Potential Spatial Distribution of the Newly Introduced Long-horned Tick, Haemaphysalis longicornis in North America.

The North American distributional potential of the recently invaded tick, Haemaphysalis longicornis, was estimated using occurrence data from its geographic range in other parts of the world and relevant climatic data sets. Several hundred candidate models were built using a correlative maximum entropy approach, and best-fitting models were selected based on statistical significance, predictive ability, and complexity. The median of the best-fitting models indicates a broad potential distribution for this species, but restricted to three sectors-the southeastern United States, the Pacific Northwest, and central and southern Mexico.

Rapid Bactericidal Action of Propolis against Porphyromonas gingivalis.

Propolis, a resinous substance produced by bees, is used as a folk medicine for treatment of periodontal diseases. However, its mode of the action and the compounds responsible for its activities remain obscure. In the present study, we comprehensively investigated the antibacterial activities of ethanol-extracted propolis (EEP) and EEP-derived compounds toward Porphyromonas gingivalis, a keystone pathogen for periodontal diseases. Broth microdilution and agar dilution assays were used to determine the minimum inhibitory concentrations of EEP against a range of oral bacterial species, of which P. gingivalis showed a higher level of sensitivity than oral commensals such as streptococci. Its antibacterial activity toward P. gingivalis was maintained even after extensive heat treatment, demonstrating a high level of thermostability. EEP also induced death of P. gingivalis cells by increasing membrane permeability within 30 min. Spatiotemporal analysis based on high-speed atomic force microscopy revealed that EEP immediately triggered development of aberrant membrane blebs, followed by bleb fusion events on the bacterial surface. Furthermore, we isolated artepillin C, baccharin, and ursolic acid from EEP as antibacterial compounds against P. gingivalis. Of those, artepillin C and baccharin showed bacteriostatic activities with membrane blebbing, while ursolic acid showed bactericidal activity with membrane rupture. In particular, ursolic acid demonstrated a greater ability to affect bacterial membrane potential with increased membrane permeability, probably because of its highly lipophilic nature as compared with other compounds. Taken together, these findings provide mechanistic insight into the antibacterial activities of EEP and its exquisite membrane-targeting antibacterial compounds and imply the applicability of narrow-spectrum therapeutics with EEP for treatment of periodontitis. In addition, the advanced technology utilized in the present study to visualize the nanometer-scale dynamics of microorganisms will contribute to expanding our understanding of the activities of antimicrobials and the mechanism of drug resistance in bacteria.

Inhibition of gingipains and Porphyromonas gingivalis growth and biofilm formation by prenyl flavonoids.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis is considered a major pathogen of chronic periodontitis, which also may be implicated with systemic diseases such as atherosclerosis. Secreted cysteine proteases, gingipains Rgp and Kgp, are essential for P. gingivalis virulence. Some polyphenols and flavonoids are known to inhibit gingipain activity and interfere with biofilm formation by P. gingivalis. Many bioactive compounds have been isolated from Epimedium species, but availability of these compounds on gingipains and P. gingivalis is still unclear. Therefore, the aim of this study was to evaluate natural products from medical plants to develop a new therapeutic agent against periodontal disease. MATERIAL AND METHODS: Prenylated flavonoids were isolated from Epimedium species plant using column chromatographies. The inhibitory effect of the prenylated flavonoids against protease activity of gingipains were examined using purified gingipains and fluorogenic substrates. Anti-P. gingivalis activity was evaluated to analyze planktonic growth and biofilm formation in brain heart infusion medium in the presence of the prenylated flavonoids. RESULTS: We isolated 17 prenylated flavonoids (Limonianin, Epimedokoreanin B, etc.) from Epimedium species. We found that some prenylated flavonoids inhibited gingipain activity in a non-competitive manner with Ki values at µm order. The prenylated flavonoids also hindered growth and biofilm formation of P. gingivalis, in a manner independent of gingipain inhibition by the compounds. CONCLUSION: The results indicated an inhibitory effect of the prenylated flavonoids against P. gingivalis and would provide useful information for future development of periodontitis treatment that suppresses gingipains, P. gingivalis growth and biofilm formation.

