Two-dimensional temperature-responsive chromatography using a poly(N-isopropylacrylamide) brush-modified stationary phase for effective therapeutic drug monitoring.

Therapeutic drug monitoring (TDM) is an effective pharmacological approach for controlling drug concentration in a patient's serum. Herein, a new two-dimensional chromatography system was developed using two poly(N-isopropylacrylamide) (PNIPAAm)-modified bead-packed columns for effective and safe drug monitoring. PNIPAAm-modified silica beads were prepared as packing materials using atom transfer radical polymerization of NIPAAm. The increase in the retention times of the drugs requiring TDM with increasing temperature, was attributed to enhanced hydrophobic interactions at elevated temperatures. The drugs and serum proteins were separated on the prepared column at 40 °C using an all-aqueous mobile phase. Differences in the hydrophobic interactions accounted for the elution of the serum proteins and drugs at short and long retention times, respectively, and a primary column was employed to separate the serum proteins and drugs. After eluting the serum proteins from the column, the drug was introduced into the secondary column, leading to a peak of its purified form and enabling determination of the drug concentration. Two-dimensional temperature-responsive chromatography can benefit TDM by allowing the drug concentration in the serum to be measured in all-aqueous mobile phases without sample preparation.

Temperature responsive chromatography for therapeutic drug monitoring with an aqueous mobile phase.

Therapeutic drug monitoring is a key technology for effective pharmacological treatment. In the present study, a temperature-responsive chromatography column was developed for safe and simple therapeutic drug monitoring without the use of organic solvents. Poly(N-isopropylacrylamide) (PNIPAAm) hydrogel-modified silica beads were prepared via a condensation reaction and radical polymerization. The temperature-dependent elution behavior of the drugs was observed using a PNIPAAm-modified silica-bead packed column and an all-aqueous mobile phase. Sharp peaks with reproducible retention times were observed at temperatures of 30 °C or 40 °C because the PNIPAAm hydrogel on the silica beads shrinks at these temperatures, limiting drug diffusion into the PNIPAAm hydrogel layer. The elution behavior of the sample from the prepared column was examined using a mixture of serum and model drugs. The serum and drugs were separated on the column at 30 °C or 40 °C, and the concentration of the eluted drug was obtained using the calibration curve. The results show that the prepared chromatography column would be useful for therapeutic drug monitoring because the drug concentration in serum can be measured without using organic solvents in the mobile phase and without any need for sample preparation.

Temperature-responsive spin column for sample preparation using an all-aqueous eluent.

We present a temperature-responsive spin column using an all-aqueous eluent. The method is intended as a simple sample preparation method for protein removal from serum, which is required for serum drug analysis. As packing materials for the spin column, we prepared two types of silica beads via surface-initiated radical polymerization. The large beads (diameter, 40-63 µm) were grafted with a temperature-responsive cationic copolymer, poly(N-isopropylacrylamide-co-N,N-dimethylaminopropyl acrylamide-co-n-butyl methacrylate) (P(NIPAAm-co-DMAPAAm-co-BMA)), and the small beads (diameter, 5 µm) were grafted with a temperature-responsive hydrophobic copolymer, P(NIPAAm-co-BMA). The beads were packed into the spin column as a double layer: P(NIPAAm-co-BMA) silica beads on the bottom and P(NIPAAm-co-DMAPAAm-co-BMA) silica beads on the top. The sample purification efficacy of the prepared spin column was evaluated on a model sample analyte (the antifungal drug voriconazole mixed with blood serum proteins). At 40 °C, the serum proteins and voriconazole were adsorbed on the prepared spin column via hydrophobic and electrostatic interactions. When the temperature was decreased to 4 °C, the adsorbed voriconazole was eluted from the column with the pure water eluent, while the serum proteins remained in the column. This temperature-responsive spin column realizes sample preparation simply by changing the temperature.

Hierarchical Spatial Concept Formation Based on Multimodal Information for Human Support Robots.

In this paper, we propose a hierarchical spatial concept formation method based on the Bayesian generative model with multimodal information e.g., vision, position and word information. Since humans have the ability to select an appropriate level of abstraction according to the situation and describe their position linguistically, e.g., "I am in my home" and "I am in front of the table," a hierarchical structure of spatial concepts is necessary in order for human support robots to communicate smoothly with users. The proposed method enables a robot to form hierarchical spatial concepts by categorizing multimodal information using hierarchical multimodal latent Dirichlet allocation (hMLDA). Object recognition results using convolutional neural network (CNN), hierarchical k-means clustering result of self-position estimated by Monte Carlo localization (MCL), and a set of location names are used, respectively, as features in vision, position, and word information. Experiments in forming hierarchical spatial concepts and evaluating how the proposed method can predict unobserved location names and position categories are performed using a robot in the real world. Results verify that, relative to comparable baseline methods, the proposed method enables a robot to predict location names and position categories closer to predictions made by humans. As an application example of the proposed method in a home environment, a demonstration in which a human support robot moves to an instructed place based on human speech instructions is achieved based on the formed hierarchical spatial concept.

