Novel ex vivo screening assay to preselect farm specific pre- and probiotics in pigs

A novel rapid ex vivo assay was developed as part of a concept to determine potential tailor-made combinations of pre- and probiotics for individual farms. Sow faecal slurries from 20 German pig farms were anaerobically incubated with pre- and probiotics or their combinations together with pathogenic strains that are of interest in pig production. Aliquots of these slurries were then incubated with media containing antibiotic mixtures allowing only growth of the specific pathogen. Growth was monitored and lag time was used to determine the residual fitness of the pathogenic strains. The background growth could be inhibited for an Escherichia coli- and a Clostridium difficile- but not for a Clostridium perfringens strain. The prebiotic fructo-oligosaccharides (FOS) and its combination with probiotics reduced the residual fitness of the E. coli strain in some farms. However, notable exceptions occurred in other farms where FOS increased the fitness of the E. coli strain. Generally, combinations of pre- and probiotics did not show additive effects on fitness for E. coli but displayed farm dependent differences. The effects of pre- and probiotics on the residual fitness of the C. difficile strain were less pronounced, but distinct differences between single application of prebiotics and their combination with probiotics were observed. It was concluded that the initial composition of the microbiota in the samples was more determinative for incubations with the C. difficile strain than for incubations with the E. coli strain, as the presumed fermentation of prebiotic products showed less influence on the fitness of the C. difficile strain. Farm dependent differences were pronounced for both pathogenic strains and therefore, this novel screening method offers a promising approach for pre-selecting pre- and probiotics for individual farms. However, evaluation of farm metadata (husbandry, feed, management) will be crucial in future studies to determine a tailor-made solution for combinations of pre- and probiotics for individual farms. Also, refinement of the ex vivo assay in terms of on-farm processing of samples and validation of unambiguous growth for pathogenic strains from individual farms should be addressed.

In vitro and ex vivo antiparasitic effect of Rheum ribes L. extract against the hydatid cyst protoscoleces

Background: Cystic echinococcosis is a zoonotic infection in humans and herbivorous animals with worldwide distribution which caused by larva stage of Echinococcus granulosus. Rhubarb (Rheum ribes L.) as an herbal medicines has various therapeutics properties such as the antioxidant, anticancer, and antimicrobial ones. With respect to the potential of the biological activities of this plant in traditional and modern medicine, we aim to examine its protoscolicidal effects against E. granulosus protoscolecess in vitro and ex vivo. Methods: Collected protoscoleces from liver hydatid cysts of infected sheep were exposed to the different concentrations of the extract (225, 450, 900 mg/mL) for 5-60 min in vitro and ex vivo. Then using the eosin exclusion assay the viability of protoscoleces was studied. Results: R. ribes extract had a potent protoscolicidal activity in vitro so that at the 450 and 900 mg/ml killed 56.3 and 100% of protoscoleces after 10 min exposure. Ex vivo assay, the extract needed more time to kill the protoscoleces than the in vitro; so that at the concentration of 900 mg/mL, all protoscoleces were killed after 15 minutes Conclusion: The obtained results exhibited the potent protoscolicidal effects of R. ribes extract particularly at the concentration of 900 mg/ml which completely killed the parasite after <15 min exposure. However, more and supplementary studies are required to verify these findings through assessing in animal models and clinical subjects.

In Vitro and Ex Vivo Evaluation of Capparis Spinosa Extract to Inactivate Protoscoleces During Hydatid cyst Surgery

Background:: Hydatidosis is one of the most dangerous zoonosis diseases in the world caused by the larval stage of the broad-worm or Echinococcus granulosus parasite. Today, cysts' rupture or content leakage during surgery and in-volvement of organs adjacent to the organ involved, and consequently secondary cysts, are the major concern for hydatid cyst surgeons. Therefore, using scolicidal substances such as hypertonic saline 20%, silver nitrate and formalin has been considered to reduce the risk of protoscoleces spread and recurrence of disease in recent years. The current work designed to assess the antiparasitic effects of Capparis spinose L. extract against hydatid cyst protoscoleces. Methods:: Collected protoscoleces from liver fertile hydatid cysts of infected sheep were exposed to the different concentra-tions of the essential oil (150, 300, 600 mg/mL) for 5-60 min in vitro and ex vivo. Then by using the eosin exclusion assay the viability of protoscoleces was studied. The primary phytochemical analysis of the C. spinosa extract was done to assess the presence of tannins, alkaloids, saponins, flavonoids, terpenoids and glycosides. Results:: C. spinosa extract had a powerful protoscolicidal activity in vitro so that at the 300 and 600 mg/ml entirely elimi-nates the parasite after 10 and 5 minutes; whereas at lower doses demonstrated weak protoscolicidal activity. Ex vivo assay, no similar effect with in vitro was observed, so that requiring a more time to show a potent protoscolicidal activity. C. spi-nosa extract at the concentrations of 300 and 600 mg/mL after exposure time of 20 and 12 min, killed 100% of protoscole-ces within the hydatid cyst, respectively. The findings of primary phytochemical screening of the C. spinosa extract demon-strated the existance of flavonoids, tannins, terpenoids, glycosides and alkaloids in this plant. Conclusion:: The obtained results in vitro and ex vivo exhibited that potent protoscolicidal effects of C. spinosa extract particu-larly at the concentrations of 600 and 300 mg/ml which entirely eliminates the parasite after 5-20 min exposure. However, more and supplementary works are required to verify these findings through assessing in animal models and clinical subjects.

