Bronchial hyperresponsiveness and analysis of induced sputum cells in Crohn's disease

With the aim of investigating the presence of latent inflammatory process in the lungs of patients with Crohn's disease, 15 patients with Crohn's disease were evaluated by spirometry, the methacholine challenge test, induced sputum, and skin tests for inhaled antigens. Serum IgE, erythrocyte sedimentation rate and hematocrit were also determined. The patients were compared with 20 healthy controls by the Mann-Whitney and Fisher exact tests. Their respiratory physical examination was normal. None had a personal or family history of clinical atopy. None had a previous history of pulmonary disease, smoking or toxic bronchopulmonary exposure. None had sinusitis, migraine, diabetes mellitus, or cardiac failure. Four (26.6%) of the patients with Crohn's disease had a positive methacholine challenge test whereas none of the 20 controls had a positive methacholine test (P = 0.026, Fisher exact test). Patients with Crohn's disease had a higher level of lymphocytes in induced sputum than controls (mean 14.59%, range 3.2-50 vs 5.46%, 0-26.92%, respectively; P = 0.011, Mann-Whitney test). Patients with Crohn's disease and a positive methacholine challenge test had an even higher percentage of lymphocytes in induced sputum compared with patients with Crohn's disease and a negative methacholine test (mean 24.88%, range 12.87-50 vs 10.48%, 3.2-21.69%; P = 0.047, Mann-Whitney test). The simultaneous findings of bronchopulmonary lymphocytosis and bronchial hyperresponsiveness in patients with Crohn's disease were not reported up to now. These results suggest that patients with Crohn's disease present a subclinical inflammatory process despite the absence of pulmonary symptoms.

