RESUMEN
Acute myeloid leukaemia (AML) is a severe haematological neoplasm that originates from the transformation of haematopoietic stem cells (HSCs) into leukaemic stem cells (LSCs). The bone marrow (BM) microenvironment, particularly that of mesenchymal stromal cells (hMSCs), plays a crucial role in the maintenance of HSCs. In this context, we explored whether alterations in the secretome of hMSCs derived from AML patients (hMSC-AML) could impact HSC gene expression. Proteomic analysis revealed that the secretome of coculture assays with hMSC-AMLs and HSC from healthy donor is altered, with increased levels of secretory leukocyte protease inhibitor (SLPI), a protein associated with important processes for maintenance of the haematopoietic niche that has already been described to be altered in several tumours. Increased SLPI expression was also observed in the BM plasma of AML patients. Transcriptome analysis of HSCs cocultured with hMSC-AML in comparison with HSCs cocultured with hMSC-HD revealed altered expression of SLPI target genes associated with the cell cycle, proliferation, and apoptosis. Important changes were identified, such as increased expression levels of CCNA2, CCNE2, CCND2, CD133 and CDK1 and decreased levels of CDKN2A and IGFBP3, among others. Overall, these findings suggest that the altered secretome of coculture assays with hMSC-AMLs and HSC from healthy donor, particularly increased SLPI expression, can contribute to gene expression changes in HSCs, potentially influencing important molecular mechanisms related to AML development and progression.
Asunto(s)
Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Células Madre Mesenquimatosas , Inhibidor Secretorio de Peptidasas Leucocitarias , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Células Madre Mesenquimatosas/metabolismo , Células Madre Hematopoyéticas/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/genética , Técnicas de Cocultivo , Transcriptoma , Femenino , Masculino , Perfilación de la Expresión Génica , Persona de Mediana Edad , Proteómica/métodos , Regulación Leucémica de la Expresión Génica , Anciano , Adulto , Proliferación Celular/genéticaRESUMEN
Aim: It is important to find biomarkers that identify the graft quality in kidney transplantation. Results & methodology: The level of SLPI in the cold preservation solution was used as a marker to predict early kidney graft function after transplantation. Before transplantation, kidneys were washed and SLPI was measured in the discarded solution. A retrospective analysis showed that patients with delayed graft function or rejection episodes in post-trasplant, had higher SLPI concentrations in the perfusion solution than patients without delayed graft function or rejections. Furthermore, SLPI could discriminate between patients with better or worse estimated glomerular filtration rate among low-risk patients (kidney donor profile index <80). Discussion & conclusion: These results suggest that the SLPI concentration in the perfusion solutions could be a predictor of short-term organ function and a complement to the kidney donor profile index score.
Asunto(s)
Riñón/química , Perfusión/instrumentación , Inhibidor Secretorio de Peptidasas Leucocitarias/análisis , Anciano , Biomarcadores/análisis , Funcionamiento Retardado del Injerto , Femenino , Tasa de Filtración Glomerular , Humanos , Riñón/metabolismo , Riñón/fisiopatología , Riñón/cirugía , Trasplante de Riñón , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismoRESUMEN
Secretory Leukocyte Proteinase Inhibitor (SLPI) is an antiinflammatory peptide that blocks the activity of serine proteases, primarily the neutrophil elastase. In an attempt to direct the activity of SLPI on inflamed sites, a chimera consisting of the transglutaminase II substrate domain of trappin 2 (cementoin), and the mature SLPI protein was constructed. Cell attachment and biological activity were compared between SLPI and this chimera. By using whole cell ELISA, fluorescence microscopy and flow cytometry assays we observed that the cementoin-SLPI fusion protein (FP) but not SLPI attached to a human lung epithelial cell line and monocytes. A maximum attachment was achieved 15 min after FP was added to the cell cultures. In an elastase activity assay, we observed that FP retained its antiprotease activity and that at equimolar amount of proteins, FP was more efficient than SLPI in the inhibition. Both, FP and SLPI inhibits IL-2-induced lymphocyte proliferation, however, lower amounts of FP were required to achieve this inhibition. Furthermore, FP binds to mycobacteria and maintained the bactericidal activity observed for SLPI. Overall, these results show that this new chimera is able to attach to the cell surfaces retaining and improving some biological activities described for SLPI.
