Exposure to anthrax toxin alters human leucocyte expression of anthrax toxin receptor 1.

Anthrax is a toxin-mediated disease, the lethal effects of which are initiated by the binding of protective antigen (PA) with one of three reported cell surface toxin receptors (ANTXR). Receptor binding has been shown to influence host susceptibility to the toxins. Despite this crucial role for ANTXR in the outcome of disease, and the reported immunomodulatory consequence of the anthrax toxins during infection, little is known about ANTXR expression on human leucocytes. We characterized the expression levels of ANTXR1 (TEM8) on human leucocytes using flow cytometry. In order to assess the effect of prior toxin exposure on ANTXR1 expression levels, leucocytes from individuals with no known exposure, those exposed to toxin through vaccination and convalescent individuals were analysed. Donors could be defined as either 'low' or 'high' expressers based on the percentage of ANTXR1-positive monocytes detected. Previous exposure to toxins appears to modulate ANTXR1 expression, exposure through active infection being associated with lower receptor expression. A significant correlation between low receptor expression and high anthrax toxin-specific interferon (IFN)-γ responses was observed in previously infected individuals. We propose that there is an attenuation of ANTXR1 expression post-infection which may be a protective mechanism that has evolved to prevent reinfection.

Differential Toll-like receptor-dependent collagenase expression in chondrocytes.

OBJECTIVES: To characterise the catabolic response of osteoarthritic chondrocytes to Toll-like receptor (TLR) ligands. METHODS: Induction of the collagenases, matrix metalloproteinase (MMP)1 and MMP13, by TLR ligands was assessed in chondrocytes by real-time reverse transcriptase (RT)-PCR. TLR signalling pathway activation and their involvement in collagenase induction were confirmed by immunoblotting and use of pathway inhibitors and siRNA. TLR expression was compared in the femoral head cartilage of normal controls and patients with osteoarthritis (OA) by real-time RT-PCR. RESULTS: Ligands for TLR6/2 and TLR3 showed the greatest upregulation of MMP1 and MMP13 respectively, although all TLR ligands upregulated these MMPs. MMP1 and MMP13 induction by TLR3 and TLR1/2 or TLR6/2 ligands were dependent on Trif and MyD88, respectively. These inductions were dependent upon the nuclear factor (NF)kappaB pathway, but were differentially inhibited by various mitogen-activated protein kinase inhibitors, with MMP13 induction most reliant on the extracellular signal-regulated kinase pathway. In addition, ligands for TLR1/2 and TLR6/2, but not TLR3, induced significant collagenolysis in a cartilage resorption assay. Finally, TLR2 was significantly downregulated and TLR3 upregulated in OA, compared to normal, cartilage. CONCLUSIONS: Activation of chondrocyte TLRs leads to differential collagenase gene activation. Treatment of chondrocytes with TLR1/2 or TLR6/2 ligands resulted in collagen resorption. The modulated expression of chondrocyte TLR2 and TLR3 in OA cartilage, compared to normal, may reflect a response to repair cartilage or prevent further extracellular matrix destruction. These data suggest modulation of TLR-mediated signalling as a potential therapeutic strategy for the treatment of OA.

The fraction 1 and V protein antigens of Yersinia pestis activate dendritic cells to induce primary T cell responses.

The F1 and V antigens of Yersinia pestis, despite acting as virulence factors secreted by the organism during infection, also combine to produce an effective recombinant vaccine against plague, currently in clinical trial. The protective mechanisms induced by rF1 + rV probably involve interactions with dendritic cells (DC) as antigen uptake, processing and presenting cells. To study such interactions, naive ex vivo DC from bone marrow, spleen and lymph node were cultured with rF1, rV or combined antigens and demonstrated to secrete interleukin (IL)-4 and IL-12 into the culture supernatant. Cytokine production in response to pulsing was dependent on the maturity of the bone marrow-derived DC culture, so that pulsed 8-day-old cultures had accumulated significantly more intracellular IL-4 and IL-12 than unpulsed cells. DC, pulsed with rF1 + rV for 2-24 h, were able to prime naive autologous lymph node T cells to proliferate in an antigen dose-dependent manner, with an order of potency of 3d bone marrow-derived DC (BMDC) > 7d BMDC > splenic DC. Significantly, cell-free supernatants from rF1 + rV-pulsed BMDC and splenic DC were also able to induce specific primary responses effectively in naive T cells, suggesting that these supernatants contained stimulatory factor(s). This study suggests an important role for DC, or factors secreted by them, in the induction of protective immunity to plague by the rF1 and rV antigens.

