In situ hybridisation of chick embryos with p53-specific probe and their immunostaining with anti-p53 antibodies

Tumor-suppressor protein p53 is an important regulator of cell cycle and apoptosis. On the level of embryo extracts it has been shown earlier that both p53 protein and mRNA are expressed in developing chicken. Here we describe the expression patterns of p53 mRNA and protein in developing chicken embryos (stages 2–12) using in situ hybridisation and immunostaining with p53-specific monoclonal antibody Mab421. p53 mRNA is equally localised all over the embryo in the stages observed. According to electron microscopy data a subfraction of p53 mRNA is bound to dissolving yolk granules expressing acid phosphatase activity characteristic for lysosomes. Protein p53 is synthesised starting from the medium primitive streak stage (stage 3) and reaches its maximum level at the full primitive streak stage. During these stages protein p53 is distributed evenly across the embryos. After gastrulation p53 protein remains visible at higher levels only in certain anlages and areas. In developing nervous system the expression is observable in neuroectoderm, during the closure of the neural tube and in mesenchyme in the area of migrating neural crest cells. In cardiogenesis protein p53 is expressed during formation of tubular heart in the epimyocardium, endocardium and cardiac jelly. p53 protein localises in the neurocoele (obviously connected with cellular debris) and cardiac jelly. Our data support the role of p53 in early development, especially during embryo gastrulation, the development of central nervous system, neural crest and heart. In some cases increased p53 amounts colocalise with the areas of intensive epithelium-mesenchyme transition. Accepted: 22 May 2001     

A simple manual rosetting method for absolute CD4+ lymphocyte counting in resource-limited countries

A CD4 monoclonal antibody which reacts with CD4+ lymphocytes but does not react significantly with monocytes was generated and used to develop a simple method for CD4 count. In a comparison with standard flow cytometry, high correlation was obtained. The developed method can be an alternative to flow cytometry for resource-limited countries.     

Method for analysis of surface molecule alteration upon phagocytosis by flow cytometry

In this study, we introduce an application of flow cytometry for the concurrent detection of phagocytotic cells and surface molecules involved in the phagocytic process. E. coli expressing green fluorescent protein (GFP) were applied as the phagocytosable particles. Blood samples were incubated with E. coli expressing GFP, followed by indirect immunofluorescence using four candidate monoclonal antibodies (mAbs). Granulocytes that had phagocytosed E. coli exhibited high levels of GFP intensity, in contrast to the nonphagocytosed cells. By comparing the level of expression of molecules expressed on phagocytosed granulocytes with that of nonphagocytosed cells by flow cytometry, it enabled the determination of the expression and alteration of the cell surface molecules upon phogocytosis. Of the four mAbs used in this study, upon phagocytosis, molecules recognized by mAbs WK13, COSA5A and COSA33NL were up-regulated. However, CD15 recognized by mAb VIMD5 was downregulated. The proposed method will benefit the study of phagocytic mechanisms in the future.     

Methods of intraoperative localization in the surgery treatment of persistent and recurrent secondary hyperparathyroidism

The intraoperative localization of residual parathyroid tissue during re-operations for persistent or recurrent secondary hyperparathyroidism (HPT) could have difficult due to cicatricial phenomena, anatomic modifications, ectopic or super-numerous glands. The use of intraoperative localisatin methods, as ultrasonographic and gamm-camera, permits a great level of success in identifying residual gland tissue. The Authors report their experience in the treatment of 5 cases of persisten HPT and 3 cases of recurrent HPT using gamma-camera and ultrasonography intraoperatively. Gamma-camera presented a sensitivity of 88% while ultrasonography did 100%. According to the experience, even numerically limited, of the Authors, intraoperative ultrasonography offers better results than gamma-camera, with less technical complexity.     

Association of the -1438 G/A and 102 T/C polymorphism of the 5-Ht2A receptor gene with irritable bowel syndrome 5-Ht2A gene polymorphism in irritable bowel syndrome

