Persurf,a New Method to Improve Surfactant Delivery: A Study in Surfactant Depleted Rats

Purpose

Exogenous surfactant is not very effective in adults with ARDS, since surfactant does not reach atelectatic alveoli. Perfluorocarbons (PFC) can recruit atelectatic areas but do not replace impaired endogenous surfactant. A surfactant-PFC-mixture could combine benefits of both therapies. The aim of the proof-of-principal-study was to produce a PFC-in-surfactant emulsion (Persurf) and to test in surfactant depleted Wistar rats whether Persurf achieves I.) a more homogenous pulmonary distribution and II.) a more homogenous recruitment of alveoli when compared with surfactant or PFC alone.

Methods

Three different PFC were mixed with surfactant and phospholipid concentration in the emulsion was measured. After surfactant depletion, animals either received 30 ml/kg of PF5080, 100 mg/kg of stained (green dye) Curosurf™ or 30 ml/kg of Persurf. Lungs were fixated after 1 hour of ventilation and alveolar aeration and surfactant distribution was estimated by a stereological approach.

Results

Persurf contained 3 mg/ml phospholipids and was stable for more than 48 hours. Persurf-administration improved oxygenation. Histological evaluation revealed a more homogenous surfactant distribution and alveolar inflation when compared with surfactant treated animals.

Conclusions

In surfactant depleted rats administration of PFC-in-surfactant emulsion leads to a more homogenous distribution and aeration of the lung than surfactant alone.     

Combining the Finite Element Method with Structural Connectome-based Analysis for Modeling Neurotrauma: Connectome Neurotrauma Mechanics

This article presents the integration of brain injury biomechanics and graph theoretical analysis of neuronal connections, or connectomics, to form a neurocomputational model that captures spatiotemporal characteristics of trauma. We relate localized mechanical brain damage predicted from biofidelic finite element simulations of the human head subjected to impact with degradation in the structural connectome for a single individual. The finite element model incorporates various length scales into the full head simulations by including anisotropic constitutive laws informed by diffusion tensor imaging. Coupling between the finite element analysis and network-based tools is established through experimentally-based cellular injury thresholds for white matter regions. Once edges are degraded, graph theoretical measures are computed on the "damaged" network. For a frontal impact, the simulations predict that the temporal and occipital regions undergo the most axonal strain and strain rate at short times (less than 24 hrs), which leads to cellular death initiation, which results in damage that shows dependence on angle of impact and underlying microstructure of brain tissue. The monotonic cellular death relationships predict a spatiotemporal change of structural damage. Interestingly, at 96 hrs post-impact, computations predict no network nodes were completely disconnected from the network, despite significant damage to network edges. At early times ([Formula: see text]) network measures of global and local efficiency were degraded little; however, as time increased to 96 hrs the network properties were significantly reduced. In the future, this computational framework could help inform functional networks from physics-based structural brain biomechanics to obtain not only a biomechanics-based understanding of injury, but also neurophysiological insight.     

Characterization of neutralizing antibody responses elicited by clade A envelope immunogens derived from early transmitted viruses

The vast majority of studies with candidate immunogens based on the human immunodeficiency virus envelope (Env) have been conducted with Env proteins derived from clade B viruses isolated during chronic infection. Whether non-clade B Env protein immunogens will elicit antibodies with epitope specificities that are similar to those of antibodies elicited by clade B Envs and whether the antibodies elicited by Envs derived from early transmitted viruses will be similar to those elicited by Envs derived from viruses isolated during chronic infection are currently unknown. Here we performed immunizations with four clade A Envs, cloned directly from the peripheral blood of infected individuals during acute infection, which differed in lengths and extents of glycosylation. The antibody responses elicited by these four Envs were compared to each other and to those elicited by a well-characterized clade B Env immunogen derived from the SF162 virus, which was isolated during chronic infection. Only one clade A Env, the one with the fewer glycosylation sites, elicited homologous neutralizing antibodies (NAbs); these did not target the V1, V2, or V3 regions. In contrast, all four clade A Envs elicited anti-V3 NAbs against "easy-to-neutralize" clade B and clade A isolates, irrespective of the variable region length and extent of glycosylation of the Env used as an immunogen. These anti-V3 NAbs did not access their epitopes on homologous and heterologous clade A, or B, neutralization-resistant viruses. The length and extent of glycosylation of the variable regions on the clade A Env immunogens tested did not affect the breadth of the elicited NAbs. Our data also indicate that the development of cross-reactive NAbs against clade A viruses faces similar hurdles to the development of cross-reactive anti-clade B NAbs.     

