Recombinant Human GM-CSF Research Articles

Abstract 6744: Activating anti-MDS immunity with polarized dendritic cell vaccines

Abstract Less than 50% of patients with higher-risk myelodysplastic syndromes (MDS) show clinical response to standard-of-care hypomethylating (HMA) therapy. Significant enthusiasm existed for immunotherapy/HMA combinations for these patients, but studies have failed to deliver on the initial promise. We have demonstrated that patients with MDS have a deficiency in antigen-presenting cells, called CD141Hi dendritic cells (DC). DC-deficient MDS patients have reduced response to HMAs, worse overall survival, and show less response to cancer vaccination. We hypothesized that restoration of this population using a novel DC vaccination approach - alpha-type-1 polarized dendritic cells (αDC1) - might compensate for this deficiency and restore immunity. To generate αDC1s, we collected monocytes from MDS patients and healthy donors (HD) and cultured them for 6 days in vitro in the presence of recombinant human GM-CSF and IL-4 followed by an additional 18-24 hours in the presence of αDC1 maturation factors (poly-I:C/IFNα/IFNγ/TNFα/IL-1β). Overall, we attempted to generate αDC1s from 15 MDS samples; 13/15 exhibited intermediate to very high-risk disease and 10/15 are currently receiving HMA therapy. We successfully cultured cells from 8/15 samples (53%). Of the 8 MDS samples, 5 were collected from patients receiving HMA therapy (62.5%). We were unable to successfully culture αDC1s from the other 5 patients receiving HMA therapy, and thus the effect of HMA therapy on aDC1 generation remains unclear. To test the function of MDS αDC1s, we focused on two signals necessary to activate immune responses: 1) the expression of co-stimulatory molecules and 2) the production of IL-12p70. Flow cytometric analysis on αDC1s and immature DCs from MDS samples revealed intra-patient variation in the fold-change surface expression of CD40/80/83/86 and HLA-DR proteins. Additionally, there was a trend towards decreased up-regulation of CD80 and CD86 in MDS specimens compared to HD. To assess production of IL-12p70, we stimulated immature DCs and αDC1s with CD40L and performed ELISA analysis on the media supernatant. We found that 3 out of 4 samples could produce IL-12p70 (range 27 - 5,480 pg/ml). To date, our results suggest that generation of MDS patient-derived αDC1 cells is feasible and additional work is being performed to fully characterize their ability to activate anti-MDS immune responses. Since αDC1s have an established safety profile in patients with solid tumors, we anticipate that we will be able to rapidly translate our results to the clinic. Citation Format: Daniel Alvarado, Jennifer Peresie, Adam Brinkman, Cheryl Allen, Weijian Jiang, Pawel Kalinski, Tiffany Tanaka, Elizabeth Griffiths, Michael Nemeth. Activating anti-MDS immunity with polarized dendritic cell vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6744.

Sargramostim for Prophylactic Management of Gastrointestinal Immune-Related Adverse Events of Immune Checkpoint Inhibitor Therapy for Cancer

Immune checkpoint inhibitor (ICI) therapy improves outcomes in several cancers. Unfortunately, many patients experience grade 3–4 treatment-related adverse events, including gastrointestinal (GI) toxicities which are common. These GI immune-related adverse events (irAEs) induced by ICIs present significant clinical challenges, require prompt intervention, and result in treatment delays or discontinuations. The treatment for these potentially severe and even fatal GI irAEs which include enterocolitis, severe diarrhea, and hepatitis may interfere with the anti-cancer approach. Sargramostim (glycosylated, yeast-derived, recombinant human GM-CSF) is an agent that has been used in clinical practice for more than 30 years with a well-recognized safety profile and has been studied in many therapeutic areas. The mechanism of action of sargramostim may treat moderate-to-severe GI irAEs without impairing the anti-cancer therapy. Some early data also suggest a potential survival benefit. Through the differentiation/maturation of monocytes, macrophages, and neutrophils and induction of anti-inflammatory T cell responses, GM-CSF aids in GI homeostasis, mucosal healing, and mucosal immunity. GM-CSF knockout mice are susceptible to severe colitis which was prevented with murine GM-CSF administration. For some patients with GI mucosa and immune cell function impairment, e.g., Crohn’s disease, sargramostim reduces disease severity. In a prospective, randomized study (ECOG 1608), advanced melanoma patients had a reduction in grade 3–5 GI irAEs and less frequent colonic perforation in the sargramostim plus ipilimumab arm compared to ipilimumab alone. Sargramostim continues to be studied with ICIs for the prophylactic management of irAEs while also potentially providing a survival benefit.

