Synovial fluid OX40T lymphocytes of patients with rheumatoid arthritis display a Th2/Th0 polarization.

Rheumatoid arthritis (RA) is an autoimmune disease in which T-cell activation plays a pivotal role in the induction of articular damage. CD4+/OX40+ T cells accumulate in the synovial fluid (SF) of RA patients, which suggests that they are involved in the pathogenesis of the disease. In this study, we assessed the intracellular cytokine production of peripheral blood and SF CD4+ and CD4+/OX40+ T cells from RA patients in order to evaluate their role in this disorder. Our results show that SF CD4+ cells are predominantly interferon gamma (IFN-gamma)-positive and express a Th1-like cytokine pattern. In SF, significantly more CD4+/OX40+ T cells expressed interleukin-4 (IL-4) and IL4/IFN-gamma than IFN-gamma alone. Our data demonstrate that SF CD4+/OX40+ T cells express a Th2/Th0 cytokine profile, which suggests that they are involved in inflammatory responses in RA joints.

Effects of interferon beta, cyclophosphamide and azathioprine on cytokine profile in patients with multiple sclerosis.

We assessed the in vitro effects of interferon beta-1b (IFNbeta-1b), cyclophosphamide (CY), and azathioprine (AZA) alone and of the combination of IFNbeta-1b with CY or AZA on the production of Th1 and Th2 cytokines in 10 patients with multiple sclerosis. Cytokine levels were determined at baseline and after stimulation with IFNbeta-1b, CY, and AZA alone or with the combination of IFNbeta-1b with CY or AZA. The combination of IFNbeta-1b with CY resulted in a statistically significant decrease in the production of interleukin-2 (IL-2) (P=0.003) and tumor necrosis factor alpha (TNF-alpha) (P=0.03). An additive effect on the production of interferon gamma (IFN-gamma) (P=0.2) and interleukin-10 (IL-10) (P=0.6), and a positive interaction on the production of interleukin-4 (IL-4) (P=0.08) were observed although the findings were not statistically significant. The combination of IFNbeta-1b with AZA resulted in a significant negative effect on the production of IL-2 (P=0.006), whereas TNF-alpha (P=0.02), IFN-gamma (P=0.03), IL-4 (P=0.2), and IL-10 (P=0.3) were not statistically impacted. Our data show that CY was able to improve the effects of IFNbeta-1b on the ratio of Th1/Th2 cytokines.

Combination therapy with cyclosporine and methotrexate in patients with early rheumatoid arthritis soon inhibits TNFalpha production without decreasing TNFalpha mRNA levels. An in vivo and in vitro study.

OBJECTIVE: To evaluate the ability of two different combination therapies with prednisone (PDN), methotrexate (MTX) and cyclosporine (CSA) to modulate both TNFalpha transcription and production in early rheumatoid arthritis (RA). METHODS: 24 patients with early RA received a step-down bridge therapy with MTX and PDN (group A). Twelve patients out of the 24 randomly received also CSA (group B). Blood samples and peripheral blood mononuclear cells (PBMC) were collected at different times. TNFalpha levels were measured both in sera and in PBMC supernatants. TNFalpha mRNA was assessed by use of RT-PCR. RESULTS: 10 patients in group A and 9 in group B improved. At baseline, RA patients serum TNFalpha levels were increased compared to controls (p < 0.001) and did not correlate with clinical and serological parameters. These levels decreased within the first month of therapy in both groups, the lower levels being observed in the sera of CSA treated patients. After 30 days of therapy, TNFalpha levels in group B supernatants were significantly lower than those observed in group A, both after 24 and 48 hours of PHA stimulation (p < 0.03 and p < 0.05 respectively). TNFalpha mRNA levels never differed between patients and controls, independently of both the clinical picture and the assigned therapy. CONCLUSION: The addition of CSA to a treatment regimen of PDN + MTX lowers TNFalpha production in vitro without decreasing TNFalpha mRNA expression. This effect could help to induce early immunosoppressive and therapeutic effects during RA.

Circulating gamma/delta T lymphocytes from systemic sclerosis (SSc) patients display a T helper (Th) 1 polarization.

