Early occurrence of red blood cell alloimmunization in patients with sickle cell disease.

Red blood cell (RBC) alloimmunization is a major complication of transfusion therapy in sickle cell disease (SCD). Identification of high-risk patients is hampered by lack of studies that take the cumulative transfusion exposure into account. In this retrospective cohort study among previously non-transfused SCD patients in the Netherlands, we aimed to elucidate the association between the cumulative transfusion exposure, first alloimmunization and independent risk factors. A total of 245 patients received 11 952 RBC units. Alloimmunization occurred in 43 patients (18%), half of them formed their first alloantibody before the 8th unit. In patients with exposure to non-extended matched transfusions (ABO and RhD) the cumulative alloimmunization risk increased up to 35% after 60 transfused units. This was significantly higher compared to a general transfused population (HR 6.6, CI 4.2-10.6). Receiving the first transfusion after the age of 5 was an independent risk factor for alloimmunization (HR 2.3, CI 1.0-5.1). Incidental, episodic transfusions in comparison to chronic scheme transfusions (HR 2.3, CI 0.9-6.0), and exposure to non-extended matched units in comparison to extended matching (HR 2.0, CI 0.9-4.6) seemed to confer a higher alloimmunization risk. The majority of first alloantibodies are formed after minor transfusion exposure, substantiating suggestions of a responder phenotype in SCD and stressing the need for risk factor identification. In this study, older age at first transfusion, episodic transfusions and non-extended matched transfusions appeared to be risk factors for alloimmunization. Am. J. Hematol. 91:763-769, 2016. © 2016 Wiley Periodicals, Inc.

NADP+ -dependent IDH1 R132 mutation and its relevance for glioma patient survival.

The isocitrate dehydrogenase 1 (IDH1) mutation occurs in high frequency in glioma and secondary glioblastoma (GBM). Mutated IDH1 produces the oncometabolite 2-hydroxyglutarate rather than α-ketoglutarate or isocitrate. The oncometabolite is considered to be the major cause of the association between the IDH1 mutation and gliomagenesis. On the other hand, the IDH1 mutation in GBM is associated with prolonged patient survival. This association is not well understood yet but IDH1 involvement in epigenetic silencing of O-6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme is considered to be an important mechanism. However, it was shown recently that the IDH1 mutation and MGMT silencing are independent prognostic factors. Here, we hypothesize that the IDH1 mutation reduces the capacity to produce NADPH and thus reduces the capacity to scavenge reactive oxygen species that are generated during irradiation and chemotherapy. IDH1 activity is responsible for two-thirds of the NADPH production capacity in normal brain, whereas the IDH1 mutation reduces this capacity by almost 40%. Therefore, we hypothesize that the reduced NADPH production capacity due to the IDH1 mutation renders GBM cells more vulnerable to irradiation and chemotherapy thus prolonging survival of the patients.

A monoclonal antibody to a cytoskeletal protein selectively recognizing malignant neuroectodermal tumors.

Fusion of myeloma (P3X63-Ag 8.653) cells with spleen cells from BALB/c mice immunized with human neuroblastoma (SK-N-SH) cells yielded a hybridoma clone, referred to as 3XB7, with a unique pattern of reactivity to malignant neuroectodermal tumors except gliomas of low-grade malignancy. Indirect immunofluorescence staining under different conditions and Western blot analysis indicate that the 3XB7 MAb recognizes an intracellular cytoskeletal protein of M(r) 52K. Immunohistochemical studies with cryostat and paraffin-embedded sections from tumor biopsies revealed that the 3XB7 MAb specifically recognizes malignant neuroectodermal tumors and reacts negatively with other epithelial and mesenchymal tumors, e.g., carcinomas, lymphomas, and sarcomas as well as with normal adult and fetal brain tissues. Negative reaction was also observed with other small round cell tumors of childhood. Thus the 3XB7 antigen can be used for diagnosis of all stages of neuroblastomas, and its specific expression in gliomas with high-grade malignancy (grades III and IV) confer on it additional prognostic value.

Monoclonal antibodies to rat brain astroglial cells-Their characterization and application for isolation of astroglial subpopulations.

