The immunomodulatory nutritional intervention NR100157 reduced CD4+ T-cell decline and immune activation: a 1-year multicenter randomized controlled double-blind trial in HIV-infected persons not receiving antiretroviral therapy (The BITE Study).

BACKGROUND: The immunomodulatory nutritional product NR100157 was developed for human immunodeficiency virus (HIV)-infected individuals. We hypothesized that targeting the compromised gastrointestinal tract of HIV-infected individuals would result in systemic immunological benefits. METHODS: In a multicenter, randomized, controlled, double-blind trial, 340 HIV-1-positive adults not on antiretroviral therapy, with CD4(+) T-cell counts <800/µL, were given either NR100157 or an isocaloric and isonitrogenous control for 52 weeks. Primary outcome was CD4(+) T-cell count. Secondary outcomes included plasma viral load (pVL), safety, and tolerability. In a pilot study (n = 20), levels of CD4(+)CD25(+) and CD8(+)CD38(+) activation were measured (n = 20). The trial is registered at the Dutch Trial Register (NTR886) and ISRCTN81868024. RESULTS: At 52 weeks, CD4(+) T-cell decline showed a 40-cell/µL difference (P = .03) in the intention-to-treat population in favor of the immunomodulatory NR100157 (control vs active, -68 ± 15 vs -28 ± 16 cells/µL/year). The change in pVL from baseline was similar between groups (P = .81). In the pilot study, the percentage of CD4(+)CD25(+) was lower in the active group (P < .05) and correlated with changes in CD4(+) T-cell count (r = -0.55, P < .05). The percentage of CD8(+)CD38(+) levels was unaffected. CONCLUSIONS: The specific immunonutritional product NR100157 significantly reduces CD4(+) decline in HIV-1-infected individuals, and this is associated with decreased levels of CD4(+)CD25(+). (This nutritional intervention is likely to affect local gut integrity and gut-associated lymphoid tissue homeostasis, which in turn translates positively to systemic effects.) Clinical Trials Registration. ISRCTN81868024.

Bifidobacterium breve - HT-29 cell line interaction: modulation of TNF-α induced gene expression.

To provide insight in the molecular basis for intestinal host-microbe interactions, we determined the genome-wide transcriptional response of human intestinal epithelial cells following exposure to cells of Bifidobacterium breve. To select an appropriate test system reflecting inflammatory conditions, the responsiveness to TNF-α was compared in T84, Caco-2 and HT-29 cells. The highest TNF-α response was observed in HT-29 cells and this cell line was selected for exposure to the B. breve strains M-16V, NR246 and UCC2003. After one hour of bacterial pre-incubation followed by two hours of additional TNF-α stimulation, B. breve M-16V (86%), but to a much lesser extent strains NR246 (50%) or UCC2003 (32%), showed a strain-specific reduction of the HT-29 transcriptional response to the inflammatory treatment. The most important functional groups of genes that were transcriptionally suppressed by the presence of B. breve M-16V, were found to be involved in immune regulation and apoptotic processes. About 54% of the TNF-α induced genes were solely suppressed by the presence of B. breve M-16V. These included apoptosis-related cysteine protease caspase 7 (CASP7), interferon regulatory factor 3 (IRF3), amyloid beta (A4) precursor proteinbinding family A member 1 (APBA1), NADPH oxidase (NOX5), and leukemia inhibitory factor receptor (LIFR). The extracellular IL-8 concentration was determined by an immunological assay but did not change significantly, indicating that B. breve M-16V only partially modulates the TNF-α pathway. In conclusion, this study shows that B. breve strains modulate gene expression in HT-29 cells under inflammatory conditions in a strain-specific way.

Specific prebiotics modulate gut microbiota and immune activation in HAART-naive HIV-infected adults: results of the "COPA" pilot randomized trial.

