Biomedical subjects

M E Klut

Publications and source records attributed to M E Klut.

14 recordsLinked to original sources

PubMed2000

Partial liquid ventilation with perfluorocarbon in acute lung injury: light and transmission electron microscopy studies.

Liquid ventilation using perfluorocarbon has been shown to improve gas exchange in animal models of acute lung injury as well as in children with acute respiratory distress syndrome. This study was designed to define structural features of lung injury following partial liquid ventilation (PLV) using light and transmission electron microscopy in a rabbit model of acute respiratory distress. Animals were treated with either conventional mechanical ventilation (CMV-gas) (n = 6) or PLV (n = 5) for 4 h after the induction of acute lung injury with saline lavage. Control animals were killed after the lung injury. PLV significantly improved alveolar-arterial oxygen tension and the oxygen index compared with CMV (P < 0.05). Morphometric studies using light microscopy show less alveolar hemorrhage, less edema, and fewer hyaline membranes in the PLV group (P < 0.05). Polymorphonuclear leukocyte sequestration in lung capillaries (11.4 +/- 1.5 versus 19.2 +/- 3 x 10(8)/ml, P < 0.05, PLV versus CMV) and migration into airspaces (3.1 +/- 1.2 versus 4.5 +/- 1.1 x 10(8)/ml, P < 0.05, PLV versus CMV) were lower in the gravity-dependent lung regions. There were fewer alveolar macrophages in the PLV group compared with other groups (P < 0.05). Fluorescence microscopy analysis shows fewer type II alveolar epithelial cells in the CMV group and brighter type II cells in the PLV group. Transmission electron microscopy studies show more alveolar wall damage in the CMV group, with type II cells detached from their basement membrane with fewer surfactant-containing lamellar bodies. We conclude that quantitative histologic analysis shows less lung damage and inflammation when perfluorocarbon is combined with CMV in the management of acute respiratory distress syndrome.

Acute Disease↗

PubMed1999

The use of flow cytometry to measure neutrophil function.

Neutrophils are important professional phagocytic cells that provide the host with a first line of defense against acute bacterial and fungal diseases and recurrent, severe or unusual infections are associated with inherited defects of neutrophil function. Furthermore, abundant evidence links inappropriate neutrophil-mediated tissue damage to the pathogenesis of conditions such as acute respiratory distress syndrome, septicemia with multiorgan failure, ischemia-reperfusion injury and rheumatoid arthritis. Flow cytometry has been increasingly used to evaluate the functional capabilities of neutrophils. In this review, we discuss the use of flow cytometry to assess neutrophil functional responses including calcium mobilization, F-actin assembly, adhesion, aggregation, degranulation, phagocytosis and reactive oxygen species (ROS) production. The use of flow cytometry to identify neutrophil priming is also discussed. The advantage of flow cytometry is that the majority of neutrophil functions can be measured using a small volume of whole blood that reduces artifactual changes in function caused by purification procedures. The advent of numerous new fluorochromes and multiparametric analysis allows the simultaneous measurement of several neutrophil functions in the same population of cells. Flow cytometric analysis provides a rapid screen for abnormalities of neutrophil function and reflects more accurately their behavior in vivo.

Animals↗

PubMed1999

Nitric oxide reduces the sequestration of polymorphonuclear leukocytes in lung by changing deformability and CD18 expression.