Nebulised sargramostim in pulmonary alveolar proteinosis.

Five patients, comprising nine treatment courses of sargramostim use in pulmonary alveolar proteinosis, are described. The prevailing standard of treatment, whole lung lavage (WLL), is highly invasive, resource intensive and carries some procedural risk. Nebulised recombinant human GM-CSF (sargramostim) offers a pharmacological treatment option, allowing patients to be treated at home, possessing potential advantages in patient experience and wider health resourcing. The majority of reported patients described subjective improvement in symptoms along with radiographical improvement, although this did not translate into significant improvement in pulmonary function testing. Drug scarcity and high drug cost remain potential barriers to accessing this treatment, and so careful patient selection and treatment outcome assessment remain as challenging needs. Incorporating the routine assessment of validated patient symptom scores with objective physiological measures will allow prediction of response to treatment and help guide management. This report describes the largest published experience of sargramostim use in Australia.

Sargramostim and immune checkpoint inhibitors: combinatorial therapeutic studies in metastatic melanoma.

The use of immune checkpoint inhibitors in patients with metastatic melanoma generates clinical benefit, including improved survival. Yet disease resistance and immune-related adverse events persist as unmet needs. Sargramostim, a yeast-derived recombinant human GM-CSF, has shown clinical activity against diverse solid tumors, including metastatic melanoma. Here we review the use of sargramostim for treatment of advanced melanoma. Potential sargramostim applications in melanoma draw on the unique ability of GM-CSF to link innate and adaptive immune responses. Wereview preclinical and translational data describing the mechanism of action of sargramostim and synergy with immune checkpoint inhibitors to enhance efficacy and reduce treatment-related toxicity.

Human type 2 macrophages biologically active soluble products in the editing of stress-induced depressive-like behavior

IntroductionIn the scientific world widely discussed phenomenon of “cytokine-induced depression”. Macrophages have high plasticity and are able to control the inflammatory response; in particular, anti-inflammatory type-2 macrophages have a pronounced potential due to complex soluble factors production.ObjectivesWe have developed an original method for the type-2 macrophages generation; the resulting macrophages are characterized by the high level of a whole range of neurotrophic, neuroprotective, proangiogenic and anti-inflammatory factors production. The aim of the study was to investigate effects of human type-2 macrophages soluble products on behavioral phenotype and brain cytokines synthesis in depressive-like animals.MethodsType-2 macrophages were generated by culturing an adherent fraction of mononuclear cells with 50 ng/ml recombinant human GM-CSF in serum deprivation conditions for 7 days. (CBA x C57Bl/6)F1 depressive-like male mice, developed under the long-term social stress, were undergoing the human type-2 macrophages conditioned medium intranasal administration (60 ml twice daily for one animal) for 6 days. Mice behavioral phenotyping was carried out using an automatic registration system (Noldus Information Technology). Cytokines were determined by ELISA.ResultsDepressive-like mice behavioral phenotyping after type-2 macrophages conditioned medium administration revealed anhedonia decrease, motor activity stimulation in the open field and forced swimming tests, anxiety reduction in elevated plus maze. Behavioral changes were recorded against the pro-inflammatory cytokines (TNF-α, IL-1β, IL- 6, INFγ) decrease in striatum and hippocampus, as well as anti-inflammatory IL- 10 increase in hippocampus and hypothalamus.ConclusionsResults demonstrated the effectiveness of human type-2 macrophages biologically active soluble products in relation to the stress-induced depressive-like behavior editingDisclosureNo significant relationships.

Abstract 5602: BT-001, an oncolytic vaccinia virus armed with a Treg-depletion-optimized recombinant human anti-CTLA4 antibody and GM-CSF to target the tumor microenvironment

