RESUMO
Ischemic stroke is a debilitating neurological disease with few effective therapeutics. Previous work has shown that oral probiotic treatment prior to stroke can attenuate cerebral infarction and neuroinflammation, highlighting the gut-microbiota-brain axis as a novel therapeutic target. Whether a more clinically relevant, post-stroke, administration of probiotics can improve stroke outcomes is unknown. In this study, we examined the effect of post-stroke oral probiotic therapy on motor behavior in the pre-clinical mouse endothelin-1 (ET-1) model of sensorimotor stroke. We found that post-stroke oral probiotic therapy with Cerebiome® (Lallemand, Montreal, Canada), containing B. longum R0175 and L. helveticus R0052, improved functional recovery and changed the composition of the post-stroke gut microbiota. Interestingly, oral Cerebiome® administration did not result in alterations of lesion volume or the number of CD8+/Iba1+ cells in the injured tissue. Overall, these findings suggest that probiotic treatment following injury can improve sensorimotor function.
Assuntos
Probióticos , Acidente Vascular Cerebral , Camundongos , Animais , Roedores , Acidente Vascular Cerebral/tratamento farmacológico , Probióticos/farmacologia , Probióticos/uso terapêuticoRESUMO
AIMS: Classical microbiology techniques are the gold standard for probiotic enumeration. However, these techniques are limited by parameters of time, specificity and incapacity to detect viable but nonculturable (VBNC) micro-organisms and nonviable cells. The aim of the study was to evaluate flow cytometry as a novel method for the specific quantification of viable and nonviable probiotics in multistrain products. METHODS AND RESULTS: Custom polyclonal antibodies were produced against five probiotic strains from different species (Bifidobacterium bifidum R0071, Bifidobacterium longum ssp. infantis R0033, Bifidobacterium longum ssp. longum R0175, Lactobacillus helveticus R0052 and Lactobacillus rhamnosus R0011). Evaluation of specificity confirmed that all antibodies were specific at least at the subspecies level. A flow cytometry method combining specific antibodies and viability assessment with SYTO® 24 and propidium iodide was applied to quantify these strains in three commercial products. Analyses were conducted on two flow cytometry instruments by two operators and compared with classical microbiology using selective media. Results indicated that flow cytometry provides higher cell counts than classical microbiology (P < 0·05) in 73% of cases highlighting the possible presence of VBNC. Equivalent performances (repeatability and reproducibility) were obtained for both methods. CONCLUSIONS: This study showed that flow cytometry methods can be applied to probiotic enumeration and viability assessment. Combination with polyclonal antibodies can achieve sufficient specificity to differentiate closely related strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Flow cytometry provides absolute and specific quantification of viable and nonviable probiotic strains in a very short time (<2 h) compared with classical techniques (>48 h), bringing efficient tools for research and development and quality control.
Assuntos
Bifidobacterium longum subspecies infantis/crescimento & desenvolvimento , Citometria de Fluxo/métodos , Lacticaseibacillus rhamnosus/crescimento & desenvolvimento , Lactobacillus helveticus/crescimento & desenvolvimento , Probióticos/química , Bifidobacterium longum subspecies infantis/química , Bifidobacterium longum subspecies infantis/isolamento & purificação , Lactobacillus helveticus/química , Lactobacillus helveticus/isolamento & purificação , Lacticaseibacillus rhamnosus/química , Lacticaseibacillus rhamnosus/isolamento & purificação , Viabilidade Microbiana , Reprodutibilidade dos TestesRESUMO
Lactobacillus helveticus CNRZ 32 is recognized for its ability to decrease bitterness and accelerate flavor development in cheese, and has also been shown to release bioactive peptides in milk. Similar capabilities have been documented in other strains of Lb. helveticus, but the ability of different strains to affect these characteristics can vary widely. Because these attributes are associated with enzymes involved in proteolysis or AA catabolism, we performed comparative genome hybridizations to a CNRZ 32 microarray to explore the distribution of genes encoding such enzymes across a bank of 38 Lb. helveticus strains, including 2 archival samples of CNRZ 32. Genes for peptidases and AA metabolism were highly conserved across the species, whereas those for cell envelope-associated proteinases varied widely. Some of the genetic differences that were detected may help explain the variability that has been noted among Lb. helveticus strains in regard to their functionality in cheese and fermented milk.
