Culture-independent species typing of neotropical Leishmania for clinical validation of a PCR-based assay targeting heat shock protein 70 genes

PCR-restriction fragment length polymorphism analysis of heat shock protein 70 genes discriminates most neotropical Leishmania species, as well as Trypanosoma cruzi. The assay, combined with capillary electrophoresis in a microchip device, may be applied directly on clinical samples with a high sensitivity, hence supporting clinical and epidemiological monitoring of leishmaniasis.     

急性颅脑损伤早期血浆CNP和脑脊液乳酸测定的临床意义

目的 :探讨了血浆C -型利钠多肽 (CNP)和脑脊液乳酸水平在急性颅脑损伤早期的病理、生理作用。方法 :分别应用放射免疫分析血浆CNP水平和生化法测定脑脊液乳酸水平。结果 :在治疗前颅脑损伤患者血浆CNP水平非常显著地低于正常人组 (p <0 0 1 ) ,脑脊液乳酸水平明显高于正常人水平 (p <0 0 1 ) ,治疗 1 5天后与正常人比较无显著差异 (p >0 0 5 )。 结论 :观察血浆CNP和脑脊液乳酸水平的变化对研究急性颅脑损伤早期的病理生理变化、判断疗效及预后观察具有十分重要的临床意义     

Real-time PCR assay for detection of fluoroquinolone resistance associated with grlA mutations in Staphylococcus aureus

Resistance to fluoroquinolones among clinical isolates of Staphylococcus aureus has become a clinical problem. Therefore, a rapid method to identify S. aureus and its susceptibility to fluoroquinolones could provide clinicians with a useful tool for the appropriate use of these antimicrobial agents in the health care settings. In this study, we developed a rapid real-time PCR assay for the detection of S. aureus and mutations at codons Ser-80 and Glu-84 of the grlA gene encoding the DNA topoisomerase IV, which are associated with decreased susceptibility to fluoroquinolones. The detection limit of the assay was 10 genome copies per reaction. The PCR assay was negative with DNA from all 26 non-S. aureus bacterial species tested. A total of 85 S. aureus isolates with various levels of fluoroquinolone resistance was tested with the PCR assay. The PCR assay correctly identified 100% of the S. aureus isolates tested compared to conventional culture methods. The correlation between the MICs of ciprofloxacin, levofloxacin, and gatifloxacin and the PCR results was 98.8%. The total time required for the identification of S. aureus and determination of its susceptibility to fluoroquinolones was about 45 min, including DNA extraction. This new rapid PCR assay represents a powerful method for the detection of S. aureus and its susceptibility to fluoroquinolones.     

A polymorphic major histocompatibility complex class II‐like locus maps outside of both the chicken B‐system and Rfp‐Y‐system

Chickens have two major regions encoding major histocompatibility complex (MHC) class Iα genes and MHC class IIß genes, the serological and functional B‐system and the Rfp‐Y‐system. Recently, they have been shown to assort in a genetically independent way although still located on the same microchromosome. Moreover, the monomorphic MHC class IIα gene maps at a third locus located 5 c m from the nearest class IIß genes, located in the B‐system ( Kaufman et al., 1995 ). A pedigree family was studied in three generations in order to assign MHC class IIß restriction fragments observed in Southern blot analyses to either the B‐system, the Rfp‐Y‐system or the B‐Lα locus. In this study, we demonstrate by classical genetic testing of chickens within this fully pedigreed family the existence of an MHC class II‐like polymorphic restriction fragment that segregates independently of the B‐system, the Rfp‐Y‐system and of the B‐Lα locus.     

Invasive pulmonary aspergillosis: high incidence of disseminated intravascular coagulation in fatal cases.

BACKGROUND AND PURPOSE: Disseminated intravascular coagulation (DIC) is a rarely described finding in invasive pulmonary aspergillosis (IPA) with unclear impact on mortality. METHODS: This study included patients with positive cultures of Aspergillus spp. from respiratory specimens, serological evidence of aspergillosis, or lung biopsy findings supporting aspergillosis treated at National Taiwan University Hospital from January 1999 to June 2005. IPA was defined based on the consensus of the European Organization for Research and Treatment of Cancer, and the Mycosis Study Group of the National Institute of Allergy and Infectious Diseases. Univariate logistic regression analysis was used to evaluate the factors associated with mortality. RESULTS: Proven or probable IPA was diagnosed in 26 patients. Hematological malignancy was found in 11 patients (42%) and immunosuppressive agents had been administered to 17 patients (65%). Among 20 culture-proven infections (77%), the most frequently encountered fungi were Aspergillus fumigatus (46%) and Aspergillus flavus (23%). The overall mortality rate was 62%. Univariate and multivariate analyses revealed that DIC was the only factor that was significantly associated with death attributable to IPA (p<0.01). CONCLUSIONS: IPA is associated with a high mortality rate, particularly for patients with DIC.     

