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Small intestine bacterial overgrowth (SIBO) has been associated with enteric inflammation, linear growth stunting, and neurodevelopmental delays in children from low-income countries. Little is known about the histologic changes or epithelial adherent microbiota associated with SIBO. We sought to describe these relationships in a cohort of impoverished Bangladeshi children. Undernourished 12-18-month-old children underwent both glucose hydrogen breath testing for SIBO and duodenoscopy with biopsy. Biopsy samples were subject to both histological scoring and 16s rRNA sequencing. 118 children were enrolled with 16s sequencing data available on 53. Of 11 histological features, we found that SIBO was associated with one, enterocyte injury in the second part of the duodenum (R = 0.21, p = 0.02). SIBO was also associated with a significant increase in Campylobacter by 16s rRNA analysis (Log 2-fold change of 4.43; adjusted p = 1.9 x 10-6). These findings support the growing body of literature showing an association between SIBO and enteric inflammation and enterocyte injury and further delineate the subgroup of children with environmental enteric dysfunction who have SIBO. Further, they show a novel association between SIBO and Campylobacter. Mechanistic work is needed to understand the relationship between SIBO, enterocyte injury, and Campylobacter.
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Infecções Bacterianas , Intestino Delgado , Criança , Humanos , Lactente , RNA Ribossômico 16S/genética , Intestino Delgado/microbiologia , Duodeno/microbiologia , Infecções Bacterianas/complicações , Inflamação/complicações , BiópsiaRESUMO
Inhibition of the dihydroorotate dehydrogenase (DHODH) has been successful at the preclinical level in controlling myeloid leukemia. However, poor clinical trials warrant the search for new potent DHODH inhibitors. Herein we present a novel DHODH inhibitor SBL-105 effective against myeloid leukemia. Chemical characteristics were identified by 1H NMR, 13C NMR, and mass spectroscopy. Virtual docking and molecular dynamic simulation analysis were performed using the automated protocol with AutoDock-VINA, GROMACS program. Human-recombinant (rh) DHODH was used for enzyme inhibition study. THP-1, TF-1, HL-60, and SKM-1 cell lines were used. MTT assay was used to assess cell viability. Flow cytometry was employed for cell cycle, apoptosis, and differentiation analysis. Chemical analysis identified the compound to be 3-benzylidene-6,7-benz-chroman-4-one (SBL-105). The compound showed high binding efficacy toward DHODH with a Gbinding score of 10.9 kcal/mol. Trajectory analysis indicated conserved interactions of SBL-105DHODH to be stable throughout the 200-ns simulation. SBL-105 inhibited rh DHODH with an IC50 value of 48.48 nM. The GI50 values of SBL-105 in controlling THP-1, TF-1, HL-60, and SKM-1 cell proliferations were 60.66, 45.33, 73.98, and 86.01 nM, respectively. A dose-dependent increase in S-phase cell cycle arrest and total apoptosis was observed by SBL-105 treatment in both cell types, which were reversed in the presence of uridine. The compound also increased the differentiation marker CD11b-positive populations in both THP-1 and TF-1 cells, which were decreased under uridine influence. SBL-105, a novel DHODH inhibitor, identified using computational and in vitro analysis, was effective in controlling AML cells and needs attention for further preclinical developments.
