Biomimetic collagen scaffolds with anisotropic pore architecture.

Sponge-like matrices with a specific three-dimensional structural design resembling the actual extracellular matrix of a particular tissue show significant potential for the regeneration and repair of a broad range of damaged anisotropic tissues. The manipulation of the structure of collagen scaffolds using a freeze-drying technique was explored in this work as an intrinsically biocompatible way of tailoring the inner architecture of the scaffold. The research focused on the influence of temperature gradients, imposed during the phase of crystallisation of collagen suspensions, upon the degree of anisotropy in the microstructures of the scaffolds produced. Moulding technology was employed to achieve differences in heat transfer rates during the freezing processes. For this purpose various moulds with different configurations were developed with a view to producing uniaxial and multi-directional temperature gradients across the sample during this process. Scanning electron microscopy analysis of different cross-sections (longitudinal and horizontal) of scaffolds revealed that highly aligned matrices with axially directed pore architectures were obtained where single unidirectional temperature gradients were induced. Altering the freezing conditions by the introduction of multiple temperature gradients allowed collagen scaffolds to be produced with complex pore orientations, and anisotropy in pore size and alignment.

Distribution and dynamics of Japanese beetles along the Indianapolis Airport perimeter and the influence of land use on trap catch.

As part of a program to minimize the accidental transportation of Japanese beetles (Popillia japonica) through cargo aircraft to areas where they are not established, a 4-yr trapping project was initiated to study the relative distribution and dynamics of the beetles along a trap line around the Indianapolis International Airport. Land use influence on beetle abundance (trap catch) was assessed using a geographic information system. Trap catch was consistently high in some locations and low in others. In general, high trap catches occurred near agronomic land planted with corn or soybeans, which are both preferred hosts of adult beetles. Low trap catches generally occurred in areas lacking preferred host plants. The amount of agronomic land within 500 m of the traps was always positively correlated with trap catch. Average trap catches were highly correlated by location from year to year, indicating stability of the relative distribution of the beetles along the trap line. Because high trap catches consistently occurred in the same locations, it can be inferred that trapping can be an effective method to monitor Japanese beetle populations. Taking airport-owned agronomic land out of corn and soybean production near the cargo terminals may reduce beetle activity in these areas.

African swine fever virus multigene family 360 and 530 genes affect host interferon response.

African swine fever virus (ASFV) multigene family 360 and 530 (MGF360/530) genes affect viral growth in macrophage cell cultures and virulence in pigs (L. Zsak, Z. Lu, T. G. Burrage, J. G. Neilan, G. F. Kutish, D. M. Moore, and D. L. Rock, J. Virol. 75:3066-3076, 2001). The mechanism by which these novel genes affect virus-host interactions is unknown. To define MGF360/530 gene function, we compared macrophage transcriptional responses following infection with parental ASFV (Pr4) and an MGF360/530 deletion mutant (Pr4 Delta 35). A swine cDNA microarray containing 7,712 macrophage cDNA clones was used to compare the transcriptional profiles of swine macrophages infected with Pr4 and Pr4 Delta 35 at 3 and 6 h postinfection (hpi). While at 3 hpi most (7,564) of the genes had similar expression levels in cells infected with either virus, 38 genes had significantly increased (>2.0-fold, P < 0.05) mRNA levels in Pr4 Delta 35-infected macrophages. Similar up-regulation of these genes was observed at 6 hpi. Viral infection was required for this induced transcriptional response. Most Pr Delta 35 up-regulated genes were part of a type I interferon (IFN) response or were genes that are normally induced by double-stranded RNA and/or viral infection. These included monocyte chemoattractant protein, transmembrane protein 3, tetratricopeptide repeat protein 1, a ubiquitin-like 17-kDa protein, ubiquitin-specific protease ISG43, an RNA helicase DEAD box protein, GTP-binding MX protein, the cytokine IP-10, and the PKR activator PACT. Differential expression of IFN early-response genes in Pr4 Delta 35 relative to Pr4 was confirmed by Northern blot analysis and real-time PCR. Analysis of IFN-alpha mRNA and secreted IFN-alpha levels at 3, 8, and 24 hpi revealed undetectable IFN-alpha in mock- and Pr4-infected macrophages but significant IFN-alpha levels at 24 hpi in Pr4 Delta 35-infected macrophages. The absence of IFN-alpha in Pr4-infected macrophages suggests that MGF360/530 genes either directly or indirectly suppress a type I IFN response. An inability to suppress host type I IFN responses may account for the growth defect of Pr4 Delta 35 in macrophages and its attenuation in swine.