The Duisburg birth cohort study: influence of the prenatal exposure to PCDD/Fs and dioxin-like PCBs on thyroid hormone status in newborns and neurodevelopment of infants until the age of 24 months

Prenatal exposure to polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) can affect neurobehavioral development of infants and children. This effect may be mediated through disruption of thyroid hormone homeostasis. However, epidemiological studies reveal no consistent influence of PCDD/Fs and PCBs on thyroid status and neurodevelopment at environmental background levels. The effects may resolve with time of further decreasing exposure to these compounds. The aim of this study was to find out if there are still effects related to prenatal PCDD/F and PCB observable at the meanwhile decreased levels of exposure by using the same methods which have been applied in similar studies during the last 10 years in Europe. The birth cohort study was initiated in the year 2000 in the industrialized city of Duisburg, Germany. 232 healthy mother-infant pairs were recruited between 2000 and 2002. Dioxins, dioxin-like PCBs and six indicator PCBs were analyzed in maternal blood during pregnancy and in maternal milk following extraction and sample clean-up by HRGC/HRMS. Thyroid stimulating hormone (TSH), total thyroxine (T4), total triiodothyronine (T3), free thyroxine (FT4) and free triiodothyronine (FT3) were measured in serum samples of the pregnant women and in cord serum samples by chemiluminescent immunometric assay. Neurological examinations were performed at ages 2 weeks and 18 months using the neurological optimality score (NOS), mental and motor development were assessed using the Bayley Scales of Infant Development (BSID) at ages 12 and 24 months. Multiple linear regression analysis was used to describe the association of PCDD/F and PCB in maternal blood or milk with the outcome measurements after adjustment for confounding. Blood levels (n=182) of WHO 2005 toxic equivalents (TEQ) (PCDD/F+PCB) were in the range of 3.8-58.4 pg/glipid base (median: 19.3 pg/glipid base). The corresponding data for human milk (n=149) were 2.6-52.4 pg/glipid base (median: 19.7 pg/glipid base). Multiple regression analysis showed no decrease of thyroid hormones related to WHO 2005 TEQ in blood and milk of mothers and their newborns. Furthermore, no associations between exposure and neurological and developmental measures were observed. This study supports the view that the current decreased exposure to PCDD/Fs and PCBs does not impair thyroid function of newborns and neurodevelopment of infants until the age of 24 months.     

Dynamic spatial coding within the dorsal frontoparietal network during a visual search task

To what extent are the left and right visual hemifields spatially coded in the dorsal frontoparietal attention network? In many experiments with neglect patients, the left hemisphere shows a contralateral hemifield preference, whereas the right hemisphere represents both hemifields. This pattern of spatial coding is often used to explain the right-hemispheric dominance of lesions causing hemispatial neglect. However, pathophysiological mechanisms of hemispatial neglect are controversial because recent experiments on healthy subjects produced conflicting results regarding the spatial coding of visual hemifields. We used an fMRI paradigm that allowed us to distinguish two attentional subprocesses during a visual search task. Either within the left or right hemifield subjects first attended to stationary locations (spatial orienting) and then shifted their attentional focus to search for a target line. Dynamic changes in spatial coding of the left and right hemifields were observed within subregions of the dorsal front-parietal network: During stationary spatial orienting, we found the well-known spatial pattern described above, with a bilateral hemifield representation in the right hemisphere and a contralateral preference in the left hemisphere. However, during search, the right hemisphere had a contralateral preference and the left hemisphere equally represented both hemifields. This finding leads to novel perspectives regarding models of visuospatial attention and hemispatial neglect.     