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Leucemia Mieloide Aguda , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Ciclo Celular , Di-Hidro-Orotato Desidrogenase , Inibidores Enzimáticos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológicoRESUMO
Coordinated effects of glucose and oleic acid on glucagon-like peptide-1 (GLP-1) mediated differentiation of insulin-positive differentiating umbilical cord mesenchymal stromal cells (dUCBMSCs) was studied using a co-culture of NCI-H716 (GLP-1+) and UCBMSCs (insulin+). The addition of 2.5 mM glucose increased the proliferation of NCI-H716 cells by 30% and induced transformation of UCBMSCs into insulin-secreting cells in 18 days as compared to 22 days in control cells. Oleic acid (25 µM) showed decrease in cell proliferation, autophagy, and apoptosis in NCI-H716 cells while no effect was observed in dUCBMSCs. Prolonged glucose and oleic acid resulted in apoptosis and cell cycle changes in dUCBMSCs after day 18 while higher concentrations resulted in cell death. Additionally, the expression of FAS and ACC mRNA was observed in NCI-H716 and dUCBMSCs post 24-hr addition of glucose and/or oleic acid. Absorption of oleic acid was high in NCI-H716 compared to dUCBMSCs. Taken together, optimal concentrations of glucose and oleic acid could be a key factor in stimulating intrinsic GLP-1, which in turn stimulates differentiating MSCs in a glucose-dependent manner. PRACTICAL APPLICATIONS: The aim of this article was to study whether differentiating or differentiated MSCs after mobilization or post-transplant would require optimal glucose and oleic acid to naturally stimulate intrinsic GLP-1, or otherwise, the high or long-term overload of glucose or oleic acid could result in inhibition of differentiated cells resulting in failure of insulin secretion.
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Peptídeo 1 Semelhante ao Glucagon , Insulina , Linhagem Celular , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Glucose , Insulina/metabolismo , Ácido OleicoRESUMO
Asthma prevalence is increasing worldwide, and surveys indicate that most patients in developed and developing countries, including South Africa, do not receive optimal care and are therefore not well controlled. Standard management guidelines adapted to in-country realities are important to support optimal care. The South African Thoracic Society (SATS) first published a guideline for the management of chronic persistent asthma in 1992, which has subsequently been revised several times. The main aim of the present document was to revise and update SATS' statement on the suggested management of chronic asthma, based on the need to promote optimal care and control of asthma, together with the incorporation of new concepts and drug developments. This revised document reinforces optimal care and incorporates the following primary objectives to achieve the recent advances in asthma care: continued emphasis on the use of inhaled corticosteroids (ICS) as the foundation of asthma treatmentto reduce the reliance on short-acting beta-2 agonist (SABA) monotherapy for asthma symptomsto incorporate the evidence and strategy for the use of the combination of an ICS and formoterol for acute symptom relief (instead of a SABA)to incorporate the evidence and strategy for the use of as-needed ICS-long-acting beta agonists (LABA) for patients with infrequent symptoms or 'mild' asthmato incorporate the evidence and strategy for the use of a long-acting muscarinic antagonist (LAMA) in combination with ICS-LABA; andto incorporate the evidence and strategy for the use of and management with a biologic therapy in severe asthma.
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Asthmatics do not appear to have increased susceptibility to COVID-19.Uncontrolled severe asthma may be associated with worsened COVID-19 outcomes, especially in asthmatics managed with oral corticosteroids. Risk mitigation measures such as hand hygiene, social distancing and wearing of face masks must be observed at all times. Asthma should be managed as outlined in local and international guidelines.Ensure an adequate supply of medication, and inhaled corticosteroids should not be withdrawnChronic obstructive pulmonary disease (COPD) is associated with severe COVID-19 disease and poor outcomes. Maintenance of background medication is important to avoid exacerbations of COPD.Vaccination against influenza is strongly advised for all patients with asthma and COPDVaccination against pneumococcal infection is advisable for patients with COPD. Patients with obstructive airway disease on oral corticosteroids and/or with impaired lung function should take stringent safety precautions. This statement will be updated when more data become available Asthma and COPD occur commonly in South Africa. SARS-CoV-2 is a novel coronavirus, which can result in COVID-19-associated severe respiratory infection with respiratory failure and the need for mechanical ventilation. The South African Thoracic Society has prepared a guidance statement to assist clinicians and patients with asthma and COPD during the current epidemic.
