Evaluation of the Hologic Panther Transcription-Mediated Amplification Assay for Detection of Mycoplasma genitalium.

The detection of Mycoplasma genitalium was evaluated on 1,080 urine samples by the use of a Panther instrument. Overall sensitivity, specificity, positive predictive values, and negative predictive values were 100%, 99.4%, 93.6%, and 100%, respectively. Detection of M. genitalium by the use of the Panther transcription-mediated amplification assay offers a simple, accurate, and sensitive platform for diagnostic laboratories.

Pharmacokinetics, pharmacodynamics and safety of QGE031 (ligelizumab), a novel high-affinity anti-IgE antibody, in atopic subjects.

BACKGROUND: Using a monoclonal antibody with greater affinity for IgE than omalizumab, we examined whether more complete suppression of IgE provided greater pharmacodynamic effects, including suppression of skin prick responses to allergen. OBJECTIVE: To explore the pharmacokinetics, pharmacodynamics and safety of QGE031 (ligelizumab), a novel high-affinity humanized monoclonal IgG1κ anti-IgE. METHODS: Preclinical assessments and two randomized, placebo-controlled, double-blind clinical trials were conducted in atopic subjects. The first trial administered single doses of QGE031 (0.1-10 mg/kg) or placebo intravenously, while the second trial administered two to four doses of QGE031 (0.2- 4 mg/kg) or placebo subcutaneously at 2-week intervals. Both trials included an open-label omalizumab arm. RESULTS: Sixty of 73 (82%) and 96 of 110 (87%) subjects completed the intravenous and subcutaneous studies, respectively. Exposure to QGE031 and its half-life depended on the QGE031 dose and serum IgE level. QGE031 had a biexponential pharmacokinetic profile after intravenous administration and a terminal half-life of approximately 20 days. QGE031 demonstrated dose- and time-dependent suppression of free IgE, basophil FcεRI and basophil surface IgE superior in extent (free IgE and surface IgE) and duration to omalizumab. At Day 85, 6 weeks after the last dose, skin prick wheal responses to allergen were suppressed by > 95% and 41% in subjects treated subcutaneously with QGE031 (2 mg/kg) or omalizumab, respectively (P < 0.001). Urticaria was observed in QGE031- and placebo-treated subjects and was accompanied by systemic symptoms in one subject treated with 10 mg/kg intravenous QGE031. There were no serious adverse events. CONCLUSION AND CLINICAL RELEVANCE: These first clinical data for QGE031, a high-affinity IgG1κ anti-IgE, demonstrate that increased suppression of free IgE compared with omalizumab translated to superior pharmacodynamic effects in atopic subjects, including those with high IgE levels. QGE031 may therefore benefit patients unable to receive, or suboptimally treated with, omalizumab.

A quantitative approach to histopathological dissection of elastin-related disorders using multiphoton microscopy.

BACKGROUND: Multiphoton microscopy (MPM) is a novel imaging technology that has recently become applicable for diagnostic purposes. The use of (near) infrared light in MPM allows for deep tissue imaging. In addition, this modality exploits the autofluorescent nature of extracellular matrix fibres within the skin. OBJECTIVES: To quantitate the structure and abundance of elastic fibres in human dermis in three dimensions utilizing autofluorescent signals generated by MPM for the objective examination of elastin-related skin disorders. METHODS: Cross-sections of skin samples from elastin-related disorders were analysed by MPM and correlated to histopathology. In situ visualization of elastic fibres by MPM was conducted by en face imaging of ex vivo skin samples through the intact epidermis. Image analysis software was used to quantify elastic fibres in three dimensions. RESULTS: Based on the MPM-detected elastin-specific autofluorescence, we developed the Dermal Elastin Morphology Index (DEMI), calculated as the ratio of elastic fibre surface area and volume. This enabled objective three-dimensional quantification of elastic fibres. Quantitative scoring of sun-damaged skin using DEMI correlated with qualitative histopathological grading of the severity of solar elastosis. Furthermore, this approach was applied to changes in elastic fibre architecture in other disorders, such as pseudoxanthoma elasticum (PXE), PXE-like syndrome, elastofibroma, focal dermal elastosis, anetoderma, mid-dermal elastolysis and striae distensae. We imaged elastic fibres in intact ex vivo skin imaged en face through the epidermis, indicating that this approach could be used in vivo. CONCLUSIONS: MPM has the potential for noninvasive in vivo visualization of elastic fibres in the dermis with near histological resolution. DEMI allows objective assessment of elastic fibres to support diagnosis and monitoring of disease progress or therapy of elastin-related skin disorders.

