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S C McLoon

Publications and source records attributed to S C McLoon.

16 recordsLinked to original sources
PubMed1992

Subtractive immunization techniques for the production of monoclonal antibodies to rare antigens.

Traditional techniques for the production of monoclonal antibodies usually result in generation of monoclonal antibodies to immunodominant molecules. To enhance the production of monoclonal antibodies to rare or less immunodominant antigens, subtractive immunization techniques have been employed. This study compared the ability of several subtractive immunization techniques to suppress the immune system to a given antigen. Neonatal tolerization, chemical immunosuppression with cyclophosphamide, a combination of the two and various permutations of these techniques were compared. The results from this study indicated that chemical immunosuppression with cyclophosphamide was the most effective subtractive immunization technique and that the cyclophosphamide regime employed was a critical determinant in the success of chemical immunosuppression.

Animals↗
PubMed1991

Morphological patterns in the developing vertebrate retina.

Changes in the morphology of the early optic cup were observed in embryos of two distantly-related vertebrate species, a teleost fish, northern pike (Esox lucius), and chicken (Gallus gallus). A similar morphological pattern was noted to appear in both species shortly after the involution of the optic vesicle and the formation of the inner retinal layer. At a gross level, three notches were observed in the retinal margin at approximately nasal, dorsal, and temporal positions, while in histological sections a sharp constriction was found in the thickness of the dorsal retinal layer. In both species, this dorsal constriction appeared to be continuous with the central or dorsal notch. The time of appearance and configuration of this morphological pattern is intriguingly similar to the specification and polarity of retinal positional markers, and suggest a segmentation hypothesis for the origin of retinal polarity.

Animals↗
PubMed1991

Elimination of the transient ipsilateral retinotectal projection is not solely achieved by cell death in the developing chick.

During development of the projection from the retina to the brain in the chick, a transient ipsilateral retinotectal projection forms and disappears. This disappearance is coincident with a wave of ganglion cell death in the retina. The contribution of cell death to the disappearance of this projection, as opposed to another mechanism such as axon retraction, was examined. Retinal ganglion cells with a projection to the ipsilateral tectum were retrogradely labeled by injection of long-lasting fluorescent dyes into the tectum prior to the onset of ganglion cell death. Large injections of fast blue labeled approximately 1800 ganglion cells in the ipsilateral retina before the period of cell death began. If the injected embryos were allowed to survive past the peak period of ganglion cell death, the average number of labeled ganglion cells in the ipsilateral retina was reduced by somewhat more than half. It is possible that the remaining labeled ganglion cells projected only to nontectal visual nuclei and were labeled by fast blue that had diffused out of the tectum. This was tested by repeating the study using very localized injections of 1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate or fluorescent microspheres into the tectum. These small injections confirmed that cells with transient projections to the ipsilateral tectum survived past the elimination of this projection. Thus, ipsilaterally projecting ganglion cells have, at most, a slightly greater propensity for death than the average ganglion cell, and elimination of the transient ipsilateral retinotectal projection in chick can be explained only, in part, by the mechanism of cell death.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

A monoclonal antibody that distinguishes between temporal and nasal retinal axons.

A monoclonal antibody was developed that recognizes an antigen with an asymmetric distribution in the chick retina. Immunohistochemistry showed that this antigen, temporal retinal axon protein (TRAP), was present on most if not all axons that arose from the temporal side of the retina. Very few of the axons from the nasal side of the retina were positive for TRAP. The nasal-temporal difference appeared to be in the number of axons that stained with this antibody rather than in the intensity of staining. The transition between nasal and temporal retina based on TRAP distribution appeared to be a vertical line centered on the optic fissure. A competition-based ELISA was developed to quantify the average amount of TRAP on axons in different regions of the retina. This assay also suggested that the pattern of TRAP distribution across the retina was a step function, though the results did not completely rule out the possibility of a continuous concentration gradient oriented circumferentially around the retina. Explants of embryonic nasal and temporal retina had a similar dichotomy in TRAP expression during the first 1 or 2 d in culture. The antibody to TRAP bound to retinal neurites in culture without the cell membrane being made permeable, which suggests that TRAP is a cell-surface molecule. In culture, TRAP was also expressed on the growth cones. Immunoblots showed that TRAP is trypsin sensitive and has an approximate molecular mass of 135 kDa. This is the first molecule identified with an asymmetric distribution in the nasal-temporal axis of the retina.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Early differentiation of retinal ganglion cells: an axonal protein expressed by premigratory and migrating retinal ganglion cells.

