Using WHO-FRAX to describe fracture risk: experience in primary care.

Ideally those at highest risk of fracture should be identified prior to fracture occurrence to reduce mortality, morbidity and costs. Case-finding strategies for those at high risk of first fracture or systematic case-finding strategies following fracture are recommended in the UK, rather than population-based screening to identify individuals at high fracture risk. General practices in the UK hold relevant data on individuals beyond fracture history that could allow identification of a wider group of patients at highest risk of fracture. The aim of the paper is to evaluate the feasibility of applying the WHO-FRAX fracture risk calculator to general practice populations using existing recorded data. A cross-sectional study of 2467 women aged 50 years and older (mean 66.2 years, standard deviation = 11.3) registered with two Scottish General Practices with low deprivation (one semi-rural, one urban) was undertaken. Patient data were extracted from the two general practices' patient information databases and the WHO-FRAX calculator was applied to these data. WHO-FRAX calculation was possible on 1872 patients. Of these, 687 patients were found to have a high fracture risk (risk of major facture ≥15% and or risk of hip fracture ≥3% - 37% of the WHO-FRAX assessed cohort) and should be considered for follow-up. In conclusion, use of the WHO-FRAX calculator using general practice-held data is feasible and can help to identify a patient group at higher fracture risk. Further evaluation and treatments can then be targeted at this group.

Gas chromatography-tandem mass spectrometry analysis of red blood cells from Göttingen minipig following whole-body vapor exposure to VX.

A method to detect fluoride ion generated O-ethyl methylphosphonofluoridate (VX-G) in Göttingen minipig red blood cells (RBC) following whole-body exposure to VX vapor utilizing a gas chromatograph-tandem mass spectrometer (GC-MS-MS) has been developed. Dose-response curves for VX exposure were generated after applying the fluoride ion reactivation assay to the RBC fraction of serially collected whole blood samples that were taken after whole-body exposures that varied in both duration and concentration. GC-MS-MS analysis of minipig RBC samples following 180-min exposures at two different concentrations was a more precise indicator for severity of exposure than the analysis of acetylcholinesterase (AChE) inhibition for the same samples. AChE enzyme activity recovered faster than indicated by the apparent elimination rate of VX-G. GC-MS-MS analyses of RBC samples following VX exposure demonstrate this technique has both adequate sensitivity and specificity to indicate the severity of exposure.

Quantification of sarin and cyclosarin metabolites isopropyl methylphosphonic acid and cyclohexyl methylphosphonic acid in minipig plasma using isotope-dilution and liquid chromatography- time-of-flight mass spectrometry.

An analysis method for determining isopropyl methylphosphonic acid (IMPA) and cyclohexyl methylphosphonic acid (CMPA), the metabolic hydrolysis products of toxic organophosphorus nerve agents isopropyl methylphosphonofluoridate (sarin, GB) and cyclohexyl methylphosphonofluoridate (cyclosarin, GF), respectively, has been developed and validated using high-performance liquid chromatography-mass spectrometry with negative ion electrospray ionization with time-of-flight detection (LC-ESI-MS-TOF). The linear range of quantitation was 5 to 125 ng/mL in plasma with a method detection limit of 2 ng/mL for each compound. This method was developed to determine the amount of metabolic hydrolysis that was formed during and after nerve agent exposure in minipigs to account for a major pathway of GB and GF elimination that had not been previously characterized in the bloodstream, particularly during low-level whole-body inhalation experiments. Metabolic hydrolysis accounted for 70% to 90% of the recoverable agent in the bloodstream during exposure, when compared to both unbound and cholinesterase bound agent recovered by fluoride ion reactivation analysis for the same samples. The estimated half-life of IMPA and CMPA in plasma was determined to be 44 and 61 min, respectively. The method utilizes the mass selectivity of LC-ESI-MS-TOF using a bench-top instrument to achieve a detection limit that is consistent with reported LC-MS-MS methods analyzing blood samples.

Biomarkers of low-level exposure to soman vapor: comparison of fluoride regeneration to acetylcholinesterase inhibition.

