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Β2-Glycoprotein I (β2GPI) is an important anti-thrombotic protein and is the major auto-antigen in the antiphospholipid syndrome (APS). The clinical relevance of nitrosative stress in post translational modification of β2GPI was examined.The effects of nitrated (n)β2GPI on its anti-thrombotic properties and its plasma levels in primary and secondary APS were determined with appropriate clinical control groups. β2-glycoprotein I was nitrated at tyrosines 218, 275 and 309. β2-glycoprotein I binds to lipid peroxidation modified products through Domains IV and V. Nitrated β2GPI loses this binding (p

Obstetric antiphospholipid syndrome (APS) is now being recognized as a distinct entity from vascular APS. Pregnancy morbidity includes >3 consecutive and spontaneous early miscarriages before 10weeks of gestation; at least one unexplained fetal death after the 10th week of gestation of a morphologically normal fetus; a premature birth before the 34th week of gestation of a normal neonate due to eclampsia or severe pre-eclampsia or placental insufficiency. It is not well understood how antiphospholipid antibodies (aPLs), beyond their diagnostic and prognostic role, contribute to pregnancy manifestations. Indeed aPL-mediated thrombotic events cannot explain the obstetric manifestations and additional pathogenic mechanisms, such as a placental aPL mediated complement activation and a direct effect of aPLs on placental development, have been reported. Still debated is the possible association between aPLs and infertility and the effect of maternal autoantibodies on non-vascular manifestations in the babies. Combination of low dose aspirin and unfractionated or low molecular weight heparin is the effective treatment in most of the cases. However, pregnancy complications, in spite of this therapy, can occur in up to 20% of the patients. Novel alternative therapies able to abrogate the aPL pathogenic action either by interfering with aPL binding at the placental level or by inhibiting the aPL-mediated detrimental effect are under active investigation.

The emergence and transmission of drug-resistant human immunodeficiency virus-1 (HIV-1) compromises antiretroviral treatment for HIV-1. Thus, testing for drug resistance is recommended at diagnosis and before initiating highly active antiretroviral treatment. We conducted an epidemiological study enrolling newly diagnosed patients between 2003 and 2008 in our nationwide surveillance network. In the 6-year study period, the prevalence of drug-resistant HIV-1 among 2573 patients, consisting mainly of Japanese men in their late-30s and infected through male-to-male sexual contacts, followed an increasing trend from 5.9% (16/273) in 2003 to 8.3% (50/605) in 2008. Nucleoside reverse transcriptase inhibitor-associated mutations predominated in each year, with T215 revertants being the most abundant. The predictive factor for drug-resistant HIV-1 transmission was subtype B (OR=2.36; p=0.004), and those for recent HIV-1 infection were male gender (OR=3.79; p=0.009), MSM behavior (OR=1.67; p=0.01), Japanese nationality (OR=2.31; p=0.008), and subtype B (OR=5.64; p

Cytomegalovirus (CMV) infection is 1 of the major causes of morbidity in patients undergoing allogeneic stem cell transplantation (allo-SCT). The incidences of CMV antigenemia and CMV disease in 43 patients who received allogeneic bone marrow transplantation (BMT) using a reduced-intensity conditioning (RIC) regimen, which mainly consisted of fludarabine (Flu), busulfan (Bu), and total body irradiation (TBI), were compared with those in 68 patients who received a myeloablative conditioning (MAC) regimen, and risk factors for CMV antigenemia and CMV disease were identified. Before engraftment, grade 3-4 mucosal injury because of the conditioning regimen was significantly decreased in RIC patients (stomatitis: P = .02; diarrhea: P

Erratum is published in Biology of Blood and Marrow Transplantation, Volume 14, Issue 9, 2008, p.1079., Adult T cell leukemia/lymphoma (ATL) is a highly aggressive T cell malignancy, and has a poor prognosis. Recently, allogeneic-hematopoietic stem cell transplantation (allo-HSCT) has been suggested to improve the outcome. We retrospectively analyzed 15 patients with ATL who had received allo-HSCT in 2 institutions in Hokkaido, Japan. The median age of the patients was 57 years. The estimated 3-year overall survival (OS) and progression-free survival (PFS) rates were 73.3% and 66.7%, respectively. Calcineurin inhibitor dosage was reduced and administration was discontinued abruptly in 6 of the 15 patients for disease control; as a result, 4 (66.7%) of the 6 patients achieved complete response (CR) or partial response. Therefore, a graft-versus-leukemia/lymphoma (GVL) effect might be induced by discontinuation of immunosuppression. Thirteen of the 15 patients were followed up by monitoring HTLV-1 proviral DNA levels. In 10 of the 11 patients with positive HTLV-1 proviral DNA before allo-HSCT, HTLV-1 proviral DNA became undetectable at least once after allo-HSCT, and only 1 of the 5 patients in whom HTLV-1 proviral DNA became detectable after allo-HSCT relapsed. Compared to the results of past studies, these results show that allo-HSCT greatly improved the prognosis of ATL and suggest a contribution of the induction of a GVL effect.

