Loss of mucosal CD103+ DCs and IL-17+ and IL-22+ lymphocytes is associated with mucosal damage in SIV infection.

Human immunodeficiency virus (HIV) and Simian immunodeficiency virus (SIV) disease progression is associated with multifocal damage to the gastrointestinal tract epithelial barrier that correlates with microbial translocation and persistent pathological immune activation, but the underlying mechanisms remain unclear. Investigating alterations in mucosal immunity during SIV infection, we found that damage to the colonic epithelial barrier was associated with loss of multiple lineages of interleukin (IL)-17-producing lymphocytes, cells that microarray analysis showed expressed genes important for enterocyte homeostasis, including IL-22. IL-22-producing lymphocytes were also lost after SIV infection. Potentially explaining coordinate loss of these distinct populations, we also observed loss of CD103+ dendritic cells (DCs) after SIV infection, which associated with the loss of IL-17- and IL-22-producing lymphocytes. CD103+ DCs expressed genes associated with promotion of IL-17/IL-22+ cells, and coculture of CD103+ DCs and naïve T cells led to increased IL17A and RORc expression in differentiating T cells. These results reveal complex interactions between mucosal immune cell subsets providing potential mechanistic insights into mechanisms of mucosal immune dysregulation during HIV/SIV infection, and offer hints for development of novel therapeutic strategies to address this aspect of AIDS virus pathogenesis.

Compromised gastrointestinal integrity in pigtail macaques is associated with increased microbial translocation, immune activation, and IL-17 production in the absence of SIV infection.

Pigtail macaques (PTMs) rapidly progress to AIDS after simian immunodeficiency virus (SIV) infection. Given the strong association between human immunodeficiency virus (HIV) and SIV disease progression and microbial translocation and immune activation, we assessed whether high basal levels of immune activation and microbial translocation exist in PTMs. We found that before SIV infection, PTMs had high levels of microbial translocation that correlated with significant damage to the structural barrier of the gastrointestinal tract. Moreover, this increased microbial translocation correlated with high levels of immune activation and was associated with high frequencies of interleukin-17-producing T cells. These data highlight the relationship among mucosal damage, microbial translocation and systemic immune activation in the absence of SIV replication, and underscore the importance of microbial translocation in the rapid course of disease progression in SIV-infected PTMs. Furthermore, these data suggest that PTM may be an ideal model to study therapeutic interventions aimed at decreasing microbial translocation-induced immune activation.

Evaluation of objects and food for environmental enrichment of NZW rabbits.

The Guide for the Care and Use of Laboratory Animals states that both structural and social environments should be considered when addressing the husbandry needs of laboratory animals. The purpose of this study was to investigate environmental enrichment strategies that could potentially enhance the well-being of rabbits. Male and female 6-week old New Zealand White rabbits were divided into three groups: food-enriched (Bunny Stix, Bunny Blocks, or celery), non-food enriched (Jingle Ball, Kong toy, or Nylabone), and not enriched. Animals were given a particular enrichment for 1 h daily for 15 days. Home cages were fitted with specially designed plexiglass doors, which allowed the animals' interactions with the objects to be videotaped. The amount of time the animal interacted with each object and the total activity during the 1-h taped session were recorded for each rabbit. Rabbits were weighed weekly. Rabbits spent significantly more time interacting with the Bunny Stix than any other food item or non-food object. In addition, total activity time was significantly greater for all rabbits enriched with food versus any of the non-food items. Weight gains after 15 days did not differ significantly, but there was a trend towards increased weight gains in food-enriched rabbits. In this study, food was a stronger, more sustained enrichment device than were non-food objects.

Autologous conjunctival resurfacing of leaking filtering blebs.

PURPOSE: To present a case series of a new technique to repair late bleb leaks. DESIGN: Retrospective, noncomparative, consecutive case series. PARTICIPANTS: Forty-seven autologous conjunctival resurfacings of late bleb leaks were performed by four surgeons at two institutions. METHODS: Autologous conjunctival grafts were placed over existing de-epithelialized leaking blebs. MAIN OUTCOME MEASURES: Leak-free, Seidel-negative blebs and controlled glaucoma. RESULTS: After a mean follow-up of 14 +/- 12 months, one patient continued to have bleb leak at the last follow-up, and one frank leak resolved with aqueous suppression. Intraocular pressure increased from 6.6 +/- 4.4 mmHg (0.13 glaucoma medications) to 11.9 +/- 4.1 mmHg (0.41 glaucoma medications). CONCLUSIONS: Conjunctival resurfacing with autologous tissue is an effective technique to repair late bleb leaks.

