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Background The majority of pediatric human immunodeficiency virus (HIV) cases in Africa reflect maternal-to-child transmission. HIV exposed but uninfected (HEU) children have increased rates of morbidity and mortality when compared to HIV unexposed and uninfected (HUU) children. The mechanisms behind these unexpected trends are only partially understood but could be explained by the differences in the immune response to infections triggered by an altered immune system state. Methods Using quantitative reverse transcription polymerase chain reaction, we compared the nasopharyngeal carriage prevalence and density of Streptococcus pneumoniae (SP) and Pneumocystis jirovecii (PJ) between children living with HIV and HEU or HUU cases (pneumonia) and controls (without pneumonia). Results The cohort included 1154 children (555 cases and 599 matched controls). The SP carriage prevalence rates were similar between cases and controls. Among SP carriers with pneumonia, carriage density was increased among children living with HIV, versus HEU or HUU children (15.8, 4.7, and 3.6 × 105 copies/mL, respectively). The rate of PJ carriage was significantly higher among children living with HIV than among HEU and HUU children (31%, 15%, and 10%, respectively; P Conclusions Carriage prevalences and densities for SP and PJ show different kinetics in terms of their relationship with HIV exposure and clinical status, particularly for Pneumocystis jirovecii. This supports the theory that the increased morbidity and mortality observed among HEU children may reflect deficits not just in humoral immunity but in cell-mediated immunity as well.

Author(s): Weinstein, John R; Diaz-Artiga, Anaite; Benowitz, Neal; Thompson, Lisa M | Abstract: BackgroundHousehold air pollution from solid fuels is a leading risk factor for morbidity and mortality worldwide. Pregnant women's exposure to polycyclic aromatic hydrocarbons (PAHs) and volatile organic compounds (VOCs), two components of solid-fuel smoke, is associated with adverse birth outcomes. Even with improved solid-fuel stoves, exposure to PAHs and VOCs remains high. Therefore, cleaner cooking fuels need to be prioritized.ObjectiveWe aimed to quantify exposure reduction to PAHs and VOCs among pregnant women in rural Guatemala with a liquefied petroleum gas (LPG) stove intervention.Materials and methodsUrine from pregnant women (N = 50) was collected twice: at l20 weeks gestation, when women cooked exclusively with wood, and 6-8 weeks after receiving an LPG stove. Metabolites of four PAHs and eight VOCs were analyzed. Concurrent with urine collection, personal 48-h PM2.5 exposure was measured.ResultsWomen cooking exclusively with wood were exposed to high levels of particulate matter (PM2.5), which was reduced by 57% with the LPG stove. Urinary concentrations of total PAH metabolites (-37%), PMA (benzene metabolite; -49%), and CNEMA (acrylonitrile metabolite; -51%) were reduced. However, recent use of a wood-fired sauna bath led to large increases in excretion of urinary toxicant metabolites (+66-135%).ConclusionsThis is the first study to report PAH and VOC reductions from an LPG stove intervention introduced during pregnancy. However, other sources of air pollution minimized the gains seen from using an LPG stove. Thus, all sources of air pollution must be addressed in concert to reduce exposures to levels that protect health.