Meteorological factors affecting scrub typhus occurrence: a retrospective study of Yamagata Prefecture, Japan, 1984-2014.

Climate change, by its influence on the ecology of vectors might affect the occurrence of vector-borne diseases. This study examines the effects of meteorological factors in Japan on the occurrence of scrub typhus, a mite-borne zoonosis caused by Orientia tsutsugamushi. Using negative binomial regression, we analysed the relationships between meteorological factors (including temperature, rainfall, snowfall) and spring-early summer cases of scrub typhus in Yamagata Prefecture, Japan, during 1984-2014. The average temperature in July and August of the previous year, cumulative rainfall in September of the previous year, snowfall throughout the winter, and maximum depth of snow cover in January and February were positively correlated with the number of scrub typhus cases. By contrast, cumulative rainfall in July of the previous year showed a negative relationship to the number of cases. These associations can be explained by the life-cycle of Leptotrombidium pallidum, a predominant vector of spring-early summer cases of scrub typhus in northern Japan. Our findings show that several meteorological factors are useful to estimate the number of scrub typhus cases before the endemic period. They are applicable to establish an early warning system for scrub typhus in northern Japan.

The timing of worm exclusion in dogs repeatedly infected with the cestode Echinococcus multilocularis.

Experimental Echinococcus multilocularis infection and deworming was repeated three or five times in nine dogs at various re-infection schedules. The mean number of worms decreased more than 91% in dogs with repeated infection, compared to first infection controls (n= 6). The copro-antigen assay and the egg count in the faeces suggested that the worm burden gradually decreased each time the dogs were re-infected. To examine whether such worm exclusion was a non-specific response, five dogs were sequentially infected with the parasite four times and subsequently fed freely for 6 months. Even after the 6-month interval, the five dogs that were infected five times with the parasite were still able largely to exclude the adult worms. The results suggested that the ability of worm exclusion in dogs that developed a resistance did not become rapidly extinct. Observation of the condition of faeces and the excretion of hooks in the faeces of repeatedly infected dogs revealed that the exclusion of worms started at the first week after the re-infection, and it continued during the patent period. Serum antibodies specific to the parasite antigen increased gradually until the third infection and significantly decreased during the 6-month interval. There was little enhancement of serum antibodies after the fifth infection in most dogs, although no clear correlation was observed between the antibody response and the worm burden. These findings suggested the possibility of developing a vaccine.

Molecular screening for Rickettsia, Anaplasmataceae and Coxiella burnetii in Rhipicephalus sanguineus ticks from Malaysia.

A total of 44 Rhipicephalus sanguineus ticks collected from 23 dogs from Malaysia were screened for Rickettsia, Anaplasmataceae and Coxiella burnetii. Coxiella burnetii was detected in 59% (26/44) of ticks however Rickettsia and Anaplasmataceae were not detected in any of the ticks. In order to genotype the strains of C. burnetii, multispacer sequence typing (MST) was carried out using three different spacers. One of the spacers; Cox2 successfully amplified a fragment for which the full length sequence of 397 bp was obtained. The sequenced product revealed only a single nucleotide difference with the Cox2.3 type sequence.

Inhibiting effects of Streptococcus salivarius on competence-stimulating peptide-dependent biofilm formation by Streptococcus mutans.

