Identification of preferential CD4+ T-cell targets for HIV infection in the cervix.

A better understanding of the cellular targets of HIV infection in the female genital tract may inform HIV prevention efforts. Proposed correlates of cellular susceptibility include the HIV co-receptor CCR5, peripheral homing integrins, and immune activation. We used a CCR5-tropic pseudovirus to quantify HIV entry into unstimulated endocervical CD4(+) T cells collected by cytobrush. Virus entry was threefold higher into cervix-derived CD4(+) T cells than blood, but was strongly correlated between these two compartments. Cervix-derived CD4(+) T cells expressing CD69, α(4)ß(7), or α(4)ß(1) were preferential HIV targets; this enhanced susceptibility was strongly correlated with increased CCR5 expression in α(4)ß(7)(+) and CD69(+) CD4(+) T cells, and to a lesser extent in α(4)ß(1)(+) CD4(+) T cells. Direct binding of gp140 to integrins was not observed, integrin inhibitors had no effect on virus entry, and pseudotypes with an env that preferentially binds α(4)ß(7) still demonstrated enhanced entry into α(4)ß(1)(+) cells. In summary, a rapid and sensitive HIV entry assay demonstrated enhanced susceptibility of activated endocervical CD4(+) T cells, and those expressing α(4)ß(7) or α(4)ß(1). This may relate to increased CCR5 expression by these cell subsets, but did not appear to be due to direct interaction of α(4)ß(7) or α(4)ß(1) with HIV envelope.

Disentangling trophic relationships in a High Arctic tundra ecosystem through food web modeling.

Determining the manner in which food webs will respond to environmental changes is difficult because the relative importance of top-down vs. bottom-up forces in controlling ecosystems is still debated. This is especially true in the Arctic tundra where, despite relatively simple food webs, it is still unclear which forces dominate in this ecosystem. Our primary goal was to assess the extent to which a tundra food web was dominated by plant-herbivore or predator-prey interactions. Based on a 17-year (1993-2009) study of terrestrial wildlife on Bylot Island, Nunavut, Canada, we developed trophic mass balance models to address this question. Snow Geese were the dominant herbivores in this ecosystem, followed by two sympatric lemming species (brown and collared lemmings). Arctic foxes, weasels, and several species of birds of prey were the dominant predators. Results of our trophic models encompassing 19 functional groups showed that <10% of the annual primary production was consumed by herbivores in most years despite the presence of a large Snow Goose colony, but that 20-100% of the annual herbivore production was consumed by predators. The impact of herbivores on vegetation has also weakened over time, probably due to an increase in primary production. The impact of predators was highest on lemmings, intermediate on passerines, and lowest on geese and shorebirds, but it varied with lemming abundance. Predation of collared lemmings exceeded production in most years and may explain why this species remained at low density. In contrast, the predation rate on brown lemmings varied with prey density and may have contributed to the high-amplitude, periodic fluctuations in the abundance of this species. Our analysis provided little evidence that herbivores are limited by primary production on Bylot Island. In contrast, we measured strong predator-prey interactions, which supports the hypothesis that this food web is primarily controlled by top-down forces. The presence of allochthonous resources subsidizing top predators and the absence of large herbivores may partly explain the predominant role of predation in this low-productivity ecosystem.

Lower predation risk for migratory birds at high latitudes.

Quantifying the costs and benefits of migration distance is critical to understanding the evolution of long-distance migration. In migratory birds, life history theory predicts that the potential survival costs of migrating longer distances should be balanced by benefits to lifetime reproductive success, yet quantification of these reproductive benefits in a controlled manner along a large geographical gradient is challenging. We measured a controlled effect of predation risk along a 3350-kilometer south-north gradient in the Arctic and found that nest predation risk declined more than twofold along the latitudinal gradient. These results provide evidence that birds migrating farther north may acquire reproductive benefits in the form of lower nest predation risk.

Identification of a novel HLA-DQA1 null allele, DQA1*0403N, from an East African woman.

