Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors.

The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCD vaccine expressing HIV gp120 that retained stable full-length expression after 10(24)-fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >10(68)-fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches.

Transgenic animals as models for human disease.

Transgenic animals, especially mice, have been used quite extensively as models for various human diseases. At first, the level of scientific inquiry was driven by the need to establish the model. In many cases, these models may be considered quite crude because of their limitations. More recently, transgenic models of disease have become more refined and are currently being used to study the pathological mechanisms behind the disease rather than to just provide a model of the disease. Using some examples from the recent literature, we will document the current level and complexity of inquiry using transgenic animals. New techniques and techniques that may prove promising will be discussed.

Membrane skeleton in cultured chick cardiac myocytes revealed by high resolution immunocytochemistry.

Distribution of cytoskeletal proteins with emphasis on the membrane-cytoskeleton interface was examined in cultured cardiac myocytes. Using specific antibodies recognizing alpha-sarcomeric actin, desmin, beta-tubulin, spectrin/alpha-fodrin and ankyrin, respectively, the cellular localization of these cytoskeletal proteins was detected by laser scanning confocal microscopy. In addition, the fine filamentous structure of these proteins was identified by combining silver-enhanced immunogold labelling with electron microscopy. The latter technique employed the sequence of quick-freezing, deep-etching and rotary shadowing of the specimens. Conventional transmission electron microscopy of the spherical cardiac myocytes revealed a filamentous submembranous layer, approximately 100 nm thick. Specific immunolabelling of alpha-sarcomeric actin and spectrin/alpha-fodrin as well as ankyrin was seen beneath the plasmalemma. A three-dimensional meshwork of spectrin/alpha-fodrin was shown. Numerous desmin filaments that exhibited a tortuous course throughout the cells were also observed running in parallel with the surface in the submembranous area, whereas beta-tubulin was infrequently detected in these areas. In conclusion, the present study shows that spherical cardiac myocytes contain a distinct and complex three-dimensional membrane skeleton. Major constituents of this distinct submembranous layer were spectrin/alpha-fodrin fibres as well as actin and desmin filaments.

Familial subepithelial corneal amyloidosis (gelatinous drop-like corneal dystrophy): exclusion of linkage to lactoferrin gene.

PURPOSE: Because corneal tissue with familial subepithelial corneal amyloidosis (FSCA; gelatinous drop-like dystrophy of the cornea) contains lactoferrin the possibility that the FSCA gene was the human lactoferrin (hLF) gene was investigated. Due to contradictory published information we also mapped the hLF gene. METHODS: We mapped the hLF gene using a genomic clone of the entire hLF gene as a probe by fluorescence in situ hybridization (FISH). Utilizing PCR primers that are specific to the hLF gene, we also mapped the hLF via radiation somatic cell hybrid analysis. Linkage of the FSCA gene to the hLF gene was evaluated by genetic linkage analysis using polymorphic markers within and in the vicinity of the hLF gene. RESULTS: The hLF gene mapped to the short arm of chromosome 3 at 3p21. Linkage analysis using polymorphic markers for hLF and haplotype analysis of the 3p21 loci indicates that the FSCA gene is not linked to the 3p21 locus. CONCLUSIONS: The gene for FSCA is not the hLF gene in these families.

Genetically engineered large animal model for studying cone photoreceptor survival and degeneration in retinitis pigmentosa.

Patients with retinitis pigmentosa (RP) typically develop night blindness early in life due to loss of rod photoreceptors. The remaining cone photoreceptors are the mainstay of their vision; however, over years or decades, these cones slowly degenerate, leading to blindness. We created transgenic pigs that express a mutated rhodopsin gene (Pro347Leu). Like RP patients with the same mutation, these pigs have early and severe rod loss; initially their cones are relatively spared, but these surviving cones slowly degenerate. By age 20 months, there is only a single layer of morphologically abnormal cones and the cone electroretinogram is markedly reduced. Given the strong similarities in phenotype to that of RP patients, these transgenic pigs will provide a large animal model for study of the protracted phase of cone degeneration found in RP and for preclinical treatment trials.

Comparative anatomy: in praise of a powerful approach to elucidate mechanisms translating cardiac excitation into purposeful contraction.

