A 51-year-old woman developed very severe aplastic anemia complicated by chronic renal failure. She underwent ATG therapy, resulting in a transient effect. Thereafter, renal insufficiency progressed, followed by the induction of CAPD therapy. Although rabbit ATG (rATG) was administered as the next immunotherapy for aplastic anemia, pancytopenia persisted and she experienced repeated episodes of severe infection including MRSA and fungal infections. She was transplanted with peripheral blood stem cells from her HLA-haploidentical son after a reduced-intensity conditioning regimen that included cyclophosphamide (three days of 30 mg/kg), fludarabine (two days of 20 mg/m(2)) and rATG (three days of 2.5 mg/kg). FK506 (0.03 mg/kg/24H) was administered for the prophylaxis of GVHD. Prompt trilineage engraftment occurred, resulting in the improvement of infections. Three months after grafting, she succumbed to cerebrovascular disease, although there was no apparent GVHD and she had remained well with stable hematopoiesis.

Quantitative assay for serum free light chains (sFLC) is reported as a useful test for diagnosis and monitoring of patients with nonsecretory multiple myeloma (NSM). We performed serial sFLC assays in a patient with NSM with light chain cast nephropathy (LCCN) and light chain deposition disease (LCDD). After 3 cycles of VAD induction therapy, while plasma cells in the marrow decreased from 93.0% to 0.2%, sFLCkappa/lambda ratio remained abnormal. Flow cytometry assay also showed that these plasma cells were CD19 negative. After the subsequent high dose melphalan therapy followed by autologous peripheral blood stem cell transplantation (PBSCT), the sFLCkappa/lambda ratio returned to normal and the patient achieved a stringent complete response (sCR). One year after PBSCT, the patient remained in sCR with improved renal function. The quantitative FLC assay was useful for the diagnosis and monitoring of NSM and LCDD in this patient.

A 46-year-old Japanese man was admitted to our hospital because of prolonged fever. Laboratory examination demonstrated leukopenia, thrombocytopenia, marked liver dysfunction, and elevation of serum ferritin. A bone marrow examination showed several hemophagocytic macrophages, and a diagnosis of hemophagocytic syndrome was made. He was treated using HLH-94 protocol, and his clinical symptoms and laboratory data were rapidly improved. After 5 weeks, fever and liver dysfunction reappeared. A repeat bone marrow examination demonstrated that 28.4% of marrow nucleated cells were atypical lymphocytes, which were positive for CD2, CD7, CD16, CD56, and HLA-DR. Clonality of these proliferating NK cells was confirmed by an analysis of EB virus terminal repeat sequence and cytogenetic analysis, and final diagnosis of aggressive NK-cell leukemia was made. After induction chemotherapy consisting of dexamethasone, etoposide, ifosfamide, and L-asparaginase, the patient achieved partial remission. He received allogeneic peripheral blood stem cell transplantation from his one locus mismatched son, and is alive with no evidence of disease 20 months after transplantation.

Crow-Fukase syndrome is also called POEMS (polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes) syndrome and is a rare cause of demyelinating and axonal mixed neuropathy with multiorgan involvement. The pathogenesis of Crow-Fukase syndrome is not well understood, but overproduction of vascular endothelial growth factor (VEGF), probably mediated by monoclonal proliferation of plasma cells, is likely to be responsible for most of the characteristic symptoms. However, other cytokines are also upregulated and could contribute to the pathophysiology of this syndrome. The etiopathophysiology of peripheral neuropathy is unclear, but VEGF may affect the blood-nerve barrier and allow some neurotoxic substances in the serum to access the nerve parenchyma, resulting in nerve demyelination. Moreover, microangiopathy due to proliferative endothelial cells and hypercoagulability may contribute to the development of neuropathy. A recent molecular biological study has shown oligoclonal usage of V(lambda) subfamily in light chain of the M-protein, suggesting that particular patterns of V(lambda) gene are associated with the development of Crow-Fukase syndrome. There is no established treatment regimen for this syndrome. In appropriate candidates, high-dose chemotherapies with autologous peripheral blood stem cell transplantation is highly recommended, because this treatment can cause obvious improvement in neuropathy as well as other symptoms, with a significant decrease in serum VEGF levels. The indication for this treatment has not yet been established, and the long-term prognosis is unclear. Potential future therapies include the administration of thalidomide or lenalidomide, and anti-VEGF monoclonal antibody (bevacizumab).

