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Biomedical subjects

S N Breit

Publications and source records attributed to S N Breit.

At least 55 records · Page 3Linked to original sources
PubMed1992

Restoring antibody activity of a monoclonal anti-RNP antibody by dissociative HPLC. Demonstration of blocking antibody binding sites with antigen released from effete hybridoma cells.

In biomedical research, monoclonal anti-nuclear antibodies have a number of advantages over polyclonal antibodies in terms of both specificity and reproducibility. However, there are some potential problems in the preparation of monoclonal antibodies. A well characterized mouse monoclonal anti-ribonucleoprotein antibody (anti-RNP antibody, 2.73) known to function in Western blotting was found to lose this activity when produced in vitro from long term hybridoma cell culture. Whilst it could no longer detect RNP antigen by Western blotting, it could still function effectively in affinity purification of RNP antigen. Further studies suggested that this was due to blocking of antibody binding sites by RNP antigen released from effete hybridoma cells in culture. The activity of the antibody in affinity purification was retained because the antigen was stripped away by repeated elutions with 6 M urea. HPLC gel filtration in the presence of 6 M guanidine was able to restore the antibody activity of the protein A purified monoclonal antibody. This finding has important general consequences for the preparation of monoclonal antibodies against antigens present in hybridoma cell culture media.

Animals↗
PubMed1992

Monocyte proliferation in a cytokine-free, serum-free system.

The aim of this study was to establish a cytokine-free, serum-free system which would enable the long-term survival and proliferation of human peripheral blood monocytes. Monocytes were isolated from peripheral blood mononuclear cells (PBMC) by adherence to untreated plastic petri dishes and maintained up to 6 weeks in serum-free medium (SFM) consisting of IMEM, insulin, transferrin, sodium selenite and BSA. Maximal cell proliferation occurred during the first 2 weeks of culture and corresponded to the appearance of large numbers of pure, nonadherent culture-derived macrophages. Monocyte maturation was characterised by the modulation of specific cell surface antigens. The percentage of cells staining for the transferrin receptor increased with time, whereas the percentages of cells expressing CD11b, CD11c and HLA-DR remained greater than 60% for the 15 days studied. The mean fluorescent intensities (MFI) of all these antibodies increased significantly with time. The only differences found between the adherent and nonadherent cells, using the above antibodies, were with the MFI for CD11b and CD11c. In both cases, the intensity of staining was significantly greater in the adherent cells. Estimation of cytokine production by cells maintained for 5 weeks in SFM found that they constitutively produced large amounts of macrophage colony-stimulating factor (M-CSF) in the absence of any exogenous stimuli. These cells were also found to secrete high levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) during the 1st week and granulocyte macrophage colony-stimulating factor (GM-CSF) during the 3rd week. However, the addition of exogenous GM-CSF (5 U/ml, S5) was found to significantly inhibit monocyte proliferation up to 17 days. This is the first report of proliferation associated with long-term survival of culture derived macrophages in a serum-free, cytokine-free system.

Antigens, Surface↗
PubMed1992

Quantitative pulmonary gallium scanning in interstitial lung disease.

The mechanisms responsible for gallium uptake in chronic, non-infective, diffuse lung disease are not completely understood. This study attempted to clarify some of them. A lung/liver gallium index was calculated in 113 subjects, some normal and some with various interstitial lung diseases, predominantly those associated with connective tissue disease. The mean gallium index was significantly higher in the groups with active interstitial lung disease (5.7) and non-infective bronchiolitis (4.1) compared with non-smoking normals (3.0; P less than 0.05). To investigate the mechanisms responsible for gallium uptake, the gallium index was correlated with bronchoalveolar lavage findings, respiratory function tests and clinical features. Significant correlations (P less than 0.05) were found with age in non-smoking normals; lavage macrophages in smoking normals; age but no other parameter in bronchiolitis; lavage lymphocytes, lavage albumin and improvement in diffusion capacity for carbon monoxide in those with active interstitial lung disease. It is concluded that in normal smokers gallium uptake may be due to a macrophage-mediated process. Gallium uptake in active interstitial lung disease associated with connective tissue disease appears to be an immunological process in which transport and retention of gallium is associated with that of albumin.

