Sensitivity to perioperative ischemia/reperfusion injury in male and female donor myocardium.

Many functions of the cardiovascular apparatus are affected by gender. The aim of our study was find out whether markers of cell death present in the donor myocardium differ in male and female hearts. The study involved 81 patients undergoing heart transplantation from September 2010 to January 2013. Patients were divided into two groups: male allograft (n=49), and female allograft (n=32). Two types of myocardial cell death were analyzed. High-sensitive cardiac troponin T as a necrosis marker and protein bcl-2, caspase 3 and TUNEL as apoptosis markers were measured. We observed a significantly higher level of high-sensitive cardiac troponin T after correcting for predicted ventricular mass in female donors before transplantation as well as in the female allograft group after transplantation throughout the monitored period (P=0.011). There were no differences in apoptosis markers (bcl-2, caspase 3, TUNEL) between male and female hearts before transplantation. Both genders showed a significant increase of TUNEL-positive myocytes one week after transplantation without differences between the groups. Moreover, there were no differences in caspase 3 and bcl-2 expression between the two groups. Our results demonstrated the presence of necrotic and apoptotic cell death in human heart allografts. High-sensitive cardiac troponin T adjusted for predicted ventricular mass as a marker of myocardial necrosis was higher in female donors, and this gender difference was even more pronounced after transplantation.

Lipasin/betatrophin is differentially expressed in liver and white adipose tissue without association with insulin resistance in Wistar and Goto-Kakizaki rats.

Lipasin is a recently identified lipokine expressed predominantly in liver and in adipose tissue. It was linked to insulin resistance in mice and to type 1 and type 2 diabetes (T1D, T2D) in humans. No metabolic studies concerning lipasin were performed yet in rats. Therefore, we used rat model of T2D and insulin resistance, Goto-Kakizaki (GK) rats, to determine changes of lipasin expression in liver and in white adipose tissue (WAT) over 52 weeks in the relation to glucose tolerance, peripheral tissue insulin sensitivity and adiposity. GK rats were grossly glucose intolerant since the age of 6 weeks and developed peripheral insulin resistance at the age of 20 weeks. Expression of lipasin in the liver did not differ between GK and Wistar rats, declining with age, and it was not related to hepatic triacylglycerol content. In WAT, the lipasin expression was significantly higher in Wistar rats where it correlated positively with adiposity. No such correlation was found in GK rats. In conclusion, lipasin expression was associated neither with a mild age-related insulin resistance (Wistar), nor with severe genetically-based insulin resistance (GK).

Severe Epidermal Nerve Fiber Loss in Diabetic Neuropathy Is Not Reversed by Long-Term Normoglycemia After Simultaneous Pancreas and Kidney Transplantation.

Whether nerve fiber loss, a prominent feature of advanced diabetic neuropathy, can be reversed by reestablishment of normal glucose control remains questionable. We present 8-year follow-up data on epidermal nerve fiber (ENF) density and neurological function in patients with type 1 diabetes after simultaneous pancreas and kidney transplantation (SPK) with long-term normoglycemia. Distal thigh skin biopsies with ENF counts, vibration perception thresholds (VPTs), autonomic function testing (AFT) and electrophysiological examinations were performed at time of SPK and 2.5 and 8 years after SPK in 12 patients with type 1 diabetes. In comparison to controls, baseline ENF density, VPT and AFT results of patients indicated severe neuropathy. At follow-up, all SPK recipients were insulin independent with excellent glycemic control and kidney graft function; however, the severe ENF depletion present at baseline had not improved, with total ENF absence in 11 patients at 8-year follow-up. Similarly, no amelioration occurred in the VPT and AFT results. Numerical improvement was seen in some electrophysiological parameters; however, statistical significance was achieved only in median motor nerve conduction velocity. ENF loss and functional deficits in advanced diabetic peripheral neuropathy are rarely reversible, even by long-term normoglycemia, which underscores the importance of neuropathy prevention by early optimal glycemic control.

Biomarkers of cellular apoptosis and necrosis in donor myocardium are not predictive of primary graft dysfunction.

