Effects of combined oral contraceptive ethinylestradiol (30 microg) and dienogest (2 mg) on carbohydrate metabolism during 1 year of conventional or extended-cycle use.

The effects of extended regimens of combined oral contraceptives (COCs) on carbohydrate metabolism are largely unknown. The present study compared the effects of a COC containing 30 microg ethinylestradiol and 2 mg dienogest (EE/DNG) in conventional and extended-cycle regimen over 1 year. Parameters of carbohydrate metabolism were measured in 59 women treated with EE/DNG either conventionally (13 cycles of 21+7 days) or in extended-cycle regimen (4 cycles of 84+7 days). Blood samples were taken in a control cycle, and at 3 and 12 months of treatment. The mean levels of HbA1c and fasting glucose levels remained stable in both conventional and extended-regimen of EE/DNG. The mean levels of fasting insulin and C-peptide underwent comparable increases in both regimens, suggesting a similar readjustment of glucose metabolism via slightly increased insulin secretion. For both regimens, the response to the oral glucose tolerance test (OGTT) showed a slightly impaired glucose tolerance and insulin resistance at 3 months. These changes improved or returned to baseline at 12 months. Accordingly, the mean index for insulin resistance (homeostasis model assessment of insulin resistance, HOMA-IR) increased and the mean insulin sensitivity index [ISI (composite)] decreased modestly in both groups. The present study demonstrates that there are no statistically significant differences between the effects of conventional and extended-cycle treatment on carbohydrate metabolism over 1 year of treatment. In general, the effects of both regimens were moderate and mostly transient.

Influence of a continuous combined HRT (2 mg estradiol valerate and 2 mg dienogest) on postmenopausal depression.

OBJECTIVE: This randomized, double-blind, placebo-controlled study was planned to investigate the effects of continuous combined hormone replacement therapy (HRT) with 2 mg estradiol valerate and 2 mg dienogest (Climodien/Lafamme) over 24 weeks on postmenopausal depression. METHOD: A total of 129 patients with a mild to moderate depressive episode according to ICD-10: F32.0, F32.1 in the context of a postmenopausal syndrome (ICD-10: N95.1) and a baseline score in the Hamilton depression scale (HAMD) > or =16 were included in the study. The primary target variable was depression severity as measured by the HAMD after 24 weeks of treatment. A four-point difference between HRT and placebo at the end of the study and, in addition, a final score < or =8 (corresponding to an improvement of > or =50% as compared to baseline) for the individual patient (responders analysis) were considered clinically relevant. Clinical global impression (CGI) of investigators (therapeutic and side-effects) at the end of the study was investigated. Secondary effects of HRT on depression severity caused by its effect on vasomotor symptoms or sleep disturbances (domino hypothesis) were taken into consideration. Also, the study addressed the question of whether the effect of HRT on depression severity depends on a history of premenstrual syndrome (PMS) or postnatal depression (PND). RESULTS: The results showed a clear and clinically relevant reduction of depression severity under HRT after 24 weeks of treatment and superiority over placebo (p < 0.0005) in spite of a strong placebo effect. The effects of the estrogen-progestin combination thereby seemed only partially to be dependent on the improvement of vasomotor symptoms and sleep disturbances. Also, the effects of HRT could not be shown to be dependent on a history of PMS and/or PND, even though women with and without this history clearly differed in baseline depression scores (p < 0.0001). The assessment of CGI was positive: whereas HRT was clearly superior to placebo with regard to therapeutic effects (p = 0.0014), there were no differences with regard to side-effects (p = 0.35). CONCLUSION: The combination of 2 mg estradiol valerate and 2 mg dienogest can be regarded as an effective and safe treatment option for women with mild to moderate depression in the context of postmenopausal syndrome.

Minimal contribution of cell-bound antibodies to the immunoscintigraphy of inflamed joints with 99mTc-anti-CD4 monoclonal antibodies.

