Detection and first characterization of an uncommon haptoglobin in porcine saliva of pigs with rectal prolapse by using boronic acid sample enrichment.

Salivary glycoprotein profiles, obtained after boronic acid enrichment, were studied for the first time in pigs in order to search for specific overall alterations related to acute inflammatory condition. Five healthy pigs and five pigs suffering from rectal prolapse were used, and the levels of acute phase proteins were measured to determine the degree of inflammation of the animals. The enriched glycoprotein profiles, achieved by two-dimensional gel electrophoresis (2DE) were statistically evaluated and spots that appeared differentially regulated between states were subjected to MS analysis for protein identification. Spots from three unique proteins were identified: carbonic anhydrase VI (CA VI), α-1-antichymotrypsin and haptoglobin (Hp). CA VI appeared as two adjacent horizontal spot trains in the glycoprotein profile of healthy animals in its regular isoelectric points (pI). One spot of α-1-antichymotrypsin was found in saliva from pigs with rectal prolapse in an unusual basic pI, and was considered as a breakdown product. Hp was identified as several spot trains in saliva from pigs with rectal prolapse in an unusual alkaline pI and was consequently further investigated. SDS-PAGE and 2DE of paired serum and saliva samples combined with Western blot analysis showed that the unusual Hp position observed in saliva samples was absent in serum. Furthermore, N-glycans from serum and saliva Hp glycopatterns were evaluated from SDS-PAGE Hp bands and showed that the serum N-glycan distribution in Hp ß-chain was comparable in quantity and quality in both groups of animals. In saliva, no Hp ß-chain derived N-glycans could unambiguously be identified from this sample set, thus needing further detailed investigations in the future.

Educational differences in associations of noticing anti-tobacco information with smoking-related attitudes and quit intentions: findings from the International Tobacco Control Europe Surveys.

This study examined educational differences in associations of noticing anti-tobacco information with smoking-related attitudes and quit intentions among adult smokers. Longitudinal data (N = 7571) from two waves of six countries of the International Tobacco Control (ITC) Europe Surveys were included. Generalized estimating equation analyses and multiple linear and logistic regression analyses were conducted. Higher educated smokers noticed anti-tobacco information slightly more often than lower educated smokers (F(2) = 25.78, P < 0.001). Noticing anti-tobacco information was associated with more negative smoking-related attitudes (ß = 0.05, P < 0.001) and more quit intentions (OR = 1.08, P < 0.001). Among smokers without a quit intention at baseline, a positive association was found for noticing anti-tobacco information at baseline with follow-up quit intention (OR = 1.14, P = 0.003). No other longitudinal associations were found. No educational differences were found in the association of noticing anti-tobacco information with smoking-related attitudes but associations with quit intentions were found only among low (OR = 1.12, P = 0.001) and high educated respondents (OR = 1.11, P < 0.001) and not among moderate educated respondents (OR = 1.02, P = 0.43). Noticing anti-tobacco information may positively influence quit intentions and possibly smoking-related attitudes. Lower educated smokers were as likely to be influenced by anti-tobacco information as higher educated smokers but noticed anti-tobacco information less often; increasing reach of anti-tobacco information may increase impact in this group.

Serum 25(OH)D, PTH and correlates of suboptimal 25(OH)D levels in persons with chronic spinal cord injury.

STUDY DESIGN: Cross-sectional cohort study. To describe: (1) the prevalence of suboptimal 25-hydroxyvitamin D status (serum 25(OH)D <75 nmol l(-1)) and to identify correlates of vitamin D deficiency; (2) the prevalence of secondary hyperparathyroidism (serum intact parathyroid hormone (PTH)>7.0 pmol l(-1)); and (3) the relationships between serum PTH and 25(OH)D in adult men and women with chronic spinal cord injury (SCI). SETTING: Outpatient services, including an osteoporosis clinic at a tertiary spinal cord rehabilitation hospital in Ontario. METHODS: Serum levels of 25(OH)D and intact PTH were acquired at enrollment. Clinical correlates of suboptimal vitamin D status were collected via interview and chart abstraction, and identified by univariate logistic regression analysis. Pearson correlations were run to assess the relationships between serum PTH and 25(OH)D. Significance was P<0.05. RESULTS: Thirty-nine percent of the cohort, comprised of 62 adult men and women with chronic SCI, had suboptimal serum 25(OH)D levels. Factors associated with suboptimal vitamin D levels included having vitamin D assessed in the winter months (odds ratio (OR)=7.38, P=0.001), lack of a calcium supplement (OR=7.19, P=0.003), lack of a vitamin D supplement (OR=7.41, P=0.019), younger age (OR=0.932, P=0.010), paraplegia (OR=4.22, P=0.016), and lack of bisphosphonate (OR=3.85, P=0.015). Significant associations were observed between serum PTH and 25(OH)D (r=-0.304, P=0.032) and between PTH and C-telopeptide of type I collagen (CTX-I) (r=0.308, P=0.025). Disruption of the vitamin D-PTH axis may contribute to the bone loss seen in the chronic SCI population. The threshold for optimal serum 25(OH)D levels in the chronic SCI population may be higher than in the non-SCI population. Serum 25(OH)D level are likely important risk factors contributing to declining bone mass and increased fracture risk post-SCI.

