Autoantibodies Neutralizing GM-CSF in HIV-Negative Colombian Patients Infected with Cryptococcus gattii and C. neoformans.

BACKGROUND: Cryptococcosis is a life-threatening disease caused by Cryptococcus neoformans or C. gattii. Neutralizing autoantibodies (auto-Abs) against granulocyte-macrophage colony-stimulating factor (GM-CSF) in otherwise healthy adults with cryptococcal meningitis have been described since 2013. We searched for neutralizing auto-Abs in sera collected from Colombian patients with non-HIV-associated cryptococcosis in a retrospective national cohort from 1997 to 2016. METHODS: We reviewed clinical and laboratory records and assessed the presence of neutralizing auto-Abs against GM-CSF in 30 HIV negative adults with cryptococcosis (13 caused by C. gattii and 17 caused by C. neoformans). RESULTS: We detected neutralizing auto-Abs against GM-CSF in the sera of 10 out of 13 (77%) patients infected with C. gattii and one out of 17 (6%) patients infected with C. neoformans. CONCLUSIONS: We report eleven Colombian patients diagnosed with cryptococcosis who had auto-Abs that neutralize GM-CSF. Among these patients, ten were infected with C. gattii and only one with C. neoformans.

Treatment with Granulocyte-Macrophage Colony-Stimulating Factor Reduces Viral Titers in the Brains of West Nile Virus-Infected Mice and Improves Survival.

West Nile virus (WNV) is the leading cause of epidemic arboviral encephalitis in the United States. As there are currently no proven antiviral therapies or licensed human vaccines, understanding the neuropathogenesis of WNV is critical for rational therapeutic design. In WNV-infected mice, the depletion of microglia leads to enhanced viral replication, increased central nervous system (CNS) tissue injury, and increased mortality, suggesting that microglia play a critical role in protection against WNV neuroinvasive disease. To determine if augmenting microglial activation would provide a potential therapeutic strategy, we administered granulocyte-macrophage colony-stimulating factor (GM-CSF) to WNV-infected mice. Recombinant human GM-CSF (rHuGMCSF) (sargramostim [Leukine]) is an FDA-approved drug used to increase white blood cells following leukopenia-inducing chemotherapy or bone marrow transplantation. Daily treatment of both uninfected and WNV-infected mice with subcutaneous injections of GM-CSF resulted in microglial proliferation and activation as indicated by the enhanced expression of the microglia activation marker ionized calcium binding adaptor molecule 1 (Iba1) and several microglia-associated inflammatory cytokines, including CCL2 (C-C motif chemokine ligand 2), interleukin 6 (IL-6), and IL-10. In addition, more microglia adopted an activated morphology as demonstrated by increased sizes and more pronounced processes. GM-CSF-induced microglial activation in WNV-infected mice was associated with reduced viral titers and apoptotic activity (caspase 3) in the brains of WNV-infected mice and significantly increased survival. WNV-infected ex vivo brain slice cultures (BSCs) treated with GM-CSF also showed reduced viral titers and caspase 3 apoptotic cell death, indicating that GM-CSF specifically targets the CNS and that its actions are not dependent on peripheral immune activity. Our studies suggest that stimulation of microglial activation may be a viable therapeutic approach for the treatment of WNV neuroinvasive disease. IMPORTANCE Although rare, WNV encephalitis poses a devastating health concern, with few treatment options and frequent long-term neurological sequelae. Currently, there are no human vaccines or specific antivirals against WNV infections, so further research into potential new therapeutic agents is critical. This study presents a novel treatment option for WNV infections using GM-CSF and lays the foundation for further studies into the use of GM-CSF as a treatment for WNV encephalitis as well as a potential treatment for other viral infections.

Hematopoietic colony-stimulating factors in head and neck cancers: Recent advances and therapeutic challenges.

