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Three-dimensional arrangement and subsequent transplantation of chondrocytic cells in resorbable polymers has been shown to be a promising technique for the treatment of cartilaginous defects. Engineering of artificial cartilage tissue includes dedifferentiation of chondrocytes in monolayer culture, the use of biodegradable matrices and polymer scaffolds, and re-expression of chondrocytic marker genes in three-dimensional culture. The aim of this study was to characterize molecularly the phenotypic changes occurring with autologous cartilage tissue engineering. Human articular chondrocytes were isolated, cultured in medium containing human serum, and expanded up to passage 3. Chondrocytes were embedded in human fibrinogen and in polyglactin-polydioxanon fleeces and cultured three-dimensionally up to 4 weeks. Dedifferentiation of chondrocytes in monolayers and formation of cartilage tissue in vitro or after subcutaneous transplantation into nude mice was assessed by gene expression analysis of typical chondrocytic genes, histology, and immunohistochemistry. The expansion of chondrocytes with human serum resulted in the induction of type I and type III collagens, whereas cartilage-specific type II collagen, cartilage oligomeric matrix protein, cartilage link protein, and aggrecan were repressed and induced again after three-dimensional arrangement of chondrocytes in polyglactin-polydioxanon. Transplantation experiments documented the synthesis of proteoglycan and cartilage-specific type II collagen in vivo. Three-dimensional arrangement of human articular chondrocytes in resorbable polyglactin-polydioxanon fleeces supports chondrogenic differentiation and the formation of a hyaline-like cartilaginous matrix in vitro and in vivo.

Two new technologies (tissue microarrays (TMAs) and proteomics) have generated a great amount of data in life science. High-density TMAs allow for the simultaneous analysis of proteins and RNA by various methods (immunohistochemistry, in situ hybridization, FISH) on a large scale and under highly standardized conditions. Proteomics includes a variety of techniques that are partly high throughput. These techniques aim at the innovation of proteins, the description of the domain structure, the determination of protein sequences and epitope characterization, and ultimately the definition of protein function and protein reactivities in immunologic processes. Proteins that have been characterized accordingly require validation mostly at the morphologic level of defined tissue, linking proteomics to TMAs. In autoimmune diseases, array-based antigenic fingerprinting of autoantibodies will drive the development and the selection of antigen-specific diagnostic tools and therapies. The powerful combination of genomics and proteomics formed in tissue arrays has the potential to change the way the biology of autoimmunity is studied. Novel targets of drug discovery, based on antigen-specific therapies to induce anergy, or regulatory T-cells using the targeted autoantigens of individual patients could be developed in the coming decades.

Vasodilator-stimulated phosphoprotein (VASP) and mammalian enabled (Mena) are actin cytoskeleton and signaling modulators. Ena-VASP proteins share an identical domain organization with an NH2-terminal Ena VASP homology (EVH1) domain, which mediates the binding of these proteins to FPPPP-motif containing partners such as zyxin and vinculin. VASP and Mena are abundantly expressed in the heart. However, previous studies showed that disruption by gene targeting of VASP or Mena genes in mice did not reveal any cardiac phenotype, whereas mice lacking both VASP and Mena died during embryonic development. To determine the in vivo function of Ena-VASP proteins in the heart, we used a dominant negative strategy with cardiac-specific expression of the VASP-EVH1 domain. Transgenic mice with cardiac myocyte-restricted, alpha-myosin heavy chain promoter-directed expression of the VASP-EVH1 domain were generated. Overexpression of the EVH1 domain resulted in specific displacement of both VASP and Mena from cardiac intercalated disks. VASP-EVH1 transgenic mice developed dilated cardiomyopathy with myocyte hypertrophy and bradycardia, which resulted in early postnatal lethality in mice with high levels of transgene expression. The results demonstrate that Ena-VASP proteins may play an important role in intercalated disk function at the interface between cardiac myocytes.

Background: STR/ort mice spontaneously develop degenerative changes of the knee joints resembling human osteoarthritis (OA), with the males being more severely affected than the females., Objective: To analyse the early changes leading to OA by examining the articular cytokine expression and degenerative changes in STR/ort mice., Methods: 122 STR/ort mice of both sexes aged between 2 and 15.5 months were included. Thin sections of the knees were analysed for osteoarthritic changes by haematoxylin/eosin staining. The articular cytokine expression was investigated by immunohistochemical staining using monoclonal antibodies specific for interleukin (IL)6, tumour necrosis factor alpha, transforming growth factor beta1 (TGFbeta1), IL1beta, IL4, and IL10, respectively., Results: Both cartilage degeneration and articular cytokine expression differ between the sexes. The protection from cartilage degeneration in the female mice correlates with an increased expression of TGFbeta1 and IL4 at 2 months of age., Conclusion: The increased expression of TGFbeta1 and IL4 in young STR/ort female mice suggests that the sexual dimorphism is mediated through the articular expression of cytokines involved in cartilage metabolism.

Ziehl-Neelsen (ZN) staining is the key technique for diagnosis of mycobacterial infections; however, a high percentage of patients exhibit positive signs of tuberculosis, as indicated by pathology, culture of mycobacteria, and polymerase chain-reaction analysis, and yet show negative results on ZN staining. In this report we present evidence that such ZN-negative specimens represent Mycobacterium tuberculosis bacilli in a dormant state with distinct cell-wall alterations: the classical cell-wall composition-dependent ZN staining of M. tuberculosis in lung sections gradually discontinued with persistence of infection, both in mice and in human patients; in contrast, detection of mycobacteria by cell-wall composition-independent staining using a polyclonal anti-M. bovis Bacille-Calmette-Guérin serum continued with persistence of infection. These findings have important implications for diagnosis, as well as for both chemotherapy and development of vaccine strategies.'

About 10% of hip endoprostheses will loosen after 10 years. Prosthesis loosening is caused by two different pathomechanisms: aseptic loosening (AL) and septic loosening (SL). This study evaluated differences in gene expression in AL and SL. Eight hybridizations were performed on PIQOR cDNA arrays. Objects of the study were periprosthetic interface tissue samples from two patients with SL and three patients with AL. Tissue parts directly adjacent to the site of RNA isolation were analyzed immuno/histopathologically in order to overcome the problem of tissue heterogeneity. Thirty-three genes were found constantly differentially expressed, among which were cd11b, cd18, cd68, osteopontin and ferritin heavy-chain upregulated in AL and collagen types 1alpha-1, 3alpha-1, integrin alpha-1, thrombospondin2 and nidogen upregulated in SL. The most striking finding was the strong upregulation (from 20-fold to 323-fold) of megakaryocyte stimulating factor (msf) in all aseptic cases and one of the two septic cases, which was confirmed by real-time reverse transcription-polymerase chain reaction. In this study, msf is linked to prosthesis loosening for the first time. The upregulation in AL suggests an important pathogenetic role: the msf splice product lubricin is responsible for the lubrication of healthy joints, but its excellent lubrication ability may disturb the tight interaction between bone and prosthesis and thereby contribute to prosthesis loosening.

