Hideki Abe

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.

Analysis of viral DNA, protein and envelope damage after methylene blue, phthalocyanine derivative or merocyanine 540 photosensitization.

Abstract— Although numerous photosensitizers have been used experimentally to decontaminate viruses in cellular blood components, little is known about their mechanisms of photoinactivation. Using M13 bacteriophage and vesicular stomatitis virus (VSV) as model viruses, we have investigated alteration of the viral genome, protein and envelope after phototreatment. Methylene blue (MB) and aluminum phthalocyanine tetrasulfonate (AlPcS4) phototreatment inactivated bacteriophage M13 and decreased the fraction of single‐stranded circular genomic DNA (sc‐DNA) by converting it to linear form. This conversion was enhanced by treating the extracted DNA with piperidine at 55°C. Piperidine‐labile breaks were well correlated to phage survival (5.1% sc‐DNA at 1.7% phage survival for MB) under conditions where only minor differences were seen in the relative abundance of M13 coat protein on sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS‐PAGE). Neither aluminum phthalocyanine (AlPc) nor merocyanine 540 (MC540) inactivated M13 nor were there significant changes observed in DNA and coat protein. Methylene blue, AlPcS4 and AlPc inactivated VSV and inhibited fusion of the virus envelope to Vero cells at pH 5.7 (i.e. with plasma membrane). However, the degree of this inhibition was small compared to the extent of virus inactivation (43% inhibition vs 4.7 log10 or 99.998% inactivation, for MB). In contrast, an antibody to VSV G‐spike protein inhibited fusion at pH 5.7 by 52% with a concomitant decline in VSV infectivity of 0.15 log10 (30%). Few changes were observed in the relative abundance of G protein for MB and AlPcS4 phototreated samples and no additional protein bands were observed on SDS‐PAGE. Phototreatment did not appreciably change the relative fusion ability at pH 7.2 (via the endocytotic pathway). These results collectively suggest that nucleic acid may be an important target for photoinactivation of these model viruses by MB and AlPcS4.

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

Effects of hemoglobin vesicles, a liposomal artificial oxygen carrier, on hematological responses, complement and anaphylactic reactions in rats

Hemoglobin vesicle (HbV), a liposomal oxygen carrier containing human hemoglobin, was intravenously infused into rats. After the infusion of saline, the HbV or empty vesicle (EV), numbers of red cells, leukocytes and platelets in peripheral blood were unchanged during the observation period of one week in addition to each time point among three groups. However, the lymphocyte ratio transiently decreased and the granulocyte ratio increased in the HbV and EV groups at 6 h after the infusion. Those changes returned to the initial value one day after the infusion and those were maintained for the subsequent observation period. No dramatic change was seen in the ratio of CD4+/CD8+ T cells. A transient decrease of the complement titer was observed three days after the infusion of HbV and EV, although the consumption of complement titer was not detected in rat serum by mixing HbV or EV in vitro, indicating that the transient decrease of complement titer in vivo was not due to the consumption of complement due to the interaction with HbV or EV. Multiple infusions of HbV caused the decrease of complement titer only after the first infusion and no allergic reaction was observed. No anaphylactic shock was observed in rats administered with EV several times, while ovalbumin (OVA) sensitized rats died with symptoms of respiratory distress after the second OVA administration. These results indicate that HbV could be administered without serious clinical symptoms or adverse reactions.

Interaction of Hemoglobin Vesicles, a Cellular-Type Artificial Oxygen Carrier, with Human Plasma: Effects on Coagulation, Kallikrein-Kinin, and Complement Systems

Hemoglobin vesicles (HbVs), cellular-type artificial oxygen carriers containing human hemoglobin, were assessed for their biocompatibility by mixing with human plasma in vitro. Among three kinds of HbVs (PEG-DPEA-HbV, PEG-DPPG-HbV and DPPG-HbV), PEG-DPEA-HbV did not affect the extrinsic or intrinsic coagulation activities of the plasma, while PEG-DPPG-HbV and DPPG-HbV tended to shorten the intrinsic coagulation time. The kallikrein-kinin cascade of the plasma was slightly activated by PEG-DPPG-HbV and DPPG-HbV, but not by PEG-DPEA-HbV. The complement consumption of the plasma was observed by incubation with DPPG-HbV, but not with PEG-DPEA-HbV or PEG-DPPG-HbV. These results indicate that PEG-DPEA-HbV has a higher biocompatibility with human plasma.

