Laparoscopic ovum pick-up followed by in vitro embryo production for the reproductive rescue of aged goats of high genetic value.

The efficiency of laparoscopic ovum pick-up (LOPU) followed by in vitro embryo production (IVEP) in the propagation of aged goats with poor reproductive performance was evaluated in the present study. Follicular development was stimulated in donor goats with 80 mg follicle-stimulating hormone and 300 IU equine chorionic gonadotrophin administered 36 h before LOPU. In addition, goats were heat synchronised with intravaginal sponges containing 60 mg medroxyprogesterone acetate for 10 days and a luteolytic injection of 125 microg cloprostenol 36 h before sponge removal and LOPU. Following in vitro maturation (IVM), oocytes were fertilised in vitro with frozen-thawed semen produced using the egg yolk-free Bioxcell extender (IVM, L'Aigle, France). The average number of follicles aspirated (17.9 +/- 8.0 per goat), oocytes recovered (15.7 +/- 8.4 per goat) and cleavage after IVM/in vitro fertilisation followed by a short 24-h in vitro culture in modified synthetic oviduct fluid medium (72 +/- 7%) were similar to results reported previously by our group and others in younger goats. A total of 296 embryos was transferred into 50 heat-synchronised recipients, of which 40 became pregnant (80%) and 38 progressed all the way to term, delivering 86 live kids. The present study indicates that LOPU-IVEP can be used successfully to extend the reproductive life of valuable goats that have acquired difficulties becoming pregnant by artificial insemination after multiple kiddings.

Assessment of mumps virus growth on various continuous cell lines by virological, immunological, molecular and morphological investigations.

Vero cells have been used as a convenient laboratory substrate for the isolation of mumps virus but may not be very sensitive and may select for particular adapted variants from clinical specimens. Continuous cell lines were evaluated for their ability to support the replication of mumps virus. Criteria included the production of infectious virus, detection of intracellular mumps proteins by immunofluorescence and electron microscopy and detection of specific nucleic acid by RT-PCR. Of the cells tested, CaCo-2, PLC/PRF/5, and Vero cells produced infectious virus, with Vero and CaCo-2 being the most permissive. The other substrates tested included cells of murine, canine and human origin showed signs of intracellular proteins and RNA but the amounts produced were much lower, and no infectious virus was detected in some cases. The virus use was a low passage of a Vero derived wild type strain, and it will ultimately be necessary to continue the studies with an unpassaged clinical specimen to identify a cell line able to isolate mumps virus at high efficiency and in unmodified form.

Development of a continuous IL-7-dependent murine pre-B cell line PB-1 suitable for the biological characterisation and assay of human IL-7.

Human interleukin-7 (IL-7) is a cytokine that appears to be critical for early T- and B-cell development and although IL-7 is currently under investigation as a therapeutic agent in a variety of hematolymphopoietic disorders, there have been few instances of the detection or investigation of this cytokine using a biological assay. This has been due, in the main, to the lack of a widely available, stable, easy to maintain and use, IL-7 responsive cell line. We have developed a pre-B-cell line, PB-1, from murine bone marrow, that is dependent on IL-7 for growth and has been maintained continually for up to 1 year without loss of responsiveness. The cells survive freezing and reviving, having been stored for periods of up to 4 years. The IL-7 bioassay is reproducible and sensitive, able to reliably detect 50 pg/ml IL-7. The assay is completely unresponsive to any other stimulatory cytokines tested and is not affected by a wide variety of inhibitory cytokines, with the exception of high levels of interferon alpha. The assay can be made completely specific for human IL-7 by including specific neutralizing antibodies for IL-7 and has been shown to be suitable for the estimation of IL-7 in both plasma and serum samples.

Primary retinal degeneration: evidence of normal phagocytosis in the retinal pigment epithelium.

In rats with primary retinal degeneration, lens extraction combined with total retinal detachment provided a model for injection of a tracer of colloidal carbon into the subretinal space. Electron microscopy and acid phosphatase cytochemistry were subsequently used to analyze the ingestion of tracer by the retinal pigment epithelium. It was found that the attachment, ingestion, and digestion phases of the phagocytic process were apparently preserved. From this evidence it is suggested that there is no lack of phagocytic power in the retinal pigment epithelium of affected rat strains.

