Rituximab treatment in patients with active Graves' orbitopathy: effects on proinflammatory and humoral immune reactions.

In active Graves' orbitopathy (GO), proinflammatory cytokines predominate. Circulating thyroid stimulating hormone (TSH)-receptor antibodies (TRAb) have been correlated with GO clinical activity and severity. In preliminary studies rituximab (RTX), an anti-CD 20 monoclonal antibody, has induced clinical improvement of active GO without a change in serum anti-thyroid antibodies. We have studied whether RTX in GO acts by affecting proinflammatory cytokines and thyroid and orbital-directed antibodies. Ten patients with GO were treated with RTX, administered twice intravenously (i.v.) (1000 mg) at days 1 and 15, and 20 with methylprednisolone, administered weekly i.v. (500 mg), for 16 weeks. Patients were studied before treatment, at B cell depletion and at 4, 8, 16, 20, 30 and 50 weeks. Peripheral lymphocytes, serum interleukin (sIL)-6, sIL-6r, chemokine (C-X-C motif) ligand 10 (CXCL10), TRAb and stimulating antibodies (TSAb) and autoantibodies against orbital calsequestrin, collagen XIII and flavoprotein subunit of succinate dehydrogenase (FP-SDH) were measured at baseline and after treatment. Serum IL-6 and sIL-6R concentrations did not change after RTX [P = not significant (n.s.)]. Serum CXCL10 increased after RTX at B cell depletion and at 30 weeks (P < 0·003). Serum TSAb did not change in relation to TRAb, nor did antibodies against orbital antigens (P = n.s.). In conclusion, this study shows that RTX in GO does not affect humoral reactions. The observed increase of serum CXCL10 concentrations at B cell depletion may result from cell lysis. We suggest that RTX may exert its effect in GO by inhibiting B cell antigen presentation.

Characterization of the T-cell epitopes of the major peach allergen Pru p 3.

BACKGROUND: Pru p 3 is the major peach allergen recognized by more than 90% of peach-allergic individuals of the Mediterranean area. Identification of the dominant Pru p 3 T-cell epitopes can improve our understanding of the immune responses against this protein and could be helpful in the development of hypoallergenic immunotherapy. For this purpose, we examined the phenotypes, specificities and cytokine secretion profiles of proliferating T cells in response to Pru p 3 in peach-allergic individuals. METHODS: Peripheral blood mononuclear cells from 15 peach-allergic patients were incubated with Pru p 3. The proliferation of antigen-specific T-cell lines (TCLs) was assessed by tritiated methylthymidine incorporation. T-cell epitopes were identified by analyzing the reactivity of TCLs against 8 overlapping peptides spanning the entire length of Pru p 3. We characterized the phenotype of Pru-p-3-specific TCLs by flow cytometry and analyzed their production of interleukin (IL) 4 and gamma-interferon (IFN-gamma) by ELISA. RESULTS: Ninety-two Pru-p-3-specific TCLs were isolated (stimulation index > or =5). These TCLs proliferated mainly in response to Pru p 3(12-27) and Pru p 3(57-72). Pru-p-3-specific TCLs were mainly CD4+ (81%) and expressed cell surface CD30. In addition, TCLs produced high levels of IL-4 and low levels of IFN-gamma, indicating a Th2 phenotype. CONCLUSIONS: Two immunodominant T-cell-reactive regions of Pru p 3 were identified: Pru p 3(12-27) and Pru p 3(57-72). These peptides showed a differential ability to elicit a Th2 response. Taken together, our results provide a better understanding of the immunological T-cell reactivity against Pru p 3.

NK cells in patients with peripheral neuropathy and IgM monoclonal protein reacting with the myelin-associated glycoprotein (MAG).

We studied Leu 7+ cell distribution and natural killer (NK) activity in the peripheral blood of six patients who had peripheral neuropathy and monoclonal IgM protein directed against myelin-associated glycoprotein (anti-MAG IgM). We did not find any difference between patients and control subjects (healthy or polyneuropathic, some with IgM monoclonal paraprotein but without anti-MAG activity). The presence of autologous sera did not interfere with these results. We noted an increase in Leu 11+ cell percentages after pre-incubation of the patient cells with autologous sera but the phenotypes of cells from control subjects did not change after incubation with autologous or patient sera.

Novel mode of action of iloprost: in vitro down-regulation of endothelial cell adhesion molecules.

