RESUMO
STUDY QUESTION: What effect does multigenerational (F2) and transgenerational (F3) cigarette smoke exposure have on female fertility in mice? SUMMARY ANSWER: Cigarette smoking has a multigenerational effect on female fertility. WHAT IS KNOWN ALREADY: It has been well established that cigarette smoking decreases female fertility. Furthermore, a growing body of evidence suggests that smoking during pregnancy decreases the fertility of daughters and increases cancer and asthma incidence in grandchildren and great-grandchildren. STUDY DESIGN, SIZE, DURATION: Six-week-old C57BL/6 female mice were exposed nasally to cigarette smoke or room air (controls) for 5 weeks prior to being housed with males. Females continued to be exposed to smoke throughout pregnancy and lactation until pups were weaned. A subset of F1 female pups born to these smoke and non-smoke exposed females were bred to create the F2 grandmaternal exposed generation (multigenerational). Finally, a subset of F2 females were bred to create the F3 great-grandmaternal exposed generation (transgenerational). The reproductive health of F2 and F3 females was examined at 8 weeks and 9 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian and oocyte quality was examined in smoke exposed and control animals. A small-scale fertility trial was performed before ovarian changes were examined using ovarian histology and immunofluorescence and/or immunoblotting analysis of markers of apoptosis (TUNEL) and proliferation (proliferating cell nuclear antigen (PCNA) and anti-Mullerian hormone (AMH)). Oocyte quality was examined using immunocytochemistry to analyze the metaphase II spindle and ploidy status. Parthenogenetic activation of oocytes was used to investigate meiosis II timing and preimplantation embryo development. Finally, diestrus hormone serum levels (FSH and LH) were quantified. MAIN RESULTS AND THE ROLE OF CHANCE: F2 smoke exposed females had no detectable change in ovarian follicle quality at 8 weeks, although by 9 months ovarian somatic cell proliferation was reduced (P = 0.0197) compared with non-smoke exposed control. Further investigation revealed changes between control and smoke exposed F2 oocyte quality, including altered meiosis II timing at 8 weeks (P = 0.0337) and decreased spindle pole to pole length at 9 months (P = 0.0109). However, no change in preimplantation embryo development was observed following parthenogenetic activation. The most noticeable effect of cigarette smoke exposure was related to the subfertility of F2 females; F2 smoke exposed females displayed significantly increased time to conception (P = 0.0042) and significantly increased lag time between pregnancies (P = 0.0274) compared with non-smoke exposed F2 females. Conversely, F3 smoke exposed females displayed negligible oocyte and follicle changes up to 9 months of age, and normal preimplantation embryo development. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: This study focused solely on a mouse model of cigarette smoke exposure to simulate human exposure. WIDER IMPLICATIONS OF THE FINDINGS: Our results demonstrate that grandmaternal cigarette smoke exposure reduces female fertility in mice, highlighting the clinical need to promote cessation of cigarette smoking in pregnant women. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Australian Research Council, National Health and Medical Research Council, Hunter Medical Research Institute, Newcastle Permanent Building Society Charitable Trust, and the University of Newcastle Priory Research Centers in Chemical Biology, Healthy Lungs and Grow Up Well. The authors declare no conflict of interest.