Colony-stimulating factors (CSFs) are key cytokines responsible for the production, maturation, and mobilization of the granulocytic and macrophage lineages from the bone marrow, which have been gaining attention for playing pro- and/or anti-tumorigenic roles in cancer. Head and neck cancers (HNCs) represent a group of heterogeneous neoplasms with high morbidity and mortality worldwide. Treatment for HNCs is still limited even with the advancements in cancer immunotherapy. Novel treatments for patients with recurrent and metastatic HNCs are urgently needed. This article provides an in-depth review of the role of hematopoietic cytokines such as granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), and interleukin-3 (IL-3; also known as multi-CSF) in the HNCs tumor microenvironment. We have reviewed current results from clinical trials using CSFs as adjuvant therapy to treat HNCs patients, and also clinical findings reported to date on the therapeutic application of CSFs toxicities arising from chemoradiotherapy.

Oxidized low-density lipoproteins impair the pro-atherosclerotic effect of granulocyte-macrophage-colony-stimulating factor-producing T helper cells on macrophages.

T cells contribute to the pathogenesis of atherosclerosis. However, the presence and function of granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (ThGM) cells in atherosclerosis development is unknown. This study aims to characterize the phenotype and function of ThGM cells in experimental atherosclerosis. Atherosclerosis was induced by feeding apolipoprotein E knockout (ApoE-/-) mice with a high-fat diet. Aortic ThGM cells were detected and sorted by flow cytometry. The effect of oxidized low-density lipoprotein (oxLDL) on ThGM cells and the impact of ThGM cells on macrophages were evaluated by flow cytometry, quantitative RT-PCR, oxLDL binding/uptake assay, immunoblotting and foam cell formation assay. We found that GM-CSF+IFN-γ- ThGM cells existed in atherosclerotic aortas. Live ThGM cells were enriched in aortic CD4+CCR6-CCR8-CXCR3-CCR10+ T cells. Aortic ThGM cells triggered the expression of interleukin-1ß (IL-1ß), tumour necrosis factor (TNF), interleukin-6 (IL-6) and C-C motif chemokine ligand 2 (CCL2) in macrophages. Besides, aortic ThGM cells expressed higher CD69 than other T cells and bound to oxLDL. oxLDL suppressed the cytokine expression in ThGM cells probably via inhibiting the signal transducer and activator of transcription 5 (STAT5) signalling. Furthermore, oxLDL alleviated the effect of ThGM cells on inducing macrophages to produce pro-inflammatory cytokines and generate foam cells. The nuclear receptor subfamily 4 group A (NR4A) members NR4A1 and NR4A2 were involved in the suppressive effect of oxLDL on ThGM cells. Collectively, oxLDL suppressed the supportive effect of ThGM cells on pro-atherosclerotic macrophages.

In vivo transfection of cytokine genes into tumor cells using a synthetic vehicle promotes antitumor immune responses in a visceral tumor model.

The tumor microenvironment (TME) strongly affects the clinical outcomes of immunotherapy. This study aimed to activate the antitumor immune response by manipulating the TME by transfecting genes encoding relevant cytokines into tumor cells using a synthetic vehicle, which is designed to target tumor cells and promote the expression of transfected genes. Lung tumors were formed by injecting CT26.WT intravenously into BALB/c mice. Upon intravenous injection of the green fluorescent protein-coding plasmid encapsulated in the vehicle, 14.2% tumor-specific expression was observed. Transfection of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and CD40 ligand (L)-plasmid combination and interferon gamma (IFNγ) and CD40L-plasmid combination showed 45.5% and 54.5% complete remission (CR), respectively, on day 60; alternate treatments with both the plasmid combinations elicited 66.7% CR, while the control animals died within 48 days. Immune status analysis revealed that the density of dendritic cells significantly increased in tumors, particularly after GM-CSF- and CD40L-gene transfection, while that of regulatory T cells significantly decreased. The proportion of activated killer cells and antitumoral macrophages significantly increased, specifically after IFNγ and CD40L transfection. Furthermore, the level of the immune escape molecule programmed death ligand-1 decreased in tumors after transfecting these cytokine genes. As a result, tumor cell-specific transfection of these cytokine genes by the synthetic vehicle significantly promotes antitumor immune responses in the TME, a key aim for visceral tumor therapy.

