Improved Canine and Human Visceral Leishmaniasis Immunodiagnosis Using Combinations of Synthetic Peptides in Enzyme-Linked Immunosorbent Assay

Visceral leishmaniasis is endemic in many areas of tropical and subtropical America where it constitutes a significant public health problem. It is usually diagnosed by enzyme-linked immunosorbent assays (ELISA) using crude Leishmania antigens, but a variety of other immunological methods may also be applied. Although these approaches are useful, historically their sensitivity and specificity have often been compromised by the use of complex mixtures of antigens. In this context, the use of combinations of purified, well-characterized antigens appears preferable and may yield better results. In the present study, combinations of peptides derived from the previously described Leishmania diagnostic antigens A2, NH, LACK and K39 were used in ELISA against sera from 106 dogs and 44 human patients. Improved sensitivities and specificities, close to 100%, for both sera of patients and dogs was observed for ELISA using some combinations of the peptides, including the detection of VL in dogs with low anti-Leishmania antibody titers and asymptomatic infection. So, the use of combinations of B cell predicted synthetic peptides derived from antigens A2, NH, LACK and K39 may provide an alternative for improved sensitivities and specificities for immunodiagnostic assays of VL

Bioavailability of Orally Administered rhGM-CSF: A Single-Dose, Randomized, Open-Label, Two-Period Crossover Trial

BACKGROUND: Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is usually administered by injection, and its oral administration in a clinical setting has been not yet reported. Here we demonstrate the bioavailability of orally administered rhGM-CSF in healthy volunteers. The rhGM-CSF was expressed in Bombyx mori expression system (BmrhGM-CSF). METHODS AND FINDINGS: Using a single-dose, randomized, open-label, two-period crossover clinical trial design, 19 healthy volunteers were orally administered with BmrhGM-CSF (8 microg/kg) and subcutaneously injected with rhGM-CSF (3.75 microg/kg) respectively. Serum samples were drawn at 0.0h, 0.5h ,0.75h,1.0h,1.5h,2.0h ,3.0h,4.0h,5.0h,6.0h,8.0h,10.0h and 12.0h after administrations. The hGM-CSF serum concentrations were determined by ELISA. The AUC was calculated using the trapezoid method. The relative bioavailability of BmrhGM-CSF was determined according to the AUC ratio of both orally administered and subcutaneously injected rhGM-CSF. Three volunteers were randomly selected from 15 orally administrated subjects with ELISA detectable values. Their serum samples at the 0.0h, 1.0h, 2.0h, 3.0h and 4.0h after the administrations were analyzed by Q-Trap MS/MS TOF. The different peaks were revealed by the spectrogram profile comparison of the 1.0h, 2.0h, 3.0h and 4.0h samples with that of the 0.0h sample, and further analyzed using both Enhanced Product Ion (EPI) scanning and Peptide Mass Fingerprinting Analysis. The rhGM-CSF was detected in the serum samples from 15 of 19 volunteers administrated with BmrhGM-CSF. Its bioavailability was observed at an average of 1.0%, with the highest of 3.1%. The rhGM-CSF peptide sequences in the serum samples were detected by MS analysis, and their sizes ranging from 2,039 to 7,336 Da. CONCLUSIONS: The results demonstrated that the oral administered BmrhGM-CSF was absorbed into the blood. This study provides an approach for an oral administration of rhGM-CSF protein in clinical settings. TRIAL REGISTRATION: www.chictr.orgChiCTR-TRC-00000107