Identification of Leishmania infantum chagasi proteins in urine of patients with visceral leishmaniasis: a promising antigen discovery approach of vaccine candidates.

Visceral leishmaniasis (VL) is a serious lethal parasitic disease caused by Leishmania donovani in Asia and by Leishmania infantum chagasi in southern Europe and South America. VL is endemic in 47 countries with an annual incidence estimated to be 500,000 cases. This high incidence is due in part to the lack of an efficacious vaccine. Here, we introduce an innovative approach to directly identify parasite vaccine candidate antigens that are abundantly produced in vivo in humans with VL. We combined RP-HPLC and mass spectrometry and categorized three L. infantum chagasi proteins, presumably produced in spleen, liver and bone marrow lesions and excreted in the patients' urine. Specifically, these proteins were the following: Li-isd1 (XP_001467866.1), Li-txn1 (XP_001466642.1) and Li-ntf2 (XP_001463738.1). Initial vaccine validation studies were performed with the rLi-ntf2 protein produced in Escherichia coli mixed with the adjuvant BpMPLA-SE. This formulation stimulated potent Th1 response in BALB/c mice. Compared to control animals, mice immunized with Li-ntf2+ BpMPLA-SE had a marked parasite burden reduction in spleens at 40 days post-challenge with virulent L. infantum chagasi. These results strongly support the proposed antigen discovery strategy of vaccine candidates to VL and opens novel possibilities for vaccine development to other serious infectious diseases.

Improved method to calculate the antibody avidity index.

The methods currently used to determine the immunoglobulin avidity index (AI) require the choice of a reference point in the ELISA titration curve. Since both curves, with and without denaturating reagents, seldom run in parallel, the AI value becomes highly dependent on this reference. The new method for AI calculation presented here takes into account the whole data of the ELISA titration curve in which the final numerical AI is the average of each point.

Pulmonary surfactant protein A isolation as a by-product of porcine pulmonary surfactant production

A pulmonary surfactant reduces surface tension at the air/liquid interface of the alveoli and stabilizes alveoli at low lung volumes. Surfactant deficiency and dysfunction were shown to be present in a number of pulmonary diseases, and surfactant replacement therapy is the common clinical conduct. The hydrophilic SP-A (surfactant protein A) is absent when solvent extraction was used during exogenous surfactant production. Addition of SP-A to the surfactant preparation increases the surface activity and completely counteracts inhibition by blood proteins. SP-A recognizes and binds to carbohydrate structures on the surfaces of pathogenic micro-organisms, and acts as opsonins or cross-linking molecules by binding to a variety of cells that participate in the pulmonary immune response. The purification procedure yielded 206 mg of high-purity SP-A/kg of porcine lung, as judged by gel filtration, SDS/PAGE and Western blotting. The electrophoretic profiles obtained showed that pure SP-A consists of proteins of wide molecular mass in the range 26-36 kDa and a dimer in the range 56-60 kDa. The Western-blot results displayed the same band pattern profile after incubating the membrane using a commercially available polyclonal anti-SP-A antibody produced in goat. Gel-filtration experiments confirmed the molecular mass of SP-A in 10 mM NaCl solution. The isolated SP-A showed mannose-binding ability, representative of its functionality.

Multiserotype protection of mice against pneumococcal colonization of the nasopharynx and middle ear by killed nonencapsulated cells given intranasally with a nontoxic adjuvant

Intranasal challenge of C57BL/6 mice with Streptococcus pneumoniae serotypes 6B, 14, and 23F produced colonization of the middle ear and NP. Intranasal vaccination with ethanol-killed nonencapsulated cells with adjuvant protected both sites. Of four nontoxic adjuvants tested, the cholera toxin B subunit was most effective and least nonspecifically protective

Ultrastructural chemical reaction to detect saturated phospholipids of a natural surfactant from pig lungs

The ultrastructure of the natural surfactant from pig lungs fixed by the tricomplex method with Pb(NO 3) 2 and K 3Fe(CN) 6 reveals its lamellar phase (ordered configuration). The lamellar forms are usually associated with being surface active

[Susceptibility of Vero cell line to vaccine strains of the measles virus]. / Suscetibilidade da linhagem de células Vero a cepas vacinais do vírus do sarampo.

Vero cells used by distinct measles vaccine control laboratories had their susceptibility to Moraten, Schwarz and Biken CAM-70 vaccine strains assayed. Of a total of 72 lots of measles vaccine whose potency was titrated by microtechnique in two Vero cell samples (Vero IB and Vero INCQS), 25 had been produced with Moraten strain, 24 with Schwarz and 23 with Biken CAM-70. The statistical analysis of the results demonstrated that both Vero cells assayed presented comparable susceptibility to Moraten and Biken CAM-70 strains. As to the Schwarz strain, Vero IB cells were more susceptible than the other cell sample tested, thus confirming the existence of different sensitivities of Vero cells to some measles vaccine strains, or even to viruses derived from the same strain but with different passage histories. An altered cell susceptibility to virus replication may significantly alter the results in potency testing. Such alteration may be caused not only by the adoption of distinct protocols for the maintenance of cell cultures by different control laboratories but also by the methodology followed in the vaccine titration. In order to minimize the differences existing among the results obtained in the potency testing, it is suggested that all control laboratories should use the same protocols for cell culture maintenance as well as for vaccine potency testing.

Caracterizacao de duas culturas celulares da linhagem BHK-21 de procedencias diferentes pela morfologia, cariotipo, crescimento populacional e suscetibilidade ao virus da febre aftosa. / Characterization of two different cultures of BHK-21 cell lines by morphology karyotype, populational rate and susceptibility to foot-and-mouth disease virus

Foram estudados a morfologia, o cariotipo, o crescimento populacional e a suscetibilidade de duas culturas celulares da linhagem BHK-21 obtidas de dois laboratorios e mantidas sob as mesmas condicoes laboratoriais. Uma das culturas celulares, a que apresentava celulas maiores do que a outra, foi mais suscetivel ao virus da febre aftosa, com relacao ao numero e tamanho de placas e efeito citopatico.O crescimento populacional foi distinto para ambas. Nas populacoes celulares das duas culturas estudadas, foram observadas variacoes qualitativas e quantitativas nos cromossomos