Asunto(s)
Membrana Celular/metabolismo , Células Epiteliales/metabolismo , Monocitos/metabolismo , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Biomarcadores , Línea Celular , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Humanos , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Monocitos/efectos de los fármacos , Péptidos/genética , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Inhibidor Secretorio de Peptidasas Leucocitarias/genética , Inhibidor Secretorio de Peptidasas Leucocitarias/farmacologíaRESUMEN
After severe skeletal muscle damage, communication between inflammatory macrophages, myogenic cells, and modulatory secretion factors is essential to induce re-establishment of skeletal muscle structure. To analyze when characteristic gene expression of macrophages, myogenic cells, and SLPI are modulated after an exercise-induced muscle damage (EIMD) downhill protocol. Twenty-six rats were exposed to an experimental protocol of exercise and euthanized before (CTRL), immediately after (G0), and 24 (G24) and 48 (G48) hours after the exercise. After euthanasia, the Triceps brachii were dissected and analyzed by enzyme-linked immunosorbent assay and real time polymerase chain reaction. The CD68 expression was higher in the G24 when compared with all groups (p value < 0.05), whereas the CD163 was inhibited compared with G0 (p value < 0.05). MyoD and Myogenin were higher in the G24 when compared with G0 and G48 (p value < 0.05). The mRNA Secretory Leukocyte Protease Inhibitor (SLPI) was higher in the G48 when compared with the CTRL and G0 (p value < 0.05). IL-6 and TNF-α cytokines did not significantly change, but IL-10 presented a trend to be lower in the G0 when compared with G24 (p value = 0.054). A significant negative correlation was observed between CD68/CD163 (C.C = -0.71) and positive correlations between CD68/Myogenin (C.C = 0.65); MyoD/Myogenin (C.C = 0.72); IL-10/MyoD (C.C = 0.46), IL-10/MYOGENIN (C.C = 0.59); and IL-6/IL-10 (C.C = 0.64). A higher expression of CD68, concomitant with an elevation in MyoD and Myogenin 24 h after exercise, along with some correlations, suggests macrophage communication with myogenic cells independent of CD163 elevation. Additionally, the reestablishment of IL-10 in 24 h with the SLPI increased until 48 h indicate that these molecules are involved with anti-inflammatory transition after downhill exercise in the TBIH of Wistar rats.
Asunto(s)
Músculo Esquelético/metabolismo , Condicionamiento Físico Animal/métodos , ARN Mensajero/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Animales , Masculino , Ratas , Ratas WistarRESUMEN
OBJECTIVE: The aim of this study was to evaluate secretory leukocyte protease inhibitor (SLPI) expression in anal biopsies from HIV-positive (HIV+) individuals, and compare that to anal intraepithelial neoplasia (AIN) diagnoses and human papillomavirus (HPV) status. DESIGN: This is a cross-sectional study of a cohort of 54 HIV+ (31 males and 23 females) from an AIDS clinic in Rio de Janeiro, Brazil. METHODS: The study material consisted of anorectal tissue biopsies obtained from HIV+ subjects, which were used to construct tissue microarray paraffin blocks for immunohistochemical analysis of SLPI expression. Biopsies were evaluated by an expert pathologist and classified as low-grade AIN1, high-grade AIN2/3, or normal squamous epithelium. In addition, DNA from the biopsies was extracted and analyzed for the presence of low- or high-risk HPV DNA. RESULTS: Histologically, normal squamous epithelium from the anorectal region showed strong positive SLPI staining in 17/20 (85%) samples. In comparison, 9/17 (53%) dysplastic squamous epithelial samples from AIN1 patients showed strong SLPI staining, and only 5/17 (29%) samples from AIN2/3 patients exhibited strong SPLI staining, which both were significantly fewer than those from normal tissue (P = 0.005). Furthermore, there was a significantly higher proportion of samples in which oncogenic high-risk HPV genotypes were detected in low SLPI-expressing tissues than that in tissues with high SLPI expression (P = 0.040). CONCLUSIONS: Taken together these results suggest that low SLPI expression is associated with high-risk HPV infections in the development of AIN.