Assuntos
Apoptose , Fumar Cigarros/efeitos adversos , Desenvolvimento Fetal/efeitos dos fármacos , Infertilidade Feminina/etiologia , Exposição Materna/efeitos adversos , Oócitos/patologia , Ovário/patologia , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Ectogênese , Feminino , Imunofluorescência , Imuno-Histoquímica , Infertilidade Feminina/metabolismo , Infertilidade Feminina/patologia , Infertilidade Feminina/fisiopatologia , Lactação , Camundongos Endogâmicos C57BL , Oócitos/metabolismo , Oogênese , Ovário/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Índice de Gravidade de Doença , Tempo para EngravidarRESUMO
STUDY QUESTION: What are the effects on fertility of cigarette smoke-induced toxicity on male offspring exposed during the gestational/weaning period? SUMMARY ANSWER: Maternal cigarette smoke exposure during the gestational/weaning period causes long-term defects in male offspring fertility. WHAT IS KNOWN ALREADY: Cigarette smoke is a well-known reproductive toxicant which is particularly harmful to both fetal and neonatal germ cells. However, recent studies suggest a significant portion of young mothers in the developed world still smoke during pregnancy. In the context of male reproductive health, our understanding of the effects of in utero exposure on offspring fertility is limited. STUDY DESIGN, SIZE, DURATION: In this study, 27 C57BL/6 5-week-old female mice were exposed via the nose-only to cigarette smoke (treatment) or 27 were exposed to room air (control) for 6 weeks before being housed with stud males to produce litters. In the treatment group, smoke exposure continued throughout mating, pregnancy and lactation until weaning of pups at 21 days post birth. Male offspring were examined at post-natal days 3, 6, 12, 21 and 98 (adult). PARTICIPANTS/MATERIALS, SETTING, METHODS: Approximately 108 maternal smoke-exposed C57BL/6 offspring and controls were examined. Spermatogenesis was examined using testicular histology and apoptosis/DNA damage was assessed using caspase immunohistochemistry and TUNEL. Sertoli cell morphology and fluctuations in the spermatogonial stem cell population were also examined using immunohistochemistry. Microarray and QPCR analysis were performed on adult testes to examine specific long-term transcriptomic alteration as a consequence of maternal smoke exposure. Sperm counts and motility, zona/oolemma binding assays, COMET analysis and mitochondrial genomic sequencing were also performed on spermatozoa obtained from adult treated and control mice. Fertility trials using exposed adult male offspring were also performed. MAIN RESULTS AND THE ROLE OF CHANCE: Maternal cigarette smoke exposure caused increased gonocyte and meiotic spermatocyte apoptosis (P < 0.01) as well as germ cell depletion in the seminiferous tubules of neonatal and juvenile offspring. Aberrant testicular development characterized by abnormal Sertoli and germ cell organization, a depleted spermatogonial stem cell population (P < 0.01), atrophic seminiferous tubules and increased germ cell DNA damage (P < 0.01) persisted in adult offspring 11 weeks after exposure. Microarray analysis of adult offspring testes associated these defects with meiotic germ cell development, sex hormone metabolism, oxidative stress and Sertoli cell signalling. Next generation sequencing also revealed a high mitochondrial DNA mutational load in the testes of adult offspring (P < 0.01). Adult maternal smoke-exposed offspring also had reduced sperm counts with spermatozoa exhibiting morphological abnormalities (P < 0.01), affecting motility and fertilization potential. Odf2, a spermatozoa flagellum component required for coordinated ciliary beating, was also significantly down-regulated (P < 0.01) in maternal smoke-exposed adult offspring, with aberrant localization along the spermatozoa flagellum. Adult maternal smoke-exposed offspring took significantly longer to impregnate control females and had a slight but significant (P < 0.01) reduction in litter size. LIMITATIONS, REASONS FOR CAUTION: This study examined only one species (mouse) using a smoking model which only simulates human cigarette smoke exposure. WIDER IMPLICATIONS OF THE FINDINGS: This study represents the first comprehensive animal model of maternal smoking on male offspring reproductive function, suggesting that exposure during the gestational/weaning period causes long-term defects in male offspring fertility. This is due to a compromised spermatogonial stem cell population resulting from gonocyte apoptosis and impaired spermatogenic development. This results in significant germ cell damage and Sertoli cell dysfunction, impacting germ cell number, tubule organization, DNA damage and spermatozoa in adult offspring. This study strengthens the current literature suggesting that maternal exposure impairs male offspring fertility, which is currently debated due to conflicting studies. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the Australian Research Council, Hunter Medical Research Institute, National Health and Medical Research Council of Australia and the Newcastle Permanent Building Society Charitable Trust. The authors declare no conflict of interest.
