Dramatic Clinical Response of Relapsed Metastatic Extramammary Paget's Disease to Trastuzumab Monotherapy.

We report the first case of 68-year-old Japanese woman with metastatic HER2-positive extramammary Paget's disease that showed the validity of trastuzumab monotherapy. We administered trastuzumab at a loading dose of 8 mg/kg i.v., followed by a 6 mg/kg maintenance dose every three weeks according to a protocol for HER2-positive metastatic breast cancers and a near-complete response was achieved after the tenth infusion. The patient experienced a moderate headache and flushing during the first infusion, but had no advanced effects during subsequent infusions with ibuprofen and d-chlorpheniramine maleate. Given the dramatic response, the patient has had 17 infusions of trastuzumab with no disease progression. Thus, trastuzumab has few side effects and is well tolerated for elderly patients. It may become a new choice of the adjubant therapy of this disease.

Elevation of serum high molecular weight adiponectin in patients with Type 2 diabetes and orthostatic hypotension: association with arterial stiffness and hypercoagulability.

AIM: Orthostatic hypotension is a hallmark of diabetic autonomic neuropathy and is associated with increased mortality. The serum level of adiponectin is elevated in patients with heart failure or renal failure. In the present study, we measured serum levels of total and high molecular weight adiponectin in patients with Type 2 diabetes and orthostatic hypotension. We also investigated the relationship between the presence of orthostatic hypotension and various clinical variables in patients with Type 2 diabetes. METHODS: We studied 105 patients with Type 2 diabetes. Orthostatic hypotension was defined as a decrease of 20 mmHg or more in systolic blood pressure and/or 10 mmHg in diastolic blood pressure when blood pressure was measured for 3 min while standing. The brachial-ankle pulse-wave velocity was also measured as an index of arterial stiffness. RESULTS: Orthostatic hypotension was found in 30 patients with diabetes (28.6%). The haematocrit and estimated glomerular filtration rate were significantly lower in patients with orthostatic hypotension than in those without it. Brachial-ankle pulse-wave velocity and serum total and high molecular weight adiponectin were significantly higher in patients with orthostatic hypotension than in those without. Furthermore, the high molecular weight/total adiponectin ratio was higher in patients with orthostatic hypotension than in those without and hypertension was more common in patients with orthostatic hypotension. Plasma prothrombin F1 + 2, a coagulation maker, was higher in patients with orthostatic hypotension than in those without, while there were no differences of fibrinolytic markers between the two groups. Multivariate analysis showed that HDL cholesterol, haematocrit, F1 + 2, brachial-ankle pulse-wave velocity and a decline of systolic blood pressure on standing were independent determinants of high molecular weight adiponectin. CONCLUSIONS: Patients with Type 2 diabetes and orthostatic hypotension had an elevated serum level of high molecular weight adiponectin, which was associated with the simultaneous presence of renal dysfunction, anaemia, arterial stiffness and hypercoagulability.

Positional relationship between recurrent intracerebral hemorrhage/lacunar infarction and previously detected microbleeds.

BACKGROUND AND PURPOSE: Although MBs, ICH, and LI are secondary to cerebral microangiopathy, it remains unclear whether the location of subsequent ICH/LI corresponds to the previous location of MBs. We performed this study to clarify the positional relationship between recurrent ICH/LI and previously detected MBs. MATERIALS AND METHODS: We evaluated patients with recurrent ICH/LI who had MBs, as shown on prior T2*-weighted MR imaging. We assessed retrospectively whether the location of recurrent ICH/LI corresponded to that of the prior MB. Patients with ICH were divided into the deep ICH group and the lobar ICH group, and the positional relationship between hematoma and previously detected MBs was evaluated. RESULTS: A total of 55 patients, including 34 with recurrent ICH and 21 with recurrent LI were evaluated. Although the location of the LI corresponded to prior MBs in only 1 patient (4.8%), the location of ICH corresponded to prior locations of MBs in 21 patients (61.8%) (OR, 32.3; 95% CI, 3.86-270.3; P < .001). Among the patients with ICH, the correspondence ratio was higher in the deep ICH group (19 of 24 patients, 79.2%) than in the lobar ICH group (2 of 10 patients, 20%) (OR, 15.2; 95% CI, 2.42-95.3; P < .002). CONCLUSIONS: The close positional association between recurrent ICH and prior MBs suggests that MBs represent hemorrhage-prone microangiopathy. In addition, different correspondence ratios between the deep ICH group and the lobar ICH group may be attributable to their different pathogenesis.

