A phase I/II study of cancer peptide vaccine S-288310 in patients with advanced urothelial carcinoma of the bladder.

Background: S-288310, a cancer peptide vaccine composed of two HLA-A*24:02-restricted peptides derived from two oncoantigens, DEP domain-containing 1 (DEPDC1) and M-phase phosphoprotein 1 (MPHOSPH1), was investigated in urothelial carcinoma (UC) of the bladder. Patients and methods: Thirty eight HLA-A*24:02-positive patients with progressive UC were enrolled in this study. In the phase I part of the study, three patients each were treated with S-288310 at 1 mg or 2 mg/peptide subcutaneously once a week to evaluate safety and tolerability. In the phase II, 32 patients were randomized to receive either 1 mg or 2 mg to evaluate the difference in cytotoxic T lymphocytes (CTL) induction and safety. Results: S-288310 was safe and well tolerated in the phase I. Of 27 patients evaluable for immune responses in the phase II, there was no difference in CTL induction rate between the 1 mg (100%) and 2 mg (80.0%) groups. Of 32 patients receiving S-288310 in the phase II, the most frequent drug-related AE was the injection site reaction that was observed in 29 patients (90.6%), but none of the patients discontinued administration due to these reactions and no dose relationship in the frequency and severity was observed. The objective response rate of the 32 patients was 6.3% and the disease control rate was 56.3%. The median overall survival (OS) rates for patients vaccinated with S-288310 after one regimen of chemotherapy, 2 regimens, or 3 or more were 14.4, 9.1 and 3.7 months, respectively, and 32.2% of patients post first-line treatment were alive at 2 years. OS of patients who showed CTL induction to both peptides was longer than that of those with CTL induction to no or one peptide. Conclusion: S-288310 was well-tolerated and effectively induced peptide-specific CTLs, which were correlated with longer survival for patients with UC of the bladder. Trial registration ID: JapicCTI-090980.

Receiver circuit improvement of dual frequency-comb ka-band Doppler backscattering system in the large helical device (LHD).

Doppler-backscattering (DBS) has been used in several fusion plasma devices because it can measure the perpendicular velocity of electron density perturbation v⊥, the radial electric field Er, and the perpendicular wavenumber spectrum S(k⊥) with high wavenumber and spatial resolution. In particular, recently constructed frequency comb DBS systems enable observation of turbulent phenomena at multiple observation points in the radial direction. A dual-comb microwave DBS system has been developed for the large helical device plasma measurement. Since it is desirable to control the gain of each frequency-comb separately, a frequency-comb DBS system was developed with a function to adjust the gain of the scattered signal intensity of each channel separately. A correction processing method was also developed to correct the amplitude ratio and the phase difference between the in-phase and quadrature-phase signals of the scattered signals. As a result, the error in Doppler-shift estimation required to observe vertical velocity and the radial electric field was reduced, which enables more precise measurements.

3D metal powder additive manufacturing phased array antenna for multichannel Doppler reflectometer.

Measuring the time variation of the wavenumber spectrum of turbulence is important for understanding the characteristics of high-temperature plasmas, and the application of a Doppler reflectometer with simultaneous multi-frequency sources is expected. To implement this diagnostic in future fusion devices, the use of a phased array antenna (PAA) that can scan microwave beams without moving antennas is recommended. Since the frequency-scanning waveguide leaky-wave antenna-type PAA has a complex structure, we have investigated its characteristics by modeling it with 3D metal powder additive manufacturing (AM). First, a single waveguide is fabricated to understand the characteristics of 3D AM techniques, and it is clear that there are differences in performance depending on the direction of manufacture and surface treatment. Then, a PAA is made, and it is confirmed that the beam can be emitted in any direction by frequency scanning. The plasma flow velocity can be measured by applying the 3D manufacturing PAA to plasma measurement.

Prevention of hypercholesterolemia and atherosclerosis in the hyperlipidemia- and atherosclerosis-prone Japanese (LAP) quail by taurine supplementation.