Assuntos
Infertilidade Masculina/etiologia , Efeitos Tardios da Exposição Pré-Natal , Fumar/efeitos adversos , Animais , Apoptose , Dano ao DNA , Feminino , Lactação , Masculino , Camundongos Endogâmicos C57BL , Gravidez , Células de Sertoli/citologia , EspermatogêneseRESUMO
Cigarette smoke is a reproductive hazard associated with pre-mature reproductive senescence and reduced clinical pregnancy rates in female smokers. Despite an increased awareness of the adverse effects of cigarette smoke exposure on systemic health, many women remain unaware of the adverse effects of cigarette smoke on female fertility. This issue is compounded by our limited understanding of the molecular mechanisms behind cigarette smoke induced infertility. In this study we used a direct nasal exposure mouse model of cigarette smoke-induced chronic obstructive pulmonary disease to characterise mechanisms of cigarette-smoke induced ovotoxicity. Cigarette smoke exposure caused increased levels of primordial follicle depletion, antral follicle oocyte apoptosis and oxidative stress in exposed ovaries, resulting in fewer follicles available for ovulation. Evidence of oxidative stress also persisted in ovulated oocytes which escaped destruction, with increased levels of mitochondrial ROS and lipid peroxidation resulting in reduced fertilisation potential. Microarray analysis of ovarian tissue correlated these insults with a complex mechanism of ovotoxicity involving genes associated with detoxification, inflammation, follicular activation, immune cell mediated apoptosis and membrane organisation. In particular, the phase I detoxifying enzyme cyp2e1 was found to be significantly up-regulated in developing oocytes; an enzyme known to cause molecular bioactivation resulting in oxidative stress. Our results provide a preliminary model of cigarette smoke induced sub-fertility through cyp2e1 bioactivation and oxidative stress, resulting in developing follicle depletion and oocyte dysfunction.
Assuntos
Oócitos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Caspases/metabolismo , Dano ao DNA/efeitos dos fármacos , Feminino , Fertilidade/efeitos dos fármacos , Fertilização/efeitos dos fármacos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Infertilidade Feminina/induzido quimicamente , Peroxidação de Lipídeos/efeitos dos fármacos , Tamanho da Ninhada de Vivíparos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oócitos/patologia , Folículo Ovariano/patologia , Ovário/patologia , RNA/biossíntese , RNA/isolamento & purificação , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND AND PURPOSE: Chronic obstructive pulmonary disease (COPD) is a major cause of illness and death, often induced by cigarette smoking (CS). It is characterized by pulmonary inflammation and fibrosis that impairs lung function. Existing treatments aim to control symptoms but have low efficacy, and there are no broadly effective treatments. A new potential target is the ectoenzyme, semicarbazide-sensitive mono-amine oxidase (SSAO; also known as vascular adhesion protein-1). SSAO is elevated in smokers' serum and is a pro-inflammatory enzyme facilitating adhesion and transmigration of leukocytes from the vasculature to sites of inflammation. EXPERIMENTAL APPROACH: PXS-4728A was developed as a low MW inhibitor of SSAO. A model of COPD induced by CS in mice reproduces key aspects of human COPD, including chronic airway inflammation, fibrosis and impaired lung function. This model was used to assess suppression of SSAO activity and amelioration of inflammation and other characteristic features of COPD. KEY RESULTS: Treatment with PXS-4728A completely inhibited lung and systemic SSAO activity induced by acute and chronic CS-exposure. Daily oral treatment inhibited airway inflammation (immune cell influx and inflammatory factors) induced by acute CS-exposure. Therapeutic treatment during chronic CS-exposure, when the key features of experimental COPD develop and progress, substantially suppressed inflammatory cell influx and fibrosis in the airways and improved lung function. CONCLUSIONS AND IMPLICATIONS: Treatment with a low MW inhibitor of SSAO, PXS-4728A, suppressed airway inflammation and fibrosis and improved lung function in experimental COPD, demonstrating the therapeutic potential of PXS-4728A for this debilitating disease.
