[Analysis of Non-serotype b Encapsulated Haemophilus influenzae Isolated from Pediatric Patients].

We analyzed non-serotype b encapsulated Haemophilus influenzae (non-b Hi) isolated from pediatric patients at Chiba Children's Hospital during 2000-2012. Among 3,532 clinical isolates of H. influenzae, there were 57 (1.6%) strains of non-b Hi, 152 (4.3%) of serotype b H. influenzae (Hib), and 3,323 (94.1%) of non-typeable H. influenzae (NTHi). Fifty-seven strains of non-b Hi were serotyped useing the slide agglutination test and PCR. Twenty-nine strains were identified as type e (Hie) and 28 as type f (Hif), and the results according to the slide agglutination test and PCR were completely identical. Whereas 52 of 57 strains (91.2%) were isolated from respiratory specimen, only one Hif strain (1.8%) was isolated from a sterile site. There were 47 (82.4%) ß-lactamase-non-producing ampicillin (ABPC)-sensitive strains (BLNAS), 5 (8.8%) ß-lactamase-producing strains (BLP), and only 1 (1.8%) ß-lactamase-non-producing ABPC-resistant strain (BLNAR). Thus the frequency of non-b Hi was lower than that of Hib. The source of non-b Hi was similar to that of NTHi, which was mainly isolated from respiratory specimen. Antimicrobial resistant pattern of non-b Hi was different from that of Hib in which the frequency of BLP was relatively high, and NTHi in which that of BLNAR was high. An increase of invasive H. influenzae infections caused by NTHi, Hie, and Hif was reported in the countries where Hib vaccine had been widely used. Because it is assumed that invasive non-Hib infection will be predominant in the near future in Japan, serotyping of invasive strains is crucial. Continuous monitoring of distribution of non-b Hi in the clinical isolates of H. influenzae is also important.

Tenc1-deficient mice develop glomerular disease in a strain-specific manner.

BACKGROUND/AIMS: Tenc1 (also known as tensin2) is an integrin-associated focal adhesion molecule that is broadly expressed in mouse tissues including the liver, muscle, heart and kidney. A mouse strain carrying mutated Tenc1, the ICR-derived glomerulonephritis (ICGN) strain, develops severe nephrotic syndrome. METHODS: To elucidate the function of Tenc1 in the kidney, Tenc1(ICGN) was introduced into 2 genetic backgrounds, i.e. DBA/2J (D2) and C57BL/6J (B6), strains that are respectively susceptible and resistant to chronic kidney disease. RESULTS: Biochemical and histological analysis revealed that homozygous Tenc1(ICGN) mice develop nephrotic syndrome on the D2 background (D2GN) but not on the B6 background (B6GN). Initially, abnormal assembly and maturation of glomerular basement membrane (GBM) were observed, and subsequently effacement of podocyte foot processes was noted in the kidneys of D2GN but not B6GN mice. These defects are likely to be involved in the integrin signaling pathway. CONCLUSION: This study suggests that Tenc1 contributes to the maintenance of GBM structures and that the genetic background influences the severity of nephrotic syndrome.

Diabetes-accelerated memory dysfunction via cerebrovascular inflammation and Abeta deposition in an Alzheimer mouse model with diabetes.

Recent epidemiological studies suggest that diabetes mellitus is a strong risk factor for Alzheimer disease. However, the underlying mechanisms remain largely unknown. In this study, to investigate the pathophysiological interaction between these diseases, we generated animal models that reflect the pathologic conditions of both diseases. We crossed Alzheimer transgenic mice (APP23) with two types of diabetic mice (ob/ob and NSY mice), and analyzed their metabolic and brain pathology. The onset of diabetes exacerbated Alzheimer-like cognitive dysfunction without an increase in brain amyloid-beta burden in double-mutant (APP(+)-ob/ob) mice. Notably, APP(+)-ob/ob mice showed cerebrovascular inflammation and severe amyloid angiopathy. Conversely, the cross-bred mice showed an accelerated diabetic phenotype compared with ob/ob mice, suggesting that Alzheimer amyloid pathology could aggravate diabetes. Similarly, APP(+)-NSY fusion mice showed more severe glucose intolerance compared with diabetic NSY mice. Furthermore, high-fat diet feeding induced severe memory deficits in APP(+)-NSY mice without an increase in brain amyloid-beta load. Here, we created Alzheimer mouse models with early onset of cognitive dysfunction. Cerebrovascular changes and alteration in brain insulin signaling might play a pivotal role in this relationship. These findings could provide insights into this intensely debated association.