Iloprost is a stable prostacyclin analog commonly employed in the treatment of peripheral vascular disease and also indicated in the treatment of patients affected by systemic sclerosis (SSc) in the presence of severe Raynaud's phenomenon (RP). Several mechanisms of action of the drug other than vasodilation and antiplatelet effect have been demonstrated that may be involved in the exertion of its clinical efficacy. Aim of the present study was to investigate whether iloprost down-regulated lymphocyte adhesion to endothelium through a modulation of adhesion molecule expression on the surface of endothelial cells. In the presence of iloprost, both lymphocyte adhesion and IL-1 stimulated expression of ICAM-1 and ELAM-1 exhibited a significant reduction, while unstimulated adhesion molecule expression was not significantly affected. Our results confirm that iloprost is able to down-regulate lymphocyte adhesion to endothelial cells and indicate that endothelium itself could be target of iloprost administration. Attenuation of the inflammatory response through modulation of cellular interactions could be suggested as a potential mechanism of action of iloprost, when used in the treatment of pathological conditions characterized by endothelial activation.

High frequency of T-cell lines responsive to immunodominant epitopes of thyrotropin receptor in healthy subjects.

In this study we analyzed the proliferative response to the extracellular domain of thyrotropin receptor (TSHR-ECD) of T-cell lines raised from healthy subjects. We found high frequencies of cell lines reactive to TSHR-ECD, ranging from 12% to 37%. The response of the cell lines to a set of overlapping peptides of TSHR-ECD showed that the most recognized epitopes by T lymphocytes are on the C-terminal portion. In particular, the regions of residues 360-396 and 258-277 are immunodominant in T-lymphocyte reactivity. A group of cell lines specific for the peptides of TSHR-ECD lost the response to the peptides during time in culture. However, these lines were still responsive to TSHR extracellular domain. The cloning of one of these lines showed three types of T-cell clones: (1) CD4+ clones (n = 4) highly responsive to the TSHR-ECD; (2) CD4+ clones (n = 4) low responsive to TSHR-ECD; (3) CD8+ clones (n = 9) not responsive to TSHR-ECD. The first group of clones was stable during time in culture, while the second group was characterized by the loss of the specific response to TSHR-ECD after some weeks from the first analysis. The observation of a spontaneous anergy in the second group of CD4+ clones suggests that mechanisms of control of the lymphocyte response to TSHR-ECD could be activated in vitro.

Lymphocyte-endothelium interaction in systemic sclerosis and Raynaud's phenomenon.

OBJECTIVE: To investigate interactions of immune cells with vascular endothelium in patients with systemic sclerosis (SSc) and in patients with idiopathic or autoimmune Raynaud's phenomenon (RP). METHODS: Lymphocytes obtained from 11 patients with SSc, 9 with RP and 14 control subjects were pre-stimulated in vitro with alloantigens and cultured together with human umbilical vein endothelial cells (HUVECs). Lymphocyte adhesion and induction of endothelial HLA-class 11 molecules were measured by flow cytometry. Lymphocyte cytotoxicity against HUVECs was also evaluated. In some cases cells were cultured under experimental conditions of hypoxia and reoxygenation. RESULTS: Lymphocyte adhesion and induction of endothelial cell expression of HLA-DR molecules were similar in controls and SSc patients, but significantly lower in RP (p < 0.05 and p < 0.03, respectively). Cytotoxic activity of lymphoblasts against endothelial cells was negligible in all patient groups. Under experimental conditions of hypoxia and reoxygenation lymphocyte adhesion was significantly greater than in normoxic conditions in SSc patients, while it was similar to normoxia in control subjects and RP patients. CONCLUSION: These results suggest that in RP patients there may be regulatory mechanisms of lymphocyte response able to control the processes that lead to lymphocyte adhesion and endothelial HLA-DR molecule induction. These mechanisms could play an important role in RP, and might possibly be lost in clinically evident SSc.

Relationship between anti-phospholipid and anti-endothelial cell antibodies III: beta 2 glycoprotein I mediates the antibody binding to endothelial membranes and induces the expression of adhesion molecules.

