Betaine reduces the expression of inflammatory adipokines caused by hypoxia in human adipocytes.

Obesity is characterised by a state of chronic low-grade inflammation and the elevated circulating and tissue levels of inflammatory markers, including inflammation-related adipokines, released from white adipose tissue. The expression and release of these adipokines generally rises as the adipose tissue expands and hypoxic conditions start to develop within the tissue. Here, the effect of betaine, a trimethylglycine having a biological role as an osmolyte and a methyl donor, on the expression of inflammation-related markers was tested in human adipocytes under hypoxia. Differentiated adipocytes were cultivated under low (1 %) oxygen tension for 8-20 h. The expression of different adipokines, including IL-6, leptin, PPARγ, TNF-α and adiponectin, was measured by quantitative PCR by determining the relative mRNA level from the adipocytes. Hypoxia, in general, led to a decrease in the expression of PPARγ mRNA in human adipocytes, whereas the expression levels of leptin and IL-6 mRNA were substantially increased by hypoxia. The cultivation of adipocytes under hypoxia also led to a reduction in the expression of TNF-α mRNA. The results showed that hypoxia increased the relative quantification of leptin gene transcription, and that betaine (250 µmol/l) reduced this effect, caused by low oxygen conditions. Under hypoxia, betaine also reduced the mRNA level of the pro-inflammatory markers IL-6 and TNF-α. These results demonstrate that the extensive changes in the expression of inflammation-related adipokines in human adipocytes caused by hypoxia can be diminished by the presence of physiologically relevant concentrations of betaine.

The effect of feeding essential oils on broiler performance and gut microbiota.

1. In this study the effect of a blend of essential oils (EO) comprising 15 g/tonne thymol and 5 g/tonne cinnamaldehyde on the performance and intestinal microbiota of broilers was investigated. 2. A total of 720 male Ross broilers were divided into two dietary treatments with 12 replicate pens per treatment. Broilers were given a control soybean-wheat-based diet with or without added EO in two diet phases (0-21 d and 22-42 d). 3. The blend of EO increased body weight gain of broilers from 0 to 42 d by 45%. 4. Caecal microbiota were affected by the EO blend; in particular increases in the proportions of Lactobacillus and Escherichia coli at 41 d was observed. 5. The EO blend had major effects on caecal metabolites. The proportion of caecal butyrate at 20 and 41 d of age increased, whereas the proportion of caecal acetic acid at 20 d, and propionic acid and isovaleric acid at 41 d, decreased with the EO blend. In addition, the caecal proportion of spermine increased and tyramine decreased at 41 d of age with the EO treatment. 6. The present study shows that EO supplementation exerts a positive effect on intestinal microbiota with a concomitant enhancement in growth performance. The study suggests that modulation of broiler gut microbiota composition and activity through the administration of EO offers an effective means for improving broiler performance.

Xylo-oligosaccharides enhance the growth of bifidobacteria and Bifidobacterium lactis in a simulated colon model.

A semi-continuous, anaerobic colon simulator, with four vessels mimicking the conditions of the human large intestine, was used to study the fermentation of xylo-oligosaccharides (XOS). Three XOS compounds and a xylan preparation were fermented for 48 hours by human colonic microbes. Fructo-oligosaccharides (FOS) were used as a prebiotic reference. As a result of the fermentation, the numbers of Bifidobacterium increased in all XOS and xylan simulations when compared to the growth observed in the baseline simulations, and increased levels of Bifidobacterium lactis were measured with the two XOS compounds that had larger distribution of the degree of polymerisation. Fermentation of XOS and xylan increased the microbial production of short chain fatty acids in the simulator vessels; especially the amounts of butyrate and acetate were increased. XOS was more efficient than FOS in increasing the numbers of B. lactis in the colonic model, whereas FOS increased the Bifidobacterium longum numbers more. The selective fermentation of XOS by B. lactis has been demonstrated in pure culture studies, and these results further indicate that the combination of B. lactis and XOS would form a successful, selective synbiotic combination.