Asunto(s)
Alphapapillomavirus/aislamiento & purificación , Enfermedades del Ano/complicaciones , Infecciones por VIH/complicaciones , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Adulto , Enfermedades del Ano/metabolismo , Enfermedades del Ano/patología , Enfermedades del Ano/virología , Biopsia , Brasil , Femenino , Infecciones por VIH/metabolismo , Humanos , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: To study the effect of topical administration of a fusion protein (PF-MC) made up of N-terminal portion of the protease inhibitor Trappin-2 (which is a substrate of transglutaminasa-2) and SLPI (protein with anti-inflammatory, anti-bacterial and anti-viral ability), in an animal model of corneal inflammation and angiogenesis. METHODS: An alkali injury was produced with a filter paper of 3 mm with 1 N NaOH during 40 seconds on the right cornea of 36 male Sprague Dawley rats, under general anesthesia. Animals were divided into three groups according to treatment. Group 1 was treated with 10 ul of PF-MC (200 ug/ml; n = 12), Group 2, with 10 ul of SLPI (200 ug/ml; n = 12) and Group 3 was treated with buffer (10 ul; n = 12) topically administered four times a day for up to 7 days. Half of the animals were sacrificed at day 3 before making a re-epithelialization time analysis with fluorescein staining at 18 and 24 hours. In the remaining animals corneal opacity was studied and digital photographs were taken at day 7 before doing euthanasia. Eyes were processed for histology and immunofluorescence. RESULTS: Corneal ulcerated area was significantly lower in PF-MC treated animals compared to SLPI and buffer-treated animals at 18 hours and 24 hours postinjury. A clear cornea and fundus red reflex was only found among PF-MC treated animals. Histological analysis revealed a stratified corneal epithelium with at least three layers in all PF-MC animals at day 7. In this group there was a reduced number of PMNs in the corneal stroma at 3 and 7 days of follow-up. Besides, corneal neovascularization was much more extended in SLPI and Buffer animals than in animals treated with PF-MC. CONCLUSIONS: The binding of SLPI with Cementoin to transglutaminase seems to be an effective strategy to treat corneal inflammation and angiogenesis.
Asunto(s)
Quemaduras Químicas/tratamiento farmacológico , Neovascularización de la Córnea/tratamiento farmacológico , Quemaduras Oculares/inducido químicamente , Proteínas de Unión al GTP/genética , Queratitis/tratamiento farmacológico , Proteínas Recombinantes de Fusión/administración & dosificación , Inhibidor Secretorio de Peptidasas Leucocitarias/genética , Transglutaminasas/genética , Administración Tópica , Animales , Quemaduras Químicas/metabolismo , Quemaduras Químicas/patología , Recuento de Células , Neovascularización de la Córnea/metabolismo , Neovascularización de la Córnea/patología , Modelos Animales de Enfermedad , Epitelio Corneal/fisiología , Técnica del Anticuerpo Fluorescente Indirecta , Queratitis/metabolismo , Queratitis/patología , Masculino , Proteína Glutamina Gamma Glutamiltransferasa 2 , Ratas , Ratas Sprague-Dawley , Repitelización , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVES: A variety of inflammatory cytokines have been demonstrated to participate in tumorigenesis and progression. Secretory leukocyte protease inhibitor (SLPI) has been demonstrated to show a broad-spectrum of anti-inflammatory effects. This study investigates the expression of SLPI in human pancreatic cancer tissues and cells as well as its biological effects in human pancreatic cancer cells. METHODS: Reverse transcription-polymerase chain reaction, immunohistochemistry, and Western blot were used to detect SLPI mRNA and protein levels in human pancreatic cancer tissues, adjacent tissues, and pancreatic cancer Bxpc-3 and Panc-1 cells. Knockout of SLPI expression was established by recombinant viral vector expressing short hairpin RNA (shRNA) targeting SLPI. Cell viability was analyzed by MTT assay. Cell apoptosis was detected by Hochest33258 staining and flow cytometry assay. RESULTS: Higher SLPI expression was observed in pancreatic tissues, Bxpc-3 cells, and Panc-1 cells compared to the peritumoral tissues (p < 0.01). SLPI expression in Bxpc-3 and Panc-1 cells was effectively silenced by shRNA (p < 0.001). Silencing of SLPI expression significantly reduced cell viability, inhibited cell proliferation, and induced cell apoptosis (p < 0.001). CONCLUSIONS: Abnormal over-expression of SLPI in pancreatic cancer cells may be associated with the development of disease through its roles in promoting cancer cell survival and proliferation as well as anti-apoptosis. SLPI can be used as a target for developing targeted therapy of pancreatic cancer.
