Inhibitory receptor expression on neonatal immune cells.

Neonates are born with quantitative and qualitative defects in both adaptive and innate immune responses. The immune system is regulated by several mechanisms, including the signalling of inhibitory receptors. Increased expression of inhibitory receptors may result in a higher threshold for activation and suppressed function of neonatal cells. The aim of this study was to determine whether the expression of seven inhibitory receptors is increased on neonatal immune cells compared to adult immune cells. In a healthy birth cohort, we examined the expression of seven inhibitory immune receptors on neonatal neutrophils, monocytes, natural killer (NK) cells, CD4(+) and CD8(+)T cells. The expression of leucocyte-associated immunoglobulin (Ig)-like receptor-1 (LAIR-1), signal inhibitory receptor on leucocytes-1 (SIRL-1), CD31, signal-regulatory protein alpha (SIRPα), Siglec-9, CD200R, immune receptor expressed on myeloid cells-1 (IREM-1) and the membrane-bound ligand CD200 was studied by flow cytometry on leucocytes in cord blood (n = 14), neonatal venous blood (n = 24) and adult venous blood (n = 22). Expression of LAIR-1, CD31 and CD200 was increased consistently across all neonatal T cell subsets. Neonatal monocytes exhibited decreased expression of LAIR-1 and IREM-1 compared to adults. Furthermore, cord blood and neonatal venous blood samples contained a distinct LAIR-1-positive neutrophil population, which was not detected in adult blood. We demonstrated distinct expression of inhibitory receptors on neonatal peripheral blood immune cells in a healthy birth cohort. This is the first evidence that inhibitory receptors play a role in regulation of the neonatal immune system. Consistently increased inhibitory receptor expression on T cells may be an important mechanism in preventing the development of allergy and autoimmunity.

Breastfeeding modulates neonatal innate immune responses: a prospective birth cohort study.

BACKGROUND: Neonatal Toll-like receptor (TLR) responses are biased toward Th2-polarizing responses at birth and rapidly mature toward more balanced responses during the first month of life. Postnatal TLR maturation may be guided by environmental exposure. AIMS: To determine the environmental determinants of neonatal TLR function. MATERIALS AND METHODS: A prospective birth cohort study was performed in 291 healthy term neonates. Mode of delivery, breastfeeding, birth month, siblings, pets and parental smoking were analyzed in relation to neonatal innate immune parameters at the age of 1 month. Whole blood concentrations of innate immune cells were measured by flow cytometry. In vitro TLR-mediated cytokine production was determined by ELISA. RESULTS: Breastfeeding was the major determinant of neonatal innate immunity, associated with 5 (31%) of neonatal innate immune parameters, of which the association with TLR7-mediated IL-10 production was most significant (76 pg/ml in breastfed neonates vs. 293 pg/ml in formula-fed neonates, p = 0.001). Of innate immune variables, TLR3-mediated IL-12p70 production was highly associated with environmental exposures (pets, breastfeeding and mode of delivery), whereas TLR9-mediated cytokine responses were not associated with any environmental factor. CONCLUSION: Neonatal innate immune responses are differentially modulated by environmental exposure in the first month of life. The protective effect of breastfeeding against subsequent infections and atopy might be explained by its innate immune modulatory effects in the first month of life.

The effects of selected probiotic strains on the development of eczema (the PandA study).

BACKGROUND: Modification of the intestinal microbiota by administration of probiotic bacteria may be a potential approach to prevent allergic disease. We aimed to study primary prevention of allergic disease in high-risk children by pre- and postnatal supplementation of selected probiotic bacteria. METHODS: In a double-blind, randomized, placebo-controlled trial, a mixture of probiotic bacteria selected by in-vitro experiments (Bifidobacterium bifidum, Bifidobacterium lactis, and Lactococcus lactis; Ecologic Panda) was prenatally administered to mothers of high-risk children (i.e. positive family history of allergic disease) and to their offspring for the first 12 months of life. RESULTS: Parental-reported eczema during the first 3 months of life was significantly lower in the intervention group compared with placebo, 6/50 vs 15/52 (P = 0.035). After 3 months, the incidence of eczema was similar in both groups. Cumulative incidence of parental-reported eczema at 1 and 2 years was 23/50 (intervention) vs 31/48 (placebo) and 27 (intervention) vs 34 (placebo), respectively. The number needed to treat was 5.9 at age 3 and 12 months and 6.7 at age 2 years. The intervention group was significantly more frequently colonized with higher numbers of Lc. lactis. Furthermore, at age 3 months, in vitro production of IL-5 (146 pg/ml vs 72 pg/ml; P = 0.04) was decreased in the probiotic-group compared with the placebo-group. CONCLUSIONS: This particular combination of probiotic bacteria shows a preventive effect on the incidence of eczema in high-risk children, which seems to be sustained during the first 2 years of life. In addition to previous studies, the preventive effect appears to be established within the first 3 months of life.