H2-M3 major histocompatibility complex class Ib-restricted CD8 T cells induced by Salmonella enterica serovar Typhimurium infection recognize proteins released by Salmonella serovar Typhimurium.

Salmonella enterica serovar Typhimurium causes a typhoid-like disease in mice which has been studied extensively as a model for typhoid fever in humans. CD8 T cells contribute to protection against S. enterica serovar Typhimurium in mice, but little is known about the specificity and major histocompatibility complex (MHC) restriction of the response. We report here that CD8 T-cell lines derived from S. enterica serovar Typhimurium-infected BALB/c mice lysed bone marrow macrophages infected with S. enterica serovar Typhimurium or pulsed with proteins from S. enterica serovar Typhimurium culture supernatants. Cytoxicity was beta-2-microglobulin dependent and largely TAP dependent, although not MHC class Ia restricted, as target cells of several different MHC haplotypes were lysed. The data suggested the participation of class Ib MHC molecules although no evidence for the presence of Qa1-restricted T cells could be found, unlike in previous reports. Instead, the T-cell lines lysed H2-M3-transfected fibroblasts infected with S. enterica serovar Typhimurium SL3261 or treated with Salmonella culture supernatants. Thus, this report increases the number of MHC class Ib antigen-presenting molecules known for Salmonella antigens to three: Qa-1, HLA-E, and now H2-M3. It also expands the range of pathogens that induce H2-M3-restricted CD8 T cells to include an example of gram-negative bacteria.

Distribution of productive antigen-processing activity for MHC class II presentation in macrophages.

We demonstrated that an epitope from the recombinant protective antigen (rPA) of Bacillus anthracis was presented by mature major histocompatibility complex class II (MHC-II) molecules, whereas an epitope from the recombinant virulent (rV) antigen of Yersinia pestis was presented by newly synthesized MHC-II. We addressed which endosomal compartments were involved in the antigen processing of each epitope. Bone-marrow-derived macrophages were subjected to subcellular fractionation; fractions were analysed for the expression of endosomal markers and used as a source of enzyme activity for the processing of rPA and rV antigens. The rPA epitope was productively processed by dense lysosomal fractions and light membrane fractions expressing early endosomal markers Rab5 and early endosomal antigen-1 as well as markers of antigen-presenting compartments (MHC-II, DM, DO and Ii chain). In contrast, the rV epitope was productively processed only by dense fractions with lysosomal activity. No productive antigen-processing activity was associated with fractions of intermediate density expressing Rab7 and Rab9, characteristic of late endosomes. The data suggest that endosomal compartments expressing Rab5 guanosine triphosphatase can productively process protein antigens for presentation by mature MHC class II molecules.

Differential efficacy of vaccinia virus envelope proteins administered by DNA immunisation in protection of BALB/c mice from a lethal intranasal poxvirus challenge.

DNA vaccines might offer an alternative to the live smallpox vaccine in providing protective efficacy in an orthopoxvirus (OPV) lethal respiratory challenge model. BALB/c mice were immunised with DNA vaccines coding for 10 different single vaccinia virus (VACV) membrane proteins. After an intranasal challenge with the VACV IHD strain, three gene candidates B5R, A33R and A27L produced > or =66% survival. The B5R DNA vaccine consistently produced 100% protection and exhibited greatest efficacy after three 50 microg intramuscular doses in this model. Sero-conversion to these vaccines was often inconsistent, implying that antibody itself was not a correlate of protection. The B5R DNA vaccine induced a strong and consistent gamma interferon (IFNgamma) response in BALB/c mice given a single DNA vaccine dose. Strong IFNgamma responses were also measured in pTB5R immunised C57BL6 mice deficient for MHC class I molecules, suggesting that the memory response was mediated by a CD4+ T cell population.

