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Abdominal normothermic regional perfusion (aNRP) is an in situ normothermic oxygenated donor perfusion technique before procurement during controlled donation after circulatory death (cDCD) procedures and allows for organ quality evaluation. There are few data on the effect of aNRP on pancreatic islet isolation and subsequent transplantation outcomes. We aim to evaluate the impact of aNRP on cDCD pancreatic islet isolation and transplantation. A retrospective analysis was performed on pancreatic islet isolation outcomes from aNRP, cDCD, and donation after brain death pancreases. Isolations were compared to previous donor age (60-75 years) matched isolations. Islet function was assessed by a dynamic glucose-stimulated insulin secretion. Donor baseline characteristics did not differ among groups. Isolations from aNRP pancreases (471 739 islet equivalents [IEQ] [655 435-244 851]) yielded more islets compared to cDCD (218 750 IEQ [375 951-112 364], P < .01) and to donation after brain death (206 522 IEQ [385 544-142 446], P = .03) pancreases. Dynamic glucose-stimulated insulin secretion tests in 7 aNRP islet preparations showed a mean stimulation index of 4.91, indicating good functionality. Bilirubin and alanine aminotransferase during aNRP correlated with islet yield (r 2 = 0.685, P = .002; r 2 = 0.491, P = .016, respectively). Islet isolation after aNRP in cDCD donors results in a high islet yield with viable functional islets. aNRP could increase the utilization of the pancreases for islet transplantation., Competing Interests: Declaration of competing interest The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: In an effort to reduce waitlist mortality, extended criteria donor organs, including those from donation after circulatory death (DCD), are being used with increasing frequency. These donors carry an increased risk for postoperative complications, and balancing donor-recipient risks is currently based on generalized nomograms. Abdominal normothermic regional perfusion (aNRP) enables individual evaluation of DCD organs, but a gold standard to determine suitability for transplantation is lacking. This study aimed to incorporate individualized and predictive measurements of the liver maximum capacity (LiMAx) test to objectively grade liver function during aNRP and prevent post-op complications., Methods: aNRP was performed to salvage 18 DCD liver grafts, otherwise discarded. Continuous variables were presented as the median with the interquartile range., Results: The liver function maximum capacity (LiMAx) test was successfully performed within the aNRP circuit in 17 aNRPs (94%). Donor livers with good lactate clearance during aNRP demonstrated significantly higher LiMAx scores (396 (301-451) µg/kg/h versus those who did not 105 (70-158) µg/kg/h; P  = 0.006). This was also true for manifesting stress hyperglycemia > 20 mmol/l ( P  = 0.032). LiMAx score correlated with alanine aminotransferase (ALT; R  =  - 0.755) and aspartate transaminase (AST; R  =  - 0.800) levels during perfusion and distinguished livers that were selected for transplantation (397 (346-453) µg/kg/h) from those who were discarded (155 (87-206) µg/kg/h; P  < 0.001). Twelve livers were accepted for transplantation, blinded for LiMAx results, and all had LiMAx scores of > 241 µg/kg/h. Postoperatively, LiMAx during aNRP displayed correlation with 24-h lactate levels., Conclusions: This study shows for the first time the feasibility to assess liver function during aNRP in individual donor livers. LiMAx presents an objective tool to predict donor liver function and risk of complications in the recipient, thus enabling individualized matching of donor livers for an individual recipient. The LiMAx test may present a valuable test for the prediction of donor liver function, preventing post-transplant complication, and personalizing the selection of donor livers for individual recipients., Supplementary Information: The online version contains supplementary material available at 10.1007/s13167-024-00371-7., Competing Interests: Competing interestsThe authors have no conflict of interest., (© The Author(s) 2024.)

Objectives: The human oral microbiome may play a role in the development of oral squamous cell carcinoma. The aim of this scoping review was to examine microbial diversity and differences in the composition of the oral microbiome between OSCC patients and healthy controls., Methods: A literature search (in PubMed and Embase.com) was performed on January 9, 2023. The outcome variables used from the included studies of this review were alpha- and beta diversity and oral microbiome composition profiles for each taxonomic level (phylum-, class-, order-, genus- and species level)., Results: Thirteen out of 423 studies were included in this review compromising 1,677 subjects, of which 905 (54.0%) were OSCC patients and 772 (46.0%) were healthy controls. Most studies found a higher alpha diversity in the OSCC patient group and significantly different beta diversities between OSCC patient samples and healthy control samples. Studies reported more abundant Fusobacteria (on phylum level), Fusobacterium (on genus level), Fusobacterium nucleatum, Porphyromonas endodontalis and Prevotella intermedia (on species level) in OSCC patients. The healthy control group had more abundant Actinobacteria (on phylum level), Streptococcus and Veilonella (on genus level) and Veilonella parvula (on species level) according to most studies., Conclusions: Our findings show differences in oral microbiome diversity and composition in OSCC patients. Clinical implications demand continuing study. Development of internationally accepted standard procedures for oral sample collection and oral microbiota analysis is needed for more conclusive and clinically relevant comparisons in future research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 van Dijk, Petersen, Raber-Durlacher, Epstein and Laheij.)

Iron deficiency anemia can be treated with oral or intravenous Fe supplementation. Such supplementation has considerable effects on the human microbiome, and on opportunistic pathogenic micro-organisms. Molecular understanding of the control and regulation of Fe availability at the host-microbe interface is crucial to interpreting the side effects of Fe supplementation. Here, we provide a concise overview of the regulation of Fe by the opportunistic pathogen Staphylococcus aureus . Ferric uptake regulator (Fur) plays a central role in controlling Fe uptake, utilization and storage in order to maintain a required value. The micro-organism has a strong preference for heme iron as an Fe source, which is enabled by the Iron-regulated surface determinant (Isd) system. The strategies it employs to overcome Fe restriction imposed by the host include: hijacking host proteins, replacing metal cofactors, and replacing functions by non-metal dependent enzymes. We propose that integrated omics approaches, which include metalloproteomics, are necessary to provide a comprehensive understanding of the metal tug of war at the host-microbe interface down to the molecular level., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 van Dijk, de Kruijff and Hagedoorn.)

Fragile perinatal and fetal brains are the rule rather than the exception for developmental neuropathologists. Retrieving the fresh brain from the skull and examining early fetal, macerated or severely hydrocephalic brains after fixation can be a challenge. Textbooks on neurodevelopmental pathology mention these challenges to macroscopic examination of the developing central nervous system only in passing, but many perinatal pathologists recognize this diagnostic problem. We reviewed protocols and publications on the removal, fixation, slicing and sampling of these fetal- and perinatal brains. In addition, we describe a technique to facilitate the removal of severely hydrocephalic brains with very thin cerebral walls from the skull by replacing the intraventricular fluid with agar in-situ . Furthermore, we present a method for post-fixation pre-embedding in agar to facilitate slicing, macroscopic examination and sampling of fragile and macerated brains.

Importance: Application of deep learning algorithms to whole-slide pathology images can potentially improve diagnostic accuracy and efficiency., Objective: Assess the performance of automated deep learning algorithms at detecting metastases in hematoxylin and eosin-stained tissue sections of lymph nodes of women with breast cancer and compare it with pathologists' diagnoses in a diagnostic setting., Design, Setting, and Participants: Researcher challenge competition (CAMELYON16) to develop automated solutions for detecting lymph node metastases (November 2015-November 2016). A training data set of whole-slide images from 2 centers in the Netherlands with (n = 110) and without (n = 160) nodal metastases verified by immunohistochemical staining were provided to challenge participants to build algorithms. Algorithm performance was evaluated in an independent test set of 129 whole-slide images (49 with and 80 without metastases). The same test set of corresponding glass slides was also evaluated by a panel of 11 pathologists with time constraint (WTC) from the Netherlands to ascertain likelihood of nodal metastases for each slide in a flexible 2-hour session, simulating routine pathology workflow, and by 1 pathologist without time constraint (WOTC)., Exposures: Deep learning algorithms submitted as part of a challenge competition or pathologist interpretation., Main Outcomes and Measures: The presence of specific metastatic foci and the absence vs presence of lymph node metastasis in a slide or image using receiver operating characteristic curve analysis. The 11 pathologists participating in the simulation exercise rated their diagnostic confidence as definitely normal, probably normal, equivocal, probably tumor, or definitely tumor., Results: The area under the receiver operating characteristic curve (AUC) for the algorithms ranged from 0.556 to 0.994. The top-performing algorithm achieved a lesion-level, true-positive fraction comparable with that of the pathologist WOTC (72.4% [95% CI, 64.3%-80.4%]) at a mean of 0.0125 false-positives per normal whole-slide image. For the whole-slide image classification task, the best algorithm (AUC, 0.994 [95% CI, 0.983-0.999]) performed significantly better than the pathologists WTC in a diagnostic simulation (mean AUC, 0.810 [range, 0.738-0.884]; P < .001). The top 5 algorithms had a mean AUC that was comparable with the pathologist interpreting the slides in the absence of time constraints (mean AUC, 0.960 [range, 0.923-0.994] for the top 5 algorithms vs 0.966 [95% CI, 0.927-0.998] for the pathologist WOTC)., Conclusions and Relevance: In the setting of a challenge competition, some deep learning algorithms achieved better diagnostic performance than a panel of 11 pathologists participating in a simulation exercise designed to mimic routine pathology workflow; algorithm performance was comparable with an expert pathologist interpreting whole-slide images without time constraints. Whether this approach has clinical utility will require evaluation in a clinical setting.