Background: Metastatic gastric cancer is usually treated with cisplatin- and 5-FU-based chemotherapy regimens. There are good data for the combination regimen ECF (epirubicin, cisplatin and 5-FU), which is therefore often regarded as a reference treatment. Docetaxel shows promising activity against gastric cancer as single agent and in combinations. To develop a well-tolerable combination chemotherapy for an ambulant setting we initiated a randomized phase II study, comparing docetaxel and 5-FU continuous infusion (DF) with ECF., Case Report: A 66-year-old patient with the history of a curatively resected gastric cancer 2 years previously presented with abdominal masses and lesions in his spleen. Histology proved metastases of gastric adenocarcinoma. The patient was treated with docetaxel (75 mg/m2, d1) and 5-FU continuous infusion (200 mg/m2/d, d1-21, q3w) within our study. Already after 2 cycles of chemotherapy he showed symptomatic improvement and partial remission of his tumor, which was confirmed after the 3rd cycle. In our ongoing study so far 50 patients are evaluable for response. Objective tumor response (CR + PR) could be documented in 44% of patients in the DF arm as well as in the ECF arm., Conclusion: Docetaxel and 5-FU continuous infusion is an active regimen which could possibly be used as an alternative to established treatment protocols., (Copyright 2003 S. Karger GmbH, Freiburg)

Objective: The pathogenesis of periprosthetic bone loss in aseptic and septic prosthesis loosening is unclear. There is considerable evidence that macrophages and osteoclasts play a key role in focal bone erosion and osteolysis around the prosthesis. RANKL (receptor activator of nuclear factor kappaB ligand) was shown to be a potent osteoclastogenic factor, and to be involved in bone destruction of myeloma and rheumatoid arthritis patients. Osteoprotegerin (OPG) is the natural RANKL inhibitor and may prevent periprosthetic bone loss., Methods: The presence and distribution of RANKL, its receptor RANK and OPG in the periprosthetic interface of septically (n = 5) and aseptically (n = 6) loosened prostheses was examined by immunohistochemistry and immunoblotting. Additionally, the immunophenotype of the inflammatory infiltrate was determined [CD3, CD68, Ki-67, tartrate-resistant acid posphatase (TRAP)]., Results: Aseptic and septic cases revealed a different histopathologic pattern. However, in all cases RANKL and RANK could be demonstrated in macrophages and giant cells. In addition, RANKL detected by immunoblot analysis proved to have the same molecular weight as a recombinant RANKL used as a control (31 kD and approximately 48 kD). OPG was detected in aseptic loosening, where macrophages showed a strong staining, but multinucleated giant cells were only weakly stained. A weak OPG staining was also observed in septic loosening., Conclusion: The pathogenesis of bone loss in septic loosening remains unclear, because the septic membrane bears few macrophages and giant cells, and half of them express OPG. In aseptic loosening, macrophages might not be stimulated by RANKL as a result of OPG expression. But multinucleated giant cells may be activated, as they hardly express OPG. They might be responsible for periprosthetic bone loss in aseptic loosening as a result of their RANKL and RANK expression.

About 5 to 12 % of hip endoprostheses will loosen after ten years. The periprosthetic membran between bone and prosthesis plays a crucial role in prosthesis loosening. Different pathomechanisms lead to the growth of such a membran, which can be discriminated by different histomorphologies: wear particle induced type, infectious type, combined type, indifferent type. 8 hybridizations were performed on PIQOR cDNA arrays. Objects of the study were periprosthetic interface tissue samples from 3 patients with particle induced and 2 patients with infectious prosthesis loosening. Tissue parts directly adjacent to the site of RNA-isolation were analyzed immuno-/ histopathologically in order to overcome the problem of tissue heterogeneity. 34 genes were found constantly differentially expressed, among which were cd9, cd11b, cd18, cd68, osteopontin, ferritin heavy-chain upregulated in the particle induced membrane and collagen types 1alpha-1, 3alpha-1, integrin alpha-1, thrombospondin 2 and nidogen upregulated in the infectious membrane. The most striking finding was the strong upregulation (from 20 fold to 323 fold) of megakaryocyte stimulating factor (msf) in all wear particle cases and 1 out of 2 infectious cases, which was confirmed by real-time RT-PCR. The upregulation of msf suggests an important pathogenetic role: The msf splice product lubricin is responsible for the lubrification of healthy joints, but its excellent lubrification ability may disturb the tight interaction between bone and prosthesis and thereby contribute to prosthesis loosening.

The pathogenesis of psoriatic arthritis (PA), which occurs in 5-7% of patients with psoriasis vulgaris, is enigmatic. There are no molecular data about synovial B-cells of chronic synovitis of PA. In order to understand the B-cell response in PA, we analysed IgVH genes and specified replacement to silent mutation (R/S) ratios of the complementarity determining regions (CDR) and framework regions (FR) as well as VH families. To prove the existence of a common pattern we additionally analysed the IgVH genes at the amino-acid level. From 5 PA patients we took cryo-tissue sections with somatically mutated IgVH genes (sum R/sum S in the CDRs: 2.5-6.8), 62 of which were analysed. In one patient two cases of clonally related IgVH genes were observed. Identical amino-acid replacements in IgVH1 and IgVH4 were found at the same mutational "cold spot". These data indicate that antigen-activated B-cells participate in the formation of chronic synovitis of PA. Since, neither histopathologically nor immunohistochemically, no germinal centres could be detected, the clonally related IgVH genes may be interpreted as residues of a germinal centre reaction that occurred before synovectomy. The existence of identical amino-acid replacement mutations in IgVH1 and IgVH4 genes suggests that a limited number of antigens are common to all PA patients analysed. The recombinant expression of the IgVH could help to define B-cell specificities underlying the pathogenesis of chronic synovitis and dermatitis of PA.

The synovitis of rheumatoid arthritis (RA) was long regarded merely as an unspecific chronic inflammatory process of minor diagnostic value and therefore did not play a major role in the understanding of the pathogenesis of RA. It is only in recent years, along with the observation that T and B cells are expanded oligoclonally in synovial tissue and that B cells are able to undergo a local germinal center (GC) reaction, that the synovial tissue has come to be regarded as a site of specific immune processes. The analysis of the immunoglobulin (Ig) gene repertoire had great impact on the understanding of B cell response in lymphatic organs and was subsequently applied to B cells from RA patients. The analyses of the variable (V) regions of the Ig heavy (H) and light (lambda) chains suggested that an antigen specific activation and differentiation of B cells into plasma cells (Plc) takes place in the chronically inflamed synovial tissue of patients with RA. It seems that in a subset of RA patients the synovial tissue develops into an ectopic lymphoid tissue that supports a local GC reaction. Ectopic GC are characteristic of RA; however, they are in general absent from synovitis of osteoarthritis (OA). Here the accumulation of Plc follows a different mechanism. Highly mutated VH genes suggest that in OA memory B cells migrate into the synovial tissue with subsequent differentiation into Plc but without further V gene diversification. Therefore in synovitis two patterns of B cell activation can be differentiated: the maturative and the accumulative type. These two patterns are not definitely disease linked. The maturative type is only found in RA whereas the accumulative type occurs in both diseases. Clinically RA is defined via serum antibodies to the constant region of Ig, so-called rheumatoid factor. However, the spectrum of autoreactive B cells in RA patients is wide and is based on the study of antibody specificities in serum, in synovial fluid and B cell lines derived from peripheral blood, bone marrow, synovial fluid and synovial tissue. These analyses defined non-organ-specific and organ-specific antigens. One can reasonably assume that the disease is far too complex to be explained by only a single antigen. There is a whole combination of antigens acting in a multistep manner that is responsible for RA pathogenesis. It can be hypothesized that chronic synovitis, which is the underlying mechanism of joint destruction, follows a three-step process: (a) initiation, (b) destruction, and (c) perpetuation. The characterization of antigens driving the local synovial B cell maturation and accumulation could lead to an understanding of the process perpetuating the disease. Identification of arthritogenic antigens may yield new avenues for diagnostics and immunotherapy but also a new approach for prevention by vaccines with antigens probably defined by synovial B cell reactivity.

Purpose: To correlate power Doppler ultrasonographic (US) findings of the vascularity of synovial tissue of the hip joint with the results of histopathologic examination of the same tissue to assess the value of power Doppler US in the visualization of synovitis., Materials and Methods: The hip joints of 24 patients with osteoarthritis (n = 15) or rheumatoid arthritis (n = 9) of the hip joint were examined with US before arthroplasty. The vascularity of the synovial membrane was classified qualitatively by using power Doppler US. During surgery, a section of the synovial tissue examined at power Doppler US preoperatively was resected. The vascularity of the tissue specimen was investigated and graded qualitatively by a pathologist who was not aware of the US findings. Visual qualitative grading was controlled by means of analysis of the US images and histopathologic specimens with a digital image evaluation system. Correlations between power Doppler US and histopathologic examination findings were calculated by using Spearman rank correlation and Pearson correlation tests., Results: The correlation between the qualitative power Doppler US results and the qualitative vascularity grades was 0.92 (P <.01, Spearman rho). The correlation between quantitative and qualitative results was 0.93 (P <.01, Spearman rho) for US imaging and 0.97 (P <.01, Spearman rho) for histopathologic examination., Conclusion: Study results showed power Doppler US to be reliable for qualitative grading of the vascularity of synovial tissue of the hip.