Analysis of the human intestinal epithelial cell transcriptional response to Lactobacillus acidophilus, Lactobacillus salivarius, Bifidobacterium lactis and Escherichia coli.

The complex microbial population residing in the human gastrointestinal tract consists of commensal, potential pathogenic and beneficial species, which are probably perceived differently by the host and consequently could be expected to trigger specific transcriptional responses. Here, we provide a comparative analysis of the global in vitro transcriptional response of human intestinal epithelial cells to Lactobacillus acidophilus NCFM™, Lactobacillus salivarius Ls-33, Bifidobacterium animalis subsp. lactis 420, and enterohaemorrhagic Escherichia coli O157:H7 (EHEC). Interestingly, L. salivarius Ls-33 DCE-induced changes were overall more similar to those of B. lactis 420 than to L. acidophilus NCFM™, which is consistent with previously observed in vivo immunomodulation properties. In the gene ontology and pathway analyses both specific and unspecific changes were observed. Common to all was the regulation of apoptosis and adipogenesis, and lipid-metabolism related regulation by the probiotics. Specific changes such as regulation of cell-cell adhesion by B. lactis 420, superoxide metabolism by L. salivarius Ls-33, and regulation of MAPK pathway by L. acidophilus NCFM™ were noted. Furthermore, fundamental differences were observed between the pathogenic and probiotic treatments in the Toll-like receptor pathway, especially for adapter molecules with a lowered level of transcriptional activation of MyD88, TRIF, IRAK1 and TRAF6 by probiotics compared to EHEC. The results in this study provide insights into the relationship between probiotics and human intestinal epithelial cells, notably with regard to strain-specific responses, and highlight the differences between transcriptional responses to pathogenic and probiotic bacteria.

Xylo-oligosaccharides and lactitol promote the growth of Bifidobacterium lactis and Lactobacillus species in pure cultures.

The current screening study aimed at identifying promising prebiotic and synbiotic candidates. The fermentation of xylo-oligosaccharides, xylan, galacto-oligosaccharide, fructo-oligosaccharide, polydextrose, lactitol, gentiobiose and pullulan was investigated in vitro. The ability of these established and potential prebiotic candidates to function as a sole carbon source for probiotic (Bifidobacterium and Lactobacillus), intestinal and potential pathogenic microbes (Eubacterium, Bacteroides, Clostridium, Escherichia coli, Salmonella, and Staphylococcus) was assessed in pure cultures. Xylo-oligosaccharides were fermented with high specificity by the tested Bifidobacterium lactis strains and lactitol by lactobacilli, whereas galacto-oligosaccharides, fructo-oligosaccharides and gentiobiose were utilised by a larger group of microbes. Xylan, polydextrose and pullulan were utilised to a limited extent by only a few of the tested microbes. The results of this screening study indicate that xylo-oligosaccharides and lactitol support the growth of a limited number of beneficial microbes in pure cultures. Such a high degree of specificity has not been previously reported for established prebiotics. Based on these results, the most promising prebiotics and synbiotic combinations can be selected for further testing.

Synbiotic effects of lactitol and Lactobacillus acidophilus NCFM™ in a semi-continuous colon fermentation model.

The effects of Lactobacillus acidophilus NCFM™, lactitol, and the combination of lactitol and L. acidophilus NCFM™ were studied with a semi-continuous colon fermentation simulation; consisting of compartments mimicking, ascending, transverse, descending and sigmoid colon and their conditions with faecal inoculation. L. acidophilus NCFM™ was detected throughout the colon simulator. Lactitol was utilised early on by the microbes in the proximal part of the simulator. Lactitol increased the total numbers of microbes and bifidobacteria, and decreased clostridia cluster IV, while L. acidophilus NCFM™ alone decreased the numbers of clostridia cluster XIV. Combination treatment increased the numbers of bifidobacteria. Furthermore, concentrations of acetic acid, butyric acid and the sum of total short-chain fatty acids were increased by both lactitol-including treatments. The treatment with L. acidophilus NCFM™ alone increased the concentration of propionic acid and butyric acid. L. acidophilus NCFM™ tended to increase the total concentrations of biogenic amines, while lactitol suppressed production of biogenic amines also in the presence of L. acidophilus NCFM™. True synergistic effects are suggested in stimulation of the production of butyrate, an important microbial metabolite for colon health. In conclusion, lactitol as well as the combination of lactitol and L. acidophilus NCFM™ were found to exhibit complementary beneficial effects on the colon microbial composition and activity.

