Interferon Gamma-Induced Protein (IP-10) as Potential Biomarker for Cancer-Related-Fatigue: Results from a 6-month Randomized Controlled Trial.

We examined if serum concentrations Interferon gamma-induced protein (IP-10) is a potential clinical biomarker for cancer-related-fatigue (CRF). Fatigue scores and IP-10 concentrations were measured from curatively treated fatigued cancer patients randomized to either cognitive behavioral therapy (CBT, n = 26) or waiting-list (WL, n = 13). No correlation was found between baseline IP-10 level and fatigue severity and no significant differences in IP-10 serum levels were observed between fatigued and matched non-fatigued patients (n = 22). Relative changes in IP-10 concentrations from baseline to six-month follow-up were not significantly different between the CBT and WL conditions. In this study, IP-10 showed low potential as clinical CRF biomarker. TRIAL REGISTRATION: This study is registered at ClinicalTrials.gov (NCT01096641).

Biological pathways, candidate genes, and molecular markers associated with quality-of-life domains: an update.

BACKGROUND: There is compelling evidence of a genetic foundation of patient-reported quality of life (QOL). Given the rapid development of substantial scientific advances in this area of research, the current paper updates and extends reviews published in 2010. OBJECTIVES: The objective was to provide an updated overview of the biological pathways, candidate genes, and molecular markers involved in fatigue, pain, negative (depressed mood) and positive (well-being/happiness) emotional functioning, social functioning, and overall QOL. METHODS: We followed a purposeful search algorithm of existing literature to capture empirical papers investigating the relationship between biological pathways and molecular markers and the identified QOL domains. RESULTS: Multiple major pathways are involved in each QOL domain. The inflammatory pathway has the strongest evidence as a controlling mechanism underlying fatigue. Inflammation and neurotransmission are key processes involved in pain perception, and the catechol-O-methyltransferase (COMT) gene is associated with multiple sorts of pain. The neurotransmitter and neuroplasticity theories have the strongest evidence for their relationship with depression. Oxytocin-related genes and genes involved in the serotonergic and dopaminergic pathways play a role in social functioning. Inflammatory pathways, via cytokines, also play an important role in overall QOL. CONCLUSIONS: Whereas the current findings need future experiments and replication efforts, they will provide researchers supportive background information when embarking on studies relating candidate genes and/or molecular markers to QOL domains. The ultimate goal of this area of research is to enhance patients' QOL.

The influence of the AA 16 beta 2-adrenoceptor polymorphism on systemic and airway responses in asthma.

BACKGROUND: The impact of the polymorphic amino acids 16 and 27 of the beta 2-adrenoceptor (beta 2-AR) on the susceptibility to bronchodilator tolerance remains unclear since clinical studies thus far have shown discordant results. Tolerance towards the effects of inhaled beta 2-AR agonists generally is more easily shown for systemic parameters than for airway effects and can be substantial. This study evaluates whether differences exist between position 16 homozygous genotyped asthmatics, in tolerance development towards airway responses and the systemic effect hypokalemia. METHODS: Twenty patients were genotyped for amino acids 16 and 27 of the beta 2-AR gene. Time-effect curves for FEV1 and serum potassium concentration were constructed after s.c. administration of terbutaline after two-week treatment periods with either terbutaline inhalation or matching placebo in a double-blind, randomised and cross-over design. Statistical analysis was done by a repeated measures multivariate analysis on area under time-effect curve (AUC). MAIN RESULTS: Pre-treatment with inhaled terbutaline did not influence the improvement in FEV1 in response to s.c. terbutaline and there were no significant differences between Arg-16 and Gly-16 individuals in this respect. Pre-treatment with inhaled terbutaline resulted in an overall increase of baseline plasma potassium before administration of s.c. terbutaline (3.78-3.95 mmol/L, p=0.034). However, this effect appeared to be solely confined to the Arg-16 homozygous individuals, leading to a statistically highly significant difference between the Arg-16 and Gly-16 subjects (p=0.005). However, there was no genotype related difference in the decrease in plasma potassium response to s.c. terbutaline relative to baseline. CONCLUSION: In patients carrying the Arg-16 genotype the development of hypokalemia by s.c. terbutaline is attenuated after pre-treatment with inhaled terbutaline, be it on the basis of higher baseline values.

