Francesca Guia Imperiali

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 patients 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.

Cytokine modulation of nuclear factor-κB activity in B-chronic lymphocytic leukemia

OBJECTIVE Dysregulation of the apoptotic mechanisms plays a key role in the accumulation of malignant B-chronic lymphocytic leukemia (B-CLL) cells. The transcription nuclear factor (NF)-kappaB is important for cell survival by regulating the expression of anti-apoptotic genes. Several cytokines can modulate leukemic growth and apoptosis in B-CLL. The aim of this study was to determine whether cytokine-mediated regulation of apoptosis occurs via modulation of NF-kappaB activity in peripheral blood mononuclear cells from B-CLL patients. PATIENTS AND METHODS We evaluated NF-kappaB activity in peripheral blood mononuclear cells from 15 untreated B-CLL patients and 11 controls in resting conditions and in the presence of phorbol-12-myristate-13-acetate (PMA) and different cytokines by electrophoretic mobility shift assay. Apoptosis was studied by spectrophotometric analysis of DNA fragmentation. RESULTS We found a constitutive high NF-kappaB activity not induced by PMA in B-CLL patients, in contrast with a normal inducible NF-kappaB activity in controls. In B-CLL cultures, addition of interleukin (IL)-4 and IL-13 increased, whereas transforming growth factor (TGF)-beta reduced NF-kappaB activity compared with unstimulated cultures. Accordingly, IL-4 and IL-13 decreased, whereas TGF-beta increased DNA fragmentation compared with unstimulated cultures. IL-13 and IL-4 production was increased, whereas TGF-beta was reduced in PMA-stimulated and unstimulated cultures from B-CLL patients compared with controls. CONCLUSIONS B-CLL patients have a constitutive high NF-kappaB activity, which is modulated by cytokines. In particular, TGF-beta displays a pro-apoptotic activity, whereas IL-4 and IL-13 have opposite effects. These cytokine alterations could be responsible for a positive autocrine circuit that maintains leukemic cells in a pre-apoptotic state.

Hereditary red cell membrane defects: diagnostic and clinical aspects

The plasma membrane of the erythrocyte accounts for all of this cell’s antigenic, transport, and mechanical characteristics, particularly its ability to undergo large passive deformations during repeated passage through the narrow capillaries of the microvasculature, throughout its 120-day life span. The determinant of normal membrane cohesion is the system of “vertical” linkages between the phospholipid bilayer and membrane skeleton, formed by the interactions of the cytoplasmic domains of various membrane proteins with the spectrin-based skeletal network. Band 3 and Rh-associated glycoprotein (RhAG) provide such links by interacting with ankyrin, which in turn binds to β-spectrin. Protein 4.2 binds to both band 3 and ankyrin and can regulate the avidity of the interaction between band 3 and ankyrin. Glycophorin C, band 3, XK, Rh, and Duffy all bind to protein 4.1R, the third member of the ternary junctional complex with β-spectrin and actin1–2. Red cell membrane disorders are inherited diseases due to mutations in various membrane or skeletal proteins, resulting in decreased red cell deformability, reduced life span and premature removal of the erythrocytes from the circulation. The red cell membrane disorders include hereditary spherocytosis, hereditary elliptocytosis, hereditary ovalocytosis and hereditary stomatocytosis.

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

To 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.

Increased prevalence of autoimmune phenomena in myelofibrosis: Relationship with clinical and morphological characteristics, and with immunoregulatory cytokine patterns

Autoimmune phenomena and cytokines were investigated in 100 patients with myelofibrosis (MF) and related to marrow fibrosis and clinical risk. Anti-erythrocyte antibodies by mitogen-stimulated direct antiglobulin test (MS-DAT) were positive in 45%, anti-platelets in 15% and organ/non organ-specific in 57% of cases, without clinically overt disease, and mostly in low-risk/intermediate-risk-1 and MF-0/MF-1. TGF-β and IL-8 were increased in MS-DAT positive cases, and IFN-γ in patients with serological autoantibodies. TGF-β and IL-17 were elevated in early clinical and morphological stages, while IL-8 increased in advanced stages. These data suggest that autoimmune phenomena and cytokine disregulation are particularly relevant in early MF.

Comparison of traditional methods and mitogen-stimulated direct antiglobulin test for detection of anti-red blood cell autoimmunity

The diagnosis of autoimmune hemolytic anemia (AIHA) is based on a positive direct antiglobulin test (DAT), which is performed using various methods with different sensitivities. Recently, mitogen-stimulated (MS)-DAT was suggested to be able to identify latent anti-erythrocyte autoimmunity. Traditional methods (tube, microcolumn, and solid phase) and MS-DAT were compared in 54 consecutive cases of suspected AIHA, 28 idiopathic AIHA in clinical remission, and 12 difficult-to-diagnose cases of DAT-negative AIHA, and the results (all cases) were correlated with hematologic and hemolytic parameters. DAT tube was confirmed as the gold standard to diagnose AIHA since almost all positive cases showed hemolytic anemia and positive eluates; 10 out of 26 tube-negative cases were positive on microcolumn and solid phase antiglobulin tests, and 22 out of 26 using MS-DAT, although only half of them showed clear signs of hemolysis. Mitogen stimulation increased the amount of IgG bound to red blood cells in all groups; moreover, MS-DAT was the only positive test in 10 cases of AIHA, and mitogen stimulation facilitated the identification of autoantibody specificity in culture supernatants. We conclude that a battery of tests rather than a single test is useful for the diagnosis of AIHA, including MS-DAT as an additional test for selected cases, although the results have to be cautiously interpreted based on the overall clinical context.