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Small intestine bacterial overgrowth (SIBO) is prevalent among children living in low-income settings, leading to impaired growth and development. The aim of this study was to assess linear and ponderal growth parameters between malnourished SIBO-positive and SIBO-negative children aged 12-18 months who prospectively underwent a nutritional intervention. A glucose hydrogen breath test to detect SIBO was performed in 194 stunted (length-for-age Z score [LAZ] < -2 standard deviations) or at-risk of stunting (LAZ score between < -1 and -2 standard deviations) children. Participants received nutritional supplementation (egg and milk) in addition to their regular family meals 6 days per week for 90 days. Small intestine bacterial overgrowth was defined as a ≥ 12-ppm rise in breath hydrogen over the patient's baseline during the 3-hour test. Small intestine bacterial overgrowth status before intervention was forced into a multivariable linear regression model to examine its effects on anthropometric changes in response to the intervention. Sociodemographic data at enrollment was analyzed through multivariable logistic regression in an attempt to predict SIBO positivity. Overall, 14.9% (29/194) children were diagnosed with SIBO before the nutritional intervention. No statistically significant difference was observed among SIBO-positive and SIBO-negative groups in terms of their response to the nutritional intervention (SIBO-positive coefficient [95% confidence interval (CI)], P-value for ∆length-for-age Z score -0.003 [-0.14, 0.13], 0.96; ∆weight-for-age Z score -0.05 [-0.20, 0.09], 0.46; and ∆weight-for-length Z score -0.10 [-0.31, 0.10], 0.33). This study demonstrated that a noteworthy proportion of malnourished children living in a disadvantaged urban community were SIBO positive; however, it failed to reveal an association between SIBO status and response to nutritional intervention.
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Bactérias/crescimento & desenvolvimento , Suplementos Nutricionais , Enteropatias/microbiologia , Intestino Delgado/microbiologia , Desnutrição/dietoterapia , Adulto , Bangladesh/epidemiologia , Testes Respiratórios , Criança , Fenômenos Fisiológicos da Nutrição Infantil , Feminino , Humanos , Lactente , Modelos Logísticos , Masculino , Desnutrição/epidemiologia , Prevalência , Estudos Prospectivos , População Urbana , Adulto JovemRESUMO
Millions of children are suffering from tuberculosis (TB) worldwide and often end-up with fatal outcome especially in resource-poor settings. Tuberculin skin test (TST) is a conventionally used diagnostic test, less sensitive but highly specific for the diagnosis of clinical TB especially in undernourished children. However, we do not have any data on the role of TST positivity among the children who received nutritional intervention. Our aim was to examine the growth differences between TST-positive and TST-negative undernourished children aged 12 to 18 months who received nutritional intervention prospectively for 90 feeding days. Our further aim was to explore the determinants of TST positivity at enrollment. TB screening as one of the secondary causes of malnutrition was performed on 243 stunted [length for age Z score (LAZ) <-2 standard deviations] or at-risk of stunting (LAZ score between <-1 and -2 standard deviations) children in a community-based intervention study designed to improve their growth parameters. Differences of growth between TST-positives (n = 29) and TST-negatives (n = 214) were compared using paired samples t-test and multivariable linear regression from anthropometric data collected before and after nutritional intervention. Multivariable logistic regression was used to find out possible predictors of TST positivity using baseline sociodemographic data. Of the 243 children screened, 29 (11.9%) were TST-positive and 11 (4.5%) had clinically diagnosed pulmonary TB. Statistically significant improvement of LAZ and weight for age Z-score (WAZ) were observed among the TST-negative participants at the end of intervention period (p = 0.03 for LAZ and p = 0.01 for WAZ). However, we did not find any association between TST status and response to nutritional intervention in our multivariable linear regression models. Our study findings demonstrated a positive impact of nutritional intervention on growth parameters among TST-negative participants.
