Granulocyte-macrophage Colony-stimulating Factor Antibody Research Articles

Repeated inhalation of GM-CSF by nonhuman primates induces bronchus-associated lymphoid tissue along the lower respiratory tract

BackgroundRepeated inhalation of granulocyte-macrophage colony-stimulating factor (GM-CSF) was recently approved in Japan as a treatment for autoimmune pulmonary alveolar proteinosis. However, the detailed physiological and pathological effects of repeated inhalation in the long term, especially at increasing doses, remain unclear.MethodsIn this chronic safety study, we administered 24 cynomolgus monkeys (Macaca fascicularis) aged 2–3 years with aerosolized sargramostim (a yeast-derived recombinant human GM-CSF [rhGM-CSF]) biweekly for 26 weeks across four dosing groups (0, 5, 100, and 500 µg/kg/day). We measured the serum GM-CSF antibody (GM-Ab) concentration by an ELISA and assessed the neutralizing capacity of GM-Ab using the GM-CSF-dependent cell line TF-1. We subjected lung tissue samples taken from all monkeys at 27 weeks to histopathological assessment using a sargramostim-specific monoclonal antibody to detect localization of residual sargramostim.ResultsAll the animals maintained good body condition and showed steady weight gain throughout the study. The pathological analyses of the lung revealed the formation of induced bronchus-associated lymphoid tissue (iBALT) in the lower respiratory tract, even at the clinical dose of 5 µg/kg/day. There was a relationship between the number or size of BALT and sargramostim dose or the serum GM-Ab levels. Immunohistochemical analyses revealed GM-Ab–producing cells in the follicular region of iBALT, with residual sargramostim in the follicles. Leucocyte counts were inversely correlated with GM-Ab levels in the high-dose groups. Additionally, serum GM-Ab from the treated animals significantly suppressed the alveolar macrophage proliferation activity of both Cynomolgus recombinant and rhGM-CSF in vitro.ConclusionLong-term repeated inhalation of sargramostim led to iBALT formation in the lower respiratory tract, even at the clinical dose of 5 µg/kg/day, with the extent of iBALT formation increasing in a dose-dependent manner. Inhaled sargramostim was localized to the follicular region of iBALT nodules, which may induce the production of GM-Ab.

De novo granulocyte–macrophage colony-stimulating factor antibody production has been linked to acute graft-versus-host disease following hematopoietic stem cell transplantation from an HLA-matched, unrelated donor

The role of non-HLA antibodies in hematopoietic stem cell transplantation (HSCT) and acute graft-versus-host disease (aGVHD) is not established. Serum samples collected from 58 adult patients before and after HSCT were examined for non-HLA antibodies. Following HSCT, 47 out of 58 patients (81.0 %) had various antibody patterns, with 23 of them (39.7 %) producing denovo antibodies. The most prevalent antibodies were directed against granulocyte–macrophage colony-stimulating factor (GM-CSF). The Fisher exact test revealed a statistically significant correlation between the incidence of acute graft-versus-host disease and denovo production of GM-CSF antibodies in patients fully HLA-matched with their donors (p = 0.001). There were no cases of denovo GM-CSF antibody production seen in non-permissively HLA-mismatched patients. Consequently, we hypothesize that the development of aGVHD after HLA-matched HSCT may differ from that following HLA-mismatched HSCT.

European Respiratory Society guidelines for the diagnosis and management of pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is a rare syndrome caused by several distinct diseases leading to progressive dyspnoea, hypoxaemia, risk of respiratory failure and early death due to accumulation of proteinaceous material in the lungs. Diagnostic strategies may include computed tomography (CT) of the lungs, bronchoalveolar lavage (BAL), evaluation of antibodies against granulocyte-macrophage colony-stimulating factor (GM-CSF), genetic testing and, eventually, lung biopsy. The management options are focused on removing the proteinaceous material by whole lung lavage (WLL), augmentation therapy with GM-CSF, rituximab, plasmapheresis and lung transplantation. The presented diagnostic and management guidelines aim to provide guidance to physicians managing patients with PAP. A European Respiratory Society Task Force composed of clinicians, methodologists and patients with experience in PAP developed recommendations in accordance with the ERS Handbook for Clinical Practice Guidelines and the GRADE (Grading of Recommendations, Assessment, Development and Evaluations) approach. This included a systematic review of the literature and application of the GRADE approach to assess the certainty of evidence and strength of recommendations. The Task Force formulated five PICO (Patients, Intervention, Comparison, Outcomes) questions and two narrative questions to develop specific evidence-based recommendations. The Task Force developed recommendations for the five PICO questions. These included management of PAP with WLL, GM-CSF augmentation therapy, rituximab, plasmapheresis and lung transplantation. Also, the Task Force made recommendations regarding the use of GM-CSF antibody testing, diagnostic BAL and biopsy based on the narrative questions. In addition to the recommendations, the Task Force provided information on the hierarchy of diagnostic interventions and therapy. The diagnosis of PAP is based on CT and BAL cytology or lung histology, whereas the diagnosis of specific PAP-causing diseases requires GM-CSF antibody testing or genetic analysis. There are several therapies including WLL and augmentation therapy with GM-CSF available to treat PAP, but supporting evidence is still limited.