Three monoclonal antibodies (MAbs) specifically recognizing rat astrocyte cell surface proteins have been characterized and their antigen binding specificities determined. One of these MAbs has been employed to isolate a distinct subpopulation of astroglial cells using immunoaffinity chromatography. MAbs to rat astroglial cell surface proteins, generated by fusion of mouse Sp2/O-Ag 14 myeloma cells and spleen cells from Balb/C mice immunized with purified astroglial cells, were screened for their cell binding specificities using ELISA and indirect immunofluorescence assay. The antigen binding specificity of three of these clones, which displayed specific binding to astrocytes, was determined by radioiodination of whole astrocytes and precipitation of the iodinated surface proteins by the MAbs. Immunoaffinity chromatography, using IgG from one of the clones coupled to CNBr activated Sepharose 6MB, demonstrated the potential usefulness of such MAbs in isolating a specific subpopulation of astroglial cells.

An ELISA method for quantitation of tubulin using poly-1-lysine coated microtiter plates.

A new, sensitive and convenient ELISA method has been developed for quantitation of tubulin using poly-1-lysine (PLL) coated multiwell microtiter plates. Binding of tubulin to untreated plastic surface of microtiter plates was extremely poor. Coating of wells with PLL enhanced the binding and facilitated quantitation by ELISA. Binding of tubulin was followed by stepwise additions of rabbit anti-tubulin IgG, HRP-conjugated goat anti-rabbit IgG and colour reagent. The method has been successfully applied to quantitate the tubulin content of extracts from rat brain and liver as evident from the excellent correlation of the results with those obtained from 3H-colchicine binding assay. The detection limit is as low as 5 ng, which is relatively better than that of the previous RIA methods. The ELISA method does not involve the use of any radioactive compound and all reagents required for this assay are commercially available.

Triiodothyronine regulated tubulin biosynthesis in oligodendrocytes during myelinogenesis.

l-Triiodothyronine (T(3)) is shown to stimulate the biosynthesis of tubulin in oligodendrocytes isolated from rat brains at different stages of myelinogenesis. The hormone sensitivity appears around day 11, reaches a maximum at day 15 and disappears by day 25 after birth. Dose response studies show that the optimal stimulation of tubulin in the oligodendrocytes from 15 day rat brain occurs with 0.045 nM T(3) in contrast to 4.5 nM T(3) that was previously shown to promote a similar age-dependent induction of tubulin in the astroglial cells from neonatal rat brain. Exposure of the oligodendrocytes to optimal dose of T(3) (0.045 nM) for 2 h elicits a marked and almost selective increase in the level of tubulin. Higher concentrations of T(3) induce additional proteins resulting in a loss of this sensitivity. Studies on the synthesis and turnover of (35)S-labeled tubulin show that the stimulation of tubulin by T(3) is primarily due to enhanced synthesis of the protein. Pulse chase experiments reveal that the half life of tubulin is about 5.5 h and that it remains unaffected by T(3). The crucial role of thyroid hormones and the possible function of tubulin in the two most critical phases of brain maturation viz. synaptogenesis and myelinogenesis is discussed.

Comparative analysis of the membrane proteins and their specificities in neurons, protoplasmic astrocytes, and oligodendrocytes from rat brain.

Plasma membranes from neuronal perikarya (N), protoplasmic astrocytes (A) and oligodendrocytes (O) of rat brains were analysed with respect to their protein and glycoprotein contents and specificities. SDS-polyacrylamide gel electrophoresis revealed a total number of 23, 17, and 17 major proteins in N, A, and O respectively. Periodate-Schiff's staining showed that approximately 40-60% of these proteins were glycoproteins. The reactivity of these glycoproteins to Con A and WGA was also studied. Selective iodination of whole cells followed by electrophoresis and autoradiography indicated that of the major proteins, only 25% of neuronal and 60% of astroglial and oligodendroglial membrane proteins were exposed outside the cell surface. The overall results suggest that membrane proteins of each of the three cell types studied here have characteristically different internal and external markers differing in size, glycoprotein content, and reactivity of the glycoproteins to lectins.