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.

Split tolerance to a viral antigen expressed in thymic epithelium and keratinocytes

When expressed as a transgene from the keratin 14 (K14) promoter in an MHC class II-deficient mouse, I-Ab expressed in thymic cortical epithelium promotes positive but not negative selection of I-Ab-restricted CD4(+) T cells (Laufer, T. M. et al., Nature 1996. 383:81-85). Transgenic mice expressing the E7 protein of human papilloma virus 16 from the K14 promoter were studied to determine the consequence of expression of a cytoplasmic/nuclear protein from the K14 promoter. K14E7-transgenic mice express E7 in the thymus and skin without evidence for autoimmunity to E7. Repeated immunization of FVB(H-2(q)) or F1(C57BV6JxFVB) mice with E7 elicited similar antibody responses to the defined B cell epitopes of E7 in K14E7-transgenic and non-transgenic animals. In contrast, for each genetic background, a single immunization with E7 elicited demonstrable T cell proliferative responses to the major promiscuous T helper epitope of E7 in the transgenic but not the non-transgenic animals. Further,E7-immunized non-transgenic F1 (FVBxC57BL/6J) animals developed strong E7-specific cytotoxic T lymphocyte (CTL) responses and were protected against challenge with E7(+) tumors, whereas similarly immunized K14E7-transgenic animals had a markedly reduced CTL response to E7 and no E7-specific tumor protection was observed, although the antibody and CTL response to ovalbumin was normal. Expression of E7 protein as a transgene from the K14 promoter in the skin and thymus thus induces E7-specific tolerance in the cytotoxic T effector repertoire, together with expansion of the E7-specific T helper repertoire. These findings demonstrate that limited tissue distribution of an autoantigen may result in split tolerance to that autoantigen.

Distribution of interleukin-2,-4,-10, tumour necrosis factor-alpha and transforming growth factor-beta mRNAs in oral lichen planus

In the present study. MRNA for the cytokines interleukin-2 (IL-2), IL-4, IL-10 tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor beta-1 (TGF-beta-1) were investigated in oral lichen planus (OLP) lesions using in situ hybridization with S-35-labelled oligonucleotide probes on frozen tissue sections. In addition, the expression of interferon-gamma (IFN-gamma), IL-10 and IL-4 mRNAs was analysed in cultured lesional T lymphocytes from oral lichen planus by polymerase chain reaction. Cells expressing mRNA for IL-2, IL-4, IL-10, TNF-alpha and TGF-beta(1) were found in all the biopsies studied. Approximately 1-2% of the total number of infiltrating cells in the lesions were positive for each of the different cytokine mRNAs. Most biopsies contained basement membrane-oriented, mRNA-positive cells. In the cultured T-cell lines, message for IFN-gamma was detected in all the patients, IL-10 in all but one, and IL-4 in just one of the seven patients investigated. The results suggest that mRNA for both pro- and anti-inflammatory cytokines, i.e., mixed T-helper 1 (T(H)1) and T(H)2 cytokine profiles, are generated simultaneously by a limited number of cells in chronic lesions of OLP. (C) 1999 Elsevier Science Ltd. All rights reserved.

Systemic coadministration of chloramphenicol with intravenous but not intracerebroventricular morphine markedly increases morphine antinociception and delays development of antinociceptive tolerance in rats

Chloramphenicol, an in vitro inhibitor of the glucuronidation of morphine to its putative antianalgesic metabolite, morphine-3-glucuronide (M3G), was coadministered with morphine in adult male Sprague-Dawley rats to determine whether it inhibited the in vivo metabolism of morphine to M3G, thereby enhancing morphine antinociception and/or delaying the development of antinociceptive tolerance. Parenteral chloramphenicol was given acutely (3-h studies) or chronically (48-h studies). Morphine was administered by the i.v. or i.c.v. route. Control rats received chloramphenicol and/or vehicle. Antinociception was quantified using the hotplate latency test. Coadministration of chloramphenicol with i.v. but not i.cv. morphine increased the extent and duration of morphine antinociception by approximate to 5.5-fold relative to rats that received i.v. morphine alone. Thus, the mechanism through which chloramphenicol enhances i.v. morphine antinociception in the rat does not directly involve supraspinal opioid receptors. Acutely, parenteral coadministration of chloramphenicol and morphine resulted in an approximate to 75% increase in the mean area under the serum morphine concentration-time curve but for chronic dosing there was no significant change in this curve, indicating that factors other than morphine concentrations contribute significantly to antinociception. Antinociceptive tolerance to morphine developed more slowly in rats coadministered chloramphenicol, consistent with our proposal that in vivo inhibition of M3G formation would result in increased antinociception and delayed development of tolerance. However, our data also indicate that chloramphenicol inhibited the biliary secretion of M3G. Whether chloramphenicol altered the passage of M3G and morphine across the blood-brain barrier remains to be investigated.

