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 investigator's 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.

Effect of Multiple Firings on the Microshear Bond Strength Between a Translucent Zirconia and a Resin Cement.

PURPOSE: This study investigated the microshear bond strength between a resin cement and a translucent zirconia subjected to multiple characterization firings. METHODS AND MATERIALS: 5Y-PSZ zirconia blocks (Katana UTML) were sliced, sintered (1550°C, 2 h), and randomly divided into six groups (n=10) according to the number of characterization firings (0, 1, 2, 3, 5, or 10) and aging (baseline or after thermocycling). Each characterization firing was performed at 750°C for 1 minute. The ceramic surfaces were all sandblasted with 50 µm Al2O3 and silanized. Then, cylinders of resin cement (0.96 mm diameter × 2 mm height) were bonded onto their surfaces. The baseline samples were immersed in distilled water for 24 hours before the microshear bond strength (µSBS) tests. The aged samples were tested after 5000 thermocycles in water (5°C-55°C). The failure modes were classified as adhesive, predominantly adhesive, or cohesive. Scanning electron microscope images of the failure modes and the ceramic surfaces after the firings were taken. The µSBS data were analyzed by two-way ANOVA and Tukey's test. RESULTS: The number of characterization firings and aging affected the bond strength. The highest bond strength values were observed from the 2-firing group at baseline. The µSBS results after 1, 2, or 3 characterization firings were similar at baseline and after aging. On the other hand, 0, 5, and 10 firings revealed the lowest bond strengths. The most frequent failures were adhesive and predominantly adhesive. Zirconia grains were not affected by the multiple firings. CONCLUSION: One to three characterization firings after sintering improve the bond strength of 5Y-PSZ to the resin cement when compared to none or several (five or ten) firing cycles.

Quality of life in patients with oral potentially malignant disorders: oral lichen planus and oral epithelial dysplasia.

OBJECTIVE: The aim of this study was to evaluate and compare quality of life (QoL) parameters in patients with oral potential malignant disorders (OPMDs), namely, oral lichen planus (OLP) and oral epithelial dysplasia (OED). STUDY DESIGN: A cross-sectional study was completed at the oral maxillofacial surgery/oral medicine practices at University of Pennsylvania. Patients with clinical and histopathologic confirmation of OLP or OED from January to June 2021 were included in the study. The primary predictor variable was the OPMD type. The primary outcome variable was the score of 3 separate surveys: the Chronic Oral Mucosal Disease Questionnaire-26 (COMDQ-26), Oral Potential Malignant Disorder QoL Questionnaire (OPMDQoL), and Hospital Anxiety and Depression Scale. Multiple linear regression was used to determine independent predictors of increased/decreased questionnaire scores. RESULTS: The final study sample consisted of 100 patients:53 patients had OLP (53.0%), 39 patients had OED (39.0%), and 8 patients had OLP with OED (8.0%). Relative to OED, OLP added 15.7 points to the COMDQ-26 survey score (P < .001). Relative to OED, OLP added 8.9 points to the OPMDQoL survey score (P = .003). CONCLUSIONS: Oral lichen planus shows significantly poorer QoL specifically within the COMD-26 and OPMDQoL questionnaires, compared with OED. Additionally, patients with OPMDs aged 40 to 64 years were independently associated with higher COMD-26 scores compared with older patients (>65 years).

Nonadipose tissue production of leptin: leptin as a novel placenta-derived hormone in humans.

Leptin is a circulating hormone that is expressed abundantly and specifically in the adipose tissue. It is involved in the regulation of energy homeostasis, as well as the neuroendocrine and reproductive systems. Here, we demonstrate production of leptin by nonadipose tissue, namely, placental trophoblasts and amnion cells from uteri of pregnant women. We show that pregnant women secrete a considerable amount of leptin from the placenta into the maternal circulation as compared with nonpregnant obese women. Leptin production was also detected in a cultured human choriocarcinoma cell line, BeWo cells, and was augmented during the course of forskolin-induced differentiation of cytotrophoblasts into syncytiotrophoblasts. Plasma leptin levels were markedly elevated in patients with hydatidiform mole or choriocarcinoma and were reduced after surgical treatment or chemotherapy. Leptin is also produced by primary cultured human amnion cells and is secreted into the amniotic fluid. The present study provides evidence for leptin as a novel placenta-derived hormone in humans and suggests the physiologic and pathophysiologic significance of leptin in normal pregnancy and gestational trophoblastic neoplasms.

