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L Strande

Publications and source records attributed to L Strande.

11 recordsLinked to original sources
PubMed2001

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.

Arteriosclerosis↗
PubMed1999

Effects of microgravity on growing cultured skin constructs.

Understanding the cellular, chemical, and physical responses of cells to stimuli is critical to successfully engineering tissue. The effect of culturing a living skin equivalent (LSE) in a submerged microgravity environment was investigated. LSEs were developed by culturing normal human epidermal keratinocyte (NHEK) on a submerged fibroblast and type 1 collagen gel matrix. Once formed, LSEs were brought up to the air/liquid interface, and after 4 days, the cultures were maintained in either (a) a normal air/liquid interface (S), (b) resubmerged in media (R), (c) folded on themselves to enclose the keratinized layer (F/R), or (d) cut into 2-4-mm fragments and suspended in a state of microgravity in a NASA-designed bioreactor (B). All groups were cultured for an average of 3 additional days. LSEs were processed for histologic evaluation. Skin cells were stained for cytokeratin to evaluate function. Images were digitally captured and processed for analysis. Parameters, including epithelial thickness, cellular areas, nuclear number, nuclear area, cytoplasmic area, and stained cytokeratin areas were measured. Removing the air/media interface significantly increased the number of NHEKs present in the skin; microgravity greatly enhanced this effect (p < 0.0001). No significant difference in cellular function as measured by protein expression [stained cytokeratin area (micro(2)) per cell] was found among the groups, though the ratio of nuclear area was significantly increased in all three groups as compared to the S group (p = 0.00227). In the case of the R and F/R groups, this appears due to the loss of the NHEK layer associated with those groups. Additionally, significant nuclear hypertrophy was demonstrated in the B group (p < 0.0001), and cellular hyperplasia was measured in all submerged groups as compared to static (p < 0.0001). Elimination of the air/liquid interface enhanced proliferation of keratinocytes. This effect was further enhanced in the presence of microgravity. No significant effect on cell function was noted with the use of this microgravity environment. We hypothesize that the increased epidermal contact plays a role in this proliferation. Microgravity is also associated with nuclear and cellular hypertrophy over and above that of the submersion methods.

Air↗
PubMed1998

Induction of apoptosis in a neuroblastoma and hepatocyte coculture model.

BACKGROUND: The dysregulation of apoptosis may alter the progression of tumor growth and explain the clinical dichotomy observed in children with neuroblastoma (NB). An overexpression of the bcl-2 proto-oncogene induces resistance to apoptosis and has been observed in unfavorable NB. We hypothesized that alterations in apoptosis may be a result of the interactions between NB and the tissues surrounding it. MATERIALS AND METHODS: Human Chang liver cells (HCL, 10(4) cells/cm2) were plated in two-chamber slides for 3 days. Human NB cells (10(5) cells/cm2) were added to one of the chambers and incubated for 3 more days. Control NB were plated under identical conditions in its own medium and in the HCL medium with growth curves measured. DNA fragmentation was detected via the TUNEL method (TdT-mediated nick end-labeling) and bcl-2 expression was determined by immunostaining. RESULTS: NB growth was unaltered by the change in medium. NB stained mildly positive for bcl-2 when plated alone but became markedly positive in coculture. Histologically, HCL and NB appeared healthy when plated alone, but a halo of apoptotic HCL was seen around NB in the coculture. When plated alone, both NB and HCL demonstrated minimal apoptotic activity as detected via the TUNEL method. In the coculture, a halo of HCL surrounding the NB exhibited markedly increased DNA fragmentation and this intensity diminished in cells distant from the NB. CONCLUSIONS: The regulation of apoptosis was altered in this coculture model of NB and HCL. HCL stimulated NB to overexpress bcl-2 and presumably become resistant to apoptosis. Conversely, NB induced the surrounding HCL to undergo apoptosis. The interaction between the local tissue and NB induced alterations in apoptosis in both cell types and resulted in a survival advantage for NB.

Apoptosis↗
PubMed1998

A novel in vitro model for xenorejection and immune mechanisms using bioengineered living skin equivalents.

We hypothesized that an in vitro bioengineered skin (LSE) could be used for the study of xenogeneic inflammatory or immunosuppressive mechanisms. Murine fibroblasts (10(4)/mL) were mixed with type 1 rat-tail collagen to form a matrix (approximately 5 days) on which human keratinocytes (NHEK, 10(5)/75 microL) were seeded. The xeno-LSE was used as a rejection target organ in vitro. Rejection was achieved by the addition of human lymphocytes (10(6)/75 microL). At various intervals of growth, control LSEs without lymphocytes (CON) and rejecting LSEs with immunocytes (REJ) were analyzed. Slides were stained with hematoxylin & eosin and examined under light microscopy. REJ xenocomposite LSEs showed classic histologic signs of rejection. There was separation of the D-E junction with the presence of dyskeratotic and necrotic cells with a concomitant inflammatory infiltrate. The CON LSEs showed essentially normal dermis (collagen fibroblast matrix) and keratinocytes. A significant finding was the separation of the D-E junction in the absence of an inflammatory infiltrate. Previous studies have shown that human keratinocytes can be grown with human fibroblasts that are histologically similar to natural human skin. This discohesive nature of the murine fibroblasts and human keratinocytes may represent an important intercellular interaction associated with xeno-cellular interactions and is worthy of further study. Furthermore, the ability to grow xeno-composite tissues has profound implications in the face of organ donor shortages. They may represent an eventual in vitro replacement of skin and even solid organs.

