Currently, the European Centre for the Validation of Alternative Methods in the EU appears to be at the forefront of the development of alternative methods for developmental toxicity test (reproductive/developmental toxicity test). Why is it difficult to develop alternative methods for developmental toxicity test in comparison with other toxicity tests? In developmental toxicity test, chemical substances first enter the bloodstream and then reach the placenta via metabolism in the liver and other organs. After further metabolism in the placenta, chemical substances finally reach the fetus, where they affect fetal development. The difference in the in vivo route of chemical substances is an important reason for the difficulty in the establishment of new methods for developmental toxicologic test in comparison with general toxicity tests. According to the EU, the use of "in silico" techniques for developmental toxicity test may be difficult, and I agree with this. The in silico technique is basically a method for prediction of toxicologic effects from existing data, and cannot predict new effects, because data obtained by developmental toxicologic test are too complex. Three techniques are now being examined to overcome the difficulty in changing the method of developmental toxicologic test: the technique utilizing embryonic stem cells; micromass culture technique; and the whole embryonic culture technique. In this symposium, the current status of developmental toxicity tests and the three techniques being examined in the EU are introduced, and opinions on future progress are presented.

Interleukin-6 (IL-6) has been demonstrated to play a pivotal role in CNS trauma as a proinflammatory cytokine which regulates inflammatory response. Not only being a mediator of inflammation, but IL-6 induces neural stem/progenitor cells to undergo astrocytic differentiation selectively in the injured CNS. These effects are considered to coordinate to prevent the CNS repair after traumatic injury. Consistently, we previously reported that the administration of anti-IL-6 receptor antibody (MR16-1) immediately after spinal cord injury in mice decreased the number of invading inflammatory cells and the severity of connective tissue scar formation, and led to improved functional recovery. These findings suggest that neutralization of IL-6 signaling in the acute phase of SCI will be beneficial for CNS repair. A critical merit of this anti-IL-6 receptor antibody is that humanized antibody to human IL-6 receptor (MRA; Atlizumab) has already been reshaped. However, there are several studies which show beneficial aspects of IL-6 signaling in the pathology of CNS trauma. Further investigation of the mechanisms how MR16-1 reduces tissue damage should be required for clinical application.

Adipocytes and osteoblasts are derived from mesenchymal stem cells, and some adipocyte differentiation regulators suppress osteoblast differentiation. PPAR-gammaplays a pivotal role for adipocyte differentiation and glucose tolerance. PPAR-gammais a member of nuclear receptor super family and regulates mRNA expression level of target genes by binding to fatty acid derivatives and thiazolidinediones. Recently, it was found that PPAR-gammainhibits osteoblast differentiation and regulates bone metabolism. In this report, we show recent studies about the extracellular signals regulating the transactivation function of PPAR-gamma.

Lissencephaly is a devastating neurological disorder characterized by smooth cerebral surface, thick cortex and dilated lateral ventricules due to defective neuronal migration. Lis1 was identified as a mutated gene in classical lissencephaly patients, and turned out to be a beta-subunit of platelet activating factor acetylhydrolase. Studies in model organisms, particularly Aspergillus nidulans, as well as those in the mouse, have uncovered an evolutionarily conserved pathway that involves LIS1 and cytoplasmic dynein. LIS1 was subsequently found to associate physically with two NudE orthologs, Ndel and its isoform Ndell. These observations also strengthen that LIS1 gene has been implicated in regulating cytoplasmic dynein. Lis1+/- neurons displayed increased and more variable separation between the nucleus and the preceding centrosome during migration. Dynein inhibition resulted in similar defects in both nucleus-centrosome (N-C) coupling and neuronal migration, suggesting that defects in this coupling may contribute to migration defects in lissencephaly. Recent report suggests that LIS1 also plays an important role on the determination cleavage plane of neuronal progenitor cells. Controlled gene deletion of Lis1 in vivo in neuroepithelial stem cells, where cleavage is uniformly vertical and symmetrical, provokes rapid apoptosis of those cells. Ndell is also involved in the regulation of microtubule organization, and becomes the target of various kinases and phosphatases, including CDK5/CDK1, Aurora-A and PP4. Coordinated regulation of cytoplasmic dynein and microtubule organization is vital for proper cell division and cell positioning, which is an important research problem for understanding of corticogenesis and promoting the development of new therapies for lissencephaly and related disorders.

In the developing nervous system, neural stem cells initially proliferate extensively by symmetric cell division and then give rise to neurons by asymmetric cell division. After production of neurons, neural stem cells finally differentiate into glial cells. These processes are regulated by basic helix-loop-helix (bHLH) transcription factors: the repressor-type bHLH factors promote maintenance of neural stem cells and differentiation of glial cells, whereas the activator-type bHLH factors induce production of neurons. There are many subtypes of neurons, and bHLH factors alone are not sufficient but other factors such as homeodomain factors are required for specification of these neuronal subtypes. These transcription factors will be useful for regeneration of the nervous system.

