Enzyme-liked Immunosorbent Assay Research Articles

Epidemiological evaluation of patients submitted to the galactomannan test with suspected invasive aspergillosis

Invasive aspergillosis (IA) increases dramatically when there is potential risk in many patient groups, in particular with hematological malignancies. The purpose of the study was to trace the epidemiological profile of patients who underwent galactomannan test by ELISA with suspected IA and to determine the factors that contribute to the development of the disease. We evaluated 264 patients who underwent galactomannan test with suspected IA from 2013 to 2015.The clinical-epidemiological characteristics were determined using descriptive statistics. The variables were evaluated using the chi-square test (χ2) and the G-test, with p-value considered significant below 0.05. According to the classification for IA by the European Organization for Research and Treatment of Cancer, the disease was considered proven in 7.3%, defined by positive culture for the fungus, 6.4% as probable through detection of galactomannan and the presence of pulmonary infiltrates and 5.1% as possible by radiological alterations suggestive of IA and negative galactomannan test. The mortality rate was 31.6% of all patients and 61.3% for proven / probable / possible IA indicating that the disease was significantly associated with the risk of death. According to these result indications and considering the high mortality rate caused by the development of IA, as well as the fact that early therapy promotes significant improvement in the patients’ prognosis, we conclude that the detection of galactomannan may be considered an effective method to aid the identification of IA. 
 KEY WORDS: Invasive pulmonary aspergillosis; neutropenia; enzyme-liked immunosorbent assay; ELISA test searching for Aspergillus; galactomannan.

Clinical Experience in Treatment of Alzheimer's Disease with Jiannao Yizhi Formula () and Routine Western Medicine.

To investigate the long-term therapeutic effects of the Chinese medicine Jiannao Yizhi Formula (, JYF) in the treatment of Alzheimer's disease (AD). Sixty mild-to-moderate AD participants were recruited and randomly allocated to the treatment (30 with JYF) and the control groups (30 with donepezil) for 6 months with the random numbers. The primary outcomes were scores of Alzheimer's Disease Rating Scale-Cognitive (ADAS-Cog) and Chinese Medicine Symptom Scale (CM-SS). The secondary outcomes were scores of Mini Mental State Examination (MMSE), Montreal Cognitive Assessment (MoCA), and Activities of Daily Living (ADL). Safety assessments were conducted at baseline and the 6th month of treatment. Serum levels of acetylcholine (Ach), amyloid-β protein 42 (Aβ42), and the microtubule-associated protein tau (Tau) were also determined by enzyme-liked immunosorbent assay. Fifty-one participants were included in the final analyses (JYF n=27; donepezil n=24). Compared with baseline, both JYF and donepezil increased the MoCA and MMSE scores and decreased the ADAS-Cog and CM-SS scores (P<0.05 or P<0.01). Both drugs increased the serum levels of Ach and decreased the serum levels of Aβ42 and Tau (all P<0.05). There was no significant difference in these variables between the two groups, which showed that JYF was not inferior to donepezil. No obviously significant changes were observed in the ADL. No severe adverse events were observed in both groups. The effect and safety of JYF for the treatment of AD were not inferior to those of donepezil.

Prevalence of Visceral Leishmaniasis in Hospitalized Patients as an increasing Public Health Problem

Introduction: Globally, Visceral Leishmaniasisan increasing public health problem, as evidenced by markedly increasing incidence rates and multiple outbreaks in all continents. Visceral Leishmaniasisis probably the most widespread and prevalent zoonotic disease in the world. World Health Organisation estimates the worldwide prevalence to be approximately 12 million cases, with annual mortality of about 60 000. The size of population at risk is about 350 million. Methods: Sera of 150 patients were analysed by indirect enzyme liked immunosorbent assay (ELISA). Serum was collected from patients with VL before the onset of treatment. Serum was separated from 1mL and stored at -20 °C. Results: For two years from January 2011 to December 2012 in our laboratory were examined 150 cases (hospitalized patients for IgG antibodies. The mean age was 15.44 with minimum 1 and maximum 69, standart deviation 16.577. The city of Durres represents 40.7 % of all cases. The prevalence for antibodies IgG was 17.3%. There was no significant relationship between antibodies IgG and sex, age and residence of patients. Conclusion: The prevalence of Leishmania in our patients is high but was not found an association regarding residence, age’s group and sex.