Monoclonal antibodies which block cellular receptors of poliovirus.

The isolation and properties of monoclonal antibodies which specifically inhibit the binding of poliovirus types 1, 2 and 3 to cells are described. The antibodies were of the IgG class and blocked infection of cells by all strains of the three poliovirus serotypes, but by none of a wide range of other viruses examined, including nine human enteroviruses. The antibodies prevented poliovirus growth in all susceptible human and primate cells tested. We conclude that the antibodies are directed against the receptor site for poliovirus which is distinct from those required by other picornaviruses, and which seems to be antigenically well conserved between cells of human and primate origin.

The effect of interleukin-1 on venous endothelium--an ultrastructural study.

The effect of systemic interleukin-1 (IL-1) on venous endothelium in the presence and absence of stasis has been studied by scanning electron microscopy (SEM). Recombinant human IL-1 beta at a concentration of 1 micrograms/kg or saline was injected intravenously into rabbits and allowed to circulate for 0.5 or 4.0 h after which complete stasis was induced for 1 h in an isolated segment of each jugular vein. One vein segment was then excised and the contents examined macroscopically for thrombi, while the other segment was fixed for SEM examination. When examined by SEM the endothelium from rabbits injected with IL-1 beta was perturbed with increased surface microvilli, blebs and gaps at cell junctions when compared with saline controls. Fibrin deposition was also observed after IL-1 beta, as was the adherence of essentially non-activated platelets to intact endothelium. However, macroscopic thrombi were not formed in isolated vein segments. We conclude that although fibrin strands and platelets were deposited on the endothelium, IL-1 is not a sufficiently powerful procoagulant stimulus to lead to an occlusive thrombus in acute experiments.

Screening for fibrin specific monoclonal antibodies: the development of a new procedure.

The acquisition of monoclonal antibodies specific for human fibrin has been impaired by the similarity in chemical composition between fibrinogen and fibrin and the conformational difference between immobilised and soluble fibrinogen. Five monoclonal antibodies (mabs) with a known affinity for fibrin have been subjected to screening procedures which involved the presentation of different forms of both fibrinogen and fibrin to the test mabs. It was observed by scanning electron microscopy that dried fibrin (denoted fibrin D), immobilised on the wells of PVC plates was morphologically similar to the fibrin found in human clots whereas PVC-immobilised fibrin monolayers (fibrin M) and a homogenised form of fibrin (fibrin FF) presented two very different morphological appearances. It was shown that lack of cross reactivity of a mab with an antigen (e.g. fibrinogen) was validly demonstrated only when both mab and antigen were present in the soluble state. These findings have allowed the generation of a screening procedure which involves the use of fibrin D on PVC plates in conjunction with whole human plasma incubated with the test antibody. This screening procedure has been validated using two mabs, one of which has an exclusive fibrin affinity while the other has a broad spectrum crossreactivity with both fibrinogen and fibrin. This procedure would ensure the acquisition of all the five fibrin-specific mabs used in this study while other less reliable screening procedures might not.

Further evidence of icosahedral symmetry in human and simian immunodeficiency virus.

Specimens of HIV and SIV have been examined by electron microscopy, using the techniques of conventional thin sectioning, freeze-substitution, cryosectioning, and cryomicroscopy of frozen hydrated specimens. In addition freeze-drying and critical point drying were used for both shadowed replicas and scanning electron microscopy. Thin sections revealed hexagonal, pentagonal, or near-spherical profiles. Angular particles were seen in shadowed replicas and also by scanning electron microscopy. The images observed were consistent with an icosahedral shape of the virus. It is proposed that mature HIV (SIV) is an icosadeltahedron with flat triangular facets. Size measurements of the specimens showed a wide range of values for conventional embedding, but a narrow range for specimens prepared by low-temperature techniques.

