DNA methylation regulates the expression of CXCL12 in rheumatoid arthritis synovial fibroblasts.

In the search for specific genes regulated by DNA methylation in rheumatoid arthritis (RA), we investigated the expression of CXCL12 in synovial fibroblasts (SFs) and the methylation status of its promoter and determined its contribution to the expression of matrix metalloproteinases (MMPs). DNA was isolated from SFs and methylation was analyzed by bisulfite sequencing and McrBC assay. CXCL12 protein was quantified by enzyme-linked immunosorbent assay before and after treatment with 5-azacytidine. RASFs were transfected with CXCR7-siRNA and stimulated with CXCL12. Expression of MMPs was analyzed by real-time PCR. Basal expression of CXCL12 was higher in RASFs than osteoarthritis (OA) SFs. 5-azacytidine demethylation increased the expression of CXCL12 and reduced the methylation of CpG nucleotides. A lower percentage of CpG methylation was found in the CXCL12 promoter of RASFs compared with OASFs. Overall, we observed a significant correlation in the mRNA expression and the CXCL12 promoter DNA methylation. Stimulation of RASFs with CXCL12 increased the expression of MMPs. CXCR7 but not CXCR4 was expressed and functional in SFs. We show here that RASFs produce more CXCL12 than OASFs due to promoter methylation changes and that stimulation with CXCL12 activates MMPs via CXCR7 in SFs. Thereby we describe an endogenously activated pathway in RASFs, which promotes joint destruction.

Expression of cathepsin K and matrix metalloproteinase 1 indicate persistent osteodestructive activity in long-standing ankylosing spondylitis.

BACKGROUND: Ankylosing spondylitis (AS) is a common, largely genetically determined, rheumatic disease that is characterised by spinal inflammation and new bone formation. However, the exact pathogenesis and pathology are still not clear. OBJECTIVE: To analyse tissue obtained at spinal surgery by immunohistochemistry and compare the specimen of patients with AS to those with degenerative disc disease (DDD). METHODS: Bony and soft tissue specimens of 30 patients with AS and 20 with DDD were obtained during spinal osteotomy from different anatomic regions including articular and spinous processes, interspinous ligaments and intervertebral disks. Immunohistolochemistry was performed with established markers for cathepsin K, matrix metalloproteinase (MMP)1, MMP3 and receptor activator for nuclear factor kappaB (RANK) ligand. RESULTS: Cathepsin K and MMP1-positive cells were only observed in AS specimens. Cathepsin K-positive multinucleated cells were detected at articular processes adjacent to fibrous tissues. MMP1 was expressed in smaller mononuclear cells attached to bone. Invasion of bone by MMP1 cells was seen at entheseal sites. In the intervertebral disks, most mononuclear cells were cathepsin K-positive. Isolated cells expressing these matrix-degrading enzymes found in DDD never showed signs of invasion. No differences were found for MMP3 between AS and DDD. Clear expression of RANK ligand was only detected in one patient with AS. CONCLUSIONS: Cathepsin K is strongly expressed in different regions of the spine in AS. Cathepsin K was mainly expressed by mononuclear cells, fibroblast-like cells and cells attached to bone and at sites of bone remodelling, suggestive of high osteoclastic activity. This supports the role of persistent inflammation in the pathogenesis of AS. How these changes relate to osteoproliferation remains to be determined.

The metastasis associated protein S100A4: a potential novel link to inflammation and consequent aggressive behaviour of rheumatoid arthritis synovial fibroblasts.

The metastasis-associated protein S100A4 belongs to the large family of S100 calcium-binding proteins that appear to play regulatory roles in diverse biological activities. Moreover, a prognostic role of S100A4 has been suggested for patients with several types of cancer. Cancer promoting properties for S100A4 have been demonstrated, particularly through its regulation of cell motility, proliferation and apoptosis, as well as by stimulation of angiogenesis and remodelling of the extracellular matrix. Increased expression of S100A4 mRNA has been detected in proliferating synovial fibroblasts in rheumatoid arthritis. Furthermore, strong upregulation of the S100A4 protein in rheumatoid arthritis synovial tissue compared with osteoarthritis and control tissues has been demonstrated recently, especially at sites of joint invasion. Several immune and vascular cells were also identified to be producing S100A4 within the synovium. The local upregulation of S100A4 was accompanied by high plasma and synovial fluid concentrations of the S100A4 protein existing in the bioactive oligomeric form in patients with rheumatoid arthritis. Consistent with data from cancer studies, the extracellular S100A4 oligomer appears to be involved in regulation of several matrix-degrading enzymes and modulation of the transcriptional activation function of the tumour suppressor protein p53 in rheumatoid arthritis synovial fibroblasts. Taken together, one can speculate that increased S100A4 protein in circulation and locally at sites of inflammation, particularly at sites of joint destruction, might be linked to the process of aggressive fibroblast behaviour contributing to the pathogenesis of chronic autoinflammatory diseases such as rheumatoid arthritis.