Abstract Oncolytic virotherapy is a clinically validated class of immune modulatory drugs with activity in solid cancer. The mode of action of armed oncolytic Vaccinia viruses (oVV) includes cell death induction following selective replication in tumor cells, local inflammation and priming of adaptive immune responses, and intra-tumoral delivery of therapeutic modalities e.g. antibodies, cytokines, enzymes and engineered receptor ligands. BioInvent and Transgene are collaborating in the development of a next generation armed oncolytic viruses, bringing respectively their n-CoDeR F.I.R.S.T antibody (mAb) discovery and Invir.IO oVV platforms. The first product, named BT-001, consists of the Copenhagen oVV strain deleted in J2R and I4L viral genes allowing restricted replication in proliferating cells, and the human IgG1 4-E03 recognizing human CTLA4. 4-E03 shows improved Treg-depleting activity compared with ipilimumab. BT-001 also encodes GM-CSF, the cytokine expressed in clinically advanced products like T-Vec (Imlygic) or Pexa-Vec. A surrogate mAb with similar pronounced Treg-depleting properties to 4-E03 was vectorized into the same oVV (mBT-1) allowing for functional and mechanistic in vivo studies. Since virus replication and expression of recombinant transgenes are tightly linked, the armed oVV is an interesting design for intra-tumoral delivery of therapeutic proteins exhibiting toxicological limitation (e.g. anti-CTLA4) and/or pharmacokinetic issues (e.g. GM-CSF). Oncoviral tumor-localized delivery of anti-CTLA4 may also allow better tolerated and more effective combination therapy with approved antibodies targeting the PD-1/PDL1 axis. Our studies demonstrate that 4-E03 and GM-CSF were expressed at high level as functional molecules after infection by BT-001 of several human tumoral cell lines in vitro. Moreover, following intratumoral administration in immune competent and immune deficient mice transplanted with mouse or human tumors, transgene expression was sustained at levels associated with receptor saturation for days to weeks. In contrast, and supporting the tumor-selective nature of oVV, blood concentrations of anti-CTLA4 mAb were lower compared to those observed following I.V. administration of therapeutic doses (3 mg/kg) of mAb. The in vivo anti-tumor activity of mBT-1 was assessed in multiple syngeneic mouse tumor models including CT26, EMT6, A20, and C38. mBT-1 conferred cures in the majority of challenged mice irrespective of tumor origin. The excellent anti-tumoral profile depends on anti-CTLA4 expression and could be boosted by co-administration of anti-PD-1 mAb. A clinical batch of BT-001 has been produced and toxicological evaluation is ongoing. Transgene and BioInvent are preparing CTA and IND filings for a multicentre clinical trial targeting injectable superficial tumors. Citation Format: Jean-Baptiste Marchand, Monika Semmrich, Laetitia Fend, Matilda Rehn, Nathalie Silvestre, Ingrid Teige, Johann Foloppe, Linda Mårtensson, Eric Quéméneur, Björn Frendeus. BT-001, an oncolytic vaccinia virus armed with a Treg-depletion-optimized recombinant human anti-CTLA4 antibody and GM-CSF to target the tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5602.

Optimization of Key Factors in Serum Free Medium for Production of Human Recombinant GM-CSF Using Response Surface Methodology.

Researchers add serum to a classical medium at concentrations of 5 to 10% (v/v) to grow cells in-vitro culture media. Unfortunately, serum is a poorly defined culture medium component as its composition can vary considerably while serum-free cell culture media are an excellent alternative to standard serum-containing media and offer several major advantages. Advantages of using serum-free media include a lower risk of infectious agents, lower risk of interfering components, less contaminant, avoids ethical issues. According to previous studies insulin, selenium, transferrin and glucose are important component of serum that affect cell growth. In the present study, we optimized amount of these factors in order to serum free culture medium fabrication. Response surface methodology (RSM) was employed for optimization of key factors in serum free medium to enhance recombinant human GM-CSF (rhGM-CSF) production in CHO cell line. Four important process parameters including insulin concentration (0-2 g/L), transferrin concentration (0-1 g/L), selenium concentration (0-0.001 g/L) and glucose concentration (0-5 g/L) were optimized to obtain the best response of rhGM-CSF production using the statistical Box–Behnken design. The experimental data obtained were analyzed by analysis of variance (ANOVA) and fitted to a second-order polynomial equation using multiple regression analysis. Numerical optimization applying desirability function was used to identify the optimum conditions for maximum production of rhGM-CSF. The optimum conditions were found to be insulin concentration = 1.1 g/L, transferrin concentration = 0.545 g/L, selenium concentration = 0.000724 g/L and glucose = 1. 4 g/L. Maximum rhGM-CSF production was found to be 3.5 g/L.

