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Cytogenetic and molecular investigations of Acute Lymphoblastic Leukaemia (ALL) have identified the existence of distinct clinical subgroups. Molecular monitoring of clonal Immunoglobulin and T cell receptor (IG/TR) gene rearrangements has become an important tool in stratification of therapy of ALL. In order to determine whether certain features of the patient-specific rearrangements could hold further prognostic clues or provide information on the cell of origin of ALL, a comprehensive analysis of structural and biological features (V gene usage, coding frame and mutational status and complementarity-determining region -III length) of 473 IG/TR rearrangements identified in 229 adults with ALL was carried out. Distinct variable-gene usage profiles were identified between ALL subgroups, particularly for patients positive for BCR-ABL1 compared to MLL-AFF1 positive leukaemias; suggesting that the former is derived from a more mature B progenitor. Interestingly, occurrence of TRGV1- TRGV8 was prognostic for better event-free survival (31% at 4 years with vs. 0% at 4 years without, P = 0·05). The heterogeneity in clinical outcome is suggested by the basic molecular processes of antigen receptor gene rearrangements as shown in this work. [ABSTRACT FROM AUTHOR]

CD52 and CD20 antigens are important therapeutic targets for the monoclonal antibodies (mAbs) alemtuzumab and rituximab respectively. Circulating CD52 (cCD52) and CD20 (cCD20) have prognostic utility in lymphoid malignancies. The efficacy of mAb therapy in patients with chronic lymphocytic leukaemia (CLL) may be adversely affected by cCD52 or cCD20. In this report, blood and bone marrow (BM) cCD52 and cCD20 were measured at response assessment in previously treated ( N = 235) patients with CLL who received fludarabine, cyclophosphamide, and rituximab (FCR). Univariate and multivariate statistical models evaluated correlations of pre- and response variables with progression-free (PFS) and overall survival (OS). Response variables included 1996 National Cancer Institute-Working Group (NCI-WG) response, polymerase chain reaction (PCR) for immunoglobulin heavy chain ( IGHV) in BM, and cCD52 and cCD20 levels (blood and BM) at response assessment. Using multivariate analysis, response blood and BM cCD52, blood cCD20, and NCI-WG response were significant independent predictors of PFS. At the time of response assessment, BM cCD52 correlated with OS in univariate analysis. cCD52 and cCD20, therefore appear useful in predicting survival and may be important for monitoring patients following salvage FCR (fludarabine, cyclophosphamide, rituximab) therapy. These data further indicate that plasma may be a good target to evaluate for minimal residual disease using cCD52/cCD20 levels. [ABSTRACT FROM AUTHOR]

Kawasaki syndrome (KS) is an acute febrile vasculitis of childhood. Coronary artery abnormalities (CAA) are a significant problem in KS patients. High dose intravenous immunoglobulin (IVIG) is effective for reducing the occurrence of CAA. Clinical and histopathological findings suggest that vascular endothelial growth factor (VEGF) is involved in CAA. In circulating blood, newly activated platelets are the major source of VEGF, which is released in large amounts in vascular inflammation. The present study analysed 80 KS patients (69 IVIG responders and 11 IVIG non-responders) and evaluated the role of platelet VEGF in KS vasculitis. Serum VEGF and platelet VEGF levels were significantly higher in KS patients than controls ( P < 0·001). Platelet VEGF reflected the reactivity of IVIG treatment and was decreased in responders ( P < 0·001), but remained increased in non-responders ( P = 0·01). Platelet VEGF levels, but not serum VEGF levels, before IVIG were significantly correlated with the maximum CAA z-score ( r = 0·524, P = 0·02). Our findings demonstrate that platelet VEGF may reflect the severity of vasculitis related to the pathological development of CAA in KS. Platelet VEGF may be an important feature of KS pathophysiology. [ABSTRACT FROM AUTHOR]

Umbilical cord blood transplantation (CBT) is widely accepted, but one critical issue for adult patients is a low engraftment rate, of which one cause is haemophagocytic syndrome (HPS). We aimed to identify the contribution of HPS to engraftment failure after CBT, following preparative regimens containing fludarabine phosphate, in 119 patients (median age, 55 years; range; 17–69 years) with haematological diseases. Graft- versus-host disease prophylaxis comprised continuous infusion of a calcineurin inhibitor with or without mycophenolate mofetil. Of the 119 patients, 20 developed HPS within a median of 15 d (cumulative incidence; 16·8%) and 17 of them did so before engraftment. Donor-dominant chimaerism was confirmed in 16 of 18 evaluable patients with HPS. Despite aggressive interventions including corticosteroid, ciclosporin, high-dose immunoglobulin and/or etoposide, engraftment failed in 14 of 18 patients. Of these 14 patients, four received second rescue transplantation and all resulted in successful engraftment. Overall survival rates significantly differed between patients with and without HPS (15·0% vs. 35·4%; P < 0·01). Univariate and multivariate analysis identified having fewer infused CD34+ cells as a significant risk factor for the development of HPS ( P = 0·01 and 0·006, respectively). We concluded that engraftment failure closely correlated with HPS in our cohort, which negatively impacted overall survival after CBT. [ABSTRACT FROM AUTHOR]

The article presents a guideline to provide healthcare professionals with clear guidance for the effective clinical investigation of patients with newly detected M-proteins. It offers them the practical management of patients with monoclonal gammopathy of undetermined significance (MGUS). This article also discusses the background of MGUS and provides information on M-proteins.

SGN-30, a chimeric anti-CD30 monoclonal antibody, has demonstrated potent preclinical antitumour activity in both Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL). We conducted an open-label, Phase II study to determine the safety and objective response rate of SGN-30 in 79 patients with refractory/recurrent HL ( n = 38) or systemic ALCL ( n = 41). Each course of SGN-30 comprised 6 weekly intravenous infusions, followed by a 2-week treatment-free period. Patients had received a median of 3 (range 1–5) prior regimens of chemotherapy or systemic therapy. The initial 40 patients received 6 mg/kg weekly; the latter 39 patients received 12 mg/kg weekly. In the ALCL group, two patients achieved a complete response and five additional patients achieved a partial response, with response durations ranging from 27 to 1460+ d. No objective responses were observed in the HL group; however, 11 patients (29%) had stable disease (duration 62–242 days). Although adverse events were common, most were mild or moderate, and no specific pattern of adverse events was observed in either disease group. These results demonstrate that weekly administration of SGN-30 is safe, with modest clinical activity in patients with ALCL. [ABSTRACT FROM AUTHOR]

In children, one-third of immune thrombocytopenic purpura (ITP) patients follow a chronic course. The present study investigated whether treatment with intravenous immunoglobulin (IVIG) at the time of diagnosis of ITP is of prognostic significance, using data from 1984 children entered in Registry I of the Intercontinental Cooperative ITP Study Group. A matched pairs analysis compared children with thrombocytopenia (platelet count <150 × 109/l) 6 months following diagnosis with children whose platelet count was normal 6 months after diagnosis. It was found that children initially treated with IVIG were more likely to have a normal platelet count 6 months after diagnosis than children not receiving IVIG (odds ratio 1·81; 95% confidence interval: 1·25–2·64). This result was independent of age, gender, country of origin, platelet count at diagnosis or infection preceding the diagnosis of ITP. In a similar analysis, comparing children with a platelet count <50 × 109/l 6 months after diagnosis with children whose platelet count was ≥50 × 109/l at that time point, the former group was less often treated with IVIG than with steroids ( P = 0·02). Prospective studies are required to further explore this potential effect of IVIG. [ABSTRACT FROM AUTHOR]

