Cells In Synovial Fluid Of Patients Research Articles

ЕКСПРЕСІЯ ГЕНІВ PTGS2 ТА NOS2 У СИНОВІАЛЬНІЙ РІДИНІ ХВОРИХ НА ОСТЕОАРТРИТ ПІСЛЯ SARS-CoV2-ІНФЕКЦІЇ

Background. The emergence of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has created a serious global threat to the health of people in various countries. The 2019 coronavirus disease (Coronavirus disease 2019, COVID-19) has caused the development of many complications in the population of most countries of the world. Currently, many people have felt the unpleasant consequences of the coronavirus infection. Among them, a special group consists of patients with chronic diseases, particularly osteoarthritis. The development of inflammation and intensification of free radical processes is a leading link in the pathogenesis of osteoarthritis. Inflammatory and degenerative processes that develop in osteoarthritis cause modification of the molecular and cellular composition of the synovial fluid. Methods. The aim of the work was to determine the expression of (Prostaglandin-endoperoxide synthase 2) and NOS2 (Nitric Oxide Synthase 2) genes in synovial fluid cells of patients with osteoarthritis after SARS-CoV2 infection. All study participants were divided into two groups. The first group (n = 22) is patients with osteoarthritis of the knee joints II-III degree. The second group (n = 14) is patients with osteoarthritis of the knee joints II–III degree, who suffered a mild and moderate form of COVID-19 6–9 months ago. Expression of PTGS2 and NOS2 genes was determined in nuclear cells of synovial fluid. RNA was obtained by the Chomczynski method. Synthesis of cDNA and quantitative polymerase chain reaction in real time (Real-time PCR, qPCR) using the commercial kit "Thermo Scientific Verso SYBR Green 1-Step qRT-PCR ROX Mix" ("Thermo Scientific, Lithuania). Processing of research results was carried out using generally accepted methods of variational statistics. Results. In patients with osteoarthritis of the knee joints after SARS-CoV2 infection, the expression of PTGS2 and NOS2 genes increases in nuclear cells of the synovial fluid compared to patients diagnosed with osteoarthritis. Conclusions. Our results indicate that patients with osteoarthritis may develop a more severe course of synovial inflammation after infection with COVID-19.

CD14+ Cells with the Phenotype of Infiltrated Monocytes Consist of Distinct Populations Characterized by Anti-inflammatory as well as Pro-inflammatory Activity in Gouty Arthritis.

It has been suggested that inflammasome-mediated IL-1β production in monocytic cells is responsible for the acute inflammatory response in gouty arthritis. However, phenotypical and functional analyses of monocytes during gouty arthritis have yet to be conducted. Therefore, we investigated the characteristics of monocytes/macrophages in the synovial fluid cells of patients with acute gout. The number and frequency of monocytes/macrophages in the synovial fluid mononuclear cells (SFMCs) of patients was examined. The expression of markers for monocyte recruitment and tissue-resident macrophages, the production of pro-inflammatory and anti-inflammatory cytokines, and phagocytosis were analyzed in the monocytes/macrophages of patients with acute gout attacks. The number and frequency of CD14+CD3−CD19−CD56− monocytes/macrophages was markedly increased in the SFMCs of patients with gout compared to those of patients with rheumatoid arthritis (RA). CD14+ cells showed the phenotypes of infiltrated monocytes rather than tissue-resident macrophages, characterized by a high expression of CCR2, MRP8, and MRP14, but a low expression of MERTK and 25F9. These cells had the capacity to produce pro-inflammatory cytokines such as TNF-α and IL-1β after stimulation with lipopolysaccharides. In addition, anti-inflammatory features, including CD163 expression and IL-10 production from CD14+ cells, were significantly higher in patients with gout than in those with RA. CD14+ cells with phenotype of M2 macrophages had high phagocytic activity for monosodium urate crystals. Thus, our results indicate that monocytes/macrophages from patients with gout have the phenotype of infiltrated monocytes, and these cells consist of different populations characterized by anti-inflammatory activities as well as pro-inflammatory functions.

Differential gene and protein expression of chemokines and cytokines in synovial fluid of patients with arthritis.

