Recombinant Human Granulocyte-macrophage Colony Research Articles

Study of the adjuvant properties of preparations containing recombinant human granulocyte-macrophage colony stimulating factor

The relevance of the search for new vaccine adjuvants is growing along with the increase in the number of current vaccine preparations, especially those developed on the basis of proteins. Some cytokines are known to exert adjuvant properties. The present work is devoted to the study of adjuvant activity of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) and constructs based on it. Earlier, we developed a technology for isolation and purification of GM-CSF from the E. coli SG20050/p280_2GM producer strain, as well as a technology for conjugating polyglucin:spermidine complexes with rhGM-CSF. Double-stranded RNA was used to obtain molecular constructs on the basis of rhGM-CSF conjugate. To assemble constructs, the ratio of the components was calculated for one dose of the preparation to contain 5-40 mg of rhGM-CSF and 100 mg of double-stranded RNA. The effectiveness of the formation of molecular constructs was evaluated by dsRNA electrophoretic mobility shift in a 1% agarose gel. The effectiveness of the resulting adjuvants was determined in ELISA assays by measuring the titers of specific antibodies in mouse sera against ovalbumin or recombinant receptor-binding domain of the surface S protein of the severe acute respiratory syndrome coronavirus 2 (Delta variant (B.1.617.2). The experiments were carried out in 100 male BALB/c mice weighing 16-18 g. Mice were immunized twice, with a 14-day interval, by intramuscular injection of 200 mL per animal. Recombinant receptor-binding domain of the surface protein of SARS-CoV-2 was administered at a dose of 50 mg/animal, ovalbumin – at two doses – 1 mg or 5 mg/animal. Corresponding antigen was used as a positive control, a saline solution – as a negative control. It was shown that the maximum effect was achieved by immunization with a construct based on double-stranded RNA and rhGM-CSF conjugated to polyglucin-spermidine. The use of a conjugate without double-stranded RNA as an adjuvant also improved humoral response. The use of native rhGM-CSF did not increase the titers of specific antibodies. Thus, it was found that rhGM-CSF being a part of a polysaccharide conjugate or a molecular construct exerted an ability to enhance the humoral immune response to protein antigens.

Effect of Xijiao Dihuang Combined Prescription on Human Dendritic Cell Function Induced by Lipopolysaccharide

To observe the effects of drug-containing serum of Xijiao Dihuang combined prescription(XJDH) on the related functions of dendritic cells(DCs) induced in vitro, and to explore the mechanisms underlying the effectiveness of XJDH treatment on primary immune thrombocytopenia(ITP). Peripheral blood samples were colle-ted from 6 healthy volunteers. Mononuclear cells were isolated by density gradient centrifugation, and CD14+ mononuclear cells were collected by the magnetic separation technique. CD14+ mononuclear cells were induced into immature DCs by recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and recombinant human interleukin 4 (IL-4). Immature DCs were divided into three groups: control group, model group and XJDH group. CCK-8 assay was used to determine the intervention concentration and time of drug-containing serum. Lipopolysaccharide(LPS) with the final concentration of 1 μg/ml was added to model group and XJDH group respectively for 24 h to induce DCs maturation. Normal rat serum was added to control group and model group, and XJDH was added to XJDH group for 24 h. Flow cytometry was used to detect the levels of CD80, CD83 and HLA-DR on the surface of DCs. Western blot was used to detect the expression of TLR4 and NF-κB, and levels of IL-6, IL-12 and TNF-α in cell supernatant was detected by ELISA. Compared with the control group, LPS stimulation increased the expression of CD80, CD83 and HLA-DR, with subsequent increasing expression of TLR4 and NF-κB, as well as IL-6, IL-12 and TNF-α increased(P<0.05). In comparison with model group, the expression of DCs surface molecules CD80, CD83 and HLA-DR, DCs' expression of TLR4 and NF-κB protein, and the levels of IL-6, IL-12 and TNF-α in the cell supernatant of XJDH group decreased after the intervention of XJDH (P<0.05). Drug containing serum of Xijiao Dihuang combined prescription can down-regulate TLR4/NF-κB signaling pathway related protein expression, inhibit DCs maturation, and reduce proinflammatory factor secretion, which may be one of the mechanisms of drug-containing serum of Xijiao Dihuang combined prescription in the treatment of immune thrombocytopenia.

