Optimization of the viability of stem cells derived from umbilical cord blood after maternal supplementation with DHA during the second or third trimester of pregnancy: study protocol for a randomized controlled trial.

BACKGROUND: Umbilical cord blood (UCB) is an important source of hematopoietic stem cells (HSCs). However, the concentration of cells in cord blood units is limited and this may represent the main restriction to their therapeutic clinical use. The percentage of metabolically active stem cells provides a measure of the viability of cells in an UCB sample. It follows that an active cellular metabolism causes a proliferation in stem cells, offering an opportunity to increase the cellular concentration. A high cell dose is essential when transplanting cord stem cells, guaranteeing, in the receiving patient, a successful outcome.This study is designed to evaluate the impact of docosahexaenoic acid (DHA) supplementation in pregnant women, in order to increase the quantity and viability of the cells in UCB samples. METHODS/DESIGN: The metabolic demand of DHA increases in the course of pregnancy and reaches maximum absorption during the third trimester of pregnancy. According to these observations, this trial will be divided into two different experimental groups: in the first group, participants will be enrolled from the 20th week of estimated stage of gestation, before the maximum absorption of DHA; while in the second group, enrolment will start from the 28th week of estimated stage of gestation, when the DHA request is higher. Participants in the trial will be divided and randomly assigned to the placebo group or to the experimental group. Each participant will receive a complete set of capsules of either placebo (250 mg of olive oil) or DHA (250 mg), to take one a day from the 20th or from the 28th week, up to the 40th week of estimated gestational age. Samples of venous blood will be taken from all participants before taking placebo or DHA, at the 20th or at the 28th week, and at the 37th to 38th week of pregnancy to monitor the level of DHA. Cell number and cellular viability will be evaluated by flow cytometry within 48 hours of the UCB sample collection. TRIAL REGISTRATION: International Standard Randomised Controlled Trial Number Register: ISRCTN58396079. Registration date: 8 October 2013.

Blood gas values in clamped and unclamped umbilical cord at birth.

AIM: To determine the reliability of the cord blood gas analysis on the unclamped cord compared to the standard technique of sampling on double clamped cord. STUDY DESIGN: Prospective observational study conducted on 46 singleton neonates vaginally delivered at term. Matched pairs of umbilical artery and vein blood samples were collected from unclamped cord within 90s after birth and from the same cord after clamping, with the clamping occurring immediately after the first blood collection. A blood gas analysis was performed on each collected sample. OUTCOME MEASURES: Arterial and venous blood samples were analyzed for pH, PO2, pCO2, SaO2, hemoglobin concentration (ctHb) and base excess (BE). The values were compared between the two groups (clamped vs unclamped) using a Wilcoxon test. RESULTS: No significant difference was found in pH, PO2, pCO2, SaO2 and ctHb values on arterial blood between unclamped and clamped cord. The only significant difference was related to BE (p<0.001). For the venous blood, the values of pH, PO2, pCO2 were comparable between unclamped and clamped cord, while the values of SaO2, ctHb and BE were significantly different (p<0.05). CONCLUSION: No significant difference was found in almost all the arterial blood gas parameters and in the main venous blood gas parameters between unclamped and clamped cord. Sampling of cord blood for gas analysis may be performed on the unclamped cord right after birth without reducing the accuracy of the analysis.

Mucosal lymphoid infiltrate dominates colonic pathological changes in murine experimental shigellosis.

BACKGROUND: Shigella species are invasive human pathogens that cause acute rectocolitis by triggering a dysregulated inflammatory reaction in the colonic and rectal mucosa. Because mice are naturally resistant to shigellosis, there is no mouse model that mimics human disease. We explore the susceptibility of intestinal flora-depleted mice to shigellosis after intragastric infection with Shigella strains. METHODS: Mice given 5 g/L streptomycin as a beverage were infected intragastrically with 1 x 108 cfu of either invasive or noninvasive Shigella strains. RESULTS: We found that invasive Shigella strains persist up to 30 days in feces, whereas the persistence of noninvasive Shigella strains was reduced. Colonization primarily involves the colon and the cecum and, to a lesser extent, the ileum. The hallmark of inflammation in the intestinal tissue is a dramatic expansion of the lymphoid follicles, in which a high apoptotic index is recorded. CONCLUSIONS: We provide a murine model in which shigellae are able to reach their natural tissue target: the colon. Moreover, the absence of polymorphonuclear leukocyte recruitment and of epithelial cell lesions reveal some aspects of shigellosis that are usually hidden by the prevalence of this cell population. This novel model may contribute to the identification of new targets for vaccines and therapies.

Intravenous infection of virulent shigellae causes fulminant hepatitis in mice.

Shigella spp. are pathogenic bacteria responsible for bacillary dysentery in humans. The major lesions in colonic mucosa are intense inflammation with apoptosis of macrophages and release of pro-inflammatory cytokines. The study of shigellosis is hindered by the natural resistance of rodents to oral infection with Shigella. Therefore, animal models exploit other routes of infection. Here, we describe a novel murine model in which animals receive shigellae via the caudal vein. Mice infected with 5 x 10(6) (LD(50)) virulent shigellae died at 48 h post infection, whereas animals receiving non-invasive mutants survived. The liver is the main target of infection, where shigellae induce microgranuloma formation. In mice infected with invasive bacteria, high frequency of apoptotic cells is observed within hepatic microgranulomas along with significant levels of mRNA for pro-inflammatory cytokines such as IL-1beta, IL-18, IL-12 and IFN-gamma. Moreover, in the blood of these animals high levels of IL-6 and transaminases are detected. Our results demonstrate the intravenous model is suitable for pathogenicity studies and useful to explore the immune response after Shigella infection.

Analysis of virulence and inflammatory potential of Shigella flexneri purine biosynthesis mutants.

Several Shigella flexneri mutants with defects in aromatic amino acid and/or purine biosynthesis have been evaluated as vaccines in humans or in animal models. To be suitable as a vaccine, a mutant has to show virulence attenuation, minimal reactogenicity, and a good immunogenic potential in animal models. With this aim, we have constructed five S. flexneri 5 (wild-type strain M90T) mutants with inactivation of one or two of the loci purEK, purHD, and guaBA, governing early or late steps of purine biosynthesis. The mutants have been analyzed in vitro in cell cultures and in vivo in the Sereny test and in the murine pulmonary model of shigellosis. M90T guaBA, M90T guaBA purEK, M90T guaBA purHD, and M90T purHD purEK gave a negative result in the Sereny test. In contrast, in the murine pulmonary model all of the strains had the same 50% lethal dose as the wild type, except M90T guaBA purHD, which did not result in death of the animals. Nevertheless, bacterial counts in infected lungs, immunohistochemistry, and reverse transcription-PCR analysis of mRNAs for tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), interleukin-1beta (IL-1beta), IL-6, IL-12, and inducible nitric oxide synthase (iNOS) revealed significant differences among the strains. At 72 h postinfection, M90T guaBA purHD still induced proinflammatory cytokines and factors such as IL-1beta, IL-6, TNF-alpha, and iNOS, along with cytokines such as IL-12 and IFN-gamma. Moreover, in the absence of evident lesions in murine tissues, this mutant highly stimulated major histocompatibility complex class II expression, showing a significant ability to activate the innate immunity of the host.