Intestinal mucosal immune system is an early target for human immunodeficiency virus type 1 (HIV-1) infection, resulting in CD4(+) T-cell depletion, deterioration of gut lining, and fecal microbiota composition. We evaluated the effects of a prebiotic oligosaccharide mixture in highly active antiretroviral therapy (HAART)-naive HIV-1-infected adults. In a pilot double-blind, randomized, placebo-controlled study, 57 HAART-naive HIV-1-infected patients received a unique oligosaccharide mixture (15 or 30 g short chain galactooligosaccharides/long chain fructooligosaccharides/pectin hydrolysate-derived acidic oligosaccharides (scGOS/lcFOS/pAOS) daily) or a placebo for 12 weeks. Microbiota composition improved significantly with increased bifidobacteria, decreased Clostridium coccoides/Eubacterium rectale cluster, and decreased pathogenic Clostridium lituseburense/Clostridium histolyticum group levels upon prebiotic supplementation. In addition, a reduction of soluble CD14 (sCD14), activated CD4(+)/CD25(+) T cells, and significantly increased natural killer (NK) cell activity when compared with control group were seen in the treatment group. The results of this pilot trial highly significantly show that dietary supplementation with a prebiotic oligosaccharide mixture results in improvement of the gut microbiota composition, reduction of sCD14, CD4(+) T-cell activation (CD25), and improved NK cell activity in HAART-naive HIV-infected individuals.

Oral treatment with probiotics reduces allergic symptoms in ovalbumin-sensitized mice: a bacterial strain comparative study.

BACKGROUND/AIM: Evidence demonstrating an important role of the intestinal microbiota in the incidence of allergic disorders has led to the concept of using probiotics as possible antiallergic therapy. This study aimed to select a bacterial strain with the best antiallergic treatment effects from a panel of 6 bacterial strains in a mouse model of ovalbumin(OVA)-allergic asthma. METHODS: OVA-sensitized BALB/c mice were orally administered the bacterial strains Bifidobacterium breve M-16V, B. infantis NumRes251, B. animalis NumRes252 and NumRes253, Lactobacillus plantarum NumRes8 and L. rhamnosus NumRes6. After challenge by OVA inhalation in the lungs, the response to methacholine was measured. Pulmonary inflammation was assessed by analyzing bronchoalveolar lavage fluid for the presence of eosinophils, neutrophils, macrophages and lymphocytes and for interleukin 4, interleukin 5, interleukin 10 and interferon-gamma. OVA-specific IgE, IgG1 and IgG2a were measured in serum. Next, the effect on acute allergic skin reaction was measured after treatment with B. breve M-16V and L. plantarum NumRes8. RESULTS: Of the panel of 6 strains, B. breve M-16V and L. plantarum NumRes8 inhibited (1) the response to methacholine, (2) reduced the number of eosinophils in the bronchoalveolar lavage fluid, (3) reduced both OVA-specific IgE and (4) OVA-specific IgG1, whereas the other strains did not affect all these parameters simultaneously. B. breve M-16V but not L. plantarum NumRes8 reduced interleukin 4, interleukin 5 and interleukin 10. Furthermore, B. breve M-16V but not L. plantarum NumRes8 reduced acute allergic skin reactions to OVA. CONCLUSION: B. breve M-16V was identified as the most potent antiallergic strain.

Preoperative supplementation with a carbohydrate mixture decreases organ dysfunction-associated risk factors.