Effects of recombinant human granulocyte macrophage colony stimulating factor gel on treatment of full-thickness frostbite wounds on foot and hand

Objective: To explore the effects of recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) gel on treatment of thefull-thickness frostbite wounds on foot and hand. Methods: From November 2013 to April 2017, a total of 45 patients of 71 full-thickness frostbite wounds on foot and hand meeting the inclusion criteria were admitted to the First Hospital of Jilin University and the prospective randomized controlled study was done. The patients were divided into rhGM-CSF group of 24 patients with 35 wounds and control group of 21 patients with 36 wounds according to the random number table. There were 20 males and 4 females, aged (38±13) years among patients in rhGM-CSF group, and there were 19 males and 2 females, aged (36±14) years among patients in control group. Patients in 2 groups were performed with the same systemic treatment of rewarming, anti-inflammation, pain relief, anti-infection, anti-coagulation, and thrombolysis. Wounds of patients in rhGM-CSF group and control group were respectively treated with rhGM-CSF gel and aloe vera gel for external usage with 10 mg for every square centimeter and dressing change once every 24 hours, until wounds healed completely. The wound inflammatory response was scored on treatment day (TD) 1, 3, 7, 14, wound secretion was collected for bacteria culture and positive bacteria detection rate was calculated before treatment and on TD 6 and 12, adverse drug reaction after drug use was observed, and the complete wound healing time was recorded. Data were processed with Fisher's exact probability test, analysis of variance for repeated measurement, t test, and Bonferroni correction. Results: The scores of wound inflammatory response of patients in 2 groups on TD 1 and 3 were close (t=0.37, 2.93, P>0.05). The scores of wound inflammatory response of patients on TD 7 and 14 in rhGM-CSF group were significantly higher than those in control group (t=5.77, 5.83, P<0.01). The results of bacteria culture of wound secretion of patients in 2 groups before treatment were negative. The positive bacteria detection rates of wound secretion of patients in rhGM-CSF group on TD 6 and 12 were 5.71% (2/35) and 22.86% (8/35), which were slightly lower than 13.89% (5/36) and 30.56%(11/36) in control group respectively, but there was no significantly statistical difference (P>0.05). No adverse drug response occurred in patients in rhGM-CSF group, while 1 patient in control group had adverse drug response, with symptoms of redness and swelling of wounds and patchy erythema on skin around wounds, which were alleviated by irrigating with normal saline. The complete wound healing time of patients in rhGM-CSF was (12.3±0.5) d, which was significantly shorter than (16.5±0.8) d in control group (t=24.89, P<0.05). Conclusions: The topical rhGM-CSF gel has effects of shortening time of wound healing and reducing inflammatory response of wound on treatment of full-thickness frostbite wounds on foot and hand, which is safe in clinical application.

Boosting Abscopal Response to Radiotherapy with Sargramostim: A Review of Data and Ongoing Studies.

Drug development in oncology today routinely focuses on approaches that utilize the patients’ immune system to destroy the malignancy. Combinatorial approaches of antineoplastic agents, both new and old, are being incorporated in the armamentarium of cancer treatments. The overarching goal of therapy remains the achievement of a complete and durable response with long term remission or cure. One approach in advancing treatment is aimed at strategies that improve immunological memory to induce long lasting immunity against the tumor. Although radiation therapy has not traditionally been thought to elicit an immunological effect, an increasing number of reports document the induction of an immune response against a tumor that kills cancer cells distant to the original site of treatment after local irradiation to a tumor. This phenomenon is called an abscopal effect. Since radiation alone is rarely associated with such a response, it is being combined with immuno-oncology drugs in an attempt to enhance response. One such strategy combines sargramostim, a recombinant human granulocyte macrophage colony stimulating factor (rhu GM-CSF), with radiotherapy. GM-CSF is a cytokine secreted by multiple cells types that promotes maturation of dendritic cells and enables the presentation of tumor-associated antigens to generate a T-cell response. This review article discusses the outcomes of clinical trials and case reports examining the efficacy and safety of combining radiation therapy with this immunomodulatory agent. We will also examine future studies and challenges facing the translation of this therapeutic approach.