Systemic sclerosis (SSc) is a connective tissue disease in which immune system activation is evidenced by high levels of different cytokines in the sera and/or in the supernatants of cultured peripheral blood mononuclear cells (PBMC) and by the presence of specific autoantibodies. gamma/delta T cells accumulate in the lung and the skin of SSc patients suggesting their potential role in the development and maintenance of the disease. The aim of this study was to assess cytokine production and cytotoxic activity of circulating gamma/delta T lymphocytes obtained from SSc patients and to evaluate their potential role during this disorder. Our results showed that both the proportion and the absolute number of IFN-gamma gamma/delta-producing cells (i.e. displaying a Th1 polarization) in SSc was significantly higher than either the proportion and the absolute number of IL-4 gamma/delta-producing cells in SSc or the proportion and the absolute number of IFN-gamma gamma/delta-producing cells in healthy controls (P < 0.05 for both groups). Furthermore, the cytotoxic activity of enriched gamma/delta T cells was significantly increased in SSc patients compared with controls. The results concerning the Vdelta1+ T cell subset paralleled those of total gamma/delta T lymphocytes. In contrast, alpha/beta T cells from SSc and control subjects displayed Th2 cytokine production. All these findings were independent of both disease subset and clinical status. Our data demonstrate that, although SSc is generally considered a Th2 autoimmune disease, Th1 polarization of gamma/delta T cells and an increase in their cytotoxic activity is observed in SSc, suggesting that gamma/delta T cells could have a relatively autonomous role in the pathogenesis in this disease.

T lymphocytes in the synovial fluid of patients with active rheumatoid arthritis display CD134-OX40 surface antigen.

OBJECTIVE: To assess the percentage of T lymphocytes, bearing CD134, a member of the TNF receptor superfamily, primarily found on autoreactive CD4+ T cells in the peripheral blood (PB) and synovial fluid (SF) of rheumatoid arthritis (RA) patients. METHODS: The surface expression of CD134 on SF and PB mononuclear cells was performed by flow cytometry in 25 RA patients and correlated to the disease activity. RESULTS: CD134 expression on CD3+, CD4+, CD8+ and CD25+ cells was higher in SF than in PB of RA patients (P < 0.001). No differences were observed in the percentage of CD134+/CD4+ T lymphocytes in the PB of RA patients and controls. Patients with active RA had significantly higher percentage of CD3+/CD134+, CD4+/CD 134+, CD8+/CD134+ and CD25+/CD 134+ than those with inactive disease. CONCLUSION: These findings suggest that CD134+ T cells are involved in the immunopathological process of RA synovitis, maybe mirroring some other autoimmune disease in which autoreactive T cell infiltrating the target tissues largely coexpress CD134.

Circulating soluble factor-inhibiting natural killer (NK) activity of fresh peripheral blood mononuclear cells (PBMC) from inflammatory bowel disease (IBD) patients.

This study was performed in order to assess the cytotoxic activity, both natural (NK) and antibody-dependent (ADCC), of PBMC from 38 IBD patients and correlate it with their clinical features. Cytotoxicity assays were performed using sensitive target cells for NK and ADCC activities. In some experiments, highly purified NK cells, obtained both by Percoll density gradient and by co-culturing non-adherent PBMC with RPMI 8866 feeder cells, were used as effector cells. Furthermore, we evaluated NK cell parameters such as number, surface expression of adhesion molecules (CD11a/CD18, CD49d and CD54) and response to different stimuli. We observed a decreased NK cytotoxicity of PBMC from IBD patients, both in ulcerative colitis (UC) and Crohn's disease (CD), independently of the clinical activity of disease. In contrast, the ADCC lytic activity was within normal range. The lower NK cytotoxic activity observed in our IBD patients cannot be related to a decreased number of NK cells, surface expression of adhesion molecules, defective response to IL-2 and maturative defect. Decreased NK activity was induced in PBMC of controls when serum of patients was added and this was unrelated to monocyte-derived modulating factor(s). Our data show a decreased natural killing by fresh PBMC from IBD patients. This lower activity seems to be unrelated to a primary NK cell defect, since purified NK cells exhibited normal levels of killing. It might be hypothesized that serum factors, possibly derived from lymphocytes, with inhibitory properties on NK activity, might be functionally active in the blood of IBD patients, thus modulating NK activity.

The Italian version of the Family Assessment Device.

