Investigation of selective JAK1 inhibitor GSK2586184 for the treatment of psoriasis in a randomized placebo-controlled phase IIa study.

BACKGROUND: GSK2586184 is a selective oral Janus kinase (JAK)1 inhibitor being evaluated as a treatment for moderate-to-severe plaque-type psoriasis. OBJECTIVES: To assess the relationship between dose of GSK2586184 and clinical response, primarily by the Psoriasis Area Severity Index (PASI). METHODS: Sixty patients with moderate-to-severe plaque psoriasis were randomized to cohort A: 100 mg, 200 mg or 400 mg GSK2586184 twice daily or placebo; and eight were randomized to open-label cohort B, a small exploratory cohort treated with 400 mg GSK2586184 twice daily, to explore differential gene expression. RESULTS: At week 12, a 75% reduction in PASI (PASI 75) response rates in the intent-to-treat population were 0% in the placebo group compared with 13%, 25% and 57% in the 100 mg, 200 mg and 400 mg GSK2586184 twice-daily groups, respectively. Increases in the proportion of PASI 75 responses were seen across all dose levels by week 4. Improvement in itch and quality of life were observed at all doses relative to placebo with the greatest improvement seen in the 400-mg dose group. Overall, the incidence of adverse events (AEs) was similar across treatment groups, and no relationship between frequency of AE and GSK2586184 dose was identified. Differential gene expression was observed in involved and uninvolved skin at baseline and in involved skin after 2 weeks of treatment with GSK2586184. CONCLUSIONS: Our study demonstrates that 12 weeks of treatment with GSK2586184 resulted in clinical improvement and was generally well tolerated in patients with moderate-to-severe plaque-type psoriasis.

Evidence of epistasis between interleukin 1 and selenoprotein-S with susceptibility to rheumatoid arthritis.

OBJECTIVE: Selenoprotein-S (SELS) is involved in the stress response within the endoplasmic reticulum (ER) and inflammation. Recently, promoter variants in the SELS gene were shown to be associated with plasma levels of interleukin (IL)6, IL1beta and tumour necrosis factor (TNF). It was hypothesised that these variants could influence rheumatoid arthritis (RA) susceptibility and may interact with functional single nucleotide polymorphisms (SNPs) in the genes for IL1, IL6 and TNF. METHODS: Genotyping was performed in 988 unrelated healthy controls and 965 patients with RA. Stratified analysis was used to test for interactions. Single gene effects and evidence of epistasis were investigated using the Mantel-Haenszel (M-H) test and the linkage disequilibrium (LD)-based statistic. RESULTS: No association of SELS -105 genotype and RA susceptibility was detected. Stratification of SELS -105 genotypes by IL1 -511 genotypes showed that the disease risk (comparing AA/GA to GG at the SELS -105 locus) in individuals with the GG/AG genotype at the IL1beta -511 locus was significantly lower than that in individuals having the AA genotype at the IL1beta -511 locus (odds ratio (OR): 0.9 and 2.3, respectively; p = 0.004 by M-H test). Significant epistasis was also detected using the LD-based statistic (p = <0.001). No interaction was observed between SELS -105 and IL6 or TNF variants. CONCLUSION: Our results reveal evidence of strong epistasis in two genes in the IL1 production pathway and highlight the potential importance of gene-gene interactions in the pathogenesis of RA.

Association between radiographic severity of rheumatoid arthritis and shared epitope alleles: differing mechanisms of susceptibility and protection.