We retrospectively evaluated the outcomes of 37 adult patients with acute lymphoblastic leukemia (ALL) undergoing allogeneic hematopoietic stem cell transplantation (allo-SCT) conditioned with medium-dose VP-16 (VP, 30 mg/kg), cyclophosphamide (CY, 120 mg/kg), and fractionated total-body irradiation (TBI, 12 Gy) (medium-dose VP/CY/TBI). The median age of the patients was 26 years. Thirteen patients underwent transplantation from HLA-matched related donors (MRD), 18 patients underwent transplantation from HLA-matched unrelated donors (MUD), and 6 patients underwent transplantation from HLA-mismatched donors (MMD). Thirty-two patients received bone marrow and 4 patients received peripheral blood stem cells. Ten patients were Philadelphia chromosome-positive (Ph+) and 35 patients were in complete remission (CR) at transplantation. All of the patients achieved engraftment, and grade 3 organ toxicity before engraftment occurred in 27 patients. Grade II-III acute graft-versus-host disease (GVHD) and chronic GVHD (cGVHD) occurred in 15 and 18 patients, respectively. No patient developed grade IV acute GVHD (aGVHD) or died of GVHD. At median follow-up of 35.1 months, 32 patients were alive and all Ph+ patients were alive. Three patients died of relapse and 2 died of transplant-related mortality (TRM). The actuarial 3-year overall survival (OS) rate, relapse rate, and TRM rate were 89.2%, 8.1%, and 5.4%, respectively. Non-CR at transplantation, MRD, and no aGVHD were significant adverse prognostic factors for survival. Medium-dose VP/CY/TBI for adult ALL patients was associated with lower relapse rate and no increase in toxicity, resulting in better survival.

Hematopoietic stem cell transplantation (HSCT) is an effective treatment for refractory autoimmune diseases. The safety and long-term outcome have been also acceptable. Infectious diseases under immune suppressive state after autologous HSCT are common transplantation related complications whereas autoimmune diseases are uncommon. Organ specific autoimmune diseases, such as immune mediated thrombocytopenia and thyroid dysfunction, are the most common after autologous HSCT. Systemic autoimmune diseases can also develop after autologous HSCT in patients with hematological disorders with genetic predisposition to autoimmune diseases. Although the mechanism of autoimmunity after HSCT is not well-known, long-term follow-up is essential in patients with autoimmune diseases treated with autologous HSCT.

Background Fibrin monomer (FM) and its complex (sFC) exist at high concentrations in hypercoagulable state blood. Two novel immunoassays for sFC (SF and FMC) using specific monoclonal antibodies (IF-43 and F405) were recently developed. Methods We measured the concentrations of thrombotic markers in 103 patients with DIC and thrombotic disorders. Results We found that the concentration of FMC was approximately 3.35-fold greater than that of SF. In patients with a high FMC/SF ratio, FDP and D dimer concentrations were increased, suggesting that the discrepancy in sFC concentrations was caused by fibrinolytic activity. Further, plasma samples from those patients were found to contain the X- and Y-fragments of FM in addition to FM and sFC in a Western blotting assay using F405, which binds with those fragments. In an in vitro study, FM formed from pooled plasma containing EDTA was degraded to the X- and Y-fragments of FM by fibrinolytic activity, and we termed those FM degradation products (FMDP). Conclusion Determination of FMDP is important for diagnosis of thrombogenic conditions associated with fibrinolysis, such as in patients with DIC, and it may serve as a useful marker for hypercoagulable states with accelerated fibrinolysis.

We examined the frequency of cognitive impairment using the mini-mental-status examination (MMSE), as well as cerebral perfusion using single photon emission computed tomography (SPECT) in elderly diabetic patients. Written consent was obtained from all patients prior to their inclusion in this study. An MMSE score of 26 or less was adopted as an indication of cognitive impairment. Following an initial study, a 3-month study incorporating the use of MMSE and SPECT was performed in subjects, some of whom were taking donepezil hydrochloride. Of the 92 subjects enrolled in this study, 38% exhibited cognitive functional impairment and 18% earned MMSE scores of 23 or lower that were indicative of dementia. With regard to their cerebral blood flow pattern as determined by SPECT 217, 31.4 and 34.2% of subjects showed parieto-temporal hypoperfusion, asymmetrical hypoperfusion and fronto-temporal hypoperfusion patterns of abnormalities, respectively; 11.4% displayed unclassifiable findings and 8.5% showed no detectable abnormalities. No significant differences were seen in patients that were taking donepezil hydrochloride compared to those who were not. The incidence of cognitive functional impairment in elderly, diabetic patients was significantly elevated and was accompanied by a reduction in cerebral blood flow in the fronto-temporal region, as determined by SPECT.