Immune thrombocytopenic purpura (ITP) is characterized by decreased platelet numbers secondary to platelet destruction and reduced platelet production. Even if the ITP persists, it typically responds to ‘ITP-specific’ therapies, such as intravenous immunoglobulin, steroids, intravenous anti-D, and splenectomy. Several reports, including our previous study, have implicated cytomegalovirus (CMV) in the pathogenesis of infrequent cases of ITP that were not severe in nature. A recent study from China suggested that CMV is the aetiology of some cases of acute ITP of childhood and may require different treatment. We report two adult and two paediatric patients with refractory, severe, symptomatic thrombocytopenia, who were diagnosed with ITP and found to have active CMV infection. Their presentations included fever, transaminitis, neutropenia, and atypical lymphocytosis, but in particular, treatment-refractory, severe ITP. Treatment with steroids appeared to worsen the CMV-ITP. All four cases showed improvement in platelet counts within two weeks of starting ganciclovir and cytogam and tapering steroids. Based on the four patients described here, we believe that, in certain cases, CMV infection will result in symptomatic, severe, refractory ITP, which may be indistinguishable from typical ITP. Eradication of CMV with antiviral therapy improved the ITP in these cases. [ABSTRACT FROM AUTHOR]

The common variable immunodeficiency disorders are a mixed group of heterogeneous conditions linked by lack of immunoglobulin production and primary antibody failure. This variability results in difficulty in making coherent sense of either immunopathogenesis or the role of various genetic abnormalities reported in the literature. The recent attempt to collate the varied complications in these conditions and to define particular clinical phenotypes has improved our understanding of these diseases. Once refined and confirmed by other studies, these definitions will facilitate improved accuracy of prognosis and better management of clinical complication. They may also provide a method of analysing outcomes as related to new immunopathological and genetic findings. [ABSTRACT FROM AUTHOR]

Antibody-based therapies, such as rituximab and alemtuzumab, have contributed significantly to the treatment of Chronic Lymphocytic leukaemia (CLL). The CD40 antigen is expressed predominantly on B-cells and represents a potential target for immune-based therapies. SGN-40 is a humanized IgG1 monoclonal antibody currently in Phase I/II clinical trials for indolent lymphomas, diffuse large B cell lymphomas and Multiple Myeloma. Its biological effect on CLL cells has not been studied. The present study demonstrated that SGN-40 mediated modest apoptosis in a subset of patients with secondary cross-linking but did not mediate complement-dependent cytotoxicity. SGN-40 also mediated antibody-dependent cellular cytotoxicity (ADCC) predominantly through natural killer (NK) cells. Previous studies by our group and others have demonstrated that lenalidomide upregulates CD40 expression on primary B CLL cells and activates NK-cells. We therefore examined for the combinatorial effect of lenalidomide and SGN-40 and demonstrated that both enhanced direct apoptosis and ADCC against primary CLL B cells. These data together provide justification for clinical trials of SGN-40 and lenalidomide in combination for CLL therapy. [ABSTRACT FROM AUTHOR]

Immunoglobulin light chain amyloidosis (AL) and non-amyloid light chain deposition disease (NALCDD) are different forms of protein aggregation disorders accompanied by a monoclonal gammopathy. Monoclonal free light chains (FLCs) are precursors of the pathological light chain tissue deposits that are fibrillar in AL and granular in NALCDD. However, direct biochemical examination of plasma FLC precursors, which would allow comparison and better understanding of these two diseases, is still lacking. In this study, we examined FLCs in plasma of patients with AL and NALCDD by employing separation on Sep-PaK C18 cartridges, micro-preparative electrophoresis, Western blotting and mass spectrometry. Comparative analysis of AL versus NALCDD and control plasma samples showed new evidence of increased level and heterogeneity of circulating disulfide-bound FLC species in AL. In addition to full length monomers comprising the disulfide-linked FLCs, the monoclonal disulfide-bound FLC fragments were typically revealed in AL plasma. We hypothesized that enhanced disulfide binding of FLCs in AL interferes with their normal clearance and metabolism, which in turn might play a role in amyloid formation. The applied methods might be useful to diagnose or predict the pathological form of the disease and shed light on the mechanisms involved in light chain aggregation in tissues. [ABSTRACT FROM AUTHOR]

A fraction of chronic lymphocytic leukaemia (CLL) cases carry highly homologous B-cell receptors (BCR), i.e. characterized by non-random combinations of immunoglobulin heavy-chain variable ( IGHV) genes and heavy-chain complementarity determining region-3 (HCDR3), often associated with a restricted selection of IGVK/L light chains. Such ‘stereotyped’ BCR occur more frequently in CLL with unmutated (UM) than mutated (M) IGHV genes. We analysed 1426 IG rearrangements (from 1398 CLL cases) by a clustering driven by HCDR3 similarities. Molecular findings were correlated to time-to-treatment (TTT) and presence of known prognosticators. Sixty-nine clusters (319 IG-rearrangements, 22·4%) with stereotyped BCR were identified. Among 30 confirmed clusters (≥3 IG-rearrangements/cluster), we found 14 novel clusters, of which 11 had M IG rearrangements (M clusters) and predominantly (8/11) used IGHV3 subgroup genes. Recurrent cluster-biased amino acid changes were found throughout IGHV sequences of these ‘M clusters’. Regarding clinical outcome: (i) UM CLL from the IGHV1-2/1-3/1-18/1-46/7-4-1/IGKV1-39 cluster had poorer prognosis than UM/M cases, or UM cases using the same IGHV genes but not in clusters; (ii) M CLL from the IGHV3-21/IGLV3-21 cluster had TTT similar to UM CLL, and shorter than M CLL expressing IGHV3-21 but not in cluster. Altogether, our analysis identified additional molecular and clinical features for CLL expressing stereotyped BCR. [ABSTRACT FROM AUTHOR]

The article focuses on a research related to association of Dutcher bodies in multiple myeloma with waldenstrom's macroglobulinemia. Topics discussed include invagination of immunoglobulin inclusions or Dutcher bodies into the nucleus, an indicated relationship between cytogenetics and morphology due to presence of Dutcher bodies, and correlation of immature morphologies with high tumour mass.

Killer immunoglobulin-like receptor (KIR)-ligand mismatched natural killer (NK) cells play a key role in achieving durable remission after haplo-identical transplantation for acute myeloid leukaemia. We investigated the feasibility of transfusing haplo-identical, T-cell depleted, KIR-ligand mismatched NK cells, after conditioning therapy with melphalan and fludarabine, to patients with advanced multiple myeloma (MM) followed by delayed rescue with autologous stem cells. No graft-versus-host disease or failure of autologous stem cells to engraft was observed. There was significant variation in the number of allo-reactive NK cells transfused. However, all NK products containing allo-reactive NK cells killed the NK cell target K562, the MM cell line U266, and recipient MM cells when available. Post NK cell infusion there was a rise in endogenous interleukin-15 accompanied by increasing donor chimaerism. Donor chimaerism was eventually lost, which correlated with the emergence of potent host anti-donor responses indicating that the immunosuppressive properties of the conditioning regimen require further optimization. Further, blocking of inhibitory KIR-ligands with anti-human leucocyte antigen antibody substantially enhanced killing of MM cells thus highlighting the potential for modulating NK/MM cell interaction. Encouragingly, 50% of patients achieved (near) complete remission. These data set the stage for future studies of KIR-ligand mismatched NK cell therapy in the autologous setting. [ABSTRACT FROM AUTHOR]

