Alfonso Ruiz-Bravo

Administration of Lactobacillus casei and Lactobacillus plantarum affects the diversity of murine intestinal lactobacilli, but not the overall bacterial community structure.

Lactobacilli are normal inhabitants of the gastrointestinal tract (GIT) of many mammalian hosts. Their administration as probiotics in functional foods is currently a frequent practice, mainly because of their benefits to host health. It is therefore of interest to study the impact of administration of exogenous strains of Lactobacillus normally used as probiotics upon endogenous microbial populations. For this purpose, fecal and intestinal tissue samples were analyzed in a mouse model fed with a mixture of Lactobacillus plantarum and Lactobacillus casei isolated from commercially available dairy products. The murine intestinal microbiota was studied by means of cultivation-independent 16S rRNA gene-targeted techniques, namely denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis of clone libraries. Multivariate statistical analysis was used to integrate datasets obtained from the different techniques applied. Whereas no differences were detected in the composition of the overall fecal bacterial community, changes were observed for intestinal tissue samples. Moreover, an increase in the diversity of gut lactobacilli was observed in fecal as well as intestinal tissue samples when mice received the mixture of L. casei and L. plantarum.

Modulation of antibody-forming cell and mitogen-driven lymphoproliferative responses by dietary nucleotides in mice.

Several studies have demonstrated that dietary nucleotides play a role in maintaining T-cell dependent immunity. In this work, we investigated the effects of nucleotide supplementation of a nucleotide-free diet (NFD) on some immunity parameters in BALB/c mice. Twenty day old mice were maintained on diets for 30 days prior to use in experiments. The addition of nucleotide mixtures to NFD resulted in an increase in the response of hemolytic IgG-forming cells induced by previous immunization with sheep erythrocytes. When NFD was supplemented with single nucleotides, AMP, GMP, or UMP increased the IgG response, whereas CMP and IMP were without effect. GMP was the only nucleotide that increased the hemolytic IgM-forming cell response. Neither the contact hypersensitivity response to dinitrofluorobenzene nor the time of death after transplantation of a syngenic lymphoma was modified by nucleotide addition to NFD. The in vitro proliferative response of splenocytes to LPS was not affected by nucleotide supplementation of NFD, but the ConA-driven proliferative response was increased in mice fed NFD supplemented with nucleotide mixtures or with UMP. These data show that dietary mononucleotides stimulated at least some T-cell dependent immunity mechanisms. Moreover, these stimulatory effects may be obtained by supplementing a nucleotide-free diet with a mixture in which mononucleotides are at the same levels as in murine breast milk.

Effects of Telithromycin in In Vitro and In Vivo Models of Lipopolysaccharide-Induced Airway Inflammation

BACKGROUND The ketolide antibiotic telithromycin (TEL) exerts immunomodulatory and antiinflammatory effects in vitro and in a mouse model of septic shock. We studied the antiinflammatory activity of TEL in in vitro and in vivo models of airway inflammation induced by lipopolysaccharide (LPS). METHODS We measured the effects of TEL on the response of RAW 264.7 macrophages to LPS and of murine lung epithelial (MLE)-12 cells to supernatants of LPS-stimulated RAW 264.7 macrophages. Macrophage inflammatory protein (MIP)-2 and tumor necrosis factor (TNF)-alpha production, nuclear factor (NF)-kappaB activation, and apoptosis were determined. Acute airway inflammation was induced in untreated and TEL-treated BALB/c mice by nebulization with LPS. Total number of leukocytes, macrophages, and neutrophils, the protein concentration, and nitrite and cytokine levels were determined in the BAL fluid. RESULTS TEL inhibited in a dose-dependent manner the production of MIP-2 and TNF-alpha by LPS-stimulated RAW 264.7 macrophages, and the production of MIP-2 by MLE-12 epithelial cells to supernatants of LPS-stimulated RAW 264.7 macrophages. NF-kappaB activation was inhibited and apoptosis was increased in both cell lines by TEL. The LPS-induced influx of neutrophils in BAL fluid was decreased by TEL pretreatment. TEL also reduced protein, nitrite, MIP-2, and TNF-alpha levels in the BAL fluid of LPS-nebulized animals. CONCLUSIONS We have provided evidence that TEL exerts potent antiinflammatory effects in LPS-induced airways injury. We propose that TEL acts in the early phase of inflammation by reducing the release of inflammatory mediators through NF-kappaB inhibition, and in the later phase through enhancement of inflammatory cell apoptosis.

