Showing papers by "Hermann Josef Gröne published in 2007"

Normal development and function of invariant natural killer T cells in mice with isoglobotrihexosylceramide (iGb3) deficiency.

Abstract: CD1d-restricted natural killer T (NKT) cells, expressing the invariant T cell antigen receptor (TCR) chain encoded by Vα14-Jα18 gene segments in mice and Vα24-Jα18 in humans [invariant NKT (iNKT) cells], contribute to immunoregulatory processes, such as tolerance, host defense, and tumor surveillance. iNKT cells are positively selected in the thymus by CD1d molecules expressed by CD4+/CD8+ cortical thymocytes. However, the identity of the endogenous lipid(s) responsible for positive selection of iNKT cells remains unclear. One candidate lipid proposed to play a role in positive selection is isoglobotrihexosylceramide (iGb3). However, no direct evidence for its physiological role has been provided. Therefore, to directly investigate the role of iGb3 in iNKT cell selection, we have generated mice deficient in iGb3 synthase [iGb3S, also known as α1–3galactosyltransferase 2 (A3galt2)]. These mice developed, grew, and reproduced normally and exhibited no overt behavioral abnormalities. Consistent with the notion that iGb3 is synthesized only by iGb3S, lack of iGb3 in the dorsal root ganglia of iGb3S-deficient mice (iGb3S−/−), as compared with iGb3S+/− mice, was confirmed. iGb3S−/− mice showed normal numbers of iNKT cells in the thymus, spleen, and liver with selected TCR Vβ chains identical to controls. Upon administration of α-galactosylceramide, activation of iNKT and dendritic cells was similar in iGb3S−/− and iGb3S+/− mice, as measured by up-regulation of CD69 as well as intracellular IL-4 and IFN-γ in iNKT cells, up-regulation of CD86 on dendritic cells, and rise in serum concentrations of IL-4, IL-6, IL-10, IL-12p70, IFN-γ, TNF-α, and Ccl2/MCP-1. Our results strongly suggest that iGb3 is unlikely to be an endogenous CD1d lipid ligand determining thymic iNKT selection.

Impairment of Sodium Balance in Mice Deficient in Renal Principal Cell Mineralocorticoid Receptor

Abstract: Germline inactivation of the mineralocorticoid receptor (MR) gene in mice results in postnatal lethality as a result of massive loss of sodium and water. The knockout mice show impaired epithelial sodium channel (ENaC) activity in kidney and colon. For determination of the role of renal MR in aldosterone-driven ENaC-mediated sodium reabsorption, mice with principal cell MR deficiency were generated using the Cre-loxP system. For driving Cre recombinase expression in principal cells, the regulatory elements of the mouse aquaporin 2 (AQP2) gene were used. Mutant mice (MR AQP2Cre ) were obtained by crossing AQP2Cre mice with mice that carried a conditional MR allele. Under standard diet, MR AQP2Cre mice develop normally and exhibit unaltered renal sodium excretion but show strongly elevated aldosterone levels. Increased renal sodium and water excretion, resulting in continuous loss of body weight, occur under low-sodium diet. Immunofluorescence revealed that the loss of MR and apical ENaC staining is restricted to principal cells of the collecting duct (CD) and late connecting tubule (CNT) and that MR is crucial for ENaC trafficking to the apical membrane. These results demonstrate that inactivation of MR in CD and late CNT can be compensated under standard diet but no longer when sodium supply is limited. Because the mutant mice show preserved renal ENaC activity, this study provides evidence that the late distal convoluted tubule and early CNT can compensate to a large extent deficient ENaC-mediated sodium reabsorption in late CNT and CD.

