The effect of B-cell depletion therapy on serological evidence of B-cell and plasmablast activation in patients with rheumatoid arthritis over multiple cycles of rituximab treatment.

B-cell depletion therapy (BCDT) based on rituximab (RTX) induces clinical remission in a majority of seropositive patients with Rheumatoid arthritis (RA). However, all patients eventually relapse. The aim of this study was to determine whether dynamic changes in combinations of serological measures of B-cell activation were associated over up to three cycles of BCDT. We included only RA patients who gave an adequate clinical response, as measured by DAS28. Twenty three patients were studied over 1 cycle, 21 over 2, and 15 over 3 cycles of BCDT. Serum analytes including isotypes of Rheumatoid factors (RhF) and anti-citrullinated protein/peptide antibodies (ACPA), B-cell activating factor (BAFF), serum free light chains (SFLC), soluble CD23 (sCD23), antibodies to tetanus toxoid (TT) and to pneumococcal capsular polysaccharide (PCP) were measured by ELISA at 4 key points in each cycle, namely: Baseline (pre-RTX in each cycle); when B-cell depleted (CD19+B-cells < 5/µl); at B-cell return (CD19+B-cells ≥ 5/µl); and at clinical relapse (ΔDAS28 > 1.2). SFLC were used as a measure of plasmablast activity. As sCD23 is cleaved from naïve B-cells coincident with attaining CD27 expression, levels were used as a novel measure of maturation of B-cells to CD27+. The most consistent changes between baseline and B-cell depletion within all 3 cycles were in SFLC, sCD23 and IgM-RhF which fell and in BAFF levels which rose. After 3 complete cycles of BCDT, both IgM autoantibodies and IgG-CCP had decreased, BAFF levels were higher (all p < 0.05); other analytes remained unchanged compared with baseline. Dynamic changes in λSFLC, sCD23, IgM-RhF and BAFF were also consistently associated with relapse in patients with longer clinical responses after B-cell return. Incremental rises in sCD23 levels in cycles 2 and 3 were correlated with time to relapse. Repopulation of the periphery after BCDT is initiated by naïve B-cells and precedes relapse. Our study showed that differentiation into plasmablasts, attended by sCD23 and SFLC production and IgM-RhF specificity may be required to precipitate relapse in patients experiencing longer responses after RTX. These studies also provide novel information related to the resumption of autoimmune responses and their association with B-cell kinetics following BCDT.

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.

Joint cytokine quantification in two rodent arthritis models: kinetics of expression, correlation of mRNA and protein levels and response to prednisolone treatment.

Biomarker quantification in disease tissues from animal models of rheumatoid arthritis (RA) can help to provide insights into the mechanisms of action of novel therapeutic agents. In this study we validated the kinetics of IL-1beta, TNF-alpha and IL-6 mRNA and protein expression levels in joints from DBA/1OlaHsd murine collagen-induced arthritis (CIA) and Lewis rat Streptococcal cell wall (SCW)-induced arthritis by real-time polymerase chain reaction (PCR) TaqMan and Enzyme-linked immunosorbent assay (ELISA). Prednisolone was used as a reference to investigate any correlation between clinical response and cytokine levels at selected time-points. To our knowledge this is the first report showing a close pattern of expression between mRNA and protein for IL-1beta and IL-6, but not for TNF-alpha, in these two models of RA. The kinetics of expression for these biomarkers suggested that the optimal sampling time-points to study the effect of compounds on both inflammation and cytokine levels were day 4 postonset in CIA and day 3 after i.v challenge in SCW-induced arthritis. Prednisolone reduced joint swelling through a mechanism associated with a reduction in IL-1beta and IL-6 protein and mRNA expression levels. At the investigated time points, protein levels for TNF-alpha in arthritic joints were lower than the lower limit of detection of the ELISA, whereas mRNA levels for this cytokine were reliably detected. These observations suggest that RT-PCR TaqMan is a sensitive technique that can be successfully applied to the quantification of mRNA levels in rodent joints from experimental arthritis models providing insights into mechanisms of action of novel anti-inflammatory drugs.

Identification of a novel beta-tubulin subfamily with one member (TUBB4Q) located near the telomere of chromosome region 4q35.