Primary graft dysfunction (PGD) is a life-threatening complication among heart transplant recipients and a major cause of early mortality. Although the pathogenesis of PGD is still unclear, ischemia/reperfusion injury has been identified as a predominant factor. Both necrosis and apoptosis contribute to the loss of cardiomyocytes during ischemia/reperfusion injury, and this loss of cells can ultimately lead to PGD. The aim of our prospective study was to find out whether cell death, necrosis and apoptosis markers present in the donor myocardium can predict PGD. The prospective study involved 64 consecutive patients who underwent orthotopic heart transplantation at our institute between September 2010 and January 2013. High-sensitive cardiac troponin T (hs-cTnT) as a marker of minor myocardial necrosis was detected from arterial blood samples before the donor's pericardium was opened. Apoptosis (caspase-3, active + pro-caspase-3, bcl-2, TUNEL) was assessed from bioptic samples taken from the right ventricle prior graft harvesting. In our study, 14 % of transplant recipients developed PGD classified according to the standardized definition proposed by the ISHLT Working Group. We did not find differences between the groups in regard to hs-cTnT serum levels. The mean hs-cTnT value for the PGD group was 57.4+/-22.9 ng/l, compared to 68.4+/-10.8 ng/l in the group without PGD. The presence and severity of apoptosis in grafted hearts did not differ between grafts without PGD and hearts that subsequently developed PGD. In conclusion, our findings did not demonstrate any association between measured myocardial cell death, necrosis or apoptosis markers in donor myocardium and PGD in allograft recipients. More detailed investigations of cell death signaling pathways in transplanted hearts are required.

Tacrolimus inhibits intimal hyperplasia in arterialised veins in rats.

OBJECTVES: We investigated whether tacrolimus (FK506) can inhibit neointimal formation in arterialised vein grafts in rats. METHODS: Lewis iliolumbar veins were implanted into the abdominal aorta of isogeneic rats. Animals in the treatment groups had daily intramuscular injections of tacrolimus at 0.2 mg/kg (group B) and 0.1 mg/kg (Group C), respectively. The control group A had no treatment. Light microscope evaluations of arterialised vein grafts were performed 30 days after operation. We determined the presence of endothelial cells, the thickness of intima and media, and the degree of infiltration by MHC class II positive, CD4 positive, and CD8 positive cells into the adventitia. RESULTS: The intimal thickness in group B (5.0±1.0 µm) was statistically lower (P < 0.05) when compared to group C (7.0±3.0 µm). The intimal thickness in untreated group A (12.7±7.0 µm) was statistically higher (P < 0.01) when compared to both treated groups B and C, respectively. The medial thickness and degree of adventitial infiltration by MHC class II positive, CD8 positive, and CD4 positive cells did not differ between groups. CONCLUSION: Treatment with tacrolimus (FK506) showed a dose dependant inhibition of neointimal hyperplasia in arterialised vein grafts in rats (Tab. 1, Fig. 3, Ref. 22).

Detection of pancreatic islet allograft impairment in advance of functional failure using magnetic resonance imaging.

This study evaluated the ability of magnetic resonance imaging (MRI) to predict failure of pancreatic islets (PI) transplanted into the hepatic portal vein. Brown-Norway (n = 18) and Lewis (n = 6) rats received islets isolated from Lewis donors. The rejection process in Brown-Norway recipients was mitigated by two different immunosuppressive regimens [tacrolimus + hydrocortisone for 3 months (n = 6) or tacrolimus for 12 days (n = 12)]. Longitudinal MRI monitoring of recipients at post-transplantation weeks 1, 2, 3, 4, 6, 8, 10, and 12 confirmed the ability to detect SPIO labeled PI after transplantation into the liver. The relative number of MRI signals related to PI isografts remained stable up to study completion. Recipients of PI allografts were normoglycemic until the end of study; signals declined gradually to 44 ± 17% in these animals. In animals with islets failure during post-transplant week 12, the number of signals decreased to 25 ± 10% of initial values. The difference between groups (islet function/failed) became significant post-transplant week 3. Our data demonstrate that the MRI changes attributable to rejection become apparent within 3 weeks after transplantation, i.e. at least 8 weeks before functional allograft failure.

A periadventitial sirolimus-releasing mesh decreased intimal hyperplasia in a rabbit model.