BackgroundPsoriatic arthritis (PsA), an inflammatory musculoskeletal disease, develops in approximately 30% of patients with psoriasis. Previously, chemokine (C-X-C motif) ligand 10 (CXCL10) was identified as a predictive biomarker of PsA in patients with psoriasis and was reduced after development of PsA. The purpose of the present study was to explore messenger RNA (mRNA) and protein expression of CXCL10 and its receptor, chemokine (C-X-C motif) receptor 3 (CXCR3), in the joints of patients with PsA to gain insight into their role in the pathogenesis of the disease.MethodsSera from 47 patients with PsA and 33 healthy control subjects were compared for expression of CXCL10 by Luminex assay. Synovial fluid (SF) was obtained from patients with PsA (n = 40), osteoarthritis (OA; n = 14), gout (n = 8), and rheumatoid arthritis (RA; n = 11) during clinical care. SF mRNA and protein expression of CXCL10, interleukin-17A (IL-17A), CXCR3, TBX21, RORC and/or interferon γ (IFNγ) were compared among the above-mentioned disease groups, as well as in paired SF and serum samples from patients with PsA using real-time polymerase chain reaction and Luminex assays, respectively.ResultsSerum CXCL10 was significantly higher in patients with PsA than in control subjects (p = 0.0007). CXCL10, IL-17A, and TBX21 expression were elevated in SF cells of patients with PsA compared with those of patients with OA and gout, but not those of patients with RA. CXCR3 and RORC were elevated in PsA SF cells compared with all other patient groups. Concordant results were obtained for CXCL10 and IL-17A protein expression. IFNγ was elevated in PsA SF compared with OA SF (p = 0.015). CXCL10 protein expression was substantially increased in SF (median 7283.9 pg/ml, interquartile range [IQR] 1330–10,362 pg/ml) compared with paired serum samples (median 282.06, IQR 180.7–395.8 pg/ml; p = 0.001), whereas IFNγ was significantly reduced (SF median 6.03 pg/ml, IQR 4.47–8.94 pg/ml; versus serum median 23.70 pg/ml, IQR 3.2–104.6 pg/ml; p = 0.001).ConclusionsCXCL10 may have an important etiological role in PsA that is analogous to that in RA, and it is a candidate biomarker to distinguish PsA from healthy individuals and from patients with OA and gout.

Induction of osteoclast-like cells derived from the synovial lavage fluids of patients with temporomandibular joint disorders

Although biochemical studies have examined the synovial fluid (SF) of patients with temporomandibular joint (TMJ) disorders (TMDs), the details of the molecular mechanism of bone destruction and remodeling remain unknown. In this study, we induced and characterized osteoclast-like cells from the SF of patients with TMD and investigated the participation of these cells in the pathogenesis of TMD. We collected SF cells from patients with TMD after a pumping procedure, cultured osteoclast-like cells, and examined their characteristics, including osteoclast markers and bone resorption activities. In addition, we obtained fibroblastic cells from the SF of TMD patients by continuous sub-culturing. Using these fibroblastic cells, we examined fibroblast markers using immunocytochemical staining and analyzed the receptor activator of nuclear-factor-kappaB ligand (RANKL) mRNA levels. Detection of soluble form of RANKL (sRANKL) in the SF was measured by enzyme-linked immunosorbent assay (ELISA). Osteoclast-like cells were induced from the SF cells of patients with TMD by adding recombinant human (rh) macrophage colony stimulating factor (M-CSF) and either 1,25-dihydroxy vitamin D3 [1,25(OH)2D3] or prostaglandin E2 (PGE2). These multinucleated giant cells were positive for tartrate-resistant acid phosphatase (TRAP) and had the ability to absorb bone. The fibroblastic cells from the SF of TMD patients were positive for fibroblast markers and RANKL mRNA was up-regulated. Detection of sRANKL in SF of patient group was significantly higher than control group. The results suggest that the joint-infiltrating SF cells from TMD patients play important roles in the pathogenesis of these disorders, which is characterized by progressive bone destruction or remodeling.