Association between cerebral microbleeds on T2*-weighted MR images and recurrent hemorrhagic stroke in patients treated with warfarin following ischemic stroke.

BACKGROUND AND PURPOSE: Although accumulating evidence suggests the presence of microbleeds as a risk factor for intracerebral hemorrhage (ICH), little is known about its significance in anticoagulated patients. The aim of this study was to determine whether the presence of microbleeds is associated with recurrent hemorrhagic stroke in patients who had received warfarin following atrial fibrillation-associated cardioembolic infarction. MATERIALS AND METHODS: A total of 87 consecutive patients with acute recurrent stroke, including 15 patients with ICH and 72 patients with cerebral infarction, were enrolled in this study. International normalized ratios (INRs), vascular risk factors, and imaging characteristics, including microbleeds on T2*-weighted MR images and white matter hyperintensity (WMH) on T2-weighted MR images, were compared in the 2 groups. RESULTS: Microbleeds were noted more frequently in patients with ICH than in patients with cerebral infarction (86.7% versus 38.9%, P = .0007). The number of microbleeds was larger in patients with ICH than in patients with cerebral infarction (mean, 8.4 versus 2.1; P = .0001). INR was higher in patients with ICH than in patients with cerebral infarction (mean, 2.2 versus 1.4; P < .0001). The frequency of hypertension was higher in patients with ICH than in patients with cerebral infarction (86.7% versus 45.8%, P = .0039). Multivariate analysis revealed that the presence of cerebral microbleeds (odds ratio, 7.383; 95% confidence interval, 1.052-51.830) was associated with ICH independent of increased INR and hypertension. CONCLUSION: The presence of cerebral microbleeds may be an independent risk factor for warfarin-related ICH, but more study is needed because of strong confounding associations with elevated INR and hypertension.

Comparison of the effects of pioglitazone and voglibose on circulating total and high-molecular-weight adiponectin, and on two fibrinolysis inhibitors, in patients with Type 2 diabetes.

BACKGROUND: To investigate short-term effects of pioglitazone and voglibose on serum concentrations of both total and high-molecular-weight (HMW) adiponectin measured with a novel sandwich enzyme-linked immunosorbent assay (ELISA) ,and on plasma fibrinolysis indicators, in Type 2 diabetic patients with inadequate glycaemic control on sulphonylureas. METHODS: Thirty-four diabetic patients were randomized to receive pioglitazone or voglibose treatment for 12 weeks, after which serum HMW adiponectin was measured. Plasma plasminogen activator inhibitor (PAI) 1 and thrombin-activatable fibrinolysis inhibitor (TAFI), a recently identified inhibitor of fibrinolysis, were measured as fibrinolysis inhibitors. RESULTS: At baseline, serum HMW adiponectin correlated negatively with plasma TAFI in all patients with Type 2 diabetes (r = -0.367, P = 0.0423). Both groups showed similar improvements in glycaemic control. Serum total and HMW adiponectin increased in patients treated with pioglitazone, but did not change in patients treated with voglibose. The HMW : total adiponectin ratio increased significantly after treatment with pioglitazone (P = 0.0004). The change in HbA(1c) correlated negatively with changes in serum HMW adiponectin in patients treated with pioglitazone (r = -0.694, P = 0.0034). Plasma PAI-1 and TAFI did not change with pioglitazone treatment. CONCLUSION: Increased serum HMW adiponectin may contribute to the improvement in glycaemic control after pioglitazone treatment. Plasma PAI-1 and TAFI were unchanged by either drug.

Purification of branched-chain amino acid aminotransferase from Helicobacter pylori NCTC 11637.

