Apoptotic U1–70 kd is antigenically distinct from the intact form of the U1–70‐kd molecule

Objective

To determine whether immune responses to an apoptotically modified form of a human lupus autoantigen can be distinguished from immune responses to the intact form of the same antigen.

Methods

Immunoblot and enzyme‐linked immunosorbent assay techniques were used to test human autoimmune sera for the presence of antibodies to apoptotic forms of the U1– 70‐kd small nuclear RNP antigen, while antibody recognition of intact U1–70 kd was blocked.

Results

Apoptosis‐specific U1–70‐kd antibodies were identified by immunoblot in 15 of 29 sera with antibodies to intact U1–70 kd and in 2 of 25 sera without measurable antibodies to intact U1–70 kd. Bacterially produced, purified, caspase‐cleaved U1–70 kd without additional posttranslational modifications was a target of apoptosis‐specific antibodies in 3 of 9 U1–70‐kd–positive sera tested.

Conclusion

The apoptotic form of U1–70 kd displays B cell epitopes that are not displayed on the intact form of U1–70 kd. Caspase cleavage in the absence of additional posttranslational modifications is sufficient to induce the display of some of these epitopes. Immunity to apoptotically modified proteins can develop against caspase‐cleaved forms or against forms that undergo additional posttranslational modification.

     

Rosiglitazone induces interleukin‐1 receptor antagonist in interleukin‐1β–stimulated rat synovial fibroblasts via a peroxisome proliferator–activated receptor β/δ–dependent mechanism

Objective

To study the potency of 2 peroxisome proliferator–activated receptor γ (PPARγ) agonists, 15‐deoxy‐Δ^12,14‐prostaglandin J₂ (15‐deoxy‐PGJ₂) and rosiglitazone, to modulate the expression of interleukin‐1 receptor antagonist (IL‐1Ra) in rat synovial fibroblasts.

Methods

Levels of messenger RNA for IL‐1Ra and PPAR isotypes (α, β/δ, γ) were assessed by real‐time polymerase chain reaction in rat synovial fibroblasts exposed to 10 ng/ml of IL‐1β. PPAR levels were assessed by Western blotting and secreted IL‐1Ra levels by immunoassay. The potency of PPARγ agonists and the PPARβ/δ agonist GW‐501516 on IL‐1Ra levels was tested in the range of 1–10 μM and at 100 pM, respectively. The contribution of PPARγ to the effects of rosiglitazone on IL‐1Ra secretion was examined either by its overexpression or by inhibition using wild‐type or dominant‐negative constructs and the antagonist GW‐9662 (10 μM), respectively. The dominant‐negative strategy was also performed to investigate the possible contribution of PPARβ/δ and NF‐κB activation.

Results

IL‐1β–induced IL‐1Ra production was increased by 10 μM rosiglitazone but was reduced dose‐dependently by 15‐deoxy‐PGJ₂. Both agonists lowered IL‐1β secretion, but rosiglitazone alone reduced the imbalance of IL‐1β/IL‐1Ra toward basal levels. Enhancement of IL‐1β–induced IL‐1Ra production by rosiglitazone was not affected by PPARγ overexpression or by its inhibition with dominant‐negative PPARγ or GW‐9662. Inhibition of NF‐κB was also ineffective against rosiglitazone but abolished the stimulating effect of IL‐1β on IL‐1Ra. All PPAR isotypes were expressed constitutively in rat synoviocytes, but PPARγ decreased dramatically upon IL‐1β exposure, whereas PPARβ/δ remained stable. Dominant‐negative PPARβ/δ abolished the enhancement of IL‐1Ra by rosiglitazone, whereas GW‐501516 reproduced the effect of rosiglitazone on IL‐1Ra secretion.

Conclusion

Rosiglitazone stimulates IL‐1Ra production by a PPARβ/δ mechanism in activated rat synovial fibroblasts, further contributing to its potential antiarthritic properties and opening new perspectives for the modulation of inflammatory genes by specific PPAR agonists in articular cells.

