L-Arginine ethylester enhances in vitro amplification of PrP(Sc) in macaques with atypical L-type bovine spongiform encephalopathy and enables presymptomatic detection of PrP(Sc) in the bodily fluids.

Protease-resistant, misfolded isoforms (PrP(Sc)) of a normal cellular prion protein (PrP(C)) in the bodily fluids, including blood, urine, and saliva, are expected to be useful diagnostic markers of prion diseases, and nonhuman primate models are suited for performing valid diagnostic tests for human Creutzfeldt-Jakob disease (CJD). We developed an effective amplification method for PrP(Sc) derived from macaques infected with the atypical L-type bovine spongiform encephalopathy (L-BSE) prion by using mouse brain homogenate as a substrate in the presence of polyanions and L-arginine ethylester. This method was highly sensitive and detected PrP(Sc) in infected brain homogenate diluted up to 10(10) by sequential amplification. This method in combination with PrP(Sc) precipitation by sodium phosphotungstic acid is capable of amplifying very small amounts of PrP(Sc) contained in the cerebrospinal fluid (CSF), saliva, urine, and plasma of macaques that have been intracerebrally inoculated with the L-BSE prion. Furthermore, PrP(Sc) was detectable in the saliva or urine samples as well as CSF samples obtained at the preclinical phases of the disease. Thus, our novel method may be useful for furthering the understanding of bodily fluid leakage of PrP(Sc) in nonhuman primate models.

Bioaccessibility of Cd and Pb in tailings from a zinc smelting in Brazil: implications for human health.

Soils and wastes enriched with heavy metals may present ecological and human health risks. A considerable number of mining areas exist in Brazil, where high levels of metals have been found. However, studies of bioaccessibility of metals in soils/tailings from these areas are scarce, despite their potential informational contribution concerning exposure risks of residents near these areas. This study evaluated tailings collected from four sites of a zinc smelting area located in Brazil with aims to: (1) evaluate the presence of metals of potential concern; (2) investigate Cd and Pb bioaccessibility; and (3) determine the desorption kinetics of Cd and Pb. High concentrations of total Cd and Pb (up to 1743 mg Cd kg(-1) and 8675 mg Pb kg(-1)) and great variability were found in the tailings, indicating the importance of adequate planning for their final disposal, in order to avoid contamination in the surrounding environment. Cadmium and Pb bioaccessibility percentages in the intestinal phase were less than 47 and 4 %, respectively, which represents significant fractions not available for absorption in the intestinal tract. However, this material has to be monitored since its bioaccessibility may increase with eventual physicochemical changes, releasing Cd and Pb. Desorption kinetics experiments revealed that Pb in the samples remained in less labile fractions, whereas Cd was found in more labile fractions, which is in accordance with the bioaccessibility results.

Clinical and immunological profiles in 17 Japanese patients with drug-induced pemphigus studied at Kurume University.

BACKGROUND: Drug-induced pemphigus (DIP) shows clinical, histopathological and immunological features of pemphigus. However, little is known about immunological profiles in DIP. OBJECTIVES: To characterize clinical and immunological profiles in patients with DIP. METHODS: We studied 17 Japanese patients with DIP who were treated at Kurume University Hospital or who consulted from other hospitals between 1997 and 2012. Complicated diseases, clinical and histopathological manifestations, responsible drugs and findings in immunofluorescence, enzyme-linked immunosorbent assays (ELISAs), immunoblotting (IB) and prognosis were analysed. RESULTS: Eight of the 17 patients with DIP showed pemphigus foliaceus-like appearance, three showed pemphigus herpetiformis-like appearance, and six showed atypical bullous lesions. Responsible drugs were thiol-containing drugs in 16 patients (bucillamine in nine cases, d-penicillamine in four cases, and cetapril, thiopronine and captopril in one patient each), and a nonthiol drug, sulfasalazine, in one patient. By ELISAs and/or IB analyses, nine patients reacted only with desmoglein 1 (Dsg1), four reacted with Dsg1 and Dsg3, and four showed no specific reactivity. By IB of normal human epidermal extracts, in addition to positive reactivity with Dsg1, four patients with no detectable malignancy showed paraneoplastic pemphigus-like reactivity with the 210-kDa envoplakin and the 190-kDa periplakin. Four cases showed anti-Dsg3 antibodies without mucosal lesions. While 11 cases recovered after discontinuation of the causative drugs, six patients had a very protracted or intractable disease course, and might develop true pemphigus. CONCLUSIONS: The present study indicated that the majority of the patients with DIP studied showed a pemphigus foliaceus-type phenotype with anti-Dsg1 autoantibodies, caused by thiol-containing drugs.

