Biomechanical effects of environmental and engineered particles on human airway smooth muscle cells.

The past decade has seen significant increases in combustion-generated ambient particles, which contain a nanosized fraction (less than 100 nm), and even greater increases have occurred in engineered nanoparticles (NPs) propelled by the booming nanotechnology industry. Although inhalation of these particulates has become a public health concern, human health effects and mechanisms of action for NPs are not well understood. Focusing on the human airway smooth muscle cell, here we show that the cellular mechanical function is altered by particulate exposure in a manner that is dependent upon particle material, size and dose. We used Alamar Blue assay to measure cell viability and optical magnetic twisting cytometry to measure cell stiffness and agonist-induced contractility. The eight particle species fell into four categories, based on their respective effect on cell viability and on mechanical function. Cell viability was impaired and cell contractility was decreased by (i) zinc oxide (40-100 nm and less than 44 microm) and copper(II) oxide (less than 50 nm); cell contractility was decreased by (ii) fluorescent polystyrene spheres (40 nm), increased by (iii) welding fumes and unchanged by (iv) diesel exhaust particles, titanium dioxide (25 nm) and copper(II) oxide (less than 5 microm), although in none of these cases was cell viability impaired. Treatment with hydrogen peroxide up to 500 microM did not alter viability or cell mechanics, suggesting that the particle effects are unlikely to be mediated by particle-generated reactive oxygen species. Our results highlight the susceptibility of cellular mechanical function to particulate exposures and suggest that direct exposure of the airway smooth muscle cells to particulates may initiate or aggravate respiratory diseases.

Evaluation of the risk of PRRSV transmission via ingestion of muscle from persistently infected pigs.

The objectives of this experiment were to determine how long porcine reproductive and respiratory syndrome virus (PRRSV) could be detected in muscle tissues of experimentally infected pigs and to evaluate the transmissibility of PRRSV to pigs via ingestion of quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR)-positive muscle tissues. Serum, lymphoid tissues, and muscle (M. longissimus dorsi) samples were collected from 135 pigs (89 PRRSV-inoculated pigs and 46 negative control). Between 28 and 202 days post-inoculation, 13 of 89 (14.6%) muscle samples were positive by qRT-PCR. Among these 13, PRRSV was isolated from four of the 13 corresponding serum samples and three of 13 lymphoid tissue samples. In addition, infectious virus was detected in lymphoid tissue homogenates of six of 13 pigs by intramuscular bioassay. Swine transmissibility studies were performed by feeding thirteen 3-week-old PRRSV-naive pigs (recipient pigs) qRT-PCR-positive muscle and then monitoring recipients for evidence of PRRSV viremia by qRT-PCR. No transmission of PRRSV to recipient pigs via consumption of muscle samples was observed. These data suggested that qRT-PCR detected non-infectious PRRSV in pig meat and/or PRRSV is not highly transmissible to susceptible pigs via consumption of PRRSV-contaminated meat.

Immune response against porcine reproductive and respiratory syndrome virus during acute and chronic infection.

A significant obstacle to the prevention and control of porcine reproductive and respiratory syndrome virus (PRRSV) is the inability of current diagnostic tests to provide information concerning the stage of PRRSV infection. To explore possible prognostic combinations of cell-mediated and humoral immune responses, 3-week-old pigs (n=10) were intramuscularly (IM) inoculated with PRRSV isolate VR-2332 and followed for 193 days post-inoculation (DPI). Negative control pigs (n=10) were IM inoculated with minimum essential medium (MEM). At approximately 2-week intervals, blood samples were collected from all animals and tested for the number of interferon (IFN)-gamma-secreting peripheral blood mononuclear cells (enzyme-linked immunosorbent spot, Elispot), PRRSV viremia (quantitative reverse-transcriptase polymerase chain reaction, qRT-PCR), and serum antibodies using PRRSV protein ELISAs (N, GP5 3', GP5 5', M 5', M 3', GP5-M, and nsp2p) and a commercial PRRSV ELISA (IDEXX Laboratories Inc.). All pigs were viremic by 7 days post-inoculation, with 50% of the pigs resolving viremia by 56 DPI. A PRRSV-specific IFN-gamma response was detected at DPI 28, reached a plateau at 42 DPI, declined slightly, and remained relatively stable from 56 to 193 DPI. On the basis of ROC area under the curve (AUC) analysis, the ELISAs that most reliably differentiated PRRSV-inoculated pigs from negative control pigs were the commercial ELISA (AUC=0.97), the N ELISA (AUC=0.96), and the M 3' ELISA (AUC=0.93). Multivariate analyses were performed to evaluate the relationship between the immune response and the duration and level of viremia. With all antibody assays and Elispot included in the models, the analysis determined that the serum-virus neutralizing antibody response was the best predictor of both level and duration of viremia. It was concluded that humoral antibody responses, particularly the commercial ELISA, N ELISA, and M 3' ELISA were good predictors of prior exposure to PRRSV, but provided little information regarding the ontogeny of the protective immune response. Likewise, cell-mediated immunity based on the number of IFN-gamma-secreting lymphocytes was a poor prognosticator of PRRSV infection status.

