Mature and immature ovarian teratomas share methylation profiles of imprinted genes: a MS-MLPA analysis.

Immature teratomas are a subset of ovarian teratomas, and the pathogenic relationship between mature and immature ovarian teratomas is unclear. Mature ovarian teratomas are parthenogenetic tumors that arise from a single oocyte/ovum, whereas the origin of immature ovarian teratomas has not been extensively investigated. Since parthenogenetic tumors contain only maternal genomes, genome imprinting in these tumors usually follows a maternal pattern. DNA methylation is among the most important mechanisms of genome imprinting. Therefore, we analyzed the methylation profile of imprinted genes by performing methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) of 25 imprinting control regions (ICRs) in 10 imprinted genes/gene clusters from formalin-fixed, paraffin-embedded samples obtained from 4 immature ovarian teratomas, 8 mature ovarian teratomas, and 4 ovarian yolk sac tumors (YSTs). Both the immature and mature components showed similar methylation levels in each ICR in immature teratomas. Overall, immature ovarian teratomas showed maternal methylation patterns of imprinted genes in concordance with their parthenogenetic origin. However, they also showed aberrant methylation levels in a few imprinted genes, suggesting that genome imprinting in immature teratomas may partially differ from that in mature teratomas. Microscopic foci of YST were seen in one immature teratoma; the YST component also showed a maternal methylation pattern, unlike the pure YSTs that showed irregular patterns. Thus, teratoma-associated YST and pure YST may have different pathogenic mechanisms.

Combination chemoradiotherapy with temozolomide, vincristine, and interferon-ß might improve outcomes regardless of O6-methyl-guanine-DNA-methyltransferase (MGMT) promoter methylation status in newly glioblastoma.

BACKGROUND: This investigator-initiated, open-label, single-arm, single-institute study was conducted to investigate the effectiveness of induction combination chemoradiotherapy and long-term maintenance therapy with temozolomide (TMZ) plus interferon (IFN)-ß for glioblastoma. METHODS: The initial induction combination chemoradiotherapy comprised radiotherapy plus TMZ plus vincristine plus IFN-ß. Maintenance chemotherapy comprised monthly TMZ, continued for 24-50 cycles, plus weekly IFN-ß continued for as long as possible. The primary endpoint was 2-year overall survival (2y-OS). The study protocol was to be considered valid if the expected 2y-OS was over 38% and the lower limit of the 95% confidence interval (CI) was no less than 31.7% compared with historical controls, using Kaplan-Meier methods. Secondary endpoints were median progression-free survival (mPFS), median OS (mOS), 5-year OS rate (5y-OS), and mPFS and mOS classified according to MGMT promoter methylation status. RESULTS: Forty-seven patients were analyzed. The 2y-OS was 40.7% (95%CI, 27.5-55.4%). The mPFS and mOS were 11.0 months and 18.0 months, respectively, and 5y-OS was 20.3% (95%CI, 10.9-34.6%). The mPFS in groups with and without MGMT promoter methylation in the tumor was 10.0 months and 11.0 months (p = 0.59), respectively, and mOS was 24.0 months and 18.0 months (p = 0.88), respectively. The frequency of grade 3/4 neutropenia was 19.1%. CONCLUSIONS: The 2y-OS with induction multidrug combination chemoradiotherapy and long-term maintenance therapy comprising TMZ plus IFN-ß tended to exceed that of historical controls, but the lower limit of the 95%CI was below 31.7%. Although the number of cases was small, this protocol may rule out MGMT promoter methylation status as a prognostic factor. TRIAL REGISTRATION: University Hospital Medical Information Network (number UMIN000040599 ).

Methylation profile of imprinted genes provides evidence for teratomatous origin of a subset of mucinous ovarian tumours.