The nerve agent O-pinacolyl methylphosphonofluoridate, also known as soman or by its military designation GD, is a highly toxic organophosphorous compound that exerts its effects through inhibition of the enzyme acetylcholinesterase (AChE). In the present study, a fluoride ion based regeneration assay was developed to quantify the level of soman present in the blood of rats following a low-level whole-body inhalation exposure. It was hypothesized that the amount of regenerated nerve agent in the blood would be dose dependent in rats subjected to a whole-body inhalation exposure to a low-level dose of soman vapor, and that the fluoride ion-based regeneration method would be more sensitive for the detection of a low-level exposure to soman vapor than the measurement of whole blood AChE activity. Regenerated soman was dose-dependently detected in both the red blood cells (RBCs) and plasma of exposed rats at all concentrations tested (0.033-0.280 mg/m(3) for a 240-min exposure). Significant inhibition of whole blood AChE activity did not occur below a concentration of 0.101 mg/m(3), and was only depressed by approximately 10-25% at concentrations ranging from 0.101 mg/m(3) to 0.280 mg/m(3). This study is the first to utilize a fluoride ion-based regeneration assay to demonstrate the dose-dependent increases in soman in the blood following whole-body inhalation exposure to low levels of vapor. Additionally, the results of the present study demonstrate that the fluoride ion based regeneration assay was approximately threefold more sensitive than the measurement of AChE activity in the blood for the detection of exposure to soman, and also that miosis is a more sensitive marker of soman exposure than inhibition of AChE activity.

Effects of whole-body VX vapor exposure on lethality in rats.

Male and female rats were whole-body exposed to VX vapor in a 1000-L single-pass exposure chamber. Estimated exposure dosages producing lethal (LCT50) effects in 50% of exposed male and female rats were established for 10, 60, and 240 min exposure durations. A potency comparison with GB and GF shows that VX becomes increasingly more potent than these G agents with increasing exposure duration. VX is approximately 4-30 times more potent than GB and 5-15 times more potent than GF. Gender differences in the estimated median dosages were not significant at the 10, 60, and 240 min exposure durations. An empirical toxic load model was developed and the toxic load exponent for lethality (n) in the equation Cn x T = k was determined to be n = 0.92. The VX-G regeneration assay was successfully used as a biomarker for the presence of VX in the blood plasma and RBC fractions of the blood 24 h postexposure.

Microarray analysis of differentially expressed genes of primary tumors in the canine central nervous system.

The pathophysiologic similarities of many human and canine cancers support the role of the domestic dog as a model for brain tumor research. Here we report the construction of a custom canine brain-specific cDNA microarray and the analysis of gene expression patterns of several different types of canine brain tumor. The microarray contained 4000 clones from a canine brain specific cDNA library including 2161 clones that matched known genes or expressed sequence tags (ESTs) and 25 cancer-related genes. Our study included 16 brain tumors (seven meningiomas, five glial tumors, two ependymomas, and two choroid plexus papillomas) from a variety of different dog breeds. We identified several genes previously found to be differentially expressed in human brain tumors. This suggests that human and canine brain tumors share a common pathogenesis. In addition, we also found differentially expressed genes unique to either meningiomas or the glial tumors. This report represents the first global gene expression analysis of different types of canine brain tumors by cDNA microarrays and might aid in the identification of potential candidate genes involved in tumor formation and progression.

Quantitation of fluoride ion released sarin in red blood cell samples by gas chromatography-chemical ionization mass spectrometry using isotope dilution and large-volume injection.

A new method for measuring fluoride ion released isopropyl methylphosphonofluoridate (sarin, GB) in the red blood cell fraction was developed that utilizes an autoinjector, a large-volume injector port (LVI), positive ion ammonia chemical ionization detection in the SIM mode, and a deuterated stable isotope internal standard. This method was applied to red blood cell (RBC) and plasma ethyl acetate extracts from spiked human and animal whole blood samples and from whole blood of minipigs, guinea pigs, and rats exposed by whole-body sarin inhalation. Evidence of nerve agent exposure was detected in plasma and red blood cells at low levels of exposure. The linear method range of quantitation was 10-1000 pg on-column with a detection limit of approximately 2-pg on-column. In the course of method development, several conditions were optimized for the LVI, including type of injector insert, injection volume, initial temperature, pressure, and flow rate. RBC fractions had advantages over the plasma with respect to assessing nerve agent exposure using the fluoride ion method especially in samples with low serum butyrylcholinesterase activity.