The effects of taurine supplementation on the serum cholesterol levels and the progression of atherosclerosis were investigated in the hyperlipidemia- and atherosclerosis-prone Japanese (LAP) quail. The ingestion of a high-cholesterol diet containing 1% cholesterol by LAP quails for 60 days resulted in a marked elevation in serum non-HDL cholesterol and triglyceride, as well as severe aortic lesions with lipid droplets. An immunohistochemical study showed that the lesion consisted of mainly lipid-rich macrophages and T cells. Sixty-day taurine supplementation (1% in drinking tap water) to LAP quails fed high-cholesterol diet containing 1% cholesterol significantly reduced serum non-HDL cholesterol from 4,549 to 2,350 mg/dl. The serum triglyceride level also decreased after taurine supplementation from 703 to 392 mg/dl. Although the HDL cholesterol level significantly decreased due to the high-cholesterol diet, it recovered to the control level fed a regular diet in response to taurine. Bile acid production was stimulated and hepatic cholesterol was reduced by taurine supplementation. A quantitative analysis using aortic cross-sections showed that areas of oil-red O positive lipid accumulation significantly decreased by 74% after taurine supplementation. These results demonstrated the lipid-lowering and anti-atherosclerotic effects of taurine in a diet-induced hyperlipidemic LAP quail model. The prevention of atherosclerosis by taurine is mainly attributed to an improvement in the serum cholesterol and triglyceride levels, which may be related to changes in the hepatic cholesterol metabolism.

Involvement of CPI-17 downregulation in the dysmotility of the colon from dextran sodium sulphate-induced experimental colitis in a mouse model.

The mechanism of gastrointestinal dysmotility in inflammatory bowel disease has not been clarified. In this study, we examined the mechanism involved in the inflamed distal colon isolated from a mouse model of dextran sodium sulphate-induced ulcerative colitis (DSS-treated mouse). Although substance P-induced contraction was not changed, carbachol-induced contraction was reduced in the DSS-treated mouse colon. Pre-incubation with the NO synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA) or the cyclooxygenase inhibitor indomethacin did not reverse the carbachol-induced contraction in the DSS-treated mouse colon. In semi-quantitative reverse transcription-polymerase chain reaction experiments and Western blot analysis, muscarinic M3 receptor expressions were not changed. The Ca2+ -sensitization of contractile elements induced by carbachol with GTP or GTPgammaS was reduced in the beta-escin-permeabilized DSS-treated mouse colon. Although the expression of proteins such as rhoA, ROCK1, ROCK2 or MYPT1 in smooth muscles was not changed, the expression of CPI-17, the functional protein involved in smooth muscle Ca2+ -sensitization, was significantly decreased in the DSS-treated mouse colon. These results suggest that the suppression of carbachol-induced contraction in mice with colitis is attributable at least partially to the increased activity of myosin phosphatase following the downregulation of CPI-17.

An epidemiological survey of echinococcosis in intermediate and definitive hosts in Qinghai Province, China.

In order to understand the epidemiological status of alveolar and cystic echinococcosis in intermediate and definitive hosts in Qinghai Province, China, during the period 2007-2011, we investigated the infection in humans and animals, including yaks, Tibetan sheep, Tibetan dogs, and wild foxes distributed in different counties around the province. Sera from local residents were examined using a rapid serodiagnostic kit to detect specific antibodies against Echinococcus. Seropositive samples were confirmed with B-scan ultrasonography and X-ray examinations. Yaks and Tibetan sheep were checked at slaughterhouses, and cysts and suspicious lesions were collected for analysis. A rapid diagnostic strip was used to detect Echinococcus adults in Tibetan dogs. Positive dogs were dewormed and the parasites collected. Wild foxes were trapped and necropsies performed with particular attention to the intestine. Forty-eight of 735 (6.4%) humans tested were positive and 475 of 854 (55.6%) Tibetan sheep and 85 of 352 (24.15%) yaks were infected with Echinococcus. Across different counties, 214 of 948 (22.57%) Tibetan dogs were positive, and five of 36 (13.9%) wild foxes were infected with Echinococcus. Molecular studies showed that all the infections detected in humans, domestic yaks, and Tibetan sheep were the G1 genotype (E. granulosus), whereas the parasites from Tibetan foxes and Tibetan dogs were E. shiquicus and E. multilocularis, respectively. In conclusion, Echinococcosis is hyperendemic in Qinghai Province in both its intermediate and definitive hosts and the G1 genotype of cystic Echinococcus is the dominant strain.