Bronchiolitis↗
PubMed1992

Microplate reader-based quantitation of collagens.

By using a picrosirius dye, sensitive and specific staining of collagens plated in microtiter wells was achieved. The range of detection was from 0.5 to 20 micrograms. Human collagen types I, III, IV, and V were tested and able to be detected by the method. The dye did not bind to acetylcholinesterase or elastin. It did bind to C1q to some extent but this is not surprising since the molecule contains some triple helical collagen-like structures. A comparison performed between this assay and a colorimetric assay for hydroxyproline using tissue culture supernatants gave similar results for both samples. Due to its simplicity and sensitivity this assay will be most useful in laboratories where large numbers of samples must be screened for collagen production.

Acetylcholinesterase↗
PubMed1992

Mitotic activity in the dermis of mice during graft versus host disease: the role of fibroblast replication in dermal fibrosis.

When immunocompetent cells are transferred to an allogeneic, immunologically compromised host a complex series of cellular events are initiated, referred to as graft versus host disease. This results in widespread organ damage with fibrosis being a prominent feature. The pathologic fibrosis may result from an increase in fibroblast numbers, an increase in collagen produced from individual fibroblasts, or a combination of the two processes. The relative contribution of fibroblast replication to the pathologic fibrosis seen in graft versus host disease has not been directly determined previously, and this is the main object of this paper. Graft versus host disease was induced by the transfer of lymphoid cells from B10D2 mice to irradiated Balb/c recipients. In order to study the mitotic activity of dermal cells following bone marrow transplantation, a thymidine anologue, bromo-deoxyuridine (BrDU), was administered to mice using an osmotically driven, implantable infusion device. The labeled cells were visualized immuno-histochemically and studied at weekly intervals. There is intense mitotic activity in the basal layer of the epidermis and the acrosyringal epithelium from the second week. Evidence of increased mitotic activity in the epidermis persisted until the fifth week post-transplantation. Fibroblast replication was seen from the end of the third post-transplant week. Dermal collagen deposition also occurred at this time. Peak mitotic activity was present at the end of the fourth week and was less pronounced by the fifth week. It was especially evident in the upper dermis where the developing collagen layer was being deposited. To our knowledge this is the first direct demonstration of fibroblast proliferation in an immunologically mediated fibrotic disorder. It is concluded that fibroblast replication is an important mechanism leading to the pathologic fibrosis seen in graft versus host disease and, by analogy, probably other types of immunologically mediated fibrosis.

Animals↗
PubMed1992

Fibroblast growth factors in connective tissue disease associated interstitial lung disease.

Fibrosis is a major cause of morbidity and mortality in chronic inflammatory diseases, especially interstitial pulmonary disorders. Fibroproliferation is an important part of this fibrotic response, and is mediated largely through growth factors such as platelet-derived growth factor (PDGF), insulin-like growth factor (IGF) I and tumour necrosis factor-alpha (TNF-alpha). Although there is some evidence implicating these cytokines in fibrotic disorders, strong evidence in vivo is almost nonexistent. In order to ascertain the role that these factors play in inflammatory lung disorders associated with connective tissue diseases, alveolar mononuclear cells have been obtained from subjects by bronchoalveolar lavage and assessed for the spontaneous release of fibroblast growth factors. The study population consisted of subjects with a variety of different connective tissue disorders, both with and without inflammatory pulmonary complications. It was found that lavage cells spontaneously secreted fibroblast growth factor activity over 24 h with maximum activity detected at 6 to 12 h. Growth factor activity could be detected in most subjects with connective tissue disease-associated inflammatory lung disease and some normal subjects, but the amount of growth factor activity was much higher in the former than in the latter. By means of antibody depletion experiments all growth factor activity from lavage cells of normal patients was attributable to TNF-alpha while patients with interstitial lung disease secreted large amounts of PDGF and fibronectin in addition to TNF-alpha. Approximately 40-50% of the total released growth factor activity could be accounted for by PDGF, and 100% by the combination of PDGF, TNF-alpha and fibronectin. While TNF-alpha is released from the bronchoalveolar lavage cells of many subjects, in addition, many patients with interstitial lung disease also release spontaneously, large amounts of fibroblast growth factor activity attributable to PDGF and fibronectin.