The M-protein is the major reference measure for response in multiple myeloma (MM) and its correct interpretation is key to clinical management. The emergence of oligoclonal banding is recognized as a benign finding in the postautologous stem cell transplantation setting (ASCT) for MM but its significance during non-myeloablative therapy is unknown. In a study of the immunomodulatory combination BiRD, (lenalidomide and dexamethasone with clarithromycin), we frequently detected the emergence of mono- and oligo-clonal immunoglobulins unrelated to the baseline diagnostic M-protein. The new M-proteins seen on serum immunofixation electrophoresis were clearly different in either heavy or light chain component(s) from the original M-spike protein and were termed atypical serum immunofixation patterns (ASIPs). Overall, 24/72 (33%) patients treated with BiRD developed ASIPs. Patients who developed ASIPs compared with patients treated with BiRD without ASIPs, had a significantly greater overall response (100% vs. 85%) and complete response rates (71% vs. 23%). ASIPs were not associated with new clonal plasma cells or other lymphoproliferative processes, and molecular remissions were documented. This is the first time this phenomenon has been seen with regularity in non-myeloablative therapy for MM. Analogous to the ASCT experience, ASIPs do not signal incipient disease progression, but rather herald robust response. [ABSTRACT FROM AUTHOR]

The eradication of inhibitory antibodies in patients with haemophilia A can be accomplished by frequent administration of high or intermediate doses of factor VIII (FVIII), so-called immune tolerance induction (ITI). This study monitored the distribution of IgG subclasses of anti-FVIII antibodies during ITI. FVIII-specific antibodies of subclass IgG1 were detected in all inhibitor patients tested, anti-FVIII IgG4 in 16, IgG2 in 10 and IgG3 in one of 20 patients analysed. Levels of anti-FVIII IgG1 and IgG4 correlated well with inhibitor titres as measured by Bethesda assay. In low-titre inhibitor patients, anti-FVIII antibodies consisted primarily of subclass IgG1 whereas, anti-FVIII antibodies of subclass IgG4 were more prominent in patients with high titre inhibitors who needed prolonged treatment or who failed ITI. Longitudinal analysis of 14 patients undergoing ITI revealed that the relative contribution of IgG subclasses was constant for most of the patients analysed. In two patients, the relative contribution of IgG4 increased during ITI. Overall, our findings document the distribution and dynamics of anti-FVIII IgG subclasses during ITI. Future studies will need to address whether monitoring the relative contribution of anti-FVIII subclasses IgG1 and IgG4 may be useful for the identification of patients who are at risk of failing ITI. [ABSTRACT FROM AUTHOR]

The identification of galectin-9 as a ligand for T-cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3), expressed on T-helper type-1 (Th1) cells, has established the Tim-3-galectin-9 pathway as a regulator of Th1 immunity. Whereas there is compelling evidence for the effects of galectin-9 on T-cell fate, limited information is available on the impact of galectin-9 on B lymphocytes. We found that protease-resistant galectin-9, hG9NC(null), but not galectin-1 or -8, prevented cell growth of malignant B cells, such as Burkitt lymphoma (BL) and Hodgkin lymphoma (HL). β-galactoside binding was essential for galectin-9-induced cell growth suppression. hG9NC(null) induced cell cycle arrest by reducing the expression of cyclin D1, D2, B1, Cdk4, Cdc25C and c-Myc, and apoptosis by reducing the expression of XIAP, c-IAP2 and survivin. Most of the genes that encode these proteins are regulated by nuclear factor-κB (NF-κB), and constitutive activation of NF-κΒ is a common characteristic of both types of malignancies. hG9NC(null) inhibited IκBα phosphorylation, resulting in suppression of NF-κB. AP-1 has also been implicated in the control of cell survival. hG9NC(null) inhibited the expression of JunD, resulting in the suppression of AP-1. Our results suggest that hG9NC(null) is a potentially suitable agent for the management of BL and HL. [ABSTRACT FROM AUTHOR]

Previous studies demonstrated that 20% of haemoglobin is lost from circulating erythrocytes during their total lifespan by vesiculation. To study whether removal molecules other than membrane-bound haemoglobin were present in erythrocyte-derived vesicles, flow cytometry and immunoblot analysis were employed to examine the presence of phosphatidylserine (PS) and IgG, and senescent cell antigens respectively. It was demonstrated that 67% of glycophorin A-positive vesicles exposed PS, and that half of these vesicles also contained IgG. Immunoblot analysis revealed the presence of a breakdown product of band 3 that reacted with antibodies directed against senescent erythrocyte antigen-associated band 3 sequences. In contrast, only the oldest erythrocytes contained senescent cell antigens and IgG, and only 0·1% of erythrocytes, of all ages, exposed PS. It was concluded that vesiculation constitutes a mechanism for the removal of erythrocyte membrane patches containing removal molecules, thereby postponing the untimely elimination of otherwise healthy erythrocytes. Consequently, these same removal molecules mediate the rapid removal of erythrocyte-derived vesicles from the circulation. [ABSTRACT FROM AUTHOR]

Autoantibodies to ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I motif, member 13) play an important role in the development of microthrombosis in thrombotic thrombocytopenic purpura (TTP). In severe cases of antiphospholipid syndrome (APS), microthrombosis can occur similar to that seen in TTP, suggesting possible mutual pathogenic factors. However, the role of ADAMTS13 in APS is unknown. We hypothesised that aberrations in ADAMTS13 may occur in APS and evaluated ADAMTS13 and von Willebrand factor (VWF) in 68 patients with antiphospholipid antibodies (aPA) including 52 with APS. Thirty-three (49%) had IgG anti-ADAMTS13 with 12 of these patients having reduced ADAMTS13 activity, suggesting neutralising antibodies. Low ADAMTS13 activity (median 34%) was demonstrated in 22/68 (33%), all with normal ADAMTS13 antigen levels consistent with dysfunctional ADAMTS13. Reduced ADAMTS13 activity was not secondary to elevated von Willebrand factor (VWF), or increased VWF secretion (normal VWF propeptide), although a reduced VWF clearance was noted in APS. Analysis found no associations between the ADAMTS13 abnormalities and any aPA profile or thrombotic/obstetric complications, although this study was not adequately powered to address clinical associations. Nevertheless, these findings highlight that ADAMTS13 autoantibodies and ADAMTS13 dysfunction can occur in APS, and although the clinical significance remains undetermined, ADAMTS13 dysfunction may be contributory to thrombogenesis in autoimmune conditions other than TTP. [ABSTRACT FROM AUTHOR]

We evaluated the dose requirements for sustained in vivo activity of ofatumumab, a human anti-CD20 antibody under development for the treatment of B cell-mediated diseases. In a mouse xenograft model, a single dose, resulting in an initial plasma antibody concentration of 5 μg/ml, which was expected to result in full target saturation, effectively inhibited human B-cell tumour development. Tumour growth resumed when plasma concentrations dropped below levels that are expected to result in half-maximal saturation. Notably, tumour load significantly impacted antibody pharmacokinetics. In monkeys, initial depletion of circulating and tissue residing B cells required relatively high-dose levels. Re-population of B-cell compartments, however, only became detectable when ofatumumab levels dropped below 10 μg/ml. We conclude that, once saturation of CD20 throughout the body has been reached by high initial dosing, plasma concentrations that maintain target saturation on circulating cells (5–10 μg/ml) are probably sufficient for sustained biological activity. These observations may provide a rationale for establishing dosing schedules for maintenance immunotherapy following initial depletion of CD20 positive (tumour) cells. [ABSTRACT FROM AUTHOR]