Development and evaluation of a fluorogenic 5'-nuclease assay to identify Marburg virus.

The ability to rapidly recognize Marburg virus infections is critical to quickly institute proper barrier nursing precautions and limit further spread of the disease. A rapid, sensitive, and specific laboratory diagnostic test is necessary to confirm outbreaks of Marburg virus and to distinguish it from other diseases that can present with similar clinical symptoms. A one-tube reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the identification of Marburg virus was developed and evaluated using the ABI PRISM 7700 Sequence Detection System and TaqMan chemistry. The sensitivity and specificity of the newly designed primer/probe set (MBGGP3) was evaluated. MBGGP3 was equivalent to or 10-100-fold more sensitive than previously designed primer sets as determined by limit of detection experiments. In addition, the MBGGP3 assay was able to detect all strains of Marburg virus tested, but gave negative results with other haemorrhagic fever and genetically related viruses. The results of this study indicate that the MBGGP3 primer/probe set is both sensitive and specific. In addition, this assay is compatible with emerging rapid nucleic acid analysis platforms and therefore may prove to be a useful diagnostic tool for the control and management of future outbreaks.

Development and evaluation of a fluorogenic 5' nuclease assay to detect and differentiate between Ebola virus subtypes Zaire and Sudan.

The ability to rapidly recognize Ebola virus infections is critical to quickly limit further spread of the disease. A rapid, sensitive, and specific laboratory diagnostic test is needed to confirm outbreaks of Ebola virus infection and to distinguish it from other diseases that can cause similar clinical symptoms. A one-tube reverse transcription-PCR assay for the identification of Ebola virus subtype Zaire (Ebola Zaire) and Ebola virus subtype Sudan (Ebola Sudan) was developed and evaluated by using the ABI PRISM 7700 sequence detection system. This assay uses one common primer set and two differentially labeled fluorescent probes to simultaneously detect and differentiate these two subtypes of Ebola virus. The sensitivity of the primer set was comparable to that of previously designed primer sets, as determined by limit-of-detection experiments. This assay is unique in its ability to simultaneously detect and differentiate Ebola Zaire and Ebola Sudan. In addition, this assay is compatible with emerging rapid nucleic acid analysis platforms and therefore may prove to be a very useful diagnostic tool for the control and management of future outbreaks.

Impact of four insecticides on Japanese beetle (Coleoptera: Scarabaeidae) egg predators and white grubs in turfgrass.

Field experiments were conducted to measure the effects of four commonly used turfgrass insecticides (isofenphos, diazinon, imidacloprid, halofenozide) on white grubs (Coleoptera: Scarabaeidae) and ant predators of white grub eggs. Ant populations were measured over time with canned tuna, whereas predation by the ants was measured with artificially placed Japanese beetle, Popillia japonica Newman, eggs. The effectiveness of each insecticide at controlling Japanese beetle grubs, when applied at different times during the growing season, also was measured. Isofenphos and diazinon significantly reduced both ant numbers and white grub egg predation, whereas imidacloprid and one halofenozide treatment did not significantly impact either measurement. A second halofenozide treatment significantly reduced white grub egg predation. Isofenphos and diazinon were ineffective at controlling Japanese beetle grubs when applied in June but were highly efficacious when applied in August. Evidence of enhanced biodegradation was found in plots that received both June and August applications of diazinon. Both June and August applications of imidacloprid and halofenozide provided good control of white grubs.

