Effect of storage temperature on the keeping quality of commercially pasteurized milk

Pour étudier l'effet de la température sur la durée de conservation du lait pasteurisé, du lait fraîchemf:'nt pasteurisé et non homogénéisé provenant de deux laiteries industrielles, a été conservé à 7 et à 25°C. L'évolution du pH, des caractéristiques sensorielles, et de la flore totale du lait a été étudiée durant la période de conservation. L'altération du lait dont le taux microbien initial était de 10 3 UFC/ml a été observée entre le 5 ème et 6ème jour de conservation à 7°C. A 25°C, cette durée n'a été que de 20 à 24 heures. Au début de l'altération, le taux microbien a dépassé 10 7 UFC/ml. Les deux laits ont montré les mêmes signes d'altération à savoir la putréfaction, l'amertume, le caractère fruité, et la rancidité à 7°C; tandis qu'à 25°C, les défauts détectés étaient l'acidité, le surissement, et la rancidité. Les échantillons de lait ont montré également le signe de coagulation après ébullition, 2 heures avant l'altération à 25°C alors qu'à 7°C la coagulation du lait a eu lieu le même jour du début de l'altération. To study the effect of temperature on the shelf-life, freshly pasteurized and unhomogenized whole milk from two dairy plants was stored at 7 and 25°C. Microbial, sensory, and pH changes were monitored during storage. Milk of 1000 CFU/ml, as initial microbial count, showed evidence of spoilage between 5th and 6th day of storage at 7°C. When stored at 25°C, the shelf-life was only 20 to 24 hours. The microbial count at the time of spoilage exceeded 10000000 CFU/ml. Both brands ofmilk showed similar spoilage characteristics of putrid, bitter, fruity and rancid at 7°C and acid, sour, and rancid defects at 25°C. Milk samples showed clotting on boiling 2 hours before evidence of spoilage at 25°C and simultaneously with spoilage at 7°C

Identification of Candida glabrata genes involved in pH modulation and modification of the phagosomal environment in macrophages

notes: PMCID: PMC4006850types: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov'tCandida glabrata currently ranks as the second most frequent cause of invasive candidiasis. Our previous work has shown that C. glabrata is adapted to intracellular survival in macrophages and replicates within non-acidified late endosomal-stage phagosomes. In contrast, heat killed yeasts are found in acidified matured phagosomes. In the present study, we aimed at elucidating the processes leading to inhibition of phagosome acidification and maturation. We show that phagosomes containing viable C. glabrata cells do not fuse with pre-labeled lysosomes and possess low phagosomal hydrolase activity. Inhibition of acidification occurs independent of macrophage type (human/murine), differentiation (M1-/M2-type) or activation status (vitamin D3 stimulation). We observed no differential activation of macrophage MAPK or NFκB signaling cascades downstream of pattern recognition receptors after internalization of viable compared to heat killed yeasts, but Syk activation decayed faster in macrophages containing viable yeasts. Thus, delivery of viable yeasts to non-matured phagosomes is likely not triggered by initial recognition events via MAPK or NFκB signaling, but Syk activation may be involved. Although V-ATPase is abundant in C. glabrata phagosomes, the influence of this proton pump on intracellular survival is low since blocking V-ATPase activity with bafilomycin A1 has no influence on fungal viability. Active pH modulation is one possible fungal strategy to change phagosome pH. In fact, C. glabrata is able to alkalinize its extracellular environment, when growing on amino acids as the sole carbon source in vitro. By screening a C. glabrata mutant library we identified genes important for environmental alkalinization that were further tested for their impact on phagosome pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinization in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosylation may play a key role in alterations of phagosomal properties caused by C. glabrata.Deutsche ForschungsgemeinschaftNational Institutes for HealthWellcome TrustBBSR

Suboptimal Activation of Antigen-Specific CD4+ Effector Cells Enables Persistence of M. tuberculosis In Vivo