Platelet protein disulphide isomerase (PDI) has a role in platelet aggregation, probably targeting a thiol-containing platelet surface protein. The thiol-containing P2Y12 ADP receptor is involved in aggregation induced by most agonists and may be the target of PDI. By excluding the P2Y12 pathway and using the anti-PDI antibody RL90 this study showed that PDI targets a non-P2Y12 thiol-protein in aggregation. Anti-PDI inhibited signalling-independent activation of the thiol-containing fibrinogen receptor αIIbβ3 by Mn2+, suggesting that PDI directly interacts with αIIbβ3. The thiol-containing form of PDI increased on the platelet surface with platelet activation, suggesting that active PDI readily becomes available for redox regulation of αIIbβ3. Finally, using purified proteins PDI had greater ability to isomerize disulphide bonds than the αIIbβ3 integrin, which also has PDI-like activity. In summary, a mechanism exists in platelets to increase the functional form of surface PDI and this PDI has a non-P2Y12 target that may be αIIbβ3. [ABSTRACT FROM AUTHOR]

CD19 and CD21 (CR2) are co-receptors found on B-cells and various B-cell lymphomas, including non-Hodgkin lymphoma. To evaluate their suitability as targets for therapy of such lymphomas using internalization-dependent antibody-drug conjugates [such as antibody-4-( N-maleimidomethyl)cyclohexane-1-carboxylate, ( N2′-deacetyl- N2′-(3-mercapto-1-oxopropyl)-maytansine) (MCC-DM1) conjugates, which require lysosomal degradation of the antibody moiety for efficacy], we examined uptake of antibodies to CD19 and CD21 in a panel of B-cell lines. Anti-CD21 antibodies were not sufficiently internalized even in the highest CD21-expressing Raji cells, resulting in lack of efficacy with anti-CD21-MCC-DM1 conjugates. Anti-CD19 antibody uptake was variable, and was unexpectedly negatively correlated with CD21 expression. Thus, high CD21-expressing Raji, ARH77 and primary B-cells only very slowly internalized anti-CD19 antibodies, while CD21-negative or low expressing cells, including Ramos and Daudi, rapidly internalized these antibodies in clathrin-coated vesicles followed by lysosomal delivery. Anti-CD19-MCC-DM1 caused greater cytotoxicity in the faster anti-CD19-internalizing cell lines, implying that the rate of lysosomal delivery and subsequent drug release is important. Furthermore, transfection of Ramos cells with CD21 impeded anti-CD19 uptake and decreased anti-CD19-MCC-DM1 efficacy, suggesting that CD21-negative tumours should respond better to such anti-CD19 conjugates. This may have possible clinical implications, as anti-CD21 immunohistochemistry revealed only approximately 30% of 54 diffuse large B-cell lymphoma patients lack CD21 expression. [ABSTRACT FROM AUTHOR]

Monoclonal B-cell populations have been detected in the peripheral blood of apparently healthy individuals by flow cytometry. In 2005, the term monoclonal B-cell lymphocytosis (MBL) was proposed to describe these findings. MBL may be immunophenotypically similar to chronic lymphocytic leukaemia (CLL) and, like CLL, the prevalence is higher in males and older individuals. We studied the prevalence of MBL in blood donors from the Midwestern United States. Samples from 5141 donors were examined and seven (0·14%) were found to have immunophenotypic characteristics of MBL or CLL. Immunoglobulin heavy chain analysis yielded monoclonality or oligoclonality. Prior and subsequent to the study, an additional undetermined number of blood donors were screened and seven of these expressed immunophenotypic characteristics of MBL or CLL. We thus found a total of 14 healthy blood donors with monoclonal expansions of B-lymphocyte populations. Of these, 12 were presumptively classified as MBL and two as CLL. All but two of the donors were male; the mean age was 59 years. The clinical importance of these findings with regard to transfusion medicine has not been established. [ABSTRACT FROM AUTHOR]

Haemolytic disease of the newborn (HDN) can be prevented by the passive administration of anti-D to the mother. The most accepted theory to describe this activity of anti-D is based upon its ability to clear opsonized erythrocytes before their recognition by the maternal immune system. We examined this hypothesis using a murine model of immunity to foreign erythrocytes. Whereas transfusion of foreign erythrocytes into mice induced immunoglobulin (Ig)M and IgG antibodies specific for the erythrocytes, these humoral immune responses were inhibited when the erythrocytes were opsonized with IgG. To specifically determine if immunological evasion occurs with these opsonized erythrocytes, we examined T-cell responses from these mice. An erythrocyte-specific T-cell response was clearly detected. We then tested whether phagocytosis of opsonized erythrocytes is sufficient to prevent the antibody response. We exposed mononuclear phagocytic cells to sheep red blood cells (SRBC) in vitro and then adoptively transferred the phagocytic cells to recipient mice; opsonized SRBC unexpectedly increased, rather than decreased, the antibody response. These data indicate that removal of opsonized erythrocytes by phagocytic cells does not prevent their immunological recognition and suggest that antigen clearance may not be the predominant mechanism of anti-erythrocyte action in downregulating the humoral immune response. [ABSTRACT FROM AUTHOR]

Idiopathic CD4+ T lymphocytopenia (ICL) is defined as a CD4+ T-cell count <0·3 × 109/l or <20% of the total T-cell count on two occasions in the absence of any immunodeficiency disorder or therapy associated with reduced CD4+ T-cell count. Although several mechanisms of ICL have been reported, the pathophysiology is still largely unknown. This case report describes a patient who presented with cryptococcal meningitis and was subsequently discovered to meet the criteria for ICL. Flow cytometric analysis of the patient's peripheral blood mononuclear cells revealed antibodies coating a much larger proportion of his CD4+ T cells (33·61%) than the CD4+ T cells of normal donors (3·94 ± 1·77%). The reasons behind the development of these autoantibodies are explored. [ABSTRACT FROM AUTHOR]

Current knowledge of the haematological and onco-haematological complications of type 1 Gaucher disease has been reviewed with the aim of identifying best clinical practice for treatment and disease management. It was concluded that: (i) Awareness of typical patterns of cytopenia can help clinicians distinguish haematological co-morbidities. (ii) Red blood cell studies and complete iron metabolism evaluation at baseline are recommended. (iii) Haemoglobin levels defining anaemia should be raised and used in Gaucher disease treatment and monitoring. (iv) Surgeons should be aware of potential bleeding complications during surgery in Gaucher patients. The higher incidence of multiple myeloma in Gaucher disease suggests that Gaucher patients should have their immunoglobulin profile determined at diagnosis and monitored every 2 years (patients <50 years) or every year (patients >50 years). If monoclonal gammopathy of undetermined significance (MGUS) is found, general MGUS guidelines should be followed. Future studies should focus on the utility of early treatment to prevent immunoglobulin abnormalities and multiple myeloma. [ABSTRACT FROM AUTHOR]

