Analysis of the intracellular traffic of IgG in the context of Down syndrome (trisomy 21).

Persons with Down syndrome (DS, trisomy 21) have widespread cellular protein trafficking defects. There is a paucity of data describing the intracellular transport of IgG in the context of endosomal-lysosomal alterations linked to trisomy 21. In this study, we analyzed the intracellular traffic of IgG mediated by the human neonatal Fc receptor (FcRn) in fibroblast cell lines with trisomy 21. Intracellular IgG trafficking studies in live cells showed that fibroblasts with trisomy 21 exhibit higher proportion of IgG in lysosomes (~ 10% increase), decreased IgG content in intracellular vesicles (~ 9% decrease), and a trend towards decreased IgG recycling (~ 55% decrease) in comparison to diploid cells. Amyloid-beta precursor protein (APP) overexpression in diploid fibroblasts replicated the increase in IgG sorting to the degradative pathway observed in cells with trisomy 21. The impact of APP on the expression of FCGRT (alpha chain component of FcRn) was investigated by APP knock down and overexpression of the APP protein. APP knock down increased the expression of FCGRT mRNA by ~ 60% in both diploid and trisomic cells. Overexpression of APP in diploid fibroblasts and HepG2 cells resulted in a decrease in FCGRT and FcRn expression. Our results indicate that the intracellular traffic of IgG is altered in cells with trisomy 21. This study lays the foundation for future investigations into the role of FcRn in the context of DS.

Contribution of DNA methylation to the expression of FCGRT in human liver and myocardium.

FcRn mediates recycling and transcytosis of IgG and albumin in various cell types. The MHC-class-I-like protein of the FcRn heterodimer is encoded by FCGRT. Few determinants of variable FCGRT expression in humans have been identified so far. In this study, we investigated the presence of DNA methylation in regulatory regions of FCGRT in samples of human liver and myocardium tissue, and we examined the impact of FCGRT methylation on FcRn expression in model cell lines. Quantitative DNA methylation analysis of the FCGRT locus revealed differentially methylated regions in DNA from liver and myocardium. Methylation status in individual CpG sites correlated with FCGRT mRNA expression. Data from model cell lines suggest that differential methylation in the -1058 to -587 bp regulatory region of FCGRT contributes to FcRn expression. Chromatin immunoprecipitation assays indicate that CpG site methylation impacts the binding of the methylation sensitive transcription factors Zbtb7a and Sp1. This study provides a foundation to further define the contribution of epigenetic factors during the control of FcRn expression and IgG traffic in human tissues.

DNA sequence variants in the carbonyl reductase 1 (cbr1) gene in seven breeds of Canis lupus familiaris.

The anticancer anthracyclines doxorubicin and daunorubicin are used to treat a variety of cancers in dogs. The therapeutic utility of anthracyclines is limited by cardiotoxicity in some cases. Synthesis of anthracycline alcohol metabolites by carbonyl reductase 1 (CBR1) is crucial for the pathogenesis of cardiotoxicity. We hypothesize that genetic polymorphisms in canine cbr1 contribute to the variable pharmacodynamics of anthracyclines in dogs. DNA sequence variants in canine cbr1 were investigated in DNA samples from dogs of seven breeds. Thirteen SNPs were detected in canine cbr1. A 10-bp deletion in the 5'-untranslated region (5'-UTR) was found in specimens from the Labrador Retriever, Beagle, Siberian Husky, and Boxer breeds. The 5'-UTR also included a polymorphic "hot spot" region immediately downstream of the 10-bp deletion. DNA sequence variants in the "hot spot region" ranged from 1 to 21 bp in length. Bioinformatics searches identified a cluster of three to six potential binding sites for the transcription factor Sp1 in the DNA segment containing both the "hot spot" region and the 10-bp deletion. This information provides a foundation to allow us to investigate whether DNA sequence variants in the 5'-UTR of canine cbr1 impact the pharmacodynamics of anticancer anthracyclines in dogs.

Sensitization to serum albumins in children allergic to cow's milk and epithelia.

Patients with persistent milk allergy and specific immunoglobulin E (IgE) to bovine serum albumin (BSA) have a greater risk of rhinoconjunctivitis and asthma because of animal dander. To prove the cross-reactivity between serum albumin (SA) of different mammals in milk, meat, and epithelia and determine if heat treatment of meats decrease the allergenicity of albumins. The study was performed using SDS-PAGE and IgE-immunoblotting using sera from eight patients sensitized to milk, BSA, and animal danders. Sera from non-allergic and only animal dander allergic subjects served as a control. With one exception, all patients' sera recognized SA in different meats (beef, lamb, deer, and pork), epithelia (dog, cat, and cow), and cow's milk. Some patients even were only sensitized to SA in meat and epithelia. Danders' allergic only recognized other proteins in epithelia but not SA. No patients reacted to SA from heated meat extracts. Serum albumin is an important allergen involved in milk, meat, and epithelia allergy. The first contact with SA was through cow's milk and patients developed sensitization to epithelia SA even without direct contact with animals. Patients with both BSA and cow's milk allergy must avoid raw meats and furry pets.