Fouling potentials of polysaccharides in membrane bioreactors (MBRs) assessed by lectin affinity chromatography.

In this study, the fouling potentials of polysaccharides contained in mixed liquor suspension in a membrane bioreactor (MBR) treating municipal wastewater were investigated using lectin affinity chromatography. This investigation was carried out with different membranes to assess the effects of membrane materials on fouling potentials of polysaccharides. In lectin affinity chromatography, some polysaccharides with high affinity to the lectin in the column can be retained in the column. The fouling potentials of the retained polysaccharides were evaluated by dead-end filtration test. Degree of reduction in fouling potential differed considerably when different lectins were used in affinity chromatography indicating that fouling potentials of polysaccharide differed depending on types of polysaccharide. Trends in the reduction of fouling potential were different depending on membrane materials. Characteristics of the organic matter associated with polysaccharides removed by lectins were investigated by means of excitation-emission matrices (EEM). The results of EEM analysis indicate that the characteristics of the organic matter eluted from different lectins were different as long as elution reagents for the lectin were different. Characteristics of the organic matter eluted from the lectins which have the same elution reagent were similar in terms of shapes of EEM fluorescence spectra. However, the trends in reduction of fouling potentials could not be explained by the characteristics of organic matter assessed by EEM analysis. On the basis of the results obtained in this study, it can be concluded that characteristics of membrane and structures or properties of sugar chain would play an important role in determining fouling potentials.

Discovery of a potent, metabolically stabilized resorcylic lactone as an anti-inflammatory lead.

With bioactivity-guided phenotype screenings, a potent anti-inflammatory compound f152A1 has been isolated, characterized and identified as the known natural product LL-Z1640-2. Metabolic instability precluded its use for the study on animal disease models. Via total synthesis, a potent, metabolically stabilized analog ER-803064 has been created; addition of the (S)-Me group at C4 onto f152A1 has resulted in a dramatic improvement on its metabolic stability, while preserving the anti-inflammatory activities.

Near-edge structures from first principles all-electron Bethe-Salpeter equation calculations.

We obtain x-ray absorption near-edge structures (XANES) by solving the equation of motion for the two-particle Green's function for the electron-hole pair, the Bethe-Salpeter equation (BSE), within the all-electron full-potential linearized augmented plane wave method (FPLAPW). The excited states are calculated for the Li K-edge in the insulating solids LiF, Li(2)O and Li(2)S, and absorption spectra are compared with independent particle results using the random phase approximation (RPA), as well as supercell calculations using the core-hole approximation within density functional theory (DFT). The binding energies of strongly bound excitations are determined in the materials, and core-exciton wavefunctions are demonstrated for LiF.

Adrenomedullin inhibits connective tissue growth factor expression, extracellular signal-regulated kinase activation and renal fibrosis.

Systemic administration of the potent vasodilating peptide adrenomedullin reduces cardiac and renal fibrosis in hypertensive animals. Here, we investigated the effects of kidney-specific adrenomedullin gene delivery in normotensive rats after unilateral ureteral obstruction, an established model of renal tubulointerstitial fibrosis. Overexpression of exogenous adrenomedullin in the renal interstitium following ureteral obstruction significantly prevented fibrosis and proliferation of tubular and interstitial cells. In this model, there is upregulation of connective tissue growth factor (CTGF) mRNA expression and extracellular signal-regulated kinase (ERK) phosphorylation, and adrenomedullin overexpression suppressed both of these activities without altering the blood pressure. In NRK-49F renal fibroblasts, adrenomedullin reduced transforming growth factor-beta-induced CTGF and fibronectin mRNA upregulation through the cyclic AMP/protein kinase A signaling pathway, and suppressed ERK phosphorylation and cell proliferation. In the kidneys with an obstructed ureter, adrenomedullin receptor gene expression was upregulated along with cyclic AMP production in kidney slices. The latter effect was partially blocked by a neutralizing antibody to adrenomedullin, indicating that an endogenous peptide-receptor system was activated. Our results show that overexpression of exogenous adrenomedullin in the ureteral-obstructed kidney prevents tubulointerstitial fibrosis and cell proliferation through the cyclic AMP-mediated decrease of CTGF induction and ERK phosphorylation.

Clinical and prognostic significance of cytokine receptor expression in adult acute lymphoblastic leukemia: interleukin-2 receptor alpha-chain predicts a poor prognosis.