Differentially methylated alleles in a distinct region of the human interleukin-1alpha promoter are associated with allele-specific expression of IL-1alpha in CD4+ T cells.

Cytokine secretion profiles of activated T cells are critical for maintaining the immunologic balance between protection and tolerance. In mice, several cytokines have been reported to exhibit monoallelic expression. Previously, we found that the human interleukin-1 alpha (IL1A) gene exhibits a stable allele-specific expression pattern in CD4+ T-cell clones. We investigated whether DNA methylation is involved in the allele-specific expression of IL-1alpha. Here, we show that differential methylation of CpGs in the proximal promoter region is associated with allele-specific expression of IL-1alpha in CD4+ T cells. The differential methylation pattern is already observed in naive T cells. In keratinocytes, which constitutively produce IL-1alpha, the proximal promoter is hypomethylated. CpGs located further upstream and in intron 4 were almost all methylated, irrespective of expression. Treatment of nonexpressing cells and of T-cell clones with 5-aza-2'deoxycytidine induced IL-1alpha expression in the nonexpressing cells and induced expression of the formerly silent allele in T-cell clones. In addition, electrophoretic mobility shift assays showed that methylation of CpGs in the proximal promoter resulted in direct inhibition of binding of nuclear factor(s). Taken together, these results suggest that allele-specific expression of IL-1alpha in CD4+ cells is achieved, at least in part, by differential methylation of the promoter.

Limited beta2-adrenoceptor haplotypes display different agonist mediated airway responses in asthmatics.

BACKGROUND: In vitro and some in vivo studies suggested that genetic haplotypes may have an impact on beta2-agonist mediated airway responses in asthmatics. Due to strong linkage disequilibrium the single nucleotide polymorphisms (SNPs) in the beta2-adrenoceptor gene result in only a limited number of haplotypes. We intended to evaluate the impact of beta2-adrenoceptor haplotypes on beta2-agonist mediated airway responses and the development of tolerance in mild to moderate asthmatics. METHODS: Patients were genotyped for the part of the beta2-adrenoceptor gene with a known bearing on receptor function and regulation. Cumulative dose response curves of fenoterol versus PD20 methacholine and FEV1 were constructed after 2 week treatment periods with either terbutaline or placebo in a double blind, randomised and cross-over design. Analysis of the dose response curves was based on a repeated measurement analysis of covariance. RESULTS: In our study population comprising 45 asthmatic patients, we found three limited allelic haplotypes, resulting in six different genotypes. Our data support the existence of differences between these six genotypes both in the shape of the dose response relationship of the beta2-adrenoceptor agonist fenoterol as well as in the propensity to develop tolerance for these effects by pre-treatment with terbutaline. However, this could only be substantiated for the endpoint PD20 methacholine. CONCLUSION: Between beta2-adrenoceptor genotypes differences exist both in baseline beta2-agonist induced airway responses as well as in the propensity to develop tolerance during maintenance beta2-agonist therapy. The net differences after two weeks of therapy are, however, of magnitudes that are unlikely to be of clinical significance.

Selective probiotic bacteria induce IL-10-producing regulatory T cells in vitro by modulating dendritic cell function through dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin.

