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Biomedical subjects

David C Young

Publications and source records attributed to David C Young.

At least 19 recordsLinked to original sources

Mycobacterium tuberculosis SigM positively regulates Esx secreted protein and nonribosomal peptide synthetase genes and down regulates virulence-associated surface lipid synthesis.

The Mycobacterium tuberculosis genome encodes 12 alternative sigma factors, several of which regulate stress responses and are required for virulence in animal models of acute infection. In this work we investigated M. tuberculosis SigM, a member of the extracytoplasmic function subfamily of alternative sigma factors. This sigma factor is expressed at low levels in vitro and does not appear to function in stress response regulation. Instead, SigM positively regulates genes required for the synthesis of surface or secreted molecules. Among these are genes encoding two pairs of Esx secreted proteins, a multisubunit nonribosomal peptide synthetase operon, and genes encoding two members of the proline-proline-glutamate (PPE) family of proteins. Genes up regulated in a sigM mutant strain include a different PPE gene, as well as several genes involved in surface lipid synthesis. Among these are genes involved in synthesis of phthiocerol dimycocerosate (PDIM), a surface lipid critical for virulence during acute infection, and the kasA-kasB operon, which is required for mycolic acid synthesis. Analysis of surface lipids showed that PDIM synthesis is increased in a sigM-disrupted strain and is undetectable in a sigM overexpression strain. These findings demonstrate that SigM positively and negatively regulates cell surface and secreted molecules that are likely to function in host-pathogen interactions.

Bacterial Proteins↗

Loss of the acyl-CoA binding protein (Acbp) results in fatty acid metabolism abnormalities in mouse hair and skin.

Proper fatty acid metabolism is critical for hair and skin development and maintenance. The acyl-CoA binding protein (Acbp) is a widely expressed protein that binds long-chain fatty acyl-CoA esters and plays a role in fatty acyl-CoA transport and pool formation. However, loss of function of Acbp in the whole animal has not been investigated. Here, we show that deletion of Acbp in mouse results in sebocyte hyperplasia and sparse, matted hair with a greasy appearance. Consistent with these gross abnormalities, Acbp is highly expressed in the pilosebaceous units of mouse skin as determined by Northern analysis and in situ hybridization. Loss of Acbp also results in fatty acid metabolism abnormalities, with hair lipid profiles showing altered levels of triacylglycerols and nearly co-migrating lipids. These data suggest that Acbp plays a role in triacylglycerol biosynthesis, and that regulation of this process is important for proper hair and skin development and maintenance in the mouse.

Animals↗

Role of lipid trimming and CD1 groove size in cellular antigen presentation.

Cellular CD1 proteins bind lipids that differ in length (C(12-80)), including antigens that exceed the capacity of the CD1 groove. This could be accomplished by trimming lipids to a uniform length before loading or by inserting each lipid so that it penetrates the groove to a varying extent. New assays to detect antigen fragments generated within human dendritic cells showed that bacterial antigens remained intact, even after delivery to lysosomes, where control lipids were cleaved. Further, recombinant CD1b proteins could bind and present C(80) lipid antigens using a mechanism that did not involve cellular enzymes or lipid cleavage, but was regulated by pH in the physiologic range. We conclude that endosomal acidification acts directly, rather than through enzymatic trimming, to insert lipids into CD1b. Lipids are loaded in an intact form, so that they likely protrude through a portal near the bottom of the groove, which represents an escape hatch for long lipids from mycobacterial pathogens.

Antigen Presentation↗

T-cell recognition of glycolipids presented by CD1 proteins.

The most well-known molecular paradigm of antigen recognition by T cells involves partial digestion of proteins to generate small peptides, which bind to major histocompatibility complex (MHC) proteins. Recent studies of CD1, an MHC class I homolog encoded outside the MHC, have revealed that it presents diverse glycolipids to T cells. The molecular mechanism for lipid antigen recognition involves insertion of the lipid portion of antigens into a hydrophobic groove to form CD1-lipid complexes, which contact T-cell receptors (TCRs). Here, we examine the known antigen structures presented by CD1, the majority of which have sugar moieties that are capable of interacting with TCRs. Recognition of carbohydrate epitopes is precise, and lipid-reactive T cells alter systemic immune responses in models of infectious and autoimmune disease. These findings provide a previously unrecognized mechanism by which the cellular immune system can recognize alterations in many types of carbohydrate structures.

