Human Granulocyte-macrophage Colony-stimulating Factor Research Articles

1083P A pilot study of engineered adenovirus ONCOS-102 in combination with pembrolizumab (pembro) in checkpoint inhibitor refractory advanced or unresectable melanoma

Patients (pts) with advanced melanoma resistant to immune checkpoint inhibitors (ICI) may benefit from oncolytic viruses. ONCOS-102 (Ad5/3-D24-GM-CSF), a chimeric oncolytic adenovirus expressing human granulocyte-macrophage colony stimulating factor, has demonstrated innate and adaptive immune responses in solid tumors. This study assessed the safety, clinical and immune responses of sequential (Part 1) or combined (Part 2) ONCOS-102 and pembro for pts with advanced melanoma after prior PD-1 blockade.

Abstract 1148: SynOV1.1: A synthetic gene circuit controlled oncolytic adenovirus demonstrating high tumor specificity, potent antitumor efficacy and high synergy with an anti-PD-L1 monoclonal antibody in preclinical hepatocellular carcinoma models

Abstract Hepatocellular carcinoma (HCC) is yet the fourth most common cause of cancer-related death although its treatment has been significantly advanced in recent years. Oncolytic viruses are among a novel class of immunotherapies that have shown promise to HCC patients with convincing abilities to selectively replicate in and kill cancer cells as well as to boost tumor immunogenicity. In order to further enhance the safety and efficacy of oncolytic viruses, SynOV1.1, a recombinant replication-competent serotype 5-based adenovirus, was constructed by deleting E1B and partial E3 genes, and incorporating a synthetic sensory switch circuit. The gene circuit was designed to enhance tumor-specific expression of E1A gene and an immune effector, human granulocyte-macrophage colony-stimulating factor (hGM-CSF) by sensing cancer-specific alpha-fetoprotein (AFP) promoter and two microRNA inputs. The specificity of SynOV1.1 to selectively kill target tumor cells and the ability to specifically express hGM-CSF in the target cells were confirmed in numerous human and mouse HCC cell lines with an IC50 approximately 3 to 35-fold less than that of normal cell lines. In vivo, intratumoral injections of SynOV1.1 in immune-deficient mice bearing human HCC cells resulted in a significant reduction of tumor volume (approximately 80%) as compared with the controls (approximately 30% for sorafenib). To further evaluate the effects of antitumor and immune stimulation, SynOV1.1m (SynOV1.1 murine surrogate) was intratumorally injected to the immune-competent mice bearing mouse HCC cells (ie., mHepa1-6), which showed SynOV1.1m induced a greater antitumor effect as well as a more robust tumor-specific immune response as compared to the controls. In addition, SynOV1.1m could significantly inhibit the growth of non-injected distal tumors after it was injected into a mHepa1-6 tumor on the other side of the mouse body, indicating that a systemic immune response was induced by SynOV1.1m. The combination of SynOV1.1m and an anti-mPD-L1 monoclonal antibody (mAb) was further explored and showed a synergistic anti-tumor effect as compared to SynOV1.1m or anti-mPD-L1 mAb alone. Preclinical safety of SynOV1.1 was examined in various toxicology species including golden hamsters, tumor-bearing mice and cynomolgus monkey, in all of which SynOV1.1 was found safe. Overall, preclinical pharmacology and toxicology data support clinical investigation of SynOV1.1 alone or in combination with atezolizumab in advanced HCC patients with positive serum AFP levels. Citation Format: Qiang Liu, Yubing Cao, Man Zhang, Shuguang Peng, Yiqi Liu, Huiya Huang, Bin Chen. SynOV1.1: A synthetic gene circuit controlled oncolytic adenovirus demonstrating high tumor specificity, potent antitumor efficacy and high synergy with an anti-PD-L1 monoclonal antibody in preclinical hepatocellular carcinoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1148.

