Suppression of lymphoma and epithelial malignancies effected by interferon gamma

The immunosurveillance of transformed cells by the immune system remains one of the most controversial and poorly understood areas of immunity. Gene-targeted mice have greatly aided our understanding of the key effector molecules in tumor immunity. Herein, we describe spontaneous tumor development in gene-targeted mice lacking interferon (IFN)-gamma and/or perform (pfp), or the immunoregulatory cytokines, interleukin (IL)-12, IL-18, and tumor necrosis factor (TNF). Both IFN-gamma and pfp were critical for suppression of lymphomagenesis, however the level of protection afforded by IFN-gamma was strain specific. Lymphomas arising in IFN-gamma deficient mice were very nonimmunogenic compared with those derived from pfp-deficient mice, suggesting a comparatively weaker immunoselection pressure by IFN-gamma. Single loss of IL-12, IL-18, or TNF was not sufficient for spontaneous tumor development. A significant incidence of late onset adenocarcinoma observed in both IFN-gamma- and pfp-deficient mice indicated that some epithelial tissues were also subject to immunosurveillance.

Altered kinetics and benzodiazepine sensitivity of a GABA(A) receptor subunit mutation [gamma(2)(R43Q)] found in human epilepsy

The gamma-aminobutyric acid type A (GABA(A)) receptor mediates fast inhibitory synaptic transmission in the CNS. Dysfunction of the GABA(A) receptor would be expected to cause neuronal hyperexcitability, a phenomenon linked with epileptogenesis. We have investigated the functional consequences of an arginine-to-glutamine mutation at position 43 within the GABA(A) gamma(2)-subunit found in a family with childhood absence epilepsy and febrile seizures. Rapid-application experiments performed on receptors expressed in HEK-293 cells demonstrated that the mutation slows GABA(A) receptor deactivation and increases the rate of desensitization, resulting in an accumulation of desensitized receptors during repeated, short applications. In Xenopus laevis oocytes, two-electrode voltage-clamp analysis of steady-state currents obtained from alpha(1)beta(2)gamma(2) or alpha(1)beta(2)gamma(2)(R43Q) receptors did not reveal any differences in GABA sensitivity. However, differences in the benzodiazepine pharmacology of mutant receptors were apparent. Mutant receptors expressed in oocytes displayed reduced sensitivity to diazepam and flunitrazepam but not the imiclazopyricline zolpidem. These results provide evidence of impaired GABA(A) receptor function that could decrease the efficacy of transmission at inhibitory synapses, possibly generating a hyperexcitable neuronal state in thalamocortical networks of epileptic patients possessing the mutant subunit.

Innate immune surveillance of spontaneous B cell lymphomas by natural killer cells and gamma delta T cells

Few studies have demonstrated that innate lymphocytes play a major role in preventing spontaneous tumor formation. We evaluated the development of spontaneous tumors in mice lacking beta-2 microglobulin (beta2m; and thus MHC class I, CD1d, and CD16) and/or perform, since these tumor cells would be expected to activate innate effector cells. Approximately half the cohort of perform gene-targeted mice succumbed to spontaneous disseminated B cell lymphomas and in mice that also lacked beta2m, the lymphomas developed earlier (by more than 100 d) and with greater incidence (84%). B cell lymphomas from perforin/beta2m gene-targeted mice effectively primed cell-mediated cytotoxicity and perform, but not IFN-gamma, IL-12, or IL-18, was absolutely essential for tumor rejection. Activated NK1.1(+) and gammadeltaTCR(+) T cells were abundant at the tumor site, and transplanted tumors were strongly rejected by either, or both, of these cell types. Blockade of a number of different known costimulatory pathways failed to prevent tumor rejection. These results reflect a critical role for NK cells and gammadeltaTCP(+) T cells in innate immune surveillance of B cell lymphomas, mediated by as yet undetermined pathway(s) of tumor recognition.

Interferon-gamma is required for killing keratinocytes in vitro and in vivo

Epithelial malignancies are common in immunosuppressed individuals and the general population. However the mechanisms by which the adaptive immune system can eliminate immunogenic epithelial cells remain undefined. The aim of this project was to determine the effector molecules required for induction of apoptosis in murine epidermal keratinocytes (MEKs) in vitro and in vivo. HPV16E7-specific CTL lines and T cell receptor transgenic (E7TCRtg) effector cells were obtained from wild type (wt)-C57 and syngeneic mice rendered functionally inactive for perforin (Pfp), interferon-g (IFN-g) or FasL. CTLs or E7TCRtg spleen cells were co-cultured with primary MEKs in vitro or transferred into skin graft recipients. Inhibition of colony formation and skin graft rejection were used as indicators of T cell:KC interaction. Wt E7-specific CTLs and CTLs deficient in perforin, FasL or IFN-g produced mean reductions in colony formation of 67% (62.4–71.3%), 72% (71.1–72%), 76% (73–78%) and 21.5% (14– 34%) respectively. Wt, perforin deficient or FasL deficient CTLs all induced rejection of skin grafts (wt: 6/12; Pfp: 9/15; FasL: 3/13 survival). Transfer and immunisation of wt E7TCRtg spleen cells induces rejection of 50% of grafts (4/8 survival). In contrast, perforin or IFN-g deficient E7TCRtg failed to induce graft rejection (5/6; 4/4 survival). FasL deficient E7TCRtg induced nonspecific rejection of grafts (E7- 2/6 survival; C57- 4/7 survival). Therefore IFN-g production by CTL is necessary and sufficient in vitro and in vivo to kill epithelial cells which express a nonself antigen. Assessment of immunotherapies directed against epithelial tissues may be more effectively achieved by assaying the amount of IFN-g production by CD8 T cells, and the number and affinity of those cells, in conjunction with quantitation of perforin mediated effects in short term assays.