We quantitatively assessed the expression of cytokine receptors (interleukin-2 receptor (IL-2R), IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, granulocyte-macrophage colony-stimulating factor R (GM-CSFR), G-CSFR, c-fms, c-mpl, c-kit and FLT3) in cells from 211 adults with acute lymphoblastic leukemia (ALL) by flow cytometry and determined their prevalence and clinical significance. Although all cytokine receptors were expressed to various degrees, the levels of IL-3R alpha-chain (IL-3Ralpha), IL-2Ralpha, IL-2Rbeta, IL-7Ralpha, common-Rgamma(gammac), c-mpl, c-kit and FLT3 exhibited a wide spectrum > or =2000 sites/cell. Among them, IL-3Ralpha, IL-2Ralpha and FLT3 were highly expressed in B-lineage ALL, whereas IL-7Ralpha, gammac and c-kit predominated in T-lineage ALL. Higher levels of IL-3Ralpha, IL-2Ralpha, c-kit and FLT3 correlated with the expression of CD13/33. Increased IL-2Ralpha levels related to the presence of Philadelphia chromosome (Ph), leukocytosis and shorter event-free survival (EFS). C-kit preferred in male. Elevated FLT3 levels correlated with age > or =60 years. Multivariate analysis in B-lineage ALL revealed only IL-2Ralpha (P=0.028) and Ph (P=0.020) as independent factors for EFS. These findings suggest that several cytokine receptors associated with certain cellular and clinical features, but IL-2Ralpha solely had a prognostic value and should be considered as a major prognostic factor for adult ALL that is comparable with Ph.

Casein hydrolysate formula-induced liver dysfunction in a neonate with non-immunoglobulin E-mediated cow's milk allergy.

A 10-day-old male neonate was admitted with bilious vomiting and gross hematochezia. Peripheral eosinophilia, delayed positive skin prick test to artificial milk, and elevated eosinophil cationic protein levels suggested cow's milk allergy. Fluid infusion with prohibition of oral intake improved the digestive symptoms. Breast-feeding was resumed on hospital day 3 and only casein hydrolysate formula was fed from day 7 onward. Nevertheless, eosinophilia and elevated transaminase levels developed on day 14. Liver dysfunction associated with casein hydrolysate formula was suspected and the infant was transferred to soy formula. Eosinophil counts decreased and transaminase levels were normalized on day 19. A cow's milk protein-specific lymphocyte proliferation test was positive for alpha-casein, beta-lactoglobulin, and bovine serum albumin, indicating sensitization of T cells to cow's milk proteins. These observations suggest that careful attention should be paid to liver dysfunction in non-immunoglobulin E-mediated cow's milk allergy, even when hypoallergenic formula is used.

Effects of Continuous Gamma-Ray Exposure In Utero in B6C3F1 Mice on Gestation Day 18 and at 10 Weeks of Age.

Pregnant C57BL/6JJcl mice were exposed to γ rays at low dose rate (20 mGy/day, LDR) or medium dose rate (200 and 400 mGy/day, MDR) from gestation day (GD) 0-18 to total accumulated doses of 360, 3,600 and 7,200 mGy, respectively. An additional group of pregnant mice were acutely exposed to 2 Gy at high dose rate (HDR) of 0.77 Gy/min on GD 11. In experiment 1, fetuses collected via cesarean section on GD 18 were examined for external and skeletal abnormalities. While the results of LDR exposure (20 mGy/day) did not significantly differ from the nonirradiated controls in all parameters examined, MDR (200 and 400 mGy/day) and acute HDR (2 Gy) exposure caused growth retardation and significantly increased incidence of unossified bones. Increased incidence of external abnormalities was observed only in the acute HDR group. In experiment 2, the dams were allowed to give birth and the pups were clinically monitored and weighed periodically until 10 weeks of age when they were sacrificed and subjected to pathological examination. Pups exposed at MDRs of 200 and 400 mGy/dayand at acute HDR of 0.77 Gy/min had lower bodyweights from weaning (3 weeks) to 10 weeks of age except for females exposed to 400 mGy/day MDR. None of the pups exposed to an acute 2 Gy dose on GD 11 survived to 10 weeks of age. Histopathological changes were not significantly different between the nonirradiated control and the 20 mGy/day LDR groups. However, at both MDR exposures of 200 and 400 mGy/day. gonadal (testes and ovary) hypoplasia/atrophy was observed in all the 10-week-old pups. Our results show that in utero LDR exposure to 20 mGy/day for the entire gestation period did not cause any significant effect in pups when compared to the nonirradiated controls up to 10 weeks of age. However, pups exposed in utero to MDRs showed dose-related growth retardation with delayed ossifications (400 mGy/day) and gonadal hypoplasia/atrophy. These findings suggest that increased post-implantation loss in dams exposed at MDR is due to early embryonic deaths resulting in early resorption.