LB0001 MAVRILIMUMAB IMPROVES OUTCOMES IN PHASE 2 TRIAL IN NON-MECHANICALLY-VENTILATED PATIENTS WITH SEVERE COVID-19 PNEUMONIA AND SYSTEMIC HYPERINFLAMMATION

Background:Granulocyte/macrophage-colony stimulating factor (GM-CSF) is a cytokine both vital to lung homeostasis and important in regulating inflammation and autoimmunity1,2,3 that has been implicated in the pathogenesis of respiratory failure and death in patients with severe COVID-19 pneumonia and systemic hyperinflammation.4-6 Mavrilimumab is a human anti GM-CSF receptor α monoclonal antibody capable of blocking GM-CSF signaling and downregulating the inflammatory process.Objectives:To evaluate the effect of mavrilimumab on clinical outcomes in patients hospitalized with severe COVID-19 pneumonia and systemic hyperinflammation.Methods:This on-going, global, randomized, double-blind, placebo-controlled seamless transition Phase 2/3 trial was designed to evaluate the efficacy and safety of mavrilimumab in adults hospitalized with severe COVID-19 pneumonia and hyperinflammation. The Phase 2 portion comprised two groups: Cohort 1 patients requiring supplemental oxygen therapy without mechanical ventilation (to maintain SpO2 ≥92%) and Cohort 2 patients requiring mechanical ventilation, initiated ≤48 hours before randomization. Here, we report results for Phase 2, Cohort 1: 116 patients with severe COVID- 19 pneumonia and hyperinflammation from USA, Brazil, Chile, Peru, and South Africa; randomized 1:1:1 to receive a single intravenous administration of mavrilimumab (10 or 6 mg/kg) or placebo. The primary efficacy endpoint was proportion of patients alive and free of mechanical ventilation at Day 29. Secondary endpoints included [1] time to 2-point clinical improvement (National Institute of Allergy and Infectious Diseases COVID-19 ordinal scale), [2] time to return to room air, and [3] mortality, all measured through Day 29. The prespecified evidentiary standard was a 2-sided α of 0.2 (not adjusted for multiplicity).Results:Baseline demographics were balanced among the intervention groups; patients were racially diverse (43% non-white), had a mean age of 57 years, and 49% were obese (BMI ≥ 30). All patients received the local standard of care: 96% received corticosteroids (including dexamethasone) and 29% received remdesivir. No differences in outcomes were observed between the 10 mg/kg and 6 mg/kg mavrilimumab arms. Results for these groups are presented together. Mavrilimumab recipients had a reduced requirement for mechanical ventilation and improved survival: at day 29, the proportion of patients alive and free of mechanical ventilation was 12.3 percentage points higher with mavrilimumab (86.7% of patients) than placebo (74.4% of patients) (Primary endpoint; p=0.1224). Mavrilimumab recipients experienced a 65% reduction in the risk of mechanical ventilation or death through Day 29 (Hazard Ratio (HR) = 0.35; p=0.0175). Day 29 mortality was 12.5 percentage points lower in mavrilimumab recipients (8%) compared to placebo (20.5%) (p=0.0718). Mavrilimumab recipients had a 61% reduction in the risk of death through Day 29 (HR= 0.39; p=0.0726). Adverse events occurred less frequently in mavrilimumab recipients compared to placebo, including secondary infections and thrombotic events (known complications of COVID-19). Thrombotic events occurred only in the placebo arm (5/40 [12.5%]).Conclusion:In a global, diverse population of patients with severe COVID-19 pneumonia and hyperinflammation receiving supplemental oxygen therapy, corticosteroids, and remdesivir, a single infusion of mavrilimumab reduced progression to mechanical ventilation and improved survival. Results indicate mavrilimumab, a potent inhibitor of GM-CSF signaling, may have added clinical benefit on top of the current standard therapy for COVID-19. Of potential importance is that this treatment strategy is mechanistically independent of the specific virus or viral variant.

Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b'a' Domain.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b′a′ domain of protein disulfide isomerase (PDIb′a′) greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb′a′-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC50 of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.

Construction of Recombinant Human GM-CSF and GM-CSF-ApoA-I Fusion Protein and Evaluation of Their Biological Activity.