Role of Dendrtic Cells in IgA Nephropathy Pathogenesis

Objective to discuss the possible role and mechanism of DCs in IgAN attack. Method Stimulating factors such as recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF), recombinant human interleukin-4 (rhIL-4) and tumor necrosis factor-a (TNF-a) etc. were used in vitro jointly to induce and culture DCs, a flow cytometry was used to detect expression of HLA-DR, CD83 and CDla of DCs membrane surface molecules, the MTT method was used to detect capacity of DCs of the IgAN patient group to stimulate proliferation of allogeneic T cells and the difference between the levels of interleukin 6 (IL-6) and interleukin 12 (IL-12) secreted by DCs and that of the normal control group. Result Combined application of cytokines GM-CSF, IL-4 and TNF-a is able to induce proliferation and differentiation of peripheral blood mononuclear cell into a mature dendritic cell. The surface of mature dendritic cells highly expresses human leucocyte antigen HLA-DR and surface maturity markers of relative specificity of dendritic cells, CD83 and CD1a. The capacity of DCs of the IgAN patient group to stimulate allogeneic T lymphocyte proliferation is higher than that of the normal control group and the difference has statistical significance (P<0.05). The capacity of DCs of the patient group influenced by lipopolysaccharide (LPS) to stimulate allogeneic T lymphocyte proliferation significantly increases compared with that of DCs of the patient group not influenced by lipopolysaccharide (LPS) and the difference has statistical significance (P<0.05). The IL-6 secreted by the DCs of the IgAN patient group is higher than that of the normal control group and the difference between the two groups has statistical significance (P<0.05). The IL-12 secreted by the DCs of the IgAN patient group is lower than that of the normal control group and the difference between the two groups has statistical significance (P<0.05). Conclusion DCs may regulate the balance between Thl/Th2 cells by secreting cytokines so as to play an imporat role in occurence and progression of IgAN and such factors as infection etc. may strengthen the functions of DCs thus easily triggering IgAN.

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.

Recombinant human granulocyte macrophage colony stimulating factor in deep second-degree burn wound healing.

Background:The aim of this study was to explore the effects of recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) on deep second-degree burn wound healing.Methods:In this study, 95 patients with a total of 190 burn wounds were treated with either rhGM-CSF or placebo, separated into 2 groups by treatment type. Wound healing rate, wound healing time, histopathological condition, and scar scale were all compared between the 2 groups.Results:The healing rates in the rhGM-CSF group were remarkably higher than those in the placebo group (P < .01). The wound healing time in the rhGM-CSF group (18.8 ± 7.6 days) was significantly shorter than that in the placebo group (25.5 ± 4.6 days, P < .01). On the 14th day and 28th day, the average optical density of vascular endothelial factor (VEGF) in the rhGM-CSF group was larger than that in the placebo group. Meanwhile, the average optical density of fibroblast growth factor (FGF) in the rhGM-CSF group was also larger than that in the placebo group. Furthermore, the Vancouver scar scale scores of pigmentation, pliability, height, and vascularity were notable lower in the rhGM-CSF group than those in the placebo group (P < .01).Conclusion:The results suggest that rhGM-CSF can significantly accelerate deep second-degree burn wound healing.

The role of DCs in the immunopathogenesis of chronic HBV infection and the methods of inducing DCs maturation.

Chronic hepatitis B virus (HBV) infection is the result of an inadequate immune response towards the virus. Dendritic cells (DCs), as the most efficient professional antigen-presenting cells (APCs), possess the strongest antigen presenting the effect in the body and can stimulate the initial T cell activation and proliferation. DCs of patients with chronic HBV infection are impaired, resulting in more tolerogenic rather than immunogenic responses, which may contribute to viral persistence. Recently, numerous methods have been developed to induce DCs maturation. To date, recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) combined with interleukin-4 (rhIL-4) has been a classic culture combination to DCs. The recently classified type III interferon group interferon-λ (IFN-λ) displays antiviral, antitumor, and immunoregulatory activity. In our laboratory, we demonstrate that IFN-λ1 combined with rhGM-CSF and rhIL-4 can significantly increase the expression of DC surface molecules and the secretion of interleukin-12 (IL-12) and interferon-γ (IFN-γ) in patients with chronic hepatitis B infection. In this review, we emphasize on the role of DCs in the immunopathogenesis of chronic HBV infection. Importantly, we systematic review that the latest update in the current status of knowledge on the methods of inducing DCs maturation in anti-HBV immunity. What's more, we conclude that IFN-λ1 combined with GM-CSF and IL-4 can induce DCs maturation, which could become a possibility to be applied to the autologus dendritic cell vaccine to treat chronic hepatitis B.

