Granulocyte-macrophage colony-stimulating factor elicits bone marrow-derived cells that promote efficient colonic mucosal healing.

BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF) therapy is effective in treating some Crohn's disease (CD) patients and protects mice from colitis induced by dextran sulfate sodium (DSS) administration. However, its mechanisms of action remain elusive. We hypothesized that GM-CSF affects intestinal mucosal repair. METHODS: DSS colitic mice were treated with daily pegylated GM-CSF or saline and clinical, histological, and inflammatory parameters were kinetically evaluated. Further, the role of bone marrow-derived cells in the impact of GM-CSF therapy on DSS colitis was addressed using cell transfers. RESULTS: GM-CSF therapy reduced clinical signs of colitis and the release of inflammatory mediators. GM-CSF therapy improved mucosal repair, with faster ulcer reepithelialization, accelerated hyperproliferative response of epithelial cells in ulcer-adjacent crypts, and lower colonoscopic ulceration scores in GM-CSF-administered mice relative to untreated mice. We observed that GM-CSF-induced promotion of mucosal repair is timely associated with a reduction in neutrophil numbers and increased accumulation of CD11b(+) monocytic cells in colon tissues. Importantly, transfer of splenic GM-CSF-induced CD11b(+) myeloid cells into DSS-exposed mice improved colitis, and lethally irradiated GM-CSF receptor-deficient mice reconstituted with wildtype bone marrow cells were protected from DSS-induced colitis upon GM-CSF therapy. Lastly, GM-CSF-induced CD11b(+) myeloid cells were shown to promote in vitro wound repair. CONCLUSIONS: Our study shows that GM-CSF-dependent stimulation of bone marrow-derived cells during DSS-induced colitis accelerates colonic tissue repair. These data provide a putative mechanism for the observed beneficial effects of GM-CSF therapy in Crohn's disease.

The roles of granulocyte-macrophage colony-stimulating factor and interleukin 3 in stromal cell-mediated hemopoiesis in vivo.

Adherent cells from murine long-term marrow cultures (LTMC) were examined for presence of mRNA for granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (Il-3). Six hours after medium replacement, GM-CSF mRNA was detected but was no longer detectable 24 h after feeding; Il-3 mRNA was not detected at any time. Neutralizing antibodies against these factors had no effect on hemopoiesis. Exogenous Il-3 increased cell production, notably mature erythroid progenitors, whereas GM-CSF had little long-term effect even at high concentrations. Furthermore, GM-CSF appeared to be specifically removed from the medium, whereas virtually all of the Il-3 could be recovered under identical incubation conditions. These results show that Il-3 is not required for maintaining long-term hemopoiesis in vitro, whereas the precise role of GM-CSF in this system remains unclear.

Atopic dermatitis-like disease and associated lethal myeloproliferative disorder arise from loss of Notch signaling in the murine skin.

BACKGROUND: The Notch pathway is essential for proper epidermal differentiation during embryonic skin development. Moreover, skin specific loss of Notch signaling in the embryo results in skin barrier defects accompanied by a B-lymphoproliferative disease. However, much less is known about the consequences of loss of Notch signaling after birth. METHODOLOGY AND PRINCIPAL FINDINGS: To study the function of Notch signaling in the skin of adult mice, we made use of a series of conditional gene targeted mice that allow inactivation of several components of the Notch signaling pathway specifically in the skin. We demonstrate that skin-specific inactivation of Notch1 and Notch2 simultaneously, or RBP-J, induces the development of a severe form of atopic dermatitis (AD), characterized by acanthosis, spongiosis and hyperkeratosis, as well as a massive dermal infiltration of eosinophils and mast cells. Likewise, patients suffering from AD, but not psoriasis or lichen planus, have a marked reduction of Notch receptor expression in the skin. Loss of Notch in keratinocytes induces the production of thymic stromal lymphopoietin (TSLP), a cytokine deeply implicated in the pathogenesis of AD. The AD-like associated inflammation is accompanied by a myeloproliferative disorder (MPD) characterized by an increase in immature myeloid populations in the bone marrow and spleen. Transplantation studies revealed that the MPD is cell non-autonomous and caused by dramatic microenvironmental alterations. Genetic studies demontrated that G-CSF mediates the MPD as well as changes in the bone marrow microenvironment leading to osteopenia. SIGNIFICANCE: Our data demonstrate a critical role for Notch in repressing TSLP production in keratinocytes, thereby maintaining integrity of the skin and the hematopoietic system.

