Iranian Journal of Basic Medical Sciences
Year:2004 | Volume:6 | Issue:4 (20)|Papers Count:11

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies in Iran. ESCC patients are asymptomatic until later stages of disease which makes most interventions unsuccessful with survival rate of 5-20%. New tumor markers for early detection and/or identification of predisposing factors in ESCC may improve the life expectancy for this disease. p16, an inhibitor of cyclin D-dependent protein kinases, is a tumor-suppressor gene, with mutation and deletion reported in a variety of tumors. p16 promoter methylation is an important mechanism for inactivation of this gene and may be studied in serum DNA of cancer patients as a tumor marker. DNA isolated from serum, blood and endoscopic tissue of 30 ESCC patients and 30 normal volunteers were examined for p16 hypermethylation in province of Khorasan, North east of Iran. DNA sequences of methylated and unmethylated genomic regions after bisulfite conversion was distinguishable by sequence-specific PCR primers using methylation specific PCR (MSP). p16 hypermethylation was found in 8/30 (26.6%) in serum samples, 13/30 (43.3%) in blood samples and 22/30 (73.3%) in tissue samples and none (0%) in normal volunteers. The 8 cases with aberrant p16 methylation in their serum DNA showed similar changes in their blood and tumor tissues. These results indicate that p16 hypermethylation may be found in the circulation with the origin of esophageal tumor DNA. Because methylation abnormalities have been found in many other genes and tumor types, this approach may have implications for noninvasive detection of a wide variety of cancers and this assay offers a potential means for the blood and serum-based detection and/or monitoring of ESCC.        

The establishment of a standard and well-studied animal tumor model in order to evaluate the effects of new anti-cancer therapeutics and the study of tumor biology is crucial to cancer research institutes in Iran. In this study, we established a standard tumor model in Balb/c mice. After the evaluation of the available cell lines, WEHI-164 cells were used for tUl110rinduction in male and female four-week inbred Balb/c mice. On the day 0, mice were inoculated with 7 × 106 WEHI-164 cells subcutaneously and tumor dimensions were measured daily. Statistical analysis of the results indicated that there is no significant difference between male and female groups and the yield of 82% about tumor induction was determined. From the day 8, tumor regression was observed.      

Regards to the weak immunigenicity of tumor cells as well as recombinant antigenic markers (like BLP25), adjuvants are needed for enhancement of immune response and its skewing toward cellular immunity. In this research poly (lactide-co-glycolide) (PLGA) microspheres were used as delivery system for a recombinant mucin named mucin 1 (MUC1, BLP25) as a recombinant antigenic cancer marker, and CpG-ODN was used for enhancement of immune response and its biasing toward cellular immunity. PLGA microspheres encapsulated with BLP25 and CpG-ODN were prepared using a w/o/w emulsion method. Encapsulation of BLP25 was determined by a HPLC method and spectrophotometry at 260 nm was used for quantification of encapsulated CpG-ODN. In vivo immunization studies were performed by SC injections of 20µg BLP25 and 4 µg CpG-ODN in mice (4 animal per group). Group I) Mice immunized with microspheres co-encapsulated with BLP25 and CpG; Group II)Mice immunized with microspheres encapsulated with BLP25 mixed with CpG solution; Group III) Mice immunized with microspheres encapsulated with BLP25. For evaluation of specifity of immune response, T lymphocytes separated from different groups of mice were incubated with different antigens (T Cell Proliferation Assay). IFN-γ and IL-4 cytokines were assayed by a sandwich ELISA method. T lymphocytes separated from group I, showed high proliferation (stimulation index = 25) and high levels of IFN-y interferon (11200±172 pg/ml) which were significantly higher than other two groups (P<0.0001). Co-encapsulation and co-delivery of MUC1 and CpG-ODN produced high cellular (Th1) immunity responses (high levels of lFN-y and no IL-4), indicating the high adjuvanticity potential of CpG-ODN for immunization against cancer markers. Importance of co-encapsulation of antigen and adjuvant in the same delivery system for better adjuvant effect was also approved.      

