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H R Duncker

Publications and source records attributed to H R Duncker.

At least 19 recordsLinked to original sources
PubMed2001

Comparison of different detection methods in quantitative microdensitometry.

Quantitative evaluation of immunohistochemical staining has become a focus of attention in research applications and in pathological diagnosis, such as Her-2/neu assessment in mammary carcinoma. Reproducibility of immunostaining techniques and microscopical evaluation are prerequisites for a standardized and reliable quantitation of immunostaining intensity. In the present study, different staining and microscopical techniques, including fluorescence microscopy, epipolarization microscopy of immunogold-silver, and absorbance microdensitometry were compared concerning suitability for quantitative evaluation. We describe a staining procedure using alkaline phosphatase-based immunohistochemistry with the substrate Vector Red and subsequent microdensitometry with a custom-designed absorbance filter. We have characterized linearity of the staining intensity in dependence of development time, antibody concentration, and section thickness by means of artificial standards consisting of agarose blocks into which immunogold- or alkaline phosphatase-conjugated antibodies were incorporated. Applicability of the different techniques was tested by anti-CD45 immunostaining of leukocytes within rat lung tissue detected by immunofluorescence, immunogold-silver epipolarization microscopy, as well as alkaline phosphatase-based Vector Red absorbance or fluorescence measurement. Excellent qualities of Vector Red for quantitative microdensitometric evaluation of staining intensity were particularly obvious for absorbance microscopy. Applicability in paraffin-embedded tissue as well as in cryosections, excellent segmentation, linearity over a wide range, light stability, and feasibility for permanent mounting and for long-term storage are the outstanding features of this technique for use in routine quantitative evaluation.

Alkaline Phosphatase↗
PubMed2000

Rat pulmonary cyclooxygenase-2 expression in response to endotoxin challenge: differential regulation in the various types of cells in the lung.

Cyclooxygenase (Cox), the key enzyme of prostanoid synthesis, consists of the two isoforms Cox-1 and Cox-2, both recently noted to be constitutively expressed in rat lungs with a distinct profile of cellular distribution. The responsiveness of pulmonary Cox-1 and Cox-2 expression to intravascular endotoxin lipopolysaccharide (LPS) administration was investigated in isolated, ventilated rat lungs, buffer-perfused with or without admixture of rat plasma. Immunohistochemical staining intensity was measured by a previously described method of silver enhancement and epipolarization image analysis. Both the Cox-1 mRNA, quantified in the whole lung homogenate, and the cellular localization of Cox-1 were unchanged in response to LPS. In contrast, time- and dose-dependent up-regulation of Cox-2 mRNA (lung homogenate) occurred, and differential LPS reactivity at the cellular level was observed. Up-regulation of Cox-2 in cell types expressing this enzyme already under baseline conditions was noted in bronchial epithelial cells, bronchial and vascular smooth muscle cells, cells within the BALT and myocytes of the large hilar veins. De novo induction of Cox-2 occurred in endothelial cells and the majority of alveolar macrophages. Down-regulation of Cox-2 was observed in perivascular and peribronchial macrophage-like cells. Moreover, differential impact of plasma components was noted: for the large majority of cells, CD14 surface expression correlated with Cox-2 responsiveness to LPS independent of plasma, whereas the presence of plasma components was a prerequisite for the LPS response in CD14-negative cells. LPS did not provoke physiological changes in the perfused lungs, but markedly enhanced baseline prostanoid generation. We conclude that LPS-induced Cox-2 regulation occurs in a complex, cell-specific manner, which may be relevant for pathogenetic sequelae in septic lung injury and acute respiratory failure.

Animals↗
PubMed2000

In situ localization and regulation of thromboxane A(2) synthase in normal and LPS-primed lungs.