Virus characterization, clinical presentation, and pathology associated with H7N3 avian influenza in British Columbia broiler breeder chickens in 2004.

Low-pathogenicity avian influenza (LPAI) subtype H7N3 was diagnosed on a two-age broiler breeder farm in Abbotsford, British Columbia (BC), in early February 2004. The presenting complaint in the older index flock was feed refusal, with 0.5% mortality over 72 hr that resolved over the following week Ten days after the initial complaint in the index flock, a younger flock in an adjacent barn experienced an abrupt spike in mortality (25% in 48 hr). The gross lesions of tracheal hyperemia and hilar pulmonary consolidation were subtle and nonspecific, and the diagnosis of avian influenza required laboratory confirmation. Two different viruses were isolated from the index farm: a LPAI (H7N3) was isolated from the older flock and a high-pathogenicity avian influenza (HPAI) (H7N3), which had an additional 21 base insertion at the hemagglutinin-cleavage site, was isolated from the younger flock. The presence of this insertion sequence and the similarity of adjacent sequences indicate that the LPAI had mutated into HPAI at some point between the first and second barn. Despite enhanced on-farm biosecurity measures, the virus was not contained on the index farm and eventually spread to over 40 commercial poultry facilities before massive depopulation efforts enabled its eradication.

Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry. II. Limitations of complex mixture analyses.

With an emphasis on obtaining a multitude of high quality tandem mass spectrometry spectra for protein identification, instrumental parameters are described for the liquid chromatography-tandem mass spectrometry analysis of trypsin digested unfractionated urine using a hybrid quadrupole-time-of-flight (Q-TOF) mass spectrometer. Precursor acquisition rates of up to 20 distinct precursors/minute in a single analysis were obtained through the use of parallel precursor selection (four precursors/survey period) and variable collision induced dissociation integration time (1 to 6 periods summed). Maximal exploitation of the gas phase fractionated ions was obtained through the use of narrow survey scans and iterative data-dependent analyses incorporating dynamic exclusion. The impact on data fidelity as a product of data-dependent selection of precursor ions from a dynamically excluded field is discussed with regards to sample complexity, precursor selection rates, survey scan range and facile chemical modifications. Operational and post-analysis strategies are presented to restore data confidence and reconcile the greatest number of matched spectra.

Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry. I. Profiling an unfractionated tryptic digest.

The proteome of normal male urine from a commercial pooled source has been examined using direct liquid chromatography-tandem mass spectrometry (LC-MS/MS). The entire urinary protein mixture was denatured, reduced and enzymatically digested prior to LC-MS/MS analysis using a hybrid-quadrupole time-of-flight mass spectrometer (Q-TOF) to perform data-dependent ion selection and fragmentation. To fragment as many peptides as possible, the mixture was analyzed four separate times, with the mass spectrometer selecting ions for fragmentation from a subset of the entire mass range for each run. This approach requires only an autosampler on the HPLC for automation (i.e, unattended operation). Across these four analyses, 1.450 peptide MS/MS spectra were matched to 751 sequences to identify 124 gene products (proteins and translations of expressed sequence tags). Interestingly, the experimental time for these analyses was less than that required to run a single two-dimensional gel.

Identification of incompletely processed potential carboxypeptidase E substrates from CpEfat/CpEfat mice.

In an attempt to identify peptides that may be involved in the obese phenotype observed in CpEfat/CpEfat mice (deficient in Carboxypeptidase E, CpE) samples from fourteen neuroendocrine tissues in wild-type and CpEfat/CpEfat mice were obtained. Peptides were purified from these tissues and potential CpE substrate peptides were enriched using an anhydrotrypsin column that captures peptides with basic C-termini. Bound peptides were subjected to tryptic digestion and followed by liquid chromatography-mass spectrometry analysis. The relative levels of CpEfat/CpEfat versus wild-type peptides were determined by comparison of the ion intensities. Peptide ions elevated in the CpEfat/CpEfat samples were identified by targeted liquid chromatography-tandem mass spectrometry. From those ions, 27 peptides derived from known neuropeptides (including CpE substrates) were identified, together with another 25 peptides from proteins not known to be components of the neuropeptide processing pathway. The known CpE substrates identified included the recently discovered proSAAS, granin-like neuroendocrine peptide precursor that inhibits prohormone processing. The approach demonstrated the feasibility of using an affinity-based method for identifying differences in specific classes of peptides between normal and mutant mice.