A recombinant vaccinia virus encoding the interferon-inducible T-cell alpha chemoattractant is attenuated in vivo.

Murine interferon-inducible T-cell alpha chemoattractant (I-TAC) is a potent non-ELR CXC chemokine that predominantly attracts activated T lymphocytes, binds to the receptor CXCR3 and is induced by interferon-gamma (IFN-gamma). We analysed I-TAC expression by reverse transcriptase-polymerase chain reaction during three different virus-infection models in mice, respiratory syncytial virus (RSV), influenza A and vaccinia virus western reserve (VV-WR). In the lungs from mice infected with RSV or influenza A viruses, peak expression of I-TAC coincided with peak viraemia. Surprisingly, there was no expression in the lungs of mice infected with vaccinia, unlike the elevated expression shown previously for other interferon-regulated chemokines, such as Crg2 and Mig. To further investigate the importance of this difference during vaccinia infection in mice, a recombinant virus encoding I-TAC (rVV I-TAC) was generated. Studies in C57BL/6 and Swiss nude mice showed that I-TAC expression caused increased mononuclear cell infiltration and significantly attenuated the VV. Infection of the footpads of naïve or already immune (with VV-WR) mice with either rVV I-TAC or VV-WR demonstrated that I-TAC expression reduced overall inflammation during infection and that this reduction was more pronounced in already immune mice. The data presented here show that I-TAC can have an important role during virus infections and that vaccinia has evolved ways to avoid inducing I-TAC expression.

Combinations of IFN-gamma and IL-4 induce distinct profiles of dendritic cell-associated immunoregulatory properties.

Interferon gamma (IFN-gamma) and interleukin-4 (IL-4) are not only generated during cell-mediated immunity (CMI) and humoral immunity (HI), but are also generated by innate immune cells in response to pathogenic factors. How these cytokines differentially effect the development of dendritic cell (DC)-associated immunoregulatory properties from progenitor cells during innate immunity is unresolved. To address this we have utilized a homogeneous DC progenitor-like cell line, MTHC-D2, as a model to examine cytokine-induced maturation of DCs. By 6 h IFN-gamma induced genes that are important for antiviral activity and development of CMI, whereas IL-4 induced genes involved in cellular adhesion, uptake of extracellular antigen, suppression of cytotoxic T-cell responses, and that repair the extracellular matrix. By 48 h the cytokine stimulus had induced many properties characteristic of immature DCs; however, these were differentially effected by IFN-gamma and IL-4. IFN-gamma induced the greatest levels of costimulatory/ activation marker expression, and the highest levels of T-cell proliferation, whereas IL-4 induced the greatest levels of phagocytic activity. Stimulation of the cells with CD40 Ab enhanced the levels of costimulatory marker expression and T-cell stimulatory capacity of cells exposed to IFN-gamma, but had little effect on cells exposed to IL-4 in the absence of IFN-gamma.

QSAR studies on P-glycoprotein-regulated multidrug resistance and on its reversal by phenothiazines.

Multidrug resistance is brought about largely by membrane transport proteins such as P-glycoprotein (P-gp). We have developed a quantitative structure-activity relationship (QSAR) for P-gp-associated ATPase activity for a diverse set of 22 drugs, and found that such activity is related to substrate molecular size and polarity. We have also developed a QSAR for drug efflux from the blood-brain barrier of another diverse set of 22 drugs, and found that such efflux is a function of drug size and polarisability. Thirdly, we have carried out a QSAR analysis of the ability of 157 phenothiazines and related drugs to reverse multidrug resistance. We were unable to obtain a good QSAR for the whole data-set, but when we divided the data-set into sub-sets of closely related structures, a series of good correlations was obtained, most of which incorporated descriptors that model molecular size and polarity/polarisability. In no instance did we find any evidence that hydrogen bonding or hydrophobicity play a part in multidrug resistance or its reversal, despite that fact that several other workers have reported that these effects appear to be important here.