Decorin-Mediated Regulation of Fibrillin-1 in the Kidney Involves the Insulin-Like Growth Factor-I Receptor and Mammalian Target of Rapamycin

Abstract: Decorin, a small leucine-rich proteoglycan, affects the synthesis of the elastic fiber component fibrillin-1 in the kidney via hitherto unknown mechanisms. Here, we show that decorin binds to and induces phosphorylation of insulin-like growth factor-I (IGF-I) receptor in renal fibroblasts. Inhibition of the IGF-I receptor tyrosine kinase and its downstream target phosphoinositide-3 kinase prevented decorin-mediated synthesis of fibrillin-1. Furthermore, decorin induced phosphorylation of phosphoinositide-dependent kinase 1, protein kinase B/Akt, mammalian target of rapamycin (mTOR), and p70 S6 kinase. Accordingly, the enhanced synthesis of fibrillin-1 was blocked by rapamycin, an inhibitor of mTOR. Notably, IGF-I, which signals through the same pathway, also stimulated fibrillin-1 synthesis. Systemic administration of rapamycin to mice subjected to unilateral ureteral obstruction, a model of renal fibrosis and increased fibrillin-1 synthesis, markedly reduced the number of interstitial fibroblasts and fibrillin-1 deposition. In streptozotocin-induced diabetes, IGF-I receptor was up-regulated in the kidneys from decorin-null mice. However, this could not compensate for the decorin deficiency, resulting ultimately in decreased fibrillin-1 content. This study provides evidence for the involvement of decorin and the IGF-I receptor/mTOR/p70 S6 kinase signaling pathway in the translational regulation of fibrillin-1.

Clinical and mechanistic aspects of glucocorticoid-induced chemotherapy resistance in the majority of solid tumors

Abstract: Background: Glucocorticoids have been used widely in conjunction with cancer therapy due to their ability to induce apoptosis in hematological cells and to prevent nausea and emesis. However, recent data including ours, suggest induction of therapy-resistance by glucocorticoids in solid tumors, although it is unclear whether this happens only in few carcinomas or is a more common cell type specific phenomenon.Material and Methods: We performed an overall statistical analysis of our new and recent data obtained with 157 tumor probes evaluated in vitro, ex vivo and in vivo. The effect of glucocorticoids on apoptosis, viability and cell cycle progression under diverse clinically important questions was examined.Results: New in vivo results demonstrate glucocorticoid-induced chemotherapy resistance in xenografted prostate cancer. In an overall statistical analysis we found glucocorticoid-induced resistance in 89% of 157 analysed tumor samples. Resistance is common for several cytotoxic treatments and for seve...

Expression analysis of imbalanced genes in prostate carcinoma using tissue microarrays

Abstract: To identify candidate genes relevant for prostate tumour prognosis and progression, we performed an exhaustive gene search in seven previously described genomic-profiling studies of 161 prostate tumours, and four expression profiling studies of 61 tumours. From the resulting list of candidate genes, six were selected for protein-expression analysis based on the availability of antibodies applicable to paraffinised tissue: fatty acid synthase (FASN), MYC, β-adrenergic receptor kinase 1 (BARK1, GRK2) the catalytic subunits of protein phosphatases PP1α (PPP1CA) and PP2A (PPP2CB) and metastasis suppressor NM23-H1. These candidates were analysed by immunohistochemistry (IHC) on a tissue microarray containing 651 cores of primary prostate cancer samples and benign prostatic hyperplasias (BPH) from 175 patients. In univariate analysis, expression of PP1α (P=0.001) was found to strongly correlate with Gleason score. MYC immunostaining negatively correlated with both pT-stage and Gleason score (P<0.001 each) in univariate as well as in multivariate analysis. Furthermore, a subgroup of patients with high Gleason scores was characterised by a complete loss of BARK1 protein (P=0.023). In conclusion, our study revealed novel molecular markers of potential diagnostic and therapeutic relevance for prostate carcinoma.

