Clinical utility of the basophil activation test for analysis of allergic transfusion reactions: a pilot study.

BACKGROUND AND OBJECTIVES: In previous studies, we demonstrated that the basophil-activating effects of supernatants found in residual-transfused platelet concentrates (PC-SNs) on whole blood basophils in cases of allergic transfusion reactions (ATRs) could be assessed by the basophil activation test (BAT) in terms of allergen/IgE dependency. However, in these studies, the basophils were derived from third-party healthy volunteers. In this study, we performed BAT using patients' own blood basophils to analyse ATRs. MATERIALS AND METHODS: The BAT was performed in two cases of severe ATRs using residual PC-SNs and the patients' own basophils in the presence and absence of dasatinib, an inhibitor of IgE-mediated basophil activation. RESULTS: In both cases, PC-SNs exhibited basophil-activating activity against the patients' basophils, but not against basophils from third-party healthy volunteers. In addition, basophil activation was inhibited in the presence of dasatinib, indicating that the basophils were activated in an allergen/IgE-dependent manner. Of note, the basophils in Case 2, but not in Case 1, were activated by PC-SNs from some unrelated non-haemolytic transfusion reaction cases. CONCLUSION: This pilot study indicates that the BAT may be useful in clarifying the causal relationship between ATRs and transfused blood as well as in elucidating the mechanisms behind ATRs considering the allergen/IgE-dependent pathway.

A hollow-fibre column system to effectively prepare washed platelets.

BACKGROUND AND OBJECTIVES: We developed a hollow-fibre column system specifically adapted to prepare washed platelet concentrates (WPCs). This study was performed to evaluate the efficacy of the hollow-fibre column system for preparing WPCs. MATERIALS AND METHODS: First, the percentages of platelet (PLT) recovery and remaining plasma proteins were calculated by determining the PLT count, volume and plasma protein levels in both the prewash and postwash. Secondly, washed PLTs and unwashed control PLTs were stored for 5 days, and the changes during this 5-day storage of in vitro PLT characteristics were determined. RESULTS: The hollow-fibre column system effectively removed >98% of plasma in platelet concentrates (PCs), and the PLT recovery was 97% on an average. The CD62P-expression level on washed PLTs immediately after washing was approximately twofold higher than that on prewashed PLTs as well as on PLTs washed via manual methods or cell washing devices. Until day 5 during storage, PLT aggregability, hypotonic shock response and swirling scores of washed PLTs were not significantly different from those of the control PCs. CONCLUSION: Our novel hollow-fibre column system proved valuable in preparing washed PLTs with <2% of residual plasma proteins and high recovery of PLTs.

A novel mutation of the GATA site in the erythroid cell-specific regulatory element of the ABO gene in a blood donor with the Am B phenotype.

The Am and Bm phenotypes are characterized by weak expression of the A or B antigens, respectively, by red blood cells with a normal expression by the saliva of secretors. Deletion of the regulatory element in the first intron of the ABO gene and disruption of the GATA motif in the element were found to be responsible. In this study, we identified a novel mutation within the GATA motif (G>C substitution at position c.28 + 5830) in the regulatory element of the A allele that might diminish transcription activity causing the generation of the Am B phenotype.

A new transfectant panel cell line-based MoAb-independent antigen capture assay system for detection of CD36 antibody.

BACKGROUND AND OBJECTIVES: CD36 antibody (Ab) causes several disorders: neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and non-haemolytic transfusion reactions. However, there is no gold-standard test for CD36 Ab. MATERIALS AND METHODS: We developed a transfectant panel cell line-based MoAb-independent antigen capture assay system for detection of CD36 Ab and compared it with the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) system in terms of sensitivity and specificity. RESULTS: Our new system was characterized by (1) gene-transfected cell lines, but not panel platelets; (2) not being hampered by HLA Abs; and (3) no need to use CD36 MoAbs to ensure the antigen specificity of this detection system. In addition, it showed a much better receiver operating characteristic curve than the MAIPA system. CONCLUSIONS: The present results indicate that our new system permits highly sensitive and specific detection of CD36 Ab.

Evaluation of a blood group genotyping platform (BLOODchip(®) Reference) in Japanese samples.