Assuntos
Lactobacillus helveticus/genética , Aminoácidos/metabolismo , Queijo/microbiologia , DNA Bacteriano/genética , Genes Bacterianos/genética , Variação Genética/genética , Lactobacillus helveticus/enzimologia , Lactobacillus helveticus/metabolismo , Hibridização de Ácido Nucleico/genética , Peptídeo Hidrolases/genética , Filogenia , Homologia de Sequência de AminoácidosRESUMO
In the absence of established best practice standards in the probiotic field for reducing the risk of bacterial transfer between experimental groups, we developed protocols and methods to ensure the highest quality and interpretability of results from animal studies, even when performed in non-conventional animal care facilities. We describe easily implementable methods for reducing cross-contamination during animal housing, behavioural testing, and euthanasia, along with highlighting protocols for contamination detection in experimental subjects and laboratory areas using qPCR. In light of the high cross-contamination risks between animals during experiments involving probiotics, constant vigilance in animal care and research protocols is critical to ensure valid and reliable research findings.
Assuntos
Experimentação Animal , Ciência dos Animais de Laboratório/normas , Probióticos/administração & dosagem , Roedores/microbiologia , Animais , Diarreia/microbiologia , Modelos AnimaisRESUMO
Microbial metabolism in the gut may alter human bile acid metabolism in a way that beneficially affects lipid homeostasis and therefore cardiovascular disease risk. Deconjugation of bile acids by microbes is thought to be key to this mechanism but has yet to be characterised in blood and stool while observing lipid markers. The aim of this study was to determine the effect of 3 different probiotic strains on plasma and stool bile acids in the context of lipid and glucose metabolism. In this 18-week, randomised, double-blind crossover study, healthy adults (53±8 years) with a high waist circumference underwent a 1-week pre-baseline period and were then randomised to receive 1 capsule/day of Bacillus subtilis R0179 (2.5×109 cfu/capsule; n=39), Lactobacillus plantarum HA-119 (5×109 cfu/capsule; n=38), Bifidobacterium animalis subsp. lactis B94 (5×109 cfu/capsule; n=37) or placebo for 6 weeks. Following a 3-week washout and second pre-baseline week, participants were crossed to the other intervention for 6 weeks followed by a 1-week post-intervention period. Blood and stool samples were collected at the beginning and end of each intervention to measure bile acids, serum lipid profiles, and glucose and insulin levels. Data from the placebo intervention were combined for all participants for analyses. In obese participants, the difference (final-baseline) in the sum of deconjugated plasma bile acids was greater with consumption of B. subtilis (691±378 nmol/l, P=0.01) and B. lactis (380±165 nmol/l, P=0.04) than with placebo (98±176 nmol/l, n=57). No significant differences were observed for any probiotics for stool bile acids, serum lipids, blood glucose or insulin. These data suggest that B. subtilis and B. lactis had no effect on glucose metabolism or serum cholesterol but increased deconjugated plasma bile acids in obese individuals. Additional studies should be conducted to confirm these findings and explore potential mechanisms. This trial was registered at clinicaltrials.gov as NCT01879098.