The t(14;18)(q32;q21) is the characteristic chromosomal translocation of follicular lymphoma (FL). Highly sensitive polymerase chain reaction (PCR) techniques can also detect t(14;18)-sequences in the blood and lymphoid tissues of healthy individuals (HI). The aim of this study was to determine the immunophenotypic markers of t(14;18)-positive cells in HI and to relate these features to lymphocyte maturation. B cells from 10 subjects with t(14;18)-positive and three subjects with t(14;18)-negative peripheral blood mononuclear cells (PBMC) were fluorescence-activated cell sorted for antigen-naïve (CD27−), immunoglobulin M (IgM) memory (IgM+CD27+) and switched memory (IgM− CD27+) cells. t(14;18)-recombinations were detected by quantitative PCR. Among PBMC-positive subjects, t(14;18)-frequency was significantly higher in IgM memory (median: 380/106) than in antigen-naïve (median: 16/106) or switched memory (median: 5/106) B cells. All PBMC-negative subjects nevertheless had detectable t(14;18) in sorted B cells; levels were lower than in PBMC-positive subjects, but had the same relative predominance. These results suggest that t(14;18) is generated during early B-cell development in the bone marrow and that affected cells may mature and expand in germinal centres. t(14;18)-frequency was highest in IgM memory cells, a B-cell subset that shares immunophenotypic similarities with FL. The significance of these cells as lymphoma precursors or indicators of lymphoma risk remains to be established. [ABSTRACT FROM AUTHOR]

Human intravenous immunoglobulin (IVIg) and anti-D immunoglobulin preparations are used in the treatment of immune thrombocytopenic purpura (ITP). One mechanism proposed to explain their therapeutic effects in ITP patients is the induction of expression of anti-inflammatory cytokines, such as interleukin (IL)-10 or IL-1ra, leading to a reduction of phagocytic activity of the reticuloendothelial system. However, increased expression of pro-inflammatory cytokines was also noted following treatment of ITP patients, raising doubt on the actual contribution of anti-inflammatory cytokines in the therapeutic effects of IVIg and anti-D immunoglobulins. The present study evaluated the in vivo modulation of expression of a large array of inflammatory cytokines using a mouse model of thrombocytopenia. IVIg was not found to modulate cytokine expression although it efficiently prevented thrombocytopenia. In contrast, protective (M1/69) and non-protective (TER-119) anti-mouse red blood cell (RBC) antibodies (mimicking anti-D treatment) both increased the expression of CXCL-1 and CXCL-5. Thus, there was no relationship between inflammatory cytokine expression and prevention of thrombocytopenia by IVIg or anti-mouse RBC in the ITP mouse model. These results suggest that the increase in cytokine expression observed in ITP patients following IVIg or anti-D infusion is not required for their therapeutic effects but may rather represent a side-effect of the treatment. [ABSTRACT FROM AUTHOR]

Immunoglobulin light chain amyloidosis (AL) and non-amyloid light chain deposition disease (NALCDD) are different forms of protein aggregation disorders that may occur in plasma cell dyscrasias with dysproteinemia. In systemic AL, the deposits are fibrillar and patchy in distribution, within and amongst different organs, whereas in NALCDD, the deposits are granular and diffusely distributed in systemic basement membranes, suggesting different mechanisms of aggregation and deposition. Previous evidence, that charge differences between the light chains in AL and NALCDD might account for their different phenotypes, prompted the present study, which compared the isoelectric points (pIs) of AL and NALCDD protein deposits extracted from human tissues. The pI profiles (5·2–8·8) of polypeptides in AL deposits were heterogenous in four cases, with a spread of both anionic and cationic isoforms; in contrast, in three of NALCDD the pI profiles (8·2–8·8) were homogeneous and restricted in the cationic range. These in vivo findings in human disease, together with other reported in vitro and in vivo experimental data, suggest that the fibrillar deposits in AL may form by electrostatic interaction between oppositely charged polypeptides, whereas the granular deposits in NALCDD form by the binding of cationic polypeptides to anionic proteoglycans sites in basement membranes. [ABSTRACT FROM AUTHOR]

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder and plasma exchange (PEX) remains the primary treatment modality. Twenty-five patients with acute refractory/relapsing idiopathic TTP received rituximab in conjunction with PEX because of progressive clinical disease or deterioration in laboratory parameters, despite intensive standard therapy. In relapsing TTP, rituximab was started if antibody to ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin motif-13) was demonstrated during previous episodes. All 25 patients attained complete clinical and laboratory remission in a median of 11 d after initiating rituximab. In 21 cases, ADAMTS-13 activity was within the normal range following rituximab. Inhibitors were detected in 24/25 patients by mixing studies and/or immunoglobulin G (IgG) antibodies to ADAMTS-13 pre-rituximab. There was no evidence of inhibitors and/or IgG activity <10% in 23/25 patients following rituximab. In acute refractory cases, the median number of PEX pre-rituximab and following the first rituximab infusion was 13 and 9, respectively. There have been no infectious complications, despite low CD 19 levels and no relapses. In patients with acute refractory/relapsing idiopathic TTP, rituximab appears to be a safe, effective, targeted therapy with a significant reduction in the requirement for PEX. [ABSTRACT FROM AUTHOR]

After decades of stagnation, the prognosis of patients with follicular lymphomas (FL) has changed substantially within the last few years due to new and effective therapeutic modalities. These include myeloablative therapy followed by autologous stem cell transplantation (ASCT) in younger patients in first remission, which showed a significant prolongation of remission duration in three prospective randomised trials while the impact on overall survival still needs to be determined. Adding the anti-CD 20 antibody Rituximab to conventional chemotherapy resulted in a significant increase in remission rate, remission duration and, in two of four currently available prospective randomised studies, even in a longer overall survival. A prolongation of remission duration was also seen when Rituximab was given as maintenance after cytoreductive therapy including Rituximab. Radio-immunotherapy (RIT) with radioisotopes coupled to monoclonal antibodies produced encouraging data in several phase II studies. New therapeutic perspectives have also emerged from increasing insights into the biology of the disease that unravel molecular targets for novel agents, some of which have entered clinical evaluation already. [ABSTRACT FROM AUTHOR]

Fluorescence in situ hybridisation (FISH) is an effective technique for the cytogenetic analysis of Waldenström macroglobulinemia (WM), but the potential impact of molecular cytogenetics on disease evolution and as a prognostic marker is still unknown. Deletion of the long arm of chromosome 6 (6q—) is the most frequent cytogenetic abnormality in WM. This study analysed the prevalence of this aberration in 102 WM patients, and correlated it with disease characteristics. The incidence of 6q21 deletion was 7% by conventional cytogenetics and 34% when analysed by FISH (54% when cytoplasmic immunoglobulin M-FISH was used). Patients with deletion of 6q displayed features of adverse prognosis, such as higher levels of β2-microglobulin and monoclonal paraprotein and a greater tendency to display anaemia and hypoalbuminemia. Interestingly, there was a correlation between the presence of 6q deletion and the International Staging System prognostic index (incidence of 6q) among patients stratified in stages 1, 2 and 3 was 24%, 42% and 67% respectively). Those patients diagnosed with smouldering WM who displayed the abnormality showed a trend to an earlier requirement of treatment. Finally, the survival analysis did not show differences between the two groups of patients, probably due to the short follow up of our series. [ABSTRACT FROM AUTHOR]

