Patterns of adversity and post-traumatic stress among children adopted from care.

BACKGROUND: Children adopted from care are more likely to have experienced early adversity, but little is known about the impact of early adversity on later post-traumatic stress (PTS) symptoms. OBJECTIVE: To investigate sub-groups of adversity in a sample of adopted children and examine the association with later PTS symptoms. PARTICIPANTS AND SETTING: A study of British children adopted from care using social worker records (N = 374) and questionnaire-based longitudinal study of n = 58 children over 4-years post adoptive placement. METHODS: We used latent class analysis to identify subgroups of children based on commonalities in perinatal and postnatal adversity experienced prior to adoption and examined differences in PTS symptoms at 4-years post-placement between subgroups. RESULTS: Nearly one in five (19 %) children were in the clinical or borderline ranges for symptoms of PTS arousal, 14 % for PTS avoidance and 8 % for PTS intrusion. The 5-class solution fitted the data best, with one class characterized by children with a low probability of experiencing any adversity, one perinatal adversity class and three classes capturing different patterns of adversity. The multiple complex adversity class involving both perinatal and postnatal adversity had significantly higher symptoms of PTS avoidance and arousal than other sub-groups. CONCLUSIONS: The prevalence and complexity of PTS symptoms among adoptive children highlights the need for effective interventions considering different profiles of early adversity.

Factor VIII pharmacokinetics associates with genetic modifiers of VWF and FVIII clearance in an adult hemophilia A population.

BACKGROUND: Factor VIII (FVIII) pharmacokinetics (PK) in adult hemophilia A populations are highly variable and have been previously determined to be influenced by von Willebrand factor:antigen (VWF:Ag), ABO blood group, and age. However, additional genetic determinants of FVIII PK are largely unknown. OBJECTIVES: The contribution of VWF clearance, VWF-FVIII-binding activity, and genetic variants in VWF clearance receptors to FVIII PK in adult patients were assessed. METHODS: FVIII PK assessment was performed in 44 adult subjects (age 18-61 years) with moderate or severe hemophilia A. VWF:Ag, VWF propeptide (VWFpp), VWFpp/VWF:Ag, and VWF:FVIII binding activity were measured. The VWF modifying loci CLEC4M, SCARA5, STAB2, and ABO, and the D'D3 FVIII-binding region of the VWF gene were genotyped. RESULTS: VWF:Ag, VWFpp, and VWF:FVIIIB positively correlated with FVIII half-life and negatively correlated with FVIII clearance. VWFpp/VWF:Ag negatively correlated with FVIII half-life and positively correlated with FVIII clearance. The correlation between VWFpp/VWF:Ag and FVIII half-life was stronger for type non-O patients than for type O patients, suggesting that slower VWF clearance increases FVIII half-life. Patients heterozygous for the CLEC4M rs868875 variant had increased FVIII clearance when compared with individuals homozygous for the reference allele. The CLEC4M variable number of tandem repeat (VNTR) alleles were also associated with the rate of FVIII clearance. When compared with the quartile of patients with the fastest FVIII clearance, the quartile of patients with the slowest FVIII clearance had a decreased frequency of the CLEC4M 5-VNTR. CONCLUSIONS: VWF-FVIII binding activity and genetic determinants of VWF clearance are important contributors to FVIII pharmacokinetics in adult patients.

The endothelial cell receptor stabilin-2 regulates VWF-FVIII complex half-life and immunogenicity.

Quantitative abnormalities of the von Willebrand factor-factor VIII (VWF-FVIII) complex associate with inherited bleeding or thrombotic disorders. Receptor-mediated interactions between plasma VWF-FVIII and phagocytic or immune cells can influence their hemostatic and immunogenic activities. Genetic association studies have demonstrated that variants in the STAB2 gene, which encodes the scavenger receptor stabilin-2, associate with plasma levels of VWF-FVIII. However, the mechanistic basis and pathophysiological consequences of this association are unknown. We have demonstrated that stabilin-2-expressing cells bind and internalize human VWF and FVIII in a VWF-dependent manner, and stabilin-2-deficient mice displayed prolonged human VWF-FVIII half-life compared with controls. The stabilin-2 variant p.E2377K significantly decreased stabilin-2 expression and impaired VWF endocytosis in a heterologous expression system, and common STAB2 variants associated with plasma VWF levels in type 1 von Willebrand disease patients. STAB2-deficient mice displayed a decreased immunogenic response to human VWF-FVIII complex, while coinfusion of human VWF-FVIII with the stabilin-2 ligand hyaluronic acid attenuated the immune response to exogenous FVIII. Collectively, these data suggest that stabilin-2 functions as both a clearance and an immunoregulatory receptor for VWF-FVIII, making stabilin-2 a novel molecular target for modification of the half-life of VWF-FVIII and the immune response to VWF-FVIII concentrates.