Orthopaedic evaluation in children with severe haemophilia A or B submitted to primary prophylaxis therapy in a coagulopathy treatment centre.

There is a lack of publications concerning the use of primary prophylaxis in developing countries. The aim of this study was to evaluate the effectiveness of primary prophylaxis therapy in preventing the development of arthropathy in children with severe haemophilia A or B. From January 1999 to April 2009, a prospective study was carried out involving 39 patients with severe haemophilia A or B. These haemophilia A and haemophilia B patients received 20-40 UI kg(-1) of factors VIII and IX, three and two times per week, respectively. The patients were followed up by a multidisciplinary team. The analysis was carried out in 23 patients who had been on prophylaxis therapy for at least 12 months. The orthopaedic evaluation was performed according to the recommendations of the Orthopedic Advisory Committee of the World Federation of Hemophilia, by evaluating pain and bleeding, and by conducting physical examination and radiological assessment (Pettersson's Joint Score and magnetic resonance): 82.6% of patients who had used the factor regularly did not present any clinical or radiographic changes in the studied joints; 17.4% used the factor irregularly at the beginning of the treatment and of those, most patients presented mild changes in the joints; and 4.3% presented transient knee and ankle pain in spite of regular factor use. The preliminary results of primary prophylaxis confirm its effectiveness in preventing haemophilic arthropathy. Socioeconomic factors did not play a significant role.

Neural expression of a sodium channel gene requires cell-specific interactions.

In the protochordate Halocynthia roretzi, voltage-activated sodium current undergoes a change in kinetics within 48 hr of fertilization. Molecular cloning and microinjection of antisense DNA into single cells suggest that the kinetic changes are due to the increased expression of a putative neural-specific sodium channel gene, TuNa I. TuNa I gene transcription is first induced in late stage gastrulae, preceding the appearance of the rapidly inactivating sodium current unique to neural cells. In cleavage-arrested and intact embryos, cell interactions between specific animal and vegetal blastomeres are required for induction of TuNa I gene expression. Our results implicate cell contact, prior to neurulation, as a mechanism for selectively activating the TuNa I gene expressed in cells of the neural lineage.

Cone signals in monostratified and bistratified amacrine cells of adult zebrafish retina.

Strata within the inner plexiform layer (IPL) of vertebrate retinas are suspected to be distinct signaling regions. Functions performed within adult zebrafish IPL strata were examined through microelectrode recording and staining of stratified amacrine types. The stimulus protocol and analysis discriminated the pattern of input from red, green, blue, and UV cones as well as the light-response waveforms in this tetrachromatic species. A total of 36 cells were analyzed. Transient depolarizing waveforms at ON and OFF originated with bistratified amacrine types, whose dendritic planes branched either in IPL sublaminas a & b, or only within sublamina a. Monophasic-sustained depolarizing waveforms originated with types monostratified in IPL s4 (sublamina b). OFF responses hyperpolarized at onset, depolarized at offset, and in some cases depolarized during mid-stimulus. These signals originated with types monostratified in s1 or s2 (sublamina a). Bistratified amacrines received depolarizing signals only from red cones, at both ON and OFF, while s4 stratified ON cells combined red and green cone signals. The s1/s2 stratified OFF cells utilized hyperpolarizing signals from red, red and green, or red and blue cones at ON, but only depolarizing red cone signals at OFF. ON and OFF depolarizing transients from red cones appear widely distributed within IPL strata. "C-type" physiologies, depolarized by some wavelengths, hyperpolarized by others, in biphasic or triphasic spectral patterns, originated with amacrine cells monostratified in s5. Collectively, cells in this stratum processed signals from all cone types. J. Comp. Neurol. 525:1532-1557, 2017. © 2016 Wiley Periodicals, Inc.

