RESUMO
Cancer is a general name for more than 100 malignant diseases. It is postulated that all cancers start from a single abnormal cell that grows out of control. Untreated cancers can cause serious consequences and deaths. Great progress has been made in cancer research that has significantly improved our knowledge and understanding of the nature and mechanisms of the disease, but the origins of cancer are far from being well understood due to the limitations of suitable model systems and to the complexities of the disease. In view of the fact that cancers are found in various species of vertebrates and other metazoa, here, we suggest that cancer also occurs in parasitic protozoans such as Trypanosoma brucei, a blood parasite, and Toxoplasma gondii, an obligate intracellular pathogen. Without treatment, these protozoan cancers may cause severe disease and death in mammals, including humans. The simpler genomes of these single-cell organisms, in combination with their complex life cycles and fascinating life cycle differentiation processes, may help us to better understand the origins of cancers and, in particular, leukemias.
Assuntos
Neoplasias/patologia , Parasitos/fisiologia , Toxoplasma/fisiologia , Trypanosoma brucei brucei/fisiologia , Animais , Proliferação de Células , Humanos , Estágios do Ciclo de Vida , Modelos Biológicos , Mutação/genética , Metástase Neoplásica , Neoplasias/genética , Toxoplasma/genética , Toxoplasma/crescimento & desenvolvimento , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
Mouse models differ considerably from humans with regard to clinical symptoms of toxoplasmosis caused by Toxoplasma gondii and, by comparison, the rat model is more representative of this disease in humans. In the present study, we found that different strains of adult and newborn rats (Lewis, Wistar, Sprague Dawley, Brown Norway and Fischer 344) exhibited remarkable variation in the number of brain cysts following inoculation with the T.gondii Prugniaud strain. In adult rats, large numbers of cysts (1231 ± 165.6) were observed in Fischer 344, but none in the other four. This situation was different in newborn rats aged from 5 to 20 days old. All Fischer 344 and Brown Norway newborns were cyst-positive while cyst-positive infection in Sprague Dawley neonates ranged from 54.5% to 60% depending on their age at infection. In Wistar and Lewis rat neonates, however, cyst-positivity rates of 0-42.9% and 0-25% were found respectively. To investigate whether rat strain differences in infectivity could be related to inherent strain and genetic differences in the host immune response, we correlated our data with previously reported strain differences in iNOS/Arginase ratio in adult rats and found them to be linked. These results show that interactions between host genetic background and age of rat influence T.gondii infection.
Assuntos
Arginase/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose Animal/genética , Toxoplasmose Animal/metabolismo , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Encéfalo/parasitologia , Distribuição de Qui-Quadrado , Modelos Animais de Doenças , Resistência à Doença/genética , Suscetibilidade a Doenças , Feminino , Masculino , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Ratos Wistar , Especificidade da Espécie , Toxoplasma/patogenicidade , Toxoplasmose Animal/enzimologia , Toxoplasmose Cerebral/genética , Toxoplasmose Cerebral/parasitologiaRESUMO
Toxoplasma gondii is an intracellular Apicomplexan parasite that infects a wide range of warm blooded animals, including human, and has complex life cycle and pathogenic mechanisms. Although T. gondii is the only species recognized in the Toxoplasma genus, research on population genetic structure has shown its geographic genetic diversity. So far 232 genotypes have been identified by multilocus polymerase chain reaction-restriction fragment length polymorphism or microsatellite genotyping from both animals and human. T. gondii strains in North America typically possess types 2, 3 and 12 (found mainly in wild animals) clonal lineages, while types 2, 3, and 1 are common in Europe, and types 2 and 3 are common in Africa. These findings suggest a strongly clonal population structure in these regions. However, strains in South America are genetically more diverse, predominated by types Br I , Br II, Br III, and Br IV. Recent research has shown that the Chinese 1 (ToxoDB#9) genotype is dominantly circulating in the mainland of China, and shares the polymorphic ROP16I/III with types 1 and 3, and GRA15II with type 2. In this review, we summarized geographically the genotypes, host immune responses, and the pathogenic mechanisms of T. gondii strains, to provide basis for further research on genotype/effector-related pathogenic mechanism as well as biological and epidemiological studies of T. gondii.