The human beta-tubulin supergene family consists of several isotypes with many associated pseudogenes. Here we report the identification of yet another beta-tubulin sequence designated TUBB4Q. This tubulin maps 80 kb proximal to the facioscapulohumeral muscular dystrophy (FSHD1) associated D4Z4 repeats on chromosome 4q35. The genomic structure contains four exons encoding a putative protein of 434 amino acids. The TUBB4Q nucleotide and protein sequence show 87% and 86% homology to beta2-tubulin, respectively. Although the genomic structure shows all functional aspects of a genuine gene, no transcript could be detected. TUBB4Q-related sequences were identified on multiple chromosomes. Since these sequences mutually exhibit a high nucleotide sequence homology, they presumably belong to a novel subfamily of beta-tubulin genes. Although the chromosome 4q35 tubulin-member probably represents a pseudogene, ectopic expression due to a postulated position effect variegation (PEV), makes TUBB4Q an ideal dominant-negative candidate gene for FSHD1.

Fluid shear stress induction of the tissue factor promoter in vitro and in vivo is mediated by Egr-1.

Hemodynamic forces such as fluid shear stress have been shown to modulate the activity of an expanding family of genes involved in vessel wall homeostasis and the pathogenesis of vascular disease. We have investigated the effect of shear stress on tissue factor (TF) gene expression in human endothelial cells (ECs) and in a rat arterial model of occlusion. As measured by reverse transcriptase polymerase chain reaction, exposure of ECs to 1.5 N/m2 shear stress resulted in a time-dependent induction of endogenous TF transcripts of over 5-fold. Transient transfection of TF promoter mutants into cultured ECs suggests the involvement of the transcription factor Egr-1 in mediating the response of the TF promoter to shear stress. To address the importance of flow induction of Egr-1 in vivo, we have established a flow-restricted rat arterial model and determined the level of expressed Egr-1 and TF at the site of restricted flow using immunohistochemistry. We report an increase in the level of Egr-1 and TF protein in ECs expressed at the site of restricted flow. Elevated expression of Egr-1 and TF is restricted to a highly localized area, as evidenced by the fact that no significant increase in level can be detected at arterial sites distal to the site of occlusion. These findings suggest a direct role for Egr-1 in flow-mediated induction of TF and further substantiate the importance of shear stress as a modulator of vascular endothelial gene function in vivo.

High-throughput RT-PCR analysis of multiple transcripts using a microplate RNA isolation procedure.

We have developed a high-throughput, multiplex reverse transcription PCR (RTPCR) assay that is suitable for the analysis of medium-to low-copy cellular RNA transcripts from small numbers of cells (10(4)). High throughput was attained by utilizing microplate-based RNA extraction and RTPCR protocols, followed by PCR product visualization of a multiwelled agarose gel, stained with SYBR Green I dye. The transcriptional assay was unaffected by solvents (dimethyl sulfoxide and methanol) routinely used in high-throughput drug screens at concentrations required for compound solubilization. Furthermore, it has been used successfully for the investigation of differential mRNA expression levels of tumor necrosis factor alpha (TNF-alpha) and Interleukin-1 beta (IL-1 beta) in lipopolysaccharide (LPS)-stimulated THP-1 cells (a human monocytic cell line) and the identification of specific IL-1 beta transcriptional inhibitors.

Defective haematopoiesis and vasculogenesis in transforming growth factor-beta 1 knock out mice.

Transforming growth factor beta 1 (TGF beta 1) is shown here to be required for yolk sac haematopoiesis and endothelial differentiation. Mice with a targeted mutation in the TGF beta 1 gene were examined to determine the cause of prenatal lethality, which occurs in 50% of homozygous TGF beta 1 null (TGF beta 1-/-) conceptions. 50% of TGF beta 1-/- and 25% of TGF beta 1-+-) conceptions. 50% of TGF beta 1-/- and 25% of TGF beta 1+/- conceptuses were found to die at around 10.5 dpc. The primary defects were restricted to extraembryonic tissues, namely the yolk sac vasculature and haematopoietic system. The embryos per se showed developmental retardation, oedema and necrosis, which were probably secondary to the extraembryonic lesions. The defect in vasculogenesis appeared to affect endothelial differentiation, rather than the initial appearance and outgrowth of endothelial cells. Initial differentiation of yolk sac mesoderm to endothelial cells occurred, but defective differentiation resulted in inadequate capillary tube formation, and weak vessels with reduced cellular adhesiveness. Defective haematopoiesis resulted in a reduced erythroid cell number within the yolk sac. Defective yolk sac vasculogenesis and haematopoiesis were present either together, or in isolation of each other. The phenotypes are consistent with the observation of abundant TGF beta 1 gene expression in both endothelial and haematopoietic precursors. The data indicate that the primary effect of loss of TGF beta 1 function in vivo is not increased haematopoietic or endothelial cell proliferation, which might have been expected by deletion of a negative growth regulator, but defective haematopoiesis and endothelial differentiation.

