Comparison of the Immunomodulatory Properties of Three Probiotic Strains of Lactobacilli Using Complex Culture Systems: Prediction for In Vivo Efficacy

Background

While the use of probiotics to treat or prevent inflammatory bowel disease (IBD) has been proposed, to this point the clinical benefits have been limited. In this report we analyzed the immunological activity of three strains of Lactobacillus to predict their in vivo efficacy in protecting against experimental colitis.

Methodology/Principal Findings

We compared the immunological properties of Lactobacillus plantarum NCIMB8826, L. rhamnosus GG (LGG), L. paracasei {"type":"entrez-nucleotide","attrs":{"text":"B21060","term_id":"2396114","term_text":"B21060"}}B21060 and pathogenic Salmonella typhimurium (SL1344). We studied the stimulatory effects of these different strains upon dendritic cells (DCs) either directly by co-culture or indirectly via conditioning of an epithelial intermediary. Furthermore, we characterized the effects of these strains in vivo using a Dextran sulphate sodium (DSS) model of colitis.We found that the three strains exhibited different abilities to induce inflammatory cytokine production by DCs with L. plantarum being the most effective followed by LGG and L. paracasei. L. paracasei minimally induced the release of cytokines, while it also inhibited the potential of DCs to both produce inflammatory cytokines (IL-12 and TNF-α) and to drive Th1 T cells in response to Salmonella. This effect on DCs was found under both direct and indirect stimulatory conditions – i.e. mediated by epithelial cells - and was dependent upon an as yet unidentified soluble mediator. When tested in vivo, L. plantarum and LGG exacerbated the development of DSS-induced colitis and caused the death of treated mice, while, conversely L. paracasei was protective.

Conclusions

We describe a new property of probiotics to either directly or indirectly inhibit DC activation by inflammatory bacteria. Moreover, some immunostimulatory probiotics not only failed to protect against colitis, they actually amplified the disease progression. In conclusion, caution must be exercised when choosing a probiotic strain to treat IBD.     

Pattern of cytokine and chemokine production by THP-1 derived macrophages in response to live or heat-killed Mycobacterium bovis bacillus Calmette-Guérin Moreau strain

Tuberculosis has great public health impact with high rates of mortality and the only prophylactic measure for it is the Mycobacterium bovisbacillus Calmette-Guérin (BCG) vaccine. The present study evaluated the release of cytokines [interleukin (IL)-1, tumour necrosis factor and IL-6] and chemokines [macrophage inflammatory protein (MIP)-1α and MIP-1β] by THP-1 derived macrophages infected with BCG vaccine obtained by growing mycobacteria in Viscondessa de Moraes Institute medium medium (oral) or Sauton medium (intradermic) to compare the effects of live and heat-killed (HK) mycobacteria. Because BCG has been reported to lose viability during the lyophilisation process and during storage, we examined whether exposing BCG to different temperatures also triggers differences in the expression of some important cytokines and chemokines of the immune response. Interestingly, we observed that HK mycobacteria stimulated cytokine and chemokine production in a different pattern from that observed with live mycobacteria.     

Insights into the Mechanisms of Protective Immunity against Cryptococcus neoformans Infection Using a Mouse Model of Pulmonary Cryptococcosis