In this study, two strains of the yeast P. pastoris were constructed, one of which produced authentic recombinant human granulocyte-macrophage colony-stimulating factor (ryGM-CSF), and the other was a chimera consisting of ryGM-CSF genetically fused with mature human apolipoprotein A-I (ApoA-I) (ryGM-CSF-ApoA-I). Both forms of the cytokine were secreted into the culture medium. The proteins’ yield during cultivation in flasks was 100 and 60 mg/L for ryGM-CSF and ryGM-CSF-ApoA-I, respectively. Both forms of recombinant GM-CSF stimulated the proliferation of human TF-1 erythroleukemia cells; however, the amount of chimera required was 10-fold that of authentic GM-CSF to induce a similar proliferative effect. RyGM-CSF exhibited a 2-fold proliferative effect on BFU-E (burst-forming units—erythroid) at a concentration 1.7 fold less than non-glycosylated E. coli-derived GM-CSF. The chimera together with authentic ryGM-CSF increased the number of both erythroid precursors and BMC granulocytes after 48 h of incubation of human bone marrow cells (BMCs). In addition, the chimeric form of ryGM-CSF was more effective at increasing the viability of the total amount of BMCs, decreasing apoptosis compared to the authentic form. ryGM-CSF-ApoA-I normalized the proliferation, maturation, and segmentation of neutrophils within the physiological norm, preserving the pool of blast cells under conditions of impaired granulopoiesis. The chimera form of GM-CSF exhibited the properties of a multilinear growth factor, modulating the activity of GM-CSF and, perhaps, it may be more suitable for the normalization of granulopoiesis.

B cell\u2010activating factors in autoimmune pulmonary alveolar proteinosis

BackgroundAutoimmune pulmonary alveolar proteinosis (APAP) results from the suppression of granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling by a neutralizing autoantibody against GM-CSF. B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) are involved in immunoglobulin G production and are overproduced in various autoimmune disorders. We hypothesized that BAFF and/or APRIL levels would be elevated in serum and bronchoalveolar lavage fluid (BALF) and serum and BALF levels of BAFF and APRIL respond to the treatments (whole lung lavage (WLL) or inhalation of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF)) in patients with APAP.Subjects and methods BAFF and APRIL levels in serum and BALF from 110 patients with APAP were measured at baseline and during and after treatment, using an enzyme-linked immunosorbent assay kit. We enrolled 34 healthy volunteers as serum cytokine controls, and 13 disease controls for BALF. Associations of BAFF and APRIL levels with clinical measures were assessed to clarify their clinical roles.ResultsIn patients with APAP, serum BAFF and APRIL levels were significantly increased relative to healthy volunteers (p < 0.0001 and p < 0.05, respectively), and BALF BAFF and APRIL levels were significantly increased versus disease controls (p < 0.0001 and p < 0.01, respectively). Serum BAFF levels (but not APRIL levels) were significantly correlated with Krebs von den Lungen-6 (KL-6), surfactant protein (SP)-D, SP-A, and lactate dehydrogenase (p < 0.0001). There was no significant correlation between serum BAFF or APRIL levels and anti-GM-CSF autoantibody. BAFF and APRIL were negatively correlated with single-breath diffusion capacity for carbon monoxide (DLco) (p = 0.004) and forced vital capacity (p = 0.04), respectively. BAFF (but not APRIL) in BALF was negatively correlated with vital capacity (p = 0.04) and DLco (p = 0.006). There were significant correlations between disease severity and BAFF levels in serum (p = 0.04) and BALF (p = 0.007). Serum levels of anti-GM-CSF autoantibody, BAFF, and APRIL were not significantly affected by WLL or inhalation of recombinant human GM-CSF.ConclusionsBAFF and APRIL levels of sera and BALF in APAP were significantly increased compared with healthy volunteer and disease control, and the BAFF and APRIL pathway might have important specific roles in pathogenesis of APAP. Our data suggest a new perspective of future treatment for APAP.

Масштабирование процесса получения рекомбинантного гранулоцитарно-макрофагального колониестимулирующего фактора человека и получение конструкций на его основе

Abstract-A step-by-step scaling-up of the production of recombinant human granulocyte-macrophage colony-stimulating factor has been carried out. The tenfold expantion of the technology made it possible to obtain 5 mg of the target protein with a purity of more than 96% from 1 g of wet cells. The produced protein series meets the requirements for recombinant proteins of the XIV edition of the State Pharmacopoeia of the Russian Federation. Conjugates of recombinant human granulocyte-macrophage colony-stimulating factor with dextran and molecular construct of the conjugates and double-stranded RNA were obtained. The adjuvant activity of the resulting preparations was shown in different models. Key words: granulocyte-macrophage colony-stimulating factor, chromatography, molecular construct We express our gratitude to the staff of the Laboratory of Immunochemistry of the SRC VB Vector N. V. Volkova, M. B. Borgoyakova, A. A. Isaeva and S. V. Belenkaya for assessing immunogenicity in the RBD model.

