Detection of Candida albicans fructose bisphosphate aldolase antigen-specific T cells in healthy individuals by ELISPOT assay / 医学研究生学报

Objective Invasive candidiasis is associated with a significant mortality clinically.The purpose of this study was to observe the T cells immune response to fructose bisphosphate aldolase (Fba), an immunodominant antigen of Candida albicans, and determine whether the antigen has the possibility of priming cellular immune protection in invasive candiasis.Methods Using ELISPOT assay, we determined the frequencies of positive spot-forming cells (SFCs) of Fba antigen-specific T cells secreting IFN-γ, IL-4 and IL-17A in the peripheral blood mononuclear cells (PBMCs) of 26 healthy individuals.Results After Fba stimulation, the frequencies of positive SFCs of IFN-γ, IL-4 and IL-17A in the 26 healthy subjects were 23 (9.75, 42.50), 0 (0, 0.25) and 1.5 (0.75, 8.25), respectively, with statistically significant differences among the three (P<0.01).The response rates of IFN-γ (100% [26/26]) and IL-17A (76.92% [20/26]) were significantly higher than that of IL-4 (15.38% [4/26]) (P<0.01).Fba-induced strong response (SCFs ≥20) for IFN-γ was observed in 57.69% (15/26) of the healthy individuals, that for IL-17A in only 1, while that for IL-4 in none.Responses of both Th1 and Th17 cells to Fba were found in 65.38% (17/26) of the subjects, that of Th1 cells in 19.23% (5/26), but that of Th2 cells in none.Conclusion Fba of Candida albicans can induce immunodominant responses of Th1 and Th17 cells and is a potential vaccine against invasive candiasis.

Can dida colonizait on associated with the humoral immune anamnestci respon se oft he dominant antigen in the early stage of invasive Candidosis / 医学研究生学报

Objective Rapid elevation of the IgG antibody against Candida Enolase ( Eno ) has been observed in patients with invasive candidosis in an early stage .The present study was to confirm the association of Candida colonization with humoral im-mune anamnestic response of the dominant antigen . Methods Twenty-four mice were randomized into group 1 treated by oral Candi-da colonization plus intraperitoneal infection ( immunocompetent , n=8) , 2 treated with immunosuppressant in addition to the treatment of group 1 ( immunocompromised , n=8) , 3 treated by oral Candida colonization only ( immunocompetent , n=4) and 4 treated by in-traperitoneal injection only( immunocompetent, n=4).The number of Eno-specific memory B-cells in the spleen and the levels of IgG , IgM and IgA antibodies were determined in the peripheral blood of the immunocompetent and immunocompromised invasive candidiasis mice . Results At 7 days after invasive infection , there were significantly more Eno-specific memory B-cells in the mice of groups1 ( 47.25 ± 13.81) and 2 (43.14±15.95) than in groups 3 (8.00±3.74) and 4(8.50±2.38) (P0.05).Eno-IgG antibodies were detected in the serum of the mice of the first two groups in the early stage of invasive infection and positively corre -lated with antigen-specific memory B-cells (r=0.737,P <00.1 ). Conclusion Rapid elevation of the Eno-IgG antibody level in the early stage of invasive infection after Candida colonization may be attributed to the rapid proliferation of humoral immune memory cells.

Evaluation of Diagnostic Value of Antibody Against Aspergillus Fumigatus Pectate Lyase A for Invasive Aspergillosis / 现代检验医学杂志

Objective To establish an ELISA for detecting antibody against Aspergillus fumigatus pectate lyase A(anti-PlyA),and evaluate its diagnostic value for invasive aspergillosis (IA).Methods An indirect ELISA for IgG antibody a-gainst PlyA was established using PlyA as coating antigen.The serum from 97 IA patients,80 non-IA patients and 200 healthy donors were tested,the results were compared with anti-DPPV (antibody against Aspergillus fumigatus dipeptidyl peptidase V fragment)and anti-TR (antibody against Aspergillus fumigatus thioredoxin reductase).Results The intra-as-say coefficients of variation of the ELISA method for detecting anti-PlyA was 5.3%,and inter-assay coefficients of variation was 10.9%.The sensitivity and specificity of diagnosis of IA were 62.9% and 90.4%,respectively.The positive rates of an-ti-PlyA in non-neutropenic and neutropenic IA patients were 43.8% and 72.3%,respectively (χ2 =7.493,P 0.05).When combined anti-PlyA,anti-TR,and anti-DPPV,the diagnostic sensitivity for IA patients in-creased to 92.8%.Conclusion An ELISA for detecting anti-PlyA was successfully established.The diagnostic value of these three kinds of antibody was superior in non-neutropenic IA patientsto that in neutropenic IA patients.The combined detec-tion of three antibodies could provide higher sensitivity.