ЛЕГЕНЕВИЙ АЛЬВЕОЛЯРНИЙ ПРОТЕЇНОЗ: ВИПАДОК СУБТОТАЛЬНОГО УРАЖЕННЯ ПАРЕНХІМИ ЛЕГЕНЬ)

Pulmonary alveolar proteinosis (PAP) is a rare pulmonary disease, characterized by the intraalveolar accumulation of surfactant-like lipoprotein substance. There are three forms of the disease: autoimmune (formerly known as idiopathic), secondary and congenital PAP. The disturbances of surfactant metabolism (protein-lipoid complex secreted by type II pneumocytes) play the crucial role in the development of the disease. Surfactant decreases a surface tension of alveoli and prevents its collapse at the end of the expiration and takes part in protection against infections as well. Surfactant is inactivated through the transformation into the surface-inactive aggregates, which are mainly absorbed by type II pneumocytes. The rest of surfactant are catabolized by alveolar macrophages (AM). This process is regulated by cytokines, especially granulocyte-macrophage colony-stimulating factor (GM-CSF) being the most important of them. Disturbances of interrelation between GM-CSF and cellular receptors blocks the signals to effector cells for surfactant deactivation. This is resulted in excessive accumulation of surfactant in alveoli. General lavage of the lung is a standard method of symptomatic management of PAP. Contemporary pathogenesis treatment uses two approaches — increasing the clearance of surfactant by means of exogenous GM-CSF administration and decreasing the level of GM-CSF antibodies using the rituximab and plasmapheresis. Due to the use of broncho-alveolar lavage (BAL) 5-year survival rate of th patients reaches 95 %. The mortality depends on the area of lung lesion and reversely correlated with the timely diagnosis and treatment. We report the case of subtotal lung parenchyma lesion associated with severe respiratory failure in 46 years old patient. Three series of BAL (5 procedures in each with 1-2 months interval) were performed using the limited volume of lavage fluid due to the severity of the patient’s condition. The results was an improvement of clinical condition, resolution of respiratory failure, significant reduction of the size of the lesions, improved lung transparency on CT scan, restoration of lung diffusion and total lung capacity. Conclusion. Therapeutic BAL is an effective method of PAP management. The outcome of clinical case presented and our many years experience confirm not only the effectiveness of conventional BAL with reduced lavage volume application but also a safety of fiberoptic bronchoscopy, especially in patients with subtotal involvement of lung parenchyma. Key words: pulmonary alveolar proteinosis, subtotal pulmonary parenchyma involvement, pulmonary bronchoalveolar lavage, efficiency.

Efficacy and safety of granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies in COVID-19 patients: a meta-analysis.

This study aims to determine the efficacy and safety of granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies in COVID-19 patients. We searched Cochrane Library, PubMed, Embase, and ClinicalTrials.gov databases until July 27, 2022. Both randomized control trials (RCTs) and cohort studies were included and analyzed separately. The outcomes included mortality, incidence of invasive mechanical ventilation (IMV), ventilation improvement rate (need oxygen therapy to without oxygen therapy), secondary infection, and adverse events (AEs). The odds ratio (OR) with a 95% confidence interval (CI) was calculated by a random-effects meta-analysis model. Five RCTs and 2 cohort studies with 1726 COVID-19 patients were recruited (n = 866 in the GM-CSF antibody group and n = 891 in the control group). GM-CSF antibodies treatment reduced the incidence of IMV, which was supported by two cohort studies (OR 0.16; 95% CI 0.03, 0.74) and three RCTs (OR 0.62; 95% CI 0.41, 0.94). GM-CSF antibodies resulted in slight but not significant reductions in mortality (based on two cohort studies and five RCTs) and ventilation improvement (based on one cohort study and two RCTs). The sensitive analysis further showed the results of mortality and ventilation improvement rate became statistically significant when one included study was removed. Besides, GM-CSF antibodies did not increase the risks of the second infection (based on one cohort study and five RCTs) and AEs (based on five RCTs). GM-CSF antibody treatments may be an efficacious and well-tolerant way for the treatment of COVID-19. Further clinical evidence is still warranted.

