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1.  Micro-Flow Imaging: Flow Microscopy Applied to Sub-visible Particulate Analysis in Protein Formulations 
The AAPS Journal  2010;12(3):455-464.
The need to monitor, measure, and control sub-visible proteinaceous particulates in biopharmaceutical formulations has been emphasized in recent publications and commentaries. Some of these particulates can be highly transparent, fragile, and unstable. In addition, for much of the size range of concern, no practical measurement method with adequate sensitivity and repeatability has been available. A complication in measuring protein particulates in many formulations is the simultaneous presence of other particle types such as silicone micro-droplets, air bubbles, and extrinsic contaminants. The need has therefore been identified for new analytical methods which can accurately measure and characterize sub-visible particulates in formulations. Micro-flow imaging has been shown to provide high sensitivity in detecting and imaging transparent protein particles and a unique capability to independently analyze such populations even when other particle types are present.
PMCID: PMC2895433  PMID: 20517661
light obscuration; micro-flow imaging; particle sizing; protein aggregation; protein formulation
2.  Flow Cytometry: A Promising Technique for the Study of Silicone Oil-Induced Particulate Formation in Protein Formulations 
Analytical biochemistry  2010;410(2):191-199.
Subvisible particles in formulations intended for parenteral administration are of concern in the biopharmaceutical industry. However, monitoring and control of subvisible particulates can be complicated by formulation components, such as the silicone oil used for the lubrication of prefilled syringes, and it is difficult to differentiate microdroplets of silicone oil from particles formed by aggregated protein. In this study, we demonstrate the ability of flow cytometry to resolve mixtures comprising subvisible bovine serum albumin (BSA) aggregate particles and silicone oil emulsion droplets with adsorbed BSA. Flow cytometry was also utilized to investigate the effects of silicone oil emulsions on the stability BSA, lysozyme, abatacept or trastuzumab formulations containing surfactant, sodium chloride or sucrose. To aid in particle characterization, the fluorescence detection capabilities of Flow cytometry were exploited by staining silicone oil with BODIPY® 493/503 and model proteins with Alexa Fluor® 647. Flow cytometric analyses revealed that silicone oil emulsions induced the loss of soluble protein via protein adsorption onto the silicone oil droplet surface. Addition of surfactant prevented protein from adsorbing onto the surface of silicone oil droplets. There was minimal formation of homogeneous protein aggregates due to exposure to silicone oil droplets, although oil droplets with surface-adsorbed trastuzumab exhibited flocculation. The results of this study demonstrate the utility of flow cytometry as an analytical tool for monitoring the effects of subvisible silicone oil droplets on the stability of protein formulations.
PMCID: PMC3040987  PMID: 21146492
protein aggregation; adsorption; silicone oil; formulation; fluorescence; flow cytometry
3.  Measurement of Subvisible Particulates in Lyophilised Erwinia chrysanthemil-asparaginase and Relationship with Clinical Experience 
The AAPS Journal  2014;16(4):784-790.
In order to generate further characterisation data for the lyophilised product Erwinia chrysanthemil-asparaginase, reconstituted drug product (DP; marketed as Erwinase or Erwinaze) was analysed for subvisible (2–10 μm) particulate content using both the light obscuration (LO) method and the newer flow-imaging microscopy (FIM) technique. No correlation of subvisible particulate counts exists between FIM and LO nor do the counts correlate with activity at both release and on stability. The subvisible particulate content of lyophilised Erwinia l-asparaginase appears to be consistent and stable over time and in line with other parenteral biopharmaceutical products. The majority (ca. 75%) of subvisible particulates in l-asparaginase DP were at the low end of the measurement range by FIM (2–4 μm). In this size range, FIM was unable to definitively classify the particulates as either protein or non-protein. More sensitive measurement techniques would be needed to classify the particulates in lyophilised l-asparaginase into type (protein and non-protein), so the LO technique has been chosen for on-going DP analyses. E. chrysanthemil-asparaginase has a lower rate of hypersensitivity compared with native Escherichia coli preparations, but a subset of patients develop hypersensitivity to the Erwinia enzyme. A DP lot that had subvisible particulate counts on the upper end of the measurement range by both LO and FIM had the same incidence of allergic hypersensitivity in clinical experience as lots at all levels of observed subvisible particulate content, suggesting that the presence of l-asparaginase subvisible particulates is not important with respect to allergic response.
PMCID: PMC4070265  PMID: 24854894
Erwinia; flow-imaging microscopy; l-asparaginase; light obscuration; subvisible particulates
4.  Flow Imaging Microscopy for Protein Particle Analysis—A Comparative Evaluation of Four Different Analytical Instruments 
The AAPS Journal  2013;15(4):1200-1211.
Flow imaging microscopy was introduced as a technique for protein particle analysis a few years ago and has strongly gained in importance ever since. The aim of the present study was a comparative evaluation of four of the most relevant flow imaging microscopy systems for biopharmaceuticals on the market: Micro-Flow Imaging (MFI)4100, MFI5200, Flow Cytometer And Microscope (FlowCAM) VS1, and FlowCAM PV. Polystyrene standards, particles generated from therapeutic monoclonal antibodies, and silicone oil droplets were analyzed by all systems. The performance was critically assessed regarding quantification, characterization, image quality, differentiation of protein particles and silicone oil droplets, and handling of the systems. The FlowCAM systems, especially the FlowCAM VS1, showed high-resolution images. The FlowCAM PV system provided the most precise quantification of particles of therapeutic monoclonal antibodies, also under impaired optical conditions by an increased refractive index of the formulation. Furthermore, the most accurate differentiation of protein particles and silicone oil droplets could be achieved with this instrument. The MFI systems provided excellent size and count accuracy (evaluated with polystyrene standards) especially the MFI5200 system. This instrument also showed very good performance for protein particles, also in case of an increased refractive index of the formulation. Both MFI systems were easier to use and appeared more standardized regarding measurement and data analysis as compared to the FlowCAM systems. Our study shows that the selection of the appropriate flow imaging microscopy system depends strongly on the main output parameters of interest and it is recommended to decide based on the intended application.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-013-9522-2) contains supplementary material, which is available to authorized users.
PMCID: PMC3787219  PMID: 23996547
flow imaging; particle analysis; protein particles; silicone oil droplets; subvisible particles
5.  Air Cleaning Technologies 
Executive Summary
This health technology policy assessment will answer the following questions:
When should in-room air cleaners be used?
How effective are in-room air cleaners?
Are in-room air cleaners that use combined HEPA and UVGI air cleaning technology more effective than those that use HEPA filtration alone?
What is the Plasmacluster ion air purifier in the pandemic influenza preparation plan?
The experience of severe acute respiratory syndrome (SARS) locally, nationally, and internationally underscored the importance of administrative, environmental, and personal protective infection control measures in health care facilities. In the aftermath of the SARS crisis, there was a need for a clearer understanding of Ontario’s capacity to manage suspected or confirmed cases of airborne infectious diseases. In so doing, the Walker Commission thought that more attention should be paid to the potential use of new technologies such as in-room air cleaning units. It recommended that the Medical Advisory Secretariat of the Ontario Ministry of Health and Long-Term Care evaluate the appropriate use and effectiveness of such new technologies.
Accordingly, the Ontario Health Technology Advisory Committee asked the Medical Advisory Secretariat to review the literature on the effectiveness and utility of in-room air cleaners that use high-efficiency particle air (HEPA) filters and ultraviolet germicidal irradiation (UVGI) air cleaning technology.
Additionally, the Ontario Health Technology Advisory Committee prioritized a request from the ministry’s Emergency Management Unit to investigate the possible role of the Plasmacluster ion air purifier manufactured by Sharp Electronics Corporation, in the pandemic influenza preparation plan.