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Desenvolvimento Infantil , Teste Tuberculínico , Populações Vulneráveis , Bangladesh , Criança , Feminino , Humanos , Lactente , Masculino , Programas de Rastreamento , Estado Nutricional , Tuberculose Pulmonar/diagnósticoRESUMO
BACKGROUND.: Giardia are among the most common enteropathogens detected in children in low-resource settings. We describe here the epidemiology of infection with Giardia in the first 2 years of life in the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development Project (MAL-ED), a multisite birth-cohort study. METHODS.: From 2089 children, 34916 stool samples collected during monthly surveillance and episodes of diarrhea were tested for Giardia using an enzyme immunoassay. We quantified the risk of Giardia detection, identified risk factors, and assessed the associations with micronutrients, markers of gut inflammation and permeability, diarrhea, and growth using multivariable linear regression. RESULTS.: The incidence of at least 1 Giardia detection varied according to site (range, 37.7%-96.4%) and was higher in the second year of life. Exclusive breastfeeding (HR for first Giardia detection in a monthly surveillance stool sample, 0.46 [95% confidence interval (CI), 0.28-0.75]), higher socioeconomic status (HR, 0.74 [95% CI, 0.56-0.97]), and recent metronidazole treatment (risk ratio for any surveillance stool detection, 0.69 [95% CI, 0.56-0.84]) were protective. Persistence of Giardia (consecutive detections) in the first 6 months of life was associated with reduced subsequent diarrheal rates in Naushahro Feroze, Pakistan but not at any other site. Giardia detection was also associated with an increased lactulose/mannitol ratio. Persistence of Giardia before 6 months of age was associated with a -0.29 (95% CI, -0.53 to -0.05) deficit in weight-for-age z score and -0.29 (95% CI, -0.64 to 0.07) deficit in length-for-age z score at 2 years. CONCLUSIONS.: Infection with Giardia occurred across epidemiological contexts, and repeated detections in 40% of the children suggest that persistent infections were common. Early persistent infection with Giardia, independent of diarrhea, might contribute to intestinal permeability and stunted growth.
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Giardíase/epidemiologia , Fatores Etários , Aleitamento Materno/estatística & dados numéricos , Pré-Escolar , Países em Desenvolvimento/estatística & dados numéricos , Diarreia/etiologia , Fezes/parasitologia , Feminino , Giardíase/complicações , Giardíase/etiologia , Transtornos do Crescimento/etiologia , Humanos , Incidência , Lactente , Recém-Nascido , Masculino , Prevalência , Fatores de Risco , Fatores Socioeconômicos , Magreza/etiologiaRESUMO
Experiments were undertaken to evaluate the antitumor effects of 131I-labeled goat antibody immunoglobulin G prepared against carcinoembryonic antigen in hamsters bearing the carcinoembryonic antigen-producing GW-39 human colonic carcinoma. At a single injection of 1 mCi 131I and higher, a marked growth inhibition of GW-39 tumors, as well as a considerable increase in the survival time of the tumor-bearing hamsters, could be achieved. At a dose of 1 mCi, the radioactive affinity-purified antibody appeared to be superior to radioactive normal goat immunoglobulin G in influencing tumor growth and survival time, but no significant difference could be seen at the higher dose of 2 mCi given. Radiobiological calculations indicated that the tumors received, at up to 20 days after therapy, 1325 rads for the specific antibody and only 411 rads for the normal immunoglobulin G preparation. These findings encourage the further evaluation of antibodies to tumor markers for isotopic cancer therapy.