BACKGROUND: Lactobacilli are probiotic bacteria that are frequently tested in the management of allergic diseases or gastroenteritis. It is hypothesized that these probiotics have immunoregulatory properties and promote mucosal tolerance, which is in part mediated by regulatory T cells (Treg cells). On the basis of pathogenic or tissue-specific priming, dendritic cells (DC) acquire different T cell-instructive signals and drive the differentiation of naive T H cells into either T H 1, T H 2, or regulatory effector T cells. OBJECTIVE: We studied in what way different species of lactobacilli prime human DCs for their ability to drive Treg cells. METHODS: Human monocyte-derived DCs were cultured in vitro with lactobacilli of different species. RESULTS: Two different species of lactobacilli, Lactobacillus reuteri and Lactobacillus casei , but not Lactobacillus plantarum, prime monocyte-derived DCs to drive the development of Treg cells. These Treg cells produced increased levels of IL-10 and were capable of inhibiting the proliferation of bystander T cells in an IL-10-dependent fashion. Strikingly, both L reuteri and L casei , but not L plantarum , bind the C-type lectin DC-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN). Blocking antibodies to DC-SIGN inhibited the induction of the Treg cells by these probiotic bacteria, stressing that ligation of DC-SIGN can actively prime DCs to induce Treg cells. CONCLUSIONS: The targeting of DC-SIGN by certain probiotic bacteria might explain their beneficial effect in the treatment of a number of inflammatory diseases, including atopic dermatitis and Crohn's disease.

Limited impact of multiple 5' single-nucleotide polymorphisms on the transcriptional control of the human beta 2-adrenoceptor gene.

The human beta(2)-adrenoceptor (beta(2)-AR) is subject to agonist-induced down-regulation. The degree of down-regulation is associated with certain single-nucleotide polymorphisms (SNPs) through yet unknown mechanisms. The 5'-leader sequence of the beta(2)-AR gene contains several SNPs that are in strong linkage disequilibrium. The -367 T/C polymorphism, in particular, has been shown to affect transcriptional activity in reporter gene assays. In the present study we analysed the impact of this -367 SNP on the transcription rate of the beta(2)-AR gene in the context of the complete natural locus. Taking advantage of the additional full disequilibrium with the +79 SNP in the beta(2)-AR coding sequence, allele-specific transcript quantification was performed in PBMCs of six -367 heterozygous mild asthmatic patients. Our data are in line with the reported impact of the -367 SNP and give no indication of additional haplotype-related effects on beta(2)-AR transcription. We further show that the -367 SNP affects the binding of a yet unidentified transcription factor complex, whose binding activity is not modulated by pharmacological compounds that induce or down-regulate beta(2)-AR expression, suggesting a role in constitutive steady state expression rather than in inducible expression. As the beta(2)-AR allele with a higher transcription rate associates with stronger agonist-induced down-regulation, it is not likely that the -367 SNP is causally related to the degree of down-regulation.

ICOS expression by activated human Th cells is enhanced by IL-12 and IL-23: increased ICOS expression enhances the effector function of both Th1 and Th2 cells.

Previous mouse studies have shown that IL-4 increases the expression of ICOS on activated Th cells, resulting in enhanced ICOS expression on Th2 cells. In this study, we show that ICOS expression on human Th cells is not increased by IL-4, but by IL-12 and by IL-23 instead. Consequently, ICOS expression during IL-12-driven Th1 cell polarization was transiently increased compared with the levels on Th0 cells and IL-4-driven Th2 cells. Addition of IL-12 and/or IL-23 during restimulation increased ICOS expression to the same extent on pre-established Th1, Th2, and Th0 cells, indicating that ICOS levels are not stably imposed by prior polarization. In contrast to the findings in the mouse, IL-4 significantly suppressed the ICOS-enhancing effects of IL-12 and IL-23. The functional consequence of variable ICOS levels was shown in coculture experiments with cells expressing the ICOS-ligand B7-related protein 1 (either transfected Chinese hamster ovary cells or autologous dendritic cells). Ligation of ICOS on 2-day-preactivated effector cells increased their cytokine production to an extent proportional to their ICOS expression levels. As the ICOS-enhancing potentials of IL-12 and IL-23 were maintained for several days after stimulation, both on Th1 and Th2 cells, we propose the concept that local regulation of ICOS expression on activated Th cells by IL-12 and/or IL-23 may provide a powerful means to amplify effector T cell responses in peripheral tissues, independently of the polarized state of the Th cells.