Clinical Need
Airborne transmission of infectious diseases depends in part on the concentration of breathable infectious pathogens (germs) in room air. Infection control is achieved by a combination of administrative, engineering, and personal protection methods. Engineering methods that are usually carried out by the building’s heating, ventilation, and air conditioning (HVAC) system function to prevent the spread of airborne infectious pathogens by diluting (dilution ventilation) and removing (exhaust ventilation) contaminated air from a room, controlling the direction of airflow and the air flow patterns in a building. However, general wear and tear over time may compromise the HVAC system’s effectiveness to maintain adequate indoor air quality. Likewise, economic issues may curtail the completion of necessary renovations to increase its effectiveness. Therefore, when exposure to airborne infectious pathogens is a risk, the use of an in-room air cleaner to reduce the concentration of airborne pathogens and prevent the spread of airborne infectious diseases has been proposed as an alternative to renovating a HVAC system.
Airborne transmission is the spread of infectious pathogens over large distances through the air. Infectious pathogens, which may include fungi, bacteria, and viruses, vary in size and can be dispersed into the air in drops of moisture after coughing or sneezing. Small drops of moisture carrying infectious pathogens are called droplet nuclei. Droplet nuclei are about 1 to 5μm in diameter. This small size in part allows them to remain suspended in the air for several hours and be carried by air currents over considerable distances. Large drops of moisture carrying infectious pathogens are called droplets. Droplets being larger than droplet nuclei, travel shorter distances (about 1 metre) before rapidly falling out of the air to the ground. Because droplet nuclei remain airborne for longer periods than do droplets, they are more amenable to engineering infection control methods than are droplets.
Droplet nuclei are responsible for the airborne transmission of infectious diseases such as tuberculosis, chicken pox (varicella), measles (rubeola), and dessiminated herpes zoster, whereas close contact is required for the direct transmission of infectious diseases transmitted by droplets, such as influenza (the flu) and SARS.
The Technology
In-room air cleaners are supplied as portable or fixed devices. Fixed devices can be attached to either a wall or ceiling and are preferred over portable units because they have a greater degree of reliability (if installed properly) for achieving adequate room air mixing and airflow patterns, which are important for optimal effectiveness.
Through a method of air recirculation, an in-room air cleaner can be used to increase room ventilation rates and if used to exhaust air out of the room it can create a negative-pressure room for airborne infection isolation (AII) when the building’s HVAC system cannot do so. A negative-pressure room is one where clean air flows into the room but contaminated air does not flow out of it. Contaminated room air is pulled into the in-room air cleaner and cleaned by passing through a series of filters, which remove the airborne infectious pathogens. The cleaned air is either recirculated into the room or exhausted outside the building. By filtering contaminated room air and then recirculating the cleaned air into the room, an in-room air cleaner can improve the room’s ventilation. By exhausting the filtered air to the outside the unit can create a negative-pressure room. There are many types of in-room air cleaners. They vary widely in the airflow rates through the unit, the type of air cleaning technology used, and the technical design.
Crucial to maximizing the efficiency of any in-room air cleaner is its strategic placement and set-up within a room, which should be done in consultation with ventilation engineers, infection control experts, and/or industrial hygienists. A poorly positioned air cleaner may disrupt airflow patterns within the room and through the air cleaner, thereby compromising its air cleaning efficiency.
The effectiveness of an in-room air cleaner to remove airborne pathogens from room air depends on several factors, including the airflow rate through the unit’s filter and the airflow patterns in the room. Tested under a variety of conditions, in-room air cleaners, including portable or ceiling mounted units with either a HEPA or a non-HEPA filter, portable units with UVGI lights only, or ceiling mounted units with combined HEPA filtration and UVGI lights, have been estimated to be between 30% and 90%, 99% and 12% and 80% effective, respectively. However, and although their effectiveness is variable, the United States Centers for Disease Control and Prevention has acknowledged in-room air cleaners as alternative technology for increasing room ventilation when this cannot be achieved by the building’s HVAC system with preference given to fixed recirculating systems over portable ones.
Importantly, the use of an in-room air cleaner does not preclude either the need for health care workers and visitors to use personal protective equipment (N95 mask or equivalent) when entering AII rooms or health care facilities from meeting current regulatory requirements for airflow rates (ventilation rates) in buildings and airflow differentials for effective negative-pressure rooms.
The Plasmacluster ion technology, developed in 2000, is an air purification technology. Its manufacturer, Sharp Electronics Corporation, says that it can disable airborne microorganisms through the generation of both positive and negative ions. (1) The functional unit is the hydroxyl, which is a molecule comprised of one oxygen molecule and one hydrogen atom.
Plasmacluster ion air purifier uses a multilayer filter system composed of a prefilter, a carbon filter, an antibacterial filter, and a HEPA filter, combined with an ion generator to purify the air. The ion generator uses an alternating plasma discharge to split water molecules into positively and negatively charged ions. When these ions are emitted into the air, they are surrounded by water molecules and form cluster ions which are attracted to airborne particles. The cluster ion surrounds the airborne particle, and the positive and negative ions react to form hydroxyls. These hydroxyls steal the airborne particle’s hydrogen atom, which creates a hole in the particle’s outer protein membrane, thereby rendering it inactive.
Because influenza is primarily acquired by large droplets and direct and indirect contact with an infectious person, any in-room air cleaner will have little benefit in controlling and preventing its spread. Therefore, there is no role for the Plasmacluster ion air purifier or any other in-room air cleaner in the control of the spread of influenza. Accordingly, for purposes of this review, the Medical Advisory Secretariat presents no further analysis of the Plasmacluster.
Review Strategy
The objective of the systematic review was to determine the effectiveness of in-room air cleaners with built in UVGI lights and HEPA filtration compared with those using HEPA filtration only.
The Medical Advisory Secretariat searched the databases of MEDLINE, EMBASE, Cochrane Database of Systematic Reviews, INAHATA (International Network of Agencies for Health Technology Assessment), Biosis Previews, Bacteriology Abstracts, Web of Science, Dissertation Abstracts, and NIOSHTIC 2.
A meta-analysis was conducted if adequate data was available from 2 or more studies and where statistical and clinical heterogeneity among studies was not an issue. Otherwise, a qualitative review was completed. The GRADE system was used to summarize the quality of the body of evidence comprised of 1 or more studies.
Summary of Findings
There were no existing health technology assessments on air cleaning technology located during the literature review. The literature search yielded 59 citations of which none were retained. One study was retrieved from a reference list of a guidance document from the United States Centers for Disease Control and Prevention, which evaluated an in-room air cleaner with combined UVGI lights and HEPA filtration under 2 conditions: UVGI lights on and UVGI lights off. Experiments were performed using different ventilation rates and using an aerosolized pathogen comprised of Mycobaterium parafortuitum, a surrogate for the bacterium that causes tuberculosis. Effectiveness was measured as equivalent air changes per hour (eACH). This single study formed the body of evidence for our systematic review research question.
Experimental Results
The eACH rate for the HEPA-UVGI in-room air cleaner was statistically significantly greater when the UV lights were on compared with when the UV lights were off. (P < .05). However, subsequent experiments could not attribute this to the UVGI. Consequently, the results are inconclusive and an estimate of effect (benefit) is uncertain.
The study was reviewed by a scientific expert and rated moderate for quality. Further analysis determined that there was some uncertainty in the directness of the outcome measure (eACH); thus, the GRADE level for the quality of the evidence was low indicating that an estimate of effect is very uncertain.
There is uncertainty in the benefits of using in-room air cleaners with combined UVGI lights and HEPA filtration over systems that use HEPA filtration alone. However, there are no known risks to using systems with combined UVGI and HEPA technology compared with those with HEPA alone. There is an increase in the burden of cost including capital costs (cost of the device), operating costs (electricity usage), and maintenance costs (cleaning and replacement of UVGI lights) to using an in-room air cleaner with combined UVGI and HEPA technology compared with those with HEPA alone. Given the uncertainty of the estimate of benefits, an in-room air cleaner with HEPA technology only may be an equally reasonable alternative to using one with combined UVGI and HEPA technology
In-room air cleaners may be used to protect health care staff from air borne infectious pathogens such as tuberculosis, chicken pox, measles, and dessiminated herpes zoster. In addition, and although in-room air cleaners are not effective at protecting staff and preventing the spread of droplet-transmitted diseases such as influenza and SARS, they may be deployed in situations with a novel/emerging infectious agent whose epidemiology is not yet defined and where airborne transmission is suspected.
