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.

Surface oxide and the role of magnesium during the sintering of aluminum

The effect of trace additions of magnesium on the sintering of aluminum and its alloys is examined. Magnesium, especially at low concentrations, has a disproportionate effect on sintering because it disrupts the passivating Al2O3 layer through the formation of a spinel phase. Magnesium penetrates the sintering compact by solid-state diffusion, and the oxide is reduced at the metal-oxide interface. This facilitates solid-state sintering, as well as wetting of the underlying metal by sintering liquids, when these are present. The optimum magnesium concentration is approximately 0.1 to 1.0 wt pet, but this is dependent on the volume of oxide and, hence, the particle size, as well as the sintering conditions. Small particle-size fractions require proportionally more magnesium than large-size fractions do.

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.

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.

The influence of the atmosphere on the sintering of aluminum

Alloys of Al, Al-0.15Mg, and Al-12Sn made using air atomized aluminum powder and pressed to green densities of 75 to 98 pet were sintered under argon or nitrogen. Sintering in argon is only effective at high green densities when magnesium is present. In contrast, highly porous aluminum can be sintered in nitrogen without the need for magnesium. The oxygen concentration in the gas is reduced by the aluminum through a self-gettering process. The outer layers of the porous powder compact serve as a getter for the inner layers such that the oxygen partial pressure is reduced deep within the pore network. Aluminum nitride then forms, either by direct reaction with the metal or by reduction of the oxide layer, and sintering follows.

Seedling responses of four Australian tree species to toxic concentrations of manganese in solution culture

Little is known about the responses of Australian plants to excess metal, including Mn. It is important to remedy this lack of information so that knowledgeable decisions can be made about managing Mn contaminated sites where inhabited by Australian vegetation. Acacia holosericea, Melaleuca leucadendra, Eucalyptus crebra and Eucalyptus camaldulensis were grown in dilute solution culture for 10 weeks. The seedlings ( 42 days old) were exposed to six Mn treatments viz., 1, 8, 32, 128, 512 and 2048 muM. The order of tolerance to toxic concentrations of Mn was A. holosericea congruent to = E. crebra < M. leucadendra < E. camaldulensis, the critical external concentrations being approximately 5.1, 5.0, 21 and 330 muM, respectively. The critical tissue Mn concentrations for the youngest fully expanded leaf and total shoots were, respectively, 265 and 215 mug g(-1) DM for A. holosericea, 445 and 495 mug g(-1) DM for M. leucadendra, 495 and 710 mug g(-1) DM for E. crebra and 7230 and 6510 mug g(-1) DM for E. camaldulensis. The high tolerance of E. camaldulensis ( as opposed to the sensitivity of E. crebra) to excess Mn raises concern about fauna feeding on the plant and is consistent with hypotheses suggesting the Eucalyptus subgenus Symphomyrtus is particularly tolerant of stress, including excess Mn. The results from this paper provide the first comprehensive combination of growth responses, critical external concentrations, critical tissue concentrations and plant toxicity symptoms for three important Australian genera, viz., Eucalyptus, Acacia and Melaleuca, for use in the management of Mn toxic sites.

Cytokine expanded myeloid precursors function as regulatory antigen presenting cells and induce tolerance through IL-10 producing regulatory T cells

The initiation of graft vs. host disease (GVHD) after stem cell transplantation is dependent on direct antigen presentation by host antigen presenting cells (APC) while the effect of indirect antigen presentation by donor APC is unknown. We have studied the role of indirect antigen presentation in allogenic responses by adding populations of cytokine-expanded donor APC to haematopoietic grafts that would otherwise induce lethal GVHD. Progenipoietin-1 (a synthetic G-CSF/Flt-3 L molecule) and G-CSF expanded myeloid DC, plasmacytoid DC and a novel granulocyte-monocyte precursor population (GM) that differentiate into class IIpos, CD80/CD86pos, CD40neg APC during GVHD. Whereas addition of plasmacytoid and myeloid donor DC augmented GVHD, GM cells induced transplant tolerance via MHC class II restricted generation of IL-10-secreting regulatory T cells. Thus a population of cytokine expanded granulocyte-monocyte precursors function as regulatory antigen presenting cells, suggesting that G-CSF derivatives may have application in disorders characterised by a loss of self-tolerance.

Nestmate recognition in a Neotropical swarm-founding wasp: no effect of seasonality on tolerance of alien conspecifics

Previous study revealed that the swarm-founding wasp Polybia paulista is accurately able to distinguish nestmates from non-nestmates in the summer. However, the risk of accepting alien intruders is considered to be low in winter colonies, and additionally brood production is limited in 30-40% of colonies during the winter in this species. Thus, it is expected that colonies might lower their acceptance threshold and accept some conspecific wasps from alien colonies in winter. We conducted field experiments to examine tolerance of conspecific (nestmate and non-nestmate) females in winter. In contrast to our prediction, our colonies did not accept any individuals from alien colonies. We suggest that P. paulista exhibits the colony-specific acceptance threshold in winter, and colonies that produced brood in their nests may have raised the acceptance threshold even if the risk of accepting alien intruders is low in winter.

