Investigation of therapeutic effects in the wound healing of chitosan/pGM-CSF complexes

Abstract Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to promote the growth, proliferation, and migration of endothelial and keratinocyte cells. Chitosan has been widely used as a biopolymer in wound-healing studies. The aim of this study was to investigate the in vitro proliferative effects of chitosan/pGM-CSF complexes as well as the therapeutic role of the complexes in an in vivo rat wound model. The effect of complexes on cell proliferation and migration was examined. Wounds were made in Wistar-albino rats, and examined histopathologically. The cell proliferation and migration were increased weight ratio- and time-dependently in HaCaT and NIH-3T3 cell lines. Wound healing was significantly accelerated in rats treated with the complexes. These results showed that the delivery of pGM-CSF using chitosan complexes could play an accelerating role in the cell proliferation, migration, and wound-healing process.

The influence of protein malnutrition on the production of GM-CSF and M-CSF by macrophages

ABSTRACT It is well established that protein malnutrition (PM) impairs immune defenses and increases susceptibility to infection. Macrophages are cells that play a central role in innate immunity, constituting one of the first barriers against infections. Macrophages produce several soluble factors, including cytokines and growth factors, important to the immune response. Among those growth factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). GM-CSF and M-CSF are important to monocyte and macrophage development and stimulation of the immune response process. Knowing the importance of GM-CSF and M-CSF, we sought to investigate the influence of PM on macrophage production of these growth factors. Two-month-old male BALB/c mice were subjected to PM with a low-protein diet (2%) and compared to a control diet (12%) mouse group. Nutritional status, hemogram and the number of peritoneal cells were evaluated. Additionally, peritoneal macrophages were cultured and the production of GM-CSF and M-CSF and mRNA expression were evaluated. To determine if PM altered macrophage production of GM-CSF and M-CSF, they were stimulated with TNF-α. The PM animals had anemia, leukopenia and a reduced number of peritoneal cells. The production of M-CSF was not different between groups; however, cells from PM animals, stimulated with or without TNF-α, presented reduced capability to produce GM-CSF. These data imply that PM interferes with the production of GM-CSF, and consequently would affect the production and maturation of hematopoietic cells and the immune response.

Análise comparativa da imunoexpressão de GLUT-1, GLUT-3 e M-CSF em lesão periférica e central de células gigantes / Comparative analysis of GLUT-1, GLUT-3 and M-CSF immunoexpression in peripheral and central giant cell lesions

A lesão periférica de células gigantes (LPCG) e a lesão central de células gigantes (LCCG) são patologias histologicamente semelhantes que acometem a região de cabeça e pescoço. O estudo objetivou analisar a expressão imuno-histoquímica dos marcadores GLUT-1, GLUT-3 e M-CSF em uma série de casos de LPCG e LCCG, na tentativa de compreender os diferentes comportamentos biológicos destas entidades patológicas. A amostra foi constituída por 20 espécimes teciduais de LPCG, 20 de lesão central de células gigantes não agressivas (LCCGNA) e 20 de lesão central de células gigantes agressivas (LCCGA), oriundos do Serviço de Anatomia Patológica da Disciplina de Patologia Oral do Departamento de Odontologia da UFRN. Foi realizada a análise semiquantitativa e qualitativa da expressão imuno-histoquímica dos marcadores nas células gigantes e nas células mononucleares. Em relação ao GLUT-1, verificou-se uma diferença estatisticamente significativa na quantidade de células mononucleares imunomarcadas entre a LPCG e a LCCGNA e entre a LPCG e a LCCGA. Em relação à intensidade da marcação também foi verificado uma diferença estatisticamente significativa tanto para as células mononucleares quanto para as células gigantes entre LPCG e LCCGNA e entre LPCG e LCCGA, nas células gigantes também ocorreu uma diferença estatisticamente significativa entre a LCCGNA e a LCCGA. Em relação ao GLUT-3, foi encontrada uma diferença estatisticamente significativa entre LPCG e LCCGA e entre LCCGNA e LCCGA na quantidade de células mononucleares imunomarcadas. No que concerne à intensidade de marcação para a referida proteína foi verificado uma diferença estatisticamente significativa nas células gigantes entre LPCG e LCCGA. Para o M-CSF foi observada apenas uma diferença estatisticamente significativa na intensidade de marcação nas células mononucleares entre LPCG e LCCGNA e entre LPCG e LCCGA. Com base nestes resultados, pode-se concluir a participação do GLUT-1, GLUT-3 e do M-CSF na patogênese das lesões estudadas. A maior imunomarcação destas proteínas nas células mononucleares evidenciam que tais células desempenham uma maior atividade metabólica e osteoclastogênica, principalmente nas LCCGA. Constatou-se que as células mononucleares estavam mais relacionadas à patogênese das lesões estudadas do que propriamente as células gigantes (AU).