Free Clinics in North Carolina: A Network of Compassion, Volunteerism, and Quality Care for Those without Healthcare Options

**Background:** Antibodies directed to proteins containing the non-standard amino acid citrulline, are extremely specific for rheumatoid arthritis (RA). Peptidylcitrulline can be generated by post-translational conversion of arginine residues. This process, citrullination, is catalysed by a group of calcium dependent peptidylarginine deiminase (PAD) enzymes. **Objective:** To investigate the expression and activity of four isotypes of PAD in peripheral blood and synovial fluid cells of patients with RA. **Results:** The data presented here show that citrullination of proteins by PAD enzymes is a process regulated at three levels: *transcription*---in peripheral blood PAD2 and PAD4 mRNAs are expressed predominantly in monocytes; PAD4 mRNA is not detectable in macrophages, *translation*---translation of PAD2 mRNA is subject to differentiation stage-specific regulation by its 3′ UTR, and *activation*---the PAD proteins are only activated when sufficient Ca^2+^ is available. Such high Ca^2+^ concentrations are normally not present in living cells. In macrophages, which are abundant in the inflamed RA synovium, vimentin is specifically citrullinated after Ca^2+^ influx. **Conclusion:** PAD2 and PAD4 are the most likely candidate PAD isotypes for the citrullination of synovial proteins in RA. Our results indicate that citrullinated vimentin is a candidate autoantigen in RA.

Trisomy 7 in synovial fluid cells of patients with rheumatoid arthritis.

Recent studies revealed trisomy 7 as a chromosomal abnormality in non-neoplastic disorders such as rheumatoid arthritis (RA). In the present study, we investigated the presence of trisomy 7 in the synovial fluid cells of patients with RA using fluorescence in situ hybridisation (FISH) analysis. Synovial fluid from 15 patients with RA was collected from knee joints. The control group consisted of seven patients with traumatic synovial effusion in their knee joints. The arthrocenteses were performed under aseptic conditions. Dual-colour FISH analysis was performed using chromosome-7-specific LSI D7S522 (7q31) and chromosome-5-specific LSI EGR1 (5q31)/D5S721 (5p15.2) probes on the slides prepared from synovial fluid of RA patients and controls. The slides of our cases were analysed using two different DNA probes. When the slides hybridised with chromosome-5-specific probes were analysed, no trisomic or monosomic cells were revealed in both patients and controls. However, in eight of 15 patients, trisomy 7 occurred in variable percentages of cells (23% to 48%) of synovial fluid. No monosomic 7 cells were detected in these specimens. All control cases were disomic for chromosome 7. The results of the present investigation suggest that trisomy 7 may play a role in the pathogenesis of synovial hyperproliferation in RA.

Absence of inducible retroviruses from synovial fibroblasts and synovial fluid cells of patients with rheumatoid arthritis.

Absence of inducible retroviruses from synovial fibroblasts and synovial fluid cells of patients with rheumatoid arthritis.

Bacterial DNA in synovial fluid cells of patients with juvenile onset spondyloarthropathies.

To identify bacterial DNA in synovial fluid cells of patients with active juvenile onset spondyloarthropathy (SpA). The main group of study constituted 22 patients with juvenile onset SpA. In addition, five patients with adult onset SpA and nine with rheumatoid arthritis (RA) were studied. Polymerase chain reaction (PCR) with either genus- or species-specific primers was performed on synovial fluid cells to detect DNA sequences of Chlamydia trachomatis, Yersinia enterocolitica, Salmonella sp., Shigella sp., Campylobacter sp. and Mycobacterium tuberculosis. The presence of antibacterial antibodies in sera and synovial fluid was also determined by enzyme-linked immunoassay. The synovial fluid of nine patients with juvenile onset SpA, three with adult onset SpA and one with RA contained bacterial DNA. Five juvenile onset SpA samples had DNA of one single bacterium; two juvenile onset SpA and three adult onset SpA had DNA of two bacteria and two juvenile onset SpA had DNA of three bacteria. Overall, Salmonella sp. DNA was detected in seven synovial fluid samples, Shigella sp., Campylobacter sp. and M. tuberculosis were found in four samples each, and C. trachomatis was found in two. The bacterial DNA findings correlated with neither diagnosis nor disease duration. One RA synovial fluid had DNA of Campylobacter sp. Neither serum nor synovial fluid antibacterial antibodies correlated with DNA findings or clinical diagnosis. In this study, single and several combinations of bacterial DNA were identified in the synovial fluid of patients with long-term undifferentiated and definite juvenile onset SpA and adult onset SpA. Of relevance is that bacterial DNA corresponds to bacteria producing endemic disease in our population.