Assuntos
Alilamina/análogos & derivados , Amina Oxidase (contendo Cobre)/antagonistas & inibidores , Benzamidas/farmacologia , Inibidores Enzimáticos/farmacologia , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Alilamina/farmacologia , Amina Oxidase (contendo Cobre)/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Doença Pulmonar Obstrutiva Crônica/enzimologia , FumarRESUMO
Malignant mesothelioma is an aggressive tumor, usually induced by asbestos exposure, that has a poor prognosis and is unresponsive to conventional therapy. The present study was aimed at assessing the potential for interferon-alpha (IFN-alpha)-based therapies in a murine model for malignant mesothelioma. The effect of recombinant human IFN-alpha B/D on tumor growth, alone and in combination with either of two immunomodulatory and antiproliferative agents beta-carotene or alpha-difluoromethylornithine (DFMO), was assessed. The data suggest that IFN-alpha treatment is most efficacious when commenced early in tumor development. Combination of IFN-alpha with either DFMO or dietary beta-carotene supplementation improved the effect of an otherwise suboptimal IFN-alpha therapy regimen. Both IFN-alpha and beta-carotene had in vivo stimulatory effects on immune cells, perhaps indirectly by inhibiting TGF-beta generation. The immunomodulatory effects may contribute, at least in part, to the positive antitumor and clinical activities of the treatments in this model.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Interferon Tipo I/uso terapêutico , Mesotelioma/terapia , Adjuvantes Imunológicos/uso terapêutico , Animais , Carotenoides/administração & dosagem , Carotenoides/uso terapêutico , Eflornitina/administração & dosagem , Eflornitina/uso terapêutico , Feminino , Interferon Tipo I/administração & dosagem , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Mesotelioma/genética , Mesotelioma/imunologia , Camundongos , Camundongos Endogâmicos CBA , Camundongos Endogâmicos , RNA Mensageiro/biossíntese , Proteínas Recombinantes , Fator de Crescimento Transformador beta/metabolismo , Células Tumorais Cultivadas , beta CarotenoRESUMO
The mechanisms whereby tumors escape immunosurveillance remain poorly understood. De-activation or deviation of T lymphocyte responses may occur following exposure to tumor-associated or -derived signals. In the present study it is demonstrated that during development of syngeneic malignant mesothelioma in mice, the relative CD3 delta, CD3 gamma and CD3 zeta mRNA levels expressed by tumor infiltrating lymphocytes (TIL) decrease, while CD3 epsilon mRNA levels remain relatively constant. Expression of IFN gamma mRNA by TIL decreased during tumor development, while IL-2 mRNA levels showed slight increases. IL-3 mRNA was not detected at any time during tumor development and IL-4 transcripts were detected in the later stages of tumor development. In the spleens of tumor-bearing mice, IL-2 transcripts were detected throughout the time course from days 1 to 22(24), while IFN gamma mRNA was only detected at early times from days 0-13. Previous work demonstrated a role for tumor cell-derived TGF beta in the immunobiology of mesothelioma. Here it is shown that the suppression of CD3-subunit expression by TIL was ameliorated in tumors where TGF beta -expression was reduced by inducible TGF beta-specific antisense-RNA, thus, suggesting that lymphocytes may become de-activated upon infiltration of the tumor micro-environment.
Assuntos
Citocinas/biossíntese , Expressão Gênica , Linfócitos do Interstício Tumoral/imunologia , Mesotelioma/imunologia , Complexo Receptor-CD3 de Antígeno de Linfócitos T/biossíntese , Linfócitos T/imunologia , Animais , Sequência de Bases , Primers do DNA , Feminino , Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Interferon gama/biossíntese , Interleucina-3/biossíntese , Interleucina-4/biossíntese , Substâncias Macromoleculares , Mesotelioma/patologia , Camundongos , Camundongos Endogâmicos CBA , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Antissenso/farmacologia , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Linfócitos T/patologia , Fatores de Tempo , Transcrição Gênica , Fator de Crescimento Transformador beta/imunologiaRESUMO
BACKGROUND: The responses of mice to the mite allergen Der p 1 have been used to study the mechanisms of allergic sensitization and the development of new types of immunotherapy. Many of the studies require a knowledge of the T cell epitopes, and because Der p 1 is polymorphic, the effect of natural amino acid substitution in the allergen. The intranasal administration of peptides containing T cell epitopes can induce a mucosal tolerance but it is not known if the major activity is limited to stimulatory peptides and if, as found for autoimmunity, some epitopes are not inhibitory. OBJECTIVE: To determine and compare the sequences of Der p 1 which contain stimulatory epitopes for the high responding H-2(b) and H-2(q) mice and the sequences which induce tolerance by intranasal administration of peptides. METHODS: T cell responses of mice immunized with Der p 1 were measured by in vitro T cell stimulation assays so an extensive study of epitope recognition and intranasal tolerance could be made. Synthetic peptides were used to examine the stimulatory and inhibitory ability of all Der p 1 sequences and to map the major H-2(b) epitope in detail. This included the effect of the common polymorphic amino acid 124 substitution found within this epitope. RESULTS: Three and two regions, respectively, were found to contain stimulatory T cell epitopes for H-2(b) and H-2(q) mice. The peptides in these regions were also the most active at inducing intranasal tolerance for the responding haplotype. The correspondence between inhibitory and stimulatory peptides was maintained for the fine mapping of the major H-2(b) epitope. This was found about a core region of 118-126 which was overlapping but separate to a consensus sequence for the binding of endogeneous peptides. Peptides with alanine at the naturally polymorphic residue 124 stimulated and inhibited responses to Der p 1 more effectively, while peptides with the valine 124 variant were immunogenic but poorly cross-reactive. CONCLUSIONS: The intranasal administration of peptides representing each of five epitopes recognized by two strains of mice were able to induce mucosal tolerance and the major tolerizing activity was limited to these epitopes. The position of the core major epitope for C57 mice, which differs from a previously predicted epitope, and its specificity for the natural alanine 124 variant is described.
