RESUMO
To understand the differences in immune responses between early feathering (EF) and late feathering (LF) chickens after infection with avian leukosis virus, subgroup J (ALV-J), we monitored the levels of prolactin, growth hormone and the immunoglobulins IgG and IgM in the serum of LF and EF chickens for 8 weeks. Moreover, we analysed the expression of immune-related genes in the spleen and the expression of PRLR, SPEF2 and dPRLR in the immune organs and DF-1 cells by qRT-PCR. The results showed that ALV-J infection affected the expression of prolactin, growth hormone, IgG and IgM in the serum. Regardless of whether LF and EF chickens were infected with ALV-J, the serum levels of the two hormones and two immunoglobulins in EF chickens were higher than those in LF chickens (P < 0.05). However, the expression of immune-related genes in the spleen of positive LF chickens was higher than that in the spleen of positive EF chickens. In the four immune organs, PRLR and SPEF2 expression was also higher in LF chickens than in EF chickens. Furthermore, the dPRLR expression of positive LF chickens was higher than that of negative LF chickens. After infection with ALV-J, the expression of PRLR in DF-1 cells significantly increased. In addition, overexpression of PRLR or dPRLR in DF-1 cells promoted replication of ALV-J. These results suggested that the susceptibility of LF chickens to ALV-J might be induced by dPRLR.
Assuntos
Vírus da Leucose Aviária , Leucose Aviária , Doenças das Aves Domésticas , Receptores da Prolactina , Animais , Leucose Aviária/imunologia , Vírus da Leucose Aviária/imunologia , Galinhas , Hormônio do Crescimento , Imunidade , Imunoglobulina G , Imunoglobulina M , Prolactina , Receptores da Prolactina/imunologiaRESUMO
Endometriosis in an estrogen-dependent disease that is characterized by the presence of endometrial tissue outside the uterine cavity leading to pain and infertility in many affected women. Highly efficient treatment options which create a hypo-estrogenic environment can cause side effects such as hot flushes and bone mass loss that are not favorable for premenopausal women. Previous work has demonstrated that increased local or systemic prolactin seems to be involved in the pathogenesis of endometriosis. Here we examined two prolactin receptor (PRLR) blocking antibodies in a murine endometriosis interna model which relies on the induction of systemic hyperprolactinemia in female SHN mice. The severity of the disease is determined by the degree of endometrial invasion into the myometrium. In this model, endometriosis was inhibited by clinical gold standards such as progestins and anti-estrogenic approaches. PRLR blockade completely inhibited endometriosis in this mouse model to the same extent as the anti-estrogen faslodex or the GnRH antagonist cetrorelix. In contrast to cetrorelix and faslodex, the PRLR antibodies did not decrease relative uterine weights and were thus devoid of anti-estrogenic effects. We therefore hypothesize that PRLR antibodies may present a novel and highly efficient treatment option for endometriosis with a good safety and tolerability profile. Clinical studies are on the way to test this hypothesis.
Assuntos
Anticorpos/farmacologia , Endometriose/terapia , Antagonistas de Hormônios/farmacologia , Receptores da Prolactina/antagonistas & inibidores , Animais , Anticorpos/toxicidade , Modelos Animais de Doenças , Endometriose/imunologia , Feminino , Fulvestranto/farmacologia , Fulvestranto/toxicidade , Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/farmacologia , Hormônio Liberador de Gonadotropina/toxicidade , Antagonistas de Hormônios/toxicidade , Camundongos , Receptores da Prolactina/imunologiaRESUMO
Glucocorticoids (GCs) are widely used to treat a variety of autoimmune and inflammatory diseases; however, systemic delivery of GCs is associated with side effects that affect essentially every organ system, reflecting the nearly ubiquitous expression of the glucocorticoid receptor (GR). Targeted delivery of GCs to diseased tissues using antibody-glucocorticoid conjugates (GC-ADCs) offers a therapeutic alternative to overcome these adverse effects. Herein, we describe novel classes of GCs that exhibited greater potency than dexamethasone and budesonide, a 100-fold selectivity toward the GR over other nuclear receptors, and no in vitro safety liability in pharmacology assays (hERG, AMES) and that demonstrated a substantial reduction in tumor necrosis factor-α (TNF-α) release in mice challenged with lipopolysaccharide (LPS). The site-specific conjugated GC-ADCs via cathepsin-cleavable linkers were highly stable in plasma and specifically released GCs in antigen-positive cells, suggesting that these novel GCs can serve as ADC payloads to treat autoimmune and inflammatory diseases.