Comparison of adjuvants with Leishmania antigens in a guinea pig model to induce delayed-type hypersensitivity responses.

BACKGROUND AND PURPOSE: Guinea pigs have been a traditional model for studies of delayed-type hypersensitivity. They are the natural host of Leishmania enriettii and have been experimentally infected with other species of Leishmania. They have been used as a skin-test model to screen potential antigens for use in diagnostic tests for Leishmania. Use of complete Freund's adjuvant (CFA), along with whole promastigote Leishmania antigen, was necessary to sensitize guinea pigs to invoke a sufficient cell-mediated immune response. However, use of CFA has come under scrutiny by Animal Care and Use Committees due to the pathologic changes associated with its use. METHODS: Thirty-two specific-pathogen-free male Hartley guinea pigs were inoculated with Leishmania antigens alone or mixed with one of three adjuvants (CFA, TiterMax, and liposomes), and were skin tested 2 weeks later. RESULTS: For the Leishmania antigens tested, guinea pigs that received liposomes as an adjuvant had skin-test responses comparable to those of guinea pigs that received CFA. TiterMax was also tested, but cellular responses at antigen test sites were poor. CONCLUSIONS: Liposomes can be used in this model as a safe, effective adjuvant.

The mouse mitotic checkpoint gene bub1b, a novel bub1 family member, is expressed in a cell cycle-dependent manner.

A search for genes differentially expressed in normal and leukemic mouse thymocytes yielded a homolog of the yeast mitotic checkpoint protein Bub1. This novel protein ("mBub1b") has 40% sequence similarity to the mouse Bub1 ("mBub1a") previously described by Taylor and McKeon (1997, Cell 89, 727-735) over four extended domains. Differences between the Bub1 sequences suggest that the two proteins may have different substrate specificities and that Bub1b alone has a putative "destruction" box that can target proteins for degradation by proteosomes during mitosis. Northern blots of normal tissues show that mouse Bub1a and Bub1b genes are expressed in thymus and spleen, but not in nondividing tissues. In synchronized cells, expression of both Bub1 genes is undetectable in G1; Bub1 gene expression peaks in G2/M with Bub1b delayed by 6 h relative to Bub1a. This cell cycle-dependent expression explains the tissue distribution and the abundance of Bub1 mRNAs in rapidly dividing cell lines. The human equivalent of mBub1b was isolated and mapped to chromosome 15q15. The existence in mammals of two separate Bub1 genes encoding distinct proteins, coupled with the different timing of peak expression, suggests that Bub1a and Bub1b have distinct roles in the mitotic checkpoint.

Open pore biodegradable matrices formed with gas foaming.

Engineering tissues utilizing biodegradable polymer matrices is a promising approach to the treatment of a number of diseases. However, processing techniques utilized to fabricate these matrices typically involve organic solvents and/or high temperatures. Here we describe a process for fabricating matrices without the use of organic solvents and/or elevated temperatures. Disks comprised of polymer [e.g., poly (D,L-lactic-co-glycolic acid)] and NaCl particles were compression molded at room temperature and subsequently allowed to equilibrate with high pressure CO2 gas (800 psi). Creation of a thermodynamic instability led to the nucleation and growth of gas pores in the polymer particles, resulting in the expansion of the polymer particles. The polymer particles fused to form a continuous matrix with entrapped salt particles. The NaCl particles subsequently were leached to yield macropores within the polymer matrix. The overall porosity and level of pore connectivity were regulated by the ratio of polymer/salt particles and the size of salt particles. Both the compressive modulus (159+/-130 kPa versus 289+/-25 kPa) and the tensile modulus (334+/-52 kPa versus 1100+/-236 kPa) of the matrices formed with this approach were significantly greater than those formed with a standard solvent casting/particulate leaching process. The utility of these matrices was demonstrated by engineering smooth muscle tissue in vitro with them. This novel process, a combination of high pressure gas foaming and particulate leaching techniques, allows one to fabricate matrices with a well controlled porosity and pore structure. This process avoids the potential negatives associated with the use of high temperatures and/or organic solvents in biomaterials processing.

UvsY protein of bacteriophage T4 is an accessory protein for in vitro catalysis of strand exchange.