Stem cell collection in patients with multiple myeloma: impact of induction therapy and mobilization regimen.

Lenalidomide is an active treatment for multiple myeloma (MM) and is increasingly used as part of the initial treatment of this disease. Recent reports have suggested decreases in the number of CD34+ cells collected and increases in the failure rate among patients whose initial therapy contained lenalidomide when mobilized with G-CSF alone. A retrospective data analysis of 364 patients with MM who underwent stem cell mobilization and attempted harvest at the Hospital of the University of Pennsylvania between January 2002 and December 2007 was performed. Forty-three of the patients received lenalidomide in their induction regimen, and were mobilized with either CY and G-CSF or G-CSF alone. The number of apheresis cycles and the failure rate were lower, whereas the mean number of collected stem cells was higher in patients who were mobilized with CY and G-CSF in comparison with G-CSF alone. This suggests that lenalidomide does not prevent the harvest of adequate numbers of CD34 cells for autologous stem cell transplant, but mobilization with G-CSF and CY may be required to obtain adequate numbers of stem cells. Finally, in our study, the number of lenalidomide cycles did not correlate with stem cell yield.

Proteomic analysis of porcine saliva.

Saliva contains a number of proteins that may be useful as biomarkers of health and disease and can be easily obtained from large numbers of animals in a non-invasive, stress-free way. The objective of this study was to explore the protein composition of porcine saliva from 10 specific pathogen free pigs using first one-dimensional SDS-PAGE and then two-dimensional electrophoresis and mass spectrometry. A reference proteome pattern for porcine saliva was established with the identification of 13 different, mainly saliva-specific, proteins. These reference data will facilitate the investigation of salivary proteins potentially altered in disease and could serve as novel diagnostic biomarkers.

Long-term event-free survivors after high-dose therapy and autologous stem-cell transplantation for low-grade follicular lymphoma.

Although follicular lymphoma (FL) is generally responsive to conventional-dose chemotherapy, improved survival in patients with this disease has been difficult to demonstrate. High-dose chemo/radiotherapy followed by autologous stem-cell transplantation (ASCT) can improve response rates, although its effects on survival remain controversial. Between 1990 and 2003, we transplanted 49 patients with low-grade FL at our institution. Twenty-two patients (45%) had undergone histologic transformation at the time of ASCT. In all, 44 patients (90%) had relapsed disease and five patients (10%) were resistant to chemotherapy at the time of transplantation. After ASCT, 30 patients (61%) were in complete remission (CR). The median overall survival (OS) has not been reached, while the median event-free survival (EFS) is 2.4 years. At a median follow-up of 5.5 years (longest 12.4 years), a plateau has been reached with 56% of patients remaining alive, and 35% event-free. ASCT was well tolerated except for two (4%) treatment-related deaths. In multivariable analysis, CR after ASCT and age less than 60 years are the best predictors of EFS and OS. ASCT is thus a safe therapeutic approach in FL, resulting in long-term EFS and OS for some patients, even with transformed disease.

Characterisation of the cell type-specificity of collagenase 3 mRNA expression in comparison with membrane type 1 matrix metalloproteinase and gelatinase A in the synovial membrane in rheumatoid arthritis.

