Evaluation of circadian phenotypes utilizing fibroblasts from patients with circadian rhythm sleep disorders.

We evaluated the circadian phenotypes of patients with delayed sleep-wake phase disorder (DSWPD) and non-24-hour sleep-wake rhythm disorder (N24SWD), two different circadian rhythm sleep disorders (CRSDs) by measuring clock gene expression rhythms in fibroblast cells derived from individual patients. Bmal1-luciferase (Bmal1-luc) expression rhythms were measured in the primary fibroblast cells derived from skin biopsy samples of patients with DSWPD and N24SWD, as well as control subjects. The period length of the Bmal1-luc rhythm (in vitro period) was distributed normally and was 22.80±0.47 (mean±s.d.) h in control-derived fibroblasts. The in vitro periods in DSWPD-derived fibroblasts and N24SWD-derived fibroblasts were 22.67±0.67 h and 23.18±0.70 h, respectively. The N24SWD group showed a significantly longer in vitro period than did the control or DSWPD group. Furthermore, in vitro period was associated with response to chronotherapy in the N24SWD group. Longer in vitro periods were observed in the non-responders (mean±s.d.: 23.59±0.89 h) compared with the responders (mean±s.d.: 22.97±0.47 h) in the N24SWD group. Our results indicate that prolonged circadian periods contribute to the onset and poor treatment outcome of N24SWD. In vitro rhythm assays could be useful for predicting circadian phenotypes and clinical prognosis in patients with CRSDs.

Initial Report for the Radiation Effects Research Foundation F1 Mail Survey.

To study the full health effects of parental radiation exposure on the children of the atomic bomb survivors, the Radiation Effects Research Foundation developed a cohort of 76,814 children born to atomic bomb survivors (F1 generation) to assess cancer incidence and mortality from common adult diseases. In analyzing radiationassociated health information, it is important to be able to adjust for sociodemographic and lifestyle variations that may affect health. In order to gain this and other background information on the F1 cohort and to determine willingness to participate in a related clinical study, the F1 Mail Survey Questionnaire was designed with questions corresponding to relevant health, sociodemographic, and lifestyle indicators. Between the years 2000 and 2006, the survey was sent to a subset of the F1 Mortality Cohort. A total of 16,183 surveys were completed and returned: 10,980 surveys from Hiroshima residents and 5,203 from Nagasaki residents. The response rate was 65.6%, varying somewhat across parental exposure category, city, gender, and year of birth. Differences in health and lifestyle were noted in several variables on comparison across city and gender. No major differences in health, lifestyle, sociodemographics, or disease were seen across parental exposure categories, though statistically significant tests for heterogeneity and linear trend revealed some possible changes with dose. The data described herein provide a foundation for studies in the future.

Steroid hormone measurements from different types of assays in relation to body mass index and breast cancer risk in postmenopausal women: Reanalysis of eighteen prospective studies.

Epidemiological studies have examined breast cancer risk in relation to sex hormone concentrations measured by different methods: "extraction" immunoassays (with prior purification by organic solvent extraction, with or without column chromatography), "direct" immunoassays (no prior extraction or column chromatography), and more recently with mass spectrometry-based assays. We describe the associations of estradiol, estrone and testosterone with both body mass index and breast cancer risk in postmenopausal women according to assay method, using data from a collaborative pooled analysis of 18 prospective studies. In general, hormone concentrations were highest in studies that used direct assays and lowest in studies that used mass spectrometry-based assays. Estradiol and estrone were strongly positively associated with body mass index, regardless of the assay method; testosterone was positively associated with body mass index for direct assays, but less clearly for extraction assays, and there were few data for mass spectrometry assays. The correlations of estradiol with body mass index, estrone and testosterone were lower for direct assays than for extraction and mass spectrometry assays, suggesting that the estimates from the direct assays were less precise. For breast cancer risk, all three hormones were strongly positively associated with risk regardless of assay method (except for testosterone by mass spectrometry where there were few data), with no statistically significant differences in the trends, but differences may emerge as new data accumulate. Future epidemiological and clinical research studies should continue to use the most accurate assays that are feasible within the design characteristics of each study.

