RESUMEN
Increasing levels of estrogen and progesterone are suggested to play a role in the gender switch in asthma prevalence during puberty. We investigated whether the process of sexual maturation in mice affects the development of lung inflammation in adulthood and the contributing roles of estrogen and progesterone during this process. By inducing ovalbumin-induced lung inflammation in sexually mature and immature (ovariectomized before sexual maturation) adult mice, we showed that sexually immature adult mice developed more eosinophilic lung inflammation. This protective effect of "puberty" appears to be dependent on estrogen, as estrogen supplementation at the time of ovariectomy protected against development of lung inflammation in adulthood whereas progesterone supplementation did not. Investigating the underlying mechanism of estrogen-mediated protection, we found that estrogen-treated mice had higher expression of the anti-inflammatory mediator secretory leukoprotease inhibitor (SLPI) and lower expression of the proasthmatic cytokine IL-33 in parenchymal lung tissue and that their expressions colocalized with type II alveolar epithelial cells (AECII). Treating AECII directly with SLPI significantly inhibited IL-33 production upon stimulation with ATP. Our data suggest that estrogen during puberty has a protective effect on asthma development, which is accompanied by induction of anti-inflammatory SLPI production and inhibition of proinflammatory IL-33 production by AECII.
Asunto(s)
Estrógenos/metabolismo , Neumonía/metabolismo , Maduración Sexual/fisiología , Animales , Asma/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Ratones Endogámicos BALB C , Inhibidor Secretorio de Peptidasas Leucocitarias/biosíntesisRESUMEN
PROBLEM: Elafin and secretory leukocyte peptidase inhibitor (SLPI) are unique among antimicrobial peptides (AMPs). This study aimed to determine the expression levels of these AMPs at the cervix during pregnancy and to investigate their association with preterm labor. METHOD OF STUDY: Cervical epithelial cells were swabbed from normal pregnant women to evaluate the physiological expression of elafin and SLPI. Cross-sectional analysis was conducted to compare cervical expression levels for SLPI and elafin among three women's groups, controls (n = 26), women with threatened preterm labor who delivered at term (t-TPL, n = 23) and TPL who ended in preterm labor (p-TPL, n = 19). RESULTS: Elafin and SLPI proteins were detected in the squamous and glandular cells of the cervix. Cervical SLPI expression levels increased over the course of pregnancy, whereas elafin levels remained unchanged. Cervical mRNA expression levels of elafin and SLPI were significantly higher in p-TPL compared with t-TPL and control groups. CONCLUSION: Constitutive expression of elafin and SLPI in cervical cells during pregnancy suggests their essential roles in local tissue homeostasis and immune defense. The elevations in cervical elafin and SLPI expression in the women with preterm delivery might reflect the local response to the pathogen invasion into the cervix preceding preterm labor.
Asunto(s)
Cuello del Útero/inmunología , Elafina/inmunología , Regulación de la Expresión Génica/inmunología , Trabajo de Parto Prematuro/inmunología , Inhibidor Secretorio de Peptidasas Leucocitarias/inmunología , Adulto , Cuello del Útero/metabolismo , Cuello del Útero/patología , Elafina/biosíntesis , Femenino , Humanos , Trabajo de Parto Prematuro/metabolismo , Trabajo de Parto Prematuro/patología , Embarazo , Inhibidor Secretorio de Peptidasas Leucocitarias/biosíntesisRESUMEN
Recently, we demonstrated a significant inverse correlation between HPV-infection and SLPI-expression suggesting that SLPI protects against HPV-infection of HNSCC. Here we analyzed in a single lab setting 307 formalin-fixed paraffin-embedded HNSCC cases (tonsillar n = 135; non-tonsillar: n = 172) from eight health care centers. Samples were analyzed for SLPI gene- and protein-expression. Annexin A2, its heterotetramer A2t, putatively facilitating HPV- and SLPI-cell entry, was measured to study the correlation between SLPI and annexin A2. Data were correlated with tobacco consumption and HPV-status. Overall, HPV-DNA prevalence was 23.5% (72/307); attributed to: 43.7% (59/135) tonsillar and 7.6% (13/172) non-tonsillar cases. Smoking resulted in 6.44-fold increased and HPV-infection in 3.46-fold decreased SLPI-gene expression in all HNSCC with similar significant results obtained in tonsillar and non-tonsillar SCC separately. Correlating annexin A2- and SLPI-gene expression showed a significant surplus of annexin A2 in HPV-positive tumors (4.21× more annexin A2) and 6.72× more annexin A2 than SLPI in nonsmokers in all HNSCCs and similar significant results for both tumor entities separately. The surplus of annexin A2 in non-smokers and HPV-positive patients supports our hypothesis that decreased SLPI levels facilitate HPV-infection i.e., increased SLPI-expression may protect against HPV-infection of tonsillar and non-tonsillar SCC.