[Antimicrobial susceptibility of Haemophilus influenzae isolated from pediatric patients at a pediatric facility between 2009 and 2012].

We examined the antimicrobial susceptibility of 1,208 Haemophilus influenzae isolates obtained at a pediatric facility between 2009 and 2012. The percentage distribution of beta-lactamase-non-producing ampicillin (ABPC)-sensitive (BLNAS) strains was 38.2%, that of beta-lactamase-non-producing ABPC-intermediately resistant (BLNAI) strains was 13.9%, that of beta-lactamase-non-producing ABPC-resistant (BLNAR) strains was 38.2%, that of beta-lactamase-producing ABPC-resistant (BLPAR) strains was 5.2%, and that of beta-lactamase-producing clavulanic acid/amoxicillin-resistant (BLPACR) strains was 4.5%. Although the percentage of BLNAR strains increased dramatically from 13.9% (2000-2003; period I) to 32.7% (2004-2008; period II), it increased more slowly from period II to the present period (2009-2012). However, the percentage of BLNAI strains, which had decreased from 10.6% (period I) to 8.9% (period II), began to increase during the present period. Tosufloxacin (< or = 0.06 microg/mL) and tazobactam/piperacillin (< or = 0.13 microg/mL) exhibited a low 90% minimum inhibitory concentration for H. influenzae, as well as for BLNAR strains. A decreased susceptibility to cephems was also observed throughout all 3 periods. Serotype b strains (Hib) were observed in 54 of the 1,208 isolates (4.5%); their distribution decreased since period II (6.3%). In Hib, the percentage distribution of strains from patients less than 2 years of age, who are recommended to be vaccinated against Hib, decreased from 56.8% to 29.6%, and this reduction seems to have been achieved by increasing the percentage of Hib vaccine inoculations in Japan. Non-serotype b-capsulated strains were identified in 21 isolates (1.7%), and 11 of them were serotyped as type e, whereas the other 10 were serotyped as type f.

[Concentration of tazobactam/piperacillin in the cerebrospinal fluid of patients with Haemophilus influenzae type B meningitis].

While the incidence of Haemophilus influenzae type b (Hib) meningitis is expected to decrease with the widespread use of the Hib vaccine, the resistance of Hib has actually increased. Therefore, selection of the initial antibiotics used for treatment must be performed with resistant bacteria, including beta-lactamase negative ampicillin resistant H. influenzae (BLNAR), in mind. Tazobactam/piperacillin (TAZ/PIPC) has a satisfactory minimum inhibitory concentration (MIC) against BLNAR and is a beta-lactamase inhibitor. Although there is no insurance coverage for its use in patients with meningitis, the penetration of TAZ/PIPC into cerebrospinal fluid (CSF) in animal experiments promises a satisfactory result, and we have been using a combination of ceftriaxone (CTRX) and TAZ/PIPC as an initial treatment and a resistant bacteria countermeasure in patients with Hib meningitis at our hospital since 2008. We examined the concentration of TAZ/PIPC in CSF to further investigate the possibility of using TAZ/PIPC as an antibiotic treatment against bacterial meningitis. In cases treated with a 1: 8 drug formulation of TAZ/PIPC against Hib meningitis at our hospital, we used the remaining portion of a CSF sample collected after the initiation of TAZ/PIPC administration and then measured the concentrations of TAZ and PIPC in the CSF. Six specimens from 5 patients between the ages of 6 and 59 months were examined. The dosage of TAZ/PIPC was 95.7-113.6 mg/kg/dose x 3 times/day, and the CSF concentrations at 0-105 minutes after the completion of the administration were 0.319-1.32 microg/mL for TAZ and 2.54-7.74 microg/mL for PIPC. With the approved dosage, the peak concentration level during the acute period indicated a sufficient CSF concentration level for the antibacterial and beta-lactamase inhibition effects against Hib. As an antibiotic treatment for H. influenzae meningitis, the combined usage of TAZ/PIPC is likely to be effective as a resistant bacteria countermeasure, in addition to third-generation cephem drugs and meropenem.

[Antimicrobial susceptibility and serotype distribution in perinatal group B Streptococcus isolates--a 1999-2009 multicenter study].

