Virus entry and innate immune activation.

Human cytomegalovirus (HCMV) exhibits an exceptionally broad cellular tropism as it is capable of infecting most major organ systems and cell types. Definitive proof of an essential role for a cellular molecule that serves as an entry receptor has proven very challenging. It is widely hypothesized that receptor utilization, envelope glycoprotein requirements and entry pathways may all vary according to cell type, which is partially supported by the data. What has clearly emerged in recent years is that virus entry is not going undetected by the host. Robust and rapid induction of innate immune response is intimately associated with entry-related events. Here we review the state of knowledge on HCMV cellular entry mediators confronting the scientific challenges by accruing a definitive data set. We also review the roles of pattern recognition receptors such as Toll-like receptors in activation of specific innate immune response and discuss how entry events are tightly coordinated with innate immune initiation steps.

Constant pressure fluid infusion into rat neocortex from implantable microfluidic devices.

Implantable electrode arrays capable of recording and stimulating neural activity with high spatial and temporal resolution will provide a foundation for future brain computer interface technology. Currently, their clinical impact has been curtailed by a general lack of functional stability, which can be attributed to the acute and chronic reactive tissue responses to devices implanted in the brain. Control of the tissue environment surrounding implanted devices through local drug delivery could significantly alter both the acute and chronic reactive responses, and thus enhance device stability. Here, we characterize pressure-mediated release of test compounds into rat cortex using an implantable microfluidic platform. A fixed volume of fluorescent cell marker cocktail was delivered using constant pressure infusion at reservoir backpressures of 0, 5 and 10 psi. Affected tissue volumes were imaged and analyzed using epifluorescence and confocal microscropies and quantitative image analysis techniques. The addressable tissue volume for the 5 and 10 psi infusions, defined by fluorescent staining with Hoescht 33342 dye, was significantly larger than the tissue volume addressed by simple diffusion (0 psi) and the tissue volume exhibiting insertion-related cell damage (stained by propidium iodide). The results demonstrate the potential for using constant pressure infusion to address relevant tissue volumes with appropriate pharmacologies to alleviate reactive biological responses around inserted neuroprosthetic devices.

Confocal Scanning Microscopy in Assessment of Cardiac Allograft Rejection--A Pilot Study.

Cardiac allograft rejection is typically diagnosed on the basis of hematoxylin and eosin (H&E) histology of endomyocardial biopsies. This diagnosis is made based on the degree of immune cell infiltrate and associated myocyte damage. However, considerable variability in rejection grading between pathologists can occur. Confocal microscopy provides high contrast and high resolution imaging that has the potential to provide detailed views of pathological features of allograft rejection. In this pilot study we sought to determine if confocal microscopy could be used to detect features of cardiac rejection. This was achieved by collection of additional sample at 30 biopsy procedures from 15 heart transplant patients. Routine pathological grading of H&E histology identified 5 gradings of 0R, 21 gradings of 1R, and 3 gradings of 2R. From these gradings, 3 samples for 0R, 9 samples for 1R, and 3 samples for 2R were imaged by confocal microscopy. This was achieved by fluorescently labeling sections with DAPI, wheat germ agglutinin, and phalloidin, to visualize the cell nuclei, cell border and extracellular matrix, and muscle cell actin, respectively. Labeling with these fluorescent markers was of high contrast. However, we did note variability in DAPI and phalloidin labeling of tissue sections. Confocal imaging of these labels revealed the following features at high resolution: perivascular and/or interstitial infiltrate, myocyte damage, and Quilty lesions. In particular increased detail of damaged myocytes reveals distortion in myofilament organization that could be exploited to distinguish between 1R and 2R grades. In conclusion, confocal microscopy provided high contrast and resolution imaging of cardiac biopsies that could be explored further to aid assessment of cardiac allograft rejection.

Evaluation of the Urotest AB antibacterial substance detection test.

The Urotest AB was used to detect antimicrobial substances in urine samples. Of 1022 urine specimens evaluated, Urotest AB detected inhibitors in 38.9%. Of 159 urine specimens from patients thought to be taking an antibiotic, inhibitors were detected in 80.5%. This test may help to explain culture negative urine samples from symptomatic patients, and could help elucidate treatment failures and the epidemiology of antibiotic resistance.

Evaluation of the Cult-Dip Plus dip slide method for urinary tract infection.