Assuntos
Budesonida/análogos & derivados , Budesonida/uso terapêutico , Glucocorticoides/uso terapêutico , Imunoconjugados/uso terapêutico , Inflamação/tratamento farmacológico , Animais , Budesonida/metabolismo , Budesonida/farmacocinética , Catepsina B/metabolismo , Glucocorticoides/síntese química , Glucocorticoides/metabolismo , Glucocorticoides/farmacocinética , Humanos , Imunoconjugados/química , Imunoconjugados/imunologia , Imunoconjugados/metabolismo , Inflamação/induzido quimicamente , Inflamação/imunologia , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Estrutura Molecular , Receptores de Glucocorticoides/metabolismo , Receptores da Prolactina/imunologia , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Prolactin receptor (PRLR) is highly expressed in a subset of human breast cancer and prostate cancer, which makes it a potential target for cancer treatment. In clinical trials, the blockade of PRLR was shown to be safe but with poor efficacy. It is therefore urgent to develop new therapies against PRLR target. Bispecific antibodies (BsAbs) could guide immune cells toward tumor cells, and produced remarkable effects in some cancers. METHODS: In this study, a bispecific antibody targeting both tumor antigen PRLR and T cell surface CD3 antigen (PRLR-DbsAb) was constructed by split intein mediated protein transsplicing (BAPTS) system for the first time. Its binding activity was determined by Biacore and Flow cytometry, and target-dependent T cell mediated cytotoxicity was detected using LDH release assay. ELISA was utilized to study the secretion of cytokines by immune cells. Subcutaneous tumor mouse models were used to analyze the in vivo anti-tumor effects of PRLR-DbsAb. RESULTS: PRLR-DbsAb in vitro could recruit and activate T cells to promote the release of Th1 cytokines IFN- γ and TNF- α, which could kill PRLR expressed breast cancer cells. In xenograft models with breast cancer cell line T47D, NOD/SCID mice intraperitoneally injected with PRLR-DbsAb exhibited significant inhibition of tumor growth and a longer survival compared to mice treated with PRLR monoclonal antibody (PRLR mAb). CONCLUSIONS: Both in vitro and in vivo experiments showed PRLR-DbsAb had a potential therapy of cancer treatment potential therapy for cancer. Immunotherapy may be a promising treatment against the tumor target of PRLR.
Assuntos
Anticorpos Biespecíficos/farmacologia , Neoplasias da Mama/terapia , Complexo CD3/imunologia , Receptores da Prolactina/imunologia , Animais , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Células Jurkat , Ativação Linfocitária , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Receptores da Prolactina/biossíntese , Linfócitos T/imunologia , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BAY 1158061 is a potent monoclonal prolactin (PRL) receptor antibody, blocking PRL receptor (PRLR)-mediated signaling in a noncompetitive manner, which was tested in a randomized, placebo-controlled multiple dose study in postmenopausal women. The objective was to investigate safety, tolerability, pharmacokinetic characteristics, and effects of BAY 1158061 on serum PRL level. The study consisted of 4 parallel groups receiving up to 3 subcutaneous (sc) administrations of BAY 1158061 or placebo in 2 different dosing regimens. Twenty-nine healthy postmenopausal women were randomized and treated with BAY 1158061 or placebo: 30 mg at 14-day interval (7 participants), 60 mg at 28-day interval (8 participants), 90 mg at 14-day interval (7 participants), and placebo (7 participants). To keep the blinding, all randomized participants received sc injections biweekly (14-day interval) on 3 occasions in the lower abdomen. The PRLR antibody showed a favorable safety and tolerability profile in postmenopausal women with no distinct differences in occurrence of adverse events in BAY 1158061 or placebo-treated participants. BAY 1158061 displayed low immunogenicity with low titers of antidrug antibodies and absence of neutralizing antidrug antibodies. Pharmacokinetics were characterized by slow absorption after sc administration with median peak plasma concentrations 7 to 11 days after first dose and about 2-fold accumulation after repeated dosing every 2 weeks. The apparent mean elimination half-life was 9 to 16 days. The PRL concentration-time profiles over 24 hours showed no differences between verum- and placebo-treated participants. Based on the data obtained, BAY 1158061 is considered a good candidate for further development in endometriosis or other PRL-mediated disease conditions.
