Speciation of arsenic metabolites in the urine of occupational workers and experimental rats using an optimised hydride cold-trapping method

A hydride cold-trapping technique was developed and optimised for the measurement of urinary arsenic metabolites. The analytical precision of the method was found to be 6.1, 4.0 and 4.8% (n = 5) for inorganic arsenic (As-i), monomethylarsonate (MMA) and dimethylarsinate (DMA), respectively, with recoveries close to 100%, The detection limits were 1.0, 1.3 and 3 ng for As-i, MMA and DMA, respectively. The method was then used to analyse urine samples obtained from three groups of workers for occupational exposure in three companies where copper chrome arsenate was used for timber treatment. The results were compared with those for a normal control group of laboratory workers. Arsenic and its metabolites were also measured in experimental rats given 5 mg As kg(-1) body mass by oral gavage in the form of sodium arsenite, calcium arsenite or sodium arsenate. Occupational workers showed a significantly higher excretion of As-i, Up to two fold increases of urinary As-i excretion in rats compared with control rats were also observed in animals dosed with various forms of arsenicals. The method is suitable for the measurement of arsenic metabolites in urine of both humans and experimental animals.

Delivery of multiple CD8 cytotoxic T cell epitopes by DNA vaccination

Development of CD8 alpha beta 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.

Mice expressing the E7 oncogene of HPV16 in epithelium show central tolerance, and evidence of peripheral anergising tolerance, to E7-encoded cytotoxic T-lymphocyte epitopes

In order to derive mice which expressed both the E7 open reading frame transgene of human papillomavirus type 16 in skin and MHC class 1 restriction elements for several E7-encoded cytotoxic T-lymphocyte (CTL) epitopes, K14.HPV16E7 mice which express E7 in basal keratinocytes were crossed to the F1 generation with A2.1 K-b transgenic mice which express the MHC binding cleft domains of human HLA A*0201, and murine H-2(b). F1 mice (denoted K14E7xA2.1) expressed E7 in the thymus at least as early as 2-5 days before birth. Immunisation of FVBxA2.1 control mice (transgenic for HLA A*0201 and H-2(b) but not for E7), with two HLA A*0201-restricted epitopes of E7 and one H-2(b)-restricted CTL epitope of E7, gave strong primary CTL responses recognising epitope-pulsed or constitutively E7-expressing syngeneic target cells. In contrast, in immunised K14E7xA2.1 mice, the CTL responses to the H-2(b) epitope and one of the HLA A*0201 CTL epitopes were strongly down-regulated, and to the other HLA A*0201 epitope, completely abolished, as demonstrated by percentage specific killing by bulk splenocyte cultures in cyrotoxicity assays, and by CTL precursor frequency analysis, In thymus-transplanted bone marrow radiation chimeras in which the immune system of K14E7xA2.1 mice was replaced by a FVBxA2.1 immune system, specific immunisation did not result in reemergence of strong E7-directed CTL responses. In agreement with these in vitro findings, specific immunisation failed to significantly alter the course of E7-associated tumour development in K14E7xA2.1 mice. These data are consistent with a model of central deletional CTL tolerance to E7-encoded epitopes recognised in the context of two distinct MHC class 1 restriction elements, and with the possibility of peripheral T-cell anergy maintained by expression of E7 in the skin. (C) 1998 Academic Press.

The haliclonacyclamines, cytotoxic tertiary alkaloids from the tropical marine sponge Haliclona sp

2D-NMR spectroscopic data is reported for the haliclonacyclamines A - D (1)-(4) and for two bismethiodide adducts (5) and (6). The structures of two new alkaloids, haliclonacyclamines C (3) and D (4), which are the 15,16-dihydro analogues of the haliclonacyclamines A (1) and B (2) are described. Revised assignments deduced by 2D-INADEQUATE spectroscopy are presented for (1) and (2). The alkene substituent in the C,, spacer group of (2) and (4) is positioned between C27-C28 by NMR, and confirmed by x-ray structural analysis for (2). Metabolite (3) has a C25-C26 double bond. (C) 1998 Elsevier Science Ltd. All rights reserved.

