An amebic anti-inflammatory peptide down-regulates ex vivo IL-1β expression in patients with rheumatoid arthritis

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"documento" => "article" "crossmark" => 0 "subdocumento" => "fla" "cita" => "Reumatol Clin. 2012;8:315-20" "abierto" => array:3 [ "ES" => true "ES2" => true "LATM" => true ] "gratuito" => true "lecturas" => array:2 [ "total" => 4822 "formatos" => array:3 [ "EPUB" => 156 "HTML" => 3383 "PDF" => 1283 ] ] "en" => array:13 [ "idiomaDefecto" => true "cabecera" => "Original article" "titulo" => "An amebic anti-inflammatory peptide down-regulates ex vivo IL-1β expression in patients with rheumatoid arthritis" "tienePdf" => "en" "tieneTextoCompleto" => "en" "tieneResumen" => array:2 [ 0 => "en" 1 => "es" ] "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "315" "paginaFinal" => "320" ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Un péptido antiinflamatorio amibiano disminuye en pacientes con artritis reumatoide la expresión ex vivo de IL-1β" ] ] "contieneResumen" => array:2 [ "en" => true "es" => true ] "contieneTextoCompleto" => array:1 [ "en" => true ] "contienePdf" => array:1 [ "en" => true ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0015" "etiqueta" => "Figure 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 1620 "Ancho" => 2222 "Tamanyo" => 148002 ] ] "descripcion" => array:1 [ "en" => "Effect of MLIF in PBMC from healthy and patients with RA. Healthy donors (n=13) and patients with RA (n=11) PBMC were cultured during 4h in the presence of LPS, MLIF or both. qPCR analysis for IL-1β transcripts was carried out in each case and comparison between groups established by a normalized expression ratio (ner). (a) Patients with AR constitutively express more IL-1β than healthy donors. IL-1β ner between them was statistically different (p=0.03). (b) MLIF shows a down-regulatory effect on IL-1β expression in patients with RA which ner is <1 in 91% of cases (p=0.01). (c) Capacity of LPS to up-regulate IL-1β in both healthy donors and patients with RA was not significantly different. (d) MLIF was able to down-regulate LPS-induction in all patients with RA (p=0.001)." ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Juan R. Velázquez, Lizeth Garibay-Martínez, Pedro Martínez-Tejada, Yelda A. Leal" "autores" => array:4 [ 0 => array:2 [ "nombre" => "Juan R." "apellidos" => "Velázquez" ] 1 => array:2 [ "nombre" => "Lizeth" "apellidos" => "Garibay-Martínez" ] 2 => array:2 [ "nombre" => "Pedro" "apellidos" => "Martínez-Tejada" ] 3 => array:2 [ "nombre" => "Yelda A." "apellidos" => "Leal" ] ] ] ] ] "idiomaDefecto" => "en" "Traduccion" => array:1 [ "en" => array:9 [ "pii" => "S2173574312001311" "doi" => "10.1016/j.reumae.2012.07.013" "estado" => "S300" "subdocumento" => "" "abierto" => array:3 [ "ES" => false "ES2" => false "LATM" => false ] "gratuito" => false "lecturas" => array:1 [ "total" => 0 ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S2173574312001311?idApp=UINPBA00004M" ] ] "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S1699258X12001362?idApp=UINPBA00004M" "url" => "/1699258X/0000000800000006/v1_201305061930/S1699258X12001362/v1_201305061930/en/main.assets" ] ] "itemSiguiente" => array:19 [ "pii" => "S2173574312000962" "issn" => "21735743" "doi" => "10.1016/j.reumae.2012.07.003" "estado" => "S300" "fechaPublicacion" => "2012-11-01" "aid" => "459" "copyright" => "Elsevier España, S.L." "documento" => "article" "crossmark" => 0 "subdocumento" => "fla" "cita" => "Reumatol Clin. 2012;8:321-7" "abierto" => array:3 [ "ES" => false "ES2" => false "LATM" => false ] "gratuito" => false "lecturas" => array:2 [ "total" => 3746 "formatos" => array:3 [ "EPUB" => 55 "HTML" => 3152 "PDF" => 539 ] ] "en" => array:13 [ "idiomaDefecto" => true "cabecera" => "Original Article" "titulo" => "Analysis of the rs20541 (R130Q) Polymorphism in the IL-13 Gene in Patients With Elderly-associated Chronic Inflammatory Diseases" "tienePdf" => "en" "tieneTextoCompleto" => "en" "tieneResumen" => array:2 [ 0 => "en" 1 => "es" ] "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "321" "paginaFinal" => "327" ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Análisis del polimorfismo rs20541 (R130Q) del gen de la IL-13 en pacientes con enfermedades inflamatorias crónicas asociadas al envejecimiento" ] ] "contieneResumen" => array:2 [ "en" => true "es" => true ] "contieneTextoCompleto" => array:1 [ "en" => true ] "contienePdf" => array:1 [ "en" => true ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0015" "etiqueta" => "Fig. 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 2057 "Ancho" => 2961 "Tamanyo" => 258109 ] ] "descripcion" => array:1 [ "en" => "Duration in months of steroid treatment (CS, left), cumulative steroid dose (in middle) and number of relapses (right) in relation to the SNP R130Q of the IL-13 gene. Data is presented separately for active giant cell arteritis (GCA, n=55; superior panel) and polymyalgia rheumatica (PMR, n=120; inferior panel)." ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Lorena Álvarez-Rodríguez, Marcos López-Hoyos, Eugenio Carrasco-Marín, Cristina Mata, Jaime Calvo-Alén, Elena Aurrecoechea, Ricardo Blanco, Teresa Ruiz, Pedro Muñoz Cacho, Ignacio Villa, Víctor Manuel Martínez-Taboada" "autores" => array:11 [ 0 => array:2 [ "nombre" => "Lorena" "apellidos" => "Álvarez-Rodríguez" ] 1 => array:2 [ "nombre" => "Marcos" "apellidos" => "López-Hoyos" ] 2 => array:2 [ "nombre" => "Eugenio" "apellidos" => "Carrasco-Marín" ] 3 => array:2 [ "nombre" => "Cristina" "apellidos" => "Mata" ] 4 => array:2 [ "nombre" => "Jaime" "apellidos" => "Calvo-Alén" ] 5 => array:2 [ "nombre" => "Elena" "apellidos" => "Aurrecoechea" ] 6 => array:2 [ "nombre" => "Ricardo" "apellidos" => "Blanco" ] 7 => array:2 [ "nombre" => "Teresa" "apellidos" => "Ruiz" ] 8 => array:2 [ "nombre" => "Pedro" "apellidos" => "Muñoz Cacho" ] 9 => array:2 [ "nombre" => "Ignacio" "apellidos" => "Villa" ] 10 => array:2 [ "nombre" => "Víctor Manuel" "apellidos" => "Martínez-Taboada" ] ] ] ] ] "idiomaDefecto" => "en" "Traduccion" => array:1 [ "es" => array:9 [ "pii" => "S1699258X12001325" "doi" => "10.1016/j.reuma.2012.04.006" "estado" => "S300" "subdocumento" => "" "abierto" => array:3 [ "ES" => true "ES2" => true "LATM" => true ] "gratuito" => true "lecturas" => array:1 [ "total" => 0 ] "idiomaDefecto" => "es" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S1699258X12001325?idApp=UINPBA00004M" ] ] "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S2173574312000962?idApp=UINPBA00004M" "url" => "/21735743/0000000800000006/v1_201305061648/S2173574312000962/v1_201305061648/en/main.assets" ] "itemAnterior" => array:19 [ "pii" => "S2173574312001621" "issn" => "21735743" "doi" => "10.1016/j.reumae.2012.10.005" "estado" => "S300" "fechaPublicacion" => "2012-11-01" "aid" => "438" "copyright" => "Elsevier España, S.L." "documento" => "article" "crossmark" => 0 "subdocumento" => "fla" "cita" => "Reumatol Clin. 2012;8:310-4" "abierto" => array:3 [ "ES" => false "ES2" => false "LATM" => false ] "gratuito" => false "lecturas" => array:2 [ "total" => 2900 "formatos" => array:3 [ "EPUB" => 57 "HTML" => 2387 "PDF" => 456 ] ] "en" => array:13 [ "idiomaDefecto" => true "cabecera" => "Original Article" "titulo" => "Situation of Spanish Echography in Spanish Rheumatology 2012" "tienePdf" => "en" "tieneTextoCompleto" => "en" "tieneResumen" => array:2 [ 0 => "en" 1 => "es" ] "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "310" "paginaFinal" => "314" ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Situación de la ecografía en la reumatología española 2012" ] ] "contieneResumen" => array:2 [ "en" => true "es" => true ] "contieneTextoCompleto" => array:1 [ "en" => true ] "contienePdf" => array:1 [ "en" => true ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0015" "etiqueta" => "Fig. 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 724 "Ancho" => 1405 "Tamanyo" => 63324 ] ] "descripcion" => array:1 [ "en" => "Area of echography examination (%)." ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Eugenio de Miguel, Jose Luis Andreu, Esperanza Naredo, Ingrid Möller" "autores" => array:5 [ 0 => array:2 [ "nombre" => "Eugenio" "apellidos" => "de Miguel" ] 1 => array:2 [ "nombre" => "Jose Luis" "apellidos" => "Andreu" ] 2 => array:2 [ "nombre" => "Esperanza" "apellidos" => "Naredo" ] 3 => array:2 [ "nombre" => "Ingrid" "apellidos" => "Möller" ] 4 => array:1 [ "colaborador" => "Grupo de Trabajo de Ecografía de la Sociedad Española de Reumatología (ECOSER)" ] ] ] ] ] "idiomaDefecto" => "en" "Traduccion" => array:1 [ "es" => array:9 [ "pii" => "S1699258X12000848" "doi" => "10.1016/j.reuma.2012.03.004" "estado" => "S300" "subdocumento" => "" "abierto" => array:3 [ "ES" => true "ES2" => true "LATM" => true ] "gratuito" => true "lecturas" => array:1 [ "total" => 0 ] "idiomaDefecto" => "es" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S1699258X12000848?idApp=UINPBA00004M" ] ] "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S2173574312001621?idApp=UINPBA00004M" "url" => "/21735743/0000000800000006/v1_201305061648/S2173574312001621/v1_201305061648/en/main.assets" ] "en" => array:20 [ "idiomaDefecto" => true "cabecera" => "Original article" "titulo" => "An amebic anti-inflammatory peptide down-regulates ex vivo IL-1β expression in patients with rheumatoid arthritis" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "315" "paginaFinal" => "320" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "Juan R. Velázquez, Lizeth Garibay-Martínez, Pedro Martínez-Tejada, Yelda A. Leal" "autores" => array:4 [ 0 => array:4 [ "nombre" => "Juan R." "apellidos" => "Velázquez" "email" => array:1 [ 0 => "velazquez_juan@hotmail.com" ] "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "¿" "identificador" => "cor0005" ] ] ] 1 => array:3 [ "nombre" => "Lizeth" "apellidos" => "Garibay-Martínez" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "b" "identificador" => "aff0010" ] ] ] 2 => array:3 [ "nombre" => "Pedro" "apellidos" => "Martínez-Tejada" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "c" "identificador" => "aff0015" ] ] ] 3 => array:3 [ "nombre" => "Yelda A." "apellidos" => "Leal" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "d" "identificador" => "aff0020" ] ] ] ] "afiliaciones" => array:4 [ 0 => array:3 [ "entidad" => "Departamento de Inmunogénetica y Alergia, INER, México, D.F., Mexico" "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:3 [ "entidad" => "Unidad de Investigación Médica Zacatecas, IMSS, Zacatecas, Zacatecas, Mexico" "etiqueta" => "b" "identificador" => "aff0010" ] 