The peripheral giant cell lesion (PGCL) and the central giant cell lesion (CGCL) are lesions histologically similar affecting the head and neck region. The study aimed to analyze the immunohistochemical expression of markers GLUT-1, GLUT-3 and MCSF in a series of cases of PGCL and CGCL, in trying to understand the different biological behavior of these pathologies. The sample consisted of 20 tissue specimens of PGCL 20 central lesion of not aggressive giant cell (CLNAGC) and 20 central lesion of aggressive giant cell (CLAGC), coming from the Pathology Unit of Oral Pathology of the Department of Dentistry of UFRN. Was performed the semi-quantitative and qualitative analysis of immunohistochemical expression of the markers in giant cells and mononuclear cells. In relation to the GLUT-1, it was found a statistically significant difference (p <0.05) in the number of mononuclear cells immunomarked between the PGCL and the CLNAGC and between the PGCL and CLAGC. Regarding the intensity of staining was also observed a statistically significant difference both at the mononuclear cells as in giant cells between PL and CLNAGC and between PGCL and CLAGC, at the giant cells there was also a statistically significant difference between the CLNAGC and CLAGC. In relation to GLUT-3, was found a statistically significant difference between PGCL and CLAGC and between CLAGC and CLNAGC in amount of mononuclear cells immunomarked. Regarding the intensity of labeling for such protein was found a statistically significant difference at the giant cells between PL and CLAGC. To the M-CSF was observed only a statistically significant difference in the intensity of labeling at the mononuclear cells between PGCL and CLNAGC and between PGCL and CLAGC. Based on these results, we can conclude the participation of GLUT-1, GLUT-3 and M-CSF in the pathogenesis of the lesions studied. The bigger immunostaining of these proteins in mononuclear cells show that these cells perform a higher metabolic activity and osteoclastogenic, especially in CLAGC. It was found that the mononuclear cells were more related to the pathogenesis of the studied lesions than properly the giants cells (AU).

An in vitro comparison of implant materials cell attachment, cytokine and osteocalcin production

Bone deposition, for any implant system, is the deciding factor for the success. The biochemical signals at the cellular level will help elucidate the direction of host response. In this report, intercellular messenger, cytokines, that are regulatory for osteoblast and osteoclast function, were measured. Production of osteocalcin, a marker for osteoblast maturation was also estimated. Human osteoblast-like cells from osteosarcoma cell line MG 63 were grown in wells in the presence of titanium (Ti), titanium alloy (Ti6A14V) and stainless steel implant materials incubated at 37 degrees C. Interleukin-1alpha (IL-1alpha), IL-6, IL-8, IL-11 and osteocalcin were quantitated using standard enzyme linked immunosorbant assay (ELISA) kits from the growth media extracted at specific intervals over the critical ten day period. In all dishes, cells were seen adhering to the base after 24 hours and to confluence at 96 hours. Both IL-1alpha and IL-11 were not produced in sufficient quantities to be measured in the assay (< pg/ml). Interleukin-6 production was significantly higher for stainless steel than for titanium and the alloy. There was a progressive rise in osteocalcin production for titanium contrasted to a basal rate for stainless steel and alloy. Interleukin-8 levels for all metals and controls increased markedly after two days implicating inherent cellular characteristics. A relatively high constant range for macrophage colony stimulating factor from the first day was seen for all metals, including the controls. In conclusion, it appears that titanium implants activate osteocalcin production while stainless steel activates IL-6.