Asunto(s)
Carcinoma de Células Escamosas/microbiología , Neoplasias de Cabeza y Cuello/microbiología , Papillomaviridae/crecimiento & desarrollo , Infecciones por Papillomavirus/prevención & control , Inhibidor Secretorio de Peptidasas Leucocitarias/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Anexina A2/biosíntesis , Anexina A2/genética , Anexina A2/metabolismo , Carcinoma de Células Escamosas/metabolismo , Femenino , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Infecciones por Papillomavirus/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/genética , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
The precise molecular mechanisms enabling cancer cells to metastasize from the primary tumor to different tissue locations are still largely unknown. Secretion of some proteins by metastatic cells could facilitate metastasis formation. The comparison of secreted proteins from cancer cells with different metastatic capabilities in vivo might provide insight into proteins involved in the metastatic process. Comparison of the secreted proteins from the mouse breast cancer cell line 4T1 and its highly metastatic 4T1.2 clone revealed a prominent differentially secreted protein which was identified as SLPI (secretory leukocyte protease inhibitor). Western blotting indicated higher levels of the protein in both conditioned media and whole cell lysates of 4T1.2 cells. Additionally higher levels of SLPI were also observed in 4T1.2 breast tumors in vivo following immunohistochemical staining. A comparison of SLPI mRNA levels by gene profiling using microarrays and RT-PCR did not detect major differences in SLPI gene expression between the 4T1 and 4T1.2 cells indicating that SLPI secretion is regulated at the protein level. Our results demonstrate that secretion of SLPI is drastically increased in highly metastatic cells, suggesting a possible role for SLPI in enhancing the metastatic behavior of breast cancer cell line 4T1.
Asunto(s)
Neoplasias Mamarias Experimentales/patología , Inhibidor Secretorio de Peptidasas Leucocitarias/biosíntesis , Animales , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Metástasis de la Neoplasia , Inhibidor Secretorio de Peptidasas Leucocitarias/genética , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismoRESUMEN
A hallmark of cigarette smoking is a shift in the protease/antiprotease balance, in favor of protease activity. However, it has recently been shown that smokers have increased expression of a key antiprotease, secretory leukoprotease inhibitor (SLPI), yet the mechanisms involved in SLPI transcriptional regulation and functional activity of SLPI remain unclear. We examined SLPI mRNA and protein secretion in differentiated nasal epithelial cells (NECs) and nasal lavage fluid (NLF) from nonsmokers and smokers and demonstrated that SLPI expression is increased in NECs and NLF from smokers. Transcriptional regulation of SLPI expression was confirmed using SLPI promoter reporter assays followed by chromatin immunoprecipitation. The role of STAT1 in regulating SLPI expression was further elucidated using WT and stat1(-/-) mice. Our data demonstrate that STAT1 regulates SLPI transcription in epithelial cells and slpi protein in the lungs of mice. Additionally, we reveal that NECs from smokers have increased STAT1 mRNA/protein expression. Finally, we demonstrate that SLPI contained in the nasal mucosa of smokers is proteolytically cleaved but retains functional activity against neutrophil elastase. These results demonstrate that smoking enhances expression of SLPI in NECs in vitro and in vivo, and that this response is regulated by STAT1. In addition, despite posttranslational cleavage of SLPI, antiprotease activity against neutrophil elastase is enhanced in smokers. Together, our findings show that SLPI regulation and activity is altered in the nasal mucosa of smokers, which could have broad implications in the context of respiratory inflammation and infection.
Asunto(s)
Inhibidor Secretorio de Peptidasas Leucocitarias/biosíntesis , Fumar/genética , Adulto , Animales , Células Epiteliales/metabolismo , Femenino , Humanos , Elastasa de Leucocito/antagonistas & inhibidores , Pulmón/metabolismo , Masculino , Ratones , Líquido del Lavado Nasal , Mucosa Nasal/metabolismo , Factor de Transcripción STAT1/biosíntesisRESUMEN
We previously showed that secretory leukocyte protease inhibitor (SLPI) gene and protein expression is significantly lower in metastatic versus non-metastatic head and neck squamous cell carcinoma (HNSCC). However, we did not assess the human papillomavirus (HPV) status of these cases. Since SLPI plays a role in HIV and herpes simplex virus (HSV) infections, we hypothesized that SLPI may be involved in HPV-infected HNSCC. In HNSCC tissue (n=54), HPV DNA was determined and correlated with SLPI expression. Additionally, to investigate a possible role of smoking on SLPI expression in clinically normal mucosa, 19 patients treated for nonmalignant diseases (non-HNSCC) were analyzed for SLPI expression and correlated with smoking habits. In HNSCC patients, SLPI expression showed a significant inverse correlation with HPV status. In patients with moderate/strong SLPI expression (n=19), 10.5% were HPV-positive. By contrast, patients with absent/weak SLPI expression (n=35), 45.7% were HPV-positive. Low SLPI expression was correlated with metastasis (P=0.003) independent of HPV status. HPV-positivity was clearly associated with lymph node status (81.3% N1-3 cases). In smoking non-HNSCC patients (n=7), 42.9% showed absent/weak and 57.1% moderate/strong SLPI staining. In non-smoking non-HNSCC patients (n=10) 83.3% showed absent/weak and 16.7% moderate/strong SLPI expression. For the first time, a correlation between SLPI downregulation and HPV infection was demonstrated, suggesting that high levels of SLPI, possibly induced by environmental factors such as tobacco smoking, correlate with protective effects against HPV infection. SLPI may be a potential biomarker identifying head and neck cancer patients not at risk of developing metastases (SLPI-positive), and those at risk to be infected by HPV (SLPI-negative) and likely to develop metastases.