Bronchoalveolar Lavage Fluid↗
PubMed1992

The differing patterns of interstitial lung involvement in connective tissue diseases.

Connective tissue disorders (CTD) have a relatively high incidence of pulmonary complications but their delineation has been hampered by difficulties inherent in the diagnostic techniques. One fresh approach to this problem is based on a clustering method that uses data from 8 separate investigations to cluster the patients into 4 distinctly separate categories representing normal nonsmoker, normal smokers, those with active interstitial lung disease and those with bronchiolitis. Using this method, a large group of patients with CTD have been examined to assess the nature and extent of their pulmonary complications. Subjects in the first cluster had a normal test profile across all variables, and included no current active smokers. The normal smoking cluster contained only smokers who had a high total lavage cell number with a relative increase in macrophages and a decrease in lymphocytes. In this group, all respiratory variables were normal with the exception of a mildly depressed DLCO, also known to be associated with smoking. The general characteristics of the active interstitial lung disease cluster was a markedly depressed DLCO indicating impaired gas exchange, and elevated gallium index, bronchoalveolar lavage (BAL) cell number and neutrophil and lymphocyte percentages, all indicating an active inflammatory process. However more careful analysis of this group suggested the presence of 2 subgroups, one with a lymphocytic alveolitis, and another with a neutrophilic alveolitis. The bronchiolitis cluster characteristics were a markedly depressed maximal mid expiratory flow rate and raised BAL lymphocyte percentage and gallium index. The distribution of patients within the cluster groupings suggested that Sjögren's syndrome, often an accompaniment of other CTD, is most frequently associated with pulmonary disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Identification of the major fibroblast growth factors released spontaneously in inflammatory arthritis as platelet derived growth factor and tumour necrosis factor-alpha.

Rheumatoid arthritis is characterized by chronic inflammation and proliferation of a number of important elements within the joint including the synovial fibroblasts. Elevated levels of a number of cytokines such as Il-1, IL-2, IL-6, interferon-gamma (IFN-gamma), transforming growth factor-beta and tumour necrosis factor-alpha (TNF-alpha) have been detected in the synovial fluid of patients with rheumatoid arthritis and other inflammatory arthritides. It seems likely that local release of such mediators may be responsible for the proliferation and overgrowth of connective tissue elements in these disorders. In order to ascertain whether there was evidence to suggest local production or release of fibroblast growth factors in the joint in inflammatory arthritis, and to determine their identity, cells were obtained from the synovial fluid of 15 patients with chronic inflammatory arthritides. All subjects' synovial fluid cells spontaneously released growth factor activity for fibroblasts. This was present in large amounts, being detectable in culture supernatants diluted to a titre of at least 1/625. By a series of depletion experiments using solid-phase bound antibodies to cytokines, it was possible to demonstrate that this activity was due to TNF-alpha and platelet-derived growth factor (PDGF). Thus, this study showed for the first time that functionally active PDGF was released from synovial fluid cells. Both PDGF and TNF-alpha appeared to contribute in approximately equal amounts to this fibroblast growth factor activity, and were synergistic in effect. Thus this study provides evidence for the local production and release of these two cytokines and suggests that together they are the dominant factors in fibroblast proliferation within the synovial cavity.

Arthritis, Rheumatoid↗
PubMed1991

Antibody penetration of viable human cells. I. Increased penetration of human lymphocytes by anti-RNP IgG.

