The cytoskeletal protein LASP-1 differentially regulates migratory activities of choriocarcinoma cells.

PURPOSE: During healthy pregnancy, a distinct but limited invasion of trophoblast cells into the uterus occurs. In contrast, excessive trophoblast invasion is associated with placental choriocarcinoma (CC). Overexpression of the cytoskeletal protein LASP-1 was shown to contribute to cancer aggressiveness. Here, the yet unknown role of LASP-1 in CC cells is analysed. METHODS: Expression of LASP-1 in human primary carcinoma was assessed by immunohistochemistry and confirmed in CC-derived cell lines by immunocytochemistry, RT-PCR and Western blot. After down-regulation of LASP-1 expression with specific si-RNA in CC-derived cell lines, migratory and proliferative activities were analysed by matrigel migration assay and WST-8 test. RESULTS: LASP-1 expression was detected in human primary choriocarcinoma and in JEG-3, JAR and BeWo cells. Knock down of LASP-1 resulted in a decreased expression of LASP-1 protein in JEG-3 and JAR cells accompanied by a diminished migration and a decreased proliferative activity of these two cell lines. Knockdown of LASP-1 in BeWo cells failed. In consequence, migratory function and proliferation was unaffected. CONCLUSION: This is the first study describing LASP-1 expression in CC cells. Detecting an affection of migratory processes after LASP-1 silencing, we propose that LASP-1 could impact on metastasis of CC cells.

High Estradiol Levels During Postmenopause - Pitfalls in Laboratory Analysis.

A 54-year-old woman was admitted with a result of high serum estradiol levels (> 4300 pg/ml) and typical postmenopausal symptoms. She had a history of an adnexectomy (normal histopathology) due to the elevated estradiol levels. After surgery, estradiol levels were as high as before. Analyzing the anti-mullerian hormone (AMH), inhibin B, DHEA-S and estrone, typical postmenopausal levels were found. Serum estradiol levels were controlled several times with rabbit-derived polyclonal as well as monoclonal antibodies to optimize the selectivity of the test system. Secondary, a radioimmunoassay was performed to exclude interferences of the detection system where lower, but still elevated estradiol levels (186 pg/ml) were found. Hypothesizing that our patient underwent a cross reaction with irregular antibodies, a control was done using sheep-derived antibodies, which proved a postmenopausal hormone level (estradiol level < 5 pg/ml). This result was confirmed using a fluorescence enzyme immunoassay (FEIA) revealing high levels of irregular antibodies (> 200 mg/l; reference < 30 mg/l). This case depicts the pitfalls of estradiol measurement detecting false elevated estradiol levels in a postmenopausal woman.

Thrombopoietin modulates the proliferation, migration and cytokine profile of decidual cell subsets during early gestation.

During early gestation, a considerable increase in different leukocyte subsets can be observed in the decidualized endometrium concomitantly to the invasion of cytotrophoblast cells (CTB). To date, it is still in question which factors induce this accumulation of immune cells and whether it is evoked by an in situ proliferation or by a migratory process. Studies on hepatoblastoma cells identified thrombopoietin (TPO) as a novel factor, which elicits dose-dependent chemotactic and chemokinetic effects. However, the impact and function of TPO on decidual cells has not been clarified yet. This study analyses the expression and function of TPO and its receptor c-Mpl in decidua during early gestation. Applying western blot analysis, we detected that TPO is expressed by decidual immune cells (uNK cells and CD14+ monocytes) as well as CTB and decidual stromal cells (DSCs). Expression of the different isoforms of c-Mpl was found in uNK cells, CD14+ monocytes and DSC. Studying the signalling pathway proteins in the uNK cells, an activation of STAT3/Tyr by TPO, was detected. The investigation of the proliferative effects of TPO on the decidual cell subsets revealed that TPO enhances the proliferation of uNK cells and CTB. No change of the proliferative activity after TPO incubation was found in DSC and even a decrease in CD14+ monocytes. In addition, TPO was observed to induce significantly the migratory activity of uNK cells, CD14+ monocytes and CTB. Investigating the effects of TPO on the cytokine profile of the isolated decidual cells, we observed a decrease in the secretion of IL-8, IL-10 and IL-1ß of isolated uNK cells, CD14+ monocytes and CTB, although these changes did not reach statistical significance. Thus, we here identified TPO as a novel factor modulating the proliferation, migration and possibly cytokine secretion of decidual cell subsets.

