Alterations in pulmonary structure by elastase administration in a model of emphysema in mice is associated with functional disturbances.

Several experimental studies of pulmonary emphysema using animal models have been described in the literature. However, only a few of these studies have focused on the assessment of ergometric function as a non-invasive technique to validate the methodology used for induction of experimental emphysema. Additionally, functional assessments of emphysema are rarely correlated with morphological pulmonary abnormalities caused by induced emphysema. The present study aimed to evaluate the effects of elastase administered by tracheal puncture on pulmonary parenchyma and their corresponding functional impairment. This was evaluated by measuring exercise capacity in C57Bl/6 mice in order to establish a reproducible and safe methodology of inducing experimental emphysema. Thirty six mice underwent ergometric tests before and 28 days after elastase administration. Pancreatic porcine elastase solution was administered by tracheal puncture, which resulted in a significantly decreased exercise capacity, shown by a shorter distance run (-30.5%) and a lower mean velocity (-15%), as well as in failure to increase the elimination of carbon dioxide. The mean linear intercept increased significantly by 50% in tracheal elastase administration. In conclusion, application of elastase by tracheal function in C57Bl/6 induces emphysema, as validated by morphometric analyses, and resulted in a significantly lower exercise capacity, while resulting in a low mortality rate.

The triterpenoid lupeol attenuates allergic airway inflammation in a murine model.

Asthma is a chronic inflammatory disease of the airways associated with a Th2 immune response. Despite their side effects, corticosteroids are the most used and effective drugs for treatment of asthma. In this work we investigated the efficacy of lupeol, a triterpenoid isolated from Lonchocarpus araripensis [corrected] Benth. (Fabaceae), in the treatment of bronchial asthma in BALB/c mice immunized with ovalbumin. Administration of lupeol caused the reduction of cellularity and eosinophils in the bronchoalveolar lavage fluid. Treatment with lupeol also reduced the production of mucus and overall inflammation in the lung. Levels of Type II cytokines IL-4, IL-5 and IL-13 were significantly reduced in mice treated with lupeol, an effect that was similar to that observed in dexamethasone-treated mice. In contrast, IgE production was not significantly altered after treatment with lupeol. In conclusion, our results demonstrate that lupeol attenuates the alterations' characteristics of allergic airway inflammation. The investigation of the mechanisms of action of this molecule may contribute for the development of new drugs for the treatment of asthma.

Characterization of cardiopulmonary function and cardiac muscarinic and adrenergic receptor density adaptation in C57BL/6 mice with chronic Trypanosoma cruzi infection.

Circulating antibodies in chagasic patients interact with myocardial beta adrenergic and muscarinic cholinergic receptors, triggering intracellular signals that alter cardiac function along the course of the disease. However, until now, experimental data in models of chronically infected chagasic mice linking the effects on myocardial beta adrenergic and muscarinic receptors to cardiopulmonary dysfunction is lacking. Thus, we studied C57BL/6 mice 8 months after intraperitoneal injection of 100 trypomastigote forms of the Colombian strain of T. cruzi. Uninfected mice, matched in age, were used as controls. Histopathological analyses (inflammation and fibrosis) and radio-ligand binding assays for estimation of muscarinic and adrenergic receptor density were performed in myocardium tissue samples. When compared to controls, infected mice had electrical conduction disturbances, diastolic dysfunction, lower O2 consumption and anaerobic threshold. In addition, hearts of chronic chagasic mice had intense inflammation and fibrosis, and decreased beta adrenergic and increased muscarinic receptor densities than normal controls. Our data suggest that chronic T. cruzi infection causes alterations in cardiac receptor density and fibrosis deposition which can be associated with cardiac conduction abnormalities, diastolic dysfunction and lower exercise capacity, associating for the first time all these functional and histopathological alterations in chagasic mice.

A new chapter opens in anti-inflammatory treatments: the antidepressant bupropion lowers production of tumor necrosis factor-alpha and interferon-gamma in mice.