IL-5 and IL-5 receptor in asthma

Eosinophils, along with mast cells are key cells involved in the innate immune response against parasitic infection whereas the adaptive immune response is largely dependent on lymphocytes. In chronic parasitic disease and in chronic allergic disease, IL-5 is predominantly a T cell derived cytokine which is particularly important for the terminal differentiation, activation and survival of committed eosinophil precursors. The human IL-5 gene is located on chromosome 5 in a gene cluster that contains the evolutionary related IL-4 family of cytokine genes. The human IL-5 receptor complex is a heterodimer consisting of a unique a subunit (predominantly expressed on eosinophils) and a beta subunit which is shared between the receptors for IL-3 & GM-CSF (more widely expressed). The a subunit is required for ligand-specific binding whereas association with the beta subunit results in increased binding affinity. The alternative splicing of the alphaIL-5R gene which contains 14 exons can yield several alphaIL-5R isoforms including a membrane-anchored isoform (alphaIL-5Rm) and a soluble isoform (alphaIL-5Rs). Cytokines such as IL-5 produce specific and non-specific cellular responses through specific cell membrane receptor mediated activation of intracellular signal transduction pathways which, to a large part, regulate gene expression. The major intracellular signal transduction mechanism is activation of non-receptor associated tyrosine kinases including JAK and MAP kinases which can then transduce signals via a novel family of transcriptional factors named signal transducers and activators of transcription (STATS). JAK2, STAT1 and STAT 5 appear to be particularly important in IL-5 mediated eosinophil responses. Asthma is characterized by episodic airways obstruction, increased bronchial responsiveness, and airway inflammation. Several studies have shown an association between the number of activated T cells and eosinophils in the airways and abnormalities in FEV1, airway reactivity and clinical severity in asthma. It has now been well documented that IL-5 is highly expressed in the bronchial mucosa of atopic and intrinsic asthmatics and that the increased IL-5 mRNA present in airway tissues is predominantly T cell derived. Immunocytochemical staining of bronchial biopsy sections has confirmed that IL-5 mRNA transcripts are translated into protein in asthmatic subjects. Furthermore, the number of activated CD 4 + T cells and IL-5 mRNA positive cells are increased in asthmatic airways following antigen challenge and studies that have examined IL-5 expression in asthmatic subjects before and after steroids have shown significantly decreased expression following oral corticosteroid treatment in steroid-sensitive asthma but not in steroid resistant and chronic severe steroid dependent asthma. The link between T cell derived IL-5 and eosinophil activation in asthmatic airways is further strengthened by the demonstration that there is an increased number of alphaIL-5R mRNA positive cells in the bronchial biopsies of atopic and non-atopic asthmatic subjects and that the eosinophil is the predominant site of this increased alphaIL-5R mRNA expression. We have also shown that the subset of activated eosinophils that expressed mRNA for membrane bound alpha IL5r inversely correlated with FEV1, whereas the subset of activated eosinophils that expressed mRNA for soluble alphaIL5r directly correlated with FEV1. Hence, not only does this data suggest that the presence of eosinophils expressing alphaIL-5R mRNA contribute towards the pathogenesis of bronchial asthma, but also that the eosinophil phenotype with respect to alphaIL-5R isoform expression is of central importance. Finally, there are several animal, and more recently in vitro lung explant, models of allergen induced eosinophilia, late airway responses(LARS), and bronchial hyperresponsiveness(BHR) - all of which support a link between IL-5 and airway eosinophila and bronchial hyperresponsiveness. The most direct demonstration of T cell involvement in LARS is the finding that these physiological responses can be transferred by CD4+ but not CD8+ T cells in rats. The importance of IL-5 in animal models of allergen induced bronchial hyperresponsiveness has been further demonstrated by a number of studies which have indicated that IL-5 administration is able to induce late phase responses and BHR and that anti-IL-5 antibody can block allergen induced late phase responses and BHR. In summary, activated T lymphocytes, IL5 production and eosinophil activation are particularly important in the asthmatic response. Human studies in asthma and studies in allergic animal models have clearly emphasised the unique role of IL-5 in linking T lymphocytes and adaptive immunity, the eosinophil effector cell, and the asthma phenotype. The central role of activated lymphocytes and eosinophils in asthma would argue for the likely therapeutic success of strategies to block T cell and eosinophil activation (eg steroids). Importantly, more targeted therapies may avoid the complications associated with steroids. Such therapies could target key T cell activation proteins and cytokines by various means including blocking antibodies (eg anti-CD4, anti-CD40, anti-IL-5 etc), antisense oligonucleotides to their specific mRNAs, and/or selective inhibition of the promoter sites for these genes. Another option would be to target key eosinophil activation mechanisms including the aIL5r. As always, the risk to benefit ratio of such strategies await the results of well conducted clinical trials.

Role of immunoglobulin E and mast cells in murine models of asthma

Immunoglobulin E (IgE) and mast cells are believed to play important roles in allergic inflammation. However, their contributions to the pathogenesis of human asthma have not been clearly established. Significant progress has been made recently in our understanding of airway inflammation and airway hyperresponsiveness through studies of murine models of asthma and genetically engineered mice. Some of the studies have provided significant insights into the role of IgE and mast cells in the allergic airway response. In these models mice are immunized systemically with soluble protein antigens and then receive an antigen challenge through the airways. Bronchoalveolar lavage fluid from mice with allergic airway inflammation contains significant amounts of IgE. The IgE can capture the antigen presented to the airways and the immune complexes so formed can augment allergic airway response in a high-affinity IgE receptor (FcepsilonRI)-dependent manner. Previously, there were conflicting reports regarding the role of mast cells in murine models of asthma, based on studies of mast cell-deficient mice. More recent studies have suggested that the extent to which mast cells contribute to murine models of asthma depends on the experimental conditions employed to generate the airway response. This conclusion was further supported by studies using FcepsilonRI-deficient mice. Therefore, IgE-dependent activation of mast cells plays an important role in the development of allergic airway inflammation and airway hyperresponsiveness in mice under specific conditions. The murine models used should be of value for testing inhibitors of IgE or mast cells for the development of therapeutic agents for human asthma.

