Iranian journal of immunology
Volume:2 Issue: 3, Summer 2005

There are two subclasses of human IgA (IgA1 and IgA2) that differ inantigenic properties and in chemical composition. The constant domains of α1 and α2 heavy chains have >95% sequence homology though major structural differences exist in the hinge region. Quantitation of IgA subclass levels depends on the availability ofmonoclonal antibodies (MAbs) specific for conserved conformational or linear epitopes restricted to each subclass. To produce, select and characterize monoclonal antibodies specific for human IgA2. Splenocytes from BALB/C mice immunized with a human IgA2 myeloma protein were fused with SP2/0 myeloma cells. Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by limiting dilution assay. Antibody (Ab) secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed, using a panel of purified myeloma proteins and some animal sera by ELISA and immunoblotting. The affinity constant (Kaff) was also determined by ELISA. Four murine hybridoma clones designated 2F20G5, 2F20B5, 3F20E3 and 6F20H11 were obtained that secreted MAbs specific for the human IgA2. 2F20G5 and 6F20H11 MAbs react with linear epitope(s) while 2F20B5 and 3F20E3 react withconformational epitope(s) located to human IgA2 subclass. 2F20G5 MAb displays a weak cross-reactivity with monkey and rabbit sera and a strong cross-reactivity with cat and dog sera while the other three MAbs showed no cross-reactivity with the animal sera tested. These MAbs, especially 6F20H11 with high affinity constant (6.03 ×109 M-1) are useful tools for quantitation of human IgA2 subclass levels in various diseases. Cross-reactivity of 2F20G5 MAb with some animal sera suggests phylogenic conservation of the epitope recognized by this MAb.

DNA immunization with plasmid DNA encoding bacterial, viral, parasitic and tumor antigens has been reported to trigger protective immunity. To evaluate the use of a DNA immunization strategy for protection against anthrax, a plasmid was constructed. The partial sequence of protective antigen of Bacillus anthracis, amino acids 175-764, as a potent immunogenic target was selected. The DNA encoding this segment was utilized in the construction of pcDNA3.1+PA plasmid. After intramuscular injection of rats with pcDNA3.1+PA plasmid, the expression of PA was assessed by RT-PCR and immunohistochemistry at RNA and protein levels, respectively. We also evaluated the presence of anti-PA antibodies in sera of immunized mice with pcDNA3.1+PA construct using immunoblotting. The integrity of pcDNA3.1+PA construct was confirmed with restriction analysis and sequencing. The expression of PA was detected at RNA and protein levels. The presence of anti-PA antibodies in immunized mice with pcDNA3.1+PA construct was also confirmed. Our results indicate that pcDNA3.1+PA eukaryotic expressing vector could express PA antigen, induce antibody response and may be used as a candidate for DNA vaccine against anthrax.

The glutathione S-transferase (GST) family of metabolising enzymesplays an important role in the detoxification of mutagens and carcinogens. The expression of many of these cancer susceptibility enzymes is genetically polymorphic. An increased frequency of GST-null genotypes has been associated with several malignancies. To investigate the rate of GSTT1 and GSTM1 null genotypes in AML patients and to determine its importance in prognosis of the disease. DNA was extracted by phenol/chloroform method from peripheral blood or bone marrow of 180 white Caucasian patients. A multiplex PCR method was used simultaneously to amplify regions of GSTM1, GSTT1, and b-globin genes in genomicDNA. The survival curves were analyzed by the Kaplan-Meier method and compared by the log-rank test (Mantel-Cox) using the SPSS software program. Of the total of 180 patients, 23 cases (12.8%) showed null genotypes in both genes, while in 52 patients (28.9%) both genes were wild-types. GSTM1 null-GSTT1 wild-type was detected in 91 patients (50.6%) and GSTM1 wild-type-GSTT1 null genotype was detected in 14 patients (7.8%). These rates are within the upper limit of the rates detected in the normal European population. There was no significant difference in the overall survival and in disease free survival between different groups. These observations suggest that the inherited absence of the GSTT1 and GSTM1 carcinogen detoxification pathway may be related to carcinogenesis but it is not an important determinant of prognosis in AML.

Alteration in peripheral blood lymphocytes (PBLs) is usually investigatedto provide an evidence of the host immune responses to tumor antigens. The tumor infiltrating NK cells interact most closely with the tumor cells and more accurately reflect tumor host interactions. To analyze peripheral blood and tumor associated Natural Killer (NK) cells in patients with breast cancer by immunophenotyping. Twenty women suffering from breast cancer were examined; 12 of them were confirmed histologically to be invasive ductal carcinoma. PBL and tissue samples from patients and matched control group were processed for analysis by flow cytometry. Results of PBL analysis indicated a significant (P<0.05) increase in both the total number and activated NK cells in invasive ducal carcinoma patients compared to normal controls. No significant differences were noticed in the percent of NK cells and their activation marker expression in intra tumor lesion of the invasive ductal carcinoma and other tumors compared to benign lesions, however a decrease in the total NK number and activated NK cells was observed with progression of the tumor. Data of this investigation conclude that the total and activated NK cell number increase in peripheral blood of patients with breast cancer. The relationship between peripheral blood and intratumor NK cells needs more clarification, however, a decrease in intratumor NK cell number and their activation status occurs with tumor progression.

IL-10 is an anti-inflammatory cytokine which is involved in tumorigenesis.Over production of IL-10 and elevated number of IL-10 generating mononuclear cells in breast tumor tissue has already been shown. To determine the association of IL-10 promoter polymorphisms with increased risk of breast cancer and its association with breast cancer prognostic factors. Peripheral blood samples from 275 female breast cancer patients and 320 cancer free controls were used to detect three single nucleotide polymorphisms in IL-10 promoter region (-1082, -819, -592) by PCR method. The frequency of genotypes and alleles of three mentioned regions of IL-10 promoter and their haplotypes (GCC, ATA, and ACC) showed no statistically significant difference between patients and controls. In the case of prognostic factors, progesterone receptor (PR) status exhibited significant relation with -1082 genotypes (P=0.03) and haplotypes (P=0.02). -1082 AA genotype was associated with negative PR expression whereas AG and GG genotypes of this site were positively associated with PR expression. Similarly GCC haplotype correlated with positive PR expression and ATA and ACC with negative PR expression. The data of this study showed that IL-10 promoter gene polymorphisms may not be considered as one of the risk factors for breast cancer in Iranian patients.

The incidence of allergic and asthmatic diseases has been continuouslyincreased in both industrial and developing countries. Extracts from various known allergens are used for the diagnostic and therapeutic purposes. To investigate the effects of an extract prepared from Chenopodium album (Ch.A.) pollen to induce allergic asthma in BALB/C mice. BALB/C mice were sensitized by i.p. injection of Ch.A. extract and alum, and an intratracheal instillation of the extract. The bronchoalveolar lavage (BAL) fluids were obtained by cannulating the trachea and lavaging the lungs and examined for eosinophilia. Splenocytes were incubated with Ch.A. extract and cell supernatants were examined for IL-4 and IL-5 by ELISA. We demonstrated that Ch.A. extract treatment in mice increased serum levels of specific IgE and production of IL-4 and IL-5 from splenocytes. An airway eosinophilia was also demonstrated in mice. These results suggest that Ch.A. allergen extract is a potential agent in inducing characteristics of allergic asthma in a mouse model useful in investigational studies.