Biomedical subjects

J Rawn

Publications and source records attributed to J Rawn.

7 recordsLinked to original sources

PubMed2001

Stochastic regulation of cell migration from the efferent lymph to oxazolone-stimulated skin.

The systemic immune response is a dynamic process involving the trafficking of lymphocytes from the Ag-stimulated lymph node to the peripheral tissue. Studies in sheep have demonstrated several phases of cell output in the efferent lymph after Ag stimulation. When skin contact sensitizers are used as Ag, the efferent lymph cell output peaks approximately 96 h after Ag stimulation and is temporally associated with the recruitment of cells into the skin. To investigate the relative contribution of this high-output phase of efferent lymphocytes to lymphocytic inflammation in the skin, we used a common contact sensitizer 2-phenyl-4-ethoxymethylene-5-oxazolone (oxazolone) to stimulate the skin and draining prescapular lymph node of adult sheep. The efferent lymph ducts draining the Ag-stimulated and contralateral control lymph nodes were cannulated throughout the experimental period. The lymphocytes leaving the lymph nodes during the 72-h period before maximum infiltration were differentially labeled with fluorescent tracers, reinjected into the arterial circulation, and tracked to the site of Ag stimulation. Quantitative tissue cytometry of the skin at the conclusion of the injection period (96 h after Ag stimulation) demonstrated more migratory cells derived from the Ag-stimulated lymph node than the contralateral control (median 18.5 vs 15.5 per field; p < 0.05). However, when corrected for total cell output of the lymph node, the Ag-stimulated migratory cells were 3.8-fold more prevalent in the skin than the contralateral control cells. These results suggest that the in situ immune response generally mirrors the frequency of recruitable lymphocytes in the peripheral blood.

Adjuvants, Immunologic↗

PubMed2001

Molecular identification using flow cytometry histograms and information theory.

Flow cytometry is a common technique for quantitatively measuring the expression of individual molecules on cells. The molecular expression is represented by a frequency histogram of fluorescence intensity. For flow cytometry to be used as a knowledge discovery tool to identify unknown molecules, histogram comparison is a major limitation. Many traditional comparison methods do not provide adequate assessment of histogram similarity and molecular relatedness. We have explored a new approach applying information theory to histogram comparison, and tested it with histograms from 14 antibodies over 3 cell types. The information theory approach was able to improve over traditional methods by recognizing various non-random correlations between histograms in addition to similarity and providing a quantitative assessment of similarity beyond hypothesis testing of identity.

Animals↗

PubMed2000

Angiocentric recruitment of lymphocytes into the lung after the intrabronchial instillation of antigen.

The pathogenesis of acute lymphocytic inflammation in the lower respiratory tract appears to involve the recruitment of lymphocytes out of the blood stream and into the extravascular lung tissue. To investigate the membrane molecules regulating this process, we used the intrabronchial instillation of cellular antigen to trigger lymphocyte recruitment into the lower respiratory tract. Sheep presensitized 6 to 10 weeks earlier at a remote site were intrabronchially challenged with 1-5 x 10(7) cells from a B lymphoblastoid cell line. The cells were instilled into a subsegmental bronchus through a bronchial catheter. The stimulated and contralateral control segments were studied at a peak of inflammation, approximately 72 hours after antigen stimulation. Gross and microscopic studies of the stimulated segment demonstrated localized inflammation characterized by the perivascular infiltration of lymphocytes. In contrast, control areas of the lung demonstrated only scattered perivascular lymphocytes. Immunohistochemistry of the stimulated lung showed that the majority of these perivascular cells were CD3+ CD4+ lymphocytes. The T lymphocytes expressed high levels of the cell adhesion molecules beta 1 integrin and LFA-1, but low levels of the L-selectin membrane molecule. Immunohistochemistry of the endothelial cells associated with the lymphocyte infiltrates demonstrated intense staining of the ICAM-1, and beta 1 integrin adhesion molecules. Electron microscopic studies of the endothelial cells in the antigen stimulated areas of the lung confirmed morphologic changes consistent with endothelialitis. These results suggest that the intrabronchial instillation of cellular antigen stimulates an angiocentric T-cell infiltration regulated by activated pulmonary endothelial cells. The histologic and morphologic findings are remarkably similar to those observed during acute lung transplant rejection.

Animals↗

PubMed1999

Fetal response to neutral gas and liquid media for intraamniotic distension.

