Renate Kain

The ephrin-A1 ligand and its receptor, EphA2, are expressed during tumor neovascularization

Eph receptor tyrosine kinases and their ephrin ligands have been implicated in embryonic vascular development and in in vivo models of angiogenesis. Eph proteins may also regulate tumor neovascularization, but this role has not been previously investigated. To screen for Eph proteins expressed in tumor blood vessels, we used tumor xenografts grown in nude mice from MDA-MB-435 human breast cancer cells or KS1767 human Kaposis sarcoma cells. By immunohistochemistry, the ephrin-A1 ligand and one of its receptors, EphA2, were detected throughout tumor vasculature. Double-labeling with anti-CD34 antibodies demonstrated that both ephrin-A1 and EphA2 were expressed in xenograft endothelial cells and also tumor cells. Furthermore, EphA2 was tyrosine-phosphorylated in the xenograft tumors, indicating that it was activated, presumably by interacting with ephrin-A1. Ephrin-A1 and EphA2 were also detected in both the vasculature and tumor cells of surgically removed human cancers. In an in vitro angiogenesis model, a dominant negative form of EphA2 inhibited capillary tube-like formation by human umbilical vein endothelial cells (HUVECs), demonstrating a requirement for EphA receptor signaling. These data suggest that ephrin-A1 and EphA2 play a role in human cancers, at least in part by influencing tumor neovascularization. Eph proteins may represent promising new targets for antiangiogenic cancer treatments.

Tenascin-W Is a Novel Marker for Activated Tumor Stroma in Low-grade Human Breast Cancer and Influences Cell Behavior

This is the first report about human tenascin-W, the fourth and final member of the extracellular matrix protein family of tenascins. Sixty-three human breast tumor extracts were analyzed by Western blotting for the presence of tenascin-W and compared with tenascin-C, an established marker of tumor stroma. Interestingly, we found tenascin-W expression in the majority of the tumor tissues, but no detectable expression in the normal mammary parenchyma. Eighty-one percent of the breast tumor samples were tenascin-W positive and 86% showed expression of tenascin-C. However, tenascin-W and tenascin-C amounts varied greatly between tumors and some contained either tenascin-W or tenascin-C exclusively, indicating independent mechanisms regulating their expression. Although there was no difference between high- or low-grade tumors with respect to the presence of tenascin-C, tenascin-W was more prominent in low-grade tumors. For 42 of the breast cancer tissues, a frozen tumor microarray was available to confirm the Western blot data by immunohistochemistry. Similar to tenascin-C, tenascin-W was detected in the tumor stroma. Fibroblasts adhered to tenascin-W in a beta(1) integrin-dependent manner and spread with a distinctive morphology under conditions where they remained round on tenascin-C. CHOB2 cells expressing alpha(v)beta(1) or alpha4beta(1) integrins were able to spread on tenascin-W. Furthermore, addition of tenascin-W to the culture medium increased migration of breast cancer cells toward a fibronectin substratum in vitro. These data imply that tenascin-W expression in the activated tumor stroma facilitates tumorigenesis by supporting the migratory behavior of breast cancer cells.

A novel variant of lysosomal acid lipase in cholesteryl ester storage disease associated with mild phenotype and improvement on lovastatin.

Cholesterol ester storage disease (CESD) is a rare congenital disorder of lipid metabolism, with mutation of the lysosomal acid lipase gene, causing chronic liver disease, usually before adolescence. We here describe three adult siblings with CESD diagnosed by light microscopic demonstration of excessive lysosomal storage of lipids with accumulation of foamy cells in liver biopsies and by a decrease in acid lipase activity (2-3% of controls). One patient (male, 46a) had extensive liver fibrosis, another (female, 58a) had cirrhosis of the liver. The third patient had died from variceal haemorrhage (female, 56a). Using sequence analysis of RT-PCR products of LAL mRNA, the patients were identified as compound heterozygotes for a G-->A substitution at position -1 of the exon 8 splice donor site and a point mutation at the second allele, resulting in a His108-->Pro shift. In two patients, therapy with lovastatin was initiated, which led to normalisation of serum cholesterol and triglyceride levels. After 12 months, liver biopsy demonstrated a significant decrease in vacuolisation of hepatocytes, with fewer and smaller droplets. Semi-automated computer-assisted image analysis of electron microscopic sections demonstrated a decrease in the hepatocellular lysosomal area from 20.5+/-7.1% to 11.7+/-6.5% (p<0.05) and 41.7+/-5.1% to 33.4+/-4.4% (p<0.01). We conclude that in two siblings with a novel LAL variant and mild phenotype of CESD, lovastatin decreased both serum lipid concentrations and hepatocellular lysosomal content.