Granulocyte macrophage colony stimulating factor improves mucosal repair in a mouse model of acute colitis (P278)

Background and Aims: Granulocyte-macrophage colonystimulating factor (GM-CSF), a cytokine modulating the number and function of innate immune cells, has been shown to provide symptomatic benefit in some patients with Crohn's disease (CD). Since, it becomes widely appreciated that a timely and spatially regulated action of innate immune cells is critical for tissue regeneration, we tested whether GM-CSF therapy may favours intestinal mucosal repair in the acute mouse model of dextran sulfate sodium (DSS)-induced colitis. Methods: Mice treated with GM-CSF or saline were exposed for 7 days to DSS to induce colitis. On day 5, 7 and 10, mice were subjected to colonoscopy or sacrificed for evaluation of inflammatory reaction and mucosal healing. Results: GM-CSF therapy prevented body weight loss, diarrhea, dampened inflammatory reactions and ameliorated mucosal damages. Mucosal repair improvement in GM-CSF-treated mice was observed from day 7 on both by colonoscopy (ulceration score 1.2}0.3 (GM-CSF-treated) vs 3.1}0.5 (untreated), p = 0.01) and histological analysis (percentage of reepithelialized ulcers 55%}4% (GM-CSF-treated) vs 18%}13% (untreated), p = 0.01). GM-CSF therapy can still improve the colitis when hematopoietic, but not non-hematopoietic cells, are responsive to GM-CSF, as shown in WT→GM-CSFRKO chimeras. Lastly, we observed that GM-CSF-induced promotion of wound healing is associated with a modification of the cellular composition of DSS-induced colonic inflammatory infiltrate, characterized by the reduction of neutrophil numbers and early accumulation of CD11b+Gr1lo myeloid cells. Conclusion: Our study shows that GM-CSF therapy accelerates the complex program leading to tissue repair during acute colitis and suggests that GM-CSF promotion of mucosal repair might contribute to the symptomatic benefits of GM-CSF therapy observed in some CD patients.

Autologous Stem Cell Transplantation In Multiple Myeloma

Background. Multiple myeloma (MM) is the second most common hematologic malignancy after lymphomas In Finland: the annual incidence of MM is approximately 200. For three decades the median survival remained at 3 to 4 years from diagnosis until high-dose melphalan treatment supported by autologous stem cell transplantation (ASCT) became the standard of care for newly diagnosed MM since the mid 1990’s and the median survival increased to 5 – 6 years. This study focuses on three important aspects of ASCT, namely 1) stem cell mobilization, 2) single vs. double ASCT as initial treatment, and 3) the role of minimal residual disease (MRD) for longterm outcome. Aim. The aim of this series of studies was to evaluate the outcomes of MM patients and the ASCT procedure at the Turku University Central Hospital, Finland. First, we tried to identify which factors predict unsuccessful mobilization of autologous stem cells. Second, we compared the use of short-acting granulocyte-colony stimulating factor (GCSF) with long-acting G-CSF as mobilization agents. Third, one and two successive ASCTs were compared in 100 patients with MM. Fourth, for patients in complete response (CR) after stem cell transplantation (SCT), patient-specific probes for quantitative allele-specific oligonucleotide polymerase-chain reaction (qASO-PCR) measurements were designed to evaluate MRD and its importance for long-term outcome. Results. The quantity of previous chemotherapy and previous interferon use were significant pre-mobilization factors that predicted mobilization failure, together with some factors related to mobilization therapy itself, such as duration and degree of cytopenias and occurrence of sepsis. Short-acting and long-acting G-CSF combined with chemotherapy were comparable as stem cells mobilizers. The progression free (PFS) and overall survival (OS) tended to be longer after double ASCT than after single ASCT with a median follow-up time of 4 years, but this difference disappeared as the follow-up time increased. qASO-PCR was a good and sensitive divider of the CR patients into two prognostic groups: MRD low/negative (≤ 0.01%) and MRD high (>0.01%) groups with a significant difference in PFS and suggestively also in OS. Conclusions. When the factors prediciting a poor outcome of stem cell mobilization prevail, it is possible to identify those patients who need specific efforts to maximize the mobilization efficacy. Long-acting pegfilgrastim is a practical and effective alternative to short-acting filgrastim for mobilization therapy. There is no need to perform double ASCT on all eligible patients. MRD assessment with qASO-PCR is a sensitive method for evaluation of the depth of the CR response and can be used to predict long-term outcome after ACST.

