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PubMed1985

Changes in the surface fine structure of ependyma of the rat third ventricle following operative leakage of cerebrospinal fluid.

The role of the ventricular ependyma in the elaboration of cerebrospinal fluid under resting conditions is well documented. In an attempt to elucidate its response to cerebrospinal fluid leakage, male young adult Wistar rats were subjected to lumbar spinal transection under intraperitoneal Nembutal anaesthesia. Control animals received a sham operation, involving opening up the cerebrospinal fluid space and its closure. A scanning electron microscopical study of the third ventricular ependyma on the seventh postoperative day revealed pronounced surface modifications in the experimental animal which included (i) bulbous dilatations in the ciliary shafts with frequent apical blebbing, and an overall turgid appearance of most cilia; (ii) a profusion of tall and stout microvilli in the non-ciliated zones; (iii) an increase in the size and number of blebs; and (iv) a greater number of supraependymal cells especially on the ventricular floor. The significance of these changes is discussed in the light of available studies. The results, considered collectively, seem to reflect an augmented activity of the ventricular ependyma as a compensatory measure to counteract the effects of cerebrospinal fluid leakage caused by surgical trauma. Such activity may be both absorptive and secretory in nature.

Animals↗

PubMed1979

An ultrastructural comparison of human endometrial adenocarcinoma with normal postmenopausal endometrium.

Although electron microscopy has been of limited value in detecting malignant neoplasms, some neoplasms do exhibit characteristic ultrastructural features. Intensive study of these features may offer insight into the etiology and activity of such tumors. In an attempt to characterize the ultrastructure of endometrial adenocarcinoma, tissue was examined with the electron microscope and compared with normal postmenopausal endometrium. Endometria from biopsy, hysterectomy, or curettage were processed routinely for light and electron microscopy. Several ultrastructural features of the adenocarcinoma were common both to previous descriptions of endometrium of the postovulatory phase and to the normal postmenopausal endometrium, described here, viz, atypical mitochondrial forms and cell surface modifications. Ribosomes were abundant in adenocarcinoma of the endometrium, normal postmenopausal endometrium, and normal cyclic endometrium in the preovulatory phase.

Adenocarcinoma↗

PubMed1979

Extraskeletal Ewing's sarcoma. Histologic and ultrastructural observations in three cases.

The histologic and ultrastructural morphology of three cases of Ewing's sarcoma of soft tissue are described and the fine structural features of extraskeletal Ewing's sarcoma are compared to those of similar round-cell tumors that are considered in the differential diagnosis. By light microscopy, these tumors are indistinguishable from Ewing's sarcoma of bone. Ultrastructurally, the salient features are also comparable to Ewing's sarcoma of bone and include: 1) absence of surface modifications; 2) cell-contact sites in the form of small thickenings of apposed membranes and large desmosome-like specializations; 3) undifferentiated cytoplasm usually containing abundant glycogen and occasionally nonspecific microfilaments; 4) significant variation in shape and irregularity of nuclear profiles. The ultrastructural features of extraskeletal Ewing's sarcoma are sufficiently distinctive to allow separation from from other small-cell malignant neoplasms in the majority of cases.

Adult↗

PubMed1984

Primary culture of chick embryo skeletal muscle on dextran microcarrier.

We report the conditions to obtain primary suspension cultures using embryonic skeletal muscle from 12-day chick breast muscle. Further, the conditions are described to obtain scanning electron micrographs of whole cells and transmission electron micrographs of sections of plastic-embedded cells on microcarriers. A positively charged hydrated dextran microcarrier, Cytodex I (Pharmacia), provided support for the cells; the myogenic stages of proliferation, myoblast alignment and fusion to form myotubes coincided temporally with replicate cultures grown on gelatin-coated plastic dishes. Microcarrier-grown cells, including non-muscle cells, had microvilli, lamellipodia, bleb, and other surface modifications but no ruffling membranes. Myoblasts and myotubes on beads had fewer microvilli compared to homologous cells grown in the static culture medium of plastic dishes. Myoblasts aligned laterally during fusion, starting at 48 h. Myotube cytodifferentiation proceeded to myofibril formation by day 4 of microcarrier culture. The sarcomeres of aligned myofibrils had normal banding with an hexagonal lattice of thick and thin myofilaments in the A-bands. Caveolae intracellulares and sarcoplasmic reticulum were evident. Scaling-up to larger volumes promises to provide a cost-effective way to obtain a large harvest of cultured skeletal muscle which may prove especially useful for studies of minor constituents.

