Induced Pluripotent Stem Cell-Derived Hematopoietic Embryoid Bodies Improve Mouse Status in Septic Peritonitis.

We examined the efficacy of embryoid bodies from 6-day induced pluripotent stem cells an in vivo sepsis model. Injection of embryoid bodies to septic mice improved the condition of their lungs and significantly increased their survival rate. Although embryoid bodies secretedsphingosine-1-phosphate in vitro, its serum levels in mouse plasma were significantly reduced compared to that in the control (untreated mice receiving PBS). Low concentrations of sphingosine-1-phosphate protected endothelial cells, while high concentrations disrupted endothelial barrier integrity. Therefore, exogenous sphingosine-1-phosphate secreted by embryoid bodies during early stage of sepsis might down regulate endogenous production of sphingosine-1-phosphate. Inhibition of excessive sphingosine-1-phosphate release protects against endothelial injury and suppresses a vicious cycle of inflammatory reactions. The obtained results open new prospects in induced pluripotent stem cells-based therapy for sepsis.

Induced Pluripotent Stem Cell-Derived Hematopoietic Embryoid Bodies Secrete Sphingosine-1-Phosphate and Revert Endothelial Injury.

The possibility of sphingosine-1-phosphate production by induced pluripotent stem cells is examined to assess their potential in treatment of sepsis. The hematopoietic embryoid bodies were derived from the culture of 6-day-old differentiated induced pluripotent stem cells. These embryoid bodies secreted sphingosine-1-phosphate, an important bioactive lipid that regulates integrity of the pulmonary endothelial barrier, prevents elevation of its permeability, and impedes the formation of stress fibers in human endotheliocytes derived from umbilical vein. The data attest to potentiality of induced pluripotent stem cells in treatment of sepsis.

Low absolute peripheral blood CD4+ T-cell count predicts poor prognosis in R-CHOP-treated patients with diffuse large B-cell lymphoma.

The absolute peripheral blood lymphocyte count at diagnosis is known to be a strong prognostic factor in patients with diffuse large B-cell lymphoma (DLBCL) treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP), but it remains unclear as to which peripheral blood lymphocyte population is reflective of DLBCL prognosis. In this cohort, 355 patients with DLBCL treated with R-CHOP from 2006 to 2013 were analyzed. The low absolute CD4+ T-cell count (ACD4C) at diagnosis negatively correlated with the overall response rate and the complete response rate significantly (P<0.00001). An ACD4C<343 × 106/l had a significant negative impact on the 5-year progression-free survival and the overall survival as compared with an ACD4C⩾343 × 106/l (73.7% (95% confidence interval (CI)=66.7-79.5) versus 50.3% (95% CI=39.0-60.6), P<0.00001 and 83.3% (95% CI=77.1-88.0) versus 59.0% (95% CI=47.9-68.5), P<0.00000001, respectively). Multivariate analysis revealed that the ACD4C was an independent prognostic marker (hazard ratio=2.2 (95% CI=1.3-3.7), P<0.01). In conclusion, a low ACD4C at diagnosis served as an independent poor prognostic marker in patients with DLBCL.

Establishment of optimized ELISA system specific for HLA-G in body fluids.

Recently, human leukocyte antigen-G (HLA-G) has been a focus in the field of reproductive immunology, tumor progression and transplantation, because of its inhibitory function as ligand to the inhibitory receptors leukocyte immunoglobulin-like receptors (LILR) B1 and LILRB2. The HLA-G is expressed in distinct mRNA isoforms, one of which encodes a soluble HLA-G (sHLA-G) protein, detectable by sandwich ELISA. Therefore, sHLA-G ELISAs have been used as a noninvasive diagnosis system. While a number of sHLA-G-specific ELISAs have been described, our prior studies showed that data obtained by the conventional ELISA system detecting sHLA-G in body fluids was not consistent with the data obtained from immunoprecipitation (IP)/immunoblotting (IB). Therefore, we established an optimized ELISA system described in this report, which yields results consistent with IP/IB analysis. Using this system, we determined sHLA-G protein in amniotic fluids, and found that sHLA-G levels at preterm (∼36 weeks) were clearly higher than those at term (37-41 weeks). These data and supporting experiments showed that the ELISA system we established can be an useful tools for the detection of sHLA-G protein in body fluids than the conventional ELISA system.

