Sadayoshi Sekiguchi

A New Platelet-Specific Antigen, Naka, Involved in the Refractoriness of HLA-Matched Platelet Transfusion

Abstract. Serum from a thrombocytopenic patient who was refractory to the transfusions of HLA‐matched platelets contained a platelet‐specific alloantibody, anti‐Naka. Immunofluorescence analyses revealed that the Naka antigen defined by the serum was expressed exclusively on platelets and its distribution was different from PlA1, Baka, Yuka or Yukb. Analysis by Dr. von dem Bornes group revealed the Naka was also different from Koa, Kob or Zwb. Family studies showed that the Naka antigen was inherited as an autosomal codominant trait. Its antigen frequency in the Japanese population was over 97%. The results of the enzyme immunoassay using monoclonal antibodies for antigen immobilization showed that the Naka epitope did not appear to reside on GPIIb/IIIa or Ib. The transfusions of Naka‐compatible platelets improved the patients thrombocytopenia.

The Particle Size Of Hepatitis C Virus Estimated By Filtration Through Microporous Regenerated Cellulose Fibre

To estimate the particle size of hepatitis C virus (HCV), a major causative agent of post-transfusion non-A, non-B hepatitis, we filtered plasma or serum samples through microporous cellulose fibres with different pore sizes. The amount of HCV particles in samples before and after filtration was determined by a quantitative reverse transcriptase polymerase chain reaction (PCR) method. Since there is no quantitative biological assay for HCV, except for that in chimpanzees, the HCV titre obtained from the PCR method was used in an equation constructed previously for application to filtration experiments with a flavivirus which is distantly related to HCV. The particle was estimated to be between 30 and 38 nm in diameter, although the possibility remained that larger HCV particles or HCV aggregates with a diameter of more than 39 nm might exist. Double-step filtration through microporous cellulose fibres with a pore size of 35 nm reduced the HCV content to below levels detectable by our PCR method, indicating that it is possible to eliminate HCV particles by simple filtration techniques.

Prevalence of Borna disease virus RNA in peripheral blood mononuclear cells from blood donors

The presence of Borna disease virus (BDV) in peripheral blood mononuclear cells (PBMC) of 100 blood donors from Sapporo and 72 blood donors from Tokyo was examined using nested reverse transcriptase/polymerase chain reaction amplification with specific-primers for BDV p24. Anti-BDV p24 antibodies in the plasma of the 100 blood donors from Sapporo also were studied by enzyme-linked immunosorbent assay and by Western blot. BDV RNA was detected in 3 (4.2%) of the 72 PBMC samples from Tokyo, and in 5 (5%) of the 100 PBMC samples from Sapporo. In contrast, anti-p24 antibodies were found in only 1 (1%) of the donors from Sapporo. These results suggest that BDV infection in humans may be more widespread than previously thought.

Photoinactivation of Virus Infectivity by Hypocrellin A

Abstract— We investigated the photoinactivation of virus infectivity by hypocrellin A and its mechanism. The titers of vesicular stomatitis virus (VSV) and human immunodeficiency virus type 1 (HIV‐1), both of which are enveloped viruses, were reduced upon illumination with hypocrellin A in a concentration‐dependent manner, whereas canine parvovirus, a nonenveloped virus, was not killed. The removal of oxygen or addition of sodium azide or bT‐carotene both inhibited VSV inactivation. Mannitol and superoxide dismutase had no effect on VSV inactivation. These results indicate that singlet oxygen was involved in the process of VSV inactivation. Of the three major VSV membrane proteins, peripheral membrane protein M was most damaged by the hypocrellin A phototreatment.

Bradykinin generation during filtration of platelet concentrates with a white cell-reduction filter