Autologous vein grafts used as aortocoronary bypasses are often prone to intimal hyperplasia, which results in stenosis and occlusion of the vein. The aim of this study was to prevent intimal hyperplasia using a newly developed perivascular system with sustained release of sirolimus. This system of controlled drug release consists of a polyester mesh coated with a copolymer of L-lactic acid and epsilon-caprolactone that releases sirolimus. The mesh is intended for wrapping around the vein graft during surgery. The mesh releasing sirolimus was implanted in periadventitial position onto arteria carotis communis of rabbits, and neointimal hyperplasia was then assessed. We found that implanted sirolimus-releasing meshes reduced intima thickness by 47+/-10 % compared to a vein graft after 3 weeks. The pure polyester mesh decreased vein intima thickness by 35+/-9 %. Thus, our periadventitial system for controlled release of sirolimus prevented the development of intimal hyperplasia in autologous vein grafts in vivo in rabbits. A perivascularly applied mesh releasing sirolimus is a promising device for preventing stenosis of autologous vein grafts.

[How to improve the histopathological diagnosis of hepatocellular carcinoma in daily practice?]. / Jak v praxi zlepsit histopatologickou diagnostiku hepatocelulárního karcinomu.

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world with an increasing incidence. Recently, an East-West consensus on the histopathologic criteria for the diagnosis of high-grade dysplastic nodules and early hepatocellular carcinoma was published. In addition to classical morphological criteria such as nucleocytoplasmic ratio, thickness of cell plates, mitotic index, and architectural disturbance, a new one--the stromal invasion--was recognized as a crucial criterion for the diagnosis of early HCC. Immunohistochemical detection of glypican--3 was shown as a specific marker for HCC that can be used to distinguish between the benign hepatocellular lesions and HCC.

[Autoimmune pancreatitis with biliary tree and liver involvement as a part of IgG4-related autoimmune sclerosing disease. Case report]. / Autoimunní pankreatitida s postizením zlucovodu a jater jako soucást IgG4 pozitivního onemocnení (IgG4-related autoimmune sclerosing disease). Kazuistika.

We report a rare case of autoimmune pancreatitis associated with sclerosing cholangitis, kidney, and salivary glands involvement. Abdominal ultrasound and CT scan showed enlarged pancreas, multiple nodular mass in the liver and kidney. ERCP showed features compatible with primary sclerosing cholangitis. The working clinical diagnosis considered malignant tumor with liver metastases. Histological examination of the liver biopsy sample revealed inflammatory process with numerous IgG4 positive plasma cells. The diagnostic conclusion of IgG4-related autoimmune sclerosing disease was drawn. The serum IgG level was elevated. Treatment with steroids improved the clinical course, all masses in the liver and kidneys disappeared, and laboratory tests were normalized. Now, 4 years after diagnosis the patient is free of all symptoms.

Hepatocyte growth factor and antibodies to HLA and MICA antigens in heart transplant recipients.

Recent unconfirmed literature data suggest that elevated concentrations of the multifunctional cytokine hepatocyte growth factor (HGF) might be a marker of increased incidence of acute rejection after organ transplantation. The aim of this study was to test the hypothesis that HGF levels may correlate with the rejection and/or with the production of HLA and MHC Class I chain-related antigens A (MICA) specific antibodies. Sixty-three heart transplant recipients were included into the study. Hundred and eighty-five endomyocardial biopsies (EMB) obtained up to 6 months after transplantation were retrospectively analyzed for signs of cellular (CR) and antibody-mediated rejection (AMR). Pre- and post-transplant sera were tested for HGF concentrations and antibodies to HLA class I, class II and MICA antigens by xMap technology (Luminex). Pre-transplant HGF did not correlate with the incidence of CR or AMR. However, higher HGF concentrations correlated significantly with HLA antibody production before and after transplantation (P = 0.006 and P < 0.0001 respectively). Patients with both HLA class I and class II antibodies before transplantation had significantly lower AMR-free survival. Furthermore, recipients with pre-transplant donor-specific antibodies (DSA) had significantly lower AMR-free survival (50%) than recipients without pre-transplant HLA antibodies (90%) and patients with antibodies not specific to donor antigens (92%) (P = 0.005). Post-transplant MICA antibodies tended to be more frequent in patients with AMR (P = 0.063). In conclusion, elevated HGF concentrations in our study were not associated with the incidence of CR and/or AMR but with the presence of HLA-specific antibodies. Testing for DSA before heart transplantation by Luminex may be helpful for the identification of patients with increased risk of AMR.

Leflunomide derivate FK 778 in accelerated renal injury in transgenic rat.