A monoclonal antibody, RA4, was developed that recognizes retinal ganglion cell axons in the mature retina. Between embryonic days 3 and 9, the RA4 antigen was associated with cell bodies in certain regions of the retina in addition to the ganglion cell axons. The RA4-positive cells were of 3 types: an apolar cell adjacent to the ventricular surface, a bipolar cell that spanned the thickness of the retina, and a monopolar cell in the ganglion cell layer. Evidence suggests that these cells are premigratory and migrating retinal ganglion cells. The expression of the RA4 antigen is the earliest indicator of ganglion cell differentiation yet reported. The existence of RA4-positive apolar cells along the outer surface of the retina suggests that the ganglion cell phenotype is expressed as soon as the cell becomes postmitotic. Approximately 20% of the migrating ganglion cells were in pairs. The paired cells most likely arose from the terminal division of a germinal cell. One possibility suggested by these data is that a ganglion cell-specific germinal cell arises from a pluripotent germinal cell. Immunoblots and other analyses revealed the RA4 antigen to be a 140 kDa cytoplasmic protein in the retina. RA4 also recognized many long tract axons in the brain. In the brain, the RA4 epitope was observed on proteins with at least 7 different molecular weights. Evidence suggests that different cell types may express the RA4 antigen with slightly different molecular weights.

Animals↗
PubMed1988

Schwann cell-conditioned medium promotes neurite outgrowth from explants of fetal rat retina and tectum in vitro.

We have examined the ability of Schwann cell-conditioned medium (SCCM) to promote neurite growth from embryonic retina and tectum in explant culture. Both retinal and tectal explants adhered to polylysine substrates, but neurite outgrowth from the explants was minimal when grown in the presence of non-conditioned serum-free defined medium. The addition of SCCM resulted in a significant extension of neurites from both types of explants. The neurite outgrowth from the retinal explants was mainly radial and appeared to be in fascicles of small diameter. Neurite outgrowth from the tectal explants stimulated by the SCCM tended to be in larger fascicles and in a more convoluted pattern than that seen from the retinal explants. After serial passaging of the SCCM to remove any substrate-adsorbable factors all neurite-promoting activity was lost. Neurite growth from 100% of the explants was seen on the tissue culture plates that had been preincubated with the SCCM. SCCM was analyzed by immunoblot to determine whether it contained laminin, a potent promoter of neurite outgrowth. Samples of media were electrophoresed on a sodium dodecyl sulphate (SDS)-polyacrylamide gel alongside purified laminin, transferred to nitrocellulose and stained with an antibody to laminin. Purified laminin ran in two bands at 400 kDa and 200 kDa. The conditioned media had a laminin positive band at 200 kDa. Antibody to laminin, when added to the conditioned media, resulted in a loss of the neurite promoting activity of the SCCM. These results suggest that the conditioned media contained laminin or a laminin-like molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Transient expression of laminin in the optic nerve of the developing rat.

The optic nerve of the developing rat was examined for the presence of laminin, an adhesive glycoprotein, to assess whether it might serve as a substrate for retinal axon growth in vivo. The optic stalk and nerve of developing rats were screened immunohistochemically for the presence of laminin before, during, and after the period of retinal axon growth. On embryonic day 14 (E14), laminin immunoreactivity was present in the ventral portion of the optic stalk, the same region in which the first retinal axons grow. Between E16 and postnatal day 10 (P10), cells positive for laminin were distributed throughout the cross-sectional area of the nerve. There was a progressive appearance of glial fibrillary acidic protein (GFAP) immunoreactivity, a marker for astrocytes, from the optic nerve head towards the chiasm beginning on E20. At the advancing front of GFAP immunoreactivity, cells were positive for both laminin and GFAP. Behind the front, laminin immunoreactivity disappeared from the cells. By P12, the only laminin immunoreactivity that remained within the optic nerve surrounded the vasculature. This is a time after the last retinal axons grow through the optic nerve. Monolayer cell cultures were prepared from perinatal rat optic nerves and processed for immunohistochemistry to determine which astrocyte type was laminin-positive. Type 1 astrocytes, which primarily compose the immature nerve, are GFAP-positive, A2B5-negative, and laminin-positive. Type 2 astrocytes, a major component of the mature optic nerve, were GFAP-positive, A2B5-positive and laminin-negative. An extract of developing optic nerve was analyzed by immunoblot along with laminin purified from Engelbreth-Holm-Swarm (EHS) sarcoma. Purified laminin ran with SDS-PAGE under reducing conditions as 2 bands with Mrs of 200,000 and 4000,000. Both bands reacted with antibodies to laminin. A low-salt extraction of whole optic nerve from E18 rats resulted in 2 bands with the same Mr as seen with laminin from EHS sarcoma. When only the inside of the optic nerve (which lacked the basal lamina and meninges that surround the outside) was processed, there was a dark 200,000 D band, but the 400,000 D band was virtually absent. These results are consistent with the hypothesis that laminin, or a variant form of laminin, serves as a substrate for retinal axon growth in the developing rat optic nerve.

Animals↗
PubMed1986

Distribution of laminin in the developing peripheral nervous system of the chick.