The uvsX and uvsY genes are essential to genetic recombination, recombination-dependent DNA synthesis and to the repair of DNA damage in bacteriophage T4. Purified UvsX protein has been shown to catalyze strand exchange and D-loop formation in vitro, but the role of UvsY protein has been unclear. We report that UvsY protein enhances strand exchange by UvsX protein by interacting specifically with UvsX protein: gene 32 protein (gp32) is not necessary for this effect and UvsY protein has no similar effect on the RecA protein of E. coli. UvsY protein, like UvsX protein, protects single-stranded DNA from digestion by nucleases, but, unlike UvsX protein, shows no ability to protect double-stranded DNA. UvsY protein enhances the rate of single-stranded-DNA-dependent ATP hydrolysis by UvsX protein, particularly in the presence of gp32 or high concentrations of salt, factors that otherwise reduce the ATPase activity of UvsX protein. The enhancement of ATP hydrolysis by UvsY protein is shown to result from the ability of UvsY protein to increase the affinity of UvsX protein for single-stranded DNA.

Formation of D loops by the UvsX protein of T4 bacteriophage: a comparison of the reaction catalyzed in the presence or absence of gene 32 protein.

The UvsX protein of T4 bacteriophage will catalyze the formation of D loops between linear single-stranded DNA (ssDNA) and homologous supercoiled double-stranded DNA (dsDNA) in the absence of T4 gene 32 protein (gp32). This reaction requires one monomer of UvsX protein per three nucleotides of ssDNA so that the ssDNA is completely covered with UvsX protein. Under these conditions, high rates of ATP hydrolysis are observed, and one-third of the products are joined paranemically. The reaction proceeds through a mechanism that creates homology-independent coaggregates of UvsX protein, dsDNA, and ssDNA. When UvsX protein is added to only 1 monomer per 8 nucleotides, but with 1 monomer of gp32 per 12 nucleotides, the rate of ATP hydrolysis is depressed, but D-loop formation is enhanced. Nearly all of the product is bound in plectonemic joints, and no coaggregated intermediates are formed. Coaggregate formation at high concentrations of UvsX protein is not inhibited by the presence of gp32; gp32 simply allows for efficient formation of D loops at such low concentrations of UvsX protein that coaggregates are not constructed. Electron microscopic visualization of the joint structures in this reaction reveals that both gp32 and UvsX protein are bound to the ssDNA. The single-stranded DNA binding (SSB) protein of Escherichia coli will substitute only partially for gp32: in the presence of SSB protein, D-loop formation can be catalyzed at one UvsX protein monomer per eight nucleotides, and it is accomplished without the formation of coaggregates, but a major portion of the product is joined paranemically.

DNA strand exchanges.

Biochemical and electron microscopic studies of the strand exchange reactions catalyzed by the RecA protein of Escherichia coli and the UvsX protein of T4 phage reveal that these reactions proceed in three distinct steps. The first step, termed joining, involves the assembly of RecA (or UvsX) protein onto a single-stranded DNA (ssDNA) molecule and the subsequent search for homology with a double-stranded DNA (dsDNA) partner and formation of a stable synapsis. In the second step (envelopment/exchange), the exchange of DNA strands occurs fueled by the hydrolysis of ATP. The third step (release of products) entails the resolution of the complexes and dissociation of the protein from the DNAs. The structure of the intermediates in the in vitro reactions catalyzed by the RecA and UvsX proteins is emphasized in this review. The results of pairing different DNA molecules in vitro (such as linear ssDNA pairing with linear or supertwisted dsDNA) are described. Paranemic joints represent a major pathway of joining between two DNA molecules which may involve, in some cases, most of the DNA substrate molecules. Since the nature of paranemic joints has only recently begun to be understood, the nature, role, and possible in vivo function of paranemic joining are considered.

Visualization of the homologous pairing of DNA catalyzed by the bacteriophage T4 UvsX protein.

The uvsX gene product is essential for DNA repair and general recombination in T4 bacteriophage. The ability of UvsX protein to catalyze the homologous pairing of single-stranded DNA (ssDNA) with double-stranded DNA (dsDNA) in vitro was examined by electron microscopic (EM), nitrocellulose filter binding, and gel electrophoretic methods. Optimal joining was observed at ratios of UvsX protein:ssDNA of 2 nucleotides/protein monomer. At this level, the ssDNA was fully covered by UvsX protein as seen by EM, while the dsDNA appeared protein-free. Using this stoichiometry, the pairing of circular ssDNA with homologous supertwisted dsDNA was found to produce a high frequency of complexes in which a supertwisted dsDNA molecule was joined to a UvsX protein-ssDNA filament over a distance of less than 100 base pairs. These joints were labile to deproteinization and must have been paranemic. Pairing of linear ssDNA containing buried homology to the dsDNA produced identical structures. Pairing of fully homologous linear ssDNA and supertwisted dsDNA yielded D-loop joints (plectonemic) as seen by EM following deproteinization. Both the paranemic and the plectonemic joints were at sites of homology, as demonstrated by restriction cleavage of the complexes. Visualization of the joined complexes prior to deproteinization showed that 50% of the joints had the architecture of the paranemic joints, whereas in the remainder, a topologically relaxed dsDNA circle merged with the UvsX protein-ssDNA filament for a distance of 450 base pairs. The structure of the filament was not visibly altered in this region. These observations are similar, but not identical, to findings in parallel studies utilizing the RecA protein of Escherichia coli.