Ethanol attenuates vasorelaxation via inhibition of inducible nitric oxide synthase in rat artery exposed to interleukin-1ß.

Nitric oxide produced by inducible nitric oxide synthase (iNOS) regulates sepsis-induced hypotension. During septic shock, interleukin (IL)-1ß is synthesized in endothelial cells and smooth muscle cells by endotoxin. Ethanol (EtOH) suppresses endotoxin-induced hypotension. The present study aimed to elucidate the effect of EtOH on gradual relaxation and iNOS expression induced by IL-1ß in isolated rat superior mesenteric arteries (SMAs). Exposure to IL-1ß-induced contraction in SMA rings, followed by a gradual relaxation of phenylephrine precontracted tone. Contraction was abolished by indomethacin (IM), cycloheximide (Chx), and endothelium denudation. In contrast, the gradual relaxation was abolished by NOS inhibitors, Chx, endothelium denudation, and inhibited by EtOH (50 and 100 mM). However, IM had no effect on relaxation. Western blot analysis demonstrated that iNOS expression was induced by IL-1ß and was inhibited by EtOH and endothelium denudation. Furthermore, messenger RNA expression of iNOS, but not endothelial NOS, was inhibited by EtOH. These data suggest that IL-1ß-induced contraction is mediated by thromboxane A2, whereas IL-1ß-induced relaxation occurs via NO derived from iNOS. The endothelium plays an important role in vasorelaxation. Taken together, EtOH inhibits IL-1ß-mediated vasorelaxation by suppressing endothelium iNOS expression. This study provides the first evidence of EtOH -induced inhibition of IL-1ß-mediated vasorelaxation.

Abnormalities in chromosome 6q24 as a cause of early-onset, non-obese, non-autoimmune diabetes mellitus without history of neonatal diabetes.

AIMS: Abnormalities in the imprinted locus on chromosome 6q24 are the most common causes of transient neonatal diabetes mellitus (6q24-related transient neonatal diabetes). 6q24-Related transient neonatal diabetes is characterized by the patient being small-for-gestational age, diabetes mellitus at birth, spontaneous remission within the first few months and frequent recurrence of diabetes after childhood. However, it is not clear whether individuals with 6q24 abnormalities invariably develop transient neonatal diabetes. This study explored the possibility that 6q24 abnormalities might cause early-onset, non-autoimmune diabetes without transient neonatal diabetes. METHODS: The 6q24 imprinted locus was screened for abnormalities in 113 Japanese patients with early-onset, non-obese, non-autoimmune diabetes mellitus who tested negative for mutations in the common maturation-onset diabetes of the young (MODY) genes and without a history of transient neonatal diabetes. Positive patients were further analysed by combined loss of heterozygosity / comparative genomic hybridization analysis and by microsatellite analysis. Detailed clinical data were collected through the medical records of the treating hospitals. RESULTS: Three patients with paternal uniparental isodisomy of chromosome 6q24 were identified. None presented with hyperglycaemia in the neonatal period. Characteristically, these patients were born small-for-gestational age, representing 27.2% of the 11 patients whose birth weight standard deviation score (SDS) for gestational age was below -2.0. CONCLUSIONS: Abnormalities in the imprinted locus on chromosome 6q24 do not necessarily cause transient neonatal diabetes. Non-penetrant 6q24-related diabetes could be an underestimated cause of early-onset, non-autoimmune diabetes in patients who are not obese and born small-for-gestational age.

The identification of irreversible rituximab-resistant lymphoma caused by CD20 gene mutations.