Immunoreactive beta-endorphin and adrenocorticotropin in human cerebrospinal fluid.

To elucidate the significance of beta-endorphin in human cerebrospinal fluid (CSF), CSF levels of beta-endorphin-like immunoreactivity (beta-EP-LI) in various diseases were determined by a specific radioimmunoassay and compared with simultaneously determined ACTH-like immunoreactivity (ACTH-LI) levels in CSF. CSF beta-EP-LI and ACTH-LI in the control group, consisting of 5 normal subjects and 19 patients with nonendocrine diseases, were 22.2+/-1.3 and 14.6+/-0.4 fmol/ml, respectively. CSF levels of these peptides in patients with schizophrenia (n = 19) and acromegaly (n = 10) were not significantly different from those in the control group. Patients with Cushing's disease (n = 7) had significantly lower CSF beta-EP-LI and ACTH-LI levels than those in the control group. Four of them showed a parallel increase in CSF beta-EP-LI and CSF ACTH-LI levels after the complete removal of pituitary microadenomas (P < 0.05). Gel chromatography of CSF beta-EP-LI from a normal volunteer, a control patient, and one patient each with catatonia, Nelson's syndrome, Cushing's syndrome (adrenal adenoma), and acromegaly gave similar patterns consisting of three peaks with the elution positions comparable to those of authentic beta-endorphin, beta-lipotropin, and possibly their precursor molecule. Gel chromatographic patterns of CSF beta-EP-LI and ACTH-LI were compared in a normal volunteer. The first peaks of beta-EP-LI and ACTH-LI eluted at the same position and the second peak of ACTH-LI coincided with the elution position of authentic ACTH.CSF beta-EP-LI and ACTH-LI levels determined every 5 min over a period of 80 min in three normal volunteers did not show moment-to-moment variability.A significant correlation (r = 0.75, P < 0.001) was seen between CSF beta-EP-LI and ACTH-LI levels in normal subjects and patients studied (n = 73). This suggests that beta-endorphin and ACTH in human CSF share the common regulatory mechanism in normal and pathologic conditions.

Cause of death and neoplasia in mice continuously exposed to very low dose rates of gamma rays.

Four thousand 8-week-old SPF B6C3F1 mice (2000 of each sex) were divided into four groups, one nonirradiated (control) and three irradiated. The irradiated groups were exposed to (137)Cs gamma rays at dose rates of 21, 1.1 and 0.05 mGy day(-1) for approximately 400 days with total doses equivalent to 8000, 400 and 20 mGy, respectively. All mice were kept until natural death, and pathological examination was performed to determine the cause of death. Neoplasms accounted for >86.7% of all deaths. Compared to the nonirradiated controls, the frequency of myeloid leukemia in males, soft tissue neoplasms and malignant granulosa cell tumors in females, and hemangiosarcoma in both sexes exposed to 21 mGy day(-1) were significantly increased. The number of multiple primary neoplasms per mouse was significantly increased in mice irradiated at 21 mGy day(-1). Significant increases in body weights were observed from 32 to 60 weeks of age in males and females exposed to 1.1 mGy day(-1) and 21 mGy day(-1), respectively. Our results suggest that life shortening (Tanaka et al., Radiat. Res. 160, 376-379, 2003) in mice continuously exposed to low-dose-rate gamma rays is due to early death from a variety of neoplasms and not from increased incidence of specific neoplasms.

Biopersistence of inhaled nickel oxide nanoparticles in rat lung.

In order to investigate whether or not airborne nanoparticles with a minimum agglomeration could be used for exposure tests on animals, we developed a nanoparticle generation system and examined the biological effects of the particles in an inhalation study. The generation system was composed of an ultrasonic nebulizer and diffusion dryers, and 30 Wistar male rats were exposed to nickel oxide (NiO) nanoparticles for 4 wk (6 h/day). The geometric mean diameter of the particles and the daily average exposure concentration determined by a combination of a differential mobility analyzer and a condensation nucleus counter in the exposure chamber were 139 +/- 12 nm and 1.0 +/- 0.5 x 10(5) particles/cm3, respectively. At 4 days and 1 and 3 mo after the inhalation, each group of 10 rats were sacrificed and NiO nanoparticles deposited in the lung were determined by chemical analysis and the biopersistence (biological half time) was calculated. The deposited amount of NiO nanoparticles in the rat lungs at 4 days after the inhalation was 29 +/- 4 microg. The retained particle amount in the rat lungs after the inhalation exponentially decreased and the calculated biological half time was 62 days. The histopathological change was not severe just after the inhalation nor throughout the observation time. We concluded that nanoparticles with a minimum agglomeration were dispersed stably in the chamber and exposed to rats for 4 wk and that deposited amounts in the rat lungs and the biopersistence of the particles and the biological response in lung were detected.

