mDia1/3-dependent actin polymerization spatiotemporally controls LAT phosphorylation by Zap70 at the immune synapse.

The mechanism by which the cytosolic protein Zap70 physically interacts with and phosphorylates its substrate, the transmembrane protein LAT, upon T cell receptor (TCR) stimulation remains largely obscure. In this study, we found that the pharmacological inhibition of formins, a major class of actin nucleators, suppressed LAT phosphorylation by Zap70, despite TCR stimulation-dependent phosphorylation of Zap70 remaining intact. High-resolution imaging and three-dimensional image reconstruction revealed that localization of phosphorylated Zap70 to the immune synapse (IS) and subsequent LAT phosphorylation are critically dependent on formin-mediated actin polymerization. Using knockout mice, we identify mDia1 and mDia3, which are highly expressed in T cells and which localize to the IS upon TCR activation, as the critical formins mediating this process. Our findings therefore describe previously unsuspected roles for mDia1 and mDia3 in the spatiotemporal control of Zap70-dependent LAT phosphorylation at the IS through regulation of filamentous actin, and underscore their physiological importance in TCR signaling.

Present status and perspective of laboratory hematology in Japan: On the standardization of blood cell morphology including myelodysplasia: On behalf of the Japanese Society for Laboratory Hematology.

The Japanese Society for Laboratory Hematology (JSLH) was launched in 2000 and has been developed by a mutual collaboration of hematologists, medical technologists, and the companies involved in hematological laboratory testing. The aim of JSLH is the progress and development of laboratory hematology by academic conferences, periodic publication of academic journal, training and education (in the meeting, the journal, or the website), promotion of the standardization of laboratory hematology, and certification of the laboratory hematology specialists. Among 3 specialized committees organized for the standardization of laboratory hematology, the standardization committee on blood cell morphology has been dealing with the various projects on peripheral/bone marrow blood cells and normal/abnormal morphology. Another independent organization, the Japanese National Research Group on idiopathic bone marrow failure syndromes (BMF), has raised the importance of the dysplasia of myelodysplastic syndromes (MDS) and has been conducting the prospective registration, central review, and follow-up study of MDS. This group recently proposed the grading system for diagnostic accuracy of MDS, and the detailed procedure of morphological diagnosis of MDS is presented in the specialized color atlas with typical photographs of various dysplasia. JSLH has also approved the grading system for diagnostic accuracy of MDS and adopted this standardized diagnostic system to the educational item for certification of the laboratory hematology specialists, aiming at a nationwidely expanding morphological evaluation of myelodysplasia. Further and significant progress in the standardization of blood cell morphology will be expected in Japan through the activity of JSLH.

Gliomatosis cerebelli in a Saint Bernard dog.

A 6-year-old, neutered male Saint Bernard dog was presented with a 1-month history of ataxia, hypermetria and head tilt. High-field magnetic resonance imaging revealed a mass in the cerebellar vermis. During necropsy examination, a cream-coloured irregular area was observed in the cerebellar white matter. Microscopically, the mass comprised a diffuse neoplastic proliferation of spindle cells with oval pleomorphic nuclei in the white and grey matter of the cerebellum and pons and in the subpial area. Neoplastic infiltration was not found in the cerebrum. Immunohistochemistry revealed that the neoplastic cells were positive for vimentin and partially positive for glial fibrillary acidic protein. Based on these findings, the neoplastic lesion was diagnosed as gliomatosis cerebelli, without involvement of the cerebrum.

Lenalidomide induces cell death in an MDS-derived cell line with deletion of chromosome 5q by inhibition of cytokinesis.

Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell disorders characterized by refractory cytopenias and susceptibility to leukemic transformation. On a subset of MDS patients with deletion of the long arm of chromosome 5 (del(5q)), lenalidomide exerts hematological and cytogenetic effects, but the underlying pharmacological mechanisms are not fully understood. In this study, we have investigated the in vitro effects of lenalidomide on an MDS-derived cell line, MDS-L, which carries del(5q) and complex chromosome abnormalities. We found that the growth of MDS-L cells was specifically suppressed mainly by apoptosis, and in addition, multinucleated cells were frequently formed and finally died out in the presence of lenalidomide. Time-lapse microscopic observation and the DNA ploidy analysis revealed that lenalidomide does not affect DNA synthesis but inhibits cytokinesis of MDS-L cells. The gene expression profile showed decreased expression of M phase-related genes such as non-muscle myosin heavy-chain 10, polo-like kinase 1, aurora kinase B, citron kinase and kinesin family member 20A(KIF20A). Interestingly, KIF20A is located at 5q31. These data contribute to the understanding of action mechanisms of lenalidomide on MDS with del(5q) and complex abnormalities.