Panose, a new prebiotic candidate.

AIMS: To investigate the prebiotic potential of two novel candidates, sophorose and panose, with in vitro methods. METHODS AND RESULTS: The growth of single microbial strains was first assessed for both substrates in pure cultures, and panose was further analysed in the simulated colon model with mixed human faecal culture. Quantitative PCR and flow cytometry were used to determine the microbial group and strain densities after the simulated colonic fermentation of panose, and chromatographic methods were utilized to analyse metabolite concentrations. In pure cultures, sophorose and panose were both fermented only by few beneficial strains, and in the colon simulator, panose gave a significant increase in the numbers of Bifidobacterium and Bifidobacterium lactis, concomitantly decreasing Bacteroides group. Butyrate and acetate production was significantly increased together with decreased markers of protein fermentation as a result of panose fermentation. CONCLUSIONS: Panose had bifidogenic activities in vitro, and these potential beneficial effects should be further assessed in vitro and in vivo. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study has provided the first data on pure panose fermentation by the endogenous microbiota and extends our knowledge of the selective fermentation of oligosaccharides by the intestinal microbes.

The effects of polydextrose and xylitol on microbial community and activity in a 4-stage colon simulator.

This study focused on the effects of candidate prebiotics polydextrose (PDX) and xylitol on the microbial community and its metabolic activity in a colon simulator. A semicontinuous, anaerobic culture system was used with 4 vessels mimicking the conditions in the human large intestine from proximal to distal colon. Bacterial inocula for the independent simulations were obtained from fecal samples of different donors. Synthetic medium, mimicking the contents of the small intestine, containing either 2% of the prebiotic candidate or no added carbohydrates as a control, was fed to the system. After 48 h of simulation samples were collected and analyzed. A sustained degradation of polydextrose throughout the colon model and a more rapid degradation of xylitol were observed. The fermentation of both compounds was characterized by a significantly increased production of short-chain fatty acids (SCFA). Polydextrose increased the concentrations of all SCFA, especially acetate and propionate, and xylitol especially the concentration of butyrate. Branched-chain fatty acids (BCFA) levels decreased significantly as a result of polydextrose and xylitol supplementation, whereas biogenic amine levels remained mostly unchanged. Thus, a beneficial shift in the metabolic patterns of the colon microbes was measured with both of the tested products. These in vitro studies provide evidence to the prebiotic characteristics of polydextrose; also, further beneficial properties of xylitol were demonstrated in the colon model.

Intron 1 retaining cyclooxygenase 1 splice variant is induced by osmotic stress in human intestinal epithelial cells.

The biological roles of intron 1 retaining cyclooxygenase (Cox) 1 splice variants Cox-3 and PCox-1a (Cox-1ir) are not known. In humans, Cox-3 transcription has previously been shown to occur in the brain and in the aorta. However, conclusive evidence regarding the existence of a human Cox-3 protein is lacking. We studied the expression of intron 1 retaining cyclooxygenase 1 splice variants in the human colon cancer cell line Caco-2 and in human colonic tissue samples. In Caco-2 cells, their transcription was induced up to 47-fold by osmotic stress. The corresponding protein, however, could not be detected by Western blotting. In human colonic tissue samples derived from intact and inflamed areas, a low level of Cox-1ir mRNA (1500 +/- 1280 copies per 100 ng total RNA; mean+/-standard deviation; n = 20) was also found. In Caco-2 cells, induction of Cox-1ir under osmotic stress was reversed by addition of the organic osmolyte betaine. Under hypertonic but not under isotonic conditions, splice variant-specific degradation of Cox-1ir mRNA using RNA interference resulted in increased production of fully spliced Cox-1 and Cox-2 mRNA (P = 0.002). In summary, our results indicate that the intron 1 retaining Cox-1 splice variant RNA molecules are expressed by human intestinal epithelial cells in a controlled manner, are most likely not translated and play a regulatory role in the cyclooxygenase mediated epithelial osmoregulation.