Background Most pediatric HIV cases in Africa reflect maternal to child transmission (MTCT). The persistently high seroprevalence of HIV in pregnant women (16.6%) and the low rates of mother to child HIV transmission (~2%) have resulted in a high number of children who are HIV exposed but uninfected (HEU). HIV exposed but uninfected (HEU) children have increased rates of morbidity and mortality vs. HIV unexposed and uninfected (HUU). Methods To explore the mechanisms behind this phenomenon, each case and control was sampled using a nasopharyngeal NP swab and an oropharyngeal (OP). A multiplex real-time polymerase chain reaction (PCR) assay was used to determine the presence of Streptococcus pneumoniae and Pneumocystis jirovecii in the NP/OP specimens. We compared nasopharyngeal carriage rates of S. pneumoniae and P. jirovecii between HIV-infected, HEU, or HUU case (pneumonia) and control (without pneumonia) children. In bivariate analyses, using carriage as a dichotomous outcome, proportions we compared using chi-square or odds ratios with 95% confidence intervals. In multivariate models, we created logistic and linear regression models adjusting for nutritional status as possible mediators of carriage status. Results SP carriage rates were similar between cases and controls. However, density was increased among HIV-infected children vs. HEU or HUU children (15.8 vs. 4.7 vs. 3.6 × 105 copies/mL, respectively). Among cases, PJ carriage among HIV positive, HEU and HUU children was 31%, 15% and 10%,, respectively, (P < 0.05) and carriage density was 63.9, 20.9, and 4.8 × 103 copies/mL, respectively (P < 0.05). In adjusted logistic regression models, HIV-infected cases were slightly more likely to be an S. pneumoniae carrier compared with HUU cases (aOR = 1.1; 95% CI: 0.57–2.14). In contrast, all other case/exposure combinations were less likely to be S. pneumoniae carriers with the lowest adjusted odds among HIV-infected controls children (aOR = 0.34; 95% CI: 0.17–0.71). The odds of being a PJ carrier was almost 6 times higher in HIV-infected cases than in HIV unexposed cases (aOR = 6.63; 95% CI: 3.24–13.54). Conclusions We demonstrate that HIV infection and HIV exposure without infection each have an impact on carriage rates and density for S. pneumoniae and P. jirovecii, though the magnitude and nature of these effects differs substantially between the two pathogens. This may be explained in part by differences in immune responses to these two different pathogens. Our analysis provides further evidence of fundamental differences in immune function among HIV exposed but uninfected infants compared with HIV unexposed, uninfected infants.

Background: Exposure to ambient and household air pollution is the fourth and fifth leading cause of death worldwide and in Guatemala, respectively. In urban Guatemala, over half of households use ...

Household air pollution (HAP) is the sixth leading risk factor for premature mortality in Guatemala. Households in Guatemala are gradually adopting liquefied petroleum gas (LPG) stoves, but a strong tradition of woodstove use persists. We conducted a mixed-methods study of LPG stove use in peri-urban Guatemala. We used Knowledge, Attitudes and Practices surveys with 187 LPG stove owners who also used woodstoves to identify perceptions of stove and cooking practices. Barriers to sustained use of LPG stoves were evaluated through focus groups, participant observations with stove users, and key informant interviews with community leaders. Seven themes emerged that explain household decisions to use LPG stoves: (1) the “new technology” should be framed in terms of what the “old technology” lacks, (2) income is not a predictor of gas stove acquisition but may predict sustained use, (3) men are key decision-makers but messages about LPG do not target them, (4) when stoves are viewed as “prize possessions” they may not be used, (5) collective fear about gas stoves is not based on personal experience, but on “stories we hear”, (6) sustained LPG use is hampered by two major factors, seasonally available wood and LPG retailers who are perceived as dishonest, and (7) wood fuel collection is a time to enjoy the company of friends and family and is not “drudgery”. National policies should promote the use of clean cookstove technologies in peri-urban and rapidly urbanizing areas in Guatemala where LPG stoves are in use, but used intermittently, instead of the current plan to install 100,000 “improved” woodstoves by 2032. This could be done by improving dependable cylinder distribution services, targeting gas safety and promoting positive health messages that appeal to men, as well as women.

BackgroundThree billion people use solid cooking fuels, and 4 million people die from household air pollution annually. Shifting households to clean fuels, like liquefied petroleum gas (LPG), may protect health only if stoves are consistently used. Few studies have used an implementation science framework to systematically assess "de-implementation" of traditional stoves, and none have done so with pregnant women who are more likely to adopt new behaviors. We evaluated an introduced LPG stove coupled with a phased behavioral intervention to encourage exclusive gas stove use among pregnant women in rural Guatemala.MethodsWe enrolled 50 women at

Background/Aim: Household air pollution is the 8th leading cause of death and disability in Guatemala where over 95% of rural households use woodstoves for cooking. Even the best performing biomass...

