The role of synovial fibroblasts in mediating joint destruction in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic, inflammatory joint disease with systemic involvement that affects about 1% of the Western population. The progressive destruction of affected joints is a major characteristic of the disease and distinguishes RA from other acute and chronic arthritides. The etiology of RA is unknown, and a variety of genetic and environmental factors are being discussed as potential causes of the disease. However, in contrast to our incomplete understanding of the etiology, the knowledge about molecular mechanisms leading to joint destruction has advanced considerably over the past years. Thus, a large number of studies have investigated the presence and interplay of several types of cells in rheumatoid synovium, such as lymphocytes, macrophages and fibroblasts. They have led to the understanding that cells in the rheumatoid synovium form a network, which interacts through direct cell-to cell contacts as well as the release of a multitude of cytokines. The use of novel molecular techniques together with the development of new animal models has revised our concept on the pathogenesis of RA and specifically on the role of fibroblasts in initiation and progression of joint destruction. This article will review current data and hypotheses on disease mechanisms by which fibroblasts are involved in the destruction of joints in RA.     

Genome Scanning of the HXB/BXH Sets of Recombinant Inbred Strains of the Rat for Quantitative Trait Loci Associated with Conditioned Taste Aversion

In the current study, we searched for quantitative trait loci (QTL) responsible for a conditioned taste aversion (CTA) measured as a decrease in the intake of a saccharin conditioned stimulus followed by an i.p. injection of 0.15 M LiCl (lithium chloride) (2 ml/100 g body weight). A genome scanning for QTL associated with CTA was performed in the HXB/BXH sets of recombinant inbred (RI) strains derived from the Brown Norway (BN-Lx) rat and the spontaneously hypertensive rat (SHR). The BN-Lx progenitor showed a significantly stronger CTA (8.3 ± 2.8%) than the SHR progenitor (27.8 ± 3.3%, p < .0001). The distribution of CTA values among RI strains was continuous, suggesting a polygenic mode of inheritance. Genome scanning of RI strains with more than 700 gene markers revealed a significant association of CTA with the D2Cebr11s4 marker on chromosome 2 (LRS = 22.7) and with the D4Cebrp149s8 marker on chromosome 4 (LRS = 23.4). The chromosome 2 putative QTL was confirmed by detecting a significant difference in CTA between the SHR progenitor (27.8 ± 3.3%) and the SHR-2 (SHR.BN-D2Rat171/D2Arb24) congenic strain (13.1 ± 4.4%, p < .01) that are genetically identical except for a segment of chromosome 2 that was transferred onto the genetic background of the SHR from the BN strain.     

Nitric oxide production by neutrophils obtained from patients during acute coronary syndromes: expression of the nitric oxide synthase isoforms

OBJECTIVES: To analyze the differences in the nitric oxide (NO) forming system between neutrophils obtained from patients during unstable angina (UA) and during acute myocardial infarction (AMI). BACKGROUND: Neutrophils are involved in the regulation of thrombus formation through the release of active substances such as NO. Acute myocardial infarction is the result of an occlusive thrombus; unstable angina is attributed to intermittent thrombus formation. METHODS: We studied 49 patients admitted to hospital within 24 h after the onset of chest pain: 31 experienced AMI and 18 experienced UA. Acute myocardial infarction was defined as CK greater than two-fold the upper limit of normal value of biochemical laboratory, with CK-MB >10% total CK. Unstable angina was defined as transient ST segment changes without significant increases in CK and CK-MB. RESULTS: The amount of NO generated by neutrophils from AMI patients was significantly higher than that generated by neutrophils from UA patients. Neutrophils from UA and AMI patients showed low levels of endothelial-like NO synthase protein expression and a marked expression of the inducible NO synthase (iNOS) isoform. Although neutrophils from patients during acute coronary syndromes generated high amounts of NO, they did not demonstrate an increased ability to stimulate cyclic guanosine monophosphate (cGMP) synthesis in platelets. This lack of activity to release NO by neutrophils from patients during AMI was unrelated to a defect in the platelet cGMP-forming system; sodium nitroprusside, an exogenous NO donor, similarly increased cGMP levels in platelets from AMI patients and healthy donors. CONCLUSIONS: Neutrophils from patients during AMI and UA showed an increased production of NO and a marked expression of the iNOS isoform. However, NO released from these neutrophils showed a deficient functionality. These findings could have clinical implications because they show differences in thrombus growth in patients with UA versus patients with AMI.     

Generation of DNA cleavage specificities of type II restriction endonucleases by reassortment of target recognition domains

Type II restriction endonucleases (REases) cleave double-stranded DNA at specific sites within or close to their recognition sequences. Shortly after their discovery in 1970, REases have become one of the primary tools in molecular biology. However, the list of available specificities of type II REases is relatively short despite the extensive search for them in natural sources and multiple attempts to artificially change their specificity. In this study, we examined the possibility of generating cleavage specificities of REases by swapping putative target recognition domains (TRDs) between the type IIB enzymes AloI, PpiI, and TstI. Our results demonstrate that individual TRDs recognize distinct parts of the bipartite DNA targets of these enzymes and are interchangeable. Based on these properties, we engineered a functional type IIB REase having previously undescribed DNA specificity. Our study suggests that the TRD-swapping approach may be used as a general technique for the generation of type II enzymes with predetermined specificities.     

Ribozymes that inhibit the production of matrix metalloproteinase 1 reduce the invasiveness of rheumatoid arthritis synovial fibroblasts

OBJECTIVE: To investigate whether retroviral gene transfer of ribozymes targeting matrix metalloproteinase 1 (MMP-1) inhibits the production of MMP-1 in rheumatoid arthritis synovial fibroblasts (RASFs) and reduces the invasiveness of these cells in vivo. METHODS: MMP-1-specific ribozymes (RzMMP-1) were designed and cloned into the pLNSX retroviral vector. Cleavage of MMP-1 was determined in vitro, and the most effective ribozyme was selected for further investigation. RASFs were transduced with replication-deficient viruses carrying RzMMP-1 or with empty viruses (mock). Quantitative polymerase chain reaction with cleavage site-spanning fluorescent probes was used to measure the levels of MMP-1, MMP-9, and MMP-13 messenger RNA. In addition, protein levels of MMP-1 in cell culture supernatants were determined by enzyme linked immunosorbent assay. The effects of stimulation with lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFalpha) on the production of MMP-1 were assessed accordingly. The invasiveness of RzMMP-1-transduced, mock-transduced, and untransduced RASFs was analyzed in the SCID mouse in vivo model of RA. RESULTS: Transduction of RASFs with RzMMP-1 significantly decreased the production of MMP-1 in RASFs without affecting other MMPs, such as MMP-9 and MMP-13. RzMMP-1 not only reduced the spontaneous production of MMP-1, but also prevented the LPS- and TNFalpha-induced increase in MMP-1 production. Inhibition of MMP-1 was maintained for at least 2 months and was accompanied by a significant reduction of the invasiveness of RASFs in the SCID mouse model of RA. CONCLUSION: Intracellular expression of ribozymes constitutes a feasible tool for inhibiting the production of matrix-degrading enzymes. Inhibition of MMP-1 alone results in a significant reduction of cartilage invasion by RASFs.