Relationship Between Adalimumab Concentrations, Antidrug Antibodies, and Disease Activity in Rheumatoid Arthritis: A Cross-Sectional Observational Study.

OBJECTIVES: To determine the influence of patient characteristics and disease activity on adalimumab (ADA) concentrations; to assess the relationships between ADA concentrations, the presence of antidrug antibodies (ADAb), and disease activity in rheumatoid arthritis (RA); and to determine the association between cytokine concentrations and ADA concentrations. METHODS: A cross-sectional study of people with RA receiving ADA for at least 4 weeks was undertaken. Disease activity was assessed by the Disease Activity Score in 28 joints (DAS28), with responders defined as DAS28 ≤ 3.2. Serum and plasma were obtained for ADA concentrations and ADAb, and a panel of cytokines were obtained for a subgroup. ADA concentrations were compared between demographic and clinical subgroups using ANOVA. The independent associations between clinical and demographic features were analyzed using a general linear model. Variables significantly associated with ADA concentrations from the univariate analyses were entered into multivariate analyses. RESULTS: Of the 156 participants, 69.2% were female and the mean age was 57.4 (SD 12.7) years. Multivariate analysis revealed that higher C-reactive protein (P < 0.001) and higher weight (P < 0.004) were independently associated with lower ADA concentrations. ADA concentrations were higher in those with DAS28 ≤ 3.2 compared to those with DAS28 > 3.2 (median 10.8 [IQR 6.4-20.8] mg/L vs 7.1 [IQR 1.5-12.6] mg/L, P < 0.001). There was a significant negative correlation between interleukin 6 (IL-6) and ADA concentrations (r = -0.04, P < 0.01). CONCLUSION: ADA concentration correlates negatively with markers of inflammatory disease activity in RA, including IL-6. ADA concentration in the range 5 to 7 mg/L over the dose interval are associated with better disease control.

Activated CD90/Thy-1 fibroblasts co-express the Δ133p53ß isoform and are associated with highly inflamed rheumatoid arthritis.

BACKGROUND: The p53 isoform Δ133p53ß is known to be associated with cancers driven by inflammation. Many of the features associated with the development of inflammation in rheumatoid arthritis (RA) parallel those evident in cancer progression. However, the role of this isoform in RA has not yet been explored. The aim of this study was to determine whether Δ133p53ß is driving aggressive disease in RA. METHODS: Using RA patient synovia, we carried out RT-qPCR and RNAScope-ISH to determine both protein and mRNA levels of Δ133p53 and p53. We also used IHC to determine the location and type of cells with elevated levels of Δ133p53ß. Plasma cytokines were also measured using a BioPlex cytokine panel and data analysed by the Milliplex Analyst software. RESULTS: Elevated levels of pro-inflammatory plasma cytokines were associated with synovia from RA patients displaying extensive tissue inflammation, increased immune cell infiltration and the highest levels of Δ133TP53 and TP53ß mRNA. Located in perivascular regions of synovial sub-lining and surrounding ectopic lymphoid structures (ELS) were a subset of cells with high levels of CD90, a marker of 'activated fibroblasts' together with elevated levels of Δ133p53ß. CONCLUSIONS: Induction of Δ133p53ß in CD90+ synovial fibroblasts leads to an increase in cytokine and chemokine expression and the recruitment of proinflammatory cells into the synovial joint, creating a persistently inflamed environment. Our results show that dysregulated expression of Δ133p53ß could represent one of the early triggers in the immunopathogenesis of RA and actively perpetuates chronic synovial inflammation. Therefore, Δ133p53ß could be used as a biomarker to identify RA patients more likely to develop aggressive disease who might benefit from targeted therapy to cytokines such as IL-6.

Plasma interleukin-23 and circulating IL-17A+IFNγ+ ex-Th17 cells predict opposing outcomes of anti-TNF therapy in rheumatoid arthritis.