Potential Involvement of Both Type I and Type II Mechanisms in M13 Virus Inactivation by Methylene Blue Photosensitization

We have investigated the mechanism of virus photoinactivation with methylene blue (MB) by conducting deuterium oxide (D2O), azide ion (N3‐) and oxygen‐dependent, studies. Inactivation of M13 bacteriophage and singlet oxygen (1O2) generation by MB photosensitization were irradiation dose dependent. Inactivation of M13 was enhanced by D2O and inhibited by N3‐, suggesting that 1O2 participates in M13 inactivation by MB photosensitization. However, N3‐ did not inhibit M13 inactivation completely. On the other hand, deoxygenating the reaction solution still caused 52‐67% of M13 inactivation observed in the presence of oxygen. These results suggest that 102‐mediated (Type II) and sensitizer‐mediated (Type I) reactions may both play roles in M13 inactivation by MB photosensitization.

Inactivation of parvovirus B19 in coagulation factor concentrates by UVC radiation: assessment by an in vitro infectivity assay using CFU–E derived from peripheral blood CD34+ cells

BACKGROUND: Nonenveloped and thermostable viruses such as parvovirus B19 (B19) can be transmitted to patients who are receiving plasma‐derived coagulation factor concentrates treated by the S/D method for inactivating enveloped viruses. Therefore, it is important to develop and validate new methods for the inactivation of nonenveloped viruses.

Effects of hemoglobin vesicles on resting and agonist-stimulated human platelets in vitro

Hemoglobin vesicles (HbV) are artificial oxygen carriers that encapsulate a concentrated hemoglobin (Hb) solution with a phospholipid bilayer membrane. The oxygen transporting ability of HbV in vivo has been demonstrated by the transfusion of HbV into hemorrhagic shock rodent models. However, the compatibility of HbV with human blood cells must be evaluated. Preincubation of platelets with concentrations of 20% or 40% HbV had no effect on the binding of PAC-1, a monoclonal antibody that detects activation-dependent conformational changes in αIIbβ3 on platelets, or the surface expression of CD62P in whole blood. ADP-induced increases in PAC-1 binding were significantly enhanced by exposing the platelets to concentrations of either 20% or 40% HbV, whereas the ADP-induced increases in CD62P expression were not affected by HbV treatment at either concentration. Preincubation of platelet-rich plasma (PRP) with HbV minimally reduced the spontaneous release of TXB2 and RANTES, but did not significantly affect the formation of TXB2 or the release of RANTES and β-TG in platelets stimulated with ADP. Similarly, preincubation of PRP with HbV minimally reduced the spontaneous release of RANTES but did not significantly affect the formation of TXB2 or the release of RANTES and β-TG in platelets stimulated with collagen, although collagen-induced serotonin release tended to decrease with HbV pretreatment. These data suggest that the exposure of human platelets to high concentrations of HbV (up to 40%) in vitro did not cause platelet activation and did not adversely affect the formation and secretion of prothrombotic substances or proinflammatory substances triggered by platelet agonists, although one of the earliest events in ADP-induced platelet activation was slightly potentiated by HbV pretreatment at the doses tested. Taken together, these results imply that HbV, at concentrations of up to 40%, do not have any aberrant interactions with either unstimulated or agonist-induced platelets.

Virus removal from hemoglobin solution using Planova Membrane

Abstract Hemoglobin (Hb) vesicles are artificial oxygen carriers that encapsulate concentrated purified Hb with a phospholipid bilayer membrane. They have been confirmed to have sufficient oxygen-transporting ability. Even though strictly inspected outdated red cells are used as a source of Hb, it is necessary to introduce an additional process that inactivates or removes viruses in the process of Hb purification in order to guarantee the utmost safety from infection. In this study, Hb filtration to remove viruses was tested with Planova 35N and 15N (virus removal fitters with a Bemberg microporous membrane). The permeation flux (LMH) and the permeated ratio of Hb solution ([Hb] = 5.6 g/dl) through Planova 35N at 13°C were 36 l/m2/h and almost 100%, respectively. The values for Planova 15N at 13°C were 15 l/m2/h and 95%, respectively. The permeation flux increased to 18 l/m2/h when the temperature was raised to 25°C. Under the same conditions, a high efficiency of removal of a bacteriophage, φX174, was confirmed (>7.7 log). These results indicate that Planova 15N is effective for the process of virus removal from Hb solution.