Downregulation of AKT reverses platinum resistance of human ovarian cancers in vitro.

Platinum resistance is the most crucial problem for treatment of ovarian cancer. Increasing evidence points towards AKT overexpression as a mechanistic reason for this clinical condition. The present study evaluates the effect of overexpression and downregulation of AKT on the sensitivity to cisplatin in a platinum-resistant human ovarian cancer cell line and the corresponding platinum-sensitive parental cell line. A2780 and A2780cis ovarian cancer cell lines were stably transfected with an AKT-sense and AKT-antisense plasmid. Successful transfection was evaluated by western blot analysis. Cytotoxic effects of cisplatin were evaluated by metabolic (MTT) and clonogenicity assays as well as by FACS analysis. AKT overexpression (confirmed by western blotting) converted platinum-sensitive A2780 into platinum-resistant cells as shown by MTT assay. Importantly, platinum resistance of A2780cis cells could be reversed by downregulation of AKT, as demonstrated by MTT and clonogenicity assays and FACS analysis. Our data provide strong evidence that cisplatin resistance in ovarian cancer is mediated by AKT overexpression and can be overcome by AKT downregulation, thus, providing a rationale for clinical phase II/III studies combining AKT inhibitors with cisplatin.

Peptidomimetic GnRH antagonist AEZS-115 inhibits the growth of ovarian and endometrial cancer cells.

BACKGROUND: AEZS-115 (Aeterna Zentaris GmbH, Frankfurt/M, Germany) is an orally active peptidomimetic antagonist of gonadotropin-releasing hormone (GnRH). In various tumors, an autocrine growth-promoting loop has been described for GnRH. The current study evaluates the antitumor activity and mechanism of action of AEZS-115 in models of ovarian and endometrial cancer. MATERIALS AND METHODS: Human A2780, Acis2780, OAW-42, Ovcar-3, SKOV-3, Hec1A and Ishikawa cells were analyzed for GnRH receptor expression by reverse transcription polymerase chain reaction (RT-PCR). These cell lines were incubated with AEZS-115 at 1, 10 and 100 µM for 24 h, 48 h, and 72 h and the number of viable cells was determined. Fluorescence activated cell sorting (FACS) cell cycle analyses were performed with increasing concentrations of AEZS-115. Co-treatment experiments of cancer cells with GnRH antagonist cetrorelix and peptidomimetic GnRH antagonist AESZ-115 were carried out. RESULTS: A2780, Acis2780, OAW-42, Ovcar-3, SKOV-3, Hec1A and Ishikawa cells expressed GnRH receptors as demonstrated by RT-PCR. GnRH antagonist AEZS-115 inhibited growth of all cell lines in a dose- and time-dependent manner. Half maximal inhibitory concentration (IC(50)) values at 48 h of incubation were between 7 and 17.5 µM and for 72 h between 4.5 and 12.5 µM. IC(50) values for ovarian and endometrial cancer cells were rather similar. These results were obtained by tetrazolium salt [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTT] assay and confirmed by additional crystal violet staining. Cell cycle FACS analysis revealed that AEZS-115 dose-dependently increased the fraction of apoptotic cells. Co-treatment experiments carried out with AEZS-115 and peptidic GnRH-antagonist cetrorelix suggest that the antitumor effect of AEZS-115 is not mediated by blockade of the GnRH receptor. CONCLUSION: GnRH antagonist AEZS-115 exhibited substantial antitumor activity in ovarian as well as endometrial cancer cell lines. However, this antitumor effect was not mediated by the tumoral GnRH receptors. To identify the mechanism of action of this compound, further research is warranted. Its in vitro antitumor activity makes AEZS-115 a promising candidate for in vivo studies of ovarian and endometrial cancer.

Overexpression of polycomb protein BMI-1 in human specimens of breast, ovarian, endometrial and cervical cancer.