IFN-gamma regulates murine interferon-inducible T cell alpha chemokine (I-TAC) expression in dendritic cell lines and during experimental autoimmune encephalomyelitis (EAE).

Murine interferon-inducible T cell alpha chemokine (I-TAC) is a potent non-ELR Cys-X-Cys (CXC) chemokine that predominantly attracts activated T lymphocytes and binds to the receptor CXCR3. Using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) we analysed murine I-TAC expression in two different progenitor dendritic cell (DC) lines, MTHC-D2 and JAWS II which were exposed to various cytokines, and Con A-activated splenocytes from a panel of knockout mice. Analysis of the progenitor DC lines and Con A cultures demonstrated that murine I-TAC is primarily regulated by interferon (IFN)-gamma via interferon regulatory factor (IRF)-1. It has been proposed that I-TAC may have a role in autoimmune diseases such as multiple sclerosis (MS). Because I-TAC appears to be secreted from antigen-presenting cells (APCs) and attracts activated T cells, we examined the level of murine I-TAC mRNA in the central nervous system (CNS) of wild-type and IFN-gamma-receptor knockout (IFN-gammaR-/-) mice with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE). Peak I-TAC expression was detected in wild-type mice on day 14 when the mice begin to recover, whereas very low levels of I-TAC were detected in the CNS of IFN-gammaR-/- mice which develop severe EAE and die. The expression characteristics of murine I-TAC suggest an important mediator of immune cell communication that could augment vaccines and autoimmune therapies.

Chromosome 17 loss-of-heterozygosity studies in benign and malignant tumors in neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant condition characterized by benign tumor (neurofibroma) growth and increased risk of malignancy. Dermal neurofibromas, arising from superficial nerves, are primarily of cosmetic significance, whereas plexiform neurofibromas, typically larger and associated with deeply placed nerves, extend into contiguous tissues and may cause serious functional impairment. Malignant peripheral nerve sheath tumors (MPNSTs) seem to arise from plexiform neurofibromas. The NF1 gene, on chromosome segment 17q11.2, encodes a protein that has tumor suppressor function. Loss of heterozygosity (LOH) for NF1 has been reported in some neurofibromas and NF1 malignancies, but plexiform tumors have been poorly represented. Also, the studies did not always employ the same markers, preventing simple comparison of the frequency and extent of LOH among different tumor types. Our chromosome 17 LOH analysis in a cohort of three tumor types was positive for NF1 allele loss in 2/15 (13%) dermal neurofibromas, 4/10 (40%) plexiform neurofibromas, and 3/5 (60%) MPNSTs. Although the region of loss varied, the p arm (including TP53) was lost only in malignant tumors. The losses in the plexiform tumors all included sequences distal to NF1. No subtle TP53 mutations were found in any tumors. This study also reports the identification of both NF1 "hits" in plexiform tumors, further supporting the tumor suppressor role of the NF1 gene in this tumor type.

Genetic vaccination strategies for enhanced cellular, humoral and mucosal immunity.

In this article, we describe several novel genetic vaccination strategies designed to facilitate the development of different types of immune responses. These include: i) the consecutive use of DNA and fowlpoxvirus vectors in "prime-boost" strategies which induce greatly enhanced and sustained levels of both cell-mediated immunity and humoral immunity, including mucosal responses; ii) the co-expression of genes encoding cytokines and cell-surface receptors, and the use of immunogenic carrier molecules, for immune modulation and/or improved targeting of vector-expressed vaccine antigens; and iii) the expression of minimal immunogenic amino acid sequences, particularly cytotoxic CD8+ T-cell determinants, in "polytope" vector vaccines. The capacity to modulate and enhance specific immune responses by the use of approaches such as these may underpin the development of vaccines against diseases for which no effective strategies are currently available.

A new hereditary cylindromatosis family associated with CYLD1 on chromosome 16.