In renal transplantation, use of calcineurin inhibitors (CNIs) is associated with nephrotoxicity and immunosuppression with malignancies and infections. This trial aimed to minimize CNI exposure and total immunosuppression while maintaining efficacy. We performed a randomized controlled, open-label multicenter trial with early cyclosporine A (CsA) elimination. Patients started with basiliximab, prednisolone (P), mycophenolate sodium (MPS), and CsA. At 6 months, immunosuppression was tapered to P/CsA, P/MPS, or P/everolimus (EVL). Primary outcomes were renal fibrosis and inflammation. Secondary outcomes were estimated glomerular filtration rate (eGFR) and incidence of rejection at 24 months. The P/MPS arm was prematurely halted. The trial continued with P/CsA (N = 89) and P/EVL (N = 96). Interstitial fibrosis and inflammation were significantly decreased and the eGFR was significantly higher in the P/EVL arm. Cumulative rejection rates were 13% (P/EVL) and 19% (P/CsA), (p = 0.08). A post hoc analysis of HLA and donor-specific antibodies at 1 year after transplantation revealed no differences. An individualized immunosuppressive strategy of early CNI elimination to dual therapy with everolimus was associated with decreased allograft fibrosis, preserved allograft function, and good efficacy, but also with more serious adverse events and discontinuation. This can be a valuable alternative regimen in patients suffering from CNI toxicity., (© Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Percutaneous coronary intervention is widely adopted to treat patients with coronary artery disease. However, restenosis remains an unsolved clinical problem after vascular interventions. The role of the systemic and local immune response in the development of restenosis is not fully understood. Hence, the aim of the current study was to investigate the role of the human immune system on subsequent neointima formation elicited by vascular injury in a humanized mouse model. Immunodeficient NOD.Cg-Prkdc scid IL2rg tm1Wjl (NSG) mice were reconstituted with human (h)PBMCs immediately after both carotid wire and femoral cuff injury were induced in order to identify how differences in the severity of injury influenced endothelial regeneration, neointima formation, and homing of human inflammatory and progenitor cells. In contrast to non-reconstituted mice, hPBMC reconstitution reduced neointima formation after femoral cuff injury whereas hPBMCs promoted neointima formation after carotid wire injury 4 weeks after induction of injury. Neointimal endothelium and smooth muscle cells in the injured arteries were of mouse origin. Our results indicate that the immune system may differentially respond to arterial injury depending on the severity of injury, which may also be influenced by the intrinsic properties of the arteries themselves, resulting in either minimal or aggravated neointima formation.

Background: Data on the outcome of renal transplantation in antineutrophil cytoplasmic antibody-associated glomerulonephritis (AAGN) patients are still limited. In particular, how disease recurrence in the renal allograft defines graft outcome is largely unknown. Therefore, we conducted a multicenter observational clinical and histopathological study to establish recurrence rate of AAGN in the allograft and the impact of recurrence on allograft survival., Methods: Using the nationwide Dutch Pathology Registry (PALGA), we retrospectively collected clinical and histopathological data of consecutive AAGN patients who had developed end-stage renal failure and received a kidney allograft in 1 of 6 Dutch university hospitals between 1984 and 2011. Transplant biopsies were scored using the Banff '09 classification. Renal disease recurrence was scored using the histopathological classification of AAGN., Results: The posttransplantation recurrence rate of AAGN was 2.8% per patient year, accumulating to recurrence in a total of 11 of 110 AAGN patients within the first 5 years after transplantation. Four of these 11 patients lost their graft, with 1-year and 5-year graft survival rates of 94.5% and 82.8%, respectively. By multivariate analysis, AAGN recurrence was independently associated with subsequent graft loss., Conclusions: In this study in 110 Dutch patients, the recurrence rate of AAGN within 5 years after kidney transplantation appeared slightly higher than in previous reports. Moreover, recurrence of AAGN contributed independently to kidney allograft loss, emphasizing the importance of clinical vigilance, because early treatment might be critical to rescuing the allograft.

Syndecan-1 is a transmembrane heparan sulfate proteoglycan involved in regenerative growth and cellular adhesion. We hypothesized that the induction of tubular syndecan-1 is a repair response to incipient renal damage in apparently stable, uncomplicated renal transplant recipients. We quantified tubular syndecan-1 in unselected renal protocol biopsies taken 1 yr after transplantation. Spearman rank correlation analysis revealed an inverse correlation between tubular syndecan-1 expression and creatinine clearance at the time of biopsy (r = -0.483, P < 0.03). In a larger panel of protocol and indication biopsies from renal transplant recipients, tubular syndecan-1 correlated with tubular proliferation marker Ki67 (r = 0.518, P < 0.0001). In a rat renal transplantation model, 2 mo after transplantation, mRNA expression of syndecan-1 and its major sheddase, A disintegrin and metalloproteinase-17, were upregulated (both P < 0.03). Since shed syndecan-1 might end up in the circulation, in a stable cross-sectional human renal transplant population (n = 510), we measured plasma syndecan-1. By multivariate regression analysis, we showed robust independent associations of plasma syndecan-1 with renal (plasma creatinine and plasma urea) and endothelial function parameters (plasma VEGF-A, all P < 0.01). By various approaches, we were not able to localize syndecan-1 in vessel wall or endothelial cells, which makes shedding of syndecan-1 from the endothelial glycocalyx unlikely. Our data suggest that early damage in transplanted kidneys induces repair mechanisms within the graft, namely, tubular syndecan-1 expression for tubular regeneration and VEGF production for endothelial repair. Elevated plasma syndecan-1 levels in renal transplantation patients might be interpreted as repair/survival factor related to loss of tubular and endothelial function in transplanted kidneys., (Copyright © 2015 the American Physiological Society.)

In chronic transplant dysfunction (CTD), persistent (allo)immune-mediated inflammation eventually leads to tissue remodeling including neointima formation in intragraft arteries. We previously showed that recipient-derived neointimal α-SMA(+) smooth muscle-like cells are present in human renal allografts with CTD. Human PBMC contain myeloid cells capable of differentiating into α-SMA(+) cells in vitro; the phenotype of the ancestral subset is as yet unknown. This study aimed to investigate whether monocyte subsets contain cells with smooth muscle-like cell differentiation capacity and whether CTD in renal transplant recipients is associated with a shift in these monocyte subsets. To accomplish this goal, monocyte subsets from healthy controls were sorted based on CD14 and CD16 expression to investigate gene expression levels of mesenchymal markers α-SMA and SM22α. CD14(+)/CD16(++) monocytes displayed increased α-SMA and SM22α mRNA expression compared with CD14(++)/CD16(-) monocytes, suggesting increased differentiation potential toward smooth muscle-like cells. Flow cytometry revealed that in non-CTD transplant recipients the percentage of CD14(+)/CD16(++) monocytes was reduced, with an even further reduction in patients with CTD. To determine a potential correlation between CD14(+)/CD16(++) monocytes and α-SMA(+) cell outgrowth potential in vitro, PBMC of healthy controls and transplant recipients with and without CTD were cultured under fibrotic culture conditions, and indeed a significant correlation (p=0.0002, r=0.62) was observed. Finally, double staining for α-SMA and CD16 revealed presence of α-SMA(+)CD16(+) cells in kidney explants from CTD patients, albeit at very low numbers. Our data represent evidence that, compared to CD14(++)CD16(-) monocytes, CD14(+)CD16(++) monocytes have an increased expression of smooth muscle cell-associated genes. This monocyte subpopulation is reduced in renal transplant patients with CTD, possibly due to selective migration into the allograft., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Background: A "thrombus in transit" is a relatively rare diagnosis involving a thrombus in a patent foramen ovale. Patent foramen ovale occurs in about 25% of the population. A thrombus in transit may lead to paradoxical arterial emboli in the cerebral circulatory system and the extremities, as well as other locations., Case Description: A 60-year-old male patient with severe pneumonia sepsis appeared to have a thrombus in the right atrium, extending into the left atrium through a patent foramen ovale. The patient was treated with therapeutic anticoagulants. Cerebral embolization occurred despite this, with extensive cerebral ischaemia. The patient ultimately died from multiple organ failure., Conclusion: A thrombus in transit may be treated with heparins, thrombolysis or by surgical removal of the thrombus. The optimum treatment must be decided for each individual patient. The mortality rate of this condition is high (16-36%).

