Levels Of G-CSF Research Articles

The inflamed microenvironment: role on MSCs immunobiology and cancer

Inflammation and cancer are an inseparable binomial. The majority of cancers are triggered by somatic mutations and environmental factors with a common element: inflammation. Inflammation creates a microenvironment in which neoplastic cells can profit from the trophic factors secreted by inflammatory cells, useful to interfere with the anti-tumor response. Among the others, mesenchymal stem cells (MSCs) participate to microenvironment creation by a strong paracrine effect. The linkage between MSCs and inflammation is bidirectional: the inflamed microenvironment affects the complex MSCs immunobiology, but also MSCs can sustain inflammation. Here, we tried to clarify the influence of inflammation on the immunobiology of MSCs and deepen the paracrine effect of MSCs on tumor growth. MSCs were isolated from periprosthetic capsule caused by breast implant, affected by inflammation (I-MSCs). The contralateral part of the same patient, not inflamed, was used as control (C-MSCs). A panel of selected cytokines were analyzed by Real-Time PCR and ELISA. The cytokines expression was different in I-MSCs compared to C-MSCs, revealing that inflammation affects MSCs immunobiology. Then, C- and I-MSCs were indirectly co-cultured with MCF7 cells from breast adenocarcinoma. New analyses on proliferation rate and cytokines expression were performed. C- and I-MSCs gave almost the same results. The over-secretion of all the cytokines referred to the Th1 pathway and the decrease of those belonging to the Th2 pathway revealed the absence of a switch from Th1 to Th2 important to induce a chronic inflammation. The levels of TGF-β and G-CSF linked to the skill to damage the antigen-presenting cell function were decreased. In conclusion, even if MCF-7 proliferation increased after co-culture with I-MSCs, MSCs-derived paracrine effect does not sustain breast adenocarcinoma. These results absolve the breast implants from the insult to enhance adenocarcinoma onset.

VASCULAR ENDOTHELIAL GROWTH FACTOR SERUM LEVELS IN WOMEN WITH ADVANCED ENDOMETRIOSIS.

Endometriosis is a common gynecological disease, characterized by ectopic deposits of endometrial tissue outside of the uterine cavity, and it is associated with pelvic pain and infertility, with an important impact on the quality of life. At this point there is a controversy regarding the etiology and pathophysiology of endometriosis and it seems that pro-angiogenic growth factors might be involved, but their role is not completely understood. To evaluate the serum concentration of the main growth factors in patients with diagnosed endometriosis compared to healthy controls. A total of 157 women were divided into two study groups (Group I - endometriosis; Group 2 - healthy women). Serum levels of VEGF, G-CSF, GM-CSF, b-FGF, EGF, and HGF were measured with Human Multiplex Cytokine Panels. VEGF serum levels were significantly lower in women with endometriosis compared to controls (1.924±0.145 compared to 1.806±0.078 pg/mL, p<0.001). Serum levels of GM-CSF, b-FGF, EGF, and HGF respectively did not differ significantly between patients with endometriosis and healthy controls. G-CSF had a very low detection rate. The present study showed that VEGF serum levels are significantly lower in endometriosis patients compared to healthy controls, indicating a possible role in endometriosis pathogenesis.

Environmental exposure to zinc and copper influences sperm quality in fertile males

The presented study was designed to investigate the associations between environmental exposure to zinc and copper and levels of the parameters of oxidative stress and antioxidant defence system and selected cytokines in the seminal plasma of fertile males. The study population consisted of 65 fertile male volunteers from the southern region of Poland. Based on the medians of the levels of copper and zinc in seminal plasma, the study subjects were divided into 4 subgroups: groups with low and high environmental exposure to copper (Cu-L and Cu-H), groups with low and high environmental exposure to zinc (Zn-L and Zn-H). Semen volume, pH, count, motility and morphology of sperm cells in the Cu-L and Cu-H groups did not differ significantly. In the Cu-H group, TOS was increased significantly by 243% when compared to the Cu-L group. Similarly, median of IL-10 level in the Cu-H group was increased by 144% compared to the Cu-L group. Spearman correlation showed positive correlations between the levels of copper and TOS and IL-10. Negative correlations between copper and G-CSF and GM-CSF were also shown. In the Zn-L group, the percentage of progressively motile sperm cells after 1 hour was significantly higher by 17%, compared to the Zn-L group. In the Zn-H group, levels of G-CSF and MCP-1 were significantly higher by 70% and 145%, respectively, compared to the Zn-L group. The level of IL-10 was significantly lower in the Zn-H group by 60%, compared to the Zn-L group. Spearman correlation indicated that there is a positive correlation between the level of zinc and thiol groups, G-CSF and GM-CSF. Negative correlations between zinc and TOS and IL-10 were also shown. Zinc enhances motility in fertile men. This beneficial effect of zinc may be due to zinc-induced reduction in the plasma oxidative stress intensity and modulations of the immune response. This study confirms the antagonistic relationship between zinc and copper.

