Interaction of a plasminogen activator proteinase, LV-PA with human alpha2-macroglobulin.

Lachesis venom plasminogen activator (LV-PA) is a 33-kDa serine proteinase isolated from bushmaster (Lachesis muta muta) snake venom, which activates the fibrinolytic system in vitro. This study has examined the effect of the plasma proteinase inhibitor alpha2-macroglobulin (alpha2-M) towards LV-PA and compares it with the effect on tissue type plasminogen activator (t-PA). The proteolytic activity of LV-PA alone or previously incubated with human plasminogen (Plg) on the large molecular mass protein substrates, dimethylcasein (DMC) and fibrinogen (Fg) was completely inhibited by human alpha2-M. However, the synthetic peptides Tos-Gly-Pro-Lys-pNA and H-D-Pro-Phe-Arg-pNA (S-2302) were hydrolyzed with almost no reduction in rate. At pH 7.4 and 37 degrees C the proteinase (0.15 microM over 15 min) interacted with alpha2-M, and each mole of alpha2-M bound 2 mol of enzyme. Sodium dodecyl sulfate gel electrophoresis of reduced samples showed that the interaction of alpha2-M with either LV-PA or t-PA preincubated with Plg resulted in the formation of approximately 90 kDa fragments and high molecular mass complexes (Mr 180 kDa), generated by the incubation mixture (LV-PA or t-PA) and Plg. The data suggest that LV-PA is a direct-type PA and its fibrinolytic effect can be reduced by alpha2-M in vivo.     

Induction of mosquitocidal activity in mice immunized with Anopheles gambiae midgut cDNA

Vaccines that induce mosquito-killing (mosquitocidal) activity could substantially reduce the transmission of certain mosquito-borne diseases, especially vaccines against African malaria vectors, such as the mosquito Anopheles gambiae. To generate and characterize antimosquito immunity we immunized groups of mice with two individual A. gambiae midgut cDNAs, Ag-Aper1 (a secreted peritrophic matrix protein) and AgMuc1 (a midgut-bound mucin), and an A. gambiae midgut cDNA library from blood-fed mosquitoes. We observed significantly increased mortality among mosquitoes that fed on either the AgMuc1- or the cDNA library-immunized mice compared to that of controls, but no differences were observed among those fed on Ag-Aper1-immunized mice. Analysis of the humoral and cellular immune responses from mice showed that the induced mosquitocidal effect was associated with immune profiles characterized by elevated tumor necrosis factor alpha and gamma interferon cytokine levels and very low antibody titers. Furthermore, an additional immunization of cDNA library-immunized mice with midgut protein shifted immunity toward a Th2-type immune response, characterized by elevated antibody titers and high interleukin-5 and interleukin-10 cytokine levels; importantly, mosquitoes feeding on these mice exhibited no undue mortality. Finally, when immune sera was ingested by mosquitoes through a membrane feeder, no effect on mosquito mortality was observed, indicating that serum factors alone were not responsible for the mosquitocidal effect. Our results demonstrate that mosquitocidal immunity in mice can be consistently generated by midgut cDNA immunization and suggest this cDNA-induced mosquitocidal immunity is cell mediated.     

Vaccination with chimeric protein induces protection in murine model against ascariasis

An estimated 400 million people are infected by parasites of the genus Ascaris and the existing control measures are inefficient. Vaccine development using B cell antigens is a promising strategy for increased protection against this parasite. The present study aimed at developing a chimeric protein capable of conferring protection against infection by Ascaris sp. For this purpose, we performed B-cell epitope predictions on previously described vaccine candidate proteins from Ascaris suum and the corresponding peptides were used to construct a chimeric protein. Female BALB / c mice were immunized subcutaneously in three doses at 10 day intervals with a vaccine formulation comprised of the chimeric protein together with monophosphoryl lipid A (MPLA). Control groups included protein alone, MPLA, or PBS. After challenge infection, animals vaccinated with chimeric protein plus MPLA showed a reduction of 73.54% of larval load in the lung compared to control group animals. Animals immunized with chimeric protein plus MPLA also display higher IgG response and a reduction in lung inflammation. Our study highlights how chimeric proteins containing more than one B cell epitope can enhance immune protection against helminthic infection and offer new approaches to the development of Ascaris vaccines.     

Resolution of isoforms of mutalysin II, the metalloproteinase from bushmaster snake venom

Mutalysin II, a zinc endopeptidase possessing direct-acting fibrinolytic activity has been previously purified from bushmaster (Lachesis muta muta) snake venom. We now report a method to isolate two isoforms of natural mutalysin II (mut IIa and mut IIb) using chromatographies on Sephacryl S-200, CM Sepharose CL 6B and Sephadex G-50. The two proteins are monomeric non-glycosylated proteinases with similar molecular masses of 23 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tryptic peptide mapping of the two native enzymes suggested a large degree of structural similarity. Both isoforms showed high similarity in all enzymatic properties using fibrinogen, fibrin and dimethylcasein as substrates. Thus, the specific fibrinolytic activity was estimated as 12±1.04 and 11.5±1.02 U/μg for mut IIa and mut IIb, respectively. The antigenic cross-reactivity of both isoforms was examined using rabbit hyperimmune serum or immunoglobulin G anti-mut IIa assays on immunodiffusion microscope slides, indirect enzyme-linked immunoabsorbent assay and western blots. From these experiments it was concluded that the two metalloproteinases mut IIa and mut IIb share identical antigenic structures. Since the stability of mutalysin II is dependent upon the presence of zinc, we examined the EDTA sensitivity of the isoforms of mutalysin II. Thus, the IC₅₀ values (concentration of EDTA to produce 50% inhibition of dimethylcasein hydrolysis) for mut IIa is 180 μM and 165 μM for mut IIb.     

Assessment of drug addiction. The experience of the Medico Legal Institute of Oporto

Since 1993 in Portugal, it is possible, by legal enforcement, to request forensic examination to study possible conditions of drug addiction. On the basis of examinations carried out in the Medico Legal Institute of Oporto, we present a study of 494 cases involving the relation to the demographic data of the examined individuals, their family background, legal situation, age at which drug consumption began, consumption profile at the beginning and at time of forensic examination and our conclusions.     

Adenosquamous Carcinoma of Hypopharynx with Intestinal-Phenotype

Adenosquamous carcinomas of the head and neck (ADSCs) are rare locally aggressive malignancies characterized by the presence of two distinctive components, a squamous cell carcinoma and an adenocarcinoma. The immunophenotype of the glandular component of ADSCs has only been rarely studied but has been reported as being positive for keratin 7 (CK7) and carcinoembryonic antigen (CEA) and negative for keratin 20 (CK20). Herein, we report a case of an ADSCs of the hypopharynx composed of a superficial squamous cell carcinoma and an adenocarcinoma with an intestinal phenotype. The patient was a 62 year-old male with a T2 N0 M0 squamous cell carcinoma (SCC) of uvula and palate and a T1 N0 M0 of right hypopharynx. The ADSCs of the hypopharynx was composed of a minimally invasive SCC and an adenocarcinoma with tubulo-glandular and cribriform architecture. The neoplastic glands were positive for CK7, CK20, CDX2, CEA and Villin. The patient underwent radiotherapy to both tumors and remains well with no evidence of recurrent disease 19 months after treatment. To the best of our knowledge, this is the first report of an ADSCs of the head and neck with an intestinal phenotype in its glandular component.