ICOS-mediated signaling regulates cytokine production by human T cells and provides a unique signal to selectively control the clonal expansion of Th2 helper cells.

The CD28 homologue inducible costimulator (ICOS) has been demonstrated to regulate a number of T cell-dependent immune responses in vivo. However, the expression and functional importance of ICOS during APC-Th cell interaction in the human is not fully understood. Here, we demonstrate that ICOS-mediated signaling plays an important role in the production of selective cytokines during both primary and subsequent Th cell responses upon allospecific or superantigen activation. In contrast, ICOS does not play a role in the differentiation of naive cells into Th1 or Th2 effector cells, nor does it determine the type of effector function of memory cells upon subsequent allogeneic challenge. In addition, our data demonstrate that ICOS provides a novel and unique role in regulating DC-mediated Th2, but not Th1 cell clonal expansion. These data suggest that ICOS-mediated signaling plays a discrete role in the regulation of human T helper cell responses.

Commensal Gram-negative bacteria prime human dendritic cells for enhanced IL-23 and IL-27 expression and enhanced Th1 development.

Dendritic cells (DC) are the main orchestrators of specific immune responses. Depending on microbial information they encounter in peripheral tissues, they promote the development of Th1, Th2 or unpolarized Th cell responses. In this study we have investigated the immunomodulatory effect of non-pathogenic intestinal Gram-negative (Escherichia coli, Bacteroides vulgatus,Veillonella parvula, Pseudomonas aeruginosa) and Gram-positive (Bifidobacterium adolescentis, Enterococcus faecalis, Lactobacillus plantarum and Staphylococcus aureus) bacteria on human monocyte-derived DC (moDC). None of the Gram-positive bacteria (GpB) primed for Th1 or Th2 development. In contrast, despite the low levels of IL-12 they induce, all Gram-negative bacteria (GnB) primed moDC for enhanced Th1 cell development, which was dependent on IL-12 and an additional unidentified cofactor. Strikingly, GnB-matured moDC expressed elevated levels of p19 and p28 mRNA, the critical subunits of IL-23 and IL-27, respectively, suggesting that the IL-12 family members may jointly be responsible for their Th1-driving capacity. Purified major cell wall components of either GnB or GpB did not yield Th cell profiles identical to those obtained with whole bacteria, and could not explain the induction of the IL-12 family members nor Th1 priming by GnB. Importantly, this study gives indications that the expression of the different IL-12 family members is dictated by different priming conditions of immature DC.

Allele-specific expression of the IL-1 alpha gene in human CD4+ T cell clones.

A number of reports have described the monoallelic expression of murine cytokine genes. Here we describe the monoallelic expression of the human IL-1alpha gene in CD4+ T cells. Analysis of peripheral blood T cell clones derived from healthy individuals revealed that the IL-1alpha gene shows predominantly monoallelic expression. Monoallelic expression was observed in Th0, Th1, and Th2 cell clones. In addition, we demonstrate monoallelic expression in T cell clones from rheumatoid arthritis patients derived from synovial fluid of the knee joint, suggesting that the occurrence of this phenomenon is not different from that in clones derived from healthy individuals. The finding of monoallelic expression of a cytokine gene in human CD4+ T cell clones provides evidence for allele-specific silencing/activation as another layer of regulation of IL-1alpha gene expression.

Analysis of allelic expression patterns of IL-2, IL-3, IL-4, and IL-13 in human CD4+ T cell clones.

The occurrence of monoallelic expression of cytokine genes in single cells has been convincingly demonstrated, but there have been few reports of this phenomenon in T cell clones. Here we describe studies on the expression of alleles of the human genes encoding IL-2, IL-3, IL-4, and IL-13 in human CD4(+) T cell clones. In contrast to the results reported in mouse T cell clones and single human T cells, we found no evidence for the monoallelic expression of the IL-2, IL-3, and IL-13 genes. The gene for IL-4 showed an imbalance in expression from each allele, indicating differential expression of IL-4 alleles within or between IL-4-expressing cells.