OBJECTIVE: To study the pattern and cell type-specificity of collagenase 3, membrane-type 1 matrix metalloproteinase (MT1-MMP), and gelatinase A mRNA expression in the synovial membrane in rheumatoid arthritis (RA). METHODS: The mRNA expression of collagenase 3, MT1-MMP, and gelatinase A was characterised by northern blot analysis, reverse transcriptase-polymerase chain reaction, and in situ hybridisation. In situ hybridisation was performed in combination with the immunohistochemical detection of cell type-specific antigens. RESULTS: Synovial membrane specimens from 19 of 21 patients with RA expressing collagenase 3 mRNA were positive for MT1-MMP and gelatinase A mRNA. In control samples from patients without destructive inflammatory joint diseases collagenase 3 mRNA was not expressed and only in two of seven cases was a coexpression of MT1-MMP and gelatinase A mRNA detected. Fibroblast-like cells of the synovial membrane were found to be the predominant source of collagenase 3, MT1-MMP, and gelatinase A mRNA expression in lining and sublining layers as well as at the synovial membrane-cartilage interface. Additionally, the expression of MT1-MMP mRNA was detected in endothelial cells. Collagenase 3 mRNA expression was found in about 5% of CD68 positive macrophages. CONCLUSIONS: Collagenase 3 mRNA is expressed simultaneously with MT1-MMP and gelatinase A mRNA in fibroblast-like cells of the synovial membrane in RA. These results suggest (a) a broad extracellular proteolytic potential of fibroblast-like cells and (b) an important role of cell surface associated procollagenase 3 activation by MT1-MMP and gelatinase A for cartilage degradation by invading fibroblast-like cells.

Expression of the T-cell chemoattractant chemokine lymphotactin in Crohn's disease.

Recruitment of lymphocytes is a prominent feature of the inflammatory process in Crohn's disease (CD). The present study was undertaken to investigate the expression of the novel lymphocyte-specific chemoattractant lymphotactin (Lptn) as a potential regulatory factor for the recruitment of T cells in CD. The expression of Lptn mRNA was quantified in resection specimens of patients with CD in comparison to normal controls without signs of inflammation by real-time quantitative reverse transcriptase-polymerase chain reaction and localized by nonradioactive in situ hybridization. Furthermore, the phenotype of cells expressing Lptn mRNA was characterized. In contrast to normal controls Lptn mRNA was significantly increased in tissue samples affected by CD. Cells expressing Lptn were identified as T cells, mast cells, and unexpectedly dendritic cells. Lptn mRNA was found to be up-regulated on stimulation with phorbol-12-myristate-13-acetate and concanavalin A in T cells isolated from peripheral blood, which could be prevented by dexamethasone, cyclosporine A, and FK506. A similar regulation mechanism could be identified for the Lptn receptor GPR-5 in peripheral T cells. In addition, Lptn mRNA expression could be induced in mature monocyte-derived dendritic cells. The results indicate that local expression of Lptn by activated T cells and to a lesser extent by mast cells and dendritic cells represents a key regulator for lymphocyte trafficking and maintenance of the inflammatory process observed in CD, which might be partly mediated through an autocrine/paracrine pathway of activated T cells.

Synovial tissue protease gene expression and joint erosions in early rheumatoid arthritis.

OBJECTIVE: To relate the expression of proteases in the lining and sublining layers of the synovial membrane to the rate of joint damage during 1 year in patients with early inflammatory arthritis. METHODS: Samples of synovial membrane were obtained by closed-needle biopsy or needle arthroscopy from inflamed knees of 20 patients with early inflammatory polyarthritis (mean disease duration 9.6 months, range 2 weeks to 18 months). Expression of matrix metalloproteinase 1 (MMP-1), cathepsin B (CB), and cathepsin L (CL) was examined using in situ hybridization. Immunohistochemistry was used to identify infiltrating mononuclear cell populations. Radiographs of the hands and feet, performed at presentation and after 1 year, were evaluated for the development of new erosions. RESULTS: Twelve patients had rheumatoid arthritis (RA), 6 had psoriatic arthritis (PsA), 1 had gout, and 1 had an undifferentiated arthritis. Six patients had erosions at presentation. Eleven patients (10 with RA, 1 with PsA) demonstrated at least 1 new erosion after 1 year of followup. MMP-1, CB, and CL messenger RNA (mRNA) were expressed in the synovial membrane of all patients and were present throughout the lining layer, as well as in perivascular cellular infiltrates and endothelial cells in the sublining layer. In the lining layer, the mean percentages of protease mRNA-positive cells per high-power field were higher in those patients who developed new joint erosions than in those without evidence of joint damage. A similar pattern was observed in the sublining layer, where mean numbers of protease mRNA-positive cells were also greater in patients with new joint erosions. There were significant differences between the two groups in MMP-1 mRNA expression in both the lining and sublining layers (P = 0.0007 and P = 0.0027, respectively), as well as in sublining layer CL mRNA expression (P = 0.017), but not in CB mRNA expression. Numbers of lining layer CD68+ cells correlated positively with lining layer MMP-1 mRNA expression (P = 0.043) and with the development of new joint erosions (P = 0.002). CONCLUSION: The detection of MMP-1, CB, and CL in the synovium soon after the onset of symptoms highlights the potential for early joint destruction in patients with RA. High levels of MMP-1 mRNA expression in the lining layer distinguished patients with more rapidly progressive erosive disease. This is the first study to demonstrate features of early synovial pathophysiology that may identify patients at increased risk of developing new joint erosions.