Role of Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor (rhGM-CSF) in Wound Healing in Critical Care Settings

Role of Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor (rhGM-CSF) in Wound Healing in Critical Care Settings

Влияние типа посевного материала и условий глубинного культивирования рекомбинантных штаммов Escherichia coli на накопление и качество целевого белка на примере рекомбинантных колониестимулирующих факторов человека

Определены оптимальные параметры воздухообмена для культивирования рекомбинантных штаммов Escherichia coli SG 20050/pGGF8 и SG 20050/p280 2GМ — продуцентов гранулоцитарного и гранулоцитарно-макрофагального колониестимулирующих факторов человека в зависимости от размеров и степени заполняемости средой ферментеров типа «Биотрон». Выбраны режимы культивирования, позволяющие получать биомассы с удельным выходом от 2,2 до 3,5 г с 1 л культуральной жидкости и содержанием целевого белка от 19,8 до 26,5 % независимо от типа посевного материала. Выход целевого белка рчГ-КСФ составил от 3,2 до 4,6 мг, рчГМ-КСФ — от 4,0 до 5,5 мг с 1 г биомассы. Выбранные условия позволяют получать белки фармацевтической чистоты, отвечающие современным требованиям к препаратам на основе рекомбинантных цитокинов.

Malignant Progenitors From Patients With Acute Myelogenous Leukemia Are Sensitive to a Diphtheria Toxin–Granulocyte-Macrophage Colony-Stimulating Factor Fusion Protein

AbstractWe have previously demonstrated that human granulocyte-macrophage colony-stimulating factor (GM-CSF) fused to a truncated diphtheria toxin (DT388-GMCSF) kills acute myelogenous leukemia (AML) cell lines bearing the GM-CSF receptor. We now report that exposure of malignant cells from 50 different patients with AML for 48 hours in culture to DT388-GMCSF reduces by a median of 1.6 logs (range, 0 to 3.7 logs) the number of leukemic cells capable of forming colonies in semisolid media (leukemic colony-forming cells [CFU-L]) with a median IC50 of 3 × 10−12 mol/L (range, 5 to >4,000 × 10−12 mol/L). Furthermore, the cell kill is dependent on the presence of high-affinity GM-CSF receptors on leukemic blasts, because CFU-L from 27 of 28 AML samples expressing ≥35 GM-CSF receptors per cell were inhibited by the toxin, whereas the colony growth from all 4 leukemic samples (2 AML, 1 acute lymphoblastic leukemia [ALL], and 1 prolymphocytic leukemia [PLL]) that had less than 35 receptors per cell was unaffected by the drug. Sensitivity of CFU-L to DT388-GMCSF was seen regardless of the clinical responsiveness of the patient's leukemia to standard chemotherapy agents. In contrast, clonogenic cells from normal bone marrow formed colonies at near control numbers after exposure to much higher toxin concentrations (4 × 10−9 mol/L) than those required to kill CFU-L from most patients. Thus, leukemic progenitors isolated directly from the peripheral blood of most AML patients show the same sensitivity to DT388-GMCSF as previously demonstrated for AML cell lines. Under the same conditions of exposure, normal hematopoietic progenitors are relatively unaffected by DT388-GMCSF, suggesting its potential as a therapeutic agent in AML.

Effect of Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-3 on Interleukin-8 Production by Human Neutrophils and Monocytes

