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A new abortifacient protein, named karasurin, was isolated from fresh root tubers of Trichosanthes kirilowii MAXIMOWICZ var. japonicum KITAMURA (Cucurbitaceae, Japanese name: kikarasuuri) by the procedure involving acetone fractionation and ion-exchange chromatography on Toyopearlpak SP 650S. Homogeneity of Karasurin was demonstrated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and high performance liquid chromatography (HPLC). Karasurin was a highly basic protein of pI 10.1 and the molecular weight was estimated as 28000 by SDS-polyacrylamide gel electrophoresis. Karasurin showed a strong abortion effect in pregnant mice. |
The contribution of hydrophobic interaction to the protein binding of acidic drugs has been evaluated in terms of a new hydrophobic index (r-value), defined as the slope of the log-log plots of capacity factor vs. reciprocal of methanol concentration in an aqueous binary mobile phase, measured by the reversed-phase high-performance liquid chromatography. The logarithms of the binding constants (log K1) of the selected acidic drugs and the related aromatic carboxylic acids indicated linear relationship with their r-values, suggesting that the effect of hydrophobicity on protein binding can be explained similarly to that on the retention onto the reversed-phase stationary ligand. |
7-Alkylaminocoumarin-4-acetic acids I-IX having alkylamino groups different in alkylchain lengths were synthesized as fluorescence probes for characterization of drug-binding sites on human serum albumin (HSA). The fluorescences of I-IX were quenched or enhanced in the presence of HSA with shifts of the emission maxima to shorter wavelength. The binding constants and the number of binding sites were determined by the spectral changes of the probes I-IX bound to HSA through analysis of Scatchard's and Job's plots. The primary binding sites of the tested probes were found to be site 2 (diazepam site) on HSA from the results of competitive displacement studies. The polarity of site 2 was estimated from the relationship between the emission maximum of the probe of IV and Z-values, and was found to be comparable to that of acetonitrile. Simple attempts to estimate the site 2 region from the molecular size of the probe of VIII obtained using the Corey-Pauling-Koltun molecular model suggest that the hydrophobic cleft at site 2 is about 21-25 A in depth. The distance between the lone tryptophan residue in HSA and probes bound to site 2 was estimated to be 15-17 A using Förster's equation on the basis of fluorescence energy transfer. The present data suggest that I-IX are useful as fluorescence probes for the characterization of site 2 on HSA. |
Induction and inhibition of a novel sulfotransferase produced by Eubacterium sp. A-44 isolated from human feces have been studied. Production of the enzyme was induced by phenylsulfate esters, sulfate donor substrates, but not by phenols, sulfate acceptor substrates, or inorganic sulfate. p-Nitrophenylsulfate (PNS), a good donor substrate, stimulated enzyme production more than 10-fold. Sulfotransferase production was strongly inhibited by phenylphosphate esters. Enzyme activity was competitively inhibited by phenylphosphate esters, but not by inorganic phosphate. High yields of sulfotransferase from sonicated cells were obtained when the bacteria were grown in a media containing 0.6% (w/v) or less of glycine. |
Phospholipase D from Streptomyces sp. AA586, PLDP, was modified with methoxypolyethylene glycol succinimidylsuccinate (ss-PEG), an active derivative of polyethylene glycol. By titration with trinitrobenzene sulfonate (TNBS), approximately 70% of the free amino groups in the enzyme protein were shown to be modified by treatment with ss-PEG. By this modification, the molecular weight of the enzyme was increased, judging from the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration with Toyopearl HW-55F and TNBS titration. Due to loss of cationic charges, the enzyme protein became eluted faster in high performance liquid chromatography with CM-Toyopearl. By modification with ss-PEG, the enzyme became fairly thermostable, while pH-stability and optimal pH were not influenced. The value of Km for phosphatidylcholine of the hydrolytic reaction increased 2-fold, whereas that of the transphosphatidyl reaction was not significantly altered. |
The renal responses of magnesium lithospermate B were investigated in the presence or absence of pretreatment with the converting enzyme (kininase II) inhibitor, captopril, in rats with adenine-induced renal failure. Magnesium lithospermate B (10 mg/kg body weight) caused a marked increase in the levels of the renal functional parameters (glomerular filtration rate, renal plasma flow and renal blood flow), accompanied by significant increases in urinary excretions of prostaglandin E2 (PGE2), kallikrein, sodium and creatinine. The administration of magnesium lithospermate B in combination with captopril (2 mg/kg body weight, 2 times) caused a further increase in renal functional parameters, urinary sodium and creatinine excretions. However, the kallikrein activity was similar to the control level. There were no significant changes between urinary PGE2 following magnesium lithospermate B alone, or in combination with captopril. In addition, angiotensin converting enzyme activity did not change following the administration of magnesium lithospermate B alone, but was significantly decreased in rats given captopril, both alone and in combination with magnesium lithospermate B. The captopril administration group (captopril alone or in combination with magnesium lithospermate B) showed a significant decrease in blood pressure. From these results, it seems that the combination of magnesium lithospermate B and captopril induces a further increase in renal function by improving the renal circulatory state. |
We examined the effect of staurosporine on cytosolic calcium response in rat basophilic leukemia (RBL-2H3) cells using fura-2 as a fluorescent indicator of calcium ion. Staurosporine at a dose of 30 nM inhibited antigen-stimulated Ca2(+)-influx into the cells from the extracellular environment. In contrast, the drug at this concentration inhibited neither the mobilization of Ca2+ from intracellular stores nor inositol 1,4,5-trisphosphate (IP3) formation. At a high concentration (300 nM), however, staurosporine completely inhibited the cytosolic calcium responses as well as IP3 formation. These results indicate that staurosporine, if used at an appropriate concentration, can be used to discriminate Ca2(+)-influx from extracellular environment from mobilization of the ion from intracellular stores. These results also suggest that protein kinases, possibly protein kinase C, are involved in the calcium influx of RBL-2H3 cells from the extracellular environment. Serotonin release was strongly inhibited by the drug at 30 nM staurosporine. Since the inhibition of serotonin release and suppression of cytosolic calcium increase in response to the antigen were in parallel, we concluded that the inhibition of serotonin release from RBL-2H3 cells caused by the drug was elicited by the suppression of Ca2(+)-influx into the cells. |
A fragment analog, [D-Arg30]prothymosin alpha fragment 1-30, containing D-arginine in place of arginine residue at position 30 was synthesized by the liquid phase procedure and studied for immunological effect on the impaired blastogenic response of T-lymphocytes isolated from uremic patients after treatment of human serum. Deacetyl-thymosin alpha 1, a synthetic octaeicosapeptide corresponding to deacetyl-prothymosin alpha fragment 1-28, has restoration ability for the impaired blastogenic response of T-lymphocytes of uremic patients but is susceptible to proteolytic digestion. On the other hand, the fragment analog, [D-Arg30]prothymosin alpha fragment 1-30 retained activity and was shown to exhibit a high degree of stability when incubated in human serum. These results indicate that N-terminal acetylation and the introduction of D-residue into the C-terminal residue of prothymosin alpha fragment 1-30 increase resistance to proteolytic degradation by exopeptidases. |
Eubacterium sp. strain BAR, isolated from human feces, transformed barbaloin to aloe-emodin anthrone in a basal medium lacking carbohydrate. Barbaloin remarkably stimulated the growth of strain BAR in the basal medium, the stimulative extent of the growth depending on the amount of barbaloin added. The addition of D-glucose, D-galactose, maltose, cellobiose, sucrose or D-amygdalin to the basal medium containing barbaloin caused a decrease of the growth stimulated by barbaloin to the growth level with each sugar, resulting in a complete inhibition of the barbaloin transformation. On the other hand, the addition of D-fructose, which itself stimulated the growth of strain BAR, further increased the growth in the presence of barbaloin and little inhibited barbaloin transformation. Nojirimycin bisulfite, a specific inhibitor of glucosidases, potently inhibited the growth with barbaloin, but did not affect the growth with glucose or cellobiose. Also, nojirimycin bisulfite completely inhibited the transformation of barbaloin to aloe-emodin anthrone. These results indicate that a unique enzyme capable of cleaving the C-glycosyl bond is induced in strain BAR by barbaloin and, consequently, strain BAR grows by utilizing as a nutrient the carbohydrate liberated from barbaloin. It is further suggested that the barbaloin-cleaving enzyme is inhibited by nojirimycin bisulfite and that the induction of the enzyme is repressed with D-glucose and D-galactose. |
The cross-reactivity of imipenem (IPM) in a delayed-type hypersensitivity (DTH) reaction was investigated by intradermal skin reaction, macrophage migration inhibition tests (MIT) and lymphocyte stimulation tests (LST) in guinea pigs. The animals were immunized with IPM, ampicillin (ABPC) or cephalexin (CEX) using Freund's complete adjuvant. Three sensitized-drugs exhibited the same immunogenicity in the skin reaction. The cross-reactivities in skin reaction among IPM, sulbactam, aztreonam, ABPC and 6-aminopenicillanic acid as penam, and CEX, ceftizoxime, 7-aminocephalosporanic acid as cephem were examined. IPM did not show cross-reactions with any of the tested drugs in three sensitized groups, indicating that the drugs possessed no cross-reactivity with tested beta-lactams in DTH. In MIT, 4 or 5 of 6 animals reacted to be sensitized drugs in each group. The cross-reactivity of IPM- and ABPC-sensitized groups in MIT was similar to that of a skin reaction, however, the CEX-sensitized animals showed broad cross-reactions. Using a kind of test drug, one or two animals in the ABPC-sensitized group showed migration enhancement, but IPM- or CEX-sensitized animals did not exhibit migration enhancement. In LST, most of the animals tested exhibited lymphocyte prolifration by sensitized drugs but a cross-reaction was scarcely observed. The above results suggest that among beta-lactams, IPM has a very low cross-reactivity. LST is considered to be more a useful assay than MIT for in vitro identification of causative drugs in animal models. |
An equation for dissolution from the whole surface of a nondisintegrating single component tablet under the sink condition was derived. Also, equations for several dissolution manners of the tablet under the sink condition were derived in the postulation of the dominant dissolution rate constant which determines the dissolution manner. The applicability or validity of these equations were examined by the dissolution measurements with nondisintegrating single component tablets. About one-tenth the amount of the amount needed to saturate the solution was used to prepare a tablet, and dissolution measurements were carried out with the tablet whose flat or side surface was masked with an adhesive tape in accordance with the conditions for derivation of equations. Among the derived equations, dissolution from the whole surface of a tablet was expressed by a form similar to the cube root law equation for particles. Hence, a single component tablet compressed by the use of a suitable amount was thought to behave like a single crystal. Also, equations derived for several dissolution manners were thought to be applicable for the dissolution of a nonspherical particle and crystal concerning the crystal's habit and its dissolution property, and the extended applicability was examined by converting the crystal into a simplified or idealized form, i.e., rectangle or plate. |
When doxorubicin was encapsulated into liposomes by freeze-thawing, the percentage of encapsulated doxorubicin (EN%) was found to vary according to the type of buffer solution used. The reason for this was investigated in the present report. Drug-free liposomes prepared by hydration were mixed with doxorubicin dissolved in a certain type of buffer solution that shows a pH decrease on freezing, and this mixture was subjected to freeze-thawing. Doxorubicin was encapsulated by the liposomes due to the difference in pH between freezing and thawing. EN% depended on the pH of the buffer solution before freezing and increased significantly at over pH 7. About 60% of doxorubicin was encapsulated into liposomes after the Ist freeze-thawing cycle, and EN% was increased gradually with the number of freeze-thawing cycles. The addition of sugar to the experimental system was seen to affect doxorubicin encapsulation and the particle size of liposomes. |
