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.

Development of an algorithm for improving quality and information processing capacity of MathSpeak synthetic speech renderings.

PURPOSE: MathSpeak is a set of rules for non speaking of mathematical expressions. These rules have been incorporated into a computerised module that translates printed mathematics into the non-ambiguous MathSpeak form for synthetic speech rendering. Differences between individual utterances produced with the translator module are difficult to discern because of insufficient pausing between utterances; hence, the purpose of this study was to develop an algorithm for improving the synthetic speech rendering of MathSpeak. METHOD: To improve synthetic speech renderings, an algorithm for inserting pauses was developed based upon recordings of middle and high school math teachers speaking mathematic expressions. Efficacy testing of this algorithm was conducted with college students without disabilities and high school/college students with visual impairments. Parameters measured included reception accuracy, short-term memory retention, MathSpeak processing capacity and various rankings concerning the quality of synthetic speech renderings. RESULTS: All parameters measured showed statistically significant improvements when the algorithm was used. CONCLUSION: The algorithm improves the quality and information processing capacity of synthetic speech renderings of MathSpeak. This increases the capacity of individuals with print disabilities to perform mathematical activities and to successfully fulfill science, technology, engineering and mathematics academic and career objectives.

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.

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.

Dexamethasone treatment reduces astroglia responses to inserted neuroprosthetic devices in rat neocortex.

Microfabricated neural prosthetic devices hold great potential for increasing knowledge of brain function and treating patients with lost CNS function. Time-dependent loss of brain-device communication limits long-term use of these devices. Lost CNS function is associated with reactive responses that produce an encapsulating cellular sheath. Since early reactive responses may be associated with injuries produced at the time of device insertion, for example, vascular damage and disruption of the blood-brain barrier, we tested the effectiveness of the synthetic glucocorticoid, dexamethasone, in controlling insertion- and device-associated reactive responses. Dexamethasone (200 microg/kg) was administered as subcutaneous injections for 1 or 6 days beginning on the day of device insertion. Single shank microfabricated silicon devices were inserted into pre-motor cortex of adult rats. Reactive responses were assessed by immunohistochemistry for glial fibrillary acidic protein (astrocytes), CD11b (microglia), and laminin that labeled extracellular protein deposited around the insertion site and in association with vascular elements. Data were collected by confocal microscopy imaging of 100-microm-thick tissue slices. Reactive responses in vehicle control animals were similar to non-injected control animals. Dexamethasone treatment profoundly effected early and sustained reactive responses observed 1 and 6 weeks following device insertion, respectively. Dexamethasone treatment greatly attenuated astroglia responses, while microglia and vascular responses appeared to be increased. The 6-day treatment was more effective than the single injection regime. These results suggest that anti-inflammatory agents can be used to control reactive responses around inserted neural prosthetic devices and may provide a means to insure their long-term function.

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.

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.

Factors contributing to the emergence of Escherichia coli O157 in Africa.

In 1992, a large outbreak of bloody diarrhea caused by Escherichia coli O157 infections occurred in southern Africa. In Swaziland, 40,912 physician visits for diarrhea in persons ages >5 years were reported during October through November 1992. This was a sevenfold increase over the same period during 1990-91. The attack rate was 42% among 778 residents we surveyed. Female gender and consuming beef and untreated water were significant risks for illness. E. coli O157:NM was recovered from seven affected foci in Swaziland and South Africa; 27 of 31 patient and environmental isolates had indistinguishable pulsed-field gel electrophoresis patterns. Compared with previous years, a fivefold increase in cattle deaths occurred in October 1992. The first heavy rains fell that same month (36 mm), following 3 months of drought. Drought, carriage of E. coli O157 by cattle, and heavy rains with contamination of surface water appear to be important factors contributing to this outbreak.

Viral hemorrhagic fever hazards for travelers in Africa.