Secondary Pulmonary Alveolar Proteinosis Due to Sirolimus

Introduction Pulmonary alveolar proteinosis (PAP) is a rare, progressive lung disease characterized by the accumulation of lipoproteinaceous material in alveoli due to impaired clearance by alveolar macrophages. Although most cases are due to the disruption of granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling, secondary PAP from immunodeficiency is described in solid organ recipients. We present a treatment conundrum in a heart transplant (OHT) patient with a history of graft rejection who develops secondary PAP while on sirolimus. Case Report A 63-year-old male 6 years post re-do OHT, initially transplanted for ischemic cardiomyopathy then re-do for antibody mediated rejection presented with new hypoxia requiring 2L O2. His immunosuppression regimen included myfortic, prednisone, sirolimus, and tacrolimus. A CT scan demonstrated central and basilar ground glass opacities with “crazy paving”. He underwent a bronchoscopy with bronchoalveolar lavage (BAL) showing 90% macrophages, negative cultures, and cytology with hemosiderin pigment and proteinaceous material. He was empirically placed on levofloxacin and weaned to room air. At 5-month follow-up a repeat CT re-demonstrated similar findings. Repeat BAL, performed for concern for PAP, was Periodic acid-Schiff (PAS) stain positive for proteinaceous material. Serum GM-CSF antibodies were absent and the patient was diagnosed with secondary PAP due to sirolimus. Given the patient's prior history of graft failure, his sirolimus dose was unchanged and he was treated with inhaled sargramostim. Summary PAP is a rare disease caused by disorders in surfactant homeostasis further divided into three distinct etiologies: autoimmune (90%), secondary (5-10%), and congenital (2%). Secondary PAP is due to functional impairment in alveolar macrophages from immunodeficiency and is well-described in kidney and lung transplant recipients. To our knowledge, this is the first case described in a heart transplant recipient. The prognosis of transplant-associated secondary PAP is difficult to establish due to the limited number of published cases, though resolution is typically observed 2-4 months after removal of the offending agent. We present a case treated with GM-CSF due to difficulties reducing immunosuppression. Pulmonary alveolar proteinosis (PAP) is a rare, progressive lung disease characterized by the accumulation of lipoproteinaceous material in alveoli due to impaired clearance by alveolar macrophages. Although most cases are due to the disruption of granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling, secondary PAP from immunodeficiency is described in solid organ recipients. We present a treatment conundrum in a heart transplant (OHT) patient with a history of graft rejection who develops secondary PAP while on sirolimus. A 63-year-old male 6 years post re-do OHT, initially transplanted for ischemic cardiomyopathy then re-do for antibody mediated rejection presented with new hypoxia requiring 2L O2. His immunosuppression regimen included myfortic, prednisone, sirolimus, and tacrolimus. A CT scan demonstrated central and basilar ground glass opacities with “crazy paving”. He underwent a bronchoscopy with bronchoalveolar lavage (BAL) showing 90% macrophages, negative cultures, and cytology with hemosiderin pigment and proteinaceous material. He was empirically placed on levofloxacin and weaned to room air. At 5-month follow-up a repeat CT re-demonstrated similar findings. Repeat BAL, performed for concern for PAP, was Periodic acid-Schiff (PAS) stain positive for proteinaceous material. Serum GM-CSF antibodies were absent and the patient was diagnosed with secondary PAP due to sirolimus. Given the patient's prior history of graft failure, his sirolimus dose was unchanged and he was treated with inhaled sargramostim. PAP is a rare disease caused by disorders in surfactant homeostasis further divided into three distinct etiologies: autoimmune (90%), secondary (5-10%), and congenital (2%). Secondary PAP is due to functional impairment in alveolar macrophages from immunodeficiency and is well-described in kidney and lung transplant recipients. To our knowledge, this is the first case described in a heart transplant recipient. The prognosis of transplant-associated secondary PAP is difficult to establish due to the limited number of published cases, though resolution is typically observed 2-4 months after removal of the offending agent. We present a case treated with GM-CSF due to difficulties reducing immunosuppression.

Granulocyte-macrophage colony-stimulating factor initiates amniotic membrane rupture and preterm birth in a mouse model.