OBJECTIVE: To investigate the association of a recently described classification of Human leukocyte antigen (HLA)-DRB1 shared epitope alleles with rheumatoid factors (RF) and anti-cyclic citrullinated peptide (CCP) production and radiological severity in rheumatoid arthritis (RA). METHODS: Patients with RA (n = 962) were studied. Genotyping of DRB1 alleles and assays for RF and anti-CCP were performed. Radiological severity was measured using the modified Larsen score. RESULTS: In accordance with previous reports, we found carriage of S2 alleles (K-R-A-A at positions 71-74) to be associated with more severe disease with a gene-dose effect (p = 0.0059), and also associated with the presence of anti-CCP and RF (p<0.001). Carriage of S1 alleles (D-E-R-A-A at positions 70-74) was associated with less severe disease (p = 0.01), however there was no association between S1 and either anti-CCP or RF, suggesting that the basis for this possible protective effect was not related to autoantibody-producing B cells. CONCLUSIONS: These data suggest that multiple biological mechanisms underlie the DRB1 association with rheumatoid arthritis severity.

Association of interleukin-6 and interleukin-10 genotypes with radiographic damage in rheumatoid arthritis is dependent on autoantibody status.

OBJECTIVE: Recent evidence has highlighted a major genetic contribution to radiographic damage in rheumatoid arthritis (RA). The objective of this study was to determine whether genetic variants in the loci for interleukin-1 (IL-1), IL-6, IL-10, protein tyrosine phosphatase N22 (PTPN22), and selenoprotein S are associated with radiographic damage. METHODS: Modified Larsen scores of radiographic damage were determined in a cross-sectional population of patients with RA (n = 964). Rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) were also assayed. The Kruskal-Wallis nonparametric test was used to compare median radiographic damage scores across genotype groups, followed by the Cuzick nonparametric test for trend to assess gene-dose effects. RESULTS: An allele-dose association of IL-6 -174G with increasing radiographic damage was present (P = 0.005), but only in patients who were RF positive (P = 0.004) or anti-CCP positive (P = 0.01). Patients with the IL-10 -592CC genotype had more extensive radiographic damage than did those with the AC or AA genotype (P = 0.006), but this was observed only among patients who were RF negative (P = 0.002) or anti-CCP negative (P = 0.002). However, RF status and anti-CCP status were not associated with the IL-6 or IL-10 genotype. No other genetic associations were detected, apart from a marginal association of PTPN22 +1858T with increased radiographic damage. CONCLUSION: The reported associations of IL-6 -174G with high IL-6 production and IL-10 -592 with low IL-10 production and our own results support a role of genetically determined dysregulated cytokine production in disease severity. The lack of association of these genotypes with RF and anti-CCP antibody status suggests that they act downstream of autoantibody production. We conclude that IL-6 and IL-10 genotypes may be useful in predicting disease severity in autoantibody-positive and autoantibody-negative patients, respectively.

Novel molecular mechanisms of dendritic cell-induced T cell activation.

In this study we have re-examined the molecular mechanisms involved in activation of T cells by dendritic cells (DC). Human peripheral blood DC (PBDC) were derived by 2 h adhesion followed by 7 day culture in a combination of granulocyte macrophage colony stimulating factor and IL-4, and depletion of residual T and B cells. These PBDC were used to induce autologous T cell proliferation in a CD3-dependent response, and antibodies against CD11a/18 and CD86 were used as control inhibitors of accessory function. Antibodies against five of the cell surface molecules that we have recently identified on the surface of DC, CD13, CD87, CD98, CD147 and CD148, and an antibody which recognizes a molecule that has not as yet been identified, all inhibited the CD3-induced T cell proliferation. These findings were observed not only when antibodies were present throughout the culture, but also when they were prepulsed on to the surface of the DC, suggesting the inhibition was mediated via the antigen-presenting cells rather than the T cell. The same set of antibodies also inhibited an allospecific mixed lymphocyte reaction, confirming that the inhibitory effect was not dependent on the use of a CD3 antibody as the stimulating agent. All the antibodies of known specificity inhibited both CD4 and CD8 T cells equally. Unlike CD87, CD98 and CD147 antibodies, which inhibited activation of both CD45RA (naive) T cells and CD45RO (memory) T cells, CD13 and CD148 appeared to be involved in activation of naive cells only. The molecules identified in this study have not previously been demonstrated to play a role as accessory molecules on DC, the cells that are pivotal for immune induction. Therefore they may provide new potential targets for modulation of the immune response at the APC level.