AIM: To evaluate the Cult-Dip Plus (Merck, Germany), a bacteriological culture test for detecting uropathogens. METHODS: Cult-Dip Plus consists of Brolacin (CLED) and MacConkey agar, each containing methylumbelliferylglucuronide (MUG). Using 1022 urine samples, this product was compared with the routine method of calibrated loop inoculated CLED and blood agar for screening urine for uropathogens. The MUG test for identifying Escherichia coli was also evaluated. RESULTS: Compared with the routine method, Cult-Dip Plus has a sensitivity, specificity, positive predictive value and negative predictive value of 88.3%, 98.0%, 91.9%, and 97.1%, respectively. The MUG test correctly identified 92% of E coli isolates with a sensitivity, specificity and positive predictive value of 91.6%, 95.2%, and 93.6%, respectively. CONCLUSION: Cult-Dip Plus appears to be an alternative method to the calibrate loop method for detecting uropathogens. The MUG test permits rapid, reliable and inexpensive identification of E coli.

The effect of iron on the toxigenicity of Vibrio cholerae.

In vitro and in vivo studies were conducted to assess the response of cholera toxin (CT) production to increasing iron concentrations in an aquatic environment. Production of CT by seven of eight Vibrio cholerae strains tested, including the Bengal strain (O139), was significantly enhanced in the presence of iron concentrations of 1.0 and 10 g/L. The exception (El Tor Ogawa) had a significant CT response only in the presence of 10 g of iron/L. Enhancement of CT production also occurred at iron concentrations less than 1.0 g/L, but not to a statistically significant degree. The high iron concentrations, which in this study were found to stimulate CT production, have been described by others in association with sediments, water plants, and chitinous fauna. Other investigators have shown a predilection by V. cholerae to attach to these sites in the aquatic environment. The importance of excess in vivo iron with respect to the pathogenicity of several gram-negative bacilli is well recognized. However, the possible impact of environmental iron on the in vitro toxigenicity of a microorganism, in this case V. cholerae in its aquatic environment, is to the best of our knowledge a new finding with important epidemiologic implications. These findings, coupled with the fact that iron concentration is considerably enhanced in industrially polluted waters and sediments, may reflect a causal link between the concurrent global upsurge of industrialization and pandemic occurrence of cholera during the latter half of the 20th century. Enhanced toxigenicity may also cause clinical disease following ingestion of lower than usual infective doses of cholera vibrios, thereby increasing the incidence of symptomatic cases and, possibly, of severe cases.

An outbreak of hepatitis E in Northern Namibia, 1983.

In 1983 in Namibia's Kavango region, epidemic jaundice affected hundreds of people living in settlements lacking potable water and waste disposal facilities. Many were Angolan refugees. The disease, which after investigation was designated non-A non-B hepatitis, was most common in males (72%), in persons aged 15-39 years, and was usually mild except in pregnant women, who incurred 6 (86%) of the 7 fatal infections. Fifteen years later, archived outbreak-associated samples were analyzed. Hepatitis E virus (HEV) was detected by reverse transcription-polymerase chain reaction in feces from 9 of 16 patients tested. Total Ig and IgM to HEV were quantitated in serum from 24 residents of an affected settlement at the outbreak's end: 42% had IgM diagnostic of recent infection and 25% had elevated total Ig without IgM, consistent with past HEV infection. The Namibia outbreak was typical hepatitis E clinically and epidemiologically. This first report of hepatitis E confirmed by virus detection from southern Africa extends the known range of HEV and highlights its risk for refugees.

A radioimmunoassay for the diagnosis of malaria.

A newly developed radioimmunoassay for the diagnosis of malaria has been tested in South Africa. The radioimmunoassay is an antibody binding-inhibition assay, based on a monoclonal antibody (D5) cross-reacting with Plasmodium berghei and P. residual binding activity was tested on antigen-coated microtiter plates. A sample was considered positive if it inhibited binding of the antibodies to an extent exceeding that of the microscopically negative blood samples. Blood was collected on 3 separate occasions from a total of 530 individuals living in a malaria-endemic area and was examined by radioimmunoassay and microscopy. Group 1, consisting of 194 samples, yielded 12 samples positive by microscopy and 10 of these (83%) were also positive by radioimmunoassay. One sample in this group was "positive" in the radioimmunoassay but negative on microscopy (false positive). In the 320 samples of group 2, 13 were positive by microscopy and 6 (46%) by radioimmunoassay. Group 3, which included 16 samples preselected as positive by microscopic examination and 16 controls, was examined after 4 weeks storage at -20 degrees C. Twelve samples (75%) were positive by radioimmunoassay. Tests carried out to determine the effect of blood storage on the activity of the antigen indicated that activity was preserved with little loss over a 3-month period.

