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Mucopolysaccharidosis type I (MPS I) due to deficient alpha-L-iduronidase (IDUA) activity results in the accumulation of glycosaminoglycans (GAGs) in many of the cells of affected patients. Stable gene replacement by in vivo administration of lentiviral vectors (LVs) has therapeutic potential for metabolic disorders and other systemic diseases. We have previously shown in a murine model the therapeutic potential of lentiviral IDUA vector-mediated gene therapy, in which human IDUA cDNA was driven by the cytomegalovirus promoter. However, the major limitation of this approach was the induction of an immune response against the therapeutic protein, which limited the efficacy and long-term duration of treatment. In this study, we evaluate the potential of liver-directed gene therapy, that is, programming of murine hepatocytes to secrete the enzyme with mannose 6-phosphate (M6P), which can be taken up by distant cells. Eight- to 10-week-old mice were injected via the tail vein with a lentiviral vector expressing human IDUA cDNA driven by the albumin gene promoter selectively expressed in hepatocytes. One month after treatment, IDUA activity was present in the liver and spleen of treated mice; an expression level of 1% normal IDUA activity was sufficient to reduce the GAG level in liver, spleen, kidney, heart, and lung. Interestingly, 6 months after a single injection of this vector, IDUA activity was detectable in several murine tissues; the level of enzyme activity was low but sufficient to maintain the decrease in GAG levels in liver, spleen, kidney, heart, and lung. Also, the level of enzyme-specific antibodies reached at 6 months postinjection was nearly null, and real-time polymerase chain reaction analysis showed high levels of vector DNA content in liver and spleen. Thus, these results show that the use of LV with the albumin gene promoter selectively expressed in hepatocytes limited the immune response to the transgene and allowed stable and prolonged expression of the IDUA enzyme and a partial correction of the pathology.

Mucopolysaccharidosis type IIIA, also known as Sanfilippo A disease, results from mutations in the sulfamidase gene. To date, a total of 62 mutations have been described underlying this lysosomal disorder. Expression studies on missense mutations have shown that each alteration was disease-causing and helped to clarify the genotype-phenotype correlation. In this report we describe a large pedigree where the mutations have been identified in two second cousins: one with severe disease (E369K/R433Q) and the other with a mild form of the illness (E369K/P128L). This study places R433Q as a severe mutation underlying Sanfilippo A disease.

Mucopolysaccharidosis type IIIB (MPS IIIB, Sanfilippo type B syndrome) is a lysosomal disorder caused by mutations in the gene encoding for alpha-N-acetylglucosaminidase (NAGLU), a glycosidase required for the degradation of heparan sulfate. The disease is characterized, clinically, by profound neurological deterioration with behavioral disturbances, but relatively mild somatic manifestations. At present, there is no treatment for MSP IIIB patients and the pathogenetic bases of the neurological disease remain unclear. We showed the efficacy of lentiviral-NAGLU vector mediated gene therapy in the visceral organs of the murine MPS IIIB model (1). More recently, we demonstrated, in the same model, that imbalance of cytokines, neurotrophins and oxidative stress can be involved in the pathogenesis of brain disease. In particular, Bdnf gene resulted to be down-regulated in the affected brains, while Cbln1, Ccl3, Casp11 and gp91phox, p67phox and p47phox (three components of the phagocyte NADPH oxidase) showed an increased expression in the affected mice vs normal controls (2). In the present study we evaluated the effect of intracranial injections of lentiviral-NAGLU vector in the MPS IIIB mice on cytokines, neurotrophins and oxidative stress. First, we used a lentiviral-GFP vector to evaluate the area of GFP gene transduction into the mice brain. The vector suspension was injected stereotactically in the right fimbria of 10-week-old animals, and treated mice were examined 1 month after treatment. Fluorescence microscopy for GFP expression on serial coronal sections showed positive cells for GFP in a large area surrounding the infusion site, and in some areas on the controlateral side of the brain. Subsequently, we injected the lentiviral-NAGLU vector in affected mice, obtaining a normal expression of active NAGLU enzyme throughout a large portion of the brain and a significant presence of vector genome copies. Real time RT PCR analysis performed on RNA from three regions (frontal, parietal and occipital) of brains from affected mice at one month from treatment, showed that Ccl3, Casp11 and gp91phox expression was unaffected; conversely, a normalization of neurotrophins Bdnf and Cbln1 was obtained. Our data suggest that gene therapy by intracranial injections of lentiviral-NAGLU vector can be effective for treatment of the brain pathology in the murine MPS IIIB model.