Renal ischaemia/reperfusion (I/R) injury and hypertension represent major alloantigen-independent risk factors contributing to the development of chronic allograft nephropathy. In a model of accelerated major histocompatibility complex-independent renal injury, we evaluated the effect of leflunomide derivate - FK778 - on the progression of accelerated nephropathy. Thirty-six uninephrectomized hypertensive transgenic (m-REN-2)-27 rats received a clip on renal pedicle for 45 minutes. Animals were treated with FK778 3 mg/kg/day (I/R 3 mg, N = 12), 10 mg/kg/day (I/R 10 mg, N = 12) or placebo (N = 12) via gavage for 16 weeks. Eighteen animals were sham-operated and treated with FK778 3 mg/kg/day (sham 3 mg, N = 6), 10 mg/kg/day (sham 10 mg, N = 6) or were untreated (sham, N = 6). Proteinuria and blood pressure were evaluated throughout and the kidneys were harvested for morphological and immunohistochemical analysis at the end of the experiment. At week 16, rats with I/R injury and FK778 treatment had lower proteinuria compared with placebo-treated rats (I/R 3 mg: 48.42 +/- 26.16, I/R 10 mg 27.28 +/- 21.86 vs. Placebo: 70.13 +/- 50.19 mg/day, P < 0.05). The untreated sham group exhibited lower proteinuria compared with FK778-treated sham groups (Sham 3 mg: 24.23 +/- 10.89; Sham 10 mg: 17.37 +/- 4.13; Sham: 14.23 +/- 1.18) There was no difference in glomerulosclerosis and interstitial fibrosis among the treated groups. In the untreated animals the rate of interstitial fibrosis decline reached statistical significance (Placebo vs. Sham: 1.125 +/- 0.641 % vs. 0.250 +/- 0.500 %, P < 0.05). There was higher CD5+ leukocyte infiltration in the placebotreated group. FK778-treated rats displayed amelioration of some changes induced by the I/R injury. Our observation also suggests potential nephrotoxicity of FK778.

The positive effect of immunosuppression on adaptation of venous allografts to arterialisation in rats.

OBJECTIVES AND DESIGN: We investigated whether immunosuppression was necessary for transplanted allogeneic veins to adapt to arterialisation. We used a transplant rat model with or without immunosuppression. MATERIAL AND METHODS: Iliolumbar veins from Lewis (LEW) or Brown-Norway (BN) rats were transplanted into the abdominal aorta of isogeneic (LEW to LEW; group A) or allogeneic (BN to LEW; groups B and C) rats. Group C had daily intramuscular injections of 0.2mgkg(-1) FK506. Light microscope evaluations of grafts were performed at 30 days following transplantation. We determined the presence of endothelial cells, the intensity of intimal proliferation and the degree of infiltration by Lewis major histocompatibility complex (MHC) class II positive, CD4-positive and CD8-positive cells into the adventitia. RESULTS: Groups A and C displayed similar results in intimal thickness (12.7+/-7.0microm vs. 15.0+/-8.4 mum, respectively) and degree of adventitial infiltration by MHC class II positive (16.6+/-7.5 vs. 14.6+/-6.2, respectively), CD8-positive (0.8+/-1.7 vs. 1.8+/-2.6, respectively) and CD4-positive (12.5+/-7.7 vs. 5.8+/-4.6, respectively) cells. In contrast, allogeneic rats without immunosuppression (group B) showed infiltration of host immunocompetent cells and destruction of the venous wall with no histological signs of arterialisation. CONCLUSION: Immunosuppressive therapy is necessary for venous allograft adaptation to arterialisation in rats.

[Experimental small intestine transplantation]. / Transplantace tenkého streva v experimentu.

AIM: Transplantation of the small intestine is a standard treatment method in patients with small intestinal failures. The aim of this study was to master the surgical technique, optimalize immunosuppression regimes, diagnose acute cellular graft rejection based on cellular and humoral indicators. METHODS: The authors performed a total of 43 transplantation procedures in pigs. The first, surgical part of the experiment was aimed at mastering two principal methods of vascular anastomosis- firstly, connecting the graft with mesenteric vessels (Group n1 = 18) and secondly, connecting the graft with the aorta and the inferior vena cava (Group n2 = 25). The second part of the experiment included assessment of rejection changes in various immunosuppression regimes. Only animals who did not die because of a technical failure of the procedure or due to internal reasons (n = 24) were assessed. The study animals were assigned to four groups (A (n = 3)--autotransplantation, without immunosuppresion; B (n = 7) and C (n = 8)--allotransplantation with immunosuppression using tacrolimus, resp. in a combination with sirolimus; D (n = 6)--allotransplantation without immunosuppression. Rejection was diagnosed based on histological examination of the grafts@ biopsy samples. Plasmatic citruline was used as a non-invasive humoral indicator of the graft impairment. RESULTS: Procedural complications were observed in 12 (67%) study animals from the first group, and in 3 (12%) animals from the second group. In the assessment of rejection changes, the longest survival was observed with autotransplantations, the shortest survival period was shown with allotransplantations without immunosuppression. No significant survival differences were demonstrated between the both treated groups. (p < 0.05). Group C showed lower rates of cellular rejections, compared to Group B and D. CONCLUSION: During the experiment, the authors managed to master the graft collection, as well as the transplantation technique. Lower rates of surgical complications were observed when the graft was supplied by the central vascular system. No significant differencies were observed between the tacrolimus monoterapy regimen and the combination therapy with sirolimus. Histological examination is the golden standard for the cellular rejection diagnostics. Plasmatic citruline has no signifiance in the rejection assessment.