Asunto(s)
Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/virología , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/virología , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Adulto , Anciano , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , ADN Viral/genética , Regulación hacia Abajo/genética , Femenino , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Humanos , Leucocitos/metabolismo , Leucocitos/virología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Ganglios Linfáticos/virología , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Papillomaviridae/genética , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/patología , Inhibidores de Proteasas/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/biosíntesis , Inhibidor Secretorio de Peptidasas Leucocitarias/genética , Fumar/efectos adversos , Fumar/genética , Fumar/metabolismoRESUMEN
BACKGROUND: There are 2 new phenotypes of HIV-1-positive individuals who exhibit a spontaneous and sustained control of viral replication at least for 1 year without antiretroviral therapy (elite controllers <50 copies/mL and viremic controllers <2000 copies/mL). Mechanisms related to this spontaneous control of viral replication are poorly understood. METHODS: The study included HIV-1 controllers (patients with at least 1 year of HIV-1 diagnosis, highly active antiretroviral therapy naive, and with viral loads less than 2000 copies/mL) and HIV-1 progressors without antiretroviral therapy (viral load >2500 copies/mL, and CD4 T-cell count >250 cells/µL at the time of sampling). The expression of soluble factors, leukocyte protease inhibitor (SLPI) and human α-defensins-1 (HAD-1), was measured by real-time polymerase chain reaction from neutrophil cultures with or without HIV stimulation; the frequency and phenotype of innate and adaptive immune cells were determined by flow cytometry, and frequency of human leukocyte antigen alleles was determined by polymerase chain reaction sequence-specific oligonucleotide typing. RESULTS: As expected, HIV-1 controllers had higher CD4 T-cell counts and lower viral load when compared with HIV-1 progressor individuals; in addition, they exhibited lower expression of activation markers, higher frequency of myeloid dendritic cell, lower percentage of regulatory T cells and natural killer cells, and higher expression of SLPI. CONCLUSIONS: All together, these findings suggest that the control of the immune activation status and the production of antiviral proteins by innate immune cells could be associated to the mechanisms involved in the control of HIV-1 replication and better preservation of the CD4 T-cell count.
Asunto(s)
Infecciones por VIH/inmunología , Sobrevivientes de VIH a Largo Plazo , VIH-1/inmunología , Inmunidad Innata , Leucocitos/inmunología , Inhibidor Secretorio de Peptidasas Leucocitarias/biosíntesis , Adulto , Alelos , Células Cultivadas , Colombia , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Antígenos HLA/análisis , Antígenos HLA/genética , Humanos , Inmunofenotipificación , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Carga Viral , alfa-Defensinas/biosíntesisRESUMEN
PURPOSE: To identify the novel gene signatures and molecular markers of nasopharyngeal carcinoma (NPC) by integrated bioinformatics analysis of multiple gene expression profiling datasets. EXPERIMENTAL DESIGN: Seven published gene expression profiling studies and one of our unpublished works were reanalyzed to identify the common significantly dysregulated (CSD) genes in NPC. Overrepresentation analysis of cytogenetic bands, Gene Ontology (GO) categories, pathways were used to explore CSD genes functionally associated with carcinogenesis. The protein expressions of selected CSD genes were examined by immunohistochemistry on tissue microarrays, and the correlations of their expressions with clinical outcomes were evaluated. RESULTS: Using the criteria (genes reported deregulated in more than one study), a total of 962 genes were identified as the CSD genes in NPC. Four upregulated (BUB1B, CCND2, CENPF, and MAD2L1) and two downregulated (LTF and SLPI) genes were markedly reported in six studies. The enrichments of chromosome aberrations were 2q23, 2q31, 7p15, 12q15, 12q22, 18q11, and 18q12 in upregulated genes and 14q32 and 16q13 in downregulated genes. The activated GO categories and pathways related to proliferation, adhesion, invasion, and downregulated immune response had been functionally associated with NPC. SLPI significantly downregulated in nasopharyngeal adenocarcinoma. Furthermore, the high expression of BUB1B or CENPF was associated with poor overall survival of patients. CONCLUSION: It was first clearly identified the dysregulated expression of BUB1B and SLPI in NPC tissues. IMPACT: Further studies of the CSD genes as gene signatures and molecular markers of NPC might improve the understanding of the disease and identify new therapeutic targets.