OBJECTIVES: TNF-α inhibitors are widely used in rheumatoid arthritis (RA) with varying success. Response to TNF-α inhibition may reflect the evolution of rheumatoid inflammation through fluctuating stages of TNF-α dependence. Our aim was to assess plasma concentrations of Th-17-related cytokines and the presence of circulating effector T-cells to identify predictors of response to TNF-α inhibitors. METHODS: Ninety-three people with RA were seen prior to and 4-6 months after commencing etanercept or adalimumab. Plasma concentrations of Th17-related cytokines, circulating effector T-cells, their production of relevant transcription factors and intracellular cytokines were measured at baseline. EULAR response criteria were used to define poor (ΔDAS28 ≤ 1.2 and/or DAS28 > 3.2) and good (ΔDAS28 > 1.2 and DAS28 ≤ 3.2) responders. Multivariate logistic regression was used to identify predictors of response. RESULTS: Participants with plasma IL-23 present at baseline were more likely to be poor responders [15/20 (75%) of IL-23+ versus 36/73 (49.3%) of IL-23-; p = 0.041]. While frequencies of Th1, Th17, ex-Th17 and Treg cell populations were similar between good and poor responders to anti-TNF therapy, IL-17A+IFNγ+ ex-Th17 cells were more prevalent in good responders (0.83% of ex-TH17 cells) compared to poor responders (0.24% of ex-Th17 cells), p = 0.023. Both plasma IL-23 cytokine status (OR = 0.17 (95% CI 0.04-0.73)) and IL-17A+IFNγ+ ex-Th17 cell frequency (OR = 1.64 (95% CI 1.06 to 2.54)) were independently associated with a good response to anti-TNF therapy. Receiver operator characteristic (ROC) analysis, including both parameters, demonstrated an area under the ROC curve (AUC) of 0.70 (95% CI 0.60-0.82; p = 0.001). CONCLUSIONS: Plasma IL-23 and circulating IL-17A+IFNγ+ ex-Th17 cells are independently associated with response to anti-TNF therapy. In combination, plasma IL-23 and circulating IL-17A+IFNγ+ ex-Th17 cells provide additive value to the prediction of response to anti-TNF therapy in RA.

The contribution from interleukin-27 towards rheumatoid inflammation: insights from gene expression.

We aimed to assess expression of genes encoding the heterodimeric IL-27 cytokine and constituent subunits of the Il-27 receptor in rheumatoid arthritis (RA), including in extra-articular, subcutaneous rheumatoid nodules. Comparing between nodules and joint synovia, significantly elevated expression of IL27A within nodules, and comparable IL27B expression, identified nodules as a significant source of IL-27 in RA. T-lymphocytes were the main source of IL27RA transcript, and IL27RA expression correlated with a number of plasma cytokines, as well as tissue TNF expression in both nodules and RA synovia. In synovia, correlations between IL27A, IL27RA IL17A and CD21L expression, and significantly elevated expression of the genes encoding IL-27, associated the presence of IL-27 with B cell-dominated synovial inflammation. Impact from nodule derived IL-27 on systemic or synovial inflammation in RA remains unknown and further study of these implications is required. Our study raises questions regarding the appropriate circumstances for the blockade or administration of IL-27 as a potential therapeutic adjunct in RA.

Co-expression of CD21L and IL17A defines a subset of rheumatoid synovia, characterised by large lymphoid aggregates and high inflammation.

OBJECTIVE: To determine whether the expression of IL17A and CD21L genes in inflamed rheumatoid synovia is associated with the neogenesis of ectopic lymphoid follicle-like structures (ELS), and if this aids the stratification of rheumatoid inflammation and thereby distinguishes patients with rheumatoid arthritis that might be responsive to specific targeted biologic therapies. METHODS: Expression of IL17A and CD21L genes was assessed by RT-PCR, qRT-PCR and dPCR in synovia from 54 patients with rheumatoid arthritis. A subset of synovia (n = 30) was assessed by immunohistology for the presence of CD20+ B-lymphocytes and size of CD20+ B-lymphocyte aggregates as indicated by maximum radial cell count. The molecular profiles of six IL17A+/CD21L+ and six IL17A-/CD21L- synovia were determined by complementary DNA microarray analysis. RESULTS: By RT-PCR, 26% of synovia expressed IL17A and 52% expressed CD21L. This provided the basis for distinguishing four subgroups of rheumatoid synovia: IL17A+/CD21L+ (18.5% of synovia), IL17A+/CD21L- (7.5%), IL17A-/CD21L+ (33.3%) and IL17A-/CD21L- (40.7%). While the subgroups did not predict clinical outcome measures, comparisons between the synovial subgroups revealed the IL17A+/CD21L+ subgroup had significantly larger CD20+ B-lymphocyte aggregates (P = 0.007) and a gene expression profile skewed toward B-cell- and antibody-mediated immunity. In contrast, genes associated with bone and cartilage remodelling were prominent in IL17A-/CD21L- synovia. CONCLUSIONS: Rheumatoid synovia can be subdivided on the basis of IL17A and CD21L gene expression. Ensuing molecular subgroups do not predict clinical outcome for patients but highlight high inflammation and the predominance of B-lymphocyte mediated mechanisms operating in IL17A+/CD21L+ synovia. This may provide a rationale for more refined therapeutic selection due to the distinct molecular profiles associated with IL17A+/CD21L+ and IL17A-/CD21L- rheumatoid synovia.