AbstractInterleukin-8 (IL-8) is a major neutrophil chemoattractant and functional stimulant that is induced by IL-1, tumor necrosis factor alpha (TNFα), and lipopolysaccharide (LPS). We report that recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and rhlL-3 are also potent inducers of IL-8 messenger RNA (mRNA) accumulation and protein secretion by normal peripheral blood monocytes. Neutrophils produce IL-8 in response to GM-CSF but not to IL-3. In contrast, recombinant human granulocyte-CSF (rhG-CSF), at concentrations as high as 100 ng/mL, does not induce IL-8 in either cell type. rhGM-CSF also induces IL-8 mRNA expression and IL-8 protein in the promonocytic cell line, U-937, whereas rhG-CSF does not. IL-8 secretion by monocytes was stimulated within 2 hours after incubation with rhGM-CSF or rhlL-3. Stimulation of neutrophils with rhGM-CSF resulted in an increase in cell-associated IL-8 at 4 hours. At 24 hours, cell-associated IL-8 levels declined, whereas secreted IL-8 levels increased. In contrast, virtually all IL-8 induced in monocytes appeared as secreted protein. Neither rhGM-CSF nor rhlL-3 induced detectable secretion of IL-1, TNFα, or IL-6 protein by monocytes. rhGM-CSF, and to a lesser degree rhlL-3, potently stimulated IL-8 secretion in cultures of heparinized whole blood, whereas rhG-CSF had no significant effect on IL-8 secretion. Induction of IL-8 by GM-CSF may be physiologically important in enhancing the acute inflammatory response.

Diphtheria Toxin Fused to Granulocyte-Macrophage Colony-Stimulating Factor Is Toxic to Blasts From Patients With Juvenile Myelomonocytic Leukemia and Chronic Myelomonocytic Leukemia

AbstractWe have previously demonstrated that human granulocyte-macrophage colony-stimulating factor fused to a truncated diphtheria toxin (DT388-GM-CSF) is toxic to patient acute myeloid leukemia progenitors bearing the GM-CSF receptor, but not normal marrow progenitors. We now report that exposure of mononuclear cells from five of seven (71%) juvenile myelomonocytic leukemia (JMML) patients and from 12 of 20 (60%) adult chronic myelomonocytic leukemia (CMML) patients to 10-9 mol/L DT388-GM-CSF for 48 hours in culture reduces the number of cells capable of forming colonies in semisolid medium (colony-forming units–leukemia) 10-fold to 300-fold (1 to 2.5 log decrease). In contrast, normal myeloid progenitors (colony-forming unit–granulocyte-macrophage) from six different donors treated and assayed under identical conditions were consistently insensitive to the same fusion toxin even when treated as highly purified CD34+ cells. The leukemic progenitors from the two other JMML patients showed intermediate sensitivity to DT388-GM-CSF and the leukemic progenitors from eight of the 20 (40%) CMML patients were not different from normal progenitors. Parallel measurements of the number and affinity of GM-CSF receptors on cells from the same samples showed no consistent differences between JMML, CMML, and normal light density or CD34+ bone marrow cells. The increased sensitivity of leukemic progenitors from all JMML progenitors and some CMML patients to the fusion toxin is therefore not likely to be explained by an increased density of GM-CSF receptors on these cells. We also examined the DT388-GM-CSF sensitivity of two murine cell lines transfected with cDNAs encoding varying portions of the human GM-CSF receptor α and/or β chains. These studies showed that high-affinity ligand binding was sufficient for DT388-GM-CSF–induced toxicity, as this could occur even in the absence of functional signal transduction and that the background of the host cell had a major influence on the degree to which this decreased the toxicity of DT388-GM-CSF. The selective sensitivity to DT388-GM-CSF of leukemic progenitors from a majority of JMML and CMML patients suggests that this agent could have therapeutic potential for some patients with these diseases.

A Placebo-Controlled Study of Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor Administered During and After Induction Treatment for De Novo Acute Myelogenous Leukemia in Elderly Patients