INTRODUCTION: The polycomb group (PcG) proteins form chromatin-modifying complexes that are commonly deregulated in cancer. The PcG protein BMI-I is overexpressed by various tumours and thus may contribute to malignant transformation. The current study investigated the expression of BMI-I in human specimens of breast, ovarian, endometrial and cervical cancer. MATERIALS AND METHODS: Expression of BMI-I was evaluated in human ovarian cancer samples by Western blot analysis and immunohistochemistry (IHC) and compared to healthy ovarian tissue. BMI-I expression in human specimens of breast, endometrial and cervical cancer was evaluated by IHC and then compared with the respective benign tissues. RESULTS: BMI-I was significantly (p<0.05) overexpressed in human breast, ovarian, endometrial and cervical cancer specimens as compared to benign controls. BMI-I expression was also more pronounced in the ovarian cancer samples as demonstrated by Western blot analysis. In human breast cancer samples, BMI-I expression was most pronounced in the invasion front of the tumour. CONCLUSION: The current study showed for the first time that the BMI-I protein is significantly overexpressed in ovarian, endometrial and cervical cancer and may thus be a potential target for novel antitumor therapies.

Cerebral angiitis in four patients with chronic GVHD.

There is growing evidence that GVHD affects the central nervous system (CNS). In this study, we describe the long-term follow-up of four allogeneic BM recipients who developed cerebral angiitis-like disease probably due to GVHD. The patients developed focal neurological signs, cognitive deficits and/or coma in association with GVHD, 2-18 years after transplantation, following reduction of immunosuppressive therapy. Magnetic resonance imaging was variable, showing generalized brain atrophy, ischemic lesions or leukoencephalopathy. Diagnosis of cerebral angiitis was confirmed by histopathological analysis of bioptic brain tissue and response to immunosuppressive therapy. By means of immunohistochemistry and immunofluorescence, perivascular lymphomononuclear cerebral infiltrates were shown to express the adhesion receptor, CD11a, and the chemokine receptor, CCR5. Our findings imply that GVHD should be considered in the differential diagnosis of noninfectious angiitis-like disease of the CNS in long-term survivors after allogeneic BMT. Infiltrating cells, in analogy to typical target organs of GVHD such as skin or liver, expressed CD11a and CCR5. These findings could be of etiopathological, diagnostic and therapeutic relevance.

Podocytes produce homeostatic chemokine stromal cell-derived factor-1/CXCL12, which contributes to glomerulosclerosis, podocyte loss and albuminuria in a mouse model of type 2 diabetes.

AIMS/HYPOTHESIS: Chemokine (C-X-C motif) ligand 12 (CXCL12) (also known as stromal cell-derived factor-1 [SDF-1]-alpha) is a homeostatic chemokine with multiple roles in cell homing, tumour metastasis, angiogenesis and tissue regeneration after acute injuries. However, its role in chronic diseases remains poorly defined, e.g. in chronic glomerular diseases like diabetic glomerulosclerosis. We hypothesised that CXCL12 may have a functional role during the evolution of diabetic glomerulosclerosis, either by assisting glomerular repair or by supporting the maladaptive tissue remodelling in response to hyperglycaemia and glomerular hyperfiltration. METHODS: To define the functional role of CXCL12 in the progression of glomerular disease, we used the CXCL12-specific inhibitor NOX-A12, an L: -enantiomeric RNA oligonucleotide (Spiegelmer). A mouse model of type 2 diabetes (db/db mice) was used. Male db/db mice, uni-nephrectomised at 6 weeks of age, received subcutaneous injections with a PEGylated form of NOX-A12, non-functional control Spiegelmer or vehicle on alternate days from 4 to 6 months of age. RESULTS: Immunostaining localised renal CXCL12 production to glomerular podocytes in db/db mice with early or advanced diabetic nephropathy. CXCL12 inhibition significantly reduced the degree of glomerulosclerosis, increased the number of podocytes, prevented the onset of albuminuria and maintained the peritubular vasculature without affecting blood glucose levels, body weight or glomerular macrophage infiltration. CONCLUSIONS/INTERPRETATION: We conclude that podocytes produce CXCL12, which contributes to proteinuria and glomerulosclerosis in our mouse model of type 2 diabetes. This novel pathomechanism provides the first evidence that CXCL12 could be a therapeutic target in (diabetic) glomerulosclerosis.

The source matters: no impact of the CCL2/MCP-1-1-2518G polymorphism of the donor on renal allograft outcome during the first year after transplantation.