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Antígeno Carcinoembrionário/imunologia , Neoplasias do Colo/radioterapia , Radioisótopos do Iodo/uso terapêutico , Animais , Linhagem Celular , Neoplasias do Colo/imunologia , Cricetinae , Humanos , Imunoglobulina G/administração & dosagem , Transplante de Neoplasias , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/radioterapia , Dosagem RadioterapêuticaRESUMO
BACKGROUND/OBJECTIVE: The role of micronutrients particularly zinc in childhood diarrhoea is well established. Immunomodulatory functions of vitamin-D in diarrhoea and its role in the effect of other micronutrients are not well understood. This study aimed to investigate whether vitamin-D directly associated or confounded the association between other micronutrient status and diarrhoeal incidence and severity in 6-24-month underweight and normal-weight children in urban Bangladesh. SUBJECTS/METHODS: Multivariable generalised estimating equations were used to estimate incidence rate ratios for incidence (Poisson) and severity (binomial) of diarrhoea on cohorts of 446 normal-weight and 466 underweight children. Outcomes of interest included incidence and severity of diarrhoea, measured daily during a follow-up period of 5 months. The exposure of interest was vitamin-D status at enrolment. RESULTS: Normal-weight and underweight children contributed 62 117 and 62 967 day observation, with 14.2 and 12.8 days/child/year of diarrhoea, respectively. None of the models showed significant associations of vitamin-D status with diarrhoeal morbidity. In the final model, zinc-insufficient normal-weight children had 1.3 times more days of diarrhoea than sufficient children (P<0.05). Again zinc insufficiency and mother's education (1-5 and >5 years) had 1.8 and 2.3 times more risk of severe diarrhoea. In underweight children, older age and female had 24-63 and 17% fewer days of diarrhoea and 52-54 and 31% fewer chances of severe diarrhoea. CONCLUSION: Vitamin-D status was not associated with incidence and severity of diarrhoea in study children. Role of zinc in diarrhoea was only evident in normal-weight children. Our findings demonstrate that vitamin-D is not a confounder of the relationship between zinc and diarrhoea.
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Diarreia/etiologia , Peso Corporal Ideal/fisiologia , Magreza/sangue , Vitamina D/sangue , Zinco/sangue , Bangladesh/epidemiologia , Pré-Escolar , Diarreia/sangue , Diarreia/epidemiologia , Feminino , Humanos , Incidência , Lactente , Masculino , Análise Multivariada , Distribuição de Poisson , Índice de Gravidade de Doença , Magreza/complicações , População Urbana , Deficiência de Vitamina D/sangue , Deficiência de Vitamina D/etiologia , Zinco/deficiênciaRESUMO
We report the first attempts to describe thermophilic bacterial communities in Indonesia's thermal springs using molecular phylogenetic analyses. 16S rRNA genes from laboratory cultures and DNA directly amplified from three hot springs in West Java were sequenced. The 22 sequences obtained were assignable to the taxa Proteobacteria, Bacillus and Flavobacterium, including a number of clades not normally associated with thermophily.
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Bacillus/isolamento & purificação , Flavobacterium/isolamento & purificação , Temperatura Alta , Proteobactérias/isolamento & purificação , RNA Ribossômico 16S/genética , Microbiologia da Água , Bacillus/classificação , Bacillus/genética , Ecologia , Flavobacterium/classificação , Flavobacterium/genética , Indonésia , Dados de Sequência Molecular , Filogenia , Proteobactérias/classificação , Proteobactérias/genética , Análise de Sequência de DNARESUMO
Live cell enzyme-linked immunosorbent (ELISA) and fixed cell indirect immunofluorescence (IF) assays were compared to screen mouse hybridomas producing immunoreactive monoclonal antibodies against cell membrane antigens expressed on Ha22T, a human hepatoma cell line. While performing live cell ELISA, two parameters were tested to improve the viability of the target cells. The first parameter was the inclusion of growth medium in the assay buffers, and the second was performing the assay incubations at 37 degrees C in an incubator containing 5% CO2 in the air. Fixed cell IF detected and classified 46% of the hybridomas secreting monoclonal antibodies reactive with membrane, cytoplasm, cytoskeleton, and nuclear antigens of Ha22T cells. Fixed cell IF was able to reveal mixtures of two or more hybridomas growing in the same well secreting antibodies to different cell organelles. The live cell ELISA, on the other hand, identified 12 additional membrane reactive monoclonal antibodies from the hybridoma supernatants that were not reactive by IF. These results disclose that cell fixation procedures used for IF either completely or partially inactivated some of the cell membrane antigens. We, therefore, propose the use of a combination of immunoassays to select the maximum number of hybridomas secreting useful monoclonal antibodies from somatic cell fusions.