Hereditary cylindromatosis (HC; MIM 132700) is an autosomal dominant condition characterized by benign skin appendage tumors most commonly on the scalp and face. Previously, the HC gene (CYLD1) was linked to chromosome 16q12-13, and tumors showed loss of heterozygosity (LOH), suggesting that CYLD1 is a tumor suppressor gene. Here we report a new multi-generation cylindromatosis family whose condition maps to that region, with 7/13 tumors showing LOH on 16q.

Immunogenicity of a human immunodeficiency virus (HIV) polytope vaccine containing multiple HLA A2 HIV CD8(+) cytotoxic T-cell epitopes.

Compelling evidence now suggests that alphabeta CD8 cytotoxic T lymphocytes (CTL) have an important role in preventing human immunodeficiency virus (HIV) infection and/or slowing progression to AIDS. Here, we describe an HIV type 1 CTL polyepitope, or polytope, vaccine comprising seven contiguous minimal HLA A2-restricted CD8 CTL epitopes conjoined in a single artificial construct. Epitope-specific CTL lines derived from HIV-infected individuals were able to recognize every epitope within the construct, and HLA A2-transgenic mice immunized with a recombinant virus vaccine coding for the HIV polytope also generated CTL specific for different epitopes. Each epitope in the polytope construct was therefore processed and presented, illustrating the feasibility of the polytope approach for HIV vaccine design. By simultaneously inducing CTL specific for different epitopes, an HIV polytope vaccine might generate activity against multiple challenge isolates and/or preempt the formation of CTL escape mutants.

Comparison of the gating behaviour of human and murine cystic fibrosis transmembrane conductance regulator Cl- channels expressed in mammalian cells.

1. To investigate the function of the murine cystic fibrosis transmembrane conductance regulator (CFTR), a full-length cDNA encoding wild-type murine CFTR was assembled and stably expressed in Chinese hamster ovary (CHO) cells. 2. Like human CFTR, murine CFTR formed Cl- channels that were regulated by cAMP-dependent phosphorylation and intracellular ATP. However, murine CFTR Cl- channels had a reduced single-channel conductance and decreased open probability (Po) compared with those of human CFTR. 3. Analysis of the dwell time distributions of single channels suggested that the reduced Po of murine CFTR was caused by both decreased residence in the open state and transitions to a new closed state, described by an intermediate closed time constant. 4. For both human and murine CFTR, ATP and ADP regulated the rate of exit from the long-lived closed state. 5. 5'-Adenylylimidodiphosphate (AMP-PNP) and pyrophosphate, two compounds that disrupt cycles of ATP hydrolysis, stabilized the open state of human CFTR. However, neither agent locked murine CFTR Cl- channels open, although AMP-PNP increased the Po of murine CFTR. 6. The data indicate that although human and murine CFTR have many properties in common, some important differences in function are observed. These differences could be exploited in future studies to provide new understanding about CFTR.

Targeting a polyepitope protein incorporating multiple class II-restricted viral epitopes to the secretory/endocytic pathway facilitates immune recognition by CD4+ cytotoxic T lymphocytes: a novel approach to vaccine design.

The role of CD4+ and CD8+ cells in the generation of an effective immune response against viral infections is well established. Moreover, there is an increasing realization that subunit vaccines which include both CD4+- and CD8+-T-cell epitopes are highly effective in controlling viral infections, as opposed to those which are designed to activate a CD8+- or CD4+-T-cell response alone. One of the major limitations of epitope-based vaccines designed to stimulate virus-specific CD4+ T cells is that endogenously expressed class II-restricted minimal cytotoxic-T-lymphocyte (CTL) epitopes are poorly recognized by CD4+ CTLs. In the present study we attempted to enhance the efficiency of class II-restricted endogenous presentation of minimal class II-restricted CTL epitopes by specifically targeting a polyepitope protein to class II processing compartments through the endosomal and/or lysosomal pathway. A significantly enhanced stimulation of virus-specific CD4+-T-cell clones by antigen-presenting cells (APC) expressing the recombinant polyepitope protein targeted to the endocytic/secretory pathway was readily demonstrated in cytotoxicity assays. In addition, in vitro activation of Epstein-Barr virus- and influenza virus-specific CD4+ memory CTLs by the recombinant constructs encoding the polyepitope protein, specifically targeted to the lysosomal compartment, was also demonstrated. The enhanced stimulatory capacity of APC expressing a lysosome-targeted polyepitope protein has important implications for vaccine design.