The complete remission (CR) rate after intensive chemotherapy for acute myelogenous leukemia (AML) remains low in elderly patients, mainly because of a higher infectious mortality rate related to neutropenia and an increased incidence of adverse prognostic factors. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to potentially recruit leukemic blasts into cell cycle and improve cytotoxic effects when given during chemotherapy, and to shorten the duration of neutropenia when administered after chemotherapy. Two hundred forty patients aged 55 to 75 years who had newly diagnosed AML were randomly assigned to receive placebo or Escherichia coli-derived GM-CSF (5 micrograms/kg/d by 6-hour intravenous infusion) starting during induction chemotherapy on day 1 and continued through and after chemotherapy until recovery of neutrophils, or evidence of regrowth of leukemia, or up to day 28. Induction chemotherapy consisted of idarubicin (8 mg/m2/d on days 1 to 5) and cytarabine (100 mg/m2/d on days 1 to 7). The study drug was not administered subsequent to the induction course. Patients who achieved a CR received continuous maintenance therapy for 1 year with four quarterly reinduction courses; in the 55- to 64-year age subgroup, patients were randomly assigned to receive or not a consolidation course before maintenance therapy. The CR rate was similar in the GM-CSF and placebo groups (63% and 60.5%, respectively; P = .79). The mortality, rate of resistant disease, and rate of regrowth of leukemia were also similar in both groups. The time to neutrophil recovery was shorter in patients who received GM-CSF (24 v 29 days; P = .0001), but the incidence and characteristics of infectious events were not different. The 2-year disease-free survival (DFS) rate was significantly improved in the GM-CSF group (48% v 21% in the placebo group; P = .003). This effect was highly significant in the cohort of patients aged 55 to 64, but only marginal in patients >/=65 years of age. There was a trend toward a longer overall survival (OS) in the GM-CSF group (P = .082). In summary, the administration of GM-CSF, concomitantly with chemotherapy and thereafter during induction course in AML, shortened the time to neutrophil recovery, but did not improve the CR rate in patients aged 55 to 75. Nonetheless, DFS and OS were significantly prolonged in patients aged 55 to 64 treated with GM-CSF. These results are promising and further evaluation of myeloid growth factors in AML is warranted.

High-affinity autoantibodies specifically eliminate granulocyte-macrophage colony-stimulating factor activity in the lungs of patients with idiopathic pulmonary alveolar proteinosis

AbstractDeficiency of granulocyte-macrophage colony-stimulating factor (GM-CSF) in mice results in pulmonary alveolar proteinosis (PAP) from impaired surfactant catabolism by alveolar macrophages (AMs). Recently, we have shown that neutralizing anti-GM-CSF autoantibodies develop specifically in patients with idiopathic pulmonary alveolar proteinosis (iPAP). Analogous to murine PAP models, it is plausible that the autoantibodies reduce GM-CSF activity, resulting in AM dysfunction and surfactant accumulation. To examine this hypothesis, we estimated the neutralizing activity of the autoantibodies in the lungs of patients and characterized their biologic properties. GM-CSF bioactivity was completely abrogated in the bronchoalveolar lavage fluid (BALF) of patients with iPAP but not in healthy subjects. Autoantibodies were present in the alveoli in high concentrations and colocalized with GM-CSF. They recognized human GM-CSF with high avidity (KAV = 20.0 ± 7.5 pM) and high specificity, reacting with its superstructure and neutralizing GM-CSF activity to a level 4000 to 58 000 times the levels of GM-CSF normally present in the lung. Although target epitopes varied among patients, GM-CSF amino acids 78 to 94 were consistently recognized. Thus, autoantibodies bind GM-CSF with high specificity and high affinity, exist abundantly in the lung, and effectively block GM-CSF binding to its receptor, inhibiting AM differentiation and function. Our data strengthen the evidence associating anti-GM-CSF autoantibodies with the pathogenesis of this disease. (Blood. 2004;103:1089-1098)

Study on Hemostimulating Properties of Granulocyte-Macrophage Colony Stimulating Factor

The hemostimulating properties of granulocyte-macrophage colony-stimulating factor (GM-CSF) make possible its clinical use in alleviating side effects of anti-cancer radio- and chemotherapy, in bone marrow transplantation, and in the treatment of some primary immunodeficiency conditions associated with leukopenia. The State Research Center of Virology and Biotechnology “Vector” of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing has developed a high-performance technology for production of recombinant human GM-CSF (rhGM-CSF) based on a recombinant E. coli strain.The aim of the study was to assess hemostimulating activity of the rhGM-CSF preparation obtained using the new developed technology, as observed in cell culture and in the mice model of myelosuppression induced by cyclophosphamide administration.Materials and methods: in vitro evaluation of rhGM-CSF hemostimulating activity was performed by MTT assay in the commercial HL-60 promyelocytic leukemia cell culture with preliminary suppression of cell growth rate by adding a low concentration of dimethyl sulfoxide to the medium. In vivo studies were carried out in CBA/CaLac mice with cyclophosphamide-induced myelosuppression. The hemostimulating properties of the drug were evaluated after subcutaneous administration of 1–175 µg/kg doses for 4–5 days, following administration of a cytostatic agent. The total number of leukocytes and the content of their morphological forms were determined in blood samples taken at different time points after the drug administration. The statistical processing of the experimental data was based on analysis of variance using Statgraphics v. 5.0 software.Results: the proliferative activity of HL-60 cells incubated with the rhGM-CSF preparation for 72 hours was shown to be dose-dependent. The highest values of the increase in proliferative activity associated with an increase in the drug dose were observed in the concentration range from 0.04 to 0.64 ng/mL (proliferative activity increased by 11–18% when the dose was increased twofold). The experiments in mice demonstrated a two-phase pattern of the dose-dependent effect. The drug showed the highest hemostimulating effect at the dose of 90 µg/kg.Conclusions: the rhGM-CSF preparation obtained using the new developed technology has a pronounced hemostimulating activity confirmed by both in vitro and in vivo test systems.