In a wide range of human diseases of inflammatory nature like Crohn's disease, pathology is mediated in part by pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF) or interferon-gamma. We show here that a commonly used generic antidepressant bupropion, in wide use worldwide to treat depression in humans for a decade now, profoundly lowers levels of TNF, interferon-gamma, and interleukin-1 beta in vivo, in a mouse lipopolysaccharide (LPS) induced inflammation model. Mice challenged with an otherwise lethal dose of LPS were protected by bupropion and levels of the anti-inflammatory cytokine interleukin-10 were increased. Previous data in rodents and humans indicate antidepressant effects of bupropion are mediated by its weak reuptake inhibition of norepinephrine and dopamine. Concordant with this, TNF suppression by bupropion in our mouse LPS model was largely abrogated by beta-adrenergic or dopamine D1 receptor antagonists but not by a D2 antagonist. TNF synthesis is controlled by an inverse relationship with intracellular cyclic adenosine monophosphate (cAMP) and stimulation of either beta-adrenoreceptors or D1 dopaminergic receptors result in increased cAMP but stimulation of D2 receptors lowers cAMP. We conclude that bupropion may suppress TNF synthesis by mediating increased signaling at beta-adrenoreceptors and D1 receptors, resulting in increased cAMP that inhibits TNF synthesis. Bupropion is well tolerated also in non-psychiatric populations and has less risk with long term use than current anti-inflammatory, immunosuppressive or TNF suppressive treatments such as prednisone, azathioprine, infliximab, or methotrexate. New anti-inflammatory treatments are needed. We believe a new chapter in antiinflammatory, TNF lowering treatment of disease has been opened. Bupropion's use for this in humans should be explored.

Physalins B, F and G, seco-steroids purified from Physalis angulata L., inhibit lymphocyte function and allogeneic transplant rejection.

Physalis angulata is a solanaceae widely used in folk medicine in various tropical countries in the world. We have previously described that seco-steroids (physalins) purified from P. angulata are potent inhibitors of macrophage activation, blocking the production of pro-inflammatory cytokines and LPS-induced lethality. Herein we investigated the immunomodulatory activities of these substances in lymphocyte proliferation and cytokine production and in transplantation. The addition of physalins B, F or G to concanavalin A-activated splenocyte cultures induced a concentration-dependent inhibition of proliferation. Physalin B also inhibited IL-2 production by Con A-activated spleen cells. The addition of 2 mug/ml physalin B to mixed lymphocyte reaction (MLR) caused a 100% inhibition of proliferation. More importantly, treatment of mice with physalin B, F or G prevented the rejection of allogeneic heterotopic heart transplant. Our results demonstrate the suppressive activity of physalins B, F and G in lymphocyte function and indicate the potential use of physalins as immunosuppressive agents for treatments of pathologies in which inhibition of immune responses is desired.

Decreased humoral and pathologic responses in undernourished mice infected with Schistosoma mansoni.

We previously demonstrated that mice subjected to a hypoproteinic diet showed milder chronic lesions on infection with Schistosoma mansoni than normally fed mice. Here we compare the immune response of well-nourished and undernourished mice with chronic S. mansoni infection. The proliferative response and cytokine (IFN-gamma and IL-5) production of splenocytes from undernourished mice against the soluble egg antigen (SEA) of S. mansoni or concanavalin A was similar to that of well-nourished mice. The levels of SEA-specific IgG1, IgG2b and IgG3 antibodies were significantly higher in the sera of well-nourished mice in comparison with undernourished mice. Undernourished animals also exhibited diminished periovular granuloma size compared to well-nourished infected controls. Our results support the importance of host nutritional status in the humoral immune response of mice and its effects on the development of periovular granulomas in malnourished animals infected with S. mansoni.

The pathogenesis of Chagas' disease: when autoimmune and parasite-specific immune responses meet.

Chagas' disease is a major health problem in Latin America, where it constitutes one of the leading causes of heart failure. About one fourth of Trypanosoma cruzi-infected individuals develop chronic chagasic cardiomyopathy (CChC), the most severe form of the disease. CChC is histologically characterized by the presence of multifocal inflammatory infiltrates in the heart, composed mainly by mononuclear cells, usually adhered to myocytes and leading to myocytolysis, and frequently by interstitial fibrosis. The pathogenesis of CChC is still unclear, despite intense investigations both in human beings and in animal models of the disease. Although tissue parasitism is rare in the chronic phase of infection, an immune response targeted to persistent parasites or parasite antigens is suggested, by some authors, as the pathogenic mechanism of CChC. Other researchers affirm that the lack of correlation between tissue parasitism and intensity of inflammation suggests, along with the presence of autoreactive immune responses, that CChC results from the action of an autoimmune response. Herein we review reports from the literature and our own data, which together indicate, on one hand, the participation of parasite-specific immune responses and, on the other hand, clearly demonstrate the participation of heart-specific immune responses in the pathogenesis of CChC. Moreover, multiple factors may determine whether an individual in the indeterminate form of the disease will develop CChC. The mechanisms by which T. cruzi breaks immunological tolerance to heart antigens are also discussed.

Modulation of chagasic cardiomyopathy by interleukin-4: dissociation between inflammation and tissue parasitism.