Effects of smoking/nicotine on performance and event-related potentials during a short-term memory scanning task.

RATIONALE: Nicotine absorbed from cigarette smoke shortens reaction time (RT) in a wide variety of cognitive tasks. However, relatively few studies have tried to isolate the specific stage(s) of information processing affected by smoking/nicotine. OBJECTIVE: The present study was designed to investigate the effect of smoking/nicotine on the short-term memory (STM) scanning stage of information processing in minimally abstaining smokers. Both RT and event-related potentials (ERPs) were measured. METHODS: A Sternberg-type STM-scanning task was performed before and after smoking each of two cigarettes. One cigarette had a 0.05-mg nicotine yield ("denicotinized") and the other had a 1.1-mg yield ("nicotine-yielding"). On each trial, either 2, 3, or 4 consonants were displayed as a memory set. After a brief interval, a single probe consonant was displayed. If the probe was in the memory set (positive probe) a right button press was required, and if the probe was not in the memory set (negative probe) the left button was pressed. RESULTS: Smoking the nicotine-yielding cigarette but not the denicotinized cigarette shortened RT. However, memory-scanning speed, as estimated from the increase in RT as a function of increasing set size, was not differentially affected by the two types of cigarettes. For the ERPs, smoking the nicotine-yielding but not the denicotinized cigarette (a) reduced N200 latency to both the memory-set stimuli and negative probes, (b) increased N200 amplitude to negative probes and P300 amplitude to both types of probes, and (c) produced a sustained negative shift in memory-set ERP amplitude beginning around 600 ms post-stimulus. CONCLUSION: While smoking/nicotine shortened probe RT, it did not affect the speed of STM scanning. Moreover, the ERP-latency effects obtained for the probes were small relative to the effects of smoking/nicotine on RT, suggesting that smoking/nicotine shortens RT primarily by affecting response-related processes.

Caffeine antagonism of alcohol-induced driving impairment.

The extent to which caffeine antagonizes alcohol-induced impairment of simulated automobile driving at the current lowest legal American limit (0.08% BrAC) was the focus of this study. Fifteen adults swallowed a capsule (0, 200, or 400 mg caffeine) then drank a beverage (0.0 or 0.6 g/kg ethanol) in a within-subject, double-blind, randomized procedure. Forty-five minutes later, participants completed a test battery of subjective effects scales, dynamic posturography, critical flicker fusion (CFF), choice reaction time (CRT), divided attention (Stroop test), and simulated driving. Alcohol alone increased ratings of 'dizzy', 'drug effect', and 'high', slowed CRT and brake latency, and increased body sway. Caffeine alone increased ratings of 'alert' and 'jittery', but did not significantly affect body sway or psychomotor performance. Both caffeine doses comparably counteracted alcohol impairment of brake latency but not CRT or body sway. Brake latency with either alcohol-caffeine combination remained significantly longer than that with placebo. Stroop and CFF performance were unaffected by any drug condition. The results suggest that caffeine may increase alertness and improve reaction time after alcohol use but will not completely counteract alcohol impairment in a driver.

Automated LC-LC-MS-MS platform using binary ion-exchange and gradient reversed-phase chromatography for improved proteomic analyses.

A simple multidimensional liquid chromatography system utilizing an isocratic pump and a HPLC system is described for the comprehensive proteomic analysis of complex peptide digest mixtures by coupled LC-LC-MS-MS techniques. A binary ion-exchange separation was achieved through the use of a strong cation-exchange column followed by a reversed-phase column for data-dependent LC-MS-MS analysis of the unbound analytes, and following salt elution (and concomitant column reequilibration), the bound analytes. Off-line validation of the platform showed near quantitative recovery of fractionated peptides and essentially complete ion-exchange partitioning. In comparative analyses of a highly complex peptide digest mixture a >40% increase in the number of peptide and protein identifications was achieved using this multidimensional platform compared to an unfractionated control.

