Gestational flu exposure induces changes in neurochemicals, affiliative hormones and brainstem inflammation, in addition to autism-like behaviors in mice.

The prevalence of neurodevelopmental disorders such as autism is increasing, however the etiology of these disorders is unclear and thought to involve a combination of genetic, environmental and immune factors. A recent epidemiological study found that gestational viral exposure during the first trimester increases risk of autism in offspring by twofold. In mice gestational viral exposures alter behavior of offspring, but the biological mechanisms which underpin these behavioral changes are unclear. We hypothesized that gestational viral exposure induces changes in affiliative hormones, brainstem autonomic nuclei and neurotransmitters which are associated with behavioral alterations in offspring. To address this hypothesis, we exposed pregnant mice to influenza A virus (H3N2) on gestational day 9 and determined behavioral, hormonal and brainstem changes in male and female offspring. We found that gestational flu exposure induced dose-dependent alterations in social and aggressive behaviors (p≤0.05) in male and female offspring and increases in locomotor behaviors particularly in male offspring (p≤0.05). We found that flu exposure was also associated with reductions in oxytocin and serotonin (p≤0.05) levels in male and female offspring and sex-specific changes in dopamine metabolism. In addition we found changes in catecholaminergic and microglia density in brainstem tissues of male flu exposed offspring only (p≤0.05). This study demonstrates that gestational viral exposure induces behavioral changes in mice, which are associated with alterations in affiliative hormones. In addition we found sex-specific changes in locomotor behavior, which may be associated with sex-specific alterations in dopamine metabolism and brainstem inflammation. Further investigations into maternal immune responses are necessary to unravel the molecular mechanisms which underpin abnormal hormonal, immune and behavioral responses in offspring after gestational viral exposure.

Host defense peptides in the oral cavity and the lung: similarities and differences.

Peptides with broad-spectrum antimicrobial activity are found in the mucosal surfaces at many sites in the body, including the airway, the oral cavity, and the digestive tract. Based on their in vitro antimicrobial and other immunomodulatory activities, these host defense peptides have been proposed to play an important role in the innate defense against pathogenic microbial colonization. The genes that encode these peptides are up-regulated by pathogens, further supporting their role in innate immune defense. However, the differences in the local microbial environments between the generally sterile airway and the highly colonized oral cavity suggest a more complex role for these peptides in innate immunity. For example, beta-defensin genes are induced in the airway by all bacteria and Toll-like receptor (TLR) agonists primarily through an NF-kappaB-mediated pathway. In contrast, the same genes are induced in the gingival epithelium by only a subset of bacteria and TLR ligands, via different pathways. Furthermore, the environments into which the peptides are secreted--specifically saliva, gingival crevicular fluid, and airway surface fluid--differ greatly and can effect their respective activities in host defense. In this review, we examine the differences and similarities between host defense peptides in the oral cavity and the airway, to gain a better understanding of their contributions to immunity.

Antimicrobial peptides in the airway.

The airway provides numerous defense mechanisms to prevent microbial colonization by the large numbers of bacteria and viruses present in ambient air. An important component of this defense is the antimicrobial peptides and proteins present in the airway surface fluid (ASF), the mucin-rich fluid covering the respiratory epithelium. These include larger proteins such as lysozyme and lactoferrin, as well as the cationic defensin and cathelicidin peptides. While some of these peptides, such as human beta-defensin (hBD)-1, are present constitutively, others, including hBD2 and -3 are inducible in response to bacterial recognition by Toll-like receptor-mediated pathways. These peptides can act as microbicides in the ASF, but also exhibit other activities, including potent chemotactic activity for cells of the innate and adaptive immune systems, suggesting they play a complex role in the host defense of the airway. Inhibition of antimicrobial peptide activity or gene expression can result in increased susceptibility to infections. This has been observed with cystic fibrosis (CF), where the CF phenotype leads to reduced antimicrobial capacity of peptides in the airway. Pathogenic virulence factors can inhibit defensin gene expression, as can environmental factors such as air pollution. Such an interference can result in infections by airway-specific pathogens including Bordetella bronchiseptica, Mycobacterium tuberculosis, and influenza virus. Research into the modulation of peptide gene expression in animal models, as well as the optimization of peptide-based therapeutics shows promise for the treatment and prevention of airway infectious diseases.

