A comparative study of human exposures to household air pollution from commonly used cookstoves in Sri Lanka.

Solid fuel burning cookstoves are a major source of household air pollution (HAP) and a significant environmental health risk in Sri Lanka. We report results of the first field study in Sri Lanka to include direct measurements of both real-time indoor concentrations and personal exposures of fine particulate matter (PM2.5 ) in households using the two most common stove types in Sri Lanka. A purposive sample of 53 households was selected in the rural community of Kopiwatta in central Sri Lanka, roughly balanced for stove type (traditional or improved 'Anagi') and ventilation (chimney present or absent). At each household, 48-h continuous real-time measurements of indoor kitchen PM2.5 and personal (primary cook) PM2.5 concentrations were measured using the RTI MicroPEM™ personal exposure monitor. Questionnaires were used to collect data related to household demographics, characteristics, and self-reported health symptoms. All primary cooks were female and of an average age of 47 years, with 66% having completed primary education. Median income was slightly over half the national median monthly income. Use of Anagi stoves was positively associated with a higher education level of the primary cook (P = 0.026), although not associated with household income (P = 0.18). The MicroPEM monitors were well-received by participants, and this study's valid data capture rate exceeded 97%. Participant wearing compliance during waking hours was on average 87.2% on Day 1 and 83.3% on Day 2. Periods of non-compliance occurred solely during non-cooking times. The measured median 48-h average indoor PM2.5 concentration for households with Anagi stoves was 64 µg/m3 if a chimney was present and 181 µg/m3 if not. For households using traditional stoves, these values were 70 µg/m3 if a chimney was present and 371 µg/m3 if not. Overall, measured indoor PM2.5 concentrations ranged from a minimum of 33 µg/m3 to a maximum of 940 µg/m3 , while personal exposure concentrations ranged from 34 to 522 µg/m3 . Linear mixed effects modeling of the dependence of indoor concentrations on stove type and presence or absence of chimney showed a significant chimney effect (65% reduction; P < 0.001) and an almost significant stove effect (24% reduction; P = 0.054). Primary cooks in households without chimneys were exposed to substantially higher levels of HAP than those in households with chimneys, while exposures in households with traditional stoves were moderately higher than those with improved Anagi stoves. As expected, simultaneously measuring both indoor concentrations and personal exposure levels indicate significant exposure misclassification bias will likely result from the use of a stationary monitor as a proxy for personal exposure. While personal exposure monitoring is more complex and expensive than deploying simple stationary devices, the value an active personal PM monitor like the MicroPEM adds to an exposure study should be considered in future study designs.

Coarse particulate matter and airborne endotoxin within wood stove homes.

Emissions from indoor biomass burning are a major public health concern in developing areas of the world. Less is known about indoor air quality, particularly airborne endotoxin, in homes burning biomass fuel in residential wood stoves in higher income countries. A filter-based sampler was used to evaluate wintertime indoor coarse particulate matter (PM10₋2.5) and airborne endotoxin (EU/m³, EU/mg) concentrations in 50 homes using wood stoves as their primary source of heat in western Montana. We investigated number of residents, number of pets, dampness (humidity), and frequency of wood stove usage as potential predictors of indoor airborne endotoxin concentrations. Two 48-h sampling events per home revealed a mean winter PM10₋2.5 concentration (± s.d.) of 12.9 (± 8.6) µg/m³, while PM2.5 concentrations averaged 32.3 (± 32.6) µg/m³. Endotoxin concentrations measured from PM10₋2.5 filter samples were 9.2 (± 12.4) EU/m³ and 1010 (± 1524) EU/mg. PM10₋2.5 and PM2.5 were significantly correlated in wood stove homes (r = 0.36, P < 0.05). The presence of pets in the homes was associated with PM10₋2.5 but not with endotoxin concentrations. Importantly, none of the other measured home characteristics was a strong predictor of airborne endotoxin, including frequency of residential wood stove usage.

Essential roles of Jab1 in cell survival, spontaneous DNA damage and DNA repair.