Background CD34 + cell-selected autologous PBSC transplantation (CD34 + APBSCT) is a procedure used for the treatment of patients with malignant disease that is intended to eliminate residual tumor cells from autologous grafts. However, frequent infectious complications after CD34 + APBSCT can occur. A delay of recovery of the absolute number ofCD4 + T cells after transplantation was reported to be one disadvantageous factor. As data on T-cell function after CD34 + APBSCT are scanty, we analyzed changes in T-helper cell 1 (Th1) and T-helper cell 2 (Th2) after CD34 + APBSCT to evaluate immune reconstitution. Methods Twelve patients underwent APBSCT (CD34 + APBSCT group, n =4, and unselected APBSCT, n =8). Peripheral blood (PB) samples were obtained at 2, 4, 8, 12 and 16 weeks after the transplantation. The dynamics of the Th1 and Th2 were analyzed at a single-cell level, using flow cytometry. Results In the CD34 + APBSCT group, not only the absolute count ofCD4 + T cells but also the proportion of Th1 cells in CD4 T cells and the ratio of Th1 to Th2 after transplantation were significantly decreased at 2 and 4 weeks after transplantation compared with findings in the unselected APBSCT group. Discussion We suggest that higher rates of infectious complications after CD34 + APBSCT may be due to the inability of residual T cells from the CD34 + cell selection to generate mature T cells that function adequately against infection. Although further study would be required, our preliminary data provide some information on the immune reconstitution after CD34 + APBSCT and differentiation of T lymphocytes into Th1 and Th2 in vivo .

The preliminary classification criteria for definite antiphospholipid syndrome (APS) include the presence of anticardiolipin antibodies (aCL) and/or lupus anticoagulant (LA) as laboratory criteria. However, antiphospholipid antibodies (aPL) are a heterogeneous group of antibodies comprising also antibodies against phospholipid-binding proteins or their complexes with phospholipids. Prothrombin is one of the antigen recognized by aPL. In the last decade, there has been increasing interest in antibodies against prothrombin alone and those against phosphatidylserine-prothrombin complex. The latter, phosphatidylserine-dependent antiprothrombin antibodies (aPT), have been closely associated with APS and LA. In this paper, we review the properties of antiprothrombin antibodies.

Objective The inhibitory effects of tumor necrosis factor-α (TNF-α) on cytokine-induced proliferation and differentiation of normal human erythroid progenitors have been characterized extensively, yet little is known about the maturation level of erythroid progenitors that are sensitive to TNF-α or of the expression of TNF receptors (TNFRs) in erythroid lineage. The aim of this study was to determine the extent to which human erythroid progenitor cells are sensitive to TNF-α, and to relate this to the expression of TNFRs in the erythroid lineage. Materials and Methods Highly purified human CD34 + cells underwent erythroid differentiation, with or without TNF-α. We used colony assay as well as a method by which colony-forming unit-erythroid (CFU-E) and glycophorin A (GPA; a specific marker for erythroid lineage) positive cells can be generated in liquid phase from purified human CD34 + cells in the presence of multiple cytokines, including stem cell factor (SCF), interleukin-3 (IL-3), and erythropoietin (EPO). During erythroid differentiation of CD34 + cells, TNFRs expression were monitored. Results TNF-α inhibited the generation of GPA + cells by CD34 + cells as well as the proliferative capacity of GPA + cells supported by EPO, IL-3, and SCF. Erythroid progenitors became resistant to the inhibitory effect of TNF-α as they matured. The detectable expression of TNFR-I was transient in the early phase of erythroid differentiation, whereas TNFR-II was expressed through the entire course of erythroid differentiation of CD34 + cells. Conclusions TNF-α suppresses erythropoiesis by inhibiting the generation of GPA + cells derived from CD34 + cells as well as by inhibiting the proliferative capacity of GPA + cells. Although the presence of TNFRs does not directly indicate that the receptor(s) mediates death signaling, altered expression of TNFRs depending on the level of maturation may imply altered sensitivities to TNF-α in various stage of erythroid progenitors.

