The contribution of A3 adenosine receptor signaling to pulmonary inflammation and mucus secretion in the mouse

Adenosine has been implicated in chronic lung diseases such as asthma and COPD. Most physiological actions of adenosine are mediated through G-protein coupled adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B, and A 3. However, the specific roles of the various adenosine receptors in processes central to asthma and COPD are not well understood in part due to the lack of adequate animal models that examine the effect of adenosine on the development of lung disease. In this study we have investigated the expression and function of the A3 adenosine receptor in pulmonary eosinophilia and mucus production/secretion in adenosine deaminase (ADA)-deficient mice in which adenosine levels are elevated. ADA-deficient mice develop features of asthma and COPD, including lung eosinophilia and mucus hyperplasia in association with elevated lung adenosine levels. The A3 receptor was found to be expressed in eosinophils and mucus producing cells in the airways of ADA-deficient. Disruption of A3 receptor signaling in ADA-deficient mice by genetic removal of the receptor or treatment with MRS 1523, a selective A3 adenosine receptor antagonist, prevented airway eosinophilia and mucus production. Although eosinophils were decreased in the airways of ADA-deficient mice with disrupted A3 receptor signaling, elevations in circulating and lung interstitial eosinophils persisted, suggesting signaling through the A3 receptor is needed for the migration of eosinophils into the airways. Further examination of the role of the A3 receptor in mucus biology demonstrated that the A3 receptor is neither required nor is overexpression of the receptor in clara cells sufficient for mucus production in naive mice. Transgenic overexpression of the A3 receptor did elucidate a role for the A3 receptor in the secretion of mucus into the airways of ovalbumin challenged mice. These findings identify an important role for the A3 adenosine receptor in regulating lung eosinophilia and mucus secretion in inflammatory lung diseases. Therefore, the A3 adenosine receptor may represent a novel therapeutic target for the treatment and prevention of asthma. ^

The role of adenosine in pulmonary angiogenesis

Angiogenesis is a feature of chronic lung diseases such as asthma and pulmonary fibrosis; however, the pathways controlling pathological angiogenesis during lung disease are not completely understood. Adenosine is a signaling nucleoside that accumulates as a result of tissue hypoxia and damage. Adenosine has been implicated in the exacerbation of chronic lung disease and in the regulation of angiogenesis; however, the relationship between these factors has not been investigated. The work presented in this dissertation utilized adenosine deaminase (ADA)-deficient mice to determine whether chronic elevations of adenosine in vivo result in pulmonary angiogenesis, and to identify factors that could potentially mediate this process. Results demonstrate that there is substantial angiogenesis in the tracheas of ADA-deficient mice in association with adenosine elevations. Replacement enzyme therapy with pegylated ADA resulted in a lowering of adenosine levels and reversal of tracheal angiogenesis, indicating that the increases in vessel number are dependent on adenosine elevations. Levels of the ELR+ angiogenic chemokine CXCL1 were found to be elevated in an adenosine-dependent manner in the lungs of ADA-deficient mice. Neutralization of CXCL1 and its putative receptor, CXCR2, in ADA-deficient lung lysates resulted in the inhibition of angiogenic activity suggesting that CXCL1 signaling through the CXCR2 receptor is responsible for mediating the observed increases in angiogenesis. Taken together, these findings suggest that adenosine plays an important role, via CXCL1, in the induction of pulmonary angiogenesis and may therefore represent an important therapeutic target for the treatment of pathological angiogenesis. ^

Factors associated with diabetes in tuberculosis patients in Harris County, Texas 1995--2004

