Hyaluronan oligosaccharide treatment of chondrocytes stimulates expression of both HAS-2 and MMP-3, but by different signaling pathways.

OBJECTIVE: Small hyaluronan (HA) oligosaccharides displace HA from the cell surface and induce cell signaling events. In articular chondrocytes this cell signaling is mediated by the HA receptor CD44 and includes stimulation of genes involved in matrix degradation such as matrix metalloproteinases (MMPs) as well as matrix repair genes including collagen type II, aggrecan and HA synthase-2 (HAS-2). The objective of this study was to determine whether stimulation of HAS-2 and MMP-3 by HA oligosaccharides is due to the activation of a single, cascading pathway or multiple signaling pathways. METHOD: Bovine articular chondrocytes were pre-treated with a variety of inhibitors of major signaling pathways prior to the addition of HA oligosaccharides. Changes in HA were monitored by real time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of HAS-2 mRNA, HA ELISA and HA accumulation at the cell surface. A 1900 base pair sequence containing the proximal promoter of HAS-2 was inserted into a luciferase reporter construct, transfected into human immortalized chondrocytes and assayed in a similar fashion. RESULTS: While our previous studies demonstrated that HA oligosaccharides stimulate MMP-13 activity via activation of p38 MAP kinase and NF-kappaB, inhibitors of these pathways did not affect the stimulation of HAS-2 mRNA expression. However, inhibiting the phosphatidylinositol-3-kinase pathway blocked HA oligosaccharide-mediated stimulation of HAS-2 yet had no effect on MMP-3. Wortmannin and LY294002 also blocked HA oligosaccharide-induced serine and threonine Akt phosphorylation. Treatment of transfected immortalized chondrocytes with HA oligosaccharides resulted in stimulation of HAS-2 mRNA, activation of Akt and enhanced luciferase activity-activity that was blocked by inhibitors of Akt phosphorylation. CONCLUSIONS: Changes in chondrocyte-matrix interactions by HA oligosaccharides induce altered matrix metabolism by the activation of least two distinct signaling pathways.

Mutational analyses of c-FLIPR, the only murine short FLIP isoform, reveal requirements for DISC recruitment.

Cellular FLICE-inhibitory protein (c-FLIP) proteins are known as potent inhibitors of death receptor-mediated apoptosis by interfering with caspase-8 activation at the death-inducing signaling complex (DISC). Among the three human isoforms, c-FLIP(long), c-FLIP(short) and c-FLIP(R), the latter isoform is poorly characterized. We report here the characterization of murine c-FLIP(R) and show that it is the only short c-FLIP isoform expressed in mice. By generating several mutants, we demonstrate that both death effector domains (DEDs) are required for DISC binding and the antiapoptotic function of c-FLIP(R). Surprisingly, the C-terminal tail is important for both protein stability and DISC recruitment. Three-dimensional modeling of c-FLIP(R) revealed a substantial similarity of the overall structures and potential interaction motifs with the viral FLIP MC159. We found, however, that c-FLIP(R) uses different structural motifs for its DISC recruitment. Whereas MC159 interferes with interaction and self-oligomerization of the DISC component FADD by its extensive hydrophilic surface, a narrow hydrophobic patch of c-FLIP(R) on the surface of DED2 is crucial for DISC association. Thus, despite the presence of similar tandem DEDs, viral and cellular FLIPs inhibit apoptosis by remarkably divergent mechanisms.

CD95 ligand mediates T-cell receptor-induced apoptosis of a CD4+ CD8+ double positive thymic lymphoma.

Tumors in the thymus can be of different cellular origin. Among the most common tumors are thymoma and lymphoma, which are derived from transformed thymic epithelial cells and transformed lymphocytes, respectively. Thymic lymphoma and their response to apoptotic stimuli are poorly characterized. Here, we analyse apoptosis events in the thymic lymphoma cell line Thy278, which expresses cell surface antigens characteristic of immature double positive thymocytes. Upon T-cell receptor (TCR)/CD3 stimulation, Thy278 cells die by apoptosis, similar as primary thymocytes during negative selection. Caspases are crucial for deletion of both Thy278 cells and normal thymocytes. Moreover, we show that deletion of primary thymocytes and Thy278 cells upon CD3 stimulation is considerably impaired by neutralizing CD95L antibody. Thus, our results not only demonstrate that TCR-induced apoptosis is still functional in transformed thymocytes, but also suggest that Thy278 cells are a helpful model for the molecular analysis of negative selection.

The role of CAP3 in CD95 signaling: new insights into the mechanism of procaspase-8 activation.

