Amplification of extracellular matrix and oncogenes in tat-transfected human salivary gland cell lines with expression of laminin, fibronectin, collagens I, III, IV, c-myc and p53.

Considerable progress has been made in the transfer of foreign genes into salivary glands in vivo using adenovirus vectors in rats. In an attempt to avoid the transient expression inherent, when using these vectors, retroviral vectors and human cell lines where used here in attempt to develop an in vitro model of HIV-associated salivary gland disease. The HIV-1-tat protein is increasingly implicated in the pathogenesis of the AIDS through altering the expression of strategic cellular genes. The purpose of this study was to transfect human salivary gland (HSG) cell lines in vitro, with the pHIV-1/LTR-tat plasmid, and examine the effect of tat on expression of matrix and basement membrane genes known to be important in the pathogenesis of salivary gland disease. HSG cells were transfected with HIV-1-tat plasmid by the lipofection method. Transfection was confirmed by polymerase chain reaction (PCR) and Southern blot, which verified that tat-specific DNA was present. Tat-mRNA was analysed by Northern blotting and quantified by reverse transcriptase polymerase chain reaction (RT-PCR) to demonstrate its expression. Numerous clones were found to contain integrated tat DNA sequences and analysis of mRNA showed stable expression of tat-specific RNA. Further analysis of mRNA expression for various marker proteins important in HIV pathogenesis showed that the HSG cell line transfected with HIV-1-tat, was associated with significant induction of mRNA expression for extracellular matrix protein. Tat-amplified transcription of the major basement membrane protein laminin, as well as of fibronectin, collagen I and III, and c-myc oncogene was demonstrated. Conversely, expression of p53 suppressor gene mRNA was reduced. Post-transfection expression of collagen IV was erratic and inconclusive. It was concluded that the presence of HIV-tat in this in vitro model of salivary ductal epithelial cell model alters the mRNA expression of several matrix, basement membrane and oncoproteins known to be involved in HIV pathogenesis. These cell lines provide a useful system for studying the role of tat in the immunopathogenesis of HIV-associated salivary gland disease.

Characteristics of salivary diffuse infiltrative lymphocytosis syndrome in West Africa.

OBJECTIVE: To determine the prevalence of diffuse infiltrative lymphocytosis syndrome (DILS) in the minor salivary glands of 30 African Cameroonian adults with the acquired immunodeficiency syndrome (AIDS). DESIGN: Salivary gland tissue was analyzed using a modified classification system that was developed to aid the diagnosis of Sjögren syndrome. The advantages and disadvantages of this approach are discussed. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded, hematoxylin-eosin-stained biopsy sections were prepared for 30 patients with AIDS, 26 healthy individuals who declined human immunodeficiency virus (HIV) testing, and 4 seronegative healthy controls. Tissues were immunostained for CD4/CD8+ lymphocytes and cytomegalovirus (CMV), and transmission electron microscopy was performed to locate viral particles. Patients were tested for HIV-1 and HIV-2 by the HIV/Chek System 3 or CAMSTIX-HIV-1 and HIV-2 assay. RESULTS: Severe salivary ductal atypia (96%) was the feature most strongly associated with AIDS, and the lymphocytic focus score was the second histologic feature most strongly correlated with AIDS. Forty-eight percent of patients with HIV-1 infection had more than 1 lymphocytic focus in a minor salivary gland. These lymphocytes were primarily CD8+. We report, to the best of our knowledge, the first case of multinucleated salivary duct epithelial cells in minor salivary glands also containing enveloped virus particles. All cases were negative for CMV. CONCLUSIONS: The prevalence of DILS in West Africans with AIDS appears higher than the prevalence reported in whites from the United States and Europe and in blacks from the United States, a group that has been reported to have a greater incidence of DILS than whites. This discrepancy may be related to differences in patient selection criteria. The determination of lymphocytic focus score, as used in the diagnosis of Sjögren syndrome, with the adjunct of ductal atypia is useful for assessing DILS. The impact of patient selection, drug therapy, and parasites on salivary gland pathology is discussed.

Interferon-mediated block in cell cycle and altered integrin expression in a differentiated salivary gland cell line (HSG) cultured on Matrigel.