Assuntos
Ácidos e Sais Biliares/sangue , Fármacos Gastrointestinais/administração & dosagem , Obesidade/terapia , Plasma/química , Probióticos/administração & dosagem , Adulto , Bacillus subtilis/crescimento & desenvolvimento , Bifidobacterium animalis/crescimento & desenvolvimento , Estudos Cross-Over , Método Duplo-Cego , Fezes/química , Feminino , Humanos , Lactobacillus plantarum/crescimento & desenvolvimento , Masculino , Pessoa de Meia-Idade , Placebos/administração & dosagem , Resultado do TratamentoRESUMO
The aim of the studies was to determine the effects of calcium carbonate and calcium phosphate supplementation on faecal Lactobacillus spp., with and without a probiotic supplement, in healthy adults. Study 1 comprised of a randomised, double-blind, crossover design; participants (n=15) received 2 capsules/d of 250 mg elemental calcium as calcium carbonate (Ca1) and calcium phosphate (Ca2) each for 2-week periods, with 2-week baseline and washout periods. Study 2 was a randomised, double-blind, crossover design; participants (n=17) received 2 capsules/d of Lactobacillus helveticus R0052 and Lactobacillus rhamnosus R0011 (probiotic) alone, the probiotic with 2 capsules/d of Ca1, and probiotic with 2 capsules/d of Ca2 each for 2-week periods with 2-week baseline and washout periods. In both studies, stools were collected during the baseline, intervention and washout periods for Lactobacillus spp. quantification and qPCR analyses. Participants completed daily questionnaires of stool frequency and compliance. In Study 1, neither calcium supplement influenced viable counts of resident Lactobacillus spp., genome equivalents of lactic acid bacteria or stool frequency. In Study 2, faecal Lactobacillus spp. counts were significantly enhanced from baseline when the probiotic was administered with Ca2 (4.83±0.30, 5.79±0.31) (P=0.02), but not with Ca1 (4.98±0.31) or with the probiotic alone (5.36±0.31, 5.55±0.29) (not significant). Detection of L. helveticus R0052 and L. rhamnosus R0011 was significantly increased with all treatments, but did not differ among treatments. There were no changes in weekly stool frequency. Calcium phosphate co-administration may increase gastrointestinal survival of orally-administered Lactobacillus spp.
Assuntos
Fosfatos de Cálcio/farmacologia , Fezes/microbiologia , Lacticaseibacillus rhamnosus/efeitos dos fármacos , Lactobacillus helveticus/efeitos dos fármacos , Probióticos/farmacologia , Adolescente , Adulto , Estudos Cross-Over , Suplementos Nutricionais , Método Duplo-Cego , Feminino , Humanos , Lactobacillus helveticus/isolamento & purificação , Lacticaseibacillus rhamnosus/isolamento & purificação , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
The modulation of a brain phosphoinositide-specific phospholipase C-alpha activity was studied using a variety of compounds of different charge. Detergents such as sodium deoxycholate and cetyltrimethylammonium bromide stimulated the phospholipase C activity when used alone but when used together the effects were not additive. Spermine was an effective inhibitor of the enzyme activity while the cationic peptide, Melittin, had no effect. The inositol phosphates produced by hydrolysis with phosphoinositide-specific phospholipase C were inhibitory while diacylglycerol and inositol did not affect the phospholipase activity. Myelin basic protein, which was previously shown to stimulate phospholipase C activity by 2.5-fold, did not interact with the anionic inositol phosphatases to any significant extent. Thus we concluded that the mechanism of stimulation was not due to relief of product inhibition. Crosslinking studies with the photoactivatable reagent, N-hydroxysuccinimidyl-4-azidosalicylic acid, showed that peptide 24-33 of myelin basic protein, which stimulated the activity almost as much as the native protein, interacted specifically with the phospholipase C. Thus the mechanism by which myelin basic protein stimulated the enzyme appeared to be through specific protein-protein interaction.
Assuntos
Encéfalo/efeitos dos fármacos , Proteína Básica da Mielina/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/enzimologia , Bovinos , Cetrimônio , Compostos de Cetrimônio/farmacologia , Ácido Desoxicólico/farmacologia , Ativação Enzimática , Meliteno/farmacologia , Dados de Sequência Molecular , Fosfatidilinositol Diacilglicerol-Liase , Espermina/farmacologiaRESUMO
BACKGROUND: Myocardial infarction (MI) is associated with apoptosis in the amygdala and, ultimately, with clinical signs of depression. Different treatments have proven to be beneficial in preventing depression, including combination of the probiotics Lactobacillus helveticus and Bifidobacterium longum for prophylaxis. We have speculated previously that the benefit of these probiotics is due to their anti-inflammatory properties, and evidence suggests that an intact vagus nerve is important for this effect to occur. This study was designed to ascertain vagus nerve involvement in the beneficial influence of probiotics on caspase activities in our post-MI animal model of depression. METHODS: Probiotics and/or vehicle were administered daily to male adult rats, 14 days before MI and until euthanasia. Vagotomy was performed in subgroups of rats 40 min before MI. They were sacrificed after 3 days of reperfusion, and MI size was assessed along with caspase-3 and -8 activities in the amygdala. KEY RESULTS: Probiotics had no effect on infarct size but vagotomy increased it. Caspase-3 and caspase-8 activities in the amygdala were higher in MI than in sham-operated rats, and this outcome was reversed by probiotics. The beneficial influence of probiotics was abolished by vagotomy. CONCLUSIONS & INFERENCES: Our data indicate that the effect of probiotics on caspase activities in the amygdala after MI depends on an intact vagus nerve.
Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Caspase 3/efeitos dos fármacos , Caspase 8/efeitos dos fármacos , Depressão/psicologia , Infarto do Miocárdio/psicologia , Probióticos/farmacologia , Vagotomia , Tonsila do Cerebelo/metabolismo , Animais , Bifidobacterium , Caspase 3/metabolismo , Caspase 8/metabolismo , Depressão/metabolismo , Modelos Animais de Doenças , Coração/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Lactobacillus helveticus , Masculino , Infarto do Miocárdio/metabolismo , Peroxidase/metabolismo , RatosRESUMO
BACKGROUND: Several probiotic strains have been shown to enhance human resistance to infectious disease. It is speculated that these strains may impose this effect by excretion of anti-microbial components, by competing with pathogens for intestinal nutrients and/or mucosal adhesion sites or modulating the immune system. OBJECTIVE: A parallel, double-blind, placebo-controlled 4-week intervention was performed in healthy males, to study the effect of a blend of probiotic bacteria (Lactobacillus helveticus Rosell-52, Lactobacillus rhamnosus Rosell-11, Bifidobacterium longum ssp. longum Rosell-175) and a probiotic yeast (Saccharomyces cerevisiae var boulardii CNCM I-1079) on enterotoxigenic Escherichia coli (ETEC) challenge. Primary outcomes studied were fecal ETEC excretion and total fecal output per day. SUBJECTS/METHODS: Subjects were randomized to the probiotic (5 × 10(9) colony-forming units (CFUs); twice daily; n=30) or placebo group (twice daily; n=30). After 2 weeks, subjects were orally challenged with a live attenuated ETEC (3 × 10(9) CFU), previously demonstrated to induce mild, short-lived symptoms of a foodborne infection. Before and after ETEC challenge, subjects collected 24 h fecal samples. Compliance to study guidelines, stool consistency (Bristol Stool Score), stool frequency, and frequency and severity of gastrointestinal (GI) complaints were recorded by the subjects on a Daily Record Questionnaire. RESULTS: ETEC challenge induced a significant increase in fecal ETEC excretion in both groups. However, a statistically significant increase in fecal output was only observed in the probiotic group. ETEC challenge resulted in a decrease in the percentage of fecal dry weight, and an increase in reported Bristol Stool Score, stool frequency and GI complaints. Dietary probiotics significantly decreased the percentage of fecal dry weight. In addition, ETEC increased C-reactive protein, total secretory Immunoglobulin A (IgA) and Immunoglobulin G Colonization Factor Antigen II. CONCLUSION: Dietary probiotics did not increase resistance to oral attenuated ETEC challenge in human subjects.