The Mechanism of Type I Interferon-Mediated Polarization of Tumor-Associated Neutrophils in Mice and Human

Neutrophils are the most abundant cells of all white blood cells and play a key role in host inflammatory responses. Importantly, inflammation has been associated with increased susceptibility for cancer and neutrophils, as a crucial component of this process, play essential role in inflammation-driven tumorigenesis. Neutrophils also represent an independent prognostic marker in a broad variety of neoplasias. The tumor microenvironment represents a special niche that is extremely influencing infiltrating immune cells. The concept of immune cell polarization was first described for macrophages (anti-tumor M1/pro-tumor M2). Recently neutrophil polarization has been postulated. Neutrophils appear to have diverse phenotypes in the tumor microenvironment i.e. tumor promoting (N2) or inhibiting (N1).Previously, we could show that significantly elevated numbers of neutrophils accumulate in tumors of mice that lack endogenous type I IFNs (Ifnb1-/-). Such tumor associated neutrophils (TANs) do not only efficiently support tumor angiogenesis and growth by up-regulating pro-angiogenic molecules (VEGF and MMP9), but also secrete higher amounts of neutrophil-attracting chemokines and display prolonged survival, compared to their WT counterparts thus representing pro-tumor N2 phenotype. Moreover, we could show that these N2 neutrophils efficiently support metastatic processes, due to up-regulation of pro-metastatic proteins, like Bv8, MMP9, S100A8 and S100A9, and due to impaired tumor cell killing. Treatment of such cells with rmIFNb reversed such an effect leading to anti-tumor N1 polarization.Here, we add further evidence emphasizing the importance of type I IFNs for neutrophil polarization in tumor microenvironment and reveal possible mechanism responsible for this phenomenon. In Ifnb1-/- mice, we observe a significant down-regulation of anti-tumor neutrophil markers, like ICAM1 and TNF-α. Moreover, neutrophils show reduced formation of NETs, accompanied by lower tumor killing capacity. Under these conditions, massively enhanced neutrophil turnover in combination with accumulation of immature neutrophils is observed. Importantly, therapeutic intervention in both Ifnb1-/- and WT mice using low dose IFN-β, induced anti-tumor activation of neutrophils. Correspondingly, in human melanoma patients undergoing type I IFN therapy, neutrophil anti-tumor characteristics were augmented, compared to untreated patients, suggesting effective outcome of this therapy.Further, we evaluated the mechanism of prolonged neutrophil survival in the absence of endogenous IFN-β. Importantly, we found that G-CSF mRNA expression levels in Ifnb1-/- neutrophils from different anatomical compartments as well as G-CSF blood serum levels were markedly up-regulated in such mice. G-CSF-expression levels were strongly reduced when the Ifnb1-/- neutrophils were incubated with rmIFN-β suggesting involvement of type I interferons in G-CSF down-regulation. Notably, we could recently show that G-CSF induces synthesis of enzyme Nicotinamide phosphoribosyltransferase (NAMPT), which is a rate-limiting enzyme converting nicotinamide (NA) into NAD+ that in turn activates NAD+ -dependent protein deacetylases sirtuins (SIRTs). NAMPT serves as an inhibitor of neutrophil apoptosis and as neutrophil chemoattractant by upregulation of CXCL8. It is a potent pro-inflammatory factor (upregulation of ROS release) and pro-angiogenic factor (smooth muscle maturation). At the same time, NAMPT was found to be strongly overexpressed in tumors and serum of leukemia patients. Analysis of NAMPT and SIRTs levels in tumor bearing Ifnb1-/- mice revealed highly upregulated levels of NAMPT and SIRT1 in blood neutrophils of these animals, in comparison to WT mice, which was in line with elevated levels of G-CSF. It also correlates with enhanced tumor angiogenesis, growth and metastasis. Based on these observations, we identified a new mechanism of interferon-mediated activation of pro-tumor neutrophils. Since tumor associated neutrophils represent a highly potent therapeutic target, these data highlight the therapeutic potential of interferons and NAMPT inhibitors, suggesting optimization of their clinical use as potent anti-tumor agent. DisclosuresNo relevant conflicts of interest to declare.

