Vascular Remodeling of Clinically Used Patches and Decellularized Pericardial Matrices Recellularized with Autologous or Allogeneic Cells in a Porcine Carotid Artery Model.

Background: Cardiovascular surgery is confronted by a lack of suitable materials for patch repair. Acellular animal tissues serve as an abundant source of promising biomaterials. The aim of our study was to explore the bio-integration of decellularized or recellularized pericardial matrices in vivo. Methods: Porcine (allograft) and ovine (heterograft, xenograft) pericardia were decellularized using 1% sodium dodecyl sulfate ((1) Allo-decel and (2) Xeno-decel). We used two cell types for pressure-stimulated recellularization in a bioreactor: autologous adipose tissue-derived stromal cells (ASCs) isolated from subcutaneous fat of pigs ((3) Allo-ASC and (4) Xeno-ASC) and allogeneic Wharton's jelly mesenchymal stem cells (WJCs) ((5) Allo-WJC and (6) Xeno-WJC). These six experimental patches were implanted in porcine carotid arteries for one month. For comparison, we also implanted six types of control patches, namely, arterial or venous autografts, expanded polytetrafluoroethylene (ePTFE Propaten® Gore®), polyethylene terephthalate (PET Vascutek®), chemically stabilized bovine pericardium (XenoSure®), and detoxified porcine pericardium (BioIntegral® NoReact®). The grafts were evaluated through the use of flowmetry, angiography, and histological examination. Results: All grafts were well-integrated and patent with no signs of thrombosis, stenosis, or aneurysm. A histological analysis revealed that the arterial autograft resembled a native artery. All other control and experimental patches developed neo-adventitial inflammation (NAI) and neo-intimal hyperplasia (NIH), and the endothelial lining was present. NAI and NIH were most prominent on XenoSure® and Xeno-decel and least prominent on NoReact®. In xenografts, the degree of NIH developed in the following order: Xeno-decel > Xeno-ASC > Xeno-WJC. NAI and patch resorption increased in Allo-ASC and Xeno-ASC and decreased in Allo-WJC and Xeno-WJC. Conclusions: In our setting, pre-implant seeding with ASC or WJC had a modest impact on vascular patch remodeling. However, ASC increased the neo-adventitial inflammatory reaction and patch resorption, suggesting accelerated remodeling. WJC mitigated this response, as well as neo-intimal hyperplasia on xenografts, suggesting immunomodulatory properties.

Molecular diagnostics identifies risks for graft dysfunction despite borderline histologic changes.

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.

Molecular patterns of subclinical and clinical rejection of kidney allograft: quantity matters.

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-ß 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.

External Support of Autologous Internal Jugular Vein Grafts with FRAME Mesh in a Porcine Carotid Artery Model.

BACKGROUND: Autologous vein grafts are widely used for bypass procedures in cardiovascular surgery. However, these grafts are susceptible to failure due to vein graft disease. Our study aimed to evaluate the impact of the latest-generation FRAME external support on vein graft remodeling in a preclinical model. METHODS: We performed autologous internal jugular vein interposition grafting in porcine carotid arteries for one month. Four grafts were supported with a FRAME mesh, while seven unsupported grafts served as controls. The conduits were examined through flowmetry, angiography, macroscopy, and microscopy. RESULTS: The one-month patency rate of FRAME-supported grafts was 100% (4/4), whereas that of unsupported controls was 43% (3/7, Log-rank p = 0.071). On explant angiography, FRAME grafts exhibited significantly more areas with no or mild stenosis (9/12) compared to control grafts (3/21, p = 0.0009). Blood flow at explantation was higher in the FRAME grafts (145 ± 51 mL/min) than in the controls (46 ± 85 mL/min, p = 0.066). Area and thickness of neo-intimal hyperplasia (NIH) at proximal anastomoses were similar for the FRAME and the control groups: 5.79 ± 1.38 versus 6.94 ± 1.10 mm2, respectively (p = 0.558) and 480 ± 95 vs. 587 ± 52 µm2/µm, respectively (p = 0.401). However, in the midgraft portions, the NIH area and thickness were significantly lower in the FRAME group than in the control group: 3.73 ± 0.64 vs. 6.27 ± 0.64 mm2, respectively (p = 0.022) and 258 ± 49 vs. 518 ± 36 µm2/µm, respectively (p = 0.0002). CONCLUSIONS: In our porcine model, the external mesh FRAME improved the patency of vein-to-carotid artery grafts and protected them from stenosis, particularly in the mid regions. The midgraft neo-intimal hyperplasia was two-fold thinner in the meshed grafts than in the controls.