Branched-chain amino acid aminotransferase was purified by several column chromatographies from Helicobacter pylori NCTC 11637, and the N-terminal amino acid sequence was analyzed. The enzyme gene was sequenced based on a putative branched-chain amino acid aminotransferase gene, ilvE of H. pylori 26695, and the whole amino acid sequence was deduced from the nucleotide sequence. The enzyme existed in a homodimer with a calculated subunit molecular weight (MW) of 37,539 and an isoelectric point (pI) of 6.47. The enzyme showed high affinity to 2-oxoglutarate (K (m) = 0.085 mM) and L-isoleucine (K (m) = 0.34 mM), and V (max) was 27.3 micromol/min/mg. The best substrate was found to be L-isoleucine followed by L-leucine and L-valine. No activity was shown toward the D-enantiomers of these amino acids. The optimal pH and temperature were pH 8.0 and 37 degrees C, respectively.

Plasma total homocysteine levels are associated with advanced leukoaraiosis but not with asymptomatic microbleeds on T2*-weighted MRI in patients with stroke.

Both leukoaraiosis and asymptomatic microbleeds are associated with small-artery diseases. Although an association between hyperhomocysteinemia and leukoaraiosis has been reported, no studies have evaluated the association between total homocysteine (tHcy) level and presence of microbleeds in stroke patients. We evaluated the association between tHcy level and leukoaraiosis or microbleeds in stroke patients. In 102 patients with stroke (69.5 +/- 10.3 years old, 54 men and 48 women), microbleeds on T2*-weighted MR images were counted, leukoaraiosis on T2-weighted images was graded and fasting plasma tHcy concentrations were measured. Plasma tHcy level was significantly higher in patients with advanced leukoaraiosis than in those without advanced leukoaraiosis (13.9 +/- 4.6 micromol/l vs. 10.2 +/- 3.4 micromol/l, P < 0.0001). Plasma tHcy level was not significantly different in patients with microbleeds and those without microbleeds (11.3 +/- 4.1 micromol/l vs. 11.4 +/- 4.3 micromol/l, P = 0.9441). Elevated tHcy level is significantly and independently associated with advanced leukoaraiosis [odds ratio (OR), 1.330; 95% CI, 1.130-1.565] but not with the presence of microbleeds. Elevated tHcy level appears to be associated with ischemic small-artery disease rather than with bleeding-prone small-artery disease.

Effects of 30-m nitrox saturation dive on the immune system in man.

Hyperbaria reportedly affects the immune system, but the role of psychological factors arising from confinement has not been taken into consideration. We investigated the immune changes in 4 subjects exposed to a 9-day simulated 30-m (400-kPa) nitrogen-oxygen (nitrox) saturation dive, and compared the results with those of our previous study that showed immune and mood changes in normobaric confinement. Blood samples were taken before, during, and after the dive or confinement, and activated with an anti-CD2 agonistic antibody. The percentages of granulocytes, natural killer (NK) cells, and cells positive for CD69, an early activation marker, were analyzed by flow cytometry. Reduction of CD69 expression percentage was observed under both hyperbaric and normobaric conditions. Percentages of innate immune cells, such as granulocytes and NK cells decreased or remained mostly unchanged, contrasting with our previous study, which demonstrated increases in both percentages coordinate with mood improvement. We conclude that these changes may have been triggered by suppression of sympathetic nerve activity that occurs in 30-m nitrox saturation hyperbaria.

Combinations of the presence or absence of cerebral microbleeds and advanced white matter hyperintensity as predictors of subsequent stroke types.

BACKGROUND AND PURPOSE: Previous studies have shown microbleeds to be a risk factor for intracerebral hemorrhage and white matter hyperintensity (WMH) to be a risk factor for ischemic stroke. This study was performed to determine whether combinations of the presence or absence of microbleeds and advanced WMH are risk factors for subsequent recurrent stroke types. METHODS: In 266 patients with stroke, microbleeds on T2*-weighted MR images were counted, and WMH on T2-weighted images was graded. Patients were divided into 4 groups by the combinations of the presence or absence of microbleeds and advanced WMH and were followed up for stroke recurrence. RESULTS: During a mean follow-up period of 564.8 +/- 220.5 days, 26 patients developed recurrent strokes, including 10 intracerebral hemorrhages and 16 ischemic strokes. Patients with microbleeds without advanced WMH (n = 42) developed only intracerebral hemorrhages (n = 8), and the recurrence rate of intracerebral hemorrhage in those patients estimated by the Kaplan-Meier method was the highest in the 4 groups (14.3% in 1 year and 21.2% in 2 years). In contrast, patients with advanced WMH without microbleeds (n = 39) developed only ischemic strokes (n = 6), and the estimated recurrent rate of ischemic stroke in those patients was the highest in the 4 groups (10.5% in 1 year and 17.4% in 2 years). Cox proportional hazards regression analysis revealed that microbleeds were associated with intracerebral hemorrhage (hazard ratio [HR], 85.626; 95% confidence interval [CI], 6.344-1155.649) and that advanced WMH was negatively associated with intracerebral hemorrhage (HR, 0.016; 95% CI, 0.001-0.258). Advanced WMH was associated with ischemic stroke (HR, 10.659; 95% CI, 2.601-43.678). CONCLUSION: It appears that patients at high risk of subsequent intracerebral hemorrhage or ischemic stroke can be identified by combinations of the presence or absence of microbleeds and advanced WMH.