Assuntos
Toxoplasma , China , Genética Populacional , GenótipoRESUMO
It is well known that toxoplasmosis can be life threatening to immunocompromised individuals such as AIDS and organ transplantation patients. Glucocorticoids (GCs) are widely used in the clinic for the treatment of autoimmune diseases and organ transplantation resulting in acute toxoplasmosis in these patients. However, the interaction and mechanism between the development of acute toxoplasmosis and GC therapy are still unknown. The aims of this study were to investigate the infection of Toxoplasma gondii in the peritoneal macrophages of rats treated with glucocorticoids. Our results showed that the growth rate of T. gondii RH strain was significantly increased in the peritoneal macrophages of rats treated with glucocorticoids in vivo. For instance, 242 (±16) tachyzoites were found in 100 macrophages from the rats treated with methylprednisolone (MP), while only 16 (±4) tachyzoites were counted in the macrophages from the non-treated control rats 24 h after infection (P < 0.01). We also demonstrated that a significant inhibition of nitric oxide (NO) production was detected in the macrophages collected from the rats post-treated with GCs with 12.90 µM (±0.99 µM) of nitrite production from the rats treated with MP, while 30.85 µM (±1.62 µM) was found in the non-treated control rats 36 h after incubation (P < 0.01). Furthermore, glucocorticoids could significantly inhibit the expression of inducible nitric oxide synthase mRNA and its protein in the rat peritoneal macrophages. Our results strongly indicate that the decrease of NO in the rat peritoneal macrophages is closely linked to the cause of acute toxoplasmosis in the host. Additionally, there was a significant increase in the number of cysts produced by the naturally cyst forming, T. gondii Prugniaud strain with an average of 2,795 (±422) cysts of the parasite being detected in the brains of the rats treated with dexamethasone, while only 1,356 (±490) cysts were found in the non-treated control animals (P < 0.01). As rats and humans are both naturally resistant to T. gondii infection, these novel data could lead to a better understanding of the development of acute toxoplasmosis during glucocorticoid therapy in humans.
Assuntos
Glucocorticoides/farmacologia , Macrófagos Peritoneais/parasitologia , Toxoplasma/crescimento & desenvolvimento , Animais , Encéfalo/parasitologia , Células Cultivadas , Dexametasona/farmacologia , Hidrocortisona/farmacologia , Masculino , Metilprednisolona/farmacologia , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Toxoplasmose Animal/imunologiaRESUMO
OBJECTIVE: To investigate the seroprevalence of Toxoplasma gondii infection and identify the genotypes of T. gondii isolates from cancer patients in Anhui Province. METHODS: Three hundred and fifty-six blood samples were collected from outpatients and hospitalized cancer patients in Hefei, Anhui Province. IgG and gM antibodies specific to T gondii were determined by ELISA. The ELISA positive samples were subjected to detection of Toxoplasma DNA with PCR targeting a 529-bp repeat element of T. gondii. Genotyping of T. gondii isolates was performed using multilocus PCR-RFLP and 10 genetic markers, including 9 nuclear loci, sagl, sag2, sag3, btub, gra6, L358, pkl, c22-8, c29-2, and apico. RESULTS: Among 356 cancer patients, 21 (5.9%) cases were found to be IgG-positive and 8 (2.3%) were IgM-positive, and five of them were found to have both IgG and IgM antibodies. The total seroprevalence of Toxoplasma infection was 6.8%. Six PCR-positive samples were genotyped at 10 loci and two of them obtained all genetic markers and identified as the genotype of Chinese 1 (ToxoDB#9). CONCLUSION: In this study, latent and active toxoplasmosis exist in the patients with malignant tumors, and two isolates are genotyped as the type of Chinese 1 (ToxoDB#9) in Anhui, China.
Assuntos
Neoplasias/complicações , Toxoplasma/genética , Toxoplasmose/epidemiologia , Animais , China , Ensaio de Imunoadsorção Enzimática , Marcadores Genéticos , Genótipo , Humanos , Reação em Cadeia da Polimerase , Estudos Soroepidemiológicos , Toxoplasmose/sangue , Toxoplasmose/complicaçõesRESUMO
As one of food-borne parasitic diseases, toxoplasmosis entails the risk of developing reactivation in immunocompromised patients. The synthetic dipeptide pidotimod is a potent immunostimulating agent that improves the immunodefenses in immunodepression. To investigate the efficacy of pidotimod as a preventive treatment, we used a murine model of reactivated toxoplasmosis with cyclophosphamide (CY)-induced immunosuppression. Pidotimod administration significantly restored the body weight and spleen organ index, increased survival time (from 70 to 90%), and decreased the parasitemia (from 80 to 35%) of CY-induced mice with reactivated toxoplasmosis. Cytokine profiles and CD4(+) T cells subpopulation analyses by Cytometric Bead Array and flow cytometry demonstrated that pidotimod treatment resulted in a significant upregulation of pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-2) and Th1 cells (from 3.73 ± 0.39 to 5.88 ± 0.46%) after CY induction in infected mice. Additionally, histological findings and parasite DNA quantification revealed that mice administered with pidotimod had a remarkable reduction of parasite burden (two-log) and amelioration of histopathology in the brains. The in vitro studies showed that pidotimod significantly restored concanavalin A-induced splenocyte proliferation and pro-inflammatory cytokines in the supernatants of splenocyte culture. It could be concluded that the administration of pidotimod in immunocompromised mice significantly increases the Th1-biased immune response, prolongs survival time, and ameliorates the load of parasites in the blood. This is the first report of the preventive effect of pidotimod on reactivated toxoplasmosis.