Background: Renal aging is characterized by functional and structural changes like decreased glomerular filtration rate, and glomerular, tubular and interstitial damage. To gain insight in pathways involved in renal aging, we studied aged mouse strains and used genetic analysis to identify genes associated with aging phenotypes., Methods: Upon morphological screening in kidneys from 20-month-old mice from 26 inbred strains we noted intracapillary PAS-positive deposits. The severity of these deposits was quantified by scoring of a total of 50 glomeruli per section (grade 0-4). Electron microscopy and immunohistochemical staining for apoE, apoB, apoA-IV and perilipin-2 was performed to further characterize the lesions. To identify loci associated with these PAS-positive intracapillary glomerular deposits, we performed haplotype association mapping., Results: Six out of 26 mouse strains showed glomerular PAS-positive deposits. The severity of these deposits varied: NOD(0.97), NZW(0.41), NON(0.30), B10(0.21), C3 H(0.9) and C57BR(0.7). The intracapillary deposits were strongly positive for apoE and weakly positive for apoB and apoA-IV. Haplotype association mapping showed a strong association with a 30-Kb haplotype block on Chr 1 within the Esrrg gene. We investigated 1 Mb on each site of this region, which includes the genes Spata17, Gpatch2, Esrrg, Ush2a and Kctd3., Conclusions: By analyzing 26 aged mouse strains we found that some strains developed an intracapillary PAS and apoE-positive lesion and identified a small haplotype block on Chr 1 within the Esrrg gene to be associated with these lipoprotein deposits. The region spanning this haplotype block contains the genes Spata17, Gpatch2, Esrrg, Ush2a and Kctd3, which are all highly expressed in the kidney. Esrrg might be involved in the evolvement of these glomerular deposits by influencing lipid metabolism and possibly immune reponses.

Background and Objectives: The objective of this study was to characterize CD4(+) and CD8(+) T-cell populations in blood and urine of renal transplant patients with BK virus (BKV) infection or allograft rejection., Materials and Methods: Percentages and absolute numbers of CD4(+) and CD8(+) effector memory T-cell subtype (TEM ) and terminal differentiated T cells (TTD ) in renal transplant patients with BKV infection (n = 14), with an episode of allograft rejection (n = 9), and in uncomplicated renal transplant patients with a stable kidney function (n = 12) were measured and compared using 4-color fluorescence-activated cell sorting. Results were correlated with the number of CD4(+) and CD8(+) T cells in renal biopsies., Results: In patients with allograft rejection, the number of urinary CD4(+) TEM and CD8(+) TEM cells was significantly increased compared to patients with BKV infection or patients without complications. Positive correlation was found between the number of CD4(+) and CD8(+) cells in the renal biopsies and the number of CD4(+) and CD8(+) cells in urine. In patients with rejection, after 2 months of immunosuppressive therapy, a reduction in urinary CD8(+) TEM cells was found., Conclusions: CD4(+) TEM and CD8(+) TEM cells in urine could be a marker to distinguish allograft rejection from BKV-associated nephropathy and to monitor therapy effectiveness in renal transplant patients with allograft rejection., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Diabetes is associated with a high incidence of macrovascular disease (MVD), including peripheral and coronary artery disease. Circulating soluble-Klotho (sKlotho) is produced in the kidney and is a putative anti-aging and vasculoprotective hormone. Reduced Klotho levels may therefore increase cardiovascular risk in diabetes. We investigated if sKlotho levels are decreased in type 2 diabetes and associate with MVD in the absence of diabetic nephropathy, and whether hyperglycemia affects renal Klotho production in vitro and in vivo., Methods: sKlotho levels were determined with ELISA in diabetic and non-diabetic patients with and without MVD, and healthy control subjects. Human renal tubular epithelial cells (TECs) were isolated and exposed to high glucose levels (15 and 30 mM) in vitro and Klotho levels were measured with qPCR and quantitative immunofluorescence. Klotho mRNA expression was quantified in kidneys obtained from long term (3 and 8 months) diabetic Ins2Akita mice and normoglycemic control mice., Results: No significant differences in sKlotho levels were observed between diabetic patients with and without MVD (527 (433-704) pg/mL, n = 35), non-diabetic MVD patients (517 (349-571) pg/mL, n = 27), and healthy control subjects (435 (346-663) pg/mL, n = 15). High glucose (15 and 30 mM) did not alter Klotho expression in TECs. Long-term hyperglycemia in diabetic Ins2Akita mice (characterized by increased HbA1c levels [12.9 ± 0.3% (3 months) and 11.3 ± 2.0% (8 months)], p < 0.05 vs. non-diabetic mice) did not affect renal Klotho mRNA expression., Conclusions: These data indicate that sKlotho levels are not affected in type 2 diabetes patients with and without MVD. Furthermore, hyperglycemia per se does not affect renal Klotho production. As type 2 diabetes does not alter sKlotho levels, sKlotho does not seem to play a major role in the pathogenesis of MVD in type 2 diabetes.

Background: Ischemia-reperfusion injury (IRI) is a major cause of cardiac damage following various pathological processes. Gaseous hydrogen sulfide (H2S) is protective during IRI by inducing a hypometabolic state in mice which is associated with anti-apoptotic, anti-inflammatory and antioxidant properties. We investigated whether gaseous H2S administration is protective in cardiac IRI and whether non-hypometabolic concentrations of H2S have similar protective properties., Methods: Male C57BL/6 mice received a 0, 10, or 100 ppm H2S-N2 mixture starting 30 minutes prior to ischemia until 5 minutes pre-reperfusion. IRI was inflicted by temporary ligation of the left coronary artery for 30 minutes. High-resolution respirometry equipment was used to assess CO2-production and blood pressure was measured using internal transmitters. The effects of H2S were assessed by histological and molecular analysis., Results: Treatment with 100 ppm H2S decreased CO2-production by 72%, blood pressure by 14% and heart rate by 25%, while treatment with 10 ppm H2S had no effects. At day 1 of reperfusion 10 ppm H2S showed no effect on necrosis, while treatment with 100 ppm H2S reduced necrosis by 62% (p<0.05). Seven days post-reperfusion, both 10 ppm (p<0.01) and 100 ppm (p<0.05) H2S showed a reduction in fibrosis compared to IRI animals. Both 10 ppm and 100 ppm H2S reduced granulocyte-influx by 43% (p<0.05) and 60% (p<0.001), respectively. At 7 days post-reperfusion both 10 and 100 ppm H2S reduced expression of fibronectin by 63% (p<0.05) and 67% (p<0.01) and ANP by 84% and 63% (p<0.05), respectively., Conclusions: Gaseous administration of H2S is protective when administered during a cardiac ischemic insult. Although hypometabolism is restricted to small animals, we now showed that low non-hypometabolic concentrations of H2S also have protective properties in IRI. Since IRI is a frequent cause of myocardial damage during percutaneous coronary intervention and cardiac transplantation, H2S treatment might lead to novel therapeutical modalities.