Study of the use of recombinant human granulocyte-macrophage colony-stimulating factor hydrogel externally to treat residual wounds of extensive deep partial-thickness burn

ObjectiveThe objective of this study was to observe the clinical effects of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) hydrogel in the treatment of residual wounds of extensive deep partial-thickness burn. MethodsIn this study, 21 subjects who sustained deep partial-thickness burns, which did not heal over 8 weeks, were observed. These were randomly assigned to two paired groups: the experimental group (using rhGM-CSF, n=21) and the control group (using mupirocin ointment, n=21). The wound dressings were changed once daily. Wound secretion, inflammation, granulation tissues, local and general side effects of the drug, wound healing time, and healing rate at different times were observed and compared between the two groups. The differences in the pathology of new vessels and fibroblasts between the two groups were observed, and their number in immunohistochemistry was detected. ResultsThe wound healing time was 17.28±6.70 days in the experimental group. It was significantly shorter than that of the control group (22.14±7.38 days). The healing rates at 10 and 14 days in the experimental group were 54±27% and 60±36%, respectively. These healing rates were remarkably higher than those of the control group (43±27% and 48±30%). On the 3rd, 7th, 10th, and 14th day, the experimental group was obviously superior to the control group in wound inflammation, secretion, and granulation tissues. Furthermore, on the 7th, 10th, 14th, 21st, and 28th day, the bacterial clearance rates of the experimental group (42.85%, 52.38%, 90.47%, 95.24%, and 95.24%) were higher than those of the control group (4.76%, 4.76%, 38.10%, 76.19%, and 80.95%). On the 14th day, the average optical density of the vascular endothelial factor (VEGF) of the experimental group (0.21±0.01) is bigger than that of the control group (0.18±0.02) (P<0.05), and the average optical density of the fibroblast growth factor (FGF) of the experimental group (0.25±0.01) is also larger than that of the control group (0.18±0.02) (P<0.05). ConclusionrhGM-CSF hydrogel effectively promotes the healing process of residual wounds of extensive deep partial-thickness burns. The hydrogel removed most of the bacteria or inhibited growth, and the local and general side reactions of the drug were mild during the study.

Genome-wide transcription profiling in neutrophils in acute respiratory distress syndrome

BackgroundAcute respiratory distress syndrome (ARDS) is characterised by diffuse neutrophil-mediated alveolar inflammation. Recently, we demonstrated that blood polymorphonuclear leucocytes (PMNs) in ARDS are basally activated, and exhibit aberrant oxidative burst and survival responses. The molecular mechanisms governing ARDS PMN function and longevity are incompletely understood. We aimed to use genome-wide transcriptional profiling of ARDS blood PMNs to explore underlying disease mechanisms and identify therapeutic targets aimed at manipulating PMN function and longevity. MethodsGeneChip Affymetrix oligonucleotide arrays were used to assess global transcriptional profiles in highly pure PMNs from ventilated patients fulfilling the Berlin ARDS definition (n=10), in freshly isolated PMNs from age-matched and sex-matched healthy volunteers (n=10), and in healthy volunteer PMNs exposed in vitro to recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) (1 ng/mL for 6 h). Ingenuity Pathway Analysis software was used to map probes identified as important onto specific pathways. FindingsTranscriptomic analysis showed that 1319 genes were altered in ARDS PMNs relative to healthy volunteer PMNs. Compared with well established reference databases, the gene expression profile in ARDS PMNs showed near-complete correlation to datasets derived from patients with sepsis and burns. Transcripts enriched in ARDS PMNs were differentially expressed in known functional network pathways associated with cancer, cellular compromise, apoptotic mechanisms, and chemotaxis. Of the observed gene changes, only 292 (22%) were seen in healthy volunteer PMNs after exposure to rhGM-CSF, of which 216 showed the same directional change as ARDS PMNs. InterpretationExisting genome-wide studies in ARDS use total blood leucocytes; our study is the first, to our knowledge, to use unbiased global genomic profiling of highly pure ARDS blood PMNs in parallel with age-matched and gender-matched healthy volunteer PMNs treated with rhGM-CSF. Collectively our results show that ARDS PMNs display important de-novo transcriptional activity. The global transcriptomic changes were consistent with the observed aberrant ARDS PMN survival and functional phenotype that we have previously reported, and show near-complete correlation to existing sepsis and burns datasets, but only limited transcriptomic overlap with healthy volunteer PMNs treated with rhGM-CSF. FundingNational Institute for Health Research, GlaxoSmithKline.