Mucinous ovarian tumours are sometimes associated with mature teratomas. It is suggested that the mucinous tumours in this setting are derived from teratomas, but there remains the possibility of collision or metastasis from extra-ovarian sites. Because mature ovarian teratomas are considered to be parthenogenetic tumours that arise from a single oocyte/ovum, they have only a maternal genome and therefore show maternal genome imprinting. If mucinous ovarian tumours originate from teratomas, their genome imprinting is theoretically maternal. One of the most important mechanisms of genome imprinting is DNA methylation. In the present study, we analysed a total of 28 mucinous ovarian tumours (7 with teratomas, 21 without teratomas; 14 malignant, 14 borderline) to clarify the methylation profiles of their imprinted genes using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) of 21 imprinting control regions (ICRs) of nine imprinted genes/gene clusters using formalin-fixed, paraffin-embedded samples. All cases lacked evidence of an extra-ovarian primary mucinous tumour. In all seven mucinous tumours with teratomas, the overall methylation profile of mucinous tumours was comparable to that of teratomas, although some ICRs showed aberrant methylation. In contrast, all but one of the mucinous tumours without teratomas showed somatic or irregular methylation patterns. Morphologically, there was little teratomatous tissue in some mucinous tumours carrying teratoma-type methylation profiles, suggesting that mucinous tumours overwhelmed ancestral teratomas. In conclusion, the methylation profile of imprinted genes provides evidence that a subset of mucinous ovarian tumours originated from mature teratomas. Genome imprinting-based analysis is a promising strategy to verify the teratomatous origin of human tumours. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Methylation profiles of imprinted genes are distinct between mature ovarian teratoma, complete hydatidiform mole, and extragonadal mature teratoma.

Mature ovarian teratoma is considered to be a parthenogenetic tumor that arises from a single oocyte/ovum. Conversely, complete hydatidiform mole (CHM) is androgenetic in origin: classic CHM arises from a single or two sperm. Since mature ovarian teratoma and CHM have only maternal and paternal genomes, respectively, their genome imprinting is theoretically reverse, but this has yet to be investigated. Genome imprinting in struma ovarii, a special form of mature teratoma, remains unclear. Although a mature teratoma can rarely arise in extragonadal sites, its genome imprinting, as well as cell origin, is poorly understood. One of the most important mechanisms of genome imprinting is DNA methylation. To investigate the methylation profile of imprinted genes, we performed methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) of 21 imprinting control region (ICRs) of 9 imprinted genes/gene clusters in formalin-fixed, paraffin-embedded samples obtained from 12 mature ovarian teratomas, 6 struma ovarii, 10 CHMs, and 7 extragonadal (1 sacrococcygeal, 6 mediastinal) mature teratomas of females. In mature ovarian teratomas, ICRs of maternally and paternally imprinted genes showed high and low levels of methylation, respectively, and this pattern was almost reverse in CHMs. In CHMs, however, some ICRs showed aberrant methylation. The methylation profile of struma ovarii was comparable to that of mature ovarian teratomas, except for an adenomatous tumor. In extragonadal mature teratomas, the methylation pattern was somatic or irregular. In conclusion, mature ovarian teratomas/struma ovarii, CHMs, and extragonadal mature teratomas showed distinct methylation profiles of imprinted genes. Ovarian teratomas and CHMs are most likely to inherit their methylation profiles from their ancestral germ cells, although some aberrant methylation suggests a relaxation of imprinting in CHMs and a subset of struma ovarii. Extragonadal mature teratomas may carry a methylation profile of misplaced primordial germ cells or possibly somatic cells that have been reprogrammed in vivo.

Comparative molecular analysis of primary and recurrent oligodendroglioma that acquired imbalanced 1p/19q codeletion and TP53 mutation: a case report.

Oligodendroglioma is defined by IDH mutation and 1p/19q codeletion. Normal TP53 status is also its molecular feature. We report a case of oligodendroglioma that acquired imbalanced 1p/19q codeletion and TP53 mutation at recurrence after temozolomide therapy. The primary and recurrent tumors shared IDH1 and TERT promoter mutations. Although 1p/19q was codeleted in the primary tumor, it was imbalanced in the recurrent tumor harboring TP53 mutation. The copy-neutral loss of heterozygosity might have imbalanced the 1p/19q codeletion, while temozolomide therapy possibly caused the TP53 mutation. Such phenomena, although rare, should be noted during the clinical treatment of oligodendrogliomas.

Giant cell glioblastoma is a distinctive subtype of glioma characterized by vulnerability to DNA damage.