Genome-wide association studies reported SLC22A2 variants to be associated with serum creatinine. As SLC22A2 encodes the organic cation transporter 2 (OCT2), the association might be due to an effect on tubular creatinine handling. To test this hypothesis we studied the association of SLC22A2 polymorphisms with phenotypes of net tubular creatinine secretion: fractional creatinine excretion (FEcreat) and bias of estimated glomerular filtration rate (eGFR). We also studied the association with end-stage renal disease (ESRD) and graft failure (GF) in renal transplant recipients. SLC22A2 single nucleotide polymorphisms (SNPs), rs3127573 and rs316009, were genotyped in 1,142 ESRD patients receiving renal transplantation and 1,186 kidney donors as controls. GFR was measured with (125)I-iothalamate clearance. Creatinine clearance was also assessed. FEcreat was calculated from the simultaneous clearances of creatinine and (125)I-iothalamate. Donor rs316009 was associated with FEcreat (beta -0.053, P = 0.024) and with estimated [modification of diet in renal disease (MDRD) and Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI)] but not measured GFR. In line with this, donor rs316009 was associated with bias of the MDRD and CKD-EPI but not the Cockroft-Gault equation. Both SNPs were associated with ESRD: odds ratios [95% CI] 1.39 [1.16-1.67], P = 0.00065, and 1.23 [1.02-1.48], P = 0.042, for rs3127573 and rs316009, respectively. Neither SNP was associated with GF. Thus, SLC22A2 is associated with phenotypes of net tubular creatinine secretion and ESRD.

Aims: The complement system, and especially C5a, plays an important role in the pathophysiology of renal diseases and post-transplant renal injury. The two receptors for C5a are C5a receptor (C5aR) and C5a-like-receptor-2 (C5L2). Only renal C5aR expression has been reported, although exact localization and alterations in expression after transplantation are unknown., Materials and Results: Renal C5aR and C5L2 expression and localization were analyzed immunohistochemically. C5aR and C5L2 expression was analyzed in human kidney biopsies obtained from living donors and patients suffering from acute tubular necrosis, acute cellular and vascular rejection or IF/TA. C5aR was expressed in the thick ascending limb of Henle's loop and first part of the distal convoluted tubule (DCT). Under inflammatory conditions, C5aR was de novo expressed in proximal tubuli. C5L2 was expressed in the kidney and localized to DCT1, DCT2 and connecting tubule. Persistent distal tubular expression of both receptors was demonstrated after renal transplantation., Conclusions: This study shows distinct renal expression patterns for C5aR and C5L2. Our findings suggest a functional role for renal C5L2 rather than being a C5a decoy receptor. Future studies focusing on renal C5a-C5aR interaction should take differential C5aR and C5L2 expression into account, alongside abundant C5aR expression on infiltrating cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Introduction: Lupus nephritis (LN) is a severe and frequent manifestation of systemic lupus erythematosus (SLE). Early detection of initial renal manifestations and relapses during follow-up is pivotal to prevent loss of renal function. Apart from renal biopsies, current urinary and serological diagnostic tests fail to accurately demonstrate the presence of active LN. Previously, we demonstrated that effector memory T-cells (CD45RO+CCR7-;TEM) migrate into the urine during active LN. The objective of this study was to assess the diagnostic value of urinary T-cells in comparison with traditional markers of active LN., Methods: T-cells in the urine during active LN and remission were investigated. Twenty-two, in most cases biopsy-proven, active LN patients and 24 SLE patients without active LN were enrolled and serial measurements were performed in 16 patients., Results: Analysis of the urinary sediment in active renal disease showed an increased number of CD8+ T-cells and absence of these cells during remission. Enumerating T-cell counts in LN patients with a history of renal involvement was a superior marker of active LN in comparison to traditional markers, such as proteinuria and s-creatinine., Conclusions: In conclusion, urinary T-cells, in particular CD8+ T cells, are a promising marker to assess renal activity in LN patients, in particular in those with prior renal involvement.

Despite numerous studies reporting deregulated microRNA (miRNA) and gene expression patterns in clear cell renal cell carcinoma (ccRCC), no direct comparisons have been made to its presumed normal counterpart: the renal proximal tubular epithelial cells (PTECs). The aim of this study was to determine the miRNA expression profiles of 10 ccRCC-derived cell lines and short-term cultures of PTEC and to correlate these with their gene expression and copy-number profiles. Using microarray-based methods, a significantly altered expression level in ccRCC cell lines was observed for 23 miRNAs and 1630 genes. The set of miRNAs with significantly decreased expression levels include all members of the miR-200 family known to be involved in the epithelial to mesenchymal transition process. Expression levels of 13 of the 47 validated target genes for the downregulated miRNAs were increased more than twofold. Our data reinforce the importance of the epithelial to mesenchymal transition process in the development of ccRCC., (Copyright © 2012 Wiley Periodicals, Inc.)

Background: In recent genetic association studies, common variants including rs12917707 in the UMOD locus have shown strong evidence of association with eGFR, prevalent and incident chronic kidney disease and uromodulin urinary concentration in general population cohorts. The association of rs12917707 with end-stage renal disease (ESRD) in a recent case-control study was only nominally significant., Methods: To investigate whether rs12917707 associates with ESRD, graft failure (GF) and urinary uromodulin levels in an independent cohort, we genotyped 1142 ESRD patients receiving a renal transplantation and 1184 kidney donors as controls. After transplantation, 1066 renal transplant recipients were followed up for GF. Urinary uromodulin concentration was measured at median [IQR] 4.2 [2.2-6.1] yrs after kidney transplantation., Results: The rs12917707 minor allele showed association with lower risk of ESRD (OR 0.89 [0.76-1.03], p = 0.04) consistent in effect size and direction with the previous report (Böger et al, PLoS Genet 2011). Meta-analysis of these findings showed significant association of rs12917707 with ESRD (OR 0.91 [0.85-98], p = 0.008). In contrast, rs12917707 was not associated with incidence of GF. Urinary uromodulin concentration was lower in recipients-carriers of the donor rs12917707 minor allele as compared to non-carriers, again consistent with previous observations in general population cohorts., Conclusions: Our study thus corroborates earlier evidence and independently confirms the association between UMOD and ESRD.

Introduction: Lupus nephritis (LN) is a severe and frequent manifestation of systemic lupus erythematosus (SLE). Its pathogenesis has not been fully elucidated but immune complexes are considered to contribute to the inflammatory pathology in LN. High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein which is secreted from different types of cells during activation and/or cell death and may act as a pro-inflammatory mediator, alone or as part of DNA-containing immune complexes in SLE. Urinary excretion of HMGB1 might reflect renal inflammatory injury. To assess whether urinary HMGB1 reflects renal inflammation we determined serum levels of HMGB1 simultaneously with its urinary levels in SLE patients with and without LN in comparison to healthy controls (HC). We also analyzed urinary HMGB1 levels in relation with clinical and serological disease activity., Methods: The study population consisted of 69 SLE patients and 17 HC. Twenty-one patients had biopsy proven active LN, 15 patients had a history of LN without current activity, and 33 patients had non-renal SLE. Serum and urine levels of HMGB1 were both measured by western blotting. Clinical and serological parameters were assessed according to routine procedures. In 17 patients with active LN a parallel analysis was performed on the expression of HMGB1 in renal biopsies., Results: Serum and urinary levels of HMGB1 were significantly increased in patients with active LN compared to patients without active LN and HC. Similarly, renal tissue of active LN patients showed strong expression of HMGB1 at cytoplasmic and extracellular sites suggesting active release of HMGB1. Serum and urinary levels in patients without active LN were also significantly higher compared to HC. Urinary HMGB1 levels correlated with SLEDAI, and showed a negative correlation with complement C3 and C4., Conclusion: Levels of HMGB1 in urine of SLE patients, in particular in those with active LN, are increased and correlate with SLEDAI scores. Renal tissue of LN patients shows increased release of nuclear HMGB1 compared to control renal tissue. HMGB1, although at lower levels, is, however, also present in the urine of patients without active LN. These data suggest that urinary HMGB1 might reflect both local renal inflammation as well as systemic inflammation.

Hydrogen sulfide (H2 S) can induce a reversible hypometabolic state, which could protect against hypoxia. In this study we investigated whether H2 S could protect livers from ischemia/reperfusion injury (IRI). Male C57BL/6 mice were subjected to partial hepatic IRI for 60 min. Animals received 0 (IRI) or 100 ppm H2 S (IRI + H2 S) from 30 min prior to ischemia until 5 min before reperfusion. Core body temperature was maintained at 37° C. Animals were sacrificed after 1, 6 or 24 h. Hepatic ischemia caused extensive hepatic necrosis in the IRI animals which coincided with an increase in ALT and AST serum levels. Animals treated with H2 S showed attenuated serum ALT and AST levels and reduced necrotic lesions after 24 h. IRI animals had increased Bcl-2 mRNA expression and increased active Caspase 3 protein, which were both significantly lower in H2 S treated animals. Increased TNFα and IL-6 mRNA in the IRI livers was significantly attenuated by H2 S treatment, as was hepatic influx of Ly-6G positive granulocytes. Hepatic superoxide production after ischemia was attenuated by H2 S treatment. In hepatic ischemia/reperfusion injury, gaseous H2 S treatment is highly protective, substantially reducing necrosis, apoptosis and inflammation. Gaseous H2 S is therefore a very promising treatment for reducing IRI during hepatic transplantation., (© 2012 The Authors. Transplant International © 2012 European Society for Organ Transplantation.)