Photo-induced magnetization and first-principles calculations of a two-dimensional cyanide-bridged Co-W bimetal assembly.

A two-dimensional cyanide-bridged Co-W bimetal assembly, (H5O2+)[Co(4-bromopyridine)2{W(CN)8}], was prepared. A synchrotron radiation (SR) X-ray single-crystal measurement shows that the crystal structure is monoclinic in the P21/c space group. Magnetic and spectroscopic measurements show that this assembly takes Co(S = 0)-WIV(S = 0) in the temperature range of 2-390 K. Such a wide temperature range Co-WIV phase has not been reported so far. First-principles calculations show that the band gap is composed of a WIV valence band and a CoIII conduction band. 785 nm light irradiation causes photo-induced magnetization with a Curie temperature of 27 K and a coercive field of 2000 Oe. The crystal structure of the photo-induced phase was determined to have larger lattice constants in the two-dimensional layer (bc-plane) by 3% compared to the original phase, which is due to the expansion of the distance of Co-N. The photo-induced phase returns to the original phase upon thermal treatment. First-principles calculations, and magnetic, and optical measurements prove that this photomagnetism is caused by the optical charge-transfer-induced spin transition from Co(S = 0)-WIV(S = 0) to Co(S = 3/2)-WV(S = 1/2).

The major low molecular weight apolipoprotein from normal and hyperlipidemia atherosclerosis-prone (LAP) Japanese quail.

The low molecular weight (LMW) apolipoprotein of apo C plays an important role in the metabolism of triglyceride-rich lipoproteins. This study aimed at a characterization of the major LMW apolipoproteins from normal quail strain, and also from LAP (hyperlipidemia atherosclerosis-prone) strain to identify its genetic disorder. The major LMW apoprotein cDNA clone from normal quail comprised of approximately 500 bp, and encoded polypeptide of 78 amino acid residues containing 57 amino acids as a mature apolipoprotein. Although the quail LMW apoprotein showed a low homology to either apo C-I, C-II, or C-III of other animals, it retained a well-developed amphipathic alpha-helix structure. There was no difference in the deduced primary structure of the quail LMW apoprotein between LAP and normal strain. An analysis of the mRNA expression showed that the quail LMW apoprotein was only expressed in the liver of both LAP and normal Japanese quail. No difference was noted in the hepatic expression of the quail LMW apoprotein mRNA between normal and LAP strains with neither normal nor atherogenic dietary conditions. The structure and expression of the major LMW apoprotein thus had no relevance to higher susceptibility of LAP strain to the experimental atherosclerosis.

Production of polyclonal antibody specific for human natriuretic peptide receptor B.

Polyclonal antibody against human natriuretic peptide receptor B (NPR-B) was produced using as immunogen a soluble chimeric protein consisting of the extracellular domain of the receptor fused with Fc portion of human IgG. The antibody was purified with protein A column, and then subjected to an adsorption of anti-Fc antibody using IgG column. The purified antibody recognized human NPR-B but not the related receptor NPR-A. The antibody inhibited C-type natriuretic peptide (CNP)-mediated intracellular cGMP accumulation in a dose-dependent manner. With regard to specific activity for the neutralization, the antibody purified with IgG column was significantly stronger than that before the adsorption step, indicating that the purification of the antibody with IgG column was extremely effective to remove the contaminating anti-Fc antibody from anti-NPR-B antibody. Western blot analysis using the purified antibody revealed that while the native NPR-B exists as an oligomer, the truncated NPR-B lacking most of its cytoplasmic domain is a monomer. This finding suggests that the cytoplasmic region may be involved in the oligomerization of the receptor. The results in this study demonstrate that soluble IgG fusion protein is very effective and useful for generating specific antibodies to the proteins expressed on cell surface.

