Improvement in clinical features of hypercortisolism during osilodrostat treatment: findings from the Phase III LINC 3 trial in Cushing's disease.

PURPOSE: Cushing's disease is associated with substantial morbidity and impaired quality of life (QoL) resulting from excess cortisol exposure. The current study explored improvements in clinical signs and additional specific manifestations of hypercortisolism during osilodrostat (potent oral 11ß-hydroxylase inhibitor) therapy by degree of control of mean urinary free cortisol (mUFC). METHODS: LINC 3 (NCT02180217) was a prospective, open-label, 48-week study of osilodrostat (starting dose: 2 mg bid; maximum: 30 mg bid) that enrolled 137 adults with Cushing's disease and mUFC > 1.5 times the upper limit of normal (ULN). mUFC (normal range 11‒138 nmol/24 h), cardiometabolic parameters (blood pressure, weight, waist circumference, body mass index, total cholesterol, fasting plasma glucose, glycated haemoglobin), physical manifestations of hypercortisolism (facial rubor, striae, fat distribution, bruising, hirsutism [females], muscle atrophy) and QoL were evaluated. mUFC was defined as controlled if ≤ ULN, partially controlled if > ULN but ≥ 50% reduction from baseline, and uncontrolled if > ULN and < 50% reduction from baseline. Concomitant medications were permitted throughout the study. RESULTS: At weeks 24 and 48, respectively, mUFC was controlled in 93 (67.9%) and 91 (66.4%) patients, partially controlled in 20 (14.6%) and 13 (9.5%), and uncontrolled in 24 (17.5%) and 33 (24.1%). Overall, mean improvements from baseline in cardiometabolic at week 24 were greater in patients with controlled or partially controlled versus uncontrolled mUFC; at week 48, improvements occurred irrespective of mUFC control. Generally, physical manifestations and QoL progressively improved from baseline irrespective of mUFC control. CONCLUSIONS: Improvements in clinical signs and additional specific manifestations of hypercortisolism associated with Cushing's disease occurred alongside decreases in mUFC. Trial registration NCT02180217 (first posted July 2014).

Adipose stromal cells mediated switching of the pro-inflammatory profile of M1-like macrophages is facilitated by PGE2: in vitro evaluation.

OBJECTIVE: To define if adipose mesenchymal stromal cell (ASC) treatment mediated switching of the pro-inflammatory profile of M1-like macrophages as a means to develop a tailored in vitro efficacy/potency test. DESIGN: We firstly performed immunohistochemical analysis of CD68, CD80 (M1-like) and CD206 (M2-like) macrophages in osteoarthritic (OA) synovial tissue. ASC were co-cultured in contact and in transwell with activated (GM-CSF + IFNγ)-M1 macrophages. We analyzed IL1ß, TNFα, IL6, MIP1α/CCL3, S100A8, S100A9, IL10, CD163 and CD206 by qRT-PCR or immunoassays. Prostaglandin E2 (PGE2) blocking experiments were performed using PGE2 receptor antagonist. RESULTS: In moderate grade OA synovium we did not always find a higher percentage of CD80 with respect to CD206. M1-like-activated macrophage factors IL1ß, TNFα, IL6, MIP1α/CCL3, S100A8 and S100A9 were down-modulated both in contact and in transwell by ASC. However, in both systems ASC induced the typical M2-like macrophage markers IL10, CD163 and CD206. Activated-M1-like macrophages pre-treated with PGE2 receptor antagonist failed to decrease secretion of TNFα, IL6 and to increase that of IL10, CD163 and CD206 when co-cultured with ASC confirming a PGE2 specific role. CONCLUSIONS: We demonstrated that ASC are responsible for the switching of activated-M1-like inflammatory macrophages to a M2-like phenotype, mainly through PGE2. This evidenced that activated-M1-like macrophages may represent a relevant cell model to test the efficacy/potency of ASC and suggests a specific role of ASC as important determinants in therapeutic dampening of synovial inflammation in OA.

Analysis of mesenchymal stem cells grown on a three-dimensional HYAFF 11-based prototype ligament scaffold.