Detection of apoptosis-specific autoantibodies directed against granzyme B-induced cleavage fragments of the SS-B (La) autoantigen in sera from patients with primary Sjögren's syndrome.

The objective of this study was to detect autoantibodies against granzyme B cleavage products in sera from patients with primary Sjögren's syndrome (SS). Cell lysates derived from human salivary gland (HSG) cell lines were incubated with granzyme B. The susceptibility to the generation of cleavage fragments of SS autoantigens was assayed by immunoblotting using sera from 57 primary SS patients, 17 primary SS patients with malignant lymphoma (ML), 28 systemic lupus erythematosus (SLE) patients, and 20 healthy controls. A 27 kD protein was recognized by serum autoantibodies in 8 (14.0%) of 57 primary SS patients, 5 (29.4%) of 17 SS patients with ML, 2 (7.1%) of 28 SLE patients, but not in 20 normal subjects. This protein was recognized by anti-SSB (La) monoclonal antibodies. Granzyme B-treated recombinant La protein was also shown to migrate as a discrete 27 kD protein by SDS PAGE. Blocking studies demonstrated the existence of an apoptosis-specific B cell epitope present in sera from 2 of 8 primary SS patients and in 2 of 5 primary SS patients with ML which recognized the 27 kD protein. Granzyme B-induced La fragments are generated during cytotoxicity in vitro. This is the first report describing autoantibodies in sera from primary SS patients that specifically recognize fragments of the La protein that are produced by the granzyme B protease.

Anti-apoptogenic function of TGFbeta1 for human synovial cells: TGFbeta1 protects cultured synovial cells from mitochondrial perturbation induced by several apoptogenic stimuli.

OBJECTIVE: To investigate anti-apoptogenic mechanism of transforming growth factor beta1 (TGFbeta1) towards synovial cells. METHODS: Isolated synovial cells, treated or not with TGFbeta1, were cultured in the presence or absence of anti-Fas IgM, proteasome inhibitor Z-Leu-Leu-Leu-aldehyde (LLL-CHO), etoposide, or C2-ceramide. After cultivation, apoptosis of synovial cells was examined by the presence of hypodiploid DNA(+) cells, the presence of terminal deoxy (d)-UTP nick end labelling(+) cells (TUNEL(+) cells), activation of caspases, and disruption of mitochondrial transmembrane potential (DeltaPsim). RESULTS: Activation of caspase-9 and DeltaPsim was found in anti-Fas IgM treated synovial cells. The increment of both hypodiploid DNA(+) cells and TUNEL(+) cells accompanied by the activation of caspase-8 and caspase-3 was also determined in anti-Fas IgM treated synovial cells. These hallmarks for apoptosis induced by anti-Fas IgM were significantly suppressed in TGFbeta1 treated synovial cells. LLL-CHO, etoposide, and C2-ceramide also caused DeltaPsim, the increment of both hypodiploid DNA(+) cells and TUNEL(+) cells, and the activation of both Leu-Glu-His-Asp ase (LEHDase; caspase-9 like activity) and Asp-Glu-Val-Asp ase (DEVDase; caspase-3 like activity) in synovial cells. As determined in anti-Fas IgM treatment, TGFbeta1 significantly reduced apoptotic cell death of synovial cells induced by the above chemicals. CONCLUSIONS: The protective effect of TGFbeta1 for mitochondrial homoeostasis may be important in the anti-apoptogenic function of TGFbeta1 for synovial cells.

New disease-modifying antirheumatic drug 2 acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (KE-298) selectively augments activation-induced T cell death.