     

Human platelet antigens – 2013

To date, 33 human platelet alloantigens (HPAs) have been identified on six functionally important platelet glycoprotein (GP) complexes and have been implicated in alloimmune platelet disorders including foetal and neonatal alloimmune thrombocytopenia (FNAIT), posttransfusion purpura (PTP) and multitransfusion platelet refractoriness (MPR). The greatest number of recognized HPA (20 of 33) resides on the GPIIb/IIIa complex, which serves as the receptor for ligands important in mediating haemostasis and inflammation. These include HPA‐1a, the most commonly implicated HPA in FNAIT and PTP in Caucasian populations. Other platelet GP complexes, GPIb/V/IX, GPIa/IIa and CD109, express the remaining 13 HPAs. Of the recognized HPAs, 12 occur as six serologically and genetically defined biallelic ‘systems’ where the –a form designates the higher frequency allele and the –b form, the lower. Twenty‐one other HPAs are low‐frequency or rare antigens for which postulated higher frequency –a alleles have not yet been identified as antibody specificities. In addition to the HPA markers, platelets also express ABO and human leucocyte antigen (HLA) antigens; antibodies directed at the former are occasionally important in FNAIT, and to the latter, in MPR.     

Haemoglobin H disease due to (– –SEA) α‐globin gene deletion and α2‐codon 30 (ΔGAG) mutation: a family study

A Chinese family in which two siblings suffer from haemogloblin (Hb) H disease due to (– –^SEA) α‐globin gene deletion and α2‐codon 30 (ΔGAG) mutation is described. Both siblings are transfusion‐independent and have survived to adulthood. In contrast to previous report of hydrops fetalis associated with ζ‐α‐thal‐1 and α2‐codon 30 (ΔGAG) mutation, the ζ‐globin genes are intact in the two siblings, which most probably alleviates the γ‐chain excess and protects the fetus from severe anaemia. Correlation of genotype with phenotype in Hb H disease is important for genetic counselling, especially in the antenatal setting.     

Endoplasmic reticulum stress in the regulation of liver diseases: Involvement of Regulated IRE1α and β‐dependent decay and miRNA

Compromised protein folding capacity in the endoplasmic reticulum (ER) leads to a protein traffic jam that produces a toxic environment called ER stress. However, the ER smartly handles such a critical situation by activating a cascade of proteins responsible for sensing and responding to the noxious stimuli of accumulated proteins. The ER protein load is higher in secretory cells, such as liver hepatocytes, which are thus prone to stress‐mediated toxicity and various diseases, including alcohol‐induced liver injury, fatty liver disease, and viral hepatitis. Therefore, we discuss the molecular cues that connect ER stress to hepatic diseases. Moreover, we review the literature on ER stress‐regulated miRNA in the pathogenesis of liver diseases to give a comprehensive overview of mechanistic insights connecting ER stress and miRNA in the context of liver diseases. We also discuss currently discovered regulated IRE1 dependent decay in regulation of hepatic diseases.     

Association of α‐actinin–binding anti–double‐stranded DNA antibodies with lupus nephritis

Objective

Anti–double‐stranded DNA (anti‐dsDNA) antibodies may contribute to the pathogenesis of glomerulonephritis (GN) by cross‐reacting with α‐actinin in murine models and in some patients with systemic lupus erythematosus (SLE). We therefore sought to determine possible disease associations with serologic and clinical features and to characterize this new autoantibody specificity.

Methods

One hundred patients with SLE were recruited into this multicenter study, as well as 100 rheumatic disease controls and 2,100 healthy blood donors. Clinical disease was evaluated by the SLE Disease Activity Index (SLEDAI; excluding the anti‐DNA component). Anti‐dsDNA antibodies were detected by conventional enzyme‐linked immunosorbent assay (ELISA) and by a commercial enzyme immunoassay (EIA). Anti–α‐actinin antibodies were detected by ELISA, and their specificity was confirmed by Western blotting and by indirect immunofluorescence using rat kidney sections and mesangial cells as substrates. Highly positive sera were selected for absorption experiments and were affinity‐purified for cross‐reactivity studies and measurement of antibody avidity.

Results

Sera from 62 of the SLE patients had anti‐dsDNA antibodies; 21 of these sera also had anti–α‐actinin antibodies, as compared with 1 of the 38 sera without anti‐dsDNA antibodies. Of the 22 patients with anti–α‐actinin antibodies, 10 had GN, as compared with 14 of the 78 without anti–α‐actinin antibodies (P < 0.01). In patients with GN, anti–α‐actinin, but not anti‐dsDNA, antibodies correlated with the SLEDAI score (minus the anti‐DNA component) and with treatment. The fraction of serum anti‐dsDNA antibodies that cross‐reacted with α‐actinin exhibited high avidity for dsDNA, as determined using a commercial EIA for high‐avidity anti‐dsDNA antibodies and an in‐house conventional ELISA.