Animals↗

Socioeconomic status and receipt of obstetric services in Canada.

OBJECTIVE: To examine differences in labor induction and cesarean delivery rates by socioeconomic status in Nova Scotia, Canada. METHODS: We studied all women in Nova Scotia who delivered between 1988 and 1995 after a singleton pregnancy. Information was obtained from the Nova Scotia Atlee Perinatal Database and the federal income tax (T1) Family Files maintained by the Small Area and Administrative Data Division of Statistics Canada (n = 76,440). Labor induction and cesarean delivery rates were contrasted by family income and other indices, with adjustment for risk factors carried out using logistic models. RESULTS: Maternal characteristics and other factors varied by socioeconomic status; in the lowest income group 4.4% of women were aged 35 years or older, and 49.4% were nonsmokers, whereas in the highest income group 17.7% were aged 35 years or older, and 88.7% were nonsmokers. Crude induction rates were similar across income groups (17.7% compared with 18.1% in the lowest compared with highest income groups), but there were significant differences in crude cesarean (17.7% compared with 21.2%) and crude primary cesarean rates (12.5% compared with 17.0%). Controlling for differences in risk factors altered these relationships. Adjusted rate ratios, 95% confidence intervals (CIs) and P values contrasting the lowest compared with highest income groups were labor induction 1.09 (95% CI 1.00-1.19), P = .05; overall cesarean delivery 1.12 (95% CI 1.03-1.23), P = .01; primary cesarean delivery 1.09 (95% CI 0.98-1.21), P = .12. CONCLUSION: Affluent women in Canada are not more likely to have labor induction or cesarean delivery compared with less affluent women. LEVEL OF EVIDENCE: II-2.

Adult↗

Determinants of perinatal mortality and serious neonatal morbidity in the second twin.

OBJECTIVE: To identify potential determinants of perinatal mortality and neonatal morbidity among second twins relative to first twins. METHODS: A retrospective cohort design was used to study twin deliveries in Nova Scotia from 1988 to 2002. Monoamniotic or conjoined twins and twin pairs with major congenital anomaly or antepartum fetal death of either twin were excluded. The primary outcome was a composite measure of perinatal mortality and neonatal morbidity, including birth asphyxia, respiratory distress, neonatal trauma, and infection. Risk of adverse outcome of second twins relative to first-born co-twins was determined by matched-pair analysis. RESULTS: Of 1,542 twin pairs, the second twin was at greater risk of composite adverse outcome (relative risk [RR] 1.62, 95% confidence interval [CI] 1.38-1.9) than the first twin. This excess risk was evident independent of presentation, chorionicity, or infant sex but was associated with planned vaginal delivery, birth weight discordance, and prolonged interdelivery interval. Term second twins were less likely to suffer excess morbidity with elective cesarean (RR 1.0, 95% CI 0.14-7.10) than with planned vaginal delivery (RR 3.0, 95% CI 1.47-6.11). The major contributors to neonatal morbidity in the second twin were birth asphyxia at 37 weeks or later and respiratory distress syndrome at less than 37 weeks. CONCLUSION: The second twin is at greater risk of adverse perinatal outcome than the first twin, independent of presentation, chorionicity, or infant sex. Planned vaginal delivery, birth weight discordance, and prolonged interdelivery interval increase this infant risk. Elective cesarean delivery at term may improve perinatal outcome for the second twin. However, the number of cesarean births required to prevent one case of composite adverse outcome, assuming causality, was 33.

Adult↗

Molecular mechanism of lipopeptide presentation by CD1a.

