Lack of evidence for an involvement of Epstein-Barr virus infection of synovial membranes in the pathogenesis of rheumatoid arthritis

OBJECTIVE: To test the hypothesis that Epstein-Barr virus (EBV) infection of cells within the synovial membrane contributes to the pathogenesis of rheumatoid arthritis (RA). METHODS: Biopsy samples of synovial membrane from 37 patients with RA and from 51 patients with other joint diseases were studied for evidence of EBV infection using in situ hybridization specific for the EBV-encoded RNAs (EBERs). Latent membrane protein 1 (LMP1) and the lytic-cycle BZLF1 protein were detected by immunohistochemistry. RESULTS: Rare EBER-positive B lymphocytes were detected in 7 RA biopsy samples. EBV was not detectable in any other cells. Expression of the LMP1 and BZLF1 proteins of EBV was not observed in any of the samples. No EBV infection was detected in synovial membranes from patients with other joint diseases. CONCLUSION: Our data indicate that EBV infection is not directly involved in the pathogenesis of RA. Any contribution of EBV to the pathogenic process leading to RA is likely to be indirect.     

Epstein-Barr virus association is rare in esophageal squamous cell carcinoma

Background. A critical role of Epstein-Barr virus (EBV) in carcinogenesis of nasopharyngeal squamous cell carcinoma and gastric adenocarcinoma is strongly suspected. We analyzed the possible EBV association for Japanese squamous cell carcinoma (SCC)-dominant esophageal cancer cases. Methods. We retrospectively screened 36 surgically resected esophageal cancer lesions from 36 patients maily with SCC using in situ hybridization (ISH) for EBV-encoded small RNA1 (EBER-1). EBV DNA analysis using real-time quantitative polymerase chain reaction (Q-PCR) was performed for three recent cases. Results. We found no EBER-1-positive cancer cell in any tested esophageal cancer lesion. There were many EBER-1-positive tumor-infiltrating lymphocytes in the basaloid SCC lesion and a small number of positive lymphocytes in the other five advanced SCC lesions (14.7% of SCC). One SCC lesion with a highcopy number of EBV DNA had EBER-1-positive lymphocytes. Conclusions. EBV is rarely associated with esophageal SCC, and may appear through tumor-infiltrating lymphocytes in some advanced lesions.     

Hepatocyte growth factor (HGF), HGF activator, and c-Met in synovial tissues in rheumatoid arthritis and osteoarthritis

OBJECTIVE: Hepatocyte growth factor (HGF) is a multifunctional polypeptide that has been implicated in cancer growth, tissue development, and wound repair. Its actions are dependent on activation by HGF activator (HGFA) and its binding to a specific HGF receptor (c-Met). We examined the role of HGF, HGFA, and c-Met in synovial tissues in rheumatoid arthritis (RA) and osteoarthritis (OA), and their localization and mRNA expression. METHODS: Immunohistochemical staining, Western blotting, RT-PCR, and in situ hybridization (ISH) for HGF, HGFA, and c-Met were performed on synovial tissue specimens from 10 patients with RA and 4 with OA, and 2 healthy controls. RESULTS: Immunohistochemical staining revealed that HGFA and c-Met were strongly expressed in fibroblasts, macrophages, endothelial cells, and synovial lining cells. HGF was expressed only faintly in macrophages and fibroblasts, and not at all in the endothelial cells of RA and OA synovial tissue. HGFA was detected near 73 and 34 kDa on Western blot analysis, corresponding to inactive and active HGFA, respectively. RT-PCR showed HGF, HGFA, and c-Met mRNA in RA, OA, and control synovial tissue. ISH and immunohistochemistry revealed mRNA expression for HGF, HGFA, and c-Met in the cell types mentioned above. CONCLUSION: HGFA, HGF, and c-Met mRNA are expressed in synovial tissue in RA and OA, and HGF is activated by HGFA and binds to c-Met on endothelial cells, inducing angiogenesis.     