C-terminal mutations of CD20 constitute part of the mechanisms that resist rituximab therapy. Most CD20 having a C-terminal mutation was not recognized by L26 antibody. As the exact epitope of L26 has not been determined, expression and localization of mutated CD20 have not been completely elucidated. In this study, we revealed that the binding site of L26 monoclonal antibody is located in the C-terminal cytoplasmic region of CD20 molecule, which was often lost in mutated CD20 molecules. This indicates that it is difficult to distinguish the mutation of CD20 from under expression of the CD20 protein. To detect comprehensive CD20 molecules including the resistant mutants, we developed a novel monoclonal antibody that recognizes the N-terminal cytoplasm region of CD20 molecule. We screened L26-negative cases with our antibody and found several mutations. A rituximab-binding analysis using the cryopreserved specimen that mutation was identified in CD20 molecules indicated that the C-terminal region of CD20 undertakes a critical role in presentation of the large loop in which the rituximab-binding site locates. Thus, combination of antibodies of two kinds of epitope permits the identification of C-terminal CD20 mutations associated with irreversible resistance to rituximab and may help the decision of the treatment strategy.

Galectin-9 exhibits anti-myeloma activity through JNK and p38 MAP kinase pathways.

Galectins constitute a family of lectins that specifically exhibit the affinity for beta-galactosides and modulate various biological events. Galectin-9 is a tandem-repeat type galectin with two carbohydrate recognition domains and has recently been shown to have an anti-proliferative effect on cancer cells. We investigated the effect of recombinant protease-resistant galectin-9 (hGal9) on multiple myeloma (MM). In vitro, hGal9 inhibited the cell proliferation of five myeloma cell lines examined, including a bortezomib-resistant subcell line, with IC(50) between 75.1 and 280.0 nM, and this effect was mediated by the induction of apoptosis with the activation of caspase-8, -9, and -3. hGal9-activated Jun NH(2)-terminal kinase (JNK) and p38 MAPK signaling pathways followed by H2AX phosphorylation. Importantly, the inhibition of either JNK or p38 MAPK partly inhibited the anti-proliferative effect of hGal9, indicating the crucial role of these pathways in the anti-MM effect of hGal9. hGal9 also induced cell death in patient-derived myeloma cells, some with poor-risk factors, such as chromosomal deletion of 13q or translocation t(4;14)(p16;q32). Finally, hGal9 potently inhibited the growth of human myeloma cells xenografted in nude mice. These suggest that hGal9 is a new therapeutic target for MM that may overcome resistance to conventional chemotherapy.

Statin-independent prognosis of patients with diffuse large B-cell lymphoma receiving rituximab plus CHOP therapy.

BACKGROUND: A recent laboratory study indicated that statins impaired the antitumor effects of rituximab by inducing conformational changes in CD20. Although these findings raised significant concerns about statin use during rituximab treatment, their clinical significance is unclear. PATIENTS AND METHODS: We conducted a retrospective study investigating the effects of statins on the prognosis of diffuse large B-cell lymphoma (DLBCL) treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP). Newly diagnosed DLBCL patients were analyzed (n = 256), including 35 patients taking statins. RESULTS: The 3-year progression-free survival rates were 84% and 73% (P = 0.38), while the overall survival rates were 89% and 78% (P = 0.28) for those patients treated with and without statins, respectively. After adjusting for the International Prognostic Index and serum cholesterol level, statin use was not associated with prognosis. CONCLUSIONS: These results indicate that statins do not influence the clinical prognosis of DLBCL treated with RCHOP. Further studies with larger numbers of patients are warranted to confirm the prognostic significance of statins for patients with DLBCL receiving rituximab-containing chemotherapy.

Bone metastasis and poor performance status are prognostic factors for survival of carcinoma of unknown primary site in patients treated with systematic chemotherapy.