Autoimmunity to glutamic acid decarboxylase in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED).

Antibodies to glutamic acid decarboxylase (GAD) occur frequently in patients with APECED, although clinical insulin-dependent diabetes mellitus (IDDM) is seen only in a subgroup of the patients. We studied the cellular immunity to GAD, antibodies to GAD and their association with the HLA DQB1 risk alleles for IDDM in patients with APECED. Proliferation responses to GAD were enhanced in the patients with APECED when compared with the control subjects (P = 0.004), but autoimmunity to GAD was not associated with IDDM in APECED. The levels of interferon-gamma (IFN-gamma) secreted by GAD-stimulated T cells were higher in the patients than in control subjects (P = 0. 001). A negative correlation (r = - 0.436, P = 0.03) existed between the antibody levels and the stimulation indices (SIs) to GAD. In 14 non-diabetic patients no difference in insulin secretion was observed in intravenous glucose tolerance test (IVGTT) between the patients with and without T cell reactivity to GAD. We conclude that cellular immunity to GAD detected as T cell proliferation response to GAD or IFN-gamma secretion by GAD-stimulated T cells was frequent in patients with APECED (69%) and was not restricted to the patients with clinically detectable beta-cell damage.

Acquired immunity to Chlamydia pneumoniae is dependent on gamma interferon in two mouse strains that initially differ in this respect after primary challenge.

The role of gamma interferon (IFN-gamma) in a Chlamydia pneumoniae mouse model was studied by in vivo neutralization in two inbred mouse strains. During primary C. pneumoniae infection, neutralization of IFN-gamma increased both the numbers of bacteria and the pneumonia score in the lungs of C57BL/6 mice but not BALB/c mice. During reinfection, the bacterial counts in the lungs were increased by IFN-gamma neutralization in both mouse strains. Thus, the effect of IFN-gamma neutralization was dependent on the genetic background in primary infection. However, IFN-gamma appeared to be equally important in both mouse strains during reinfection.

Immunity to Chlamydia pneumoniae induced by vaccination with DNA vectors expressing a cytoplasmic protein (Hsp60) or outer membrane proteins (MOMP and Omp2).

Immune responses induced by intramuscular DNA immunization with Chlamydia pneumoniae genes coding for the major outer membrane protein (MOMP), cysteine-rich outer membrane protein 2 (Omp2) or the heat shock protein 60 (Hsp60) were studied. BALB/c mice were vaccinated intramuscularly three times at 3-week intervals and challenged intranasally 2 weeks after the last injection. Immunization with pmomp or phsp60 showed 1.2-1.5 log reduction in the mean lung bacterial counts after the challenge. Specific antibodies were detected only in sera of the mice immunized with pomp2 and phsp60. Although immunization with pomp2 resulted in a strong serum antibody response against Omp2 protein, it failed to protect the mice. Immunization with any of the three vaccines did not reduce the severity of histologically assessed pneumonia, but resulted in significantly higher lymphoid reaction in the lung indicating immunological memory.

Depletion of CD8+ cells abolishes memory in acquired immunity against Chlamydia pneumoniae in BALB/c mice.