Animals↗

PubMed1984

Naturally occurring autoantibodies to exoplasmic and cryptic regions of band 3 protein, the major integral membrane protein of human red blood cells.

Immunoglobulin G (IgG) of healthy human blood donors and IgG from pooled sera (Sandoglobulin) contain natural (auto)antibodies to band 3 protein, the major integral membrane protein of human red blood cells. Affinity-purified and 125I-iodinated anti-band 3 antibodies bound specifically to band 3 protein on immunoblots from membrane proteins in the presence of unlabeled, absorbed IgG. Purified (auto)antibodies also bound nonspecifically to band 4.2 and weakly to band 5 and 6, when assayed with second antibody and 125I-iodinated protein A. The antibodies were directed to regions of band 3 protein that were cryptic and in part exoplasmic but with a low accessibility to surface modifications. The antigenic sites were located within the 65K, but not the 38K-dalton chymotryptic fragment of band 3 protein. Antigenic band 3 protein was equally present in membranes of young and senescent red cells. Hence, if these antibodies were involved in recognizing a few exoplasmic sites of band 3 protein on senescent red cells, antigen exposure would require alterations in band 3 accessibility (conformation, topology) rather than an enzymatic generation of antigenic sites.

Adult↗

PubMed1984

Response of ependyma of the rat third ventricle to operative loss of cerebrospinal fluid: a transmission electron microscopical study.

The ependyma of the cerebral ventricles has been implicated in absorption and secretion of cerebrospinal fluid under resting and experimental conditions by various workers. To investigate the response of the ventricular ependyma to experimental leakage of the fluid, spinal transection was carried out in young adult Wistar rats in the lumbar region. The ependyma of the third ventricle was processed for transmission electron microscopy on the 7th post-operative day. The ependymal cells in the sham-operated control animal exhibited regional variations in respect of cilia, microvilli and in their relationship to the subjacent subependymal zone. However, most cells showed apical mitochondria and Golgi cisternae with few profiles of granular and smooth endoplasmic reticulum. Pinocytotic and dense-coated vesicles were not prominent features. In the operated rat, a few ultrastructural changes were noted. A profusion of microvilli and blebs with secondary surface modifications was characteristically seen on the ventricular face. Pinocytotic and dense-coated vesicles appeared to be consistent features. Proliferation of mitochondria, Golgi cisternae, polyribosomes and of smooth and granular endoplasmic reticulum was evident throughout the cytoplasm. The mitochondria, in many cases, presented a pleomorphic appearance with increased electron density. The above modifications in the fine structure, considered collectively, point to an increased ependymal activity. The evidences of both absorptive (presence of microvilli, pinocytotic and dense-coated vesicles) and secretory features (presence of blebs, proliferation in membrane components and in polyribosomes) within the same cell suggest its simultaneous involvement in both absorption and secretion. The observed changes may be interpreted as a compensatory attempt by the ependymal cell toward conservation and replenishment of cerebrospinal fluid.

Animals↗

PubMed1980

Effects of cytochalasin B on the ependyma.

In order to analyse whether or not surface morphology and intracellular skeleton are interdependent, Cytochalasin B (CB), an agent which interferes with microfilaments, was introduced ino the brain ventricular system of adult rats. Following intraventricular application of CB for different periods of time (up to 6h) the animals were sacrificed and the ventricular wall examined with both transmission and scanning electron microscopes. The first signs of cellular alteration after short periods of CB application were an increase in the number of microfilaments within the cytoplasm and the appearance of a multitude of small knobs on the surface of the cilia of the cuboidal ependymal cells, whereas the tanycytes exhibited extensive blebbing at their apical poles. When the duration of CB perfusion was lengthened, entire ciliated cells became rounded up and were loosened from the epithelium, especially in the transition zone of the third ventricle where the ependyma is composed of both tanycytes and ciliated cells. The significance of the CB-induced changes in the ependyma are discussed in relation to the ependymal surface modifications seen under physiological and other experimental conditions.