Preterm premature rupture of membranes is associated with 30% of all preterm births. The weakening of amniotic membranes is associated with an increase in matrix metallopeptidases (MMPs) along with a decrease in their inhibitors, tissue inhibitor metallopeptidases (TIMPs). Additionally, granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to weaken fetal membranes in-vitro. We hypothesize pregnant mice treated with GM-CSF lead to increased MMPs:TIMPs resulting in membrane rupture and preterm birth. Pregnant CD-1 mice on gestational day 17 received either an intrauterine injection of GM-CSF or vehicle control. A second series of mice were administered an intrauterine injection of Lipopolysaccharide along with either anti-mouse GM-CSF or control antibody. Mice were evaluated for rupture of membranes and/or preterm birth and the uterus, amniotic fluid, and serum were collected for analysis. 87.5% of GM-CSF mice exhibited evidence of membrane rupture or preterm birth, compared with 0% in control mice (p<.001). Treatment with GM-CSF decreased the expression of TNFα (p<.05) while increasing the ratio of MMP2:TIMP1 (p<.05), MMP2:TIMP2 (p<.05), MMP2:TIMP3 (p<.001), MMP9:TIMP1 (p<.01), MMP9:TIMP2 (p<.05), MMP9:TIMP3 (p<.001), and MMP10:TIMP1 (p<.05). Mice treated with LPS and the GM-CSF antibody resulted in a decrease in the ratio of MMP2:TIMP1 (p<.0001) compared with controls. These studies demonstrate GM-CSF will result in membrane rupture and preterm birth by increasing the ratio MMPs:TIMPs in our animal model. By increasing our understanding of the molecular pathways associated with GM-CSF, we may be able to develop future therapies to prevent preterm birth and reduce neonatal morbidity.

A meta-analysis of granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody treatment for COVID-19 patients.

Objective:This meta-analysis aims to assess the efficacy and safety of granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies on COVID-19.Methods:Relevant literatures about GM-CSF antibody treatment in COVID-19 patients were searched from the PubMed, Cochrane Library, Embase, Google scholar, and Baiduscholar databases from the COVID-19 outbreak in December 2019 until 1 January 2021. The primary outcomes included the death, intensive care unit (ICU) admission risk, ventilation requirement, and secondary infection.Results:A total of 12 eligible literature involving 8979 COVID-19 patients were recruited, and they were divided into experimental group (n = 2673) and control group (n = 6306). Using a random-effect model, it is found that the GM-CSF antibody treatment was associated with a 23% decline of the risk of death [odd’s ratio (OR): 0.34, 95% confidence interval (CI): 0.21–0.56, p < 0.0001] and a 20% enhancement of ventilation (OR: 1.47, 95% CI: 1.19, 1.80, p = 0.0002). GM-CSF antibody treatment did not have a significant correlation to secondary infection and increased risk of ICU admission in COVID-19 patients, which may be attributed to the older age and the length of stay.Conclusions:Severe COVID-19 patients can benefit from GM-CSF antibodies.

Block of Granulocyte-Macrophage Colony-Stimulating Factor Prevents Inflammation-Induced Preterm Birth in a Mouse Model for Parturition

A multitude of factors promotes inflammation in the reproductive tract leading to preterm birth. Macrophages peak in the cervix prior to birth and their numbers are increased by the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). We hypothesize GM-CSF is produced from multiple sites in the genital tract and is a key mediator in preterm birth. Ectocervical, endocervical, and amniotic fluid mesenchymal stem cells were treated with lipopolysaccharide (LPS), and the concentration and expression of GM-CSF was measured. Pregnant CD-1 mice on gestational day 17 received LPS and an intravenous injection of either anti-mouse GM-CSF or control antibody. After 6 hours, the preterm birth rate was recorded. Treatment with LPS increased the GM-CSF concentration and messenger RNA expression after 24 hours in all 3 cell lines ( P < .01). Mice treated with LPS and the GM-CSF antibody had a preterm birth rate of 25%, compared to a 66.7% preterm birth rate in controls, within 6 hours ( P < .05, χ2). Treatment with the anti-mouse GM-CSF antibody decreased the concentration of GM-CSF in the mouse serum ( P < .01) but did not alter the number of macrophages or collagen content in the cervix. These studies demonstrate that GM-CSF is produced from multiple sites in the genital tract and that treatment with an antibody to GM-CSF prevents preterm birth. Curiously, the anti-mouse GM-CSF antibody did not decrease the number of macrophages in the cervix. Further research is needed to determine whether antibodies to GM-CSF can be utilized as a therapeutic agent to prevent preterm birth.