Background: Interstitial fibrosis and tubular atrophy (IF/TA) is an important cause of renal function loss and ischaemia-reperfusion (I/R) injury is considered to play an important role in its pathophysiology. The aim of the present study was to investigate the role of a disintegrin and metalloproteinase 17 (ADAM17) in human renal allograft disease and in experimental I/R injury of the kidney., Methods: We studied the expression of ADAM17 messenger RNA (mRNA) in IF/TA and control kidneys by reverse transcription-polymerase chain reaction and in situ hybridization. Moreover, we assessed ADAM17-mediated heparin-binding epidermal growth factor (HB-EGF) shedding in immortalized human cells. Finally, we studied the effect of pharmacological ADAM17 inhibition in a model of renal I/R injury in rats., Results: ADAM17 mRNA was up-regulated in IF/TA when compared to control kidneys. In normal kidneys, ADAM17 mRNA was weakly expressed in proximal tubules, peritubular capillaries, glomerular endothelium and parietal epithelium. In IF/TA, tubular, capillary and glomerular ADAM17 expression was strongly enhanced with de novo expression in the mesangium. In interstitial fibrotic lesions, we observed co-localization of ADAM17 with HB-EGF protein. In vitro, inhibition of ADAM17 with TNF484 resulted in a dose-dependent reduction of HB-EGF shedding in phorbol 12-myrisate 13-acetate-stimulated cells and non-stimulated cells. In vivo, ADAM17 inhibition significantly reduced the number of glomerular and interstitial macrophages at Day 4 of reperfusion., Conclusions: In conclusion, HB-EGF co-expresses with ADAM17 in renal interstitial fibrosis, suggesting a potential interaction in IF/TA. Targeting ADAM17 to reduce epidermal growth factor receptor phosphorylation could be a promising way of intervention in human renal disease.

Syndecan-1, a heparan sulfate proteoglycan, has an important role in wound healing by binding several growth factors and cytokines. As these processes are also crucial in damage and repair after renal transplantation, we examined syndecan-1 expression in human control kidney tissue, renal allograft protocol biopsies, renal allograft biopsies taken at indication, and non-transplant interstitial fibrosis. Syndecan-1 expression was increased in tubular epithelial cells in renal allograft biopsies compared with control. Increased epithelial syndecan-1 in allografts correlated with low proteinuria and serum creatinine, less interstitial inflammation, less tubular atrophy, and prolonged allograft survival. Knockdown of syndecan-1 in human tubular epithelial cells in vitro reduced cell proliferation. Selective binding of growth factors suggests that syndecan-1 may promote epithelial restoration. Bilateral renal ischemia/reperfusion in syndecan-1-deficient mice resulted in increased initial renal failure and tubular injury compared with wild-type mice. Macrophage and myofibroblast numbers, tubular damage, and plasma urea levels were increased, and tubular proliferation reduced in the kidneys of syndecan-1 deficient compared with wild-type mice 14 days following injury. Hence syndecan-1 promotes tubular survival and repair in murine ischemia/reperfusion injury and correlates with functional improvement in human renal allograft transplantation.

In kidney transplantation, complement activation was found to be induced by donor brain death, renal ischemia-reperfusion injury and allograft rejection. There are three known pathways of complement activation: the classical, lectin and the alternative pathway. The lectin complement pathway can be activated upon pattern recognition by mannan binding lectin (MBL) or ficolins (FCN). Single nucleotide polymorphisms (SNPs) in the genes encoding the lectin pathway proteins determine their functional activity and serum levels. The aim of this study was to investigate the role of the lectin gene profile of the donor and recipient on post-transplant outcome. A total of 12 functional SNPs in the MBL2, FCN2 and MBL-associated serine proteases 2 (MASP2) genes of 1271 donor-recipient pairs were determined. Lectin genotypic variants were analyzed for association with primary non-function (PNF), delayed graft function (DGF), biopsy proven acute rejection, death-censored graft survival and patient survival. Multivariate analyses found no association of donor and recipient MBL2 and MASP2 genotype with allograft outcome. Analysis of separate functional SNPs and haplotypes in the FCN2 gene of the donor and recipient did not reveal an association with transplant outcome. Also, the joint effect of the MBL2 and FCN2 genotype was not associated with allograft outcome.This study shows that the genetic profile of the lectin pathway of complement activation of the donor and recipient is not associated with allograft outcome after kidney transplantation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Chronic kidney disease (CKD) is a complex disorder. As genome-wide association studies identified cubilin gene CUBN as a locus for albuminuria, and urinary protein loss is a risk factor for progressive CKD, we tested the hypothesis that common genetic variants in CUBN are associated with end-stage renal disease (ESRD) and proteinuria. First, a total of 1142 patients with ESRD, admitted for renal transplantation, and 1186 donors were genotyped for SNPs rs7918972 and rs1801239 (case-control study). The rs7918972 minor allele frequency (MAF) was higher in ESRD patients comparing to kidney donors, implicating an increased risk for ESRD (OR 1.39, p = 0.0004) in native kidneys. Second, after transplantation recipients were followed for 5.8 [3.8-9.2] years (longitudinal study) documenting ESRD in transplanted kidneys--graft failure (GF). During post-transplant follow-up 92 (9.6%) cases of death-censored GF occurred. Donor rs7918972 MAF, representing genotype of the transplanted kidney, was 16.3% in GF vs 10.7% in cases with functioning graft. Consistently, a multivariate Cox regression analysis showed that donor rs7918972 is a predictor of GF, although statistical significance was not reached (HR 1.53, p = 0.055). There was no association of recipient rs7918972 with GF. Rs1801239 was not associated with ESRD or GF. In line with an association with the outcome, donor rs7918972 was associated with elevated proteinuria levels cross-sectionally at 1 year after transplantation. Thus, we identified CUBN rs7918972 as a novel risk variant for renal function loss in two independent settings: ESRD in native kidneys and GF in transplanted kidneys.

Background: Urinary uromodulin (UMOD) predicts renal prognosis in native kidneys, but data are conflicting. We investigated its prognostic impact for graft failure (GF) in renal transplant recipients (RTR; n = 600)., Methods: UMOD concentration was measured cross-sectionally in RTR at 6.0 years [2.6-11.4] post-transplant, in matched patients with native chronic kidney disease (CKD) and healthy subjects. In 59 cases, RTR allograft biopsies were reviewed., Results: During a follow-up of 5.3 years [4.5-5.7], GF had occurred in 7% of RTR. Median UMOD excretion (mg/24 h) was 20.4 in RTR, 11.6 in CKD and 5.7 in controls (p < 0.001). There was a curvilinear association between UMOD excretion and baseline renal function (p < 0.003) and death-censored GF, with 5.5, 11.5 and 4.0% of the cases in subsequent UMOD excretion tertiles, respectively (p = 0.002). On multivariate Cox regression analysis, hazard ratios for GF for the 1st and 3rd tertiles were 0.37 (p = 0.01) and 0.21 (p = 0.001), respectively. Interstitial fibrosis and tubular atrophy were more severe in the middle tertile (p = 0.007)., Conclusions: Urinary UMOD is elevated in RTR and associated with graft function, morphology and outcome in a bimodal fashion. Dissection of the disparate mechanisms of GF prediction by urinary UMOD might provide new clues for its alleged pathogenetic significance in progressive renal function loss., (Copyright © 2011 S. Karger AG, Basel.)

