Genetic sequence variations and <Emphasis Type="Italic">ADPRT</Emphasis> haplotype analysis in French Canadian families with high risk of breast cancer

The poly(ADP-ribose) polymerase (PARP/ADPRT) protein family catalyzes the synthesis of cellular poly(ADP-ribose) following DNA damage and is involved in genomic integrity by regulating cellular responses to DNA damage and apoptosis. Moreover, ADPRT inhibition contributes to a protective effect against cancer development. These findings render ADPRT an attractive candidate susceptibility gene for breast cancer, and thus the goal of this study was to evaluate the possible involvement of ADPRT sequence variations in breast cancer susceptibility. The complete sequence of the 23 exons and flanking intronic sequences of the ADPRT gene was analyzed in 54 affected individuals from distinct high-risk non-BRCA1/2 French Canadian families. No deleterious truncating mutation was identified in the coding region. However, 34 sequence variations were identified, among which seven are coding variants and seven are novel changes. All coding variants and intronic changes located in the vicinity of the coding variants identified in the case series were also analyzed in a cohort of 73 unrelated healthy French Canadian individuals. Interestingly, one missense variant (Pro377Ser) was observed in three different breast cancer cases but was not present among unaffected individuals. We have conducted here an exhaustive detailed mutation and haplotype tagging analysis of the ADPRT gene with regard to breast cancer, providing useful data for other large-scale association studies. Additional studies in other cohorts and other populations are however needed to further evaluate the implication of the Pro377Ser missense variant with regard to breast cancer susceptibility. Other members of INHERIT BRCAs involved in clinical aspects of the study are listed in the Appendix. J. Simard holds a Canada Research Chair in Oncogenetics.     

Analysis of the Bactericidal Response to an Experimental Neisseria meningitidis Vesicle Vaccine

Rabbit immunogenicity studies on an experimental trivalent native outer membrane vesicle vaccine derived from three serogroup B strains were conducted to evaluate the effectiveness of this vaccine at inducing an antibody response with serum bactericidal activity against meningococcal strains of other serogroups in addition to serogroup B strains. The results showed that the vaccine was capable of inducing an effective broad-based bactericidal antibody response in rabbits against a small sample of Neisseria meningitidis strains of serogroups C, W135, and X and, to a lesser extent, serogroups A and Y. Analysis of antibody specificity using a bactericidal depletion assay revealed that antibodies to lipooligosaccharide (LOS), PorA, and NadA induced in rabbits by the experimental trivalent outer membrane vesicle vaccine were responsible for most of the bactericidal activity against strains of the other N. meningitidis serogroups. In the case of serogroup A N. meningitidis strains, the outer membrane antigen NadA was primarily responsible for protection. The outer membrane antigens fHbp and OpcA were also effective in removing some bactericidal activity from the sera.     

Pain behavior and spinal cell activation due to carrageenan-induced inflammation in two inbred rat strains with differential hypothalamic–pituitary–adrenal axis reactivity

Lewis (LEW) and Fischer (FIS) inbred rats were used to study the relationship of hypothalamic–pituitary–adrenal (HPA) axis reactivity with inflammation-related pain behavior. LEW rats are susceptible to the development of autoimmune and chronic inflammatory disorders, whereas FIS rats are resistant. Since contradictory data have previously been collected under conditions of acute inflammation, we investigated the onset and maintenance of thermal and mechanical hyperalgesia and spinal activation of neurons and glia cells in a model of ongoing inflammation in both strains. Hind paw volumes and mechanical and thermal pain thresholds were measured prior to and during one week after intraplantar injection of carrageenan. The activation of nociceptive neurons (FosB), astroglia (GFAP) and microglia (OX-42) in the spinal cord of segments L5/L6 was assessed using immunohistochemistry. Inflammation increased paw volume, pain sensitivity and cell activation in both strains. FIS rats were more sensitive to sensory stimulation and developed a more severe edema on day 1, but recovered faster up to day 7 than LEW rats. At that time a higher amount of activated nociceptive neurons and corticosterone was seen in FIS rats, but microglial activation was more pronounced in LEW rats. Our results suggest a biphasic role of the HPA axis in pain behavior and spinal cell activation associated with ongoing inflammation. In the acute stage, the stronger reaction in FIS rats might be explained by an activating effect of corticosteroids on neutrophil function. Under ongoing inflammatory conditions the immunosuppressive actions of corticosteroids may dominate and lead to a quicker recovery of paw volume and pain sensitivity in FIS rats.     