We report here the successful establishment of callus, cell and root cultures from explants of in-vitro-grown plantlets of the soapwort Saponaria officinalis L. The production of saporin in the different tissue systems was evaluated by determining the capability of crude extracts to inactivate protein synthesis and by Western blotting analysis. Protein synthesis inhibition varied in callus and derived cell suspensions and in cultured roots, the latter, in particular, showing the lowest specific activity. The ribosome-inactivating principle from root cultures was purified to homogeneity by cation exchange chromatography.

The adhesion of ADP-stimulated platelets to immobilized fibrinogen induces the tyrosine phosphorylation of multiple proteins which include pp72 syk and pp125 FAK . The phosphorylation of these two proteins increases as function of time of platelet adhesion to fibrinogen; however, pp72 syk results strongly phosphorylated already after 15 min. whereas pp125 FAK reaches high levels of phosphorylation after 1 h of platelet adhesion. Phosphorylation of both proteins is only slightly detectable when platelets are held in suspension or when platelets are allowed to adhere to bovine serum albumin, a non-specific substrate. Echistatin, an Arg-Gly-Asp (RGD)-containing snake-venom protein, affects protein tyrosine phosphorylation promoted by platelet adhesion to fibrinogen, by causing an approximately 44% and 39% decrease of pp72 syk and pp125 FAK phosphorylation, respectively. The interaction of echistatin with fibrinogen receptor glycoprotein Ilb-Illa on platelet surface might be responsible for the block of integrin-mediated signaling cascade, including pp72 syk and pp125 FAK inactivation.

Bovine lactoperoxidase (LPO) is taken as a model protein of mammalian peroxidases to investigate the activity and the stability of the enzyme in the presence of different surfactants. The cationic benzalkonium chloride (Bz) has proved efficient in preserving the enzymatic activity for over 10 days, while the native enzyme completely lost its activity within 3-4 days. The presence of Bz allows the enzyme to preserve its secondary structure for a long time, as shown in CD spectra, and creates a more hydrophobic environment for the enzyme, as indicated in fluorescence studies. Moreover, this surfactant at a concentration of 0.01% (0.3 mM) increases the lactoperoxidase activity in the first 2 h of incubation at 37 degrees C. Both hydrophobic and electrostatic interactions of the cationic surfactant seem to be responsible for the enzyme activation and stabilization, and this is a promising result in view of industrial applications of enzymes.

Exposure of fibrinogen to the Fe3+/ascorbate oxidative system resulted in structural modifications and altered functionality of the glycoprotein. The overnight treatment of fibrinogen by oxidants caused a 20-fold increase of carbonyl content with respect to the native protein. Formation of dityrosines as well as loss of tryptophan following fibrinogen oxidation were observed. The occurrence of conformational changes of the fibrinogen molecule as a consequence of the oxidative treatment was also established. Oxidized fibrinogen showed a distinct capability from the native molecule to mediate platelet aggregation and adhesion. The percentage of ADP-induced platelet aggregation decreased as a function of fibrinogen oxidative damage. Further, both unstimulated platelets and ADP-activated platelets showed a reduced ability to adhere to oxidized fibrinogen than to the native protein. These results suggest that oxidative treatment alters fibrinogen domains involved in the recognition and the binding of this molecule by the platelet receptor GP IIb/IIIa.

The study of bioethics in Argentina owes much to the efforts of José Alberto Mainetti, who in 1972 founded the Institute of Medical Humanities, devoted to researching, teaching, and publicizing the discipline. Because of growing interest in the field of bioethics, a Bioethics Center was created in the Institute in 1987 and it has stimulated the organization of several groups in various provinces in the country. One of the most important of them, located at the National University of Mar del Plata, is responsible for designing a graduate-level specialization in bioethics and for organizing the First Mar del Plata Bioethics Workshop. This interdisciplinary group examines the bioethical implications of concrete scientific and professional activities as well as legislative measures and decisions affecting the public.