Assuntos
Anticorpos Monoclonais/farmacologia , Receptores da Prolactina/antagonistas & inibidores , Anticorpos Monoclonais/sangue , Esquema de Medicação , Endometriose/prevenção & controle , Feminino , Humanos , Injeções Subcutâneas , Pessoa de Meia-Idade , Pós-Menopausa , Receptores da Prolactina/imunologiaRESUMO
Antibodies labeled with positron-emitting isotopes have been used for tumor detection, predicting which patients may respond to tumor antigen-directed therapy, and assessing pharmacodynamic effects of drug interventions. Prolactin receptor (PRLR) is overexpressed in breast and prostate cancers and is a new target for cancer therapy. We evaluated REGN2878, an anti-PRLR monoclonal antibody, as an immunoPET reagent. REGN2878 was labeled with Zr-89 after conjugation with desferrioxamine B or labeled with I-131/I-124. In vitro determination of the half-maximal inhibitory concentration (IC50) of parental REGN2878, DFO-REGN2878, and iodinated REGN2878 was performed by examining the effect of the increasing amounts of these on uptake of trace-labeled I-131 REGN2878. REGN1932, a non-PRLR binding antibody, was used as a control. Imaging and biodistribution studies were performed in mice bearing tumor xenografts with various expression levels of PRLR, including MCF-7, transfected MCF-7/PRLR, PC3, and transfected PC3/PRLR and T4D7v11 cell lines. The specificity of uptake in tumors was evaluated by comparing Zr-89 REGN2878 and REGN1932, and in vivo competition compared Zr-89 REGN2878 uptake in tumor xenografts with and without prior injection of 2 mg of nonradioactive REGN2878. The competition binding assay of DFO-REGN2878 at ratios of 3.53-5.77 DFO per antibody showed IC50 values of 0.4917 and 0.7136 nM, respectively, compared to 0.3455 nM for parental REGN2878 and 0.3343 nM for I-124 REGN2878. Imaging and biodistribution studies showed excellent targeting of Zr-89 REGN2878 in PRLR-positive xenografts at delayed times of 189 h (presented as mean ± 1 SD, percent injected activity per mL (%IA/mL) 74.6 ± 33.8%IA/mL). In contrast, MCF-7/PRLR tumor xenografts showed a low uptake (7.0 ± 2.3%IA/mL) of control Zr-89 REGN1932 and a very low uptake and rapid clearance of I-124 REGN2878 (1.4 ± 0.6%IA/mL). Zr-89 REGN2878 has excellent antigen-specific targeting in various PRLR tumor xenograft models. We estimated, using image-based kinetic modeling, that PRLR antigen has a very rapid in vivo turnover half-life of â¼14 min from the cell membrane. Despite relatively modest estimated tumor PRLR expression numbers, PRLR-expressing cells have shown final retention of the Zr-89 REGN2878 antibody, with an uptake that appeared to be related to PRLR expression. This reagent has the potential to be used in clinical trials targeting PRLR.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Imunoconjugados/administração & dosagem , Neoplasias/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/administração & dosagem , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Linhagem Celular Tumoral , Feminino , Humanos , Imunoconjugados/química , Imunoconjugados/imunologia , Imunoconjugados/farmacocinética , Camundongos , Camundongos Nus , Imagem Molecular/métodos , Neoplasias/patologia , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/imunologia , Compostos Radiofarmacêuticos/farmacocinética , Receptores da Prolactina/imunologia , Receptores da Prolactina/metabolismo , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Growth hormone receptor (GHR) and prolactin (PRL) receptor (PRLR) are homologous transmembrane class I cytokine receptors. In humans, GH interacts with GHR homodimers or PRLR homodimers and PRL interacts with only PRLR homodimers to promote signaling. In human breast cancer cells endogenously expressing both receptors, GHR and PRLR specifically coimmunoprecipitate. We previously devised a split luciferase complementation assay to study GHR and PRLR assemblages. In this technique, firefly luciferase is split into two fragments (N- and C-terminal fragments of the luciferase), each without enzyme activity and tethered to the tails of two receptors. The fragments restore luciferase activity when brought close to each other by the receptors. Real-time ligand-induced complementation changes reflect the arrangement of receptors and indicate that GHR/PRLR is arranged as a heteromultimer comprised of GHR-GHR homodimers and PRLR-PRLR homodimers. We now dissect determinants for GHR and PRLR homodimerization versus heteroassociation. GHR and PRLR have extracellular domains comprised of the ligand-binding N-terminal subdomain 1 and a membrane-proximal subdomain 2 (S2), which fosters receptor-receptor contact. Based on previous studies of S2 versus the transmembrane domain (TMD) in GHR dimerization, we constructed GHR(PRLRS2), GHR(PRLRS2-TMD), and GHR(PRLRTMD), replacing GHR's S2 alone, S2 plus TMD, and TMD alone with PRLR's counterpart. We tested by complementation the ability of these chimeras and GHR or PRLR to homodimerize or heteroassociate. Comparing various combinations, we found GHR(PRLRS2) and GHR(PRLRS2-TMD) behaved as PRLR, whereas GHR(PRLRTMD) behaved as GHR regarding their dimerization partners. We conclude that S2 of GHR and PRLR, rather than their TMDs, determines their dimerization partner.