Regulatory CD25(+)CD4(+) T cells are considered as important players in T cell homeostasis and self-tolerance. Here we report that the integrin alpha(E)beta(7), which recognizes epithelial cadherin, identifies the most potent subpopulation of regulatory CD25(+) T cells. Strikingly, CD25-negative alpha(E)+CD4(+) T cells displayed regulatory activity. Both alpha(E)+ subsets, CD25(+) and CD25(-), express CTLA-4, suppress T cell proliferation in vitro, and protect mice from colitis in the severe combined immunodeficient model (SCID) in vivo. Whereas alpha(E)+CD25(+) T cells produce almost no cytokines, alpha(E)+CD25(-) T cells represent a unique subset in which high IL-2, IFN-gamma and T helper 2-cytokine production is linked with suppressive function. Thus, the integrin alpha(E)beta(7) can be regarded as a novel marker for subsets of highly potent, functionally distinct regulatory T cells specialized for crosstalk with epithelial environments.

The antibody SC-1 is a human IGM molecule, which binds to a tumor specific receptor. This SC-1 receptor is detectable on biopsies, it is present in about 50% of gastric cancers. After binding of the antibody to the receptor the tumor cells go into apoptosis. 50 patients expressing the SC-1 receptor on their tumors have been treated with SC-1 prior to gastrectomy. In 80% of cases apoptosis induction could be demonstrated in the tumors. The only side effect of the SC-1 therapy was a reversible episode of fever during antibody infusion in 8% of our patients.

The presence of immunoglobulin crystal inclusions in plasma cells from plasmacytomas and B-NHLs (linked to overstimulation and overproduction) has been frequently reported. Our case describes a lymphoplasmacellular synovitis in a patient with osteoarthritis (OA) showing an unusually high plasma cell infiltration and for the first time crystals in plasma cells. Using immunohistochemistry. these crystals were identified as being IgG with a balanced lambda/kappa ratio. IgV(H) gene analysis (n = 5 clones) showed that they were somatically mutated (R/S of CDR > 3): in one case, an insertion of 9 nucleotides on the CDR2 region was observed. High R/S values in the CDR indicated antigen selectivity and affinity (4/5). Since no germinal centers could be detected and the analyzed B cells showed antigen selectivity, it may be concluded that already antigenically activated B cells migrated into the synovium and locally differentiated into plasma cells, leading to the extensive infiltration observed. Rheumatoid fibroblasts were shown to support terminal B cell differentiation. Our data suggests that the ability of fibroblasts to activate B cells is not only restricted to RA, but also occurs in OA. The intense plasma cell infiltration contributed to further cartilage damage by altering the microenvironment of the nourishing synovial tissue or by the local production of pathogenic autoantibodies.

The following is a proposition for a simple histopathological classification system to measure inflammation in synovial tissue. This synovitis-score is employed in conventionally stained routine sections, and is applicable to every kind of synovitis, irrespective of etiology and including the following relevant morphological alterations. First: hyperplasia/enlargement of synovial lining cell layer. Second: activation of resident cells/synovial stroma. Third: inflammatory infiltration. All defined histopathological qualities are graded from absent (0), slight (1) and moderate (2) to strong (3), with summaries ranging from 0 to 9. 0 to 1 corresponds to no synovitis (inflammatory grade = 0), 2 to 3 to a slight synovitis (inflammatory grade 1), 4 to 6 to a moderate synovitis (inflammatory grade 2), and 7 to 9 to a strong synovitis (inflammatory grade 3). Using this score, we analyzed 308 random specimens of synovial tissue from degenerative (osteoarthritis (OA)) and inflammatory joint diseases - reactive arthritis (ReA), psoriasis arthritis (PA) and rheumatoid arthritis (RA) - as well as synovial tissue from healthy individuals. The mean grade given to synovitis of RA turned out to be significantly higher than the mean grade of OA (p < 0.0005) and of healthy controls (p < 0.0005). On the contrary, no significant differences could be found between the mean grades of synovitis scores from patients with RA and those with PA and ReA. Another comparison between RA-synovitis types I and II according to the Stiehl classification resulted in type I (p < 0.0005), showing significantly higher values of inflammatory infiltration, and type II (p = 0.037), showing significantly higher values of stroma activation. Since in OA, synovitis is regarded as a result of degenerative cartilage destruction whereas in inflammatory joint diseases (RA, PA, ReA), synovitis is regarded to be the cause of cartilage destruction, it can be concluded that scores with considerable high values indicate the pathogenetic potential of synovitis and that the inflammatory score may be helpful in estimating the destructive potential of synovitis at the same time. Furthermore, the comparison of the score data with the Stiehl RA-synovitis types shows that the score enables us to discriminate the morphological peculiarities of the synovitis types. In experimental pathology, it could provide standardized information on molecular synovial tissue analyses where a correlation of molecular with morphological data is essential. In diagnostic pathology, this synovitis score (in combination with other typing systems) could provide basic and standardized information concerning the degree of inflammatory alterations in synovial tissue.

Synovial fibroblasts were infected with Yersinia enterocolitica or Salmonella enterica serovar Enteritidis and analyzed by electron microscopy and fluorescence in situ hybridization. Intracellular bacterial replication was followed by degradation leading to "ghosts" possessing lipopolysaccharides but not DNA. However, single bacteria survived for more than 2 weeks. Therefore, transient intra-articular infection might be the missing link between initial intestinal infection and late synovial inflammation in the pathogenesis of reactive arthritis.

The human monoclonal antibody SC-1 was isolated from a patient with a diffuse-type adenocarcinoma of the stomach using somatic cell hybridization. The immunoglobulin (Ig)M antibody reacts specifically with diffuse- (70%) and intestinal-type (25%) gastric adenocarcinoma and induces apoptosis in vitro and in vivo. When used in clinical trials with stomach carcinoma patients, significant apoptotic and regressive effects in primary tumors have been observed with the antibody SC-1. The SC-1 receptor is a new 82 kd membrane-bound isoform of glycosylphosphatidylinositol (GPI)-linked CD55 (decay-accelerating factor, DAF). CD55 is known to protect cells from lysis through autologous complement and is coexpressed with the ubiquitously distributed 70 kd isoform. The SC-1-specific CD55 isoform is up-regulated shortly after antibody binding, followed by an internalization of the antibody/receptor-complex, whereas the membranous expression of wild-type CD55 remains unchanged. The apoptotic process is marked by cleavage of cytokeratin 18, indicating the involvement of caspase-6 in the apoptotic process. In contrast to other apoptotic pathways, a cleavage of poly(ADP-ribose)polymerase (PARP) is not observed. The expression of the cell-cycle regulator c-myc becomes up-regulated, whereas expression of topoisomerase IIalpha is down-regulated. Induction of apoptosis leads to an increase in the internal Ca(2+) concentration, which is not necessary for the apoptotic process but for the transport of newly synthesized SC-1-specific CD55 isoform to the membrane.

(NZB x NZW)F1 (NZB / W) mice develop a disease similar to human systemic lupus erythematosus (SLE), including autoantibody production, hypergammaglobulinaemia and inflammation of the kidneys. It is known that large numbers of lymphocytes infiltrate the kidneys of these mice. Here, we compare the roles of bone marrow, spleen and inflamed kidneys of NZB / W mice in the activation of B cells and the persistence of antibody-secreting cells (ASC). ASC are present in the kidneys of NZB / W mice with full-blown disease, as many as in the spleen and bone marrow. The specificity of the ASC in the inflamed kidneys is not restricted to self-antigens. After immunization of NZB / W mice with ovalbumin (OVA) the OVA-specific ASC are found initially in the spleen. Weeks later, OVA-specific ASC are found in high numbers in the bone marrow and the kidneys of these mice, but no longer in the spleen. As determined by FACS, B cells with a germinal center phenotype (B220(+) / PNA(+)) are found only in very low numbers in the kidneys, but in high numbers in the spleen of NZB / W mice. Germinal centers could not be detected in the kidneys, but in the spleen, and plasma cells appear to be scattered over the tissue. These data suggest that in autoimmune NZB / W mice, plasma cells generated in immune reactions of secondary lymphoid organs, later accumulate and persist in the inflamed kidneys, were they enhance the local concentrations of Ab and immunocomplexes. These experiments identify the inflamed kidneys of NZB / W mice as a site of prime relevance for the homeostasis of plasma cells, irrespective of their specificity.

