Evidence of reversible bradycardia and arrhythmias caused by immunogenic proteins secreted by T. cruzi in isolated rat hearts.

RATIONALE: Chagas cardiomyopathy, caused by the protozoan Trypanosoma cruzi, is characterized by alterations in intracellular ion, heart failure and arrhythmias. Arrhythmias have been related to sudden death, even in asymptomatic patients, and their molecular mechanisms have not been fully elucidated. OBJECTIVE: The aim of this study is to demonstrate the effect of proteins secreted by T. cruzi on healthy, isolated beating rat heart model under a non-damage-inducing protocol. METHODS AND RESULTS: We established a non-damage-inducing recirculation-reoxygenation model where ultrafiltrate fractions of conditioned medium control or conditioned infected medium were perfused at a standard flow rate and under partial oxygenation. Western blotting with chagasic patient serum was performed to determine the antigenicity of the conditioned infected medium fractions. We observed bradycardia, ventricular fibrillation and complete atrioventricular block in hearts during perfusion with >50 kDa conditioned infected culture medium. The preincubation of conditioned infected medium with chagasic serum abolished the bradycardia and arrhythmias. The proteins present in the conditioned infected culture medium of >50 kDa fractions were recognized by the chagasic patient sera associated with arrhythmias. CONCLUSIONS: These results suggest that proteins secreted by T. cruzi are involved in Chagas disease arrhythmias and may be a potential biomarker in chagasic patients.

ß-defensin-2 in breast milk displays a broad antimicrobial activity against pathogenic bacteria / ß-defensina 2 no leite materno mostra uma ampla atividade antimicrobiana contra bactérias patogênicas

OBJECTIVE: To describe the antimicrobial activity of ß-defensin-2 produced in the mammary gland and secreted in human breast milk. METHODS: The peptide production was performed by DNA cloning. ß-defensin-2 levels were quantified in 61 colostrum samples and 39 mature milk samples from healthy donors, by an indirect enzyme-linked immunosorbent assay (ELISA). Using halo inhibition assay, this study assessed activity against seven clinical isolates from diarrheal feces of children between 0 and 2 years of age. The activity of ß-defensin-2 against three opportunistic pathogens that can cause nosocomial infections was determined by microdilution test. RESULTS: The peptide levels were higher in colostrum (n = 61) than in mature milk samples (n = 39), as follows: median and range, 8.52 (2.6-16.3) µg/ml versus 0.97 (0.22-3.78), p < 0.0001; Mann-Whitney test. The recombinant peptide obtained showed high antimicrobial activity against a broad range of pathogenic bacteria. Its antibacterial activity was demonstrated in a disk containing between 1-4 µg, which produced inhibition zones ranging from 18 to 30 mm against three isolates of Salmonella spp. and four of E. coli. ß-defensin-2 showed minimum inhibitory concentrations (MICs) of 0.25 µg/mL and 0.5 µg/mL for S. marcescen and P. aeruginosa, respectively, while a higher MIC (4 µg/mL) was obtained against an isolated of multidrug-resistant strain of A. baumannii. CONCLUSIONS: To the authors' knowledge, this study is the first to report ß-defensin-2 levels in Latin American women. The production and the activity of ß-defensin-2 in breast milk prove its importance as a defense molecule for intestinal health in pediatric patients. .

OBJETIVO: Descrever a atividade antimicrobiana da defensina-beta 2 na glândula mamária e secretada no leite materno humano. MÉTODOS: A produção de peptídeos foi realizada por clonagem de DNA. Os níveis de defensina-beta 2 foram quantificados em 61 amostras de colostro e 39 de leite maduro de doadoras saudáveis pelo teste ELISA indireto. Por um ensaio de halo de inibição, avaliamos a atividade contra sete isolados clínicos diarreicos de crianças entre 0 e 2 anos. A atividade da defensina 2 contra três patógenos oportunistas que podem causar infecções nosocomiais foi determinada pelo teste de microdiluição. RESULTADOS: Os níveis de peptídeos estavam significativamente maiores nas amostras de colostro (n = 61) que de leite maduro (n = 39), como segue: 8,52 (2,6-16,3 µg/mL) mediana e faixa em comparação a 0,97 (0,22-3,78), p < 0,0001; teste de Mann-Whitney. O peptídeo recombinante foi obtido da alta atividade antimicrobiana demonstrada contra uma ampla gama de bactérias patogênicas. Sua atividade antibacteriana foi demonstrada em um disco contendo entre 1-4 µg, que produziu zonas de inibição entre 18 e 30 mm contra três isolados de Salmonella spp. e quatro de E. coli. A defensina-beta 2 demonstrou concentrações inibitórias mínimas (CIMs) de 0,25 µg/mL e 0,5 µg/mL para S. marcescen and P. aeruginosa, ao passo que uma CIM maior (4 µg/mL) foi obtida contra um isolado de cepa multirresistente de A. baumannii. CONCLUSÕES: Até onde sabemos, este estudo é o primeiro a relatar níveis de defensina em mulheres da América Latina. A produção e a atividade da defensina 2 no leite materno comprovam sua importância como uma molécula de defesa para a saúde intestinal em pacientes pediátricos. .