Microglia-like Cells Derived from Hematopoietic Stem and Progenitor Cells Are a Model System to Investigate Chronic Pain in Sickle Cell Disease

Background: Patients with sickle cell disease (SCD) experience acute pain episodes in early childhood; over 50% of adult SCD patients go on to develop chronic pain. In animal models, microglia, derived from hematopoietic stem cells, play an important role in the transition from acute to chronic pain; in patients with chronic pain microglia are often activated with minimal stimuli, releasing TNF-alpha and stimulating neurons. Research involving microglia has been stymied by difficulty in obtaining and culturing microglia, particularly from humans. We therefore propose to culture microglia-like cells from peripheral blood mononuclear cells (PBMC) from pediatric patients with SCD with and without chronic pain (defined as pain for more than 50% of days for at least 3 months) and from normal blood donors.Objectives: Our goal was to determine if our cultured cells exhibited microglia morphology and surface markers, could be activated with inflammatory stimuli, and if donor characteristics were retained in the culture derived microglia-like cells.Methods: PBMC were obtained from peripheral blood by Ficoll separation from three unique patients with SCD and no history of chronic pain (SCD CP-), peripheral blood from three unique patients with SCD with history of chronic pain (SCD CP+), or buffy coat from three human blood donors. PBMC were cultured with recombinant human GM-CSF (10 ng/ml; R&D Systems, MN), recombinant human IL-34 (100 ng/ml; R&D Systems), as previously described (Ohgidani et al, 2014) to induce ramified microglia. On day 7 of culture, cells were collected and morphology analyzed by phase contrast microscopy, phenotyped by flow cytometry by staining with CD11b and CD45 antibodies, indirect immunofluorescence performed with anti-CX3CR1antibodies and imaging by confocal laser scanning microscope. Microglia-like cells from 3 normal blood donors, 3 SCD CP- and 3 SCD CP+, were stimulated by 100 ng/mL LPS for 24 hours, beginning on day 6 of culture. Deramification (activation) was assessed by immunofluorescence using anti-Iba1, and by flow using anti-CD68.Results: PBMCs from patients with SCD ± CP and normal blood donors developed microglial morphology, namely, soma bodies with branched collaterals, after incubation with GM-CSF and IL-34. When subjected to flow cytometry, treated cells were CD11bhigh and CD45low; fluorescence microscopy revealed CX3CR1 positivity in the cultured cells, all consistent with accepted microglia phenotype. PBMCs untreated with GM-CSF and IL-34 maintained their round morphology, and were CD11blow, CD45high by FACS.Microglia-like morphology differed significantly between PBMC sources. Microglia-like cells from SCD CP+ had shorter and fewer branches than normal individuals; branching of microglia-like cells from SCD CP- were intermediate in number and length, greater than to SCD CP+ but less than normal individuals.When treated with LPS, nearly 100% of microglia-like cells derived from patients with SCD CP+ became amoeboid in shape, indicating microglial deramification, or activation, compared to approximately 25% of microglial-like cells derived from normal human blood donors or SCD CP-.We also assessed deramification through flow measurement of CD68, a marker found on activated microglia and found that the microglia-like cells derived from HSPCs from SCD CP+ patients had significantly higher CD68 positivity compared to microglia-like cells derived from HSPCs from normal human blood donors or SCD CP-, and significantly higher Iba1 positivity as measured by immunofluorescence.Conclusions: We established the microglia-like nature of the cultured PBMCs derived from patients with SCD±CP and normal blood donors through morphologic examination and confirmation of characteristic surface markers via flow and fluorescence microscopy, and through demonstration of activation with LPS. Our microglial-like cells derived from patients with SCD CP+ were activated at higher percentages by LPs treatment than microglial-like cells derived from normal human blood donors and SCD CP-, suggesting that donor characteristics are retained by the microglial-like cells developed in culture. We propose to use this model system to derive mechanistic insights into the development of chronic pain in SCD, and to screen pharmacologic agents to treat and prevent chronic pain. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Assay system development to measure the concentration of sargramostim with high specificity in patients with autoimmune pulmonary alveolar proteinosis after single-dose inhalation

During a clinical trial of a Saccharomyces cerviciae-derived recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF), sargramostim, in patients with autoimmune pulmonary alveolar proteinosis (aPAP), we conducted a pharmacokinetic study of single-dose sargramostim inhalation. Several problems were encountered whereby sargramostim formed an immune-complex with GM-CSF autoantibodies (GMAbs) immediately after entering the body; thus, we could not measure the concentration of sargramostim using a commercial high sensitivity enzyme-linked immunosorbent assay (ELISA). Moreover, the ELISA could not discriminate inhaled sargramostim from intrinsic GM-CSF. To solve these problems, we developed a novel ELISA system with a capture antibody that is specific for sargramostim and a detection antibody capable of binding with GM-CSF. This system quantified the serum sargramostim concentration, but not E. coli-, CHO-, or HEK293T-derived human recombinant GM-CSF. Using this system, serum pharmacokinetics were estimated in five patients after inhalation of 250 μg sargramostim, with a mean Cmax of 9.7 ± 2.85 pg/ml at a Tmax of 2 ± 1.22 h.

