Accurate sensitivity of quantum dots for detection of HER2 expression in breast cancer cells and tissues.

Here we introduce novel optical properties and accurate sensitivity of Quantum dot (QD)-based detection system for tracking the breast cancer marker, HER2. QD525 was used to detect HER2 using home-made HER2-specific monoclonal antibodies in fixed and living HER2(+) SKBR-3 cell line and breast cancer tissues. Additionally, we compared fluorescence intensity (FI), photostability and staining index (SI) of QD525 signals at different exposure times and two excitation wavelengths with those of the conventional organic dye, FITC. Labeling signals of QD525 in both fixed and living breast cancer cells and tissue preparations were found to be significantly higher than those of FITC at 460-495 nm excitation wavelengths. Interestingly, when excited at 330-385 nm, the superiority of QD525 was more highlighted with at least 4-5 fold higher FI and SI compared to FITC. Moreover, QDs exhibited exceptional photostability during continuous illumination of cancerous cells and tissues, while FITC signal faded very quickly. QDs can be used as sensitive reporters for in situ detection of tumor markers which in turn could be viewed as a novel approach for early detection of cancers. To take comprehensive advantage of QDs, it is necessary that their optimal excitation wavelength is employed.

C-reactive protein (CRP) gene polymorphisms: implication in CRP plasma levels and susceptibility to acute myocardial infarction.

C-reactive protein (CRP) is one of the many molecular factors involved in pathogenesis of coronary artery disease which its plasma levels are associated with increased risk of cardiovascular events. The present study designed to determine whether polymorphisms in the CRP gene are associated with plasma CRP levels and susceptibility to acute myocardial infarction (AMI). Plasma CRP levels were measured in patients with AMI and control subjects and genomic DNA and peripheral blood mononuclear cells (PBMCs) were extracted. The -717A/G and 1059G/C CRP polymorphisms were detected. The mRNA expression of CRP gene and plasma levels of CRP and interleukin-6 (IL-6) were also analyzed. The -717A/G variation was significantly associated with higher CRP levels, but 1059G/C variation was associated with lower CRP levels. The AA genotype frequency of -717A/G variation was significantly more frequent in the patients than control subjects. By contrast, the genotype and allele distribution in 1059G/C of patient were not statistically different between patients and controls. There were significant differences in circulating levels of CRP and IL-6 in the patients than in controls. The mRNA expression levels of CRP were significantly higher in the patient plasma compared with controls. Our results indicate relationship between many polymorphisms in CRP gene and risk of AMI which suggest that genetic variations in CRP might be helpful for determining susceptibility to AMI in Iranian patients. In addition, CRP gene polymorphisms are associated with plasma CRP levels and susceptibility to AMI might be related to CRP gene expression which affects its plasma levels.

Tumor necrosis factor-α: investigation of gene polymorphism and regulation of TACE-TNF-α system in patients with acute myocardial infarction.

A polymorphism within tumor necrosis factor-α (TNF-α) gene promoter and contribution of TNF-α converting enzyme (TACE) have been reported to be associated with TNF-α production which may increase susceptibility to heart failure such as acute myocardial infarction (AMI). However, the relationship between this polymorphism and susceptibility to AMI and the mechanism of TACE-TNF-α system regulation has poorly been studied. Genomic DNA and peripheral blood mononuclear cells (PBMCs) of patients with AMI and control subjects was extracted. The -308 G/A TNF-α polymorphism was detected. The mRNA transcription and protein expression levels of TNF-α and TACE were analyzed by real time RT-PCR and flow cytometry respectively as well as plasma TNF-α by ELISA. The 'A' allele frequency of TNF-α was significantly more frequent in the patients than controls (P < 0.001). There were statistically significant differences in TNF-α and TACE mRNA and protein levels as well as circulating TNF-α in the patients. However, these levels were higher in the patients who carry 'A' allele. There were significant positive correlation between these mRNAs and protein expression levels (r = 0.66, P < 0.001, r = 0.78, P < 0.001 respectively). These data suggest that genetic polymorphism in TNF-α might be helpful for determining susceptibility to AMI in Iranian patients. The TACE-TNF-α system in circulating leucocytes is stimulated which these results demonstrate that in patients with AMI, TACE expression in PBMC increases with TNF-α expression and processing of TNF-α in PBMC might be regulated by TACE at transcriptional, translational, and post-translational levels in AMI.