Greenshell™ Mussels: A Review of Veterinary Trials and Future Research Directions.

The therapeutic benefits of Greenshell™ mussel (GSM; Perna canaliculus) preparations have been studied using in vitro test systems, animal models, and human clinical trials focusing mainly on anti-inflammatory and anti-arthritic effects. Activity is thought to be linked to key active ingredients that include omega-3 polyunsaturated fatty acids, a variety of carotenoids and other bioactive compounds. In this paper, we review the studies that have been undertaken in dogs, cats, and horses, and outline new research directions in shellfish breeding and high-value nutrition research programmes targeted at enhancing the efficacy of mussel and algal extracts. The addition of GSM to animal diets has alleviated feline degenerative joint disease and arthritis symptoms, and chronic orthopaedic pain in dogs. In horses, GSM extracts decreased the severity of lameness and joint pain and provided improved joint flexion in limbs with lameness attributed to osteoarthritis. Future research in this area should focus on elucidating the key active ingredients in order to link concentrations of these active ingredients with their pharmacokinetics and therapeutic effects. This would enable consistent and improved efficacy from GSM-based products for the purpose of improved animal health.

Digital-PCR for gene expression: impact from inherent tissue RNA degradation.

Subtle molecular differences indicate the heterogeneity present in a number of disease settings. Digital-PCR (dPCR) platforms achieve the necessary levels of sensitivity and accuracy over standard quantitative RT-PCR (qPCR) that promote their use for such situations, detecting low abundance transcript and subtle changes from gene expression. An underlying requisite is good quality RNA, principally dictated by appropriate tissue handling and RNA extraction. Here we consider the application of dPCR to measures of gene expression in pathological tissues with inherent necrosis, focusing on rheumatoid subcutaneous nodules. Variable RNA fragmentation is a feature of RNA from such tissues. Increased presence of transcript fragmentation is reflected in a proportionate decrease in Agilent DV200 metric and downstream, a reduction in endogenous control genes' expression, measured by RT-dPCR. We show that normalisation of target gene expression to that for endogenous control genes sufficiently corrects for the variable level of fragmented RNA. Recovery of target gene values was achieved in samples comprising as much as 50 percent fragmented RNA, indicating the suitability and appropriate limitation of such data treatment when applied to samples obtained from inherently necrotic tissues.

Expression of the genes facilitating methotrexate action within subcutaneous rheumatoid nodules.

OBJECTIVES: We sought further understanding of the association between methotrexate (MTX) therapy and accelerated development of subcutaneous rheumatoid nodules. The objective was to establish expression of genes involved in the transport, metabolism, and mechanism of action of MTX within nodule tissue. We also examined for differences in gene expression between nodules from patients actively receiving MTX compared to those not receiving MTX. METHODS: Subcutaneous nodule tissues (n=23) were obtained from 21 patients with RA, undergoing elective surgery. Expression of genes important to the transport (SLC19A1, ABCB1, ABCC1, ABCG2), metabolism (FPGS, GGH), and mechanism of action (TYMS, MTR, MTRR) of MTX, including for the adenosine receptors ADORA1, ADORA2A, ADORA2B, ADORA3 and ADORA3variant were quantitated by real-time PCR in each nodule sample. RESULTS: Transcripts for all genes were found in all nodules. Expression of MTR was significantly reduced in nodules from patients receiving MTX therapy. Patterns of gene expression differed, with those metabolising MTX more prominent in nodules from patients receiving MTX when compared to nodules from those not receiving MTX, where genes involved in MTX transport were more prominent. CONCLUSIONS: Genes involved in MTX handling are expressed in rheumatoid nodules, providing further evidence that metabolism of MTX within nodules could exert a local effect. Furthermore the profile of gene expression in nodules differed from that previously observed in rheumatoid synovial membrane. The significant reduction of MTR expression in nodules has implications for MTR- and MTRR-mediated re-methylation reactions. Our data suggest that in contrast to synovium, downstream methylation reactions involving methionine and the biosynthesis of S-adenosylmethionine (SAM) could be reduced in nodule tissue. This could help explain differing responses to MTX in rheumatoid nodules and synovium and warrants further investigation.