Chemically modified thrombin and anhydrothrombin that differentiate macromolecular substrates of thrombin.

BACKGROUND: Thrombin is a primary inducer of thrombus formation by activations of coagulation cascade and platelet aggregation. Hitherto, several types of thrombin inhibitors have been developed for therapeutic purpose. OBJECTIVES: We prepared modified thrombin (M-thrombin) and modified anhydrothrombin (M-anhydrothrombin) by chemical modification of carboxyl groups of thrombin and anhydrothrombin, respectively, to present a new strategy for a potent antiplatelet-anticoagulant agent and new tools for investigation of thrombin functions. RESULTS: M-anhydrothrombin retained high affinity for factor VIII (FVIII), but demonstrated lower affinity than anhydrothrombin for fibrinogen and factor V (FV). Both M-anhydrothrombin and anhydrothrombin prolonged activated partial thromboplastin time (APTT) without affecting prothrombin time, and M-anhydrothrombin prolonged APTT much more than anhydrothrombin. M-anhydrothrombin also retained affinity for the recombinant extracellular domain peptide of protease-activated receptor 1 (PAR1). M-thrombin exhibited marginal clotting activity (4% of thrombin), but induced platelet aggregation in platelet-rich plasma without forming a fibrin clot, which was completely suppressed by anti-PAR1 antibody (ATAP2) and by M-anhydrothrombin, but not by anhydrothrombin. These results indicate that M-thrombin induced platelet aggregation through the activation of PAR1, and M-anhydrothrombin inhibited this process completely. In contrast, neither M-anhydrothrombin nor anhydrothrombin apparently inhibited thrombin-induced platelet aggregation. Only in the presence of the Gly-Pro-Arg-Pro (GPRP) peptide that inhibits polymerization of fibrin, M-anhydrothrombin completely inhibited thrombin-induced platelet aggregation. CONCLUSION: M-thrombin is PAR1-specific and M-anhydrothrombin is FVIII- and PAR1-specific derivatives, and thereby, are new tools as specific agonist and antagonist, respectively, of PAR1. Furthermore, M-anhydrothrombin may be an attractive model for development of a potent anticoagulant-antiplatelet agent.

Small low-density lipoprotein and small low-density lipoprotein/total low-density lipoprotein are closely associated with intima-media thickness of the carotid artery in Type 2 diabetic patients.

BACKGROUND: The intima-media thickness (IMT) of the carotid artery, as determined by ultrasonography, is useful for reflecting the extent of subclinical atherosclerosis. We investigated the relationship between IMT and the serum concentrations of small low-density lipoprotein (LDL) in diabetic patients. METHODS: The study was conducted with 27 Type 2 diabetic patients (14 males and 13 females; mean age=62.6+/-8.3 years) and 12 age-matched healthy controls. The LDL subfraction was measured using a polyacrylamide gel electrophoresis method. Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) concentrations were measured by an enzyme immunoassay. The IMT was expressed as the maximum IMT (Max-IMT) and average IMT (Ave-IMT) of the carotid artery, measured by ultrasonography. RESULTS: Both the IMT and the small LDL concentrations were significantly increased in the diabetic patients compared with the healthy participants. The IMTs were significantly correlated with small LDL concentration and small LDL/total LDL more than LDL concentrations by multivariate analysis. The IMTs were not significantly correlated with the serum VEGF or PDGF concentrations. The patients with a larger IMT had a significantly higher prevalence of hypertension or ischemic heart disease than did the patients with a normal IMT. CONCLUSIONS: The increased small LDL concentrations and small LDL/total LDL, in addition to total LDL concentrations, in Type 2 diabetic patients are closely associated with increased IMT of the carotid artery.