Animals↗

PubMed1978

The structure of the germinal disc region of the hen's ovarian follicle during the rapid growth phase.

The structure of the ovarian follicle in the region of the germinal disc, which appears as a white plaque at the surface of the oocyte, was examined by electron microscopy and compared with the non-disc region which overlies the yellow yolk mass of the oocyte in the final growth phase. The main differences concerned the granulosa cell layer and the surface layer of the oocyte. In the disc the granulosa cells were less regularly arranged and the spaces between them varied in width. Their mitotic rate was higher than that in the non-disc region, where cell division was seldom observed at maturity. The perivitelline layer was comparatively poorly developed at the periphery of the germinal vesicle in 15 mm follicles, but eventually attained a uniform thickness throughout the follicle. In the intercellular and perivitelline spaces there were smaller amounts of granular material. Marked differences were observed in the ooxyte surface layer. In 15 mm follicles the surface of the germinal disc was thrown into numerous microvilli and some narrow indentations containing macrovilli from the granulosa cells. Coated vesicles, 120 nm diameter, appeared to be invaginating from the oolemma, whereas 70 nm coated vesicles were present in the deeper cytoplasm. In follicles of more than 25 nm diameter these structural conformations were evident only at the periphery of the disc; for the most part the 120 nm coated vesicles were absent, and over the germinal vesicle microvilli were of rare occurrence. On the other hand, the bulk of the oocyte surface was highly convoluted throughout this period of growth, numerous granulosa cell macrovilli extended into deep pouches associated with 300 nm coated vesicles, and the oolemma possessed a coating of fuzzy material. These observations suggest that there is a restricted passage of yolk precursors to the surface of the germinal disc, and that the inability to transport yellow yolk into the disc is related to differences in the oolemmal surface coat and the population of coated vesicles. The surface modifications, as well as the proliferation of the granulosa cells, are likely to be influenced by the presence of the germinal vesicle.

Animals↗

PubMed1980

Effect of 12-O-tetradecanoylphorbol-13-acetate on the morphology and growth of C3H/10T1/2 mouse embryo cells.

The effects of the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the morphology and growth properties of C3H/10T1/2 clone 8 cells were examined. The morphology of these cells was changed within 30 min following treatment with 0.1 microgram of TPA per ml; they became smaller and refractile with long beady processes. Such changes were observed in both logarithmic and confluent cultures and lasted about 72 hr. Subsequent treatments were much less effective in inducing these changes. Scanning electron microscopy showed cell retraction and rounding as the most significant immediate effects of TPA treatment; many cells remained partially rounded 48 hr afterwards. Long-term surface modifications ascribable to TPA treatment were not detected. TPA had only minor effects on the growth of cultured C3H/10T1/2 cells in the presence of 10% fetal calf serum. Slight increases in plating efficiencies and saturation densities were generally observed in the presence of TPA but not with the related non-tumor-promoting compound phorbol. The cells grew slowly in 1% fetal calf serum and demonstrated serum batch-dependent alterations in their growth properties when exposed to TPA. Under conditions that produce doubling times of 70 hr or greater, TPA, but not phorbol, reduced the doubling time to about 50 hr. Saturation densities were also increased by TPA in 1% fetal calf serum. The effects of TPA on the growth of an oncogenically transformed variant of C3H/10T1/2 were quite different. While minimal effects of TPA were observed quite different. While minimal effects of TPA were observed when transformed cells were treated in the presence of 10% total calf serum, TPA treatment in 1% fetal calf serum significantly inhibited cell growth.

Animals↗

PubMed1995

Altered in vivo and in vitro behavior of butanol-modified bone marrow cells.