Epstein-Barr virus infection of the colon with inflammatory bowel disease

OBJECTIVE: Epstein-Barr virus (EBV)-infected cells can evoke severe host immune responses, as shown in infectious mononucleosis and EBV-associated gastric carcinoma. To investigate the possible pathological role of EBV in inflammatory bowel disease (IBD), we tested for the presence of EBV in the colon in IBD patients. METHODS: Surgically resected colonic specimens of 11 patients with Crohn's disease, five patients with ulcerative colitis, nine noninflammatory controls (disease-free area of the colorectal carcinoma), and 10 appendicitis cases were tested using highly sensitive in situ hybridization for EBV-encoded small RNA1 (EBER-1). RESULTS: EBER-1 was detected in 63.6% of Crohn's disease cases and 60% of ulcerative colitis cases, but not at all in noninflammatory controls and appendicitis cases. EBER-1-positive cells were very rare in the noninflammatory areas of colonic specimens from IBD patients. EBER-1-positive cells were nonepithelial cells (mainly B lymphocytes and a few histiocyte-shaped cells) located in erosive or ulcerative areas of the colonic specimens. CONCLUSION: The limited presence of EBV-infected cells in the diseased areas of IBD colonic specimens indicated that EBV infection may be related to such diseases.     

Bacterial components in the synovial tissue of patients with advanced rheumatoid arthritis or osteoarthritis: analysis with gas chromatography-mass spectrometry and pan-bacterial polymerase chain reaction

OBJECTIVE: To study the presence of bacterial components in the synovial tissue (ST) of patients with advanced rheumatoid arthritis (RA). METHODS: ST was collected during joint surgery from 41 RA patients. Tissue from 39 patients with osteoarthritis (OA), 4 patients with undifferentiated inflammatory arthritis (UA), and 3 cases of accidental deaths served as controls. The pan-bacterial polymerase chain reaction (PCR) with primers for the 23S ribosomal RNA (rRNA) and 16S rRNA genes was used to detect bacterial DNA. In addition, synovial fluid (SF) samples from patients with chlamydial reactive arthritis (ReA) were also examined by the same method. The positive controls, bacterial DNA or ST spiked with different living bacteria, were analyzed alongside clinical samples. Most of the ST samples were also analyzed by gas chromatography-mass spectrometry (GC-MS) for determining the presence of bacteria-derived muramic acid. Strict precautions were followed in the clinics and the laboratory to prevent contamination. RESULTS: In GC-MS analysis, muramic acid was observed in the ST from 4 of 35 RA patients and from 2 of 14 OA patients, but not in ST from 2 patients with UA and 3 cadavers. Bacterial DNA was not detected by either one of the PCR primers used in ST from 42 patients with RA and 39 patients with OA. However, 5 of 15 SF samples from ReA patients were PCR positive. The sensitivity of GC-MS to detect muramic acid was 2 pg/injected amount (227 pg muramic acid/mg ST), and that of the pan-bacterial PCR was 2-20 bacteria colony forming units/reaction. CONCLUSION: These results indicate that a bacterial component, muramic acid, is detectable by GC-MS in ST from a few patients with advanced RA or OA. However, no bacterial DNA was detectable by PCR.     

Analysis of cartilage oligomeric matrix protein (COMP) in synovial fibroblasts and synovial fluids

We investigated the expression of cartilage oligomeric matrix protein (COMP) in normal and rheumatoid arthritis (RA) synovial fibroblasts. In situ hybridization (ISH) was conducted on synovial specimens from five RA patients applying specific probes for COMP or fibroblast collagen type I. ISH was combined with immunohistochemistry, applying antibodies to the macrophage marker CD68. Ribonuclease protection assay (RPA) and rapid amplification of 3'-cDNA ends (3'-RACE) were performed on total RNA from normal and RA synovial fibroblast cultures. Protein extracts from fibroblasts and culture supernatants were compared with synovial fluids and protein extracts from isolated chondrocytes by Western blot utilizing polyclonal and monoclonal antibodies (18-G3 mAb) to COMP. COMP mRNA was detected in fibroblasts of RA synovium by ISH, and in normal and RA synovial fibroblast cultures by RPA. 3'-RACE demonstrated sequence homology of chondrocyte and synovial fibroblast COMP along the coding sequence. COMP protein was detected in synovial fibroblasts and culture supernatants by immunoblot. Using polyclonal antibodies, the major portion of COMP from fibroblasts and culture supernatants was present as low-molecular-weight (LMW) bands, corresponding to those found in synovial fluids. These LMW COMP bands, however, were not detected in any of the cells or tissues tested using 18-G3 mAb. In protein extracts from chondrocytes and in COMP purified from cartilage, these LMW bands could not be detected. In conclusion, the data suggest that certain forms of COMP detected in synovial fluid are secreted from synovial fibroblasts and could be distinguished by specific mAbs from COMP secreted by chondrocytes.      