It is preferable that in-room air cleaners be used with a fixed and permanent room placement when ventilation requirements must be improved and the HVAC system cannot be used. However, for acute (temporary) situations where a novel/emerging infectious agent presents whose epidemiology is not yet defined and where airborne transmission is suspected it may be prudent to use the in room air cleaner as a portable device until mode of transmission is confirmed. To maximize effectiveness, consultation with an environmental engineer and infection control expert should be undertaken before using an in-room air cleaner and protocols for maintenance and monitoring of these devices should be in place.
If properly installed and maintained, in room air cleaners with HEPA or combined HEPA and UVGI air cleaning technology are effective in removing airborne pathogens. However, there is only weak evidence available at this time regarding the benefit of using an in-room air cleaner with combined HEPA and UVGI air cleaner technology instead of those with HEPA filter technology only.
PMCID: PMC3382390  PMID: 23074468
6.  Protein Particulate Detection Issues in Biotherapeutics Development—Current Status 
AAPS PharmSciTech  2012;13(2):732-746.
Formation of aggregates and particulates in biopharmaceutical formulation continues to be one of the major quality concerns in biotherapeutics development. The presence of large quantities of aggregates is believed to be one of the causes of unwanted immunogenic responses. Protein particulates can form in a wide range of sizes and shapes. Therefore, a comprehensive characterization of particulates in biologics formulation continues to be challenging. The quantity of small size aggregates (e.g., dimer) in a stable biologics formulation is well controlled using precision analytical techniques (e.g., high-performance liquid chromatography). Particulate in clinical and commercial formulations is monitored using visual inspection and subvisible particulate counting assays. While visual inspection (by human eye or automated systems) is intended to detect particulates (intrinsic and extrinsic) of ~100 μm or larger, the subvisible counting methods cover smaller size ranges down to 10 μm. It is well recognized that research of particulates in the submicron (<1 μm) and low-micron (1–10 μm) ranges may provide important clues to understand the mechanism of particulate formation. The recent years have seen a significant increase in the development of newer technologies for more comprehensive characterization of particulates. This is attributed to increased awareness in this field of research over the past 5 years, stimulated by scholarly articles, commentaries, and robust discussions in various forums. This article provides an overview of emerging detection technologies that provide complementary characterization data encompassing a wider size range of particulates. It also discusses their advantages and limitations in the context of applications in biotherapeutics development.
PMCID: PMC3364383  PMID: 22566174
biotherapeutics; formulation development; laser diffraction; particulate matter; protein aggregation
7.  Air Pollution and the Microvasculature: A Cross-Sectional Assessment of In Vivo Retinal Images in the Population-Based Multi-Ethnic Study of Atherosclerosis (MESA) 
PLoS Medicine  2010;7(11):e1000372.
Sara Adar and colleagues show that residing in locations with higher air pollution concentrations and experiencing daily increases in air pollution are associated with narrower retinal arteriolar diameters in older individuals, thus providing a link between air pollution and cardiovascular disease.
Long- and short-term exposures to air pollution, especially fine particulate matter (PM2.5), have been linked to cardiovascular morbidity and mortality. One hypothesized mechanism for these associations involves microvascular effects. Retinal photography provides a novel, in vivo approach to examine the association of air pollution with changes in the human microvasculature.
Methods and Findings
Chronic and acute associations between residential air pollution concentrations and retinal vessel diameters, expressed as central retinal arteriolar equivalents (CRAE) and central retinal venular equivalents (CRVE), were examined using digital retinal images taken in Multi-Ethnic Study of Atherosclerosis (MESA) participants between 2002 and 2003. Study participants (46 to 87 years of age) were without clinical cardiovascular disease at the baseline examination (2000–2002). Long-term outdoor concentrations of PM2.5 were estimated at each participant's home for the 2 years preceding the clinical exam using a spatio-temporal model. Short-term concentrations were assigned using outdoor measurements on the day preceding the clinical exam. Residential proximity to roadways was also used as an indicator of long-term traffic exposures. All associations were examined using linear regression models adjusted for subject-specific age, sex, race/ethnicity, education, income, smoking status, alcohol use, physical activity, body mass index, family history of cardiovascular disease, diabetes status, serum cholesterol, glucose, blood pressure, emphysema, C-reactive protein, medication use, and fellow vessel diameter. Short-term associations were further controlled for weather and seasonality. Among the 4,607 participants with complete data, CRAE were found to be narrower among persons residing in regions with increased long- and short-term levels of PM2.5. These relationships were observed in a joint exposure model with −0.8 µm (95% confidence interval [CI] −1.1 to −0.5) and −0.4 µm (95% CI −0.8 to 0.1) decreases in CRAE per interquartile increases in long- (3 µg/m3) and short-term (9 µg/m3) PM2.5 levels, respectively. These reductions in CRAE are equivalent to 7- and 3-year increases in age in the same cohort. Similarly, living near a major road was also associated with a −0.7 µm decrease (95% CI −1.4 to 0.1) in CRAE. Although the chronic association with CRAE was largely influenced by differences in exposure between cities, this relationship was generally robust to control for city-level covariates and no significant differences were observed between cities. Wider CRVE were associated with living in areas of higher PM2.5 concentrations, but these findings were less robust and not supported by the presence of consistent acute associations with PM2.5.
Residing in regions with higher air pollution concentrations and experiencing daily increases in air pollution were each associated with narrower retinal arteriolar diameters in older individuals. These findings support the hypothesis that important vascular phenomena are associated with small increases in short-term or long-term air pollution exposures, even at current exposure levels, and further corroborate reported associations between air pollution and the development and exacerbation of clinical cardiovascular disease.
Please see later in the article for the Editors' Summary
Editors' Summary
Cardiovascular disease (CVD)—disease that affects the heart and/or the blood vessels—is a common cause of illness and death among adults in developed countries. In the United States, for example, the leading cause of death is coronary heart disease, a CVD in which narrowing of the heart's arteries by atherosclerotic plaques (fatty deposits that build up with age) slows the blood supply to the heart and may eventually cause a heart attack (myocardial infarction). Other types of CVD include stroke (in which atherosclerotic plaques interrupt the brain's blood supply) and peripheral arterial disease (in which the blood supply to the limbs is blocked). Smoking, high blood pressure, high blood levels of cholesterol (a type of fat), having diabetes, being overweight, and being physically inactive all increase a person's risk of developing CVD. Treatments for CVD include lifestyle changes and taking drugs that lower blood pressure or blood cholesterol levels.
Why Was This Study Done?
Another risk factor for CVD is exposure to long-term and/or short-term air pollution. Fine particle pollution or PM2.5 is particularly strongly associated with an increased risk of CVD. PM2.5—particulate matter 2.5 µm in diameter or 1/30th the diameter of a human hair—is mainly produced by motor vehicles, power plants, and other combustion sources. Why PM2.5 increases CVD risk is not clear but one possibility is that it alters the body's microvasculature (fine blood vessels known as capillaries, arterioles, and venules), thereby impairing the blood flow through the heart and brain. In this study, the researchers use noninvasive digital retinal photography to investigate whether there is an association between air pollution and changes in the human microvasculature. The retina—a light-sensitive layer at the back of the eye that converts images into electrical messages and sends them to the brain—has a dense microvasculature. Retinal photography is used to check the retinal microvasculature for signs of potentially blinding eye diseases such as diabetic retinopathy. Previous studies have found that narrower than normal retinal arterioles and wider than normal retinal venules are associated with CVD.
