RESUMO
Fibrin forms the structural scaffold of blood clots and has great potential for biomaterial applications. Creating recombinant expression systems of fibrinogen, fibrin's soluble precursor, would advance the ability to construct mutational libraries that would enable structure-function studies of fibrinogen and expand the utility of fibrin as a biomaterial. Despite these needs, recombinant fibrinogen expression systems, thus far, have relied on the time-consuming creation of stable cell lines. Here we present tests of a transient fibrinogen expression system that can rapidly generate yields of 8-12 mg/L using suspension HEK Expi293TM cells. We report results from two different plasmid systems encoding the fibrinogen cDNAs and two different transfection reagents. In addition, we describe a novel, affinity-based approach to purifying fibrinogen from complex media such as human plasma. We show that using a high-affinity peptide which mimics fibrin's knob 'A' sequence enables the purification of 50-75% of fibrinogen present in plasma. Having robust expression and purification systems of fibrinogen will enable future studies of basic fibrin(ogen) biology, while paving the way for the ubiquitous use of fibrin as a biomaterial.
Assuntos
Fibrinogênio , Trombose , Materiais Biocompatíveis , Cromatografia de Afinidade , Fibrina/metabolismo , Fibrinogênio/metabolismo , HumanosRESUMO
BACKGROUND: Undernutrition remains a significant problem worldwide, with environmental enteropathy implicated as a contributing factor. An understanding of the pathogenesis and identification of children at risk are critical to the design of more-effective interventions. OBJECTIVE: The stool regenerating gene 1ß (REG1B) protein, which is a putative measure of intestinal injury and repair, was tested as a noninvasive biomarker of future childhood stunting. DESIGN: A total of 222 children from Bangladesh and 97 children from Peru, who were from impoverished communities, were followed from birth through 24 mo of age with anthropometric measures obtained every 3 mo. Stool REG1B protein concentrations were obtained by using an REG1B polyclonal-polyclonal ELISA at 3 mo of age. We tested for the ability of REG1B to forecast future anthropometric shortfalls, independent of known predictors of undernutrition of family income and baseline height and weight. RESULTS: In the Bangladesh cohort of 222 children, higher REG1B concentrations at month 3 were significantly and independently associated with a growth shortfall in a linear regression analysis at months 9, 12, 18, 21, and 24 and, in the Peru cohort, at months 12, 15, 18, 21, and 24. With the use of a mixed model for repeated measurements, higher stool REG1B concentrations at 3 mo were also independently predictive of a lower future length-for-age z score through 24 mo of age (Bangladesh P = 0.006; Peru P = 0.058). CONCLUSION: The ability of fecal REG1B to predict growth shortfall in independent cohorts of impoverished children from the developing world offers promise as a malnutrition biomarker and supports a role for environmental enteropathy in the pathogenesis of growth shortfall.
Assuntos
Transtornos do Crescimento/epidemiologia , Transtornos do Crescimento/genética , Litostatina/genética , Desnutrição/epidemiologia , Bangladesh/epidemiologia , Estatura , Peso Corporal , Pré-Escolar , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática , Feminino , Transtornos do Crescimento/etiologia , Humanos , Modelos Lineares , Litostatina/metabolismo , Masculino , Desnutrição/complicações , Estado Nutricional , Peru/epidemiologia , Prevalência , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Risco , Fatores SocioeconômicosRESUMO
Entamoeba histolytica, a protozoan parasite, is an important cause of diarrhea and colitis in the developing world. Amebic colitis is characterized by ulceration of the intestinal mucosa. We performed microarray analysis of intestinal biopsies during acute and convalescent amebiasis in order to identify genes potentially involved in tissue injury or repair. Colonic biopsy samples were obtained from 8 patients during acute E. histolytica colitis and again 60 days after recovery. Gene expression in the biopsies was evaluated using microarray, and confirmed by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). REG 1A and REG 1B were the most up-regulated of all genes in the human intestine in acute versus convalescent E. histolytica disease: as determined by microarray, the levels of induction were 7.4-fold and 10.7 fold for REG 1A and B; p=0.003 and p=0.006 respectively. Increased expression of REG 1A and REG 1B protein in the colonic crypt epithelial cells during acute amebiasis was similarly observed by immunohistochemistry. Because REG 1 protein is anti-apoptotic and pro-proliferative, and since E. histolytica induces apoptosis of the intestinal epithelium as part of its disease process, we next tested if REG 1 might be protective during amebiasis by preventing parasite-induced apoptosis. Intestinal epithelial cells from REG 1-/- mice were found to be more susceptible to spontaneous, and parasite-induced, apoptosis in vitro (p=0.03). We concluded that REG 1A and REG 1B were upregulated during amebiasis and may function to protect the intestinal epithelium from parasite-induced apoptosis.