Pathogenesis of experimental Ebola Zaire virus infection in BALB/c mice.

Guinea-pigs and non-human primates have traditionally been used as animal models for studying Ebola Zaire virus (EBO-Z) infections. The virus was also recently adapted to the stage of lethal virulence in BALB/c mice. This murine model is now in use for testing antiviral medications and vaccines. However, the pathological features of EBO-Z infection in mice have not yet been fully described. To identify sites of viral replication and characterize sequential morphological changes in BALB/c mice, adult female mice were infected with mouse-adapted EBO-Z and killed in groups each day for 5 days post-infection. Tissues were examined by light microscopy, immunohistochemistry, electron microscopy and in-situ hybridization. As in guinea-pigs and non-human primates, cells of the mononuclear phagocytic system were the earliest targets of infection. Viral replication was observed by day 2 in macrophages in lymph nodes and spleen. By the time of onset of illness and weight loss (day 3), the infection had spread to hepatocytes and adrenal cortical cells, and to macrophages and fibroblast-like cells in many organs. Severe lymphocytolysis was observed in the spleen, lymph nodes and thymus. There was minimal infection of endothelial cells. All of these changes resembled those observed in EBO-Z-infected guinea-pigs and non-human primates. In contrast to the other animal models, however, there was little fibrin deposition in the late stage of disease. The availability of immunodeficient, "gene-knockout" and transgenic mice will make the mouse model particularly useful for studying the early steps of Ebola pathogenesis.

Apoptosis induced in vitro and in vivo during infection by Ebola and Marburg viruses.

Induction of apoptosis has been documented during infection with a number of different viruses. In this study, we used transmission electron microscopy (TEM) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling to investigate the effects of Ebola and Marburg viruses on apoptosis of different cell populations during in vitro and in vivo infections. Tissues from 18 filovirus-infected nonhuman primates killed in extremis were evaluated. Apoptotic lymphocytes were seen in all tissues examined. Filoviral replication occurred in cells of the mononuclear phagocyte system and other well-documented cellular targets by TEM and immunohistochemistry, but there was no evidence of replication in lymphocytes. With the exception of intracytoplasmic viral inclusions, filovirus-infected cells were morphologically normal or necrotic, but did not exhibit ultrastructural changes characteristic of apoptosis. In lymph nodes, filoviral antigen was co-localized with apoptotic lymphocytes. Examination of cell populations in lymph nodes showed increased numbers of macrophages and concomitant depletion of CD8+ T cells and plasma cells in filovirus-infected animals. This depletion was particularly striking in animals infected with the Zaire subtype of Ebola virus. In addition, apoptosis was demonstrated in vitro in lymphocytes of filovirus-infected human peripheral blood mononuclear cells by TEM. These findings suggest that lymphopenia and lymphoid depletion associated with filoviral infections result from lymphocyte apoptosis induced by a number of factors that may include release of various chemical mediators from filovirus-infected or activated cells, damage to the fibroblastic reticular cell conduit system, and possibly stimulation by a viral protein.

Interaction between halofenozide and the entomopathogenic nematode Heterorhabditis marelatus for control of Japanese beetle (Coleoptera: Scarabaeidae) larvae.

Japanese beetle, Popillia japonica Newman is a major pest of turf and ornamentals. Laboratory bioassays were conducted to evaluate the potential interactions between a biological control agent, Heterorhabditis marelatus (Nematoda: Heterorhabditidae) IN strain and the insecticide halofenozide against both overwintered and nonoverwintered 3rd instars of Japanese beetle. Treatments consisted of all combinations of 2 rates of halofenozid with H. marelatus nematodes Imidacloprid was used as a standard. Percentage larval mortality was evaluated at 7, 14, and 21 d after treatment. No deleterious effects were observed. The nematode treatments generally produced significantly greater larval mortality relative to both chemical treatments. Twenty-one days after treatment, both rates of nematodes resulted in 100% mortality, whereas insecticide treatments did not surpass 60% mortality. No synergism was detected in any of the combination treatments. There were no significant differences in nematode reproduction in larvae exposed to halofenozide and nematodes versus larvae exposed to only nematodes.