Clinical research on dendritic cell vaccines to prevent postoperative recurrence and metastasis of liver cancer.

We aimed to evaluate dendritic cell (DC) tumor vaccines for preventing liver cancer recurrence and metastasis. DCs were induced from mononuclear cells in the peripheral blood with recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) and recombinant human interleukin 4 (rhIL-4), followed by sensitization with lysis of autologous liver cancer cells. One hundred and sixty patients with hepatocellular carcinoma were randomly divided into two groups of 80. One group was treated postoperatively with six cycles of the DC tumor vaccine. The other group was treated postoperatively with six cycles of FOLFOX 6, beginning 1 week after surgery. After treatment with DC tumor vaccines, the levels of CD3+, CD4+, and CD8+, the ratio of CD4+ to CD8+ DC, and the serum levels of IL-12 and IFN-γ were significantly increased both in comparison to the pre-treatment levels (P < 0.001) and to the chemotherapy group (P < 0.001). After a postoperative follow-up of 18 months, the metastatic recurrence rate in the DC tumor vaccine group was significantly lower than that in the chemotherapy group (17.50 vs 48.75%, P < 0.005), and the survival rate of the patients in the DC tumor vaccine group was higher than that of the chemotherapy treatment group (86.25 vs 52.50%, P < 0.005). Treatment with DC tumor vaccines was safe and feasible. It can enhance the immunity of the patients, reduce the rates of metastasis and recurrence, and improve survival rates. This is a promising treatment for the prevention of postoperative recurrence in patients with liver cancer.

The killing effect of NY-ESO-1-sensitzed dendritic cells-induced cellular toxic lymphocyte on human retinoblastoma cell

Background Cell immunologic therapy for retinoblastoma （RB）is becoming a hot research topic. Cancer-testis antigen is a human immunogenic protein and is used to treat some tumors. However,its effect on RB has not been investigated. Objective The present study was to discuss the antigen specific anti-tumor effect of cytotoxic T lymphocytes （CTL）induced by the cancer-testis antigen, NY-ESO-l-sensitized dendritic cells （DCs）, on human RB. Methods PCR was performed to amplify target gene fragments from the NY-ESO-1 plasmids,and then the target gene fragments were digested with the restriction enzymes SalI and EcoRI. Harvested fragments were inserted into the pDC316 plasmid to construct the recombinant plasmid pDC316/NY-ESO-1. The expression of NY-ESO-1 protein in human RB cells strain, HXO-RB44,was detected by immunofluoreseence and Western blot. Monoeytes wereisolated from 60 ml of peripheral blood from a healthy donor using Ficoll density-gradient centrifugation with a cell density of 1 x 107/ml. DCs isolated from blood were stimulated with recombinant human granulocyte-macrophage colony stimulating factor （rhGM-CSF） and recombinant human interleukin-4 （ rhIL-4 ）. The recombinant plasmid pDC316/NY-ESO-1 was transfected into DCs and the DCs were co-cultured with T lymphocytes. The resultant CTL were used as effector ceils. The growth of the CTL was detected by MTT assay. The CTL were then added into the growth medium used for culturing HXO-RB44 cells and the vitality of the HXO-RB44 cells was assayed by MTT assay. Results The sequence of the cloned DNA fragment of the recombinant plasmid pDC316/NY-ESO-lwas conforms with the sequence of the NY-ESO-1 gene. The expression of the NY-ESO-1 protein in HXO-RB44 cells was tested by immunofluorescence and Western blot. DCs were successfully induced with rhIL-4 and rhGM-CSF from PBMC. The recombinant expression plasmid pDC316/NY-ESO-1 was successfully transferred into DCs. These DCs had high expression of surface molecules such as HLA-DR （42. 1% ） , CD80 （54.2%） , CD83 （39.7%） and CD86 （94.8%）. The CTL that was induced by DCs-sensitized with NY-ESO-1 specifically killed HXO-RB44 cells. CTL induced by the sensitized DCs had a stronger cytotoxic effect against HXO-RB44 cells compared with un-sensitized DCs and CTL un-induced with DCs, as shown by MTT asssay（ P〈0.05 ）. The anti-tumor activity was highest when the ratio of effector to target was 75 ： 1 （ P〈0. 05 ）. Conclusions DCs transfected by the recombinant plasmid pDC316/ NY-ESO-1 can induce the proliferation of allogenic CTLs, which showed a specific anti-tunmr effect against HXORB44 cells. These results present a new type of immunotherapy for the treatment of RB. Key words: Retinoblastoma; Ddendritic cell; Cytotoxic T lymphocyte; NY-ESO-1