Serum miR-146a and miR-150 as potential new biomarkers for acute lung injury induced by hip fracture

BackgroundThe elderly hip fracture is one of the most common injuries and represents a high incidence of complications and mortality. Among these complications, acute lung injury (ALI) and subsequent pulmonary dysfunction are the most serious and fatal. It is still uncertain why elderly patients more easily suffer it and younger do not under almost similar clinical conditions. For early diagnosis and prevention of ALI after fracture, it is very necessary to find some biomarkers for it. ObjectiveThis study aims to examine the differential expression of miR-146a, miR-150 and cytokines (TNF-a, IL-6, and IL-10) between younger and elderly rats suffering from hip fracture and to investigate the possible meaning of them in early diagnosis and prognosis of ALI after hip fracture. Methods and subjectsForty elderly rats and 40 younger rats were randomly divided into two groups respectively (sham group and fracture group). Two sham groups only received anaesthesia, cannulation and observation. Two fracture groups received hip fracture operations. The rats were respectively sacrificed at 4, 12, 24, and 48h after hip fracture or anaesthesia. Blood samples and lung tissues were collected for further examination. The damage degree of ALI was evaluated by histological observation and pathological score on lung tissue slices. Cytokines were measured by enzyme-liked immunosorbent assay (ELISA); miR-146a and miR-150 were analysed by quantitative real-time PCR (qRT-PCR). ResultsAt 4, 12, 24, and 48h after treatment, the pulmonary histological score and serum concentrations of miR-146a, miR-150, and cytokines (TNF-a, IL-6, and IL-10) had no significant difference between elderly sham group and younger sham group; however, compared with the corresponding sham groups (elderly fracture group vs. elderly sham group, younger fracture group vs. younger sham group), the pulmonary histological score and serum levels of TNF-a, IL-6, IL-10, and miR-146a were significantly higher (whereas the miR-150 were lower) in fracture groups (except the TNF-a and IL-10 of younger fracture group at 48h). At 24 and 48h after fracture, between two fracture groups, the pulmonary histological score and serum levels of TNF-a, IL-6, IL-10, and miR-146a were higher (whereas the miR-150 were lower) in elderly fracture group (except the TNF-a at 48h). The results of linear regression analysis shown that the serum levels of cytokines (TNF-a, IL-6, and IL-10) were not significantly related with pulmonary histological score, however, the serum levels of miR-146a and miR-150 were significantly associated with it. ConclusionHip fracture can result in significant systemic inflammation and ALI in rats. Compared to younger, the elderly rats suffered a more remarkable ALI after hip fracture. It may be related to the abnormal expression of miR-146a and miR-150. Serum miR-146a and miR-150 are potential biomarkers for early diagnosis and prognosis of ALI after hip fracture.

Analysis of cytotoxicity induced by proinflammatory cytokines in the human alveolar epithelial cell line A549