Assuntos
Disenteria Amebiana/parasitologia , Entamebíase/parasitologia , Litostatina/genética , Adolescente , Adulto , Animais , Apoptose , Colo/parasitologia , Colo/patologia , Disenteria Amebiana/genética , Entamoeba histolytica/patogenicidade , Entamebíase/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Intestinos/parasitologia , Intestinos/patologia , Litostatina/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , RNA/genética , Adulto JovemRESUMO
Malnutrition substantially increases susceptibility to Entamoeba histolytica in children. Leptin is a hormone produced by adipocytes that inhibits food intake, influences the immune system, and is suppressed in malnourished children. Therefore we hypothesized that diminished leptin function may increase susceptibility to E. histolytica infection. We prospectively observed a cohort of children, beginning at preschool age, for infection by the parasite E. histolytica every other day over 9 years and evaluated them for genetic variants in leptin (LEP) and the leptin receptor (LEPR). We found increased susceptibility to intestinal infection by this parasite associated with an amino acid substitution in the cytokine receptor homology domain 1 of LEPR. Children carrying the allele for arginine (223R) were nearly 4 times more likely to have an infection compared with those homozygous for the ancestral glutamine allele (223Q). An association of this allele with amebic liver abscess was also determined in an independent cohort of adult patients. In addition, mice carrying at least 1 copy of the R allele of Lepr were more susceptible to infection and exhibited greater levels of mucosal destruction and intestinal epithelial apoptosis after amebic infection. These findings suggest that leptin signaling is important in mucosal defense against amebiasis and that polymorphisms in the leptin receptor explain differences in susceptibility of children in the Bangladesh cohort to amebiasis.
Assuntos
Entamoeba histolytica/metabolismo , Entamebíase/genética , Entamebíase/parasitologia , Predisposição Genética para Doença , Mutação , Receptores para Leptina/genética , Alelos , Apoptose , Pré-Escolar , Estudos de Coortes , Feminino , Glutamina/genética , Homozigoto , Humanos , Abscesso Hepático/metabolismo , Masculino , Estudos ProspectivosRESUMO
OBJECTIVE: We sought to compare the tuberculin skin test (TST) to the QuantiFERON-TB Gold In-Tube assay (QFT-IT) and assess the effects of malnourishment and intestinal helminth infection on QFT-IT results. METHODS: In this population-based cross-sectional study from Dhaka, Bangladesh, we screened children for latent tuberculosis infection with the QFT-IT and TST. We assess the agreement between the TST and QFT-IT, risk factors associated with indeterminate QFT-IT results, and magnitude of interferon γ (IFN-γ) production. RESULTS: Three hundred and two children (aged 11-15.3 years) were enrolled, including 93 (30.8%) who were malnourished. Of 251 participants who provided stool samples, 117 (46.6%) were infected with Ascaris lumbricoides and/or Trichuris trichiura. TST results were positive (≥10 mm) for 101 (33.4%) children and negative for 201 (66.6%) children. QFT-IT results were positive for 107 (35.4%) children, negative for 121 (40.1%) children, and indeterminate for 74 (24.5%) children. Agreement between the tests was moderate (κ = 0.55 [95% confidence interval: 0.44-0.65]; P < .0001) when excluding indeterminate results. Children with indeterminate QFT-IT results were separately compared with children with positive and negative QFT-IT results; malnutrition (P = .0006 and .0003), and helminth infection (P = .05 and .02), and the statistical interaction between these 2 terms (P = .03 and .004) were associated with indeterminate results. Higher levels of IFN-γ in response to tuberculosis antigens were associated with positive TST results (P < .0001); lower levels were associated with malnutrition (P = .02). CONCLUSIONS: Malnutrition and helminth infections were associated with indeterminate QFT-IT results. Therefore, the presence of such conditions may limit the interpretability of QFT-IT results in children.