IgG4-related systemic disease is a new clinical entity with a large variety of clinical symptoms that can affect almost all organs. The best known manifestations are retroperitoneal fibrosis and autoimmune pancreatitis. We present 3 patients aged 71, 83 and 70 years, with malaise, fatigue and swellings suggestive of a malignancy. However, histopathology of these swellings showed infiltration with plasma cells. Increased serum IgG4-levels confirmed the diagnosis 'IgG4-related systemic disease'. All patients responded well to treatment with glucocorticoids. IgG4-related systemic disease is often mistaken for malignancy because of similar presenting symptoms. The diagnosis can easily be confirmed by high serum protein levels, high serum IgG4-levels and infiltrates of IgG4-positive plasma cells. Response to treatment with glucocorticoids is good, as is the prognosis. IgG4-related systemic disease should be part of the differential diagnosis when patients present with malaise, high protein-levels and multi-organ involvement. Rapid diagnosis can prevent unnecessary surgical procedures for malignancy.

Background: Systemic lupus erythematosus (SLE) is accompanied by alterations in T cell homeostasis including an increased effector response. Migrated effector memory T cells (CD45RO(+)CCR7⁻; T(EM)) appear to be involved in tissue injury. The objective of this study was to investigate the distribution and phenotype of effector memory T cells in the peripheral blood (PB), and their presence in renal biopsies and urine of patients with SLE. The hypothesis that these T(EM) cells migrate to the kidney during active disease was tested., Methods: A total of 43 patients with SLE and 20 healthy controls were enrolled. CD4(+)T(EM) cells and CD8(+)T(EM) cells were analysed in PB and urine using flow cytometric analysis. In 10 patients with active lupus nephritis a parallel analysis was performed on the presence of T(EM) cells in kidney biopsies., Results: The percentage of circulating CD8(+)T(EM) cells in patients with SLE was significantly decreased versus healthy controls (33.9±18.3% vs 42.9±11.0%, p=0.008). In patients with active renal involvement (n=12) this percentage was further decreased to 30.4±15.9%, p=0.01. Analysis of the urinary sediment in active renal disease showed increased numbers of CD4(+)T cells (134±71 cells/ml) and CD8(+)T cells (287±220 cells/ml), respectively, while in healthy controls and patients without active renal disease almost no T cells were present. In all, 73.6±8.3% of urinary CD4(+)T cells and 69.3±26.0% of urinary CD8(+)T cells expressed the T(EM) phenotype. CD8(+) cells were also found in renal biopsies., Conclusions: The data presented are compatible with the hypothesis that CD8(+) effector memory cells migrate from the PB to the kidney and appear in the urine during active renal disease in patients with SLE. These cells could serve as an additional marker of renal activity in patients with SLE.

It is often very difficult to confidently distinguish benign and malignant Spitz lesions, and a diagnosis of Spitz tumor of unknown malignant potential (STUMP) is rendered. To address this problem, we performed molecular genetic analysis in a large group of Spitz tumors (93 Spitz nevi and 77 STUMPs) and identified a subgroup of 24 lesions harboring a HRAS mutation. This subgroup lay predominantly in the dermis, had a relatively low cellularity, showed desmoplasia (with single cells interspersed between the collagen bundles), and had an infiltrating base. In 7 of these 24 cases (29%) melanoma had been the initial diagnosis, or an important differential diagnostic consideration, mainly based on the presence of multiple or deeply located mitotic figures, especially in adult patients. In our series none of the patients with the HRAS-mutated lesions developed recurrences or metastases (mean and median follow-up: 10.5 y). This was in accordance with the literature: review showed that no HRAS mutations had so far been reported in Spitzoid melanomas. We therefore conclude that HRAS mutation analysis may be a useful diagnostic tool to help differentiate between Spitz nevus and Spitzoid melanoma, thereby reducing the frequency of overdiagnosis of melanoma, and to help predict the biological behavior of a STUMP. Moreover, this might be a first step toward a more reproducible classification of Spitz tumors combining histological and genetic data.

Malignant transformation of giant congenital nevi (GCN) is very rare, but may already occur in childhood. Benign proliferative nodules in GCN may clinically and histologically mimic a malignant melanoma. We report a newborn infant with large proliferative nodules and multiple satellite nevi in a GCN of the bathing suit type. The diagnosis in our patient was based on the smooth transition of the pre-existing lesion and the nodule, the absence of strong atypical nuclei, the atypical mitotic figures, the ascension of melanocytes in the epidermis and the absence of necrosis. A review of the literature showed that the clinical and the histological features of benign (proliferative) nodules are variable. The awareness and the identification of this entity by clinicians and pathologist are imperative to prevent diagnostic errors.

This review intends to update current knowledge regarding molecular cytogenetics in melanocytic tumours with a focus on cutaneous melanocytic lesions. Advantages and limitations of diverse, already established methods, such as (fluorescence) in situ hybridization and mutation analysis, to detect these cytogenetic alterations in melanocytic tumours are described. In addition, the potential value of more novel techniques such as multiplex ligation-dependent probe amplification is pointed out. This review demonstrates that at present cytogenetics has mainly increased our understanding of the pathogenesis of melanocytic tumours, with an important role for activation of the mitogen-activated protein kinase (MAPK) signalling pathway in the initiation of melanocytic tumours. Mutations in BRAF (in common naevocellular naevi), NRAS (congenital naevi), HRAS (Spitz naevi) and GNAQ (blue naevi) can all cause MAPK activation. All these mutations seem early events in the development of melanocytic tumours, but by themselves are insufficient to cause progression towards melanoma. Additional molecular alterations are implicated in progression towards melanoma, with different genetic alterations in melanomas at different sites and with varying levels of sun exposure. This genetic heterogeneity in distinct types of naevi and melanomas can be used for the development of molecular tests for diagnostic purposes. However, at the moment only few molecular tests have become of diagnostic value and are performed in daily routine practice. This is caused by lack of large prospective studies on the diagnostic value of molecular tests including follow-up, and by the low prevalence of certain molecular alterations. For the future we foresee an increasing role for cytogenetics in the treatment of melanoma patients with the increasing availability of targeted therapy. Potential targets for metastatic melanoma include genes involved in the MAPK pathway, such as BRAF and RAS. More recently, KIT has emerged as a potential target in melanoma patients. These targeted treatments all need careful evaluation, but might be a promising adjunct for treatment of metastatic melanoma patients, in which other therapies have not brought important survival advantages yet.

Tumors are complex tissues composed of neoplastic cells, soluble and insoluble matrix components and stromal cells. Here we report that in melanoma, turn-over of type I collagen (Col(I)), the predominant matrix protein in dermal stroma affects melanoma progression. Fibroblasts juxtaposed to melanoma cell nests within the papillary dermis display high levels of Col(I) mRNA expression. These nests are enveloped by collagen fibers. In contrast, melanoma-associated fibroblasts within the reticular dermis express Col(I) mRNA at a level that is comparable to its expression in uninvolved dermis and reduced amount of collagen protein can be observed. To determine the significance of Col(I) expression in melanoma, we pharmacologically inhibited its transcription in a porcine cutaneous melanoma model by oral administration of halofuginone. When administered before melanoma development, it reduced melanoma incidence and diminished the transition from microinvasive toward deeply invasive growth by limiting the development of a tumor vasculature. Whereas invasive melanoma growth has been correlated with increased blood vessel density previously, our data for the first time demonstrate that the proangiogenic effect of Col(I) expression by fibroblasts and vascular cells precedes the development of invasive melanomas in a de novo tumor model., ((c) 2007 Wiley-Liss, Inc.)

Aims: To assess the type of problems encountered in diagnosing melanocytic lesions and to evaluate the contribution of expert review., Methods and Results: Data from 1887 lesions submitted for consultation to one of the expert pathologists of the Dutch Melanoma Working Group Pathology Panel between 1991 and 2004 were analysed. Referring pathologists can voluntarily submit lesions which are difficult to classify to the panel. Most cutaneous melanocytic lesions (n = 1217) were submitted with a presumed diagnosis by the referring pathologists. Relevant underdiagnoses of melanoma (in situ) and overdiagnoses of naevi were prevented in 12% (144/1217) and 15% (178/1217) of cases, respectively. Problematic melanocytic lesions were (i) spitzoid and dysplastic lesions, (ii) lesions with histological features that hampered the diagnosis such as regression, lymphocytic infiltrate, or a combination with other melanocytic lesions, and (iii) lesions with unusual clinical features, e.g. childhood melanoma. Remarkably, the features of the lesions that were submitted and the types of over- and under-diagnosis remained consistent from 1991 to 2004., Conclusions: A second opinion from an expert pathologist on problem-prone melanocytic lesions improves patient care, in our series in 27% of cases.