Formation of the CD95 (APO-1/Fas) death inducing signaling complex (DISC) plays a central role in CD95 signaling. Previously, CD95 DISC composition was analyzed by two-dimensional gel electrophoresis and four major cytotoxicity-associated proteins (CAP1-4) were found. CAP1 and CAP2 were defined to be unmodified and phosphorylated FADD, respectively. CAP4 was identified as procaspase-8a. CAP3, however, has remained elusive. In this study, we demonstrate that CAP3 is an intermediate of procaspase-8 processing. CAP3 is generated within seconds of DISC formation and subsequently processed to the prodomain of procaspase-8a that is known as p26 (CAP5). These findings lead to new insights into the mechanism of procaspase-8 processing and apoptosis initiation.

Analysis of biodegradation of copolymer dermis substitutes in the dorsal skinfold chamber of balb/c mice.

PEGT/PBT-block-copolymer dermis substitutes were inserted into dorsal skinfold chambers of balb/c mice (n=36). Scaffolding matrices with 3 different pore diameters (pore diameter: <75 micro m, 75-212 micro m and 250-300 micro m) were analyzed on days 7, 14, and 21 post implantation by scanning electron and light microscopy. The quantification of matrix fragmentation was performed using image-analytical software analySIS(R). The fragmentation rate in scaffolding matrices with a pore size of < 75 micro m was observed to be higher than in matrices of larger pore sizes. Image-analytical evaluation over 21 days revealed a reduction of the copolymer matrix by approximately 32% for the <75 micro m matrices, 23% for the 75-212 micro m matrices and 18% for the matrices, where pore size ranged between 250 micro m and 300 micro m. Twenty-one days after implantation, the matrix pores of 75-212 micro m and 250-300 micro m scaffolds were totally filled by vascularized fibrous tissue. Contrarily, an increased formation of foreign-body giant cells was observed in matrices with pore size <75 micro m. The pore size of the scaffolding PEGT/PBT dermis substitutes affects their degradative behaviour in vivo.

Induction of apoptosis in 9-nitrocamptothecin-treated DU145 human prostate carcinoma cells correlates with de novo synthesis of CD95 and CD95 ligand and down-regulation of c-FLIP(short).

Stimulation of CD95 leads to oligomerization of this receptor and the recruitment of the Fas-associated death domain (FADD) and procaspase-8 to form the death-inducing signaling complex (DISC). Subsequent proteolytic activation of caspase-8 at the DISC leads to the activation of downstream caspases and execution of apoptosis. The anticancer drug 9-nitrocamptothecin (9NC) inhibits the nuclear enzyme topoisomerase I (Top1), an event followed by apoptosis of cancer cells. We investigated whether other mechanisms downstream of the DNA-Top1-9NC complexing step regulate the apoptotic ability of 9NC in DU145 cells. We demonstrate that induction of apoptosis in DU145 cells, upon exposure to 9NC, is associated with de novo expression of CD95 and CD95L, suggesting that 9NC-induced apoptosis is mediated by the CD95 system. In this line, we observed early activation of procaspase-3, -7, and -8, but not -1, -9, and -10. Moreover, 9NC treatment resulted in the dramatic down-regulation of c-FLIP(short) expression, but not that of c-FLIP(long) or FADD. Furthermore, incubation of DU145 cells with a neutralizing antibody (NOK-1) to CD95L or transient transfection of a c-FLIP(short) expression vector into DU145 cells partially abrogated 9NC-triggered apoptosis. We propose that 9NC triggers apoptosis by driving DU145 cells from a nonapoptotic status (c-FLIP(short)(high), CD95(low), CD95L(low)) toward a proapoptotic status (c-FLIP(short)(low), CD95(high), CD95L(high)). These findings indicate that in addition to a Top1-mediated effect, 9NC can additionally activate a CD95/CD95L-dependent apoptotic pathway.

Regulation of death receptor-mediated apoptosis pathways.

Apoptosis or programmed cell death can be induced by a variety of stimuli including activation of death receptors. This subgroup of the TNF/NGF-receptor-superfamily activates caspases, a family of aspartyl-specific cysteine-proteases, which are the main executioners of apoptosis. Depending on the cell type, signalling pathways downstream of the death receptors can be modulated by different proteins such as Bcl-2, FLIPs, chaperones and kinases. Deregulation of apoptosis has been associated with diseases as cancer, autoimmunity and AIDS. Therefore, the identification of modulators of apoptosis has several therapeutic implications.