Changes in the immune functions and susceptibility to Listeria monocytogenes infection in mice fed dietary lipids

The direct examination of the effects that fish oil diets (composed of long‐chain n‐3 polyunsaturated fatty acids) exert on immune system function indicates a reduction of host natural resistance to infectious diseases mainly because of a suppression of immune function generated by the fatty acids contained in this diet. Here, we evaluated the concentration of IL‐12, IL‐4, prostaglandin E2 and leukotriene B4 in the serum from BALB/c mice receiving four different diets. Each group was fed a diet that differed only in the source of fat: a low‐fat diet (2.5% by weight), an olive oil diet (20% by weight), a fish oil diet (20% by weight) or a hydrogenated coconut oil diet (20% by weight). Mice were fed for 4 weeks and then infected with the intracellular pathogen Listeria monocytogenes. An initial reduction in the Th1‐type response as a result of a decrease in IL‐12p70 secretion, an inefficient action of IL‐4 (Th2‐type response) and no modification of pro‐inflammatory lipid‐mediator production could be, at least in part, the key events responsible for the inadequate elimination of L. monocytogenes from the spleens of mice fed a fish oil diet. Furthermore, our results suggest that the type of dietary lipids may affect the circulating concentration of IL‐12p70 and IL‐4, leading to a modulation in the protective cellular immune response to L. monocytogenes infection.

Orally administered Lactobacillus plantarum reduces pro-inflammatory interleukin secretion in sera from Listeria monocytogenes infected mice

Lactic acid bacteria have traditionally been thought to have immunomodulating effects. To verify this property, Lactobacillus plantarum was orally administered to mice (5 x 107 colony forming units (c.f.u.)), prior to infection with Listeria monocytogenes in order to evaluate the host resistance against an infectious micro-organism and to better define the influence of L. plantarum on such responses. Balb/c mice were treated daily with L. plantarum or received PBS (sham-treated mice as controls) for 4 weeks. Subsequently, mice were intravenously infected with a clinical isolate of L. monocytogenes. Our study revealed that the administration of L. plantarum did not significantly increase the survival (P = 0.13) of mice (fifteen in each group) after L. monocytogenes infection (106 c.f.u./ml), whereas a sub-lethal dose of L. monocytogenes (105 c.f.u./ml) was eliminated from liver and spleen 5 d after the challenge in both L. plantarum- and sham-treated mice (n 5). Nevertheless, the levels of IL-1beta and IL-6 from sera of orally administered L. plantarum were drastically reduced at 0, 4 (P < 0.01) and 6 d after L. monocytogenes infection, whereas TNF-alpha production was unaltered. In conclusion, administration of L. plantarum reduced pro-inflammatory IL production after challenge with L. monocytogenes, although it did not significantly impact the survival of mice. We speculate that L. plantarum could exert anti-inflammatory effects, which may represent an important model to reduce inflammatory disorders. Therefore, further studies in human subjects should determine the role of L. plantarum as an immunomodulatory micro-organism and its relationship in the host protection to pathogens.