From sabotage to camouflage: viral evasion of cytotoxic T lymphocyte and natural killer cell-mediated immunity

The outcome of a virus infection is strongly influenced by interactions between host immune defences and virus 'anti-defence' mechanisms. For many viruses, their continued survival depends on, the speed of their attach: their capacity to replicate and transmit to uninfected hosts prior to their elimination by an effective immune response. In contrast, the success of persistent viruses lies in their capacity for immunological subterfuge: the evasion of host defence mechanisms by either mutation (covered elsewhere in this issue, by Gould and Bangham, pp. 321-328) or interference with the action of host cellular proteins that are important components of the immune response. This review will focus on the strategies employed by persistent viruses against two formidable host defences against virus infection: the CD8+ cytotoxic T lymphocyte (CTL) and natural killer (NK) cell responses.

Human and murine cytomegalovirus evasion of cytotoxic T lymphocyte and natural killer cell-mediated immune responses

Herpesviruses, such as human and murine cytomegalovirus, possess an impressive array of genes believed to assist in virus survival against the host immune response. In this review, we cover the rapidly growing area of cytomegalovirus evasion of cellular immunity, specifically cytotoxic T lymphocytes and natural killer cells. The proposed mechanisms of action of viral proteins involved in blocking peptide presentation to CD8(+) T cells, namely, interference with peptide generation, inhibition of peptide assembly with class I MHC and retention/destabilization of class I MHC complexes, are described. In addition, recent evidence implicating the viral class I MHC-like proteins as inhibitors of natural killer cell-mediated clearance is reviewed, (C) 1998 Academic Press.

Clathrins A-C: Metabolites from a southern Australian marine sponge, Clathria species

A Clathria sp. collected in the Great Australian Bight has yielded the novel metabolites clathrins A (6), B (7), and C (8). Structures were assigned to clathrins A-C on the basis of spectroscopic analysis. Clathrin A (6) represents a plausible biosynthetic intermediate that provides an inferred link between marine sesquiterpene/benzenoids and mixed terpene/shikimate biosynthesis.

Papillomavirus virus-like particles for the delivery of multiple cytotoxic T cell epitopes

Chimeric papillomavirus (PV) virus-like particles (VLPs) based on the bovine papillomavirus type 1 (BPV-1) L1 protein were constructed by replacing the 23-carboxyl-terminal amino acids of the BPV1 major protein L1 with an artificial polytope minigene, containing known CTL epitopes of human PV16 E7 protein, HIV IIIB gp120 P18, Nef, and reverse transcriptase (RT) proteins, and an HPV16 E7 linear B epitope. The CTL epitopes were restricted by three different MHC class 1 alleles (H-2(b), H-2(d), HLA-A*0201). The chimeric L1 protein assembled into VLPs when expressed in SF-9 cells by recombinant baculovirus. After immunization of mice with polytope VLPs in the absence of adjuvant, serum antibodies were detected which reacted with both polytope VLPs and wild-type BPV1L1 VLPs, in addition to the HPV16E7 linear B cell epitope. CTL precursors specific for the HPV16 E7, HIV P18, and RT CTL epitopes were also detected in the spleen of immunized mice. Polytope VLPs can thus deliver multiple B and T epitopes as immunogens to the MHC class I and class II pathways, extending the utility of VLPs as self-adjuvanting immunogen delivery systems. (C) 2000 Academic Press.