Chronic chagasic cardiomyopathy (CChC) is characterized by an inflammatory reaction which may eventually lead to heart enlargement, arrythmia, and death. As described herein, interleukin-4-deficient mice mount increased specific T helper (Th) 1 immune responses when infected with Trypanosoma cruzi, as compared to wild-type mice. Interestingly, these mice had reduced parasitism and mortality and exacerbated inflammation in their hearts, demonstrating a clear dissociation between inflammation and parasite load. The modulation of these phenomena so as to maximize host and parasite survivals may depend on a fine balance between Th responses, in which a Th1 response will, on one hand, control parasitism and, on the other hand, enhance heart inflammation throughout the course of the infection.

A heart-specific CD4+ T-cell line obtained from a chronic chagasic mouse induces carditis in heart-immunized mice and rejection of normal heart transplants in the absence of Trypanosoma cruzi.

To study the role of autoreactive T cells in the pathogenesis of cardiomyopathy in Chagas' disease, we generated a cell line by repeated in vitro antigenic stimulation of purified splenic CD4+ T lymphocytes from a chronically Trypanosoma cruzi-infected mouse. Cells from this line were confirmed to be CD4+ CD8- and proliferated upon stimulation with soluble heart antigens from different animal species, as well as with T. cruzi antigen, in the presence of syngeneic feeder cells. In vitro antigen stimulation of the cell line produced a Th1 cytokine profile, with high levels of IFNgamma and IL-2 and absence of IL-4, IL-5 and IL-10. The cell line also terminated the beating of fetal heart clusters in vitro when cocultured with irradiated syngeneic normal spleen cells. In situ injection of the cell line into well established heart transplants also induced the cessation of heart beating. Finally, adoptive transfer of the cell line to heart-immunized or T. cruzi-infected BALB/c nude mice caused intense heart inflammation.

Evaluation of anti-inflammatory-related activity of essential oils from the leaves and resin of species of Protium.

The resins and leaves of species of Protium are commonly used by folk medicine. In the present study, we analyse the pharmacological effects of essential oils obtained by steam distillation (leaves and resin) from Protium species. Analysis by gas chromatography (GC) coupled to mass spectrometry and retention indices calculations demonstrate that the resin oil is constituted mainly of monoterpenes and phenylpropanoids: alpha-terpinolene (22%), p-cymene (11%), p-cimen-8-ol (11%), limonene (5%) and dillapiol (16%), whereas sesquiterpenes predominate as the volatile constituents of the leaves. The resin of Protium heptaphyllum (PHP) and leaves of P. strumosum (PS), P. grandifolium (PG), P. lewellyni (PL) and P. hebetatum (PHT) were screened for anti-inflammatory activity by the use of mouse pleurisy model induced by zymosan (500 microg/cavity) and lipopolysaccharide (LPS) (250 ng/cavity), for antinociceptive effect (by means of preventing mice abdominal writhings), as well as NO production from stimulated macrophages and proliferation of neoplasic cell lines: Neuro-2a (mouse neuroblastoma), SP2/0 (mouse plasmocytoma) and J774 (mouse monocytic cell line). The oils from PHP, PS and PL were able to inhibit protein extravasation but no sample inhibited total or differential leucocyte counts after administrating p.o. (100 mg/kg) 1 h before stimulation with zymosan. The oils from PG, PL and PHT inhibited neutrophil accumulation whereas PHP and specially PL inhibited LPS-induced eosinophil accumulation in mouse pleural cavity. PHT was also able to inhibit mononuclear cells accumulation. Antinociceptive effect was not observed, when animals received oral administration of the essential oils (100 mg/kg). In vitro treatment with essential oils (100 microg/well) changed the NO production from stimulated mouse macrophages. PHP inhibited in 74% and PS in 46% the LPS-induced NO production. In contrast, treatment with PL was able to increase in 49% the NO production. Cell lines proliferation was affected by the oils assayed in the range of 60-100% for Neuro-2a, 65-95% for SP2/0 and 70-90% for J774. Taken together these results showed that essential oils could be useful as efficient pharmacological tools.

Morphological characterization of lung and kidney lesions in C3H/HeJ mice infected with Leptospira interrogans serovar icterohaemorrhagiae: defect of CD4+ and CD8+ T-cells are prognosticators of the disease progression.