Abstract 3546: Extracellular vesicles and exosome-associated microRNAs in squamous cell carcinoma of the head and neck disrupt dendritic cell differentiation and maturation

Abstract Squamous cell carcinomas (SCCs) remain among the most common malignancies of the head and neck region. Plasma microRNAs (miRNAs) are appealing biomarker candidates for diagnosis or therapy response monitoring since they may be associated with clinical and pathological features of the disease. Increasing evidence also points to a role for these molecules, mostly when transported by extracellular vesicles (EVs), in signaling between cancer and immune cells. The aims of this study are to search for potential miRNA biomarkers for SCCs in plasma, to evaluate the presence of these markers within EVs using SCC-derived cell lines as study models and to study the effect of EVs on monocyte-derived dendritic cells functions. Total RNA was isolated from plasma samples from head and neck SCC patients and healthy donors following optimized protocols. EVs from the culture medium of SCC cell lines were collected by differential centrifugation and visualized by transmission electron microscopy. The expression profile of miRNAs in plasma and EVs was evaluated by qRT-PCR. CD14+ cells from healthy donors were selected from peripheral blood using magnetic selection columns. Monocyte-derived immature dendritic cells were obtained following CD14+ cells treatment with human recombinant IL-4 and GM-CSF for six days and the maturation signal consisted on treatment of immature dendritic cells with LPS for 24 hours. Both treatments were carried out either in the presence or absence of EVs. Immunophenotyping of monocytes and monocyte-derived dendritic cells was based on the detection and expression levels of cell surface markers CD14, CD33, CD45, CD80, CD86, CD209 and HLA-DR by flow cytometry. A subset of miRNAs was consistently detected in SCC patients' plasma and EVs. Among these, miR-24 is of special interest since it has been associated with antigen processing and presentation by dendritic cells. The percentage of CD14+ cells expressing CD33 when cultured in the presence of EVs was significantly lower, and the same effect was seen for the CD209 and CD80 markers, suggesting alterations of antigen presentation functions. The percentage of cells expressing HLA-DR was similar both when CD14+ cells were cultured in the presence or without the addition of EVs, but the level of HLA-DR expression, as measured by fluorescence intensity, was lower when EVs were present, following both differentiation and maturation signals. The detection of miRNAs both in plasma and in association with EVs corroborates their potential as cancer biomarkers but also their possible implication in cell signaling. Differences in surface markers of monocyte-derived dendritic cells in the presence of EVs suggest an effect of EV content, including miRNAs, on differentiation and maturation processes, essential aspects for the proper function of dendritic cells in the tumor microenvironment. Citation Format: Luciana C. Marti, Elisangela P. Silva, Mariana B. Rizzo, Rossana V. Lopez, Otavio A. Curioni, Raquel A. Moyses, Fabio D. Nunes, Patricia Severino. Extracellular vesicles and exosome-associated microRNAs in squamous cell carcinoma of the head and neck disrupt dendritic cell differentiation and maturation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3546.

Induction of Increased Levels of Matrix Metalloproteinase-2 (MMP-2) and -9 in Human Breast Cancer Cell Lines by Activation of GM-CSF Receptor Bc via C-Fos-ERK 1/2 Signaling