BACKGROUND: Cancer of unknown primary site (CUP) generally has a poor prognosis, and there is no established standard therapy. There have been no reports of a prognostic model for CUP patients treated with a single regimen of systemic chemotherapy. METHODS: Univariate and multivariate prognostic factor analysis for overall survival (OS) were conducted retrospectively in 58 consecutive CUP patients treated with carboplatin plus paclitaxel (Taxol) therapy as a first-line treatment. RESULTS: Univariate prognostic factor analysis revealed baseline performance status (PS) of two or more, low serum albumin level, pleural effusion, bone metastasis, and liver metastasis as adverse prognostic factors. Cox proportional hazards analysis showed that poor PS and bone metastasis had the most powerful adverse impact on survival. We developed a prognostic model using those two variables-a good-risk group (PS 0-1 without bone metastasis) and a poor-risk group (PS > or =2 or bone metastasis). The poor-risk group showed significantly poorer OS than the good-risk group (1 year OS 36.8% versus 67.1%, P = 0.0003). CONCLUSIONS: Poor PS and bone metastasis were identified as independent adverse prognostic factors in CUP. A simple prognostic model was developed and seems useful for decision making as to whether chemotherapy is indicated for CUP patients.

Soluble interleukin-2 receptor retains prognostic value in patients with diffuse large B-cell lymphoma receiving rituximab plus CHOP (RCHOP) therapy.

BACKGROUND: Soluble interleukin-2 receptor (SIL-2R) is known to be a prognostic parameter in patients with diffuse large B-cell lymphoma (DLBCL) receiving cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) therapy. However, its prognostic value has not been well known since the introduction of rituximab. PATIENTS AND METHODS: We retrospectively evaluated the prognostic impact of SIL-2R in 228 DLBCL patients, comparing 141 rituximab-combined CHOP (RCHOP)-treated patients with 87 CHOP-treated patients as a historical control. RESULTS: Patients with high serum SIL-2R showed significantly poorer event-free survival (EFS) and overall survival (OS) than patients with low SIL-2R in both the RCHOP group (2-year EFS, 66% versus 92%, P<0.001; OS, 82% versus 95%, P=0.005) and the CHOP group (2-year EFS, 40% versus 82%; OS, 61% versus 90%, both P<0.001). Multivariate analysis including the five parameters of International Prognostic Index (IPI) and two-categorized IPI revealed that SIL-2R was an independent prognostic factor for EFS and OS in the RCHOP group as well as in the CHOP group. CONCLUSIONS: Our results demonstrate that SIL-2R retains its prognostic value in the rituximab era. The prognostic value of SIL-2R in DLBCL patients receiving rituximab-combined chemotherapy should be reassessed on a larger scale and by long-term follow-up.

CD5 expression is potentially predictive of poor outcome among biomarkers in patients with diffuse large B-cell lymphoma receiving rituximab plus CHOP therapy.

BACKGROUND: Several biomarkers indicating poor prognosis have been reassessed in patients receiving rituximab combination chemotherapy for diffuse large B-cell lymphoma (DLBCL). However, few studies have investigated outcome in relation to a combination of these biomarkers. In addition, no large-scale studies have reassessed the outcome of patients with CD5-positive DLBCL treated with rituximab. PATIENTS AND METHODS: We conducted a retrospective study and investigated the predictive value of three biomarkers -- BCL2, germinal center (GC) phenotype and CD5 -- in 121 DLBCL patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone. RESULTS: CD5-positive patients showed significantly poorer event-free survival (EFS) and overall survival (OS) than CD5-negative patients (2-year EFS, 18% versus 73%, P < 0.001; 2-year OS, 45% versus 91%, P = 0.001). However, no significant difference in outcome according to BCL2 or GC phenotype was observed. Multivariate analysis revealed that CD5 expression was a significant prognostic factor for EFS [hazard ratio 14.2, 95% confidence interval (CI) 4.7-43.2] and OS (hazard ratio 20.3, 95% CI 3.6-114.4). CONCLUSIONS: CD5 expression was the only significant prognostic factor among the biomarkers examined in this study. Further studies with larger numbers are warranted to confirm the prognostic significance of CD5 expression for patients with DLBCL receiving rituximab-containing chemotherapy.

Establishment of embryonic stem cells secreting human factor VIII for cell-based treatment of hemophilia A.

