Wagner V. Yotov

Absence of Association of Thrombophilia Polymorphisms with Intrauterine Growth Restriction

BACKGROUND Previous data have demonstrated associations between thrombophilia polymorphisms in pregnant women and an increased risk of intrauterine growth restriction in their offspring, but this finding remains uncertain. METHODS We performed a hospital-based case-control study and a family-based study including 493 newborns with intrauterine growth restriction (defined by birth weight below the 10th percentile for gestational age and sex according to Canadian norms) and 472 controls (with birth weight at or above the 10th percentile). We determined the presence or absence in newborns and their parents of the following polymorphisms: methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, factor V Leiden G1691A, and prothrombin G20210A. Mothers were interviewed to obtain information on other risk factors for intrauterine growth restriction. RESULTS The risk of intrauterine growth restriction was not increased among mothers carrying a polymorphism associated with thrombophilia. In the case-control study, the odds ratios associated with two copies of the variant, after adjustment for newborn genotype and other risk factors, were 1.55 for MTHFR C677T (95 percent confidence interval, 0.83 to 2.90) and 0.49 for MTHFR A1298C (95 percent confidence interval, 0.25 to 0.93); heterozygotes for factor V Leiden had an odds ratio of 1.18 (95 percent confidence interval, 0.54 to 2.55), and heterozygotes for prothrombin G20210A had an odds ratio of 0.92 (95 percent confidence interval, 0.36 to 2.35). These polymorphisms in the newborn were not associated with an increased risk. Newborns who were homozygous for the MTHFR C677T variant had a decreased risk of intrauterine growth restriction (odds ratio after adjustment for mothers genotype and other confounders, 0.52 [95 percent confidence interval, 0.29 to 0.94]). The results of the family-based study supported those of the case-control study. CONCLUSIONS Our findings do not indicate that there are associations between maternal or newborn polymorphisms associated with thrombophilia and an increased risk of intrauterine growth restriction.

Human papillomavirus (HPV) study of 691 pathological specimens from Quebec by PCR-direct sequencing approach.

Human papillomaviruses (HPV) are etiological agents of cervical cancer. In order to address clinical demand for HPV detection and sequence typing, mostly in pre‐cancerous cervical lesions, we applied our two‐tier PCR‐direct sequencing (PCR‐DS) approach based on the use of both MY09/MY11 and GP5 + /GP6 + sets of primers. We tested 691 pathological specimens, all of which were biopsies, 75% of which were diagnosed histologically as cervical intraepithelial neoplasia (CIN) grades I–III. In total, 484 samples (70%) tested HPV‐positive, yielding 531 HPV sequences from 47 HPV types, including two novel types. Four most frequently found HPV types accounted for 52.9% of all isolates: HPV6, 16, 11, and 31 (21.5%, 20.0%, 7.0%, and 4.5%, respectively). Some interesting results are the following: all currently known high‐risk HPV (14 types) and low‐risk HPV (6 types) were detected; HPV18 was not the 1st or 2nd but rather the 4th–5th most frequent high‐risk HPV type; the highest detection rate for HPV (86%) among samples suspected to be HPV‐infected was found in the youngest age group (0–10 years old), including 70% (44/63) “genital” HPV types; HPV types of undetermined cervical cancer risk represented 19% and of the total HPV isolates but were strongly increased in co‐infections (36.5% of all isolates). To our knowledge, this is the largest sequencing‐based study of HPV. The HPV types of unknown cancer risk, representing the majority of the known HPV types, 27 of the 47 types detected in this study, are not likely to play a major role in cervical cancer because their prevalence in CIN‐I, II, and III declines from 16% to 8% to 2.5%. The two‐tier PCR‐DS method provides greater sensitivity than cycle sequencing using only one pair of primers. It could be used in a simple laboratory setting for quick and reliable typing of known and novel HPV from clinical specimens with fine sequence precision. It could also be applied to anti‐cancer vaccine development. J. Med. Virol. 63:284–292, 2001.

Modulation of intestinal and liver fatty acid-binding proteins in Caco-2 cells by lipids, hormones and cytokines

Intestinal and liver fatty acid binding proteins (I‐ and L‐FABP) are thought to play a role in enterocyte fatty acid (FA) trafficking. Their modulation by cell differentiation and various potential effectors was investigated in the human Caco‐2 cell line. With the acquisition of enterocytic features, Caco‐2 cells seeded on plastic progressively increased L‐FABP quantities, whereas I‐FABP was not detectable even very late in the maturation process. On permeable filters that improved differentiation markers (sucrase, alkaline phosphatase, transepithelial resistance), Caco‐2 cells furthered their L‐FABP content and expressed I‐FABP. Western blot analysis showed a significant increase in I‐ and L‐FABP expression following an 8‐hour incubation period with butyric acid, oleic acid, and phosphatidylcholine. However, in all cases, I‐FABP levels were higher than L‐FABP concentrations regardless of the lipid substrates added. Similarly, hydrocortisone and insulin enhanced the cellular content of I‐ and L‐FABP whereas leptin triggered I‐FABP expression only after an 8‐hour incubation. Finally, tumor necrosis factor‐α was more effective in increasing the cytosolic amount of I‐FABP levels. In conclusion, our data demonstrate that I‐FABP expression is limited to fully differentiated Caco‐2 cells and can be more easily regulated than L‐FABP by lipids, hormones, and cytokines. J. Cell. Biochem. 81: 613–620, 2001.