INTRODUCTION: The effects of Streptococcus salivarius on the competence-stimulating peptide (CSP)-dependent biofilm formation by Streptococcus mutans were investigated. METHODS: Biofilms were grown on 96-well microtiter plates coated with salivary components in tryptic soy broth without dextrose supplemented with 0.25% sucrose. Biofilm formations were stained using safranin and quantification of stained biofilms was performed by measuring absorbance at 492 nm. RESULTS: S. mutans formed substantial biofilms, whereas biofilms of S. salivarius were formed poorly in the medium conditions used. Furthermore, in combination cultures, S. salivarius strongly inhibited biofilm formation when cultured with S. mutans. This inhibition occurred in the early phase of biofilm formation and was dependent on inactivation of the CSP of S. mutans, which is associated with competence, biofilm formation, and antimicrobial activity of the bacterium, and is induced by expression of the comC gene. Comparisons between the S. mutans clinical strains FSC-3 and FSC-3DeltaglrA in separate dual-species cultures with S. salivarius indicated that the presence of the bacitracin transport ATP-binding protein gene glrA caused susceptibility to inhibition of S. mutans biofilm formation by S. salivarius, and was also associated with the regulation of CSP production by com gene-dependent quorum sensing systems. CONCLUSION: It is considered that regulation of CSP by glrA in S. mutans and CSP inactivation by S. salivarius are important functions for cell-to-cell communication between biofilm bacteria and oral streptococci such as S. salivarius. Our results provide useful information for understanding the ecosystem of oral streptococcal biofilms, as well as the competition between and coexistence of multiple species in the oral cavity.

Evaluation of plasma C-terminal atrial natriuretic peptide in healthy cats and cats with heart disease.

BACKGROUND: The clinical implications of evaluating C-terminal atrial natriuretic peptide (ANP) concentration in cats are still controversial. HYPOTHESIS: The objective of this study was to investigate the relationship between plasma C-terminal ANP concentration and left atrial pressure (LAP) in healthy cats with volume overload (study 1), and to compare plasma C-terminal ANP in normal cats and cats with cardiomyopathy (study 2). ANIMALS: Five healthy adult cats were used in study 1, and clinically healthy cats (n=8) and cats with cardiomyopathy (n=14) were used in study 2. METHODS: In study 1, cats were anesthetized and given acetated Ringer's solution (100 mL/kg/h for 60 minute) via the cephalic vein. Hemodynamic measurements and blood samples, collected from the jugular vein, were performed at 10-min intervals. In study 2, blood samples from normal cats and cats with cardiomyopathy were collected from the cephalic vein. The plasma C-terminal ANP concentration was determined by radioimmunoassay for human alpha-ANP. RESULTS: In study 1, volume overload significantly increased the C-terminal ANP concentration and LAP from baseline. The C-terminal ANP concentration was strongly correlated with the mean LAP. In study 2, age, E wave velocity, and the ratios of the left atrium to aorta were significantly higher in the cats with cardiomyopathy compared with the normal cats. The C-terminal ANP concentration was significantly higher in the cats with cardiomyopathy compared with the normal cats. CONCLUSIONS AND CLINICAL IMPORTANCE: Our results suggest that the measurement of plasma C-terminal ANP in cats may provide additional information for the diagnosis of heart disease.

Laboratory exposure to 17beta-estradiol fails to induce vitellogenin and estrogen receptor gene expression in the marine invertebrate Mytilus edulis.

To investigate the effect of estrogenic compounds on the marine mussel Mytilus edulis, an assay was developed to measure the expression of two vertebrate estrogen responsive genes-estrogen receptor (ER) and vitellogenin (VTG) genes. Expression was measured in M. edulis gonads following a 10-day exposure to 200 ng/l 17beta-estradiol (estradiol). The concentrations of esterified estradiol in mussel tissue increased 15-fold in a time-dependent manner-confirming uptake of the compound by the mussels, however there was no significant increase of free estradiol in mussel tissues during the exposure period. The ER and VTG mRNA levels in the gonads of both sexes were measured at days 1-3, 5, and 10 in control and exposed mussels. However, no significant change in the expression of either the ER or VTG genes was recorded at any of the sampled time points. The results suggest that either a regulatory mechanism exists in a mussel that is able to maintain constant levels of free estradiol by converting the excess estradiol into esterified products which may have reduced affinity for the estrogen receptor, or alternatively, that the ER and VTG genes are unresponsive to estrogens in these organisms. The significance of these findings in terms of the utility of ER and VTG as biomarkers of endocrine disruption in bivalve species is discussed.

Human T-cell responses to oral streptococci in human PBMC-NOD/SCID mice.