Myelodysplastic syndrome with chromosome 5 abnormalities: a nationwide survey in Japan.

Chromosome 5 abnormalities, deletion of the long arm of chromosome 5 (del(5q)) or monosomy 5 (-5), arise in about 10% of myelodysplastic syndromes (MDS), either as the sole cytogenetic abnormality or as part of complicated karyotype, and has distinct clinical implications for MDS. However, the prognostic factors of MDS patients with chromosome 5 abnormalities are not determined yet. In this study, 183 Japanese MDS patients with chromosome 5 abnormalities were analyzed. Estimated incidence of del(5q) and 5q- syndrome among MDS patients was 8.4 and 1.3%, respectively. Significant shorter overall survival (OS) and leukemia-free survival (LFS) were observed in -5 patients than del(5q) patients. Among del(5q) patients, addition of monosomy 7 or complex karyotype with more than three abnormalities were significantly related to shorter OS. LFS of del(5q) patients was divided into two risk groups by international prognostic scoring system (IPSS): low/intermediate (Int)-1 and Int-2/high groups. LFS sorted by World Health Organization classification-based prognostic scoring system (WPSS) was also divided into two groups: very low/low/Int and high/very high, and WPSS was able to predict the outcome of del(5q) patients more clearly than IPSS. Together with additional cytogenetic data, WPSS might be useful for clinical decision making in MDS patients with del(5q).

Activation of the Keap1/Nrf2 pathway for neuroprotection by electrophilic [correction of electrophillic] phase II inducers.

Electrophilic neurite outgrowth-promoting prostaglandin (NEPP) compounds protect neurons from oxidative insults. At least part of the neuroprotective action of NEPPs lies in induction of hemeoxygenase-1 (HO-1), which, along with other phase II enzymes, serve as a defense system against oxidative stress. Here, we found that, by using fluorescent tags and immunoprecipitation assays, NEPPs are taken up preferentially into neurons and bind in a thiol-dependent manner to Keap1, a negative regulator of the transcription factor Nrf2. By binding to Keap1, NEPPs prevent Keap1-mediated inactivation of Nrf2 and, thus, enhance Nrf2 translocation into the nucleus of cultured neuronal cells. In turn, Nrf2 binds to antioxidant/electrophile-responsive elements of the HO-1 promoter to induce HO-1 expression. Consistent with this notion, NEPP induction of an HO-1 reporter construct is prevented if the antioxidant-responsive elements are mutated. We show that NEPPs are neuroprotective both in vitro from glutamate-related excitotoxicity and in vivo in a model of cerebral ischemia/reperfusion injury (stroke). Our results suggest that NEPPs prevent excitotoxicity by activating the Keap1/Nrf2/HO-1 pathway. Because NEPPs accumulate preferentially in neurons, they may provide a category of neuroprotective compounds, distinct from other electrophilic compounds such as tert-butylhydroquinone, which activates the antioxidant-responsive element in astrocytes. NEPPs thus represent a therapeutic approach for stroke and neurodegenerative disorders.

Neurotropism of swine haemagglutinating encephalomyelitis virus (coronavirus) in mice depending upon host age and route of infection.

Mice aged 1, 4 or 8 weeks were inoculated with haemagglutinating encephalomyelitis virus (HEV), strain 67N, by the intracerebral (i.c.), intranasal (i.n.), intraperitoneal (i.p.), subcutaneous (s.c.), intravenous (i.v.) or oral route, with different doses. In 1-week-old mice, mortality and mean time to death were mostly the same regardless of the inoculation route, except for the oral route, which appeared to be the least effective. The virus killed 4-week-old mice readily by all routes of inoculation except the oral, and 8-week-old mice by i.c., i.n. or s.c. inoculation. In descending order of efficacy, the routes of HEV infection were: i.c., i.n., s.c., i.p., i.v. and oral. To follow the spread of HEV from peripheral nerves to the central nervous system (CNS), the virus was inoculated subcutaneously into the right hind leg of 4-week-old mice. The virus was first detected in the spinal cord on day 2, and in the brain on day 3. The brain titres became higher than those of the spinal cord, reaching a maximum of 10(7)PFU/0.2 g when the animals were showing CNS signs. Viral antigen was first detected immunohistochemically in the lumbar spinal cord and the dorsal root ganglion ipsilateral to the inoculated leg; it was detected later in the pyramidal cells of the hippocampus and cerebral cortex, and in the Purkinje cells of the cerebellum but not in the ependymal cells, choroid plexus cells or other glial cells. The infected neurons showed no cytopathological changes.