Effects of ethanol and other alkanols on passive proton influx in the yeast Saccharomyces cerevisiae.

Ethanol, isopropanol, propanol and butanol enhanced the passive influx of protons into deenergized cells of Saccharomyces cerevisiae. The influx followed first-order kinetics with a rate constant that increased exponentially with the alkanol concentration. The exponential enhancement constants increased with the lipid solubility of the alkanols, which indicated hydrophobic membrane regions as the target sites. While the enhancement constants were independent of pH over the range tested (3.3-5.0), the rate constants decreased linearly with increasing extracellular proton concentration, indicating the presence of an additional surface barrier against proton penetration, the effectiveness of which increased with protonation. The alkanols affected the acidification curves of energized yeast suspensions in such a way that the final pH values were linear functions of the alkanol concentrations. These results were consistent with a balance between active and passive proton movements at the final pH, the exponential enhancement constants calculated from the slopes being nearly identical with those obtained with deenergized cells. It was concluded that passive proton influx contributes to the kinetics of acidification in S. cerevisiae and that uncoupling contributes to the overall kinetics of alkanol-inhibited secondary active transport across the yeast plasma membrane.

Role of de novo protein synthesis in the interconversion of glucose transport systems in the yeast Pichia ohmeri.

Glucose-repressed cells of the yeast Pichia ohmeri IGC 2879 transported glucose by facilitated diffusion. Derepression led to the formation of a glucose/proton symport and the simultaneous reduction of the facilitated diffusion capacity by about 70%. Cycloheximide prevented this interconversion indicating its dependence on de novo protein synthesis (proteosynthetic interconversion). In buffer with 2% glucose the glucose/proton symport suffered irreversible inactivation while the facilitated diffusion system was simultaneously restored. This reverse interconversion process did not require de novo protein synthesis as indicated by its lack of sensitivity to cycloheximide (degradative interconversion). Thus the glucose/proton symport system appeared to consist of about 70% of the facilitated diffusion proteins turned silent through association with additional protein(s) the latter being sensitive to glucose-induced repression and glucose-induced inactivation.

Selection of probiotic bacteria for prevention of allergic diseases: immunomodulation of neonatal dendritic cells.

Modification of intestinal microbiota early in life by administration of probiotic bacteria may be a potential approach to prevent allergic disease. To select probiotic bacteria for in vivo purposes, we investigated the capacity of probiotic bacteria to interact with neonatal dendritic cells (DC) and studied the ensuing T cell polarizing effect. Immature DC were generated from cord blood-derived monocytes and maturation was induced by maturation factors (MF), lipopolysaccharide (LPS) plus MF and Bifidobacterium bifidum, B. infantis, Lactobacillus salivarius, Lactococcus lactis alone or combined with MF. After 12 days of co-culture with DC and Staphylococcus aureus enterotoxin B (SEB) as antigenic stimulus, cytokine production by autologous T cells was determined by intracellular cytokine staining. Additionally, cells were stimulated with CD3 and CD28 monoclonal antibodies and cytokines were measured in supernatants by multiplex assay. The probiotic strains induced partial maturation of DC. Full maturation of DC was induced for all strains tested when MF was added. The percentage of interleukin (IL)-4 producing T cells was lower in T cell cultures stimulated with B. bifidum matured DC compared to MF and LPS matured DC, which coincided with a higher percentage of interferon (IFN)-gamma-producing T cells. Furthermore, T cells stimulated by B. bifidum matured DC produced significantly more IL-10 compared to MF matured DC. Selected species of the Bifidobacterium genus prime in vitro cultured neonatal DC to polarize T cell responses and may therefore be candidates to use in primary prevention of allergic diseases.

Identification of strong interleukin-10 inducing lactic acid bacteria which down-regulate T helper type 2 cytokines.