AIM: The cellular joint infiltrate in rheumatoid arthritis patients is rich in CD4-positive T-helper lymphocytes and macrophages, rendering anti-CD4 monoclonal antibodies (mAbs) suitable for specific immunoscintigraphy of human/experimental arthritis. Following intravenous injection, however, mAbs are present both in the free form and bound to CD4-positive, circulating monocytes and T-cells. Thus, the present study aimed at analyzing the relative contribution of the free and the cell-bound component to the imaging of inflamed joints in experimental adjuvant arthritis (AA). METHODS: AA rat peritoneal macrophages or lymph node l-cells were incubated in vitro with saturating amounts of 99mTc-anti-CD4 mAb (W3/25) and injected i.v. into rats with AA. RESULTS: In vitro release of 99mTc-anti-CD4 mAb from the cells was limited (on average 1.57%/h for macrophages and 0.84%/h for T-cells). Following i.v. injection, whole body/joint scans and tissue measurements showed only negligible accumulation of radioactivity in inflamed ankle joints (tissue: 0.22 and 0.34% of the injected activity, respectively), whereas the radioactivity was concentrated in liver (tissue: 79% and 71%, respectively), kidney, and urinary bladder. Unlike macrophages, however, anti-CD4 mAb-coated T-cells significantly accumulated in lymphoid organs, the inflamed synovial membrane of the ankle joints, as well as in elbow and knee joints. CONCLUSION: While the overall contribution of cell-bound mAbs to the imaging of arthritic joints with anti-CD4 mAbs is minimal, differential accumulation of macrophages and T-cells in lymphoid organs and the inflamed synovial membrane indicates preferential migration patterns of these 2 cell populations in arthritic rats. Although only validated for 99mTc-anti-CD4 mAbs, extrapolation of the results to other anticellular mAbs with similar affinity for their antigen may be possible.

Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture--primary culture cells markedly differ from fourth-passage cells.

To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies. This method yielded highly enriched SFB (85% prolyl-4-hydroxylase+/74% Thy-1/CD90+ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1beta. This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.

Macrophages in rheumatoid arthritis.

The abundance and activation of macrophages in the inflamed synovial membrane/pannus significantly correlates with the severity of rheumatoid arthritis (RA). Although unlikely to be the 'initiators' of RA (if not as antigen-presenting cells in early disease), macrophages possess widespread pro-inflammatory, destructive, and remodeling capabilities that can critically contribute to acute and chronic disease. Also, activation of the monocytic lineage is not locally restricted, but extends to systemic parts of the mononuclear phagocyte system. Thus, selective counteraction of macrophage activation remains an efficacious approach to diminish local and systemic inflammation, as well as to prevent irreversible joint damage.

123I-antileukoproteinase scintigraphy reveals microscopic cartilage alterations in the contralateral knee joint of rats with "monarticular" antigen-induced arthritis.

OBJECTIVE: To assess the involvement of the contralateral knee joint in monarticular antigen-induced arthritis (AIA) by scintigraphy with the cationic (pI >10), 123I-labeled, serine proteinase inhibitor antileukoproteinase (123I-ALP) and to compare the scintigraphic findings with those of radiography and high-resolution ex vivo magnetic resonance imaging (MRI). METHODS: Lewis rats with chronic AIA were examined 2.5 months following arthritis induction (injection of 500 microg of methylated bovine serum albumin/saline into the ipsilateral [arthritic] knee joint and injection of phosphate buffered saline into the contralateral knee joint following systemic immunization). 123I-ALP was injected intravenously into normal rats (n = 4) or rats with AIA (n = 6). The ipsilateral and contralateral knee joints and both ankles were examined by scintigraphy and radiography. Joint cartilage was examined by high-resolution ex vivo MRI, histopathology, and measurement of tissue radioactivity. RESULTS: ALP accumulation (typically observed in normal articular cartilage) was lost in both the ipsilateral and the contralateral knee joints, but not in the clinically unaffected ankles of rats with AIA. In both knee joints, 123I-ALP target:background ratios and cartilage radioactivity correlated negatively with the loss of toluidine blue staining in cartilage, which documents the depletion of charged matrix molecules. Findings of histopathology confirmed mild alterations in the ipsilateral knee joint and even milder alterations in the contralateral knee joint, while the ankles were normal. Radiography and high-resolution ex vivo MRI failed to detect abnormalities in the contralateral knee joint. CONCLUSION: Loss of ALP accumulation appears to document proteoglycan depletion, even in the microscopically altered cartilage of the contralateral knee joint in AIA. These findings underscore the high sensitivity of 123I-ALP for in vivo detection of biochemical cartilage alterations in arthritis, and furthermore, question the use of the contralateral knee joint as a normal control in AIA.