Value of serum procalcitonin determination for the etiological diagnosis and prognosis of systemic inflam-matory response syndrome in surgical ICU / 医学研究生学报

Objective The systemic inflammatory response syndrome ( SIRS) can be caused by infection and non-infection factors, which have similar clinical features but differ in treatment and prognosis .Rapid synthesis of procalcitonin ( PCT) during infec-tion can be used as a biomarker for the early diagnosis of sepsis .The present study aims to assess the value of the serum PCT level in the etiological diagnosis and prognosis of SIRS in the surgical ICU . Methods We retrospectively analyzed the data on 166 cases of SIRS from the surgical ICU in Jinling Hospital between June 2014 and June 2015 .The data obtained were associated with the patients'demograph-ics, primary diseases, laboratory results, and clinical outcomes.We analyzed the serum PCT values , blood culture results , and clinical outcomes. Results Totally, 131 of the patients were diagnosed with sepsis, with a median value of serum PCT of 2.43 (0.81-10.51) ng/mL, of whom 109 were PCT-positive (≥0.47 ng/mL), with a positive rate of 83.2%.Among the 35 non-infection SIRS patients, the mean level of serum PCT was 0.23 (0.1 -0.39) ng/mL, with a positive rate of 17.14%(6/35).There were statistically significant differences between the two groups in both the ser-um PCT level and positive rate (P<0.05).The PCT-positive rate was significantly higher in the bacteria-infected than in the fungi-in-fected group (86.5%[83/96] vs 74.3%[26/35], P<0.05), with a median value of 4.28 (1.05-14.59) ng/mL and 0.89 (0.37-1.59) ng/mL, respectively, so was it in the survivors than in the non-survivors (94.4%[34/36] vs 78.9%[75/95], P<0.05), with a median value of 12.89 (4.76-47.73) ng/mL and 1.41 (0.54-4.00) ng/mL, respectively. Conclusion The se-rum PCT level might be used to distinguish between sepsis and non-infection SIRS, significantly higher in bacteria-infected and survival groups than in fungi-infected and non-survival groups .Serum PCT determination contributes to the etiological diagnosis and prognosis of SIRS.

Development of an immunomagnetic bead assay for quantitative detection enolase of Candida albicans / 医学研究生学报

Objective To develop an immunomagnetic bead ( IMB) assay for quantitative detection enolase ( Eno) of Candi-da albicans, and to improve the diagonosis of invasive candidiasis . Methods The immunomagnetic bead was prepared by conjuga-ting with Anti Eno of Candida albicans monoclonal antibody .HRP-conjugated goat polyclonal antibody against Candida albicans Eno was employed as detecting antibody .The performance parameters of the IMB assay including precision , specificity, linear range and limit of detection were verified by using recombinant Candida albicans Eno.Then the developed assay was applied to determine Eno levels in supernatant of pathogenic fungi cultures . Results The intra and inter-coefficient of variation was 4.54%, 5.87% and 5.26%, 8.82%at the concentration of 25 ng/mL and 5 ng/mL, respectively.The limit of detection was 0.5 ng/mL.The linear range was(0.5-50) ng/mL.The level of Eno in Candida albicans culture after incubated in 37℃for 24 h was 3.89 ng/mL and gradually in-creased to 37.89 ng/mL at 120 h.There was a positive correlation between the level of Eno and growth hyphae of Candiad albicans. There was weak cross reaction with Candida parapsilosis and no cross reaction with Candida tropicalis, Candida guilliermondii, Candida glabrata, Cryptococcus neoformans and Saccharomyces cerevisiae. Conclusion An IMB assay for quantitative detection Eno of Candida albicans was developed , which was more sensitive , rapid and reliable than previous qualitative ELISA .The IMB assay has the potential to be applied to the research in invasive candidasis .

