Rüdiger Waldherr

Tissue factor controls the balance of angiogenic and antiangiogenic properties of tumor cells in mice.

Meth-A sarcoma cells were stable transfected to overexpress (sense construct) or underexpress (antisense construct) tissue factor. In vitro, there was no difference in plating efficiency or growth between these cell lines. In vivo, tumor cells transfected to overexpress tissue factor grew more rapidly, and established larger and more vascularized tumors than control transfectants. Antisense transfectants grew the slowest and were the least vascularized. Anticoagulation of mice with warfarin did not alter the difference between these tumor lines. Tumor cells over-expressing tissue factor released more (compared with control transfectants) mitogenic activity for endothelial cells in parallel with enhanced transcription of vascular permeability factor/vascular endothelial cell growth factor (VEGF/VPF), and diminished transcription of thrombospondin (TSP2), a molecule with anti-angiogenic properties. Antisense tissue factor transfectants, while releasing the lowest amount of mitogenic activity, had increased thrombospondin and decreased VEGF/VPF transcription compared with control transfectants or wild-type cells. Experiments with these sense, antisense, truncated sense, or vector tumor lines gave comparable results in complete medium, serum free medium or in the presence of hirudin, indicating that the activation of the coagulation mechanism was not likely to be responsible for changes in tumor cell properties. These results suggest that tissue factor regulates angiogenic properties of tumor cells by altering the production of growth regulatory molecules of endothelium by a mechanism distinct from tissue factor activation of the coagulation mechanism.

Role of NFkappaB in the mortality of sepsis.

Binding activity for nuclear factor kappa B (NFkappaB) consensus probes was studied in nuclear extracts from peripheral blood mononuclear cells of 15 septic patients (10 surviving and 5 not surviving). Nonsurvivors could be distinguished from survivors by an increase in NFkappaB binding activity during the observation period (P < 0.001). The increase in NFkappaB binding activity was comparable to the APACHE-II score as a predictor of outcome. Intravenous somatic gene transfer with an expression plasmid coding for IkappaBalpha was used to investigate the role of members of the NFkappaB family in a mouse model of endotoxemia. In this model, increased NFkappaB binding activity was present after injection of LPS. Intravenous somatic gene transfer with IkappaBalpha given before LPS attenuated renal NFkappaB binding activity and increased survival. Endothelial cells and monocytes/macrophages were the major target cells for somatic gene transfer, transfected with an average transfection efficiency of 20-35%. Tissue factor, a gene under regulatory control of NFkappaB, was induced by LPS. Somatic gene transfer with a reporter plasmid containing the functional tissue factor promoter demonstrated NFkappaB-dependent stimulation by LPS. Intravenous somatic gene transfer with IkappaBalpha reduced LPS-induced renal tissue factor expression, activation of the plasmatic coagulation system (decrease of thrombin-antithrombin III complexes) and renal fibrin/fibrinogen deposition. Somatic gene transfer with an expression plasmid with tissue factor cDNA in the antisense direction (in contrast to sense or vector alone) also increased survival. Furthermore, antisense tissue factor decreased renal tissue factor expression and the activation of the plasmatic coagulation system.

Histopathologic Classification of ANCA-Associated Glomerulonephritis

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis is the most common cause of rapidly progressive glomerulonephritis worldwide, and the renal biopsy is the gold standard for establishing the diagnosis. Although the prognostic value of the renal biopsy in ANCA-associated glomerulonephritis is widely recognized, there is no consensus regarding its pathologic classification. We present here such a pathologic classification developed by an international working group of renal pathologists. Our classification proposes four general categories of lesions: Focal, crescentic, mixed, and sclerotic. To determine whether these lesions have predictive value for renal outcome, we performed a validation study on 100 biopsies from patients with clinically and histologically confirmed ANCA-associated glomerulonephritis. Two independent pathologists, blinded to patient data, scored all biopsies according to a standardized protocol. Results show that the proposed classification system is of prognostic value for 1- and 5-year renal outcomes. We believe this pathologic classification will aid in the prognostication of patients at the time of diagnosis and facilitate uniform reporting between centers. This classification at some point might also provide means to guide therapy.

