Feasibility of using cryopreserved lymphoblastoid cells to diagnose some lysosomal storage diseases.

OBJECTIVE: The Epstein-Barr virus (EBV) is utilized as a tool in the study of cellular biology because of its capacity to transform B-lymphocytes. For this reason, EBV is used in conservation of human B-lymphocytes for long periods for subsequent evaluation of lysosomal hydrolase activity. Lymphoblastoid cell lines have several advantages for use over other cell types, such as prompt availability and possibility to develop, characterize and standardize cell banks, to test effects of promising pharmaceutical reagents. The study below presents biochemical data that demonstrate validity of lymphoblastoid cell lines for diagnosis of GM1-gangliosidosis, Gaucher, Fabry and Pompe diseases and mucopolysaccharidosis type I. MATERIALS AND METHODS: Cultures were prepared from peripheral blood, collected from 25 normal subjects and 13 affected individuals. Enzyme activities and immunohistochemistry (IHC) were measured. Activities of enzymes beta-galactosidase, beta-glucosidase, alpha-iduronidase, alpha-galactosidase and alpha-glucosidase were measured before and after cryopreservation for 180 days. Enzymatic activity was measured when transformation was confirmed by IHC. RESULTS: We observed some significant alterations in enzymatic activity of non-cultured cells when compared to others that had been cultured for 12 days and kept frozen for 180 days. CONCLUSIONS: However, these alterations did not invalidate use of the technology of transformation of lymphoblastoid cell lines with EBV, to diagnose the diseases mentioned above, in view of the fact that the cultured cells, before and after freezing, demonstrated similar enzymatic activities.

Safety and efficacy of enzyme replacement therapy with agalsidase beta: an international, open-label study in pediatric patients with Fabry disease.

OBJECTIVE: To evaluate the safety and explore the efficacy of enzyme replacement therapy with agalsidase beta (recombinant human alpha-galactosidase A; Fabrazyme [Genzyme Corporation, Cambridge, MA]) in pediatric patients with Fabry disease, a genetic disorder in which deficient endogenous enzyme causes pathogenic tissue accumulation of globotriaosylceramide (GL-3). STUDY DESIGN: Fourteen male and 2 female patients, 8 to 16 years old, were treated in this open-label study. A 12-week observation period to collect baseline data preceded the 48-week treatment period when agalsidase beta (1 mg/kg) was infused intravenously every 2 weeks. No primary efficacy end point was specified. RESULTS: Before treatment, results of skin biopsies from 12 male patients showed moderate or severe GL-3 accumulation in superficial dermal capillary endothelial cells; with treatment, these cells were completely cleared of GL-3 in week-24 biopsies from all 12 male patients and in all available week-48 biopsies. With treatment, reports of gastrointestinal symptoms declined steadily. Patient diaries documented significant reductions in school absences due to sickness. Agalsidase beta was generally well tolerated; most treatment-related adverse events were mild or moderate infusion-associated reactions involving rigors, fever, or rhinitis. CONCLUSIONS: Agalsidase beta safely and effectively reduced the GL-3 accumulation in dermal endothelium already evident in children with Fabry disease. Early intervention may prevent irreversible end-organ damage from chronic GL-3 deposition.

High incidence of autoantibodies in Fabry disease patients.

Fabry disease (FD) is an X-linked disorder of glycosphingolipid catabolism that results from a deficiency of the lysosomal enzyme alpha-galactosidase A. This defect leads to the accumulation of its substrates, mainly globotriaosylceramide, in lysosomes of cells of different tissues. Different studies have shown the involvement of immunopathologies in different sphingolipidoses. The coexistence of FD and immune disorders such as systemic lupus erythematosus, rheumatoid arthritis and IgA nephropathy, has been described in the literature. The aim of this study was to evaluate the prevalence of a group of autoantibodies in a series of Argentine FD patients. Autoantibodies against extractable nuclear antigens (ENAs), double-stranded DNA, anticardiolipin and phosphatidylserine were assayed by ELISA. Lupus anticoagulants were also tested. Fifty-seven per cent of the samples showed reactivity with at least one autoantigen. Such reactivities were more frequent among males than among females. Antiphospholipid autoantibodies were detected in 45% of our patients. The high rate of thrombosis associated with FD could be related, at least in part, to the presence of antiphospholipid autoantibodies in Fabry patients. We found the presence of ENAs, which are a characteristic finding of rheumatological diseases, previous a frequent misdiagnosis of FD, in around 39% of the cases. The detection of a high level of autoantibodies must be correlated clinically to determine the existence of an underlying autoimmune disease. With the recent development of therapy, the life expectancy in FD will increase and autoimmune diseases might play an important role in the morbidity of FD.

Respuesta inmune humoral contra el epitopo alfa-galactosil en pacientes con neoplasia intraepitelial cervical / Immunity humoral answer against the a galactosyl epitopes in patients with cervical intraepithelial neoplasia

Anti-Gal es un anticuerpo natural policlonal que interactúa con un gluconjugado no expresado en células humanas. En condiciones fisiológicas especiales y en infecciones por kinetoplástidos anti-gal asume un rol protector. Evidencias señalan que este anticuerpo y otros relacionados como el que reacciona contra el residuo glucosídico Gal (&1-2) Gal, forman parte del sistema de vigilancia antitumoral. La finalidad de este estudio fue confirmar que pacientes con carcinoma in situ de cuello uterino (NIC 3) tienen niveles séricos anormalmente elevados de estos anticuerpos, comparar estos niveles a los de pacientes con lesiones más tempranas (infección por virus de papiloma humano, NIC 1 y NIC 2) y estudiar si el editopo se expresa en las células atípicas. Se encontraron niveles séricos elevados de anticuerpos anti Gal (&1-2)Gal en 21 por ciento de los pacientes (p=0.006, Mann-Whitney), sin diferencia entre pacientes con infección por VHP y carcinoma in situ. Veintiocho por ciento de la serie tuvo inmunoreactividad sérica elevada de antiGal (p=0.001, Mann-Whitney), y esta diferencia se mantuvo al comparar los subgrupos NIC con el control (p=0.004, Kruskal-Wallis). El subgrupo con carcinoma invasor tuvo niveles iguales al control e inferiores al de pacientes con lesiones más tempranas (p<0.05, Bonferroni). Utilizando un anticuerpo monoclonal como anticuerpo primario y el método de inmunoperoxidasa se demostró la expresión del editopo en 5/12 casos, todos con niveles normales de anticuerpos. La inmunotinción fue positiva en los sectores con cambios coilocíticos sugestivos de infección por VPH y no en el epitelio normal ni en el glandular. Se propone que el editopo galactosil se expresa en células del epitelio cervical infectadas por VPH y que los niveles anormales son parte de la respuesta inmune a la infección.