Background and Objectives: Matrix metalloproteinase (MMP) -2 and -9 play important roles in the invasion and metastasis of breast cancer, but the mechanism of their regulation is not clearly understood. GM-CSF has been shown to be associated with cancer invasion and metastasis. The goal of our study was to examine the stimulation of GM-CSF/ interleukin 3 (IL-3)/IL-5 receptor common β-chain (βc) and its effects on MMP-2 and -9 regulation in human breast cancer cells. Methods: The constitutive expression of the GM-CSF/IL-3/IL-5 receptor common βc and GM-CSF production were analyzed in BT 549, MCF-7, and MDA-MB 231 human breast cancer cell lines. We studied the effects of recombinant IL-3, IL-5 and GM-CSF on the gene expression and enzyme activity of MMP-2, and -9 in the aforementioned cell lines. The signaling pathway activated by these cytokines, the blocking of this pathway, and the effect on MMP-2 and -9 productions were also assessed. The downregulation of the GM-CSF receptor βc gene (CSF2RB) expression and its response to cytokine stimulation were also studied. Results: We observed that the human breast cancer cell lines BT 549, MCF-7, and MDA-MB 231 constitutively produce GM-CSF and express the GM-CSF/IL-3/IL-5 receptor common βc. When these cell lines were treated with recombinant human (rh) GM-CSF, IL-3, and IL-5, enzyme activity and gene expression of MMP-2, and -9 were increased. Conclusions: Our findings indicate that the activation of the c-Fos – ERK 1/2 signaling pathway upregulates MMP-2 in response to exogenous GM-CSF, IL-3 or IL-5 cytokines. Clinically relevant concentrations of GM-CSF (as low as 10 ng/mL) were sufficient to stimulate MMP-2 and -9. Our results suggest a potential mechanism by which GM-CSF may promote tumor invasion and metastases.

O-glycans and O-glycosylation sites of recombinant human GM-CSF derived from suspension-cultured rice cells, and their structural role

Recombinant human GM-CSF (rhGM-CSF) from yeast has been clinically applied to immunosuppressed patients. The production of suspension-cultured rice-cell-derived rhGM-CSF (rrhGM-CSF), which has a longer blood clearance time and the same bioactivity as yeast-derived rhGM-CSF, and the analysis of its N-glycans have been reported recently. However, there are no previous reports of the O-glycosylation of rhGM-CSF from plant cells, and so this study investigated O-glycans, O-glycosylation sites, and their structural role in rrhGM-CSF. Monosaccharide analysis revealed the presence of O-glycans comprising arabinose and galactose. Eight O-glycans comprising four arabinose residues with zero to seven galactose residues along with their relative quantities were analyzed. Analysis of pronase-digested glycopeptides indicated that the O-glycans are partially attached to Ser 5, Ser 7, Ser 9, or Thr 10 residues, and glycan heterogeneity was confirmed at each site. Pro-to-hydroxyproline conversions occurred at Pro 2, Pro 6, and Pro 8 residues. The preparation of deglycosylated rrhGM-CSFs revealed that deglycosylation greatly affects their α-helix structures. These findings indicate that O-glycans of rrhGM-CSF are essential for maintaining its structural stability and result in an extended in vivo half-life, but without affecting its biological function. This is the first report on the O-glycosylation of rhGM-CSF derived from plant cells.

Inhaled Sargramostim Induces Resolution of Pulmonary Alveolar Proteinosis in Lysinuric Protein Intolerance.

Pulmonary alveolar proteinosis (PAP) is a potentially fatal complication of lysinuric protein intolerance (LPI), an inherited disorder of cationic amino acid transport. The patients often present with mild respiratory symptoms, which may rapidly progress to acute respiratory failure responding poorly to conventional treatment with steroids and bronchoalveolar lavations (BALs). The pathogenesis of PAP in LPI is still largely unclear. In previous studies, we have shown disturbances in the function and activity of alveolar macrophages of these patients, suggesting that increasing the activity and the number of macrophages by recombinant human GM-CSF (rhuGM-CSF) might be beneficial in this patient group.Two LPI patients with complicated PAP were treated with experimental inhaled rhuGM-CSF (sargramostim) after poor response to maximal conventional therapy. BAL fluid and cell samples from one patient were studied with light microscopy and transmission electron microscopy.Excellent response to therapy was observed in patient 1 with no compliance problems or side effects. Macrophages with myelin figure-like structures were seen in her BAL sample. Slight improvement of the pulmonary function was evident also in patient 2, but the role of sargramostim could not be properly evaluated due to the complicated clinical situation.In conclusion, inhaled rhuGM-CSF might be of benefit in patients with LPI-associated PAP.