Expression of methotrexate transporters and metabolizing enzymes in rheumatoid synovial tissue.

OBJECTIVE: To determine whether methotrexate (MTX) affects the expression of genes involved in the transport [SLC19A1 (RFC1), ABCB1 (MDR1), ABCC1 (multidrug resistance proteins 1), ABCG2 (BCRP)], metabolism [γ-glutamyl hydrolase (GGH), folylpolyglutamate synthetase (FPGS)], and mechanism of action of MTX [thymidylate synthase, MTR, MTRR] in rheumatoid synovium. METHODS: Synovial tissue samples were obtained from 20 patients with rheumatoid arthritis (RA). Gene expression was undertaken using quantitative real-time PCR. RESULTS: All the genes examined were expressed in all samples. Expression of SLC19A1, GGH, FPGS, ABCC1, and MTRR was significantly higher in patients receiving MTX compared to those not receiving MTX (p < 0.05). The ratio of FPGS:GGH gene expression was 2.7 ± 0.51 ng/ml GAPDH (range 0.67-9.58). CONCLUSION: Genes involved in the transport, metabolism, and mechanism of action of MTX are expressed in rheumatoid joint synovium. These data provide evidence that MTX has the potential to be polyglutamated within the joint. The higher expression of FPGS compared to GGH in synovial tissue might favor production of long-chain MTX polyglutamates. Thus MTX has the potential to exert its therapeutic effects at the primary site of the inflammatory process in RA.

Dendritic cells provide a potential link between smoking and inflammation in rheumatoid arthritis.

INTRODUCTION: Smoking increases the risk of developing rheumatoid arthritis (RA) and affects the severity of established RA. Smoking can impact on Th17 lymphocyte differentiation and function through activation of the aryl hydrocarbon receptor (AHR), a process with implications for the pathogenic mechanisms in RA that involve the cytokine, interleukin (IL)-17A. The objective of this study was to establish any effect of smoking on the inflammatory tissue lesions of rheumatoid arthritis via the AHR and IL-17A. METHODS: Twenty synovial and eighteen subcutaneous nodule tissue samples from 31 patients with RA were studied. Patient smoking status at the time of tissue collection was established. Expression of AHR, CYP1A1, AHRR, IL6, IL17A, IL17F, IL22, IL23, IL23R, IFNG, TBX21, IDO1 and FOXP3 genes were assessed in tissues and cultured cells using real-time PCR. Two-colour immunofluorescence was used to co-localise AHR and CYP1A1 protein in synovial tissues. The response of monocytes and monocyte-derived dendritic cells (mo-DCs) to the AHR agonist, benzo(a)pyrene (BaP) was compared in vitro. RESULTS: AHR gene expression was demonstrated in rheumatoid synovial tissues and nodules with significantly greater expression in synovia. Expression was not influenced by smoking in either tissue. Evidence of AHR activation, indicated by CYP1A1 and AHRR gene expression, was found only in synovia from patients who smoked. However, IL17A gene expression was lower in synovia from smokers. TBX21 and FOXP3 expression was not affected by smoking. Within the synovial tissues of smokers the principal cell type with evidence of AHR activation was a subset of synovial DCs. This observation was consistent with the sensitivity of human mo-DCs to BaP stimulation demonstrated in vitro. Exposure to BaP affected mo-DC function as demonstrated by decreased IL6 expression induced by PolyI:C, without affecting indoleamine 2,3 dioxygenase (IDO)1 expression. CONCLUSION: Our findings show that one effect of smoking on inflamed rheumatoid synovial tissue involves activation of the AHR pathway. A subset of synovial DCs is important in the response to cigarette smoke. The potential for smoking to affect DC behaviour in joint tissues has relevance to both early and late phases of RA pathogenesis and warrants further investigation.