[Biologic changes in MDS-L cell line induced by As2O3 and/or TRAIL].

OBJECTIVE: To investigate the biological changes in myelodysplastic syndromes (MDS) myeloid blast cell line MDS-L after different duration and concentration of As2O3/TRAIL (TNF related apoptosis inducing ligand) treatment. METHODS: MDS-L cells were treated with As2O3 and TRAIL at 9 different concentrations and the treated cells were detected at 24 h, 48 h and 72 h for biologic indexes. The same detections were conducted in untreated MDS-L cells and normal and MDS marrow cells as controls. Apoptosis was assayed by flow cytometry after Annexin V-FITC labelling. Differentiation-induction effect of these drugs on the cells were detected by morphologic examination and CD34(+) proportion analysis after 24 hours treatment and further agar culture for 18 days; P15(ink4b) mRNA expression were detected by RT-PCR and its protein expression by DAB immunocytochemistry, P15(ink4b) DNA methylation by methylation specific PCR (Msp). RESULTS: As2O3/TRAIL treatment promoted MDS-L cells to undergo apoptosis and As2O3 plus TRAIL showed obvious differentiation-induction effect on MDS-L. P15(ink4b) mRNA expression was upregulated in MDS-L cell line after different drug treatment but almost no protein expression increased. Increased P15 expression seemed to be related with DNA demethylation effect of these drugs. CONCLUSIONS: As2O3 or/and TRAIL treatment could promote apoptosis of the clonal cells and induce incomplete cell differentiation. The drugs treatment could also increase P15(ink4b) expression in MDS-L cell line through their demethylation effects.

Adhesion via CD43 induces Syk activation and cell proliferation in TF-1 cells.

The effect of adhesion via CD43 (leukosialin, sialophorin) on cell proliferation and phosphorylation signaling were examined in a growth factor-dependent hematopoietic progenitor cell line, TF-1. TF-1 cells promptly resulted in death after withdrawal of growth factors. However, the viable cell number increased when TF-1 cells were cultured on anti-CD43 monoclonal antibody-coated plates. In this case, sustained activation of protein tyrosine kinase Syk and extracellular signal-regulated kinase (Erk) 1/2 were detected. Overexpression of exogenous Syk on TF-1 cells by the adenovirus vector system induced enhancement of the cell proliferation accompanied with enhancement of the Erk activation by a dominant-positive effect. The signal transducer and activator of transcription (STAT) 5 seemed not to be associated with the CD43-mediated cell proliferation. These results indicated that adhesion via CD43 induces the proliferation of TF-1 cells in the absence of growth factors in part by Syk-dependent Erk 1/2 signaling.

Basolateral sorting of human poliovirus receptor alpha involves an interaction with the mu1B subunit of the clathrin adaptor complex in polarized epithelial cells.

Poliovirus receptor (hPVR/CD155) is a cell surface glycoprotein that belongs to the immunoglobulin superfamily but its natural function remains unknown. Two membrane-bound isoforms, hPVRalpha and hPVRdelta, are known to date, and they differ only in the amino acid sequence of their cytoplasmic domains. To gain an insight into the possible function of the cytoplasmic domains, we examined the localization of introduced hPVRalpha and hPVRdelta in polarized epithelial cells deficient of native hPVRs. Basolateral sorting of hPVRalpha was observed in Madine-Darby canine kidney cells expressing mu1B, but not in LLC-PK1 porcine kidney cells deficient in mu1B. Distribution of hPVRdelta, however, occurred both on the apical and basolateral plasma membranes of these two cell lines. Basolateral sorting of hPVRalpha was also seen in LLC-PK1 cells that expressed an intact exogenous mu1B, but not in the cells that expressed a mutant mu1B lacking binding ability to tyrosine-containing signals. These results indicate that mu1B is involved in the distribution of hPVRalpha to the basolateral membrane. Comparative distribution analysis of hPVRalpha using a series of mutants with truncations and substitutions in the cytoplasmic tail demonstrated that determinant for the basolateral sorting resided in the tyrosine-containing motif of the cytoplasmic tail. Furthermore, yeast two hybrid analysis strongly suggested that the tyrosine motif directly interacted with mu1B protein. Thus, basolateral sorting of hPVRalpha appears to involve the interaction with mu1B through a tyrosine motif existing in the cytoplasmic domain.

