Identification of an N-cadherin motif that can interact with the fibroblast growth factor receptor and is required for axonal growth.

In this study, we show that the neurite outgrowth response stimulated by N-cadherin is inhibited by a recently developed and highly specific fibroblast growth factor receptor (FGFR) antagonist. To test whether the N-cadherin response also requires FGF function, we developed peptide mimetics of the receptor binding sites on FGFs. Most mimetics inhibit the neurite outgrowth response stimulated by FGF in the absence of any effect on the N-cadherin response. The exceptions to this result were two mimetics of a short FGF1 sequence, which has been shown to interact with the region of the FGFR containing the histidine-alanine-valine motif. These peptides inhibited FGF and N-cadherin responses with similar efficacy. The histidine-alanine-valine region of the FGFR has previously been implicated in the N-cadherin response, and a candidate interaction site has been identified in extracellular domain 4 of N-cadherin. We now show that antibodies directed to this site on N-cadherin inhibit the neurite outgrowth response stimulated by N-cadherin, and peptide mimetics of the site inhibit N-cadherin and FGF responses. Thus, we can conclude that N-cadherin contains a novel motility motif in extracellular domain 4, and that peptide mimetics of this motif can interact with the FGFR.

Selective inhibition of fibroblast growth factor (FGF)-stimulated mitogenesis by a FGF receptor-1-derived phosphopeptide.

The activated fibroblast growth factor receptor (FGFR)-1 is phosphorylated on five tyrosine residues outside the catalytic site. Although one such residue, Tyr730, is flanked by potential binding sites for phosphotyrosine-interacting molecules, a physiological role for this region is still controversial. We report that a cell-permeant phosphopeptide mimic of this site, FGFR730(p)Y, inhibits FGF-mediated mitogenesis in cells with no effect on responses stimulated by other growth factors. A similar phosphopeptide corresponding to the phospholipase Cgamma binding site on the receptor had no effect on the mitogenic response. The FGFR730(p)Y peptide did not inhibit phosphorylation of p90/FRS2 or Erk, suggesting that it does not act by inhibiting the Erk-kinase cascade. However, the FGFR730(p)Y peptide bound Shc in a manner requiring both phosphorylated tyrosine and a putative PTB domain binding determinant. These data suggest that the peptide might inhibit mitogenesis by competing with the corresponding site on the FGFR for the ability to bind SHC.

Soluble N-cadherin stimulates fibroblast growth factor receptor dependent neurite outgrowth and N-cadherin and the fibroblast growth factor receptor co-cluster in cells.

A chimeric molecule consisting of the extracellular domain of the adhesion molecule, N-cadherin, fused to the Fc region of human IgG (NCAD-Fc) supports calcium-dependent cell adhesion and promotes neurite outgrowth following affinity-capture to a tissue culture substrate. When presented to cerebellar neurons as a soluble molecule, the NCAD-Fc stimulated neurite outgrowth in a manner equivalent to that seen for N-cadherin expressed as a cell surface glycoprotein. Neurons expressing a dominant-negative version of the fibroblast growth factor (FGF) receptor did not respond to soluble NCAD-Fc. In cells transfected with full-length N-cadherin and the FGF receptor, antibody-clustering of N-cadherin resulted in a co-clustering of the FGF receptor to discrete patches in the cell membrane. The data demonstrate that the ability of N-cadherin to stimulate neurite outgrowth can be dissociated from its ability to function as a substrate associated adhesion molecule. The N-cadherin and the FGF receptor co-clustering in cells provides a basis for the neurite outgrowth response stimulated by N-cadherin being dependent on FGF receptor function.

Selective inhibition of growth factor-stimulated mitogenesis by a cell-permeable Grb2-binding peptide.

The activation of the mitogen-activated protein kinase (MAPK) cascade by a variety of growth factors and other agents is central to a mitogenic response. In the case of polypeptide growth factors such as the epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), the steps leading to activation of MAPK require the function of the adaptor protein Grb2 (growth factor receptor binding protein 2), which can bind either directly or indirectly via its Src homology 2 domain to activated receptor tyrosine kinases. A cell-permeable mimetic of the EGF receptor Grb2 binding site has been investigated for its ability to inhibit biological responses stimulated by a variety of growth factors. Pretreatment of cells with this peptide results in the accumulation of the peptide in cells and its association with Grb2. This is associated with a complete inhibition of the mitogenic response stimulated by EGF and PDGF. In contrast, the peptide has no effect on the mitogenic response stimulated by fibroblast growth factor. The peptide could also inhibit the phosphorylation of MAPK stimulated with EGF and PDGF in the absence of an effect on the fibroblast growth factor response. These data demonstrate that cell-permeable mimetics of Src homology 2 binding sites can selectively inhibit growth factor-stimulated mitogenesis, and also directly demonstrate specificity in the coupling of activated receptor tyrosine kinases to the MAPK cascade.

