Inmunodeficiencias secundarias relacionadas con la presencia de autoanticuerpos anticitocinas / Secondary immunodeficiencies related to the presence of anti-cytokine autoantibodies

Resumen Los autoanticuerpos anticitocinas (ACAA) han sido reportados como causa importante de inmunodeficiencias secundarias. Altos títulos de autoanticuerpos neutralizantes pueden causar susceptibilidad a diferentes enfermedades infecciosas potencialmente mortales. Por ejemplo, se ha informado que autoanticuerpos neutralizantes contra IFNγ se correlacionan con susceptibilidad a infecciones micobacterianas y patógenos fúngicos intracelulares. Autoanticuerpos contra IL-6 se detectaron en pacientes con abscesos subcutáneos y celulitis estafilocócica recurrente; asimismo, pacientes con criptococosis, nocardiosis y proteinosis alveolar pulmonar fueron positivos a autoanticuerpos contra GM-CSF. También se ha establecido una relación entre los autoanticuerpos contra IL-17 e IL-22 y las infecciones crónicas por Candida en mucosas, que se han identificado en pacientes con poliendocrinopatía autoinmune tipo 1 o timoma. Recientemente se han reportado autoanticuerpos contra interferón tipo I durante el inicio de COVID-19 aguda. Estos ACAA se asemejan a defectos genéticos en citocinas o en sus rutas de señalización. Por ello, pueden considerarse fenocopias de inmunodeficiencias primarias. De esta forma, la detección de ACAA podría ser importante en el diagnóstico, particularmente en pacientes con enfermedades de aparición tardía, para decidir los tratamientos apropiados. Esta revisión presenta una descripción general de la comprensión actual de las inmunodeficiencias secundarias asociadas a ACAA.

Abstract Anti-cytokine autoantibodies (ACAA) have been reported to be an important cause of secondary immunodeficiencies. High titers of neutralizing autoantibodies may cause susceptibility to different life-threatening infectious diseases. For example, neutralizing autoantibodies against IFNγ have been reported to be correlated with susceptibility to mycobacterial infections and intracellular fungal pathogens. Autoantibodies against IL-6 were detected in patients with subcutaneous abscesses and recurrent staphylococcal cellulitis; on the other hand, patients with cryptococcosis, nocardiosis, and pulmonary alveolar proteinosis were positive for autoantibodies to GM-CSF. A relationship has also been established between autoantibodies against IL-17 and IL-22 and chronic mucosal Candida infections, which have been identified in patients with APECED or thymoma. Autoantibodies against type-I IFN have been recently reported during the onset of acute COVID-19. These ACAAs resemble genetic defects in cytokines or their signaling pathways. Therefore, they may be considered to be primary immunodeficiencies phenocopies. Consequently, the detection of ACAA could be important in the diagnosis of patients, particularly in the case of late-onset diseases, in order to decide appropriate treatments. This review presents an overview of current understanding of ACAA-associated secondary immunodeficiencies.

Granulocyte-Macrophage colony stimulating factor in asthmatic patients infected with respiratory syncytial virus

@#Introduction: It is estimated that at least 30 to 40% of asthma attacks in adults are related to respiratory infections with viruses. The majority of asthma-related viruses include respiratory syncytial virus (RSV), rhinovirus, and parainfluenza. Inflammatory cytokines are supposed to play a vital role in causing inflammation of the respiratory tract as regulators of proliferation, chemotaxis, and activation of inflammatory cells. Objectives: The aim of this study is to assess the role of Granulocyte Macrophage-Colony Stimulating Factor (GMCSF) in asthmatic airway hyper-responsiveness associated with RSV infections. Materials and Methods: Forty five asthmatic cases and 45 healthy individuals were studied in a cross-sectional design. All asthmatics underwent symptom score assessment.GMCSF concentrations in sputum and RSV-IgM/IgG in serum samples were measured for all participants by Enzyme Linked Immuno-Sorbent Assay (ELISA). Results: The GM-CSF concentration level was significantly higher in asthmatics (270.27± 194.87pg/mL) especially among moderate and severe disease with mean concentration of 197.33±98.47 and 521.08± 310.04 respectively, compared to healthy controls (22.20±21.27 pg/ mL) (p=0.0001). The sputum level of GM-CSF in asthmatics is highly significant associated with positive anti-RSV IgG sera which represents 35/45(77.8%) with mean GM-CSF concentration of (276.99± 86.42) compared with controls at about 31/45 (68.9%) with GM-CSF mean concentration of (22.84±23.47). On the other hand, positive anti-RSV IgM in asthma cases was 8 out of 45(17.8 %) with GM-CSF mean concentration of (307.25± 306.65). Furthermore, GM-CSF sputum level was significantly correlated with eosinophil count especially in moderate and severe asthma. Conclusions: This study revealed that GM-CSF level is associated with eosinophilia and indicates asthma severity that might be evident during RSV infection .The distinctive GM-CSF features observed in the sputum from asthmatics with RSV may be useful as a diagnostic methods to help match patients with antibody therapy.