Assuntos
Fatores Imunológicos/uso terapêutico , Ácido Pirrolidonocarboxílico/análogos & derivados , Tiazolidinas/uso terapêutico , Toxoplasmose Animal/prevenção & controle , Animais , Ciclofosfamida/farmacologia , Citocinas/genética , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Imunossupressores/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Parasitemia , Ácido Pirrolidonocarboxílico/uso terapêutico , Organismos Livres de Patógenos Específicos , Baço/citologia , Baço/efeitos dos fármacos , Toxoplasmose Animal/imunologiaRESUMO
Toxoplasmosis is a disease caused by the protozoan Toxoplasma gondii, which is widely prevalent in animals and human throughout the world. It causes serious harm to human health and the development of animal husbandry. T. gondii isolates were considered a single species without geographical boundaries. However, high diversity has been revealed within and between T. gondii populations collected from around the world defined by the multi-locus enzyme electrophoresis (MLEE), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or microsatellite analysis. Different strains of T. gondii may exhibit differences in virulence to mice. This paper summarizes the research progress on the genotypes from T. gondii isolates in different geographic regions around the world, and the relationship between genotype and virulence of T. gondii.
Assuntos
Toxoplasma/genética , Toxoplasma/patogenicidade , Toxoplasmose/parasitologia , DNA de Protozoário/genética , Genótipo , Geografia , VirulênciaRESUMO
OBJECTIVE: To screen the host cell proteins that can interact with Toxoplasma gondii ROP18 by using yeast two-hybrid system. METHODS: The ROP18 gene fragments were amplified by RT-PCR from mRNA of T. gondii RH strain. The product of RT-PCR was digested with double restriction enzyme and was subcloned into the bait vector pGBKT7. The recombinant plasmid was transferred into yeast AH109 strain. Its toxicity and the autonomous activating activity were tested. The human fetal brain cDNA library was screened with pGBKT7-ROP18(25-251aa) as the bait plasmid by yeast two-hybrid system. RESULTS: The bait was constructed and AH109/PGBKT7-ROP18 showed an autonomous activity. The yeast strain AH109/pGBKT7-ROP18(25-251aa) line was then mated with the Mate & PlateTM Human Fetal Brain cDNA library. Using the selection procedures, eight novel host cell proteins were obtained: damage-specific DNA binding protein 1 (DDB1), torsin A interacting protein 1 (TOR1AIP1), integrin beta 1, solute carrier family 3 (SLC3A2), tyrosyl protein sulfotransferase (TPST2), OCIA domain containing 1 (OCIAD1), Derl-like domain family member 2 (DERL2), in addition to Homo sapiens activating transcription factor 6 beta(ATF6). CONCLUSION: Eight novel host cell proteins have been obtained via yeast two-hybrid system, which can interact with TgROP18.
Assuntos
Proteínas de Ligação a DNA/isolamento & purificação , Interações Hospedeiro-Parasita , Proteínas Serina-Treonina Quinases/metabolismo , Toxoplasma , Técnicas do Sistema de Duplo-Híbrido , Biblioteca Gênica , Vetores Genéticos , Humanos , Cadeias beta de Integrinas/isolamento & purificação , Chaperonas Moleculares/isolamento & purificação , Plasmídeos , Proteínas de Protozoários , Toxoplasma/genéticaRESUMO
OBJECTIVE: To study the inhibition effect of pidotimod (PT) on dexamethasone (Dem)-induced reactivated toxoplasmosis in mice. METHODS: A total of 96 female BALB/C mice were infected orally with 30 cysts of Toxoplasma gondii TgCtwh6 strain (genotype Chinese 1). 4 weeks later the mice were divided into three groups (A, B, and C). Mice of group A (Dem+NS) were given Dem [6 mg/(kg x d)] intraperitoneally and 200 microl normal saline given orally. Mice of group B (Dem+PT) were orally given pidotimod [100 mg/(kg x d)] and intraperitoneally injected with Dem[6 mg/(kg x d)] simultaneously. Each mouse in group C received 200 microl normal saline intraperitoneally. The mice were injected and given by gavage for 5 weeks. After treatment, three mice in each group were scarified weekly, and the survival time of the mice was recorded in days. Brain parasite burden and T. gondii DNA copies in serum were detected by quantitative real-time PCR. T cell subsets, cytokine profiles in each group were analyzed by flow cytometry, and CBA kit, respectively. RESULTS: On the second week after Dem administration, parasitemia appeared in group A; in 5 weeks 50% mice had parasitemia again, and 17 mice died. Comparatively, in group B parasitemia appeared on the third week after PT and Dem administration, in 5 weeks 25% mice had parasitemia again, and 7 mice died. Parasitemia did not appear in Group C. On the 21st day after Dem administration, T. gondii DNA copies in brain tissues of group A was (209 +/- 12) x 10(9), significantly higher than (62 +/- 10) x 10(9) in group B treated with PT (n = 3, P < 0.01). Flow cytometry test showed that on the 21st day after Dem administration, the proportions of Th1, Th2 and Treg cells in groups A and B were (4.0 +/- .5)% and (6.1 +/- 1.0)%, (0.6 +/- 0.1)% and (0.5 +/- 0.2)%, and (5.0 +/- 0.9)% and (7.0 +/- 1.2)%, respectively. There was significant difference in the percentages of Th1 and Treg between group B and A (P < 0.01). The levels of IFN-gamma, TNF-alpha in group A were (2.2 +/- 0.7) pg/ml and (20.1 +/- 5.0) pg/ml, respectively, lower than that of group B [(3.6 +/- 0.6) pg/ml and (32.0 +/- 8.0) pg/ml] (P < 0.01). No statistical significance was found in the levels of IL-4 and IL-10 between group A [(2.6 +/- 0.4) pg/ml, (39.0 +/- 6.0) pg/ml] and group B [(2.7 +/- 0.7) pg/ml, (40.0 +/- 8.0) pg/ml] (P > 0.05). CONCLUSION: Pidotimod can inhibit activation of latent Toxoplasma gondii infection induced by dexamethasone in mice. Th1 and Treg cells may contribute to the pidotimod/dexamethasone-induced immunoregulation.