The dual inhibitor of lipoxygenase and cyclooxygenase ML3000 decreases the expression of CXCR3 ligands.

OBJECTIVE: To find previously unknown properties of ML3000, a competitive inhibitor of the cyclooxygenase and the lipoxygenase (LO) pathway. METHODS: Gene expression of ML3000 treated and untreated rheumatoid arthritis synovial fibroblasts were measured with Affymetrix gene arrays. Downregulation of chemokine (C-X-C motif) ligands CXCL9, CXCL10 and CXCL11 was verified with Real-time polymerase chain reaction, CXCL10 protein levels were determined with ELISA. Rheumatoid arthritis synovial fibroblasts were treated with the cyclooxygenase inhibitor naproxen, the 5-LO inhibitor BWA4C and the 5-lipoxygenase-activating protein (FLAP) inhibitor MK886, and consecutive changes in CXCL10 protein levels measured. 5-LO expression was determined by polymerase chain reaction and Western blot. RESULTS: In synovial fibroblasts and monocyte-derived macrophages ML3000 inhibited the tumour necrosis factor induced expression of CXCL9, CXCL10 and CXCL11, which are all ligands of the chemokine receptor CXCR3. No effect was observed in monocytes. Whereas inhibition of the cyclooxygenase pathway or the FLAP protein showed no effect, blockade of 5-LO significantly downregulated CXCL10 protein levels. 5-LO mRNA was detected in monocytes and in monocyte-derived macrophages. All tested cell types expressed 5-LO protein. CONCLUSIONS: ML3000 effectively downregulates CXCR3 ligands. This study confirms that a thorough analysis of the impact of a drug on its target cells cannot only reveal unexpected properties of a substance, but also helps to understand the underlying molecular mechanisms. Accordingly, our data provide the basis for further clinical studies testing the application of ML3000 in diseases such as rheumatoid arthritis or multiple sclerosis.

Bromocriptine microcapsules inhibit ornithine decarboxylase activity induced by Freund's complete adjuvant in lymphoid tissues of male rats.

Arthritis is produced in male rats within 9-10 days after a single injection of Freund's complete adjuvant (FCA) at the base of the tail. When bromocriptine, a dopaminomimetic that suppresses PRL secretion, was given in form of long-acting microcapsules (CBLA) 3 days before FCA, the hind limb swelling was significantly reduced by 70%. Here, we showed that plasma PRL levels were significantly elevated (by 150% over controls) during the 6-day period after FCA, particularly at night. Further, within 1-4 days after FCA inoculation, marked increases in ornithine decarboxylase (ODC) activity occurred in bone marrow, thymus, spleen, and lymph nodes (by 190%, 160%, 80% and 75% over control values, respectively). In FCA-treated rats, the circadian rhythm of thymic ODC showed that an important enhancement of activity occurred during the dark phase, which correlated with the peak of PRL secretion (between 2200-0400 h). Finally, pretreatment with CBLA significantly inhibited the induction of ODC in response to FCA in thymus, spleen, and lymph nodes (by 65%, 80%, and 45%, respectively) and inhibited it more weakly in the bone marrow. This in vivo study leaves little doubt about the existence of a PRL-dependent immuno-stimulatory mechanism, probably involved in the pathogenesis of adjuvant arthritis.

Freund's complete adjuvant induces ornithine decarboxylase activity in the central nervous system of male rats and triggers the release of pituitary hormones.

