Direct detection and differentiation of Legionella spp. and Legionella pneumophila in clinical specimens by dual-color real-time PCR and melting curve analysis

A dual-color LightCycler PCR assay targeting the 16S rDNA gene of Legionella spp. was established. By using two pairs of hybridization probes, Legionella spp. and Legionella pneumophila could be detected and differentiated simultaneously. With 26 culture-positive and 42 culture-negative respiratory specimens from patients with atypical pneumonia, 100% sensitivity and specificity was observed for L. pneumophila.     

Global deletion of thrombospondin-1 increases cardiac and skeletal muscle capillarity and exercise capacity in mice

Thrombospondin-1 (TSP-1) is a known inhibitor of angiogenesis; however, a skeletal muscle phenotype of TSP-1 null mice has not been investigated. The purposes of this study were to compare and contrast TSP-1 null and wild-type mice by examining the following: (1) capillarity in the skeletal and cardiac muscles; (2) fibre type composition and oxidative enzyme activity in the hindlimb; and (3) the consequences of TSP-1 gene deletion for exercise capacity. In TSP-1 null mice, maximal running speed was 11% greater and time to exhaustion during submaximal endurance running was 67% greater compared with wild-type mice. Morphometric analyses revealed that TSP-1 null mice had higher ( P < 0.05) capillarity in the heart and skeletal muscle than wild-type mice, whereas no differences for fibre type composition or oxidative enzyme activity were present between the two groups. Cardiac function, as measured by transthoracic echocardiography, revealed no difference in myocardial contractility but greater left ventricular end-diastolic and systolic dimensions, corresponding to an elevated heart mass in the TSP-1 null mice. The results of this study indicate that TSP-1 is an important endogenous negative regulator of angiogenesis that prevents excessive capillarization in the heart and skeletal muscles. The increased capillarity alone was sufficient to increase ( P < 0.05) exercise capacity. These data demonstrate that the capillary-to-muscle interface is a critical factor that limits oxygen transport during exercise.     

Tropical calcific pancreatitis: strong association with SPINK1 trypsin inhibitor mutations

BACKGROUND & AIMS: Tropical calcific pancreatitis (TCP) is a chronic pancreatitis unique to developing countries in tropical regions. The cause of TCP is obscure. Whereas environmental factors, such as protein energy malnutrition and ingestion of cassava, have been implicated, a genetic predisposition to the disease also may be important. In the present study we report on mutations in the serine protease inhibitor, Kazal type 1 (SPINK1) gene in north Indian patients with TCP. METHODS: We studied 66 unrelated TCP patients (44 men, 49 with diabetes, and 6 with family history of TCP), 25 relatives, and 92 healthy control subjects. Samples were analyzed for SPINK1 variants (-53C>T, L14P, N34S, P55S, and 272T>C) and cationic trypsinogen (PRSS1) variants (A16V, K23R, N29I, and R122H) by melting curve analysis. RESULTS: Twenty-nine patients (44%) carried the N34S missense mutation, of whom 9 (14%) were homozygotes. In contrast, only 2 (2.2%) control subjects were N34S heterozygotes (prevalence ratio 20.2; 95% confidence interval 5.0-81.8; P < 0.0001 vs. TCP). The severity of pancreatitis did not differ between TCP patients with or without N34S, or among those heterozygous or homozygous for N34S. Among TCP patients with or without diabetes, the frequency of N34S carriers (43% vs. 47%) and N34S homozygotes (14% vs. 12%) was similar. CONCLUSIONS: TCP is highly associated with the SPINK1 N34S mutation. The high prevalence of N34S in TCP patients with and without diabetes suggests that these 2 subtypes have a similar genetic predisposition. The genetic predisposition to TCP resembles, at least in part, the idiopathic chronic pancreatitis found in industrialized countries.     

H5N1 Oseltamivir-resistance detection by real-time PCR using two high sensitivity labeled TaqMan probes

A single amino acid substitution, from histidine to tyrosine at position 274 of the neuraminidase gene has converted Oseltamivir sensitive H5N1 influenza A virus into a resistant strain. Currently, Oseltamivir is being stockpiled in many countries potentially affected by the influenza A virus subtype H5N1 epidemic. To identify this change in Oseltamivir-treated patients, a method based on real-time PCR using two labeled TaqMan probes was developed for its rapid detection. In order to validate the method, Oseltamivir specimen from treated (Oseltamivir-resistant strain from a Vietnamese patient, two Oseltamivir-treated tigers) and untreated subjects have been used for this study. The results thus obtained as well as those derived from clone selection and sequencing showed that TaqMan probes could clearly discriminate wild type H274 from the mutant 274Y variant. The sensitivity of this assay was as low as 10 copies/microl and allowed the detection of the mutation in a mixture of wild type and mutant. Overall, the assay based on real-time PCR with two labeled TaqMan probes described here should be useful for detecting Oseltamivir-resistant H274Y H5N1 influenza A virus in many species and various sources of specimens with high sensitivity and specificity. Such studies can address potential differences in the diagnostic outcomes between patients who develop detectable Oseltamivir resistance and those who retain only the wild type strain of H5N1.     

