Fatima Laraba-Djebari

Oral delivery of insulin from alginate/chitosan crosslinked by glutaraldehyde

Insulin is mainly administered via subcutaneous route by injection which is the cause of painful and possible infections. Oral insulin administration would present a more convenient form of application because it is less invasive. Oral delivery of insulin to the gastrointestinal tract is one of the most challenging issues, because it numerous barriers to overcome in order to create an effective system for insulin delivery. In the present study, insulin-loaded alginate/chitosan blend gel beads were prepared with different mass ratios. Chitosan was depolymerized by gamma irradiation at a dose of 80 kGy reducing its molecular weight for ideal blend with sodium alginate. The homogeneous solution of alginate and chitosan was dripped into CaCl2 solution (2%), the resultant calcium crosslinked beads were dipped in glutaraldehyde (2%) solution sequentially to prepare dual crosslinked beads with improved mechanical properties so as to withstand the simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Morphological structure, FTIR analysis, thermogravimetry analysis, specific surface area, gel fraction, swelling kinetics in SGF and SIF, loading efficiency, insulin release behavior, mucoadhesivity of the alginate/chitosan beads were investigated. The cumulative insulin release of pure alginate beads (10:0) reached as maximum level 100% in 3h after they were dipped in SIF. Concerning the beads Alg/Chi (8:2), Alg/Chi (7:3) and Alg/Chi (6:4) the cumulative release of insulin reached 90.5%, 89.2% and 70.2%, respectively in 6h. The rate of 100% was reached after 24h for Alg/Chi (8:2), Alg/Chi (7:3) and after 73 h for Alg/Chi (6:4). The presence of chitosan in the blend beads decreased the cumulative insulin release in gastric media and enhanced behavior of alginate/chitosan beads in intestinal medium due to the crosslinking. The alginate/chitosan beads crosslinked by glutaraldehyde may be considered as potential insulin carriers for oral drug delivery system.

A fibrinogen-clotting serine proteinase from Cerastes cerastes (horned viper) venom with arginine-esterase and amidase activities. Purification, characterization and kinetic parameter determination

An enzyme displaying proteolytic activity toward the natural substrate casein as well as clotting activity on fibrinogen was purified to homogeneity from Cerastes cerastes (horned viper) venom and characterized. The enzyme is constituted of two identical subunits of mol. wt 48,500 as determined by SDS-polyacrylamide gel electrophoresis, and has an isoelectric point of 3.75. N-terminal sequencing up to the 33rd residue evidenced a high homology with other snake venom proteinases. The proteinase is of serine-type as indicated by high sensitivity to DFP and shows both arginine-ester hydrolase and amidase activities on synthetic substrates. Both specific activities were 30-fold higher than the respective activities found in the crude venom. The Km value determined for arginine-containing substrate BAEE was 3.0 x 10(-4) M and the Km for chromogenic substrate CBS 34-47 0.65 x 10(-4) M. The Vm/Km ratio, however, was two-fold higher for BAEE than for CBS 34-47; the arginine-esterase activity of this enzyme is thus slightly higher than its amidase activity.

Combination of two antibody fragments F(ab')(2)/Fab: an alternative for scorpion envenoming treatment.

Immunotherapy is the only effective treatment for scorpion stings. However the efficiency of this treatment varies depending on the forms of the antibodies and route of administration used. The antibodies are mostly injected as F(ab )(2) fragments. In this study, we investigated damage to the heart and lung tissue and the inflammatory response caused by Androctonus australis hector venom, its toxic fraction after molecular filtration or the isolated main alpha toxin (Aah II) in the presence or absence of different antibody molecules. A mixture of antibody fragments, F(ab )(2) and Fab, significantly reduced local leukocytosis, hemorrhage and inflammatory oedema induced by the A. australis hector venom and its toxins.

Irradiated Cerastes cerastes Venom as a Novel Tool for Immunotherapy

Immunotherapy is the most effective treatment for the snake bites. The antivenoms are commonly obtained by hyperimmunization of animals that suffer from venom toxicity. The present report describes gamma irradiation effects on Cerastes cerastes venom. Doses of 1 kGy and 2 kGy gamma radiations were used for venom detoxification. These treated venoms did not have any residual lethal effects until 10 LD50. Immunological analysis of sera raised against native and irradiated venoms, showed that elicited antibodies to irradiated venoms were able to recognize native venom. Anti-2 kGy irradiated venom had more protective ability than anti-native venom, as tested in mice.