Assuntos
Multimerização Proteica , Receptores da Prolactina/química , Receptores da Somatotropina/química , Anticorpos Monoclonais/farmacologia , Neoplasias da Mama/química , Linhagem Celular Tumoral , Membrana Celular/química , Hormônio do Crescimento/metabolismo , Humanos , Ligantes , Luciferases , Medições Luminescentes , Receptores da Prolactina/imunologia , Receptores da Prolactina/metabolismo , Receptores da Somatotropina/imunologia , Receptores da Somatotropina/metabolismo , Proteínas Recombinantes de FusãoRESUMO
Over the past decades, a number of prolactin receptor (PRLR) antagonists have been developed, which can be divided into two categories, PRLR analogue and anti-PRLR antibody. However, until now, there have been no commercially available PRLR antagonists. Here, we described a new approach for the preparation of PRLR antagonist, namely internal image anti-idiotypic antibody strategy. The hybridoma technique was used to generate anti-idiotypic antibodies to PRL. Competitive ELISA, competitive receptor-binding analysis and immunofluorescence assay (IFA) were then used to screen and characterize anti-idiotypic antibodies to PRL. One internal image anti-idiotypic antibody, termed MG7, was obtained. A series of experiments demonstrated that MG7 behaved as a typical internal image anti-idiotypic antibody (Ab2ß). MG7 exhibited effective antagonistic activity, which not only inhibited PRL binding to PRLR in a dose-dependent manner but also inhibited PRLR-mediated intracellular signalling. Furthermore, MG7 also blocked Nb2 cell proliferation induced by PRL. The current study suggests that MG7 has the potential application in the PRL/PRLR-related studies in future. In addition, this work also suggests that the internal image anti-idiotypic antibody may represent a novel strategy for the development of PRLR antagonist.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Receptores da Prolactina/antagonistas & inibidores , Receptores da Prolactina/imunologia , Animais , Anticorpos Monoclonais/imunologia , Ligação Competitiva/imunologia , Células CHO , Proliferação de Células/fisiologia , Cricetinae , Cricetulus , Hibridomas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação/imunologia , Prolactina/imunologia , Ligação Proteica/imunologia , Transdução de Sinais/imunologiaRESUMO
The Prolactin Receptor (PRLR) is a type 1 cytokine receptor that is expressed in a subset of breast cancers and may contribute to its pathogenesis. It is relatively overexpressed in approximately 25% of human breast tumors while expressed at low levels in some normal human tissues including the mammary gland. We developed an anti-PRLR antibody-drug conjugate (ADC), to target PRLR-positive breast cancer. REGN2878-DM1 is comprised of a fully human high-affinity function-blocking anti-PRLR IgG1 antibody (REGN2878) conjugated via a noncleavable SMCC linker to the cytotoxic maytansine derivative DM1. Both unconjugated REGN2878 and conjugated REGN2878-DM1 block PRL-mediated activation in vitro and are rapidly internalized into lysosomes. REGN2878-DM1 induces potent cell-cycle arrest and cytotoxicity in PRLR-expressing tumor cell lines. In vivo, REGN2878-DM1 demonstrated significant antigen-specific antitumor activity against breast cancer xenograft models. In addition, REGN2878-DM1 showed additive activity when combined with the antiestrogen agent fulvestrant. These results illustrate promising antitumor activity against PRLR-positive breast cancer xenografts and support the evaluation of anti-PRLR ADCs as potential therapeutic agents in breast cancer. Mol Cancer Ther; 16(7); 1299-311. ©2017 AACR.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Imunoconjugados/administração & dosagem , Receptores da Prolactina/imunologia , Animais , Anticorpos Anti-Idiotípicos/administração & dosagem , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Imunoconjugados/imunologia , Camundongos , Receptores da Prolactina/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In this work, we developed a new prolactin receptor (PRLR) antagonist using the hybridoma technique. A series of monoclonal antibodies against prolactin receptor (PRLR) was prepared, from which we characterized and selected one anti-PRLR antibody, F56. Epitome mapping showed that F56 and prolactin (PRL) share a common binding epitope on PRLR, and therefore, F56 could compete with prolactin (PRL) for binding to PRLR. Subsequent experiments indicated that F56 could effectively neutralize PRLR-mediated intracellular signalling molecules, such as signal transducer and activator of transcription (STAT) and extracellular signal-regulated kinase-1 and kinase 2 (ERK1/2), either by endogenously expressed PRLR or in a cell model transfected with PRLR. In addition, further experiments showed that F56 could effectively inhibit PRL-induced cell proliferation. The current study suggests that F56 has potential applications in PRLR-related studies.