In vivo effects of chicken interferon-gamma during infection with Eimeria

Newly hatched chickens are highly susceptible to infection by opportunistic pathogens during the first 1 or 2 weeks of life, The use of cytokines as therapeutic agents has been studied in animal models as well as in immunosuppressed patients, This approach has become more feasible in livestock animals, in particular poultry, with the recent cloning of cytokine genes and the development of new technologies, such as live delivery vectors, We have recently cloned the gene for chicken interferon-gamma (Ch-IFN-gamma), Poly-HIS-tagged recombinant Ch-IFN-gamma was expressed in Escherichia coil, was purified by Ni chromatography, and was found to be stable at 4 degrees C and an ambient temperature for at least several months and Several weeks, respectively, Ch-IFN-gamma was capable of protecting chick fibroblasts from undergoing virus-mediated lysis, induced nitrite secretion from chicken macrophages in vitro, and enhanced MHC class II expression on macrophages, Administration of recombinant Ch-IFN-gamma to chickens resulted in enhanced weight gain over a 12-day period, Furthermore, the therapeutic potential of Ch-IFN-gamma was assessed using a coccidial challenge model, Birds were treated with Ch-IFN-gamma or a diluent control and then infected with Eimeria acervulina. Infected birds treated with Ch-IFN-gamma showed improved weight gain relative to noninfected birds, The ability of Ch-IFN-gamma to enhance weight gain in the face of coccidial infection makes it an excellent candidate as a therapeutic agent.

A study of the vacuum gamma-radiolysis of two aromatic polyimide fluoropolymers containing phenylphosphine oxide

The vacuum gamma -radiolysis of two fluorinated polyimides containing phenylphosphine oxide units, TOR-RC and TOR-RC ODPA, have been studied at 77 K and 300 K. The phenyl phosphine oxide units provide protection of the polymers towards oxidation by oxygen atoms and the bulky fluoromethyl groups reduce the colouration of the polymers by limiting donor-acceptor complex formation through the aromatic units. At 77 K the radicals formed were identified to be a mixture of neutral radicals (60%) and anion radicals. At 300 K only neutral radicals were found. The G-values for radical formation were found to be 0.50 and 0.42 at 77 K and 0.051 and 0.052 at 300 K for TOR-RC and TOR-RC ODPA, respectively. Little change was observed in the visible spectra of the polyimides following vacuum radiolysis at 300 K up to a dose of 3.3 MGy, and the polymers were shown to undergo net cross linking with a gel dose of 0.45 MGy.

A study of the oxidation of two aromatic polyimide fluoropolymers containing phenylphosphine oxide by a water plasma and gamma-radiolysis in air

The oxidation of two fluorinated polyimides containing phenylphosphine oxide units, TOR-RC and TOR-RC ODPA, have been studied at 300 K for treatment by a water plasma and gamma -radiolysis in air. The changes in the O 1s/C 1s ratios obtained from x-ray photoelectron spectroscopy (XPS) analysis showed that for exposure to the water plasma the ratio increases at short exposure times and then levels to a constant value. Evidence for the formation of phosphate species was also obtained from the XPS analyses. Similar observations were made for gamma -radiolysis of the polymers in air. The polymers containing phenylphosphine oxide were found to be more resistant to oxidation in the water plasma than Kapton(R). Radiolysis of the polymers in air to high doses were also accompanied by a red shift in the visible absorption spectra.

An ESR study of the gamma radiolysis of aromatic polyesters containing isomeric naphthalene links

Six polyesters were synthesised from 4,4 ' -oxy-bis(benzoyl chloride) and 1,4-, 1,5-, 1,6-, 2,3-, 2,6-, and 2,7-naphthalenediol isomers. The structures of the polyesters were characterised by means of IR, inherent viscosities in tetrachloroethane (TCE), solutions at 303 K and thermal analysis. The glass transition temperatures were in the range of 425-494 K by DSC thermal analysis. All of the polyesters were irradiated in an AECL Gammacell 220 unit at a dose rate of approximately 6.7 kGy/h to doses in the range of 0-15 kGy at 77 and 300 K. ESR spectroscopy was used to examine the radicals formed during radiolysis and to measure their yields. The G-values for radical formation in the polyesters were found to be in the range 0.18-1.41 at 77 K and 0.19-0.78 at 300 K. At 77 K, up to 15% of the radicals formed on radiolysis were found to be photo-bleachable anion radicals. Annealing experiments were carried out in order to identify the neutral radicals, which were assigned to naphthyl- or phenyl- and phenoxyl-type radicals. (C) 2001 Elsevier Science Ltd. All rights reserved.