BACKGROUND: Blood-group genotyping arrays have been widely used in Caucasian and African American populations, but have not been thoroughly tested in Japanese subjects. AIM: To evaluate, using the BLOODchip(®) Reference genotyping system, the concordance of previously typed samples with expected phenotypes and the coverage of the Japanese variants. METHODS: Blood samples from 100 Japanese donors were obtained. DNA was extracted with QIAsymphony (Qiagen, Hilden, Germany). Samples were typed by serological methods and processed with the BLOODchip(®) . When a non-concordant result was identified, further sequencing by polymerase chain reaction-single specific primer (PCR-SSP) was performed. RESULTS: Concordance between systems was 98% (736/751), and 98.8% (742/751) if only non-software-related non-concordances were considered. In the ABO group, 6 'No Call' (NC, inability of the BLOODchip(®) to assign a result) were ascribed to a variant of blood subtype A1 (A102; 467C>T), a common subtype in Asian populations, whereas three NC presented additional polymorphisms not contained in the BLOODchip(®) (A102/A205, A102/O06 and A204/O02). In the RhD group, one discrepancy was correctly genotyped as RHD*1227A (Del phenotype) by the BLOODchip(®) (phenotyped as partial D, RHD*DIVb). Another was phenotyped as D+ by the BLOODchip(®) (phenotyped weak D by serology) and confirmed as RHD*D-CE(2)-D heterozygous by sequencing. The 3 RhD NC can be solved by further software update. For RhCE, one discrepancy was correctly genotyped for both systems; however, only the BLOODchip(®) was able to detect RHCE*CX allele. CONCLUSIONS: By programming the A102 ABO variant into the system software with the new allele combinations, the BLOODchip(®) Reference is a suitable genotyping tool to be applied to Asian samples.

Approach of using established and new laboratory tests to more comprehensively investigate noninfectious and nonhemolytic transfusion reactions--along with the experience in Japan.

BACKGROUND AND OBJECTIVES: Noninfectious and nonhaemolytic transfusion reactions are the most common type of transfusion reactions. Several new tests have been made, helping diagnosis and understanding of their pathogenesis. This manuscript provides a review of the literature on currently available tests in association with the approach in Japan. MATERIALS & METHODS: Primarily by using key words, more than 100 pertinent articles in the Medline database were identified and reviewed. RESULTS: Numbers of laboratory tests are available including those for plasma protein levels, plasma protein antibodies, leucocyte and platelet antibodies, serum N-terminal-pro-brain natriuretic peptide levels, serum tryptase levels and genetic microchimerism. Cross-match tests, such as basophil activation test and neutrophil activation test, are also available to determine a causal relationship between the reaction and transfusion. CONCLUSIONS: Several tests should help to confirm diagnosis and determine causal relationship between adverse reactions and transfusion and to gain an insight into the mechanism of the reaction in some cases, although some of the recently developed tests have not been completely validated.

A standardized immunofluorescence test method with human neutrophil antigen-expressing cell lines to enhance antibody detection.

There is an international need for a large-scale human neutrophils antigen (HNA) antibody screening platform to minimize the risk of antibody-mediated transfusion-related acute lung injury. However, sourcing a substantial, reliable source of HNA, as well as the scarcity of well-characterized HNA antisera for validating new screening platforms, remain as major obstacles. This short communication presents an improved protocol for the effective use of HNA-expressing KY cells as a screening platform using eight well-characterized HNA antisera of a single defined specificity.

Prevalence and risk factors for third- and fourth-degree perineal lacerations during vaginal delivery: a multi-country study.