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening pneumonia and meningoencephalitis in immune compromised individuals. Previous studies have shown that immunization of BALB/c mice with an IFN-γ-producing C. neoformans strain, H99γ, results in complete protection against a second pulmonary challenge with an otherwise lethal cryptococcal strain. The current study evaluated local anamnestic cell-mediated immune responses against pulmonary cryptococcosis in mice immunized with C. neoformans strain H99γ compared to mice immunized with heat-killed C. neoformans (HKC.n.). Mice immunized with C. neoformans strain H99γ had significantly reduced pulmonary fungal burden post-secondary challenge compared to mice immunized with HKC.n. Protection against pulmonary cryptococcosis was associated with increased pulmonary granulomatous formation and leukocyte infiltration followed by a rapid resolution of pulmonary inflammation, which protected the lungs from severe allergic bronchopulmonary mycosis (ABPM)-pathology that developed in the lungs of mice immunized with HKC.n. Pulmonary challenge of interleukin (IL)-4 receptor, IL-12p40, IL-12p35, IFN-γ, T cell and B cell deficient mice with C. neoformans strain H99γ demonstrated a requirement for Th1-type T cell-mediated immunity, but not B cell-mediated immunity, for the induction of H99γ-mediated protective immune responses against pulmonary C. neoformans infection. CD4⁺ T cells, CD11c⁺ cells, and Gr-1⁺ cells were increased in both proportion and absolute number in protected mice. In addition, significantly increased production of Th1-type/pro-inflammatory cytokines and chemokines, and conversely, reduced Th2-type cytokine production was observed in the lungs of protected mice. Interestingly, protection was not associated with increased production of cytokines IFN-γ or TNF-α in lungs of protected mice. In conclusion, immunization with C. neoformans strain H99γ results in the development of protective anti-cryptococcal immune responses that may be measured and subsequently used in the development of immune-based therapies to combat pulmonary cryptococcosis.     

Effects of the Oral Administration of Viable and Heat-Killed Streptococcus bovis HC5 Cells to Pre-Sensitized BALB/c Mice

Antimicrobial peptides have been suggested as an alternative to classical antibiotics in livestock production and bacteriocin-producing bacteria could be added to animal feeds to deliver bacteriocins in the gastrointestinal (GI) tract of ruminant and monogastric animals. In this study, viable (V) and heat-killed (HK) Streptococcus bovis HC5 cells were orally administered to pre-sensitized mice in order to assess the effects of a bacteriocin-producing bacteria on histological parameters and the immune response of the GI tract of monogastric animals. The administration of V and HK S. bovis HC5 cells during 58 days to BALB/c mice did not affect weight gain, but an increase in gut permeability was detected in animals receiving the HK cells. Viable and heat killed cells caused similar morphological alterations in the GI tract of the animals, but the most prominent effects were detected in the small intestine. The oral administration of S. bovis HC5 also influenced cytokine production in the small intestine, and the immune-mediated activity differed between V and HK cells. The relative expression of IL-12 and INF-γ was significantly higher in the small intestine of mice treated with V cells, while an increase in IL-5, IL-13 and TNF-α expression was only detected in mice treated with HK cells. Considering that even under a condition of severe challenge (pre-sensitization followed by daily exposure to the same bacterial immunogen) the general health of the animals was maintained, it appears that oral administration of S. bovis HC5 cells could be a useful route to deliver bacteriocin in the GI tract of livestock animals.     

Kinetics of IL-23 and IL-12 Secretion in Response to Toxoplasma gondii Antigens from THP-1 Monocytic Cells

IL-23 and IL-12 are structurally similar and critical for the generation of efficient cellular immune responses. Toxoplasma gondii induces a strong cell-mediated immune response. However, little is known about IL-23 secretion profiles in T. gondii-infected immune cells in connection with IL-12. We compared the patterns of IL-23 and IL-12 production by THP-1 human monocytic cells in response to stimulation with live or heat-killed T. gondii tachyzoites, or with equivalent quantities of either T. gondii excretory/secretory proteins (ESP) or soluble tachyzoite antigen (STAg). IL-23 and IL-12 were significantly increased from 6 hr after stimulation with T. gondii antigens, and their secretions were increased with parasite dose-dependent manner. IL-23 concentrations were significantly higher than those of IL-12 at the same multiplicity of infection. IL-23 secretion induced by live parasites was significantly higher than that by heat-killed parasites, ESP, or STAg, whereas IL-12 secretion by live parasite was similar to those of ESP or STAg. However, the lowest levels of both cytokines were at stimulation with heat-killed parasites. These data indicate that IL-23 secretion patterns by stimulation with various kinds of T. gondii antigens at THP-1 monocytic cells are similar to those of IL-12, even though the levels of IL-23 induction were significantly higher than those of IL-12. The detailed kinetics induced by each T. gondii antigen were different from each other.     

