[Consequences of autopsies for the living : Causes of death in the clinical diagnosis "septic and toxic shock"]. / Konsequenzen von Autopsiebefunden für die Lebenden : Todesursachenfeststellungen bei der klinischen Diagnose "septisch-toxischer Schock".

BACKGROUND: There is reason to believe that the diagnosis of septic and toxic shock, as indicated on the death certificate, cannot be confirmed as the cause of death without autopsy and subsequent histological analysis. The external examination of the corpse can therefore not represent the sole basis for a reliable statement about the infection status of a corpse, e. g. as a prerequisite for embalming. MATERIAL AND METHODS: The validity of autopsy in determining septic and toxic shock as the cause of death is demonstrated in 7 exemplary cases. RESULTS: Decades of experience in a university pathology institute have shown that an external examination of the corpse alone is not suitable for certifying the cause of death if an infectious disease is suspected. Consequently, only autopsy with subsequent histological analysis provides reliable statements on the etiopathogenesis of the underlying process. Possible problems and discrepancies between clinical and pathological diagnoses are discussed on the basis of several cases with or without autoptic confirmation of the septic shock. The case of a missionary from Africa infected with Lassa virus serves to point out the seriousness of the threat an undiagnosed infection may represent to the attending staff. CONCLUSION: During the treatment of patients suspected to have an infectious cause of fever of unknown origin, compliance with the usual safety regulations, including adequate disinfecting measures, is essential. In cases with fatal outcome, not infrequently under the clinical picture of a septic and toxic shock, autopsy should be regularly performed to confirm the type of infection and the infectious cause of death. Rapid and open communication between the professional groups involved plays a crucial role in this process.

ETS-dependent p16INK4a and p21waf1/cip1 gene expression upon endothelin-1 stimulation in malignant versus and non-malignant proximal tubule cells.

AIM: Cellular senescence, leading to cell death through prevention of regular cell renewal, is associated with the upregulation of the tumor suppressor gene p16(INK4a). While this mechanism has been described as leading to progressive nephron loss, p16(INK4a) upregulation in renal cell carcinoma has been linked to a disease-specific improved patient survival rate. While in both conditions endothelin-1 is also upregulated, the signaling pathway connecting ET-1 to p16(INK4a) has not been characterized until this study. MAIN METHODS: Cell culture, qRT-PCR, Western Blot, immunoprecipitation (IP), proximity ligation assay (PLA), and non-radioactive electrophoretic mobility shift assay (EMSA). KEY FINDINGS: In malignant renal proximal tumor cells (Caki-1), an activation of p16(INK4a) and p21(waf1/cip1) was observed. An increased expression of E-26 transformation-specific (ETS) transcription factors was detectable. Using specific antibodies, a complex formation between ETS1 and extracellular signal-regulated kinase-2 (ERK2) was shown. A further complex partner was Mxi2. EMSA with supershift analysis for ETS1 and Mxi2 indicated the involvement of both factors in the protein-DNA interaction. After specifically blocking the endothelin receptors, ETS1 expression was significantly downregulated. However, the endothelin B receptor dependent downregulation was stronger than that of the A receptor. In contrast, primary proximal tubule cells showed a nuclear decrease after ET-1 stimulation. This indicates that other ETS members may be involved in the observed p16(INK4a) upregulation (as described in the literature). SIGNIFICANCE: ETS1, ERK2 and Mxi2 are important complex partners initiating increased p16(INK4a) and p21w(af1/cip1) activation in renal tumor cells.

Recovery of whisking function after manual stimulation of denervated vibrissal muscles requires brain-derived neurotrophic factor and its receptor tyrosine kinase B.