Investigation of arsenic species in tailings and windblown dust from a gold mining area.

Research has shown the presence of high levels of arsenic (up to 2666 mg As kg(-1)) in tailings from a gold mining area of Brazil. This is an important point of attention, generating concerns about impacts on human health. Yet, a recent study showed that As bioaccessibility in the same area was very low (<4.4%). Thus, determination of the direct solid-phase speciation of As in the mine tailings and windblown dust is needed to explain this low bioaccessibility. Mine samples were collected from four subareas and windblown dust from eight sites. Synchrotron-based bulk-X-ray absorption near-edge structure (bulk-XANES) spectroscopy, micro-X-ray absorption near-edge structure (µ-XANES), and µ-X-ray fluorescence (µ-SXRF) spectroscopy were applied to determine As speciation. Bulk-XANES spectra indicated that As occurs as the As(V) oxidation state. Micro-XANES and µ-SXRF analyses revealed that As was also present as arsenopyrite (FeAsS) and its weathering products, but mostly it was As(V) as poorly crystalline ferric arsenate. This supports the findings of low bioaccessible As and highlights the importance of Fe oxides in immobilizing As in the terrestrial environment. All air particulate samples exhibited As-rich particles (up to 313 mg As kg(-1)). The air particulates exhibited solid-phase As species very similar to those found in the mine samples, which indicates that As in the windblown dust is not easily available.

Subfamily-specific posttranscriptional mechanism underlies K(+) channel expression in a developing neuronal blastomere.

Na(+) and K(+) channels are the two key proteins that shape the action potentials in neurons. However, little is known about how the expression of these two channels is coordinated. To address this issue, we cloned a Shab-related K(+) channel gene from ascidian Halocynthia roretzi (TuKv2). In this animal, a blastomere of neuronal lineage isolated from the 8-cell embryo expresses single Na(+) channel and K(+) channel genes after neural induction. Expression of a dominant negative form of TuKv2 eliminated the native delayed rectifier K(+) currents, indicating that the entire delayed rectifier K(+) current of the neuronal blastomere is exclusively encoded by TuKv2. TuKv2 transcripts are expressed more broadly than Na(+) channel transcripts, which are restricted to the neuronal lineages. There is also a temporal mismatch in the expression of TuKv2 transcript and the K(+) current; TuKv2 transcripts are present throughout development, whereas delayed rectifier K(+) currents only appear after the tailbud stage, suggesting that the functional expression of the TuKv2 transcript is suppressed during the early embryonic stages. To test if this suppression occurs by a mechanism specific to the TuKv2 channel protein, an ascidian Shaker-related gene, TuKv1, was misexpressed in neural blastomeres. A TuKv1-encoded current was expressed earlier than the TuKv2 current. Furthermore, the introduction of the TuKv2-expressing plasmid into noninduced cells did not lead to the current expression. These results raise the possibility that the expression of TuKv2 is post-transcriptionally controlled through a mechanism that is dependent on neural induction.

Paralytic zebrafish lacking acetylcholine receptors fail to localize rapsyn clusters to the synapse.

Physiological analysis of two lines of paralytic mutant zebrafish, relaxed and sofa potato, reveals defects in distinct types of receptors in skeletal muscle. In sofa potato the paralysis results from failed synaptic transmission because of the absence of acetylcholine receptors, whereas relaxed mutants lack dihydropyridine receptor-mediated release of internal calcium in response to the muscle action potential. Synaptic structure and function appear normal in relaxed, showing that muscle paralysis per se does not impede proper synapse development. However, sofa potato mutants show incomplete development of the postsynaptic complex. Specifically, in the absence of ACh receptors, clusters of the receptor-aggregating protein rapsyn form in the extrasynaptic membrane but generally fail to localize to the subsynaptic region. Our results indicate that, although rapsyn molecules are capable of self-aggregation, interaction with ACh receptors is required for proper subsynaptic localization.