Assuntos
Anticorpos Monoclonais/biossíntese , Epitopos/química , Receptores da Prolactina/antagonistas & inibidores , Receptores da Prolactina/química , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/farmacologia , Ligação Competitiva , Células CHO , Proliferação de Células , Cricetulus , Mapeamento de Epitopos , Epitopos/imunologia , Regulação da Expressão Gênica , Humanos , Hibridomas/imunologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Prolactina/metabolismo , Ligação Proteica , Receptores da Prolactina/imunologia , Receptores da Prolactina/metabolismo , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/imunologia , Transdução de Sinais , TransfecçãoRESUMO
In this work, we prepared a panel of monoclonal antibodies directed against prolactin receptor (PRLR) using the hybridoma technique. Of these monoclonal antibodies (Mabs), the Mab designated B6 was chosen for further characterization based on its biological activity. We first demonstrated that B6 can specifically bind to the prolactin receptor (PRLR) expressed on target cells by immunoprecipitation and Western blotting analysis. Subsequently, epitope mapping studies using a competitive receptor-binding assay indicated that B6 epitopes partially overlapped with those of prolactin (PRL). We then examined the resulting signal transduction pathways activated by this antibody in T-47D and CHO-PRLR cells and found that B6 induced different intracellular signalling compared with prolactin, which activates serine-threonine kinase (AKT), extracellular signal-regulated kinase 1/2 (ERK1/2), signal transducer and activator of transcription1 (STAT1) and STAT3 but not STAT5. The present study suggests that: (a) B6 may be a signal-specific prolactin receptor (PRLR) agonist; (b) B6 may be a biological reagent that can be used to explore the mechanism of PRLR-mediated intracellular signalling. In addition, this work also implies a strategy for preparing signal-specific cytokine agonists.
Assuntos
Anticorpos Monoclonais/farmacologia , Receptores da Prolactina/antagonistas & inibidores , Receptores da Prolactina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Células CHO , Linhagem Celular Tumoral , Cricetulus , Epitopos/imunologia , Epitopos/metabolismo , Feminino , Humanos , Espaço Intracelular/metabolismo , Janus Quinase 2/metabolismo , Camundongos , Ligação Proteica , Receptores da Prolactina/imunologiaRESUMO
The prolactin receptor (PRLR) has been implicated in a variety of physiological processes (lactation, reproduction) and diseases (breast cancer, autoimmune diseases). Prolactin synthesis in the pituitary and extrapituitary sites is regulated by different promoters. Dopamine receptor agonists such as bromocriptine can only interfere with pituitary prolactin synthesis and thus do not induce a complete blockade of PRLR signaling. Here we describe the identification of a human monoclonal antibody 005-C04 that blocks PRLR-mediated signaling at nanomolar concentrations in vitro. In contrast to a negative control antibody, the neutralizing PRLR antibody 005-C04 inhibits signal transducer and activator of transcription 5 phosphorylation in T47D cells and proliferation of BaF3 cells stably expressing murine or human PRLRs in a dose-dependent manner. In vivo application of this new function-blocking PRLR antibody reflects the phenotype of PRLR-deficient mice. After antibody administration female mice become infertile in a reversible manner. In lactating dams, the antibody induces mammary gland involution and negatively interferes with lactation capacity as evidenced by reduced milk protein expression in mammary glands and impaired litter weight gain. Antibody-mediated blockade of the PRLR in vivo stimulates hair regrowth in female mice. Compared with peptide-derived PRLR antagonists, the PRLR antibody 005-C04 exhibits several advantages such as higher potency, noncompetitive inhibition of PRLR signaling, and a longer half-life, which allows its use as a tool compound also in long-term in vivo studies. Therefore, we suggest that this antibody will help to further our understanding of the role of auto- and paracrine PRLR signaling in health and disease.