Toll-like receptor 4 and 9 expression in B-chronic lymphocytic leukemia: relationship with infections, autoimmunity and disease progression

Abstract Toll-like receptors (TLRs) represent major agents of innate immunity and initiators of adaptive immunity. TLR4 and TLR9 gene expression was related to the occurrence of infections, autoimmunity and disease progression in 95 patients with B-chronic lymphocytic leukemia (B-CLL), grouped according to stage, therapy and known prognostic markers, and followed prospectively (median 33.6 months, range 25–50). A retrospective analysis (median 6.8 years, range 6–26) was also performed. TLR4 gene expression was decreased and TLR9 increased in patients versus controls, the former being more pronounced in advanced and multi-treated disease, and in patients with unmutated immunoglobulin heavy chain variable (IgVH) region and unfavorable cytogenetics. Patients with reduced TLR4 had an increased risk of disease progression and development of autoimmune complications. No relationship was found between reduced TLR4 expression and infectious episodes, which were observed in advanced stages and treated patients. These findings suggest that impaired innate immunity identifies patients with B-CLL with a poor prognosis and reduced ability to silence autoreactive phenomena.

Detection of erythroblast antibodies in mitogen-stimulated bone marrow cultures from patients with myelodysplastic syndromes

Low‐risk myelodysplastic syndromes (MDS) show several immunologic abnormalities, including increased frequency of autoimmune manifestations and/or overt autoimmune diseases, whose prognostic significance still remains controversial.

Detection of red blood cell antibodies in mitogen-stimulated cultures from patients with hereditary spherocytosis.

Hereditary spherocytosis (HS) is a congenital hemolytic anemia caused by defects in red blood cell (RBC) membrane proteins leading to premature RBC clearance in the spleen. The presence of RBC autoantibodies has never been extensively investigated in HS.

Hematological, molecular and cytokine changes after reduced intensity bone marrow transplantation for paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder of one or several hematopoietic stem cells (HSCs), characterised by intravascular haemolysis, increased risk of venous thrombotic events, recurrent infections and bone marrow failure and may be complicated by myelodysplastic, aplastic and leukaemic conversion. PNH phenotype is due to the abnormality of the glycosylphosphatidylinositol (GPI) biosynthetic pathway, caused at the molecular level by somatic mutations in the X-linked PIG-A gene. This results in the absence or decreased surface expression of GPI-anchored proteins (GPI-AP), as assessed by cytofluorimetric determination of CD55/ CD59-negative cells [1]. There is convincing evidence that the non-malignant expansion of GPInegative HSCs occurs as a consequence of a selective growing advantage due to an underlying bone marrow failure. It has been suggested that autoreactive cytotoxic T cells cause the selective destruction of normal HSCs in PNH, whereas HSCs with a PIG-A mutation and consequently with a GPInegative phenotype can escape T-cell-mediated damage, thus being able to survive and expand [2,3]. In line with this hypothesis, the expansion of GPI-negative lymphocytes has been demonstrated by ourselves and other groups after campath-1H therapy [4,5]. Moreover, the existence of a T-cell receptor Vbeta-chain skewing [6] together with the resistance of GPI-negative clones to the inhibitory effect of IFN-g and TNF-a [7] is consistent with a T-cell mediated process leading to suppression of hematopoietic function in PNH. The therapy of PNH includes supportive care, prevention of complications and, in severe cases, bone marrow transplantation (BMT). The use of reduced intensity, nonmyeloablative conditioning regimens (RIC) allowed exploitation of the graft-versus-leukemia effect without the toxicity of myeloablative therapy. In addition RIC provides the opportunity to investigate the role of the immune system on the growth of the PNH clone [8,9]. Here we describe the cytokine profile, bone marrow precursor activity and the mutational status of PIG-A gene in a PNH patient before and after RIC-BMT. Bone marrow (BM) aspirates were collected from the patient and controls during diagnostic procedures after obtaining informed consent and approval from the institutional Human Research Committee, in accordance with the Helsinki Declaration of 1975. The control group consisted of 21 subjects with miscellaneous non-malignant hematological conditions. BM mononuclear cells (BMMC) from the PNH patient and controls were isolated on density gradient and plated (10 cells) on methylcellulose medium, MethoCult GF-H4434 (Stemcell Technologies, Vancouver, BC, Canada). Burst-forming unit-erythroid (BFU-E) and colony-forming unit granulocyte-macrophage (CFU-GM) were counted