Asunto(s)
Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Nasofaríngeas/genética , Biomarcadores de Tumor/biosíntesis , Carcinoma , Proteínas Cromosómicas no Histona/biosíntesis , Proteínas Cromosómicas no Histona/genética , Biología Computacional/métodos , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Análisis por Micromatrices , Proteínas de Microfilamentos/biosíntesis , Proteínas de Microfilamentos/genética , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patología , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/genética , Inhibidor Secretorio de Peptidasas Leucocitarias/biosíntesis , Inhibidor Secretorio de Peptidasas Leucocitarias/genéticaRESUMEN
OBJECTIVES: Secretory leukocyte protease inhibitor (SLPI) is amplified in serous ovarian cancer. We have dissected its function, showing it is a survival factor for ovarian cancer and promotes tumorigenesis and paclitaxel-resistance. We hypothesized that the protease inhibitory function was responsible for modulating SLPI's invasive capacity. METHODS: Stable HEYA8 ovarian cancer transfectants expressing vector, wild type SLPI, and protease inhibitor null (F-)SLPI were examined in vitro and in xenografts. Invasion, enzyme activity, and MMP production and function assays were applied. SLPI and MMP immunoexpression was graded on tissue microarray and clinical samples. Statistical comparisons used unpaired t test and ANOVA, where appropriate. RESULTS: SLPI and F-SLPI cells caused greater parenchymal and peritoneal dissemination over control cells in xenografts and invasion assays (p<0.001). MMP-9 protease activity was increased in SLPI and F-SLPI cells over control. SLPI, but not F-SLPI, inhibited plasmin activity, necessary for MMP-9 activation and release, and inhibited activation of MMP-9. However, paradoxically, both induced quantitative MMP-9 transcription (p<0.05) and protein (p<0.008), yielding an increased net MMP-9 activity in the face of plasmin inhibition. SLPI and MMP-9 expression were strongly correlated in serous ovarian cancers (r(2)=0.986) and a set of ovarian cancers (p<0.02). SLPI expression was greater in serous than endometrioid ovarian cancers (p=0.04). CONCLUSIONS: SLPI stimulates ovarian cancer invasion, modulated in part by its serine protease inhibitory activity attenuating MMP-9 release. However, SLPI induction of MMP-9, independent of protease inhibition activity, is greater yielding a net pro-invasive behavior. These findings further support SLPI as a molecular target for ovarian cancer.
Asunto(s)
Metaloproteinasa 9 de la Matriz/biosíntesis , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Cistadenocarcinoma Seroso/enzimología , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patología , Femenino , Fibrinolisina/antagonistas & inhibidores , Fibrinolisina/metabolismo , Humanos , Metaloproteinasa 9 de la Matriz/genética , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Inhibidor Secretorio de Peptidasas Leucocitarias/biosíntesis , Inhibidor Secretorio de Peptidasas Leucocitarias/genética , Transcripción Genética , Transfección , Regulación hacia ArribaRESUMEN
Secretory leukocyte protease inhibitor (SLPI) is an anti-inflammatory protein that is observed at high levels in asthma patients. Resiquimod, a TLR7/8 ligand, is protective against acute and chronic asthma, and it increases SLPI expression of macrophages in vitro. However, the protective role played by SLPI and the interactions between the SLPI and resiquimod pathways in the immune response occurring in allergic asthma have not been fully elucidated. To evaluate the role of SLPI in the development of asthma phenotypes and the effect of resiquimod treatment on SLPI, we assessed airway resistance and inflammatory parameters in the lungs of OVA-induced asthmatic SLPI transgenic and knockout mice and in mice treated with resiquimod. Compared with wild-type mice, allergic SLPI transgenic mice showed a decrease in lung resistance (p < 0.001), airway eosinophilia (p < 0.001), goblet cell hyperplasia (p < 0.001), and plasma IgE levels (p < 0.001). Allergic SLPI knockout mice displayed phenotype changes significantly more severe compared with wild-type mice. These phenotypes included lung resistance (p < 0.001), airway eosinophilia (p < 0.001), goblet cell hyperplasia (p < 0.001), cytokine levels in the lungs (p < 0.05), and plasma IgE levels (p < 0.001). Treatment of asthmatic transgenic mice with resiquimod increased the expression of SLPI and decreased inflammation in the lungs; resiquimod treatment was still effective in asthmatic SLPI knockout mice. Taken together, our study showed that the expression of SLPI protects against allergic asthma phenotypes, and treatment by resiquimod is independent of SLPI expression, displayed through the use of transgenic and knockout SLPI mice.