Pioderma gangrenoso paraneoplásico en una paciente con recaída de leucemia mieloide aguda / Paraneoplastic bullous pyoderma gangrenosum occurring in a patient with a relapse of an acute myeloid leukemia

Resumen El pioderma gangrenoso ampolloso fue descrito por primera vez en 1972. Se presenta el caso de una paciente con pioderma gangrenoso asociado a una recaída de leucemia mieloide aguda y se hace una revisión de la literatura sobre el tema.

Abstract Bullous pyoderma gangrenosum was first described by Perry in 1972. We present a case of a patient with paraneoplastic pyoderma gangrenosum associated to relapse of an acute myelogenous leukemia and we review the literature on pyoderma gangrenosum.

Immunohistochemical identification of GM-CSF in testis of rats with oligozoospermia with the intervention of Cuscuta chinensis flavonoids / Identificación inmunohistoquímica de GM-CSF en testículos de ratas con oligozoospermia con la intervención de flavonoides de Cuscuta chinensis

Oligozoospermia is a common infertility disease, and the incidence rate is increasing year by year. Cuscuta chinensis is a commonly used medicine for the treatment of oligozoospermia in Chinese medicine. Flavonoids are its main component. GM-CSF is a multifunctional cytokine that plays an important role in the inflammatory response. In this paper, we performed HE staining and immunohistochemical staining on the testis of rats with oligozoospermia. We intend to study the expression changes of GM-CSF in rats with oligospermia and the effect of flavonoids on the expression of GM-CSF in testis of rats with oligozoospermia.

La oligozoospermia es una enfermedad común de infertilidad, con una tasa de incidencia que aumenta año tras año. Cuscuta chinensis es un medicamento de uso común para el tratamiento de la oligozoospermia en la medicina china. Los flavonoides son su componente principal. GM-CSF es una citocina multifuncional que tiene un rol importante en la respuesta inflamatoria. En este trabajo, realizamos tinción con hematoxilina y eosina y tinción inmunohistoquímica en testículos de ratas con oligozoospermia. TNuestro objetivo fue estudiar los cambios de expresión de GM-CSF en ratas con oligozoospermia y el efecto de los flavonoides en la expresión de GM-CSF en testículos de ratas con oligozoospermia.

Influences on induction and maturation of mouse bone marrow-derived dendritic cells by varying combinations of cytokines / 中华微生物学和免疫学杂志

