Education debate: clinical diagnostic reasoning.

Whilst it is clear that experienced clinicians adopt a rational approach of diagnosis, the logic of their clinical reasoning has been difficult to define. I outline here an approach based on the four categories of a complete diagnosis: Anatomical diagnosis (system involved); Pathological diagnosis (nature of the condition); Physiological diagnosis (functional consequences) and Aetiological diagnosis (background cause). Each phrase of the history and examination data is assigned to one or other of these categories as the case unfolds, with interpretations and interactions allowing sub-conclusions that gradually build to a final clinical diagnosis overall. The system has the advantage of facilitating a diagnosis individualized to the patient, rather than to some previously learned diagnostic 'checklist'. As such, it should provide an advance over current problem-based approaches to clinical diagnosis.

An evolution-based hypothesis on the origin and mechanisms of autoimmune disease.

The pathogenesis of autoimmune disease remains an enigma. Here, the condition is analysed from an evolutionary standpoint, and the thesis developed that viruses, in particular retroviruses, are important to our evolution, and that their inappropriate re-expression by repetitive (? ischaemic) cell damage in individuals of appropriate major histocompatibility type, leads to autoimmune disease. Such a view requires a slight adjustment to traditional ways of seeing Darwinian evolution, but one which makes real sense of the MHC-restricted nature of the adaptive immune response.

Species variation in the metabolism of 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one to its 3,4-dihydrodiol, the proximate carcinogen.

The title compound is a strong carcinogen, similar in potency to benzo[a]pyrene in mouse skin assay. This paper describes a comparison of its in vitro metabolism by hepatic microsomal preparations from mouse, rat, rabbit, hamster, dog, monkey and man. Metabolites were isolated by preparative high pressure liquid chromatography from the ethyl acetate extractable material and their structures tentatively assigned on the basis of their retention times and ultraviolet spectra, when possible by direct comparison with authentic synthetic specimens. Mass spectrometry was then used to confirm these assignments. All these animals produce the same range of metabolites derived exclusively from oxidation at the benzo-ring A, the five-membered ring D, and at the 11-methyl group. However, the amounts of individual metabolites varied substantially. In particular all the animals yielded the proximate carcinogen 3,4-dihydroxy-11-methyl-3,4,15, 16-tetrahydrocyclopenta[a]phenanthren-17-one, from which it is reasoned that all might be susceptible to its carcinogenic action. A rationalization for the observed distribution of the metabolites is proposed on the basis of a molecular model of the active site of cytochrome P450 1A1, the oxidative enzyme involved.

The presence of a trifluoromethyl rather than a methyl substituent in the bay-region greatly decreases the DNA-binding and tumour-initiating activity of the cyclopenta[alpha]phenanthren-17-ones.

The increase in carcinogenicity of polycyclic aromatic compounds following bay-region methyl group substitution involves a steric component: increasing the size of the alkyl substituent decreases the carcinogenic activity of the compound. To determine whether there is also an electronic component to this effect, we synthesized a bay-region 11-trifluoromethyl analogue of 15,16-dihydrocyclopenta[alpha]phenanthren-17-one which is sterically similar but electronically very different from the 11-methyl derivative. This trifluoromethyl derivative bound to DNA in cultures of the human mammary carcinoma cell line MCF-7 to a much lower extent than the methyl-substituted compound. The trifluoromethyl derivative did not form detectable levels of DNA adducts in the epidermis of Sencar mice and was inactive as an initiator after promotion with 12-O-tetradecanoylphorbol-13-acetate for 20 weeks. In contrast, the 11-methyl derivative formed > 3 pmol adducts/mg DNA and initiated eight papillomas per mouse. These data indicate that both the steric configuration and the electronic nature of a bay-region substituent are important in determining the overall effect of the substituent on the biological activity of the molecule.

The influence of dihydrodiol conformation on the metabolic activation of cyclopenta[a]phenanthrenes.

