The Essential Role of Circulating Thyroglobulin in Maintaining Dominance of Natural Regulatory T Cell Function to Prevent Autoimmune Thyroiditis.

Several key findings from the late 1960s to mid-1970s regarding thyroid hormone metabolism and circulating thyroglobulin composition converged with studies pertaining to the role of T lymphocytes in autoimmune thyroiditis. These studies cemented the foundation for subsequent investigations into the existence and antigenic specificity of thymus-derived natural regulatory T cells (nTregs). These nTregs prevented the development of autoimmune thyroiditis, despite the ever-present genetic predisposition, autoantigen (thyroglobulin), and thyroglobulin-reactive T cells. Guided by the hypothalamus-pituitary-thyroid axis as a fixed set-point regulator in thyroid hormone metabolism, we used a murine model and compared at key junctures the capacity of circulating thyroglobulin level (raised by thyroid-stimulating hormone or exogenous thyroglobulin administration) to strengthen self-tolerance and resist autoimmune thyroiditis. The findings clearly demonstrated an essential role for raised circulating thyroglobulin levels in maintaining the dominance of nTreg function and inhibiting thyroid autoimmunity. Subsequent identification of thyroglobulin-specific nTregs as CD4(+)CD25(+)Foxp3(+) in the early 2000s enabled the examination of probable mechanisms of nTreg function. We observed that whenever nTreg function was perturbed by immunotherapeutic measures, opportunistic autoimmune disorders invariably surfaced. This review highlights the step-wise progression of applying insights from endocrinologic and immunologic studies to advance our understanding of the clonal balance between natural regulatory and autoreactive T cells. Moreover, we focus on how tilting the balance in favor of maintaining peripheral tolerance could be achieved. Thus, murine autoimmune thyroiditis has served as a unique model capable of closely simulating natural physiologic conditions.

EDTA is superior to DTT treatment for overcoming the prozone effect in HLA antibody testing.

A limitation of solid-phase human leukocyte antigen (HLA) antibody assays is the falsely low/negative result of samples with high-titer antibodies, a phenomenon known as the prozone effect. Here we compared the efficacy of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) treatment of serum samples in overcoming the prozone effect. A total of 21 serum samples were treated with either EDTA or DTT before HLA single antigen bead assay. The efficacy of prozone effect reversal, compared with untreated samples, was examined on fourfold, serially diluted samples, from neat to 1:256, using PBS as diluent. EDTA reversed the prozone effect in all tested samples, with an efficiency of greater than 84%, estimated by the ratio of undiluted sample mean fluorescence intensity (MFI) to peak MFI, for any given dilution. In contrast, the efficiency of DTT treatment was as low as 47%. These results show superior prozone effect reversal with EDTA treatment, compared with DTT.

Nontreatment and aggressive narcotic therapy among hospitalized pancreatic cancer patients.

OBJECTIVES: Strong feelings about patient autonomy as expressed in living wills, polls, and legislative referenda have been challenging the medical establishment to increase nontreatment, defined as foregoing a life-prolonging treatment, and even to provide treatments having life-shortening potential to selected patients. Because there are little data about the actual practice of these procedures, including aggressive narcotic therapy as defined herein, we studied the terminal management of 417 pancreatic cancer patients. DESIGN AND PARTICIPANTS: The medical records of 417 residents of King County, Washington, who died of pancreatic cancer in the time periods 1959-1962, 1969-1972, and 1985-1990, were reviewed to study the frequency of, and risk factors for, end-of-life nontreatment decisions and aggressive narcotic therapy decisions, defined here as the decision to administer treatment doses of narcotics or major sedatives to already comatose patients within 4 hours of death. RESULTS: Antibiotics were not provided to 71% of the 70 febrile patients (two readings >38.33-38.83 degrees C or one reading of 38.88 degrees C), intravenous fluid was not provided to 43% of 294 dehydrated patients (oral intake <500 mL/24 hours), transfusions were not provided to 39% of 57 severely anemic patients (hematocrit <20%), and laparotomy was not performed for 86% of 36 patients with abdominal emergencies (obstruction, bleeding, dehiscence). Also, 46% of the 118 patients who were comatose for at least 24 hours before death received aggressive narcotic therapy, as defined above. A total of 335 of the 417 patients had documentation of at least one of the above life-threatening conditions or were comatose for at least 24 hours before death, and 289 (86%) of these patients experienced nontreatment of one or more of these conditions or received aggressive narcotic therapy. Nontreatment decisions for febrile, dehydrated, or anemic patients tended to be more frequent if the patient was comatose (P=.004, .010, and .065, respectively), if there was a nontreatment statement in the medical record (P=.009, .035, and .001, respectively), or if the patient was described as terminal (P=.262, .029, and .002, respectively). Aggressive narcotic therapy in comatose patients was more common among patients who had regular visitors (P=.002), who had pre-coma pain (P=.006), who had nontreatment statements in their charts (P=.031), whose in-charge physician was an oncologist (P < .001), who were treated in a community nonprofit hospital compared with a Catholic hospital (P=.007), or who were treated in recent years (P=.011). CONCLUSION: Both nontreatment and aggressive narcotic therapy forms of medical management have been occurring commonly in terminal pancreatic cancer patients in King County, Washington, during the past 3 decades, the latter with greater frequency in recent years.