Microarray analysis of newly synthesized RNA in cells and animals

Abstract: Current methods to analyze gene expression measure steady-state levels of mRNA. To specifically analyze mRNA transcription, we have developed a technique that can be applied in vivo in intact cells and animals. Our method makes use of the cellular pyrimidine salvage pathway and is based on affinity-chromatographic isolation of thiolated mRNA. When combined with data on mRNA steady-state levels, this method is able to assess the relative contributions of mRNA synthesis and degradation/stabilization. It overcomes limitations associated with currently available methods such as mechanistic intervention that disrupts cellular physiology, or the inability to apply the techniques in vivo. Our method was first tested in serum response of cultured fibroblast cells and then applied to the study of renal ischemia reperfusion injury, demonstrating its applicability for whole organs in vivo. Combined with data on mRNA steady-state levels, this method provided a detailed analysis of regulatory mechanisms of mRNA expression and the relative contributions of RNA synthesis and turnover within distinct pathways, and identification of genes expressed at low abundance at the transcriptional level.

Sphingosine-1-Phosphate Analogue FTY720 Causes Lymphocyte Redistribution and Hypercholesterolemia in ApoE-Deficient Mice

Abstract: Objective— Resident immune cells are a hallmark of atherosclerotic lesions. The sphingolipid analogue drug FTY720 mediates retrafficking of immune cells and inhibits their homing to inflammatory sites. We have evaluated the effect of FTY720 on atherogenesis and lipid metabolism. Methods and Results— ApoE −/− mice on a normal laboratory diet received oral FTY720 for 12 weeks, which led to a 2.4-fold increase in serum cholesterol (largely VLDL fraction) and a 1.8-fold increase in hepatic HMGCoA reductase mRNA. FTY720 increased plasma sphingosine-1-phosphate and induced marked peripheral blood lymphopenia. A discoordinate modulation of B, T and monocyte cell numbers was found in peripheral lymphoid organs. Overall depletion of T cells was accompanied by a relative (2-fold) increase in regulatory T cell content paralleled by a similar increase in effector memory T cells (CD4+CD44hiCD62lo) as absolute numbers of both subpopulations remained essentially unchanged. Lymphocyte function was unaltered as indicated by anti-OxLDL antibodies and T cell proliferation. There were no changes in atherosclerotic lesions in early and established atherosclerosis. Conclusions— FTY720 mediated peripheral lymphocyte depletion and retrafficking without altering function and overall balance of pro- and antiatherogenic lymphocyte populations. A net decrease in lymphocyte numbers occurred concomitantly with a more proatherogenic hypercholesterolemia resulting in unaltered atherogenesis.

Met-RANTES reduces endothelial progenitor cell homing to activated (glomerular) endothelium in vitro and in vivo

Abstract: The chemokine RANTES (regulated upon activation normal T-cell expressed and secreted) is involved in the formation of an inflammatory infiltrate during glomerulonephritis. However, RANTES receptor ...

Sulfated Glycosphingolipid as Mediator of Phagocytosis: SM4s Enhances Apoptotic Cell Clearance and Modulates Macrophage Activity

Abstract: Sulfoglycolipids are present on the surface of a variety of cells. The sulfatide SM4s is increased in lung, renal, and colon cancer and is associated with an adverse prognosis, possibly due to a low immunoreactivity of the tumor. As macrophages significantly contribute to the inflammatory infiltrate in malignancies, we postulated that SM4s may modulate macrophage function. We have investigated the effect of SM4s on the uptake of apoptotic tumor cells, macrophage cytokine profile, and receptor expression. Using flow cytometry and microscopic analyses, we found that coating apoptotic murine carcinoma cells from the colon and kidney with SM4s promoted their phagocytosis by murine macrophages up to 3-fold ex vivo and in vivo. This increased capacity was specifically inhibited by preincubation of macrophages with oxidized or acetylated low density lipoprotein and maleylated albumin, indicating involvement of scavenger receptors in this interaction. The uptake of SM4s-coated apoptotic cells significantly enhanced macrophage production of TGF-β1, expression of P-selectin, and secretion of IL-6. These data suggest that SM4s within tumors may promote apoptotic cell removal and alter the phenotype of tumor-associated macrophages.