The present study was undertaken in order to rationalise the apparent biological inactivity of 15,16-dihydro-6-methylcyclopenta[a]phenanthren-17- one (4) when other methyl isomers of 15,16-dihydrocyclopenta[a]phenanthren- -17-one, e.g. the 11-methyl derivative (2), display appreciable tumorigenicity. In vitro metabolism of the 6-methyl-ketone-17-one (4) demonstrated that its principal metabolite was the 3,4-dihydro-3,4-diol (3,4-dihydroxy-6-methyl-3,4,15,16- tetrahydrocyclopenta[a]phenanthren-17-one) (5) which, in the case of the active 11-methyl derivative, is the proximate genotoxin. Thus the inactivity of this 6-methyl-17-ketone cannot be ascribed to lack of formation of the 3,4-dihydro-3,4-diol, the precursor of the 3,4-diol-1,2-epoxides (the ultimate mutagens in this series). However, the 6-methyl-3,4-dihydro-3,4-diol exists in a pseudo-diaxial rather than a pseudo-diequatorial conformation characteristic of the 3,4-dihydro-3,4-diols of the other members of the series. It is therefore suggested that a diequatorial conformation in the dihydrodiol is essential to the metabolic activation of the cyclopenta[a]phenanthren-17-ones.

CYP1 induction, binding to the hepatic aromatic hydrocarbon receptor and mutagenicity of a series of 11-alkoxy cyclopenta[a]phenanthren-17-ones: a structure activity relationship.

A series of four 11-alkoxy cyclopenta[a]phenanthren-17-ones, ranging from the methoxy to the butoxy derivative, has been synthesised in order to investigate the effect of the size of the 11-substituent on the mutagenicity and ability of these compounds to induce hepatic CYP1 activity in rats. The latter was monitored by using as diagnostic probes methoxy and ethoxy-resorufin, and immunologically in Western blots employing anti-CYP1A1 antibodies. All four members of the series induced both CYP1A1 and CYP1A2 activities and apoprotein levels, but the methoxy- and ethoxy-CPP-17-ones were clearly the most potent. Of the four isomers, only 11-methoxy-CPP-17-one displaced 3H-TCDD from the cytosolic Ah receptor. Similarly only 11-methoxy-CPP-17-one elicited a positive mutagenic response in the Ames test in the presence of an Aroclor 1254-induced activation system. The relevance of these findings to the carcinogenicity of these compounds in the mouse skin painting model is discussed.

Cytochrome P450-dependent metabolism and mutagenicity of 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one and their implications in its carcinogenicity.

Methylation of the non-carcinogen 15,16-dihydrocyclopenta[a]phenanthren-17-one (CPP-17-one) at the bay region to form 11-CH3-CPP-17-one confers carcinogenic potential. In the present study we have investigated the in vitro metabolism and mutagenicity of the methylated compound by hepatic microsomal preparations from rats pretreated with various prototype inducers of cytochrome P450 proteins in order to provide a rationale for this marked difference in carcinogenic activity. The most effective metabolism of 11-CH3-CPP-17-one occurred in the presence of Aroclor 1254-induced microsomes, the principal metabolites being oxidative products of the A- and D-rings and of the methyl substituent. When benzo[a]pyrene-induced microsomes served as the metabolising system, the major A-ring metabolite was the 3,4-diol. A similar metabolic pattern was seen with microsomes from rats treated with 11-CH3-CPP-one itself, but the overall effect of metabolism was lower than that observed with benzo[a]pyrene-treated microsomes but higher than that of control animals. In contrast, microsomes from rats treated with clofibrate, dexamethasone, isoniazid and phenobarbitone failed to enhance the metabolism of 11-CH3-CPP-17-one when compared with control microsomes and the metabolites reflected primarily oxidation of the D-ring. When 11-CH3-CPP-17-one was employed as a promutagen in the Ames test, a mutagenic response was evident only in the presence of microsomes from benzo[a]pyrene-induced rats, but induction with phenobarbitone, isoniazid, dexamethasone, clofibrate and the compound itself, failed to elicit a positive mutagenic response. When 3,4-dihydroxy-11-CH3-CPP-17-one served as the promutagen, a mutagenic response was observed in the presence of benzo[a]pyrene-induced and, to a lesser extent, 11-CH3-CPP-17-one-induced microsomes. Treatment of rats with 11-CH3-CPP-17-one caused a marked increase in the O-deethylation of ethoxyresorufin and, to a much lesser extent in epoxide hydrolase activity. It is concluded that (i) 11-CH3CPP-17-one is an inducer of the CYP1 family; (ii) under the present experimental conditions only the CYP1 family can oxidise the A-ring to form the 3,4-dihydroxy-11-CH3-CPP-17-one, the precursor of the ultimate carcinogen and (iii) only the CYP1 family oxidizes the diol to generate the ultimate carcinogen.(ABSTRACT TRUNCATED AT 400 WORDS)

The in vitro metabolic activation of the 11-trifluoromethyl analogue of the potent carcinogen 15,16-dihydro-11-methyl-cyclopenta[a]-phenanthren-17-one to mutagens.