Pure populations of transduced primary human cells can be produced using GFP expressing herpes virus vectors and flow cytometry.

Herpes simplex virus (HSV) has often been suggested as a vector for gene delivery to the nervous system although it is also capable of infecting many other cell types. HSV also has the ability to package large genetic insertions so the expression of multiple genes from a single virus is possible. Here we show that a green fluorescent protein (GFP) expressing HSV1 vector can transduce two primary human cell types--quiescent human CD34+ hematopoietic progenitor cells and dendritic cells--which are both hard to transduce by other means. We also show that GFP is an effective marker when expressed from an HSV vector in vivo in the mouse brain. When GFP is expressed together with a second gene (in this case lacZ) from a single virus, transduced GFP-positive CD34+ hematopoietic progenitor cells or dendritic cells can both be generated at an effective efficiency of 100% for the second gene. Here transduction with the vector is combined with flow cytometry allowing GFP-positive cells to be sorted from the untransduced population. Such completely transduced populations of quiescent CD34+ hematopoietic progenitor and dendritic cells cannot easily be achieved by other means, and might thus allow experimental or therapeutic protocols to be carried out requiring high-level transduction which would not otherwise be possible. Such an approach using HSV vectors might also be applicable to other cell types for which transduction is as yet unreliable or of low efficiency.

From sentinel to messenger: an extended phenotypic analysis of the monocyte to dendritic cell transition.

The transitional stages in the relationship between sentinel monocytes and messenger dendritic cells that are active in adaptive immunity, are, as yet, unclear. To explore these events, 2-hr adherent peripheral blood mononuclear cells were used either as monocytes, or cultured for 7 days with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) to generate dendritic cells, and the phenotypic features and relationship of the two cell populations was investigated using an extensive panel of monoclonal antibodies (mAbs). The features of the shift from monocyte to dendritic cell were also examined by daily phenotyping during the 7-day culture period. Twenty-five mAbs, most of which recognized known CD molecules, bound both monocytes and dendritic cells equally, whereas 19 mAbs exhibited differential staining. Four molecules not previously reported on dendritic cells were documented: CD87, CD98, CD147 and CD148. Seven cell-surface molecules (HLA-DQ, CD1a, CD13, CD30, CD43, CD63 and CD86) were expressed either at very low levels or not at all on monocytes, but had a strikingly increased expression on dendritic cells, suggesting a role in antigen presentation. The kinetics of monocyte to dendritic cell transition revealed a rapid activation phase within the first 24 hr, with a considerable increase in expression of the activation markers HLA-DR, CD13, CD14 and CD98; this was followed by a down-regulation of CD14 and a more gradual development of the other dendritic cell features over the remaining 6 days, with steady increases in CD1a, CD18, CD43, CD86, HLA-DR and HLA-DQ. Thus, these studies have demonstrated four novel components of the dendritic cell, and have documented the dynamic multistep nature of the process whereby an antigen-presenting dendritic cell phenotype may emerge from a monocyte precursor.

Synthesis of tumor necrosis factor-alpha mRNA in bronchoalveolar lavage cells from human immunodeficiency virus-infected persons with Pneumocystis carinii pneumonia.

Bronchoalveolar lavage fluid cells from a cohort of 34 human immunodeficiency virus-infected persons with established Pneumocystis carinii pneumonia were examined for expression of tumor necrosis factors (TNF-alpha) mRNA by fluorescence in situ hybridization with an antisense riboprobe. Video image analysis was used to develop a quantitative assay that evaluates relative single-cell levels of mRNA. The resulting data were analyzed as an antisense-to-sense ratio and examined for correlation between TNF-alpha mRNA expression and other measures of disease severity. Higher levels of TNF-alpha mRNA were seen in persons who had higher levels of arterial oxygen.