Satoshi Higasa

Relationship of CD4+CD25+ regulatory T cells to immune status in HIV-infected patients.

Objective:To determine whether the frequencies of CD4+CD25+ regulatory T cells (T Reg) were related to immune status in HIV-infected patients. Methods:Peripheral blood CD4 T-cell populations were examined for T-helper 1 cells (Th1), T-helper 2 cells (Th2), and T Reg by intracellular staining for interferon (IFN)-γ and interleukin (IL)-4, and surface staining for CD25, respectively. The immunoregulatory properties of T Reg were assessed by measurement of the inhibitory effects of isolated CD4+CD25+ T Reg on CD4+CD25− T-cell proliferation. Results:Isolated CD4+CD25+ T Reg from both HIV-infected patients and healthy controls strongly expressed CD45RO, HLA-DR, and FoxP3. HIV-infected patients with detectable plasma HIV-1 RNA showed a statistically significant increase in CD4+CD25high T Reg frequencies (P < 0.05) compared to healthy controls, with T Reg frequency inversely proportional to CD4 T-cell count (P < 0.01). However, in HIV-infected patients with undetectable plasma HIV-1 RNA, CD4+CD25high T Reg frequencies were not increased and were not related to CD4 T-cell counts. In both HIV-infected patient groups, T Reg frequency was inversely related to Th1 frequency (detectable HIV-1 RNA: P < 0.05; undetectable: P < 0.001), but positively related to Th2 frequency (detectable HIV-1 RNA: P < 0.01; undetectable: P < 0.001). T Reg activity was lower in patients with detectable plasma HIV-1 RNA than in patients with undetectable plasma HIV-1 RNA. Conclusions:Increased T Reg frequencies in peripheral blood were related to low peripheral blood CD4 T-cell counts and polarization toward Th2 immune responses in HIV-infected patients.

Pregnancy-induced thrombocytopenia and TTP, and the risk of fetal death, in Upshaw-Schulman syndrome: a series of 15 pregnancies in 9 genotyped patients

Upshaw–Schulman syndrome (USS) is a congenital thrombotic thrombocytopenic purpura (TTP) due to mutations in the gene that encodes for ADAMTS13 (ADAMTS13), but its clinical signs may be mild or absent during childhood. We have identified 37 patients with USS (24 females, 13 males) belonging to 32 families. The nine women from six families who were diagnosed during their first pregnancy are the focus of this report. Six of the nine women had episodes of thrombocytopenia during childhood misdiagnosed as idiopathic thrombocytopenic purpura. Thrombocytopenia occurred during the second–third trimesters in each of their 15 pregnancies, with 16 babies (one twin pregnancy), often followed by TTP. Of 15 pregnancies, eight babies were stillborn or died soon after birth, and the remaining seven were all premature except one, who was born naturally following plasma infusions to the mother that had started at 8 weeks’ gestation. All nine USS women had severely deficient ADAMTS13 activity. ADAMTS13 analyses demonstrated that eight women were compound heterozygotes of Y304C/G525D (2 siblings), R125VfsX6/Q1302X (2 siblings), R193W/R349C (2 siblings), I178T/Q929X, and R193W/A606P; one woman was homozygous for R193W. Only the R193W mutation has been previously reported. These observations emphasize the importance of measuring ADAMTS13 activity in the evaluation of thrombocytopenia during childhood and pregnancy.

Enhancing effect of advanced glycation end products on serotonin-induced platelet aggregation in patients with diabetes mellitus.

Advanced glycation end products (AGEs) are thought to be responsible for some complications of diabetes mellitus (DM), including microangiopathy. Plasma serotonin is increased in diabetes mellitus patients, and this increase is related, at least in part, to platelet hyperfunction. In order to clarify the relationship between advanced glycation end products, serotonin, and thrombotic complications in diabetes mellitus patients, we examined the effect of advanced glycation end products on serotonin-induced platelet aggregation. In diabetic patients, although serotonin-induced platelet aggregation was enhanced with an increase in serum-advanced glycation end products, there was no correlation between platelet aggregation and either hemoglobin A1c or fasting blood sugar. To examine the direct effect of advanced glycation end products on platelet aggregation, we prepared advanced glycation end products by in vitro incubation of human albumin with glucose (250 mM) at 37 degrees C for 8 weeks. Serotonin-induced platelet aggregation was dose-dependently increased by advanced glycation end products. Adenosine diphosphate-induced platelet aggregation also was increased by advanced glycation end products, but this increment was diminished by addition of sarpogrelate, a selective serotonin receptor antagonist. These results suggest that advanced glycation end products enhance platelet aggregation through the serotonin receptor, and perhaps influencing the development of thrombotic complications in diabetic patients.

The prevalence of neutralizing antibodies against adeno-associated virus capsids is reduced in young Japanese individuals.