2 => array:3 [ "entidad" => "Hospital Dr. Emilio Varela-Luján, IMSS, Zacatecas, Zacatecas, Mexico" "etiqueta" => "c" "identificador" => "aff0015" ] 3 => array:3 [ "entidad" => "Unidad de Investigación Médica Yucatán (UIMY), Unidad Médica de Alta Especialidad de Mérida “Dr. Ignacio García Tellez”, IMSS, Mérida, Yucatán, Mexico" "etiqueta" => "d" "identificador" => "aff0020" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Un péptido antiinflamatorio amibiano disminuye en pacientes con artritis reumatoide la expresión ex vivo de IL-1β" ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1111 "Ancho" => 1689 "Tamanyo" => 91407 ] ] "descripcion" => array:1 [ "en" => "Time-course expression of IL-1β in U-937 cells. Triplicate cultures of 4×106 U-937 cells in the presence of MLIF, LPS or both were harvested at 0.3, 1, 2, 4, 6, 8 and 10h. RNA was obtained from pellets and used for real time PCR. Comparative expression was established using unstimulated cells as reference. LPS induced a strong up-regulation of IL-1β which peaks at 4h. MLIF per se did not affect IL-1β basal expression but MLIF was able to reduce significantly the stimulatory effect of LPS when amebic peptide was added simultaneously to LPS (p=0.05). Bars of SEM are <1 in all points." ] ] ] "textoCompleto" => "IntroductionEntamoeba histolytica produces a heat-stable pentapeptide (Met-Gln-Cys-Asp-Ser, MQCNS) that was originally identified by its effects in delaying mononuclear phagocytes migration to inflammation sites. This peptide was hereby denominated by the MLIF and was shown to have several anti-inflammatory properties.<a class="elsevierStyleCrossRef" href="#bib0005">1</a>In vitro, it inhibits induction of macrophage inflammatory proteins (MIP-1α, -1β), IL-1β, IL-8, and chemokine CCL1 (I-309), and the chemokine receptor 1 (CCR1) in phorbol myristate acetate (PMA)-stimulated promonocytic human cell line (U-937) cells.<a class="elsevierStyleCrossRefs" href="#bib0010">2,3</a> In umbilical vein endothelial cells (UVEC), MLIF reduces vascular cell adhesion molecule (VCAM)-1 and E-selectin expression (personal communication). In vivo it delays mononuclear cells migration to the inflammatory focus in human skin1. The peptide is able to inhibit expression of cell adhesion molecules very late antigen (VLA)-4 and VCAM-1 in a dinitrochlorobenzene (DNCB)-hypersensitivity, guinea pig model.<a class="elsevierStyleCrossRef" href="#bib0020">4</a> In U-937 cells and in the CIA model, analysis of gene expression profiles employing microarrays reveals a regulatory capacity of MLIF on numerous inflammatory genes including IL-1β.<a class="elsevierStyleCrossRef" href="#bib0010">2</a> In addition, the amebic peptide has exhibited a notorious anti-inflammatory effect in carragenin-induced inflammation model in BALB/c mice (personal communication). The MLIF molecular mechanisms of action are poorly understood; however, there is evidence of its involvement in nuclear factor-kappa B (NF-κB) signal transduction; MLIF appears to induce nuclear translocation of p50 homodimers, affecting a number of inflammatory transcripts.<a class="elsevierStyleCrossRef" href="#bib0025">5</a>RA is a multisystemic chronic disease of unknown etiology. During its development, there are periods of activity and reminiscence that could be diagnosed on the basis of a battery of clinical and laboratory tests.<a class="elsevierStyleCrossRefs" href="#bib0030">6,7</a> One hallmark of RA is the inflammatory process that occurs in the joint. The resulting inflammatory response induced by pro-inflammatory cytokines (mainly tumor necrosis factor [TNF]α and IL-1β) leads to synovial fibroblast hyperplasia, increased vascularity, and inflammatory cell infiltrate.<a class="elsevierStyleCrossRefs" href="#bib0040">8–10</a> Messenger RNA (mRNA) and protein analysis of patients with RA has shown abundant expression of IL-1β and TNFα cytokines.<a class="elsevierStyleCrossRefs" href="#bib0055">11,12</a> In this work we tested the effect of anti-inflammatory peptide MLIF upon the constitutive and LPS-induced synthesis of IL-1β in U-937 and PBMC from healthy subjects and from patients with RA.Materials and methodsCellsThe promonocytic human cell line U-937 (American Type Culture Collection, ATCC, Manassas, VA, USA) was propagated in RPMI-1640 endotoxin-free medium (In vitro, Life Technologies, Eggenstein, Germany) supplemented with 2mM l-glutamine, 1.5g/L sodium bicarbonate, 4.5g/L d-glucose, 10mM HEPES, 1.0mM sodium pyruvate and 10% heat-inactivated, endotoxin-free fetal bovine serum (FBS, Gibco BRL Life Technologies, Grand Island, NY, USA). Cells were maintained at 37°C in a humidified atmosphere with 5% CO2. PBMC were obtained from healthy individuals or patients with AR by Böyum method.<a class="elsevierStyleCrossRef" href="#bib0065">13</a> Briefly, 4mL of diluted blood sample was carefully layered on 3mL of Ficoll-Paque PLUS (GE Heathcare Bio-Sciences Corp, Piscatawa, NJ, USA), tubes were centrifuged at 400×g for 30min at room temperature. Using a Pasteur pipette we take off the mononuclear layer and washed it with balanced salt solution. Typically recovered cells were ≥95% mononuclear cells with ≥95% viability.CulturesTime-course experiments were carried out in triplicate groups of 4×106mL−1. U-937 cells incubated at 37°C in a humidified atmosphere with 5% CO2 during 0–10h were treated as follows: (a) in RPMI-1640 medium alone (unstimulated); (b) MLIF (50g/mL) added (per se effect); (c) LPS (10 ng/mL) added (induced expression), and (d) MLIF and LPS added simultaneously (interference). After 0.3, 1, 2, 4, 6, 8 and 10h, cells were harvested by centrifugation. All cell pellets were used for RNA isolation, and 4, 6 and 8h supernatant fluids employed for IL-1β immunoreactivity. Triplicate groups of 4×106mL−1 human PBMC were incubated during 4 and 6h without stimulus, MLIF, LPS or both. After 4h, the cell pellet (for RNA isolation) or after 6h, the supernatant fluid (for IL-1β immunoreactivity) was harvested by centrifugation. At the conclusion of all experiments, cell viability was ≥90% by trypan blue dye (Sigma Chemical Co., St. Louis, MO) exclusion. The concentration of MLIF employed in these studies was based on previous in vitro functional experiments.<a class="elsevierStyleCrossRefs" href="#bib0010">2,3</a> Simultaneous addition of MLIF to assays was employed, because pilot studies have revealed that MLIF mainly affected cytokine induction when added prior to (weak) or simultaneously (strong), but less after LPS stimulation.Chemical substancesA single batch, 96% pure MLIF (Met-Gln-Cys-Asp-Ser, MQCNS) endotoxin-free sample was commercially obtained (American Peptide Co., Sunnyvale, CA, USA). MLIF was dissolved in sterile PBS and tested for endotoxin activity using Limulus amoebocyte lysate KTA (Charles River Endosafe Inc., Charleston, SC, USA). Endotoxin activity was ≤0.0625UIEmL/endotoxin. LPS Escherichia coli serotype 0111:B4 was obtained from Sigma.SubjectsFemales, 35–59 years of age, were recruited from the Blood Bank (healthy donors) and Rheumatology Clinic of “Dr. Emilio Varela-Lujan” General Hospital, Mexican Institute of Social Security (IMSS) in Zacatecas City, Mexico. Patients with RA were classified as such when they fulfilled four or more American College of Rheumatology (ACR) criteria. Subjects were under Anti-inflammatory non-steroid treatment (AINES), or glucocorticoids at doses ≤10mg/day and were free of biologic therapy (anti-TNFα, anti-IL-1β or anti CD20) at least 6 months prior to the study. The study included 13 healthy subjects and 11 patients with RA. All study participants gave written informed consent to participate in the study. The procedure complies with the international ethics for human research and was approved by the Dr. Emilio Varela Luján Hospital Ethics Committee.RNA extraction and reverse transcriptionTotal RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) as described.<a class="elsevierStyleCrossRef" href="#bib0015">3</a> RNA purity was established in 2.5% denaturing agarose gels, and the concentration determined in a NanoDrop® ND-1000 spectrophotometer (Thermo Scientific Inc., Wilmington, DE, USA). For reverse transcription, 1μg of total RNA was mixed with 1μL oligo-dT (12–18-mer, Invitrogen), 200U SuperScript™ reverse transcriptase (Invitrogen), 1× First Stand Buffer (Invitrogen), and 0.5mM mixed dNTPs (dATP, dCTP, dGTP, dTTP) (Promega, Co., Madison, WI, USA). The mixture was incubated at 42°C for 1h. Reverse transcription was heat-inactivated at 80°C for 10min and the resulting cDNA was employed for real-time polymerase chain reaction (PCR).Real-time polymerase chain reaction analysisReal-time PCR was performed in capillaries in a 20μL reaction mix containing Light Cycler Taqman (Roche Applied Science, Salt Lake City, UT, USA), 0.2μM specific primer mix, and 0.1μM of gene-specific hydrolysis probes from the Universal Probe Library (Roche) and cDNA. PCR reaction included 45 cycles consisting of denaturation at 95°C for 10s, annealing at 60°C for 20s, and extension at 72°C for 1s. All reactions were performed in a LightCycler 2.0 instrument (Roche) and data analysis was conducted with LightCycler software 4.05 (Roche).We obtained the relative expression of IL-1β induced by LPS, MLIF and LPS+MLIF with respect to unstimulated cells using the ΔΔCP method.<a class="elsevierStyleCrossRef" href="#bib0070">14</a> Briefly, we copied the CP values for each sample in an Excel spreadsheet, the first delta CP was calculated from IL-1β CP minus glyceraldehyde 3-phosphate dehydrogenase (GAPDH) CP for each sample accordingly. Deltadelta CP for each condition was equal to stimulated sample delta CP minus unstimulated sample CP. Relative expression was calculated using Relative expression=2(−deltadelta CP) formula.We considered IL-1β as target gene and GAPDH as housekeeping gene.For comparison among study groups (healthy subjects vs patients with RA), real-time PCR calibration curves for IL-1β and GAPDH genes were produced from purified specific amplicons. In all groups we obtained normalized expression ratio (ner) of each condition (unstimulated, LPS, MLIF and LPS+MLIF) employing the formula:<elsevierMultimedia ident="eq0005"></elsevierMultimedia>Comparison among different ner was used to evaluate basal IL-1β, and responses induced by LPS and MLIF, as well as MLIF capacity to antagonize LPS response.Enzyme-linked immunoabsorbent assaysIL-1β proteins in supernatant fluids were measured using a commercial ELISA kit DuoSet® (R & D Systems, Inc., Minneapolis, MN, USA). Plates were incubated overnight at 4°C, washed and blocked. Final development employed ExtrAvidin™ HRP and substrate 3,3′,5,5′-tetramethyl-benzidine (Sigma). Microtiter plates were read at 450nm in a BIO-RAD microplate Benchmark densitometer (Richmond, CA, USA). Results were expressed as ng/mL. Sensitivity for IL-1β was ≥2pg/mL.Statistical analysisResults are expressed as mean ranks. Statistical significance of differences in mean values between different groups was evaluated by Kruskal–Wallis test, followed by U-Mann–Whitney test for inter-group comparisons. A value of p<0.05 was considered to be significant. All analyses were performed with SPSS software version 15.0 (SPSS Inc., Chicago, IL).ResultsCellular response to lipopolysaccharide and the antagonist effect of monocyte locomotion inhibitory factorPrevious work utilizing U-937 demonstrated that MLIF was able to decrease PMA-induced expression of chemokines MIP-1α and MIP-1β, as well as that of cytokine IL-1β.