Assuntos
Alérgenos/imunologia , Antígenos de Dermatophagoides/imunologia , Epitopos de Linfócito T/imunologia , Mucosa Nasal/imunologia , Administração Intranasal , Alérgenos/administração & dosagem , Aminoácidos/análise , Aminoácidos/genética , Aminoácidos/imunologia , Animais , Antígenos de Dermatophagoides/administração & dosagem , Antígenos de Dermatophagoides/análise , Proteínas de Artrópodes , Cisteína Endopeptidases , DNA Complementar/análise , DNA Complementar/genética , DNA Complementar/imunologia , Imunização , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Peptídeos/administração & dosagem , Peptídeos/imunologia , Polimorfismo GenéticoRESUMO
Although the intranasal administration of peptides containing T cell epitopes has been shown to be a potent method of inhibiting responses to the allergen Der p 1, the experiments to date have concentrated on their ability to regulate immune responses to the injection of antigen in a T(h)1-type adjuvant. Their ability to regulate responses to a T(h)2-type immunization and to sensitization via the respiratory mucosa has not been examined. Here it is shown that peptide used in doses required to block delayed-type hypersensitivity can also readily inhibit IgE responses to Der p 1 injected in alum. To examine responses induced in the respiratory mucosa, mice pretreated with intranasal peptide were sensitized with an intranasal dose of Der p 1 in conjunction with a mutated enterotoxin adjuvant. Intranasal peptide even in very high doses did not reduce IgE titers, but the ability of cells from the draining lymph nodes to release IL-4 and IL-13 but not IL-2, IL-5, IL-10 or IFN-gamma was reduced. These are the first reports on the effect of intranasal peptides containing T cell epitopes on IgE in T(h)2 immunization and on responses to respiratory immunization. Thus the effect of the peptide-induced mucosal tolerance differs depending on the type of immunization used for sensitization, but the potential to inhibit T(h)2 responses and responses to respiratory sensitization as well as T(h)1 responses has been demonstrated.
