Can folic acid protect against congenital heart defects in down syndrome?

BACKGROUND: Several studies have suggested a protective effect of folic acid (FA) on congenital heart anomalies. Down syndrome (DS) infants are known to have a high frequency of heart anomalies. Not all children with DS suffer from heart anomalies, which raises the question whether maternal factors might affect the risk of these anomalies. Our objectives were to investigate whether first-trimester FA use protects against heart anomalies among DS children. METHODS: Women with liveborn DS children participating in the Slone Epidemiology Center Birth Defects Study between 1976 and 1997 were included. We performed case-control analyses using DS, with heart anomalies as cases and DS, without heart anomalies as controls. Subanalyses were performed for defects that have been associated with FA in non-DS populations (conotruncal, ventricular septal [VSD]) and for those that are associated with DS (ostium secundum type atrial septal defects [ASD] and endocardial cushion defects [ECD]). Exposure was defined as the use of any FA-containing product for an average of at least 4 days per week during the first 12 weeks of pregnancy, whereas no exposure was defined as no use of FA in these 12 weeks. RESULTS: Of the 223 cases, 110 (49%) were exposed versus 84 (46%) of the 184 controls. After adjustment for possible confounders, no protective effect of FA was found on heart anomalies overall (OR 0.95, 95% CI: 0.61-1.47) nor separately for conotruncal defects, VSDs, ASDs, or ECDs. CONCLUSIONS: Our study does not show a protective effect of FA on heart anomalies among infants with DS.     

Dynamic remodeling of dendritic arbors in GABAergic interneurons of adult visual cortex

Despite decades of evidence for functional plasticity in the adult brain, the role of structural plasticity in its manifestation remains unclear. To examine the extent of neuronal remodeling that occurs in the brain on a day-to-day basis, we used a multiphoton-based microscopy system for chronic in vivo imaging and reconstruction of entire neurons in the superficial layers of the rodent cerebral cortex. Here we show the first unambiguous evidence (to our knowledge) of dendrite growth and remodeling in adult neurons. Over a period of months, neurons could be seen extending and retracting existing branches, and in rare cases adding new branch tips. Neurons exhibiting dynamic arbor rearrangements were GABA-positive non-pyramidal interneurons, while pyramidal cells remained stable. These results are consistent with the idea that dendritic structural remodeling is a substrate for adult plasticity and they suggest that circuit rearrangement in the adult cortex is restricted by cell type–specific rules.     

Dinosaur fossils predict body temperatures

Perhaps the greatest mystery surrounding dinosaurs concerns whether they were endotherms, ectotherms, or some unique intermediate form. Here we present a model that yields estimates of dinosaur body temperature based on ontogenetic growth trajectories obtained from fossil bones. The model predicts that dinosaur body temperatures increased with body mass from approximately 25 °C at 12 kg to approximately 41 °C at 13,000 kg. The model also successfully predicts observed increases in body temperature with body mass for extant crocodiles. These results provide direct evidence that dinosaurs were reptiles that exhibited inertial homeothermy.     

Early Selection in Gag by Protective HLA Alleles Contributes to Reduced HIV-1 Replication Capacity That May Be Largely Compensated for in Chronic Infection

