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Mosquito borne flaviviruses, including dengue (DENV) and Zika (ZIKV) viruses, have caused global epidemics in areas with high HIV prevalence due to the expanded geographic range of arthropod vectors. Despite the occurrence of large flavivirus outbreaks in countries with high HIV prevalence, there is little knowledge regarding the effects of flavivirus infection in people living with HIV (PLWH). Here, we use a pigtail macaque model of HIV/AIDS to investigate the impact of simian immunodeficiency virus (SIV)-induced immunosuppression on ZIKV replication and pathogenesis. Early acute SIV infection induced expansion of peripheral ZIKV cellular targets and increased innate immune activation and peripheral blood mononuclear cells (PBMC) from SIV infected macaques were less permissive to ZIKV infection in vitro . In SIV-ZIKV co-infected animals, we found increased persistence of ZIKV in the periphery and tissues corresponding to alterations in innate cellular (monocytes, neutrophils) recruitment to the blood and tissues, decreased anti-ZIKV immunity, and chronic peripheral inflammatory and innate immune gene expression. Collectively, these findings suggest that untreated SIV infection may impair cellular innate responses and create an environment of chronic immune activation that promotes prolonged ZIKV viremia and persistence in the gastrointestinal tract. These results suggest that PLWH or other immunocompromised individuals could be at a higher risk for chronic ZIKV replication, which in turn could increase the timeframe of ZIKV transmission. Thus, PLWH are important populations to target during the deployment of vaccine and treatment strategies against ZIKV. Author Summary: Flaviviruses, including Zika virus (ZIKV), cause global epidemics in areas with high HIV prevalence. Yet questions remain as to whether ZIKV disease is altered during an immunocompromised state and the potential immune mechanisms contributing to enhanced disease. This is essential to our understanding of ZIKV disease in people living with HIV (PLWH). Here, we use a non-human primate (NHP) model of HIV/AIDS to investigate the impact of immune suppression on ZIKV replication and pathogenesis. The use of the NHP model was critical for the assessment of longitudinal specimens across tissues that are active sites of flavivirus replication and host immune responses. This study broadly demonstrates that ZIKV pathogenesis is altered and more persistent in states of immunosuppression. Collectively, this study suggests that in PLWH and immunocompromised individuals, other arboviruses, including dengue and West Nile viruses, could similarly alter pathogenesis and/or viral peristance in tissues. Furthermore, this study highlights the need to prioritize immunocompromised individuals in the design and rollout of vaccines against arboviral diseases.
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The global SARS-CoV-2 pandemic prompted rapid development of COVID-19 vaccines. Although several vaccines have received emergency approval through various public health agencies, the SARS-CoV-2 pandemic continues. Emergent variants of concern, waning immunity in the vaccinated, evidence that vaccines may not prevent transmission and inequity in vaccine distribution have driven continued development of vaccines against SARS-CoV-2 to address these public health needs. In this report, we evaluated a novel self-amplifying replicon RNA vaccine against SARS-CoV-2 in a pigtail macaque model of COVID-19 disease. We found that this vaccine elicited strong binding and neutralizing antibody responses against homologous virus. We also observed broad binding antibody against heterologous contemporary and ancestral strains, but neutralizing antibody responses were primarily targeted to the vaccine-homologous strain. While binding antibody responses were sustained, neutralizing antibody waned to undetectable levels in some animals after six months but were rapidly recalled and conferred protection from disease when the animals were challenged 7 months after vaccination as evident by reduced viral replication and pathology in the lower respiratory tract, reduced viral shedding in the nasal cavity and lower concentrations of pro-inflammatory cytokines in the lung. Cumulatively, our data demonstrate in pigtail macaques that a self-amplifying replicon RNA vaccine can elicit durable and protective immunity to SARS-CoV-2 infection. Furthermore, these data provide evidence that this vaccine can provide durable protective efficacy and reduce viral shedding even after neutralizing antibody responses have waned to undetectable levels.
