Leptin receptor q223r polymorphism influences neutrophil mobilization after Clostridium difficile infection.

Clostridium difficile is the leading cause of nosocomial infections in the United States. Clinical disease outcomes after C. difficile infection (CDI) are dependent on intensity of host inflammatory responses. Specifically, peak peripheral white blood cell (WBC) count >20 × 109 l-1 is an indicator of adverse outcomes in CDI patients, and is associated with higher 30-day mortality. We show that homozygosity for a common single nucleotide polymorphism (Q to R mutation in leptin receptor that is present in up to 50% of people), significantly increases the risk of having peak peripheral WBC count >20 × 109 l-1 (odds ratio=5.41; P=0.0023) in CDI patients. In a murine model of CDI, we demonstrate that mice homozygous for the same single nucleotide polymorphism (RR mice) have more blood and tissue leukocytes (specifically neutrophils), exaggerated tissue inflammation, and higher mortality as compared with control mice, despite similar pathogen burden. Further, we show that neutrophilia in RR mice is mediated by gut microbiota-directed expression of CXC chemokine receptor 2 (CXCR2), which promotes the release of neutrophils from bone marrow reservoir. Overall these studies provide novel mechanistic insights into the role of human genetic polymorphisms and gut microbiota in regulating the fundamental biological process of CDI-induced neutrophilia.

Utility of a routine urinalysis in children who require clean intermittent catheterization.

BACKGROUND: Children who require clean intermittent catheterization (CIC) frequently have positive urine cultures. However, diagnosing a urinary tract infection (UTI) can be difficult, as there are no standardized criteria. Routine urinalysis (UA) has good predictive accuracy for UTI in the general pediatric population, but data are limited on the utility of routine UA in the population of children who require CIC. OBJECTIVE: To determine the utility of UA parameters (e.g. leukocyte esterase, nitrites, and pyuria) to predict UTI in children who require CIC, and identify a composite UA that has maximal predictive accuracy for UTI. METHODS: A cross-sectional study of 133 children who required CIC, and had a UA and urine culture sent as part of standard of care. Patients in the no-UTI group all had UA and urine cultures sent as part of routine urodynamics, and were asymptomatic. Patients included in the UTI group had growth of ≥50,000 colony-forming units/ml of a known uropathogen on urine culture, in addition to two or more of the following symptoms: fever, abdominal pain, back pain, foul-smelling urine, new or worse incontinence, and pain with catheterization. Categorical data were compared using Chi-squared test, and continuous data were compared with Student's t-test. Sensitivity, specificity, and positive and negative predictive values were calculated for individual UA parameters, as well as the composite UA. Logistic regression was performed on potential composite UA models to identify the model that best fit the data. RESULTS: There was a higher proportion of patients in the no-UTI group with negative leukocyte esterase compared with the UTI group. There was a higher proportion of patients with UTI who had large leukocyte esterase and positive nitrites compared with the no-UTI group (Summary Figure). There was no between-group difference in urinary white blood cells. Positive nitrites were the most specific (84.4%) for UTI. None of the parameters had a high positive predictive value, while all had high negative predictive values. The composite model with the best Akaike information criterion was >10 urinary white blood cells and either moderate or large leukocyte esterase, which had a positive predictive value of 33.3 and a negative predictive value of 90.4. CONCLUSION: Routine UA had limited sensitivity, but moderate specificity, in predicting UTI in children who required CIC. The composite UA and moderate or large leukocyte esterase both had good negative predictive values for the outcome of UTI.

The ecology and pathobiology of Clostridium difficile infections: an interdisciplinary challenge.

Clostridium difficile is a well recognized pathogen of humans and animals. Although C. difficile was first identified over 70 years ago, much remains unknown in regards to the primary source of human acquisition and its pathobiology. These deficits in our knowledge have been intensified by dramatic increases in both the frequency and severity of disease in humans over the last decade. The changes in C. difficile epidemiology might be due to the emergence of a hypervirulent stain of C. difficile, ageing of the population, altered risk of developing infection with newer medications, and/or increased exposure to C. difficile outside of hospitals. In recent years, there have been numerous reports documenting C. difficile contamination of various foods, and reports of similarities between strains that infect animals and strains that infect humans as well. The purposes of this review are to highlight the many challenges to diagnosing, treating, and preventing C. difficile infection in humans, and to stress that collaboration between human and veterinary researchers is needed to control this pathogen.