Granulocyte-macrophage colony-stimulating factor does not increase the potency or efficacy of a foot-and-mouth disease virus subunit vaccine

A febre aftosa é uma das doenças mais temidas nos rebanhos em todo o mundo. A vacinação tem sido uma arma eficiente no controle da doença, no entanto há preocupações com as vacinas atualmente utilizadas incluindo a necessidade de instalações de alta segurança para a produção dessas vacinas e a falta de um teste de diagnóstico aprovado que faça distinção precisa entre animais vacinados dos infectados. Várias vacinas têm sido testadas contra a febre aftosa e uma dessas utiliza como vetor um vírus defectivo para replicação, derivado do adenovírus humano tipo 5 (Ad5), o qual contém as proteínas que compõe capsídeo do vírus da febre aftosa (P1-2A) e a protease 3C, protegeu completamente suínos contra o desafio de uma cepa homóloga (A12 e A24). Uma vacina com o Ad5-P1-2A+3C proveniente da cepa O1 Campos (Ad5-O1C), no entanto, somente induziu um baixo título de anticorpos neutralizantes específicos em testes de potência vacinal em suínos. O fator estimulante de colônias de granulócitos e macrófagos (GM-CSF) tem sido utilizado com sucesso na formulação de vacinas para estimular a resposta imune contra inúmeras doenças, incluindo HIV, Hepatite C e B. Na tentativa de melhorar a resposta imune específica contra a febre aftosa induzida pelo Ad5-O1C, suínos foram vacinados com Ad5-O1C juntamente com Ad5-GM-CSFporcino. Entretanto nas, condições utilizadas nesse teste, o GM-CSF suíno não melhorou a resposta imune do Ad5-O1C e adversamente afetou o nível de proteção de suínos desafiados com o vírus homólogo da febre aftosa.

Citocina. ¿La terapéutica del futuro? / Citokines. The therapy in the future?

Se definen qué son las citocinas y se describe el papel de la interleucina-1 como un ejemplo de las fuciones que desempeñan. Actualmente se han encontrado más de 40 citocinas que se han agrupado en 1) factores de crecimiento, 2) interleucinas, 3) interferones, 4) quimiocinas y 5) otras. Hoy en día, mediante transfección genética, se han preparado 11 citocinas para aplicación terapéutica; de éstas, la eritropoyetina, el factor estimulante de colonias granulocito-monocito y el factor estimulante de colonias granulocito-monocito y el factor estimulante de colonias de granulocitos se utilizan con buen éxito para estimular la producción de glóbulos rojos, granulocitos y de monocitos en algunas patologías con daño a la médula ósea. Después de varios años de aplicación clínica, el uso de los interferones alfa, beta y gamma se han visto limitados a infecciones como la hepatitis B o C y algunos tipos de cáncer como la leucemia de células peludas y la leucemia granolocítica crónica. Las otras citocinas, como las interleucinas 2, 3, 4, 6 y el factor estimulante de colonias de monocitos, empiezan a mostrar su utilidad terapéutica en algunos ensayos clínicos. Se espera que en el futuro algunas de ellas, solas o combinadas con otras u otros activadores inmunológicos curen enfermedades como el síndrome de inmunodeficiencia adquirida o el cáncer

Macrófagos en el asma / Macrophages in asthma

Cada vez existen más demostraciones del papel que desempeña la línea monocito-macrófago en la patogénesis del asma bronquial. Las células fagocíticas mononucleares, como los macrófagos alveolares, también pueden activarse durante procesos alérgicos. Los monocitos macrófagos son posibles inductores potentes de la inflamación; ello debido a que pueden secretar mediadores inflamatorios, entre los que se cuentan los gránulos preformados de péptidos, metabolitos de activación de oxidación, factor activador de plaquetas y metabolitos del ácido araquidónico. La identificación de IL-1 en el liquido del lavado bronquial de enfermos asmáticos, así como la identificación de IL.1 en el líquido de ronchas de sitios de prueba de alergenos cutáneos, apoya el concepto de activación mononuclear de células fagociticas en padecimientos alérgicos

Activation of macrophage functions by growth factor present in uterus and embryo extract of pregnant mouse