What Did the Researchers Do and Find?
The researchers used digital retinal photography to measure the diameters of retinal blood vessels in the participants of the Multi-Ethnic Study of Atherosclerosis (MESA). This study is investigating CVD progression in people aged 45–84 years of various ethnic backgrounds who had no CVD symptoms when they enrolled in the study in 2000–2002. The researchers modeled the long-term outdoor concentration of PM2.5 at each participant's house for the 2-year period preceding the retinal examination (which was done between 2002 and 2003) using data on PM2.5 levels collected by regulatory monitoring stations as well as study-specific air samples collected outside of the homes and in the communities of study participants. Outdoor PM2.5 measurements taken the day before the examination provided short-term PM2.5 levels. Among the 4,607 MESA participants who had complete data, retinal arteriolar diameters were narrowed among those who lived in regions with increased long- and short-term PM2.5 levels. Specifically, an increase in long-term PM2.5 concentrations of 3 µg/m3 was associated with a 0.8 µm decrease in arteriolar diameter, a reduction equivalent to that seen for a 7-year increase in age in this group of people. Living near a major road, another indicator of long-term exposure to PM2.5 pollution, was also associated with narrowed arterioles. Finally, increased retinal venular diameters were weakly associated with long-term high PM2.5 concentrations.
What Do These Findings Mean?
These findings indicate that living in areas with long-term air pollution or being exposed to short-term air pollution is associated with narrowing of the retinal arterioles in older individuals. They also show that widening of retinal venules is associated with long-term (but not short-term) PM2.5 pollution. Together, these findings support the hypothesis that long- and short-term air pollution increases CVD risk through effects on the microvasculature. However, they do not prove that PM2.5 is the constituent of air pollution that drives microvascular changes—these findings could reflect the toxicity of another pollutant or the pollution mixture as a whole. Importantly, these findings show that microvascular changes can occur at the PM2.5 levels that commonly occur in developed countries, which are well below those seen in developing countries. Worryingly, they also suggest that the deleterious cardiovascular effects of air pollution could occur at levels below existing regulatory standards.
Additional Information
Please access these Web sites via the online version of this summary at 10.1371/journal.pmed.1000372.
The American Heart Association provides information for patients and caregivers on all aspects of cardiovascular disease (in several languages), including information on air pollution, heart disease, and stroke
The US Centers for Disease Control and Prevention has information on heart disease and on stroke
Information is available from the British Heart Foundation on cardiovascular disease
The UK National Health Service Choices website provides information for patients and caregivers about cardiovascular disease
MedlinePlus provides links to other sources of information on heart disease and on vascular disease (in English and Spanish)
The AIRNow site provides information about US air quality and about air pollution and health
The Air Quality Archive has up-to-date information about air pollution in the UK and information about the health effects of air pollution
The US Environmental Protection Agency has information on PM2.5
The following Web sites contain information available on the MESA and MESA Air studies
PMCID: PMC2994677  PMID: 21152417
8.  Number concentration and size of particles in urban air: effects on spirometric lung function in adult asthmatic subjects. 
Environmental Health Perspectives  2001;109(4):319-323.
Daily variations in ambient particulate air pollution are associated with variations in respiratory lung function. It has been suggested that the effects of particulate matter may be due to particles in the ultrafine (0.01-0.1 microm) size range. Because previous studies on ultrafine particles only used self-monitored peak expiratory flow rate (PEFR), we assessed the associations between particle mass and number concentrations in several size ranges measured at a central site and measured (biweekly) spirometric lung function among a group of 54 adult asthmatics (n = 495 measurements). We also compared results to daily morning, afternoon, and evening PEFR measurements done at home (n = 7,672-8,110 measurements). The median (maximum) 24 hr number concentrations were 14,500/cm(3) (46,500/cm(3)) ultrafine particles and 800/cm(3) (2,800/cm(3)) accumulation mode (0.1-1 microm) particles. The median (maximum) mass concentration of PM(2.5) (particulate matter < 2.5 microm) and PM(10) (particulate matter < 10 microm in aerodynamic diameter) were 8.4 microg/m(3) (38.3 microg/m(3)) and 13.5 microg/m(3) (73.7 microg/m(3)), respectively. The number of accumulation mode particles was consistently inversely associated with PEFR in spirometry. Inverse, but nonsignificant, associations were observed with ultrafine particles, and no associations were observed with large particles (PM(10)). Compared to the effect estimates for self-monitored PEFR, the effect estimates for spirometric PEFR tended to be larger. The standard errors were also larger, probably due to the lower number of spirometric measurements. The present results support the need to monitor the particle number and size distributions in urban air in addition to mass.
PMCID: PMC1240270  PMID: 11335178
9.  Free radical activity and pro-inflammatory effects of particulate air pollution (PM10) in vivo and in vitro. 
Thorax  1996;51(12):1216-1222.
BACKGROUND: Epidemiological evidence has implicated fine particulate air pollution, particularly particles less than 10 microns in diameter (PM10), in the development of exacerbations of asthma and chronic obstructive pulmonary disease (COPD) although the mechanism is unknown. The hypothesis that PM10 particles induce oxidant stress, causing inflammation and injury to airway epithelium, was tested. METHODS: The effects of intratracheal instillation of PM10 was assessed in rat lungs (three per group). Inflammatory cell influx was measured by bronchoalveolar lavage (BAL) and air space epithelial permeability was assessed as the total protein in BAL fluid in vivo. The oxidant properties of PM10 particles were determined by their ability to cause damage to plasmid DNA and by changes in reduced (GSH) and oxidised (GSSG) glutathione. The effects of PM10 particles were compared in some experiments with those of fine (CB) and ultrafine (ufCB) carbon black particles. RESULTS: Six hours after intratracheal instillation of PM10 there was an influx of neutrophils (up to 15% of total cells in BAL fluid) into the alveolar space, increased epithelial permeability, the mean (SE) total protein in the BAL fluid increasing from 0.39 (0.01) to 0.62 (0.01) mg/ml, and increased lactate dehydrogenase (LDH) concentrations in the BAL fluid. An even greater inflammatory response was seen following intratracheal instillation of ufCB but not following CB instillation. PM10 particles had free radical activity in vivo, as shown by a decrease in GSH levels in the BAL fluid from 0.36 (0.05) to 0.25 (0.01) nmol/ml following instillation. The free radical activity of PM10 was confirmed in vitro by its ability to deplete supercoiled plasmid DNA, an effect which could be reversed by mannitol, a specific hydroxyl radical scavenger. BAL fluid leucocytes from rats treated with PM10 produced greater amounts of nitric oxide (NO), measured as nitrite (control 3.07 (0.33), treated 4.45 (0.23) microM/1 x 10(6) cells), and tumour necrosis factor alpha (control 21.0 (3.1), treated 179.2 (29.4) units/l x 10(6) cells) in culture than those obtained from control animals. Since the PM10 preparation was contaminated with small amounts of filter fibres due to the extraction process, the effects of instillation of filter fibres alone was assessed. These studies showed that filter fibres did not account for the proinflammatory and injurious effects of the PM10 suspension. CONCLUSIONS: These findings provide evidence that PM10 has free radical activity and causes lung inflammation and epithelial injury. These data support the proposed hypothesis for the mechanism by which particulate air pollution causes adverse effects in patients with airways diseases.
PMCID: PMC472766  PMID: 8994518
10.  Metals in Particulate Pollutants Affect Peak Expiratory Flow of Schoolchildren 
Environmental Health Perspectives  2006;115(3):430-434.
The contribution of the metal components of particulate pollutants to acute respiratory effects has not been adequately evaluated. Moreover, little is known about the effects of genetic polymorphisms of xenobiotic metabolism on pulmonary function.
This study was conducted to assess lung function decrement associated with metal components in particulate pollutants and genetic polymorphisms of glutathione S-transferase M1 and T1.