534: Anti-granulocyte macrophage-colony stimulating factor (GM-CSF) antibody prevents the upregulation of cyclooxygenase-2 in a mouse model

Preterm birth is believed to be a final common pathway of a multitude of instigating factors, and inflammation plays a critical role in preterm birth. Previous work from our laboratory has shown granulocyte macrophage-colony-stimulating factor (GM-CSF), a pro-inflammatory cytokine, is upregulated in the uterus in a mouse model of preterm birth. We have also shown treatment with an antibody to GM-CSF will prevent preterm birth within six hours. Therefore, we hypothesized treatment with an antibody to GM-CSF will prevent the upregulation of contraction associated proteins in the uterus leading to preterm birth. For this study, we utilized a mouse model of preterm birth. On embryonic day 17, pregnant CD1 mice were anesthetized and received an intrauterine injection of lipopolysaccharide (LPS) along with either an intravenous injection of anti-mouse GM-CSF or control antibody. After six hours the uteri were harvested and analyzed for expression of the oxytocin transmembrane receptor (OXTR), cyclooxygenase-2 (COX-2), or connexin-43 (Cx43) using quantitative polymerase chain reaction (qPCR). LPS increased the expression of COX-2 (p<0.001) compared to mice receiving an intrauterine injection of saline, and treatment with the anti-mouse GM-CSF antibody resulted in a decreased expression of COX-2 (p<0.05). Treatment with LPS did not increase the expression of OXTR or Cx43 compared to control mice, although the GM-CSF antibody did decrease the expression of Cx43 compared to mice treated with the control antibody. These studies demonstrate treatment with the anti-mouse GM-CSF antibody prevents upregulation of the contraction associated protein COX-2 in the uterus utilizing an inflammatory mouse model. Further research is needed to determine if antibodies to GM-CSF can be utilized as a therapeutic agent to prevent preterm birth.

PULMONARY ALVEOLAR PROTEINOSIS PRESENTING AS INTRACEREBRAL NOCARDIOSIS

SESSION TITLE: Diffuse Lung Disease SESSION TYPE: Med Student/Res Case Report PRESENTED ON: 10/09/2018 07:30 am - 08:30 am INTRODUCTION: Pulmonary alveolar proteinosis (PAP) is a rare disorder with an incidence of 0.5 / million people [1]. PAP is caused by the accumulation of surfactant in the alveoli, leading to hypoxemic respiratory failure and increased risk of opportunistic infections [2]. First-line therapy after infection eradication is whole-lung lavage. Treatment with exogenous granulocyte macrophage colony stimulating factor (GM-CSF) is under investigation. PAP can be a diagnostic challenge due to its low prevalence and nonspecific presentation. Here we describe a unique presentation that was diagnosed as PAP. CASE PRESENTATION: A 56 year old male farmer, never smoker, with no past medical history presented with right-sided weakness, two months of dyspnea, and dry cough. Brain MRI demonstrated multiple cerebral rim-enhancing abscesses (Fig 1A). He underwent a craniotomy and evacuation of cerebral abscesses with acid fast cultures positive for Nocardia farcinica. Chest CT showed a "crazy paving" pattern consistent with PAP (Fig 1B, C). Serum anti GM-CSF antibody was positive. Bronchoscopy showed opaque fluid on bronchoalveolar lavage, with negative cultures, and clumps of amorphous proteinaceous material consistent with PAP. Disseminated Nocardia was treated with ciprofloxacin for 1 year and sulfamethoxazole/ trimethoprim for 6 months. He underwent a whole lung lavage with resolution of symptoms. DISCUSSION: PAP is caused by surfactant accumulation due to incomplete removal by alveolar macrophages. Autoimmune PAP (primary PAP) accounts for the majority of cases [1]. Autoimmune PAP is caused by anti GM-CSF antibodies that disrupt macrophage activation, antigen presentation, and phagocytosis [2]. Secondary PAP is thought due to a relative deficiency of GM-CSF usually in the setting of hematologic diseases or dust exposures. The ensuing macrophage dysfunction leads to increased infection risk. Opportunistic infections including Nocardia, Mycobacterium, and fungi in otherwise immunocompetent patients with PAP have been previously reported [2]. In a report of 7 patients with disseminated Nocardiosis without PAP, 5 patients were found to have anti GM-CSF antibodies; two of these patients were also found to have radiographic characteristics of PAP [3]. CONCLUSIONS: Primary PAP is a rare autoimmune disorder that confers an immunocompromised state due to impaired alveolar macrophage activation. Opportunistic infections such as Nocardiosis are frequently seen with PAP. Eradication of the opportunistic infections, and targeted whole lung lavage are effective at relieving hypoxia in PAP. A high diagnostic suspicion is essential in the work-up of PAP. Reference #1: Clinics in Chest Medicine. 2016;37(3):431-440. https://doi.org/10.1016/j.ccm.2016.04.006 Reference #2: Journal of Infectio. 2012;65(2):173-179. https://doi.org/10.1016/j.jinf.2012.03.020 Reference #3: Clinical Infectious Disease. 2014;60(7):1017-1025. https://doi.org/10.1093/cid/ciu968 DISCLOSURES: No relevant relationships by David Dennis, source=Web Response No relevant relationships by Ulysses Magalang, source=Web Response No relevant relationships by Virgil Secasanu, source=Web Response