Nucleophilic attack of tert-butyl/phenylpyridines 3 on 1-chloro-2,4-dinitrobenzene 4 results in the formation of tert-butyl/phenyl substituted 2,4-dinitrophenylpyridinium chlorides 5. Benzoyl hydrazide and pyridyl acid hydrazides 6 were reacted with the pyridinium chlorides 5 furnishing the 2,4-dinitroanilino derivatives 7, which were subsequently hydrolyzed with water: p-dioxane to yield N-[pyridyl(phenyl)carbonylimino]-tert-butyl/phenylpyridinium ylides 8. The title compounds 9, N-[pyridyl(phenyl)carbonylamino]-tert-butyl/phenyl-1,2,3,6- tetrahydropyridines, were obtained by sodium borohydride reduction of the pyridinium ylides 8. The anti-inflammatory activities of compounds 9a-p were determined using the carrageenan-soaked sponge model of inflammation in Sprague Dawley rats. All compounds tested showed moderate to good anti-inflammatory effects compared to indomethacin. Compounds 9b, 9c and 9p were the most active analogs of the group in this model. |
Two pyrazine derivatives [fructosazine (3) and deoxyfructosazine (6)] were simultaneously formed in a solution of D-glucosamine hydrochloride under various conditions. They showed deoxyribonucleic acid (DNA) strand breakage activity in plasmid pBR322 comparable to that of D-glucosamine. The DNA strand breakage by fructosazine (3) was stimulated by Cu2+. |
Derivatives of 7-ethoxycarbonyl-4-formyl-6,8-dimethyl-1(2H)-phthalazinone and closely related compounds were synthesized using Wittig and epoxidation reactions. Ring opening amination of the epoxides were carried out using dimethylaluminum amide reagents under mild reaction conditions. beta-Keto ester and beta-diketone moieties were introduced through diazo derivatives. These moieties were reacted with hydrazine hydrate to produce 4-pyrazolyl derivatives. The derivatives were tested for their inhibitory effect on platelet aggregation, and their relaxing effect on blood vessels. |
Two water-insoluble glucans, U-3-N ([alpha]D +1.0 degree, 0.5 M sodium hydroxide) and U-3-AP1 ([alpha]D +2.5 degrees, 1 M sodium hydroxide) were isolated from hot-water extract of the fruiting bodies of Yũ ĕr (Chinese name) (Auricularia sp.). U-3-N and U-3-AP1 were investigated by a combination of chemical and spectroscopic methods. The results indicated that U-3-N (molecular weight, 6.1 x 10(5)) was similar to beta-(1----6)-branched (1----3)-beta-D-glucan (N-5P: molecular weight, 5.6 x 10(5)) isolated from the alkaline extract of the fruiting bodies, and U-3-AP1 (molecular weight, 6.3 x 10(4)) was beta-(1----6)-branched (1----3)-beta-D-glucan containing beta-(1----6)-linked D-glucopyranosyl residues. U-3-N showed potent anti-tumor activity against the solid form of sarcoma 180, although U-3-AP1 had little effect on the tumor. |
Treatment of calf thymus deoxyribonucleic acid (DNA) in vitro with a methylating carcinogen, N-methyl-N-nitrosourea (MNU), in phosphate buffer (pH 7.2) resulted in formation of O6,7-dimethylguanine residues in DNA besides the well-known methylated DNA adducts, 7-methylguanine, O6-methylguanine and 3-methyladenine. The product ratio (%) of O6,7-dimethylguanine versus 7-methylguanine was 0.32 after one MNU treatment. The significance of formation of O6,7-dimethylguanine residues in DNA is discussed briefly in relation to the carcinogenicity of MNU. |
Microdialysis sampling method coupled to high-performance liquid chromatography with UV detection was applied to continuous monitoring of in vivo unbound flomoxef concentration in rat blood. By comparison with ultrafiltration method, it was demonstrated that it gave reliable results for the unbound drug monitoring in blood. Furthermore, a new method was presented for the calculation of pharmacokinetic parameters from the data obtained by the microdialysis method. |
To identify chemical contaminant(s) associated with eosinophilia-myalgia syndrome (EMS), case and control lots of tryptophan were analyzed by HPLC with both UV and FL detection. Numerous contaminant peaks appeared on the chromatograms and some of them were identified as 5-hydroxytryptophan, indol aldehyde, indol, etc; from the retention time of authentic compounds. Among these, three peaks were significantly associated with case lots. One corresponds to di-tryptophan aminal of aldehyde (peak E). Others are unknown contaminants, UV-5 (FL-7) and UV-28 (FL-36). The structural elucidation and toxicological implication of UV-5 (FL-7) are currently in progress. |
A chiral building block, (R)-2-(2,2-diethoxyethyl)-1,3-propanediol monoacetate was synthesized in high optical and chemical yields by lipase-catalyzed transesterification. From this compound, we synthesized chiral 3-substituted gamma-lactones and a new nucleoside with antiviral activity. |
The presence of the viable yellow Avy and lethal yellow Ay genes has been demonstrated to accelerate the process of tumorigenesis in mice bearing genetically, chemically or virally initiated cells. This promoting effect has been reported in the liver, lung, breast, bladder and skin. Although it has been demonstrated that the accelerated process is associated with systemic, pathophysiologic alterations such as an alteration in the lipogenic pathway, which in turn leads to obesity, alteration in glucose metabolism and/or random immunologic alterations, no examination of the in vitro 'tumorigenic' susceptibility of the cells of animals bearing the yellow genes has yet been reported. In the current study, spontaneous and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced transformation was determined in skin-derived fibroblasts from C57BL/6N-Avy/a mice (Hy) and their black a/a litter mates (B1-). The growth rate of untreated fibroblasts was greater for those from the yellow mice than from the black, while susceptibility to MNNG toxicity was equivalent. However, the rate of spontaneous and chemically induced transformation was consistently and significantly higher in the cells obtained from the black a/a mice than in those from yellow, Avy/a litter mates. These findings, with others from the literature, suggest that the Avy gene may suppress transformation and carcinogenic susceptibility in specific cells, such as fibroblasts, while the systemic effects of the gene are promotional in other cells. |
Reactive intermediates (ultimate mutagens/carcinogens) generated by alkylating agents are unstable and difficult to characterize by chemical means. We have used a genetic system to distinguish the in vivo interactions of eight carcinogenic methylating agents and five ethylating agents by the patterns of induced mutations at different target sites in Escherichia coli WU3610. For this multiple locus assay, target sites were an amber (TAG) and an ochre (TAA) triplet, DNA encoding five suppressor tRNA anticodons, and one unidentified locus. Most of the mutations could be classified as specific sequence changes at the target loci by suppressor analysis using T4 bacteriophage. Ratios of the slopes of dose-response curves for induced mutations were used to generate a profile of preferred sites for mutagenesis independent of mutagen potency. 'Mutational fingerprints' derived from different methylating and ethylating agents were compared, as evidence for the existence of common intermediates responsible for their biological effects. Six methylating agents thought to act via SN1 mechanisms were found to generate similar mutational patterns, indicative of a common mechanism, while two methylating agents reacting via SN2 mechanisms gave different patterns. The mutational fingerprints of SN1- and SN-type ethylating agents were also distinct. Mutational fingerprints may be useful in distinguishing the interactions of different ultimate mutagens. |
Tumor initiating potential was tested for eight pyrolysates of carbohydrates in a two-stage mouse skin carcinogenesis model using 12-O-tetradecanoylphorbol-13-acetate (TPA) as the promoter. The pyrolysates were levoglucosan (LG-I), levoglucosenone (LG-II), furfural (FF), 5-(hydroxymethyl)-2-furfural (HMF), glyoxal (GL), methylglyoxal (MGL), 3-deoxy-D-glucosone (DG) and thiazolidine (TZ). The total initiating doses were 200 mumol for LG-I and DG, 25 mumol for LG-II and 500 mumol for FF, HMF, GL, MGL and TZ. 7,12-Dimethylbenz[a]anthracene (DMBA) was used as a positive control agent applied to a total dose of 100 micrograms. All compounds were topically administered to the dorsal skin twice weekly for 5 weeks with or without TPA treatment for the following 47 weeks. In conjunction with promotion TZ induced skin tumors in 40% of the mice (average 0.65 tumors/mouse), FF in 25% (0.40/mouse), LG-I in 25% (0.35/mouse) and LG-II, HMF, DG and GL in 10-20% (0.11-0.25/mouse) respectively whereas DMBA induced skin tumors in 100% of the animals (6.7/mouse). MGL did not induce any tumors during the experiment and no tumors appeared in any of the groups treated with test chemicals alone. As assessed by Fisher's exact test, tumor incidences were significant in the TZ (0.01 less than P less than 0.05) and DMBA (P less than 0.01) groups as compared with the dimethylsulfoxide (DMSO) followed by TPA groups (5%, 0.05/mouse). Statistical analyses with Peto's trend test revealed significant tumor development in the LG-I (P less than 0.01), LG-II (0.05 less than P less than 0.01), FF (0.01 less than P less than 0.05) and TZ (P less than 0.01) followed by TPA groups (compared with DMSO + TPA). The results indicate that LG-I, LG-II, FF and TZ potentially possess tumor initiating activity, while HMF, GL, MGL and DG do not, as judged by the statistical analysis based on the incidences and development of the skin papillomas and/or carcinomas. A positive correlation between tumor initiating potential and clastogenic activity based on calculated ID50 (50% initiating dose) and published CD20 (20% clastogenic dose) values was evident for LG-II, FF and TZ, while LG-I was an exception. |
We have examined DNA adduct formation and cytotoxicity in HL-60 cells treated with either hydroquinone (HQ) or p-benzoquinone (p-BQ). Treatment of HL-60 cells with either HQ or p-BQ produced the same DNA adduct. The DNA adduct level varied from 0.05 to 10 adducts per 10(7) nucleotides as a function of treatment time and concentration for both compounds. To achieve the same DNA adduct level required higher concentrations and longer treatment times with HQ compared to p-BQ. p-BQ was also more cytotoxic to HL-60 cells than HQ. Reaction of calf thymus DNA with a p-BQ/HQ mixture produced five adducts as detected by 32P-postlabeling. Two isomers of (hydroxy)-1,N2-benzetheno-2'- deoxyguanosine-3'-phosphate were isolated from the reaction of 2'-deoxyguanosine-3'-phosphate with a p-BQ/HQ mixture and one of the isomers was identified as adduct no. 1 of the DNA reaction. The DNA adduct formed in HL-60 cells treated with HQ or p-BQ did not correspond to any of the principal adducts formed in DNA reacted with p-BQ/HQ. This result suggests that cellular mechanisms modify DNA adduct formation by HQ and p-BQ. |
Dietary quercetin (QU) and rutin (RU), phenolic flavonoids commonly found in many fruits and vegetables, were provided to CF1 female mice for 50 weeks to assess the ability of these compounds to inhibit azoxymethanol (AOM)-induced colonic neoplasia. In addition to a control group fed an AIN 76A diet, five other groups received that diet to which was added either 0.1, 0.5 or 2.0% QU and 1.0 or 4.0% RU. Acute studies revealed that, among saline controls, no alteration of any proliferative parameters of colonic epithelial cells was observed among those groups receiving any dose of QU or RU. However, among the AOM-treated mice, both 2% QU and 4% RU significantly reduced hyperproliferation and inhibited the shift of S-phase cells to the middle and upper portion of crypts. Moreover, mice fed these concentrations of QU and RU had significantly fewer AOM-induced focal areas of dysplasia (FADs) than those fed the control diet (0.2 +/- 0.4 and 0.4 +/- 0.5 versus 3.6 +/- 2.3 respectively). Tumors occurred more frequently in the distal half of the colon, regardless of treatment. Compared with controls, mice fed 2% QU had a significantly reduced tumor incidence (25.0% versus 5.9%, P = 0.03). Those fed 4% RU showed only a trend toward inhibition (25% versus 9.7%, P = 0.11). Nevertheless, both 2% QU and 4% RU suppressed tumor multiplicity, i.e. fewer tumors/animal arose in these groups than in the AOM-treated control mice (1.2 versus 2.3, P = 0.005; 1.1 versus 2.3, P = 0.003 respectively). Clearly, QU and RU exhibit significant activity in reducing AOM-induced hyperproliferation of colonic epithelial cells and FAD incidence. This behavior successfully forecast the ability of both flavonoids to suppress tumor multiplicity and ultimately tumor development. |
We have developed a human B-lymphoblastoid cell line, designated 2D6/Hol, which stably expresses human cytochrome P450 CYP2D6 cDNA. This cell line exhibits bufuralol 1'-hydroxylase activity and immunologically detectable CYP2D6 protein. The specific activity of (+)-bufuralol 1'-hydroxylase in microsomes from 2D6/Hol cells was comparable to that observed in human liver microsomes. This cell line was used to examine the mutagenicity activation of three tobacco smoke-derived nitrosamines, N-nitrosonornicotine (NNN), 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal) (NNA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), by CYP2D6. Exposure of 2D6/Hol cells to NNK concentrations of 30-90 micrograms/ml induced a concentration-dependent decrease in relative survival and increase in mutant fraction at the hypoxanthine guanine phosphoribosyl transferase (hprt) locus. In contrast, NNK was non-mutagenic and non-cytotoxic to control cells at exposure concentrations up to 150 micrograms/ml. NNK mutagenicity in 2D6/Hol cells was compared to the responses observed in isogenic cell lines expressing human CYP1A2 (1A2/Hol), human CYP2A3 (2A3/Hol) and human CYP2E1 (2E1/Hol). These three additional human cytochrome P450-expressing cell lines were also found to be sensitive to NNK-induced mutagenicity and cytotoxicity. We found no evidence for CYP2D6-mediated activation of NNN or NNA. NNN was non-cytotoxic and non-mutagenic to both control and 2D6/Hol cells. NNA was equally cytotoxic and mutagenic to control cells and 2D6/Hol cells. The activation of NNA to a mutagen may have been carried out by P450 native to the AHH-1 TK +/- cell line. The 2D6/Hol cell line, in conjunction with the control cell line and other isogenic cell lines expressing other human cytochrome P450 cDNAs provides a useful system for the examination of the role of the polymorphic CYP2D6 in human procarcinogen activation and drug metabolism. |
