Effects of dietary supplementation of different levels of vitamin B12 on the liver metabolism of laying hens.

BACKGROUND: Vitamin B12 plays an important role in lipid, protein, carbohydrate and nucleic acid metabolism. We investigated the effect of supplementing layers' diets with different vitamin B12 levels on liver metabolism using a liquid chromatography-mass spectrometry-based metabolomic approach to observe and analyse wide-target metabolomics in the liver. RESULTS: We assigned hens to three groups, namely blank control group without vitamin B12 diet (BCG), normal control group with 25 µg kg-1 vitamin B12 (NCG) and vitamin B12 supplement group I with 100 µg kg-1 vitamin (VBSG I). The VBSG I group layers had higher (P < 0.05) vitamin B12 concentration than those from other groups. The egg yolk vitamin B12 concentration increased (P < 0.01) with the increasing vitamin B12 dietary supplemental level. Between the NCG versus BCG, VBSG I versus BCG, and VBSG I versus NCG groups, 11, 20 and 11 metabolites were significantly changed, respectively. The KEGG pathway of vitamin B6 metabolism was significantly impacted in the NCG layers than those from BCG; seven and five pathways were significantly impacted in the VBSG I layers compared with those from BCG and NCG, including pyrimidine metabolism, vitamin B6 metabolism, glycerophospholipid metabolism, etc. CONCLUSION: We concluded that 25 µg kg-1 vitamin B12 supplementation in corn-soybean meal-based layer diet increased the egg yolk vitamin B12 concentration and impacted the vitamin B6 metabolic pathway, and 100 µg kg-1 of it increased the egg yolk and liver vitamin B12 concentrations and impacted vitamin B6 , lipid, nucleic acid and amino acid metabolic pathways. © 2022 Society of Chemical Industry.

Estudo com vacina BCG investiga resposta imune à Covid-19

Liderado pela Austrália, um estudo investigará se é possível prever quem permanece suscetível às variantes do Sars-CoV-2, mesmo tendo recebido uma vacina contra a Covid-19 ou de já ter adoecido pelo vírus.

La tuberculosis: un viejo problema aún sin resolver ¿para cuándo una nueva vacuna? / Tuberculosis: an old unresolved problem when will we have a new vaccine?

No disponible

Ausência de efeito da memória imunológica e antígenos micobacterianos sobre a produção de IgE e citocinas em resposta a Dermatophagoides pteronyssinus / No effect of mycobacterial antigens and immunological memory on the production of cytokines and IgE in response to Dermatophagoides pteronyssinus

Introdução: As doenças respiratórias alérgicas, tais como rinite e asma, afetam elevada proporção da população brasileira. Estima-se que mais de 58 mil pessoas foram afetadas por alguma destas condições no Brasil em 2002-2003. Estudos realizados em humanos e animais sugerem que a exposição ambiental ao Mycobacterium tuberculosis ou imunização com o M. bovis (vacina BCG), podem estar relacionadas à proteção contra doenças alérgicas. Objetivo: Investigar a influência da resposta Th1 a antígenos micobacterianos sobre a modulação da resposta do tipo Th2 ao ácaro Dermatophagoides pteronyssinus (Derp). Métodos: O estudo compreendeu duas fases. Para avaliar o efeito da resposta à revacinação com o BCG sobre a modulação de uma resposta do tipo Th2 ao Derp, foi realizado um estudo de intervenção randomizado com coorte prospectiva, e os voluntários que participaram compuseram a Amostra 1. Para avaliar o efeito da resposta à infecção latente com M. tuberculosis sobre a modulação de uma resposta do tipo Th2 ao Derp, foi feito um estudo de caso-controle e os voluntários que participaram compuseram a Amostra 2. A população foi composta por adultos jovens com idade entre 19 a 33 anos. Todos responderam ao questionáro ISAAC...

