Granulocyte-macrophage Colony-stimulating Factor Levels Research Articles

Correlation between Blood and CSF Compartment Cytokines and Chemokines in Subjects with Cryptococcal Meningitis.

Background Though peripheral blood is a crucial sample to study immunology, it is unclear whether the immune environment in the peripheral vasculature correlates with that at the end-organ site of infection. Using cryptococcal meningitis as a model, we investigated the correlation between serum and cerebrospinal fluid biomarkers over time. Methods We analyzed the cerebrospinal fluid and serum of 160 subjects presenting with first episode cryptococcal meningitis for soluble cytokines and chemokines measured by Luminex assay. Specimens were collected at meningitis diagnosis, 1-week, and 2-week post cryptococcal diagnosis. We compared paired samples by Spearman's correlation and the p value was set at <0.01. Results Of the 21 analytes tested at baseline, there was no correlation detected between nearly all analytes. A weak negative correlation was found between serum and cerebrospinal fluid levels of interferon-gamma (Rho = −0.214; p = .007) and interleukin-4 (Rho = −0.232; p = .003). There was no correlation at 1-week post cryptococcal diagnosis. However, at 2-week post cryptococcal diagnosis, there was a weak positive correlation of granulocyte-macrophage colony-stimulating factor levels (Rho = 0.25; p = .007) in serum and cerebrospinal fluid. No cytokine or chemokine showed consistent correlation overtime. Conclusion Based on our analysis of 21 biomarkers, serum and cerebrospinal fluid immune responses do not correlate. There appears to be a distinct immune environment in terms of soluble biomarkers in the vasculature versus end-organ site of infection. While this is a model of HIV-related cryptococcal meningitis, we postulate that assuming the blood compartment is representative of the immune function at the end-organ site of infection may not be appropriate.

Engineered Bacteriophage T7 as a Potent Anticancer Agent in vivo.

Oncolytic viruses (OVs) induce antitumor effect by both direct lysis of target cells and eliciting immunogenic response to the virus and ultimately to the target cells. These viruses are usually natural human pathogens. Bacteriophages are natural pathogens of bacteria that do not infect human and have greater advantages in safety, manipulation, and production over human viruses. We constructed an engineered bacteriophage T7 displaying a peptide, which targets murine melanoma cells and harbors a mammalian expression cassette of the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) in viral genomic DNA. The engineered phage was successfully transduced to B16F10 melanoma cells both in vitro and in vivo. GM-CSF was expressed from the transduced phage DNA. All mice treated with the phage intravenously survived for 25 days until the end of experiment, while only 40% of those not treated survived. During the 16 days of phage treatment, phage T7 displaying homing peptide and expressing GM-CSF inhibited tumor growth by 72% compared to the untreated control. Serum cytokine levels of IL-1α, TNF-α, and GM-CSF were seen to increase during the treatment. Immunohistochemical analysis of tumor tissue revealed infiltration by macrophages, dendritic cells (DCs), and CD8+ T cells. Migration of murine macrophages to bacteriophages was also observed in in vitro transwell assays in both time- and dose-dependent manners. Taken together, the recombinant bacteriophage T7 efficiently inhibited tumor growth by changing the tumor microenvironment and recruiting anti-tumor immune cells.

Enhanced Intestinal Immune Response in Mice after Oral Administration of Korea Red Ginseng-Derived Polysaccharide.

(1) Background: The immunostimulatory role of the polysaccharide fraction (KRG-P) of Korea red ginseng (KRG) was studied in cells. However, its immunomodulatory activity is unknown. Therefore, we investigated the chemical properties of KRG-P and its intestinal immune responses in vitro and in vivo. (2) Methods: KRG-P monosaccharide composition and molecular weight were determined using high-performance liquid and size-exclusion chromatography systems. Immunoglobulin A (IgA) and α-defensin-1 transcript levels were measured using a SYBR Green qRT-PCR; defensin-1, Granulocyte-macrophage colony-stimulating factor (GM-CSF), and IgA protein levels were determined using Western blotting and ELISA kits. (3) Results: The molecular weight of KRG-P was estimated to be 106 kDa, and it contained neutral sugar (74.3%), uronic acid (24.6%), and proteins (1%). In vitro studies of intestinal immunomodulatory activity of KRG-P indicated that GM-CSF and IgA levels increased in Peyer’s patch cells to higher levels than those obtained with KRG and induced bone marrow cell proliferation. In in vivo study, oral KRG-P administration to mice upregulated the expression of α-defensin-1 and IgA in the small intestinal tissue and that of secreted IgA in the feces. (4) Conclusions: KRG-P contributed to the modulation of intestinal immunity and maintenance of intestinal homeostasis against intestinal infection.