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Antígenos de Neoplasias/análise , Carcinoma Hepatocelular/imunologia , Ensaio de Imunoadsorção Enzimática , Neoplasias Hepáticas/imunologia , Animais , Anticorpos Monoclonais , Anticorpos Antineoplásicos , Imunofluorescência , Humanos , Hibridomas/imunologia , CamundongosRESUMO
A series of human-human hybridomas derived from a single fusion of UC 729-6 with lymph node lymphocytes were examined for the type and nature of macromolecules synthesized and secreted. One hybrid, VLN3G2, secreted fourfold higher IgG than that present in the cytoplasm over 4 days of growth, while the IgM distribution was opposite to that of IgG. VLN5C7, contrary to VLN3G2, contained several-fold more cytoplasmic IgG as well as IgM than the amounts secreted over the same period of time. Of the secreted IgG and IgM by both of these hybridomas, only the IgG showed immunoreactivity against target A431 cell surface antigen(s). Another hybridoma, termed VLN1H12, secreted immunoreactive IgM against target A431 cells, but no detectable IgG. Cytoplasmic proteins prepared by repeated freeze-thaw of the hybridoma cells, membrane proteins obtained by NP-40 extraction of the cell membrane, and secreted proteins present in the supernates of the various hybridomas were assayed by an enzyme-linked immunoassay (ELISA), to understand the discrepancy observed in the immunoglobulins of the cellular and extracellular compartments. The parental UC 729-6 cell line used in these cell fusions produced only trace amounts of immunologically inactive IgM and no detectable IgG. Molecular sieving column chromatography of these hybridoma supernates suggested the presence of intact IgG and IgM molecules and the absence of free heavy chains or hybrid antibodies containing both mu and gamma heavy chains. Intrinsic labeling of VLN3G2 hybridoma cells with 35S-methionine demonstrated the presence of not only a nonimmunoglobulin protein but also a small molecular weight protein-A-binding polypeptide in the culture supernatant. 35S-methionine-incorporated IgG and IgM antibodies, isolated from spent media, cytoplasm, and cell membranes of VLN3G2, also showed binding to protein-A-bearing bacteria. In conclusion, the differences observed in the amounts of secreted MAbs by the human-human hybridomas were not due to the decreased synthesis of these molecules.
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Anticorpos Monoclonais/biossíntese , Hibridomas/metabolismo , Anticorpos Monoclonais/classificação , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Linhagem Celular , Membrana Celular/imunologia , Cromatografia em Gel , Meios de Cultura/análise , Citoplasma/análise , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/análise , Imunoglobulina M/análise , Cariotipagem , Metionina/metabolismo , Fenótipo , Ligação Proteica , Solubilidade , Proteína Estafilocócica A/análise , Radioisótopos de EnxofreRESUMO
Two novel murine monoclonal antibodies that bind to intracellular antigens, designated TNT-1 and TNT-2, have been generated by immunizing mice with nuclear extracts from human lymphoma cells. The monoclonal antibodies were initially identified by indirect immunofluorescence on lymphoma cell lines and subsequently were found to stain the nucleus of all cell types from several species including plants by indirect immunofluorescence techniques. Immunoblot analysis demonstrated that TNT-1 bound to a protein of 22 kD and TNT-2 bound to two proteins of 15 and 22 kD, consistent with the known molecular weight of histones. To characterize their immunoreactivity, competition radioimmunoassays were performed using purified histone fractions H1, H2a, H2b and H3. By these assays, TNT-1 was found to bind to histone fraction H1 and TNT-2 to an epitope common to histone fractions H1 and H3. Histones are found in abundance in the nucleus of the cell and are known to play a major role in chromosome structure and gene expression. Upon cell death, histones remain tightly bound to DNA and consequently provide an abundant intracellular antigen that can be exploited in targeting necrotic cells, such as those found in tumors.