GM-CSF Antibodies and Peripheral Treg Cells as Potential Markers of Disease Activity to Guide Therapeutic Decisions in Pediatric IBD

Objectives: 1. To measure and compare granulocytemacrophage colony-stimulating factor (GM-CSF) antibodies and peripheral regulatory T cells (Tregs) cells as non-invasive biomarkers in active inflammatory bowel disease (IBD) and remission in pediatric IBD. 2. To determine the ability of these biomarkers to predict severity/type and choice of therapy in pediatric IBD. Methods: We hypothesized that patients with severe IBD requiring biologics will have higher circulating GM-CSF antibodies and lower Foxp3+ Tregs at baseline, with subsequent improvement in response to therapy. 28 patients were prospectively analyzed. Blood collected preand post-treatment was analyzed for GM-CSF antibodies by ELISA and Tregs by flow cytometry. Clinically, PCDAI and PUCAI were scored. Wilcoxon Rank Sum tests were used to compare baseline values between groups, and paired signed-rank tests used to compare pre- and posttreatment values. Results: Median GM-CSF antibodies trended higher (0.50 µg/mL vs. 0.37) and Tregs lower (5.23% vs. 6.71%) in the biologic cohort with no significant difference between cohorts. In response to biologics, 37% showed decrease in GM-CSF antibodies and 75% showed increase in Tregs. GM-CSF antibodies trended higher in Crohn’s disease (CD) than ulcerative colitis (UC) (0.81 vs. 0.31, p=0.073). Activity indices showed significant improvement in the biologic cohort of UC patients (p=0.04). Conclusion: GM-CSF antibodies and peripheral Tregs may reflect disease activity and thereby guide IBD therapy. Although no individual biomarker could predict need for biologics, their trend favors their use in a predictive model to identify the most effective treatment strategy in pediatric IBD.

673: Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) antibody prevents inflammation-induced preterm birth and effects on cervix remodeling using a mouse model

The convergence of a multitude of factors promotes inflammation in the reproductive tract as part of a final common pathway leading to preterm birth. Granulocyte macrophage colony stimulating factor (GM-CSF), a pro-inflammatory cytokine, stimulates stem cells to produce monocytes leading to an increase in macrophages, which have been shown to increase in the cervix prior to preterm birth. Previously, our laboratory has shown GM-CSF is upregulated in the cervix and uterus in a mouse model for preterm birth. Therefore, we hypothesized an antibody to GM-CSF will prevent preterm birth by quelling the inflammatory response. Pregnant CD-1 mice on gestational day 17 received either an intrauterine injection of saline or lipopolysaccharide (LPS). Additionally, mice receiving LPS also received an intravenous injection of either anti-mouse GM-CSF or a nonspecific control IgG. After 6 hours, the animals were evaluated for preterm birth and cervices were harvested and processed to assess the density of cell nuclei and resident mature F4/80 macrophages as previously described. Mice treated with LPS + control IgG had a preterm birth rate of 66.7% (started to deliver) compared to a 25% preterm birth rate for mice treated with LPS + anti-mouse GM-CSF (p<0.05 chi square). Moreover, LPS treatment alone reduced cell nuclei density in the cervix, an effect blocked by the anti-mouse GM-CSF antibody treatment. This suggests the LPS-induced cervical remodeling was prevented by GM-CSF blockade due to a reduced presence of macrophages in the LPS + anti-mouse GM-CSF vs LPS only group. These studies demonstrate an antibody to GM-CSF prevents preterm birth through a mechanism that includes effects on cervix remodeling. Further research is needed to determine if antibodies to GM-CSF can be utilized as a therapeutic agent to prevent preterm birth.

Clinical Utility of Anti- Granulocyte Macrophage-Colony Stimulating Factor Antibodies in Inflammatory Bowel Disease

Clinical Utility of Anti- Granulocyte Macrophage-Colony Stimulating Factor Antibodies in Inflammatory Bowel Disease

Cytokine amplification and macrophage effector functions in aortic inflammation and abdominal aortic aneurysm formation