Epithelial cell injury under hyperinflammatory conditions is critical in the development of septic acute lung injury (ALI). The aim of the present study is to analyze the cytotoxic effects of a mixture of proinflammatory cytokines in the human alveolar epithelial cell line A549. The cytotoxicity of proinflammatory cytokines were assessed in A549 cells by measuring lactate dehydrogenase released into the culture medium and by crystal violet staining of surviving cells. Activation of the caspase-dependent apoptotic pathway was evaluated by monitoring cleavage of cytokeratin 18 by caspases using enzyme-liked immunosorbent assay (ELISA). To estimate the cytotoxic signaling pathways responsible for epithelial injury, agents with antiinflammatory or antioxidative properties were extensively screened for cytoprotective effects in the inflammation-associated epithelial injury model. The present study revealed that inflammatory cytokines exerted cytotoxicity in A549 cells. A mixture of interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), designated as cytomix, augmented cytotoxicity compared with each individual cytokine. Treatment with glucocorticoid (dexamethasone), tetracycline-derived antiinflammatory antibiotics (minocycline or doxycycline), angiotensin II receptor blockers (losartan or telmisartan), or antioxidants (dimethyl sulfoxide, catalase) attenuated cytomix-induced cytotoxicity, including caspase activation. These results implied that inflammatory cytokines alone could cause alveolar epithelial injury in the pathophysiology of septic ALI. Caspase-dependent apoptosis was speculated to be one mechanism responsible for the cytokine-induced cytotoxicity. Agents with antiinflammatory or antioxidative properties such as glucocorticoid, tetracycline-derived antibiotics, angiotensin II receptor blockers, or direct antioxidants showed substantial effect in attenuating cytokine-induced cytotoxicity and may be candidates for treatment options.

A case report of visceral leishmaniasis in red fox (Vulpes vulpes)

A survey of 52 red foxes, a single two year old male weighing about 6 kg showed clinical signs including hair loss, impotence, local or general lymphadenopathy, keratitis, hepatosplenomegaly, lymphadenopathy, hair shedding, dermal lesions, onychogriposis and cachexia. The studied fox IFA titer was larger or equal to 1:1280 and light absorption in enzyme liked immunosorbent assay (ELISA) was equal to 2.127. In the slides prepared from impression smear of the liver and spleen, Leishman body was observed. The culture results were positive. Parasites isolated from seropositive fox with specific primers K-DNA and ITS-18sRNA was tested and polymerase chain reaction (PCR) identification of parasites isolated from seropositive fox were 99% homologous with Donovani complex (Leishmania infantum, Leishmania donovani, Leishmania chagasi ).

Effect of CD-14 and toll like receptors on the expression of interleukin-6 induced by lipopolysaccharides of Porphyromonas endodontalis

To evaluate the effect of cluster of differentiation 14 (CD-14) and Toll like receptors (TLR) on the expression of interleukin-6 (IL-6) mRNA induced by Porphyromonas endodontalis (Pe) lipopolysaccharides (LPS). MC3T3-E1 cells were treated with 10 mg/L Pe-LPS for different hours, and the cells uninvolved by anything as the blank group. The expression of IL-6 was detected by reverse transcription polymerse chain reaction (RT-PCR) and enzyme-liked immunosorbent assay (ELISA). The expression of CD-14, TLR-2 and TLR-4 mRNA was observed at different time point (0 - 24 h) by RT-PCR. The protein of CD-14, TLR-2 and TLR-4 was analyzed with a flow cytometer. MC3T3-E1 cells were pretreated with anti-CD-14, anti-TLR-2 and anti-TLR-4 antibody for 1 h, and then cells were stimulated with 10 mg/L Pe-LPS for 6 h. The expression of IL-6 mRNA was examined by RT-PCR. Statistical analysis was performed using one-way ANOVA Dunnett-t test with SPSS 11.0 software package. The IL-6 mRNA and proteins increased significantly after treatment with Pe-LPS. When MC3T3-E1 cells treated by Pe-LPS for 6 h, the expression of proteins soared from (11.696 ± 0.672) ng/L to (36.534 ± 0.574) ng/L (P < 0.01); In the control group, the CD-14 and TLR-4 mRNA are ambly-expression, and the ratios of CD-14 and TLR-4 positive cells were (39.038 ± 3.131)% and (11.438 ± 0.385)% respectively in MC3T3-E1. After treatment by Pe-LPS, the expression of CD-14 and TLR-4 mRNA increased significantly, and the ratios of CD-14 and TLR-4 positive cells markedly increased to (62.407 ± 1.800)% and (21.367 ± 2.271)%. TLR-2 expression did not change apparently after Pe-LPS treatment. The expression of IL-6 mRNA was partly inhibited by anti-CD-14 or anti-TLR-4 antibody, but not by TLR-2. Pe-LPS can induce the expression of IL-6 in osteoblast MC3T3-E1 through CD-14 and TLR-4, but not TLR-2.