This review wishes to illustrate and, thus, reemphasize the importance of descriptive comparative anatomy for the elucidation of mechanisms driving cardiac function at different levels of spatial resolution. The following examples have been chosen: 1. the cardiac conduction system; and 2. the sarcoplasmic reticulum of cardiac and skeletal muscle. Both examples demonstrate that anatomy and geometry dictate the mechanistic behaviour of the systems under discussion, and that precise knowledge of the architecture of biological systems, in general, is crucial for an understanding of how function is consummated. The detailed comparative display and discussion of the sarcoplasmic reticulum's architecture serves the additional purpose of exposing important anatomical and geometric features of this organelle at a time when considerable efforts are being expended toward the unravelling of the mechanism of excitation-contraction coupling; anatomy is manifestly critical to these efforts.

Extended junctional sarcoplasmic reticulum of avian cardiac muscle contains functional ryanodine receptors.

The ryanodine receptor (RYR)/Ca2+ release channel of avian cardiac muscle was localized by immunocytochemical techniques and biochemically characterized using isolated membrane and receptor protein fractions. Monoclonal antibody C3-33 raised against the canine cardiac RYR bound to the junctional sarcoplasmic reticulum of pigeon and finch hearts, both at peripheral couplings and at extended junctional sarcoplasmic reticulum (EJSR). Immunoblots of sarcoplasmic reticulum vesicles from pigeon and finch hearts showed this antibody recognized a single high molecular weight protein, which co-migrated with the canine M(r) 565,000 RYR/Ca2+ release channel polypeptide. The pigeon heart RYR bound [3H]ryanodine with high affinity in a Ca(2+)-dependent manner, comparable to the canine cardiac RYR. Purification of the pigeon RYR yielded a 30 S protein complex, which bound the maximum calculated amount of [3H]ryanodine ((440 +/- 60) pmol/mg protein), assuming one high affinity site/tetrameric 30 S RYR comprised of M(r) 565,000 polypeptides. Autoradiography of isolated finch cardiac myocytes indicated [3H]ryanodine binding throughout the cells. These results suggest that avian heart contains a single population of RYRs, and thereby support the hypothesis that avian EJSR contains functional calcium release channels which, because of the absence of transverse tubules, can be located micrometers away from the surface membrane in avian heart.

Quick-freezing of cultured cardiac cells in situ with special attention to the mitochondrial ultrastructure.

A new method has been developed which allows quick-freezing in situ of primary, cardiac cell cultures grown to confluence on gas-permeable membranes (Petriperm dishes). Small pieces of the growth substratum, with rhythmically beating myocardial cells, were slam-frozen, without cryoprotectants, against the surface of a helium-cooled copper block at approximately 16 K. The quality of the cellular cryopreservation, as judged by ultrastructural criteria, was studied in freeze-substituted specimens processed for transmission electron microscopy. The ultrastructure of cryofixed cardiac cells was compared with that of unfrozen, chemically fixed samples. The severity of cryodistortions increased progressively with increasing distance from the point of first impact. Of particular interest were the dramatic alterations of the mitochondrial ultrastructure. The concept that the reticular and the outer mitochondrial membranes are intimately and strongly associated was clearly demonstrated. Optimally frozen material revealed cryopreserved ultrastructure of high quality. The method described appears to offer an ideal model system for correlating the information gained by phase-contrast microscopy of living cell cultures with the ultrastructure of the same samples fixed in situ by chemical or physical techniques. Cryofixation would be particularly useful for studying dynamic cellular processes associated with physiological and pathophysiological conditions, e.g. metabolic inhibition, anoxia and substrate deprivation.

A stimulus timing device for capturing fast physiologic events by quick-freezing.

A timing device was designed that, in conjunction with an impact type of quick-freezing apparatus and an externally-triggerable stimulus generator, allows the application of an electrical stimulus to a muscle preparation at a selected time interval before quick-freezing and the measurement of the interval with submillisecond precision. This is needed for stopping fast physiological events in calcium release and excitation-contraction coupling and allows studying the morphological parameters (by freeze-fracture and freeze-substitution) and elemental distributions (by x-ray microanalysis) as a function of time after stimulation. The device should be adaptable for use with most equipment designed for quick-freezing electrically excitable tissue by impact on a cold solid surface.

