Unsupervised Learning for Hydrogen Breath Tests.

Hydrogen breath tests are a well-established method to help diagnose functional intestinal disorders such as carbohydrate malabsorption or small intestinal bacterial overgrowth. In this work we apply unsupervised machine learning techniques to analyze hydrogen breath test datasets. We propose a method that uses 26 internal cluster validation measures to determine a suitable number of clusters. In an induced external validation step we use a predefined categorization proposed by a medical expert. The results indicate that the majority of the considered internal validation indexes was not able to produce a reasonable clustering. Considering a predefined categorization performed by a medical expert, a novel shape-based method obtained the highest external validation measure in terms of adjusted rand index. The predefined clusterings constitute the basis of a supervised machine learning step that is part of our ongoing research.

LPS-induced NF-kappaB expression in THP-1Blue cells correlates with neopterin production and activity of indoleamine 2,3-dioxygenase.

Neopterin production is induced in human monocyte-derived macrophages and dendritic cells upon stimulation with Th1-type cytokine interferon-gamma (IFN-gamma). In parallel, IFN-gamma induces the tryptophan-(trp)-degrading enzyme indoleamine 2,3-dioxygenase (IDO) and triggers the formation of reactive oxygen species (ROS). Translocation of the signal transduction element nuclear factor-kappaB (NF-kappaB) is induced by ROS and accelerates the pro-inflammatory response by activation of other pro-inflammatory pathways. Therefore, a close relationship between NF-kappaB expression, the production of neopterin and the degradation of trp can be assumed, although this has not been demonstrated so far. In the present in vitro study we compared the influence of lipopolysaccharide (LPS) on NF-kappaB activation, neopterin formation and the degradation of trp in THP-1Blue cells, which represent the human myelomonocytic cell line THP-1 stably transfected with an NF-kappaB inducible reporter system. In cells stimulated with LPS, a significant induction of NF-kappaB was observed, and this was paralleled by an increase of kynureunine (kyn) and neopterin concentrations and a decline of trp. The increase of the kyn to trp quotient indicates accelerated IDO activity. Higher LPS concentrations and longer incubation of cells were associated with higher activities of all three biochemical pathways and significant correlations existed between NF-kappaB activation, neopterin release and trp degradation (all p<0.001). We conclude that there is a parallel induction of NF-kappaB, neopterin formation and trp degradation in monocytic THP-1 cells, which is elicited by pro-inflammatory triggers like LPS during innate immune responses.

Immunomodulatory effects of Turkish propolis: changes in neopterin release and tryptophan degradation.

In most of the diseases which are considered to benefit from propolis, cellular immune reaction is activated, neopterin levels in body fluids are increased and enhanced tryptophan degradation is observed. In this study, the immunomodulatory effects of six Turkish propolis samples were evaluated by using the in vitro model of peripheral blood mononuclear cells (PBMC). Concentrations of neopterin, tryptophan, kynurenine and pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were determined and also the viability of the cells was checked with trypan blue and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test. In PBMC treated with mitogen phytohaemagglutinin, neopterin production and tryptophan degradation by enzyme indoleamine 2,3-dioxygenase (IDO) as well as release of cytokines was significantly enhanced and upon treatment with propolis extracts all these effects were dose-dependently suppressed. Results show an immunomodulatory effect of propolis extracts which includes down-regulation of IDO activity. IDO enzyme is considered to play an important role in the development of immunodeficiency and neuropsychiatric symptoms in patient with chronic inflammation. The suppression of tryptophan degradation by propolis extracts may therefore be related with some of its beneficial health properties in humans.

Gliadin IgG antibodies and circulating immune complexes.