A strongly electronegative, bay-region analogue of the potent carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one, namely 15,16-dihydro-11-trifluoromethylcyclopenta[a]phenanthren-17-one, is mutagenic to Salmonella typhimurium TA100. Also it is metabolized at the 1,2- and 3,4-positions in the A-ring as well as C-15 in the D-ring to give 3,4-dihydroxy-3,4,15,16-tetrahydro-11-trifluoromethyl- cyclopenta[a]phenanthren-17-one as the only mutagenic metabolite. In these respects its behaviour is closely similar to that of the 11-methyl compound, suggesting that the electronic nature of the bay-region substituent is rather less critical than its spatial configuration in influencing metabolism to genotoxic intermediates. It remains to be seen, however, whether the trifluoromethyl compound is also a carcinogen.

The mutagenicity of chemically synthesized metabolites of 16,17-dihydro-15H-cyclopenta[a]phenanthrene and its carcinogenic 11-methyl homologue.

Putative synthetic metabolites of the hydrocarbon 16,17-dihydro-15H-cyclopenta[a]phenanthrene and its carcinogenic 11-methyl analogue, namely trans-3,4-dihydroxy-3,4,16,17-tetrahydro-15H- cyclopenta[a]phenanthrene and its 11-methyl derivative, together with the four associated trans-3,4-dihydroxy-syn- and anti-1,2-epoxides, were assayed for mutagenicity in the Ames test with Salmonella typhimurium TA100 with and without microsomal activation. The hydrocarbons were weakly mutagenic and the 3,4-diols were more strongly so, but all required activation to express their mutagenic potential. All four diol-epoxides were much more potent mutagens, even in the absence of activation. This is in accord with the anticipated metabolic activation sequence: hydrocarbons-->3,4-diols-->3,4-diol-1,2-epoxides.

The metabolism and activation of 15,16-dihydrocyclopenta[a]phenanthren-17-one by cytochrome P-450 proteins.

The in vitro metabolism and activation to mutagens of 15,16-dihydrocyclopenta[a]phenanthren-17-one (CPP-17-one) were investigated using hepatic preparations from rats pretreated with prototype inducers of the cytochrome P-450-dependent mixed-function oxidases. Aroclor 1254-induced microsomes were the most effective metabolisers of this compound, the major metabolites being oxidation products of the bay region A ring. To a lesser extent hydroxylation of the non-aromatic D ring occurred, the products being the 15- and 16-hydroxyderivatives. Oxidation of the A ring was also achieved with microsomes from benzo[a]pyrene-treated rats but not with those from rats treated with clofibrate, phenobarbitone, isoniazid, dexamethasone and CPP-17-one itself, where the metabolites were primarily the oxidation products of the D ring. When CPP-17-one was used as a promutagen in the Ames test, only microsomes from Aroclor 1254-treated rats could elicit a positive mutagenic response. When 3,4-dihydrodihydroxy-CPP-17-one, the precursor of the ultimate mutagen, was used as the promutagen, a positive response was observed with microsomes from Aroclor 1254- and benzo[a]pyrene-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Investigating optimal bull:heifer ratios required for estrus-synchronized heifers.

The objectives of this study were to determine 1) the effect of bull:heifer ratio on reproductive performance and associated costs and return on heifers in synchronized estrus and 2) the effect of estrus synchronization on reproductive performance and economic variables, in a multiple-sire, pasture breeding situation. Eight hundred yearling beef heifers and 28 mature, sexually experienced beef bulls were allotted to four treatments (two replicates per treatment) at bull:heifer ratios of 2 per 100 (1:50; Treatment 1), 2 per 100 (1:50; Treatment 2), 4 per 100 (1:25; Treatment 3), and 6 per 100 (1:16; Treatment 4). Treatment 1 (control) used nonsynchronized heifers, whereas heifers in Treatments 2, 3, and 4 were synchronized using the 33-d melengestrol acetate (MGA)-prostaglandin F2 alpha (PGF2 alpha) program. Pregnancy results after a 28-d breeding season indicate that there may be a limit to how many estrus-synchronized heifers bulls can impregnate. Treatment 2 showed a 6% decrease (P < .10) in pregnancy rate (77%) compared with Treatment 3 (83%), indicating that the bulls probably were not able to service all the synchronized heifers. Treatments 3 and 4 had similar pregnancy rates (83 and 84%, respectively). Treatment 4 had a 3-d advantage (P < .01) over Treatment 3 in average day of conception. However, based on economic analysis, Treatment 3 exhibited greater returns. Estrus synchronization failed to provide any advantage in pregnancy rate or day of conception. For unknown reasons, the control, nonsynchronized heifers cycled and conceived as if they were synchronized.(ABSTRACT TRUNCATED AT 250 WORDS)

Haemodynamic changes in the Moncada model of atherosclerosis.

