Romina Comín

High affinity of anti-GM1 antibodies is associated with disease onset in experimental neuropathy.

High antibody affinity has been proposed as a disease determinant factor in neuropathies associated with anti‐GM1 antibodies. An experimental model of Guillain‐Barré syndrome, induced by immunization of rabbits with bovine brain gangliosides or GM1, was described recently (Yuki et al. [ 2001 ] Ann. Neurol. 49:712–720). We searched plasma from these rabbits, taken at disease onset and 1 or 2 weeks prior to onset, for the presence of high‐affinity anti‐GM1 IgG antibodies. Affinity was estimated by soluble antigen binding inhibition. High‐affinity antibodies (binding inhibition by 10–9 M GM1) were detected at disease onset but not before. No such difference was found for other antibody parameters such as titer, fine specificity, and population distribution. These findings support the proposed role of high affinity as an important factor in disease induction by anti‐GM1 antibodies.

Differential endocytic trafficking of neuropathy-associated antibodies to GM1 ganglioside and cholera toxin in epithelial and neural cells

Gangliosides are glycolipids mainly present at the plasma membrane (PM). Antibodies to gangliosides have been associated with a wide range of neuropathy syndromes. Particularly, antibodies to GM1 ganglioside are present in patients with Guillain-Barré syndrome (GBS). We investigated the binding and intracellular fate of antibody to GM1 obtained from rabbits with experimental GBS in comparison with the transport of cholera toxin (CTx), which binds with high affinity to GM1. We demonstrated that antibody to GM1 is rapidly and specifically endocytosed in CHO-K1 cells. After internalization, the antibody transited sorting endosomes to accumulate at the recycling endosome. Endocytosed antibody to GM1 is recycled back to the PM and released into the culture medium. In CHO-K1 cells, antibody to GM1 colocalized with co-endocytosed CTx at early and recycling endosomes, but not in Golgi complex and endoplasmic reticulum, where CTx was also located. Antibody to GM1, in contraposition to CTx, showed a reduced internalization to recycling endosomes in COS-7 cells and neural cell lines SH-SY5Y and Neuro2A. Results from photobleaching studies revealed differences in the lateral mobility of antibody to GM1 in the PM of analyzed cell lines, suggesting a relationship between the efficiency of endocytosis and lateral mobility of GM1 at the PM. Taken together, results indicate that two different ligands of GM1 ganglioside (antibody and CTx) are differentially endocytosed and trafficked, providing the basis to gain further insight into the mechanisms that operate in the intracellular trafficking of glycosphingolipid-binding toxins and pathological effects of neuropathy-associated antibodies.

Preparation of a three-dimensional extracellular matrix by decellularization of rabbit livers

INTRODUCTION the availability of transplantable livers is not sufficient to fulfill the current demand for grafts, with the search for therapeutic alternatives having generated different lines of research, one of which is the use of decellularized three-dimensional biological matrices and subsequent cell seeding to obtain a functional organ. OBJECTIVE to produce a decellularization protocol from rabbit liver to generate a three-dimensional matrix. METHODS a combination of physical, chemical (Triton X-100 and SDS) and enzymatic agents to decellularize rabbit livers was used. After 68 h of retrograde perfusion, a decellularized translucent matrix was generated. To evaluate if the decellularization protocol was successful, with the extracellular matrix being preserved, we carried out histological (light microscopy and scanning electron microscopy) and biochemical (DNA quantification) studies. RESULTS the decellularization process was verified by macroscopic observation of the organ using macroscopic staining, which revealed a correct conservation of bile and vascular trees. A microscopic observation corroborated these macroscopic results, with the hematoxylin-eosin staining showing no cells or nuclear material and the presence of a portal triad. Wilde´s staining demonstrated the conservationof reticulin fibers in the decellularized matrix. In addition, scanning electron microscopy revealed a preserved Glisson´s capsule and a decellularized matrix, with the DNA quantification being less than 10 % in the decellularized liver compared to control. Finally, the time taken to develop the decellularization protocol was less than 96 hours. CONCLUSIONS the proposed decellularization protocol was correct, and was verified by an absence of cells. The hepatic matrix had preserved vascular and bile ducts with a suitable three-dimensional architecture permitting further cell seeding.

Validation of a rabbit model of neuropathy induced by immunization with gangliosides

The induction of neurological signs by immunization of rabbits with gangliosides has been a controversial topic for many years. Recently, Yuki et al. [N. Yuki, M. Yamada, M. Koga, M. Odaka, K. Susuki, Y. Tagawa, et al. Animal model of axonal Guillain-Barré syndrome induced by sensitization with GM1 ganglioside. Ann Neurol 2001;49:712-720.] described an immunization protocol, including keyhole lympet hemocyanin in addition to ganglioside that induced a neurological disease resembling human Guillain-Barré syndrome. We employed this protocol in our laboratory and succeeded in reproducing the disease. Five different experiments were performed during a period of two years by different operators, using different batches of drugs, in a total of 26 rabbits. Despite minor variations in onset time and severity of the induced disease, the model proved to be reproducible. Both gangliosides and keyhole limpet hemocyanin are required for induction of disease.

Cytotoxicity of hydroxyapatite and morphology in composites with Ti

Hydroxyapatite (Ca10(PO4)6(OH)2 is the material used to improve osseointegration of implants in humans. Traditionally Ti-based implant is coated with hydroxyapatite, but this presents several problems such as, low fracture toughness of hydroxyapatite, generation of interfacial cracks during the deposition step and low adhesion ceramic /metal. Furthermore some studies reported that some excellent Ti¿HA bio-composites could be fabricated by the powder metallurgy (PM) technology. In present study it was investigate the cytotoxicity of coralline and bovine hydroxyapatite. Topography of Ti-HA composites obtained by powder metallurgy was evaluated by scanning electron microscopy. The results showed that HA samples analyzed did not affect cell viability. Furthermore, a good integration of HA to the matrix of Ti was observed by microscopy studies.

Titanium-hydroxyapatite composites sintered at low temperature for tissue engineering: in vitro cell support and biocompatibility

Background In clinical orthopedics, a critical problem is the bone tissue loss produced by a disease or injury. The use of composites from titanium and hydroxyapatite for biomedical applications has increased due to the resulting advantageous combination of hydroxyapatite bioactivity and favorable mechanical properties of titanium. Powder metallurgy is a simple and lower-cost method that uses powder from titanium and hydroxyapatite to obtain composites having hydroxyapatite phases in a metallic matrix. However, this method has certain limitations arising from thermal decomposition of hydroxyapatite in the titanium-hydroxyapatite system above 800°C. We obtained a composite from titanium and bovine hydroxyapatite powders sintered at 800°C and evaluated its bioactivity and cytocompatibility according to the ISO 10993 standard. Methods Surface analysis and bioactivity of the composite was evaluated by X-ray diffraction and SEM. MTT assay was carried out to assess cytotoxicity on Vero and NIH3T3 cells. Cell morphology and cell adhesion on the composite surface were analyzed using fluorescence and SEM. Results We obtained a porous composite with hydroxyapatite particles well integrated in titanium matrix which presented excellent bioactivity. Our data did not reveal any toxicity of titanium-hydroxyapatite composite on Vero or NIH3T3 cells. Moreover, extracts from composite did not affect cell morphology or density. Finally, NIH3T3 cells were capable of adhering to and proliferating on the composite surface. Conclusions The composite obtained displayed promising biomedical applications through the simple method of powder metallurgy. Additionally, these findings provide an in vitro proof for adequate biocompatibility of titanium-hydroxyapatite composite sintered at 800°C.