Adaptive immunity to Mycobacterium tuberculosis controls progressive bacterial growth and disease but does not eradicate infection. Among CD4+ T cells in the lungs of M. tuberculosis-infected mice, we observed that few produced IFN-γ without ex vivo restimulation. Therefore, we hypothesized that one mechanism whereby M. tuberculosis avoids elimination is by limiting activation of CD4+ effector T cells at the site of infection in the lungs. To test this hypothesis, we adoptively transferred Th1-polarized CD4+ effector T cells specific for M. tuberculosis Ag85B peptide 25 (P25TCRTh1 cells), which trafficked to the lungs of infected mice and exhibited antigen-dependent IFN-γ production. During the early phase of infection, ∼10% of P25TCRTh1 cells produced IFN-γ in vivo; this declined to <1% as infection progressed to chronic phase. Bacterial downregulation of fbpB (encoding Ag85B) contributed to the decrease in effector T cell activation in the lungs, as a strain of M. tuberculosis engineered to express fbpB in the chronic phase stimulated P25TCRTh1 effector cells at higher frequencies in vivo, and this resulted in CD4+ T cell-dependent reduction of lung bacterial burdens and prolonged survival of mice. Administration of synthetic peptide 25 alone also increased activation of endogenous antigen-specific effector cells and reduced the bacterial burden in the lungs without apparent host toxicity. These results indicate that CD4+ effector T cells are activated at suboptimal frequencies in tuberculosis, and that increasing effector T cell activation in the lungs by providing one or more epitope peptides may be a successful strategy for TB therapy

Regulation of Inflammatory Gene Expression in PBMCs by Immunostimulatory Botanicals

Many hundreds of botanicals are used in complementary and alternative medicine for therapeutic use as antimicrobials and immune stimulators. While there exists many centuries of anecdotal evidence and few clinical studies on the activity and efficacy of these botanicals, limited scientific evidence exists on the ability of these botanicals to modulate the immune and inflammatory responses. Using botanogenomics (or herbogenomics), this study provides novel insight into inflammatory genes which are induced in peripheral blood mononuclear cells following treatment with immunomodulatory botanical extracts. These results may suggest putative genes involved in the physiological responses thought to occur following administration of these botanical extracts. Using extracts from immunostimulatory herbs (Astragalus membranaceus, Sambucus cerulea, Andrographis paniculata) and an immunosuppressive herb (Urtica dioica), the data presented supports previous cytokine studies on these herbs as well as identifying additional genes which may be involved in immune cell activation and migration and various inflammatory responses, including wound healing, angiogenesis, and blood pressure modulation. Additionally, we report the presence of lipopolysaccharide in medicinally prepared extracts of these herbs which is theorized to be a natural and active component of the immunostimulatory herbal extracts. The data presented provides a more extensive picture on how these herbs may be mediating their biological effects on the immune and inflammatory responses

Alteration of deacylated lipopolysaccharide antagonistic properties by interaction with plasma factors

International audienceThe acyl poly(1,3)galactoside (APG) from Klebsiella pneumoniae is a bis-acylated Lipopolysaccharide(LPS) devoid of eater-linked fatty acids, APG interacts with CD14 and CD11b/CD18 on monocytes, This study addressed the role of serum proteins in the binding and functional properties of APG as a candidate LPS antagonist, In the absence of serum, APG did not induce tumor necrosis factor alpha (TNF-alpha) synthesis by human mononuclear cells and dose-dependently inhibited their activation induced by different LPS, Conversely, in the presence of 5% autologous plasma, APG activated cells and did not antagonize LPS, Serum decreased APG but not LPS binding to monocytes, Binding competition experiments indicated that APG and LPS competed for the same receptors in serum-free conditions but bound to different receptors in the presence of plasma, The data indicate that serum-dependent LPS receptors do contribute to LPS activation of monocytes but do hot recognize deacylated LPS analogues

The animal lectin galectin-3 interacts with bacterial lipopolysaccharides via two independent sites.