Leonurine protects against tumor necrosis factor-α-mediated inflammation in human umbilical vein endothelial cells

ObjectiveLeonurine, a bioactive alkaloid compound in Herba leonuri, has various pharmacological activities, including antioxidant and anti-apoptotic capacities. This study was conducted to test the hypothesis that leonurine was able to attenuate tumor necrosis factor (TNF)-α-induced human umbilical vein endothelial cells (HUVEC) activation and the underlying molecular mechanisms. MethodsMitogen-activated protein kinases (MAPK) activation, nuclear factor-κB (NF-κB) activation, and inflammatory mediators expression were detected by Western blot or enzyme-liked immunosorbent assay, intracellular reactive oxygen species (ROS) and NF-κB p65 translocation were measured by immunofluorescence, endothelial cell–monocyte interaction was detected by microscope. ResultsLeonurine inhibited U937 cells adhesion to TNF-α-activated HUVEC in a concentration dependent manner. Treatment with leonurine blocked TNF-α-induced mRNA and protein expression of adhesion molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1), cyclooxygenase-2, and monocyte chemoattractant protein-1 in endothelial cells. In addition, leonurine attenuated TNF-α-induced intracellular ROS production in HUVEC. Furthermore, leonurine also suppressed the TNF-α-activated p38 phosphorylation and IκBα degradation. Subsequently, reduced NF-κB p65 phosphorylation, nuclear translocation, and DNA-binding activity were also observed. ConclusionsOur results demonstrated for the first time that the anti-inflammatory properties of leonurine in endothelial cells, at least in part, through suppression of NF-κB activation, which may have a potential therapeutic use for inflammatory vascular diseases.

Patterns of cytokine release and evolution of remote organs from proximal femur fracture in COPD rats

BackgroundChronic obstructive pulmonary disease (COPD) is at increased risk for developing osteoporosis (OP) with subsequent proximal femur fracture. The presence of COPD is suggested to be a strong risk factor for proximal femur fracture or hip fracture. However, what happen behind it is not clearly understood. ObjectiveTo investigate the pattern of cytokine (TNF-a, IL-6, and IL-10) releases in pulmonary and hepatic in rats with COPD suffering from proximal femur fracture, and its possible adverse effect on pulmonary and hepatic. Methods and subjectiveThis paper has two parts. In the first part, we describe the procedure of COPD model in detail. In the second part, we study the influences of fracture on the COPD rats. 5 months WISTAR rats with 37 weeks cigarette smoking exposure (CS group) were dynamically determined for pulmonary function, inflammatory response in bronchoalveolar lavage fluid (BALF), histological changes in pulmonary in the first part. When the COPD model is proved to be successful, we begin the second part. COPD rats were euthanized at 2, 24, 48, 72, and 96h after proximal femur fracture (fracture group) or anaesthesia (control group). Cytokines (TNF-a, IL-6, and IL-10) and myeloperoxidase activity of pulmonary and hepatic (MPO) were measured with enzyme-liked immunosorbent assay technique. Permeability changes of the lung were assessed via bronchoalveolar lavage, and those of the liver via assessment of oedema formation. Tissues were further examined microscopically. ResultsThe current sidestream cigarette smoke induced rat COPD model has been proved an adequate animal model with several advantages as assessed by dynamically monitored lung mechanics and pathological changes for 37 weeks. In the second part, TNF-a, IL-6, and IL-10 levels of pulmonary tissue were significantly increased after proximal femur fracture compared to control rats. TNF-a, and IL-6 levels in pulmonary peaked at 2h, 24h in fracture group, whereas IL-10 level peaked at 24h and 96h. Pulmonary myeloperoxidase activity, permeability and histological score in fracture group were remarkably elevated, and peaked at 24h. In addition to TNF-a, all above parameters did not return to normal through our study. Hepatic in COPD rats showed notable increase of cytokines (TNF-a, IL-6, and IL-10), myeloperoxidase activity, histological score, and permeability in fracture group compared to control rats, and severity of these changes were much lower than in pulmonary. Apart from TNF-a, the peak of these parameters was at 24h after fracture. Changes of cytokines, MPO activity, permeability and histological score in pulmonary and hepatic in control rat were little changed. ConclusionCOPD rats produced a remarkably increase of inflammatory response (TNF-a, IL-6, IL-10) in lung (liver) after proximal femur fracture, which lead to lung (liver) injury, as evidence by changes of MPO, permeability, and histological scores in local organs.