To the Editor: White cell (WBC)-reduction filters are widely used in blood transfusion to prevent transfusion-associated complications,’ such as nonhemolytic febrile reaction, alloimmunization, and cell-associated virus transmission, but some acute side effects are still observed in transfused patients. Recently, anaphylactoid hypotension has been reported in patients receiving platelet concentrates (PCs) filtered at the bedside.* In patients receiving an angiotensin-converting enzyme (ACE) inhibitor, anaphylactoid reactions, including hypotension, have been observed during hemodialysis with polyacrylonitrile membranes3 and during low-density lipoprotein apheresis with dextran ~ u l f a t e . ~ Further studies showed that the cause of these reactions is bradykinin, generated through contact activation with the negatively charged materials of the devices used.5 These reports led us to investigate whether similar bradykinin generation occurs when PCs are filtered with WBC-reduction filters. We examined two filters currently used worldwide. The PCs obtained by apheresis from single donors were divided into two parts (approx. 120 mLeach) and filtered through Filter A, which has a negative charge (PXL8, Pall Biomedical Products Corp., Glen Cove, NY), and through Filter B, which has a positive charge (Sepacell PLSIOA, Asahi Medical Co., Tokyo, Japan). The bradykinin levels were measured by radioimmunoassayh before and during the filtration. The average prefiltration level of bradykinin was 37.1 f 22.0 pg per mL (mean ? SD, n = 12). The bradykinin level increased significantly after Filter A was primed with PCs ( ~ ~ 0 . 0 1 , Wilcoxon’s signed-rank test); that level was maintained during filtration of the first SO to 60 mL, and then it decreased. The average and highest levels of bradykinin at the beginning of filtration were 6,7945 and 28,800 pg per mL, respectively. The average level of bradykinin at the end of filtration was 2,502.9 pg per mL. On the other hand, at the end of filtration, no significant amount of bradykinin was observed in filtrates from Filter B (p>0.05), and the highest level of filtrate was 53.9 pg per mL. To investigate the mechanism of bradykinin generation by Filter A, we studied the influences of an ACE inhibitor and a proteinase inhibitor. In each of three experiments, a PC was divided into three bags. The first bag contained no inhibitors. The second contained the ACE inhibitor, captopril, dissolved in saline, to a final captopril concentration of 50 ng per mL. The third contained the proteinase inhibitor, nafamostat mesilate, in saline, to a final concentration of 0.2 mg per mL. All aliquots were then filtered with filter A. In one experiment, the level of bradykinin rose to 21,900 pg per mL at the beginning of filtration without an inhibitor and then decreased to 9,150 pg per mL in the middle of filtration. The presence of captopril increased bradykinin to 36,000 pg per mL, and the high level was maintained during filtration. In contrast, the proteinase inhibitor prevented such generation and the level of bradykinin averaged 355.3 pg per mL. We obtained similar results from the other two experiments. These findings suggest that bradykinin is generated through activation of the contact system. Nuclear magnetic resonance study indicated that the material of Filter A has an anionic carboxyl group, while that of Filter B has a cationic amino group. The bradykinin generated by Filter A, and concentrated through liquid chromatography, was biologically a ~ t i v e . ~ It induced dose-dependent contraction of rat uterus treated with the bradykinin antagonist D-kg, [Hyp3, Thi5.8, ~-Phe~]-bradykinin and dose-dependent depression of blood pressure in rats given captopril in advance. The clinical significance of this bradykinin generation is still not clear. However, the transfusion of PCs filtered at the bedside may be more risky than that of PCs filtered in the laboratory, where the initially high levels of bradykinin will be diluted in the final component. In addition, bradykinin is degraded during storage by kinase I and 11. ’ h o well-known disadvantages of bedside filtration of blood components are the inability to exercise quality control and the inconsistency in such aspects of administration as the speed and temperature of transfusion. Our data additionally suggest that patients receiving PCs that are WBC-reduced at the bedside with a filter consisting of negatively charged polyester, particularly those patients being treated with ACE inhibitors, may be at risk for anaphylactoid reactions.

Higher prevalence of Borna disease virus infection in blood donors living near thoroughbred horse farms

It is believed that Borna disease virus (BDV), an etiological agent of progressive polioencephalomyelitis in horses and sheep, is closely associated with psychiatric disorders in humans since the prevalence of BDV is higher in psychiatric patients than in blood donors. We investigated whether or not BDVs in humans are derived from infected domestic animals, by characterizing the BDVs in blood donors and horses derived from the same region of Hokkaido island, Japan. The seroprevalences (2.6 to 14.8%) of BDV were significantly higher in the blood donors from four regions where most horse farms are concentrated, compared with only 1% in the blood donors from Sapporo, the largest city in Hokkaido. BDV RNA was also detected in peripheral blood mononuclear cells from most of the seropositive horses and blood donors by nested reverse transcriptase‐polymerase chain reaction. These findings support that BDV may be horizontally transmitted, at least in part, from infected horses to humans. J. Med. Virol. 52:330–335, 1997.