Impact of labile and recalcitrant carbon treatments on available nitrogen and plant communities in a semiarid ecosystem.

In a 10-year study, we assessed the influence of five carbon (C) treatments on the labile C and nitrogen (N) pools of historically N-enriched plots on the Shortgrass Steppe Long Term Ecological Research site located in northeastern Colorado. For eight years, we applied sawdust, sugar, industrial lignin, sawdust + sugar, and lignin + sugar to plots that had received N and water additions in the early 1970s. Previous work showed that past water and N additions altered plant species composition and enhanced rates of nutrient cycling; these effects were still apparent 25 years later. We hypothesized that labile C amendments would stimulate microbial activity and suppress rates of N mineralization, whereas complex forms of carbon (sawdust and lignin) could enhance humification and lead to longer-term reductions in N availability. Results indicated that, of the five carbon treatments, sugar, sawdust, and sawdust + sugar suppressed N availability, with sawdust + sugar being the most effective treatment to reduce N availability. The year after treatments stopped, N availability remained less in the sawdust + sugar treatment plots than in the high-N control plots. Three years after treatments ended, reductions in N availability were smaller (40-60%). Our results suggest that highly labile forms of carbon generate strong short-term N sinks, but these effects dissipate within one year of application, and that more recalcitrant forms reduce N longer. Sawdust + sugar was the most effective treatment to decrease exotic species canopy cover and increase native species density over the long term. Labile carbon had neither short- nor long-term effects on exotic species. Even though the organic amendments did not contribute to recovery of the dominant native species Bouteloua gracilis, they were effective in increasing another native species, Carex eleocharis. These results indicate that organic amendments may be a useful tool for restoring some native species in the shortgrass steppe, though not all.

Audit of a sexual health website email link for general practitioners.

The aim of this review was to evaluate an 'Email a Clinician' link on a medically reviewed sexual health website, which was established to allow general practitioners (GPs) to communicate remotely with sexual health clinic specialists. The website was developed in consultation with GPs and extensively promoted throughout the relevant professional primary health-care networks. Despite this, the email link appeared to fail in its objective of facilitating GP access to specialist sexual health physician opinion within five working days. An audit examining use of the email link was conducted for a one-year period, during which time 324 emails were received. Results showed that the bulk of the emails (93.2%) were spam, and only 6.8% were genuine enquiries. Of the 22 genuine emails, 21 (95%) originated from the general public and there were no enquiries from the GPs, who were the target audience of the website, resulting in removal of the email link from the site. Direct survey of local GPs to evaluate reasons for non-utilization of the link was not possible. However, discomfort with the technology, time added to existing workload, lack of direct perceived benefit and lack of immediate response have been cited as contributing factors that may limit widespread adoption of other telemedicine services. As a new generation of recently graduated GPs enters the Australian workforce, who might be expected to be skilled and comfortable with electronic medical communication, the option of a direct email link to a sexual health clinic, with a faster turnaround time, may be worth re-visiting in the future.

Alcohol-induced IL-17A production in Paneth cells amplifies endoplasmic reticulum stress, apoptosis, and inflammasome-IL-18 activation in the proximal small intestine in mice.

Gut microbial translocation contributes to alcoholic hepatitis. Using a mouse model of alcoholic hepatitis, we investigated the effects of chronic alcohol plus binge and found increased abundance of Paneth cells and IL-17A in the proximal small intestine (PSI). Alcohol increased IL-17A production and pro-apoptotic signaling evidenced by Bax, Bim, caspase-3, and caspase-8 increases via endoplasmic reticulum (ER) stress indicated by C/EBP homologous protein (CHOP) upregulation; this was prevented by the ER stress inhibitor, 4-PBA, in isolated crypts in vitro and in vivo. Mechanistically, IL-17 augmented alcohol-induced ER stress in isolated crypts. In vivo IL-17A blocking antibody administration in alcohol-treated mice attenuated ER stress-mediated apoptosis and IL-18 induction and prevented alcohol-induced impairment of tight junctions in the PSI and LPS translocation to the liver. Acute-on-chronic alcohol resulted in inflammasome activation, caspase-1 cleavage, and IL-18 production in the PSI. In vivo treatment with antibiotics or 4-PBA prevented CHOP upregulation and inflammasome activation. Our data suggest that alcohol upregulates innate immune mechanisms by increasing Paneth cell numbers and IL-17A release contributing to apoptosis amplification, inflammasome activation, and gut leakiness in the PSI. Binge alcohol-induced Paneth cell expansion, ER stress, and inflammasome activation in the PSI are modulated by the gut microbiome.