BACKGROUND & AIMS: Recently, both asymmetrical dimethylarginine and IL-6 have been suggested to be associated with the induction and severity of single and multiple organ dysfunction. The aims of the present study were to elucidate if these factors were increased in an ischemia reperfusion (IR) model and whether pre-operative carbohydrate supplementation can reduce the risk factors along with the IR injury. METHODS: One group of male Wistar rats was fasted for 16 h (water ad libitum) prior to clamping the superior mesenteric artery (IR fasted n=14). A second group had ad libitum access to a carbohydrate solution prior to clamping (IR fasted CHO group n=11). Sham-fasted animals, which only received laparotomy and no clamping, served as controls (n=4). RESULTS: Plasma urea and ALAT activity were both increased in the IR fasted animals when compared to the sham rats (P=0.007 and P<0.02, respectively). Furthermore, it was shown that IR fasted rats had increased ADMA and IL-6 concentration in plasma when compared to sham animals (P<0.02). Moreover, the GSH level in lung was significantly decreased in the IR fasted animals (P=0.014). IR CHO supplemented showed no significant increase of ALAT activity and decrease of lung GSH. Furthermore, significantly lower plasma urea, ADMA and IL-6 concentration was seen in the IR CHO supplemented group when compared to the IR fasted rats (P=0.028, P<0.01 and P<0.02, respectively). The liver glycogen concentration in IR fasted rats was 48% of that IR rats supplemented the carbohydrate mixture. CONCLUSION: The present rat intestinal ischemia reperfusion model not only induces organ injury indicated by the classical parameters such as plasma urea and ALAT activity, but also increased plasma IL-6 and ADMA and decreased lung GSH concentration in IR fasted rats. Pre-operative supplementation with the carbohydrate mixture significantly lowered the plasma urea, IL-6 and ADMA concentrations and maintained lung GSH concentration. This indicates that pre-operative carbohydrate supplementation reduces post-operative organ injury.

Altered gene expression in Staphylococcus aureus upon interaction with human endothelial cells.

Staphylococcus aureus is isolated from a substantial number of patients with infective endocarditis who are not known to have predisposing heart abnormalities. It has been suggested that the infection is initiated by the direct binding of S. aureus to human vascular endothelium. To determine the mutual response of the endothelial cells and the bacteria, we studied the interaction between S. aureus and human vascular endothelium. Scanning electron microscopic analyses showed that binding of S. aureus to human umbilical vein endothelial cells (HUVEC) mainly occurred via thread-like protrusions extending from the cell surface. Bound bacteria appeared to be internalized via retraction of the protrusions into newly formed invaginations of the endothelial cell surface. The growth phase of S. aureus had a major impact on the interaction with HUVEC. Logarithmically growing bacteria showed increased binding to, and were more readily internalized by, HUVEC compared to stationary-phase bacteria. To assess the bacterial response to the cellular environment, an expression library of S. aureus was used to identify genes whose expression was induced after 4 h of exposure to HUVEC. The identified genes could be divided into different categories based on the functions of the encoded proteins (transport, catabolism, biosynthesis, and DNA repair). Further analyses of five of the S. aureus transposon clones showed that HUVEC as well as human serum are stimuli for triggering gene expression in S. aureus.

Broad-host-range shuttle vectors for screening of regulated promoter activity in viridans group streptococci: isolation of a pH-regulated promoter.

Viridans group streptococci are major constituents of the normal human oral flora and are also identified as the predominant pathogenic bacteria in native valve infective endocarditis. Little information is available regarding the regulation of gene expression in viridans group streptococci, either in response to changes in the oral environment or during development of endocarditis. We therefore constructed a set of broad-host-range vectors for the isolation of promoters from viridans group streptococci that are activated by specific environmental stimuli in vitro or in vivo. A genomic library of Streptococcus gordonii strain CH1 was constructed in one of the new vectors, and this library was introduced into a homologous bacterium by using an optimized electroporation protocol for viridans group streptococci. Because viridans group streptococci entering the bloodstream from the oral cavity encounter an increase in pH, we selected promoters upregulated by this specific stimulus. One of the selected promoter sequences showed homology to the promoter region of the hydA gene from Clostridium acetobutylicum, the expression of which is known to be regulated by the environmental pH. The isolation of this pH-regulated promoter shows that S. gordonii can sense an increase in the environmental pH, which serves as a signal for bacterial gene activation. Furthermore, this demonstrates the usefulness of these new selection vectors in research on adaptive gene expression of viridans group streptococci and possibly also of other gram-positive bacteria.

A shift from oral to blood pH is a stimulus for adaptive gene expression of Streptococcus gordonii CH1 and induces protection against oxidative stress and enhanced bacterial growth by expression of msrA.