Objective. To determine the prevalence and factors associated with diabetes in tuberculosis patients in Harris County, Texas. ^ Background. Tuberculosis and diabetes mellitus are two diseases of immense public health significance. Various epidemiologic studies have established an association between the two conditions. While many studies have identified factors associated with the conditions individually, few have looked at factors associated with their co-occurrence particularly in the United States. Furthermore, most of those studies are hospital-based and may not be representative of the population. The aim of this study was to determine the prevalence and distribution of diabetes among tuberculosis patients in Harris County, Texas and to identify the factors associated with diabetes in tuberculosis. ^ Methods. A population-based case control study was performed using secondary data from the Houston Tuberculosis Initiative (HTI) collected from October 1995 to September 2004. Socio-demographic characteristics and clinical variables were compared between tuberculosis patients with diabetes and non-diabetic tuberculosis patients. Logistic regression analysis was performed to identify associations. Survival at 180 days post tuberculosis diagnosis was assessed by Cox regression. ^ Results. The prevalence of diabetes among the tuberculosis (TB) population was 14.4%. The diabetics (cases) with a mean age 53 ± 13.3 years were older than the non-diabetics (controls) with a mean age of 39 ± 18.5 years (p<0.001). Socio-demographic variables that were independently associated with the risk of diabetes were age (OR 1.04, p<0.001) and Hispanic ethnicity (OR 2.04, p<0.001). Diabetes was associated with an increased risk of pulmonary tuberculosis disease (OR 1.33, p<0.028). Among individuals with pulmonary TB, diabetes was associated with positive sputum acid-fast bacilli (AFB) smear (OR 1.47, p<0.005) and culture (OR 1.83, p<0.018). Diabetics were more likely to have cavitary lung disease than non-diabetics (OR 1.50, p<0.002). After adjustment for age and HIV status, the risk of dying within 180 days of TB diagnosis was significantly increased in the diabetics (HR 1.51, p<0.002). ^ Conclusion. Diabetes mellitus was more prevalent in our tuberculosis patients than in the general population. The tuberculous diabetic may be more infectious and has a higher risk of death. It is therefore imperative to screen diabetics for TB and TB patients for diabetes. ^

Adenosine induced pulmonary fibrosis and the contribution of the adenosine A2B receptor to the induction of osteopontin in a mouse model of pulmonary fibrosis

Pulmonary fibrosis is a devastating and lethal lung disease with no current cure. Research into cellular signaling pathways able to modulate aspects of pulmonary inflammation and fibrosis will aid in the development of effective therapies for its treatment. Our laboratory has generated a transgenic/knockout mouse with systemic elevations in adenosine due to the partial lack of its metabolic enzyme, adenosine deaminase (ADA). These mice spontaneously develop progressive lung inflammation and severe pulmonary fibrosis suggesting that aberrant adenosine signaling is influencing the development and/or progression of the disease in these animals. These mice also show marked increases in the pro-fibrotic mediator, osteopontin (OPN), which are reversed through ADA therapy that serves to lower lung adenosine levels and ameliorate aspects of the disease. OPN is known to be regulated by intracellular signaling pathways that can be accessed through adenosine receptors, particularly the low affinity A2BR receptor, suggesting that adenosine receptor signaling may be responsible for the induction of OPN in our model. In-vitro, adenosine and the broad spectrum adenosine receptor agonist, NECA, were able to induce a 2.5-fold increase in OPN transcripts in primary alveolar macrophages. This induction was blocked through antagonism of the A2BR receptor pharmacologically, and through the deletion of the receptor subtype in these cells genetically, supporting the hypothesis that the A2BR receptor was responsible for the induction of OPN in our model. These findings demonstrate for the first time that adenosine signaling is an important modulator of pulmonary fibrosis in ADA-deficient mice and that this is in part due to signaling through the A2BR receptor which leads to the induction of the pro-fibrotic molecule, otseopontin. ^

Respiratory syncytial virus infection in hematopoietic stem cell transplant patients