Immunomodulation of the Inflammatory Response Induced by Androctonus australis hector Neurotoxins: Biomarker Interactions

Objective:Androctonus australis hector (Aah) is the most dangerous scorpion in the Maghreb countries. Its venom contains three major neurotoxins (Aah I, Aah II and Aah III), which are responsible for almost all the lethal effects caused in mammals. These toxins act on the voltage-gated sodium channels of excitable cells. The targets and the lethal effects of these toxins have been extensively studied. However, their effects on the induced immune response after envenoming have not deeply elicited. We therefore investigated the effects induced by Aah venom and its toxic components, mainly its main toxin Aah II, on the activation of the inflammatory process. Methods: Wistar rats were injected by intraperitoneal route with a sublethal dose of Aah venom, FTox-G50, the purified Aah II toxin or with 400 µl of sterile physiological saline solution. Immunological biomarkers such as MPO, NO and ICAM-1 were analyzed in serum in lung tissue. Cytokine levels were also determined in serum at 3, 6 and 24 h after envenoming. Results: We report in this study that intraperitoneal injection of the venom or its toxins (the whole toxic fraction or Aah II toxin) caused an inflammatory reaction involving increased neutrophil release into blood and neutrophil accumulation in lung tissue. This cell infiltration was associated with the release of NO, histamine, cytokines (IL-1, IL-6, IL-12, IL-4 and IL-5) and ICAM. Conclusion: Aah II binding to its targets, in this case Na+ channels, may induce a cascade of events such as inflammatory mediator release and neutrophil migration that could contribute to the exacerbation of the systemic inflammatory response and the development of lung injury following scorpion envenoming.

Modulation of Tissue Inflammatory Response by Histamine Receptors in Scorpion Envenomation Pathogenesis: Involvement of H4 Receptor

The inflammatory response caused by scorpion venoms is a key event in the pathogenesis of scorpion envenomation. This response was assessed in the cardiac, pulmonary, and gastric tissues of envenomed mice. The results reveal an increase of permeability in cardiac, pulmonary, and gastric vessels accompanied by an edema-forming, inflammatory cell infiltration, and imbalanced redox status. These effects are correlated with severe tissue alterations and concomitant increase of metabolic enzymes in sera. Pretreatment of mice with antagonists of H1, H2, or H4 receptors markedly alleviated these alterations in the heart and lungs. Nevertheless, the blockade of the H3 receptor slightly reduced these disorders. Histamine H2 and H4 receptors were the most pharmacological targets involved in the gastric oxidative inflammation. These findings could help to better understand the role of histamine in scorpion venom-induced inflammatory response and propose new therapy using as targets the H4 receptor in addition to histamine H1 and H2 receptors to attenuate the induced inflammatory disorders encountered in scorpion envenoming.

Development and characterization of a new carrier for vaccine delivery based on calcium-alginate nanoparticles: Safe immunoprotective approach against scorpion envenoming.

To enhance humoral defense against diseases, vaccine formulation is routinely prepared to improve immune response. Studies in nanomaterials as a carrier of vaccine delivery are promising and interesting. In this study, attenuated Androctonus australis hector (Aah) venom and its toxic fraction were encapsulated into different formulations inside calcium-alginate nanoparticles (Ca-Alg Nps), and used as a vaccine delivery system against scorpion envenomation. Ca-Alg Nps were prepared by ionic gelation and characterized. An immunization schedule was undertaken in rabbits in order to study how Aah venom entrapped in Ca-Alg Nps might induce protective immunity. Results showed the influence of different parameters on the suitable nanoparticle formation. They also showed no toxicity of free Ca-Alg Nps and a different inflammatory profile depending on the nanovaccine formulations. More interestingly, evaluation of specific IgG titer and IgG1/IgG2a isotype balance revealed a protective effect with the nanoparticles encapsulating the attenuated antigens. Challenge up to 6 LD 50 of native venom, allowed to an important immunoprotection of all immunized rabbits, with no recorded death. Taken together and with respect to the properties of nanoparticles and high immunogenicity, calcium-alginate nanoparticles could be considered as a new promising adjuvant system and a vaccine delivery against scorpion envenomation.