Specific detection of human coagulation factor IX in cynomolgus macaques.

After screening for species-specific antihuman factor (F)IX monoclonal antibodies, we found that antibody 3A6 did not bind to cynomolgus FIX. The 3A6 epitope was found to include Ala262 of human FIX. The 3A6 antibody was used as a catching antibody in an enzyme immunoassay (EIA) for specific detection of human FIX in cynomolgus macaque plasma. No significant increase of substrate hydrolysis was observed when EIA buffer containing cynomolgus macaque plasma was subjected to the 3A6-based EIA. Addition of up to 30% cynomolgus macaque plasma or canine plasma to the assay did not alter detection of human FIX. Three cynomolgus macaques were injected with human FIX (10 U kg-1; i.v.) and the circulating human FIX was quantified in the macaque plasma. The FIX level in the circulation increased to 470 +/- 37.6 ng mL-1 at 1 h after the injection and gradually decreased to 1.79 +/- 1.1 ng mL-1 by day 5, which is approximately 0.06% of the normal human plasma FIX concentration. These data suggest that the cynomolgus macaque can be used as a primate model for studying hemophilia B gene therapy by transduction of macaque organs with vectors to express human FIX in vivo and detection of human FIX using the 3A6 monoclonal antibody.

Synthesis of new heparinoids with high anticoagulant activity.

New heparinoids were synthesized by the chemical method starting from ring-opening polymerization of anhydro sugar derivatives. Sulfation of synthetic (1----6)-alpha-linked 3-amino-3-deoxy-D-glucopyranan and its copolymers gave dextran-type heparinoids having a sulfamide group on the C-3 carbon of the sugar unit. Heparinoids with different sulfamide contents indicated that the anticoagulant activity (35.3-41.3 units/mg) is independent of the sulfamide content, while an increase in sulfamide content lowered the toxicity. Sulfation of (1----5)-alpha-D-xylofuranan and -ribofuranan provided furanan-type heparinoids the anticoagulant activities of which were higher than those of the corresponding sulfated pyranan-type polysaccharides (1----4)-beta-D-xylopyranan and -ribopyranan. The highest activity (69.1 units/mg) was shown by sulfated (1----5)-alpha-D-xylofuranan. The dextran-type heparinoid having a sulfamide group showed a high anticoagulant activity also in vivo and high lipemia-clearing activity.

Induction of MHC-IIDR expression on circulating CD8+ lymphocytes in macaques infected with SIVmac239 nef-open but not with its nef-deletion mutant.

We examined the expression kinetics of activation antigens CD38 and MHC-IIDR (DR) on circulating CD8+ lymphocytes in rhesus macaques infected with pathogenic simian immunodeficiency virus strain SIVmac239 nef-open (239) or its nonpathogenic nef-deletion mutant (delta nef). In the longitudinal study, we found for the first time the induction of DR expression on CD8+ lymphocytes in 239-infected macaques. The induction of DR was in parallel with an increasing viral load and a decreasing CD4+ lymphocyte level. In the macaques with the high viral load and low CD4 level, a considerable proportion of the DR+CD8+ subpopulation was CD69+, indicating an activated state. On the other hand, no significant increase in the DR+CD8+ subpopulation level was observed in delta nef-infected macaques. These data indicate that the evaluation of activation markers such as DR and/or CD69 on circulating CD8+ cells may be valuable as a surrogate marker in the SIV-macaque model.

Simian T cell leukemia virus type I-induced malignant adult T cell leukemia-like disease in a naturally infected African green monkey: implication of CD8+ T cell leukemia.

