RESUMEN
Leishmaniasis (Kala-azar) from different endemic regions of China expresses different clinic and epidemiological features, and traditionally is classified as hilly, plain and desert types/foci. We concentrated our review on whether the pathogens from those foci were different at molecular level, if so, whether there are were molecular markers readily identifiable by molecular technologies. This was a review of a 20-year search for such markers by using kinetoplastic DNA (kDNA), nDNA hybridization, PCR-SSCP, RAPD and sequence analysis of SSU rDNA variable regions and LACK gene. The results showed that heterogeneities at molecular level exist in Leishmania isolated from different foci of China, which could be used as markers for different types of Leishmaniasis in China.
Asunto(s)
Dermatoglifia del ADN , ADN Protozoario/genética , Leishmania donovani/genética , Leishmaniasis Visceral/parasitología , China , ADN Protozoario/análisis , Genotipo , Humanos , Leishmania donovani/clasificación , Leishmania donovani/aislamiento & purificación , Leishmaniasis Visceral/clasificación , MutaciónRESUMEN
Host macrophage migration inhibitory factor (MIF) has been implicated in the pathogenesis of malaria infections. Several Plasmodium parasite-derived MIFs were identified to have the potential to regulate host immune response. However, the role of Plasmodium MIFs in the immunopathogenesis of malaria infection and the relationships between these mediators and inflammatory cytokines remained unclear. In this study, we have investigated two Plasmodium MIFs in peripheral blood of uncomplicated malaria patients and analyzed their correlations with several major factors during malaria infection. We found that both Plasmodium falciparum MIF (PfMIF) and Plasmodium vivax MIF (PvMIF) levels in patients were positively correlated with parasitemia, tumor necrosis factor alpha, interleukin-10 (IL-10), and monocyte chemoattractant protein 1 but were not correlated with transforming growth factor ß1 and IL-12. Of interest was that the PvMIF level was positively correlated with host body temperature and human MIF (HuMIF) concentrations. Moreover, multiple stepwise regression analysis also showed that parasitemia, IL-10, and HuMIF expression were significant predictors of Plasmodium MIF production. In addition, during antimalarial drug treatment, the decreasing of Plasmodium MIF concentrations was followed by parasitemia in most patients. Our results suggested that the Plasmodium MIF circulating level reflects the level of parasitemia and thus was closely correlated with disease severity in uncomplicated malaria. Therefore, this factor has the potential to be a promising disease predictor and is applicable in clinical diagnosis.
Asunto(s)
Factores Inhibidores de la Migración de Macrófagos/sangre , Malaria Falciparum/inmunología , Malaria Falciparum/patología , Malaria Vivax/inmunología , Malaria Vivax/patología , Parasitemia , Proteínas Protozoarias/sangre , Adolescente , Adulto , Biomarcadores , Niño , Preescolar , Femenino , Humanos , Interleucina-10/biosíntesis , Interleucina-12/sangre , Factores Inhibidores de la Migración de Macrófagos/inmunología , Malaria Falciparum/diagnóstico , Malaria Falciparum/parasitología , Malaria Vivax/diagnóstico , Malaria Vivax/parasitología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Plasmodium falciparum/inmunología , Plasmodium falciparum/patogenicidad , Plasmodium vivax/inmunología , Plasmodium vivax/patogenicidad , Proteínas Protozoarias/inmunología , Análisis de Secuencia de ADN , Índice de Severidad de la Enfermedad , Factor de Crecimiento Transformador beta1/sangre , Factor de Necrosis Tumoral alfa/sangre , Adulto JovenRESUMEN
Host-derived macrophage migration inhibitory factor (MIF) has been implicated in the pathogenesis of malaria infection, especially in malarial anemia. Although two Plasmodium parasite-derived MIF orthologs, Plasmodium falciparum MIF and P. berghei MIF were identified recently, the crystal structure and the precise roles of Plasmodium-derived MIFs, particularly in combination with the host MIF, remain unknown. In this study, we identified another MIF ortholog from a rodent-specific P. yoelii (PyMIF). This molecule shares a conserved three-dimensional structure with murine MIF (MmMIF), but with a different substrate binding pattern and much lower tautomerase activity. It could activate host cells via several signaling pathways in vitro, and inhibiting macrophage apoptosis, also similarly to MmMIF. However, we found that PyMIF and MmMIF acted synergistically to activate the MAPK-ERK1/2 signaling pathway at very low concentration but acted antagonistically at higher concentration. Furthermore, we detected PyMIF in the sera of infected mice and found that injection of recombinant PyMIF (rPyMIF) during infection could up-regulate several pro-inflammatory cytokines in vivo and slightly delay the death of infected mice. These data suggest that PyMIF modulates host immune responses together with host MIF and has potential to prolong parasitemia or the chronicity of malaria infection.