Soluble Human Leukocyte Antigen G Level in Fluid from Single Dominant Follicle and the Association with Oocyte Competence

PurposeTo investigate the direct relationship between the follicular fluid (FF) level of soluble human leukocyte antigen G (HLA-G) and fertilizability of the corresponding oocyte as well as the morphological quality of the corresponding embryo.Materials and MethodsSixty-three patients were stimulated with recombinant FSH combined with gonadotropin-releasing hormone (GnRH) agonist long (n=5) or antagonist protocol (n=58) for standard in vitro fertilization (IVF). At the time oocyte retrieval, follicular fluid was obtained from single dominant follicle in 63 patients, and the level of soluble HLA-G was measured by sandwich enzyme-liked immunosorbent assay (ELISA). Normal fertilization and individual embryo quality were evaluated, and were graded to four categories by morphological criteria (the embryo with symmetrical blastomeres and no fragmentation were assigned as grade A). Good-quality embryo was defined as those with grade A or B.ResultsSoluble HLA-G was not detected in 15 FF samples. In the group with positive FF soluble HLA-G (sHLA-G) (n=48), high levels of sHLA-G (>117.758 U/mL) could predict the failure of fertilization with statistical significance {area under the curve (AUC) 0.676, 95% confidence interval (CI) 0.525-0.804}. However, the FF sHLA-G level was not related with the formation of good-quality embryo.ConclusionHigh level of FF sHLA-G could predict the fertilization failure of the corresponding oocyte, but was not related with the formation of good-quality embryo.

A sensitive immunoassay based on direct hapten coated format and biotin–streptavidin system for the detection of chloramphenicol

A novel enzyme liked immunosorbent assay (ELISA) was developed for the detection of chloramphenicol (CAP). In this assay, the small molecular hapten (Hap) was directly coated on the surface of microtiter plates and biotin–streptavidin system (BSAS) was employed to improve the sensitivity of immunoassay (BSAS-direct Hap coated ELISA). The surface of microtiter plates was treated with glutaraldehyde (GA) polymer network to introduce aldehyde group, which was used to cross-link with amino group of CAP. Compared with conventional ELISA (the plates were coated with Hap–carrier protein conjugates), the modified plates presented significantly high antibody and antigen (Ab–Ag) affinity and showed excellent stability. And then the biotinylated monoclonal antibody (mAb) and HRP-labeled streptavidin were employed in this assay for amplification of signals. The sensitivity of BSAS-direct Hap coated ELISA was increased by approximately 20-folds and the stability was also improved greatly compared to conventional ELISA. Its 50% inhibition concentration (IC 50) for CAP was 10.5 ng mL −1 and the limit of detection (LOD) was 0.2 ng mL −1 after optimization of reaction conditions. To our knowledge, this was one of the most sensitive immunoassay for CAP yet reported. In sample analysis, the results of CAP detected by this assay were in accordance with which obtained by conventional ELISA and high performance liquid chromatography (HPLC). Therefore, it is an attractive alternative compared to conventional immunoassays in routine supervision for residue detection in food and environment.