The urinary inflammatory profile in gluten free diet—adherent adolescents with type 1 diabetes and celiac disease

AimsOur objective was to characterize urinary cytokine/chemokine excretion in adolescents with type 1 diabetes (T1D) and celiac disease (CD) adhering to gluten free diet (GFD) compared to matched T1D patients and healthy control (HC) group from an existing cohort. MethodsEighteen T1D+CD+GFD patients aged 10–16years were identified and matched 2:1 for age, sex, diabetes duration and glycated hemoglobin to 36 T1D subjects and 36 HC. T1D+CD+GFD patients were adherent with a GFD. Urine and serum levels of cytokines/chemokines as well as baseline clinical and laboratory variables were assessed. ResultsT1D+CD+GFD patients exhibited lower levels of urinary IL-1B, IL-4, IL-5 (p<0.05) and IFN-γ, IL-8 and G-CSF levels (p<0.07) compared with T1D patients. Urinary biomarker levels between T1D+CD+GFD and HC were mostly similar. In contrast, urinary FGF-2, Flt-3, IL-1B, IL-1RA, IL-4, IL-5, IL-9, IL-10, IL-12p40, IL-15, MIP-1β, and TNF-β (p<0.05) were higher in T1D patients compared to HC. Similar levels of inflammatory markers were seen in the serum for all 3 groups. ConclusionsT1D+CD+GFD patients demonstrated decreased urinary inflammatory cytokine/chemokines compared to T1D and some similar to HC, which is suggestive of a potential modulatory role of treated CD on urinary markers.

Regulation of inflammatory biomarkers by intravenous methylprednisolone in pediatric ARDS patients: Results from a double-blind, placebo-controlled randomized pilot trial

ObjectiveA double-blind, randomized controlled trial showed that low-dose glucocorticoid therapy in pediatric ARDS patients is feasible and may improve both ventilation and oxygenation indices in these patients. However, the molecular mechanisms underlying potential changes in outcomes remain unclear. Based on these clinical findings, this study was designed to examine the effects of intravenous methylprednisolone on circulating inflammatory biomarkers in pediatric ARDS patients. DesignDouble-blind, placebo-controlled randomized trial with blood collection on study entry and day 7. SettingTertiary care children’s hospital. PatientsChildren (0–18years) with ARDS undergoing mechanical ventilation. Interventions35 children were randomized within 72h of mechanical ventilation. The glucocorticoid group received methylprednisolone 2mg/kg loading dose followed by 1mg/kg/day continuous infusion from days 1 to 7. Both groups were ventilated following the ARDSnet recommendations. WBC and differential cell counts, plasma cytokines and CRP levels, and coagulation parameters were analyzed on days 0 and 7. ResultsAt study entry, the placebo group had higher IL-15 and basophil levels. On day 7, in comparison to study entry, the placebo group had lower IL-1α, IFN-γ and IL-10 levels. The glucocorticoid group had lower INF-α, IL-6, IL-10, MCP-1, G-CSF and GM-CSF levels, and higher IL-17α levels on day 7 in comparison to study entry. Total and differential cell counts remained unchanged within the placebo group between days 0 and 7, whereas in the glucocorticoid group total WBC and platelets counts were increased on day 7. Pearson’s correlation studies within the placebo and glucocorticoid groups revealed positive and negative correlations between cytokine levels, cell counts, coagulation parameters and relevant clinical parameters of disease severity identified in our previous study. Multiple regression models identified several cytokines as predictors for alterations in clinical parameters of disease severity. ConclusionThis pilot study shows the feasibility of simultaneously measuring multiple inflammatory cytokines, cell counts and coagulation parameters in pediatric ARDS patients. We report statistical models that may be useful for future, larger trials to predict ARDS severity and outcomes.

Temporal changes of serum cytokine/chemokine levels in patients of Nakajo-Nishimra syndrome treated with tocilizumab

In Nakajo-Nishimura syndrome (NNS), proteasome disability due to a loss-of-function PSMB8 mutation induces storage of ubiquitinated proteins and overproduction of inflammatory cytokines and chemokines. However, the precise mechanisms causing complex phenotypes of the disease, including pernio-like eruptions, lipodystrophy and calcification of basal ganglia, is mostly unclear. As IL-6 overproduction in association with p38 hyperactivation was supposed to have a role in NNS (Arima et al, PNAS 2011), tocilizumab, a monoclonal antibody for IL-6 receptor, has recently been applied for two patients with NNS after informed consents were obtained. Decreased serum CRP and CPK levels in both patients and improved myalgia and arthralgia in one patient have been observed, whereas none of decrease in serum LDH level or improvement of fever and eruptions have been achieved. By analysis of serum cytokine/chemokine levels, IL-6, G-CSF and MCP-1 levels have changed in accordance to the CRP level, whereas IP-10 has shown constantly high levels independent of the CRP level. Furthermore, both the patients-derived peripheral blood monocytes and monocytes differentiated from a patient-derived iPS cells produced higher level of IP-10 than control cells after IFNgamma stimulation. These findings suggest that monocyte-derived IP-10 has a major role in pathogenesis of the sustained/progressing phenotypes in NNS.