Protective effect of bronchial challenge with hypertonic saline on nocturnal asthma

Inhalation of hypertonic saline (HS) causes bronchoconstriction in asthmatic subjects. Repeated inhalation of HS leads to substantially reduced bronchoconstriction, known as the refractory period. Refractoriness due to different stimuli has also been described (cross-refractoriness). Nocturnal asthma is defined as an increase in symptoms, need for medication, airway responsiveness, and/or worsening of lung function that usually occurs from 4 to 6 am. Our objective was to determine the effect of refractoriness on nocturnal asthma. The challenge test consisted of inhalations of 4.5% saline with increasing durations until a reduction of 20% in forced expiratory volume in 1 s (FEV1) (PD20HS) or total time of 15.5 min. Twelve subjects with nocturnal asthma were challenged with HS at 16:00 and 18:00 h and FEV1 was measured at 4:00 h. One to 2 weeks later, FEV1 was determined at 16:00 and 4:00 h. LogPD20HS at 18:00 h was significantly greater than logPD20HS at 16:00 h, 0.51 ± 0.50 and 0.69 ± 0.60 mg, respectively (P = 0.0033). When subjects underwent two HS challenges in the afternoon, mean (± SD) FEV1 reduction was 206 ± 414 mL or 9.81 ± 17.42%. On the control day (without challenge in the afternoon) FEV1 reduction was 523 ± 308 mL or 22.75 ± 15.40% (P = 0.021). Baseline FEV1 values did not differ significantly between the control and study days, 2.48 ± 0.62 and 2.36 ± 0.46 L, respectively. The refractory period following HS challenges reduces the nocturnal worsening of asthma. This new concept may provide beneficial applications to asthmatic patients.

Sildenafil decreases rat tracheal hyperresponsiveness to carbachol and changes canonical transient receptor potential gene expression after antigen challenge

Inhibition of type-5 phosphodiesterase by sildenafil decreases capacitative Ca2+ entry mediated by transient receptor potential proteins (TRPs) in the pulmonary artery. These families of channels, especially the canonical TRP (TRPC) subfamily, may be involved in the development of bronchial hyperresponsiveness, a hallmark of asthma. In the present study, we evaluated i) the effects of sildenafil on tracheal rings of rats subjected to antigen challenge, ii) whether the extent of TRPC gene expression may be modified by antigen challenge, and iii) whether inhibition of type-5 phosphodiesterase (PDE5) may alter TRPC gene expression after antigen challenge. Sildenafil (0.1 µM to 0.6 mM) fully relaxed carbachol-induced contractions in isolated tracheal rings prepared from naive male Wistar rats (250-300 g) by activating the NO-cGMP-K+ channel pathway. Rats sensitized to antigen by intraperitoneal injections of ovalbumin were subjected to antigen challenge by ovalbumin inhalation, and their tracheal rings were used to study the effects of sildenafil, which more effectively inhibited contractions induced by either carbachol (10 µM) or extracellular Ca2+ restoration after thapsigargin (1 µM) treatment. Antigen challenge increased the expression of the TRPC1 and TRPC4 genes but not the expression of the TRPC5 and TRPC6 genes. Applied before the antigen challenge, sildenafil increased the gene expression, which was evaluated by RT-PCR, of TRPC1 and TRPC6, decreased TRPC5 expression, and was inert against TRPC4. Thus, we conclude that PDE5 inhibition is involved in the development of an airway hyperresponsive phenotype in rats after antigen challenge by altering TRPC gene expression.