Adenosine receptor expression in rheumatoid synovium: a basis for methotrexate action.

INTRODUCTION: Methotrexate (MTX) exerts at least part of its anti-inflammatory effects through adenosine receptors (ADOR). The aims of this study were to determine the expression of all four adenosine receptor genes (ADORA1, ADORA2A, ADORA2B, ADORA3 and ADORA3variant) in rheumatoid synovial tissue and any influence of MTX exposure on this expression. Furthermore, we investigated whether polymorphisms within ADORA3 were associated with response and/or adverse effects associated with MTX. METHODS: Adenosine receptor gene expression was undertaken using PCR in 20 rheumatoid arthritis (RA) synovial samples. A separate cohort of 225 RA patients receiving MTX was genotyped for SNPs in the ADORA3 receptor gene. Double immunofluorescence was used to identify cells expressing ADOR protein. RESULTS: All ADOR genes were expressed in all synovial samples. ADORA3 and A3variant were the dominant subtypes expressed irrespective of MTX therapy. Expression of ADORA2A and ADORA2B was increased in patients receiving MTX compared to those not receiving MTX. There was no association between the ADORA3 rs1544224 SNP and high and low disease activity or MTX-associated adverse effects. ADORA2B protein expression was most obvious in vascular endothelial cells whereas ADORA3 protein was more abundant and expressed by synovial fibroblasts. CONCLUSIONS: We have shown that adenosine receptors are expressed in RA synovium. There is differential expression of receptors such that ADORA3 is expressed at significantly higher levels. This evidence demonstrates the potential for MTX to exert its anti-inflammatory effects at the primary site of pathology within the joints of patients with RA.

The G82S polymorphism promotes glycosylation of the receptor for advanced glycation end products (RAGE) at asparagine 81: comparison of wild-type rage with the G82S polymorphic variant.

Interaction between the receptor for advanced glycation end products (RAGE) and its ligands amplifies the proinflammatory response. N-Linked glycosylation of RAGE plays an important role in the regulation of ligand binding. Two potential sites for N-linked glycosylation, at Asn(25) and Asn(81), are implicated, one of which is potentially influenced by a naturally occurring polymorphism that substitutes Gly(82) with Ser. This G82S polymorphic RAGE variant displays increased ligand binding and downstream signaling. We hypothesized that the G82S polymorphism affects RAGE glycosylation and thereby affects ligand binding. WT or various mutant forms of RAGE protein, including N25Q, N81Q, N25Q/G82S, and N25Q/N81Q, were produced by transfecting HEK293 cells. The glycosylation patterns of expressed proteins were compared. Enzymatic deglycosylation showed that WT RAGE and the G82S polymorphic variant are glycosylated to the same extent. Our data also revealed N-linked glycosylation of N25Q and N81Q mutants, suggesting that both Asn(25) and Asn(81) can be utilized for N-linked glycosylation. Using mass spectrometry analysis, we found that Asn(81) may or may not be glycosylated in WT RAGE, whereas in G82S RAGE, Asn(81) is always glycosylated. Furthermore, RAGE binding to S100B ligand is affected by Asn(81) glycosylation, with consequences for NF-κB activation. Therefore, the G82S polymorphism promotes N-linked glycosylation of Asn(81), which has implications for the structure of the ligand binding region of RAGE and might explain the enhanced function associated with the G82S polymorphic RAGE variant.

Cell-specific expression of TLR9 isoforms in inflammation.