Antiviral properties of Ro 31-8959, an inhibitor of human immunodeficiency virus (HIV) proteinase.

Ro 31-8959 inhibits the spread of HIV infection and the production of cytopathic effects in cultures of acutely infected cells. IC50 values for these effects are in the range 0.5-6.0 nM and IC90 values are in the range 6.0-30.0 nM. This inhibitor is effective even when added to cultures at a late stage of infection, after syncytia have started to form. Virus antigen, virus particles and virus cytopathic effects can largely be cleared from cultures treated with compound from 3 days until 6 days post infection. In chronically-infected cells, inhibition of virus maturation can be detected after 24 hours' treatment with 10 nM Ro 31-8959. In addition, a significant reduction of the proteolytic processing of p56 to p24 can be demonstrated in these cells with compound at picomolar concentrations. These properties indicate that Ro 31-8959 is highly effective against HIV with the potential to inhibit acute, established acute and chronic infections.

Resorbable lamellar particles of polylactide as adjuvants for influenza virus vaccines.

Lamellar particles and microspheres were produced by precipitation from solutions of resorbable, biocompatible, semi-crystalline poly(L-lactide)[PLA] and amorphous poly(DL lactide co-glycolide)[PLG] copolymer, respectively, to investigate their adjuvanticity towards adsorbed influenza virus. Both types of substrate were capable of adsorbing large quantities of virus (> 15% w/w) and retaining virus (> 60% of the initial load) over an 8 week time scale in-vitro. Potent immune responses were obtained in mice after the intra-muscular injection of adsorbed vaccine systems. The response to virus adsorbed on PLA lamellar particles was almost five times that obtained using PLG microspheres and fourteen times that using aqueous vaccine. The lamellar forms of PLA may function as an immunomodulator enhancing phagocytic activity due to their irregular shape and may be useful in improving the immune response to a variety of protein and viral antigens.

Chemical properties, distribution, and physiology of plant and algal carbonic anhydrases.

Plant carbonic anhydrases (CAs) have a range of molecular weights (MW). Among flowering plants, dicotyledons with C3 photosynthesis have two isoenzymes of 140-250K each with 6 subunits, while monocotyledons have two isoenzymes of 42-45K. Plant and animal CAs have a similar amino acid content, subunit size and zinc content, suggesting they are homologous proteins, although the higher plant CAs have no esterase activity and are not strongly inhibited by sulfonamides. Algal CAs vary widely in MW and some are highly sensitive to sulfonamides like the animal enzymes. The two plant isoenzymes, from the chloroplast and cytosol, can be separated by gradient polyacrylamide gel electrophoresis and subsequently visualized by enzymic H+ ion production. In plants, CAs probably facilitate diffusion of CO2 to the site of photosynthetic fixation; they may also have a role in pH regulation, in the use of bicarbonate by aquatic plants and in concentrating inorganic carbon within the chloroplast.

Experimental retinal branch vein occlusion in rhesus monkeys. III. Histopathological and electron microscopical studies.

Experimental retinal vein occlusion in monkeys was followed by an immediate increase in capillary permeability which was accompanied by retinal oedema. This functional capillary change was followed at about 6 hours after occlusion by structural damage to the capillary wall. Thrombus formation occurred in the damaged vessels, and areas of stasis were thus produced with associated retinal haemorrhages. Finally, there was complete loss of the capillary endothelium and pericytes, and the acellular capillaries were invaded by proliferating glial cell processes, so producing permanent capillary closure.

A morphological and immunolabelling study of freeze-substituted human and simian immunodeficiency viruses.