Objective To investigate the influences of culture conditions on the in vitro induction and maturation of dendritic cells by using different combinations of cytokines. -ethods Mouse bone mar-row cells were isolated and cultured in media containing varying combinations of cytokines, including granu-locyte-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4) and fms-like tyrosine kinase 3 ligand (Flt3L). After cultured at 37℃ for seven days, the attached bone marrow cells were collected and stained by fluorescence-labeled monoclonal antibodies (McAb) against CD11c, MHCⅡ and CD86 for flow cytometry analysis. In the parallel group, LPS was added on day 5 to a final concentration of 1μg/ml for DC maturation analysis by flow cytometry. Results In group Flt3L (20 ng/ml)/GM-CSF (20 ng/ml)/IL-4 (10 ng/ml), 90％ of bone marrow cells were CD11c-positive. Flt3L (100 ng/ml) could induce 88％ of bone marrow cells to express CD11c. Bone marrow cells positive for MHCⅡ accounted for 35. 4％ and 36. 1％ in group Flt3L/GM-CSF/IL-4 and group Flt3L/GM-CSF, where both Flt3L and GM-CSF were used at a concentration of 20 ng/ml. After LPS stimulation, the positive rates of MHCⅡ in group Flt3L/GM-CSF/IL-4 and group Flt3L/GM-CSF were 58. 1％ and 59. 6％, which increased by 22. 7％ and 23. 5％, re-spectively. The percentages of CD86-positive bone marrow cells were 7. 1％ and 5. 5％ in group Flt3L/GM-CSF/IL-4 and group Flt3L/GM-CSF. Bone marrow cells positive for CD86 grew by 7. 1％ and 6. 2％ in group Flt3L (20 ng/ml) and group GM-CSF/IL-4 after LPS stimulation. Conclusions Flt3L and GM-CSF probably dominated the differentiation and maturation of bone marrow-derived dendritic cells with a synergis-tic effect. Combined usage of Flt3L and GM-CSF at the concentration of 20 ng/ml would be an optimal proto-col for DC research.

A study to assess the therapeutic role of GM-CSF (EMGRAST -M) on augmentation of total leucocyte count and total platelet count in cancer patients after chemotherapy

Background: The aim of the present study was to assess the therapeutic role of GM-CSF (EMGRAST -M) on augmentation of total leucocyte count and total platelet count in cancer patients after chemotherapy.Methods: The total leucocyte count (TLC) and total platelet count (TPC) of thirty patients on chemotherapy were obtained before and after the administration of GM-CSF. The results were analysed retrospectively for the effect of GM-CSF on these parameters. Statistical analysis was done, and graphs were made by Libre office calc and Student’s T Test was used for comparison of data.Results: The study showed that EMGRAST-M had an impressive effect on both the platelet count and the leucocyte count.Conclusions: GM-CSF has a great therapeutic role in the enhancement of platelet count and leucocyte count in patients of cancer chemotherapy.

Evaluation of monocytes and dendritic cells changes in peripheral blood of diffuse large B-cell lymphoma patients before and after GM-CSF treatment / 国际检验医学杂志

Objective To evaluate the changes of monocytes,dendritic cells(DC),myeloid dendritic cells (mDC)and plasma dendritic cells(pDC)of peripheral blood in diffuse large B-cell lymphoma(DLBCL)pa-tients after 3 courses of GM-CSF treatment.Methods 33 patients diagnosed with DLBCL in the hospital from October 2015 to February 2016 were enrolled in the study.All the patients were treated with GM-CSF for 3 courses.Before each course,3 mL venous blood samples were extracted and the monocytes,dendritic cells,my-eloid dendritic cells and plasma dendritic cells were analyzed by using flow cytometer within 24 h.All the data was analyzed with SPSS17.0 software.Results After 3 courses′ treatment,the monocytes decreased signifi-cantly,from(9.85 ± 2.31)% to(3.67 ± 0.78)%.Dendritic cells increased significantly,from(0.24 ± 0.04)% to(0.58 ± 0.13)%.mDCs increased significantly from(0.11 ± 0.02)% to(0.36 ± 0.08)%,however, pDCs only increased from(0.13 ± 0.03)% to(0.21 ± 0.04)% with no statistically significance.Conclusion After 3 treatment courses with GM-CSF,the monocytes decreased obviously while DCs especially mDCs in-creased obviously in DLBCL patients.GM-CSF might activate the anti-tumor immunity of DLBCL patients.