The aim of the study was to evaluate in a heterogeneous. Italian sample (n = 340) the psychometric properties of the Italian version of the Family Assessment Device (FAD), a 60-item questionnaire assessing family functioning. The questionnaire was administered to psychiatric (n = 116), medical (n = 114) and non-clinical samples (n = 110). In a sample of 30 non-clinical subjects the temporal stability of the FAD was investigated. The results showed a good temporal stability for Problem Solving, General Functioning. Communication, and Affective Responsiveness scales, and a good internal reliability of the scale. Factor analysis of the Italian version provided discrepancies with the hypothesized structure of the instrument, leading to the identification of seven slightly different dimensions. The proposed seven-factor model of the instrument did not provide a good fit to our data. The results of our study suggest the need for a major improvement in the adaptation of the FAD in the Italian setting.

Serum levels of soluble CD30 are increased in ulcerative colitis (UC) but not in Crohn's disease (CD).

Imbalance in Th1 and Th2 subsets and their derived cytokines seems to be involved in the immune abnormalities underlying UC and CD. CD30 is a member of the tumour necrosis factor/nerve growth receptor superfamily expressed on T cells producing Th2 cytokines and released as a soluble form. In this study high levels of soluble CD30 were found in sera of UC patients independently of disease activity. Furthermore, increased titres of soluble CD30 molecule were shown, in the same patients, by mitogen-stimulated cultures of peripheral blood mononuclear cells. Our data seem to indicate that an activation of Th2 immune response is involved in the pathogenesis of UC, but not of CD. Furthermore, this finding indicates that serum soluble CD30 measurement may be helpful for differentiating these two forms of inflammatory bowel disease.

Circulating Vdelta1+ T cells are activated and accumulate in the skin of systemic sclerosis patients.

OBJECTIVE: An increased percentage of Vdelta1+/gamma/delta T cells has been detected both in the peripheral blood and bronchoalveolar lavage fluid of patients with systemic sclerosis (SSc). This study evaluated the subset distribution, activation status, and expression of cellular adhesion molecules, such as intercellular adhesion molecule 1 (CD54), very late activation antigen alpha4 (CD49d), and lymphocyte function-associated antigen 1alpha (CD11a), on circulating gamma/delta T cells, as well as their presence in the skin of SSc patients. METHODS: We studied 12 patients with SSc and 16 healthy volunteer donors. The distribution, activation status, and expression of cellular adhesion molecules were studied by flow cytometry; their presence in SSc patient skin was evaluated by immunohistochemistry. RESULTS: We found that the percentages and absolute numbers of peripheral blood gamma/delta T cells, CD16, CD8, CD45RO, CD25, HLA-DR, CD54, and CD11a coexpression did not differ significantly from those of the controls. CD49d gamma/delta T cells were significantly increased in SSc patients (2.3%) compared with controls (0.5%). A marked increase in the ratio of Vdelta1+ cells to gamma/delta cells was observed in the patients (72%) compared with the controls (31%). The Vdelta1+ subset showed a significant expression of both HLA-DR (83% of total Vdelta1+ cells) and CD49d (90% of total Vdelta1+ cells) compared with the controls (20.5% and 60%, respectively). In the skin, the absolute numbers of gamma/delta T cells were found in striking amounts in perivascular areas, particularly in the early edematous phase of SSc (22.58 in patients and 0 in controls); the majority of gamma/delta T cells were Vdelta1+ (19 in patients and 0 in controls). In the advanced phase of SSc, Vdelta1+ T cells were also increased compared with controls (3.5 versus 0). CONCLUSION: Our results show that Vdelta1+ T cells express both adhesion molecules and activation markers, and strongly support gamma/delta T cell homing to sites of inflammation. The increase in the Vdelta1 subset suggests a selective V gene subset expansion.

Circulating levels of soluble CD30 are increased in patients with systemic sclerosis (SSc) and correlate with serological and clinical features of the disease.

Activated Th2 lymphocytes express the surface molecule CD30 and release a soluble form of the same molecule which can be detected both in vivo and in vitro. In the present study, high levels of soluble CD30 were found in the peripheral blood of patients with SSc, and a significant correlation with skin score and erythrocyte sedimentation rate (ESR) was detected. Furthermore, we observed a higher spontaneous release of soluble CD30 in the supernatants of unstimulated cultures of peripheral blood mononuclear cells from our patients compared with healthy controls. Taken together, these data suggest a possible involvement of Th2 cells in the immunopathogenesis of SSc, and the dosage of CD30 soluble in the peripheral blood may be helpful in following the outcome of the disease.