Giant cell glioblastoma (GC-GBM) consists of large cells with pleomorphic nuclei. As a contrast to GC-GBM, we defined monotonous small GBM (MS-GBM) as GBM that consists of small cells with monotonous small nuclei, and compared the DNA damage as well as other pathological features. GC-GBM showed minimal invasion (< 2 mm) and focal sarcomatous areas. TERTp was wild type in GC-GBM but mutant in MS-GBM. OLIG2 expression was significantly higher in MS-GBM (P < 0.01) (77% in MS-GBM and 7% in GC-GBM). GC-GBM showed significantly higher DNA double-strand breaks (DSBs) compared with MS-GBM (P < 0.01) (76% in GC-GBM and 15% in MS-GBM). Nearly, all large cells in GC-GBM underwent DSBs. Thus, significant DSBs in GC-GBM might be induced by an innate lesser stemness characteristic and be followed by mitotic slippage, resulting in polyploidization and the large pleomorphic nuclei. We conclude that GC-GBM is a distinctive subtype of glioma characterized by its vulnerability to DNA damage and that wild-type TERTp and lower OLIG2 function might induce this feature. Notably, even large pleomorphic nuclei with severe DSBs demonstrated Ki67 positivity, which alerts pathologists to the interpretation of Ki67 positivity, because cells with large nuclei undergoing severe DSBs cannot be recognized as proliferating cells that contribute to tumor aggressiveness.

Nurse-like cells in rheumatoid arthritis: Formation of survival niches cooperating between the cell types.

Nurse-like cells (NLCs) established from bone marrow and synovial tissue of rheumatoid arthritis (RA) patients were found to promote maturation and differentiation of B lineage cells as well as T cells. In co-culture of RA-NLCs and B cells, tight physical interactions (pseudoemperipolesis) developed, which resulted in activation of both cell types. RA-NLCs also supported myeloid cell maturation, promoting their differentiation into tartrate-resistant acid phosphatase-positive mononuclear cells, which are precursor cells of osteoclasts. In RA synovial tissue, the characteristic dendritic-shaped cells (the DCs) were electron microscopically found to form direct physical interactions with adjacent plasma cells (PCs) suspecting to be pseudoemperipolesis. The numbers of PCs accumulating in various areas tended to correlate with the numbers of the DCs, which appeared to have RA-NLC functions forming survival niches for PCs. Immunohistochemical staining analysis indicated that CD14+ cells including the DCs formed survival niches for CD138+ PCs by RA-NLC functions. Quantitative dual immunofluorescence staining studies of these areas indicated that the majority of CD14+ cells were of myeloid lineage. These survival niches promoted by RA-NLCs appear to play important roles in supporting immunological functions in RA bone marrow and synovial tissues.

PIK3CA hotspot mutations and cyclooxygenase-2 expression in ovarian clear cell carcinomas: a close association with stromal features.

Ovarian clear cell carcinomas (CCCs) have 2 distinct stromas: a hyalinized/mucoid stroma and a plasma cell-rich inflammatory stroma. Clinically, CCC is the most common ovarian cancer associated with thromboembolism. Recent studies suggested a potential role of PIK3CA mutation in the cyclooxygenase (COX) pathway, which mediates inflammation or hemostasis. In the present study, 54 ovarian CCCs and 3 CCC cell lines were analyzed for PIK3CA hotspot mutation and COX-2 expression with special reference to stromal features. Among the 54 CCCs, 20 (37.0%) and 8 (14.8%) were classified as CCCs with a hyalinized/mucoid stroma and an inflammatory stroma, respectively. PIK3CA mutation was identified in 11 (55%) of the 20 CCCs with a hyalinized/mucoid stroma, but not in any of the 8 CCCs with an inflammatory stroma. In contrast, COX-2 expression was frequent in CCCs with an inflammatory stroma (1/20 [5%] versus 7/8 [87.5%], respectively). Such a relationship between the PIK3CA mutation, COX-2 expression, and stromal features was repeated in the 3 CCC cell lines. Thromboembolism was noted in 9 (16.7%) of the 54 CCC patients, and it was more frequent in CCCs with a hyalinized/mucoid stroma (7/20 [35%]) than in those with an inflammatory stroma (0/8 [0%]). In conclusion, there is a difference in PIK3CA mutation, COX-2 expression, and paraneoplastic thromboembolism between CCCs with a different stroma. It is suggested that a different stromal feature, either hyalinized/mucoid change or inflammation, represents a different molecular genetic background or hemostatic potential in ovarian CCCs.