Pseudomonas aeruginosa ExoT inhibits in vitro lung epithelial wound repair.

The nosocomial pathogen Pseudomonas aeruginosa causes clinical infection in the setting of pre-existing epithelial tissue damage, an association that is mirrored by the increased ability of P. aeruginosa to bind, invade and damage injured epithelial cells in vitro. In this study, we report that P. aeruginosa inhibits the process of epithelial wound repair in vitro through the type III-secreted bacterial protein ExoT, a GTPase-activating protein (GAP) for Rho family GTPases. This inhibition primarily targets cells at the edge of the wound, and causes actin cytoskeleton collapse, cell rounding and cell detachment. ExoT-dependent inhibition of wound repair is mediated through the GAP activity of this bacterial protein, as mutations in ExoT that alter the conserved arginine (R149) within the GAP domain abolish the ability of P. aeruginosa to inhibit wound closure. Because ExoT can also inhibit P. aeruginosa internalization by phagocytes and epithelial cells, this protein may contribute to the in vivo virulence of P. aeruginosa by allowing organisms both to overcome local host defences, such as an intact epithelial barrier, and to evade phagocytosis by immune effector cells.

Prevention of influenza-induced lung injury in mice overexpressing extracellular superoxide dismutase.

Reactive oxygen and nitrogen species such as superoxide and nitric oxide are released into the extracellular spaces by inflammatory and airway epithelial cells. These molecules may exacerbate lung injury after influenza virus pneumonia. We hypothesized that enhanced expression of extracellular superoxide dismutase (EC SOD) in mouse airways would attenuate the pathological effects of influenza pneumonia. We compared the pathogenic effects of a nonlethal primary infection with mouse-adapted Hong Kong influenza A/68 virus in transgenic (TG) EC SOD mice versus non-TG (wild-type) littermates. Compared with wild-type mice, EC SOD TG mice showed less lung injury and inflammation as measured by significant blunting of interferon-gamma induction, reduced cell count and total protein in bronchoalveolar lavage fluid, reduced levels of lung nitrite/nitrate nitrotyrosine, and markedly reduced lung pathology. These results demonstrate that enhancing EC SOD in the conducting and distal airways of the lung minimizes influenza-induced lung injury by both ameliorating inflammation and attenuating oxidative stress.

Exposure to ultraviolet radiation enhances mortality and pathology associated with influenza virus infection in mice.

Ultraviolet radiation (UVR) causes systemic immune suppression, decreasing the delayed type and contact hypersensitivity responses in animals and humans and enhancing certain mycobacterial, parasitic and viral infections in mice. This study tests the hypothesis that prior exposure to UVR enhances influenza infections in mice. BALB/c female mice were exposed to 0-8.2 kJ/m2 of UVR. Exposed and unexposed mice were infected intranasally three days later with 150-300 plaque-forming units/mouse (lethal dose (LD)20-LD40) of mouse-adapted Hong Kong Influenza A/68 (H3N2) virus or sham infected with 50 microL Hanks' balanced salt solution/mouse. Mortality from viral infection ranged from 25-50%. UVR exposure increased virus-associated mortality in a dose-dependent manner (up to a two-fold increase at 8.2 kJ/m2). The increased mortality was not associated with bacterial pneumonia. The highest dose of UVR also accelerated the body weight loss and increased the severity and incidence of thymic atrophy associated with influenza infection. However, UVR treatment had little effect on the increase in lung wet weight seen with viral infection, and, to our surprise, did not cause an increase in virus titers in the lung or dissemination of virus. The mice died 5-6 days after infection, too early for adaptive immune responses to have much impact. Also, UVR did not interfere with the development of protective immunity to influenza, as measured by reinfection with a lethal challenge of virus. Also, cells adoptively transferred from UVR or untreated mice were equally protective of recipient mice challenged with a lethal dose of virus. The mice resemble mice succumbing to endotoxin, and influenza infection increased the levels of tumor necrosis factor alpha (TNF-alpha) in bronchoalveolar lavage fluid and serum cortisol levels; however, UVR preexposure did not increase either of these responses to the virus. The results show that UVR increased the morbidity, mortality and pathogenesis of influenza virus in mice without affecting protective immunity to the virus, as measured by resistance to reinfection. The mechanism of enhanced mortality is uncertain, but the data raises concerns that UVR may exacerbate early responses that contribute to the pathogenesis of a primary viral infection.