Chemokines are involved in the recruitment of inflammatory cells to vascularized allografts. The chemokine CCL2/MCP-1 is expressed during allograft dysfunction, which is associated with the recruitment of inflammatory cells. Both intrinsic renal cells (donor origin) as well as infiltrating inflammatory cells (recipient origin) can be a source of CCL2/MCP-1. We previously demonstrated that the recipient MCP-1-2518G polymorphism is associated with increased CCL2/MCP-1 production by inflammatory cells and decreased renal allograft survival. We evaluated the impact of the MCP-1-2518G polymorphism in donor cells on renal allograft outcomes. We enrolled 252 recipients of kidney allografts in this retrospective study who had received grafts from 152 cadaveric donors. The CCL2/MCP-1 genotype was assessed using genomic DNA isolated from cryopreserved donor splenocytes. Outcome parameters studied were acute biopsy proven rejection (Banff criteria), serum creatinine, and glomerular filtration rate (GFR) at 1 year after transplantation, allograft loss, and death. MCP-1-2518 genotypes were in HW equilibrium. A/A was present in 125 (49.6%), A/G in 107 (42.5%), and G/G in 20 (7.9%) donor kidneys. There were no significant differences in the number of rejection episodes, the number of allograft losses, serum creatinine, GFR, or overall survival 1 year after transplantation. In contrast with the detrimental effect of the CCL2/MCP-1 polymorphism of the recipient, the CCL2/MCP-1 polymorphism of the donor has no impact on the allograft outcome during the first year after transplantation. The impact on the long-term outcomes needs further evaluation.

SDF-1 expression is elevated in chronic human renal allograft rejection.

The exact mechanism of acute and chronic allograft rejection still remains unclear. The chemokine SDF-1 as mediator of allograft rejection has been under intensive investigation in liver, cardiac and bone marrow transplantation, whereas in renal transplantation, there are no reports about SDF-1 to date. This study was performed to evaluate if SDF-1 might also play an important role in human renal graft biopsies. One hundred and ninety formalin-fixed, paraffin-embedded renal allograft biopsies were included in the analysis from patients with normal renal graft morphology (according to Banff 97 classification grade 1, n = 84), with acute interstitial rejection (Banff grade 4 type I, n = 10), with acute vascular rejection (Banff grade 4 type II, n = 21), with chronic allograft nephropathy (CAN, Banff grade 5, n = 23), and without rejection but with various other lesions (Banff grade 6, n = 42). SDF-1 was localized by immunohistochemistry. In biopsies with CAN, SDF-1 expression was significantly elevated in interstitial infiltrates and infiltrating neointimal cells of arteries compared with biopsies with normal renal graft morphology. This is the first study describing a role of SDF-1 in human renal allograft rejection. We were able to demonstrate in a large number of biopsies an upregulation of SDF-1 in patients with CAN. Whether SDF-1 has pro-inflammatory or protective properties in this setting has to be evaluated in further trials.

Multipotent mesenchymal stem cells reduce interstitial fibrosis but do not delay progression of chronic kidney disease in collagen4A3-deficient mice.

Multipotent mesenchymal stem or stromal cells (MSC) have shown to improve outcome of acute renal injury models, but whether MSC can delay renal failure in chronic kidney disease is not known. We injected primary MSC or saline into mice that lack the alpha3-chain of type IV collagen (COL4A3), a model of chronic kidney disease with close similarities to human Alport disease. Weekly injections of MSC from week 6 to 10 of life prevented the loss of peritubular capillaries and reduced markers of renal fibrosis, that is, interstitial volume, numbers of smooth muscle actin-positive interstitial cells, and interstitial collagen deposits as compared to saline-injected COL4A3-deficient mice. However, renal function, that is, blood urea nitrogen, creatinine levels, proteinuria as well as survival of COL4A3-deficient mice were not affected by MSC injections. Although MSC were found to localize to kidneys of COL4A3-deficient mice after injection, differentiation into renal cells was not detected. However, MSC expressed growth factors, that is, vascular endothelial growth factor (VEGF) and bone morphogenetic protein-7 under basal culture conditions. In fact, VEGF mRNA levels were increased in kidneys of MSC-injected COL4A3-deficient mice and MSC supernatants enhance endothelial cell proliferation in vitro. Thus, weekly injections with MSC prevent loss of peritubular capillaries possibly owing to local production of growth factors rather than by differentiation into renal cells. The maintenance of interstitial vasculature is associated with less interstitial fibrosis but, is insufficient to delay renal failure and survival of COL4A3-deficient mice.