Asunto(s)
Factores Inhibidores de la Migración de Macrófagos/química , Factores Inhibidores de la Migración de Macrófagos/inmunología , Plasmodium yoelii/química , Plasmodium yoelii/inmunología , Homología Estructural de Proteína , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Activación Enzimática/efectos de los fármacos , Interacciones Huésped-Parásitos , Mediadores de Inflamación/metabolismo , Inyecciones Intravenosas , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Activación de Macrófagos/efectos de los fármacos , Factores Inhibidores de la Migración de Macrófagos/sangre , Factores Inhibidores de la Migración de Macrófagos/genética , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Malaria/sangre , Malaria/parasitología , Ratones , Datos de Secuencia Molecular , Plasmodium yoelii/efectos de los fármacos , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Members of tetraspanin family expressed on the tegument of Schistosoma mansoni have been regarded as potential protective antigens. In this work, we were surprised to identify seven tetraspanin-2 (TSP-2) homologs of the protective antigen Sm-TSP-2 in Schistosoma japonicum and found that the transcription profiles of Sj-tsp-2 subclasses were highly variable in individual adult worms. RT-PCR revealed that Sj-tsp-2 genes were transcribed in cercariae, schistosomula, adult worms, and eggs; however, Western blot analysis indicated that the Sj-TSP-2 proteins were not expressed in eggs. Immunolocalization assays showed that the Sj-TSP-2 proteins localized on the tegument of schistosomula and adult worms, but exposed only on the surface of adult worms. Mice immunized with the recombinant protein of a single TSP-2 subclass showed no protection, while immunized with a mixture of seven recombinant TSP-2 subclasses provided a moderate protection. Those data implicate that the tegument protein Sj-TSP-2 is involved in immune evasion and the high polymorphism of this molecule must affect its potential as a vaccine candidate.
Asunto(s)
Proteínas del Helminto/genética , Proteínas del Helminto/inmunología , Schistosoma japonicum/genética , Schistosoma japonicum/inmunología , Esquistosomiasis Japónica/prevención & control , Secuencia de Aminoácidos , Animales , Clonación Molecular , Femenino , Genes de Helminto , Proteínas del Helminto/química , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Polimorfismo Genético , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Schistosoma japonicum/crecimiento & desarrollo , Transcripción Genética , Vacunación , Vacunas/genética , Vacunas/inmunologíaRESUMEN
A polyepitope chimeric antigen incorporating multiple protective and conservative epitopes from multiple antigens of Plasmodium falciparum has been considered to be more effective in inducing multiple layers of immunity against malaria than a single stage- or single antigen-based vaccine. By modifying the molecular breeding approach to epitope shuffling, we have constructed a polyepitope chimeric gene that encodes 11 B-cell and T-cell proliferative epitope peptides derived from eight key antigens mostly in the blood stage of Plasmodium falciparum. A 35-kDa antigen encoded by this gene, named Malaria RCAg-1, was purified from an E. coli expression system. Immunization of rabbits and mice with the purified protein in the presence of Freund's adjuvant strongly generated long-lasting antibody responses that recognized the corresponding individual epitope peptide in this vaccine as well as blood stage parasites. CD4(+) T-cell responses were also elicited as shown by the enhancement of T-cell proliferation, IFN-gamma and IL-4 level. In vitro assay of protection revealed that vaccine-elicited antibodies could efficiently inhibit the growth of blood-stage parasites. Additionally, the chimeric antigen was recognized by human serum specimens from malaria patients and individuals living in the endemic area. Our studies indicate the potential of M.RCAg-1 recombinant protein as malaria candidate vaccines as well as the rationale of the epitope shuffling technology applied in designing malaria vaccines.
Asunto(s)
Antígenos de Protozoos/inmunología , Epítopos/inmunología , Vacunas contra la Malaria/inmunología , Plasmodium falciparum/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/sangre , Formación de Anticuerpos/inmunología , Antígenos de Protozoos/genética , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Proliferación Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Epítopos/genética , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoglobulina G/sangre , Interferón gamma/metabolismo , Interleucina-4/metabolismo , Vacunas contra la Malaria/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Plasmodium falciparum/genética , Plasmodium falciparum/ultraestructura , Conejos , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
OBJECTIVE: To develop multivalent DNA vaccine PV-IL12-Sj23 which co-expresses Sj23 and cytokine IL-12, and investigate its protective efficacy in BALB/c mice against challenge infection. METHODS: On the basis of the reconstructed plasmid PV-IL12-Sj23 and plasmid PV-IL12, blank plasmid PV and plasmid PV-Sj23 only expressing Sj23 were constructed. Fifty BALB/c male mice were divided into five groups, which were immunized intramuscularly with multivalent DNA vaccine PV-IL12-Sj23, plasmid PV-Sj23 expressing Sj23, plasmid PV-IL12 expressing cytokine IL-12, blank plasmid PV and saline, respectively. Each mouse was immunized with 100 microg DNA only once. All the mice were challenged with 40 cercariae at week 4, killed and perfused for collection of worms at week10. The number of recovered worms and eggs in the liver were counted. RESULTS: Blank plasmid PV and plasmid PV-Sj23 expressing Sj23 were successfully constructed. The worm reduction rate in PV-IL12-Sj23 group and PV-Sj23 group was 45.5% and 27.2% (P< 0.05) respectively. The number of eggs in liver tissue was reduced by 58.4% and 33.9% respectively. CONCLUSION: Multivalent DNA vaccine PV-IL12-Sj23 can induce protective immunity against Schistosoma japonicum in BALB/c mice significantly, with a better protective efficacy than the monovalent DNA vaccine PV-Sj23.