Antibody penetration of viable cells and interaction with intracellular antigens may have major consequences for immunopathological processes in connective tissue diseases. We have reported previously that antibody can penetrate viable human lymphocytes. To assess further the role of antinuclear antibodies in this process, peripheral blood lymphocytes (PBMC) were incubated with FITC-conjugated IgG fractions from sera containing anti-RNP (anti-RNP IgG), Ro(SS-A), La(SS-B) and dsDNA antibodies and control sera for 24 h. Using crystal violet to quench cell surface staining, intracellular fluorescence of viable lymphocytes was quantified on the flow cytometer. It was noted that anti-RNP IgG entered 46.4 +/- 7.2% of lymphocytes which was significantly higher than anti-Ro(SS-A) (29.9 +/- 4.1%, P less than 0.05), La(SS-B) (22.0 +/- 7.5%, P less than 0.01) IgG and control IgG (28.8 +/- 2.1%, P less than 0.05) and not statistically different from anti-dsDNA IgG (32.6 +/- 14.3%). Inhibition experiments showed that the increased number of cells penetrated by anti-RNP IgG was a specific process. Time-course studies showed that anti-RNP IgG entry into cells was different from pooled control IgG. With anti-RNP IgG, positive-staining lymphocytes gradually increased in number from 12 to 24 h incubation, whilst with pooled control IgG, the peak was reached within 5 min. Dual staining experiments suggested that whereas both anti-RNP IgG and pooled control IgG entered B and NK cells, anti-RNP IgG also entered T cells. Using IgG F(ab')2 and Fc fragments from either anti-RNP IgG or pooled control IgG to compete with their FITC-conjugated counterparts indicated that the entry of anti-RNP IgG into-viable cells appeared to involve both F(ab')2 and Fc fragments, and pooled control IgG depended exclusively on the Fc portion of IgG. Further investigation by incubating anti-RNP IgG with 35S-methionine-labelled monocyte-depleted PBMC (MD-PBMC) suggested that anti-RNP IgG might react with the corresponding antigens either on the cell surface or within the cytoplasm.

Animals↗
PubMed1991

Interstitial lung disease associated with connective tissue diseases. The use of statistical structure analysis in model development.

Interstitial lung disorders associated with the connective tissue diseases are thought to be quite common, but their precise analysis is fraught with difficulty because of the absence of a Gold Standard that, short of open lung biopsy, is not available. Analysis is hampered by the biologic variability in test results, large overlap between the normal and the disease population for individual tests, the confounding effect of smoking, and the complexities of viewing multidimensional data. In order to better define the pattern of the lung involvement, an entirely different approach has been adopted that is based on the use of graphic and clustering techniques to define the multivariate structure inherent in the data, then discriminant analysis to assign patients into distinct clusters. Using this approach it has been possible to group patients into four distinct clusters based on the result of respiratory function studies, gallium lung scan, bronchoalveolar lavage, and smoking status. These clusters are a normal smoking cluster and a normal nonsmoking cluster, a cluster of patients with active ILD, and a cluster with bronchiolitis. The validity of this method has been verified, and an algorithm has been developed that allows the assignment of any new patient entering the study into one of these clusters. This type of analysis offers a valuable new approach to the categorization of patients into distinct groups based on the results of multiple investigations.

Algorithms↗
PubMed1991

Antibodies to tubulin and actin bind to the surface of a human monocytic cell line, U937.

Intermittent reports of cytoskeleton proteins (actin and tubulin) on the cell surface have appeared over the last 13 years. Whereas most have concentrated on lymphocytes, this study provides evidence for the presence of these proteins on the surface of a human cultured monocyte-like cell line, U937. Both actin and tubulin were detected on the surface of U937 cells by flow cytometry, using an indirect staining procedure based on biotin-streptavidin-phycoerythrin, chosen for greater sensitivity. By use of this procedure, the majority of viable unstimulated U937 cells stained positively for actin and tubulin, although the level of fluorescence intensity was low. With an antibody specific for tyrosine-tubulin, most of the surface tubulin was also found to be tyrosinylated. For vimentin, an intermediate filament protein abundantly present in the cytoplasm of U937 cells, no staining could be detected. Confirmation of the flow cytometry data for surface actin and tubulin on unstimulated U937 cells was achieved by direct vesualization using a confocal laser scanning microscope. When U937 cells were activated with PMA and LPS, a marked reduction in the level of cell surface actin and tubulin occurred. The role of cell surface actin and tubulin on unstimulated U937 cells, in terms of monocyte function, remains to be elucidated.

