p < 0.01; sham 28.1 ± 1.4 mg/day) and reduced glomerulosclerosis (MMF 10: 11.4 ± 7.8, MMF 20: 5.2 ± 2.7 vs. 20.9 ± 10.9; p < 0.05; sham 15.7 ± 9.2%). There were no differences in systolic blood pressure among groups. MMF-treated rats had lower CD4+ (MMF 10: 61.2 ± 46.4, MMF 20: 29.3 ± 18.2 vs. 125.3 ± 42.8; p < 0.01, sham 84.9 ± 6.1 cells/field of view) and CD8+ (MMF 10: 13.7 ± 10.2, MMF 20: 10.0 ± 8.1 vs. 37.8 ± 14.3; p < 0.01; sham: 31.8 ± 7.6 cells/field of view) lymphocytes infiltration and ED-1 macrophages infiltration (MMF 10: 5.5 ± 6.4, MMF 20: 2.5 ± 2.8 vs. 16.7 ± 4.1; p < 0.01; sham 12.2 ± 4.6 cells/field of view) than placebo-treated rats. Conclusion: Our results thus support the hypothesis about the key role of immune mechanisms in progression of chronic nephropathies.

Interleukin 18 (IL-18) is a potent proinflammatory cytokine involved in the host defence by upregulating both innate and acquired immune responses and may be of particular importance also in mechanisms of kidney allograft rejection. Immunohistochemical staining of protocol biopsies showed constitutive IL-18 expression in the epithelium of distal tubules with the induction of immunoreactivity in acute rejection patients where also proximal tubules, infiltrating leukocytes, and endothelium were strongly positive. Furthermore, serum levels of IL-18 were significantly elevated in patients with acute rejection of kidney allograft (1247+/-389 pg/l) as compared to patients with uncomplicated outcome of kidney transplantation (444+/-164 pg/l) and subjects with acute tubulointerstitial nephropathy (385+/-155 pg/l, p

Background. Renal ischaemia/reperfusion (I/R) injury and hypertension represent major alloantigen-independent factors contributing to the development of chronic allograft nephropathy of renal allografts. In the present study, we investigated the effect of the anti-proliferative immunosuppressant, sirolimus (SIR), in a model of accelerated renal injury in hypertensive transgenic rats (TGRs). Twenty anaesthetized uninephrectomized TGRs with renin overproduction [TGR(mREN2)27] and 20 normotensive Han SD (SD) rats as genetic controls had their renal pedicles clipped for 45 min and were subsequently treated with either SIR (0.5 mg/kg per day, orally) or placebo (n=10 in each group) for 16 weeks, after which time the kidneys were harvested for morphological and immunohistochemical analysis. High-renin hypertension aggravated the functional and structural changes induced by I/R in SD animals: both SIR-treated and untreated TGRs exhibited significantly greater proteinuria and suffered from more severe glomerulosclerosis (P

Background. Malakoplakia is an unusual chronic inflammatory disease with distinctive histopathological features rarely involving the parenchyma of a transplanted kidney, and to date less than ten cases have been reported. Methods and results. We present a case of malakoplakia of a kidney graft in a 31 year old woman after simultaneous kidney and pancreas transplantation, which was successfully treated with quinolones. After the treatment of malakoplakia, she was monitored regularly, and her renal and pancreas grafts functioned well for the following 9 years, which is 12 years post transplantation. Moreover, 1 year after treatment of malakoplakia she became pregnant and gave birth to a healthy child. Conclusion. Evaluation of a kidney biopsy sample represents the key to diagnosis of malakoplakia which is important for correct patient management. Treatment with antibiotics with intracellular penetration (quinolone type) may result in curing the disease. According to our knowledge, this is the first case of allograft renal malakoplakia after combined kidney and pancreas transplantation.

The significance of borderline changes in kidney allograft biopsies is widely debated. To help resolve this, we studied differences in intrarenal gene expression patterns between early clinical and 3-month protocol biopsies, all of which had borderline histologic changes. The gene expression profiles in training set of patients by microarray analysis and data were validated in a larger cohort using RT-qPCR. There was greater expression of immunity- and inflammation-related genes in the early clinical biopsies compared to the 3-month protocol biopsies with borderline changes. In early clinically manifested borderline changes, graft deterioration within 24 months due to chronic rejection was associated with increased activation of immune, defense, and inflammatory processes. Regression modeling identified higher donor age and expression of macrophage receptor CLEC5A as risk factors for progression. In the 3-month protocol biopsies with borderline changes, graft dysfunction was associated with increased expression of fibrinogen complex transcripts. The discrimination power of fibrinogen was confirmed by cross-validation on two independent cohorts. Thus, our study highlights variations in gene expression between clinical and subclinical borderline changes despite similar histological findings. The data also support a recommendation for frequent patient monitoring, especially in those with borderline changes who received grafts from older donors.