[Immunosuppressive therapy of vasculitis: current aspects and perspectives]. / Terapia immunosoppressiva delle vasculiti. Attualità e prospettive.

Vasculitides are a set of serious diseases of unknown aetiology with various immunopathogenetic mechanisms, characterized by inflammation and necrosis of the vessel wall with consequent lumen obliteration. They may be primitive or associated with other diseases, have heterogeneous clinical manifestations and different degrees of severity which may be related to the localization of the interested vessels. Although in the last years many classifications have been proposed, a standardized nomenclature of vasculitides is unquestionably still needed to facilitate the diagnosis and management of patients with the disease. Steroids and immunosuppressant are the conventional therapy, whereas other therapeutic strategies are reserved for the refractory vasculitides to conventional therapies or for intolerant recipients to cytotoxic drugs. New approaches are represented by monoclonal antibodies and drugs which could be effective in the treatment of the trigger factors which activate the immunopathological mechanisms. Current data suggest that, rather than pursuing the idea of a single therapy for vasculitides, an oncological model of combined therapy, to induce both the disease control and maintenance of remission, might be adopted. An improvement of our knowledges on the mechanisms underlying the different entities associated to standardized criteria of activity and remission of disease will lead to an improvement of our therapeutic strategies.

Peripheral blood mononuclear cells of patients with systemic sclerosis produce increased amounts of interleukin 6, but not transforming growth factor beta 1.

OBJECTIVE: To assess the ability of peripheral blood mononuclear cells (PBMC) of patients with systemic sclerosis (SSc) to produce interleukin 6 (IL-6), transforming growth factor beta 1 (TGF-beta 1), to identify the IL-6 producer cells in the in vitro model, and to correlate these data with the clinical evidence of our patients. METHODS: We used a sandwich ELISA to quantitate IL-6 and TGF-beta 1 levels in sera, plasma, and supernatants, and an imunofluorescence technique to evaluate IL-6 producing cells in our patients. RESULTS: IL-6 was detected in sera from 8 of 20 patients and no controls (p < 0.05). A significant increase of IL-6 production was observed in both spontaneous and phytohemagglutinin (PHA) induced cultures of PBMC from patients with SSc vs controls. No differences in TGF-beta 1 production were observed, either in sera or supernatants, between patients and controls. A significant increase of IL-6 synthesizing cells was observed after 3 h of PHA stimulation in patients vs controls (p < 0.05). CONCLUSION: Spontaneous IL-6 production and the higher number of IL-6 producing cells in patients with SSc suggest that these cells have been already primed in vivo. The absence of PBMC primed for TGF-beta 1 production supports the hypothesis that cells other than lymphocytes produce and secrete this cytokine in the skin of patients. Higher serum levels of IL-6 observed in a subset of patients did not correlate with either severity or duration of disease.

Increase of circulating gamma/delta T lymphocytes in the peripheral blood of patients affected by active inflammatory bowel disease.

In order to study the role of gamma/delta T cells in the pathogenesis of inflammatory bowel disease (IBD) in humans, we measured the percentage of these cells in the peripheral blood, assessed the ratio of the non-disulphide-linked (delta TCS1) type of T cell receptor (TCR) in the total gamma/delta T cells, studied the co-expression of gamma/delta TCR and accessory molecules CD8 and CD16, and compared these data with both the type and the activity of the disease. Percentage levels and absolute numbers of gamma/delta+ T cells were higher in active patients than in controls (P < 0.05), mainly as a result of an increase of V delta 1+ (delta TCS1) T cell subset (P < 0.05). This trend was strongly retained independently of disease activity and clinical picture. An increased percentage of TCR delta 1+/CD16+ cells was observed in our patients compared with controls (P < 0.05). In contrast, no difference was observed as far as the TCR delta 1+/CD8+ cells were concerned. These results suggest that IBD is associated with an expansion of gamma/delta T cells in peripheral blood, which may play a role in the pathogenesis of these disorders.

The syndrome of idiopathic CD4+ lymphocytopenia.