Spontaneous T cell leukemia was found in an African green monkey (Cercopithecus aethiops, AGM) naturally infected with simian T cell leukemia virus type I (STLV-I). The hematological features and the evidence for monoclonal integration of provirus DNA in the leukemic cells revealed that the leukemia was an ATL-like disease. The expression of surface markers on the leukemic cells indicated that they were defined as an activated CD8+ T cell subset. Together with the finding that seven in vitro spontaneously STLV-I-transformed cell lines were CD4-CD8+, it is likely that CD8+ T cells are transformed by STLV-I in AGMs, in contrast with human ATL. Finally, we assessed characteristics of the CD8 chains on these transformed cells. The result indicated that the leukemic cells expressed only the alpha chains but not the beta chains. However, in the case of in vitro-transformed cell lines the expression pattern of the CD8 chains varied in individual monkeys. Thus, STLV-I may preferentially transform CD8+ (both alphaalpha+ and alphabeta+) T cells in AGMs.

Widespread distribution of adenovirus-transduced monkey amniotic epithelial cells after local intracerebral injection: implication for cell-mediated therapy for lysosome storage disorders.

Cell-mediated therapy for mucopolysaccharidosis type VII (MPSVII) was studied using monkey amniotic epithelial cells (mAEC). The cells were transduced with a recombinant adenovirus expressing human beta-glucuronidase (GUSB), and cells overexpressing GUSB were generated. The cells expressed 2000-fold higher activities than the endogenous GUSB activities of nontransduced mAEC, demonstrating that mAEC were successfully transduced with adenoviral vectors. These cells also secreted high levels of GUSB. To clarify the cross-correction of GUSB secreted from mAEC, the conditioned medium containing high levels of GUSB was added into the medium for culturing human or murine fibroblasts established from an MPSVII patient or a mouse model of the disease. Dramatic increases in GUSB activities were observed in both fibroblasts. We then transplanted the cells transduced with an adenovirus expressing LacZ into the caudate-putamen of monkey brain. Survival and distribution of the transplanted cells 1 month after the treatment were evaluated. Histochemical analysis showed that LacZ-positive cells were widely distributed in the brain, suggesting that the transplanted cells had migrated and were distributed even at regions far from the implantation site. These findings suggest that local intracerebral engraftment of genetically engineered amniotic epithelial cells is favorable for the treatment of lysosome storage disorders, whose pathological abnormalities are not restricted to specific regions of the brain.

Low prevalence of activated protein C resistance and coagulation factor V Arg506 to Gln mutation among Japanese patients with various forms of thrombosis, and normal individuals.

Resistance to activated protein C (APC), recently reported to be the most prevalent inherited cause of thrombosis among Caucasians, is associated with a single point mutation in the coagulation factor V gene. We investigated the prevalence of APC resistance and the factor V gene mutation (R506Q) in 34 consecutive Japanese patients with venous thrombosis or pulmonary thromboembolism and 63 control subjects. Three of the 33 patients examined (9%) had an APC ratio below the 5th percentile of control values (2.27), but all were above 2.0. The factor V mutation (R506Q) was not detected in the 29 patients studied, including the 3 patients whose APC ratios were below 2.27, or in 53 controls. In a tissue factor-based factor V assay to detect APC resistance recently described by Le et al. (Blood 1995;85:1704-1711), all patients studied were found to be normal including the three with a low APC ratio. We conclude that APC resistance and factor V gene mutation are less prevalent in Japan than in several European countries.

Age-related changes in the localization of presenilin-1 in cynomolgus monkey brain.

Age-related changes in PS-1 localization were examined in the brains of 22 cynomolgus monkeys ranging in age from embryonic day 87 to 35 years. In embryonic monkey brains, anti-PS-1 antibody N12, which recognizes the PS-1 N-terminal fragment (Ntf) and holo protein, stained immature neuronal cells. In juvenile monkeys, N12 stained large pyramidal neurons, cerebral neocortical neurons, and cerebellar Purkinje's cells. Cytoplasmic staining of these cells was granular in appearance. In aged monkeys, N12 stained neurons in all layers of the neocortex. In contrast, regardless of the age of the animals examined, M5, an anti-PS-1 antibody that specifically recognizes only the PS-1 C-terminal fragment (Ctf), stained neurons in all layers of the neocortex and neurons in the cerebellum. M5 also stained neuropil and white matter, and in aged monkeys, M5 stained swollen neurites of mature senile plaques. Age-related changes in PS-1 expression were further examined using Western blot analysis of mitochondrial, myelin, microsomal, nuclear, synaptosomal, and cytosol fractions isolated from 10 monkey brains ranging in age from embryonic day 87 to 32 years. In all brains, Ntf and Ctf were expressed most abundantly in the microsome fraction. The amount of PS-1 in the nuclear fraction dramatically increased with age. We conclude that the transport of PS-1 diminished with age and that PS-1 fragments accumulated in endoplasmic reticulum (ER) associated with the nuclear membrane.