Importance and accuracy of intraoperative frozen section diagnosis of the resection margin for effective carmustine wafer implantation.

For effective implantation of carmustine (BCNU) wafers, it is important to determine the order of priority with reference to the intraoperative frozen section diagnosis of the resection margin (IOFM). The accuracy of IOFM and patterns of tumor recurrence with implantation of BCNU wafers were studied retrospectively. Forty-six cases of newly diagnosed malignant glioma were evaluated. Tumors were resected after intraoperative frozen section diagnosis (IOFD). IOFM was performed for resection walls and evaluated on a three-level scale (-, no tumor invasion; 1+, minor cell invasion; 2+, evident cell invasion). The results were used for effective BCNU wafer implantation. The IOFM sections were then thawed, frozen-paraffin marginal (FPM) sections were prepared, and IOFM was evaluated with FPM sections. The accuracy of IOFD grading was compared to that of the formalin fixed paraffin-embedded section and was 76.1%. The accuracy of IOFM was compared with the FPM section in 148 specimens from 42 patients. The IOFM accuracy was 80.4%. BCNU wafers were implanted in 25 patients and there was recurrence in 15. Local recurrence was seen in 40% (6 patients). However, there was no recurrence immediately below the BCNU wafers. With properly performed IOFM, BCNU wafers can be efficiently implanted, and local recurrence immediately below the BCNU wafers can be inhibited.

Genetic zygosity of mature ovarian teratomas, struma ovarii, and ovarian carcinoids.

Although ovarian monodermal teratomas, including struma ovarii and carcinoids, are closely associated with mature teratomas, their genetic basis is poorly understood. A series of mature and monodermal ovarian teratomas were analyzed by short tandem repeat genotyping to evaluate their genetic zygosity and its associations. Informative DNA genotyping data were obtained for ten mature teratomas, six struma ovarii, and three carcinoids (one insular, one trabecular, and one mucinous). A homozygous genotype was present in five of the ten (50%) mature teratomas, three of the six (50%) struma ovarii, and one of the three (33%) ovarian carcinoids. There was no significant difference in genetic zygosity between mature and monodermal teratomas. Patients' age was not correlated with the genetic zygosity: the youngest age in the homozygous tumor group of patients was 4 years. It is suggested that an oocyte after meiosis I, which has escaped from meiotic arrest, is a significant cause of these tumors. Although one mature teratoma was a rare case with lactating adenoma-like breast tissue, its genetic zygosity was concordant with that of the surrounding teratomatous tissue. In one ovarian carcinoid, the carcinoid and accompanying teratomatous components showed matching zygosity at all but one locus: the carcinoid was heterozygous but teratoma was homozygous at one pericentromeric locus. This suggests that not all carcinoids are secondary neoplasms arising from a fully developed mature teratoma: some are neoplasms deviating from a developing mature teratoma.

DICER1 hotspot mutations in ovarian Sertoli-Leydig cell tumors: a potential association with androgenic effects.

Sertoli-Leydig cell tumors (SLCTs) are representative of androgenic ovarian tumors, and they show diverse histologic differentiation, including heterologous differentiation. Genetically, SLCTs are characterized by the presence of DICER1 mutations. In the present study, we analyzed the correlation between somatic DICER1 hotspot mutations and clinicopathological features in 10 ovarian SLCTs. Six of the 10 (60%) SLCTs harbored a DICER1 hotspot mutation. Five of the 6 DICER1-mutated SLCT patients showed androgenic manifestations, including amenorrhea and hirsutism, and 4 of the 6 were associated with the significant elevation of serum testosterone. In contrast, none of the 4 DICER1 wild-type SLCT patients showed virilization. The patient age at diagnosis was lower in those with DICER1-mutated SLCTs (average, 24.7; range, 17-43) than in those with DICER1 wild-type tumors (average, 64.8; range, 47-77). Histologically, heterologous differentiation was found in 4 SLCTs, all of which were DICER1 mutant. Heterologous components included gastrointestinal-type mucinous epithelium (n=3), carcinoid (n=1), and rhabdomyosarcoma (n=1). In the latter, the rhabdomyosarcomatous component was dominant to the SLCT component. In summary, DICER1 hotspot mutations are closely associated with androgenic effects in ovarian SLCTs. It is suggested that DICER1 mutations are involved in the dysregulation of sex hormone synthesis in SLCT patients. Somatic DICER1 hotspot mutations are more common in SLCT patients during the reproductive years than in those during the postreproductive years. DICER1 hotspot mutations may support the pathological diagnosis of SLCTs in cases wherein the heterologous component overwhelms and masks the SLCT component.