Expression cloning of human globoside synthase cDNAs. Identification of beta 3Gal-T3 as UDP-N-acetylgalactosamine:globotriaosylceramide beta 1,3-N-acetylgalactosaminyltransferase.

By using a eukaryocytic cell expression cloning system, we have isolated cDNAs of the globoside synthase (beta1, 3-N-acetylgalactosaminyltransferase) gene. Mouse fibroblast L cells transfected with SV40 large T antigen and previously cloned Gb3/CD77 synthase cDNAs were co-transfected with a cDNA library prepared from mRNA from human kidney together with Forssman synthase cDNA, and Forssman antigen-positive cells were panned using an anti-Forssman monoclonal antibody. The isolated cDNAs contained a single open reading frame predicting a type II membrane protein with 351 amino acids. Surprisingly, the cDNA clones turned out to be identical with previously reported beta3Gal-T3, which had been cloned by sequence homology with other galactosyltransferases. Substrate specificity analysis with extracts from cDNA-transfected L cells confirmed that the gene product was actually beta1, 3-N-acetylgalactosaminyltransferase that specifically catalyzes the transfer of N-acetylgalactosamine onto globotriaosylceramide. Results of TLC immunostaining of neutral glycolipids from the cDNA-transfected cells also supported the identity of the newly synthesized component as globoside. The results show that glycosyltransferases apparently belonging to a single glycosyltransferase family do not necessarily catalyze reactions utilizing the same acceptor or even the same sugar donor. The globoside synthase gene was expressed in many tissues, such as heart, brain, testis, etc. We propose the designation beta3GalNAc-T1 for the cloned globoside synthase gene.

HEDJ, an Hsp40 co-chaperone localized to the endoplasmic reticulum of human cells.

Hsp40 co-chaperones, characterized by the presence of a highly conserved J domain, are involved in nearly all aspects of protein synthesis, folding, and secretion. Within the lumen of the endoplasmic reticulum, these chaperones are also involved in reverse translocation and degradation of misfolded proteins. We describe here the cloning and characterization of a novel Hsp40 chaperone, which we named HEDJ. Epitope-tagged HEDJ was demonstrated by confocal microscopy to be localized to the endoplasmic reticulum. Protease susceptibility, glycosidase treatment, and detergent solubility assays demonstrated that the molecule was luminally oriented and membrane-associated. In vitro experiments demonstrated that the J domain interacted with the endoplasmic reticulum-associated Hsp70, Bip, in an ATP-dependent manner and was capable of stimulating its ATPase activity. HEDJ mRNA expression was detected in all human tissues examined. Highly homologous sequences were found in mouse, Drosophila, and Caenorhabditis elegans data bases. These results suggest potential roles for HEDJ in protein import, folding, or translocation within the endoplasmic reticulum.

Characterization of the human Forssman synthetase gene. An evolving association between glycolipid synthesis and host-microbial interactions.

Differences in glycolipid expression between species contribute to the tropism of many infectious pathogens for their hosts. For example, we demonstrate that cultured human and monkey urinary epithelial cells fail to bind a canine Escherichia coli uropathogenic isolate; however, transfection of these cells with the canine Forssman synthetase (FS) cDNA enables abundant adherence by the same pathogen, indicating that addition of a single sugar residue to a glycolipid receptor has marked effects on microbial attachment. Given the contribution of glycolipids to host-microbial interactions, we sought to determine why human tissues do not express Forssman glycolipid. Query of the GenBank(TM) data base yielded a human sequence with high identity to the canine FS cDNA. Reverse transcription polymerase chain reaction and Northern blotting demonstrated the presence of FS mRNA in all tissues examined. A human FS cDNA was characterized, revealing identities with the canine FS gene of 86 and 83% at the nucleotide and predicted amino acid sequences, respectively. In contrast to the canine FS cDNA, transfection of COS-1 cells with the human FS cDNA resulted in no detectable FS enzyme activity. These results suggest that variability in glycolipid synthesis between species is an important determinant of microbial tropism. Evolutionary pressure from pathogenic organisms may have contributed to diversity in glycolipid expression among species.