A pregnant mouse uterus and embryo extract (PMUE) that contains growth hematopoietic factor (M-CSF or CSF-1), was used to test its action on the phagocytic and digestive functions of macrophage. Macrophages incubated with and without PMUE for 24 hours previous to each experiment were compared. A good phagocytosis of Trypanosoma cruzi by macrophages incubated with PMUE, was observed on video microscopy. No phagocytic activity was observed in the macrophages deprived of PMUE 24 hours before. The studies of phagocytic and degradative behavior of macrophages by both soluble and particulated (S. aureus) complex 125I-antibodies showed that total binding of soluble ligands was almost double in the group of macrophages incubated with PMUE. Both the soluble and particulated ligands were digested more efficiently by the macrophages stimulated by PMUE. Counting the macrophages with trypan blue, an equal viability was found, of the cells incubated with and without PMUE. From the experimental data obtained, we may conclude that the hematopoietic growth factor present in PMUE is essential for phagocytic and degradative functions of macrophages.

Canales iónicos en células no excitables / Ion channels in non excitable cells

Several distinct types of voltage-gated and second-messenger-operated K+, Ca2+, Na+ and Cl- channels exist in electrically non excitable cells such as those of the hematopoietic lineage. In these cells ion channels mediate cellular functions involving intraceiiuiar biochemical responses, rather than rapid electrical signaling. The presence of the channels is required for several basic functions, such as activation, secretion of lymphokines, mitogenesis, the regulation of cell volume and the mechanisms of resistance to chemotherapeutic agents. Here IN we review the patch-clamp method for studying many characteristics of these ionic channels, particularly in blood cells.

Estudo sobre eventual influencia do estimulo para producao de macrofagos, por meio da proteose-peptona, na infeccao experimental de camundongos pelo Plasmodium berghei / Study about eventual influence of macrophage stimulation by means of proteose-peptone in experimental infection caused by Plasmodium berghei in mice

A disponibilidade de modelo experimental, baseado na infeccao de camundongos pelo Plasmodium berghei, permitiu a realizacao do presente estudo, no qual procurou-se verificar possivel diminuicao da parasitemia e consequente alteracao quanto a mortalidade como decorrencia de exacerbacao macrofagica inespecifica, apos estimulo por meio de proteose-peptona a 10 por cento. Os resultados nao demonstraram a cogitada acao sobre os protozoarios, mantendo-se o nivel de parasitemia e a mortalidade proximos ao verificado no grupo de animais infectados, sem participacao do indutor da producao de macrofagos. Assim, pelo menos de acordo com a metodologia empregada, nao ocorreu a contencao da protozoose, sugerida por informacoes registradas em publicacoes anteriores.

Effects of deltamethrin on the growth and differentiation of bone marrow hematopoietic stem cells

1. The effects of deltamethrin on mouse bone marrow and spleen progenitor cell responsiveness to granulocyte and macrophage colony-stimulating factors (CSFs) were evaluated. 2. Deltamethrin (1-5 mg/kg) was administered four times subcutaneously on alternate days for one week to male BALB/c mice, 5-8 weeks old (N = 6 mice/group), raised under pathogen-free conditions and maintained in conventional animal rooms for four weeks before use. Soft agar colony formation (CFU-C), marrow and spleen cell counts as well as body, spleen and thymus weights were determined. 3. Although treatment with the lowest dose (1 mg kg-1 48 h-1) produced no significant effect on CFU-C, the administration of 3 and 5 mg kg-1 48 h-1 caused a more than two-fold increase in the formation of granulocyte and macrophage colonies in the marrow, but not in the spleen (control value = 100.5 +/- 12 for N = 6). Colony numbers returned to normal values within five days after the end of deltamethrin administration. 4. No changes were observed in the total (range: 1-3 x 10(8) per spleen) and differential marrow and spleen cell counts, nor was there any alteration in spleen weight. However, treatment with the three doses resulted in a dramatic reduction in thymus weight. 5. These effects were not due to the liberation of endotoxin, because if endotoxin had been present it would have been < 0.060 ng/ml, a concentration that would not have a biological effect. 6. In vitro addition of 0.10 to 10 microM deltamethrin to marrow cell cultures obtained from untreated mice did not induce any response. 7. These data indicate that the CSF-driven granulocyte and macrophage development provides a useful model for the study of the effects of toxicants on myelopoiesis