We investigated cellular and humoral immune responses to oral biofilm bacteria, including Streptococcus mutans, Streptococcus anginosus, Streptococcus sobrinus, and Streptococcus sanguinis, in NOD/SCID mice immunized with human peripheral blood mononuclear cells (hu-PBMC-NOD/SCID mice) to explore the pathogenicity of each of those organisms in dental and oral inflammatory diseases. hu-PBMC-NOD/SCID mice were immunized by intraperitoneal injections with the whole cells of the streptococci once a week for 3 weeks. FACS analyses were used to determine the percentages of various hu-T cell types, as well as intracellular cytokine production of interleukin-4 and interferon-gamma. Serum IgG and IgM antibody levels in response to the streptococci were also determined by enzyme-linked immunosorbent assay. S. anginosus induced a significant amount of the proinflammatory cytokine interferon-gamma in CD4(+) and CD8(+) T cells in comparison with the other streptococci. However, there was no significant differences between the streptococci in interleukin-4 production by CD4(+) and CD8(+) T cells after inoculation. Further, S. mutans significantly induced human anti-S. mutans IgG, IgG(1), IgG(2), and IgM antibodies in comparison with the other organisms. In conclusion, S. anginosus up-regulated Th1 and Tc1 cells, and S. mutans led to increasing levels of their antibodies, which was associated with the induction of Th2 cells. These results may contribute to a better understanding of human lymphocyte interactions to biofilm bacteria, along with their impact on dental and mucosal inflammatory diseases, as well as endocarditis.

Factors affecting quality control of [18F]FDG injection: bacterial endotoxins test, aluminum ions test and HPLC analysis for FDG and CIDG.

High concentrations of citrates and phosphates which are often used in the manufacturing of [18F] fluro-D-glucose (FDG) preparations and wide deviation in the pH value from the neutral level often disturb the detection of endotoxins and aluminum ions using the turbidimetric and aluminum ion paper test method. The column temperature was found to be a major factor influencing the sensitivity of ClDG detection with the HPAEC/PAD method.

Stability of 11C-labeled PET radiopharmaceuticals.

The stability of widely used 11C-radiopharmaceuticals was investigated. The purity of many 11C-radiopharmaceuticals was found to be low even at the end of synthesis and to lower with time by radiolysis. Examinations of the effects of radical scavengers showed that the radiopharmaceuticals were classified into four groups: Group 1 showing excellent stability; Group 2 showing suppressed radiolysis in the presence of a selective scavenger of hydroxyl radicals; Group 3 showing suppression in the presence of a selective scavenger of hydrated electrons; Group 4 showing suppression in the presence of both scavengers. Using appropriate additives according to these classes, 11C-radiopharmaceuticals were prepared with excellent radiochemical purity even with high levels of radioactivity and specific activity.

Role of peptide antigen for induction of inhibitory antibodies to Streptococcus mutans in human oral cavity.

The alanine-rich repeating region (A-region) in the surface protein antigen (PAc) of Streptococcus mutans has received much attention as an antigenic component for vaccines against dental caries. The PAc (residue 361-386) peptide in the A-region possesses a multiple binding motif (L- -V-K- -A) to various HLA-DR molecules and a B-cell core epitope (- Y- - -L- -Y- - - -) that recognizes the inhibiting antibody to S. mutans. In the present study, we investigated the immunogenicity of the PAc (361-386) peptide in humans and regulators of induction of the anti-PAc (361-386) peptide IgA antibody (aPPA) in saliva. The PAc (361-386) peptide was confirmed as an ideal peptide antigen for induction of the inhibiting antibody to S. mutans in 151 healthy human subjects (36.6 +/- 12.6 years old) by quantitative analyses of oral bacteria and ELISA, as the aPPA titre in human saliva decreased significantly in an age-dependent manner. Homozygous DRB1*0405 and 1502, and heterozygous DRB1*0405/1502 showed a negative association with production of aPPA and tended to reduce the number of total streptococci in saliva. In contrast, the DRB1*1501 allele was significantly correlated with a high level of induction of the antibodies, and also tended to reduce lactobacilli and mutans streptococci. Further, peptide immunogenicity was confirmed in NOD-SCID mice grafted with human peripheral blood mononuclear cells. Our results indicate that the interplay between regulators such as age, DRB1 genotype, cytokines, and peptide immunogenicity may provide a potential means for developing a vaccine useful for the prevention of dental caries as well as their diagnosis.