Cell-surface molecules, particularly glycoconjugates, appear to be involved in the in vivo homing of hematopoietic stem and progenitor cells and in their interactions with hematopoietic stromal cells. To study the role of cell-surface molecules of hematopoietic stem cells, the expression of some surface molecules was altered using n-butanol treatment. We examined the in vivo and in vitro colony-forming abilities, in vivo homing patterns, and cell-surface lectin receptor expression of butanol-treated bone marrow cells (BMC) from BDF1 mice. The butanol-treated/-modified BMC formed an increased number of significantly larger spleen colonies (CFU-S) in lethally irradiated (1050 rad) mice. The butanol-treated BM formed significantly larger in vitro granulocyte-macrophage progenitor cell colonies (CFU-C) and in vitro fibroblastic colonies (CFU-F), although the number of such colonies was not significantly altered. The homing pattern of butanol-treated BMC was studied by comparing the distribution in lethally irradiated mice of intravenously injected 51Cr-labeled butanol-treated BMC with that of untreated cells. The butanol treatment altered the in vivo homing pattern of these cells, with increased homing to liver, spleen, and bone marrow and decreased homing to thymus, lung, and mesenteric lymph nodes. Flow-cytometric analyses of butanol-treated BMC showed an increased expression of receptors for the lectins concanavalin A (conA) and wheat germ agglutinin (WGA), indicating an increased expression of mannosyl and galactosyl residues, which are known sugar moieties in hematopoietic stem/progenitor cell homing. These results indicate that cell surface modifications can influence homing and growth of transplanted BMC and that butanol treatment is a useful tool for studying the mechanisms of hematopoietic stem/progenitor cell homing in vivo and for further characterizing the molecules involved in this process.

Animals↗

PubMed1994

[Assays of cytotoxicity of the Nickel-Titanium shape memory alloy].

The equiatomic Nickel-Titanium (NiTi) alloy has exceptional mechanical properties such as shape memory and superelasticity. It already has applications in orthodontics and is a promising orthopaedic biomaterial. Cytocompatibility studies must therefore be undertaken. The objective of this study is to determine the biological response that NiTi elicits compared to other orthopaedic metals currently used in orthopaedic surgery. Cytotoxicity tests constitute an efficient first step in a biocompatibility study and contribute to reduce animal use in laboratory. Direct contact and agar diffusion cytotoxicity assays were performed following ASTM standards #F813-83 and #F895-84 respectively. Confluent L-929 fibroblasts culture plates were incubated (directly or under an agar bed) in presence of NiTi, titanium (Ti), vitallium (Co-Cr-Mo) and 316L stainless steel discs. Following exposition to specimens, a vital dye was added to the plates. All cultures were evaluated for cytotoxic reactions, under light microscopy. Direct contact and agar diffusion assays indicated that all metals tested induced a mild biological reaction. Specimens were ranked according to an index of biological response, they are enumerated here in decreasing order of cytotoxicity: NiTi approximately Co-Cr-Mo >> pure grade 4 Ti approximately pure grade 1 Ti approximately Ti 6A1 4V approximately 316L stainless steel. Furthermore, plasma surface modification increased the cytocompatibility of NiTi.

Biocompatible Materials↗

PubMed1993

Pathological sequelae of implantation of intracorporeal left ventricular assist devices in the calf.

Thromboembolic complications have previously been a major pathological complication of left ventricular assist device (LVAD) insertion and appear to be a consequence of the use of smooth polyurethane linings. Textured biomaterial surfaces are designed to attract a stable coagulum which becomes organized to form a neointima. We describe the pathological changes following experimental implantation of textured-surface LVADs in calves. The incidence of systemic embolization from such linings appears to be low. Surface modifications with cell seeding offer the possibility of further reduction in incidence of this problem. However, degradation of prosthetic valves may be a potential source of emboli and infection. With these complications minimized, the most significant pathological changes observed appear to relate to the device haemodynamics. At present renovascular changes associated with systemic hypertension appear to be the greatest potential problem for the long-term use of these devices.

Animals↗

PubMed1996

Photodamaging effects of tetraphenylporphycene in a human carcinoma cell line.