OBJECTIVE: Circulating immune complexes (CICs) in blood are associated with autoimmune-diseases such as systemic lupus erythematosus, immune complex glomerulonephritis, rheumatoid arthritis and vasculitis. However, slightly increased serum concentrations of such CICs are sometimes also found in healthy individuals. The objective of the current study was to assess whether food antigens could play a role in the formation of CICs. MATERIAL AND METHODS: A total of 352 (265 F, 87 M), so far, healthy individuals were tested for CICs containing C1q and immunoglobulin G (IgG) as well as for gliadin IgG antibodies using the ELISA technique. Additionally, fructose and lactose malabsorption was assessed using hydrogen breath tests. RESULTS: In our study, 15.3% (54/352) of the patients presented with elevated CIC concentrations (above 50 microg/ml) and 6.5% (23/352) of the study population were positive for gliadin IgG antibodies (above 20 U/ml). CIC concentration levels were significantly higher in the group with elevated gliadin IgG antibodies (CIC median: 49.0 microg/ml) compared with the group with normal levels of gliadin IgG antibodies (CIC median: 30.0 microg/ml; Mann-Whitney U-test, U=1992; p <0.001). As expected, there was no difference in CIC concentrations (Mann-Whitney U-test, U=6106; p=0.783) and gliadin IgG (Mann-Whitney U-test, U=3761; p=0.411) between patients in the fructose or lactose malabsorber groups and the subjects without malabsorption. CONCLUSIONS: The results of this study indicate that certain food antigens (e.g. gluten) could play a role in the formation of CICs. An association between CICs and fructose or lactose malabsorption seems to be improbable.

Serum phenylalanine concentrations in patients with ovarian carcinoma correlate with concentrations of immune activation markers and of isoprostane-8.

Increased blood concentrations of essential amino acid phenylalanine are common in patients with HIV infection, in trauma and sepsis and in patients with cancer. The reason for this phenomenon is still unclear. However, all these clinical conditions are known to be linked with inflammation and immune activation. Oxidative stress resulting from chronic immune activation and inflammation could impair activity of phenylalanine (4)-hydroxylase (PAH) and thus give rise to increased phenylalanine concentrations. We therefore examined in 20 patients with ovarian cancer a possible association of serum concentrations of phenylalanine and tyrosine with immune activation markers 75 kDa soluble tumor necrosis factor-alpha receptor (sTNF-R75) and neopterin, and of oxidative stress marker isoprostane-8. Phenylalanine concentrations were higher in patients with higher FIGO stage and correlated with concentrations of sTNF-R75 (rs=0.441) and neopterin (rs=0.346; both p<0.05). No such correlations existed for tyrosine levels. The phenylalanine to tyrosine ratio (phe/tyr), an estimate of PAH activity, correlated somewhat stronger with sTNF-R75 (rs=0.549; p<0.01) and neopterin (rs=0.497; p=0.01). Finally, phenylalanine concentrations correlated with isoprostane-8 concentrations (rs=0.450, p=0.02). Correlations of phenylalanine and phe/tyr with immune activation markers point to a potential role of inflammation and immune activation in the accumulation of phenylalanine. The relationship between oxidative stress marker isoprostane-8 and phenylalanine as well as sTNF-R75 concentrations suggests a link between reactive oxygen species formed during chronic immune activation and inflammation and the decline of PAH activity, which might underlie the increase of phe/tyr (248 words).

Evaluation of a novel reverse-hybridization StripAssay for typing DNA variants useful in diagnosis of adult-type hypolactasia.

BACKGROUND: Adult-type hypolactasia is a genetically determined inability to digest lactose after weaning. Two single-nucleotide polymorphisms (C-13910T, G-22018A) located upstream of the lactase gene (LCT) within the gene MCM6 are associated with the lactase persistence/non-persistence trait in patients of European descent. Therefore, the genotyping of these SNPs has been established as a diagnostic tool for adult-type hypolactasia. We have recently shown that several novel allelic variants located in close proximity to the C-13910T SNP interfere with the diagnostic accuracy of real-time PCR-based genotyping methods. METHODS: We describe here the validation of a comprehensive reverse-hybridization teststrip-based assay for the detection of common and novel LCT SNPs (C-13907G, C-13910T, T-13913C, G-13914A, T-13915G, and G-22018A). This assay is based on multiplex DNA amplification and ready-to-use membrane teststrips containing variant-specific oligonucleotide probes immobilized as an array of parallel lines. RESULTS: We evaluated the novel reverse-hybridization StripAssay on 125 DNA samples in comparison to LightCycler analysis and sequencing. The outcome of StripAssay genotyping was found to be completely concordant with that obtained by sequencing. CONCLUSIONS: The StripAssay represents an accurate and robust screening tool to identify multiple LCT/MCM6 variants in a rapid manner. It overcomes diagnostic pitfalls that were reported and allows the simultaneous genotyping of closely spaced LCT variant sites in a single-step diagnostic approach.