An Immunochromatographic Lateral-flow Device(LFD) for Rapid Diagnosis of Aspergillosis / 现代检验医学杂志

An immunochromatographic Lateral-Flow Device (LFD)was invented for rapid serodiagnosis of Invasive Asper-gillosis by foreign investigator.A monoclonal antibody against Aspergillus extracellular glycoprotein is used as capture anti-body and detection antibody in LFD.LFD is a simple,rapid,single sample performance technique by which Aspergillus ex-tracellular glycoprotein in serum and/or bronchoalveolar lavage fluid (BAL)of patients can be detected.The principle of LFD and the features of antigen and antibody involved in the technique will be described.The diagnositic value of novel LED is compared with that of GM test and PCR.

Detection of Aspergillus fumigatus thioredoxin reductase GliT antigen-specific T cells by ELISPOT assay in healthy individuals and result evaluation / 医学研究生学报

Objective The purpose of this study was to evaluate the protective potential of the Aspergillus fumigatus thiore -doxin reductase GliT ( TR) antigen by establishing and optimizing ELISPOT assay for TR antigen-specific T cells ( TR/AST) secreting IFN-γand IL-4 in peripheral blood mononuclear cells ( PBMCs ) and explore the role of TR/AST in invasive aspergillosis ( IA ) . Methods We optimized the reaction conditions of ELISPOT by preliminary checkerboard titration and determined the frequencies of positive spot-forming cells ( SFCs) specifically secreting IFN-γand IL-4 in the PBMCs of 20 healthy individuals with TR as specific stimulant and with PHA and PMA as positive controls ,. Results Checkerboard titration demonstrated the best result of ELISPOT with the TR antigen at the final concentration of 10μg/well and PBMCs at 3 ×105/well.The median frequency of IFN-γSFCs was sig-nificantly higher (15 [3.5, 59.5]) than that of IL-4 SFCs (0 [0, 0]) (P20/3 ×105 PBMCs), accounting for 45%, but failed to induce IL-4 response in 19 of the healthy individuals . Conclusion The Aspergillus fumigatus TR antigen could induce an immunodominant Th1 response , and therefore might be a potential protective antigen .

Biological activity assays and cellular imaging of anti-human sperm protein 17 immunomagnetic nanoparticles / 细胞与分子免疫学杂志

AIM: To prepare anti-Sperm protein 17 (Sp17) immunomagnetic nanoparticles (IMNPs), and make foundation for target diagnosis of ovarian cancer by magnetic resonance imaging. METHODS: The anti-human Sp17 IMNPs were prepared by grafting anti-Sp17 antibodies on the surface of chitosan-coated magnetic nanoparticles （MNPs） using the linker of EDC/NHS (1-ethyl-3-［3-dimethylaminopropyl］carbodiimide/N-hydroxysuccinimide). The morphology and properties of the nanoparticles were characterized by transmission electronic microscopy (TEM), the conjugation of the antibodies was evaluated by native-polyacrylamide gel electrophoresis, the immunologic activity of IMNPs was evaluated by enzyme linked immunosorbent assay (ELISA). A set of in vitro magnetic resonance imaging (MRI) experiments were performed after incubated the IMNPs with human Sp17 gene transfected ovarian cancer HO-8910 cells. RESULTS: We had successfully grafted the MNPs with anti-Sp17 antibody and the IMNPs kept good bioactivity. The MRI showed that the IMNPs were targeted successfully to the positive cells, and no obviously non-specific adsorption was observed. CONCLUSION: The anti-Sp17 IMNPs with good specificity can used for further study of ovarian cancer target therapy.

