IGF-I induz proliferação e diferenciação celular. Neste estudo visamos sua participação na imunidade específica na leishmaniose. Estudamos efeito de citocinas Th1 (IFN-g) e Th2 (IL- 4 mais IL-13) na modulação da produção de IGF-I e seu reflexo no parasitismo de macrófago (MØ) de camundongos BALB/c e C57BL/6, infectados por amastigotas e promastigotas de Leishmania (L.) major. Iniciamos avaliando os efeitos de IGF-I no modelo, onde no desenvolvimento da lesão IGF-I induziu aumento maior de lesão em camundongos BALB/c. Nos camundongos C57BL/6 o efeito foi marcante com evolução progressiva da lesão enquanto que sem o fator, a lesão se controlava. Como a produção de IGF-I por MØ é modulada por citocinas, onde IFN-g diminui e IL-4 e IL-13 aumentam a sua expressão, fomos estudar seus efeitos no modelo proposto onde MØ foram infectados com amastigotas ou promastigotas de L.(L.) major (parasitos/célula = 2:1) e incubados com IFN-g (200U/mL) ou IL-4 (2ng/mL) mais IL-13 (5ng/mL). IFN-g induziu aumento significante na produção de NO nos dois modelos estudados. O efeito de citocinas Th1 e Th2 sobre o parasitismo em MØ foi distinto em BALB/c e C57BL/6. Quando infectados por amastigotas, o parasitismo aumentou sob estímulo com IL-4 mais IL-13 e diminuiu com IFN-g como era esperado. No entanto, quando infectado por promastigota, o parasitismo aumentou com IL-4 mais IL-13 somente em células BALB/c. Por outro lado, o parasitismo diminuiu com IFN- g somente em células C57BL/6. Mesmo na ausência do estímulo por citocinas, observou-se aumento na expressão de RNA de IGF-I nos MØ de animais BALB/c infectados por formas amastigotas ou promastigotas e de C57BL/6 infectados por promastigotas. Um resultado destoante foi à diminuição da expressão de IGF-I em MØ de C57BL/6 infectados por amastigotas. Em células BALB/c, essa expressão aumentada de IGF-I ocorrida em função da infecção, somente se alterou com IL-4 mais IL-13 quando infectadas por amastigotas. Em células C57BL/6, a expressão aumentada de IGF-I com a infecção por prormastigota, sofreu diminuição sob estímulo com IFN-g, não se alterando com citocinas Th2. A expressão diminuída de IGF-I com a infecção por amastigotas diminuiu ainda mais com IFN-g e aumentou com citocinas TH2. Os dados sugerem a possibilidade de modulação do efeito de citocinas pela expressão diferenciada de IGF-I em macrófagos infectados por L. (L.) major / Insulin-like growth factor (IGF)-I induces cell proliferation and differentiation. In this study, we focused on its participation on specific immunity. We studied the effect of Th1 (IFN-g) and Th2 (IL-4 plus IL-13) cytokines on the modulation of the production of IGF-I and its influence on BALB/c and C57BL/6 macrophage (MØ) parasitism using Leishmania (L.) major amastigote and promastigote. IGF-I expression was increased in infected macrophages. Initially, we substantiated the effect of IGF-I on in vitro growth of Leishmania (L.) major promastigote, on cutaneous lesion development in vivo and diminished production of nitric oxide in MØ. On development of the lesion, IGF-I induced greater increase of the lesion in BALB/c mice. In C5BL/6 mice, its effect was more pronounced with progression of the lesion whilst without the factor it was controlled. Since IGF-I production by MØ is modulated by cytokines where IFN-g decreases and IL-4 plus IL-13 increase its expression, we studied their effects in BALB/c and C57BL/6 MØ. Macrophages were infected with L.(L.) major amastigotes or promastigotes (parasites/cell = 2:1) and incubated with IFN-g (200U/mL) or IL-4 (2ng/mL) plus IL-13 (5ng/mL). IFN-g induced a significant increase of NO production by MØ from both models. The effect of Th1 and Th2 cytokines on parasitism was distinct when MØ infected with amastigotes, the parasitism increased upon IL-4 plus IL-13 stimuli and diminished with IFN-g as expected. However, when infected with promastigotes, the parasitism increased with IL-4 plus IL-13 only in BALB/c cells. Conversely, the parasitism diminished with IFN-g only in C57BL/6. Even in the absence of cytokine stimuli, an increase of the expression of IGF-I mRNA was observed in L (L.) major amastigote- and promastigote- infected BALB/c MØ and in promastigoteinfected C57BL/6 MØ. A dissonant result was a diminished IGF-I expression in amastigoteinfected C57BL/6 MØ. In BALB/c this increased IGF-I expression with infection only altered with IL-4 plus IL-13 in amastigote-infected cells. In C57BL/6 cells, the increased IGF-I expression upon prormastigote infection diminished under IFN-g stimulus that was not altered with Th2 cytokines. A decreased IGF-I expression with amastigote-infection diminished even more with IFN-g and increased with Th2 cytokines. These data suggest a possibility of modulation of the effect of cytokines on distinct expression of IGF-I in L. (L.) major-infected MØ