Assuntos
Anticorpos Neutralizantes/administração & dosagem , Fenótipo , Receptores da Prolactina/imunologia , Animais , Relação Dose-Resposta a Droga , Feminino , Camundongos , Camundongos Knockout , Receptores da Prolactina/genética , Receptores da Prolactina/metabolismoRESUMO
Tumor necrosis factor (TNF)-α is one of the major proinflammatory mediators of rheumatic arthritis (RA); the regulatory factors for TNF-α release is not fully understood. This study aims to investigate the role of prolactin receptor (PRLR) activation in regulating the expression and release of TNF-α from CD14(+) monocytes. The results showed that the expression of PRLR was detectable in CD14(+) monocytes of healthy subjects, which was markedly increased in RA patients. Exposure to PRL in the culture increased the expression and release of TNF-α from CD14(+) monocytes, which was abolished by the PRLR gene silencing or blocking the mitogen activated protein (MAPK) pathway. We conclude that exposure to PRL increases TNF-α release from CD14(+) monocytes of RA patients, which can be abolished by PRLR gene silencing or treating with MAPK inhibitor.
Assuntos
Artrite Reumatoide/imunologia , Monócitos/imunologia , Receptores da Prolactina/genética , Fator de Necrose Tumoral alfa/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Flavonoides/farmacologia , Regulação da Expressão Gênica/imunologia , Inativação Gênica , Humanos , Receptores de Lipopolissacarídeos/biossíntese , Sistema de Sinalização das MAP Quinases/imunologia , Prolactina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptores da Prolactina/imunologia , Fator de Necrose Tumoral alfa/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
Numerous lines of evidence suggest that the polypeptide hormone prolactin (PRL) may contribute to breast and prostate tumorigenesis through its interactions with the prolactin receptor (PRLR). Here, we describe the biologic properties of LFA102, a humanized neutralizing monoclonal antibody directed against the extracellular domain of PRLR. This antibody was found to effectively antagonize PRL-induced signaling in breast cancer cells in vitro and in vivo and to block PRL-induced proliferation in numerous cell line models, including examples of autocrine/paracrine PRL activity. A single administration of LFA102 resulted in regression of PRL-dependent Nb2-11 tumor xenografts and significantly prolonged time to progression. Finally, LFA102 treatment significantly inhibited PRLR signaling as well as tumor growth in a carcinogen-induced, estrogen receptor-positive rat mammary cancer model as a monotherapy and enhanced the efficacy of the aromatase inhibitor letrozole when administered in combination. The biologic properties of LFA102, elucidated by the preclinical studies presented here, suggest that this antibody has the potential to be a first-in-class, effective therapeutic for the treatment of PRL-dependent cancers.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Receptores da Prolactina/imunologia , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Células MCF-7 , Camundongos , Terapia de Alvo Molecular , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Prolactina/metabolismo , Ratos , Receptores da Prolactina/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Disorders in the hypothalamic-pituitary-adrenal axis are associated with the pathogenesis of Trypanosoma cruzi infection. During the acute phase of this disease, increased levels of circulating glucocorticoids (GCs) correlate with thymic atrophy. Recently, we demonstrated that this phenomenon is paralleled by a decrease of prolactin (PRL) secretion, another stress hormone that seems to counteract many immunosuppressive effects of GCs. Both GCs and PRL are intrathymically produced and exhibit mutual antagonism through the activation of their respective receptors, GR, and PRLR. Considering that GCs induce apoptosis and inhibit double-positive (DP) thymocyte proliferation and that PRL administration prevents these effects, it seems plausible that a local imbalance of GR-PRLR crosstalk underlies the thymic involution occurring in acute T. cruzi infection. In this respect, preserving PRLR signaling seems to be crucial for protecting DP from GC-induced apoptosis.