Pre‐existing antibodies against adeno‐associated virus (AAV), caused by natural AAV infections, interfere with recombinant AAV vector‐mediated gene transfer. We studied the prevalence of neutralizing antibodies against AAV serotypes 1, 2, 5, 8, and 9 in healthy subjects (n = 85) and hemophilia patients (n = 59) in a Japanese population. For healthy subjects, the prevalence of neutralizing antibodies against AAV serotypes 1, 2, 5, 8, and 9 was 36.5%, 35.3%, 37.6%, 32.9%, and 36.5%, respectively, while that in hemophilia patients was 39.7%, 28.8%, 35.6%, 32.9%, and 27.4%, respectively. There was no difference in the prevalence of neutralizing antibody against each AAV serotype between the healthy subjects and the hemophilia patients. The prevalence of neutralizing antibodies against all AAV serotypes increased with age in both healthy subjects and hemophilia patients. High titers of neutralizing antibodies against AAV2 (≥1:224) and AAV8 (≥1:224) were more evident in older individuals (≥42 years old). Approximately 50% of all screened individuals were seronegative for neutralizing antibodies against each AAV tested, while approximately 25% of individuals were seropositive for each AAV serotype tested. The prevalence of seronegativity for all AAV serotypes was 67.0% (healthy subjects, 68.6%; hemophilia patients, 65.0%) and 18.6% (healthy subjects, 20.5%; hemophilia patients, 15.7%) in young (<42 years old) and older subjects (≥42 years old), respectively. The findings from this study suggested that young subjects are more likely to be eligible for gene therapy based on AAV vectors delivered via an intravascular route because of the low prevalence of antibodies to AAV capsids. J. Med. Virol. 86:1990–1997, 2014.

Clinical pharmacological study of a plasma-derived factor VIIa and factor X mixture (MC710) in haemophilia patients with inhibitors – Phase I trial

Summary.  MC710, a combined product of plasma‐derived activated factor VII (FVIIa) and factor X (FX) at a protein weight ratio of 1:10, is a novel bypassing agent for haemostasis in haemophilia patients with inhibitors. In this study, pharmacokinetic (PK), pharmacodynamic (PD) parameters and safety of single doses of MC710 were investigated in 11 male haemophilia patients with inhibitors in a non‐bleeding state. This was a multi‐centre, open‐labelled, non‐randomized, active controlled crossover, dose‐escalation study of five doses (20–120 μg kg−1 of FVIIa) with re‐administration of different MC710 dosages to the same subjects. The active controls were NovoSeven (120 μg kg−1) and/or FEIBA (50 and 75 U kg−1) which were used to compare PD parameters. The area under the curve (AUC) and maximum plasma concentration (Cmax) of MC710 active ingredients increased dose‐dependently within the range of 20 and 120 μg kg−1. After administration of MC710, activated partial thromboplastin time (APTT) was dose‐dependently improved and prothrombin time (PT) was shortened to approximately 6 s at 10 min, and APTT improvement and PT shortening effects were maintained until 12 h after administration of MC710 at all doses. No serious or severe adverse event was observed after administration of MC710; furthermore, several diagnostic marker values and those changes did not indicate any signs of disseminated intravascular coagulation (DIC). These results suggest that MC710 would have haemostatic potential equal to or greater than NovoSeven and FEIBA and was be tolerable when given at doses up to 120 μg kg−1.

Suitability of four polymorphic DNA markers for indirect genetic diagnosis of haemophilia A in Japanese subject

Indirect genetic diagnosis using polymorphic DNA markers can detect carriers of haemophilia A (HA). These markers include CA repeat polymorphisms at intron 13 (CA-13) and CT-AG at intron 22 of the coagulation factor VIII (FVIII) gene, and also certain restriction fragment length polymorphisms (RFLPs) such as HindIII at intron 19 and BclI at intron 18. Recently, CA repeat polymorphism at intron 6 (CA-6) was also reported. We compared usefulness of the BclI RFLP, the HindIII RFLP, and CA-13 to that of CA-6. Heterozygosity of markers was examined in 282 X chromosomes obtained from 205 subjects including HA patients. Expected heterozygosity of the HindIII and BclI RFLPs was 30.0% and 27.9%, while observed heterozygosity was 28.0% and 26.5%. Expected heterozygosity of CA-13 was 45.6%, and observed heterozygosity was 40.0%. CA-6 showed two alleles with repeat numbers of 13 and 14, expected and observed heterozygosity were low (1.4% and 2.6%, respectively). When we used these markers in a HA lineage where the mother was a carrier according to coagulation factor assays, carrier diagnosis was possible using CA-13, the HindIII RFLP, and the BclI RFLP. This was not true for CA-6, for which the mother was homozygous. Although CA-13, and the HindIII and BclI RFLPs were useful for indirect genetic diagnosis of HA, CA-6 proved less useful because of low heterozygosity.

Results of clot waveform analysis and thrombin generation test for a plasma-derived factor VIIa and X mixture (MC710) in haemophilia patients with inhibitors--phase I trial: 2nd report.