The porcine insulin-like growth factor-I gene: characterization and expression of alternate transcription sites.

Genomic DNA encoding the 5' region of the porcine IGF-I gene was cloned and sequenced and shown to be highly homologous to that of man, rats and sheep. Two leader exons (exons 1 and 2), which are alternately spliced to exon 3 (encoding part of the mature IGF-I molecule), were identified by RNase protection analysis. In both cases, transcription initiates upstream from exons 1 and 2 at multiple dispersed start sites to yield two distinct IGF-I mRNA transcript classes (1 and 2) which differ in the precursor peptides predicted from their individual leader sequences. The expression of class 1 and 2 transcripts was measured in liver and muscle RNA from two groups of 2-month-old pigs whose energy status had been manipulated within physiological limits to produce marked differences in plasma IGF-I levels and growth rates. For this purpose, RNase protection probes were developed that contained the individual leader exons 1 and 2 linked separately to the common exon 3, so that class-specific and total IGF-I gene expression could be determined in a single assay. At normal plasma IGF-I concentrations (200 ng/ml), class 1 and 2 transcripts comprised 81 and 19% respectively of total liver IGF-I mRNA, while at a lower plasma concentration (90 ng/ml) the corresponding values were 95 and 5% respectively. Although both classes of transcript declined with the decrease in plasma IGF-I, the relative drop in levels of class 2 transcripts (84%) was substantially greater than that of class 1 (54%). In longissimus dorsi, cardiac and soleus muscles IGF-I mRNA was predominantly of class 1 and did not change in response to decreased plasma IGF-I. This suggests that liver-derived endocrine IGF-I has an important function in the regulation of muscle growth and that class 2 IGF-I transcripts are more sensitive to conditions that promote optimal growth.

RNA and protein localisations of TGF beta 2 in the early mouse embryo suggest an involvement in cardiac development.

We have performed a detailed analysis of the localisations of RNAs for TGF beta 2 and beta 3, and of TGF beta 2 protein in mouse embryos from 6.5 to 9.5 days post coitum, using in situ hybridisation and immunohistochemistry on serial sections, and whole-mount in situ hybridisation to complete embryos. TGF beta 3 RNA was not seen in any of the tissue sections, but very low levels of the RNA were seen by whole-mount in situ hybridisation around the outflow tract of the heart at 8.5 days post coitum. TGF beta 2 RNA is expressed at high levels in all cells with the potential to differentiate into cardiomyocytes. Additionally, the foregut endoderm, juxtaposed to the heart, and the neuroepithelium at the rostral extremity of the foregut, express very high levels of TGF beta 2 RNA, between 8.5 and 9.5 days post coitum. As cardiomyogenesis proceeds, TGF beta 2 RNA levels diminishes within the myocytes, with a concomitant increase in staining for TGF beta 2 protein. TGF beta 2 protein staining of cardiomyocytes persists throughout development and in the adult, in the absence of detectable levels of the corresponding RNA. Superimposed upon this myocardial pattern of expression, there is an upregulation of TGF beta 2 RNA in the myocardium of the outflow tract and atrioventricular canal between 8.5 and 9.5 days post coitum, which returns to low levels by 11.5 days post coitum. The results are discussed in terms of a potential role of TGF beta 2 in controlling cardiomyogenesis and in inductive interactions leading to cardiac cushion tissue formation.

Rapid induction and clearance of TGF beta 1 is an early response to wounding in the mouse embryo.