The importance of T cells in Chlamydia pneumoniae infection in mice was assessed by comparing wild-type BALB/c mice with nude mice and mice depleted in vivo of either CD4+ or CD8+ T cells. Whereas wild-type mice cleared the primary infection in 3 weeks, nude mice were only able to restrict the infection and could not clear it during the observation period of 56 days. Nude mice exhibited a greater number of macrophages in their lungs and the pulmonary cells secreted a higher level of tumour necrosis factor-alpha (TNF-alpha) than wild-type mice. Depletion of CD4+ cells did not change the overall infection kinetics of the primary infection. However, depletion of CD8+ cells resulted in a slightly impaired clearance of the bacteria in the late stages of primary infection. To assess the role of the two T-cell subsets in the acquired immunity that develops during primary infection in wild-type BALB/c mice, in vivo depletions were performed during reinfection. Prior to reinfection, immunocompetent wild-type mice were infected and natural immunity was allowed to form. During reinfection, depletion of CD4+ cells did not have any effect on infection kinetics, whereas depletion of CD8+ cells abolished the protection, reverting the infection kinetics and bacterial load to the same levels found in wild-type mice during primary infection. These results show that T cells are necessary for clearing C. pneumoniae infection in mice. Furthermore, whereas neither of the two main T-cell subsets, separately, were essential for clearance of primary infection, the induced protective immunity was strongly CD8 dependent.

Mononuclear cell subpopulations in preterm and full-term neonates: independent effects of gestational age, neonatal infection, maternal pre-eclampsia, maternal betamethason therapy, and mode of delivery.

Blood samples from 29 preterm (24-32 weeks of gestation) and 21 full-term (37-42 weeks of gestation) neonates were analysed for surface markers of lymphocyte subtypes and macrophages, and the effects of gestational age, neonatal infection, maternal pre-eclampsia, maternal betamethason therapy and mode of delivery were assessed with multiple regression analysis. Gestational age alone had few independent effects (increase in CD3+, CD8+CD45RA+, and CD11alpha+ cells, and decrease in CD14+, HLA-DR- cells) during the third trimester on the proportions of the immune cell subtypes studied. Neonatal infection and mother's pre-eclampsia had the broadest and very opposite kinds of effects on the profile of immune cells in the blood. Infection of the neonate increased the proportions of several 'immature' cells (CD11alpha-CD20+, CD40+CD19-, and CD14+HLA-DR-), whereas mother's pre-eclampsia decreased the proportions of naive cell types (CD4+CD8+, CD5+CD19+). In addition, neonatal infection increased the proportion of T cells (CD3+, CD3+CD25+, and CD4+/CD8+ ratio, and CD45RA+ cells), while maternal pre-eclampsia had a decreasing effect on the proportion of CD4+ cells, CD4+/CD8+ ratio, and proportions of CD11alpha+, CD14+ and CD14+HLA-DR+ cells. Maternal betamethason therapy increased the proportion of T cells (CD3+) and macrophages (CD14+, CD14+HLA-DR+), but decreased the proportion of natural killer (NK) cells. Caesarean section was associated with a decrease in the proportion of CD14+ cells. We conclude that the 'normal range' of proportions of different mononuclear cells is wide during the last trimester; further, the effect of gestational age on these proportions is more limited than the effects of other neonatal and even maternal factors.

Local immune responses to Chlamydia pneumoniae in the lungs of BALB/c mice during primary infection and reinfection.

Cell-mediated immune (CMI) responses play a major role in protection as well as pathogenesis of many intracellular bacterial infections. In this study, we evaluated the infection kinetics and assessed histologically the lymphoid reactions and local, in vitro-restimulated CMI responses in lungs of BALB/c mice, during both primary infection and reinfection with Chlamydia pneumoniae. The primary challenge resulted in a self-restricted infection with elimination of culturable bacteria by day 27 after challenge. A mild lymphoid reaction characterized the pathology in the lungs. In vitro CMI responses consisted of a weak proliferative response and no secretion of gamma interferon (IFN-gamma). The number of lung-derived mononuclear cells increased substantially during the primary infection; the largest relative increase was observed in B cells (B220(+)). After reinfection, the number of lung-derived mononuclear cells increased further, and the response consisted mainly of T cells. The reinfection was characterized in vivo by significant protection from infection (fewer cultivable bacteria in the lungs for a shorter period of time) but increased local lymphoid reaction at the infection site. In vitro, as opposed to the response in naive mice, acquired immunity was characterized by a strongly Th1-biased (IFN-gamma) CMI response. These results suggest that repeated infections with C. pneumoniae may induce Th1-type responses with similar associated tissue reactions, as shown in C. trachomatis infection models.