<a class="elsevierStyleCrossRefs" href="#bib0010">2,3</a> We observed this effect only when the peptide was administered prior to or simultaneously with the activator agent. The neutralizing effect of MLIF on such an event was outstanding, if we take into account that PMA is a powerful cell activator that disrupts several cell signaling pathways. On the other hand, because MLIF is involved in the NF-κB signaling, we were interested in observing the amebic peptide effect on the production of LPS/TLR4 signaling-dependent IL-1β.In a triplicate time-course experiment using U-937 cells, LPS-induced expression of IL-1β was followed with real-time PCR. Specific IL-1β mRNA was detected in a 1–10h window of time (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>). Maximum response to LPS peaked at 4h post-stimulation with 94±1.15 (mean±SEM) folds, and afterwards there was a symmetrically decreasing production. When MLIF was administrated simultaneously with LPS, the amebic peptide exhibited an astonishing antagonist effect at 4h (relative expression 94±1.15-fold with LPS vs 8±0.03-fold with LPS+MLIF (p<0.05)). On the other hand, amebic peptide per se showed no IL-1β regulatory capacity.<elsevierMultimedia ident="fig0005"></elsevierMultimedia>Immunoreactivity of lipopolysaccharide-induced interleukin-1βSupernatants from mentioned triplicate experiment were used for measuring IL-1β immunoreactivity released by U-937 cells stimulated with LPS, MLIF or both at three different times (4, 6, and 8h). A higher amount of IL-1β was detected after 6h of incubation where LPS-induction was maximum with 18.5±0.74pg/mL, statistical difference between LPS vs MLIF (18.5±0.4 vs 13.2±0.4), and LPS vs unstimulated cells (18.5±0.4 vs 9.6±0.5) was significant with p<0.05 for both. MLIF showed no effect on IL-1β release by LPS stimulation. At 4 and 8h IL-1β response to LPS was poor, and in most conditions immunoreactivity was near the assay detection limit (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>). There is a trend for MLIF to interfere with LPS effect at 4h and 8h but statistics showed it not to be significant. Once again, the peptide per se did not affect the production of IL-1β.<elsevierMultimedia ident="fig0010"></elsevierMultimedia>Monocyte locomotion inhibitory factor effect on immunocompetent cellsWe were interested in testing the observed MLIF effects in differentiated immunocompetent cells. PBMC cultures from healthy subjects and patients with RA were used to look at basal expression of IL-1β. As expected, patients with RA exhibited higher expression of IL-1β mRNA in comparison with healthy subjects. 91% of patients with RA (10 of 11 patients) IL-1β ner was >1; meanwhile in 54% of healthy subjects (9 of 13) IL-1β ner was >1 (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 3</a>a). Statistical data showed a significantly higher expression of IL-1β in patients with RA compared with healthy donors (p=0.03).<elsevierMultimedia ident="fig0015"></elsevierMultimedia>The capacity of MLIF to affect the expression of IL-1β was determined as the ratio of MLIF ner to unstimulated ner. The presence of MLIF in PBMC cultures decreased IL-1β expression (ratio <1) in 91% of patients with AR (10 of 11) and in 46% of healthy donors (6 of 13) (p=0.01) (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 3</a>b).Ratios of LPS-induced ner to unstimulated ner were employed to evaluate LPS stimulatory capacity in PBMC cultures. We considered an increase of >2-folds in IL-1β expression as a biologically effective criterion. Studied groups responded in similar fashion to LPS stimulus; 64% (7 of 11) patients with RA and 62% (8 of 13) healthy donors responded to LPS, and no significant differences were found among them (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 3</a>c).We considered that MLIF could antagonize with LPS when ratios of LPS+MLIF ner to LPS-induced ner were <1. Expression of IL-1β decreased in 100% (11 of 11) of patients with RA and in 38% (5 of 13) of healthy subjects (p=0.001) (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 3</a>d).Inmunoreactivity of interleukin-1β in peripheral blood mononuclear cellsIL-1β inmunoreactivity in PBMC cultures was in general low (below the detection limit in the majority of cases). It was only possible to detect IL-1β in 36% (4 of 11) of patients with RA and in 8% (1 of 13) of healthy donors. Apparently, RA group releases more IL-1β than healthy group (18±2.1 vs 8±1.6pg/mL, respectively). However, because the low number of data, no significant differences were found between them. In the group of patients with RA, we observed a MLIF trend to diminish the amount of IL-1β spontaneously released in culture from 18.0 to 5.1pg/mL, as well as an antagonist effect on LPS-stimulation in both RA and healthy groups (from 14.6 to 9.0; and from 19.2 to 7.1pg/mL, respectively) (<a class="elsevierStyleCrossRef" href="#fig0020">Fig. 4</a>). This result is interesting but not conclusive due the low number of immunoreactive samples.<elsevierMultimedia ident="fig0020"></elsevierMultimedia>DiscussionSeveral studies have shown the MLIF modulatory capacity on inflammatory genes. The antagonist effect of the amebic peptide when administered prior to or simultaneously with LPS appears to involve it in early signaling events linked with either putative or non- putative membrane receptors.<a class="elsevierStyleCrossRef" href="#bib0025">5</a> Data on LPS activation by means of TLR4 in U-937 cells had revealed that MLIF is able to interfere with MyD88 membrane recruitment in response to LPS, thus affecting an array of MyD88-dependent genes 5. Ongoing studies in our laboratory with respect to IL-1β synthesis utilizing U-937 cells have shown that MLIF increase the degradation of specific mRNA when induced with LPS. The use of ERK, JNK, p38 and NF-κB inhibitors have brought to light the participation of a NF-κB inhibitory dimmer induced by MLIF that is perhaps responsible for abrogating IL-1β transcription and participating in a cross-talk between NF-κB and mitogen-activated protein kinase (MAPK) signaling cascades. Differential expression profiles of U-937 stimulated with PMA demonstrated a remarkable interfering capacity of MLIF on induced transcription of IL-1β. In this report when using U-937 cells, we found that MLIF per se did not affect IL-1β expression, but that it indeed interferes with LPS in a narrow window of time. MLIF shows no capacity in regulating the LPS-induced immunoreactivity of IL-1β in U-937 cells; however, it is important to consider the poor response of these cells to LPS. Regulation of MLIF on IL-1β transcripts but not on IL-1β protein of U-937 cells could be due to genetic alterations in the cell line, lack of synchronicity in the synthesis, storage, and further secretion of mature IL-1β. Assessment of IL-1β requires the presence in the medium of inmunoreactive cytokine which is generated in a cyclic and complicated process. The immunoreactivity detected is closely related with the cell's ability to generate and to secrete caspase-1-processed IL-1β. Clearly, additional studies on the extracellular bioavailability and intracellular storage of IL-1β in stimulated cells in the presence of MLIF are required.On the other hand, the effect of MLIF on primed cells is slightly different in immunocompetent PBMC from an inflammatory environment such as RA. In arthritic PBMC, there is an over-expression of basal IL-1β, which is in agreement with other reports.<a class="elsevierStyleCrossRef" href="#bib0075">15</a> MLIF down-regulated IL-1β basal expression and interfered with LPS-induction. IL-1β immunoreactivity levels in PBMC culture supernatants exhibited in general a lower detection limit, but in the few subjects with detected activity MLIF acted mainly on patients with RA, decreasing spontaneous release of IL-1β and interfering with LPS-induction. An increased number of subjects would aid in clarifying this issue.We are not certain about MLIF action only in transcription but not in translation of IL-1β. For example, we have evidence that administration of the amebic peptide in a CIA model ameliorates the inflammation process and modulates gene expression profiles (IL-1β included among other dozens of genes).