BACKGROUND: Decreased exposure to microbial stimuli has been proposed to be involved in the increased prevalence of atopic disease. Such a relationship was indicated by enhanced presence of typical probiotic bacteria in the intestinal flora correlating with reduced prevalence of atopic disease. Recent clinical trials suggested that probiotic bacteria may decrease and prevent allergic symptoms, but which (different) species or strains may contribute is poorly understood. OBJECTIVE: We sought to select probiotic bacteria by their ability to modulate in vitro production of cytokines by peripheral blood mononuclear cells (PBMCs), to make a rational choice from available strains. METHODS: PBMCs, purified monocytes, and lymphocytes from healthy donors were co-cultured with 13 different strains of probiotic bacteria. The effect of lactic acid bacteria (LAB) on different cell populations and effects on cytokine production induced by the polyclonal T cell stimulator phytohaemagglutinin (PHA) was evaluated by measuring T helper type 1, T helper type 2 (Th2), and regulatory cell cytokines in culture supernatants by multiplex assay. RESULTS: PBMCs cultured with different strains produced large amounts of IL-10 and low levels of IL-12p70, IL-5, and IL-13. In PHA-stimulated PBMC cultures, the tested strains decreased the production of Th2 cytokines. Neutralizing IL-10 production resulted in partial to full restoration of Th2 cytokine production and concurred with an increase in pro-inflammatory cytokines such as IL-12p70 and TNF-alpha. Within the PBMCs, the CD14(+) cell fraction was the main source of IL-10 production upon interaction with LAB. CONCLUSION: Our results indicate that certain strains of lactobacilli and bifidobacteria modulate the production of cytokines by monocytes and lymphocytes, and may divert the immune system in a regulatory or tolerant mode. These specific strains may be favorable to use in prevention or treatment of atopic disease.

Interconversion and glucose-induced inactivation of glucose transport systems in Candida shehatae.

During starvation (derepression) glucose-grown cells of Candida shehatae IGC 3607 displayed total interconversion of facilitated diffusion of glucose into a glucose-proton symport, dependent on de novo protein synthesis (proteosynthetic interconversion). The reverse process, inactivation of the proton symport induced by glucose or 2-deoxyglucose, was not accompanied by reemergence of the facilitated diffusion function. The inactivation process had a rapid initial and a slow second phase. The rapid inactivation depended on the external sugar concentration and was reversible while the subsequent slow inactivation was irreversible and independent of the external concentration of the signalling sugar. Interaction of the latter with a surface receptor was indicated by the range of sugar concentrations that affected rapid inactivation.

Leucosporidium fellii sp. nov., a basidiomycetous yeast that degrades L(+)-tartaric acid.

A new species of basidiomycetous yeast Leucosporidium fellii was isolated from soil in Portugal on a selective L(+)-tartaric acid medium. This yeast is self-sporulating but forms dikaryotic hyphae with clamp connections and is presumably homothallic. It differs from the type strain of Leucosporidium scottii in its life cycle, assimilation pattern and guanine-cytosine content and from the other described Leucosporidium species by additional characteristics.

Yield and maintenance relations of yeast growth in the chemostat at superoptimal temperatures.

A model is proposed that accounts for the decreases in yield which occur in chemostat cultures of mesophilic yeasts at superoptimal growth temperatures. Two yield depressing effects were identified, one due to increased maintenance requirements by the viable fraction of the population, the other due to energy substrate dissipation by the nonviable function. The two effects are functions of the dilution rate, as is the fraction of nonviable cells. Experimental results were obtained on the yield, maintenance, and dissipation of energy substrate in a glucose-limited chemostat culture of a respiration-deficient mutant of Saccharomyces cerevisiae at 39 degrees C. The rates of glucose utilization for maintenance and for dissipation constituted, respectively, 33-28% and 15-9% of the total glucose utilization rate over the range of dilution rates tested (0.038-0.064 hr-1), while the yield varied over this range from 0.066-0.085 g of biomass (dry wt) per gram of glucose.

Dependence of the maximum temperature for growth of Saccharomyces cerevisiae on nutrient concentration.

Saccharomyces cerevisae was grown in a chemostat under glucose limitation at three superoptimal temperatures. In each steady state the specific growth rate was the sum of the dilution rate and the specific death rate, exponential death occurring with exponential growth. The specific death rate was a function of both the temperature and the concentration of the limiting nutrient. Each superoptimal temperature was characterized by a critical glucose concentration below which net growth was not possible. The critical glucose concentration increased with the temperature. Consequently the maximum temperature for growth was a function of the concentration of the limiting nutrient and approached the optimum temperature for growth with decreasing glucose concentrations.