Nitric oxide (NO) influences polymorphonuclear leukocytes (PMN)-endothelial cell interactions. The aim of this study was to evaluate this effect in the lung and investigate this mechanism. PMN sequestration in the lung was evaluated in vivo after the infusion of complement fragments. Rabbits (n = 9) that inhaled 40 ppm of NO were compared with control rabbits (n = 9) over a 2-h period following infusion of complement fragments. Circulating PMN counts immediately decreased after infusion of complement fragments in both groups followed by a recovery to baseline. This recovery was maintained in the NO-treated group compared with the control rabbits (p < 0.05). NO reduced PMN sequestration in the lung measured by both arteriovenous PMN difference across the lung (p < 0.01) and the myeloperoxidase (MPO) content of the lung tissue (p < 0.01). NO had no effect on the complement fragments-induced PMN release from the bone marrow. In vitro studies showed that NO partially inhibited F-actin assembly (p < 0.01) reduced the change in deformability (p < 0.05) and inhibited CD18 upregulation (p < 0.05) but had no effect on the L-selectin shedding of PMN stimulated by complement fragments. We conclude that NO reduces the sequestration of activated PMN by reducing deformability change via inhibition of F-actin assembly and inhibiting the upregulation of CD18.

Actins↗

PubMed1999

Cigarette smoking causes sequestration of polymorphonuclear leukocytes released from the bone marrow in lung microvessels.

Studies from our laboratory have shown that chronic cigarette smoke exposure causes a neutrophilia associated with a shortening of the mean transit time of polymorphonuclear leukocytes (PMN) though the postmitotic pool of the marrow. The present study was designed to test the hypothesis that PMN newly released from bone marrow by smoke exposure preferentially sequestered in pulmonary microvessels. The thymidine analogue 5'-bromo-2'-deoxyuridine (BrdU) was used to label dividing PMN in the marrow of rabbits; their appearance in the circulation was measured using immunocytochemistry, and their sequestration in lung tissue was determined using standard morphometric techniques. Animals exposed to 11 d of cigarette smoke (n = 6) compared with sham-exposed control animals (n = 4) showed no increase in circulating PMN counts but showed an increase in both the percentage of band cells (smoking, 9.8 +/- 1.1% versus control, 5.5 +/- 0.9%; P < 0.05) and BrdU-labeled PMN (PMNBrdU) in the circulation (smoking, 10.8 +/- 0.6% versus control, 7.5 +/- 0.3%; P < 0.05). There were more PMN sequestered in the lungs of smoke-exposed animals (51.7 +/- 3.4 x 10(7)/ml tissue) than in those of control animals (25.1 +/- 1.8 x 10(7)/ ml tissue) (P < 0.05) and a higher percentage of these cells were PMNBrdU (smoking, 16.9 +/- 2. 3% versus control, 9.6 +/- 0.4%; P < 0.05). The percentage of PMNBrdU in the gravity-independent regions (11.7 +/- 1.9%) of the lung was higher than gravity-dependent regions (7.8 +/- 1.8%) in the smoke-exposure group (P < 0.05). Transmission electron microscopy showed pulmonary capillary endothelial damage with adherent PMN in the smoke-exposure group. We conclude that younger PMN released from the bone marrow by cigarette smoking preferentially sequestered in pulmonary microvessels and speculate that these PMN may contribute to the alveolar wall damage associated with smoke-induced lung emphysema.

Animals↗

PubMed1998

Leukocyte activation does not mediate myocardial leukocyte retention during endotoxemia in rabbits.

Our goal was to determine whether coronary leukocyte retention after endotoxin infusion was due primarily to leukocyte activation. Leukocytes were activated by infusion of endotoxin into 12 blood donor rabbits. Separately, 12 isolated rabbit hearts were perfused with blood from an endotoxemic support rabbit to expose coronary endothelium to an inflammatory stimulus. During an infusion of 20 ml of donor blood into the isolated heart, the coronary transit time of leukocytes was determined by deconvolution of multiple measurements of injectate and collected leukocyte concentrations. With no leukocyte activation or inflammatory stimulation of endothelium, leukocyte transit time was 9.2 +/- 3.5 s, and 11.6 +/- 4.1 x 10(6) leukocytes were retained in the coronary circulation. Leukocyte activation alone did not alter transit time (9.8 +/- 3.2 s) or retention (9.3 +/- 4.6 x 10(6) leukocytes). Inflammatory stimulation of endothelium with and without leukocyte activation increased transit time (18.0 +/- 3.6 and 18.9 +/- 3.8 s, respectively; P < 0. 05) and retention (24.8 +/- 8.4 and 25.3 +/- 6.8 x 10(6) leukocytes, respectively; P < 0.05) to the same extent. Differential counts showed that neutrophils (but not lymphocytes) were slowed and retained. Inflammatory stimulation of endothelium caused coronary capillary endothelial swelling and pseudopod formation. Thus increased coronary neutrophil transit time and retention are due to structural changes of coronary endothelial cells or other effects of the inflammatory response occurring within coronary capillaries, not only due to activation of leukocytes.