Pulmonary tumor thrombotic microangiopathy with pulmonary hypertension

Pulmonary tumor thrombotic microangiopathy is characterized by fibrocellular intimal proliferation of small pulmonary arteries and arterioles in patients with metastastic carcinoma. Its morphologic features, including precursor lesions, were studied in 21 patients diagnosed in 630 consecutive autopsy cases with carcinoma (3.3%). Nineteen of 21 patients had adenocarcinoma and 11 of these 19 patients had gastric carcinoma. The pathogenetic events start with microscopic tumor cell embolism. Tumor emboli do not occlude affected vessels but induce both local activation of coagulation and fibrocellular intimal proliferation, which lead into stenosis or occlusion. Hemodynamically, an increase in vascular resistance results in pulmonary hypertension. In three patients, metastatic carcinoma was unknown before death, and the condition was diagnosed as pulmonary hypertension of unknown origin. Thus, pulmonary tumor thrombotic microangiopathy should be considered in the differential diagnosis of primary pulmonary hypertension, particularly in patients with well‐known carcinoma who develop acute or subacute cor pulmonale.

Clinical and Histologic Determinants of Renal Outcome in ANCA-Associated Vasculitis: A Prospective Analysis of 100 Patients with Severe Renal Involvement

This study aimed to identify clinical and histologic prognostic indicators of renal outcome in patients with ANCA-associated vasculitis and severe renal involvement (serum creatinine >500 micromol/L). One hundred patients who were enrolled in an international, randomized, clinical trial to compare plasma exchange with intravenous methylprednisolone as an additional initial treatment were analyzed prospectively. Diagnostic renal biopsies were performed upon entry into the study. Thirty-nine histologic and nine clinical parameters were determined as candidate predictors of renal outcome. The end points were renal function at the time of diagnosis (GFR0) and 12 mo after diagnosis (GFR12), dialysis at entry and 12 mo after diagnosis, and death. Multivariate analyses were performed. Predictive of GFR0 were age (r = -0.40, P = 0.04), arteriosclerosis (r = -0.53, P = 0.01), segmental crescents (r = 0.35, P = 0.07), and eosinophilic infiltrate (r = -0.41, P = 0.04). Prognostic indicators for GFR12 were age (r = -0.32, P = 0.01), normal glomeruli (r = 0.24, P = 0.04), tubular atrophy (r = -0.28, P = 0.02), intraepithelial infiltrate (r = -0.26, P = 0.03), and GFR0 (r = 0.29, P = 0.01). Fibrous crescents (r = 0.22, P = 0.03) were predictive of dialysis at entry. Normal glomeruli (r = -0.30, P = 0.01) and treatment arm (r = -0.28, P = 0.02) were predictive of dialysis after 12 mo. No parameter predicted death. Both chronic and acute tubulointerstitial lesions predicted GFR12 in severe ANCA-associated glomerulonephritis, whereas plasma exchange was a positive predictor of dialysis independence after 12 mo for the entire patient group. Plasma exchange remained a positive predictor when patients who were dialysis dependent at presentation were analyzed separately (r = -0.36, P = 0.01). Normal glomeruli were a positive predictor of dialysis independence and improved renal function after 12 mo, indicating that the unaffected part of the kidney is vital in determining renal outcome.

Mechanism of the Tumor Necrosis Factor α-mediated Induction of Endothelial Tissue Factor

This study examines the regulation of the human tissue factor (TF) promotor in vitro and in vivo. Transient transfections were performed in bovine aortic endothelial cells to investigate the role of two fundamentally different AP-1 sites and a closely located NF-κB site in the human TF promotor. The NF-κB site is functionally active, since overexpression of NF-κB(p65) resulted in induction of TF mRNA and activity. Promotor analysis showed that NF-κB induction was dependent on the integrity of the region from base pair −188 to −181. Overexpression of Jun/Fos resulted in TF induction of transcription and protein/activity. Functional studies revealed that the proximal AP-1 site, but not the distal, was inducible by Jun/Fos heterodimers. The distal AP-1 site, which has a G → A switch at position 4, was inducible by Jun homodimers. Electrophoretic mobility shift assays, using extracts of tumor necrosis factor α (TNFα)-stimulated bovine aortic endothelial cells, demonstrated TNFα-inducible binding to the proximal AP-1 site, comprising JunD/Fos heterodimers. At the distal AP-1 site, only minor induction of binding activity, characterized as proteins of the Jun and ATF family, was observed. Consistently, this site only marginally participates in TNFα induction. Functional studies with TF promotor plasmids confirmed that deletion of the proximal AP-1 or the NF-κB site decreased TNFα-mediated TF induction to a higher extend than loss of the distal AP-1 site. However, integrity of both AP-1 sites and the NF-κB site was required for optimal TNFα stimulation. The relevance of these in vitro data was confirmed in vivo in a mouse tumor model. Expression plasmids for a dominant negative Jun mutant or I-κB were packaged in liposomes. When either mutated Jun or I-κB were injected intravenously 48 h before TNFα, a reduction in TNFα-mediated TF expression in the tumor endothelial cells was observed. Simultaneously, fibrin/fibrinogen deposition decreased and free blood flow could be restored. Thus, TNFα-induced up-regulation of endothelial cell TF depends on a concerted action of members of the bZIP and NF-κB family.