Asunto(s)
Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Inhibidor Secretorio de Peptidasas Leucocitarias/genética , Adulto , Anciano , Apoptosis , Western Blotting/métodos , Proliferación Celular , Supervivencia Celular , Femenino , Citometría de Flujo , Silenciador del Gen , Vectores Genéticos , Humanos , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transfección , Células Tumorales Cultivadas , Adulto JovenRESUMEN
BACKGROUND INFORMATION: Epithelial cadherin (E-cadherin) is involved in cell-cell adhesion through its extracellular domain, whereas the intracellular domain interacts with adaptor proteins, i.e. ß-catenin, links E-cadherin to the actin cytoskeleton and participates in signal transduction events. E-cadherin protects mammary epithelial cells from apoptosis and its loss during tumour progression has been documented. Secretory Leukocyte Protease Inhibitor (SLPI) has anti- and pro-tumourigenic activities but its role in breast cancer has not been fully elucidated. Notwithstanding its relevance, how SLPI affects E-cadherin in breast cancer is still unknown. This study evaluated the effect of SLPI upon E-cadherin/ß-catenin expression and apoptosis-related markers in murine (F3II) and human (MCF-7) breast tumour cells either treated with exogenous recombinant human SLPI (rhSLPI) or stably transfected with a plasmid encoding its sequence. RESULTS: Addition of rhSLPI to F3II cells caused a decrease (P < 0.05) in E-cadherin transcript and protein levels. Similar results were observed in SLPI-stable F3II transfectants (2C1), and treatment of 2C1 cells with a siRNA toward SLPI restored E-cadherin to control levels. SLPI-expressing cells showed disruption of E-cadherin/ß-catenin complex and increased (P < 0.05) percentage of cells depicting nuclear ß-catenin localisation. Associated to these changes, 2C1 cells showed increased Bax/Bcl-2 ratio and p21 protein levels, decreased c-Myc protein levels and decreased Cyclin D1 and Claudin-1 transcript levels. No differences in N- and P-cadherin were observed between SLPI-transfected cells and controls. Addition of rhSLPI to MCF-7 cells or stable transfection with SLPI caused a decrease (P < 0.05) in E-cadherin expression (transcript/protein) and its redistribution to the cytoplasm, as well as ß-catenin re-localisation to the cell nucleus. CONCLUSIONS: Expression of SLPI was associated to a decrease in E-cadherin expression and re-localisation of E-cadherin to the cell cytoplasm and ß-catenin to the cell cytoplasm and nucleus, and had pro-apoptotic and cell cycle-arrest effects.
Asunto(s)
Apoptosis , Neoplasias de la Mama , Cadherinas/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , beta Catenina/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Transporte de Proteínas , Inhibidor Secretorio de Peptidasas Leucocitarias/genéticaRESUMEN
OBJECTIVES: The aim of this study was to evaluate the effect of SLPI on the growth and biological processes of Candida albicans. METHODS: Two C. albicans strains were used in this study, a clinical isolate resistant to fluconazole (PRI) and a reference strain ATCC 24433. The minimal inhibitory concentration (MIC) was determined according to the CLSI methodology. The influence of SLPI on secreted serine proteinase activities (SSP) was measured by the cleavage of specific substrate, and surface hydrophobicity was determined by the aqueous-hydrocarbon biphasic separation method. Flow cytometry was performed to investigate receptors for SLPI and variations in the cell wall mannoprotein expression. Interaction between yeast and epithelium was assessed using the MA-104 cells lineage. Ultrastructure was analyzed by transmission electron microscopy (TEM). RESULTS: MIC values were calculated as 18 and 18.9µM for the PRI and ATCC 24433, respectively. SSP activity was reduced by 48.8% by 18µM of SLPI and cell surface hydrophobicity increased by 11.1%. Flow cytometry suggest the existence of SLPI binding sites on the surface of the yeast. Results showed a reduction in the expression of mannoproteins in 20.8% by the cells treated with 80µM of SLPI, and 18µM reduced the adhesion of yeasts to mammalian cells in 60.1%. TEM revealed ultrastructural changes in cells treated with 80µM of SLPI, such as the presence of membrane-like structures within the cytoplasm. CONCLUSIONS: SLPI exerts a significant influence on C. albicans viability and biological processes. Considering its constitutive and physiologic features, SLPI may become a promising tool for the development of new methodologies for the treatment and control of candidiasis.