Implementation and interpretation of hydrogen breath tests.

Hydrogen breath tests are non-invasive and safe diagnostic tools used to investigate functional intestinal disorders. For the diagnosis of fructose or lactose malabsorption as well as for the detection of small intestinal bacterial overgrowth syndrome, hydrogen breath tests are even regarded as gold standard. However, standardization of the testing procedure and the interpretation of the test results are still lacking. In this paper, reliable information on the implementation of the most common hydrogen breath tests and precise guidelines for the interpretation of the test results are presented.

Hydrogen breath testing versus LCT genotyping for the diagnosis of lactose intolerance: a matter of age?

BACKGROUND: Two single nucleotide polymorphisms (-13910 C/T and -22018 G/A) upstream of the lactase gene (LCT) have been found to be associated with lactose tolerance in Europeans. METHODS: In one hundred and twenty Austrian outpatients, who visited the physician's office for symptoms of irritable bowel syndrome (IBS), hydrogen breath testing (HBT) and LCT genotyping by polymerase chain reaction and reverse-hybridisation were performed in parallel. RESULTS: The coincidence between a genotype suggesting lactase non-persistence (lactose intolerance) and a positive HBT result was almost perfect (97.4% for LCT-13910 C/T and 100% for LCT-22018 G/A). Between a genotype indicating lactase persistence (lactose tolerance) and a negative HBT result the coincidence was lower (72% and 71.4%, respectively). Among heterozygotes, there was a statistically significant increase in the proportion of positive HBT results with age. Both SNPs were in accordance in 117/120 (97.5%) patients. CONCLUSION: Genetic analysis of LCT-13910 C/T and LCT-22018 G/A is a good indicator for the presence of lactose intolerance. Because age, as well as a number of secondary causes (e.g. celiac disease), can influence HBT results, it is useful to combine HBT and genetic analysis in the diagnostic assessment of IBS.

Interaction of 5-hydroxytryptamine (serotonin) against Aspergillus spp. in vitro.

This study examined the direct interaction of serotonin (5-hydroxytryptamine (5-HT)) with Aspergillus species. Accumulation of 5-HT in aspergilli was investigated by immunofluorescence staining and laser confocal scanning microscopy. The influence of 5-HT on fungal ergosterol content, cell membrane integrity, fungal growth and hyphal elongation was determined. 5-HT was localised in the cytoplasm of Aspergillus spp., as 5-HT fluorescent signals appeared after 30min at 4 degrees C and in the presence of inhibitors of oxidative phosphorylation. 5-HT treatment of Aspergillus spp. significantly affected ergosterol synthesis, fungal cell membrane integrity and hyphal elongation (P<0.05). 5-HT treatment for 4h resulted in a lag of re-growth (post-antifungal effect). In conclusion, our findings suggest that 5-HT affects hyphal growth and diminishes fungal cell membrane integrity.

Antioxidants may increase the probability of developing allergic diseases and asthma.

In addition to genetic predisposition, a lack of triggers for Th1 immune response like exposure to infections, endotoxins and dirt in childhood are supposed to be responsible for the higher incidence of allergic rhinitis and asthma (hygiene hypothesis). In vitro, beverages rich in antioxidants like green tea and wine were found to suppress formation of Th1-type cytokine interferon-gamma. Due to the existing cross-regulatory interplay between Th1- and Th2-type immune response, these beverages may thus slow-down Th1-type immune response and thereby favour an over-production of Th2-type cytokines. Also food rich in antioxidants may increase the risk of atopic disease. Thus, not only a lack of triggers for Th1 type immune response, but also a nutrition rich in antioxidants suppressing interferon-gamma would result in a persistence of Th2-type immune response and increase the susceptibility for allergic reactions and asthma. In addition to improved hygienic standards in the past decades, also social changes including the availability of functional food and food enriched in antioxidants may have increased the prevalence of atopic diseases in Western countries.