In male rats, inoculation of Freund's complete adjuvant (FCA, 0.5 mg/rat of Mycobacterium butyricum in paraffin oil) induced high levels of ornithine decarboxylase (ODC) in the hypothalamus and pituitary gland (285% and 245% of controls, respectively, within 12 h to 2 days). ODC activity also was altered in the cerebellum and left neocortex, but not in the right neocortex. This activity reflected a dynamic equilibrium which is influenced by ODC synthesis, degradation, activation, etc. The circadian rhythms of pituitary ODC activity and plasma prolactin level, 3-4 days after FCA, showed that enhancement of enzymatic activity during the dark phase correlated with a marked release of prolactin (Prl). During this early period after FCA, changes in plasma levels of other pituitary hormones were not significant or were less important. Pretreatment with bromocriptine microcapsules inhibited both basal and FCA-induced pituitary ODC activity, as well as Prl secretion. Further, significant increases in plasma luteinizing hormone and adrenocorticotropic hormone were noted from days 4 and 8, respectively, and onwards. Finally, a phase of reduced corticosterone secretion occurred during the latency period. This study shows that FCA influences central nervous system pathways and supports the idea that endogenous Prl is involved in some early events which lead to the development of adjuvant arthritis.

Prospective randomized study comparing the efficacy and safety of ciprofloxacin with cefaclor in the treatment of patients with purulent bronchitis.

We compared safety and efficacy of ciprofloxacin and cefaclor in the treatment of patients with purulent bronchitis. Fifty-five patients were randomized prospectively to receive ciprofloxacin with a dose of 500 mg orally twice daily or cefaclor 250 mg over 8 hr for 5 days or longer. Patient groups did not differ with respect to age, duration of illness, severity of infection, or number of other concomitant disease states. A significantly larger number of patients in the ciprofloxacin group had poor health status (39.3% vs 7.4% for the ciprofloxacin and cefaclor groups, respectively, p = 0.02). The response to therapy did not differ between groups. Infection was completely resolved in 71.4% vs 66.7% and markedly improved in 7.1% and 11.1% for the ciprofloxacin and cefaclor groups, respectively. The response to therapy and adverse reaction rate did not differ between groups. Seven patients treated with ciprofloxacin and five patients treated with cefaclor developed adverse reactions. We conclude that ciprofloxacin is a useful agent for the treatment of purulent bronchitis.

Bromocriptine has little direct effect on murine lymphocytes, the immunomodulatory effect being mediated by the suppression of prolactin secretion.

We investigated whether the immunosuppressive effect of bromocriptine in mice is due to its direct effect on lymphocyte functions or through inhibition of prolactin secretion. Incubation of mouse Babl/c splenic lymphocytes with bromocriptine in vitro at a concentration of around 0.5 to 1 microgram/mL causes an inhibition of antigen- or mitogen-induced proliferation. However, bromocriptine in vitro has no effect on lymphokine production (gamma-interferon and interleukin-2), expression of interleukin-2 receptor or lymphocyte cytotoxic function. Furthermore, treatment of Babl/c mice with bromocriptine inhibits the mixed lymphocyte reaction and mitogen stimulation, as well as primary and secondary antibody production. However, we postulated that the inhibition of ex vivo functional activity could not account for a direct cytostatic or cytotoxic effect of bromocriptine. This is supported by the in vitro data, which shows that bromocriptine has no effect on proliferating P-815 mastocytoma tumor cells. Finally, (NZB/NZW) F1 mice spontaneously develop a disease similar to systemic lupus erythematosus. In both non-autoimmune Babl/c mice and (NZB/NZW) F1 lupus-mice, the serum level of bromocriptine achieved by a treatment with 5 mg/kg on average is 2-6 ng/mL. On the one hand, this dose is sufficient to significantly alter the ex vivo functional tests in Babl/c mice and to show a beneficial effect in the in vivo model of female lupus-mice. On the other hand, the lowest concentration which could have an inhibitory effect on antigen- and mitogen-induced proliferation in vitro is 200 ng/mL, ie 50 times more than that required in vivo to obtain significant reductions of proteinurea, glomerular membrane proliferation and immune deposits in lupus-mice. The serum levels of gamma-interferon and interleukin-2 are reduced in lupus-mice when compared with Babl/c mice. The treatment with bromocriptine does not influence these parameters. In conclusion, our data demonstrate that the major immunosuppressive activity of bromocriptine is probably dependent on its hypoprolactinemic effect.

Prolactin and prolactin-like polypeptides in rheumatoid arthritis.