We report a novel DQA1 allele (DQA1*0403N) identified during sequence-based HLA-DQA1 typing of a Kenyan population. The new allele is identical to DQA1*0401 at exon 2 except for a single-nucleotide substitution at codon 53, changing it from lysine to a stop codon (CAA-->TAA). The substitution at codon 53 was confirmed by sequencing two separate polymerase chain reaction products and by sequencing multiple clones obtained following TOPO-TA cloning. The resulting stop codon at position of codon 53 in exon 2 is predicted to produce a non-functional DQA1 alpha-chain. The new allele has been named by the WHO nomenclature committee as DQA1*0403N. This is the first report of a null allele detected in the DQA1 gene.

Photoprotection, not increased growth, characterizes the response of Engelmann spruce (Picea engelmannii) seedlings to high light, even when resources are plentiful.

• To investigate the effects of resource limitation on the use of light by Engelmann spruce seedlings (Picea engelmannii), we examined the effects of nitrogen (N) supply on growth and physiological acclimation. • Seedlings were grown under a factorial combination of two levels of light (100%, 33% full light) and two levels of N-supply (100 mg l-1 and 10 mg l-1 ). Biomass, foliage physiology, and pigment composition were measured. • No significant differences were found in growth or photosynthetic capacity between seedlings grown under high and low light, regardless of whether seedlings were grown under conditions of high or low N-supply. Both a decrease in the capacity for light capture and an increase in the capacity for thermal dissipation of excess absorbed light occurred with growth at high relative to low light as well as at low relative to high N-supply. • Damage to foliage from excess light appeared to be avoided through a combination of downward adjustments in chlorophyll and upward adjustments in photoprotective xanthophyll cycle carotenoids.

Longitudinal outcome of verbal discourse in children with traumatic brain injury: three-year follow-up.

PURPOSE: This study compared changes in discourse ability between two groups of children age 5 to 10 years after brain injury: those with severe traumatic brain injury (TBI) and those with mild/moderate injury over 3-year follow-up testing. MATERIALS AND METHODS: Forty-three children with TBI were recruited from a larger research project examining cognitive and linguistic recovery after injury. Twenty-two of these patients had severe injuries and 21 sustained mild/moderate injuries. All children were presented an ordered sequence of pictures and asked to verbally produce a story/narrative discourse. Each child was then asked to produce a lesson relating to the story. RESULTS: The severe group performed significantly worse than the mild/moderate group when performance across all four discourse domains was considered. Both groups improved across time on selected discourse measures. Qualitative analysis suggested that the severe group showed differential rates of improvement across the individual discourse variables over the 3-year interval. CONCLUSIONS: Severe TBI can have a pernicious effect on discourse abilities in children years after injury compared with children with mild/moderate injuries. The major caveat is that the discourse measures must be sufficiently challenging when used to assess older children and children with milder forms of TBI.

Developmental expression of muscarinic acetylcholine receptors in chick retina: selective induction of M2 muscarinic receptor expression in ovo by a factor secreted by muller glial cells.

Muscarinic acetylcholine receptors (mAChRs) play an important role in signal processing in the retina. We have used subtype-specific antibodies to identify the changes in the localization of mAChR expression during embryonic development of the retina in vivo and their relationship to the changes in mAChRs in retinal cells in culture. We have demonstrated previously that treatment of fresh retinal cultures with conditioned media from mature retinal cultures specifically induces expression of the M(2) mAChR (McKinnon et al., 1998). We show that the M(2)-inducing activity, which we tentatively have called MARIA (muscarinic acetylcholine receptor-inducing activity) is produced by Müller glial cells in culture, because significant activity can be found in media conditioned by essentially neuron-free cultures of Müller glia, as well as by a Müller glial cell line but not several neuroblastoma cell lines. We also demonstrate that the appearance of the M(2) receptor in vivo occurs concomitantly with the appearance of significant numbers of Müller glial cells in the developing retina. Furthermore, the administration of crude or partially purified preparations of MARIA to developing chick embryos in ovo induces precocious expression of M(2) mAChRs in the appropriate cell types in the retina. These results show that a factor secreted by cultured retinal Müller glia can regulate M(2) mAChR expression in vivo and in vitro and suggest that the secretion of MARIA by Müller glia in vivo may be responsible for the normal induction of M(2) mAChR expression during embryonic development.