The Mechanistic Target of Rapamycin Complex 1 Pathway Contributes to the Anti-Tumor Effect of Granulocyte-Macrophage-Colony-Stimulating Factor-Producing T Helper Cells in Mouse Colorectal Cancer.

INTRODUCTION: The role of granulocyte-macrophage-colony-stimulating factor-producing T helper (ThGM) cells in colorectal cancer (CRC) development remains unclear. This study characterizes the function of ThGM cells in mouse CRC. METHODS: Mouse CRC was induced by administrating azoxymethane and dextran sulfate sodium. The presence of ThGM cells in CRC tissues and the mechanistic target of rapamycin complex 1 (mTORC1) signaling in ThGM cells was detected by flow cytometry. The impact of mTORC1 signaling on ThGM cell function was determined by in vitro culture. The effect of ThGM cells on CRC development was evaluated by adoptive transfer assays. RESULTS: ThGM cells, which expressed granulocyte-macrophage-colony-stimulating factor (GM-CSF), accumulated in CRC tissues. mTORC1 signaling is activated in CRC ThGM cells. mTORC1 inhibition by rapamycin suppressed ThGM cell differentiation and proliferation and resulted in the death of differentiating ThGM cells. mTORC1 inhibition in already differentiated ThGM cells did not induce significant cell death but decreased the expression of GM-CSF, interleukin-2, and tumor necrosis factor-alpha while impeding cell proliferation. Furthermore, mTORC1 inhibition diminished the effect of ThGM cells on driving macrophage polarization toward the M1 type, as evidenced by lower expression of pro-inflammatory cytokines, major histocompatibility complex class II molecule, and CD80 in macrophages after co-culture with rapamycin-treated ThGM cells. Lentivirus-mediated knockdown/overexpression of regulatory-associated protein of mTOR (Raptor) confirmed the essential role of mTORC1 in ThGM cell differentiation and function. Adoptively transferred ThGM cells suppressed CRC growth whereas mTORC1 inhibition abolished this effect. CONCLUSION: mTORC1 is essential for the anti-CRC activity of ThGM cells.

Autochthonous Cryptococcus gattii genotype VGIIb infection in a Japanese patient with anti-granulocyte-macrophage colony-stimulating factor antibodies.

A 31-year-old Japanese man presented with cerebral and pulmonary cryptococcosis. Cryptococcus gattii (C. gattii) genotype VGIIb was detected in the patient's sputum and cerebrospinal fluid specimens. The serum levels of anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies were elevated in this patient, which has been associated with pulmonary alveolar proteinosis and is considered a risk factor for C. gattii infection. After undergoing >12 months of antifungal treatments, the patient showed improvements in symptoms and findings on brain and lung imaging. Several Japanese patients who develop C. gattii infection have also been reported; however, most of these patients have been infected outside Japan, as C. gattii infection is rare in Japan. Only one patient with C. gattii genotype VGIIb infection has been reported in Japan, and it is believed that this patient contracted the infection in China. In the present case, our patient has never been outside Japan, indicating that the infection originated in Japan. Our findings suggest that C. gattii might be spreading in Japan. Therefore, patients with positive serum anti-GM-CSF antibodies should be thoroughly monitored for C. gattii infection, even those living in Japan.

A case of autoimmune pulmonary alveolar proteinosis during the course of treatment of rapidly progressive interstitial pneumonia associated with anti-MDA5 antibody-positive dermatomyositis.