Breastfeeding is of crucial importance in the developing world, not only to the health of the mother and her infant, but to the community in general. The importance of breastfeeding of infants lies not only in its nutritional and hygienic properties, but also because of its immunological effects and its influence in the control of fertility. The literature review is combined with a discussion of aspects of research undertaken by the authors under the headings of length of breastfeeding, bottlefeeding and breastfeeding and fertility. Most of the examples come from Nigeria, but the problems discussed are common to much of the developing world. Under a discussion of social and policy implications, the need for governments in the developing countries to adopt the recent World Health Organisation's code of practice to curtail the sale of artificial infant milks, is stressed. It is suggested that health agencies in countries like Nigeria should go to the rural areas now, to instruct the women and men in the dangers of bottle feeding and the benefits of breastfeeding, before the 'modernisation' of infant feeding, so evident in the towns, spreads to the rural areas.

Breastfeeding is of crucial importance in the developing world, not only to the health of the mother and her infant, but to the community in general. The importance of breastfeeding of infants lies not only in its nutritional and hygienic properties, but also because of its immunological effects and its influence in the control of fertility. The literature review is combined with a discussion of aspects of research undertaken by the authors under the headings of length of breastfeeding, bottlefeeding and breastfeeding and fertility. Most of the examples come from Nigeria, but the problems discussed are common to much of the developing world. Under a discussion of social and policy implications, the need for governments in the developing countries to adopt the recent World Health Organisation's code of practice to curtail the sale of artificial infant milks, is stressed. It is suggested that health agencies in countries like Nigeria should go to the rural areas now, to instruct the women and men in the dangers of bottle feeding and the benefits of breastfeeding, before the 'modernisation' of infant feeding, so evident in the towns, spreads to the rural areas.

Platelets represent a target of reactive oxygen species produced under oxidative stress conditions. Controversial data on the effect of these species on platelet functions have been reported so far. In this study we evaluated the effect of a wide range of H 2 O 2 concentrations on platelet adhesion to immobilized fibrinogen and on pp72 syk and pp125 FAK tyrosine phosphorylation. Our results demonstrate that: (1) H 2 O 2 does not affect the adhesion of unstimulated or apyrase-treated platelets to immobilized fibrinogen; (2) H 2 O 2 does not affect pp72 syk phosphorylation induced by platelet adhesion to fibrinogen-coated dishes; (3) H 2 O 2 reduces, in a dose-dependent fashion, pp125 FAK phosphorylation of fibrinogen-adherent platelets; (4) concentrations of H 2 O 2 near to physiological values (10–12 μM) are able to strengthen the subthreshold activation of pp125 FAK induced by epinephrine in apyrase-treated platelets; (5) H 2 O 2 doses higher than 0.1 mM inhibit ADP-induced platelet aggregation and dense granule secretion. The ability of H 2 O 2 to modulate pp125 FAK phosphorylation suggests a role of this molecule in physiological hemostasis as well as in thrombus generation.

Sanfilippo B syndrome (Mucopolysaccharidosis IIIB, MPS IIIB) is a lysosomal storage disease due to mutations in the gene encoding alpha-N-acetylglucosaminidase and is characterized by a severe neurological disorder. Although several studies have been reported for the murine model of the disease, the molecular basis and the sequence of events leading to neurodegeneration remain to be clarified. We previously suggested the possible involvement of the reactive oxygen species in the disease pathogenesis. In the present paper we extended the analysis of oxidative stress by evaluating the production of superoxide ions throughout the CNS and by evaluating the effect of the stress on the cellular macromolecules. These approaches applied to one-month-old, three-month-old and six-month-old mice revealed that oxidative stress is present in the affected cerebrum and cerebellum tissues from one month from birth, and that it results primarily in protein oxidation, both in the cerebrum and cerebellum, with lipid peroxidation, and especially DNA oxidation, appearing milder and restricted essentially to the cerebellum. We also identified additional genes possibly associated with the neuropathology of MPS IIIB disease. Real time RT-PCR analysis revealed an altered expression of the Sod1, Ret, Bmp4, Tgfb, Gzmb and Prf1 genes. Since Gzmb and Prf1 are proteins secreted by NK/cytotoxic T-cells, these data suggest the involvement of cytotoxic cells in the neuronal pathogenesis. Extending our previous study, findings reported in the present paper show that oxidative stress and all the analyzed stress-related pathological changes occur very early in the disease course, most likely before one month of age.