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Inhibidor Secretorio de Peptidasas Leucocitarias/farmacología , Fenómenos Biológicos/efectos de los fármacos , Candida albicans/ultraestructura , Adhesión Celular/efectos de los fármacos , Farmacorresistencia Fúngica , Citometría de Flujo , Fluconazol/farmacología , Humanos , Técnicas In Vitro , Glicoproteínas de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica , Nistatina/farmacologíaRESUMEN
Interferon (IFN)-γ displays a critical role in tuberculosis (TB), modulating the innate and adaptive immune responses. Previously, we reported that secretory leukocyte protease inhibitor (SLPI) is a pattern recognition receptor with anti-mycobacterial activity against Mycobacterium tuberculosis (Mtb). Herein, we determined whether IFN-γ modulated the levels of SLPI in TB patients. Plasma levels of SLPI and IFN-γ were studied in healthy donors (HDs) and TB patients. Peripheral blood mononuclear cells from HDs and patients with TB or defective IFN-γ receptor 1* were stimulated with Mtb antigen and SLPI, and IFN-γR expression levels were measured. Both SLPI and IFN-γ were significantly enhanced in plasma from those with TB compared with HDs. A direct association between SLPI levels and the severity of TB was detected. In addition, Mtb antigen stimulation decreased the SLPI produced by peripheral blood mononuclear cells from HDs, but not from TB or IFN-γR patients. Neutralization of IFN-γ reversed the inhibition of SLPI induced by Mtb antigen in HDs, but not in TB patients. Furthermore, recombinant IFN-γ was unable to modify the expression of SLPI in TB patients. Finally, IFN-γR expression was lower in TB compared with HD peripheral blood mononuclear cells. These results show that Mtb-induced IFN-γ down-modulated SLPI levels by signaling through the IFN-γR in HDs. This inhibitory mechanism was not observed in TB, probably because of the low expression of IFN-γR detected in these individuals.
Asunto(s)
Interferón gamma/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Índice de Severidad de la Enfermedad , Tuberculosis/metabolismo , Tuberculosis/patología , Adulto , Estudios de Casos y Controles , Humanos , Interferón gamma/sangre , Inhibidor Secretorio de Peptidasas Leucocitarias/sangre , Tuberculosis/sangreRESUMEN
Protease-activated receptor 2 (PAR2) is implicated in the pathogenesis of chronic inflammatory diseases, including periodontitis; it can be activated by gingipain and produced by Porphyromonas gingivalis and by neutrophil protease 3 (P3). PAR2 activation plays a relevant role in inflammatory processes by inducing the release of important inflammatory mediators associated with periodontal breakdown. The effects of periodontal treatment on PAR2 expression and its association with levels of proinflammatory mediators and activating proteases were investigated in chronic periodontitis patients. Positive staining for PAR2 was observed in gingival crevicular fluid cells and was reflective of tissue destruction. Overexpression of PAR2 was positively associated with inflammatory clinical parameters and with the levels of interleukin-6 (IL-6), IL-8, tumor necrosis factor alpha, matrix metalloprotease 2 (MMP-2), MMP-8, hepatocyte growth factor, and vascular endothelial growth factor. Elevated levels of gingipain and P3 and decreased levels of dentilisin and the protease inhibitors secretory leukocyte protease inhibitor and elafin were also associated with PAR2 overexpression. Healthy periodontal sites from individuals with chronic periodontitis showed diminished expression of PAR2 mRNA and the PAR2 protein (P < 0.05). Furthermore, periodontal treatment resulted in decreased PAR2 expression and correlated with decreased expression of inflammatory mediators and activating proteases. We concluded that periodontal treatment resulted in decreased levels of proteases and that proinflammatory mediators are associated with decreased PAR2 expression, suggesting that PAR2 expression is influenced by the presence of periodontal infection and is not a constitutive characteristic favoring periodontal inflammation.