Cysteine protease activity is up-regulated in inflamed ankle joints of rats with adjuvant-induced arthritis and decreases with in vivo administration of a vinyl sulfone cysteine protease inhibitor.

OBJECTIVE: Cysteine proteases are postulated to play a role in tissue destruction in the joints of animals with arthritis. The purpose of the present study was to confirm the concept that cysteine proteases are enzymes involved in the pathology of rheumatoid arthritis (RA). METHODS: Arthritis was induced in Lewis rats by adjuvant injection (adjuvant-induced arthritis [AIA] model) and scored for inflammation. At necropsy, the rear paws were either fixed in formalin and assigned a histologic score (based on synovial cell proliferation, cartilage erosion, bone erosion, and fibroproliferative pannus) or frozen, cryosectioned, and assayed for enzyme activity either by in situ cytochemical staining with a post-azo-coupling method using a chromogenic substrate (Z-arg-arg-MNA) or by a novel assay placing the tissue section directly in a cuvette using the fluorogenic substrate Z-arg-arg-AMC. RESULTS: Enzymatic activity, measured either in frozen sections in situ or in the cuvette assay, was positively correlated with joint destruction (r = 0.7) and inflammation (r = 0.8). Activity was not inhibited significantly by Pefabloc (a serine protease inhibitor), EDTA (a metalloprotease inhibitor), or pepstatin A (an aspartyl protease inhibitor) but was inhibited by E-64 and vinyl sulfone irreversible inhibitors of cysteine proteases. The effect of one of the vinyl sulfone cysteine protease inhibitors, Mu-Leu-HomoPhe-vinylsulfone, was tested in vivo by dietary administration at 2.2 mg/kg/day in the AIA model; this resulted in a significant decrease in inflammation and in the amount of cysteine protease activity measured in the joint tissue. CONCLUSION: Cysteine protease activity levels increase in the diseased state and may be an important target for designing small molecule inhibitors to reduce the inflammation and tissue destruction associated with RA.

Expression of osteopontin messenger RNA and protein in rheumatoid arthritis: effects of osteopontin on the release of collagenase 1 from articular chondrocytes and synovial fibroblasts.

OBJECTIVE: Osteopontin (OPN) is an extracellular matrix protein that has been implicated in the interactions between tumor cells and host matrix, including those involved in invasion and spread of tumor cells. Because joint destruction in rheumatoid arthritis (RA) is mediated by the invasive growth of synovial tissue through its attachment to cartilage, we examined the expression of OPN in the synovia of patients with RA and the effect of OPN on the production of collagenase 1 in rheumatoid synovial fibroblasts and articular chondrocytes. METHODS: The expression of OPN messenger RNA (mRNA) and protein in synovia from 10 RA patients was examined by in situ hybridization and immunohistochemistry. Synovial fibroblasts from RA patients and articular chondrocytes from patients without joint disease were cultured in the presence of various concentrations of OPN, and levels of collagenase 1 in the culture supernatants were measured by enzyme-linked immunosorbent assay. RESULTS: The expression of OPN mRNA and protein was observed in 9 of 10 specimens obtained from patients with RA. OPN was expressed in the synovial lining and sublining layer and at the interface of cartilage and invading synovium. Double labeling revealed that the majority of OPN-expressing cells were positive for the fibroblast-specific enzyme prolyl 4-hydroxylase and negative for the macrophage marker CD68, while only a few, single OPN-expressing cells were positive for CD68 at sites of synovial invasion into cartilage. OPN staining was not observed in lymphocytic infiltrates or leukocyte common antigen (CD45)-positive cells. Three of 3 cultures of human articular chondrocytes secreted detectable basal amounts of collagenase, with a dose-dependent increase upon OPN stimulation, while synovial fibroblast cultures produced much lower levels of collagenase, with only 2 of 4 fibroblast cultures responding in a dose-dependent manner. CONCLUSION: These findings suggest that OPN produced by synovial fibroblasts in the synovial lining layer and at sites of cartilage invasion not only mediates attachment of these cells to cartilage, but also contributes to matrix degradation in RA by stimulating the secretion of collagenase 1 in articular chondrocytes.