Detection of sperm protein 17 in serum and its significance in patients with gynecology cancer / 临床检验杂志

Objective To quantify the level of human sperm protein 17(Sp17)in serum of the healthy and patients with gynecological cancers and to evaluate whether Sp17 is a suitable serum marker for the diagnosis of gynecological cancers.Methods The recombinant human Sp17 was radioiodinated by the chloramine T method.125I-rhSp17 was used to develop a radioimmunoassay to determine Sp17 in serum of the healthy and the patients.Meanwhile,Sp17 levels in serum were compared with the expression of Sp17 in tissues from gynecological cancers.Results The level of serum Sp17 was(15.60?7.66)?g/L in healthy subjects(n=59),which was significantly lower than those in twenty-two patients(64.36?34.44)?g/L.Sera from nine of twenty-two patients(40.9%)were positive for Sp17 by radioimmunoassay assay.Tumor tissues from twelve of twenty-two patients(54.5%)were positive for Sp17 by immunohistochemistry assay.Conclusions Markedly elevated serum levels of Sp17 were found in patients with gynecological cancers.However,there was no significant correlation between serum level of Sp17 and expression of Sp17 in tumor tissues in patients with gynecological cancer.

Application of fluorescence imaging in the research of tumor / 医学研究生学报

Fluorescence imaging of animal in vivo is to tag cell or DNA with fluorescence reporter gene, and then detect the fluorescence using sensitive optics apparatus. With this system, researcher can observe tumor growth, metastasis and angiogenesis in vivo, and reliable information for tumor diagnosis and treatment will be provided for researcher.

Organophosphorus pesticide-induced immunotoxicity and its updated mechanisms / 医学研究生学报

Organophosphorus pesticides(OP) are used as insecticides in agriculture throughout the world.The main toxicity of OP is neurotoxicity,which is caused by the inhibition of acetylcholinesterase.OPs also affect the immune response and suppress antibody production,T cell proliferation and the activity of natural killer(NK) cells and cytotoxic T lymphocytes(CTL).However,there have been few studies on the mechanism of OP-induced immunotoxicity,especially the mechanism of OP-induced inhibition of the cytolytic activity of killer cells.This study updates the mechanisms of pesticide-induced immunotoxicity,including its direct and indirect effects on the immune system.

Construction of the eukaryotic expression vector containing the human perforin gene and Its expression in COS-7 cells / 医学研究生学报

Objective: To clone human perforin(pfn) full-length DNA,construct the eukaryotic expression vector and observe the expression of the pfn gene in the transfected COS-7 cells.Methods: Full-length DNA of pfn was obtained by PCR from rPCR2.1/pfn,inserted into the pMD-18T vector and subcloned to the pcDNA3.1(+) vector to construct the recombinant eukaryotic expression vector pcDNA3.1(+)/pfn.The recombinant plasmid was transfected into COS-7 cells and the expression of the pfn gene in the transfected cells was detected by RT-PCR.Results: The pfn full-length DNA was successfully cloned and inserted into the pcDNA3.1(+) vector.The expression of pfn mRNA in the transfected COS-7 cells was confirmed by RT-PCR. Conclusion: The full-length human pfn gene can be expressed in transfected COS-7 cells.

Advances in research on cancer/testis antigens / 医学研究生学报

Cancer/testis(CT) antigens,of which more than 40 kinds have now been identified,are encoded by genes that are expressed normally in the human germ line,and are also expressed in various tumor types,including melanoma,and carcinomas of the bladder,lung and liver.These immunogenic proteins are being vigorously pursued as targets for therapeutic cancer vaccines.In this paper,the classification,expression and function of CT antigens are reviewed.

Granulysin / 医学研究生学报

Granulysin is a cytotoxic granule protein,colocalizes with perforin and granzymes in the cytolytic granules of human CTL and NK cells. The released granulysin was found in the sera of healthy individuals and patients. Granulysin exhibits potent cytotoxic activity against tumors and a broad panel of microbes, including bacteria, fungi, and parasites. The serum levels of granulysin is well associated with diverse activities of NK cells and CTL in physiological and pathological settings and could be a useful novel serum marker for acute rejection of grafts and the overall status of host cellular immunity.