Real-time quantitative elemental analysis and mapping: microchemical imaging in cell physiology.

Recent advances in widely available microcomputers have made the acquisition and processing of digital quantitative X-ray maps of one to several cells readily feasible. Here we describe a system which uses a graphics-based microcomputer to acquire spectrally filtered X-ray elemental image maps that are fitted to standards, to display the image in real time, and to correct the post-acquisition image map with regard to specimen drift. Both high-resolution quantitative energy-dispersive X-ray images of freeze-dried cyrosections and low-dose quantitative bright-field images of frozen-hydrated sections can be acquired to obtain element and water content from the same intracellular regions. The software programs developed, together with the associated hardware, also allow static probe acquisition of data from selected cell regions with spectral processing and quantification performed on-line in real time. In addition, the unified design of the software program provides for off-line processing and analysing by several investigators at microcomputers remote from the microscope. The overall experimental strategy employs computer-aided imaging, combined with static probes, as an essential interactive tool of investigation for biological analysis. This type of microchemical microscopy facilitates studies in cell physiology and pathophysiology which focus on mechanisms of ionic (elemental) compartmentation, i.e. structure-function correlation at cellular and subcellular levels; it allows investigation of intracellular concentration gradients, of the heterogeneity of cell responses to stimuli, of certain fast physiological events in vivo at ultrastructural resolution, and of events occurring with low incidence or involving cell-to-cell interactions.

Cryopreservation evaluated with mitochondrial and Z line ultrastructure in striated muscle.

Single, intact, frog skeletal muscle fibres and whole frog hearts were quick-frozen on a polished, liquid-He-cooled copper block and examined in the electron microscope after freeze-substitution and freeze-fracture. In both kinds of striated muscle, collapse of the peripheral and intracristal membrane spaces in mitochondria was found to increase with increasing distance from the point of first impact (PFI) of the muscle cells on the cold copper block. The changes correlated with a previously described gradient of Z line and A band cryodamage occurring with distance from the PFI. The findings in thin sections from freeze-substituted preparations were confirmed by freeze-fracture preparations. It is concluded that, since the mitochondrial membrane changes are concurrent with, and follow the same spatial distribution of, other manifest cryoartefacts, the cryoartefactual nature of the mitochondrial changes must be excluded before functional significance is attributed to them. The collapse of mitochondrial membrane spaces as a sensitive indicator of quality of cryopreservation may apply to non-muscle cells as well.

To excite a heart: a bird's view.

Ultrastructural investigations of avian cardiac muscle, including ratite hearts, have provided great insights into the mechanisms as to how excitation leads to contraction in the heart. The geometry of the conduction fibers of ratite hearts confirms earlier observations on birds showing that the geometry of the conduction system and its component cells is adapted to hearts of different sizes and rates of contraction so as to maintain a differential in conduction velocities between the conduction system and the working fibers. The study of the ratite conduction fibers bears out the idea of an inverse relationship between the size of the gap junctions and the input resistance of cardiac cells. The anomalous extended junctional SR typical of all avian hearts, proscribes the notion of direct contact transduction into calcium release for contraction of an excitatory signal propagating at the cell surface. Couplings appear well suited to maintain direct, if transitory, connections to the extracellular space in addition to harboring channels for intracellular calcium release.

Morphology of Leishmania braziliensis: changes during reversible heat-induced transformation from promastigote to an ellipsoidal form.

Leishmania braziliensis, growing axenically at 26 C and transferred to 34 C, changes within 3 hr from the long slender motile promastigote form to an ellipsoidal form with a nonmotile flagellum. This transformation is reversible for heat treatments of up to 12 hr. In this study we show by light microscopic measurements that the cells decrease in length and increase in diameter at constant volume. Quantitative morphometry of electron micrographs further demonstrates that: the distance between nucleus and kinetoplast decreases; the kinetoplast enlarges slightly; the distance between adjacent subpellicular microtubules decreases; and that after 3 hr of heat treatment there is no change in mitochondrial morphology, but after 6 hr of heat treatment the mitochondria lose their cristae and no longer possess a clearly defined double membrane. These observations are compared with the morphological changes that occur normally in the gut of a sandfly and in the in vivo transformation occurring during infection of the mammalian host and of macrophage cultures.