The objective of this open label, phase 1–2, multicentre trial was to evaluate the safety of AMG 531, a novel thrombopoiesis-stimulating peptibody, and its effect on platelet counts in adults with immune thrombocytopenic purpura. Four patients were assigned to each of four unit-dose cohorts: 30, 100, 300 or 500 μg, administered subcutaneously on days 1 and 15 (or day 22 if the day 15 platelet count was >50 × 109/l). Safety was assessed by adverse event (AE) monitoring, clinical laboratory studies and antibody assays. Platelet response was defined as a platelet count double the baseline value and between 50 and 450 × 109/l. Sixteen patients (10 women) were enrolled. The 500- μg cohort was discontinued because the first patient's platelet count became unacceptably high. AEs were generally expected and mild or moderate; the most frequent was headache (eight of 16 patients). Two patients experienced serious AEs related to AMG 531 (severe headache and elevated serum lactic dehydrogenase; thrombocytopenia). Platelet responses occurred with all doses and with a dose equivalent to ≥1 μg/kg in eight of 11 patients. In summary, patients tolerated AMG 531 well at the doses tested. No anti-AMG or antithrombopoietin antibodies were detected. Doses equivalent to ≥1 μg/kg increased platelet counts. [ABSTRACT FROM AUTHOR]

The prothrombotic mechanisms associated with antiphospholipid antibodies remain incompletely defined. Antibody binding to endothelial cells in vitro is a feature of antiphospholipid antibody-positive sera. We hypothesised that impairment of endothelium-dependent fibrinolysis by antiphospholipid/anti-endothelial antibodies is a contributory factor in the pathogenesis of thrombosis. We also aimed to confirm the displacement of annexin-V from endothelial cells and enhanced fibrin formation. Binding of immunoglobulin (Ig) from antiphospholipid antibody-positive sera to endothelial cells was examined using a cell-based enzyme-linked immunosorbent assay. Effects on fibrin formation and lysis were examined on cultured endothelial cell monolayers. Plasminogen activator inhibitor-1 (PAI-1) was assayed in supernatants. We confirmed antibody binding to endothelial cells. With four of 14 antiphospholipid antibody-positive sera there was some prolongation of fibrin clot lysis time, consistent with impairment of endothelial fibrinolytic activity. Secretion of PAI-1 was significantly correlated with clot lysis time on endothelial cell monolayers incubated with antiphospholipid/anti-endothelial antibody-positive sera, but not with control sera. IgG from antiphospholipid antibody-positive sera had little effect on endothelial cell surface annexin-V expression. We conclude that impaired endothelial fibrinolysis is a potential prothrombotic mechanism in subjects with antiphospholipid antibodies. We were unable to confirm enhanced displacement of annexin-V from endothelium by antiphospholipid antibodies. [ABSTRACT FROM AUTHOR]

Intraclonal diversification of immunoglobulin (Ig) variable (V) genes was evaluated in leukaemic cells from a B-cell chronic lymphocytic leukaemia (B-CLL) case over a 2-year period at four time points. Intraclonal heterogeneity was analysed by sequencing 305 molecular clones derived from polymerase chain reaction amplification of B-CLL cell IgV heavy (H) and light (C) chain gene rearrangements. Sequences were compared with evaluating intraclonal variation and the nature of somatic mutations. Although IgV intraclonal variation was detected at all time points, its level decreased with time and a parallel emergence of two more represented VHDJH clones was observed. They differed by nine nucleotide substitutions one of which only caused a conservative replacement aminoacid change. In addition, one VLJL rearrangement became more represented over time. Analyses of somatic mutations suggest antigen selection and impairment of negative selection of neoplastic cells. In addition, a genealogical tree representing a model of clonal evolution of the neoplastic cells was created. It is of note that, during the period of study, the patient showed clinical progression of disease. We conclude that antigen stimulation and somatic hypermutation may participate in disease progression through the selection and expansion of neoplastic subclone(s). [ABSTRACT FROM AUTHOR]

Extranodal marginal zone B-cell lymphomas (EMZL) are thought to develop from reactive infiltrates that represent immune responses to external or auto-antigens. Except for gastric EMZL, the antigenic triggers of EMZL development are mostly unknown, although a subset of cutaneous EMZL have been associated with Borrelia burgdorferi infections. To further evaluate whether a common antigen may be promoting the development of cutaneous EMZL, the immunoglobulin heavy chain variable (VH) genes from eight USA cases were sequenced and analysed. All used VH3 family gene segments, with 2/8 using the same V3–30 segment, 2/8 using the closely related V3–30·3 or V3–33 segments, 6/8 containing mutations and 2/7 showing evidence of ongoing mutation. Many of the complimentarity-determining region 3s (CDR3s) also showed similarities in length and displayed conserved amino acid motifs in the non-templated areas between the diversity and joining segments. The use of similar VH gene segments and conserved CDR3 amino acid motifs suggests that some of these cutaneous EMZL may bind the same or similar antigen via their surface immunoglobulin receptor. Analysis of the somatic mutations present in many of the VH genes was also consistent with antigen directly stimulating the growth of cutaneous EMZL. [ABSTRACT FROM AUTHOR]

Lymphoma-derived immunoglobulin idiotype (Id) is a tumour-specific antigen used for antitumour vaccination in follicular lymphoma (FL). However, FL Ids are subject to hypermutation and subclones may escape antitumour cytotoxic T-cell response. To investigate the intraclonal epitope diversity, we sequenced the FL heavy chain gene (consensus Id gene) and subclones of 24 patients. The derived polypeptide sequences were analysed by bioinformatics for human leucocyte antigen (HLA)-A0201-restricted epitope prediction. Epitopes were classified according to BIMAS and SYFPEITHI scores. Surprisingly in these highly mutated polypeptides, the epitopes concentrated in short hotspots in the conserved framework regions (FRs), both in HLA-A0201+ and HLA-A0201− patients. Similar hotspots have been observed by others in chronic lymphocytic leukaemia Ids which in contrast to FL have low mutation frequency. FR3 amino acids 78–88 displayed the best-score epitopes in Ids containing a VH3-family segment, the most represented in FL Ids. Such VH3-FR378−88 epitopes were previously demonstrated as immunogenic. Modifications of the epitope pattern in subclones of HLA-A0201+ patients were generally absent from high-score peptides, including VH3-FR378−88 epitopes (83% unmodified). Therefore, no tendency for loss of HLA-A0201-restricted epitopes was evidenced and, given their limited intraclonal diversity, VH3-FR378−88 epitopes may provide a useful target for the induction of cytotoxic response in Id-vaccinated FL patients. [ABSTRACT FROM AUTHOR]

We investigated the feasibility, safety and efficacy of two B-cell purging methods in patients with CD20+ non-Hodgkin lymphoma (NHL) receiving autologous stsem cell transplantation. Myeloid and immune recoveries between the methods were compared. Twenty-seven patients were randomised to either in vivo purging with rituximab or ex vivo purging by CD34+ cell selection. Both purging methods were efficient at eliminating B-cells in infusates. When compared with in vivo purging, ex vivo purging was associated with CD34+ cell loss and delayed median neutrophil (10 d vs. 11 d) and platelet (12·5 d vs. 17 d) count recoveries. Lymphocyte recovery was similar in both groups, but immunoglobulin recovery was delayed after in vivo purging. Late-infectious complications were few in both arms. At a median follow-up of 27 months, 2-year probabilities of event-free survival (EFS) rates were 81% for in vivo purging and 76% for ex vivo purging ( P = 0·66). When compared with 53 unpurged patients, all 27 purged patients had improved 3-year probabilities of overall survival (89% vs. 70%, P = 0·014) and a trend for improved EFS (78% vs. 57%, P = 0·075). In conclusion, although both purging methods were feasible and safe, rituximab purging was superior as it did not impair CD34+ cell mobilisation and was associated with faster myeloid recovery. Further studies are needed to determine whether rituximab purging is more effective than the use of unpurged autografts. [ABSTRACT FROM AUTHOR]