Asunto(s)
Líquido del Surco Gingival/citología , Periodontitis/metabolismo , Periodontitis/terapia , Receptor PAR-2/metabolismo , Adhesinas Bacterianas/metabolismo , Adulto , Proteínas Bacterianas , Quimotripsina/metabolismo , Cisteína Endopeptidasas/metabolismo , Elafina/metabolismo , Femenino , Cisteína-Endopeptidasas Gingipaínas , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 8 de la Matriz/metabolismo , Persona de Mediana Edad , Mieloblastina/metabolismo , Péptido Hidrolasas , Bolsa Periodontal/inmunología , Periodontitis/inmunología , Porphyromonas gingivalis/metabolismo , ARN Mensajero/biosíntesis , Receptor PAR-2/biosíntesis , Receptor PAR-2/genética , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adulto JovenRESUMEN
Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor that was related to cancer development and metastasis dissemination on several types of tumors. However, it is not known the effect of SLPI on mammary and colon tumors. The aim of this study was to examine the effect of SLPI on mammary and colon tumor growth. The effect of SLPI was tested on in vitro cell apoptosis and in vivo tumor growth experiments. SLPI over-expressing human and murine mammary and colon tumor cells were generated by gene transfection. The administration of murine mammary tumor cells over-expressing high levels of SLPI did not develop tumors in mice. On the contrary, the administration of murine colon tumor cells over-expressing SLPI, developed faster tumors than control cells. Intratumoral, but not intraperitoneal administration of SLPI, delayed the growth of tumors and increased the survival of mammary but not colon tumor bearing mice. In vitro culture of mammary tumor cell lines treated with SLPI, and SLPI producer clones were more prone to apoptosis than control cells, mainly under serum deprivation culture conditions. Herein we demonstrated that SLPI induces the apoptosis of mammary tumor cells in vitro and decreases the mammary but not colon tumor growth in vivo. Therefore, SLPI may be a new potential therapeutic tool for certain tumors, such as mammary tumors.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias Mamarias Animales/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Femenino , Silenciador del Gen , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Mamarias Animales/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB C , Inhibidor Secretorio de Peptidasas Leucocitarias/farmacología , Transfección , Neoplasias del Cuello Uterino/metabolismoRESUMEN
Samples of exhaled breath condensate (EBC) provide a convenient and non-invasive method to study inflammation in lung diseases. The aim of the present study was to evaluate and compare the inflammatory protein mediator levels in EBC from healthy donors (HD) and from patients with exacerbation of chronic obstructive pulmonary disease (COPD) using an EBC collection device with and without a coating of albumin as a carrier. We studied 13 HD and 26 patients with exacerbation of COPD. The concentrations of myeloperoxidase (MPO), IFNγ and secretory leukocyte protease inhibitor (SLPI) in EBC were measured by immunoassays. The EBC samples from HD and COPD patients showed higher concentrations of MPO when samples were recovered with an albumin-coated device. Furthermore, levels of MPO in COPD patients were significantly higher than in HD. An inverse correlation was observed between MPO and spirometric parameters (FVC and FEV1). Almost all samples collected with the albumin-coated device showed higher amounts of IFNγ and SLPI than those collected with the uncoated device. The levels of SLPI in COPD patients were significantly higher than in HD. A direct correlation was observed between FVC% predicted and SLPI. We concluded that coating the collection device with albumin increased the sensitivity of the technique, at least for measurements of MPO, SLPI and IFNγ. Furthermore, the higher levels of MPO and SLPI and lower levels of IFNγ in EBC from COPD patients could reflect the immunological status and the response of lung parenchyma to treatment during the exacerbation of the illness.
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Pruebas Respiratorias , Mediadores de Inflamación/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Adulto , Estudios de Casos y Controles , Femenino , Humanos , Interferón gamma/metabolismo , Masculino , Persona de Mediana Edad , Peroxidasa/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , EspirometríaRESUMEN
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
The exposure to human immunodeficiency virus type 1 (HIV-1) does not always result in infection. Indeed, there are individuals who have been repeatedly exposed to HIV-1 but do not exhibit clinical or serological evidence of infection; they are known as HIV-exposed seronegative individuals (HESN). To determine if secretory leukocyte protease inhibitor (SLPI), a soluble factor secreted by epithelial cells lining mucosal surfaces that showed anti-HIV activity in vitro, was associated with natural resistance to HIV infection, we measured by real time RT-PCR the expression of SLPI in oral mucosa of a cohort of Colombian HESN, in chronically HIV-1-infected individuals and in healthy controls. The HESN expressed significantly higher levels of SLPI mRNA than healthy controls (p=0.033) and chronically infected subjects (p=0.011). These findings suggest an association between SLPI expression and the natural resistance to HIV-1 infection exhibited by our HESN cohort.