Antimicrobial and immunomodulatory activity of host defense peptides,clavanins and LL-37, in vitro: An endodontic perspective

Endodontic treatment is mainly based on root canal disinfection and its failure may be motivated by microbial resistance. Endodontic therapy can be benefitted by host defense peptides (HDPs), which are multifunctional molecules that act against persistent infection and inflammation. This study aimed to evaluate the antimicrobial, cytotoxic and immunomodulatory activity of several HDPs, namely clavanin A, clavanin A modified (MO) and LL-37, compared to intracanal medication Ca(OH)₂. HDPs and Ca(OH)₂ were evaluated by: (1) antimicrobial assays against Candida albicans and Enterococcus faecalis, (2) cytotoxicity assays and (3) cytokine tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, interleukin (IL)-1α, IL-6, IL-10 and IL-12 and nitric oxide (NO) production by RAW 264.7 cells incubated with or without heat-killed (HK) C. albicans or E. faecalis combined or not with interferon-γ. The minimum inhibitory concentration (MIC) was established only for E. faecalis (LL-37, 57 μM). Considering cytotoxicity, clavanin MO was able to reduce cell viability in many groups and demonstrated lowest LC₅₀. The Ca(OH)₂ up-regulated the production of MCP-1, TNF-α, IL-12 and IL-6 and down-regulated IL-1α, IL-10 and NO. Clavanins up-regulated the TNF-α and NO and down-regulated IL-10 production. LL-37 demonstrated up-regulation of IL-6 and TNF-α production and down-regulation in IL-10 and NO production. In conclusion, LL-37 demonstrated better antibacterial potential. In addition, Ca(OH)₂ demonstrated a proinflammatory response, while the HDPs modulated the inflammatory response from non-interference with the active cytokines in the osteoclastogenesis process, probably promoting the health of periradicular tissues.     

Lactobacillus paracasei DSMZ16671 Reduces Mutans Streptococci: A Short-Term Pilot Study

Reducing the burden of pathogenic mutans streptococci is a goal of oral health. Lactobacillus paracasei DSMZ16671, even after heat-killing, specifically co-aggregates mutans streptococci in vitro and retains this activity in human saliva. In rats, it reduces mutans streptococcal colonization of teeth and caries scores. This pilot study sought to assess the potential of heat-killed L. paracasei DSMZ16671 (pro-t-action^®) to reduce levels of salivary mutans streptococci in humans, using sugar-free candies as a delivery vehicle. A randomized, placebo-controlled, double-blind in vivo study of three groups examined the short-term effect of sugar-free candies containing 0 (placebo), 1, or 2 mg/candy piece of heat-killed L. paracasei DSMZ16671 on the levels of salivary mutans streptococci determined before and after consumption of the candies. The candies were consumed 4 times during 1.5 consecutive days. Compared to the placebo group, the test groups’ saliva had significantly reduced mutans streptococci as an immediate effect. These results suggest the use of heat-killed L. paracasei DSMZ16671 in suckable candies as a method to reduce mutans streptococci in the mouth and, thereby, caries risk. We think this a new concept and strategy for caries prevention and management.     

Lactobacillus plantarum Lp62 exerts probiotic effects against Gardnerella vaginalis ATCC 49154 in bacterial vaginosis

The severe side-effects elicited by conventional antibiotic therapy and the recurrence of Bacterial vaginosis-associated bacteria and bacterial resistance have led to the development of novel alternative therapies, among which genital probiotics are widely used. In this study, we aimed to evaluate the antimicrobial activities of Lactobacillus plantarum Lp62 and its supernatant against Gardnerella vaginalis, using both in vitro and in vivo approaches. In vitro assays were used to evaluate the viability of the strain and the antimicrobial activities of the supernatant in different pH ranges. An in vivo assay was performed on female BALB/c mice, wherein the animals were divided into eight groups: four control groups and four treated groups (for curative and preventive therapies). After infecting and treating the mice, the animals were killed to quantify the bacterial load using qPCR, evaluate leucocyte cellular response, determine vaginal cytokine levels and perform cytokine tissue gene expression. Our analyses revealed significant activity of the strain and its supernatant against G. vaginalis. Preliminary in vitro tests showed that the strain grew with equal efficiency in different pH ranges. Meanwhile, the presence of halo and inhibition of pathogen growth established the significant activity of the supernatant against G. vaginalis. We observed that both micro-organisms are resident bacteria of mouse microbiota and that the lactobacilli population growth was affected by G. vaginalis and vice versa. We also observed that the treated groups, with their low bacterial load, absence of leucocyte recruitment, reduced cytokine levels in the vaginal lavage and normalized cytokine gene expression, successfully controlled the infection.     

<Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> strain 06TCa19 suppresses inflammatory chemokine induced by <Emphasis Type="Italic">Helicobacter pylori</Emphasis> in human gastric epithelial cells

Helicobacter (H.) pylori infection is an important risk factor for gastric cancer that causes gastric inflammation. Inflammatory chemokines such as interleukin (IL)-8 and regulated on activation normal T cell expressed and secreted (RANTES) are elevated in the gastric mucosa by H. pylori. This study aimed to investigate the effects of Lactobacillus paracasei strain 06TCa19, a probiotic strain, on IL-8 and RANTES expression and production induced by H. pylori using human gastric epithelial cell lines. Strain 06TCa19 was shown to suppress H. pylori-mediated elevation of gene expression related to these chemokines in MKN45 cells. The strain also suppressed the increase in IL-8 and RANTES products induced by H. pylori in AGS cells as well as in MKN45 cells. In MKN45 cells inoculated with H. pylori, strain 06TCa19 was shown to downregulate the activation of NF-κB and p38 MAPK signaling pathways. Additionally, the level of the CagA virulence protein of H. pylori in the MKN45 cells and the number of viable H. pylori adhering to MKN45 cells decreased with the addition of strain 06TCa19. Moreover, the strain 06TCa19 notably increased lactic acid in the supernatant of MKN45 cells. Thus, lactic acid released from strain 06TCa19 might have inhibited the adhesion of H. pylori to MKN45 cells and prevented the insertion of H. pylori CagA into the cells, and elevation of IL-8 and RANTES genes and proteins might be suppressed by downregulating the NF-κB and p38 MAPK pathways. Therefore, use of strain 06TCa19 may prevent H. pylori-associated gastric inflammation.     

The Functional Response of B Cells to Antigenic Stimulation: A Preliminary Report of Latent Tuberculosis

Mycobacterium tuberculosis (M.tb) remains a successful pathogen, causing tuberculosis disease numbers to constantly increase. Although great progress has been made in delineating the disease, the host-pathogen interaction is incompletely described. B cells have shown to function as both effectors and regulators of immunity via non-humoral methods in both innate and adaptive immune settings. Here we assessed specific B cell functional interaction following stimulation with a broad range of antigens within the LTBI milieu. Our results indicate that B cells readily produce pro- and anti-inflammatory cytokines (including IL-1β, IL-10, IL-17, IL-21 and TNF-α) in response to stimulation. TLR4 and TLR9 based stimulations achieved the greatest secreted cytokine-production response and BCG stimulation displayed a clear preference for inducing IL-1β production. We also show that the cytokines produced by B cells are implicated strongly in cell-mediated communication and that plasma (memory) B cells (CD19⁺CD27⁺CD138⁺) is the subset with the greatest contribution to cytokine production. Collectively our data provides insight into B cell responses, where they are implicated in and quantifies responses from specific B cell phenotypes. These findings warrant further functional B cell research with a focus on specific B cell phenotypes under conditions of active TB disease to further our knowledge about the contribution of various cell subsets which could have implications for future vaccine development or refined B cell orientated treatment in the health setting.     

