Iranian Biomedical Journal
Volume:5 Issue: 1, Jan 2001

We have previously shown that the proliferative response of T cells to antigenic or mitogenic stimulus decreased with age and that caloric resection (CR) attenuated the age-related decline in proliferation and IL-2 expression. Because activation-induced apoptosis is known to regulate cell proliferation and eliminate the high number of activated cells during an immune response, it was of interest to determine what effect aging or CR has on activation-induced apoptosis in T cells. Splenic T cells isolated from young (6-month) and old (25-month) mice fed ad libitum (control group) and from old (24-month) mice fed a restricted diet (40% caloric restriction) that began at 6 weeks of age. T cells were stimulated with superantigen staphylococcal enterotoxin B (SEB) or anti-CD3 antibody (primary stimulus) for 72 to 96 h, followed by restimulation with anti-CD3 (secondary stimulus). Activation-induced apoptosis was assessed by DNA fragmentation assay and the expression Fas/CD95 and Fas-ligand (Fas-L) was measured by flow cytometry. We found that the amount of DNA fragmentation was significantly (p<0.05) increased in the stimulated and restimulated T cells from old control mice and old caloric restricted mice compared to young control mice. The increase in DNA fragmentation with age was paralleled with an increase in the proportion of the cells expressing Fas and Fas-L. However, CR had no significant effect on the age-related increase in DNA fragmentation, Fas, or Fas-L expression. We also measured the Bcl-2 and Bax protein level and found that the level of Bcl-2 decreased and Bax increased with age and that CR had no effect on the age-related changes in the level of Bcl-2 or Bax protein. These results demonstrate that aging but not CR alters activation-induced apoptosis in mice T cells.

Increased number of mast cells at the site of infection is widely regarded as important host defense against parasites. The kinetics of mucosal mast cells and connective tissue mast cells responses were studied in the intestines of 68 female CFLP mice infected with 100 Schistosoma mansoni cercariae. The number of mucosal mast cells and the connective tissue mast cells increased from week 3 and week 6 respectively. While the number of connective tissue mast cells was still increasing during the chronic phase of the infection, the number of mucosal mast cells continuously decreased as the infection matured. The number of both cells was always greater in the proximal part of the small intestine. Their distribution within the injured areas appeared to depend other factors than parasites.

The early cytokines production in response to live logarithmic and stationary phase promastigotes of Leishmania major was examined on the peripheral blood cells of the healthy individuals. Whole blood cultures were stimulated by either logarithmic or stationary phase promastigotes. IFN-g and IL-10 productions were assessed by specific sandwich ELISA. The results showed that the logarithmic promastigotes were more potent than the stationary promastigotes in induction of IFN-g. In contrast, IL-10 production was significantly higher in the supernatants of the cells stimulated by the stationary promastigotes compared to the logarithmic phase of the parasites. The effect of BCG on IL-10 and IFN-g productions induced by these two types of promastigotes was also studied. BCG had augmenting effect on the cytokine production. However, the difference between the logarithmic and the stationary promastigotes was still observed, since logarithmic parasites induced higher amount of IFN-g and lower amount of IL-10 compared to the stationary parasites. In parallel, intracellular expression of IL-12 and IL-10 in the CD14+ cells was studied and the same results were obtained. The logarithmic phase parasites induced significantly higher expression of IL-12 and lower expression of IL-10 compared to the stationary phase parasites which induced lower expression of IL-12 and higher expression of IL-10. These results demonstrated that logarithmic promastigotes of L. major are more potent to induce Th1 response than stationary promastigotes which might have implication in vaccine preparation.

Glutathione S-transferases (GSTs) are encoded by a superfamily of genes and play a role in the detoxification of potential carcinogens. The human GSTs are divided into four classes: alpha, mu, pi and theta. Previous studies indicated that the absence of the Glutathione S-Transferase M1 (GSTM1) protein correlated with an increased risk of developing some types of cancers. Association between specific genotype and the development of breast cancer is still an open question. In the present study, the association between genetic polymorphism in the GSTM1 and susceptibility to breast cancer was investigated. The genetic polymorphism of the GSTM1 in exon 5 to exon 6 segment was detected by PCR in 59 patients with breast cancer and 59 normal subjects. The frequency of the GSTM1 null genotype in control and patient groups was 39.0% and 50.8% respectively. There was no significant association between null genotype of the GSTM1 and susceptibility to breast cancer (X2 = 1.63, df = 1, p >0.05).