The Ha-ras protein, p21, is modified by a derivative of mevalonate and methyl-esterified when expressed in the insect/baculovirus system.

Using the insect/baculovirus expression system, we demonstrate the incorporation of [3H]mevalonate and [3H]methyl groups into recombinant c-Ha-ras protein (p21). Unlike the post-translational palmitoylation of p21 expressed in this system, the modification by mevalonate is not removed by hydroxylamine suggesting the absence of a thioester linkage. It is highly likely that the insect expression system recognizes the C-terminal CAAX Motif in p21, incorporates the mevalonate into the recently described polyisoprenylation modification and carboxyl-methylates the protein.

Cloning and characterisation of cDNAs encoding a novel non-receptor tyrosine kinase, brk, expressed in human breast tumours.

Using a polymerase chain reaction based differential screening approach, we have isolated and characterised a cDNA from a human metastatic breast tumour representing a novel protein tyrosine kinase (brk). Sequencing of brk cDNAs isolated from T-47D and MCF-7 human breast tumour cell lines indicate that they encode a protein with the features of a novel nonreceptor tyrosine kinase, including amino terminal SH3 and SH2 domains. When synthesised in recombinant baculovirus and bacterial expression systems, brk protein products are capable of autophosphorylation on tyrosine residues. Initial expression studies have detected low levels of brk transcripts in some human breast tumours and breast tumour cell lines, but not in normal breast tissue.

Rifampicin binding as a probe for subunit interactions in Escherchia coli RNA polymerase.

The binding of the inhibitor rifampicin to RNA polymerase (alpha2betabeta') and its deficient subunit mixtures was investigated. The ability of beta to bind stoichiometric amounts of rifampicin was restored by formation of the alpha2beta subassembly. beta,beta' alpha, betabeta' and alpha2beta' were unable to bind rifampicin. RNA polymerase denatured with 6 M guanidine hydrochloride and dialysed against a renaturing buffer at 0degrees C ("renatured inactive enzyme") bound stoichiometric amounts of rifampicin but had lost the ability of bind dna. compared with native RNA polymerase "renatured inactive" enzyme possessed a markedly different tertiary structure as judged by limited proteolysis.

Fluorescence anisotropy from diphenylhexatriene in rat liver plasma membranes.

Rat livers were fractionated to obtain intracellular membrane preparations and a highly purified preparation of bile canaliculi. The fraction containing bile canaliculi was homogenized and subfractionated to give fractions representing fragments of contiguous membrane and of canalicular microvilli. The relative purity and extent of contamination of each preparation was determined. When the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene was incorporated into aliquots of each fraction at the same probe: lipid ratio and the steady-state anisotropy of its fluorescence measured, it was found that the plasma membrane preparations were much more ordered than the intracellular membrane preparations. Of the plasma membrane preparations, that containing the canalicular microvilli was the most ordered, even allowing for any contribution of contaminants. Thus the microvillus membrane of the bile canaliculus appears to be the most ordered domain of the plasma membrane of the hepatocyte. The high order in this domain may be a factor in reducing the susceptibility to bile salt damage during bile secretion, since it is this region which is exposed to high concentrations of bile salts in vivo.

Membrane fluidity and bile salt damage.