Cytokine gene activation in synovial membrane, regional lymph nodes, and spleen during the course of rat adjuvant arthritis.

Cytokine gene activation was assessed during rat adjuvant arthritis (AA) in synovial membrane (SM), popliteal lymph node (popl-LN), and spleen, using semiquantitative, competitive RT-PCR. Changes in the popl-LN were considerably higher than in spleen or SM. In the preclinical phase (day 6), cytokine mRNA elevations occurred exclusively in the popl-LN and included IFN-gamma, IL-1beta, IL-5, IL-6, and IL-10. In the acute phase (days 13-16) all three organs became involved: (i) in the SM, significant elevations were limited to IL-1beta and IL-6, which, notably, correlated positively with the degree of arthritis; (ii) in the popl-LN, IFN-gamma, IL-1beta, IL-6, and IL-10 (but not IL-5) were still elevated, while IL-2 rose significantly; (iii) in the spleen, TNF-alpha peaked simultaneously with the arthritis score (day 16) and dramatically dropped thereafter. Upon transition into the chronic phase (day 20) the following phenomena were observed: (i) IL-1beta and IL-6 were still significantly increased in the SM; (ii) IFN-gamma, IL-1beta, IL-2, IL-6, and IL-10 were still elevated in the popl-LN; and (iii) there was a progressive rise of IL-5 mRNA in the spleen, positively correlated with the arthritis score. In conclusion, cytokines with pro- and anti-inflammatory functions overlap throughout disease, but in different organ-related patterns. Local (SM) and regional (popl-LN) IL-1beta and IL-6, elevated throughout the entire course of AA, may directly contribute to disease severity. While in AA spleen TNF-alpha appears to be a systemic marker of acute disease, spleen IL-5 may be involved in disease resolution.

Rat antigen-induced arthritis: cartilage alterations assessed with iodine-123-antileukoproteinase.

UNLABELLED: Imaging of cartilage alterations was attempted in joints of rats with chronic antigen-induced arthritis (AIA) using the cationic 123I-labeled serine proteinase inhibitor antileukoproteinase (123I-ALP; pI > 10), which selectively accumulates in normal cartilage, presumably through interaction with negatively charged proteoglycans. METHODS: Iodine-123-ALP or 123I-myoglobin, a control protein of comparable size but with different isoelectric point (pI=7.3) was injected intravenously into normal or AIA rats. Joint accumulation was followed by scintigraphy for 14 hr. Tissue radioactivity was assessed by well-counter measurements after dissection. The content of charged molecules in articular cartilage was determined by toluidine blue staining; the degree of joint destruction was assessed in parallel by x-ray, ex vivo MRI and histopathology. RESULTS: In intact articular cartilage, ALP accumulated to a significantly higher degree than myoglobin. This preferential accumulation was lost in rats with chronic AIA. The target-to-background ratio for 123I-ALP negatively correlated with the loss of toluidine blue staining in cartilage, which documents depletion of charged matrix molecules (r=-0.92, p < 0.01 at 4 hr; r=-0.97, p < 0.01 at 13 hr). ALP scintigraphy was sensitive in detecting cartilage alterations, even though the degree of joint destruction and inflammatory infiltration was mild, as demonstrated by x-ray, MRI and histopathology. CONCLUSION: In rat AIA, loss of ALP accumulation appears to document proteoglycan depletion in mildly altered arthritic cartilage. ALP scintigraphy may represent a functional assay for early, premorphological cartilage alterations in human arthritis as well.