We examined in this study whether the newly developed disease-modifying antirheumatic drug (DMARD) 2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (KE-298) augments activation-induced T cell death. Peripheral blood (PB) T cells, isolated from healthy donors, were activated by incubation with interleukin-2 (IL-2) followed by further culture with 12-0-tetradecanoyl phorbol 13-acetate (PMA) and ionomycin in the presence or absence of KE-298. The apoptosis of activated T cells was examined by flow cytometric determination of hypodiploid DNA. Fas expression and caspase-3 activity in activated T cells were also examined by flow cytometry, and expression of Fas ligand (FasL), Bcl-2-related proteins, and X chromosome-linked inhibitor of apoptosis protein (XIAP) was determined by Western blot analysis. Apoptosis was not obvious in resting T cells and was not augmented by KE-298. In contrast, apoptosis was clearly detected in activated T cells (activation-induced T cell death) with the increment of caspase-3 activity, and incubation of these cells with KE-298 further enhanced apoptosis. Treatment of activated T cells with KE-298 increased Bax expression but decreased XIAP expression without affecting the expression of Fas/FasL. Thus caspase-3 activity in activated T cells appeared to be increased by KE-298. Our results suggest that the newly developed DMARD, KE-298, selectively augmented activation-induced T cell death. This finding may contribute to the therapeutic efficacy of KE-298 in rheumatoid arthritis (RA) patients and provide new insight into the pharmacologic action of DMARDs.

Importance of NF-kappaB in rheumatoid synovial tissues: in situ NF-kappaB expression and in vitro study using cultured synovial cells.

OBJECTIVES: To examine whether inhibition of NF-kappaB induces apoptosis of human synovial cells stimulated by tumour necrosis factor alpha (TNFalpha), interleukin 1beta (IL1beta), and anti-Fas monoclonal antibody (mAb). METHODS: The expression of proliferating cell nuclear antigen (PCNA), NF-kappaB, and the presence of apoptotic synovial cells were determined in synovial tissues. Apoptosis of cultured synovial cells was induced by inhibition of NF-kappaB nuclear translocation by Z-Leu-Leu-Leu-aldehyde (LLL-CHO). The activation of caspase-3 and expression of XIAP and cIAP2 in synovial cells in LLL-CHO induced apoptosis was also examined. RESULTS: Abundant PCNA+ synovial cells were found in rheumatoid arthritis (RA) synovial tissue, though a few apoptotic synovial cells were also detected in the RA synovial tissues. Nuclear NF-kappaB was expressed in RA synovial cells. Electrophoretic mobility shift assay showed that treatment of cells with TNFalpha or IL1beta significantly stimulated nuclear NF-kappaB activity. A small number of apoptotic synovial cells expressing intracellular active caspase-3 were found after treatment of cells with LLL-CHO. Although treatment of RA synovial cells with TNFalpha or IL1beta alone did not induce apoptosis, apoptosis induced by LLL-CHO and caspase-3 activation were clearly enhanced in TNFalpha or IL1beta stimulated synovial cells compared with unstimulated synovial cells. Furthermore, induction of apoptosis of synovial cells with caspase-3 activation by anti-Fas mAb was clearly increased by LLL-CHO. The expression of cIAP2 and XIAP in synovial cells may not directly influence the sensitivity of synovial cells to apoptosis induced by LLL-CHO. CONCLUSION: The results suggest that NF-kappaB inhibition may be a potentially important therapeutic approach for RA by correcting the imbalance between apoptosis and proliferation of synovial cells in RA synovial tissue.

Regulation of cyclooxygenase-2 expression in human osteoblastic cells by N-acetylcysteine.

Cyclooxygenase (COX) plays a pivotal role in the inflammatory process of inflammatory arthropathies. Inflammatory cytokines induce COX-2 expression in osteoblasts of inflamed joints, followed by osteoclast activation. The inhibition of COX-2 expression could help prevent prostaglandin E2 secretion, followed by osteoclast activation for bone destruction and resorption. We examined whether the antioxidant N-acetylcysteine (NAC) inhibited COX-2 expression induced in the human osteoblastic cell line MG63 by interleukin-1beta (IL-1beta). According to Western blot and reverse transcription-polymerase chain reaction (RT-PCR) test results, NAC inhibited IL-1beta-induced COX-2 expression in protein and messenger RNA. We also demonstrated immunohistochemically that NAC inhibited NFkappaB nuclear translocation. These results suggested that NAC inhibited both COX-2 expression and NFkappaB nuclear translocation in MG63, which in turn indicated that NAC could inhibit the inflammatory process involved in bone resorption by regulating COX-2 expression at the level of transcription.