Early diagnosis of HIV-1: infected infants in Brazil using nested-PCR.

The polymerase chain reaction (PCR) has been the most promising test for HIV-1 early diagnosis in infants suspected of perinatal transmission. The first and second reactions of the amplification in 41 infants (under 18 months old) suspected of HIV-1 perinatal infection, were standardized and carried out in the present study. The first and the second PCR were carried out with the sets of primers JA4-JA7, JA9-JA12, JA13-JA16, and JA17-JA20 for the first reaction of amplification (outer primers) and JA5-JA6, JA10-JA11, JA14-JA15, and JA18-JA19 for the second reaction of amplification (inner primers), resulting in amplification of 131, 341, 172, and 129 pb, respectively. From 41 patients analysed, 12 patients presented positive to HIV-1 infection by PCR. The gag, env (region 1), and pol regions presented a greater sensitivity. The first and the second reactions of the amplification were performed with the same concentration of MgCl2 for all sets of primers. The results agree with several studies that affirm that the PCR is the indicated method for HIV-1 early diagnosis in infants suspected of perinatal infection.

Effects of a perfluorochemical emulsion on the fate of circulating Pseudomonas aeruginosa.

Because mononuclear phagocytes take up perfluorochemical emulsions (PFCE), we examined how prior treatment with PFCE affects the fate of circulating bacteria. Rats were preinjected with three daily intravenous injections of PFCE (2.0 ml/100 g) containing 12.5% (vol/vol) of a 4:1 mixture of F-dimethyl adamantane and F-trimethylbicyclo-nonane, 2.5% (wt/vol) Pluronic F-68 as the emulsifying agent, and 3% (wt/vol) hydroxyethyl starch as the oncotic agent. Pseudomonas aeruginosa or Staphylococcus aureus were injected 4 h after the third PFCE injection. PFCE pretreatment decreased the rate and extent of vascular clearance of P. aeruginosa, with decreased uptake by the liver. Importantly, there were significant decreases in killing of P. aeruginosa in the liver, lungs, spleen, and kidneys of PFCE animals. PFCE did not alter the clearance of S. aureus from the circulation. However, hepatic uptake was reduced, with concomitant increases in lung and kidney uptake. Ultrastructure of Kupffer cells revealed PFCE inclusions and extensive vacuolization. These experiments demonstrate that the clearance kinetics and organ distribution of circulating P. aeruginosa and their subsequent killing are altered by PFCE. Diminished hepatic phagocyte function leads to a decrease in vascular clearance of circulating bacteria, increased uptake in other reticuloendothelial organs, and decreased bactericidal activity versus P. aeruginosa.

Pulmonary intravascular macrophages: their contribution to the mononuclear phagocyte system in 13 species.

The organ uptake of intravenously injected particles was examined in 13 species. All animals were injected intravenously with 198Au colloid and magnetic iron oxide particles. Vascular clearance kinetics of 198Au colloid was similar in all species. Pulmonary uptake of 198Au colloid ranged from 17 to 60% in sheep, calves, pigs, and cats but was <1.1% in monkeys, hyraxes, rabbits, guinea pigs, rats, mice, and chickens. For iron oxide particles, pulmonary uptake ranged from 80 to 99% in sheep, calves, pigs, goats, and cats and 15 to 18% in hamsters, hyraxes, and monkeys and was <10% in rabbits, chicken, mice, rats, and guinea pigs. In all species, the bulk of the remainder of particle uptake was in the liver. Pulmonary intravascular macrophages are the cellular site of lung uptake in calves, cats, pigs, goats, and sheep, whereas monocytes and neutrophils predominate in other species. Kupffer cells were the site of uptake in the liver. Our data show marked species differences in the fate of circulating particles; ruminants, pigs, and cats have extensive pulmonary localization due to phagocytosis by pulmonary intravascular macrophages.

Gadolinium induces macrophage apoptosis.

