Use of multiple pharmacodynamic measures to deconstruct the Nix-TB regimen in a short-course murine model of tuberculosis.

A major challenge for tuberculosis (TB) drug development is to prioritize promising combination regimens from a large and growing number of possibilities. This includes demonstrating individual drug contributions to the activity of higher-order combinations. A BALB/c mouse TB infection model was used to evaluate the contributions of each drug and pairwise combination in the clinically relevant Nix-TB regimen [bedaquiline-pretomanid-linezolid (BPaL)] during the first 3 weeks of treatment at human equivalent doses. The rRNA synthesis (RS) ratio, an exploratory pharmacodynamic (PD) marker of ongoing Mycobacterium tuberculosis rRNA synthesis, together with solid culture CFU counts and liquid culture time to positivity (TTP) were used as PD markers of treatment response in lung tissue; and their time-course profiles were mathematically modeled using rate equations with pharmacologically interpretable parameters. Antimicrobial interactions were quantified using Bliss independence and Isserlis formulas. Subadditive (or antagonistic) and additive effects on bacillary load, assessed by CFU and TTP, were found for bedaquiline-pretomanid and linezolid-containing pairs, respectively. In contrast, subadditive and additive effects on rRNA synthesis were found for pretomanid-linezolid and bedaquiline-containing pairs, respectively. Additionally, accurate predictions of the response to BPaL for all three PD markers were made using only the single-drug and pairwise effects together with an assumption of negligible three-way drug interactions. The results represent an experimental and PD modeling approach aimed at reducing combinatorial complexity and improving the cost-effectiveness of in vivo systems for preclinical TB regimen development.

Use of Multiple Pharmacodynamic Measures to Deconstruct the Nix-TB Regimen in a Short-Course Murine Model of Tuberculosis.

A major challenge for tuberculosis (TB) drug development is to prioritize promising combination regimens from a large and growing number of possibilities. This includes demonstrating individual drug contributions to the activity of higher-order combinations. A BALB/c mouse TB infection model was used to evaluate the contributions of each drug and pairwise combination in the clinically relevant Nix-TB regimen (bedaquiline-pretomanid-linezolid [BPaL]) during the first three weeks of treatment at human equivalent doses. RS ratio, an exploratory pharmacodynamic (PD) marker of ongoing Mycobacterium tuberculosis rRNA synthesis, to-gether with solid culture CFU and liquid culture time to positivity (TTP) were used as PD markers of treatment response in lung tissue; and their time course profiles were mathematically modeled using rate equations with pharmacologically interpretable parameters. Antimicrobial interactions were quantified using Bliss independence and Isserlis formulas. Subadditive (or antagonistic) and additive effects on bacillary load, assessed by CFU and TTP, were found for bedaquiline-pretomanid and linezolid-containing pairs, respectively. In contrast, subadditive and additive effects on rRNA synthesis were found for pretomanid-linezolid and bedaquiline-containing pairs, respectively. Additionally, accurate predictions of the response to BPaL for all three PD markers were made using only the single-drug and pairwise effects together with an assumption of negligible three-way drug interactions. The results represent an experimental and PD modeling approach aimed at reducing combinatorial complexity and improving the cost-effectiveness of in vivo systems for preclinical TB regimen development.

The professionalisation of Irish medicine in the generations before Charles Lucas.

The educational opportunities and qualifications, corporate structures and regulation that had become accepted features of the Irish medical profession in the era of Charles Lucas were the fruits of a slow process of professionalization initiated by a handful of Irish medics who, from the 1620s, began tackling poor standards of medical practice in an era of medical pluralism. This paper begins by reviewing the standard of practice in early seventeenth-century Ireland. Through the examples of Thomas Arthur, M.D. and John Clavell, a self-styled medic, differences in the approaches adopted by university-trained physicians and unorthodox practitioners are highlighted. The succession of significant steps taken in this professionalization process are traced, with particular emphasis on the failed attempt to establish a Royal College of Physicians in Dublin during the mid-1620s and the importance of the influx of English physicians in the 1650s for the creation of permanent corporate structures and regulation.

Chemotaxis toward carbohydrates and peptides by mixed ruminal protozoa when fed, fasted, or incubated with polyunsaturated fatty acids.