Actins↗
PubMed1990

Interaction of immune and connective tissue cells: I. The effect of lymphokines and monokines on fibroblast growth.

In order to determine if mononuclear cells may be secreting factors capable of modulating fibroblast growth, the in vitro proliferative response of fibroblasts to cytokines known to be secreted by mononuclear cells was measured, using both growth arrested and proliferating cells. Of the cytokines tested, which included interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-6 (IL-6), interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma), transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), platelet derived growth factor (PDGF), and tumor necrosis factor-alpha (TNF-alpha), only TNF-alpha and PDGF had demonstrable growth factor activity. Neither IL-1 alpha nor beta showed any true growth factor activity but were able to enhance the replication of already proliferating cells. No inhibition of proliferation was noted by any of the cytokines with the exception of TNF-alpha and TGF-beta. TNF-alpha in doses greater than 500 ng/ml caused fibroblast death, probably by a prostaglandin related mechanism as fibroblasts remained viable, although non proliferative, when assayed in the presence of indomethacin, a known inhibitor of prostaglandin E2 (PGE2) synthesis. TGF-beta was inhibitory to proliferation at doses greater than 100 ng/ml, while fibroblasts remained viable. This effect was not influenced by indomethacin and hence is unlikely to be PGE2 related.

Cell Division↗
PubMed1990

Hypoxia during bronchoalveolar lavage.

The aim of this study was to determine the degree and duration of hypoxaemia during bronchoalveolar lavage (BAL) and to examine the effect of supplemental oxygen on this response. Transcutaneous oxygen tension (PO2) was recorded continuously in 22 patients having bronchoscopy alone (Group 1), and during BAL in patients with a variety of connective tissue disorders. Thirty eight of these patients were breathing room air (Group 2) and 28 were given supplemental oxygen (Group 3). The mean fall in PO2 in Group 1 was 12 +/- 3 mmHg and the PO2 in these subjects rose promptly to the initial value once the bronchoscopy was finished. The mean falls in Groups 2 and 3 were 24 +/- 4 and 32 +/- 5 mmHg and the mean times taken for the PO2 values to return to baseline after the procedure were 47 +/- 9 and 53 +/- 10 minutes respectively. The PO2 fell to less than 60 mmHg in 76% of the patients in Group 2 but in only 25% of those in Group 3. It is recommended that supplemental oxygen be given to all patients having BAL during and for one hour after the procedure and that oxygenation be monitored continuously throughout the bronchoscopy. Arterial blood gases should be always measured prior to bronchoscopy and BAL. In those cases where it is not possible to maintain the PaO2 at 70 mmHg or more the procedure should be undertaken with great care and with due consideration of the risk of the hypoxaemia that may occur.

Adult↗
PubMed1989

Response of microvascular endothelial cells to biological response modifiers.

This study investigated in vitro biological response modifiers (BRM) possibly involved in initiation and regulation of human capillary endothelial cell (HCEC) growth. An indicator assay was developed using tritiated thymidine to measure increased DNA turnover. Purified or recombinant BRMs tested singly and in combination included: interferon (IFN; alpha and gamma), tumour necrosis factor (TNF; alpha and beta), transforming growth factor (TGF; alpha and beta), rIL-1, rIL-2, pIL-2, platelet derived growth factor (PDGF) and retinoic acid. Under limiting serum conditions only rIL-2 (greater than or equal to 10 U/mL) caused proliferation of the cells. gamma IFN together with TNF alpha(500 U/mL of each) was cytotoxic. Under maximal stimulus conditions, a cytostatic effect resulted from exposing HCEC to: alpha or gamma IFN (greater than or equal to 1000 U/mL), TGF beta (greater than or equal to 5 ng/mL), rIL-1 (greater than or equal to 0.5 U/mL) and rIL-1 plus gamma IFN. gamma IFN (500 U/mL) plus TNF alpha exhibited synergism in the inhibition of proliferation and produced a cytotoxic effect at TNF alpha concentrations greater than or equal to 500 U/mL. By contrast, rIL-2 enhanced proliferation at greater than 5 U/mL. When rIL-2 was combined with gamma IFN, an inhibitory effect on proliferation was observed, although to a lesser extent than gamma IFN alone. Pretreating the cells with 100 U/mL gamma IFN prior to rIL-1 or IL-2 exposure produced no change in the trends observed above.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Division↗
PubMed1989

