Maria Jimenez-Valera

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.

Modification of immune response in mice by ciprofloxacin.

Some studies have suggested that the addition of ciprofloxacin to in vitro cultures of mitogen-stimulated lymphocytes exerts inhibitory effects on cell cycle progression and immunoglobulin (Ig) secretion. We tested the effects of this drug on some immunity parameters in BALB/c mice. Mice treated intraperitoneally with ciprofloxacin (10 mg/kg of body weight per day) for 3 consecutive days and immunized with sheep erythrocytes 24 h after the last injection showed significant suppression of hemolytic IgG-forming cells, whereas the response of IgM-forming cells remained unchanged. When treatment lasted 7 days the response of antibody-forming cells was not modified. When the 3-day treatment was started at 24 h after immunization with sheep erythrocytes, the response of IgM-forming cells was increased, whereas the response of IgG-forming cells was suppressed. Delayed-type hypersensitivity to sheep erythrocytes was significantly suppressed in animals that received the 3-day treatment with ciprofloxacin and were immunized subcutaneously 24 h after the last injection. In vitro proliferation of lymphocytes from ciprofloxacin-treated mice in response to either lipopolysaccharide or concanavalin A was also suppressed. Leukopenia and an increase in the level of granulocyte-macrophage colony-forming cells in bone marrow were also observed in ciprofloxacin-treated mice. These results, together with those from other reports, suggest that modification of the biological responses by ciprofloxacin is a complex phenomenon that may be influenced by several factors.

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.

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.

Telithromycin inhibits the production of proinflammatory mediators and the activation of NF-κB in in vitro-stimulated murine cells

Telithromycin is a ketolide antibiotic with anti-inflammatory properties. To investigate the mechanisms of these effects, we examined the in vitro immunomodulatory activity of telithromycin on murine splenocytes and the murine macrophage cell line RAW 264.7. Spleen cells from BALB/c-untreated mice and RAW 264.7 macrophages were cultured in the presence of telithromycin. Proliferation and apoptosis (colorimetric assay), and cytokine production (enzyme immunoassay) of spleen cells in response to lipopolysaccharide and concanavalin A (Con A), and nitric oxide (NO) (colorimetric assay) and cytokine production by lipopolysaccharide-stimulated RAW 264.7 cells were determined. Telithromycin moderately enhanced lymphocyte proliferation in response to lipopolysaccharide and Con A, and enhanced apoptosis induced by camptothecin in mitogen-stimulated splenocytes. Con A-induced IFN-gamma production was suppressed and lipopolysaccharide-induced IL-10 production was increased in spleen cell cultures with telithromycin. Lipopolysaccharide-induced secretion of NO and tumor necrosis factor-alpha (TNF-alpha) was suppressed by telithromycin in RAW 264.7 cultures. Lipopolysaccharide-induced activation of NF-kappaB transcription factor and TNF-alpha promoter in RAW 264.7 macrophages transitorily transfected with luciferase reporter constructs was also inhibited by the ketolide. The suppressive effect of telithromycin on NF-kappaB activation was confirmed by Western blot and enzyme immunoassay. These results suggest that telithromycin exerts anti-inflammatory activity mediated by the inhibition of activation of NF-kappaB.

Experimental Infection of Mice with Yersinia enterocolitica Serotype O9 by Oral and Parenteral Routes: Spreading and Enterotropism of Virulent Yersiniae

Abstract. An isogenic pair of Yersinia enterocolitica serotype O9 strains, with and without virulence plasmid, was used to study the plasmid role in the infection of BALB/c mice by oral, intraperitoneal, and intravenous routes. The plasmid-bearing strain, but not its plasmid-less derivative, caused enteric infection after challenge by all three routes. The virulence plasmid did not influence the peritoneal clearance of yersiniae, but only the plasmid-bearing yersiniae were able to move from the peritoneal cavity to the bloodstream, and thus they spread to spleen and liver. Moreover, plasmid-bearing yersiniae were able to move from the liver to the gallbladder, and they shed in bile into the intestine. Western blot analysis of antibody responses to chromosomally encoded outer membrane proteins revealed similar patterns with sera from mice challenged with each one of the two strains by intraperitoneal route. In contrast, only the plasmid-bearing strain elicited an antibody response to these antigens in mice challenged by oral route. Although mice experimentally infected with plasmid-bearing O9 yersiniae developed an enteric infection, irrespective of the inoculation route, differences between the first steps in infection by oral and parenteral routes may be important, especially when the infection model is used as an approach to study the yersinia-host interactions.

A Lactobacillus plantarum strain isolated from kefir protects against intestinal infection with Yersinia enterocolitica O9 and modulates immunity in mice.

Lactobacillus plantarum C4, previously isolated from kefir and characterized as a potential probiotic strain, was tested for its protective and immunomodulatory capacity in a murine model of yersiniosis. The inoculation of BALB/c mice with a low pathogenicity serotype O9 strain of Yersinia enterocolitica results in a prolonged intestinal infection with colonization of Peyers patches. Pretreatment with C4 was without effect on fecal excretion of yersiniae, but shortened the colonization of Peyers patches. This protective effect was associated with pro-inflammatory status in the intestinal mucosa (TNF-α production in infected mice was increased by C4) and an increase in total IgA secretion. At a systemic level, C4 did not promote a pro-inflammatory response, although production of the immunoregulatory cytokine IFN-γ was enhanced. These findings suggest that L. plantarum C4 can increase resistance to intestinal infections through its immunomodulatory activity.