Respiratory Syncytial Virus (RSV) is a major cause of respiratory tract infections in immunocompromised patients such as children less than 2 years, premature infants with congenital heart disease and chronic lung disease, elderly patients and patients who have undergone hematopoietic stem cell transplant (HSCT). HSCT patients are at high risk of RSV infection, at increased risk of developing pneumonia, and RSV-related mortality. Immunodeficiency can be a major risk factor for severe infection & mortality. Therapy of RSV infection with Ribavirin, Palivizumab and Immunoglobulin has shown to reduce the risk of progression to LRI and mortality, especially if initiated early in the disease. Data on RSV infection in HSCT patients is limited, especially at various levels of immunodeficiency. 323 RSV infections in HSCT patients have been identified between 1/1995 and 8/2009 at University of Texas M D Anderson Cancer Center (UTMDACC). In this proposed study, we attempted to analyze a de-identified database of these cases and describe the epidemiologic characteristics of RSV infection in HSCT patients, the course of the infection, rate of development of pneumonia and RSV-related mortality in HSCT patients at UTMDACC.^ Key words: RSV infections, HSCT patients ^

Seasonality of respiratory syncytial virus infection in Texas

Respiratory syncytial virus (RSV) is a common cause of respiratory infection in infants and children that can result in bronchiolitis or pneumonia. Each year in the United States, it causes up to 400 deaths and 125,000 hospitalizations among children less than one year of age. RSV is transmitted by direct or close contact with contaminated secretions, which may involve droplets and fomites. Monthly administration of a monoclonal RSV antibody, palivizumab (Synagis™, MedImmune, Gaithersburg, MD), in premature infants, infants with chronic lung disease, or congenital heart disease has been shown to significantly reduce the risk of severe RSV infection. The Centers for Disease Control and Prevention's (CDC) National Respiratory and Enteric Virus Surveillance System (NREVSS) is a laboratory based passive reporting system that collects state, regional, and national RSV data. The CDC defines the RSV season onset as “the first of 2 consecutive weeks during which the mean percentage of specimens testing positive for RSV antigen is 10%.” RSV season offset is defined as the last of 2 consecutive weeks during which the percentage of positive specimens is less than or equal to 10%. Annual RSV epidemics generally occur during the winter and early spring months, but the RSV season is known to vary by national regions. Precise delineation of the RSV epidemiology by region could maximize protection from RSV and minimize the cost of RSV immune prophylaxis. ^ The purpose of this thesis is to define the RSV season in Texas over time; compare the RSV season of the state of Texas and its regions with the national norms; and to compare RSV seasonality between the various regions in Texas. ^ This study was a retrospective analysis of data reported to NREVSS to evaluate potential disparities in the onset weeks, offset weeks, and duration of the annual RSV season in Texas. Data were collected from 70 reporting sites, and includes information from the 2004–2005 to 2009–2010 RSV seasons. ^ The observed median onset (week 44) and offset week (week 8) for the Texas were consistent with national estimates for the South. Regional estimates and statistical analysis suggested that the RSV season in Texas would be better represented by regions. Regional seasonal comparisons revealed considerable variation in season offset and duration between many of the geographic regions within Texas. This trend should be studied further.^

Significado de la pérdida de peso en pacientes hospitalizados

La pérdida involuntaria de peso es un predictor independiente de morbimortalidad, especialmente en ancianos, pacientes con cáncer, SIDA y postoperatorios. Con el objeto de determinar la significación clínica de la pérdida de peso en pacientes internados, se estudiaron 100 pacientes. La edad media fue de 57.6 años (DS±11.04); 38% mayores de 65 años y 62% hombres. La permanencia hospitalaria media fue de 13 días, superior a la media del servicio (7,3 días). El 61% pertenecían a clase social baja y 25% eran desocupados. En el 100% fue involuntaria y en ninguno fue causa de hospitalización. Tenían hiporexia 61 pacientes y 57 malnutrición. El IMC fue inferior a 20 en el 50% de los casos. La causa fue determinada en el 70% y en 72% se relacionó con la enfermedad de base, en 27% con trastornos alimentarios y con fármacos en 1%. Las etiologías más frecuentes fueron: neoplasias (34 pacientes), enfermedades crónicas (24), TBC (3) y SIDA (3). El 46% desarrollaron infecciones nosocomiales y el 100% tenían comórbidas (alcoholismo 26%, depresión 22%, diabetes 20%, EPOC 11%, insuficiencia cardiaca, cirrosis y demencia 8% c/u e insuficiencia renal 6%). La mortalidad fue del 18% y las causas más frecuentes fueron sepsis severa, fallo multiorgánico y neoplasias. Conclusiones: La pérdida significativa de peso en el paciente hospitalizado se caracterizó por ser involuntaria, asociada a clase social baja, a malnutrición, a alta taza de comorbilidad, a predisposición a infecciones nosocomiales, secundaria a enfermedades crónicas, neoplasias, tuberculosis y SIDA y a una tasa de mortalidad elevada.