<a class="elsevierStyleCrossRef" href="#bib0080">16</a> Furthermore, MLIF is able to reduce the immunoreactivity of IL-1β and matrix metalloproteinases in joints of arthritic mice (paper in progress).Concluding remarksMLIF was able to distinguish between healthy subjects and patients with RA, based perhaps upon a differential cell-activation degree and the signaling pathways in use. It is plausible to think that synovial cells would be a more representative entity for testing the interesting effects described in this work, and further studies are required to resolve this matter. The MLIF effect on caspase-1, translation, storage and release of mature IL-1β is unknown.The results presented in this work provide evidence of MLIF's most exquisite anti-inflammatory properties on prime and arthritic cells and encourage its research in other inflammatory processes.Ethical disclosuresProtection of human and animal subjects. The authors declare that no experiments were performed on humans or animals for this investigation.Confidentiality of Data. The authors declare that no patient data appears in this article.Right to privacy and informed consent. The authors have obtained the informed consent of the patients and /or subjects mentioned in the article. The author for correspondence is in possession of this document.FundingThis work was supported by Fondo de Investigación en Salud of IMSS, Mexico, grant FIS/IMSS/PROT/356.Conflicts of interestThe authors have no conflicts of interest to declare." "textoCompletoSecciones" => array:1 [ "secciones" => array:14 [ 0 => array:2 [ "identificador" => "xres125980" "titulo" => array:5 [ 0 => "Abstract" 1 => "Objectives" 2 => "Material and methods" 3 => "Results" 4 => "Conclusions" ] ] 1 => array:2 [ "identificador" => "xpalclavsec113275" "titulo" => "Keywords" ] 2 => array:2 [ "identificador" => "xres125979" "titulo" => array:5 [ 0 => "Resumen" 1 => "Objetivo" 2 => "Material y Métodos" 3 => "Resultados" 4 => "Conclusiones" ] ] 3 => array:2 [ "identificador" => "xpalclavsec113274" "titulo" => "Palabras clave" ] 4 => array:2 [ "identificador" => "sec0005" "titulo" => "Introduction" ] 5 => array:3 [ "identificador" => "sec0010" "titulo" => "Materials and methods" "secciones" => array:8 [ 0 => array:2 [ "identificador" => "sec0015" "titulo" => "Cells" ] 1 => array:2 [ "identificador" => "sec0020" "titulo" => "Cultures" ] 2 => array:2 [ "identificador" => "sec0025" "titulo" => "Chemical substances" ] 3 => array:2 [ "identificador" => "sec0030" "titulo" => "Subjects" ] 4 => array:2 [ "identificador" => "sec0035" "titulo" => "RNA extraction and reverse transcription" ] 5 => array:2 [ "identificador" => "sec0040" "titulo" => "Real-time polymerase chain reaction analysis" ] 6 => array:2 [ "identificador" => "sec0045" "titulo" => "Enzyme-linked immunoabsorbent assays" ] 7 => array:2 [ "identificador" => "sec0050" "titulo" => "Statistical analysis" ] ] ] 6 => array:3 [ "identificador" => "sec0055" "titulo" => "Results" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "sec0060" "titulo" => "Cellular response to lipopolysaccharide and the antagonist effect of monocyte locomotion inhibitory factor" ] 1 => array:2 [ "identificador" => "sec0065" "titulo" => "Immunoreactivity of lipopolysaccharide-induced interleukin-1β" ] 2 => array:2 [ "identificador" => "sec0070" "titulo" => "Monocyte locomotion inhibitory factor effect on immunocompetent cells" ] 3 => array:2 [ "identificador" => "sec0075" "titulo" => "Inmunoreactivity of interleukin-1β in peripheral blood mononuclear cells" ] ] ] 7 => array:2 [ "identificador" => "sec0080" "titulo" => "Discussion" ] 8 => array:2 [ "identificador" => "sec0085" "titulo" => "Concluding remarks" ] 9 => array:2 [ "identificador" => "sec0100" "titulo" => "Ethical disclosures" ] 10 => array:2 [ "identificador" => "sec0090" "titulo" => "Funding" ] 11 => array:2 [ "identificador" => "sec0095" "titulo" => "Conflicts of interest" ] 12 => array:2 [ "identificador" => "xack38135" "titulo" => "Acknowledgments" ] 13 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2011-10-11" "fechaAceptado" => "2012-03-28" "PalabrasClave" => array:2 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec113275" "palabras" => array:4 [ 0 => "Amebic peptide" 1 => "Rheumatoid arthritis" 2 => "Inflammation" 3 => "Interleukin-1β" ] ] ] "es" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec113274" "palabras" => array:4 [ 0 => "Artritis reumatoide" 1 => "Inflamación" 2 => "IL-1β" 3 => "Péptido amibiano" ] ] ] ] "tieneResumen" => true "resumen" => array:2 [ "en" => array:2 [ "titulo" => "Abstract" "resumen" => "The monocyte locomotion inhibitory factor (MLIF) is a heat-stable pentapeptide produced by Entamoeba histolytica in culture. This factor displays several anti-inflammatory properties (i.e., inhibition of locomotion and respiratory burst in monocytes, reduction of skin hypersensitivity and delay of mononuclear cells in human Rebuck skin windows) with inhibition of adhesion molecules, chemokines, and other genes including interleukin-1β (IL-1β). In animal models, it reduces carragenin-induced inflammation and delays the inflammatory process in murine collagen-induced arthritis (CIA). ObjectivesTo test, in vitro, the anti-inflammatory capacity of MLIF on a promonocytic human cell line (U-937) cells and peripheral blood mononuclear cells (PBMC) from healthy subjects and from patients with rheumatoid arthritis (RA). Material and methodsIL-1β gene expression was evaluated in cell cultures either in the presence of MLIF, lipopolysaccharide (LPS), or both. Relative gene expression and immunoreactivity of IL-1β were assayed in cells and supernatants, respectively. ResultsAmebic peptide was able to down-regulate LPS-induced expression of IL-1β, in U-937 cells without a detectable effect upon the bioavailability of the cytokine. In similar culture conditions, MLIF was capable to down-regulate baseline and LPS-induced expression of IL-β only in PBMC from patients with RA. Peptide effect on immunoreactivity of IL-1β was not statistically significant. ConclusionsMLIF exerts, in primed cells, exquisite anti-inflammatory properties that deserve to be explored mechanistically." ] "es" => array:2 [ "titulo" => "Resumen" "resumen" => "El factor inhibidor de la locomoción de monocitos (FILM) es un pentapéptido termoestable producido por Entamoeba histolytica en cultivo. Este factor presenta diversas propiedades antiinflamatorias, a saber: inhibición de la locomoción y estallido respiratorio en monocitos, abatimiento de la hipersensibilidad por contacto al dinitroclorobenceno y retraso de la quimitoaxis de células mononucleares, disminución en la expresión de moléculas de adhesión y quimiocinas entre otros genes. En ratones el FILM reduce la inflamación inducida por carragenina y retrasa el proceso inflamatorio de la artritis inducida por colágena. ObjetivoEvaluar in vitro el efecto del FILM sobre la expresión de IL-1β en la línea celular promonocítica humana (U-937) y en células mononucleares de sangre periférica provenientes de donadores sanos y de pacientes con artritis reumatoide. Material y MétodosSe realizaron cultivos celulares en presencia de FILM, lipopolisacárido o ambos. Después del cultivo se determinó expresión relativa e inmunoreactividad de IL-1β en los botones celulares y sobrenadantes respectivamente. ResultadosEl péptido amibiano pudo reducir la expresión de IL-1β inducida por LPS en células U937, sin mostrar un efecto detectable sobre la biodisponibilidad de la citocina. En condiciones de cultivo similares, el FILM fue capaz de disminuir la expresión de IL-1β, basal e inducida por LPS, sólo en células mononucleares provenientes de pacientes con artritis. Su efecto sobre la inmunoreactividad de la citocina no fue significativo estadísticamente. ConclusionesEl FILM ejerce en las células activadas propiedades antiinflamatorias exquisitas que merecen ser exploradas mecanísticamente." ] ] "multimedia" => array:5 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1111 "Ancho" => 1689 "Tamanyo" => 91407 ] ] "descripcion" => array:1 [ "en" => "Time-course expression of IL-1β in U-937 cells. Triplicate cultures of 4×106 U-937 cells in the presence of MLIF, LPS or both were harvested at 0.3, 1, 2, 4, 6, 8 and 10h. RNA was obtained from pellets and used for real time PCR. Comparative expression was established using unstimulated cells as reference. LPS induced a strong up-regulation of IL-1β which peaks at 4h. MLIF per se did not affect IL-1β basal expression but MLIF was able to reduce significantly the stimulatory effect of LPS when amebic peptide was added simultaneously to LPS (p=0.05). Bars of SEM are <1 in all points." ] ] 1 => array:7 [ "identificador" => "fig0010" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 932 "Ancho" => 1513 "Tamanyo" => 54236 ] ] "descripcion" => array:1 [ "en" => "Time-course of IL-1β immunoreactivity in U-937 cells. Triplicate cultures of 4×106 U-937 cells in the presence of MLIF, LPS or both were harvested at 4, 6 and 8h by centrifugation. Supernatant fluids were employed for IL-1β immunoreactivity. In general IL-1β released to medium was low at 4 and 8h, response to LPS was higher at 6h *(p=0.05), MLIF per se had no effect over IL-1β release and did not interfere with LPS stimulation." ] ] 2 => array:7 [ "identificador" => "fig0015" "etiqueta" => "Figure 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 1631 "Ancho" => 2222 "Tamanyo" => 152202 ] ] "descripcion" => array:1 [ "en" => "Effect of MLIF in PBMC from healthy and patients with RA. Healthy donors (n=13) and patients with RA (n=11) PBMC were cultured during 4h in the presence of LPS, MLIF or both. qPCR analysis for IL-1β transcripts was carried out in each case and comparison between groups established by a normalized expression ratio (ner). (a) Patients with AR constitutively express more IL-1β than healthy donors. IL-1β ner between them was statistically different (p=0.03). (b) MLIF shows a down-regulatory effect on IL-1β expression in patients with RA which ner is <1 in 91% of cases (p=0.01). (c) Capacity of LPS to up-regulate IL-1β in both healthy donors and patients with RA was not significantly different. (d) MLIF was able to down-regulate LPS-induction in all patients with RA (p=0.001)." ] ] 3 => array:7 [ "identificador" => "fig0020" "etiqueta" => "Figure 4" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr4.jpeg" "Alto" => 813 "Ancho" => 1483 "Tamanyo" => 47461 ] ] "descripcion" => array:1 [ "en" => "Immunoreactivity of IL-1β in PBMC from healthy donors and patients with RA IL-1β immunoreactivity was detected by ELISA in supernatant fluids of 6h cultures of PBMC from healthy donors (n=11) and patients with RA (n=13). IL-1β was not detected in most patients with RA and healthy donors. Patients with RA show higher spontaneous release of IL-1β than healthy donors, and a poor response to LPS stimulus. MLIF per se seems to down-regulate IL-1β in patients with RA, and interferes with LPS induction in both healthy donors and patients with RA. Statistics were not available because of the low number of immunoreactive samples." ] ] 4 => array:5 [ "identificador" => "eq0005" "tipo" => "MULTIMEDIAFORMULA" "mostrarFloat" => false "mostrarDisplay" => true "Formula" => array:5 [ "Matematica" => "ner=fg IL-1βfg GAPDH" "Fichero" => "si1.jpeg" "Tamanyo" => 1846 "Alto" => 38 "Ancho" => 119 ] ] ] "bibliografia" => array:2 [ "titulo" => "References" "seccion" => array:1 [ 0 => array:2 [ "identificador" => "bibs0005" "bibliografiaReferencia" => array:16 [ 0 => array:3 [ "identificador" => "bib0005" "etiqueta" => "1" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Inhibition of human monocyte locomotion by products of axenically grown E. histolytica" "autores" => array:1 [ 0 => array:2 [ "etal" => true "autores" => array:6 [ 0 => "R. Kretschmer" 1 => "M.L. Collado" 2 => "M.G. Pacheco" 3 => "M.C. Salinas" 4 => "M. Lopez-Osuna" 5 => "M. 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Original article