Generalized Wegener's granulomatosis responding to sulfamethoxazole-trimethoprim monotherapy.

Wegener's granulomatosis (WG) has two different clinical phases: the initial phase and generalized phase. In patients with generalized WG, both steroids and cyclophosphamide have generally been used. We report a case of generalized WG that was temporarily, but successfully treated with sulfamethoxazole-trimethoprim (S/T) alone. S/T therapy reduced the elevated levels of soluble IL-2 receptor and IL-6 in parallel with improvement of the patient's symptoms and urinary protein excretion. In view of the high incidence of lethal adverse effects of cytotoxic drugs, S/T monotherapy may be worth trying not only for initial phase WG but also for generalized WG with careful follow-up when the patient is not acutely ill.

Susceptibility of rats of different ages to inoculation with swine haemagglutinating encephalomyelitis virus (a coronavirus) by various routes.

Haemagglutinating encephalomyelitis virus, strain 67N, was used to inoculate 1-, 2-, 4- and 8-week-old rats by the intracerebral (i.c.), intranasal (i.n.), intraperitoneal (i.p.), subcutaneous (s.c.), intravenous (i.v.) and oral routes with graded doses. The routes of infection, in descending order of efficacy, were: i.ci.ns.ci.pi.v. and oral. Rats aged 1 and 2 weeks were generally similar in terms of mortality and mean time to death, regardless of inoculation route, except for the oral route, which had little effect. In comparison with the 1- and 2-week-old rats, the 4-week-old rats were less susceptible to the virus by all routes. Eight-week-old rats inoculated by the i.ci.n. or s.c. routes died, but all those inoculated by other routes survived. To follow the spread of virus in the central nervous system, 4-week-old rats inoculated by the i.c. route were examined. The virus was first detected in the brain on day 1 and in the spinal cord on day 2. The viral titres in both tissues reached a plateau of 10(7) plaque-forming units (PFU)/0.2 g by day 4, at which time clinical signs had developed. By immunohistochemical analysis, virus-specific antigen was found first in the pyramidal cells of the hippocampus and cerebral cortex, and later in the large-sized neurons of the pons and spinal cord. Still later (day 4) immunolabelling was found in Purkinje cells of the cerebellum, but not in the ependymal cells, choroid plexus or other glial cells.

Caspase-3 activation by lysosomal enzymes in cytochrome c-independent apoptosis in myelodysplastic syndrome-derived cell line P39.

In most cases, apoptosis is considered to involve mitochondrial dysfunction with sequential release of cytochrome c from mitochondria, resulting in activation of caspase-3. However, we found that etoposide induced apoptosis in P39 cells, a myelodysplastic syndrome-derived cell line, without the release of cytochrome c. Furthermore, in etoposide-treated P39 cells, no changes in mitochondrial membrane potential (delta psi m) were detected by flow cytometry. Flow cytometry using a pH-sensitive probe demonstrated that lysosomal pH increased during early apoptosis in P39 cells treated with etoposide. A reduction in the ATP level preceded the elevation of lysosomal pH. In addition, specific inhibitors of vacuolar H+-ATPase induced apoptosis in P39 cells but not in HL60 cells. Although etoposide-induced activation of caspase-3 was followed by DNA ladder formation in P39 cells, E-64d, an inhibitor of lysosomal thiol proteases, specifically suppressed etoposide-induced activation of caspase-3. Western blotting analysis provided direct evidence for the involvement of a lysosomal enzyme, cathepsin L. These findings indicate that lysosomal dysfunction induced by a reduction in ATP results in leakage of lysosomal enzymes into the cytosolic compartment and that lysosomal enzyme(s) may be involved in activation of caspase-3 during apoptosis in P39 cells treated with etoposide.

Early and progressive accumulation of reactive microglia in the Huntington disease brain.