Recently, planarians have received much attention because of their contributions to research on the basic science of stem cell systems, neural regeneration, and regenerative medicine. Planarians can regenerate complete organs, including a well-organized central nervous system (CNS), within about 7 days. This high regenerative capacity is supported by pluripotent stem cells present in the mesenchymal space throughout the body. Interestingly, planarians can regenerate their brain via a molecular mechanism similar to that of mammalian brain development. The regeneration process of the planarian brain can be divided into five steps: (1) anterior blastema formation, (2) brain rudiment formation, (3) brain pattern formation, (4) neural network formation, and (5) functional recovery, with several kinds of genes and molecular cascades acting at each step. Recently, we have identified a planarian tyrosine hydroxylase (TH) gene, a rate-limiting enzyme for dopamine (DA) biosynthesis, and produced TH-knockdown planarians by the RNA interference technique. Studies of TH-knockdown planarians showed that DA has an important role of the modification in behavioral movement in planarians. Using monoclonal anti-planarian TH antibody, we also found that dopaminergic neurons are mainly localized in the planarian brain. When the planarian body was amputated, newly generated TH-immunopositive neurons were detected in the anterior region at day 3 of regeneration (i.e., the period of neural network formation), and the TH-immunopositive axonal and dendritic neural network in the CNS was reconstructed during day 5-7 of regeneration. In this article, recent advances in elucidating the molecular mechanism of planarian brain regeneration and dopaminergic neurons are reviewed, and its future prospects for contribution of this system to basic science and medical science research are described.

We discussed the usefulness of routine technologies of laboratory medicine in blood transfusion and transplantation medicine. New parameters that can be measured by automated hematology analyzers have been clinically evaluated and proven to be useful so far. Based on our experience, detection systems for fragmented red cells (FRC), immature platelets (immature platelet function, IPF), and hematopoietic progenitor cells (HPC) are useful for the diagnosis of thrombotic microangiopathy, differential diagnosis of thrombocytopenia, and decision regarding the optimal timing to collect peripheral stem cells, respectively. Moreover, IPF were suggested to be an indicator of the platelet transfusion requirement. The establishment of non invasive assaying technology has been eagerly anticipated. We evaluated a hemoglobin measurement tool, and revealed that it might be applicable in predeposited, autologous blood donation. Some adverse transfusion reactions are related to neutrophil activation. Thus, we investigated the effects of serum from patients and blood donors, in the context of adverse reactions, on adhesion molecule expressions of neutrophils from volunteers using flow-cytometry. This kind of simple technology is expected to be useful in future studies to clarify the mechanisms and prevent adverse reactions.

We report a case of Bickerstaff's brainstem encephalitis (BBE) with positive findings on head MRI. Abnormal lesions on head MRI were detected in approximately 30% cases of BBE. An 81-year-old female presented with disturbance of consciousness (Japan Coma Scale 200), ophthalmoplegia (downward deviation) and tetraplegia. Cerebrospinal fluid examination revealed mild pleocytosis 244 cells in 3 microl of CSF with normal glycorrhachia. Head MRI showed high-intensity abnormalities extending over the brain stem. The patient was diagnosed with BBE. It was thought that her symptoms were believed to arise from the lesion in reticular formation, rostral interstitial nucleus of the medial longitudinal fasciculus, and the pyramidal tract. At discharged after 1 month, she had no sequela.

Bone marrow-derived cells, which are recruited to peripheral tissues, have been shown to play a major role as angiogenic factor supplier. Moreover, bone marrow-derived "hemangiocytes" through graded chemokines production, can function as the molecular hub for hematopoietic cytokine-induced neovascularization. Current focus on cancer metastasis has centered on the intrinsic factors regulating the cell autonomous homing of the tumor cells to the metastatic site : Bone marrow-derived cells expressing vascular endothelial growth factor receptor 1 can be mobilized into the circulation due to growth factors released by the primary tumor. These cells cluster at distant sites, "the premetastatic niche" , where they set up a microenvironment ideal for tumor cells to seed, thereby completing the metastatic spread. Focus on the early cellular and molecular events in cancer infiltration, metastasis and proliferation will likely lead to new approaches to detect and prevent metastasis at its earliest inception.

A 69-year-old Japanese man was diagnosed as having primary plasma cell leukemia. His malignant plasma cells had a chromosomal translocation t(11;14)(q13;q32) that created overexpression of cyclin D1. Two courses of VAD (vincristine, doxorubicin, dexamethasone) therapy failed to achieve complete remission. Three subsequent courses of hyper-CVAD (cyclophosphamide, vincristine, doxorubicin, dexamethasone) therapy successfully induced remission with negative FISH test for t(11;14)(q13;q32). Thereafter, the patient received high-dose melphalan (125 mg/m(2)) followed by autologous peripheral blood stem cell transplantation. Cyclin D1 that was present prior to the high-dose chemotherapy, was no longer detected by qualitative PCR analysis. Despite complete cytogenetic remission, the disease relapsed 6 months later, and the patient eventually died 16 months following the diagnosis. Plasma cell leukemia is a rare hematological malignancy with a poor prognosis. The treatment has not been standardized yet. The present case suggested the effectiveness of the combination of hyper-CVAD and high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation. Nevertheless, because of the short remission duration, intensification using tandem high-dose chemotherapy or maintenance using new agents such as bortezomib and thalidomide should be considered for improving the prognosis.