The germline coded human monoclonal IgM antibody 103/51 was isolated from a gastric carcinoma patient. This antibody binds to a 130-kd membrane molecule and has a mitotic effect on tumor cells in vitro. To characterize the target, we sequenced the protein and showed that the antibody binds to the cysteine-rich fibroblast growth factor receptor (CFR)-1, which is highly homologous to MG-160 and the E-selectin-ligand (ESL)-1. The epitope was determined by glycosidase-digestion experiments to be an N-linked carbohydrate side chain. Immunohistochemistry was used to investigate the tissue distribution of CFR-1. Different healthy tissues were tested and only the collecting tubes of the kidney, the Golgi apparatus, and the glomerular and fascicular zones of the adrenal gland stained positive. However, on malignant tissue the receptor is overexpressed in nearly all tested stomach cancers (12 of 15) and other tested carcinomas (13 of 15). Most interestingly, the receptor is also present in Helicobacter pylori gastritis and gastric dysplasia, but absent on uninflamed stomach mucosa. This restricted tissue pattern indicates that antibody 103/51 reacts with a membrane-bound variant of CFR-1, which is mainly expressed on transformed cells and precursor lesions and is essential for proliferation processes. The possible activity of antibody 103/51 as an activating ligand in these proliferative changes of gastric epithelial mucosa is discussed.

A 69-year-old male presented with symptoms of fulminant lung embolism and, despite immediate therapy with plasminogen activator, died of acute right heart failure. At autopsy multiple tumor cell emboli were detected in small pulmonary vessels in addition to widespread liver metastases from an urothelial carcinoma. - In a 23-year-old female a malignant gastric ulcer and multiple liver metastases were diagnosed at initial presentation. She too died from pulmonary hypertension due to a series of lung embolisms which occurred despite heparin therapy. At autopsy, many small pulmonary arteries were filled with adenocarcinoma cells; the primary gastric tumor and liver metastases were confirmed. These cases demonstrate that the shedding of tumor cells from hepatic metastases can obstruct the pulmonary vessels and lead to acute cor pulmonale. Tumor cell emboli should be considered in the differential diagnosis of acute pulmonary hypertension, especially in patients with a known tumor. They may, however, also represent the first clinical signs of previously unrecognized malignancy.

The ability to control gene expression in a temporal and spatial manner provides a new tool for the study of mammalian gene function particularly during development and oncogenesis. In this study the suitability of the tet-system for investigating embryogenesis was tested in detail. The tTACMV(M1) and rTACMV-3 (reverse Tc-controlled transactivator) transgenic mice were bred with NZL-2 bi-reporter mice containing the vector with a tTA/rTA responsive bidirectional promoter that allows simultaneous regulation of expression of two reporter genes encoding luciferase and beta-galactosidase. In both cases reporter genes were found to be expressed in a wide spectrum of tissues of double transgenic embryos and adult mice. The earliest expression was detected in tTACMV(M1)/NZL-2 embryos at embryonic day 10.5 (E10.5) and rTACMV-3/NZL-2 embryos at E13.5. Doxycycline abolished beta-gal expression in tTACMV(M1)/NZL-2 but induced it in rTACMV-3/NZL-2 embryos including late stages of embryo-genesis. The tTA and rtTA transactivators thus revealed a partially complementary mode of action during second half of embryonic development. These experiments demonstrated that both Tet regulatory systems function during embryonic development. We conclude that the Tet systems allows regulation of gene expression during embryonic development and that 'double reporter' animals like the NZL-2 mice are useful tools for the characterization of newly generated tet transactivator lines expressing tTA (or rtTA) in embryonic as well as in adult tissues.

Joint-specific self-Ags are considered to play an important role in the induction of synovial T and B cell expansion in human rheumatoid arthritis (RA). However, the nature of these autoantigens is still enigmatic. In this study a somatically mutated IgG2 lambda B cell hybridoma was established from the synovial membrane of an RA patient and analyzed for its Ag specificity. A heptameric peptide of cartilage oligomeric matrix protein (COMP) could be characterized as the target structure recognized by the human synovial B cell hybridoma. The clonotypic V(H) sequences of the COMP-specific hybridoma could also be detected in synovectomy material derived from five different RA patients but in none of the investigated osteoarthritis cases (n = 5), indicating a preferential usage of V(H) genes closely related to those coding for a COMP-specific Ag receptor in RA synovial B cells. Moreover, the COMP heptamer was preferentially recognized by circulating IgG in RA (n = 22) compared with osteoarthritis patients (n = 24) or age-matched healthy controls (n = 20; both p < 0.0001). Hence, the COMP-specific serum IgG is likely to reflect local immune responses toward a cartilage- and tendon-restricted Ag that might be crucial to the induction of tissue damage in RA.

Objective: To examine the significance of power Doppler sonography (PDS) in the diagnosis of synovial hypertrophy of the knee joint by verifying and comparing the PDS findings with histopathologic findings of synovial membrane vascularity., Methods: The knee joints of 23 patients who were undergoing arthroplasty of the knee joint because of osteoarthritis or rheumatoid arthritis were examined with ultrasound before arthroplasty. The vascularity of the synovial membrane was classified semiquantitatively using PDS. A sample of synovial tissue was obtained during the arthroplasty, and the vascularity of the synovial tissue was evaluated by immunohistochemistry (factor VIII) and was graded qualitatively by a pathologist who was unaware of the PDS findings. The visual qualitative grading by the examiner was controlled by analyzing PDS images and histologic samples using a digital image evaluation system., Results: The correlation between the qualitative PDS results and the qualitative grading of the vascularity by the pathologist was 0.89 by Spearman's rho (P < 0.01). The Pearson correlation coefficient between the digital analysis of the PDS images and the digital analysis of the tissue sections was 0.81 (P < 0.01). Digital image analysis and qualitative grading by the examiner had a correlation of 0.89 by Spearman's p (P < 0.01) for the PDS images. The correlation between the qualitative estimation of vascularity by the pathologist and the digital image analysis was 0.88 by Spearman's rho (P < 0.01)., Conclusion: In the present study, PDS proved to be a reliable diagnostic method for qualitative grading of the vascularity of the synovial tissue. In clinical practice, PDS allows further differentiation of the hypertrophic synovium.

B-cells of the rheumatoid synovial tissue are a constant part of and, in some histopathological subtypes, the dominant population of the inflammatory infiltrate, located in the region of tissue destruction. The pattern of B-cell distribution and the relationship to the corresponding antigen-presenting cells (follicular dendritic reticulum cells: FDCs) show a great variety. B-cells may exhibit (i) a follicular organization forming secondary follicles; (ii) follicle-like patterns with irregularly formed FDC networks, and (iii) a diffuse pattern of isolated FDCs. Molecular analysis of immunoglobulin VH and VL genes from human synovial B-cell hybridomas and synovial tissue demonstrates somatic mutations due to antigen activation. The FDC formations in the synovial tissue may therefore serve as an environment for B-cell maturation, which is involved in the generation of autoantibodies. An autoantibody is defined as "pathogenic" if it fulfills the Witebsky-Rose-Koch criteria for classical autoimmune diseases: definition of the autoantibody; induction of the disease by transfer of the autoantibody; and isolation of the autoantibody from the disease-specific lesion. B-cells from rheumatoid synovial tissue show specificity for FcIgG, type II collagen, COMP, sDNA, tetanus toxoid, mitochondrial antigens (M2), filaggrin and bacterial HSPs. The contributions of these antigens to the pathogenesis of RA are still hypothetical. A possible contribution could derive from crossreactivity and epitope mimicry: due to crossreaction, an antibody directed originally against a foreign infectious agent could react with epitopes from articular tissues, perpetuating the local inflammatory process. The characteristic distribution pattern, the localisation within the area of tissue destruction, the hypermutated IgVH and IgVL genes, and their exclusive function to recognize conformation-dependent antigens suggest a central role for B-cells in the inflammatory process of rheumatoid arthritis. Therefore, the analysis of synovial B-cell hybridomas and experimental expression of synovial IgVH and IgVL genes will help to characterise the antigens responsible for the pathogenesis of rheumatoid arthritis.