Regulación de la hematopoyesis / Hematopoyesis regulation

Las señales positivas y negativas juegan un papel trascendental en la regulación del sistema hematopoyético. En los últimos 30 años se purificaron más de 20 moléculas (glicoproteínas) con actividad biológica sobre las células progenitoras del sistema hematopoyético y sobre las células maduras circulantes. Los factores de crecimiento hematopoyéticos son las más conocidas de estas biomoléculas (citocinas como los factores estimulantes de colonias y las interleucinas), las cuales son capaces de estimular a las células de la médula ósea para producir descendencia madura. Hasta el presente se ha podido determianr no sólo la secuencia de la mayoría de estas glicoproteínas y los gene que las codifican, sino también caracterizar a las células blanco de cada uno de los factores y a sus receptores celulares. Los estudios de la interacción entre las células progenitoras hematopoyéticas y los factores que estimulan y/o inhiben su proliferación, han demostrado ser muy útiles en el desarrollo de terapias clínicas y podrían ser herramientas importantes en el análisis de la patogenia de muchas enfermedades hematológicas. Sin embargo, los mecanismos subyacentes en la producción de factores estimulantes o inhibidores y la proliferación de las células progenitoras hematopoyéticas continúan siendo escasamente conocidos

Sistema monocitico-macrofagico y Mycobacterium leprae: su importancia en la interpretacion patogenica de las distintas formas clinico-patologicas de la lepra y de las reacciones leprosas / Monocyte-macrophage system and Mycobacterium leprae: its importance in the patogen interpretation of the different clinic-patology forms of the leprosy and the leprous reactions

Se realiza una revision de la patogenia de la lepra (Hanseniasis), en sus vinculaciones con el "sistema monotitico-macrofagico" y se efectua el estudio histopatologico, histoquimico (lipidico) y con microscopia electronica de cuatro enfermos de lepra, dos lepromatosos (sin y con eritema nudoso) y otros dos dimorfos ("Borderline"). Se concluye que: 1) La lepra presenta sus manifestaciones clinico-patologicas mas ostensibles, en vinculacion con lesiones del "sistema manocitico-macrofagico". 2) Existem distintos comportamientos de los macrofagos frente al Mycobacterium leprae: a) en personas Mitsuda-positivos de puede demostrar el predominio de "macrofagos lisadores del M. leprae" con muerte y desaparicion de los bacilos; b)en individuos Mitsuda-negativos predominan los "macrofagos no-lisadores del M. leprae", que tambiem producen al muerte de los bacilos, pero con persistencia de la "envoltura lipidica bacilar", que se deposita en las vacuolas de los virchowianos: y c) en enfermos Mitsuda-negativos, luego de fromado el "granuloma virchowiano", se desarrollan los "macrofagos lisadores de las celulas de Virchow" que permitiram el recambio celular de los virchowianos. 3) los "macrofagos lisadores del M. leprae" obtienen informacion antigenica del "bacilo aislado" que puedem transmitir al sistema de inmunidad celular, cuyos linfocitos T activados generan los "granulomas epitelioides con celulas de Langhans" (granulomas tuberculoides), de la Lepra tuberculoide. 4) Los "macrofagos no-lisadores del M. leprae" no obtendrian infromacion antigenica adecuada y no tendrian capacidad para estimular ningun sistema inmunologico (celular, o humoral), originando los "granulomas virchowianos" de la lepra lepromatosa. 5) Luego del envejecimiento y muerte de los virchowianos, los "macrofagos lisadores de las celulas de Virchow" obtendrian informacion antigenica de los "bacilos degenerados asociados a lipidos y glucoproteinas citoplasmaticas", con capacidad de estimulacion del sistema inmunologico humoral cuyos linfocitos B activados generarian anticuerpos complejos: a) contra bacilos ubicados en los virchowianos en la reaccion leprosa tipo 2 (eritema nudoso leprotico); b) contra la pared de vasos en la reaccion leprosa tipo 3 (fenomeno de Lucio); y c) contra tejidos del huesped (enfermedad autoagresiva hanseniana-Azulay)