Effectiveness of recombinant human granulocyte macrophage colony-stimulating factor for treating deep second-degree burns: a systematic review and meta-analysis

IntroductionIt is uncertain whether treatment by recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) can promote healing of deep second-degree burns. This meta-analysis aimed to systematically review and assess randomised controlled...

The Tumor Milieu Promotes Functional Human Tumor-Resident Plasmacytoid Dendritic Cells in Humanized Mouse Models.

Particular interest to harness the innate immune system for cancer immunotherapy is fueled by limitations of immune checkpoint blockade. Plasmacytoid dendritic cells (pDC) are detected in a variety of solid tumors and correlate with poor clinical outcome. Release of type I interferons in response to toll-like-receptor (TLR)7 and TLR9 activation is the pDC hallmark. Mouse and human pDC differ substantially in their biology concerning surface marker expression and cytokine production. Here, we employed humanized mouse models (HIS) to study pDC function. We performed a comprehensive characterization of transgenic, myeloid-enhanced mouse strains (NOG-EXL and NSG-SGM3) expressing human interleukin-3 (hIL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) using identical humanization protocols. Only in HIS-NOG-EXL mice sufficient pDC infiltration was detectable. Therefore, we selected this strain for subsequent tumor studies. We analyzed pDC frequency in peripheral blood and tumors by comparing HIS-NOG-EXL with HIS-NOG mice bearing three different ovarian and breast tumors. Despite the substantially increased pDC numbers in peripheral blood of HIS-NOG-EXL mice, we detected TLR7/8 agonist responsive and thus functional pDCs only in certain tumor models independent of the mouse strain employed. However, HIS-NOG-EXL mice showed in general a superior humanization phenotype characterized by reconstitution of different myeloid subsets, NK cells and B cells producing physiologic IgG levels. Hence, we provide first evidence that the tumor milieu but not genetically introduced cytokines defines intratumoral (i.t.) frequencies of the rare pDC subset. This study provides model systems to investigate in vivo pro- and anti-tumoral human pDC functions.

GRANULOCYTE-COLONY STIMULATING FACTOR (G-CSF) AND GRANULOCYTE-MACROPHAGE CSF (GM-CSF) ADMINISTRATION ENRICHES FOR HIGHLY SUPPRESSIVE CD4+CD45RA-FOXP3HI CELLS IN LEUKAPHERESIS PRODUCTS OF RHESUS MONKEYS