Assuntos
Vacinas contra COVID-19 , Vacinas de mRNA , Vacinas contra COVID-19/imunologia , Macaca nemestrina , Pulmão/imunologia , Pulmão/virologia , SARS-CoV-2/fisiologia , Animais , Anticorpos Neutralizantes/imunologia , COVID-19/transmissãoRESUMO
The global SARS-CoV-2 pandemic prompted rapid development of COVID-19 vaccines. Although several vaccines have received emergency approval through various public health agencies, the SARS-CoV-2 pandemic continues. Emergent variants of concern, waning immunity in the vaccinated, evidence that vaccines may not prevent transmission and inequity in vaccine distribution have driven continued development of vaccines against SARS-CoV-2 to address these public health needs. In this report, we evaluated a novel self-amplifying replicon RNA vaccine against SARS-CoV-2 in a pigtail macaque model of COVID-19 disease. We found that this vaccine elicited strong binding and neutralizing antibody responses. While binding antibody responses were sustained, neutralizing antibody waned to undetectable levels after six months but were rapidly recalled and conferred protection from disease when the animals were challenged 7 months after vaccination as evident by reduced viral replication and pathology in the lower respiratory tract, reduced viral shedding in the nasal cavity and lower concentrations of pro-inflammatory cytokines in the lung. Cumulatively, our data demonstrate in pigtail macaques that a self-amplifying replicon RNA vaccine can elicit durable and protective immunity to SARS-CoV-2 infection. Furthermore, these data provide evidence that this vaccine can provide durable protective efficacy and reduce viral shedding even after neutralizing antibody responses have waned to undetectable levels.
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The ongoing COVID-19 vaccine rollout is critical for reducing SARS-CoV-2 infections, hospitalizations, and deaths worldwide. Unfortunately, massive disparities exist in getting vaccines to vulnerable populations, including people living with HIV. Preliminary studies indicate that COVID-19 mRNA vaccines are safe and immunogenic in people living with HIV that are virally suppressed with potent antiretroviral therapy but may be less efficacious in immunocompromised individuals. This raises the concern that COVID-19 vaccines may be less effective in resource poor settings with limited access to antiretroviral therapy. Here, we evaluated the immunogenicity of a single dose COVID-19 replicon RNA vaccine expressing Spike protein (A.1) from SARS-CoV-2 (repRNA-CoV2S) in immunocompromised, SIV infected and immune competent, naïve pigtail macaques. Moderate vaccine-specific cellular Th1 T-cell responses and binding and neutralizing antibodies were induced by repRNA-CoV2S in SIV infected animals and naïve animals. Furthermore, vaccine immunogenicity was elicited even among the animals with the highest SIV viral burden or lowest peripheral CD4 counts prior to immunization. This study provides evidence that a SARS-CoV-2 repRNA vaccine could be employed to induce strong immunity against COVID-19 in HIV infected and other immunocompromised individuals.
Assuntos
Vacinas contra COVID-19/administração & dosagem , COVID-19/prevenção & controle , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Imunogenicidade da Vacina , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Glicoproteína da Espícula de Coronavírus/administração & dosagem , Eficácia de Vacinas , Vacinas de mRNA/administração & dosagem , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , COVID-19/imunologia , COVID-19/virologia , Vacinas contra COVID-19/genética , Vacinas contra COVID-19/imunologia , Células Cultivadas , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Hospedeiro Imunocomprometido , Macaca nemestrina , Masculino , Síndrome de Imunodeficiência Adquirida dos Símios/sangue , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/patogenicidade , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/virologia , Fatores de Tempo , Vacinação , Vacinas de mRNA/genética , Vacinas de mRNA/imunologiaRESUMO
Coccidioidomycosis is a common fungal infection in people living with HIV-1, particularly in southwest regions of the United States where the Coccidioides sp. is endemic, but rates of infection have significantly declined in the era of potent combination antiretroviral therapy (cART). Natural coccidioidomycosis also occurs in outdoor-housed macaques residing in the southwestern states that are utilized in biomedical research. Here, we report on a recrudescent case of previously treated, naturally occurring coccidioidomycosis in a pigtail macaque that was experimentally infected with simian immunodeficiency virus (SIV) and virally suppressed on cART. Coccidioides IgG antibody titer became detectable before discontinuation of cART, but symptomatic coccidioidomycosis developed subsequent to cART withdrawal. This animal was screened and treated in accordance with the guidelines for the prevention and treatment of coccidioidomycosis, suggesting that macaques with a history of coccidioidomycosis should be excluded from enrollment in HIV studies. Continual monitoring for known endemic pathogens based on the colony of origin is also recommended for animals utilized for HIV/AIDS research.