In contrast to the well-characterized chemotaxis and migratory behavior between the dorsal and ventral locations of the rumen by isotrichids, we hypothesized that chemotaxis toward soluble nutrients maintains entodiniomorphid protozoa in the particulate fraction. The objectives of these experiments were to compare the dose-responsive chemotaxis (1) toward different glucose concentrations when ruminal samples were harvested from fed versus fasted cows; (2) toward increasing concentrations of glucose compared with xylose when protozoa were harvested from a fed cow; (3) toward peptides of bacterial, protozoal, and soy origin; and (4) toward glucose when mixed ruminal protozoa were previously incubated for 0, 3, or 6h in the presence of emulsified polyunsaturated fatty acids (PUFA; Liposyn II, Hospira, Lake Forest, IL). In experiment 1, isotrichid protozoa decreased chemotaxis toward increasing glucose concentration when cows were fasted. Entodiniomorphids exhibited chemotaxis to similar concentrations of glucose as did isotrichids, but to a lesser magnitude of response. In experiment 2, xylose was chemotactic to both groups. Xylose might draw fibrolytic entodiniomorphid protozoa toward newly ingested feed. In contrast, even though isotrichids should not use xylose as an energy source, they were highly chemoattracted to xylose. In experiment 3, entodiniomorphids were not selectively chemoattracted toward bacterial or protozoal peptides compared with soy peptides. In experiment 4, despite isotrichid populations decreasing in abundance with increasing time of incubation in PUFA, chemotaxis to glucose remained unchanged. In contrast, entodiniomorphids recovered chemotaxis to glucose with increased time of PUFA incubation. Current results support isotrichid chemotaxis to sugars but also our hypothesis that a more moderate chemotaxis toward glucose and peptides explains how they swim in the fluid but pass from the rumen with the potentially digestible fraction of particulates.

A physiologically based pharmacokinetic model for capreomycin.

The emergence of multidrug-resistant tuberculosis (MDR-TB) has led to a renewed interest in the use of second-line antibiotic agents. Unfortunately, there are currently dearths of information, data, and computational models that can be used to help design rational regimens for administration of these drugs. To help fill this knowledge gap, an exploratory physiologically based pharmacokinetic (PBPK) model, supported by targeted experimental data, was developed to predict the absorption, distribution, metabolism, and excretion (ADME) of the second-line agent capreomycin, a cyclic peptide antibiotic often grouped with the aminoglycoside antibiotics. To account for interindividual variability, Bayesian inference and Monte Carlo methods were used for model calibration, validation, and testing. Along with the predictive PBPK model, the first for an antituberculosis agent, this study provides estimates of various key pharmacokinetic parameter distributions and supports a hypothesized mechanism for capreomycin transport into the kidney.

Mapping cholesterol gallstone susceptibility (Lith) genes in inbred mice.

The individual risk for developing cholesterol gallstones in response to specific environmental factors is determined by complex genetics involving multiple genes. In this review, we introduce inbred mice as a model to localise and identify the murine genes that harbour cholesterol gallstone susceptibility alleles (Lith genes). These genes are associated with increased risk of gallstone formation when mice are fed a lithogenic diet containing cholesterol and cholic acid. We summarise the steps involved in localising the chromosomal regions that harbour Lith genes, focusing particularly on the initial step known as quantitative trait locus mapping, which employs breeding crosses of gallstone-susceptible and gallstone-resistant inbred mouse strains. Subsequent steps to narrow the chromosomal regions of the quantitative trait loci and identify the underlying Lith genes are outlined, with particular reference to the examples of Lith1 and Lith2, the first discovered quantitative trait loci associated with murine cholesterol cholelithiasis. We have now reported five quantitative trait loci for murine cholelithogenesis, which are officially named Lith1 through Lith5. Once the genes underlying these quantitative trait loci and other chromosomal loci from ongoing mouse crosses are identified and confirmed, the 'road-map' for discovery of orthologous human LITH genes will be available and, thereafter, their putative roles in cholesterol gallstone formation can be tested in selected human populations.

Trans fatty acids affect lipoprotein metabolism in rats.