Jun activation domain-binding protein 1 (JAB1) is a multifunctional protein that participates in the control of cell proliferation and the stability of multiple proteins. JAB1 overexpression has been implicated in the pathogenesis of human cancer. JAB1 regulates several key proteins and thereby produces varied effects on cell cycle progression, genome stability and cell survival. However, the biological significance of JAB1 activity in these cellular signaling pathways is unclear. Therefore, we developed mice that were deficient in Jab1 and analyzed the null embryos and heterozygous cells. This disruption of Jab1 in mice resulted in early embryonic lethality due to accelerated apoptosis. Loss of Jab1 expression sensitized both mouse primary embryonic fibroblasts and osteosarcoma cells to γ-radiation-induced apoptosis, with an increase in spontaneous DNA damage and homologous recombination (HR) defects, both of which correlated with reduced levels of the DNA repair protein Rad51 and elevated levels of p53. Furthermore, the accumulated p53 directly binds to Rad51 promoter, inhibits its activity and represents a major mechanism underlying the HR repair defect in Jab1-deficient cells. These results indicate that Jab1 is essential for efficient DNA repair and mechanistically link Jab1 to the maintenance of genome integrity and to cell survival.

Selective inhibition of choline kinase simultaneously attenuates MAPK and PI3K/AKT signaling.

Choline is an essential anabolic substrate for the synthesis of phospholipids. Choline kinase phosphorylates choline to phosphocholine that serves as a precursor for the production of phosphatidylcholine, the major phospholipid constituent of membranes and substrate for the synthesis of lipid signaling molecules. Nuclear magnetic resonance (NMR)-based metabolomic studies of human tumors have identified a marked increase in the intracellular concentration of phosphocholine relative to normal tissues. We postulated that the observed intracellular pooling of phosphocholine may be required to sustain the production of the pleiotropic lipid second messenger, phosphatidic acid. Phosphatidic acid is generated from the cleavage of phosphatidylcholine by phospholipase D2 and is a key activator of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT survival signaling pathways. In this study we show that the steady-state concentration of phosphocholine is increased by the ectopic expression of oncogenic H-Ras(V12) in immortalized human bronchial epithelial cells. We then find that small interfering RNA (siRNA) silencing of choline kinase expression in transformed HeLa cells completely abrogates the high concentration of phosphocholine, which in turn decreases phosphatidylcholine, phosphatidic acid and signaling through the MAPK and PI3K/AKT pathways. This simultaneous reduction in survival signaling markedly decreases the anchorage-independent survival of HeLa cells in soft agar and in athymic mice. Last, we confirm the relative importance of phosphatidic acid for this pro-survival effect as phosphatidic acid supplementation fully restores MAPK signaling and partially rescues HeLa cells from choline kinase inhibition. Taken together, these data indicate that the pooling of phosphocholine in cancer cells may be required to provide a ready supply of phosphatidic acid necessary for the feed-forward amplification of cancer survival signaling pathways.

The Medical College of Georgia Fitkid project: the relations between program attendance and changes in outcomes in year 1.

OBJECTIVE: To investigate the relations of attendance of children in an after-school physical activity (PA) program to changes in body composition and cardiovascular fitness (CVF). DESIGN: Eight-month after-school PA-based intervention. SUBJECTS: In all, 278 third-grade boys and girls from nine elementary schools (age, 8.7 y (s.d. = 0.6 y), body mass index (BMI) 19.1 kg/m2 (s.d. = 4.4) and percent body fat (%BF) 26.0 (s.d. = 9.0)). MEASUREMENTS: Body composition (from dual-energy X-ray absorptiometry), BMI (from height and weight), waist circumference (WC) and CVF (from the YMCA submaximal bench-stepping test). RESULTS: There was a significant negative linear trend between level of attendance in the after-school program and change in %BF and fat mass; there was also a significant positive linear trend between program attendance and change in CVF. There was a marginally significant linear trend between program attendance and fat-free mass. Greater increases in bone mineral density were observed with higher program attendance. Changes in BMI and WC were not influenced by program attendance. CONCLUSION: Understanding the dose-response effect of PA on health outcomes, particularly body composition, in children is crucial in our effort to prevent overweight and its health consequences. Since there is limited data available to base PA dose recommendations for youths, findings from this study are relevant, and suggest that greater health benefits can be obtained in young children with more frequent participation in PA.