The serine proteinase inhibitor antileukoproteinase specifically accumulates in normal but not in arthritic cartilage.

OBJECTIVE: To investigate whether the serine proteinase inhibitor antileukoproteinase (aLP) specifically accumulates in articular and extraarticular cartilage in normal and arthritic rats after intravenous (i.v.) injection. METHODS: [123I] or [125I] radiolabeled aLP and a control protein of comparable size were injected iv into normal rats or rats with chronic, antigen induced arthritis (AIA). Joint accumulation of the 2 proteins was followed by scintigraphy and organ tissue radioactivity was assessed by autoradiography and well counter measurements. Immunoprecipitation of aLP from articular cartilage was also performed and the content of charged molecules in normal and arthritic cartilage determined using toluidine blue staining. RESULTS: The accumulation of both radiolabeled aLP and control protein in the normal joint was clearly detectable by scintigraphy, with significant differences between the 2 proteins. In accordance with the scintigraphic data, direct tissue radioactivity measurements, immunoprecipitation, and autoradiography showed highly specific accumulation of radiolabeled aLP in articular and extraarticular cartilage of normal rats. The specific accumulation of aLP in articular cartilage was lost in rats with AIA in parallel with a loss of charged matrix molecules. CONCLUSION: I.v. injected serine protease inhibitor aLP specifically accumulates in articular and extraarticular cartilage of normal rats. This physiological pathway of cartilage accumulation, lost in proteoglycan depleted arthritic cartilage, may serve to maintain the local balance between proteinase function and inhibition.

Apoptotic cell death in activated monocytes following incorporation of clodronate-liposomes.

The present study was performed to elucidate whether sterically stabilized liposomes laden with clodronate, which lead to depletion of macrophages (Mphis) and amelioration of experimental autoimmune arthritis in vivo, selectively affect cells of the mphi lineage in vitro. The rates of incorporation of drug-free, fluorescent liposomes and the rates of cell death following exposure to clodronate-liposomes were assessed in human peripheral blood monocytes, as well as in polymorphonuclear leukocytes (PMNs), T cells, endothelial cells, and fibroblasts, both at rest and following activation. Gel electrophoresis of nuclear extracts and ultrastructural analyses were performed to identify the modality of cell death. Monocytes, particularly upon activation, were more efficient in incorporating sterically stabilized liposomes than all other cells except PMNs. Twenty percent of resting monocytes and up to 65% of activated monocytes died within 24 h of exposure to clodronate-liposomes, whereas the other cell types, including PMNs, remained unaffected. Activated monocytes exposed to clodronate-liposomes, but not resting or activated monocytes exposed to drug-free liposomes, showed clear signs of apoptotic cell death. In most of the assays, sterically stabilized liposomes were more efficient than conventional phosphatidylcholine-liposomes. Sterically stabilized clodronate-liposomes preferentially affect cells of the mphi lineage, particularly if activated. Selective elimination of activated Mphis by apoptosis may explain both therapeutic efficacy and safety of clodronate-liposomes in experimental models of autoimmunity.

Rat adjuvant arthritis: imaging with technetium-99m-anti-CD4 Fab' fragments.