Epidemiologic investigation of Marburg virus disease, Southern Africa, 1975.

During the first 10 days of February 1975, an Australian hitchhiker contracted Marburg virus disease while traveling through Rhodesia and died; the infection was subsequently passed to two other persons, who recovered. Investigators retraced the hitchhiker's steps in March and again in June 1975 in an effort to uncover the natural reservoir of the virus and determine how it was transmitted. Serum samples were collected from humans and animals wherever the patient had come in close contact with animals or insects. Arthropods of various types were collected in June 1975 and again in February 1976 for virus isolation attempts; at no time did the patient come in direct contact with nonhuman primates of any kind, or any other animals. Indirect contact with bats, monkeys, and birds through aerosols was possible, though at some distance. Direct contact with arthropods occurred throughout the trip; on several occasions it was notably severe. We believe that during this outbreak the first Marburg virus infection occurred by vector-borne transmission from an arthropod yet to be identified, and that patients 2 and 3 acquired the disease by exposure to the oropharyngeal secretions of patients 1 and 2, respectively. Studies are underway to identify the species of arthropod involved in this transmission.

A micromachined silicon multielectrode for multiunit recording.

A 16-channel multielectrode was used to record propagating action potentials from multiple units in the ventral nerve cord of the cricket Gryllus bimaculatus. The multielectrode was fabricated using photolithographic and bulk silicon etching techniques. The fabrication differs from standard methods in its use of deep reactive ion etching (DRIE) to form the bulk electrode structure. This technique enables the fabrication of relatively thick (>100 microm), rigid structures whose top surface can have any form of thin film electronics. The multielectrode tested in this paper consists of 16 narrow silicon bridges, 150 microm wide and 350 microm tall, spaced evenly over a centimeter, with passive rectangular gold recording sites on the top surface. The nerve cord was placed perpendicularly across the bridges. In this geometry, the nerve spans a 350 microm deep, 450 microm wide trench between each recording site, permitting adequate isolation of recording sites from each other and a platinum ground plane. Spike templates for eight neurons were formed using principle component analysis and clustering of the concatenated multichannel waveforms. Clean templates were generated from a 40 s recording of stimulus evoked activity. Conduction velocities ranged from 2.59+/-0.05 to 4.99+/-0.12 m/s. Two limitations of extracellular electrode arrays are the resolution of overlapping spikes and relation of discriminated units to known anatomy. The high density, precise positioning, and controlled impedance of recording sites achievable in microfabricated devices such as this one will aid in overcoming these limitations. The rigid devices fabricated using this process offer stable positioning of recording sites over relatively large distances (several millimeters) and are suitable for clamping or squeezing of nerve cords.

A selective genetic analysis of the Syracuse high- and low-avoidance (SHA/Bru and SLA/Bru) strains of rats (Rattus norvegicus).

Selective breeding of Long-Evans rats for good and poor avoidance learning in a two-way shuttle box resulted in the Syracuse strains that differ markedly in the selected phenotypes. These phenotypes have many associated traits, five of which are studied here: emotionality (open-field defecation), Pavlovian fear conditioning (CER suppression), passive avoidance training (punishment), size (weight) of the adrenal glands and adrenal concentration of corticosterone. Specifically, animals of the low-avoidance strain are more emotional, show greater fear conditioning, exhibit faster passive avoidance learning, and have larger adrenal glands in which adrenal corticosterone levels are lower than those of the high-avoidance strain. A reciprocal dihybrid cross of the two strains produced F1 hybrids, which were used to produce the segregating second filial and high and low backcross generations from which animals displaying the extreme high- and low-avoidance phenotypes were selected for study of the associated traits. Measurement of the five traits in these high and low phenotypic animals indicated that all five remain significantly associated with the avoidance phenotypes, in the expected direction, and comparably in all three segregating generations. The results indicate that the hypothesis of a major gene controlling avoidance learning must be rejected and that the few (2-3) genetic units thought to be involved may be closely linked to those that mediate these five associated characters, or express all five pleiotropically.

Brain responses to micro-machined silicon devices.