Experimental study on establishing tumor bearing nude mice model with different ovarian adenocarcinoma cell lines / 医学研究生学报

Objective:To establish the best way of tumor-bearing nude mice model with epithelial ovarian adenocarcinoma cell lines. Methods:CAOV3 (wall-attached cell) and COC2 (suspended cell) cell lines were transplanted subcutaneously to the nude mice by injection with different cell concentration or tissue block implantation. Results:The rate and time of tumor formation in the two cell lines were not related to cell concentration in the range from 5?10 6/0.2 ml to 6?10 7/0.2 ml. Time of tumor formation was earlier(especially in the manner of tissue block implantation),the successful rate of tumor formation was higher and the speed of tumor growth was relatively retarded in the nude mice bore CAOV3 cell line's tumor than that in COC2 cell line's. But once tumors were formed in nude mice bearing COC2 cell line, the mice died too fast to make experiment because of very quick speed of tumor growth. Conclusion:Wall-attached cell line, tissue block implantation with trocar may be the best way to establish tumor-bearing nude mice model in epithelial ovarian adenocarcinoma cell lines.

Advances in the research of sperm protein 17 / 中华男科学杂志

Sperm protein 17 (Sp17) is a testis-specific protein involved in acrosome reaction in spermatozoa. However, the Sp17 gene has been recently detected in normal non-testis tissues and malignant neoplasias. Therefore Sp17 may be a potential target for immunocontraception and a suitable target for tumor immunotherapy. This paper reviews the advances in the protein characterization, expression and distribution, and biological function of Sp17 and its clinical research.

The state of non-culture-based laboratory diagnostic assays of invasive aspergillosis / 医学研究生学报

In high-risk patients,such as patients with hematologic malignancies,or patients after allogeneic stem-cell or solid-organ transplantation,early diagnosis of invasive aspergillosis(IA) is essential,as missing or delayed diagnosis of IA results in increasing rates of mortality.However,diagnosis of IA is difficult because classic tests have low sensitivity and specificity.The limited sensitivity and specificity of conventional assays for the detection of IA and the growing number of IA patients have led to the development of new assays.These methods include antigen detection systems,such as galactomannan(GM) assay,and different molecular methods(PCR assays).The GM assay is commercially available.However,it still need to be evaluated in large patient cohorts,especially children.A range of different PCR assays have been developed,targeting different gene regions,including a variety of amplicon detection methods.These molecular assays provide high potential in terms of sensitivity and specificity,but vary widely in their feasibility and up to now have not been standardised.Taken together,new non-culture-based diagnostic assays are noninvasive,simple,rapid and highly sensitive.Thus,they might be valuable tools to make early diagonosis,to reduce empirical antifungal therapy and to monitor the therapy.

Roles of macrophages in resisting Aspergillus fumigatus infection / 医学研究生学报

Aspergillus fumigatus,as an opportunistic pathogenic fungus that causes high morbidity and mortality among immunocompromised patients,has aroused wide attention.As a major component of the innate immune system,macrophages play a pivotal role in resisting Aspergillus fungal infection.This review focuses on the contributions of macrophages in Aspergillus fungal infection.

Combined use of near-infrared and other technologies in tumor research / 医学研究生学报

Near-infrared technology can be used as an auxiliary means for the early diagnosis of cancer and for surgical resection of tiny lesion that can not be discriminated by naked eyes,but it can not satisfy the purpose when used alone.Thus,we have developed an integrated technique by combining near-infrared and other technologies,and extended the scope of its application.This article reviews the advances of several integrated techniques applied to tumor research,including double labeled technology of nuclide and near-infrared,the combination of near-infrared and fluorescence resonance energy transfer,the combined use of near-infrared and endoscopy,and the integration of near-infrared with carbon nanotubes.

Discovery of a tumor resistance mouse and investigation of its mechanism / 医学研究生学报

A colony of mice with a unique trait of host resistance to tumorigenesis induced by transplantable cells has been established and studied by Cui Zheng et al.These spontaneous regression/complete resistance(SR/CR) mice possess a trait of resistance to a broad spectrum of tumors and can be transferred to cancer-sensitive mice,with an age-dependent spontaneous regression mediated mainly by innate leukocytes.This article reviews the discovery of the SR/CR mouse model and related study of its anti-cancer mechanism.