Modelling cell turnover in a complex tissue during development.

The growth of organs results from proliferation within distinct cellular compartments. Organ development also involves transitions between cell types and variations in cell cycle duration as development progresses, and is regulated by a balance between entry into the compartment, proliferation of cells within the compartment, acquisition of quiescence and exit from that cell state via differentiation or death. While it is important to understand how environmental or genetic alterations can perturb such development, most approaches employed to date are descriptive rather than quantitative. This is because the identification and quantification of such parameters, while tractable in vitro, is challenging in the context of a complex tissue in vivo. Here we present a new framework for determining cell turnover in developing organs in vivo that combines cumulative cell-labelling and quantification of distinct cell-cycle phases without assuming homogeneity of behaviour within that compartment. A mathematical model is given that allows the calculation of cell cycle length in the context of a specific biological example and assesses the uncertainty of this calculation due to incomplete knowledge of cell cycle dynamics. This includes the development of a two population model to quantify possible heterogeneity of cell cycle length within a compartment and estimate the aggregate proliferation rate. These models are demonstrated on data collected from a progenitor cell compartment within the developing mouse kidney, the cap mesenchyme. This tissue was labelled by cumulative infusion, volumetrically quantified across time, and temporally analysed for the proportion of cells undergoing proliferation. By combining the cell cycle length predicted by the model with measurements of total cell population and mitotic rate, this approach facilitates the quantification of exit from this compartment without the need for a direct marker of that event. As a method specifically designed with assumptions appropriate to developing organs we believe this approach will be applicable to a range of developmental systems, facilitating estimations of cell cycle length and compartment behaviour that extend beyond simple comparisons of mitotic rates between normal and perturbed states.

Mite dispersal among the Southern Ocean Islands and Antarctica before the last glacial maximum.

It has long been maintained that the majority of terrestrial Antarctic species are relatively recent, post last glacial maximum, arrivals with perhaps a few microbial or protozoan taxa being substantially older. Recent studies have questioned this 'recolonization hypothesis', though the range of taxa examined has been limited. Here, we present the first large-scale study for mites, one of two dominant terrestrial arthropod groups in the region. Specifically, we provide a broad-scale molecular phylogeny of a biologically significant group of ameronothroid mites from across the maritime and sub-Antarctic regions. Applying different dating approaches, we show that divergences among the ameronothroid mite genera Podacarus, Alaskozetes and Halozetes significantly predate the Pleistocene and provide evidence of independent dispersals across the Antarctic Polar Front. Our data add to a growing body of evidence demonstrating that many taxa have survived glaciation of the Antarctic continent and the sub-Antarctic islands. Moreover, they also provide evidence of a relatively uncommon trend of dispersals from islands to continental mainlands. Within the ameronothroid mites, two distinct clades with specific habitat preferences (marine intertidal versus terrestrial/supralittoral) exist, supporting a model of within-habitat speciation rather than colonization from marine refugia to terrestrial habitats. The present results provide additional impetus for a search for terrestrial refugia in an area previously thought to have lacked ice-free ground during glacial maxima.

Phenotype-environment mismatches reduce connectivity in the sea.

The connectivity of marine populations is often surprisingly lower than predicted by the dispersal capabilities of propagules alone. Estimates of connectivity, moreover, do not always scale with distance and are sometimes counterintuitive. Population connectivity requires more than just the simple exchange of settlers among populations: it also requires the successful establishment and reproduction of exogenous colonizers. Marine organisms often disperse over large spatial scales, encountering very different environments and suffering extremely high levels of post-colonization mortality. Given the growing evidence that such selection pressures often vary over spatial scales that are much smaller than those of dispersal, we argue that selection will bias survival against exogenous colonizers. We call this selection against exogenous colonizers a phenotype-environment mismatch and argue that phenotype-environment mismatches represent an important barrier to connectivity in the sea. Crucially, these mismatches may operate independently of distance and thereby have the potential to explain the counterintuitive patterns of connectivity often seen in marine environments. We discuss how such mismatches might alter our understanding and management of marine populations.

Induced polarization: a geophysical method for locating cultural metallic refuse.

The problem of delineating cultural refuse sites (dumps) arises in civil engineering studies. Induced polarization measurements have been successfully applied in several cases. Laboratory tests on synthetic samples indicate that the effect is due to the metal content of the dumps. The method may be applicable to archeological investigations.

Recent advances in skeletal-muscle-based gene therapy.