Effect of one-week treatment with vaginal estrogen preparations on serum estrogen levels in postmenopausal women

OBJECTIVE: Approximately 50% of postmenopausal women suffer from vaginal atrophy, and a large proportion of them choose intravaginal estrogen preparations administered for local action to avoid systemic exposure to estrogens and its associated risk of breast and uterine cancer. The primary objective of this study was the evaluation of the systematic bioavailability of estradiol and estrone and the pharmacokinetics of two of the most frequently used intravaginal estrogen preparations, namely Vagifem and Premarin cream. DESIGN: While immunobased assays could not previously provide accurate measurement of serum estrogen concentrations in postmenopausal women, we have used validated mass spectrometry assays to measure the pharmacokinetics of serum estradiol and estrone during the 24 hours following the seventh daily application of 25 microg estradiol (Vagifem) and 1 g (0.625 mg) conjugated estrogens (Premarin) cream in 10 postmenopausal women in each group. RESULTS: Serum estradiol was increased on average by 5.4-fold from 3 to 17 pg/mL during the 24-hour period after daily administration of 25 microg estradiol or 1 g (0.625 mg) conjugated estrogens cream. Serum estrone, conversely, increased 150% with Vagifem and 500% with Premarin cream. CONCLUSIONS: The present data using validated, accurate, and sensitive mass spectrometry assays of estrogens show that the Vagifem pill and Premarin cream, after 1 week of daily treatment, cause an approximately fivefold increase in serum estradiol in postmenopausal women, thus indicating that the effects are unlikely to be limited to the vagina and that systemic actions are expected after application of these intravaginal estrogen preparations.     

Characteristics of Radioimmunoassays for the α- and β-Subunits of Human Luteinizing Hormone

Abstract

The binding characteristics and specificities of the National Hormone and Pituitary Program (NHPP) kits for the radioimmunoassay of the alpha- and beta-subunits of human luteinizing hormone (hLH-α and hLH-β) were studied, as well as the specificities of the anti-hLH and anti-human follicle stimulating hormone (anti-hFSH) antisera distributed by the same organization. The affinity constants of the anti-hLH-α and anti-hLH-β antisera were calculated at 157 ± 8.4 nM^?1 and 109 ± 7.4 nM^?1, respectively. Both antisera were highly specific with regard to the other subunit. However, in the homologous hLH-α RIA, native hLH cross-reacted at 21.9%, hFSH at 17.5% and hTSH at 7.9%. The alpha-subunit of the human chorionic gonadotropin, hCG-α, was equipotent with the hLH-α standard in this assay. In the homologous hLH-β RIA, hLH showed a cross-reactivity of 14.7% while the cross-reactivities of hCG-β, hFSH and hTSH were 3.5%, 1.2% and 0.6%, respectively. The anti-hFSH antiserum was highly specific, while the anti-hLH antiserum showed non parallel competition curves. With this knowledge of the specificity of each antiserum, corrections can be properly made for the assays of hLH, hLH-α and hLH-β while the hFSH RIA can be used without correction for the presence of the three other components.     

Identification of an inducible bacteriophage in a virulent strain of Streptococcus suis serotype 2

Streptococcus suis infection is considered to be a major problem in the swine industry worldwide. Most virulent Canadian isolates of S. suis serotype 2 do not produce the known virulence markers for this pathogen. PCR-based subtraction hybridization was adapted to isolate unique DNA sequences which were specific to virulent strains of S. suis isolated in Canada. Analysis of some subtracted DNA clones revealed significant homology with bacteriophages of gram-positive bacteria. An inducible phage (named Ss1) was observed in S. suis following the incubation of the virulent strain 89-999 with mitomycin C. Phage Ss1 has a long noncontractile tail and a small isometric nucleocapsid and is a member of the Siphoviridae family. Ss1 phage DNA appears to be present in most Canadian S. suis strains tested in this study, which were isolated from diseased pigs or had proven virulence in mouse or pig models. To our knowledge, this is the first report of the isolation of a phage in S. suis.     

Role for macrophage migration inhibitory factor in acute respiratory distress syndrome

The critical role of macrophage migration inhibitory factor (MIF) in mediating inflammatory lung injury in acute respiratory distress syndrome (ARDS) has been raised recently. The present study has identified enhanced MIF protein expression in alveolar capillary endothelium and infiltrating macrophages in lung tissues from ARDS patients. The possibility that MIF up-regulates its synthesis in an autocrine fashion in ARDS was tested using cultured endothelial cells stimulated with MIF and a murine model of lipopolysaccharide (LPS)-induced acute lung injury. MIF induced significant MIF and tumour necrosis factor (TNF)-alpha synthesis in cultured endothelial cells and the effect was blocked by neutralizing anti-MIF antibody. A similar blocking effect was observed when MIF-stimulated endothelial cells were pretreated with neutralizing anti-TNF-alpha antibody or glucocorticoid, supporting the notion that MIF induced TNF-alpha production via an amplifying pro-inflammatory loop. Treatment with anti-MIF or glucocorticoid effectively attenuated pulmonary pathology and the synthesis of MIF or TNF-alpha in mice with LPS-induced acute lung injury. Mildly augmented expression of aquaporin 1 (AQP1) was also detected in alveolar capillary endothelium in ARDS. In vitro studies revealed that both MIF and TNF-alpha induced a small increase of AQP1 synthesis in cultured endothelial cells. These findings suggest that MIF plays a crucial pathological role leading to alveolar inflammation in ARDS. Anti-MIF and early glucocorticoid therapy may represent a novel therapeutic approach for reducing alveolar inflammation in ARDS.     