Increased serum levels of non-collagenous matrix proteins (cartilage oligomeric matrix protein and melanoma inhibitory activity) in marathon runners.

OBJECTIVE: Marathon runners have an increased risk of developing joint disease. During and after a 42-km run, elevation of multiple cytokines occurs in the blood, reflecting inflammatory processes. We compared this cytokine response with serum levels of cartilage oligomeric matrix protein (COMP) and melanoma inhibitory activity (MIA), two markers for joint metabolism and/or damage. METHODS: Serum from eight endurance-trained runners was collected shortly before the start of a marathon run, after 31 km, 42 km, 2 h after the end, on the first and on the second morning after the run. For comparison, serum was obtained from 35 healthy controls and 80 patients with knee joint injury, rheumatoid arthritis or osteoarthritis. Serum levels of C-reactive protein (CRP), interleukin-1beta (IL-1beta), interleukin-1 receptor antagonist (IL-1RA), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), soluble interleukin-6 receptor (sIL-6R, gp80), soluble tumor necrosis factor receptor II (sTNFRII, p75), COMP and MIA were measured by ELISA. RESULTS: Compared with healthy controls, the runner's baseline serum levels of TNF-alpha, sIL-6R, COMP and MIA were significantly increased. COMP and MIA levels, higher than the upper normal limits of 5 microg/ml and 6 ng/ml respectively, were found in seven and five of eight runners. The elevated levels of COMP were similar to those found in joint injury or osteoarthritis, and the elevated levels of MIA were comparable to those reported in rheumatoid arthritis. During the run, the serum levels of IL-1RA, IL-6, TNF-alpha and COMP rose significantly, and gradually returned to baseline within 24 h. Only modest changes of CRP, sIL-6R, sTNFRII and MIA occurred during the run. Late elevations of CRP and MIA were observed after 24 and 48 h. The correlation analysis suggests associations between COMP, sIL-6R, TNF-alpha, IL-1RA on one hand and sTNFRII, and MIA and CRP on the other hand. CONCLUSIONS: Elevated baseline levels of COMP and MIA might reflect increased joint matrix turnover and/or damage due to prior extreme physical training. During the run, COMP was increasing possibly due to the severe physical strain on joint structures, associated with the early inflammation. After the run, MIA and CRP increased within 24 h, suggesting a correlation with later inflammatory processes. Thus, our data suggest that COMP and MIA are markers for distinct aspects of joint metabolism and/or damage in both disease and sport.

Basic fibroblast growth factor expression is increased in human renal fibrogenesis and may mediate autocrine fibroblast proliferation.