To determine the accuracy of Fine Needle Aspiration (FNA) cytology in the diagnosis of bone lesions, fifty bone lesions after clinical and radiographic correlation were evaluated by FNA. The results were compared with the histopathology of the open biopsis. Accuracy rates of 75% and 88% were achieved by FNA of non-malignant and malignant bone lesions respectively. Chondrosarcoma and non-malignant lesions such as unicameral bone cyst gave the greatest diagnostic difficulties. Osteosarcoma, Ewing's sarcoma and metastasis could be easily identified when interpreted with clinical and radiographic findings. FNA plays an important role in early diagnosis of most of the malignant bone lesions. Cytologic evaluation together with clinical and radiographic findings has a high accuracy rate for diagnosis of bone lesions.      

Present studies have shown that gap junction channels could be a novel target to reduce brain damage during cerebral ischemia. Carbenoxolone, a gap junction channel blocker decreased the spread of cell death in in vitro brain ischemic model and inhibition of lipid peroxidation has been of much interest to ameliorate excitotoxic neuronal damage. Thus the aim of the present study was to determine the effect of carbenoxolone on lipid peroxidation in experimental global ischemic-reperfusion in the rat hippocampus. The brain damages were determined using the measurement malondialdehyde (MDA). Cerebral ischemia was induced by four-vessel-occlusion (4VO). Carbenoxolone (50-200mg/kg), phenytoin (50 mg/kg) and normal saline (10ml/kg) were administered intraperitoneally immediately after reperfusion. The malondialdehyde (MDA) was measured using the thiobarbituric acid (TBA) test. The MDA level was higher in the saline group during ischemic-reperfusion than the sham group. The MDA levels recovered significantly upon phenytoin and carbenoxolone (100-200 mg/kg) therapy in the ischemic-reperfusion. These results suggest that carbenoxolone may protect the brain against lipid peroxidation in cerebral ischemia.    

Helicobacter pylori is the etiologic agent of chronic-active gastritis, gastroduodenal ulcers in humans, and a co-factor in the occurrence of gastric cancer and mucosa-associated lymphoid tumors. Adherence of H. pylori to the gastric mucosa is a critical, initial step in the pathogenesis of the disease. So bacterial adhesion inhibitory agents provide a novel pharmacologic approach to the management of infectious diseases. 22 H. pylori strains, obtained from the antral or duodenal biopsies of 49 patients with dyspepsia, gastritis, gastric ulcer, duodenal ulcer, etc. were assayed by ELISA reader (UPR: Urea Phenol Red) to investigate the diversity of attachment to 7 mamalian cell lines. The concentration of H. pylori and cell suspension, the condition and temperature, can alter the attachment rate. H. pylori can attach to all 7 cell lines. There are no significant differences between 22 H. pylori strains in attachment to cells. The attachment pattern of H. pylori to the cells showed significant reduction respectively from HepII, HeLa, SW742, AGS, HT29/219, HT29 to Caco-2. Best attachment was seen to HepII, HeLa and SW742 cells, and among these HepII were the best cells for this purpose. Our studies suggest that HepII, HeLa and SW742 cells could serve as a suitable in-vitro model for the study of H. pylori adhesions, attachment, inhibition of attachment and detachment assays and among these HepII cell is preferably recommended.      