Evidence for the presence of dopamine D1 receptor mRNA and binding sites in monkey amniotic epithelial cells.

In this study we examined the presence of dopamine D1 receptors in monkey amniotic epithelial cells (MAEC) using RT-PCR and radioligand binding experiments. We found that MAEC express D1 receptor mRNA that is having 99% homology with human dopamine D1 receptors. Saturation binding studies using [3H]SCH-23390 showed a high affinity D1 site with K(D) and Bmax values of 0.82 +/- 0.12 nM and 20.77 +/- 4.22 fmol/mg protein, respectively. Competition experiments showed that selective D1, but not D2, antagonists are potent displacers of [3H]SCH 23390 binding with a rank order of potency that is consistent with the pharmacology of the dopaminergic D1 site. These data provide, for the first time, compelling evidence that MAEC natively express D1 mRNA and binding sites and suggest that it may be a potential primate cell model to study D1 receptors and to explore new selective drugs active at these receptors.

Carboxyl end-specific monoclonal antibodies to amyloid beta protein (A beta) subtypes (A beta 40 and A beta 42(43)) differentiate A beta in senile plaques and amyloid angiopathy in brains of aged cynomolgus monkeys.

Senile plaques (SPs) and cerebral amyloid angiopathy (CAA) in the brains of five aged (20-26 years old) cynomolgus monkeys were investigated immunohistochemically using two monoclonal antibodies (anti-A beta 40 (BA27) and anti-A beta 42(43) (BC05)) that can differentiate the carboxyl termini of amyloid beta protein (A beta) subtypes. In four of five animals, all types of SPs (i.e. diffuse, primitive, and classical plaques; DPs, PPs, and CPs, respectively) were identified by BC05. However, BA27 did not label DPs and stained only about one third of PPs and CPs, mainly labeling granular structures and cored portions, respectively. In CAA, lesions of cortical capillaries reacted to BC05 in four of five cases, but rarely and weakly to BA27 in two of five cases. On the other hand, lesions of parenchymal and meningeal arterioles were stained by both BA27 and BC05. These staining profiles of SPs in cynomolgus monkeys correspond well to those in humans, although there are two remarkable features in cynomolgus monkeys. First, BA27 stained PPs associated with granular structures. Secondly, capillary A beta reacted intensely to BC05 but only slightly to BA27. Despite these unique features, the results suggest that aged cynomolgus monkeys can be used to investigate the pathogenesis of A beta deposition in SPs and CAA.

Detection of hypercoagulability by the measurement of the dynamic loss modulus of clotting blood.

The dynamic loss modulus of clotting whole blood was measured in thrombotic patients to characterize the physical properties of coagulation in the hypercoagulable state. The dynamic loss modulus was measured by a Sonoclot. Thrombotic patients consisted of 30 with deep vein thrombosis and 25 with arterial thrombosis. An accelerated increment rate of the dynamic loss modulus at the beginning of gelling was the characteristic of hypercoagulability. This characteristic occurred more frequently than other abnormalities in other tests for hypercoagulability (beta-thromboglobulin, antithrombin III and TEG). Only in deep vein thrombosis, a moderately positive correlation was noted between the increment rate of the dynamic loss modulus and the plasma fibrinogen level.

Possible relation of small pox vaccination to multiple sclerosis-like disease. A personal note.

My multiple sclerosis-like disease seemed to be an allergy to small pox vaccination. Repeated small pox vaccinations apparently arrested the disease.