Object: Routine microbiological and chemical analysis of cerebrospinal fluid (CSF) is often performed to diagnose external drainage-related bacterial meningitis (ED-BM) at an early stage. A cohort study was performed to investigate the value of several commonly used CSF parameters for the prediction and diagnosis of ED-BM., Methods: In a cohort of 230 consecutive patients in whom external drains had been placed, CSF samples were collected daily, prospectively evaluated for the presence of bacteria using Gram stain and microbiological culture, and analyzed for leukocyte count, protein concentration, glucose concentration, and ratio of CSF glucose to blood glucose. In addition, the CSF concentration of interleukin-6 (IL-6) was determined. The definition of ED-BM was based on positive culture results in combination with clinical symptoms. A matched case-control study was performed to evaluate the cohort longitudinally and to control for biasing factors such as duration of external drainage. External drainage-related bacterial meningitis developed in 22 patients (9.6%). Results from analyses of 1516 CSF samples showed no significant differences between the patients in whom ED-BM developed and a control group without ED-BM during the first 3 days of infection or during the 3 days preceding the infection with regard to leukocyte count, protein concentration, glucose concentration, and CSF/blood glucose ratio. No significant difference between groups was found for the CSF IL-6 concentration during the 3 days preceding the infection. In the matched case-control study, none of the parameters had significant predictive or diagnostic value for ED-BM in analyses using absolute values, ratios, and differences between the current and previous day's values. A comparison of the results from Gram stains and CSF cultures showed that the Gram staining had a very high specificity (99.9%) but a low sensitivity (18% [four of 22 patients] on the 1st day of infection and 60% [nine of 15 patients] on the 2nd day)., Conclusions: Severe disturbances in the CSF of patients with external drains limit the value of routine CSF analysis for prediction or diagnosis of ED-BM. Routine Gram stain of CSF has also limited predictive or diagnostic value due to its low sensitivity in screening for ED-BM.

A definite diagnosis cannot be established based on histologic features alone in a large number of Spitz nevi and spitzoid melanomas. In a vast majority of common benign and malignant melanocytic lesions, B-RAF and N-RAS mutations were described, but these were not detected in Spitz nevi. In contrast, H-RAS mutations were frequently encountered in Spitz nevi, but only rarely in melanomas. To date, B-RAF mutation analysis has not been reported in atypical Spitz nevi, and there are only a few reports of it in spitzoid melanomas. We analyzed 96 formalin-fixed, paraffin-embedded spitzoid melanocytic lesions for hotspot mutations in B-RAF, N-RAS, and H-RAS genes to test the assumption whether mutation analysis would assist a more accurate diagnosis of spitzoid melanocytic lesions, which are notoriously difficult to classify. B-RAF or N-RAS mutations were observed in 31 of 36 (86%) spitzoid melanomas, and in 6 of 7 (86%) spitzoid melanoma metastases. In contrast, none of the 14 Spitz nevi and none of the 16 atypical Spitz nevi had mutations in any of the three genes. A B-RAF or N-RAS mutation was found in 8 of 23 (35%) spitzoid lesions suspected for melanoma. H-RAS mutations were detected in 4 of 14 (29%) Spitz nevi, in 3 of 22 (14%) atypical Spitz nevi, in 1 of 15 (7%) spitzoid tumors suspected for melanoma, but in none of the spitzoid melanomas. These results strongly indicate that Spitz nevi and spitzoid melanomas are genetically unrelated entities. Furthermore, we can conclude that mutation analysis may be useful as an additional diagnostic tool to distinguish between benign and malignant spitzoid lesions.

Molecular analysis on formalin-fixed paraffin-embedded tissue is of increasing importance in diagnostic histopathology and tumor research. Multiplex ligation-dependent probe amplification (MLPA) is a technique that can be used for detection of copy number alterations of up to 45 different DNA sequences in one experiment. It can be performed on partially degraded DNA, which makes this technique very suitable for analysis of formalin-fixed lesions. We tested the reliability of MLPA by analyzing DNA isolated from formalin-fixed melanomas that were previously characterized by comparative genomic hybridization (CGH), and additionally the applicability of MLPA was tested by analyzing 29 routinely processed melanocytic lesions. MLPA appears to be a reliable and efficient method to evaluate DNA copy number changes as 86% of the loci tested revealed concordant CGH results. Discordance mainly involved alterations that were detected by MLPA and not by CGH probably due to a combination of lower resolution of CGH and occasionally false positive MLPA results. For application of MLPA in a diagnostic setting, different probes on a specific region of interest should be used to prevent false positive MLPA results. In a research setting as well as in a diagnostic setting, MLPA is a fast technique to screen large numbers of formalin-fixed lesions for DNA gains and losses.

Background: Laser microbeam microdissection has greatly facilitated the procurement of specific cell populations from tissue sections. However, the fact that a coverslip is not used means that the morphology of the tissue sections is often poor., Aims: To develop a mounting method that greatly improves the morphological quality of tissue sections for laser microbeam microdissection purposes so that the identification of target cells can be facilitated., Methods: Fresh frozen tissue and formalin fixed, paraffin wax embedded tissue specimens were used to test the morphological quality of mounted and unmounted tissue. The mounting solution consisted of an adhesive gum and blue ink diluted in water. Interference of the mounting solution with DNA quality was analysed by the polymerase chain reaction using 10-2000 cells isolated by microdissection from mounted and unmounted tissue., Results: The mounting solution greatly improved the morphology of tissue sections for laser microdissection purposes and had no detrimental effects on the isolation and efficiency of amplification of DNA. One disadvantage was that the mounting solution reduced the cutting efficiency of the ultraviolet laser. To minimise this effect, the mounting solution should be diluted as much as possible. Furthermore, the addition of blue ink to the mounting medium restores the cutting efficiency of the laser., Conclusions: The mounting solution is easy to prepare and apply and can be combined with various staining methods without compromising the quality of the DNA extracted.

Aim: To report on eight patients with severe idiopathic intermediate uveitis (IU) and granuloma annulare (GA), a self limiting cutaneous condition of unknown aetiology., Methods: Retrospective case series. Clinical ophthalmic and dermatological data were studied and fluorescein angiography and skin biopsies were reviewed., Results: All patients with idiopathic IU had similar ocular features (eight with vitritis, seven with retinal vasculitis) and developed complications such as cystoid macular oedema (n=5), cataract (n=4), and glaucoma (n=3). Systemic diseases were not found, but a localised type of GA was observed in all., Conclusion: Seven out of eight patients with IU and GA developed severe retinal vasculitis. Further studies are needed for a better understanding of this association, a common pathogenesis, and its eventual clinical consequences.

The histopathological diagnosis of cutaneous melanocytic lesions may be difficult to assess. Frequently encountered diagnostic problems include: 1) Dysplastic nevus or melanoma in situ?; 2) Melanoma in situ or superficial spreading melanoma?; 3) Lentigo maligna or lentigo maligna melanoma?; 4) Compound nevocellular nevus or nevoid melanoma?; and 5) Spitz nevus or Spitzoid melanoma? Moreover, less frequently encountered diagnostic challenges are discussed: 1) Deep penetrating nevus or nodular melanoma?; and 2) Cellular blue nevus or melanoma metastasis? In this contribution, these problems are discussed after a systematic approach involving a concise histopathological description of the classic lesions considered in the differential diagnoses, a presentation of the deviating histopathological features that give rise to the diagnostic problems, and finally diagnostic recommendations on the classification of the problematic lesions. We also briefly discuss the contribution of additional immunohistochemistry and molecular pathology in aiding to establish a correct diagnosis.

To test the diagnostic usefulness of allelic imbalance (AI) analysis based on routinely paraffin-embedded tissue, a series of 55 benign Spitz naevi, Spitz tumours with uncertain malignant potential, and malignant Spitzoid melanomas was investigated. Laser microdissection was used to ensure representative sampling of lesional cells and to investigate AI in separate tumour areas of four melanomas. AI was found in 2/12 (17%) typical Spitz naevi, 3/9 (33%) atypical Spitz tumours, 12/17 (65%) atypical Spitz tumours suspicious for melanoma and 15/17 (88%) Spitzoid melanomas. Additional immunohistochemical staining for Ki-67 using the MIB-1 antibody revealed positive deeply situated lesional cells in 0/6 (0%) Spitz naevi, 1/8 (13%) atypical Spitz tumours, 5/14 (35%) atypical Spitz tumours suspicious for melanoma, and 7/14 (50%) Spitzoid melanomas, respectively. Two of the melanomas examined for AI in separate tumour areas showed intratumoural genetic heterogeneity. In view of the finding of AI and deeply situated Ki-67 positive cells not only in melanomas but also in Spitz tumours with uncertain malignant potential, these approaches appear to have no direct diagnostic applicability for the distinction between benign and malignant Spitz tumours. Further molecular studies will be required to determine whether Spitz tumours and Spitzoid melanomas are unrelated entities, or whether there is a true spectrum of tumour progression., (Copyright 2002 John Wiley & Sons, Ltd.)