Asunto(s)
Alérgenos/administración & dosificación , Asma/enzimología , Asma/inmunología , Inhibidor Secretorio de Peptidasas Leucocitarias/fisiología , Enfermedad Aguda , Animales , Asma/patología , Hiperreactividad Bronquial/enzimología , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/fisiopatología , Movimiento Celular/genética , Movimiento Celular/inmunología , Modelos Animales de Enfermedad , Inflamación/enzimología , Inflamación/genética , Inflamación/prevención & control , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Inhibidor Secretorio de Peptidasas Leucocitarias/biosíntesis , Inhibidor Secretorio de Peptidasas Leucocitarias/deficienciaRESUMEN
Oral transmission of human immunodeficiency virus (HIV) in adult populations is rare. However, HIV spread across fetal/neonatal oropharyngeal epithelia could be important in mother-to-child transmission. Analysis of HIV transmission across polarized adult and fetal oral epithelial cells revealed that HIV transmigrates through both adult and fetal cells. However, only virions that passed through the fetal cells - and not those that passed through the adult cells - remained infectious. Analysis of expression of anti-HIV innate proteins beta-defensins 2 and 3, and secretory leukocyte protease inhibitor in adult, fetal, and infant oral epithelia showed that their expression is predominantly in the adult oral epithelium. Retention of HIV infectivity after transmigration correlated inversely with the expression of these innate proteins. Inactivation of innate proteins in adult oral keratinocytes restored HIV infectivity. These data suggest that high-level innate protein expression may contribute to the resistance of the adult oral epithelium to HIV transmission.
Asunto(s)
Células Epiteliales/virología , VIH/fisiología , Migración Transendotelial y Transepitelial , Inactivación de Virus , Adulto , Células Cultivadas , Células Epiteliales/inmunología , Feto , Expresión Génica , Perfilación de la Expresión Génica , VIH/crecimiento & desarrollo , VIH/inmunología , VIH/patogenicidad , Humanos , Recién Nacido , Inhibidor Secretorio de Peptidasas Leucocitarias/biosíntesis , beta-Defensinas/biosíntesisRESUMEN
Secretory leukocyte protease inhibitor (SLPI) is produced at mucosal sites where it plays an important role in the homeostatic control of local inflammation. In addition to its anti-protease activity SLPI is able to reduce LPS activity by interfering with the transfer of LPS to CD14. In addition SLPI can be taken up by cells where it can prevent signaling via the NF-κB route. It is preferentially expressed in dendritic cells from mucosal sites, suggesting a role in the maintenance of a tolerogenic environment, but it is unclear how this differential expression is regulated. Here we analyzed the regulation of SLPI expression in dendritic cells and found that activation by TLR ligands but not via antiCD40 leads to its expression, which is predominantly dependent on p38 activation. This induced expression is late compared to the induction of cytokines and co-stimulatory molecules, is not dependent on factors that are secreted by the cell itself and may be related to cellular feedback mechanisms involved in inflammation and immunity. In correlation with the differential expression by TLR ligands and antiCD40, SLPI deficient mice show enhanced specific immunity when antigen is co-injected with LPS, but not with antiCD40. The results underscore the importance of SLPI as a modulator of specific immunity that can also function at peripheral sites under pathogenic pressure.
Asunto(s)
Células Dendríticas/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/biosíntesis , Inmunidad Adaptativa/efectos de los fármacos , Animales , Células de la Médula Ósea/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/enzimología , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Cinética , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Modelos Inmunológicos , FN-kappa B/metabolismo , Estructura Secundaria de Proteína , Inhibidor Secretorio de Peptidasas Leucocitarias/química , Inhibidor Secretorio de Peptidasas Leucocitarias/genética , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The human secretory leukocyte protease inhibitor (SLPI) is an 11.7 kD cysteine-rich protein that has been shown to possess anti-protease, anti-inflammatory, and antimicrobial properties. By using a Pichia pastoris strain that overproduces protein disulfide isomerase (PDI), we obtained greater than fivefold higher levels of SLPI than in strains expressing normal levels of PDI and containing multiple copies of the SLPI gene. Elevated levels of PDI also enhanced the specific activity of the secreted SLPI by helping it achieve a proper tertiary structure. Mass spectrometry analysis indicated a greater number of disulfide bonds in the SLPI produced by the PDI overexpression strain compared to the SLPI produced in strains with normal PDI levels. Although others have utilized a similar strategy to increase yield, we believe that this is the first example of PDI overexpression being demonstrated to enhance the folding and thus increase the biological activity of a protein produced in the yeast P. pastoris.