Conclusion

The α‐actinin–binding antibodies are significantly associated with GN in SLE. Whether such autoantibodies may anticipate the development of this complication of SLE remains to be verified.

     

Pediatric influenza‐associated myositis – Nebraska, 2001–2007

Objective Influenza‐associated myositis (IAM), characterized by severe lower‐extremity myalgia and reluctance to walk, is a complication of influenza among children. We investigated IAM in Nebraska during six influenza seasons, 2001–2007. Methods During 2006–2007, we requested reports of severe influenza illness among persons aged <18 years and investigated medical records to identify and confirm IAM cases defined as severe myalgia with elevated serum creatinine kinase level in a patient aged <18 years, occurring within 7 days of laboratory confirmed influenza illness onset. Statewide hospital discharge data (HDD) were reviewed to identify retrospectively confirmed IAM cases during 2006–2007 and five previous seasons, by using surveillance data to define periods of influenza activity. Statewide IAM incidence was estimated for 2001–2002 through 2006–2007. Results During 2006–2007, a total of 13 IAM cases were confirmed by enhanced surveillance. Median age was 6 years (range, 4–11 years). Influenza diagnosis was established by viral isolation from six patients (one influenza A and five influenza B) and rapid diagnostic tests for seven. Twelve (92%) patients, including one who died, were hospitalized for a median of 3 days (range, 1–4 days). Review of HDD identified 12 retrospectively confirmed IAM cases during 2006–2007, including four not reported through enhanced surveillance, and only one during five previous seasons (2003–2004). The HDD‐derived, retrospectively confirmed statewide IAM incidence estimates/1 00 000 population aged <18 years were 2·693 and 0·225 during 2006–2007 and 2003–2004, respectively. Conclusion An IAM epidemic occurred in Nebraska during the 2006–2007 influenza season.     

Estrogen receptor–related receptor α regulation by interleukin‐1β in prostaglandin E2– and cAMP‐dependent pathways in osteoarthritic chondrocytes

Objective

We reported previously that the orphan nuclear receptor, estrogen receptor–related receptor α (ERRα), is expressed in articular chondrocytes and is dysregulated in a mouse model of inflammatory arthritis. The aim of this study, therefore, was to determine whether ERRα is also dysregulated in patients with osteoarthritis (OA).

Methods

ERRα messenger RNA (mRNA) and protein were quantified in normal and OA cartilage samples and in OA chondrocytes in vitro, with and without short‐term treatment with a variety of OA‐associated factors and signaling pathway agonists and inhibitors.

Results

ERRα expression was lower in OA than in normal articular cartilage. Interleukin‐1β (IL‐1β) markedly up‐regulated ERRα expression in OA chondrocytes in vitro, and agonist or inhibitor treatment indicated that the up‐regulation was dependent on cyclooxygenase 2 (COX‐2; NS398), prostaglandin E₂, cAMP (8‐bromo‐cAMP), and protein kinase A (PKA; KT5720). Treatment with the ERRα inverse agonist XCT790 decreased the expression of SOX9 and the up‐regulation of ERRα by IL‐1β, suggesting autoregulation of ERRα in the IL‐1β pathway. Matrix metalloproteinase 13 (MMP‐13) expression was also decreased by treatment with XCT790 plus IL‐1β versus IL‐1β alone, and the down‐regulation of MMP‐13 mRNA and protein observed with XCT790 alone suggests that the up‐regulation of MMP‐13 by IL‐1β is ERRα‐dependent.

Conclusion

We report the first evidence that ERRα expression is regulated by IL‐1β in COX‐2–, cAMP‐, and PKA‐dependent pathways in OA chondrocytes. We confirmed that SOX9 is an ERRα target gene in human, as in rodent, chondrocytes and identified MMP‐13 as a potential new target gene, which suggests that ERRα may both respond to the healing signal and contribute to extracellular degradation in OA cartilage.