1. The application of a non-constricting silastic cuff to the rabbit common carotid artery (CCA; n = 5) results in intimal thickening within 7 days. 2. Ultrasonography showed kinking of the CCA at the cuff edges, as well as a 13% arterial narrowing (P less than 0.02) within the cuffed segment, both at days 1 and 7. Correspondingly, the time averaged diastolic Doppler velocity (TAV) was 68.8 +/- 12.8% higher (P less than 0.025) in the cuffed region in comparison with that 1 cm proximal (P 1 cm) on day 1, and 54.2 +/- 11.5% higher at day 7 (P less than 0.05). TAV values along control silastic strips were not significantly changed. 3. There was a significant increase (P less than 0.025) in intimal area within the cuffed region (0.098 +/- 0.024 mm3) compared with both the proximal control (0.014 +/- 0.001 mm3) and with that over control silastic strips (0.021 +/- 0.004 mm3, P less than 0.01). 4. Medial area within the cuff (0.433 +/- 0.017 mm3) was decreased (P less than 0.005) compared with P 1 cm control (0.602 +/- 0.069 mm3). 5. There was gross peri-arteritic thickening involving the adventitia along the non-constricted cuffed segment. Importantly, it was also noted alongside the control silastic strip. 6. Kinking of the CCA and associated vasoconstriction cause changes in blood flow velocity along even a non-constricting cuff, and this may explain the intimal thickening previously noted in this experimental model. The peri-arteritic changes, on the other hand, appear to be a reaction to the silastic itself.

Prevention of arterial disease in experimental renal hypertension.

1. This study examined the effect of various antihypertensive agents on the development of polyarteritis nodosa lesions along the mesenteric artery system over a 10 week period after renal artery clipping in uninephrectomized rats (lKlC). 2. Of the agents, only hydralazine, enalapril and diltiazem significantly inhibited systolic blood pressure (SBP) rise over the 10 week period (P less than 0.001). 3. All agents except hydralazine reduced the severity of arteritic lesions compared with lKlC rats, but only with enalapril (P less than 0.001), nifedipine (P less than 0.001), diltiazem (P less than 0.005), propranolol (P less than 0.001) and reserpine (P less than 0.05) was this reduction statistically significant. 4. There was a positive correlation between the degree of arteritic change and SBP, but the correlation coefficient was neither high (r = 0.68) nor highly significant (P = 0.03, d.f. = 9). On examining the data, this was due on the one hand to nifedipine, propranolol and reserpine reducing the severity of lesions without significantly inhibiting SBP, and on the other to hydralazine reducing SBP without significantly affecting the extent of arteritic change. 5. These findings suggest that factors other than mere SBP alone are involved in the pathogenesis of these arteritic lesions.

Hypothesis: ischaemic plaque necrosis as the initiator of unstable angina/acute myocardial infarction?

1. Coronary atherosclerotic plaque complications are important in precipitating acute coronary events such as unstable angina and myocardial infarction. 2. The hypothesis is put forward that plaque complications are initiated by increases in local coronary artery tone sufficient to cut off the vasa vasorum blood supply to the plaque and result in its ischaemic necrosis.

Effects of natural mating stimuli on serum luteinizing hormone, testosterone and estradiol-17 beta in yearling beef bulls.

The objective of this study was to determine the effect of natural mating stimuli on serum concentrations of LH, testosterone (T) and estradiol-17 beta (E2) in beef bulls. Twenty sexually experienced, yearling beef bulls were bled every 15 min during a 9-h period, 4 h before and 5 h after exposure to estrual females. For exposure, each bull was placed individually in an isolated pen with two restrained estrual heifers for 10 min or until one service was achieved. Timing and number of all behavioral events, including flehmen responses, abortive mounts and services, were recorded for each bull by two independent observers. Of the 20 bulls, 9 bulls mounted and were removed immediately after achieving a service, 8 bulls mounted without achieving a service and 3 bulls exhibited no interest during exposure. Twelve bulls achieved fewer than three and eight bulls achieved three or more flehmen responses during exposure. Postexposure responses in LH, T and E2 were not consistently correlated with number of mounts or presence or absence of a service. However, postexposure LH and T, but not E2, responses were highly correlated with number of flehmen responses achieved (r = .40 to .66; P = .08 to .001). In bulls that achieved three or more flehmen responses, serum LH increased within 30 min after exposure (P = .02) and serum T was increased dramatically within 1 h after exposure (P less than .01), compared with preexposure hormone concentrations, regardless of the number of mounts and regardless of the presence or absence of a service. Natural mating stimuli had no effect on serum E2, and mounting activity alone and mounting that culminated in a service did not necessarily result in increased LH or T in beef bulls. However, number of flehmen responses achieved during exposure to females dramatically influenced postexposure serum LH and T concentrations in beef bulls.