Studies on tumor-cell-induced platelet aggregation in human lung cancer cell lines.

We investigated the ability of human lung cancer cells of different histological subtypes to cause platelet aggregation. Tumor-cell-induced platelet aggregation (TCIPA) was studied in vitro in 13 human lung cancer cell lines [small-cell lung cancer (SCLC), squamous-cell lung cancer, large-cell lung cancer, adenocarcinoma and alveolar-cell lung cancer]. Three tumor cell lines failed to aggregate platelets in platelet-rich plasma, whereas platelet aggregation was induced by 12 cell lines when added to washed platelets and minimal amounts of platelet-poor plasma (0.5% v/v). The thrombin antagonist hirudin inhibited TCIPA in non-small-cell lung cancer cell lines (NSCLC). In SCLC, TCIPA was fully abolished only when the ADP scavenger apyrase was added to hirudin. Thus ADP and thrombin generation by these tumor cell lines are responsible for platelet aggregation. The ability to activate platelets independently of coagulation factors VII and X was demonstrated for 8 cell lines. Electron-microscopically, direct tumor-cell/platelet contact was found to be the initiating mechanism of TCIPA in SCLC, whereas tumor-cell/platelet contacts in NSCLC could only be observed at the peak of the aggregation curve. Lung cancer cells activate platelets in vitro by generation of thrombin and/or ADP.

A comparative study of clinically well-characterized human atherosclerotic plaques with histological, chemical, and ultrastructural methods.

Atherosclerotic plaques (six cases) with well-documented clinical history were analysed using histology, scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray (EDX) spectroscopy, infrared spectroscopy (IR), thermogravimetry (TG), and high-resolution synchrotron X-ray diffraction. All samples contained about 60-70 wt% biological carbonated apatite (in dry state) in a nanocrystalline form with particle sizes of about 20 nm. Structurally, there are strong similarities to bone mineral. Ultrastructural investigations documented typical calcospherites, mineralisation processes starting at collagen fibrils and ring-shaped crystalline mineralised structures. There were no significant ultrastructural or chemical differences between the calcifications of individual patients.

Introduction of new crosslinks into proteins.

Analysis of the crosslinks epsilon-(gamma-glutamyl) lysine and epsilon- (beta-aspartyl) lysine present in treated wool has been improved by modifying the enzymic digestion. Treatment of wool with either monocarboxylic acid chlorides in dimethylsulfoxide or with 1-fluoro-2,4-dinitrobenzene in the presence of acetate considerably decreased epsilon-amino groups and solubility. Since no information of interchain amide crosslinks was observed, the hypothesis of so-called self-crosslinking postulated by ZAHN has to be withdrawn. The effects of both treatments are explained in the light of new results. The reaction of wool with glutaraldehyde leads to a stabilization of the fiber. Experiments with glutaraldehyde and primary alkyl amines as model componds revealed that the cyclic form of the aldehyde gave the unstable N-alkyl-2,6-dihydroxypiperidine, which either looses water to give N-alkyldihydropyridine or condenses with 2,6-dihydroxytetrahydropyran to yield a copolyether which was isolated. According to recent publications, crosslinking of proteins by glutaraldehyde is due to the formation of quaternary pyridinium compounds.

Multiresidue determination of pesticides in drinking and related waters by gas chromatography/mass spectrometry after solid-phase extraction: interlaboratory study.

As part of a project funded by the European Commission (EC) for the development and evaluation of multiresidue methods for analysis of drinking and related waters, 15 European laboratories evaluated a method using styrene-divinylbenzene co-polymer solid-phase extraction followed by gas chromatography/mass spectrometry. The main aim of the study was to evaluate whether the method meets the requirements of EC Directive 98/83 in terms of accuracy, precision, and detection limit for 22 pesticides according to the following requirements: limit of detection, < or = 0.025 microg/L; accuracy, expressed as recovery between 75 and 125%; and precision, expressed as repeatability relative standard deviation of the method of < 12.5% and as reproducibility relative standard deviation of the method of < 25%. Analyses for unknown concentrations were performed with fortified commercial bottled and tap waters. All laboratories were able to achieve detection limits of 0.01 microg/L for all pesticides except dimethoate and desisopropylatrazine (0.02 microg/L). The criteria for repeatability were met for all compounds except trifluralin, dimethoate, and lindane in bottled water and chlorpyrifos, dimethoate, and lindane in tap water. The criteria for reproducibility were met for all compounds except trifluralin, dimethoate, and lindane in bottled water and pendimethalin, chlorpyrifos, dimethoate, terbutryn, and lindane in tap water. In terms of accuracy, the method meets the requirements for all pesticides in both matrixes, except for lindane in bottled water and lindane and chlorpyrifos in tap water.

