Effect of injury to the microvessels on the frequency of tumor cell lodgment in them

The action of local laser injury to the microvessels on the frequency of lodgment of tumor cells and on the ability of the cells to migrate from the lumen of the vessel was studied. A television microscope with video tape recorder and UV-laser were used. Cells of a Zajdela's ascites hepatoma were injected into the blood stream of a rat. Lodgment of tumor cells in the mesenteric vessels was observed infrequently (11%). The lodged cells adhered to the vessel wall and migrated from its lumen. Injury to the vessel wall by the laser beam led to the formation of microthrombi and to an increase in the frequency of lodging of the tumor cells in the vessels: 82% instead of 11%. The frequency of migration of cells from the lumen of the vessel under these circumstances, however, was reduced. The results confirm the role of microthrombus formation in metastasization; however, the role of microthrombus formation mav be exerted in different directions for although the frequency of adhesion of the tumor cells is increased, their mobility is reduced for a few a few hours after lodging in the microvessels.Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 7, pp. 859–861, July, 1976.     

Tissue-specific expression of the tight junction proteins claudins and occludin in the rat salivary glands

Tight junctions (TJs) are essential features of endothelial barrier membranes and of fluid-secreting epithelial cells, such as in the salivary glands. Novel integral membrane proteins have been identified as components of TJs, namely claudins and occludin. The aim of the present study was to determine the distribution of occludin and claudins in the large salivary glands of the rat. The parotid, submandibular and sublingual salivary glands were harvested from adult Sprague-Dawley rats and cryostat sections were stained using immunoperoxidase and immunofluorescence methods. Claudin-1 was expressed in endothelial cells of microvessels and in short selected segments of the duct system. Claudin-3 was expressed principally in the acinar cells and intercalated ducts, while claudin-4 was principally expressed by the striated and interlobular ducts. Claudin-5 was specific to endothelial cells of microvessels. Occludin was ubiquitously detected in the duct system. Double labelling and confocal microscopy showed some co-localization of claudin-3 with claudin-4, and minimal co-localization of occludin with claudin-4, in the striated ducts. Claudin 2 was not detected in any of the salivary glands. The results indicate specificity of the chemical composition of tight junctions in the rat salivary glands, and may reflect different physiological roles for TJs in the glandular and duct epithelial cells, and in endothelial cells of salivary gland microvessels.     

Bronchopulmonary Dysplasia: A Correlative Study by Light, Scanning, and Transmission Electron Microscopy

The sequential stages of bronchopulmonary dysplasia occurring in 18 infants after intensive respiratory therapy supplemented by oxygen in high concentrations were studied by correlative light, scanning, and transmission electron microscopy. Infant survival ranged from 3 to 225 days. The earliest stage was an exudative reaction with a predominance of hyaline membranes. This merged with a subacute reparative response that was replaced by a chronic fibroproliferative stage in infants of longest survival; this stage was complicated by pulmonary fibrosis and emphysema. Correlative scanning and transmission electron microscopy demonstrated that type 2 pneumocytes contributed significantly to the reparative fibroproliferative response by organization of hyaline membranes and reepithelialization of damaged septal walls.     

Kupffer cell-mediated cytotoxicity against hepatoma cells occurs through production of nitric oxide and adhesion via ICAM-1/CD18

Rat Kupffer cell (KC)-mediated cytotoxicity against both thesyngeneic hepatoma cell line AH70 and hepatocytes was evaluatedby changes in mitochondrial function, and the possible roleof ICAM-1/CD18 in the interaction between the cells was studied.Rhodamine 123 fluorescence, a marker of the mitochondrial membranepotential, decreased in AH70 cells after co-culture with KC,while that in hepatocytes was unchanged by co-culture. Thisdecrease was blocked by anti-ICAM-1, anti-CD18 and the Inhibitionof nitric oxide synthesis. Cytometric studies demonstrated thatICAM-1 expression on AH70 cells increased after addition ofIFN-, IL-1ß, tumor necrosis factor (TNF)- or KC, whilein hepatocytes ICAM-1 was not increased. Anti-ICAM-1 pretreatmentinhibited the increase in ICAM-1 expression and the decreasein rhodamine 123 fluorescence on AH70 cells after co-culturewith KC. CD18 on KC was increased only after co-culture withAH70. TNF- but not IFN- was detected in the supernatant of co-culturebetween KC and AH70 cells, and this production was partiallyinhibited by anti-ICAM-1 and anti-CD18. The activity of Induciblenitric oxide synthase in Kupffer cells and the levels of nitritesand nitrates in the co-culture supernatant increased over time,and this increase was attenuated either by addition of NO synthesisinhibitors, anti-ICAM-1 or anti-CD18. These results indicatethat the rat KC causes mitochondrial dysfunction in cancer cellsvia the production of NO and cell-to-cell adhesion via ICAM-1/CD18has an Important role in this cytotoxic process.     