Introduction: Allergic respiratory diseases such as asthma and rhinitis, affecting a high proportion of the Brazilian population. More than 58.000 people have been affected by some of these conditions in Brazil in 2002-2003. Studies in humans and animals suggest that environmental exposure to Mycobacterium tuberculosis or immunization with Mycobacterium bovis (BCG), may be related to protection against allergic diseases. Objective: To investigate the influence of Th1 response to mycobacterial antigens on the modulation of Th2-type response to aeroallergen Dermatophagoides pteronyssinus (Derp). Methods: The study comprised two phases. To evaluate the effect of the response to revaccination with BCG on the modulation of a Th2-type response to Derp, we conducted a randomized intervention study with prospective cohort, and the volunteers composed the Sample 1. To evaluate the effect of latent response to infection with M. tuberculosis on the modulation of a Th2-type response to Derp, a study was made of case-control and the volunteers composed the Sample 2. The population consisted of young adults aged 19 to 33 years. All responded to questionnaire ISAAC...

Spectrophotometric determination of lamotrigine in pharmaceutical preparations and urine by charge-transfer complexation.

Rapid and sensitive spectrophotometric methods are developed for the determination of lamotrigine (LTG) in pharmaceutical dosage forms and urine samples, based on the formation of the charge-transfer (CT) complexes between LTG as an n-donor and the acceptors: bromocresol green (BCG), bromocresol purple (BCP), and chlorophenol red (CPR). These complexes are studied spectrophotometrically in chloroform solution in order to obtain some information about their stoichiometry and stability of complexation. The analytical parameters and their effects on the extraction of drug from urine samples are investigated. The reactions were extremely rapid at room temperature, and the absorbance values remained unchanged after 24 h for all reactions. Beer's law was obeyed in the concentration ranges 0.15-19.8, 0.15-19.8 and 0.05-34.1 microg x ml(-1) for CPR, BCP and BCG, respectively. The proposed methods were applied successfully for the determination of LTG in pharmaceutical formulations, and human urine samples in the presence of other antiepileptic drugs such as carbamazepine, oxcarbazepine and phenobarbital, with good accuracy and precision.

Development and validation of an ATP method for rapid estimation of viable units in lyophilised BCG Danish 1331 vaccine.

An assay for quantifying viability in BCG vaccine by determining intracellular ATP content was developed and validated. ATP content was determined by measuring bioluminescence in the presence of luciferin/luciferase. During development and validation the ATP method was compared to the conventional viable count method. A key step to obtain correlation between ATP content and CFU was found to be a period of pre-incubation in a growth medium before ATP determination. During the validation, the robustness, linearity, accuracy, precision, and range were studied. The method validation study showed that the method applied was robust and applicable to determine ATP content in lyophilised BCG for estimating viability in the BCG samples. By comparison with a conventional viable count method, a high correlation between ATP content and the viable count was found; this relationship can be applied in routine quality control to estimate viable count from the ATP content determined in a sample.

Epitope-tagging vectors for the expression and detection of recombinant proteins in mycobacteria.

New tools are required to study the growing number of uncharacterised genes derived from genome sequence projects that are specific to bacterial pathogens such as Mycobacterium tuberculosis. We have developed a series of vectors that permit the specific detection of recombinant proteins expressed in mycobacterial species. Gene expression in these vectors is driven by the strong hsp60 promoter of Mycobacterium bovis BCG and detection of expressed products is facilitated by C-terminal fusion of residues 409-419 of the human c-myc proto-oncogene. Using the M. tuberculosis Ag85B as a reporter of gene expression, we demonstrate that the vectors permit the specific detection of recombinant products expressed in the host species M. bovis BCG. BCG over-expressing Ag85B was a potent inducer of Ag85B-specific T cells in immunised mice, indicating that the C-terminal c-myc tag did not alter the characteristics of the recombinant protein. The versatility of the epitope-tagging vectors was demonstrated by the efficient secretion and detection of recombinant products in BCG. The vectors described in this study will facilitate the expression of foreign proteins in mycobacterial host systems.

[Examination of protein MPB 70 properties in BCG substrains used for the production of Polish antituberculosis vaccines]. / Badanie obecnosci bialka MPB 70 w podszczepie BCG uzywanym do produkcji Polskiej szczepionki przeciwgruzliczej.