Common measures of egg quality have been survival to specific developmental stages, higher hatching rate of fertilized eggs and final production of fry. Determinants of egg quality are variable among and between teleost species and no common unified criteria have been established. Maternally inherited genes influence egg quality and early embryo development is partially programmed by the messenger ribonucleic acid (mRNA). Among the genes, the insulin family is important for growth functions and the presence of their transcripts in the ovary, oocytes and embryos implies their involvement during the reproductive process and their relevance to egg quality. The insulin-like growth factor (IGF) system has three components, the ligands IGF-I and II, the IGFBPs (insulin-like growth factor binding proteins) and the IGF receptors that mediate biological activity of the ligands. Vitellogenin (Vtg) is the major source of nutrients for the developing embryo and elevated levels in female fish plasma signals gonadal development preceding spawning. In oviparous fish where the developing embryo is dependent on the stored food in the yolk, vitellogenin levels in the egg could indicate its capability to support embryonic growth. This study aimed to develop molecular tools, specifically probes for IGF-I, IGF-II and IGF-IR, for the evaluation of fish egg quality. These probes would be used to determine expression levels of IGF-I, IGF-II and IGF-IR during egg development to assess their potential as molecular indicators for egg quality. In addition, this study also aimed to establish an enzyme-linked immunoassay (ELISA) for quantifying Vtg in fish eggs and determine if differences in Vtg levels could be linked to fertilization and hatching success. Through reverse-transcription polymerase chain reaction (RT-PCR) putative complementary deoxyribonucleic acid (cDNA) fragments of IGF-I, IGF-II and IGF-IR were cloned and sequenced from mullet (Mugil cephalus) and grouper (Epinephelus coioides). The relative expression ratio of the three genes in the eggs of mullet and grouper were assayed by quantitative PCR (QPCR) and calculated using the Pfaffl method (Pfaffl, 2001). Levels of vitellogenin in different batches of mullet eggs were quantified by ELISA. Spawned eggs of grouper were grouped into low (<60%) or high (>60%) fertilization rate (FR) and the fertilized eggs that were incubated until hatching were grouped into medium (>90%) or high (>90%) hatching rate (HR). Samples were categorized into sinking eggs, late embryo and hatched larvae. Relative expression ratio of IGF-II was significantly high (P<0.01) compared to IGF-I and IGF-IR in all samples examined. All three genes were strongly expressed in sinking eggs compared to either late embryo or hatched larvae. However, there was no significant interaction effect between the genes and the samples analyzed. Mullet samples all came from a high FR and high HR group and were categorized into sinking, multicell stage, blastula, gastrula, late embryo and hatched larvae. There was a significant interaction effect (P<0.01) between gene and stage, showing that genes are differentially expressed during embryonic development. IGF-II was strongly expressed relative to the other genes in all stages examined and was highest during the gastrula stage. Vtg levels were examined in mullet oocytes and egg samples that were grouped into 4; oocytes from females that subsequently spawned, had fertilized eggs which hatched (Group A); oocytes from females that did not spawn, therefore no fertilization and no hatching (Group B); eggs that were stripped, artificially fertilized but no hatching (Group C); and eggs that were spawned, assumed to be fertilized but did not hatch (Group D). Group A showed a trend of higher Vtg levels than the other three but this result was not statistically significant.