A bidirectional communication network exists between the neuroendocrine and immune systems, and a dysfunctional communication may contribute to the development of autoimmune diseases in various species, including humans. Experimental, epidemiological, and clinical data suggest that breast feeding and hyperprolactinemia constitute a risk factor for the development of diseases with autoimmune components, including rheumatoid arthritis (RA). We hypothesized that the anterior pituitary hormone prolactin (Prl) and locally produced Prl-like polypeptides may act as endocrine, autocrine, and paracrine regulators of synovial cell functions. They may participate not only in enhancing T-lymphocyte immune reactivity, but also in the exacerbation of RA lesions through their influence on synovial fibroblasts. In RA synovial tissue, Prl-like polypeptides could participate in a bidirectional communication between immunocytes and fibroblasts. Both Prl and Prl-like polypeptides might act via proto-oncogenes and transcriptional factors, leading to cell proliferation, i.e., synovial tissue hyperplasia, neo-angiogenesis, and the production of catabolic enzymes such as matrix metalloproteinases and cathepsins. In such cases, they could represent important regulators of the T-cell independent mechanism of joint destruction.

[Cerebral protection by retrograde perfusion in the treatment of acute aortic dissection]. / Protection cérébrale par rétroperfusion dans le traitement des dissections aiguës de l'aorte.

Four consecutive patients underwent resection and graft replacement of ascending aorta or aortic arch for acute dissection. Retrograde cerebral perfusion (RCP) was used during circulatory arrest. RCP at 15 degrees C was administered through the superior vena cava. Duration of cerebral ischaemia and cardiopulmonary bypass averaged 33 and 156 minutes respectively. Retrograde perfusion flow was regulated from 100 to 800 ml/minute to maintain an internal jugular vein pressure of about 25 cm H2O. All patients survived. Three patients awoke neurologically intact. Minor neurological disturbance was found in 1 patient, he was discharged from hospital at day 11 without any detectable neurological deficit. This technique was attractive because it provided a dry operative field unencumbered by perfusion cannulas or clamps, facilitated construction of a more secure distal anastomosis, and avoided the risk of further injury resulting from the aortic cross clamp. It seems that RCP allows longer circulatory arrest time.

Functional autoantibodies against serpin E2 in rheumatoid arthritis.

OBJECTIVE: To search for novel autoantibodies in patients with rheumatoid arthritis (RA) in an effort to better understand the processes of joint destruction in this disease. METHODS: Using a modified SEREX technique and complementary DNA derived from RA synovium, serpin E2 was identified as a novel autoantigen and was analyzed by immunohistochemistry. Levels of anti-serpin E2 autoantibodies in serum and synovial fluid from patients with RA, osteoarthritis (OA), psoriatic arthritis, and ankylosing spondylitis, and/or from healthy individuals were assessed by enzyme-linked immunosorbent assay. Since serpin E2 is an inhibitor of serine proteases, we studied the inhibitory activity of serpin E2 toward its target, urokinase plasminogen activator (uPA), in vitro in the presence of isolated anti-serpin E2 autoantibodies and in vivo using the uPA activity assay. RESULTS: We identified autoantibodies against serpin E2 by the SEREX technique. Serpin E2 was overexpressed in RA synovial tissues as compared with OA synovial tissues. Significantly higher levels of anti-serpin E2 autoantibodies were present in samples of synovial fluid (28%) and serum (22%) from RA patients as compared with OA patients (0 and 6%, respectively) or with healthy individuals (6% of sera). Most importantly, anti-serpin E2 autoantibodies isolated from RA sera reversed the inhibitory activity of serpin E2 by 70%. Furthermore, the levels of anti-serpin E2 autoantibodies correlated with the uPA activity in vivo. CONCLUSION: This study characterizes a functional property of a novel autoantibody in RA. Since anti-serpin E2 autoantibodies interfere with the inhibitory activity of serpin E2 toward serine proteases, they might facilitate the joint destruction in RA.

S100A4 is expressed at site of invasion in rheumatoid arthritis synovium and modulates production of matrix metalloproteinases.