Ligaments are complex structures that maintain the mechanical stability of the joint. Healing of injured ligaments involves the interactions of different cell types, local cellular environment, and the use of devices. To gain new information on the complex interactions between mesenchymal stem cells (MSCs) and a specific hyaluronan-based prototype scaffold (HYAFF, useful for ligament tissue engineering, short time-course experiments were performed to analyze the proliferation, vitality, and phenotype of MSCs grown on the scaffold. MSC proliferation was analyzed using the MTT test, during the early time points (2, 4, 6, days). Viability was assessed using calcein/acetyloxymethylester immunofluorescence dye and confocal microscopy analysis. Hyaluronic acid receptor (CD44), typical matrix ligament proteins (collagen type I, type III, laminin, fibronectin, actin), and chondrogenic/osteogenic markers (collagen type II and bone sialoprotein) were evaluated by immunohistochemistry. Our data demonstrated that MSC growth and viability were cell density-dependent. MSCs completely wrapped the fibers of the scaffold, expressed CD44, collagen type I, type III, laminin, fibronectin, and actin, and were negative to collagen type II and bone sialoprotein. These data demonstrate that MSCs survive well in the hyaluronan-based prototype ligament scaffold, as assessed after 2 days from seeding, and express CD44, a receptor important for scaffold interaction, and proteins responsible for the functional characteristics of the ligaments.

A fluorescent in situ hybridization method in flow cytometry to detect HIV-1 specific RNA.

In HIV+ patients, the presence of HIV-RNA in plasma and circulating cells has been reported to be a marker of progression but the percentage of transcriptionally active infected cells remains unclear. We have developed a reliable fluorescent in situ hybridization method for the detection of HIV specific RNA by flow cytometry. The procedure was applied to a panel of chronically infected cell lines and to an acutely infected cell line mimicking normal peripheral blood lymphocytes in susceptibility to HIV-1. The cells were fixed in suspension and hybridized by means of an HIV-1 genomic probe labeled with digoxigenin-11-dUTP. An FITC-labeled anti-digoxigenin antiserum was then applied and the resulting fluorescence signals were analyzed both by flow cytometry and confocal microscopy. Different procedures for double staining HIV-RNA together with virus induced proteins or surface markers were also developed. Flow cytometric detection of in situ hybridization offers the possibility of analyzing thousands of cells in a few seconds and of collecting multiparametric information at the single cell level, thus providing a potential tool for detecting the rare HIV-RNA expressing cells in peripheral blood samples.

Basic fibroblast growth factor enhances in vitro mineralization of rat bone marrow stromal cells grown on non-woven hyaluronic acid based polymer scaffold.

A biodegradable non-woven hyaluronic acid polymer scaffold (Hyaff 11) was analysed in vitro as a carrier vehicle for differentiation and mineralization of rat bone marrow stromal cells (BMSC). BMSC were grown on Hyaff 11 in a mineralizing medium in the presence/absence of basic fibroblast growth factor (bFGF). Osteoblastic differentiation was investigated by light and electron microscopy analysing the expression of osteogenic markers: calcium, alkaline phosphatase (AP), osteopontin (OP), bone sialoprotein (BSP) and collagen type 1. We also measured proliferation, AP activity and mRNA expression of AP and osteocalcin (OC). Electron microscopy and Toluidine-blue staining demonstrated that bFGF accelerated (day 20 vs. day 40) and increased mineralization. With bFGF, calcium, OP and BSP were strongly enhanced at day 40, whereas AP decreased. Our in vitro results demonstrate that Hyaff 11 is a useful vehicle for growth, differentiation and mineralization of rat BMSC, and that it permits bone development.

Outcome of chronic opioid therapy for non-cancer pain.