PURPOSE: This study was aimed at comparing the effects of a neutral liquid and a neutral gas used as intraamniotic media on umbilical blood flow, O2 delivery, blood pressure, acid-base status, and electrolytes in the fetus at escalating intraamniotic pressures. METHODS: Eight fetal lambs underwent invasive monitoring of common umbilical blood flow, blood pressure, blood gases, sodium, and hematocrit, as intraamniotic pressure was raised from 0 to 30 mm Hg. The animals were divided equally in two groups depending on the intraamniotic medium used (group I, warmed saline and group II, air). Maternal systemic blood pressure, O2 saturation, and temperature were kept constant. RESULTS: In each group, a threshold level of intraamniotic pressure was evident, above which there was a significant decrease in the common umbilical artery blood flow, with concomitant fetal hypoxemia and hypercarbia. This intraamniotic pressure threshold was 20 mm Hg in group I (saline), but only 15 mm Hg in group II (air). CONCLUSIONS: Although both a neutral liquid and a neutral gas can safely be used as intraamniotic media, a neutral liquid medium allows for a wider range of safe intrauterine working pressure (0 to 20 mm Hg), as compared with a neutral gas (0-15 mm Hg).

Air↗

PubMed1996

Hybridoma screening for cell adhesion molecules using multiple parallel comparisons in conditions of flow.

Cell adhesion is a complex biophysical process that plays a central role in immunophysiology. Because of the complex force-energy relationships involved, insights into the mechanism of cell adhesion largely depend on comparative measurements. In this report, we describe a comparative approach to the measurement of cell adhesion under conditions of flow. The assay system perfuses fluorescently labeled lymphocytes over a cell monolayer in commercially available multiwell culture plates. The fluorescently labeled cells demonstrate a reproducible flow pattern within the well. Videomicroscopic recordings of cell movement have demonstrated rolling behavior over a wide range of cell velocities. This technique permits the measurement of cell adhesion over a variety of flow velocities, time courses, and treatment conditions. The ability to vary treatment conditions and provide multiple parallel conditions suggests the utility of this approach in the development of monoclonal antibodies (MAb) recognizing cell adhesion molecules.

Animals↗

PubMed1996

LFA-1 and L-selectin regulation of recirculating lymphocyte tethering and rolling on lung microvascular endothelium.

Recirculating lymphocytes migrate into areas of lung inflammation by binding to microvascular endothelium and transmigrating into extravascular tissue. In this report, we examined the multiple-step paradigm using a unique system: recirculating lymphocytes from sheep peripheral lymphatics adhering to activated lung microvascular endothelium in conditions of physiologic flow. Video microscopy demonstrated that recirculating lymphocytes formed abrupt adhesions, without requisite rolling, on the lung microvascular endothelial cells. Lymphocyte velocity was unchanged within 100 ms of the development of firm adhesions. To dissect the adhesion mechanism, the lymphocytes were pretreated with anti-LFA-1 or anti-L-selectin monoclonal antibody (mAb). Both mAb decreased the incidence of firm adhesions. The mechanism of this inhibition was investigated using time-lapse topographic reconstructions of cell movement after pretreatment with mAb. Time-lapse analysis of the movement of lymphocytes pretreated with anti-LFA-1 mAb suggested that abortive adhesion was manifested by a characteristic saltatory movement and a sustained reduction in cell velocity (rolling) to <25 microns/s. In contrast, abortive adhesions of lymphocytes pretreated with anti-L-selectin mAb demonstrated transient arrest (tethering) but minimal rolling before resumption of baseline velocity in the flow stream. These observations provide insights into selectin and integrin regulation of lymphocyte transmigration into the lung. Further, the results of mAb inhibition suggest that the mechanism of lymphocyte migration may have some unique features not observed in studies of neutrophil transmigration.

Animals↗

PubMed1995

In situ fluorescence labeling of sheep lung microvascular endothelium.

Endothelial cells are intimately involved in a variety of biological processes such as inflammatory disorders, wound healing, and tumor invasion. The finding of endothelial heterogeneity in various tissues has led to major efforts to isolate and culture microvascular endothelial cells in human and animal tissue. In this report we have used phosphatidyl ethanolamine (PE)-labeled liposomes to fluorescently label the sheep lung microvasculature in situ. Using normotensive perfusion pressure, the PE-labeled liposomes did not extravasate into extravascular lung tissue. Mechanical and enzymatic digestion of the lung tissue demonstrated that the PE-labeled liposomes provided a stable label of the vascular lining cells during ex vivo processing. After digestion, the overwhelming majority of the fluorescent label appeared in cellular aggregates. Approximately 80% of these cells demonstrated an in vitro phenotype consistent with microvascular endothelium. A novel monoclonal antibody selective for sheep endothelial cells was developed to confirm the presence of lung endothelium in the fluorescently labeled cellular aggregates. We conclude that in situ fluorescence labeling of vascular lining cells provides an anatomic marker for relevant vascular lining cells and an opportunity to study these cells in vitro.

Animals↗