We studied 43 schoolchildren who were in the 3rd to 6th grades. Each student measured peak expiratory flow rate three times a day for 42 days. Particulate air concentrations were monitored every day, and the concentrations of iron, manganese, lead, zinc, and aluminum in the particles were measured. Glutathione S-transferase M1 and T1 genetic polymorphisms were determined using DNA extracted from participant buccal washings. We used a mixed linear regression model to estimate the association between peak expiratory flow rate and particulate air pollutants.
We found significant reduction in the peak expiratory flow rate after the children’s exposure to particulate pollutants. The effect was shown most significantly 1 day after exposure to the ambient particles. Manganese and lead in the particles also reduced the peak expiratory flow rate. Genetic polymorphisms of glutathione S-transferase M1 and T1 did not significantly affect peak expiratory flow rate.
This study demonstrated that particulate pollutants and metals such as manganese and lead in the particles are associated with a decrement of peak expiratory flow rate. These effects were robust even with consideration of genetic polymorphisms of glutathione S-transferase.
PMCID: PMC1849935  PMID: 17431494
air pollution; genetic polymorphism; lung function; metals; particles
11.  Toxic evaluation of organic extracts from airborne particulate matter in Puerto Rico. 
Environmental Health Perspectives  2000;108(7):635-640.
In recent years, several hypotheses have emerged to explain the toxicologic activity of particulate matter. Organic compounds, ultrafine particles, biologic components, and transition metals are some of the constituents that reportedly exert some type of adverse effect on human health. A considerable fraction of the urban particulate matter consists of carbon compounds, which originate mostly from anthropogenic sources. The toxicity of organic fractions from particulate matter have been mainly evaluated by considering their mutagenic activity. This research expands on the toxicologic profile of organic compounds adsorbed to particulate matter, specifically in Puerto Rico, by using the cytotoxic neutral red bioassay (NRB). The NRB uses normal human epidermal keratinocytes or other types of cells to measure the effect on cell viability when exposed to organic compounds associated to the particles in the air. We validated the NRB for particulate matter by using a standard reference material (SRM 1649). We used the NRB to determine toxicologic differences of extracts between an urban industrialized site with anthropogenic activity versus a coastal region with less human activity. The cytotoxicity associated with organic compounds in particulate matter collected at the urban industrialized site was detected in both the particulate matter (3/4) 10 microm in aerodynamic diameter (PM(10)) and particulate matter (3/4) 100 microm in aerodynamic diameter (PM(100)). Greater toxic effects were observed in PM(10) extracts than in PM(100) extracts, but PM(10) toxic effects were not significantly different from those in PM(100). The extracts from the industrialized site were more cytotoxic than the extracts from coastal reference site, although in the summer, extracts from both sites were significantly cytotoxic to normal human epidermal keratinocytes. In addition, the nonpolar extracts of both PM(10) and PM(100) exerted the greatest cytotoxicity, followed by the polar, and, finally, the moderately polar extract. This study demonstrates that extracts from the Guaynabo industrialized site were more toxic than similar extracts obtained from a reference coastal site in Fajardo, Puerto Rico.
PMCID: PMC1638202  PMID: 10903617
12.  Issues and Challenges of Subvisible and Submicron Particulate Analysis in Protein Solutions 
The AAPS Journal  2012;14(2):236-243.
The analysis of particulates has been a longstanding challenge in biopharmaceutical drug product development and quality control because the active constituents themselves may form particulate matter as a degradation product that may be difficult to quantify. These analytical challenges were met with success as long as the definition of particulate matter remained well within the capabilities of the instruments and methods used to measure it. The current testing as per USP <788> for parenterals at ≤100 mL stipulates that the sample “passes” the test if the average number of particles present does not exceed 6,000 per container at ≥10 μm and does not exceed 600 per container at ≥25 μm. The new challenge, posed by regulatory direction and academic research, is to count and to characterize subvisible particulates that are ≤10 μm with the goal of providing higher resolution information about the particulate levels and potential consequences of this product quality attribute in vivo. The present discussion focuses on two parallel efforts: (a) to develop a model system for protein subvisible particulates in samples with high protein concentrations and (b) to evaluate the capabilities and limitations of different technologies available (at the time these studies were conducted) for subvisible and submicron particle (<1 μm in diameter) sizing and counting. Our findings illustrate the importance of using appropriate instrumentation that is adapted to the characteristics of the samples to be analyzed. Any sample manipulation to meet the capabilities and to accommodate the limitations of the analytical technique should be carefully evaluated.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-012-9335-8) contains supplementary material, which is available to authorized users.
PMCID: PMC3326173  PMID: 22391789
light-scattering methods for particle characterization; particle analysis in high-concentration protein solutions; particle formation; particle size and distribution analysis; submicron particle characterization; subvisible particle characterization
13.  Exposure to Concentrated Ambient Particles Does Not Affect Vascular Function in Patients with Coronary Heart Disease 
Environmental Health Perspectives  2008;116(6):709-715.
Exposure to fine particulate air pollution is associated with increased cardiovascular morbidity and mortality. We previously demonstrated that exposure to dilute diesel exhaust causes vascular dysfunction in humans.
We conducted a study to determine whether exposure to ambient particulate matter causes vascular dysfunction.
Twelve male patients with stable coronary heart disease and 12 age-matched volunteers were exposed to concentrated ambient fine and ultrafine particles (CAPs) or filtered air for 2 hr using a randomized, double-blind cross-over study design. We measured peripheral vascular vasomotor and fibrinolytic function, and inflammatory variables—including circulating leukocytes, serum C-reactive protein, and exhaled breath 8-isoprostane and nitrotyrosine—6–8 hr after both exposures.
Particulate concentrations (mean ± SE) in the exposure chamber (190 ± 37 μg/m3) were higher than ambient levels (31 ± 8 μg/m3) and levels in filtered air (0.5 ± 0.4 μg/m3; p < 0.001). Chemical analysis of CAPs identified low levels of elemental carbon. Exhaled breath 8-isoprostane concentrations increased after exposure to CAPs (16.9 ± 8.5 vs. 4.9 ± 1.2 pg/mL, p < 0.05), but markers of systemic inflammation were largely unchanged. Although there was a dose-dependent increase in blood flow and plasma tissue plasminogen activator release (p < 0.001 for all), CAPs exposure had no effect on vascular function in either group.
Despite achieving marked increases in particulate matter, exposure to CAPs—low in combustion-derived particles—did not affect vasomotor or fibrinolytic function in either middle-aged healthy volunteers or patients with coronary heart disease. These findings contrast with previous exposures to dilute diesel exhaust and highlight the importance of particle composition in determining the vascular effects of particulate matter in humans.
PMCID: PMC2430224  PMID: 18560524
air pollution; blood flow; endothelium; fibrinolysis; inflammation
14.  Response of a Concentrated Monoclonal Antibody Formulation to High Shear 
Biotechnology and bioengineering  2009;103(5):936-943.
There is concern that shear could cause protein unfolding or aggregation during commercial biopharmaceutical production. In this work we exposed two concentrated immunoglobulin-G1 (IgG1) monoclonal antibody (mAb, at >100 mg/mL) formulations to shear rates of between 20,000 and 250,000 s-1 for between 5 minutes and 30 ms using a parallel-plate and capillary rheometer respectively. The maximum shear and force exposures were far in excess of those expected during normal processing operations (20,000 s-1 and 0.06 pN respectively). We used multiple characterization techniques to determine if there was any detectable aggregation. We found that shear alone did not cause aggregation, but that prolonged exposure to shear in the stainless steel parallel-plate rheometer caused a very minor reversible aggregation (<0.3%). Additionally, shear did not alter aggregate populations in formulations containing 17% preformed heat-induced aggregates of a mAb. We calculate that that the forces applied to a protein by production shear exposures (<0.06 pN) are small when compared with the 140 pN force expected at the air-water interface or the 20 to 150 pN forces required to mechanically unfold proteins described in the atomic force microscope (AFM) literature. Therefore, we suggest that in many cases air-bubble entrainment, adsorption to solid surfaces (with possible shear synergy), contamination by particulates, or pump cavitation stresses could be much more important causes of aggregation than shear exposure during production.