Salinity tolerance in Schinopsis quebracho colorado: Seed germination, growth, ion relations and metabolic responses

Soil salinity is a major abiotic stress influencing plant productivity worldwide. Schinopsis quebracho colorado is one of the most important woody species in the Gran Chaco, an and and salt-prone subtropical biome of South America. To gain a better understanding of the physiological mechanisms that allow plant establishment under salt conditions, germination and seedling growth of S. quebracho colorado were examined under treatment with a range of NaCl solutions (germination: 0-300 mmol l(-1) NaCl; seedling growth: 0-200 mmol l(-1) NaCl). The aim was to test the hypothesis that S. quebracho colorado is a glycophite that shows different salt tolerance responses with development stage. Proline content, total soluble carbohydrates and Na+, K+ and Cl- concentrations in leaves and roots of seedlings, and the chlorophyll concentration and relative water content of leaves were measured. Germination was not affected by 100 mmol l(-1) NaCl, but decreased at a concentration of 200 mmol l(-1). At 300 mmol l(-1) NaCl, germination did not occur. Seedling growth decreased drastically with increasing salinity. An increase in NaCl from 0 to 100 mmol l(-1) also significantly reduced the leaf relative water content by 22% and increased the proline concentration by 60% in roots. In contrast, total soluble carbohydrates were not significantly affected by salinity. Seedlings showed a sodium exclusion capacity, and there was an inverse correlation between Cl- concentration and the total chlorophyll concentration. S. quebracho colorado was more tolerant to salinity during germination than in the seedling phase. The results suggest that this increased tolerance during germination might, in part, be the result of lower sensitivity to high tissue Na+ concentrations. The significant increment of proline in the roots suggests the positive role of this amino acid as a compatible solute in balancing the accumulation of Na+ and Cl- as a result of salinity. These results help clarify the physiological mechanisms that allow establishment of S. quebracho colorado on salt-affected soils in arid and semi-arid Gran Chaco. (c) 2008 Elsevier Ltd. All rights reserved.

Photodynamic therapy with aluminum-chloro-phtalocyanine induces necrosis and vascular damage in mice tongue tumors

In this paper we describe the efficacy of the liposomal-AlClPc (aluminum-chloro-phthalocyanine) formulation in PDT study against Ehrlich tumor cells proliferation in immunocompetent swiss mice tongue. Experiments were conduced in sixteen tumor induced mice that were divided in three control groups: (1) tumor without treatment; (2) tumor with 100 J/cm(2) laser (670 nm) irradiation; and (3) tumor with AlClPc peritumoral injection; and a PDT experimental group when tumors received AlClPc injection followed by tumor irradiation. Control groups present similar macroscopically and histological patterns after treatments, while PDT treatment induced 90% of Ehrlich tumor necrosis after 24 h of one single showing the efficacy of liposome-AlClPc (aluminum-chloro-phthalocyanine) mediated PDT application, on the treatment of oral cancer. (C) 2008 Elsevier B.V. All rights reserved.

Photophysical and Complexation Studies of Chloro-Aluminum Phthalocyanine with Beta-Cyclodextrin and Hydroxypropyl-Beta-Cyclodextrin

A variety of nanostructures are being investigated as functional drug carriers for treatment of a wide range of diseases, most notably cardiovascular defects, autoimmune diseases, and cancer. The aim of this present contribution is to evaluate potentially applicable nanomaterials in the diagnosis and treatment of cancer due to their photophysical and photobiological properties and complexation behavior. The delivery systems consisted of chloro-aluminum phthalocyanine associated with beta-cyclodextrin and hydroxypropyl-beta-cyclodextrin. The preparation of the complex and its stoichiometry in an ethanol/buffer (3:1) solution were studied by spectroscopic techniques, which were defined as 1:2. The inclusion complex in the nanometer scale was observed on the basis of changes to the spectroscopic properties. The singlet oxygen production and complex photophysical parameters were determined by measuring luminescence at 1270 nm and by steady state and time resolved spectroscopic, respectively. The preparation of the complex was tested and analyzed with regard to cellular damage by visible light activation. The inclusion complex showed a higher singlet oxygen quantum yield compared with other systems and other photoactive dyes. There was also a reduction in the fluorescence quantum yield compared with the results obtained for zinc phthalocyanine in organic medium. The results reported clearly that the inclusion complex chloro-aluminum phthalocyanine/cyclodextrin showed some changes in its spectroscopy properties leading to better biodistribution and biocompatibility with a potential application in photodynamic therapy, especially in the case of neoplasy. Additionally, it also has non-oncological applications as a drug delivery system.

Modification-related porosity formation in hypoeutectic aluminum-silicon alloys

A series of aluminum-10 wt pet silicon castings were produced in sand molds to investigate the effect of modification on porosity formation. Modification with individual additions of either strontium or sodium resulted in a statistically significant increase in the level of porosity compared to unmodified castings. The increase in porosity with modification is due to the presence of numerous dispersed pores, which were absent in the unmodified casting. It is proposed that these pores form as a result of differences in size of the aluminum-silicon eutectic grains between unmodified and modified alloys. A geometric model is developed to show how the size of eutectic grains can influence the amount and distribution of porosity. Unlike traditional feeding-based models, which incorporate the effect: of microstructure on permeability, this model considers what happens when liquid is isolated from the riser and can no longer flow. This simple isolation model complements rather than contradicts existing theories on modification-related porosity formation and should be considered in the development of future comprehensive models.