Expression cloning of Forssman glycolipid synthetase: a novel member of the histo-blood group ABO gene family.

A phenotypic cloning approach was used to isolate a canine cDNA encoding Forssman glycolipid synthetase (FS; UDP-GalNAc:globoside alpha-1,3-N-acetylgalactosaminyltransferase; EC 2.4.1.88). The deduced amino acid sequence of FS demonstrates extensive identity to three previously cloned glycosyltransferases, including the enzymes responsible for synthesis of histo-blood group A and B antigens. These three enzymes, like FS, catalyze the addition of either N-acetylgalactosamine (GalNAc) or galactose (Gal) in alpha-1,3-linkage to their respective substrates. Despite the high degree of sequence similarity among the transferases, we demonstrate that the FS cDNA encodes an enzyme capable of synthesizing Forssman glycolipid, and demonstrates no GalNAc or Gal transferase activity when closely related substrates are examined. Thus, the FS cDNA is a novel member of the histo-blood group ABO gene family that encodes glycosyltransferases with related but distinct substrate specificity. Cloning of the FS cDNA will allow a detailed dissection of the roles Forssman glycolipid plays in cellular differentiation, development, and malignant transformation.

The amino-terminal domain of the P-pilus adhesin determines receptor specificity.

Pyelonephritic isolates of Escherichia coli commonly express P-pili, which mediate bacterial attachment to glycolipids on epithelial cell surfaces. Three classes of P-pili have been defined, based on varying specificity for galabiose-containing glycolipids. Variation in adhesive capacity is correlated with a shift in preferred host, suggesting that host tropism depends largely on detailed specificity for the globoseries glycolipids. In this study we examined the importance of the PapG adhesin in determining receptor specificity. Translational fusions were constructed between the amino-terminus of the PapG adhesin from each of the three pilus classes and a reporter protein. The binding specificity of the purified fusion proteins in vitro was identical to that seen with whole bacteria. Adherence of intact bacteria to cultured kidney cells was markedly reduced by a monoclonal antibody specific for the Class III adhesin (previously denoted PrsG), confirming the importance of the amino-terminus of PapG in mediating attachment to a receptor when presented on the eukaryotic cell surface. These results suggest that the detailed receptor specificity resides solely within the amino-terminus of the PapG adhesin and is independent of the complex pilus architecture.

Disseminated measles infection after vaccination in a child with a congenital immunodeficiency.

An infant boy with a congenital immunodeficiency had fatal disseminated measles after administration of a live attenuated measles vaccine. This rare complication was confirmed with molecular virologic techniques. Although efforts to expand availability of vaccinations are critically important, caution is warranted in children with potentially severe immunologic dysfunction.

Improved direct molecular diagnosis and rapid fetal sexing.

Adaptations of the techniques of modern molecular biology to prenatal diagnosis has opened new avenues for the detection of genetic diseases. We have taken advantage of the rapid adhesion of colony forming cells in cultured amniotic fluid samples to develop an improved method for molecular diagnosis. By employing the cell adherence regime sickle cell diagnosis using Mst II can be undertaken directly. In addition, hybridization with a cloned repetitive sequence that is of Y origin and has limited autosomal homology permits rapid fetal sexing in 3 to 4 days without compromising conventional cytogenetic or biochemical analysis. This combination of techniques provides a useful adjunct to convential prenatal genetic diagnosis in the second trimester.

Prenatal identification of a deleted Y chromosome by cytogenetics and a Y-specific repetitive DNA probe.

A very small sex chromosome was identified prenatally as a Y chromosome by using molecular hybridization in conjunction with conventional cytogenetics techniques. The combination of R-banding, Q-banding, distamycin-DAPI staining suggested that the chromosome might be a de novo deletion of the Y chromosome as the father's Y chromosome was normal. Restriction enzyme analysis of amniotic fluid cell DNA using a Y chromosome repetitive probe confirmed the origin of this chromosome.