PMCID: PMC2724069  PMID: 19370772
protein aggregation; shear; monoclonal antibody; stability; pumping
15.  Acute respiratory effects of particles: mass or number? 
OBJECTIVES—To determine whether associations might be found, in patients with chronic airflow obstruction, between symptoms, peak flow rate (PEF), and particle mass and numbers, and to assess which measure was most closely associated with changes in health. Epidemiological studies have shown associations between particulate air pollution and cardiovascular and respiratory disease, and it has been proposed that these may be mediated by particles of nm size (ultrafine).
METHODS—Relations were investigated between symptom scores, PEF, and bronchodilator use in 44 patients aged ⩾50 years with chronic obstructive pulmonary disease, and daily measurements of both mass of ambient particles of aerodynamic diameter less than 10 µm (PM10) and numbers of ultrafine particles (<100 nm), allowing for meteorological variables. Symptom scores, bronchodilator use, and PEF were recorded daily for 3 months. Counts of ultrafine particles were made by the TSI model 3934 scanning mobility particle sizer (SMPS) and PM10 measurements by the tapered element oscillating microbalance (TEOM).
RESULTS—Ultrafine particle counts indoors and outdoors were significantly correlated, those indoors being about half of those outdoors. No associations were found between actual PEF and PM10 or ultrafine particles. However, there was a 19% increase in the rate of 10% decrements in daytime PEF with increases in PM10 from 10 to 20 µg/m3 which was of borderline significance (p=0.05). A change in PM10 from 10 to 20 µg/m3 was significantly associated with a 14% increase in the rate of high scores of shortness of breath (p=0.003). A similar change in PM10 as a moving average of the same day and 2 previous days was associated with a 31% increase in the rate of high scores for cough (p=0.02). Cough symptoms were also associated with lower temperatures (p=0.02). Higher use of medicines was also associated with higher PM10, but the increases were very small in clinical terms.
CONCLUSIONS—Evidence was not found to support the hypothesis that the component of particulate pollution responsible for effects on respiratory symptoms or function resides in the fraction below 100 nm diameter. The consistent associations between symptoms and PM10 suggest that a contribution of the coarser fraction should not be dismissed. Further studies will be needed before the conclusions of this specific project may be generalised.

Keywords: air pollution; ultrafine particles; chronic obstructive lung disease
PMCID: PMC1740106  PMID: 11171927
16.  Multiscale image-based modeling and simulation of gas flow and particle transport in the human lungs 
Improved understanding of structure and function relationships in the human lungs in individuals and sub-populations is fundamentally important to the future of pulmonary medicine. Image-based measures of the lungs can provide sensitive indicators of localized features, however to provide a better prediction of lung response to disease, treatment and environment, it is desirable to integrate quantifiable regional features from imaging with associated value-added high-level modeling. With this objective in mind, recent advances in computational fluid dynamics (CFD) of the bronchial airways - from a single bifurcation symmetric model to a multiscale image-based subject-specific lung model - will be reviewed. The interaction of CFD models with local parenchymal tissue expansion - assessed by image registration - allows new understanding of the interplay between environment, hot spots where inhaled aerosols could accumulate, and inflammation. To bridge ventilation function with image-derived central airway structure in CFD, an airway geometrical modeling method that spans from the model ‘entrance’ to the terminal bronchioles will be introduced. Finally, the effects of turbulent flows and CFD turbulence models on aerosol transport and deposition will be discussed.
CFD simulation of airflow and particle transport in the human lung has been pursued by a number of research groups, whose interest has been in studying flow physics and airways resistance, improving drug delivery, or investigating which populations are most susceptible to inhaled pollutants. The three most important factors that need to be considered in airway CFD studies are lung structure, regional lung function, and flow characteristics. Their correct treatment is important because the transport of therapeutic or pollutant particles is dependent on the characteristics of the flow by which they are transported; and the airflow in the lungs is dependent on the geometry of the airways and how ventilation is distributed to the peripheral tissue. The human airway structure spans more than 20 generations, beginning with the extra-thoracic airways (oral or nasal cavity, and through the pharynx and larynx to the trachea), then the conducting airways, the respiratory airways, and to the alveoli. The airways in individuals and sub-populations (by gender, age, ethnicity, and normal vs. diseased states) may exhibit different dimensions, branching patterns and angles, and thickness and rigidity. At the local level, one would like to capture detailed flow characteristics, e.g. local velocity profiles, shear stress, and pressure, for prediction of particle transport in an airway (lung structure) model that is specific to the geometry of an individual, to understand how inter-subject variation in airway geometry (normal or pathological) influences the transport and deposition of particles. In a systems biology – or multiscale modeling – approach, these local flow characteristics can be further integrated with epithelial cell models for the study of mechanotransduction. At the global (organ) level, one would like to match regional ventilation (lung function) that is specific to the individual, thus ensuring that the flow that transports inhaled particles is appropriately distributed throughout the lung model. Computational models that do not account for realistic distribution of ventilation are not capable of predicting realistic particle distribution or targeted drug deposition. Furthermore, the flow in the human lung can be transitional or turbulent in the upper and proximal airways, and becomes laminar in the distal airways. The flows in the laminar, transitional and turbulent regimes have different temporal and spatial scales. Therefore, modeling airway structure and predicting gas flow and particle transport at both local and global levels require image-guided multiscale modeling strategies.
In this article, we will review the aforementioned three key aspects of CFD studies of the human lungs: airway structure (conducting airways), lung function (regional ventilation and boundary conditions), and flow characteristics (modeling of turbulent flow and its effect on particle transport). For modeling airway structure, we will focus on the conducting airways, and review both symmetric vs. asymmetric airway models, idealized vs. CT-based airway models, and multiscale subject-specific airway models. Imposition of physiological subject-specific boundary conditions (BCs) in CFD is essential to match regional ventilation in individuals, which is also critical in studying preferential deposition of inhaled aerosols in sub-populations, e.g. normals vs. asthmatics that may exhibit different ventilation patterns. Subject-specific regional ventilation defines flow distributions and characteristics in airway segments and bifurcations, which subsequently determines the transport and deposition of aerosols in the entire lungs. Turbulence models are needed to capture the transient and turbulent nature of the gas flow in the human lungs. Thus, the advantages and disadvantages of different turbulence models as well as their effects on particle transport will be discussed. The ultimate goal of the development is to identify sensitive structural and functional variables in sub-populations of normal and diseased lungs for potential clinical applications.
PMCID: PMC3763693  PMID: 23843310
17.  Respiratory exposures associated with silicon carbide production: estimation of cumulative exposures for an epidemiological study. 
Silicon carbide is produced by heating a mixture of petroleum coke and silica sand to approximately 2000 degrees C in an electric furnace for 36 hours. During heating, large amounts of carbon monoxide are released, sulphur dioxide is produced from residual sulphur in the coke, and hydrocarbon fume is produced by pyrolysis of the coke. Loading and unloading furnaces causes exposures to respirable dust containing crystalline silica, silicon carbide, and hydrocarbons. In the autumn of 1980 extensive measurements were made of personal exposures to air contaminants. Eight hour time weighted exposures to sulphur dioxide ranged from less than 0.1 ppm to 1.5 ppm and respirable participate exposures ranged from 0.01 mg/m3 to 9.0 mg/m3. Geometric mean particulate exposures for jobs ranged from 0.1 mg/m3 to 1.46 mg/m3. The particulate contained varying amounts of alpha-quartz, ranging from less than 1% to 17%, and most quartz exposures were substantially below the threshold limit value of 100 micrograms/m3. Only traces of cristobalite (less than 1%) were found in the particulate. Median exposures to air contaminants in each job were estimated. Since the operations at the plant had been stable over the past 30 years, it was possible to estimate long term exposures of workers to sulphur dioxide, respirable particulate, quartz, total inorganic material, and extractable organic material. Cumulative exposure (average concentration times exposure duration) for each of the air contaminants was estimated for each worker using his job history. There was sufficient independent variability in the sulphur dioxide and respirable particulate cumulative exposures to make an assessment of their independent effects feasible. The theoretical basis for using the cumulative exposure index and its shortcomings for epidemiological applications were presented.