An amebic anti-inflammatory peptide down-regulates ex vivo IL-1β expression in patients with rheumatoid arthritis

Un péptido antiinflamatorio amibiano disminuye en pacientes con artritis reumatoide la expresión ex vivo de IL-1β

Juan R. Velázqueza,

Corresponding author

velazquez_juan@hotmail.com

Corresponding author.

, Lizeth Garibay-Martínezb, Pedro Martínez-Tejadac, Yelda A. Leald

a Departamento de Inmunogénetica y Alergia, INER, México, D.F., Mexico

b Unidad de Investigación Médica Zacatecas, IMSS, Zacatecas, Zacatecas, Mexico

c Hospital Dr. Emilio Varela-Luján, IMSS, Zacatecas, Zacatecas, Mexico

d Unidad de Investigación Médica Yucatán (UIMY), Unidad Médica de Alta Especialidad de Mérida “Dr. Ignacio García Tellez”, IMSS, Mérida, Yucatán, Mexico

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cells&#46;<a class="elsevierStyleCrossRefs" href="#bib0010"><span class="elsevierStyleSup">2&#44;3</span></a> In umbilical vein endothelial cells &#40;UVEC&#41;&#44; MLIF reduces vascular cell adhesion molecule &#40;VCAM&#41;-1 and E-selectin expression &#40;personal communication&#41;&#46; <span class="elsevierStyleItalic">In vivo</span> it delays mononuclear cells migration to the inflammatory focus in human skin1&#46; The peptide is able to inhibit expression of cell adhesion molecules very late antigen &#40;VLA&#41;-4 and VCAM-1 in a dinitrochlorobenzene &#40;DNCB&#41;-hypersensitivity&#44; guinea pig model&#46;<a class="elsevierStyleCrossRef" href="#bib0020"><span class="elsevierStyleSup">4</span></a> In U-937 cells and in the CIA model&#44; analysis of gene expression profiles employing microarrays reveals a regulatory capacity of MLIF on numerous inflammatory genes including IL-1&#946;&#46;<a class="elsevierStyleCrossRef" href="#bib0010"><span class="elsevierStyleSup">2</span></a> In addition&#44; the amebic peptide has exhibited a notorious anti-inflammatory effect in carragenin-induced inflammation model in BALB&#47;c mice &#40;personal communication&#41;&#46; The MLIF molecular mechanisms of action are poorly understood&#59; however&#44; there is evidence of its involvement in nuclear factor-kappa B &#40;NF-&#954;B&#41; signal transduction&#59; MLIF appears to induce nuclear translocation of p50 homodimers&#44; affecting a number of inflammatory transcripts&#46;<a class="elsevierStyleCrossRef" href="#bib0025"><span class="elsevierStyleSup">5</span></a></p><p id="par0010" class="elsevierStylePara elsevierViewall">RA is a multisystemic chronic disease of unknown etiology&#46; During its development&#44; there are periods of activity and reminiscence that could be diagnosed on the basis of a battery of clinical and laboratory tests&#46;<a class="elsevierStyleCrossRefs" href="#bib0030"><span class="elsevierStyleSup">6&#44;7</span></a> One hallmark of RA is the inflammatory process that occurs in the joint&#46; The resulting inflammatory response induced by pro-inflammatory cytokines &#40;mainly tumor necrosis factor &#91;TNF&#93;&#945; and IL-1&#946;&#41; leads to synovial fibroblast hyperplasia&#44; increased vascularity&#44; and inflammatory cell infiltrate&#46;<a class="elsevierStyleCrossRefs" href="#bib0040"><span class="elsevierStyleSup">8&#8211;10</span></a> Messenger RNA &#40;mRNA&#41; and protein analysis of patients with RA has shown abundant expression of IL-1&#946; and TNF&#945; cytokines&#46;<a class="elsevierStyleCrossRefs" href="#bib0055"><span class="elsevierStyleSup">11&#44;12</span></a> In this work we tested the effect of anti-inflammatory peptide MLIF upon the constitutive and LPS-induced synthesis of IL-1&#946; in U-937 and PBMC from healthy subjects and from patients with RA&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Materials and methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Cells</span><p id="par0015" class="elsevierStylePara elsevierViewall">The promonocytic human cell line U-937 &#40;American Type Culture Collection&#44; 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4<span class="elsevierStyleHsp" style=""></span>mL of diluted blood sample was carefully layered on 3<span class="elsevierStyleHsp" style=""></span>mL of Ficoll-Paque PLUS &#40;GE Heathcare Bio-Sciences Corp&#44; Piscatawa&#44; NJ&#44; USA&#41;&#44; tubes were centrifuged at 400<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">g</span> for 30<span class="elsevierStyleHsp" style=""></span>min at room temperature&#46; Using a Pasteur pipette we take off the mononuclear layer and washed it with balanced salt solution&#46; Typically recovered cells were &#8805;95&#37; mononuclear cells with &#8805;95&#37; viability&#46;</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Cultures</span><p id="par0020" class="elsevierStylePara elsevierViewall">Time-course experiments were carried out in triplicate groups of 4<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">6</span><span class="elsevierStyleHsp" style=""></span>mL<span class="elsevierStyleSup">&#8722;1</span>&#46; 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MLIF&#44; LPS or both&#46; After 4<span class="elsevierStyleHsp" style=""></span>h&#44; the cell pellet &#40;for RNA isolation&#41; or after 6<span class="elsevierStyleHsp" style=""></span>h&#44; the supernatant fluid &#40;for IL-1&#946; immunoreactivity&#41; was harvested by centrifugation&#46; At the conclusion of all experiments&#44; cell viability was &#8805;90&#37; by trypan blue dye &#40;Sigma Chemical Co&#46;&#44; St&#46; Louis&#44; MO&#41; exclusion&#46; The concentration of MLIF employed in these studies was based on previous <span class="elsevierStyleItalic">in vitro</span> functional experiments&#46;<a class="elsevierStyleCrossRefs" href="#bib0010"><span class="elsevierStyleSup">2&#44;3</span></a> Simultaneous addition of MLIF to assays was employed&#44; because pilot studies have revealed that MLIF mainly affected cytokine induction when added prior to &#40;weak&#41; or simultaneously &#40;strong&#41;&#44; but less after LPS stimulation&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Chemical substances</span><p id="par0025" class="elsevierStylePara elsevierViewall">A single batch&#44; 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Carlsbad&#44; CA&#44; USA&#41; as described&#46;<a class="elsevierStyleCrossRef" href="#bib0015"><span class="elsevierStyleSup">3</span></a> RNA purity was established in 2&#46;5&#37; denaturing agarose gels&#44; and the concentration determined in a NanoDrop<span class="elsevierStyleSup">&#174;</span> ND-1000 spectrophotometer &#40;Thermo Scientific Inc&#46;&#44; Wilmington&#44; DE&#44; USA&#41;&#46; For reverse transcription&#44; 1<span class="elsevierStyleHsp" style=""></span>&#956;g of total RNA was mixed with 1<span class="elsevierStyleHsp" style=""></span>&#956;L oligo-dT &#40;12&#8211;18-mer&#44; Invitrogen&#41;&#44; 200<span class="elsevierStyleHsp" style=""></span>U SuperScript&#8482; reverse transcriptase &#40;Invitrogen&#41;&#44; 1&#215; First Stand Buffer &#40;Invitrogen&#41;&#44; and 0&#46;5<span class="elsevierStyleHsp" style=""></span>mM mixed dNTPs &#40;dATP&#44; dCTP&#44; dGTP&#44; dTTP&#41; &#40;Promega&#44; Co&#46;&#44; Madison&#44; WI&#44; USA&#41;&#46; The mixture was incubated at 42<span class="elsevierStyleHsp" style=""></span>&#176;C for 1<span class="elsevierStyleHsp" style=""></span>h&#46; Reverse transcription was heat-inactivated at 80<span class="elsevierStyleHsp" style=""></span>&#176;C for 10<span class="elsevierStyleHsp" style=""></span>min and the resulting cDNA was employed for real-time polymerase chain reaction &#40;PCR&#41;&#46;</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Real-time polymerase chain reaction analysis</span><p id="par0040" class="elsevierStylePara elsevierViewall">Real-time PCR was performed in capillaries in a 20<span class="elsevierStyleHsp" style=""></span>&#956;L reaction mix containing Light Cycler Taqman &#40;Roche Applied Science&#44; Salt Lake City&#44; UT&#44; USA&#41;&#44; 0&#46;2<span class="elsevierStyleHsp" style=""></span>&#956;M specific primer mix&#44; and 0&#46;1<span class="elsevierStyleHsp" style=""></span>&#956;M of gene-specific hydrolysis probes from the Universal Probe Library &#40;Roche&#41; and cDNA&#46; PCR reaction included 45 cycles consisting of denaturation at 95<span class="elsevierStyleHsp" style=""></span>&#176;C for 10<span class="elsevierStyleHsp" style=""></span>s&#44; annealing at 60<span class="elsevierStyleHsp" style=""></span>&#176;C for 20<span class="elsevierStyleHsp" style=""></span>s&#44; and extension at 72<span class="elsevierStyleHsp" style=""></span>&#176;C for 1<span class="elsevierStyleHsp" style=""></span>s&#46; All reactions were performed in a LightCycler 2&#46;0 instrument &#40;Roche&#41; and data analysis was conducted with LightCycler software 4&#46;05 &#40;Roche&#41;&#46;</p><p id="par0045" class="elsevierStylePara elsevierViewall">We obtained the relative expression of IL-1&#946; induced by LPS&#44; MLIF and LPS<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>MLIF with respect to unstimulated cells using the &#916;&#916;CP method&#46;<a class="elsevierStyleCrossRef" href="#bib0070"><span class="elsevierStyleSup">14</span></a> Briefly&#44; we copied the CP values for each sample in an Excel spreadsheet&#44; the first delta CP was calculated from IL-1&#946; CP minus glyceraldehyde 3-phosphate dehydrogenase &#40;GAPDH&#41; CP for each sample accordingly&#46; Deltadelta CP for each condition was equal to stimulated sample delta CP minus unstimulated sample CP&#46; Relative expression was calculated using Relative expression<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>2<span class="elsevierStyleSup">&#40;&#8722;deltadelta CP&#41;</span> formula&#46;</p><p id="par0050" class="elsevierStylePara elsevierViewall">We considered IL-1&#946; as target gene and GAPDH as housekeeping gene&#46;</p><p id="par0055" class="elsevierStylePara elsevierViewall">For comparison among study groups &#40;healthy subjects <span class="elsevierStyleItalic">vs</span> patients with RA&#41;&#44; real-time PCR calibration curves for IL-1&#946; and GAPDH genes were produced from purified specific amplicons&#46; In all groups we obtained normalized expression ratio &#40;ner&#41; of each condition &#40;unstimulated&#44; LPS&#44; MLIF and LPS<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>MLIF&#41; employing the formula&#58;<elsevierMultimedia ident="eq0005"></elsevierMultimedia></p><p id="par0060" class="elsevierStylePara elsevierViewall">Comparison among different ner was used to evaluate basal IL-1&#946;&#44; and responses induced by LPS and MLIF&#44; as well as MLIF capacity to antagonize LPS response&#46;</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Enzyme-linked immunoabsorbent assays</span><p id="par0065" class="elsevierStylePara elsevierViewall">IL-1&#946; proteins in supernatant fluids were measured using a commercial ELISA kit DuoSet<span class="elsevierStyleSup">&#174;</span> &#40;R &#38; D Systems&#44; Inc&#46;&#44; Minneapolis&#44; MN&#44; USA&#41;&#46; Plates were incubated overnight at 4<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; washed and blocked&#46; Final development employed ExtrAvidin&#8482; HRP and substrate 3&#44;3&#8242;&#44;5&#44;5&#8242;-tetramethyl-benzidine &#40;Sigma&#41;&#46; Microtiter plates were read at 450<span class="elsevierStyleHsp" style=""></span>nm in a BIO-RAD microplate Benchmark densitometer &#40;Richmond&#44; CA&#44; USA&#41;&#46; Results were expressed as ng&#47;mL&#46; Sensitivity for IL-1&#946; was &#8805;2<span class="elsevierStyleHsp" style=""></span>pg&#47;mL&#46;</p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Statistical analysis</span><p id="par0070" class="elsevierStylePara elsevierViewall">Results are expressed as mean ranks&#46; Statistical significance of differences in mean values between different groups was evaluated by Kruskal&#8211;Wallis test&#44; followed by <span class="elsevierStyleItalic">U</span>-Mann&#8211;Whitney test for inter-group comparisons&#46; A value of <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05 was considered to be significant&#46; All analyses were performed with SPSS software version 15&#46;0 &#40;SPSS Inc&#46;&#44; Chicago&#44; IL&#41;&#46;</p></span></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Results</span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Cellular response to lipopolysaccharide and the antagonist effect of monocyte locomotion inhibitory factor</span><p id="par0075" class="elsevierStylePara elsevierViewall">Previous work utilizing U-937 demonstrated that MLIF was able to decrease PMA-induced expression of chemokines MIP-1&#945; and MIP-1&#946;&#44; as well as that of cytokine IL-1&#946;&#46;<a class="elsevierStyleCrossRefs" href="#bib0010"><span class="elsevierStyleSup">2&#44;3</span></a> We observed this effect only when the peptide was administered prior to or simultaneously with the activator agent&#46; The neutralizing effect of MLIF on such an event was outstanding&#44; if we take into account that PMA is a powerful cell activator that disrupts several cell signaling pathways&#46; On the other hand&#44; because MLIF is involved in the NF-&#954;B signaling&#44; we were interested in observing the amebic peptide effect on the production of LPS&#47;TLR4 signaling-dependent IL-1&#946;&#46;</p><p id="par0080" class="elsevierStylePara elsevierViewall">In a triplicate time-course experiment using U-937 cells&#44; LPS-induced expression of IL-1&#946; was followed with real-time PCR&#46; Specific IL-1&#946; mRNA was detected in a 1&#8211;10<span class="elsevierStyleHsp" style=""></span>h window of time &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>&#41;&#46; Maximum