RESUMO
BACKGROUND: Alveolar macrophages (AM) in COPD have fundamentally impaired responsiveness to Toll-like receptor 2 (TLR2) and TLR4 ligands of non-typeable Haemophilus influenzae (NTHI). However, the contribution of innate immune dysfunction to exacerbations of COPD is unexplored. We hypothesised that impaired innate AM responses in COPD extend beyond NTHI to other pathogens and are linked with COPD exacerbations and severity. METHODS: AMs, obtained by bronchoalveolar lavage from 88 volunteers with stable-to-moderate COPD, were incubated with respiratory pathogens (NTHI, Moraxella catarrhalis (MC), Streptococcus pneumoniae (SP) and TLR ligands lipopolysaccharide, Pam3Cys) and elicited IL-8 and TNF-α were measured by microsphere flow cytometry. NF-κB nuclear translocation was measured by colorimetric assay. AM TLR2 and TLR4 expression was determined by immunolabeling and quantitation of mean fluorescent indices. Participants were monitored prospectively for occurrence of COPD exacerbations for 1â year following bronchoscopy. Non-parametric analyses were used to compare exacerbation-prone and non-exacerbation-prone individuals. RESULTS: 29 subjects had at least one exacerbation in the follow-up period (exacerbation-prone) and 59 remained exacerbation-free (non-exacerbation-prone). AMs of exacerbation-prone COPD donors were more refractory to cytokine induction by NTHI (p=0.02), MC (p=0.045) and SP (p=0.046), and to TLR2 (p=0.07) and TLR4 (p=0.028) ligands, and had diminished NF-κB nuclear activation, compared with non-exacerbation-prone counterparts. AMs of exacerbation-prone subjects were more refractory to TLR2 upregulation by MC and SP (p=0.04 each). CONCLUSIONS: Our results support a paradigm of impaired innate responses of COPD AMs to respiratory pathogens, mediated by impaired TLR responses, underlying a propensity for exacerbations in COPD.
Assuntos
Imunidade Inata , Macrófagos Alveolares/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Células Cultivadas , Técnicas de Cocultura , Progressão da Doença , Feminino , Haemophilus influenzae/imunologia , Humanos , Interleucina-8/imunologia , Interleucina-8/metabolismo , Ligantes , Lipopolissacarídeos , Lipoproteínas/farmacologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiologia , Masculino , Pessoa de Meia-Idade , Moraxella catarrhalis/imunologia , NF-kappa B/metabolismo , Doença Pulmonar Obstrutiva Crônica/microbiologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Índice de Gravidade de Doença , Transdução de Sinais/efeitos dos fármacos , Streptococcus pneumoniae/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
Immune signatures in breast tumors differ by estrogen receptor (ER) status. The purpose of this study was to assess associations between ER phenotypes and circulating levels of cytokines that co-ordinate cell-mediated [T-helper type 1 (Th1)] and humoral [T-helper type 2 (Th2)] immunity. We conducted a case-case comparison of 523 women with newly diagnosed breast cancer to evaluate associations between 27 circulating cytokines, measured using Luminex XMap technology, and breast cancer phenotypes [ER(-) vs. ER(+); triple negative breast cancer (TNBC) vs. luminal A (LumA)]. Ratios of Th1 to Th2 cytokines were also evaluated. Levels of interleukin (IL)-5, a Th-2 cytokine, were higher in ER(-) than in ER(+) tumors. The highest tertile of IL-5 was more strongly associated with ER(-) (OR = 2.33, 95 % CI 1.40-3.90) and TNBCs (OR = 2.78, 95 % CI 1.53-5.06) compared to ER(+) and LumA cancers, respectively, particularly among premenopausal women (OR = 4.17, 95 % CI 1.86-9.34, ER(-) vs. ER(+); OR = 5.60, 95 % CI 2.09-15.01, TNBC vs. LumA). Elevated Th1 cytokines were also detected in women with ER(-) and TNBCs, with women in the highest tertile of interferon α2 (OR = 2.39, 95 % CI 1.31-4.35) or tumor necrosis factor-α (OR = 2.27, 95 % CI 1.21-4.26) being twice as likely to have TNBC versus LumA cancer. When cytokine ratios were examined, women with the highest ratios of Th1 cytokines to IL-5 levels were least likely to have ER(-) or TNBCs compared to ER(+) or LumA cancers, respectively. The strongest associations were in premenopausal women, who were up to 80 % less likely to have TNBC than LumA cancers (IL-12p40/IL-5, OR = 0.19, 95 % CI 0.07-0.56). These findings indicate that immune function is associated with ER(-) and TNBC and may be most relevant among younger women, who are likely to be diagnosed with these aggressive phenotypes.