EGCG induces G-CSF expression and neutrophilia in experimental sepsis.

A major green tea component, epigallocatechin-3-gallate (EGCG), has been proven protective against lethal sepsis in experimental setting, but its protective mechanisms remain incompletely understood. Here, we provide evidence to support EGCG's capacities in stimulating G-CSF production and neutrophilia in vivo. In an animal model of sepsis, EGCG significantly elevated peritoneal levels of G-CSF and several chemokines (e.g., MCP-1/CCL2 and MIP-1γ/CCL9), and consequently increased peritoneal neutrophil numbers (neutrophilia) at a late stage. In vitro, EGCG divergently affected HMGB1-mediated production of several chemokines: reducing CXCL15 and RANTES/CCL5, but elevating G-CSF and MIP-1α/CCL3 production by peritoneal macrophages. Similarly, it significantly induced the expression and secretion of G-CSF and MIP-1α/CCL3 in human peripheral blood mononuclear cells. Based on our preliminary data, it may be important to search for anti-inflammatory and G-CSF-stimulating agents for the clinical management of inflammatory diseases.

Abstract 38: ERK5 inhibitor XMD8-92 for malignant mesothelioma treatment: A preclinical study

Abstract Malignant mesothelioma (MM) is an aggressive cancer in dire need of successful treatment. We are first to demonstrate that Extracellular signal regulated kinase 5 (ERK5) plays an important role in MM pathogenesis (Clinical Cancer Research, 19, 2013). In this study we used genetically manipulated (ERK5 inhibited) MM cell lines to show that MM tumorigenesis is influenced by ERK5. In the present study we used recently discovered specific ERK5 inhibitor (XMD8-92) to show that it regulates MM tumor growth both in vitro and in vivo. First, we show that XMD8-92 inhibits ERK5 phosphorylation in 3 out of 4 MM cell lines showing constitutive activation of ERK5. Next, time and dose dependent study demonstrated significant cytotoxicity of XMD8-92 on 3 different MM cell lines. We also showed that soft agar colony forming efficiency of H2373 MM cells was significantly reduced in response to XMD8-92 treatment. Furthermore, in vivo studies using syngeneic mouse model of intraperitoneal mesothelioma showed that XMD8-92 treatment (50 mg/kg, ip, 1x daily for 3 weeks) resulted in significant decreases in tumor weights as compared to vehicle treated mice. Total and differential cell counts in peritoneal lavage fluid (PLF) were also inhibited by XMD8-92 suggesting the role of inflammation in MM tumor growth and their inhibition by ERK5 inhibitor. Cytokine profile assessment in PLF demonstrated the reduced levels of VEGF and IL-6 in XMD8-92 treated animals as compared to control animals receiving saline. FGF2, MCP-1, G-CSF, KC and RAGE levels were, however, not significantly impacted by the ERK5 inhibition. Taken together these data suggest that ERK5 inhibitor/ERK5 inhibition could be used for inhibiting the growth of MM tumors. Currently we are exploring the role of XMD8-92 alone as well as in combination with chemotherapeutic drugs for the treatment of MM using various mouse models. We are also exploring the role(s) of ERK5 inhibitor in inflammasome regulation by chemotherapeutics. This work is supported by NIEHS RO1 ES021110 and VCC/LCCRO grants. Citation Format: Arti Shukla, Joyce Thompson, Anurag Shukla, Alan Leggett, Maximilian MacPherson, Stacie Beuschel. ERK5 inhibitor XMD8-92 for malignant mesothelioma treatment: A preclinical study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 38. doi:10.1158/1538-7445.AM2015-38

Interleukin-8 level as a prognostic marker in patients with hepatitis B virus-associated hepatocellular carcinoma treated with transarterial chemoembolization