CD1a is expressed on Langerhans cells (LCs) and dendritic cells (DCs), where it mediates T cell recognition of glycolipid and lipopeptide antigens that contain either one or two alkyl chains. We demonstrate here that CD1a-restricted T cells can discriminate the peptide component of didehydroxymycobactin lipopeptides. Structure analysis of CD1a cocrystallized with a synthetic mycobactin lipopeptide at 2.8 A resolution further reveals that the single alkyl chain is inserted deep within the A' pocket of the groove, whereas its two peptidic branches protrude along the F' pocket to the outer, alpha-helical surface of CD1a for recognition by the TCR. Remarkably, the cyclized lysine branch of the peptide moiety lies in the shallow F' pocket in a conformation that closely mimics that of the alkyl chain in the CD1a-sulfatide structure. Thus, this structural study illustrates how a single chain lipid can be presented by CD1 and that the peptide moiety of the lipopeptide is recognized by the TCR.

Antigen Presentation↗

Mycobacterium tuberculosis pks12 produces a novel polyketide presented by CD1c to T cells.

CD1c-mediated T cells are activated by a mycobacterial phospholipid antigen whose carbohydrate structure precisely corresponds to mammalian mannosyl beta-1-phosphodolichol (MPD), but contains an unusual lipid moiety. Here, we show that this T cell antigen is a member of a family of branched, alkane lipids that vary in length (C30-34) and are produced by medically important mycobacteria such as M. tuberculosis and M. bovis Bacille-Calmette-Guerin. The alkane moiety distinguished these mycobacterial lipid antigens from mammalian MPDs and was necessary for activation of CD1c-restricted T cells, but could not be accounted for by any known lipid biosynthetic pathway. Metabolic labeling and mass spectrometric analyses suggested a mechanism for elongating lipids using alternating C2 and C3 units, rather than C5 isopentenyl pyrophosphate. Inspection of the M. tuberculosis genome identified one candidate gene, pks12, which was predicted to encode the largest protein in M. tuberculosis, consisting of 12 catalytic domains that correspond to key steps in the proposed pathway. Genetic deletion and complementation showed that Pks12 was necessary for antigen production, but did not affect synthesis of true isoprenols. These studies establish the genetic and enzymatic basis for a previously unknown type of polyketide, designated mycoketide, which contains a lipidic pathogen-associated molecular pattern.

Antigen Presentation↗

CD1d-restricted T cell activation by nonlipidic small molecules.

In addition to NK T cells expressing invariant Valpha14 or Valpha24 T cell receptors (TCRs), the CD1d-restricted T cell repertoire is comprised of T cells with diverse TCRs that mediate inflammation during autoimmune and infectious disease. Here we describe the isolation of human Valpha24(-) T cells that are activated by antigen and CD1d. Mass spectrometric and NMR studies revealed that the stimulatory compounds were neither peptidic nor lipidic but instead were composed of sulfur and aromatic hydrocarbon rings, corresponding to the general structure of phenyl pentamethyldihydrobenzofuran sulfonates. Studies of the molecular mechanism of T cell activation showed that a clonotypic Valpha2/Vbeta21 TCR transmitted activating signals, which were highly specific for hydroxylation and methylation patterns at the terminal structures of stimulatory compounds. These studies provide evidence for noninvariant CD1d-restricted T cells in humans and identify the complete molecular structure of a nonlipidic small molecule that activates T cells through an alphabeta TCR.

Antigens, CD1↗

T cell activation by lipopeptide antigens.

Unlike major histocompatibility proteins, which bind peptides, CD1 proteins display lipid antigens to T cells. Here, we report that CD1a presents a family of previously unknown lipopeptides from Mycobacterium tuberculosis, named didehydroxymycobactins because of their structural relation to mycobactin siderophores. T cell activation was mediated by the alphabeta T cell receptors and was specific for structure of the acyl and peptidic components of these antigens. These studies identify a means of intracellular pathogen detection and identify lipopeptides as a biochemical class of antigens for T cells, which, like conventional peptides, have a potential for marked structural diversity.

Antigen Presentation↗

Structural features of the acyl chain determine self-phospholipid antigen recognition by a CD1d-restricted invariant NKT (iNKT) cell.