Matrix metalloproteinase: investigation from gene to protein as effective factor in myocardial infarction.

Current evidence indicates that extracellular matrix (ECM) remodeling is a component of acute myocardial infarction (AMI) and matrix metalloproteinase (MMP) has a role in early atherosclerosis, plaque rupture and myocardial infarction (MI). The necessity of inhibition of ECM remodeling and subsequent injuries in patients with AMI suggests that MMP might be involved in this task. Therefore, we investigated the activities of MMP-1, -2, -3, and -9 which play an important role in AMI. Plasma and peripheral blood mononuclear cells (PBMCs) of 50 patients with AMI were isolated from peripheral blood after the onset of AMI within 24 h, comparing with 50 control subjects. The active form of MMPs was measured by enzyme linked immunosorbent assay (ELISA); MMP proteins presence and expression by immunoblotting and zymography analysis; and mRNA expression of MMPs by real time reverse transcriptase polymerase chain reaction. Plasma concentrations of MMPs increase in patients rather than control subjects. Gel zymography revealed 43, 66, 45, and 83 kDa molecular weight bands which consistent with active MMP-1, -2, -3, and -9, respectively, exhibiting gelatin-degrading activity in both patient and control subjects. No up-regulation of mRNA expression was found. To our knowledge, it is the first monitoring of MMP gene and protein expression and also circulating active MMPs in Iranian patients with AMI and normal subjects. Up-regulation of MMPs activity is common in the falling myocardium and missing up-regulation of transcription indicates that protein levels of MMPs were regulated at the post transcriptional level.

Quantum Dot-labeled Tags Improve Minimal Detection Limit of CA125 in Ovarian Cancer Cells and Tissues.

In recent years, a lot of attention has been paid to quantum dot (QD) nanoparticles as fluorescent sensors for sensitive and accurate detection of cancer biomarkers. Here, using a homemade specific monoclonal antibody against CA125 and QD525- or FITC-labeled probes, expression of this marker in an ovarian cancer cell line and cancer tissues were traced and optical properties of fluorophores were compared qualitatively and quantitatively. Our results clearly showed that besides lower background and exceptionally higher photobleaching resistance, QD525 exhibited higher fluorescent intensity for both ovarian cancer cell and tissues at different exposure times (p<0.0001) and excitation filter sets (p<0.0001) exemplified by significantly higher staining index (p<0.016). More importantly, the FITC-labeled probe detected antigen-antibody complex at minimum concentration of 0.3 mg/mL of anti-CA125, while reactivity limit decreased to 0.078 mg/mL of anti-CA125 when QD525-labeled probe was applied showing four times higher reactivity level of QD525 probe compared to the same probe labeled with FITC. Based on our results, it seems that QDs are inimitable tags for sensitive detection and localization of ovarian cancer micrometastasis and molecular demarcation of cancer tissues in surgical practice, which subsequently figure out accurate therapeutic approaches.

Genetic polymorphism of interleukin-6 gene and susceptibility to acute myocardial infarction.

OBJECTIVES: Inflammatory processes play a pivotal role in the pathogenesis of atherosclerosis. Genes coding for cytokines such as interleukin-6 (IL-6) are candidates for predisposing to the risk of coronary artery disease. The aim of this study was to investigate whether molecular polymorphism of the IL-6 gene is involved in the predisposition to acute myocardial infarction (AMI). METHODS: Genomic DNA and peripheral blood mononuclear cells of patients with AMI and controls were extracted. IL-6 gene variations were evaluated by polymerase chain reaction followed by restriction enzyme analysis. The mRNA expression of IL-6 gene and plasma levels of IL-6 and C-reactive protein (CRP) were analyzed. RESULTS: The prevalence of 'C' allele in -174 G/C variation was higher in patients with AMI than in controls. The IL-6 -174 'C' allele is associated with high levels of IL-6 in the patients, of which the patients with CC and GC genotypes significantly have higher IL-6 concentrations, respectively. Increased CRP concentrations were associated with -174 G/C variation in the patients compared with controls. The mRNA expression levels of IL-6 were significantly higher in the patient compared with controls (P<0.001). CONCLUSION: The findings of this study indicate the relationship between IL-6 gene polymorphism and the risk of AMI, which suggests that genetic polymorphism in IL-6 gene, might be helpful for determining susceptibility to AMI in Iranian patients. In addition, susceptibility to AMI might be related to IL-6 gene expression, which affects its plasma levels. CRP plasma levels also were associated with IL-6 gene variation in the patients.

