Cytokine message and protein expression during lung granuloma formation and resolution induced by the mycobacterial cord factor trehalose-6,6'-dimycolate.

Trehalose-6,6'-dimycolate (TDM), or cord factor, is a mycobacterial cell wall component that induces granuloma formation and proinflammatory cytokine production in vivo and in vitro. The purpose of this work was to better understand the mechanisms by which TDM promotes lung granuloma formation. This was accomplished by characterizing cytokine mRNA expression during TDM-induced alveolitis culminating in cohesive granuloma development. A single intravenous injection of TDM given to C57BL/6 mice produced lung granulomas that peaked in number 5 days after challenge and were nearly resolved by 14 days. mRNA in whole lung preparations was quantitated by bioluminescent RT-PCR. Tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 were significantly elevated during granuloma development and decreased during granuloma resolution. There were no detectable changes in mRNA for interferon-y (IFN-y), IL-2, IL-4, IL-5, IL-10, and IL-12(p40). The level of TNF-alpha protein extracted from lung minces highly correlated with morphologic indices of granulomatous inflammation, indicating that it may be an important modulator of the inflammatory intensity induced by TDM. TDM may interact specifically with macrophages in vivo, as evidenced by induction of TNF-alpha, IL-1beta, and IL-6, but not IFN-gamma, protein in bone marrow-derived macrophages from C57BL/6 mice. TDM may therefore play an important role early in macrophage activation during the host granulomatous response to mycobacteria.

Fiesta as tradition, fiesta as change: ritual, alcohol and violence in a Mexican community.

AIMS: To explore the traditional Mexican religious fiesta as a primary locus for community-based excessive drinking and violence against women. The research argues that the pattern of drinking and violence cannot be placed within explanations that tie a breakdown in social norms to drinking nor drinking to a breakdown in social norms. DESIGN: The data were gathered over 14 continuous months of participant observation in 1995 and 1996, followed by two summer research projects in 1997 and 1998. The researcher documented the activities of the participants at each fiesta and followed-up each event with interviews of the parties involved in violent confrontations. In addition, life stories and archival data on violence were conducted and used to situate current findings against historical data. SETTING: All data were collected in the community of Santa Maria Atzompa. Atzompa is a cabécera or municipal seat, for six colonias (large neighborhoods), and three ranchos (agriculturally based communities) and has a population of just over 5200. The main community of Atzompa, where most of the research was conducted, has a population of 2700+. PARTICIPANTS: Over 1000 individuals participated in community religious fiestas that the researcher attended. More than half of these were men, and almost all the men present participated in the binge drinking. MEASUREMENTS: Sixteen religious fiestas and 13 non-religious fiestas were documented through participant observation and photographs. FINDINGS: In every religious-based fiesta, violence erupted and was directed primarily against women. Husband and wife abuse accounted for 10 of the 16 violent disruptions (63%) while male/male abuse accounted for six (38%). CONCLUSIONS: The perpetuation of binge drinking and violence are part of a historic cycle of male dominance that dates back to the introduction of alcohol distillation during colonization by the Spaniards in the 16th century, compounded today by frustration over their inability to control the economic and political aspects of their households and community. Women's acquiescence to the violence is a form of mediation over male frustrations that allow women to continue in their roles as economic providers.

The effects of chronic alcohol abuse on pulmonary glutathione homeostasis.

The incidence and severity of the acute respiratory distress syndrome (ARDS) is increased in critically ill patients with a prior history of chronic alcohol abuse; however, the specific mechanisms responsible for this association are unknown. Recently, we determined that chronic ethanol ingestion in rats decreased the alveolar epithelial lining fluid (ELF) concentration of the antioxidant glutathione (GSH), which is a characteristic finding in patients with ARDS. However, the effects of chronic alcohol abuse on the human alveolar epithelium are essentially unknown. Therefore, as a first step we asked if chronic alcohol abuse, independent of other comorbid conditions, decreases the concentration of GSH in the human lung. We determined that otherwise healthy chronic alcoholics had significantly decreased ELF concentrations of GSH compared with nonalcoholic control subjects (79 micromol [48 to 118 micromol] versus 576 micromol [493 to 728 mmol], p < 0.001). Furthermore, the percentage of GSH in the oxidized form was higher in the chronic alcoholics (9.8% [2.2 to 14.8%] versus 2.8% [0.4 to 4.0%] p = 0.05), indicative of increased utilization of GSH. This is the first report that chronic alcohol abuse alters GSH homeostasis in the human lung, and suggests a potential mechanism by which chronic alcohol abuse predisposes susceptible patients to develop ARDS.