Little is known about the antigen specificity of CD1d-restricted T cells, except that they frequently recognize CD1d-expressing antigen-presenting cells in the absence of exogenous antigen. We previously demonstrated that the 24.8.A iNKT cell hybridoma was broadly reactive with CD1d-transfected cell lines and recognized the polar lipid fraction of a tumor cell extract. In the present study, the antigen recognized by the 24.8.A iNKT cell hybridoma was purified to homogeneity and identified as palmitoyl-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1 PE). The 24.8.A iNKT cell hybridoma recognized synthetic 16:0-18:1[cis] PE, confirming that this phospholipid is antigenic. Recognition correlated with the degree of unsaturation of the acyl chains. Using a panel of synthetic PEs, the 24.8.A iNKT cell hybridoma was shown to be activated by PEs that contained at least one unsaturated acyl chain. The configuration of the double bonds was important, as the 24.8.A iNKT cell hybridoma recognized unsaturated acyl chains in the cis, but not the trans, configuration. PEs with multiple double bonds were recognized better than those with a single double bond, and increasing acyl chain unsaturation correlated with increased binding of PE to CD1d. These data illustrate the potential importance of the acyl chain structure for phospholipid antigen binding to CD1d.

Animals↗

Effects of IgM from rheumatic fever patients on intracellular calcium levels of neonatal rat cardiac myocytes.

Rheumatic fever (RF), a potential sequela of Streptococcus pyogenes pharyngitis, sometimes results in myocarditis and heart failure. Antibodies have been implicated in the pathogenesis of RF and anti-cardiac myosin antibody levels are elevated in RF patients. Since myocarditis is associated with altered cardiomyocyte calcium transients it was of interest to determine the direct effects of RF patient antibodies on calcium transients in cultured myocytes. RF patient polyclonal IgM treatment caused increased calcium retention by neonatal rat heart cells in vitro as determined with isotopically labeled calcium. Therefore, to further characterize this finding, calcium transients were evaluated by real time fluorescence spectroscopy and deconvolution imaging. RF patient polyclonal IgM produced increased calcium retention during the relaxation stage of the contraction cycle leading to a slowing of contraction rate, disorganized calcium transients, and eventual tetany. In contrast, calcium transient studies of cardiomyocytes following treatment with monoclonal anti-myosin antibodies revealed declining intracellular calcium levels, accompanied by disorganized transients and tetany. Treatment with both antibodies led to myocyte dysfunction and these novel findings suggest a role for antibodies in the pathogenesis of the myocarditis associated with rheumatic carditis.

Animals↗

Spontaneous versus induced labor after a previous cesarean delivery.

OBJECTIVE: To compare maternal and neonatal outcomes in spontaneous versus induced labor after one previous cesarean delivery. METHODS: Women with one previous cesarean delivery who had spontaneous labor between January 1992 and January 2000 were compared with those whose labor was induced. RESULTS: Three thousand seven hundred forty-six patients had a trial of labor (2943 spontaneous, 803 induced). Those induced had more frequent early postpartum hemorrhage (7.3% versus 5.0%; odds ratio [OR] 1.66; 95% confidence interval [CI] 1.18, 2.32), cesarean delivery (37.5% versus 24.2%; OR 1.84; 95% CI 1.51, 2.25), and neonatal intensive care unit (NICU) admission (13.3% versus 9.4%; OR 1.69; 95% CI 1.25, 2.29). There was a trend toward higher uterine rupture rates in those with induced versus spontaneous labor (0.7% versus 0.3%, P =.128) and for patients undergoing dinoprostone (prostaglandin E(2)) induction versus other methods (1.1% versus 0.6%, P =.62), although neither difference achieved statistical significance. CONCLUSION: Induced labor is associated with an increased rate of early postpartum hemorrhage, cesarean delivery, and neonatal ICU admission. The higher rate of uterine rupture in those who had labor induced was not statistically significant.

Adult↗

Changes in maternal characteristics and obstetric practice and recent increases in primary cesarean delivery.