Gadolinium (Gd) suppresses reticuloendothelial functions in vivo by unknown mechanisms. In vitro exposure of rat alveolar macrophages to GdCl3.6H20 caused cell death, as measured by trypan blue permeability, in both dose- and time-dependent fashions. Even a 10-min exposure to Gd caused significant cell death by 24 h. The morphology of Gd-treated cells, pyknosis and karyorrhexis prior to loss of membrane integrity, suggested apoptosis. Upon flow cytometric examination, Gd-treated propidium iodide-excluding cells demonstrated light scatter changes characteristic of apoptotic cells (decreased forward and increased right angle scatter). Gel electrophoresis of DNA from Gd-treated macrophages clearly showed the ladder pattern unique to apoptotic cells. Electron-dense structures containing Gd were observed via electron spectroscopic imaging within phagosomes and also within nuclei (associated with condensed chromatin). Gadolinium, endocytosed by macrophages and distributed to nuclei, causes apoptosis of macrophages in vitro.

Influence of the nature of the reporter gene and selection pressure on stability of avian retrovirus vectors.

We have compared the long-term stability of 2 avian non-replicative retroviral vectors in an infected permanent cell line from quail fibroblasts (QT6). Vectors NL53 and NPL, expressing both the neo and LacZ genes under control of cis-acting elements originated from avian erythroblastosis virus (AEV), are similar to each other except for the presence of the phleomycin-resistance SHble gene fused upstream the reporter LacZ gene, in NPL vector. The use of such vectors, with an uniform backbone, to infect QT6 cells, allowed us to demonstrate that stability of the beta-galactosidase activity encoded by the SHble-LacZ fusion gene remains higher than that encoded by the native LacZ gene, as determined in the same conditions of culture. Moreover, stability of the provirus was dependent on the selection pressure. Here we show that stability of beta-galactosidase activity in infected QT6 cells was obtained with high dose selection for the selectable SHble-LacZ fusion gene.

Influence of expression and cis-acting sequences from avian leukosis viruses (ALVs) on stability of (ALV)-based retrovirus vectors.

Defective avian leukosis virus (ALV)-based vectors expressing the neo and LacZ genes were constructed under the control of cis-acting elements originated from 4 avian retroviruses: avian erythroblastosis virus (AEV), Rous associated viruses 1 (RAV-1) and 2 (RAV-2), and the Schmidt Ruppin strain of Rous sarcoma virus subgroup D (SR-RSV-D). We used these vectors to study the long-term stability of beta-galactosidase expression (encoded by the LacZ gene) in a permanent cell line from quail fibroblasts (QT6). Infection of the immortalized QT6 cell line with these vectors resulted in unstable beta-galactosidase expression. We determined whether this instability of provirus expression was correlated with: (1) presence of G418 selection; (2) deletion in the proviral genome; (3) hypermethylation of the proviral genome; (4) position of the neo and LacZ genes in the proviral genome; and (5) the transcriptional activity of the long terminal repeat (LTR) elements of proviral vectors. We observed that G418 selection pressure applied to infected QT6 cells lead to a more stable LacZ gene expression. Moreover, our results suggest a correlation between the stability of proviral gene expression and the level of gene expression driven by the LTR elements and depending on the strain origin of these.

Patterns of integration and expression of retroviral, non-replicative vectors in avian embryos: embryo developmental stage and virus subgroup envelope modulate tissue-tropism.

We previously demonstrated that Avian Leukemia Viruses (ALV) carrying the v-myc gene specifically induce two types of tumors, cardiomyocytic tumors when the virus is injected before embryonic day 3 (E3), skin tumors when the virus is injected at E3 or E5. Aiming to elucidate the mechanisms which determine this time-dependent change in target, we infected chick and quail embryos at E3 and E5 with replication-deficient, lacZ gene-carrying, ALV-based viruses produced by a packaging cell line. Three constructs driven by 3 different Long Terminal Repeats (LTRs) were tested and yielded similar results. When the constructs were inoculated at E3 and the lacZ gene product revealed 5 days later, around 70% of the embryos carried lacZ+ clones in the heart, around 50% had positive clones in the skin anywhere on the body, while a few embryos displayed clones in internal organs (liver, stomach, lungs). Immunocytological identification of the heart cell type(s) expressing the virus revealed that the only cells infected were cardiomyocytes. When the constructs were inoculated at E5, no lacZ+ clones appeared in the heart but all were located in the cephalic skin. In order to examine the relationship between viral integration and expression, DNA of different organs or tissues from lacZ stained embryos was analyzed by PCR. A tight correlation between integration and expression in the heart and in the skin was revealed in most cases. In contrast, a significant PCR signal was often detected in the liver or the stomach despite weak or absent expression as revealed by lacZ+ clones. We then investigated the influence of envelope glycoprotein subgroups on the tropism of these constructs. The lacZ vector driven by RAV-2 LTRs was packaged as subgroups A, B or E viral particles. The A subgroup, used in the part of the study described above, infects both chick and quail while the B and E subgroups are specific for chick or quail respectively. These B and E subgroups induced lacZ+ clones in the heart (after E3 injection) while no clones or only a few were detected in the skin either after E3 or E5 injection. The following conclusions can be drawn: 1) cardiomyocytes are at E3 the major target for integration and expression of ALV-derived viruses in vivo; 2) targets change rapidly with embryonic age; and 3) tissue-specific infections depend on the envelope subgroup, thus presumably on the presence of the cognate receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Use of helper cells with two host ranges to generate high-titer retroviral vectors.