OBJECTIVE: To investigate the prevalence and risk factors of third- and fourth-degree perineal lacerations in 24, mainly developing, countries. DESIGN: Analysis using cross-sectional data from the WHO Global Survey on Maternal and Perinatal Health. SETTING: Seven African, nine Asian and eight Latin American countries. POPULATION: Women at admission to hospital for delivery in 373 facilities between 2004 and 2008. METHODS: We estimated the country-wise prevalence of third- and fourth-degree perineal lacerations, and conducted region-wise multivariate logistic regression analyses to identify its risk factors. MAIN OUTCOME MEASURES: Prevalence and risk factors of third- and fourth-degree perineal lacerations. RESULTS: A total of 214,599 women who underwent vaginal delivery were analysed. The prevalence of third- and fourth-degree perineal lacerations ranged widely across countries [from 0.1% (China, Cambodia, India) to 15.0% (Philippines)] and facilities (from null to 76.3%). After the deletion of facilities reporting no third- or fourth-degree perineal lacerations, and also highly outlying facilities, the range in prevalence was 0.1% (Uganda) to 1.4% (Japan). Forceps-assisted delivery, nulliparity and high birthweight were significant risk factors in all three regions. Vacuum-assisted delivery was also a significant risk factor in Africa and Asia. CONCLUSIONS: Misdiagnosis of third- and fourth-degree perineal lacerations in developing countries may be common. Correct recognition and diagnosis may lead to timely treatment and fewer sequelae. Risk factors of third- and fourth-degree perineal lacerations in developing countries were similar to those previously reported from developed countries.

Role of mitochondria in the maintenance of platelet function during in vitro storage.

AIMS/OBJECTIVES: Platelets undergo structural and biochemical alterations during in vitro storage and these are collectively called platelet storage lesions (PSL). The mitochondrion is an important cell organelle involved not only in energy production but also in the regulation of cellular functions and viability. This implies that some platelet functions may be regulated by mitochondria; hence, preservation of mitochondrial functions may be important for the maintenance of platelet quality in stored platelet concentrates (PCs). This work describes the effects of various compounds on mitochondrial functions important for the maintenance of platelet quality in in vitro stored PCs. METHODS: PCs were stored at 22 °C with gentle agitation in the presence or absence of 2,4-dinitrophenol, antimycin A, acetyl-l-carnitine and ascorbic acid. The effects of these products on platelet quality were assessed by analysing glucose and lactate concentrations, pH of the storage medium, shape of the platelets, mitochondrial membrane potential and depolarisation, surface expression of CD62P and collagen-induced platelet aggregation. RESULTS: 2,4-Dinitrophenol and antimycin A increased PSL levels, whereas acetyl-l-carnitine reduced the level of changes in pH and mitochondrial depolarisation. Ascorbic acid in the storage medium resulted in improved levels of collagen-induced platelet aggregation. However, none of the examined reagents suppressed CD62P expression in platelets. CONCLUSIONS: These results suggest that preservation of mitochondrial function is fundamental, but not fully sufficient, for the maintenance of platelet in vitro quality during storage. Further research is necessary to develop methods for preserving both mitochondrial and platelet functions in in vitro stored PCs.

Establishment of a cell line panel as an alternative source of platelet antigens for a screening assay of anti-human platelet antibodies.

BACKGROUND: A panel of platelets expressing various human platelet antigens (HPAs) for a platelet antibody screening assay is difficult to prepare because some antigens are rarely expressed. Therefore, an alternative method without using platelets would be helpful in detecting HPA antibodies. This study describes the establishment of cell lines that stably express specific HPAs and their application for detecting specific antibodies. METHODS: Wild-type ß3, HPA-1b, -6b, -7b and -7 variant cDNA as well as wild-type αIIb and HPA-3b cDNA were individually co-transduced with wild-type αIIb and ß3 cDNA in the K562 cell line. We performed an immunobead monoclonal antibody immobilisation of platelet antigens (MAIPA) assay to evaluate this cell line panel for antibody detection using identified sera containing HPA antibodies, whose specificities had been determined by the mixed passive haemagglutination test. RESULTS AND CONCLUSION: Of the 12 sera containing HPA-1a (n = 2), HPA-3a (n = 6), HPA-6b (n = 3) or HPA-7 variant (n = 1) antibodies, all antibodies were detected and determined by our new method, except for two HPA-3a antibodies. One of the two antibodies was also negative for conventional platelet MAIPA, suggesting that the cell line panel might be used as an alternative source of platelet antigens in the MAIPA assay.

Frequency-Domain Multiplexing Readout with a Self-Trigger System for Pulse Signals from Kinetic Inductance Detectors.