We investigated the association between serum interleukin (IL)-8 levels and post-transarterial chemoembolization (TACE) outcomes in patients with hepatitis B virus (HBV)-associated HCC. We enrolled 119 TACE-treated patients with HBV-associated HCC; TACE refractoriness and liver transplantation (LT)-free survival were evaluated during follow-up. Pre-TACE serum levels of various cytokines (epidermal growth factor [EGF], fibroblast growth factor 2, granulocyte-colony stimulating factor [G-CSF], interferon-γ, IL-8, IL-12, IL-17A, interferon-γ-inducible protein-10, monocyte chemotactic protein-1, tumor necrosis factor-α and vascular endothelial growth factor) were analyzed. During a mean follow-up of 24.3 (1–79) months, 91 patients (76.5%) exhibited TACE refractoriness. In multivariate analyses, multiple tumors (hazard ratio [HR], 2.37; 95% confidence interval [CI], 1.28–4.39; P=0.006), large tumor size (HR, 2.36; 95% CI, 1.38–4.03; P=0.002), and combination of alpha-fetoprotein and IL-8 levels (AFP>400ng/mL or IL-8>32pg/mL; HR, 1.72; 95% CI, 1.03–2.85; P=0.037) independently predicted overall TACE refractoriness. Higher EGF (>35pg/mL) and lower G-CSF levels (⩽12.5pg/mL) were associated with early TACE refractoriness (<1year; HR, 3.47; 95% CI, 1.01–11.96; P=0.049 and HR, 6.25; 95% CI, 1.62–23.81; P=0.008, respectively). Furthermore, high IL-8 level (>32pg/mL; HR, 1.68; 95% CI, 1.09–2.59; P=0.020) was associated with poor LT-free survival. In conclusion, pretreatment serum IL-8 is a useful prognostic marker for TACE refractoriness and LT-free survival in TACE-treated patients with HBV-associated HCC.

Staphylococcal enterotoxin A regulates bone marrow granulocyte trafficking during pulmonary inflammatory disease in mice

Pulmonary neutrophil infiltration produced by Staphylococcal enterotoxin A (SEA) airway exposure is accompanied by marked granulocyte accumulation in bone marrow (BM). Therefore, the aim of this study was to investigate the mechanisms of BM cell accumulation, and trafficking to circulating blood and lung tissue after SEA airway exposure. Male BALB/C mice were intranasally exposed to SEA (1μg), and at 4, 12 and 24h thereafter, BM, circulating blood, bronchoalveolar lavage (BAL) fluid and lung tissue were collected. Adhesion of BM granulocytes and flow cytometry for MAC-1, LFA1-α and VLA-4 and cytokine and/or chemokine levels were assayed after SEA-airway exposure. Prior exposure to SEA promoted a marked PMN influx to BAL and lung tissue, which was accompanied by increased counts of immature and/or mature neutrophils and eosinophils in BM, along with blood neutrophilia. Airway exposure to SEA enhanced BM neutrophil MAC-1 expression, and adhesion to VCAM-1 and/or ICAM-1-coated plates. Elevated levels of GM-CSF, G-CSF, INF-γ, TNF-α, KC/CXCL-1 and SDF-1α were detected in BM after SEA exposure. SEA exposure increased production of eosinopoietic cytokines (eotaxin and IL-5) and BM eosinophil VLA-4 expression, but it failed to affect eosinophil adhesion to VCAM-1 and ICAM-1. In conclusion, BM neutrophil accumulation after SEA exposure takes place by integrated action of cytokines and/or chemokines, enhancing the adhesive responses of BM neutrophils and its trafficking to lung tissues, leading to acute lung injury. BM eosinophil accumulation in SEA-induced acute lung injury may occur via increased eosinopoietic cytokines and VLA-4 expression.

Tumor inoculation site affects the development of cancer cachexia and muscle wasting.

The phenotype and severity of cancer cachexia differ among tumor types and metastatic site in individual patients. In this study, we evaluated if differences in tumor microenvironment would affect the development of cancer cachexia in a murine model, and demonstrated that body weight, adipose tissue and gastrocnemius muscle decreased in tumor-bearing mice. Interestingly, a reduction in heart weight was observed in the intraperitoneal tumor group but not in the subcutaneous group. We evaluated 23 circulating cytokines and members of the TGF-β family, and found that levels of IL-6, TNF-α and activin A increased in both groups of tumor-bearing mice. Eotaxin and G-CSF levels in the intraperitoneal tumor group were higher than in the subcutaneous group. Atrogin 1 and MuRF1 mRNA expressions in the gastrocnemius muscle increased significantly in both groups of tumor-bearing mice, however, in the myocardium, expression of these mRNAs increased in the intraperitoneal group but not in subcutaneous group. Based on these results, we believe that differences in microenvironment where tumor cells develop can affect the progression and phenotype of cancer cachexia through alterations in various circulating factors derived from the tumor microenvironment.

Cytokine profiles in interstitial fluid from chronic atopic dermatitis skin.