The syndrome of idiopathic CD4+ lymphocytopenia has recently been recognized and referred to as the persistent depletion of peripheral blood CD4+ T lymphocytes below 300 cells per cubic millimeter or less than 20% of total lymphocytes in the absence of either HIV infection or other known causes of immunodeficiency. The available literature indicates that neither human retroviruses (HIV-1, HIV-2, HTLV-I, HTLV-II) nor other transmissible agents play any clear-cut role in the pathogenesis. Furthermore, the epidemiologic, immunologic and clinical features of this syndrome differ substantially from those of HIV infection. The heterogeneity of both immunologic abnormalities, in addition to CD4+ depletion, and clinical course in patients with this disorder points out no common cause although in at least a subset of patients the pathogenetic pathways could be shared with common variable immunodeficiency.

Evaluation of the morphological and functional damage to human sperm subjected to freezing at -196 degrees C and to refrigeration at +4 degrees C.

A study was carried out on five healthy, fertile donors to evaluate refrigeration at +4 C compared to cryopreservation at -196 degrees C. These donors had produced more than two pregnancies in different women with their cryopreserved semen in an AID program. The following parameters for evaluation and comparison were used: (i) the percentage of forward sperm motility, (ii) the percentage of swollen sperm after hypoosmotic stress (swelling test) and (iii) the sperm morphology observed both with a light microscope after staining and with an electron microscope. After 48 hours of refrigeration the result obtained were comparable with those observed after one week of cryopreservation. After 72 hours of refrigeration, a sharp and significant decrease of these values was noted. Our data underlined the fact that there is an individual variability in subject response to the method of preservation employed. Our findings show the possibility of using sperm refrigerated for up to 48 hours in AIH programs.

Natural killer cell frequency and function in patients with monoclonal gammopathies.

We evaluated the frequency and the functional activity of peripheral blood mononuclear cells (PBMCs) with natural killer (NK) cell phenotype in patients with monoclonal gammopathies. CD16+ and CD56+ PBMCs were strongly increased in monoclonal gammopathies of undetermined significance (MGUS) and multiple myeloma (MM). Furthermore, increased frequency of CD16+/CD3+ PBMCs was found in 7/15 patients with MGUS, indicating that T lymphocytes with NK-like phenotype are expanded in at least a subset of these patients. However, despite the increased frequency of PBMCs with natural killer phenotype, the functional NK activity was as comparable in both MGUS and MM patients as in normal individuals. The discrepancy between the expansion of circulating NK cells and the normal NK activity in patients with monoclonal gammopathies requires further investigation. However, at least in some MGUS patients, this discrepancy could be accounted for by the expansion of PBMCs with the rare phenotype CD16/CD3 which have been reported not to mediate significant NK activity.

[The expansion of NK CD56+ cells with reduced cytotoxicity in a female patient with systemic sclerosis]. / Espansione delle cellule NK CD56+ con ridotta citotossicità in una paziente affetta da sclerosi sistemica.

An absolute and relative increase of circulating CD56+ (NKH1, Leu19) natural killer cells has been found in a patient with systemic sclerosis. The expanded natural killer cells exhibited reduced natural killer activity and antibody-dependent cell-mediated cytotoxicity. Furthermore, the limiting dilution analysis of spontaneous in vitro immunoglobulin synthesis demonstrated that the precursor frequency of immunoglobulin-secreting cells and the "single-hit" kinetics of the titration curve were similar to healthy controls, but strongly different from previously reported patients in whom CD16+NK cell subset was expanded. Thus, our findings might suggest that expanded CD56+ natural killer cells exhibit unique regulatory properties on B cell function in systemic sclerosis.

Modulation by ST 789 of in vitro lymphocyte activation and cytokine production.

Our results indicate that ST 789 exhibits complex immunomodulant properties. In fact, we found that ST 789 inhibits the expression of activation antigens, such as interleukin-2 and transferrin receptors by peripheral blood mononuclear cells (PBMCs) from healthy subjects following mitogen stimulation, but we were not able to detect under the same experimental conditions any effect on the in vitro production of soluble CD8 antigen and of interleukin-4 as well as on the proliferative response of antigen-specific and autoreactive human T cell lines. Finally, we showed that ST 789 is able to strongly enhance the in vitro production of interleukin-6 by PHA-stimulated PBMCs. The reduced expression of activation antigens in the presence of ST 789 does not seem to be mediated by CD8+ suppressor T lymphocytes, as indicated by the normal soluble CD8 levels in culture media, but rather reflects a direct inhibitory action on T helper proliferation and likely on interleukin-2 secretion. The strong enhancement by ST 789 of the in vitro interleukin-6 production seems to indicate the most relevant possibility of clinical applications in human diseases.