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

Foot-and-mouth disease (FMD) is one of the most feared diseases of livestock worldwide. Vaccination has been a very effective weapon in controlling the disease, however a number of concerns with the current vaccine including the inability of approved diagnostic tests to reliably distinguish vaccinated from infected animals and the need for high containment facilities for vaccine production, have limited its use during outbreaks in countries previously free of the disease. A number of FMD vaccine candidates have been tested and a replication-defective human adenovirus type 5 (Ad5) vector containing the FMDV capsid (P1-2A) and 3C protease coding regions has been shown to completely protect pigs against challenge with the homologous virus (FMDV A12 and A24). An Ad5-P1-2A+3C vaccine for FMDV O1 Campos (Ad5-O1C), however, only induced a low FMDV-specific neutralizing antibody response in swine potency tests. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been successfully used to stimulate the immune response in vaccine formulations against a number of diseases, including HIV, hepatitis C and B. To attempt to improve the FMDV-specific immune response induced by Ad5-O1C, we inoculated swine with Ad5-O1C and an Ad5 vector containing the gene for porcine GM-CSF (pGM-CSF). However, in the conditions used in this trial, pGM-CSF did not improve the immune response to Ad5-O1C and adversely affected the level of protection of swine challenged with homologous FMDV.

Contribution des protéines issues du liquide synovial dans la protection et la survie des PMN humains : chimioprotection : étude comparative des mécanismes d’action impliqués par rapport au GM-CSF

Les polymorphonucléaires neutrophiles (PMNs) représentent une arme primordiale dans la défense contre divers agents pathogènes; notamment les bactéries, les champignons, les cellules tumorales de même que les cellules infectées par des virus. Cependant, certaines pathologies reliées à l’inflammation chronique soulèvent l’implication des neutrophiles notamment dans l’arthrite rhumatoïde. La réponse inflammatoire persistante générée par l’activation et la survie des neutrophiles engendre une destruction des tissus environnants suite à la sécrétion non contrôlée de leurs produits cytotoxiques. Même si l’activation chronique des neutrophiles est néfaste dans plusieurs pathologies, elle pourrait s’avérer un bon outil en cas de neutropénie, comme c’est souvent le cas les patients ayant reçu des traitements de chimiothérapie. Ce projet fait suite aux travaux doctoraux de Lagraoui (1999). Il vise à identifier le(s) facteur(s) du liquide synovial qui augmente la survie des neutrophiles ainsi que le mécanisme d’action impliqué dans ce processus. Similairement au facteur semi-pur isolés par Lagraoui (1999), le milieu conditionné concentré (MCC) augmente la survie des PMNs de 75% (39% ± 9.5 vs 68% ± 2.5, p<0.01). Suivant le séquençage du MCC parallèlement au facteur semi-pur actif, deux protéines ont été identifiées à la fois dans le MCC et dans le facteur semi-pur soient : l’albumine et la fétuine. Notre projet vise donc à comparer les effets de l’albumine et de la fétuine à ceux du GM-CSF dans l’optique d’une thérapie alternative au GM-CSF en tant qu’adjuvant de chimiothérapie. La présence d’albumine, de fétuine ou de GM-CSF chez les PMNs incubés 24 heures avec la Mutamycin® induit une diminution du nombre de cellules en apoptose par rapport à la Mutamycin® (Ctrl : 43% ± 10; A : 74% ± 3; F : (82% ± 6 et GM : 74% ± 7; p<0.01). L’effet de l’albumine dépend de la voie de la kinase PI3 mais également celle la kinase ERK, alors que celle de la fétuine dépend de la kinase PI3. Similairement l’EPO, l’albumine et la fétuine supporte la différentiation des HSCs en précurseurs érythrocytaires de type BFU-E. Dans un modèle murin de chiomioprotection, l’albumine augmente la concentration cellulaire rapport au groupe contrôle des leukocytes de la rate (66 ±8 x106c/ml vs 81 ±16 x106c/ml) et du sang (3.6 ±0.4 x106c/ml vs 5.7 ±2.3 x106c/ml). Donc, in vitro, l’albumine et la fétuine sont comparables au GM-CSF au niveau fonctionalité et mécansimes d’action. Cependant, vu leur manque de spécificité, l’application thérapeutique en tant qu’adjuvant de chiomiothérapie de l’albumine et la fétuine est peu prometteuse. Par contre, les maladies dégénératives et les évènements ischémiques pourraient s’avérer de bonnes cibles thérapeutiques, principalement pour l’albumine.