BACKGROUND: Autoimmune pulmonary alveolar proteinosis (APAP) is a diffuse lung disease that causes abnormal accumulation of lipoproteins in the alveoli; however, its pathogenesis remains unclear. Recently, APAP cases have been reported during the course of dermatomyositis. The combination of these two diseases may be coincidental; however, it may have been overlooked because differentiating APAP from a flare-up of interstitial pneumonia associated with dermatomyositis is challenging. This didactic case demonstrates the need for early APAP scrutiny. CASE PRESENTATION: A 50-year-old woman was diagnosed with anti-melanoma differentiation-associated gene 5 (anti-MDA5) antibody-positive dermatitis and interstitial pneumonia in April 2021. The patient was treated with corticosteroids, tacrolimus, and cyclophosphamide pulse therapy for interstitial pneumonia complicated by MDA5 antibody-positive dermatitis, which improved the symptoms and interstitial pneumonia. Eight months after the start of treatment, a new interstitial shadow appeared that worsened. Therefore, three additional courses of cyclophosphamide pulse therapy were administered; however, the respiratory symptoms and interstitial shadows did not improve. Respiratory failure progressed, and 14 months after treatment initiation, bronchoscopy revealed turbid alveolar lavage fluid, numerous foamy macrophages, and numerous periodic acid-Schiff-positive unstructured materials. Blood test results revealed high anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody levels, leading to a diagnosis of APAP. The patient underwent whole-lung lavage, and the respiratory disturbance promptly improved. Anti-GM-CSF antibodies were measured from the cryopreserved serum samples collected at the time of diagnosis of anti-MDA5 antibody-positive dermatitis, and 10 months later, both values were significantly higher than normal. CONCLUSIONS: This is the first report of anti-MDA5 antibody-positive dermatomyositis complicated by interstitial pneumonia with APAP, which may develop during immunosuppressive therapy and be misdiagnosed as a re-exacerbation of interstitial pneumonia. In anti-MDA5 antibody-positive dermatomyositis, APAP comorbidity may have been overlooked, and early evaluation with bronchoalveolar lavage fluid and anti-GM-CSF antibody measurements should be considered, keeping the development of APAP in mind.

Anti-GM-CSF Neutralizing Autoantibodies in Colombian Patients with Disseminated Cryptococcosis.

BACKGROUND: Cryptococcosis is a potentially life-threatening fungal disease caused by encapsulated yeasts of the genus Cryptococcus, mostly C. neoformans or C. gattii. Cryptococcal meningitis is the most frequent clinical manifestation in humans. Neutralizing autoantibodies (auto-Abs) against granulocyte-macrophage colony-stimulating factor (GM-CSF) have recently been discovered in otherwise healthy adult patients with cryptococcal meningitis, mostly caused by C. gattii. We hypothesized that three Colombian patients with cryptococcal meningitis caused by C. neoformans in two of them would carry high plasma levels of neutralizing auto-Abs against GM-CSF. METHODS: We reviewed medical and laboratory records, performed immunological evaluations, and tested for anti-cytokine auto-Abs three previously healthy HIV-negative adults with disseminated cryptococcosis. RESULTS: Peripheral blood leukocyte subset levels and serum immunoglobulin concentrations were within the normal ranges. We detected high levels of neutralizing auto-Abs against GM-CSF in the plasma of all three patients. CONCLUSIONS: We report three Colombian patients with disseminated cryptococcosis associated with neutralizing auto-Abs against GM-CSF. Further studies should evaluate the genetic contribution to anti-GM-CSF autoantibody production and the role of the GM-CSF signaling pathway in the immune response to Cryptococcus spp.

GM-CSF improves endometrial receptivity in a thin endometrium rat model by upregulating HOXA10.

Endometrial receptivity is a prerequisite for the success of assisted reproduction. Patients with a consistently thin endometrium frequently fail to conceive, owing to low endometrial receptivity, and there are currently very few therapeutic options available. Our previous study demonstrated that intrauterine granulocyte-macrophage colony-stimulating factor (GM-CSF) administration resulted in a significant improvement in clinical pregnancy and implantation rates and was an effective means of increasing endometrial thickness on the day of embryo transfer in patients with thin endometrium. In order to explore the underlying process, an animal model with a thin endometrium was constructed, the homeobox A10 gene (HOXA10) was downregulated, and an inhibitor of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway (MAPK/ERK) was employed. Our findings strongly suggest a marked decrease in GM-CSF levels in the thin endometrial rat model, and the suppression of HOXA10 impeded the therapeutic efficacy of GM-CSF in this model. Moreover, we showed that GM-CSF significantly increases endometrial receptivity in the rat model and upregulates HOXA10 via the MAPK/ERK pathway. Our data provide new molecular insights into the mechanisms underlying formation of a thin endometrium and highlight a novel, potential clinical treatment strategy as well as directions for further research.