Humanization of a mouse neutralizing monoclonal antibody against tumor necrosis factor-alpha (TNF-alpha).

An anti-human tumor necrosis factor-alpha (TNF-alpha) monoclonal antibody, designated as 3B10, inhibits the biological activity of human TNF-alpha. In the present study, we constructed humanized version of the antibody by grafting its complementarity-determining regions (CDRs) onto a human antibody, HBS-1. Using a molecular model of mouse 3B10, framework residues affecting the CDR conformation were identified. Thus, these residues were also introduced into the framework together with the CDRs in a stepwise manner, depending on the degree of the possible importance of the residues. As a result, one humanized version (h3B10-9) which possesses nine mouse framework residues showed the same binding activity as that of the chimeric version. This humanized anti-TNF-alpha antibody is expected to be less immunogenic and thus more suitable for possible clinical use.

5,6-Cis-penems: broad-spectrum anti-methicillin-resistant Staphylococcus aureus beta-lactam antibiotics.

5,6-cis-Penem derivatives have been synthesized and evaluated as anti-MRSA antibiotics. The cis-penems 5 and 6 showed potent activities against not only MRSA but also a wide variety of bacteria including beta-lactamase-producing microorganisms. These compounds were designed to have high affinity to the penicillin-binding protein 2a of MRSA and to form stable acyl intermediates with beta-lactamases by blocking the deacylating water molecule.

Epitope mapping of monoclonal antibodies to tumor necrosis factor-alpha by synthetic peptide approach.

A monoclonal antibody (mAb) against human tumor necrosis factor-alpha (TNF-alpha), designated 3B10, neutralizes biological activity of TNF-alpha, while another anti-TNF-alpha mAb 10F10 does not. In Western blot analysis, both mAbs bound to SDS-denatured TNF-alpha, indicating that the epitopes recognized by the mAbs are sequential but not conformational. To map precisely the epitopes of the mAbs, 76 overlapping octapeptides corresponding to an entire sequence of TNF-alpha were synthesized and their abilities to react with the mAbs were examined by enzyme-linked immunosorbent assay (ELISA). 3B10 bound to only one peptide at position 81-88 of TNF-alpha, SRIAVSYQ, whereas 10F10 was reactive with three overlapping peptides, ANALLANG (33-40), ALLANGVE (35-42), and LANGVELR (37-44). These results demonstrate that the 81-88 and 37-40 regions are important for the recognition of TNF-alpha by 3B10 and by 10F10, respectively. In solid-phase ELISA, 3B10 inhibited the binding of TNF-alpha to soluble TNF receptors, sTNF-RI and sTNF-RII. In contrast, 10F10 exerted little effect on the binding. TNF-alpha was detected by sandwich-type ELISA where 3B10 alone was used for both capture and detection, suggesting that 3B10 did not interfere with the trimer formation of TNF-alpha. The results obtained in this study suggest that the 81-88 region of TNF-alpha may participate in the receptor binding and that 3B10 neutralizes the activities of TNF-alpha by blocking the region.

Production and characterization of monoclonal antibodies against human natriuretic peptide receptor-A or -B.