IGF-I induz proliferação e diferenciação celular. Neste estudo visamos sua participação na imunidade específica na leishmaniose. Estudamos efeito de citocinas Th1 (IFN-g) e Th2 (IL- 4 mais IL-13) na modulação da produção de IGF-I e seu reflexo no parasitismo de macrófago (MØ) de camundongos BALB/c e C57BL/6, infectados por amastigotas e promastigotas de Leishmania (L.) major. Iniciamos avaliando os efeitos de IGF-I no modelo, onde no desenvolvimento da lesão IGF-I induziu aumento maior de lesão em camundongos BALB/c. Nos camundongos C57BL/6 o efeito foi marcante com evolução progressiva da lesão enquanto que sem o fator, a lesão se controlava. Como a produção de IGF-I por MØ é modulada por citocinas, onde IFN-g diminui e IL-4 e IL-13 aumentam a sua expressão, fomos estudar seus efeitos no modelo proposto onde MØ foram infectados com amastigotas ou promastigotas de L.(L.) major (parasitos/célula = 2:1) e incubados com IFN-g (200U/mL) ou IL-4 (2ng/mL) mais IL-13 (5ng/mL). IFN-g induziu aumento significante na produção de NO nos dois modelos estudados. O efeito de citocinas Th1 e Th2 sobre o parasitismo em MØ foi distinto em BALB/c e C57BL/6. Quando infectados por amastigotas, o parasitismo aumentou sob estímulo com IL-4 mais IL-13 e diminuiu com IFN-g como era esperado. No entanto, quando infectado por promastigota, o parasitismo aumentou com IL-4 mais IL-13 somente em células BALB/c. Por outro lado, o parasitismo diminuiu com IFN- g somente em células C57BL/6. Mesmo na ausência do estímulo por citocinas, observou-se aumento na expressão de RNA de IGF-I nos MØ de animais BALB/c infectados por formas amastigotas ou promastigotas e de C57BL/6 infectados por promastigotas. Um resultado destoante foi à diminuição da expressão de IGF-I em MØ de C57BL/6 infectados por amastigotas. Em células BALB/c, essa expressão aumentada de IGF-I ocorrida em função da infecção, somente se alterou com IL-4 mais IL-13 quando infectadas por amastigotas. Em células C57BL/6, a expressão aumentada de IGF-I com a infecção por prormastigota, sofreu diminuição sob estímulo com IFN-g, não se alterando com citocinas Th2. A expressão diminuída de IGF-I com a infecção por amastigotas diminuiu ainda mais com IFN-g e aumentou com citocinas TH2. Os dados sugerem a possibilidade de modulação do efeito de citocinas pela expressão diferenciada de IGF-I em macrófagos infectados por L. (L.) major / Insulin-like growth factor (IGF)-I induces cell proliferation and differentiation. In this study, we focused on its participation on specific immunity. We studied the effect of Th1 (IFN-g) and Th2 (IL-4 plus IL-13) cytokines on the modulation of the production of IGF-I and its influence on BALB/c and C57BL/6 macrophage (MØ) parasitism using Leishmania (L.) major amastigote and promastigote. IGF-I expression was increased in infected macrophages. Initially, we substantiated the effect of IGF-I on in vitro growth of Leishmania (L.) major promastigote, on cutaneous lesion development in vivo and diminished production of nitric oxide in MØ. On development of the lesion, IGF-I induced greater increase of the lesion in BALB/c mice. In C5BL/6 mice, its effect was more pronounced with progression of the lesion whilst without the factor it was controlled. Since IGF-I production by MØ is modulated by cytokines where IFN-g decreases and IL-4 plus IL-13 increase its expression, we studied their effects in BALB/c and C57BL/6 MØ. Macrophages were infected with L.(L.) major amastigotes or promastigotes (parasites/cell = 2:1) and incubated with IFN-g (200U/mL) or IL-4 (2ng/mL) plus IL-13 (5ng/mL). IFN-g induced a significant increase of NO production by MØ from both models. The effect of Th1 and Th2 cytokines on parasitism was distinct when MØ infected with amastigotes, the parasitism increased upon IL-4 plus IL-13 stimuli and diminished with IFN-g as expected. However, when infected with promastigotes, the parasitism increased with IL-4 plus IL-13 only in BALB/c cells. Conversely, the parasitism diminished with IFN-g only in C57BL/6. Even in the absence of cytokine stimuli, an increase of the expression of IGF-I mRNA was observed in L (L.) major amastigote- and promastigote- infected BALB/c MØ and in promastigoteinfected C57BL/6 MØ. A dissonant result was a diminished IGF-I expression in amastigoteinfected C57BL/6 MØ. In BALB/c this increased IGF-I expression with infection only altered with IL-4 plus IL-13 in amastigote-infected cells. In C57BL/6 cells, the increased IGF-I expression upon prormastigote infection diminished under IFN-g stimulus that was not altered with Th2 cytokines. A decreased IGF-I expression with amastigote-infection diminished even more with IFN-g and increased with Th2 cytokines. These data suggest a possibility of modulation of the effect of cytokines on distinct expression of IGF-I in L. (L.) major-infected MØ