GM-CSF induces noninflammatory proliferation of microglia and disturbs electrical neuronal network rhythms in situ

BackgroundThe granulocyte-macrophage colony-stimulating factor (GM-CSF) (or CSF-2) is involved in myeloid cell growth and differentiation, and, possibly, a major mediator of inflammation in body tissues. The role of GM-CSF in the activation of microglia (CNS resident macrophages) and the consequent impacts on neuronal survival, excitability, and synaptic transmission are widely unknown, however. Here, we focused on electrical neuronal network rhythms in the gamma frequency band (30–70 Hz). Gamma oscillations are fundamental to higher brain functions, such as perception, attention, and memory, and they are exquisitely sensitive to metabolic and oxidative stress.MethodsWe explored the effects of chronic GM-CSF exposure (72 h) on microglia in male rat organotypic hippocampal slice cultures (in situ), i.e., postnatal cortex tissue lacking leukocyte invasion (adaptive immunity). We applied extracellular electrophysiological recordings of local field potential, immunohistochemistry, design-based stereology, biochemical analysis, and pharmacological ablation of microglia.ResultsGM-CSF triggered substantial proliferation of microglia (microgliosis). By contrast, the release of proinflammatory cytokines (IL-6, TNF-α) and nitric oxide, the hippocampal cytoarchitecture as well as the morphology of parvalbumin-positive inhibitory interneurons were unaffected. Notably, GM-CSF induced concentration-dependent, long-lasting disturbances of gamma oscillations, such as slowing (beta frequency band) and neural burst firing (hyperexcitability), which were not mimicked by the T lymphocyte cytokine IL-17. These disturbances were attenuated by depletion of the microglial cell population with liposome-encapsulated clodronate. In contrast to priming with the cytokine IFN-γ (type II interferon), GM-CSF did not cause inflammatory neurodegeneration when paired with the TLR4 ligand LPS.ConclusionsGM-CSF has a unique role in the activation of microglia, including the potential to induce neuronal network dysfunction. These immunomodulatory properties might contribute to cognitive impairment and/or epileptic seizure development in disease featuring elevated GM-CSF levels, blood-brain barrier leakage, and/or T cell infiltration.

GM-CSF Dependent Differential Control of Mycobacterium tuberculosis Infection in Human and Mouse Macrophages: Is Macrophage Source of GM-CSF Critical to Tuberculosis Immunity?

Although classically associated with myelopoiesis, granulocyte-macrophage colony-stimulating factor (GM-CSF) is being increasingly recognized for its potential role in innate resistance against tuberculosis (TB). While the GM-CSF is produced by a variety of host cells, including conventional and non-conventional T cells, macrophages, alveolar epithelial cells, the cell population that promotes GM-CSF mediated innate protection against Mycobacterium tuberculosis infection remains unclear. This is because studies related to the role of GM-CSF so far have been carried out in murine models of experimental TB, which is inherently susceptible to TB as compared to humans, who exhibit a resolution of infection in majority of cases. We found a significantly higher amount of GM-CSF production by human macrophages, compared to mouse macrophages, after infection with M. tuberculosis in vitro. The higher levels of GM-CSF produced by human macrophages were also directly correlated with their increased life span and ability to control M. tuberculosis infection. Other evidence from recent studies also support that M. tuberculosis infected human macrophages display heterogeneity in their antibacterial capacity, and cells with increased expression of genes involved in GM-CSF signaling pathway can control intracellular M. tuberculosis growth more efficiently. Collectively, these emerging evidence indicate that GM-CSF produced by lung resident macrophages could be vital for the host resistance against M. tuberculosis infection in humans. Identification of GM-CSF dependent key cellular pathways/processes that mediate intracellular host defense can lay the groundwork for the development of novel host directed therapies against TB as well as other intracellular infections.

Serum Levels of Granulocyte-Macrophage-colony-stimulating Factor and Stem-cell Factor During Liver Regeneration after Partial Hepatectomy in Humans.