Diagnosis and Management of Grain-Induced Asthma

Grain-induced asthma is a frequent occupational allergic disease mainly caused by inhalation of cereal flour or powder. The main professions affected are bakers, confectioners, pastry factory workers, millers, farmers, and cereal handlers. This disorder is usually due to an IgE-mediated allergic response to inhalation of cereal flour proteins. The major causative allergens of grain-related asthma are proteins derived from wheat, rye and barley flour, although baking additives, such as fungal α-amylase are also important. This review deals with the current diagnosis and treatment of grain-induced asthma, emphasizing the role of cereal allergens as molecular tools to enhance diagnosis and management of this disorder. Asthma-like symptoms caused by endotoxin exposure among grain workers are beyond the scope of this review. Progress is being made in the characterization of grain and bakery allergens, particularly cereal-derived allergens, as well as in the standardization of allergy tests. Salt-soluble proteins (albumins plus globulins), particularly members of the α-amylase/trypsin inhibitor family, thioredoxins, peroxidase, lipid transfer protein and other soluble enzymes show the strongest IgE reactivities in wheat flour. In addition, prolamins (not extractable by salt solutions) have also been claimed as potential allergens. However, the large variability of IgE-binding patterns of cereal proteins among patients with grain-induced asthma, together with the great differences in the concentrations of potential allergens observed in commercial cereal extracts used for diagnosis, highlight the necessity to standardize and improve the diagnostic tools. Removal from exposure to the offending agents is the cornerstone of the management of grain-induced asthma. The availability of purified allergens should be very helpful for a more refined diagnosis, and new immunomodulatory treatments, including allergen immunotherapy and biological drugs, should aid in the management of patients with this disorder.

Production of β-defensins by human airway epithelia

Human β-defensins (HBDs) are antimicrobial peptides that may play a role in mucosal defense. Diminished activity of these peptides has been implicated in the pathogenesis of cystic fibrosis (CF) lung disease. We show that HBD-1 and HBD-2 mRNAs are expressed in excised surface and submucosal gland epithelia from non-CF and CF patients. The pro-inflammatory cytokine interleukin-1β stimulated the expression of HBD-2 but not HBD-1 mRNA and peptide in primary cultures of airway epithelia. HBD-1 was found in bronchoalveolar lavage (BAL) fluid from normal volunteers, CF patients, and patients with inflammatory lung diseases, whereas HBD-2 was detected in BAL fluid from patients with CF or inflammatory lung diseases, but not in normal volunteers. Both HBD-1 and HBD-2 were found in BAL fluid in concentrations of several ng/ml, and both recombinant peptides showed salt-sensitive bactericidal activity. These data suggest that in the lung HBD-2 expression is induced by inflammation, whereas HBD-1 may serve as a defense in the absence of inflammation.