BACKGROUND: Interstitial fibroblasts play a critical role in renal fibrogenesis, and autocrine proliferation of these cells may account for continuous matrix synthesis. Basic fibroblast growth factor (FGF-2) is mitogenic for most cells and exerts intracrine, autocrine, and paracrine effects on epithelial and mesenchymal cells. The aims of the present studies were to localize and quantitate the expression of FGF-2 in normal and pathologic human kidneys and to study the in vitro effects of FGF-2 on proliferation, differentiation, and matrix production of isolated cortical kidney fibroblasts. METHODS: FGF-2 protein expression was localized by immunofluoresence double labelings in normal and fibrotic human kidneys. Subsequently, interstitial FGF-2 labeling was determined semiquantitatively in 8 normal kidneys and 39 kidneys with variable degrees of interstitial fibrosis and was correlated with the morphometrically determined interstitial cortical volume. In addition, FGF-2 expression was quantitated by immunoblot analysis in three normal and six fibrotic kidneys. FGF-2 mRNA was localized by in situ hybridizations. Seven primary cortical fibroblast lines were established, and expression of FGF-2 and FGF receptor-1 (FGFR-1) were examined. The effects of FGF-2 on cell proliferation were determined by bromodeoxyuridine incorporation and cell counts, those on differentiation into myofibroblasts by staining for alpha-smooth muscle actin, and those on matrix synthesis by enzyme-linked immunosorbent assay for collagen type I and fibronectin. Finally, proliferative activity in vivo was evaluated by expression of MIB-1 (Ki-67 antigen). RESULTS: In normal kidneys, FGF-2 expression was confined to glomerular, vascular, and a few tubular as well as interstitial fibroblast-like cells. The expression of FGF-2 protein was increased in human kidneys, with tubulointerstitial scarring correlating with the degree of interstitial fibrosis (r = 0.84, P < 0.01). Immunoblot analyses confirmed a significant increase in FGF-2 protein expression in kidneys with interstitial scarring. In situ hybridization studies demonstrated low-level detection of FGF-2 mRNA in normal kidneys. However, FGF-2 mRNA expression was robustly up-regulated in interstitial and tubular cells in end-stage kidneys, indicating that these cells are the source of excess FGF-2 protein. Primary cortical fibroblasts express FGF-2 and FGFR-1 in vitro. FGF-2 induced a robust growth response in these cells that could be blocked specifically by a neutralizing FGF-2 antibody. Interestingly, the addition of the neutralizing antibody alone did reduce basal proliferation up to 31.5%. In addition, FGF-2 induced expression of alpha-smooth muscle actin up to 1.6-fold, but no significant effect was observed on the synthesis of collagen type I and fibronectin. Finally, staining for MIB-1 revealed a good correlation of interstitial FGF-2 positivity with interstitial and tubular proliferative activity (r = 0.71, P < 0.01 for interstitial proliferation, N = 30). CONCLUSIONS: Interstitial FGF-2 protein and mRNA expression correlate with interstitial scarring. FGF-2 is a strong mitogen for cortical kidney fibroblasts and may promote autocrine fibroblast growth. Expression of FGF-2 correlates with interstitial and tubular proliferation in vivo.

Increased expression of CD40 ligand (CD154) on CD4+ T cells as a marker of disease activity in rheumatoid arthritis.

OBJECTIVES: The interaction between the activation induced surface glycoprotein CD40L (ligand) (CD154) on CD4+ T cells and its receptor CD40, which is expressed on various cell types, plays a crucial part in numerous cell mediated and humoral immune reactions that may be of pathogenetic importance in rheumatoid arthritis (RA). To further evaluate the pathogenetic role of CD40L in RA, expression of CD40L and various other T cell activation antigens as well as costimulatory molecules was investigated on CD4+ T cells in RA by flow cytometry. METHODS: Two colour flow cytometry was used to determine the percentage of CD4+ T cells expressing CD40L, CD69, CD25, HLA-DR, CD39, CD27 and CD28 in peripheral blood (PB) of 62 RA patients in comparison to 20 healthy controls (HC). Disease activity was assessed by clinical, laboratory and radiological examination. Status of clinical remission of RA was evaluated according to the ACR preliminary criteria for complete clinical remission of RA. RESULTS: CD40L was expressed on > 10% of CD4+ T cells in 29% of RA patients thus defining a CD40L(high+) patient group. Disease activity as estimated by C reactive protein, rheumatoid factor and status of clinical remission of disease (p = 0.049) was higher in this subgroup than in the RA CD40L(low+) group. Expression of CD69, CD25, and HLA-DR was significantly increased in both RA patient groups in comparison with HC. However, the percentage of CD39+ CD4+ T cells was increased only in the RA CD40L(high+) subgroup (versus HC p = 0.019, versus RA CD40L(low+) p = 0.044). Furthermore, expression of CD40L and CD39 on CD4+ T cells correlated positively as estimated by Spearman rank correlation (p<0.001). The percentage of CD4+ T cells lacking the costimulatory molecules CD27 (p = 0.002) and CD28 (p = 0.026) was increased in RA CD40L(low+) patients in comparison with HC. CONCLUSIONS: These data suggest that increased expression of CD40L on CD4+ T cells in RA indicates prolonged and increased activation of CD4+ T lymphocytes and is associated with active disease and possibly an unfavourable prognosis. Whether this phenotypically defined RA CD40L(high+) subgroup will preferentially respond to an anti-CD40L antibody treatment remains to be elucidated.

Expression of sentrin, a novel antiapoptotic molecule, at sites of synovial invasion in rheumatoid arthritis.