The photosensitizing effects of tetraphenylporphycene (TPPo) and light on HeLa cells, with emphasis on cell viability and the microtubular network, have been investigated. The survival of the cells incubated with TPPo was dependent on both drug concentration and light dose. The integrity of microtubules (MTs) was evaluated by immunofluorescence staining of alpha-tubulin. Interphasic and mitotic MTs were altered after 30 min of incubation with 5 microM TPPo followed by light irradiation. The degree of damage depended on light exposure time: 3 or 15 min corresponded to survival rates of approximately 50% or < 5%, respectively. Sublethal treatment led to the gradual accumulation of cells in metaphase, which caused an increase in the mitotic index (MI), with a maximum being found 6 h later. The number of cells in metaphase, as well as the MI, were within control values 24 h after sublethal photodynamic treatment. Lethal treatment provoked irreversible damage of interphasic and mitotic MTs. In addition, cell surface modifications such as bleb projections of the plasma membrane were also observed immediately after lethal treatment. It is concluded that both cellular structures, plasma membrane and MTs, constitute important targets for the phototoxic action of TPPo.

Cell Survival↗

PubMed1996

The potential of electroanalytical techniques in pharmaceutical analysis.

With the considerable progresses observed in analytical instrumentation, it was of interest to survey recent trends in the field of electroanalysis of drugs. Potentiometric, voltammetric and amperometric techniques were scrutinized both in terms of historical evolution and in terms of potentialities with respect to the analysis of drugs in various matrices. With regard to the former, it appeared that numerous original selective electrodes (for drugs and ions) have been studied and several ion-selective electrodes have been successfully commercialized. Improvements are still expected in this field in order to find more robust membrane matrices and to minimize the surface fouling. Electrochemistry is well suited for trace metal analysis. A renewed interest in potentiometric stripping analysis is observed and is stimulated by the power of computers and microprocessors which allow rapid signal recording and data handling. Polarography and its refinements (Pulsed Waveform, Automation,...) is ideally applied for trace metal analysis and speciation. The technique is still useful in the analysis of drug formulations and in biological samples provided that the method is adequately validated (selectivity!). The same holds for solid electrodes which are currently routinely applied as sensitive detectors after chromatographic separation. New instrumentation is soon expected as regard electrochemical detection in capillary electrophoresis. Actually, in order to increase the responses and improve the selectivity, solid electrodes are facing exponential research dedicated to surface modifications. Perm-selectivity, chelations catalysis, etc. may be considered as appropriate strategies. Microelectrodes and screen printed (disposable) sensors are of considerable interest in cell culture e.g. for single cell excretion analysis and in field (decentralized) assays, respectively. Finally several biosensors and electrochemical immunoassays have been successfully development for the selective and sensitive analysis of drugs.

Automation↗

PubMed1996

Immobilization of L-asparaginase into a biocompatible poly(ethylene glycol)-albumin hydrogel: I: Preparation and in vitro characterization.

The feasibility of the immobilization of Escherichia coli L-asparaginase into a hydrogel matrix made of poly-(ethylene glycol) (PEG) and BSA was demonstrated. After immobilization a 200-fold increase in the Km value was observed. The use of an L-aspartic acid analogue, carbobenzoxy-L-aspartic acid and surface modification by methoxy-PEG of molecular mass 5 kDa cause a only a slight gain in affinity of the enzyme for its natural substrate. The immobilized L-asparaginase has an optimal activity over a larger range of pH than the native enzyme, owing to the effect of the matrix. At a physiological pH of 7.3, the immobilized enzyme retained 90% of its activity compared with only 43% for the native form. The immobilized enzyme retained a high proportion of its initial activity, more than 90% after 50 days of incubation at 37 degrees C, even in the presence of its substrate. This may be compared with a half-life of 2 days observed for native enzyme incubated under the same conditions. These results suggest that the BSA-PEG matrix can be very useful for enzyme immobilization and, taking into account the good biocompatibility of the matrix, one can expect that this matrix will provide a functional bioreactor for use in vivo.

Animals↗

PubMed1996

Targeting to intestinal M cells.