Zymographic analysis using gelatin-coated film of the effect of etanercept on the extracellular matrix-degrading activity in synovial fluids of rheumatoid arthritis patients.

AIM: Rheumatoid arthritis (RA) is a chronic inflammatory disease. Most RA patients develop cartilage and bone destruction, and various proteinases are involved in the destruction of extracellular matrix of cartilage and bone. The aim of this study is to evaluate the utility of our newly developed method to measure total gelatinolytic activity. We adopted this method for measurement in synovial fluid from RA patients treated by the anti-rheumatic drug etanercept (ETN), a recombinant human soluble tumor necrosis factor receptor fusion protein, and compared the findings with clinical and laboratory data. METHODS: Enzymatic activity of synovial fluid was analyzed by zymography using gelatin-coated film, and compared with the index of Disease Activity Score of 28 joints - C-reactive protein (DAS28-CRP), CRP and matrix metalloproteinase (MMP)-3 level before and after ETN therapy. RESULTS: Synovial fluids of 19 patients were collected before and after administration of ETN therapy. In nine of 19 patients, who showed a decrease in gelatin-degrading activity in synovial fluid, the index of DAS28-CRP (4.85-2.85, ΔDAS = -2.00) and CRP (3.30-0.94 mg/dL, ΔCRP = -2.36) was alleviated after ETN therapy, while cases with no change or an increase in gelatin-degrading activity showed a modest improvement in clinical data: DAS28-CRP (4.23-3.38, ΔDAS = -0.85) and CRP (1.70-0.74 mg/dL, ΔCRP = -0.96). CONCLUSION: Our newly developed method for measurement of gelatin-degrading activity in synovial fluid from RA patients is highly practicable and useful for predicting the effect of ETN therapy.

World's first telepathology experiments employing WINDS ultra-high-speed internet satellite, nicknamed "KIZUNA".

BACKGROUND: Recent advances in information technology have allowed the development of a telepathology system involving high-speed transfer of high-volume histological figures via fiber optic landlines. However, at present there are geographical limits to landlines. The Japan Aerospace Exploration Agency (JAXA) has developed the "Kizuna" ultra-high speed internet satellite and has pursued its various applications. In this study we experimented with telepathology in collaboration with JAXA using Kizuna. To measure the functionality of the Wideband InterNet working engineering test and Demonstration Satellite (WINDS) ultra-high speed internet satellite in remote pathological diagnosis and consultation, we examined the adequate data transfer speed and stability to conduct telepathology (both diagnosis and conferencing) with functionality, and ease similar or equal to telepathology using fiber-optic landlines. MATERIALS AND METHODS: We performed experiments for 2 years. In year 1, we tested the usability of the WINDS for telepathology with real-time video and virtual slide systems. These are state-of-the-art technologies requiring massive volumes of data transfer. In year 2, we tested the usability of the WINDS for three-way teleconferencing with virtual slides. Facilities in Iwate (northern Japan), Tokyo, and Okinawa were connected via the WINDS and voice conferenced while remotely examining and manipulating virtual slides. RESULTS: Network function parameters measured using ping and Iperf were within acceptable limits. However; stage movement, zoom, and conversation suffered a lag of approximately 0.8 s when using real-time video, and a delay of 60-90 s was experienced when accessing the first virtual slide in a session. No significant lag or inconvenience was experienced during diagnosis and conferencing, and the results were satisfactory. Our hypothesis was confirmed for both remote diagnosis using real-time video and virtual slide systems, and also for teleconferencing using virtual slide systems with voice functionality. CONCLUSIONS: Our results demonstrate the feasibility of ultra-high-speed internet satellite networks for use in telepathology. Because communications satellites have less geographical and infrastructural requirements than landlines, ultra-high-speed internet satellite telepathology represents a major step toward alleviating regional disparity in the quality of medical care.