UNLABELLED: The abundance of CD4 molecules on inflammatory cells in the synovial membrane renders anti-CD4 monoclonal antibodies (MAbs) or their fragments very promising for specific imaging of arthritic joints. METHODS: Joint uptake and body distribution of a 99mTc-labeled Fab', derived from the anti-rat CD4 MAb W3/25 (IgG1), were investigated following intravenous injection in normal and adjuvant arthritic rats. An isotype-matched Fab' (anti-human nonspecific crossreacting antigen-90) was used as control. RESULTS: A 14-hr sequential pinhole scan of the ankle joints revealed that both the anti-CD4 and the control Fab' accumulated to a higher degree in arthritic than in normal ankle joints; however, accumulation of the anti-CD4 Fab' in arthritic joints exceeded that of the control Fab' (approximate to 1.6 fold). Preferential joint accumulation of anti-CD4 Fab' was confirmed by whole-body scans at 14 hr and by direct well counter measurements of tissue samples at 16 hr following injection. Unlike the control Fab', the anti-CD4 Fab' preferentially accumulated in the liver and lymph nodes, organs rich in CD4-positive cells, as observed by direct tissue measurements. CONCLUSION: Despite its monovalency, the anti-CD4 Fab' retains the in vivo selectivity for CD4-positive cell-rich tissues, previously reported for the complete anti-CD4 MAb, and improves imaging of inflamed joints in experimental adjuvant arthritis.

Long-term amelioration of rat adjuvant arthritis following systemic elimination of macrophages by clodronate-containing liposomes.

OBJECTIVE: To determine whether systemic elimination of macrophages by means of clodronate-containing liposomes counteracts inflammation and joint destruction in rats with established adjuvant arthritis (AA). METHODS: Rats with AA received a total of 2.7 mg of clodronate encapsulated in liposomes in 3 intravenous doses on days 10, 11, and 12 of arthritis. Phosphate buffered saline (PBS), PBS-laden liposomes, or free clodronate were used as negative controls. Clinical, hematologic, and histopathologic signs of AA were monitored, and depletion of macrophages by clodronate-liposomes was evaluated both in the synovial membrane (SM) and in organs of the mononuclear phagocyte system (MPS). RESULTS: Clodronate-laden liposomes led to significant, long-term amelioration of the clinical signs of AA, a reduction in the erythrocyte sedimentation rate (ESR), and counteraction of joint destruction, not only immediately after treatment, but also for 2 weeks thereafter. Free clodronate induced moderate clinical improvement and a significant decrease in the ESR, but only during the late phase of AA. Drug-free vesicles even aggravated the joint destruction. Clodronate-laden liposomes did not induce significant depletion of resident macrophages in the SM, but rather, in the paracortical region of popliteal lymph nodes, in the liver, and in the marginal zone and periarteriolar lymphatic sheaths of the spleen. CONCLUSION: Clodronate-laden liposomes induce long-term amelioration of AA, even if administered for a brief period during the florid phase of the disease. The amelioration is paralleled by the elimination of macrophages in immunocompetent areas of the spleen and draining lymph nodes, but not locally in the SM. This suggests an influence of the treatment on the immunoregulatory rather than effector, functions of macrophages.

Joint uptake and body distribution of a technetium-99m-labeled anti-rat-CD4 monoclonal antibody in rat adjuvant arthritis.

Joint uptake and body distribution of a 99mTc-labeled monoclonal antibody (Mab) to the rat CD4 molecule (W3/25; IgG1) were investigated after intravenous injection in normal rats and in animals with experimentally induced adjuvant arthritis. An isotype-matched Mab with irrelevant specificity (anti-human carcino-embryonic-antigen) was used as control. A 4 hr sequential gamma-camera imaging revealed that both anti-CD4 and control Mab accumulated to a higher degree in arthritic than in normal ankle joints; the accumulation was comparable for the two Mabs. In contrast to the inflamed joints, a specific accumulation of the anti-CD4 Mab was found in organs rich in CD4-positive cells, i.e. spleen, bone marrow and lymph nodes, as assessed by direct well counter measurements 16 hr after injection. The control Mab displayed no preferential organ accumulation in either normal or diseased animals. These results indicate that a specific accumulation of anti-CD4 Mabs occurs in CD4-positive-cell-rich tissues in both normal and diseased animals and that immunoglobulins accumulate preferentially in inflamed joints regardless of their antibody specificity.