Glatiramer acetate (copolymer-1, copaxone) promotes Th2 cell development and increased IL-10 production through modulation of dendritic cells.

Glatiramer acetate (GA; copolymer-1, Copaxone) suppresses the induction of experimental autoimmune encephalomyelitis and reduces the relapse frequency in relapsing-remitting multiple sclerosis. Although it has become clear that GA induces protective degenerate Th2/IL-10 responses, its precise mode of action remains elusive. Because the cytokine profile of Th cells is often regulated by dendritic cells (DC), we studied the modulatory effects of GA on the T cell regulatory function of human DC. This study shows the novel selective inhibitory effect of GA on the production of DC-derived inflammatory mediators without affecting DC maturation or DC immunostimulatory potential. DC exposed to GA have an impaired capacity to secrete the major Th1 polarizing factor IL-12p70 in response to LPS and CD40 ligand triggering. DC exposed to GA induce effector IL-4-secreting Th2 cells and enhanced levels of the anti-inflammatory cytokine IL-10. The anti-inflammatory effect of GA is mediated via DC as GA does not affect the polarization patterns of naive Th cells activated in an APC-free system. Together, these results reveal that APC are essential for the GA-mediated shift in the Th cell profiles and indicate that DC are a prime target for the immunomodulatory effects of GA.

Monocyte-derived dendritic cells exposed to Der p 1 allergen enhance the recruitment of Th2 cells: major involvement of the chemokines TARC/CCL17 and MDC/CCL22.

Dendritic cells (DC) are potent antigen - presenting cells that can orientate the immune response towards a Th1 or a Th2 type. DC produce chemokines that are involved in the recruitment of either Th1 cells, such as IP10 (CXCL10), Th2 cells such as TARC (CCL17) and MDC (CCL22), or non-polarized T cells such as RANTES (CCL5) and MIP-lalpha (CCL3). We investigated whether monocyte-derived DC (MD-DC) generated from healthy donors or from patients sensitive to Dermatophagoides pteronyssinus (Dpt) and exposed to the cysteine-protease Der p 1(allergen of Dpt), could upregulate the expression of chemokines involved in type 1 or type 2 T cell recruitment. MD-DC were pulsed with either Der p 1 or with LPS as the control and the chemokines produced were evaluated using ELISA and chemotaxis assays. Der p 1-pulsed DC from allergic patients showed increased TARC (CCL17) and MDC (CCL22) production without modifying IP-10 (CXCL10) release. Der p 1-pulsed DC from healthy donors showed only increased IP-10 (CXCL10) secretion. RANTES (CCL5) and MIP-lalpha (CCL3) production were similarly increased when DC were from healthy or allergic donors. The selective Th2 clone recruitment activity of supernatants from Der p 1-pulsed DC of allergic patients was inhibited by anti-TARC (CCL17) and anti-MDC (CCL22) neutralizing Abs. By using anti-IP10 (CXCL10) blocking Abs, supernatants of Der p 1-pulsed DC from healthy donors were shown to be involved in the recruitment of Th1 cells. These results suggest that in allergic patients exposed to house dust mites, DC may favour the exacerbation of the Th2 response via the increase in type 2 chemokine production.

CD26/DPPIV signal transduction function, but not proteolytic activity, is directly related to its expression level on human Th1 and Th2 cell lines as detected with living cell cytochemistry.