Lactobacillus plantarum 299v Prevents Caspase-Dependent Apoptosis In Vitro

Selective microbes used as probiotics can enhance epithelial cell protection. We have previously shown that a Lactobacillus plantarum strain 299v (Lp299v) has the ability to induce mucin genes. In the current study, we utilized a cytokine model of inflammation in cell culture to study the modulation of apoptosis by this probiotic. HT-29 cells were pre-incubated with the Lp299v or L. plantarum strain adh- (Lpadh-), a non-adherent derivative of Lp299v. Cells were challenged with a mixture of cytokines (TNF-α, IFN-γ, and IL-1a) to imitate conditions of inflammation. To assess for cell death, we evaluated TUNEL, multi-caspase, and caspase-3 and caspase-7 activity assays. There was a marked decrease in apoptosis as measured by TUNEL⁺ cells in samples pre-treated with Lp299v (18.7 ± 4.1%, p < 0.01) and Lpadh- (16.6 ± 3.2%, p < 0.05) prior to cytokine exposure when compared to cells (43.6 ± 6.2%) exposed to the cytokine mixture. Lp299v pre-incubation with HT-29 cells reduced caspase⁺ cells in the multi-caspase activity assay (3.6 ± 0.6%, p < 0.05) compared to cells exposed to cytokines (68.9 ± 5.1%) whereas Lpadh- did not (46.8 ± 17.5%, p > 0.05). Similarly, caspase-3, caspase-7 activity was also reduced by Lp299v. Selected probiotics may confer an exogenous protective effect at the mucosal–luminal interface for intestinal epithelial cells via alteration of caspase-dependent apoptotic pathways.     

Role of IL-17A on resolution of pulmonary C. neoformans infection

The current studies evaluated the role of interleukin (IL)-17A in the induction of protective immunity against pulmonary cryptococcosis in mice. Protection against pulmonary infection with C. neoformans strain H99γ was associated with increased IL-17A production. Signaling through the IFN-γ receptor (R) was required for increased IL-17A production, however, a Th17-type cytokine profile was not observed. Neutrophils were found to be the predominant leukocytic source of IL-17A, rather than T cells, suggesting that the IL-17A produced was not part of a T cell-mediated Th17-type immune response. Depletion of IL-17A in mice during pulmonary infection with C. neoformans strain H99γ resulted in an initial increase in pulmonary fungal burden, but had no effect on cryptococcal burden at later time points. Also, depletion of IL-17A did not affect the local production of other cytokines. IL-17RA^−/− mice infected with C. neoformans strain H99γ survived the primary infection as well as a secondary challenge with wild-type cryptococci. However, dissemination of the wild-type strain to the brain was noted in the surviving IL-17RA^−/− mice. Altogether, our results suggested that IL-17A may be important for optimal protective immune responsiveness during pulmonary C. neoformans infection, but protective Th1-type immune responses are sufficient for protection against cryptococcal infection.     

Role of NK cells in host defense against pulmonary type A Francisella tularensis infection

Pneumonic tularemia is a potentially fatal disease caused by the Category A bioterrorism agent Francisella tularensis. Understanding the pulmonary immune response to this bacterium is necessary for developing effective vaccines and therapeutics. In this study, characterization of immune cell populations in the lungs of mice infected with the type A strain Schu S4 revealed a significant loss in natural killer (NK) cells over time. Since this decline in NK cells correlated with morbidity and mortality, we hypothesized these cells contribute to host defense against Schu S4 infection. Depletion of NK cells prior to Schu S4 challenge significantly reduced IFN-γ and granzyme B in the lung but had no effect on bacterial burden or disease progression. Conversely, increasing NK cell numbers with the anti-apoptotic cytokine IL-15 and soluble receptor IL-15Rα had no significant impact on Schu S4 growth in vivo. A modest decrease in median time to death, however, was observed in live vaccine strain (LVS)-vaccinated mice depleted of NK1.1⁺ cells and challenged with Schu S4. Therefore, NK cells do not appear to contribute to host defense against acute respiratory infection with type A F. tularensis in vivo, but they play a minor role in protection elicited by LVS vaccination.     