A comparison of the accumulation of ricin by hepatic parenchymal and non-parenchymal cells and its inhibition of protein synthesis.

Rat liver non-parenchymal cells in vivo were found to accumulate 125I-labelled ricin to a much greater extent than parenchymal cells. Similarly, in monolayer cell cultures, the rate of ricin uptake by non-parenchymal Kupffer cells was several times that by parenchymal cells. Evidence is provided also to suggest that ricin is primarily recognized by Kupffer cells via terminal mannose residues in the toxin, whereas ricin uptake by parenchymal cells was consistent with a role of the previously postulated galactosyl-containing cell receptors. Protein synthesis in Kupffer cells in vitro, although observed to occur at a lower rate than in parenchymal cells, was 100--1000-times more sensitive to inhibition by ricin. The selective damage known to be caused to liver sinusoids by ricin, therefore, may reflect both the relative efficiency with which the toxin is taken up by these cells and the extreme sensitivity of protein synthesis in the cells to inhibition by ricin.

Evidence that the loss of rat liver cytochrome P450 in vitro is not solely associated with the use of collagenase, the loss of cell-cell contacts and/or the absence of an extracellular matrix.

Two methods avoiding the widespread technique of collagenase perfusion have been employed to study the regulation of total cytochrome P450 content in rat hepatocyte culture. One technique required the perfusion of the liver with the chelating agent EDTA to dissociate the parenchymal cells prior to culture. Over a period of 48 hr, cultured hepatocytes isolated by EDTA perfusion showed comparable losses of cytochrome P450 as cells isolated by perfusion with collagenase. The second technique involved the culture of 210-240 microns thick "precision cut" liver slices. The results presented here indicate that the liver slices remain viable for 24 hr of culture, but that liver slices also lose their cytochrome P450 content at a comparable rate to collagenase prepared cells in culture. Collectively the results suggest that there is not a direct causal relationship between the loss of cytochrome P450 and one or a combination of the use of collagenase; the loss of cell-cell contacts and the absence of an extracellular matrix.

Cytochrome P450 maintenance and diazepam metabolism in cultured rat hepatocytes.

Diazepam metabolism has been investigated in rat hepatocytes cultured for 3, 24, 48 and 72 hr under five different conditions. Although four of the treatments studied reduced markedly the spontaneous loss of cytochrome P450, they had different effects on the metabolism of diazepam (DZ) presumably by affecting the relative proportions of cytochrome P450 isozymes during the period of culture. Thus P450 medium or dimethyl sulphoxide-supplemented medium maintained the rate of disappearance of DZ from the culture medium and metabolite profile in 24 hr cultures at the initial levels found in 3 hr cultures, while culture at 30 degrees or in metyrapone-containing medium resulted in the production of oxazepam, a metabolite normally only produced by dog, monkey and human hepatocytes. These findings indicate that the well recognized phenotypic alteration of cytochrome P450-dependent mono-oxygenase activities that occurs when rat hepatocytes are cultured in different media can result in a range of metabolic options that are normally only available in other animal species.

Developmental changes in the constitutive and inducible expression of cytochrome P450 3A2.

Using a CYP3A2-specific oligonucleotide and an antipeptide antibody raised against the C terminus of CYP3A2 (VINGA) it is demonstrated that metyrapone administration to adult (12 weeks old) but not immature (3 weeks old) male Sprague Dawley rats induces the hepatic expression of CYP3A2 mRNA and protein. The constitutively expressed level of CYP3A2 protein in adult male rats is markedly lower than the levels expressed in immature rats as determined using the anti-VINGA antibody, in contrast to previous reports using antibodies that do not discriminate between CYP3A forms. Hepatic microsomal CYP3A2 protein expression, examined between 3 and 15 weeks of age, is extinguished between 9 and 12 weeks of age in contrast to immunoreactive CYP3A protein (determined using a nonselective antibody) and CYP3A-dependent androstenedione 6beta-hydroxylase activity. These data suggest that the regulation of the induction of CYP3A2 is developmentally controlled and that the major expressed adult form(s) of constitutively expressed CYP3A is not CYP3A2.

