Nicotine induces endothelial TNF-alpha expression, which mediates growth retardation in vitro.

PURPOSE: Atherosclerosis is understood as the common pathologic manifestation of arterial injury caused by a variety of etiologies. One well-established etiologic agent is nicotine. We hypothesized that cytokines of endothelial origin are involved with the pathologic changes found in atherosclerosis associated with smoking. We chose to assay for TNF-alpha due to its many biologic actions that are similar to those found in peripheral vascular disease. METHODS: Human umbilical vein endothelial cells (HUVEC) were plated in endothelial growth medium (EGM-2) on plastic coverslips until 75% confluent. Free base nicotine (FBN) was diluted in EGM-2 to a concentration of 10(-8) M and added to experimental cells. At 1, 3, and 24 h, coverslips were removed and fixed. Immunohistochemical staining was performed using anti-TNF-alpha. Digital image analysis (DIA) was performed to quantify expression of TNF-alpha. An intensity stain index measuring area and intensity of stain/total cellular area was determined for each time point (n = 5). Additional HUVEC were plated in 12-well plates in EGM-2 at 2 x 10(3) cells/cm(2) on T(-2) day. FBN was diluted in medium to 10(-9) M and added to wells with and without 0.9 microg/ml anti-TNF-alpha on T(0) day. Cell counts were performed in triplicate on days T(2-5) utilizing hemocytometry. Data was analyzed using Student's t test and ANOVA, with a 95% confidence interval. RESULTS: Dose response determinations showed that the minimal concentration required to show statistically significant cell retardation is 10 (-9) M. Accordingly, this concentration was used for subsequent proliferation studies. DIA showed a threefold increase in TNF-alpha activity at 1 h and a twofold increase at 3 h. Activity returned to baseline by 24 h. Cell growth was significantly decreased in cells exposed to nicotine when compared to controls on days T(2)-T(5) (P < 0.05). In cells exposed to anti-TNF-alpha and nicotine there was inhibition of the growth retardation seen in the cells containing nicotine alone. Differences between the control group and the anti-TNF-alpha group were not statistically significant. CONCLUSION: These data demonstrate the ability of endothelial cells to secrete TNF-alpha in response to nicotine at levels found in serum after smoking and also shows that endothelial cell growth retardation as a consequence of nicotine exposure may be TNF-alpha mediated.

Adrenocortical carcinoma in two female children.

Adrenocortical carcinoma is a rare tumor in children. This tumor is more likely to be hormonally active in children than in adults and tends to cause a variety of symptoms, which may mimic other benign endocrinopathies. These tumors are usually diagnosed at advanced stages and portend a dismal prognosis. We describe two cases of adrenocortical carcinoma. One child presented with Cushingoid symptoms secondary to hypercortisolism, including amenorrhea, hirsutism and weight gain. The other child presented with precocious puberty. Both children underwent resection of the tumors. We describe their presenting symptoms, postoperative course, adjuvant therapy and clinical course. Pertinent literature regarding the anatomy of the adrenal gland, pathology of adrenocortical carcinoma, factors influencing outcome, diagnostic modalities and treatment, are discussed.

Age-adjusted outcomes in traumatic flail chest injuries in the elderly.

Severe chest trauma does not independently predict poor outcome in elderly patients. We chose a specific injury, flail chest, to determine whether age factored into outcome of these patients. A retrospective chart review of all trauma admissions to our Level I trauma center between January 1994 and January 1998 sustaining flail chest was undertaken. Sixty-eight patients were identified, but ten patients were excluded because of death on arrival. Fifty-eight patients were included in the study and separated into groups. The first group comprised those under the age of 55 (n = 32) and the second comprised those over age 55 (n = 26). Parameters evaluated were age, Injury Severity Score (ISS), neurologic injury, the need for mechanical ventilation, need for tracheostomy, length of stay, and death. Statistical analysis was performed with Wilcoxon t test, chi2, and logistic regression where appropriate. A 95 per cent confidence interval was sought as determinant of significance. Of the 58 surviving patients analyzed there was no significant difference between the groups regarding ISS, length of stay, days on the ventilator, head injury, tracheostomy, or development of pneumonia or adult respiratory distress syndrome. The likelihood of death was shown to increase by 132 per cent for every 10 years starting at the second decade and continuing to the eighth decade of life. The likelihood of death also increased by 30 per cent for each unit increase in ISS. The likelihood of death decreased by 23 per cent for every day survived in the hospital. Blunt chest trauma directly impacts respiratory mechanics. Elderly patients are more likely to have comorbid conditions and less likely to tolerate traumatic respiratory compromise. Age (and its effects on the body) is the strongest predictor of outcome with flail chest and is associated with an increased mortality (P < or = 0.05).

Characterization of beta-carotene 15,15'-dioxygenase activity in TC7 clone of human intestinal cell line Caco-2.