Potential iatrogenic mood and cognitive declines associated with long-acting opioid therapy were examined in 19 patients receiving long-acting oral opioid medications and compared to ten patients receiving usual care. Pain, mood, and cognitive function were measured before and after achieving stable doses. In addition to reducing pain, long-acting opioid medication reduced anxiety and hostility. No declines in cognitive function were associated with the long-acting opioid medications, and the group receiving long-acting opioid medications showed significant improvement on a measure of psychomotor speed and sustained attention. Both patient groups reported significant reductions in perceived impairment in daily activities due to pain. Treatment responders taking long-acting opioid medications (63%) were taking a significantly lower dose at follow-up than the treatment non-responder group. These findings suggest that long-acting opioid medications can improve mood and do not impair cognitive functioning in patients with chronic non-cancer pain.

Restrained and nonrestrained eaters' orienting responses to food and nonfood odors.

Dietary restraint was assessed by Stunkard and Messick's (1985) three-factor eating questionnaire, using the restraint subfactor score only in normal-weight college students (n = 41). The subjects were than assessed for skin conductance orienting responses (ORs) to food and nonfood odors when hungry and sated (after a standard breakfast and after an overnight fast). Subjects also rated their hunger and each odorant for pleasantness on separate 7-point scales. Results indicated that restrained eaters oriented less to odors than did nonrestrained subjects. Food deprivation did not differentially affect the ORs in restrained and nonrestrained eaters. The ORs, however, tended to be decreased in all of subjects who had had breakfast. Finally, nonrestrained subjects rated food and nonfood odors approximately equal in pleasantness, while the restrained eaters rated food odors as more pleasant than the nonfood odors. These results suggest that restrained eaters must certainly process odor stimuli related to foods, but also suggests that orienting to these salient (informative) cues is restricted. Perhaps in defense of the diet, restrained eaters learn methods/responses (cognitive strategies, instructional sets) to block orienting to food related cues such as odors.

Psychiatric symptoms and distress differ between patients with postherpetic neuralgia and peripheral vestibular disease.

OBJECTIVE: No previous studies have investigated the psychiatric characteristics of patients with postherpetic neuralgia (PHN). Similarly, no studies have been performed on patients with different chronic somatic symptoms due to a defined medical disease to compare the characteristics of psychiatric morbidity associated with each etiology. METHODS: After completing the subscales of the Symptom Checklist 90-R, a psychiatrist administered the Diagnostic Interview Schedule to all subjects. The psychiatric comorbidity in 35 patients with pain due to PHN was compared with a control group of 34 patients with the nonpainful aversive symptom of vertigo due to a peripheral vestibular disorder that caused unilateral hypofunction. RESULTS: PHN patients had significantly more symptoms of major depression and somatization disorder. No significant differences were found between groups for psychiatric diagnoses. Patients with PHN reported significantly less acutely distressing somatic symptoms. CONCLUSION: These results suggest that the psychiatric symptoms of patients with PHN are distinct from nonspecific acute distress and may be related to the experience of suffering from chronic neuropathic pain. Patients with PHN may not meet criteria for a psychiatric diagnosis, but their psychiatric comorbidity places them at substantial risk for increased pain, suicidal ideation, sustained disability, and the numerous complications of excessive medical evaluation and treatment. Patients with PHN should be evaluated specifically for psychiatric symptoms to reduce potential negative consequences through appropriate treatment.

Cell cycle synchronization of FRTL5 cells. A physiological model system.

We describe a "physiological" cell cycle synchronization model system. FRTL5 cells, TSH-dependent for proliferation, were starved from TSH. The cell cycle phases and the expression of markers associated to different cycle phases were evaluated. TSH starvation blocks proliferation without provoking death and induces virtually all the cells to accumulate in G0/G1 phase. TSH readdition allows 30% of these cells to enter the S phase. DNA topoisomerase II 170-kDa isoform is not expressed in G0/G1 synchronized cells while it is expressed in logarithmic growing cells. The 180-kDa isoform is not expressed in G0/G1 synchronized cells while it is expressed in 20% of logarithmic growing cells regardless of the cycle phase. c-myc mRNA is not expressed in G0/G1 synchronized cells while it is detectable upon TSH readdition. This system provides a tool for the analysis of events associated with the G0/G1 phase and the transition from G0/G1 to S phase.