Serial block-face scanning electron microscopy combined with double-axis electron beam tomography provides new insight into cellular relationships.

To evaluate the advantages of combination of two advanced electron microscopic technologies such as serial block-face scanning electron microscopy and double-axis electron beam tomography, we analyzed the three-dimensional morphology of cellular relationships between dendritic and plasma cells in the synovial membrane from patients with rheumatoid arthritis, using the combined approach.

Development of a teledermatopathology consultation system using virtual slides.

BACKGROUND: An online consultation system using virtual slides (whole slide images; WSI) has been developed for pathological diagnosis, and could help compensate for the shortage of pathologists, especially in the field of dermatopathology and in other fields dealing with difficult cases. This study focused on the performance and future potential of the system. METHOD: In our system, histological specimens on slide glasses are digitalized by a virtual slide instrument, converted into web data, and up-loaded to an open server. Using our own purpose-built online system, we then input patient details such as age, gender, affected region, clinical data, past history and other related items. We next select up to ten consultants. Finally we send an e-mail to all consultants simultaneously through a single command. The consultant receives an e-mail containing an ID and password which is used to access the open server and inspect the images and other data associated with the case. The consultant makes a diagnosis, which is sent to us along with comments. Because this was a pilot study, we also conducted several questionnaires with consultants concerning the quality of images, operability, usability, and other issues. RESULTS: We solicited consultations for 36 cases, including cases of tumor, and involving one to eight consultants in the field of dermatopathology. No problems were noted concerning the images or the functioning of the system on the sender or receiver sides. The quickest diagnosis was received only 18 minutes after sending our data. This is much faster than in conventional consultation using glass slides. There were no major problems relating to the diagnosis, although there were some minor differences of opinion between consultants. The results of questionnaires answered by many consultants confirmed the usability of this system for pathological consultation. (16 out of 23 consultants.) CONCLUSION: We have developed a novel teledermatopathological consultation system using virtual slides, and investigated the usefulness of the system. The results demonstrate that our system can be a useful tool for international medical work, and we anticipate its wider application in the future. VIRTUAL SLIDES: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1902376044831574.

Role of secretory phospholipase A(2) in rhythmic contraction of pulmonary arteries of rats with monocrotaline-induced pulmonary arterial hypertension.

Excessive stretching of the vascular wall in accordance with pulmonary arterial hypertension (PAH) induces a variety of pathogenic cellular events in the pulmonary arteries. We previously reported that indoxam, a selective inhibitor for secretory phospholipase A(2) (sPLA(2)), blocked the stretch-induced contraction of rabbit pulmonary arteries by inhibition of untransformed prostaglandin H(2) (PGH(2)) production. The present study was undertaken to investigate involvement of sPLA(2) and untransformed PGH(2) in the enhanced contractility of pulmonary arteries of experimental PAH in rats. Among all the known isoforms of sPLA(2), sPLA(2)-X transcript was most significantly augmented in the pulmonary arteries of rats with monocrotaline-induced pulmonary hypertension (MCT-PHR). The pulmonary arteries of MCT-PHR frequently showed two types of spontaneous contraction in response to stretch; 27% showed rhythmic contraction, which was sensitive to indoxam and SC-560 (selective COX-1 inhibitor), but less sensitive to NS-398 (selective COX-2 inhibitor); and 47% showed sustained incremental tension (tonic contraction), which was insensitive to indoxam and SC-560, but sensitive to NS-398 and was attenuated to 45% of the control. Only the rhythmically contracting pulmonary arteries of MCT-PHR produced a substantial amount of untransformed PGH(2), which was abolished by indoxam. These results suggest that sPLA(2)-mediated PGH(2) synthesis plays an important role in the rhythmic contraction of pulmonary arteries of MCT-PHR.