Thromboxane (Tx) A(2) synthase catalyzes the conversion of prostaglandin H(2) to the unstable metabolite TxA(2), which is a potent mediator of vasoconstriction and bronchoconstriction. The cellular localization of TxA(2) synthase was examined by immunohistochemistry and in situ hybridization in human and rat lung tissues. Bronchial epithelial cells, bronchial smooth muscle cells, peribronchial nerve fibers, single cells of bronchus-associated lymphoid tissue, single cells located in the alveolar septum, and alveolar macrophages exhibited positive immunostaining for TxA(2) synthase protein in lung tissue of both species. In addition, vascular smooth muscle cells of muscular and partially muscular vessels displayed strong (rat) and moderate (human) immunostaining for TxA(2) synthase. In situ hybridization performed in the rat lungs demonstrated TxA(2) synthase mRNA localization in accordance with the immunostaining pattern. Perfusing isolated rat lungs with endotoxin for 1 and 2 h resulted in a marked increase in TxA(2) synthase protein staining intensity in most cell types as measured by quantitative image analysis, whereas the in situ hybridization signal was unchanged. We conclude that the pulmonary distribution of TxA(2) synthase displays close similarity between rat and human lung tissues and matches well with the previously described immunolocalization of cyclooxygenase-1 and cyclooxygenase-2 in this tissue. Endotoxin challenge is suggested to cause a rapid upregulation of TxA(2) synthase at the posttranscriptional level. These data provide a morphological basis for the understanding of the role of TxA(2) in the regulation of lung bronchial and vascular tone and in immunologic events.

Animals↗
PubMed1997

Ultrastructural changes of lung capillary endothelium in response to botulinum C2 toxin.

The role of the endothelial cytoskeleton for the structural integrity of the pulmonary gas exchange area was probed with the use of Clostridium botulinum C2 toxin. This agent causes selective loss of nonmuscle F-actin. In buffer-perfused rabbit lungs, vascular pressures were kept within physiological ranges. In different groups, low-dose [0.3 (C2,I)/0.6 (C2,II) ng/ml] and high-dose [10 (C2,I)/20 (C2,II) ng/ml] toxin were applicated into the buffer fluid; experiments were terminated after a total weight gain of either 1 or 7.5 g. Electron microscopy revealed extensive attenuations, undulations, and protrusions of the endothelial layer, suggestive of "remodeling" and "flowing" of the cell membrane in low C2 toxin-treated lungs accompanied by few disruptions of the endothelial layer and edema formation. In addition, endothelial cells displayed vesiculation and bleb formation. Lungs that were exposed to high-toxin doses displayed marked attenuations of the endothelial layer in addition to large endothelial cell disruptions, which did not include interendothelial junctions. Interestingly, type II epithelial cells displayed fusion of lamellar bodies. Collectively, these data suggest that the actin microfilament system is instrumental in supporting endothelial cell membrane configuration and integrity and maintains the intimal barrier function of the lung microvasculature.

Animals↗
PubMed1995

Role of endothelial cytoskeleton in high-permeability edema due to botulinum C2 toxin in perfused rabbit lungs.

The cytoskeleton of the endothelial cell has been suggested to regulate endothelial barrier function. We investigated the role of actin in the maintenance of pulmonary capillary integrity in perfused rabbit lungs. As a tool for selective perturbation of actin, we employed Clostridium botulinum C2 toxin, which is composed of a membrane translocation component (C2II) and a component (C2I) effecting ADP-ribosylation of nonmuscle G-actin. ADP-ribosylated actin no longer capable of polymerization but acts as a barbed end-capping protein, thereby effecting selective loss of the nonmuscle F-actin content. In buffer-perfused rabbit lungs, combined application of both toxin components (range 50 pg/ml-5 ng/ml C2I) resulted in a time- and dose-dependent increase in the capillary filtration coefficient (Kfc) with concomitant edema formation. Only 300:600 pg/ml C2I:II sufficed to induce a > 10-fold rise of Kfc values within 110 min. This severe lung permeability increase occurred in the absence of vasomotor responses and potassium release or lactate dehydrogenase release. Application of each single toxin component displayed markedly reduced efficacy. Similar to the C2 toxin effect, severe permeability increase without concomitant hemodynamic changes was evoked by cytochalasin D, known to possess F-actin-disrupting properties. Preloading of lung cells with phallacidin, which in opposition to C2 toxin decreases F-actin depolymerization, significantly reduced the C2 toxin-induced increase in vascular permeability. Electron microscopic examination of C2 toxin-poisoned lungs showed early, extensive endothelial cell attenuations, followed by disruptions of the endothelial layer and marked interstitial edema formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Morphometric analysis of pulmonary intracapillary leukocyte pools in ex vivo-perfused rabbit lungs.