Gene transfer into skeletal muscle holds promise for the treatment of a variety of serum protein deficiencies, muscular dystrophies, and chronic ischemic limb syndromes. The past two years have seen the development of new and improved vectors for programming recombinant gene expression in skeletal muscle. Important advances include first, novel plasmid DNA, adenovirus, and adeno-associated virus vectors that can be used to stably express therapeutic levels of recombinant proteins in the skeletal muscle of immunocompetent hosts and second, the development of vector systems that enable regulated and tissue-specific transgene expression in skeletal muscle in vivo.

Intestinal neoplasia in the ApcMin mouse: independence from the microbial and natural killer (beige locus) status.

We have tested the hypothesis that enteric bacteria are necessary for formation of intestinal adenomas in C57BL/6-ApcMin/+ mouse. Germ-free mice developed 2-fold fewer adenomas than conventional controls in the medial small intestine (7.3 versus 14.9; P < 0.003), but there were no significant differences in the rest of the intestinal tract. We conclude that microbial status does not strongly alter the adenoma phenotype in this mouse model of familial adenomatous polyposis. In parallel, we have found that C57BL/6-ApcMin/+ mice mutated at the beige locus, which controls natural killer activity, are also unaltered in adenoma multiplicity.

Chemoprevention of spontaneous intestinal adenomas in the Apc Min mouse model by the nonsteroidal anti-inflammatory drug piroxicam.

C57BL/6J-Min/+mice (n = 56), heterozygous for a nonsense mutation in the Apc gene, were randomized at weaning to seven groups, including groups treated with piroxicam at 0, 50, 100, and 200 ppm in the AIN93G diet. After only 6 weeks of treatment, intestinal adenomas and aberrant crypt foci were counted, and serum levels of piroxicam and thromboxane B2 were quantitated. Tumor multiplicity was decreased in a dose-dependent manner from 17.3 +/- 2.7 in the control to 2.1 +/- 1.1 (12%) in the high-dose piroxicam group (P < 0.001). Thromboxane B2 levels in plasma also decreased monotonically in parallel to the decrease in tumor multiplicity, consistent with the prostaglandin inhibitory effect of piroxicam. The Min mouse model demonstrates that the nonsteroidal anti-inflammatory drug piroxicam has strong biological and therapeutic effects, potentially useful for prevention of the early adenoma stage of tumor development.

Efficient and stable transduction of cardiomyocytes after intramyocardial injection or intracoronary perfusion with recombinant adeno-associated virus vectors.

BACKGROUND: The delivery of recombinant genes to cardiomyocytes holds promise for the treatment of a variety of cardiovascular diseases. Previous gene transfer approaches that used direct injection of plasmid DNA or replication-defective adenovirus vectors have been limited by low transduction frequencies and transient transgene expression due to immune responses, respectively. In this report, we have tested the feasibility of using intramyocardial injection or intracoronary infusions of recombinant adeno-associated virus (rAAV) vectors to program transgene expression in murine cardiomyocytes in vivo. METHODS AND RESULTS: We constructed an rAAV containing the LacZ gene under the transcriptional control of the cytomegalovirus (CMV) promoter (AAVCMV-LacZ). We then injected 1x10(8) infectious units (IU) of this virus into the left ventricular myocardium of adult CD-1 mice. Control hearts were injected with the AdCMV-LacZ adenovirus vector. Hearts harvested 2, 4, and 8 weeks after AAVCMV-LacZ injection demonstrated stable beta-galactosidase (beta-gal) expression in large numbers of cardiomyocytes without evidence of myocardial inflammation or myocyte necrosis. In contrast, the AdCMV-LacZ-injected hearts displayed transient beta-gal expression, which was undetectable by 4 weeks after injection. Explanted C57BL/6 mouse hearts were also perfused via the coronary arteries with 1.5x10(9) IU of AAVCMV-LacZ and assayed 2, 4, and 8 weeks later for beta-gal expression. beta-Gal expression was detected in <1% of cardiomyocytes at 2 weeks after perfusion but was detected in up to 50% of cardiomyocytes 4 to 8 weeks after perfusion. CONCLUSIONS: Direct intramyocardial injection or coronary artery perfusion with rAAV vectors can be used to program stable transgene expression in cardiomyocytes in vivo. rAAV appears to represent a useful vector for the delivery of therapeutic genes to the myocardium.

The role of the immune response in MULV-induced lymphomagenesis.