Microglia may contribute to cell death in neurodegenerative diseases. We studied the activation of microglia in affected regions of Huntington disease (HD) brain by localizing thymosin beta-4 (Tbeta4), which is increased in reactive microglia. Activated microglia appeared in the neostriatum, cortex, and globus pallidus and the adjoining white matter of the HD brain, but not in control brain. In the striatum and cortex, reactive microglia occurred in all grades of pathology, accumulated with increasing grade, and grew in density in relation to degree of neuronal loss. The predominant morphology of activated microglia differed in the striatum and cortex. Processes of reactive microglia were conspicuous in low-grade HD, suggesting an early microglia response to changes in neuropil and axons and in the grade 2 and grade 3 cortex, were aligned with the apical dendrites of pyramidal neurons. Some reactive microglia contacted pyramidal neurons with huntingtin-positive nuclear inclusions. The early and proximate association of activated microglia with degenerating neurons in the HD brain implicates a role for activated microglia in HD pathogenesis.

Correlation between putative inhibitory molecules at the dorsal root entry zone and failure of dorsal root axonal regeneration.

The molecular mechanisms involved in preventing regenerating dorsal root axons from entering the spinal cord at the dorsal root entry zone (DREZ) are obscure. We used immunohistochemistry, in situ hybridization, and electron microscopy to study axonal regeneration after dorsal rhizotomy in adult rats and its relationship to cellular changes and the distribution of putative growth inhibitory molecules in this region. Astrocyte processes, ending as bulb-shaped expansions, grew up to 700 microm into the basal lamina tubes of injured roots, where regenerating axons were also present. Some of these axons approached or reached the DREZ but grew no further; others turned back toward the ganglion, suggesting the presence of repulsive cues in or near the DREZ. Tenascin-C mRNA and protein and CSPG stub immunoreactivity were strongly upregulated in the roots after rhizotomy, but were only weakly expressed in the DREZ. Tenascin-R immunoreactivity was confined to CNS tissue, and unaffected by rhizotomy. Large, rounded GFAP-negative, NG2-immunoreactive cells, a few of which were OX42 positive, were found in the DREZ following rhizotomy. Astrocyte processes projecting into the roots were tenascin-R and NG2 negative. Hence, only NG2-expressing cells and tenascin-R were appropriately situated to inhibit regeneration through the DREZ.

Retrovirus-mediated gene transfer of granulocyte colony-stimulating factor receptor (G-CSFR) cDNA into MDS cells and induction of their differentiation by G-CSF.

Myelodysplastic syndromes (MDS) are clonal disorders in which the proper differentiation of hematopoietic stem cells is impaired. There is no effective treatment for this stem cell disorder at present. In an attempt to find a new strategy that promotes the differentiation of MDS blast cells, we tried retroviral transduction of granulocyte colony-stimulating factor receptor (G-CSFR) into an interleukin-3-dependent MDS cell line, MDS-L, since expression of G-CSFR is known to be essential for the differentiation of myeloid progenitor cells and this expression is impaired in most MDS cells. Ectopic expression of human G-CSFR cDNA in MDS-L cells gave rise to granulocytic differentiation by G-CSF stimulation. G-CSF caused the transformants expressing G-CSFR to display a morphological characteristic of mature granulocytes, upregulated CD11b on the cell surface, and improved NBT reduction activity. These results demonstrate that MDS-L cells ecopically expressing G-CSFR are induced to granulocytic differentiation upon exposure to G-CSF, and shed light on the molecular mechanisms of maturation arrest in MDS cells.

[Effect of heparin cofactor II on the antithrombin III activities measured by thrombin methods or factor Xa methods--fundamental studies and clinical studies using the plasma of pregnant women].

We evaluated the effects of heparin cofactor II(HC II) on the antithrombin III(AT III) activities measured by the methods of thrombin or factor Xa. Reagents A and B were using the method of thrombin and reagent C was based on the method of Xa. Purified HC II was directly measured or indirectly measured after the dilution with control plasma. Cross reaction of HC II in AT III assay were negligible in reagent C, but substantial amount of AT III activities were measured in reagent A and B. Plasma AT III activities from full-term pregnant women were significantly higher than those from non pregnant control women in reagent A, but comparable in reagent B or C. These results indicate that AT III activities measured by thrombin methods by thrombin were overestimated in pregnant women due to the cross-reactivities of HC II. It is recommended that AT III activities would be measured by the methods of factor Xa.