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Coccidioidomicose , Infecções por HIV , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , Coccidioidomicose/tratamento farmacológico , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Humanos , Macaca nemestrina , Recidiva , Síndrome de Imunodeficiência Adquirida dos Símios/complicações , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Carga ViralRESUMO
A safe, effective, and scalable vaccine is needed to halt the ongoing SARS-CoV-2 pandemic. We describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 SARS-CoV-2 spike receptor-binding domains (RBDs) in a highly immunogenic array and induce neutralizing antibody titers 10-fold higher than the prefusion-stabilized spike despite a 5-fold lower dose. Antibodies elicited by the RBD nanoparticles target multiple distinct epitopes, suggesting they may not be easily susceptible to escape mutations, and exhibit a lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the assembled nanoparticles suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms and have launched cGMP manufacturing efforts to advance the SARS-CoV-2-RBD nanoparticle vaccine into the clinic.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Nanopartículas/química , Domínios Proteicos/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química , Vacinação , Adolescente , Adulto , Idoso , Animais , COVID-19/virologia , Chlorocebus aethiops , Estudos de Coortes , Epitopos/imunologia , Feminino , Células HEK293 , Humanos , Macaca nemestrina , Masculino , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero , Adulto JovemRESUMO
The coronavirus disease 2019 (COVID-19) pandemic, caused by infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is having a deleterious impact on health services and the global economy, highlighting the urgent need for an effective vaccine. Such a vaccine would need to rapidly confer protection after one or two doses and would need to be manufactured using components suitable for scale up. Here, we developed an Alphavirus-derived replicon RNA vaccine candidate, repRNA-CoV2S, encoding the SARS-CoV-2 spike (S) protein. The RNA replicons were formulated with lipid inorganic nanoparticles (LIONs) that were designed to enhance vaccine stability, delivery, and immunogenicity. We show that a single intramuscular injection of the LION/repRNA-CoV2S vaccine in mice elicited robust production of anti-SARS-CoV-2 S protein IgG antibody isotypes indicative of a type 1 T helper cell response. A prime/boost regimen induced potent T cell responses in mice including antigen-specific responses in the lung and spleen. Prime-only immunization of aged (17 months old) mice induced smaller immune responses compared to young mice, but this difference was abrogated by booster immunization. In nonhuman primates, prime-only immunization in one intramuscular injection site or prime/boost immunizations in five intramuscular injection sites elicited modest T cell responses and robust antibody responses. The antibody responses persisted for at least 70 days and neutralized SARS-CoV-2 at titers comparable to those in human serum samples collected from individuals convalescing from COVID-19. These data support further development of LION/repRNA-CoV2S as a vaccine candidate for prophylactic protection against SARS-CoV-2 infection.