Enhanced blood coagulation and fibrinolysis in mice lacking histidine-rich glycoprotein (HRG).

Histidine-rich glycoprotein (HRG) is a serum protein belonging to the cystatin superfamily. HRG may play a regulatory role in hemostasis and innate immunity. However, this role is uncertain because of a lack of rigorous testing in an animal model. We generated mice lacking the translation start point of exon 1 of the Hrg gene, effectively resulting in a null mutation (Hrg-/-). The mice were viable and fertile but had no HRG in their blood. Antithrombin activity in the plasma of Hrg-/- mice was higher than in the plasma of heterozygous Hrg+/- or wild-type Hrg+/+ mice. The prothrombin time was shorter in Hrg-/- mice than in Hrg+/- and Hrg+/+ mice. Bleeding time after tail tip amputation in Hrg-/- mice was shorter than in Hrg+/+ mice. The spontaneous fibrinolytic activity in clotted blood of Hrg-/- mice was higher than in Hrg+/+ mice. These findings suggest that HRG plays a role as both an anticoagulant and an antifibrinolytic modifier, and may regulate platelet function in vivo.

A phosphodiesterase inhibitor, pentoxifylline, enhances the bone morphogenetic protein-4 (BMP-4)-dependent differentiation of osteoprogenitor cells.

Bone morphogenetic protein-4 (BMP-4), a member of the transforming growth factor-beta superfamily, is capable of initiating differentiation of uncommitted mesenchymal cells into a chondro/osteogenic pathway. This study reports the effects of pentoxifylline (PTX), a nonspecific inhibitor of phosphodiesterases (PDEs), that causes elevation of the intracellular cyclic adenosine monophosphate (cAMP) level on the BMP-4-induced chondro/osteogenic differentiation of a mesenchymal cell line, C3H10T1/2; a bone marrow stromal cell line, ST2; and an osteoblastic cell line, MC3T3-E1. It was found that PTX enhanced BMP-4-induced chondro/osteogenic differentiation in C3H10T1/2 and ST2 cells. Similar effects were observed when adding dibutyryl-cAMP and forskolin. These results indicate that cAMP may potentiate the action of BMP-4 on osteoprogenitor cells, highlighting the possibility that PDE inhibitors could be used as therapeutic agents to enhance bone formation through this effect.

Effect of phosphodiesterase inhibitor-4, rolipram, on new bone formations by recombinant human bone morphogenetic protein-2.

Collagen sponge disks (6 mm diameter, 1 mm thickness) were impregnated with recombinant human bone morphogenetic protein-2 (rhBMP-2) (5 microg/disk) and implanted onto the back muscles of mice. Ten or 20 mg/kg per day of Rolipram, a selective inhibitory agent to phosphodiesterase type 4 (PDE-4), or vehicle, was injected subcutaneously into the host mice for 3 weeks. After treatment, rhBMP-2-induced ectopic ossicles were harvested and examined by radiographic and histologic methods to determine the size, bone quality, and mineral content of the ossicles. The ossicles from a group treated with 20 mg/kg per day Rolipram were significantly larger in size and higher in bone mineral density (BMD) and bone mineral content (BMC) than the control samples. No significant differences were noted in mice treated with 10 mg/kg per day of Rolipram. Histologically, ossicles from the high-dose (20 mg/kg per day) Rolipram-treated group showed densely packed, thicker trabeculae when compared with those from the control group. These experimental results indicate that the PDE-4 inhibitor, Rolipram, may enhance the bone-inducing capacity of BMP-2 in mesenchymal cells. This in turn may result in increased responsiveness to BMP-2 and point to a potential use of PDE-4 inhibitors for the promotion of rhBMP-dependent bone repair.

NHE6 protein possesses a signal peptide destined for endoplasmic reticulum membrane and localizes in secretory organelles of the cell.