Characterization and quantification of lung intracapillary leukocytes is of interest for a better understanding of immunological and inflammatory features in this organ. We developed a technique of computer-assisted measurement of digitalized electron-microscopic images and electronic data processing for morphometry of intracapillary leukocyte pools in rabbit lungs (L. Ermert, W. Seeger, and H.-R. Duncker, Cell Tissue Res. 271: 469-476, 1993). Measurements were undertaken in buffer-perfused isolated lungs (avoiding any reentry of washed-out cells); perfusion fixation was performed 7.5, 35, and 185 min after onset of artificial circulation (n = 5 each). Data were compared with that of nonperfused lungs fixed by tracheal instillation (baseline). Total lung capillary neutrophil counts were 1.41 x 10(9), 1.35 x 10(9), 1.37 x 10(9), and 0.69 x 10(9) (baseline, 7.5, 35, and 185 min perfusion, respectively). Corresponding data for intracapillary lymphocytes were 1.07 x 10(9), 0.84 x 10(9), 0.81 x 10(9), and 0.57 x 10(9); and for microvascular monocytes, data were 0.21 x 10(9), 0.19 x 10(9), 0.18 x 10(9), and 0.08 x 10(9). Ratios of cell volume and surface variables of the different intracapillary leukocyte types did not change during ex vivo lung perfusion. We conclude that the rabbit pulmonary capillary bed harbors large pools of different leukocytes, which surpass pool sizes of corresponding circulating cells and display very slow washout kinetics under conditions of lung-buffer perfusion. A major impact of these intracapillary leukocyte pools on immunological and inflammatory events in isolated-perfused and transplanted lungs must be assumed.

Animals↗
PubMed1993

Computer-assisted morphometry of the intracapillary leukocyte pool in the rabbit lung.

Computer-assisted morphometry was performed to evaluate the number and cell characteristics of capillary and alveolar leukocytes in rabbit lungs. An image-processing system and a programmable spread-sheet program were used, which allowed morphometric analysis of a large reference area. Neutrophils represented the largest intracapillary leukocyte population (2.2 x 10(7)/ml parenchyma, which corresponds to an approximately 104-fold microvascular enrichment of this cell type related to cell counts calculated for the capillary blood volume). In addition, large numbers of intracapillary lymphocytes (1.7 x 10(7)/ml parenchyma; 47-fold enrichment) and monocytes (0.3 x 10(7)/ml parenchyma; 86-fold enrichment) were detected. The total count of pulmonary leukocytes thus approximated the total number of pulmonary endothelial cells; and the total circulating pools of the different leukocytes were surpassed by the corresponding lung capillary pools, 3.2-fold for neutrophils, 1.2-fold for lymphocytes and 4.8-fold for monocytes. In contrast, alveolar cell numbers ranged from 1-2% of the capillary counts for all types of leukocytes. We conclude that the rabbit lung microvasculature harbours large pools of immunocompetent cells, which may contribute to host-defense mechanisms at the gas-exchange area.

Animals↗
PubMed1992

Induction of severe vascular leakage by low doses of Escherichia coli hemolysin in perfused rabbit lungs.