Evaluation of a computerized polysomnographic system.

Computerized polysomnographic systems have came into common use in sleep laboratories around the world. Despite potential advantages over standard paper polysomnography, these computerized systems have been minimally evaluated as to accuracy, analysis time, or cost effectiveness when compared to paper. We evaluated the Healthdyne ALICE 3 system for comparability to paper polysomnography in sleep quantification and technician analysis time. Fifty patients were recorded simultaneously both on paper and on the ALICE 3 system and analyzed blindly with summary data from these records being quantified and compared. Five additional patients were studied for epoch-by-epoch analysis. Score-rescore assessments were accomplished for both groups. The results indicate that when allowed to autoscore, this computerized system produced substantial errors in sleep staging (REM sleep time 56.4 + 4.9 minutes vs 73.2 + 8.4 minutes for paper versus computer). This was the case for respiratory (AHI of 26.5 + 4.3 vs 15.3 + 2.6 for paper vs computer) and arousal assessment as well. However, with editing, similar results to those obtained with paper were achieved (REM sleep time -56.4 + 4.9 vs 59.0 + 4.6; AHI -26.5 + 4.3 vs 26.1 + 4.7 for paper and computer respectively), with differences rarely exceeding score-rescore discrepancies. Analysis time was substantially reduced by use of the computer (172.6 + 9.9 vs 79.7 + 4.8 minutes for paper vs computer). Epoch-by-epoch analysis revealed a trend to score toward wakefulness or lighter sleep on computer compared to paper although the differences were small. Respiratory, arousal and PLM scoring were quite similar. In conclusion, this study suggests that the ALICE 3 system with editing can produce results similar to those obtained with paper.

Evaluation of the Healthdyne NightWatch system to titrate CPAP in the home.

Although a number of devices have been developed to monitor sleep and breathing in the home, there are few publications on methodologies by which CPAP can be titrated in the home setting. This study was conducted to determine the outcome of CPAP titration in the home using the Healthdyne NightWatch (NW) system. This home sleep-evaluation system was used to diagnose sleep apnea in 30 patients using a previously described methodology. These patients subsequently underwent CPAP titration in the home using the NW system, with modem technology allowing the transfer of data from the home to the laboratory. This group was compared with 30 patients who were diagnosed with sleep apnea using standard in-lab polysomnography and had CPAP titrated on a full night in the laboratory. Both groups were subsequently placed on CPAP at the appropriate pressure for 6-8 weeks, after which a full in-lab study was completed to assess CPAP efficacy at the prescribed pressure. Compliance was also determined using a pressure-activated monitor. No differences in any variable assessed could be found between the two groups. Mean compliance was 4.6 + 0.5 (SEM) and 4.3 + 0.5 hours of CPAP use per night for the home and in-lab groups respectively. Mean AHIs on the follow-up study were 7.4 + 1.2 and 7.6 + 1.6 events per hour for the home versus in-lab groups. Sleep stage distribution was also quite comparable between groups. As a result, this study suggests that sleep apnea can be diagnosed and CPAP titrated in the home with a similar outcome, at least at 6 to 8 weeks, to standard in-laboratory testing.

Assessment of accuracy and analysis time of a novel device to monitor sleep and breathing in the home.

