Effect of culture parameters on the heat tolerance and inorganic polyphosphate accumulation by Lacticaseibacillus rhamnosus CRL1505, a multifunctional bacterium.

Lacticaseibacillus rhamnosus CRL1505 can be used in functional products as a probiotic powder (dried live cells) or as a postbiotic intracellular extract containing inorganic polyphosphate as a functional biopolymer. Thus, the aim of this work was to optimize the production of Lr-CRL1505 depending on the target of the functional product (probiotic or postbiotic). For this purpose, the effect of culture parameters (pH, growth phase) on cell viability, heat tolerance and polyphosphate accumulation by Lacticaseibacillus rhamnosus CRL1505 was evaluated. Fermentations at free pH produced less biomass (0.6 log units) than at controlled pH while the growth phase affected both polyphosphate accumulation and cell heat tolerance. Exponential phase cultures showed 4-15 times greater survival rate against heat shock and 49-62% increased polyphosphate level, compared with the stationary phase. Results obtained allowed setting the appropriate culture conditions for the production of this strain according to its potential application, i.e., as live probiotic cells in powder form or postbiotic. In the first case, running fermentations at pH 5.5 and harvesting the cells at the exponential phase are the best conditions for obtaining a high live biomass yield capable of overcoming heat stress. Whereas the postbiotic formulations production requires fermentations at free pH and harvesting the cells in exponential phase to increase the intracellular polyphosphate level as a first step.

Inorganic Additive Improves the Survival of the Probiotic Lacticaseibacillus rhamnosus CRL1505 During Spray Drying, Rehydration, and Storage.

In previous in vitro studies, an inorganic additive (MCM3) showed a thermo-protective effect on the cell viability of Lacticaseibacillus rhamnosus CRL1505 (Lr-CRL1505). In this work, cultures of this probiotic strain were spray dried at lab scale using two carriers: maltodextrin (powder MA) and maltodextrin plus MCM3 (powder MA/MCM3). The cell survival was higher in powder MA/MCM3 (72.8%) than in powder MA (42.8%). Different rehydration media, including the additive MCM3, and two temperatures (37 °C and 45 °C) were evaluated. The best results were obtained in cells rehydrated at 37 °C in MCM3. During the storage of the powders, the highest cell counts were observed in the MA/MCM3 powder. Our results demonstrated that the presence of MCM3 in the carrier and in reconstitution media benefits the spray drying process and the recovery of dehydrated cells. Thus, the use of this additive of inorganic nature and low cost represents a promising technological alternative.

Oral Vaccination with Hepatitis E Virus Capsid Protein and Immunobiotic Bacterium-Like Particles Induce Intestinal and Systemic Immunity in Mice.

The hepatitis E virus (HEV) genotype 3 (GT3) is an emergent pathogen in industrialized countries. It is transmitted zoonotically and may lead to chronic hepatitis in immunocompromised individuals. We evaluated if the major antigen of HEV, the capsid protein, can be used in combination with immunobiotic bacterium-like particles (IBLP) for oral vaccination in a mouse model. We have cloned and expressed the RGS-His5-tagged HEV GT3 capsid protein (ORF2) in E. coli and purified it by NiNTA. IBLP were obtained from two immunobiotic Lactobacillus rhamnosus strains acid- and heat-treated. ORF2 and the IBLP were orally administered to Balb/c mice. After three oral immunizations (14-day intervals), blood, intestinal fluid, Peyer´s patches, and spleen samples were drawn. IgA- and IgG-specific antibodies were determined by ELISA. Mononuclear cell populations from Peyer's patches and spleen were analyzed by flow cytometry, and the cytokine profiles were determined by ELISA to study cellular immunity. Orally administered recombinant ORF2 and IBLP from two L. rhamnosus strains (CRL1505 and IBL027) induced both antigen-specific humoral and cellular immune responses in mice. IBLP027 was more effective in inducing specific secretory IgA in the gut. IFN-γ, TNF-α, and IL-4 were produced by Peyer's plaques lymphocytes stimulated with ORF2 ex vivo suggesting a mixed Th1/Th2-type adaptive immune response in immunized mice. Oral vaccines are not invasive, do not need to be administered by specialized personal, and elicit both systemic and local immune responses at the port of entry. Here, we present an experimental oral vaccine for HEV GT3, which could be further developed for human and/or veterinary use.