Interleukin-17 receptor C is essential for the pro-inflammatory pathogenicity of granulocyte-macrophage-colony-stimulating factor-producing T helper cells in experimental autoimmune uveitis.

Autoimmune uveitis is an inflammatory disorder of the eye triggered by the responses of autoreactive T cells to ocular autoantigens. This study aims to understand the role of granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (ThGM) cells in the pathophysiology of mouse experimental autoimmune uveitis (EAU). We established an EAU model by immunizing mice with interphotoreceptor retinoid-binding protein (IRBP) 651-670. Splenic or eye-infiltrating ThGM cells were analyzed and enriched by flow cytometry according to the levels of an array of surface markers, transcription factors, and cytokines. Lentiviral transduction was conducted to silence or overexpress the target gene in differentiated ThGM cells. The adoptive transfer was applied to determine the pathogenicity of ThGM cells in vivo. We found that ThGM cells were present in the spleen and the eye after EAU induction. Both splenic and eye-infiltrating ThGM cells were phenotypically CD4+CCR7-CXCR3-CCR6-CCR10hi. Eye-infiltrating ThGM cells up-regulated interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and IL-17 receptor C (IL-17RC) relative to splenic ThGM cells. IL-17RC overexpression enabled interleukin-17A (IL-17A)-induced up-regulation of IL-1ß and IL-6 production in ThGM cells. Adoptive transfer of IL-17RC overexpressing ThGM cells exacerbated EAU severity, as evidenced by a higher histology score as well as increased pro-inflammatory cytokines and inflammatory cells in the eye. However, IL-17RC-silenced ThGM cells did not impact EAU. Therefore, for the first time, this study unveils the essential pro-inflammatory role of IL-17RC-expressing ThGM cells in EAU pathophysiology. We discovered a novel mechanism underlying the pathophysiology of autoimmune uveitis.

GM-CSF is a marker of compartmentalised intrathecal inflammation in multiple sclerosis.

BACKGROUND: Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pro-inflammatory cytokine secreted by various immune cells. Several studies have demonstrated an expansion of GM-CSF producing T cells in the blood or CSF of people with MS (pwMS). However, whether this equates to greater concentrations of circulating cytokine remains unknown as quantification is difficult with traditional assays. OBJECTIVE: To determine whether GM-CSF can be quantified and whether GM-CSF levels are elevated in pwMS. METHODS: We employed Single Molecule Array (Simoa) to measure GM-CSF in both CSF and blood. We then investigated relationships between GM-CSF levels and measures of blood-CSF-barrier integrity. RESULTS: GM-CSF was quantifiable in all samples and was significantly higher in the CSF of pwMS compared with controls. No association was found between CSF GM-CSF levels and Q-Albumin - a measure of blood-CSF-barrier integrity. CSF GM-CSF correlated with measures of intrathecal inflammation, and these relationships were greater in primary progressive MS compared with relapsing-remitting MS. CONCLUSION: GM-CSF levels are elevated specifically in the CSF of pwMS. Our results suggest that elevated cytokine levels may reflect (at least partial) intrathecal production, as opposed to simple diffusion across a dysfunctional blood-CSF-barrier.

Design of a Tumor Binding GMCSF as Intratumoral Immunotherapy of Solid Tumors.