Tissue engineering for cartilage repair: in vitro properties of a hyaluronan-derivative.

Association of biomaterials with autologous cells can provide a new generation of implantable devices for cartilage and bone repair. Such scaffolds should provide a performed three-dimensional shape, prevent cells from floating out of the defect, have sufficient mechanical strength, facilitate uniform spread of cells, and stimulate the phenotype of transplanted cells. Hyaff-11 is a recently developed hyaluronic-acid based biodegradable polymer, that has been shown to provide successful cell scaffolds for tissue-engineered repair. The aim of this study was to evaluate in vitro the potential of Hyaff-11 to support the growth of human chondrocytes and to maintain their original phenotype. Our data indicate that human chondrocytes seeded on Hyaff-11 express and produce collagen type II and aggrecan and downregulate the production of collagen type I. These results provide an in vitro demonstration of therapeutic potential of Hyaff-11 as a delivery vehicle in tissue-engineered repair of articular cartilage defects.

Hyaluronan-based biomaterial (Hyaff-11) as scaffold to support mineralization of bone marrow stromal cells.

Various techniques are widely used to repair bone defects, association of hyaluronan-based biodegradable polymers (Hyaff-11) with bone marrow stromal cells (BMSC) promises to provide successful cell scaffolds for tissue-engineered repair of bone tissue. We evaluate in vitro and in vivo the potential of Hyaff-11 to facilitate mineralization of BMSC. Rat BMSC were seeded on Hyaff-11 and their differentiation were assessed at different time points. Osteogenic differentiation was investigated in vitro analysing the expression of alkaline phosphatase and osteocalcin. Mineralization of bone defects was evaluated also in vivo implanting Hyaff-11 scaffold combined with BMSC in large segmental radius defects. In vitro, we found a decrease expression of alkaline phosphatase and an increase of osteocalcin. In vivo, our data showed that mineralization was induced and basic fibroblast growth factor contributed to this process. These results provide a demonstration to therapeutic potential of Hyaff-11 as appropriate carrier vehicle for differentiation and mineralization of BMSC and for the repair of bone defects.

Development of a sensitive, direct luminescent enzyme immunoassay for plasma estradiol-17 beta.

A rapid, sensitive, precise, chemiluminescent enzyme immunoassay for estradiol-17 beta has been developed and validated. Antibodies were produced in rabbits using estradiol-17 beta-6-(O-carboxymethyl)oxime coupled to bovine serum albumin, purified and immobilized on polystyrene beads (6.4 mm diameter). The same derivative was used to prepare the enzymatic tracer by coupling with horseradish peroxidase. The assay, direct on the serum sample, featured a 4-h binding step at 4 degrees C followed by the chemiluminescent detection using luminol/H2O2. The detection limit was 0.15 pg/tube and the assay was carried out on 20-100 microliter of sample, allowing measurement of estradiol-17 beta in plasma concentrations from 1.5 to 500 pg/ml. The method fulfills all the standard requisites of precision and accuracy and the results agree well with a radioimmunoassay procedure on extracted serum.

Antifungal activity of ketoconazole and other azoles against Malassezia furfur in vitro and in vivo.

The in vitro activity of several antifungal agents (ketoconazole, miconazole, econazole, fenticonazole, itraconazole, fluconazole) in routine clinical use against Malassezia furfur infections has been studied with freshly isolated strains of M. furfur from pityriasis versicolor lesions. The results indicate that the drugs tested exert a good activity, and both ketoconazole and itraconazole appear very active (0.8 mg/l respectively). Hair samples from the beards of volunteer patients affected by pityriasis versicolor but otherwise healthy were examined to determine ketoconazole levels during oral therapy (one or two 200 mg tablets daily). It was shown that the drug progressively accumulates in the beard, reaching levels proportional to the dose administered, although blood levels did not increase in parallel. The study of drug concentration profile has evidenced a long ketoconazole persistence in the beard at therapeutic levels. In conclusion, the possibility of reaching high and lasting ketoconazole levels in the keratin layer of the epidermis indicates that systemic ketoconazole therapy could be useful for eradication of M. furfur in patients affected by pityriasis versicolor.