Obstructive sleep apnea is increasingly recognized as a common and debilitating disorder. As a result, a variety of diagnostic technologies have evolved to potentially decrease cost and improve access and ease of assessment. In this study we compared the Healthdyne NightWatch (NW) System (a home sleep diagnostic methodology) to standard polysomnography (PSG) in two sleep centers. Two separate studies were completed. NW was compared to a simultaneously obtained PSG in 30 patients (IN-LAB study). Seventy additional patients were studied in both the home with NW and in the laboratory with PSG (HOME-LAB study). The NW system records eye movement, leg movement, SaO2, nasal-oral airflow, chest and abdominal wall motion, body position and heart rate on a solid state recorder, which permits sleep staging based on body and eye movement and standard respiratory assessment. For the PSG, standard paper recording techniques were used. The IN-LAB study revealed a correlation between NW and PSG for total sleep time of r = 0.72, with NW tending to score some awake time as nonrapid eye movement sleep. The correlation for apnea-hypopnea index (AHI) was r = 0.94 between systems, with a sensitivity of 100% and specificity of 63.6% at an AHI threshold of 10. The HOME-LAB study demonstrated understandably poor correlations between NW and PSG for most measures of sleep, which is likely a product of night-to-night variability in sleep, home versus laboratory effects and the differences in sleep staging methodology. However, the correlation for AHI was r = 0.92, with a sensitivity of 90.7% and a specificity of 70.4% at an AHI threshold of 10. Using a new methodology to assess agreement between diagnostic systems, we observed 78.6% diagnostic agreement between NW and PSG in the HOME-LAB study, with NW underestimating AHI 4.3% of the time and overestimating it in 17.1% of cases. This may relate to night-to-night variability in AHI or greater NW computer sensitivity to subtle hypopneas. We conclude that NW provides an accurate determination of AHI in both the home and laboratory, using limited instrumentation. The analysis time for NW is also reduced compared to PSG, and patients generally prefer the NW evaluation.

Evaluation and significance of the pupillary light reflex in trauma patients.

The pupillary light reflex is an important component of the neurologic examination of the trauma victim. Although the normal reflex can be predictably altered by specific head injuries, a variety of other factors common to trauma patients such as alcohol, illicit drugs, narcotics, paralyzing agents, hypothermia, and orbital or ophthalmic injury can confound the evaluation of the pupillary light reflex. This report reviews the anatomy and neurophysiology of the pupillary light reflex and discusses the impact these confounding variables may have on this key component of the initial trauma evaluation.

The effect of capsular venting on glenohumeral laxity.

Anesthetized shoulders are frequently stable against forces applied during drawer and sulcus tests, even though the shoulder muscles are inactive and do not contribute to stability. This passive stability is also evident in the glenohumeral joints of anatomic specimens. The translational laxity of anatomic specimen shoulders was measured, and it was demonstrated that this laxity was substantially increased when air was admitted into the capsule. Eight shoulders, aged 57-87 years, including six contralateral pairs, were analyzed using a six degrees-of-freedom force transducer and a six degrees-of-freedom spatial tracker. Capsules were vented by admitting air ad libitum through an 18-gauge needle. Venting reduced the force necessary to translate the humeral head with respect to the glenoid fossa by an average of 15.3 N (55%) for anterior forces, 10.8 N (43%) for posterior forces, and 19.0 N (57%) for inferior forces. It is likely that passive stability will also be diminished by a similar mechanism in patients with intact but excessively lax capsules. The principle of limited joint volume should be considered and tested when investigating glenohumeral stability.

Translation of the humeral head on the glenoid with passive glenohumeral motion.

We have demonstrated that certain passive motions of the glenohumeral joint are reproducibly accompanied by translation of the head of the humerus on the glenoid. We investigated the relationship of these translations to the position of the glenohumeral joint and to applied torques and forces in seven isolated glenohumeral joints from fresh cadavera, using a six-degrees-of-freedom position sensor and a six-axis force and torque transducer. Reproducible and significant translation occurred in an anterior direction with glenohumeral flexion and in a posterior direction with extension. We also observed translation with cross-body movement. The translation occurring with flexion was obligate in that it could not be prevented by the application of an oppositely directed force of thirty to forty newtons. Operative tightening of the posterior portion of the capsule increased the anterior translation on flexion and cross-body movement and caused it to occur earlier in the arc of motion compared with the intact glenohumeral joint. Operative tightening of the posterior part of the capsule also resulted in significant superior translation with flexion of the glenohumeral joint.