Assuntos
Alérgenos/imunologia , Glicoproteínas/imunologia , Ácaros/imunologia , Células Th2/imunologia , Administração Intranasal , Alérgenos/administração & dosagem , Animais , Antígenos de Dermatophagoides , Glicoproteínas/administração & dosagem , Tolerância Imunológica , Imunidade Celular , Epitopos Imunodominantes , Isotipos de Imunoglobulinas/análise , Interferon gama/análise , Interleucinas/análise , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/administração & dosagem , Peptídeos/imunologia , Mucosa Respiratória/imunologia , Células Th1/imunologia , VacinaçãoRESUMO
BACKGROUND: Sequence diversity is a common feature of mite allergens. Previous studies, using predominantly commercial mite clones, have described several polymorphic residues for Der p 1 and Der p 2. OBJECTIVE: This study aimed at determining the occurrence of sequence diversity in environmental mite isolates. METHODS: Mites were isolated from houses in Perth and Sydney, Australia. Total RNA was extracted from 1 to 30 Perth mites, and cDNA was synthesized by reverse transcriptase PCR. Der p 1 and Der p 2 cDNAs were PCR amplified and sequenced. Genomic Der p 1 DNA was amplified from whole Sydney mites directly by PCR and then sequenced. RESULTS: Twelve Der p 1 and 9 Der p 2 cDNA clones and 3 Der p 1 genomic DNA were analyzed and showed a high frequency of amino acid polymorphisms. Der p 2 displayed a clear pattern of divergence toward 2 alleles that differed by 4 amino acids and had characteristic silent nucleotide changes. The pattern for Der p 1 was different and unusual, with almost no silent nucleotide substitutions but frequent sporadic missense changes. Proliferative responses of peripheral blood mononuclear cells to peptides containing polymorphic residues of Der p 1 were detected in 8 of 19 subjects, with stimulation being found only for either one of the variant forms of the peptides. However, the responses to variants of whole recombinant allergens were similar, as shown for 4 variants of Der p 2. CONCLUSION: Two clones for each of the allergens were identified as containing sequences that were largely representative of environmental isolates. A small-scale reverse transcriptase PCR used to produce cDNA from individual mites isolated from house dust will have wide application for studies on mite genetics and the production of recombinant mite allergens. Differences in T-cell responses to peptides representing variant epitopes were found, but responses to variants of whole recombinant allergens were similar. The GenBank and Swiss Prot database entries for Der p 1 (U11695) and Der p 2 (P49278) have been updated with the inclusion of the sequence polymorphisms described in this study.
Assuntos
Poeira , Glicoproteínas/genética , Glicoproteínas/imunologia , Ácaros/imunologia , Polimorfismo Genético , Animais , Antígenos de Dermatophagoides , Sequência de Bases , DNA Complementar , Epitopos/química , Epitopos/imunologia , Glicoproteínas/química , Habitação , Humanos , Ativação Linfocitária , Ácaros/genética , Peptídeos/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologiaRESUMO
Mucosal administration of antigens in experimental animals leads to the induction of peripheral T cell tolerance. We have previously reported that in H-2b mice, intranasal (i.n.) or oral administration of a peptide containing the immunodominant T cell epitope will down-regulate the function of CD4+ T cells reactive with Der P 1, a major target antigen in both B and T cell responses to house dust mite. In the present study we have investigated the tolerogenicity of peptides containing both dominant and subdominant determinants when given i.n. to nalve mice. Induction of tolerance by the nasally administered immunodominant peptide leads to a diminution in all T cell-derived cytokines and modulation of delayed-type hypersensitivity responses, but IgE production did not seem to be affected, furthermore the induction of T cell tolerance was stable, lasting beyond 6 months. We have also examined the specificity of intramolecular epitope suppression which is a feature of mucosal tolerance induced by nasally administered peptides and demonstrate that regulatory CD4+ T cells may exert their suppressive effect by linked recognition of epitopes on the same or neighbouring antigen-presenting cells.
Assuntos
Anergia Clonal/imunologia , Epitopos de Linfócito T/imunologia , Glicoproteínas/imunologia , Glicoproteínas/farmacologia , Linfocinas/metabolismo , Ácaros/imunologia , Peptídeos/farmacologia , Linfócitos T/efeitos dos fármacos , Administração Intranasal , Animais , Apresentação de Antígeno/imunologia , Antígenos de Dermatophagoides , Linfócitos T CD4-Positivos/imunologia , Relação Dose-Resposta Imunológica , Regulação para Baixo , Camundongos , Camundongos Endogâmicos C57BL , Anafilaxia Cutânea Passiva , Peptídeos/administração & dosagem , Peptídeos/síntese química , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
A role for interleukin-6 (IL-6) in malignant mesothelioma has been suggested by the clinically presenting symptoms of mesothelioma patients, which include fever, weight loss and thrombocytosis. A murine model of malignant mesothelioma was therefore used to examine the potential role of IL-6 in this cancer type and whether the effect of interferon alpha (IFN alpha) therapy on mesothelioma might be mediated, in part, by regulating IL-6 levels and/or IL-6-induced pathobiology. A panel of human and murine mesothelioma cell lines was assayed for endogenous IL-6 production in a bioassay, and for IL-6-mRNA expression. Four out of 5 human and 5 out of 15 murine mesothelioma cell lines produced moderate to high levels of bioactive IL-6 in vitro. This result was corroborated by mRNA detection. One of the representative murine cell lines, AB22, was chosen for further in vivo studies in the murine mesothelioma model. In AB22-inoculated mice detectable serum IL-6 levels were found to precede macroscopically detectable tumour growth, clinical signs (cachexia, abdominal distension, diarrhoea) and changes in the peripheral lymphoid organs (cell depletion and functional depression). Treatment with anti-IL-6 antibody curtailed the clinical symptoms (P < 0.001), as did treatment with recombinant human (rhu) IFN alpha (P < 0.001). Neither anti-IL-6 antibody nor rhuIFN alpha had a direct growth-inhibitory effect on the AB22 mesothelioma cell line in vitro, however, in vivo rhuIFN alpha treatment of mice inoculated with AB22 cells attenuated both IL-6 mRNA expression in the tumours and serum IL-6 levels, ameliorated the depression of lymphocyte activities, and enhanced the number of tumour-infiltrating lymphocytes and macrophages. On the basis of these results it is suggested that IL-6 mediates some of these effects, directly or indirectly, and that a combination therapy of rhuIFN alpha and anti-IL-6 antibody may be an improved palliative treatment for patients with malignant mesothelioma.