Hyaluronan does not affect cytokine and chemokine expression in osteoarthritic chondrocytes and synoviocytes.

OBJECTIVE: Many studies have evidenced the clinical efficacy of hyaluronan (HA) in the treatment of osteoarthritis (OA). However, human and animal studies have described proinflammatory effects of HA on cells not involved in OA. We therefore investigated whether different molecular weight HA preparations can affect proinflammatory cytokine (IL1beta and TNFalpha) or chemokine (IL8, MCP-1 and RANTES) expression in human chondrocytes and synoviocytes isolated from OA patients. DESIGN: Human chondrocytes and synoviocytes were cultured in vitro in the presence or absence of three different purified HA pharmaceutical preparations (1x10(6) Kd, 5x10(5) Kd and 6.5x10(4) Kd) and assessed for the production of proinflammatory cytokines and chemokines and their mRNA expression. RESULTS: basal conditions, both chondrocytes and synoviocytes produce only MCP-1 and IL8, along with low quantities of IL1beta and TNFalpha, but not RANTES. IL8 production was generally about 100 times higher in chondrocytes than in synoviocytes, while MCP-1 was roughly twice as high in synoviocytes than in chondrocytes. At the mRNA level, expression of IL1beta, TNFalpha, IL8, MCP-1 and RANTES did not change in the presence of the three HA preparations either in synoviocytes or in chondrocytes with respect to basal condition. None of the three different HA preparations significantly affected production of IL8 or MCP-1. CONCLUSIONS: These data demonstrate that preparations of HA of the same origin but with different MWs do not induce proinflammatory cytokines and chemokines expressed by chondrocytes and synoviocytes that are either directly or indirectly involved in OA progression.

Contribution of interleukin 17 to human cartilage degradation and synovial inflammation in osteoarthritis.

OBJECTIVE: To compare the effect of interleukin (IL)-17, IL-1beta and TNF-alpha on chemokine production by human chondrocytes and synovial fibroblasts isolated from patients with osteoarthritis (OA). The expression of IL-1beta mRNA by OA chondrocytes was also assessed, as well as the presence and expression of IL-17 receptor (IL-17R) in OA chondrocytes and synovial fibroblasts after stimulation with IL-17, IL-1beta and TNF-alpha. DESIGN: Synovial fibroblasts and chondrocytes isolated from patients with OA were stimulated in vitro with IL-17, IL-1beta or TNF-alpha. Supernatants were collected and immunoassayed for the presence of IL-8, GRO-alpha (CXC chemokines) and MCP-1, RANTES (CC chemokines). The cells were used to detect the presence of IL-17R and the expression of IL-17R mRNA. Stimulated chondrocytes were also used to detect IL-1beta production and mRNA expression. RESULTS: IL-17 upregulated the release of IL-8 and GRO-alpha both by synovial fibroblasts and chondrocytes, and the release of MCP-1 only by chondrocytes. IL-17 was a weaker stimulator than IL-1beta and TNF-alpha, except for GRO-alpha release which was maximally upregulated by IL-1beta, less by IL-17 and minimally by TNF-alpha. When compared to IL-1beta, IL-17 was more active on chondrocytes than on fibroblasts. In chondrocytes the expression of IL-1beta mRNA was enhanced by IL-17 and TNF-alpha, with a maximum level reached by IL-1beta. IL-17 and TNF-alpha stimulated IL-1beta release in few subjects. Neither IL-17, IL-1beta nor TNF-alpha modulated the presence of IL-17R and the expression of IL-17R mRNA. CONCLUSIONS: These data suggest that IL-17 could contribute to cartilage breakdown and synovial infiltration in OA by inducing both the release of chemokines by chondrocytes and synovial fibroblasts and, in a less extent, the synthesis of IL-1beta by chondrocytes.

Epitope specificity of Th0/Th2 CD4+ T-lymphocyte clones induced by vaccination with rHBsAg vaccine.