Chimerism analysis by polymerase chain reaction amplification of short tandem repeats (PCR-STR) has become a routine diagnostic procedure for evaluating grafts and assessing the likeliness of original disease recurrence after allogeneic stem cell transplantation. Following a sex-mismatched hematopoietic stem cell transplantation (HSCT), we monitored the clinical course of a 61-year old male AML M6 patient with trisomy 8 using PCR-STR with a TH01 locus on 11p15 and fluorescence in situ hybridization (FISH) analysis specific for alpha satellite DNA on chromosome 8. Ten months after HSCT, FISH analysis showed 24.8% recipient cells, but PCR-STR demonstrated 100% donor type chimerism. Further XY FISH analysis of May-Grünwald-Giemsa-stained bone marrow samples clearly demonstrated relapse of the original disease and G-banding analysis of bone marrow samples at relapse showed that an additional chromosomal abnormality, del(11) (p10), had deleted the PCR-STR detection site in all recipient type cells. As such, clinicians should consider the possibility that unexpected karyotype changes may invalidate PCR-STR analysis findings, especially when conflicting results appear among chimerism analyses.

We retrospectively analyzed the clinical course and prognosis of 11 patients with angioimmunoblastic T-cell Lymphoma (AILT). Median patient age was 62 years old (range 39 to 85). All patients were in clinical stage III or IV. Clinical features included B symptoms, hepatosplenomegaly, skin rushes, pleural effusion, ascites and polyclonal hypergammaglobulinemia. The disease can be classified into three categories based on histological findings: 3 cases of AILT with hyperplastic germinal centers, 4 cases of typical AILT, and 4 cases of AILT with numerous clear cells. As the initial therapy, 10 patients received combination chemotherapy and only 1 patient received autologous peripheral blood stem cell transplantation. Seven patients achieved CR and 4 showed PD. The response rate was 63% and the median survival time was 20 months. One patient survived in CR for 122 months. Patients with AILT demonstrating hyperplastic germinal centers and no bone marrow infiltration were able to achieve long-term survival. The survival time of AILT demonstrated a wide range. It was thought that further consideration of the prognostic factors and stratification was required.

Paroxysmal nocturnal hemoglobinuria is an acquired hematopoietic stem cell disorder. The most common clinical manifestations of PNH include intravascular hemolysis, venous thrombosis, and bone marrow failure. Hematopoietic stems cells with mutant PIG-A are present in normal marrow, but they are not noticed because they have no advantage under normal circumstances. In the setting of immune-mediated bone marrow injury, such as that seen in aplastic anemia, PIG-A mutant cells are selected because they have a survival advantage based on deficiency of one or more GPI-anchored proteins. Expansion of the PIG-A-mutant clones occurs when other events that enhance the growth properties of the cells work together with the effects of the PIG-A mutation to enhance further the growth properties of the mutant cells.

Fanconi anemia (FA) is a genetically heterogeneous inherited disorder characterized by progressive bone marrow failure, development of hematopoietic and solid malignancies and genomic instability. 13 FA proteins, identified to date, closely cooperate with familial breast cancer susceptibility proteins such as BRCA2 and PALB2, thereby forming 'the FA/BRCA molecular network'. Here I summarize our recent understanding of the molecular network and its significance in the pathogenesis of FA. I emphasize that FA provides an excellent genetic model for studying senescence and malignant transformation of human hematopoietic stem cells.

Stem cell transplantation is an effective treatment for anemia associated with stem cell disorders such as severe aplastic anemia, myelodysplastic syndrome. Long-term survival rates after transplantation for those diseases range from less than 40% to more than 80%. The rates are significantly affected by recipient's age, HLA disparity between donor and recipient, sources of stem cells, and disease status. Results are best in younger patients transplanted with bone marrow from HLA-identical sibling at the early course of the diseases. The decision of whether and at what point to proceed to transplantation should be made early after diagnosis, based upon the consideration of a survival rate with reasonable quality of life expected with transplantation and other therapeutic approaches.

Anemia of chronic disease (ACD) is a mild to moderate anemia seen with many infections and inflammatory disorders. These patients have low serum iron, but high serum ferritin levels. As for the pathogenesis of ACD, previous studies have reported several abnormalities, such as insufficient EPO production, impaired growth response of erythroid progenitors to EPO, and shortened survival of erythrocytes, due to the inflammatory cytokines. However, recent analyses have clearly shown that hepcidin, of which expression is induced by inflammatory cytokines such as IL-1beta and IL-6, suppresses the expression of the iron transporter, ferroportin-1, thereby inhibiting the absorption of iron from the duodenum, the release of iron from the reticulo-endothelial system. So, the abnormal expression of hepcidin alone may be able to explain the unique iron metabolism in ACD.