Sjögren's syndrome (SS), an idiopathic, autoimmune exocrinopathy, is partly characterized by diminished salivary flow, acinar cell atrophy, and increased expression of several cytokines. Several in vivo characteristics of the sialoadenitis are also evident in a human salivary gland ductal epithelial cell line (HSG) treated with cytokines. HSG cells differentiate to the acinar phenotype when cultured on Matrigel (Becton Dickinson, Bedford, MA), a basement membrane extract. To elucidate mechanisms of salivary gland pathology, the effects of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on cell cycle progression and integrin expression were evaluated in HSG acinarlike cells. Flow cytometry experiments showed that cytokine treatment for 2 days arrested cells in G(1) phase of the cell cycle, and this preceded significant morphologic changes and decreased viability. Whereas only modest cytokine-mediated increases in protein expression for the alpha 3 and beta 1 integrin subunits were seen by immunoprecipitation, a form of alpha 3 integrin displaying enhanced electrophoretic mobility was evident after 6 days of cytokine treatment. To our knowledge, this is the first report demonstrating an IFN-mediated alteration in the electrophoretic mobility of integrin subunits. From this study, it was evident that the combination of IFN-gamma and TNF-alpha resulted in a block in G(1) phase for acinar cells before accumulation of the alpha 3 integrin variant or significant degenerative cellular changes. Information from the present and previous studies suggests that cytokines may alter the pattern of integrin expression and block cell cycle progression in salivary gland cells grown in three-dimensional acinarlike clusters. These experiments may provide a new cell culture model to study the effects of cytokines in normal and diseased salivary glands, including SS.

Gonadotropin-releasing hormone increases CD4 T-lymphocyte numbers in an animal model of immunodeficiency.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) possesses immunostimulatory properties. We have previously demonstrated that GnRH antagonists decrease lymphocyte numbers in an animal model of autoimmune disease. We speculated that the converse might be true, that GnRH administration would increase lymphocyte numbers or alter lymphocyte subsets in an immunodeficiency state. OBJECTIVE: Our purpose was to test the hypothesis that GnRH agonist would increase IgG and CD4 counts in a rat model of immunodeficiency independently of gonadal steroids. METHODS: We used diabetes-prone (DP) BB rats. This model has been characterized to have an AIDS-like lymphocyte profile, with lymphopenia and depressed CD4 counts. Ovariectomized female DP rats were randomized to receive subcutaneous injections with GnRH or vehicle 6 times weekly. DR rats were ovariectomized and treated with vehicle as controls. We performed flow cytometric analysis and complete blood cell counts at baseline, 3.5 weeks, and 7 weeks of treatment. We also measured total serum IgG and luteinizing hormone levels. RESULTS: GnRH administration significantly increased total serum IgG levels in DP rats compared with vehicle. The percentages of CD4(+) cells in blood were also significantly increased in the GnRH-treated group compared with the vehicle-treated group and compared with baseline. Similarly, the absolute numbers of CD4(+) positive T cells were increased over controls at 7 weeks. The effects of GnRH were specific for the CD4 subset because there were no significant differences in numbers of CD8(+) positive cells between the 2 treatment groups. CONCLUSION: GnRH shows potential utility as an immunostimulatory agent in immunodeficient states manifesting diminished numbers of immunocompetent CD4(+) T lymphocytes.

Cytokine-mediated stimulation of laminin expression and cell-growth arrest in a human submandibular gland duct-cell line (HSG).