Assuntos
Imunoterapia , Interferon Tipo I/farmacologia , Interleucina-6/antagonistas & inibidores , Interleucina-6/fisiologia , Mesotelioma/patologia , Mesotelioma/terapia , Animais , Sequência de Bases , Feminino , Humanos , Interleucina-6/farmacologia , Mesotelioma/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , RNA Mensageiro/genética , Proteínas Recombinantes , Células Tumorais CultivadasRESUMO
Transforming growth factor-beta (TGF-beta) is produced by a number of tumor cell types including human malignant mesothelioma (MM), but its role as a direct or indirect factor in tumorigenesis is incompletely understood. We have investigated the expression of TGF-beta isoforms by human and murine MM cells and have analysed the effects of inducible antisense RNA-mediated inhibition of TGF-beta expression on murine MM in vitro and in vivo. The results showed that (a) TGF-beta 1 and -beta 2 were produced by both human and mouse MM cells, (b) antisense RNA against either TGF-beta 1 or -beta 2 cross-inhibited both TGF-beta 1 and -beta 2 expression, (c) inhibition of TGF-beta expression reduced the anchorage-independent growth of MM cells in vitro and the tumorigenicity of MM cells in vivo, and (d) inhibition of TGF-beta expression led to increased T lymphocyte infiltration into tumors. The data suggest that TGF-beta has multiple tumor-enhancing effects in MM.
Assuntos
Mesotelioma/etiologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Sequência de Bases , Adesão Celular/fisiologia , Divisão Celular/fisiologia , Primers do DNA/genética , Feminino , Vetores Genéticos , Humanos , Linfócitos do Interstício Tumoral/patologia , Mesotelioma/patologia , Camundongos , Camundongos Endogâmicos CBA , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Antissenso/genética , RNA Antissenso/farmacologia , RNA Mensageiro/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/fisiologia , Células Tumorais Cultivadas/patologiaRESUMO
Malignant mesothelioma (MM) is an aggressive, uniformly fatal serosal tumour, usually associated with asbestos exposure, for which there currently is no effective treatment. In order to gain insight into the mechanism(s) whereby MM might escape immune surveillance, a murine model for MM was used (a) to characterise the tumour-infiltrating lymphocytes (TIL) and macrophages (TIM) phenotypically, (b) to examine systemic immune recognition of MM, and (c) to examine the possible influence of tumour-derived cytokines on systemic and local pathobiological manifestations of MM. A profound down-regulation of lymphocyte surface markers, known to be involved in T cell activation, was found in TIL. Likewise, although TIM were present in large numbers, their expression of MHC class II antigen and integrins was weak or absent, suggestive of altered functional activity. Significant amounts of cytokines, in particular transforming growth factor beta, interleukin-6 (IL-6), IL-1 and tumour necrosis factor were produced during the course of MM tumour development-directly by the MM cells and/or indirectly in response to tumour growth. These factors may contribute both to derangement of antitumour effector mechanisms and to the clinical and pathological manifestations of the disease.