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Pichia/metabolismo , Proteínas Recombinantes/biosíntesis , Inhibidor Secretorio de Peptidasas Leucocitarias/biosíntesis , Fermentación , Glicosilación , Humanos , Pichia/genética , Pichia/crecimiento & desarrollo , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Inhibidor Secretorio de Peptidasas Leucocitarias/química , Inhibidor Secretorio de Peptidasas Leucocitarias/genéticaRESUMEN
BACKGROUND AND OBJECTIVE: The junctional epithelium attaches to the tooth enamel at the dentogingival junction. The attachment mechanisms of the junctional epithelium have been studied histologically, but the molecular functions of the junctional epithelium have not been elucidated. The aim of this study was to perform a comprehensive analysis of gene expression in the junctional epithelium and to search for specific genetic markers of the junctional epithelium. MATERIAL AND METHODS: A comprehensive analysis of genes expressed in the mouse junctional epithelium and oral gingival epithelium was performed using laser microdissection and microarray analysis. To extract high-quality RNA from these tissues, we made frozen sections using a modified film method. Confirmation of the differential expression of selected genes was performed by quantitative real-time PCR and immunohistochemistry. RESULTS: The modified method produced RNA of sufficient quality for microarray analysis. The result of microarray analysis showed that 841 genes were up-regulated in the junctional epithelium compared with the oral gingival epithelium, and five were increased more than 50-fold in the junctional epithelium. These five genes were secretory leukocyte protease inhibitor (Slpi), keratin 17 (Krt17), annexin A1 (Anxa1), myosin light peptide 6 (Myl6) and endoplasmic reticulum protein 29 (Erp29). In particular, Slpi expression in the junctional epithelium was approximately 100-fold higher than in the oral gingival epithelium by real-time PCR. Additionally, immunohistochemistry indicated that the Slpi protein is highly expressed in the junctional epithelium. CONCLUSION: We developed a method for generating fresh-frozen tissue sections suitable for extraction of good-quality RNA. We determined that Slpi is characteristically expressed in the junctional epithelium. Our results provide a substantial advance in the analysis of gene expression in the junctional epithelium.
Asunto(s)
Inserción Epitelial/metabolismo , Perfilación de la Expresión Génica/métodos , Inhibidor Secretorio de Peptidasas Leucocitarias/biosíntesis , Animales , Anexina A1/biosíntesis , Anexina A1/genética , Retículo Endoplásmico , Inserción Epitelial/enzimología , Secciones por Congelación , Encía/metabolismo , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/genética , Queratina-17/biosíntesis , Queratina-17/genética , Láseres de Gas , Ratones , Microdisección/métodos , Cadenas Ligeras de Miosina/biosíntesis , Cadenas Ligeras de Miosina/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Inhibidor Secretorio de Peptidasas Leucocitarias/genéticaRESUMEN
Heterosexual transmission of human immunodeficiency virus (HIV-1) is the predominant mode of infection worldwide. However, the early steps of transepithelial infection still need to be clarified. Using epithelial cells, originating from the female genital tract, and peripheral blood mononuclear cells as subepithelial target cells, an in vitro dual-chamber model of the female genital tract was developed. Remarkably, an intact layer of some cell types (HEC-1A, CaSki and Ect1) served as a protective barrier against cell-free but not against cell-associated HIV-1 that crossed the epithelial barrier through transmigration. Furthermore, dysfunctions of the epithelial layers were assessed by monitoring transepithelial electric resistance and transepithelial passage of FluoSpheres and HIV-1 after treatment with nonoxynol-9 (N-9). Most of the functional assays showed dysfunction of the epithelial barrier at lower concentrations compared to a widely used colorimetric toxicity assay (WST-1). Finally, N-9 treatment caused a significant increase in the production of interleukin-8 (IL-8) and macrophage inflammatory protein-3alpha (MIP-3alpha) and a decrease of Secretory Leukocyte Protease Inhibitor (SLPI) and Monocyte Chemotactic Protein-1 (MCP-1) in this model. In conclusion, this model is a useful tool to (1) study HIV-1 transmission mechanisms and (2) evaluate epithelial toxicity of candidate microbicides.
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Fármacos Anti-VIH/toxicidad , Genitales Femeninos/virología , Infecciones por VIH/transmisión , VIH-1/fisiología , Modelos Biológicos , Técnicas de Cultivo de Célula/instrumentación , Línea Celular , Cuello del Útero/efectos de los fármacos , Cuello del Útero/virología , Quimiocina CCL2/biosíntesis , Quimiocina CCL20/biosíntesis , Técnicas de Cocultivo , Endometrio/efectos de los fármacos , Endometrio/virología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Femenino , Genitales Femeninos/efectos de los fármacos , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Humanos , Interleucina-8/biosíntesis , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Nonoxinol/farmacología , Especificidad de Órganos/efectos de los fármacos , Inhibidor Secretorio de Peptidasas Leucocitarias/biosíntesis , Útero/efectos de los fármacos , Útero/virología , Vagina/efectos de los fármacos , Vagina/virología , Replicación ViralRESUMEN
INTRODUCTION: The secretory leukocyte protease inhibitor (SLPI) is a bacterial lipopolysaccharide (LPS)-induced product of macrophages that antagonizes the LPS-induced activation of a number of proinflammatory signaling factors. From our previous experiments, it was found that SLPI was expressed slightly in odontoblast-like cells (MDPC-23). Therefore, these experiments were designed to determine the function of SLPI in MDPC-23 and odontoblasts during the inflammatory response caused by infections and wounds. METHODS: MDPC-23 cells were exposed to 100 ng/mL Escherichia coli LPS, and artificial wounds were induced in the right first molar of the maxillary of rats. In addition, a morphological change in the MDPC-23 cells was observed after LPS treatment. MDPC-23 cells were transfected transiently with the nuclear factor kappa-B (NF-kappaB) promoter binding vector. RESULTS: The level of SLPI expression increased strongly 30 minutes after the LPS treatment. Scanning electron microscopy revealed many extensions of the cytoplasmic processes after LPS stimulation. SLPI was expressed along the dentinal tubules and odontoblasts layer in rat teeth after an artificial wound. SLPI also inhibited the LPS-induced activation of NF-kappaB in MDPC-23. CONCLUSIONS: We report for the first time that SLPI is expressed temporally in infected odontoblasts and may participate in the anti-inflammatory response through NF-kappaB signaling in odontoblast-like cells.