Efficacy enhancement of a Baculovirus-vectored Newcastle Disease Virus F protein vaccine by chicken GM-CSF and IL-2 / 生物工程学报

To compare with the effects of the GM-CSF and IL-2 used as adjuvants in the baculovirus vaccine, we used genetic engineering to construct the recombinant baculovirus rBV-LMI-F and with GM-CSF and IL-2 to immunized chickens. Then, we compared the concentration of the neutralizing antibody and cytokines to determine the immunostimulatory effects of GM-CSF and IL-2. GM-CSF induced higher levels of antibodies and cytokines in chickens at 28 d and 42 d post-vaccination. In conclusion, GM-CSF could elicit higher serum antibody and cytokines responses and improved the effects of Baculovirus vaccine.

Promoting effect of Ginsenoside Ro on differentiation of THP-1-derived DC / 中国免疫学杂志

Objective:To investigate the promoting effect of Ginsenoside Ro on the differentiation of THP-1-derived dendritic cells (DCs) induced by GM-CSF and IL-4.Methods: Sensitive leukemia-derived DC cell line was screened first.Then,the selected sensitive cell line THP-1 was stimulated to differentiate into DCs by cytokines (GM-CSF and IL-4) and small(5 μmol/L),middle(10 μmol/L),and large (20 μmol/L) dose of Ginsenoside Ro respectively.The expressions of CD1a,MHCⅡ and CD86 of leukemia-derived DCs were detected by flow cytometry.In addition,the transcription levels of CD1a,CD86 and MHCⅡ of leukemia-derived DCs were detected by RT-PCR.ELISA was used to measure the protein levels of TNF-α and IL-6 in the culture supernatant.Results: THP-1 was the sensitive leukemia cell line which could be induced to differentiate into DCs by cytokines.Compared with cytokine stimulation alone,the expression of CD1a,MHCⅡ and CD86 in leukemia-derived cells was significantly increased after the stimulation of Ginsenoside Ro combined with cytokine(P<0.05).The CD1a,CD86 and MHCⅡ mRNA expression was significantly increased after the treatment of Ginsenoside Ro combined with cytokine(P<0.05).Moreover,the protein levels of TNF-α and IL-6 in culture supernatant were significantly increased (P<0.05) after the stimulation of Ginsenoside Ro in combination with cytokines.Conclusion: Ginsenoside Ro can significantly promote the differentiation of leukemia-derived DCs.

Effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the anti-fertility efficacy of canine zona pellucida glycoprotein 3 (CZP3) DNA vaccine / 中华微生物学和免疫学杂志

Objective To investigate the effects of GM-CSF (granulocyte-macrophage colony-stimulating factor) as an immune adjuvant on the anti-fertility efficacy of canine zona pellucida glycoprotein 3 (CZP3) DNA vaccine.Methods RT-PCR and quantitative real-time PCR were performed to analyze the effects of GM-CSF on the maturation of antigen presenting cells (APCs) and the enrichment of APCs at injection site.Female BALB/c mice were immunized with CZP3 DNA vaccine pcDNA3-CZP3 alone or in combination with genetic adjuvant pcDNA3-GM-CSF by using the method of electrical impulses.ELISA (enzyme-linked immunosorbent assay) was used to detect the levels of IgG and sIgA (secretory IgA) antibodies in serum samples and genital tracts and the levels of IL-4 and IFN-γ in serum samples.MTT method was used to analyze the proliferation of spleen T cells in mice.The binding ability of serum anti-CZP3 antibody to native mouse oocytes was analyzed by indirect immunofluorescence assay.All of the female BALB/c mice were coupled with male mice of the same age six weeks after the first-dose vaccination.Litter size at birth in each group was counted and the differences between different groups were comparatively analyzed.ResultsImmunizing the mice with pcDNA3-CZP3 and GM-CSF significantly promoted the expression of CD80, CD83 and CD86 (P<0.05) and increased the sIgA antibody level in genital tract and IgG level in serum (P<0.01).Moreover, the levels of IL-4 and IFN-γ in serum samples were significantly up-regulated (P<0.05) and the proliferation of spleen T cells was significantly enhanced (P<0.05).Results of the indirect immunofluorescence assay showed that the fluorescence intensity and density on mouse egg surface were positively correlated with the level of antibody in serum.Results of the anti-fertility test suggested that GM-CSF significantly reduced the litter size in mice immunized with pcDNA3-CZP3 vaccine (P<0.05).Conclusion GM-CSF could be used as an effective adjuvant to enhance the anti-fertility efficacy of CZP3 DNA vaccine.

