miR-217 through SIRT1 regulates the immunotoxicity of cadmium in Cyprinus carpio.

MicroRNAs (miRNAs) play a critical role in regulating the response of animals exposed to heavy metal stress. As a globally dispersed heavy metal in aquatic ecosystems, cadmium (Cd) is highly toxic to many aquatic species. However, little is known about the miRNA response to Cd stress in fish. To investigate the regulatory effect of miRNAs in response to Cd, common carp (Cyprinus carpio) were exposed to Cd2+-containing water (0.005 mg/L, 0.05 mg/L, 0.5 mg/L) for 30 days. After exposure, Cd2+ contents were significantly higher in the kidneys of C. carpio compared to other tissues, when exposed to 0.5 mg/L Cd2+. Hematoxylin and eosin staining images revealed that elevated Cd induced inflammatory damage in the kidneys of C.carpio. Further, miRNA sequencing revealed nine differentially expressed miRNAs (miR-217, miR-205 and seven novel miRNAs) in the kidneys, between 0.5 mg/L Cd2+ exposure and control groups. Potential target mRNAs of miRNAs suggest that miR-217 is involved in immunotoxicity. miR-217 agomir was intraperitoneally administered to C. carpio and RT-PCR revealed that the expression of IL-8 and SIRT1 decreased, while TLR-4, TRAF6, NF-kB, TNF-α, IL-1ß, and TGF-ß increased in the kidneys of C.carpio. Additionally, the expression of SIRT1 decreased, while the expression of other mRNAs increased in kidneys of C. carpio exposed to Cd. According to mRNAs expression in the agomir and Cd treatment, miRNAs inhibit the expressions of target mRNAs. These results demonstrate that miR-217 via SIRT1 plays a regulatory role in the immunotoxicity of Cd to C. carpio.

A novel protein biochip screening serum anti-sperm antibody expression and natural pregnancy rate in a follow-up study in Chinese infertility.

Production of anti-sperm antibody (ASA) often suffers from autoimmune reaction against sperms in human infertility. The antibodies are measured in both blood and seminal plasma of males. Here, we reported a simple protein biochip methodology that takes advantage of a functionalized self-assembled monolayer modified by N-hydroxysuccinimide (NHS) and enables identification of anti-sperm antibody in Chinese male infertility. To validate this biochip platform, we immobilized purified sperm protein on the biochip surface and tested a variety of parameters in quality controls for the protein assay, respectively. Then, we analyzed serum samples from 368 patients with infertility and 116 healthy donors by means of this biochip simultaneously. We found that positive rate of serum ASA was 20.92% (77/368) in the cases and 1.72% (2/116) in the controls, respectively. Furthermore, we further corroborated the biochip assay in comparison with ELISA method. We found that both methods were compatible for the detection of serum ASA in the patients. In addition, a follow-up study for natural conception in ASA-positive and ASA-negative patients was conducted. The result showed a significant correlation between serum ASA expression and natural pregnancy rate 6.5% in ASA-positive patients while 18.9% in ASA-negative patients, indicating the potential roles of ASA in naturally reproductive processes.

Prediabetes is associated with genetic variations in the gene encoding the Kir6.2 subunit of the pancreatic ATP-sensitive potassium channel (KCNJ11): A case-control study in a Han Chinese youth population.

BACKGROUND: The E23K variant of the potassium voltage-gated channel subfamily J member 11 (KCNJ11) gene has been reported to be associated with type 2 diabetes (T2D) in many populations. However, little is known about the role of E23K in the development of prediabetes in Chinese youth. METHODS: To investigate the role of E23K in the development of prediabetes, 279 subjects with prediabetes and 240 normal controls (mean [± SD] age 18.1 ± 3.2 and 17.8 ± 4.3 years, respectively) were recruited to the study. Height, weight, and hip and waist circumferences were measured by trained physicians. Genotyping of KCNJ11 polymorphisms and clinical laboratory tests to determine cholesterol, triglyceride (TG), blood glucose, and insulin levels were performed. RESULTS: The carrier rate of K23 allele-containing genotypes was higher for prediabetic than control subjects (P = 0.005). Logistic regression analyses revealed that higher body mass index percentiles (P = 0.013), lower insulin levels at 30 min during an oral glucose tolerance test (P = 0.001), a higher ratio of total cholesterol: high-density lipoprotein cholesterol (P = 0.001), and a K allele-containing genotype (P = 0.019) are independent risk factors for prediabetes in Chinese Han youth. Furthermore, K23 allele-containing genotypes were associated with impaired indices of insulin secretion and ß-cell function in female youth with prediabetes. These effects were not seen in male youth with prediabetes. CONCLUSIONS: The results confirm that the common E23K polymorphism of KCNJ11 carries a higher susceptibility to the development of prediabetes in the Chinese Han population. The results suggest that E23K may have a greater effect on the development of T2D in female Chinese youth.

