Allo-anti-M: Detection peaks around 2 years of age, but may be attenuated by red blood cell transfusion.

BACKGROUND: Anti-M is frequently observed as a naturally occurring antibody of little clinical significance. Naturally occurring anti-M is often found in children although the specific triggers of production, persistence, and evanescence of anti-M have yet to be elucidated. METHODS: In a retrospective, multicenter, nationwide cohort survey conducted from 2001 to 2015, alloantibody screening was performed before and after transfusion in 18,944 recipients younger than 20 years. Recipients were categorized into six cohorts based on their age at transfusion; within and among these cohorts, allo-anti-M was analyzed in regard to its production, persistence, and evanescence. RESULTS: In 44 patients, anti-M detected before and/or after transfusion was an age-related phenomenon, with a median age of 2 years and an interquartile range of 1-3 years; anti-M was most frequently detected in a cohort of children 1 to <5 years (0.77%, 31 of 4035). At least five patients were presumed to have concurrent infections. Among 1575 adolescents/young adults (15 to <20 years), no anti-M was detected. Of 29 patients with anti-M prior to transfusion, the antibody fell to undetectable levels in 17 recipients (89.5%, of whom at least 13 received only M-negative red cells) after anywhere from 5 days to 5.8 years; anti-M persisted in 2, and was not tested in 10. Only 15 recipients (0.08%) produced new anti-M after transfusion. CONCLUSION: Naturally occurring anti-M is a phenomenon of younger ages, predominantly between 1 and 3 years. After transfusion, it often falls to undetectable levels.

Successful rituximab treatment in an elderly patient with recurrent thrombotic thrombocytopenic purpura.

An 81-year-old man presenting with fever, neurological symptoms, thrombocytopenia, and hemolytic anemia was diagnosed with acquired idiopathic thrombotic thrombocytopenic purpura (TTP). His disintegrin-like and metalloproteinase with thrombospondin type 1 motifs 13 (ADAMTS13) activity was <1% and the ADAMTS13 inhibitor titer was 3.2 BU/ml. He received plasma exchange and steroid administration until remission was achieved. Seven months later, he suffered from paralysis of the right hand, hemolytic anemia, and thrombocytopenia. We confirmed TTP recurrence based on ADAMTS13 activity <1% and an ADAMTS13 inhibitor titer of 19.4 BU/ml. Four infusions of rituximab were administered in addition to plasma exchange and steroid pulse therapy. Platelet count recovery was observed within 5 days. No severe side effects related to rituximab occurred. Although rituximab has not been approved for TTP in Japan, we report the efficacy and safety of rituximab in an elderly patient with recurrent TTP. We suggest that rituximab therapy should be started as soon as possible for recurrent TTP in patients with high titers of ADAMTS13 inhibitor.

Reference guide for the diagnosis of adult primary immune thrombocytopenia, 2023 edition.

Primary immune thrombocytopenia (ITP) is an autoimmune disorder characterized by isolated thrombocytopenia due to accelerated platelet destruction and impaired platelet production. Diagnosis of ITP is still challenging because ITP has been diagnosed by exclusion. Exclusion of thrombocytopenia due to bone marrow failure is especially important in Japan because of high prevalence of aplastic anemia compared to Western countries. Hence, we propose a new diagnostic criteria involving the measurement of plasma thrombopoietin (TPO) levels and percentage of immature platelet fraction (RP% or IPF%); 1) isolated thrombocytopenia with no morphological evidence of dysplasia in any blood cell type in a blood smear, 2) normal or slightly increased plasma TPO level (< cutoff), 3) elevated RP% or IPF% (> upper limit of normal), and 4) absence of other conditions that potentially cause thrombocytopenia including secondary ITP. A diagnosis of ITP is made if conditions 1-4 are all met. Cases in which criterion 2 or 3 is not met or unavailable are defined as "possible ITP," and diagnosis of ITP can be made mainly by typical clinical course. These new criteria enable us to clearly differentiate ITP from aplastic anemia and other forms of hypoplastic thrombocytopenia and can be highly useful in clinical practice for avoiding unnecessary bone marrow examination as well as for appropriate selection of treatments.