Discussion of developmental plasticity: factors affecting cognitive outcome after pediatric traumatic brain injury.

Current research on plasticity has altered the over simplistic view of greater capacity in the developing brain after injury. In another paper in this issue, Dennis provides a model to elucidate the complexity of the multiple factors that influence recovery after brain injury in children. The authors present a brief summary of findings from their longitudinal research in neurobehavioral recovery after traumatic brain injury in children and adolescents that elaborates on the framework of Dennis. The discussion highlights the psychobiological factors that interact to define developmental plasticity and outlines promising directions for future research to elucidate and promote long-term recovery in pediatric brain-injured populations.

Molecular analysis of the regulation of muscarinic receptor expression and function.

We have investigated the molecular mechanisms involved in the regulation of muscarinic acetylcholine receptor gene expression and localization and generated knockout mice to study the role of the M1 muscarinic receptor in vivo. We have used the MDCK cell system to demonstrate that different subtypes of mAChR can be targeted to different regions of polarized cells. We have also examined the developmental regulation of mAChR expression in the chick retina. Early in development, the M4 receptor is the predominant mAChR while the levels of the M2 and M3 receptors increase later in development. The level of M2 receptor is also initially very low in retinal cultures and undergoes a dramatic increase over several days in vitro. The level of M2 receptor can be increased by a potentially novel, developmentally regulated, secreted factor produced by retinal cells. The promoter for the chick M2 receptor gene has been isolated and shown to contain a site for GATA-family transcription factors which is required for high level cardiac expression. The M2 promoter also contains sites which mediate induction of transcription in neural cells by neurally active cytokines. We have generated knockout mice lacking the M1 receptor and shown that these mice do not exhibit pilocarpine-induced seizures and muscarinic agonist-induced suppression of the M-current potassium channel in sympathetic neurons.

Molecular mechanisms for the regulation of the expression and function of muscarinic acetylcholine receptors.

The regulation of muscarinic acetylcholine receptor expression and function was investigated in cultured cells and in knockout mice. Muscarinic agonist exposure causes m2 receptor desensitization and sequestration and decreases the expression of cardiac potassium channels. The expression of m2 receptors in chick retina is regulated by a developmentally regulated secreted factor. Mice lacking the m1 receptor exhibit a loss of muscarinic regulation of M-current potassium channel activity and pilocarpine-induced seizures.

Identification and localization of muscarinic acetylcholine receptors in the ocular tissues of the chick.

The purpose of this study was to characterize the distribution of muscarinic acetylcholine receptors (mAChRs) in the ocular tissues of hatched chicks. In the chick, different isoforms of these receptors have been detected in the brain, heart, and retina, and mAChRs in ocular tissues have been implicated in the pathogenesis of form-deprivation myopia. However, the precise anatomical distribution of mAChRs within the retina, retinal pigment epithelium, choroid, ciliary body, and ciliary ganglion remains unknown. We used affinity-purified, type-specific antibodies directed to three different chick mAChR subtypes (cm2, cm3, and cm4) to detect receptor immunoreactivity in sections and extracts of these ocular tissues. We found cm2, cm3, and cm4 in the retina, retinal pigment epithelium, choroid, and ciliary body. Within the retina, cm2 was expressed in numerous amacrine and ganglion cells; cm3 was expressed in many bipolar cells and small subsets of amacrine cells; and cm4 was found in most, if not all, amacrine and ganglion cells. Each mAChR was localized to distinct strata within the inner plexiform layer that cumulatively form three broad bands that closely match previously described localizations of subtype-nonspecific muscarinic ligand binding. Only cm3 was detected in the outer plexiform layer, and only cm4 was detected in the ciliary ganglion. We propose that each mAChR subtype has unique functions in each ocular tissue.