ß-defensin-2 in breast milk displays a broad antimicrobial activity against pathogenic bacteria.

OBJECTIVE: To describe the antimicrobial activity of ß-defensin-2 produced in the mammary gland and secreted in human breast milk. METHODS: The peptide production was performed by DNA cloning. ß-defensin-2 levels were quantified in 61 colostrum samples and 39 mature milk samples from healthy donors, by an indirect enzyme-linked immunosorbent assay (ELISA). Using halo inhibition assay, this study assessed activity against seven clinical isolates from diarrheal feces of children between 0 and 2 years of age. The activity of ß-defensin-2 against three opportunistic pathogens that can cause nosocomial infections was determined by microdilution test. RESULTS: The peptide levels were higher in colostrum (n=61) than in mature milk samples (n=39), as follows: median and range, 8.52 (2.6-16.3) µg/ml versus 0.97 (0.22-3.78), p<0.0001; Mann-Whitney test. The recombinant peptide obtained showed high antimicrobial activity against a broad range of pathogenic bacteria. Its antibacterial activity was demonstrated in a disk containing between 1-4 µg, which produced inhibition zones ranging from 18 to 30 mm against three isolates of Salmonella spp. and four of E. coli. ß-defensin-2 showed minimum inhibitory concentrations (MICs) of 0.25 µg/mL and 0.5 µg/mL for S. marcescen and P. aeruginosa, respectively, while a higher MIC (4 µg/mL) was obtained against an isolated of multidrug-resistant strain of A. baumannii. CONCLUSIONS: To the authors' knowledge, this study is the first to report ß-defensin-2 levels in Latin American women. The production and the activity of ß-defensin-2 in breast milk prove its importance as a defense molecule for intestinal health in pediatric patients.

The second sodium pump: from the function to the gene.

Transepithelial Na(+) transport is mediated by passive Na(+) entry across the luminal membrane and exit through the basolateral membrane by two active mechanisms: the Na(+)/K(+) pump and the second sodium pump. These processes are associated with the ouabain-sensitive Na(+)/K(+)-ATPase and the ouabain-insensitive, furosemide-inhibitable Na(+)-ATPase, respectively. Over the last 40 years, the second sodium pump has not been successfully associated with any particular membrane protein. Recently, however, purification and cloning of intestinal α-subunit of the Na(+)-ATPase from guinea pig allowed us to define it as a unique biochemical and molecular entity. The Na(+)- and Na(+)/K(+)-ATPase genes are at the same locus, atp1a1, but have independent promoters and some different exons. Herein, we spotlight the functional characteristics of the second sodium pump, and the associated Na(+)-ATPase, in the context of its role in transepithelial transport and its response to a variety of physiological and pathophysiological conditions. Identification of the Na(+)-ATPase gene (atna) allowed us, using a bioinformatics approach, to explore the tertiary structure of the protein in relation to other P-type ATPases and to predict regulatory sites in the promoter region. Potential regulatory sites linked to inflammation and cellular stress were identified in the atna gene. In addition, a human atna ortholog was recognized. Finally, experimental data obtained using spontaneously hypertensive rats suggest that the Na(+)-ATPase could play a role in the pathogenesis of essential hypertension. Thus, the participation of the second sodium pump in transepithelial Na(+) transport and cellular Na(+) homeostasis leads us to reconsider its role in health and disease.

Isolation and cloning of the K+-independent, ouabain-insensitive Na+-ATPase.