Differential modes of regulation of interleukin-1beta expression by extracellular matrices.

Extracellular matrices (ECMs) can stimulate human monocytic cells to express interleukin-1beta (IL-1beta), a proinflammatory cytokine implicated in the regulation of tissue inflammation. In this study, we explored the intracellular mechanisms responsible for ECM induction of IL-1beta using human promonocytic U937 cells transfected with the full-length human IL-1beta gene promoter connected to a reporter gene. Using this system, we demonstrated that the ECM molecules fibronectin (FN), type I collagen (Coll), fibrin (Fb) and laminin (Lm) induced transcription of the IL-1beta gene (which was associated with a modest increase in IL-1beta protein secretion) in suspended cells, when used in their soluble monomeric form. This effect was mimicked or blocked by anti-integrin monoclonal antibodies (mAbs) and was dependent on the activation of protein kinase C (PKC). Three of the ECMs tested (FN, Coll and Fb) induced the activation of mitogen-activated protein kinases (MAPKs), whereas Lm had no effect. FN, Coll and Fb (but not Lm) also induced DNA binding of the transcription factor activator protein-1 (AP-1), but not that of nuclear factor-kappaB. Co-transfection of U937 cells with a competing AP-1 oligomer blocked the IL-1beta response induced by FN, but not that induced by the other ECMs. By inhibiting AP-1 translocation, glucocorticoids also blocked the FN-induced response, but not that of the other ECMs. These studies suggest that the signalling pathways which mediate ECM induction of IL-1beta expression in human monocytic cells converge at the level of PKC activation. However, they diverge in other aspects, as demonstrated by the differential activation of MAPKs and the need for diverse transcription factors.

Transcriptional regulation of the interleukin-1beta promoter via fibrinogen engagement of the CD18 integrin receptor.

Fibrinogen, with or without its conversion to fibrin, in the extravascular spaces of injured and inflamed lung tissues is thought to promote inflammatory responses that can eventually lead to pulmonary fibrosis. One of these responses likely involves the elaboration of the proinflammatory cytokine interleukin (IL)- 1beta. We reported that both fibrinogen and fibrin stimulated production of IL-1beta message and protein by binding to CD18 integrin receptors on normal human monocytes (J. Immunol., 1995;154:1879-1887). The purpose of the current work was to extend our previous observations by characterizing the transcriptional regulation of fibrinogen-induced IL-1beta expression. Our model was the human monocytic cell line U937 transfected with the human IL-1beta promoter connected to reporter genes. We found that fibrinogen induced the IL-1beta promoter and that induction could be blocked by anti-CD18 antibody. Transfection with deletion constructs of the promoter and DNA electrophoresis mobility gel shift assays suggested that sequences containing activator protein (AP)-1, cyclic adenosine monophosphate response element (CRE), and nuclear factor (NF)-kappaB cis-acting motifs regulate IL-1beta gene expression by fibrinogen. In combination with competitive cotransfection studies using consensus oligonucleotides mimicking these motifs, we conclude that transactivation of an NF-kappaB-like sequence is necessary for induction of the IL-1beta gene, that activation of CRE may repress induction of the gene, and that AP-1 potentially modulates induction and repression of the gene induced by fibrinogen. This study begins to define the molecular mechanisms by which fibrin(ogen) promotes and regulates expression of the IL-1beta gene and further substantiates a role for fibrin(ogen) in tissue injury and inflammation.

Offspring of streptozotocin diabetic rats: size changes in Langerhans islets with time after birth.