In order to assess the prognostic value of inhibitory anti-ADAMTS13 antibodies in thrombotic thrombocytopenic purpura (TTP), we performed a multicentre prospective study of 33 adult patients with idiopathic acquired TTP. Patients were treated with high-dose plasma infusion and therapeutic plasma exchange. Patients without (group 1, n = 12) and with (group 2, n = 21) detectable inhibitory anti-ADAMTS13 antibodies were compared for clinical presentation, treatment and outcome. Both groups were comparable for clinical presentation. All patients in group 1 achieved a sustained complete remission within a median of 7 d [95% confidence interval (CI), 4–18], which required a median plasma volume of 235 ml/kg (range, 131–1251). In group 2, 17 patients achieved a durable complete remission within a median of 23 d (95% CI, 11–32) ( P = 0·001). Median plasma volume was 718 ml/kg (range, 219–3107) ( P = 0·02). In group 2, there was a trend for more episodes of flare-up than in group 1 (13 vs. 3, respectively, P = 0·07). Four patients, all from group 2, died ( P = not significant). The relapse rate was comparable between both groups. We suggest that TTP with detectable inhibitory anti-ADAMTS13 antibodies displays a worse prognosis, relative to a delayed platelet count recovery, a higher plasma volume requirement to achieve complete remission, and a trend for more frequent episodes of flare-up. [ABSTRACT FROM AUTHOR]

We have studied the surface expression of the Toll-like receptor family member CD180 on cells from 78 patients with B-chronic lymphocytic leukaemia (B-CLL). B-CLL cells had variable levels of CD180 expression, but this was always less than that expressed by normal blood B cells and was stable for 24 months. Significantly higher levels of CD180 were expressed by B-CLL cells with mutated IGVH genes compared with those using unmutated IGVH genes. This was in contrast to the higher levels of expression of surface immunoglobulin M by B-CLL cells using unmutated, rather than mutated IGVH genes. CD180 was functional on B-CLL cells from some of the patients, as shown by the increased expression of CD86 following incubation in vitro with anti-CD180. The differential expression of CD180 amongst B-CLL patients is one more marker that may define more precisely the different biological properties of this heterogeneous disease. [ABSTRACT FROM AUTHOR]

Infant acute lymphoblastic leukaemia (ALL) represents a rare but unique subset with poor prognosis. We analysed mixed-lineage leukaemia ( MLL) gene rearrangements and the sequences of complete and incomplete immunoglobulin heavy chain gene rearrangements ( IGH) in 14 infants (age ≤12 months at diagnosis) enrolled on Dana-Farber Cancer Institute ALL Consortium Protocol 95–01. The dynamics of the leukaemic clone were followed during the course of the disease by quantitative real-time polymerase chain reaction of IGH rearrangements. Sixteen sequences were obtained from 13 (93%) of these infants. There was marked over usage of the VH6.1 gene segment (64%) in infants compared with older children with ALL (8%), ( P < 0·001) and overusage of DH6 ( P = 0·004) and JH1 ( P = 0·004). Poor outcome was associated with MLL gene rearrangements rather than any specific VHDHJH gene usage patterns. Levels of minimal residual disease (MRD) at the end of induction appeared to be high in infants with ALL compared with older children, and although the number of infant cases studied was small, there were no differences in MRD levels after induction therapy in infant ALL with or without MLL gene rearrangements ( P = 0·41) and quantitative MRD assessment at the early time points may not be predictive of outcome. Novel treatment strategies are required to improve the outcome in this poor prognosis subset of children with ALL. [ABSTRACT FROM AUTHOR]

The development of neutralising antibodies to factor VIII (FVIII) is a major complication of haemophilia A (HA) therapy. We aimed to construct an individual risk profile for the development of inhibitors in HA and started by screening for the causative mutation in our HA patient population. A total of 109 patients and 28 carriers were screened. The analysis revealed 38 different mutations in the FVIII gene, of which 13 have not been described on the Haemophilia A Mutation, Search, Test and Resource Site (HAMSTeRS). Twenty-five mutations have been reported previously and all except two had a similar phenotype to what has been described. Three novel mutations were associated with severe HA: one non-missense mutation, a small insertion in the A2 domain, and two missense mutations, a H256R mutation in the A1 domain and a L2025P substitution in the C1 domain. One novel mutation, Y156C, was associated with moderate HA. Nine novel mutations caused mild HA. The P130R, D167E and V278M mutations are located in the A1 domain. R439C, Y511H, A544G and Q645H in the A2 domain, L1758F in the A3 domain and a S2157R mutation in the C1 domain. In conclusion, the genotypic profile of our HA population was not different from others described and is suitable to study inhibitor formation. [ABSTRACT FROM AUTHOR]

B-cell chronic lymphocytic leukaemia (B-CLL) cells display low amounts of surface immunoglobulins (sIg). To investigate the mechanisms underlying this phenomenon, we performed a thorough study of surface and intracellular expression of the B-cell receptor (BCR) components in B-CLL cells using flow cytometry. There was an heterogeneous pattern of expression. Overall, 20 of 22 samples showed reduced sIgM levels, compared with normal B cells. Among them, three (15%) had very low to undetectable intracellular IgM levels and variable amounts of CD79a and CD79b; nine (45%) had low intracellular CD79b levels but appreciable levels of IgM and CD79a; and eight (40%) had relatively normal intracellular levels of all BCR components. To investigate whether surface BCR levels could be controlled by the rate of CD79b synthesis, adenoviral vectors encoding CD79b were generated and used for gene transfer experiments. Delivery of CD79b to non-B cells transfected with IgM and CD79a lead to high-level expression of a functional BCR. Moreover, CD79b gene transfer in a B cell line derived from a B-CLL patient and characterised by low intracellular levels of endogenous CD79b consistently increased sIgM levels. These findings indicate that the phenotype of B-CLL cells in a subset of patients may depend primarily on poor CD79b expression, and suggest that upregulation of CD79b expression may correct the phenotype of these cells. [ABSTRACT FROM AUTHOR]

Several methods to detect anti-A/B antibodies based on haemagglutination and haemolysis have been described. These methods measure predominantly anti-A/B immunoglobulin (Ig)M, whereas anti-A/B IgG and IgG subclasses are less well examined. We established a flow cytometry method (ABO-fluorescence-activated cell sorting; ABO-FACS) to quantify binding of anti-A/B IgM, IgG and IgG subclasses to human A or B red blood cells. Anti-A/B IgM were present in the majority of 120 blood donors, as expected from blood group typing. The sensitivity and specificity of anti-A/B IgM to predict the blood group was 93% and 96% respectively. Anti-A/B IgG was found in 34/38 blood group O samples (89%). Anti-B IgG in blood group A or anti-A IgG in blood group B was present in 4/28 (14%) and 1/28 (4%) samples, respectively, and absent in 26 AB sera. IgG2 was the predominant IgG subclass. The correlation of anti-A/B IgM and IgG in the ABO-FACS with haemagglutination titres was 0·870 and 0·783, respectively ( n = 240; P < 0·001) whereas the comparison of ABO-FACS with ABO-enzyme-linked immunosorbent assay was less significant. In conclusion, ABO-FACS is a valid method to quantify anti-A/B IgM, IgG and IgG subclasses. It opens the possibility of isotype-specific monitoring of anti-A/B antibodies levels after ABO-incompatible solid organ and stem cell transplantation. [ABSTRACT FROM AUTHOR]