Developmental regulation of the cm2 muscarinic acetylcholine receptor gene: selective induction by a secreted factor produced by embryonic chick retinal cells.

The expression of the cm2 muscarinic acetylcholine receptor gene increases dramatically in chick retina during embryonic development in vivo. A similar developmental increase in cm2 expression occurs in embryonic chick retinal cells in culture. Conditioned medium from mature, but not young, retinal cultures contains a secreted factor that causes a selective increase in expression of cm2, but not cm3 or cm4, receptors. The secreted factor has been partially purified from serum-free medium, is protease-sensitive, and has a molecular weight >10 kDa. The cm2-inducing factor stimulates expression of a cm2 promoter/luciferase reporter gene, demonstrating that the increase in cm2 expression is attributable to increased gene transcription. Incubation of retinal cells with 14 identified neurotrophic and growth factors did not increase cm2 expression, suggesting that a novel developmentally regulated secreted factor mediates the subtype-specific induction of the cm2 receptor gene in retina.

Heterophilic antibodies remain a problem for the immunoassay laboratory.

The prevalence of heterophilic antibody interference in a modern immunochemiluminometric assay containing blocking agents was determined using thyrotropin as an illustrative example. Serum samples were obtained from 295 consecutive patients who underwent routine thyroid function testing. The following versions of the thyrotropin assay were used: protocol A (zero blocker), protocol B (routine blocker concentration), and protocol C (extra blocker). Ten patients (prevalence 3.4%) had significant levels of heterophilic antibodies (protocol A value greater than 9 SD from the protocol B value). The observed thyrotropin levels for protocols B and C were the same for all patients, consistent with the reagent blockers in routine assays adequately eliminating heterophilic antibody interference. However, seven more patients (0.03%) in series of 21,000 assessed by routine thyroid function testing had discordant results because of a concentration of heterophilic antibodies so high as to overwhelm the added blocking agents.

Regulation of muscarinic receptor expression and function in cultured cells and in knock-out mice.

We have investigated the molecular and cellular basis for the regulation of expression and function of the muscarinic acetylcholine receptors. Treatment of cultured chick cardiac cells with the agonist carbachol results in decreased levels of mRNA encoding the m2 and m4 receptors. Treatment of chick embryos in ovo with carbachol results in decreased levels of mRNA encoding the potassium channel subunits GIRK1 and GIRK4 as well as the m2 receptor. There are thus multiple pathways for the regulation of mAChR responsiveness by long-term agonist exposure. Immunoblot, immunoprecipitation, and solution hybridization analyses have been used to quantitate the regulation of mAChR expression in chick retina during embryonic development. The m4 receptor is the predominant subtype expressed early in development, while the expression of the m3 and m2 receptors increases later in development. A cAMP-regulated luciferase reporter gene has been used to demonstrate that the m2 and m4 receptors have distinct specificities for coupling to G-protein subtypes to mediate inhibition of adenylyl cyclase. This system has also been used to demonstrate that beta-arrestin1 and beta-adrenergic receptor kinase-1 act synergistically to promote receptor desensitization. We have isolated the promoter region for the chick m2 receptor gene, identified regions of the promoter required to drive high level expression in cardiac and neural cells, and have identified a region which confers sensitivity of gene expression to neurally active cytokines. Finally, in order to determine the role of individual receptor subtypes in muscarinic-mediated responses in vivo, we have used the method of targeted gene disruption by homologous recombination to generate mice deficient in the m1 receptor.

Tissue-specific regulation of muscarinic acetylcholine receptor expression during embryonic development.