BACKGROUND: Hemophilia A is an X-chromosome-linked recessive bleeding disorder resulting from an F8 gene abnormality. Although various gene therapies have been attempted with the aim of eliminating the need for factor VIII replacement therapy, obstacles to their clinical application remain. OBJECTIVES: We evaluated whether embryonic stem (ES) cells with a tetracycline-inducible system could secrete human FVIII. METHODS AND RESULTS: We found that embryoid bodies (EBs) developed under conditions promoting liver differentiation efficiently secreted human FVIII after doxycycline induction. Moreover, use of a B-domain variant F8 cDNA (226aa/N6) dramatically enhanced FVIII secretion. Sorting based on green fluorescent protein (GFP)-brachyury (Bry) and c-kit revealed that GFP-Bry(+)/c-kit(+) cells during EB differentiation with serum contain an endoderm progenitor population. When GFP-Bry(+)/c-kit(+) cells were cultured under the liver cell-promoting conditions, these cells secreted FVIII more efficiently than other populations tested. CONCLUSION: Our findings suggest the potential for future development of an effective ES cell-based approach to treating hemophilia A.

Comparison of two-year and five-year tamoxifen use in Japanese post-menopausal women.

AIM: Large multicenter, randomized trials have revealed the advantages of using tamoxifen for 5 years vs 2 years in breast cancer patients. The aim of this report is to confirm the optimal duration of tamoxifen administration in a study of Japanese breast cancer patients. METHODS: Japanese post-menopausal estrogen receptor-positive breast cancer patients treated with mastectomy were randomly assigned to either a 5-year or 2-year course of tamoxifen. The primary endpoint was disease-free survival, with secondary endpoints of overall survival and a reduction in the development of metachronous contra-lateral breast cancer. RESULTS: A total of 256 breast cancer patients were randomized to a 5-year or 2-year tamoxifen administration group. After a median follow-up time of 81 months, there were no significant differences seen in terms of disease-free or overall survival (p=0.65 and 0.56, respectively). Furthermore, the impact of the 5-year use of tamoxifen on the development of contra-lateral breast cancer did not reach statistical significance (p=0.0511). However, 5-year tamoxifen use was closely associated with gynaecological complications (p=0.0081). CONCLUSION: We could not show a beneficial effect of using tamoxifen for 5 years over 2 years in Japanese estrogen receptor-positive patients. This is likely due to the small number of patients enrolled in our study; however, racial disparity may influence this result. A reevaluation is necessary to study the advantages of the 5-year use of tamoxifen in the Japanese racial subgroup.

Elevation of circulating plasma cytokines in cancer patients with high plasma parathyroid hormone-related protein levels.

Parathyroid hormone (PTH) and PTH-related protein/peptide (PTHrP) bind to the same PTH/PTHrP receptor and stimulate osteoblasts to secrete pro-inflammatory cytokines like interleukin (IL)-6. In patients with primary hyperparathyroidism, elevation of plasma levels of tumor necrosis factor (TNF)-alpha and IL-6 was also described. We, therefore, postulated that PTHrP secreted from cancer cells stimulates the secretion of cytokines and causes increases in their blood levels. Blood concentrations of several cytokines (TNF-alpha, IL-1beta, IL-5, IL-6, IL-8, IL-11 and IL-12) in cancer-bearing patients with or without elevation of blood PTHrP were measured by ELISA. The patients with high plasma PTHrP levels (n=29, intact PTHrP: 8.5 +/- 1.4 pmol/l, normal: <1.1) had higher serum type 1 collagen C-telopeptide (ICTP). Twenty of the patients were hypercalcemic. Plasma concentrations of TNF-alpha, IL-6 and IL-8 were significantly increased in patients with high PTHrP, in either the presence or absence of hypercalcemia. The concentrations of TNF-alpha and IL-6 were also significantly correlated with those of PTHrP. Our observations indicate that high plasma levels of PTHrP in cancer-bearing patients contribute not only to the development of hypercalcemia, but also to the development of the syndrome caused by an excess of pro-inflammatory cytokines.