The invivo levels of inflammatory mediators in chronic atopic dermatitis (AD) skin are not well-defined due to the lack of a non-invasive or minimally invasive sampling technique. To investigate the cytokine milieu in interstitial fluid (ISF) collected from chronic lesional AD skin as compared to ISF from non-lesional AD skin and/or healthy donor skin. ISF was obtained using a minimally invasive technique of creating micropores in the skin by a laser, and harvesting ISF through aspiration. We determined the levels of 33 cytokines by Luminex and ELISA in ISF and plasma from sixteen AD patients and twelve healthy individuals. In seven AD patients, we analysed the IL-13, IL-31, IL-17, IL-22 and IFN-γ production by T cells isolated from lesional skin. AD patients were genotyped for the filaggrin gene (FLG)-null mutations 2282del4, R501X, R2447X and S3247X. Twenty-five of 33 examined mediators were detected in the ISF. The levels of IL-1α, IL-1β, IL-18, IL-1RA, IL-5, IL-13, IL-6, IL-8, TNF-α, RANTES(CCL-5), MIG(CXCL-9), IP-10(CXCL-10), TARC(CCL-17), VEGF and G-CSF showed significant differences between either lesional, non-lesional and/or healthy skin. IP-10 levels in ISF from lesional and non-lesional AD skin showed significant correlation with IP-10 blood levels. IP-10 also showed a significant correlation with clinical severity (SCORAD), as did IL-13. Levels of both IP-10 and IL-13 were more pronounced in patients with FLG-null mutations. Furthermore, FLG-null mutation carriers had more severe AD. The presented minimally invasive technique is a valuable tool to determine the invivo cytokine profile of AD skin.

A pre-conditioning stress accelerates increases in mouse plasma inflammatory cytokines induced by stress.

BackgroundMajor depressive disorder is a prevalent disease that is inadequately treated with currently available interventions. Stress increases susceptibility to depression in patients and rodent models. Depression is also associated with aberrant activation of inflammation, such as increases in circulating levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNFα). The two main goals of this study were (i) to identify cytokine changes measuring a broad panel of 19 cytokines, and (ii) to test if a pre-conditioning stress altered the inflammatory response to a subsequent stress.ResultStress-induced changes in mouse plasma cytokines were measured by multiplex following administration of one or two daily stresses of inescapable foot shocks using the learned helplessness paradigm for modeling depression-like behavior.Administration of inescapable foot shocks increased plasma levels of IL-1β, IL-6, TNFα, IL-3, IL-10, IL-13, IL-17A, IL-5, GM-CSF, IL-12(p70), IFN-γ, MIP-1α, MIP-1β, IL-1α, IL-2, KC, RANTES and G-CSF, with peak levels occurring in the range of 6 to 12 hr after stress. Pre-conditioning the mice 24 hr before with an equivalent inescapable foot shock stress resulted in similar magnitudes of increases in most cytokines as occurred after a single stress, but accelerated the increase, causing the levels of most cytokines to peak 1 hr after stress. These results demonstrate that a single stress induces the expression of many cytokines, and that sequential, daily stresses accelerates the rate of cytokine production.ConclusionsAcute stress broadly activates inflammation in mice, and the inflammatory response is more rapid following repeated stress, actions that may contribute to deleterious effects of stress on depression and other stress-linked diseases.

430. MVhGCSF Is Therapeutic In-Vivo B Cell Malignancies With Neutrophils (PMNs) Playing Different Roles

Measles virus (MV) has been shown to be oncolytic in various tumor models and is being evaluated in various Phase I clinical trials. The mechanisms by which MV kills cancer cells still remain obscure. We aimed to enhance the therapeutic efficacy of MV by expressing the human granulocyte colony stimulating factor (hGCSF), as an additional transcription unit of MV. We showed that MVhGCSF is therapeutic in two different in-vivo subcutaneous model of B cell malignancy, Raji (Burkitt's lymphoma) and Nalm-6 (acute lymphoblastic leukemia). In the Raji model, MVhGCSF treatment generated a highly significantly superior (p=0.0001) anti tumor effect by comparison to MVNSe and the MVhGCSF treated mice survived significantly longer than MVNSe treated mice (p=0.0098). In the Nalm-6 model, both MVhGCSF and MVNSe resulted in complete regression of the tumors in both groups with no survival advantage to MVhGCSF therapy. We then determined therapeutic efficacy of MVhGCSF in disseminated Nalm-6 xenografts as our previous data showed that approx. 60% of mice have complete regression of the tumors with IV delivered MV, offering a greater potential to observe any therapeutic benefit to MVhGCSF. Nalm-6 tumor cells expressing luciferase were used, in order to enable in-vivo imaging of disease progression by imaging. Mice were treated with MVNSe, MVhGCSF, or UV-MV control and an additional control of hGCSF alone, using pegylated hGCSF. Mice treated with the controls alone succumbed to leukemia the most quickly; in the group treated with GCSF alone, the median survival was 50 days compared to 75 days in the UV-MV treated group (p=0.012). As expected, 75% of the mice treated with MVNSe responded well and were alive at the end of the experiment. Surprisingly, mice receiving treatment with MVhGCSF had a median survival of 78.5 days compared to the MVNSe treated cohort where the median was not reached by the end of the experiment (p=0.0149). At humane end point, the spleen sizes and cellularity were similar across all the groups. hGCSF was detected at comparable levels in the serum of the hGCSF and MVhGCSF treated cohorts. Flow cytometric analysis of CD11b, Ly6G6C, NK and Mac3 cells did not show any difference in the PMN, macrophage and NK cell percentages between the different groups. Finally, to be certain that the less good outcome of mice treated with MVhGCSF did not also result from toxicity of high GCSF levels, IfnarKO * CD46Ge mice were injected IV with either MVNSe or MVhGCSF. They were monitored for 35 days after which spleen size, differential cell count (NK, macrophage, PMN percentages) and serum hGCSF levels were determined. There was no difference between the groups. The mice treated with MVhGCSF showed 200-800pg/ml of hGCSF in the serum at Day 35, but there was no toxicity. Taken together these data in the disseminated Nalm-6 model, suggests that any adverse effect of expressing hGCSF as an additional transcription unit in tumor bearing mice relates solely to promotion of tumor growth and not to toxicity of GCSF production.Our data suggest that a “one-size fits all” model of immune response to viral oncolysis is not appropriate and each tumor target will need full characterization for the potential of both direct and indirect, innate immune responses to generate benefit.