Animals↗

PubMed1998

Neutrophil structural changes associated with chronic endotoxemia and lung injury.

Previous studies showed that chronic endotoxemia induces thickening of the alveolar wall of rabbits. The present study examines cellular changes associated with this process and attempts to define the role of PMN in this response. Rabbits received i.v. injections of either Escherichia coli lipopolysaccharide (LPS) or saline (control), 2-3 times weekly, for 28 weeks. Peripheral blood mature and immature polymorphonuclear leukocyte (PMN) cell counts were determined on Wright-stained blood smears. Lung histological analysis was performed by both light and electron microscopy. FITC-Maclura pomifera was used to identify type II cells and diaminobenzidine tetrahydrochloride-H2O2 was employed to localize peroxidase. The results show that the LPS-induced neutrophilia is associated with an increase in the circulating band cells which is consistent with active bone marrow release. PMN in the pulmonary microvessels display structural features characteristic of phagocytosis and active macromolecule synthesis. Endothelial cells, adjacent to these PMN, show numerous coated pits and large inclusions suggestive of endocytosis. The LPS-induced thickening of the alveolar wall is associated with leukocyte migration into the interstitial and alveolar spaces. Some interstitial PMN are fragmented and surrounded by dispersed elements of the connective tissue, while others appear activated and are closely associated with hyperactive fibroblasts and alveolar type II cells. The number of alveolar type II cells has increased twofold. These results show that chronic endotoxemia in rabbits causes structural changes in PMN, endothelium, interstitium, and epithelium. PMN structural changes are consistent with enhanced functional properties and their close association with modified regions of the lung parenchyma suggest that PMN play an important role in the process of this lung injury and repair.

Animals↗

PubMed1997

Activation-associated changes in blood and bone marrow neutrophils.

Accelerated granulocytopoiesis and polymorphonuclear leukocyte (PMN) activation via the complement system are important events in the inflammatory response. This study tests the hypothesis that PMN of the peripheral blood and the bone marrow behave differently when stimulated with zymosan-activated plasma (ZAP). PMN were treated with ZAP and processed to determine the content and distribution of F-actin, the expression of CD18-integrins, and the cell area/shape with the use of a combination of flow cytometry, fluorescence, and light microscopy. The results show that untreated bone marrow PMN display similar F-actin content and cell area but express less CD18 than peripheral blood PMN. ZAP-activated peripheral blood PMN show a marked increase in the F-actin content, CD18 expression, cell area, and length. Highly elongated PMN develop a polar shape in which microfilaments are redistributed in cell protrusions and CD18 is located in the uropod and the lamellipodium. In comparison, activated bone marrow PMN show a lower and more transient increase in the F-actin content, express less CD18, show a reduced increase in the cell area, and display reduced polarity and microfilament redistribution. We conclude that bone marrow PMN show lower complement-activation response than peripheral blood PMN and suggest that the lower expression of surface membrane integrins and related cytoskeletal filaments may account for their reduced ability to develop the polar shape required for diapedesis and migration.

Actins↗

PubMed1997

Effect of mechanical deformation on structure and function of polymorphonuclear leukocytes.