beta(2)-Glycoprotein I (beta(2)-GPI) is a major antigen for anticardiolipin antibodies (aCL, Abs) present in patients with antiphospholipid syndrome. We recently reported that beta(2)-GPI specifically binds to oxidized LDL (oxLDL) and that the beta(2)-GPI's major ligand, oxLig-1 is 7-ketocholesteryl-9-carboxynonanoate (Kobayashi, K., E. Matsuura, Q. P. Liu, J. Furukawa, K. Kaihara, J. Inagaki, T. Atsumi, N. Sakairi, T. Yasuda, D. R. Voelker, and T. Koike. 2001. A specific ligand for beta(2)-glycoprotein I mediates autoantibody-dependent uptake of oxidized low density lipoprotein by macrophages. J. Lipid Res. 42: 697-709). In the present study, we demonstrate that omega-carboxylated 7-ketocholesteryl esters are critical for beta(2)-GPI binding. A positive ion mass spectrum of a novel ligand, designated oxLig-2, showed fragmented ions at m/z 383 and 441 in the presence of acetone, which share features of oxLig-1 and 7-ketocholesterol. In the negative ion mode, ions at m/z 627, 625, and 243 were observed. oxLig-2 was most likely 7-ketocholesteryl-12-carboxy (keto) dodecanoate. These ligands were recognized by beta(2)-GPI. Liposome binding to macrophages was significantly increased depending on the ligand's concentration, in the presence of beta(2)-GPI and an anti-beta(2)-GPI Ab. Synthesized variant, 7-ketocholesteryl-13-carboxytridecanoate (13-COOH-7KC), also showed a significant interaction with beta(2)-GPI and a similar binding profile with macrophages. Methylation of the carboxyl function diminished all of the specific ligand interactions with beta(2)-GPI. Thus, omega-carboxyl variants of 7-ketocholesteryl esters can mediate anti-beta(2)-GPI Ab-dependent uptake of oxLDL by macrophages, and autoimmune atherogenesis linked to beta(2)-GPI interaction with oxLDL.

Advanced glycation end products (AGEs) have been thought to participate in diabetic microangiopathy. However, the effects of AGEs on angiogenesis have so far been mainly examined either in vitro or by using cultured cells. In the present study, we have analyzed whether AGEs induce angiogenesis in vivo by using the chorioallantoic membrane (CAM) assay. The CAM assay was carried out in embryonated hen eggs to determine the effects of AGEs. Following generation of AGEs based on bovine serum albumin (BSA), either AGE-BSA or nonglycated BSA was administered to the CAM and their effects on angiogenesis were assessed, together with an inhibitory effect of an anti-AGE antibody against AGE-BSA-induced angiogenesis. The histological features of AGE-induced vascular lumens were examined by immunohistochemical analysis for Factor VIII and smooth muscle alpha-actin. AGE-BSA induced angiogenesis in CAM in a dose- and time-dependent manner. AGE-induced angiogenesis on CAM was neutralized by the anti-AGE antibody. Immunohistochemical analysis demonstrated that AGE-induced vascular lumens were devoid of pericytes. Our data demonstrated that AGEs are an angiogenetic factor and that our system of AGE-induced abnormal vessels in CAMs is useful in further investigations of the mechanism of diabetic retinal angiogenesis and can also be used to provide a therapeutic model for diabetic angiopathy.

To identify zinc finger genes in human primary cultured erythroid progenitor cells, RT-PCR was performed using primers specific for the conserved sequence in zinc finger domains of mouse Friend of GATA-1. We identified a novel Krüppel-associated box (KRAB) zinc finger gene, ZNF317. Evaluation of full-length cDNA obtained by RACE showed that it encoded a protein composed of 13 Krüppel-like zinc fingers and a KRAB domain. RT-PCR analysis revealed four alternatively splicing products (ZNF317-1 through ZNF317-4). The ZNF317 gene has seven exons and is located in human chromosome 19p13. Northern analysis revealed that ZNF317-1 and ZNF317-2 transcripts were ubiquitously expressed, whereas ZNF317-3 and ZNF317-4 transcripts were detected only in lymphocytes, spleen, and lung. Competitive RT-PCR analysis showed that the expression of ZNF317 mRNA significantly decreased during erythroid maturation. In lymphocytes, ZNF317 expression was reduced in response to mitogenic stimulation. We propose that ZNF317 may play an important role in erythroid maturation and lymphoid proliferation.

Objective It has been reported that stem cell factor (SCF) promotes cell survival in primary cultured human erythroid colony-forming cells (ECFC). Given the heterogeneous nature of ECFC, which may affect interpretation of the data, we purified c-kit + ECFC and investigated the specificity and mechanisms of the anti-apoptotic effects of SCF on these cells. Materials and Methods Glycophorin A + (GPA + ) c-kit + cells were purified from primary cultured ECFC derived from purified human CD34 + cells. The GPA + c-kit − and nonerythroid cells were generated from the same CD34 + cells. Apoptosis of ECFC was investigated in the absence or presence of SCF and erythropoietin (EPO) in serum-free medium. DNA fragmentation was measured with enzyme linked immunosorbent assay for oligonucleosome-sized DNA, gel electrophoresis, and annexin V labeling. Characterization of expanded cells and enriched cells was performed using multiparameter flow cytometry. For Akt assay, cells were lysed and the cleared lysates subjected to SDS-PAGE followed by Western blotting. Results In GPA + c-kit + cells, deprivation of cytokine caused rapid DNA fragmentation within 4 hours that reached a maximum at 6 hours. This was partially but clearly prevented by SCF or EPO. In contrast, no significant DNA fragmentation was seen in GPA + c-kit − and nonerythroid cells within 24 hours. PP2, a specific Src family kinase inhibitor, but not its inactive analogue PP3, reversed the anti-apoptotic effects of SCF. PP2 also inhibited SCF-induced phosphorylation of Akt. Conclusions These data indicate that SCF protects purified human GPA + c-kit + cells from apoptosis and suggest that kit-mediated Src kinase activation is involved in Akt activation and cell survival.