Viridans group streptococci (VS) from the oral cavity entering the bloodstream may initiate infective endocarditis (IE). We aimed to identify genes expressed in response to a pH increase from slightly acidic (pH 6.2) to neutral (pH 7.3) as encountered by VS entering the bloodstream from the oral cavity. Using a recently developed promoter-screening vector, we isolated five promoter fragments from the genomic DNA of Streptococcus gordonii CH1 responding to this stimulus. No common regulatory sequences were identified in these promoter fragments that could account for the coordinate expression of the corresponding genes. One of the isolated fragments contained the promoter region and 5' end of a gene highly homologous to the methionine sulfoxide reductase gene (msrA) of various bacterial and eukaryotic species. This gene has been found to be activated in S. gordonii strain V288 in a rabbit model of IE (A. O. Kiliç, M. C. Herzberg, M. W. Meyer, X. Zhao, and L. Tao, Plasmid 42:67-72, 1999). We isolated and characterized the msrA gene of S. gordonii CH1 and constructed a chromosomal insertion mutant. This mutant was more sensitive to hydrogen peroxide, suggesting a role for the streptococcal MsrA in protecting against oxidative stress. Moreover, MsrA appeared to be important for the growth of S. gordonii CH1 under aerobic and anaerobic conditions. Both these properties of MsrA may contribute to the ability of S. gordonii to cause IE.

Isolation and characterization of promoter regions from Streptococcus gordonii CH1.

We aimed to identify transcription signal sequences from Streptococcus gordonii strain CH1 by random chromosomal cloning. Five genomic fragments from a Sau3A digest, which constitutively activated transcription of a promoterless spectinomycin resistance gene in this strain, were isolated and characterized. Additionally, one promoter fragment was isolated that was specifically activated under iron-limiting conditions. A sequence motif with similarity to the consensus for Fur-binding regulatory DNA sequences (Fur box) in Escherichia coli was detected within the putative promoter region. The open reading frame downstream of this region possibly encodes a transmembrane protein involved in iron uptake.

A simple procedure for isolation of cloning vectors and endogenous plasmids from viridans group streptococci and Staphylococcus aureus.

Isolation of plasmid DNA from viridans group streptococci is difficult, and preparations are often heavily contaminated with chromosomal DNA. We developed a simple protocol to isolate pure plasmid DNA for use in different molecular techniques, including automated sequencing. The protocol is also applicable for plasmid isolation from Staphylococcus aureus. In addition, the protocol allows isolation of pure endogenous plasmids from streptococci and S. aureus.

Carbon monoxide dehydrogenase from Methanosarcina frisia Gö1. Characterization of the enzyme and the regulated expression of two operon-like cdh gene clusters.

Carbon monoxide dehydrogenase (Cdh) has been anaerobically purified from Methanosarcina frisia Gö1. The enzyme is a Ni2+-, Fe2+-, and S2--containing alpha2beta2 heterotetramer of 214 kDa with a pI of 5.2 and subunits of 94 and 19 kDa. It has a Vmax of 0.3 mmol of CO min-1 mg-1 and Km values for CO and methyl viologen of approximately 0.9 mM and 0.12 mM, respectively. EPR spectroscopy on the reduced enzyme showed two overlapping signals: one indicative for 2 (4Fe-4S)+ clusters and a second signal that is atypical for standard Fe/S clusters. The latter was, together with high-spin EPR signals of the oxidized enzyme tentatively assigned to an Fe/S cluster of high nuclearity.

Organization and growth phase-dependent transcription of methane genes in two regions of the Methanobacterium thermoautotrophicum genome.