The innate immune response of the respiratory epithelium.

The respiratory epithelium maintains an effective antimicrobial environment to prevent colonization by microorganisms in inspired air. In addition to constitutively present host defenses which include antimicrobial peptides and proteins, the epithelial cells respond to the presence of microbes by the induction two complementary parts of an innate immune response. The first response is the increased production of antimicrobial agents, and the second is the induction of a signal network to recruit phagocytic cells to contain the infection. Inflammatory mediators released by the recruited cells as well as from the epithelium itself further induce the expression of the antimicrobial agents. The result is an effective prevention of microbial colonization. The epithelial cells recognize the pathogen-associated patterns on microbes by surface receptors such as CD14 and Toll-like receptors. Subsequent signal transduction pathways have been identified which result in the increased transcription of host defense response genes. Diseases such as cystic fibrosis, or environmental exposures such as the inhalation of air pollution particles, may create an environment that impairs the expression or activity of the host defenses in the airway. This can lead to increased susceptibility to airway infections.

Expression of beta-defensin genes in bovine alveolar macrophages.

Bovine alveolar macrophages (BAM) were examined for the expression of beta-defensins and to determine whether their expression could be upregulated by bacterial lipopolysaccharide (LPS), as observed with beta-defensins expressed in bovine tracheal epithelial cells. Four beta-defensins were expressed constitutively in BAM, with bovine neutrophil beta-defensin (BNBD)-4 and BNBD-5 being the most predominant. This is the first evidence of beta-defensin gene expression in a mature myeloid cell. LPS had no effect on beta-defensin expression in BAM, even though tumor necrosis factor alpha (TNF-alpha) production was induced. Nonbacterial inflammatory particles had little effect on beta-defensin gene expression or TNF-alpha production in BAM. We hypothesize that constitutively expressed beta-defensins of alveolar macrophages may have a role in lung host defense.

Characterization of proinflammatory cytokine production and CD14 expression by murine alveolar macrophage cell lines.

Alveolar macrophages, which play a central role in lung defense, produce cytokines that help orchestrate local inflammatory responses. In sepsis and other pathological conditions, bacterial lipopolysaccharide endotoxin can induce alveolar macrophages (AM) to release proinflammatory cytokines, including tumor necrosis factor-alpha, interleukin-1, and interleukin-6. Studying the mechanisms that control alveolar macrophage cytokine production may lead to better therapies for conditions involving inflammatory lung injury. We and others have noted significant differences between alveolar macrophages and peritoneal macrophages, but large numbers of human or murine alveolar macrophages are rarely available for detailed mechanistic studies. We have obtained three murine alveolar macrophage cell lines (AMJ2C8, AMJ2C11, and AMJ2C20) and have begun to characterize their cytokine responses to proinflammatory stimuli. We measured the effects of endotoxin, interferon gamma, and the combination of the two on production of tumor necrosis factor, interleukin-1 beta, and interleukin-6 in each line. We also studied the expression of the endotoxin receptor CD14 by these cells, and investigated the effect of serum on their endotoxin responsiveness. We show here that all three of the cell lines responded in a manner comparable to that of primary murine alveolar macrophages. Observed variations between these lines may reflect the documented heterogeneity seen in populations of primary alveolar macrophages. These cell lines should expand the repertoire of tools available to investigators studying regulation of murine alveolar macrophage responses.

High-frequency intracoronary ultrasound imaging.