International audienceGalectin-3 is a beta-galactoside binding protein expressed by activated macrophages, epithelial cells, and certain other cell types. Galectin-3 has a C-terminal carbohydrate binding domain, an N-terminal part consisting of a proline- and glycine-rich repetitive domain, and a small N-terminal domain. Two independent LPS binding sites on galectin-3 were demonstrated by binding of biotinylated LPS to immobilized recombinant galectin-3. One appears to be the carbohydrate binding site in the C-terminal domain that confers binding of LPS from Klebsiella pneumoniae that has a beta-galactoside-containing polysaccharide chain. This binding is best demonstrated using galectin-3 immunocaptured by a mAb to the N-terminal part (M3/38) and is inhibited by lactose. In contrast, Salmonella minnesota R7 LPS (Rd mutant), which is devoid of beta-galactosides, appears to bind to a site within the N-terminal part of galectin-3. This interaction is best demonstrated using galectin-3 directly immobilized in wells, and it is inhibited by the Ab M3/38, but not by lactose. Binding inhibition by polymyxin B and the profile of inhibition by a panel of LPSs with different amounts of the inner and outer cores present indicate that this second binding site recognizes the lipid A/inner core region of LPSs

CD14 and CD11b mediate serum-independent binding to human monocytes of an acylpolygalactoside isolated from Klebsiella pneumoniae.

A water-soluble acylpolygalactosyl (APG) of 34 kDa was obtained from the Klebsiella pneumoniae membrane by alkaline hydrolysis and delipidation. APG comprises a poly(1,3)galactose chain, a core, and a lipid moiety made of a glucosamine disaccharide with two N-linked beta OH-myristates. The monocyte binding sites for APG were investigated by flow cytometry. Biotin-labelled APG (Biot-APG) bound to monocytes at 4 degrees C in the absence of serum, calcium, and magnesium. The binding was dose dependent, saturable, and displaced by unlabelled APG. Neither the polysaccharide chain present in APG-related molecules nor the PPi group or additional ester-linked myristates and palmitates were required for APG binding. The role of CD11b and CD14 was demonstrated by competitive inhibition with monoclonal antibodies and by the uptake of APG by these solubilized proteins. APG was rapidly internalized into monocytes at 37 degrees C while CD14 and CD11b/CD18 molecules were partially down-modulated. Lipopolysaccharides (LPS) from the same K. pneumoniae strain and from Escherichia coli and Salmonella minnesota partially competed for Biot-APG binding in the absence but not in the presence of serum. When altered by alkaline hydrolysis, those LPS became strong competitors for APG binding. It was concluded that alkaline hydrolysis of the K. pneumoniae membrane yielded molecules structurally related to LPS which bind to LPS membrane receptors in the absence of serum

Mycobacterial nucleoside diphosphate kinase blocks phagosome maturation in murine raw 264.7 macrophages

BACKGROUND: Microorganisms capable of surviving within macrophages are rare, but represent very successful pathogens. One of them is Mycobacterium tuberculosis (Mtb) whose resistance to early mechanisms of macrophage killing and failure of its phagosomes to fuse with lysosomes causes tuberculosis (TB) disease in humans. Thus, defining the mechanisms of phagosome maturation arrest and identifying mycobacterial factors responsible for it are key to rational design of novel drugs for the treatment of TB. Previous studies have shown that Mtb and the related vaccine strain, M. bovis bacille Calmette-Guérin (BCG), disrupt the normal function of host Rab5 and Rab7, two small GTPases that are instrumental in the control of phagosome fusion with early endosomes and late endosomes/lysosomes respectively. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that recombinant Mtb nucleoside diphosphate kinase (Ndk) exhibits GTPase activating protein (GAP) activity towards Rab5 and Rab7. Then, using a model of latex bead phagosomes, we demonstrated that Ndk inhibits phagosome maturation and fusion with lysosomes in murine RAW 264.7 macrophages. Maturation arrest of phagosomes containing Ndk-beads was associated with the inactivation of both Rab5 and Rab7 as evidenced by the lack of recruitment of their respective effectors EEA1 (early endosome antigen 1) and RILP (Rab7-interacting lysosomal protein). Consistent with these findings, macrophage infection with an Ndk knocked-down BCG strain resulted in increased fusion of its phagosome with lysosomes along with decreased survival of the mutant. CONCLUSION: Our findings provide evidence in support of the hypothesis that mycobacterial Ndk is a putative virulence factor that inhibits phagosome maturation and promotes survival of mycobacteria within the macrophage