OBJECTIVE: To investigate the role of antibodies reacting with beta 2 glycoprotein I (beta 2GPI) in the antiendothelial cell binding activity present in sera from patients with the anti-phospholipid syndrome. METHODS: Sera positive for anti-phospholipid, anti-endothelial and anti-beta 2 GPI antibodies were studied for their binding activity on endothelial monolayers cultured in the presence or absence of media containing bovine serum as a source of beta 2GPI. Anti-endothelial activity was also evaluated on endothelial cells cultured without serum and supplemented with exogenous human purified beta 2GPI. Affinity purified anti-beta 2 GPI antibodies were investigated under the same experimental conditions. Finally, the effect of the incubation of these affinity purified fractions on the expression of adhesion molecules (ELAM-1) was studied. RESULTS: The reactivity of the sera decreased on endothelial cells incubated in serum-free medium, while endothelial cell binding was restored in a dose dependent manner after the addition of exogenous purified human beta 2 GPI. Affinity purified anti-beta 2 GPI antibodies obtained from the same sera retained their endothelial cell binding and were able to activate endothelial cells by inducing the ex novo surface expression of adhesion molecules (ELAM-1). CONCLUSIONS: These findings indicate that the close association between anti-endothelial and anti-phospholipid antibodies is sustained by antibodies which recognize beta 2 GPI adhering to the endothelial cells, and can promote their activation.

[Case report: rheumatoid arthritis and large granular lymphocytes syndrome]. / Artrite reumatoide e leucemia a "large granular lymphocytes": descrizione di un caso clinico.

Felty's syndrome (FS) is a rare complication (less than 1%) of rheumatoid arthritis (RA), with the clinical feature of splenomegaly and neutropenia. Approximately 10-40% of FS patients have an expansion of peripheral blood large granular lymphocytes (LGL). This cell population mainly consists of two subsets: cytotoxic T cells (CD8+, CD57+) and natural killer cells (CD3-,CD8-,CD56+). It has been hypothesised that LGL expansion could be responsible for neutropenia by suppressing neutrophil precursors in the bone marrow, but various mechanisms have been proposed to explain this association. We report a case of a 60-year-old woman with rheumatoid factor positive RA who developed LGL expansion responsible for splenomegaly, but without neutropenia. In conclusion, LGL expansion is an uncommon complication of RA and may be responsible for both FS and clinical pictures resembling FS.

Rituximab induces distinct intraorbital and intrathyroidal effects in one patient satisfactorily treated for Graves' ophthalmopathy.

Hyperthyroid Graves' disease (GD) is a B-cell-mediated disease caused by antibodies stimulating the thyroid stimulating hormone (TSH) receptor (TRAb). A proportion of patients (40-60%) present with an associated ophthalmopathy (TAO), a progressive inflammatory autoimmune disease of the retroorbital tissue. We thought that the anti-CD20 monoclonal antibody rituximab (RTX), by inducing transient B-cell depletion, may potentially modify the active inflammatory phase of TAO. One patient with GD and TAO in its active phase and unresponsive to steroid, was treated with RTX. Whereas the ophthalmopathy responded to RTX therapy and a decrease in the clinical activity score from 5 to 2 was observed during the B-cell depletion, serum antithyroid antibodies, and in particular serum TRAb, were not affected by therapy. When the patient underwent total thyroidectomy, we found B-cells in the thyroid tissue specimens. The eye disease remained stable (clinical activity score=2) and the patient subsequently underwent orbital decompression to correct proptosis of the eye. At that time we did not find lymphocytes in any of the orbital tissue specimens. We believe that RTX therapy in GD may cause amelioration of ophthalmopathy by depleting total lymphocyte population in the orbit, but not lymphocyte depletion in thyroid tissue with consequent unchanged serum TRAb levels.

Unusual presentation of large B cell lymphoma: a case report and review of literature.

Diffuse large B cell lymphoma (DLBCL) is the largest subtype of non-Hodgkin's lymphomas (NHLs) and is characterized by relatively frequent extranodal presentation. In these cases, the most common extranodal localizations are stomach, CNS, bone, testis and liver. Simultaneous detection of multiple extranodal involvement at presentation is quite uncommon, with the majority of these cases characterized by gastric or intestinal disease localization. Retrospective analysis concerning multifocal extranodal NHLs never pointed out disease features such as those described here. We report a patient with an unusual presentation of DLBCL, characterized by adrenal and renal involvement, associated with symptoms and signs of the cold agglutinin disease and a hypercoagulable state. Subsequently, computed tomography (CT) and fluorodeoxyglucose-positron emission tomography (FDG-PET) scanning disclosed a rapidly extensive spread to nodes and bones. Cytofluorimetric analysis of a renal specimen showed medium-to-large lympho-monocytoid elements positive for CD20 with monoclonal expression of immunoglobulin kappa light chain. Histopathological examination confirmed a renal CD20 positive DLBCL localization.

Increased expression of C3b and C3bi receptors on human neutrophils and monocytes induced by a glycoprotein extract from Klebsiella pneumoniae (RU41740).

