RESUMO
Visceral leishmaniasis (VL) or kala-azar, the most severe form of leishmaniasis, can lead to death if not properly diagnosed and treated. Correct identification of infected patients and reservoirs is vital for controlling the spread of leishmaniasis. Current diagnostic kits for leishmaniasis show high sensitivity and specificity, but can also result in false negatives and cross reactions with related parasitic infections. New diagnostic methods with greater accuracy are urgently needed for diagnosis of leishmaniasis. In this study, we aimed to uncover a new highly effective antigen for the diagnosis of visceral leishmaniasis in dogs and humans, aiming to improve the accuracy compared with those of current methods of diagnosis. Initially, in-silico epitope prediction analyses identified several potential B-cell epitopes in the repetitive region of Leishmania infantum kinesin, which co-localized with predicted structural disordered regions, suggesting high potential for antigenicity. Based on this analysis, 8.5 genomic motifs, which encode the repetitive sequence of 39 degenerate amino acids, were selected for recombinant expression. BLASTn analysis of this repetitive region indicated that it is absent in the T. cruzi parasite, which is closely related to Leishmania, indicating the specificity of this region. This potentially antigenic protein, named recombinant kinesin degenerated derived repeat (rKDDR), was recombinantly expressed in Escherichia coli BL21-Star using the pET28a-TEV expression vector. We then evaluated the performance of rKDDR in correctly diagnosing Leishmania infection and compared this new assay with currently used diagnostic tests for leishmaniasis. rKDDR showed greater sensitivity and specificity in correctly diagnosing leishmaniasis both in human (sensitivity 92.86% and specificity 100%) and canine (sensitivity 88.54% and specificity 97.30%) sera compared with those of rK39 (human: sensitivity 90.48% and specificity 97.92%; canine: sensitivity 78.13% and specificity 90.09%). In addition, the rKDDR-ELISA outperformed the EIE-LVC kit, which is the serologic kit recommended by the Brazilian Ministry of Health for the diagnosis of canine visceral leishmaniasis. These results indicate that rKDDR is a highly promising candidate for diagnosis of visceral leishmaniasis, and is more accurate than the currently used gold-standard antigens.
Assuntos
Antígenos de Protozoários/sangue , Doenças do Cão/diagnóstico , Cinesinas/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/diagnóstico , Proteínas Recombinantes/imunologia , Testes Sorológicos/métodos , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Área Sob a Curva , Sequência de Bases , Doenças do Cão/epidemiologia , Doenças do Cão/parasitologia , Cães , Humanos , Leishmania donovani/isolamento & purificação , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/veterinária , Estudos RetrospectivosRESUMO
The serodiagnosis for tegumentary leishmaniasis (TL) presents problems related to the sensitivity and/or specificity of the tests. In the present study, an enzyme-linked immunosorbent assay (ELISA) technique was used to evaluate the performance from a Leishmania braziliensis hypothetical protein, LbHyM, in an attempt to compare its serological reactivity with a soluble Leishmania antigenic preparation (SLA) for the serodiagnosis of cutaneous (CL) and mucosal (ML) leishmaniasis. LbHyM was predicted to be a kinesin-like protein by bioinformatics tools. Serum samples were collected from both CL and ML patients, as well as from those with Chagas disease and from healthy subjects living in endemic or non-endemic areas of TL. Also, sera were collected from patients before and after the treatments, seeking to evaluate their serological follow-up in relation to the anti-protein and anti-parasite antibody levels. When an ELISA-rLbHyM assay was performed, it proved to be significantly more sensitive than ELISA-L. braziliensis SLA in detecting both CL and ML patients. Also, when using sera from Chagas disease patients, the ELISA-rLbHyM proved to be more specific than ELISA-SLA. The anti-protein and anti-parasite antibody levels were also evaluated 6 months after the treatments, and treated patients showed significantly lower levels of specific-rLbHyM antibodies, when compared to the anti-parasite antibody levels. In conclusion, the ELISA-rLbHyM assay can be considered a confirmatory serological technique for the serodiagnosis of L. braziliensis infection and can also be used in the serological follow-up of treated patients, aiming to correlate the low anti-protein antibody levels with the improvement of the healthy state of the patients.