The role of bronchoalveolar lavage in the assessment of diffuse lung diseases.

Bronchoalveolar lavage is a safe and simple technique for sampling the inflammatory cells of the lung. However, while its use in the evaluation of pulmonary pathogenic mechanisms is both well accepted and described, its clinical utility is more controversial. Marked variation in results may occur through variation in the lavage procedure. Standardisation of the lavage technique and laboratory processing of the specimen are essential for reliable results. This review examines the current clinical role of bronchoalveolar lavage in the assessment of patients with diffuse lung diseases, and immunocompromised patients with pulmonary infiltrates. In this latter category, for patients with Acquired Immunodeficiency Syndrome, lavage is of equal efficacy to lung biopsy and can establish the cause of pulmonary infiltrates in over 90% of cases. Bronchoalveolar lavage can detect abnormalities in patients with diffuse lung diseases prior to the development of irreversible fibrosis. Lavage features have been described for sarcoidosis, cryptogenic fibrosing alveolitis, extrinsic allergic alveolitis, connective tissue diseases, and asbestosis. In cryptogenic fibrosing alveolitis lavage data may be used to indicate a subsequent deterioration in the patient's condition, or predict a favourable response to therapy.

Alveolitis, Extrinsic Allergic↗
PubMed1989

The presence of Sjögren's syndrome is a major determinant of the pattern of interstitial lung disease in scleroderma and other connective tissue diseases.

A number of patients with scleroderma, Sjögren's syndrome and other connective tissue diseases (CTD) were assessed to ascertain the prevalence of respiratory abnormalities as defined by bronchoalveolar lavage (BAL), standard respiratory function studies and gallium scan of the lung, and the relationship of these abnormalities to the presence or absence of dyspnea. These results suggest that respiratory symptoms are very common in CTD and in scleroderma, particularly if Sjögren's syndrome is also present. Our findings also suggest the presence of 2 patterns of interstitial lung involvement in scleroderma. In scleroderma alone this appears to be characterized by the presence of increased neutrophil proportions in the BAL, decreased DLCO, and no increase in gallium uptake within the lung. Where scleroderma is associated with Sjögren's syndrome, there is an increase in the proportion of lymphocytes in the BAL and respiratory symptoms are very prominent, the latter associated with an increase in gallium uptake within the lung. This suggests that Sjögren's is a major determinant of the pattern of interstitial lung disease seen in CTD.

Bronchoalveolar Lavage Fluid↗
PubMed1988

Radiation-induced lung injury: a hypersensitivity pneumonitis?

Radiation pneumonitis occurs 6 to 12 weeks after thoracic irradiation, and is thought to be due to direct radiation-induced lung injury. Four patients who developed pneumonitis after unilateral thoracic irradiation for carcinoma of the breast were studied with bronchoalveolar lavage, gallium scan of the lung, and respiratory function tests. On the irradiated side of the chest, all four patients showed an increase in total cells recovered from the lavage fluid and a marked increase in the percentage of lymphocytes. When results for the unirradiated lung were compared with results for the irradiated lung, there was a comparable increase in total cells and percentage of lymphocytes. Gallium scans showed increases for both irradiated and unirradiated lungs. Prompt improvement was seen after corticosteroid therapy in all patients. The fact that abnormal findings occur equally in irradiated and unirradiated lung is inconsistent with simple direct radiation-induced injury and suggests an immunologically mediated mechanism such as a hypersensitivity pneumonitis.

Aged↗