Mastering research critique and statistical interpretation. Guidelines and golden rules.

Mastery of statistical analysis research critique is an important skill for professional nurses. A Guideline for Statistical Analysis and Golden Rules for Statistical Analysis Adequacy are presented and applied to classroom use. Students who learn how to critique research and statistics usage effectively are satisfied consumers and report using knowledge in other clinical courses. Effective strategies to teach statistical analysis critique are discussed.

Testing meningococcal vaccines for mitogenicity and superantigenicity.

Proteins with intrinsic mitogenic properties are widely represented in prokaryotes, such as in different Streptococcus species (1-3), Candida albicans (4), and Eikenella corrodens (5). Specifically, several bacterial porins of Escherichia coli, Shigella dysenteriae, Salmonella typhimurium, Fusobacterium nucleatum, and pathogenic Neisseria species have been shown to induce nonspecific proliferation of lymphocytes (6-12).

CD4 T-cell epitope mapping.

The majority of T cells recognize peptide epitopes bound to major histocompatibility complex (MHC)-encoded glycoproteins on the surface of professional antigen-presenting cells (APC), principally dendritic cells, macrophages, and B cells (1-3). Most T cells are specific for peptide epitopes in association with either classical MHC class Ia molecules (HLA-A, B, and C in humans and H2-K, D, and L in mice) in the case of CD8(+) T cells, or class II molecules (HLA-DR, DP, and DQ in humans and H2-A and E in mice) for CD4(+) T cells. However, a significant proportion of T cells recognize peptide antigens bound to nonclassical MHC class Ib molecules such as the human HLA-E (mouse analog Qa1) (4) and mouse H2-M3 (5). In addition, some T cells recognize not peptides but lipid or glycolipid antigens bound to nonclassical MHC class Ib molecules such as CD1 in both humans and mice (6).

Increasing students' cultural sensitivity. A step toward greater diversity in nursing.

Increasing minority representation in nursing is essential to assure culturally competent care in the next century. Academic institutions need to recruit, encourage, and retain minority students and faculty. Students need minority mentors in their academic and clinical environments. Euro-American faculties teaching in predominantly white institutions need to explore effective ways to facilitate aggregate understanding and appreciation of cultural diversity within the profession. The author explains a teaching strategy to increase awareness and sensitivity of graduate nursing students to the role of minority nurses within the culture of nursing. Becoming culturally sensitive is a prerequisite to increasing diversity and culturally competent care.

Distinguishing withdrawal relief from absolute facilitation: commentary on Bell et al.

A recent article in this journal by Bell and colleagues (Bell SL, Taylor RC, Singleton EG, Henningfield JE, Heishman SJ, Nicotine & Tobacco Research 1:45-52, 1999) studied the effects of smoking on cognitive performance using an overnight smoking abstention design. They interpreted their results for a two-letter search task (poorer performance following abstention that was improved by smoking) as indicating 'nicotine withdrawal-induced cognitive impairment' (p. 50). However, in this commentary, we point out that overnight-abstention experimental designs cannot distinguish withdrawal relief from absolute facilitation of performance (or a combination of the two). We suggest two approaches to resolving the issue of the nature of smoking/nicotine's effects on human cognitive performance.

Intraneural perineurioma involving the median nerve.

Intraneural perineurioma is a rare clinical entity, which tends to affect major nerve trunks in the upper extremities. On light microscopy, numerous pseudo-onion-bulb structures having a central clear area are surrounded by concentric layers of eosinophilic elongate cells having spindled nuclei. Immunohistochemistry of concentric cells stains positive for epithelial membrane antigen but negative for S100 protein. Because of the small number of cases, no consensus has been made on proper treatment of this entity. Although none of the patients who have had excision of tumor with nerve grafting have had sensory nerve recovery, we believe each patient should be individualized until more data are available regarding this tumor.