The aim of the study was examination of MPB70 protein production by different BCG substrains and testing this protein as reagent in allergenic skin test on BCG vaccinated guinea pigs. Three BCG substrains: Danish 1331 (D), Japanese 172 (J) and Polish BCG Moreau (P) were used for the study. The protein MPB70 received from dr Nagai from Osaka University was used as reference preparation. It has been shown that MPB70 protein of molecular weight about 21,500 Da was present only in BCG Moreau (P) and Japanese (J) substrains. In immunoblotting test monoclonal antibodies reacted additionally with 43,000 Da protein in all tested substrains. We suspect that the detected additional molecule was a dimer of MPB70 protein. The development of skin reactions to MPB70 protein was seen in guinea pigs vaccinated with J and P substrains. The peak of alergy to MPB70 protein was observed 9 weeks after vaccination. In sera of vaccinated animals antibodies against MPB70 protein were detected by ELISA method. The possibility of using MPB70 protein for diagnostic purpose is discussed.

O método polarográfico na determinaçäo da viabilidade da vacina BCG liofilizada / The polarographic method in the assays of the viability of the freezedried BCG vaccines

Estudou-se métodos alternativos a serem empregados em procedimentos de controle de qualidade da vacina BCG liofilizada. Com esse objetivo foi desenhado e contruído um polarógrafo de eletrodo de oxigênio constituído de eletrodo de platina e o de referência de Ag-AgCl embutido em peça única e dotado de sensor de temperatura, com circuito eletrônico permitindo a medida de consumo de oxigênio. Levou-se a efeito, estudo comparativo de viabilidade de vacina BCG liofilizada pela medida polarográfica de consumo de oxigênio em comparaçäo à manométrica pelo clássico método de Warburg e o método de contagem de colônias. (UFC). A análise de 66 diferentes lotes de vacina BCG liofilizada produzidas pelo Instituto Butantan, Säo Paulo, e pela Fundaçäo Ataulpho de Paiva, Rio de Janeiro, demonstrou que as técnica polarográfica revelou ser altamente reprodutível e muito mais conveniente que os métodos manométrico e de contagem de colônias. Verificou-se que entre o método polarográfico e o manométrico e UFC os resultados de regressäo linear mostraram correlaçäo positiva de alta intensidade e significante ao nível de *=0,05, permitindo indicar o método polarográfico, como procedimento de escolha para operaçöes de controle de qualidade em laboratórios de produçäo de vacina BCG bem como em Unidades de Saúde, para determinaçöes adequadas de viabilidade de lotes de vacinas

Cell mass of Mycobacterium bovis BCG estimated by gas chromatography.

The presence of additives and large cellular aggregates in freeze-dried BCG vaccines precludes accurate measurement of total cell content by traditional methods. The possibility that extraction and quantitation of a cell membrane fatty acid may provide a suitable means of cell mass determination was tested. The palmitic acid methyl ester peak area determined by gas chromatography was directly proportional to the wet weight of freshly grown Tice-, Pasteur-, and Glaxo-substrain BCG, as well as the dry weight of the ampoule contents after removal of soluble material. Extraction of palmitic acid from Tice BCG vaccine was not appreciably affected by lyophilization and the calculated dry cell mass values of freeze-dried vaccine samples correlated well with particle number. This method, therefore, may be useful in measuring BCG cell mass during all stages of vaccine manufacture and storage.

Experimental research on utilization of bioluminescent technique to substitute the current viability count for BCG vaccine.

The possibility of using the bioluminescent (BL) technique to substitute the traditional viability count of colony forming units (CFU) of BCG vaccine was investigated. The results showed there is a significant dose-dependent correlation between the concentration of standard adenosine triphosphate (ATP) and ATP BL value. The ultrasonic-chloroform method designed by us yielded the best results. The correlation coefficient values (r) of BL of the liquid and lyophilized vaccine were found to be 0.8 155 and 0.8 484 respectively (P less than 0.05). The coefficient of variation (CV) between the BL value of different lots of ATP obtained was 3.2-4.4%, much lower than that of CFU (CV = 10.4-11.2%). The presence of bacterial clumps within the vaccine had great influence on the bacterial ATP value and on the CFU viability count. 2.8 fg of ATP was found in each CFU formed in the vaccine with clumps, while it was only 0.94 fg in the vaccine without clumps, indicating the superiority of the BL method. The BL method has shown a high sensitivity, good reproducibility and simplicity in handling with quick results and high accuracy. Therefore, we consider that the BL method can be used to substitute the CFU method.