Expression of the chemokine receptor CXCR1 in human glomerular diseases.

Leukocyte infiltration, a hallmark of renal diseases, is orchestrated in part by the actions of chemokines. The chemokine CXCL8/interleukin (IL)-8 is expressed during renal diseases and allograft rejection, whereas the corresponding receptor CXCR1 has not been described previously. Expression of CXCR1 was characterized in peripheral blood using multicolor fluorescence-activated cell sorter analysis (FACS). CXCR1 was localized in 81 formalin-fixed, paraffin-embedded renal specimens by immunohistochemistry using a monoclonal antibody against human CXCR1. Included were biopsies with crescentic glomerulonephritis (CGN, n = 22), immunoglobulin (Ig) A nephropathy (n = 15), membranoproliferative glomerulonephritis (MPGN, n = 17), lupus nephritis (n = 12), membranous nephropathy (n = 11), and non-involved parts of tumor nephrectomies (n = 4). Consecutive tissue sections of human tonsils, allograft explants, and renal biopsies were stained for CD15- and CD68-positive cells. Expression of CXCR1 and CXCL8/IL-8 mRNA was quantified by real-time reverse transcriptase-polymerse chain reaction of microdissected renal biopsies (n = 35) of the same disease entities. By FACS CXCR1 expression was found on polymorphonuclear CXCR1 expression by polymorphonuclear leukocytes (PMNs), natural killer cells, and a subpopulation of monocytes. By immunohistochemistry, CXCR1 expression was found on infiltrating inflammatory cells (predominantly PMNs), as well as on intrinsic renal cells (arterial smooth muscle cells, endothelial cells of peritubular capillaries). The distribution pattern of CXCR1 differed between disease entities. The highest numbers of glomerular CXCR1-positive cells were present in biopsies with MPGN, followed by lupus nephritis, and CGN. CXCR1 might be involved in the recruitment of PMNs to the glomerular tuft, which could be targeted by CXCR1-blocking agents.

Expression of DARC, CXCR3 and CCR5 in giant cell arteritis.

OBJECTIVES: Leucocyte infiltration is the hallmark of vasculitis, chemokines being mainly responsible for leucocyte migration into inflamed tissues. The objective was to evaluate the local expression of chemokines and chemokine receptors in biopsies of patients with giant cell arteritis (GCA) compared with arteries from patients with polymyalgia rheumatica (PMR). We studied the expression of CCR5, CXCR3 and that of the Duffy antigen/receptor of chemokine (DARC), a chemokine internalizing receptor (interceptor), in parallel to the expression of the CCR5 ligand RANTES/CCL5. METHODS: Paraffin-embedded tissue sections from six patients with GCA and five patients with PMR were available for immunohistological analysis of chemokine receptor expression. RANTES/CCL5 mRNA was detected in tissue sections by in situ hybridization. RESULTS: In patients with biopsy-proven giant cell arteritis, CCR5 and CXCR3 were highly expressed by infiltrating leucocytes in involved tissue sections. Predominant clustering of CCR5+ and CXCR3+ leucocytes was found in the adventitia and was co-localized with the expression of CCL5/RANTES mRNA. Interestingly, we found marked expression of DARC on adventitial high endothelial venules in vasculitis lesions of patients with GCA, while in arteries from patients with PMR DARC was only expressed on a low number of vessels with flat lining endothelium. CONCLUSIONS: The co-localization of infiltrating CCR5+ and CXCR3+ leucocytes together with CCL5/RANTES and DARC in vasculitis lesions suggests a role for these chemokine receptors in leucocyte infiltration, possibly supported by DARC-mediated vascular presentation of chemokines.

Osteopontin expression in human cyclosporine toxicity.