Serum proteolytic activity during the growth of C6 astrocytoma.

Tumor growth is dependent on the ability of neoplastic cells to induce angiogenesis. Blood-vessel remodeling requires the reconstruction of the nonfibrous proteins and type IV collagen components of the basement membrane. This study has assessed the influence of the growth of C6 astrocytoma cells in the rat spheroid implantation model on serum general protease and type IV collagenase activity. The results demonstrate that general protease activity increased in serum, reaching maximum values on Day 6 and Day 13 following spheroid implantation, and that type IV collagenase activity increased in serum, obtaining maximum values on Day 8 and Day 15. The measurement of serum proteolytic activity may be of value in the detection of recurrent tumors.

Comparative in vitro metabolism of the cannabinoids.

The metabolism of delta-9-tetrahydrocannabinol (delta-9-THC), delta-8-THC, delta-11-THC, cannabidiol (CBD), cannabinol (CBN), cannabichromene (CBC), cannabigerol (CBG) and the equatorial-isomer of hexahydrocannabinol (HHC) was studied in microsomal preparations obtained from rats, mice, guinea pigs, rabbits, hamsters, gerbils and a cat. Identification of metabolites was by GC/MS and quantification by gas chromatography. Major metabolites were monohydroxylated compounds but the pattern of hydroxylation varied considerably between the species, no doubt reflecting the variable nature of the cytochrome P-450 mixed-function oxidases. Although the primary carbon allylic to the endocyclic double bond of tricyclic cannabinoids was usually the major site of attack, the 4' (side-chain, omega-1 position) and the terpene ring were usually favoured by the cat and hamster respectively. The guinea pig generally produced more metabolites hydroxylated in the side-chain (all positions) than did the other species. The results from HHC were very similar to those from THC, namely hydroxylation at C-11 in most species, and the production of high concentrations of 8 alpha-hydroxy-HHC in the mouse and 8 beta-hydroxy-HHC in the hamster. As this molecule lacks the double bond of the THCs and, hence, the allylic nature of C-11 and C-8, the results suggest that it is the orientation of the molecule to the active site of the cytochrome P-450 mixed-function oxidase rather than the reactivity of the C-H bond that governs the position of hydroxylation.

Clinical judgments of high-risk behavior during recovery.

This study utilized focus group research to explore high-risk behavior during recovery from drugs and alcohol. Participants in the focus group were professional substance abuse counselors. The findings identified specific high-risk behaviors and began an exploration of the processes that support them. Specific information is included on such issues as causes, time frames, developmental issues, and other factors associated with high-risk behavior. Attention was paid to existential issues in recovery, as well as childhood factors such as sexual abuse. High-risk behavior was seen as a means to avoid the existential dilemma of continuing in recovery or returning to drug use and as a possible means to leave this crisis unresolved without actual relapse to drug use, or dealing with the issues of advancing in recovery.

Metabolism of n-hexyl-homologues of delta-8-tetrahydrocannabinol and delta-9-tetrahydrocannabinol in the mouse.

n-Hexyl-delta-8-tetrahydrocannabinol (n-hexyl-delta-8-THC) and n-hexyl-delta-9-THC were synthesized by condensation of (1S)-cis-verbenol with 5-n-hexyl-1,3-dihydroxybenzene and administered intraperitoneally to male Charles-River CD-1 mice. Hepatic metabolites were isolated by solvent extraction and chromatography on Sephadex LH-20 and identified by GC/MS. Eleven metabolites were identified from n-hexyl-delta-8-THC and sixteen from n-hexyl-delta-9-THC. The pattern of metabolites was intermediate between that previously observed from the pentyl homologues and that from n-heptyl-delta-9-THC with the major biotransformation pathway being hydroxylation and oxidation at C-11. Other metabolites were mainly hydroxylated derivatives of these compounds. Metabolites containing two hydroxy groups in the side-chain were present in low concentration. These have not been observed from lower homologues but are major metabolites of n-heptyl-delta-9-THC. Compared with the metabolism of the n-pentyl homologue, there was a trend towards the production of more hydroxy metabolites at the expense of carboxylic acids, in keeping with the general reduction of oxidation observed with other homologous cannabinoids as the chain length increases.