Efficacy of a universal screening program for the prevention of neonatal group B streptococcal disease

Universal antepartum vaginal cultures for group B streptococcus (GBS) were initiated in a Spanish hospital in 1994 using Granada medium. Infants born to carriers were monitored closely, and blood, urine and mucocutaneous areas were cultured for GBS. Group B streptococcus was detected in 543 of 4,525 women (12 %). Of these, 454 gave birth vaginally, of whom 201 (44 %) received intrapartum ampicillin. Prophylaxis was not administered to 253 women (56 %). In this group, infants of 120 women were colonized and 1 case of neonatal GBS disease occurred. Using this protocol, most GBS carriers with risk factors received intrapartum prophylaxis. This protocol also led to early identification of colonized newborns.

Immunomodulation- in Mice by Experimental Infection with Yersinia enterocolitica

Intraperitoneal infection of mice with two strains of Yersinia enterocolitica resulted in an inflammatory response and immunomodulation which appeared to be related to the invasive properties of the bacteria. The primary antibody response to sheep erythrocytes was enhanced by noninvasive cultures of Y. enterocolitica (serotype O:4–33 grown at 22 C and at 37 C, and serotype O:3 grown at 37 C), when given at the same time or two days after the antigen (invasiveness was tested on HeLa cells). In contrast, invasive cultures of serotype O:3 grown at 22 C, injected three days before the antigen suppressed the antibody response; enhancement was caused by these cultures only when given on the day of immunization. Delayed‐type hypersensitivity to sheep erythrocytes was also suppressed by invasive cultures of Y. enterccolitica. These data indicate that the temperature of growth as well as some serotype‐linked factors play a role in immunomodulation by Y. enterocolitica.

Biological Response Modifier Activity of an Exopolysaccharide from Paenibacillus jamilae CP-7

ABSTRACT An extracellular polysaccharide was purified from culture supernatants of Paenibacillus jamilae CP-7, a gram-positive bacillus that was isolated from compost prepared with olive mill wastewaters. The extracellular polysaccharide was produced under aerobic conditions in a medium containing olive mill wastewaters (80% [vol/vol]). This exopolymer had a low level of acute toxicity when it is administered to BALB/c mice by the intraperitoneal route. Interesting immunomodulatory effects were detected when mice were given 10 mg of exopolysaccharide per kg of body weight; the proliferative responses of splenocytes to B-cell and T-cell mitogens were suppressed, the in vitro levels of production of gamma interferon and granulocyte-macrophage colony-stimulating factor by unstimulated and lipopolysaccharide-stimulated splenocytes were enhanced, and the levels of resistance to the intracellular pathogen Listeria monocytogenes was increased in mice. Also, the exopolysaccharide was able to induce lymphocyte proliferation in vitro. We conclude thatP. jamilae produces an exopolysaccharide with interesting immunomodulatory properties.

The Probiotic Bacterial Strain Lactobacillus fermentum D3 Increases In Vitro the Bioavailability of Ca, P, and Zn in Fermented Goat Milk

We determined calcium, magnesium, phosphorus and zinc levels in a total of 27 samples of commercial goat- and cow-milk fermented products and 9 samples of a goat-milk fermented product with addition of a probiotic bacterial strain, Lactobacillus fermentum D3, manufactured experimentally by our research group. Atomic absorption spectroscopy with flame atomization and UV/VIS spectrophotometry were used as analytic techniques. The results of an in vitro digestion process showed that the bioavailability of calcium, phosphorus, and zinc was significantly higher in our fermented milk containing the probiotic bacterial strain than it was in commercial goat-milk fermented products. Furthermore, our product showed a significantly higher bioavailability of calcium and zinc compared to goat- and cow-milk fermented products made with other microorganisms. We conclude that, in in vitro assays, strain D3 seems to increase the bioavailability of these minerals and that this new product may constitute a better source of bioavailable minerals compared to other products already on the market.

Effects of concentrated supernatants recovered from Lactobacillus plantarum on Escherichia coli growth and on the viability of a human promyelocytic cell line

Aims:  The ability of concentrated supernatants from Lactobacillus plantarum to produce a disruption of plasma membrane in eukaryotic and prokaryotic cells has been examined.