Assuntos
Bifidobacterium , Defecação , Diarreia/microbiologia , Escherichia coli , Fezes , Lactobacillus , Probióticos , Adulto , Diarreia/prevenção & controle , Método Duplo-Cego , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Fezes/microbiologia , Humanos , Intestinos/microbiologia , Lactobacillus acidophilus , Lactobacillus helveticus , Lacticaseibacillus rhamnosus , Masculino , Probióticos/uso terapêutico , Adulto JovemRESUMO
A probiotic formulation of Enterococcus faecium R0026 and Bacillus subtilis R0179 has been evaluated in previous clinical trials. However, B. subtilis R0179 has not been evaluated as a single probiotic strain or in combination with other strains at doses higher than 0.1×109 cfu. To establish oral dose-response tolerance and gastrointestinal (GI) viability of B. subtilis R0179, a randomised, double-blind, placebo-controlled trial in healthy adults (n=81; 18-50 years old) was conducted. Participants received B. subtilis R0179 at 0.1, 1.0 or 10×109 cfu/capsule/day or placebo for four weeks. General wellness was assessed using a daily questionnaire evaluating GI, cephalic, ear-nose-throat, behavioural, emetic, and epidermal symptoms. GI symptoms were further evaluated using a weekly gastrointestinal symptom rating scale (GSRS). GI transit viability of B. subtilis R0179 was assessed by plating and microbiota analysis by 16S rRNA at baseline, week 4 of the intervention and washout. General wellness and GI function were not affected by oral consumption of B. subtilis R0179 at any dose. Daily questionnaire syndrome scores were not different from baseline and did not exceed a clinically significant score of 1. GSRS syndrome scores were not different from baseline and ranged from 1.1±0.1 to 1.9±0.2. Faecal viable counts of B. subtilis R0179 demonstrated a dose response: the placebo group (1.1±0.1 log10 cfu/g) differed from 0.1×109 (4.6±0.1 log10 cfu/g), 1×109 (5.6±0.1 log10 cfu/g) and 10×109 (6.4±0.1 log10 cfu/g) (P<0.0001). No significant changes in phyla were observed, but sequence reads binned to multiple operational taxonomic units matching closest to Ruminococci increased during probiotic supplementation. B. subtilis R0179 survives passage through the human GI tract and is well tolerated by healthy adults at intakes from 0.1 to 10×109 cfu/day. The trial has been registered at www.clinicaltrials.gov under NCT01802151.
Assuntos
Bacillus subtilis/fisiologia , Fezes/microbiologia , Viabilidade Microbiana , Probióticos/administração & dosagem , Probióticos/efeitos adversos , Administração Oral , Adulto , Contagem de Colônia Microbiana , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Método Duplo-Cego , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Trato Gastrointestinal/microbiologia , Humanos , Placebos/administração & dosagem , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
The value of exogenously supplied live bacteria for the maintenance of health in humans has been recognized both scientifically in the published literature and commercially in the availability of probiotic products. Although many bacteria characterized as probiotics are strains of Lactobacillus or Bifidobacterium, sporeforming bacteria, primarily of the genus Bacillus and related genera, have also been studied and commercialized as probiotics. This article reviews the characterization, efficacy, and safety of sporeformers used as probiotics.
RESUMO
Enterohaemorrhagic Escherichia coli O157:H7 and adherent-invasive Escherichia coli are two groups of enteric bacterial pathogens associated with haemorrhagic colitis and Crohn's Disease, respectively. Bacterial contact with host epithelial cells stimulates an immediate innate immune response designed to combat infection. In this study, immune responses of human epithelial cells to pathogens, either alone or in combination with probiotic bacteria were studied. Industrially prepared Lactobacillus helveticus strain R0052 was first examined by microarray analysis and then compared to broth-grown strains of R0052 and Lactobacillus rhamnosus strain GG using quantitative realt-time polymerase chain reaction. Results showed host immune activation responses increased following pathogen exposure, which were differentially ameliorated using probiotics depending on both the preparation of probiotics employed and conditions of exposure. These findings provide additional support for the concept that specific probiotic strains serve as a promising option for use in preventing the risk of enteric bacterial infections.