Distinct Phenotypes of Islet Antigen-Specific CD4+ T Cells Among the 3 Subtypes of Type 1 Diabetes

Context Type 1 diabetes (T1D) is classified into 3 subtypes: acute-onset (AT1D), slowly progressive (SP1D), and fulminant (FT1D). The differences in the type of cellular autoimmunity within each subtype remain largely undetermined. Objective To determine the type and frequency of islet antigen-specific CD4+ T cells in each subtype of T1D. Participants Twenty patients with AT1D, 17 with SP1D, 18 with FT1D, and 17 persons without diabetes (ND). Methods We performed an integrated assay to determine cellular immune responses and T-cell repertoires specific for islet antigens. This assay included an ex vivo assay involving a 48-hour stimulation of peripheral blood mononuclear cells with antigen peptides and an expansion assay involving intracytoplasmic cytokine analysis. Results The results of the ex vivo assay indicated that glutamic acid decarboxylase 65 (GAD65)-specific interleukin-6 and interferon-inducible protein-10 (IP-10) responses and preproinsulin (PPI)-specific IP-10 responses were significantly upregulated in AT1D compared with those of ND. Furthermore, GAD65- and PPI-specific granulocyte colony-stimulating factor responses were significantly upregulated in FT1D. Expansion assay revealed that GAD65- and PPI-specific CD4+ T cells were skewed toward a type 1 helper T (Th1)- cell phenotype in AT1D, whereas GAD65-specific Th2 cells were prevalent in SP1D. GAD65-specific Th1 cells were more abundant in SP1D with human leukocyte antigen-DR9 than in SP1D without DR9. FT1D displayed significantly less type 1 regulatory T (Tr1) cells specific for all 4 antigens than ND. Conclusions The phenotypes of islet antigen-specific CD4+ T cells differed among the three T1D subtypes. These distinct T-cell phenotypes may be associated with the manner of progressive β-cell destruction.

Synthesis of β-d-galactopyranoside-Presenting Glycoclusters, Investigation of Their Interactions with Pseudomonas aeruginosa Lectin A (PA-IL) and Evaluation of Their Anti-Adhesion Potential

Pseudomonas aeruginosa is an opportunistic human pathogen associated with cystic fibrosis. This bacterium produces, among other virulence factors, a soluble d-galactose-specific lectin PA-IL (LecA). PA-IL plays an important role in the adhesion to the host cells and is also cytotoxic. Therefore, this protein is an interesting therapeutic target, suitable for inhibition by carbohydrate-based compounds. In the current study, β-d-galactopyranoside-containing tri- and tetravalent glycoclusters were synthesized. Methyl gallate and pentaerythritol equipped with propargyl groups were chosen as multivalent scaffolds and the galactoclusters were built from the above-mentioned cores by coupling ethylene or tetraethylene glycol-bridges and peracetylated propargyl β-d-galactosides using 1,3-dipolar azide-alkyne cycloaddition. The interaction between galactoside derivatives and PA-IL was investigated by several biophysical methods, including hemagglutination inhibition assay, isothermal titration calorimetry, analytical ultracentrifugation, and surface plasmon resonance. Their ability to inhibit the adhesion of P. aeruginosa to bronchial cells was determined by ex vivo assay. The newly synthesized multivalent galactoclusters proved to be significantly better ligands than simple d-galactose for lectin PA-IL and as a result, two representatives of the dendrimers were able to decrease adhesion of P. aeruginosa to bronchial cells to approximately 32% and 42%, respectively. The results may provide an opportunity to develop anti-adhesion therapy for the treatment of P. aeruginosa infection.