Induction of rat hepatic glucocorticoid-inducible cytochrome P450 3A by metyrapone.

The bipyridyl compound metyrapone is a potent inhibitor of cytochromes P450, a gene superfamily of haemoproteins involved in the metabolism of many xenobiotics as well as endogenous compounds such as steroid hormones. Administration of metyrapone to male rats induces the expression of the cytochrome P450 sub-family 3A (CYP3A). In order to determine whether metyrapone was causing the induction of CYP3A by blocking endogenous glucocorticoid metabolism, CYP3A levels were examined in rat hepatocytes cultured in serum-free medium supplemented with hydrocortisone 21-hemisuccinate plus or minus metyrapone. Western blotting indicated that metyrapone alone induces CYP3A and that hydrocortisone 21-hemisuccinate is ineffective. However, hydrocortisone 21-hemisuccinate enhanced the levels of CYP3A induced by metyrapone. In contrast, glucocorticoid-inducible tyrosine aminotransferase (TAT) activity was unaffected by metyrapone but metyrapone enhanced the levels induced by hydrocortisone 21-hemisuccinate. An examination of the metabolism of hydrocortisone by rat hepatocytes in vitro indicated that metyrapone perturbed the catabolism of hydrocortisone under conditions which give rise to an enhancement of hydrocortisone 21-hemisuccinate and hydrocortisone-dependent TAT induction. However, evidence is presented to suggest that such a perturbation of hydrocortisone metabolism could not account for the glucocorticoid potency amplifying property of metyrapone. Thus the induction of CYP3A and the enhancement of glucocorticoid-mediated TAT induction appears not to be associated with any perturbation in glucocorticoid metabolism but with some other as yet undefined mechanism(s).

Anti-leishmanial activity of neolignans from Virola species and synthetic analogues.

Surinamensin, a neolignan isolated from Virola surinamensis, 3,4,5-trimethoxy-8-[2',6'-dimethoxy-4'-(E)-propenylphenoxy]-phenylpropane, a neolignan isolated from Virola pavonis, and 25 of its synthetic analogues or correlated substances with ether linkages and their corresponding C-8 sulphur and nitrogen analogues, were tested for activity against Leishmania donovani amastigotes and promastigotes in vitro. Some were active against L. donovani promastigotes at 30 microM but inactive against intracellular amastigotes. The natural neolignan from V. pavonis was active against promastigotes at 100 microM. The highest selective activity was found in those compounds with sulphur bridges. The beta-ketosulfide (3,4-dimethoxy)-8-(4'-methylthiophenoxy)-propiophenone produced 42% inhibition of L. donovani amastigotes in the liver of BALB/c mice at 100 mg/kg given once daily for five consecutive days (P>0.05).

The maintenance of cytochrome P-450 in liver cell culture: recent studies on P-450 mediated mechanisms of toxicity.

Rat hepatocytes have been cultured under conditions that result in low and high cytochrome P-450 concentrations and then used as a "screening test" to determine the involvement of cytochrome P-450 in toxicity. The results show that a limitation to the use of cultured hepatocytes in toxicity tests is the lack of extrahepatic release mechanisms of toxic compounds.

The maintenance of cytochrome P-450 in rat hepatocyte culture: some applications of liver cell cultures to the study of drug metabolism, toxicity and the induction of the P-450 system.

Treatments affecting the loss of cytochrome P-450 in rat hepatocyte culture are reviewed and the way in which these have produced an understanding of the mechanisms involved are discussed extensively. A simple way to prevent the loss of P-450 in hepatocytes is to culture them with 0.5 mM metyrapone which appears to restore the cytochromes' synthesis and degradation to steady state values. Knowledge of this mechanism has led to the formulation of special culture medium and the application of both culture systems to the study of drug metabolism and toxicity are described. Finally the effect of these culture systems on the expression of the multiple forms of cytochrome P-450 are presented to illustrate the potential of cultured hepatocytes in induction studies.

Effect of amino acids and inducers on the activity of the microsomal mono-oxygenase system in rat liver cell culture.