Three-dimensional display and analysis of tomographic volume images utilizing a varifocal mirror.

Described is a system for the multidimensional display and analysis of tomographic images utilizing the principle of variable focal (varifocal) length optics. The display system uses a vibrating mirror in the form of an aluminized membrane stretched over a loudspeaker, coupled with a cathode ray tube (CRT) display monitor suspended face down over the mirror, plus the associated digital hardware to generate a space filling display. The mirror is made to vibrate back and forth, as a spherical cap, by exciting the loudspeaker with a 30 Hz sine wave. "Stacks" of 2-D tomographic images are displayed, one image at a time, on the CRT in synchrony with the mirror motion. Because of the changing focal length of the mirror and the integrating nature of the human eye-brain combination, the time sequence of 2-D images, displayed on the CRT face, appears as a 3-D image in the mirror. The system simplifies procedures such as: reviewing large amounts of 3-D image information, exploring volume images in three dimensions, and gaining an appreciation or understanding of three-dimensional shapes and spatial relationships. The display system facilitates operator interactivity, e.g., the user can point at structures within the volume image, remove selected image regions to more clearly visualize underlying structure, and control the orientation of brightened oblique planes through the volume.

Three-dimensional cardiac anatomy and function in heart disease in adults: initial results with the dynamic spatial reconstructor.

The dynamic spatial reconstructor, or DSR, is a unique high-speed volume-imaging x-ray scanner based on computed tomographic principles. In this report, we present data obtained from the first feasibility DSR studies of adult patients with heart disease. Information from three patients--one with hypertrophic obstructive cardiomyopathy, one with calcific aortic valvular disease, and one with a left ventricular aneurysm--is described in detail. The mean DSR scanning time for each patient was 20 seconds, and the mean total irradiation to the sternum was 15.3 R. Transverse cross sections were reconstructed and then retrospectively reformatted to provide operator-selected oblique sections in space (for example, long-axis and short-axis sections of the left ventricle), to follow these sections through time (such as from end-diastole through end-systole), and to create three-dimensional displays (for instance, of the left ventricular chamber). Unique quantitative measurements of structure and function were made by using these images. For generation of most imaging data, only one injection of contrast material into the right side of the heart is necessary. Clinically useful three-dimensional dynamic imaging data can be acquired from adult patients with heart disease by using the DSR. Compared with conventional angiocardiography, DSR studies can provide information with less x-ray exposure and fewer angiographic injections.

Quantitative analysis of a vascular tree model with the dynamic spatial reconstructor.

The accuracy in determining the three-dimensional anatomy of a vessel network by computed tomography (CT) is evaluated using a glass model of a pulmonary artery. The dynamic spatial reconstructor (DSR), a high temporal resolution, volumetric, roentgenographic, CT scanner, was used to scan the model. The glass of the model had a roentgen attenuation coefficient mu = 0.55 cm-1, which is approximately equivalent to the 20% dilution of contrast medium to be expected in the pulmonary arterial tree following a contrast agent bolus injection of 2 ml/kg in the right atrium. The model was scanned inside a 20 cm diameter Plexiglas cylinder with a 1 cm thick wall (mu congruent to 0.2 cm-1) to simulate the chest wall of a 20 kg dog, and it was filled with potato flakes to simulate lung parenchyma (mu congruent to 0.06 cm-1). In one 0.011 s scan, information for reconstruction of a stack of images of transaxial sections was recorded. Sequential scans were performed to obtain data for either maximum transaxial resolution (14 angles of view every 0.0167 s, 120 parallel slices each 1.8 mm thick) or maximum axial resolution (eight angles of view every 0.0167 s, 240 parallel slices each 0.9 mm thick) reconstructions. Estimated detectable "vessel" size, cross-sectional area, branching angle, and interbranch segment length were determined as a function of imaged slice thickness, orientation of section image, and number of angles of view (i.e., scan duration) used to make images. Retrospective selection of 0.05 s duration scan apertures at sequential 0.5 s intervals was used to simulate a typical, retrospectively gated reconstruction from a DSR scan. Using these reconstructed images, 2 mm diameter "vessels" could be readily detected and their structure quantitated. Comparing direct measurements and DSR estimates, cross-sectional area (SEE = 3 mm2), branching angles (SEE = 2 degrees), and segment length (SEE = 1 mm) all had a correlation coefficient greater than 0.99, and the regression lines showed no significant differences from the lines of identity (p greater than 0.05).