Asunto(s)
Antígenos Helmínticos/inmunología , Proteínas del Helminto/inmunología , Interleucina-12/inmunología , Proteínas de la Membrana/inmunología , Schistosoma japonicum/inmunología , Esquistosomiasis Japónica/prevención & control , Vacunas de ADN/inmunología , Animales , Masculino , Ratones , Ratones Endogámicos BALB C , Plásmidos/inmunología , Esquistosomiasis Japónica/inmunologíaRESUMEN
OBJECTIVE: To develop a Schistosoma japonica integral membrane protein Sm23 or Sj23 combined with murine IL-12 DNA-base vaccine against schistosomiasis. METHODS: Plasmids pVIVO2-Sj23 and pVIVO2-IL12-Sj23, expressing the integral membrane protein Sj23 of Schistosoma japonica and/or murine IL-12 were constructed. The plasmid pVIVO2-IL12-Sj23 was transfected into the human embryonic kidney cells of the line 293. RT-PCR was used to detect the expression of Sj23 mRNA in the 293 cells. Indirect immunofluorescence test was used to detect the expression of Sj23 protein. Fifty BALB/c mice were randomly divided into 5 equal groups to be injected with pVIVO2-IL12-Sj23 plasmid DNA, pVIVO2-Sj23 plasmid DNA, pVIVO2-IL12 plasmid DNA, pVIVO2 blank vector, and normal saline respectively into the quadriceps muscle of thigh. Four weeks after each mouse were infested with 40 +/- 2 cercariae of Schistosoma japonica. Six weeks after the infestation the mice were killed to calculate the load of schistosoma and the amount of eggs per gram (EPG) of liver so as to calculate the worm reduction rate and egg reduction rate after the vaccination. Before immunization, 4 weeks after immunization, and 6 weeks after immunization blood samples were collected from the caudal veins of mice. With soluble egg antigen (SEA) and adult worm antigen (AWA) ELISA was used to detect the serum IgG level. Western blotting was used to detect the serum specific anti-Sj23 IgG level. Six weeks after the cercaria challenge single splenocyte suspension was prepared. Splenocytes were cultured with SEA, and concanavalin A (ConA). ELISA was used to detect the levels of IL-4 and IFN-gamma in the supernatant. Flow cytometry was used to analyze the subgroups of splenocyte. RESULTS: + Forty-eight hours after the transfection, RT-PCR and indirect immunofluorescence test showed expression of Sj23 mRNA and protein in the HEK-293 cells. The worm reduction rate was 45.53% and the egg reduction was 58.35% in the pVIVO2-IL12-Sj23 group, significantly higher than those in the monovalent vaccine pVIVO2-Sj23 group (27.23% and 33.93% respectively, both P <0.05). ELISA and Western blotting analysis showed that the level of IgG specific for Sj23 significantly increased 4 weeks after vaccination in the pVIVO2-IL12-Sj23 and pVIVO2-Sj23 groups without significant difference between these 2 groups (P > 0.05). After stimulation of ConA and SEA the level of Th1 type cell factor IFN-gamma was higher and the level of the Th2 type cellular factor IL-4 was low in the supernatant of suspension of splenocytes of the pVIVO2-IL12-Sj23 group. FCM showed the percentages of CD4+ and CD8+ subgroups of the murine splenocytes of all experimental groups were all significantly lower than those of the normal mice (all P < 0.001 approximately 0.02). However, there was no significant difference in the CD4+/CD8+ ratio among the experimental groups (all CONCLUSION: pVIVO2-IL12-Sj23 is sufficient to elicit significant levels of protective immunity against Schistosoma japonica challenge infection. IL-12, a cytokine and a gene adjuvant, is able to induce Th1 responses and hence the protective immunity.