It has been demonstrated that, in the diabetic rat, pregnancy and lactation are severely altered: in this study, we have measured the size of Langerhans islets of rat pups, the offspring of experimental diabetic mothers and nondiabetic controls. Diabetes was induced through streptozotocin administration (dose, 60 mg/kg body wt.). This drug was injected in every animal; their blood sugar was measured 1 week later (Haemo-Glukotest, Boehringer Mannheim), and they were then separated into three groups according to their fasting blood sugar levels: (a) severe diabetics (above 16.5 mM/l); (b) mild diabetics (6.5-16.5 mM/l); and (c) nondiabetic normals. They received insulin therapy (2-4 I.U./day) as the mild diabetics exhibited a slightly higher than normal fasting blood sugar, and the diabetic ones, above 15 mM/l. The areas of Langerhans islets of pups were measured 1 and 5 days after parturition; pancreas sections were dyed (haematoxylin-eosin) and morphometry was then performed using a digitalized magnetic tabloid connected to a Zeiss Morphomat 30 (Kontron). On the first day after parturition, the pancreas section areas in pups from mildly and severely diabetic mothers were smaller than those in neonates from nondiabetic controls (P < 0.001). The areas in neonates from severely diabetic mothers showed a more intense decrease than those from mildly diabetic animals (P < 0.01). On day 5 after delivery, the areas of Langerhans islets in offspring from normal mothers decreased and those in pups from diabetic mothers tended to normalize (P < 0.01), particularly those from the severely sick group (P < 0.01). We conclude that after parturition the offspring is no longer exposed to the high blood sugar levels found in both diabetic groups of mothers, thereby no hyperinsulinemia is needed; as time elapses, then, the area of their Langerhans islets tends to normalization.

Quantitation of insulin stores in Langerhans islets by combined immunohistochemistry and computerized microspectrophotometric analysis.

Histology, size, and insulin content of Langerhans islets from normal and recent experimental hyperthyroid (REH) dogs were studied. Insulin localization in the islets was revealed by immunohistochemistry, and the remaining two variables were analyzed by computerized microspectrophotometry according to an original technique described here. Observed under the light microscope, the REH dog pancreas section shows larger than normal islets whose scarce beta-granules are mainly located near B-cell borders and grouped along capillaries. The brown areas occupied by insulin in Langerhans islets from REH and normal dogs (mean +/- SEM) are 5182 +/- 311 and 4236 +/- 287 microns2, the total mean insulin amount per respective islet section-as expressed in absorbance arbitrary units-is 1108 and 1846, and the light absorbances per such area units are 0.214 +/- 0.070 and 0.436 +/- 0.060, respectively. Measuring these variables in dog and human (large) pancreases by the conventional methods successfully used for small pancreases would have been technically impossible.

Distribution of extracellular matrices, matrix receptors, and transforming growth factor-beta 1 in human and experimental lung granulomatous inflammation.

Aberrant deposition of extracellular matrices (ECMs) may affect lung inflammation by influencing cell adhesion, migration, and activation. Little is known about the expression of ECMs in lungs with granulomatous inflammation. Therefore the authors investigated the distribution of ECMs, matrix receptors of the integrin family, and transforming growth factor-beta 1 (TGF-beta 1) in lungs from patients with pulmonary sarcoidosis and animals with experimental granulomatosis. Immunohistochemistry revealed increased deposition of type I collagen and fibronectin in human lung granulomas when compared with healthy human lungs. Procollagen type I and cellular fibronectin also were increased, suggesting local synthesis of ECM in sarcoid granulomas. These findings were accompanied by increased staining for fibronectin (alpha 5 beta 1) and collagen (alpha 2 beta 1) integrin receptors. The matrix-inducing cytokine TGF-beta 1 was co-distributed with the aforementioned molecules in the granulomas, whereas no significant staining for TGF-beta 1 was found in healthy lungs. Similar to sarcoid lungs, analysis of lung sections obtained from a murine model of granuloma formation revealed increased expression of fibronectin, collagen, integrin receptors, and TGF-beta 1 within granulomas. Based on these observations, there is increased expression of ECM and matrix receptors in both human and experimental lung granulomas. Such alterations may influence the recruitment and activation of inflammatory cells and fibroblasts, promoting granuloma formation and remodeling of tissue by fibrosis. Activation of mononuclear cells resulting in production of TGF-beta 1 is likely to contribute to the changes described.