Pharmacokinetic genes do not influence response or tolerance to citalopram in the STAR*D sample

Background

We sought to determine whether clinical response or tolerance to the Selective Serotonin Reuptake Inhibitor (SSRI) citalopram is associated with genetic polymorphisms in potentially relevant pharmacokinetic enzymes.

Methodology

We used a two-stage case-control study design in which we split the sample of 1,953 subjects from the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) trial into a discovery (n = 831) and validation set (n = 1,046). Fifteen polymorphisms from five (CYP2D6, ABCB1, CYP2C19, CYP3A4, and CYP3A5) pharmacokinetic genes were genotyped. We examined the associations between these polymorphisms and citalopram response and tolerance. Significant associations were validated in the second stage for those polymorphism found to be statistically significant in the first stage.

Conclusions

No genetic polymorphism in the pharmacokinetic genes examined was significantly associated with our response or tolerance phenotypes in both stages. For managing pharmacological treatment with citalopram, routine screening of the common pharmacokinetic DNA variants that we examined appears to be of limited clinical utility.     

Vegetation control effects on untreated wood,crude cellulose and holocellulose δ<Superscript>13</Superscript>C of early and latewood in 3- to 5-year-old rings of Douglas-fir

The stable carbon (C) composition of tree rings expressed as δ¹³C, is a measure of intrinsic water-use efficiency and can indicate the occurrence of past water shortages for tree growth. We examined δ¹³C in 3- to 5-year-old rings of Douglas-fir (Pseudotsuga menziesii (Mirb) Franco) trees to elucidate if decreased water supply or uptake was a critical factor in the observed growth reduction of trees competing with understory herb and shrub vegetation compared to those growing without competition. We hypothesized that there would be no differences in δ¹³C of earlywood in trees growing in plots with competing vegetation and those in plots receiving complete vegetation control during 5 years because earlywood formed early in the growing season when soil water was ample. We also hypothesized that δ¹³C in latewood which was formed during the later half of the growing season when precipitation was low, would be greater (less negative) in trees in plots without vegetation control. We then separated early and latewood from rings for three consecutive years and analyzed their δ¹³C composition. No significant differences in earlywood δ¹³C in years 3–5 were observed for trees in the two vegetation control treatments. δ¹³C of untreated latewood separated from wood cores was greater in 4- and 5-year-old rings of trees growing with competing vegetation compared to trees growing without vegetation competition (i.e., −25.5 vs. −26.3‰ for year 4, and −26.1 vs. −26.8‰ for year 5). Results suggest that water shortages occurred in Douglas-fir trees on this coastal Washington site in the latewood-forming portion of the growing season of years 4 and 5 in the no-vegetation control treatment. We also compared δ¹³C from untreated wood, crude cellulose extracted with the Diglyme–HCl method, and holocellulose extracted with toluene–ethanol to see if the extraction method would increase the sensitivity of the analysis. δ¹³C values from the two extraction methods were highly correlated with those from untreated samples (r ² = 0.97, 0.98, respectively). Therefore, using untreated wood would be as effective as using crude cellulose or holocellulose to investigate δ¹³C patterns in young Douglas-fir.     

The <Emphasis Type="Italic">Helicobacter pylori mutY</Emphasis> homologue HP0142 is an antimutator gene that prevents specific C to A transversions

Extensive genetic variability resulting from a high mutation rate and frequent recombination is a characteristic of Helicobacter pylori. Its average mutation rate is 1 × 10⁻⁶, similar to that of Escherichia coli mutator strains. Few genes involved in DNA repair have been functionally characterized in H. pylori. In E. coli, the DNA glycosylase MutY is a part of the base excision repair system. The H. pylori mutY homolog HP0142 was analyzed in this study. HP0142 was disrupted by inserting a kanamycin resistance cassette. Mutation rates were determined by measuring the frequency of point mutations in rpoB conferring resistance against rifampicin. Inactivation of mutY in H. pylori resulted in an increase of the mutation frequency by a factor of up to 34. Sequence analysis of rpoB in rifampicin-resistant clones selected from the mutY mutant showed a modest increase of G:C/T:A transversions in comparison to clones selected from wild type strains. In contrast, inactivation of mutY had a profound impact on the distribution of mutations within rpoB. This finding suggests that the efficiency with which mutY prevents transversions is strongly dependent upon the sequence context. Inactivation of mutY was associated with a stationary phase fitness deficit in competitive cultures with the wild type strain.     