We determined temporary changes in group B Streptococcus antimicrobial susceptibility and serotype distribution from perinatal strains. We examined invasive microbiological isolates from neonates with early-onset group B streptococcal disease (n = 14), and colonized isolates from those born uneventfully (n = 55) and from the genital tracts of pregnant and puerperal women (n = 198), collected between 1999 and 2009. All isolates were susceptible to penicillin. No significant differences were seen in susceptibility of 12 antimicrobial agents examined between invasive and colonized isolates. MIC50, MIC90, and resistance did not differ between stage I (1999-2005) and II (2006-2009) isolates. Serotype distribution significantly differed, however, serotypes III and Ia predominated among invasive isolates, while serotypes Ib and VI were common among their colonized counterparts. These findings suggest that to date, penicillin remains effective in intrapartum prophylactic use in colonized pregnant women.

[Susceptibility testing for Haemophilus influenzae isolated from pediatric cases during 2004-2008].

We tested for antimicrobial susceptibility of 1,317 clinical isolates of Haemophilus influenzae at a pediatric facility during 2004-2008. The percentage distribution of beta-lactamase-non-producing ampicillin (ABPC)-sensitive strain (BLNAS) was 47.8%, that of beta-lactamase-non-producing ABPC-resistant strain (BLNAR) 32.7% that of intermediately resistant strain (BLNAI) 8.9% that of beta-lactamase producing ABPC resistant strain (BLPAR) 6.8% and that of clavulanic acid/amoxicillin resistant strain (BLPACR) 3.7%. BLNAR prevalence was 30% between 2005 and 2008, increasing slowly. Though reduced susceptibility was seen in most beta-lactams, piperacillin and tazobactam/piperacillin showed good susceptibility for H. influenzae. Of 1,317 strains, 83 (6.3%) were serotype b (Hib). The frequency of Hib was high in sterilized site.

Elevation of plasma beta-amyloid level by glucose loading in Alzheimer mouse models.

With the emergence of a promising approach to treat Alzheimer disease (AD) targeting the beta-amyloid (Abeta) pathway, it is necessary to establish new diagnostic biomarkers that enable the antemortem diagnosis of AD. Although plasma Abeta has been suggested as a non-invasive biomarker, its significance has been inconclusive. Thus, it is important to improve the diagnostic potential of plasma Abeta. One of the potential approaches is to modify plasma Abeta level using various modulators. In this study, we evaluated the influence of glucometabolic status on plasma Abeta level in two lines of AD transgenic mouse. The present study demonstrated that plasma Abeta level rapidly increased after glucose loading. More importantly, the magnitude of the increase in plasma Abeta was significantly larger in AD transgenic mice than in wild-type littermates. These findings might provide a novel diagnostic tool for AD using the elevation of plasma Abeta level after glucose loading.

Expression of macrophage metalloelastase (MMP-12) in podocytes of hereditary nephrotic mice (ICGN strain).

The Institute for Cancer Research (ICR)-derived glomerulonephritis (ICGN) mouse is a good model for renal fibrosis. In the glomeruli and tubulointerstitium of ICGN mouse kidneys, the components of the extracellular matrix (ECM) accumulated, and matrix metalloproteinases (MMPs) participated in this process. To clarify the mechanism of renal fibrosis, we investigated the expression and localization of macrophage metalloelastase (MMP-12), whose functions in kidney diseases are not fully understood, and its regulatory molecules, monocyte chemoattractive protein-1 (MCP-1) and CC chemokine receptor 2 (CCR2), in the kidneys of ICGN mice by RT-PCR, Western blotting and immunohistochemical staining, respectively. Extensive expression of MMP-12 mRNA and its protein was noted in ICGN mice with progressed nephrotic syndrome. The increase in MMP-12 expression occurred predominantly in podocytes. Furthermore, MCP-1 and CCR2 were also increased in podocytes of the ICGN strain. These results suggest that the expression of MMP-12 is involved in the progression of nephrotic syndrome in ICGN mice.

[Clinical and bacterial analysis of pediatric urinary tract infection].

We analyzed the clinical and bacterial backgrounds of 120 patients with pediatric urinary tract infection (UTI). Escherichia coli was the main pathogen recovered from 98 patients (81.7%). All causative agents isolated from 50 uncomplicated UTI cases were E. coli. Of 98 cases of E. coli UTI, 71 were treated with second-generation cephems, whose therapeutic effect was equal to that of third and fourth-generation cephems. MIC50 and MIC90 (microg/mL) for E. coli were as follows: cefazolin :2, 4; cefmetazole: < or = 0.5, 2; and ceftazidime: < or = 0.25, < or = 0.25. Yearly decline in susceptibility was not observed, but MIC elevation for third generation cephems (< or = 2 microg/mL) including ceftazidime was seen in six isolates. Careful monitoring of susceptibility trends is therefore necessary for appropriate antimicrobial therapy.

