To create new in vitro culture models for extrapolating the cell response in vivo, we attempted to devise culture substrata of anchorage-dependent cells. The first substratum, tissue/organ sections for histopathology(TOSHI)-substratum was found to conserve both tissue composition and microarchitecture in an in vivo environment. Collagen vitrigel membrane, the second substratum investigated, possesses excellent strength and protein permeability. Both substrata were developed and utilized for the culture of various anchorage-dependent cells. TOSHI-substratum prepared from regenerative mouse livers after carbon tetrachloride intoxication efficiently induced the differentiation of mouse embryonic stem cells into hepatocyte-like cells. Also, the time-course cell behavior of two different cell lines on various TOSHI-substrata prepared from rat mature organs was successfully converted into a three-dimensional graph chart, i.e. a mathematical model. These data suggest that the analysis of interactions between different cell types and various TOSHI-substrata will play an important role for a novel approach to study both cellomics and histomics. Meanwhile, the collagen vitrigel membrane is easy to handle by forceps, resulting in double surface-culture of different cell types by the manipulation of two-dimensional cultures. In the crosstalk model between PC-12 pheochromocytoma cells and L929 fibroblasts, nerve growth factor secreted from L929 cells permeated the collagen vitrigel membrane and induced neurite outgrowth of PC-12 cells via a paracrine effect. Futhermore, the function of rat primary hepatocytes was well maintained on the collagen vitrigel membrane. These data suggest that the collagen vitrigel membrane-substratum has many advantages for the reconstruction of culture models.

Magnetic nanoparticles for medical applications have been developed by many researchers. Since these nanoparticles have unique magnetic features not present in other materials, they can be applied to special medical techniques. Magnetite cationic liposomes (MCLs), one group of the cationic magnetic particles, can be used as carriers to introduce magnetite nanoparticles into target cells since their positively charged surface interacts with the negatively charged cell surface. Magnetite nanoparticles conjugated with antibodies (antibody-conjugated magnetoliposomes, AMLs) are applicable to introduce magnetite nanoparticles specifically into target cells, even when target cells coexist with other kinds of cells. Since the cells labeled with magnetite nanoparticles could be manipulated using magnets, we applied this technique to tissue engineering and termed it ;magnetic force-based tissue engineering (Mag-TE)'. Both magnetic force and functionalized magnetite nanoparticles were used in a process of tissue engineering: construction of multilayered cell sheet-like structures and tubular structures. Thus, the applications of these functionalized magnetite nanoparticles with their unique features will further improve tissue engineering techniques.

Stem cell-based therapy has received attention as a possible alternative to organ transplantation, owing to the ability of stem cells to repopulate and differentiate at the engrafted site. We transplanted bone marrow-derived mesenchymal stem cells (BMSCs) into liver-injured rats to test the therapeutic effect. Rat bone marrow cells were cultured in the presence of hepatocyte growth factor (HGF). RT-PCR and immunocytochemical analysis indicated that the BMSCs expressed the albumin mRNA and the production of protein after cultivation with HGF for 2 weeks. The BMSCs appeared to differentiate into hepatocyte-like cells in response to the culture with HGF. After labeling with a fluorescent marker, the BMSCs were transplanted into CCl(4)-injured rats by injection through the caudal vein. The liver was excised and blood samples were collected 4 weeks later. Engraftment of the transplanted BMSCs was seen with significant fluorescence in the injured liver. Transplantation of the BMSCs into liver-injured rats restored their serum albumin level and suppressed transaminase activity and liver fibrosis. Therefore, BMSCs were shown to have a therapeutic effect on liver injury. Recently, we have been trying to use mesenchymal stem cells isolated from dental papilla of discarded human wisdom teeth. Autologous transplantation of mesenchymal stem cells from bone marrow and dental papilla could be ethically and functionally promising for stem cell-based therapy.

Recently, the neural mechanism of respiratory control in the brainstem has been extensively analyzed mainly in vitro. A neuronal group in the ventrolateral medulla, the ventral respiratory group (VRG), is important in respiratory rhythm and pattern generation. A small region in the rostral VRG, the pre-Bötzinger Complex (pre-BötC), is the kernel of respiratory rhythmogenesis. A novel region ventrolateral to the facial nucleus, the para-facial respiratory group (pFRG), was found and has been considered to also generate respiratory rhythm. These two oscillators, pre-BötC and pFRG, are coupled and synchronized. In central chemoreception, small cells surrounding fine vessels in the most superficial layer in the rostral ventral medulla are considered to be primary chemoreceptor cells. Currently, several kinds of neurotransmitters, including glutamic acid, serotonin, ATP and acetylcholine, are considered to play important roles in the signal transduction from chemoreceptor cells to the VRG and other parts of the respiratory neuronal network. The mechanism of respiratory suppression by opioids is the blockade of excitatory drive to the pre-BötC. Although recently we have elucidated that propofol, widely used intravenous anesthetics, suppresses respiratory output through the activation of GABAA receptor, the mechanism of respiratory depression by inhalation anesthetics remains unknown.