Moderate hyperhomocysteinaemia and immune activation in patients with rheumatoid arthritis.

BACKGROUND: Moderate hyperhomocysteinaemia related to folate deficiency has been described in patients with cardiovascular risk and also in patients with autoimmune diseases including rheumatoid arthritis (RA). METHODS: In 33 patients with RA, serum concentrations of homocysteine and cysteine, of B-vitamins folate and vitamin B(12), and of immune activation markers neopterin and soluble 75-kDa TNF-receptor (sTNF-R75) were measured. RESULTS: A significant proportion of patients presented with elevated homocysteine and cysteine concentrations in comparison to reference ranges of healthy control persons. Moderate hyperhomocysteinaemia coincided with decreased serum folate and with higher concentrations of sTNF-R75 and neopterin, but it was rather independent from methotrexate (MTX) therapy. CONCLUSIONS: The coincidence of higher homocysteine and lower folate concentrations with increased concentrations of immune activation markers in patients with RA suggests that immune activation could be involved in the development of hyperhomocysteinaemia.

Increased degradation of tryptophan in blood of patients with rheumatoid arthritis.

OBJECTIVE: Activation of the enzyme indoleamine-(2,3)-dioxygenase (IDO) by interferon (IFN)-g leads to enhanced tryptophan conversion to kynurenine. In consequence of chronic immune activation, tryptophan availability is reduced, leading to inhibition of cell proliferation as protein synthesis is affected. Tryptophan deprivation due to IDO activation could therefore be effective in abrogating processes with high metabolic turnover, thus modulating cellular immune response. METHODS: Concentrations of tryptophan, kynurenine, and neopterin were measured by HPLC in the sera of 38 patients with rheumatoid arthritis (RA). The kynurenine:tryptophan ratios (kyn/trp) were calculated to estimate IDO activity. RESULTS: Tryptophan concentrations were lower in patients with RA (median, interquartile range: 44.95 microM, 40.31-49.95 microM) compared to healthy blood donors (62.62 microM, 57.27-74.61 microM; p < 0.001). Kynurenine in patients (1.86 microM, 1.54-2.31 microM) did not differ from controls (2.06 microM, 1.58-2.65 microM; NS). The kyn/trp ratio was higher in patients (42.39 mM/M, 37.02-48.60 mM/M) than in controls (31.72 mM/M; 27.95-35.03 mM/M; p < 0.001). Kynurenine concentrations (rs = 0.611; p < 0.001) and kyn/trp ratios (rs = 0.621; p < 0.001) correlated with neopterin concentrations, which indicate stimulated cellular immune response in patients with RA. CONCLUSIONS: The data point to a role of immune activation and Th1-type cytokine INF-g to induce elevated tryptophan degradation in patients with RA.

Association between increased serum cholesterol and signs of depressive mood.

Hypercholesterolemia is associated with an increased risk of atherosclerosis and coronary heart disease. Therefore, therapeutic lowering of cholesterol is an important preventive measure of cardiac morbidity and death. As one side effect, cholesterol-lowering drugs appear to increase the mortality due to suicides or violence, and low lipid concentrations were found to be associated with trait measures of depression. We compared serum cholesterol concentrations and the Beck Depression Rating Scale (Beck's score) in 604 otherwise healthy outpatients who visited the physician's office for a medical health check-up; 65.4% of individuals presented with serum cholesterol concentrations > or = 5.2 mmol/l (> 200 mg/dl) and 5.3% had elevated Beck's score (> 19), indicative for depression. Beck's score was higher in patients with cholesterol concentrations above the 75th percentile (= 6.2 mmol/l; U = 31221, p < 0.02, Mann-Whitney U-test), and Beck's score correlated with cholesterol concentrations and with age. Thus, in contrast to the widely accepted view, in our study, higher cholesterol concentrations were associated with signs of depressive mood. Hypercholesterolemia may not necessarily increase the risk of depressive mood, conversely, increased intake of fat and carbohydrates by individuals with depressive mood may increase cholesterol levels.