Acute motor-sensory axonal Guillain-Barré syndrome with unilateral facial nerve paralysis after rotavirus gastroenteritis in a 2-year-old boy.

Guillain-Barré syndrome (GBS) is well known as presenting with acute immune-mediated polyneuropathies, with strong associations with antecedent infections. Several variant forms of GBS have been described, including acute inflammatory demyelinating polyneuropathy, acute motor axonal neuropathy, acute motor-sensory axonal neuropathy, and sensory GBS. We present a rare case of 2-year-old boy with acute motor and sensory polyneuropathy and left-sided facial nerve paralysis after rotavirus infection. He received immunoglobulin i.v. with subsequent satisfactory recovery.

Critical tooth number without subjective dysphagia.

AIM: To assess the critical tooth number (CTN) required for prevention of subjective dysphagia caused by oral impairments and to evaluate the relationship between this CTN and mortality. METHODS: The baseline study was performed with residents aged 40-89years (n=5,643; 2224 men and 3419 women) in Miyako Islands, Okinawa, in 1987. Dental examinations were performed by dentists, and subjective dysphagia was asked. We recorded the cause and year of death of participants who died during the next 15years. RESULTS: The average functional tooth number of participants with and without subjective dysphagia declined with age in both sexes. The CTN for each respective age group (40s, 50s, 60s, 70s and 80s) not including the denture group were 20.0, 17.5, 14.0, 11.8 and 10.1 in men and 19.0, 14.7, 12.7, 9.5 and 4.0 in women. These CTN were significant factors of 15-year mortality in both sexes (P<0.05). The hazard ratios were 0.72 (95% confidence interval=0.55-0.93) in men and 0.71 (0.51-0.99) in women. CONCLUSION: In aged people, the minimum number of functional teeth needed to avoid subjective dysphagia might not be as high as in young people.

Evaluation of Japanese practice guidelines for newly diagnosed childhood idiopathic thrombocytopenic purpura.

BACKGROUND: Practice guidelines for childhood idiopathic thrombocytopenic purpura (ITP) were proposed in 1998 and 2004 in Japan. It is important to evaluate the feasibility and validity of the guidelines because the guidelines are based not on evidence but on opinion. METHODS: Records of 30 consecutive hospitalized patients aged 0-15 years (median, 3 years; 17 boys, 13 girls) were retrospectively reviewed. The feasibility and validity of the 2004 guidelines were evaluated by calculating an implementation rate, a response rate to first treatments, and a final clinical outcome. RESULTS: Deviation from the guidelines was found in two patients. Management of the other 28 patients followed the guidelines. The implementation rate was 93.3%. Twenty-one of the 28 eligible patients (75%) responded to the first treatment, whereas seven patients (25%) required additional treatment. In 23 patients (76.7%) platelet counts returned to normal within 6 months. Seven patients (23.3%) developed chronic conditions. All patients had no complications and achieved a platelet count >150 000/µL within 6 years. CONCLUSION: The 2004 Japanese guidelines are feasible and valid for the management of newly diagnosed childhood ITP patients.

[Clinical investigation of nine pediatric Japanese spotted fever cases].

To clarify the clinical manifestations of pediatric Japanese spotted fever (JSF), which remain unclear, we retrospectively reviewed the records of 9 consecutive hospitalized children 5 boys and 4 girls aged 0-15 years (median: 2.3) whose diagnosis was patients with JSF who were serologically confirmed from April 2008 to October 2009. We initially studied the polymerase chain reaction (PCR) assay validity for specific Rickettsia japonica DNA in the blood. We also studied febrile duration, the history of contact with tick-infested areas, body temperature, eschars at tick bite sites, skin rash, treatment drugs, and laboratory data. Five of the 9 (56%) had positive PCR tests. Prehospitalization febrile duration was 1-5 days. Five had had contact with tick-infested areas and 4 had not despite living near such areas. Body temperature was 40 degrees C in 7. Only 4 had eschars at bite sites. Characteristic spotted palmar and/or plantar erythema seen in 8 was useful in diagnosis. Laboratory studies showed typical hyponatremia of < 135mEq/L in 6 JSF was diagnosed easily at hospitalization in 7. Diagnosis in a 2-month-old infant proved difficults, however, worsening the child's condition and causing hepatosplenomegaly, thrombocytopenia, anemia, and hyperferritinema. The infant was treated with high-dose gamma-globulin and azithromycin (AZM) followed by minocycline (MINO). Another case was difficult to diagnose due to clinical manifestations consistent with Kawasaki disease. The child was treated with high-dose gamma-globulin and AZM. Three of the 9 were treated with MINO alone and 4 with combined MINO and new quinolones. Fever was resolved within 2 days of treatment in all cases. Our findings show that children with high fever and spotted palmar and/or plantar erythema should be treated immediately for JSF in prevalent areas, even in the absence of eschars.