BACKGROUND & AIMS: Different amino acid sequences of hepatitis B virus surface antigen (HBsAg) are involved in the activation of CD4+ lymphocytes needed to induce an optimal antiviral function. The aim of this study was to characterize the CD4-mediated response to immunodominant HBsAg epitopes in hepatitis B virus (HBV) vaccine recipients by defining minimal sequences recognized by T cells, cytokine profiles, and HLA restriction of peptide recognition. METHODS: T-lymphocyte lines and clones specific for HBsAg were isolated from the peripheral blood of subjects immunized with recombinant HBsAg and stimulated in vitro with synthetic peptides spanning the whole HBsAg sequence. RESULTS: Four immunodominant epitopes (sequences 21-40, 136-155, 156-175, and 211-226) were identified. Using panels of truncated peptides of different length, sequences 21-28, 165-172, and 215-223 were shown to correspond to the minimal epitopes recognized by T cells. The antigen-specific T-lymphocyte proliferation was HLA class II restricted, and each peptide could be presented in association with different HLA class II determinants. Th0/Th2 cytokine patterns were induced on peptide stimulation. CONCLUSIONS: These results indicate the presence of at least four immunodominant epitopes within HBsAg that represent potential candidates for the design of anti-HBV synthetic vaccines.

Osteoblasts and stromal cells isolated from femora in rheumatoid arthritis (RA) and osteoarthritis (OA) patients express IL-11, leukaemia inhibitory factor and oncostatin M.

We investigated both in vitro and ex vivo the role of mature osteoblasts (OB) and bone marrow stromal cells (BMSC) in RA and OA by analysing the expression of the following IL-6-type cytokines: IL-11, leukaemia inhibitory factor (LIF), oncostatin M (OSM) and IL-6. OB and BMSC were isolated from femora of RA, OA and post-traumatic (PT) patients, cultured in vitro in the presence or absence of IL-1beta and tumour necrosis factor-alpha (TNF-alpha), and assessed for the production and mRNA expression of IL-6-type cytokines. Trabecular bone biopsies were obtained from the inner portions of femoral heads and used for cytokine in situ immunostaining. Cultured OB and BMSC from different patients constitutively secreted IL-11 and IL-6 but not OSM. LIF was secreted only by BMSC, at very low levels. Interestingly, IL-11 basal production was significantly higher in BMSC than in OB in all three groups tested. IL-1beta and TNF-alpha strongly stimulated IL-6-type cytokine release (except for OSM) by both OB and BMSC. OSM was expressed only at mRNA levels in all groups studied. Cytokine immunostaining on bone biopsies confirmed the data obtained on cultured cells: IL-11, IL-6 and LIF proteins were detected both in mesenchymal (BMSC and OB) and mononuclear cells; OSM was found only in mononuclear cells. These data demonstrate that IL-6-type cytokines are constitutively expressed in the bone compartment in RA, OA and PT patients and can be secreted by bone cells at different stages of differentiation (BMSC and OB). This suggests that these cytokines may be involved in the mechanisms of bone remodelling in OA and RA.

Chemokine production by human chondrocytes.