The present studies were designed to test the hypothesis that mechanical deformation of polymorphonuclear leukocytes (PMN) leads to functional changes that might influence their transit in the pulmonary capillaries. Human leukocytes were passed through 5- or 3-micron-pore polycarbonate filters under controlled conditions. Morphometric analysis showed that the majority of PMN were deformed and that this deformation persisted longer after filtration through 3-micron filters than through 5-micron filters (P < 0.05) but did not result in the cytoskeletal polarization characteristic of migrating cells. Flow cytometric studies of the filtered PMN showed that there was a transient increase in the cytosolic free Ca2+ concentration after both 3- and 5-micron filtration (P < 0.01) with an increase in F-actin content after 3-micron filtration (P < 0.05). Although L-selectin expression on PMN was not changed by either 5- or 3-micron filtration, CD18 and CD11b were increased by 3-micron filtration (P < 0.05). Priming of the PMN with N-formyl-methionyl-leucyl-phenylalanine (0.5 nM) before filtration resulted in an increase of CD11b by both 5 (P < 0.05)- and 3-micron (P < 0.01) filtration. Neither 5- nor 3-micron filtration induced hydrogen peroxide production. We conclude that mechanical deformation of PMN, similar to what occurs in the pulmonary microvessels, induces both structural and functional changes in the cells, which might influence their passage through the pulmonary capillary bed.

Actins↗

PubMed1997

Polymorphonuclear leukocytes released from the bone marrow preferentially sequester in lung microvessels.

OBJECTIVE: A hallmark of the systemic response to an inflammatory stimulus is the release of polymorphonuclear leukocytes (PMNs) from the bone marrow. This study was designed to measure the release of PMNs from the bone marrow and to determine their sequestration in the lung after an intravenous injection of either endotoxin (n = 5) or saline (n = 5). METHODS AND RESULTS: The thymidine analogue 5'-bromo-2-deoxyuridine (BrdU) was used to pulse label dividing PMNs in the bone marrow of rabbits (n = 13), and immunohistochemistry and morphometry were used to detect the release of BrdU-labeled PMNs into the circulation and to determine their sequestration in the lung. Endotoxin treatment caused a drop in the circulating PMN counts (3.3 +/- 0.08 at baseline to 0.12 +/- 0.02 x 10(9)/L at 1 hour after endotoxin), which was followed by a neutrophilia at 8 hours (6.3 +/- 1.1 x 10(9)/L, P < 0.01), an increase in circulating band cells (0.12 +/- 0.01 at baseline to 2.18 +/- 0.4 x 1(9)/L at 8 hours, p < 0.001), and an increase in the percentage of BrdU-labeled PMNs (0.01% +/- 0.004% at baseline to 26.1% +/- 3.2% at 8 hours, p < 0.001). Endotoxemia caused an arteriovenous difference in BrdU-labeled PMNs across the lung (35.9% +/- 2.9% versus 26.1% +/- 3.1%, mixed venous versus arterial, p < 0.02). Morphometric studies showed that endotoxin caused sequestration of PMNs in the lung (2.2 +/- 0.4 versus 1.0 +/- 0.2 x 10(10), endotoxin versus saline, p < 0.03) with preferential retention of BrdU-labeled PMNs (0.79 +/- 0.21 versus 0.039 +/- 0.016 x 10(10), endotoxin versus saline, p < 0.05). The percentage of BrdU-labeled PMNs in the alveolocapillary walls was higher than in circulating blood (64.01% +/- 4.3% versus 26.1% +/- 3.2%, p < 0.01) in the endotoxin group. In vitro filtration of cells through 5-mm pore size filters showed that circulating BrdU-labeled PMNs, 8 hours after endotoxin, were preferentially retained in the filters (p < 0.01). CONCLUSIONS: We conclude that endotoxemia stimulates the bone marrow to release mature and immature PMNs. Compared to PMNs released from the bone marrow during normal turnover, these PMNs are less deformable and preferentially sequester in the lung microvessels.

Animals↗

PubMed1993

Activation of neutrophils within pulmonary microvessels of rabbits exposed to cigarette smoke.