Assuntos
Dexametasona/efeitos adversos , Ácido Pirrolidonocarboxílico/análogos & derivados , Tiazolidinas/farmacologia , Toxoplasma/efeitos dos fármacos , Toxoplasmose Animal/induzido quimicamente , Animais , Encéfalo/parasitologia , Citocinas/imunologia , DNA de Protozoário/análise , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Ácido Pirrolidonocarboxílico/farmacologia , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th2/imunologia , Toxoplasmose Animal/imunologia , Toxoplasmose Animal/parasitologiaRESUMO
OBJECTIVE: To observe the role of CD8+ T cells in the tumor growth delay induced by Toxoplasma gondii excreted-secreted antigens (TgESA) in B16FI0 mouse melanoma model in the early stage. METHODS: TgESA were prepared by incubating T. gondii tachyzoites for 12 h in vitro. 15 C57BL/6 mice were randomly assigned to group A, B, and C (5 mice per group). Each mouse in group B and C was subcutaneously injected in right flank with 2 x 10(5) B16F10 cells. Mice in group C were intraperitoneally injected with TgESA (100 microl per mouse) at 7d after B16F10 cells injection. Mice of group A were only injected with PBS. On the 13th day after melanoma cell injection, the mice were sacrificed and spleen was removed. The percentage of CD8+ T cells in the spleen was analyzed by flow cytometry. CD8+ T cells were isolated from spleen cells by using immunomagnetic beads. The activity of CD8+ T cells against B16F10 melanoma cells was determined by LDH release assay at different effect-to-target cell ratios (2.5:1, 5:1, and 10:1). Other 30 C57BL/6 mice were randomly divided into group E, F, and G. Each mice were injected with 2 x 10(5) B16F10 cells. At the same time, mice in group F and G were simultaneously injected via the tail vein with CD8+ T cells isolated from mice in group B and C. Tumor growth, mortality and survival time of mice were observed and recorded during 35-d observation period. RESULTS: The percentage of CD3+CD8+T cells in the spleen cells of group C [(15.74 +/- 0.28)%] was significantly higher than that of group B [(14.18 +/- 0.27)%] and A [(13.86 +/- 0.13)%] (P < 0.05). At different effect-to-target cell ratios, the activity of CD8+ T cells against B16F10 cells in group C was significantly higher than that of group B (P < 0.05). The average time of tumor formation in group G [(14.9 +/- 1.2) d] was longer than that in group F [(11.9 +/- 0.7) d] and E [(9.4 +/- 1.2) d] (P < 0.05). The tumor size in these groups increased, but there was no obvious difference in the tumor growth rate among the three groups. The tumor size of group G was significantly smaller than the other two groups (P < 0.05). In group E, F and G, mice began to die on the 26th day, the 29th day and the 30th day after tumor inoculation, and the number of survival mice was 3, 5 and 7, respectively, at the 35th day after injection. CONCLUSIONS: TgESA may up-regulate the quantity and function of CD8+ T cell in B16F10 melanoma mouse model, which plays a role of delaying tumor growth in early stage.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Melanoma Experimental/patologia , Toxoplasma/imunologia , Animais , Antígenos de Protozoários/imunologia , Linfócitos T CD8-Positivos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Toxoplasmose AnimalRESUMO
OBJECTIVE: To observe the immune response induced by ROP2 protein of Toxoplasma gondii with cimetidine in mice. METHODS: Eighty BALB/c mice were randomly divided into 4 groups: PBS (group A), ROP2 protein (group B), ROP2 protein-Freund's adjuvant (group C) and ROP2 protein-cimetidine (group D). Mice were immunized with PBS, ROP2 protein, ROP2 protein and Freund's adjuvant, ROP2 protein and cimetidine [200 mg/(kg x d)] by subcutaneous injection three times at an interval of 2 weeks, respectively. Experimental mice were immunized with 100 microg ROP2 proteins, which were diluted to a final volume of 200 microl in PBS. On day 13, 27 and 41 after immunization, mice sera were collected for determination of antibody IgG and cytokine IFN-gamma by ELISA. Two weeks after the final immunization, T cells subpopulation was detected by flow cytometry and splenocyte proliferation activity was determined with CCK-8. Another 12 immunized mice in each group were intraperitoneally challenged with 5 x 10(4) tachyzoites of T. gondii and the survival time was observed. RESULTS: Two weeks after final immunization, compared with groups A [(659.750 +/- 239.962) pg/ml] and B [(872.750 +/- 197.011) pg/ml], the level of IFN-gamma significantly increased in groups C [(1 600.750 +/- 480.680) pg/ml] and D [(1494.375 +/- 451.655) pg/ml] (P < 0.01). Similarly, compared with groups A (0.636 +/- 0.108) and B (0.871 +/- 0.089), the level of IgG was also higher in groups C (1.068 +/- 0.111) and D (1.046 +/- 0.147) (P < 0.01). The proliferation of splenocytes rose in group C (0.831 +/- 0.130) after immunization, and similarly in group D (0.762 +/- 0.089), and were both significantly higher than that of groups A (0.504 +/- 0.078) and B (0.592 +/- 0.160) (P < 0.01). Moreover, ratio of CD4+/CD8+ in groups C (0.831 +/- 0.130) and D (0.762 +/- 0.089) were higher than that of groups A (0.504 +/- 0.078) and B (0.592 +/- 0.160) (P < 0.05). After challenge with violent virulence strain of tachyzoites, the median survival time of mice in groups A, B, C, and D were 96, 108, 132, and 132 h, respectively. The mean survival time of mice in groups C and D were longer than that of groups A and B (P < 0.05). There was no significant difference in 5 parameters between C and D: the level of IFN-gamma and IgG, CD4+/CD8+ ratio, splenocyte proliferation, and survival time of mice (P > 0.05). CONCLUSION: Cimetidine can enhance the humoral and cellular immune response induced by ROP2 protein.