Cloning, periplasmic expression and purification of hGM-CSF (Human Granulocyte - Macrophage ColonyStimulating Factor) in Escherichia coli

Human GM-CSF is a cytokine consisting of 127 amino acid residues, with four cysteines being involved in two disulfide bonds. Although GM-CSF is glycosylated in its natural form, the glycosylation perhaps has not been involved in its biological function. GM-CSF stimulates the survival, proliferation, and differentiation of hem at opoietic progenitor cells and also enhances the functional properties of mature myeloid cells. GM-CSF is used as a therapeutic agent in various clinical cases such as neutropenia following chemotherapy, bone marrow transplantation, acute myeloid leukemia… In this study, we report the results on the cloning and expression of recombinant human GM-CSF in the periplasmic space of Escherichia coli. The hGM-CSF gene was amplified by polymerase chain reaction using two oligonucleotide primers containing NcoI and XhoI restriction sites. This DNA fragment was successfully cloned between the NcoI and Xho I sites of the plasmid pET-22b, in frame with the pelB signal peptide sequence. The expression vector pET-hGM was transformed into E. coli BL21(DE3) and the transformants were induced by IPTG and examined for hGM-CSF production. Periplasmic proteins were released by osmotic shock treatment. The expression of recombinant hGM-CSF was evaluated by SDS–PAGE in total, cytoplasmic and periplasmic fractions. The recombinant hGMCSF in periplasmic fraction was then subjected to ion exchange chromatography using Q Sepharose FF column with saltincrement elution step. SDS-PAGE showed there was a visible expression of recombinant hGM-CSF in the periplasmic fraction of the E. coli BL21(DE3)/pET-hGM and a purified band with the purity of 97.4% after ion exchange chromatography. This result was further confirmed by Western blot using anti-hGM-CSF antibody.

Human IL10 Gene Repression by Rev-erbα Ameliorates Mycobacterium tuberculosis Clearance

Nuclear receptors modulate macrophage effector functions, which are imperative for clearance or survival of mycobacterial infection. The adopted orphan nuclear receptor Rev-erbα is a constitutive transcriptional repressor as it lacks AF2 domain and was earlier shown to be present in macrophages. In the present study, we highlight the differences in the relative subcellular localization of Rev-erbα in monocytes and macrophages. The nuclear localization of Rev-erbα in macrophages is subsequent to monocyte differentiation. Expression analysis of Rev-erbα elucidated it to be considerably more expressed in M1 phenotype in comparison with M2. Rev-erbα overexpression augments antimycobacterial properties of macrophage by keeping IL10 in a basal repressed state. Further, promoter analysis revealed that IL10 promoter harbors a Rev-erbα binding site exclusive to humans and higher order primates and not mouse, demonstrating a species barrier in its functionality. This direct gene repression is mediated by recruitment of co-repressors NCoR and HDAC3. In addition, our data elucidate that its overexpression reduced the survival of intracellular pathogen Mycobacterium tuberculosis by enhancing phagosome lysosome maturation, an event resulting from IL10 repression. Thus, these findings suggest that Rev-erbα bestows protection against mycobacterial infection by direct gene repression of IL10 and thus provide a novel target in modulating macrophage microbicidal properties.

Enhanced SH3/Linker Interaction Overcomes Abl Kinase Activation by Gatekeeper and Myristic Acid Binding Pocket Mutations and Increases Sensitivity to Small Molecule Inhibitors*

Multidomain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their noncatalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3/linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3/linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with chronic myelogenous leukemia to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multidomain kinase systems that use multiple small molecules to exploit natural mechanisms of kinase control.

A Simplified Method for the Efficient Refolding and Purification of Recombinant Human GM-CSF

Human granulocyte macrophage colony-stimulating factor (hGM-CSF) is a haematopoietic growth factor and proinflammatory cytokine. Recombinant hGM-CSF is important not only as a research tool but also as a biotherapeutic. However, rhGM-CSF expressed in E. coli is known to form inclusion bodies of misfolded, aggregated protein. Refolding and subsequent purification of rhGM-CSF from inclusion bodies is difficult with low yields of bioactive protein being produced. Here we describe a method for the isolation, refolding and purification of bioactive rhGM-CSF from inclusion bodies. The method is straightforward, not requiring extensive experience in protein refolding nor purification and using standard laboratory equipment.

Activated Apoptotic Cells Induce Dendritic Cell Maturation via Engagement of Toll-like Receptor 4 (TLR4), Dendritic Cell-specific Intercellular Adhesion Molecule 3 (ICAM-3)-grabbing Nonintegrin (DC-SIGN), and β2 Integrins