GOALS: The aim of this study is to investigate whether there were any association between the 102 T/C and -1438 G/A polymorphisms of the 5-HT2A receptor gene and IBS, and abdominal pain, anxiety and depression. BACKGROUND: Genes involved in serotonin (5-HT) metabolism are good candidates for the pathogenesis of irritable bowel syndrome (IBS). Recently, a silent polymorphism in the 5-HT2A receptor gene was identified that is defined by a T to C transition at position 102. Also, a novel G to A base change at position -1438 of the promoter region has been detected in 5-HT2A receptor gene. STUDY: Fifty-four patients with IBS diagnosed according to the Rome 1 criteria and 107 healthy individuals were included in the study. PCR was used to amplify a 468-bp (G-->A) and 342-bp (T-->C) fragment of genomic DNA containing the polymorphism. Hospital anxiety and depression scale was used to assess the risk of depression and anxiety. Severity of chronic abdominal pain was determined by visual analogue scale (VAS). RESULTS: It was shown that there was a high incidence of homozygote C allele of the 102T/C polymorphism (%22.2; OR: 7.89, P = 0.04) and homozygote A allele of the -1438 G/A promoter region (%%37; OR: 11.14, P = 0.01) in patients with IBS. The risk of having an anxiety disorder was 83.3% in patients with C/C genotype, which was higher than other allele carrying patients, and overall mean (%52.7). (chi = 8.56, P = 0.014). The patients with T/T genotype had a VAS score of 54.93 +/- 2.59 mm, which was significantly higher than that of the patients with other genotypes (p1 = 0.02, p2 = 0.001). CONCLUSION: This study suggests that the patients with homozygote C allele of the 102 T/C polymorphisms or homozygote A allele of the -1438 G/A polymorphism of the 5-HT2A receptor gene, have a high risk of IBS. On the other hand, T/T genotype of 102 T/C polymorphism may be associated with more severe pain in patient with IBS.     

Analysis of hematopoietic progenitor cells in patients with myelodysplastic syndromes according to their cytogenetic abnormalities

The present work analyzes the hematopoietic progenitor cells (HPC) in myelodysplastic syndrome (MDS) patients using both an immunophenotypical and a functional approaches in order to know whether they are similar in patients with or without cytogenetic abnormalities. Among CD34+ HPC, the proportion of myeloid committed progenitors was higher in patients with an abnormal karyotype. Ninety MDS patients were studied. Patients with abnormal karyotype showed a similar platting efficiency than patients with normal cytogenetics. Trisomy 8 and 5q- showed a significant higher P.E. than patients with normal karyotype or monosomy 7. We observed that when the most immature HPC were studied, the total number of granulo-monocytic colonies produced by LTBMC was higher in the normal karyotype group. In summary, the present study shows that in MDS the HPC are impaired; this impairment is deeper in patients with abnormal karyotype.     

Cytogenetic biomonitoring in children with chronic tonsillitis: micronucleus frequency in exfoliated buccal epithelium cells

OBJECTIVE: To investigate the possible harmful cytogenetic effects associated with chronic tonsillitis by analyzing the micronucleus frequency and other nuclear abnormalities in exfoliated buccal epithelial cells. MATERIALS AND METHODS: The study consisted of 20 children with chronic tonsillitis, and 20 control subjects with similar age and sex. The ages ranged between 5 and 12 years old (mean age: 7.5). The patients were diagnosed as having chronic tonsillitis on the basis of history, throat culture and clinical examinations. Buccal cell samples were collected with a wooden spatula. The samples were then applied to clean microscope slides. Smears were air dried and fixed in methanol:acetic acid. Then slides were stained by the Feulgen reaction technique. Three slides were prepared for each subject and 1000 cells were evaluated per slide to determine the frequencies of micronucleus and other nuclear abnormalities (binucleats, karyorrhexis and karyolysis). Statistically, Mann-Whitney U-test was used to analyze and compare the data. RESULTS: The mean micronucleus frequencies in patient and control groups were 5.29+/-1.67 and 1.58+/-0.33, respectively. In the patient group, mean binucleus, karyorrhexis and karyolysis frequencies were 3.13+/-1.2, 2.04+/-0.64, and 1.74+/-0.47, respectively. However, in the control group, mean binucleus, karyorrhexis and karyolysis frequencies were 1.43+/-0.47, 1.26+/-0.45, and 0.88+/-0.27, respectively. The mean frequencies of all parameters in the patient group were higher than the control values, and the difference was found to be statistically significant (p<0.001). CONCLUSION: Our results revealed that children with chronic tonsillitis could be under risk of significant cytogenetic damage.     