Next-generation cancer immunotherapies may utilize immunostimulants to selectively activate the host immune system against tumor cells. Checkpoint inhibitors (CPIs) like anti-PD1/PDL-1 that inhibit immunosuppression have shown unprecedented success but are only effective in the 20-30% of patients that possess an already "hot" (immunogenic) tumor. In this regard, intratumoral (IT) injection of immunostimulants is a promising approach since they can work synergistically with CPIs to overcome the resistance to immunotherapies by inducing immune stimulation in the tumor. One such immunostimulant is granulocyte macrophage-colony-stimulating factor (GMCSF) that functions by recruiting and activating antigen-presenting cells (dendritic cells) in the tumor, thereby initiating anti-tumor immune responses. However, key problems with GMCSF are lack of efficacy and the risk of systemic toxicity caused by the leakage of GMCSF from the tumor tissue. We have designed tumor-retentive versions of GMCSF that are safe yet potent immunostimulants for the local treatment of solid tumors. The engineered GMCSFs (eGMCSF) were synthesized by recombinantly fusing tumor-ECM (extracellular matrix) binding peptides to GMCSF. The eGMCSFs exhibited enhanced tumor binding and potent immunological activity in vitro and in vivo. Upon IT administration, the tumor-retentive eGMCSFs persisted in the tumor, thereby alleviating systemic toxicity, and elicited localized immune activation to effectively turn an unresponsive immunologically "cold" tumor "hot".

Quantitative Evaluation of Changes in Three-Dimensional CT Density Distributions in Pulmonary Alveolar Proteinosis after GM-CSF Inhalation.

BACKGROUND: A previous clinical trial for autoimmune pulmonary alveolar proteinosis (APAP) demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) inhalation reduced the mean density of the lung field on computed tomography (CT) across 18 axial slice planes at a two-dimensional level. In contrast, in this study, we challenged three-dimensional analysis for changes in CT density distribution using the same datasets. METHODS: As a sub-study of the trial, CT data of 31 and 27 patients who received GM-CSF and placebo, respectively, were analyzed. To overcome the difference between various shooting conditions, a newly developed automatic lung field segmentation algorithm was applied to CT data to extract the whole lung volume, and the accuracy of the segmentation was evaluated by five pulmonary physicians independently. For normalization, the percent pixel (PP) in a certain density range was calculated as a percentage of the total number of pixels from -1,000 to 0 HU. RESULTS: The automatically segmented images revealed that the lung field was accurately extracted except for 7 patients with minor deletion or addition. Using the change in PP from baseline to week 25 (ΔPP) as the vertical axis, we created a histogram with 143 HU bins set for each patient. The most significant difference in ΔPP between GM-CSF and placebo groups was observed in two ranges: from -1,000 to -857 and -143 to 0 HU. CONCLUSION: Whole lung extraction followed by density histogram analysis of ΔPP may be an appropriate evaluation method for assessing CT improvement in APAP.

Autoimmune Pulmonary Alveolar Proteinosis.

Autoimmune pulmonary alveolar proteinosis (PAP) is a rare disease characterized by myeloid cell dysfunction, abnormal pulmonary surfactant accumulation, and innate immune deficiency. It has a prevalence of 7-10 per million; occurs in individuals of all races, geographic regions, sex, and socioeconomic status; and accounts for 90% of all patients with PAP syndrome. The most common presentation is dyspnea of insidious onset with or without cough, production of scant white and frothy sputum, and diffuse radiographic infiltrates in a previously healthy adult, but it can also occur in children as young as 3 years. Digital clubbing, fever, and hemoptysis are not typical, and the latter two indicate that intercurrent infection may be present. Low prevalence and nonspecific clinical, radiological, and laboratory findings commonly lead to misdiagnosis as pneumonia and substantially delay an accurate diagnosis. The clinical course, although variable, usually includes progressive hypoxemic respiratory insufficiency and, in some patients, secondary infections, pulmonary fibrosis, respiratory failure, and death. Two decades of research have raised autoimmune PAP from obscurity to a paradigm of molecular pathogenesis-based diagnostic and therapeutic development. Pathogenesis is driven by GM-CSF (granulocyte/macrophage colony-stimulating factor) autoantibodies, which are present at high concentrations in blood and tissues and form the basis of an accurate, commercially available diagnostic blood test with sensitivity and specificity of 100%. Although whole-lung lavage remains the first-line therapy, inhaled GM-CSF is a promising pharmacotherapeutic approach demonstrated in well-controlled trials to be safe, well tolerated, and efficacious. Research has established GM-CSF as a pulmonary regulatory molecule critical to surfactant homeostasis, alveolar stability, lung function, and host defense.