Multiple biological functions have been recognized regarding Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) and Stem Cell Factor (SCF). To evaluate the serum changes of GM-CSF and SCF in patients undergoing surgical resection for liver tumor, in the regenerative phase after surgery in order to identify the possible relationship with the patient, tumor or surgical variables. Thirty-two consecutive patients (50% male, median age 66), undergoing hepatic resection of liver neoplasm, were evaluated. The liver tumor was Hepatocellular Carcinoma (HCC) in 44% of cases. Other tumors were cholangiocarcinoma and metastasis. Serum levels of GM-CSF and SCF were assessed at baseline and 2 days, 7 days and 4 weeks after surgery. Personal and clinical patient data were also recorded. The statistical analysis was carried out using t-test for unpaired data or ANOVA (repeated measure) for continuous variables and Fisher test for discrete variables. GM-CSF levels remained constant after surgery and were compared to baseline values. SCF levels, on the other hand, increased during the time, after surgery. The evaluation of SCF levels (fold increase) according to surgical, patient and tumor variables evidenced some differences. At day 7 and week 4, SCF levels were statistically increased: i) in patients undergoing a large resection in comparison with others (p<0.05); ii) in patients non-cirrhotic in comparison with cirrhotic ones (p=0.02) and finally; iii) in patients with non-HCC tumor in comparison with HCC ones (p=0.02). During liver regeneration in humans, SCF serum levels are increased allowing to hypothesize a possible role of this chemokine during tissue growth and remodeling.

FRI0010 GM-CSFR PATHWAY IS IMPLICATED IN PATHOGENIC INFLAMMATORY MECHANISMS IN GIANT CELL ARTERITIS

Background:Giant Cell Arteritis (GCA) is characterized by inflammation of large and medium arteries. Classic symptoms include headaches, malaise and, in severe cases, blindness and aortic aneurysms. Corticosteroids (CS) are the first line of treatment. Relapsing disease patients undergo multiple courses of CS therapy increasing their CS exposure and toxicity. A significant unmet need for disease-modifying CS-sparing therapy remains in GCA as the efficacy of current treatment options, including tocilizumab have limitations.We have previously reported elevated expression of granulocyte-macrophage colony stimulating factor (GM-CSF) pathway transcriptomic signature in GCA vessels. GM-CSF may contribute to underlying disease mechanisms by regulating inflammatory macrophages, dendritic cells (DCs) and T helper (TH1/TH17) cells which are involved in GCA pathogenesis. GM-CSF produced by T cells1can promote polarization of inflammatory macrophages2and recruitment and differentiation of monocytes into inflammatory DCs2that can in turn recruit T cells and stimulate TH1/TH17 differentiation creating a feedback loop. GM-CSF may also exert direct effects on angiogenesis3and vessel wall remodeling4.Objectives:To demonstrate the contributing role of GM-CSF pathway to inflammation in GCA arteries.Methods:Immunostaining was used to examine expression of GM-CSF and GM-CSF-Rα proteins in temporal artery biopsies (TABs) from GCA and controls (patients with suspected but not confirmed GCA and a negative TAB). Costaining with cell markers such as CD31, CD3, and CD68 allowed visualization of cells expressing GM-CSF and GM-CSF-Rα. Expression of GM-CSF pathway molecules such as phospho-JAK2 and PU.1 proteins was detected by immunohistochemical staining of GCA and control TABs.Ex vivocultured GCA arteries treated (10 each) with mavrilimumab (anti-GM-CSF-Rα) or placebo for 5 days were assayed for gene expression by qPCR, and culture supernatants were analyzed by ELISA.Results:Endothelial cells and macrophages were the main cell types expressing GM-CSF and GM-CSF-Rα. Increased expression of phospho-JAK2 (activated signaling molecule) and nuclear-localized PU.1 (transcription factor) in GCA TABs compared to controls indicated the presence of active GM-CSF signaling pathway in GCA.Inhibition of PU.1 mRNA expression inex vivocultures of GCA arteries treated with mavrilimumab indicated blockade of GM-CSFR signaling pathway. Mavrilimumab induced decrease in mRNA expression of key cell type markers including DC and macrophage activation markers CD83 and HLA-DRA, monocyte markers CD14 and CD16, T cell marker CD3ε, and B cell marker CD20 in these GCA artery cultures. Expression of inflammatory TH1/TH17 factors IFNγ (mRNA), TNFα, CXCL10 (IFNγ-stimulated chemokine) and IL-6 (mRNA and protein) was also inhibited by mavrilimumab in GCA artery cultures.Conclusion:Increased GM-CSF, GM-CSF-Rα, and downstream pathway-associated protein levels in GCA biopsies were consistent with previously-observed increased transcriptome signature. Expression of genes associated with inflammatory cells was suppressed by mavrilimumab in cultured GCA arteries. These data implicate the GM-CSF pathway in GCA pathophysiology and increase confidence in rationale for targeting the GM-CSF pathway in GCA.