Although the exact mechanisms of murine leukemia virus (MuLV)-induced lymphomagenesis have yet to be elucidated, it is clear that the immune reponse to viral proteins plays a critical role in this disease process. The parameters for lymphomagenesis are governed by an inverse relationship between viral persistence and immune responsiveness. MuLV have evolved ways to avoid immune detection either by altering their own genome or by altering the host environment. In addition, the intrathymic replication of MuLV during thymocyte maturation and immune selection plays an important role in T cell repertoire development and immune inhibition. These viruses have served as a highly effective experimental model in understanding the many pathways by which MuLV have overcome immune detection and thereby led to lymphomagenesis.

Manipulation of the immune response by foreign gene expression in the thymus.

Retroviral gene transfer vectors have been developed for optimal in vivo gene therapy. Ideally, these vectors should target gene expression specifically to selected tissues or organs. Our studies focus on the development of retroviral vectors for gene delivery to the thymus. The goal of these studies is to utilize thymic expression of exogenous genes to manipulate the immune repertoire. We have characterized the selective thymic tropism of a molecular clone of Gross murine leukemia virus, GD-17, to thymic medullary epithelial cells using immunohistochemical staining and confocal microscopy. Specific expression of viral antigens in the thymus lead to the induction of immunologic tolerance to GMuLV proteins. This tissue specific vector may thus be used to study the requirements of epithelial mediated tolerance induction, and provide a more efficient tool for gene therapy.

FERMI-LAT OBSERVATIONS OF HIGH- AND INTERMEDIATE-VELOCITY CLOUDS: TRACING COSMIC RAYS IN THE HALO OF THE MILKY WAY.

It is widely accepted that cosmic rays (CRs) up to at least PeV energies are Galactic in origin. Accelerated particles are injected into the interstellar medium where they propagate to the farthest reaches of the Milky Way, including a surrounding halo. The composition of CRs coming to the solar system can be measured directly and has been used to infer the details of CR propagation that are extrapolated to the whole Galaxy. In contrast, indirect methods, such as observations of γ-ray emission from CR interactions with interstellar gas, have been employed to directly probe the CR densities in distant locations throughout the Galactic plane. In this article we use 73 months of data from the Fermi Large Area Telescope in the energy range between 300 MeV and 10 GeV to search for γ-ray emission produced by CR interactions in several high- and intermediate-velocity clouds (IVCs) located at up to ~7 kpc above the Galactic plane. We achieve the first detection of IVCs in γ rays and set upper limits on the emission from the remaining targets, thereby tracing the distribution of CR nuclei in the halo for the first time. We find that the γ-ray emissivity per H atom decreases with increasing distance from the plane at 97.5% confidence level. This corroborates the notion that CRs at the relevant energies originate in the Galactic disk. The emissivity of the upper intermediate-velocity Arch hints at a 50% decline of CR densities within 2 kpc from the plane. We compare our results to predictions of CR propagation models.

Restricted isotype, distinct variable gene usage, and high rate of gp120 specificity of HIV-1 envelope-specific B cells in colostrum compared with those in blood of HIV-1-infected, lactating African women.

A successful HIV-1 vaccine must elicit immune responses that impede mucosal virus transmission, though functional roles of protective HIV-1 Envelope (Env)-specific mucosal antibodies remain unclear. Colostrum is a rich source of readily accessible mucosal B cells that may help define the mucosal antibody response contributing to prevention of postnatal HIV-1 transmission. To examine the HIV-1 Env-specific colostrum B-cell repertoire, single B cells were isolated from 17 chronically HIV-infected, lactating women, producing 51 blood and 39 colostrum HIV-1 Env-specific B-cell antibodies. All HIV-1 Env-specific colostrum-derived antibodies were immunoglobulin (Ig)G1 isotype and had mean heavy chain complementarity-determining region 3 (CDR3) lengths and mutation frequencies similar to those isolated from blood. However, variable heavy chain (VH) gene subfamily 1(∼)69 usage was higher among colostrum than blood HIV-1 Env-reactive antibodies (49% vs. 20%, P=0.006, Fisher's exact test). Additionally, more HIV-1 Env-specific colostrum antibodies were gp120 specific than those isolated from blood (44% vs. 16%, P=0.005, Fisher's exact test). One cross-compartment HIV-1 Env-specific clonal B-cell lineage was identified. These unique characteristics of colostrum B-cell antibodies suggest selective homing of HIV-1-specific IgG1-secreting memory B cells to the mammary gland and have implications for targeting mucosal B-cell populations by vaccination.

Synthesis and estrogenic properties of 7 alpha,8 alpha-epoxy- and 7 alpha, 8 alpha-methyleneestradiols.