Comparison of Karl Fischer titrimetric and gravimetric methods for determination of moisture content in BCG vaccine.

A model 447 Coulomatic K-F titrimeter was used to determine the water content of seventeen lots of freeze-dried Tice-substrain BCG vaccine. The results were compared with corresponding moisture contents determined by a standard gravimetric method at the time of manufacture. The advantages of the titrimetric method include simplicity, rapidity, convenience, sensitivity, reproducibility and specificity, whereas the gravimetric method is tedious and time-consuming. Although moisture content determined by the K-F titrimeter tended to be higher than that determined by the gravimetric method, the results correlated significantly (r = 0.882, P less than 10(-5]. Alteration of national and international regulations to permit use of the K-F titrimeter is recommended.

Size characterization of Mycobacterium bovis BCG (Bacillus Calmette Guérin) vaccine, Tice substrain.

Reconstituted, lyophilized, attenuated Mycobacterium bovis, Bacillus Calmette Guérin (BCG) vaccine, Tice substrain, was characterized using a Coulter Multisizer and a HIAC/Royco counter. The primary organism has an equivalent spherical diameter approximating 1 micron but the BCG cell suspension is heavily aggregated. The cumulative size distribution of the suspension fits a log-probit plot and this information can be used to determine the total number of particles per ampoule. The instrumental count may be related to the viable count. The state of dispersion was unaffected by mild shear (syringe aspiration or ultrasound) and only slightly affected by the addition of cetylpyridinium chloride or sodium tauroglycolate.

Eficacia de la vacunacion con BCG evaluada mediante el método de casos y testigos en Buenos Aires, Argentina / Effectiveness of BCG vaccination evaluated by means of the case control method in Buenos Aires, Argentina

A retrospective case control study was carried out between 1981 and 1984 in three hospitals in the western part of Greater Buenos Aires to determine protection provided by BCG tuberculosis vaccine in children under six years of age residing in a region of Argentina in which vaccination coverage is approximately 55 percent. A total of 175 cases of tuberculosis were included in the study. For each case five controls were selected (a total of 875) from among patients being treated in the same hospital for other causes unrelated to tuberculosis. The controls were selected on the basis of their similarity with the cases with respect to age, socioeconomic status, nutritional status, and place of residence. Information on the presence or absence of vaccination scarring and other data related to vaccination of both the cases and controls was collected by an independent examiner. In 152 of the 175 cases location of tuberculosis was pulmonary; in 15, miliary; and in six there were pleural complications. Of the remaining 23 cases, 18 were meningeal, two gangliar, two osteoarticular, and one otic. Diagnosis was based on bacteriological and histopathological analysis, computerized tomography, radiological and clinical examination, endoscopy, and verification of a focus of infection. The protective effect of BCG vaccine in vaccinated patients was 73 percent on average (CI95 percent = 62 to 85 percent

Antitumor activity of the DNA fraction from Mycobacterium bovis BCG. II. Effects on various syngeneic mouse tumors.