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Seronegatividad para VIH/inmunología , VIH-1/inmunología , Inmunidad Innata , Mucosa Bucal/inmunología , Inhibidor Secretorio de Peptidasas Leucocitarias/inmunología , Adolescente , Adulto , Colombia , Relación Dosis-Respuesta Inmunológica , Epitelio , Femenino , Humanos , Inmunidad Mucosa , Masculino , Persona de Mediana Edad , ARN Mensajero , Reacción en Cadena en Tiempo Real de la Polimerasa , Asunción de Riesgos , Encuestas y Cuestionarios , Adulto JovenRESUMEN
AIMS: We previously reported that recombinant human Secretory Leukocyte Protease Inhibitor (SLPI) inhibits mitogen-induced proliferation of human peripheral blood mononuclear cells. To determine the relevance of this effect in vivo, we investigated the immuno-regulatory role of SLPI in an experimental autoimmune orchitis (EAO) model. MAIN METHODS: In order to increase SLPI half life, poly-ε-caprolactone microspheres containing SLPI were prepared and used for in vitro and in vivo experiments. Multifocal orchitis was induced in Sprague-Dawley adult rats by active immunization with testis homogenate and adjuvants. Microspheres containing SLPI (SLPI group) or vehicle (control group) were administered s.c. to rats during or after the immunization period. KEY FINDINGS: In vitro SLPI-release microspheres inhibited rat lymphocyte proliferation and retained trypsin inhibitory activity. A significant decrease in EAO incidence was observed in the SLPI group (37.5%) versus the control group (93%). Also, SLPI treatment significantly reduced severity of the disease (mean EAO score: control, 6.33±0.81; SLPI, 2.72±1.05). In vivo delayed-type hypersensitivity and ex vivo proliferative response to testicular antigens were reduced by SLPI treatment compared to control group (p<0.05). SIGNIFICANCE: Our results highlight the in vivo immunosuppressive effect of released SLPI from microspheres which suggests its feasible therapeutic use.
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Inmunidad Celular/efectos de los fármacos , Factores Inmunológicos/farmacología , Orquitis/tratamiento farmacológico , Inhibidor Secretorio de Peptidasas Leucocitarias/farmacología , Animales , Enfermedades Autoinmunes/inmunología , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Composición de Medicamentos , Hipersensibilidad Tardía/tratamiento farmacológico , Inmunidad Celular/inmunología , Terapia de Inmunosupresión , Linfocitos/efectos de los fármacos , Masculino , Microesferas , Orquitis/inmunología , Ratas , Ratas Sprague-Dawley , Proteínas RecombinantesRESUMEN
We have demonstrated previously that the inoculation of murine mammary tumor cells genetically modified to express high levels of secretory leukocyte protease inhibitor (2C1) do not develop tumors in immunocompetent mice and these cells are more prone to apoptosis than control cells. The aim of the present study was to evaluate the role of the adaptive immune response in the lack of tumor growth of 2C1 cells and the possibility of using these cells for immunotherapy. The s.c. administration of mock transfected F3II cells induces tumor in BALB/c and Nude mice. However, the inoculation of 2C1 cells develops tumor in Nude but not in BALB/c mice. The inoculation of mock transfected F3II cells to 2C1 immunized BALB/c mice by repeated administration of 2C1 cells (once a week for 3 weeks) developed significantly smaller tumors than those observed in non-immunized mice. Remarkably, survival of tumor-bearing immunized mice was higher than non-immunized animals. Herein, we demonstrate that an immunotherapy with SLPI over-expressing non-irradiated tumor cells which do not develop tumor in immunocompetent mice, partially restrain the tumor growth induced by F3II cells and increase the survival of the mice.
Asunto(s)
Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/terapia , Inhibidor Secretorio de Peptidasas Leucocitarias/inmunología , Animales , Procesos de Crecimiento Celular/inmunología , Femenino , Inmunoterapia , Inmunoterapia Adoptiva/métodos , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Inhibidor Secretorio de Peptidasas Leucocitarias/genética , TransfecciónRESUMEN
We have previously shown that neutrophilic elastase converts human immature dendritic cells (DCs) into TGF-ß secreting cells and reduces its allostimulatory ability. Since TGF-ß has been involved in regulatory T cells (Tregs) induction we analyzed whether elastase or neutrophil-derived culture supernatant treated DCs induce CD4(+)FOXP3(+) Tregs in a mixed lymphocyte reaction (MLR). We found that elastase or neutrophil-derived culture supernatant treated DCs increased TGF-ß and decreased IL-6 production. Together with this pattern of cytokines, we observed a higher number of CD4(+)FOXP3(+) cells in the MLR cultures induced by elastase or neutrophil-derived culture supernatant treated DCs but not with untreated DCs. The higher number of CD4(+)FOXP3(+) T cell population was not observed when the enzymatic activity of elastase was inhibited with an elastase specific inhibitor and also when a TGF-ß1 blocking antibody was added during the MLR culture. The increased number of CD4(+) that express FOXP3 was also seen when CD4(+)CD25(-) purified T cells were cocultured with the TGF-ß producing DCs. Furthermore, these FOXP3(+) T cells showed suppressive activity in vitro. These results identify a novel mechanism by which the tolerogenic DCs generated by elastase exposure contribute to the immune regulation and may be relevant in the pathogenesis of several lung diseases where the inflammatory infiltrate contains high numbers of neutrophils and high elastase concentrations.