Increased expression of laminin and various cytokines, including interferon-y (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) has been demonstrated in minor salivary glands from patients with Sjögren's syndrome. Previous reports state that exposure of a human salivary-gland cell line (HSG) to IFN-gamma results in cellular changes similar to those in vivo Sjögren's syndrome. To begin studies of the cause of increased laminin expression in salivary glands in Sjögren's syndrome and laminin's role in the pathological process, the effects of IFN-gamma on laminin expression and growth of HSG cells were examined here. Subconfluent cultures of HSG cells were treated or not with IFN-gamma (1000 units/ml) for 1, 3 or 6 days. Immunoprecipitation showed that the expression of cell-associated laminin was significantly greater in IFN-gamma-treated cells at 3 or 6 days than in untreated cells, while no significant differences in laminin counts precipitated from the media were evident among any of the IFN-gamma-treated or untreated samples. Western blot analysis strongly suggested that this immunoprecipitated product is a dimer of the beta- and gamma-chains of laminin. Intracellular laminin was demonstrated immunocytochemically in a distinct, perinuclear pattern in both cytokine-treated and untreated cells. However, only faint staining for type IV collagen, and no staining for fibronectin were evident in untreated and cytokine-treated cells. An RNase protection assay showed only slight upregulation of the laminin beta-chain mRNA at 3 days, but no significant difference at 6 days of treatment. Taken together, these data suggest enhanced accumulation of a dimer of laminin beta- and gamma-chains in the cytoplasm of cytokine-treated HSG cells. However, mRNA for glyceraldehyde 3-phosphate dehydrogenase was significantly reduced at 6 days of treatment, suggestive of cytokine-mediated metabolic abnormalities. IFN-gamma treatment also resulted in significant reductions in cell numbers over time, in agreement with previous reports. Treatment of HSG cells for 3 days with IFN-gamma (1000 U/ml) and TNF-alpha (20 U/ml) resulted in no significant changes in cell proliferation or laminin protein and/or mRNA species compared to cells treated with IFN-gamma alone. Karyotype analysis of HSG cells revealed human chromosomes with triploid chromosome numbers and rearrangements, characteristic of transformed cells. These data demonstrate that IFN-gamma increases the amount of intracellular laminin beta-gamma dimers while decreasing cell growth. Further studies are required to define an interaction between laminin expression and the growth and viability of HSG cells.

Sjögren's syndrome salivary gland immunopathology: increased laminin expression precedes lymphocytic infiltration.

Previous studies have demonstrated increased expression of a laminin-like protein in labial salivary glands from Sjögren's syndrome (SS) patients. The objective of the present study was to verify the identity of this protein and to compare lymphocytic infiltration and laminin expression in minor salivary gland ductal epithelium. This was carried out by comparing laminin protein and laminin mRNA expression in 13 SS patients, 11 normal controls, and 12 patients with non-specific sialoadenitis. Laminin protein and laminin mRNA expression was determined using immunoperoxidase (IP) and in situ hybridization (ISH) techniques, respectively. In addition, the relationship between lymphocytic infiltration and laminin expression was evaluated on adjacent serial salivary gland sections from SS patients, using routine histological methods and IP immunohistochemistry. Biopsies from SS patients showed significant increases in staining for both laminin protein and laminin mRNA compared to normal controls. On seven of the eight SS samples that showed significant laminin protein staining, the ductal epithelial staining occurred in the absence of periductal lymphocytic foci. Results of the ISH assay strongly suggest that the increased expression of the laminin-like protein is laminin, since the cDNA probe was specific for the B1 chain of laminin. In addition, this increased expression in ductal epithelial cells occurs without significant lymphocytic infiltration. These studies provide further evidence that altered laminin expression is an early event associated with salivary gland pathology in SS, since these data demonstrate a potential pathologic event prior to the arrival of lymphocytes. Further studies are underway to examine the relationship between laminin and lymphocytic infiltration in salivary gland pathology.

Dental professional's role in diagnosing Sjögren's syndrome.

In this investigation, a written questionnaire was used to assess patient's perceptions of health care professionals' knowledge about Sjögren's syndrome. Results show that dental and other health care professionals need to be better informed about the nature of Sjögren's and its symptoms so they can diagnose patients. Early diagnosis may help mollify symptoms and reduce anxiety or other perceived harmful consequences.

Rare primary signet ring carcinoma of the pancreas.

Signet ring carcinoma of the pancreas is a rare mucin-producing variant occurring in less than 1% of pancreatic carcinoma. The fourth leading cause of cancer deaths in the U.S., pancreatic carcinoma is increasing in incidence and mortality. It is difficult to detect during the early stages of the disease and usually become apparent when extrahepatic biliary obstruction occurs. Early radiologic findings may be negative and there is no well defined tumor marker for this neoplasm.

Acquired immunodeficiency syndrome--a review and update. History, characteristics of human immunodeficiency virus infection, its replication, biologic factors, and pathogenicity.