Interleukin-6 upregulates expression of ADAMTS-4 in fibroblast-like synoviocytes from patients with rheumatoid arthritis.

AIM: A disintegrin-like and metalloproteinase with thrombospondin type 1 motif (ADAMTS)-4 and ADAMTS-5 play crucial roles in the cleavage of aggrecan. Several recent studies have demonstrated the effect of cytokines such as interleukin (IL)-1ß, tumor necrosis factor-α and transforming growth factor-ß on the expression of ADAMTS-4 and ADAMTS-5 in fibroblast-like synoviocytes (FLS). However, the effect of IL-6 remains unclear. The aim of this study is to investigate the expression of ADAMTS-4 and ADAMTS-5 in FLS of rheumatoid arthritis (RA) patients after IL-6 stimulation. METHOD: Immunohistochemical staining was performed to examine the expression and localization of ADAMTS-4 and ADAMTS-5 in RA synovium. FLS isolated from RA patients were stimulated by IL-6 and soluble IL-6 receptor (sIL-6R) in the presence or absence of anti-IL-6R antibody, and the expression levels of ADAMTS-4 and ADAMTS-5 messenger RNA (mRNA) were assessed using real-time polymerase chain reaction. Furthermore, the IL-6 signaling pathway for regulation of ADAMTS-4 and ADAMTS-5 was also examined. RESULTS: Staining for ADAMTS-4 and ADAMTS-5 was mainly observed in the sublining layer of synovial membrane and pannus. The expression of ADAMTS-4 mRNA was increased by IL-6/sIL-6R, whereas that of ADAMTS-5 was decreased. Anti-IL-6R antibody suppressed the effect of IL-6/sIL-6R. Mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) kinase (MEK)1/2 inhibitor U0126 inhibited the effect of IL-6/sIL-6R on ADAMTS-4 mRNA expression in FLS. Signal transducer and activator of transcription 3 (STAT3) inhibitor parthenolide inhibited the effect of IL-6/sIL-6R on ADAMTS-4, and downregulated ADAMTS-5 expression. CONCLUSION: These results suggest IL-6 may participate in cartilage destruction in RA as an inducer of ADAMTS-4 expression from FLS.

Quantitative measurement of serum hyaluronic acid molecular weight in rheumatoid arthritis patients and the role of hyaluronidase.

AIM: Hyaluronic acid (HA) is a glycosaminoglycan and is essential for protecting the cartilage surface by its physical property. It is known that serum HA concentration in rheumatoid arthritis (RA) patients is higher than in healthy volunteer. However, molecular weight (MW) of serum HA in RA patients is not clear, since it needs a large sample volume to assay serum HA MW. The aim of this study is to establish the method for measuring serum HA MW in small sample sizes and to assess the association between serum HA MW and hyaluronidase (HAase) activity. METHODS: MW of serum HA in RA patients was measured using high-performance liquid chromatography and HA-binding protein. Additionally, the correlation between serum HA and HAase activity was examined using zymographic measurements. RESULTS: Serum HA MW peaked at 1-2 × 10(5) Da in all cases. However, in certain cases two peaks were observed, one each at low (1-2 × 10(5) Da) and high (8-14 × 10(5) Da) MW. HAase activity was lower in cases exhibiting this two-peaked serum HA MW pattern than in those cases with only a single peak. CONCLUSION: The novel method developed for this study permits accurate measurement of serum HA MW. The correlation observed between serum HA MW and HAase activity suggests that serum HA MW may reflect the condition of subjects' joints.

A histopathological study of pulmonary hypertension in connective tissue disease.

Connective tissue diseases (CTD), such as systemic sclerosis (SSc), systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCTD), develop pulmonary hypertension (PH). Generally all PH cases associated with any CTD are classified into the same PH group. However, histological examination shows both common and specific lesions for each disease. In patients with SLE, fibrosis is generally rare and mild. The findings of PH in SLE are similar to those in primary pulmonary hypertension. Many cases of SSc are accompanied by fibrosis. MCTD is rather close to SSc. Arterial and arteriolar lesions of MCTD are characterized by fibrous intimal thickening. In this review, we describe the pathological features of PH associated with each CTD.