[Antimicrobial susceptibility testing of clinical isolates of Haemophilus influenzae derived from Japanese children].

We examined the antimicrobial susceptibility testing of 1,024 Haemophilus influenzae isolates recovered from Japanese children. Percent distribution of beta-lactamase-negative ampicillin (ABPC)--resistant strain (BLNAR; ABPC-MIC > or = 4 microg/ml) was 15.0%. Prevalence of BLNAR increased remarkably from 12.7 to 22.1% in recent 4 years. Furthermore, the frequency of highly resistant strains (ABPC-MIC > or = 8 microg/ml) increased from 28.6 to 56.9% among BLNAR. Decreased susceptibility for cephems was observed in non-BLNAR strains including serotype b strain isolated from the cerebrospinal fluid.

Involvement of the mevalonate pathway in the antiproliferative effect of zoledronate on ACHN renal cell carcinoma cells.

Renal cell carcinoma (RCC) has been shown to be resistant to chemotherapy and radiotherapy. In order to examine the potential of zoledronate (ZOL), a bisphosphonate, as an anticancer agent, we investigated the effects of ZOL on RCC cells and the involvement of the mevalonate pathway in antiproliferative effects, as well as the effects of ZOL administration on mice inoculated with RCC. ACHN cells were used and cell viability was measured via intra-cellular reductase activity. Chromatin condensation was detected by Hoechst 33342 staining. Proteins were detected by western blot analysis. Tumor volume was measured bidimensionally in mice inoculated with ACHN cells after vehicle or ZOL subcutaneous administration. ZOL exhibited antiproliferative effects with an IC50 value of 2.29±0.53 µM in ACHN cells and chromatin condensation was observed when treated with ZOL. Farnesol (FOH) and geranylgeraniol (GGOH), precursors of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, exhibited potency to rescue cells treated with ZOL. Additionally, Ras and RhoA proteins located in the membrane fraction decreased when treated with ZOL and recovered by FOH or GGOH treatment, suggesting that ZOL inhibited the mevalonate pathway, thereby suppressing the translocation of prenylated Ras and RhoA proteins to membrane fractions. An in vivo study showed the inhibitory potential of ZOL on tumor growth in mice without changes in body weight. Our study showed that ZOL could be useful as an anticancer agent for the treatment of RCC, and the mevalonate pathway could be an efficient target for novel therapeutic agents against RCC.

Cytotoxicity of troglitazone through PPARγ-independent pathway and p38 MAPK pathway in renal cell carcinoma.

Agonists of peroxisome proliferator-activated receptor gamma (PPARγ) have been examined as chemopreventive and chemotherapeutic agents. The aim was to investigate the cytotoxicity of troglitazone (TGZ) and its mechanisms in terms of PPARγ dependency and the p38 mitogen-activated protein kinase (MAPK) pathway in three human renal cell carcinoma (RCC) cell lines, 786-O, Caki-2 and ACHN cells. TGZ induced apoptosis and exerted cytotoxicity in a PPARγ-independent manner. We demonstrated that TGZ activated the p38 MAPK pathway and was involved in the cytotoxicity of TGZ. It was also revealed that TGZ induced G(2)/M cell cycle arrest through activation of p38 MAPK.

Bacterial colonization in respiratory secretions from acute and recurrent wheezing infants and children.