Adult↗
PubMed1997

Rat renal allograft tolerance is associated with local TGF-beta and absence of IL-2r expression within chimeric immunocytic foci.

Tolerance was induced in Lewis (LEW) rat renal allograft recipients of Brown Norway kidneys by multiple pretransplant donor-blood transfusions and prior limited cyclosporine A. Rat renal allograft tolerance was associated with the induction of systemic donor T cells (10%), an early phase of nonspecific suppressor-cell generation, followed by maturation of systemic antigen-specific suppressor cells, and renal cellular infiltrates that develop long-term in situ in the kidney graft model. It was hypothesized that these infiltrates represent chimeric immunocytic foci that are locally regulated via a TGF-beta-dependent mechanism. Both immunohistochemical staining and digital image analysis for cellular and extracellular TGF-beta, IL-2 receptor (CD25), and the BN Class I-MHC marker (OX-27) were performed. Control rejecting (REJ) kidneys did not demonstrate any differences with respect to levels of infiltrating immunocyte area vs long-term surviving (TOL) kidneys (3.9% vs 4.5%, P = .303). Immunostaining with the BN Class I MHC marker (OX-27) demonstrated high levels of chimerism within immunocyte foci of the tolerant grafts (OX-27 BN+immunocytes 49.0% +/- 5.1%). In situ cellular IL-2 receptor (CD25) expression was demonstrated in REJ kidney infiltrates but not within TOL immunocytic infiltrating foci, when measured as percent of total lymphocytes (REJ = 5.0% vs TOL = 0.4%, P = .031). Conversely, TGF-beta expression was significantly higher in immunocytes of TOL kidneys when measured as the number of DAB chromogen-staining pixels per total immunocyte area (TOL = .076 vs REJ = .047, P = .003). In conclusion, these results suggested that stable mixed immune chimerism (SMIC) plays an important role in DST-CyA-induced tolerance in situ. SMIC-induced tolerance may involve a local TGF-beta-dependent mechanism that is associated with in situ TGF-beta (+) and IL-2r (-) immunocytes.

Animals↗
PubMed1996

Beneficial effects of cyclosporine and rapamycin in small bowel ischemic injury.

Gut ischemia has been implicated in the pathogenesis of necrotizing enterocolitis. Cyclosporine A and rapamycin, both potent novel immunosuppressants which act on signal transduction pathways in CD4+ T-cells, could potentially modulate immune/inflammatory cellular reactions involved in tissue ischemia/reperfusion injury. We hypothesized that cyclosporine A and rapamycin would preserve mucosal cell function and attenuate inflammatory T-cell-mediated cellular changes associated with small bowel ischemic injury. Forty Sprague-Dawley rats underwent 60 min of gut ischemia by vascular occlusion of the superior mesenteric vessels. Animals were randomized to four groups (n = 10): cyclosporine A (CSA, 5 mg/kg/day SQ), rapamycin (RAP, 2 mg/kg/day SQ), cyclosporine A and rapamycin (C&R), and vehicle given to controls (CON). Following 1 hr of reperfusion, small bowel was harvested for xanthine oxidase (XO, units/mg protein) and maltase (MALT, mM substrate degraded/min/g protein) assays. Blood was obtained from the portal vein for tumor necrosis factor-alpha (TNF-alpha, pg/ml) assay. The results of the study are presented below (mean +/- SEM, *, P < 0.05 versus controls). (Table in text) The results indicate that cyclosporine and rapamycin each play a significant role in attenuating ischemia/reperfusion injury in the gut. These data suggest that there are cytoprotective and anti-inflammatory mechanisms of these drugs independent of T-cell signal transduction that provide some protective effect in small bowel ischemia. Furthermore, T-cell-mediated immune mechanisms may not be associated with the adverse effects of small bowel ischemia/reperfusion injury. Additional investigation will be necessary in order to define the role of T-cell-mediated immune injury in the gut and how this relates to the beneficial effect of immunosuppression in small bowel mucosal ischemic injury.

Animals↗
PubMed1996

The effect of cyclosporine in gut ischemic injury: a computerized morphometric and enzymatic analysis.