Hormonal and biochemical changes during treatment of endometriosis with the luteinizing hormone-releasing hormone (LH-RH) agonist [D-Trp6,des-Gly-NH2(10)]LH-RH ethylamide

The effect of medical oophorectomy induced by treatment with the luteinizing hormone-releasing hormone (LH-RH) agonist [D-Trp6,des-Gly-NH2(10)]LH-RH ethylamide was studied in 34 patients with laparoscopically proven endometriosis. Tamoxifen was administered during the 1st month of therapy to prevent flare-up of the disease during the estrogen surge. Fifteen women had a decrease of their laparoscopy scores translated into an improvement in the stage of disease, whereas in 12 others, the decrease in their scores was not enough to allow a change of disease stage. The 2nd laparoscopy was not performed in 7 women. Medical oophorectomy, after daily injection of the LH-RH agonist (LH-RH-a), was accompanied by low levels of circulating estradiol. The serum concentration of all delta 4-3-ketosteroids was significantly decreased during medical oophorectomy, whereas the level of circulating delta 5-3 beta-hydroxysteroids was not altered except for pregnenolone. The present data indicate that medical oophorectomy induced by an LH-RH-a in association with tamoxifen is a very efficient and well tolerated therapy in endometriosis.     

Inhibition of ovulation during discontinuous intranasal luteinizing hormone-releasing hormone agonist dosing in combination with gestagen-induced bleeding

Four groups of eight or nine normal cycling volunteers with regular menstrual cycles had daily blood sampling during two pretreatment, two treatment, and two posttreatment cycles. Intranasal doses of 100, 200, and 300 micrograms of (D-Ser[TBU]6-des-Gly-NH210) luteinizing hormone-releasing hormone (LH-RH) ethylamide were administered every 12 hours and compared with a 400-micrograms dose given every 24 hours during two periods of 21 days followed by a drug-free interval of 7 days. Five milligrams of medroxyprogesterone acetate was taken orally on days 17 to 21. Serum luteinizing hormone was elevated during the first 2 weeks of treatment, and serum follicle-stimulating hormone was increased only during the first 2 days of treatment. At 100 to 300 micrograms/12 hours serum estradiol was stimulated up to preovulatory levels, whereas at 400 micrograms/24 hours most values were in the early follicular phase range. Ovarian ultrasonography revealed the transient development of preovulatory-like follicles in 8 of 12 studied cycles. Serum progesterone values were less than 2 ng/ml in 57.3%, between 2 and 5 ng/ml in 27.9%, and greater than 5 ng/ml in 14.7%. Withdrawal bleeding occurred during the pause in 97% of treatment cycles. Nine of 13 breakthrough bleedings happened in the groups given 100-micrograms and 300-micrograms/12 hours. Recovery cycles showed slightly prolonged follicular phases with normal luteal phases. No changes were observed in routine laboratory measurements. In conclusion, intermittent administration of appropriate LH-RH agonist dosing in combination with a progestogen would effectively block ovulation while preserving adequate cyclic estradiol secretion and could be an alternative contraceptive approach.     

Platelet-activating factor in vasoobliteration of oxygen-induced retinopathy

PURPOSE: To test whether platelet-activating factor (PAF) directly causes retinovascular endothelial cell (EC) death. METHODS: Retinovascular density was calculated in rat pups exposed to 80% O(2) from postnatal days (P)6 to P14 (to produce oxygen-induced retinopathy [OIR]), using the adenosine diphosphatase (ADPase) technique, in animals treated with distinct PAF receptor blockers (PCA-4248, BN52021, or THG315). PAF levels were then measured in the retinas. Viability of ECs from piglets and humans in response to C-PAF (a stable PAF analogue) was determined by the reduction of the tetrazolium salt 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) by viable cells, incorporation of propidium iodide (PI), TUNEL assay, and release of lactate dehydrogenase. Release of thromboxane (TX) was measured in the cell media. RESULTS: PAF levels in retina were markedly increased by exposure of isolated rat retinas to H(2)O(2) (1 micro M) and of rat pups placed in 80% O(2). Exposure to 80% O(2) induced retinal vasoobliteration, which was equally significantly inhibited ( approximately 60%) by all PAF receptor blockers tested. C-PAF increased incorporation of PI by isolated rat retinal microvasculature. Also, C-PAF caused time- and concentration-dependent death of cultured retinal ECs, which was prevented by the PAF receptor antagonist CV-3988. This effect of C-PAF was selective on retinal and neurovascular ECs, but not on other ECs. DNA fragmentation (TUNEL) was hardly detected, and inhibition of apoptosis-related processes by nicotinamide, cyclosporin A, and Z-DEVD-FMK and Z-VAD-FMK (caspase inhibitors) barely protected against death in EC, whereas C-PAF increased release of lactate dehydrogenase, implying that necrosis is the nature of EC death. Finally, C-PAF-induced cell death was preceded by an increase in TXB(2) levels and was prevented by TXA(2) synthase inhibition (with CGS12970). CONCLUSIONS: The data suggest PAF plays a major role in vasoobliteration in OIR by triggering death of neuroretinal microvascular ECs. The cell death seems to be mediated at least in part by TXA(2). These effects of PAF may participate in ischemic retinopathies such as diabetes and retinopathy of prematurity.