Sicca syndrome in patients infected with human immunodeficiency virus-1.

Abstract We investigated human immunodeficiency virus-1 (HIV-1)-associated sicca syndrome. The average saliva production in HIV-infected patients was 15.9 ± 6.3 ml, and the average tear production was 9.8 ± 4.5 mm. In particular, 6 patients (42.9%) showed a significant decrease in tear production. This sicca syndrome mimicked autoimmune Sjögren's syndrome (SS) because of the presence of dry eye, dry mouth, hyperamylasemia, and hypergammaglobulinemia; however, no antinuclear antibodies, anti-SS-A, or anti-SS-B were detected in sera from HIV-1-infected patients. In addition, no relationship was observed between saliva and tear production and CD4, HIV-RNA. Hepatitis C virus (HCV) and human T-lymphotrophic virus (HTLV-1) are considered to be possible causative agents of SS. However, coinfection with HCV did not affect the decrease of saliva and tear production, and only one patient was coinfected with HTLV-1. Epstein-Barr virus (EBV) and cytomegalovirus (CMV) are also potential causative agents of SS, and they are sometimes detected in the saliva of HIV-1-infected patients. However, the detection of EBV and CMV in the saliva was not related to the decrease in saliva production. Furthermore, HIV therapy (highly active anti-retroviral therapy; HAART) did not affect the state of sicca syndrome. The pathogenesis of sicca syndrome in HIV-1-infected patients is not clear, but we did find some infiltration of CD8 lymphocytes in salivary gland biopsy. Usually, CD8 lymphocytosis is found in peripheral blood in HIV-infected patients. Diffuse infiltrative lymphocytosis syndrome by predominant CD8 lymphocytes is occasionally found in HIV-infected patients. Such CD8 infiltration may induce the destruction of both the salivary and lacrimal glands.

Pathophysiological roles of endogenous endothelin-1 in dogs with chronic heart failure produced by rapid right ventricular pacing.

This study was designed to analyze the pathophysiological role of the endogenous endothelin (ET) system and the therapeutic approach to congestive heart failure (CHF) with ET(A)/ET(B) receptor antagonists in a canine CHF model. After 3 weeks of rapid right ventricular pacing (240 beats/min), concentrations of immunoreactive ET-1 in dogs increased approximately 2-fold in plasma and in the left and right ventricles but not in the lung. There were no meaningful changes in the density and affinity of total ET receptors, or in the ratio of ET(A) to ET(B) receptors. To clarify the functional role of endogenous ET, we examined the effects of acute injection of J-104132 (1 and 3 mg/kg i.v.), an ET(A)/ET(B) receptor antagonist, on cardiovascular and renal function in dogs with CHF. Compared with vehicle, J-104132 at both doses significantly decreased pulmonary artery pressure (PAP), pulmonary capillary wedge pressure (PCWP), and mean arterial pressure (MAP), and increased cardiac output (CO) and renal blood flow. J-104132 had no effects on heart rate and cardiac contractility. In addition, we examined whether J-104132 has an additive effect in the presence of enalaprilat. J-104132 (1 mg/kg i.v.) administered after enalaprilat (0.05 mg/kg i.v.) induced further decreases in MAP, PCWP and PAP, and further increases in CO, resulting in further decreases in total peripheral resistance. These results indicate that the endogenous ET system is exaggerated in CHF and has a detrimental effect on cardiac function. Therefore, J-104132 given alone or as combination therapy may play a beneficial role in the treatment of CHF in humans.

4-(6,7-Dihydro-5,8-dioxothiazolo[4,5-g]phthalazin-2-yl)benzoic acid N-hydroxysuccinimide ester as a highly sensitive chemiluminescence derivatization reagent for amines in liquid chromatography.