Introduction: In humans, CD4+CD45RA-Foxp3hi T cells comprise a regulatory T cell (Treg) subset with highly suppressive function. We have previously shown that granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF) administration enhances the incidence of peripheral blood CD4+CD25hiFoxp3hi Treg in non-human primates. Here, we examined whether GM-CSF/G-CSF administration enhances the incidence of CD4+CD45RA-Foxp3hi Treg in leukapheresis products (LP) of rhesus monkeys. Methods: Rhesus monkeys peripheral blood mononuclear cells (PBMC, n=3) were cultured with either GM-CSF (1ng/ml) or G-CSF (1ng/ml). Treg in PBMC were assessed based on their expression of CD45RA, CD25 and Foxp3. The percentages and phenotype of CD4+CD45RA-Foxp3hi Treg and CD4+CD45RA-Foxp3lo populations were evaluated. Naïve rhesus monkeys (n=6; 5-7kg) were treated sequentially with recombinant human GM-CSF (10 mg/kg/day for 4 days), then recombinant human G-CSF (10 mg/kg/day for 4 days), followed by leukapheresis. Similarly, Treg in PBMC, obtained before treatment, and in LP obtained after GM-CSF/GCSF administration were assessed. Additionally, LP CD4+CD45RA-CD25lo and CD4+CD45RA-CD25hi populations were flow-sorted (n=5) and assessed for their suppressive function. Results: In vitro, G-CSF did not enhanced Treg percentages. Meanwhile, GM-CSF enhanced the percentages of both CD4+CD25hiFoxp3hi and CD4+CD45RA-Foxp3hi Treg populations. In vivo, while G-CSF administration increased lymphocyte numbers, no increase in Treg percentages was observed. On the other hand, GM-CSF administration augmented the percentages of both CD4+CD25hiFoxp3hi and CD4+CD45RA-Foxp3hi Treg populations in peripheral blood. After combined G-CSF/GM-CSF administration, the incidences of CD4+CD45RA-Foxp3hi Treg increased significantly in LP (p<0.01), compared to peripheral blood before treatment. Furthermore, the ratio of CD4+CD45RA-Foxp3hi to CD4+CD45RA-Foxp3lo cells increased significantly (p<0.05). In LP, The CD4+CD45RA-CD25hi population expressed significantly higher levels of Foxp3, CTLA4 and Helios, compared to the CD4+CD45RA-CD25lo population (p<0.05). In 5 animals, LP flow-sorted CD4+CD45RA-CD25hi cells exhibited higher suppressive capacity of T cell proliferation in response to anti-CD2/CD3/CD28 stimulation, compared to the CD4+CD45RA-CD25hi population. Conclusions: G-CSF/GM-CSF administration enhances the incidences of highly suppressive CD4+CD45RA-Foxp3hi T cells in LP of mobilized rhesus monkeys. These observations further support the therapeutic potential of Treg-enriched LP from mobilized individuals. NIH grant U19 AI131453.

Topical treatment for facial burns.

Background Burn injuries are an important health problem. They occur frequently in the head and neck region - the area central to a person's identity, that provides our most expressive means of communication. Topical interventions are currently the cornerstone of treatment of partial-thickness burns to the face. Objectives To assess the effects of topical interventions on wound healing in people with facial burns of any depth. Search methods We searched the Cochrane Wounds Group Specialised Register (searched 12 November 2012); the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2012, Issue 10); Ovid MEDLINE (1950 to November Week 1 2012); Ovid MEDLINE - In-process & Other Non-Indexed Citations (searched November 12, 2012); Ovid EMBASE (1980 to 2012 Week 45); and EBSCO CINAHL (1982 to 9 November 2012) for relevant trials. We did not apply date or language restrictions. Selection criteria Randomised controlled trials (RCTs) that evaluated the effects of topical treatment for facial burns were eligible for inclusion in this review. Data collection and analysis Two review authors independently assessed and included the references identified by the search strategy. Included trials were assessed using a risk of bias form, and data were extracted using a standardised data extraction sheet. For dichotomous and continuous outcomes, we calculated risk ratios and mean differences, respectively, both with 95% confidence intervals (CI). Main results We included five RCTs, comprising a total of 119 participants. Two studies compared two different antimicrobial agents and three compared a biological or bioengineered skin substitute with an antimicrobial agent. All studies had small sample sizes and were at high risk of bias. Heterogeneity of interventions and outcomes prevented pooling of data. In three studies time to complete wound healing was significantly shorter for those using a skin substitute than for those using an antibacterial agent, but the quality of the evidence was low. Pain was significantly reduced with the use of skin substitutes in both studies that reported this outcome in all groups, range mean differences -2.00 (95% CI -3.82 to -0.18) to -4.00 (95% CI -5.05 to -2.95) on a 10-point scale. Authors' conclusions There is insufficient high quality research and evidence to enable conclusions to be drawn about the effects of topical interventions on wound healing in people with facial burns.

Puerperal Sepsis Caused by Clostridium Innocuum in a Patient with Placenta Accreta and Literature Review

Puerperal Sepsis Caused by Clostridium Innocuum in a Patient with Placenta Accreta and Literature Review