Collagen type VIII expression in human diabetic nephropathy

Abstract: BACKGROUND Collagen type VIII is a non-fibrillar short-chain collagen that may modulate migration, proliferation and adherence of various cells. Only very sparse information exists on collagen type VIII expression in human diabetic nephropathy. MATERIAL AND METHODS We retrospectively studied mRNA expression for the two collagen type VIII chains (COL8A1 and COL8A2) in 20 biopsies with histologically confirmed diabetic nephropathy by real-time PCR, and compared glomerular and tubular expression with normal kidney [pre-transplant biopsies (n = 10)]. Expression of collagen type VIII was also studied in biopsies from patients with benign nephrosclerosis (BNS; n = 16) and focal-segmental glomerulosclerosis (FSGS; n = 9). RESULTS A strong specific induction of COL8A1 mRNA was found in diabetic nephropathy in both glomerular and tubular compartments. There was also a robust induction of COL8A2 in diabetic nephropathy, but overall expression was lower than that of COL8A1 transcripts. No significant increase in COL8A1 and COL8A2 mRNAs expression was found in biopsies from patients with BNS and FSGS compared with normal kidneys. The cross-reactivity of the used anti-alpha1(VIII) antibody with human tissue was confirmed by Western blots. Immunohistological analysis revealed only little staining for collagen type VIII in the normal kidney, localized to vessels. There was an up-regulation of collagen type VIII protein expression as shown by immunohistochemistry in the diabetic nephropathy biopsies mainly localized to mesangial cells, tubules and the interstitium. Proteinuria and serum creatinine did not correlate with glomerular or tubular COL8A1 and COL8A2 mRNA expression in diabetic patients. CONCLUSION Our study systemically investigates collagen type VIII expression in human biopsies. Induction of collagen type VIII was specific for diabetic nephropathy and did not occur in the other renal diseases studied. More specific factors of the diabetic environment are likely involved in the stimulated expression because there was no correlation of collagen type VIII mRNA expression with proteinuria. Since collagen type VIII may influence proliferation and migration of cells, it is possible that an increase in renal expression of collagen type VIII initiates other pathophysiological processes (e.g. proliferation of renal fibroblasts) involved in diabetic nephropathy.

Anti-Inflammatory Effects of αv Integrin Antagonism in Acute Kidney Allograft Rejection

Abstract: Integrin-mediated cell adhesion and signaling is essential to vascular development and inflammatory processes. Elevated expression of integrin αvβ3 has been detected in ischemia-reperfusion injury and rejecting heart allografts. We thus hypothesized that the inhibition of αv-associated integrins may have potent anti-inflammatory effects in acute kidney allograft rejection. We studied the effects of a peptidomimetic antagonist of αv integrins in two rat models of renal allotransplantation, differing in degree of major histocompatibility complex mismatch. Integrin αvβ3 was up-regulated in rejecting renal allografts. Integrin antagonist reduced the histological signs of acute rejection, the intensity of the mononuclear cell infiltration, and cell proliferation in the grafted kidneys. This could be correlated to a reduced leukocyte-endothelial interaction and an improved peritubular microcirculation observed by intravital microscopy. In vitro under laminar flow conditions, the arrest of monocytes to interleukin-1β-activated endothelium was decreased. Furthermore, in co-culture models the proliferation and transmigration of monocytes/macrophages, endothelium, and fibroblasts induced by renal tubular epithelia was efficiently inhibited by αv integrin antagonism. These data reveal an important role of this integrin subclass in leukocyte recruitment and development and maintenance of acute rejection; blockade of αv integrins may provide a new therapeutic strategy to attenuate acute allograft rejection.