Cytomegalovirus infection accelerates inflammation in vascular tissue overexpressing monocyte chemoattractant protein-1.

Cardiovascular disease is the leading cause of mortality in the United States. Atherosclerosis is responsible for most of this pathology and is an inflammatory disease with multiple cytokines and adhesion molecules expressed during atherogenesis. Cytomegalovirus (CMV), monocytes, and monocyte chemoattractant protein-1 (MCP-1) have all been implicated in human atherogenesis. A transgenic mouse overexpressing MCP-1 in the myocardium and pulmonary arteries develops myocarditis and pulmonary vascular inflammation. We infected MCP-1 transgenic mice with a sublethal dose of murine cytomegalovirus (MCMV) to look for evidence of accelerated inflammation in vascular tissues overexpressing MCP-1 to determine if MCMV could interact with monocytes and MCP-1 in a manner similar to what may occur in atherogenesis. MCMV infection of MCP-1 transgenic mice caused ascites, myocarditis, and pulmonary artery inflammation, which was not present in mock-infected MCP-1 or MCMV-infected wild-type mice. Inflammatory infiltrates in these tissues consisted of macrophages and T lymphocytes similar to the infiltrates seen in atherosclerosis. Virus presence in inflamed tissues was demonstrated by infecting transgenic mice with MCMV recombinant virus containing the gene sequence for the enhanced green fluorescent protein (EGFP). Human CMV could be involved in atherogenesis in a similar manner by interacting with monocytes and MCP-1 specifically expressed in vascular walls.

Contribution of monocytes/macrophages to compensatory neovascularization: the drilling of metalloelastase-positive tunnels in ischemic myocardium.

In a transgenic model of ischemic cardiomyopathy in which monocytes are attracted to the myocardium by the targeted overexpression of monocyte chemoattractant protein-1 (MCP-1), we have observed the presence of endothelial NO synthase and platelet endothelial cell adhesion molecule-1-negative tunnels, occasionally containing blood-derived cells, that probe the cardiac tissue. Immunohistochemical data show that monocytes/macrophages (MCs/Mphs) drill tunnels using the broad-spectrum mouse macrophage metalloelastase. 5-Bromo-2'-deoxyuridine incorporation and neo-endothelial markers present in the microvasculature of MCP-1 mouse hearts suggest an active angiogenic process. Further studies will be required to establish that the MC-/Mph-drilled tunnels evolve to become capillaries, connected to the existing vessels and colonized by circulating endothelial cell progenitors. This possibility is supported by the availability of these cells, which is demonstrated by cell tagging with beta-galactosidase placed under an active endothelial Tie-2 promoter. This phenomenon might represent another mechanism, in addition to the secretion of the angiogenic factors, by which MCs/MPhs may participate in the elaboration of new blood vessels in adult tissues.

Size distribution of mist generated during metal machining.

Mist generated by machining processes is formed by three mechanisms: impaction, centrifugal force, and evaporation/condensation. This study characterized the size distribution of soluble and mineral oil mists that resulted from these formation mechanisms. Salient parameters influencing the particle size distributions also were identified. Variables investigated included metalworking fluid and machining characteristics. The size distribution of the mist generated on a small lathe by each mechanism was measured using an Aerosizer LD. For impaction, only the mineral oil viscosity influenced the mass median diameter of the mist. No parameter affected the geometric standard deviation. High-viscosity mineral oil mist had a mass median diameter of 6.1 microns and a geometric standard deviation of 2.0. Low-viscosity mineral oil mist had a mass median diameter of 21.9 microns and a geometric standard deviation of 2.2. The mass median diameter of the mist generated by centrifugal force depended on the type of metalworking fluid, fluid flow, and rotational speed of the lathe. Mass median diameters for low-viscosity mineral oil mist ranged from 5 to 110 microns. Mass median diameters for soluble oil mist varied between 40 and 80 microns. The average geometric standard deviation was 2.4, and was not affected by any parameter. The mass median diameter and geometric standard deviation of the mist generated by evaporation/condensation varied with the type of metalworking fluid. The mineral oil mist and soluble oil mist mass median diameters were 2.1 microns and 3.2 microns, respectively. No machining or fluid parameter was important because the mist size distribution depended on the rate of condensation, coagulation processes, and the dynamics of the apparatus. Using the size distribution data from all three mechanisms, the estimated inhalable, thoracic, and respirable fractions of the total mass generated for each metalworking fluid were 60 percent, 12 percent, and 8 percent, respectively. To minimize exposure to the inhalable mass fraction, the amount of mist generated by centrifugal force must be reduced or the size of the drops generated must be increased. Altering the machining or fluid parameters did not change the mist size distribution and reduce exposure to the respirable mass fraction.