The influence of protein malnutrition on the production of GM-CSF and M-CSF by macrophages

ABSTRACT It is well established that protein malnutrition (PM) impairs immune defenses and increases susceptibility to infection. Macrophages are cells that play a central role in innate immunity, constituting one of the first barriers against infections. Macrophages produce several soluble factors, including cytokines and growth factors, important to the immune response. Among those growth factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). GM-CSF and M-CSF are important to monocyte and macrophage development and stimulation of the immune response process. Knowing the importance of GM-CSF and M-CSF, we sought to investigate the influence of PM on macrophage production of these growth factors. Two-month-old male BALB/c mice were subjected to PM with a low-protein diet (2%) and compared to a control diet (12%) mouse group. Nutritional status, hemogram and the number of peritoneal cells were evaluated. Additionally, peritoneal macrophages were cultured and the production of GM-CSF and M-CSF and mRNA expression were evaluated. To determine if PM altered macrophage production of GM-CSF and M-CSF, they were stimulated with TNF-α. The PM animals had anemia, leukopenia and a reduced number of peritoneal cells. The production of M-CSF was not different between groups; however, cells from PM animals, stimulated with or without TNF-α, presented reduced capability to produce GM-CSF. These data imply that PM interferes with the production of GM-CSF, and consequently would affect the production and maturation of hematopoietic cells and the immune response.

Tos e hipoxemia como manifestación clínica de la proteinosis alveolar: Reporte de caso clínico / Cough and hypoxemia as clinical manifestation of pulmonary alveolar proteinosis: Clinical case report

La proteinosis alveolar es una enfermedad pulmonar crónica poco frecuente, especialmente en pediatría, caracterizada por la acumulación anormal de lipoproteínas y derivados del surfactante en el espacio intraalveolar, que genera una grave reducción del intercambio gaseoso. La forma de presentación idiopática constituye más del 90% de los casos relacionados con un fenómeno de autoinmunidad, con producción de anticuerpos dirigidos contra el receptor del factor estimulante de colonias de granulocitos y macrófagos. Se presenta un caso clínico de una niña de 4 años de edad tratada por neumonía atípica con evolución desfavorable por hipoxemia persistente. El diagnóstico se obtuvo a través del estudio anatomopatológico de la biopsia pulmonar por toracotomía. Se llevaron a cabo 17 lavados broncopulmonares mediante endoscopía respiratoria y la paciente evidenció franca mejoría clínica.

Alveolar proteinosis is a rare chronic lung disease, especially in children, characterized by abnormal accumulation of lipoproteins and derived surfactant in the intra-alveolar space that generates a severe reduction of gas exchange. Idiopathic presentation form constitutes over 90% of cases, a phenomenon associated with production of autoimmune antibodies directed at the receptor for granulocyte-macrophage colony-stimulating factor. A case of a girl of 5 years of age treated because of atypical pneumonia with unfavorable evolution due to persistent hypoxemia is presented. The diagnosis is obtained through pathologic examination of lung biopsy by thoracotomy, as treatment is carried out by 17bronchopulmonary bronchoscopy lavages and the patient evidences marked clinical improvement.

The immunogenicity and anti-tumor effects of a lung cancer DNA vaccine harboring a MUC-1 and GM-CSF fusion gene

ABSTRACT DNA vaccines have been shown to be an effective approach to induce antigen-specific cellular and humoral immunity. However, the inability of DNA vaccines to elicit strong immune responses in clinical trials limits the application of DNA vaccines. Here, we developed a new DNA vaccine based on MUC1, which has been suggested as a potential target for lung cancer therapy, and we enhanced the potency of the DNA vaccine by including granulocyte-macrophage colony-stimulating factor (GM-CSF) as an adjuvant. A series of DNA plasmids encoding MUC1, human GM-CSF and their conjugates were constructed and injected into female mice intramuscularly (i.m.). This action was followed by an electric pulse. The humoral and cellular immune responses after immunization were examined by ELISA and ELISPOT, respectively. To evaluate the therapeutic efficacy of the plasmids, a mouse model with a MUC1-expressing tumor was designed. Mice vaccinated with the MUC1-GM-CSF plasmid generated the strongest MUC1-specific humoral and cellular immune responses. Furthermore, these vaccinations inhibited the growth of MUC1-expressing tumors and prolonged mouse survival. These observations emphasize the potential of GM-CSF as an adjuvant for DNA vaccines and of vaccines based on MUC1 and GM-CSF as a promising treatment for lung cancer.