beta(2)-Glycoprotein I (beta(2)-GPI) is a major antigen for antiphospholipid antibodies (Abs) present in patients with the antiphospholipid syndrome (APS). We previously reported that beta(2)-GPI specifically binds to oxidized low density lipoprotein (oxLDL), but not to native low density lipoprotein (LDL). In the present study, a ligand specific for beta(2)-GPI, oxLig-1, was purified from the extracted lipids of oxLDL. The structure of oxLig-1 was shown to be identical to that of synthesized 7-ketocholesteryl-9-carboxynonanoate by mass spectroscopy and nuclear magnetic resonance analyses. Both purified and synthesized oxLig-1 were recognized by beta(2)-GPI and subsequently by anti-beta(2)-GPI auto-Abs, either in enzyme-linked immunosorbent assay (ELISA) or in ligand blot analysis. Binding of liposomes containing oxLig-1 (oxLig-1-liposomes) to mouse macrophages, J774A.1 cells, was relatively low, as compared with that of phosphatidylserine (PS)-liposomes. In contrast, binding of oxLig-1-liposomes was enhanced more than 10-fold in the presence of both beta(2)-GPI and an anti-beta(2)-GPI auto-Ab (WB-CAL-1), derived from (NZW x BXSB) F1 mouse, an animal APS model. Anti-beta(2)-GPI auto-Abs derived from APS patients with episodes of arterial thrombosis were detected in ELISA, using a solid phase oxLig-1 complexed with beta(2)-GPI. We suggest that autoimmune atherogenesis linked to beta(2)-GPI interaction with oxLDL and Abs may be present in APS.

We examined the expression of macrophage migration inhibitory factor (MIF) mRNA in murine macrophage cell line (RAW 264.7 cells) in response to oxidized low-density lipoprotein (oxLDL), and investigated the influence of MIF on the uptake and degradation of oxLDL by RAW 264.7 cells. MIF mRNA expression was markedly upregulated in the presence of oxLDL. Consistent with this, the MIF level of the culture medium was increased by stimulation with oxLDL in dose- and time-dependent manners. Next, we added recombinant rat MIF to the culture medium and examined its effects on the uptake of(125)I-labelled oxLDL. Pretreatment with MIF enhanced both the uptake and degradation of(125)I-oxLDL. Taken together, these results suggest that MIF released from macrophages in response to oxLDL stimulates oxLDL uptake and degradation in an autocrine and paracrine fashion, which potentially results in atherosclerosis.

β 2 -glycoprotein I (β 2 -GPI=apolipoprotein H) is an important autoantigen in patients with the antiphospholipid syndrome. It also plays a role in lipoprotein metabolism, such as anti-atherogenic property, triglyceride removal, and enhancement of lipoprotein lipase. Serum β 2 -GPI concentration of 812 apparently healthy Japanese individuals was measured by sandwich EIA. Two families with complete β 2 -GPI deficiency were identified. In one family, all affected had increased serum LDL-cholesterol levels or smaller particle sizes of LDL, while the other had no apparent abnormality in lipid metabolism. Individuals investigated had no history of thrombosis or overt abnormalities in hemostatic tests. A thymine corresponding to position 379 of the β 2 -GPI cDNA was deleted in every β 2 -GPI deficient individual. The incidence of this heterozygous deficiency determined by RFLP was 6.3% in Japanese and none in Caucasians. Heterozygotes had significantly lower concentrations of serum β 2 -GPI than did those without the mutation, yet no significantly different lipid profiles, such as total cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol, apoA-I, apoB and Lp(a), were observed. A low concentration of β 2 -GPI seemed not to be associated with apparent abnormality in lipoprotein metabolism.

To determine whether human endogenous retroviruses are implicated in the pathogenesis of inflammatory vascular diseases of unknown etiology, we examined mRNA expression of a human endogenous retrovirus, HERV-R, which has a long open reading frame in the env region, in cultured human vascular endothelial and smooth muscle cells stimulated in the presence of various cytokines. mRNA of HERV-R was always evident in these cells but not in fibroblastic cells. Levels of expression in vascular endothelial cells were significantly regulated by treatment with tumor necrosis factor-alpha, interleukin (IL)-1alpha, and IL-1beta as up-regulators and interferon-gamma as a down-regulator. These observations are interpreted to mean that HERV-R expression may be up- or down-regulated at sites of inflammation in vessels in vivo and hence may play a pathogenetic role in inflammatory vascular diseases in humans, perhaps similar to endogenous retroviruses in mouse models of polyarteritis nodosa in humans.

