Infection by Mycobacterium tuberculosis promotes human alveolar macrophage apoptosis

The effect of Mycobacterium tuberculosis infection on the viability of healthy (control) human alveolar macrophages was evaluated by staining with ethidium homodimer and calcein to discriminate live from dead cells. Infection with M. tuberculosis H37Ra or H37Rv increased macrophage mortality at 6 days from the control level of 3.8% +/- 0.7% to 28.7% +/- 6.9% or 12.6% +/- 3.1%, respectively (P < 0.001 for comparisons of all conditions). A role for tumor necrosis factor alpha (TNF-alpha) in the M. tuberculosis-induced cytolysis of alveolar macrophages was demonstrated by increased cytotoxicity following the addition of exogenous TNF-alpha to the cultures and by enhancement of macrophage survival when M. tuberculosis-infected alveolar macrophages were treated with pentoxifylline or anti-TNF-alpha antibody. The cytolytic mechanism was determined to be apoptosis by the demonstration of a characteristic internucleosomal ladder of genomic DNA by agarose gel electrophoresis, by finding nuclear fragmentation and condensation by electron microscopy, and by in situ terminal transferase-mediated nick end labeling of fragmented DNA in alveolar macrophages infected with M. tuberculosis in vitro. The latter technique was employed to reveal extensive apoptosis within caseating granulomas from lung tissue samples from clinical tuberculosis cases. The induction of apoptosis in alveolar macrophages by M. tuberculosis may play a role in the macrophage-pathogen interaction of tuberculosis in vivo.     

Copy number-dependent expression of a YAC-cloned human CFTR gene in a human epithelial cell line

INTRODUCTION: The reference values (RV) of biological indicators are used in the interpretation of the results of such indicators in individuals occupationally exposed to chemical agents. The Brazilian Group for the Establishment of Reference Values has worked on these definitions for the purpose of establishing RVs for several bioindicators in various regions of the country. In the present study, the RV for carboxyhemoglobin (COHb) was determined for the South of Minas Gerais. MATERIAL AND METHOD: The COHb was analyzed by the Beutler and West (1984) spectrophotometric method, optimized in our laboratory. In all the samples, analyses of some biochemical and hematological parameters were made to evaluate the health condition of a population of 200 volunteer non-smokers occupationally not exposed to CO. Each individual answered a questionnaire to obtain data pertinent to the interpretation of the results. The reference values were expressed as mean values +/- standard deviation, with a 95% confidence interval, and an upper reference value. The statistical distribution of the results was made so as to enable comparisons between the results of groups of workers, rather than individual evaluations, to be made. RESULTS AND CONCLUSIONS: The mean value +/- standard deviation was 1.0% +/- 0.75; the 95% confidence interval was 0.9-1.1% and the upper reference value was 2.5%. By the t Student test (p < or = 0.05), no difference was detected between the values related to sex, age or ingestion of alcoholic beverages. The reference values obtained were close to those reported for others countries.     

Association between an early humoral response to Mycobacterium tuberculosis antigens and later development of tuberculosis in human immunodeficiency virus-infected individuals

We describe four cases of inflammatory pseudotumor seen at our institution in the past 4 years. Four children were each found to have a large extraperitoneal mass on imaging studies, three of which were in the pelvis. Malignant sarcomatous tumors were suspected. Surgical biopsy of each mass, however, revealed inflammatory pseudotumor.     

Inhibition of adhesion molecules by budesonide on a human epithelial cell line (lung carcinoma)

Inhaled corticosteroids in the treatment of asthma have been shown to produce marked reductions in the number of inflammatory cells (mainly mast cells and eosinophils) and their products at bronchial level (such as cytokines). Recently, it has been demonstrated that epithelial cells express ICAM-1/CD54 in allergic patients both during natural allergen exposure and after allergen challenge. We have previously demonstrated that deflazacort (a systemic steroid) reduces the expression of ICAM-1 on conjunctival epithelial cells. The present study aimed to evaluate the effects exerted by budesonide on adhesion molecule expression by a human epithelial cell line (lung carcinoma: DM) and on soluble ICAM-1. Budesonide was added at concentrations corresponding to 10(-8), 10(-7), and 10(-6) mol/l in cultured epithelial cells, either in the absence of any stimulus or in the presence of interferon-gamma (IFN-gamma) at 500 U/ml. After 24 h of incubation, cytofluorometric analysis was performed for ICAM-1 and CD29/VLA beta 1. The 24-h supernatants of the same cultures were collected and then evaluated for soluble ICAM-1 (sICAM-1). The results showed that budesonide inhibits ICAM-1 and CD29 basal expression on the cells studied (P < 0.05): budesonide was effective in a dose-dependent manner. In addition, budesonide reduced surface ICAM-1 upregulation induced by IFN-gamma at 500 U/ml (P < 0.05). Finally, cell cultures with budesonide showed decreased levels of soluble ICAM-1 in basal condition, but not after IFN-gamma stimulation.     