Light microscopic and scanning electron microscopic analyses of dental implants retrieved from humans.

This paper reports analyses obtained from 51 implant cases retrieved from humans and submitted to the AAIDRF-MCG Implant Retrieval Center. The undecalcified samples were embedded in PMMA and examined with scanning electron microscopy and with routine light or Nomarski microscopy. Cases included individual implants as well as 2 mandibles obtained at autopsy. Retrieved implants were sometimes shown to be encapsulated with connective tissue (CT), whereas other implants were apposed by bone, with only minimal CT association. In the latter cases, the implants were apposed by substantial amounts of viable bone. Nomarski microscopy disclosed the orientation and close apposition of the collagen bundles comprising the interfacial bone. In these cases where close bone apposition was observed to the implants, implant fracture was often the cause of failure. Periodontal lesions were reported around some implants showing a marked degree of inflammatory cell infiltrate (ICI). This study underscores the need for evaluation of failed human dental implants. Failure of implants placed longer than 10 years ago (perhaps loaded immediately) may be due to loss of bone support, CT encapsulation, and ICI (i.e., biological failure). Failure of more recently placed implants could also be due to this scenario, but failure was more often ascribed to biomaterial failure.

The growth inhibition of human breast cancer cells by a novel synthetic progestin involves the induction of transforming growth factor beta.

Recent experimental work has identified a novel intracellular binding site for the synthetic progestin, Gestodene, that appears to be uniquely expressed in human breast cancer cells. Gestodene is shown here to inhibit the growth of human breast cancer cells in a dose-dependent fashion, but has no effect on endocrine-responsive human endometrial cancer cells. Gestodene induced a 90-fold increase in the secretion of transforming growth factor-beta (TGF-beta) by T47D human breast cancer cells. Other synthetic progestins had no effect, indicating that this induction is mediated by the novel Gestodene binding site and not by the conventional progesterone receptor. Furthermore, in four breast cancer cell lines, the extent of induction of TGF-beta correlated with intracellular levels of Gestodene binding site. No induction of TGF-beta was observed with the endometrial cancer line, HECl-B, which lacks the Gestodene binding site, but which expresses high levels of progesterone receptor. The inhibition of growth of T47D cells by Gestodene is partly reversible by a polyclonal antiserum to TGF-beta. These data indicate that the growth-inhibitory action of Gestodene may be mediated in part by an autocrine induction of TGF-beta.

Anti-oestrogens induce the secretion of active transforming growth factor beta from human fetal fibroblasts.

The clinical use of anti-oestrogens in breast cancer therapy has traditionally been restricted to tumours that contain measurable oestrogen receptor protein. However, it is now widely recognised that the clinical response to adjuvant anti-oestrogen therapy appears to be independent of the oestrogen receptor content of the primary tumour. The study reported here was designed to investigate the possibility that human stromal cells can respond to anti-oestrogens by an increased synthesis of the inhibitory growth factor, transforming growth factor beta (TGF-beta). Two established human fetal fibroblast strains were used as models for the breast cancer stromal fibroblasts. These cells were found to respond to the addition of anti-oestrogens by a large increase in their synthesis of biologically active TGF-beta. Despite the application of ligand binding, immunoassay and Northern analysis, no oestrogen receptor or oestrogen receptor mRNA was detected in either of the human fetal fibroblasts strains. These observations may provide a mechanism of action of anti-oestrogens that is independent of the presence of oestrogen receptor in the tumour epithelial cells, and thus provide an explantation for the counter-intuitive results of adjuvant anti-oestrogen action.

A novel binding site for a synthetic progestagen in breast cancer cells.