In rat liver cell culture both benzanthracene and phenobarbitone induce the activity of benzypyrene hydrxylase while only phenobarbitone increases NADPH cytochrome c reductase activity. Benzpyrene hydroxylase but not NADPH cytochrome c reductase activity is dependent on the amino acid concentration of the culture medium in a similar manner to the regulation of the hepatic hydroxylase activity by dietary protein intake in the whole animal. Of all the amino acids present in the culture medium, only tryptophan induced benzpyrene hydroxylase when added singly to the medium. However, tryptophan also induced the activity of the reductase suggesting that its inducing effect is unrelated to raising the concentration of all the amino acids of the culture medium. It is proposed that tryptophan may only be an inducer because the cells have low levels of tryptophan pyrrolase activity.

Comparison of the photochemical and enzymic generation of excited states of oxygen on the induction of benzo(alpha)pyrene mono-oxygenase in liver cell cultures.

The photochemical generation of excited states of oxygen such as the superoxide ion(O-2) and singlet oxygen (1o2) by the mild illumination of culture medium containing riboflavin induces benzo(alpha)pyrene mono-oxygenase in 3 different cell lines derived from rat liver. Similar rates of O-2 generation can be produced by the action of xanthine oxidase on xanthine yet this system does not induce the mono-oxygenase. This result confirms that the mono-oxygenase induction is not mediated by O-2 is not mediated by O-2 and that 1O2 is the most likely candidate for stimulating the mono-oxygenase activity.

Relative toxicities of particulate and soluble forms of beryllium to a rat liver parenchymal cell line in culture and possible mechanisms of uptake.

The relative toxicities of particulate beryllium phosphate, soluble beryllium sulphate and a beryllium sulphosalicylate complex to a rat liver parencymal derived cell line have been examined in culture. Due to the propensity of beryllium salts to form beryllium phosphate in solution the incubation medium used was free of inorganic phosphate. Cell death measured by the loss of cellular lactate dehydrogenase into the medium can be produced within 76 h from beryllium phosphate and beryllium sulphosalicylate or 48 h from beryllium sulphate provided the cells have, irrespective of the form of added beryllium, taken up a minimum of 2--5 nmol Be/10(6) cells. Whilst beryllium phosphate was readily taken up as a particle, beryllium complexed with excess sulphosalicylate was not so markedly accumulated by the cells except possibly by formation of small amounts of beryllium phosphate in the medium as a result of inorganic phosphate lost from the cells. The extent of beryllium uptake from beryllium sulphate quantitatively most resembled that observed for beryllium phosphate but was largely independent of beryllium phosphate formation in the medium and not accompanied by the uptake of the SO42- anion. However, the accumulation of beryllium derived from beryllium sulphate did appear to be associated with the production of a sedimentable from believed most probably to be colloidal beryllium hydroxide. The uptake of all forms of beryllium was temperature sensitive and metabolic inhibitor studies and treatment of the cells with trypsin or neuraminidase supported the view that the distinct behaviour of beryllium derived from beryllium sulphate may be related to the enhanced toxicity of this form both under the conditions used and when administered to experimental animals.

The induction of benzo[a]pyrene-3-mono-oxygenase by singlet oxygen in liver cell culture is mediated by oxidation products of histidine.

The photochemical generation of excited states of oxygen by the mild illumination of culture medium containing 15 microM riboflavin results in a typical induction of benzo[a]pyrene-3-mono-oxygenase in cell lines derived from liver. However, the induction of the mono-oxygenase is not due to an excited state of oxygen directly activating the inducing mechanism inside the cell but is due to the oxidation of a component of the culture medium forming a stable inducer. The present work unequivocably shows that the component oxidised is histidine. The mild illumination of culture medium containing riboflavin therefore converts a physiological component of the medium which is not normally an inducer of the mono-oxygenase into a compound which is as effective an inducer as the classical inducer. The finding that singlet oxygen will oxidise a cell constituent into a powerful inducer is compatible with the hypothesis that excited states of oxygen and their oxidation products may play a central role in the induction of cytochrome P-450 and associated enzyme activities by many chemically unrelated inducers.

Interaction of [14C]dimethylnitrosamine with albumin produced by rat hepatocytes in culture.