Modulation of the immune response by probiotic strains in a mouse model of gluten sensitivity

Probiotic strains play an important role in modulating activities in the gut-associated lymphoid tissue. Elucidation of the mechanisms that mediate probiotic-driven immunomodulation may facilitate their therapeutic application for specific immune-mediated diseases or for prophylaxis. In this study, we explored the effect of different Lactobacillus spp. and Bifidobacterium lactis in transgenic mice expressing the human DQ8 heterodimer, a HLA molecule linked to Celiac Disease (CD). In vitro analysis on immature bone marrow-derived dendritic cells (iBMDCs) showed that all strains up-regulated surface B7-2 (CD86), indicative of DC maturation, however, with different intensity. No strain induced appreciable levels of IL-10 or IL-12 in iBMDCs, whereas TNF-α expression was essentially elicited by Lactobacillus paracasei and Lactobacillus fermentum. Interestingly, these strains were found also to increase the antigen-specific TNF-α secretion in vivo, following co-administration of probiotic bacteria in mice mucosally immunized with the gluten component gliadin. Together these findings highlighted the ability of probiotics to exert strain-specific inductive rather than suppressive effects both on the innate and adaptive immunity in a mouse model of food antigen sensitivity.     

Development of Probiotic Tablets Using Microparticles: Viability Studies and Stability Studies

Alternative vectors to deliver viable cells of probiotics, to those conferring limited resistance to gastrointestinal conditions, still need to be sought. Therefore the main goal of the study was to develop tablets able to protect entrapped probiotic bacteria from gastric acidity, thus providing an easily manufacturing scale-up dosage form to deliver probiotics to the vicinity of the human colon. Whey protein concentrate microparticles with Lactobacillus paracasei L26 were produced by spray-drying and incorporated in tablets with cellulose acetate phthalate and sodium croscarmellose. The viability of L. paracasei L.26 throughout tableting as well as its gastric resistance and release from the tablets were evaluated. Storage stability of L. paracasei L26 tablets was also performed by evaluation of viable cells throughout 60 days at 23°C and 33% relative humidity. A decrease of approximately one logarithmic cycle was observed after the acid stage and the release of L. paracasei L26 from the tablets occurred only after 4 h in the conditions tested. Microencapsulated L. paracasei L26 in tablets revealed some susceptibility to the storage conditions tested since the number of viable cells decreased 2 log cycles after 60 days of storage. However, the viability of L. paracasei L26 after 45 days of storage did not reveal significant susceptibility upon exposure to simulated gastrointestinal conditions. The developed probiotic tablets revealed to be potential vectors for delivering viable cells of L. paracasei L26 and probably other probiotics to persons/patients who might benefit from probiotic therapy.     

Induction of cellular and humoral responses by autoclaved and heat-killed antigen of Leishmania donovani in experimental visceral leishmaniasis

The potential of autoclaved and heat-killed antigen of Leishmania donovani to induce cell-mediated and humoral response has been evaluated in the present study. The vaccines were delivered thrice subcutaneously at an interval of 2 weeks. Two weeks after second booster, BALB/c mice were challenged with 10⁷ stationary phase promastigotes of L. donovani. Significant protection was achieved in immunized mice against L. donovani challenge with 69% to 76% and 59% to 64% reduction in parasite load in the liver and spleen respectively. Immunization induced significantly higher level of delayed type hypersensitivity (DTH) response in mice immunized with heat-killed antigen followed by autoclaved antigen. The immune response was assessed by quantifying Leishmania-specific antibodies and cytokine production. The antibody response was predominantly of IgG type with increased IgG2a production and lesser amount of IgM. The immunization preferentially stimulates the production of IFN-γ and IL-2 in splenocytes which suggests a Th1 type response with a concomitant down-regulation of IL-10 and IL-4. These results indicate a potential for the heat-killed and autoclaved antigen as a vaccine which could trigger cell-mediated immune response.     