PMCID: PMC1009243  PMID: 6691927
18.  Particulate matter and atherosclerosis: role of particle size, composition and oxidative stress 
Air Pollution has been associated with significant adverse health effects leading to increased morbidity and mortality. Cumulative epidemiological and experimental data have shown that exposure to air pollutants lead to increased cardiovascular ischemic events and enhanced atherosclerosis. It appears that these associations are much stronger with the air particulate matter (PM) component and that in urban areas, the smaller particles could be more pathogenic, as a result of their greater propensity to induce systemic prooxidant and proinflammatory effects. Much is still unknown about the toxicology of ambient particulates as well as the pathogenic mechanisms responsible for the induction of adverse cardiovascular health effects. It is expected that better understanding of these effects will have large implications and may lead to the formulation and implementation of new regulatory policies. Indeed, we have found that ultrafine particles (<0.18 μm) enhance early atherosclerosis, partly due to their high content in redox cycling chemicals and their ability to synergize with known proatherogenic mediators in the promotion of tissue oxidative stress. These changes take place in parallel with increased evidence of phase 2 enzymes expression, via the electrophile-sensitive transcription factor, p45-NFE2 related transcription factor 2 (Nrf2). Exposure to ultrafine particles also results in alterations of the plasma HDL anti-inflammatory function that could be indicative of systemic proatherogenic effects. This article reviews the epidemiological, clinical and experimental animal evidence that support the association of particulate matter with atherogenesis. It also discusses the possible pathogenic mechanisms involved, the physicochemical variables that may be of importance in the greater toxicity exhibited by a small particle size, interaction with genes and other proatherogenic factors as well as important elements to consider in the design of future mechanistic studies.
Extensive epidemiological evidence supports the association of air pollution with adverse health effects [1-3]. It is increasingly being recognized that such effects lead to enhanced morbidity and mortality, mostly due to exacerbation of cardiovascular diseases and predominantly those of ischemic character [4]. Indeed, in addition to the classical risk factors such as serum lipids, smoking, hypertension, aging, gender, family history, physical inactivity and diet, recent data have implicated air pollution as an important additional risk factor for atherosclerosis. This has been the subject of extensive reviews [5,6] and a consensus statement from the American Heart Association [7]. This article reviews the supporting epidemiological and animal data, possible pathogenic mechanisms and future perspectives.
PMCID: PMC2761850  PMID: 19761620
19.  The role of particulate size and chemistry in the association between summertime ambient air pollution and hospitalization for cardiorespiratory diseases. 
Environmental Health Perspectives  1997;105(6):614-620.
In order to address the role that the ambient air pollution mix, comprised of gaseous pollutants and various physical and chemical measures of particulate matter, plays in exacerbating cardiorespiratory disease, daily measures of fine and coarse particulate mass, aerosol chemistry (sulfates and acidity), and gaseous pollution (ozone, nitrogen dioxide, sulfur dioxide, and carbon monoxide) were collected in Toronto, Ontario, Canada, in the summers of 1992, 1993, and 1994. These time series were then compared with concurrent data on the number of daily admissions to hospitals for either cardiac diseases (ischemic heart disease, heart failure, and dysthymias) or respiratory diseases (tracheobronchitis, chronic obstructive long disease, asthma, and pneumonia). After adjusting the admission time series for long-term temporal trends, seasonal variations, the effects of short-term epidemics, day of the week effects, and ambient temperature and dew point temperature, positive associations were observed for all ambient air pollutants for both respiratory and cardiac diseases. Ozone was least sensitive to adjustment for the gaseous and particulate pollution measures. However, the association between the health outcomes and carbon monoxide, fine and coarse mass, sulfate levels and aerosol acidity could be explained by adjustment for exposure to gaseous pollutants. Increases in ozone, nitrogen dioxide, and sulfur dioxide equivalent to their interquartile ranges corresponded to an 11% and 13% increase in daily hospitalizations for respiratory and cardiac diseases, respectively. The inclusion of any one of the particulate air pollutants in multiple regression models did not increase these percentages. Particle mass and chemistry could not be identified as an independent risk factor for the exacerbation of cardiorespiratory diseases in this study beyond that attributable to climate and gaseous air pollution. We recommend that effects of particulate matter on health be assessed in conjunction with temporally covarying gaseous air pollutants.
PMCID: PMC1470088  PMID: 9288496
20.  Characterization of Particles in Protein Solutions: Reaching the Limits of Current Technologies 
The AAPS Journal  2010;12(4):708-715.
Recent publications have emphasized the lack of characterization methods available for protein particles in a size range comprised between 0.1 and 10 μm and the potential risk of immunogenicity associated with such particles. In the present paper, we have investigated the performance of light obscuration, flow microscopy, and Coulter counter instruments for particle counting and sizing in protein formulations. We focused on particles 2–10 μm in diameter and studied the effect of silicon oil droplets originating from the barrel of pre-filled syringes, as well as the effect of high protein concentrations (up to 150 mg/ml) on the accuracy of particle characterization. Silicon oil was demonstrated to contribute significantly to the particle counts observed in pre-filled syringes. Inconsistent results were observed between different protein concentrations in the range 7.5–150 mg/ml for particles <10 μm studied by optical techniques (light obscuration and flow microscopy). However, the Coulter counter measurements were consistent across the same studied concentration range but required sufficient solution conductivity from the formulation buffer or excipients. Our results show that currently available technologies, while allowing comparisons between samples of a given protein at a fixed concentration, may be unable to measure particle numbers accurately in a variety of protein formulations, e.g., at high concentration in sugar-based formulations.
PMCID: PMC2977008  PMID: 20953747
biopharmaceuticals; protein; sub-visible particles
21.  Characterization of Particles in Protein Solutions: Reaching the Limits of Current Technologies 
The AAPS Journal  2010;12(4):708-715.
Recent publications have emphasized the lack of characterization methods available for protein particles in a size range comprised between 0.1 and 10 μm and the potential risk of immunogenicity associated with such particles. In the present paper, we have investigated the performance of light obscuration, flow microscopy, and Coulter counter instruments for particle counting and sizing in protein formulations. We focused on particles 2–10 μm in diameter and studied the effect of silicon oil droplets originating from the barrel of pre-filled syringes, as well as the effect of high protein concentrations (up to 150 mg/ml) on the accuracy of particle characterization. Silicon oil was demonstrated to contribute significantly to the particle counts observed in pre-filled syringes. Inconsistent results were observed between different protein concentrations in the range 7.5–150 mg/ml for particles <10 μm studied by optical techniques (light obscuration and flow microscopy). However, the Coulter counter measurements were consistent across the same studied concentration range but required sufficient solution conductivity from the formulation buffer or excipients. Our results show that currently available technologies, while allowing comparisons between samples of a given protein at a fixed concentration, may be unable to measure particle numbers accurately in a variety of protein formulations, e.g., at high concentration in sugar-based formulations.
PMCID: PMC2977008  PMID: 20953747
biopharmaceuticals; protein; sub-visible particles
22.  Personal PM2.5 exposure and markers of oxidative stress in blood. 
Environmental Health Perspectives  2003;111(2):161-166.