To investigate lymphocyte and monocyte recruitment-specific chemokine expression in synovial tissues from patients with rheumatoid arthritis (RA), psoriatic arthritis (PA) and osteoarthritis (OA), synovial membranes and cytocentrifuge preparations of 7 RA, 8 PA and 10 OA patients were examined by in situ hybridisation with antisense probes of Mig, GRO alpha and RANTES and by immunohistochemistry. Patients' local disease activity (swelling and tenderness) in order to was graded and histological evaluation was performed compare these data with their chemokine expression profiles. Mig and RANTES hybridisation signals were detected in the synovial lining layer and in cellular infiltrates, whereas GRO alpha expression was localised exclusively in the lining layer of PA and RA. Cytological analysis revealed Mig and GRO alpha mRNA mainly in monocytic cells expressing KIM6, while RANTES mRNA was demonstrated predominantly in lymphocytic cells expressing CD3. In OA synovial membranes, significantly fewer hybridisation signals were present than in RA and PA synovial membranes. Patients with PA and RA had mild to severe local disease activity, whereas OA patients showed only mild disease activity. Histologically, PA and RA inflammatory scores ranged from 1 to 5, while OA synovium was consistently graded 1. Therefore, we conclude that the differential expression of Mig, GRO alpha and RANTES in resident and in inflammatory cells has an important role in regulating leucocyte traffic in inflammatory arthropathies. The diverse leucocyte specificity of Mig, GRO alpha and RANTES may thus regulate the recruitment of different leucocyte populations, as detected in PA and RA. Therefore, the pattern of cellular infiltration in human synovitis and the corresponding clinical signs of inflammation basically reflect the localisation and expression intensity of chemokines, which may be an important target for future disease modulation.

In the present study 55 IgVH genes amplified from three different anatomical regions of a rheumatoid arthritis (RA) patient were analyzed, adding further information on synovial B-cell maturation and recirculation in RA. This analysis demonstrated somatically mutated IgVh genes in all regions studied, with amino acid deletions and mixed IgVh molecules, suggesting the existence of a novel pathway to generate (auto) antibody specificities. Comparison of amino acid sequences of amplified genes that belong to the VH1 family (with predominantly the same germline counterpart) exhibited strong homology, indicating an apparently conserved mutational pattern. This suggests that the number of antigens that activate B cells in different locations is restricted. The most striking result was the finding of clonally related sequences in different anatomical regions, indicating a recirculation of activated B cells between the different affected joints.

In osteoarthritis (OA), the synovial tissue exhibits a nonfollicular inflammatory infiltration with a characteristic arrangement of lymphocytes and plasma cells. These arrangements are either small perivascular aggregates with plasma cells surrounding the lymphocytes or small groups of plasma cells, located in the vicinity of small blood vessels. These patterns suggest that B lymphocytes directly differentiate into plasma cells. To understand the B-cell response in OA, we analyzed the V(H) genes from B cells of synovial tissue of nine OA patients (average age, 71.5+/-10.5 years; six female and three male). V(H) gene repertoires were determined from RNA prepared from tissue cryosections and from DNA of single isolated B lymphocytes and plasma cells. The inflammatory infiltrate was analyzed immunohistochemically by detecting CD20, Ki-M4 (follicular dendritic cells), CD4, IgG, IgM, IgA, Ki-67, and by simultaneous demonstration of the plasma-cell-specific antigen CD138 (syndecan-1) and factor VIII. The molecular data demonstrate B cells with a high number of somatic mutations (average, 16.5 to 19.8), and high ratios of replacement to silent mutations in the small lymphocytic/plasmacellular aggregates of OA. In the tissue cryosections, the values of the sigmaR/sigmaS at the complementarity determining regions were 5.3 and 2.0 in the framework regions. For both the isolated B lymphocytes and plasma cells, the value of this ratio in the complementarity determining regions was 3.5. In the framework regions, the values of this ratio were 2.0 for the isolated B cells and 1.8 for the plasma cells. B lymphocytes and plasma cells exhibited a distribution not described thus far. Two patterns of B-cell distribution could be observed: (a) Centrally located CD20+ B and CD4+ and CD8+ T lymphocytes were surrounded directly by IgG (predominantly) or IgA and IgM plasma cells. No proliferating Ki-67-positive cells and no follicular dendritic cells (germinal centers) could be detected in the aggregates; (b) Plasma cells (predominantly IgG) were located directly near endothelial cells of small blood vessels. The finding of highly mutated V(H) genes in B lymphocytes and the characteristic arrangement of B lymphocytes and plasma cells suggests that B cells, which participate in OA synovialitis, have undergone germinal center reaction at different sites. This may explain the low inflammatory infiltration without germinal centers in OA, which is a feature of this primarily degenerative joint disease.

Erosive human/murine (hu/mu) SCID arthritis, caused by unilateral engrafting of human rheumatoid arthritis synovial membrane (RA-SM) in the knee joints of SCID mice, was monitored for up to 18 weeks by scintigraphic, radiological, morphological and immunohistochemical analyses.(99m)Tc-DPD scintigraphy and histology revealed secondary, oligoarticular spreading of arthritis to contralateral knees and hips, but not to forelimb joints. Also, there were no extraarticular manifestations. At 18 weeks, surviving human cells were found within the pannus, but not directly at the cartilage erosion front, where fibroblast-like cells and macrophages of murine origin predominated. The latter cells also predominated in secondarily affected joints, where no human cells were detectable. Preventive depletion of murine NK-cells by anti-asialo-GMI antibodies, to check the influence of NK cells independently of strain and MHC system, combined with application of autologous human PBMN cells, had virtually no effects on the disease process. The completeness of the SCID defect was not critical, i.e. T cells were completely absent in the organs examined, and the presence of a few B cells in the spleen did not correspond to particular disease features. The SCID defect itself had a clear impact, since, in the chronic phase, SCID.bg and RAG-2(-/-)knockout mice developed less consistent pathological/scintigraphic signs of disease than SCID mice. Thus, unilaterally-induced hu/mu SCID arthritis is an oligoarticular disorder of the hindlimbs. Murine macrophages and fibroblast-like cells appear responsible for tissue destruction in engrafted and non-engrafted arthritic joints., (Copyright 1999 Academic Press.)

Vasodilator-stimulated phosphoprotein (VASP), a substrate of cAMP- and cGMP-dependent protein kinases, is associated with focal adhesions, cell-cell contacts, microfilaments, and highly dynamic membrane regions. VASP, which is expressed in most cell types and in particularly high levels in human platelets, binds to profilin, zyxin, vinculin, F-actin, and the Listeria monocytogenes surface protein ActA. VASP is a member of the enabled (Ena)/VASP protein family and is thought to be involved in actin filament formation and integrin alphaIIbbeta3 inhibition in human platelets. To gain further insight into the in vivo function of this protein, VASP-deficient mice were generated by homologous recombination. VASP-/- mice demonstrated hyperplasia of megakaryocytes in bone marrow and spleen but exhibited no other macroscopic or microscopic abnormalities. Activation of platelets with thrombin induced a more than 2-fold higher surface expression of P-selectin and fibrinogen binding in VASP-deficient platelets in comparison to wild type. These data support the concept that VASP is a negative modulator of platelet and integrin alphaIIbbeta3 activation. Although the limited phenotypic differences between wild-type and VASP-/- mice suggested functional compensation of VASP by members of the Ena/VASP family, alterations in the expression levels of mammalian enabled (Mena) and Ena-VASP-like (Evl) protein were not detected. VASP-deficient mice may provide an interesting model system for diseases in which enhanced platelet activation plays a major role.