Assuntos
Helmintíase/complicações , Interferon gama/metabolismo , Desnutrição/etiologia , Adolescente , Animais , Bangladesh/epidemiologia , Portador Sadio , Criança , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Fezes/parasitologia , Feminino , Seguimentos , Helmintíase/sangue , Helmintíase/epidemiologia , Helmintos/isolamento & purificação , Humanos , Incidência , Interferon gama/sangue , Masculino , Desnutrição/sangue , Desnutrição/epidemiologia , Programas de Rastreamento , Reprodutibilidade dos Testes , Estudos Retrospectivos , Fatores de Risco , Teste Tuberculínico/métodos , Tuberculose/sangue , Tuberculose/complicações , Tuberculose/epidemiologiaRESUMO
An association between tumor necrosis factor alpha (TNF-alpha) and Entamoeba histolytica diarrhea was assessed in a cohort of 138 non-related Bangladeshi children who have been prospectively followed since 2001. Peripheral blood mononuclear cells (PBMCs) obtained at study entry were purified, cultured, and stimulated with soluble amebic antigen before cytokine measurement from supernatant. Higher levels of TNF-alpha were associated with increased risk of first (P = 0.01) and recurrent E. histolytica-related diarrheal episodes (P = 0.005). Children who developed E. histolytica diarrhea had significantly higher TNF-alpha protein levels than those who experienced asymptomatic E. histolytica infection (P value = 0.027) or no infection (P value = 0.017). Microarray studies performed using RNA isolated from acute and convalescent whole blood and colon biopsy samples revealed higher but non-significant TNF-alpha messenger RNA (mRNA) levels in subjects with acute E. histolytica diarrhea compared with convalescence. We conclude that there is an association between higher TNF-alpha production and E. histolytica diarrhea.
Assuntos
Diarreia/parasitologia , Disenteria Amebiana/metabolismo , Entamoeba histolytica/isolamento & purificação , Fator de Necrose Tumoral alfa/sangue , Bangladesh/epidemiologia , Estudos de Coortes , Disenteria Amebiana/sangue , Disenteria Amebiana/epidemiologia , Regulação da Expressão Gênica , Humanos , Leucócitos Mononucleares , Mucosa/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Observation of a cohort of preschool children in Dhaka, Bangladesh, is beginning to reveal the contributions of environment, host, and parasite to amebiasis. Reviewed here are the associations and interactions of malnutrition, IgA and interferon-gamma, human leukocyte antigen alleles, and parasite genotypes to the outcome of infection. Future efforts aimed at understanding the mechanisms of these effects are described.
Assuntos
Transtornos da Nutrição Infantil/imunologia , Transtornos da Nutrição Infantil/parasitologia , Disenteria Amebiana/complicações , Entamoeba histolytica/patogenicidade , Interações Hospedeiro-Parasita , Alelos , Animais , Bangladesh , Pré-Escolar , Disenteria Amebiana/imunologia , Entamoeba histolytica/genética , Entamebíase/complicações , Entamebíase/imunologia , Genótipo , Humanos , Imunoglobulina A/sangue , Interferon gama/sangueRESUMO
BACKGROUND: Because studies suggest that the dietary supplement conjugated linoleic acid (CLA) has immunomodulatory activities that might benefit common colds, we performed two studies of CLA effects in experimental human rhinovirus (HRV) infection. METHODS: The first study explored whether CLA supplementation (Safflorin; Loders Croklaan, BV, Wormerveer, the Netherlands) altered the virological or clinical course of experimental HRV infection, and the second explored whether CLA affected the frequency and severity of HRV cold-associated sore throat and cough. The trials were randomized, double-blinded and placebo-controlled. In total, 50 healthy volunteers aged 18-45 years and susceptible to HRV type-39 (serum neutralizing antibody titre < or = 1:2) participated in study 1 and 80 similar volunteers susceptible to Hank's HRV participated in study 2. Participants ingested CLA 2 g/day or placebo for 4 weeks before and 4 days following intranasal HRV inoculation. The primary endpoint for study 1 was the frequency of colds and for study 2 was the symptom severity scores for sore throat and cough. RESULTS: In study 1, 10/24 (42%) placebo compared with 7/21 (33%) CLA participants developed colds (P = 0.53). CLA was associated with significant reductions in mean scores for cough (0 CLA versus 0.9 placebo) and sore throat (0.8 CLA versus 2.9 placebo). In study 2, clinical colds developed in 19/33 (58%) placebo and 27/43 (63%) CLA participants. Symptom scores for cough (0.9 CLA versus 1.0 placebo) and sore throat (2.6 CLA versus 3.2 placebo) were not significantly different. Similarly no differences in nasal viral titres or serological responses were found. CONCLUSIONS: CLA dietary supplementation had no consistent effects on the virological or clinical course of experimental HRV colds. A larger study would be required to detect more subtle effects of CLA on HRV cold-associated symptoms.