Cardiac muscle following quick-freezing: preservation of in vivo ultrastructure and geometry with special emphasis on intercellular clefts in the intact frog heart.

Intact frog, mouse and finch hearts were quick-frozen on a liquid He-cooled copper block. Adjacent frozen samples from the same heart were processed by freeze-substitution (followed by embedding and thin sectioning), freeze-fracture/etch (followed by platinum/carbon replication) and frozen sectioning (followed by freeze-drying), respectively, and examined with the electron microscope for fidelity of reproducing the in vivo state of heart muscle geometry, especially that of the narrow intercellular clefts between frog cardiac muscle cells. It was concluded that quick-freezing followed by the above procedures accomplishes that and that, therefore, narrow intercellular clefts are an invariant feature of the normal anatomy of frog cardiac muscle, which must be considered in physiological experiments. The methodology showed that quick-freezing through the epicardial surface is capable of producing superb cryopreservation for ultrathin cryosections, as well.

The quick-freezing of single intact skeletal muscle fibers at known time intervals following electrical stimulation.

Single intact frog skeletal muscle fibers quick-frozen after known time intervals following electrical stimulation are examined electron microscopically in thin sections, after freeze-substitution, in freeze-fracture/etch preparations, and in cryosections prepared for x-ray microprobe analysis. Techniques are described to perform these operations on a single fiber. Notable morphological differences between conventionally fixed and cryopreserved muscle fibers, and between fibers quick-frozen at different post-stimulation intervals are demonstrated.

Geometry of cell and bundle appositions in cardiac muscle: light microscopy.

A strand each of cardiac conduction and working cells of the left ventricle is studied in serial sections with the light microscope to define the geometry of cell appositions that form networks of cardiac muscle cells. Anatomic and thus electrical coupling is very frequent among all cells; it is accomplished within a few hundred micrometers axially regardless at which point of the strand electrical current is assumed to originate. Most individual cardiac myocytes are not only connected in longitudinal direction but also make lateral contacts. Only a few bundles of varying diameters remain unconnected over appreciable distances of greater than 200 micron (so-called unit bundles). Thus abnormal current vectors are averted, at least in normal cardiac tissue, even if excitation were to originate from a point. Plastic thick sections studied with the light microscope were unsuitable to define cell lengths.

Corbular sarcoplasmic reticulum of rabbit cardiac muscle.

The structure of corbular sarcoplasmic reticulum as part of the sarcoplasmic reticulum (SR) in perfusion-fixed rabbit cardiac muscle was studied by thin sections and freeze fracture. In thin sections, processes on the surface of corbular SR have all the anatomical features of junctional processes of junctional SR. By freeze fracture, the E face of corbular SR was particle poor and showed deep pits; the P face was particle rich. The demonstrated structural homology of corbular SR to all forms of junctional SR justifies its inclusion in that group.

Ultrastructural comparison of ion beam and radiofrequency plasma etching effects on biological tissue sections.

Three dry etching techniques (Ar+ ion beam, O2+ ion beam, O2 radiofrequency electrodeless discharge) were compared with respect to preferential etching and damage to the ultrastructure of glutaraldehyde-fixed Epon-embedded frog skeletal muscle sections. SEM and TEM studies were performed on both unstained and stained (osmium tetroxide, uranyl acetate) sections. Etching effects were observed to differ for the various ion beam or plasma etching techniques. Whereas selective retention of electron dense structures (e.g. Z lines, nuclear heterochromatin) was observed for oxygen plasma etching, preferential etching of these components was observed using O2+ ion beam bombardment. Selectively etched Z lines and etch-resistant nucleoli were observed for both reactive (O2+) and inert (Ar+) ion beam sputtering after sufficiently high ion doses. The above suggest that selective etching under keV ion beam irradiation is related more to physical sputtering processes (momentum transfer) than to the chemical reactivity of the incident ion. Heavy metal post-fixation and staining had no qualitative effect on the nature of the selective etching phenomena. The above findings are significant in that they potentially influence both electron and ion microprobe measurements of etched biological specimens.