Ectopic overexpression of engrailed-2 in cerebellar Purkinje cells causes restricted cell loss and retarded external germinal layer development at lobule junctions.

Members of the En and Wnt gene families seem to play a key role in the early specification of the brain territory that gives rise to the cerebellum, the midhindbrain junction. To analyze the possible continuous role of the En and Wnt signaling pathway in later cerebellar patterning and function, we expressed En-2 ectopically in Purkinje cells during late embryonic and postnatal cerebellar development. As a result of this expression, the cerebellum is greatly reduced in size, and Purkinje cell numbers throughout the cerebellum are reduced by more than one-third relative to normal animals. Detailed analysis of both adult and developing cerebella reveals a pattern of selectivity to the loss of Purkinje cells and other cerebellar neurons. This is observed as a general loss of prominence of cerebellar fissures that is highlighted by a total loss of sublobular fissures. In contrast, mediolateral patterning is generally only subtly affected. That En-2 overexpression selectively affects Purkinje cells in the transition zone between lobules is evidenced by direct observation of selective Purkinje cell loss in certain fissures and by the observation that growth and migration of the external germinal layer (EGL) is selectively retarded in the deep fissures during early postnatal development. Thus, in addition to demonstrating the critical role of Purkinje cells in the generation and migration of granule cells, the heterogeneous distribution of cellular effects induced by ectopic En expression suggests a relatively late morphogenetic role for this and other segment polarity proteins, mainly oriented at lobule junctions.

A functional model of hepatocyte transplantation for in vivo immunologic studies.

BACKGROUND: Previous studies to determine the significant donor and host factors which contribute to immune responses to allogeneic hepatocytes have been pursued with in vitro and in vivo studies. However, in order to further characterize in vivo host immune responses to transplanted hepatocytes in conjunction with their consequences upon allograft survival, a functional model of hepatocyte transplantation in which the function and survival of transplanted hepatocytes could be serially assessed was pursued. METHODS: A transgenic mouse line expressing the human alpha1-antitrypsin (hA1AT) gene was developed in an FVB/N (H2q) mouse (hA1AT-FVB/N). Hepatocytes from these "donor" transgenic mice were transplanted into host mice by intrasplenic injection, and subsequent survival of the transgenic hepatocytes was determined by assay for the secreted hA1AT protein in host serum by enzyme-linked immunosorbent assay. RESULTS: Transplantation of transgenic "donor" hepatocytes (hA1AT-FVB/N) into (a) syngeneic FVB/N (H2q), (b) allogeneic immunoincompetent C57BL/6.SCID (H2b), or (c) allogeneic T cell-deficient (outbred) hosts resulted in long-term survival (> 16 weeks) of hepatocytes. Transplantation of allogeneic hepatocytes (hA1AT-FVB/N, H2q) into C57BL/6 (H2b), C3H (H2k), or BALB/c (H2d) hosts resulted in loss of hA1AT in host serum by day 10 after transplant. Likewise, transplantation of allogeneic hepatocytes into hosts deficient in B cells, CD8+ T cells, or CD4+ T cells resulted in loss of hA1AT by day 10 after transplant. CONCLUSIONS: This functional model of hepatocyte transplantation is validated for the study of host immune responses to hepatocellular grafts and to assess efficacy of strategies designed to alter these in vivo immune responses. The immunologic rejection of allogeneic hepatocytes appears to be T cell-mediated.