Mass of left ventricular myocardium estimated with dynamic spatial reconstructor.

Using the Dynamic Spatial Reconstructor (DSR), a unique multiple X-ray source, high-repetition-rate CAT scanner, we estimated left ventricular (LV) myocardial volume and chamber volume of eight dogs ranging from 2.5 to 32.5 kg. Dogs were given subcutaneous morphine (3 mg/kg) and anesthetized with intravenous pentobarbital sodium (22 mg/kg). A bolus of 1 ml/kg body wt contrast medium was injected into the superior vena cava and 60/s scans repeated over 7 s were performed. Each 0.0167-s scan generated image data for 120 1.8-mm-thick transverse slices, in the dextro and levo phases of the angiograms. Retrospective reformatting of the scan data was used to generate images of thin slices perpendicular to the aortoapical axis of the LV. The LV muscle and chamber volumes were estimated from their outlines in each imaged slice using a manually operated trackball interfaced to a computer. Values of the LV muscle ranged from 18.0 to 146.8 cm3 by DSR and showed a good correlation with the postmortem values (r = 0.99, y = 0.94x + 4.1). Ratios of volume of the myocardium to chamber volume ranged from 1.19 to 3.10.

Visualization of SSB-ssDNA complexes active in the assembly of stable RecA-DNA filaments.

We have demonstrated that SSB binds to ssDNA in a complex manner, producing two fundamentally different structures: one that appears as a nucleosomal chain of beads and linkers, and the other as a smooth-contoured, extended nucleoprotein filament. Under physiologic salt conditions, only the beaded complexes were observed. Experiments indicate that the highly beaded forms are the most active in the assembly of the stable RecA-ssDNA filaments. These results further support our previous suggestion (Chrysogelos and Griffith 1982) that the protein-free linker regions are important in two ways. First, they provide access to the DNA template, and second, they provide a means for the two proteins to associate one with the other when bound to ssDNA. We suggest here that SSB should be considered as an assembly factor for RecA in its binding to ssDNA. Furthermore, our results argue that once RecA has associated with both the ssDNA template and SSB, some structural alteration in the (ATP-primed) RecA must occur that nucleates the formation of the very ordered, helical RecA filament along the ssDNA.

The Dynamic Spatial Reconstructor: investigating congenital heart disease in four dimensions.

The Dynamic Spatial Reconstructor (DSR) is a high-temporal resolution, three-dimensional (3-D) X-ray scanning device based on computed tomography (CT) principles. It was designed for investigation of some problems inherent in current diagnostic imaging techniques, and to allow quantitative studies of cardiovascular structure and function. One of the research protocols in which DSR is currently used involves studying selected pediatric patients with complex congenital heart disease. Initial results show that 3-D dynamic images can be obtained from these patients with minimal invasiveness and that these images may provide useful diagnostic information.

Esophageal manometry in systemic amyloidosis. A study of 30 patients.

The motility of the esophagus was studied by esophageal manometry in 24 patients with primary amyloidosis and six with secondary amyloidosis. Resting lower esophageal sphincter pressure was decreased in 12 patients with primary amyloidosis and two with secondary amyloidosis; 12 of these 14 patients complained of heartburn. Abnormalities in the motility of the body of the esophagus were found in nine patients with primary amyloidosis and one with secondary amyloidosis. No abnormality of the upper esophageal sphincter was demonstrated in any of the 30 patients. Six of the nine patients with primary amyloidosis exhibiting the most marked esophageal motor dysfunction had striking evidence of peripheral and/or autonomic nervous system involvement. No consistent pattern of motility disorder was observed in either group. The manometric abnormalities observed are consistent with a random deposition of amyloid in the esophagus involving a myopathic and/or neuropathic component.

Mayo Clinic and Medical School Biodynamics Research Unit.

The facilities that make up the Mayo Biodynamics Research Unit include the dynamic spatial reconstructor (DSR), which when fully operational will generate raw data at 200 million samples per second. Processing of these data will require a computer capable of several billion arithmetic operations per second.