Background. In renal transplant patients with stable graft function, triple-drug immunosuppression may not be necessary, while withdrawal of steroids may eliminate side effects. The primary aim of this study was to assess the risk of rejection after steroid withdrawal. Methods. A total of 88 patients with stable graft function and serum creatinine < 160 μmol/l. treated with cyclosporin A, azathioprine and prednisone were randomized into group A (n=46) with a gradual prednisone reduction to zero in the course of 6 months, and group B (n=42) on triple-drug therapy without change. At the time of randomization, fine-needle aspiration biopsy (FNAB) was carried out in all of the patients. After stopping steroids, the patients were followed up for a period of 12 months. Results. Four patients failed to complete steroid withdrawal, three due to rejection, and one due to leukopenia. The proportion of rejection in three patients in group A (6.6%) was not significantly different from rejection in two patients in group B (4.8%). The mean value of serum creatinine was not significantly different in both groups in the course of follow-up. A finding of some degree of immunological activity in FNAB was made in four patients in each group, but none of these patients developed rejection. Compared with group B, significant decreases in serum cholesterol and blood leukocytes were observed in group A. Prednisone withdrawal did not have any influence on hypertension and serum triglycerides. Conclusions. Gradual withdrawal of steroids is not associated with a higher risk for rejection and has a beneficial effect on serum total cholesterol levels. FNAB was not a useful tool for predicting rejection.

The main problem of currently used xenogeneic biological heart valves is the development of degenerative changes leading to valve failure. Reoperation is required in approximately 65% of patients at 15 years after implantation. The challenge of heart valve tissue engineering is to create a new type of biological prosthesis for clinical use. The aim of our study is to construct a living autologous human pericardial heart valve that will have optimal mechanical properties and a similar histological structure as the normal aortic heart valve. Three leaflet heart valve constructs made from human pericardium were attached onto a plastic holder and cultured under dynamic conditions for up to four weeks. After this time conditioned pericardial samples were compared to control unconditioned pericardial samples from the same patient and to that of the normal aortic heart valve. Histological, immunohistochemical and biomechanical assessments were performed. Pericardial interstitial cells (PICs) are able to respond to mechanical stresses by proliferating and differentiating into an active (myofibroblast-like) phenotype and are able to produce new extracellurar matrix (ECM). A threefold increase in PIC number and a twofold increase in smooth muscle actin (SMA) positive cells was observed after dynamic conditioning. These measurements were statistically significant (p

The objective of our study was to compare the histological structure and cellular organization of autologous human pericardium to that of the human aortic heart valve. Collagen, elastin and glycosaminoglycans are responsible for the mechanical properties of aortic heart valve leaflets. Aortic valve leaflets are composed primarily of collagen representing 50% of total extracellular matrix (ECM). The main collagen types in the aortic heart valve are collagen I (74% of total collagen) and collagen III (24% of total collagen). Elastin represents 13% and is responsible for the elastic properties of the valve leaflet. Collagen has a specific architecture that endows heart valve tissue with the ability to withstand circulatory forces over the course of a lifetime. Its fiber orientation, density and cell associations are very important for this purpose. Normal aortic heart valves were obtained during heart transplantation and compared to autologous human pericardium before and after dynamic conditioning using classical histological assessment, immunohistochemical analysis and confocal microscopy. The architecture of pericardial tissue is very similar to that of the normal aortic heart valve possessing well organized collagen fibers with embedded pericardial interstitial cells (PICs) forming a three dimensional network. Instead of the trilaminar histological organization present in the aortic heart valve, the pericardium possesses one layer whose densely packed collagen bundles closely resemble that of the lamina fibrosa of the native aortic heart valve by confocal microscopy. Elastin fibers are evenly distributed throughout the entire thickness of the pericardium in comparison to the specialized elastin containing layer in the lamina ventricularis of the aortic heart valve. PICs are also evenly distributed throughout the pericardium. In the inner pericardial layer facing the heart these cells have a more spindle-like shape similar to that of valvular interstitial cells (VICs) in the lamina fibrosa, while in the outer part of the pericardium these cells have a more spread-out cytoplasmic morphology interacting with more loosely distributed collagen bundles. Like in the aortic heart valve, PICs show cell-cell interactions in addition to cell-matrix interactions. Our study confirmed similarities in the cellular and ECM organization of human pericardium and the native aortic heart valve. It may be concluded that human autologous pericardium may be a favorable tissue for heart valve replacement. Autologous pericardial tissue may also avoid a negative immune system response that could adversely affect recipient graft uptake and downstream ECM remodeling events.