Depletion of langerin+ cells enhances cutaneous wound healing.

Langerin is a C-type lectin receptor that is expressed on Langerhans cells and langerin-positive dermal dendritic cells in the skin. Little is known about the function of langerin+ cells in wound healing. In this study, the effects of ablation of langerin+ cells on healing of a full-thickness excision wound were investigated using the langerin-DTR depletable mouse. Strikingly, depletion of langerin+ cells resulted in more rapid reduction in wound area. Accelerated wound healing in the langerin+ -cell-depleted group was characterized by enhanced neo-epidermis and granulation tissue formation, and increased cellular proliferation within the newly formed tissues. Accelerated healing in the absence of langerin+ cells was associated with increased levels of granulocyte-macrophage colony-stimulating factor, F4/80+ cells and blood vessels within the granulation tissue. These data support an inhibitory role for langerin+ cells during wound healing. Therapies that suppress langerin+ cells or their function may therefore have utility in progressing the healing of wounds in humans.

Early single-dose exosome treatment improves neurologic outcomes in a 7-day swine model of traumatic brain injury and hemorrhagic shock.

Early single-dose treatment with human mesenchymal stem cell-derived exosomes promotes neuroprotection and promotes blood-brain barrier integrity in models of traumatic brain injury (TBI) and hemorrhagic shock (HS) in swine. The impact of an early single dose of exosomes on late survival (7 days), however, remains unknown. We sought to evaluate the impact of early single-dose exosome treatment on neurologic outcomes, brain lesion size, inflammatory cytokines, apoptotic markers, and mediators of neural plasticity in a 7-day survival model. Yorkshire swine were subjected to a severe TBI (8-mm cortical impact) and HS (40% estimated total blood volume). After 1 hour of shock, animals were randomized (n = 4/cohort) to receive either lactated Ringer's (5 mL) or lactated Ringer's with exosomes (1 × 10 exosome particles). After an additional hour of shock, animals were resuscitated with normal saline. Daily neurologic severity scores were compared. At 7 days following injury, lesion size, inflammatory markers, and mediators of inflammation (NF-κB), apoptosis (BAX), and neural plasticity (brain-derived neurotrophic factor) in brain tissue were compared between groups. Exosome-treated animals had significantly lower neurologic severity scores (first 4 days; p < 0.05) and faster neurologic recovery. At 7 days, exosome-treated animals had significantly smaller (p < 0.05) brain lesion sizes. Exosome-treated animals also had significantly lower levels of inflammatory markers (interleukin [IL]-1, IL-6, IL-8, and IL-18) and higher granulocyte-macrophage colony-stimulating factor levels compared with the control animals, indicating specific impacts on various cytokines. The BAX and NF-κB levels were significantly lower (p < 0.05) in exosome-treated animals, while brain-derived neurotrophic factor levels were significantly higher (p < 0.05) in the exosome-treated animals. In a large animal model of TBI and HS, early single-dose exosome treatment attenuates neurologic injury, decreases brain lesion size, inhibits inflammation and apoptosis, and promotes neural plasticity over a 7-day period.