Ambient particulate air pollution assessed as outdoor concentrations of particulate matter less than or equal to 2.5 micro m in diameter (PM(2.5)) in urban background has been associated with cardiovascular diseases at the population level. However, the significance of individual exposure and the involved mechanisms remain uncertain. We measured personal PM(2.5) and carbon black exposure in 50 students four times in 1 year and analyzed blood samples for markers of protein and lipid oxidation, for red blood cell (RBC) and platelet counts, and for concentrations of hemoglobin and fibrinogen. We analyzed protein oxidation in terms of gamma-glutamyl semialdehyde in hemoglobin (HBGGS) and 2-aminoadipic semialdehyde in hemoglobin (HBAAS) and plasma proteins (PLAAS), and lipid peroxidation was measured as malondialdehyde (MDA) in plasma. Median exposures were 16.1 micro g/m(3) for personal PM(2.5) exposure, 9.2 micro g/m(3) for background PM(2.5) concentration, and 8.1 X 10(-6)/m for personal carbon black exposure. Personal carbon black exposure and PLAAS concentration were positively associated (p < 0.01), whereas an association between personal PM(2.5) exposure and PLAAS was only of borderline significance (p = 0.061). A 3.7% increase in MDA concentrations per 10 micro g/m(3) increase in personal PM(2.5) exposure was found for women (p < 0.05), whereas there was no significant relationship for the men. Similarly, positive associations between personal PM(2.5)exposure and both RBC and hemoglobin concentrations were found only in women (p < 0.01). There were no significant relationships between background PM(2.5) concentration and any of the biomarkers. This suggests that exposure to particles in moderate concentrations can induce oxidative stress and increase RBCs in peripheral blood. Personal exposure appears more closely related to these biomarkers potentially related to cardiovascular disease than is ambient PM(2.5) background concentrations.
PMCID: PMC1241344  PMID: 12573899
23.  Particulate emissions from diesel engines: correlation between engine technology and emissions 
In the last 30 years, diesel engines have made rapid progress to increased efficiency, environmental protection and comfort for both light- and heavy-duty applications. The technical developments include all issues from fuel to combustion process to exhaust gas aftertreatment. This paper provides a comprehensive summary of the available literature regarding technical developments and their impact on the reduction of pollutant emission. This includes emission legislation, fuel quality, diesel engine- and exhaust gas aftertreatment technologies, as well as particulate composition, with a focus on the mass-related particulate emission of on-road vehicle applications. Diesel engine technologies representative of real-world on-road applications will be highlighted.
Internal engine modifications now make it possible to minimize particulate and nitrogen oxide emissions with nearly no reduction in power. Among these modifications are cooled exhaust gas recirculation, optimized injections systems, adapted charging systems and optimized combustion processes with high turbulence. With introduction and optimization of exhaust gas aftertreatment systems, such as the diesel oxidation catalyst and the diesel particulate trap, as well as NOx-reduction systems, pollutant emissions have been significantly decreased. Today, sulfur poisoning of diesel oxidation catalysts is no longer considered a problem due to the low-sulfur fuel used in Europe. In the future, there will be an increased use of bio-fuels, which generally have a positive impact on the particulate emissions and do not increase the particle number emissions.
Since the introduction of the EU emissions legislation, all emission limits have been reduced by over 90%. Further steps can be expected in the future. Retrospectively, the particulate emissions of modern diesel engines with respect to quality and quantity cannot be compared with those of older engines. Internal engine modifications lead to a clear reduction of the particulate emissions without a negative impact on the particulate-size distribution towards smaller particles. The residual particles can be trapped in a diesel particulate trap independent of their size or the engine operating mode. The usage of a wall-flow diesel particulate filter leads to an extreme reduction of the emitted particulate mass and number, approaching 100%. A reduced particulate mass emission is always connected to a reduced particle number emission.
PMCID: PMC3973853  PMID: 24606725
Diesel engine technology; Diesel engine emissions; Particulate mass emission; Particle number emission; Technological progress
24.  Latex allergens in tire dust and airborne particles. 
Environmental Health Perspectives  1996;104(11):1180-1186.
The prevalence and severity of latex allergy has increased dramatically in the last 15 years due to exposure to natural rubber products. Although historically this health risk has been elevated in hospital personnel and patients, a recent survey has indicated a significant potential risk for the general population. To obtain a wide-spread source for latex exposure, we have considered tire debris. We have searched for the presence of latex allergens in passenger car and truck tire tread, in debris deposited from the atmosphere near a freeway, and in airborne particulate matter samples representative of the entire year 1993 at two sites in the Los Angeles basin (California). After extraction of the samples with phosphate buffered saline, a modified-ELISA inhibition assay was used to measure relative allergen potency and Western blot analyses were used to identify latex allergens. The inhibition studies with the human IgE latex assay revealed inhibition by the tire tread source samples and ambient freeway dust, as well as by control latex sap and latex glove extracts. Levels of extractable latex allergen per unit of protein extracted were about two orders of magnitude lower for tire tread as compared to latex gloves. Western blot analyses using binding of human IgE from latex-sensitive patients showed a band at 34-36 kDa in all tire and ambient samples. Long Beach and Los Angeles, California, air samples showed four additional bands between 50 and 135 kDa. Alternative Western blot analyses using rabbit IgG raised against latex proteins showed a broad band at 30-50 kDa in all samples, with additional bands in the urban air samples similar to the IgE results. A latex cross-reactive material was identified in mountain cedar. In conclusion, the latex allergens or latex cross-reactive material present in sedimented and airborne particulate material, derived from tire debris, and generated by heavy urban vehicle traffic could be important factors in producing latex allergy and asthma symptoms associated with air pollution particles.
PMCID: PMC1469526  PMID: 8959407
25.  Activation of the Stress Axis and Neurochemical Alterations in Specific Brain Areas by Concentrated Ambient Particle Exposure with Concomitant Allergic Airway Disease 
Environmental Health Perspectives  2006;114(6):870-874.
Exposure to ambient particulate matter (PM) has been linked to respiratory diseases in people living in urban communities. The mechanism by which PM produces these diseases is not clear. We hypothesized that PM could act on the brain directly to stimulate the stress axis and predispose individuals to these diseases. The purpose of this study was to test if exposure to PM can affect brain areas involved in the regulation of neuroendocrine functions, especially the stress axis, and to study whether the presence of preexisting allergic airway disease aggravates the stress response.
Adult male rats (n = 8/group) with or without ovalbumin (OVA)-induced allergic airway disease were exposed to concentrated air particles containing PM with an aerodynamic diameter ≤ 2.5 μm (PM2.5) for 8 hr, generated from ambient air in an urban Grand Rapids, Michigan, community using a mobile air research laboratory (AirCARE 1). Control animals were exposed to normal air and were treated with saline.
A day after PM2.5 exposure, animals were sacrificed and the brains were removed, frozen, and sectioned. The paraventricular nucleus (PVN) and other brain nuclei were micro-dissected, and the concentrations of aminergic neurotransmitters and their metabolites were measured using high-performance liquid chromatography with electrochemical detection. Serum corticosterone levels were measured using radioimmunoassay.
A significant increase in the concentration (mean ± SE, pg/μg protein) of norepinephrine in the PVN was produced by exposure to concentrated ambient particles (CAPs) or OVA alone (12.45 ± 2.7 and 15.84 ± 2.8, respectively) or after sensitization with OVA (19.06 ± 3.8) compared with controls (7.98 ± 1.3; p < 0.05). Serum corticosterone (mean ± SE, ng/mL) was significantly elevated in the OVA + CAPs group (242.786 ± 33.315) and in the OVA-presensitized group (242.786 ± 33.315) compared with CAP exposure alone (114.55 ± 20.9). Exposure to CAPs (alone or in combination with OVA pretreatment) can activate the stress axis, and this could probably play a role in aggravating allergic airway disease.
PMCID: PMC1480501  PMID: 16759987
CAPs; corticosterone; hypothalamus; norepinephrine; stress axis

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