Protein biochip-based semiquantitative detection for plasma leptin.

PURPOSE: Plasma leptin is secreted from adipose tissues and plays pivotal roles in human physiological and pathological processes. Here, we aimed at conducting a protein biochip-based sandwich-like approach for detection of plasma leptin among healthy individuals, obesity, and diabetes patients. EXPERIMENTAL DESIGN: Totally, 96 plasma samples, including 45 healthy individuals with standard body mass index (BMI), 28 obesity and 23 diabetes patients, were recruited in the study. Plasma leptin was detected by a well-established protein biochip. Meanwhile an ELISA was also performed for assessment of the leptin detection by the protein biochip. RESULTS: We found that the plasma leptin level in the obesity and diabetes patients was significantly higher than that in healthy individuals with standard body mass index (p < 0.001). The limit detection concentration of leptin was as low as 0.006 µg/mL. The plasma leptin could be semiquantitatively detected by the protein biochip. The compatibility of the biochip-based detection approach seemed acceptable in comparison with the ELISA assay (R2 = 0.948). CONCLUSIONS: We provided a protein biochip-based approach for plasma detection. This approach would be a potential substitution for the ELISA assay.

[Preparation, identification and application of monoclonal antibody against matrix metalloproteinases-2].

AIM: To prepare and characterize monoclonal antibodies against matrix metalloproteinase-2 (MMP-2), check its expression in the tissues of human ovarian cancer and transplanted tumors in nude mice. METHODS: MMP-2 were linked to the carrier protein bovine serumalbumin (BSA) and keyhole limpet hemocyanin (KLH) using glutaraldehyde method to obtain MMP-2-BSA and MMP-2-KLH, respectively. The anti-MMP-2 monoclonal antibody was obtained through hybridoma technique. We established the cell strains secreting mAb by hybridoma technique and prepared the mAb by induction of ascites in vivo. The prepared mAb was purified by salting out with ammonium sulfate and identified by ELISA and Western blotting. We compared the mAb and commercial polyclonal antibody by immunohistochemistry and detected the expressions of MMP-2 and CA125 in ovarian cancer issues and transplanted tumor. RESULTS: The artificial antigen and 3 hybridoma cell lines secreting monoclonal antibodies (mAb) against MMP-2 were obtained. The subclasses of mAb were all IgG1. The titer of peritoneal exudates was 1:1×10(6);. The expressions of MMP-2 and CA125 in transplanted tumor and ovarian cancer tissues were all high. The positive expression rate of MMP-2 checked using generated antibody was 71.2%(57/80) in ovarian cancer tissues and 16.67% (5/30) in normal tissues, with significant difference between them (P<0.01). In early stage, the positive rate of MMP-2 and CA125 combined detection was higher than that of CA125 detection alone (P<0.01). The mAb was suitable for detecting the expression of MMP-2 in human tissues and gave results consistent with commercial polyclonal antibody. The mAb was more specific than commercial mAb (P<0.01). CONCLUSION: The anti-human MMP-2 mAb is successfully prepared, which may serve as a valuable tool in the functionaI studies of ovarian cancer.

Development of a protein biochip to identify 6 monoclonal antibodies against subtypes of recombinant human interferons.