Intravascular ultrasound is playing an increasingly important role in the clinical management of coronary interventions. In the past few years the technology for intracoronary ultrasound, in response to clinical pressure, has moved towards lower profile probes with improved handling. While new catheter designs are markedly improved on their predecessors, image quality has not seen significant gains due to the primitive nature of the ultrasound transducer designs. In this article, the potential for improving image quality by increasing the frequency and focusing the ultrasound beam is explored. Basic aspects of transducer implementation are discussed and the acoustic properties of vascular tissues and blood are reviewed. A variety of instruments are used to image coronary and femoral arteries at frequencies ranging from 40 to 200 MHz. These studies serve to illustrate the trade-offs in the development of high frequency IVUS systems. There would appear to be no fundamental reason why frequencies in excess of 50 MHz could not be implemented. Studies using prototype IVUS instruments in the 50 MHz range demonstrate significant improvements in image quality.

Arterial imaging: comparison of high-resolution US and MR imaging with histologic correlation.

The potential roles of intravascular ultrasound (US) and magnetic resonance (MR) imaging in evaluating the artery wall and atherosclerotic plaque were compared. Excised human femoral and carotid arteries were imaged with a 42-MHz intravascular US system and a 1.5-T MR imager equipped with enhanced gradients. In-plane resolution was 40-280 microns for US and 156 microns for MR imaging. Stained histologic tissue sections were obtained for correlation with the imaging findings. Intravascular US and MR imaging both had sufficient resolution and contrast to demonstrate arterial layers and allow distinction of atheroma. Correspondence between structures identified with the two modalities was excellent and in agreement with histologically defined arterial structures. Findings on state-of-the-art intravascular US and MR images correlate well with histologic findings in normal and diseased arteries. Intravascular US has the advantages of speed and resolution, whereas MR imaging demonstrates superior contrast in the depiction of atheroma.

Tissue equivalent vessel phantoms for intravascular ultrasound.

A tissue equivalent arterial vessel phantom has been developed for use in intravascular ultrasound imaging studies. The phantom material is constructed from a cross-linked gelatin matrix to which amorphous silica scattering particles are added. The ultrasonic properties (speed of sound, frequency-dependent attenuation coefficient and frequency-dependent backscatter coefficient) of the phantom material have been characterized at 42 MHz and correspond well with in vitro measurements of excised human arterial tissue. The mechanical properties of the cast vessel phantoms are controlled by varying the concentration of gelatin used in the matrix. Experimentally measured values of the circumferential Young's elastic modulus of hard and soft vessel phantoms agree well with values reported in the literature for human and canine arterial tissue for transmural pressures up to 100 mmHg. The phantoms are therefore suitable models for use in the development of new applications of intravascular ultrasound imaging, such as the assessment regional arterial elasticity.

Ultrasonic measurement of differential displacement and strain in a vascular model.

The potential in intravascular ultrasound imaging for characterizing regional arterial elasticity was examined in an experimental tissue-equivalent vessel model. Differential intrawall displacement measurement, the first step in regional elasticity determination, was investigated using a crosscorrelation tracking algorithm. Calibration studies showed that tracking accuracy varied significantly with tracking direction (axial versus lateral) and position in the field of the transducer. Midfield geometric error in the axial direction for a nominal displacement of 100 microns was 5.5 microns whereas the corresponding error in the lateral direction was 31.7 microns. Displacement was tracked in serial intravascular images of vessel phantoms acquired during stepwise pressurization experiments from 0-250 mmHg. Two-dimensional grey scale maps of axial, lateral and net intrawall displacement components over the full pressurization range were generated. Displacement profiles demonstrated successful detection of differential radial displacement and good correlation with theoretical profiles (root mean square difference 3%). The corresponding experimental strain profiles were significantly noisier (root mean square difference 76%) due to small fluctuations in the displacement data. This work demonstrates that, with further refinement, regional strain mapping in vessel walls with intravascular ultrasound imaging is feasible. Mechanical characterization of arteries may provide a new tool to aid and treating atherosclerotic lesions.

Alveolar macrophages from C3H/HeJ mice show sensitivity to endotoxin.