Micro-machined neural prosthetic devices can be designed and fabricated to permit recording and stimulation of specific sites in the nervous system. Unfortunately, the long-term use of these devices is compromised by cellular encapsulation. The goals of this study were to determine if device size, surface characteristics, or insertion method affected this response. Devices with two general designs were used. One group had chisel-shaped tips, sharp angular corners, and surface irregularities on the micrometer size scale. The second group had rounded corners, and smooth surfaces. Devices of the first group were inserted using a microprocessor-controlled inserter. Devices of the second group were inserted by hand. Comparisons were made of responses to the larger devices in the first group with devices from the second group. Responses were assessed 1 day and 1, 2, 4, 6, and 12 weeks after insertions. Tissues were immunochemically labeled for glial fibrillary acidic protein (GFAP) or vimentin to identify astrocytes, or for ED1 to identify microglia. For the second comparison devices from the first group with different cross-sectional areas were analyzed. Similar reactive responses were observed following insertion of all devices; however, the volume of tissue involved at early times, <1 week, was proportional to the cross-sectional area of the devices. Responses observed after 4 weeks were similar for all devices. Thus, the continued presence of devices promotes formation of a sheath composed partly of reactive astrocytes and microglia. Both GFAP-positive and -negative cells were adherent to all devices. These data indicate that device insertion promotes two responses-an early response that is proportional to device size and a sustained response that is independent of device size, geometry, and surface roughness. The early response may be associated with the amount of damage generated during insertion. The sustained response is more likely due to tissue-device interactions.

Investigation of contaminated parenteral nutrition fluids associated with an outbreak of Serratia odorifera septicaemia.

An outbreak of fatal septicaemia caused by Serratia odorifera biotype 1 involved infants at several hospitals; the common vehicle of infection was contaminated parenteral nutrition fluid. The transfusate had been made up in a flexible film isolator system. The implicated organism was recovered from surfaces inside the isolator, despite routine decontamination procedures having been carried out shortly before. Our investigation into the origin of contamination revealed several shortcomings in the infusate compounding process. We noted deficiencies in cleaning and decontamination procedures, and in storage and sterility testing policies, but the origin and mechanism of the contamination were unclear. Withdrawal of parenteral nutrition products and revision of decontamination procedures terminated the outbreak. The efficacy of peracetic acid treatment of flexible film isolators, given the circumstances of this outbreak, may need further investigation. Regular training and assessment of admixture technicians is important.

The effect of iron on the survival of Vibrio cholerae O1 in dechlorinated tap water.

Many factors, such as temperature, pH, organic nutrients, types of water storage containers, etc., determine the survival of Vibrio cholerae in water. Since the survival of V. cholerae O1 has been shown to be much longer in metal drums used as household water storage containers than in clay pots and plastic drums, the present study was designed to explore the possible role played by insoluble iron on the survival of V. cholerae O1 in water. The possibility of iron acting as particulate matter for the organisms to adhere to was also examined by using inert glass beads in water. Survival of V. cholerae O1 in dechlorinated tap water, with and without inert glass beads, ranged from < 24 h to 10 d. The number of surviving bacteria was, however, very low. In the presence of impure ferric oxide (Fe2O3), survival in tap water ranged from 4 to 12 d and the numbers of surviving bacteria were very high. Iron was thought to play an important role in the survival of V. cholerae O1 in water. Differences between the numbers of bacteria and the length of survival in surface water and in sediment were unremarkable. The El Tor and classical biotypes gave similar results.

Effect of iron and pH on the survival of Vibrio cholerae in water.

Many physicochemical factors affect the survival of Vibrio cholerae in the aquatic environment. An attempt was made to study the combined effect of pH and iron on the survival of V. cholerae in water in a laboratory environment. None of the 6 strains of V. cholerae used survived at pH 5.0; survival of all strains increased with increasing pH. The effect of ferric oxide on survival was significant for V. cholerae O1 only, not for non O1 strains. The longest survival of V. cholerae non O1 was 82 d, of El Tor V. cholerae 68 d, and of classical V. cholerae 56 d.

Prepuberal social rearing conditions and sexual behavior in control and neonatally castrated male rats.

Male and female sexual behaviors were assessed in control and neonatally castrated male rats that had been housed with a female, a control male, or a neonatally castrated male from day 16 until adulthood. Prepuberal housing conditions had no differential effect on lordosis or ejaculatory potentials of neonatally castrated males tested as adults. A smaller percentage of control males raised with a neonatal castrate ejaculated than did animals housed with a female or a control male. However, a greater proportion of control males caged with a female showed lordosis than did those living with another control male or with a neonatal castrate. The data demonstrate the modulating effects which specific types of social stimulation experienced during early life have on sexual behaviors displayed in adulthood.