Effect of vitamin K2 on osteoblast apoptosis: vitamin K2 inhibits apoptotic cell death of human osteoblasts induced by Fas, proteasome inhibitor, etoposide, and staurosporine.

Vitamin K2 is used for the treatment of osteoporosis, but the precise mode of action is still not clear. We investigated the effects of vitamin K2 on apoptosis of human osteoblasts. Human osteoblastic cell line MG63 cells and human primary osteoblast-like cells obtained from bone fragments in corrective surgery were used as human osteoblasts. Cells were cultured with or without various concentrations of vitamin K2 and tumor necrosis factor-alpha (TNF-alpha). We then determined the proliferative response, expression of Fas and Bcl-2-related proteins, and Fas-mediated apoptosis of these cells induced by anti-Fas immunoglobulin M (IgM). In addition, the effect of vitamin K2 in osteoblast apoptosis induced by Z-Leu-Leu-Leu-aldehyde (LLL-CHO), etoposide, or staurosporine was also examined. Human osteoblasts did not show spontaneous apoptosis in culture, even in the presence of vitamin K2 or TNF-alpha. Furthermore, proliferation of the cells was not influenced by vitamin K2 or TNF-alpha. Fas was functionally expressed on human osteoblasts, and the treatment with TNF-alpha significantly enhanced both Fas expression and Fas-mediated apoptosis of osteoblasts. The addition of vitamin K2 to the culture resulted in a dose-dependent inhibition of functional Fas expression on osteoblasts, in the presence or absence of TNF-alpha. Treatment of human osteoblasts with vitamin K2 clearly suppressed Bax expression of the cells, although the expression of Bcl-2 was not influenced by vitamin K2. Fas ligand (FasL) cDNA transformants were cytotoxic against osteoblasts, and the cytotoxicity was increased when osteoblasts were treated with TNF-alpha. The addition of vitamin K2 to osteoblasts significantly decreased the cytotoxic effects of FasL cDNA transformants. Furthermore, apoptosis of human osteoblasts induced by LLL-CHO, etoposide, or staurosporine was also clearly suppressed in vitamin K2-treated osteoblasts. Our results suggest that vitamin K2 inhibits apoptotic cell death of osteoblasts and maintains the number of osteoblasts. These actions may explain the therapeutic efficacy of vitamin K2 in osteoporosis.

The human and mouse Period1 genes: five well-conserved E-boxes additively contribute to the enhancement of mPer1 transcription.

The clock gene, Period1, from human and mouse was sequenced and characterized. Both human PERIOD1 (human PER1) and mouse Period1 (mouse Per1) consisted of 23 exons spanning approximately 16 kb, and their structures showed strong similarity to each other. For example, six highly conserved regions were identified in the 5' upstream sequences. These conserved segments exhibited 77-88% identity and possessed several potential regulatory elements including five E-boxes (the binding site of the CLOCK-BMAL1 complex) and four cyclic AMP response elements. Transient transfection assays using a mPer1-luciferase fusion gene revealed that each of the conserved E-boxes additively functions as an enhancer for the transactivation of mPer1 by mCLOCK and mBMAL1.

Nuclear factor-kappaB and caspases co-operatively regulate the activation and apoptosis of human macrophages.