Assuntos
Antígenos de Protozoários/imunologia , Cimetidina/farmacologia , Proteínas de Membrana/imunologia , Proteínas de Protozoários/imunologia , Toxoplasma/imunologia , Toxoplasmose Animal/imunologia , Animais , Feminino , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Protozoárias/imunologiaRESUMO
DNA in dried blood spots of 39 vivax malaria patients (2009-2010) from Anhui (Bengbu urban district and counties of Wuhe, Huaiyuan, Mengcheng and Lixin) was extracted. The Plasmodium vivax LDH (PvLDH) gene was amplified, cloned and sequenced. The sequences were subjected to NCBI Blast program. The results showed that the targeted DNA fragment size was 951 bp without difference among the 39 samples (accession No. GU078391), and was more than 99% homologous to the PvLDH sequences in other strains from GenBank. There was only one different amino acid in the protein sequences between the isolates from Anhui and EJEU60134 or MIA061251 strains.
Assuntos
L-Lactato Desidrogenase/genética , Plasmodium vivax/enzimologia , China , DNA de Protozoário/genética , Humanos , Análise de Sequência de DNA , Homologia de Sequência , Homologia de Sequência de AminoácidosRESUMO
OBJECTIVE: To investigate microglial activation and inflammatory cytokine expression in chronic Toxoplasma gondii infection. METHODS: Thirty mice were randomly divided into chronic T. gondii infection group and normal control group. Each mouse in infection group was infected orally with 30 cysts of the TgCtwh6 strain. Normal group received 0.3 ml normal saline. On the 60th day after infection, immunohistochemical staining was performed to assess the number of microglia and morphological change. The expression of inflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) was measured by RT-PCR. The expression of iNOS was determined by Western blotting and immunofluorescence. RESULTS: Immunohistochemistry analysis showed that the number of Iba-1 positive cells in the cortex and hippocampus of infection group (16.5 +/- 0.8 and 17.9 +/- 1.1) was higher than that of the control (8.4 +/- 0.2 and 10.3 +/- 0.8)(P < 0.05). Iba-1 positive cells (i.e. microglia) had larger cell bodies and ramified morphology. RT-PCR result indicated that mRNA level of IL-1beta, IL-6, and TNF-alpha in infection group (0.862 +/- 0.169, 0.407 +/- 0.158, and 0.305 +/- 0.073) was significantly higher than that of the control (0.149 +/- 0.030, 0.037 +/- 0.008, and 0.001 +/- 0.001) (P < 0.05). The iNOS protein expression in infection group (0.252 +/- 0.164) was higher than that of the control (0.0433 +/- 0.004) (P < 0.05). Immunofluorescence demonstrated that iNOS protein released by activated microglia. CONCLUSION: Chronic T. gondii infection caused microglial activation, which up-regulate the level of IL-1beta, IL-6, TNF-alpha, and iNOS.