A Factor analysis of hidrochemical composition of Llobregat river basin

Hydrogeological research usually includes some statistical studies devised to elucidate mean background state, characterise relationships among different hydrochemical parameters, and show the influence of human activities. These goals are achieved either by means of a statistical approach or by mixing models between end-members. Compositional data analysis has proved to be effective with the first approach, but there is no commonly accepted solution to the end-member problem in a compositional framework. We present here a possible solution based on factor analysis of compositions illustrated with a case study. We find two factors on the compositional bi-plot fitting two non-centered orthogonal axes to the most representative variables. Each one of these axes defines a subcomposition, grouping those variables that lay nearest to it. With each subcomposition a log-contrast is computed and rewritten as an equilibrium equation. These two factors can be interpreted as the isometric log-ratio coordinates (ilr) of three hidden components, that can be plotted in a ternary diagram. These hidden components might be interpreted as end-members. We have analysed 14 molarities in 31 sampling stations all along the Llobregat River and its tributaries, with a monthly measure during two years. We have obtained a bi-plot with a 57% of explained total variance, from which we have extracted two factors: factor G, reflecting geological background enhanced by potash mining; and factor A, essentially controlled by urban and/or farming wastewater. Graphical representation of these two factors allows us to identify three extreme samples, corresponding to pristine waters, potash mining influence and urban sewage influence. To confirm this, we have available analysis of diffused and widespread point sources identified in the area: springs, potash mining lixiviates, sewage, and fertilisers. Each one of these sources shows a clear link with one of the extreme samples, except fertilisers due to the heterogeneity of their composition. This approach is a useful tool to distinguish end-members, and characterise them, an issue generally difficult to solve. It is worth note that the end-member composition cannot be fully estimated but only characterised through log-ratio relationships among components. Moreover, the influence of each endmember in a given sample must be evaluated in relative terms of the other samples. These limitations are intrinsic to the relative nature of compositional data

High frequency of immature dendritic cells and altered in situ production of interleukin-4 and tumor necrosis factor-alpha in lung cancer

Introduction Antigen-presenting cells, like dendritic cells (DCs) and macrophages, play a significant role in the induction of an immune response and an imbalance in the proportion of macrophages, immature and mature DCs within the tumor could affect significantly the immune response to cancer. DCs and macrophages can differentiate from monocytes, depending on the milieu, where cytokines, like interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) induce DC differentiation and tumor necrosis factor (TNF)-alpha induce DC maturation. Thus, the aim of this work was to analyze by immunohistochemistry the presence of DCs (S100+ or CD1a+), macrophages (CD68+), IL-4 and TNF-alpha within the microenvironment of primary lung carcinomas. Results Higher frequencies of both immature DCs and macrophages were detected in the tumor-affected lung, when compared to the non-affected lung. Also, TNF-alpha-positive cells were more frequent, while IL-4-positive cells were less frequent in neoplastic tissues. This decreased frequency of mature DCs within the tumor was further confirmed by the lower frequency of CD14-CD80+ cells in cell suspensions obtained from the same lung tissues analyzed by flow cytometry. Conclusion These data are discussed and interpreted as the result of an environment that does not oppose monocyte differentiation into DCs, but that could impair DC maturation, thus affecting the induction of effective immune responses against the tumor.