The effect of different health education methods on treatment of oral mucosal inflammafion caused by chemo - radiotherapy

Objective To explore the effect of different health education methods on the treatment of oral mucosal inflammation caused by chemo - radiotherapy.Methods A total of 100 cases of patients with head and neck malignant tumor undergone radiation and chemotherapy were randomly divided into two groups.The test group received personalized health education,and the control group received traditional health education.The effect of different health education methods was evaluated on the patients treated with recombinant human granulocyte macrophage colony stimulating factor for their oral mucosal inflammation caused by chemo-radiotherapy.Results The patients in test group had lighter oral mucosa reaction than those in control group.The difference between the two groups was significant (P＜0.05)The patients and familyin test group understood nursing skills and health care better than those in control group.The difference was statistically significant (P＜0.05) There was significantly different satisfaction degree toward health education between two groups.Conclusions The systemic personalized health education model can play a better and specific role in health education. Key words: Health education; Recombinant human granulocyte macrophage colony stimulating factor; Oral mucosal inflammation caused by chemotherapy; Nursing

A randomized trial of recombinant human granulocyte-macrophage colony stimulating factor for patients with acute lung injury*

Despite recent advances in critical care and ventilator management, acute lung injury and acute respiratory distress syndrome continue to cause significant morbidity and mortality. Granulocyte-macrophage colony stimulating factor may be beneficial for patients with acute respiratory distress syndrome. To determine whether intravenous infusion of granulocyte-macrophage colony stimulating factor would improve clinical outcomes for patients with acute lung injury/acute respiratory distress syndrome. A randomized, double-blind, placebo-controlled clinical trial of human recombinant granulocyte-macrophage colony stimulating factor vs. placebo. The primary outcome was days alive and breathing without mechanical ventilatory support within the first 28 days after randomization. Secondary outcomes included mortality and organ failure-free days. Medical and surgical intensive care units at three academic medical centers. One hundred thirty individuals with acute lung injury of at least 3 days duration were enrolled, out of a planned cohort of 200 subjects. Patients were randomized to receive human recombinant granulocyte-macrophage colony stimulating factor (64 subjects, 250 μg/M) or placebo (66 subjects) by intravenous infusion daily for 14 days. Patients received mechanical ventilation using a lung-protective protocol. There was no difference in ventilator-free days between groups (10.7 ± 10.3 days placebo vs. 10.8 ± 10.5 days granulocyte-macrophage colony stimulating factor, p = .82). Differences in 28-day mortality (23% in placebo vs. 17% in patients receiving granulocyte-macrophage colony stimulating factor (p = .31) and organ failure-free days (12.8 ± 11.3 days placebo vs. 15.7 ± 11.9 days granulocyte-macrophage colony stimulating factor, p = .16) were not statistically significant. There were similar numbers of serious adverse events in each group. In a randomized phase II trial, granulocyte-macrophage colony stimulating factor treatment did not increase the number of ventilator-free days in patients with acute lung injury/acute respiratory distress syndrome. A larger trial would be required to determine whether treatment with granulocyte-macrophage colony stimulating factor might alter important clinical outcomes, such as mortality or multiorgan failure. (ClinicalTrials.gov number, NCT00201409 [ClinicalTrials.gov]).

A randomized trial of recombinant human granulocyte-macrophage colony stimulating factor for patients with acute lung injury

A randomized trial of recombinant human granulocyte-macrophage colony stimulating factor for patients with acute lung injury

Formulation of sustained-release microspheres of granulocyte macrophage colony stimulating factor by freezing-induced phase separation with dextran and encapsulation with blended polymers

This study aimed to assess the potential merits of formulating sustained-release microspheres of recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) via freezing-induced phase separation (FIPS) of the protein with dextran followed by encapsulation with binary mixture of poly(lactic-co-glycolic acid) (PLGA) 2A (MW∼12K) and 3A (MW∼47K) or of PLGA2A and polylactic acid (PLA; MW∼83K). The formulated dextran particles and microspheres were characterized in vitro for loading, aggregation, bioactivity and release behavior of the protein where appropriate. rhGM-CSF retained about 60% of bioactivity with no significant aggregation after each formulation step. Encapsulation of protein-loaded dextran particles attained only 80% with the PLGA2A and PLGA3A blend, but 100% with the PLGA2A and PLA mixture. The former formulation exhibited a triphasic in-vitro release profile typical of PLGA microspheres while the latter revealed a much lower initial burst followed by a steady and complete release of rhGM-CSF with preserved bioactivity over a 15-day period.