We reported the results of a clinical pharmacological study of MC710 (a mixture of plasma‐derived FVIIa and FX) in haemophilia patients with inhibitors during a non‐haemorrhagic state. This report provides the results of a clot waveform analysis (CWA) and thrombin generation test (TGT) using blood samples obtained in this study. CWA and TGT were conducted using blood samples obtained from a pharmacokinetic and pharmacodynamic study in which MC710 (five dose rates: 20, 40, 80, 100 and 120 μg kg−1) was compared with NovoSeven (120 μg kg−1) and FEIBA (two dose rates: 50 and 75 U kg−1) as control drugs in 11 haemophilia patients with inhibitors without haemorrhagic symptoms. CWA showed that MC710 provided significantly greater improvement than the control drugs in activated partial thromboplastin time (APTT) at 80 μg kg−1; maximum clot velocity and maximum clot acceleration were more enhanced by MC710 than by control drugs. TGT revealed that MC710 significantly shortened the initiation time of thrombin generation in comparison to FEIBA and induced greater thrombin generation potency than NovoSeven. It was not clear whether or not MC710 caused significant dose‐dependent changes in the two measurements; however, differences between MC710 and the control drugs were clarified. MC710 was confirmed to have superior coagulation activity and thrombin productivity and is expected to have superior bypassing activity.

An analysis of factors affecting the incidence of inhibitor formation in patients with congenital haemophilia in Japan

Summary.  Studies conducted in European and North American countries have demonstrated that various factors including races affect the frequency of inhibitor formation in haemophilia patients. The present study was undertaken to analyse factors affecting the incidence of inhibitor formation in Japanese haemophilia A and B patients. Analytical data were retrospectively collected from haemophilia A and B patients born after 1988, the year when monoclonal antibody‐purified factor VIII products were first marketed in Japan. Various data were collected from 184 patients (153 cases of haemophilia A; 31 cases of haemophilia B). The sample size of haemophilia B cases was too small to reveal any significant differences between the inhibitor formation group and the inhibitor‐free group in any of background variables. For patients with haemophilia A, on the other hand, univariate analysis identified the severity of haemophilia and a positive family history of inhibitor development as risk factors for the formation of inhibitors. In analyses of the clotting factor products used, the incidence of inhibitor formation did not differ significantly between the group treated with plasma‐derived products (29.7%) and the group treated with recombinant products (25.0%). When background variables were compared, age was higher in the group treated with plasma‐derived products but none of the other background variables differed between the two groups. These results suggest that in Japanese haemophilia patients, the type of clotting factor preparations used for therapy has not influenced the incidence of inhibitor formation.

A Phase II clinical trial of a mixture of plasma-derived factor VIIa and factor X (MC710) in haemophilia patients with inhibitors: haemostatic efficacy, safety and pharmacokinetics/pharmacodynamics.

MC710, a mixture of plasma‐derived activated factor VII and factor X at a protein weight ratio of 1:10, is a novel bypassing agent for haemostasis in haemophilia patients with inhibitors. In a Phase II trial, we evaluated the haemostatic efficacy and safety of single doses of MC710, and investigated pharmacokinetic and pharmacodynamic parameters in nine joint bleeding episodes in six male haemophilia patients with inhibitors. This trial was a multi‐centre, open‐label, non‐randomized study of two doses (60 and 120 μg kg−1 as FVIIa dose), allowing the re‐administration of different MC710 dosages to the same subjects. Haemostatic efficacy was assessed by evaluating reduction in pain and swelling, as well as increase in range of motion in a bleeding joint. The results of the study showed that in nine bleeding episodes, seven treatments were rated as ‘excellent’ or ‘effective’ according to investigators rating system of efficacy at 8 h after administration. No serious or severe adverse events were observed after administration; furthermore, measurement of several diagnostic markers revealed no signs or symptoms of disseminated intravascular coagulation (DIC). The haemostatic potential of MC710 was confirmed at doses of 60 and 120 μg kg−1 in this trial. MC710 is thus expected to be a safe and efficacious novel bypassing agent for controlling bleeding in haemophilia patients with inhibitors.

Serum macrophage colony-stimulating factor (M-CSF) level is elevated in patients with old cerebral infarction related to vascular damage.

We measured the serum levels of macrophage colony‐stimulating factor (M‐CSF) in 37 patients with an old cerebral infarction who had been surmised to have a damaged vessel wall and who had been in a stable condition for over three months after stroke onset, and those of 41 healthy control subjects. The M‐CSF levels in the patients were significantly higher (P < 0.01) than those of the controls at 1320.4 ± 410.6 unit/ml and 853.9 ± 180.3 unit/ml, respectively. The plasma levels of von Willebrand factor (vWF) antigen (P < 0.01) and thrombomodulin (TM) (P < 0.05), as well as those of thrombin‐antithrombin III (TAT) complex (P < 0.05), prothrombin fragment 1+2 (F1+2) (P < 0.02), D‐dimer products of crosslinked fibrin degradation products (D‐dimer) (P < 0.01), and plasmin‐antiplasmin (PAP) complex (P < 0.05) in the patients were also significantly higher than those in the controls. Significant positive correlations (P < 0.01) were found between these parameters and the M‐CSF level, but there was no significant correlation between the M‐CSF level and the white blood cell count, serum lipids, or blood pressure. Based on these results, we suggest that an increased M‐CSF level indicates vascular damage or a thrombotic state in patients with an old cerebral infarction. Am. J. Hematol. 60:185–190, 1999.