Clara Spinel

Schwann cell-enriched cultures from adult human peripheral nerve: a technique combining short enzymatic dissociation and treatment with cytosine arabinoside (Ara-C)

Attempts to design the nerve cellular prostheses have focused on the production of autologous Schwann cells expanded in vitro as the essential component in the regeneration process of injured peripheral nerves. To obtain human Schwann cells of high quality we tested a short enzymatic dissociation protocol that optimized cellular viability levels. We also assessed patterns of bromodeoxyuridine (BrdU) incorporation in both Schwann cells and fibroblasts in the presence or absence of the antimitotic Ara-C, an enrichment option for adult human Schwann cell cultures. The Ara-C treated cultures showed a significantly higher Schwann cell percentage (95%), compared with that obtained in the absence of Ara-C (70%), indicating that this antimitotic acts to eliminate fibroblasts in each one of the applied pulses (four pulses). However, we have observed that the use of this antimitotic during prolonged periods of time produced a cumulative effect causing Schwann cell cytotoxicity. Therefore, we consider that our enzymatic dissociation technique and the application of only two pulses of Ara-C to the cultures are enough to achieve enrichment of adult human Schwann cells in culture.

Characterizing cellular immune response to kinetoplastid membrane protein‐11 (KMP‐11) during Leishmania (Viannia) panamensis infection using dendritic cells (DCs) as antigen presenting cells (APCs)

In vitro peptide binding assays and DCs pulsed with recombinant KMP‐11 (rKMP‐11) plus six 20‐mer overlapping peptides covering the entire protein of Leishmania (Viannia) panamensis (L(V)p) promastigotes were used to identify T‐cell epitopes in this protein. Such in vitro binding assays, using HLA DRB1* 0101, ‐0401, ‐0701 and ‐1101 alleles, demonstrated that two peptide sequences ( DEEFNKKMQEQNAKFFADKP and FKHKFAELLEQQKAAQYPSK) exhibited high HLA DRB1* 0401 allele binding capacity. rKMP‐11 specific T‐cell proliferation and cytokine production, derived from 13 volunteers exposed to the parasite, suggested that using autologous DCs as APCs becomes advantageous in uncovering T‐cell epitopes promoting proliferation and differences in IFN‐γ and IL‐4 production in T‐cells from volunteers with ACTIVE and CURED undetectable disease when other APCs were used. The two peptides which bound in vitro to the HLA DRB1* 0401 allele were immunogenic in HLA DRB1* 04 volunteers, thus validating the use of in vitro binding assays for predicting epitopes in this protein. The experimental approach used here may prove useful for characterizing T‐cell epitopes in a protein useful in designing peptide‐based vaccine candidates for Leishmania and other intracellular pathogens.