response to LPS peaked at 4<span class="elsevierStyleHsp" style=""></span>h post-stimulation with 94<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;15 &#40;mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>SEM&#41; folds&#44; and afterwards there was a symmetrically decreasing production&#46; When MLIF was administrated simultaneously with LPS&#44; the amebic peptide exhibited an astonishing antagonist effect at 4<span class="elsevierStyleHsp" style=""></span>h &#40;relative expression 94<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;15-fold with LPS <span class="elsevierStyleItalic">vs</span> 8<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;03-fold with LPS<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>MLIF &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#41;&#46; On the other hand&#44; amebic peptide <span class="elsevierStyleItalic">per se</span> showed no IL-1&#946; regulatory capacity&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Immunoreactivity of lipopolysaccharide-induced interleukin-1&#946;</span><p id="par0085" class="elsevierStylePara elsevierViewall">Supernatants from mentioned triplicate experiment were used for measuring IL-1&#946; immunoreactivity released by U-937 cells stimulated with LPS&#44; MLIF or both at three different times &#40;4&#44; 6&#44; and 8<span class="elsevierStyleHsp" style=""></span>h&#41;&#46; A higher amount of IL-1&#946; was detected after 6<span class="elsevierStyleHsp" style=""></span>h of incubation where LPS-induction was maximum with 18&#46;5<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;74<span class="elsevierStyleHsp" style=""></span>pg&#47;mL&#44; statistical difference between LPS <span class="elsevierStyleItalic">vs</span> MLIF &#40;18&#46;5<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;4 <span class="elsevierStyleItalic">vs</span> 13&#46;2<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;4&#41;&#44; and LPS <span class="elsevierStyleItalic">vs</span> unstimulated cells &#40;18&#46;5<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;4 <span class="elsevierStyleItalic">vs</span> 9&#46;6<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;5&#41; was significant with <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05 for both&#46; MLIF showed no effect on IL-1&#946; release by LPS stimulation&#46; At 4 and 8<span class="elsevierStyleHsp" style=""></span>h IL-1&#946; response to LPS was poor&#44; and in most conditions immunoreactivity was near the assay detection limit &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>&#41;&#46; There is a trend for MLIF to interfere with LPS effect at 4<span class="elsevierStyleHsp" style=""></span>h and 8<span class="elsevierStyleHsp" style=""></span>h but statistics showed it not to be significant&#46; Once again&#44; the peptide <span class="elsevierStyleItalic">per se</span> did not affect the production of IL-1&#946;&#46;</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Monocyte locomotion inhibitory factor effect on immunocompetent cells</span><p id="par0090" class="elsevierStylePara elsevierViewall">We were interested in testing the observed MLIF effects in differentiated immunocompetent cells&#46; PBMC cultures from healthy subjects and patients with RA were used to look at basal expression of IL-1&#946;&#46; As expected&#44; patients with RA exhibited higher expression of IL-1&#946; mRNA in comparison with healthy subjects&#46; 91&#37; of patients with RA &#40;10 of 11 patients&#41; IL-1&#946; ner was &#62;1&#59; meanwhile in 54&#37; of healthy subjects &#40;9 of 13&#41; IL-1&#946; ner was &#62;1 &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>a&#41;&#46; Statistical data showed a significantly higher expression of IL-1&#946; in patients with RA compared with healthy donors &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;03&#41;&#46;</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia><p id="par0095" class="elsevierStylePara elsevierViewall">The capacity of MLIF to affect the expression of IL-1&#946; was determined as the ratio of MLIF ner to unstimulated ner&#46; The presence of MLIF in PBMC cultures decreased IL-1&#946; expression &#40;ratio &#60;1&#41; in 91&#37; of patients with AR &#40;10 of 11&#41; and in 46&#37; of healthy donors &#40;6 of 13&#41; &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;01&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>b&#41;&#46;</p><p id="par0100" class="elsevierStylePara elsevierViewall">Ratios of LPS-induced ner to unstimulated ner were employed to evaluate LPS stimulatory capacity in PBMC cultures&#46; We considered an increase of &#62;2-folds in IL-1&#946; expression as a biologically effective criterion&#46; Studied groups responded in similar fashion to LPS stimulus&#59; 64&#37; &#40;7 of 11&#41; patients with RA and 62&#37; &#40;8 of 13&#41; healthy donors responded to LPS&#44; and no significant differences were found among them &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>c&#41;&#46;</p><p id="par0105" class="elsevierStylePara elsevierViewall">We considered that MLIF could antagonize with LPS when ratios of LPS<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>MLIF ner to LPS-induced ner were &#60;1&#46; Expression of IL-1&#946; decreased in 100&#37; &#40;11 of 11&#41; of patients with RA and in 38&#37; &#40;5 of 13&#41; of healthy subjects &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>d&#41;&#46;</p></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Inmunoreactivity of interleukin-1&#946; in peripheral blood mononuclear cells</span><p id="par0110" class="elsevierStylePara elsevierViewall">IL-1&#946; inmunoreactivity in PBMC cultures was in general low &#40;below the detection limit in the majority of cases&#41;&#46; It was only possible to detect IL-1&#946; in 36&#37; &#40;4 of 11&#41; of patients with RA and in 8&#37; &#40;1 of 13&#41; of healthy donors&#46; Apparently&#44; RA group releases more IL-1&#946; than healthy group &#40;18<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>2&#46;1 <span class="elsevierStyleItalic">vs</span> 8<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;6<span class="elsevierStyleHsp" style=""></span>pg&#47;mL&#44; respectively&#41;&#46; However&#44; because the low number of data&#44; no significant differences were found between them&#46; In the group of patients with RA&#44; we observed a MLIF trend to diminish the amount of IL-1&#946; spontaneously released in culture from 18&#46;0 to 5&#46;1<span class="elsevierStyleHsp" style=""></span>pg&#47;mL&#44; as well as an antagonist effect on LPS-stimulation in both RA and healthy groups &#40;from 14&#46;6 to 9&#46;0&#59; and from 19&#46;2 to 7&#46;1<span class="elsevierStyleHsp" style=""></span>pg&#47;mL&#44; respectively&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0020">Fig&#46; 4</a>&#41;&#46; This result is interesting but not conclusive due the low number of immunoreactive samples&#46;</p><elsevierMultimedia ident="fig0020"></elsevierMultimedia></span></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Discussion</span><p id="par0115" class="elsevierStylePara elsevierViewall">Several studies have shown the MLIF modulatory capacity on inflammatory genes&#46; The antagonist effect of the amebic peptide when administered prior to or simultaneously with LPS appears to involve it in early signaling events linked with either putative or non- putative membrane receptors&#46;<a class="elsevierStyleCrossRef" href="#bib0025"><span class="elsevierStyleSup">5</span></a> Data on LPS activation by means of TLR4 in U-937 cells had revealed that MLIF is able to interfere with MyD88 membrane recruitment in response to LPS&#44; thus affecting an array of MyD88-dependent genes 5&#46; Ongoing studies in our laboratory with respect to IL-1&#946; synthesis utilizing U-937 cells have shown that MLIF increase the degradation of specific mRNA when induced with LPS&#46; The use of ERK&#44; JNK&#44; p38 and NF-&#954;B inhibitors have brought to light the participation of a NF-&#954;B inhibitory dimmer induced by MLIF that is perhaps responsible for abrogating IL-1&#946; transcription and participating in a cross-talk between NF-&#954;B and mitogen-activated protein kinase &#40;MAPK&#41; signaling cascades&#46; Differential expression profiles of U-937 stimulated with PMA demonstrated a remarkable interfering capacity of MLIF on induced transcription of IL-1&#946;&#46; In this report when using U-937 cells&#44; we found that MLIF <span class="elsevierStyleItalic">per se</span> did not affect IL-1&#946; expression&#44; but that it indeed interferes with LPS in a narrow window of time&#46; MLIF shows no capacity in regulating the LPS-induced immunoreactivity of IL-1&#946; in U-937 cells&#59; however&#44; it is important to consider the poor response of these cells to LPS&#46; Regulation of MLIF on IL-1&#946; transcripts but not on IL-1&#946; protein of U-937 cells could be due to genetic alterations in the cell line&#44; lack of synchronicity in the synthesis&#44; storage&#44; and further secretion of mature IL-1&#946;&#46; Assessment of IL-1&#946; requires the presence in the medium of inmunoreactive cytokine which is generated in a cyclic and complicated process&#46; The immunoreactivity detected is closely related with the cell&#39;s ability to generate and to secrete caspase-1-processed IL-1&#946;&#46; Clearly&#44; additional studies on the extracellular bioavailability and intracellular storage of IL-1&#946; in stimulated cells in the presence of MLIF are required&#46;</p><p id="par0120" class="elsevierStylePara elsevierViewall">On the other hand&#44; the effect of MLIF on primed cells is slightly different in immunocompetent PBMC from an inflammatory environment such as RA&#46; In arthritic PBMC&#44; there is an over-expression of basal IL-1&#946;&#44; which is in agreement with other reports&#46;<a class="elsevierStyleCrossRef" href="#bib0075"><span class="elsevierStyleSup">15</span></a> MLIF down-regulated IL-1&#946; basal expression and interfered with LPS-induction&#46; IL-1&#946; immunoreactivity levels in PBMC culture supernatants exhibited in general a lower detection limit&#44; but in the few subjects with detected activity MLIF acted mainly on patients with RA&#44; decreasing spontaneous release of IL-1&#946; and interfering with LPS-induction&#46; An increased number of subjects would aid in clarifying this issue&#46;</p><p id="par0125" class="elsevierStylePara elsevierViewall">We are not certain about MLIF action only in transcription but not in translation of IL-1&#946;&#46; For example&#44; we have evidence that administration of the amebic peptide in a CIA model ameliorates the inflammation process and modulates gene expression profiles &#40;IL-1&#946; included among other dozens of genes&#41;&#46;<a class="elsevierStyleCrossRef" href="#bib0080"><span class="elsevierStyleSup">16</span></a> Furthermore&#44; MLIF is able to reduce the immunoreactivity of IL-1&#946; and matrix metalloproteinases in joints of arthritic mice &#40;paper in progress&#41;&#46;</p></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Concluding remarks</span><p id="par0130" class="elsevierStylePara elsevierViewall">MLIF was able to distinguish between healthy subjects and patients with RA&#44; based perhaps upon a differential cell-activation degree and the signaling pathways in use&#46; It is plausible to think that synovial cells would be a more representative entity for testing the interesting effects described in this work&#44; and further studies are required to resolve this matter&#46; The MLIF effect on caspase-1&#44; translation&#44; storage and release of mature IL-1&#946; is unknown&#46;</p><p id="par0135" class="elsevierStylePara elsevierViewall">The results presented in this work provide evidence of MLIF&#39;s most exquisite anti-inflammatory properties on prime and arthritic cells and encourage its research in other inflammatory processes&#46;</p></span><span id="sec0100" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Ethical disclosures</span><p id="par0155" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">Protection of human and animal subjects</span>&#46; The authors declare that no experiments were performed on humans or animals for this investigation&#46;</p><p id="par0160" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">Confidentiality of Data</span>&#46; The authors declare that no patient data appears in this article&#46;</p><p id="par0165" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">Right to privacy and informed consent</span>&#46; The authors have obtained the informed consent of the patients and &#47;or subjects mentioned in the article&#46; The author for correspondence is in possession of this document&#46;</p></span><span id="sec0090" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Funding</span><p id="par0140" class="elsevierStylePara elsevierViewall">This work was supported by <span class="elsevierStyleGrantSponsor" id="gs0005">Fondo de Investigaci&#243;n en Salud of IMSS&#44; Mexico</span>&#44; grant <span class="elsevierStyleGrantNumber" refid="gs0005">FIS&#47;IMSS&#47;PROT&#47;356</span>&#46;</p></span><span id="sec0095" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Conflicts of interest</span><p id="par0145" class="elsevierStylePara elsevierViewall">The authors have no conflicts of interest to declare&#46;</p></span></span>"
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          "identificador" => "xpalclavsec113274"
          "palabras" => array:4 [
            0 => "Artritis reumatoide"
            1 => "Inflamaci&#243;n"
            2 => "IL-1&#946;"
