Pea-protein alginate encapsulation adversely affects development of clinical signs of Citrobacter rodentium-induced colitis in mice treated with probiotics.

The efficacy of two strains of Lactobacillus probiotics (Lactobacillus rhamnosus R0011 and Lactobacillus helveticus R0052) immobilized in microcapsules composed of pea protein isolate (PPI) and alginate microcapsules was assessed using a mouse model of Citrobacter rodentium-induced colitis. Accordingly, 4-week-old mice were fed diets supplemented with freeze-dried probiotics (group P), probiotic-containing microcapsules (group PE) (lyophilized PPI-alginate microcapsules containing probiotics), or PPI-alginate microcapsules containing no probiotics (group E). Half of the mice (controls, groups P, PE, and E) received C. rodentium by gavage 2 weeks after initiation of feeding. Daily monitoring of disease symptoms (abnormal behavior, diarrhea, etc.) and body weights was undertaken. Histopathological changes in colonic and cecal tissues, cytokine expression levels, and pathogen and probiotic densities in feces were examined, and the microbial communities of the distal colon mucosa were characterized by 16S rRNA sequencing. Infection with C. rodentium led to marked progression of infectious colitis, as revealed by symptomatic and histopathological data, changes in cytokine expression, and alteration of composition of mucosal communities. Probiotics led to changes in most of the disease markers but did not have a significant impact on cytokine profiles in infected animals. On the basis of cytokine expression analyses and histopathological data, it was evident that encapsulation materials (pea protein and calcium alginate) contributed to inflammation and worsened a set of symptoms in the cecum. These results suggest that even though food ingredients may be generally recognized as safe, they may in fact contribute to the development of an inflammatory response in certain animal disease models.

BCG vaccination-induced emergency granulopoiesis provides rapid protection from neonatal sepsis.

Death from sepsis in the neonatal period remains a serious threat for millions. Within 3 days of administration, bacille Calmette-Guérin (BCG) vaccination can reduce mortality from neonatal sepsis in human newborns, but the underlying mechanism for this rapid protection is unknown. We found that BCG was also protective in a mouse model of neonatal polymicrobial sepsis, where it induced granulocyte colony-stimulating factor (G-CSF) within hours of administration. This was necessary and sufficient to drive emergency granulopoiesis (EG), resulting in a marked increase in neutrophils. This increase in neutrophils was directly and quantitatively responsible for protection from sepsis. Rapid induction of EG after BCG administration also occurred in three independent cohorts of human neonates.

Systems Biology Methods Applied to Blood and Tissue for a Comprehensive Analysis of Immune Response to Hepatitis B Vaccine in Adults.

Conventional vaccine design has been based on trial-and-error approaches, which have been generally successful. However, there have been some major failures in vaccine development and we still do not have highly effective licensed vaccines for tuberculosis, HIV, respiratory syncytial virus, and other major infections of global significance. Approaches at rational vaccine design have been limited by our understanding of the immune response to vaccination at the molecular level. Tools now exist to undertake in-depth analysis using systems biology approaches, but to be fully realized, studies are required in humans with intensive blood and tissue sampling. Methods that support this intensive sampling need to be developed and validated as feasible. To this end, we describe here a detailed approach that was applied in a study of 15 healthy adults, who were immunized with hepatitis B vaccine. Sampling included ~350 mL of blood, 12 microbiome samples, and lymph node fine needle aspirates obtained over a ~7-month period, enabling comprehensive analysis of the immune response at the molecular level, including single cell and tissue sample analysis. Samples were collected for analysis of immune phenotyping, whole blood and single cell gene expression, proteomics, lipidomics, epigenetics, whole blood response to key immune stimuli, cytokine responses, in vitro T cell responses, antibody repertoire analysis and the microbiome. Data integration was undertaken using different approaches-NetworkAnalyst and DIABLO. Our results demonstrate that such intensive sampling studies are feasible in healthy adults, and data integration tools exist to analyze the vast amount of data generated from a multi-omics systems biology approach. This will provide the basis for a better understanding of vaccine-induced immunity and accelerate future rational vaccine design.

Probiotic Studies in Neonatal Mice Using Gavage.