A novel, high-affinity saturable binding site for the synthetic 19-nor testosterone progestagen, 17 alpha-ethinyl-13 beta-ethyl 17 beta-hydroxy-4,15-oestradiene-3-one (gestodene) has been detected using a sensitive affinity chromatography technique. This binding site is present in a range of malignant breast-derived cells lines, regardless of the presence of oestrogen and progesterone receptors, but is absent from endometrial carcinoma cells that contain both oestrogen and progesterone receptors. Competition studies show that this binding is not attributable to the receptors for the progestagens, androgens, glucocorticoids or mineralocorticoids. Cytosolic gestodene binding is refractory to competition with oestradiol but nuclear gestodene binding is completely abolished by oestradiol. The binding of oestradiol to the oestrogen receptor is reduced 40-50% by competition with gestodene. Non-dissociating polyacrylamide gel electrophoresis and size-exclusion high performance liquid chromatography reveal that this binding activity is associated with a protein of mean molecular mass 47 +/- 9 kDa. Ligand binding studies with a range of other cell lines indicates that this binding site appears to be specific to breast cancer cells. These data show the presence of a partly oestrogen competable novel binding protein in breast cancer cells which does not appear to be due to any of the conventional steroid receptors.

Treatment of primary Sjögren's syndrome with hydroxychloroquine.

Sjögren's syndrome is an autoimmune disease characterized by lymphocytic infiltration of the salivary/lacrimal glands, autoantibody production, and polyclonal hyperglobulinemia. In view of the efficacy and relative safety of hydroxychloroquine in other autoimmune disorders, the potential benefit of hydroxychloroquine (200 mg per day for 12 months) in 10 patients with Sjögren's syndrome was evaluated. Changes in levels of total immunoglobulin, antibody against Sjögren's syndrome-associated antigen B, rheumatoid factor, and in vitro production of immunoglobulin in the serum were evaluated. For comparison, 10 patients matched according to age and sex, who did not receive hydroxychloroquine were studied. In the hydroxychloroquine-treated group, the following observations were made: (1) significantly decreased total immunoglobulin G (IgG) and IgA levels with little change in IgM levels; (2) significant decrease in IgA-rheumatoid factor with a smaller decrease in IgM-rheumatoid factor; (3) decreased IgG anti-Sjögren's syndrome-associated antigen B autoantibody; and (4) decreased erythrocyte sedimentation rate and increased hemoglobin level. Further, a specific idiotype present on their rheumatoid factor (defined by monoclonal antibody 17-109) was significantly decreased, with disappearance of detectable circulating paraprotein in two hydroxychloroquine-treated patients. Finally, rheumatoid factor production in vitro by lymphocytes from hydroxychloroquine-treated patients using a T cell-dependent mitogen was significantly decreased. These results suggest that hydroxychloroquine modulates lymphoproliferation in patients with Sjögren's syndrome and may prevent progression to extraglandular sites of neoplastic transformation.

Reactivation of Epstein-Barr virus in Sjögren's syndrome.

Sjögren's syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration and destruction of salivary and lacrimal glands. This condition may be limited to glandular tissues or may be associated with other autoimmune disorders such as rheumatoid arthritis or systemic lupus erythematosus. Since the environmental factors that initiate SS are unknown, we have investigated the potential role of Epstein-Barr virus (EBV), cytomegalovirus (CMV) and other viruses. We observed that epithelial cells in salivary gland biopsies of SS patients contained antigens reactive with monoclonal antibodies against EBV-associated antigens. These antigens were not found in other tissues of SS patients and were absent in salivary gland biopsies from normals and patients with other autoimmune diseases lacking SS. Also, the content of EBV DNA in the saliva of SS patients was significantly greater than in age- and sex-matched controls or individuals with other autoimmune disorders. These studies provide one of the first examples where a specific viral agent may be implicated in perpetuating a chronic autoimmune disease. However, great caution must be used before an etiologic role can be attributed to an ubiquitous agent such as EBV.

Potential role of Epstein-Barr virus in Sjögren's syndrome.

Sjögren's syndrome is an autoimmune disease characterized by lymphocytic infiltration and destruction of salivary and lacrimal glands. This condition may occur as a primary condition or may be associated with other autoimmune disorders such as rheumatoid arthritis or systemic lupus. Because the environmental factors that initiate SS are unknown, we have investigated the potential role of EBV, CMV, and other viruses. We observed that epithelial cells in salivary gland biopsies of patients with SS contained antigens reactive with monoclonal antibodies against EBV-associated antigens. These antigens were not found in other tissues of patients with SS and were not detectable in salivary gland biopsies from normal persons and patients with other autoimmune diseases lacking SS. The molecular weight of the antigens present in the SS salivary gland extracts was similar to that expressed in cells containing reactivated EBV. Also, the content of EBV DNA in the saliva of patients with SS was significantly greater than in age-, sex-matched controls or persons with other autoimmune disorders. These studies provide one of the first examples where a specific viral agent may be implicated in perpetuating a chronic autoimmune disease. These results also may provide insight into other autoimmune diseases where the target organ is less accessible to biopsy.