(+)-Catechin is a plant flavonoid which decreases the mutagenicity of several mutagens and carcinogens. In this study, we have investigated how (+)-catechin could inhibit the metabolism and DNA damage induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific carcinogen. Addition of 5 to 1000 microM (+)-catechin to rat hepatocytes cultured with 4.5 mM NNK caused a concentration-dependent reduction of alpha-carbon hydroxylation which is the activation pathway of NNK. Under the same conditions, (+)-catechin had a less significant effect on pyridine N-oxidation, which is a deactivation pathway. Reduction of the carbonyl group of NNK was not inhibited by (+)-catechin. We had previously shown that NNK induced single-strand breaks (SSBs) in primary culture of hepatocytes. In this study, we observed that 1.0 mM (+)-catechin inhibited the DNA SSBs induced by 1 mM NNK by 31%. With 1 mM N-nitrosodimethylamine, the inhibition of DNA SSBs was 30%. We concluded that (+)-catechin selectively inhibits the enzymes involved in the activation of NNK. Rats were gavaged with (+)-catechin (1.5 mmol/kg), injected s.c. 1 h later with NNK (0.39 mmol/kg) and killed 4 h after NNK treatment. (+)-Catechin significantly reduced DNA SSBs induced by NNK. Rats were injected s.c. with 0.39 mmol/kg NNK. (+)-Catechin reduced the methylation of liver DNA at the O6-guanine and N-7 guanine sites by 28 and 34% respectively. These results demonstrate that (+)-catechin inhibits the formation of DNA-damaging intermediates by selectively impairing the enzymatic activation of NNK. They suggest that (+)-catechin could be an effective preventive agent against NNK hepatocarcinogenicity. |
For assessment of the carcinogenic potential and the mutagenicity of dipyrone, an antipyretic anodyne, -[(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-yl) methylamino]-methanesulfonic acid sodium salt monohydrate, three experiments were conducted using dipyrone A produced in Japan and/or dipyrone B obtained from the Federal Republic of Germany. (i) Carcinogenic potential of dipyrone A for rat liver: 8 week old male F344 rats were pretreated with 0.01% diethylnitrosamine (DEN) in drinking water for 2 weeks and, after 1 week of resting, administered 0.4% dipyrone in drinking water, 5 days a week, for 72 weeks. After an 8 week recovery period, all surviving rats were killed at 83 weeks. Hepatocellular carcinomas developed at a higher incidence in the DEN + dipyrone group (18 of 29 rats, 62%) than in the DEN alone group (9 of 29 rats, 31%), the difference being statistically significant (P less than 0.05). No carcinogenic activity of dipyrone was demonstrated in the groups given 0.4% dipyrone for 72 weeks or 0.4% dipyrone for 25 weeks, followed by 0.05% phenobarbital (PB) for 50 weeks. However, glutathione S-transferase P positive (GST-P+) preneoplastic hepatic foci in these groups were observed at a higher incidence than in the untreated control group (P less than 0.01). (ii) Effect of dipyrone A and dipyrone B on induction of DEN-initiated GST-P+ hepatic foci in a medium-term bioassay system: 0.4% dipyrone A in drinking water and 0.57% dipyrone A or dipyrone B in powdered diet after DEN initiation had similar enhancing effects on the development of GST-P+ foci (P less than 0.001). (iii) The Ames mutation test in Salmonella: both dipyrone A and dipyrone B proved weakly mutagenic for strain TA100 in the presence or absence of S9 fraction. |
The effect of truncal vagotomy (TV) on pancreatic carcinogenesis initiated with N-nitrosobis(2-oxopropyl)amine (BOP) was investigated in 81 female Syrian golden hamsters. The animals were divided into four groups according to the treatment, groups 1 and 2 serving as non-initiated controls receiving a single s.c. injection of 0.9% NaCl followed by either a sham operation or TV respectively, at week 2. Groups 3 and 4 were given a single s.c. injection of 70 mg/kg body wt of BOP before the sham operation or TV. All hamsters were killed at week 24, and the pancreas, liver and gall bladder tissues were examined histologically. While TV itself caused no significant change in pancreatic weight, the incidence of pancreatic carcinomas in hamsters from group 4 was 48.4%, significantly higher than the 16.7% evident in hamsters from group 3 (P less than 0.05). GLC analysis of the bile acid composition of gall bladder bile from hamsters not receiving carcinogen 1 and 4 months after TV revealed significantly decreased secondary bile acids. The results thus indicated that changes in bile acid composition may be involved in enhancement of BOP-initiated pancreatic carcinogenesis in hamsters by TV. |
Human breast carcinoma cell lines MCF-7 and MDA-MB231 were transplanted s.c. to female athymic nude mice at 3-4 weeks of age. At 7-10 days after transplantation, the mice were divided into groups and fed for 6-8 weeks one of the following semi-purified diets containing different amounts and types of fat, i.e. 5% corn oil, 20% corn oil, 20% butter, 19% beef tallow/1% corn oil and 19% fish (Menhaden) oil/1% corn oil. In addition experiments, the fish oil diets were supplemented with antioxidants (vitamin E, 8 g or 2000 IU/kg diet plus tertiary butyl hydroquinone, TBHQ, 4 g/kg diet) or ferric citrate (3 g/kg diet). Tumor peroxidation product levels were assessed by measuring 2-thiobarbituric acid reactants (TBA assay). At the termination of the studies (6-8 weeks of diet feeding) mean human breast carcinoma volume (MCF-7 and MDA-MB231) was the largest in mice fed the 20% corn oil diet, intermediate in mice fed the butter or beef tallow diets and the least in mice fed the fish oil diet. The difference in mean tumor volumes among mice fed the 20% corn oil diet and those fed the fish oil diet was significant (P less than 0.01). When comparing low (5% corn oil) and high (20% corn oil) fat diets, numerical increases in human breast carcinoma volume (MCF-7 and MDA-MB231) were consistently observed in the high-fat diet groups but these differences were not always significant. Tumor lipid peroxidation product levels were determined on the MDA-MB231 tumors; tumor lipid peroxidation levels were significantly (P less than 0.01) increased only in mice fed the fish oil diets. Supplementation of the fish oil diets with antioxidants (vitamin E + TBHQ) significantly reduced the level of tumor peroxidation products and significantly increased tumor volume (P less than 0.05). When tumor lipid peroxidation product levels in the fish oil plus antioxidant fed mice were reduced to the level of that observed in the tumors of the corn oil fed mice, no significant differences in tumor volumes were observed in these two groups. In contrast, supplementation of the fish oil diets with ferric citrate, significantly (P less than 0.05) increased tumor lipid peroxidation product levels and decreased tumor volume. Thus, the type of dietary fat can clearly affect the growth of human breast carcinomas (MCF-7 and MDA-MB231) maintained in athymic nude mice.(ABSTRACT TRUNCATED AT 400 WORDS) |
The most widely studied model of plasmacytomagenesis is the induction of plasmacytomas in BALB/c mice by i.p. injections of the isoalkane pristane (2,6,10,14-tetramethylpentadecane). Employing a simple quantitative and well-established short-term bacterial genotoxicity assay, the SOS chromotest, as a model system, we have investigated whether pristane may potentially be involved in causing or modulating the genotoxic events thought to induce plasma cell tumorigenesis. We found that incorporation of pristane into the cell membranes enhance the SOS response in Escherichia coli PQ37 and PQ300 induced by gamma-radiation under hyperoxic conditions. Moreover, the oxidation of pristane by radiolytically generated reactive oxygen intermediates yielded a stable, genotoxic product active on E. coli PQ300, a SOS tester strain designed to detect oxidative genotoxins. We discuss these findings in relation to the tumor-promoting role of the chronic i.p. inflammation that accompanies plasmacytomagenesis and conclude that, under these specific conditions, pristane may possess a previously unrecognized genotoxic activity in its tumorigenic potential. |
Previous studies have shown that platinum-DNA adduct level in leukocyte DNA (measured by antibody methodology) is directly related to disease response in ovarian cancer and testicular cancer. To determine if this principle could be more broadly applied, platinum-DNA damage was studied in a blinded fashion in leukocyte DNA of 21 cancer patients who received carboplatin (day 1) and cisplatin (day 3) in a phase 1 clinical trial. Fifteen different tumor types were included in this cohort. Using atomic absorption spectrometry with Zeeman background correction, DNA-bound platinum was measured during cycles 1 (C1) and 2 (C2) of therapy for most patients. For each of two cycles of therapy, most patients developed measurable levels of adduct after carboplatin, and in most patients adduct levels increased further after cisplatin, often in a supra-additive fashion. Total mg dose levels varied by less than 2-fold, whereas individual patients differed by as much as 10(3) in their adduct measurements after C1 and after C2, and by 29-fold after the very first carboplatin dose. All patients had refractory disease at the initiation of therapy, and 19 patients were evaluable for disease response. Adduct determinations were made 24 h after the first dose of platinum therapy in 17 of these individuals. Mean adduct levels after the first dose of carboplatin were higher in six responders (50 fmol/micrograms DNA +/- 26) than in 11 non-responders (14 fmol/micrograms DNA +/- 10); Wilcoxon two sample test two-sided P = 0.0071. The six responders were patients with pleural mesothelioma (2), breast cancer, buccal mucosa cancer, esophageal cancer and ovarian cancer. Adduct levels were consistently higher in the group of responders on each day that adduct was measured, with a summary two-sided P value of 0.00011. We conclude that analysis of platinum-DNA adduct formation may help determine whether pharmacogenetics are important in cancer drug resistance; and may help to determine the relationship between DNA damage in the peripheral blood compartment and internal organ response to in vivo exposures to DNA-damaging agents. |
The previously observed sex difference in the growth rate of enzyme-altered foci (male greater than female) in rats treated according to the resistant hepatocyte model (RH model) occurs during selection/promotion of diethylnitrosamine-initiated cells with dietary 2-acetylaminofluorene (2-AAF) and partial hepatectomy (PH). The secretory pattern of growth hormone (GH) is sex dependent in the adult rat and is a major determinant for sex-differentiated liver functions. In the present study the capacity for liver regeneration following PH in the RH model was studied in rats of both sexes, in castrated males and in males receiving GH infusion, both treatments leading to a feminine pattern of GH secretion. Mitoinhibition during treatment with 2-AAF was shown to be more pronounced in liver from male than from female rats, both in initiated and non-initiated animals. Castration of male rats and continuous infusion of GH to males during the selection period, previously shown to decrease the focal growth rate towards that in female rats, 'feminized' also the degree of mitoinhibition. Autoradiography of sections from animals receiving continuous infusion of [3H]thymidine for 1 week, starting at the time of PH, demonstrated a lower labeling index in surrounding liver from male rats treated in the RH model than in surrounding liver from female rats. Males treated with continuous infusion of human GH during 2-AAF/PH treatment had an intermediary labeling index in the surrounding tissue. No differences in labeling index in enzyme-altered foci was observed between males, females or males plus hGH. These findings support the concept that sex-differentiated promotion in the RH model is exerted by selective mitoinhibition and that this feature is regulated by GH. |
The naturally-occurring anthraquinones (AQs), alizarin (1,2-dihydroxyanthraquinone) and lucidin (1,3-dihydroxy-2-hydroxymethylanthraquinone), were incubated with DNA in the presence of S9 mix. The isolated DNA was analysed by 32P-postlabelling for the presence of aromatic adducts. Only lucidin formed up to five different DNA adducts in the range from 0.995 to 3.05 adducts/10(8) nucleotides. Lucidin was also incubated with polynucleotides poly[d(A-T)] and polydG*polydC in the presence of S9 mix. Analysis of polydG*polydC revealed a similar adduct pattern to that obtained with lucidin-modified DNA. Alizarin, lucidin, a glycoside mixture containing alizarinprimeveroside and lucidinprimeveroside, and Rubia Teep (a herbal drug made from Rubia tinctorum containing lucidin) were incubated with primary rat hepatocytes for 24 h and the isolated DNA was analysed by 32P-postlabelling. Lucidin, the glycoside mixture and Rubia Teep gave rise to DNA adducts, but alizarin did not. Male Parkes mice were treated orally for 4 days with alizarin (10 mg/d), lucidin (2 mg/d), the glycoside mixture (20 mg/d) or Rubia Teep (1/2 tablet/d) and DNA was isolated from liver, kidney, duodenum and colon. Analysis by 32P-postlabelling revealed that lucidin, the glycoside mixture and Rubia Teep, but not alizarin, formed DNA adducts in all the tissues examined but that the adduct patterns were organ-specific. |