The metastasis-associated protein S100A4 promotes the progression of cancer by regulating the remodelling of the extracellular matrix. The expression of S100A4 in vivo is shown and the functional role of S100A4 in the pathogenesis of osteoarthritis and rheumatoid arthritisis is explored. The expression of S100A4 in rheumatoid arthritis, osteoarthritis and normal synovial tissues was determined by immunohistochemistry. The expression of matrix metalloproteinase (MMP) mRNA was measured in rheumatoid arthritis and osteoarthritis synovial fibroblasts treated and untreated with S100A4 oligomer by real-time polymerase chain reaction. Levels of released MMPs were confirmed by ELISA in cell culture supernatants. S100A4 protein was expressed in rheumatoid arthritis and osteoarthritis synovial tissues, in contrast with normal synovium. S100A4 up regulated MMP-3 mRNA in rheumatoid arthritis synovial fluid, with a peak after 6 h. This resulted in release of MMP-3 protein. MMP-1, MMP-9 and MMP-13 mRNA were also up regulated in synovial fluid, but with different kinetics. MMP-14 mRNA showed no change. Thus, S100A4 protein is expressed in synovial tissues of patients with rheumatoid arthritis and osteoarthritis in contrast with healthy people. It induces the expression and release of MMP-3 and other MMPs from synovial fluid. The data suggest that S100A4-producing cells could be involved in the pathogenesis of osteoarthritis and rheumatoid arthritis, including pannus formation and joint destruction.

Trichostatin A sensitises rheumatoid arthritis synovial fibroblasts for TRAIL-induced apoptosis.

BACKGROUND: Histone acetylation/deacetylation has a critical role in the regulation of transcription by altering the chromatin structure. OBJECTIVE: To analyse the effect of trichostatin A (TSA), a streptomyces metabolite which specifically inhibits mammalian histone deacetylases, on TRAIL-induced apoptosis of rheumatoid arthritis synovial fibroblasts (RASF). METHODS: Apoptotic cells were detected after co-treatment of RASF with TRAIL (200 ng/ml) and TSA (0.5, 1, and 2 micromol/l) by flow cytometry using propidium iodide/annexin-V-FITC staining. Cell proliferation was assessed using the MTS proliferation test. Induction of the cell cycle inhibitor p21Waf/Cip1 by TSA was analysed by western blot. Expression of the TRAIL receptor-2 (DR5) on the cell surface of RASF was analysed by flow cytometry. Levels of soluble TRAIL were measured in synovial fluid of patients with RA and osteoarthritis (OA) by ELISA. RESULTS: Co-treatment of the cells with TSA and TRAIL induced cell death in a synergistic and dose dependent manner, whereas TRAIL and TSA alone had no effect or only a modest effect. RASF express DR5 (TRAIL receptor 2), but treatment of the cells with TSA for 24 hours did not change the expression level of DR5, as it is shown for cancer cells. TSA induced cell cycle arrest in RASF through up regulation of p21Waf1/Cip1. Levels of soluble TRAIL were significantly higher in RA than in OA synovial fluids. CONCLUSION: Because TSA sensitises RASF for TRAIL-induced apoptosis, it is concluded that TSA discloses sensitive sites in the cascade of TRAIL signalling and may represent a new principle for the treatment of RA.

Galectin-3 is induced in rheumatoid arthritis synovial fibroblasts after adhesion to cartilage oligomeric matrix protein.

BACKGROUND: Galectin-3 is expressed in the synovial tissue of patients with rheumatoid arthritis (RA), particularly at sites of joint destruction. OBJECTIVE: To explore the possibilities that galectin-3 is induced either by proinflammatory cytokines or by adhesion to cartilage components. METHODS: Cell culture plates were coated with fibronectin, collagens I-VI, or cartilage oligomeric matrix protein (COMP), and the suspended cells were then added. The medium was changed after 1 hour at 37 degrees C. Adherent cells were further incubated for 18 hours in the presence or absence of tumour necrosis factor alpha (TNF alpha) or interleukin 1 beta. Cells were pretreated with murine IgG1, anti-CD29, -CD51, -CD61 (integrins), or -CD3 monoclonal antibodies and transferred to culture plates coated with COMP. Adherent cells were counted by light microscopy. The expression of intracellular galectin-3, or cell surface CD29, CD51, and CD61 was determined by flow cytometry before and after adhesion. RESULTS: Four times more RA synovial fibroblasts (SF) than osteoarthritis SF adhered to COMP. RA SF presented more cell surface integrins, and monoclonal antibodies against CD51 inhibited the adhesion to COMP by 80%. TNF alpha reduced the expression of CD61 and the adhesion to COMP, but did not reverse the adhesion once it had taken place. The adhesion of RA SF to COMP was found to increase the intracellular level of galectin-3. In contrast, intracellular galectin-3 decreased after exposure to TNF alpha. CONCLUSION: The increase of galectin-3 occurs after adhesion to COMP, and the alpha V beta 3 receptor (CD51/CD61) has a pivotal role in this process.