We have recently described a new member of the PMP22/gas3 family of plasma membrane proteins referred to as THW. This gene is located on chromosome 6q and preliminary data have indicated a possible tumor suppressor gene function. We have therefore investigated LOH for gene THW in a panel of cancer cell lines and in a series of primary human melanomas as well as in melanoma metastases. We have detected LOH for gene THW in cell lines derived from melanoma, breast, pancreas, cervical, prostate and colon carcinoma with different prevalence, whereas the ovary carcinoma cell lines (n = 3) were negative. For melanomas we found a prevalence of LOH for gene THW of 10-20% in primary tumors, whereas in melanoma metastases we found a score of 50%. These data and the fact that the recently identified murine homologue PERP of gene THW mediates cell death in murine fibroblasts support the possible tumor suppressor function of gene THW.

14-3-3 Proteins may function as adapters or scaffold in signal-transduction pathways. We found previously that protein kinase C-zeta (PKC-zeta) can phosphorylate and activate Raf-1 in a signalling complex [van Dijk, Hilkmann and van Blitterswijk (1997) Biochem. J. 325, 303-307]. We report now that PKC-zeta-Raf-1 interaction is mediated by 14-3-3 proteins in vitro and in vivo. Co-immunoprecipitation experiments in COS cells revealed that complex formation between PKC-zeta and Raf-1 is mediated strongly by the 14-3-3beta and -theta; isotypes, but not by 14-3-3zeta. Far-Western blotting revealed that 14-3-3 binds PKC-zeta directly at its regulatory domain, where a S186A mutation in a putative 14-3-3-binding domain strongly reduced the binding and the complex formation with 14-3-3beta and Raf-1. Treatment of PKC-zeta with lambda protein phosphatase also reduced its binding to 14-3-3beta in vitro. Preincubation of an immobilized Raf-1 construct with 14-3-3beta facilitated PKC-zeta binding. Together, the results suggest that 14-3-3 binds both PKC-zeta (at phospho-Ser-186) and Raf-1 in a ternary complex. Complex formation was much stronger with a kinase-inactive PKC-zeta mutant than with wild-type PKC-zeta, supporting the idea that kinase activity leads to complex dissociation. 14-3-3beta and -&theta; were substrates for PKC-zeta, whereas 14-3-3zeta was not. Phosphorylation of 14-3-3beta by PKC-zeta negatively regulated their physical association. 14-3-3beta with its putative PKC-zeta phosphorylation sites mutated enhanced co-precipitation between PKC-zeta and Raf-1, suggesting that phosphorylation of 14-3-3 by PKC-zeta weakens the complex in vivo. We conclude that 14-3-3 facilitates coupling of PKC-zeta to Raf-1 in an isotype-specific and phosphorylation-dependent manner. We suggest that 14-3-3 is a transient mediator of Raf-1 phosphorylation and activation by PKC-zeta.

Phosphatidylcholine (PC) hydrolysis induced by basic fibroblast growth factor (bFGF) was studied in rat L6 myoblasts expressing the wild-type FGF receptor-1 (FGFR-1) or a mutant (Y766F) that is incapable of activating phospholipase C-gamma (PLCgamma). Stimulation of FGFR-1 activated phospholipase D (PLD) rapidly and transiently, but did not induce PC-specific PLC activity. Downregulation of protein kinase C blocked bFGF-induced PLD activation but not phosphatidic acid formation by diacylglycerol (DG) kinase. Only phosphoinositide (PI)-derived DG, not PC-derived DG, appeared to be a substrate for DG kinase. Stimulation of FGFR-1(Y766F) did not activate PLD or DG kinase, both of which apparently require initial PLCgamma activation. The Y766F mutation reduced mitogen-activated protein kinase activation but not cell proliferation. We conclude that both PI turnover and PC hydrolysis are dispensable for bFGF-induced mitogenesis., (Copyright 1998 Elsevier Science B.V.)

Background: Phospholipase D (PLD) hydrolyzes phospholipids to generate phosphatidic acid (PA) and a free headgroup. PLDs occur as both intracellular and secreted forms; the latter can act as potent virulence factors. Exogenous PLD has growth-factor-like properties, in that it induces proto-oncogene transcription, mitogenesis and cytoskeletal changes in target cells. The underlying mechanism is unknown, although it is generally assumed that PLD action is mediated by PA serving as a putative second messenger., Results: In quiescent fibroblasts, exogenous PLD (from Streptomyces chromofuscus) stimulated accumulation of the GTP-bound form of Ras, activation of mitogen-activated protein (MAP) kinase and DNA synthesis, through the pertussis-toxin-sensitive inhibitory G protein Gi. Furthermore, PLD mimicked bioactive lysophospholipids (but not PA) in inducing Ca2+ mobilization, membrane depolarization and Rho-mediated neurite retraction. PLD action was mediated by Iysophosphatidic acid (LPA) derived from Iysophosphatidylcholine acting on cognate G-protein-coupled LPA receptor(s). There was no evidence for the involvement of PA in mediating the effects of exogenous PLD., Conclusions: Our results provide a molecular explanation for the multiple cellular responses to exogenous PLDs. These PLDs generate bioactive LPA from pre-existing Iysophosphatidylcholine in the outer membrane leaflet, resulting in activation of G-protein-coupled LPA receptors and consequent activation of Ras, Rho and Ca2+ signaling pathways. Unscheduled activation of LPA receptors may underlie, at least in part, the known pathogenic effects of exogenous PLDs.

The mechanism of Raf-1 activation by platelet-derived growth factor (PDGF) is poorly defined. We previously reported that, in Rat-1 fibroblasts, PDGF activates a phosphatidylcholine-specific phospholipase C (PC-PLC) and that the product, diacylglycerol, somehow activates protein kinase C-zeta (PKC-zeta). Both PC-PLC and PKC-zeta activities were required for PDGF activation of mitogen-activated protein kinase (MAPK). Now we report that MAPK activation by exogenous PC-PLC depends on Raf-1 activation. PKC-zeta co-immunoprecipitates with, phoshorylates and activates Raf-1, suggesting that in the PDGF- and PC-PLC-activated MAPK pathway, PKC-zeta operates in a signalling complex as a direct activator of Raf-1.

The role of phosphatidylcholine (PC) hydrolysis in activation of the mitogen-activated protein kinase (MAPK) pathway by platelet-derived growth factor (PDGF) was studied in Rat-1 fibroblasts. PDGF induced the transient formation of phosphatidic acid, choline, diacylglycerol (DG), and phosphocholine, the respective products of phospholipase D (PLD) and phospholipase C (PC-PLC) activity, with peak levels at 5-10 min. PLD-catalyzed transphosphatidylation (with n-butyl alcohol) diminished DG formation at 5 min but not at later stages of PDGF stimulation. Phorbol ester-induced down-regulation of protein kinase C (PKC) completely blocked PLD activation but not the formation of DG and phosphocholine at 10 min of PDGF stimulation. Collectively, these data indicate that PDGF activates both PLD and PC-PLC. In contrast, epidermal growth factor did not activate PC-PLC in these cells, and it activated PLD only weakly. DG formation by itself, through Bacillus cereus PC-PLC treatment of cells, was sufficient to mimic PDGF in activation of MAPK independent of phorbol ester-sensitive PKC. Since PKC down-regulation blocked PDGF-induced PLD but not MAPK activation, we conclude that PLD is not involved in MAPK signaling. In contrast, MAPK activation by exogenous (bacterial) PLD was not affected by PKC down-regulation, indicating that signals evoked by exogenous PLD differ from endogenous PLD. D609 (2-10 microg/ml), an inhibitor of PC-PLC, blocked PDGF- but not epidermal growth factor-induced MAPK activation. However, D609 should be used with caution since it also affects PLD activity. The results suggest that PC-PLC rather than PLD plays a critical role in the PDGF-activated MAPK pathway.