Epidermal cell kinetics and DNA adduct levels, and skin morphological changes were measured following weekly topical applications for 29 weeks of high (16, 32 and 64 micrograms) benzo[a]pyrene (B[a]P) doses to female ICR/Harlan mice, in order to investigate the relationship of these parameters to the timing, incidence and morphology of the elicited tumors. During the tumor latency period, [3H]thymidine labeling index, mitotic index, epidermal cell stacking, incidence of pyknotic and dark basal keratinocytes and labeled mitoses were periodically measured, as were nuclear area and DNA content. DNA adducts in skin epidermis were measured by an ELISA method over a period of 9 weeks of single weekly applications of 64, 32, 16 or 8 micrograms B[a]P. There was an initial linear increase in DNA adducts with dose in the epidermis but the increase was much less steep above 32 micrograms/week. This did not correlate with the sharp rise in tumor response above the 32 micrograms/week dose rate. Cell kinetic changes in response to the 64 micrograms/week dose reached a plateau in the first few weeks of the tumor latent period. There was little epidermal hyperplasia but an associated dose-dependent increase in [3H]thymidine labeling index, mitotic index and incidence of pyknotic and dark cells. This evidence indicated that B[a]P produced extensive cytotoxicity and cell death with regenerative proliferation under these conditions. Giant keratinocytes occurred in all dose groups. Analysis of a labeled mitosis curve indicated that B[a]P produced a G2/M block. There was a marked inflammatory response in the dermis at all B[a]P doses. Mice were observed weekly for tumor formation. Virtually all of the tumors were papillomas on initial appearance and required an average of 8 weeks to convert to carcinomas. The substantial cell killing and regenerative proliferation, and the correspondence between the dose-response patterns for epidermal damage and tumors, together with the initial appearance of tumors in the benign form, a characteristic of the action of promoting agents, provided evidence that the tissue damage associated with the high dose levels of B[a]P used in this study reflected tumor-promoting activity in this mouse epidermal tumorigenesis model. The implication of the results for mathematical models of tumor formation are discussed. |
Cytotoxicity, morphological neoplastic transformation, intracellular retention and metabolic behaviour have been investigated in BALB/3T3 Cl A 31-1-1 cells for arsenobetaine, the main form of arsenic in certain seafoods, in comparison to inorganic sodium arsenite. In order to avoid false results, particular attention was paid to the purity, checking for the presence of any trace amounts of inorganic arsenic as well as methylated contaminants in the chemically synthesized arsenobetaine. Cytotoxicity and morphological transformation assays gave obvious positive results for sodium arsenite at a dose exposure of 10 microM. On the other hand, concentrations of arsenobetaine as high as 500 microM failed to induce either cytotoxic effects or neoplastic transformations. The absence of cytotoxicity and transforming potential of arsenobetaine in comparison to inorganic arsenite can be explained by the different degree of retention and the intracellular behaviour of the two arsenic species. Cellular retention of arsenobetaine was dose dependent for exposure concentrations ranging from 1 to 500 microM with a mechanism resembling a simple diffusion (1.4 and 760 pmol of As/10(6) cells were cell associated for the two concentrations at 24 h respectively). About 95% of the intracellular arsenobetaine was present in the cytosol fraction and the attempt to detect any intracellular degradation of the organoarsenic compound failed. Thus, the low retention efficiency of arsenobetaine, its inability to interact with intracellular components and the absence of biotransformation in the cell could explain the lack of cytotoxicity and transforming potential observed in the BALB/3T3 cells. These findings reinforce the view that in humans exposed to different chemical species of arsenic the contribution to the total health risk, including the carcinogenic potential, of arsenobetaine ingested with marine foodstuffs would be negligible. |
The in vitro V79/metabolic cooperation assay measures the extent of gap-junctional transfer of metabolites from wild-type to mutant V79 cells. The assay is currently being explored as a short-term test to screen for tumor promoting chemicals, many of which inhibit metabolic cooperation. In this study, the assay was used to determine whether chemical interactions affect detection of tumor promoters in mixtures and to investigate types of interactions that may occur between chemicals. Several two-chemical mixtures were examined. The effects of phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate, two inhibitors of metabolic cooperation that operate through the same receptor-mediated pathway, were additive at concentrations below the maximally effective concentrations of either. A summation effect was observed in mixtures of two other inhibitors of metabolic cooperation, the pesticide aldrin and the principal metabolite of sodium cyclamate, cyclohexylamine. Synergistic effects were noted when PMA was combined with either aldrin or cyclohexylamine, demonstrating that chemicals in a mixture may yield a much stronger response than expected based on individual chemical exposures. Interactions were also examined between PMA, aldrin, cyclohexylamine and 2,4-diaminotoluene, a chemical that appears to enhance metabolic cooperation. 2,4-Diaminotoluene reversed effects of all inhibiting chemicals to some extent, although the pattern of response was different for each combination. In the most dramatic case, the powerful tumor promoter PMA was completely masked by 2,4-diaminotoluene. These results suggest that the V79/metabolic cooperation assay must be applied with caution in mixture testing because detection of tumor promoting chemicals can depend on other chemicals present. |
In order to compare pulmonary DNA adducts and aryl hydrocarbon hydroxylase (AHH) activity, we have measured these two parameters in non-neoplastic surgical lung parenchymal samples from four ex-smokers and 19 smokers, out of 20 patients operated for lung cancer, and three for nonmalignant lung diseases. DNA adducts were determined by scintillation counting after 32P-postlabelling analysis. The microsomal fractions of the same lung specimen were assayed for AHH activity by a fluorometric method. Autoradiograms of DNA adducts found in lungs of smokers revealed two distinct diagonal radioactive zones that were absent in ex-smokers. The smokers had significantly higher levels (1.68-13.4 DNA adducts/10(8) nucleotides; mean +/- SD 5.38 +/- 3.19) than ex-smokers (0.23-2.21; 1.09 +/- 0.84). AHH activity in smokers ranged from 0.01 to 0.69 pmol/min/mg. This activity was significantly (P less than 0.05) higher in smokers (0.26 +/- 0.26) who had smoked until 1 week before surgery than in those who had stopped smoking for greater than 7 days (0.11 +/- 0.11). A positive linear correlation between DNA adduct levels and AHH activity (r = 0.69; P less than 0.001; n = 19) was found in smokers. This relationship could explain why AHH inducibility appears to be a crude marker for lung cancer risk in smokers. |
We have reported earlier that hypomethylated DNA is rapidly induced in the livers of male Fischer rats fed an extremely methyl-deficient diet (MDD). The early effects of dietary methyl deficiency on the expression of several genes in the livers of such animals have now been investigated. Poly(A)+ RNA was isolated from the livers of rats fed MDD or a similar diet supplemented with adequate supplies of choline, methionine, folic acid and vitamin B12 (CSD) for periods ranging from 1 to 4 weeks. The levels of mRNAs for the c-myc and c-fos protooncogenes in livers of rats given either MDD or the liver carcinogen, 2-acetylaminofluorene (AAF), were compared by Northern blot analysis with those in livers of animals given control diets. Both AAF and MDD induced significant elevations in levels of mRNAs specific for these two genes. After 1 week of MDD intake, large increases in the levels of c-myc and c-fos mRNAs and a smaller increase in the levels of c-Ha-ras mRNAs were observed. In contrast, there were marked decreases in the levels of mRNAs for epidermal growth factor receptor and for epidermal growth factor. These effects on mRNA accumulation persisted and were further enhanced during a 4 week period of MDD feeding. The appearance of hypomethylated DNA in the livers of these MDD-fed rats coincided with the observed changes in levels of mRNA for these genes associated with the regulation of cell growth. Increases in levels of mRNA for c-fos, c-Ha-ras and c-myc were correlated with loss of methylation at specific sites within these genes as early as 1 week after the start of MDD feeding. These combined observations are consistent with the hypothesis that methyl-deficient diets are cancer promoting and/or carcinogenic, at least in part, because they induce hypomethylation of DNA with concomitant alterations in the regulation of gene expression. |
Previous studies have shown that in about one-fifth of human tumor cell strains, the activity of O6-methylguanine-DNA methyltransferase (MGMT), which can repair O6-alkylguanine in DNA produced by alkylating agents, is deficient. These strains are termed Mer- cells. To see if there is any human tumor lacking MGMT activity, we measured the MGMT activity in extracts from liver tumors of 21 patients, and compared it to the activity in normal peritumoral tissues derived from the same patients. The MGMT activity was assayed by measuring the 3H radioactivity transferred from the substrate DNA containing [methyl-3H]-labeled O6-methylguanine to an acid-insoluble protein fraction. There was considerable variation in MGMT activity among individual extracts; the interindividual variation was approximately 6-fold in normal liver tissue and much larger in liver tumors. Although in many cases similar high levels of MGMT activity were found both in liver tumors and in the normal counterpart, six tumors had greater than 3-fold less activity compared with the normal liver tissue from the same patient. Liver tumors from two patients did not have any detectable level of MGMT activity by the present method used, in spite of the fact that the corresponding normal liver samples demonstrated significant activities. We also measured in the same tissue extracts the activities of two common enzymes, glutamic pyruvic transaminase (GPT) and lactic dehydrogenase (LDH). The activities of GPT and LDH in the liver tumor samples that showed undetectable levels of MGMT activity were similar to those in the surrounding normal liver tissues. These results may suggest the existence of human Mer- tumors, deficient or very low MGMT activity. |
Dose-response curves for the O6-methylation of guanine in the hepatic DNA of Wistar and Sprague-Dawley rats were determined after administration of N-nitrosomethylbenzylamine (NMBzA) or N-nitrosodimethylamine (NDMA). Similar results were obtained for both rat strains but methylation of hepatic DNA by NDMA was approximately 9-fold more efficient than with NMBzA when doses were compared on a molar basis. Comparison by immunohistochemical analysis of the distribution of nuclei containing O6-methylguanine within the liver lobules showed that both agents tended to alkylate cells close to the central veins at the lower doses. With increasing doses, the band width of alkylated cells around the central vein increased, spreading in the case of NDMA virtually into the portal zones, whereas with NMBzA the zone of alkylated nuclei reached little more than halfway from the central vein to the portal zone. These differences in the distribution of alkylated cells may explain the differing hepatic responses to these two nitrosamines. |
The modifying effects of the phenolic antioxidant catechol (CC) and its analogs hydroquinone (HQ) and resorcinol (RN) on pancreatic carcinogenesis were evaluated in 146 female Syrian golden hamsters. Groups of animals received either saline or 70 mg/kg body wt N-nitrosobis(2-oxopropyl)amine (BOP) s.c. injections, twice with a 2 week interval, followed by basal diet or diet containing 1.5% of CC, HQ or RN, and 0.75% CC from week 4. All hamsters were killed at week 20 and histopathologically examined for development of pancreatic, liver and gall bladder lesions. The total numbers of pancreatic lesions comprising carcinomas, atypical ductal hyperplasias and ductal hyperplasias per hamster were significantly decreased in animals receiving BOP followed by CC, HQ and RN when compared to those in hamsters given BOP followed by basal diet. Incidence values for atypical ductal hyperplasias were also significantly decreased by the RN or 0.75% CC treatments. The results thus suggest that pancreatic carcinogenesis initiated by BOP in Syrian hamsters can be inhibited by treatments with phenolic antioxidants such as CC, HQ and RN for a relatively short experimental period. |
The di-tert-butyl-substituted hydroxyanisoles 2,5-di-tert-butyl-4-hydroxyanisole (2,5-DTBHA) and 3,5-di-tert-butyl-4-hydroxyanisole (3,5-DTBHA) are contaminants of the commercial antioxidant butylated hydroxyanisole. The tumorigenicity of 2,5-DTBHA, 3,5-DTBHA, 2,5-di-tert-butyl hydroquinone (2,5-DTBHQ), a demethylated analog of 2,5-DTBHA, and 3-tert-butyl-4-hydroxyanisole (3-BHA) were investigated. Dietary 3-BHA, which has been shown previously to induce neoplastic lesions in the hamster forestomach, was found to cause the development of forestomach papilloma in 75% of the hamsters in 24 weeks at 1% addition to the diet. General thickening of the forestomach lining with moderate to severe hyperkeratosis and hyperplasia were observed in 14 (88%) of the 16 hamsters in this group. Inflammation was generally found in the forestomach of these animals. Similar observations were found in 14 (88%) of the 16 hamsters in the group that was fed 1% DTBHQ. Severe hyperplasia and papilloma found in the forestomach of these hamsters were similar in severity to those in the 3-BHA group. Unlike the 3-BHA and 2,5-DTBHQ groups, the forestomachs of 1% 2,5-DTBHA and 3,5-DTBHA treated animals were not different from the hamsters in the control group. Only mild hyperplasia at or near the limiting ridge was found in the forestomach of some of these animals. Hyperkeratosis or inflammation were not observed. Steric factors may play a role in the lack of tumorigenicity of 2,5-DTBHA and 3,5-DTBHA, while the formation of a stable free radical from 2,5-DTBHQ may be the activated species responsible for its tumorigenicity. |
