Antigenicity of fusion proteins from sarcoma-associated chromosomal translocations.

Synovial sarcoma (SS), clear cell sarcoma (CCS), and desmoplastic small round cell tumor (DSRCT) are soft-tissue malignancies occurring primarily in adolescents and young adults. These tumors contain specific chromosomal translocations that fuse the 5' region of one gene with the 3' region of another, resulting in the formation of characteristic fusion proteins. These translocations are unique to tumor cells and may be required for persistence, thereby serving as targets for immunotherapy. It was hypothesized that the fusion breakpoint sequences associated with SS, CCS, and DSRCT can serve as tumor-specific neoantigens. To test this, peptides corresponding to the fusion breakpoints were designed and assessed for ability to bind to various class I HLA molecules. Two peptides derived from the SS breakpoint specifically bind the HLA-B7 antigen, and a 10-amino acid minimal epitope was identified for this interaction. Specific binding of a SS peptide and a CCS peptide to HLA-B27 molecule was also observed. Finally, a peptide designed from the DSRCT breakpoint specifically binds the HLA-A3 molecule, and a 9-amino acid optimal epitope was identified for this interaction. The physiological/immunological relevance of these peptide/MHC interactions was demonstrated by the induction of SS-specific CTLs from normal donor lymphocytes using in vitro stimulation with autologous, peptide-pulsed dendritic cells and by the ability of these CTLs to lyse human SS tumor cells endogenously expressing the full-length fusion protein. These results suggest that sequences in the fusion region of sarcoma-associated chimeras can bind class I HLA molecules and serve as neoantigens. These may be useful for the development of novel immunotherapies for sarcoma patients with appropriate HLA molecules and tumors bearing these translocations.

Direct Ni2+ antigen formation on cultured human dendritic cells.

The possible direct antigen formation of Ni2+ on antigen-presenting cells (APCs) was studied with cultured human dendritic cells (DCs) obtained from 10 subjects contact allergic to Ni2+ and six non-allergic control individuals. All contact allergic subjects showed a significantly increased peripheral blood mononuclear cell (PBMC) response in vitro to Ni2+. DCs were expanded from the plastic-adherent cell fraction of PBMCs by culturing with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 7 days to obtain immature DCs, and with the addition of monocyte-conditioned medium for another 4 days, for DC maturation. The DCs were pulsed for 20 min with Ni2+ (50 micrometers) in protein-free Hank's balanced salt solution (HBSS) and added to freshly prepared autologous responder PBMCs. With five allergic subjects, immature DCs pulsed with Ni2+ demonstrated a significant capacity to activate Ni2+-reactive lymphocytes. With the remaining five patients and the six controls no difference in lymphocyte proliferation was observed between Ni2+-pulsed and non-pulsed immature DCs. In contrast, with mature Ni2+-pulsed DCs from both 'positive responder' (n=4) and 'non-responder' (n=4) patients, there was a significantly stimulated PBMC proliferation, whereas with the controls (n=4) still no activation was observed. Our results indicate that direct formation of the antigenic determinant of Ni2+ on APCs is possible and that Ni2+ uptake and processing mechanisms may not play a major role. Differences in the ease of activation of Ni2+-reactive lymphocytes are discussed in terms of a possible heterogeneity in the availability of Ni2+-reactive groups presented on endogenous peptides bound in the antigen binding groove of human leucocyte antigen (HLA) class-II molecules.

Free radicals as potential mediators of metal-allergy: Ni2+- and Co2+-mediated free radical generation.

The generation of free radicals by Ni(2+) and Co(2+) was studied at physiological pH in H(2)O(2)-containing solutions in the absence and presence of various radical-mediating ligands and in human peripheral blood mononuclear cell (PBMC) cultures. With ESR spectroscopy, free radical species were identified and quantitated by spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Co(2+) generated hydroxyl radicals from H(2)O(2) in PBS solutions containing glutathione (GSH) or histidine (His). Omission of GSH or His from the reaction mixture significantly reduced the ESR-signal, indicating the importance of metal-chelation in free radical generation. Carnosine did not significantly enhance the reactivity of Co(2+) toward H(2)O(2), whereas cysteine (Cys) and N-acetylcysteine (NAC) suppressed free radical generation. Under identical reaction conditions, Ni(2+) was markedly less reactive toward H(2)O(2) in comparison with Co(2+). GSH, His, Cys and NAC did not enhance free radical generation of Ni(2+) from H(2)O(2). However, in the presence of carnosine weak but significantly enhanced ESR intensities were found. Incubation of PBMC cultures from healthy subjects with Co(2+) (10-50 microM) yielded the DMPO-.OH adduct, suggesting Co(2+)-mediated hydroxyl radical generation. In contrast, incubation of PBMC cultures with Ni(2+) (10-50 microM) did not produce a detectable ESR-signal. Ascorbic acid efficiently inhibited Co(2+)-mediated free radical generation in PBS solutions and PBMC cultures. The observed difference in free radical generating capacity between Ni(2+) and Co(2+) is of interest with respect to the absence of cross-reactivity between the two metal-ions in experimental allergic contact dermatitis.