Vaccine-associated paralytic poliomyelitis in an infant with perianal abscesses.

We describe a case of vaccine-associated paralytic poliomyelitis (VAPP) in a 7-month-old infant with perianal abscesses. The infant had suffered from perianal abscesses from 3 weeks after birth. The abscesses repeatedly developed and spontaneously drained through the orifice. Twenty-seven days before admission, a live attenuated oral poliovirus vaccine (OPV) was given to the infant for the first time for routine immunization. His body temperature rose to 38°C 19 days after receiving the OPV and fell 4 days later. Flaccid paralysis of the right leg appeared 26 days after receipt of the OPV. A Sabin type 3 poliovirus was isolated from a stool obtained at admission. The DNA sequences of the VP1 region of the isolated virus were more than 99% identical with those of the vaccine strain. Mild muscle atrophy with moderate motor impairment in the right leg persisted at 18 months of age. One VAPP case provoked by a perianal abscess has been reported from the United Kingdom. Database search revealed that one of nine VAPP cases reported during 2003-2008 in Japan had a perianal abscess. Taken together, these reports and our case imply that we should give OPV with caution to infants with a perianal abscess.

Associations between functional tooth number and physical complaints of community-residing adults in a 15-year cohort study.

BACKGROUND: Oral function influences various general health and organ diseases. We wondered if physical complaints of unknown origin were related to oral function. METHODS: Five thousand five hundred and eighty-four community residents (2206 men and 3378 women, aged 40-89 years) on the Miyako Islands, Okinawa Prefecture, Japan, were recruited for the baseline study in 1987 and followed up for 15 years. Physical complaints and functional tooth numbers were assessed for the baseline study in 1987 by dentists and followed up for 15 years. Physical complaints of unknown origin included shoulder stiffness, lower back pain, pain of upper extremity, pain of lower extremity, neuralgia, ear ringing, difficult of hearing, dizziness and sleeping difficulties. RESULTS: Physical complaints were significantly associated with functional tooth number in the baseline study. There were no systematic differences between physical complaints and functional tooth number among different age groups. Physical complaints did not influence the survival rates in either men or women. CONCLUSION: Oral function may be related to physical complaints. Dental care may be one of the targets to treat these physical complaints of unknown origin.

A retrospective evaluation of hospitalizations for acute gastroenteritis at 2 sentinel hospitals in central Japan to estimate the health burden of rotavirus.

BACKGROUND: Two rotavirus vaccines have recently been licensed for use in >80 countries worldwide but not in Japan. To assess the value of introducing rotavirus vaccination in Japan, data on the burden of rotavirus disease are needed. METHODS: To describe the epidemiology of severe rotavirus disease among Japanese children aged <5 years, we examined retrospective demographic, clinical, and laboratory data from the period 2003-2007 for children hospitalized with acute gastroenteritis (AGE) at 2 sentinel hospitals in Japan. RESULTS: At each of the 2 hospitals, 17%-21% of all pediatric hospitalizations were for AGE. Three-fourths of all AGE-related admissions occurred during the winter (December-May). Rotavirus testing was performed for approximately three-fourths of patients admitted with AGE in the winter, of which 55% at one hospital and 59% at the other tested positive. By extrapolating the test results to those patients with AGE admitted in the winter who were not tested, we estimated that 39%-44% of year-round and 52%-57% of winter hospitalizations were attributable to rotavirus. The annual incidence of hospitalization for rotavirus AGE in the 2 cities served by the hospitals was estimated to be 3.8 and 4.9 per 1000 person-years. CONCLUSIONS: The burden of severe rotavirus disease among Japanese children is substantial and warrants consideration of vaccination as a prevention strategy.

Nosocomial bacteremia caused by biofilm-forming Bacillus cereus and Bacillus thuringiensis.