PURPOSE: Gut ischemia has been implicated in the pathogenesis of necrotizing enterocolitis. Cyclosporine A (CSA), a potent immunosuppressant, attenuates immune/inflammatory cellular reactions. CSA also might be useful for inhibiting cellular immune responses involved in tissue ischemia/reperfusion injury. The authors hypothesized that CSA would attenuate inflammatory cellular changes associated with gut ischemic injury and that these effects could be quantified by computerized morphometry. METHODS: Twenty Sprague-Dawley rats underwent 60 minutes of gut ischemia by vascular occlusion of the superior mesenteric vessels. After 1 hour of reperfusion, the ischemic small bowel was harvested for histopathological examination and computerized morphometry, as well as xanthine oxidase (XO, U/mg protein) and maltase (MALT, mmol/L substrate degraded/min/mg protein) assays. CSA (5 mg/kg/d subcutaneously) was given to experimental animals (CSA, n = 10) for 5 days before ischemia, and vehicle was given to controls (CON, n = 10). The computer morphometric parameters studied were: surface index (SI, mucosal surface length per linear unit of intestine), average villous thickness (AVT), and average villous height (AVH). RESULTS: Results are provided in Table 1. CONCLUSION: The results of this study show that CSA may play a role in attenuating ischemia/reperfusion injury in the gut. Enzymatic analysis showed a beneficial role in the preservation of mucosal cell function after gut ischemia/reperfusion injury, as demonstrated by an elevated maltose level. Computerized morphometry demonstrated significant differences in all parameters in the experimental group, showing that CSA does confer gut mucosal protection during ischemia.

Animals↗
PubMed1995

Morphometry and histochemistry of pulmonary arteries in a hypoplastic lung model.

Persistent pulmonary hypertension (PPH) is a common consequence of many neonatal respiratory diseases. The pathophysiology of PPH remains unknown. To study PPH, a rat model of pulmonary hypoplasia was used. Lung mass and body mass were recorded and lungs were prepared for frozen section examination and stained with hematoxylin and eosin, elastin, anti-Factor VIII, and nitro blue tetrazolium to identify nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase. The lungs were analyzed for air space volume, pulmonary artery wall thickness, total pulmonary arterial cross-sectional area, and density of tissue NADPH diaphorase staining. The mass of hypoplastic lungs was less than that of normal lungs (mean mass 85.93 mg vs 142.97 mg, P < 0.0001). The measured fraction of airspace volume was significantly less in hypoplastic lungs compared to controls (17.7% vs 30.8%, P < 0.0001). There was a significant difference in the pulmonary artery wall thickness ratio between the two groups (control 0.46 vs hypoplastic 0.487, P = 0.001). The arterial cross-sectional area was identical (control 1.25% vs hypoplastic 1.37%, P = 0.47). Staining density for NADPH diaphorase activity was determined using an intensity staining index (ISI). The experimental group showed increased staining for NADPH diaphorase (ISI = 54 in hypoplastic lungs vs 38 in controls, P < 0.01). Lung mass, appearance, and measured volume of airspace and tissue were all consistent with hypoplasia. In this model, arterial wall thickness was measurably greater in the hypoplastic group, while arterial cross-sectional area was not different. Staining for NADPH diaphorase showed significantly greater levels of enzyme in the hypoplastic lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Calibration of endoscopic images.

In pediatric airway surgery, endoscopic evaluation of the lesions and treatment is important. However, there are no objective and reproducible methods for measurement. The purpose of this study was to define the optical effects of a Storz-Hopkins system and develop a method of calibration and measurement. Known geometric images (grids and circles) were viewed through the Storz-Hopkins system and recorded. These images were then analyzed morphometrically with a computer image analyzer (Jandel Scientific). The distortion of the endoscope could be identified, and the severity was found to be a function of the distance from the center of the field (r = .962). When this distortion factor (range, 0% to 25% shrinkage) was used mathematically, known circles could be measured by means of an endoscopic image. The error was reduced from 17.6% to 4.3% (p < .003). With morphometric programs the optical distortion of the bronchoscope can be calibrated. This can then be used to correct the measurement of images.

Analysis of Variance↗
PubMed1995

Airway measurement using morphometric analysis.

Measurement of airway size using endoscopic morphometry was investigated. Endoscopic images of the porcine tracheobronchial tree were obtained via a Storz system at a known distance from the telescope. The bronchi were marked with transluminal wires, transected, and imaged. All images were analyzed morphometrically with a computer image at multiple sites (N = 20). The endoscopic measurements were plotted against the macro lens images so that a straight line with a slope of 1 would indicate a consistent correlation. Area measurements for all images had a slope of 0.67 (r = .885); however, for images with area < 80 mm2 the slope was 1 (r = .879), for images with area < 80 mm2 of 0.93 (r = .875), and for images with area > 80 mm2 of -0.34 (r = .482), and major axis had a slope for all images of 0.73 (r = .904), for images with area < 80 mm2 of 0.88 (r = .923), and for images with area > 80 mm2 of -0.59 (r = .771). Accurate area measurements as well as major and minor axis measurements can be obtained for airways with area < 80 mm2 by means of this system. Each bronchoscope-telescope unit has an optimal target size for which measurements are accurate.

Bronchi↗