OBJECTIVE: Sentrin, a novel antiapoptotic molecule, has been shown to interact with the signal-competent form of Fas/APO-1 and tumor necrosis factor receptor I (TNFRI), and thereby, to protect cells against anti-Fas/APO-1- and TNF-induced cell death. Since reduced apoptosis in the synovial lining is supposed to contribute to synovial hyperplasia in rheumatoid arthritis (RA), we searched for the expression of sentrin-1 messenger RNA (mRNA) in synovium from patients with RA. METHODS: The expression of sentrin-1 mRNA was examined by in situ hybridization on snap-frozen sections of normal and RA synovial tissues as well as on paraffin-embedded RA synovial specimens, including the interface of cartilage-bone and invading synovium. Immunohistochemical double labeling after in situ hybridization was performed to further characterize sentrin-1 mRNA-expressing cells. In addition, quantitative analysis of sentrin-1 mRNA expression in RA synovial fibroblasts (RASF), osteoarthritis synovial fibroblasts (OASF), and normal fibroblasts was performed by quantitative real-time polymerase chain reaction. Expression levels were standardized to the expression of GAPDH. The in vivo maintenance of sentrin expression in RASF aggressively invading human cartilage was explored in the SCID mouse model of RA. RESULTS: A marked expression of sentrin-1 mRNA could be seen in all RA synovial specimens, predominantly in SF of the lining layer and at sites of invasion of RA synovium into cartilage. In normal synovial tissues, no sentrin-1 mRNA was detectable. RASF showed a maximum 32.5-fold (mean +/- SD 14.9 +/- 11.6) increase of sentrin-1 mRNA expression compared with normal fibroblasts and a maximum 31.4-fold (mean +/-SD 14.3 +/- 10.9) increase compared with OASF. When coimplanted with normal human cartilage in the SCID mouse model, invading RASF maintained their sentrin-1 mRNA expression for at least 60 days in vivo. CONCLUSION: The marked expression of sentrin in rheumatoid synovial tissue, but not in normal or OA synovial tissue, may contribute to the modulation of Fas- and TNFR-mediated apoptosis in RA synovium, and thereby extend the lifespan of invasive, cartilage-destructive SF.

Expression of lymphotoxin-alpha by keratinocytes: a further mediator for the lichenoid reaction.

OBJECTIVE: Lichen planus (LP) represents a disease in which autoimmune mechanisms mediated by Th1 T cells are involved. Lymphotoxin-alpha (LT-alpha) represents a Th1 cytokine with proinflammatory activities in LP, as has recently been demonstrated for interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). METHODS: Expression of LT-alpha mRNA was investigated by RT-PCR and nonradioactive in situ hybridization. Double staining methods were applied to characterize the phenotype of cells expressing LT-alpha. Cell stimulation experiments were performed on the transformed squamous cell line HaCaT. RESULTS: In contrast to normal skin, LT-alpha-specific RT-PCR products were found in all cases of LP. Cells in the inflammatory infiltrate expressing LT-alpha were identified as mainly T cells and mast cells, as shown by in situ hybridization. Furthermore, predominant LT-alpha mRNA expression could be observed in lesional keratinocytes adjacent to the band-like inflammatory infiltrate. In cell stimulation experiments, it could be shown that IFN-gamma induces LT-alpha and TNF-alpha mRNA in the human squamous cell line HaCaT, concomitant with upregulation of MHC class II and intercellular adhesion molecule-1, which could also be observed on lesional keratinocytes in LP. CONCLUSIONS: In LP, LT-alpha mRNA is predominantly expressed by lesional keratinocytes and to a lesser extent by inflammatory cells. Induction of LT-alpha in keratinocytes is closely related to the expression of TNF-alpha and MHC class II. The loci of TNF-alpha and LT-alpha map to MHC class III on chromosome 6, which is closely linked to the MHC class II gene locus. Our results suggest that stimulation of keratinocytes with IFN-gamma results in the upregulation of proinflammatory cytokines such as LT-alpha and TNF-alpha as well as MHC class II, which map to the same gene region of immunoregulatory genes on chromosome 6 and may be involved in the induction and maintenance of the disease.

Activation of synovial fibroblasts in rheumatoid arthritis: lack of Expression of the tumour suppressor PTEN at sites of invasive growth and destruction.