Recombinant human interferons (rhIFNs) are broadly used as effective therapeutic agents with antiviral, antitumor, and immune-modulating properties. Advances in protein biochip technology have benefited the medical community greatly, making true parallelism, miniaturization, and high throughput possible. In this study, 5 rhIFN proteins (IFN-alpha1b, IFN-alpha2a, IFN-alpha2b, IFN-beta, and IFN-gamma) were immobilized onto an N-hydroxysuccinimide (NHS)-modified gold-based biochip. The protein biochip was incubated with 6 specific mouse IgG antibodies (AK1, AK2, AK3, AK4, BK1, and CK1) against the human IFNs and then with Cy3-conjugated goat anti-mouse IgG antibody. The results showed that monoclonal antibody AK1 presented a unique binding characteristic to IFN-alpha1b. AK2 reacted in immunoassays equally with IFN-alpha2a and IFN-alpha2b. AK3 detected IFN-alpha1b, IFN-alpha2a, and IFN-alpha2b. AK4 had positive immunological responses directed to both IFN-alpha1b and IFN-alpha2b. Monoclonal antibodies BK1 and CK1 recognized epitope of IFN-beta and IFN-gamma, specifically. The assay specificity of the biochip was further confirmed by enzyme-linked immunosorbent assay (ELISA) and western blotting. Finally, 88 serum samples from patients treated with rhIFN-alpha2b were simultaneously tested on a single biochip. The result demonstrated that 6.8% (6 of 88 cases) presented positive reactions to anti-IFN-alpha2b antibodies, indicating that the patients under rhIFN-alpha2b therapy produced neutralized antibody against the IFN. The biochip format would offer a competitive alternative tool not only for facilitating characterization of IFN subtypes but also potentially for enabling clinical serum detection of corresponding antibodies directed against IFNs.

[Preparation and characterization of human colon tumor-associated antigen and its clinical significance].

AIM: To purify the colon tumor-associated antigen from cultured colon tumor cells, and to investigate its expression in the sera of patients with colon cancer. METHODS: The monoclonal antibody (mAb) against human colon tumor-associated antigen 4D10 was employed as the ligand for the immunoaffinity chromatography to purify the colon tumor-associated antigen from the lysate of colorectal tumor cell LOVO. The purified antigen was identified by SDS-PAGE and Western blot. The expression of colon tumor-associated antigen in the sera of patients with colon cancer and in normal sera was detected by Sandwich ELISA. RESULTS: The purified colon tumor-associated antigen binding to mAb 4D10 was a heterodimer composed of two subunits with relative molecular mass M(r) of 30 x 10(3) and 35 x 10(3) respectively. The antigen was significantly higher expressed in sera from patients with colon cancer than that in normal sera (P<0.01). CONCLUSION: The tumor-associated antigen obtained from the colon tumor cells has been successfully purified through immunoaffinity chromatography with mAb 4D10, which may be useful for diagnosis on clinic.

Biochip as a potential platform of serological interferon alpha2b antibody assay.

The formation of antibodies against cytokines may play a major role in the generation of the immune response and may affect treatment protocols with recombinant cytokines. Interferon (IFN) is one of the effective therapeutic agents with anti-viral and anti-tumor specific effects. The appearance of IFN antibodies in patients may limit the natural and the therapeutic effect by IFNs. In contrast to conventional ELISA techniques, we here report a simple biochip methodology that enables identification of antibodies against cytokines and peptides. The method takes advantage of a functionalized self-assembled monolayer modified by N-hydroxysuccinimide (NHS). To validate this surface, four human proteins: IFNalpha2b, leptin, growth hormone and human IgG, with molecular sizes ranging between 14 and 150 kDa, were used. A number of other parameters for protein assay conditions by array technology were evaluated concomitantly. Finally, 56 serum samples from patients treated with recombinant human IFNalpha2b were simultaneously tested on single chip. In these patients, 16.1% (9 of 56 cases) were positive for IFNalpha2b antibodies. All results were confirmed in an ELISA, specific for the identification of IFNalpha specific antibodies in human samples. The potential application of this protein biochip can be amplified rapidly and reliably to test not only IFNalpha2b, but also other cytokine specific antibodies. The clinical relevance of such assays for investigations in autoimmune disorders is expected.