We report a rare case showing involvement with the cauda equina after autologous peripheral blood stem cell transplantation for primary plasma cell leukemia (PCL). A 55-year-old man was diagnosed with PCL(IgA-k type, stage III)in November of 2006. He was treated with VAD chemotherapy consisting of vincristine, doxorubicin, and dexamethasone. After achieving hematological remission, he received tandem high-dose melphalan supported by autologous peripheral blood stem cell transplantation. Five months after his second transplant, he complained of lumbago and bilateral leg pain. M-protein and Bence-Jones protein were not detected in serum or urine. An axial magnetic resonance imaging study revealed enlargement of the cauda equina nerve roots on T-1 weighted image. A sagittal T-1 weighted gadolinium-enhanced imaging study showed hyperintensities along the cauda equina. Leptomeningeal enhancement was also seen below the level of Th6. A cytological examination of the cerebrospinal fluid (CSF) with May-Giemsa stain showed atypical plasma cells. Immunoelectrophoresis of the CSF revealed monoclonal IgA-k type protein. A diagnosis of central nervous system (CNS)relapse was made. The patient died of pneumonia two months after relapse. It should be kept in mind that CNS relapse can occur during hematological remission in patients with multiple myeloma including PCL.

There are significant advances in immune-modulating treatments for Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) in the past 20 years. GBS, however, is still a serious disease with a mortality rate of 8% and 20% of the patients being unable to walk independently a year after onset For CIDP and related disorders such as multifocal motor neuropathy, and demyeinating neuropathy with anti-myelin-associated-glycoprotein (MAG) antibody, treatments should be based on individual pathophysiology. Rituximab could be a promising agent for the subtypes of CIDP refractory to conventional immune treatments. Crow-Fukase syndrome is a rare cause of demyelinating neuropathy with multiorgan involvement Overproduction of vascular endothelial growth factor (VEGF), probably mediated by monoclonal proliferation of plasma cells, is likely to be responsible for most of the characteristic symptoms. There is no established treatment regimen for Crow-Fukase syndrome. In appropriate candidates, high-dose chemotherapies with autologous peripheral blood stem cell transplantation is highly recommended, because this treatment could result in obvious improvement in neuropathy as well as other symptoms. Indication of this treatment has not yet been established, and long-term prognosis is unclear at present. Treatments that should be considered as future therapy against Crow-Fukase syndrome include thalidomide, and anti-VEGF monoclonal antibody (bevacizumab).

Improvement of therapy for severe heart failure due to ischemic cardiomyopathy and myocardial infarction is an important issue of cardiovascular medicine. Recently, regenerative therapy is seemed to be one of the possible ways to solve this problem. There are many potential cell sources for regenerative medicine, such as skeletal myocytes, bone marrow stem cells, endothelial progenitor cells, cardiac progenitor cells and embryonic stem (ES) cells. ES cells are highly proliferative and suitable for mass production and many protocols have been established to ensure selective cardiomyocyte induction. Current studies have successfully generated induced pluripotent stem(iPS) cells from human fibroblasts by the gene transfer of 4 transcription factors that are strongly expressed in ES cells: Oct3/4, Sox2, Klf4 and c-Myc. iPS cells can differentiate into all 3 germ layer-derived cells, including cardiomyocytes as same as ES cells and are syngeneic, indicating that they can become an ideal cell source for regenerative medicine.

Despite a great progress of modern reperfusion strategies in acute coronary syndrome (ACS), ischemic heart disease is still a leading course of mortality in the developed countries. Cell therapy is a promising option to treat ischemic heart disease refractory to conventional treatments. In particular, adult stem cells have been identified and used to clinical cases. Although the detail mechanisms to repair adult heart need further investigation, several clinical studies suggest its safety and feasibility. Current large-scale randomized study will reveal optimal strategy of cell therapy for the patients with ACS.

We had examined the efficacy of stem cell therapy using tissue engineered sheet technique compared to needle injection. This technique has advantages such as the ability for treatment to large area, and less invasive for host heart such as lethal arrhythmia. In vivo, implantation of autologous myoblast sheet had improved cardiac function of ischemic or dilated cardiomyopathy models using rat, hamster, canine and porcine models. We also showed that myoblast sheets provided various factors inducing angiogenesis, hematopoietic cell recruitment and anti-apoptosis, following anti remodeling. Thus, after approved by IRB of our institution, we have started the clinical trial of myoblast sheet implantation for DCM patients, and assessed the feasibility and efficacy for the first patient. In this patient, any sequelae including arrhythmia have not occurred after implantation, and the cardiac function showed recovery. Thus, stem cell sheet implantation could be safe and eligible as cardiac regeneration therapy.