G-CSF induced neutrophil invasion of the CNS is inhibited by Interferon γ (VIR9P.1150)

Abstract Inflammation of the brainstem (BS) of wildtype (wt) 129 mice due to uncontrolled invasion by Ly6Chigh inflammatory monocytes (IM) and neutrophils is causally implicated in fatal HSV induced encephalitis (HSE). Anti-viral treatment with acyclovir following virus entry into BS cannot protect mice, as inflammation continues unabated. We showed previously that IL-10 regulates IM infiltration into BS. We determined the role of IFNγ, a key pleiotropic cytokine that directs the host immune response by inducing potent inflammatory or regulatory activities, in HSE. IFNγ knockout (GKO) mice developed HSE much quicker than wt or Rag mice. Surprisingly, mortality did not correlate with the ability to control virus replication but rather with massive expansion and infiltration of neutrophils into the BS. Neutrophil expansion in the absence of IFNγ was not due to increased IL-17 but rather to augmented secretion of G-CSF especially in CNS. Depletion of G-CSF by administration of anti G-CSF Ab or 25 mg IVIG (high dose), which contains anti G-CSF Ab, but not 4 mg (low dose) inhibited progenitor proliferation and neutrophil output from bone marrow and protected GKO mice. Importantly, reduction of serum G-CSF levels resulted in renewed IL-10 secretion by ICOS+ CD4 T cells and Tregs, which limited IM expansion. This study reveals that the complex interplay of IFNγ with G-CSF and IL-10 determines the ability to skew neutrophil or monocyte output from the bone marrow in response to infection.

Abstract O.51: Effects of Anti-TNF-alpha Antibody Therapy on IVIG-resistant Patients with Kawasaki Disease

Background: Kawasaki disease (KD) patients who do not respond to initial intravenous immunoglobulin (IVIG) therapy have a higher risk of developing coronary artery lesions (CALs). Infliximab (IFX) has been reported to be effective in alleviating fever in these patients and has been increasingly used. However, little is known about how IFX suppresses inflammation in KD. We analyzed immunological parameters in KD patients who responded to IFX, as well as those who did not, after the initial IVIG therapy. Methods: Twenty-six KD patients hospitalized and treated with IFX after initial IVIG and aspirin between August 2009 and July 2014 were enrolled in this study. We analyzed electronic medical records of baseline characteristics and laboratory data (WBC, neutrophil, lymphocyte and platelet counts, levels of GOT, GPT, Alb, Na, CRP and D-dimer), and measured plasma levels of cytokines (G-CSF, IL-6, IL-8, IL-10, IL-12p70, IL-17, TNF-α, IFN-γ, sIL-2R, sTNFR1 and sTNFR2) before and after IFX. We defined responders as patients who became afebrile within two days after IFX treatment. Results: The median age of 17 responders (11 males and 6 females) and 9 non-responders (5 males and 4 females) was 31 mo (9-84 mo) and 39 mo (16-64 mo), respectively (p=0.65). The initiation of IFX treatment after the onset of fever was compatible in both the group (day 9 vs day 9 in medians) (p=0.85). The incidence of CALs one month after onset was 0% (0/17) and 22% (2/9), respectively (p=0.11). Observed only in responders, WBC and neutrophil counts, and the levels of CRP, D-dimer, G-CSF, IL-6, sIL-2Rα, TNF-α, sTNFR-1 and sTNFR-2 decreased remarkably after IFX (p&lt;0.01). In non-responders, serum level of Na was significantly lower (p=0.04) before IFX, while WBC (p=0.04) and neutrophil (p=0.01) counts, and the levels of CRP (p=0.02), IL-6 (p=0.01), IL-10 (p=0.03) were higher. After IFX, the level of Alb (p=0.02) was lower. Conclusions: After IFX, the level of TNF-α, together with many pro- and anti-inflammatory mediators, decreased in responders but not in non-responders. In particular, suppression of IL-6 and IL-10 was insufficient in non-responders, suggesting that inflammation mechanisms other than the TNF-α axis would be important in KD patients who are unresponsive to IFX.