Monoclonal antibodies (mAbs) against human natriuretic peptide receptor-A (NPR-A) or NPR-B were produced using NPR-expressing Chinese hamster ovary (CHO) cells and soluble chimeric NPRs consisting of the extracellular domain of each receptor fused to Fc region of human IgG. Three anti-NPR-A mAbs, designated as A144, A397 and A416, bound to human NPR-A but not to NPR-B, while an anti-NPR-B mAb B136 reacted with human NPR-B but not with NPR-A. Competition analysis with the anti-NPR-A mAbs revealed that two mAbs, A144 and A416, recognize an identical or the adjacent site of the receptor and that A397 is directed against another epitope. No anti-NPR-A mAb affected binding of atrial natriuretic peptide (ANP) to NPR-A, while the anti-NPR-B mAb B136 inhibited binding of C-type natriuretic peptide (CNP) to NPR-B. Inhibition of the ligand-binding by B136 is specific in that the mAb showed no effect on the binding of ANP to NPR-A. B136 also blocked CNP-mediated intracellular cGMP accumulation in NPR-B-expressing cells. These results suggest that the region recognized by B136 may be related to the ligand-binding region of NPR-B. NPR-A- and NPR-B-expressing cells were selectively detected by immunostaining using the mAbs. These findings demonstrate that the mAbs will be useful to elucidate the role of the natriuretic peptides and their receptors in normal and disease states in humans [correction of human].

Fucoidin, a potent inhibitor of L-selectin function, reduces contact hypersensitivity reaction in mice.

In order to investigate the role of L-selectin (CD62L) in delayed-type hypersensitivity (DTH) reaction, effect of fucoidin, a potent inhibitor of CD62L function, was examined in a model of mouse contact hypersensitivity reaction. Intravenous injection of fucoidin to sensitized mice just before hapten challenge resulted in a significant and dose-dependent reduction of the ear swelling in contact hypersensitivity. The ear swelling caused by the hapten challenge was also inhibited when fucoidin was administered at the sensitization phase. Histological analyses of the ear sections revealed that the fucoidin-induced suppression of contact hypersensitivity reflected a marked inhibition of the ear edema and the leukocyte infiltration. The activity of fucoidin was specific in that its related saccharides exerted little effect on the reaction. These results suggest that CD62L may play an important role in both afferent and efferent phases of cutaneous DTH reaction. Since DTH response is one of the most significant features of several chronic inflammatory diseases, our data also show that blocking of CD62L function may be beneficial for the treatment of these diseases in humans.

Construction and expression of a mouse-human chimeric antibody against human tumor necrosis factor-alpha.

A mouse anti-human tumor necrosis factor-alpha (TNF-alpha) monoclonal antibody (MoAb), designated as 3B10, has previously been produced and characterized by our laboratory. We report here the construction and the expression of mouse-human chimeric antibody derived from the MoAb. cDNAs encoding variable regions of heavy and light chains were prepared from 3B10 cells by polymerase chain reaction, and introduced to mammalian expression vectors containing cDNA for human gamma1 and kappa constant regions, respectively. Cotransfection of the vectors into CHO cells resulted in production of antibody reacting with human TNF-alpha. In SDS-PAGE analysis, the chimeric antibody, c3B10, migrated at 170 kDa under a nonreducing condition, whereas two bands with 58 and 28 kDa appeared following treatment with 2-mercaptoethanol. Both c3B10 and mouse 3B10 neutralized the cytotoxic activity of human TNF-alpha to the same level, indicating that c3B10 holds the binding activity of its original MoAb. These findings suggest that the introduced genes for chimeric heavy and light chains are transcribed and translated to produce the chimeric heavy and light chain peptides, and that the peptides are assembled to form native IgG molecule. The chimeric anti-TNF-alpha antibody described in this study is expected to be less immunogenic and thus more suitable for possible clinical use.

Epitope mapping of monoclonal antibodies against N-domain of carcinoembryonic antigen.