Abstract A60: GM-CSF modulation restricts the secretion of main cytokines associated with CAR T-cell induced cytokine release syndrome

Abstract Emerging CAR T-cell therapies have shown high remission rates in patients with hematologic malignancies. Despite the success of these novel immunotherapies, multiple adverse effects are associated with CAR T-cell infusions. Cytokine release syndrome (CRS) is one of the most common CAR T cell-associated toxicities and results from the rapid elevation of inflammatory cytokines, which can be life threatening. CRS is commonly managed in the clinic with anti-IL-6R antibodies and glucocorticoids. While efficacious, these treatments address CRS only after it has initiated; therefore, alternative therapeutic strategies preventing CRS onset remain an urgent need. Using cytokine profiling arrays, we identified that secreted granulocyte macrophage colony stimulating factor (GM-CSF) contributes to CRS, since elevated GM-CSF levels promote macrophage-dependent secretion of key CRS biomarkers including MCP-1, IL-6, and IL-8. Modulation of GM-CSF levels either via genetic engineering of CAR T-cells using TALEN® technology or via neutralizing-antibodies resulted in a marked reduction of macrophage-dependent release of CRS-associated cytokines, while preserving antitumor activity. Our work proposes a novel therapeutic strategy to improve the overall safety of CAR T-cell therapies in cancer patients by using GM-CSF knockout CAR T cells. Citation Format: Mohit Sachdeva, Beatriz Aranda-Orgilles, Philippe Duchateau, Laurent Poirot, Julien Valton. GM-CSF modulation restricts the secretion of main cytokines associated with CAR T-cell induced cytokine release syndrome [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A60.

Early predicting of preeclampsia in pregnant women after assisted reproductive technologies

Aim: to identify new prognostic criteria of potential preeclampsia (PE) in pregnant women after assisted reproductive technologies (ART) for timely PE prophylaxis.Materials and methods. A prospective study of 85 patients who entered the program of ART was conducted. All patients were examined for possible hemostatic abnormalities (genetic thrombophilia and chronic hypercoagulation) and also for granulocytemacrophage colony-stimulating factor (GM-CSF) in the serum during the most critical periods (4–6, 12–14, 22–24 и and 30–32 weeks) of the fetoplacental complex formation.Results. The lowest level of GM-CSF was observed in patients with hemostatic disorders. Thus, in pregnant women who later developed PE, there was a decrease in GM-CSF level below the physiological: in those diagnosed with genetic thrombophilia – by 79.4 %, and those with hypercoagulation – by 63.6 %.Conclusion. The determination of serum GM-CSF and identification of hemostatic abnormalities in pregnant women after ART has a prognostic importance for potential PE. This result is significant for the understanding of the pathogenesis of PE and also has a practical value: it allows the doctor to attribute the patient to a high risk group from the first trimester of her pregnancy and start preventive therapy rather early.

Evaluation of soluble expression of recombinant granulocyte macrophage stimulating factor (rGM-CSF) by three different E. coli strains.

Background and purpose:Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with a wide range of therapeutic applications although expression of GM-CSF in Escherichia coli (E. coli) usually leads to formation of insoluble aggregates mostly lack biological activity. The aim of this study was to compare the soluble expression level of GM-CSF in three E. coli strains, BL21 (DE3), SHuffle® T7 and Origami™ 2 (DE3).Experimental approach:The effect of different temperatures and inducer concentrations on soluble expression of GM-CSF was evaluated. The soluble GM-CSF was subjected to endotoxin removal and purification using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, ultrafiltration. The biological activity of produced GM-CSF was evaluated based on its growth promotion effect on TF-1 cell lines by MTT assay method.Findings / Results:A significant improvement of the soluble yield of GM-CSF (about 30% of GM-CSF was expressed as soluble proteins) was observed when protein expression was induced at 30 °C with 0.5 mM isopropyl β- d-1-thiogalactopyranoside (IPTG) in E. coli Shuffle T7. The soluble GM-CSF with a high purity up to 95 % and specific activity of 1.25 × 104 IU/μg was obtained.Conclusion and implications:The proposed strategy here can be used to improve the soluble expression of other hard-to-express proteins with similar structural properties (i.e., containing disulfide binds or cysteine).

Liver fibrosis and CD206+ macrophage accumulation are suppressed by anti-GM-CSF therapy.