Neonates and young C3H/HeJ mice were highly susceptible to lethal infection with Leptospira interrogans serovar icterohaemorrhagiae. The main pathological changes were seen by light microscopy in the lung and kidneys of 3-week-old mice at 11 days after inoculation. Lung histological lesions included small and medium-sized vasculitis with fibrinoid changes, hemorrhages, moderate infiltrate of mononuclear inflammatory cells and fibrin thrombi. In the kidney there was mild to severe acute tubular necrosis associated with interstitial nephritis. Repair of damaged tubules in surviving mice was observed within 17 days after inoculation. Pathological findings of CD4+ and CD8+ cell-depleted mice were clearly more severe than that seen in untreated animals by 17 days after inoculation. Comparatively, CD4+/CD8+ cell-depleted mice had more marked lung and kidney lesions than in the CD8+ or CD4+ cell-depleted mice. A very high level of tubular alterations was seen in the kidneys of all treated groups. Increased degrees of interstitial nephritis also reflected the T-cell subsets depletion related events. Leptospires were clearly demonstrated by immunoperoxidase close to the sites of histological damage in all infected mice. C3H/HeJ mice represent a useful model for further studies in pathogenicity of leptospires and natural resistance of the host.

The role of gammadelta T lymphocytes in lipopolysaccharide-induced eosinophil accumulation into the mouse pleural cavity.

LPS induces an accumulation of eosinophils in the pleural cavity that requires resident macrophages and lymphocytes, but is independent of IL-5 production. In the present study we investigated the involvement of different T lymphocyte subsets on the modulation of LPS-induced eosinophil accumulation into the pleural cavity of mice. Within 4 h after LPS injection the number of neutrophils in the pleural cavity increased significantly. Mononuclear cell counts increased after 12 h, while a significant rise on eosinophil counts was observed only after 24 h. T lymphocytes counts were increased in the pleural cavity 24 and 48 h after LPS administration. This T lymphocyte accumulation was accounted for by an influx of the gammadelta+ subset, while CD4+ and CD8+ subsets did not accumulate in the pleural cavity after LPS stimulation. All those changes had resolved 96 h after LPS injection. Depletion of T lymphocytes by treatment with mAb anti-Thy 1.0 inhibited the eosinophil accumulation triggered by LPS. Aiming to clarify which T lymphocyte subset would be involved in the LPS-induced eosinophil accumulation, we depleted mice of various T lymphocyte subpopulations using specific Abs. Depletion of either CD4+ or CD8+ subsets failed to inhibit LPS-induced eosinophil migration. In contrast, when mice were treated with anti-gammadelta+ T lymphocyte mAb, a significant reduction of LPS-induced eosinophil accumulation was observed. Similarly, the administration of LPS in BALB/c-nu/nu mice induced the expected significant influx of eosinophils into the pleural cavity. Our results indicate that the gammadelta+ T lymphocytes are centrally involved in LPS-induced eosinophil accumulation in mice.

Demonstration of leptospiral antigens on tissues using monoclonal antibodies and avidin-biotin peroxidase staining.

Glycolipoprotein (GLP) cytotoxin was extracted from Leptospira interrogans serovar canicola. The silver staining profile of GLP subjected to SDS-PAGE under denaturing conditions showed a number of bands in the mol. weight range of 14-66 kDa. Mouse Monoclonal Antibodies (MAbs) IgG3 recognizing a band near to 24 kDa of leptospiral GLP were produced (clone number MGLP-01). The agglutinating property of MAbs was established by microscopic agglutination test (MAT) using 25 different serovars as antigens. Only the homologous serovar was agglutinated by MAbs suggesting that the recognized epitope is a specific surface-exposed antigen. The MAbs were applied to demonstration of leptospiral antigens in tissue damage by avidin-biotin immunoperoxidase staining. Golden hamsters were experimentally infected with a virulent strain of L. interrogans serovar canicola. Histologically kidneys stained by routine hematoxylin and eosin showed changes characterized by injury of tubular epithelial cells leading to acute tubular necrosis (ATN). Typical, well-defined morphologic leptospires or finely granular deposits were found by immunoperoxidase staining near to blood vessels, within inflammatory infiltrates and intraluminal in proximal and distal parts of the nephron. Binding of leptospiral antigens to capillary endothelial cells, tubular epithelial cells and macrophages were also demonstrated. This entails a basis for further studies either in research or in diagnostic histopathology.

Antibodies directed to antigens secreted by murine epithelioid macrophages modulate BCG-induced granulomata.