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

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

Granulocyte-macrophage colony-stimulating factor: Conductor of the wound healing orchestra?

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein and is derived from both hemopoietic and nonhemopoietic sources which exert immunomodulatory properties. Various theories have been proposed to explain why some wounds become chronic and non-healing. Generalized suppression of inflammation locally or systemically may impede the body's physiological healing response by crippling the activity of reparative cells within the wound ecosystem. Thus, highlighting the importance of promoting host-directed therapeutics with immunomodulatory properties. The temporal and spatial expression of GM-CSF and GM-CSF receptors in the integumentary system suggests that epithelial-derived GM-CSF functions in an autocrine/paracrine manner. This may positively affect wound healing physiology via local inflammatory regulation promoting macrophage survival. Although diabetes negatively affects multiple aspects of wound healing GM-CSF activation is particularly impacted. Compared to acute/healthy wounds diabetic foot ulcers (DFU) only partially activate GM-CSF activity. There is a deleterious chain of events associated with this unfortunate sequala. DFUs also have a high proportion of monocytes and an absence of activated macrophages which results in an impaired inflammatory response. This may potentially serve as a vital point for GM-CSF to act as a companion diagnostic/theragnostic modality to help modulate the inflammatory response in wound healing. Correcting macrophage immune dysfunction with exogenous GM-CSF may help restore the immune balance in the wound ecosystem and jumpstart the wound healing cascade. Thus, the recognized beneficial role of GM-CSF in immune regulation across many studies provides a rationale for the initiation of the ongoing randomized controlled trials using GM-CSF.

[Relationship between promoting effect of Zuogui Pills on angiogenesis of reproductive organs and mobilization factors GM-CSF, SDF-1, and their receptors of EPCs in early-aging rats].

This study aimed to investigate the relationship between the promoting effect of Zuogui Pills on ovarian and vaginal angiogenesis in early-aging rats and mobilization factors granulocyte-macrophage colony-stimulating factor(GM-CSF), stromal cell-derived factor-1(SDF-1), and their receptors of endothelial progenitor cells(EPCs) and explore the mechanism of Zuogui Pills in improving reproductive hypofunction in early-aging rats. Ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS) was used to analyze the chemical components of the extract of Zuogui Pills. Forty 14-month-old female early-aging rats with estrous cycle disorder were randomly divided into a blank group, a conjugated estrogen group(conjugated estrogen suspension, 65 µg·kg~(-1)), and low-(11 g·kg~(-1)) and high-dose(33 g·kg~(-1)) Zuogui Pills groups, with 10 rats in each group. In addition, 10 4-month-old female rats were assigned to the youth control group. The rats in the blank group and the youth control group were treated with 20 g·kg~(-1) distilled water by gavage, while those in the groups with drug intervention were treated with corresponding drugs by gavage, once a day for 15 days. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of SDF-1 and GM-CSF in the mobilization of EPCs in serum. Hematoxylin-eosin(HE) staining was used to observe the changes in the number of ovarian follicles at all levels and corpus luteum, the number of vaginal epithelial layers, the number of vaginal folds, and the blood vessels of ovarian and vaginal tissues in the groups with drug intervention. Western blot was used to detect the expression of ER, GM-CSFR, CXCR4, and CXCR7 proteins in ovarian and vaginal tissues. As revealed by the results, the blank group showed decreased number of corpus luteum, gro-wing follicles at all levels, and blood vessels(P<0.05), decreased thickness of vaginal mucosa, the number of epithelial layers, the number of vaginal folds, and the number of vessels in the lamina propria(P<0.05), reduced content of SDF-1 and GM-CSF in the peripheral blood(P<0.05), and down-regulated levels of ER, CXCR4, CXCR7, and GM-CSFR proteins in ovarian and vaginal tissues(P<0.05). The groups with drug intervention showed increased number of growing follicles at all levels, corpus luteum, and blood vessels(P<0.05), decreased number of atresia follicles(P<0.05), increased thickness of vaginal mucosa, the number of epithelial layers, the number of vaginal mucosal folds, and the number of blood vessels in the lamina propria(P<0.05), increased content of SDF-1 and GM-CSF in the peripheral blood(P<0.05), and up-regulated levels of ER, CXCR4, CXCR7, and GM-CSFR proteins in ovarian and vaginal tissues(P<0.05). This experiment suggests that Zuogui Pills may promote ovarian and vaginal angiogenesis and improve the reproductive function of early-aging rats by up-regulating the levels of mobilization factors SDF-1, GM-CSF, and their receptors of EPCs.