S. aureus alpha-toxin and E. coli hemolysin (Hly) represent two prototypes of pore-forming cytolysins. Both are established virulence factors and have been implicated in the development of septic lung failure. Low doses of these agents cause thromboxane-mediated vasoconstriction and edema formation in isolated perfused rabbit lungs. In a preceding investigation, we observed that alpha-toxin causes overt endothelial cell damage in these lungs, as demonstrable by electron microscopy (Seeger W, Birkemeyer RG, Ermert L, Suttorp N, Bhakdi S, Duncker HR: Lab Invest 63:341, 1990). Here, we present results of a parallel study conducted with E. coli hemolysin. Thromboxane-dependent pulmonary hypertension was suppressed by the addition of acetylsalicylic acid to the perfusion fluid in all cases. Administration of 0.2 hemolytic units (HU; i.e., 20 ng/ml protein) resulted in progressive weight gain after a lag period of 10 to 15 minutes, and 30 minutes after toxin application the gravimetrically determined capillary filtration coefficients (Kfc) were increased greater than 10-fold. Perfusion was terminated when the total lung weight gain surpassed 20 gm. 0.12 HU/ml E. coli hemolysin caused 2- to 3-fold increased capillary filtration coefficients values within 110 minutes, concomitant with intermediate quantities of edema formation (9.7 +/- 2.7 gm). Potassium liberation in the absence of lactate dehydrogenase release occurred in all toxin treated lungs. Electron microscopic examination after perfusion fixation revealed interstitial edema formation in areas remote from the blood-gas exchange barrier. Increased numbers of endothelial plasmalemmal vesicles were visualized at the very onset of edema formation in lungs exposed to 0.2 HU/ml, and after a 110-minute exposure to 0.12 HU/ml of the toxin, but not in lungs exhibiting severe edema (greater than 20 gm weight gain). In contrast to our previous results with alpha-toxin, endothelial cells displayed normal electron density here and were not detached from the fused basal lamina. Hence, although both pore formers provoke severe vascular leakage in our experimental model, the underlying pathways probably divert fundamentally from each other.

Animals↗
PubMed1990

Staphylococcal alpha-toxin-induced vascular leakage in isolated perfused rabbit lungs.

alpha-Toxin, the major, pore-forming exotoxin of Staphylococcus aureus, caused acute hypertension when perfused through blood-free rabbit lungs (21). This reaction is mediated by pulmonary thromboxane generation, for which toxin-induced calcium flux into target cells with subsequent stimulation of arachidonic acid metabolism is predominantly responsible. In the present study, we investigated the effects of alpha-toxin on the integrity of the lung microvasculature. Thromboxane generation was inhibited in all experiments to suppress the development of pulmonary hypertension. Application of low alpha-toxin concentrations (5 to 40 ng/ml) induced protracted, severe vascular leakage in a dose-dependent manner. After a lag period of 40 to 120 minutes, gravimetrically determined capillary filtration coefficients progressively increased to greater than 10-fold values, and this was followed by pronounced weight gain of the isolated organs. These physiologic alterations were paralleled by dose- and time-dependent structural changes documented by electron microscopic examination of perfusion-fixed lungs. Increasing electron density of microvascular endothelial cell nuclei and subsequently of their cytoplasma was noted, followed by detachment of these cells from the mutual endoepithelial basal lamina. Edema was localized in the blood-gas exchange area, in contrast to hydrostatically induced lung fluid accumulation. These results identify pulmonary microvascular endothelium cells as highly susceptible targets for attack by alpha-toxin. Given a similar sensitivity of human endothelial cells, alpha-toxin might directly contribute to the pathogenesis of acute respiratory failure under conditions of severe infection with Staphylococcus aureus.

Animals↗
PubMed1978

Lung architecture volume and static mechanics in five species of lizards.

Compared with mammals, lizards have large, highly compliant lungs and a compliant body wall. High lung compliance is not necessarily associated with high body wall compliance but rather with the degree of development of the caudal and ventral dilatations of the lung. Comparison of the midinflation distance between inflation and deflation curves on standard volume--pressure diagrams indicates hysteresis may have an intrapulmonary component related to the degree of lung partitioning and an extrapulmonary component, attributable to forces hindering the free movement of the lung in the body cavity. Simultaneous consideration of lung structure, mechanical properties and certain nonrespiratory functions such as buoyancy and display indicate that the function of the lung in gas exchange may be only one of several factors important in determining lung structure in lizards.

Animals↗