The authors have previously shown that epithelioid cells isolated from mice secrete a factor, called macrophage deactivating factor (MDF), that promptly deactivates superoxide release by activated macrophages and neutrophils. In this paper some biological properties of a polyclonal rat antiserum directed to MDF and other substances secreted by these cells are described. The immunoglobulin fraction of this antiserum reacted, by immunocytochemical methods, with epitopes in the cell membrane of macrophages adherent to coverslips subcutaneously implanted for 14 days; but not for 5 days. It also reacted with antigens within and outside cells in BCG-induced granulomas. This antiserum blocked completely the macrophage deactivating activity of epithelioid cell culture supernatants. Anti-IL-10 monoclonal antibody, did not block MDF activity. The administration of the immunoglobulin fraction from immunized rats to C(5) deficient mice bearing BCG-induced granulomatas in the footpad, significantly reduced the size of the lesions. A marked necrosis of inflammatory cells and mononuclear cells phagocyting debris of necrotic cells were observed in these lesions.

Immunosuppression by peritoneal BCG-induced granulomas in mice.

1. Intraperitoneal injection of viable BCG resulted in the formation of peritoneal granulomas in mice. Granuloma-bearing animals presented a specific immunosuppression of delayed-type hypersensitivity (DTH) to BCG. 2. The transplantation of peritoneal granulomas induced by BCG to animals previously sensitized with BCG suppressed the DTH response to this antigen in the recipient animals to the same extent as observed in animals with granulomas induced by the injection of BCG. No suppression was observed in animals transplanted with granulomas induced by either Cytodex or talc. 3. The hypersensitivity reaction to Brucella abortus and the acute inflammatory reaction produced by carrageenin were not affected by granuloma transplantation. 4. Granulomas collected from animals treated with a lethal does of irradiation maintained the capacity to suppress DTH in recipient animals. 5. These results suggest that the response observed in granuloma recipients is modulated by cells within the granuloma.

Susceptible mice present higher macrophage activation than resistant mice during infections with myotropic strains of Trypanosoma cruzi.

The kinetics of macrophage activation were compared among inbred strains of mice (C3H, BALB, B6 and B10.A) that are known to differ in their relative resistance to infections with the myotropic strains (Colombian and CL) of Trypanosoma cruzi. The parameters utilized to measure macrophage activation were rapid spreading on glass surfaces, hydrogen peroxide release and tumour necrosis factor/cachectin production. Macrophages obtained from C3H (susceptible), BALB (intermediate) and B6 or B10.A (resistant) mice infected with both strains of T. cruzi began to spread rapidly at the onset of parasitaemia. Surprisingly, the amount of hydrogen peroxide released by peritoneal cells obtained from the more susceptible mouse strain (C3H) was significantly higher than in the other mouse strains. Also, only in the serum of C3H mice was tumour necrosis factor/cachectin detected. These results suggest that resistance against infections with myotropic strains of T. cruzi does not correlate with enhanced macrophage activation. It is also shown that the acquired macrophage activation is largely dependent on T-lymphocytes bearing the phenotypic marker CD4 (helper/inducer), since all parameters of macrophage activation were significantly inhibited in athymic mice or in C3H mice treated in vivo with monoclonal antibody anti-CD4+ T-cells.

Demonstration of the presence of IgA in the human chorioamniotic membrane.

The presence of IgA was detected by direct immunofluorescence techniques in the chorion and decidua of eight fragments of chorioamniotic membranes obtained near the rupture edge from eight normal patients. It is suggested that, similar to what occurs in other organic systems, a first barrier against infection of the amniotic chamber exists at this site.

Experimental Trypanosoma cruzi cardiomyopathy in BALB/c mice. The potential role of intravascular platelet aggregation in its genesis.

In male BALB/c mice aged 5-6 weeks inoculated three times at intervals of 15 days with 1 X 10(7) epimastigote forms of the PF strain of Trypanosoma cruzi and challenged 30 days after the last inoculation with 2 X 10(4) trypomastigote forms of the Colombia strain of T cruzi (the mice were sacrificed 80-100 days after the challenge) a cardiomyopathy very similar to that observed in the chronic phase of Chagas' disease in man develops. The cardiac syndrome is characterized grossly by cardiomegaly with hypertrophy, dilatation of ventricular chambers, and thinning of the apex of the left ventricle (apical aneurysm) and microscopically by focal areas of myocytolytic necrosis and myocardial degeneration with an inflammatory response composed of mononuclear cells (predominantly macrophages and a few lymphocytes) with concurrent interstitial fibrosis and occasional myofibers containing pseudocysts. In addition, aggregated platelets and occlusive thrombi were found in small epicardial and intramyocardial vessels of infected mice as compared with controls. The potential role of intravascular platelet aggregation in the causation of focal myocardial necrosis and degeneration and apical aneurysm in experimental T cruzi cardiomyopathy in BALB/c mice is discussed.