Rapid freezing, freeze substitution and low temperature embedding were used to obtain resin-embedded specimens of HIV and SIV for morphological and immunolabelling studies, with particular emphasis on the 'lateral bodies' and p6 protein. HIV- or SIV-infected cells were fixed in 3% paraformaldehyde and cryoprotected with 0.5 M sucrose. Cells were applied to pieces of Whatman No 1 filter paper and impact-frozen onto a liquid nitrogen cooled copper block. Specimens were freeze-substituted at -90 degrees C using one of three different media: (a) absolute methanol, (b) methanol containing 0.5% uranyl acetate, and (c) methanol containing glutaraldehyde, osmium tetroxide and uranyl acetate. Specimens substituted in methanol and uranyl acetate showed both good structural preservation and retention of antigenicity. We found that the use of filter paper for supporting the specimen was an important factor in obtaining good freezing rates and was more practical than freezing mixtures of cells and gelatin. When compared with specimens prepared by conventional fixation and embedding, freeze-substituted virus particles showed a greater uniformity of shape and size and were more dense in appearance. Distinct 'lateral bodies' were not observed in freeze-substituted viruses. The viral protein p6 was widely distributed in the centre of mature virus particles.

Ultrastructural localization of the RNA of immunodeficiency viruses using electron microscopy in situ hybridization and in vitroinfected lymphocytes.

Cells infected in vitro with immunodeficiency viruses have been examined by electron microscopy in situ hybridization (EM ISH) methods for localization of viral RNA. Techniques used for preparation of specimens and probes are described. Unambiguous positive results were obtained using a mixture of two or three single negative strand DNA oligonucleotides complementary to regions of the gag, env and nef genes, each 200-300 bases and labelled with dig-11-UTP. Positive strand probes were used as a negative control. Cells were fixed with a mixture of formaldehyde and glutaraldehyde, dehydrated in ethanol with progressive lowering of temperature and embedded in Lowicryl K4M or HM20 at -35 degrees C. Permeabilization or pre-treatment of sections with proteinase K was not essential. The hybridization mixture was applied for 3-4h at 37 degrees C and probe was visualized by direct immuno-staining with sheep anti-digoxigenin antibodies conjugated to 10nm gold. This method would be suitable for future studies of the pathogenesis of retroviral infections and as a basis for further development of the EM ISH technique. EM ISH of in vitro infections of immunodeficiency viruses has shown the location of viral RNA in immature and mature viruses and its relationship to multimerized Gag protein during viral budding. The label for RNA has also been found in the cytoplasm of infected cells; it was mainly located adjacent to the plasma membrane and unassociated with visible Gag proteins. This may indicate that viral RNA migrates to the plasma membrane independently of the Gag protein and may, in some instances, arrive at the plasma membrane prior to the Gag protein. Viral RNA has also been found in the nucleus of peripheral blood mononuclear cells (PBMC) that were showing no morphological evidence of infection. The RNA was typically located in the nucleolus and in peripheral dense chromatin. These cells, which displayed morphological features of macrophage lineage, may have been the initial cell type to be infected in the PBMC.

A freeze-fracture study of the tegumental membrane of Schistosoma mansoni (Platyhelminthes:Trematoda).

Two fracture faces in each half of the freeze-fractured tegumental membrane of adult Schistosoma mansoni indicate the presence of two trilaminate membranes. This result is compatible with the heptalaminate appearance of the tegumental membrane in ultrathin sections. Intramembranous particles are located mainly in the outermost leaflet of the outer membrane and in the cytoplasmic leaflet of the inner membrane. The tegumental membrane of the cercaria (infective larva) has a single fracture plane, which conforms with its trilaminate appearance in sections. Intramembranous particles are extremely numerous and are almost all located in the cytoplasmic leaflet.

Superovulatory response to a single subcutaneous injection of Folltropin-V in beef cattle.