Asunto(s)
Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/inmunología , Proteínas del Helminto/inmunología , Interleucina-12/genética , Proteínas de la Membrana/inmunología , Schistosoma japonicum/inmunología , Vacunas de ADN/inmunología , Animales , Antígenos Helmínticos/genética , Relación CD4-CD8 , Interleucina-12/inmunología , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Células TH1/inmunología , VacunaciónRESUMEN
BACKGROUND: The vaccination of mice with DNA encoding single candidate antigens has failed to induce significant protection against Schistosoma japonicum (S. japonicum) challenge infections. In this study, we evaluated the feasibility of using a multivalent DNA vaccine which co-expressed S. japonicum integral membrane protein Sj23 and murine cytokine IL-12 to induce protective immune responses. METHODS: The plasmid pVIVO2-IL12-Sj23, a eukaryotic expression vector expressing Sj23 and murine IL-12 simultaneously, was constructed, identified, and tested for expression in vitro. Its ability to protect against S. japonicum challenge infections was analyzed according to worm reduction rate and egg reduction rate after vaccination of BALB/c mice. The serum levels of specific IgG antibody were determined by enzyme-linked-immuno sorbent assay (ELISA) and Western blot analysis. Using cultured spleen cells, IFN-gamma and IL-4 post-stimulation were quantified by ELISA. The phenotypes of splenocyte populations were analyzed by flow cytometry (FCM). RESULTS: The plasmid DNA pVIVO2-IL12-Sj23 was proven to express well in vitro by transient transfection of HEK-293 cells. Immunization resulted in a worm reduction rate of 45.53% and egg reduction rate of 58.35%. ELISA and Western blot analysis indicated that immunized mice generated specific IgG against Sj23. Spleen cells showed significant increases in IFN-gamma but decreases in IL-4. No significant differences in CD4+ and CD8+ subgroup ratios were observed after the challenges. CONCLUSIONS: The multivalent DNA vaccine pVIVO2-IL12-Sj23 is sufficient to elicit moderate but highly significant levels of protective immunity against challenge infections. Cytokine IL-12, as a gene adjuvant, was able to enhance the Th1 responses and, hence, the protective immunity.
Asunto(s)
Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/genética , Proteínas del Helminto/genética , Interleucina-12/genética , Proteínas de la Membrana/genética , Schistosoma japonicum/inmunología , Vacunas de ADN/inmunología , Animales , Antígenos Helmínticos/inmunología , Relación CD4-CD8 , Citocinas/biosíntesis , Proteínas del Helminto/inmunología , Interleucina-12/inmunología , Masculino , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Células TH1/inmunología , VacunaciónRESUMEN
The LACK gene from Leishmania, an analogue of the receptor of activated protein kinase C, was discovered recently. In this study, the LACK gene of Leishmania donovani was obtained from the recombinant plasmid T-LACK by PCR. The gene was cloned into eukaryotic expressed plasmid pcDNA3.1(+) to construct recombinant plasmid. This recombinant plasmid then was transfected into the eukaryotic cell COS-7, and the expression of LACK gene in eukaryotic cell was detected by RT-PCR and immunofluorescent staining. Both RT-PCR and immunofluorescent staining of recombinant plasmid transfected COS-7 showed positive reaction, thus indicating that the recombinant plasmid pcDNA3-LACK can express LACK protein in euka ryotic cell COS-7.
Asunto(s)
Antígenos de Protozoos/genética , ADN Recombinante/genética , Plásmidos/genética , Proteínas Protozoarias/genética , Animales , Antígenos de Protozoos/biosíntesis , Antígenos de Protozoos/inmunología , Células COS , Clonación Molecular , ADN Recombinante/biosíntesis , Células Eucariotas/metabolismo , Vectores Genéticos , Leishmania donovani , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/inmunología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Vacunas de ADNRESUMEN
OBJECTIVE: To confirm the existence of point mutations in the SSU rDNA variable regions of 5 Leishmania donovani (L.d.) isolates from different epidemic foci in China. METHODS: Specific SSU rDNA fragments from nuclear DNA of 7 Leishmania species/isolates were amplified by PCR and then cloned into pGEM(R)-T Easy Vectors. After that, the specific fragments were sequenced by an automated DNA sequencer. RESULTS: Sequence analysis showed that the amplified DNA fragments of 7 Leishmania species/isolates were all 392 bp in length. All 5 point mutations were located in two unique sequence blocks (UQ-I and UQ-II), and no insertions or deletions were found. The identities of comparison of Leishmania in GeneBank were more than 98%. CONCLUSION: Five point mutations exist in the SSU rDNA variable region of 5 L.d. isolates from different epidemic foci of visceral leishmaniasis (VL) in China. Sequence differences of the SSU rDNA variable region exist among L.d. isolates from different foci.