Beyond ovulation: Oral contraceptives and epithelial ovarian cancer

In a case-control study in three Australian states that included 794 women with epithelial ovarian cancer and 853 community controls for whom we had adequate contraceptive and reproductive histories, Re examined the effects of oral contraceptive use after controlling for estimated number of ovulatory cycles. Other covariates included in the multiple logistic regression analysis were parity, smoking, and history of pelvic surgery. The protective effect of duration of oral contraceptive use appeared to be multiplicative, with a 7% decrease in relative risk per year [95% confidence interval (CI) = 4-9%], persisting beyond 15 years of exposure. Use for up to 1 year may have a greater effect than predicted (odds ratio = 0.57; 95% CI = 0.40-0.82), whereas use before the first pregnancy may be additionally beneficial (odds ratio = 0.95; 95% CI = 0.87-1.03, adjusted for overall duration of use). Better control for ovulatory life might attenuate these estimates somewhat. There was little evidence of waning protection with time since last exposure or of extra benefit with early commencement of oral contraceptive use. We found no convincing evidence of effect modification in any factor examined or differences in effect among the three main histologic cancer types or between borderline and malignant tumors. Oral contraceptives may act by both suppressing ovulation and altering the tumor-promoting milieu.

MDMA (Ecstasy) neurotoxicity: assessing and communicating the risks

MDMA (3,4-methylenedioxymethamphetamine) is an amphetamine analogue that produces euphoric and stimulant effects and a feeling of closeness towards others.1 and 2 For more than a decade, MDMA (colloquially known as “Ecstasy” or “E”) has been widely used by young adults as a dance-party drug. The usual recreational oral dose is 1-2 tablets (each containing about 60-120 mg of MDMA) a standard oral dose of 0·75–4·00 mg per kg in 60–80 kg people. MDMA is typically used once fortnightly or less because tolerance to the effects of MDMA develops rapidly. More frequent use requires larger doses to achieve the desired effects, but this increases the prevalence of unpleasant side-effects.3 A number of deaths have occurred as a result of malignant hyperthermia or idiosyncractic reactions to the drug, but these have been rare.4 MDMA is perceived by many users to be a safe drug.1 Few report the craving associated with opiates or cocaine3 and most MDMA users are aware of only mild and transient disruptions of functioning.3 and 5 AC Parrott and J Lasky, Ecstasy (MDMA) effects upon mood and cognition: before, during and after a Saturday night dance, Psychopharmacology 139 (1998), pp. 261–268. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (174)5 The perceived safety of MDMA is at odds with animal evidence of MDMA neurotoxicity, an increasing prevalence of hazardous patterns of use among recreational MDMA users, and emerging evidence of neurotoxicity among heavier MDMA users.

Desiccation tolerance of three moss species from continental Antarctica

Tolerance of desiccation was examined in three species of moss, Grimmia antarctici Card., Ceratodon purpureus (Hedw.) Brid. and Bryum pseudotriquetrum (Hedw.) Gaertn., Meyer et Scherb. collected from two sites of contrasting water availability in the Windmill Islands, continental Antarctica. Physiological tolerance to desiccation was measured using chlorophyll fluorescence in plugs of moss during natural drying in the laboratory. Differences in relative water content, rate of drying and the response of photosynthesis to desiccation were observed among the three species and between sites. Of the three species studied, G. antarctici showed the lowest capacity to sustain photosynthetic processes during desiccation, B. pseudotriquetrum had an intermediate response and showed the greatest plasticity and C. purpureus showed the greatest capacity to sustain photosynthesis during desiccation. These results fit well with the known distribution of the three species with G. antarctici being limited to relatively wet sites, C. purpureus being common in the driest sites and B. pseudotriquetrum showing a wide distribution between these two extremes. Levels of soluble carbohydrates were also measured in these samples following desiccation and these indicate the presence of stachyose, an oligosaccharide known to be important in desiccation tolerance in seeds, in B. pseudotriquetrum. Both gross morphology and carbohydrate content are likely to contribute to differences in desiccation tolerance of the moss species. These results indicate that if the Casey region continues to dry out, as a result of local geological uplifting or global climate change, we would expect to see not only reductions in the moss community but also changes in community composition. G. antarctici is likely to become more limited in distribution as C. purpureus and B. pseudotriquetrum expand into drying areas.