Dendritic cells (DCs) are professional antigen-presenting cells playing a central role in connecting innate and adaptive immunity. Maturation signals are, however, required for DCs to undergo phenotypic and functional changes to acquire a fully competent antigen-presenting capacity. We previously reported that activated apoptotic peripheral lymphocytes (ActApo) provide activation/maturation signals to human monocyte-derived DCs. In this paper, we have characterized the signaling pathways and molecules involved in ActApo-mediated DC maturation. We found that both cellular and supernatant fractions from ActApo are required for DC maturation signaling. ActApoSup-induced CD80 and CD86 expression was significantly blocked in the presence of neutralizing antibodies against tumor necrosis factor-α (TNF-α). Cell-cell contact-dependent signaling involved β2 integrins, dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), and TLR4 because ActApo-induced up-regulation of the maturation markers CD80 and CD86 was significantly inhibited in the presence of neutralizing antibodies against CD18, CD11a, CD11b, and DC-SIGN as well as TLR4. The role of TLR4 was further confirmed by silencing of TLR4 in DCs. In addition, the endogenous adjuvant effect exerted by activated apoptotic splenocytes (ActApoSp) was reduced after immunization with human serum albumin in TLR4(-/-) mice. We detected activation of multiple signaling pathways and transcription factors in DCs upon co-culture with ActApo, including p38, JNK, PI3K-Akt, Src family kinases, NFκB p65, and AP1 transcription factor family members c-Jun and c-Fos, demonstrating the complex interactions occurring between ActApo and DCs. These studies provide important mechanistic insight into the responses of DCs during encounter with cells undergoing immunogenic cell death.

Different cytokines induce the maturation of human monocyte-derived dendritic cells

Objective To investigate the immune effect of different cytokines on immature dendritic cells (DC) in vitro. Methods The human monocyte-derived DCs were induced in the presence of recombinant human GM-CSF (rhGM-CSF, 1000 U/ml) and interleukin (IL)-4 (500 U/ml). The effects of tumor necrosis factor (TNF) -α (1000 U/ml) , lipopolysaccharide (LPS) (1 mg/L), the cocktail of cytokines (TNF-α, IL-6, IL-1β, PGE2) , and sodium butyrate (1 mmol/L) on the DCs were detected by flow cytometry (FCM), endocytic activity, T cells stimulatory proliferation capacity, and IL-12 production. Results The cocktail of cytokines could up-regulate the major histocompatibility complex (MHC) class Ⅱ and costimulatory molecules of DCs, decrease the endocytic activity 186. 74 ±38. 66, induce a stage of Tcell stimulation 18. 23 ± 2. 22 and promote the T helper cell type 1-skewing factor IL-12 production (656. 18 ±38. 52) ng/L. But the sodium butyrate induced reverse result on DCs compared to cocktail of cytokines. Conclusion The most effective method for inducing dendritic cells maturation was cocktail of cytokines. The sodium butyrate could inhibit the development of mature DCs, resulting in impaired DC function, and modify the outcome of the subsequent immune response. Key words: Dendritic cell; Cytokines; Sodium butyrate

GM-CSF up-regulated in reumatoid arthritis reverses cognitive impairment and amyloidosis in Alzheimer mice

Rheumatoid arthritis (RA) is a negative risk factor for the development of Alzheimer's disease (AD). While it has been commonly assumed that RA patients' usage of non-steroi dal anti-inflammatory drugs (NSAIDs) helped prevent onset and progression of AD, NSAID clinical trials have proven unsuccessful in AD patients. To determine whether intrinsic factors within RA pathogenesis itself may underlie RA's protective effect, we investigated the activity of colony-stimulatin g factors, upregulated in RA, on the pathology and behavior of transgenic AD mice. 5 µg bolus injections of macrophage, granulocyte, and granulocyte-macrophage colony-stimulating factors (M-CSF, G-CSF, or GM-CSF) were administered unilaterally into the hippocampus of aged cognitively-impaired AD mice and the resulting amyloid load reductions determined one week later, using the artificial cerebrospinal fluid-injected contrala teral sides as controls. G-CSF and more significantly, GM-CS F reduced amyloidosis throughout the treated brain hemisphere one week following bolus administration to AD mice. 20 daily subcutaneous injections of 5 µg of GM-CSF (the most amyloid-reducing CSF in the bolus experiment) were administered to balanced cohorts of AD mice after assessment in a battery of cognitive tests. Reductions in amyloid load and improvements in cognitive function were assessed. Subcutaneous GM-CSF administration significant ly reduced brain amyloidosis and completely reversed the cognitive impairment, while increasing hippocampal synaptic area and microglial density. These findings, along with two decades of accrued safety data using Leukine, recombinant human GM-CSF, in elderly leukopenic patients, suggest that Leukine should be tested as a treatment to reverse cerebral amyloid pathology and cognitive impairment in AD.