[Rituximab for managing refractory thrombotic thrombocytopenic purpura: a report of three cases].

About 20% of TTP are resistant to plasma exchange. As reported in a few case reports and small case series, rituximab has been used in the treatment of TTP with some benefit. However, the optimal dosing, frequency, and timing of rituximab remain to be determined. We treated three cases of refractory TTP with rituximab. Case 1 exhibited brain sequelae probably due to the late administration of rituximab, case 2 died before the expected effect of rituximab could occur, and case 3 recovered completely because of the early administration of rituximab. These results suggest that rituximab should be given as early as possible in TTP, but large clinical studies are required to determine the optimal use of rituximab in TTP.

Role of activation-induced cytidine deaminase in the progression of follicular lymphoma.

Activation-induced cytidine deaminase (AID/AICDA) is required for somatic hypermutation and class-switch recombination of the immunoglobulin gene, and for c-myc translocation of germinal center-derived B-cell lymphoma. In the present study, we attempted to clarify the significance of AID associated with c-myc in the progression of follicular lymphoma (FL) using RT-PCR and quantitative real-time PCR. Tissues from the patients with grade 3 FL expressed relatively higher levels of c-myc and AID. The samples taken from a patient with FL who died within 2 years after the start of treatment showed either no or low expression of AID, despite expressing high levels of c-myc. In order to examine the role of AID expression in rapidly progressive FL, the full-length AID transcript was transfected into AID-negative cell lines established from different patients with rapidly progressive FL. This led to the establishment of AID-expressing transfectants with a low proliferation rate and a significantly increased incidence of G(0)/G(1) arrest compared with controls. Our results indicate that AID may act as a negative regulator of cell survival in FL when sufficient c-myc is expressed. Switch-off or low expression of AID after c-myc amplification may correlate with the clinical outcomes of FL.

Aurora-A kinase: a novel target of cellular immunotherapy for leukemia.

Aurora-A kinase (Aur-A) is a member of the serine/threonine kinase family that regulates the cell division process, and has recently been implicated in tumorigenesis. In this study, we identified an antigenic 9-amino-acid epitope (Aur-A(207-215): YLILEYAPL) derived from Aur-A capable of generating leukemia-reactive cytotoxic T lymphocytes (CTLs) in the context of HLA-A*0201. The synthetic peptide of this epitope appeared to be capable of binding to HLA-A*2402 as well as HLA-A*0201 molecules. Leukemia cell lines and freshly isolated leukemia cells, particularly chronic myelogenous leukemia (CML) cells, appeared to express Aur-A abundantly. Aur-A-specific CTLs were able to lyse human leukemia cell lines and freshly isolated leukemia cells, but not normal cells, in an HLA-A*0201-restricted manner. Importantly, Aur-A-specific CTLs were able to lyse CD34+ CML progenitor cells but did not show any cytotoxicity against normal CD34+ hematopoietic stem cells. The tetramer assay revealed that the Aur-A(207-215) epitope-specific CTL precursors are present in peripheral blood of HLA-A*0201-positive and HLA-A*2402-positive patients with leukemia, but not in healthy individuals. Our results indicate that cellular immunotherapy targeting Aur-A is a promising strategy for treatment of leukemia.

Vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay for platelet response to cilostazol.