Rat hepatocyte cultures were incubated with [14C]dimethylnitrosamine (14C-NDMA) and the utility of measuring covalent binding of the radiolabel to newly synthesized albumin as a dose monitor of in vivo alkylation was determined. Hydrolysis of albumin, followed by derivatization and high performance liquid chromatography (HPLC) analysis of its constituent amino acids, showed radioactivity to be associated with a number of peaks. As the albumin fraction isolated from rat hepatocytes cultured with [14C]NDMA or [14C]methanol (14C-CH3OH) had similar profiles of radiolabelled amino acids it is concluded that the presence of radiolabel in albumin isolated from 14C-NDMA-treated hepatocytes is due to incorporation of its metabolic products rather than to direct alkylation.

Resistance of precision-cut liver slices to the toxic effects of menadione.

The viability of precision-cut rat liver slices has been assessed in two different culture systems. Liver slices cultured in sealed vessels gassed with 5% carbon dioxide and 95% oxygen only remained viable for about 12 hr when judged by potassium content but 18 hr when judged by enzyme leakage. However, rat liver slices remained viable for up to 24 hr as judged by both these criteria when unsealed culture vessels were placed in a humidified atmosphere of 5% carbon dioxide in air. The effect of menadione (2-methyl-1,4-naphthoquinone) on the viability of slices and hepatocytes in culture was compared. The results indicate that signs of toxicity may be observed in cultured hepatocytes within 1-2 hr in medium containing 200 mum-menadione. In contrast, no signs of toxicity were observed in slices cultured in medium containing 200 mum-menadione for at least 12 hr. Higher concentrations of menadione did give rise to toxicity in cultured slices. Evidence that preserved levels of glycogen in liver slices may in part underlie their resistance to menadione-induced toxicity is presented. These results suggest that precision-cut liver slices may more accurately reflect the hepatotoxic potential of chemicals in short-term tests than do isolated hepatocytes.

Effect of oral and parenteral administration of B6 vitamers on the lymphopenia produced by feeding ammonia caramel or 2-acetyl-4(5)-(1,2,3,4-tetrahydroxy)butylimidazole to rats.

The ability of B6 vitamers to prevent the lymphopenic effects of ammonia caramel fed to rats has been evaluated. Diets containing 10 ppm pyridoxine or pyridoxal prevented the lymphopenia produced in rats consuming an 8% (w/v) solution of ammonia caramel, whereas the dietary content of pyridoxamine needed to be increased to 20 ppm to have the same effect. In contrast to the results of the enteral administration of the individual B6 vitamers, pyridoxamine was found to be the most effective vitamer in preventing the ammonia caramel-induced lymphopenia when administered parenterally. However, all the nutritionally active forms of vitamin B6 were able to prevent the depression of the peripheral blood lymphocyte count, which resulted from ingestion of ammonia caramel by rats. The proposal that oral administration of pyridoxine may prevent the intestinal absorption of the lymphopenic constituent of ammonia caramel, 2-acetyl-4(5)-(1,2,3,4-tetrahydroxy)butylimidazole (THI), is discredited, since THI was found to reduce the lymphocyte count after parenteral administration in rats fed 0.04 ppm pyridoxone in the diet and that increased amounts of dietary pyridoxine (10 ppm) could still prevent this effect. These findings further emphasise the important relationship between dietary vitamin B6 content and the lymphopenic effects of ammonia caramel/THI in the rat.

Mechanisms of chromium toxicity, carcinogenicity and allergenicity: review of the literature from 1985 to 2000.

Laboratory and clinical reports about the pathogenesis of the carcinogenicity and allergenicity of chromium compounds published between 1985 and 2000 have been reviewed as a basis for consideration of the pathogenetic mechanisms involved. There is good evidence from the clinic and the laboratory that Cr[VI] is the ion responsible for most of the toxic actions, although much of the underlying molecular damage may be due to its intracellular reduction to the even more highly reactive and short-lived chemical species Cr[III] and Cr[V]. Exposure to Cr[VI] can result in various point mutations in DNA and to chromosomal damage, as well as to oxidative changes in proteins and to adduct formation. The relative importance of these effects of chromium ions and of the free oxidising radicals they may generate in the body in causing tumours and allergic sensitisation remain to be demonstrated. Biochemical studies of the DNA-damaging effects and of the pathogenesis of the allergic reactions to chromium ions have not kept up with advances in understanding of the molecular basis of the effects of other carcinogens and allergens.