Effects of filtration and gamma radiation on the accumulation of RANTES and transforming growth factor-β1 in apheresis platelet concentrates during storage

BACKGROUND: Platelet‐derived biologic response modifiers (BRMs) including RANTES and transforming growth factor (TGF)‐β1 accumulate in platelet components during storage because of platelet activation, and they may play a causative role in nonhemolytic febrile transfusion reactions. The majority of PCs with high unit values are provided by single donor apheresis in Japan.

Culture system for extensive production of CD19+IgM+ cells by human cord blood CD34+ progenitors.

We established a co-culture system with a monolayer of the murine bone marrow (BM) stroma cell line, MS-5, in which human cord blood CD34+ cells differentiated to CD19+ cells. The addition of stem cell factor (SCF) and granulocyte colony- stimulating factor (G-CSF) highly enhanced the production of CD19+ cells. The expansion of the cell numbers was over 103-fold. Furthermore, a significant proportion (<45%) of the cells expressed surface igm (sigm) after 5 weeks of co-culture. cd34+CD19− cells also showed a similar development of CD19+ cells and CD19+sIgM+ cells. Filter separation of MS-5 cells and CD34+ cells did not inhibit the growth of CD19+ cells. However, when further purified CD34+CD19−CD13− CD33− cells were cultured in the presence of MS-5 cells with or without a separation filter, CD19+ cells did not appear in the non-contact setting. This result suggested that the highly purified CD34+CD19−CD13−CD33− progenitors require the cell–cell contact for the development of CD19+ cells, whereas other CD34+ fractions contain progenitors that do not require the contact. This co-culture system should be useful for the study of early human B-lymphopoiesis.

Growth-blocking peptide titer during larval development of parasitized and cold-stressed armyworm

Growth-blocking peptide (GBP) has been isolated for the first time from the haemolymph of the host armyworm Pseudaletia separata whose development was halted in the last larval instar stage by parasitization with the parasitoid wasp Cotesia kariyai. Recent studies demonstrated that GBP not only exists in the plasma (haemolymph without cells) of parasitized last instar larvae, but also in the plasma of nonparasitized penultimate (5th) instar larvae. Monoclonal antibodies were prepared to measure the titers of GBP in nonparasitized and parasitized larval plasma. One of three monoclonal antibodies raised against GBP, which is the most specific for GBP, was used to quantify the concentration of plasma GBP. As this antibody recognized two plasma peptides other than GBP in crude plasma fractions, each plasma peptide fraction was separated by a reversed phase HPLC, and then plasma GBP level was measured by ELISA. The highest level of plasma GBP detected on Day 0 of the penultimate instar larvae was gradually decreased throughout the larval growth except for the temporary increase on Day 0 of last larval instar. After parasitization on Day 0 of last larval instar, two peaks of plasma GBP titer were detected during the last larval instar, one day and six days after parasitization. This characteristic increase and decrease in plasma GBP level was also observed by transferring last instar larvae of the armyworm from 25 to 10 degrees C, as a result of which larvae delayed pupation by more than 15 days. From these results, it is reasonable to propose that plasma GBP in lepidopteran larvae might control certain upstream steps in a cascade of events leading to pupation; thus, an elevated level of plasma GBP interferes with normal metamorphosis from larvae to pupae.

Hypotensive reactions with a white cell‐reduction filter: activation of kallikrein‐kinin cascade in a patient

blood pressure changes have been recorded during these transfusions. We believe the above two cases are consistent with the acute generation of bradykinin during filtration of the RBC component. Bradykinin generation would ordinarily not cause symptoms, except for the presence of an inhibitor of an enzyme (ACE) that is important to its catabolism? Bradykinin causes hypotension by reducing peripheral resistanceJ; stimulation of smooth muscle of the gastrointestinal tract may well explain the abdominal cramps? The use of prestorage white cell-reduced RBCs was not associated with clinical symptoms, possibly because any bradykinin generated during filtration was catabolized during storage by carryover plasma containing ACE. It is also possible that the mannitol contained in AS-1 RBCs may be contributory, as it has been reported to have negative inotropic effects5 Mannitol is not present in AS-3. This phenomenon is rare in our experience, and the use of RBCs filtered with a similar negatively charged filter before storage appears to be effective in preventing hypotensive reactions. Joseph D. Sweeney, MD Michelle Dupuis, BS MT(ASCP1SBB Anthony J. Mega, MD Herbert C. Lichtman Blood Bank and Transfusion Medicine Research Unit The Miriam Hospital 164 SummitAvenue Providence. RI 02906