Numerous in vitro studies indicate that endothelium-mediated relaxation is reduced with development of hypertension. Considering the role of protein kinases in many metabolic processes of phosphate transferring and its importance in the cell communication and function (in the physiological and pathological states) and also the absence of any reports on the effects of these enzymes in the mediating responses to vasodilators during hypertension, this study became of interest. The objectives of this study were to measure the vascular responses to acetylcholine (ACh) and sodium nitroprusside (SNP) in the isolated rat aorta and mesenteric bed removed either from hypertensive or control rats and also to investigate the role of protein kinase C in these responses. Hypertension was induced in the male Sprague-Dawley rats (200-250 g) by DOCA-salt injection (20 mg/kg, twice weekly, for 5 weeks, s.c.) and NaCl (1 %) was added to their drinking water. Control rats received saline injection (0.5 ml/kg, twice weekly, for 5 weeks, s.c.). 5 weeks later, animals were anaesthetized with thiopental (30 mg/kg, Lp.), and arterial blood pressure was directly measured. Mean arterial blood pressure in control and hypertensive rats were: 98±7.5, 163± 3.5, mmHg, respectively (P < 0.0001). In in vitro studies, rings of descending aorta were cut and mounted for isometric tension recording in an orian chamber containg Krebs solution. After 1 h of stabilization, rings were precontracted with phenylephrine (5 × 10-8 or 10-6M), then concentration response curve to acetylcholine (ACh, 10-6-10-3M) and SNP (10-8-10-4 M) were constructed. There was a significant decrease in response to Ach and also a reduction in the maximal response in rings isolated trom hypertensive rats. Mesenteric beds were also removed either from control or hypertensive rats and perfused with Krebs solution. After 1 h of stabilization, tissue were precontracted with noradernalin (10-6M), then concentration-response curve to ACh (10-8-10-4 M) and SNP (10-8-10-4M) were constructed. Responses to ACh but not to SNP were significantly reduced in tissue removed trom hypertensive rats (eg. in response to 10-6 M of ACh: control:-41.6 ±4.9 hypertensive: 17.2 ±3.6 mmHg, P < 0.05). However, addition of chelerythrine (10µ M), a protein kinase C (PKC) inhibitor, to the organ bath significantly restored these impaired responses. These results suggest that protein kinase C is involved in the endothelial dysfunction induced by hypertension.      

The accumulation of DNA-antibody immune complexes in kidneys is associated with SLE in human and lupus in mice. The level of serum of anti-DNA antibodies is also directly related to the disease severity. Determination of structures that confers DNA specificity in these antibodies can help in designing new therapeutic strategies. In this study, computer simulation and artificial neural network (ANN) has been utilized for the first time to determine effective structure of anti-DNA antibodies in binding to DNA. VH CDR3, FR3 and CDR2 were designated the most effective regions in antibodies that bind to DNA, using an educated neural network. We have also shown that the presence of Arg in position 104, 109 and 113 of CDR3, position 63 and 64 of CDR2 and position 90 of FR3, enhances the DNA specificity of antibodies. A part of our result confirms the previous laboratory works, and the other part introduces novel structures that may be important in DNA binding. This study shows the potency of logic networks in solving medical dilemmas.

Allergenic reaction to melon, Cucumis melo (belongs to Cucurbitaceae family), has been reported in some allergic patients. Oral allergy syndrome was the most common clinical features associated with melon allergy. This study was aimed to confirm allergenicity of Mashadi melon, identify allergenic protein(s) of melon and allergenic cross reactivity of melon with other allergens. Prick test was preformed with the extract of different parts of melon (Peel, pulp and loose layer on pulp) on the 35 patients who suffered from allergic symptoms after the ingestion of melon. Total IgE and specific IgE to melon were measured in 21 sera from patients with positive skin prick test and 15 healthy controls' sera by means of ELlSA. The IgE reactive protein of melon extract was detected by western blotting, using the 12 patient's sera (with high levels of IgE). ELISA inhibition carried out in order to detect cross-reactivity between melon and kiwi, banana, Cynodon dactylon and Poa pratensis. Clinical reactions to melon were oral allergy syndrome 61% (immediate oral itching with or without angioedema of the lips and oral mucosa), rhinitis 38%, itching 19% and gastrointestinal symptoms 4.8%. Twenty one of the 35 patients showed positive skin prick test (SPT) to loose layer on pulp. Three patients also showed reaction to pulp and loose layer. Increased specific IgE levels to melon were observed in 18 patients with positive SPT to melon extract. Inhibition experiments showed a strong cross-reactivity of melon specific IgE with two species of ragweed pollen, especially with Cynodon doctylon, but banana and kiwi extract did not inhibit melon specific IgE in inhibition ELISA method. Immunoblot analyses of aqueous protein extract from melon were showed an IgE-binding protein of - 14.4 kDa with 8 of 12 melon-allergic patients' sera. In conclusion, we confirmed IgE-mediated hypersensitivity to melon with common clinical feature of oral allergy syndrome (OAS) and presence of an IgE-binding protein of - 14.4kDa in melonextract. These findings suggest that main allergen of melon could be profilin.