Accumulating evidence suggests that macrophages function as major effector cells in the pathological process of various human diseases. We examined here the role of nuclear factor-kappaB (NF-kappaB) and caspases in the regulation of activation and apoptosis of macrophages. Activation of the human monoblastic leukaemia cell line, U937, by phorbol 12-myristate 13-acetate (PMA) increased the expression of CD14/CD86, and cytokine production. PMA stimulation also increased the expression of both pro-caspase-8 and pro-caspase-3 in U937, but not apoptosis or intracellular caspase-3 activity. PMA also increased the expression of X-chromosome-linked inhibitor of apoptosis protein (XIAP) in U937, suggesting an inhibitory action for XIAP on the caspase cascade in PMA-stimulated U937. Electrophoretic mobility shift assay (EMSA) showed a significant increase of nuclear NF-kappaB activity in PMA-stimulated U937. When a potent NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC), was added to U937 cell culture in the presence of PMA, apoptosis was triggered by activation of caspase-3, which was induced by caspase-8 activation. XIAP expression was markedly suppressed in PMA-treated U937 in the presence of PDTC. The inhibitors of caspase-8 and caspase-3 mostly inhibited apoptosis of U937 treated with PMA in the presence of PDTC. Furthermore, a phenotype of U937 treated with PMA and PDTC in the presence of caspase inhibitor was almost identical to that of unstimulated U937. Our results suggest that the signalling pathways involved in the activation and apoptosis of human macrophages could be co-operatively regulated by the use of NF-kappaB and caspase inhibitors, thus enabling the control of macrophage function and number.

CD4+ T cell-mediated cytotoxicity toward thyrocytes: the importance of Fas/Fas ligand interaction inducing apoptosis of thyrocytes and the inhibitory effect of thyroid-stimulating hormone.

The accumulation of activated CD4+ T cells and antigen (Ag)-dependent cellular interactions between thyrocytes and CD4+ T cells have been determined in thyroid gland from patients with Graves' disease. The Fas/Fas ligand (FasL) interaction between antigen-presenting cells and T cells regulates the apoptosis of the former cells triggered by the latter cells. The inhibition of Fas-mediated apoptosis in thyrocytes could be a underlying mechanism of hyperplasia of thyrocytes in patients with Graves' disease. We investigated the potential role of Fas/FasL interaction between thyrocytes and CD4+ T cells in the induction of Fas-mediated apoptosis of the former cells induced by the latter cells. The presence of only a few specific T cells responsive to a putative autoantigen has hampered the investigation of specific T cell activation toward antigen-presenting cells (APCs). Therefore, we used a superantigen, staphylococcal enterotoxin B (SEB), to examine specific T cell activation toward thyrocytes in vitro since it stimulates a large proportion of T cells with particular Vbeta elements. Spontaneous apoptosis of thyrocytes in culture was not found even in the presence of various kinds of cytokines. In contrast, a clear induction of Fas-mediated apoptosis by anti-Fas IgM was determined in interferon-gamma (IFN-gamma)-stimulated thyrocytes. In addition, a significant cytotoxicity of purified CD4+ T cells toward IFN-gamma-stimulated thyrocytes in the presence of SEB was induced, and the addition of anti-HLA-DR and -DQ monoclonal antibodies (mAbs) or blockade of the Fas/FasL interaction reduced this cytotoxicity. FasL expression of CD4+ T cells cocultured with IFN-gamma-stimulated thyrocytes in the presence of SEB was clearly induced. Furthermore, the addition of mAbs against CD54 and CD58 inhibited both cytotoxicity and FasL expression of CD4+ T cells. The cytotoxicity of CD4+ T cells toward IFN-gamma-stimulated, SEB-pulsed thyrocytes was markedly inhibited when we used thyrocytes cultured with IFN-gamma in the presence of thyroid-stimulating hormone (TSH) as target cells. Our results suggest that 1) CD4+ T cells were activated by thyrocytes expressing MHC class II molecules in an SEB-dependent manner and then expressed FasL. 2) These activated FasL+ CD4+ T cells killed thyrocytes by interacting with Fas on thyrocytes and FasL on activated CD4+ T cells. The presence of costimulating molecules such as CD54 and CD58 on thyrocytes was also necessary to generate activated FasL+ CD4+ T cells. 3) Since the actions of thyroid stimulating antibody (TSAb) toward thyrocytes are similar to those of TSH, one goitrogenic activity of TSAb may, in part, be due to the inhibitory effect on Fas-mediated apoptosis of thyrocytes triggered by activated CD4+ T cells.

Relationship between Sjögren's syndrome and human T-lymphotropic virus type I infection: follow-up study of 83 patients.