The lysis, by bile salts, of membranes of different fluidities was studied; it was shown that membranes of low fluidity were less readily lysed than membranes of higher fluidity. Membrane fluidity levels were controlled (i) by the use of erythrocytes, from different species, systematically differing in their lipid composition; (ii) by using each membrane at a range of temperatures; and (iii) by incorporating into the membranes the fluidizing agent, benzyl alcohol, at a range of concentrations. Membrane fluidity (and order) in each case was monitored by measuring the degree of polarization of fluorescence from the hydrophobic probe molecule, 1,6-diphenyl-1,3,5-hexatriene. The response of lytic behaviour to modulations of membrane fluidity also indicated a difference between the bile salts, glycodeoxycholate and glycocholate; the former initiates lysis close to (at or below) its critical micellar concentrations whereas the latter only causes lysis above, and often substantially above, its critical micellar concentration. In their respective ranges of lytic concentrations, both bile salts are far less effective with membranes of low fluidity. The results are discussed with regard to the features of a membrane which would be expected to be resistant to high concentrations of bile salts in vivo, i.e., the plasma membranes of the bile canaliculus and lumenal surface of biliary tract cells.

Membrane lipid composition and susceptibility to bile salt damage.

Erythrocyte membranes with low sphingomyelin : choline-containing phospholipid ratios haemolyse at low concentrations of the bile salt, glycocholate. Erythrocytes with higher sphingomyelin : choline-containing phospholipid ratios require progressively greater concentrations of the bile salt for lysis. Sublytic concentrations of glycocholate remove phospholipid and acetylcholinesterase from the membranes. Membranes with low sphingomyelin : choline-containing phospholipid ratios lose both particulate (microvesicles of distinct composition) and 'solubilized' material, the particulate form predominating. The proportion of particulate material falls with increase of the membrane sphingomyelin : choline-containing phospholipid ratio and those membranes of highest sphingomyelin : choline-containing phospholipid ratio lose material predominantly in 'solubilized' form. Sheep erythrocytes treated to increase their content of phosphatidylcholine (and thereby reduce their membrane sphingomyelin : choline-containing phospholipid ratio) become more susceptible to lysis by glycocholate. These observations indicate a correlation between membrane lipid composition and the perturbation of membranes with bile salt; they also point to possible features of membranes capable of surviving exposure to the high bile salt concentrations of the biliary tract.

Importance of human gastric lipase for intestinal lipolysis: an in vitro study.

Using soybean triacylglycerols emulsified with egg lecithin we have studied, in vitro, the influence of substrate prehydrolysis by human gastric lipase upon subsequent degradation by the pancreatic lipase-co-lipase system. Fatty acids liberated by pure human gastric lipase or juice trigger immediate activity of human pancreatic lipase. Gastric lipolysis appears to be of prime importance for dietary lipid digestion in human.

Molecular cloning of a human gastric lipase and expression of the enzyme in yeast.

The molecular cloning of a cDNA coding for human gastric lipase and its expression in yeast is described. A lipase present in human gastric aspirates was purified and its N-terminal amino-acid sequence was determined. This was found to be homologous with the N-terminal sequence of rat lingual lipase. A cDNA library was constructed from mRNA isolated from human stomach tissue and probed with cloned rat lingual lipase DNA. One clone, pGL17, consisting of approximately 1450 base-pairs, contained the entire coding sequence for a human gastric lipase. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid polypeptide with an unglycosylated Mr of 43,162. Human gastric lipase and rat lingual lipase amino-acid sequences were closely homologous but were unrelated to porcine pancreatic lipase apart from a 6 amino-acid sequence around the essential Ser-152 of porcine pancreatic lipase. A yeast expression plasmid containing the phosphoglycerate kinase promoter and terminator sequences together with the human gastric lipase gene was constructed. Yeast transformed with this vector synthesised the lipolytically active enzyme.

T cell recognition of a point mutation in the P21 Ras protein.

Point mutations of p21 Ras proteins correlate with many human malignancies. To determine whether the mutations of Ras proteins generate immunogenic determinants which can be recognized by T cells and possibly serve as targets for immunotherapy, we studied the murine T helper responses to synthetic Ras peptides corresponding to amino acids 1-23 of normal or mutant Ras protein. Immunization of C3H/He and B10.BR mice with Ras peptides containing a valine mutation at position 12 stimulated MHC class II-restricted T helper cells which recognised specifically the Ras mutation. Surprisingly C57BL/10 mice generated T helper responses not only against mutant but also against normal Ras peptides. Importantly, natural processing of Ras protein was found to generate the epitopes recognized by the peptide-induced T cells.