Identical allelic losses in mature teratoma and other histologic components of malignant mixed germ cell tumors of the testis

Teratomas of the testis in post-pubertal patients are histologically diverse tumors that often coexist with other types of germ cell tumors. Using laser capture microdissection and loss of heterozygosity analysis, we investigated the clonality of mature teratoma and its relationship to other components of malignant mixed germ cell tumors to gain potential insight into the histogenetic relationship of teratoma with other germ cell tumor components. All 16 patients had mature teratoma as one component of their mixed germ cell tumors. The other histological subtypes included immature teratoma, seminoma, embryonal carcinoma, yolk sac tumor, and choriocarcinoma. Laser-assisted microdissection was performed on the formalin-fixed, paraffin-embedded tissue. Polymerase chain reaction was used to amplify genomic DNA at specific loci on chromosome 1p36.2 (D1S508), 2q22-32 (D2S156), 9p21-22 (D9S162), 11p13 (D11S903), 12q22-23 (D12S1051), and 18q21 (D18S46). Fourteen of 16 (88%) cases showed allelic loss in one or more components of the mixed germ cell tumors. Fourteen of 16 mature teratomas showed allelic loss in at least one of six microsatellite polymorphic markers analyzed. The frequency of allelic loss in mature teratoma was 50% (7 of 14) with D1S508, 33% (5 of 15) with D2S156, 58% (7 of 12) with D9S162, 43% (6 of 14) with D11S903, 20% (3 of 15) with D12S1051, and 33% (5 of 15) with D18S46. Completely concordant allelic loss patterns between mature teratoma and all of the other germ cell tumor components were seen in 10 of 14 tumors in which mature teratoma showed loss of heterozygosity. Our data support the common clonal origin of mature teratoma with other components of malignant mixed germ cell tumors of the testis.     

Molecular analysis of bacterial species associated with childhood caries

Although substantial epidemiologic evidence links Streptococcus mutans to caries, the pathobiology of caries may involve more complex communities of bacterial species. Molecular methods for bacterial identification and enumeration now make it possible to more precisely study the microbiota associated with dental caries. The purpose of this study was to compare the bacteria found in early childhood caries (ECC) to those found in caries-free children by using molecular identification methods. Cloning and sequencing of bacterial 16S ribosomal DNAs from a healthy subject and a subject with ECC were used for identification of novel species or uncultivated phylotypes and species not previously associated with dental caries. Ten novel phylotypes were identified. A number of species or phylotypes that may play a role in health or disease were identified and warrant further investigation. In addition, quantitative measurements for 23 previously known bacterial species or species groups were obtained by a reverse capture checkerboard assay for 30 subjects with caries and 30 healthy controls. Significant differences were observed for nine species: S. sanguinis was associated with health and, in order of decreasing cell numbers, Actinomyces gerencseriae, Bifidobacterium, S. mutans, Veillonella, S. salivarius, S. constellatus, S. parasanguinis, and Lactobacillus fermentum were associated with caries. These data suggest that A. gerencseriae and other Actinomyces species may play an important role in caries initiation and that a novel Bifidobacterium may be a major pathogen in deep caries. Further investigation could lead to the identification of targets for biological interventions in the caries process and thereby contribute to improved prevention of and treatment for this significant public health problem.     

Size fractions of ambient particulate matter induce granulocyte macrophage colony-stimulating factor in human bronchial epithelial cells by mitogen-activated protein kinase pathways

Environmental pollutants, including ambient particulate matter (PM), increase respiratory morbidity. Studies of model PM particles, including residual oil fly ash and freshly generated diesel exhaust particles, have demonstrated that PM affects inflammatory airway responses. Neither of these particles completely represents ambient PM, and therefore questions remain about ambient particulates. We hypothesized that ambient PM of different size fractions collected from an urban environment (New York City air), would activate primary culture human bronchial epithelial cells (HBECs). Because of the importance of granulocyte-macrophage colony-stimulating factor (GM-CSF) on inflammatory and immunomodulatory processes, we focused our studies on this cytokine. We demonstrated that the smallest size fraction (ultrafine/fine; < 0.18 micro m) of ambient PM (11 micro g/cm(2)), upregulated GM-CSF production (2-fold increase). The absence of effect of carbon particles of similar size, and the day-to-day variation in response, suggested that the chemical composition, but not the particle itself, was necessary for GM-CSF induction. Activation of the extracellular signal-regulated kinase and the p38 mitogen-activated protein kinase was associated with, and necessary for, GM-CSF release. These studies serve to corroborate and extend those on model particles. Moreover, they emphasize the role of the smallest size ambient particles in airway epithelial cell responses.     

Mutation analysis of PEX7 in 60 probands with rhizomelic chondrodysplasia punctata and functional correlations of genotype with phenotype

PEX7 encodes the cytosolic receptor for the set of peroxisomal matrix enzymes targeted to the organelle by the peroxisome targeting signal 2 (PTS2). Mutations in PEX7 cause rhizomelic chondrodysplasia punctata (RCDP), a distinct peroxisome biogenesis disorder. In previous work we described three novel PEX7 mutant alleles, including one, L292X, with a high frequency due to a founder effect. We have now extended our analysis to 60 RCDP probands and identified a total of 24 PEX7 alleles, accounting for 95% of the mutant PEX7 genes in our sample. Of these, 50% are L292X, 13% are IVS9+1G>C, and the remainder are mostly private. IVS9+1G>C occurs on at least three different haplotypes and thus appears to result from recurrent mutation. The phenotypic spectrum of RCDP is broader than commonly recognized and includes minimally affected individuals at the mild end of the spectrum. To relate PEX7 genotype and phenotype, we evaluated the consequence of the disease mutation on PEX7 RNA by Northern analysis and RT/PCR. We evaluated the function of the encoded Pex7 protein (Pex7p) by expressing selected alleles in fibroblasts from RCDP patients and assaying their ability to restore import of a PTS2 marker protein. We find that residual activity of mutant Pex7p and reduced amounts of normal Pex7p are associated with milder and variant phenotypes.