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Pulpa Dental/metabolismo , FN-kappa B/antagonistas & inhibidores , Odontoblastos/metabolismo , Pulpitis/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/biosíntesis , Infección de Heridas/metabolismo , Animales , Línea Celular Transformada , Pulpa Dental/citología , Dentina/metabolismo , Inducción Enzimática , Escherichia coli/química , Inmunohistoquímica , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Ratones , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidor Secretorio de Peptidasas Leucocitarias/fisiología , Transducción de Señal , Factores de TiempoRESUMEN
The human secretory leukocyte protease inhibitor (SLPI) has been shown to possess anti-protease, anti-inflammatory and antimicrobial properties. Its presence in saliva is believed to be a major deterrent to oral transmission of human immunodeficiency virus-1. The 11.7kDa peptide is a secreted, nonglycosylated protein rich in disulfide bonds. Currently, recombinant SLPI is only available as an expensive bacterial expression product. We have investigated the utility of the methylotrophic yeast Pichia pastoris to produce and secrete SLPI with C-terminal c-myc and polyhistidine tags. The post-transformational vector amplification protocol was used to isolate strains with increased copy number, and culturing parameters were varied to optimize SLPI expression. Modification of the purification procedure allowed the secreted, recombinant protein to be isolated from the cell-free fermentation medium with cobalt affinity chromatography. This yeast-derived SLPI was shown to have an anti-protease activity comparable to the commercially available bacterial product. Thus, P. pastoris provides an efficient, cost-effective system for producing SLPI for structure function analysis studies as well as a wide array of potential therapeutic applications.
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Pichia/química , Pichia/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/biosíntesis , Inhibidor Secretorio de Peptidasas Leucocitarias/química , Técnicas de Cultivo de Célula , Fermentación , Glicosilación , Humanos , Pichia/genética , Pliegue de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Inhibidor Secretorio de Peptidasas Leucocitarias/genética , Inhibidor Secretorio de Peptidasas Leucocitarias/aislamiento & purificación , Transfección , Tripsina/metabolismoRESUMEN
INTRODUCTION: In the present study, we investigated the ability of microparticles isolated from synovial fluids from patients with rheumatoid arthritis or osteoarthritis to induce the synthesis and release of key cytokines of B-lymphocyte modulation such as B cell-activating factor, thymic stroma lymphopoietin, and secretory leukocyte protease inhibitor by rheumatoid fibroblast-like synoviocytes. METHODS: Microparticles were analyzed in synovial fluids from patients with rheumatoid arthritis, osteoarthritis, microcristalline arthritis, and reactive arthritis. In addition, microparticle release after activation from various cell lines (CEM lymphocyte and THP-1 cells) was assessed. Microparticles were isolated by differential centrifugation, and quantitative determinations were carried out by prothrombinase assay after capture on immobilized annexin V. B cell-activating factor, thymic stroma lymphopoietin, and secretory leukocyte protease inhibitor release was evaluated by enzyme-linked immunosorbent assay. RESULTS: Microparticles isolated from synovial fluids obtained from rheumatoid arthritis and osteoarthritis patients or microparticles derived from activated THP-1 cells were able to induce B cell-activating factor, thymic stroma lymphopoietin, and secretory leukocyte protease inhibitor release by rheumatoid arthritis fibroblast-like synoviocytes. Conversely, CEM-lymphocytes-derived microparticles generated by treatment with a combination of PHA, PMA and Adt-D did not promote the release of B cell-activating factor but favored the secretion of thymic stroma lymphopoietin and secretory leukocyte protease inhibitor by rheumatoid arthritis fibrobast-like synoviocytes. However, microparticles isolated from actinomycin D-treated CEM lymphocytes were not able to induce B cell-activating factor, thymic stroma lymphopoietin, or secretory leukocyte protease inhibitor release, indicating that microparticles derived from apoptotic T cells do not function as effectors in B-cell activation. CONCLUSIONS: These results demonstrate that microparticles are signalling structures that may act as specific conveyors in the triggered induction and amplification of autoimmunity. This study also indicates that microparticles have differential effects in the crosstalk between B lymphocytes and target cells of autoimmunity regarding the parental cells from which they derive.
