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We analyzed the clinical features of 157 patients admitted to 11 institutions in the Kagawa Prefecture for volvulus of the sigmoid colon. The following were the background information of the patients:median age, 79.0 years;male-to-female ratio, 102:55;median body mass index, 20.0kg/m

A male patient in his sixties with a long-term history of schizophrenia had been received glycerin enema once or twice a week in a mental hospital. He was emergently transferred to our hospital due to fever, vomiting, hematuria, and dyspnea. Laboratory findings on admission showed an elevation of white blood cells indicating inflammation, hemolysis, and renal dysfunction. Plain CT showed pleural effusion and ascites, elevated levels of perirectal fat, in addition to extraintestinal gas. Based on these findings, he was diagnosed with rectal damage caused by the glycerin enema and associated hemolysis with acute renal failure. He was kept under conditions of nil by mouth and received intravenous antibiotics, diuretic drug, and haptoglobin. Eventually, his condition improved with these conservative therapies. In this case, it is assumed that the hemolysis was caused by the influx of glycerin in the cytoplasm and an increase of osmotic pressure. Care should be taken during glycerin enema, which is widely used in daily practice as well as in home care settings.

In the present investigation, we generated platelets (PLTs) from cord blood (CB) CD34(+) cells using a three-phase culture system. We first cultured 500 CB CD34(+) cells on telomerase gene-transduced human stromal cells (hTERT stroma) in serum-free medium supplemented with stem cell factor (SCF), Flt-3/Flk-2 ligand (FL), and thrombopoietin (TPO) for 14 days. We then transferred the cells to hTERT stroma and cultured for another 14 days with fresh medium containing interleukin-11 (IL-11) in addition to the original cytokine cocktail. Subsequently, we cultured the cells in a liquid culture medium containing SCF, FL, TPO, and IL-11 for another 5 days to recover PLT fractions from the supernatant, which were then gel-filtered to purify the PLTs. The calculated yield of PLTs from 1.0 unit of CB (5 x 10(6) CD34(+) cells) was 1.26 x 10(11) - 1.68 x 10(11) PLTs. These numbers of PLTs are equivalent to 2.5-3.4 units of random donor-derived PLTs or 2/5-6/10 of single-apheresis PLTs. The CB-derived PLTs exhibited features quite similar to those from peripheral blood in morphology, as revealed by electron micrographs, and in function, as revealed by fibrinogen/ADP aggregation, with the appearance of P-selectin and activated glycoprotein IIb-IIIa antigens. Thus, this culture system may be applicable for large-scale generation of PLTs for future clinical use.

Essential thrombocythaemia (ET) is characterized by the abnormal and sustained proliferation of megakaryocytes. The mechanism for this lineage-specific expansion in ET, remains unclear. We have previously reported that transforming growth factor-beta1 (TGF-beta1) is involved in negative feedback regulation of megakaryopoiesis in both healthy volunteers (HV) and patients with idiopathic thrombocytopenic purpura (ITP). The present study found that megakaryocyte colony-forming units (CFU-MK) of ET patients were less sensitive to TGF-beta1 than those of HV. The expression of Smad4 (Sma- and Mad-related protein-4) in CFU-MK of ET patients was reduced in comparison with that of HV. Finally, to confirm that the impaired TGF-beta1 sensitivity was caused by reduced expression of Smad4, we examined Smad4-transfected CFU-MK from ET patients in the presence of TGF-beta1, and verified that the transfectants were indeed as susceptible as CFU-MK from HV to TGF-beta1. Thus it was surmised that one of the mechanisms for impaired sensitivity of CFU-MK to TGF-beta1 is the reduced expression of Smad4.