Assuntos
Encéfalo/parasitologia , Citocinas/metabolismo , Microglia/metabolismo , Toxoplasmose/metabolismo , Animais , Feminino , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Microglia/citologia , Óxido Nítrico Sintase Tipo II/metabolismo , Toxoplasma , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Raf kinase inhibitor protein (RKIP) is an inflammationinhibiting mediator that is involved in several diseases; however, the potential mechanism of action of RKIP on the inflammatory response induced by influenza A virus (IAV) remains unclear. The present study aimed to investigate whether RKIP regulated the inflammatory response via the ERK/MAPK pathway. The present study detected the expression levels of RKIP and alterations in the inflammatory response in human normal bronchial epithelial BEAS2B cells, diseased human bronchial epithelial cells and primary human bronchial epithelial cells infected with IAV. Cells were treated with locostatin to inhibit the expression of RKIP. RKIP was overexpressed by lentivirus transduction and the small molecule inhibitor SCH772984 was applied to specifically inhibit activation of the ERK/MAPK pathway. In addition, C57BL/6 mice were infected with IAV to further confirm the role of RKIP in regulation of the inflammatory response via ERK/MAPK in vivo. Western blotting, reverse transcriptionquantitative PCR, ELISA, 5ethynyl-2'deoxyuridine assay, immunofluorescence staining, Cell Counting Kit8, cell cycle assay, hematoxylin and eosin staining, and immunohistochemistry were used to detect all of the changes. Notably, RKIP attenuated the inflammatory response that was triggered by IAV infection in airway epithelial cells, which was characterized by augmented inflammatory cytokines and cell cycle arrest. Furthermore, the ERK/MAPK pathway was revealed to be activated by IAV infection and downregulation of RKIP aggravated the airway inflammatory response. By contrast, overexpression of RKIP effectively ameliorated the airway inflammatory response induced by IAV. These findings demonstrated that RKIP may serve a protective role in airway epithelial cells by combating inflammation via the ERK/MAPK pathway. Collectively, the present findings suggested that RKIP may negatively regulate airway inflammation and thus may constitute a promising therapeutic strategy for airway inflammatoryrelated diseases that are induced by IAV.
Assuntos
Vírus da Influenza A , Proteína de Ligação a Fosfatidiletanolamina , Animais , Humanos , Camundongos , Inflamação , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos C57BL , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismoRESUMO
OBJECTIVE: To investigate the lethal effect of exogenous nitric oxide on adult worms of Trichinella spiralis in vitro. METHODS: Adult worms of T. spiralis isolated from the small intestine of Trichinella-infected BALB/c mice were cultured in RPMI 1640 medium with sodium nitroprusside (SNP) in different final concentration of 0, 0.02, 0.05, 0.10, 0.20, 0.50, and 1.00 mmol/L, 1.00 mmol/L SNP+0.15 mmol/L hemoglobin (Hb), 1.00 mmol/L SNP+0.15 mmol/L FeSO4, 1.00 mmol/L SNP+1.00 mmol/L L-cysteine (L-cyst), 1.00 mmol/L SNP+0.15 mmol/L FeSO4 +1.00 mmol/L L-cyst, respectively, and incubated at 37 degrees C in a humidified 5% CO2 atmosphere. On the 4th day after incubation, the adult worms were stained with safranin, and observed under light microscope. The worm mortality in the groups was analyzed. RESULTS: Under concentration of 0.02 and 0.05 mmol/L, SNP was not cytotoxic to adult T. spiralis with an inhibition of (1.4 +/- 1.2)% and (3.2 +/- 1.0)%, respectively. The worm mortality in the groups of SNP 0.10, 0.20, 0.50, and 1.00 mmol/L was (9.9 +/- 1.8)%, (37.7 +/- 2.5)%, (50.1 +/- 3.5)%, and (80.8 +/- 1.1)%, respectively, significantly higher than that of negative control group [(1.9 +/- 0.2)%, P < 0.05]. There was a positive linear correlation between the worm mortality and SNP concentration in the range of 0.02-1.00 mmol/L. Combination of hemoglobin, L-cyst, FeSO4 and FeSO4+L-cyst with 1.00 mmol/L SNP led to a decrease of the mortality from (80.8 +/- 1.1)% to (56.5 +/- 3.7)%, (69.8 +/- 2.3)%, (74.8 +/- 2.4)%, (72.7 +/- 5.6)%, respectively. CONCLUSION: Exogenous nitric oxide released from SNP can kill adult worms of Trichinella spiralis. However, hemoglobin and L-cysteine+FeSO4 can reverse its lethal effect on the parasites.
Assuntos
Óxido Nítrico/farmacologia , Trichinella spiralis/efeitos dos fármacos , Triquinelose/parasitologia , Animais , Meios de Cultura , Cisteína , Feminino , Compostos Ferrosos/farmacologia , Hemoglobinas , Intestino Delgado/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Nitroprussiato/farmacologia , Trichinella spiralis/isolamento & purificaçãoRESUMO
OBJECTIVE: To express the recombinant BSR4 protein of Toxoplasma gondii Prugniaud (PRU) strain and study its immunologic characteristics. METHODS: The recombinant plasmid pET28a(+)-BSR4 was transformed into E. coli BL21, followed by expression of BSR4 induced by IPTG and its purification. The immunoreactivity of the recombinant protein BSR4 was analyzed by Western blotting with the sera from mice infected by PRU strain of T. gondii or normal mice as the first antibody. The 3H-TdR incorporation assay was performed to determine the proliferation of splenocytes in mice infected by PRU strain stimulated by BSR4, and the stimulation index (SI) was calculated. ELISA was used to evaluate the immunoreactivity of BSR4 protein, in which 20 sera from each of acute (anti-T. gondii IgG(-)IgM+) and chronic (IgG+IgM(-)) toxoplasmosis patients, and healthy people were tested. RESULTS: The recombinant BSR4 was induced and expressed. After denaturation, renaturation and purification, the soluble protein (Mr 45 000) was obtained and detected by anti-T. gondii serum with Western blotting. BSR4 in 1, 5, and 25 microg/ml induced the proliferation of splenocytes in mice infected by PRU strain with higher SI (1.13, 0.88, and 1.17) than that of control (0.46, 0.24, and 0.49, respectively, P<0.01). ELISA showed that the recombinant BSR4 specifically reacted with the sera of toxoplasmosis patients (IgG+IgM(-), 20/20), not with that of the acute cases (IgG(-)IgM+, 0/20). CONCLUSION: The recombinant BSR4 shows specific immunogenicity and immunoreactivity.