Lack of antagonism to Ni2+ and Co2+ contact allergy from other essential divalent metal ions.

The potential antagonistic effects of Ca2+, Cu2+, Fe2+, Mg2+, Mn2+ and Zn2+ on contact allergy to Co2+ and Ni2+ were studied. The immune response was characterized by the Co2+ or Ni2+ mediated cellular [methyl-3H]thymidine uptake in peripheral blood mononuclear cell (PBMC) cultures from 6 subjects contact-allergic to Co2+ and Ni2+ and 6 non-allergic control individuals. Results from the in vitro experiments were further evaluated with Co2+-sensitized guinea pigs according to the modified Freund's complete adjuvant test. Ni2+ and Co2+ (10-50 microM) significantly increased the lymphocyte proliferation in PBMC cultures from contact-allergic subjects in comparison with those from control individuals. Pretreatment of the PBMCs with Ca2+, Fe2+, Mg2+ (10-100 microM) or Mn2+ (1-10 microM) did not influence, while Zn2+ (100 microM) enhanced, and Cu2+ (5 and 10 microM) markedly reduced the Ni2+ and Co2+ mediated cellular [methyl-3H]thymidine uptake. The inhibition of the Ni2+- and Co2+-induced cell proliferation by Cu2+ in vitro was probably related to toxicity, since the viability of the cells was significantly reduced by applied combinations of Ni2+ or Co2+ with Cu2+. Topical pretreatment of Co2+-sensitized guinea pigs with maximum non-irritating doses of CuCl2 x 2H2O (0.8%) did not affect the challenge testing to CoCl2 x 6H2O (0.1 and 0.3%). In conclusion, our combined in vitro and in vivo results indicate that Ca2+, Cu2+, Fe2+, Mg2+, Mn2+ and Zn2+ are not able to antagonise the formation of Ni2+ and Co2+ antigens.

Free radicals as potential mediators of metal allergy: effect of ascorbic acid on lymphocyte proliferation and IFN-gamma production in contact allergy to Ni2+ and Co2+.

A possible free radical mechanism in metal allergy was investigated in peripheral blood mononuclear cell (PBMC) cultures from 6 subjects, contact allergic to Ni2+ and Co2+, and 6 control individuals. Ni2+ and Co(2+)-mediated free radical generation was studied with electron spin resonance spectroscopy. The immune response was characterized by cellular [methyl-3H]thymidine uptake and interferon-gamma (IFN-gamma) production Ni2+ and Co2+ (10-50 microM) significantly increased lymphocyte proliferation and IFN-gamma production in PBMC cultures from contact allergic subjects in comparison with cultures from controls. Inhibition of Co(2+)-mediated free radical generation by ascorbic acid did not influence cellular [methyl-3H]thymidine uptake and IFN production. Detectable amounts of free radicals were not obtained with Ni2+. We therefore conclude that it is unlikely that free radicals are involved in contact allergy to Ni2+ and Co2+.

UV radiation protecting efficacy of cysteine derivatives, studies with UVA-induced binding of 8-MOP and CPZ to rat epidermal biomacromolecules in vivo.

With the aim of optimizing the UV radiation protecting efficacy of N-acetylcysteine (NAC), the following topically applied cysteine derivatives were investigated: N-acetylcysteine ethylester (NACET), S-acetylcysteine ethylester (SACET), cysteine ethylester (CYSET), N,S-diacetylcysteinamide (SNACA), N,S-diacetylcysteine (SNAC) and N,S-diacetylcysteine ethylester (SNACET). As a measure for protection the inhibition of in vivo irreversible photobinding of the labelled phototoxic drugs chlorpromazine (CPZ) and 8-methoxypsoralen (8-MOP) to rat epidermal biomacromolecules was used. The duration of protection of the cysteine derivatives was shortened by S-acetylation, N-acetylation and carboxyl derivatization. Compounds with a free thiol group showed a long-lasting presence in the stratum corneum, probably by the formation of mixed disulphides with proteins. The intrinsic protecting efficacy with respect to the total epidermis increased in the order CYSET < SNACET,SNACA,SACET < NACET, SNAC,NAC. The results of this study are discussed in view of susceptibility to oxidation, epidermal bioavailability and metabolic activation. With respect to the viable epidermis we postulate that NACET and SNAC have the most promising properties as UV protective agents.