Immunolocalization of MMP-19 in the human intervertebral disc: implications for disc aging and degeneration

Matrix metalloproteinases (MMPs) degrade components of the extracellular matrix of the disc, but the presence of MMP-19 has not been explored. In other tissues, MMP-19 is known to act in proteolysis of the insulin-like growth factor (IGF) binding protein-3, thereby exposing this protein to make it available to influence cell behavior. MMP-19 also has been shown to inhibit capillary-like formation and thus play a role in the avascular nature of the disc. Using immunohistochemistry, normal discs from six subjects aged newborn through 10 years and 20 disc specimens from control donors or surgical patients aged 15-76 (mean age 40.2 years) were examined for immunolocalization of MMP-19; six Thompson grade I discs, five Thompson grade II, eight Thompson grade III, five Thompson grade IV, and one Thompson grade V discs were analyzed. The results indicate that in discs from young subjects, MMP-19 was uniformly localized in the outer annulus. In discs from adult donors and surgical patients, outer and inner annulus cells only occasionally showed MMP-19 localization. The greatest expression of MMP-19 was observed in young discs, and little expression was seen in older or degenerating discs. Because MMP-19 has been shown to regulate IGF-mediated proliferation in other tissues, its decline in the aging/degenerating disc may contribute to the age-related decrease in disc cell numbers.     

Characterization of sequence determinants within the carboxyl-terminal domain of chemokine receptor CCR5 that regulate signaling and receptor internalization.

The CC chemokine receptor CCR5 mediates chemotaxis of leukocytes and serves as a principal co-receptor for macrophage-tropic human immunodeficiency virus type 1. To identify determinants on the CCR5 carboxyl-terminal domain that regulate receptor signaling and internalization, we generated several CCR5 mutants, which were progressively shortened from the COOH terminus or had carboxyl-terminal serine, cysteine, or leucine residues substituted by alanine and expressed them in RBL-2H3 cells. Using fluorescence resonance energy transfer between beta-arrestin and CCR5 tagged with cyan and yellow variants of green fluorescent protein, we show that high affinity association of the two molecules in living cells requires intact carboxyl-terminal serine phosphorylation sites. Phosphorylation-deficient truncation or Ser/Ala replacement mutants of CCR5 mediated a sustained calcium response and enhanced granular enzyme release in RANTES-stimulated cells. Carboxyl-terminal serine residues are critically involved in CCR5 endocytosis and a dileucine motif, similar to that implicated in the regulation of CXCR2 and CXCR4, contributes to the internalization of CCR5 in a phosphorylation-independent manner. Despite their prominent role in receptor desensitization and internalization, beta-arrestins are dispensable for the CCR5-mediated stimulation of mitogen-activated protein kinase pathways in RBL-2H3 cells. We also show that CCR5 is palmitoylated on carboxyl-terminal cysteine residues. Inhibition of CCR5 palmitoylation by alanine mutagenesis of cysteines or treatment with a palmitate analogue inhibitor profoundly reduces phorbol 12-myristate 13-acetate- and RANTES-induced receptor phosphorylation, homologous desensitization, and internalization. Alanine mutagenesis of serine, cysteine, or leucine residues or the limited carboxyl-terminal truncation of CCR5 did not impair chemokine-stimulated migration of RBL-2H3 cells. Together these results indicate that post-translational modifications of carboxyl-terminal serine and cysteine residues have a significant impact on receptor deactivation and internalization.