Adult T-cell leukaemia (ATL) is a peripheral T-cell neoplasm caused by human T-cell leukaemia virus type I (HTLV-I). Several clinical observations suggest that some tumour-associated antigens in ATL may be recognised by the immune system. In this study, we performed the serological screening of an expression library to identify ATL-associated antigens by using materials from a unique ATL patient with long-term stable disease. Among five distinct genes isolated, serine/arginine protein kinase 1 ( SRPK1), which has been reported to have a restricted normal tissue distribution, was found to be overexpressed in most acute type ATL samples, but not in chronic type ATL or in normal peripheral blood mononuclear cells by real-time reverse transcription polymerase chain reaction. Interestingly, the overexpression of SRPK1 in aggressive types of ATL was more exclusively observed at the protein level than at the mRNA level. Autologous antibody to SRPK1 was confirmed in the ATL patient using Western blot analysis with plasma, but not detected in asymptomatic HTLV-I carriers or in healthy volunteers. These results indicate that SRPK1 may be useful for the development of therapeutic and diagnostic methods for patients with ATL. [ABSTRACT FROM AUTHOR]

A recombinant bispecific single-chain fragment variable antibody (bsscFv), directed against the B-cell antigen CD19 and the low affinity Fc-receptor Fc γRIII (CD16), was designed for use in the treatment of patients with leukaemias and lymphomas. The Fc-portions of whole antibodies were deliberately eliminated in this construct to avoid undesired effector functions. A stabilised bsscFv, ds[CD19 × CD16], was generated, in which disulphide bonds bridging the respective variable light (VL) and variable heavy (VH) chains were introduced into both component single-chain (sc)Fvs. After production in 293T cells and chromatographic purification, ds[CD19 × CD16] specifically and simultaneously bound both antigens. The serum stability of ds[CD19 × CD16] was increased more than threefold when compared with the unstabilised counterpart, while other biological properties were not affected by these mutations. In antibody-dependent cellular cytotoxicity experiments, ds[CD19 × CD16] mediated specific lysis of both CD19-positive malignant human B-lymphoid cell lines and primary tumour cells from patients with B-cell chronic lymphocytic leukaemia or B-cell acute lymphoblastic leukaemia. Natural killer cells, mononuclear cells (MNCs) from healthy donors and, in some instances, MNCs isolated from patients after allogeneic stem cell transplantation, were used as effectors. Thus, ds[CD19 × CD16] holds promise for the treatment of CD19+ B-lineage malignancies. [ABSTRACT FROM AUTHOR]

The mutational status of the variable region of the immunoglobulin heavy chain gene (IgVH) is an important prognostic marker in B-cell chronic lymphocytic leukaemia (B-CLL), with mutated patients having improved outcome. To examine the impact of mutational status on VH, DH, and JH gene segment location and immunogenicity, we analysed 375 IgH sequences from 356 patients with B-CLL. Although VH and DH gene usage was different in mutated compared to unmutated patients, there was no impact of gene location on frequency of use or clinical outcome. Surprisingly, somatic mutations did not increase the immunogenicity of the Ig, as assessed by predicted binding affinity of Ig-derived peptides to major histocompatibility Class I and Class II molecules. Even excluding patients using VH1-69, cases using the VH1 gene family had a poor outcome. Both mutated and unmutated CLL patients demonstrated evidence of antigen selection. The worst outcome was seen in the subset of 14 unmutated patients with similar HCDR3 amino acid sequence using VH1-69, DH3-3 and JH6, suggesting an antigen-driven process modulating the clinical course. [ABSTRACT FROM AUTHOR]

Abnormalities ofp53have been associated with short survival and non-response to therapy in chronic lymphocytic leukaemia (CLL). We have evaluated the rate of response to fludarabine as first-line therapy in 54 patients with advanced stage CLL, analysing the cytogenetic profile, aberrations inp53, including the methylation status of its promoter, and the immunoglobulin heavy-chain variable-region (IGVH) mutation status. According to the advanced stage of the disease in this series, 75% of patients presented genetic aberrations associated with poor prognosis: del(17p) and/or del(11q), and no-mutatedIGVHgenes. Ten patients (18·5%) had methylation in the promoter region of p53. Eighty-three per cent of patients treated achieved a response, with a high rate of complete remission (47·6%). Although we found a significant correlation between failures and the presence ofp53aberrations (P = 0·0065), either with methylation (P = 0·018) or deletion (P = 0·015), 64% of the patients with aberrations in this gene responded to treatment (11/17), suggesting that fludarabine induces high remission rates, even in these patients. This is the first time that the significance of p53 promoter methylation status is described in this pathology, and our data support that this epigenetic phenomenon could be involved in the pathogenesis and clinical evolution of CLL. [ABSTRACT FROM AUTHOR]

Leucodepletion has been shown to reduce human leucocyte antigen immunization, but studies on the effect of leucodepletion on red cell alloimmunization reported discordant results. We conducted a retrospective multicentre study to determine whether prestorage filter leucodepletion alters the development of clinically significant red blood cell alloimmunization against the Rhesus, Kell, Duffy, Kidd and MSs blood group systems. Two periods were investigated, 2 years before and 2 years after universal leucodepletion. Comparisons were made between the transfused patient cohorts. To control for changes not related to leucoreduction, we compared antibody incidence with antibody prevalence in the two study periods. Newly detected antibodies (n = 4770) were found in 4115 patients from 19 participating hospitals. Of these, 857 antibodies in 659 patients were because of transfusions given in the study periods. The immunization risk was 0·13% for both periods. No differences were found regarding incidence of new antibodies, nor for patients regarding age, sex, previous antibodies, multiple antibodies, additional antibodies, number of transfusions, transfusions episodes and days from transfusion to date of immunization. In conclusion, compared with buffy-coat leucoreduction, universal prestorage filter leucodepletion did not alter the development of clinically significant red blood cell alloimmunization. [ABSTRACT FROM AUTHOR]

It is well known that the differentiation of acute promyelocytic leukaemia (APL) cells by all-trans-retinoic acid (ATRA) may be enhanced by myeloid growth factors, but the requirement for growth factors in this process is unclear. Our previous studies in multiple myeloma and non-APL acute myeloid leukaemia demonstrated that lineage-specific growth factors are required for the maximal activity of many pharmacologic differentiating agentsin vitro. Thus, we studied whether the differentiation of APL is similarly dependent on growth factors. We found that the myeloid growth factors granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor markedly increased the differentiation of NB4 cells or APL blasts from clinical samples treated with ATRA, arsenic trioxide (ATO), or bryostatin-1 as evidenced by the enhanced expression of myeloid surface antigens and the inhibition of clonogenic growth. Furthermore, myeloid growth factors were necessary for the differentiation of APL cells since the activity of each pharmacologic agent could be blocked by specific growth factor-neutralizing antibodies. Each differentiating agent was active only at concentrations that inhibited cell cycling, suggesting that this property is also required for differentiation. These data demonstrate that both pharmacologic differentiating agents and myeloid growth factors are required, but neither sufficient, for the differentiation of APL cells. The combined use of pharmacologic differentiating agents and growth factors may improve the clinical efficacy of differentiation therapy in APL. [ABSTRACT FROM AUTHOR]