We used solution hybridization, immunoprecipitation, and immunoblot analyses to examine the developmental expression of chicken m2 (cm2), cm3, and cm4 muscarinic acetylcholine receptor (mAChR) mRNA and protein in embryonic and post-hatched chick heart and retina in order to correlate developmental expression patterns with known physiological events. cm2 is the predominant mAChR subtype expressed in chick heart. cm3 and cm4 protein and mRNA expression is very low in chick heart, and cm3 expression is highest early in development. The decrease in cm3 expression correlates well with the developmental decrease in mAChR-mediated activation of phospholipase C. cm4 is the predominant mAChR subtype expressed in chick retina. The expression of both cm4 protein and mRNA is highest early in development and decreases as development progresses. cm2 and cm3 mAChR are expressed at approximately equivalent levels and have similar patterns of expression. The cm2 and cm3 protein levels increase throughout development, while cm2 and cm3 mRNA levels peak at embryonic day 15 and then decrease after hatching. Our data indicate that the three mAChR subtypes are differentially regulated in chick heart and retina and that the patterns of expression of mAChR may be important in the development and physiology of these tissues.

Molecular analysis of the regulation of muscarinic receptor expression and function.

Several systems are being used to determine the molecular and cellular basis for the regulation of expression and function of the muscarinic receptors. Treatment of chick heart cells in culture results in decreased levels of mRNA encoding the cm2 and cm4 receptors. This probably results from decreased gene transcription which requires concomitant mAChR-mediated inhibition of adenylyl cyclase and mAChR-mediated stimulation of phospholipase C. Site-directed mutagenesis was used to demonstrate that the single tyrosine residue in the carboxyl-terminal cytoplasmic tail of the m2 receptor is involved in agonist-induced down-regulation but not sequestration. Activation of heterologous receptors in chick heart cells can also regulate mAChR mRNA levels. A cAMP-regulated luciferase reporter gene, has been used to demonstrate that the m4 receptor preferentially couples to Gi alpha-2 or Go alpha over Gi alpha-1 or Gi alpha-3 to mediate inhibition of adenylyl cyclase activity. Finally, in order to determine the role of individual receptor subtypes in muscarinic-mediated responses in vivo, we are beginning to use the method of targeted gene disruption by homologous recombination to generate mice deficient in specific receptor subtypes.

Prediction and prevention of anastomotic complications of esophageal atresia and tracheoesophageal fistula.

We analyzed our experience with 64 infants with esophageal atresia (EA) and tracheoesophageal fistula (TEF), to determine the possibility of prediction and prevention of anastomotic complications (leak, stricture, and recurrent TEF). In most of the infants, the anatomical level of the fistula was documented preoperatively by bronchoscopy. The level of the fistula, in turn, correlated with the esophageal anatomy at thoracotomy, ie, carinal fistulas had a wide gap between esophageal pouches, whereas midtracheal or cervical fistulas had a minimal gap. Major anastomotic complications were defined as leak requiring reoperation, symptomatic strictures requiring four or more dilatations, or a recurrent TEF. The complication rates wre: leak (major and minor), 21%; major stricture, 15%; and recurrent TEF, 5%. Major complications occurred in 42% (11/26) of infants with wide gaps, compared with 8% (3/36) of infants with minimal gaps. Route of repair (transpleural or retropleural) made no difference in incidence of anastomotic complications. No infant died of an anastomotic complication. Survival was 100% for Waterston A and B infants, 83% for Waterston C, and 90% overall. Severe gastroesophageal reflux, requiring Nissen fundoplication, was more common among infants with wide gaps than those with minimal gaps (32% v 3%). The most important pathogenetic factor, present in 79% (11/14) of major anastomotic complications, was anastomotic tension, determined by the gap between esophageal pouches, and predicted by preoperative bronchoscopy. Thus the bronchoscopic finding of a carinal fistula signals the need for technical measures that may limit anastomotic morbidity, such as myotomy, patching the anastomosis, retropleural approach, or delayed repair. Assuming precise technique and gentle handling of tissues, the anatomy of the anomaly determines the anastomotic morbidity of EA and TEF.

Equipment maintenance.

Basic to the success of the perinatal intensive care unit is a cooperative liasion between the medical and nursing staff and the clinical engineering staff. To this end the functions of the clinical engineering staff are preventive maintenance, inventory documentation, pre-purchase evaluation, emergency repair, personnel education, and management integration.