The purpose of this study was to identify mammalian cell line(s) which possess intrinsic enzymatic activity of beta-carotene 15, 15'-dioxygenase. This enzyme (EC1.13.11.21) converts beta-carotene to retinal (precursor of retinol and retinoic acid). To assess activity, cellular enzyme preparations were incubated with beta-carotene for 60 min; retinal formed was quantified by HPLC. Activity was not detected in IPEC-1, HepG2, HL60, Wurzburg, or parent Caco-2 cell lines. However, two subclones of Caco-2, PF11 and TC7, possessed activity (2.5 and 14.7 pmol/h.mg, respectively). Using the enzyme preparation of TC7 cells, retinal formation was linear with incubation time and protein concentration; Km and Vm values were 1.6 microM and 23.8 pmol/h.mg, respectively. In addition, when TC7 cells were maintained in serum-free medium, activity was increased 8.2-fold after 19 days of postconfluency. Finally, 48 h incubation with beta-carotene (delivered to TC7 cells in Tween 40) resulted in a 1.7-fold increase of dioxygenase activity and the appearance of vitamin A (9.3 pmol/mg protein). However, retinoic acid was not detected under our experimental conditions. In sum, the TC7 subclone of the Caco-2 cell line possesses beta-carotene 15, 15'-dioxygenase activity and thus can be useful in future investigations of human carotenoid metabolism.

Isolation of exfoliated colonic epithelial cells, a novel, non-invasive approach to the study of cellular markers.

Human stool is a heterogeneous mixture of non-digestible food residues, bacteria, cells exfoliated from the gastrointestinal mucosa and other secretory products. We have demonstrated that fresh human stools dispersed in a buffered saline solution can be fractionated over Percoll/BSA gradients to yield 9 discrete bands of cells in the density range of rho 1.033 to 1.139 and which could be further purified over Histopaque 1077. Enzyme-linked immunoassays (ELISA) for colon-specific antigen (CSA) and cytokeratins (CK) were positive. Western blot analysis showed the presence of 3 cytokeratin bands in the 40-kDa to 60-kDa range suggestive of cytokeratins 8, 18, and 19. Fluorescence flow-cytometric analysis of these cells using antibodies against CSA, CK, the blood-group antigens, carcinoembryonic antigen (CEA), non-mucus-secreting columnar-epithelium-specific MAb PR1A3, and to mucus-secreting colonic-epithelium-specific MAb PR5D5 showed varying degrees of reactivity. Expression of the blood-group phenotype suggests that cells from the proximal half of the colon had survived the transit, since in the adult expression of this marker is limited to cells from the proximal region of the colon. In this report we demonstrate the feasibility of studying, non-invasively, cell-specific markers on exfoliated cells isolated from stools. The evidence strongly suggests that almost all the cells are of colonic origin.

Human stools as a source of viable colonic epithelial cells.

Human stools consist of a mixture of undigested food residues, colonic microflora, and cellular components shed from the walls of the gastrointestinal tract. The cellular components are made up mostly of terminally differentiated colonic epithelial cells. Using a combination of Percoll density gradient centrifugation and countercurrent centrifugal elutriation, it is now possible to recover these cells as an enriched fraction from fresh human stools. Cells can be visualized on heat-fixed smears of the enriched fractions stained with modified Wright's stain. The enrichment process is optimized by following the segregation of eukaryotic cells as determined by an ELISA technique using monoclonal antibodies against human double-stranded DNA. This work, demonstrating the feasibility of isolating intact colonic cells from stools, has important applications as a noninvasive approach to the biology of exfoliated cells from the gastrointestinal tract.

Regulation of the expression of α-amylase gene by sodium butyrate.

Sodium butyrate exerts a pronounced inhibition on the gibberellic acid-induced synthesis and secretion of α-amylase by aleurone cells of barley seeds. This inhibition, which is reversible and non-competitive with cespect to gibberellic acid, is concentration dependent, with virtually total inhibition being accomplished between 4 and 5 mM sodium butyrate. The pattern of inhibition of α-amylase formation correlates well with a decrease in the accumulation of its messenger RNA. The addition of butyrate 12 h after the addition of gibberellic acid to half-seeds, has no effect on the formation and secretion of α-amylase. It has been shown in earlier studies that the synthesis of α-amylase mRNAs takes about 12 h for completion. The conclusion that butyrate interferes with some step in the transcriptional process is supported by a decrease observed in the RNAs that hybridize with a cloned α-amylase cDNA. The results of in vitro translation confirm the inhibition of the formation of several translatable mRNAs. Further, immunological probing detected only trace amounts of α-amylase proteins in the secretion of butyrate-treated seeds. Translation of functional mRNAs, post-translational modifications and the secretion α-amylase are not affected by sodium butyrate. It is concluded that butyrate selectively inhibits the transcription of several genes that are under the influence of gibberellic acid. This report is the first one documenting the inhibitory effect of sodium butyrate on a hormone-induced synthesis and accumulation of mRNAs in a plant system.

Modulation by abscisic acid and S-2-aminoethyl-L-cysteine of α-amylase mRNA in barley aleurone cells.

The changes in the levels of α-amylase mRNA is barley aleurone layers in response to addition of plant growth regulators have been studied using a cloned α-amylase cDNA as the hybridization probe. An increase in gibberellic acid (GA) concentration in the incubation medium from 10(-9) M to 10(-6) M results in a progressive increase in α-amylase mRNA concentration in the aleurone cells. Detectable levels of α-amylase mRNA appear in the aleurone cells as early as 1 h after addition of GA. The concentration of this mRNA increases for several hours and then declines rapidly. Abscisic acid (ABA) and the amino acid analog S-2-aminoethyl-L-cysteine (AEC) suppress the GA-mediated induction of α-amylase. These compounds appear to affect the level of α-amylase mRNA in aleurone cells as measured byin vitro translation assays and by analysis of RNA blots with α-amylase cDNA probes. It is concluded that the regulation of α-amylase gene expression by ABA is at the level of transcription. Further, a protein factor appears to be required in addition to GA for transcription of α-amylase genes.