Animal models are indispensable for glaucoma research, because intraocular pressure (IOP) regulation, IOP reduction by ocular hypotensive drugs, and chronic progressive glaucomatous optic neuropathy stem from the eye and its adnexa. The mouse, an excellent animal model available as transgenic mice which are an outstanding experimental tool, has been recently applied for glaucoma research. Many regulating factors such as diurnal variation, body position, and clock genes have been elucidated since accurate measurements of mouse IOP have been published in this decade. These advances were followed by clarification of the molecular mechanism of IOP reduction by eye drops and of the aqueous humor dynamics using transgenic mice. Now, the pathogenesis of glaucomatous optic neuropathy is being investigated using glaucoma mouse models and transgenic mice expressing fluorescent proteins in retinal ganglion cells. By comparing the differences between mouse and human eyes, the mechanism of IOP regulation and retinal ganglion cell damage will be gradually revealed.

In order to develop new therapeutic modalities for corneal diseases, it is essential to combine cutting-edge translational research based upon liberal original ideas obtained from clinical experience with state-of-the-art basic science and technology. Here, I describe seven important research projects on which our group has been working. 1. Elucidation of the pathogenesis in gelatinous drop-like corneal dystrophy(GDLD). Due to loss of function of the tumor-associated calcium signal transducer 2 (TACSTD2), a responsible gene for this dystrophy, tight-junction-related proteins cease to function, resulting in severe corneal epithelial barrier impairment. As a result, various proteins contained in tear fluid continuously penetrate into the corneal stroma, promoting the development of massive amyloid deposits. 2. The development of cultivated mucosal epithelial transplantation: A landmark surgery, involving the transplantation of cultivated mucosal epithelial cells from in vitro to in vivo, now recognized as the next generation of ocular surface reconstruction. We began performing cultivated allocorneal epithelial transplantations in 1999, and cultivated auto-corneal and auto-oral mucosal epithelial transplantations in 2002. These proved to be very effective in the reconstruction of both the corneal surface and the conjunctival fornix. 3. Elucidation of the pathogenesis of Stevens-Johnson syndrome: Studies have shown that there is a close relationship between corneal epithelial stem cell loss and the associated degree of visual impairment. We discovered that a steroid pulse therapy at the acute phase aimed at minimizing stem cell loss is very effective in restoring visual acuity. This implies that inhibition of the cytokine storm is essential for the treatment of acute-phase Stevens-Johnson syndrome. The innate immunity abnormality seems to be heavily involved at the onset of this devastating disease. 4. Elucidation of the involvement of EP3 and toll like receptor 3 (TLR3) in inflammatory ocular surface reactions : We discovered that EP3, one of the prostanoid receptors expressed by ocular surface epithelium, has a dramatic inhibitory effect on ocular surface inflammation in a mouse model. Since EP3 is also expressed in human ocular surface epithelium, and since abnormality of its single nucleotide polymorphisms (SNPs) is involved in some ocular surface inflammatory diseases, we theorized that an allergic reaction may be negatively regulated by EP3 which is predominantly expressed by the ocular surface epithelium. Our findings show that this is similarly true for TLR3, which, conversely, upregulates ocular surface inflammation. 5. Functional regulation of the ocular surface epithelium: Our findings show that intracellular glutathione (GSH) content in the ocular surface epithelium regulates its intracellular redox state. For instance, the GSH content of the conjunctival epithelium decreases in dry eye diseases, yet recovers after the surgical insertion of a punctal plug. Since various amino acids are also heavily involved in the regulation of cellular functions, we investigated the profile of amino acids contained in tear fluids. Our results indicate that there is a marked difference in amino acid profiles between tear fluids and plasma. Furthermore, we found that several amino acids are up-regulated in inflamed eyes, probably due to an oxidative redox response. 6. The development of new therapeutic modalities for corneal edema: We are developing a new therapeutic modality of cultivated corneal endothelial transplantation using methods based on regenerative medicine. For instance, our findings show that cultivated corneal endothelial sheet transplantation in monkeys maintains corneal transparency for at least four years after transplantation. The supplementation of a Rho kinase (ROCK) inhibitor in the culture media produces an excellent result in culturing human corneal endothelium, maintaining a normal-looking endothelial cell morphology. The use of a ROCK inhibitor, both for cultivated endothelial cell injection into the anterior chamber and for use as a topical application, may prove to be a potential tool for the treatment of corneal endothelial dysfunction. 7. The development of a new type of tear function test : The results of our investigations show that the time-dependent changes of tear film lipid layer (TFLL) spread are compatible with the Voigt model of viscoelasticity, and that the initial velocity of the TFLL spread after a blink decreases in proportion to the decrease in tear volume. Thus, a lipid-layer analysis will become an important tear analysis tool. The above are projects representing the way we believe new treatments for severe corneal diseases are heading.