MOLECULAR CLONING AND EXPRESSION OF A NOVEL HUMAN TRANS-GOLGI NETWORK GLYCOPROTEIN, TGN51, THAT CONTAINS MULTIPLE TYROSINE-CONTAINING MOTIFS

Previously, it has been shown that glycoproteins with ∼130-kDa molecular mass react with antisera from patients with renal vasculitis (Kain, R., Matsui, K., Exner, M., Binder, S., Schaffner, G., Sommer, E. M., and Kerjaschki, D. (1995) J. Exp. Med. 181, 585–597). To search for a molecule that reacts with the antibodies, we screened a λgt11 human placental cDNA library. Two of the isolated clones were found to encode a putative counterpart of the rodent trans-Golgi network (TGN) glycoprotein 38, hTGN46, which has the tyrosine containing motif YQRL shared by mouse and rat TGN38. Moreover, reverse transcription-polymerase chain reaction analysis of hTGN46transcripts and genomic analysis of a cDNA deposited as an expressed sequence tag in dbEST Data Base revealed that additional cDNAs exist that are produced by alternate usage of 3′-splice sites of intron III. Alternative splicing results in frame shifts and leads to novel larger translation products with one (for hTGN48) or two (for hTGN51) additional tyrosine-containing motifs. hTGN51 expressed in Chinese hamster ovary cells were localized to thetrans-Golgi network, overlapping with β-1,4-galactosyltransferase even after mutating the tyrosine-containing motif common to hTGN46. In contrast, mutated hTGN48 and hTGN46 are no longer retrieved to the TGN. These results strongly suggest that hTGN51 may have a unique function compared with hTGN46 or hTGN48 in shuttling between the cell surface and the TGN.

Immunological risk factors are solely responsible for primary non‐funetion of renal allografts

Abstract Primary non‐function (PNF) of renal allografts has been attributed to various risk factors, among them immunological ones, as well as unfavourable preservation conditions. To investigate the impact of these risk factory on the occurrence of PNF, 1335 consecutive kidney transplants performed at a single centre over a 10‐year period were analysed. All patients received immunosuppression based on cyclosporine. As the method of analysis a conditional stepwise logistic regression model was chosen, comparing each graft suffering PNF with its partner kidney retrieved from the same donor. Thus, all donor‐related variables could be omitted from the analysis, as they are the same in every pair of grafts. Risk factors analysed included panel‐reactive antibodies, number of pretransplant transfusions, pregnancies, number of prior transplants, cold and second warm ischaemia time, mismatches on HLA loci A, B and DR and recipient age. The overall incidence of PNF was 87 grafts (6.5%). One patient suffered immediate rejection due to transplantation of an ABO incompatible graft. This case was excluded from further analysis. PNF occurred three times in recipients of living related grafts, twice in recipients of en‐bloc grafts and four times in grafts, in which the paired kidney was either not transplanted or shipped outside the Eurotransplant region, so that no paired graft was available for matched case‐control analysis. Of the remaining 77 pairs, twice both organs of one donor failed immediately. The remaining 73 complete pairs were analysed. Two of the investigated risk factors have independently a significant impact on the occurrence of PNF. Increasing the number of pretransplant transfusions raises the relative risk of graft failure up to six fold (P=0.02), while a history of prior transplants bears a felative risk of 0.21E05 (P=0.005). Ischaemia has no significant impact on the occurrence of PNF. Our data strongly suggest that immunological rather than donor risk factors are responsible for the non‐function of kidney grafts.

Diagnostik glomerulärer und tubulointerstitieller Erkrankungen

Dieses Kapitel behandelt die technischen und klinischen Vorraussetzungen und Qualitatsmasstabe der Nierenbiopsie, die zu den wichtigsten Untersuchungsmethoden des Nephrologen gehort. Im Einzelnen wird zu den zur Verfugung stehenden immunhistochemischen Nachweismethoden Stellung genommen und ein standardisierter Befundbericht empfohlen.

Hereditäre und genetisch bedingte Glomerulopathien

Das Kapitel behandelt die primaren Glomerulopathien mit ihren meist monogenen Ursachen sowie die glomerularen Lasionsmuster bei syndromalen Erkrankungen. Es werden neueste genetische Erkenntnisse beleuchtet, wobei sowohl monogene als auch komplexe Mutationen in Korrelation zum Phanotyp der Nierenerkrankung gestellt werden. Da diese Befunde zunehmend zur Reklassifizierung der genetischen Glomerulopathien fuhren, wird bereits auf die neuesten Klassifikationen eingegangen. Fur die diagnostische Praxis stehen exemplarisches Bildmaterial und differentialdiagnostische Tabellen zur Verfugung.

Ontogenese, Anatomie und Funktion

Das Kapitel behandelt die entwicklungsgeschichtliche Entstehung der Nieren, ihre molekulare und anatomische Entwicklung sowie den anatomischen und feingeweblichen Aufbau. Neben morphologischen Gesichtspunkten wird ein besonderes Augenmerk auf die vergleichende Genese und die bei der Entwicklung und Funktion der Niere wichtigen molekularen Mechanismen gelegt sowie die wichtigsten Begriffe erlautert.