Elevated serum prolactin or elevated prolactin/cortisol ratio are associated with autoimmune processes in systemic lupus erythematosus and other connective tissue diseases.

OBJECTIVE: . We investigated whether serum prolactin (Prl) alone or an increased Prl/cortisol ratio correlated with the autoimmune processes in systemic lupus erythematosus (SLE) and other connective tissue diseases (CTD). METHODS: Serum Prl, cortisol, cytokines, and autoantibodies were measured in 29 patients with SLE and 29 patients with other CTD by ELISA. The patients were clinically assessed. RESULTS: Serum Prl was elevated in both SLE (p < 0.05) and other CTD (p < 0.01). Some patients with SLE had reduced serum cortisol levels. In SLE only, the Prl and cortisol levels were correlated (p <0.05), suggesting a parallel activation of the different neuroendocrine pathways. Both groups of patients showed significantly increased Prl/cortisol ratios (p < 0.01 in SLE and p < 0.05 in other CTD). In CTD other than SLE, increased tumor necrosis factor-alpha correlated with the elevated Pri/cortisol ratio (p < 0.05). In SLE, other CTD and control sera, elevated levels of IgG anti-cardiolipin antibody (aCL) were accompanied by increased Prl (0.01

Prolactin in autoimmune diseases.

The immune system is still regarded by many as autonomous, and prolactin (Prl) has traditionally been considered as a lactogenic hormone. Over the last 10 years, the total number of publications considering Prl is decreasing, while the number of those investigating its role in immunity sustainly increased. In addition to the pituitary gland, Prl-like peptides can be produced by activated leukocytes and fibroblasts. Elevated serum levels of Prl in (rat) adjuvant arthritis, (murine) collagen type II-induced arthritis, (murine and human) systemic lupus erythematosus (SLE), and (murine and rat) autoimmune type I diabetes may influence the outcome of the disease. It is suggested that mild hyperprolactinemia is a risk factor for the development of autoimmunity. This can occur under certain circumstances, for example adrenocortical deficiency or postpartum. In human SLE, Prl appears to favor the production of anti-double stranded DNA. While glucocorticoids would damp the immune reactivity, Prl constitutes a stimulatory link between the neuroendocrine and immune systems. Future directions should include: 1) multicenter projects for evaluation of the therapy with Prl-inhibiting compounds in SLE, considering for example the HLA-DRB1 *0301 status; and 2) the regulation of extra-pituitary Prl-like cytokines ("proliferins") (e.g., in rheumatoid arthritis synovium) and their role in the production of catabolic enzymes.

Effects of Freund's complete adjuvant on the diurnal rhythms of neuroendocrine processes and ornithine decarboxylase activity in various tissues of male rats.

The aim of this study was to investigate the effects of Freund's complete adjuvant (FCA) on the diurnal rhythms of hormonal parameters in serum and ornithine decarboxylase (ODC) activity in various tissues of male rats. On days 1-2 after FCA, increase of ODC activity (used to evaluate the level of activation) was observed in the hypothalamus, pituitary gland, adrenal medulla, adrenal cortex, liver and lymphoid tissues, while the ODC activity in the kidney was reduced. This was accompanied by an increase in serum corticosterone. On days 3-4 after FCA, ODC activity remained elevated in the pituitary gland, liver and lymphoid tissues, while the ODC activity in the testes and pancreas was reduced; kidney ODC activity returned to baseline. This was associated with increased serum levels of prolactin (Prl) and luteinizing hormone, but decreased growth hormone, testosterone and insulin. The increase in ODC activity in the thymus, as well as the reduced ODC activity in the testes and kidney, can be obtained with paraffin. Furthermore, bromocryptine microcapsules (CBLA) reduced the FCA-induced increase of ODC activity in the pituitary gland, liver and lymphoid tissues (days 3-4) but did not affect the changes in other tissues. The increase in ODC activity in the pituitary gland, liver and lymphoid tissues is specific for FCA. A role for Prl in the induction of ODC in liver and lymphoid tissues is suggested by the fact that CBLA suppresses this enhancement.

Synergism between long-acting bromocryptine microcapsules and cyclosporine A in the prevention of various autoimmune diseases in rats.