Myocarditis induced by targeted expression of the MCP-1 gene in murine cardiac muscle.

To explore the possible role of monocyte chemotactic protein (MCP-1) in inflammatory diseases of the heart, we expressed the murine MCP-1(JE) gene under the control of the alpha-cardiac myosin heavy chain promoter to attempt to target MCP-1 expression to the adult heart muscle. The five lines of transgenic mice thus produced showed targeted expression of MCP-1 transcripts and protein in the adult heart muscle and pulmonary vein but not in skeletal muscle. MCP-1 level in the transgenic hearts increased up to 30 to 45 days of age, and leukocyte infiltration into interstitium between cardiomyocytes increased up to 60 to 75 days. The infiltrate was mainly macrophages but not T cells. The presence of MCP-1 in the transgenic hearts did not induce cytokine production indicative of leukocyte activation. Echocardiographic analysis of 1-year-old mice that express MCP-1 in the myocardium and of age-matched controls revealed cardiac hypertrophy and dilation, increases in left ventricular (LV) mass, and systolic and diastolic left ventricular internal diameters. A significant decline in M-mode shortening fraction showed depressed contractile function. Transgenic hearts were 65% heavier, and histological analysis showed moderate myocarditis, edema, and some fibrosis. Thus, MCP-1 expression in the heart muscle may provide a model to investigate myocarditis and cardiomyopathy.

Role of intramolecular disulfide bond formation in the assembly and secretion of apolipoprotein B-100-containing lipoproteins.

Apolipoprotein B-100 (apoB) is essential for the hepatic assembly and secretion of triglyceride-rich very low density lipoprotein (VLDL). The mechanism of VLDL assembly was explored by perturbing apoB folding in HepG2 cells with the thiol reducing agent dithiothreitol (DTT). Although apoB contains eight known disulfide bonds, seven of which are positioned in the amino-terminal 21% of the protein, its assembly and secretion was only partially blocked in cells treated with 2 mM DTT, a condition that fully blocks the secretion of other disulfide-bonded proteins. Nonreducing gel electrophoresis of an apoB-derived proteolytic peptide revealed that apoB escapes the secretory block normally caused by DTT because its amino-terminal disulfide bonds undergo maturation to a DTT-resistant form after completing synthesis of only the first approximately 20-25% of the protein. If, however, DTT was used under conditions that prevented the initial formation of amino-terminal disulfide bonds, lipoprotein secretion was blocked. Reduced forms of apoB were extremely labile and, unlike other disulfide-bonded proteins, incapable of achieving secretion competence posttranslationally. These results indicate that disulfide bond formation within the amino-terminus of apoB is essential for the proper folding and assembly of its downstream lipophilic sequences. The onset of DTT resistance while still a nascent polypeptide chain is consistent with a model in which the amino-terminal domain of apoB undergoes an independent folding and maturation process, the completion of which may represent an initiation phase of triglyceride-rich lipoprotein assembly.

Targeted expression of the ret/PTC1 oncogene induces papillary thyroid carcinomas.

The ret/PTC oncogene, a rearranged form of the ret proto-oncogene, has been found to be restricted to human papillary thyroid carcinomas. This report shows that transgenic mice with thyroid-targeted expression of the ret/PTC1 oncogene developed thyroid carcinomas with considerable similarities to human papillary thyroid carcinomas, particularly in the nuclear cytologic features and the presence of local invasion. Our findings indicate that ret/PTC2 is not only a biomarker associated with papillary thyroid carcinomas, but is also the only proven specific genetic event leading to the development of papillary thyroid carcinoma.

Dietary fatty acid modification of HDL phospholipid molecular species alters lecithin: cholesterol acyltransferase reactivity in cynomolgus monkeys.