Background Preterm birth is the leading cause of death among children

Background Low rates of mother to child HIV transmission in Zambia, translates into a high number of children who are HIV exposed but uninfected (HEU) who have increased mortality and morbidity when compared with children HIV unexposed and uninfected (HUU). We performed a secondary analysis on The Pneumonia Etiology Research in Child Health (PERCH), a case–control study focused on identifying the etiologies of pediatric pneumonia including two pathogens, Streptococcus pneumoniae and Pneumocystis jirovecii in Zambian children to evaluate if HIV exposure status influences carriage rates and density for these pathogens. Methods Children ages 1–59 months were enrolled as cases if they met the World Health Organization (WHO) definition of severe or very severe pneumonia. Controls did not have a diagnosis of pneumonia and were matched by age and HIV status to cases. Each case and control had a nasopharyngeal (NP) swab and an oropharyngeal (OP) swab specimen. A multiplex real-time polymerase chain reaction (PCR) assay was used to test the NP/OP specimens for S. pneumoniae and P. jirovecii. A density of log10 copies/mL in microbiology confirmed cases compared with controls was used to define positive infection with S. pneumoniae and P. jirovecii. Results The highest S. pneumoniae carrier rates were seen in HIV unexposed controls and the lowest carrier rates seen in HIV-infected controls. HIV-infected children who were S. pneumoniae carriers and were classified as controls had the highest S. pneumoniae density of all groups. Overall, the HIV-infected group had the highest S. pneumoniae density rates. There was minimal variation in the S. pneumoniae density of those in the HIV exposed and HIV unexposed. P. jirovecii was present only in 31% of HIV-infected cases and 7% of the same group controls. HIV exposed cases had half the carrier rates of their counterparts in the HIV-infected group, but the P. jirovecii carriage rates were the same as the carriage rates in HIV-infected controls. The P. jirovecii carriage density in HIV-infected and HIV-exposed cases was similar. Conclusion HIV exposure status in children can be a predictor factor in S. pneumoniae and P. jirovecii carriage and density. The results of our analysis could potentially explain the high rates of pneumonia in children exposed to HIV but uninfected. Our findings open the door to more in-depth studies about the immunological status in children exposed to HIV but uninfected. Disclosures All authors: No reported disclosures.

© 2017 Elsevier GmbH Background Household air pollution is a major contributor to death and disability worldwide. Over 95% of rural Guatemalan households use woodstoves for cooking or heating. Woodsmoke contains carcinogenic or fetotoxic polycyclic aromatic hydrocarbons (PAHs) and volatile organic compounds (VOCs). Increased PAHs and VOCs have been shown to increase levels of oxidative stress. Objective We examined PAH and VOC exposures among recently pregnant rural Guatemalan women exposed to woodsmoke and compared exposures to levels seen occupationally or among smokers. Methods Urine was collected from 23 women who were 3 months post-partum three times over 72 h: morning (fasting), after lunch, and following dinner or use of wood-fired traditional sauna baths (samples = 68). Creatinine-adjusted urinary concentrations of metabolites of four PAHs and eight VOCs were analyzed by liquid chromatography–mass spectrometry. Creatinine-adjusted urinary biomarkers of oxidative stress, 8-isoprostane and 8-OHdG, were analyzed using enzyme-linked immunosorbent assays (ELISA). Long-term (pregnancy through 3 months prenatal) exposure to particulate matter and airborne PAHs were measured. Results Women using wood-fueled chimney stoves are exposed to high levels of particulate matter (median 48 h PM2.5 105.7 μg/m3; inter-quartile range (IQR): 77.6–130.4). Urinary PAH and VOC metabolites were significantly associated with woodsmoke exposures: 2-naphthol (median (IQR) in ng/mg creatinine: 295.9 (74.4–430.9) after sauna versus 23.9 (17.1–49.5) fasting; and acrolein: 571.7 (429.3–1040.7) after sauna versus 268.0 (178.3–398.6) fasting. Urinary PAH (total PAH: ρ = 0.89, p 0.85) or PAH and VOC biomarkers (ρ = -0.20 to 0.38, p > 0.07). Urinary metabolite concentrations were significantly greater than those of heavy smokers (mean cigarettes/day = 18) across all PAHs. In 15 (65%) women, maximum 1-hydroxypyrene concentrations exceeded the occupational exposure limit of coke-oven workers. Conclusions The high concentrations of urinary PAH and VOC metabolites among recently pregnant women is alarming given the detrimental fetal and neonatal effects of prenatal PAH exposure. As most women used chimney woodstoves, cleaner fuels are critically needed to reduce smoke exposure.