Molecular emergence of acute myeloid leukemia during treatment for acute lymphoblastic leukemia.

Therapy-related acute myeloid leukemias (t-AML) with translocations of the MLL gene are associated with the use of topoisomerase II inhibitors. We established the emergence of the malignant clone in a child who developed t-AML with a t(11;19) (q23;p13.3) during treatment for acute lymphoblastic leukemia (ALL). The MLL-ENL and the reciprocal ENL-MLL genomic fusions and their chimeric transcripts were characterized from samples collected at the time of t-AML diagnosis. We used PCR with patient-specific genomic primers to establish the emergence of the MLL-ENL fusion in serially obtained DNA samples. The MLL-ENL fusion was not detectable in bone marrow at the time of ALL diagnosis or after 2 months of chemotherapy (frequency <8.3 x 10(-7) cells(-1)). The genomic fusion was first detected in bone marrow after 6 months of treatment at a frequency of one in 4,000 mononuclear bone marrow cells; the frequency was one in 70 cells after 20 months of therapy. At the first detection of MLL-ENL, the only topoisomerase II inhibitors the patient had received were one dose of daunorubicin and two doses of etoposide. The MLL-ENL fusion was not detectable in blood at the time of ALL diagnosis or after 0.7, 2, 8, 10, and 12 months of therapy but was detectable in blood at 16 months (one in 2.3 x 10(4) cells). Recombinogenic Alu sequences bracketed the breakpoints in both fusions. These data indicate that the malignant clone was not present before therapy, arose early during chemotherapy, and was able to proliferate even during exposure to antileukemic therapy.

Differing contribution of thiopurine methyltransferase to mercaptopurine versus thioguanine effects in human leukemic cells.

Thioguanine and mercaptopurine are prodrugs requiring conversion into thiopurine nucleotides to exert cytotoxicity. Thiopurine S-methyltransferase (TPMT), an enzyme subject to genetic polymorphism, catabolizes thiopurines into inactive methylated bases, but also produces methylthioguanine nucleotides and methylmercaptopurine nucleotides from thioguanine and mercaptopurine nucleotides, respectively. To study the effect of TPMT on activation versus inactivation of mercaptopurine and thioguanine, we used a retroviral gene transfer technique to develop human CCRF-CEM cell lines that did (TPMT+) and did not (MOCK) overexpress TPMT. After transduction, TPMT activities were 14-fold higher in the TPMT+ versus the MOCK cell lines (P < 0.001). TPMT+ cells were less sensitive to thioguanine than MOCK cells (IC(50) = 1.10+/- 0.12 microM versus 0.55 +/- 0.19 microM; P = 0.02); in contrast, TPMT+ cells were more sensitive to mercaptopurine than MOCK cells (IC(50) = 0.52 +/- 0.20 microM versus 1.50 +/- 0.23 microM; P < 0.01). The lower sensitivity of TPMT+ versus MOCK cells to thioguanine was associated with lower thioguanine nucleotide concentrations (917 +/- 282 versus 1515 +/- 183 pmol/5 x 10(6) cells; P = 0.01), higher methylthioguanine nucleotide concentrations (252 +/- 34 versus 27 +/- 10 pmol/5 x 10(6) cells; P = 0.01), less inhibition of de novo purine synthesis (13 versus 95%; P < 0.01), and lower deoxythioguanosine incorporation into DNA (2.0 +/- 0.6% versus 7.2 +/- 2.0%; P < 0.001). The higher sensitivity of TPMT+ cells to mercaptopurine was associated with higher concentrations of methylmercaptopurine nucleotide (2601 +/- 1055 versus 174 +/- 77 pmol/5 x 10(6) cells; P = 0.01) and greater inhibition of de novo purine synthesis (>99% versus 74%; P < 0.01) compared with MOCK cells. We conclude that methylation of mercaptopurine contributes to the antiproliferative properties of the drug, probably through inhibition of de novo purine synthesis by methylmercaptopurine nucleotides, whereas thioguanine is inactivated primarily by TPMT.

Freezing protects against allergy to Anisakis simplex.