Topically applied N-acetylcysteine as a protector against UVB-induced systemic immunosuppression.

The potential protective efficacy of N-acetylcysteine against systemic immunosuppression in mice, as a result of UVB exposure, was investigated. The contact hypersensitivity response to trinitrochlorobenzene applied at a distant, non-irradiated site, was used to assess the systemic immunosuppression. Topical application of N-acetylcysteine (0.4-3.2 mumol cm-2), 30 min prior to irradiation (15 kJ m-2), markedly inhibited the UVB-induced immunosuppression. Because N-acetylcysteine does not absorb UVA or UVB radiation, the mechanism of protection must be different from that of sunscreens. The results of this study may have important practical implications in protecting human beings against the deleterious effects of UVB radiation.

The effect of N-acetylcysteine on the UVB-induced inhibition of epidermal DNA synthesis in rat skin.

The effect of N-acetylcysteine (NAC), on the UVB-induced inhibition of epidermal DNA synthesis in rat skin was investigated. Topical application of NAC, 30 min prior to UVB irradiation (20 kJ m-2), significantly reduced the UVB-induced inhibition of the epidermal (methyl-1',2'-3H)-thymidine uptake. These results indicate that NAC affords protection against at least some of the damaging effects of UVB radiation on epidermal DNA, probably by neutralization of UVB induced reactive species.

UV-radiation protecting efficacy of thiols, studied with UVA-induced binding of 8-MOP and CPZ to rat epidermal biomacromolecules in vivo.

The following topically-applied thiols were investigated with regard to their possible UV-radiation protective properties: captopril, cysteamine, ergothioneine, mesna, mercaptopropionylglycine, N-acetyl-cysteine and penicillamine. As a measure for protection the inhibition of in vivo irreversible photobinding of the labelled phototoxic drugs chlorpromazine (CPZ) and 8-methoxypsoralen (8-MOP) to rat epidermal biomacromolecules was used. Ergothioneine, mesna and penicillamine did not show any effect; probably, as a result of their charge they are not able to enter the stratum corneum. Captopril, cysteamine, mercaptopropionylglycine and N-acetylcysteine showed a considerable inhibition of CPZ and 8-MOP photobinding. Captopril and N-acetylcysteine were clearly the most potent whereas cysteamine was the least effective. Captopril, mercaptopropionylglycine and N-acetylcysteine appeared to have a wider action range and to be a more effective protector than dl-alpha-tocopherol and di-butyl-hydroxytoluene. Cysteamine and mercaptopropionylglycine were only capable of protecting the stratum corneum. Captopril and N-acetylcysteine on the other hand showed an additional dose-dependent inhibition of photobinding to the viable epidermis. Gradually with increasing time after application, the protecting efficacy with regard to the viable layer of the epidermis decreased; the duration of protection depending on the dose.

Thiols as potential UV radiation protectors: an in vitro study.

The following thiols were investigated with regard to their possible UV-radiation protective properties: captopril, cysteamine, ergothioneine, mesna, mercaptopropionylglycine, N-acetylcysteine, and penicillamine. As a measure for protection, the inhibition of in vitro irreversible photobinding of the labeled phototoxic drugs chlorpromazine (CPZ) and 8-methoxypsoralen (8-MOP) to protein and DNA was used. Besides photobinding to biomacromolecules, the photodegradation of CPZ and the formation of promazine (PZH) and hydroxypromazine (PZOH) were measured as well. Because of the H-atom and electron donating capacity of the thiols, the ratio [PZOH]/[PZH] was expected to be decreased and the photodegradation of CPZ was expected to be higher in the presence of thiols. Maximum inhibition of CPZ photobinding ranged for the different thiols between 21-100% (DNA) and 17-87% (human serum albumin). All thiols enhanced the photodegradation of CPZ (19-84%) and inhibited the ratio [PZOH]/[PZH] (90-97%). 8-MOP photobinding to human serum albumin was also clearly inhibited (75-96%), but remarkably less to DNA (2-41%). This study indicates that thiols are able to cope with a variety of reactive species. Scavenging of radicals, quenching of singlet molecular oxygen species and reaction with excited states seem to be essential mechanisms involved with this process.