Sjögren's syndrome. Proposed criteria for classification.

The term "Sjögren's syndrome" (SS) is frequently used to describe the occurrence of keratoconjunctivis sicca and xerostomia in association with an autoimmune disorder. However, well-defined criteria for the classification of SS have not been established, and this diagnosis is being applied to a wide spectrum of conditions, ranging from clear "autoimmune" disease in some patients, to sicca complaints without evidence of a systemic immune process in elderly patients. Here, we review the clinical and laboratory features of patients referred for evaluation of sicca symptoms. In particular, we emphasize the need for care in choosing the site for minor salivary gland biopsy, and we describe the histologic features that aid in the evaluation of these biopsy specimens. In an attempt to identify a population of patients whose conditions might have a common etiopathogenesis and, thus, a common treatment program, we propose the following criteria for a diagnosis of SS: 1) objective evidence of keratoconjunctivis sicca, as documented by rose bengal or fluorescein dye staining; 2) objective evidence of diminished salivary gland flow; 3) minor salivary gland biopsy, obtained through normal mucosa, with the specimen containing at least 4 evaluable salivary gland lobules, and having an average of at least 2 foci/4 mm2; 4) evidence of a systemic autoimmune process, as manifested by the presence of autoantibodies, such as rheumatoid factor and/or antinuclear antibody. The diagnosis of "definite SS" would be made when all 4 criteria are met; the diagnosis of "possible SS" would be made when 3 criteria are present. Specific exclusions for this diagnosis are preexisting lymphoma, graft-versus-host disease, sarcoidosis, and acquired immunodeficiency disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Ocular and oral problems in arthritis. How to recognize, when to refer.

Recognition of oral and ocular problems is important in the management of arthritis. Although the focus of attention is usually on joint problems, patients with these chronic disorders require a comprehensive approach to their total medical care. Physicians need to watch for physical signs of significant inflammation and should alert their patients to watch for significant symptoms.

Sjogren's syndrome: a persistent clinical problem.

One hundred sixty patients with Sjogren's syndrome have been evaluated and managed at Scripps Clinic. Objective diagnosis has relied heavily on rose-bengal vital staining and corneal slit lamp examination to establish the presence of KCS and lip biopsy. The role of the head and neck surgeon in evaluating the patient with "dry mouth" is discussed. Usually Sjogren's syndrome is managed nonsurgically, but problems of abscess, recurrent infection, disfigurement, and malignant transformation may result in the need for total parotidectomy with nerve preservation. Radiation for Sjogren's syndrome is rarely, if ever, indicated. The etiology of Sjogren's syndrome may be closely tied to the homogeneous genetic background of its patients and the presence of a chronic immunogenic stimulus well recognized in the secondary form but less clear in the primary.

Primary Sjogren syndrome: clinical and immunopathologic features.

Primary Sjogren syndrome is an autoimmune condition in which dry eyes (keratoconjunctivitis sicca) and dry mouth (xerostomia) result from lymphocytic infiltration of lacrimal and salivary glands. Clinical and laboratory features of 60 primary Sjogren syndrome patients seen at our clinic during the past three years are presented. These patients illustrate the wide spectrum of extraglandular features that may occur as a result of lymphoid infiltration of lung, kidney, skin, stomach, liver, and muscle. They further emphasize the difficulty in classifying a patient as primary or secondary Sjogren syndrome (ie, sicca symptoms associated with systemic lupus erythematosus, rheumatoid arthritis, or scleroderma), particularly early in the disease course. As an initial step in understanding the pathogenesis, the lymphocytes that infiltrate the salivary glands and lymph nodes were characterized by using monoclonal antibodies that recognize distinct lymphocyte subsets and by using in vitro functional assays. These studies have demonstrated that affected tissues have infiltrates of T cells with helper/inducer activity and with a high frequency of "activation antigens." The immunohistologic techniques are useful in differentiating "benign" and "pseudolymphoma" lesions (both due predominantly to T cells) from non-Hodgkin lymphoma (usually due to B-cell infiltrates). Although there is no "cure" for primary Sjogren syndrome patient's symptoms may be significantly improved by measures aimed at prevention of ocular and dental complications and by the recognition of extraglandular features that may be amenable to specific treatment.

Lymphocyte phenotype and function in pseudolymphoma associated with Sjögren's syndrome.