The failure of axonal regeneration after central nervous system (CNS) injury is thought to be due in part to the expression of molecules inhibitory for axonal growth and/or the lack of neurotrophic factors. Antibody treatment to neutralize axon growth inhibitory activity, and delivery of neurotrophic factor have been attempted extensively to overcome inhibition and augment regeneration of spinal motor pathways. Local delivery of neurotrophins can counteract pathological events and induce a regenerative response after both acute and chronic spinal cord injury. Furthermore, genetically modified cells that deliver neurotrophins, olfactory ensheathing glia that facilitate nerve regeneration and neural stem/progenitor cells that generate neurons or glia have been studied. These experiments result in a substantial level of restoration of motor function, and give evidence for possible regeneration of particular CNS axons related locomotion activity. However, more practical and promising methods to induce more drastic axonal regeneration are desired at present for clinical use. Fibroblast growth factors (FGFs) have been implicated in numerous cellular processes. FGF-2 stimulates the growth of blood vessels via proliferation of endotherial cells and smooth muscle cells, and enhances neurogenesis via mitotic activity of neural stem/progenitor cells. As angiogenesis is crucial for forming nervous system, FGF-2 may play roles for nerve regeneration in the injured spinal cord. We examined the effects on locomotor function of the FGF-2 injected into the completely transected rat spinal cord. The locomotor function of the FGF-2-treated animals was substantially recovered up to extent where the joint of the hind limb moves 6 weeks after transection, but the recovery was not seen at all on the locomotor activity of the vehicle-treated animals. This regeneration might be facilitated by prominant cell growth of fibroblast-like cells markedly enhanced by FGF-2 around the lesion site, because the cells have properties advantageous for neurite outgrowth. FGF-2-induced cells may become a crucial and promising tool to attain successful axonal regeneration.

A 44-year-old man was admitted to hospital because of respiratory distress and progressive edema in the lower extremities. He was diagnosed as having congestive heart failure, but his condition improved following intensive care. Echocardiogram revealed a thickened interventricular septum, insufficient diastolic function, and granular sparkling pattern in the ventricular wall. Pathological examination of a myocardial biopsy specimen showed the deposition of AL amyloid, resulting in a diagnosis of AL amyloidosis. He was then referred to our hospital for treatment. After a course of high-dose dexamethasone therapy, peripheral blood stem cells induced by the administration of granulocyte colony stimulating factor were harvested. He then received high-dose melphalan (HDM) with autologous peripheral blood stem cell transplantation (auto-PBSCT) support, leading to complete remission. He has been well for more than three years after the transplantation and enjoys the same daily life as before the onset of symptoms. HDM/auto-PBSCT for AL amyloidosis confers a higher response rate and longer survival than conventional chemotherapies; however, treatment-related toxicity is also high. Refinements of treatment strategies are urgently needed. This case provides insights into appropriate strategies for HDM/auto-PBSCT for AL amyloidosis with regard to patient selection, the best induction therapy, and the risk-adjusted melphalan conditioning dose; all of which should be confirmed by randomized controlled trials.

The results of allogeneic stem cell transplantation for patients with chemotherapy-resistant non-remission acute leukemia have been very poor. We have used a melphalan-preceding intensified preparative regimen in which a six-day interval is set between melphalan 70 mg/m2 and the main part of the preparative regimen to avoid toxicity in 15 consecutive pediatric patients with refractory acute leukemia. Only one patient died of transplant-related toxicity within 100 days of transplant. One patient had refractory anemia originating from donor cells at three months after transplant. Eight patients relapsed at a median of six months after transplant; therefore, five of 15 patients have been in complete remission (CR) for a median of 61 months. Four of six patients who did not have blasts in their peripheral blood before melphalan are in CR This method seems to be safe and effective for refractory acute leukemia.

Typical multiple myeloma can be diagnosed precisely through lots of evaluations including the confirmation of the presence of increased clonal myeloma cells in bone marrow accompanied by monoclonal protein, cytopenia, bone diseases and renal disturbance. However, there still exists not a little difficulity during diagnostic process. In this article, representative problems are presented. Among them, the following issues are included; the confirmation or identification of minimal or 'masked' monoclonal proteins in several conditions (eg. myeloma following polyclonal hypergammaglobulinemic conditions), the differential diagnosis between Waldenström macroglobulinemia and IgM multiple myeloma, the differential diagnosis between MGUS with various complications and myeloma, the differential diagnosis of tumors in refractory phase of myeloma (eg. extramedullary plasmacytoma or transition to lympho -proliferative disorders from myeloma), and finally the interpretation of new M-component or oligo-clonal protein band in some periods after stem cell transplantation for myeloma.

Multiple myeloma(MM) is a neoplasia of plasma cells in bone marrow. High dose chemotherapy followed by stem cell transplantation and new drugs such as thalidomide and bortezomib have improved the survival in MM. However, most of patients with myeloma are incurable so there is a need for the new therapeutic approaches that have been developed against molecular targets and pathway. It has been reported that activation of NF-kappaB pathway was required to survival of myeloma cells and this pathway was the potential target for anti -MM therapy. Recently we reported diverse genetic aberrations that activated NF-kappaB signaling in MM. In this section, the molecular pathogenesis of myeloma, especially the new findings related with NF-kappaB activation, will be reviewed in Japanese.