Photoactivation of 2-nitrofluorene in vitro and in the rat in vivo. UVA-induced formation of reactive intermediates that bind covalently to RNA and protein.

2-Nitrofluorene (2-NF), an environmental pollutant, can be activated by UV light to reactive intermediates that bind covalently to RNA and protein in vitro: high levels of covalent binding were obtained. This covalent binding was not dependent on the presence of oxygen in the solution and could be decreased by glutathione. Hydrolysis of the in vitro modified RNA and subsequent analysis of the liberated bases by HPLC revealed that approximately 15% of the covalent binding of 2-NF to RNA could be attributed to the formation of a guanosine adduct of nitroreduced 2-NF, N-(deoxyguanosin-8-yl)-2-aminofluorene. Many other adducts are formed of which the structure and mechanism of formation are as yet unknown. The possibility of photoactivation of 2-NF in the rat in vivo was also investigated. Photoactivation increased covalent binding of 2-NF in the skin but not in other organs. The mechanism of the photoactivation of 2-NF is discussed.

Effects of monounsaturated fatty acids v complex carbohydrates on serum lipoproteins and apoproteins in healthy men and women.

The effects of a high-carbohydrate, high-fiber diet and an olive-oil-rich diet on the distribution of cholesterol over the various lipoproteins, on serum apolipoproteins, and on the composition of HDL2 and HDL3 were studied under strict dietary control. Forty-eight healthy subjects first consumed a high-saturated-fat diet [proportion of energy, en%] (saturated fat 20 en%, total fat 38 en%) for 17 days. For the next 36 days, 24 subjects consumed a diet high in complex carbohydrates (monounsaturated fat 9 en%, total fat 22 en%) and the other 24 consumed a high-fat, olive-oil-rich diet (monounsaturated fat 24 en%, total fat 41 en%). The amounts of protein (12% to 14 en%), polyunsaturated fat (4 to 5 en%), and cholesterol (31 to 35 mg/MJ) were similar in all three diets. Serum cholesterol levels fell by 0.44 mmol/L in subjects consuming the carbohydrate diet and by 0.52 mmol/L for those receiving the olive-oil-rich diet. VLDL-cholesterol levels rose by 0.08 mmol/L in the carbohydrate group and fell by 0.08 mmol/L in the olive oil group (P less than .05 for difference between test diets). HDL2 and LDL cholesterol levels fell to the same extent on both diets. HDL3 cholesterol fell by 0.09 mmol/L on the high-carbohydrate diet and increased by 0.01 mmol/L on the olive oil diet (P less than .05). There was no change in the composition of HDL3, suggesting that the fall was due to a decrease in the total number of circulating particles.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug-induced protoporphyria in the olfactory mucosa of the hamster.

Administration of 3,5-diethoxycarbonyl-4-ethyl-1,4-dihydro-2,6-dimethylpyridine (4-ethyl-DDC) to hamsters resulted in a marked loss of cytochrome P-450-dependent reactions (peroxidase, 7-ethoxycoumarin O-deethylase, and 7-ethoxyresorufin O-deethylase) in both liver and olfactory epithelium within 2 hr. This inactivation of cytochrome P-450 was accompanied by inhibition of ferrochelatase (FK), stimulation of 5-aminolevulinate synthase (ALA-S), and accumulation of protoporphyrin both in the liver and to a lesser degree, in the olfactory epithelium. These results suggest that the mechanism of induction of protoporphyria in nasal tissues is similar to that occurring in the liver, namely, suicidal metabolism of 4-ethyl DDC by cytochrome P-450 resulting in formation of N-ethylprotoporphyrin, a potent inhibitor of FK. The consequent depletion of heme leads to stimulation of ALA-S and, thus, porphyrin accumulation. Investigation of the dose-response to 4-ethyl DDC demonstrated that, in liver, maximal inhibition of FK and accumulation of protoporphyrin occurred at a dose of 50 mg/kg while ALA-S activity continued to increase up to a dose of 100 mg/kg. This is compatible with an additional effect of the drug on ALA-S involving induction of cytochrome P-450 and, thus, further depletion of heme. In the olfactory epithelium, stimulation of ALA-S was significantly less marked, suggesting that this secondary effect does not operate in nasal tissue. This is consistent with reports that olfactory cytochrome P-450s are noninducible.