The influence of sulfate-reducing bacteria colonization of 2 different bioresorbable barrier membranes for GTR. An 18-month case-controlled microbiologic and clinical study.

The purpose of the present microbiologic and case-controlled clinical study was to examine the colonization of 2 different resorbable barrier membranes by sulfate-reducing bacteria (SRB). The barrier membranes tested were Guidor matrix barrier and Resolut regenerative material. Ten patients exhibiting 3 Class II furcation defects and 7 intrabony defects were included in the study. The probing depth and the clinical attachment level at 4 surfaces per tooth were taken at the beginning of the study. Microbiologic samples were taken from the experimental sites and from the approximal sites of the adjacent teeth. Both types of resorbable membranes were positive for SRB colonization. The detection of SRB in 2 of 7 intrabony defects and in all defects with furcation involvement before the membrane placement indicated that these organisms are a common inhabitant of sites showing periodontal destruction and are associated with guided tissue regeneration (GTR). According to the clinical criteria for healing tendencies used in this study, the GTR procedures were less successful in the presence of SRB. There were no significant clinical effects of different resorbable membrane materials or membrane layout on attachment level changes for either the intrabony defect or furcation groups after 18 months. There were no statistical differences for sites that became exposed to SRB when compared to sites that remained unexposed after 18 months. The numeric significance of SRB in relation to the total microbial count needs to be determined to gain insight into the ecologic role of membrane resorption rates.

Long-term stent implantation in a case of relapsing intrathoracic Ewing-sarcoma.

The overall prognosis of relapsing Ewing sarcoma is poor and therapeutic options can be limited by extensive chemotherapeutic pretreatment. We report on a case of a 27-year-old male, presenting with a large mediastinal mass and malignant pleural effusions. 5 years prior peripheral Ewing sarcoma had been treated according to the CESS 86 protocol. Relapse chemotherapy was initiated (CESS protocol) but tumor progression led to stenoses of both main bronchi. At this critical point, 2 Strecker tantal stents were placed endoscopically to prevent suffocation and provide the time for further chemotherapy, regardless of the poor overall prognosis. Complete remission was achieved by high-dose ifosfamide, surgery, radiotherapy and adjuvant ifosfamide. In spite of possible complications of long-term stent implantation, the stents were not removed until 4 years later when stent dislocation occurred. After removal, the stents were epithelialized and electron microscopy demonstrated structural integrity of the stent. The patient has remained in complete remission since (6 years).

[Ultrastructural findings in pleural cysts in spontaneous pneumothorax]. / Ultrastrukturelle Befunde pleuraler Cysten beim Spontanpneumothorax.

Lung biopsies obtained by conventional thoracotomy from five patients (mean age 33.6 years, range 21-45) following recurrence of spontaneous pneumothorax were studied by light, scanning, and transmission electron microscopy. The purpose of the study was to define abnormalities that predispose to air penetration through the intact wall of pleural blebs. Two types of blebs were identified either with a complete or with an incomplete layer of mesothelial cells. In those areas where the mesothelial cells were lacking, the walls only consisted of irregular, discontinuous collagen fibers. Increasing intraalveolar pressure may distort the net of collagen fibers and air penetration appears to be possible. The wall of pleural blebs is generally weakened by degenerative changes and may predispose to recurrence of pneumothorax. Therefore, wedge resection of the blebs including the underlying lung parenchyma is suggested for surgical therapy of pneumothorax.

Chronic bronchitis--alterations of the bronchial mucosa.

The mucociliary transport system is usually an important defense system which protects the body against a variety of noxious agents. Reactions of the bronchial mucosa to chronic infections are seen in the ciliated cells, the amount of globlet cells, in modifications of the basement membrane underlying the bronchial epithelium and an altered percentage of inflammatory cells. In ciliated cells the following atypia can be seen: thickening of the ciliar membrane, swollen cilia, formation of compound cilia, disarrangement of microtubules. Common alterations of the basement membrane are: increased diameter of the basement membrane zone, inhomogeneous staining pattern of the basement membrane zone, formation of cytoplasmatic protrusions, formation of double layers of the basement membrane and increased number of cytoplasmatic bound vesicles. Structural abnormalities of the basement membrane will lead to disturbances of the zone of transition and have to be interpreted as a sign of disregulation in the process of diffusion and resorption. The inflammatory response of the epithelium during chronic bronchitis and asthma shows many similarities. The bronchial epithelium has a specific reaction pattern which supports the response against different noxious agents. So all findings have to be interpreted as unspecific pathological changes. All alterations may show different degrees of severity and are dependent on individual pattern and the severity of chronic process. Electronmicroscopical examinations in combination with lightmicroscopical findings and immuno-histochemistry and seen in context with clinical data help to understand the mechanism of the inflammatory process.