Mutations that allow escape from CD8 T-cell responses are common in HIV-1 and may attenuate pathogenesis by reducing viral fitness. While this has been demonstrated for individual cases, a systematic investigation of the consequence of HLA class I-mediated selection on HIV-1 in vitro replication capacity (RC) has not been undertaken. We examined this question by generating recombinant viruses expressing plasma HIV-1 RNA-derived Gag-Protease sequences from 66 acute/early and 803 chronic untreated subtype B-infected individuals in an NL4-3 background and measuring their RCs using a green fluorescent protein (GFP) reporter CD4 T-cell assay. In acute/early infection, viruses derived from individuals expressing the protective alleles HLA-B*57, -B*5801, and/or -B*13 displayed significantly lower RCs than did viruses from individuals lacking these alleles (P < 0.05). Furthermore, acute/early RC inversely correlated with the presence of HLA-B-associated Gag polymorphisms (R = −0.27; P = 0.03), suggesting a cumulative effect of primary escape mutations on fitness during the first months of infection. At the chronic stage of infection, no strong correlations were observed between RC and protective HLA-B alleles or with the presence of HLA-B-associated polymorphisms restricted by protective alleles despite increased statistical power to detect these associations. However, RC correlated positively with the presence of known compensatory mutations in chronic viruses from B*57-expressing individuals harboring the Gag T242N mutation (n = 50; R = 0.36; P = 0.01), suggesting that the rescue of fitness defects occurred through mutations at secondary sites. Additional mutations in Gag that may modulate the impact of the T242N mutation on RC were identified. A modest inverse correlation was observed between RC and CD4 cell count in chronic infection (R = −0.17; P < 0.0001), suggesting that Gag-Protease RC could increase over the disease course. Notably, this association was stronger for individuals who expressed B*57, B*58, or B*13 (R = −0.27; P = 0.004). Taken together, these data indicate that certain protective HLA alleles contribute to early defects in HIV-1 fitness through the selection of detrimental mutations in Gag; however, these effects wane as compensatory mutations accumulate in chronic infection. The long-term control of HIV-1 in some persons who express protective alleles suggests that early fitness hits may provide lasting benefits.The host immune response elicited by CD8⁺ cytotoxic T lymphocytes (CTLs) is a major contributor to viral control following human immunodeficiency virus type 1 (HIV-1) infection (6, 39), but antiviral pressure exerted by CTLs is diminished by the selection of escape mutations in targeted regions throughout the viral proteome (7, 18, 29, 35, 41, 45, 57). A comprehensive identification of HLA-associated viral polymorphisms has recently been achieved through population-based analyses of HIV-1 sequences and HLA class I types from different cohorts worldwide (3, 8, 13-15, 34, 43, 50, 56, 63). However, despite improved characterization of the sites and pathways of immune escape, effective ways to incorporate these findings into immunogen design remain an area of debate. A better understanding of the impact of escape mutations on viral fitness may provide novel directions for HIV-1 vaccines that are designed to attenuate pathogenesis.The development of innovative vaccine strategies that can overcome the extreme diversity of HIV is a key priority (4). One proposed approach is to target the most conserved T-cell epitopes, which presumably cannot escape from CTL pressure easily due to structural or functional constraints on the viral protein (55). Complementary approaches include the design of polyvalent and/or mosaic immunogens that incorporate commonly observed viral diversity (4, 38) or the specific targeting of vulnerable regions of the viral proteome that do escape but only at a substantial cost to viral replication capacity (RC) (1, 40). A chief target of such vaccine approaches is the major HIV-1 structural protein Gag, which is known to be highly immunogenic and to elicit CTL responses that correlate with the natural control of infection (22, 36, 66). Indeed, several lines of evidence support a relationship between the selection of CTL escape mutations and reduced HIV-1 fitness. These include the reversion of escape mutations following transmission to an HLA-mismatched recipient who cannot target the epitope (19, 24, 41) as well as reduced plasma viral load (pVL) set point following the transmission of certain escape variants from donors who expressed protective HLA alleles (17, 27). Notably, these in vivo observations have been made most often for variations within Gag that are attributed to CTL responses restricted by the protective alleles HLA-B*57 and -B*5801 (17, 19, 27, 41). Most recently, reduced in vitro RCs of clinical isolates and/or engineered strains encoding single or multiple escape mutations in Gag selected in the context of certain protective HLA alleles, including B*57, B*5801, B*27, and B*13, have been demonstrated (9, 10, 42, 53, 59, 62). Despite these efforts, the goal of a T-cell vaccine that targets highly conserved and attenuation-inducing sites is hampered by a lack of knowledge concerning the contribution of most escape mutations to HIV-1 fitness as well as a poor understanding of the relative influence of HLA on the viral RC at different stages of infection.The mutability of HIV-1 permits the generation of progeny viruses encoding compensatory mutations that restore normal protein function and/or viral fitness. Detailed studies have demonstrated that the in vitro RC of escape variants in human and primate immunodeficiency viruses can be enhanced by the addition of secondary mutations outside the targeted epitope (10, 20, 52, 59, 65). Thus, vaccine strategies aimed at attenuating HIV-1 must also consider, among other factors, the frequency, time course, and extent to which compensation might overcome attenuation mediated by CTL-induced escape. Despite its anticipated utility for HIV-1 vaccine design, systematic studies to examine the consequences of naturally occurring CTL escape and compensatory mutations on viral RC have not been undertaken.We have described previously an in vitro recombinant viral assay to examine the impact of Gag-Protease mutations on HIV-1 RC (47, 49). Gag and protease have been included in each virus to minimize the impact of sequence polymorphisms at Gag cleavage sites, which coevolve with changes in protease (5, 37). Using this approach, we have demonstrated that viruses derived from HIV-1 controllers replicated significantly less well than those derived from noncontrollers and that these differences were detectable at both the acute/early (49) and chronic (47) stages. Escape mutations in Gag associated with the protective HLA-B*57 allele, as well as putative compensatory mutations outside known CTL epitopes, contributed to this difference in RC (47). However, substantial variability was observed for viruses from controllers and noncontrollers, indicating that additional factors were likely to be involved. Benefits of this assay include its relatively high-throughput capacity as well as the fact that clinically derived HIV-1 sequences are used in their entirety. Thus, it is possible to examine a large number of “real-world” Gag-Protease sequences, to define an RC value for each one, and to identify sequences within the population of recombinant strains that are responsible for RC differences.Here, we use this recombinant virus approach to examine the contribution of HLA-associated immune pressure on Gag-Protease RC during acute/early (n = 66) and chronic (n = 803) infections in the context of naturally occurring HIV-1 subtype B isolates from untreated individuals. In a recent report (64), we employed this system to examine the Gag-Protease RC in a similar cohort of chronic HIV-1 subtype C-infected individuals. The results of these studies provide important insights into the roles of immune pressure and fitness constraints on HIV-1 evolution that may contribute to the rational design of an effective vaccine.     