Background To examine the effects of percutaneous endoscopic gastrostomy (PEG) on quality of life (QOL) in patients with dementia. Methods We retrospectively included 53 Japanese community and tertiary hospitals to investigate the relationship between the newly developed PEG and consecutive dementia patients with swallowing difficulty between Jan 1st 2006 and Dec 31st 2008. We set improvements in 1) the level of independent living, 2) pneumonia, 3) peroral intake as outcome measures of QOL and explored the factors associated with these improvements. Results Till October 31st 2010, 1,353 patients with Alzheimer’s dementia (33.1%), vascular dementia (61.7%), dementia with Lewy body disease (2.0%), Pick disease (0.6%) and others were followed-up for a median of 847 days (mean 805 ± 542 days). A total of 509 deaths were observed (mortality 59%) in full-followed patients. After multivariate adjustments, improvement in the level of independent living was observed in milder dementia, or those who can live independently with someone, compared with advanced dementia, characterized by those who need care by someone: Odds Ratio (OR), 3.90, 95% confidence interval (95%CI), 1.59 - 9.39, P = 0.003. Similarly, improvement of peroral intake was noticed in milder dementia: OR, 2.69, 95%CI, 1.17 - 6.17, P = 0.02. Such significant associations were not observed in improvement of pneumonia. Conclusions These results suggest that improvement of QOL after PEG insertion may be expected more in milder dementia than in advanced dementia.

We generated red blood cells (RBC) from cord blood (CB) CD34+ cells using a four-phase culture system. We first cultured CB CD34+ cells on telomerase gene-transduced human stromal cells in serum-free medium containing stem cell factor (SCF), Flt-3/Flk-2 ligand, and thrombopoietin to expand CD34+ cells (980-fold) and the total cells (10,400-fold) (first phase). Expanded cells from the first phase were liquid-cultured with SCF, interleukin-3 (IL-3), and erythropoietin (EPO) to expand (113-fold) and differentiate them into erythroblasts (second phase). To obtain macrophages for the next phase, we expanded CD34+ cells from a different donor using the same coculture system. Expanded cells from the first phase were liquid-cultured with granulocyte-macrophage colony stimulating factor, macrophage-colony stimulating factor (M-CSF), IL-3, and SCF to generate monocytes/macrophages (75-fold), which were incubated with type AB serum and M-CSF to fully differentiate them into macrophages. Erythroblasts were then co-cultured with macrophages in the presence of EPO to expand (threefold) and fully differentiate them (61% orthochromatic erythroblasts plus 39% RBC) (third phase). RBC were purified from erythroblasts and debris through a deleukocyting filter to generate 6.0 x 10(12) RBC from 1.0 unit of CB (3.0 transfusable units). Qualitatively, these RBC showed a hemoglobin content, oxygenation of hemoglobin, and in vivo clearance similar to those of adult peripheral RBC. Finally, an almost complete enucleation of orthochromatic erythroblasts (99.4%) was achieved by the cultivation method recently described by Miharada et al. in the absence of macrophages and cytokines (fourth phase). RBC were purified from remnant erythroblasts and debris by passage through a deleukocyting filter to generate 1.76 x 10(13) RBC from 1.0 unit of CB (8.8 transfusable units), the highest yield ever reported. Thus, this method may be useful for generating an alternative RBC supply for transfusions, investigating infectious agents that target erythroid cells, and as a general in vitro hematopoietic model system.

Administration of imatinib exacerbated psoriasis vulgaris in a case of chronic myelogenous leukemia (CML). After the cessation of imatinib therapy, the psoriasis was alleviated. Upon readministration of imatinib, the psoriasis worsened despite the improvement of hematological and cytogenetic findings in the CML. Psoriasis is known to be an autoimmune skin disease characterized by Th1 cell-mediated hyperproliferation of keratinocytes, and the type 1 helper T (Th1) cell subset increased with imatinib therapy. Thus, the exacerbation of psoriasis was likely due to the increase in Th1 cells associated with imatinib therapy.