Ric-8B interacts with G alpha olf and G gamma 13 and co-localizes with G alpha olf, G beta 1 and G gamma 13 in the cilia of olfactory sensory neurons

Olfactory sensory neurons are able to detect odorants with high sensitivity and specificity. We have demonstrated that Ric-8B, a guanine nucleotide exchange factor (GEF), interacts with G alpha olf and enhances odorant receptor signaling. Here we show that Ric-8B also interacts with G gamma 13, a divergent member of the G gamma subunit family which has been implicated in taste signal transduction, and is abundantly expressed in the cilia of olfactory sensory neurons. We show that G beta 1 is the predominant GP subunit expressed in the olfactory sensory neurons. Ric-8B and G beta 1, like G alpha olf and G gamma 13, are enriched in the cilia of olfactory sensory neurons. We also show that Ric-8B interacts with G alpha olf in a nucleotide dependent manner, consistent with the role as a GEF. Our results constitute the first example of a GEF protein that interacts with two different olfactory G protein subunits and further implicate Ric-8B as a regulator of odorant signal transduction. (C) 2008 Elsevier Inc. All rights reserved.

Granulocyte macrophage colony-stimulating factor enhances the modulatory effect of cytokines on monocyte-derived multinucleated giant cell formation and fungicidal activity against Paracoccidioides brasiliensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Uso do estimulante de colônia de granulócitos nas neutropenias em cães e gatos

As neutropenias persistentes podem ser decorrentes de alterações na granulopoiese, causadas por efeitos supressivos ou tóxicos à medula óssea, predispõem o paciente a infecções comprometendo sua sobrevida. As neutropenias intensas decorrentes de toxicidade por quimioterápicos podem requerer a suspensão temporária ou permanente do medicamento, podendo gerar resistência das células neoplásicas ao tratamento. O uso de fatores de crescimento hematopoiético recombinantes em animais tem aumentado muito nos últimos anos, devido a sua crescente disponibilidade na medicina humana. O fator estimulante de colônia para granulócitos recombinante humano (rhG-CSF) age aumentando o número de neutrófilos circulantes e possui grande potencial para amenizar ou reverter quadros de neutropenia associada a condições de mielotoxicidade e mielosupressão em cães e gatos.

Silibinin attenuates oxidative metabolism and cytokine production by monocytes from preeclamptic women.

Silibinin is a polyphenolic plant flavonoid with anti-inflammatory properties. The present study investigated the effect of silibinin on oxidative metabolism and cytokine production - tumor necrosis factor-alpha (TNF-α), interleukin (IL)12, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, IL-10, and transforming growth factor beta (TGF-β1) - by peripheral blood monocytes (PBM) from preeclamptic pregnant women. It is a case-controlled study involving women with preeclampsia (PE, n = 30) compared with normotensive pregnant (NT, n = 30) and with non-pregnant (NP, n = 30) women. Monocytes were obtained and cultured with or without silibinin (5 μM or 50 μM) for 18 h. Superoxide anion (O2-) and hydrogen peroxide (H2O2) release were determined by specific assays, and cytokine levels were determined by immunoenzymatic assays (ELISA). Monocytes from preeclamptic women cultured without stimulus released higher levels of O22, H2O2 and TNF-α, and lower levels of IL-10 and TGF-β1 than did monocytes from NT and NP women. Treatment in vitro with silibinin significantly inhibited spontaneous O2- and H2O2 release and TNF-α production by monocytes from preeclamptic women. The main effect of silibinin was obtained at 50 μM concentration. Thus, silibinin exerts anti-oxidative and anti-inflammatory effects on monocytes from preeclamptic pregnant women by inhibiting the in vitro endogenous release of reactive oxygen species and TNF-α production.

Efeito da silibinina sobre o metabolismo oxidativo e produção de citocinas por monócitos em gestantes portadores de pré-eclâmpsia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expressão de CD45+ na medula óssea e sangue periférico de cães com linfoma tratados com alta dose de ciclofosfamida suportada por transplante autólogo de medula óssea

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)