Expression and mechanism of granulocyte-macrophage colony stimulating factor in acute skin defect of the mice

Objective To investigate the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its associated mechanism during the wound healing. Methods The animal model with the full-thickness skin injury was used in the study. Fifty male mice were involved in the study and divided randomly into control group (n = 25) and GM-CSF group (n = 25). Each group had five time points (5 mice per time point). All the mice received full-thickness skin defect (1 cm × 1 cm) through the panniculus camosus on the midline of the back near the neck after anesthesia. Recombinant human granulocyte-macrophage colony stimulating factor (RhGM-CSF) gel (10 μg/cm2) were applied in the GM-CSF group and gel matrix without RhGM-CSF applied in the control group. The wound healing time and rate were observed at days 3, 5, 7, 10 and 14 after injury. The wound specimens were collected to detect the histopathological change. The microvessel density of the wound was counted based on the results of CD31 immunohistochemistry. RT-PCR was employed to detect the expression changes of GMCSF, platelet-derived growth factor (PDGF) , vascular endothelial growth factor ( VEGF) and stromal cell derived factor-1 (SDF-1). Results RT-PCR results showed that the gene expression of GM-CSF reached the peak at day 3 after injury (P＜0. 01) and kept the high level at days 3-10 after injury (P＜ 0. 05) , followed by a sharp decrease to a normal level at day 14 after wound. The wound healing time was average (2.4 ±0. 3) days earlier than the control mice after application of rhGM-CSF, with significant increase of the wound healing rate during 7-14 days after injury ( P ＜ 0. 05 ). In the GM-CSF group, the early histology of trauma wound showed a small number of neutrophils, obvious epithelial cell proliferation in the wound margin, marked hyperplasia of the granulation tissue, high cell density with quantity of spindle-shaped and oval-shaped cells and increased number of new blood vessels. The microvessel density was also increased significantly (P ＜ 0. 05) at days 7-14 after injury. The gene expressions of VEGF and SDF-1 were significantly increased at day 7 and day 10 respectively after injury (P＜0.05) and the gene expression of pro-healing factor PDGF was significantly increased in every time point (at days 5, 7 and 10,P＜0.05;at day 14,P＜0.01). Conclusion GM-CSF expresses highly in the early stage after injury and can promote the wound healing, when the mechanism may relate to the up-regulated expressions of pro-angiogenic factors and pro-healing growth factors. Key words: Wound healing; Granulocyte-macrophage colony stimulating factor; Microvessel density; Growth factor; Gene expression

Phenotype of Dendritic Cells Generated from Peripheral Blood Monocytes of Patients with Ovarian Cancer

Objectives The aim of our study was to generate dendritic cells (DCs) from peripheral blood monocytes (PBMC) of patients suffering from ovarian cancer. Materials and Methods Immature DCs were generated from PBMC cultured in RPMI 1640 medium with 2% human serum albumin (HSA), supplemented with recombinant human (rh) granulocyte macrophage colony stimulating factor (GM-CSF) and rh interleukin (IL)-4. After 5 days of culture, DC maturation was induced by the addition of an ovarian cancer cell line (CAOV3) lysate and after 6 days of rh tumor necrosis factor (TNF)-α for a further 2 days. The phenotype of the generated cells was assessed by flow cytometry for the expressions of human leukocyte antigen (HLA)-DR, costimulatory molecules (e.g., CD86, CD80), CD83, CD1a, and CD14. PBMC cultured in 2% HSA without rhIL-4, rhGM-CSF, rh-TNF-α, or tumor cell lysate formed the control group. Results The 2.41% (interquartile range, 1.51%–3.52%) of CD45 +/CD14 + cells in cultures with rhIL-4, rhGM-CSF, rhTNF-α and tumor cell lysate was significantly lower than in the control group (31.10%; interquartile range, 11.11%–64.06%). Cultures with rhIL-4, rhGM-CSF, rhTNF-α, and tumor cell lysate showed a higher percentage (19.96%; interquartile range, 9.30%–24.42%) of fully mature CD83 +/CD1a −/HLA-DR + DCs compared with control culture (6.02%; interquartile range, 3.01%–7.37%). There was no significant difference in the expression of the immature DC marker (CD1a) between the cultures. The expression of co-stimulatory markers (CD80, CD86, HLA-DR) was greater (24.29%; interquartile range, 18.68%–33.95%) on DCs from cultures with rhIL-4, rhGM-CSF, TNF-α, and tumor cell lysate versus controls (4.93%; interquartile range, 2.67%–9.09%). Conclusion Our data demonstrated that immature and mature DCs can be generated from adherent human PBMC from ovarian cancer patients cultured with rhIL-4, rhGM-CSF, rhTNF-α, and tumor cell lysates.