Background & AimsChronic liver inflammation leads to fibrosis and cirrhosis and is associated with an accumulation of intrahepatic TNFα-secreting CD206+ macrophages, which may participate in maintaining chronic liver disease in a GM-CSF-dependent manner. We aimed to elucidate the exact role of GM-CSF in the development and progression of chronic liver disease.MethodsLiver immunohistochemistry and serum quantification were performed in patients with viral and non-viral-related liver disease to compare CD206+ monocyte/macrophages, fibrosis and GM-CSF. This was followed by functional validations in vitro and in vivo in humanised mice.ResultsUsing multiplex immunofluorescence and histo-cytometry, we show that highly fibrotic livers had a greater density of CD206+ macrophages that produced more TNFα and GM-CSF in the non-tumour liver regions of patients with hepatocellular carcinoma (n = 47), independent of aetiology. In addition, the absolute number of CD206+ macrophages strongly correlated with the absolute number of GM-CSF-producing macrophages. In non-HCC chronic HCV+ patients (n = 40), circulating GM-CSF levels were also increased in proportion to the degree of liver fibrosis and serum viral titres. We then demonstrated in vitro that monocytes converted to TNFα-producing CD206+ macrophage-like cells in response to bacterial products (lipopolysaccharide) in a GM-CSF-dependent manner, confirming the in vivo normalisation of serum GM-CSF concentration following oral antibiotic treatment observed in HBV-infected humanised mice. Finally, anti-GM-CSF neutralising antibody treatment reduced intrahepatic CD206+ macrophage accumulation and abolished liver fibrosis in HBV-infected humanised mice.ConclusionsWhile the direct involvement of CD206+ macrophages in liver fibrosis remains to be demonstrated, these findings show that GM-CSF may play a central role in liver fibrosis and could guide the development of anti-GM-CSF antibody-based therapy for the management of patients with chronic liver disease.Lay summaryLiver fibrosis is a major driver of liver disease progression. Herein, we have shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in the development of liver fibrosis. Our findings support the use of anti-GM-CSF neutralising antibodies for the management of patients with chronic liver disease resulting from both viral and non-viral causes.

Impaired production of immune mediators in dengue virus type 2-infected mononuclear cells of adults with end stage renal disease

Chronic kidney disease is an epidemiologically identified risk factor for development of severe dengue in dengue-affected patients. However, available data on the immune pathogenesis in end stage renal disease (ESRD) patients affected by dengue is insufficient. We performed an in vitro study to evaluate the sequential immunological reactions and viral load in dengue virus type 2-infected mononuclear cells of patients with ESRD (n = 34) and in healthy controls (n = 30). The concentrations of interleukins (IL)-1 receptor antagonist (Ra), IL-2, IL-6, IL-8, IL-10, IL-12p40, granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1b (MIP-1b), vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-α and viral load cycle threshold (Ct) were measured in the dengue virus type 2-infected mononuclear cells at 6 h, 24 h, 48 h, and 72 h post-infection. We found in the ESRD group significantly higher GM-CSF and IL-2 levels at 6 h post-infection. However, IL-8, IL-10, IL-12p40, TNF-α, MCP-1, and MIP-1b levels were found significantly lower than in the control group. At 24 h, 48 h, and 72 h post-infection, significantly lower levels of IL-1Ra, IL-6, IL-8, IL-10, IL-12p40, TNF-α, MCP-1, and MIP-1b were detected in ESRD group. Concentration of VEGF at 24 h and 48 h, and of GM-CSF at 48 h and 72 h were also found to be lower in ESRD group than in control group. Compared with controls, the viral load Ct values were significantly lower in ESRD group at 6 h and 24 h post-infection No significant difference in viral load Ct values between two groups was found at 48 h and 72 h post-infection. Our study discloses that the expression of immune mediators of dengue-infected mononuclear cells is impaired in ESRD patients.

Multiday maintenance of extracorporeal lungs using cross-circulation with conscious swine