In vitro metabolism of cannabinol in rat, mouse, rabbit, guinea pig, hamster, gerbil and cat.

Metabolism of cannabinol (CBN) was studied in hepatic microsomal incubates from mouse, rat, rabbit, guinea pig, cat, hamster and gerbil. Metabolites were extracted with ethyl acetate, concentrated by chromatography on Sephadex LH-20 and identified by GC/MS as TMS derivatives. Six monohydroxy metabolites were identified. These had hydroxy groups at C-11 and at all positions of the pentyl side-chain. Metabolism varied considerably between the species. 11-Hydroxylation was the most prominent route in the majority of species, but in the hamster and cat the major metabolic pathway was 4'-hydroxylation. Metabolites hydroxylated in the pentyl chain were generally more abundant in guinea pig, hamster and cat.

Metabolites of the 1',2'-dimethylheptyl analogue of delta-8-tetrahydrocannabinol in the mouse and their identification by gas chromatography/mass spectrometry.

Metabolism of the 1,2-dimethylheptyl analogue of delta-8-tetrahydrocannabinol (delta-8-DMHP) was studied in vitro using mouse hepatic microsomes and in vivo in mouse liver. Metabolites were extracted with ethyl acetate, concentrated by chromatography on Sephadex LH-20 and examined by low-resolution mass spectrometry as trimethylsilyl (TMS), (2H9)TMS and methyl ester/TMS derivatives. Reduction of metabolites with lithium aluminium deuteride also provided structural information. The electron-impact-induced mass spectrum of the TMS derivative of DMHP differed from that of its unbranched side-chain analogues in that prominent ions were produced by fragmentation of the side-chain at the expense of the retro-Diels-Alder fragmentation that was prominent in the spectra of the latter compounds. This, however, was found to reduce the relative abundance of ions diagnostic of side-chain hydroxy substitution in the spectra of the metabolites. In vitro, the only significant metabolite was 11-hydroxy-delta-8-DMHP. This is in contrast with metabolism of the corresponding delta-8-tetrahydrocannabinol (delta-8-THC, n-C5-side-chain) where a number of other monohydroxy metabolites are produced. Fifteen metabolites were found in vivo, of which nine were identified. Mass spectral information was not sufficient to determine the position of one of the hydroxy groups in the other six metabolites. The major site of hydroxylation was at C-11 and the resulting hydroxy metabolite was oxidized to delta-8-DMHP-11-oic acid. In this respect metabolism paralleled that of delta-8-THC. Dihydroxylation of the double bond also occurred, presumably via the epoxide.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro metabolism of delta-11-tetrahydrocannabinol in the mouse, rat, guinea pig, rabbit, hamster, gerbil and cat.

1. Liver microsomes were prepared from rats, rabbits, guinea pigs, hamsters, gerbils, a cat and three strains of mice, and were incubated with delta-11-tetrahydrocannabinol (delta-11-THC). The extracted metabolites were separated by chromatography on Sephadex LH-20 and examined by gas chromatography and combined gas chromatography/mass spectrometry. 2. Eleven metabolites were identified; these were formed by aliphatic hydroxylation of all positions of the pentyl chain, allylic hydroxylation at C-10 and C-8 (alpha and beta), and by the epoxide-diol pathway. 3. The ratio of the metabolites varied considerably between the species. Mice and rats favoured hydroxylation at C-8-alpha with very little hydroxylation of the pentyl chain. 4. In the guinea pig, however, hydroxylation of the pentyl chain, particularly at C-4', produced the major metabolites; very little hydroxylation occurred at C-8. 5. Side-chain hydroxylation was also favoured by the gerbil. 6. In the cat and hamster, 8-beta-hydroxylation was by far the major metabolic route, accounting, in the cat, for nearly 70% of the recovered metabolites. 7. The rabbit, on the other hand, favoured the epoxide-diol pathway with over 70% of the recovered metabolites being accounted for by the 9,11-dihydro-diols. 8. The results emphasise the need to make appropriate choices of animal models for metabolic and toxicological studies in humans.