Pathology of Serially Sacrificed Female B6C3F1 Mice Continuously Exposed to Very Low-Dose-Rate Gamma Rays.

We have previously reported on life span shortening as well as increased incidence rates in several neoplasms in B6C3F1 mice that were continuously exposed to 21 mGy/day of gamma rays for 400 days. To clarify whether the life shortening was due to early appearance of neoplasms (shortened latency) or increased promotion/progression, 8-week-old female specific-pathogen-free B6C3F1 mice were gamma-ray irradiated at a low dose rate of 20 mGy/day for 400 days. At 100 days postirradiation, 60-90 mice were sacrificed, and thereafter every 100 days alongside the age-matched nonirradiated controls, for 700 days. Additional groups were allowed to live out their natural life span. Pathological examination was performed on all mice to identify lesions, non-neoplastic and neoplastic, as well as to determine the cause of death. Body weights were significantly increased in irradiated mice from sacrifice days 200-500. Incidence rates for spontaneously occurring non-neoplastic lesions, such as adrenal subcapsular cell hyperplasia, fatty degeneration of the liver, atrophy and tubulostromal hyperplasia of the ovaries, were significantly increased in irradiated mice. Significantly increased incidence rates with no shortening of latency periods were observed in irradiated mice for malignant lymphomas, hepatocellular adenomas/carcinomas, bronchioloalveolar adenomas, harderian gland adenoma/adenocarcinoma. Shortened latencies with significantly increased incidence rates were observed for adrenal subcapsular cell adenomas and ovarian neoplasms (tubulostromal adenoma, granulosa cell tumors) in irradiated mice. Life span shortening in mice exposed to 20 mGy/day was mostly due to malignant lymphomas. Multiple primary neoplasms were significantly increased in mice exposed to 20 mGy/day from sacrifice days 400-700 and in the life span group. Our results confirm that continuous low-dose-rate gamma-ray irradiation of female B6C3F1 mice causes both cancer induction (shortened latency) and promotion/progression (early death), depending on the neoplasm's organ/tissue of origin.

Manifestation of hopping conductivity and granularity within phase diagrams of LaO1-x F x BiS2, Sr1-x La x FBiS2 and related BiS2-based compounds.

Layered BiS [Formula: see text] -based series, such as LaO [Formula: see text] F [Formula: see text] BiS [Formula: see text] and Sr [Formula: see text] La [Formula: see text] FBiS [Formula: see text] , offer ideal examples for studying normal and superconducting phase diagram of a solid solution that evolves from a nonmagnetic band-insulator parent. We constructed typical [Formula: see text] phase diagrams of these systems based on events occurring in thermal evolution of their electrical resistivity, [Formula: see text]. Overall evolution of these diagrams can be rationalized in terms of (i) Mott-Efros-Shklovskii scenario which, within the semiconducting [Formula: see text] regime ([Formula: see text] metal-insulator transition), describes the doping influence on the thermally activated hopping conductivity. (ii) A granular metal (superconductor) scenario which, within [Formula: see text], describes the evolution of normal and superconducting properties in terms of conductance g, Coulomb charging energy E c and Josephson coupling J; their joint influence is usually captured within a [Formula: see text] phase diagram. Based on analysis of the granular character of [Formula: see text], we converted the [Formula: see text] diagrams into projected g - T diagrams which, being fundamental, allow a better understanding of evolution of various granular-related properties (in particular the hallmarks of normal-state [Formula: see text] feature and superconductor-insulator transition) and how such properties are influenced by x, pressure or heat treatment.

The measurement of low levels of factor VIII or factor IX in hemophilia A and hemophilia B plasma by clot waveform analysis and thrombin generation assay.