Effect of Xiaoyan Decoction on bone marrow suppression of mice with Lewis lung cancer / 中草药

Objective: To study the effects and mechanism of Xiaoyan Decoction on bone marrow suppression in mice from aspects of peripheral blood cell ratio, immune function, and cytokines. Methods: A mouse model of bone marrow suppression was established. Fifty mice were randomly divided into five groups (n = 10): control group, model group, Xiaoyan Decoction group, rhG-CSF group, and Xiaoyan Decoction combined with rhG-CS group. Lewis-tumor mice in Xiaoyan Decoction group were ig given Xiaoyan Decoction 18.2 g/(kg·d) twice daily, for 7 d. Lewis-tumor mice in rhG-CS group were treated by sc injection of rhG-CSF. The routine blood test, immunological functions of peripheral blood and cytokines (IL-3, IL-6, EPO, and GM-CSF) in serum were detected. Results: The bone marrow suppression model was successfully established. Compared with control group, the levels of WBC, RBC, Hb, and PLT in peripheral blood of model group were decreased, especially in WBC (P 0.05). The concentration of IL-3 and IL-6 was dramatically reduced in model group compared with control group (P < 0.05). And it showed an obviously increasing trend in IL-3, IL-6, EPO, and GM-CSF levels in Xiaoyan Decoction group and combined group, as well as an increase in IL-3, IL-6 levels in rhG-CS group (P < 0.05). Conclusion: It shows that Xiaoyan Decoction combined with rhG-CS can improve the level of WBC and immunological functions of peripheral blood in bone marrow suppression model mice. And the effect of combination of both drugs is more significant. This might be related to the effect of promoting the production of serum hematopoietic regulator such as IL-3, IL-6, EPO, and GM-CSF, and relieving chemotherapy-induced bone marrow suppression.

IL-17 A promotes differentiation and maturation of bone marrow-derived dendritic cells by cooperating with GM-CSF and LPS / 中国免疫学杂志

Objective:To investigate the effect of IL-17A on the differentiation and maturation of murine bone marrow-derived dendritic cells( BMDCs ) . Methods: Murine bone marrow cells were isolated and cultured in RPMI1640 complete medium in the presence of GM-CSF(20 ng/ml) for 8 days to induce differentiation of murine bone marrow cells to DC progenitors. Then these cells were treated with LPS(1 μg/ml) for 36 h which polarized immature DCs into mature DCs. Different concentrations of rmIL-17A(10 or 100 ng/ml) was added to the culture medium at different stages of BMDC differentiation and maturation. Co-stimulatory molecules expression on BMDC were analyzed by flow cytometry,and the culture supernatants were analyzed for IL-12p40 and IL-10 level by ELISA. Results:rmIL-17 could promote co-stimulatory molecules( CD40,CD80,CD86 and MHCⅡ) expression on BMDCs in a does-dependent manner,especially,the expression of CD40 and MHCⅡhad a significant increase in high concentration of rmIL-17A group;rmIL-17A was added while LPS induced maturation of BMDCs. CD40,CD80,CD86 and MHCⅡexpression on BMDC increased sharply in LPS plus rmIL-17A stimulation group,besides,CD86,MHCⅡ showed a higher level expression on BMDC with the increase of con-centration of rmIL-17A. Furthermore,secretion of IL-12p40 and IL-10 increased significantly in the group of DCs treated with LPS plus low concentration of rmIL-17 compared with the group without rmIL-17(P<0. 001). However,high concentration of rmIL-17A group showed significantly higher levels of IL-12p40(P<0. 001),but there was no difference in IL-10. Conclusion:IL-17A promotes the phe-notypic development of BMDC progenitors propagated in GM-CSF and cooperate with LPS to induce BMDC differentiation and matura-tion.