Isolation and characterization of a myotoxin from the venom of Macrovipera lebetina transmediterranea

The Macrovipera lebetina venom consists of a complex mixture of proteins belonging to a few main families according to their enzymatic and pharmacological activity. Given the serious pathophysiological effects caused by M. lebetina bites mainly induced by muscle degeneration, we decided to investigate the myotoxic activity of some venom fractions. In the present study we describe the purification and characterization of a 22.600 kDa protein, named in the following Mlp4.2, that shares myotoxic but not haemorrhagic activity in vivo. Herein we report that Mlp4.2 is a metalloproteinase belonging to the PI-SVMPS family able, in vitro, to proteolyse extracellular matrix proteins as laminin and fibronectin. Histological observations of mouse anterior tibialis Mlp4.2-treated muscle, demonstrate that this protein induces a massive degeneration of myofibers but not haemorrhage. The immunofluorescence analysis of protein-treated anterior tibialis, demonstrates that Mlp4.2 is able to disarray the laminin network surrounding muscle fibers. Finally Mlp4.2 did not show any direct cytolytic activity towards the myogenic cell line C2C12 in culture. The data reported herein suggest that the myotoxicity of Mlp4.2 is primarily linked to the disruption of the muscle fibers interaction with extracellular matrix proteins.

KTX3, the kaliotoxin from Buthus occitanus tunetanus scorpion venom: one of an extensive family of peptidyl ligands of potassium channels.

A new ligand of the K+ channels sensitive to KTX was purified from the venom of Buthus occitanus tunetanus, using two steps of high-performance-liquid-chromatography and by following its ability to compete with [125I]-KTX for binding to the KTX receptor on rat brain synaptosomes. Amino-acid analysis, amino acid sequencing and mass spectroscopy defined this new ligand. KTX3, as a 37-amino acid peptide, with three disulfide bridges. Its sequence shares 76% identity with KTX. The main differences between the two peptides are in the N-terminal region and the residue position 34 located in the region involved in channel recognition. These differences may explain the 5-fold lower binding affinity of KTX3, IC50=50 pM, than KTX to rat brain synaptosomes. Specific antibodies raised against KTX (1-37) were not able to recognize KTX3.

Systemic responses following brain injuries and inflammatory process activation induced by a neurotoxin of Androctonus scorpion venom.

Objective: Kaliotoxin 2 (KTX2), a neurotoxin isolated from Androctonus australis hector scorpion venom, presents a high affinity with the voltage-gated potassium channels. The targets of KTX2 in the brain and its toxic effects on the cerebral cortex have been extensively studied; however, its deleterious systemic effects on other organ systems have not yet been investigated. Inflammatory response induced by KTX2 is supported by cytokine release which could provoke multiple organ dysfunction and diverse biological disorders in mammals. The possibility that inflammatory response and brain injuries induced by KTX2 may lead to functional disturbances, e.g. in the pancreas and the liver, were investigated. The contribution of IL-6 and TNF-α to the modulation of pathophysiological effects induced by KTX2 was also tested. Methods: NMRI mice were injected by the intracerebroventricular route with a sublethal dose of KTX2 or saline solution. Inflammatory response and oxidative stress were assessed in sera and tissue homogenates. Biomarkers of pancreatic and hepatic functions and the correlation with tissue damage in the brain, liver and pancreas were also analyzed. Results: The obtained results revealed that KTX2 injection induced an inflammatory process activation and imbalanced redox status. It also induced severe alterations in cerebral cortex, hepatic and pancreatic tissues associated with a significant increase in pancreatic and hepatic pathological biomarkers. Cytokine antagonists injected 30 min prior to KTX2 led to a significant reduction of all disturbances induced by KTX2. Conclusion: In addition to its significant toxicity on the central nervous system, KTX2 can also affect pancreatic and hepatic functions, probably by an indirect mechanism involving activation of the inflammatory response with release of IL-6 and TNF-α.