AIMS: PTEN is a novel tumour suppressor which exhibits tyrosine phosphatase activity as well as homology to the cytoskeletal proteins tensin and auxilin. Mutations of PTEN have been described in several human cancers and associated their invasiveness and metastatic properties. Although not malignant, rheumatoid arthritis synovial fibroblasts (RA-SF) exhibit certain tumour-like features such as attachment to cartilage and invasive growth. In the present study, we analyzed whether mutant transcripts of PTEN were present in RA-SF. In addition, we used in situ hybridization to study the expression of PTEN messenger (m)RNA in tissue samples of RA and normal individuals as well as in cultured RA-SF and in the severe combined immunodeficiency (SCID) mouse model of RA. Synovial tissue specimens were obtained from seven patients with RA and from two nonarthritic individuals. Total RNA was isolated from synovial fibroblasts and after first strand complementary (c)DNA synthesis, polymerase chain reaction (PCR) was performed to amplify a 1063 base pair PTEN fragment that encompassed the coding sequence of PTEN including the phosphatase domain and all mutation sites described so far. The PCR products were subcloned in Escherichia coli, and up to four clones were picked from each plate for automated sequencing. For in situ hybridization, digoxigenin-labelled PTEN-specific RNA probes were generated by in vitro transcription. For control in situ hybridization, a matrix metalloproteinase (MMP)-2-specific probe was prepared. To investigate the expression of PTEN in the absence of human macrophage or lymphocyte derived factors, we implanted RA-SF from three patients together with normal human cartilage under the renal capsule of SCID mice. After 60 days, mice were sacrificed, the implants removed and embedded into paraffin. RESULTS: PCR revealed the presence of the expected 1063 base pair PTEN fragment in all (9/9) cell cultures (Fig.1). No additional bands that could account for mutant PTEN variants were detected. Sequence analysis revealed 100% homology of all RA-derived PTEN fragments to those from normal SF as well as to the published GenBank sequence (accession number U93051). However, in situ hybridization demonstrated considerable differences in the expression of PTEN mRNA within the lining and the sublining layers of RA synovial membranes. As shown in Figure 2a, no staining was observed within the lining layer which has been demonstrated to mediate degradation of cartilage and bone in RA. In contrast, abundant expression of PTEN mRNA was found in the sublining of all RA synovial tissues (Figs 2a and b). Normal synovial specimens showed homogeneous staining fo PTEN within the thin synovial membrane (Fig. 2c). In situ hybridization using the sense probe gave no specific staining (Fig. 2d). We also performed in situ hybridization on four of the seven cultured RA-SF and followed one cell line from the first to the sixth passage. Interestingly, only 40% of cultured RA-SF expressed PTEN mRNA (Fig. 3A), and the proportion of PTEN expressing cells did not change throughout the passages. In contrast, control experiments using a specific RNA probe fo MMP-2 revealed mRNA expression by nearly all cultured cells (Fig. 3B). As seen before, implantation of RA-SF into the SCID mice showed considerable cartilage degradation. Interestingly, only negligible PTEN expression was found in those RA-SF aggressively invading the cartilage (Fig. 3c). In situ hybridization for MMP-2 showed abundant staining in these cells (Fig. 3d). DISCUSSION: Although this study found no evidence for mutations of PTEN in RA synovium, the observation that PTEN expression is lacking in the lining layer of RA synovium as well as in more that half of cultured RA-SF is of interest. It suggests that loss of PTEN function may not exclusively be caused by genetic alterations, yet at the same time links the low expression of PTEN to a phenotype of cells that have been shown to invade cartilage aggressively. It has been proposed that the tyrosine phosphatase activity of counteracting th actions o protein tyrosine kinases. As some studies have demonstrated an upregulation of tyrosine kinase activity in RA synovial cells, it might be speculated that the lack of PTEN expression in aggressive RA-SF contributes to the imbalance of tyrosine kinases and phosphatases in this disease. However, the extensive amino-terminal homology of the predicted protein to the cytoskeletal proteins tensin and auxilin suggests a complex regulatory function involving cellular adhesion molecules and phosphatase-mediated signalling. The tyrosine phosphatase TEP1 has been shown to be identical to the protein encoded by PTEN, and gene transcription of TEP1 has been demonstrated to be downregulated by transforming growth factor (TGF)-beta. Therefore, it could be hypothesized that TGF-beta might be responsible for the downregulation of PTEN. Low expression of PTEN may belong to the features that distinguish between the activated phenotype of RA-SF and the sublining, proliferating but nondestructive cells.