Primary Na+ transport has been essentially attributed to Na+/K+ pump. However, there are functional and biochemical evidences that suggest the existence of a K+-independent, ouabain-insensitive Na+ pump, associated to a Na+-ATPase with similar characteristics, located at basolateral plasma membrane of epithelial cells. Herein, membrane protein complex associated with this Na+-ATPase was identified. Basolateral membranes from guinea-pig enterocytes were solubilized with polyoxyethylene-9-lauryl ether and Na+-ATPase was purified by concanavalin A affinity and ion exchange chromatographies. Purified enzyme preserves its native biochemical characteristics: Mg2+ dependence, specific Na+ stimulation, K+ independence, ouabain insensitivity and inhibition by furosemide (IC50: 0.5 mM) and vanadate (IC50: 9.1 µM). IgY antibodies against purified Na+-ATPase did not recognize Na+/K+-ATPase and vice versa. Analysis of purified Na+-ATPase by SDS-PAGE and 2D-electrophoresis showed that is constituted by two subunits: 90 (α) and 50 (ß) kDa. Tandem mass spectrometry of α-subunit identified three peptides, also present in most Na+/K+-ATPase isoforms, which were used to design primers for cloning both ATPases by PCR from guinea-pig intestinal epithelial cells. A cDNA fragment of 1148 bp (atna) was cloned, in addition to Na+/K+-ATPase α1-isoform cDNA (1283 bp). In MDCK cells, which constitutively express Na+-ATPase, silencing of atna mRNA specifically suppressed Na+-ATPase α-subunit and ouabain-insensitive Na+-ATPase activity, demonstrating that atna transcript is linked to this enzyme. Guinea-pig atna mRNA sequence (2787 bp) was completed using RLM-RACE. It encodes a protein of 811 amino acids (88.9 kDa) with the nine structural motifs of P-type ATPases. It has 64% identity and 72% homology with guinea-pig Na+/K+-ATPase α1-isoform. These structural and biochemical evidences identify the K+-independent, ouabain-insensitive Na+-ATPase as a unique P-type ATPase.

Differential expression of P-type ATPases in intestinal epithelial cells: identification of putative new atp1a1 splice-variant.

P-type ATPases are membrane proteins that couple ATP hydrolysis with cation transport across the membrane. Ten different subtypes have been described. In mammalia, 15 genes of P-type ATPases from subtypes II-A, II-B and II-C, that transport low-atomic-weight cations (Ca(2+), Na(+), K(+) and H(+)), have been reported. They include reticulum and plasma-membrane Ca(2+)-ATPases, Na(+)/K(+)-ATPase and H(+)/K(+)-ATPases. Enterocytes and colonocytes show functional differences, which seem to be partially due to the differential expression of P-type ATPases. These enzymes have 9 structural motifs, being the phosphorylation (E) and the Mg(2+)ATP-binding (H) motifs the most preserved. These structural characteristics permitted developing a Multiplex-Nested-PCR (MN-PCR) for the simultaneous identification of different P-type ATPases. Thus, using MN-PCR, seven different cDNAs were cloned from enterocytes and colonocytes, including SERCA3, SERCA2, Na(+)/K(+)-ATPase alpha1-isoform, H(+)/K(+)-ATPase alpha2-isoform, PMCA1, PMCA4 and a cDNA-fragment that seems to be a new cassette-type splice-variant of the atp1a1 gen. PMCA4 in enterocytes and H(+)/K(+)-ATPase alpha2-isoform in colonocytes were differentially expressed. This cell-specific expression pattern is related with the distinctive enterocyte and colonocyte functions.

Modulation of membrane type 1 matrix metalloproteinase by LPS and gamma interferon bound to extracellular matrix in intestinal crypt cells.

Membrane type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane protein that participates in the processing and degradation of cell surface proteins and the extracellular matrix (ECM). This enzyme regulates ECM turnover in wound repair, promotes cell migration and activates other MMPs, such as MMP-2, which is involved in angiogenesis, cell migration and tumoral metastasis. An increase in pro-inflammatory cytokine expression, such as gamma interferon (IFN-gamma), has been associated with chronic wounds in inflammatory bowel diseases. However, the extent to which cytokines modulate MT1-MMP has not been totally defined. In this report, the effects of the bacterial lipopolysaccharide (LPS) and ECM-bound IFN-gamma on MT1-MMP expression and MMP-2 activity were evaluated by Western blot, RT-PCR and zymography in isolated intestinal epithelial and cultured HT-29 cells. In the presence of LPS, ECM-bound IFN-gamma, but not soluble IFN-gamma, reduced the enterocyte MT1-MMP protein expression. In addition, the active form of MMP-2 was also decreased in the presence of both LPS and IFN-gamma, indicating that lower MMP-2 activity accompanied the decrease in MT1-MMP expression. These results suggest the possibility that endotoxin and ECM-bound IFN-gamma may affect matrix remodeling by modulating matrix metalloproteinase in enterocytes during wound healing.