Recent clinical trials have demonstrated that cilostazol, an antiplatelet drug competent to inhibit phosphodiesterase 3, is effective in secondary prevention of atherothrombosis including ischemic stroke, myocardial infarction, and peripheral arterial disease. However, there is no reliable assay for detection of the platelet response to cilostazol. We attempted to establish such an assay for clinical use. Phosphorylation of vasodilator-stimulated phosphoprotein (VASP) subsequent to the pharmacological action of cilostazol was measured by a platelet VASP assay kit that has been widely used for monitoring platelet response to ADP receptor antagonists in clinical settings. We modified the kit and found the optimal conditions for detection of cilostazol-dependent VASP phosphorylation. The assay could detect the in vitro and in vivo platelet responses to cilostazol effectively in the presence of 50 nM PGE1. ROC analysis showed that our assay had 97% sensitivity and 75% specificity when blood drawn between 3 and 9?h after cilostazol ingestion was subjected to the assay. The assay results were verified by immunoblotting specific for VASP phosphorylation. Standard light transmission platelet aggregometry could not detect significant inhibition of agonist-induced platelet aggregation by cilostazol except for the in vitro effect of high concentrations of cilostazol. These results demonstrate that the platelet VASP assay can detect the platelet response to cilostazol with high sensitivity and specificity.

Usage of granulocyte colony-stimulating factor every 2 days is clinically useful and cost-effective for febrile neutropenia during early courses of chemotherapy.

BACKGROUND: In order to analyze the clinical activity and cost-effectiveness of granulocyte colony-stimulating factors (G-CSF), the prophylactic usage of G-CSF in patients treated with a single chemotherapy regimen during early courses was prospectively evaluated. METHODS: Thirty patients with newly diagnosed non-Hodgkin lymphoma (NHL) treated with the first course of an R-CHOP regimen were enrolled randomly. After treatment with the first course of chemotherapy, a daily dose of G-CSF (lenograstim, 100 µg) was administered to half (15 cases) of the patients, and a dose of G-CSF (100 µg) was administered every other day to the other half of the patients when leukocytopenia (<1.5 × 10(9)/L) and/or neutropenia (<0.5 × 10(9)/L) occurred. Changes in leukocyte and neutrophil counts, prophylaxis, febrile neutropenia (FN) events, and cost performance between the two groups were analyzed. RESULTS: No significant difference between the two groups was observed in recoveries of leukocyte and neutrophil counts and evidence of FN. The only difference was the total cost of G-CSF. CONCLUSION: We concluded that every-other-day use of G-CSF was as clinically effective for the prophylaxis of FN as the daily use of G-CSF, and economically speaking, the administration of G-CSF every other day should be more beneficial for patients with NHL during early courses of R-CHOP chemotherapy.

Risk factors for skin, mucosal, and organ bleeding in adults with primary ITP: a nationwide study in Japan.

Bleeding manifestations in primary immune thrombocytopenia (ITP) range from skin petechiae to life-threatening intracranial hemorrhage (ICH). However, the relation between these various bleeding manifestations and the platelet count in ITP remains poorly characterized. Using a nationwide database of patients with ITP during the years 2005 to 2014 (10 years) in Japan, we analyzed 19 415 adult patients newly diagnosed with ITP, including 222 with ICH. The frequency of skin purpura was 64.8%, and this increased linearly with thrombocytopenia without a specific platelet count threshold. In contrast, mucosal bleeding (epistaxis and gingival bleeding) and organ bleeding (melena, hematuria, and ICH) increased exponentially with thrombocytopenia at a platelet count threshold of 10 to 15 × 109/L. Age showed a much weaker correlation than platelet count with skin and mucosal bleeding. However, the incidence of organ bleeding increased exponentially above 60 years of age. Multivariate analysis showed that the presence of mucosal bleeding was a risk factor for occurrence of melena and hematuria but not for ICH. The frequency of ICH was 1.1% and risk factors for ICH were age ≥60 years (odds ratio [OR], 3.09; 95% confidence interval [CI], 2.13-4.47; P < .001), platelet count <10 × 109/L (OR, 2.96; 95% CI, 2.11-4.15; P < .001), and the presence of hematuria (OR, 1.56; 95% CI, 1.04-2.35; P = .033). The relation between ICH and platelet count varied with age. This large-scale analysis of risk factors for bleeding in ITP has revealed distinct characteristics of skin, mucosal, and organ bleeding in adult patients with newly diagnosed ITP, thus indicating those who are at a high risk of severe organ bleeding.