            3 => "P&#233;ptido amibiano"
          ]
        ]
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    "resumen" => array:2 [
      "en" => array:2 [
        "titulo" => "Abstract"
        "resumen" => "<p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">The monocyte locomotion inhibitory factor &#40;MLIF&#41; is a heat-stable pentapeptide produced by <span class="elsevierStyleItalic">Entamoeba histolytica</span> in culture&#46; This factor displays several anti-inflammatory properties &#40;<span class="elsevierStyleItalic">i&#46;e&#46;</span>&#44; inhibition of locomotion and respiratory burst in monocytes&#44; reduction of skin hypersensitivity and delay of mononuclear cells in human Rebuck skin windows&#41; with inhibition of adhesion molecules&#44; chemokines&#44; and other genes including interleukin-1&#946; &#40;IL-1&#946;&#41;&#46; In animal models&#44; it reduces carragenin-induced inflammation and delays the inflammatory process in murine collagen-induced arthritis &#40;CIA&#41;&#46;</p> <span class="elsevierStyleSectionTitle">Objectives</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">To test&#44; <span class="elsevierStyleItalic">in vitro</span>&#44; the anti-inflammatory capacity of MLIF on a promonocytic human cell line &#40;U-937&#41; cells and peripheral blood mononuclear cells &#40;PBMC&#41; from healthy subjects and from patients with rheumatoid arthritis &#40;RA&#41;&#46;</p> <span class="elsevierStyleSectionTitle">Material and methods</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">IL-1&#946; gene expression was evaluated in cell cultures either in the presence of MLIF&#44; lipopolysaccharide &#40;LPS&#41;&#44; or both&#46; Relative gene expression and immunoreactivity of IL-1&#946; were assayed in cells and supernatants&#44; respectively&#46;</p> <span class="elsevierStyleSectionTitle">Results</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Amebic peptide was able to down-regulate LPS-induced expression of IL-1&#946;&#44; in U-937 cells without a detectable effect upon the bioavailability of the cytokine&#46; In similar culture conditions&#44; MLIF was capable to down-regulate baseline and LPS-induced expression of IL-&#946; only in PBMC from patients with RA&#46; Peptide effect on immunoreactivity of IL-1&#946; was not statistically significant&#46;</p> <span class="elsevierStyleSectionTitle">Conclusions</span><p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">MLIF exerts&#44; in primed cells&#44; exquisite anti-inflammatory properties that deserve to be explored mechanistically&#46;</p>"
      ]
      "es" => array:2 [
        "titulo" => "Resumen"
        "resumen" => "<p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">El factor inhibidor de la locomoci&#243;n de monocitos &#40;FILM&#41; es un pentap&#233;ptido termoestable producido por <span class="elsevierStyleItalic">Entamoeba histolytica</span> en cultivo&#46; Este factor presenta diversas propiedades antiinflamatorias&#44; a saber&#58; inhibici&#243;n de la locomoci&#243;n y estallido respiratorio en monocitos&#44; abatimiento de la hipersensibilidad por contacto al dinitroclorobenceno y retraso de la quimitoaxis de c&#233;lulas mononucleares&#44; disminuci&#243;n en la expresi&#243;n de mol&#233;culas de adhesi&#243;n y quimiocinas entre otros genes&#46; En ratones el FILM reduce la inflamaci&#243;n inducida por carragenina y retrasa el proceso inflamatorio de la artritis inducida por col&#225;gena&#46;</p> <span class="elsevierStyleSectionTitle">Objetivo</span><p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">Evaluar <span class="elsevierStyleItalic">in vitro</span> el efecto del FILM sobre la expresi&#243;n de IL-1&#946; en la l&#237;nea celular promonoc&#237;tica humana &#40;U-937&#41; y en c&#233;lulas mononucleares de sangre perif&#233;rica provenientes de donadores sanos y de pacientes con artritis reumatoide&#46;</p> <span class="elsevierStyleSectionTitle">Material y M&#233;todos</span><p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">Se realizaron cultivos celulares en presencia de FILM&#44; lipopolisac&#225;rido o ambos&#46; Despu&#233;s del cultivo se determin&#243; expresi&#243;n relativa e inmunoreactividad de IL-1&#946; en los botones celulares y sobrenadantes respectivamente&#46;</p> <span class="elsevierStyleSectionTitle">Resultados</span><p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">El p&#233;ptido amibiano pudo reducir la expresi&#243;n de IL-1&#946; inducida por LPS en c&#233;lulas U937&#44; sin mostrar un efecto detectable sobre la biodisponibilidad de la citocina&#46; En condiciones de cultivo similares&#44; el FILM fue capaz de disminuir la expresi&#243;n de IL-1&#946;&#44; basal e inducida por LPS&#44; s&#243;lo en c&#233;lulas mononucleares provenientes de pacientes con artritis&#46; Su efecto sobre la inmunoreactividad de la citocina no fue significativo estad&#237;sticamente&#46;</p> <span class="elsevierStyleSectionTitle">Conclusiones</span><p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">El FILM ejerce en las c&#233;lulas activadas propiedades antiinflamatorias exquisitas que merecen ser exploradas mecan&#237;sticamente&#46;</p>"
      ]
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        "tipo" => "MULTIMEDIAFIGURA"
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          "en" => "<p id="spar0055" class="elsevierStyleSimplePara elsevierViewall">Time-course expression of IL-1&#946; in U-937 cells&#46; Triplicate cultures of 4<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">6</span> U-937 cells in the presence of MLIF&#44; LPS or both were harvested at 0&#46;3&#44; 1&#44; 2&#44; 4&#44; 6&#44; 8 and 10<span class="elsevierStyleHsp" style=""></span>h&#46; RNA was obtained from pellets and used for real time PCR&#46; Comparative expression was established using unstimulated cells as reference&#46; LPS induced a strong up-regulation of IL-1&#946; which peaks at 4<span class="elsevierStyleHsp" style=""></span>h&#46; MLIF <span class="elsevierStyleItalic">per se</span> did not affect IL-1&#946; basal expression but MLIF was able to reduce significantly the stimulatory effect of LPS when amebic peptide was added simultaneously to LPS &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46; Bars of SEM are &#60;1 in all points&#46;</p>"
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          "en" => "<p id="spar0060" class="elsevierStyleSimplePara elsevierViewall">Time-course of IL-1&#946; immunoreactivity in U-937 cells&#46; Triplicate cultures of 4<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">6</span> U-937 cells in the presence of MLIF&#44; LPS or both were harvested at 4&#44; 6 and 8<span class="elsevierStyleHsp" style=""></span>h by centrifugation&#46; Supernatant fluids were employed for IL-1&#946; immunoreactivity&#46; In general IL-1&#946; released to medium was low at 4 and 8<span class="elsevierStyleHsp" style=""></span>h&#44; response to LPS was higher at 6<span class="elsevierStyleHsp" style=""></span>h &#42;&#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#44; MLIF <span class="elsevierStyleItalic">per se</span> had no effect over IL-1&#946; release and did not interfere with LPS stimulation&#46;</p>"
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          "en" => "<p id="spar0065" class="elsevierStyleSimplePara elsevierViewall">Effect of MLIF in PBMC from healthy and patients with RA&#46; Healthy donors &#40;<span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>13&#41; and patients with RA &#40;<span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>11&#41; PBMC were cultured during 4<span class="elsevierStyleHsp" style=""></span>h in the presence of LPS&#44; MLIF or both&#46; qPCR analysis for IL-1&#946; transcripts was carried out in each case and comparison between groups established by a normalized expression ratio &#40;ner&#41;&#46; &#40;a&#41; Patients with AR constitutively express more IL-1&#946; than healthy donors&#46; IL-1&#946; ner between them was statistically different &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;03&#41;&#46; &#40;b&#41; MLIF shows a down-regulatory effect on IL-1&#946; expression in patients with RA which ner is &#60;1 in 91&#37; of cases &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;01&#41;&#46; &#40;c&#41; Capacity of LPS to up-regulate IL-1&#946; in both healthy donors and patients with RA was not significantly different&#46; &#40;d&#41; MLIF was able to down-regulate LPS-induction in all patients with RA &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41;&#46;</p>"
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        "mostrarFloat" => true
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          "en" => "<p id="spar0070" class="elsevierStyleSimplePara elsevierViewall">Immunoreactivity of IL-1&#946; in PBMC from healthy donors and patients with RA IL-1&#946; immunoreactivity was detected by ELISA in supernatant fluids of 6<span class="elsevierStyleHsp" style=""></span>h cultures of PBMC from healthy donors &#40;<span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>11&#41; and patients with RA &#40;<span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>13&#41;&#46; IL-1&#946; was not detected in most patients with RA and healthy donors&#46; Patients with RA show higher spontaneous release of IL-1&#946; than healthy donors&#44; and a poor response to LPS stimulus&#46; MLIF <span class="elsevierStyleItalic">per se</span> seems to down-regulate IL-1&#946; in patients with RA&#44; and interferes with LPS induction in both healthy donors and patients with RA&#46; Statistics were not available because of the low number of immunoreactive samples&#46;</p>"
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                          "etal" => true
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                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:6 [
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                            1 => "E&#46; Arenas"
                            2 => "L&#46; Diaz"
                            3 => "G&#46; Rico"
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                            5 => "R&#46; Kretschmer"
                          ]
                        ]
                      ]
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                  ]
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                        "fecha" => "2000"
                        "volumen" => "31"
                        "numero" => "4 Suppl&#46;"
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                        "paginaFinal" => "S93"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/11070238"
                            "web" => "Medline"
                          ]
                        ]
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              "identificador" => "bib0025"
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                      "autores" => array:1 [
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                          "etal" => false
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                            0 => "J&#46;R&#46; Velazquez"
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                          ]
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                    0 => array:1 [
                      "Revista" => array:5 [
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                          "etal" => false
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                        "volumen" => "8"
                        "paginaInicial" => "1773"
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              "etiqueta" => "8"
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                      "autores" => array:1 [
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                          "etal" => false
                          "autores" => array:2 [
                            0 => "E&#46;H&#46; Choy"
                            1 => "G&#46;S&#46; Panayi"
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                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1056/NEJM200103223441207"
                      "Revista" => array:6 [
                        "tituloSerie" => "N Engl J Med"
                        "fecha" => "2001"
                        "volumen" => "344"
                        "paginaInicial" => "907"
                        "paginaFinal" => "916"
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                            "web" => "Medline"
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              "identificador" => "bib0045"
              "etiqueta" => "9"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "The role of chemokines in leucocyte&#8211;stromal interactions in rheumatoid arthritis"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:4 [
                            0 => "A&#46; Filer"
                            1 => "K&#46; Raza"
                            2 => "M&#46; Salmon"
                            3 => "C&#46;D&#46; Buckley"
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                        ]
                      ]
                    ]
                  ]
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                    0 => array:1 [
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                        "fecha" => "2008"
                        "volumen" => "13"
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                            "web" => "Medline"
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Article information