Alveolar macrophages (AM) orchestrate the release of several cytokines in the lung, including tumor necrosis factor alpha (TNF). Lipopolysaccharide endotoxin (LPS), one of the most potent stimulators of TNF production in macrophages, often contributes to the development of adult respiratory distress syndrome. The mechanism by which LPS induces TNF production in macrophages is unclear. Many studies have employed murine macrophages lavaged from the peritoneal cavity (PM), either resident cells or those obtained following elicitation with sterile thioglycollate (TGPM), as these cells are readily accessible. LPS does not induce TNF in PM or TGPM from C3H/HeJ mice, and the alteration is thought to reside at the post-transcriptional level of gene regulation. Generalization of results from PM to AM may not be warranted, however. While investigating cytokine production by AM cell lines, we observed that C3HAMSV40, a transformed cell line derived from C3H/HeJ AM, made substantial quantities of TNF when stimulated with 1 microgram/ml LPS, prompting us to study TNF production in primary C3H/HeJ AM. In the present study, readily detectable quantities of biologically active TNF were found in supernatants of C3H/HeJ AM that had been stimulated in vitro with 0.01 to 10 micrograms/ml LPS. The amount of TNF produced was not significantly different from that observed with AM from endotoxin-sensitive C3HeB/FeJ mice. LPS induction of TNF in AM from either mouse strain was completely inhibited by polymyxin B, demonstrating that the sensitivity of C3H/HeJ AM was not due to a contaminant in the LPS.(ABSTRACT TRUNCATED AT 250 WORDS)

A 45 to 55 MHz needle-based ultrasound system for invasive imaging.

The design, construction and fabrication of a high frequency needle-based ultrasound imaging system is described. A miniature lead zirconate titanate transducer was mounted opposite a parabolic mirror in a stainless steel needle. By inserting the needle into a tissue, a cross section image of the tissue can be made. Two needle probes were built, a 45 MHz 2.8 mm diameter probe with 125 microns lateral, 55 microns axial resolution and a 55 MHz 1.6 mm diameter probe with 105 microns lateral, 40 microns axial resolution. Preliminary phantom and in vitro tissue images demonstrate the feasibility of high frequency needle-based imaging.

Principles and applications of ultrasound backscatter microscopy.

The development of ultrasound backscatter microscopy (UBM) is described together with initial clinical and biological applications. UBM is essentially an extension of the powerful B-mode backscatter methods developed for clinical imaging in the 3-10-MHz frequency range. The development of new high sensitivity transducers in the 40-100-MHz range now permits visualization of tissue structures with resolution approaching 20 mum and a maximum penetration of approximately 4 mm. The performance characteristics and trade-offs of these new polymer and ceramic devices are reviewed, and the implementation of high-frequency imaging systems is described. Initial clinical applications of UBM include ophthalmic, skin, and intravascular imaging. Examples of images and progress in these areas are presented. The biological application of UBM is illustrated by studies of drug uptake in living tumor spheroids. Significant increases in backscatter levels resulting from drugs targeting oxic and hypoxic cell populations are demonstrated.

In vitro high resolution intravascular imaging in muscular and elastic arteries.

High resolution (125-microns lateral, 55-microns axial) images of 16 muscular (femoral) and 15 elastic (common carotid) human arteries were made in vitro with use of a prototype 45-MHz intravascular imaging system. Four distinct regions of scattering, excluding plaque, were identified in the ultrasound images corresponding histologically to the adventitia, media, thickened intima and elastic laminae, both internal and external. Arterial samples imaged under pressure and in a collapsed state underwent dimensional changes but exhibited similar levels of backscatter amplitude. All the elastic arteries displayed a prominent echogenic media, whereas all the muscular arteries displayed an echolucent media. Scattering from the internal elastic lamina in muscular arteries provided an excellent landmark for defining the location and extent of intimal thickening or plaque. In elastic arteries the internal elastic lamina could not be distinguished from the echogenic media; consequently, the boundary between the media and intimal layer was indistinct. Differences in the relative concentration and organization of collagen and elastin were found to provide a consistent explanation for the differences in scattering that were observed between individual layers within an artery as well as between muscular and elastic arteries.

Release of tumor necrosis factor in guinea pigs upon acute inhalation of cotton dust.