OBJECTIVE: To estimate the contribution of changes in maternal characteristics (namely, age, parity, prepregnancy weight, weight gain in pregnancy, smoking status) and obstetric practice (namely, labor induction, epidural anesthesia, delivery by an obstetrician, midpelvic forceps delivery) to recent increases in primary cesarean delivery rates. METHODS: We studied all deliveries in Nova Scotia, Canada, between 1988 and 2000 after excluding women who had a previous cesarean delivery (n = 127,564). Logistic regression was used to study the effect of changes in maternal characteristics and obstetric practice on primary cesarean delivery rates. The effect of changes in midpelvic forceps delivery was examined through ecologic Poisson regression. RESULTS: Primary cesarean delivery rates increased from 13.4% of deliveries in 1988 to 17.5% in 2000. This was due to increases in cesarean deliveries for dystocia (14% increase), breech (24% increase), suspected fetal distress (21% increase), hypertension (47% increase), and miscellaneous indications (73% increase). Adjustment for maternal characteristics reduced the temporal increase in primary cesarean delivery rates between 1988-1991 and 1998-2000 from 21% (95% confidence interval [CI] 16%, 25%) to 2% (95% CI -2%, 7%). Additional adjustment for obstetric practice factors further reduced period effects. Midpelvic forceps delivery was significantly and negatively associated with primary cesarean delivery (P =.001). CONCLUSION: Recent increases in primary cesarean delivery rates are a consequence of changes in maternal characteristics. Obstetric practice, which has altered due to changes in maternal characteristics and concerns related to fetal and maternal safety, has also contributed to increases in primary cesarean delivery.

Adult↗

Induction of labour with a favourable cervix and/or pre-labour rupture of membranes.

Premature rupture of membranes (PROM) occurs in 8% of term deliveries. In this situation labour induction with prostaglandins, compared with expectant management, results in a reduced risk of chorioamnionitis, neonatal antibiotic therapy, neonatal intensive care (NICU) admission, and increased maternal satisfaction. The use of prostaglandin is associated with an increased rate of diarrhoea and use of analgesia/anaesthesia. Compared with oxytocin, prostaglandin induction results in a lower rate of epidural use and internal fetal heart rate monitoring but a greater risk of chorioamnionitis, nausea, vomiting, more vaginal examinations, neonatal antibiotic therapy, NICU admission and neonatal infection. Women should be informed of the risks and benefits of each method of induction.Misoprostol is gaining increasing interest as an alternative induction agent. It appears to be an effective method of labour induction with term PROM. Further research is needed to identify the preferred dosage, route and interval of administration, and to assess uncommon maternal and neonatal outcomes. There has been limited research on the use of prostaglandins, including misoprostol, for induction of labour with a favourable cervix and intact membranes. Compared with intravenous oxytocin (with and without amniotomy), labour induction using vaginal prostaglandins in women with a favourable cervix (with and without PROM) results in a higher rate of vaginal delivery within 24 hours and increased maternal satisfaction. In women with a favourable cervix, artificial rupture of membranes followed by oral misoprostol has similar time to vaginal delivery compared with artificial rupture of membranes followed by oxytocin. Further research with prostaglandins, including misoprostol, is needed to evaluate other maternal and neonatal outcomes in women being induced with a favourable cervix. No form of prostaglandin induction in women with PROM or favourable cervix has proven clearly superior to oxytocin infusion.

Administration, Intravaginal↗

Prevention of Rh alloimmunization.