PREVALENCE OF BACTERIAL RESISTANCE IN HOSPITALIZED CIRRHOTIC PATIENTS IN SOUTHERN BRAZIL: A NEW CHALLENGE

Background & Aims: An increased frequency of infections by multiresistant bacteria has been described in hospitalized patients. The aim of this study was to evaluate the bacterial resistance profile in cirrhotic patients. Methods: This is a retrospective observational study. We assessed the antimicrobial susceptibility of 5,839 bacterial isolates from patients with and without cirrhosis. Regarding the multidrug resistance, we evaluated 4,505 bacterial isolates from 2,180 patients. Results: Two hundred and fifty-one patients had cirrhosis (mean age 57.6 ± 11 years; 61.8% were male, 47.8% of cases associated with hepatitis C virus). Of the isolates of patients with and without cirrhosis, 174/464 (37.5%) and 1,783/4,041 (44.1%) were multiresistant, respectively (p = 0.007). E. coli was the most common multiresistant bacteria in both groups. Approximately 20% of E. coli and Klebsiella sp. isolates were ESBL-producers and 44% of S. aureus isolates were methicillin-resistant in cirrhotic patients. In cirrhotic patients admitted to the emergency department, hospital ward, and intensive care unit, 28.3%, 50% and 40% had multiresistant isolates, respectively. In patients with and without cirrhosis, 36.2% and 33.5% of isolates were resistant to third-generation cephalosporins, respectively. Conclusions: The empirical treatment of infections in hospitalized patients using broad-spectrum antibiotics should consider the observed pattern of bacterial resistance.

Role of eosinophilic airway inflammation in models of asthma

Eosinophils play a central role in the establishment and outcome of bronchial inflammation in asthma. Animal models of allergy are useful to answer questions related to mechanisms of allergic inflammation. We have used models of sensitized and boosted guinea pigs to investigate the nature of bronchial inflammation in allergic conditions. These animals develop marked bronchial infiltration composed mainly of CD4+ T-lymphocytes and eosinophils. Further provocation with antigen leads to degranulation of eosinophils and ulceration of the bronchial mucosa. Eosinophils are the first cells to increase in numbers in the mucosa after antigen challenge and depend on the expression of alpha 4 integrin to adhere to the vascular endothelium and transmigrate to the mucosa. Blockage of alpha4 integrin expression with specific antibody prevents not only the transmigration of eosinophils but also the development of bronchial hyperresponsiveness (BHR) to agonists in sensitized and challenged animals, clearly suggesting a role for this cell type in this altered functional state. Moreover, introduction of antibody against Major Basic Protein into the airways also prevents the development of BHR in similar model. BHR can also be suppressed by the use of FK506, an immunosuppressor that reduces in almost 100% the infiltration of eosinophils into the bronchi of allergic animals. These data support the concept that eosinophil is the most important pro-inflammatory factor in bronchial inflammation associated with allergy.

Antiretroviral resistance mutations in human immunodeficiency virus type 1 infected patients enrolled in genotype testing at the Central Public Health Laboratory, São Paulo, Brazil: preliminary results

Antiretroviral resistance mutations (ARM) are one of the major obstacles for pharmacological human immunodeficiency virus (HIV) suppression. Plasma HIV-1 RNA from 306 patients on antiretroviral therapy with virological failure was analyzed, most of them (60%) exposed to three or more regimens, and 28% of them have started therapy before 1997. The most common regimens in use at the time of genotype testing were AZT/3TC/nelfinavir, 3TC/D4T/nelfinavir and AZT/3TC/efavirenz. The majority of ARM occurred at protease (PR) gene at residue L90 (41%) and V82 (25%); at reverse transcriptase (RT) gene, mutations at residue M184 (V/I) were observed in 64%. One or more thymidine analogue mutations were detected in 73%. The number of ARM at PR gene increased from a mean of four mutations per patient who showed virological failure at the first ARV regimens to six mutations per patient exposed to six or more regimens; similar trend in RT was also observed. No differences in ARM at principal codon to the three drug classes for HIV-1 clades B or F were observed, but some polymorphisms in secondary codons showed significant differences. Strategies to improve the cost effectiveness of drug therapy and to optimize the sequencing and the rescue therapy are the major health priorities.