The Sanfilippo syndrome type B (mucopolysaccharidosis IIIB) is an autosomal recessive disorder due to mutations in the gene encoding NAGLU (α-N-acetylglucosaminidase), one of the enzymes required for the degradation of the GAG (glycosaminoglycan) heparan sulphate. No therapy exists for affected patients. We have shown previously the efficacy of lentiviral-NAGLU-mediated gene transfer in correcting in vitro the defect on fibroblasts of patients. In the present study, we tested the therapy in vivo on a knockout mouse model using intravenous injections. Mice (8–10 weeks old) were injected with one of the lentiviral doses through the tail vein and analysed 1 month after treatment. A single injection of lentiviral-NAGLU vector resulted in transgene expression in liver, spleen, lung and heart of treated mice, with the highest level reached in liver and spleen. Expression of 1% normal NAGLU activity in liver resulted in a 77% decrease in the GAG content; more remarkably, an expression of 0.16% normal activity in lung was capable of decreasing the GAG level by 29%. Long-term (6 months) follow up of the gene therapy revealed that the viral genome integration persisted in the target tissues, although the real-time PCR analysis showed a decrease in the vector DNA content with time. Interestingly, the decrease in GAG levels was maintained in liver, spleen, lung and heart of treated mice. These results show the promising potential and the limitations of lentiviral-NAGLU vector to deliver the human NAGLU gene in vivo.

Mucopolysaccharidosis type I is a lysosomal disease due to mutations in the IDUA gene, resulting in deficiency of alpha- L -iduronidase and accumulation of glycosaminoglycans (GAGs). Bone marrow transplantation and enzyme replacement are two therapies considered only moderately successful for affected patients, making the development of novel treatments necessary. We have previously shown the efficacy of lentivirus-mediated gene transfer to correct patient fibroblasts in vitro. Here we tested lentiviral-IDUA vector gene therapy in vivo on a murine MPS I model. Eight- to 10 week-old mice were injected with increasing lentiviral doses via the tail vein and analyzed 1 month after treatment. A single injection of lentiviral-IDUA vector resulted in transgene expression in several murine tissues, with the highest level reached in liver and spleen. Expression of 1% normal activity was sufficient in treated animals to normalize the GAG level in urine, liver, and spleen and was able to reduce the GAG level in kidney, heart, and lung. Polymerase chain reaction assays showed integration of the viral genome only in liver and spleen of treated animals, suggesting that the correction of the pathology in other tissues was due to secretion into the plasma by liver and spleen and uptake of corrective enzyme by distant tissues. Long-term (6 months) analysis showed the presence of enzyme-specific antibodies and the loss of enzyme activity and vector sequence in the target tissue, suggesting that the transgene-specific immune response interfered with long-term therapeutic correction and led to clearance of transduced cells. In conclusion, our results show the promising potential and the limitations of lentiviral-IDUA vector-mediated gene therapy in an in vivo model.

Echistatin, a 5000-Da disintegrin, is a strong competitive inhibitor of platelet KIIbL3 binding to fibrinogen. In addition to its antiplatelet activity, echistatin also exhibits activating properties by inducing a switch of KIIbL3 conformation towards an active state. However, soluble echistatin, which is a monomeric ligand, provides only receptor affinity modulation, but it is unable to activate integrin-dependent intracellular signals. Since proteins may exhibit a multivalent functionality as a result of their absorption to a substrate, in this study we evaluated whether immobilised echistatin is able to stimulate platelet adhesion and signalling. The immobilisation process led to an increase of echistatin affinity for integrin(s) expressed on resting platelets. Unlike the soluble form, immobilised echistatin bound at comparable extent either unstimulated or ADPactivated platelets. Furthermore, echistatin presented in this manner was effective in stimulating integrin-dependent protein tyrosine phosphorylation. Platelets adhering to immobilised echistatin showed a pattern of total tyrosine phosphorylated proteins resembling that of fibrinogen-attached platelets. In particular, solid-phase echistatin induced a strong phosphorylation of tyrosine kinases pp72 syk and pp125 FAK . Inhibitors of platelet signalling, such as apyrase, prostaglandin E1, cytochalasin D and bisindolylmaleimide, while not affecting platelet adhesion to immobilised echistatin, abolished pp125 FAK phosphorylation. This suggests that signals activating protein kinase C function, dense granule secretion and cytoskeleton assembly might be involved in echistatin-induced pp125 FAK phosphorylation. fl 2000 Elsevier Science B.V.

We report a female patient with heparan N-sulphatase deficiency (MPS IIIA) whose genotype (P293T/R304L; V387M) was investigated