Previous studies have shown that polymorphonuclear leukocytes (PMN) are delayed in the pulmonary capillaries by the presence of cigarette smoke. To determine if the PMN delayed by smoking are activated, we estimated the in vivo expression of CD11/CD18 and L-selectin on the surface of PMN in lungs and peripheral blood of rabbits because these molecules are known to be upregulated and downregulated, respectively, on the surface of activated PMN. New Zealand white rabbits (3.5 +/- 0.1 kg) were exposed to either air (n = 5) or cigarette smoke (n = 5), and we used an established protocol to measure pulmonary vascular blood flow, volume, and red blood cell (RBC) transit time in the left lung. The right lungs were then fixed in 0.025% glutaraldehyde and stored in liquid nitrogen. Ultrathin sections were immuno-labeled with either the anti-CD18 monoclonal antibody 60.3 or the anti-L-selectin antibody Dreg-200, followed by a secondary antibody conjugated to 10 nm colloidal gold. The target antigens were quantified by counting the number of gold particles per micron (G/microns) of PMN surface membrane. The data show that smoke exposure had no effect on pulmonary blood flow, volume, or RBC transit time. However, it increased the expression of CD11/CD18 on intravascular PMN in the upper region of the lung (control, 7.4 +/- 1.3 G/microns; smoke-exposed, 13.2 +/- 3.3 G/microns; P < 0.05) and decreased the expression of L-selectin on intravascular PMN in both the lower (control, 5.5 +/- 2.0 G/microns; smoke-exposed, 2.6 +/- 1.5 G/microns; P = 0.05) and the upper (control, 6.8 +/- 1.4 G/microns; smoke-exposed, 2.6 +/- 1.2 G/microns; P < 0.05) regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Further use of fluorochromes in the cytochemical characterization of phytoplankton.

The rapid, specific effects of 25 fluorochromes at low concentration and physiological conditions of pH and temperature were investigated on live cells of five phytoplankton species (Prorocentrum micans, Amphidinium carterae, Dunaliella tertiolecta, Chlamydomonas moewusii and Fragilaria crotonensis). They allowed the identification of cellular components such as the plasma membrane, endoplasmic reticulum, Golgi apparatus, thecal plates, nucleus, mitochondria, trichocysts, vacuoles/lysosomes, polyphosphate and starch granules, lipid bodies and hydrolytic enzymes. Morphological alterations of some of these constituents were examined in cells at different metabolic states. It was found that the thickness of Prorocentrum thecal plates increases during cell development while surface pores appear to be formed in the early stages of thecal formation. The number and size of mitochondria varies among cells at different stages of growth. The number of trichocysts, the size of vacuoles and the quantity of polyphosphates, starch or lipid inclusions increases in nitrogen-depleted cells. Photodegradation and photoenhancement phenomena are described. Some important factors helping to avoid quenching and some applications of the fluorochroming technique are presented.

Fluorescent Dyes↗

PubMed1988

The use of fluorochromes in the cytochemical characterization of some phytoflagellates.

Sixteen fluorochromes were tested for the cytochemical characterization of two dinoflagellates (Amphidinuim carterae, Prorocentrum micans) and one chlorophycean flagellate (Dunaliella tertiolecta). Depending on the fluorochrome used, various cellular components (including the plasma membrane, thecal plates, pusule, trichocysts, nucleus, lipid bodies and vacuoles) were revealed. The different colours obtained from single or double fluorochrome staining enabled the differentiation and identification of most cellular components. Protoplasmic staining with Fluorescein diacetate suggested the occurrence of esterases in the three phytoflagellates. Rhodamine B, Neutral Red, FluoroBora P and Nile Blue revealed extensive occurrence of lipoid bodies in A. carterae, but Nile Blue showed considerable difference from the other stains in the inclusion size and intracellular location of these bodies. Chlortetracycline binding, and its inhibition by the Ca2+ionophore A23187, indicated that the plasma membrane, pusule system and trichocysts contain sites of Ca2+ binding. Calcofluor White ST proved superior to Congo Red and Lucifer Yellow in elucidating structural details of the thecal plates of P. micans. While Acridine Orange revealed the presence of surface-coat acidic polysaccharides, the fluoresceinated lectins established their glycoconjugate nature in all the three flagellates. Possible mechanisms of fluorochrome uptake are discussed.