MY-1, a fraction extracted from BCG and composed of 70.0% DNA and 28.0% RNA, was examined for its antitumor activity against 9 different syngeneic mouse tumors. Tumor regression was induced in almost all of the mice bearing any of five kinds of solid tumors by repeated intralesional injections of 100 micrograms MY-1. When cells of some tumors were inoculated intradermally together with MY-1, tumor growth was suppressed, lung metastases were inhibited, and the survival times of mice bearing 1 of 3 leukemic tumors were prolonged. Repeated sc injections with MY-1 in sites remote from tumor cell inoculation or repeated iv injections were more or less effective against three kinds of solid tumors. Mice inoculated with Lewis lung carcinoma cells in a hind footpad and whose legs were amputated 9 days later were given iv or sc injections of MY-1 every other day (8 times in total), resulting in substantial prolongation of survival. No direct cytotoxicity of MY-1 for these tumors could be shown in three kinds of experiments, which indicates that the antitumor mechanism of MY-1 is host mediated. MY-1 was equally effective in mice with or without presensitization with BCG, whereas BCG was much more effective in BCG-sensitized mice. This finding suggests that a delayed-type hypersensitivity reaction elicited by BCG protein is not required for the antitumor activity of MY-1.

Antitumor activity of deoxyribonucleic acid fraction from Mycobacterium bovis BCG. I. Isolation, physicochemical characterization, and antitumor activity.

A fraction extracted from Mycobacterium bovis strain BCG, which was composed of 70.0% DNA, 28.0% RNA, 1.3% protein, 0.20% glucose, and 0.1% lipid and of no detectable amounts of cell wall components such as alpha, epsilon-diaminopimelic acid and hexosamine, was found to possess strong antitumor activity. Repeated intralesional injection of this fraction, designated MY-1, without attachment to oil or a single intralesional injection of MY-1 emulsified in mineral oil caused the IMC carcinoma of CDF1 mice and line 10 tumor of strain 2 guinea pigs to regress and/or prevented metastasis very effectively. MY-1 after digestion with RNase, which contained 97.0% single-stranded DNA with a guanine-cytosine content of 69.8%, was more effective than undigested MY-1 against IMC and line 10 tumor, while MY-1 digested with DNase, which contained 97.0% RNA, had reduced activity, suggesting that the DNA from BCG possessed strong antitumor activity under certain conditions. Details of the extraction procedures and physicochemical characterization of MY-1 were also described.

A comparison of the rate of oxygen uptake and the adenosine triphosphate content of routine and experimental BCG preparations.

The ATP content and oxygen uptake rate, two parameters of viability of a BCG suspension, are compared. The lack of correlation between measurements made on fresh routine preparations indicates that the ATP measurements is affected by the degree of dispersion of the preparation. Experimental preparations made under standard conditions (constant semi-dry weight and stepwise dispersive grinding) from cultures of the same age as the fresh routine cultures (14 days) had a smaller ATP content, which correlated well with the respiration rate. In experimental preparations from seven-day cultures this correlation was significant. Between days 7 and 14 the ATP content declined much more rapidly than the respiration rate.

Viability, heat stability and immunogenicity of four BCG vaccines prepared from four different BCG strains.

Four BCG strains, French 1173-P2, Danish 1331, Japanese 172 and Glaxo-1077, among those most widely used throughout the world for tuberculosis vaccination, were prepared according to the respective conditions in each BCG-production unit. These 16 vaccine preparations were distributed and compared using standardized in vitro and in vivo conditions, based on optical density and bacillary mass/ml. The results presented in this report concern only the 4 freeze-dried BCG strains prepared by the Institut Pasteur BCG unit. It is shown that these 4 preparations differ in their in vitro characteristics such as growth, morphology, heat stability and viability, tested by colony-forming units and the ATP content. In addition, statistically significant differences were observed in biological control tests performed in guinea-pigs and mice when they were immunized with the same concentrations of 4 reconstituted vaccines. For instance, in tests measuring hallmarks of cell-mediated immunity--such as local granuloma formation, spleen index, delayed-type hypersensitivity to tuberculin, and non-specific immunopotentiation to unrelated antigen--a ranking order of relative immunopotency of the four strains was made, giving the following classification in decreasing order: French 1173-P2, Danish 1331, Japanese 172 and Glaxo-1077. Specific acquired resistance after BCG vaccination in mice was obtained with all four vaccines. However, higher protection was observed in mice vaccinated with Glaxo-1077 and French 1173-P2 strains. No obvious correlation existed between the intensity of cellular post-vaccination resistance and the above CMI tests, except those concerning the three-week index of BCG infection measured in the spleen, which correlated with the protection test.