Incomplete, asymmetric, and route-dependent cross-tolerance between oxycodone and morphine in the Dark Agouti rat

Our previous studies indicate that oxycodone is a putative kappa-opioid agonist, whereas morphine is a well documented mu-opioid agonist. Because there is limited information regarding the development of tolerance to oxycodone, this study was designed to 1) document the development of tolerance to the antinociceptive effects of chronically infused i.v. oxycodone relative to that for i.v. morphine and 2) quantify the degree of antinociceptive cross-tolerance between morphine and oxycodone in adult male Dark Agouti (DA) rats. Antinociceptive testing was performed using the tail-flick latency test. Complete antinociceptive tolerance was achieved in 48 to 84 h after chronic infusion of equi-antinociceptive doses of i.v. oxycodone (2.5 mg/24 h and 5 mg/24 h) and i.v. morphine (10 mg/24 h and 20 mg/24 h, respectively). Dose-response curves for bolus doses of i.v. and i.c.v. morphine and oxycodone were produced in naive, morphine-tolerant, and oxycodone-tolerant rats. Consistent with our previous findings that oxycodone and morphine produce their intrinsic antinociceptive effects through distinctly different opioid receptor populations, there was no discernible cross-tolerance when i.c.v. oxycodone was given to morphine-tolerant rats. Similarly, only a low degree of cross-tolerance (approximate to 24%) was observed after i.v. oxycodone administration to morphine-tolerant rats. By contrast, both i.v. and i.c.v. morphine showed a high degree of cross-tolerance (approximate to 71% and approximate to 54%, respectively) in rats rendered tolerant to oxycodone. Taken together, these findings suggest that, after parenteral but not supraspinal administration, oxycodone is metabolized to a mu-opioid agonist metabolite, thereby explaining asymmetric and incomplete cross-tolerance between oxycodone and morphine.

Effects of an oral vasopressin receptor antagonist (OPC-31260) in a dog with syndrome of inappropriate secretion of antidiuretic hormone

Objective The syndrome of inappropriate secretion of antidiuretic hormone is a rare disorder in dogs characterised by hypo-osmolality and persistent arginine vasopressin production in the absence of hypovolaemia and/or hypotension. The study describes the efficacy and safety of the nonpeptide selective arginine vasopressin V-2 receptor antagonist OPC-31260 in a dog with the naturally occurring syndrome. Design The detailed case history of a dog with spontaneous syndrome of inappropriate secretion of antidiuretic hormone that received long-term therapy with oral OPC-31260 is presented. Effects of the first dose of OPC-31260 and of a dose administered after a continuous dosing period of 12 days are reported. Procedure Packed cell volume, plasma sodium, total protein, arginine vasopressin, renin activity, atrial natriuretic peptide, urine specific gravity, urine output, heart rate and body weight were monitored for 2 h before, and for 4 h after, the first dose of OPC-31260. The same parameters plus plasma osmolality and urine osmolality were monitored when an identical dose was administered after 12 days of therapy. Results Oral administration of OPC-31260 at 3 mg/kg body weight resulted in marked aquaresis with increased urine output and decline in urine specific gravity within 1 h. Corresponding increases in concentrations of plasma sodium, plasma osmolality and plasma renin activity were recorded over a 4 h period. Arginine vasopressin concentration remained inappropriately elevated throughout the study. Results were similar when the trial procedure was repeated after a stabilisation period of 12 days. Long-term therapy with OPC-31260 at a dose of 3 mg/kg body weight orally every 12 h resulted in good control of clinical signs with no deleterious effects detected during a 3-year follow-up period. Despite sustained clinical benefits observed in this case, plasma sodium did not normalise with continued administration of the drug. Conclusions Treatment of a dog with naturally occurring syndrome of inappropriate secretion of antidiuretic hormone with OPC-31260 at 3 mg/kg body weight orally every 12 h resulted in marked aquaresis and significant palliation of clinical signs with no discernible side-effects detected over a 3-year period. Thus, OPC-31260 appears to offer a feasible medical alternative to water restriction for treatment of dogs with syndrome of inappropriate secretion of antidiuretic hormone. Higher doses of OPC-31260 may be required to achieve and maintain normal plasma sodium in dogs with this syndrome.

Expression of RANTES and CCR1 in oral lichen planus and association with mast cell migration

Background: T lymphocytes and mast cells infiltrate the lamina propria in oral lichen planus (OLP). Chemokines and their receptors are involved in T cell and mast cell migration and accumulation during the inflammatory process. Methods: In the present study, we investigated the role of RANTES and its receptors in OLP using immunohistochemistry, RT-PCR and an in vitro chemotaxis assay. Results: RANTES and CCR1 were expressed on T cells and mast cells in OLP, while OLP lesional T cell supernatants stimulated CCR1 mRNA expression in a human leukemia mast cell line (HMC-1). TNF-alpha stimulated CCR1, CCR4 and CCR5 mRNA expression in the same cell line. OLP lesional T cell supernatants stimulated HMC-1 migration, which was partly inhibited by anti-RANTES antibody. Conclusions: The present study shows, for the first time, the distribution of RANTES and CCR1 in OLR It is hypothesized that RANTES and CCR1 may play important roles in mast cell trafficking and related events in OLP.