In vitro production of anti-RBC antibodies and cytokines in chronic lymphocytic leukemia

B-chronic lymphocytic leukemia (B-CLL) patients have a high prevalence of autoimmune phenomena, mainly autoimmune hemolytic anemia (AIHA). Immunoregulatory cytokines play a role in the regulation of both autoimmunity and leukemic B-cell growth. Mitogen-stimulated direct antiglobulin test (MS-DAT) is a recently described test able to disclose latent anti-RBC autoimmunity in AIHA. We investigated the prevalence of anti-RBC autoimmunity by MS-DAT and the pattern of cytokine production by PHA-stimulated whole blood cultures from 69 B-CLL patients and 53 controls. Results showed that anti-RBC IgG values in unstimulated, PHA-, PMA-, and PWM-stimulated cultures were significantly higher in B-CLL patients compared with controls. In B-CLL, the prevalence of anti-RBC autoimmunity was 28.9% by MS-DAT, compared with 4.3% by the standard DAT. Production of IFN-gamma, IL-2, IL-13, TNF-alpha, sCD23, and sCD30 was significantly increased in all B-CLL patients compared with controls, whereas there was no difference in IL-4, IL-6, IL-10, and TGF-beta production. Multivariate analysis showed that IL-4 was significantly increased in MS-DAT-positive compared with -negative patients. Patients with autoantibody positivity displayed greater IFN-gamma production than negative patients. These data are in line with the hypothesis that autoimmune phenomena in B-CLL are associated with an imbalance towards a Th-2-like profile. The elevated prevalence of anti-RBC autoimmunity found by MS-DAT suggests that an underestimated latent autoimmunity exists in B-CLL

Pleural Tuberculosis in Patients with Early HIV Infection Is Associated with Increased TNF-Alpha Expression and Necrosis in Granulomas

Although granulomas may be an essential host response against persistent antigens, they are also associated with immunopathology. We investigated whether HIV co-infection affects histopathological appearance and cytokine profiles of pleural granulomas in patients with active pleural tuberculosis (TB). Granulomas were investigated in pleural biopsies from HIV positive and negative TB pleuritis patients. Granulomas were characterised as necrotic or non-necrotic, graded histologically and investigated for the mRNA expression of IL-12, IFN-γ, TNF-α and IL-4 by in situ hybridisation. In all TB patients a mixed Th1/Th2 profile was noted. Necrotic granulomas were more evident in HIV positive patients with a clear association between TNF-α and necrosis. This study demonstrates immune dysregulation which may include TNF-α-mediated immunopathology at the site of disease in HIV infected pleural TB patients

Sustained response to low-dose rituximab in idiopathic autoimmune hemolytic anemia

ObjectivesTo evaluate the sustained response to low-dose (LD) rituximab in autoimmune hemolytic anemia (AIHA), the ex vivo effect on anti-RBC antibody production by mitogen-stimulated direct antiglobulin test (MS-DAT), and the in vitro dose effect of the drug on the production of anti-RBC antibodies. MethodsThirty two patients, 18 warm (W) AIHA and 14 cold hemagglutinin disease (CHD), were treated with LD rituximab (100mg fixed dose x4 weekly infusions) along with a short course of oral prednisone. Complete clinical examination, blood counts, and hemolytic markers were performed at enrollment and at month 6, 12, 24, and 36. ResultsHematological parameters significantly improved at all time points compared to enrollment. The overall response was 90%, 100%, 100%, and 89% and the relapse-free survival 87%, 79%, 68%, and 68% at 6, 12, 24, and 36months, respectively. Response rates were slightly better in WAIHA than in CHD, and relapse risk was greater in cold than warm forms (HR 2.1, 95% CI 0.6-7.9). Four patients were retreated (one patient twice), all achieving a response, lasting a median of 18months (range 9-30). Treatment was well tolerated without adverse events or infections. Anti-RBC antibody production by MS-DAT significantly decreased over time. In vitro studies showed that rituximab effectively inhibited anti-RBC antibody production at 50g/mL, 1/6 of the drug concentration after therapy with standard doses. ConclusionsThese data confirm that LD rituximab treatment is effective and induces sustained responses in AIHA, and that a lower dose of the drug is enough to down-regulate autoantibody production

Sustained response to low-dose rituximab in idiopathic autoimmune hemolytic anemia.

ObjectivesTo evaluate the sustained response to low-dose (LD) rituximab in autoimmune hemolytic anemia (AIHA), the ex vivo effect on anti-RBC antibody production by mitogen-stimulated direct antiglobulin test (MS-DAT), and the in vitro dose effect of the drug on the production of anti-RBC antibodies. MethodsThirty two patients, 18 warm (W) AIHA and 14 cold hemagglutinin disease (CHD), were treated with LD rituximab (100mg fixed dose x4 weekly infusions) along with a short course of oral prednisone. Complete clinical examination, blood counts, and hemolytic markers were performed at enrollment and at month 6, 12, 24, and 36. ResultsHematological parameters significantly improved at all time points compared to enrollment. The overall response was 90%, 100%, 100%, and 89% and the relapse-free survival 87%, 79%, 68%, and 68% at 6, 12, 24, and 36months, respectively. Response rates were slightly better in WAIHA than in CHD, and relapse risk was greater in cold than warm forms (HR 2.1, 95% CI 0.6-7.9). Four patients were retreated (one patient twice), all achieving a response, lasting a median of 18months (range 9-30). Treatment was well tolerated without adverse events or infections. Anti-RBC antibody production by MS-DAT significantly decreased over time. In vitro studies showed that rituximab effectively inhibited anti-RBC antibody production at 50g/mL, 1/6 of the drug concentration after therapy with standard doses. ConclusionsThese data confirm that LD rituximab treatment is effective and induces sustained responses in AIHA, and that a lower dose of the drug is enough to down-regulate autoantibody production

Low-dose rituximab in adult patients with idiopathic autoimmune hemolytic anemia: clinical efficacy and biologic studies.

This prospective study investigated the efficacy, safety, and response duration of low-dose rituximab (100 mg fixed dose for 4 weekly infusions) together with a short course of steroids as first- or second-line therapy in 23 patients with primary autoimmune hemolytic anemia (AIHA). The overall response was 82.6% at month +2, and subsequently stabilized to ∼ 90% at months +6 and +12; the response was better in warm autoimmune hemolytic anemia (WAIHA; overall response, 100% at all time points) than in cold hemagglutinin disease (CHD; average, 60%); the relapse-free survival was 100% for WAIHA at +6 and +12 months versus 89% and 59% in CHD, respectively, and the estimated relapse-free survival at 2 years was 81% and 40% for the warm and cold forms, respectively. The risk of relapse was higher in CHD and in patients with a longer interval between diagnosis and enrollment. Steroid administration was reduced both as cumulative dose (∼ 50%) and duration compared with the patient's past history. Treatment was well tolerated and no adverse events or infections were recorded; retreatment was also effective. The clinical response was correlated with amelioration biologic markers such as cytokine production (IFN-γ, IL-12, TNF-α, and IL-17), suggesting that low-dose rituximab exerts an immunomodulating activity. This study is registered at www.clinicaltrials.gov as NCT01345708