Toll-like receptors (TLRs) are key pattern recognition receptors during an immune response. With five isoforms of human TLR9 described, we hypothesised that differential expression of TLR9 isoforms in different cell types would result in variable contributions to the overall input from TLR9 during inflammation. We assessed the molecular expression of the TLR9 isoforms, TLR9-A, -C and -D. In normal peripheral blood mononuclear cells, B-lymphocytes express ∼100-fold more TLR9-A transcript than monocytes or T-lymphocytes, which predominantly express the TLR9-C transcript. Switches in isoform predominance accompany B-lymphocyte development. TLR9 protein expression in rheumatoid inflammatory lesions reflected the TLR9 isoform expression by immune cells. Herein we suggest that B-lymphocytes and plasmacytoid dendritic cells contribute the ∼3-fold higher TLR9-A transcript levels observed in inflamed synovium when compared to subcutaneous rheumatoid nodules. In contrast, macrophages and T-lymphocytes contribute the ∼4-fold higher TLR9-C transcript levels seen in nodules, compared to synovia. From protein sequence, predictions of subcellular localisation suggest TLR9-B may locate to the mitochondria, whereas TLR9-D adopts an opposing orientation in the endoplasmic reticulum. Consistent with this, structure models raise the possibility of alternative ligands for the TLR9-B and TLR9-D variants. Our results highlight differences in the expression of human TLR9 isoforms in normal and inflamed tissues, with differing contributions to inflammation.

Ingestion of native and thermally oxidized polyunsaturated fats acutely increases circulating numbers of endothelial microparticles.

Circulating numbers of endothelial microparticles (EMP) are an index of endothelial injury and dysfunction; and microparticles positive to CD31 antibody increase acutely after cooked, fatty fast-food meals that are rich in saturated fatty acids (SAFA) and lipid oxidation products. The aim of this study was to determine the acute effect of meals rich in SAFA and native and thermally oxidized polyunsaturated vegetable oil on circulating numbers of EMP positive to CD144 antibody, a more specific marker of EMP. Twenty-two apparently healthy subjects received isocaloric meals rich in cream (CR), unheated sunflower oil, or heated sunflower oil in a randomized crossover study design. Circulating numbers of CD144-EMP and plasma lipids and Svedberg unit of flotation (S(f)) greater than 400 triglyceride content were measured before and 1 and 3 hours after the meals. Triglycerides in the plasma S(f) greater than 400 fraction increased significantly (P < .001) after the meals, with a significantly (P < .05) larger increase after the CR meal. Plasma CD144-EMP increased significantly (20%, P < .05) after the unheated sunflower oil and heated sunflower oil meals and did not increase significantly (P = .55) after the CR meal. This response was significantly different among the meals (P = .002) when first-visit fasting plasma glucose was a covariate. In conclusion, these data suggest that ingestion of meals rich in n-6 polyunsaturated vegetable oil irrespective of whether it has been mildly thermally oxidized may acutely alter the state of the vascular endothelium, resulting in increased shedding of CD144-EMP. The physiologic implications of these findings remain to be determined.

Monocyte derived interleukin (IL)-23 is an important determinant of synovial IL-17A expression in rheumatoid arthritis.

OBJECTIVE: To demonstrate gene expression of interleukin (IL)-17A, IL-23, and IL-12 and to determine the proximity of IL-17A and IL-23 producing cells in rheumatoid synovial tissue. METHODS: Total RNA was isolated from 25 synovial membranes obtained from 20 patients with rheumatoid arthritis (RA). Quantitative real-time polymerase chain reaction was used to measure IL-17A, IL-12p35, IL-23p19, p40, and GAPDH expression. Immunohistochemistry was utilized to determine cell type and proximity of IL-17A, IL-12, and IL-23 in rheumatoid synovium. RESULTS: IL-17A was present in 13/25 synovia. IL-12p35 was present in all samples while IL-23p19 was present in 23/25. p40 was present in 23/25 samples. Of the 2 p40- samples both were IL-23p19 and IL-12p35 positive. Mean expression of IL-23p19 was significantly higher in the IL-17A+ versus IL-17A- synovia (0.10 +/- 0.02 ng vs 0.05 +/- 0.01 ng; p < 0.05). There was no difference in IL-12p35 expression between IL-17A+ and IL-17A- synovia (0.5 +/- 0.21 ng vs 0.38 +/- 0.24 ng; p = 0.2). All IL-17A+ cells were in the vicinity of IL-23+ cells. IL-12+ cells were both close to and removed from IL-17A+ cells. Only a proportion of CD3+T cells appeared to produce IL-17A. CONCLUSION: IL-17A gene expression occurs in only a subset of rheumatoid synovial membranes. IL-23 gene expression is higher in IL-17A+ versus IL-17A- membranes. In keeping with this, IL-17A+ and IL-23+ cells colocalize in synovial membranes. IL-17 is not an absolute requirement in RA but may be important in amplifying the inflammatory response. Anti-IL23 therapies may have a role in those patients with IL-17A expression.