Research on mechanism of GM-CSF secreting liver cancer vaccine on CTL killing activity of transplanted liver cancer mice / 中国生化药物杂志

Objective To study the effect of granulocyte-macrophage colony-stimulating factor ( GM-CSF) secreting liver cancer vaccine on killing activity of cytotoxic T lymphocytes ( CTL) of transplanted liver cancer mice and its mechanism.Methods There were three groups:liver cancer vaccine group (A group), liver cancer group (B group) and PBS group (C group).The transplanted liver cancer model was builded with injection of H 22 hepatoma cells, while the GM-CSF secreting liver cancer vaccine group and PBS group was builded.GM-CSF secreting liver cancer vaccine group and PBS group were establised.The levels of CD8 +T cell in peripheral blood were detected by flow cytometry.The killing activity of cytotoxic T lymphocytes ( CTL) of spleen cells was detected by MTT method.The expression levels of tumor necrosis factor-α( TNF-α) and interferon-γ(γ-INF) were detected by Western blot.Results The flow cytometry results showed that, compared with B group, the levels of CD8 +T cell of A group significantly increased (P<0.01).MTT results showed that, compared with B group, the killing activity of cytotoxic T lymphocytes (CTL) in A group significantly increased (P<0.01).Western blot results showed that, compared with B group, the expression levels of tumor necrosis factor-α(TNF-α) and interferon-γ(γ-INF) in A group significantly decreased (P<0.01).Conclusion GM-CSF secreting liver cancer vaccine can significantly inhibit the activity of H22 cell, and its possible mechanism of action may be to activated CD8 +T expression, improve cytotoxic activity of CTL of spleen cells, and reduce TNF-αand γ-INF protein expression.

Enhanced metastasis in RNF13 knockout mice is mediated by a reduction in GM-CSF levels

RING finger protein 13 (RNF13) is a novel E3 ubiquitin ligase whose expression is associated with cancer development. However, its specific role in cancer progression and metastasis remains unclear. Here, a B16F10/LLC experimental pulmonary metastatic model was developed to examine the formation of metastatic foci in the lung. A greater number of tumor colonies were observed in the lungs of RNF13-knockout (KO) mice than in their wild-type (WT) littermates, whereas no significant differences in tumor size were observed between the two groups. In short-term experiments, the number of fluorescently-labeled B16F10 cells increased remarkably in RNF13-KO lungs at early time points, whereas clearance of tumor cells from the blood was not affected. These results indicated that RNF13 may inhibit the colonization of B16F10 cells in the lung. Assessment of the concentration of various cytokines in tumor bearing lungs and blood did not detect significant differences between the blood of RNF13-KO and WT mice; however the levels of GM-CSF were significantly reduced in RNF13-KO tumor bearing lungs, which may have guided more B16F10 cells to migrate to the lungs. This was confirmed by lower GM-CSF concentrations in conditioned media from the culture of RNF13-KO lung slices. Collectively, our results suggest that host RNF13 affects the concentration of GM-CSF in tumor-bearing lungs, leading to a reduction in the colonization of metastatic tumor cells in the lung.

Effect of M-CSF and GM-CSF on migration and VEGF-A expression of breast cancer cell line 4T1 / 肿瘤研究与临床