Assuntos
Antígenos de Protozoários/imunologia , Proteínas Recombinantes/imunologia , Toxoplasma/imunologia , Animais , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Camundongos , Camundongos Endogâmicos , Baço/citologia , Baço/imunologia , Toxoplasmose/imunologiaRESUMO
OBJECTIVE: To determine the kinetics of infection and cyst formation in CD1 mice following oral infection with cyst-forming Chinese isolate of Toxoplasma gondii TgCtwh1(genotype China 1, ToxoDB#9). METHODS: 50 CD1 female mice were obtained from specific pathogen-free (SPF) mouse colony in the Vital River Laboratories (VRL), Beijing. Mice were randomly divided into 10 groups each with 5 mice. All mice but control were peroral gavage infected with 50 cysts (1x10(4) bradyzoites) of TgCtwh1 isolate of T. gondii isolated from Wuhan, China. Cysts were isolated from the entire brain of mice infected with TgCtwh1 by density gradient centrifugation over Fycoll-paque plus. Animals were orally inoculated with cysts on day zero, and peripheral blood, lymph nodes, heart, liver, and brain of infected mice were collected on days 2, 4, 7, 10, 14, 21, 35, 50, and 72 post infection. Five mice were sacrificed by cervical dislocation under anesthesia at each time of collection, and the kinetic distribution was detected by fluorescence quantitative PCR and tissue inoculation into fresh mice. The cyst formation at various intervals after infection was also observed, as was the number of the cysts in brains and the cyst-forming rate. RESULTS: The body weight of the mice lessened (3.650 +/- 0.252)g post oral infection on day 7, and the weight was progressively decreased between day 10 [(1.730 +/- 0.017)g] and day 14 [(-0.390 +/- 0.554) g] after infection (P<0.05). In the brain tissue, cysts were first observed on day 21 post oral infection and the cyst-forming rate was 80%, and the average diameter of cysts was 20-40 microm. While on day 35 after infection, the cysts were formed in all infected mice(cyst-forming rate was 100%) and the average diameter was 50-60 microm. In chronic infection, DNA copies of parasites were first detected in blood, heart, liver and lymph node at 3.51 +/- 0.152, 4.100 +/- 0.198, 4.220 +/- 0.209 and 4.960 +/- 0.052 respectively on day 2, then in the brain on day 4 (3.800 +/- 0.154). During the early days of infection, the parasite burden in blood was progressively increased until days 7 (5.240 +/- 0.115) then gradually decreased and become undetectable on day 35. The burden of T. gondii in the heart and brain tissues increased significantly and reached their maximum on day 14 (5.640 +/- 0.214) and day 10 (5.790 +/- 0.060), respectively, and remained a stable level thereafter. Liver and lymph tissues reached their maximum on day 7 (5.310 +/- 0.038) and day 10 (6.200 +/- 0.152), then gradually decreased and become undetectable on day 50. CONCLUSION: The parasitemia in mice infected with T. gondii cyst-forming isolate lasts for 21 d at least, and cysts are detected in brain on day 21.
Assuntos
Encéfalo/parasitologia , Toxoplasma/isolamento & purificação , Toxoplasmose Animal/parasitologia , Animais , Feminino , Genes de Protozoários , Genótipo , Camundongos , Reação em Cadeia da Polimerase/métodos , Toxoplasma/genéticaRESUMO
BACKGROUND: Influenza A virus (IAV) triggers acute exacerbation of chronic obstructive pulmonary disease (AECOPD), but the molecular mechanisms remain unclear. In this study, we investigated the role of IAV induced NLRP3 inflammasome activation to increase airway inflammation response in the progression of AECOPD. METHODS: Human bronchial epithelial cells were isolated and cultured from normal and COPD bronchial tissues and co-cultured with IAV. The NLRP3 inflammasome associated genes were identified using RNA sequencing, and the expressions of NLRP3 inflammasome components were measured using qRT-PCR and western blot after cells were transfected with siRNA and treated with MCC950. Moreover, IAV-induced COPD rat models were established to confirm the results; 37 AECOPD patients were included to measure the serum and bronchoalveolar lavage fluid (BALF) of interleukin (IL)-18 and IL-1ß. RESULTS: Increased levels of NLRP3 inflammasome components were not seen until 6 h post-inoculation in normal cells. However, both cell groups reached peak NLRP3 level at 12 h post-inoculation and maintained it for up to 24 h. ASC, Caspase-1, IL-1ß and IL-18 were also elevated in a similar time-dependent pattern in both cell groups. The mRNA and protein expression of the NLRP3 inflammasome components were decreased when COPD cells treated with siRNA and MCC950. In COPD rats, the NLRP3 inflammasome components were elevated by IAV. MCC950 alleviated lung damage, improved survival time, and reduced NLRP3 inflammasome components expression in COPD rats. Additionally, the serum and BALF levels of IL-1ß and IL-18 were increased in AECOPD patients. CONCLUSIONS: NLRP3 inflammasome is activated in COPD patients as a pre-existing condition that is further exacerbated by IAV infection.