The NHE6 protein is a unique Na(+)/H(+) exchanger isoform believed to localize in mitochondria. It possesses a hydrophilic N-terminal portion that is rich in positively charged residues and many hydrophobic segments. In the present study, signal sequences in the NHE6 molecule were examined for organelle localization and membrane topogenesis. When the full-length protein was expressed in COS7 cells, it localized in the endoplasmic reticulum and on the cell surface. Furthermore, the protein was fully N-glycosylated. When green fluorescent protein was fused after the second (H2) or third (H3) hydrophobic segment, the fusion proteins were targeted to the endoplasmic reticulum (ER) membrane. The localization pattern was the same as that of fusion proteins in which green fluorescent protein was fused after H2 of NHE1. In an in vitro system, H1 behaved as a signal peptide that directs the translocation of the following polypeptide chain and is then processed off. The next hydrophobic segment (H2) halted translocation and eventually became a transmembrane segment. The N-terminal hydrophobic segment (H1) of NHE1 also behaved as a signal peptide. Cell fractionation studies using antibodies against the 15 C-terminal residues indicated that NHE6 protein localized in the microsomal membranes of rat liver cells. All of the NHE6 molecules in liver tissue possess an endoglycosidase H-resistant sugar chain. These findings indicate that NHE6 protein is targeted to the ER membrane via the N-terminal signal peptide and is sorted to organelle membranes derived from the ER membrane.

Comparative study of effects of angiotensin II receptor antagonist, KD3-671, and angiotensin converting enzyme inhibitor, enalaprilat, on cough reflex in guinea pig.

Angiotensin converting enzyme (ACE) inhibitor prevents the inactivation of bradykinin by inhibiting ACE activity, leading to side effects such as dry cough and angioedema. KD3-671 is a novel nonpeptide angiotensin II antagonist which is expected to exhibit persistent hypotensive action without these side effects. In this study, we investigated the relationship between the pharmacokinetics and cough-inducing effect of this drug in guinea-pig, compared with that of an ACE inhibitor, enalaprilat. KD3-671 was not significantly different from the vehicle treatment in the ability to induce coughing, whereas enalaprilat significantly enhanced coughing compared with the vehicle treatment. Thus, as expected from its mechanism of pharmacological action, KD3-671 did not induce coughing. We suggest that the citric acid-induced guinea pig coughing model will be useful in preclinical studies to examine the effect of drug on pulmonary function.

Acute toxicity and mutagenicity study on branched corn syrup and evaluation of its laxative effect in humans.

We developed a branched corn syrup (BCS, average molecular weight: 500, content of indigestible portion: 45%) by heat treatment of indigestible dextrin with hydrochloric acid. To confirm the safety of BCS, we conducted both an acute toxicity test and a mutagenicity test. Moreover, we observed gastroenteric effects of BCS in fifty healthy humans. The results are summarized as follows. 1) There was no death observed after oral administration of BCS in Sprague-Dawley-strain rats. Lethal dose (LD)50, value was estimated to be more than 10 g/kg body weight. 2) No mutagenicity was observed in Salmonella typhimurium TA98, TA100, TA1535, TA1537, or Escherichia coli WP2uvrA. 3) Fifty adults were divided into five groups often (five of each sex) and orally administered BCS at 0.2, 0.3, 0.4. 0.5 and 0.6 g/kg body weight as indigestible portion. Although no diarrhea was observed in females, BCS at 0.6 g/kg as indigestible portion caused diarrhea in two out of five males. The maximum non-effective dose of indigestible portion of BCS was estimated to be 0.5 g/kg in males and more than 0.6 g/kg in females.

Calcineurin homologous protein as an essential cofactor for Na+/H+ exchangers.

The Na+/H+ exchangers (NHEs) comprise a family of transporters that catalyze cell functions such as regulation of the pH and volume of a cell and epithelial absorption of Na+ and bicarbonate. Ubiquitous calcineurin B homologous protein (CHP or p22) is co-localized and co-immunoprecipitated with expressed NHE1, NHE2, or NHE3 independently of its myristoylation and Ca2+ binding, and its binding site was identified as the juxtamembrane region within the carboxyl-terminal cytoplasmic domain of exchangers. CHP binding-defective mutations of NHE1-3 or CHP depletion by injection of the competitive CHP-binding region of NHE1 into Xenopus oocytes resulted in a dramatic reduction (>90%) in the Na+/H+ exchange activity. The data suggest that CHP serves as an essential cofactor, which supports the physiological activity of NHE family members.