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Células Dendríticas/inmunología , Factores de Transcripción Forkhead/metabolismo , Elastasa de Leucocito/farmacología , Linfocitos T Reguladores/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Medios de Cultivo Condicionados/metabolismo , Medios de Cultivo Condicionados/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Humanos , Tolerancia Inmunológica/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Interleucina-6/metabolismo , Elastasa de Leucocito/antagonistas & inhibidores , Leucocitos Mononucleares/inmunología , Prueba de Cultivo Mixto de Linfocitos , Neutrófilos/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/farmacología , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/inmunología , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
RATIONALE: Human secretory leukocyte protease inhibitor (SLPI) displays bactericidal activity against pathogens such as Escherichia coli and Streptococcus. Furthermore, it has been reported that murine SLPI shows potent antimycobacterial activity. OBJECTIVES: The aim of the present study was to investigate whether human recombinant SLPI not only kills mycobacteria but also acts as a pattern recognition receptor for the host immune system. METHODS: For the in vivo experiment, BALB/c mice were infected by intranasal instillation with Mycobacterium bovis BCG and viable BCG load in lung homogenates was later determined. For the in vitro experiments, SLPI was incubated overnight with a suspension of M. bovis BCG or the virulent strain Mycobacterium tuberculosis H37Rv, and the percentage survival as well as the binding of SLPI to mycobacteria was determined. Furthermore, bacteria phagocytosis was also determined by flow cytometry. MEASUREMENTS AND MAIN RESULTS: Intranasal SLPI treatment decreased the number of colony-forming units recovered from lung homogenates, indicating that SLPI interfered with M. bovis BCG infection. Moreover, SLPI decreased the viability of both M. bovis BCG and H37Rv. We demonstrated that SLPI attached to the surface of the mycobacteria by binding to pathogen-associated molecular pattern mannan-capped lipoarabinomannans and phosphatidylinositol mannoside. Furthermore, we found that in the sputum of patients with tuberculosis, mycobacteria were coated with endogenous SLPI. Finally, we showed that phagocytosis of SLPI-coated mycobacteria was faster than that of uncoated bacteria. CONCLUSIONS: The present results demonstrate for the first time that human SLPI kills mycobacteria and is a new pattern recognition receptor for them.
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Mycobacterium tuberculosis/fisiología , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Tuberculosis Pulmonar/metabolismo , Animales , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Ratones , Ratones Endogámicos BALB C , Fagocitosis , Esputo/química , Esputo/microbiología , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiologíaRESUMEN
OBJECTIVE: Secretory leukocyte proteinase inhibitor (SLPI) is an endogenous proteinase inhibitor present in mucosal secretions. It also displays antimicrobial activity including anti-human immunodeficiency virus activity. This protease inhibitor is also expressed in submandibular glands (SMG), but there are few data on its expression in AIDS patients with infectious conditions. METHODS: We analyzed the expression of SLPI using immunohistochemistry in submandibular gland samples of 36 AIDS patients [10 with normal histology, 10 with chronic nonspecific sialadenitis, eight with mycobacteriosis, and eight with cytomegalovirus (CMV) infection] and 10 HIV-negative controls. The proteinase inhibitor was quantified using image analysis and expressed as % of positively stained area. RESULTS: There was a higher expression of SLPI in AIDS patients with CMV infection (% of stained area, mean+/-SD: 37.37+/-14.45) when compared with all other groups (P=0.009). There were no significant differences between control subjects (22.70+/-9.42%) and AIDS patients without histologic alterations (18.10+/-7.58%), with chronic nonspecific sialadenitis (17.13+/-5.36%), or mycobacterial infection (21.09+/-4.66%). CONCLUSION: Cytomegalovirus infection increases SLPI expression in the SMG of AIDS patients. Our results reveal new insights into the pathogenic association between HIV and CMV in AIDS patients.