Disposition of MDMA and Metabolites in Human Sweat Following Controlled MDMA Administration

BACKGROUND: Understanding the excretion of 3,4-methylenedioxymethamphetamine (MDMA) and metabolites in sweat is vital for interpretation of sweat tests in drug treatment, criminal justice, and workplace programs. METHODS: Placebo, low (1.0 mg/kg), and high (1.6 mg/kg) doses of oral MDMA were given double-blind in random order to healthy volunteers (n = 15) with histories of MDMA use. Participants resided on the closed clinical research unit for up to 7 days after each dose. Volunteers wore PharmChek (R) sweat patches (n = 640) before, during, and after controlled dosing. Patches were analyzed by solid phase extraction and GC-MS for MDMA, methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxyamphetamine (HMA), and 4hydroxy-3-methoxymethamphetamine (HMMA). Limits of quantification (LOQ) were 2.5 ng/patch for MDMA and 5 ng/patch for HMA, HMMA, and MDA. RESULTS: MDMA was the primary analyte detected in 382 patches (59.7%), with concentrations up to 3007 ng/patch. MDA was detected in 188 patches (29.4%) at <172 ng/patch, whereas no HMMA or HMA was detected; 224 patches (35.0%) and 60 patches (9.4%) were positive for MDMA and MDA, respectively, at the 25-ng/patch threshold proposed by the Substance Abuse and Mental Health Services Administration. CONCLUSIONS: Sweat testing was shown to be an effective and reliable method for monitoring MDMA use in this controlled MDMA administration study. However, variability in sweat excretion suggests that results should be interpreted qualitatively rather than quantitatively. These data provide a scientific database for interpretation of MDMA sweat test results. (C) 2008 American Association for Clinical Chemistry

Interaction of 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate with mimetic membranes and cytotoxic effect on leukemic cells

10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) is an alkylphospholipid that can interact with cell membranes because of its amphiphilic character. We describe here the interaction of ODPC with liposomes and its toxicity to leukemic cells with an ED-50 of 5.4, 5.6 and 2.9 pM for 72 h of treatment for inhibition of proliferation of NB4, U937 and K562 cell lines, respectively, and lack of toxicity to normal hematopoietic progenitor cells at concentrations up to 25 pM. The ED-50 for the non-malignant HEK-293 and primary human umbilical vein endothelial cells (HUVEC) was 63.4 and 60.7 mu M, respectively. The critical micellar concentration (CMC) of ODPC was 200 mu M. Dynamic light scattering indicated that dipalmitoylphosphatidylcholine (DPPC) liposome size was affected only above the CMC of ODPC. Differential calorimetric scanning (DCS) of liposomes indicated a critical transition temperature (T(c)) of 41.5 degrees C and an enthalpy (Delta H) variation of 7.3 kcal mol(-1). The presence of 25 mu M ODPC decreased T(c) and Delta H to 393 degrees C and 4.7 kcal mol(-1), respectively. ODPC at 250 mu M destabilized the liposomes (36.3 degrees C. 0.46 kcal mol(-1)). Kinetics of 5(6)-carboxyfluorescein (CF) leakage from different liposome systems indicated that the rate and extent of CF release depended on liposome composition and ODPC concentration and that above the CMC it was instantaneous. Overall, the data indicate that ODPC acts on in vitro membrane systems and leukemia cell lines at concentrations below its CMC, suggesting that it does not act as a detergent and that this effect is dependent on membrane composition. (C) 2010 Elsevier B.V. All rights reserved.

Generation and phenotypic characterization of new human ovarian cancer cell lines with the identification of antigens potentially recognizable by HLA-restricted cytotoxic T cells