We have compared expression of involucrin, E-cadherin and P-cadherin in cultures of normal keratinocytes and in five different lines derived from squamous cell carcinomas (SCCs), using Northern analysis and immunofluorescence. In normal keratinocytes there was an inverse correlation between P-cadherin and involucrin expression, whereas E-cadherin was expressed by both basal and terminally differentiating cells. In SCC lines involucrin expression was lower than in normal keratinocytes, and there was variable expression of P- and E-cadherin: E-cadherin mRNA levels tended to be lower in SCC lines than in normal keratinocytes, whereas P-cadherin levels were similar. Our results are consistent with observations of cadherin expression in vivo and suggest that the cultures provide a useful experimental model for investigating the role of cadherins in determining the spatial organization of normal and neoplastic keratinocytes. |
Recombinant human tumor necrosis factor alpha (TNF) was found to cause a significant increase in cell proliferation rates and sister chromatid exchange (SCE) frequency in phytohemagglutinin-stimulated human lymphocytes in vitro. The cells were treated for the entire cultivation time with 10, 50, 100, 500, 1000 and 5000 U/ml TNF. Cell proliferation rates, as measured by the replication index, increased significantly (P less than 0.01) in a concentration-independent manner. The maximal extent of SCE induction (15.18 +/- 0.57 versus 10.26 +/- 0.45 SCEs/cell in control, P less than 0.001) was observed with 50 U/ml TNF. |
This study examines the hypothesis that severe facial deformities may be less of a psychological burden than those that are more mild. Twenty-seven experimental children and 12 controls, plus 26 siblings of the experimental group and 12 control siblings, were assessed using tests of intelligence, self-concept and ratings of behaviour. The results provided some support for the hypothesis being considered. |
We describe a group of eight children seen over a 4-year period. After a few months of apparently normal health and development, the children developed feeding difficulty, failed to thrive and had mild diplegia. No other underlying pathology was demonstrated. After a period of careful attention to feeding in the stimulating environment of a child development centre, the feeding difficulty, developmental delay and diplegia all disappeared, but, by school age, the children showed learning or behavioural difficulty. It is suggested that these children suffered a subtle neurological insult during the intra-uterine period and that this predisposed them to the neurological difficulty and failure to thrive. It is important to distinguish this small group of children from those in whom non-organic failure to thrive is found and parental neglect is suspected. |
It has been suggested that screening for impairment in early childhood may cause anxiety to parents. Using self-administered questionnaires, we studied the attitudes and concerns of parents of infants aged 6 months and 8-9 months. Parents were randomly allocated to one of two groups. One group completed the second questionnaire after the 8-month assessment routinely performed by health visitors, and the other group completed the second questionnaire before the assessment. We showed changes in attitude and concerns over the 2-3 month period, but these were not related to the assessment. It appeared that particular concerns and anxieties were not resolved by a recent contact with a health visitor, although a high proportion of mothers stated that they found the assessment reassuring. There was some evidence of a lack of appreciation, both of the purpose of the tests and of the implication of test failure. Screening tests performed by health visitors at an age of 8 months do not appear to generate undue anxiety in parents. However, as many of the tests used are of doubtful validity, a review of the purpose and content of this early health visitor assessment is needed. |
The purposes of this study were to document the self-concept, cardiovascular endurance and isometric muscular strength of children with spina bifida, and to examine the changes in these variables in response to a structured 10-week exercise programme. Eight children trained for about 1 hour a week for 10 weeks, while the five control-group children did not participate in the physical-activity programme. The exercise group improved significantly on five of eight criterion means, while the control group showed no significant improvements. This programme represents an initial attempt to delineate the essential elements in a physical-activity programme to enhance physical fitness and self-concepts of children with spina bifida, and provides a framework for further study. |
Most smokers start experimenting with cigarettes in early adolescence. This paper describes the factors which influence the development of the smoking habit. Methods of intervention, both in and out of the classroom, are discussed. Recommendations for further legislation and increased taxation on tobacco are supported. |
The clinical, angiographic, and procedural findings in 40 in-hospital deaths among 5,000 consecutive percutaneous transluminal coronary angioplasties were reviewed. Compared to the total group, the mortality group had a higher proportion of women, older age, and more extensive coronary disease. Angioplasty was performed as an emergency procedure in 21 of the 40 patients who died. Eighteen presented with an evolving acute myocardial infarction and 17 with unstable angina. Most patients presented in critical condition prior to angioplasty: 18 patients were in cardiogenic shock and 5 patients were on cardiopulmonary resuscitation. Among 13 patients who died following elective angioplasty, the salient feature was acute vessel closure or dissection in 7 patients and failed dilatation of a saphenous vein graft in 4 patients. |
We compared Telelab Personal Blood Pressure Transmitters to mercury sphygmomanometers on a random sample of 63 patients in an office setting and on 29 different patients in a home trial. Each patient was tested with the sphygmomanometer by one of two observers. Three consecutive measurements of each patient were averaged for each method. Although some differences between observers were statistically significant, they were not clinically significant. Differences between the two methods were well within the Association for the Advancement of Medical Instrumentation's accepted range for comparable medical equipment. The 29 hypertensive outpatients used the Telelab transmitter for periods ranging from 2 to 55 weeks during a clinical validation phase. The reliability and accuracy of the monitor were again demonstrated by frequent comparisons with office mercury sphygmomanometer measurements. The high degree of patient acceptance of the monitor for repeated readings over prolonged periods clearly adds to its usefulness. |
Pulmonary dirofilariasis was diagnosed in a 70-year-old woman by tissue examination after she underwent a right thoracotomy. The preliminary clinical diagnosis was pulmonary coin lesion of probable neoplastic nature. Pulmonary dirofilariasis, caused by the dog heartworm, is occurring with increased frequency in the United States and must be considered in the differential diagnosis of pulmonary coin lesions. We describe the clinicopathological features of this case with special emphasis on the gross examination of the specimen. |
Topical testosterone is recognized as a standard treatment for vulvar lichen sclerosus et atrophicus. Systemic androgenic side effects of this medication are widely believed to be uncommon and mild. A case is reported of severe hirsutism with other signs of virilization and markedly elevated serum testosterone levels secondary to use of topical testosterone propionate for vulvar lichen sclerosus et atrophicus. Caution is necessary in the use of topical testosterone, with close monitoring for signs of virilization. Alternative treatments for this condition, such as intralesional corticosteroids or topical progesterone, are recommended to avoid the risk of androgenic side effects. |
A new renal preservation flush solution (PB-2) has been developed to minimize the ischemic injury processes that occur during hypothermic storage and reperfusion and that can decrease renal viability and survival. Development of the new formulation took into account the intracellular and biological interactions that occur pre- and post-transplantation. PB-2 was compared with the conventional standardized Collins-2 flush solution in the preservation of autografts in dogs and was found to provide significantly improved renal recovery, viability, and survival. Studies to test the new solution in human renal allograft transplant preservation are planned. |
Haemangiomas of the head and neck in children may be of several histological types, the clinical course depending on the group to which the haemangioma belongs. Treatment may be required if the haemangioma interferes with the airway, as in the subglottic group, or if the lesion becomes ulcerated with subsequent haemorrhage, as in capillary cavernous haemangiomas. Most haemangiomas require no immediate treatment as they involute spontaneously, though parental reassurance will be of paramount importance. Those lesions which persist may be amenable to treatment at a later date, the laser probably offering the best long-term results in terms of cosmesis as in the case of port-wine stains. Superselective embolization is becoming the treatment of choice for arterial haemangiomas. This paper is designed to clarify the histological and clinical features of these tumours and their management in view of considerable confusion in the literature encountered in our study. |
A restructuring of the service for provision of hearing aids has been proposed by the Royal National Institute for the Deaf. This is based on the assumption that very few patients referred for hearing aids have significant ear disease and it is not necessary for them to see an ENT specialist. The case notes of 200 consecutive patients referred to the Hearing Aid clinic were reviewed. In only half of these would a hearing aid have been prescribed without a specialist opinion. The remainder either did not need a hearing aid or required further investigation and surgical or medical treatment. In addition there was significant evidence of lack of expertise amongst General Practitioners in recognizing ear disorders. It is imperative that any patient requiring a hearing aid be seen by someone experienced in otology rather than be dealt with by the General Practitioner alone. |
The effect of bilateral tube insertion on language development was studied in a randomized controlled trial of 43 preschool children with persistent bilateral otitis media with effusion (OME). Improvement in language scores was observed in both the treatment and non-treatment groups. There was a minor difference in favour of the treatment group which did not reach statistical significance. Although there is a growing consensus that otitis media with effusion can have negative effects on language development in young children, it has not yet been proved that this effect can be reversed by treatment with ventilating tubes. |
Abnormalities in the adrenergic system are believed by some to be a major contributing factor to primary hypertension, which may explain the increasing clinical usefulness of drugs that affect the adrenergic system. The clinical effects of drugs in this class can be predicted by the location of the alpha or beta receptors that are activated or inhibited by the drug. With the wide range of antihypertensive drugs now available, including diuretics and vasodilators as well as adrenergic agents, it is possible to individualize treatment in ways more appropriate than the traditional stepped-care approach. |
Recently it has been shown that patients with atopic rhinitis and with an allergy to house dust mites have a stronger nasal response to insufflation of histamine, methacholine and phentolamine than a control group. This hyper-responsiveness could not be demonstrated in patients with perennial non-allergic rhinitis, unless the patients were selected according to the predominant symptoms in the history. Patients with rhinorrhoea ('runners') proved to be hyper-responsive to methacholine compared with normal controls. The existence of two subpopulations was emphasized by hyper-responsiveness to both histamine and methacholine in the runners group compared with the patients with a stuffy nose ('blockers'). Patients with chronic nasal infections (characterized by recurrent episodes of purulent discharge) showed no hyper-responsiveness at all, indicating that either hyper-reactivity does not play an important part in this patient population or methods to detect hyper-reactivity in this group are inadequate. In contrast to our earlier observations in patients with atopic rhinitis, increased responsiveness to phentolamine could not be detected either in the patients with perennial rhinitis or in the patients with infectious rhinitis, indicating that the possible alpha-adrenergic dysfunction found in patients with atopic rhinitis is restricted to this group. |