Functional recovery following facial nerve injury is poor. Neuromuscular junctions (NMJs) are "bridged" by terminal Schwann cells and numerous regenerating axonal sprouts. We have shown that this poly-innervation of NMJs can be reduced by manual stimulation (MS) with restoration of whisking function. In addition, we have recently reported that insulin-like growth factor-1 (IGF-1) is required to mediate the beneficial effects of MS. Here we extend our findings to brain derived neurotrophic factor (BDNF). We then examined the effect of MS after facial-facial anastomosis (FFA) in heterozygous mice deficient in BDNF (BDNF(+/-)) or in its receptor TrkB (TrkB(+/-)). We quantified vibrissal motor performance and the percentage of NMJ bridged by S100-positive terminal Schwann cells. In intact BDNF(+/-) or TrkB(+/-) mice and their wild type (WT) littermates, there were no differences in vibrissal whisking nor in the percentage of bridged NMJ (0% in each genotype). After FFA and handling alone (i.e. no MS) in WT animals, vibrissal whisking amplitude was reduced (60% lower than intact) and the percentage of bridged NMJ increased (27% more than intact). MS improved both the amplitude of vibrissal whisking (not significantly different from intact) and the percentage of bridged NMJ (11% more than intact). After FFA and handling in BDNF(+/-) or TrkB(+/-) mice, whisking amplitude was again reduced (53% and 60% lower than intact) and proportion of bridged NMJ increased (24% and 29% more than intact). However, MS failed to improve outcome in both heterozygous strains (whisking amplitude 55% and 58% lower than intact; proportion of bridged NMJ 27% and 18% more than intact). We conclude that BDNF and TRkB are required to mediate the effects of MS on target muscle reinnervation and recovery of whisking function.

Recovery of whisking function promoted by manual stimulation of the vibrissal muscles after facial nerve injury requires insulin-like growth factor 1 (IGF-1).

Recently, we showed that manual stimulation (MS) of denervated vibrissal muscles enhanced functional recovery following facial nerve cut and suture (FFA) by reducing poly-innervation at the neuro-muscular junctions (NMJ). Although the cellular correlates of poly-innervation are established, with terminal Schwann cells (TSC) processes attracting axon sprouts to "bridge" adjacent NMJ, molecular correlates are poorly understood. Since quantitative RT-PCR revealed a rapid increase of IGF-1 mRNA in denervated muscles, we examined the effect of daily MS for 2 months after FFA in IGF-1(+/-) heterozygous mice; controls were wild-type (WT) littermates including intact animals. We quantified vibrissal motor performance and the percentage of NMJ bridged by S100-positive TSC. There were no differences between intact WT and IGF-1(+/-) mice for vibrissal whisking amplitude (48 degrees and 49 degrees ) or the percentage of bridged NMJ (0%). After FFA and handling alone (i.e. no MS) in WT animals, vibrissal whisking amplitude was reduced (60% lower than intact) and the percentage of bridged NMJ increased (42% more than intact). MS improved both the amplitude of vibrissal whisking (not significantly different from intact) and the percentage of bridged NMJ (12% more than intact). After FFA and handling in IGF-1(+/-) mice, the pattern was similar (whisking amplitude 57% lower than intact; proportion of bridged NMJ 42% more than intact). However, MS did not improve outcome (whisking amplitude 47% lower than intact; proportion of bridged NMJ 40% more than intact). We conclude that IGF-I is required to mediate the effects of MS on target muscle reinnervation and recovery of whisking function.

Hepatic steatosis in organ donors: disparity between surgery and histology?

BACKGROUND: In times of organ shortage, use of marginal cadaveric livers has become increasingly important to reduce pressing organ demand and rising death rates while awaiting donations. Indisputably, fatty change in donor livers is a risk factor for poor initial function after orthotopic transplantation. However, identifying and rejecting marginal from good donor livers is one of the most difficult surgical tasks. Unfortunately, a liver biopsy with rapid histological diagnosis is rarely performed to identify marginal livers. METHODS: From 2005 to 2008, we investigated 36 livers of organ donors, which were explanted but not transplanted or underwent liver wedge biopsy during organ donation. All livers underwent standard surgical procedures and were allocated by Eurotransplant International Foundation. After unsuccessful allocation, explanted livers were photographically documented, formalin-fixed, and analyzed histopathologically. RESULTS: Seven livers were classified as good organ quality by the surgeon (19.4%); 15 were acceptable (41.6%); and 14 poor (39%). In 63.8% of livers, a frozen section was performed; 6/36 cases (16.7%) showed macrovesicular and microvesicular steatosis of less than 30%. In addition, all six cases fulfilled two or less extended donor criteria, as defined by the German Medical Association. CONCLUSION: More marginal livers from cadaveric organ donors could have been transplanted. To extend the transplant pool of liver grafts, liver biopsies should be performed in all cases of acceptable and poor livers. If frozen section analysis is performed, a wedge liver biopsy should be taken from at least two different segments of the liver to validate the histological results.