Persistence of Yersinia antigens in peripheral blood cells from patients with Yersinia enterocolitica O:3 infection with or without reactive arthritis.

To assess the persistence of bacterial antigens in peripheral blood cells from patients with Yersinia enterocolitica O:3-triggered reactive arthritis (ReA). Peripheral blood samples were obtained from 20 patients with Y. enterocolitica O:3 infection (11 with ReA and 9 without). These samples were studied by immunochemical techniques for the presence of Yersinia antigens at the beginning of infection and up to 4 years thereafter. Synovial fluid samples from 6 of the 11 ReA patients were also studied. The Yersinia antigens lipopolysaccharide and heat-shock protein (HSP) were detected in peripheral blood mononuclear cells and polymorphonuclear phagocytes from all patients studied at the early phase of the disease. They were also found in the synovial fluid cells of patients with Yersinia-triggered ReA. At 4 years after the onset of infection, these bacterial antigens were still detected in the peripheral blood cells of most of the ReA patients studied. This study has, for the first time, directly demonstrated that bacterial antigens persist for a long time in patients who develop ReA after Y. enterocolitica O:3 infection. The finding of bacterial HSP in synovial fluid cells could provide a link to the pathogenesis of ReA, since T cell responses of synovial cells have been shown to be directed against that structure. A close similarity between the bacterial and host HSP might contribute to the development of the relatively common, chronic form of this complication.

Autometallographic Demonstration of Gold Uptake in Cultured Synovial Fluid Cells from Patients with Rheumatoid Arthritis

Gold can be visualized in synoviocytes and subsynovial mononuclear cells of patients with rheumatoid arthritis (RA) treated with gold compounds (1-3), and has been found to interfere with the structure and function of monocytes/macrophages (4-8). Synovial fluid cells (SFC) have not been examined. Recently a revised version of the photochemical method for the demonstration of gold was presented (9). This autometallographic technique can be applied both to light microscopy and to electron microscopy and allows the detection of 3-6 gold atoms, theoretically 1 atom, by silver enhancement in ultraviolet (UV) radiated tissue sections. In the present study gold has been demonstrated by this method in the SFC of patients in chrysotherapy and in cultured synovial cells incubated with gold in vitro.

Immunoelectronmicroscopic characterization of intracellular inclusions in synovial fluid cells of patients with rheumatoid arthritis.

Qualitative immunoelectronmicroscopic (IEM) analysis of intracellular inclusions in synovial fluid (SF) cells in rheumatoid arthritis (RA) was performed using the peroxidase-antiperoxidase (PAP) method. Cells from patients with chronic RA were fixed in glutaraldehyde containing 0.05% saponin to permeabilize membranes before immunologic treatments. Intracellular inclusions of IgG, IgM, and C3 were observed in vacuoles of greater than 75% of both polymorphonuclear leukocytes (PMNs) and mononuclear phagocytic cells. IgA- and fibrinogen-containing inclusions were less frequent. Intracellular staining for albumin was minimal. Other membranous, vesicular, and granular unstained materials of potential importance were also often present in the same vacuoles. Stained inclusions were clearly distinct from lipid bodies, which were negative for immunostaining. Control samples had only occasional, scattered and weak stain that was easily recognizable as nonspecific and thus established the specificity of the reactions. A few lymphocytes in 2 patients showed positive staining for IgG in vacuoles. Extracellular staining of clumps of immunoglobulins, C3, and fibrinogen was also present. The various materials phagocytized by the different SF cells may be important in perpetuation of joint inflammation.