Selective Binding and Presentation of CCL5 by Discrete Tissue Microenvironments during Renal Inflammation

Abstract: T cells are differentially recruited to the tubulointerstitium during renal inflammation. The selective presentation of chemokines by surface structures may in part underlie this phenomenon. In an attempt to better characterize the presentation of chemokines by tissue environments an exemplary chemokine with a well-defined structure was selected, and a binding assay for the protein on fixed archival tissue sections was developed. This article describes the selective binding of the chemokine CCL5 to renal structures. CCL5 was shown to bind to endothelial regions, interstitial extracellular matrix, tubular epithelial cells, and tubular basement membranes but rarely to glomerular structures in well-preserved kidneys. In contrast, binding of CCL5 to glomerular components was seen in renal biopsies with acute allograft glomerulitis (in which T cells accumulate in glomeruli). The N terminus mediates receptor binding, whereas two clusters of basic amino acid residues ( 44 RKNR 47 and 55 KKWVR 59 ) are involved in the presentation of CCL5 by extracellular structures. Mutation of either loop abrogated CCL5 binding to tissue sections. Variations of the N terminus and a mutation that prevents higher order oligomerization did not change the binding pattern. The data suggest that renal compartments differ in their capacity to present chemokines, which may help explain the differential recruitment of leukocytes during allograft injury. Both clusters of basic residues in CCL5 are necessary for sufficient binding of CCL5 to tissue sections.

Histiocytosis X and renal insufficiency

Abstract: Langerhans cell histiocytosis (LCH) is a rare disordershowing a wide spectrum of clinical manifestationsranging from single cutaneous lesions to multifocalsystemic disease [1,2]. The typical infiltration consistsof pathological monoclonal Langerhans cells togetherwith lymphocytes, eosinophilic granulocytes and non-dendritic histiocytes [3]. Langerhans cells are antigen-presenting cells with features of dendritic cells, stainingpositive for CD1a [4]. One of their original functions iscutaneous immunosurveillance. LCH is rare and theincidence in adults is 1–2 cases per million, with a meanage of 35 14 years, and is slightly more prevalent inmales [5,6]. Histological diagnosis is made if lightmicroscopic morphological characteristics of the dis-ease are found with positive staining for CD1a antigen[4,7]. In addition, Birbeck granules in the lesional cellthat are detected with electron microscopy are alsosuggestive of the disease [4,7]. Typical manifestationsof LCH in adults are central diabetes insipidus, bone,pulmonary, skin disease and dental involvement.We report a case of a patient with progressive renalinsufficiency due to multi-systemic manifestation ofLangerhans cell histiocytosis including involvement ofthe kidneys. Treatment of LCH with chemotherapyresulted in stabilization of renal function.CaseA 46-year-old male patient was admitted to ournephrology clinic in May 2006, with a 1-year historyof impaired renal function with moderate proteinuriaand glomerular erythrocyturia. In 1991, the patientwas successfully treated for fibrous alveolitis withprednisolone. Two years later, the patient developedcentral diabetes insipidus. In 1999, Histiocytosis X(Langerhans cell histiocytosis, LCH) was diagnosed ina skin biopsy obtained from an axillary erythema(histological findings: cellular infiltration of the epi-dermis with histiocytes, co-expression of S100 andCD1a; negativity for lymphocytic markers and KL-1;positive PG-M1; epitheliotropism; highly-positive toMiB1). In the follow-up, he also developed bilaterallyrelapsing therapy-resistant external otitis and swollencervical and submandibular lymph nodes. CT thoraxrevealed disseminated micronodular pulmonal opaci-ties. Skeletal scintigraphy showed a suspicious focus inthe proximal left tibia, and foci in the left mandible andleft mastoid could be localized. Thin-layer magneticresonance tomography of the sella turcica providedevidence for granuloma, with contrast enhancement inthe area of the hypophyseal stalk. An intravenousprednisolone therapy with 100mg was performed andthe cutaneous focus showed a rapid diminution.Therefore, methylprednisolone (60mg/day for 4weeks) and vinblastine (7mg/m