Pasquale A. Cancilla

Cerebral microvessels and derived cells in tissue culture: isolation and preliminary characterization.

SummaryMicrovessels isolated from mouse forebrain were used as the source material for the derivation of cerebral vascular endothelium and smooth-muscle cells in culture. The microvessels were isolated by a mechanical dispersion and filtration technique, and were maintained in vitro as organoid cultures. A microvessel classification system was developed and proved to be useful as a tool in monitoring culture progress and in predicting the type(s) of microvessel(s) that would give rise to migrating and/or proliferating cells. The isolated cerebral microvessels were heterogeneous in diameter, size of individual vascular isolate, and proliferative potential. The isolated microvessels ranged in diameter from 4 μm to 25 μm and in size from a single microvascular segment to a large multibranched plexus with mural cells. The initial viability, determined by erythrosin B exclusion, was approximately 50% on a per cell basis. All microvessel classes had proliferative potential although the rate and extent of proliferation were both microvessel class- and density-dependent. The smaller microvessels gave rise to endothelial cells, whereas the large microvessels gave rise to endothelial and smooth-muscle cells. The viability and progress of a microvessel toward derived cell proliferation seemed to be directly proportional to the number of mural cells present.

Cerebral microvessels and derived cells in tissue culture: II. Establishment, identification, and preliminary characterization of an endothelial cell line.

SummaryAn endothelial cell line has been established from a primary culture of cerebral microvessels isolated from Swiss-Webster mice. The microvessels were isolated by a mechanical dispersion and filtration technique. The cells that emerged from these microvessels, maintained in organoid cultures, proliferated and formed plaques of a single or mixed cell type. The endothelial cell line, designated ME-2, was isolated from one such morphologically homogeneous cell plaque, using both cloning ring techniques and C6 glioma-conditioned medium.An endothelial specific antiserum was made in rabbits and was used immunocytochemically to confirm the cell type of origin of the ME-2 cell line. Not only did the cell type specific antiserum react exclusively with endothelial cells in vivo, but in the brain the antiserum localized preferentially to the luminal membrane of the endothelium.The ME-2 endothelial cells have retained several of their unique properties such as cytomorphology, growth characteristics, and cell type specific surface antigens throughout the life of the line (in one case 40 passages before senescence).

Neutral amino acid transport properties of cerebral endothelial cells in vitro.

An A and L system of neutral amino acid transport has been demonstrated previously in cerebral microvcssels in vivo and in isolated microvessels in vitro. This report describes the neutral amino acid transport properties of cultured cerebral endothelial cells and investigates the influence of astroglia on the transport process. These studies confirm the presence of a sodium-dependent A-system and a sodium-independent L-system of neutral amino acid transport in cultured cerebral endothelial cells. The A-system transport is slower than L-system transport and each is variably inhibited by other amino acids. Transport is enhanced in log phase cells as compared to stationary phase contact-inhibited cells. Contact with glial cells or exposure to glial-conditioned media enhances neutral amino acid transport. These in vitro studies indicate that the A- and L-systems of neutral amino acid transport arc in a dynamic state and are influenced by the phase of cell growth and contact with astroglia.

Anterior horn cell degeneration and Bunina-type inclusions associated with dementia.

SummaryThe ultrastructural features of Bunina type inclusions in the anterior horn cells of a patient dying of amyotrophic lateral sclerosis with dementia appear unique. The Bunina-type inclusions are electron dense aggregates containing, and surrounded by organelle-like membranes. These inclusions appear to be a special type of autophagic vacuole, possibly arising from altered mitochondria.

A histochemical and fine structure study of the developing rat choroid plexus

Summary1.A histochemical and fine structural study of the developing rat choroid plexus was made from 1–3 days before birth to the adult stage.2.The maturation of the choroidal epithelium is associated with a loss of cytoplasmic glycogen, increasing surface area of the plasma membrane associated with the development of an apparent adenosine triphosphatase activity, and increasing numbers of mitochondria and prominence of the rough endoplasmic reticulum associated with increasing diaphorase and dehydrogenase activity. Following birth, thiamine pyrophosphatase and adenosine mono- and diphosphatase activity appear in the blood vessels. Structures associated with pinocytotic activity are most numerous in young rats.3.It is suggested that the increased susceptibility of young rats to experimentally produced hydrocephalus is related to the potential of their choroid plexus for active transport.Zusammenfassung1.Die Entwicklung des Plexus choroideus in der Ratte vom 1.–3. Tag vor der Geburt bis zur Reife wurde mit histochemischen Methoden und mit dem Elektronen-Mikroskop untersucht.2.Die Reifung des Plexus-Epithels ist verbunden mit dem Schwund des Glykogens im Cytoplasma, mit eine Zunahme der Oberfläche der Zellmembrane zusammen mit dem Erscheinen von Adenosine-Triphosphatase-Aktivität sowie mit einer Zunahme von Mitochondrien und von endoplasmischem Reticulum zusammen mit einer Zunahme der Diaphorase- und Dehydrogenase-Aktivität. Nach der Geburt kann die Aktivität von Thiamine-Pyrophosphatase und von Adenosine-Mono- und Di-Phosphatase in den Blutgefäßen nachgewiesen werden. Zellstrukturen, die pinocytotische Aktivität besitzen, sind in größter Anzahl in jungen Ratten vorhanden.3.Es wird angenommen, daß die erhöhte Empfänglichkeit von jungen Ratten für experimentell erzeugten Hydrocephalus auf die besondere Fähigkeit von deren Plexus choroideus zu aktivem Transport zurückzuführen ist.