Lactobacillus rhamnosus CRL1505 nasal administration improves recovery of T-cell mediated immunity against pneumococcal infection in malnourished mice.

Immunobiotic lactic acid bacteria have become an interesting alternative for the prevention of respiratory infections. Previously, we demonstrated that the nasal administration of Lactobacillus rhamnosus CRL1505, during repletion of malnourished mice, resulted in diminished susceptibility to the challenge with the respiratory pathogen Streptococcus pneumoniae. Considering the known alterations induced by malnutrition on T lymphocytes and the importance of this cell population on the protection against respiratory pathogens, we aimed to study the effect of L. rhamnosus CRL1505 nasal administration on the recovery of T cell-mediated defences against pneumococcal infection in malnourished mice under nutritional recovery. Malnourished mice received a balanced conventional diet (BCD) for seven days or BCD for seven days with nasal L. rhamnosus CRL1505 supplementation during last two days of the treatment. After the treatments mice were infected with S. pneumoniae. Flow cytometry studies were carried out in bone marrow, thymus, spleen and lung to study T cells, and Th1/Th2 cytokine profiles were determined in broncho-alveolar lavages and serum. The administration of CRL1505 strain to malnourished mice under recovery reduced quantitative and qualitative alterations of CD4+ T cells in the bone marrow, thymus, spleen and lung induced by malnutrition. In addition, CRL1505 treatment augmented Th2-cytokines (interleukin 10 and 4) in respiratory and systemic compartments after pneumococcal infection. These results show that modulation of CD4+ T lymphocytes induced by L. rhamnosus CRL1505 has an important role in the beneficial effect induced by this strain on the recovery of malnourished mice. These data also indicate that nasally administered L. rhamnosus CRL1505 may represent a non-invasive alternative to modulate and improve the T cell-mediated immunity against respiratory pathogens in immunocompromised malnourished hosts.

Potentiality of standardized extract and isolated flavonoids from Zuccagnia punctata for the treatment of respiratory infections by Streptococcus pneumoniae: in vitro and in vivo studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Zuccagnia punctata Cav. (Fabaceae) is a monotypic species distributed in western Argentina and is traditionally used for the treatment of bacterial and fungal infections. The aim of this study was to demonstrated the antibacterial activity of the Zuccagnia punctata standardized extract and the structurally related non-methoxylated flavonoids with similar pattern of substitution and differences in ring C present in this plant species: 7-hydroxyflavanone (HF), 2',4'-dihydroxychalcone (DHC) and 3,7-dihydroxyflavone (DHF), against Streptococcus pneumoniae clinical isolates using in vitro and in vivo models. MATERIALS AND METHODS: MIC values of natural products were determined by agar macrodilution method. In vivo activities were investigated in a Streptococcus pneumoniae infection model in mice. Lung and blood samples were obtained for bacterial cell counts. The serum was used by biochemical analysis (alanine transaminase, aspartate transaminase, urea and creatinine) in order to evaluate the toxicity of natural products. RESULTS: All samples showed antimicrobial activity in vitro with MIC values between 50 and 500µg/ml. Zuccagnia punctata extract (1mg/mice) and HF (1mg/mice) significantly reduced the number of viable Streptococcus pneumoniae in lung (p<0.01) while lower quantities has not effect. Therefore, the present study has shown that intake once or twice a day of 1mg of Zuccagnia punctata extract or HF for seven days did not result in toxicity. CONCLUSIONS: Our results showed that Zuccagnia punctata extract as well as one of its isolated flavonoids, 7-hydroxyflavanone, could be useful for the development of a novel respiratory infections treatment.