High prevalence of Epstein-Barr virus in gastric remnant carcinoma after Billroth-II reconstruction

BACKGROUND: Epstein-Barr virus (EBV) has been detected in about 10% of gastric carcinoma cases worldwide, and a high prevalence of EBV involvement in gastric remnant carcinoma has been reported recently. Details of the background remnant stomach of EBV-positive lesions, however, have not been well clarified. METHODS: We screened 17 consecutive gastric remnant carcinoma lesions resected surgically. To detect EBV, we used in situ hybridization (ISH) for EBV-encoded small RNA1 (EBER-1) and we compared the clinicopathologic feature between EBV-positive and -negative gastric remnant carcinoma cases. RESULTS: EBV was detected in 41.8% (7 of 17) of the lesions by EBER-1 ISH. All 7 EBV-positive lesions developed in the anastomotic site had undergone Billroth-II reconstruction excess 20 years previously (mean 26.4 years). Histologically, all EBV-positive lesions were poorly differentiated adenocarcinomas with intense lymphocyte infiltration. In the adjacent mucosa of carcinomas, moderate or marked intestinal metaplasia was found in 85.7% (6 of 7) of EBV-positive lesions and in 40% (4 of 10) of EBV-negative lesions. CONCLUSIONS: EBV infection is strongly associated with gastric remnant carcinoma. Atrophic change of remnant gastritis in Billroth-II anastomoses is considered to be the carcinogenic background for EBV-positive gastric remnant carcinoma.     

GI Cancer High Prevalence of Epstein-Barr Virus in Gastric Remnant Carcinoma after Billroth-II Reconstruction

Background : Epstein-Barr virus (EBV) has been detected in about 10% of gastric carcinoma cases worldwide, and a high prevalence of EBV involvement in gastric remnant carcinoma has been reported recently. Details of the background remnant stomach of EBV-positive lesions, however, have not been well clarified. Methods : We screened 17 consecutive gastric remnant carcinoma lesions resected surgically. To detect EBV, we used in situ hybridization (ISH) for EBV-encoded small RNA1 (EBER-1) and we compared the clinicopathologic feature between EBV-positive and-negative gastric remnant carcinoma cases. Results : EBV was detected in 41.8% (7 of 17) of the lesions by EBER-1 ISH. All 7 EBVpositive lesions developed in the anastomotic site had undergone Billroth-II reconstruction excess 20 years previously (mean 26.4 years). Histologically, all EBV-positive lesions were poorly differentiated adenocarcinomas with intense lymphocyte infiltration. In the adjacent mucosa of carcinomas, moderate or marked intestinal metaplasia was found in 85.7% (6 of 7) of EBV-positive lesions and in 40% (4 of 10) of EBV-negative lesions. Conclusions : EBV infection is strongly associated with gastric remnant carcinoma. Atrophic change of remnant gastritis in Billroth-II anastomoses is considered to be the carcinogenic background for EBV-positive gastric remnant carcinoma.     

EBV-encoded small RNA1 and nonresolving inflammation in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by perpetuated inflammation in multiple joints. To date, there is no cure for RA, and the causal factor for non-resolving inflammation in RA remains unclear. In this study, we initially observed expression of Epstein–Barr virus-encoded small RNA1 (EBER1) in the synovial tissue of all five patients who showed nonresolving RA inflammation. By contrast, EBER1 was detected in the synovial tissue of only one out of seven patients with advanced osteoarthritis (OA; p < 0.01, Fisher’s exact test). To confirm this finding, we conducted a second study on synovial tissue samples taken from 23 patients with nonresolving RA inflammation and 13 patients with OA. All synovial samples from patients with nonresolving inflammation of RA showed positive expression of EBER1 (23/23, 100%), whereas none of the synovial samples from patients with OA showed expression of EBER1 (0/13, 0%; p < 0.001, by Fisher’s exact test). In vitro, transfection of RA synovial fibroblasts with EBER1 induced the production of interleukin-6. Taken together, these data strongly suggest that nonresolving RA inflammation is strongly related to the presence of EBER1, which might be, at least partially, responsible for synovial fibroblast interleukin-6 production.