Transcription factor GATA-3 alters pathway selection of olivocochlear neurons and affects morphogenesis of the ear

Patterning the vertebrate ear requires the coordinated expression of genes that are involved in morphogenesis, neurogenesis, and hair cell formation. The zinc finger gene GATA-3 is expressed both in the inner ear and in afferent and efferent auditory neurons. Specifically, GATA-3 is expressed in a population of neurons in rhombomere 4 that extend their axons across the floor plate of rhombomere 4 (r4) at embryonic day 10 (E10) and reach the sensory epithelia of the ear by E13.5. The distribution of their cell bodies corresponds to that of the cell bodies of the cochlear and vestibular efferent neurons as revealed by labeling with tracers. Both GATA-3 heterozygous and GATA-3 null mutant mice show unusual axonal projections, such as misrouted crossing fibers and fibers in the facial nerve, that are absent in wild-type littermates. This suggests that GATA-3 is involved in the pathfinding of efferent neuron axons that navigate to the ear. In the ear, GATA-3 is expressed inside the otocyst and the surrounding periotic mesenchyme. The latter expression is in areas of branching of the developing ear leading to the formation of semicircular canals. Ears of GATA-3 null mutants remain cystic, with a single extension of the endolymphatic duct and no formation of semicircular canals or saccular and utricular recesses. Thus, both the distribution of GATA-3 and the effects of null mutations on the ear suggest involvement of GATA-3 in morphogenesis of the ear. This study shows for the first time that a zinc finger factor is involved in axonal navigation of the inner ear efferent neurons and, simultaneously, in the morphogenesis of the inner ear.     

Serum levels of intercellular adhesion molecule-1 and nitric oxide in patients with chronic hepatitis related to hepatitis C virus: connection fibrosis

BACKGROUND/AIMS: Chronicity of inflammation or fibrosis of liver parenchyma in patients with hepatitis C virus infection can be related to features of immunological responses in the liver. Adhesive interactions and free radicals are two important aspects of this inflammatory response. The aim of this study was to investigate serum sICAM-1 and nitric oxide levels in the sera of patients with post-hepatitis C chronic hepatitis. METHODOLOGY: Twenty chronic hepatitis patients diagnosed histopathologically and positive for antihepatitis C virus antibody and 20 healthy carriers of hepatitis C virus were included as study and control groups, respectively. Serum hepatitis C virus-RNA, sICAM-I, nitric oxide, blb, albumin and ALT determinations were made in the specimens of each subject in the study and control group. RESULTS: The mean serum sICAM-I levels of study and control subjects were 463.85 +/- 20.42 ng/mL and 241.85 +/- 13.71 ng/mL, respectively (p < 0.01). The same values for nitric oxide levels were 53.57 +/- 3.63 mumol/L and 32.17 +/- 2.19 mumol/L in the same order and also significantly different from each other (p < 0.01). There was a close correlation between fibrosis scores and serum sICAM-I levels (r = 0.77, p < 0.001), serum albumin (r = -0.54, p = 0.014) and ALT (r = 0.49, p = 0.02). Nitric oxide levels, on the other hand, negatively correlated with fibrosis scores (r = -0.59, p = 0.006), sICAM-1 (r = -0.57, p = 0.009) and ALT (r = -0.54, p = 0.013). CONCLUSIONS: Increased serum levels of sICAM are associated with increased fibrotic changes of patients with hepatitis C virus infection, whereas, nitric oxide which may be a suppressor molecule for fibrosing levels are inversely related to fibrotic process.     

The effect of L-carnitine on the prevention of experimentally induced myringosclerosis in rats

The objective of this study was to investigate the possible effect of L-carnitine on the prevention of experimentally induced myringosclerosis. Twenty Sprague-Dawley rats were bilaterally myringotomized. The rats were divided into two groups randomly: group 1 which were intraperitoneally administered saline and group 2 which were intraperitoneally administered L-carnitine. Blood samples were collected for biochemical evaluation and the tympanic membranes were harvested after 28 days. Histopathological and immunohistochemical evaluation were done under light microscopy. The mean malondialdehyde levels were 3.9+/-0.9 in group 2, and 7.9+/-1.1 in group 1 (P<0.001), nitric oxide levels were 25.6+/-6.4 in group 2 and 30.8+/-8.2 in group 1 (P=0.14) and acetylcholinesterase was 1035+/-60 in group 2 and 678+/-35 in group 1 (P=0.001). Myringosclerosis was more frequent and severe in group 1 than group 2 (P<0.007). Immunoreactivity was seen in 16 of 20 tympanic membranes in group 2 and six of 20 tympanic membranes in group 1 (P=0.005). We conclude that L-carnitine diminishes the occurrence of myringosclerosis in rats after myringotomy possibly by antioxidant activity and decreasing the formation of reactive oxygen species.