Influenza C virus possesses specific neuraminate-O-acetylesterase as a receptor-destroying function. This enzymatic activity of the viral glycoprotein HEF (Hemagglutinin, esterase activity and fusion factor) can be visualized in situ by the use of distinct colour substrates. Hereby the localization, as well as the quantity of synthesized HEF protein is detectable. We further developed the esterase staining technique for a rapid detection and typing of influenza C progeny virus and for sensitive quantification of low viral loads. Neutralizing antibodies, interfering with virus attachment, were utilized to determine the infectivity of the inoculum in an indirect manner. The amount of unneutralized, infectious virus could easily be quantitated by in situ staining of the HEF esterase activity in infected cells. An evaluation of infectivity is presented and is put into relation to hemagglutinating virus units. Both, virus and serum antibody titers can be reliably determined by the esterase-neutralization assay. In this study a serial dilution of human and different animals (swine, dog and rabbit) sera were tested by in situ estrase neutralization assay (ENA) and hemagglutination inhibition (HI) test. The results show that in situ ENA is a sensitive method for titration of infectious rate of virus and quantification of neutralizing antibody against influenza C virus in different sera.

High morbidity and mortality of influenza virus infection makes it an important disease world-wide. Mouse is a very well-studied animal model for this disease with similar manifestation to human disease. It would be desirable to induce mucosal as well as circulating immune responses to obtain protection from infection and to decrease the spread of the virus. Cell mediated immunity (proliferative and cytolytic responses) is needed for long-term immunity. A new type of influenza subunit vaccine which can be given orally or parenterally has been developed to induce mucosal and circulating immune responses. This vaccine consists of membrane proteins rolled up in a unique phospholipid structures called protein cochleates. BALB/c mice were immunized three times with influenza glycoprotein-containing cochleates orally or intramuscularly, or they were primed orally or intramuscularly followed by two boosts with the alternate routes. Proliferation assays of spleen cells, and ELISA for IgA, IgG, and IgM of the sera and saliva from these mice shown some differences in the immune responses induced by different immunization regimens. Mucosal administration of the formulation led to secretory IgA in saliva while parenteral immunization resulted in circulating IgG. Increased proliferative responses as well as IgG following 2nd and 3rd administration, indicated the effect of boosting in all immunization regimens. Oral immunization with influenza virus envelope glycoprotein-containing cochleates led to protection from infection in the lungs and trachea following intranasal challenge with the live virus.

Leishmaniasis is a major infectious disease of considerable public health in more than 86 countries around the world. Several approaches toward vaccine development against this disease have been taken. Glycoprotein (gp63) is conserved among diverse species of Leishmania and has induced immunological responses in murine models. Therefore, this glycoprotein has been considered as a second generation vaccine for Leishmaniasis and for potential diagnostic antigen. Recombinant vaccine using gp63 in cocktail form is one of the candidates. Since, Pichia pastoris expression system is similar to that of the eukaryotic genes, refolding and glycosylation aspects of the expressed protein, gp63 gene from NIH strain of L. major cloned into BamHI site of pHIL-S1 as yeast expression vector (shuttle vector). This vector carries sequences of acid phosphatase (PHO1) signal peptide from yeast. The construction transfected into the P. pastoris using lithium chloride method. Recombinant clones were screened on histidin minus media. The expression of rgp63 was studied by methanol after induction. The expressed recombinant protein was confirmed by Western blotting and electron microscopy. The expression level of rgp63 was more than 30%. Since the rgp63 expression in P. pastoris was active in SDS-PAGE gelatin gel, therefore it should be very similar to the native form.

Cystic hydatid disease is caused by cystic larval (metacestode) stages of Echinococcus granulosus. This parasite can potentially occur all over the world especially in Mediterranean and Middle East countries and some parts of Africa, Latin America and China which have major foci of human infections. The cyst wall of metacestodes consists of inner, middle and external layers. To date, little attention has been paid to the immunological studies of the laminated (middle) layer. Because of the presence of diagnostic carbohydrate antigens, this layer is important. In this report, the characterisation of the laminated layer carbohydrate of the E. granulosus was investigated by peroxidase-labelled lectin conjugate blots. The comparison of SDS-PAGE and Western blot analysis confirms that many laminated layer bands are glycoproteins, especially 50-66 kDa and 25-29 kDa bands. These glycoproteins contain a-Methyl-D-mannoside, N-Acetyl-b-D-glucosamine, a-L-Fucose and N-Acetyl-b-D-galactosamine.