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Alphavirus/genética , Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , RNA Viral/genética , Replicon/genética , Linfócitos T/imunologia , Vacinas Virais/imunologia , Animais , Formação de Anticorpos/imunologia , COVID-19 , Vacinas contra COVID-19 , Infecções por Coronavirus/prevenção & controle , Compostos Inorgânicos/química , Lipídeos/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nanopartículas/química , Pandemias , Primatas , SARS-CoV-2RESUMO
The ongoing COVID-19 pandemic, caused by infection with SARS-CoV-2, is having a dramatic and deleterious impact on health services and the global economy. Grim public health statistics highlight the need for vaccines that can rapidly confer protection after a single dose and be manufactured using components suitable for scale-up and efficient distribution. In response, we have rapidly developed repRNA-CoV2S, a stable and highly immunogenic vaccine candidate comprised of an RNA replicon formulated with a novel Lipid InOrganic Nanoparticle (LION) designed to enhance vaccine stability, delivery and immunogenicity. We show that intramuscular injection of LION/repRNA-CoV2S elicits robust anti-SARS-CoV-2 spike protein IgG antibody isotypes indicative of a Type 1 T helper response as well as potent T cell responses in mice. Importantly, a single-dose administration in nonhuman primates elicited antibody responses that potently neutralized SARS-CoV-2. These data support further development of LION/repRNA-CoV2S as a vaccine candidate for prophylactic protection from SARS-CoV-2 infection.
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An adult male rhesus macaque (Macaca mulatta) that was enrolled in a study evaluating cognition and memory presented with suppurative exudate along the margins of a long-standing cranial implant that included a stainless-steel head post, plastic left-sided recording cylinder, and acrylic over a previously placed right-sided recording cylinder. Both cylinders were located at the level of the prefrontal cortex. After treatment comprising systemic antibiotics and daily cleaning with povidone-iodine for several months, the macaque underwent single-photon emission computed tomography-computed tomography (SPECT-CT) in which his neutrophils were labeled with 99mTc-hexamethylpropylene amine oxime ( 99m Tc-HMPAO) to evalu- ate for active infection below the implant. Soft tissue inflammation and osteomyelitis were found at the site of the previous right-sided recording cylinder. Cephalosporin and tetracycline antibiotics were administered for 12 wk. Follow-up SPECT-CT imaging was then performed to evaluate response to medical treatment. Results indicated no change in the degrees of soft tissue inflammation and osteomyelitis associated with the right-sided recording cylinder site. SPECT-CT imaging was used to guide the surgical removal of the implant and debridement of the infected tissue. On removal of the entire cranial implant, the osteomyelitis and soft tissue inflammation observed on the pre- and posttreatment SPECT-CT scans were confirmed. In addition, a large cavitary defect through the calvarium with suppurative exudate was discovered below the base of the head post. Infection in this defect was not apparent on SPECT; however, the bony defect was confirmed on reevaluation of the CT images. We concluded that the infection in this defect was silent on SPECT due to the limited vascularization of the sur-rounding bone and the chronicity of the infection. This case study is the first to describe the use of SPECT-CT for evaluating soft tissue inflammation and osteomyelitis beneath a cranial implant in a NHP.
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Macaca mulatta , Doenças dos Macacos/diagnóstico por imagem , Infecções Relacionadas à Prótese/veterinária , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único/veterinária , Crânio/diagnóstico por imagem , Crânio/cirurgia , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Inflamação/diagnóstico por imagem , Inflamação/etiologia , Inflamação/veterinária , Leucócitos , Macaca mulatta/cirurgia , Masculino , Osteomielite/diagnóstico por imagem , Osteomielite/etiologia , Osteomielite/veterinária , Próteses e Implantes , Infecções Relacionadas à Prótese/complicações , Infecções Relacionadas à Prótese/diagnóstico por imagem , Compostos Radiofarmacêuticos , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único/métodos , Crânio/patologia , Tecnécio Tc 99m ExametazimaRESUMO
An adult female beagle (Canis lupus familiaris) used in a model of doxorubicin-induced cardiomyopathy presented with epithelial desquamation on the shoulders and ventrum after receiving the 8th weekly intravenous dose of the free form of doxorubicin (20 mg/m2; total accumulation, 160 mg/m2). The lesions were empirically treated with topical disinfectants and topical and systemic antibiotics. Despite treatment, the lesions progressed and ulcerated. Bacterial culture revealed Staphylococcus aureus, but trichogram, skin scraping, and fungal culture were negative for microorganisms. Skin biopsies revealed epidermal and apocrine gland hyperplasia, apocrine gland dilation, abnormal maturation of epithelial keratinocytes, and perivascular lymphocytic infiltration. These histopathologic findings resemble those in humans and canines after chronic administration of doxorubicin-containing pegylated liposomes. Here we report a clinical presentation after chronic administration of the free form of doxorubicin. In dogs, cutaneous toxicity after administration of pegylated liposomal doxorubicin is most often localized to the footpads, limbs, and axillary and urogenital regions. In the current case, lesions affected the ventrum and trunk but did not involve the footpads or axillary or urogenital regions.