OBJECTIVE: To provide guidelines on use of anti-D prophylaxis to optimize prevention of rhesus (Rh) alloimmunization in Canadian women. OUTCOMES: Decreased incidence of Rh alloimmunization and minimized practice variation with regards to immunoprophylaxis strategies. EVIDENCE: The Cochrane Library and MEDLINE were searched for English-language articles from 1968 to 2001, relating to the prevention of Rh alloimmunization. Search terms included: Rho(D) immune globulin, Rh iso- or allo-immunization, anti-D, anti-Rh, WinRho, Rhogam, and pregnancy. Additional publications were identified from the bibliographies of these articles. All study types were reviewed. Randomized controlled trials were considered evidence of highest quality, followed by cohort studies. Key individual studies on which the principal recommendations are based are referenced. Supporting data for each recommendation is briefly summarized with evaluative comments and referenced. VALUES: The evidence collected was reviewed by the Maternal-Fetal Medicine and Genetics Committees of the Society of Obstetricians and Gynaecologists of Canada (SOGC) and quantified using the Evaluation of Evidence guidelines developed by the Canadian Task Force on the Periodic Health Exam. RECOMMENDATIONS: 1. Anti-D Ig 300 microg IM or IV should be given within 72 hours of delivery to a postpartum nonsensitized Rh-negative woman delivering an Rh-positive infant. Additional anti-D Ig may be required for fetomaternal hemorrhage (FMH) greater than 15 mL of fetal red blood cells (about 30 mL of fetal blood). Alternatively, anti-D Ig 120 microg IM or IV may be given within 72 hours of delivery, with testing and additional anti-D Ig given for FMH over 6 mL of fetal red blood cells (12 mL fetal blood). (I-A) 2. If anti-D is not given within 72 hours of delivery or other potentially sensitizing event, anti-D should be given as soon as the need is recognized, for up to 28 days after delivery or other potentially sensitizing event. (III-B) 3. There is poor evidence regarding inclusion or exclusion of routine testing for postpartum FMH, as the cost-benefit of such testing in Rh mothers at risk has not been determined. (III-C) 4. Anti-D Ig 300 microg should be given routinely to all Rh-negative nonsensitized women at 28 weeks' gestation when fetal blood type is unknown or known to be Rh-positive. Alternatively, 2 doses of 100-120 microg may be given (120 microg being the lowest currently available dose in Canada): one at 28 weeks and one at 34 weeks. (I-A) 5. All pregnant women (D-negative or D-positive) should be typed and screened for alloantibodies with an indirect antiglobulin test at the first prenatal visit and again at 28 weeks. (III-C) 6. When paternity is certain, Rh testing of the baby's father may be offered to all Rh-negative pregnant women to eliminate unnecessary blood product administration. (III-C) 7. A woman with "weak D" (also known as Du-positive) should not receive anti-D. (III-D) 8. A repeat antepartum dose of Rh immune globulin is generally not required at 40 weeks, provided that the antepartum injection was given no earlier than 28 weeks' gestation. (III-C) 9. After miscarriage or threatened abortion or induced abortion during the first 12 weeks of gestation, nonsensitized D-negative women should be given a minimum anti-D of 120 microg. After 12 weeks' gestation, they should be given 300 microg. (II-3B) 10. At abortion, blood type and antibody screen should be done unless results of blood type and antibody screen during the pregnancy are available, in which case antibody screening need not be repeated. (III-B) 11. Anti-D should be given to nonsensitized D-negative women following ectopic pregnancy. A minimum of 120 microg should be given before 12 weeks' gestation and 300 microg after 12 weeks' gestation. (III-B) 12. Anti-D should be given to nonsensitized D-negative women following molar pregnancy because of the possibility of partial mole. Anti-D may be withheld if the diagnosis of complete mole is certain. (III-B) 13. At amniocentesis, anti-D 300 microg should be given to nonsensitized D-negativeesis, anti-D 300 microg should be given to nonsensitized D-negative women. (II-3B) 14. Anti-D should be given to nonsensitized D-negative women following chorionic villous sampling, at a minimum dose of 120 microg during the first 12 weeks' gestation, and at a dose of 300 microg after 12 weeks' gestation. (II-B) 15. Following cordocentesis, anti-D Ig 300 microg should be given to nonsensitized D-negative women. (II-3B) 16. Quantitative testing for FMH may be considered following events potentially associated with placental trauma and disruption of the fetomaternal interface (e.g., placental abruption, blunt trauma to the abdomen, cordocentesis, placenta previa with bleeding). There is a substantial risk of FMH over 30 mL with such events, especially with blunt trauma to the abdomen. (III-B) 17. Anti-D 120 microg or 300 microg is recommended in association with testing to quantitate FMH following conditions potentially associated with placental trauma and disruption of the fetomaternal interface (e.g., placental abruption, external cephalic version, blunt trauma to the abdomen, placenta previa with bleeding). If FMH is in excess of the amount covered by the dose given (6 mL or 15 mL fetal RBC), 10 microg additional anti-D should be given for every additional 0.5 mL fetal red blood cells. There is a risk of excess FMH, especially when there has been blunt trauma to the abdomen. (III-B) 18. Verbal or written informed consent must be obtained prior to administration of the blood product Rh immune globulin. (III-C) VALIDATION: These guidelines have been reviewed by the Maternal-Fetal Medicine Committee and the Genetics Committee, with input from the Rh Program of Nova Scotia. Final approval has been given by the Executive and Council of the Society of Obstetricians and Gynaecologists of Canada.

Canada↗