Development of T-cell leukaemia in an ataxia telangiectasia patient following clonal selection in t(X;14)-containing lymphocytes.

Ataxia telangiectasia is a rare inherited and progressive neurological disorder in which patients show an unusual predisposition to T-cell leukaemia. We report here observations on a patient with a large cytogenetically abnormal clone showing a single t(X;14)(q28;q11) translocation which conferred a proliferative advantage on the cells. The further evolution of this clone to cytogenetically more complex clones of lymphocytes was seen in the patient. She subsequently developed a rapidly progressing T-cell leukaemia, with a CD4+CD8+ T-cell phenotype, about five years after the first appearance of additional chromosome translocations in the clone cells.

A critical analysis of the literature on the Internet and consumer health information.

A critical review of the published literature investigating the Internet and consumer health information was undertaken in order to inform further research and policy. A qualitative, narrative method was used, consisting of a three-stage process of identification and collation, thematic coding, and critical analysis. This analysis identified five main themes in the research in this area: (1) the quality of online health information for consumers; (2) consumer use of the Internet for health information; (3) the effect of e-health on the practitioner-patient relationship; (4) virtual communities and online social support and (5) the electronic delivery of information-based interventions. Analysis of these themes revealed more about the concerns of health professionals than about the effect of the Internet on users. Much of the existing work has concentrated on quantifying characteristics of the Internet: for example, measuring the quality of online information, or describing the numbers of users in different health-care settings. There is a lack of qualitative research that explores how citizens are actually using the Internet for health care.

Integrated pharmacokinetic, pharmacodynamic and immunogenicity profiling of an anti-CCL21 monoclonal antibody in cynomolgus monkeys.

QBP359 is an IgG1 human monoclonal antibody that binds with high affinity to human CCL21, a chemokine hypothesized to play a role in inflammatory disease conditions through activation of resident CCR7-expressing fibroblasts/myofibroblasts. The pharmacokinetics (PK) and pharmacodynamics (PD) of QBP359 in non-human primates were characterized through an integrated approach, combining PK, PD, immunogenicity, immunohistochemistry (IHC) and tissue profiling data from single- and multiple-dose experiments in cynomolgus monkeys. When compared with regular immunoglobulin typical kinetics, faster drug clearance was observed in serum following intravenous administration of 10 mg/kg and 50 mg/kg of QBP359. We have shown by means of PK/PD modeling that clearance of mAb-ligand complex is the most likely explanation for the rapid clearance of QBP359 in cynomolgus monkey. IHC and liquid chromatography mass spectrometry data suggested a high turnover and synthesis rate of CCL21 in tissues. Although lymphoid tissue was expected to accumulate drug due to the high levels of CCL21 present, bioavailability following subcutaneous administration in monkeys was 52%. In human disease states, where CCL21 expression is believed to be expressed at 10-fold higher concentrations compared with cynomolgus monkeys, the PK/PD model of QBP359 and its binding to CCL21 suggested that very large doses requiring frequent administration of mAb would be required to maintain suppression of CCL21 in the clinical setting. This highlights the difficulty in targeting soluble proteins with high synthesis rates.

The nucleotide sequence of cDNA coding for the structural proteins of foot-and-mouth disease virus.

The complete nucleotide sequence of cDNA coding for the structural capsid polypeptides of foot-and-mouth disease virus (FMDV) (strain A(10)61) has been determined. Portions of the flanking sequence coding for the nonstructural proteins p20a and p52 are also provided. The three larger structural polypeptides VP1, VP2 and VP3 have unmodified Mrs of 23248, 24649 and 24213, respectively. The size of the smaller polypeptide, VP4, can only be estimated at 7360 because the 5'-limit of its coding region is not yet known with certainty. The sequence data for VP1 (the major immunising antigen) and the amino-terminal quarter of p52 are compared with the data of Kurz et al. (Nucl. Acids Res. 9 (1981) 1919-1931) for a different serotype (O1K). This shows that variation is much greater in the region coding for VP1 than in that coding for p52. This is reflected in the level of amino acid sequence variation predicted for the two proteins. Analysis of relative codon usage reveals a strong bias in favour of C and G over U and A in the third base position. The dinucleotide frequencies show a bias against A-U and U-A, and for A-C and C-A.