Amplifications of DNA primase 1 (PRIM1) in human osteosarcoma.

We studied the involvement of PRIM1 in osteosarcoma by differential display, Northern and Southern hybridization, as well as fluorescence in situ hybridization (FISH) on interphase nuclei. In total, 22 pediatric oncology specimens were tested. PRIM1 was found to be amplified in 41% of the samples. PRIM1 is coamplified with the core 12q13 amplicon genes CDK4, SAS, and OS9, and was physically mapped very close to them. PRIM1 is therefore a new candidate for the role of a major target gene of 12q13 amplifications in human cancers. Genes Chromosomes Cancer 26:62–69, 1999.

Caco-2 cells and human fetal colon: a comparative analysis of their lipid transport.

Caco-2 cells and human colonic explants were compared for their ability to esterify lipid classes, synthesize apolipoproteins and assemble lipoproteins. Highly differentiated cells and colonic explants were incubated with [(14)C]oleic acid or [(35)S]methionine for 48 h. Caco-2 cells demonstrated a higher ability to incorporate [(14)C]oleic acid into cellular phospholipids (13-fold, P<0.005), triglycerides (28-fold, P<0.005) and cholesteryl ester (2-fold, P<0. 01). However, their medium/cell lipid ratio was 11 times lower, indicating a limited capacity to export newly synthesized lipids. De novo synthesis of apo B-48 and apo B-100 was markedly increased (7%0 and 240%, respectively), whereas the biogenesis of apo A-I was decreased (60%) in Caco-2 cells. The calculated apo B-48/apo B-100 ratio was substantially diminished (107%), suggesting less efficient mRNA editing in Caco-2 cells. When lipoprotein distribution was examined, it displayed a prevalence of VLDL and LDL, accompanied along with a lower proportion of chylomicron and HDL. In addition, differences in lipoprotein composition were evidenced between colonic explants and Caco-2 cells. Therefore, our findings stress the variance in the magnitude of lipid, apolipoprotein and lipoprotein synthesis and secretion between the two intestinal models. This may be due to various factors, including the origin of Caco-2 cell line, i.e., colon carcinoma.

Modulation of apo A-IV transcript levels and synthesis by n-3, n-6, and n-9 fatty acids in CACO-2 cells.

It has been postulated that apolipoprotein (apo) A‐IV plays various significant roles in lipid transport and lipoprotein metabolism. Although it is controlled by fat feeding, so far little else is known about its regulation by specific fatty acids. In this study, we focused on the modulation of apo A‐IV mRNA levels, mass, and biogenesis by mono‐ and polyunsaturated fatty acids (FA) in the human intestinal Caco‐2 cell line. In confluent cells incubated with 1 mM oleic (n‐9), linoleic (n‐6), α‐linolenic (n‐3), or docosahexaenoic (n‐3) acids for a long‐term period, both apo A‐IV protein levels and de novo synthesis were increased. The induction resulted from the up‐regulation of apo A‐IV mRNA transcripts. In contrast, an inhibitory effect was evident with short‐term incubation. FA chain length and degree of unsaturation had little effect altering apo A‐IV transcript and biogenesis. These data offer evidence that isolated fatty acids regulate gene expression and the production of apo A‐IV in the enterocyte. J. Cell. Biochem. 75:73–81, 1999.

Direct Human Papillomavirus (HPV) Sequencing Method Yields a Novel HPV in a Human Immunodeficiency Virus-Positive Quebec Woman and Distinguishes a New HPV Clade

Papillomaviruses of supergroup A exhibit genital tropism and are best known as etiologic agents for benign and malignant cervical lesions in women. A polymerase chain reaction direct sequencing approach with P-33-labeled dideoxynucleotides was used to detect and type human papillomaviruses (HPVs) in cervical biopsies. A novel sequence was found in condylomatous specimens from a human immunodeficiency virus-positive French Canadian woman. The viral gene L1 was sequenced completely, yielding a novel HPV type of supergroup A, named JC9710. This is related to a previously described HPV type from New Mexico, CP8061, and to Colobus monkey papillomavirus 1. Sequence similarity searches and phylogenetic analyses with different software packages clustered the three viral types as a new clade, for which the next available number, A15, was proposed.

Human papillomavirus PCR direct sequencing study of cervical precancerous lesions in Quebec children.

Editor,—Similarly to adult pathology, human papillomavirus (HPV) infection is the most common sexually transmitted disease in adolescent girls, whose prevalence is 16% according to one US study.1 However, little or no HPV sequencing data from paediatric specimens are available. We used our two tier polymerase chain reaction (PCR) direct sequencing (PCR-DS) approach2 to study cervical biopsies from 44 adolescent Quebec girls (14–17 years old). They originated from various social and ethnic groups, as well as geographically distinct areas of Quebec. Written informed consent about the use of the specimens was obtained from the ethics committee of this institution. All biopsies were analysed for histological changes and presence of HPV specific DNA. Most of them (n = 36) were diagnosed as cervical intraepithelial neoplasia (CIN), seven as inflammatory changes, and one as …