Expansion of a novel pulmonary CD3(-) CD4(+) CD8(+) cell population in mice during Chlamydia pneumoniae infection.

A new pulmonary T-cell-like lymphocyte population with the phenotype CD3(-) CD4(+) CD8(+) was discovered in mice. CD4(+) CD8(+) but CD3(+) cells among murine intestinal intraepithelial lymphocytes have previously been described. We describe herein a dramatic expansion of the CD3(-) CD4(+) CD8(+) cell population in response to experimental respiratory infection. After intranasal Chlamydia pneumoniae infection, CD4(+) CD8(+) cells became transiently the dominant lymphocyte type (maximum of 87% of all lymphocytes) in the lungs of NIH/S mice but remained virtually undetectable in spleen and blood. The enrichment of these cells was not a C. pneumoniae-specific event, since infection of NIH/S mice with influenza A virus also resulted in an increase in the number of CD4(+) CD8(+) cells (maximum of 42% of all lymphocytes). In addition to outbred NIH/S mice, two other mouse strains were studied: BALB/c (H-2(d)) and C57BL/6 (H-2(b)). C. pneumoniae-infected BALB/c mice responded with an intermediate increase in the number of CD4(+) CD8(+) cells in lungs, whereas C57BL/6 mice did not respond. The double-positive CD4(+) CD8(+) cells lacked a major part of the T-cell receptor complex, being both CD3(-) and TCR alpha beta-. However, when they were stimulated in vitro with a T-cell mitogen, they responded by proliferation but did not secrete gamma interferon. The dramatic expansion of this cell population at the infection site suggests an active role for them in respiratory infection, but the specification of this requires further study.

Interleukin-10 production by cord blood mononuclear cells.

The production of IL-10 by newborns has not been studied in much detail. We analyzed the IL-10 production by, and surface marker distribution of, cord blood mononuclear cells (n = 47); adult peripheral blood mononuclear cells (n = 30) were used as controls. Both the baseline (0.79 versus 1.54 ng/mL, p = 0.001) and lipopolysaccharide-stimulated (1.20 versus 2.88 ng/mL, p = 0.003) levels of IL-10 production were significantly lower in newborns than in adults. No significant differences were observed after stimulation with concanavalin A. Both cord blood and adult CD19+CD5+ cells were able to produce IL-10; however, the level of production was only an average of 16% of the total stimulated IL-10 production by unfractionated cells, indicating that CD5+ B cells are not the primary source of IL-10 in either adults or newborns. In newborns, the proportion of naive CD4+CD45RA+ cells was inversely correlated with IL-10 response to lipopolysaccharide (r = -0.49, p = 0.004) indicating a role for maturing T cells in neonatal IL-10 production; the number of macrophages was not significantly correlated with IL-10 response (r = 0.30, p = 0.10). In contrast, in adults IL-10 production correlated with the number of macrophages (r = 0.49, p = 0.01) but not CD4+CD45RA+ cells (r = -0.06, p = 0.77). We conclude that newborns produce less IL-10 than adults; the primary cells of origin and the regulatory mechanisms may be different from those observed in adults.

HIV type 1 Tat protein induces immunoglobulin and interleukin 6 synthesis by uninfected peripheral blood mononuclear cells.

Tat is a potent trans-activating protein encoded by the HIV genome. It is essential for viral replication, but has pleiotropic effects on host cells as well. We demonstrated that exogenous recombinant Tat increases immunoglobulin (Ig) and interleukin 6 (IL-6) production in vitro by normal uninfected peripheral blood mononuclear cells by 100-500%. The optimal Tat concentration was 100 ng/ml, but even a low concentration of 1 ng/ml induced a response in most subjects. The observed induction was inhibited by monoclonal anti-Tat antibodies and 2,3-dimercapto-1-propanol. Both anti-IL-6 antibodies and IL-6 antisense oligonucleotides inhibited Tat-induced IgG and IgA synthesis to some degree, whereas induction of IgM appeared to be independent of IL-6. We conclude that Tat can function in vitro in the absence of any other viral structures and induce Ig and IL-6 production; the clinical significance of these findings remains as yet undetermined.