The influence of exercise on the auditory brain-stem response (ABR) was examined in 16 healthy volunteers (8 female and 8 male). Ipsilateral ABR recordings were obtained before and after exercise on a bicycle ergonometer. The rise of body temperature so generated was 0.5-2.1 degrees C (mean, 1.3 degrees C) as measured in the contralateral external auditory meatus. Latencies of waves III and V (but not wave I) were found to be significantly lower immediately post-exercise (P less than 0.01). The temperature relations of the latency of wave V are described by the regression equation: Latency (ms) = 11.06-0.146 x temp. (degrees C). (The effects on amplitude were not significant, nor were male/female differences.) It is suggested that exercise hyperthermia could be an appropriate model for the evaluation of the ABR in fever. |
Carefully designed, monitored rehabilitation regimens can benefit patients with significant cardiac disease, such as life-threatening arrhythmias or congestive heart failure, or who have concurrent systemic disease such as diabetes. Patients with heart failure can tolerate minimal workloads but, with conditioning, they can increase their duration of exercise. Heart transplant recipients, who are usually severely deconditioned at the time of surgery, are good candidates for a comprehensive rehabilitation program; some have progressed to competition-level athletic achievements. Rehabilitation is safe for patients with arrhythmias, given appropriate monitoring, and can contribute to enhanced quality of life. Objective measures are needed to distinguish between symptomatic and functional improvement. |
The aim of this study was to evaluate whether, in the treatment of active non-cholesteatomatous chronic otitis media, the effectiveness of gentamicin with hydrocortisone ear-drops is due solely to the contained steroid or to the combination of antibiotic and steroid. Sixty-four patients presenting to the Ear, Nose and Throat outpatient department with active non-cholesteatomatous chronic otitis media were randomly allocated to receive either gentamicin with hydrocortisone ear-drops, or betamethasone ear-drops, for up to 4 weeks. Activity in the ear was assessed at 2 and 4 weeks. Gentamicin with hydrocortisone combination drops were significantly more effective than betamethasone drops (P less than 0.001) in producing inactivity of chronic otitis media, being effective in 80% as opposed to 29% of cases. The effectiveness of gentamicin with hydrocortisone ear-drops appears to be due to the combination of antibiotic and steroid. |
An assessment of the strain on the tympanic membrane caused by diving was performed using impedance measurement of the middle ear in 21 untrained young men going through a scuba-diving training programme (scuba, self-contained under-water breathing apparatus). Tympanometry was carried out just before and after diving. The divers made 104 dives between them (median 5 each, range 2-7) at depths from 2 to 12 m (median 6 m). The results showed a significant increase in middle ear compliance on diving. The increase in compliance was significant at different depths, was transient, and fell to the initial level between the dives. We conclude that the strain exerted on the tympanic membrane and middle ear from barotrauma due to diving results in a reversible impairment of the recoiling capacity of the elastic fibrils of the tympanic membrane. This transient increase in compliance, we think, is the first measurable change in elasticity of the tympanic membrane. If barotrauma continue the changes could be irreversible. |
The influence of a heat and moisture exchanger (HME) on the respiratory symptoms after total laryngectomy was studied in 42 patients. A significant reduction was found in the mean daily frequency of sputum production, forced expectoration in order to clean the airway and stoma cleaning after use of the HME for 6 weeks. Symptoms of fatigue and malaise decreased significantly, while social contact improved. Patients using oesophageal speech or an electrolarynx benefited more than patients using a voice prosthesis. The findings indicate that respiratory problems after total laryngectomy can be reduced significantly with the use of a device with heat and moisture exchanging properties. In turn, reduction of respiratory symptoms results in an improved quality of life. |
The monitoring of children with otitis media with effusion ties up considerable resources in audiology departments. Impedance audiometry is frequently used when investigating these children. It has been shown to be highly sensitive in detecting middle ear effusion, but its value in identifying those children with a significant hearing impairment secondary to this is in question because of the wide range of hearing impairments possible with a type B tympanogram. This study quantified the sensitivity and specificity of impedance audiometry in detecting a hearing impairment greater than or equal to 25 dB HL due to otitis media with effusion. The subjects were 285 children of whom 20% had hearing thresholds greater than or equal to 25 dB HL. A peaked tympanogram (types A or C) virtually eliminated the possibility (98% confidence) of such a hearing impairment. A flat type B tympanogram was satisfactorily sensitive (93%) in detecting a hearing impairment, but non-specific (76%). The positive predictive value was 49%, i.e. 51% of ears with this type of tympanogram had hearing within acceptable limits. Assuming that a sensorineural impairment has been excluded and a population is being monitored for hearing impairment associated with otitis media with effusion, it is suggested that the presence of a peaked tympanogram indicates normal hearing, whereas those children with a flat tympanogram would require their hearing to be evaluated. Increased use of impedance audiometry to monitor children with otitis media with effusion would reduce the number requiring full pure-tone audiometry with a subsequent reduction in the workload of an audiology department. |
Eight patients with a verrucous carcinoma of the larynx have been seen at the Charing Cross Hospital between 1970 and 1985. Seven patients were treated by primary radiotherapy and one by primary endoscopic laser excision. Four of those treated with radiotherapy developed recurrent disease which was treated by radical surgery in three and by endoscopic laser excision in one. The place of radiotherapy in the management of this condition requires further evaluation in view of the high rate of recurrence. Endoscopic laser excision may be a reasonable alternative to more radical surgery in this highly differentiated neoplasm. |
A family group with confirmed branchio-oto-renal (BOR) syndrome was investigated in this study. Computerized tomography of the temporal bones has demonstrated that the malformations of the inner ear consist of hypoplastic structural changes within the cochlea with reduced vertical diameters, and absent or hypoplastic semicircular canals and normal endolymphatic ducts. It is concluded that in the present cases, the Mondini malformation of the cochlea is not associated with the BOR syndrome. |
A survey of hearing amongst a population of Maori schoolchildren in the eastern North Island of New Zealand has demonstrated a high prevalence of hearing impairment. Out of 194 children undergoing audiometry an impairment of 20 dB or greater at 0.5, 1.2 and 4 kHz was found in the worse hearing ear in 29% and in the better hearing ear in 12%. Comparison with a similar survey done in the same valley in 1977 revealed an apparent reduction in the prevalence of hearing loss and the prevalence of otitis media. This improvement appears to be due to a reduced prevalence of otitis media. An unexpected finding was that at least 2% of the children had a bilateral sensorineural hearing impairment. |
A prospective assessment of the complications of submucous resection is presented. Preoperative photographs were taken and a post-operative assessment was carried out at a minimum time of 2 years by questionnaire and photographs. No examples of significant cosmetic changes were found. |
Carcinoembryonic antigen (CEA) concentrations were determined in the sera of 45 patients with a head and neck squamous cell carcinoma and of 13 controls. In 13 patients serial CEA measurements were made during the follow-up period. In 38% of the patients the serum CEA level was slightly elevated (greater than or equal to 2.5 ng/ml). Only 13% of the patients had clearly elevated CEA levels (greater than 5 ng/ml). CEA levels were significantly higher in patients with advanced, e.g. stage IV, disease but a correlation between serum CEA concentration and prognosis was not found. Patients who smoked had significantly higher serum CEA levels than non-smoking patients. In the serial determinations slight CEA elevations could be found in only 50% of patients with tumour recurrence. Combined with the data from the literature we conclude that serum CEA determination is not useful in predicting the outcome in patients with a head and neck squamous carcinoma. |
Analysis of 3445 cases of cancer of the larynx has been made with reference to primary (first) and secondary treatments. It is easy to confirm that while radiotherapy is inferior to laryngectomy in the cure of larger glottic tumours, it is preferable in many cases because salvage surgery is possible and successful. Salvage surgery cannot be shown to be successful in larger supraglottic tumours, which should be considered for primary surgery. Radiotherapy appears better than surgery for small tumours. |
A follow-up study of 104 patients with globus sensation was performed by postal questionnaire. All were asked to complete the Eysenck Personality Questionnaire (EPQ) and a 12-item Throat Symptom Questionnaire. Seventy-two of these patients were sent a follow-up General Health Questionnaire (GHQ). Replies were received from 89 patients (86%), 68 females and 21 males, a mean of 31 months after initial presentation. The feeling of something stuck in the throat had disappeared completely in 27% of patients, but only 4% were never aware of the throat. Throat symptoms showed a reduction with increasing age, but not with increasing interval since presentation. The 15 patients initially treated with antacid therapy had significantly greater dyspepsia scores at review than untreated patients, but no other difference was found between the two groups. GHQ scores showed a small but significant reduction over time. Of EPQ parameters, only the lower lie score in female patients showed any significant difference on repeat testing. Those with persistently high throat scores had significantly lower EPQ lie scores and a trend towards higher GHQ scores at follow-up. None of the psychological parameters measured at the first interview was found to be of prognostic significance. We conclude that, although there is a reduction in occult psychiatric morbidity in patients with the globus sensation over time, underlying personality traits remain stable and that there is a remarkable persistence of pharyngeal symptoms. |
Nasal patency was measured by five techniques in 24 subjects and the results compared. In addition three pulmonary parameters were measured as well as height and weight. Nasal resistance to airflow measured by active anterior rhinomanometry was found to be highly correlated with peak nasal inspiratory flow rate. Other correlations were also noted. Peak nasal inspiratory flow was itself highly correlated with pulmonary peak expiratory flow rate as well as with several other parameters. The possible reasons for these correlations are discussed in terms of fluid mechanics. |
Antroscopy has an established role in the diagnosis of maxillary sinus disease; however, its role in overall patient management requires clarification if it is to be widely adopted as an outpatient procedure. This study examined 100 consecutive attenders to an antroscopy clinic and monitored their diagnosis and management plan both before and after antroscopy. Diagnosis was altered or added to in 79% of cases and management in 73% of cases. The trend after knowledge of antroscopic findings was towards more medical treatment and less surgical intervention. Such a change would clearly have significant resource implications. While computed tomographic scanning time is limited, outpatient sinus endoscopy can be used as an intermediate investigation, with only selected patients going forward to be more comprehensively investigated. |
The Holt-Oram syndrome was diagnosed in four offspring of three mothers and the same unaffected father. One additional child lacked the characteristic clinical features of the Holt-Oram syndrome. In contrast to the general autosomal dominant inheritance with complete penetrance, our observation suggests a paternal mutation, resulting in mosaicism, probably restricted to the germline. |
We performed genetic analysis for carrier detection for several at-risk females in a four-generation Duchenne muscular dystrophy (DMD) pedigree using deletion analysis. We demonstrated that dosage analysis is a suitable alternative method to determine the carrier status of female relatives of DMD patients shown to have a deletion within the DMD gene. Subsequently, we diagnosed an affected male fetus for an at-risk female shown to be a DMD carrier by deletion analysis. The usefulness of deletion and linkage analysis are compared. In this family, linkage analysis was complicated by the unavailability of key family members, two recombination events and by previously undisclosed nonpaternity. We found that dosage analysis was more efficient than linkage for carrier evaluation in this family. |
Jarcho-Levin syndrome is a variety of autosomal recessive spondylocostal dysostosis characterized by severe deformity of the thoracic cage, leading to respiratory failure and early death. There are often associated dysmorphic features. The disease is more frequent in Puerto Ricans and rare in Europe. A Sicilian family with four affected individuals in two interrelated sibships is reported. |