Pre-treatment of male Sprague-Dawley rats with long-acting bromocryptine microcapsules (CBLA) significantly inhibited the arthritic response to Freund's complete adjuvant and reduced weight loss, thymolysis, splenomegaly and leukocytosis. In the prevention of adjuvant arthritis (AA), the combination of CBLA plus sub-optimal doses of cyclosporine A (CsA) was more efficient than either of the drugs alone. Sub-optimal doses of CsA were 0.1 and 1.0 mg/kg/day s.c. for 5 days. Furthermore, CBLA alone did not decrease the incidence of experimental allergic uveitis (EAU) in the male Lewis rats. Low-dose CsA reduced the incidence of uveitis by 50%, and with the addition of CBLA, 100% of rats were protected. Low-dose CsA was 2 mg/kg/day i.m. for 14 days. Long-term treatment of male Sprague-Dawley rats with CBLA alone reduced the incidence and severity of spontaneous autoimmune periarteritis nodosa (PN) in a dose-dependent manner; CsA was less potent than CBLA, and only additive effects were obtained. Finally, for the prevention of spontaneous autoimmune insulin-dependent diabetes (DM), the administration of CBLA did not improve the effect of a low-dose CsA in male BB rats. Nevertheless, a delay in onset of DM could be achieved. A sequential therapy using CsA plus CBLA clearly showed beneficial effects. The dose of CsA was 10 mg/kg p.o. 6 days/week for 21 weeks. Compared with Sprague-Dawley or Lewis male rats, BB male rats showed only weak prolactin suppression after the same doses of CBLA. It is suggested that the use of CBLA may be particularly beneficial in autoimmune disorders. The effectiveness of the combination therapy CBLA plus CsA, however, was dependent on the model considered. Various factors could play a role: (1) the different ways of administering CsA (s.c. in AA, i.m. in EAU and PN, oral in DM); (2) strain-dependency in the capacity of CBLA to suppress Prl secretion; and (3) at least in the BB rats, the transient increase of CsA bioavailibility which was possibly induced by CBLA.

Serum levels of interleukin-1 beta, luteinizing hormone, and prolactin correlate with the expression of CD45 isoforms on CD4+ peripheral blood T lymphocytes in healthy women.

The expected association between age and the CD45 isoforms expression on CD4+ T-PBL is much more obvious in men than in women. We investigated whether or not circulating factors influence the differentiation of CD4+ T-PBL. Peripheral blood samples were obtained from 56 healthy age-matched subjects (28 men and 28 women, 21-55 years old). Mononuclear leukocytes were analyzed by three-color flow cytometry. The serum concentrations of interleukin-1 beta (IL-1 beta), interleukin-6, tumor necrosis factor-alpha (TNF-alpha), GM colony-stimulating factor, prolactin (Prl), and luteinizing hormone (LH) were determined by ELISA. The expected age-related decrease of naive (CD45RA+,RO-) cells and increase of memory (CD45RA-,RO+) cells among CD4+ T-PBL were observed in men only (p < 0.001 and 0.005). In women, these correlations were not significant. On the other hand, in women only, elevated IL-1 beta was associated with fewer naive and more memory cells among CD4+ T-PBL (p < 0.001). In both sexes, IL-1 beta correlated with the expression of CD25 on CD4+ T-PBL (on either naive or memory cells, p < 0.001). Other cytokines or the CD8+ T-PBL showed no significant correlation. In women, the elevation of LH at mid-cycle inversely correlated with the proportion of naive CD4+ T-PBL (p < 0.01). Elevated LH was associated with more CD25 on memory CD4+ T-PBL (p < 0.01). A significant correlation exists between IL-1 beta and LH (p < 0.001). Furthermore, in both sexes, Prl correlated with the proportion of CD4+ cells among T-PBL. In men, elevated Prl was associated with more naive CD4+ T-PBL (p < 0.005), while in women, Prl correlated with more transient CD45RA+, RO+ cells among CD4+ T-PBL and increased TNF-alpha (p < 0.05 for both). Thus, circulating IL-1 beta could be involved in the expression of CD25 on CD4+ T-PBL and favors the generation of memory CD4+ T-PBL. In women, the IL-1 beta- and/or mid-cycle-dependent processes seem to overwhelm the age-related changes. Elevated Prl might exert a dual influence: it favors the development of naive CD4+ T lymphocytes and possibly acts in, synergy with other cytokines during immune stimulation.