ISSN: 21735743
Original language: English

DOI:10.1016/j.reumae.2012.07.013

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Year/Month	Html	Pdf	Total

2024 November	7	11	18
2024 October	32	27	59
2024 September	35	10	45
2024 August	53	31	84
2024 July	45	30	75
2024 June	38	21	59
2024 May	33	25	58
2024 April	41	22	63
2024 March	51	22	73
2024 February	39	19	58
2024 January	42	16	58
2023 December	41	25	66
2023 November	43	22	65
2023 October	28	24	52
2023 September	80	38	118
2023 August	26	10	36
2023 July	37	30	67
2023 June	26	24	50
2023 May	21	17	38
2023 April	22	7	29
2023 March	45	24	69
2023 February	29	24	53
2023 January	23	20	43
2022 December	47	35	82
2022 November	54	26	80
2022 October	48	21	69
2022 September	71	21	92
2022 August	72	34	106
2022 July	52	30	82
2022 June	31	25	56
2022 May	25	41	66
2022 April	30	52	82
2022 March	46	59	105
2022 February	21	31	52
2022 January	20	45	65
2021 December	25	34	59
2021 November	26	37	63
2021 October	31	47	78
2021 September	26	38	64
2021 August	32	38	70
2021 July	18	27	45
2021 June	25	31	56
2021 May	35	35	70
2021 April	26	59	85
2021 March	33	28	61
2021 February	10	15	25
2021 January	19	20	39
2020 December	22	14	36
2020 November	13	15	28
2020 October	18	11	29
2020 September	18	13	31
2020 August	17	13	30
2020 July	20	16	36
2020 June	29	14	43
2020 May	27	11	38
2020 April	19	16	35
2020 March	12	8	20
2019 January	1	0	1
2018 May	3	0	3
2018 April	26	6	32
2018 March	31	10	41
2018 February	18	3	21
2018 January	26	7	33
2017 December	20	3	23
2017 November	29	8	37
2017 October	16	9	25
2017 September	39	4	43
2017 August	44	4	48
2017 July	47	2	49
2017 June	51	13	64
2017 May	50	6	56
2017 April	41	7	48
2017 March	47	6	53
2017 February	26	6	32
2017 January	26	7	33
2016 December	61	15	76
2016 November	48	2	50
2016 October	61	10	71
2016 September	49	7	56
2016 August	45	7	52
2016 July	25	4	29
2016 January	1	0	1
2015 December	3	0	3
2015 October	1	11	12
2015 September	4	0	4
2015 August	1	0	1
2015 July	17	0	17
2015 June	19	5	24
2015 May	33	16	49
2015 April	21	15	36
2015 March	22	3	25
2015 February	19	7	26
2015 January	23	10	33
2014 December	23	14	37
2014 November	24	4	28
2014 October	24	15	39
2014 September	15	7	22
2014 August	16	8	24
2014 July	19	13	32
2014 June	39	11	50
2014 May	36	12	48
2014 April	24	12	36
2014 March	31	15	46
2014 February	21	11	32
2014 January	17	11	28
2013 December	24	10	34
2013 November	24	6	30
2013 October	30	9	39
2013 September	25	9	34
2013 August	34	8	42
2013 July	27	10	37
2013 June	22	9	31
2013 May	30	11	41
2013 April	28	11	39
2013 March	28	6	34
2013 February	22	7	29
2013 January	31	14	45
2012 December	30	14	44
2012 November	4	2	6

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