Assuntos
Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Escherichia coli/imunologia , Fatores Imunológicos/farmacologia , Lacticaseibacillus rhamnosus/imunologia , Lactobacillus helveticus/imunologia , Probióticos/farmacologia , Células CACO-2 , Perfilação da Expressão Gênica , Humanos , Análise em Microsséries , Reação em Cadeia da Polimerase em Tempo RealRESUMO
In order to understand the appropriate use of potentially probiotic Gram-positive microbes through their introduction in the gut microbiome, it is necessary to understand the influence of individual bacteria on the host-response system at a cellular level. In the present study, we have shown that lipopolysaccharides, flagellated Gram-negative bacteria, potentially probiotic Gram-positive bacteria and yeast interact differently with human intestinal epithelial cells with a custom-designed expression microarray evaluating 17 specific host-response pathways. Only lipopolysaccharides and flagellated Gram-negative bacteria induced inflammatory response, while a subset of Gram-positive microbes had anti-inflammatory potential. The main outcome from the study was the differential regulation of the central mitogen-activated protein kinase signalling pathway by these Gram-positive microbes versus commensal/pathogenic Gram-negative bacteria. The microarray was efficient to highlight the impact of individual bacteria on the response of intestinal epithelial cells, but quantitative real-time polymerase chain reaction validation demonstrated some underestimation for down-regulated genes by the microarray. This immune array will allow us to better understand the mechanisms underlying microbe-induced host immune responses.
Assuntos
Células Epiteliais/imunologia , Mucosa Intestinal/imunologia , Lipopolissacarídeos/imunologia , Sistema de Sinalização das MAP Quinases , Probióticos , Salmonella/imunologia , Transcriptoma , Aderência Bacteriana , Bifidobacterium/imunologia , Regulação para Baixo , Células Epiteliais/microbiologia , Regulação da Expressão Gênica , Genoma Humano , Células HT29 , Humanos , Inflamação/imunologia , Inflamação/microbiologia , Interleucina-9/imunologia , Mucosa Intestinal/citologia , Mucosa Intestinal/microbiologia , Lactobacillus/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Probióticos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Gênica , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Commercial literature on various probiotic products suggests that they can be taken before meals, during meals or after meals or even without meals. This has led to serious confusion for the industry and the consumer. The objective of our study was to examine the impact of the time of administration with respect to mealtime and the impact of the buffering capacity of the food on the survival of probiotic microbes during gastrointestinal transit. We used an in vitro Digestive System (IViDiS) model of the upper gastrointestinal tract to examine the survival of a commercial multi-strain probiotic, ProtecFlor®. This product, in a capsule form, contains four different microbes: two lactobacilli (Lactobacillus helveticus R0052 and Lactobacillus rhamnosus R0011), Bifidobacterium longum R0175 and Saccharomyces cerevisiae boulardii. Enumeration during and after transit of the stomach and duodenal models showed that survival of all the bacteria in the product was best when given with a meal or 30 minutes before a meal (cooked oatmeal with milk). Probiotics given 30 minutes after the meal did not survive in high numbers. Survival in milk with 1% milk fat and oatmeal-milk gruel were significantly better than apple juice or spring water. S. boulardii was not affected by time of meal or the buffering capacity of the meal. The protein content of the meal was probably not as important for the survival of the bacteria as the fat content. We conclude that ideally, non-enteric coated bacterial probiotic products should be taken with or just prior to a meal containing some fats.
Assuntos
Bactérias/crescimento & desenvolvimento , Ingestão de Alimentos , Probióticos/administração & dosagem , Probióticos/farmacocinética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Trato Gastrointestinal Superior/microbiologia , Contagem de Colônia Microbiana , Humanos , Viabilidade Microbiana , Modelos Teóricos , Probióticos/farmacologiaRESUMO
The probiotic preparation Lacidofil® has been commercially available in Europe, Asia and North America since 1995. This product is a combination of two strains, Lactobacillus helveticus R0052 and Lactobacillus rhamnosus R0011. The strains have been evaluated for safety, identity and mechanisms of probiotic action in vitro, in animal models and human clinical trials. The strains adhered to human epithelial cells, helped to maintain the barrier function and blocked the adhesion of a number of pathogens, allowing them to be cleared from the intestine. The strains also elicited an anti-inflammatory response by down-regulating IL-1ß, IL-8 and TNF-α. In various stress models, the probiotic combination facilitated better coping and outcomes which may be through the maintenance of barrier function and suppressing inflammation. Overall, pre-clinical studies suggest a potential anti-infectious role for the strains and the combination. Clinical studies, primarily in children, have identified Lacidofil as an effective supplement for various gastrointestinal diseases such as antibiotic-associated diarrhoea and acute gastroenteritis. Recent research has also indicated that Lacidofil may be beneficial for individuals with atopic dermatitis or vaginal dysbacteriosis.