Role of the cystic fibrosis transmembrane conductance regulator in innate immunity to Pseudomonas aeruginosa infections

Chronic Pseudomonas aeruginosa infection occurs in 75–90% of patients with cystic fibrosis (CF). It is the foremost factor in pulmonary function decline and early mortality. A connection has been made between mutant or missing CF transmembrane conductance regulator (CFTR) in lung epithelial cell membranes and a failure in innate immunity leading to initiation of P. aeruginosa infection. Epithelial cells use CFTR as a receptor for internalization of P. aeruginosa via endocytosis and subsequent removal of bacteria from the airway. In the absence of functional CFTR, this interaction does not occur, allowing for increased bacterial loads in the lungs. Binding occurs between the outer core of the bacterial lipopolysaccharide and amino acids 108–117 in the first predicted extracellular domain of CFTR. In experimentally infected mice, inhibiting CFTR-mediated endocytosis of P. aeruginosa by inclusion in the bacterial inoculum of either free bacterial lipopolysaccharide or CFTR peptide 108–117 resulted in increased bacterial counts in the lungs. CFTR is also a receptor on gastrointestinal epithelial cells for Salmonella enterica serovar Typhi, the etiologic agent of typhoid fever. There was a significant decrease in translocation of this organism to the gastrointestinal submucosa in transgenic mice that are heterozygous carriers of a mutant ΔF508 CFTR allele, suggesting heterozygous CFTR carriers may have increased resistance to typhoid fever. The identification of CFTR as a receptor for bacterial pathogens could underlie the biology of CF lung disease and be the basis for the heterozygote advantage for carriers of mutant alleles of CFTR.

Two cystic fibrosis transmembrane conductance regulator mutations have different effects on both pulmonary phenotype and regulation of outwardly rectified chloride currents.

Cystic fibrosis (CF), a disorder of electrolyte transport manifest in the lungs, pancreas, sweat duct, and vas deferens, is caused by mutations in the CF transmembrane conductance regulator (CFTR). The CFTR protein has been shown to function as a cAMP-activated chloride channel and also regulates a separate protein, the outwardly rectifying chloride channel (ORCC). To determine the consequence of disease-producing mutations upon these functions, mutant CFTR was transiently expressed in Xenopus oocytes and in human airway epithelial cells lacking functional CFTR. Both G551D, a mutation that causes severe lung disease, and A455E, a mutation associated with mild lung disease, altered but did not abolish CFTR's function as a chloride channel in Xenopus oocytes. Airway epithelial cells transfected with CFTR bearing either A455E or G551D had levels of chloride conductance significantly greater than those of mock-transfected and lower than those of wild-type CFTR-transfected cells, as measured by chloride efflux. A combination of channel blockers and analysis of current-voltage relationships were used to dissect the contribution of CFTR and the ORCC to whole cell currents of transfected cells. While CFTR bearing either mutation could function as a chloride channel, only CFTR bearing A455E retained the function of regulating the ORCC. These results indicate that CF mutations can affect CFTR functions differently and suggest that severity of pulmonary disease may be more closely associated with the regulatory rather than chloride channel function of CFTR.

Utilità e limiti della diagnosi di patologie polmonari e pleuriche asbesto-correlate

L’apparato respiratorio rappresenta il bersaglio di numerose sostanze tossiche aerodisperse che rivestono un ruolo chiave nella patogenesi della maggior parte delle patologie polmonari e pleuriche, sia benigne che maligne. Nonostante per alcune di esse siano noti specifici fattori di rischio, le sole attività di prevenzione primaria non sono sufficienti a limitarne la diffusione. Si rende quindi necessario attuare adeguate misure di prevenzione secondaria per la diagnosi di malattie potenzialmente curabili allo stadio iniziale, in modo da aumentare l’efficacia dei trattamenti terapeutici e le possibilità di guarigione. Un approccio non invasivo per lo studio dei meccanismi fisiopatologici alla base delle patologie polmonari e pleuriche potrebbe essere effettuato anche con nuove metodiche (es. naso elettronico), al fine di identificare e validare nuovi biomarcatori per un più specifico approccio diagnostico. Il lavoro scientifico ha riguardato inizialmente l’identificazione di un indicatore o di un gruppo di indicatori dotati di potere diagnostico sufficientemente elevato per poter discriminare precocemente, nell’ambito di soggetti con pregressa esposizone ad asbesto, patologie benigne, sia polmonari che pleuriche, da patologie maligne. Successivamente l’attenzione è stata rivolta alla diagnosi precoce di patologie neoplastiche a carico del solo parenchima polmonare, valutando il potere discriminante di un pattern di composti organici volatili (VOCs, tra cui pentano, 2-metilpentano, esano, etilbenzene, eptanale e trans-2-nonenale) raccolti con metodiche non invasive e dotati di potere diagnostico tale da discriminare patologie benigne da patologie maligne potenzialmente curabili in soggetti ad alto rischio di sviluppare cancro del polmone. Infine abbiamo tentato di ottimizzare i parametri di impostazione e raccolta di un nuovo strumento: il naso elettronico. Su di esso esistono alcuni lavori in letteratura in cui ne vengono descritte le potenzialità in ambito diagnostico per il riconoscimento di specifici pattern suggestivi di patologie polmonari, sia flogistiche (TBC, BPCO) che neoplastiche (mesotelioma, NSCLC). Purtroppo nessuno di questi lavori definisce le condizioni ottimali di utilizzo, i limiti dello strumento e le interferenze di fattori ambientali e soggettivi riguardo al segnale elaborato. Il lavoro si è concentrato soprattutto sull’indagine delle condizioni ottimali di utilizzo e sull’eventuale condizionamento del segnale da parte di determinate variabili ambientali (es. umidità) o individuali (es. fumo, cibo, alcol).