During axonal elongation in the developing peripheral nervous system, the temporal and spatial distribution of adhesive molecules in extracellular matrices and on neighboring cell surfaces may provide "choices" of pathways for growth cone migration. The extracellular matrix glycoprotein laminin appears in early embryos and mediates neuronal adhesion and neurite extension in vitro. In this study, we have examined the distribution of laminin at early periods of peripheral nervous system development. The distribution of laminin, demonstrated by immunostaining frozen sections of chick embryos, was compared to the distribution of fibronectin and of early peripheral neurites as revealed with an antibody to a neurofilament-associated protein. Laminin is present in the neural tube basement membrane, in early ganglia, and in developing dorsal and ventral roots, where the laminin staining pattern parallels that of neurofilaments. In early ganglia and nerve roots, laminin immunostaining defines loose "meshworks" rather than basement membranes, which seem to form slightly later in these structures. In contrast, fibronectin is absent in neural tube basement membrane, ganglia, and nerve roots, although it is present along neural crest migratory pathways and in intersomitic spaces. Our observations of laminin distribution are consistent with the possibility that laminin provides an adhesive surface for neurite extension at some stages of early peripheral nervous system development.

Age Factors↗
PubMed1986

Response of astrocytes in the visual system to Wallerian degeneration: an immunohistochemical analysis of laminin and glial fibrillary acidic protein (GFAP).

Eye removal in adult rats resulted in an increase in immunohistochemical staining with antibodies to glial fibrillary acidic protein in the optic nerve, optic tract, and primary visual nuclei. Astrocytes along the primary visual pathway did not stain with antibodies to laminin in response to eye removal. There was an increase in laminin immunoreactivity associated with blood vessels along the injured pathway. These blood vessels were also shown to have an increased size. The lack of laminin induction in astrocytes along the path of degenerating optic axons may be responsible in part for the inability of axons to regenerate in the primary visual system.

Animals↗
PubMed1986

The appearance of an L1-like molecule in the chick primary visual pathway.

A monoclonal antibody, 8D9, has been obtained that binds to axons in the chick nervous system. Biochemical and immunological experiments indicate that the 8D9 antigen is related to the mouse L1 cell-adhesion molecule. The results of immunohistochemical experiments using monoclonal antibody 8D9 to study the development of the chick visual system are consistent with the 8D9 antigen functioning in axon fasciculation.

Animals↗
PubMed1985

Ganglion cells in retinae transplanted to newborn rats.

Cells projecting out of retinal transplants placed over the tectum of newborn rats were studied by labelling with horseradish peroxidase 1 month or more after transplantation. Using this technique, it was found that only cells with the dendritic characteristics of ganglion cells were labelled and, furthermore, that the major classes of ganglion cells seen in normal retinae were also present in the transplants. The cell body size histograms of ganglion cells in normal and transplanted retinae compared closely with each other. Dendritic trees were closely confined by the limits of the inner plexiform layer, and if that layer was folded or distorted, they were themselves frequently abnormal. While axons usually coursed over the surface of the retinal transplants, they quite often followed an anomalous course crossing the individual layers. It appears, therefore, that this transplantation procedure has relatively little impact on the ability of ganglion cells to develop many of their characteristic morphological features. Whether the different functional responses of the various ganglion cell classes are also preserved after transplantation is a matter for further investigation.

Animals↗
PubMed1985

Evidence for shifting connections during development of the chick retinotectal projection.

The pattern in which optic axons invade the tectum and begin synaptogenesis was studied in the chick. The anterogradely transported marker, horseradish peroxidase, was injected into one eye of embryos between 5 and 16 days of development (E5 to E16). This labeled the optic axons in the brain. The first retinal axons arrived in the most superficial lamina of the tectum on E6. They entered the tectum at the rostroventral margin. During the next 6 days of development the axons grew over the tectal surface. First they filled the rostral tectum, the oldest portion of the tectum, and then they spread to the caudal pole. Shortly after the first axons entered the tectum on E6, labeled retinal axons were found penetrating from the surface into deeper tectal layers. In any given area of the tectum, optic axons were seen penetrating deeper layers shortly after arriving in that area. Electron microscopic examination showed that at least some of the labeled axons in rostral tectum formed synapses with tectal cells by E7. These results show two things which contrast with results from previous studies. First, there is no delay between the time the retinal axons enter the tectum and the time they penetrate into synaptic layers of the tectum. Second, the first retinotectal connections are formed in rostral tectum and not central tectum. Retrograde tracing showed the first optic axons that arrived in the tectum were from ganglion cells in central retina. Previous studies have shown that the ganglion cells of central retina project to the central tectum in the mature chick. This opens the possibility that the optic axons from central retina, which connect to rostral tectum in the young embryo, shift their connections to central tectum during subsequent development. As they enter the tectum the growth cones of retinal axons appear to be associated with the external limiting membrane. During the time that connections would begin to shift in the tectum a second population of axons appears at the bottom of stratum opticum, some with characteristics of growth cones. This late-appearing population may represent axons shifting their connections. These results have implications for theories on how the retinotopic pattern of retinotectal connections develops.

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