To establish an available, economical technique for the large-scale purification of CD34(+) cells we used a nylon-fiber syringe (NF-S) for depletion of adherent cells and then selected CD 34(+) cells from peripheral blood mobilized by G-CSF, using MAb and magnetic heads.PBSC were mobilized and collected from adult, healthy volunteers. With the effect of concentration of anti-CD34 MAb (9C5) on the purification of CD34(+) cells from 1 2 10(8) NF-S treated cells (NF cells), the recovery and the purity of CD34(+) cells was identical for 5, 10, 20, 40 microg of 9C5. In this study, therefore, the concentration of 9C5 was maintained at5 microg/10(8) NF cells, with a cellhead ratio of 1:10.When half to one-third of leukapheresis product containing 121.5 + or - 26.0 2 10(8) mononuclear cells (mean + or - SD; n = 6) was processed for CD34(+) cell purification, 5.26 + or - 3.01 2 10(7) total purified cells were obtained with a purity of CD34(+) cells at 94.9 - 8.5%. The numbers of CD34(+) cells and total progenitor cells recovered in this process were 4.71 + or - 2.33 2 10(7) cells and 145.9 + or - 121.8 2 10(5) cells, respectively. The total recovery of CD34(+) cells was 37.0 + or - 21.0%. The depletion of monocyte by NF-S reduced the 9C5 anti-human CD34 MAb needed for purification of CD34(+) cells to one-third. Two patients were grafted with peripheral blood CD34(+) cells selected by this procedure and achieved a rapid and consistent hematopoiesis.Our clinical data show that these cells are capable of rapid reconstitution of hematopoiesis after high-dose chemotherapy. The procedure contains an additional step; however, consistent high purity of CD34(+) cells in the purified population and cost reduction were achieved, both critical issues in the better purging of malignant cells and for a wide clinical application.

In hypertension, impairment of hyperpolarization by K+ efflux through ATP-sensitive K+ (K(ATP)) channels may contribute to the elevated renal vascular resistance. To elucidate such a role for K(ATP) channels in the renal vasculature, we used micropuncture techniques to examine the effect of K(ATP) channel opener, pinacidil (0.15 mg/h per kg body wt i.v.), on renal and glomerular haemodynamics in spontaneously hypertensive rats (SHR) and in normotensive controls (Wistar Kyoto, WKY). Since pinacidil reduced blood pressure significantly in both groups, the abdominal aorta was clamped before pinacidil administration to yield a renal perfusion pressure equivalent to that during pinacidil infusion. Pinacidil significantly decreased renal vascular resistance in both groups, but the relative change from baseline value was greater in WKY than in SHR. These effects of pinacidil were abolished by pretreatment with glibenclamide (3 mg/kg body wt i.v.). Proximal tubular stop-flow pressure (Psf), an index of glomerular capillary pressure, was significantly elevated by pinacidil infusion in WKY, a response abolished by pretreatment with glibenclamide, but not in SHR. The tubuloglomerular feedback response of Psf was not affected by pinacidil in either group. These data suggest that the activity of K(ATP) channels in SHR may be attenuated in the renal microvasculature. This may contribute to the elevated vascular tone in the renal preglomerular vasculature in SHR.

Advanced glycation end products (AGEs) have been implicated in the chronic complications of diabetes mellitus and have been reported to play an important role in the pathogenesis of Alzheimer's disease. In this study, we examined the immunohistochemical localization of AGEs, amyloid beta protein (A beta), apolipoprotein E (ApoE), and tau protein in senile plaques, neurofibrillary tangles (NFTs), and cerebral amyloid angiopathy (CAA) in Alzheimer's disease and other neurodegenerative diseases (progressive supranuclear palsy, Pick's disease, and Guamanian amyotrophic lateral sclerosis/Parkinsonism-dementia complex). In most senile plaques (including diffuse plaques) and CAA from Alzheimer's brains, AGE and ApoE were observed together. However, approximately 5% of plaques were AGE positive but A beta negative, and the vessels without CAA often showed AGE immunoreactivity. In Alzheimer's disease, AGEs were mainly present in intracellular NFTs, whereas ApoE was mainly present in extracellular NFTs. Pick's bodies in Pick's disease and granulovacuolar degeneration in various neurodegenerative diseases were also AGE positive. In non-Alzheimer neurodegenerative diseases, senile plaques and NFTs showed similar findings to those in Alzheimer's disease. These results suggest that AGE may contribute to eventual neuronal dysfunction and death as an important factor in the progression of various neurodegenerative diseases, including Alzheimer's disease.