BACKGROUND: Osteopontin is a secreted phosphoprotein that has a number of diverse biological functions, including cell signaling, mediation of cell adhesion, migration, and chemoattraction of monocytes/macrophages. Up-regulation of osteopontin expression by proximal tubular epithelium has been demonstrated in both human and rodent models of renal injury in association with macrophage influx. METHODS: We studied the expression of osteopontin protein and mRNA in renal donor biopsies (N = 7) and renal transplant biopsies with cyclosporine A toxicity (N = 23) by immunohistochemistry and in situ hybridization. Serial tissue sections were immunostained with a monocyte/macrophage marker, CD68, to demonstrate the pattern of macrophage infiltration. RESULTS: Strong osteopontin expression was observed in the majority of pretransplant donor biopsies in the absence of any macrophage infiltration. In the biopsies with cyclosporine toxicity, osteopontin expression was widespread and demonstrated moderate immunohistochemical signal intensity that did not correlate with the number of interstitial macrophages present. CONCLUSIONS: Strong osteopontin protein and mRNA expression by tubular epithelium was observed in pretransplant donor biopsies and in biopsies with cyclosporine toxicity without an inflammatory cell infiltration. Therefore, osteopontin expression alone is insufficient to serve as the principal mediator of intrarenal monocyte/macrophage influx in the transplant setting.

Expression of chemokines and chemokine receptors during human renal transplant rejection.

Infiltration of renal allografts by leukocytes is a hallmark of acute transplant rejection. Chemokines attract leukocytes bearing specific chemokine receptors, and the specific leukocyte chemokine receptor phenotype is associated with types of immune responses, ie, T helper subtype 1 (Th1; CXC chemokine receptor 3 [CXCR3], CC chemokine receptor 5 [CCR5]) versus Th2 (CCR3, CCR4, CCR8). We studied the expression of the chemokine monocyte chemoattractant protein-1 and the chemokine receptors CCR2B and CXCR4 messenger RNA (mRNA) by in situ hybridization, as well as the chemokine receptors Duffy antigen receptor for chemokines (DARC) and CCR5 protein by immunohistochemistry in renal biopsy specimens with acute cellular rejection (n = 12) and acute vascular rejection (n = 8), transplant nephrectomy specimens (n = 6), and normal areas of tumor nephrectomy specimens (n = 5). CC chemokines and CC chemokine receptor mRNA expression were evaluated by ribonuclease protection assay in specimens from four transplant nephrectomies and one tumor nephrectomy. Upregulation of mRNAs for the chemokines, interferon-inducible protein-10 (IP-10); regulated on activation normal T-cell expressed and secreted; macrophage inflammatory protein-1alpha (MIP-1alpha); MIP-1beta; and lymphotactin, as well as the chemokine receptors, CCR2 and CCR5, were documented during allograft rejection. CCR1 mRNA was detectable in both allografts and controls, but CCR3 and CCR8 were absent. The number of CXCR4, CCR5, and CCR2B mRNAs expressing leukocytes and DARC-positive vessels increased during rejection episodes. CXCR4 mRNA was the most widely expressed. Leukocytes in diffuse interstitial infiltrates were mainly CCR5 positive, but in areas in which leukocytes formed nodular aggregates of infiltrating cells, the number of CCR5-positive cells was low. Instead, leukocytes in these nodular aggregates mainly expressed CXCR4. DARC was expressed on peritubular capillaries, where it was upregulated in areas of interstitial infiltration. Induction of chemokines during renal allograft rejection is accompanied by infiltration of leukocytes bearing the respective chemokine receptors. The upregulation of the CXCR3 ligand IP-10, as well as CCR5 and its ligands, in the absence of CCR3 and CCR8 is indicative that renal allograft rejection is primarily the result of a Th1-type immune response.

The Duffy antigen receptor for chemokines is up-regulated during acute renal transplant rejection and crescentic glomerulonephritis.