OBJECTIVE: Bacterial biofilms cause serious problems, such as antibiotic resistance and medical device-related infections. Recent reports indicate that Bacillus species potentially form biofilms and cause nosocomial bacteremia via catheter infection. Our objective was to investigate the relationship between nosocomial bacteremia caused by Bacillus species and biofilm formations. METHODS: Between 2001 and 2006, Bacillus cereus and Bacillus thuringiensis were isolated from blood samples of 21 patients with nosocomial bacteremia in two hospitals. The patients had underlying diseases such as cerebrovascular damage, malignant disease, or chronic obstructive lung disease and had high fever at the onset of bacteremia. After investigation, B. cereus and B. thuringiensis were isolated from patient's catheter tip, gauze, and hospital environment. Pulsed-field gel electrophoresis (PFGE) on 32 B. cereus and 7 B. thuringiensis isolates, microtiter biofilm assay and scanning electron microscopy (SEM) on 22 B. cereus isolates from patient's blood were performed. RESULTS: Molecular analysis by PFGE showed that 32 B. cereus strains had 21 patterns and 7 B. thuringiensis strains had 3 patterns. The PFGE patterns of B. thuringiensis and B. cereus in blood samples from 2 patients blood were similar to those from the same patient's catheter tip. The PFGE pattern of B. cereus from a hospital environment was similar to that from 2 patients' blood samples, and the PFGE pattern of B. thuringiensis from 2 hospital environments was similar to that from 2 patients' blood. The biofilm formations by 22 B. cereus isolates from patients' blood were confirmed by microtiter biofilm assay and SEM even at 24 hours. CONCLUSION: Our data indicate that various types of Bacillus species exist in hospital environments and the biofilm-forming strains potentially cause nosocomial bacteremia by catheter infection.

Prevalence of Mycoplasma IgM in children with lower respiratory tract illness.

BACKGROUND: Few data are available for the recent occurrence of Mycoplasma infections in children in Japan. The purpose of the present study was therefore to identify the prevalence of Mycoplasma infections in children in Japan. METHODS: IgM antibodies to M. pneumoniae were prospectively determined using the Meridian ImmunoCard Mycoplasma test in hospitalized patients with lower respiratory tract infections between January 2004 and June 2007. A total of 858 hospitalized patients aged 0-15 years (445 male, 413 female), diagnosed as having acute pneumonia or bronchitis, were enrolled. The number of patients with pneumonia or bronchitis was 331 (male/female, 167/164) and 527 (male/female/ 278/249), respectively. Two hundred and five of the 858 patients (23.9%) were ImmunoCard positive. Of the 205 patients, 121 children and 84 children were diagnosed as having pneumonia and bronchitis, respectively. One hundred and forty-three of the 727 patients (19.7%) <5 years of age were ImmunoCard test positive. CONCLUSIONS: M. pneumoniae infection is not rare in children aged <5 years in Japan.

Mortality rates of community-residing adults with and without dentures.

AIM: To prospectively study how dental status with and without dentures could become a predictor of overall mortality risk. METHODS: Five thousand six hundred eighty-eight community residents over 40 years old in the Miyako Islands, Okinawa Prefecture, Japan, were followed up for 15 years from 1987-2002. RESULTS: We found that female subjects with less than 10 functional teeth and without dentures showed a significantly higher mortality rate than those with dentures. There was no significant difference of mortality rates in male subjects. There were no significant differences of mortality rates between subjects with 10 or more functional teeth with and without dentures. CONCLUSION: The present study suggests that dentures are one of the factors associated with mortality rates especially in female subjects with less than 10 functional teeth.

Proliferation of mitogen-stimulated human peripheral blood mononuclear cells is inhibited by extracellular arginine deiminase of Granulicatella elegans isolated from the human mouth.

Granulicatella elegans is a member of normal human oral flora and is thought to be a potent pathogen in endocarditis, especially so-called "culture-negative" endocarditis. To elucidate the pathogenicity of this microorganism in inflammatory diseases, the effect of the extracellular products of this bacteria on human peripheral blood mononuclear cells (PBMCs) was examined. Culture supernatants produced by oral isolates of G. elegans strongly inhibited the proliferation of PBMC stimulated by the T-cell mitogens phytohemagglutinin-P, phorbol 12-myristate 13-acetate, concanavalin A, and staphylococcal enterotoxin B. Purification of the active extracellular product revealed that a fraction containing proteins of approximately 47 kDa showing arginine deiminase activity contributed to the inhibition of PBMC proliferation.

Experimental infective endocarditis induced by human supragingival dental plaque in rats.