On April 29, 2015, Son and colleagues published an article entitled "Granulocyte macrophage colony-stimulating factor (GM-CSF) is required for aortic dissection/intramural haematoma" in Nature Communications. The authors observed that the heterozygous Kruppel-like transcription factor 6 (KLF6) deficiency or absence of myeloid-specific KLF6 led to upregulation of macrophage GM-CSF expression, promoted the development of aortic hematoma/dissection, and stimulated abdominal aortic aneurysm (AAA) formation when the vessel wall was subjected to an inflammatory stimulus. The additional findings of increased adventitial fibrotic deposition, marked infiltration of macrophages, and increased expression of matrix metalloprotease-9 (MMP-9) and IL-6 were blocked with neutralizing GM-CSF antibodies, or recapitulated in normal mice with excess GM-CSF administration. The authors concluded that GM-CSF is a key regulatory molecule in the development of AAA and further suggested that activation of GM-CSF is independent of the transforming growth factor β (TGFβ)-Smad pathway associated with the Marfan aortic pathology. In this perspective, we expand on this mechanism, drawing from previous studies implicating a similar essential role for IL-6 signaling in macrophage activation, Th17 expansion and aortic dissections. We propose a sequential "two-hit" model of vascular inflammation involving initial vascular injury followed by recruitment of Ly6C(hi) macrophages. Aided by fibroblast interactions inflammatory macrophages produce amplification of IL-6 and GM-CSF expression that converge on a common, pathogenic Janus kinase (JAK)-signal transducers and activations of transcription 3 (STAT3) signaling pathway. This pathway stimulates effector functions of macrophages, promotes differentiation of Th17 lymphocytes and enhances matrix metalloproteinase expression, ultimately resulting in deterioration of vascular wall structural integrity. Further research evaluating the impact of interventions modulating this common JAK-STAT3 pathway may yield new therapeutic interventions for late stages of vascular expansion in inflammation driven aortic disease.

Impact of Serum Granulocyte-Macrophage Colony-Stimulating Factor Levels among Diabetes Patients in Hilla City - Iraq

Background: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pro-inflammatory cytokine with growth factor-like properties for monocytes and dendritic cells. Methods: Study subjects were recruited from the hospital in Hilla city. Healthy controls (n=15) and D.M. patients (n=35) were included in the study. Serum levels of GM-CSF and Anti-Insulin Antibody (AIA) measured by using ELISA technique. Results: The serum GM-CSF levels were significantly higher among diabetes subjects as compared with subjects without diabetes and results showed a high significant in serum AIA and glucose levels in patient as compare with control (p=0.009, P=0.001). The serum GM-CSF levels were increased in age group (45-54 years) shows the higher percent (42.85%) of patients with diabetes, with decreased of AIA and GM-CSF level rather than other age groups. Conclusions: There is indirect relationship between AIA and GM-CSF level and that is mean increased AIA and glucose level reflect the reduced GM-CSF level among the diabetic patients may be used as biomarker or use GM-CSF as co-therapy with insulin.

Brief report: Granulocyte-macrophage colony-stimulating factor drives monosodium urate monohydrate crystal-induced inflammatory macrophage differentiation and NLRP3 inflammasome up-regulation in an in vivo mouse model.

To determine the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the differentiation of inflammatory macrophages in an in vivo model of monosodium urate monohydrate (MSU) crystal-induced inflammation. C57BL/6J mice were treated with either clodronate liposomes to deplete peritoneal macrophages or GM-CSF antibody and were then challenged by intraperitoneal injection of MSU crystals. Peritoneal lavage fluid was collected, and cellular infiltration was determined by flow cytometry. Purified resident and MSU crystal-recruited monocyte/macrophages were stimulated ex vivo with MSU crystals. The interleukin-1β (IL-1β) levels in lavage fluids and ex vivo assay supernatants were measured. GM-CSF-derived and macrophage colony-stimulating factor (M-CSF)-derived macrophages were generated in vitro from bone marrow cells. Protein expression of IL-1β, caspase 1, NLRP3, and ASC by in vitro- and in vivo-generated monocyte/macrophages was analyzed by Western blotting. Depletion of resident macrophages lowered MSU crystal-induced IL-1β and GM-CSF levels in vivo as well as IL-1β production by MSU crystal-recruited monocytes stimulated ex vivo. GM-CSF neutralization in vivo decreased MSU crystal-induced IL-1β levels and neutrophil infiltration. MSU crystal-recruited monocyte/macrophages from GM-CSF-neutralized mice expressed lower levels of the macrophage marker CD115 and produced less IL-1β following ex vivo stimulation. These monocytes exhibited decreased expression of NLRP3, pro/active IL-1β, and pro/active caspase 1. In vitro-derived GM-CSF-differentiated macrophages expressed higher levels of NLRP3, pro/active IL-1β, and pro/active caspase 1 compared to M-CSF-differentiated macrophages. GM-CSF plays a key role in the differentiation of MSU crystal-recruited monocytes into proinflammatory macrophages. GM-CSF production may therefore contribute to the exacerbation of inflammation in gout.