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Artritis Reumatoide/inmunología , Micropartículas Derivadas de Células/inmunología , Líquido Sinovial/inmunología , Anciano , Anciano de 80 o más Años , Factor Activador de Células B/biosíntesis , Factor Activador de Células B/inmunología , Linfocitos B/inmunología , Citocinas/biosíntesis , Citocinas/inmunología , Femenino , Fibroblastos/inmunología , Humanos , Interleucina-6/biosíntesis , Interleucina-6/inmunología , Interleucina-8/biosíntesis , Interleucina-8/inmunología , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Receptor Cross-Talk/inmunología , Inhibidor Secretorio de Peptidasas Leucocitarias/biosíntesis , Inhibidor Secretorio de Peptidasas Leucocitarias/inmunología , Líquido Sinovial/citología , Membrana Sinovial/citología , Membrana Sinovial/inmunología , Linfopoyetina del Estroma TímicoRESUMEN
Mice lacking surfactant protein A (SP-A) are susceptible to bacterial infection associated with an excessive inflammatory response in the lung. To determine mechanisms by which SP-A is antiinflammatory in the lung during bacterial infection, SP-A regulation of secretory leukoprotease inhibitor (SLPI), an inhibitor of serine proteases, was assessed. SLPI protein expression and antineutrophil elastase activity were reduced in bronchoalveolar fluid of SP-A(-/-) compared with SP-A(+/+) mice. Intratracheal administration of SP-A to SP-A(-/-) mice enhanced SLPI protein expression and antineutrophil elastase activity in the lung. SLPI mRNA was similar in whole lung and alveolar type II cells; however, it was significantly reduced in alveolar macrophages from SP-A(-/-) compared with SP-A(+/+) mice. In vitro, SP-A enhanced SLPI production by macrophage THP-1 cells but not respiratory epithelial A549 cells. SP-A inhibited LPS induced IkappaB-alpha degradation in THP-1 cells, which was partially reversed with knockdown of SLPI. Matrix metalloproteinase (MMP)-12 cleaved SLPI and incubation with SP-A reduced MMP-12-mediated SLPI cleavage. The collagen-like region of SP-A conferred protection of SLPI against MMP mediated cleavage. SP-A plays an important role in the lung during bacterial infection regulating protease and antiprotease activity.
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Metaloproteinasa 12 de la Matriz/fisiología , Inhibidores de la Metaloproteinasa de la Matriz , Proteína A Asociada a Surfactante Pulmonar/fisiología , Inhibidor Secretorio de Peptidasas Leucocitarias/biosíntesis , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Líquido del Lavado Bronquioalveolar/microbiología , Línea Celular Tumoral , Femenino , Infecciones por Haemophilus/enzimología , Infecciones por Haemophilus/inmunología , Infecciones por Haemophilus/metabolismo , Haemophilus influenzae/inmunología , Humanos , Hidrólisis , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/fisiología , Masculino , Metaloproteinasa 12 de la Matriz/biosíntesis , Ratones , Ratones Noqueados , Neumonía/enzimología , Neumonía/inmunología , Neumonía/metabolismo , Neumonía/patología , Proteína A Asociada a Surfactante Pulmonar/deficiencia , Proteína A Asociada a Surfactante Pulmonar/genética , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , Inhibidor Secretorio de Peptidasas Leucocitarias/antagonistas & inhibidores , Inhibidor Secretorio de Peptidasas Leucocitarias/genética , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/fisiología , Regulación hacia Arriba/inmunología , alfa 1-Antitripsina/metabolismoRESUMEN
BACKGROUND: Secretory leukocyte protease inhibitor (SLPI) and elafin are anti-protease and anti-microbial molecules with a role in innate immune defence. They have been demonstrated at multiple mucosal surfaces including those of the female reproductive tract. METHODS AND RESULTS: This study details their expression in human Fallopian tubes (ampullary region) throughout the menstrual cycle (n = 18) and from women with ectopic pregnancy (n = 6), and examined their regulation by infection with Chlamydia trachomatis in an in-vitro model. Quantitative real-time PCR analysis showed that SLPI and elafin were constitutively expressed in the Fallopian tube during the menstrual cycle but were increased in ectopic pregnancy (P < 0.05 versus early-mid luteal phase, P < 0.01 versus all phases, respectively). SLPI and elafin were immunolocalised to the Fallopian tube epithelium in biopsies from non-pregnant women and those with ectopic pregnancy. An in-vitro culture model of C. trachomatis infection of the OE-E6/E7 oviductal epithelial cell line showed that elafin mRNA expression was upregulated in response to chlamydial infection. CONCLUSION: These data suggest that SLPI and elafin have a role in the innate immune defence of the Fallopian tube in infection and ectopic pregnancy. Their role is likely to include regulation of protease activity, wound healing and tissue remodelling.