Colonization of the bacterium Helicobacter pylori of gastric mucosa plays an important role in stomach carcinogenesis, while the gastric mucosa and nearby lymphoid tissue are active sites of humoral immunity against both bacteria and tumor. In a broad study on the humoral immunity of stomach-cancer patients (5 patients with diffuse- and intestinal-type stomach carcinoma), we immortalized spleen cells by using human hybridoma technology and isolated 11 hybrid clones (9 IgM, 1 IgG and 1 IgA) which react with defined proteins on different stomach-cancer cells and, interestingly, also with distinct proteins on H. pylori; 4 of these antibodies are mitogenic and stimulate the proliferation of stomach-cancer cells in vitro. Furthermore, immunohistochemical studies define these 4 clearly as autoantibodies, in view of their reactivity to normal epithelial cells. Sequence analysis of the genes for the immunoglobulin heavy (V(H)) and light (V(L)) chain variable regions revealed that most of the human antibodies belong to the V(H)3, Vlambda I and III gene families (DP-49, DPL-5 and DPL-23) and are germ-line configured.

Plasma cells are found surrounding the inflammatory infiltrates of macrophages, T, and B cells in the synovial tissue of patients with rheumatoid and reactive arthritis. This characteristic arrangement suggests that in the synovial tissue CD20+ B cells differentiate into plasma cells. To examine clonal relationships, we have used micromanipulation to separately isolate CD20+ B cells and plasma cells from single infiltrates. DNA was extracted, and from both populations the VH/VL gene repertoires was determined. The data show that in the inflamed synovial tissue activated B cells are clonally expanded. During proliferation in the network of follicular dendritic cells, V gene variants are generated by the hypermutation mechanism. Surprisingly, we do not find identical rearrangements between CD20+ B cells and plasma cells. Nevertheless, the finding of clonally related plasma cells within single infiltrates suggests that these cells underwent terminal differentiation in the synovial tissue. These results indicate that B cell differentiation in the synovial tissue is a dynamic process. Whereas CD20+ B cells may turnover rapidly, plasma cells may well be long lived and thus accumulate in the synovial tissue. The analysis of individual B cells recovered from synovial tissue opens a new way to determine the specificity of those cells that take part in the local immune reaction. This will provide new insights into the pathogenesis of chronic inflammatory diseases like rheumatoid or reactive arthritis.

The mutational pattern of IgVH and IgVL genes from synovial tissue B cell hybridomas (n = 8) of patients (n = 4) with rheumatoid arthritis (RA) was analysed, which had been produced by the electrofusion technique without prior in vitro stimulation. The molecular data were correlated with immunohistopathological data and parameters of local disease activity. The IgVH genes of the B cell hybridomas belonged to the VH3 family (DP42; DP47, n = 2; DP53), the VH1 family (DP75), the VH4 family (DP71) and the VH5 family (DP73); 7/7 IgVH genes showed somatic mutations, the R/S ratio (CDR) was > 3 in 4/7 IgVH genes and the mean R/S ratio of all IgVH genes was 9.3 (CDR) and 1.0 (FR), suggesting an antigen-dependent selection. The IgVL/lambda genes belonged to the Vlambda1 family (DPL2, DPL5, DPL8nf), the Vlambda2 family (DPL11, n = 2) and to the Vlambda6 family (IGLV6S1); 6/6 IgVL genes showed somatic mutations, the R/S ratio (CDR) was > 3 in 3/6 IgVL genes and the mean R/S ratio of all IgVL was 3.0 (CDR) and 2.3 (FR), suggesting an antigen-dependent selection. The synovial tissue exhibited germinal centres in the follicles (3/4), with the unique distribution of Ki-M4+ follicular dendritic cells and Ki-67+ proliferating cells and a dominance of IgA+ plasma cells (3/3). All patients were positive for RF in serum and exhibited severe local symptoms (swelling 4/4; warmth 4/4; effusion 2/4), whereas the hybridomas were negative for RF. Since B cell hybridomas showed hypermutation and affinity selection for IgVH and IgVL/lambda genes and the patients exhibited severe local symptoms with germinal centres in synovial tissue, this study indicates that an antigen-driven process is behind the B cell expansion in the synovial tissue of clinically affected joints. These mutated B hybridomas were negative for RF, thus suggesting that antigens different from RF are also involved in the local B cell expansion and in the chronic synovitis of RA.

Synovial B-lymphocytes which are a constant feature of RA synovialitis are expanded in an antigen dependent manner. To understand the nature of the antigen driving the B-cell expansion it would be interesting to know whether there exists a restricted number of antigens. Therefore the usage of germline genes and the mutational pattern of Ig-VH-genes from synovial B-cells from 3 different anatomical regions with different onsets of local disease were analysed. Together with an immunohistological study characterising the inflammatory infiltrate (CD3, CD22, CD23 and Ki-M4), RNA was prepared from tissue sections taken from the right and left peroneal tendons and right elbow of a patient with seropositive RA. The corresponding cDNA was amplified using specific VH primers and the obtained variable region sequences were compared to their closest germline counterparts on the EMBL/Genbank data base. The molecular analysis demonstrated somatically mutated VH genes (total R/S ratios in CDR 1 + 2: right peroneal tendon 7.5; left peroneal tendon 3.5 and right elbow 3.0) in all three different regions, with two clones presenting aminoacid deletions. The values of total R/S ratios correlated directly to the duration of local disease. The aminoacid sequences from the different locations exhibited strong homology among each other. This homology was of 77% for the 19 clones belonging to the VH1 family. Immunohistochemistry demonstrated in two locations a dense follicle-like infiltrate with FDC-network, the third location presented a non-follicular distribution of lymphozytes without FDC. Hence the R/S values were correlated directly with the duration of local disease, it appears that here is an ongoing antigen-dependent B-cell activation in joints probably occurring in the FDC-networks. Moreover this study suggests--due to a high homology among the VH aminoacid sequences from B-cells of different anatomical regions--that a restricted number of antigens is involved in the B-cell expansion of RA patients.

In a first clinical trial with the apoptosis-inducing human antibody SC-1 eight patients with poorly differentiated stomach adenocarcinoma of diffuse-type received 20 or 30 mg of purified SC-1 antibody intravenously, followed 24 or 48 h later by gastrectomy and lymphadenectomy. In seven cases a significant induction of apoptotic activity was measured in primary tumors as compared with earlier biopsy material and in five patients a significant regression of tumor mass could be determined histopathologically. No toxic crossreactivity was observed with normal tissue or organs of patients.

To elucidate the pathogenic role of synovial B cells in rheumatoid arthritis (RA), nine human IgG/lambda-secreting B-cell hybridomas from rheumatoid synovial tissue of a patient with definite RA were screened by enzyme-linked immunosorbent assay and indirect immunofluorescence on tissue cryosections for detection of antibodies against autoantigens. One IgG2/lambda monoclonal antibody (mAb) from the B-cell hybridoma ELG211/15/63 (= hybr63) exhibited intense immunofluorescence reactivity in the cytoplasm of chondrocytes and epithelial cells of the gastrointestinal tract, especially in parietal cells of gastric mucosa (human and mouse tissue), representing a mitochondrial pattern. This result was confirmed by morphometric analysis of immunoelectron microscopy data, exhibiting a significantly higher labeling density in mitochondria (p < or = 0.001) than in the cytoplasmic background, with predominant staining in the inner mitochondrial membrane and mitochondrial matrix (p < or = 0.05). Immunoblotting experiments carried out with gastric mucosa, and a mitochondrial protein preparation revealed two major proteins of 38 and 50 kd under reducing conditions. The analysis of the IgV(H) genes from this B-cell hybridoma showed highest homology to the human germline gene DP53 (96%). The IgV(L) region gave highest homology to the human germline gene DP5 (93%). In the complementarity-determining regions, residues of the H- and L-chain variable regions replacement mutations only indicated that this B-cell clone had been antigen-selected for its affinity (ratio of replacement to silent mutations: > or = 7). To analyze the in vivo expansion of the B-cell clone, primers specific for the V(H) to D to J(H) rearrangement of this B-cell hybridoma were used. Specific amplifications could be detected within part of the synovial tissue but not within the cells of the synovial fluid and peripheral blood of the patient. The ability of the IgG2/lambda mAb to induce an inflammatory reaction was tested by intraperitoneal application in severe combined immunodeficiency (SCID) mice, which resulted in an inflammatory, predominantly granulocytic infiltration of the peritoneum. Consequently, intrasynovial cell death or cartilage destruction seems to be a possible source of liberation of mitochondrial antigens, inducing a local, antigen-driven IgG2/lambda B-cell response with the ability to induce an inflammatory reaction. These data suggest that tissue destruction may serve as a source of arthritogenic antigens that perpetuate and amplify the local pernicious inflammatory process in RA synovialitis.