Effects of chloramphenicol on the thermal profile of Saccharomyces cerevisiae.

Chloramphenicol decreased the maximum temperature for growth of a petite mutant of Saccharomyces cerevisiae, shifted the ARRHENIUS plot of thermal death to lower temperatures and shortened correspondingly, the ARRHENIUS plot of growth, while an associative thermal profile was maintained. At saturating concentrations (about 5 mg per ml) of chloramphenicol in liquid mineral medium with vitamins and glucose the final maximum temperature for growth was depressed from about 40 degrees C to about 37 degrees C. The results suggested that chloramphenicol acted in the mutant on targets other than mitochondrial ribosomes and that these targets are identical or associated with the death and Tmax sites of the yeast.

Cryptococcus yarrowii sp. nov., a novel yeast species from Portugal.

A novel species of the basidiomycetous genus Cryptococcus is described as Cr. yarrowii based on the study of an isolate from a decayed mushroom collected in Portugal. DNA-DNA homology with the type strain of the phenotypically similar species Cr. albidus was 10 +/- 2%.

The temperature profile of growth, death and yield of the starch-converting yeast Lipomyces kononenkoae.

A strain of Lipomyces kononenkoae earlier proposed for industrial starch bioconversion was found to have a dissociative temperature profile. The Arrhenius plot of sustained exponential growth displayed a single branch between the optimum (32-33 degrees C) and the maximum (about 35 degrees C) temperature for growth while the extrapolated Arrhenius plots of growth and thermal death intersected at a biologically non-significant value. The yield of L. kononenkoae on glucose did not decrease at supraoptimal temperatures while the associative yeast Saccharomyces cerevisiae suffered yield decreases above the optimum temperature for growth with increasing temperature.

Kinetics and activation energetics of death in Saccharomyces cerevisiae induced by sulfur dioxide.

Death of Saccharomyces cerevisiae induced by sulfur dioxide (K(2)S(2)O(2) was used as the SO(2) source) followed saturation kinetics. The enthalpy of activation of death was not affected by concentration over the range tested (5-150) mg/L of (K(2)S(2)O(2) at pH 3.4) and averaged 3.6 x 10(4) cal/mol as compared with 8.5 x 10(4) cal/mol for DeltaH of thermal death. The entropy of activation of death was hyperbolic function of the sulfur dioxide concentration, extrapolated at zero concentration to DeltaS(0) = 36.8 cal mol(-1) K(-1) and tended to DeltaDeltaS(max) = 13.2 cal mol(-1) K(-1) at saturating concentration, yielding a dissociation constant of 5.8 x 10(-1) M sulfur dioxide. As was predicted from these results, In K(d) (the specific rate of death induced by sulfur dioxide) was hyperbolic function of concentration under isothermic conditions and extrapolated to a finite value at zero concentration. The Arrhenuis plots and the DeltaS(+/-) plot versus concentration revealed the occurrence of substrate inhabitation of the death effect at high concentrations (above 60 mg/L K(2)S(2)O(2) at pH 3.4). A model is presented involving two types of receptor sites for sulfur dioxide on the cell surface, on directly connected with the death process, the other modulating its entropy of activation.

Effects of ethanol and other alkanols on the kinetics and the activation parameters of thermal death in Saccharomyces cerevisiae.

Ethanol, isopropanol, propanol, and butanol enhanced thermal death in Saccharomyces cerevisiae by increasing DeltaSdouble dagger, the entropy of activation of thermal death while DeltaHdouble dagger, the enthalpy of activation, was not significantly affected. The relation between DeltaSdouble dagger and alkanol concentration was linear with a different slope for each alkanol: DeltaS(double dagger) (X) = DeltaS(double dagger) (0) + C(A) (E)X, where X is the alkanol concentration and C(A) (E) the entropy coefficient for the aqueous phase defined as increase in entropy of activation per unit concentrations of the alkanol. C(A) (E) was correlated with the lipid-buffer partition coefficients of the alkanols while C(M) (E), the entropy coefficient for the lipid phase, was nearly identical for the four alkanol and averaged 37.6 entropy units per mole of alkanol per kilogram of membrane. As predicted by these results, the specific death rates (K(d)) at constant temperature were an exponential function of the alkanol concentration and behaved in agreement with the following equation: In K(X) (d) = In K(0) (d) + (C(A) (E)/R)X, where R is the gas constant. It was concluded that the alkanols enhanced thermal death through nonspecific action on membrane structure.