This study was designed to investigate the effects of oleic (CIS), palmitic (SAT) and trans fatty acids (TRANS) on cholesterol metabolism. Rats fed the TRANS diet had lower plasma total cholesterol (P < 0.005) and non-HDL-cholesterol (non HDL-C) concentrations (P < 0.005) compared with their CIS-fed counterparts. Plasma HDL-C was highest in rats fed the SAT diet (P = 0.01). An in vivo assay of reverse cholesterol transport (RCT) was performed whereby radiolabeled cholesterol was delivered to the liver as acetylated LDL and the reappearance of label into plasma and HDL was determined. Plasma radioactivity in TRANS-fed rats was lower than in their SAT-fed counterparts (P = 0.01), and consistent with the cholesterol distribution in plasma, the difference was due to lower [(3)H]-cholesterol in lower density lipoproteins. Despite diet-induced differences in the cholesterol and phospholipid concentrations and fatty acid composition of HDL, the amount of label in HDL did not differ among groups, suggesting that consumption of these diets resulted in HDL populations with similar capacity to participate in RCT. The present findings suggest that dietary trans fatty acids regulate the metabolism of apolipoprotein B-containing lipoproteins in rats and that the effect may be masked in species possessing high plasma cholesteryl ester transfer protein (CETP) activity. These results reinforce the important role of CETP activity in determining the distribution of plasma cholesterol in response to dietary trans fatty acids.

Trans fatty acids and cholesterol metabolism: mechanistic studies in rats and rabbits fed semipurified diets.

Studies were conducted in rabbits and rats to investigate the effects of diets rich in oleic (CIS diet), palmitic (SAT diet) and trans fatty acids (TRANS diet) on plasma lipids and lipoprotein metabolism. An important difference between these species is that rabbits possess plasma cholesteryl ester transfer protein (CETP) activity while rats are devoid of transfer activity. In the presence of dietary cholesterol (0.2% w/w) the change in plasma low density lipoprotein-cholesterol (LDL-C) concentration from baseline was significantly higher in rabbits fed the TRANS diet compared with those fed the CIS diet (P < 0.01). Despite this difference, the hepatic LDL-receptor activity was similar in all groups. Also, the fatty acid composition of hepatic phospholipids was affected by diet with lower proportion of palmitic (11%) and higher (19%) linoleic acid despite a similar content in the diet. These effects may represent the maintenance of membrane fluidity within narrow limits to ensure optimal function. The studies in rats showed that the plasma total cholesterol concentration was 20% lower (P < 0.01) in TRANS-fed rats compared with those fed the CIS diet. The results of an in vivo assay of reverse cholesterol transport (RCT) suggested that the three diets gave rise to high density lipoprotein (HDL) particles with similar capacity to accept cellular cholesterol. The differential effects of dietary trans fatty acids in these animal models provide another line of evidence that reinforces the significant role of CETP activity in determining the distribution of plasma cholesterol in response to dietary trans fatty acids.

Metabolism of an oxysterol, 7-ketocholesterol, by sterol 27-hydroxylase in HepG2 cells.

7-Ketocholesterol (7K) is a quantitatively important oxysterol in both atherosclerotic lesions and macrophage foam cells. We reported recently that radiolabeled 7K delivered to rodents in a modified lipoprotein or chylomicron remnant-like emulsion, both cleared predominantly by the liver, was rapidly excreted into the intestine as water-soluble products, presumably bile acids. Herein, we aimed to elucidate the early or initial reactions in 7K metabolism. The hypothesis was tested that sterol 27-hydroxylase, a mitochondrial cytochrome P450 and the first enzyme of the acidic bile acid pathway, is responsible for the initial metabolism of 7K by HepG2 cells, a human hepatoblastoma cell-line. The 27-hydroxylated product of 7K (27OH-7K) was shown to be the initial, lipid-soluble product of 7K metabolism. It was produced in mitochondrial incubations and whole cells and was readily released into the media from cells. Intact cells generated metabolites of 7K that had undergone conversion from lipid-soluble precursors to water-soluble products rapidly and extensively. Their production was ablated with cyclosporin A, a sterol 27-hydroxylase inhibitor. Furthermore, we demonstrated the effectiveness of two novel selective inhibitors of this enzyme, GW273297X and GI268267X. These inhibitors also ablated the production of water-soluble products by cells; and the inhibitor of choice, GW273297X, decreased the production of 27OH-7K in mitochondrial preparations. This is the first study to demonstrate that sterol 27-hydroxylase plays an important role in the metabolism of oxysterols such as 7K in liver cells.

Living with post-traumatic stress disorder: the wives'/female partners' perspective.