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Doenças do Cão/induzido quimicamente , Doxorrubicina/efeitos adversos , Dermatopatias/veterinária , Animais , Cardiomiopatias/induzido quimicamente , Doenças do Cão/diagnóstico , Cães , Doxorrubicina/administração & dosagem , Feminino , Dermatopatias/induzido quimicamente , Dermatopatias/microbiologiaRESUMO
Loss-of-function or inactivating mutations in the genes coding for kisspeptin and its receptor (KISS1R) or neurokinin B (NKB) and the NKB receptor (NK3R) in humans result in a delay in or the absence of puberty. However, precise mechanisms of kisspeptin and NKB signaling in the regulation of the pubertal increase in gonadotropin-releasing hormone (GnRH) release in primates are unknown. In this study, we conducted a series of experiments infusing agonists and antagonists of kisspeptin and NKB into the stalk-median eminence, where GnRH, kisspeptin, and NKB neuroterminal fibers are concentrated, and measuring GnRH release in prepubertal and pubertal female rhesus monkeys. Results indicate that (1) similar to those previously reported for GnRH stimulation by the KISS1R agonist (i.e., human kisspeptin-10), the NK3R agonist senktide stimulated GnRH release in a dose-responsive manner in both prepubertal and pubertal monkeys; (2) the senktide-induced GnRH release was blocked in the presence of the KISS1R antagonist peptide 234 in pubertal but not prepubertal monkeys; and (3) the kisspeptin-induced GnRH release was blocked in the presence of the NK3R antagonist SB222200 in the pubertal but not prepubertal monkeys. These results are interpreted to mean that although, in prepubertal female monkeys, kisspeptin and NKB signaling to GnRH release is independent, in pubertal female monkeys, a reciprocal signaling mechanism between kisspeptin and NKB neurons is established. We speculate that this cooperative mechanism by the kisspeptin and NKB network underlies the pubertal increase in GnRH release in female monkeys.
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Hormônio Liberador de Gonadotropina/metabolismo , Kisspeptinas/fisiologia , Macaca mulatta/fisiologia , Neurocinina B/fisiologia , Maturidade Sexual/fisiologia , Transdução de Sinais/fisiologia , Animais , Feminino , Kisspeptinas/agonistas , Kisspeptinas/antagonistas & inibidores , Kisspeptinas/farmacologia , Eminência Mediana/efeitos dos fármacos , Neurocinina B/agonistas , Neurocinina B/antagonistas & inibidores , Neurônios/metabolismo , Fragmentos de Peptídeos/farmacologia , Quinolinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores de Kisspeptina-1 , Receptores da Neurocinina-3/agonistas , Transdução de Sinais/efeitos dos fármacos , Substância P/análogos & derivados , Substância P/farmacologiaRESUMO
Release of gonadotropin releasing hormone (GnRH) from the medial basal hypothalamus (MBH)/median eminence region (S-ME) is essential for normal reproductive function. GnRH release is profoundly regulated by the negative and positive feedback effects of ovarian estradiol (E2). Here we report that neuroestradiol, released in the S-ME, also directly influences GnRH release in ovariectomized female monkeys, in which the ovarian source of E2 is removed. We found that (1) brief infusion of E2 benzoate (EB) to the S-ME rapidly stimulated release of GnRH and E2 in the S-ME of ovariectomized monkeys, (2) electrical stimulation of the MBH resulted in GnRH release as well as E2 release, and (3) direct infusion of an aromatase inhibitor to the S-ME suppressed spontaneous GnRH release as well as the EB-induced release of GnRH and E2. These findings reveal the importance of neuroestradiol as a neurotransmitter in regulation of GnRH release. How circulating ovarian E2 interacts with hypothalamic neuroestrogens in the control of GnRH release remains to be investigated.