Histometric assessment of renal arterioles during DOCA and post-DOCA hypertension and hydralazine treatment in rats.

It is now well established that structural changes in resistance vessels contribute to the long-term maintenance of many forms of hypertension. This study measured the time course of structural change in smaller resistance vessels during the development of deoxycorticosterone acetates (DOCA) hypertension and after cessation of DOCA in Wistar rats by histometric assessment of cross-sections of renal arterioles. The relationship of structural change to blood pressure was assessed by preventing hypertension during DOCA treatment with hydralazine. Blood pressure rose progressively during DOCA treatment reaching 192 +/- 3 mmHg compared with 132 +/- 2 mmHg in controls after 10 weeks. Five and 10 weeks after cessation of DOCA, following 10 weeks of DOCA treatment, post-DOCA reversal of hypertension was only partial. Medial area to internal elastic lamina (IEL) radius ratio, wall to lumen ratio and intimal area to IEL radius ratio of renal arterioles increased progressively during 10 weeks of DOCA treatment with partial reversal of the increased medial area and wall to lumen ratio 5 weeks post-DOCA. Hydralazine completely prevented hypertension in DOCA rats and also largely prevented structural change.

Structural change in the rat hindlimb during deoxycorticosterone acetate hypertension; its reversibility and prevention.

This study measured the time course of the development and reversal of structural change in resistance vessels during deoxycorticosterone acetate (DOCA) hypertension and after cessation of DOCA in Wistar rats by hindquarter perfusion. The relationship of structural change to blood pressure was further assessed by preventing hypertension during DOCA treatment with hydralazine. Blood pressure rose progressively during DOCA treatment reaching 198.6 +/- 2.0 (s.e.) mmHg at 10 weeks. Five weeks after the cessation of DOCA, when it had been given for 2 weeks, hypertension reversed completely but after 4, 8 and 10 weeks of treatment, post-DOCA reversal of hypertension was only partial. Hindquarter perfusion pressure at maximum vasodilatation and maximum vasoconstriction increased with increasing duration of DOCA treatment and reversed 5 weeks post-DOCA to a similar degree to the blood pressure with only partial reversal of both perfusion pressures and hypertension when DOCA had been given for 8 and 10 weeks. Hydralazine did not completely prevent heart hypertrophy in the DOCA rats and caused some cardiac hypertrophy in the control ('vehicle' only) rats, although it both prevented hypertension and evidence of vascular structural change in DOCA rats.

Structural changes vary along individual arterioles in deoxycorticosterone acetate hypertensive rats.

The variability of structural changes along individual arterioles in deoxycorticosterone acetate (DOCA) hypertensive rats was measured by coefficients of variation of: (a) serial lumen diameters along microfil casts of individual mesenteric arterioles; and (b) wall and lumen indices in serial histological cross-sections along individual renal arterioles. The mean lumen diameter of third-order mesenteric arterioles decreased with increasing duration of hypertension. There was increased variability of lumen diameter along lengths of DOCA arterioles, the coefficient of variation at 10 weeks DOCA treatment being 20.2 +/- 0.6% compared to 9.2 +/- 0.2% in controls (P less than 0.001). Medial area to internal elastic lamina (IEL) radius ratio of renal arterioles was increased in DOCA rats compared with control rats (P less than 0.025) and its variability along individual arterioles expressed as the coefficient of variation was 31.70 +/- 3.87% in DOCA rats compared with 15.29 +/- 1.72% in controls (P less than 0.005). The observed increase in variability of lumen diameter and medial area along short lengths of individual arterioles in DOCA hypertensive rats indicates that hypertensive structural changes are probably not directly related to local blood pressure. We suggest that irregular functional vasoconstriction in hypertensive rats could account for this distribution of structural changes.