Monoclonal antibodies (MoAbs) against N-domain of carcinoembryonic antigen (CEA), C249, K348, K1338, and K1444, that inhibit CEA-mediated cell adhesion, did not crossreact with nonspecific cross-reacting antigen (NCA). To determine amino acid sequences involved in the adhesion, epitopes of the MoAbs were mapped with recombinant NCAs carrying CEA-NCA chimeric N-domain. The data showed that the epitopes of C249, K1338, K1444 are located within the regions 1-32, 1-32, and 33-59 of CEA, respectively, and that two discrete regions 1-32 and 60-93 may be related to the epitope of K348. Comparison of amino acid sequences between CEA and NCA suggested that four residues (21, 27-29), eight residues (21, 27-29, 66, 78, 79, 89), and three residues (43, 44, 46) are important for recognition by C249 (or K1338), K348, and K1444, respectively. These residues seem to participate in the cell adhesion mediated by CEA.

Spasmolytic effect of cadmium and cadmium uptake in aorta.

The relationship between inhibition of tension by Cd2+ in aorta and the kinetics of cadmium uptake and efflux was studied. Cd2+ (0.01-0.5 mM) inhibited the contraction of aorta to high-K+ (30 mM) in a dose-dependent manner. The high-K+ -induced tension completely returned to control values after 60 min washing with a solution containing 5 mM disodium edetate (EDTA) or 5 mM cysteine, following a treatment with 0.5 mM Cd2+ for 30 min; after washing with normal medium only 15% of the control response returned. Cadmium uptake increased with an increase of the Cd2+ concentration (0.01-0.5 mM). Aortae preincubated with 0.5 mM Cd2+ for 60 min were washed subsequently with a medium containing 5 mM EDTA or 5 mM cysteine. About 5% of the original tissue cadmium was retained after washing with EDTA or cysteine. It is suggested that Cd2+ binds chiefly to the surface membrane of aorta. It seems possible that the quantity bound is correlated with the degree of inhibition of tension.

Juberg-Marsidi syndrome: report of an additional case.

We report on a 2-year-old boy with Juberg-Marsidi syndrome. He has mental retardation, short stature, micropenis, cryptorchidism, and minor facial abnormalities. His Leydig cells responded to the administration of human chorionic gonadotropin and there were positive responses of LH and FSH to the administration of LH-RH. He showed normal weight gain and head circumference which have not been described previously. The association of Juberg-Marsidi syndrome with HbH disease was ruled out in the propositus.

Present state of fungal infections in autopsy cases in Japan.

In order to clarify the present state of opportunistic fungal infections increasing in incidence in autopsy cases, all autopsy cases from 1966 to 1975 reported in the Annual of Pathological Autopsy Cases in Japan were reviewed. Of the total 233,130 autopsy cases, mycoses were present in 4,340 (1.86%). The incidence of mycoses has strikingly increased during the recent five-year period. In Japan, the mycoses most frequently occurring in autopsy cases were candidiasis (32.28%), aspergillosis (23.08%), cryptococcosis (9.63%), and mucormycosis (2.90%). These occurred more frequently in younger persons and were most commonly secondary and deep-seated infections (95.78%). Among the primary diseases associated with mycoses, aplastic anemia (14.36%), leukemia (9.89%), malignant lymphoma (5.73%), multiple myeloma (4.68%), and systemic lupus erythematosus (4.62%) were most frequent. The incidence of the primary diseases associated with mycoses is increasing extraordinarily, and this seems to be strongly related to the modern therapy of using high doses of anticancer or immunosuppressive agents.

Development of regulation of melatonin release in pineal cells in chick embryo.

Melatonin release in a pineal cell culture from 13- and 14-day-old chick embryos increased during the dark phase and decreased during the light phase of a 12 h light:12 h dark cycle. When the light-dark cycle was reversed, the pattern of melatonin release in the culture also reversed. 8-Bromo cyclic-AMP stimulated melatonin release in both the light and dark phases. However, no rhythm of melatonin release was detected under constant dark (DD) conditions in a cell culture from 14-day-old chick embryos. In 18-day-old chick embryos, the pineal cell culture expressed a circadian rhythm of melatonin release under DD conditions. These results indicate that mechanisms regulating melatonin synthesis in the avian pineal gland are established during embryonic life.