We have recently described Avian Leukosis Virus (ALV)-based packaging cell lines that can produce helper-free ALV-based retrovirus vectors with A, B, C, and E envelope host ranges. Here, we report that lacZ retroviral vectors of subgroup C or E can infect helper cells of subgroup A (Isolde) which are then able to produce high titers of lacZ recombinant viruses of subgroup A. Superinfection of helper cells by lacZ recombinant virus was performed by cocultivating packaging cells with two subgroup specificities (A and E), but this did not result in increased recombinant virus titers. This "ping-pong" process caused the emergence of replication-competent (RC) viruses which are shown to result from recombination between the viral sequences of the helper cell lines and the cis-acting sequences of the lacZ recombinant virus.

Packaging cells for avian leukosis virus-based vectors with various host ranges.

Using our previously described Haydée semipackaging cell line (F. L. Cosset, C. Legras, Y. Chebloune, P. Savatier, P. Thoraval, J. L. Thomas, J. Samarut, V. M. Nigon, and G. Verdier, J. Virol. 64:1070-1078, 1990) which produces avian leukosis virus gag and pol proteins, we have constructed packaging cells with subgroups B, C, and E envelope specificities. This allows us to produce helper-free avian leukosis virus particles carrying the lacZ reporter gene and the A, B, C, or E subgroup specificities. Titers of the recombinant lacZ virus are shown to be dependent upon the type of the env subgroup and the target avian cell.

Hepatic versus pulmonary uptake of particles injected into the portal circulation in sheep. Endotoxin escapes hepatic clearance causing pulmonary inflammation.

Removal of circulating particulates (bacteria, cell debris, endotoxin) is accomplished in most species by macrophages resident in the liver and spleen. We have shown that sheep and other species have phagocytic macrophages resident in their pulmonary capillaries. Moreover, these pulmonary intravascular macrophages accomplish the bulk of uptake of injected tracer particles, bacteria, or endotoxin (LPS). Because bacteria or LPS of intestinal origin enter the portal circulation, they would first encounter hepatic mononuclear phagocytes. We sought to determine the extent to which particulates injected into the portal circulation of sheep would be taken up by liver or by lung macrophages. Sheep (four per group) were injected via a mesenteric vein with radiolabeled gold colloid, magnetic iron oxide particles, live Pseudomonas aeruginosa, or 125I E. coli endotoxin. For each, the uptake pattern was determined 1 h after injection. Lung and liver were also fixed to determine the cells responsible for uptake and subsequent inflammatory changes. We found that for circulating gold colloid, iron oxide particles, or bacteria, hepatic uptake predominated, and Kupffer cells were responsible. After hepatic uptake of bacteria, inflammatory changes were confined to the liver. In contrast, nearly 50% of endotoxin escaped hepatic clearance and was subsequently removed by the lungs. We then saw inflammatory changes in both lungs and liver. Thus, hepatic macrophages are active in species with pulmonary intravascular macrophages, partially sparing the lungs from uptake and acute inflammation. Endotoxin, however, may elude hepatic uptake, be sequestered in the lungs, and initiate inflammation there.

In situ expression of helper-free avian leukosis virus (ALV)-based retrovirus vectors in early chick embryos.