Fibrin enhances the expression of IL-1 beta by human peripheral blood mononuclear cells. Implications in pulmonary inflammation.

Tissue injury is accompanied by increased vascular permeability and influx of plasma proteins including fibrinogen. Fibrinogen is converted into a fibrin matrix by procoagulants activated at the site of tissue injury and inflammation. This fibrin matrix is thought to participate early in the inflammatory response by providing a temporary protein "scaffold" for inflammatory cell adhesion and migration, and subsequent remodeling of the tissue with permanent extracellular matrix proteins such as collagen. Collagen and fibronectin have been shown to regulate the expression of inflammatory cytokines, but whether fibrin can regulate cytokine expression is not known. We hypothesized that fibrin induces the expression of the proinflammatory cytokine IL-1 beta and sought to explore mechanisms responsible for this induction. In this report, we demonstrate that fibrin stimulates human PBMCs to express IL-1 beta message and protein. We show that induction of IL-1 beta by fibrin is mediated partly by the integrin receptor CD11b/CD18 and modulated by cytoskeletal rearrangement. Fibrin also suppresses production of IL-1 receptor antagonist (IL-1ra) a non-bioactive competitor of IL-1 for IL-1Rs. We propose that injured tissues where the conditions favor coagulation and fibrin accumulation, the interaction between mononuclear cells and the newly formed fibrin matrices may elicit the production of the proinflammatory cytokine IL-1 beta. This proposal is supported by immunohistochemical studies which show the co-distribution of fibrin and IL-1 beta in granulomas in lung sections from patients with the systemic granulomatous disease, sarcoidosis.

Extravascular coagulation and fibrinolysis in murine lung inflammation induced by the mycobacterial cord factor trehalose-6,6'-dimycolate.

Diffuse pulmonary inflammation in interstitial lung diseases is associated with increased coagulation in the extravascular spaces of the lung. We hypothesized that conditions favoring coagulation over fibrinolysis in the lung are related to inflammation. Pulmonary coagulation and fibrinolysis were studied in two strains of mice susceptible or resistant to the development of lung inflammation in response to the mycobacterial cell wall glycolipid trehalose-6,6'-dimycolate (TDM). Susceptible animals treated with TDM intravenously develop well-organized collections of mononuclear cells in the lung parenchyma referred to as granulomas in this report. More granulomas were found in the susceptible ICR mice than in the resistant A/J mice after intravenous administration of TDM (7 +/- 1 granulomas/mm2 versus 1 +/- 0.3 granulomas/mm2, p = 0.005). Granuloma formation was associated with increased lung procoagulant activity (PCA) measured in bronchoalveolar lavage (BAL) cell lysates from susceptible mice. In contrast, TDM-resistant A/J mice challenged with TDM did not have a significant BAL cell PCA response, but expressed several-fold greater levels of lung BAL fluid plasminogen activator activity (PAA) than ICR mice. To examine the role of coagulation in the TDM pulmonary inflammatory response, susceptible C57Bl/10SnJ mice were anticoagulated by oral administration of warfarin prior to challenge of TDM; these mice developed fewer pulmonary granulomas than TDM-treated mice without warfarin treatment (2.6 +/- 0.5 granulomas/mm2 versus 6.5 +/- 0.8 granulomas/mm2, p < 0.001) but had similar BAL cell PCA and lung inflammatory changes as measured by lung weights and BAL cellularity.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of pulmonary granulomas, macrophage procoagulant activity, and tumor necrosis factor-alpha by trehalose glycolipids.

Trehalose 6,6' dimycolate (TDM), a mycobacterial glycolipid, induces granulomas and hemorrhagic toxic reactions when administered in oil but not as a suspension in saline. It was previously demonstrated by us that TDM forms highly structured layers at oil-water interfaces and then postulated that its toxicity derives from the adhesive properties of these layers. To test this hypothesis, an evaluation was made of the ability of TDM and two analogs, trehalose 6-monomycolate (TMM) and galactose-galactose 6, 6' dimycolate (GDM), to induce pulmonary granulomas and stimulate expression of procoagulant activity (PCA) and tumor necrosis factor-alpha (TNF-alpha). Intravenous injection in mice of oil-in-water emulsions of TDM produced more and larger pulmonary granulomas than injection of TMM or GDM. Similarly, TDM on the surface of beads induced higher levels of PCA and TNF-alpha in human mononuclear cells than the analogs. The correlation of these results with the structure of surface layers of the glycolipids strengthens the hypothesis that the particular surface structure formed by TDM is necessary for its biologic activity.