Protective effects of recombinant human granulocyte macrophage colony stimulating factor on H1N1 influenza virus-induced pneumonia in mice

Protective effects of recombinant human granulocyte macrophage colony stimulating factor (rHuGM-CSF) on H1N1 influenza virus infection was studied in vivo and in vitro. Mice were infected with H1N1 influenza A viruses and rHuGM-CSF at doses of 0.34, 0.67, and 1.34 mg kg −1 d −1 was administrated for 7 days before the mice were infected with influenza virus and continued for a further 3 days. Compared with control mice, rHuGM-CSF was demonstrated to increase the survival rate of the infected mice by 50.0%, 55.6%, and 80.0% and increased the mean survival days by 25.7%, 30.0%, and 46.8%, respectively. Histopathological study of the lungs in pneumonia mice found that pre-treatment with rHuGM-CSF significantly ameliorated lung injury induced by influenza virus infection. In vitro study demonstrated that when rHuGM-CSF were co-incubated with peripheral blood mononuclear cells (PBMCs), the PBMCs culture supernatant induced a dose-dependent reduction of virus-induced cytopathic effect (CPE) in Madin-Darby canine kidney (MDCK) cells in vitro. These results suggested that rHuGM-CSF might be an effective and potential protection for H1N1 influenza virus-induced pneumonia.

Experimental study of transfecting adenovirus carrying G250 antigen gene to dendritic cells and to activate immune effector cells in kidney cancer treatment

Objective To investigate T cell-mediated effects of dendritic cells(DC)transfected with kidney associated antigen G250 gene using adenovirus(Ad)as vector in the treatment of kidney cancer. Methods Peripheral blood mononuclear cells(PBMC)were isolated by standard FicollPaque density gradient centrifugation of heparinized blood obtained from healthy donors.After removal of the nonadherent ceils,the adherent cells were divided into gene transfer group,protein pulsed group and control group.The gene transfer group was infected with Ad/G250 virus;the protein pulsed group was added with G250 protein; the control group was added with PBS.The DCs were cultured and proliferated in vitro using recombinant human granulocyte macrophage colony stimulating factor and inlerleukin-4.The autologous T cells were added into the 3 DC groups(responders:dendritics,20:1).After 5 d co-culture,3 eytotoxic T lymphocyte(CTL)groups were collected.TheG250 mRNA expression was detected in transduced DCs using RT-PCR.The G250 protein translation products and cell surface marker of DCs were analyzed by flow cytometry.The specific cytolytic activities of CTL to different target cells(renal carcinoma 786-0 cell line and lung cancer A549 cell line)were assessed by MTT method. Results Ad/G250 transfected DC successfully and the G250 expression was confirmed by RT-PCR. The expression levels of CD_(80), CD_(83). HLA-DR and CD_(86) in the Ad/G250 group were higher than those in the other 2 groups. The cytotoxicity to 786-0 of T lymphocytes activated by Ad/G250 transfecting DCs(83. 4±2. 8)% was greater than those of T lymphocytes in the protein pulsed group(79. 6±2. 4)% and control group(77. 3±2. 1)%. There were no significant differences of the cytotyxicities to A549 among the 3 cytotoxic T lymphocyte groups(F=0. 373.P=0.693). Conclusions Ad-loading DCs may be useful for immunotherapeutic protocols against self-antigens for the kidney cancer. Key words: Dendritic cells; Carcinoma, renal cell; Adenoviridae; T-lymphocytes, cytotoxic