Constitutive expression of murine c-FLIPR causes autoimmunity in aged mice.

Death receptor-mediated apoptosis is a key mechanism for the control of immune responses and dysregulation of this pathway may lead to autoimmunity. Cellular FLICE-inhibitory proteins (c-FLIPs) are known as inhibitors of death receptor-mediated apoptosis. The only short murine c-FLIP splice variant is c-FLIPRaji (c-FLIPR). To investigate the functional role of c-FLIPR in the immune system, we used the vavFLIPR mouse model constitutively expressing murine c-FLIPR in all hematopoietic compartments. Lymphocytes from these mice are protected against CD95-mediated apoptosis and activation-induced cell death. Young vavFLIPR mice display normal lymphocyte compartments, but the lymphocyte populations alter with age. We identified reduced levels of T cells and slightly higher levels of B cells in 1-year-old vavFLIPR mice compared with wild-type (WT) littermates. Moreover, both B and T cells from aged vavFLIPR animals show activated phenotypes. Sera from 1-year-old WT and transgenic animals were analysed for anti-nuclear antibodies. Notably, elevated titres of these autoantibodies were detected in vavFLIPR sera. Furthermore, tissue damage in kidneys and lungs from aged vavFLIPR animals was observed, indicating that vavFLIPR mice develop a systemic lupus erythematosus-like phenotype with age. Taken together, these data suggest that c-FLIPR is an important modulator of apoptosis and enforced expression leads to autoimmunity.

Phosphorylation of Atg5 by the Gadd45ß-MEKK4-p38 pathway inhibits autophagy.

Autophagy is a lysosomal degradation pathway important for cellular homeostasis, mammalian development, cancer and immunity. Many molecular components of autophagy have been identified, but little is known about regulatory mechanisms controlling their effector functions. Here, we show that, in contrast to other p38 MAP kinase activators, the growth arrest and DNA damage 45 beta (Gadd45ß)-MAPK/ERK kinase kinase 4 (MEKK4) pathway specifically directs p38 to autophagosomes. This process results in an accumulation of autophagosomes through p38-mediated inhibition of lysosome fusion. Conversely, autophagic flux is increased in p38-deficient fibroblasts and Gadd45ß-deficient cells. We further identified the underlying mechanism and demonstrate that phosphorylation of the autophagy regulator autophagy-related (Atg)5 at threonine 75 through p38 is responsible for inhibition of starvation-induced autophagy. Thus, we show for the first time that Atg5 activity is controlled by phosphorylation and, moreover, that the spatial regulation of p38 by Gadd45ß/MEKK4 negatively regulates the autophagic process.

The role of c-FLIP splice variants in urothelial tumours.

Deregulation of apoptosis is common in cancer and is often caused by overexpression of anti-apoptotic proteins in tumour cells. One important regulator of apoptosis is the cellular FLICE-inhibitory protein (c-FLIP), which is overexpressed, for example, in melanoma and Hodgkin's lymphoma cells. Here, we addressed the question whether deregulated c-FLIP expression in urothelial carcinoma impinges on the ability of death ligands to induce apoptosis. In particular, we investigated the role of the c-FLIP splice variants c-FLIP(long) (c-FLIP(L)) and c-FLIP(short) (c-FLIP(S)), which can have opposing functions. We observed diminished expression of the c-FLIP(L) isoform in urothelial carcinoma tissues as well as in established carcinoma cell lines compared with normal urothelial tissues and cells, whereas c-FLIP(S) was unchanged. Overexpression and RNA interference studies in urothelial cell lines nevertheless demonstrated that c-FLIP remained a crucial factor conferring resistance towards induction of apoptosis by death ligands CD95L and TRAIL. Isoform-specific RNA interference showed c-FLIP(L) to be of particular importance. Thus, urothelial carcinoma cells appear to fine-tune c-FLIP expression to a level sufficient for protection against activation of apoptosis by the extrinsic pathway. Therefore, targeting c-FLIP, and especially the c-FLIP(L) isoform, may facilitate apoptosis-based therapies of bladder cancer in otherwise resistant tumours.