Protective cytotoxic T lymphocyte responses against paramyxoviruses induced by epitope-based DNA vaccines: involvement of IFN-gamma

Plasmid DNA vectors have been constructed with minigenes encoding a single cytotoxic T lymphocyte (CTL) epitope from either the M2 protein of respiratory syncytial virus (RSV) or from the nucleoprotein of measles virus (MV) with or without a signal sequence (also called secretory or leader sequence). Following intradermal immunization, plasmids in which the CTL epitopes were expressed in-frame with the signal sequence were more effective at inducing peptide- and virus-specific CTL responses than plasmids expressing CTL epitopes without the signal sequence. This immunization resulted in protection against MV-induced encephalitis and a significant reduction in viral load following RSV challenge. The reduction of viral load following RSV challenge was abrogated by prior injection with anti-IFN-gamma antibodies. These results highlight the ability of epitope-based DNA immunization to induce protective immune responses to well-defined epitopes and indicate the potential of this approach for the development of vaccines against infectious diseases.     

Endothelin-induced inositol phosphate formation in rat kidney. Studies on receptor subtypes, G-proteins and regulation during ontogenesis

The aim of this study was to characterize the properties of endothelin (ET)-receptor subtypes mediating inositol phosphate (IP)-formation in rat kidney and their regulation during ontogenesis. In renal cortical slices of adult rats (12-16 weeks old) ET's concentration-dependently increased IP-formation with an order of potency ET-1 > ET-3. While the non-selective ET-receptor antagonist bosentan (10 microM) completely suppressed ET-induced IP-formation, the ETA-receptor antagonist BQ-123 (10 microM) inhibited it only by 70%, the ETB-receptor antagonist IRL 1038 (1 microM) by 25%; combined application of BQ-123 + IRL 1038 caused complete inhibition of ET-1-induced IP-formation. Pretreatment of isolated renal cells with pertussis toxin (PTX, 500 ng/ml) overnight did not attenuate but significantly increased ET-1-induced IP-formation. Ontogenetic studies in renal sites from neonatal, 1, 2, 3, 6, 12 and 24 weeks old rats revealed that ET-1-induced IP-formation maturation-dependently declined being highest in neonatal rats (increase: 169% over basal) and lowest in 24 weeks old rats (increase: 47% over basal). This decline in ET-induced IP-formation was accompanied by a decrease in renal ET-receptor number and the amount of immunodetectable Gq/11 (assessed by Western-blotting using the QL-antiserum). Moreover, ET-receptor subtypes changed during the maturation process: from neonates to 12 weeks old rats number and functional responsiveness of ETA-receptors declined, while that of ETB-receptors increased. We conclude that in adult rat renal cortex ET-induced IP-formation is mediated by activation of both ETA- and ETB-receptors and does not involve a PTX-sensitive G-protein. ET-induced IP-formation declines during the maturation process; this is associated with a decrease in ET-receptor number and the immunodetectable amount of Gq/11.     

Clinical diagnosis of deep venous thrombosis after hip replacement surgery

Hip replacement surgery is associated with a high frequency of postoperative deep vein thrombosis. This prospective study was performed in order to investigate if routine bedside questioning and examination by the visiting doctor could reveal deep vein thrombosis in the legs of patients who had received a hip replacement. 258 patients were evaluated. Thromboprophylaxis (dextran-70, low molecular weight heparin and graded elastic stockings) was given during the first week after operation. Bilateral venography was performed in all patients on day seven after operation, and showed an overall deep vein thrombosis incidence of 16%. The visiting doctors had not suspected deep vein thrombosis in any of the patients. This may have been because postoperative painful and swollen legs effectively masked any signs and symptoms of deep vein thrombosis. Our results show that deep vein thrombosis during the first week after hip replacement surgery cannot be discovered by clinical diagnostics. The high subclinical frequency of deep vein thrombosis indicates the importance of improving thromboprophylaxis in order to further minimise the occurrence of deep vein thrombosis and the risk of thromboembolic complications.     