Interferon gamma bound to extracellular matrix changes the hyporesponsiveness to LPS in crypt but not villous intestinal epithelial cells.

Intestinal epithelial cells (IEC) are hyporesponsive to LPS. Responsiveness to luminal bacteria has been implicated in the pathogenesis of inflammatory bowel diseases (IBD). In support of this, previous studies have demonstrated that some intestinal epithelial cell lines are induced by IFN-gamma to respond to LPS. However, both the responsiveness to LPS and the effect of IFN-gamma in intestinal cell lines are heterogeneous. In addition, IFN-gamma may be sequestered in the extracellular matrix (ECM) compartment. The ECM-bound form is more effective than soluble IFN-gamma in producing its biological effects in several experimental models. We investigated the effect of ECM-bound and soluble IFN-gamma treatment on interleukin-8 (IL-8) secretion in response to LPS in freshly isolated villous and crypt cells. We demonstrate that ECM-bound, but not soluble IFN-gamma, induced an increase in IL-8 secretion in response to LPS in undifferentiated crypt cells. This effect was associated with an increase in TLR4 expression. In contrast, mature villous cells did not modify their response to LPS when treated with IFN-gamma (ECM-bound or soluble). These results suggest that selective changes in immature crypt cells induced by IFN-gamma bound to extracellular matrix could contribute to inappropriate responsiveness to commensal bacteria in IBD.

Polymorphonuclear leukocyte migration across intestinal epithelium induces epithelial cytokine expression

La migración de neutrófilos polimorfonucleares es un rasgo común de la inflamación activa, que precede la formación de abscesos. La contribución relativa de células epiteliales como fuente de quimioquinas en la infiltración de leucocitos durante la inflamación intestinal no ha sido estudiada. Para evaluar esta contribución nosotros diseñamos un modelo heterólogo de migración transepitelial, “in vitro”, haciendo uso de PMN de rata y células epiteliales de origen humano. Nosotros demostramos que neutrófilos polimorfonucleares luego de su activación quimiotáctica, inducen el incremento en los niveles de ARNm de IL-1ß, IL-8 en las células epiteliales de intestino, mientras que no afecta al ARNm de ENA-78. Estos resultados sugieren que quimiocinas y citoquinas sintetizados por la célula epitelial podrían jugar un papel en el mantenimiento de la respuesta inflamatoria.

Polymorphonuclear neutrophil migration is a common feature of active inflammation that precedes the formation of abscesses. The relative contribution of epithelial cells as a source of chemokines in the recruitment of leukocytes during intestinal inflammation has not been studied. To evaluate this contribution, we have designed a heterologous “in vitro” model for transepithelial PMN migration, based on the use of rat neutrophils and human epithelial cells. We show that polymorphonuclear neutrophil, upon chemotactic activation, induces an increase in IL-1ß, IL-8 levels in intestinal epithelial cells, while not changed ENA-78 mRNA. These results suggest that chemokines and cytokines synthesized by intestinal epithelial cells could play a role in the maintenance of the inflammatory response.

Backdoor phosphorylation of basolateral plasma membranes of small intestinal epithelial cells: characterization of a furosemide-induced phosphoprotein related to the second sodium pump.

Enterocyte has two different Na+-stimulated ATPases, the ouabain-sensitive Na+/K+ ATPase and a furosemide-inhibitable Na+ ATPase. To identify the polypeptide associated with the Na+-ATPase, 32Pi phosphorylation into basolateral membranes of enterocyte was investigated. Both, ouabain and furosemide induced Mg2+-dependent, vanadate-sensitive 32Pi incorporation into a 100kDa polypeptide. K(m) for Pi was 17.7+/-1.82 microM and 16.8+/-0.69 microM for ouabain-induced and furosemide-induced phosphorylation, respectively. K(m) for furosemide was 1.3+/-0.21 mM. Furosemide-induced 32Pi incorporation was sensitive to alkaline pH and hydroxylamine suggesting an acyl-phosphate bond. Na+ and K+ inhibited 32Pi incorporation induced by ouabain. In contrast, Na+ stimulated furosemide-induced phosphorylation with a K(m) of 16.5+/-5.59 mM while K+ had no effect. Purified Na+/K+ ATPase only presented ouabain-induced phosphoprotein, indicating that furosemide-induced phosphorylation is not related to this enzyme and appears to correspond to a new member of P-type ATPases associated with the second Na+ pump.