Primary resistance of HIV to antiretrovirals among individuals recently diagnosed at voluntary counselling and testing centres in the metropolitan region of Recife, Pernambuco

Determining the prevalence and type of antiretroviral (ARV) resistance among ARV-naïve individuals is important to assess the potential responses of these individuals to first-line regimens. The prevalence of primary resistance and the occurrence of recent infections among individuals with human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) were identified among recently diagnosed patients at five sexually transmitted disease/AIDS testing and counselling centres in the metropolitan region of Recife (RMR), Pernambuco, Brazil, between 2007-2009. One-hundred and eight samples were analysed using the Calypte® BED assay. Males predominated (56%), as did patients aged 31-50 years. Twenty-three percent presented evidence of a recent HIV infection. The median CD4+ T lymphocyte count was 408 cells/mm³ and the median viral load was 3.683 copies/mL. The prevalence of primary resistance was 4.6% (confidence interval 95% = 1-8.2%) based on criteria that excluded common polymorphisms in accordance with the surveillance drug resistance mutation criteria. The prevalence of resistance to non-nucleoside reverse transcriptase, nucleoside/nucleotide reverse transcriptase and protease inhibitors were 3.8%, 1.5% and 0.8%, respectively. Fifty-seven percent of strains were from clade B, 37.7% were clade F and 3.1% were clade C; there were no statistically significant differences with respect to resistance between clades. Recent infection tended to be more common in men (p = 0.06) and in municipalities in the south of the RMR (Jaboatão dos Guararapes and Cabo de Santo Agostinho) (p = 0.046). The high prevalence of recent infection and the high prevalence of non-B strains in this poor Brazilian region merit further attention.

Effect of salbutamol on pulmonary responsiveness in chronic pulmonary allergic inflammation in guinea pigs

Beta-2-agonists have been widely used by asthmatic subjects to relieve their obstructive symptoms. However, there are reports that continuous use could lead to loss of bronchial protection and exacerbation of asthma symptoms. We evaluated the effect of two regimens of salbutamol administration (twice and five times a week) in a model of chronic airway inflammation in male Hartley guinea pigs (protocol starting weight: 286 ± 30 g) induced by repeated exposures to aerosols of ovalbumin (OVA). After sensitization, guinea pigs were exposed to aerosols of 0.1 mg/ml salbutamol solution twice a week (OVA + S2x, N = 7) or five times a week (OVA + S5x, N = 8). We studied allergen-specific (OVA inhalation time) and -nonspecific (response to methacholine) respiratory system responsiveness. Seventy-two hours after the last OVA challenge, guinea pigs were anesthetized and tracheostomized, respiratory system resistance and elastance were measured and a dose-response curve to inhaled methacholine chloride was obtained. Specific IgG1 was also quantified by the passive cutaneous anaphylactic technique. OVA-sensitized guinea pigs (N = 8) showed reduction of the time of OVA exposure before the onset of respiratory distress, at the 5th, 6th and 7th exposures (P < 0.001). The OVA + S2x group (but not the OVA + S5x group) showed a significant increase in OVA inhalation time. There were no significant differences in pulmonary responsiveness to methacholine among the experimental groups. OVA + S2x (but not OVA + S5x) animals showed a decrease in the levels of IgG1-specific anaphylactic antibodies compared to the OVA group (P < 0.05). Our results suggest that, in this experimental model, frequent administration of ß2-agonists results in a loss of some of their protective effects against the allergen.

Evaluation of genotype resistance testing for salvage antiretroviral therapy at AIDS care centers from Ribeirão Preto, São Paulo, Brazil

The availability of HIV-1 genotype resistance testing (GRT) to clinicians has been insufficiently studied outside randomized clinical trials. The present study evaluated the outcome of salvage antiretroviral therapy (ART) recommended by an expert physician based on GRT in a non-clinical trial setting in Ribeirão Preto, Brazil. A prospective, open, nonrandomized study evaluating easy access to GRT at six Brazilian AIDS Clinics was carried out. This cooperative study analyzed the efficacy of treatment recommended to patients whose salvage ART was guided by GRT with that of treatment with ART based only on previous ART history. A total of 112 patients with ART failure were included in the study, and 77 of them were submitted to GRT. The median CD4 cell count and viral load for these 77 patients at baseline were (mean ± SD) 252.1 ± 157.4 cells/µL and 4.60 ± 0.5 log10 HIV RNA copies/mL, respectively. The access time, i.e., the time elapsed between ordering the GRT and receiving the result was, on average, 71.9 ± 37.3 days. The study results demonstrated that access to GRT followed by expert recommendations did not improve the time to persistent treatment failure when compared to conventional salvage ART. Access to GRT in this Brazilian community health care setting did not improve the long-term virologic outcomes of HIV-infected patients experiencing treatment failure. This result is probably related to the long time required to implement ART guided by GRT.