Assuntos
Neoplasias da Mama/metabolismo , Citocinas/sangue , Neoplasias de Mama Triplo Negativas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Receptores de Estrogênio , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Neoplasias de Mama Triplo Negativas/sangue , Neoplasias de Mama Triplo Negativas/imunologiaRESUMO
The rTSbeta protein has been hypothesized to synthesize signaling molecules that can down-regulate thymidylate synthase. These molecules share biological and chemical properties with acyl-homoserine lactones (AHL), suggesting some AHLs might act as rTS signaling mimics and down-regulate thymidylate synthase. We have determined that the AHL, 3-oxododecanoyl homoserine lactone (3-oxo-C12-(L)-HSL) can down-regulate thymidylate synthase protein at 10 micromol/L and reduce H630 (human colorectal cancer) growth by 50% at 23 micromol/L (IC50) in cell culture. At its IC50 concentration, 3-oxo-C12-(L)-HSL reduces the apparent IC50 of 5-fluorouracil (5-FU) from 1 micromol/L to 80 nmol/L (12-fold) in a colony formation assay. 3-Oxo-C12-(L)-HSL enhances the activity of 5-fluorodeoxyuridine, tomudex, and taxol but not the activity of 5-fluorouridine, methotrexate or Adriamycin. The unexpected interaction with taxol probably results from effects of the AHL on tubulin expression. Differences in taxol sensitivity, tubulin, and cellular morphology between H630 and the thymidylate synthase and rTSbeta-overproducing, 5-FU-resistant H630-1 cell line as determined by colony formation assays, Western analysis of one-dimensional and two-dimensional gels, and photomicroscopy confirm that cytoskeletal changes are induced by the AHL or by rTS signaling. Isozyme differences in thymidylate synthase and rTSbeta also exist in the two cell lines. Phosphorylation of rTSbeta amino acid S121 is shown to occur and is decreased at least 10-fold in the drug-resistant cells. The data presented provide support for further investigations of rTS signaling mimics as enhancers to thymidylate synthase-directed chemotherapy, evidence that the phosphorylation state of rTSbeta may be a marker for 5-FU resistance and a previously unrealized relationship between rTS signaling and the cytoskeleton.
Assuntos
4-Butirolactona/análogos & derivados , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/enzimologia , Fluoruracila/farmacologia , Homosserina/análogos & derivados , Timidilato Sintase/metabolismo , 4-Butirolactona/administração & dosagem , 4-Butirolactona/farmacologia , Materiais Biomiméticos/administração & dosagem , Materiais Biomiméticos/farmacologia , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Fluoruracila/administração & dosagem , Homosserina/administração & dosagem , Homosserina/farmacologia , Humanos , Isoenzimas , Fosforilação , Isoformas de Proteínas , RNA Antissenso/biossíntese , RNA Antissenso/genética , Transdução de Sinais/fisiologia , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/biossíntese , Timidilato Sintase/genética , Tubulina (Proteína)/metabolismoRESUMO
T-cell immunoglobulin and mucin domain 1 (TIM-1) is a type I transmembrane protein that was originally described as kidney injury molecule 1 (KIM-1) due to its elevated expression in kidney and urine after renal injury. TIM-1 expression is also upregulated in several human cancers, most notably in renal and ovarian carcinomas, but has very restricted expression in healthy tissues, thus representing a promising target for antibody-mediated therapy. To this end, we have developed a fully human monoclonal IgG1 antibody specific for the extracellular domain of TIM-1. This antibody was shown to bind purified recombinant chimeric TIM-1-Fc protein and TIM-1 expressed on a variety of transformed cell lines, including Caki-1 (human renal clear cell carcinoma), IGROV-1 (human ovarian adenocarcinoma), and A549 (human lung carcinoma). Internalization studies using confocal microscopy revealed the antibody was rapidly internalized by cells in vitro, and internalization was confirmed by quantitative imaging flow cytometry. An antibody-drug conjugate (ADC) was produced with the anti-TIM-1 antibody covalently linked to the potent cytotoxin, monomethyl auristatin E (MMAE), and designated CDX-014. The ADC was shown to exhibit in vitro cytostatic or cytotoxic activity against a variety of TIM-1-expressing cell lines, but not on TIM-1-negative cell lines. Using the Caki-1, IGROV-1, and A549 xenograft mouse models, CDX-014 showed significant antitumor activity in a clinically relevant dose range. Safety evaluation in nonhuman primates has demonstrated a good profile and led to the initiation of clinical studies of CDX-014 in renal cell carcinoma and potentially other TIM-1-expressing tumors. Mol Cancer Ther; 15(12); 2946-54. ©2016 AACR.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Renais/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Receptor Celular 1 do Vírus da Hepatite A/genética , Imunoconjugados/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Ovarianas/genética , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Desenho de Fármacos , Feminino , Receptor Celular 1 do Vírus da Hepatite A/química , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Macaca fascicularis , Camundongos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Domínios e Motivos de Interação entre Proteínas/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The rTS gene codes for a naturally occurring antisense RNA to thymidylate synthase (TS) mRNA and two proteins (rTSalpha and rTSbeta). The role of the major protein product of rTS, rTSbeta has been linked to alterations in TS protein expression, but the precise function of rTSbeta is unknown. In this report we demonstrate that increased expression of rTSbeta is associated with the decrease in TS protein expression due to production of novel, diffusible signal molecules. These signal molecules are produced more abundantly when rTSbeta amounts are elevated. This hypothesis is supported by the demonstration that the rTSbeta-overproducing cell line H630-1 can downregulate TS protein in other cells without direct cellular contact. These cells are shown to secrete significant amounts of lipophilic metabolites derived from methionine, in contrast to cells that do not overproduce rTSbeta. In support of the hypothesis that rTSbeta is essential for the generation of these compounds, we demonstrate that rTSbeta can catalyze the transfer of the carboxyl carbon of methionine from S-adenosylmethionine to a lipophilic acceptor molecule in vitro. We propose rTS is involved in regulation of TS through a novel methionine-based signaling pathway.