Autocrine motility factor (neuroleukin, phosphohexose isomerase) induces cell movement through 12-lipoxygenase-dependent tyrosine phosphorylation and serine dephosphorylation events

Autocrine motility factor (AMF) is one of the motility cytokines regulating tumor cell migration, therefore identification of the signaling pathway coupled with it has critical importance. Previous studies revealed several elements of this pathway predominated by lipoxygenase-PKC activations but the role for tyrosine kinases remained questionable. Motility cytokines frequently have mitogenic effect as well, producing activation of overlapping signaling pathways therefore we have used B16a melanoma cells as models where AMF has exclusive motility effect. Our studies revealed that in B16a cells AMF initiated rapid (1–5 min) activation of the protein tyrosine kinase (PTK) cascade inducing phosphorylation of 179, 125, 95 and 40/37 kD proteins which was mediated by upstream cyclo- and lipoxygenases. The phosphorylated proteins were localized to the cortical actin-stress fiber attachment zones in situ by confocal microscopy. On the other hand, AMF receptor activation induced significant decrease in overall serine-phosphorylation level of cellular proteins accompanied by serine phosphorylation of 200, 90, 78 and 65 kd proteins. The decrease in serine phosphorylation was independent of PTKs, PKC as well as cyclo- and lipoxygenases. However, AMF induced robust translocation of PKCα to the stress fibers and cortical actin suggesting a critical role for this kinase in the generation of the motility signal. Based on the significant decrease in serine phosphorylation after AMF stimulus in B16a cells we postulated the involvement of putative serine/threonine phosphatase(s) upstream lipoxygenase and activation of the protein tyrosine kinase cascade downstream cyclo- and lipoxygenase(s) in the previously identified autocrine motility signal. This revised version was published online in July 2006 with corrections to the Cover Date.     

The atopy patch test -- reproducibility and comparison of different evaluation methods

BACKGROUND: There is still a lack of standardization of the atopy patch test (APT) in test procedures and evaluation methods. Our aim was to examine the reproducibility of APT results and to compare visual evaluation to chromametry and laser Doppler imaging. METHODS: Fifty-two volunteers with atopic eczema/dermatitis syndrome (AEDS) were included. The APT was performed on tape-stripped and unstripped test fields on their backs using cat dander, house dust mite and grass pollen allergens from two different suppliers. Responders were re-tested 4-12 weeks later with the same allergens on their forearms. RESULTS: Using Allergopharma allergens, 14 (26.9%) volunteers showed one or more positive reactions. The reproducibility rate was 56.3%. The Erlangen atopy score in APT-positive and negative volunteers was 19 +/- 6 vs 15 +/- 6. The test agreement in volunteers tested with both allergens, from Allergopharma and Stallergènes, was poor. Correlation of the results between the three evaluation methods was significant (P < or = 0.001). CONCLUSIONS: The low reproducibility rate of APT results and the poor inter-test-agreement using allergens from different suppliers show that much work remains to make the APT a reliable tool in identifying relevant aeroallergens that lead to flare ups of AEDS. Compared to chromametry and laser Doppler imaging, visual scoring was superior in differentiation between irritative and allergic reactions.     

Biomedical applications of tunable diode laser spectrometry: Correlation between breath carbon monoxide and low level blood carboxyhemoglobin saturation

Tunable diode laser spectrometry was used to measure breath carbon monoxide (CO) concentrations in nonsmoking subjects. Corresponding blood carboxyhemoglobin (COHb) saturations between physiological levels and levels after 1 and 8 hours exposure to 9 ppm CO, the maximum permissible concentration in the air under federal regulations, were also determined. A good linear correlation between breath CO and blood COHb saturation for low levels representing ambient conditions was established, with a correlation coefficient better than 0.999 for the means and 0.945 for the individual data points. Similar studies on the other compounds of concern are also possible using tunable diode laser spectrometry, requiring only a breath sample and with no sample preparation. This technique will facilitate fast, noninvasive environmental health effect and biomedical studies.     

DGGE-based whole-gene mutation scanning of the dystrophin gene in Duchenne and Becker muscular dystrophy patients

Duchenne and Becker muscular dystrophy (DMD and BMD) are caused by mutations in the dystrophin gene. Large rearrangements in the gene are found in about two-thirds of DMD patients, with approximately 60% carrying deletions and 5-10% carrying duplications. Most of the remaining 30-35% of patients are expected to have small nucleotide substitutions, insertions, or deletions. To detect these subtle changes within the coding and splice site determining sequences of the dystrophin gene, we established a semiautomated denaturing gradient gel electrophoresis (DGGE) mutation scanning system. The DGGE scan covers the dystrophin gene with 95 amplicons, PCRed either individually or in a multiplex setup. PCR and pooling were performed semiautomatically, using a pipetting robot and 384-well plates, enabling concurrent amplification of DNA of four patients in one run. Amplification of individual fragments was performed using one PCR program. The products were pooled just before gel loading; DGGE requires only a single gel condition. Validation was performed using DNA samples harboring 39 known DMD variants, all of which could be readily detected. DGGE mutation scanning was applied to analyze 135 DMD/BMD patients and potential DMD carriers without large deletions or duplications. In DNA from 25 out of 44 DMD patients (57%) and from 5 out of 39 BMD patients (13%), we identified clear pathogenic changes. All mutations were different, with the exception of one DMD mutation, which occurred twice. In DNA from 10 out of 44 potential DMD carriers, including four obligate carriers, we detected causative changes, including one pathogenic change in every obligate carrier. In addition to these pathogenic changes, we detected 15 unique unclassified variants, i.e., changes for which a pathogenic nature is uncertain.