Efeitos da imunossupressão sobre a depuração mucociliar de ratos: comparação entre dois esquemas de terapia tríplice

INTRODUÇÃO: O transplante de pulmão é parte fundamental no tratamento das doenças terminais do pulmão, constituindo uma modalidade terapêutica eficaz para pacientes com doença pulmonar incapacitante, progressiva e em estágio final. No entanto, as drogas imunossupressoras usadas para evitar a rejeição do enxerto podem causar efeitos colaterais em diversos tecidos. O sistema mucociliar, presente nas vias aéreas, é um dos principais mecanismos de defesa do trato respiratório e pode ser alterado por ação das drogas imunossupressoras. Desta forma, o objetivo deste estudo foi avaliar o sistema mucociliar traqueobrônquico de ratos submetidos a dois esquemas de terapia tríplice imunossupressora. MÉTODOS: Foram utilizados 90 ratos machos Wistar distribuídos em 3 grupos conforme o tratamento: controle (C) = solução salina; terapia 1 (TI) = tacrolimus + micofenolato de mofetil + prednisona; terapia 2 (TII) = ciclosporina + azatioprina + prednisona. Após o período de tratamento (7, 15 ou 30 dias), os animais foram sacrificados e realizadas as seguintes medidas: transportabilidade do muco (TM), frequência de batimento ciliar (FBC), quantificação de muco neutro e ácido, velocidade de transporte mucociliar (VTMC), e contagem total e diferencial de células no lavado broncoalveolar (LBA). RESULTADOS: A TM não foi afetada pelas terapias em nenhum dos tempos estudados. Ambas as terapias causaram significativa redução da FBC dos animais tratados por 7 e 15 dias. A produção de muco neutro foi menor nos animais tratados com a TI por 7, 15 e 30 dias. Porém, com a TII, essa redução ocorreu apenas aos 7 dias. Por outro lado, a quantidade de muco ácido foi significativamente maior em todos os animais tratados com as duas terapias. Todos os animais tratados com as terapias imunossupressoras apresentaram redução da VTMC nos três tempos. Houve aumento do número total de células e de macrófagos e neutrófilos no grupo TI em 7 dias. CONCLUSÕES: Ambas as terapias imunossupressoras foram prejudiciais ao transporte mucociliar das vias aéreas de ratos, tanto pela redução da FBC e da VTMC, quanto pela maior produção de muco ácido e menor produção de muco neutro. A TI foi mais prejudicial ao sistema mucociliar em comparação à TII

Mecanismos celulares, stress oxidativo e balanço protease-antiprotease na DPOC : revisão de literatura

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Pneumotórax espontâneo primário : da incerteza etiológica ás dicotomias do tratamento

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014