An Acid-loading Chloride Transport Pathway in the Intraerythrocytic Malaria Parasite,Plasmodium falciparum

The intraerythrocytic malaria parasite exerts tight control over its ionic composition. In this study, a combination of fluorescent ion indicators and ³⁶Cl⁻ flux measurements was used to investigate the transport of Cl⁻ and the Cl⁻-dependent transport of “H⁺-equivalents” in mature (trophozoite stage) parasites, isolated from their host erythrocytes. Removal of extracellular Cl⁻, resulting in an outward [Cl⁻] gradient, gave rise to a cytosolic alkalinization (i.e. a net efflux of H⁺-equivalents). This was reversed on restoration of extracellular Cl⁻. The flux of H⁺-equivalents was inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and, when measured in ATP-depleted parasites, showed a pronounced dependence on the pH of the parasite cytosol; the flux was low at cytosolic pH values < 7.2 but increased steeply with cytosolic pH at values > 7.2. ³⁶Cl⁻ influx measurements revealed the presence of a Cl⁻ uptake mechanism with characteristics similar to those of the Cl⁻-dependent H⁺-equivalent flux. The intracellular concentration of Cl⁻ in the parasite was estimated to be ∼48 mm in situ. The data are consistent with the intraerythrocytic parasite having in its plasma membrane a 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid-sensitive transporter that, under physiological conditions, imports Cl⁻ together with H⁺-equivalents, resulting in an intracellular Cl⁻ concentration well above that which would occur if Cl⁻ ions were distributed passively in accordance with the parasite''s large, inwardly negative membrane potential.     