Clinical outcome of Schönlein-Henoch purpura nephritis in children.

Abstract We studied the long-term outcome of 64 children with biopsy-proven Schönlein-Henoch purpura (SHP) nephritis over 1–23 years of follow-up. Overall renal survival 10 years after onset was 73%. Multivariate logistic regression analysis identified initial renal insufficiency (P=0.004), nephrotic syndrome (P=0.037), and the severity of histological alterations, as defined by the proportion of glomerular crescents (P=0.051), as significant independent predictors of progressive renal failure. Four patients followed for more than 19 years showed glomerular damage after transient recovery. Eight children with crescentic nephritis associated with a rapidly progressive course and/or persistent nephrotic syndrome were treated by at least seven sessions of plasma exchange (PE) within 16 weeks of onset of purpura. During treatment serum creatinine levels dropped in each patient from a mean of 2.3 to 1.1 mg/dl, followed by a rebound increase. Repeated courses of PE in 5 patients produced comparable responses. Four patients undergoing PE reached end-stage renal disease at 1.2.–3.7 years after onset, whilst 3 finally were in preterminal renal failure (creatinine 3.2–6.1 mg/dl after 7–13.5 years), and 1 patient reached a normal glomerular filtration rate. Our experience suggests that initial renal insufficiency is the best single predictor of the further clinical course in children with SHP nephritis. Early PE appears to delay the progression in some patients with severe, rapidly progressive forms of the disease.

Co-expression of cytokeratin and vimentin intermediate-sized filaments in renal cell carcinomas. Comparative study of the intermediate-sized filament distribution in renal cell carcinomas and normal human kidney.

The expression of intermediate-sized filaments (IF) was examined by immunocytochemical methods in 40 primary renal cell carcinomas and compared with the IF distribution in the normal adult human kidney. All tumours stained positively with cytokeratin IF antibodies. Co-expression of cytokeratins and vimentin was observed in 21/40 (52,5%) renal carcinomas. Double immunofluorescence labelling demonstrated that in most of these cases tumour cells contained both cytokeratin and vimentin type IF. In normal human kidneys, cells of the various tubular segments disclosed a positive reaction with cytokeratin antibodies in a different intensity and intracellular localization. Co-expression of cytokeratin and vimentin IF in normal adult human kidneys has never been observed. From a histogenetic point of view, co-expression of cytokeratins and vimentin in renal cell carcinoma obviously represents an atavistic phenomenon since vimentin is re-expressed by these tumour cells during neoplastic transformation. This finding indicates the metanephric origin of the renal parenchyma. In surgical pathology the possibility of very rare co-expression of cytokeratin and vimentin IF within tumour cells should be considered, particularly in the differential diagnosis of clear cell carcinomas.

Concomitant overexpression of the EGFR and erbB-2 genes in renal cell carcinoma (RCC) is correlated with dedifferentiation and metastasis

Gene amplification or structural alteration of different erbB genes exerts a transforming effect in a variety of human neoplasms. Overexpression of the EGF receptor is associated with tumor initiation and progression of renal cell carcinoma (RCC). However, the role of erbB‐2 in these processes remains unknown. We investigated 34 renal cell carcinomas for gene amplification and expression of the EGFR and erbB‐2 genes at the mRNA and protein level and their relationship to pathological and clinical parameters. No amplification of both genes has been observed. However, high expression of the EGF receptor protein and p185erbB2 was frequent in RCC and statistically significantly related to higher tumor grades. We could demonstrate a close correlation of p185erbB2 overexpression with high EGF receptor levels. Co‐overexpression of both receptor types was significantly associated with metastatic disease. Our results suggest a synergistic involvement of both EGF receptor and p185erbB2 in the progression of RCC.

Prenatal diagnosis of autosomal dominant polycystic kidney disease with a DNA probe

A highly polymorphic DNA probe genetically linked to the locus of autosomal dominant polycystic kidney disease was used in linkage studies for prenatal diagnosis in a nine-week fetus at risk for the disease. The fetus was judged to have inherited the polycystic kidney disease mutation, and this was confirmed by microscopic examination of the fetal kidneys at necropsy.