Tumor-specific oncolytic adenoviruses expressing granulocyte macrophage colony-stimulating factor or anti-CTLA4 antibody for the treatment of cancers.

The purpose of this study was to examine the tumor specificity, cytotoxicity and the antitumor activity of two conditionally replicating oncolytic adenoviruses, SKL001 and SKL002, which expressed granulocyte macrophage colony-stimulating factor (GM-CSF) or anti-cytotoxic T lymphocyte-associated antigen-4 (CTLA4) antibody, respectively, and determine their antitumor efficacy in A549 lung tumor model, B16F10 mouse melanoma tumor model and CMT-64 mouse small lung carcinoma tumor model. Virus yield and cytotoxicity were used to determine tumor specificity and virus replication-mediated cytotoxicity of SKL001 and SKL002 in a panel of human tumor cell lines and primary cells in vitro. Two subcutaneous (s.c.) tumor nexograft tumor models were used to assess their antitumor activity. Under the control of the E2F promoter, the expression of E1a genes appeared only in tumor cells, whereas the wild-type Ad5 expressed its E1a genes in both tumor cells and normal cells. GM-CSF and anti-CTLA4 production were significantly higher in tumor cells than normal cells. SKL001 and SKL002 replicated in Rb-defective cell lines as efficiently as wild-type adenovirus but produced 100-fold less virus in normal human cells. SKL001 and SKL002 was up to 1000-fold more cytotoxic in Rb pathway-defective human tumor cells in comparison with normal human cells. Antitumor activity of SKL001 and SKL002 following intravenous administration was shown in a human lung A549 s.c. xenograft tumor model and mouse B16F10 melanoma tumor model when compared with phosphate-buffered saline treatment. In immune-competent mice, the addition of GM-CSF produced a stronger antitumor activity and induced a higher number of mature dendritic cells and macrophages, whereas additive antitumor activity was observed in the group when SKL001 and SKL002 were combined. In vitro and in vivo studies showed the selective replication, cytotoxicity, gene production and antitumor efficacy of SKL001 and SKL002 in human tumor model, suggesting a potential utility of this oncolytic agent for the treatment of human cancer. Further studies are warranted to show the role of human GM-CSF and anti-CTLA4 antibody in the antitumor efficacy of these two oncolytic viruses.

P0053 Expression of the GM-CSF gene and anti GM-CSF antibodies in egyptian adults with acute myeloid leukaemia and myelodysblastic syndromes

<h3>Background</h3> Granulocyte–macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine, which regulates myeloid progenitors. Antibodies to GM-CSF, while uncommon, are implicated in the pathogenesis of some diseases. We aimed to assess GM-CSF mRNA gene expression and anti GM-CSF antibodies in acute myeloid leukaemia (AML) and myelodysblastic syndromes (MDS) in relation to disease behaviour, patients' survival, and therapy outcome. <h3>Methods</h3> We examined GM-CSF mRNA gene expression quantitatively by real time PCR (RT-PCR) and anti GM-CSF antibodies by ELISA in sera of 50 AML/MDS Egyptian cases in comparison with 20 healthy blood donors (HD). <h3>Findings</h3> We found lower GM-CSF gene expression (<i>p</i>=0.008) and higher serum levels of anti GM-CSF antibodies (<i>p</i>=0.001) in cases irrespective of disease category when compared to HD. <h3>Interpretation</h3> Alteration in GM-CSF gene expression is implicated in the pathogenesis of AML/MDS and also the high titre of anti GM-CSF antibodies might influence myeloid development. Use of anti GM-CSF for disease monitoring and as a marker of disease activity needs larger future studies.

A Rare Occurrence of Pulmonary Alveolar Proteinosis after Lung Transplantation

We present a case of pulmonary alveolar proteinosis (PAP) initially diagnosed 28 months after left single-lung transplantation for idiopathic pulmonary fibrosis. The diagnosis was based upon the presence of periodic acid-Schiff (PAS)-positive and surfactant immunostain-positive acellular lipoproteinaceous material within alveoli seen on transbronchial biopsy as well as in bronchoalveolar lavage fluid. The patient eventually also displayed a characteristic "crazy paving" pattern on radiographic imaging. Granulocyte macrophage-colony stimulating factor antibodies were negative, consistent with secondary PAP. PAP is a rare interstitial lung disease with only a few reported cases occurring after lung transplantation. The etiology is thought to be related to a defect in macrophage function caused by immunosuppression. Reduced immunosuppression has been associated with stabilization, but not reversal, of the condition in the case reported here. PAP is an exceptionally rare cause of dyspnea and radiographic infiltrates after lung transplantation and may be related to toxicity of immune-suppressive medications.