Heart-specific Deletion of CnB1 Reveals Multiple Mechanisms Whereby Calcineurin Regulates Cardiac Growth and Function

Calcineurin is a protein phosphatase that is uniquely regulated by sustained increases in intracellular Ca²⁺ following signal transduction events. Calcineurin controls cellular proliferation, differentiation, apoptosis, and inducible gene expression following stress and neuroendocrine stimulation. In the adult heart, calcineurin regulates hypertrophic growth of cardiomyocytes in response to pathologic insults that are associated with altered Ca²⁺ handling. Here we determined that calcineurin signaling is directly linked to the proper control of cardiac contractility, rhythm, and the expression of Ca²⁺-handling genes in the heart. Our approach involved a cardiomyocyte-specific deletion using a CnB1-LoxP-targeted allele in mice and three different cardiac-expressing Cre alleles/transgenes. Deletion of calcineurin with the Nkx2.5-Cre knock-in allele resulted in lethality at 1 day after birth due to altered right ventricular morphogenesis, reduced ventricular trabeculation, septal defects, and valvular overgrowth. Slightly later deletion of calcineurin with the α-myosin heavy chain Cre transgene resulted in lethality in early mid adulthood that was characterized by substantial reductions in cardiac contractility, severe arrhythmia, and reduced myocyte content in the heart. Young calcineurin heart-deleted mice died suddenly after pressure overload stimulation or neuroendocrine agonist infusion, and telemetric monitoring of older mice showed arrhythmia leading to sudden death. Mechanistically, loss of calcineurin reduced expression of key Ca²⁺-handling genes that likely lead to arrhythmia and reduced contractility. Loss of calcineurin also directly impacted cellular proliferation in the postnatal developing heart. These results reveal multiple mechanisms whereby calcineurin regulates cardiac development and myocyte contractility.     

HTII-280, a Biomarker Specific to the Apical Plasma Membrane of Human Lung Alveolar Type II Cells

The pulmonary alveolar epithelium is composed of two morphologically distinct cell types, type I (TI) and type II (TII) cells. Alveolar TII cells synthesize, secrete, and recycle surfactant components; contain ion transporters; and secrete immune effector molecules. In response to alveolar injury, TII cells have the capacity to act as progenitor cells, proliferating and transdifferentiating into TI cells. Although various proteins are associated with TII cells, a plasma membrane marker specific to human TII cells that would be useful for identification in tissue and for isolating this cell type has not been described previously. We devised a strategy to produce a monoclonal antibody (MAb) specific to the apical surface of human TII cells and developed an MAb that appears to be specific for human TII cells. The antibody recognizes a 280- to 300-kDa protein, HTII-280, which has the biochemical characteristics of an integral membrane protein. HTII-280 is detected by week 11 of gestation and is developmentally regulated. HTII-280 is useful for isolating human TII cells with purities and viabilities >95%. HTII-280 is likely to be a useful morphological and biochemical marker of human TII cells that may help to advance our understanding of various lung pathological conditions, including the origin and development of various lung tumors. (J Histochem Cytochem 58:891–901, 2010)     

Deletion of ripA alleviates suppression of the inflammasome and MAPK by Francisella tularensis

Francisella tularensis is a facultative intracellular pathogen and potential biothreat agent. Evasion of the immune response contributes to the extraordinary virulence of this organism although the mechanism is unclear. Whereas wild-type strains induced low levels of cytokines, an F. tularensis ripA deletion mutant (LVSΔripA) provoked significant release of IL-1β, IL-18, and TNF-α by resting macrophages. IL-1β and IL-18 secretion was dependent on inflammasome components pyrin-caspase recruitment domain/apoptotic speck-containing protein with a caspase recruitment domain and caspase-1, and the TLR/IL-1R signaling molecule MyD88 was required for inflammatory cytokine synthesis. Complementation of LVSΔripA with a plasmid encoding ripA restored immune evasion. Similar findings were observed in a human monocytic line. The presence of ripA nearly eliminated activation of MAPKs including ERK1/2, JNK, and p38, and pharmacologic inhibitors of these three MAPKs reduced cytokine induction by LVSΔripA. Animals infected with LVSΔripA mounted a stronger IL-1β and TNF-α response than that of mice infected with wild-type live vaccine strain. This analysis revealed novel immune evasive mechanisms of F. tularensis.