OBJECTIVE—To assess the effect of normoglycemia following simultaneous pancreas/kidney transplantation (SPK) on neurological function and intraepidermal nerve fiber density (IENFD) in patients with type 1 diabetes. RESEARCH DESIGN AND METHODS—We performed vibration perception threshold (VPT) testing and autonomic function testing (AFT) and assessed IENFD in skin biopsies from the lower thigh and upper calf in 14 healthy control subjects and 18 patients with type 1 diabetes at the time of and at 21–40 (median 29) months post SPK. RESULTS—At baseline, significantly increased VPTs, pathological AFT results, and severe reduction in IENFD were present in SPK recipients. After SPK, an increase of IENFD in the thigh of more than one epidermal nerve fiber per millimeter was noted in three patients (median 4.1, range 1.9–10.2), but changes were not significant for the group as a whole. CONCLUSIONS—We conclude that either irreversible nerve damage might be present in some SPK recipients or that longer periods of normoglycemia might be needed to allow nerve regeneration.

Early postoperative complications after small bowel transplantation (SBT) including endotoxemia, bacterial translocations, and stimulation of the recipient's immune response have been attributed to preservation injury. Small intestine is notoriously sensitive to ischemia and there is still no general agreement as to which segment of the small bowel is preferred (jejunum or ileum) for clinical use.In our study, using light microscopy and concentrations of tissue serotonin-positive cells we sought to identify the part of the human intestine, which is more resistant to preservation injury sustained by HTK preservation solution with 1-24 hr cold ischemia time.Statistical analysis of both parameters did not reveal any significant differences between the jejunum and ileum.Judging by our data, there is no difference between jejunal and ileal grafts in susceptibility to ischemic injury due to cold ischemia within 24 hours when using HTK preservation solution. Significant difference was observed in histological pictures only after 12-hour of cold ischemia time in both experimental groups (jejunum and ileum).

Arterial allografts are used now-a-days as a modality in the treatment of vascular prosthesis infections. Prolonged administration of immunosuppressive drugs seemed to be essential for long-time patency rates of alloarterial vascular reconstructions. Nevertheless, the use of immunosuppressives if there exist an acute infection is controversial. The experimental work described herein studied effects of a delayed low-dose FK 506 administration on the development of acute rejection changes 30 days after aortal transplantation in rats. The response of the recipient's immune system to aortal wall antigens of the donor in the field of no immunosuppression resulted in an intimal proliferation and its infiltration by immunocompetent cells of the recipient, necrosis of medial smooth muscle cells, including deposition of immunoglobulins, and a massive adventitial infiltration of CD4 and CD8 positive cells. On the other hand, all the principal histological signs of rejection listed above were suppressed by FK 506 administration, no matter whether the immunosuppressive was administered on day 0 or day 7 after the transplantation.

Background/Aims: In addition to their lipid-lowering effects, HMG-CoA reductase inhibitors (statins) induce a variety of pleiotropic actions that have been recently studied in the area of cardiovascular and renal protection. In the present studies we sought to determine whether statins retain beneficial effects in the experimental model of NO deficiency achieved by chronic administration of a pressor dose of L-arginine analogue N-nitro-L-arginine-methyl ester (L-NAME). Methods: To address this issue, blood pressure (BP), renal function (GFR), and albuminuria were determined in rats treated for 4 weeks with L-NAME, L-NAME + atorvastatin (ATO), and in untreated controls. In addition, renal cortical protein expression of caveolin 1 (CAV1), vascular endothelial growth factor (VEGF), and activity of RhoA were also determined. Results:L-NAME administration resulted in sustained elevation of BP, decreased GFR, and in higher albuminuria as compared to control animals. Co-administration of ATO with L-NAME normalized albuminuria and prevented decreases in GFR in L-NAME rats without having an impact on pressor effects of L-NAME. CAV1 protein expression was similar in all groups of rats. In contrast, VEGF expression and RhoA activity was increased in L-NAME-treated animals, and normalized with co-administration of ATO. Conclusion: Treatment with ATO exerts early nephroprotective effects in the NO-deficient model of hypertension. These effects could be mediated by amelioration of VEGF expression and reduction of RhoA activity.

