Establishment of certified reference material for the potency assay in yellow fever vaccine quality control, in accordance with International Standards Organization guidance.

The attenuated yellow fever vaccine (YFV) is offered free of charge to the Brazilian population through the National Immunization Program (NIP). One of the specifications for quality control analyses of the vaccine is the potency determination. This test determines the number of plaque forming units (PFU) in Vero cells. In order to validate the results, the reference material (RM) is analysed in parallel with an established reference vaccine. The aim of this study was to establish certified RM for use as an internal control in the potency assay for the production chain of YFV. The candidate RM homogeneity and stability were determined, and characterized by a collaborative study for further certification. The RM was considered sufficiently homogeneous with average 4.68 log10 IU/HD and stable at (-20 ± 10) ºC and (22.5 ± 2.5) ºC for 715 and 183 days, respectively. When reconstituted and stored in aliquots of 0.6 mL, it was stable at (-20 ± 10) ºC for eight days. But it was not stable at (5 ± 3) ºC for three days. In a collaborative study, two independents' laboratories gave an averaged value of 4.56 ± 0.030 log10 IU/HD. After determining the expanded uncertainty of homogeneity, stability, and characterization, the certified RM lot: 195VFA020Z presented a property value of 4.56 ± 0.22 log10 IU/HD. It was concluded that the new certified RM can be used in routine analysis of a YFV producer, since it has its property value established and it is stable. The possibility of using it in aliquots after reconstitution will also allow the RM to have a much longer shelf life.

Peripheral blood fibrocytes: new information to explain the dynamics of Leishmania infection.

Fibrocytes are important for understanding the progression of many diseases because they are present in areas where pathogenic lesions are generated. However, the morphology of fibrocytes and their interactions with parasites are poorly understood. In this study, we examined the morphology of peripheral blood fibrocytes and their interactions with Leishmania (L.) amazonensis . Through ultrastructural analysis, we describe the details of fibrocyte morphology and how fibrocytes rapidly internalise Leishmania promastigotes. The parasites differentiated into amastigotes after 2 h in phagolysosomes and the infection was completely resolved after 72 h. Early in the infection, we found increased nitric oxide production and large lysosomes with electron-dense material. These factors may regulate the proliferation and death of the parasites. Because fibrocytes are present at the infection site and are directly involved in developing cutaneous leishmaniasis, they are targets for effective, non-toxic cell-based therapies that control and treat leishmaniasis.

Peripheral blood fibrocytes: new information to explain the dynamics of Leishmania infection

Fibrocytes are important for understanding the progression of many diseases because they are present in areas where pathogenic lesions are generated. However, the morphology of fibrocytes and their interactions with parasites are poorly understood. In this study, we examined the morphology of peripheral blood fibrocytes and their interactions with Leishmania (L.) amazonensis . Through ultrastructural analysis, we describe the details of fibrocyte morphology and how fibrocytes rapidly internalise Leishmania promastigotes. The parasites differentiated into amastigotes after 2 h in phagolysosomes and the infection was completely resolved after 72 h. Early in the infection, we found increased nitric oxide production and large lysosomes with electron-dense material. These factors may regulate the proliferation and death of the parasites. Because fibrocytes are present at the infection site and are directly involved in developing cutaneous leishmaniasis, they are targets for effective, non-toxic cell-based therapies that control and treat leishmaniasis.

Leishmania (Viannia) braziliensis nucleoside triphosphate diphosphohydrolase (NTPDase 1): localization and in vitro inhibition of promastigotes growth by polyclonal antibodies.

Nucleoside triphosphate diphosphohydrolase (NTPDase) activity was recently characterized in Leishmania (Viannia) braziliensis promastigotes (Lb), and an antigenic conserved domain (r82-121) from the specific NTPDase 1 isoform was identified. In this work, mouse polyclonal antibodies produced against two synthetic peptides derived from this domain (LbB1LJ, r82-103; LbB2LJ, r102-121) were used. The anti-LbB1LJ or anti-LbB2LJ antibodies were immobilized on protein A-sepharose and immunoprecipitated the NTPDase 1 of 48 kDa and depleted approximately 40% of the phosphohydrolytic activity from detergent-homogenized Lb preparation. Ultrastructural immunocytochemical microscopy identified the NTPDase 1 on the parasite surface and in its subcellular cytoplasmic vesicles, mitochondria, kinetoplast and nucleus. The ATPase and ADPase activities of detergent-homogenized Lb preparation were partially inhibited by anti-LbB1LJ antibody (43-79%), which was more effective than that inhibition (18-47%) by anti-LbB2LJ antibody. In addition, the immune serum anti-LbB1LJ (67%) or anti-LbB2LJ (33%) was cytotoxic, significantly reducing the promastigotes growth in vitro. The results appoint the conserved domain from the L. braziliensis NTPDase as an important target for inhibitor design and the potential application of these biomolecules in experimental protocols of disease control.