This short review covers 6 viral hemorrhagic fevers (VHFs) that are known to occur in Africa: yellow fever, Rift Valley fever, Crimean-Congo hemorrhagic fever, Lassa fever, Marburg virus disease, and Ebola hemorrhagic fever. All of these have at one time or another affected travelers, often the adventurous kind who are "roughing it" in rural areas, who should therefore be made aware by their physicians or travel health clinics about their potential risk of exposure to any VHF along their travel route and how to minimize the risk. A significant proportion of VHF cases involving travelers have affected expatriate health care workers who were nosocomially exposed in African hospitals or clinics. The VHFs are associated with a high case-fatality rate but are readily prevented by well-known basic precautions.

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.

Molecular characterization of a hepatitis E virus isolate from Namibia.

Hepatitis E virus (HEV) causes sporadic and epidemic acute viral hepatitis in many developing countries. In Africa, hepatitis E has been documented by virus detection (reverse transcriptase polymerase chain reaction, RT-PCR) in Egypt, Chad, Algeria, Morocco and Tunisia. Cases of presumptive hepatitis E also have been documented by detection of antibody to HEV in the Sudan, Kenya, Ethiopia, Somalia, Djibouti and South Africa. Recently, we reported the recovery of 9 isolates of HEV from feces collected during an outbreak of jaundice in Namibia. These specimens were stored frozen for many years at the South African Institute for Medical Research awaiting new methods to determine the etiology of jaundice. HEV genomic sequences were detected by antigen-capture RT-PCR with primers that amplified 2 independent regions of the HEV genome (ORF-2 and ORF-3). To further characterize the HEV 83-Namibia isolates, we determined the nucleotide (nt) sequence of the 3' end of the capsid gene (296 of 1, 980 nt in ORF-2) and ORF-3 for 1 isolate. The capsid gene sequence shared 86% identity with the prototype Burma strain and up to 96% identity with other African strains at the (nt) level, and 99% identity with Burma or other Africa strains at the amino acid level. A 188 (nt) fragment amplified from ORF-3 was also highly homologous to other HEV but was too short for meaningful comparison. Phylogenetic analysis indicated that HEV 83-Namibia is closely related to other African isolates, and differs from Burmese, Mexican and Chinese HEV. These data link the HEV causing the 1983 Namibia outbreak to more recent HEV transmission in northern and sub-Saharan Africa, suggesting this subgenotype of HEV is firmly established throughout the continent.

Attachment of astroglial cells to microfabricated pillar arrays of different geometries.

We studied the attachment of astroglial cells on smooth silicon and arrays of silicon pillars and wells with various widths and separations. Standard semiconductor industry photolithographic techniques were used to fabricate pillar arrays and wells in single-crystal silicon. The resulting pillars varied in width from 0. 5 to 2.0 micrometer, had interpillar gaps of 1.0-5.0 micrometer, and were 1.0 micrometer in height. Arrays also contained 1.0-micromter-deep wells that were 0.5 micrometer in diameter and separated by 0.5-2.0 micrometer. Fluorescence, reflectance, and confocal light microscopies as well as scanning electron microscopy were used to quantify cell attachment, describe cell morphologies, and study the distribution of cytoskeletal proteins actin and vinculin on surfaces with pillars, wells, and smooth silicon. Seventy percent of LRM55 astroglial cells displayed a preference for pillars over smooth silicon, whereas only 40% preferred the wells to the smooth surfaces. Analysis of variance statistics performed on the data sets yielded values of p > approximately.5 for the comparison between pillar data sets and < approximately.0003 in the comparison between pillar and well data sets. Actin and vinculin distributions were highly polarized in cells found on pillar arrays. Scanning electron microscopy clearly demonstrated that cells made contact with the tops of the pillars and did not reach down into the spaces between pillars even when the interpillar gap was 5.0 microm. These experiments support the use of surface topography to direct the attachment, growth, and morphology of cells. These surfaces can be used to study fundamental cell properties such as cell attachment, proliferation, and gene expression. Such topography might also be used to modify implantable medical devices such as neural implants and lead to future developments in tissue engineering.

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.

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 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.