BACKGROUND: Allergic reactions to Anisakis simplex have been described following ingestion of fish and were thought to be possible even if seafood is frozen or well-cooked. OBJECTIVE: This study aimed to confirm, by challenge test, that dead A. simplex larvae are not able to trigger allergic reactions in patients with proven hypersensitivity to this parasite. METHODS: Simple-blind, placebo-controlled, oral challenge tests with frozen A. simplex larvae were performed in 12 patients who had suffered severe anaphylactic reactions after ingestion of seafood and diagnosed of A. simplex hypersensitivity by skin prick test and specific IgE. If no reaction appeared, they were told to eat frozen seafood. 63 patients who had suffered urticaria or urticaria/angioedema by demonstratred hypersensitivity to A. simplex were also advised to eat frozen seafood. All of them were reevaluated 6 months later. RESULTS: All patients tolerated the dead larvae challenge test. After eating previously frozen seafood at least two times per week, all patients, including those who had suffered anaphylactic reactions and those who had only presented cutaneous manifestations, remained asymptomatic. CONCLUSIONS: Anisakis simplex-allergic patients tolerate ingestion of dead larvae. It is probable that these patients can eat frozen fish and that a seafood-free diet is not necessary.

Aromatase inhibition by an 11,13-dihydroderivative of a sesquiterpene lactone.

Compounds that inhibit aromatase activity are used for the treatment of breast cancer. A group of sesquiterpene lactones inhibit aromatase activity and also exert cytotoxicity through their reactive alpha-methylene-gamma-lactone group. To synthesize sesquiterpene lactones with greater specificity for aromatase inhibition and lower cytotoxicity, we chemically reduced the alpha-methylene-gamma-lactone group in the active aromatase inhibitor 10-epi-8-deoxycumambrin B (compound 1), to obtain the new compound 11betaH,13-dihydro-10-epi-8-deoxycumambrin B (compound 2). Reduction of the alpha-methylene-gamma-lactone group abrogated the cytotoxic activity of compound 1 against the JEG-3, HeLa, and COS-7 cell lines. Compound 2 had higher aromatase inhibitory activity than compound 1 (IC(50) = 2 +/- 0.5 microM versus 7 +/- 0.5 microM, K(i) = 1.5 microM versus 4.0 microM) and was a more potent type II ligand to the heme iron present in the cytochrome P450(arom) active site. Compound 2 inhibited aromatase activity in JEG-3 cells in a comparable manner to the inhibitor aminoglutethimide (AG) used clinically for the treatment of breast cancer. Additionally, compound 2 inhibited androstenedione-induced uterine hypertrophy in sexually immature mice (41% of uterine weight suppression for compound 2 versus 51% for AG). We conclude that the anti-aromatase activity of sesquiterpene lactones does not depend on the presence of the highly reactive alpha-methylene-gamma-lactone group, whereas their cytotoxicity does. These findings may facilitate the development of safer agents for breast cancer therapy.

Human cytochrome P450 maximal activities in pediatric versus adult liver.

Drug clearance is often higher in children than in adults, particularly when normalized to body weight. We previously showed that liver volume normalized to body weight was inversely related to age, but that the systemic clearance of a nonspecific cytochrome P450 (CYP) substrate (antipyrine) was higher in young children compared with adults even when normalized per liver volume. Our purpose herein was to evaluate whether P450 catalytic activities, expressed as maximal catalytic rates per milligram of microsomal protein, differed in up to 37 normal livers from subjects <10 (range 0.5-9 years of age), >10 but <60 years of age (range 10-59 years), and >60 year (range 63-93 years of age). There were no age-related differences in the oxidation of ethoxyresorufin (P =.83) (CYP1A2), ethoxycoumarin (P =.52) (CYP2E1 and other P450s), teniposide (P =. 58), midazolam (P =.47) (CYP3A4/3A5), or paclitaxel (P =.24) (at the 17alpha position, CYP2C8). Tolbutamide hydroxylation tended to be lower in children versus adults (P =.047) (CYP2C9), but did not reach statistical significance after correcting for multiple comparisons. No relationship was found to exist between age and microsomal recovery (P =.98); thus, recovery did not account for the lack of age-related differences in catalytic activity. We conclude that increased intrinsic cytochrome P450 activity is unlikely to account for increased clearance of most P450 drug substrates in children.

A novel activity for a group of sesquiterpene lactones: inhibition of aromatase.

A group of eleven sesquiterpene lactones isolated from different Asteraceae species from north-western Argentina were investigated for their inhibitory action on the estrogen biosynthesis. Seven of them, of different skeleton types, were found to inhibit the aromatase enzyme activity in human placental microsomes, showing IC50 values ranging from 7 to 110 microM. The most active were the guaianolides 10-epi-8-deoxycumambrin B (compound 1), dehydroleucodin (compound 2) and ludartin (compound 3). These compounds were competitive inhibitors with an apparent Ki = 4 microM, Ki = 21 microM and Ki = 23 microM, respectively. Compounds 1 and 2 acted as type II ligands to the heme iron present in the active site of aromatase cytochrome P450 (P450arom). Besides, all of them failed to affect the cholesterol side-chain cleavage enzyme activity on human placental mitochondrias. This is the first report on the aromatase inhibitory activity of this group of natural compounds.