ObjectivesLung remains the least-utilized solid organ for transplantation. Efforts to recover donor lungs with reversible injuries using ex vivo perfusion systems are limited to <24 hours of support. Here, we demonstrate the feasibility of extending normothermic extracorporeal lung support to 4 days using cross-circulation with conscious swine. MethodsA swine behavioral training program and custom enclosure were developed to enable multiday cross-circulation between extracorporeal lungs and recipient swine. Lungs were ventilated and perfused in a normothermic chamber for 4 days. Longitudinal analyses of extracorporeal lungs (ie, functional assessments, multiscale imaging, cytokine quantification, and cellular assays) and recipient swine (eg, vital signs and blood and tissue analyses) were performed. ResultsThroughout 4 days of normothermic support, extracorporeal lung function was maintained (arterial oxygen tension/inspired oxygen fraction >400 mm Hg; compliance >20 mL/cm H2O), and recipient swine were hemodynamically stable (lactate <3 mmol/L; pH, 7.42 ± 0.05). Radiography revealed well-aerated lower lobes and consolidation in upper lobes of extracorporeal lungs, and bronchoscopy showed healthy airways without edema or secretions. In bronchoalveolar lavage fluid, granulocyte-macrophage colony-stimulating factor, interleukin (IL) 4, IL-6, and IL-10 levels increased less than 6-fold, whereas interferon gamma, IL-1α, IL-1β, IL-1ra, IL-2, IL-8, IL-12, IL-18, and tumor necrosis factor alpha levels decreased from baseline to day 4. Histologic evaluations confirmed an intact blood–gas barrier and outstanding preservation of airway and alveolar architecture. Cellular viability and metabolism in extracorporeal lungs were confirmed after 4 days. ConclusionsWe demonstrate feasibility of normothermic maintenance of extracorporeal lungs for 4 days by cross-circulation with conscious swine. Cross-circulation approaches could support the recovery of damaged lungs and enable organ bioengineering to improve transplant outcomes.

Delayed neutrophil apoptosis in granulomatosis with polyangiitis: dysregulation of neutrophil gene signature and circulating apoptosis-related proteins

Objectives: Neutrophil apoptosis is mandatory for resolving inflammation and is regulated by expression of pro- and anti-apoptotic genes. We studied neutrophils isolated from patients with granulomatosis with polyangiitis (GPA) to investigate apoptosis alterations and to identify transcriptional and circulating factors affecting this process.Method: We enrolled 36 patients (18 in active stage, 18 in remission) and 18 healthy controls. Circulating levels of tumour necrosis factor-α (TNF-α), granulocyte–macrophage colony-stimulating factor (GM-CSF), macrophage migration inhibitory factor, plasminogen activator inhibitor-1, interferon-γ, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, platelet endothelial cell adhesion molecule-1, soluble Fas (sFas), sFas ligand, survivin, and pentraxin-3 (PTX3) were evaluated by enzyme-linked immunosorbent assay/Luminex; circulating apoptotic neutrophils by flow cytometry; and apoptosis-related gene transcripts by real-time polymerase chain reaction.Results: Patients had decreased fractions of circulating apoptotic neutrophils and delayed neutrophil apoptosis was present in vitro. Circulating levels of TNF-α, GM-CSF, sFas, and PTX3 were higher in GPA. Delayed neutrophil apoptosis was accompanied by decreased mRNA of pro-apoptotic genes and transcription factors (DIABLO, PMAIP1, BAX, CASP3, CASP7, RUNX3, E2F1, TP53) and increased anti-apoptotic CFLAR and BCL2A1 mRNA. TNF-α and sFas levels correlated with circulating apoptotic neutrophils and expression of apoptosis genes. Stimulation with TNF-α of neutrophils from controls significantly down-regulated E2F1 and CASP3 expression.Conclusions: Circulating neutrophils in GPA have anti-apoptotic phenotype involving both intrinsic and extrinsic pathways of apoptosis. This is accompanied by increased levels of circulating pro-survival factors (GM-CSF, TNF-α, sFas), independent of disease activity. Anti-apoptotic phenotype of neutrophils in GPA is reproduced by exposure to low concentrations of TNF-α.

Interleukin-17 receptor C gene polymorphism reduces treatment effect and promotes poor prognosis of ischemic stroke.

Objective: To study the relationship between Interleukin-17 receptor C (IL-17RC) gene polymorphism and ischemic stroke (IS).Methods: Three hundred cases of IS patients and 300 cases of the healthy controls were selected. Serum of IS patients and the controls was collected. The relative mRNA levels of IL-17, IL-17RC, IL-6, IL-8, G-CSF and granulocyte-macrophage colony stimulating factor (GM-CSF) by qRT-PCR. The protein expression of IL-17 and IL-17RC was determined by Western blotting. IL-17RC genotype was identified by PCR amplification. The proportion of IL-17RC, SNP and re37511 in IS and control group was determined. The treatment effect on IS and prognosis of patients with IL-17RC, SNP and re37511 was compared.Results: The relative mRNA levels of IL-17, IL-17RC, IL-6, IL-8, G-CSF and GM-CSF in IS group were significantly higher than the control group. The protein expression of IL-17 and IL-17RC in IS group was also markedly higher than the control group. The proportion of IL-17RC re37511 in IS group was much larger than control group and proportion of IL-17RC much less. The percent of poor treatment effect in re37511 was much larger than IL-17RC. The percent of death and recrudescence in patients with IL-17RC re37511 was the highest.Conclusion: IS up-regulates the expression of IL-17 and IL-17RC. IL-17RC re37511 indicates the patients have a poorer treatment effect and prognosis.