Enzyme-liked immunosorbent assay for the detection of pathogenic spiroplasma in commercially exploited crustaceans from China

Spiroplasmas are emerging pathogens in commercially exploited freshwater crustaceans from China. The bacteria infect Chinese mitten crab ( Eriocheir sinensis), crayfish ( Procambarus clarkii) and pacific white shrimp ( Litopenaeus vannamei) in Jiangsu and Zhejiang provinces of China. A rabbit antiserum was prepared against the spiroplasma isolated from a Chinese mitten crab that had obvious signs of tremor disease. An indirect enzyme-linked immunosorbent assay (ELISA) for the rapid detection of pathogen was developed. The titer of antibody against the pathogen was 1:31,250. The lowest detectable antigen protein was 0.573 μg/mL (3 × 10 5 CCU·ml − 1 pathogens) in pure culture. No color reaction was observed with seven other bacteria ( Acinetobacter spp, Bacillus subtilis, Streptomyces sp., Pseudomonas aeruginosa, Escherichia coli, Vibrio cholerae, Aeromonas hydrophila). Very weak color reaction was observed in tests with spiroplasma species from plants and honeybees. The coefficient of variability of the assay was less than 10%, with an excellent reliability in moderate and heavy infections. The whole test procedure was rapid, being completed within 3 h. The results showed that the assay is a simple and applied method for diagnosis of spiroplasma diseases in crustaceans.

Treament of traumatic vitreous hemorrhage with intravitreal triamcinolone and defibrase injection in a rabbit model.

目的 观察玻璃体腔注射蛇毒降纤酶和曲安奈德治疗兔眼外伤性玻璃体积血的效果,同时观察玻璃体腔细胞因子含量的变化.方法 建立外伤性玻璃体积血兔眼模型.右眼随机分为生理盐水组、蛇毒降纤酶组、曲安奈德组及联合用药组.直接检眼镜观察玻璃体积血指数、增生性玻璃体视网膜病变(PVR)程度;酶联免疫吸附法(ELISA)测定玻璃体腔白介素-1β(IL-1β)、Ⅲ型胶原(CⅢ)浓度.结果 联合用药组玻璃体积血指数、PVR程度、玻璃体腔IL-1β、Ⅲ型胶原浓度都低于生理盐水组.结论 玻璃体腔注射蛇毒降纤酶和曲安奈德可以促进玻璃体腔积血的吸收;同时下调玻璃体腔IL-1β浓度;减少Ⅲ型胶原的形成,从而降低外伤性PVR程度。

Effect of transforming growth factor‐beta1 on expression of the connective tissue growth factor (CCN2/CTGF) gene in normal human gingival fibroblasts and periodontal ligament cells

Connective tissue growth factor (CCN2/CTGF) plays an important role in wound healing and regulation of the extracellular matrix in periodontal tissue. However, the functional relationship between altered transforming growth factor-beta1 levels and CCN2/CTGF has not been extensively investigated in human gingival fibroblasts and periodontal ligament cells. This study investigated the effects of transforming growth factor-beta1 on the expression of the CCN2/CTGF gene in human gingival fibroblasts and periodontal ligament cells in vitro. Cells were isolated from normal periodontal tissues and cultured in Dulbecco's modified Eagle's minimal essential medium/F12 containing 10% fetal bovine serum. Subconfluent cells were maintained under serum deprivation for 24 h then treated with Dulbecco's modified Eagle's minimal essential medium/F12 containing 0.5% fetal bovine serum (control) and 0.1, 1, 5 or 10 ng/mL of transforming growth factor-beta1 for 24, 48 or 72 h. The effects of transforming growth factor-beta1 on CCN2/CTGF mRNA expression were measured by reverse transcription-polymerase chain reaction. CCN2/CTGF protein was quantitatively analyzed using enzyme-liked immunosorbent assay. Subcellular distribution of CCN2/CTGF protein in both human gingival fibroblasts and periodontal ligament cells was observed using immunofluorescence microscopy. In both human gingival fibroblasts and periodontal ligament cells, the expression of CCN2/CTGF mRNA and CCN2/CTGF protein was significantly increased, in a dose- and time-dependent manner, in the presence of transforming growth factor-beta1. Moreover, immunofluorescence analysis indicated that immunoreactivity to CCN2/CTGF showed a granular pattern of protein localization. The expression of CCN2/CTGF mRNA and protein was induced by transforming growth factor-beta1 in human gingival fibroblasts and periodontal ligament cells. These results suggest that CCN2/CTGF plays an important role in wound healing and in the regeneration of periodontal tissue.