Site-specific phosphorylation of synapsin I by Ca2+/calmodulin-dependent protein kinase II in pancreatic betaTC3 cells: synapsin I is not associated with insulin secretory granules

Increasing evidence supports a physiological role of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in the secretion of insulin from the pancreatic beta-cell, but the precise sites of action are not known. A role of this enzyme in neuroexocytosis is implicated by its phosphorylation of a vesicle-associated protein, synapsin I. Because of emerging similarities to the neuron with respect to exocytotic mechanisms, the expression and phosphorylation of synapsin I in the beta-cell have been studied. Synapsin I expression in clonal mouse beta-cells (betaTC3) and primary rat islet beta-cells was initially confirmed by immunoblot analysis. By immunoprecipitation, in situ phosphorylation of synapsin I was induced in permeabilized betaTC3 cells within a Ca2+ concentration range shown to activate endogenous CaM kinase II under identical conditions. Proteolytic digests of these immunoprecipitates revealed that calcium primarily induced the increased phosphorylation of sites identified as CaM kinase II-specific and distinct from protein kinase A-specific sites. Immunofluorescence and immunogold electron microscopy verified synapsin I expression in betaTC3 cells and pancreatic slices but demonstrated little if any colocalization of synapsin I with insulin-containing dense core granules. Thus, although this study establishes that synapsin I is a substrate for CaM kinase II in the pancreatic beta-cell, this event appears not to be important for the mobilization of insulin granules.     

Distribution and effects of polymorphic RANTES gene alleles in HIV/HCV coinfection -- a prospective cross-sectional study

AIM: Chemokines and their receptors are crucial for immune responses in HCV and HIV infection. RANTES gene polymorphisms lead to altered gene expression and influence the natural course of HIV infection. Therefore, these mutations may also affect the course of HIV/HCV coinfection. METHODS: We determined allele frequencies of RANTES-403 (G→A), RANTES-28 (C→G) and RANTES-IN1.1 (T→C) polymorphisms using real-time PCR and hybridization probes in patients with HIV (n = 85), HCV (n = 112), HIV/HCV coinfection (n = 121), and 109 healthy controls. Furthermore, HIV and HCV loads as well as CD4+ and CD8+ cell counts were compared between different RANTES genotypes. RESULTS: Frequencies of RANTES-403 A, RANTES-28 G and RANTES-IN1.1 C alleles were higher in HIV infected patients than in healthy controls (-403: 28.2% vs 15.1%, P = 0.002; -28: 5.4% vs 2.8%, not significant; IN1.1: 19.0% vs 11.0%, P = 0.038). In HIV/HCV coinfected patients, these RANTES alleles were less frequent than in patients with HIV infection alone (15.4% P = 0.002; 1.7%; P = 0.048; 12.0%; not significant). Frequencies of these alleles were not significantly different between HIV/HCV positive patients, HCV positive patients and healthy controls. CONCLUSION: All three RANTES polymorphisms showed increased frequencies of the variant allele exclusively in patients with HIV monoinfection. The finding that the frequencies of these alleles remained unaltered in HIV/HCV coinfected patients suggests that HCV coinfection interferes with selection processes associated with these alleles in HIV infection.     

Polymorphic metabolic susceptibility genes and longevity: a study in octogonarians

In order to investigate possible associations of genetic variants in genes of xenobiotic metabolism with longevity, we compared allele frequencies and genotype distributions of polymorphic genes between 205 octogenarians and a non-cancer reference group of 294 persons aged less than 80 years. We analyzed common sequence variations in the cytochrome P-450 genes CYP1A1 T461N, 3801 T > C and CYP1B1 V432L, and in the glutathione S-transferase genes GSTM1 (deletion), GSTT1 (deletion), and GSTP1 (I105V). In octogenarians, the CYP1B1 432L allele was less prevalent than in the reference group (allele frequency 0.49 versus 0.60; odds ratio, OR, 0.63, 95% confidence limits (CI) 0.40-1.00). Octogenarians turned out to have marginally significant more GSTM1 negatives (frequency 0.56 versus 0.48; OR 1.41, 95% CI 0.97-2.05), but less GSTT1 deficient genotypes (frequency 0.14 versus 0.21; OR 0.64; 95% CI 0.38-1.06). In octogenarians without cancer, GSTT1 negative carriers were less prevalent than in the aged with cancer (frequency 0.12 versus 0.27; OR 2.81; 95% CI 1.00-7.38). Polymorphic metabolic susceptibility genes could become relevant for processes of aging when toxic defense mechanisms decline.     

One-step detection of c-kit point mutations using peptide nucleic acid-mediated polymerase chain reaction clamping and hybridization probes

The prognostic significance of somatic activating codon 816 c-kit mutations in pediatric urticaria pigmentosa has not yet been established in detail. Detection of such mutations in archival paraffin-embedded biopsies is usually hampered by an abundance of surrounding normal cells. Here we describe a method for the selective amplification and specific detection of c-kit mutation Asp816-->Val in complete tissue sections cut from up to 24-year-old paraffin blocks. Peptide nucleic acid-mediated polymerase chain reaction clamping of the wild-type allele was combined with on-line mutation detection using oligonucleotide hybridization probes. In DNA extracted from HMC-1 cells heterozygously carrying the c-kit mutation Asp816-->Val, the one-tube assay allowed specific detection of this mutation in a more than 1000-fold excess of normal background DNA within 1 hour and without the need for additional analytical steps. In a series of 38 cases with pediatric urticaria pigmentosa we detected c-kit codons 815 and 816 mutations in 16 cases. Mutation detection did not correlate with clinical outcome after a mean follow-up of 11.2 years. In conclusion, the procedure described may represent an ideal screening tool for all kinds of clinical applications, using point mutations as markers of, for example, early events in carcinogenesis, circulating metastatic tumor cells, and minimal residual disease.