Interaction of sertraline with Candida species selectively attenuates fungal virulence in vitro.

This study investigated whether the interaction between isolates of Candida albicans (n=7), Candida parapsilosis (n=3), Candida krusei (n=2), Candida dubliniensis (n=1) and sertraline, a typical selective serotonin reuptake inhibitor, alters candidal virulence. Sertraline treatment of Candida spp. significantly (P<0.05) affected hyphal elongation, phospholipase activity, production of secreted aspartyl proteinases and fungal viability. In addition, monocyte-derived macrophages (MDMs) treated with sertraline reduced inhibition of blastoconidia germination in comparison to MDMs alone. In conclusion, our findings suggest that the interaction between sertraline and Candida spp. may also diminish the virulence properties of this fungal pathogen in vivo.

Antifungal properties of 5-hydroxytryptamine (serotonin) against Candida species in vitro.

In this study the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) of 5-hydroxytryptamine (5 HT, serotonin) against clinical isolates of Candida albicans (n = 11), Candida glabrata (n = 9), Candida tropicalis (n = 10) and Candida parapsilosis (ATCC 22019) using a broth microdilution test were investigated. In addition, it was examined whether delayed regrowth as a post-antifungal effect results following short exposure to 5 HT. 5 HT showed antifungal activity towards all isolates of Candida spp. The isolates yielded comparable MIC and MFC values of 5 HT in the range 0.91-7.34 mM and 1.83-14.68 mM, respectively. A lag in regrowth was dependent on the concentration tested. Treatment for 3 h at concentrations of 5 HT below and equipotent to the MFC resulted in a delayed regrowth of 8-12 h for isolates of Candida spp. In conclusion, these in vitro studies clearly demonstrate antifungal effects of 5 HT. Identifying the mode of action could be of great help in developing and researching new antifungal drugs.

Neopterin production, tryptophan degradation, and mental depression--what is the link?

The cytokine interferon-gamma stimulates human monocytes/macrophages to release large amounts of neopterin. Increased neopterin concentrations in body fluids of patients are observed during diseases with activated cellular (=TH1-type) immune response such as allograft rejection, virus infections, autoimmune disorders, or malignant tumors but also in neurodegenerative diseases or during pregnancy. In various cells interferon-gamma induces indoleamine 2,3-dioxygenase (IDO) which degrades tryptophan via the kynurenine pathway. Therefore like increased neopterin formation, enhanced tryptophan degradation is observed in diseases concomitant with cellular immune activation. Disturbed metabolism of tryptophan affects biosynthesis of neurotransmitter 5-hydroxytryptamine (serotonin), and it appears to be associated with an increased susceptibility for depression. In fact, enhanced neopterin concentrations together with increased degradation of tryptophan and low serum levels of tryptophan correlate with neuropsychiatric abnormalities like cognitive decline and depressive symptoms especially in long-lasting and chronic diseases. Activation of IDO could represent an important link between the immunological network and the pathogenesis of depression.

Antifungal properties of 5-hydroxytryptamine (serotonin) against Aspergillus spp. in vitro.

This study shows that 5-hydroxytrypatmine (5-HT, serotonin) is fungicidal towards conidia and hyphae of clinical isolates of Aspergillus (A.) fumigatus, A. flavus and A. terreus. The minimal fungicidal concentrations for Aspergillus conidia and hyphae ranged between 14.68 to 117.5 mM and 29.37 to 235 mM during 24 and 48 h of incubation. Several serotonin receptor antagonists (5-HT2, 5-HT3) studied in vitro did not influence antifungal activity.