Two regions of the Methanobacterium thermoautotrophicum genome containing genes that encode enzymes involved in methanogenesis (methane genes) have been cloned and sequenced to determine the extent of methane gene clustering and conservation. One region from the M. thermoautotrophicum strains delta H and Winter, extending approximately 13.5 kb upstream from the adjacent mvhDGAB and mrtBDGA operons that encode the methyl-viologen-reducing hydrogenase (MVH) and the methyl coenzyme M reductase II (MRII), respectively, was sequenced, and 76% sequence identity and very similar gene organizations were demonstrated. Five closely linked open reading frames were located immediately upstream of the mvh operon and were designated flpECBDA. The flpCBD genes encode amino acid sequences that are 31, 47, and 65% identical to the primary sequences of the alpha and beta subunits of formate dehydrogenase and the delta subunit of MVH, respectively. Located immediately upstream of the flp genes was the mth gene, which encodes the H2-dependent methylene-tetrahydromethanopterin dehydrogenase (MTH). In contrast to this mth-flp-mvh-mrt cluster of methane genes, a separate approximately 5.4-kb genomic fragment cloned from M. thermoautotrophicum delta H contained only one methane gene, the mtd gene, which encodes the 8-hydroxy-5-deazaflavin (H2F420)-dependent methylene-tetrahydromethanopterin dehydrogenase (MTD). Northern (RNA) blot experiments demonstrated that mth was transcribed only at early growth stages in fermentor-grown cultures of M. thermoautotrophicum delta H, whereas mtd was transcribed at later growth stages and in the stationary phase. Very similar transcription patterns have been observed by T.D. Pihl, S. Sharma, and J. N. Reeve (J. Bacteriol. 176:6384-6391, 1994) for the MRI- and MRII-encoding operons, mrtBDGA and mcrBDCGA, im M. thermoautotrophicum deltaH, suggesting coordinated regulation of methane gene expression. In contrast to the growth phase-dependent transcription of the mth/mrt and mtd/mcr genes, transcription of the mvhDGAB and frhADGB operons, which encode the two (NiFe) hydrogenases in M. thermoautotrophicum deltaH, was found to occur at all growth stages.

Mitochondrial DNA restriction fragment length polymorphisms in field isolates of the Aspergillus niger aggregate.

The mitochondrial DNAs (mtDNAs) and the ribosomal repeat unit (ribosomal DNA, rDNA) of black Aspergillus isolates collected in various parts of the world were examined. Wide-ranging mtDNA variation was observed in natural populations of the Aspergillus niger aggregate. Most isolates were classifiable as A. niger or Aspergillus tubingensis according to their rDNA and mtDNA patterns. The mtDNA variation was distributed unevenly in the populations studied. The mtDNAs of most of the isolates collected in Australia were of the A. tubingensis type, with an unexpectedly high degree of variation, while the rDNA of these isolates exhibited the same A. tubingensis pattern as that of isolates from other locations. Some other local populations displayed very little polymorphism in their mtDNA and rDNA. Hybridization experiments in which cloned A. niger and Aspergillus nidulans mtDNA fragments were used revealed that the two main mtDNA groups corresponding to A. niger and A. tubingensis are more distantly related than concluded earlier. Six of the 13 Brazilian isolates examined exhibited mtDNA and rDNA types different from those of all the other strains and could not be classified into the above species. Classical taxonomic examination of these strains is in progress.

Double-stranded RNA mycoviruses in section Nigri of the Aspergillus genus.

Double-stranded RNA bands were detected electrophoretically in about 7% of natural isolates and in 13 of 51 collection strains belonging in section Nigri of the genus Aspergillus. The identity of these bands was proved by S1 nuclease and RNase treatment. Most of the virus-containing natural isolates came from Indonesia. Electron microscopic examination of the strains revealed the presence of virus-like particles in the mycelia of the strains examined. All of the virus-like particles were isometric and their size was around 30-35 nm, while some Indonesian isolates also contained virus-like particles in the size range 23-25 nm. It was possible to cure some of these strains of virus-like particles by mutagenic treatment. The four strains tested lost their virus-like particles and also their 'arginine-proline leaky' phenotype and became prototrophic. Virus transfer was possible among these four strains by protoplast fusion. It also proved possible to transfer mycoviruses into a more distantly related Aspergillus tubingensis strain by prolonged incubation of the polyethylene glycol treated protoplasts in osmotically stabilized medium. In spite of the finding that all Aspergillus foetidus and both Aspergillus heteromorphus strains examined contained double-stranded RNA segments and virus-like particles, no phenotypes related to the presence of these VLPs have been observed so far.