We present the development of a frequency-domain multiplexing readout of kinetic inductance detectors (KIDs) for pulse signals with a self-trigger system. The KIDs consist of an array of superconducting resonators that have different resonant frequencies individually, allowing us to read out multiple channels in the frequency domain with a single wire using a microwave-frequency comb. The energy deposited to the resonators break Cooper pairs, changing the kinetic inductance and, hence, the amplitude and the phase of the probing microwaves. For some applications such as X-ray detections, the deposited energy is detected as a pulse signal shaped by the time constants of the quasiparticle lifetime, the resonator quality factor, and the ballistic phonon lifetime in the substrate, ranging from microseconds to milliseconds. A readout system commonly used converts the frequency-domain data to the time-domain data. For the short pulse signals, the data rate may exceed the data transfer bandwidth, as the short time constant pulses require us to have a high sampling rate. In order to overcome this circumstance, we have developed a KID readout system that contains a self-trigger system to extract relevant signal data and reduces the total data rate with a commercial off-the-shelf FPGA board. We have demonstrated that the system can read out pulse signals of 15 resonators simultaneously with about 10 Hz event rate by irradiating α particles from 241 Am to the silicon substrate on whose surface aluminum KID resonators are formed.

A single base insertion of the 4-alpha-galactosyltransferase gene led to the deficiency of Gb3 biosynthesis.

cDNAs for alpha 1,4 galactosyltransferase (A4GALT) have been isolated. To explore the molecular basis of the p phenotype in Japanese donors, we analyzed the A4GALT gene sequences of normal and p phenotype samples. The coding region in the A4GALT gene for DNA sequencing was amplified by PCR amplification. A4GALT expression vectors for individual were constructed by PCR amplification of the coding region using primers and subsequent subcloning into an expression vector. The expression of Gb3/CD77 antigen on the cell surface was evaluated by flow cytometry and by immunochemical techniques. All individuals with the p phenotype were found to have a single base insertion (A4GALT/insC) at the same nucleotide position. Neither the transfectant cells with a mutant gene (A4GALT/insC) of donor origin or those with a synthesized mutant gene (A4GALT/insC-Mu) expressed Gb3 antigen indicating that the presence of A4GALT/insC diminished the A4GALT enzyme activity. In addition, an allele-specific PCR (ASP) system was developed in which of the p phenotype with A4GALT/insC can be unambiguously discriminated from normal donors. Based on the finding that a single base insertion (A4GALT/insC) diminishes A4GALT activity, an ASP assay was developed to detect individuals with this particular p phenotype.

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 10(3)-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.

Stromal cell-independent differentiation of human cord blood CD34+CD38- lymphohematopoietic progenitors toward B cell lineage.

To study the cytokine regulation of early stages of human B-lymphopoiesis, we developed a stroma-free two-step culture system. Single human cord blood CD34+CD38- cells were individually cultured by micromanipulation with interleukin (IL)-3, stem cell factor (SCF), fIt3 ligand (FL), IL-6 and granulocyte colony-stimulating factor (G-CSF). About 10% of the cells formed primary colonies, which were individually tested for myeloid and B-lymphoid potentials by reculturing aliquots of the primary colony cells into secondary myeloid and B-lymphoid cultures. One third of the primary colonies proved capable of differentiation into CD19+IgM+ cells, as well as into myeloid lineage cells. RT-PCR analyses revealed that some cells in the primary culture had already matured to express B cell-specific transcripts. Thus, the combination of IL-3, SCF, FL, IL-6 and G-CSF supported the differentiation of CD34+CD38- lymphohematopoietic progenitors toward B cell lineage in addition to myeloid lineages. Screening of cytokines to identify the minimum requirement of cytokines in the primary culture revealed that IL-3 and SCF were essential and that the addition of FL, and to a lesser extent IL-6 or G-CSF, to the combination of IL3 and SCF remarkably enhanced the primary colony formation and the generation of CD19+ cells in the secondary B-lymphoid culture.

Modulation of early B lymphopoiesis by interleukin-3.