4-(6,7-Dihydro-5,8-dioxothiazolo[4,5-g]phthalazin-2-yl)benzoic acid N-hydroxysuccinimide ester was synthesized as a highly sensitive and selective chemiluminescence derivatization reagent for primary and secondary amines in liquid chromatography. Methyl-n-octylamine, n-nonylamine and n-decylamine were used as model compounds to optimize the derivatization, separation and chemiluminescence reaction conditions. This reagent reacts selectively with amines in the presence of triethylamine to give the highly chemiluminescent derivatives, which produce chemiluminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in an alkaline medium. The chemiluminescent derivatives of the three amines can be separated within 20 min by reversed-phase liquid chromatography with isocratic elution, followed by chemiluminescence detection. The detection limits (signal-to-noise ratio=3) for primary and secondary amines are at sub-fmol levels for a 20-microl injection. Furthermore, this method was applicable to the determination of amantadine in human plasma.

Antihypertensive effects of a mixed endothelin-A- and -B-receptor antagonist, J-104132, were augmented in the presence of an AT1 -receptor antagonist, MK-954.

The single oral administration of a mixed endothelin-A-and -B- (ETA/ETB) receptor antagonist, J-104132 (L-753,037) decreased the blood pressure in conscious spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP) and Dahl salt-sensitive hypertensive rats fed high-salt diet (DS-H) at doses of 3 and 10 mg/kg, with a duration of approximately 24 h. The magnitude of the antihypertensive effects was greater in DS-H than in SHR and SHRSP Preproendothelin-1 mRNA expression in the kidney and aorta was greater (about twofold) in DS-H than that in nonnotensive Dahl salt-resistant rats fed high-salt diet (DR-H), while there was no difference in preproendothelin-1 mRNA expression between SHR and Wistar Kyoto rats (WKY). An AT1-receptor antagonist, MK-954 (Losartan), also attenuated hypertension in SHR and SHRSP at oral doses of 3 and 10 mg/kg, but bad no effect in DS-H. The concomitant treatment with MK-954 and J-104132 showed additive antihypertensive effects in SHR and SHRSP. In DS-H, MK-954 potentiated the antihypertensive effects of J-104132. From these results, we suggest that: (1) the contribution of endothelin (ET) to the maintenance of hypertension is greater in salt-sensitive hypertensive models than in SHR and SHRSP; (2) the antihypertensive efficacy of ETA/ETB blockade is augmented by AT1-receptor blockade; (3) ET blockers alone or in combination with AT1 antagonists could be useful in the treatment of hypertension for patients who do not respond adequately to renin-angiotensin system inhibitors alone.

5-Amino-4-sulfanylphthalhydrazide as a chemiluminescence derivatization reagent for aromatic aldehydes in liquid chromatography.

5-Amino-4-sulfanylphthalhydrazide (ASPH) was synthesized as a chemiluminescence derivatization reagent for aromatic aldehydes in liquid chromatography (LC). Benzaldehyde, 4-tolualdehyde, 4-chlorobenzaldehyde, 4-formylbenzoic acid, 4-hydroxybenzaldehyde and vanillin were used as model compounds to optimize the derivatization conditions. This reagent, ASPH, reacts selectively with aromatic aldehydes in the presence of sodium sulfite and disodium hydrogenphoshite in acidic medium at 100 degrees C to give the corresponding highly chemiluminescent 2-arylbenzothiazole derivatives. The resulting derivatives generated intense chemiluminescence by reaction with hydrogen peroxide and potassium hexacyanoferrate(III) in alkaline solution. The ASPH derivatives of aromatic aldehydes were separated by reversed-phase liquid chromatography with isocratic elution, and detected chemiluminometrically after mixing with oxidizing agents. The detection limits (signal-to-noise ratio = 3) for aromatic aldehydes are in the range 0.2-4.0 fmol for a 20-microl injection volume. Currently, the method is not effective for aliphatic aldehydes because of interfering LC peaks.