In vivo metabolism of the n-butyl-homologues of delta 9-tetrahydrocannabinol and delta 8-tetrahydrocannabinol by the mouse.

1. n-Butyl-homologues of delta 8-tetrahydrocannabinol (delta 8-THC) and delta 9-THC were synthesized from 5-butyl-1,3-dihydroxybenzene and (1S)-cis-verbenol, and the delta 9-isomer was shown to have the same g.l.c.-mass spectral characteristics as the natural product. 2. Metabolism of these cannabinoids was studied in mice following i.p. injection. Metabolites were extracted from the livers, separated from endogenous lipids by chromatography on Sephadex LH-20 and examined by g.l.c.-mass spectrometry. 3. Thirteen metabolites were identified from both n-butyl-delta 8-THC and n-butyl-delta 9-THC. 4. Major metabolic routes were hydroxylations in the 2', 3', 8 and 11 positions and oxidation of the resulting 11-hydroxy-metabolites to carboxylic acids. 5. Metabolism was very similar to that of the pentyl homologues, the major constituents of cannabis, but with the production of a greater proportion of acidic metabolites at the expense of alcohols.

Electron impact-induced fragmentation of the trimethylsilyl derivatives of monohydroxy-hexahydrocannabinols.

Monohydroxylated derivatives of hexahydrocannabinols were synthesized by catalytic hydrogenation of hydroxytetrahydrocannabinols over a rhodium/alumina catalyst, reduction of tetrahydrocannabinol epoxides with lithium aluminium hydride, or by reaction of tetrahydrocannabinols with hydrogen peroxide. The electron impact-induced fragmentation of their trimethylsilyl ethers was investigated with the aid of deuterium labelling. Most of the compounds gave characteristically different mass spectra with abundant, diagnostically useful fragment ions. As hexahydrocannabinols containing hydroxy groups in all metabolically sensitive positions were readily prepared by the above methods, these provided reference samples for identification of new hydroxylated metabolites of isomeric tetrahydrocannabinols following hydrogenation. The method was validated by application to metabolites of delta-9(11)-tetrahydrocannabinol.

In vivo metabolism of the ethyl homologues of delta-8-tetrahydrocannabinol and delta-9-tetrahydrocannabinol in the mouse.

Ethyl-delta-8-tetrahydrocannabinol (ethyl-delta-8-THC) and ethyl-delta-9-THC were synthesized by condensation of 5-ethyl-1,3-dihydroxybenzene and 1S-cis-verbenol. The two cannabinoids were administered to male Charles River CD-1 mice and hepatic metabolites were extracted with ethyl acetate and isolated by chromatography on Sephadex LH-20. Metabolite identification was by gas chromatography/mass spectrometry as trimethylsilyl (TMS), (2H9)TMS, methyl ester/TMS and dihydro/TMS derivatives. Metabolites from ethyl-delta-8-THC, of which six were identified, were similar with respect to the positions substituted on the terpene ring to those produced by higher homologues; the major metabolite, accounting for about 95% of the metabolic fraction, was ethyl-delta-8-THC-11-oic acid. Side-chain hydroxy metabolites were not detected. Metabolism of ethyl-delta-9-THC was also similar to that of the higher homologues with the exception that less metabolism occurred at C-8 and a higher percentage of the total metabolic fraction was accounted for by the 11-oic acid metabolite. Five metabolites were identified; minor metabolites were mainly dihydroxylated compounds and hydroxylated derivatives of ethyl-delta-9-THC-11-oic acid. A dihydro-metabolite, the C-9-axial-COOH isomer of ethyl-hexahydrocannabinol-11-oic acid, was produced by both compounds and a trace of ethyl-CBN-11-oic acid was produced by ethyl-delta-9-THC.

Identification of cannabichromene metabolites by mass spectrometry: identification of eight new dihydroxy metabolites in the rabbit.

Metabolites of cannabichromene (CBC) produced by hepatic microsomal incubates from rabbits and mice were examined by gas chromatography/mass spectrometry (GC/MS) as trimethylsilyl (TMS) and (2H9)TMS derivatives. Most metabolites were hydroxylated compounds whose mass spectra gave very little information on metabolite structure as fragmentation was dominated by formation of the substituted chromenyl ion. This prevented charge localization and diagnostic fragmentation at the site of metabolic attack. This paper describes the identification of these metabolites by GC/MS techniques using both deuterium-exchange reactions and hydrogenation of the metabolites to tetrahydro derivatives; the latter method was used to suppress chromenyl ion formation and to enhance the relative abundance of diagnostic fragment ions. Twenty-one metabolites were identified. Metabolites were found hydroxylated in all positions of both aliphatic chains, with additional compounds formed by epoxidation and reduction of the aliphatic double bond in the methylpentenyl chain. Dihydroxy metabolites were hydoxylated in both the pentyl and methylpentenyl chains in positions common to those hydroxylated in the monohydroxy metabolites.