We have previously demonstrated a high prevalence of Sjögren's syndrome (SS) in patients with human T-lymphotropic virus type I-associated myelopathy (HAM) in Nagasaki prefecture. The present follow-up study compared the clinical and laboratory findings of SS with or without human T-lymphotropic virus type I (HTLV-I) antibody in this endemic area for HTLV-I infection. We investigated the clinical and laboratory manifestations in 83 patients with SS and HAM, including histologic examination of labial salivary glands and the prevalence of SS in patients with HAM. Definite SS was diagnosed in 13 out of 20 patients with HAM when the European Community criteria were used. The density of mononuclear cell infiltration in labial salivary glands was higher in HTLV-I-seropositive patients with SS (including patients with HAM) than in HTLV-I-seronegative patients. The volume of saliva and lacrima determined by the Schirmer or Saxon test was lower than normal but was not different among SS-HTLV-I-seronegative patients, HTLV-I-seropositive patients without HAM, and HTLV-I-seropositive patients with HAM. The proportions of patients positive for antinuclear antibody (ANA) and anti-SS-A (Ro) antibody or anti-SS-B (La) antibody were similar in the three groups. However, the low volume of saliva and the frequency of ANA in SS correlated with the degree of mononuclear cell infiltration in labial salivary glands. Our results suggested that HTLV-I infection is related to SS and that laboratory and clinical findings in SS closely correlate with the degree of mononuclear cell infiltration in the salivary glands.

Inhibition of caspase cascade by HTLV-I tax through induction of NF-kappaB nuclear translocation.

NF-kappaB is required for prevention of apoptosis. We examined the importance of human T-cell leukemia virus-I (HTLV-I) Tax protein to stimulate NF-kappaB nuclear translocation, thus preventing apoptosis. Jurkat cells and JPX-9 cells in which the inducible Tax expression plasmid vector was stably transfected were used in the present study. Both Jurkat and Tax(-) JPX-9 cells had small amounts of basal nuclear NF-kappaB activity. The addition of NF-kappaB inhibitors suppressed NF-kappaB nuclear translocation of the cells, thus inducing apoptosis. Sequential activation of caspases from caspase-8 to caspase-3 was shown during this process. NF-kappaB nuclear translocation in JPX-9 cells was stimulated through Tax expression, and both the activation of caspases and apoptosis induced by NF-kappaB inhibitors were significantly suppressed in the Tax(+) JPX-9 cells. The expression of Bcl-2, Bax, and Bcl-x was not changed among Jurkat, Tax(-) JPX-9, and Tax(+) JPX-9 cells in the presence or absence of NF-kappaB inhibitors. X-chromosome-linked inhibitor of apoptosis (XIAP) protein expression in Tax(-) JPX-9 cells was significantly suppressed by NF-kappaB inhibitors, however, its expression in Tax(+) JPX-9 cells was maintained even by the addition of NF-kappaB inhibitors. Our results suggest that the activation of NF-kappaB via Tax protein in HTLV-I infected cells renders the cells resistant to apoptosis. The expression of anti-apoptotic gene products such as XIAP to suppress caspase cascade, results in an increase of cytokine production and cell proliferation; one of the proposed mechanisms that promotes autoimmune disorders such as Sjögren's syndrome and rheumatoid arthritis found in HTLV-I seropositive subjects.

Regulation of synovial cell apoptosis by proteasome inhibitor.