The specialised, antigen-transporting, epithelial M cells in the follicle-associated epithelium (FAE) overlying gut-associated lymphoid tissues constitute the primary target for oral delivery of vaccines. Our studies have shown that polystyrene microspheres selectively bind to, and are efficiently transcytosed by, rabbit Peyer's patch M cells in closed intestinal loops. Binding of biodegradable poly(DL-lactide-co-glycolide) microspheres to rabbit Peyer's patch FAE is an order of magnitude lower than that of polystyrene microspheres. Although poly(DL-lactide-co-glycolide) microspheres are not selectively targeted to M cells, a high proportion of those which bind to M cells are transcytosed, supporting the potential of such microspheres as vehicles for oral vaccine delivery. Comparison of the binding of polystyrene microspheres by murine FAE revealed this to be markedly less extensive than by rabbit FAE. These data demonstrate that microsphere binding by M cells depends on the surface properties of both cells and microspheres and suggest that surface modification may enhance the efficacy of microsphere delivery vehicles. One such approach is the incorporation of molecules with inherent binding specificity for M cells. Lectin-binding studies have revealed that M cells exhibit pronounced regional and species variation in glycoconjugate expression. In murine intestine, certain lectins bind selectively to M cells either in Peyer's patches or caecum, or at both sites. Selective targeting to, and transcytosis of, lectin-conjugates by M cells in ligated segments of murine intestine have also been demonstrated. While several lectins display strong selectivity for rabbit caecal M cells, none to date have been identified with specificity for rabbit or rat Peyer's patch M cells. Knowledge of human M cells is limited and no lectin has yet been identified with specificity for these cells. However, at least one lectin exhibits binding specificity for FAE in the human ileum. In the future, knowledge of the regional patterns of M cell carbohydrate expression within a species may allow lectins to be utilised to target selectively antigenic material to the mucosal immune system at specific locations.

Administration, Oral↗

PubMed1997

[2nd generation bio-integrated keratoprosthesis. Implantation in animals].

PURPOSE: Our goal was to evaluate a second generation biointegrable keratoprosthetic device (BioKpro II). METHODS: The device consists of a porous transparent PTFE (12 mm diameter) and a soft copolymer structure for the optical system (7 mm diameter). Intraocular pression was evaluated with a Goldmann tonometer on an artificial chamber. A lamellar dissection was carried out 3 mm radially for 360 degrees creating a pocket to accommodate the skirt. The soft optic (42.5 D; 500 microns thick) was positioned through a hole (6 mm in diameter) trephined in the central cornea. A 360 degrees peritomy was performed and conjunctiva was placed in front of the prosthesis, sutured and was opened 15 days post implantation. 14 rabbits were implanted and were allowed to heal for three months. RESULTS: We are able to measure the intraocular pressure by Goldmann tonometer on an artificial chamber. We did not observed epithelialisation on the hydrophilic optical surface (14/14). Aseptic necrosis in direct contact between cornea and the soft optical system was observed when conjunctiva was not effective (4/14). As observed with the first generation devices, conjunctiva or buccal mucosa is needed to avoid local necrosis. In other cases no adverse reactions were observed after implantation. DISCUSSION: The geometry and biomechanical properties of the BioKpro II is closed to that of the human cornea. The junction between the optical and the flange was bound by polymeric interpenetration and prosthesis dislocation was not seen under pressure over 1000 mmHg. CONCLUSION: Second generation biointegrable keratoprostheses (BioKpro II) can be implanted successfully. Further tests will determine if surface modifications designed to promote epithelial cell attachment on the optical system could prevent ulceration at the junction between the fluorocarbon and the core.

Animals↗

PubMed1998

Contact lens associated bacterial keratitis: past, present, and future.

PURPOSE: This paper reviews the medical history of the pathogenesis of contact lens related bacterial keratitis and its relationship to the development of successive generations of soft contact lenses. METHODS: The Methods section presents a literature review and the author's personal experience. RESULTS: Bacterial corneal infections have been a significant complication limiting the overall success of soft contact lenses for visual correction. Experience with different types of lenses has pointed to specific causes for the frequency of infection. However, there has been a general understanding that the health of the patient's epithelium, the patient's lens care practices, and the hypoxic effects of overnight wear each significantly contribute to the development of corneal infection. CONCLUSIONS: A complete understanding of the pathogenesis of contact lens associated bacterial infection would allow the development of strategies to prevent infectious complications of lens wear. Progress appears to have been made with a trend toward fewer and less severe infections. As the possibility of a reasonably priced daily wear disposable lens with better oxygen transmissibility and surface modifications to limit deposits and bacterial adherence evolves, safer lenses will continue to be developed.

Contact Lenses, Hydrophilic↗

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