[How to improve the histopathological diagnosis of hepatocellular benign affections (adenoma versus focal nodular hyperplasia) in daily practice?]. / Jak v praxi zlepsit bioptickou diagnostiku benigních lézí jater (adenom versus fokální nodulární hyperplazie).

Adenomas of the liver and focal nodular hyperplasia (FNH) are benign hepatocellular affection and their distinguishing in a needle biopsy sample and sometimes also in a surgical specimen causes often a problem. Although it might seem that the differentiation of the benign conditions is of a low value for the clinicians and also for the patients, the opposite is true. The risk of life-threatening bleeding and risk of the malignant transformation of adenomas leads to request accurate diagnosis of these conditions. New genetic methods followed by immunohistochemical detection of several antigens enables more accurate distinction not only of the two main groups of FNH and adenomas, but allows also to distinguish subsets of adenomas with varying risk of malignant transformation. Therefore, to determine the subtype of adenoma represents now essential part of a biopsy diagnosis. Identification of the subsets of adenomas allows an individualized treatment with resection in high-risk forms and, on the other hand, allows avoiding liver resection in the case of small liver mass with a low risk of malignant transformation.

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.

Glypican-3 immunostaining significantly improves histological diagnosis of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors which occur mainly in patients with chronic liver disease. Early detection of HCC is critically important for treatment of the patients. However, most early HCC cases are asymptomatic clinically with the lack of typical radiological findings. Also histological diagnosis is often very difficult with the lack of agreement even among expert pathologists. METHODS: We studied the expression of Glypican-3 in 138 liver biopsy samples; 86 HCC, 10 hepatocellular adenomas, 12 focal nodular hyperplasias, 25 samples with liver cirrhosis without tumor, and 5 liver metastases of neuroendocrine carcinomas. RESULTS: HCC showed positive staining in 80 nodules (93%; all of the 11 needle biopsy samples, 12 out of 15 liver resection specimens, 57 out of 60 nodules in explanted livers). Glypican-3 expression was independent of the differentiation and size of the HCC. Six cases (6.9%), 3 HCC in liver resection specimens and 3 in the explanted liver were negative for Glypican-3. However, all cases with benign nodular lesions and cirrhosis without tumors were negative for Glypican-3. CONCLUSIONS: Immunohistochemical detection of Glypican-3 significantly improves the complicated routine histological diagnosis of HCC even in early lesions in needle biopsy samples.

Accelerated in vitro recellularization of decellularized porcine pericardium for cardiovascular grafts.

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.

Does the renal expression of Toll-like receptors play a role in patients with IgA nephropathy?

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.

The Effect of Butyrate-Supplemented Parenteral Nutrition on Intestinal Defence Mechanisms and the Parenteral Nutrition-Induced Shift in the Gut Microbiota in the Rat Model.

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 whether i.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.

Labeling of pancreatic islets with iron oxide nanoparticles for in vivo detection with magnetic resonance.

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 T2*-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.

Intrarenal Complement System Transcripts in Chronic Antibody-Mediated Rejection and Recurrent IgA Nephropathy in Kidney Transplantation.