RU41740 is a glycoprotein extract from Klebsiella pneumoniae with immunomodulating properties under different experimental conditions. In particular the compound is able to stimulate several functions of human phagocytes in vitro and ex vivo. Using monoclonal antibodies and flow cytometry, in this work we assessed the effect of RU41740 on surface expression of receptors for C3b (CR1) and C3bi (CR3) in human phagocytic cells in vitro. The incubation of whole blood with varying RU41740 concentrations led to a dose-dependent increase in surface expression of CR1 and CR3 on both neutrophils and monocytes when compared with control samples incubated in buffer alone. The maximal drug-induced enhancement of complement receptors was: 291% +/- 13.4% for CR1 and 265% +/- 8.5% for CR3 in neutrophils; 117% +/- 4.5% for CR1 and 98% +/- 4.1% for CR3 in monocytes. These peak effects were observed using RU41740 at a final concentration of 10 micrograms/ml and were similar to those induced by optimal concentrations of the activating compound N-formyl-methionyl-leucyl-phenylalanine (10(-7)M). Polymyxin B did not modify the RU41740-induced enhancement of CR1 and CR3 expression on phagocytes, suggesting no role for endotoxin in this activity. These results define, at least in part, the mechanism of action of RU41740 on human phagocytes in vitro and could be relevant to in vivo events during RU41740 treatment.

T cell defect in essential mixed cryoglobulinaemia.

Peripheral blood lymphocytes from untreated patients with essential cryoglobulinaemia were studied for their surface markers and for their in vitro mitogenic reactivity. No differences in lymphocyte subpopulations were observed between cryoglobulinaemic patients and normal controls. Cultures of separated lymphocytes were stimulated with different concentrations of phytohaemagglutinin, Con-A and pokeweed mitogen. Incorporation of [3H]-thymidine in patients' cultures was compared with that of normal controls. Significantly decreased reactivity to phytohaemagglutinin and Con-A, but not to pokeweed mitogen, was found in all patients studied. The depressed mitogenic reactivity to phytohaemagglutinin and Con-A might be referred to a qualitative T cell defect.

AMLR and MLR stimulating activity of human T lymphocytes activated in vitro by soluble HLA-DR antigens.

We have examined the allogeneic mixed lymphocyte reaction (MLR) and autologous mixed lymphocyte reaction (AMLR) stimulating activities of T cells precultured in vitro with soluble allogeneic or autologous HLA-DR antigens. These cells (Ts) are known to suppress the human MLR: this suppression is specific in that it occurs only when stimulator cells have the same HLA-DR antigen as that used to induce differentiation of suppressor cells. Ts cells express new membrane specificities; they can be separated by immunoabsorption into two populations: Ts enriched (Tx+; with suppressive activity) and Ts depleted (Ts-; with helper function). In the present study, we have demonstrated that both Ts cell subsets activated by soluble HLA-DR alloantigens are able to stimulate both MLR and AMLR. Ts cells activated by soluble autologous HLA-DR antigens are able to stimulate MLR, but not AMLR.

Susceptibility to HIV infection and AIDS in Italian haemophiliacs is HLA associated.

We compared the frequencies of HLA antigens in two matched groups of 31 HIV-seronegative and 31 HIV-seropositive haemophiliacs, exposed during the years 1981-85 to comparable amounts and batches of presumably infectious clotting factor concentrates. The frequency of A2 was significantly higher in HIV-seropositive than in seronegative haemophiliacs, with a relative risk (RR) of seroconversion of 3.92, whereas both Bw52 and DR4 were negatively associated with it. We also studied the distribution of HLA antigens in a larger group of 76 HIV-seropositive haemophiliacs, who were at different clinical stages of HIV infection (CDC classes II-IV) but were comparable for age and time elapsed since seroconversion. DR3 and DQw2 antigens were, particularly when concomitantly present, associated with a high risk of developing symptomatic HIV infection (RR = 11.79 and 25.33). Our data suggest that the HLA region controls susceptibility to infection with HIV and its progression to symptomatic disease in Italian haemophiliacs.

In vivo enhancement of NK-cell activity by thymopentin.

Natural killer (NK) activities of fresh and IL-2-precultured peripheral blood mononuclear cells (PBMC) were investigated in 13 elderly subjects before and after thymopentin (TP-5) treatment and in 12 age-matched controls. The NK activity of fresh PBMC was found to be significantly higher in subjects given TP-5 at 50 mg s.c. three times per week for one month. Conversely, this type of treatment with TP-5 did not significantly modify the cytotoxic capacity of IL-2-precultured PBMC.