We investigated the cause of myelofibrosis and proliferation of megakaryocytes in myelodysplastic syndrome with myelofibrosis (MDS-MF (+)). Plasma-transforming growth factor-beta1 (PTGF-beta1) concentrations closely correlated with myelofibrosis grade in MDS-MF (+) and were higher than those in idiopathic myelofibrosis (IMF), essential thrombocythemia (ET), idiopathic thrombocytopenic purpura (ITP), MDS-without MF (MDS-MF (-)) or healthy volunteers (HV). Peripheral blood mononuclear cells from MDS-MF (+) patients expressed more TGF-beta1 mRNA than those from IMF, MDS-MF (-) or HV. When we immunostained bone marrow specimens of MDS-MF (+) for TGF-beta, the intensity of blasts was apparently higher than that of megakaryocytes, while in MDS-MF (-), megakaryocytes were immunostained with a similar intensity as that in MDS-MF (+), but blasts were negative for staining. In IMF, megakaryocytes, monocytes and small mononuclear cells representing CD34+ cells were all similarly stained with a much lower intensity than that of blasts in MDS-MF (+). The number of bone marrow megakaryocytes were increased the most in MDS-MF (+), followed by ET, ITP, MDS-MF (-) and NHL and correlated with plasma thrombopoietin (TPO) levels or with plasma TGF-beta1 levels, respectively, in each disease. Thus, in MDS-MF (+), both myelofibrosis and the increased megakaryocytes were ascribed to overproduction of TGF-beta1 from blasts.

Essential thrombocythaemia (ET) is characterized by the abnormal and sustained proliferation of megakaryocytes. The mechanism for this lineage-specific expansion in ET, remains unclear. We have previously reported that transforming growth factor-beta1 (TGF-beta1) is involved in negative feedback regulation of megakaryopoiesis in both healthy volunteers (HV) and patients with idiopathic thrombocytopenic purpura (ITP). The present study found that megakaryocyte colony-forming units (CFU-MK) of ET patients were less sensitive to TGF-beta1 than those of HV. The expression of Smad4 (Sma- and Mad-related protein-4) in CFU-MK of ET patients was reduced in comparison with that of HV. Finally, to confirm that the impaired TGF-beta1 sensitivity was caused by reduced expression of Smad4, we examined Smad4-transfected CFU-MK from ET patients in the presence of TGF-beta1, and verified that the transfectants were indeed as susceptible as CFU-MK from HV to TGF-beta1. Thus it was surmised that one of the mechanisms for impaired sensitivity of CFU-MK to TGF-beta1 is the reduced expression of Smad4.

Bone-marrow minimal residual disease (MRD) causes relapse after chemotherapy in patients with acute myelogenous leukemia (AML). We postulate that the drug resistance is induced by the attachment of very late antigen (VLA)-4 on leukemic cells to fibronectin on bone-marrow stromal cells. We found that VLA-4-positive cells acquired resistance to anoikis (loss of anchorage) or drug-induced apoptosis through the phosphatidylinositol-3-kinase (PI-3K)/AKT/Bcl-2 signaling pathway, which is activated by the interaction of VLA-4 and fibronectin. This resistance was negated by VLA-4-specific antibodies. In a mouse model of MRD, we achieved a 100% survival rate by combining VLA-4-specific antibodies and cytosine arabinoside (AraC), whereas AraC alone prolonged survival only slightly. In addition, overall survival at 5 years was 100% for 10 VLA-4-negative patients and 44.4% for 15 VLA-4-positive patients. Thus, the interaction between VLA-4 on leukemic cells and fibronectin on stromal cells may be crucial in bone marrow MRD and AML prognosis.

A 63-year-old woman had previously been admitted to another hospital due to fever, abdominal pain and diarrhea. She was treated with fasting, antibiotics and G-CSF administration because of the coexistence of neutropenia, and the symptoms improved. However, discontinuation of G-CSF administration resulted in a recurrence of the neutropenia accompanied with enterocolitis. After admission to our hospital, a diagnosis for idiopathic AIN was performed as she tested positive in both granulocyte immunofluorescence and granulocyte agglutination tests. Administration of corticosteroid following G-CSF resulted in a continuous increase in the neutrophil count and the disappearance of anti-neutrophil autoantibodies.