Sarah L. PerryInstitute for Molecular Engineering, University of Chicago, Chicago, IL 60637Dept. of Bioengineering, University of California at Berkeley, Berkeley, CA 94720Surekha G. NeumannDept. of Chemistry and Biochemistry, University of California at Santa Barbara, Santa Barbara, CA 93106Thorsten NeumannDept. of Chemistry and Biochemistry, University of California at Santa Barbara, Santa Barbara, CA 93106Dept. of Bioengineering, University of California at Berkeley, Berkeley, CA 94720Karen Cheng, Jennifer Ni, and John R. WeinsteinDept. of Bioengineering, University of California at Berkeley, Berkeley, CA 94720David V. SchafferDept. of Bioengineering and Dept of Chemical and Biomolecular Engineering, University of California atBerkeley, Berkeley, CA 94720Matthew TirrellDept. of Bioengineering, University of California at Berkeley, Berkeley, CA 94720Institute for Molecular Engineering, University of Chicago, Chicago, IL 60637DOI 10.1002/aic.14198Published online August 9, 2013 in Wiley Online Library (wileyonlinelibrary.com)The use of surface-based methods for the delivery of therapeutics has recently generated increasing interest. Theseplatforms have tremendous potential to minimize detrimental side effects associated with systemic delivery by localiz-ing the therapeutic vehicle, and thus provide higher local doses for improved efficacy. Cationic lipids are one of themost commonly used synthetic carriers for the delivery of genetic cargo, such as DNA and RNA. However, reportson the use of lipid-based films for gene delivery are scarce. Here we investigate the use of a lipid-based film for thein vitro delivery of plasmid DNA. Solid DNA-lipid films show very low levels of transfection, while identical com-plexes prepared for bolus delivery provide high levels of transfection when used directly. We investigate the mecha-nism, whereby the activity of these solid-state films is lost and suggest methods for circumventing these challengesand restoring the efficacy of these films as gene delivery platforms.

The physiological components that contribute to cystic fibrosis (CF) lung disease are steadily being elucidated. Gene therapy could potentially correct these defects. CFTR-null pigs provide a relevant model to test gene therapy vectors. Using an in vivo selection strategy that amplifies successful capsids by replicating their genomes with helper adenovirus coinfection, we selected an adeno-associated virus (AAV) with tropism for pig airway epithelia. The evolved capsid, termed AAV2H22, is based on AAV2 with 5 point mutations that result in a 240-fold increased infection efficiency. In contrast to AAV2, AAV2H22 binds specifically to pig airway epithelia and is less reliant on heparan sulfate for transduction. We administer AAV2H22-CFTR expressing the CF transmembrane conductance regulator (CFTR) cDNA to the airways of CF pigs. The transduced airways expressed CFTR on ciliated and nonciliated cells, induced anion transport, and improved the airway surface liquid pH and bacterial killing. Most gene therapy studies to date focus solely on Cl– transport as the primary metric of phenotypic correction. Here, we describe a gene therapy experiment where we not only correct defective anion transport, but also restore bacterial killing in CFTR-null pig airways.

Purpose: Over 85% of rural Guatemalan households use woodstoves for cooking or heating. Smoke from these stoves and other sources, such as wood-fired sauna baths, contains polycyclic aromatic hydro...

© 2015 Macmillan Publishers Limited Adeno-associated virus (AAV) vectors have achieved clinical efficacy in treating several diseases. However, enhanced vectors are required to extend these landmark successes to other indications and protein engineering approaches may provide the necessary vector improvements to address such unmet medical needs. To generate new capsid variants with potentially enhanced infectious properties and to gain insights into AAV’s evolutionary history, we computationally designed and experimentally constructed a putative ancestral AAV library. Combinatorial variations at 32 amino acid sites were introduced to account for uncertainty in their identities. We then analyzed the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to iterative selection on a representative cell line panel. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes and, in general, ancestral libraries were broadly infectious across the cell line panel, indicating that they favored promiscuity over specificity. Interestingly, putative ancestral AAVs were more thermostable than modern serotypes and did not use sialic acids, galactose or heparan sulfate proteoglycans for cellular entry. Finally, variants mediated 19- to 31-fold higher gene expression in the muscle compared with AAV1, a clinically used serotype for muscle delivery, highlighting their promise for gene therapy.Gene Therapy advance online publication, 13 August 2015; doi:10.1038/gt.2015.74.