This study describes a simple method for long-term establishment of human ovarian tumor lines and prediction of T-cell epitopes that could be potentially useful in the generation of tumor-specific cytotoxic T lymphocytes (CTLs), Nine ovarian tumor lines (INT.Ov) were generated from solid primary or metastatic tumors as well as from ascitic fluid, Notably all lines expressed HLA class I, intercellular adhesion molecule-1 (ICAM-1), polymorphic epithelial mucin (PEM) and cytokeratin (CK), but not HLA class II, B7.1 (CD80) or BAGE, While of the 9 lines tested 4 (INT.Ov1, 2, 5 and 6) expressed the folate receptor (FR-alpha) and 6 (INT.Ov1, 2, 5, 6, 7 and 9) expressed the epidermal growth factor receptor (EGFR); MAGE-1 and p185(HER-2/neu) were only found in 2 lines (INT.Ov1 and 2) and GAGE-1 expression in 1 line (INT.Ov2). The identification of class I MHC ligands and T-cell epitopes within protein antigens was achieved by applying several theoretical methods including: 1) similarity or homology searches to MHCPEP; 2) BIMAS and 3) artificial neural network-based predictions of proteins MACE, GAGE, EGFR, p185(HER-2/neu) and FR-alpha expressed in INT.Ov lines, Because of the high frequency of expression of some of these proteins in ovarian cancer and the ability to determine HLA binding peptides efficiently, it is expected that after appropriate screening, a large cohort of ovarian cancer patients may become candidates to receive peptide based vaccines. (C) 1997 Wiley-Liss, Inc.

Impulse-response studies on tracer doses of [C-14]lignocaine and its multiple metabolites in the perfused rat liver

The outflow-concentration-time profiles for lignocaine (lidocaine) and its metabolites have been measured after bolus impulse administration of [C-14]lignocaine into the perfused rat liver. Livers from female Sprague-Dawley rats were perfused in a once-through fashion with red-blood-cell-free Krebs-Henseleit buffer containing 0 or 2% bovine serum albumin. Perfusate flow rates of 20 and 30 mL min(-1) were used and both normal and retrograde flow directions were employed. Significant amounts of metabolite were detected in the effluent perfusate soon after lignocaine injection. The early appearance of metabolite contributed to bimodal outflow profiles observed for total C-14 radioactivity. The lignocaine outflow profiles were well characterized by the two-compartment dispersion model, with efflux rate << influx rate. The profiles for lignocaine metabolites were also characterized in terms of a simplified two-compartment dispersion model. Lignocaine was found to be extensively metabolized under the experimental conditions with the hepatic availability ranging between 0.09 and 0.18. Generally lignocaine and metabolite availability showed no significant change with alterations in perfusate flow rate from 20 to 30 mt min(-1) or protein content from 0 to 2%. A significant increase in lignocaine availability occurred when 1200 mu M unlabelled lignocaine was added to the perfusate. Solute mean transit times generally decreased with increasing flow rate and with increasing perfusate protein content. The results confirm that lignocaine pharmacokinetics in the liver closely follow the predictions of the well-stirred model. The increase in lignocaine availability when 1200 mu M unlabelled lignocaine was added to the perfusate is consistent with saturation of the hydroxylation metabolic pathways of lignocaine metabolism.

Characterization of cytotoxic immune response in skin and mucosal lesions of paracoccidioidomycosis

Background: CD8+ T cells and natural killer (NK) cells are involved in the immune response against some pathogens. For this purpose, we investigated the in situ paracoccidioidomycosis (PCM) immune response addressing the participation of NK cells, CD8+ T cells, perforin and granzyme B expression. Methods: Sixty biopsies of PCM skin and mucosa were classified according to the presence of compact granulomas (G1), poorly organized granulomas (G2) and both kinds in the same lesion (G3). CD8+ T cells, NK cells, perforin and granzyme B were showed by immunohistochemistry. Results: CD8+ T cells were increased over NK cells in cutaneous G1 and G2 lesions. There was no difference regarding such cells in G3 lesions, although they were abundant in such lesions. In mucosa, CD8+ T cells were increased in number over NK cells in all groups. Granzyme B in skin increased in G2 and G3. The number of granzyme did not differ in mucosal lesions in the three groups. Conclusions: CD8+ T cells and NK cells play a role in PCM cutaneous and mucosal lesions. The predominance of CD8+ T cells over NK cells may represent an effective response against the fungi. Moreover, the high number of granzyme B expressing cells corroborates this possibility.