A 37-year-old man with the chief complaints of lumbago and fever is presented. Laboratory data showed the extreme elevation of alfa feto-protein (AFP), human chorionic gonadotropin (hCG) and lactate dehydorgenase (LDH). Computed tomography (CT) scan revealed a huge retroperitoneal tumor with multiple pulmonary nodules as well as left supraclavicular and left axillary lymph nodes enlargement. Although he was suspected the testicular tumor with metastasis, he had no testicular abnormalities including tumor and microlithiasis. Therefore, he was diagnosed as a retroperitoneal extra-gonadal germ cell tumor, which had poor prognosis because of multiple metastasis and the tremendous increase of hCG. Although he was treated with three cycles of bleomycin, etoposide, and cisplatin (BEP), he achieved partial response and no normalization of tumor markers. After three cycles of BEP, he was treated with four cycles of paclitaxel and ifosfamide plus cisplatin (TIP) immediately. During chemotherapy, he was treated with his peripheral blood stem cell transplantation (PBSCT) as well. After the completion of two regimens' chemotherapies, all his tumor markers returned to be normal. However, retroperitoneal tumor, left supraclavicular and axillary lymphnodes still remained. He underwent three operations including retroperitoneal lymphnode dissection with nephrectomy, left supraclavicular and axillary lymphnodes removal, respectively. All specimens had no viable cells, histologically, The patient has been quite well and free of disease for 24 months. It is concluded that even if far-advanced germ cell tumor is discovered, a more promising prognosis could be expected with intensive and aggressive treatment such as our case.

A 47-year-old woman was admitted to our hospital complaining of persistent fever and dry cough in June 2007. CT scan showed hepatosplenomegaly. Laboratory data revealed pancytopenia and increased levels of LDH and soluble interleukin-2 receptor. Malignant lymphoma was suspected, but histological diagnosis was difficult because superficial lymph nodes could not be palpated. Histological examination of the bone marrow biopsy specimen demonstrated the proliferation of large atypical lymphoid cells positive for CD20 and CD79a in the small capillaries, leading to the diagnosis of intravascular large B-cell lymphoma (IVLBCL). Although the results of neurological examination and CSF analysis were normal, head MRI showed a T2-hyperintense lesion in the pons. We chose R-hyper-CVAD/R-MTX-Ara-C alternating therapy with MTX intrathecal injection because CNS involvement in IVLBCL was highly suspected, and she responded well. Head MRI showed the disappearance of the abnormal signal in the pons after one cycle of R-hyper-CVAD. Five cycles of R-hyper-CVAD/R-MTX-Ara-C were performed and complete remission was obtained. R-hyper-CVAD/R-MTX-Ara-C alternating therapy was effective in an IVLBCL patient with CNS involvement.

Limitation of conventional human Ether-a-go-go Related Gene (hERG) assay and QT prolongation testing for accurate prediction of Torsades de Pointes (TdP) by compounds showed us the necessity of a new approach to evaluate global cardiac safety. As one of the advanced applications of an on-chip cellomics system, on-chip cardiomyocyte cell network-based re-entry model assay has the potential to measure the TdP probability as a pre-clinical test for cardiac safety. This system also can estimate the heart pressure, Na, K, and Ca ion channel conditions using a single cell-based optical/electrical measurement system. In this presentation, we present the system setup and then its possible application for drug discovery and toxicology.

Cultured hepatocytes are expected to be used for drug screening and bioartificial liver. Since hepatocytes lose their functions very rapidly in vitro, many attempts have been made to maintain their viability and functions. First, we want to introduce the surface modification of culture substrate using a starburst dendrimer. Addition of fructose to the terminal of the dendrimer was shown to be effective in maintaining hepatocyte function. As the second topic, we will show results of the use of a three-dimensional carrier for hepatocyte cultivation. Hepatocytes and bone marrow stromal cells were cocultured in silane beads, and packed into a radial flow-type bioreactor. The perfusion culture showed the effectiveness of bone marrow stromal cells for the maintenance of hepatocyte function. The next topic will be the trial of adenoviral gene transfer into hepatocytes. Thioredoxin gene was chosen because the products play important roles in redox control and antiapoptosis. The introduction of the gene could inhibit apoptosis and maintain the hepatocyte viability. Finally, we want to introduce the results on differentiation of stem cells into hepatocytes, because it is very difficult to obtain sufficient number of human hepatocytes. Human mesenchymal stem cells were cultured in the presence of several protein factors and the hepatocyte-specific marker was expressed after 2 weeks of induction culture. The use of human stem cells could be an important strategy for the support of a drug development system.