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult mouse models to neonatal diseases is limited. To better understand disease progression, host responses and long-term impact of interventions in neonates, a neonatal mouse model likely is a better fit. The sparse use of neonatal mouse models can in part be attributed to the technical difficulties of working with these small animals. A neonatal mouse model was developed to determine the effects of probiotic administration in early life and to specifically assess the ability to establish colonization in the newborn mouse intestinal tract. Specifically, to assess probiotic colonization in the neonatal mouse, Lactobacillus plantarum (LP) was delivered directly into the neonatal mouse gastrointestinal tract. To this end, LP was administered to mice by feeding through intra-esophageal (IE) gavage. A highly reproducible method was developed to standardize the process of IE gavage that allows an accurate administration of probiotic dosages while minimizing trauma, an aspect particularly important given the fragility of newborn mice. Limitations of this process include possibilities of esophageal irritation or damage and aspiration if gavaged incorrectly. This approach represents an improvement on current practices because IE gavage into the distal esophagus reduces the chances of aspiration. Following gavage, the colonization profile of the probiotic was traced using quantitative polymerase chain reaction (qPCR) of the extracted intestinal DNA with LP specific primers. Different litter settings and cage management techniques were used to assess the potential for colonization-spread. The protocol details the intricacies of IE neonatal mouse gavage and subsequent colonization quantification with LP.

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis.

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

Survival of probiotics in pea protein-alginate microcapsules with or without chitosan coating during storage and in a simulated gastrointestinal environment.

Pea protein-alginate microcapsules with or without a chitosan coating and containing Lactobacillus rhamnosus R0011 and L. helveticus R0052 were produced by extrusion and tested for survivability during storage and in an in vitro gastrointestinal environment. Both microcapsule formulations provided significant protection for cells incubated in synthetic stomach juice at 37°C for 2 h, followed by 3 h in simulated intestinal fluid, relative to non-encapsulated bacteria. However, evaluation of cell viability during 9 weeks of storage at room temperature revealed that chitosan coating significantly improved microcapsule performance compared to non-coated microcapsules. Refrigerated storage had no negative impact on the microcapsule protection ability of both types of microcapsules. Notably, chitosan-containing microcapsules showed much higher bacterial survival counts during challenge tests even after storage. Moreover, the addition of chitosan to the microcapsule formulation did not increase the microcapsule size.

Nature of protein-protein interactions during the gelation of canola protein isolate networks.

The nature of interactions involved during the gelation of a canola protein isolate was investigated using rheology and fractal imaging at neutral pH as a function of protein concentration (5.0-9.0% w/w). The onset of denaturation and the denaturation temperature by differential scanning calorimetry for canola protein isolate (CPI; 98.2% protein) was 78.6°C and 87.1°C, respectively. Rheological testing determined the gelation temperature (Tgel) to be ~87-90°C for all concentrations. The log % strain at break increased from 1.70 to 1.80 as CPI concentration increased from 5.0 to 7.0% (w/w). Rheological testing of CPI in the presence of destabilizing agents, NaCl (0.1 and 0.5M), urea (0.1, 0.5, 1 and 5M) and 2-ß-mercaptoethanol (0.1 and 2%), was performed. Samples with NaCl and urea (0.1-1M) had similar temperature profiles and Tgel values to CPI alone whereas no gel was formed with the addition of 5M urea and 2-ß-mercaptoethanol reduced the strength of the gel network. Fractal dimension and lacunarity was analyzed using CLSM imaging. The fractal dimension value for all CPI concentrations was ~1.5. The lacunarity of the gel decreased from 0.62 to 0.41 as the concentration of CPI increased from 5 to 7% (w/w). Mechanistic understanding of CPI aggregation and network formation will enable the food industry to better tailor food structure when CPI is present as ingredient.

Probiotic-based strategies for therapeutic and prophylactic use against multiple gastrointestinal diseases.

Probiotic bacteria offer a number of potential health benefits when administered in sufficient amounts that in part include reducing the number of harmful organisms in the intestine, producing antimicrobial substances and stimulating the body's immune response. However, precisely elucidating the probiotic effect of a specific bacterium has been challenging due to the complexity of the gut's microbial ecosystem and a lack of definitive means for its characterization. This review provides an overview of widely used and recently described probiotics, their impact on the human's gut microflora as a preventative treatment of disease, human/animal models being used to help show efficacy, and discusses the potential use of probiotics in gastrointestinal diseases associated with antibiotic administration.