RESUMO
Influenza virus infection is one of the strongest pathogenic factors for the development of acute lung injury (ALI)/ acute respiratory distress syndrome (ARDS). However, the underlying cellular and molecular mechanisms have not been clarified. In this study, we aim to investigate whether melatonin modulates macrophage polarization, oxidative stress, and pyroptosis via activating Apolipoprotein E/low-density lipoprotein receptor (ApoE/LDLR) pathway in influenza A-induced ALI. Here, wild-type (WT) and ApoE-/- mice were instilled intratracheally with influenza A (H3N2) and injected intraperitoneally with melatonin for 7 consecutive days. In vitro, WT and ApoE-/- murine bone marrow-derived macrophages (BMDMs) were pretreated with melatonin before H3N2 stimulation. The results showed that melatonin administration significantly attenuated H3N2-induced pulmonary damage, leukocyte infiltration, and edema; decreased the expression of proinflammatory M1 markers; enhanced anti-inflammatory M2 markers; and switched the polarization of alveolar macrophages (AMs) from M1 to M2 phenotype. Additionally, melatonin inhibited reactive oxygen species- (ROS-) mediated pyroptosis shown by downregulation of malonaldehyde (MDA) and ROS levels as well as inhibition of the NLRP3/GSDMD pathway and lactate dehydrogenase (LDH) release. Strikingly, the ApoE/LDLR pathway was activated when melatonin was applied in H3N2-infected macrophages and mice. ApoE knockout mostly abrogated the protective impacts of melatonin on H3N2-induced ALI and its regulatory ability on macrophage polarization, oxidative stress, and pyroptosis. Furthermore, recombinant ApoE3 (re-ApoE3) inhibited H3N2-induced M1 polarization of BMDMs with upregulation of MT1 and MT2 expression, but re-ApoE2 and re-ApoE4 failed to do this. Melatonin combined with re-ApoE3 played more beneficial protective effects on modulating macrophage polarization, oxidative stress, and pyroptosis in H3N2-infected ApoE-/- BMDMs. Our study indicated that melatonin attenuated influenza A- (H3N2-) induced ALI by inhibiting the M1 polarization of pulmonary macrophages and ROS-mediated pyroptosis via activating the ApoE/LDLR pathway. This study suggested that melatonin-ApoE/LDLR axis may serve as a novel therapeutic strategy for influenza virus-induced ALI.
Assuntos
Lesão Pulmonar Aguda , Melatonina , Infecções por Orthomyxoviridae , Animais , Camundongos , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/virologia , Apolipoproteína E3/farmacologia , Apolipoproteínas E/metabolismo , Vírus da Influenza A Subtipo H3N2 , Macrófagos/metabolismo , Melatonina/uso terapêutico , Camundongos Knockout para ApoE , Piroptose , Espécies Reativas de Oxigênio/metabolismo , Infecções por Orthomyxoviridae/tratamento farmacológicoRESUMO
Acute lung injury (ALI)/ acute respiratory distress syndrome (ARDS) is featured by intensive inflammatory responses and oxidative stress, which lead to cytokine storms and pyroptosis. Here, we aimed to investigate whether melatonin was capable of alleviating LPS-induced ALI via activating the nuclear factor erythroid 2-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signaling axis and inhibiting pyroptosis. Mice were injected with melatonin (30 mg/kg) intraperitoneally for consecutive five days before LPS instillation intratracheally, and human alveolar epithelial cell (AECâ ¡) A549 cell lines and murine macrophages Raw264.7 cell lines were pretreated with melatonin (400 µM) before LPS (10 µg/ml) stimulation. The result demonstrated that LPS induced obvious lung injury characterized by alveolar damage, neutrophil infiltration and lung edema as well as the reduction of the survival rate of mice, which were totally reversed by melatonin pretreatment. Mechanistically, melatonin pretreatment activated nuclear factor erythroid2-related factor (Nrf) 2 signaling, subsequently, drove antioxidant pathways including significant increases in the expression of Nrf2, HO-1, NQO1, Mn-SOD and Catalase in vivo and in vitro. Simultaneously, melatonin inhibited ROS and MDA overproduction, iNOS expression as well as TNF-α and IL-1ß expression and release. Furthermore, melatonin inhibited LPS-induced pyroptosis by reversing the overexpression of NLRP3, Caspase-1, IL-1ß, IL-18 and GSDMD-N, as well as LDH release and TUNEL-positive cells in A549 cells and Raw264.7 cells. Overall, the current study suggests that melatonin exerts protective roles on LPS-induced ALI and pyroptosis by inhibiting NLRP3-GSDMD pathway via activating Nrf2/HO-1 signaling axis.