Different T cell subsets in the nodule and synovial membrane: absence of interleukin-17A in rheumatoid nodules.

OBJECTIVE: To determine gene expression of the interleukin-17 (IL-17) family members (IL-17A-F) in rheumatoid subcutaneous nodules, and to assess the cytokines involved in regulating IL-17A expression. METHODS: Total RNA was isolated from 19 nodules obtained from 16 different patients with rheumatoid arthritis (RA). Reverse transcription-polymerase chain reaction (PCR) was used to screen for gene expression of the IL-17 subtypes (IL-17A-F) in all nodules. Quantitative real-time PCR was used to measure the expression of interferon-gamma (IFN gamma), IL-6, IL-23, IL-12, and transforming growth factor beta (TGFbeta), relative to GAPDH as control, in a subset of 10 nodules. RESULTS: IL-17A gene expression was present in only 1 of 19 nodules, IL-17B in 17 of 19 nodules, IL-17C in 18 of 19 nodules, IL-17D in 16 of 19 nodules, and IL-17E in 3 of 19 nodules. IL-17F was absent in all samples. Cytokines that stimulate IL-17A production (IL-6, IL-23) as well as those that inhibit IL-17A production (IL-12, IFN gamma, TGFbeta) were present in the majority of nodules. Quantitative real-time PCR showed a similar pattern of gene expression for the individual cytokines between the different nodules. The mean +/- SD expression of IL-6 relative to GAPDH was 2.28 +/- 2.2 ng, and that of TGFbeta was 2.96 +/- 1.14 ng. There was a lower relative expression of IL-23 (0.05 +/- 0.05 ng), while the expression of IFN gamma was 0.67 +/- 0.68 ng and that of IL-12 was 0.48 +/- 0.23 ng. CONCLUSION: IL-17 family members are varyingly expressed in rheumatoid nodules. The paucity of IL-17A in nodules suggests an important difference from that observed in the synovium. The expression of IL-23 below a critical threshold level seems the most likely explanation for the virtual absence of IL-17A. The presence of tissue destruction within the nodule despite the absence of IL-17A suggests that IL-17A may be an important amplifier rather than an absolute requirement for inflammation in RA.

The potential for cryopreserving larvae of the sea urchin, Evechinus chloroticus.

Larvae of the sea urchin, Evechinus chloroticus, at varying stages of development, were assessed for their potential to survive cryopreservation. Ethylene glycol (EG) and dimethyl sulphoxide (Me2SO), at concentrations of 1-2 M, were evaluated as cryoprotectants (CPAs) in freezing regimes initially based on methods established for freezing larvae of other sea urchin species. Subsequent work varied cooling rate, holding temperature, holding time, and plunge temperature. Ethylene glycol was less toxic to larvae than Me2SO. However, no larvae survived freezing and thawing in EG. Larvae frozen in Me2SO at the gastrula stage and 4-armed pluteus stage regained motility post-thawing. The most successful freezing regime cooled straws containing larvae in 1.5 M Me2SO from 0 to -35 degrees C at 2.5 degrees C min(-1), held at -35 degrees C for 5 min, then plunged straws into liquid nitrogen. Motility was high 2-4 h post-thawing using this regime but decreased markedly within 24 h. Some 4-armed pluteus larvae that survived beyond this time developed through to metamorphosis and settled. Different Me2SO concentrations and supplementary trehalose did not improve long-term survival. Large variation in post-thaw survival was observed among batches of larvae produced from different females.

Cryopreservation of sea urchin (Evechinus chloroticus) sperm.