Constitutive transduction of peptide transporter and HLA genes restores antigen processing function and cytotoxic T cell-mediated immune recognition of human melanoma cells.

Potentiation of immunogenicity of malignant cells by gene transduction provides a unique opportunity for immune targeting of human cancers in vivo. This approach is undoubtedly influenced by the ability of the malignant cells to process and present endogenously target epitopes on their cell surface for immune recognition by cytotoxic T lymphocytes (CTLs). In the present study, we have investigated potential immune-resistance pathways in human malignant melanoma by analyzing the major histocompatibility complex (MHC) gene expression and function in a panel of tumour cell lines. Our analysis showed that a large proportion of these cell lines consistently display a functional defect in the endogenous processing of CTL epitopes and are recognised poorly by specific T cells in spite of high levels of target antigen expression in the tumour cells. Molecular characterisation of this defect revealed that tumour cells under-expressed peptide transporters and surface-assembled MHC class I molecules, which constitute essential components of the class I processing pathway. Induction of peptide transporter and surface class I following treatment of these tumour cells with interferon gamma (IFN-gamma) suggested a transcriptional defect in the expression of antigen-processing genes. Endogenous processing function in these tumour cells was restored completely following simultaneous transduction of cells with peptide transporter and HLA class I genes. Our findings provide a rationale for focussing on strategies designed to improve antigen-processing function in tumour cells and, thus, may strongly influence future strategies for melanoma-specific immunotherapy.

Delivery of multiple CD8 cytotoxic T cell epitopes by DNA vaccination.

Development of CD8 alphabeta CTL epitope-based vaccines requires an effective strategy capable of co-delivering large numbers of CTL epitopes. Here we describe a DNA plasmid encoding a polyepitope or "polytope" protein, which contained multiple contiguous minimal murine CTL epitopes. Mice vaccinated with this plasmid made MHC-restricted CTL responses to each of the epitopes, and protective CTL were demonstrated in recombinant vaccinia virus, influenza virus, and tumor challenge models. CTL responses generated by polytope DNA plasmid vaccination lasted for 1 yr, could be enhanced by co-delivering a gene for granulocyte-macrophage CSF, and appeared to be induced in the absence of CD4 T cell-mediated help. The ability to deliver large numbers of CTL epitopes using relatively small polytope constructs and DNA vaccination technology should find application in the design of human epitope-based CTL vaccines, in particular in vaccines against EBV, HIV, and certain cancers.

Class I processing-defective Burkitt's lymphoma cells are recognized efficiently by CD4+ EBV-specific CTLs.

In the present study, we have isolated CD4+ CTLs that recognize an epitope from EBV nuclear Ag 2 in association with two different HLA-DQ Ags, DQA1*0501/DQB1*0201 (DQ2) or DQA1*0501/DQB1*0301 (DQ7). Both the HLA-DQ2 and HLA-DQ7 alleles displayed a similar efficiency in the endogenous and exogenous presentation of this epitope. Since earlier studies have shown that the EBV-associated malignancy, Burkitt's lymphoma (BL), escapes class I-restricted immune recognition by down-regulating the expression of peptide transporter genes, we have explored the possibility that these tumor cells can process class II-restricted CTL epitopes. The data presented in this study clearly demonstrate that BL cells were recognized efficiently by CD4+, MHC class II-restricted EBV-specific CTLs following infection with recombinant vaccinia encoding EBV nuclear Ag 2. Analysis of surface MHC class II expression on BL cells revealed high levels of HLA-DR and HLA-DQ molecules, and most of these molecules were negative for the invariant chain peptide, referred to as CLIP. Moreover, these tumor cells also showed normal levels of HLA-DMB gene expression, which has been shown previously to be an essential component of the class II processing pathway. The present finding of efficient processing function through the class II pathway in BL cells provides a novel mechanism for immune targeting of EBV-positive malignancies.