AIM OF THE STUDY: This phenomenological study examined what it was like for the wives/female partners to live with a Vietnam veteran who suffers from post-traumatic stress disorder (PTSD). PROCEDURE: Audio-taped interviews were conducted with 10 women and data were examined from three overlapping phases of the veteran/partner relationship: the early phase, the middle phase and the later phase. FINDINGS: The early phase was conceptualized as a period of adjustment in which three themes were identified: (1) attractors, (2) feelings and (3) communication. The middle phase, one of enmeshment, was characterized by six themes: (1) dealing with veteran PTSD symptoms, (2) substance abuse, (3) physical and/or emotional abuse, (4) roles, (5) feelings and (6) coping techniques. Three themes depicted the later phase of resolution/healing: (1) stress related symptoms, (2) staying or leaving and (3) activities that promoted an ongoing process of resolution/healing. CONCLUSIONS: The central meaning statement that best described the experience of wives/female partners who live with a Vietnam veteran with PTSD is that the experience is a gradual process of becoming enmeshed in the veteran's pathology, with all energies being directed at minimizing the effect on self and family, culminating in intermittent movement towards resolution/healing. This study has implications for practitioners who treat Vietnam PTSD veterans and their wives or female partners.

7-Ketocholesterol delivered to mice in chylomicron remnant-like particles is rapidly metabolised, excreted and does not accumulate in aorta.

Cholesterol oxidation products (oxysterols) have been implicated in atherogenesis due to their presence in atherosclerotic tissue and their potent effects in vitro. One of the major oxysterols currently of interest is 7-ketocholesterol (7K) and it has been suggested that the diet is an important source of this oxysterol. This investigation tested the hypothesis that 7K, delivered in a physiologically relevant vehicle, chylomicron remnant-like emulsion (CMR), would be metabolised and excreted by mice in a similar manner and to a similar extent as previously observed in rats when delivered in a chemically modified lipoprotein, acetylated low-density lipoprotein (acLDL). Indeed, the metabolism of 14C-7K delivered in CMR mirrored that of acLDL and was much more rapid than (3)H-cholesterol delivered simultaneously. The 7K-derived (14)C was cleared from the liver, appeared in the intestine and was excreted in the faeces. A substantial proportion of the 7K-derived (14)C in the intestine and faeces was aqueous-soluble, indicating metabolism to polar products, presumably bile acids. Moreover, while cholesterol-derived (3)H increased in the aorta, (14)C appeared transiently and there was no observable accumulation within 24 h. The data confirm our previous findings of rapid hepatic metabolism of 7K when delivered in acLDL and demonstrate that 7K delivered in a vehicle of dietary significance is similarly metabolised and excreted. Indeed, the data encourage further investigation into the contribution that dietary oxysterols may or may not make to atherogenesis.

Rapid hepatic metabolism of 7-ketocholesterol in vivo: implications for dietary oxysterols.

7-Ketocholesterol is a major dietary oxysterol and the predominant non-enzymically formed oxysterol in human atherosclerotic plaque. We tested the hypothesis that 7-ketocholesterol is preferentially retained by tissues relative to cholesterol in vivo. To ensure rapid tissue uptake, acetylated low density lipoprotein, labeled with esters of [(14)C]-7-ketocholesterol and [(3)H]cholesterol, was injected into rats via a jugular catheter. At timed intervals (2 min to 24 h) rats (n = 48 total) were exsanguinated and tissues were dissected and assayed for radioactivity. In two experiments the majority of both radiolabels appeared in the liver after 2 min. In all tissues, (14)C appeared transiently and did not accumulate. Rather, it was metabolized in the liver and excreted into the intestine mainly as aqueous-soluble metabolites (presumably bile acids). By 9 h, (14)C in the liver had decreased to 10% of the injected dose while 36% was present in the intestine. In contrast, at 9 h 38% of (3)H was evident in the liver while only 5% was found in the intestine. Unlike [(3)H]cholesterol, little (14)C was found to re-enter the circulation, indicating that enterohepatic recycling of 7-ketocholesterol was negligible. This is the first report of the distribution of an oxysterol relative to cholesterol, administered simultaneously, in a whole animal model. The finding that [(14)C]-7-ketocholesterol is rapidly metabolized and excreted by the liver suggests that diet may not be a major source of oxysterols in atherosclerotic plaque, and that perhaps dietary oxysterols make little or no contribution to atherogenesis.

Additive inhibitory action of chlorpyrifos and polycyclic aromatic hydrocarbons on acetylcholinesterase activity in vitro.