Sensitive Aflatoxin B1 Determination Using a Magnetic Particles-Based Enzyme-Linked Immunosorbent Assay.

A magnetic particle-based enzyme-liked immunosorbent assay (mp-ELISA) has been developed as new an alternative immunoassay for Aflatoxin B1 determination. The method is based on conventional competitive ELISA whereby the anti-Aflatoxin B1 antibody is immobilized on the magnetic particles' surface. The influence of the antibody type as well as antibody immobilization on the magnetic beads surface was investigated in detail. Also, optimum values for the general parameters of the method (e.g. tracer concentration, type of antibody, and incubation time) were established. Finally, a sensitive immunoassay method (mp-ELISA) was performed for Aflatoxin B1 determination at ppt level (LOD = 1 ppt Aflatoxin B1).

Allergen Inactivation with Colloidal Silica

We evaluated the allergen inactivating effect of colloidal silica by performing enzyme-liked immunosorbent assay (ELISA) whose wells were coated with 150 ng/mL of Japanese cedar pollen allergen (Cry j 1) or mite allergen (Der f 2). The allergens were almost 100% inactivated by 100 microg/mL of colloidal silica having a particle size 5 nm, and the inactivating effect was increased by aluminum binding to the surface of the colloidal silica. The results show that colloidal silica is a promising material for allergen inactivation. Since colloidal silica forms an insoluble nondispersive solid when dried, it is expected that airborne allergens can be reduced by binding them to colloidal silica.

A flow cytometry-based method for detecting antibody responses to murine cytomegalovirus infection

An assay based on target cells infected with green fluorescent protein labeled murine cytomegalovirus (GFP-MCMV) and dual color flow cytometry for detecting antibody to MCMV is described. After optimizing conditions for this technique, kinetics of anti-MCMV IgG antibody response was tested in susceptible (BALB/c) and resistant (C57BL/6) mouse strains following primary MCMV infection. Previously published antibody kinssetics were confirmed in susceptible mice, with peak IgG response seen ∼8 weeks after primary infection, decreasing by 20 weeks after infection. In contrast, MCMV resistant C57BL/6 mice showed significantly lower IgG antibody responses than susceptible mice. Although several techniques have been previously described to detect murine antibody responses to MCMV, including nuclear anti-complement immunofluorescence, viral immunoblotting, complement fixation, indirect immunofluorescence, indirect hemagglutination, and enzyme-liked immunosorbent assay techniques, these techniques are all time consuming and laborious. The technique presented is a simple time efficient alternative to detect previous MCMV antibody responses in experimentally infected mice.

Antitumor and antiangiogenic activities of anti-vascular endothelial growth factor hairpin ribozyme in human hepatocellular carcinoma cell cultures and xenografts

To study the effectiveness and mechanisms of anti- human vascular endothelial growth factor (hVEGF) hairpin ribozyme on angiogenesis, oncogenicity and tumor growth in a hepatocarcinoma cell line and a xenografted model. The artificial anti-hVEGF hairpin ribozyme was transfected into hepatocarcinoma cell line SMMC-7,721 and, subsequently, polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) were performed to confirm the ribozyme gene integration and transcription. To determine the effects of ribozyme ,VEGF expression was detected by semiquantitative RT-PCR and enzyme liked immunosorbent assay (ELISA). MTT assay was carried out to measure the cell proliferation. Furthermore,the transfected and control cells were inoculated into nude mice respectively, the growth of cells in nude mice and angiogenesis were observed. VEGF expression was down-regulated sharply by ribozyme in transfected SMMC-7,721 cells and xenografted tumor. Compared to the control group, the transfected cells grew slower in cell cultures and xenografts, and the xenograft formation was delayed as well. In addition, the microvessel density of the xenografted tumor was obviously declined in the transfected group. As demonstrated by microscopy,reduction of VEGF production induced by ribozyme resulted in a significantly higher cell differentiation and less proliferation vigor in xenografted tumor. Anti-hVEGF hairpin ribozyme can effectively inhibit VEGF expression and growth of hepatocarcinoma in vitro and in vivo. VEGF is functionally related to cell proliferation, differentiation and tumori-genesis in hepatocarcinoma.