OBJECTIVE: To evaluate the role of chondrocytes in producing CXC chemokines [interleukin 8 (IL-8), growth related gene product (GRO-alpha)] and CC chemokines [monocyte chemoattractant protein (MCP-1), macrophage inflammatory protein (MIP-1alpha), RANTES] in patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and subjects after traumatic injury (PT). METHODS: Articular cartilage specimens were obtained from 38 patients with OA and 18 with RA undergoing joint replacement surgery. Healthy human cartilage was obtained from femoral condyles removed after trauma in 11 subjects with no history of joint pathology (PT cases). Chondrocytes were isolated from articular cartilage by sequential enzymatic digestion and cultured in vitro. Chemokine production was investigated in unstimulated condition and after 72 h incubation with proinflammatory [IL-1beta, tumor necrosis factor-alpha (TNF-alpha)] and antiinflammatory [transforming growth factor-beta1 (TGF-beta1), IL-10] mediators. Chemokine concentrations in cell supernatants were evaluated by ELISA. RESULTS: Chondrocytes produce all these chemokines to a different extent. IL-1beta was a more potent stimulus than TNF-alpha in inducing production of all chemokines except MCP-1. We found no statistical differences among chondrocytes isolated from OA, RA, and PT for chemokine production in either basal conditions or after cytokine stimulation. IL-1beta induced chemokine production can be modulated by TGF-beta1 in different ways according to the various chemokines, while IL-10 does not affect IL-1beta induced chemokine production. CONCLUSION: Chondrocytes produce IL-8, GRO-alpha, MCP-1, MIP-1alpha, and RANTES. Proinflammatory factors (IL-1beta, TNF-alpha) effectively upregulate chemokine production, but production is scarcely modulated by the antiinflammatory mediators TGF-beta and IL-10. Chondrocyte derived chemokines may play a role in triggering the mechanisms involved in pathogenesis and persistence of joint diseases.

Proinflammatory cytokines and chemokine production and expression by human osteoblasts isolated from patients with rheumatoid arthritis and osteoarthritis.

OBJECTIVE: To evaluate whether subchondral osteoblasts (OB) are involved in the production of cytokines and chemokines in rheumatic diseases. METHODS: OB were isolated from subchondral bone of rheumatoid arthritis (RA), osteoarthritis (OA) and post-traumatic (PT) patients, cultured in vitro in the presence or absence of interleukin 1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), and assessed for the production, immunolocalization, and mRNA expression of proinflammatory cytokines (IL-1alpha, IL-1beta, TNF-alpha) and alpha and beta chemokines [IL-8, growth related gene product (GRO-alpha), monocyte chemoattractant protein 1 (MCP-1), RANTES, and macrophage inflammatory proteins MIP-1alpha, MIP-1beta]. RESULTS: Cultured OB from different patients did not release IL-1alpha, IL-1beta, or TNF-alpha, and constitutively secreted IL-8, GRO-alpha, and MCP-1, while RANTES, MIP-1alpha, MIP-1beta were undetectable or near the lower level of sensitivity of the immunoenzymatic assay. GRO-alpha was significantly higher in RA than in OA and PT patients. IL-1beta and TNF-alpha alone or in combination strongly stimulated chemokine release by OB. Only RANTES production was not increased by the combination of the 2 cytokines. IL-1alpha, IL-1beta, and TNF-alpha were expressed as cytoplasmic proteins and were not secreted by OB even after stimulation. CONCLUSION: OB from subchondral bone release chemokines that could be involved in the mechanisms that directly or indirectly cause bone remodelling and cartilage destruction.

Chemokine expression by subchondral bone marrow stromal cells isolated from osteoarthritis (OA) and rheumatoid arthritis (RA) patients.

We analysed the spontaneous and cytokine-stimulated production and expression in vitro of IL-8, GROalpha, MCP-1, RANTES, MIP-1alpha, MIP-1beta, by subchondral bone marrow stromal cells (BMSC) isolated from RA, OA, post-traumatic (PT) patients and normal donors (ND). BMSC were cultured in vitro in the presence or absence of IL-1beta and tumour necrosis factor-alpha (TNF-alpha), and assessed for chemokine production, expression and immunolocalization. BMSC from different sources constitutively released MCP-1, GROalpha and IL-8, but not MIP-1alpha or MIP-1beta, while BMSC from ND constitutively released only IL-8 and MCP-1. IL-8, GROalpha and RANTES production in basal conditions was significantly higher in RA patients than in ND. RANTES production was also higher in OA and RA than in PT patients. The combination of TNF-alpha and IL-1beta synergistically increased the production of all chemokines tested except for RANTES. Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that all chemokines not detectable in the supernatants were expressed at the mRNA level. Chemokine immunostaining was localized around the nuclei. This work demonstrates that BMSC from subchondral bone produce chemokines and indicates that these cells could actively participate in the mechanisms directly or indirectly causing cartilage destruction and bone remodelling.