Assuntos
Gastroenteropatias/terapia , Lacticaseibacillus rhamnosus/fisiologia , Lactobacillus helveticus/fisiologia , Probióticos/efeitos adversos , Probióticos/farmacologia , Vigilância de Produtos Comercializados , Ásia , Europa (Continente) , Humanos , Lactobacillus helveticus/crescimento & desenvolvimento , Lacticaseibacillus rhamnosus/crescimento & desenvolvimento , América do Norte , Probióticos/administração & dosagemRESUMO
Probiotics as dietary supplements have been readily accepted by Asian populations. Use of certain probiotic preparations is widespread and the number of clinical trials undertaken with such products is unparalleled in western scientific literature. One such preparation, containing a combination of Enterococcus faecium R0026 and Bacillus subtilis R0179, has 23 publications on post-market clinical studies involving over 1,800 adults. The majority of these publications are printed in Chinese and Korean journals. This review examines the clinical findings with this probiotic combination. As mono-therapy, it has been used to overcome symptoms associated with chronic diarrhoea and irritable bowel syndrome. It has been used as co-adjuvant therapy with sulfasalazine and mesalazine to improve remission times in mild to moderate Ulcerative Colitis and to improve compliance with conventional triple therapy for Helicobacter pylori eradication. While the much of the data is preliminary and the study designs require refinement, the contribution of these trials should not be ignored. The information derived in this review will provide practitioners with practical information on appropriate applications for probiotic supplements, expected outcomes, dosing regimes, safety and reported adverse events. Furthermore, identification of problems in these trials should help researchers design better clinical trials when investigating probiotic products.
Assuntos
Gastroenteropatias/tratamento farmacológico , Probióticos/uso terapêutico , Vigilância de Produtos Comercializados , Adulto , Animais , Ásia , Ensaios Clínicos como Assunto , Feminino , Gastroenteropatias/microbiologia , Humanos , MasculinoRESUMO
We reported previously a highly purified phosphatidylinositol-specific phospholipase C (PI-PLC) from bovine brain and from human myelin which was stimulated by myelin basic protein. In this paper we report that the stimulation of the PI-PLC activity by myelin basic protein (MBP) requires arginine residues in peptide linkage. MBP and poly-L-arginine were able to stimulate the PI-PLC activity by 250% while other basic poly amino acids were unable to stimulate the PI-PLC activity. Neither free arginine nor benzoyl-arginine ethylester was able to stimulate the activity of the enzyme. These results suggested a requirement for the guanidino group of arginine and arginine in peptidyl linkage. The arginyl residues of MBP were modified chemically with 1,2-cyclohexanedione, or enzymatically by cholera toxin which ADP-ribosylated arginyl groups, or by peptidylarginine deiminase which converted the guanidino group of arginine to the ureido group of citrulline. ADP-ribosylation did not affect the stimulation while the 1,2-cyclohexanedione modified MBP and the peptidylarginine deiminase-treated MBP showed a reduced ability to stimulate the PI-PLC activity which correlated with the number of arginyl residues modified. Sequence analysis of the peptidylarginine deiminase-treated MBP established that specific arginyl residues had been converted to citrulline to a greater extent than others. When 70% of Arg 25 and Arg31 were converted to citrulline little stimulatory activity remained, whereas the conversion of 100% of Arg 170 did not affect the ability of C1 to stimulate the enzyme. A role for "active" arginine in this MBP peptide is suggested by our data.