An increase in the nuchal translucency that can be detected at 10-14 weeks of gestation by ultrasound forms the basis for a screening test for chromosomal abnormality. Several mechanisms leading to this increase in skin thickness have been proposed, including changes of the extracellular matrix, cardiac defects and abnormalities of the large vessels. This study examines the composition of the extracellular matrix of the skin in gestational age-matched fetuses with trisomy 21, 18 and 13 from 12-18 weeks. Immunohistochemistry was applied with monoclonal and polyclonal antibodies against collagen type I, III, IV, V and VI and against laminin and fibronectin. Collagen type VI gene expression was further studied by in situ hybridization to detect differences in expression patterns of COL6A1, COL6A3 and COL1A1 between normal fetuses and those with trisomy 21. The ultrastructure of tissue samples was studied by transmission electron microscopy (TEM) and additionally by immunogold TEM. Further, we examined the morphology of the skin in an animal model for Down's syndrome, the murine trisomy 16, by light and TEM. The dermis of trisomy 21 fetuses was richer in collagen type VI than that of normal fetuses and other trisomies, and COL6A1, located on chromosome 21, was expressed in a wider area than COL6A3, which is located on chromosome 2. Collagen type I was less abundant in the skin of trisomy 18 fetuses, while the skin of all three trisomies contained a dense network of collagen type III and V in comparison with normal fetuses. Collagen type IV, of which two genes are located on chromosome 13, was expressed in the basement membranes of the skin in all fetuses and additionally in the dermal fibroblasts only of trisomy 13 fetuses. Likewise, laminin was present in all basement membranes of normal and trisomic fetuses as well as in dermal fibroblasts of fetuses with trisomy 18. LAMA1 and LAMA3 genes are located on chromosome 18. Dermal cysts were found in the skin of trisomy 18 and 13, but not in trisomy 21 and normal fetuses. Ultrastructural findings showed that an extracellular precipitate containing glycosaminoglycans was regularly present in the skin of trisomy 21 fetuses and murine trisomy 16 embryos. In conclusion, this study suggests that the skin edema in fetal trisomies is characterized by specific alterations of the extracellular matrix that may be attributed to gene dosage effects as a result of a genetic imbalance due to the condition of fetal trisomy.

The objective of this research was to investigate the cellular source of soluble ICAM-1 (siCAM-1) from rheumatoid synovial tissue (RS) and its relation to sICAM-1 in synovial fluid (SF) and serum, and to study the expression of ICAM-1 in isolated cells of RS. sICAM-1 was determined by using the enzyme-linked immunosorbent assay (ELISA) and Western blot analysis in supernatants from RS cultured for short periods (n = 19), in SF (n = 7) and in serum (n = 19). ICAM-1 expression, vascularization and inflammatory infiltration (CD3, CD68, CD22) were characterized immunohistochemically in cytospin preparations (n = 18), cryosections (n = 18) and in conventionally stained paraffin sections (n = 19) of RS. The degree of RS vascularization was analysed morphometrically in immunohistochemically stained cryosections (factor VIII related antigen). We found 90-kD sICAM-1 in supernatants of cultured cells, in SF and in sera. sICAM-1 in cellular supernatant correlated significantly (P < 0.01) with SF sICAM-1. The amount of sICAM in cellular supernatants showed no correlation to the score of inflammatory infiltration, but correlated significantly (P < 0.001) with the vascularization index of RS. The percentage of ICAM-1-expressing cells correlated significantly (P < 0.001) with the percentage of CD68-positive macrophages, but not with CD3- and CD22-positive lymphocytes. Macrophages, multinucleated giant cells and endothelial cells exhibited a higher expression of ICAM-1 as compared to lymphocytes and fibroblasts. The differential expression of ICAM-1 on infiltrating leucocytes and resident cells of RS indicates a functional role of ICAM-1 in the local inflammatory process. SF sICAM-1 originated in RS, but serum sICAM-1 did not. Shedding of sICAM-1 by RS was independent of inflammatory infiltration, but depended on the degree of vascularization, indicating that endothelial cells are the major source of sICAM-1 in RS.

Psoriatic arthritis is an inflammatory arthropathy that ultimately can lead to joint destruction. In this study, we investigated the immunophenotypes of the inflammatory cells and the expression of interleukin-8 (IL-8), which is the hallmark chemoattractant cytokine of psoriasis in synovial membranes from patients exhibiting active psoriatic synovitis (n = 9). The tissue samples were examined by immunohistochemistry, Western blot analysis and in situ hybridisation. The inflammatory infiltrate consisted predominantly of CD3+ T lymphocytes, with a higher proportion of CD4+ than CD8+ T lymphocytes in six cases. CD3+ T lymphocytes were focally distributed near small blood vessels and the enlarged synovial intima. CD1+ interdigitating reticulum cells were not detected. CD22+ B lymphocytes and plasma cells were found in small aggregates without KiM4+ follicular dendritic cells. KiM8+ macrophages were located in the synovial intima and were distributed in a diffuse pattern near the synovial lining cells. CD15+ neutrophil granulocytes were detected in four cases. They were preferentially located in the vicinity of blood vessels and the synovial intima. IL-8 was found at a high level in the synovial lining cells and to a lesser extent in cells located in the perivascular areas. Immunofluorescence double staining showed IL-8 to be expressed in KiM8+ multinucleated giant cells, KiM8+ macrophages and CD3+ T lymphocytes. IL-8 receptor A was demonstrated in the synovial lining and in macrophages and lymphocytes. IL-8 was detected by immunoblot analysis of the synovial tissue at 8.4 kD. Employing in situ hybridisation, IL-8 mRNA was strongly and preferentially expressed in the synovial intima, as well as in macrophages and lymphocytes. The immunophenotype of the psoriatic arthritis inflammatory cells shows great similarity to the inflammatory infiltrate found in the synovial tissue of patients with rheumatoid arthritis. The preferential expression of IL-8 and IL-8 mRNA in the enlarged synovial intima and in lymphocytes and macrophages suggests that IL-8 exerts its action through activated mononuclear cells and T lymphocytes. It seems to play a role in regulating leucocyte traffic into the enlarged synovial intima and may contribute to the aggressive synovitis of patients with psoriatic arthritis.

The focal adhesion and microfilament-associated protein VASP is a major substrate of both cAMP- and cGMP-dependent protein kinase in intact human platelets. The recent elucidation of the primary VASP structure and identity of VASP binding proteins suggest that VASP is an important component of focal contacts which link signal transduction pathways and elements controlling cell motility. In this study, the high expression of VASP in vascular smooth muscle and endothelial cells of human blood vessels is reported. Western blot and immunofluorescence analysis detected VASP in human heart, femoral artery and a uterine leiomyosarcoma. Within these tissues, smooth muscle cells, small capillaries and the endothelial cell layer were strongly stained by the VASP antiserum. In human heart, an overlapping cellular distribution of the cGMP-dependent protein kinase I (cGK I) and its substrate VASP was noted. Immunoelectron microscopy experiments with vascular smooth muscle cells of the vessel wall revealed that VASP is localized in close proximity to microfilaments, dense plaques and dense bodies. The data of this study and the properties of VASP as a major target of inhibitory vasoactive agents suggest that VASP is an important component which participates in the regulation of cell motility of human vessel wall cells in vivo.