Mineralocorticoid hormone receptor and the epithelial sodium channel in a human leukemic cell line

A Cell extract from the HEL (human erythroblastic leukemia) cell line was positive for both the epithelial sodium channel (ENaC) and the mineralocorticoid receptor (MCR) as glycosylated 82-84 kDa bands, and a single 102 kDa band, respectively, in Western blots using polyclonal antibodies raised against these proteins. The immunofluorescent labeling of the MCR in all cell lines showed a nucleocytoplasmic localization of the receptor whereas the ENaC was exclusively membrane-bound. These results were confirmed by confocal microscopy. The expression of the MCR in HEL cells was evident as a predicted band of 843 bp (234 amino acids) after total RNA from HEL cells had been reverse transcribed and then amplified by PCR; the ENaC was similarly evident as a predicted band of 520 bp. In both cases, near 100% identity was observed between the deduced amino acid sequences of the PCR products and those from known human sources. The multiplication of HEL cells was influenced by antagonists (RU 26752, ZK 91587) targeted for specificity to the MCR and this was reversed by the natural hormone aldosterone. These steroids also provoked chromatin condensation in the HEL population.     

Transport of paraquat by a renal epithelial cell line, MDCK

Transport of paraquat (PQ), a herbicidal cation, was previously investigated in a proximal (LLC-PK1), renal epithelial cell line using permeable collagen-coated filters. PQ was actively transported from the basolateral side via a cation transport system by the LLC-PK1 cells. In the present study, the transport of PQ was investigated in a distal renal epithelial cell line, MDCK. PQ was predominantly transported from the basolateral to apical (B to A) side. The basolateral transport of PQ in MDCK cells was not saturable with increasing concentrations and not energy dependent. The flux and uptake of PQ was much lower in the MDCK than LLC-PK1 cells. It is concluded that MDCK, a distal renal tubular cell line, does not have an active transport system for PQ.     

Regulation of nitric oxide production by rat alveolar macrophages in response to silica exposure

Research conducted primarily over the past 5-8 years on the psychosocial effects of pediatric chronic physical disorders on children and their families is reviewed. A large body of studies show that both children and their mothers, as groups, are at increased risk for psychosocial adjustment problems compared to peers, but that there is considerable individual variation in outcome. Since the last review on this topic (Eiser, 1990a), many studies have been conducted to identify risk and resistance factors associated with differences in adjustment among these children and their mothers. Improvements are noted in the theoretical basis for this work, programmatic nature of some of the research, and efforts at producing clinically relevant information. Evaluations of interventions, however, are lagging. Critical issues and future directions regarding developmental approaches, theory, method, measurement, and intervention are discussed.     

Tumor necrosis factor-alpha stimulates human Clara cell secretory protein production by human airway epithelial cells

OBJECTIVE: Low serum levels of mannan binding lectin (MBL) are associated with increased risk of recurrent infections. We determined whether there was an association between serum MBL levels and the course and prognosis of rheumatoid arthritis (RA). METHODS: MBL was analyzed in sera from 99 patients with RA who were included in a longterm prospective study. RESULTS: Compared with controls, a high fraction of patients lacked detectable MBL in serum (11 vs 3%; p = 0.025). Comparing patients with MBL serum levels above and below the median revealed that those with levels below the median were younger at onset of RA (p = 0.043) and had higher erythrocyte sedimentation rate (p = 0.006), joint swelling score (p = 0.019), limitation of joint motion score (p = 0.027), and annual increase in radiographic destruction score (p = 0.053). CONCLUSION: MBL insufficiency may be a contributing pathogenetic factor in RA.     

Increased epidermal growth factor-receptor protein in a human mesothelial cell line in response to long asbestos fibers

The burden of violence is born disproportionately by the youngest of our country. In this article, three important aspects of violence are discussed: premature death, violence recidivism, and violent criminality. The author emphasizes the role of the pediatrician in preventing these consequences.     

Perflubron decreases inflammatory cytokine production by human alveolar macrophages

OBJECTIVE: To determine whether inflammatory cytokine production by stimulated human alveolar macrophages is affected by perflubron exposure. DESIGN: Controlled laboratory investigation of alveolar macrophage function in vitro. SETTING: Research laboratory. SUBJECTS: Cultured alveolar macrophages obtained by bronchoalveolar lavage from eleven normal volunteers. INTERVENTIONS: Endotoxin-stimulated alveolar macrophages were treated with perflubron. MEASUREMENTS AND MAIN RESULTS: Alveolar macrophages were stimulated for 1 hr with lipopolysaccharide and then treated with perflubron for 23 hrs. Cell-free supernatants were collected and cytokines were assayed by enzyme-linked immunosorbent assay. Tumor necrosis factor-alpha, interleukin-1, and interleukin-6 were stimulated by lipopolysaccharide (endotoxin) and all of these cytokines were significantly (p < .05) inhibited by perflubron. Cell viability was not affected by perflubron. Basal cytokine concentrations from unstimulated alveolar macrophages were not altered by perflubron. CONCLUSIONS: Exposure of stimulated human alveolar macrophages to perflubron in vitro decreases cytokine production. This observation suggests that perflubron may have anti-inflammatory activity.     