This study tested the hypothesis that the inhibition of acetylcholinesterase is greater when the insecticide chlorpyrifos (CPF) is in the presence of several polycyclic aromatic hydrocarbons (PAHs) found in house dust. CPF-oxon (CPFO) inhibition curves of purified AChE (electric eel) were generated in the presence or absence of different concentrations of the PAHs pyrene, benzo(a)pyrene, anthracene, and fluoranthene. Without CPF-oxon, all four PAHs themselves inhibited AChE activity with IC50 values in the range 8.2-17 microM. The IC50 for benzo(a)pyrene with human recombinant AChE was 1.5 microM. When AChE was incubated with CPF-oxon together with the PAHs, the inhibitory effect on AChE was additive. This was exemplified by large (60-80%) and significant (P<0.01) inhibition in AChE activity by the PAHs when combined with nanomolar concentrations of CPF-oxon. Kinetic studies indicated that benzo(a)pyrene inhibited AChE in a noncompetitive manner, and the reduction in maximal velocity (Vmax) by benzo(a)pyrene and CPFO together was the sum of the inhibitory effect of the two inhibitors alone, further supporting an additive effect. These data suggest that some PAHs have anticholinesterase activity, and contribute in an additive manner to the inhibitory effect of CPFO on AChE in vitro. Further research is needed to determine the toxicological relevance of these findings.

7-Ketocholesterol.

7-Ketocholesterol is a major oxidation product of cholesterol found in human atherosclerotic plaque and is more atherogenic than cholesterol in some animal studies. 7-Ketocholesterol can inhibit cholesterol 7 alpha-hydroxylase, the rate-limiting step in bile acid biosynthesis, as well as strongly inhibiting HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. It has even been suggested that 7-ketocholesterol is formed enzymically as an endogenous regulator of cholesterol biosynthesis. However, when tested as a pharmacological cholesterol-lowering agent, inhibition of HMG-CoA reductase was rapidly overcome and the 7-ketocholesterol metabolised. In vitro, 7-ketocholesterol has wide-ranging and potent effects, most of which have the potential to contribute to atherosclerosis. For example, 7-ketocholesterol can be cytotoxic and can induce apoptosis in vascular cells. These effects, either individually or more likely, in combination, all implicate 7-ketocholesterol in the initiation and development of atherosclerosis, but further work is needed to establish whether or not its role is a direct causal one.

The phenomenon of compulsive overeating in a selected group of professional women.

Many data have been generated concerning individuals with eating disorders such as anorexia and bulimia. However, few data exist that have explored the phenomenon of compulsive overeating or binge eating. The purpose of this study was to determine the meaning of compulsive overeating, or binge eating, in the lives of adult professional women. Six adult women from the south-east United States were interviewed using an open-ended interview format. Interviews were audio-taped and analysed using the Giorgi (1979) method of phenomenology. Recurrent themes that emerged from data collection included those pertaining to childhood experiences with food, descriptions of the types of food most often eaten during the adult years, eating behaviours in the adult years, perceptions of loss of control, reasons for overeating, emotional consequences of overeating, compensatory behaviours, and prevention strategies. The synthesis of meaning statement elicited was that of a personal struggle to achieve or maintain an acceptable weight and gain mastery over binge eating behaviours. This struggle can be likened to an addictive process in which the individual thinks about food constantly, consciously or unconsciously, and eats compulsively in spite of the consequences.

alpha-Difluoromethylornithine inhibits liver metastasis produced by intrasplenic injection of human tumor cells into nude mice.

The purpose of these studies was to examine metastatic potentials of a human colon tumor xenograft (T6) and three different human tumor cell lines (LS174T, HT29 and A549) using the intrasplenic-nude mouse model system (ISMS model system). A further objective was to study the activity of alpha-difluoromethyl-ornithine (DFMO) against primary and metastatic growth of the xenograft and the three cell lines. DFMO is an irreversible inhibitor of ornithine decarboxylase, a rate-limiting step in polyamine biosynthesis. Tumor burdens in the liver of nude mice were observed 6 weeks after the intrasplenic injection with LS174T and 12-14 weeks after intrasplenic injections with T6, HT29 and A549. Most of the mice developed primary tumor growth in the spleens. DFMO showed significant activity against liver metastases but had little or no activity against primary tumor growth in the spleens of the ISMS model and against s.c. growth of the xenograft. The studies demonstrated that the ISMS model system is an excellent system for studying metastatic behavior of human tumors and for studying the antimetastatic activity of experimental drugs.