We analyzed the mRNA expression of somatostatin receptor subtypes 1 to 5 (SSTR1-5) in human lymphoid cell lines, human peripheral blood lymphocytes (PBL), and human lymphatic leukemia cells, using the reverse transcription-polymerase chain reaction method. In human lymphoid cell lines, SSTR2 mRNA expression was clearly detectable, and there was no evidence of SSTR1 mRNA expression. SSTR2 mRNA was barely detectable in PBL from healthy individuals but was clearly detectable in EB virus-transformed lymphocytes. Lymphocytes from some of the leukemic patients showed elevated SSTR2 mRNA expression. SSTR2 mRNA expression in PBL was upregulated upon stimulation by PHA. SSTR3 mRNA was also observed in all the cell lines examined, although in one cell line, the expression was weak. Some cell lines showed little or no SSTR4 or 5 mRNA expression. The expression pattern of SSTR2 mRNA suggests that this receptor may have some important roles in lymphocyte activation, development, and/or tumorgenesis.

As a step toward understanding the transcriptional regulation of the adrenocorticotropin receptor (ACTH-R) gene, we examined the full length cDNA sequence of the mouse ACTH-R by rapid amplification of cDNA ends, and the organization of the gene. Mouse ACTH-R mRNA consists of 374 bp in the 5′-untranslated region ( UTR ), 888 bp in the coding sequence, and 445 bp in the 3′- UTR , the 1707 bp being fairly compatible with the 1.8-kb adrenal mRNA detected by Northern analysis. The mouse ACTH-R gene consists of at least four exons; the first three exons encode 5′- UTR and the fourth exon encodes part of 5′- UTR , the entire coding region, and the whole of 3′- UTR . We also defined two mRNA species, one with and one without the 57-bp exon 2, produced by alternative splicing.

To determine the possible association between antiprothrombin antibodies and conventional antiphospholipid antibody (aPL) 1 (1.0%), 2 (2.0%) and 17 (17.0%) were found to be positive for phosphatidylserine-dependent antiprothrombin antibody (aPS/PT) IgG, beta2glycoprotein I-dependent anticardiolipin antibody, and lupus anticoagulant by activated partial thromboplastin time (LA), respectively, in 100 recurrent aborters. Because patients with aPS/PT were included in the 17 with LA, we should not add aPS/PT measurement to routine testing in addition to conventional aPL for patients with recurrent miscarriage.

Diabetic nephropathy is currently the single largest cause of endstage renal disease (ESRD) in the United States and many European countries. The primary cause for the development of diabetic complications (including diabetic nephropathy) is persistent exposure to hyperglycemia, although genetic and other incompletely understood factors also play an important role. Although much consideration has been given to the pathogenesis and genetics of the disease itself, the mechanisms by which persistent exposure to hyperglycemia cause biochemical and metabolic alterations have been very sketchily understood. Recently, a growing body of evidence has linked the accumulation of the late products of glucose-protein interaction to a variety of chronic complications, including diabetic nephropathy. The formation of irreversible advanced glycosylation endproducts (AGEs) resulting from the spontaneous reaction between glucose and proteins occur most noticeably on long-lived structural proteins. Recent studies demonstrate that the pathogenesis of diabetic nephropathy is caused by the hyperglycemia-accelerated formation of AGEs. Also, reactive AGE peptides in the circulation are thought to play a role as a new version of so called middle molecule toxic substances. This evidence is opening a new window for our understanding of the pathogenesis of diabetic nephropathy.

We studied the effect of a thromboxane A2 (TXA2) dual blocker KDI-792 on skin blood flow as well as on the peripheral nerve function of nine diabetics with neuropathy. After administration of KDI-792, there was no change in urinary TXB2; however, urinary 6-keto-prostaglandin (PG) F1 alpha increased significantly. Nerve conduction velocity (NCV) and vibration perception threshold (VT) in the four extremities improved significantly, as did deep skin temperature and skin blood flow. The degree of improvement in sensory NCV in the lower extremities correlated significantly with that of deep skin temperature in the toes and the degree of improvement of VT in the lower extremities correlated well with that of deep skin temperature in the soles and of skin blood flow in the toes. Based on these findings, treatment of diabetic neuropathy with a TXA2 dual blocker appears to increase PGI2 production, improving blood flow, and resulting in improvement of nerve functions.

Recent extensive work on apoptosis has begun to reveal its molecular mechanisms. Several genes that regulate apoptosis have been identified. Among them, the BCL2 gene is considered to be an important gene that inhibits apoptosis. However, there must be other genes, yet to be identified, which suppress apoptosis. It has been suggested that the activation of RAS function by BCR-ABL fusion protein in chronic myelogenous leukemia may be an important mechanism in the BCR-ABL mediated transformation. Therefore, in this study we have investigated whether the suppression of endogenous H-RAS function inhibits the BCR-ABL mediated transforming activity in a K562 human chronic myelogenous leukemia cell line. The induced expression of a dominant negative v-H-RAS mutant (116Y) in K562 cells has resulted in cell death. The morphological characteristics and the detection of fragmented DNA by gel electrophoresis in the dead cells have revealed that this cell death is apoptosis. These results directly indicate that the RAS gene as well as the BCL2 gene has an ability to suppress apoptosis.