[Subclinical acute rejections in protocol biopsies at 3 months after kidney transplantation]. / Subklinické akutní rejekce v protokolárních biopsiích 3 mesíce po transplantaci ledviny.

AIM: The primary aim of the study was detection of subclinical acute rejection and borderline changes in protocol biopsies at 3 months after transplantation, and assessment of possible clinical and laboratory associations. METHODS: Biopsy was carried out in 194 patients with stabilized graft function. Patients were treated with immunosuppressive regimen based on cyclosporine A (n = 34), tacrolimus (n = 152), or sirolimus/everolimus (n = 10). Samples were processed by standard paraffine technique, and stained according to laboratory protocol. All samples were tested by immunofluorescence or immunohistochemical procedures for C4d presence as a sign of humoral rejection. RESULTS: Of 192 representative samples, subclinical acute rejection and borderline changes were found in 24 samples (12.5%). In patients with this finding, the mean serum creatinine was significantly higher (185.2 +/- 2.2 micromol/L), than in patients with normal finding (128.2 +/- 28.3 micromol/L) p < 0.001. Using the ROC curve analysis of serum creatinine, the cut-off point 170 micromol/L was found to discriminate normal findings from subclinical rejection and borderline changes. A significant correlation between acute rejections before protocol biopsy and subclinical acute rejections together with borderline changes in protocol biopsy was found. C4d positivity was found in 6 samples. Immunosuppressive therapy (cyclosporine versus tacrolimus) did not have any impact on subclinical acute rejections and borderline changes prevalence. CONCLUSIONS: The main conclusion of this study is a finding, that acute rejection early after renal transplantation and serum creatinine > or = 170 micromol/l at three months after transplantation are risks for development of subclinical acute rejection, even of humoral type, or borderline changes.

Heightened expression of the cytotoxicity receptor NKG2D correlates with acute and chronic nephropathy after kidney transplantation.

The activating cytotoxicity receptor NKG2D binds to stress-regulated molecules encoded by the major histocompatibility complex class I chain-related (MIC) and UL-16-binding protein (ULBP)/retinoic acid early transcript (RAET) gene family. To assess whether acute allograft rejection leads to an induction of these inducible ligands and their receptor NKG2D, we examined the mRNA profiles in kidney transplant biopsies. Expression levels were correlated with the incidence of acute rejection (aRx) episodes and chronic allograft nephropathy (CAN) proven by histology. Whereas MICA, ULBP1/3 and RAET1-E did not display heightened gene expression, elevated levels of NKG2D mRNA could be associated with aRx (p < 0.001). Immunohistology of kidney biopsies diagnosed with aRx revealed NKG2D+ cells in tubulointerstitial areas positive for CD8+ cells. Most importantly, elevated levels of NKG2D mRNA were associated with restricted long-term graft function assessed by the glomerular filtration rate at 6, 12 and 18 months posttransplantation. Induced NKG2D mRNA expression was still observable in biopsies diagnosed with CAN (p < 0.001), demonstrating a higher sensitivity and specificity compared to CD3, granzyme B and granulysin mRNA measurement. Significant elevated levels of NKG2D mRNA could be further detected in urine sediment prior to aRx, suggesting this receptor as a new candidate marker for the diagnosis of acute and chronic allograft rejection.

Preservation injury of the small bowel graft in clinical small bowel transplantation.