Assuntos
Arginina/farmacologia , Encéfalo/enzimologia , Proteína Básica da Mielina/farmacologia , Peptídeos/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Animais , Arginina/análogos & derivados , Bovinos , Citrulina/metabolismo , Hidrolases/metabolismo , Peptídeos/metabolismo , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Diester Fosfórico Hidrolases/efeitos dos fármacos , Polilisina/farmacologia , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Análise de Sequência , Relação Estrutura-Atividade , Tripsina/metabolismoRESUMO
A phosphatidylinositol-specific phospholipase C (PI-PLC) has been isolated from bovine brain (purification factor of 5.6 x 10(4)). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it had a Mr of 57,000. Neither amino nor neutral sugars were detected in the purified enzyme. The pH optimum was 7.0-7.5, and the activity decreased only slightly at pH 8.0. When phosphatidylinositol was used as a substrate, the optimum Ca2+ requirement was 4 mM, and Km was 260 microM. When phosphatidylinositol 4,5-bisphosphate was used, the optimum Ca2+ requirement was 10(-7) M, and the Km was reduced to 90 microM. Lipid specificity studies showed that equal amounts of inositol phosphate and diacylglycerol were released from phosphatidylinositol but 4 times as much inositol 1,4,5-trisphosphate was released from phosphatidylinositol 4,5-bisphosphate. Other lipids, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, were not substrates. Failure to detect phosphatidic acid confirmed the absence of a phospholipase D activity in the purified enzyme. Myelin basic protein (MBP) stimulated the PI-PLC activity between 2- and 3-fold. Histone had a small effect only, whereas bovine serum albumin and cytochrome C had no effect. Phosphorylation of MBP reduced the stimulatory effect. Protein-protein interactions between MBP and PI-PLC have been demonstrated both immunologically and by sucrose density gradients. A stoichiometry of 1:1 has been suggested by the latter method. A number of peptides have been prepared by chemical, enzymatic, and synthetic methods. Peptides containing the MBP sequences consisting of residues 24-33 and 114-122 stimulated the PI-PLC but were less effective than the intact protein.
Assuntos
Encéfalo/enzimologia , Proteína Básica da Mielina/metabolismo , Diester Fosfórico Hidrolases/isolamento & purificação , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Cálcio/metabolismo , Bovinos , Ativação Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Peso Molecular , Proteína Básica da Mielina/química , Fragmentos de Peptídeos/farmacologia , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Fosfoproteínas/metabolismo , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Ligação Proteica , Especificidade por SubstratoRESUMO
A computational model of myelin basic protein (MBP) has been constructed based on the premise of a phylogenetically conserved beta-sheet backbone and on electron microscopical three-dimensional reconstructions. Many residues subject to post-translational modification (phosphorylation, methylation, or conversion of arginines to citrullines) were located in loop regions and thus accessible to modifying enzymes. The triproline segment (residues 99-101) is fully exposed on the back surface of the protein in a long crossover connection between two parallel beta-strands. The proximity of this region to the underlying beta-sheet suggests that post-translational modifications here might have potential synergistic effects on the entire structure. Post-translational modifications that lead to a reduced surface charge could result first in a weakened attachment to the myelin membrane rather than in a gross conformational change of the protein itself. Such mechanisms could be operative in demyelinating diseases such as multiple sclerosis.
Assuntos
Esclerose Múltipla/metabolismo , Proteína Básica da Mielina/química , Humanos , Microscopia Eletrônica , Modelos Moleculares , Proteína Básica da Mielina/metabolismo , Conformação ProteicaRESUMO
Myelin basic protein (MBP) plays an integral role in the structure and function of the myelin sheath. In humans and cattle, an 18.5-kDa isoform of MBP predominates and exists as a multitude of charge isomers resulting from extensive and varied post-translational modifications. We have purified the least modified isomer (named C1) of the 18.5-kDa isoform of MBP from fresh bovine brain and imaged this protein as negatively stained single particles adsorbed to a lipid monolayer. Under these conditions, MBP/C1 presented diverse projections whose relative orientations were determined using an iterative quaternion-assisted angular reconstitution scheme. In different buffers, one with a low salt and the other with a high salt concentration, the conformation of the protein was slightly different. In low salt buffer, the three-dimensional reconstruction, solved to a resolution of 4 nm, had an overall "C" shape of outer radius 5.5 nm, inner radius 3 nm, overall circumference 15 nm, and height 4.7 nm. The three-dimensional reconstruction of the protein in high salt buffer, solved to a resolution of 2.8 nm, was essentially the same in terms of overall dimensions but had a somewhat more compact architecture. These results are the first structures achieved directly for this unusual macromolecule, which plays a key role in the development of multiple sclerosis.