Human dental plaque is thought to contribute to disease, not only in the oral cavity but also at other body sites. To investigate the pathogenicity of dental plaque in tissues remote from the mouth, we examined the ability of human supragingival dental plaque to induce infective endocarditis (IE) in rats. In total, 15 out of 27 catheterized rats survived after intravenous injections with human supragingival dental plaque suspensions containing 3 x 10(6) colony-forming units (CFU) of bacterial cells. In surviving rats, infected vegetations were formed in all except one rat. The microbial composition of the infected vegetations was different from that of the respective dental plaque inocula, with Streptococcus oralis comprising the majority of the isolates. In rats affected with endocarditis, the aortic sinus was filled with fibrinous vegetation containing bacteria. Inflammatory cells infiltrated the aortic valve, the aorta adjacent to the valve, and the cardiac muscles. The inoculation of catheterized rats with a cell suspension of S. oralis isolate (5 x 10(6) CFU) was not lethal but capable of inducing endocarditis in all animals. The results suggest that if dental plaque were introduced into the bloodstream, it could serve as a potent source of bacteria causing IE in humans.

Experimental abscess formation caused by human dental plaque.

Human dental plaque consists of a wide variety of microorganisms, some of which are believed to cause systemic infections, including abscesses, at various sites in the body. To confirm this hypothesis experimentally, we examined the abscess-forming ability of native dental plaque in mice, the microbial features of the infectious locus produced by the plaque, and the anti-phagocytic property of microbial isolates. Aliquots of a suspension of supragingival dental plaque containing 6 x 10(6) colony-forming unit of bacteria were injected subcutaneously into the dorsa of mice. Abscess formation was induced in 76 of 85 mice using ten different plaque samples. Thirteen microorganisms were isolated from pus samples aspirated from abscess lesions. The microbial composition of pus, examined in 17 of 76 abscesses, was very simple compared to that of the plaque sample that had induced the abscess. The majority of the isolates belonged to the Streptococcus anginosus group, normally a minor component of plaque samples. S. anginosus was the most frequently detected organism and the most prevalent in seven abscesses, and Streptococcus intermedius and Streptococcus constellatus were predominant in one and three abscess samples, respectively. Each isolate of S. anginosus group produced abscesses in mice, and heat-treated supragingival dental plaque influenced the abscess-forming ability of S. anginosus isolate. These isolates possessed a high antiphagocytic capacity against human polymorphonuclear leukocytes. Our results suggest that human supragingival dental plaque itself is a source of the infectious pathogens that cause abscess formation.

Induction of immune response to Streptococcus pneumoniae by administration of oral viridans streptococci via phosphorylcholine determinant.

Expression of the phosphorylcholine (PC) epitope was examined in 48 viridans streptococcal strains, including Streptococcus pneumoniae R36a as the positive control, and their immunogenicity to induce an S. pneumoniae-cross-reactive response was evaluated in mice. Thirteen strains were found to express the PC epitope, while no obvious association was found between the taxonomic categories and PC expression. Serum antibody responses to S. pneumoniae cells were induced in mice by intraperitoneal injection of the PC-positive, but not PC-negative, strains. The cross-reactive antibodies induced by non-pneumococcal oral streptococci were readily inhibited by free hapten PC. IgM was the sole isotype of the anti-pneumococcal and anti-PC antibodies, and the phenomenon of immunological memory was not observed. Since the anti-PC antibody is critically important for resistance against pneumococcal infection in mice, the present results indicate the possibility that PC-expressing oral commensal bacteria have a significant influence on the hosts' responsiveness to S. pneumoniae.

Identification and characterization of bacterial-binding property in the type III repeat domain of fibronectin.

To characterize fibronectin binding with Granulicatella adiacens, a causative agent of infective endocarditis, monoclonal antibodies were generated against human fibronectin and selected for their capacity to inhibit the fibronectin binding of the organism. Thermolysin and lysyl-endopeptidase digests of fibronectin were characterized by Western blot. The epitope of inhibitory monoclonal antibody was found in the central portion of fibronectin known as the cell-binding domain, and not in the N-terminal portion known to be the binding region of most microbial species, e.g., Staphylococcus aureus and Streptococcus pyogenes. While these two species could bind to both the N-terminal and central portion, Escherichia coli and G. adiacens bind only to the latter. Excess amounts of free fibronectin in the solution inhibited the bacterial adherence to the N-terminal fibronectin fragment, but not to the central region, thereby suggesting the central region plays a significant role for in vivo bacterial colonization in the presence of high concentrations of soluble fibronectin.