Assuntos
Anticorpos Antiprotozoários/sangue , Doença de Chagas/diagnóstico , Cinesinas/imunologia , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/diagnóstico , Proteínas de Protozoários/imunologia , Adulto , Animais , Anticorpos Antiprotozoários/imunologia , Doença de Chagas/parasitologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Leishmania infantum/imunologia , Leishmaniose Cutânea/parasitologia , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Testes Sorológicos/métodos , Adulto JovemRESUMO
In this study we present evidence about the cellular functions of KIF4. Using subcellular fractionation techniques and immunoisolation, we have now identified a type of vesicle that associates with KIF4, an NH(2)-terminal globular motor domain kinesin-like protein. This vesicle is highly concentrated in growth cones and contains L1, a cell adhesion molecule implicated in axonal elongation. It lacks synaptic vesicle markers, receptors for neurotrophins, and membrane proteins involved in growth cone guidance. In cultured neurons, KIF4 and L1 predominantly localize to the axonal shaft and its growth cone. Suppression of KIF4 with antisense oligonucleotides results in the accumulation of L1 within the cell body and in its complete disappearance from axonal tips. In addition, KIF4 suppression prevents L1-enhanced axonal elongation. Taken collectively, our results suggest an important role for KIF4 during neuronal development, a phenomenon which may be related to the anterograde transport of L1-containing vesicles.
Assuntos
Transporte Axonal , Cinesinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso , Moléculas de Adesão de Célula Nervosa/metabolismo , Vacúolos/química , Vacúolos/metabolismo , Animais , Especificidade de Anticorpos , Axônios/metabolismo , Biomarcadores/análise , Células Cultivadas , Córtex Cerebral/citologia , Imunofluorescência , Cones de Crescimento/metabolismo , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Cinesinas/química , Cinesinas/genética , Cinesinas/imunologia , Complexo Antígeno L1 Leucocitário , Camundongos , Microssomos/química , Microssomos/metabolismo , Peso Molecular , Oligonucleotídeos Antissenso/genética , Células Piramidais/citologia , Células Piramidais/embriologia , Células Piramidais/metabolismo , Ratos , Vesículas Sinápticas/química , Vesículas Sinápticas/metabolismoRESUMO
In the present study, we present evidence about the cellular functions of KIF2, a kinesin-like superfamily member having a unique structure in that its motor domain is localized at the center of the molecule (Noda Y., Y. Sato-Yoshitake, S. Kondo, M. Nangaku, and N. Hirokawa. 1995. J. Cell Biol. 129:157-167.). Using subcellular fractionation techniques, isopicnic sucrose density centrifugation of microsomal fractions from developing rat cerebral cortex, and immunoisolation with KIF2 antibodies, we have now identified a type of nonsynaptic vesicle that associates with KIF2. This type of organelle lacks synaptic vesicle markers (synapsin, synaptophysin), amyloid precursor protein, GAP-43, or N-cadherin. On the other hand, it contains betagc, which is a novel variant of the beta subunit of the IGF-1 receptor, which is highly enriched in growth cone membranes. Both betagc and KIF2 are upregulated by NGF in PC12 cells and highly concentrated in growth cones of developing neurons. We have also analyzed the consequences of KIF2 suppression by antisense oligonucleotide treatment on nerve cell morphogenesis and the distribution of synaptic and nonsynaptic vesicle markers. KIF2 suppression results in a dramatic accumulation of betagc within the cell body and in its complete disappearance from growth cones; no alterations in the distribution of synapsin, synaptophysin, GAP-43, or amyloid percursor protein are detected in KIF2-suppressed neurons. Instead, all of them remained highly enriched at nerve terminals. KIF2 suppression also produces a dramatic inhibition of neurite outgrowth; this phenomenon occurs after betagc has disappeared from growth cones. Taken collectively, our results suggest an important role for KIF2 in neurite extension, a phenomenon that may be related with the anterograde transport of a type of nonsynaptic vesicle that contains as one of its components a growth cone membrane receptor for IGF-1, a growth factor implicated in nerve cell development.