A series of 7alpha,8alpha-expoxyestradiol derivatives with ethynyl, 2- or 3-furyl, or 2-thienyl substituents in the 17alpha position was prepared. The products were highly active orally in the Allen-Doisy test in rats, but most of them were only weakly active in the uterotrophic assay in the mouse. A 7alpha,8alpha-methylene analog and a 7alpha,8alpha-difluormethylene analog were less active than the corresponding epoxides.

Flow cytometric analyses of lymphocyte subsets in peripheral blood and lymphoid tissues of gnotobiotic calves during primary acute postnatal infections of bovine viral diarrhea virus.

Eight 1-day-old gnotobiotic calves were exposed to one of 2 noncytopathic isolates of bovine viral diarrhea virus (ncp-BVDV 7937 and ncp-BVDV 126) and 2 similar calves were not infected and served as controls. Phenotypic analyses of peripheral blood mononuclear leukocytes at 3, 7, and 10 days postinfection (PI), and cell preparations of thymus, Peyer's patch, mesenteric lymph node, spleen, and bone marrow collected at necropsy, 10 days PI, were conducted using flow cytometric techniques on cells stained by an indirect fluorescent antibody assay using monoclonal antibodies specific for mononuclear leukocyte subsets. Significant fluctuations in specific subsets of peripheral blood mononuclear leukocytes were observed in calves exposed to ncp-BVDV 7937 on day 3 PI (cells expressing MHC class II), day 7 PI (B-cells), and day 10 PI (B cells and cells expressing MHCII). Significant phenotypic differences between groups were also detected in cell preparations from Peyer's patch and thymus. Calves exposed to ncp-BVDV 7937 had a significantly higher percentage of B cells than calves exposed to ncp-BVDV 126 and calves not exposed to BVDV. Calves exposed to ncp-BVDV 126 had a significantly higher percentage of CD2 (BoCD2) positive T cells than calves not exposed to BVDV. Fragility of thymic cell preparations was attributed to infection with virus. These results highlight the importance of the tropism of BVDV for cells of the bovine immune system and its role as a significant immunosuppressive agent capable of predisposing affected animals to infection with other agents.

Experimental infection of calves with bovine viral diarrhea virus genotype II (NY-93).

To ascertain the virulence of bovine viral diarrhea virus (BVDV) genotype II, isolate NY-93 was inoculated intranasally into 3 calves, 2 of which were treated with a synthetic glucocorticoid prior to and after virus inoculation. Anorexia, fever (up to 42 C), dyspnea, and hemorrhagic diarrhea developed 6 days after intranasal inoculation with BVDV NY-93. The condition of all calves deteriorated further until the end of the study on day 14 postinoculation. The most significant postmortem macroscopic changes in all calves were limited to the gastrointestinal tract and consisted of moderate to severe congestion of the mucosa with multifocal hemorrhages. Microscopic lesions found in the gastrointestinal tract were similar to those observed in mucosal disease, including degeneration and necrosis of crypt epithelium and necrosis of lymphoid tissue throughout the ileum, colon, and rectum. The basal stratum of the epithelium of tongue, esophagus, and rumen had scattered individual necrotic cells. Spleen and lymph nodes had lymphocytolysis and severe lymphoid depletion. Severe acute fibrinous bronchopneumonia was present in dexamethasone-treated calves. Abundant viral antigen was detected by immunohistochemistry in the squamous epithelium of tongue, esophagus, and forestomachs. BVDV antigen was prominent in cells of the media of small arteries and endothelial cells. The presence of infectious virus in tissues correlated with an absence of circulating neutralizing antibodies. These findings highlight the potential of BVDV genotype II to cause severe disease in normal and stressed cattle.

Evaluation of an absorbed ELISA and an agar-gel immuno-diffusion test for ovine paratuberculosis in sheep in Australia.