Hypereosinophilic syndrome associated with acute necrotizing myocarditis and cardiomyopathy.

We report the rare case of a 55-year-old female with massive eosinophilic myocarditis and severe, however reversible, impairment of left ventricular function. The patient presented with reduced physical condition, progressive dyspnea on exertion and peripheral edema. The white blood count revealed a leukocytosis and markedly elevated peripheral blood eosinophilics (48.8%). An endomyocardial biopsy demonstrated massive myocardial infiltration with eosinophilic granulocytes and necrosis. The symptoms and laboratory parameters indicate the presence of a hypereosinophilic syndrome. The differential diagnosis of a Churg-Strauss syndrome is discussed. Medical heart failure treatment according to international guidelines and an immunosuppressive treatment with prednisolone (Decortin H) 1.5 mg/kgBW) were initiated. This therapy led to a dramatic reduction of the eosinophilic granulocyte count and normalization of the peripheral blood count, which correlated with a significant improvement of clinical symptoms. Consistently, an increase of left-ventricular function was observed. Upon successive dose reduction to a maintenance dosage of 10 mg prednisolone, the patient's clinical status and peripheral blood count remained stable.

Radiation therapy induced modulation of wound healing at experimental vein graft anastomoses.

OBJECTIVES: The aim of this study was to investigate if radiation therapy (RT) favorably modulates wound healing at vein graft anastomoses. MATERIALS AND METHODS: Jugular vein grafts were sewn into carotid arteries in 32 rats which were randomly divided into two groups: RT (gamma source, 14 Gray, n=16) and control (C, sham irradiation, n=16). Grafts and adjacent arteries were analyzed at 2 (n=8) and 8 weeks (n=8) by histology, immunohistochemistry, and morphometry. RESULTS: Although, RT did not reduce the overall occurrence of intimal hyperplasia, the distribution differed. RT led to a reduction of intimal hyperplasia in arterial segments (median: C: 41.873 microm2; RT: 6.452 microm2, p < 0.0007). In contrast, RT augmented intimal hyperplasia in vein grafts (median: C: 30.287 microm2; RT: 90.455 microm2, p < 0.014). Vein graft diameters after RT were enlarged (median: C: 2.098 microm; RT: 3.381, p < 0.031). Over 80% of the cells were of mesenchymal origin in both groups. CONCLUSIONS: RT reduced intimal hyperplasia in arterial segments. However, RT led to graft dilatation and increased intimal hyperplasia in vein grafts. RT did not favorably modulate the vascular wound healing response in this model.

High-dose azathioprine pulse therapy as a new treatment option in patients with active Wegener's granulomatosis and lupus nephritis refractory or intolerant to cyclophosphamide.

The objective of this study was to evaluate the feasibility and safety of high-dose azathioprine pulse (HAP) therapy in the induction of remission in patients with active Wegener's granulomatosis (WG) or progressive lupus nephritis (LN) refractory to or intolerant of cyclophosphamide. Four patients with antineutrophil cytoplasmic antibody (ANCA)-associated WG and two patients with progressive LN were treated with HAP (1200-1800 mg) applied monthly as continuous intravenous infusions at 50 mg/h. Patients received a total of 50 courses of intravenous azathioprine (AZA) therapy. Disease activity was assessed using the Birmingham Vasculitis Activity Score (BVAS) and the Systemic Lupus Erythematosus Activity Index (SLEDAI). As only partial remission was induced in patients with progressive LN on this regimen, an additional 18 cycles were applied in these patients in which oral AZA at 100 mg/day in weeks 2 and 3 was added between two intravenous courses. A hereditary defect in thiopurine methyltransferase activity was excluded before initiation of treatment. High-dose azathioprine pulse and the intensified HAP treatment were well tolerated. Complete remission was achieved in two patients with WG suffering from three relapses of disease on application of 2-6 courses of HAP. Remission was maintained for 16-24 months. The remaining two patients with WG were withdrawn after 2-3 courses due to unchanged disease activity. In two patients with LN, partial remission was noted on 6-9 courses of HAP; however, the patients relapsed despite therapy with methotrexate and mycophenolate mofetil. The intensified HAP regimen led to partial or complete remission in both LN patients which was confirmed by sequential renal biopsies. Our results suggest that HAP therapy represents a well-tolerated regimen in patients with active WG and LN intolerant of or refractory to cyclophosphamide. As partial or complete remission was observed in four of six patients, further studies seem warranted to assess clinical efficacy in these patients.