The TGF beta family of growth factors has been implicated as playing a significant role in many aspects of embryonic morphogenesis, and also as a mediator of adult tissue repair processes. Unlike the situation in the adult, tissue repair in the embryo does not result in scarring, and it has been suggested that this might be due, in part, to reduced levels of growth factors, particularly TGF beta, at the wound site. We have examined the expression patterns of TGF beta genes following wounding of limb bud lesions in cultured E11.5 mouse embryos. The timetable of wound closure was investigated by standard light and electron microscopy from the time of wounding until the lesion had re-epithelialised 24 hours later. The expression of transcripts for each of the three TGF beta genes was examined at various time points during the healing process using radioactive in situ hybridisation to tissue sections and wholemount non-radioactive in situ hybridisation to embryo pieces. Within 1 to 3 hours of wounding, transcripts encoding TGF beta 1 were rapidly induced within the epithelial cells of the wound margin, particularly those cells at the ventral aspect of the wound. By 3 to 6 hours post-wounding, TGF beta 1 transcripts were detectable in the mesenchyme of the wound bed. No TGF beta 3 induction was observed, and possible TGF beta 2 induction was largely obscured by endogenous expression associated with pre-cartilage mesenchymal condensation. Immunocytochemical analysis of tissue sections of the wound demonstrated a rapid induction of TGF beta 1 protein within 1 hour post-wounding, but also a subsequent rapid clearance of the protein from the wound site such that, by 18 hours post-wounding, TGF beta 1 levels had returned to near background. These data are discussed in terms of the molecular mechanisms underlying embryonic wound healing and the significance of the results to an understanding of scarring following adult tissue repair.

Natriuretic peptide receptor mRNAs in the rat and human heart.

Functional studies indicate that atrial natriuretic peptide (ANP), a member of the natriuretic peptide family, has direct effects on cardiac muscle cells. However, conventional ligand-binding studies designed to establish the presence of natriuretic peptide-binding sites in the heart have yielded conflicting results. There are discrepancies also between the latter and the receptor distribution predicted from the pattern of the mRNA transcripts localized by in situ hybridization. Here we have employed the technique of cDNA amplification with the polymerase chain reaction to confirm the presence of natriuretic peptide A, B, and C receptor mRNAs in rat and human cardiac tissue. In the rat heart, the distribution of the A and B receptor transcripts appears to be relatively homogeneous; in contrast, the C type mRNA is concentrated principally in the atria, with no difference between the left and right sides of the heart. A and B receptor DNA products were obtained after amplification of left, but not right, ventricular cDNA from the heart of a 16-yr-old male with cystic fibrosis; the yield of C receptor DNA was similar for both ventricles. If these mRNA transcripts are translated into functional receptors in the rat and human heart, ANP and the other natriuretic peptides may have direct effects on cardiac function, including regulation of natriuretic peptide release via a short feedback loop, modulation of contractility of the heart, or activation of cardiac reflexes.

Expression of a growth hormone-responsive exon of the ovine insulin-like growth factor-I gene.

The sheep insulin-like growth factor-I (IGF-I) gene encodes mRNAs containing three different 5'-untranslated sequences as a consequence of alternate splicing of leader exons. Using a combination of RNase protection and primer extension assays, we have mapped the transcriptional start sites of one of the leader exons, exon 1A. Transcription from exon 1A appeared to initiate from multiple points within a 20 bp region situated about 60 bp upstream of the exon 1A splice site. The presence of this transcript in the liver of animals treated with GH was enhanced five- to tenfold and contributed to about 95% of the total hepatic increase in IGF-I mRNA. This exon is generally expressed in a number of tissues immediately after birth; by about 4 weeks postpartum, however, expression is confined to liver. The regulation of hepatic and non-hepatic IGF-I synthesis by GH may involve different mechanisms.

The ovine insulin-like growth factor-I gene: characterization, expression and identification of a putative promoter.

Genomic DNA encoding the ovine insulin-like growth factor-I (IGF-I) gene was cloned and sequenced. The predicted amino acid sequence of the mature form of ovine IGF-I was highly homologous to that of human, rat and mouse. Analysis of the DNA sequence between exons 1 and 2 suggested the existence of an alternative 5' exon (exon 1A) and this was confirmed by polymerase chain reaction (PCR) analysis of sheep liver mRNA. Primer extension of mRNA from exon 1A indicated a class of transcripts which initiated at a point 32 nucleotides 5' to the Met codon of exon 1A to give a mRNA comprising exons 1A, 2, 3 and 5. In liver these transcripts co-existed with the alternative exon 1, 2, 3 and 5 mRNA form. Analysis by PCR of the 3' terminus of liver RNA indicated heterogeneity arising from multiple polyadenylation sites; however, of the two possible alternatively spliced 3' exons, only exon 5 could be detected. Expression of IGF-I mRNA, as measured by a solution hybridization/RNase protection assay, predominated in the liver of the neonate and the late-gestation fetus; however, lower levels of expression were seen in multiple tissues throughout fetal and neonatal development.