We recently reported that interleukin-3 (IL-3) inhibits B lymphoid lineage expression in methylcellulose culture in a dose-dependent manner. We subsequently used flow cytometric analysis of individual colonies in timed exposure to IL-3 to define more precisely the negative regulatory role of IL-3 in early B lymphopoiesis. When lymphohematopoietic progenitors isolated from 5-fluorouracil (5-FU)-treated mice were cultured in the presence of Steel factor (SF), IL-11, IL-7, and erythropoietin (Epo), B lymphopoiesis appeared to proceed through three stages: lymphohematopoietic proliferation, commitment, and early B lymphoid proliferation. When IL-3 was added to the culture for a 48-hour interval from days 4 to 6 of culture, IL-3 slightly enhanced the formation of pre-B cell colonies. These data appeared to contradict our previous observations that continued exposure to IL-3 from days 0 to 4 or longer severely inhibits B lymphoid potential of the cultured cells. A more frequently timed kinetic observation revealed that in the presence of IL-3 the peak of lymphohematopoietic progenitors was 48 hours earlier but less than one-tenth the number of lymphohematopoietic progenitors in cultures without IL-3. When added to cultures for 48 hours beyond day 6 of culture, IL-3 abrogated the B cell potential of the cultured cells. However, IL-3 failed to negatively modulate B lymphoid progenitors when added on day 14 of culture or later. These observations indicate that IL-3 is a potent negative modulator of the early B lymphopoiesis. IL-3 appears to hasten but suppress the proliferation and commitment of lymphohematopoietic progenitors to B cell lineage. It may also inhibit the proliferation of the progenitors immediately after commitment to B cell lineage.

Interleukin-4 (IL-4) in combination with IL-11 or IL-6 reverses the inhibitory effect of IL-3 on early B lymphocyte development.

We have previously described a two-step methylcellulose culture system in which individual primitive progenitors from 5-fluorouracil (5-FU)-treated mice were shown to have both myeloid and B lymphoid differentiation capacity. Highly enriched Lin-Sca+FU2d BM cells were cultured in methylcellulose in the presence of Steel factor (SF), interleukin-7 (IL-7), and pokeweed mitogen stimulated spleen cell conditioned medium (PWM-SCM). Primary mixed myeloid colonies were replated after 8-11 days into secondary cultures containing SF and IL-7, which supported the generation of B220+sIgM- pre-B cell colonies. A number of growth factors, including IL-6, IL-11, granulocyte colony-stimulating factor (G-CSF), and IL-12 were shown to be capable of substituting for PWM-SCM to support the B lymphoid potential of primary colonies. B lymphoid potential was not supported, however, in SF + IL-3 or in SF + IL-3 plus any single growth factor (IL-1 to -12, granulocyte-macrophage colony-stimulating factor [GM-CSF], G-CSF, erythropoietin [Epo], leukemia inhibitory factor [LIF], tumor necrosis factor-alpha [TNF-alpha], transforming growth factor-beta [TGF-beta], gamma interferon [IFN-gamma], or insulin-like growth factor-1 [IGF-1]), but was supported in SF + IL-3 + 5% PWM-SCM. Experiments were designed to identify the factor or factors in PWM-SCM that reverse the inhibitory effects of IL-3 on B lymphoid potential. By substituting various cytokine combinations for PWM-SCM, we determined that combinations of IL-4 + IL-6 or IL-4 + IL-11, but not IL-4 alone, can substitute for PWM-SCM to reverse the inhibitory effect of IL-3 on B lymphoid potential. Neutralizing antibodies to IL-4 completely eliminated the activity in PWM-SCM, but antibodies to IL-6 only partially inhibited the activity. IL-11 was not detected in PWM-SCM, and the activity co-purified with IL-4, but not with IL-6. Thus, IL-4 plus IL-6, IL-11, or one or more unidentified growth factors in PWM-SCM can reverse the inhibitory effects of IL-3 on early B lymphocyte development in culture.

Serum-free coculture system for ex vivo expansion of human cord blood primitive progenitors and SCID mouse-reconstituting cells using human bone marrow primary stromal cells.