Animals↗

PubMedDate not supplied

Functional changes in aging polymorphonuclear leukocytes.

BACKGROUND: Previous studies from our laboratory have shown that the expression of L-selectin on polymorphonuclear neutrophils (PMN) decreases as the cell ages in the circulation and that these older PMN have more fragmented DNA and show morphological features of apoptosis. METHODS AND RESULTS: The present study was designed to compare the functional capabilities of PMN expressing low levels of L-selectin (L-selectin[low]) and the total population of PMN they were isolated from (L-selectin[mixed]). The results show no difference of the baseline filamentous actin (F-actin) content between PMN expressing low and high levels of L-selectin. However, the ability of L-selectin(low) PMN to assemble F-actin was impaired after stimulation by n-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) (1 nmol/L fMLP: P<.02, 10 nmol/L fMLP: P<.01). The ability of L-selectin(low) PMN to change shape when stimulated (10 nmol/L fMLP) was also decreased (P<.05). Filtration studies showed no difference in baseline deformability between L-selectin(low) and L-selectin(mixed) leukocytes, but the L-selectin(low) cells showed a decreased ability to stiffen after fMLP stimulation (P<.05). L-selectin(low) cells demonstrated a decreased ability to migrate toward a chemoattractant (1, 3, and 10 nmol/L fMLP) (P<.004) but have an enhanced ability to upregulate CD18 (P<.00002) and produce hydrogen peroxide (P<.00004). CONCLUSIONS: We conclude that PMN undergo substantial functional changes as they age in the circulation.

Actins↗

PubMedDate not supplied

Neutrophil activation and lung injury associated with chronic endotoxemia in rabbits.

Chronic endotoxemia produces emphysematous lung destruction in several animal models. The present study was designed to examine changes in the polymorphonuclear leukocytes (PMN) and the lung parenchyma of rabbits that received either saline (control, n = 6) or Escherichia coli endotoxin (LPS, n = 6) 2-3 times weekly for 15 to 28 weeks. Peripheral blood was collected just before and after each intravenous injection and lung tissue was processed at the end of the experiment. PMN myeloperoxidase was stained with diaminobenzidine tetrahydrochloride (DAB)-H2O2, and CD11/CD18 was detected with immunogold. The changes in the PMN and the lung parenchyma were quantitatively analyzed. The results show that each dose of LPS produced an initial fall, followed by a rise in the circulating mature and immature PMN cell counts. Repeated doses of LPS induced PMN activation, degranulation, and an increase in the mean thickness of the alveolar wall (control, 4.1 +/- 0.2; LPS, 5.1 +/- 0.5; p < .05) at 28 weeks without evidence of alveolar septa destruction. Morphometric analysis of intravascular PMN showed an increase in the volume (V) of myeloperoxidase-containing azurophil granules (control, 6.1 +/- 1.3 microns3; LPS, 13.1 +/- 2.8 microns3; p < .05); a trend for a decrease in the V of specific granules (control, 15.8 +/- 3.4 microns3; LPS, 10.2 +/- 1.5 microns3; p = .09); an increase in the V of the cytoplasm (control, 37.3 +/- 6.4 microns3; LPS, 54.5 +/- 7.1 microns3; p < .05); and an increase in CD11/CD18 expressed as the number of gold particles per micrometer of cell surface membrane (G/micron) (control, 7.1 +/- 1.4 G/micron; LPS, 18.1 +/- 7.8 G/micron; p < .05). The results indicate that chronic endoxemia in rabbits, accelerates the release of PMN from the bone marrow, enhances the retention of both mature and immature activated PMN in the pulmonary microvessels, and causes alveolar wall thickening rather than emphysematous lung destruction.

Animals↗