Background: The complement system activation and regulation have been linked to post-transplant pathologies including chronic antibody mediated rejection (cAMR) and the recurrence of IgA nephropathy (ReIgAN) but distinct mechanisms remain to be elucidated. Methods: In this retrospective single center study, the outcome of kidney transplantation was studied in 150 patients with late histological diagnosis to be either cAMR or ReIgAN, 14 stable kidney grafts at 3 months and finally 11 patients with native kidney IgAN nephropathy. To study a role of complement cascade and regulation in cAMR and ReIgAN, the RNA was extracted from available frozen kidney biopsy samples and using RT-qPCR transcripts of 11 target genes along with clinical data were determined and compared with stable grafts at 3 months protocol biopsies or IgAN native kidney nephropathy. Immunohistologically, CD46 (MCP), and C5 proteins were stained in biopsies. Results: Interestingly, there were no differences in kidney graft survival between cAMR and ReIgAN since transplantation. cAMR was associated with significantly higher intragraft transcripts of C3, CD59, and C1-INH as compared to ReIgAN (p < 0.05). When compared to normal stable grafts, cAMR grafts exhibited higher C3, CD55, CD59, CFH, CFI, and C1-INH (p < 0.01). Moreover, ReIgAN was associated with the increase of CD46, CD55, CD59 (p < 0.01), and CFI (p < 0.05) transcripts compared with native kidney IgAN. Rapid progression of cAMR (failure at 2 years after biopsy) was observed in patients with lower intrarenal CD55 expression (AUC 0.77, 78.6% sensitivity, and 72.7 specificity). There was highly significant association of several complement intrarenal transcripts and the degree of CKD regardless the diagnosis; C3, CD55, CFH, CFI, and C1-INH expressions positively correlated with eGFR (for all p < 0.001). Neither the low mRNA transcripts nor the high mRNA transcripts biopsies were associated with distinct trend in MCP or C5 proteins staining. Conclusions: The intrarenal complement system transcripts are upregulated in progressively deteriorated kidney allografts.

Silymarin prevents acetaminophen-induced hepatotoxicity in mice.

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.

Soluble CD30 in patients with antibody-mediated rejection of the kidney allograft.

UNLABELLED: The aim of our retrospective study was to evaluate the clinical significance of measurement of the soluble CD30 (sCD30) molecule for the prediction of antibody-mediated (humoral) rejection (HR). Sixty-two kidney transplant recipients (thirty-one C4d-positive and thirty-one C4d-negative patients) were included into the study. Soluble CD30 levels were evaluated before transplantation and during periods of graft function deterioration. The median concentrations of the sCD30 molecule were identical in C4d-positive and C4d-negative patients before and after transplantation (65.5 vs. 65.0 and 28.2 vs. 36.0 U/ml, respectively). C4d+ patients who developed DSA de novo had a tendency to have higher sCD30 levels before transplantation (80.7+/-53.6 U/ml, n=8) compared with C4d-negative patients (65.0+/-33.4 U/ml, n=15). Soluble CD30 levels were evaluated as positive and negative (>or=100 U/ml and <100 U/ml respectively) and the sensitivity, specificity and accuracy of sCD30 estimation with regard to finding C4d deposits in peritubular capillaries were determined. The sensitivity of sCD30+ testing was generally below 40%, while the specificity of the test, i.e. the likelihood that if sCD30 testing is negative, C4d deposits would be absent, was 82%. C4d+ patients who developed DSA de novo were evaluated separately; the specificity of sCD30 testing for the incidence of HR in this cohort was 86%. CONCLUSION: We could not confirm in our study that high sCD30 levels (>or=100 U/ml) might be predictive for the incidence of HR. Negative sCD30 values might be however helpful for identifying patients with a low risk for development of DSA and antibody-mediated rejection.

Preservation injury to the human small bowel graft: jejunum vs. ileum.

BACKGROUND: 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. AIM OF STUDY: 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. RESULTS: Statistical analysis of both parameters did not reveal any significant differences between the jejunum and ileum. CONCLUSIONS: 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).

Multimodal Imaging Reveals Improvement of Blood Supply to an Artificial Cell Transplant Site Induced by Bioluminescent Mesenchymal Stem Cells.

PURPOSE: An artificial site for cell or pancreatic islet transplantation can be created using a polymeric scaffold, even though it suffers subcutaneously from improper vascularisation. A sufficient blood supply is crucial for graft survival and function and can be enhanced by transplantation of mesenchymal stem cells (MSCs). The purpose of this study was to assess the effect of syngeneic MSCs on neoangiogenesis and cell engraftment in an artificial site by multimodal imaging. PROCEDURES: MSCs expressing a gene for luciferase were injected into the artificial subcutaneous site 7 days after scaffold implantation. MRI experiments (anatomical and dynamic contrast-enhanced images) were performed on a 4.7-T scanner using gradient echo sequences. Bioluminescent images were acquired on an IVIS Lumina optical imager. Longitudinal examination was performed for 2 months, and one animal was monitored for 16 months. RESULTS: We confirmed the long-term presence (lasting more than 16 months) of viable donor cells inside the scaffolds using bioluminescence imaging with an optical signal peak appearing on day 3 after MSC implantation. When compared to controls, the tissue perfusion and vessel permeability in the scaffolds were significantly improved at the site with MSCs with a maximal peak on day 9 after MSC transplantation. CONCLUSIONS: Our data suggest that the maximal signal obtained by bioluminescence and magnetic resonance imaging from an artificially created site between 3 and 9 days after MSC transplantation can predict the optimal time range for subsequent cellular or tissue transplantation, including pancreatic islets.