Assuntos
Doença de Chagas/imunologia , Doença de Chagas/patologia , Timo/patologia , Animais , Apoptose/imunologia , Atrofia , Glucocorticoides/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Camundongos , Neuroimunomodulação , Sistemas Neurossecretores/imunologia , Prolactina/imunologia , Receptor Cross-Talk , Receptores de Glucocorticoides/imunologia , Receptores da Prolactina/imunologia , Transdução de Sinais , Estresse Fisiológico , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Timo/imunologiaRESUMO
Macroprolactinemia is characterized by a large molecular mass of PRL (macroprolactin) as the main molecular form of PRL in sera, the frequent elevation of serum PRL (hyperprolactinemia), and the lack of symptoms. Macroprolactin is largely a complex of PRL with immunoglobulin G (IgG), especially anti-PRL autoantibodies. The prevalence of macroprolactinemia is 10-25% in patients with hyperprolactinemia and 3.7% in general population. There is no gender difference and a long-term followup demonstrates that macroprolactinemia develops before middle age and is likely a chronic condition. Polyethylene-glycol- (PEG-) precipitation method is widely used for screening macroprolactinemia, and gel filtration chromatography, protein A/G column, and I125-PRL binding studies are performed to confirm and clarify its nature. The cross-reactivity of macroprolactin varies widely according to the immunoassay systems. The epitope on PRL molecule recognized by the autoantibodies is located close to the binding site for PRL receptors, which may explain that macroprolactin has a lower biological activity. Hyperprolactinemia frequently seen in macroprolactinemic patients is due to the delayed clearance of autoantibody-bound PRL. When rats are immunized with rat pituitary PRL, anti-PRL autoantibodies are produced and hyperprolactinemia develops, mimicking macroprolactinemia in humans. Screening of macroprolactinemia is important for the differential diagnosis of hyperprolactinemia to avoid unnecessary examinations and treatments.
Assuntos
Hiperprolactinemia/sangue , Prolactina/sangue , Animais , Autoanticorpos/sangue , Autoanticorpos/imunologia , Humanos , Hiperprolactinemia/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Receptores da Prolactina/sangue , Receptores da Prolactina/imunologiaRESUMO
The predominant prevalence of autoimmune diseases in women of reproductive age has led to the investigation of the effects of sex hormones on immune regulation and in autoimmune diseases, in particular the prototypic systemic autoimmune disease lupus. The female hormone prolactin has receptors beyond the reproductive axis including immune cells, and it is thought to promote autoimmunity in human and murine lupus. Induced hyperprolactinemia in experimental lupus models, regardless of gender, exacerbates disease activity and leads to premature death. Prolactin treatment in mice that are not prone to develop lupus leads to the development of a lupus-like phenotype. Persistent mild-moderate hyperprolactinemia alters the selection of the naïve B cell repertoire. Recent studies demonstrate that prolactin impairs all three mechanisms of B cell tolerance induction (negative selection, receptor editing, and anergy) and thereby contributes to the pathogenesis of autoimmunity. The effects of prolactin are genetically determined as shown by the differential response to the hormone in the different mice strains. Bromocriptine, a drug that inhibits prolactin secretion, abrogates some of the immune effects of this hormone. Further research is required to elucidate molecular mechanisms involved in immune effects of prolactin and to develop novel targeted treatments for SLE patients with prolactin-responsive disease.
Assuntos
Linfócitos B/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Prolactina/imunologia , Animais , Autoimunidade/genética , Autoimunidade/imunologia , Modelos Animais de Doenças , Estrogênios/imunologia , Humanos , Imunidade/genética , Imunidade/imunologia , Lúpus Eritematoso Sistêmico/induzido quimicamente , Receptores da Prolactina/imunologiaRESUMO
BACKGROUND: Prolactin is a polypeptide hormone responsible for proliferation and differentiation of the mammary gland. More recently, prolactin's role in mammary carcinogenesis has been studied with greater interest. Studies from our laboratory and from others have demonstrated that three specific isoforms of the prolactin receptor (PRLR) are expressed in both normal and cancerous breast cells and tissues. Until now, reliable isoform specific antibodies have been lacking. We have prepared and characterized polyclonal antibodies against each of the human PRLR isoforms that can effectively be used to characterize human breast cancers. METHODS: Rabbits were immunized with synthetic peptides of isoform unique regions and immune sera affinity purified prior to validation by Western blot and immunohistochemical analyses. Sections of ductal and lobular carcinomas were stained with each affinity purified isoform specific antibody to determine expression patterns in breast cancer subclasses. RESULTS: We show that the rabbit antibodies have high titer and could specifically recognize each isoform of PRLR. Differences in PRLR isoform expression levels were observed and quantified using histosections from xenografts of established human breast cancer cells lines, and ductal and lobular carcinoma human biopsy specimens. In addition, these results were verified by real-time PCR with isoform specific primers. While nearly all tumors contained LF and SF1b, the majority (76%) of ductal carcinoma biopsies expressed SF1a while the majority of lobular carcinomas lacked SF1a staining (72%) and 27% had only low levels of expression. CONCLUSIONS: Differences in the receptor isoform expression profiles may be critical to understanding the role of PRL in mammary tumorigenesis. Since these antibodies are specifically directed against each PRLR isoform, they are valuable tools for the evaluation of breast cancer PRLR content and have potential clinical importance in treatment of this disease by providing new reagents to study the protein expression of the human PRLR.