Selecting Lactic Acid Bacteria for Their Safety and Functionality by Use of a Mouse Colitis Model

Studies showed that specific probiotics might provide therapeutic benefits in inflammatory bowel disease. However, a rigorous screening of new probiotics is needed to study possible adverse interactions with the host, particularly when intended for administration to individuals with certain health risks. In this context, the objective of this study was to investigate the role of three lactobacilli (LAB) on intestinal inflammation and bacterial translocation using variations of the mouse model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced acute colitis. We first compared the in vitro ability of LAB to survive gastrointestinal tract (GIT) conditions and their ability to persist in the GIT of mice following daily oral administration. As a control, we included a nonprobiotic Lactobacillus paracasei strain, previously isolated from an endocarditis patient. Feeding high doses of LAB strains to healthy and to TNBS-treated mice did not induce any detrimental effect or abnormal translocation of the bacteria. Oral administration of Lactobacillus salivarius Ls-33 had a significant preventive effect on colitis in mice, while Lactobacillus plantarum Lp-115 and Lactobacillus acidophilus NCFM did not. None of the three selected LAB strains translocated to extraintestinal organs of TNBS-treated mice. In contrast, L. paracasei exacerbated colitis under severe inflammatory conditions and translocated to extraintestinal organs. This study showed that evaluations of the safety and functionality of new probiotics are recommended. We conclude that not all lactobacilli have similar effects on intestinal inflammation and that selected probiotics such as L. salivarius Ls-33 may be considered in the prevention or treatment of intestinal inflammation.     

Host genetic background and the innate inflammatory response of lung to influenza virus

Many studies of influenza severity have focused on viral properties that confer virulence, whereas the contributory role of the host genetic background on infection severity remains largely unexplored. In this study, we measure the impact of inoculation with influenza virus in four strains of inbred mice - BALB/cByJ, C57BL/6 J, A/J, and DBA/2 J. To evaluate the extent to which responses are inherent to lung per se, as opposed to effects of the systemic response to lung infection, we also measured cytokines and chemokines in lung slices exposed to the virus in vitro. Finally, we evaluate the in vivo responses of recombinant inbred (RI) and select consomic strains of mice to search for genomic loci that contribute to phenotypic variance in response to influenza infection. We found marked variation among mouse strains after challenge with virus strain A/HKX31(H3N2), consistent with previous reports using more virulent strains. Furthermore, response patterns differ after in vivo versus in vitro exposure of lung to virus, supporting a predominant role of the systemic host inflammatory response in generating the strain differences. These results add to the body of information pointing to host genotype as a crucial factor in mediating the severity of influenza infections.     

Protection of ornamental gold fish Carassius auratus against Aeromonas hydrophila by treating Ixora coccinea active principles

Herbals such as Ixora coccinea, Daemia extensa and Tridax procumbens were selected to screen in vitro antibacterial and immunostimulant activity against the freshwater fish pathogen Aeromonas hydrophila using different organic polar and non-polar solvents. Initial screening results revealed that, ethyl acetate extracts and its purified fraction of I. coccinea was able to suppress the A. hydrophila strains at more than 15 mm of zone of inhibition and positive immunostimulant activity. The purified active fraction, which eluted from H40: EA60 mobile phase was structurally characterized by GC–MS analysis. Two compounds such as Diethyl Phthalate (1,2-Benzene dicarboxylic acid, monobutyl ester) and Dibutyl Phthalate were characterized using NIST database search. In order to study the in vivo immunostimulant influence of the compounds, the crude extracts (ICE) and purified fractions (ICF) were incorporated to the artificial diets at the concentration of 400 mg kg⁻¹ and fed to the ornamental gold fish Carassius auratus for 30 days. After termination of feeding experiment, they were challenged with highly virulent A. hydrophila AHV-1 which was isolated from infected gold fish and studied the survival, specific bacterial load reduction, serum biochemistry, haematology, immunology and histological parameters. The control diet fed fishes succumbed to death within five days at 100% mortality whereas ICE and ICF fed groups survived 60 and 80% respectively after 10 days. The diets also helped to decrease the Aeromonas load after challenge and significantly (P ≤ 0.01) improved the serum albumin, globulin and protein. The diets also helped to increase the RBC and haemoglobin level significantly (P ≤ 0.05) from the control group. Surprisingly the immunological parameters like phagocytic activity, serum bactericidal activity and lysozyme activity were significantly increased (P ≤ 0.001) in the experimental diets. Macrophages and erythrocytes were abundantly expressed in the treated groups and the present work concluded that, the Phthalate derivatives from I. coccinea helps to stimulate the immune system against A. hydrophila challenge in C. auratus.