Magnetic resonance imaging of pancreatic islets in tolerance and rejection.

BACKGROUND: We have recently described a magnetic resonance (MR) method for detection of rat pancreatic islets transplanted into the liver after labeling with superparamagnetic iron oxide nanoparticles. The aim of this work was to study whether this technique could be applicable over a longer period after transplantation and whether it could help to detect islet rejection. METHODS: Islets from Lewis and Wistar rats were cultured in the presence of iron oxide nanoparticles. Two thousand of Lewis (n=6) or Wistar (n=8) iron-labeled islets were transplanted into the portal vein of Lewis diabetic animals. Serial MR imaging of the liver were performed at 1, 2, 3, 4, 5, and 6 weeks. RESULTS: Although all allogeneic islets were rejected by 12 days, syngeneic animals remained normoglycemic throughout the study. At week 1, the labeled islets were visualized on MR scans as distinct hypointense spots homogeneously distributed in the liver. While their number declined only insignificantly in the syngeneic group, in the allogeneic group the number of spots gradually decreased until approximately 35% of their initial count. Although syngeneic islets showed a normal histology, the allogeneic islets were completely rejected. Iron particles, localized in macrophages, were detected only in the syngeneic islets and were absent in the rejected islet structures. In vitro incubation tests did not reveal any differences in insulin secretion between labeled and nonlabeled islets. CONCLUSIONS: MR imaging of iron-labeled pancreatic islets can be used for verification of the technical success of the transplantation procedure itself and for the detection of the decreasing relative islet mass due to rejection.

Interleukin 18 (IL-18) upregulation in acute rejection of kidney allograft.

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<0.0001 for both comparisons). Tissue culture model of renal epithelial cells expressed IL-18 mRNA constitutively and released mature IL-18 in response to TNF-alpha and IFN-gamma. We assume that upregulation of epithelial IL-18 plays an important role in immune and immunopathological reactions in renal parenchyma and contributes to rejection mechanisms of kidney allograft.

Soluble CD30 and HLA antibodies as potential risk factors for kidney transplant rejection.

Recent literary data suggest that high pre- and post-transplant serum levels of the soluble CD30 (sCD30) molecule may be a risk factor for acute rejection and worse prognosis of the transplanted kidney. The aim of our study was to correlate the concentrations of sCD30 and the presence of HLA antibodies as defined by flow cytometry and ELISA with the clinical course and graft prognosis after transplantation. One hundred and seventeen kidney transplant patients were included into the study. The incidence of rejection episodes, graft function and graft survival for up to 1 year post-transplant were evaluated. Soluble CD30 levels before transplantation were virtually the same in patients who experienced rejection and in non-rejecting patients. In both patient groups, a significant decrease of sCD30 was detected 2 weeks after transplantation (104.4 U/ml before vs. 37.0 U/ml post-transplant, P < 0.001). However, there was a substantial difference in the level of decrease of sCD30 between rejecting and non-rejecting patients. Patients without rejection had lower sCD30 values (31.2 U/ml post-transplant) compared to patients who experienced rejection episodes (62.9 U/ml), P < 0.04. Multifactorial analysis showed that antibodies to HLA class II antigens and elevated concentrations of sCD30 shortly after transplantation were associated with increased risk for acute rejection in the first post-transplant year. Measurement of soluble CD30 after transplantation, taken into consideration with the presence of HLA class II antibodies, might be helpful for evaluating the potential risk for acute rejection.

Down-regulation of OATP1B proteins correlates with hyperbilirubinemia in advanced cholestasis.

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.