[Stereotaxic biopsy and 192Ir implant in intracranial tumors. A review of 7 years of experience]. / Biopsia estereotáxica e implante de Ir-192 en tumores intracraneales. Revisión de 7 años de experiencia.

INTRODUCTION: The combination of stereotaxic techniques, advances in neuroimaging and the creation of continually improving software has permitted stereotaxic biopsy of cerebral lesions at the most varied sites. Improvement in the method of permanent interstitial radiation (brachytherapy) improves the precision with which the radioactive sources may be inserted, releasing a maximum dose of radiation to the tumour with minimum radiation to the surrounding tissue. PATIENTS AND METHODS: We treated 237 patients (aged 1 to 78 years) with intracranial lesions, all included in the protocol of our centre. Stereotaxic systems of Leksell, Riechert-Mundinger, Micromar and Estereoflex were used. The procedure was in three stages: acquisition of the image, surgical planning and surgical operation. The imaging guide was the computerized axial tomography (CAT). RESULTS: Stereotaxic biopsy guided by CAT images was done in 153 patients. These were divided into three groups, taking the biopsy findings as the reference: group A (primary tumors, 128), group B (metastatic tumors, 15) and group C (non-malignant lesions, 10). Ninety six permanent implants of 192Ir were inserted, with a low dose of 4-7 cGy/h and a total dose of 80-120 Gy. CONCLUSIONS: Stereotaxic biopsy is a very effective procedure with a significantly low range of complications. The permanent implant with a low dose rate, well situated and using a source of 192Ir is a safe, simple, effective method for the treatment of primary and recurrent glial tumours, and non-glial tumours which fulfil criteria for this type of brachytherapy.

Biopsia estereotáxica e implante de 192Ir en tumores intracraneales: revisión de siete años de experiencia / Stereotaxic biopsy and implantation of 193Ir in intracranial tumors: a review of seven years experience

Introducción. La combinación de técnicas estereotáxicas, los avances en las neuroimágenes y la creación de programas informáticos cada vez más óptimos ha permitido la realización de biopsias estereotáxicas a lesiones cerebrales en las más variadas localizaciones. El perfeccionamiento del método de radiación intersticial permanente (braquiterapia) mejora la exactitud en la colocación de las fuentes radiactivas, liberando una dosis de radiciación máxima al tumor con mínima irradiación del tejido normal que lo rodea. Pacientes y métodos. Fueron tratados 237 pacientes (de 1 a 78 años) con lesiones intracraneales, todos ellos incluidos en el protocolo de nuestra institución; se utilizaron los sitemas estereotáxicos de Leksell, Riechert-Mundinger, Micromar y Estereoflex. El procedimiento constó de tres etapas: adquisición de la imagen, planificación quirúrgica e intervención quirúrgica. La guía por imagen fue la tomografía axial computarizada (TAC). Resultados. Se realizó biopsia estereotáxica guiada por imágenes de TAC a 153 pacientes, los cuales se dividieron en tres grupos tomando como referencia el hallazgo de la biopsia: grupo A (tumores primarios, 128); grupo B (tumores metastásicos, 15), y grupo C (lesiones no malignas, 10). Se realizaron 96 implantes permanentes de 192Ir con una tasa de dosis baja de 4-7 cGy/h con dosis total de 80 a 120 Gy. Conclusiones. La biopsia estereotáxica es un procedimiento muy eficaz con un margen de complicaciones significativamente bajo. El implante permanente de tasa de dosis baja, bien localizado y utilizando una fuente de 192Ir es un método simple, seguro y eficaz en el tratamiento de tumores gliales primarios y recurrentes, así como en otros no gliales que reunan los criterios para dicha modalidad de braquiterapia(AU)