Lymph node (LNL) and salivary gland lymphocytes (SGL) from three patients with pseudolymphoma and primary Sjögren's syndrome (1(0)SS) were characterized with monoclonal antibodies to demonstrate (a) a predominance of T cells (greater than 80%) reactive with anti-T cell antibodies OKT4 (greater than 70%) and OKT8 (less than 20%); (b) a high prevalence of activation antigens (greater than 50% of cells reactive with antibody OKT10 and anti-Ia antibody); (c) polyclonal B cells (8-15% of all cells expressing kappa or lambda); and (d) a specific B cell subset defined by reactivity with antibody B532 that was not present in their peripheral blood. In vitro functional studies showed that both SGL and LNL provided T helper activity for immunoglobulin synthesis and that this activity could be abolished by treatment with antibody OKT4 plus complement. The SGL and LNL exhibited little natural killer, antibody-dependent cellular cytotoxicity, or cytotoxic T cell activity. Normal karyotype was observed in SGL, LNL, and peripheral blood lymphocytes (PBL) from these patients. These findings indicate that pseudolymphoma in 1(0)SS results from the infiltration of salivary glands and extraglandular tissues by nonneoplastic T helper cells. Monoclonal antibodies provide an important tool to distinguish pseudolymphoma from non-Hodgkins (B cell) lymphomas that have a markedly elevated incidence in 1(0)SS patients. Our finding of T helper cells in pseudolymphoma tissues supports the hypothesis that chronic stimulation of B cells by helper T cells leads to eventual escape of a malignant B cell clone.

Immunohistologic analysis of lymphoid infiltrates in primary Sjogren's syndrome using monoclonal antibodies.

The characterization of lymphocytes infiltrating salivary glands in patients with primary Sjogren's syndrome (1 degree SS) yields insights to disease pathogenesis that are not revealed by studies of the corresponding peripheral blood lymphocytes (PBL) alone. We analyzed salivary gland lymphocytes (SGL) and PBL in 14 patients with untreated 1 degree SS using monoclonal antibodies that detect T cells, T cell subsets, B cells, and antigens associated with lymphocyte activation. A four-step biotin-avidin immunoperoxidase technique was used for salivary gland frozen sections; cell suspensions and PBL were stained cytofluorographically. A predominance of T cells (Leu 1 = L17F12; Leu 4 = OKT3) was found in SGL (greater than 75%) and PBL (76 +/- 9%) with the majority belonging to the Leu 3a (OKT4) subset. A minority of B cells (anti-delta, -kappa, -lambda) was present in both SGL and PBL; however, a subset of B cells defined by monoclonal antibody B532 was present in SGL (5 to 20%) but was absent from PBL. An increased prevalence of activation antigens (Ia; OKT10) was found on SGL T cells (greater than 50% positive) compared to PBL T cells (less than 15% positive). These studies demonstrate that specific antigenic markers on lymphocytes at the site of inflammation in 1 degree SS differ significantly from those of the corresponding PBL. These differences emphasize that theories of disease pathogenesis of 1 degree SS must include studies on SGL.

Characterization of the phenotype and function of lymphocytes infiltrating the salivary gland in patients with primary Sjogren syndrome.

Monoclonal antibodies directed against T-cell subsets, B-cell subsets, and monocytes were used to characterize the cells infiltrating the salivary glands of patients with primary Sjogren syndrome (1 degree SS). Analysis of stained frozen tissue sections and lymphocyte suspensions derived from salivary glands revealed the majority of infiltrating cells to be T cells (reactive with antibodies SC1 and Leu 4) of the Leu 3a+ subset (greater than 70% reactive). Of particular interest, a high frequency of Ia+ T cells (up to 50% of T cells) and B cells reactive with antibody B532 (5-15% of infiltrating cells) were found in salivary gland lymphocytes (SGL) but not in peripheral blood lymphocytes (PBL) of the same patients. Our finding that the phenotype of SGL was significantly different from PBL emphasizes the need to characterize lymphocytes at the site of tissue destruction. In vitro functional assays demonstrated that the OKT4+ SGL exhibited T-helper activity but not natural killer, antibody-dependent cellular cytotoxicity, or cytotoxic T-lymphocyte activity. Of note, rheumatoid factor (IgM anti-IgG) was produced by SGL but not by the corresponding PBL. Our studies on SG-lymphocyte function represent an early step in elucidating the cellular and subcellular events responsible for this autoimmune disease.