BACKGROUND: Precise assessment of clotting function is essential for monitoring of hemostatic treatment for hemophilias A and B. MATERIALS AND METHODS: Clot waveform analysis and thrombin generation assays were performed on factor (F) VIII- and FIX-deficient plasmas, which had been reconstituted with known amounts of recombinant FVIII (rFVIII) and affinity-purified FIX respectively. Clot waveforms were assessed qualitatively and quantitatively by measuring the parameters clotting time, maximum coagulation velocity (Min1), and maximum coagulation acceleration (Min2). The thrombin generation assay was also assessed qualitatively and measurements made of time to peak and peak height. RESULTS: Overall results obtained with both assays showed good correlation for both clotting factors confirming that the changes in clotting waveform reflected changes in thrombin generation. Both assays demonstrated a predictable dose response to the addition of FVIII or IX. However, clot waveform analysis was more sensitive than the thrombin generation assay, particularly in detecting very low levels (0-0.1 IU dL(-1)) of both factors. CONCLUSIONS: These data suggest that the application of clot waveform analysis to the routine management of the hemophiliacs could increase our understanding of the clinical significance of low levels of FVIII and FIX that cannot be measured by assays in current use. This may be particularly useful in the management of hemophiliacs with inhibitors or undergoing gene therapy.

Factor V C2 domain contains a major thrombin-binding site responsible for thrombin-catalyzed factor V activation.

Factor (F)V is converted into its active form, FVa, by limited proteolysis. Thrombin-catalyzed activation of FV is essential for its full cofactor activation. Previously, we reported that thrombin was bound to the C2 domain in the light chain of FVIII. As FV has a similar domain structure to FVIII, we focused on the FV C2 domain as a possible binding region for thrombin. Kinetic parameters, measured by surface plasmon resonance, revealed that the K(d) values of anhydro-thrombin for FV, FVa, and the FV C2 domain were 66, 240, and 670 nmol L(-1), respectively. FV activation was increased by approximately 9-fold by the addition of thrombin. In the presence of the FV C2 domain, this increase of the FV activation was inhibited. However, FV activation was not inhibited by the addition of the FVIII C2 domain. FV was cleaved into a 105-kDa heavy chain and a 71/74-kDa light chain by thrombin-catalyzed proteolysis at Arg709, Arg1018 and Arg1545. In the presence of the FV C2 domain, the cleavage was inhibited at all sites. Proteolysis was not affected by the addition of the FVIII C2 domain. These results indicated that the FV C2 domain contains a major binding site for thrombin and that this domain is necessary for the proteolysis at all cleavage sites. Furthermore, the present results also suggested that thrombin has an independent binding site for FV different from that for FVIII.

Distribution and medullary projection of respiratory neurons in the dorsolateral pons of the rat.

The dorsolateral pons around the parabrachial nucleus including the Kölliker-Fuse nucleus is closely linked with the medullary respiratory center and plays an important role in respiratory control. We aimed to elucidate the firing properties, detailed distributions, and medullary projections of pontine respiratory neurons in pentobarbitone-anesthetized, paralyzed, and artificially ventilated rats with intact vagi. A total of 235 respiratory neurons were recorded from the dorsolateral pons in and around the Kölliker-Fuse nucleus. Six types of firing patterns were identified: inspiratory, expiratory-inspiratory phase spanning, inspiratory-expiratory phase spanning, decrementing expiratory, augmenting expiratory, and whole-phase expiratory patterns. Of these, the inspiratory neurons and the expiratory-inspiratory phase spanning neurons, which constituted the largest population (61%), were characterized most carefully by changing lung inflation levels, since under some conditions both showed similar firing patterns. Many (58%) of the 133 respiratory neurons examined were antidromically activated by electrical stimulation of the medulla. They were activated from the ventrolateral medulla around the ventral respiratory group and the Bötzinger complex and from the dorsomedial medulla around the nucleus tractus solitarii and the hypoglossal nucleus. The projections to the dorsomedial medulla were bilateral in many cases, and those to the ventrolateral medulla were unilateral. Of these medullary projections, two specific projections could be characterized in detail. First, many expiratory-inspiratory phase spanning neurons projected to the hypoglossal nucleus, suggesting that these pontine neurons are important premotor neurons of the hypoglossal motoneurons. This projection explains well the hypoglossal inspiratory activity, which is often dissociated from the phrenic inspiratory activity. Second, most whole-phase expiratory neurons that were distributed medially to the KF nucleus sent their axons toward the spinal cord via the midline medulla. These findings provide a new insight into the pontine control of medullary and spinal respiratory function.

Involvement of transforming growth factor-beta and thrombopoietin in the pathogenesis of myelodysplastic syndrome with myelofibrosis.

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.