OBJECTIVE: Recent studies have shown the importance of proteasome function in the regulation of apoptosis. This study examined whether inhibition of proteasome function mediates apoptosis of synovial cells, and whether cytokines modulate this process. METHODS: Type B synovial cells (fibroblast-like synovial cells) were cultured with tumor necrosis factor alpha (TNF alpha) or transforming growth factor beta1 (TGFbeta1), and further incubated in the presence of variable concentrations of Z-Leu-Leu-Leu-aldehyde (LLL-CHO), a proteasome inhibitor. During this process, apoptosis of synovial cells was determined by Hoechst 33258 dye staining and 51Cr release assay. The involvement of caspase cascade was examined using enzyme activity assay and blocking experiments by peptide inhibitors. The expression of pro-caspases, Bcl-2-related proteins, and X chromosome-linked inhibitor of apoptosis (XIAP) in synovial cells was examined by Western blot analysis. RESULTS: Apoptosis of cultured synovial cells was induced in a dose-dependent manner by LLL-CHO. Activation of caspase cascade through caspase-8 to caspase-3 was essential during this process. Pretreatment of synovial cells with TNF alpha significantly augmented both the activation of caspases and the proportion of apoptosis in synovial cells induced by LLL-CHO, whereas TGFbeta1 pretreatment markedly suppressed these phenomena. The ratio of the expression of Bcl-2 to Bax or Bcl-xL to Bax, and XIAP expression in synovial cells may not be directly associated with the susceptibility of synovial cells to apoptosis by LLL-CHO. CONCLUSION: Apoptosis of synovial cells was induced by inhibition of proteasome function through the activation of caspase cascade, and this process was clearly modulated by cytokines. These data provide new insight into the regulatory mechanisms controlling synovial cells in rheumatoid synovitis by proteasome inhibitors, and might be useful for the design of new therapeutic strategies in rheumatoid arthritis.

Expression of metalloproteinase-2 (gelatinase A) in labial salivary glands of patients with Sjögren's syndrome with HTLV-I infection.

OBJECTIVE: To determine whether gelatinase A (MMP-2) plays a significant role in the pathogenesis of Sjögren's syndrome (SS) with or without HTLV-I infection. METHODS: We examined 24 patients with SS (14 HTLV-I-seropositive and 8 HTLV-I-seronegative). Labial salivary gland tissue samples were analysed immunohistochemically using anti-MMP-2 monoclonal antibodies. RESULTS: In normal salivary glands, MMP-2 expression was not detected. All biopsy samples of 8 SS patients with HTLV-I-associated myelopathy (HAM) and 3 of 6 HTLV-I-seropositive SS patients without manifestation of HAM stained positively for MMP-2. However, the other samples were negative for MMP-2. CONCLUSION: Our study showed the MMP-2 expression in labial salivary glands of HTLV-I seropositive SS patients, especially in all SS patients with HAM. The presence of MMP-2 in the salivary glands of these patients suggests that it may play a role in cellular infiltration and destruction in salivary glands of SS.

The effect of papaverine on morphologic differentiation, proliferation and invasive potential of human prostatic cancer LNCaP cells.

BACKGROUND: Intracellular cyclic adenosine monophosphate (AMP) level changes are thought to play an important role in inhibiting cell proliferation and inducing differentiation in several types of cells. It has been reported that cyclic AMP analogs induce terminal differentiation in human prostate cancer cells. Consequently, phosphodiesterase inhibitors may be useful in delineating the role of cyclic AMP in the differentiation of these cells. Therefore, the effect of phosphodiesterase inhibitors on morphologic differentiation, proliferation and invasive potential of human prostate cancer cells was investigated. METHODS: Three human prostate cancer cell lines PC-3, DU145 and LNCaP were treated with one of the phosphodiesterase inhibitors, papaverine, 3-isobutyl-1-methylxanthine (IBMX) or theophylline, for 6 days. Morphologic changes of these cells induced by phosphodiesterase inhibitors were observed by microscopy. Intracellular cyclic AMP levels in LNCaP cells were measured by radioimmunoassay using a cyclic AMP assay kit. The effect of papaverine on the proliferation and invasive potential of LNCaP cells were measured by cell counting and the Matrigel invasion chamber assay. RESULTS: Of the three agents, examined papaverine (10(-5) mol/L) is the most effective inducer of morphologic change and also raised intracellular cyclic AMP levels in LNCaP cells. However, unlike LNCaP cells, PC-3 and DU145 cells treated with phosphodiesterase inhibitors, including papaverine, showed little change in morphology. Additionally, proliferation and invasive potential of LNCaP cells were significantly inhibited by papaverine. CONCLUSION: The results suggest that papaverine induces terminal differentiation in LNCaP cells, which is correlated with an intracellular cyclic AMP-mediated pathway.