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Anticorpos Antinucleares/imunologia , Anticorpos Monoclonais/imunologia , Histonas/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Animais , Humanos , Hibridomas/imunologia , Immunoblotting , Camundongos , RadioimunoensaioRESUMO
BACKGROUND AND OBJECTIVE: The increased risk for coronary artery disease observed in postmenopausal (PoW) women is partly explained by a more atherogenic lipoprotein profile. Moreover, natural menopause has been associated with an altered postprandial lipid profile. This study was designed to test the hypothesis that young premenopausal (PrW) and PoW may be independently associated with postprandial lipemia and indirectly associated with atherosclerosis. PATIENTS AND METHODS: A total of 46 healthy PrW and 44 healthy PoW participated in a 5-h intervention study. Blood samples were taken at the baseline and at 1, 2, 3, and 4 h after eating. Total cholesterol, LDL-cholesterol, HDL-cholesterol, fasting, and postprandial triglycerides (PPTG) were determined sequentially in blood samples. RESULTS: PPTG presented significant higher values in PoW compared to PrW (P < 0.05), but other lipids did not significantly differ between groups. PPTG concentrations in PoW were significantly higher than in PrW (P < 0.05). There was a significant time influence (P < 0.05) in TG in PrW and PoW, while time to peak and peak concentration were significantly higher in PoW than PrW. Other lipids were also decreased more in PrW than PoW, but not significantly so. Cholesterol concentrations showed a significant reduction after 2 h, to reach values similar to the baseline after 4 h in PrW but not in PoW. HDL-cholesterol concentration was decreased more in PoW compared to PrW but it was not significant. CONCLUSIONS: Lipid postprandial response indicates a higher cardiovascular risk pattern in PoW compared to PrW.
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Asparaginase was found in the soluble fraction of cells of Azotobacter vinelandii, and its activity remained the same during growth of the organism in a nitrogen-free medium. The specific activity and the yield of A. vinelandii increased twofold in the presence of ammonium sulfate. Within limits, the temperature (30 to 37 degrees C) and pH (6.5 to 8.0) of the medium showed little effect on the levels of enzyme activity. The enzyme was purified to near homogeneity by standard methods of enzyme purification, including affinity chromatography, and had optimum activity at pH 8.6 and 48 degrees C. The approximate molecular weight was 84,000. The apparent K(m) value for the substrate was 1.1 x 10 M. Metal ions or sulfhydryl reagents were not required for enzyme activity. Cu, Zn, and Hg showed concentration-dependent inhibition, whereas amino and keto acids had no effect on the enzyme activity. Asparaginase was stable when incubated with rat serum and ascites fluid. The enzyme had no effect on the membrane of sheep erythrocytes and did not inhibit the incorporation of radioactive precursors into deoxyribonucleic acid, ribonucleic acid, and protein in Yoshida ascites sarcoma cells. Asparaginase activity was not detected in the tumor cells.
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Lym-1 and TNT-1 are two murine immunoglobulin G2a monoclonal antibodies (MAbs) which have been used for clinical trials in cancer patients. This paper describes methods for large-scale preparation of F(ab')2 fragments from 50 mg to 4 g of MAbs Lym-1 and TNT-1. Digestion of MAbs with pepsin was optimized and performed at pH 3.8, a pepsin/antibody ratio of 1:250, and 3-4 h of incubation at 37 degrees C. The F(ab')2 fragments were purified by tandem column procedures using fast protein liquid chromatography. Quality control analyses of the products included protein purity, isoelectric point, immunoreactivity, and endotoxin level. The results revealed that the chromatographic procedures are practical, simple, and effective, and can be used to produce gram quantities of clinical-grade F(ab')2 fragments for the diagnosis of cancer in patients.