N-acetyl cysteine blocks mesangial VCAM-1 and NF-kappa B expression in vivo.

Inducible vascular cell adhesion molecule-1 (VCAM-1) in glomerular mesangial cells (GMC) exposed to lipopolysaccharide (LPS) in vitro involves the activation of nuclear factor-kappa B (NF-kappa B) and its interaction with the proximal VCAM-1 promoter. We used a murine model to assess the effect of the antioxidant, N-acetyl cysteine on GMC activation in vivo. Single intraperitoneal administration of N-acetyl cysteine completely suppressed LPS-induced VCAM-1 expression on the GMC surface. When an oligonucleotide spanning the NF-kappa B binding region of the VCAM-1 promoter was incubated with extracts from the renal cortex of LPS-treated animals, a single nucleoprotein complex formed. This complex was composed of p50 and p65, but not p52, c-Rel, or RelB, and its formation was dramatically inhibited by pretreatment with N-acetyl cysteine, D,L-Buthionine-[S,R]-sulfoximide, a compound that depletes glutathione, augmented VCAM-1 expression inducible with a suboptimal amount of LPS to levels comparable with using 50 micrograms of LPS alone, D,L-Buthionine-[S,R]-sulfoximide also potentiated the p50-p65 binding activity induced with a suboptimal amount of LPS. These data provide a redox-sensitive, transcriptional link between NF-kappa B and VCAM-1 in GMC in vivo and implicate oxidative stress as an important regulatory signal in the pathogenesis of glomerular mesangial cell disorders.

Chondrodysplasia and neurological abnormalities in ATF-2-deficient mice.

Activating transcription factor-2 (ATF-2) is a basic region leucine zipper protein whose DNA target sequence is the widely distributed cAMP response element (CRE). We report here that mice carrying a germline mutation in ATF-2 demonstrated unique actions of ATF-2 not duplicated by other ATF/CREB family members. Mutant mice had decreased postnatal viability and growth, with a defect in endochondral ossification at epiphyseal plates similar to human hypochondroplasia. The animals had ataxic gait, hyperactivity and decreased hearing. In the brain, there were reduced numbers of cerebellar Purkinje cells, atrophic vestibular sense organs and enlarged ventricles. Unlike CREB alpha/delta-deficient mice whose main defect is in long-term potentiation, the widespread abnormalities in ATF-2 mutant mice demonstrate its absolute requirement for skeletal and central nervous system development, and for maximal induction of select genes with CRE sites, such as E-selectin.

Nuclear factor-kappa B mediates induction of vascular cell adhesion molecule-1 in glomerular mesangial cells.

Cultured glomerular mesangial cells (GMCs) can be activated at the transcriptional level by a variety of physiologically relevant factors including cytokines, endotoxin and glycosylated end products. The mechanism with which the signal is transduced from the membrane to the nucleus of these cells is largely unclear. In vascular endothelial cells, the signal transduction pathway involves activation of the pleuripotent transcription factor, NF-kappa B, and leads to increased expression of a variety of genes including vascular cell adhesion molecule-1 (VCAM-1). Here, we demonstrate that TNF-alpha and IL-1 beta transiently induced VCAM-1 mRNA expression in a time dependent manner. TNF-alpha also induced the specific interaction of proteins from GMC nuclei with an oligonucleotide bearing the NF-kappa B binding sites in the VCAM-1 promoter. Electrophoretic mobility shift and supershift analysis indicated that the p65 subunit of NF-kappa B is a component of this induced complex. Finally, reporter activity driven by a VCAM-1 promoter-chloramphenicol acetyltransferase reporter construct increased 8-10 fold following TNF-alpha incubation, or p65 cotransfection. Thus, the p65 subunit of NF-kappa B is activated in GMCs exposed to cytokine and can mediate induction of gene expression.

Novel cis-acting elements in the human platelet-derived growth factor B-chain core promoter that mediate gene expression in cultured vascular endothelial cells.

Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant constitutively expressed by a variety of normal and transformed cells. Transient transfection and deletion analysis of the human c-sis proto-oncogene in cultured vascular endothelial cells revealed a minimal core promoter region extending 82 base pairs upstream from the TATA box. Two novel and functional cis-acting elements were identified within the core that share considerable sequence homology with consensus binding elements for transacting factors of the ETS class and those involved in AP-1 complexes. Deletion or mutation of either the ETS-like site or the AP-1-like site resulted in significant attenuation in the ability of the core to drive transcription. Electrophoretic mobility shift assays revealed that proteins from bovine aortic and human umbilical vein endothelial nuclear extracts bound to these elements in a specific manner and that both sites were essential for protein binding. Ferguson analysis predicted a combined molecular mass of 153 kDa for these proteins. In addition, transient transfection, gel shift, and DNase I footprint analysis were used to identify a functional Sp1 binding site downstream of these elements in the core promoter. By localizing the functional cis-acting elements in the PDGF-B promoter, it may be possible to elucidate the normal transcriptional control of the gene, as well as the mechanisms that activate it in pathologic settings.

Expression of VCAM-1 and E-selectin in an in vivo model of endothelial activation.

Vascular cell adhesion molecule 1 (VCAM-1) and E-selectin (or endothelial-leukocyte adhesion molecule 1) are inducible endothelial cell adhesion molecules that play a role in the recruitment of leukocytes into sites of inflammation. Information about the spatial and temporal pattern of induced expression of these leukocyte adhesion molecules in vivo is limited. This study reports the expression profile of VCAM-1 and E-selectin in various mouse tissues after lipopolysaccharide administration. Using rat complementary DNA probes for VCAM-1 and E-selectin, Northern blot analysis showed a marked increase in transcript levels for both adhesion molecules in lung, heart, and kidney. Maximal transcript levels for both VCAM-1 and E-selectin were observed at 3-6 hours and declined to low, constitutive levels of expression at 48 hours. Consistent with the Northern blot results, immunoperoxidase analysis revealed focal endothelial cell expression of VCAM-1 in control animals. Following lipopolysaccharide administration, VCAM-1 expression increased dramatically in all vascular beds examined, although the response was heterogeneous. Widespread induced expression of VCAM-1 on cells other than vascular endothelium was not seen. Neither basal nor induced expression correlated with leukocyte adhesion. Signals other than the expression of endothelial leukocyte adhesion molecules are required in vivo for leukocyte infiltration in this murine model of systemic endothelial activation.

Nucleotide sequence of rat vascular cell adhesion molecule-1 cDNA.

Vascular cell adhesion molecule 1 (VCAM-1) is an inducible transmembrane protein which is expressed by vascular endothelium following cytokine activation. VCAM-1 mediated the adhesion of certain blood leukocytes and tumor cells via the interaction with its counter-receptor, the integrin VLA4. When initially cloned from interleukin-1 (IL-1) stimulated human umbilical vein endothelial cells, VCAM-1 was reported to contain six immunoglobulin-like domains. However, subsequent cDNA clones and structural analysis of the human gene evealed an alternatively spliced seventh immunoglobulin domain. This seven domain form appears to be the predominant transcript in IL-1 activated endothelium. In this report, the cloning and nucleotide sequence of rat VCAM-1 is described.

Gene structure, chromosomal location, and basis for alternative mRNA splicing of the human VCAM1 gene.

Vascular cell adhesion molecule 1 (VCAM-1) is a cell surface glycoprotein adhesive for certain blood leukocytes and tumor cells, which is expressed by activated endothelium in a variety of pathologic conditions including atherosclerosis. Genomic clones encoding the VCAM1 gene were isolated and the organization of the gene was determined. The gene, which is present in a single copy in the human genome, contains 9 exons spanning approximately 25 kilobases of DNA. Exons 2-8 contain C2 or H-type immunoglobulin domains. At least two different VCAM-1 precursors can be generated from the human gene as a result of alternative mRNA splicing events, which include or exclude exon 5. A consensus TATAA element is located upstream of the transcriptional start site. The VCAM1 promoter contains consensus binding sites for NF-kappa B, the GATA family of transcription factors, as well as an AP1 site. The VCAM1 gene was assigned to the 1p31-32 region of chromosome 1 based on the analysis of human-mouse hybrid cell lines and in situ hybridization. Structural analysis of the human VCAM1 gene provides the basis for alternative mRNA splicing and an initial approach to elucidating the regulation of VCAM-1 expression.