Resistance of mice immunized with killed culture trypomastigotes against infection by insect-derived trypomastigotes of Trypanosoma cruzi

Mice immunized with heat or merthiolate-killed culture trypomastigotes of the non-virulent G strain were resistant to the challenge by insect-derived trypomastigotes of the CL strain of Trypanosoma cruzi. No parasitemia was detected, by direct microscopic examination of blood samples, in 90% of immunized mice while all control animals developed a high parasitemia. Trypsinization before heat-inactivation, or fixation with paraformaldehyde, apparently reduced the immunogenicity of the G strain trypomastigotes. Mice immunized with trypomastigotes treated by either of these procedures were not protected against infection by virulent T. cruzi. Analysis of the 13I-labeled surface proteins of G strain trypomastigotes inactivated by the various methods suggests that these components are involved in eliciting protective immunity against T. cruzi infection.

: acquired resistance in mice by implantation of young irradiated worms into the portal system

In two distinct experiments, immature S. mansoni worms (LE strain, Belo Horizonte, Brazil), aged 20 days, obtained from the portal system of white outbred mice, were irradiated with 14 and 4 Krad, respectively. Afterwards, the worms were directly inoculated into the portal vein of normal mice. Inoculation was performed with 20 irradiated worms per animal. Fifty days after inoculation, the mice that received 4 and 14 Krad-irradiated worms and their respective controls were infected with S. mansoni cercariae (LE strain), by transcutaneous route. Twenty days after this challenge infection, the animals were sacrificed and perfused for mature irradiated (90-day-old) and immature (20-day-old) worm counts. Analysis of the results showed that statistically significant protection against cercariae occurred in both groups with irradiated worms.

Clonal types and antimicrobial resistance profiles of methicillin-resistant Staphylococcus aureus isolates from hospitals in south Brazil

In the present study were evaluated the DNA macrorestriction profile and SCCmec types for nine multi-resistant MRSA selected. Also antimicrobial susceptibility testing by disk diffusion method was evaluated for 68 MRSA isolates against 12 antimicrobial agents. The isolates were recovered from blood culture collected from hospitalized patients in three hospitals of Porto Alegre, Brazil. PFGE and PCR for mecA and SCCmec I, II, III, IV types genes were done on selected nine isolates with susceptibility only to vancomycin, teicoplanin and linezolid. Two clone profiles, with five subtypes, were demonstrated among multi-resistant MRSA analyzed. Eight isolates showed harbor SCCmec type III and one isolate was not typeable. The knowledge of SCCmec type, clone and antimicrobial profiles among S. aureus is essential mainly to prevention and control of dissemination of the antimicrobial resistance.

Chagas disease as a mechanistic model for testing a novel hypothesis

The association between depression and cardiovascular disease is well documented. Nevertheless, the process through which they are linked remains unknown, as does the direction of this relationship. Studies have suggested both that depression is a risk factor for heart disease and that heart disease is a risk factor for depression. A number of studies have established that a relationship exists between depression and inflammation, with alterations in the levels of inflammatory markers (IL-1, IL-6, TNF-alpha and others). Depressive symptoms have also been identified in many diseases characterized by inflammatory processes e.g. rheumatoid arthritis, bronchial asthma, diabetes, tuberculosis and cardiovascular diseases. In this brief viewpoint, we explain and propose how to use Chagas disease, a disorder characterized by inflammatory processes and leading to cardiovascular and autonomic problems, as a model for studying the directionality of the relationship between heart disease and depression.