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Anticorpos Monoclonais/análise , Cromatografia Líquida/métodos , Fragmentos de Imunoglobulinas/análise , Imunoglobulina G/imunologia , Anticorpos Monoclonais/imunologia , Eletroforese em Gel de Poliacrilamida , Fragmentos de Imunoglobulinas/imunologia , Pepsina ARESUMO
Direct and indirect radioimmunoassay (RIA) procedures to determine the amount of binding of a mouse monoclonal antibody (MoAb) reactive with an intracellular antigen present in human cells are described. In these RIAs, mouse IgG2a MoAb, designated as Tumor Necrosis Treatment (TNT-1) antibody, paraformaldehyde/acetone fixed cells, and Sephadex beads were used to standardize the assay conditions. In the direct RIA, 83% of the 125I-labeled TNT-1 MoAb was bound to the target cells within 30 min after the addition of reagents. The amount of binding of the MoAb was directly proportional to the amount of antigen present in the assay. When the direct RIA was carried out using different types of target cells, 125I-labeled TNT-1 MoAb showed greater than 70% binding. In the indirect RIA, the amount of binding of secondary 125I-labeled goat anti-mouse IgG antibody to the target cells was linear. These results suggest that the indirect RIA can be used to estimate the immunoreactivity of the unlabeled TNT-1 MoAb present in crude protein preparations. Based on the results of RIAs the following two conclusions are drawn: 1) the direct RIA can be used to estimate rapidly the amount of immunoreactive TNT-1 MoAb present in 125I-labeled antibody preparations and 2) the indirect RIA which estimates the amount of immunoreactivity of unlabeled TNT-1 MoAb can be used to monitor the purification and study the characteristics of the MoAb present in crude protein preparations. These methods enable the quantitative measurement of MoAbs reactive against intracellular antigens using standard RIA procedures.
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Anticorpos Monoclonais/análise , Anticorpos Antineoplásicos/análise , Radioimunoensaio/métodos , Animais , Antígenos de Neoplasias , Humanos , Hibridomas/imunologia , Líquido Intracelular/imunologia , Camundongos , Células Tumorais Cultivadas/imunologiaRESUMO
Experiments with the GW-39 human colonic carcinoma growing in hamsters showed that injection of radioactive antibody to a colorectal-specific, tumor-associated antigen, CSAp, results in better tumor radiolocalization than was seen previously with radioantibodies to carcinoembryonic antigen (CEA). However, a mixture of both radioactive antibodies resulted in potentiation of CEA-tumor radioimmunodetection without affecting CSAp-tumor radiolocalization. Hence, multi-marker antibody mixtures may be the method of choice in cancer radioimmunodetection.
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Antígenos de Neoplasias/imunologia , Antígeno Carcinoembrionário/imunologia , Neoplasias do Colo/diagnóstico por imagem , Animais , Anticorpos Antineoplásicos/imunologia , Neoplasias do Colo/imunologia , Cricetinae , Cabras , Humanos , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/imunologia , CintilografiaRESUMO
Intrapulmonary bacterial clearance, lung inflammatory cell recruitment and macrophage superoxide generating capacity were studied in newborn rabbits nursed by their mother and given a supplement of either high [5 g/(kg.d)] or low [0.22 g/(kg.d)] doses of fish oil, safflower oil or saline for 7 d after birth. The high dose fish and safflower oil regimens diminished lung clearance of inspired Staphylococcus aureus by approximately 50% compared with the saline controls, but they did not alter lung neutrophil recruitment or alveolar macrophage bacterial phagocytosis. Only high dose fish oil decreased macrophage superoxide anion generation (by 30%). With high dose fish or safflower oil supplementation, the fatty acid content of lung parenchyma, bronchoalveolar lavage effluent and alveolar macrophages increased significantly. Low dose supplementation did not have this effect. We conclude that pharmacologic dietary (n-6) and (n-3) fatty acid supplementation impairs the ability of the neonatal rabbit lung to kill intrapulmonary S. aureus. It has been proposed that human infant formula be supplemented with fatty acids either to alleviate dietary deficiencies or to treat pulmonary inflammatory disorders. Based on our findings, the effects of such supplementation should be monitored relative to the possible heightened risk of bacterial infection.