BACKGROUND: Recruitment of leukocytes during immune responses requires the coordinate expression of adhesion molecules in concert with chemokines and their receptors. The Duffy antigen receptor for chemokines (DARC) binds multiple chemokines and is expressed on postcapillary venules in the normal kidney. The chemokine receptor CCR5, which shares the ligand regulated upon activation, normal T-cell expressed and secreted (RANTES) with DARC, is expressed by infiltrating T cells in the renal interstitium. As DARC might be involved in the attraction of CCR5-positive cells, we studied the distribution of DARC and CCR5 in two forms of cell-mediated renal injury: renal allograft rejection and crescentic glomerulonephritis (cGN). METHODS: A total of 87 renal specimens, including 12 pretransplant biopsies, 47 transplant biopsies (Banff 1, N = 10; Banff 2, N = 19; and various other lesions N = 18), and 28 biopsies from patients with cGN, was analyzed. Immunohistochemistry for CCR5 and DARC was performed on serial sections of formalin-fixed and paraffin-embedded tissue. RESULTS: Compared with pretransplant biopsies, the mean number of DARC-positive interstitial venules was significantly increased during both transplant rejection and cGN. This was accompanied by an infiltration of CCR5-positive leukocytes. During transplant rejection, the number and distribution of CCR5-positive cells correlated with DARC-positive venules. Infiltrating CCR5-positive leukocytes were found mainly in the interstitium, often clustering around Bowman's capsules in biopsies from cGN. The number of glomerular CCR5 positive cells is low, but they are common in a subset of crescents. CONCLUSIONS: We hypothesize that the increased number of DARC-positive venules in areas of interstitial injury and the colocalization with CCR5-positive infiltrating leukocytes may indicate a role for endothelial DARC expression during leukocyte adhesion and interstitial infiltration.

Metanephrogenic mesenchyme-to-epithelium transition induces profound expression changes of ion channels.

The expression patterns of plasma membrane transporters that specify the epithelial cell type are acquired with ontogeny. To study this process during metanephrogenic mesenchyme-to-epithelium transition, branching ureteric buds with their adjacent mesenchymal blastema (mouse embryonic day E14) were dissected and explanted on a collagen matrix. In culture, induced mesenchymal cells condensed, aggregated, and converted to the comma- and S-shaped body. During in vitro condensation and aggregation, transcription factor Pax-2 protein was downregulated while the epithelial markers E-cadherin and beta-catenin proteins were upregulated. In addition, Wilms' tumor suppressor protein WT-1 was detectable upon condensation and downregulated in the S stage, where expression persisted in the long arm of the S. Patch-clamp, whole cell conductance (G, in nS/10 pF) of pre-epithelial condensed mesenchymal cells (n = 7) was compared with that of tubular proximal S-shaped-body epithelium (n = 6). Both stages expressed E-cadherin and WT-1 mRNA, as demonstrated by single-cell RT-PCR, testifying further to the epithelial as well as the nephrogenic commitment of the recorded cells. Mesenchymal cells exhibited whole cell currents (G = 6.7 +/- 1.3) with reversal potentials (V(rev), in mV) near equilibrium potential for Cl(-) (E(Cl)) (V(rev) = -40 +/- 7) suggestive of a high fractional Cl(-) conductance. Currents of the S-shaped-body cells (G = 4.0 +/- 1.1), in sharp contrast, had a V(rev) at E(K) (V(rev) = -82 +/- 6) indicating a high fractional K(+) conductance. Further, analysis of K(+)-selective whole cell tail currents and single-channel recording revealed a change in K(+) channel expression. Also, Kir6.1 K(+) channel mRNA and protein were downregulated between both stages, whereas K(v)LQT K(+) channel mRNA was abundant throughout. In conclusion, metanephrogenic mesenchyme-to-epithelium transition is accompanied by a profound reorganization of plasma membrane ion channel conductance.

[Angina abdominalis: duplex ultrasound diagnosis and percutaneous revascularization]. / Angina abdominalis: duplexsonographische Diagnostik und perkutane Revaskularisierung.

Intestinal ischemia is still a challenge for clinicians and requires a close interdisciplinary cooperation between internist, surgeon and radiologist. In the last years the diagnosis and therapy, classically invasive and surgical, was supplemented by duplex ultrasound and percutaneous techniques like angioplasty and stenting. A 56 year-old man from Greece presented with epigastric pain, which was intensified by food ingestion. These symptoms were caused by a stenosis of the superior mesenteric artery, which was diagnosed by duplex sonography and angiography. No blood flow was detected in the inferior mesenteric and the celiac artery. Occlusion of one internal carotid artery made the patient a poor candidate for surgery. Therefore an interventional approach was chosen. A good result was achieved by angioplasty and stent implantation. On the day after the intervention oral food intake was possible without any pain. 18 months after the intervention the patient was free of abdominal symptoms. Therapy of mesenteric ischemia by percutaneous angioplasty and stenting is published only in case-reports and small series. Therefore the indication is mainly restricted to patients with a high risk for a surgical intervention.