A method was developed for cryopreserving sperm of the sea urchin, Evechinus chloroticus. Sperm fertilisation ability, mitochondrial function and membrane integrity were assessed before and after cryopreservation. Highest post-thaw fertilisation ability was achieved with lower concentrations (2.5%-7.5%) of dimethyl sulphoxide (DMSO). In contrast, post-thaw mitochondrial function and membrane integrity were higher for sperm frozen in intermediate and high DMSO concentrations (5%-15%). Surprisingly, some sperm frozen in seawater only, without DMSO, were able to survive post-thawing, although the fertilisation ability (10(6) sperm/ml; approximately 50% fertilisation), mitochondrial function and membrane integrity of these sperm were notably lower than of sperm frozen with DMSO (10(6) sperm cells/ml; 2.5%-7.5% DMSO; >85% fertilisation) at the concentrations tested. Amongst sperm from individual males, fertilisation ability varied before and after cryopreservation for both males frozen with and without cryoprotectant. Specific differences among males also varied. Sperm mitochondrial function and membrane integrity was similar among males before cryopreservation but differed considerably after cryopreservation. Cryopreserved sperm were able to fertilise eggs and develop to pluteus stage larvae. This study has practical applications and will provide benefits such as reduced broodstock conditioning costs, control of parental input and opportunities for hybridisation studies.

Role of hepatic stellate cell/hepatocyte interaction and activation of hepatic stellate cells in the early phase of liver regeneration in the rat.

BACKGROUND/AIMS: Hepatic stellate cells (HSCs) are known to play a role in hepatic regeneration. We investigated hepatocyte/HSC interaction and HSC activation at various times after 70% partial hepatectomy (PHx) in the rat. METHODS: The hepatic microcirculation was studied using intravital fluorescence microscopy (IVFM). Desmin and alpha-SMA within liver tissue were detected by immunohistochemistry. In isolated parenchymal liver cells (PLCs) and HSCs, double immunostaining was used to identify activated HSC. RESULTS: Using IVFM, hepatocyte-clusters were often seen in vivo at 3 days after PHx (PHx3). Distance between HSC fell from 61.7+/-2.1 microm in controls to 36.1+/-1.4 microm (P<0.001) while the HSC/hepatocyte ratio rose (0.71+/-0.01 to 1.08+/-0.03; P<0.001). In >80% of in vivo microscopic fields in the PHx3 group, clusters of HSCs were observed especially near hepatocyte-clusters. At PHx1 and PHx3, >20% of cells in the PLC-fraction were HSCs which adhered to hepatocytes. At PHx3, in addition to desmin staining, isolated HSCs were also positive for BrdU and alpha-SMA, and formed clusters. HSCs in the HSC-fraction were only positive for desmin which indicated that adherence to hepatocytes is required for HSC activation. CONCLUSIONS: Our data suggest that HSCs are activated by adhering to hepatocytes in the early phase of liver regeneration.

Membrane permeability characteristics and osmotic tolerance limits of sea urchin (Evechinus chloroticus) eggs.

Development of effective cryopreservation protocols relies on knowledge of the fundamental cryobiological characteristics for a particular cell type. These characteristics include osmotic behaviour, membrane permeability characteristics, and osmotic tolerance limits. Here, we report on measures of these characteristics for unfertilized and fertilised eggs of the sea urchin (Evechinus chloroticus). In NaCl solutions of varying osmolalities, sea urchin eggs behaved as ideal linear osmometers. The osmotically inactive volume (vb) was similar for unfertilized and fertilised eggs, 0.367+/-0.008 (mean+/-SE) and 0.303+/-0.007, respectively. Estimates of water solubility (Lp) and solute permeability (Ps) and their respective activation energies (Ea) for unfertilized and fertilised eggs were determined following exposure to cryoprotectant (CPA) solutions at different temperatures. Irrespective of treatment, fertilised eggs had higher values of Lp and Ps. The presence of a CPA decreased Lp. Among CPAs, solute permeability was highest for propylene glycol followed by dimethyl sulphoxide and then ethylene glycol. Measures of osmotic tolerance limits of the eggs revealed unfertilized eggs were able to tolerate volumetric changes of -20% and +30% of their equilibrium volume; fertilised eggs were able to tolerate changes +/-30%. Using membrane permeability data and osmotic tolerance limits, we established effective methods for loading and unloading CPAs from the eggs. The results of this study establish cryobiological characteristics for E. chloroticus eggs of use for developing an effective cryopreservation protocol. The approach we outline can be readily adapted for determining cryobiological characteristics of other species and cell types, as an aid to successful cryopreservation.