In guinea pigs, inhalation of cotton dust results in an acute pulmonary response with symptoms of increased breathing rate, cough, bronchoconstriction, and periods of apnea. These symptoms resemble those noted in individuals upon exposure to cotton and other organic dusts. A major contaminant of cotton dust is bacterial endotoxin. Because endotoxin, or lipopolysaccharide, is recognized to be a potent stimulator of tumor necrosis factor (TNF), it was postulated that TNF might be released in the lung following cotton dust exposure and associated with the pulmonary inflammatory response. Groups of guinea pigs were exposed to an atmosphere of 33 mg/m3 cotton dust for up to 6 h. At 3, 6, 7.5, and 24 h, lungs were isolated and lavaged to assess cell populations and production of TNF. Neutrophil infiltration was apparent by 3 h as was a marked increase in TNF in bronchial alveolar lavage fluid. Alveolar macrophages (AM) isolated at 3 h showed enhanced release of TNF upon in vitro culture when compared with those isolated at the other time points. AM were found to be primed to release TNF upon ex vivo stimulation with lipopolysaccharide. The greatest effect was noted with AM isolated 1.5 h after the 6-h cotton dust exposure. These results demonstrate the ability of cotton dust to cause release of TNF in the lung and suggest a role for TNF in the inflammatory response to cotton dust.

Measurement of the ultrasonic properties of vascular tissues and blood from 35-65 MHz.

A 50 MHz ultrasound backscatter microscope has been built to measure the acoustic properties of vascular tissues and blood over the frequency range from 35-65 MHz. High resolution (45 microns) ultrasound backscatter microscope images of nine femoral and eight common carotid human artery samples were made and compared with corresponding histological sections. Individual tissue layers were selected using these images for quantitative measurement of the frequency dependent backscatter. Backscatter measurements were made in each layer of an artery at two different angles of incidence: along the axis of the artery (axial direction) and at 90 degrees to this measurement radially out from the center of the artery (radial direction). Scattering was found to be higher in elastic arteries (carotid) than in the muscular arteries (femoral). The largest difference was found in the media where the average scatter (measured in the radial direction at 50 MHz) increased from 0.002 sr-1 mm-1 in muscular arteries to 0.4 sr-1 mm-1 in elastic arteries. Large differences in scattering between measurements made in the axial and radial direction were also found. Again, the largest differences were found in the media where scattering (at 50 MHz) in carotid arteries increased from 0.003 sr-1 mm-1 measured in the axial direction to 0.4 sr-1 mm-1 measured in the radial direction. The speed of sound and attenuation in the artery wall of each sample were measured. Speed of sound measurements were found to range between 1579-1628 ms-1. The average attenuation in the artery wall increased from 4 dB mm-1 at 30 MHz to 10 dB mm-1 at 60 mHz. This is higher than the attenuation measured in blood which increased from 1.6 dB mm-1 to 5 dB mm-1 over the same frequency range. The backscatter coefficient for flowing blood was measured for flow velocities up to 36 cms-1. At flow velocities below 18 cms-1 a level of scattering of 0.0005 sr-1 mm-1 (at 50 MHz) was found. An increase in scattering of 1.6 times was measured when the flow velocity was increased to 36 cms-1. All measurements were made at 37 degrees C. The relevance of these results to clinical imaging and image interpretation is discussed.

Characterization of lead zirconate titanate ceramics for use in miniature high-frequency (20-80 MHz) transducers.

The material properties of lead zirconate titanate (PZT) ceramics for operation in the thickness mode at frequencies as high as 80 MHz are reported. Each of the ceramics tested showed a reduction in k (t) with increasing frequency. In a fine-grained PZT, values of k(t) as high as 0.44 were measured at 80 MHz. The effects of grain size were also evident in the measurement of frequency dependent mechanical losses. Experimental and theoretical analysis of a 1 mmx1 mm, 45 MHz PZT transducer verified the validity of the measurements of the properties and demonstrated excellent insertion loss and bandwidth characteristics. The minimum insertion loss of -17.5 dB is in good agreement with theory and is a marked improvement over the performance of polymer devices. Details on the fabrication and testing of high frequency ceramic transducers are described.