[Acquired hemophilia complicated with multiple muscle abscess by Nocardia].

An 82-year-old man was referred to our hospital because of bilateral leg swelling and ecchymosis. A hemostatic study showed prolonged aPTT, <1% factor VIII coagulant activity, and a high titer (30.4 Bethesda Units/ml) of factor VIII inhibitor. The diagnosis of acquired hemophilia A (AHA) was made, and treatment with prednisolone (PSL) was started. Within one month of treatment, the hemorrhagic symptom disappeared, aPTT levels returned to normal, and his factor VIII inhibitor was eradicated; however, factor VIII inhibitor was detected again when PSL was decreased to 10 mg/day. We then added cyclosporine A (CyA) to PSL as a second line salvage therapy. CyA therapy resulted in the resolution of AHA with marked and prolonged efficacy; however, hot, red tumors appeared in his right arm and left thigh. Needle aspiration of the tumors revealed muscle abscess, and Nocardia brasiliensis was isolated. We started treatment with sulfamethoxazole-trimethoprim, and the abscess healed promptly without recurrence.

[Primary osseous lymphoma with pathological fracture during therapy].

A 66-year-old woman was aware of a cervical tumor in May 2007. She was hospitalized in June 2007 because her cervical tumor had increased. A biopsy was performed and a diagnosis of CD20-positive diffuse large B-cell lymphoma was obtained. Ga-67 scintigraphy showed abnormal accumulation in the right clavicle, right femur, right knee joint, right ankle joint, and the left tibia and fibula; however, no abnormality was detected on plain radiography and CT scan, whereas MRI showed that the right femur had a low signal on the T1-weighted image, and high and low signals on the T2-weighted image. CHOP therapy was begun, and the right cervical tumor promptly reduced. She was administered rituximab seven days after initiation of the treatment. When standing up from the toilet at midnight, she suffered fractures of the left tibia and fibula, and the right neck of the femur. These regions were identical to the sites with abnormal accumulation on Ga-67 scintigraphy, so we supposed them to be chemotherapy-associated pathologic fractures. This case is reported because primary bone lymphoma is rare and followed an unusual course of pathologic fracture under treatment.

Safe childbirth for a type 1 antithrombin-deficient woman with novel mutation in the SERPINC1 gene undergoing antithrombin concentrate therapy.

: Inherited antithrombin (AT) deficiency is an autosomal dominant thrombotic disorder. We encountered a case of inherited type I AT deficiency and identified the causative mutation; a novel c.7430A>G missense mutation in the SERPINC1 gene in which tyrosine was substituted for cysteine at the 292nd amino acid. A recombinant AT protein with the 7430A>G mutation was not detected in cell lysates or culture supernatants. And then, our patient without personal or family history of thrombosis was pregnant woman with asymptomatic AT deficiency. Our patient treated with only AT concentrate therapy during pregnancy and she was able to safely give birth naturally and avoid thrombosis. We believe that this therapy for pregnant woman with asymptomatic AT deficiency is effective and safety as anticoagulant therapy during pregnancy.

Identification of an epitope derived from CML66, a novel tumor-associated antigen expressed broadly in human leukemia, recognized by human leukocyte antigen-A*2402-restricted cytotoxic T lymphocytes.

CML66 is a newly identified differentiation antigen that is expressed broadly in human leukemia and solid tumors, but its physiological function remains unknown. In the present study, to clarify the feasibility of CML66-targeted cancer immunotherapy, we attempted to identify cytotoxic T lymphocyte (CTL) epitopes derived from CML66. An immunogenic CML66-derived epitope (amino acid residues 76-84; YYIDTLGRI) capable of inducing human leukocyte antigen (HLA)-A*2402-restricted CTL specific for this peptide was identified. CML66-derived peptide-specific CTL efficiently lysed human leukemia cells, but not normal cells, in a HLA-A*2402-restricted fashion. Quantitative real-time polymerase chain reaction revealed that CML66 mRNA is expressed abundantly in primary acute myeloid leukemia cells, acute lymphoid leukemia cells, and chronic myelogenous leukemia cells in advanced phase, and that the expression level of CML66 mRNA in normal cells is low compared with that in leukemia cells. CML66-specific CTL precursors were detected in the peripheral blood of patients with acute leukemia. These data indicate that the CML66-derived epitope identified in the present study is a new target antigen for cellular immunotherapy of human leukemia.