The innate immune system stimulating cytokine GM-CSF improves learning/memory and interneuron and astrocyte brain pathology in Dp16 Down syndrome mice and improves learning/memory in wild-type mice.

Down syndrome (DS) is characterized by chronic neuroinflammation, peripheral inflammation, astrogliosis, imbalanced excitatory/inhibitory neuronal function, and cognitive deficits in both humans and mouse models. Suppression of inflammation has been proposed as a therapeutic approach to treating DS co-morbidities, including intellectual disability (DS/ID). Conversely, we discovered previously that treatment with the innate immune system stimulating cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), which has both pro- and anti-inflammatory activities, improved cognition and reduced brain pathology in a mouse model of Alzheimer's disease (AD), another inflammatory disorder, and improved cognition and reduced biomarkers of brain pathology in a phase II trial of humans with mild-to-moderate AD. To investigate the effects of GM-CSF treatment on DS/ID in the absence of AD, we assessed behavior and brain pathology in 12-14 month-old DS mice (Dp[16]1Yey) and their wild-type (WT) littermates, neither of which develop amyloid, and found that subcutaneous GM-CSF treatment (5 µg/day, five days/week, for five weeks) improved performance in the radial arm water maze in both Dp16 and WT mice compared to placebo. Dp16 mice also showed abnormal astrocyte morphology, increased percent area of GFAP staining in the hippocampus, clustering of astrocytes in the hippocampus, and reduced numbers of calretinin-positive interneurons in the entorhinal cortex and subiculum, and all of these brain pathologies were improved by GM-CSF treatment. These findings suggest that stimulating and/or modulating inflammation and the innate immune system with GM-CSF treatment may enhance cognition in both people with DS/ID and in the typical aging population.

Characterization of a novel + 70 Da modification in rhGM-CSF expressed in E. coli using chemical assays in combination with mass spectrometry.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine and a white blood cell growth factor that has found usage as a therapeutic protein. During analysis of different fermentation batches of GM-CSF recombinantly expressed in E. coli, a covalent modification was identified on the protein by intact mass spectrometry. The modification gave a mass shift of + 70 Da and peptide mapping analysis demonstrated that it located to the protein N-terminus and lysine side chains. The chemical composition of C4H6O was found to be the best candidate by peptide fragmentation using tandem mass spectrometry. The modification likely contains a carbonyl group, since the mass of the modification increased by 2 Da by reduction with borane pyridine complex and it reacted with 2,4-dinitrophenylhydrazine. On the basis of chemical and tandem mass spectrometry fragmentation behavior, the modification could be attributed to crotonaldehyde, a reactive compound formed during lipid peroxidation. A low recorded oxygen pressure in the reactor during protein expression could be linked to the formation of this compound. This study shows the importance of maintaining full control over all reaction parameters during recombinant protein production.