We report an elderly patient with paroxysmal nocturnal hemoglobinuria (PNH), having recurrent gastrointestinal infections and hemolytic episodes caused by cellular immunodeficiency due to a decreased number of T cells. On admission to our hospital, the patient had normal neutrophil count and function, but had decreased T cell count, decreased mitogenic reaction to PHA and ConA, and negative reaction to the tuberculin test. Thus, a cellular immunodeficiency state was suggested and postulated to be due to decreased T cell count caused by the loss of PNH clone of T cells. We previously reported that the administration of G-CSF to normal donors of allogeneic peripheral stem cell transplantation increases the lymphocyte count. Based on this experience, we administered G-CSF to this patient. This resulted in an increase in the T cell count, normalization of the mitogenic response to PHA and ConA, positive reaction to the tuberculin test and increases in the blood levels of Thl cytokines and Th2 cytokines, with an improvement in the gastrointestinal infections and hemolytic episodes. This suggested that G-CSF administration resulted in the recovery of cell-mediated immunity in the gastrointestinal mucosa. At 9 months after the start of G-CSF administration, these complications have not recurred. This is the first report of a patient of this type and suggests that G-CSF administration may be useful in the treatment of elderly PNH patients with cellular immunodeficiency.

We treated two chronic phase chronic myelogenous leukemia patients with imatinib mesylate. Hematological complete remission and significant regression of bone marrow fibrosis were observed in both patients. The large amount of TGF-beta produced by increased bone marrow megakaryocytes might have caused the myelofibrosis, and it was revealed that imatinib mesylate brought about regression of the myelofibrosis by reducing the number of megakaryocytes in both patients.

Summary. We report an elderly patient with paroxysmal nocturnal haemoglobinuria (PNH), having recurrent enterocolitis and haemolytic attacks associated with cellular immunodeficiency. On admission, the patient had normal neutrophil count and function but a decreased T-cell count, decreased mitogenic reactions, and a negative tuberculin test. Granulocyte colony-stimulating factor (G-CSF) was administered, resulting in an increased T-cell count, normalization of T-cell function, increased blood levels of helper T cell (Th)1 and Th2 cytokines and improvement in the enterocolitis and haemolytic attacks. This suggests that G-CSF may be useful in the treatment of elderly PNH patients with cellular immunodeficiency.

It is well known that the expression of anticancer drug-resistant factors is elevated in patients with primary refractory or relapsed chronic lymphocytic leukemia (CLL) who have been treated with chemotherapy. We report here two C(H)OP refractory patients with CLL in whom salvage chemotherapy chosen by evaluating anticancer drug-resistant factors (glutathione-S-transferase-Pi [GST-Pi], glycoprotein [GP]-170, multidrug resistance-associated protein [MRP], and lung resistance protein [LRP]) was remarkably effective. A 71-year-old male patient was refractory to induction therapy with cyclophosphamide, vincristine, and prednisone (COP), and his leukemic cells at diagnosis displayed overexpression of GST-Pi and GP-170. A 74-year-old female patient's condition had been stable; she had received ten courses of COP over 9 years. However, because systemic lymphadenopathies recurred, she was treated with chemotherapy consisting of cyclophosphamide, adriamycin, vincristine, and prednisone (CHOP) or dexamethasone, etoposide, ifosphamide, and carboplatin (DeVIC). However, she did not respond at all, and her leukemic cells at recurrence displayed overexpression of GST-Pi. Therefore, we chose for these patients a salvage therapy consisting of dexamethasone and high-dose cytosine arabinoside (Ara C), to which neither GST-Pi nor GP-170 show any drug resistance. In both patients, this salvage therapy proved effective.