The search to find a cure for acquired immunodeficiency syndrome has led to an unprecedented explosion of knowledge about viruses in general, especially retroviruses, of which the human immunodeficiency viruses (HIV) types 1 and 2 are members. We attempt to describe how retroviruses work, particularly HIV-1, and what regulates their expression in the host-cell system. Viral stability and its implications for health care workers is also discussed. One of the many mysteries of HIV is the ability it has to elude normal immune responses. Even though B- and T-cell responses are mounted by people infected with HIV, such responses are restricted and increasingly ineffective as the disease progresses. Host susceptibility plays a major role in the virus' ability to infect individuals. Finally, some current treatment options and the key role of the family physician in the battle against acquired immunodeficiency syndrome are discussed.

Monoclonal antibody detection of laminin in minor salivary glands of patients with Sjögren's syndrome.

The potential roles of the basement membrane proteins, laminin and fibronectin, and the cytoskeletal protein, tubulin, were assessed in the pathogenesis of Sjögren's Syndrome (SS) by comparing their expressions in SS with normal labial salivary gland (LSG) tissue. Laminin, fibronectin and tubulin expression were determined using well characterized monoclonal antibodies in the peroxidase anti-peroxidase technique on formalin-fixed LSG's from patients with SS and normal controls. Characteristic periductal staining for laminin occurred in the LSG's of 14/18 SS patients scored by one observer and 16/18 scored by the second observer. Staining of LSG's for laminin occurred in 2/35 control specimens consisting of 15 normal LSG's and 20 inflammatory lesions with attached normal LSG. The staining which occurred in the two controls was diffuse and 'non-specific' in one case, and indistinguishable from the characteristic periductal staining found in SS in the other case. Among the 20 controls containing inflammatory lesions, four showed diffuse staining for laminin within the actual lesion, but the adjacent LSG's did not stain. No statistically significant difference between SS and normal tissues stained by anti-fibronectin and anti-tubulin was observed. The study concluded that there was an increase in laminin or a laminin-like substance on salivary ductal epithelia of SS patients. This suggests a potential role for laminin in the pathologic mechanism and may indicate that increased laminin expression is a marker for SS.

Field evaluation of a pregnancy immunoassay for the detection of oestrone sulphate in goats.

The concentration of oestrone sulphate in whey obtained from 66 pregnant dairy goats was measured by direct radioimmunoassay. The mean time at which pregnancy was first detected was day 41 of gestation. Levels remained low (37 pmol/l-0.96 nmol/l, mean = 167 pmol/l) until week 5 of gestation when they rose rapidly. The test had an accuracy of 95.6%, was able to distinguish true from pseudopregnancy, and suggested that pregnancy in females carrying multiple fetuses could be detected earlier, possibly as a result of the fetal-placental origin of oestrone sulphate.

Enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against the H-Y antigen on murine sperm.

An enzyme-linked immunosorbent assay (ELISA) using murine spermatozoa and thymocytes is described for the detection of anti-H-Y antibodies. ELISA is more sensitive and reproducible than cytotoxicity and the PA-SRBC test. It is particularly useful for the rapid screening of a large number of samples.

Demonstration in vitro of cytotoxic T cells with apparent specificity toward tumor-specific transplantation antigens on chemically induced tumors.

Chemically induced tumors of mice exhibit apparently unique antigenicity upon syngeneic transplantation into appropriately immunized hosts. An in vitro counterpart of this pattern in terms of specificity has not been reported. Data are presented that demonstrate that immune peritoneal exudates contain cells cytotoxic for the specific immunogen tumor but with rare exceptions, not toward other syngeneic methylcholanthrene-induced fibrosarcomas. Only tumors highly immunogenic by transplantation criteria induce cytotoxic PEC regularly; nonimmunogenic tumors consistently fail to do so. The effector cell responsible is eliminated by pretreatment with anti-Thy.1 but not anti-Ig plus complement. In concomitant experiments, PEC populations cytotoxic in vitro also conveyed adoptive protection against the specific tumor in syngeneic hosts. This in vitro assay appears to provide a tool for studying T cell-mediated cytotoxicity toward a set of unique surface antigens present on chemically induced tumors.