We describe 14 patients, from 11 families, who have a progressive encephalopathy with early onset. The clinical signs of the disease are severe hypotonia, convulsions with hypsarrhythmia, profound mental retardation, hyperreflexia, transient or persistent edema, and optic atrophy. These findings and the characteristic dysmorphic features allow recognition of these patients, although no basic metabolic defect has been found. Microcephaly and atrophy of the brain develop, especially in the cerebellar and brain stem areas. An autosomal recessive mode of inheritance is likely. |
Two sibs with Beemer type short rib syndrome, one of them diagnosed in utero, are reported. Both patients had previously unreported additional abnormalities such as pyloric stenosis and short bowels. Dysplasia of pancreatic Langerhans cells was present in one sib. |
We report on a family with two sibs suffering from tuberous sclerosis. The parents were normal in all clinical tests including Wood's light examination of the skin, ophthalmoscopy, X-ray computerized tomography of brain, liver, and kidneys, cardiac echography and MR imaging of the brain. The most likely explanation is a germinal cell mosaic in one of the parents. A recurrence risk of 20 to 37% seems appropriate. The implications for risk assessment of sporadic cases are emphasized. |
In a sample of 79 pregnant women at risk offspring with neural tube defects (NTDs) and 158 controls, significantly increased median values were found for apo-transcobalamins I and II in amniotic fluid in the group at risk, thus confirming previous results. The findings may reflect a genetic disposition to NTDs associated with altered levels of apo-transcobalamins, and research on the etiology and mechanisms of NTDs should focus on these proteins. |
We examined the appearance of donor (DA) type class I antigen in the serum of rats that had received isogeneic (DA----DA) or allogeneic (DA----PVG, DA----BN, DA----LEW) liver transplants with or without cyclosporin A treatment, using two-site enzyme immunoassay. We also tested the serum titre of class I antigen in the normal DA rats with either 70% hepatectomy or cyclosporin A treatment, in order to clarify the relationship between the fluctuation in the serum titre of class I antigen in the recipient and the outcome of the transplanted liver graft. The suppression of liver graft rejection by cyclosporin A treatment significantly lowered the serum level of donor liver-derived class I antigen as compared with that of the recipient without cyclosporin A for a certain period. However, there was almost no correlation between the intensity of rejection of the liver graft and the serum level type class I among these allogeneic rejection and non-rejection liver transplantation combinations. The amount of donor-type class I antigen in the recipient's serum is dependent on whether the grafted liver is severely damaged following partial hepatectomy, whether the liver has associated biliary complications or ischaemic damage, or whether the liver has had absolute residual parenchymal cell volume or function following liver rejection. Our results suggest that the appearance of donor type class I antigen following liver transplantation is dependent on many factors, and therefore the titre of serum class 1 antigen may not always be a decisive indicator of liver graft rejection. |
After trauma, inflammatory, immunological and hormonal changes are well documented. Surgical intervention is a form of programmed trauma. Through the study of surgical patients, changes in early endogenous mediators of inflammation, immune response and tissue repair can be investigated. Here we analysed changes in serum levels of IL-1 inhibitors, IL-1 beta, IL-6, tumour necrosis factor-alpha (TNF-alpha) and cortisol in patients undergoing elective surgery. C-reactive protein (CRP) was measured as a marker of the acute-phase response. Rises in serum levels of IL-1 inhibitors, IL-6 and cortisol were detected as early as 1 h after the intervention. Peak levels were reached between 2 and 5 h. Serum levels of IL-6 and cortisol remained elevated for several days implying a persistent production. Serum levels of IL-1 and TNF did not change after the intervention. CRP levels peaked on day 2. The communication system sustained by endogenous mediators is activated after surgery as shown by selective changes in IL-1 inhibitors, IL-6 and cortisol. These mediators have different kinetics in serum and IL-6 is not the only early mediator detected. Some IL-1 inhibitors might be involved in the immunological depression observed after major surgery, in the regulation of the inflammatory response or in tissue repair. IL-6 and cortisol seem to act synergistically to activate the acute-phase response. A systemic role for IL-1 and TNF is not evident, even if the possibility that these lymphokines may act locally is not ruled out. |
To define mechanisms of vascular injury in Wegener's granulomatosis and microscopic polyarteritis, anti-endothelial cell antibodies (AECA) were sought in serum from 168 patients, all of whom had anti-neutrophil cytoplasm antibodies (ANCA) detectable by indirect immunofluorescence. Using an ELISA with human umbilical vein endothelial cells (HUVEC), IgG AECA were demonstrated in 59% and IgM AECA in 68% of patients. Pretreatment of HUVEC with tumour necrosis factor (TNF), IL-1 or interferon-gamma (IFN-gamma) led to increased binding. Adsorption of AECA/ANCA-containing serum with HUVEC or neutrophils demonstrated that AECA and ANCA recognized different targets. von Willebrand factor (vWf) antigen levels in the patient samples were markedly elevated, with a mean of 3.10 +/- 1.89 U/ml (control population mean 1.04 +/- 0.36 U/ml), suggesting widespread endothelial cell damage. Studies using an 111In-labelled HUVEC release assay with 29 AECA-containing sera did not demonstrate complement-mediated cytotoxicity, even following activation of HUVEC with TNF. Four out of 16 AECA-containing sera tested showed antibody-dependent cellular cytotoxicity with unfractionated peripheral blood mononuclear cells. These data suggest that patients with Wegener's granulomatosis or microscopic polyarteritis can develop AECA to constitutively expressed but cytokine modulated determinants on HUVEC. These antibodies do not appear to support complement-mediated cytotoxicity, but a proportion can support antibody-dependent cellular cytotoxicity, suggesting that they may contribute to vascular injury. |
The aetiology of sustained autoantibody production in human autoimmune diseases is unknown. Evidence for structural similarities and common clonal origin among autoantibodies have been demonstrated through the expression of cross-reactive idiotype (CRI). In the present study we use four monoclonal antibodies (MoAbs) with specificity for non-overlapping CRI on human rheumatoid factor (RF) autoantibodies to define the structural features of polyclonal RF characteristic of patients with autoimmune rheumatic diseases. The pattern of CRI expression in the serum of 12 patients with rheumatoid arthritis (RA), eight with systemic lupus erythematosus (SLE) and 20 with primary Sjögren's syndrome and 34 normal individuals were determined in parallel with the level of IgM RF, IgA RF and autoantibodies to the cellular antigens SS-A, SS-B, Sm, nRNP and dsDNA and cryoglobulins. The results demonstrate significant elevation in the level of IgM and IgA expressing VHI (G6 and G8) and VHIII (B6 and D12) associated CRI in the serum of patients with autoimmune rheumatic diseases compared with normal individuals. These increases paralleled, but did not equal the increase in the level of immunoglobulins and RF. However, when expressed as proportion of immunoglobulin, only the VHI-associated CRI were significantly elevated in patients compared with normal individuals. The proportion of IgM RF expressing the VHI-associated CRI was higher in patients with Sjögren's syndrome compared with SLE and RA. Furthermore, the proportion of IgA RF expressing the G6 CRI was higher than G6+ IgM RF. These findings imply that different mechanisms contribute to RF production in autoimmune diseases. It is suggested that polyconal B cell activation is likely to be a contributing mechanism. However, such polyclonal activation is unlikely to be random since a selective elevation in the level of specific autoantibodies and VHI-associated CRI is observed. Furthermore, the data demonstrate that a proportion of autoantibodies in autoimmune diseases are immunoglobulin germline gene encoded. This is more evident in some patients with primary Sjögren's syndrome, where RF is likely to be oligoclonal or monoclonal in individuals with lymphoproliferation. |
Experimental autoimmune glomerulonephritis (EAG) was induced in brown Norway (BN) rats by a single i.m. injection of collagenase-solubilized homologous glomerular basement membrane (GBM) in Freund's complete adjuvant. This model of anti-GBM disease is characterized by the development, over several weeks, of circulating and deposited anti-GBM antibodies, accompanied by albuminuria. We examined the effects of treatment with oral cyclosporin A (CsA) at different doses, starting at the time of immunization and during the course of the disease. Pretreatment with CsA 5 mg kg daily produced a moderate reduction in circulating anti-GBM antibody levels, reduced deposition of antibody on the GBM and decreased albuminuria. Doses of 10 and 20 mg/kg CsA produced a marked reduction in circulating antibody, absence of detectable deposited antibody and virtual absence of albuminuria. Renal function remained normal in CsA-treated and control animals. When CsA treatment was introduced at 2 or 4 weeks after immunization, there were significant effects on the subsequent autoimmune response and albuminuria at 10 and 20 mg/kg daily. These studies demonstrate that CsA in conventional doses has a therapeutic effect in this model of anti-GBM disease, and suggest a role for T lymphocytes in the pathogenesis of EAG. |
Autoantigens in canine autoimmune haemolytic anaemia (AIHA) were identified by immunoprecipitation using autoantibody eluted from the erythrocytes of affected dogs. At least three patterns of precipitated antigen were identified in six cases of AIHA. The most commonly precipitated antigen pattern was a combination of 42-kD and 29-kD peptides, associated with up to three other membrane components. These autoantigens may be canine glycophorins, which are of similar molecular mass, or may possibly represent an equivalent of the human Rhesus complex. An autoantigen identical in molecular mass to band 3, the erythrocyte anion channel protein, was precipitated in one case of AIHA, and unknown peptides of 37 kD and 100 kD were isolated by autoantibody from another dog. In one case, no antigens were precipitated by the eluted antibody, indicating that the autoantibody may have bound a non-protein membrane component such as phospholipid. Overall it is considered that the different patterns observed may reflect differences in the aetiology of the condition. In other studies, sera from dogs with AIHA failed to immunoprecipitate autoantigens, but were shown by immunoblotting to contain autoantibodies to proteins of the erythrocyte cytoskeleton. Such autoantibodies were also demonstrated in normal canine sera and it is suggested that they are unlikely to play a role in the pathogenesis of AIHA, but may be part of a normal clearance mechanism for damaged red blood cells. |
Pregnancy is known to influence the course of rheumatoid arthritis (RA) in women, as well as type II collagen-induced arthritis (CIA) in DBA/1 mice. A characteristic feature is the remission during gestation and the exacerbation of the diseases during the post-partum period. In the case of CIA in DBA 1 mice, two hormonal changes have been assumed to be critical for the induction of the post-partum flare: (i) the fall in steroid hormone levels from those present during pregnancy; and (ii) surges of prolactin (PRL) release at and after delivery. Our results show that treatment with oestradiol during a short period immediately after parturition protects the mouse from a post-partum flare of the disease, and that treatment with bromocriptine, a drug known to inhibit the endogenous PRL release, has a significant though less marked effect. Studies of lactating (i.e. animals with physiological stimulation of endogenous PRL release) and non-lactating arthritic mice revealed no clear-cut differences, indicating that PRL is of minor importance for the induction of the post-partum flare. Some steroids other than oestradiol, which may be implicated in the exacerbation of arthritis, namely progesterone and hydrocortisone, had no clinical effect. Analyses of agalactosyl IgG levels in mice with CIA, and anti-collagen II antibodies in sera collected at the end of the experiments revealed no significant differences between the oestradiol and the control groups. The successful oestradiol treatment of the mice indicates that the drop in endogenous oestradiol levels prior to delivery ends the oestrogen-mediated protection against arthritis during pregnancy. |
The relative contribution of different lymphoid tissues to the anti-CII antibody response was studied in rats with arthritis produced as a result of immunizing them with collagen type II (CII). Antibody production was measured by maintaining lymphoid cells in short-term culture in collagen-coated microculture wells: the antibody they secreted was determined directly by a modified ELISA. Systemic sensitization to CII was established within a week of immunization, and a stronger response in the local draining lymph nodes relative to the spleen was associated with the development of clinical disease. From experiments involving splenectomy and adoptive cell transfer, the spleen was ascribed a suppressive role in controlling both arthritis and total antibody production. The bone marrow was found to be an important site of antibody production and the greater production of antibody by cells from tibial marrow in limbs with arthritis, compared with healthy limbs, argues for a local immune response to degrading joint antigens that may have systemic suppressive or protective properties. It is concluded that local immunity reflects the state of disease and that the antibodies produced by different lymphoid tissues may be made in response to different stimuli, and that the antibodies in turn may have different pathological effects. |