Exacerbation of the inflammatory response to Mycobacterium tuberculosis after antiretroviral therapy

A patient with HIV infection was successfully treated for pulmonary tuberculosis, but pulmonary inflammation and lymphadenitis worsened dramatically after subsequent combination antiretroviral therapy. As this relapse coincided with development of a strong delayed-type hypersensitivity response to tuberculin and improved after treatment with the anti-inflammatory agent oxpentifylline, it was probably caused by restoration of pathogen-specific cellular immunity.     

Fenspiride inhibits histamine-induced responses in a lung epithelial cell line

Using the human lung epithelial WI26VA4 cell line, we investigated the capacity of fenspiride, an anti-inflammatory drug with anti-bronchoconstrictor properties, to interfere with histamine-induced intracellular Ca2+ increase and eicosanoid formation. Histamine and a histamine H1 receptor agonist elicited a rapid and transient intracellular Ca2+ increase (0-60 s) in fluo 3-loaded WI26VA4 cells. This response was antagonized by the histamine H1 receptor antagonist, diphenhydramine, the histamine H2 receptor antagonist, cimetidine, having no effect. Fenspiride (10(-7)-10(-5) M) inhibited the histamine H1 receptor-induced Ca2+ increase. In addition, histamine induced a biphasic increase in arachidonic acid release. The initial rise (0-30 s), a rapid and transient arachidonic acid release, was responsible for the histamine-induced intracellular Ca2+ increase. In the second phase release (15-60 min), a sustained arachidonic acid release appeared to be associated with the formation of cyclooxygenase and lipoxygenase metabolites. Fenspiride (10(-5) M) abolished both phases of histamine-induced arachidonic acid release. These results suggest that anti-inflammatory and antibronchoconstrictor properties of fenspiride may result from the inhibition of these effects of histamine.     

Cell-mediated HTLV-I infection of a cervix-derived epithelial cell line

There is considerable evidence that sexual transmission of human T-cell leukemia virus-I (HTLV-I) is mediated by virus-infected lymphocytes in genital tract secretions. However, it is not clear whether infection occurs through lesions in the genital tract epithelium or takes place via an intact epithelium. We have carried out experiments to test the hypothesis that sexual transmission of HTLV-I is initiated by lymphocyte-mediated infection of intact genital tract epithelia. To examine this question we added either free virus or HTLV-I producing MT-2 cells to cultures of a cervix-derived epithelial cell line, MS751. Although free virus did not infect MS751 cells, MS751 cells which had been coincubated with MT-2 cells became infected. These cultures produced about 50 pg/ml of HTLV-I p24 antigen per 10(6) cells over a 24 h period on the sixth day following exposure to donor T-cells. Proviral DNA could be detected in target MS751 epithelial cells by PCR. Infection of epithelia could be blocked, in a dose-dependent manner, by the sulfated polysaccharides dextran sulfate, heparin, and fucoidan, and by the enzymes fucosidase and mannosidase, but not by a number of other agents that were tested. Since MT-2 cells were observed to attach to the epithelial monolayer, we examined the ability of agents to inhibit adhesion. Adherence was inhibited by the same agents that inhibited infection. Based on these findings, we hypothesize that sexual transmission of HTLV-I may involve lymphocyte-mediated infection of genital tract epithelia and that lymphocyte adhesion to the epithelium is a critical event in transmission of HTLV-I. We speculate that a sugar moiety on the epithelium, possibly mannose or fucose, may be involved in adhesion of T-cells to epithelial cells. As sulfated polysaccharides block both adhesion and productive infection of the epithelium, these compounds might be used as active ingredients in a vaginal formulation to help prevent HTLV-I transmission.     

Dopaminergic phenotype induced by oestrogens in a human neuroblastoma cell line

Oestrogens are the key factor in the sexual differentiation of the mammalian brain and play an important role in the activity of selected areas of the mature brain. To pursue the study of oestrogen action on neural cells at the molecular level, we developed a human neuroblastoma cell line (SK-ER3) expressing the oestrogen receptor (ER). Treatment of these cells with 17beta-oestradiol causes growth arrest and morphological and biochemical differentiation. The aim of the present study was to investigate whether oestrogen-differentiated SK-ER3 neuroblastoma cells acquire the ability to synthesize a specific neurotransmitter and whether the growth arrest previously reported can be ascribed to the blockage of the cells at a specific stage of the cell cycle. The results presented here indicate that oestrogens induce accumulation of SK-ER3 cells in the G0 phase of the cell cycle, underscoring the acquisition of a mature neural phenotype upon hormonal treatment. Most importantly, we show that in the differentiated cells the content of tyrosine hydroxylase and Na+-dependent dopamine uptake is significantly augmented, proving that the oestrogen-differentiated SK-ER3 cells can synthesize and store a specific neurotransmitter. In addition, we prove that the dopamine accumulated in differentiated SK-ER3 cells can be released. These studies therefore suggest that oestrogen treatment results in the acquisition of a fully functional dopaminergic phenotype of SK-ER3 cells. Ample evidence shows a link between dopaminergic neurons and oestrogen activity in hypothalamic and non-hypothalamic areas of the mammalian brain. Our study indicates that oestrogens might play a primary role in committing undifferentiated neuroblasts towards the dopaminergic phenotype.