PROTEIN SYNTHESIS IN MUSCLE CULTURES FROM PATIENTS WITH DUCHENNE MUSCULAR DYSTROPHY

Muscle samples for cultures were obtained from the quadriceps by open biopsy under local anesthesia in five patients with early stage of Duchenne muscular dystrophy (DMD) and 10 controls. Primary cultures were grown in Eagles Minimum Essential Medium (MEM) with 20 per cent fetal calf serum. After 4 weeks, cells were trypsinized, counted, sub‐cultured for 5 days in MEM with 5 per cent horse serum and finally incubated for 4 h with (3H) leucine. Total protein synthesis showed a significant decrease (half of control values) only in muscle cultures from patients with DMD. Addition of calcium chloride alone or with A23187 ionophore normalized this defect in protein synthesis. By contrast, myosin heavy chain synthesis was measured and found normal in all patients.

Morphologic effects of antibody to mouse brain endothelium in vivo.

The purpose of this study was to assess the morphologic effects of antiendothelial antibodies (EAB) on mouse brain endothelium in vivo. The antigen utilized was the plasma membranes fraction of cultured mouse brain endothelial cells, which was injected into rabbits with complete Freunds adjuvant. The resultant antibody-containing serum was injected back into mice via tail veins in varying time courses and dosages, followed by perfusion of the animals. The antibody was primarily IgG, and was visualized on the brain endothelium by electron microscopy, using the peroxidase antiperoxidase (PAP) labeling technique. Normal rabbit sera and saline were used as controls. Results showed a significantly greater number of micropinocytotic vesicles in the endothelium of the test animals compared to controls. The number of multivesicular bodies and the thickness of the endothelium were also greater in the test animals. At no time was antibody visualized internal to the endothelial luminal membrane, and no lesions such as inflammation or necrosis were observed. This study shows that serum-containing antiendothelial antibodies has a direct, but apparently limited, effect on endothelium.

Altered protein synthesis and creatine kinase in breast muscle cell cultures from dystrophic chick embryos

The total protein synthesis (TPS), myosin synthesis (MS) and creatine kinase (CK) levels in muscle cell cultures obtained from 400 normal (strain 454) and 400 dystrophic chick embryos (strain 455) were investigated. The cultures were obtained from breast muscles of 12 day chick embryos by dissociation in 0.25% trypsin, preplating and plating of 5 x 10(5) floating cells on gelatin coated dishes in Minimal Essential Medium, 10% horse serum and 2% chick embryo extract. After 6 days, when electron-microscopic studies demonstrated good muscle differentiation, cell cultures were labeled with [3H]leucine. TPS and MS, respectively, showed 85% and 65% increases in breast muscle cell cultures from dystrophic chick embryos. The half-life times for total protein and myosin from dystrophics were 19 and 32 hr, respectively as compared with 36 and 48 hr from controls. Noncollagen protein content (NCP) showed 27% decrease in postfusion stage (12 days) of cell cultures from dystrophics. The CK level showed 30% lower values in the cells from dystrophics but 50% higher values in their culture medium. The addition of leupeptin plus pepstatin (50 microgram/ml) to these cultures resotred NCP content, total protein and myosin turnover to normal values and significantly increased TPS and MS. The addition of diphenylhydantoin (DPH) (20 microgram/ml) to cell cultures from dystrophics did not change the NCP content nor the turnover for total protein and myosin but significantly increased TPS, MS and CK while medium CK significantly decreased. The addition of leupeptin plus pepstatin or DPH to muscle cell cultures from normal chick embryos also significantly stimulated TPS and MS.

Partial purification and characterization of a folatebinding protein from human choroid plexus

A folate-binding protein (binder) from human choroid plexus was solubilized with Triton X-100 and partially purified in three steps: (1) affinity chromatography, (2) Sephadex G-200 column chromatography, and (3) polyacrylamide gel electrophoresis. When the partially purified binder was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the binding activity was located in the region of the gel with a molecular weight between 45,000 and 60,000. The specific activity of the binder after the three purification steps was 1.2 μg folic acid/mg protein, a 316-fold purification. Binding activity of the partially purified binder decreased below pH 6.0 and above pH 8.0 was unaffected by treatment with ribonuclease or deoxyribonuclease, but was abolished with trypsin, chymotrypsin, or protease (Streptomyces griesus). The binding of folic acid to the human binder was inhibited by folate > H4-folate > methyl-H4-folate ≃dihydrofolate ≃ pteroic acid ≫ methotrexate ≃ aminopterin.

Platelet factor and cerebral vascular endothelium: platelet-induced mitogenesis.

Abstract Intact bovine platelets or their products were added to pre-confluent or contact-inhibited monolayers of endothelial cells isolated from mouse cerebral microvessels. Mitogenic responses of these cerebral microvascular endothelial cells were determined by tritiated thymidine incorporation. In pre-confluent cultures the incorporation produced by whole platelets and platelet-rich plasma serum approached that of whole blood serum controls. Serial platelet dilutions induced a graded response, with a discrete threshold. Whole blood plasma serum, platelet-rich plasma serum, and thrombin-activated platelet supernatant augmented the low incorporation produced by platelet-poor plasma serum. Contact-inhibited, complete monolayers did not respond. These results were reproducible, even after sera were frozen and retested at varied intervals.