Genetic polymorphisms and plasma levels of matrix metalloproteinases and their relationships with developing acute myocardial infarction.

OBJECTIVES: Matrix metalloproteinases (MMPs) play an important role in early atherosclerosis, plaque rupture, extracellular matrix remodeling, and myocardial infarction (MI). MMP gene polymorphisms contribute to the risk of developing cardiovascular disease. We designed to investigate the association of acute MI (AMI) with a polymorphism in the human MMP-1, 2, 3, and 9 genes in Iranian patients with AMI. METHODS: Genomic DNA of 400 enrolled patients with AMI and 200 controls was extracted from their blood samples. The -1607 1G/2G MMP-1, -1306 C/T MMP-2, -1171 5A/6A MMP-3, -1562 C/T MMP-9 polymorphisms were detected. Plasma levels of MMPs were analyzed. RESULTS: There are significant differences in MMP-3 '5A' allele and genotype in the patients with AMI comparing with controls. However, no significant differences were observed in MMP-1, 2, and 9 allele frequencies between the patients and controls. Differences between plasma levels of MMPs were significant in the patients than in controls. There were statistically significant differences between plasma MMP-3 in carriers of 5A allele compared with 6A allele. MMP-9 plasma levels were significantly higher in the carriers of -1306 TT and -1306 CT than CC. However, there were no statistically significant association between genetic variation of MMP-1, 2, and 3 in the patients and their plasma levels. CONCLUSION: These data suggest that MMP genotyping such as genetic polymorphism in MMP-3 might be helpful in determining susceptibility to AMI in Iranian patients. In addition, susceptibility to AMI might be related to MMP-9 gene expression, which affects its plasma levels.

Heterologous immunization, a way out of the problem of monoclonal antibody production against carcinoma antigen 125.

BACKGROUND: Monoclonal antibodies (mAbs) are essential tools for many molecular immunology investigations, epitope mapping and molecular modelling, clinical laboratory diagnostic tests and immunotherapy. Humoral immune response of immunized animals largely depends on the nature of antigen and the immunization technique. Polysaccharides and heavily-glycosylated proteins are very elusive targets incapable of mounting long-lasting, high affinity antibody responses. Carcinoma antigen 125 (CA 125), a well known tumor marker of ovarian cancer, is a mucin type antigen consisting of repetitive units of heavily glycosylated moieties which render production of mAbs very difficult. OBJECTIVE: To evaluate the efficacy of heterologous antigen preparations as a way of mouse immunization in the production of anti-CA 125 mAb. METHODS: Two different protocols of immunization were used for priming of NMRI mice. In the first method, mice conventionally immunized by three intraperitoneal injections of purified CA 125 and boosted by the antigen three days before fusion. In the second approach, mice were primed by three intraperitoneal injections of living CA 125 positive cells of OVCAR-3 cell line, and boosted by intravenous injection of the purified extracellular domain of CA 125. Production of mAb was performed by standard hybridoma technology and mAbs were characterized by different immunoassays. RESULTS: The first method failed to produce stable clones despite six time fusion. A total of ten stable clones, however, were produced in the second approach. Some of the clones were characterized and found to have excellent immunoreactivity when tested by ELISA assay, western blotting, intracellular and surface immunofluorescent staining of OVCAR-3 cell line and immunohistochemical staining of ovarian cancer tissues. CONCLUSION: Altogether the results of the present study clearly showed that heterologous antigen preparation is the method of choice for immunization when production of monoclonal antibody against highly glycosylated poorly immunogenic antigens is concerned.