A series of 4 experiments were designed to evaluate the feasibility of superstimulation in beef cattle with a single sc injection of the porcine pituitary extract, Folltropin-V. In the preliminary study (Experiment 1), superovulatory response of cows (n=7) treated with a single sc injection of 400 mg NIH-FSH-P1 Folltropin-V was not different than that of cows (n=8) superstimulated with twice daily im injections over 4 d, or a single sc injection plus an injection of eCG (n=12). Experiments 2 and 3 were designed to determine the optimal site of a single sc injection. In Experiment 2, cows (n=25) with body condition scores (BCS) of 1 to 2 were used. The mean number of CL counted and ova/embryos collected was lower (P<0.05) in cows treated with the single sc injection in the neck region than in cows treated with a single sc injection behind the shoulder, or with the twice daily im injection treatment. In Experiment 3, cows (n=49) with BCS of 3 to 5 were used. There were no differences in the number of CL, total ova/embrvos collected, fertilized ova and transferable embryos whether treatments were given in the neck region or behind the shoulder, or whether the cows were implanted or not implanted with Syncro-Mate-B. Experiment 4 was designed to determine the optimal superstimulatory dosage of Folltropin-V administered by a single sc injection. Superovulatory response of cows treated with the higher doses (400 mg, 600 mg or 800 mg NIH-FSH-P1) was higher (P<0.05) than those treated with 200 mg NIH-FSH-P1. The number of unovulated (>or=10 mm) follicles at the time of ova/embryo collection was higher (P<0.05) in the 600 and 800 mg groups, and progesterone concentration at estrus was higher (P<0.05) in cows treated with 800 mg than with 400 or 200 mg. It was concluded that a single, bolus sc injection of 400 mg NIH-FSH-P1 of Folltropin-V is as efficacious as the 4-d, twice daily im treatment protocol for inducing superovulation in beef cows. The amount of subcutaneous fat and site of injection appeared to affect the efficacy of a single sc injection; a single bolus sc injection of Folltropin-V behind the shoulder resulted in the most predictable superovulatory response.

Multiphase transfer processes in waste rock piles producing acid mine drainage 1: Conceptual model and system characterization.

Acid mine drainage (AMD) results from the oxidation of sulfides, mainly pyrite, present in mine wastes, either mill tailings or waste rock. This is the first of two papers describing the coupled physical processes taking place in waste rock piles undergoing AMD production. Since the oxidation of pyrite involves the consumption of oxygen and the production of heat, the oxidation process initiates coupled processes of gas transfer by diffusion and convection as well as heat transfer. These processes influence the supply of oxygen that is required to sustain the oxidation process. This first paper describes a general conceptual model of the interaction of these coupled transfer processes. This general conceptual model is illustrated by the physicochemical conditions observed at two large sites where extensive characterization programs revealed widely different properties. The South Dump of the Doyon mine in Canada is permeable and has a high pyrite oxidation rate leading to high temperatures (over 65 degrees C), thus making temperature-driven air convection the main oxygen supply mechanism. The Nordhalde of the Ronnenberg mining district in Germany contains lower permeability material which is less reactive, thus leading to a more balanced contribution of gaseous diffusion and convection as oxygen supply mechanisms. The field characterization and monitoring data at these sites were thoroughly analyzed to yield two coherent sets of representative physical properties. These properties are used in the second paper as a basis for applications of numerical simulation in AMD-producing waste rock piles.

Multiphase transfer processes in waste rock piles producing acid mine drainage 2. Applications of numerical simulation.

Acid mine drainage (AMD) results from the oxidation of sulfides, mainly pyrite, present in mine wastes, either mill tailings or waste rock. This is the second of two papers describing the coupled physical processes taking place in waste rock piles undergoing AMD production. Since the oxidation of pyrite involves the consumption of oxygen and the production of heat, the oxidation process initiates coupled processes of gas transfer by diffusion and convection as well as heat transfer. These processes influence the supply of oxygen that is required to sustain the oxidation process. This second paper describes a numerical simulator used to represent the interaction of these coupled transfer processes. Numerical simulations are applied to two large sites with extensive characterization programs and widely different properties and behavior that were described in the first paper. The South Dump of the Doyon mine in Canada is permeable and has a high pyrite oxidation rate, thus making temperature-driven air convection the main oxygen supply mechanism. The Nordhalde of the Ronnenberg mining district in Germany contains lower permeability material which is less reactive, thus leading to a more balanced contribution of gaseous diffusion and convection as oxygen supply mechanisms. Overall, simulations allow a coherent representation of the conditions monitored within the waste rock piles and the confirmation of their physical properties. Conceptual simulations are also carried out to illustrate the potential effect of border membranes and layered co-mingling as mitigation methods used to control AMD production in either active or future waste rock piles.