Crystal structure of the Fab region of a neutralizing antibody against granulocyte-macrophage colony-stimulating factor.

An increased level of granulocyte-macrophage colony-stimulating factor has a potential role in the development of autoimmune diseases, and the neutralization of its activity by monoclonal antibodies is a promising therapy for some diseases. Here, the crystal structure of the Fab region of EV1007, a fully human antibody expressed in Chinese hamster ovary cells that was developed from human peripheral blood mononuclear cells, is described. The structure closely resembles that of MB007, which is the Fab region of the same antibody expressed in Escherichia coli [Blech et al. (2012), Biochem. J. 447, 205-215], except at the hinge regions between the immunoglobulin domains and the H3 loop region. This paper presents evidence for the flexibility of the hinge and H3 loop regions of the antibody based on the comparison of two independently solved crystal structures.

Cytokine profiling reveals increased serum inflammatory cytokines in idiopathic choroidal neovascularization

BackgroundThe exact pathogenesis of idiopathic choroidal neovascularization (ICNV) remains unclear. Cytokine-mediated inflammation has been thought to be involved in the pathophysiology of ICNV. The purpose of this study was to investigate serum cytokine profiles in patients with ICNV and to explore the relationship between serum cytokine levels and ICNV severity.MethodsThis case-control study was conducted in 32 ICNV patients and 30 healthy volunteers. Clinical and demographic information was obtained from the medical data platform and the serum was analysed with a multiplex assay to determine the levels of seven cytokines: interleukin (IL)-2, IL-10, IL-15, IL-17, basic fibroblast growth factor (basic FGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and vascular endothelial growth factor (VEGF).ResultsSerum levels of IL-2, IL-10, IL-17, basic FGF, and VEGF were elevated in ICNV patients compared to controls. Serum GM-CSF levels were positively related to central retinal thickness, and serum IL-17 levels were positively related to CNV lesion area.ConclusionSerum inflammatory cytokines were significantly elevated in ICNV patients compared to controls. This suggests that systemic inflammation may play a critical role in the physiopathology of ICNV.

Indoor PM2.5 from coal combustion aggravates ovalbumin-induced asthma-like airway inflammation in BALB/c mice.

We hypothesized that indoor PM2.5 exposure from coal combustion exaggerates airway inflammation in the lung tissue of asthmatic mice induced with ovalbumin (OVA). Forty BALB/c mice, randomly divided into four groups (n = 10 per group), were intratracheally instilled with normal saline alone, PM2.5 (2.5 mg/ml PM2.5 alone), OVA (15 μg/ml OVA alone), and PM2.5+OVA (2.5 mg/ml PM2.5 and 15 μg/ml OVA), respectively, four times at 2-wk intervals. Daily mean concentration of PM2.5 from indoor coal combustion was 156.95 μg/m3. The highest metal composition in PM2.5 was Zn (34.81 ± 1.8 μg/m3). Exposure to PM2.5+OVA significantly elevated IL-4 and decreased IFN-γ production in mice compared with the control (P < 0.05). Exposure to PM2.5+OVA showed a significant increase in the protein levels of granulocyte-macrophage colony-stimulating factor and IL-8 and a decrease in the protein level of transforming growth factor-β1 in bronchoalveolar lavage fluid of mice compared with the control (P < 0.05). The expression of IL-4 mRNA was significantly increased, whereas the expression of IFN-γ mRNA was decreased in lung tissue of the PM2.5+OVA group (P < 0.05). The expression level of Foxp3 mRNA in the PM2.5+OVA group was significantly lower than that in the control group in lung tissue (P < 0.05). Treatment with PM2.5+OVA promoted a prominent neutrophil sequestration into the lung parenchyma, goblet cell proliferation, and severe inflammatory cell infiltration in the airways. Exposure to PM2.5 from indoor coal combustion might induce airway inflammatory immune responses and exacerbate peribronchiolar inflammation due to infiltration of inflammatory cells into the airway submucosa and airway structural pathological changes.