Objective To investigate the effect of M-CSF and GM-CSF on migration and expression of VEGF-A in breast cancer cell line 4T1.Methods Real-time PCR was used to detect VEGF-A mRNA expression in 4T1 cells treated by 5 ng/ml or 10 ng/ml M-CSF or GM-CSF.Ability of migration and metastasis of 4T1 cells were analyzed by scratch and Transwell assays.Results The relative expression of VEGF-A mRNA at 12 h and 24 h in 4T1 cells treated by 5 ng/ml or 10 ng/ml M-CSF were 17.81±2.49 and 17.48± 5.43,5.15±2.59 and 5.45±4.28,respectively,while those treated by GM-CSF were 9.77±2.39 and 7.61±2.80,6.53±2.41 and 6.30±2.89,respectively.M-CSF and GM-CSF can promote the expression of VEGF-A in 4T1 cells (P ＜ 0.05).The relative expression of VEGF-A was higher in 4T1 cells treated for 12 h than that for 24 h (P ＜ 0.01).M-CSF,GM-CSF and VEGF-A can promote metastasis of 4T1 cells (all P ＜ 0.05),whereas no gross migration of 4T1 cells was showed by VEGF-A treatment.Conclusion M-CSF and GM-CSF can promote the migration and expression of VEGF-A in breast cancer cell line 4T1.

Evaluation of the immunogenicity of a rBCG expressing GM-CSF and LMP2A / 中华微生物学和免疫学杂志

Objective To identify the expression of a fusion gene GCA formed from GM-CSF gene and LMP2A gene of Epstein-Barr virus (EBV) in a recombinant BCG (rBCG) and to study its immunoge-nicity.Methods The rBCG was constructed to express the fusion gene GCA and the expressed products were detected by Western blot assay .ELISA was performed to measure specific antibody titers in serum sam-ples from mice immunized with rBCG .Lactate dehydrogenase assay was used to analyze the cellular immuni-ty of mice.A mouse model of EBV-positive gastric carcinoma was established to evaluate the therapeutic effects of rBCG.Results The target proteins of GM-CSF and LMP2A were successfully expressed in rBCG . The specific antibodies were detected in rBCG immunized mice as indicated by ELISA .The maximum anti-body titer reached 1 ∶27 900 [(326.5±7.8) pg/ml] as injection with rBCG 5×108/mouse.The rBCG in-duced cytotoxicity of cytotoxic lymphocytes (CTLs) to EBV-positive gastric carcinoma cells (GT39) (with a killing rate of 89.6%±6.8%) was significantly higher than that of control group (P<0.05) The sizes of tumor in PBS control group [(1964.0±548.7) mm3] and BCG group [(1268.65±72.4) mm3] were big-ger than those in rBCG group [(168.64±78.80) mm3].Conclusion The rBCG expressing GM-CSF and LMP2A fusion gene was successfully constructed .The rBCG could induce humoral and cellular immune re-sponses in mice and inhibit the growth of tumor .

Inactivated genotype 1 Japanese encephalitis vaccine for swine

PURPOSE: Japanese encephalitis is a reproductive disorder caused by Japanese encephalitis virus (JEV) in swine. Recent genotype (G) shift phenomenon (G3 to G1) in the Asia-wide has posed a challenge for proper prevention by the current vaccine strain. Thus, new kinds of JEV G1 vaccines with enhanced immunogenicity have been required for pigs. MATERIALS AND METHODS: Recombinant porcine granulocyte monocyte-colony stimulating factor (reporGM-CSF) protein was expressed in Spodoptera frugiperda (Sf-9) cells using baculovirus expression system. Two kinds of trials with inactivated JEV vaccines containing IMS1313 adjuvant (Seppic, France) were prepared with or without reporGM-CSF protein. Safety and immunogenicity of the pigs inoculated with the JEV vaccines via intramuscular route was evaluated for 28 days after inoculation. RESULTS: Mice, guinea pigs, and fattening pigs inoculated with the inactivated vaccine showed no signs for 14 and 21 days. Both hemagglutination inhibition and plaque reduction neutralizing antibody titers were significantly higher in pigs immunized with the vaccine containing reporGM-CSF protein after boosting. However, on the side of vaccine efficacy, most mice (87%) immunized with the inactivated JEV vaccine survived after virulent JEV challenge. Whereas the group with the vaccine containing reporGM-CSF protein showed lower protective effects than the vaccine alone for the biological activity of the GM-CSF depending on species specific. CONCLUSION: Our data indicate that animals inoculated with the JEV vaccines was safe and pigs inoculated with inactivated JEV vaccine containing reporGM-CSF protein showed higher humoral immune responses than that of inactivated JEV vaccine without reporGM-CSF protein.