Flow cytometry as a sensitive tool to assess testicular damage in rat

The aim of this study was to compare results obtained by flow cytometry (FCM) with those obtained from testicular histopathology with regard to testicular damage following acute exposure of adult rats to the known testicular toxicant, methoxyacetic acid (MAA). Special emphasis was given to defining the sensitivity of three-parameter FCM compared with testicular histopathology. Furthermore, the effect on the male reproductive system of a single oral dose of MAA was evaluated with traditional methods, e.g. testicular sperm head counts, and organ weights. Adult, male Han/Wistar rats were randomly assigned to four groups of ten animals to be treated with a single dose of 0, 65, 325 and 650 mg MAA/kg body wt. (p.o., gavage). The animals were killed 2 days after treatment, and testicular and epididymal weights were recorded. One testis and the corresponding epididymis were used for histopathology. The other testis was used partly to determine sonication-resistant, testicular sperm-head counts (SHC), and partly for enzymatic digestion followed by FCM. The results obtained in this study are in agreement with the literature, and show that, in the adult male rat, 2 days after administering a single oral dose of MAA, specific depletion of spermatocytes is evident. Detectable testicular effects were produced by the high (650 mg/kg body wt.) and mid (325 mg/kg body wt.) doses, whilst the low dose (65 mg/kg body wt.) did not produce any noticeable effect. There was a strong correlation between results obtained by FCM and those obtained by testicular histopathology, and no difference in sensitivity between the two methods was observed. In summary, three-parameter FCM represents a sensitive and reliable method for the detection of testicular injury in the rat. It requires only small amounts of tissue, and the sensitivity was shown to be similar to that of histopathology. Moreover, FCM has the advantages of being quick and objective, which permits large numbers of cells to be analysed. The potential use of this method as a fast screening tool for testicular toxicity in routine toxicology studies should be considered.     

The impact of industrial factors of oil processing plant on serum lipoproteins

The study covered serum lipoproteins in workers engaged into production of higher fatty alcohols at oil-processing plant. The industrial factors appeared to induce changes in serum lipoproteins composition: lower share of High Density Lipoproteins and higher share of Very Low Density Lipoproteins. On the background of cardiovascular and gastrointestinal diseases, the occupational hazards may precipitate and aggravate alterations of serum lipoproteins composition in the workers.     

The luminescence immunoassay S-100: a sensitive test to measure circulating S-100B: its prognostic value in malignant melanoma

In this study we measured S-100B using a recently developed luminometric immunoassay with a detection limit of 0.02 microg l(-1). By measuring serum S-100B concentrations in 58 apparently healthy individuals a reference value of 0.16 microg l(-1) was found. To assess the sensitivity of the assay we measured levels of S-100B protein in the serum of 251 patients with cutaneous malignant melanoma before the start of treatment. Only one of 179 patients with limited disease had a serum concentration higher than the reference value, whereas elevated levels were seen in 79% of patients with metastasized disease. In the latter group the NSE serum concentration was elevated in 42%. Using a receiver operating characteristic (ROC) curve it is shown that S-100B is a significantly better parameter than neuron-specific enolase (NSE) for distinguishing patients with limited disease from those with extensive melanoma. Pretreatment S-100B values were highly predictive for the period of survival. Patients with limited disease have increased risk for early death with increasing levels of S-100B protein. Within the group of patients with positive lymph nodes and/or with distant metastases, elevated S-100B levels strongly identified high-risk patients. Our study indicates that the measurement of S-100B as a tumour marker in the management of patients with cutaneous malignant melanoma has clinical significance.     

Papillary thyroid carcinoma oncogene (RET/PTC) alters the nuclear envelope and chromatin structure

Current evidence suggests the papillary thyroid carcinoma oncogene (RET/PTC) generates papillary thyroid carcinomas in one genetic step. We tested a resulting prediction that RET/PTC expression in thyroid epithelium should be sufficient to cause the changes in nuclear morphology diagnostic of this tumor. Primary cultures of human thyroid epithelial cells were infected with a RET/PTC retroviral construct. Morphological scoring by two independent cytopathologists shows RET/PTC expression by immunohistochemistry to be highly associated (p < 0.0001) with an irregular nuclear contour and a euchromatic appearance compared with non-expressing cells in the same cultures. The altered nuclear morphology is not due to gene transfer or transformation per se as primary thyroid cell cultures infected with a retroviral H-RAS construct differ from RET/PTC-infected cells by showing round nuclear envelopes and coarser chromatin, as determined by the independent scoring of two cytopathologists (p < 0.0001). In addition, RET/ PTC-transfected cells appear to disperse, whereas RAS-transfected cells grow as discrete colonies. The results provide additional support for the hypothesis that RET/PTC is sufficient to cause papillary thyroid carcinomas. A signaling pathway downstream of RET/ PTC leads to restructuring of the nuclear envelope and chromatin, and the signal does not depend entirely, if at all, on a RAS pathway.     

Karyotypic anomalies and chromosomal sites of increased fragility in colorectal cancer

Karyotypic structural aberrations in tumor cells and chromosome constitutive fragile sites (cFSs) in peripheral blood lymphocytes were studied in ten patients with colorectal adenocarcinoma Most chromosome breakpoints (38 out of 40, i.e., 95.0%) were shown to be located within cFSs in tumor cells. Expression of 24 out of 137 cFSs in patients was higher than that in healthy donors. Four of these cFSs (6q26, 7q36, 16q23, and 17q21), were involved in the formation of nonrandom tumor cell chromosome markers most characteristic of colorectal neoplasms. The frequency of damages induced within these sites was analyzed in each patient. Expression of 7q36, 16q23, and 17q21 was increased in blood cells of patients carrying specific chromosome rearrangements with the breakpoints within these sites. The association between nonrandom chromosome aberrations in tumor cells and cFSs in normal cells is discussed.