CD26/DPPIV is a cell surface glycoprotein that functions both in signal transduction and as a proteolytic enzyme, dipeptidyl peptidase IV (DPPIV). To investigate how two separate functions of one molecule are regulated, we analyzed CD26 protein expression and DPPIV enzyme activity on living human T-helper 1 (Th1) and Th2 cells that express different levels of CD26/DPPIV. DPPIV activity was specifically determined with the synthetic fluorogenic substrate ala-pro-cresyl violet and CD26 protein expression was demonstrated with an FITC-conjugated CD26-specific antibody. Fluorescence of liberated cresyl violet (red) and FITC (green) was detected simultaneously on living T-cells using flow cytometry and spectrofluorometry. Th1 cells expressed three- to sixfold more CD26 protein than Th2 cells. The signal transduction function of the CD26/DPPIV complex, tested by measuring its co-stimulatory potential for proliferation, was directly related to the amount of CD26 protein at the cell surface. However, DPPIV activity was similar in both cell populations at physiological substrate concentrations because of differences in K(m) and V(max) values of DPPIV on Th1 and Th2 cells. Western blotting and zymography of Th1 and Th2 whole-cell lysates demonstrated similar patterns. This study shows that two functions of one molecule can be controlled differentially.

Microbial compounds selectively induce Th1 cell-promoting or Th2 cell-promoting dendritic cells in vitro with diverse th cell-polarizing signals.

Upon microbial infection, specific Th1 or Th2 responses develop depending on the type of microbe. Here, we demonstrate that different microbial compounds polarize the maturation of human myeloid dendritic cells (DCs) into stably committed Th1 cell-promoting (DC1) or Th2 cell-promoting (DC2) effector DCs that polarize Th cells via different mechanisms. Protein extract derived from the helminth Schistosoma mansoni induced the development of DC2s that promote the development of Th2 cells via the enhanced expression of OX40 ligand. Likewise, toxin from the extracellular bacterium Vibrio cholerae induced development of DC2s as well, however, via an OX40 ligand-independent, still unknown mechanism. In contrast, toxin from the intracellular bacterium Bordetella pertussis induced the development of DC1s with enhanced IL-12 production, which promotes a Th1 cell development. Poly(I:C) (dsRNA, mimic for virus) induced the development of extremely potent Th1-inducing DC1, surprisingly, without an enhanced IL-12 production. The obtained DC1s and DC2s are genuine effector cells that stably express Th cell-polarizing factors and are unresponsive to further modulation. The data suggest that the molecular basis of Th1/Th2 polarization via DCs is unexpectedly diverse and is adapted to the nature of the microbial compounds.

Intercellular adhesion molecule-1/LFA-1 ligation favors human Th1 development.

Th cell polarization toward Th1 or Th2 cells is strongly driven by exogenous cytokines, in particular IL-12 or IL-4, if present during activation by Ag-presenting dendritic cells (DC). However, additional Th cell polarizing mechanisms are induced by the ligation of cell surface molecules on DC and naive Th cells. In the present study, the role of LFA-1/ICAM-1 ligation in human Th cell polarization was investigated. Triggering of LFA-1 on anti-CD3/CD28 stimulated naive Th cells with immobilized Fc-ICAM-1, in the absence of DC and exogenous cytokines, induced a marked shift toward Th1 cell development, accompanied by a dose-dependent decrease in GATA-3 expression and a dose-dependent increase in T-bet expression. Th1 polarization by LFA-1 ligation could be demonstrated only under low cytokine conditions, as it was largely overruled by IL-12 or IL-4. This IL-12-independent Th1-driving mechanism appears to be operated by certain subsets of effector DC. Maturation of DC by poly(I:C), a synthetic dsRNA, used as an in vitro model for viral infections, leads to the generation of Th1-driving effector DC (DC1), which express elevated levels of ICAM-1 but produce only low levels of IL-12p70. Blocking the ICAM-1/LFA-1 interaction in cocultures of these DC with naive Th cells attenuated their Th1-driving capacity. The molecular mechanism by which LFA-1 signaling supports Th1 differentiation is blocked by specific inhibitors of extracellular signal-regulated kinase phosphorylation. The present data indicate the existence of an IL-12-independent, extracellular signal-regulated kinase-mediated mechanism, through which high ICAM-1-expressing DC1 can drive Th1 polarization. This mechanism may be operational during viral infections.