Defective avian leukosis-based vectors expressing the bacterial lacZ gene were used as helper-free preparations to infect early stage Brown-Leghorn embryos. Both in toto X-gal staining and DNA analysis using Southern blot technique were applied to detect virus integration and expression. Our results demonstrate a low efficiency of in vitro infection in early stages of embryonic development. Southern blot analysis reveals that only 1% of embryonic cells integrate the vector genome after infection using 2 to 12 virus particle per embryonic cell. In situ expression of the lacZ marker gene was detected in only 0.06% of embryonic cells. These results lead us to conclude that only 6% of infected cells express efficiently the lacZ marker gene. This low level of expression could result from avian leukosis virus LTRs inhibition in chicken embryonic cells at an early stage of development. In spite of the low efficiency of infection, no evidence for tissue restrictive expression was observed. However, vector containing LTRs from RAV-2 virus allows preferential expression of provirus vector in neural tube tissue, whereas cardiac localization of the preferential expression was observed using vector containing the RAV-1 LTRs. The chronological analysis of the marker gene expression in terms of location of expression foci and sizes of these foci, lead us to hypothesize the putative regulation of retrovirus expression linked to embryonic development.

Improvement of avian leukosis virus (ALV)-based retrovirus vectors by using different cis-acting sequences from ALVs.

Production and expression of double-expression vectors which transduce both Neo(r) and lacZ genes and are based on the structure of avian leukosis virus were enhanced by using cis-acting sequences (long terminal repeats and noncoding sequences) from Rous-associated virus-1 and Rous-associated virus-2 rather than those of avian erythroblastosis virus previously used in our constructs. Polyclonal producer cells obtained after transfection of these vectors into the Isolde packaging cell line gave rise to titers as high as 3 x 10(5) lacZ CFU/ml, whereas it was possible to isolate clones of producer cells giving rise to titers of more than 10(6) resistance focus-forming units per ml.

Pulmonary removal of circulating endotoxin results in acute lung injury in sheep.

Endotoxemia has often been associated with the development of the adult respiratory distress syndrome. Our previous studies have shown that sheep, a popular animal model for adult respiratory distress syndrome, have abundant resident pulmonary intravascular macrophages that rapidly remove inorganic particles and live bacteria from the circulating blood. In this study, we examine the fate of circulating endotoxin in sheep and correlated the site of uptake with early morphologic evidence of tissue injury. Mature sheep and rats received intravenous 125I-labeled lipopolysaccharide (LPS). The dose was 0.8 microgram/kg in sheep and 17.0 micrograms/kg in rats. 125I-LPS clearance from the blood was assayed by gamma-counting of blood samples drawn over 1 hour and was correlated with circulating leukocyte numbers. The distribution of 125I-LPS was determined by gamma-counting of samples of major organs and tissues at time of necropsy. Lungs and liver were examined morphologically in both species. The half-life of circulating 125I-LPS was 2.38 minutes in sheep, and 12.39 minutes in rats. The endotoxin content of the lungs after injection was 77.58% of the total recovered dose in sheep, but only 2.02% in rats. Neutrophil margination occurred in the lungs of both species. In sheep, almost 25 minutes elapsed before peripheral neutrophil numbers decreased by 50%, much longer than the time required for LPS sequestration in the lungs. Rapid LPS uptake by the sheep lungs was associated with early (10-minute) ultrastructural changes including signs of pulmonary intravascular macrophage activation and microvascular congestion. By 60 minutes, many capillaries were occluded with neutrophils, platelets, and fibrin. There was interstitial edema, and endothelial cells showed evidence of severe injury. We conclude that in contrast to the rat, the sheep clears circulating endotoxin more rapidly and preferentially sequesters it in the lungs. Subsequent release of mediators by activated pulmonary intravascular macrophages may then lead to influx of other inflammatory cells and cascading injury.

Uptake of bloodborne bacteria by pulmonary intravascular macrophages and consequent inflammatory responses in sheep.

Bacteria are primarily removed from the bloodstream of most species by mononuclear phagocytic cells in the liver and spleen. We have recently described large numbers of pulmonary intravascular macrophages in several ruminant species that also remove inert particles from the bloodstream. To determine the role of these cells in removal of bacteria from the bloodstream, sheep and rats were injected intravenously with a single dose of Pseudomonas aeruginosa, and kinetics of clearance, relative uptake among organs, and morphologic changes in the lungs and liver 1 h after injection were compared. In rats, bacteria were removed primarily by the liver, and pathologic changes were found in hepatic sinusoids but not in the lungs. However, in sheep, bacteria were removed primarily by the lungs, and pulmonary capillaries became filled with neutrophils, platelets, and fibrin deposits. We propose that uptake of bacteria by pulmonary intravascular macrophages and subsequent release of inflammatory mediators are central to the pathologic changes produced in ruminant models of sepsis-induced adult respiratory distress syndrome.