A novel immunosuppressive compound, FK-506, isolated from Streptomyces has potent immunosuppressive activities. To investigate the effect of FK-506 on the course of diabetes in nonobese diabetic (NOD) mice, we gave this drug to these animals, from the age of 8 weeks, intraperitoneally, in doses of 0.1 mg (2.5 mg/kg/day) or 0.01 mg (0.25 mg/kg/day) three times a week. Overt diabetes were observed in 75.5% of control mice by the age of 20 weeks. In contrast, no diabetes occurred in mice given 0.1 mg of FK-506. Sixteen percent of mice treated with 0.01 mg of the drug became diabetic. Administration of this drug prevented the progression of insulitis in NOD mice. The mice given 0.1 mg of FK-506 lost weight, but this was reversible.

The KATP channel opener nicorandil: Effect on renal hemodynamic in spontaneously hypertensive and Wistar Kyoto rats. Adenosine triphosphate-sensitive K channels (KATP) may subserve vasodilation in the renal microvasculature. Using micropuncture techniques, we examined whether the renal vasomotor action of KATP differs in hypertensive and normotensive animals. Nicorandil (NC), a KATP opener, was given i.v. (1 mg/hr/kg) to spontaneously hypertensive rats (SHR) or Wistar Kyoto rats (WKY). Mean arterial blood pressure decreased in both groups. Renal blood flow was almost unchanged in SHR but increased significantly in WKY. This effect was completely abolished by pretreatment with glibenclamide (GC; 3 mg/kg i.v.). To investigate the effect of NC on the regulatory mechanism of renal microcirculation, we measured proximal tubular stop-flow pressure (SFP) and assessed tubuloglomerular feedback (TGF) by monitoring SFP during loop perfusion with artificial tubular fluid. NC significantly increased SFP in WKY, an effect abolished by pretreatment with GC. In SHR, however, treatment with NC elicited no significant change in SFP. TGF response was not affected by NC treatment in either group. The data suggest that KATP may modulate preglomerular vascular resistance, especially in the larger vascular segments not subject to TGF regulation, and may be attenuated in SHR. This might, at least in part, be attributable to the increased vascular resistance in SHR.

A 30-year-old Japanese woman with acquired severe aplastic anemia (SAA), diagnosed 20 years ago, was referred to our institution for allogeneic stem cell transplantation (SCT). As an unusual case of long-standing SAA, the patient was complicated with moderate heart failure due to secondary hemochromatosis. After successful SCT using a non-myeloablative conditioning regimen, she needed no transfusion. Five years after SCT, echocardiography showed a dramatic improvement of her cardiac function. This case indicates that the cardiac function in secondary hemochromatosis could be reversed once iron overload from multitransfusions is stopped.

Systemic lupus erythematosus (SLE)-related mortality has significantly declined in Japan during the past four decades. Decrease in number of deaths due to renal failure has considerably improved the prognosis of SLE. The adequate use of steroid therapy, the new advances in the second-line therapies including immunosuppressants, plasmapheresis and hemodialysis, and the appropriate control of infectious complications are the major reasons of the reduction of SLE-related mortality. The establishment of a nation-wide registration system of SLE in Japan will allow us to improve our strategy to control the disease.

To identify the correlation between incidence of anti-phospholipid antibodies and fetal prognosis in pregnant SLE patients, we measured the amount of anti-cardiolipin antibody in their sera, using solid-phase enzyme immunoassay (EIA) methods. Findings in the group having poor obstetric results (fetal loss group) and in those with a history of full-term births (live birth group) were compared with regard to other anti-phospholipid antibodies. The incidence of IgG class anti-cardiolipin antibody was 60% in the fetal loss group and 19% in the live birth group, (P less than 0.05). The incidence of the other anti-phospholipid antibodies, including lupus anticoagulant and biological false-positive serological test for syphilis (BFP-STS), did not differ significantly between the two groups. Therefore, the presence of IgG class anti-cardiolipin antibody may prove to be a useful marker for evaluating fetal risk in SLE patients.

A novel immunosuppressive compound extracted from the fermentation broth of Streptomyces tsukubaensis belongs to the macrolide family. We gave this drug (FK-506) to MRL/lpr and NZB X NZW F1 (B/W F1) mice, an animal model of human systemic lupus erythematosus (SLE), to investigate its effect on the course of the disease. This drug showed potential to prolong the lifespan, to reduce proteinuria, and to prevent the progression of nephropathy. Appreciable differences in the levels of anti-dsDNA antibodies between treated and control animals were nil.