Development of an FVIII Inhibitor in a Mild Hemophilia Patient with a Phe595Cys Mutation.

Mild hemophilia A is caused by a missense mutation in the FVIII gene that is responsible for a decrease in the FVIII:C to between 5% and 40%. The development of FVIII inhibitors has been reported in 3-13% of patients with mild hemophilia. Genetic risk factors for the development of inhibitors in mild hemophilia have been investigated. In the present study, we encountered a case of mild hemophilia with an FVIII inhibitor and identified the mutation responsible: a novel Phe595Cys mutation in the FVIII gene. In addition, this study showed that the inhibitor recognized exogenous wild-type FVIII and autologous mutant FVIII.

Absence of chlamydial infection in Japanese patients with ocular adnexal lymphoma of mucosa-associated lymphoid tissue.

Ocular adnexal extranodal marginal zone B-cell lymphomas of mucosa-associated lymphoid tissue (ocular adnexal MALT lymphoma) are predominately low-grade, small B-cell types and may be caused by several putative inflammatory agents. Twenty-three ocular adnexal MALT lymphoma cases, the monoclonality of which was confirmed by examination of immunoglobulin heavy chain gene rearrangement and/or cell surface antigens, were analyzed for evidence of several causative factors. A serologic evaluation of our series of patients showed no evidence of infection by Epstein-Barr virus, hepatitis C virus, or Chlamydophila psittaci. Two patients tested positive for serum antibodies for autoimmunity, and another 2 patients tested positive for antibodies against Chlamydia trachomatis. Polymerase chain reaction analysis did not reveal the presence of the chlamydial 16S ribosomal RNA (rRNA) gene or the 16S-23S spacer rRNA gene. These results indicate that the inflammatory agents in our series of ocular adnexal MALT lymphomas are still unknown and that some types of chlamydial infections are not associated with orbital adnexal MALT lymphoma in southern regions of Japan.

Human herpesvirus 6 impairs differentiation of monocytes to dendritic cells.

OBJECTIVE: Monocyte-derived dendritic cells (DCs) play important roles in the immune response against infections and malignancies. Human herpesvirus 6 (HHV-6) infects monocytes and is reactivated in immunodeficient patients. To clarify the mechanisms of HHV-6-induced immunodeficiency, we investigated the effect of HHV-6 infection on differentiation of monocytes to DCs. METHODS: Monocytes were inoculated with or without HHV-6 and then allowed to differentiate to myeloid DCs in culture medium containing granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4. The expression of cell surface molecules on DCs and the capacity of the DCs for antigen capture were examined by flow cytometric analysis. Alteration of antigen-presenting capacity induced by HHV-6 infection was examined. RESULTS: The morphology of HHV-6-infected monocyte-derived DCs was distinctly different from that of the DCs derived from mock-infected monocytes. Although expression levels of DC-associated surface antigens, including CD80, CD83, and CD86, were significantly higher on HHV-6-infected monocyte-derived DCs than on DCs derived from mock-infected monocytes, antigen-presenting capacity was significantly lower in the former group. Addition of culture supernatant of HHV-6-infected monocytes resulted in suppression of the T-lymphocyte proliferative response, and anti-IL-10 neutralizing antibody partly inhibited this suppressive effect. The antigen-presenting capacity of DCs generated from a patient with severe HHV-6 reactivation was significantly lower than that of DCs generated from the same patient in the recovery phase. CONCLUSIONS: HHV-6 infection induces immunodeficiency via impaired differentiation of DCs. These results present a new concept for the pathogenesis of HHV-6-induced immunodeficiency.