Clinical success of small bowel transplantation depends on quality of the preservation small bowel graft which is notoriously sensitive to ischemia. 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 a light microscopy and concentrations of tissue serotonin-positive cells, we tried to identify a part of the human intestine, which is more resistant to preservation injury sustained by HTK preservation solution with 1-24 hr of cold ischemia. Statistical analysis of both parameters did not reveal any significant differences between the jejunum and ileum. According to 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. A significant difference was observed in histological pictures only after 12-hour of cold ischemia in both groups (jejunum and ileum) (Fig. 2, Ref. 11). Full Text (Free, PDF) www.bmj.sk.

Mycophenolate mofetil ameliorates accelerated progressive nephropathy in rat.

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.

TGF-beta1 mRNA upregulation influences chronic renal allograft dysfunction.

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 (P < 0.001). TGF-beta1 expression was heightened in borderline changes (P < 0.01), recurrence of glomerulonephritis, and cyclosporine toxicity (P < 0.05). 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.

BK-virus nephropathy and simultaneous C4d positive staining in renal allografts.

The role of antibodies in rejection of transplanted kidneys was the subject of debate at the last two Banff meetings and in medical journals. Diffuse C4d positive staining of peritubular capillaries (PTCs) was recognized as a marker of antibody-mediated rejection and this morphological feature was included in the updated Banff schema. At the same time polyomavirus infection of the renal allografts has been reported more frequently and is emerging as an important cause of renal allograft dysfunction and graft loss. At the present time, BK-virus nephropathy (BKN) represents the most common viral disease affecting renal allografts. BKN was identified in 6 patients in 12 biopsies and 2 graft nephrectomy specimens of 1115 biopsies between September 2000 and December 2003. Definite virus identification was done by immunohistochemistry. The reason for graft nephrectomies was graft failure due to BKN in a recipient after kidney-pancreas transplantation with good function of his pancreas graft and the necessity of continuing immunosuppression. Detection of C4d deposits was performed by immunofluorescence or by immunohistochemistry. In graftectomy samples C4d detection was performed by immunohistochemistry and retrospectively in all cases of BKN. Focal C4d positive PTCs and BKN were found simultaneously in 9 of 12 needle biopsies and in both graft nephrectomy samples. Detection of C4d by immunohistochemistry disclosed focal C4d positive staining in kidney tissue but diffuse in the sites where BK-virus inclusions in tubular epithelial cells were found. The complement system is part of the host defense response and is crucial to our natural ability to ward off infection. In cases of BKN, virus likely gains access to the bloodstream through injured tubular walls and via PTCs. Vascular endothelium in the PTCs represents a potential target antigen for alloresponse, and simultaneously possibly represents an imprint of complement activation or complement production in the places with BK-virus infection.

Severe depletion of intraepidermal nerve fibers in skin biopsies of pancreas transplant recipients.

BACKGROUND: The minimally invasive method of skin biopsy with intraepidermal nerve fiber (IENF) counts may be used to analyze nerve regeneration in pancreas transplant (PTx) recipients. We assessed IENF counts as a database for long-term follow-up of diabetic neuropathy. METHODS: Skin biopsies were performed using a 3-mm punch from lower thigh and upper calf areas of 16 (13 pancreas/kidney, 3 pancreas alone) PTx patients (mean +/- SD: age, 45+/-8 years; type 1 diabetes duration, 27 +/- 8 years) at 1 month posttransplant. Ten healthy gender- and age-matched controls (C) were also examined. After fixation and freezing, 40-microm sections were stained using rabbit polyclonal antibody to the panaxonal marker PGP 9.5 followed by mouse antirabbit IgG antibody conjugated with rhodamine. Samples were imaged with a digital camera, mounted on a microscope, and equipped for fluorescence. The average number of IENF per millimeter length of epidermis was derived. Clinical neuropathy was assessed by foot vibration perception thresholds (VPT) with a biothesiometer (normal values < mean + 2 SD of C). RESULTS: Significantly lower IENF densities were found in skin biopsies from PTx (PTx vs C: thigh, 0.74 +/- 0.88 vs 9.74 +/- 2.41 IENF/mm; calf, 0.34 +/- 0.91 vs 7.66 +/- 3.16 IENF/mm; P < .001). IENF were totally absent from the thigh and calf samples of 7 and 12 PTxs, respectively. Clinical neuropathy (VPT > 21 V) was present in all but one PTx. CONCLUSIONS: Severe intraepidermal nerve fiber depletion is present in the lower limb area of pancreas transplant recipients with neuropathy. Long-term follow-up would probably be necessary to assess the possibility of posttransplant nerve fiber regeneration.