Aligned microcontact printing of micrometer-scale poly-L-lysine structures for controlled growth of cultured neurons on planar microelectrode arrays.

We describe a method for producing high-resolution chemical patterns on surfaces to control the attachment and growth of cultured neurons. Microcontact printing has been extended to allow the printing of micron-scale protein lines aligned to an underlying pattern of planar microelectrodes. Poly-L-lysine (PL) lines have been printed on the electrode array for electrical studies on cultured neural networks. Rat hippocampal neurons showed a high degree of attachment selectivity to the PL and produced neurites that faithfully grew onto the electrode recording sites.

The microanalysis of light elements using transmitted energy loss electrons.

The use of transmitted energy loss electrons is shown to hold considerable promise for the elemental analysis of light elements. In this technique, those electrons which have lost energy in exciting characteristic inner shell atomic levels are detected rather than X-rays resulting from the decay of the excited levels. The advantages of this technique are (1) a large fraction (0.1-1) of the information-carrying energy loss electrons can be detected, and (2) for each excited level, one energy loss electron is produced independent of the fluorescent yield. Thus the technique potentially offers higher sensitivity than X-ray analysis. We have begun a program to evaluate this technique both theoretically and experimentally for electron probe devices. First, we have developed the necessary theoretical framework to make predictions concerning relevant quantities of elemental analysis such as the minimum detectable mass (MDM) and mimimum detectable mass fraction (MMF). The results of these calculations for thin specimens indicate a potential reduction in the MDM by up to three orders of magnitude and in the MMF by up to 500 through the use of transmitted energy loss electrons rather than X-rays; the advantages over X-ray detection being greater for lower atomic number. Second, we have begun experimental measurements to verify our predictions. These experiments were performed in a field emission scanning microscope with known limitations in collection efficiency, but the results indicate the validity of the basic assumptions and also aid in the design of an instrument which can fully exploit this technique. The experimental results obtained indicate the ease of detection of characteristic K-shell energy levels in elements as light as lithium and indicate the mass detectability of less than 10(-18)g.

The use of electron energy loss spectroscopy for studying membrane architecture: a preliminary report.

Some initial measurements of interest with respect to the use of energy loss spectroscopy (ELS) in the electron microscope are reported. We have obtained energy loss spectra in the region less than 15 eV energy loss for three components of biological components; lecithin, cholesterol and spectrin. Some indications of the effect of electron beam damage on the cholesterol spectrum are shown. In addition, energy loss pictures of erythrocyte membrane fragments are demonstrated for various energy losses less than 40 eV (taken with 0.75 eV width window) and it is shown that an individual ferritin molecule can be identified on the basis of its iron MII-III excitation line. From the results of the preliminary data we have discussed the possibilities of ELS for studies of biological membranes.

Image reconstruction from electron and X-ray diffraction patterns using iterative algorithms: experiment and simulation.

The hybrid input-output iterative algorithm, which solves the phase problem for scattering from non-periodic objects, is reviewed for application to X-ray and electron diffraction data. Desirable convex constraints, including the sign of the scattering potential for electrons, and compact support, are discussed. The cases of complex and real exit-face wavefunctions, strong and weak phase objects, various supports, and the use of coherent focussed radiation are reviewed. Reconstruction of general complex objects requires accurate knowledge of the support, which should consist of two holes or a triangle in an opaque mask. The support boundaries should be as sharp as possible. Strong phase objects without absorption can be recovered if the support consists of one hole, is accurately known and has sufficiently sharp boundaries. Real and weak phase objects with absorption can be recovered without accurate knowledge of the support area if the support boundaries are sufficiently sharp and the support consists of one or more holes. A sign constraint on the scattering potential is used to recover weak phase objects. The experimental realization of theoretically desirable support conditions is discussed. A two-stage method of finding the support for complex objects is proposed. Experimental results from applying the Gerchberg-Saxton-Fienup HiO-algorithm to coherent electron diffraction patterns are presented, using specially made e-beam lithographed support structures. Images with a resolution of about 5 nm are thus recovered from the intensities alone in coherent electron diffraction patterns from non-periodic objects. Limitations of the present experiments are identified and suggestions made for development of both X-ray and electron work.