Assuntos
Anticorpos , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma Lobular/metabolismo , Imuno-Histoquímica , Receptores da Prolactina/metabolismo , Animais , Especificidade de Anticorpos , Biópsia , Western Blotting , Neoplasias da Mama/genética , Células CHO , Carcinoma Ductal de Mama/genética , Carcinoma Lobular/genética , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Feminino , Humanos , Camundongos , Camundongos Nus , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Coelhos , Receptores da Prolactina/genética , Receptores da Prolactina/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Ample evidence indicates that prolactin (PRL) secreted from the pituitary gland plays an important role in a variety of human immune responses. However, the immunoregulation of autocrine PRL in T lymphocytes is not fully understood. To evaluate the role of autocrine PRL in T lymphocyte activation, PRL receptor (PRLR) in Jurkat cells was silenced by lentivirus-mediated stable expression of PRLR shRNAi. Knockdown of PRLR resulted in a considerable reduction of phytohemagglutinin (PHA)-induced T cell proliferation. Moreover, the synthesis and secretion of CD137, CD154, IL-2 and IL-4 were significantly decreased, while the production of CD28, IFN-gamma and IL-10 was not affected in PHA-primed PRLR-deficient cells. These results demonstrate the importance of autocrine regulation of the PRL signaling in T lymphocyte growth and activation, and support a mechanism by which autocrine PRL participates in the immunoregulation through selectively influencing the expression of certain critical costimulatory molecules and cytokines.
Assuntos
Antígenos CD/metabolismo , Comunicação Autócrina , Citocinas/metabolismo , Imunomodulação , Prolactina , Receptores da Prolactina/antagonistas & inibidores , Receptores da Prolactina/metabolismo , Linfócitos T/metabolismo , Antígenos CD/genética , Antígenos CD/imunologia , Comunicação Autócrina/imunologia , Proliferação de Células , Citocinas/genética , Citocinas/imunologia , Regulação para Baixo , Técnicas de Silenciamento de Genes , Vetores Genéticos , Humanos , Células Jurkat , Lentivirus/genética , Ativação Linfocitária/genética , Prolactina/imunologia , Prolactina/metabolismo , RNA Interferente Pequeno/genética , Receptores da Prolactina/genética , Receptores da Prolactina/imunologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
The pituitary hormone PRL is involved in tumorigenesis in rodents and humans. PRL promotes proliferation, survival and migration of cancer cells acting via the PRL receptor (PRLR). Aiming to perform a large-scale immunohistochemical (IHC) screening of human mammary carcinomas for PRLR expression, we evaluated the specificity of commercially available anti-human PRLR antibodies (B6.2, U5, PRLRi pAb, 1A2B1, 250448 and H-300). The latter three antibodies were found to specifically recognise PRLR. The relative PRLR expression level detected with these antibodies closely reflected the level of (125)I-PRL binding to the cell surface. The monoclonal antibody (mAb) 250448 was specific for the N-()glycosylated form of PRLR and blocked PRL binding and signalling. The PRLRi polyclonal antibody recognised cytokeratin-18. The mAb B6.2, previously used in a number of studies, was found to lack specificity for PRLR and to rather recognise a PRLR-associated protein. The mAb U5 raised against the rat PRLR did not cross-react with the human receptor. Only one mAb, 1A2B1, was found useful for detection of PRLR in IHC applications. This antibody recognised PRLR expressed in human breast cancer cell lines and decidual cells in tissue sections of human placenta. Screening of 160 mammary adenocarcinomas demonstrated significant immunoreactivity in only four tumours, indicating that PRLR is generally not strongly upregulated in human breast cancer. However, even a very low level of PRLR expression was found to be sufficient to mediate PRL responsiveness in breast cancer cell lines.