Nontreatment of fever in extended-care facilities.

In a study of decisions not to treat febrile patients, we reviewed the medical records of 1256 people admitted to nine extended-care facilities in Seattle during 1973. Fever, defined as two temperatures of 38.33 degrees C to 38.83 degrees C (101 to 101.9 degrees F), within 24 hours or one temperature greater than or equal to 38.88 degrees C (102 degrees F), developed in 190 patients before two years of stay. Active treatment, defined as antibiotics or hospitalization (or both), was ordered for fever in 109 patients, of whom 10 (9 per cent) died. Active treatment was not ordered for 81 patients, of whom 48 (59 per cent) died. The pre-decision factors that showed a significant relation (P less than 0.05) to such nontreatment were: diagnosis, mental status, mobility, pain, narcotics prescribed, size of the facility, relation of the physician to the patient and medical-record statements documenting the patient's deterioration or plans for nontreatment in general. This pattern of nontreatment suggests that physicians and nurses did not intend to treat these patients actively and that high mortality was expected.

In vitro metabolism of cannabigerol in several mammalian species.

Microsomal incubations were prepared from the livers of male mice, rats, cats, guinea-pigs, hamsters and gerbils and both male and female rabbits and were incubated with cannabigerol (CBG), a constituent of marihuana. Metabolites were extracted with ethyl acetate, concentrated by chromatography on Sephadex LH-20 and examined as trimethylsilyl (TMS) and (2H9)TMS derivatives by gas chromatography/mass spectrometry. Structural elucidation was aided by hydrogenation of the metabolites to tetrahydro derivatives. Similar metabolites were produced by each of the species but the ratios of the individual compounds differed considerably. Twelve metabolites were identified. The major metabolites were monohydroxy compounds with the hydroxyl group at C-8', C-9', C-4' or at one of any position of the pentyl chain. Reduction of the delta-6' double bond was prominent in the cat to give 8'-hydroxy-6',7'-dihydro-CBG. The other major metabolic route was epoxidation of this double bond and hydrolysis to give 6',7'-dihydroxy-6',7'-dihydro-CBG. Although epoxidation of the other double bond was detected, the resulting metabolite was present in low concentration and hydrolysis was not observed. The mass spectral fragmentation of CBG and its metabolites was dominated by formation of the tropylium ion by cleavage of the C-1'--C-2' bond and by ions formed by cleavage of the C-3'--C-4' and C-4'--C-5' bonds. In addition, compounds containing hydroxylation at C-1"--C-4" (pentyl chain) gave rise to the same abundant diagnostic ions that have been observed for corresponding metabolites of other cannabinoids.

In vitro metabolism of cannabidiol in the rabbit: identification of seventeen new metabolites including thirteen dihydroxylated in the isopropenyl chain.

The metabolism of cannabidiol (CBD) was studied in liver microsomes from the female New Zealand white rabbit. Metabolites were extracted with ethyl acetate, concentrated by chromatography on Sephadex LH-20 and examined as trimethylsilyl (TMS), methyl ester/TMS and (2H9)TMS derivatives by gas chromatography/mass spectrometry. Thirty-nine metabolites, mainly mono-, di- and tri-hydroxy compounds, were identified; 17 of these have not been reported before. New metabolites included 8,9-dihydroxy-8,9-dihydro-CBD (two isomers) and seven monohydroxy derivatives of each of these two compounds. The mass spectra of the TMS derivatives of metabolites not hydroxylated in the isopropenyl group were generally dominated by the ion produced by retro-Diels-Alder cleavage of the terpene ring. Other structurally informative ions included the tropylium ion and fragments diagnostic of hydroxylation at C-1", C-2", C-3", C-4" and C-7. The spectra of the TMS derivatives of metabolites hydroxylated in the isopropenyl group were generally dominated by the ion at m/z 143. This involved loss of CH2OTMS and a retro-Diels-Alder fragmentation analogous to that seen in the other metabolites, but with charge retention by the other (smaller) fragment. Other, related fragment ions also characterized these metabolites.