The sensitivities and specificities of an absorbed enzyme-linked immunosorbent assay (ELISA) and an agar-gel immuno-diffusion (AGID) test for the detection of Johne's disease in sheep were estimated using data from six known infected and 12 assumed uninfected sheep flocks. Sensitivities were estimated for all histologically positive sheep, as well as by histological lesion score, lesion type (paucibacillary or multibacillary) and sheep body-condition score, with ELISA sensitivities estimated at 95 and 99% specificity. Logistic-regression analysis was used to test for significant effects of lesion score and condition score, with flock included in the model as a random effect. Estimated specificities were 95% (95% CI: 93.4, 95.6%) and 99% (98.4, 99.4%) for ELISA cut-point ratios of 2.4 and 3.6, respectively, and 100% (99.7, 100.0%) for the AGID. Estimated sensitivities for all infected sheep were 41.5% (35.0, 48.3%), 21.9% (16.6, 27.9%) and 24.6% (19.1, 30.7%) for ELISA cut-point ratios of 2.4 and 3.6 and for AGID, respectively. Sensitivities of all tests and cut-points varied significantly between flocks and between categories of lesion score and condition score. Sensitivity ranged from 25 to 73, 10 to 47 and 9.2 to 63% between flocks, for the ELISA with cut-points of 2.4 and 3.6, and for the AGID, respectively. Sensitivity was highest in thin sheep and in sheep with multibacillary lesions. The effects of lesion type and condition score on test sensitivity were significant in the logistic regressions for the AGID and ELISA at both cut-points and the flock effect was significant for the AGID but not for the ELISA at either cut-point.

The pathologic medical shelf.

It is now recognized that a previously normal fold of synovium on the medial side of the knee (a medial patellar plica) may, as a result of trauma, become pathologically inflamed and/or fibrosed, producing symptoms that may be mistakenly diagnosed as a torn meniscus or chondromalacia patellae. Clinical awareness, aided by arthroscopy, can establish the diagnosis. The correct treatment is simple and, if instituted early before irreversible changes have occurred in the femoral condyle articular cartilage, is extremely effective.

Correlations between metal uptake in the soft tissue of Perna perna and gill filament pathology after exposure to mercury.

The accumulation of metal in soft tissues, filtration rate and gill filament morphology are correlated in the southern African rock mussel, Perna perna, during exposure to mercury (24 days) and recovery (24 days). The amount of Hg in soft tissues increased from 0.13 to 87.5 microg/g dry weight after 24 days exposure, and declined to 13 microg/g during recovery. Mean filtration rate fell from 3,979 to 1,818 ml/h/g dry weight by day 2, but recovered slightly through days 4 and 8 (3,037 ml/h/g), with a higher average rate (5,030 ml/h/g) being maintained over the 24-48 days recovery period. The initial decline in filtration coincided with epithelial cell deterioration presented as interstitial oedema, neural and epithelial cell degeneration and reduced ciliation. Between days 8 and 24, cilia regenerated and there was a general improvement in cell morphology. Gill filament morphology returned to near normal during the metal-free recovery period. The usefulness of P. perna as an indicator of pollution is discussed.

An anamnestic serological test for ovine footrot.

Following recovery from ovine footrot, a proportion of sheep in a flock may carry the causative organism and spread it to other sheep if environmental conditions are favourable. Footrot affected sheep have elevated levels of serum antibody against Bacteroides nodosus, but these levels decline rapidly after clinical recovery. When challenged by subcutaneous injection with 470 micrograms of protein extracted from the cell membrane of B. nodosus, without adjuvant, sheep that had recovered clinically from virulent footrot produced a marked increase in specific serum antibody within 7 d, while antibody levels in footrot-free sheep injected with the same antigen, and in saline injected controls, did not increase over a period of 25 d. Artificial stimulation and serological detection of immune memory may be useful in footrot eradication programs by identifying sheep that have had clinical footrot infection. This procedure may be applicable to other diseases where antibody responses are inconsistent or transient.

Serum antibody responses in sheep after natural infection with Bacteroides nodosus.

Soluble outer membrane protein of Bacteroides nodosus extracted with potassium thiocyanate (KSCN) was employed as antigen in an enzyme linked immunosorbent assay (ELISA) to detect serum antibody in sheep naturally infected with a heterologous serogroup. Serum antibody responses in 55 sheep were monitored for 2 years and maximum levels were directly related to the severity of clinical foot lesions. Serum antibody levels rose 2 weeks after foot lesions developed and declined within several months of resolution of lesions. After the first footrot transmission period, antibody levels persisted significantly (P less than 0.001) longer in sheep that did not become affected in the next transmission period compared with sheep in which footrot recurred. Antibody response did not appear to result in resolution of foot lesions. ELISA using KSCN antigen gave similar results to whole cell ELISA where cells prepared from an homologous serogroup were used as antigen. Both these assays were more sensitive than ELISA in which heterologous whole cell antigen was used. Proteins extracted from the outer membrane of B. nodosus, which are known to be immunogenic in natural infection and common to different serogroups of B. nodosus, appear to be useful antigens for serological investigations of ovine footrot.