Limb muscle formation involves invasion of the limb bud mesoderm by myogenic precursor cells from the dermomyotomes at limb bud level. Directed cell migration, homing, and differentiation of myogenic cells are controlled by the stationary cells of the limb bud mesoderm. At the level of the extracellular matrix, the molecular basis of migration control has been suggested to be exerted by the distribution of hyaluronan. Here, we demonstrate that N-cadherin-mediated interactions play a role at cell-membrane level in myoblast distribution and differentiation. N-cadherin is strongly expressed by myogenic cells in the chick limb bud and more moderately expressed by stationary mesodermal cells in the myogenic zones and progress zone. After in vivo injection of antibodies and Fab-fragments against the homophilic binding site of N-cadherin into the wing bud mesoderm, aggregates of myoblasts are found predominantly in the dorsal myogenic zone 36 hr after injection apparently due to immobilization. In the same position, areas of myf-5-positive cells are also observed. In injected limb buds, Pax-3-positive cells are less evenly distributed than in uninjected limbs. They are found to spread up to the epidermis and also form loosely arranged aggregates. After prolonged reincubation periods, injected limbs show ectopic myoblasts that are rich in desmin and areas of strongly desmin-expressing myoblasts within muscle blastemas. These effects were not observed after application of antibodies against other parts of the N-cadherin molecule. We conclude that N-cadherin is involved in myoblast migration in the limb buds via homophilic interactions and that it plays a role in signal transduction during myogenesis.

Several studies indicate a pathogenetic role of T-lymphocytes with specificity for heat shock proteins (HSP) in rheumatoid arthritis (RA). Surprisingly, there are no experimental data for B-lymphocytes with specificity for HSP. To investigate whether B-lymphocytes from rheumatoid synovial tissue show a specificity for HSP 60 we immortalized synovial tissue B-lymphocytes by the electrofusion technique and tested the specificity of the B-cell clones for HSP 60 by ELISA. Tissue samples from four patients with classic, active RA were used in this study. The isolated cells were electrofused in strongly hypo-osmolar medium with cells either of the mouse strain X63-Ag8-653 (Ag8) or the heteromyeloma strain HAB-1. Clones positive for IgG, the IgG fraction of the supernatant of the isolated synovial cells and the IgG of the serum of the patients were tested in an ELISA for reactivity to the recombinant HSP 60 or Yersinia enterocolitica, which shows great homology with mycobacterial HSP 65 and human HSP 60. The expression of this HSP 60 was studied in normal and rheumatoid synovial tissue using a polyclonal rabbit serum against HSP 60 from Y. enterocolitica (Ye HSP 60). In this way we investigate differences in the expression of HSP 60 and compared the pattern of this HSP60 with the pattern of mycobacterial HSP65 and human HSP 60 described by others. In three of four patients 10 IgG secreting B-cell clones showing a specificity for HSP 60 were detected. IgG specific for HSP 60 was also detected in the supernatant of the isolated synovial cells before fusion and in the serum of these patients. HSP 60 was demonstrated immunohistochemically within the rheumatoid synovial tissue and showed stronger expression with a different distribution when compared with the expression in normal synovial tissue. B-cell clones from rheumatoid synovial tissue thus exhibit a specificity for bacterial HSP 60, and a monospecific rabbit serum against this HSP shows strong reactivity within the rheumatoid synovial tissue. It may be postulated that a humoral HSP 60 response, initially directed against an infectious agent, could react with cross-reactive epitopes of rheumatoid synovial tissue or with self-HSP perpetuating the local inflammatory process.

B-cells of the rheumatoid synovial tissue are constant and in some cases dominant elements of the inflammatory infiltrate and are located near to the site of tissue destruction. The pattern of B-cell distribution, the pattern and the relationship to the corresponding antigen presenting cells (follicular dendritical reticulum cells; FDC's) shows a great variation: B cells exhibit a follicular organisation forming secondary follicles, follicle like patterns with irregular formed FDC's networks and a diffuse pattern of and isolated FDC's. Molecular analysis of immunoglobulin genes from synovial B-cell clones and synovial tissue demonstrates the occurrence of immunoglobulin gene hypermutation as well as germline configuration. The FDC formations in the synovial tissue may therefore serve as an environment for B-cell maturation which is involved in the generation of autoantibodies. An autoantibody may be only defined as "pathogenic" if the antibody fulfills the Witebsky-Rose-Koch criteria for classical autoimmune disease: definition of the autoantibody, induction of the disease by transfer of the autoantibody and isolation of the autoantibody from the disease specific lesion. B-cells of rheumatoid synovial tissue show specificity for FcIgG, collagen 2, sDNA, tetanus toxoid, mitochondrial antigens (M2) and bacterial HSP's and the contribution of these antibodies to the pathogenesis of RA are still hypothetic. Antibody with specificity for bacterial HSP's which have arose during contact with an infectious agent and may due to crossreactivity with eukaryotic HSP of synovial tissue perpetuate the local inflammatory process. The characteristic pattern, the localisation within the area of tissue destruction and the exclusive function of B-cells to recognize conformation dependent antigens suggests a central role of B-cells in the inflammatory process. The analyzation of the synovial tissue B-cell therefore will help to characterise antigens which are responsible for the pathogenesis of RA.

We analysed the proliferative activity of synovial lining cells (SLCs), the distribution of proliferating B and T lymphocytes and the relationship of proliferating B and T lymphocytes to the pattern of antigen-presenting cells (APCs) within the rheumatoid synovial tissue (n = 21). The immunohistochemical detection of the proliferation-associated antigen Ki67 revealed low proliferative activity of SCL with and without expression of the Kim 8 (CD68) antigen. Ki67-positive B lymphocytes could be observed within secondary follicles (2/21), in small follicular dendritic reticulum cell (FDC)-containing follicle-like aggregates (7/21) and near the enlarged synovial intima (6/21). Ki67-positive T lymphocytes could be detected in T-lymphocyte aggregates (8/21), in the vicinity of blood vessels (18/21) and within the enlarged synovial intima (15/21). Semiquantitative analysis showed a strong correlation between the numbers of Ki67-positive B lymphocytes and FDCs and between the numbers of Ki67-positive T lymphocytes and interdigitating dendritic reticulum cells (IDC). There were significant differences in the number of Ki 67-positive B and T lymphocytes, IDCs and FDCs between the two groups of rheumatoid arthritis (RA) patients with different local clinical activity. These findings demonstrate a low proliferation of SLCs with and without expression of the monocyte-specific antigen Kim 8 and imply that B and T lymphocyte proliferation occurs in the presence of FDCs and IDCs. These results indicate that the RA synovial tissue is a site for antigen-dependent proliferation and maturation of B and T lymphocytes. The atypical pattern of FDC distribution within the rheumatoid synovial tissue "dysmorphic follicle" may be regarded as morphological substrate for a dysmaturation compartment of B lymphocytes leading to pathogenetic autoimmune phenomena in RA patients.

To investigate if Borrelia burgdorferi can persist in resident joint cells, an infection model using cell cultures of human synovial cells was established and compared to the interaction of Borrelia burgdorferi and human macrophages. Borrelia burgdorferi were found attached to the cell surface or folded into the cell membrane of synovial cells analysed by transmission electron and confocal laser scanning microscopy. In contrast to macrophages, morphologically intact Borrelia burgdorferi were found in the cytosol of synovial cells without engulfment by cell membrane folds or phagosomes. Borrelia burgdorferi were isolated from parallel cultures. Treatment with ceftriaxone eradicated extracellular Borrelia burgdorferi, but spirochetes were reisolated after lysis of the synovial cells. Borrelia burgdorferi persisted inside synovial cells for at least 8 weeks. These data suggested that Borrelia burgdorferi might be able to persist within resident joint cells in vivo.