In the following report, cynomolgous monkeys, fed atherogenic diets containing either saturated, monounsaturated, polyunsaturated (n-6 Poly) or fish oil (n-3 Poly) fat as 35% of total calories, provide a model for the study of dietary fat effects on plasma lipoproteins and atherosclerosis. We have previously described the ability of polyunsaturated fat diets to lower plasma described the ability of polyunsaturated fat diets to lower plasma high density lipoprotein (HDL) cholesterol levels and alter HDL subpopulation distribution in the primate model. These experiments investigate possible mechanisms responsible for such modifications. Animals fed polyunsaturated fat had significantly lower plasma concentrations of HDL cholesterol, total plasma cholesterol, and apolipoprotein A-I. Such changes were reflected in the distribution of protein among HDL subfractions, with the most remarkable modification in subclass distribution being the preponderance of small HDL particles in the n-3 Poly-fed animals. Striking alterations were also observed in the distribution of phosphatidylcholine (PC) molecular species (diet effect P < 0.0001 for all major molecular species). Phosphatidylcholine isolated from lipoproteins were used to make recombinant HDL (rHDL) particles. The reaction rate of purified lecithin:cholesterol acyltransferase (LCAT) with particles made from n-3 Poly-derived PC was 50% of that determined using rHDL formed with PC from other dietary groups (P < 0.0001). When the distribution of LCAT-derived rHDL cholesteryl esters was analyzed, LCAT demonstrated little selectivity for certain PC molecular species except in n-3 Poly-derived rHDL where 18:2-containing PC was selectively utilized. These data demonstrate that differences in dietary fat intake can significantly alter HDL PC concentration and molecular species distribution. We suggest that diet-induced alterations in HDL PC molecular species modify the type of cholesteryl esters produced during the LCAT reaction thereby affecting the plasma cholesteryl ester pool. We also propose that dietary n-3 Poly affects cholesteryl ester metabolism in part via LCAT by lowering PC (LCAT substrate) availability, altering the rate of the LCAT reaction, and decreasing HDL cholesterol concentrations; however, n-6 Poly dietary fat effects on HDL concentration appear to be through some mechanism other than LCAT.

Reading skills and auditory processing ability in children with chronic otitis media in early childhood.

The effect of recurrent middle ear disease during the first 2 years of life on auditory perceptual skills and reading ability was examined in two groups of 6- and 7-year-old children who were pair-matched by age, gender, socioeconomic status, and receptive vocabulary. Group 1 consisted of children with documented chronic otitis media at an early age, and group 2 had no history of middle ear problems. Tests of auditory perceptual skills and reading ability were administered. Significant differences in performance on all tests of auditory processing ability and reading ability were noted.

Structural and developmental analysis of two linked myosin heavy chain genes.

We have identified at the molecular level two murine myosin heavy chain (MHC) genes which encode adult skeletal isoforms. As is the case for the murine cardiac MHC genes, the genes are closely linked, head to tail. To identify the isoform encoded by each gene, we located and sequenced the 3' termini and assessed the genes' temporal and tissue-specific expression patterns using probes specific for the 3' untranslated regions. These analyses indicate that the myosin encoded by the gene located 5' in the linked pair is the murine 2A isoform. The isoform encoded by the 3'-most gene of the linked pair appears to encode an additional isoform that is expressed in a number of adult skeletal muscles. The pattern of expression of the 3'-most gene indicates that it is tightly controlled developmentally. Transcripts from this gene, when compared to those from the MHC2A and beta-MHC genes, are the predominant MHC transcripts found in the diaphragm, tongue, soleus, and masseter, indicating that it encodes a major skeletal MHC isoform.

Mutations that induce the heat shock response of Drosophila.

We have isolated a number of mutations in D. melanogaster that result in the constitutive expression of the heat shock response in a tissue-specific manner. These mutations induce alcohol dehydrogenase (ADH) when the ADH structural gene is fused to the promoter for the 70 kd heat shock protein (hsp70) gene. Flies carrying these mutations, the hsp70-Adh fusion, and a deletion in their endogenous Adh genes are ethanol tolerant and exhibit elevated ADH levels. Several of the tissue-specific mutations have also been shown to induce an hsp26-Adh fusion gene in trans. The mutation Act88FKM75, a G----A transition in the indirect flight muscle-specific actin gene, also exhibits this phenotype. Comparisons with the Act88FKM75 mutation suggest that the tissue-specific mutations induce the heat shock response by disrupting the physiology of the cells in which the variant gene product is expressed.