OBJECTIVE: In an attempt to maintain and expand human stem cells, many investigators have used xenogeneic, especially murine, stromal cells and fetal calf serum. Because of the possible transmission of infectious diseases, however, the safety of the delivery of grafts expanded in culture using xenogeneic cells and serum has been debated. Using primary human marrow stromal cells, we established a novel serum-free culture system to expand human primitive progenitors and transplantable stem cells. MATERIALS AND METHODS: Cord blood CD34(+) cells were cultured on a monolayer of human primary marrow stromal cells in the presence of thrombopoietin (TPO), flt3/flk2 ligand (FL), and/or stem cell factor (SCF) under serum-free conditions. After 2 or 4 weeks of culture, cells were examined for clonogenic progenitors and severe combined immunodeficient disorder (SCID) mouse-reconstituting cells (SRC). RESULTS: In the presence of TPO, FL, and SCF, marrow stromal cells supported more than a 100- and 1,000-fold expansion of CD34(+) cells and colony-forming units in culture after 2 and 4 weeks of incubation, respectively. In addition, cobblestone area-forming cells were expanded more than 18- and 60-fold after 2 and 4 weeks of culture, respectively. Furthermore, SRC assay demonstrated augmented engraftment by cultured cells. CONCLUSION: This ex vivo expansion system should prove valuable in clinical settings in which stromal cells are available from recipients or stem cell donors.

6A-O-[(4-biphenylyl)acetyl]-alpha-, -beta-, and -gamma-cyclodextrins and 6A-deoxy-6A-[[(4-biphenylyl)acetyl]amino]-alpha-, -beta-, and -gamma-cyclodextrins: potential prodrugs for colon-specific delivery.

Cyclodextrins (CyDs) are known to be fermented to small saccharides by colonic microflora, whereas they are only slightly hydrolyzable and thus are not easily absorbed in the stomach and small intestine. This property of CyDs is particularly useful for colon-specific delivery of drugs. In this study, an antiinflammatory 4-biphenylylacetic acid (BPAA) was selectively conjugated onto one of the primary hydroxyl groups of alpha-, beta-, and gamma-CyDs through an ester or amide linkage, 6A-O-[(4-biphenylyl)acetyl[-alpha-, -beta-, and -gamma-CyDs (1-3) and 6A-deoxy-6A-[[(4-biphenylyl)acetyl]amino]-alpha-, -beta-, and -gamma-CyDs (4-6). In rat cecal and colonic contents (10%, w/v), 1 and 3 released more than 95% of BPAA within 1-2 h, and 2 released about 50% of the drug within 12 h. The amide prodrugs, 4-6, did not release BPAA in the cecal contents, but gave BPAA/maltose or BPAA/triose conjugates linked through an amide bond. On the other hand, these prodrugs were found to be stable in the contents of rat stomachs and small intestines, in intestinal or liver homogenates, and in rat blood. The serum levels of BPAA increased about 3 h after oral administration of 1 and 3 to rats, accompanying a marked increase in the serum levels, whereas 2 and 4-6 resulted in little increase of the serum levels. These facts suggest that BPAA is released after the ring opening of CyDs followed by the ester hydrolysis, and the BPAA activation takes place site-specifically in the cecum and colon. Therefore, the present CyD prodrug approach provides a versatile means of constructing a novel colon-specific drug delivery system.

Quinolone antimicrobial agents substituted with morpholines at the 7-position. Syntheses and structure-activity relationships.

A series of novel 7-substituted 1-cyclopropyl-6,8-difluoro-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acids have been prepared and tested for antibacterial activities and for convulsive activities in combination with nonsteroidal antiinflammatory drug. Structure-activity relationships revealed that 7-(2-(aminomethyl)morpholino) derivative 28 had a better Gram-positive activity than the reference quinolones, such as ciprofloxacin, norfloxacin, and ofloxacin. Its Gram-negative activity was equipotent with those of norfloxacin and ofloxacin but was inferior to that of ciprofloxacin. In mouse systemic infection models, 28 showed an excellent therapeutic efficacy which might result from the potent antibacterial activity and suitable physicochemical properties. Convulsive activities of 7-morpholino derivatives in combination with nonsteroidal antiinflammatory drug fenbufen or its metabolite biphenylacetic acid markedly diminished as compared to those of 7-piperazino derivatives in the electrophysiological, biochemical, and behavioral experiments. These results suggest that 28 (Y-26611) is a novel quinolone with reduced neurotoxic excitatory adverse reaction.