Elevated D dimer in the lungs and blood of patients with sarcoidosis.

The hypothesis of this study was that D dimer, a specific degradation product of cross-linked fibrin, would be increased in the bronchoalveolar lavage (BAL) fluids of patients with sarcoidosis and that it would be related to other BAL parameters of disease activity. Eight of 10 sarcoidosis patients but none of 18 healthy volunteers had detectable BAL D dimer by enzyme immunoassay. Autoradiography revealed the presence of fibrinogen and D dimer in the BAL fluids from sarcoidosis patients. Bronchoalveolar lavage D dimer levels in sarcoidosis patients correlated with total BAL cells per milliliter, lymphocytes per milliliter, and total protein level, but not macrophages per milliliter. The D dimer in the BAL fluids from sarcoidosis patients did not correlate with D dimer in the blood. Our findings indicate that BAL D dimer parallels directly the lymphocytic alveolitis that characterizes pulmonary sarcoidosis.

Interstitial lung disease: state of the clinical art.

Interstitial lung diseases pose a great challenge to the clinician because of the indolent and variably active nature of these disorders and the limited number of therapeutic options. Adjunctive therapy includes supplemental oxygen in hypoxic patients, bronchodilators in patients with an obstructive lung component, and aggressive use of antibiotics in febrile patients on potent immunosuppressive therapy and suspected or confirmed infections. In younger patients who present late in their illness or deteriorate on therapy, lung transplantation is the only option. Recent advances in our knowledge of the cellular and molecular mechanisms operating in ILD and techniques which include gene amplification and cloning promise to yield more effective treatments for these diseases which currently produce a high incidence of morbidity and mortality.

Influence of estrogen-progesterone sequential administration on pancreas cytology. Serum insulin and metabolic adjustments in female dogs.

In bitches in anestrus, artificial endometrial sex cycles were induced. Estrus was induced by 17 beta-estradiol benzoate administration; matched untreated and vehicle-treated controls were studied. Early metadiestrus (6th day after appearance of metestrum cells in vaginal smears) was induced by the sequential administration of 17 beta-estradiol benzoate and progesterone: matched studies with only one hormone or vehicles were also carried out. In different groups of animals, blood sugar (BS), serum immunoreactive insulin (IRI) and serum free fatty acids (FFA) in the basal conditions and during glucose and insulin tests were studied. Insulin was immunocytolocalized in sections of pancreas from a part of these animals. Size and insulin content in Langerhans islets were measured by morphometric and cytospectrophotometric computerized analysis. Extra-pancreatic factors--space of distribution, t1/2 in blood stream--regulating serum IRI and BS levels were calculated. The hypoglycemic effect of insulin was enhanced by estrogenization, together with insulin accumulation in Langerhans islets. Progesterone treatment caused mild insulin resistance together with depletion of pancreatic insulin stores in the long run. Glucose tolerance of progesterone-injected bitches was improved after estrogen priming with greater space of distribution of glucose. Furthermore, a high basal serum FFA levels in bitches receiving the hormone sequence was observed. We may therefore conclude that the metabolic and endocrine changes induced in bitches by artificial sex cycles converge towards excellent BS homeostasis leads to the replenishing of pancreatic insulin stores, so that estrogen-progesterone administration in sequence appears to be, in this experimental condition, non-diabetogenic.

On changes in pancreas cytomorphometry, serum insulin and metabolic adjustments induced by long term orchidectomy and substitutive therapy with testosterone in dogs.