There still remains some controversy regarding the possible role of immune complexes in the pathogenesis of the late-phase skin reaction (LPSR). To assess this, skin biopsies were obtained from LPSR induced in atopic human subjects 6, 24 and 48 h after allergen challenge. Cryostat sections were stained by direct immunofluorescence for the presence of fibrinogen, immunoglobulin classes IgM and IgG and for the complement components C1q and C3c. Complement components were observed in only two of the 29 biopsies studied. In both instances, only C3c was detected. One of these subjects also had unequivocal IgG staining at 6 h. IgM staining was detected in two out of 10 subjects at 6 h but no significant deposition of immunoglobulins could be found at 24 or 48 h. Fibrinogen deposition was observed in about half of the biopsies at each time-point. This study suggests that substantial complement and immunoglobulin deposition are not overt features of the allergen-induced LPSR, although the presence of small amounts of immune complexes, below the sensitivity of the method employed cannot be excluded. Fibrin deposition occurs in the LPSR but does not appear to be a prerequisite for LPSR development. |
Vaccination with an inactivated, whole cell, Q fever vaccine (Q-vax) induces lasting antibody conversion and a positive delayed-type hypersensitivity (DTH) skin reaction in about 60% of recipients but a long-lasting positive lymphoproliferative or mitogenic response to C. burnetii antigens with peripheral blood mononuclear cells (PBMC) in 85-95% of subjects. Analysis of the lymphoproliferative response to C. burnetii antigens has now been made by fractionation-reconstitution experiments with PBMC from vaccines, from past infections, and from healthy controls. The major contributor to the response in immune subjects proved to be the T lymphocyte. T cells were stimulated by both the phase I and phase II antigens of two prototype strains of C. burnetii and responses were greatly amplified by addition of IL-2. Similar T lymphocyte stimulation profiles were obtained with the 'Priscilla' strain of C. burnetii which represents a different biotype of Coxiella isolated from Q fever endocarditis; Q-vax is therefore likely to protect against endocarditis strains. Fractionation-reconstitution experiments with T and B cells from vaccines and subjects infected in the past, using various antigenic or haptenic fractions from C. burnetii indicate that protein, non-lipopolysaccharide components of the organism are responsible for the mitogenic response of immune T cells. However, the role of the lipopolysaccharide in the protective immunogen has still to be defined. |
1. The influence of myocardial noradrenaline depletion on the incidence and severity of reperfusion arrhythmias in closed-chest anaesthetized rats was investigated. Five-minute left coronary artery occlusion was carried out via an implanted occluder. Four groups of rats were studied: controls, rats treated with reserpine (5 mg/kg, intraperitoneally) 24 h before occlusion, and rats receiving 0.2 mg/kg gallopamil intravenously 5 min before occlusion either with or without reserpine pretreatment. 2. In the control group all animals had ventricular tachycardia (VT) and fibrillation (VF) immediately after reperfusion. Gallopamil reduced VT to 50% and VF to 25% (P less than 0.05 versus control). In the reserpine group all had VT, and 67% had VF, this being not significantly different from controls. Additional treatment with gallopamil markedly reduced VT and totally prevented VF (P less than 0.05 versus control). 3. Thus, total depletion of myocardial noradrenaline stores neither prevented the occurrence nor reduced the severity of reperfusion arrhythmia in rats, while gallopamil treatment was effective. |
1. We have characterized in unanaesthetized rabbits the reflex effects of injecting nicotine into the pericardial sac or left atrium on heart rate, arterial pressure, systemic vascular resistance and the amplitude and frequency of respiration. These effects were compared with those of atrial administration of nicotine and veratridine, and intrapericardial administration of veratridine and bradykinin. 2. Injection of nicotine (6.25-400 micrograms) into the pericardial sac caused dose-dependent falls of heart rate and arterial pressure, and a brief period of hypopnoea. The fall in arterial pressure was mainly due to a fall in systemic vascular resistance. The threshold dose was 25 micrograms. Near maximal falls in heart rate (108 beats/min) and arterial pressure (47 mmHg) occurred at a dose of 200-400 micrograms. The latency between injection and the onset of bradycardia was 3.0 s. 3. The effects of intrapericardial nicotine on arterial pressure and respiration were antagonized in a dose-dependent fashion by intrapericardial mecamylamine (1-100 micrograms/kg) but were unaffected by intrapericardial hyoscine methylbromide (10 micrograms/kg) or vecuronium (1-10 micrograms/kg). The haemodynamic and respiratory effects were abolished by intrapericardial procaine. The haemodynamic effects were increased, though not significantly, by sino-aortic baroreceptor denervation. In decerebrate, artificially ventilated rabbits, bilateral cervical vagotomy converted the hypotensive and bradycardic response into a slowly developing tachycardia without change in arterial pressure. 4. Left atrial injection of nicotine (6.25-100 micrograms) caused bradycardia, a rise in arterial pressure, and prolonged hyperpnoea preceded by transient hypopnoea. After sino-aortic barodenervation it caused profound falls in heart rate and arterial pressure and transient hypopnoea, which were abolished by intrapericardial procaine. 5. Intrapericardial injection of veratridine (50-100 micrograms) had no consistent effect under control conditions. After sino-aortic barodenervation it caused falls in heart rate and arterial pressure which were abolished by intrapericardial procaine. Left atrial injection of veratridine caused highly variable haemodynamic effects. 6. Intrapericardial bradykinin (2.5-25 micrograms) caused rises in both arterial pressure and heart rate. These were abolished by intrapericardial procaine. 7. We conclude that when nicotine is injected into the pericardial sac of conscious rabbits the reflex haemodynamic and respiratory effects are due to the selective activation of neuronal-type nicotinic cholinoceptors on vagal afferents that originate in the epicardium. The reflex effects of left atrial nicotine are probably due to the excitation of a combination of carotid chemoreceptors and cardiac receptors. 8. The effects of nicotine, veratridine and bradykinin that we observed in conscious rabbits were profoundly different from those reported in anaesthetized rabbits. |
1. Effects of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an inhibitor of intracellular calcium release, on renal function were examined in anaesthetized dogs. 2. Intrarenal arterial infusion of TMB-8 (0.03 and 0.1 mg/kg per min) increased urine flow rate, urinary sodium excretion and fractional excretion of sodium without affecting blood pressure, renal blood flow or glomerular filtration rate. 3. The results suggest that TMB-8 inhibits tubular sodium reabsorption to induce natriuresis. |
To determine whether there is an intrinsic defect in T cells from patients with systemic lupus erythematosus (SLE), we studied signal transduction systems, assaying the total protein kinase C (PKC) levels and the phorbol myristate acetate (PMA)-induced activation of PKC in PHA-treated T cells. T cells from SLE patients showed a decrease in proliferation in response to PMA, but not to PHA, thereby suggesting the existence of an intrinsic abnormality in the PKC-mediated activation pathway. Total PKC activity in the T cells from SLE patients was significantly decreased. Although stimulation with PMA induced a translocation of PKC from the cytosol to the particulate fraction, translocated PKC activity after 2 nM PMA treatment was decreased in the SLE T cells. Furthermore, PMA-induced phosphorylation of 80-kDa substrates was also decreased in SLE T cells. These results suggest that there is a reduced PKC activity and an impaired PKC activation in response to PMA in the SLE T cells, a finding which may explain, if partially, the defect in T cell activation in patients with SLE. |
Rheumatoid factor cross-reactive idiotype (RF-CRI) is expressed in high concentrations in the sera of some patients with rheumatoid arthritis (RA) and juvenile rheumatoid arthritis (JRA). To determine if RF-CRI is specifically expressed in rheumatic disease or if it is secondary to polyclonal B-cell activation, we examined sera of 23 children with SLE, 16 adolescents with infectious mononucleosis (IM), and age-matched pediatric controls for RF-CRI expression. Concentrations of RF-CRI in serum, determined by an inhibition ELISA, were 24 +/- 17 micrograms/ml (mean +/- SD) in 25 normal children, 31 +/- 17 in 16 young adults with IM, and were significantly increased, 70 +/- 80 micrograms/ml, in the 23 children with SLE (p less than 0.036). Eleven of 23 SLE patients had serum RF-CRI greater than the mean +/- 2 SD for normal children. Ten of 23 SLE sera contained IgM rheumatoid factor (RF) activity. One patient with IM had a borderline elevated RF-CRI level, and 5 IM patients had RF in their sera. The serum IgM concentrations in sera were: SLE (192 +/- 93 mg/dl) and IM (234 +/- 77 mg/dl) sera. These levels were significantly elevated compared to controls (132 +/- 44 mg/dl), p less than 0.031 for SLE and p less than 0.001 for IM, suggesting that polyclonal activation of B cells was present in SLE and IM patient groups. Increased expression of RF-CRI in the SLE patients correlated directly with high titer anti-DNA antibody values (r = 0.3965, p less than 0.05) and RF activity when human IgG (r = 0.5026, p less than 0.05) was used as the RF binding substrate and inversely with serum C3 levels (r = 0.3925, p less than 0.05). RF-CRI expression did not correlate with RF that bound rabbit (r = 0.3123, p greater than 0.05). Increased serum RF-CRI expression is not a result of polyclonal B-cell activation. RF-CRI may be selectively up-regulated in patients with SLE. |
Sixteen healthy very low birth weight infants (VLBWI) were studied in a serial fashion over a 3-week period. Subjects were evaluated for lymphocyte phenotype, phytohemagglutinin (PHA) response, and metabolic status including weight, blood urea nitrogen, creatinine, albumin, pH, calcium, phosphorus, and ammonia. Lymphocyte phenotype determination showed a decreased proportion of CD3+ cells (66.8 +/- 10.4 vs 75.9 +/- 6.1, P less than 0.02) in VLBWI. When subsets of the CD4+ population were examined, VLBWI had a lower proportion of CD4+/CD29+ cells (8.2 +/- 5.8% vs 23.5 +/- 8.0%, P less than 0.0001) than adults and a higher proportion of CD4+/CD45R+ cells (35.6 +/- 12.4% vs 22.2 +/- 7.4%, P less than 0.03). The CD4+ subsets in VLBWI were similar to those seen in term infants. The peak PHA response in VLBWI was greater than that of adults (P less than 0.01). There was little change in the immune measurements over the 3-week study period. There were no strong correlations between any of the immunological measurements and the metabolic measurements except that the proportion of CD8+ cells increased with birth weight. Our findings demonstrate that immune measurements in healthy VLBWI differ from values found in adults but are similar to those of full-term infants. Lower proportions of the CD4+/CD29+ cells (the helper/inducer subset for antibody production) may contribute to some of the differences in immune function reported in neonates. |
Estradiol (E) abolished clearing of pulmonary inflammation in 2-month-old male MRL/MpJ-lpr/lpr (MRL/l) mice treated with cyclophosphamide (CY). To determine if this effect persisted in animals with advanced disease, we studied male and female MRL/l mice, aged 4 and 6 months (4M, 6M, 4F, and 6F, respectively). Mice were treated, beginning at 1 month of age, with saline, CY (12 mg/kg/day), CY + castration, CY + castration + testosterone (T) in females, and CY + castration + E in males. CY had no effect on pulmonary inflammation in 4M, possibly because of the development of relatively mild lesions. However, CY was highly effective in 6M. CY + castration + T significantly reduced overall inflammation in 6F and showed a trend in 4F. CY alone had a variable effect on bronchoalveolar lavage fluid (BALF) cells and BALF IgG in both males and females. However, concurrent treatment with T was required for histologic changes of pulmonary inflammation to fully respond to a high dose of CY in female mice. E-treated males had reduced responsiveness to CY. |
Late effects of total body irradiation and subsequent autologous bone marrow transplantation on the development of age-related monoclonal gammapathies (MG) were studied in 14 long-term surviving Rhesus monkeys. Together with 27 untreated control monkeys, they have been followed up for more than 20 years. In contrast with the control group, the experimental monkeys developed MG with aging in higher frequencies, earlier and mainly of the benign MG category. One experimental monkey developed a multiple myeloma, the first observed in the nonhuman primates so far. These results indicate an accelerated senescence of the immune system in the experimental monkeys as a late consequence of tissue or cell damage during irradiation. |