Laparoscopic surgery for colorectal cancer is not associated with an increase in the circulating levels of several inflammation-related factors

It has been hypothesized that inflammatory response triggered by surgery might induce the release of molecules that could promote proliferation, invasion and metastasis of surviving cancer cells. To test this hypothesis, the levels of multiple inflammation-related circulating factors were analyzed in patients undergoing surgery for colorectal cancer. A Luminex xMAP system was used to simultaneously assess levels of IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, FGF, eotaxin, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α and VEGF in 20 colorectal cancer patients and 10 age-matched non-neoplastic patients. In cancer patients analyses were performed at baseline (before surgery) and at different time points (up to 30 days) following laparoscopic surgery. Significantly higher levels of IL-1β, IL-7, IL-8, G-CSF, IFN-γ and TNF-α were detected in colorectal cancer patients compared to controls at baseline. In colorectal cancer patients, circulating levels decreased progressively following surgery and after day 30 post-surgery were no longer different from controls. These findings suggest that expression levels of several cytokines are higher in colorectal cancer patients compared to control subjects and no significant increase in several inflammation-related circulating factors is observed following laparoscopic surgery for cancer. Confirmation and validation in a different and larger cohort of patients are warranted.

Monophosphoryl Lipid A Induces Endotoxin Tolerance in Endothelial Cells

Monophosphoryl lipid A (MPLA) is a lipid A TLR4 agonist that exhibits a mild inflammatory effect compared to lipopolysaccharide (LPS). As endothelial cells regulate vascular inflammation, we hypothesized that MPLA treatment would induce tolerance in endothelial cells subsequently challenged with LPS. We cultured human umbilical vein endothelial cells (HUVECs) and exposed them to MPLA or LPS for 16 hours and then allowed them to sit in fresh media for 24 hours. Afterwards, cells were exposed to LPS for an additional 6 hours. Inflammation was assessed by quantifying IL‐6 and G‐CSF levels in the culture media. Pre‐treatment with MPLA reduced LPS‐induced IL‐6 secretion by 26% ± 5% compared to LPS alone. LPS pretreatment induced a similar reduction in IL‐6 after repeat LPS exposure (25% ± 7%, n=4 per group). MPLA and LPS also induced endotoxin tolerance of GCSF secretion compared to LPS alone with a reduction of GCSF levels by 46% and 38%, respectively. MPLA alone induced only minimal secretion of IL‐6 and GCSF (227.04 ± 9.68 pg/ml and 18.81 ± 1.19 pg/ml, respectively) compared to LPS alone (1634.92 ± 76.92 pg/ml for IL‐6 and 291.29 ± 33.18 pg/ml for GCSF). These studies demonstrate the ability of MPLA to induce endotoxin tolerance in HUVECs with potency equal to that of LPS, but without the associated pro‐inflammatory response.

The lack of type I interferon induces neutrophil-mediated pre-metastatic niche formation in the mouse lung.

Metastases are the major cause of death from cancer. Thus, understanding the regulation of metastatic processes is of utmost importance. Here we show that mice with impaired type I IFN signaling (Ifnar1(-/-)) develop more lung metastases in the 4T1 mammary and LLC lung carcinoma model, compared to control mice. In Ifnar1(-/-) mice, higher metastasis load is accompanied by massive neutrophil accumulation in lungs. Elevated G-CSF levels in serum and enhanced CXCR2 expression on neutrophils are most likely responsible for this phenomenon. Lung infiltrating neutrophils facilitate an improved pre-metastatic niche formation, supporting more efficient tumor cell extravasation and proliferation in this organ. This is due to the enhanced expression of pro-metastatic proteins, like Bv8, MMP9, S100A8 and S100A9. Development of pre-metastatic niche together with reduced neutrophil cytotoxicity against tumor cells results in enhanced metastatic processes in Ifnar1(-/-) mice. Overall, our findings describe a novel role for IFN during metastasis development and suggest that new treatment strategies should be considered for prevention of metastasis formation in patients.