An ESR study on gamma-irradiated poly(vinyl alcohol)

The formation of radicals in poly(vinyl alcohol), PVA, powder irradiated at 77 K by gamma -rays and the transformations of these radicals during photolysis with visible wavelengths and on thermal annealing have been studied. After irradiation a four-line ESR spectrum was observed. It was assigned to a triplet of the C-alpha-radical (38%), with a splitting of 3.27 mT, superimposed on a doublet (62%) with a splitting of 2.7 mT. The doublet appears to be composed of two radicals, one of which is photo-bleachable (58%) and the other which is not photo-bleachable (42%). This suggests that the latter radical is a neutral radical. The photo-bleachable component of the doublet has been assigned to a carbonyl anion radical. but the second doublet due to a neutral radical is unassigned. The total G-value for formation of radicals at 77 K was found to be 2.41 +/- 0.03. Upon illumination with visible light, the anion radicals were removed and the doublet components or the spectrum diminished in intensity, while the three-line spectrum of the C-alpha-radical became more clearly visible. This transition was due to the photo-detachment of electrons from traps which were proposed to be located on carbonyl groups in the polymer resulting from incomplete hydrolysis of the vinyl acetate. The photo-decay of the anion radicals could be satisfactorily described by a two-stage process. The first stage comprised the decay of approximately 80% of the anion radicals present, while the second stage was associated with the decay of the remaining 20%. Subsequent thermal annealing of a photolysed sample to 290 K led to a change in the shape of the spectrum to form a more clearly defined triplet, As the doublet of the neutral radical decays on thermal annealing between 150 and 250K, the C-alpha-radical is formed. (C) 2001 Elsevier Science Ltd. All rights reserved.

The behaviour of phenylphosphine oxide containing polyimides in a water plasma following gamma-radiolysis

The surface oxidation of two polyimides containing fluorinated phenylphosphine oxide units, TOR-RC and TOR-RC ODPA, have been studied by (XPS) spectroscopy following gamma -radiolysis under vacuum or in air and subsequent treatment in a water plasma. The changes in the O 1s/C 1s ratios obtained from (XPS) analysis showed that on exposure to the water plasma the ratio increases and then levels to a constant value which is similar to that found for exposure to the plasma without prior gamma -radiation treatment. Evidence for the formation of phosphate species was also obtained from the (XPS) analyses. (C) 2001 Elsevier Science Ltd. All rights reserved.

Molecular weight changes and scission and crosslinking in poly(dimethyl siloxane) on gamma radiolysis

The molecular weight changes which occur on the gamma -radiolysis of poly(dimethyl siloxane) under vacuum between 77 and 373 K and in air at 303 K have been investigated using triple detection GPC to obtain the complete molecular weight distributions for the irradiated samples and to determine the number and weight average molecular weights. The results have been interpreted in terms of the relative yields of scission and crosslinking. The total yields for crosslinking predominate over those for scission at all the temperatures investigated for radiolysis under vacuum. Based on a solid-state Si-29 NMR analysis of PDMS irradiated under vacuum at 303 K, which yielded a value of G(Y) of 1.70, the values of G(S) = 1.15 +/-0.2 and G(H) = 1.45 +/-0.2 were obtained for radiolysis under vacuum at 303 K. For radiolysis in air at 303 K, crosslinking was also predominant, but the nett yield of crosslinking was much less than that observed for radiolysis under vacuum. Under the conditions of the radiolysis in air at 303 K, because of the low solubility of oxygen in PDMS, it is likely that the radiation chemistry is limited by oxygen diffusion. (C) 2001 Elsevier Science Ltd. All rights reserved.

Synthesis and stereochemistry of some bicyclic gamma-lactones from parasitic wasps (Hymenoptera : Braconidae). Utility of hydrolytic kinetic resolution of epoxides and palladium(II)-catalyzed hydroxycyclization-carbonylation-lactonization of ene-diols

A palladium(II)-catalyzed hydroxycyclization-carbonylation-lactonization sequence with appropriate pent-4-ene-1,3-diols provides efficient access to the bicyclic gamma -lactones, 5-n-butyl- and 5-n-hexyltetrahydrofuro-[3,2-b]furan-2(3H)-ones (3) and (4), respectively, in both racemic and enantiomeric forms. Some of the substrate pent-4-ene-1,3-diols of high enantiomeric excess (ee) have been derived from racemic terminal epoxides by hydrolytic kinetic resolution (HKR) using cobalt (III)-salen complexes. (9Z,12R)-(+)-Ricinoleic acid also serves as a chiral pool source of other pent-4-ene-1,3-diols. These syntheses and enantioselective gas chromatography confirm the structures and absolute stereochemistry of the lactones in some species of parasitic wasps (Hymenoptera: Braconidae). The highly abundant 5-n-hexyltetrahydrofuro-[3,2-b]furan-2(3H)-one (4) in Diachasmimorpha kraussii and D. longicaudata is of high ee (> 99%) with (3aR,5R,6aR) stereochemistry.