Transforming growth factor (TGF)-beta1 is a key profibrogenic cytokine associated with the pathogenesis of chronic allograft nephropathy (CAN). The primary aim of this study was to evaluate TGF-beta1 expression in protocol kidney graft biopsy in patients treated with different immunosuppressive regimens. Protocol kidney graft biopsies were carried out in 77 patients with stable graft function at 1 year after kidney transplantation, treated with a triple-drug regimen based on cyclosporin A (CyA; n = 49) or tacrolimus (TAC; n = 28). Morphological findings were assessed using the Banff 97 classification. TGF-beta1 expression was analysed using immunochemistry, and semiquantitatively scored in different renal structures (total score 0-18). Clinical data were analysed at the time of biopsy, and 12 months thereafter. No significant relation was found between the used immunosuppressive regimen and the histomorphological picture in the graft. TGF-beta1 expression within graft tissue was significantly higher in patients treated with CyA when compared with TAC (9.94 +/- 4.2 vs. 5.0 +/- 3.2; P < 0.001). Serum creatinine and glomerular filtration rate (GFR; Cockroft-Gault calculation) were comparable in both groups but, in the course of the next 12 months, GFR significantly decreased only in the CyA-treated group (from 1.03 +/- 0.33 to 0.96 +/- 0.37 ml/s) while not changing in the TAC-treated group. Patients treated with TAC had significantly lower diastolic blood pressure and serum cholesterol. The significantly lower TGF-beta1 expression in 1-year protocol kidney graft biopsy in TAC-treated patients with stable renal function, and the different development of graft function in both groups suggest a possible benefit of TAC for long-term graft acceptance.

Using immunohistochemistry with the newly available monoclonal antibody MEM-G/1 the reaction patterns on frozen and formaldehyde-fixed paraffin-embedded sections on human placentas, lymph nodes, skeletal muscles, and kidney and liver allografts were compared. HLA-G (a nonclassical major histocompatibility complex class I molecule that is assumed to influence the immune response during pregnancy and some pathological conditions) was found within human extravillous cytotrophoblast but not within villous cytotrophoblast and placental mesenchymal tissue. No HLA-G expression on human lymph nodes, tonsils, and kidney and liver allografts was demonstrated. However, HLA-G expression was observed in all samples of skeletal muscle. The binding capacity of monoclonal antibody MEM-G/1 provides new possibilities to study physiological and pathophysiological roles of HLA-G in humans.

OBJECTIVE—To assess the effect of normoglycemia following simultaneous pancreas/kidney transplantation (SPK) on neurological function and intraepidermal nerve fiber density (IENFD) in patients with type 1 diabetes. RESEARCH DESIGN AND METHODS—We performed vibration perception threshold (VPT) testing and autonomic function testing (AFT) and assessed IENFD in skin biopsies from the lower thigh and upper calf in 14 healthy control subjects and 18 patients with type 1 diabetes at the time of and at 21–40 (median 29) months post SPK. RESULTS—At baseline, significantly increased VPTs, pathological AFT results, and severe reduction in IENFD were present in SPK recipients. After SPK, an increase of IENFD in the thigh of more than one epidermal nerve fiber per millimeter was noted in three patients (median 4.1, range 1.9–10.2), but changes were not significant for the group as a whole. CONCLUSIONS—We conclude that either irreversible nerve damage might be present in some SPK recipients or that longer periods of normoglycemia might be needed to allow nerve regeneration.

Acute rejection (AR) is a dominant risk factor for developing chronic allograft nephropathy (CAN) after kidney transplantation. CAN is characterized by progressive interstitial fibrosis. It has been associated with increased transforming growth factor (TGF)-beta1 expression, however, kinetic studies are absent. We investigated whether intragraft TGF-beta1 expression in various causes of early graft dysfunction may influence late renal allograft dysfunction. A total of 174 human renal biopsies were quantified for TGF-beta1 mRNA expression using real-time reverse transcriptase-polymerase chain reaction. Expression levels were correlated with the Banff histopathological grades, TGF-beta1 immunohistology, and clinical follow-up. TGF-beta1 was most markedly upregulated in AR, CAN, and acute tubular necrosis - delayed graft function compared to non-rejecting controls (P0.001). TGF-beta1 expression was heightened in borderline changes (P0.01), recurrence of glomerulonephritis, and cyclosporine toxicity (P0.05). There was no correlation between intragraft TGF-beta1 expression during AR and short-term outcome of a rejection episode. TGF-beta1 gene overexpression during CAN has been shown to be associated with the increased risk for renal allograft dysfunction 18 months after biopsy (odds ratios 9.9 vs 3.2, respectively). Intragraft TGF-beta1 mRNA expression is significantly upregulated in both AR and CAN. Thus, our results support the hypothesis that TGF-beta1 might play a key role in chronic allograft dysfunction.