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Estradiol/análogos & derivados , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Animais , Inibidores da Aromatase/farmacologia , Cromatografia Líquida de Alta Pressão , Estimulação Elétrica , Eletrodos Implantados , Estradiol/farmacologia , Feminino , Hipotálamo Médio/efeitos dos fármacos , Hipotálamo Médio/metabolismo , Letrozol , Macaca mulatta , Espectrometria de Massas , Eminência Mediana/efeitos dos fármacos , Eminência Mediana/metabolismo , Microdiálise , Nitrilas/farmacologia , Ovariectomia , Radioimunoensaio , Triazóis/farmacologiaRESUMO
Since the discovery of the G-protein coupled receptor 54 (kisspeptin receptor) and its ligand, kisspeptin, our understanding of the neurobiological mechanisms that govern the pituitary-gonadal axis has evolved dramatically. In this chapter, we have reviewed progress regarding the relationship between kisspeptin and puberty, and have proposed a novel hypothesis for the role of kisspeptin signaling in the onset of this crucial developmental event. According to this hypothesis, although kisspeptin neurons in the arcuate nucleus (ARC) are critical for puberty, this is simply because these cells are an integral component of the hypothalamic GnRH pulse generating mechanism that drives intermittent release of the decapeptide, as an increase in GnRH is obligatory for the onset of puberty. In our model, ARC kisspeptin neurons play no "regulatory" role in controlling the timing of puberty. Rather, as a component of the neural network responsible for GnRH pulse generation, they subserve upstream regulatory mechanisms that are responsible for the timing of puberty.
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Kisspeptinas/metabolismo , Modelos Biológicos , Puberdade/fisiologia , Transdução de Sinais/fisiologia , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Feminino , Hormônio Liberador de Gonadotropina/metabolismo , Humanos , Masculino , Neurônios/metabolismo , Hipófise/metabolismoRESUMO
Previously we have shown that a reduction in γ-amino butyric acid (GABA) inhibition is critical for the mechanism initiating puberty onset because chronic infusion of the GABA(A) receptor antagonist, bicuculline, significantly increased GnRH release and accelerated the timing of menarche and first ovulation in female rhesus monkeys. Because previous studies in our laboratory indicate that in prepubertal female monkeys, kisspeptin release in the medial basal hypothalamus is low, whereas kisspeptin-10 can stimulate GnRH release, we hypothesized that a low level of kisspeptin release prior to puberty onset is due to tonic GABA inhibition. To test this hypothesis we examined the effects of bicuculline infusion on kisspeptin release using a microdialysis method. We found that bicuculline at 1 µM dramatically stimulates kisspeptin release in the medial basal hypothalamus of prepubertal monkeys but had little effect on kisspeptin release in midpubertal monkeys. We further examined whether bicuculline-induced GnRH release is blocked by the presence of the kisspeptin antagonist, peptide 234. We found that inhibition of kisspeptin signaling blocked the bicuculline-induced stimulation of GnRH release, suggesting that kisspeptin neurons may relay inhibitory GABA signals to GnRH neurons. This implies that a reduction in tonic GABA inhibition of GnRH release is, at least in part, mediated through kisspeptin neurons.