Bone marrow (BM) minimal residual disease causes relapse after chemotherapy in AML. We have previously reported that VLA4-positive leukemic cells acquired resistance to drug-induced apoptosis through the PI-3K/AKT/Bcl-2 signaling pathway, which is activated by the interaction of VLA4 and fibronectin on BM stromal cells. This resistance was negated by mouse-anti-human VLA4Ab (mouse VLA4Ab). In human leukemia SCID mouse model, we demonstrated a 100% survival rate with combination of mouse VLA4 Ab and AraC, while with AraC alone, only slight prolongation of survival was attained. In clinical study, overall survival at 5 years was 90% for 10 VLA4− patients and 25% for 15 VLA4+ patients (Matsunaga T et al, Nature Med 2003, 9, 1158–1165). In the present study, to perform the translational research, we first examined the myelosuppressive effect of the combination of rat-anti-mouse VLA4 Ab and AraC in C57/BL6 mice, and found that CBC data were almost the same as those of the mice treated with AraC alone. We next produced humanized chimeric-anti-human VLA4 Ab (chimeric-VLA4Ab), and examined its efficacy in combination with anti-cancer drugs in vitro and in vivo (human leukemia SCID mouse model). Chimeric-VLA4Abs were produced as follows: (i) total RNA of mouse VLA4 Abs were extracted from two hybridomas (SG/17 and SG/73), (ii) cDNA were synthesized by reverse transcriptase, (iii) variable region gene of mouse VLA4 Abs were amplified by 5′RACE method, (iv) TA cloning of amplified gene was performed, (v) sequence of mouse VLA4Abs gene was determined, (v) cloned variable region gene of mouse VLA4 Abs and constant region gene of human IgG1 were inserted into expression vector, and the expression vector was transfected into 293T cells, (vi) supernatant of the 293T cells was collected and purified to obtain chimeric-VLA4 Abs. The effects of chimeric-VLA4 Abs thus obtained in in vitro and in vivo (human leukemia SCID mouse model) were comparable to those of mouse VLA4 Abs. To perform the clinical study, we are presently producing the GMP-graded chimeric-VLA4 Abs.

New sources of red blood cells (RBC) would improve the transfusion capacity of blood centers. Several investigators have previously reported that erythroblasts could be obtained from hematopoietic stem cells including those of cord blood (CB) by in vitro culture. However, transfusion of erythroblasts may not be suitable for supplementation of acute blood loss because it should need some time lag until hemoglobin (RBC) boost in circulation due to the fact that transfused erythroblasts once lodged at bone marrow where they undergo maturation into RBCs which are bound to be released into circulation. We have developed a culture system for producing large quantity of enucleated RBCs (e-RBCs) as well as erythroblasts from CB in vitro: one unit e-RBCs (2 x 1012 RBCs) was obtained from one standard CB unit (corresponding to 2 x 106 CD34+ cells) using a coculture system with hTERT-transfected human stromal cells at early phase followed by with activated macrophage in liquid culture (American Society of Hematology 45th Annual Meeting, SanDiego, 2003). In the present study, we first analyzed the function of those manufactured e-RBCs in comparison of that of adult peripheral blood RBCs (PB-RBCs) or that of eryhthroblasts. The hemoglobin (Hb) content of the e-RBCs quantified by photometric determination was almost equivalent to that of adult PBRBC. A Hb A/Hb F ratio of e-RBC analyzed by high-performance liquid chromatography (HbA: HbF = 35: 65) was between those of CB RBCs (10: 90) and adult PB-RBC (99: 1). Oxygen dissociation curves of e-RBCs measured by Hemox-Analyzer was comparable to that of fresh adult PB-RBCs. The erythroblasts showed adhesive property to stromal cells in vitro but e-RBC did not. When we injected e-RBCs into NOD/SCID mice, they were detectable in circulation while erythroblasts were not. In conclusion, the e-RBCs produced by large-scale culturing system from CB CD34+ cells may be useful for acute blood loss.