Posttranslational modification of tyrosine residues in proteins, to produce 3-nitrotyrosine (3-NT), is associated with over 50 disease states including transplant rejection, lung infection, central nervous system and ocular inflammation shock, cancer, and neurological disorders (for example, Alzheimer's disease, Parkinson's disease, and stroke). The levels of 3-NT increase in aging tissue, and levels of 3-NT in proteins are a predictor of disease risk. Here we report the evolution and characterization of an aminoacyl-tRNA synthetase/tRNA pair for the cotranslational, site-specific incorporation of 3-NT into proteins at genetically encoded sites. To demonstrate the utility of our approach for studying the effect on protein function of nitration on sites defined in vivo, we prepared manganese superoxide dismutase (MnSOD) that is homogeneously nitrated at a site known to be modified in disease-related inflammatory responses, and we measured the effect of this defined modification on protein function.

Adeno-associated virus (AAV) vectors have demonstrated clinical efficacy in treating several diseases. Improved vectors may extend these landmark successes to other indications, and protein engineering approaches are enhancing the gene delivery properties of AAV vectors to address such unmet medical needs. To access new viral sequences with potentially enhanced infectious properties, and to gain insights into AAV's evolutionary history, we computationally designed and experimentally characterized an ancestral reconstruction of the AAV capsid. To this end, we first generated a phylogenetic tree of AAV sequences through Bayesian Markov chain Monte Carlo simulations, then selected a node for reconstruction based on its relative confidence value and proximity to AAV serotypes of clinical interest. We next used HandAlign, a Markov chain Monte Carlo alignment sampler, to predict the ancestral sequence of the most likely alignment at the selected node. This generated a reconstruction where most amino acids were predicted with high confidence, while 32 of the positions were predicted with low confidence. In order to address uncertainty in these positions, we synthesized the inferred ancestral capsid as a large combinatorial library incorporating degenerate residues at the 32 variable amino acid sites. We then characterized the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to six rounds of selection on a panel of representative cell lines. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes, and in general the selected ancestral libraries were broadly infectious across the cell line panel, indicating that they favor promiscuity over specificity. Unlike many current serotypes, these ancestral AAVs do not utilize sialic acids, galactose, or heparan sulfate proteoglycans for cellular entry. Additionally, ancestral libraries were found to be as susceptible to neutralizing antibodies as extant serotypes, suggesting that such ancestral variant candidates exhibit immunogenic properties similar to modern descendants. Interestingly, ancestral AAVs retained infectivity at temperatures that completely ablated infectivity of modern serotypes; this higher thermostability may have supported increased evolvability through mutational tolerance. Finally, selected variants mediated 19-31 fold higher gene expression in muscle compared to AAV1, a serotype clinically utilized for muscle delivery, highlighting their potential promise for gene therapy.

Background Microarrays for the analysis of gene expression are of three different types: short oligonucleotide (25–30 base), long oligonucleotide (50–80 base), and cDNA (highly variable in length). The short oligonucleotide and cDNA arrays have been the mainstay of expression analysis to date, but long oligonucleotide platforms are gaining in popularity and will probably replace cDNA arrays. As part of a validation study for the long oligonucleotide arrays, we compared and contrasted expression profiles from the three formats, testing RNA from six different cell lines against a universal reference standard. Results The three platforms had 6430 genes in common. In general, correlation of gene expression levels across the platforms was good when defined by concordance in the direction of expression difference (upregulation or downregulation), scatter plot analysis, principal component analysis, cell line correlation or quantitative RT-PCR. The overall correlations (r values) between platforms were in the range 0.7 to 0.8, as determined by analysis of scatter plots. When concordance was measured for expression ratios significant at p-values of Conclusion Our results indicate that the long oligonucleotide platform is highly suitable for expression analysis and compares favorably with the cDNA and short oligonucleotide varieties. All three platforms can give similar and reproducible results if the criterion is the direction of change in gene expression and minimal emphasis is placed on the magnitude of change.