Effects of Mitogen-Activated Protein Kinase Inhibitors on Tumor Necrosis Factor-α Gene Expression and Apoptosis Induction in Cultured Human Fetal Membrane Chorion Cells Infected with Influenza Virus

Objectives: We investigated the involvement of p38 mitogen-activated protein (MAP) kinase in tumor necrosis factor (TNF)-α gene expression, apoptosis induction and virus replication in cultured human fetal membrane chorion cells infected with influenza virus. Methods: Influenza virus-infected chorion cells were incubated in the absence or presence of inhibitors of p38 MAP kinase, SB203580 and SB202190. TNF-α mRNA and hemagglutinin viral RNA (HA vRNA) were amplified with reverse transcriptase-polymerase chain reaction techniques. TNF-α protein concentrations were determined by enzyme-liked immunosorbent assay. The extent of apoptosis induction was estimated by DNA agarose gel electrophoresis. Pyrrolidine dithiocarbamate (PDTC) and ribavirin, which have been shown to inhibit apoptosis induction via the inhibition of viral gene replication, were used as positive control reagents. Results: PDTC and ribavirin inhibited the accumulation of TNF-α mRNA and HA vRNA in the virus-infected chorion cells, resulting in the suppression of TNF-α protein secretion. Both SB203580 and SB202190 suppressed TNF-α protein secretion, but not the accumulation of TNF-α mRNA as well as HA vRNA and the induction of apoptosis. Conclusions: These results suggest that p38 MAP kinase pathway is critical in TNF-α gene expression at a post-transcriptional level but not in the apoptosis induction and influenza virus replication in cultured chorion cells.

Specific Epitopes of Domains II and III of Bacillus thuringiensis Cry1Ab Toxin Involved in the Sequential Interaction with Cadherin and Aminopeptidase-N Receptors in Manduca sexta

The Bacillus thuringiensis Cry toxins are specific to different insects. In Manduca sexta cadherin (Bt-R1) and aminopeptidase-N (APN) proteins are recognized as Cry1A receptors. Previous work showed that Cry1Ab binds to Bt-R1 promoting the formation of a pre-pore oligomer that binds to APN leading to membrane insertion. In this work we characterized the binding epitopes involved in the sequential interaction of Cry1Ab with Bt-R1 and APN. A Cry1Ab immune M13 phage repertoire was constructed using antibody gene transcripts of bone marrow or spleen from a rabbit immunized with Cry1Ab. We identified antibodies that recognize domain II loop 3 (scFvL3-3) or beta16-beta22 (scFvM22) in domain III. Enzyme-linked immunosorbent assay and toxin overlay binding competition assays in the presence of scFvL3-3, scFvM22, or synthetic peptides showed that domain II loop 3 is an important epitope for interaction with Bt-R1 receptor, whereas domain III beta16 is involved in the interaction with APN. Both scFvL3-3 and scFvM22 lowered the toxicity of Cry1Ab to M. sexta larvae indicating that interaction with both receptors is important for in vivo toxicity. scFvL3-3 and anti-loop2 scFv (scFv73) promoted the formation of the pre-pore oligomer in contrast to scFvM22. In addition, scFvL3-3 and scFv73 preferentially recognized the monomeric toxin rather than the pre-pore suggesting a conformational change in domain II loops upon oligomerization. These results indicate for the first time that both receptor molecules participate in Cry1Ab toxin action in vivo: first the monomeric toxin binds to Bt-R1 through loops 2 and 3 of domain II promoting the formation of the pre-pore inducing some structural changes, then the pre-pore interacts with APN through beta-16 of domain III promoting membrane insertion and cell death.