Mongrel, male, fasted, unanesthetized dogs under the following alternative treatments: 1) nil, 2) orchidectomy 4 months before the study, 3) orchidectomy 10 months in advance, 4) orchidectomy like in (3) followed by i.m. propylenglycol treatment, 0.05 ml/kg body wt./day, 15 days (vehicle controls, and 5) testosterone hemisuccinate in propylenglycol, 0.75 mg in 0.05 ml/kg body wt./day, for 15 days, were used in this study. Pancreas slices of animals of every group were stained with immunoperoxidase. The animals were fasted, anesthetized for pancreas removal. Pancreatic section mean absorbance was estimated in a Zeiss cytospectrophotometer with a coupled computer. Blood sugar (BS) and both serum, immunoreactive insulin (IRI) and free fatty acids (FFA) were assayed in these.

On changes in pancreas cytomorphometry, serum insulin and metabolic adjustments induced by long term orchidectomy and substitutive therapy with testosterone in dogs.

Mongrel, male, fasted, unanesthetized dogs under the following alternative treatments: 1) nil, 2) orchidectomy 4 months before the study, 3) orchidectomy 10 months in advance, 4) orchidectomy like in (3) followed by i.m. propylenglycol treatment, 0.05 ml/kg body wt./day, 15 days (vehicle controls, and 5) testosterone hemisuccinate in propylenglycol, 0.75 mg in 0.05 ml/kg body wt./day, for 15 days, were used in this study. Pancreas slices of animals of every group were stained with immunoperoxidase. The animals were fasted, anesthetized for pancreas removal. Pancreatic section mean absorbance was estimated in a Zeiss cytospectrophotometer with a coupled computer. Blood sugar (BS) and both serum, immunoreactive insulin (IRI) and free fatty acids (FFA) were assayed in these.

Metabolic and histological pancreatic changes induced by ovariectomy in the female dog.

The actions of ovariectomy performed 4 months in advance on pancreas cytology and also upon the blood sugar, serum immunoreactive insulin and circulating free fatty acid changes over glucose and insulin tests, were studied in female dogs. We concluded that ovariectomy does not affect blood sugar -basally or during the tests-, glucose space and clearance rate of glucose from circulation. Conversely, the integrated insulinemic response over glucose test was highly risen (956%) by ovariectomy in such animals; the rise occurs despite they show a broadened (59%) insulin space, is slightly mediated by a reduction (132%) in insulin clearance from circulation, and appears to be chiefly mediated by a major enhancement in insulin secretion. The immunocytolocalization of insulin in the pancreatic tissue of ovariectomized female dogs showed hypertrophy of Langerhans islets, beta-degranulation but no vacuolation. However, the piling up of the beta-granules by the vascularly pole of the B-cells as well as the appearance of a pretty number of small islets and microislets widespread over the acini, absent in the pancreatic tissue of the untreated controls in anestrous, indicate for the insulin secretory potency of the pancreas of the ovariectomized female dog to be apparently high. In the female dog, ovariectomy affects serum free fatty acid levels via at least two mechanisms, viz., a) stimulation of lipid storage over the glucose test, and b) reduction in lipomobilization as insulin antagonism predominates.

Metabolic and histological pancreatic changes induced by ovariectomy in the female dog.

The actions of ovariectomy performed 4 months in advance on pancreas cytology and also upon the blood sugar, serum immunoreactive insulin and circulating free fatty acid changes over glucose and insulin tests, were studied in female dogs. We concluded that ovariectomy does not affect blood sugar -basally or during the tests-, glucose space and clearance rate of glucose from circulation. Conversely, the integrated insulinemic response over glucose test was highly risen (956

) by ovariectomy in such animals; the rise occurs despite they show a broadened (59

) insulin space, is slightly mediated by a reduction (132

) in insulin clearance from circulation, and appears to be chiefly mediated by a major enhancement in insulin secretion. The immunocytolocalization of insulin in the pancreatic tissue of ovariectomized female dogs showed hypertrophy of Langerhans islets, beta-degranulation but no vacuolation. However, the piling up of the beta-granules by the vascularly pole of the B-cells as well as the appearance of a pretty number of small islets and microislets widespread over the acini, absent in the pancreatic tissue of the untreated controls in anestrous, indicate for the insulin secretory potency of the pancreas of the ovariectomized female dog to be apparently high. In the female dog, ovariectomy affects serum free fatty acid levels via at least two mechanisms, viz., a) stimulation of lipid storage over the glucose test, and b) reduction in lipomobilization as insulin antagonism predominates.