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Kisspeptinas/metabolismo , Animais , Bicuculina/farmacologia , Feminino , Hormônio Liberador de Gonadotropina/metabolismo , Haplorrinos , Hipotálamo/metabolismo , Modelos Biológicos , Puberdade , Radioimunoensaio/métodos , Receptores de GABA-A/metabolismo , Transdução de Sinais , Ácido gama-Aminobutírico/metabolismoRESUMO
Kisspeptin (KP) signaling has been proposed as an important regulator in the mechanism of puberty. In this study, to determine the role of KP in puberty, we assessed the in vivo release pattern of KP-54 from the basal hypothalamus/stalk-median eminence in prepubertal and pubertal ovarian-intact female rhesus monkeys. We found that there was a developmental increase in mean KP-54 release, pulse frequency, and pulse amplitude, which is parallel to the developmental changes in GnRH release that we previously reported. Moreover, a nocturnal increase in KP-54 release becomes prominent after the onset of puberty. Because the pubertal increase in GnRH release occurs independent of the pubertal increase in circulating gonadal steroids, we further examined whether ovariectomy (OVX) modifies the release pattern of KP-54. Results show that OVX in pubertal monkeys enhanced mean KP-54 release and pulse amplitude but not pulse frequency, whereas OVX did not alter the release pattern of KP-54 in prepubertal monkeys. Estradiol replacement in OVX pubertal monkeys suppressed mean KP-54 release and pulse amplitude but not pulse frequency. Estradiol replacement in OVX prepubertal monkeys did not alter the KP-54 release pattern. Collectively these results suggest that the pubertal increase in KP release occurs independent of the pubertal increase in circulating estradiol. Nevertheless, the pubertal increase in KP release is not likely responsible for the initiation of the pubertal increase in GnRH release. Rather, after puberty onset, the increase in KP release contributes to further increase GnRH release during the progression of puberty.
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Envelhecimento/metabolismo , Estradiol/metabolismo , Kisspeptinas/metabolismo , Macaca mulatta/metabolismo , Maturidade Sexual/fisiologia , Animais , Estradiol/farmacologia , Feminino , Hormônio Liberador de Gonadotropina/metabolismo , Modelos Animais , Ovariectomia , Transdução de Sinais/fisiologiaRESUMO
Kisspeptin (KP) and KP-1 receptor (KISS1R) have emerged as important upstream regulators in the control of puberty. However, how developmental changes in KP-KISS1R contribute to the pubertal increase in GnRH release still remains elusive. In this study, we examined the effects of the KP agonist, human KP-10 (hKP-10), and the KP antagonist, peptide 234, on in vivo GnRH release in prepubertal and pubertal ovarian-intact female rhesus monkeys using a microdialysis method. We found that direct infusion of hKP-10 into the medial basal hypothalamus and stalk-median eminence region stimulated GnRH release in a dose-responsive manner, whereas infusion of peptide 234 suppressed GnRH release in both developmental stages. Because ovarian steroid feedback on GnRH release becomes prominent after the initiation of puberty in primates, we further examined whether ovarian steroids modify the GnRH response to hKP-10. Results demonstrate that the hKP-10-induced stimulation of GnRH release was eliminated by ovariectomy in pubertal, but not prepubertal, monkeys. Furthermore, replacement of estradiol into ovariectomized pubertal monkeys resulted in a partial recovery of the hKP-10-induced GnRH release. Collectively, these results suggest that a KISS1R-mediated mechanism, in addition to the pubertal increase in KP-54 release we previously reported, contributes to the pubertal increase in GnRH release and that there is a switch from an ovarian steroid-independent to -dependent mechanism in the response of GnRH to KP.
Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Kisspeptinas/antagonistas & inibidores , Macaca mulatta/metabolismo , Peptídeos/antagonistas & inibidores , Maturidade Sexual/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Estradiol/sangue , Estradiol/farmacologia , Feminino , Hormônio Liberador de Gonadotropina/sangue , Kisspeptinas/administração & dosagem , Kisspeptinas/agonistas , Kisspeptinas/farmacologia , Macaca mulatta/crescimento & desenvolvimento , OvariectomiaRESUMO
Neurons that produce gonadotropin-releasing hormone (GnRH) are the final common pathway by which the brain regulates reproduction. GnRH neurons are regulated by an afferent network of kisspeptin-producing neurons. Kisspeptin binds to its cognate receptor on GnRH neurons and stimulates their activity, which in turn provides an obligatory signal for GnRH secretion, thus gating down-stream events supporting reproduction. We have developed kisspeptin antagonists to facilitate the direct determination of the role of kisspeptin neurons in the neuroendocrine regulation of reproduction. In vitro and in vivo studies of analogues of kisspeptin-10 with amino substitutions have identified several potent and specific antagonists. A selected antagonist was shown to inhibit the firing of GnRH neurons in the brain of the mouse and to reduce pulsatile GnRH secretion in female pubertal monkeys; the later supporting a key role of kisspeptin in puberty onset. This analog also inhibited the kisspeptin-induced release of luteinizing hormone (LH) in rats and mice and blocked the postcastration rise in LH in sheep, rats, and mice, suggesting that kisspeptin neurons mediate the negative feedback effect of sex steroids on gonadotropin secretion in mammals. The development of kisspeptin antagonists provides a valuable tool for investigating the physiological and pathophysiological roles of kisspeptin in the regulation of reproduction and could offer a unique therapeutic agent for treating hormone-dependent disorders of reproduction, including precocious puberty, endometriosis, and metastatic prostate cancer.
Assuntos
Hormônio Liberador de Gonadotropina/fisiologia , Peptídeos/farmacologia , Proteínas Supressoras de Tumor/antagonistas & inibidores , Potenciais de Ação , Animais , Encéfalo/fisiologia , Células CHO , Castração , Cricetinae , Cricetulus , Feminino , Hormônio Liberador de Gonadotropina/metabolismo , Humanos , Técnicas In Vitro , Kisspeptinas , Hormônio Luteinizante/metabolismo , Macaca mulatta , Masculino , Camundongos , Microdiálise , Peptídeos/química , Ratos , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética , Receptores de Kisspeptina-1 , Ovinos , Relação Estrutura-Atividade , Proteínas Supressoras de Tumor/químicaRESUMO
Kisspeptin is recognized to play a critical role in eliciting the pubertal resurgence of pulsatile GnRH release, the proximal trigger of puberty in higher primates. Expression of the kisspeptin receptor (GPR54) by GnRH neurons indicates a direct action of kisspeptin on the GnRH neuronal network. The purpose of the present study was to examine the distribution of kisspeptin cell bodies in the monkey hypothalamus and to assess the structural basis for the stimulatory action of kisspeptin on the GnRH neuronal network. Three castrated male rhesus monkeys, 39-51 months of age, were deeply anesthetized and their brains perfused transcardially with 4% paraformaldehyde in PBS. Serial 25-microm coronal sections throughout the hypothalamus were prepared, and immunopositive neurons identified using a cocktail of specific primary antibodies (sheep anti-kisspeptin at 1:120,000, and rabbit anti-GnRH at 1:100,000) detected with fluorescently tagged secondary antibodies (antisheep, Alexa Fluor 488; antirabbit, Cy3) in combination with confocal microscopy. Kisspeptin perikarya were found only in the mediobasal hypothalamus (MBH) almost exclusively in the posterior two-thirds of the arcuate nucleus. Surprisingly, kisspeptin-beaded axons made only infrequent contacts with GnRH neurons (kisspeptin and GnRH profiles abutting in a 0.5- to 1.0-mum optical section) in the MBH. In the median eminence, kisspeptin and GnRH axons were found in extensive and intimate association. GnRH contacts on kisspeptin perikarya and dendrites were observed. These findings indicate that nonsynaptic pathways of communication in the median eminence should be considered as a possible mechanism of kisspeptin regulation of GnRH release, and provide an anatomical basis for reciprocal control of kisspeptin neuronal activity by GnRH.