Lower respiratory tract infection in childhood often results in airway obstruction, characterized by wheezing. However, contribution of bacterial colonization to the wheezy state in children remains unclear. Wheezing and non-wheezing children requiring hospitalization were classified into three groups: (i) wheezing children having a past history of recurrent wheezing; (ii) wheezing children without such history; and (iii) non-wheezing children as control subjects. Respiratory secretions as sputum were analyzed microscopically, and cultured. Cultured pathogenic bacterial species in sputum were categorized into two subgroups according to their amounts, i.e., dominant and non-dominant amounts of colonies. Incidence of bacterial colonization and wheezing were assessed. Hospitalized children were mainly 1- to 2-yr old, and rapidly decreased in number for older ages. Children in the three groups belonged to different clinical entities. Children in the recurrent wheezing group were highly sensitized to mite allergens, and still required hospitalization after 2 yr of age. Incidence of bacterial colonization was similar between the three groups. Dominant and non-dominant amounts of bacterial colonization were 170/997 (17.1%) and 170/997 (17.1%), respectively, in the recurrent wheezing group; 28/146 (19.2%) and 35/146 (24.0%), respectively, in the acute wheezing group; and 15/56 (26.8%) and 7/56 (12.5%), respectively, in the non-wheezing group. Regardless of the presence of wheezing, bacterial colonization commonly occurred at a young age in the three groups. In recurrent wheezing children, boys (122/611, 20.0%) carried non-dominant amounts of bacteria more frequently than girls (48/386, 12.4%) (p < 0.01). Boys showed predominant wheezing and susceptibility to bacterial colonization. Assessment of bacterial colonization allowed us to characterize asthma onset and outgrowth in childhood.

Age and sex as factors of response to RSV infections among those with previous history of wheezing.

Although enhanced immune reaction caused by the respiratory syncytial virus (RSV) in allergen-sensitized animal model has been reported, RSV illnesses in children already sensitized or having recurrent wheezing episodes have not been completely studied. In addition, the reason for male dominances in RSV infection at young ages was also inconclusive. Therefore, gender analysis in recurrent wheezing children with RSV infection can shed light on asthma pathogenesis. We studied the clinical features and the laboratory data of RSV infections in children who had recurrent wheezing histories. The subjects with RSV infection consisted of 98 boys and 58 girls. The children under 4 yr of age were 123 (78.8%) in number. Children with pneumonia were 78 and those with febrile episode were 119. Children above 1 yr of age were highly sensitized with mite antigen (75/96, 78.1%). The clinical symptoms and signs differed according to their ages. Children in each age group behaved differently in their immune reaction to RSV. Above all, 3-yr-old children deteriorated clinically during acute RSV infection, accompanied by transient elevated C-reactive protein (CRP) and suppressed blood eosinophil counts. Clinical features differed in several points between boys and girls. In general, the white blood cell count and the CRP levels were higher in girls in every age group. Blood eosinophil counts at the acute illness were significantly higher in boys than girls aged 2 and 3< yr. Age and gender comparison in already sensitized children might suggest a clue to asthma pathogenesis.

Gender analysis in acute bronchiolitis due to respiratory syncytial virus.

It is reasonable to compare immune reactions between boys and girls because many infections in the early stages are predominant in males. A relationship between immunomodulatory effects of sex hormone surge in boys at early months and infectious diseases is still unclear. We compared clinical features between boys and girls who suffered from wheezing that was initially triggered by acute respiratory syncytial virus (RSV) bronchiolitis. For systemic immune response evaluation, white blood cell (WBC) count, blood eosinophil count, and serum C-reactive protein (CRP) were measured. For local inflammation evaluation, scores for eosinophils and neutrophils in sputum were evaluated microscopically. Patients consisted of 90 boys and 51 girls. Most children were under 6 months of age. WBC counts and serum CRP levels were significantly increased in girls compared with boys. Blood eosinophilia at the acute stage was rarely observed in children after 6 months of age. For local response evaluation, sputum specimens obtained from 42 boys and 29 girls were microscopically examined. Sputum eosinophil score of 2+ and more was observed in boys (6/42) exclusively. In contrast, sputum neutrophilia was commonly observed in boys and girls. From a follow-up study, we confirmed that 28 children with RSV bronchiolitis showed wheezing episodes afterwards. However, their blood and sputum eosinophilia during RSV bronchiolitis did not reflect their subsequent wheezing. We speculated that gender-specific responses to RSV infection might account for male susceptibility. Differences in RSV pathogenicity between boys and girls should be further investigated in terms of asthma progression.

An improved synthesis of benzocycloalkanone derivatives.

A convenient and improved annulation method for the synthesis of bicyclic ketones was developed. A 2,2-dimethyl-6-(2-phenylsulfonyl)ethylcyclohexanone was converted into a sulfonylester by the addition of ethyl acetate and subsequent dehydration. A Dieckmann type condensation of the sulfonylester followed by desulfonylation afforded the 8,8-dimethyl-1,2,3,4,5,6,7,8-octahydronaphthalene-2-one in good yield. This annulation method was also applicable for the synthesis of the benzocyclooctanone derivative.