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PubMed1983

Granulocytes and phorbol myristate acetate increase permeability to albumin of cultured endothelial monolayers and isolated perfused lungs. Role of oxygen radicals and granulocyte adherence.

Human granulocytes and phorbol myristate acetate (PMA) increased permeability to albumin of monolayers of cultured endothelial cells grown on micropore filters. Granulocytes from a patient with chronic granulomatous disease and PMA did not increase endothelial permeability to albumin, demonstrating that the increase in permeability is dependent on granulocyte-derived oxygen radicals. When granulocytes were separated from the endothelial cells by a micropore filter, granulocytes and PMA no longer increased endothelial permeability to albumin, demonstrating that PMA-stimulated granulocytes must be closely approximated to endothelial cells to increase endothelial permeability. The relevance of these in vitro findings to an intact microvasculature was confirmed by demonstrating that agents that reduce granulocyte adherence to endothelium reduce edema formed in isolated lungs by granulocytes and PMA, an oxygen radical dependent process. Pretreatment of granulocytes with cytochalasin B or addition of 2% dextran to isolated lung perfusates reduced granulocyte adherence and markedly reduced edema formation in isolated lungs. These studies demonstrate that PMA-stimulated granulocytes must be closely apposed to endothelial cells to increase endothelial permeability through an oxygen-radical-dependent mechanism, and they suggest that reduction of granulocyte adherence may protect against granulocyte-dependent edema.

Animals↗

PubMed1965

THE ROLE OF SERUM COMPLEMENT IN CHEMOTAXIS OF LEUKOCYTES IN VITRO.

By the use of chambers containing two compartments with an interposed micropore filter, chemotaxis of polymorphonuclear leukocytes (PMN's) in vitro was studied employing various agents that fixed serum complement (C'). Antigen-antibody complexes, zymosan, and aggregated human gamma globulin, in the presence of fresh rabbit, guinea pig, or mouse serum resulted in the migration of PMN's through the micropore filter. Pepsin-degraded rabbit antibody or unaltered duck serum containing antibody did not exhibit such activity after addition of antigen. Heating of the serum before treatment or the presence of EDTA prevented the generation of the chemotactic factor. The chemotactic factor could not be generated in whole serum from rabbits genetically deficient in C'. However, the defect in this rabbit serum could be corrected by addition of rabbit or human C'6. Serum of B10.D2 mice deficient in hemolytic C' also yielded poor chemotactic activity. Interaction of the first four reacting components of guinea pig C' did not result in significant chemotactic activity unless guinea pig euglobulin with heat labile components was also present. In rabbit serum, C'5 and C'6, when "activated" by interaction with the first four reacting components, behaved like a protein-protein complex and exhibited marked chemotactic activity. By employing conditions favoring dissociation of the complex, the individual components were isolated and shown to be chemotactically inactive. Upon recombination of the two components, however, activity reappeared. Using another approach, the C'5-C'6 complex was isolated intact, and shown to be chemotactically active while other fractions not containing these components were not active. It is postulated that the C'5-C'6 complex is the active chemotactic factor generated in serum after the addition of C'-fixing agents.

Animals↗

PubMed1992

An image processing workstation for automatic evaluation of human granulocyte motility.

Polymorphonuclear (PMN) motility is currently determined by a method using chemotactic chambers with micropore filters. PMN locomotive capacity is measured by evaluating the maximum distance travelled by the cells, or by counting the number of cells which have moved through the filter. Although in recent years some attempts to improve the analysis of chemotaxis data by computer-assisted systems have been made no technique has been shown capable of both measurement and correlation of these parameters in real time. In the present paper we describe an automated technique, based on a workstation capable of exploring microporous filters employed in chemotactic chambers and of measuring PMN motility in a reliable, reproducible, rapid manner, independent of the subjectivity of the operator. The measurement is carried out by custom software capable of undertaking computerized image analysis of microscopic fields acquired by a TV camera and of driving the motorized microscopic table. Depth of migration, cell distribution at each plane, correlation index of the random behaviour with the model described by a gaussian distribution and data about the patient or assay under study, are computed, displayed, stored in a data base and printed in real time.

Algorithms↗

PubMed1982

[Effects of protizinic acid on the prostaglandins system and the production of oxygen radicals].

Effects of protizinic acid (PRT) on prostaglandins (PG) and the production of oxygen radicals were compared with those of other non-steroidal anti-inflammatory agents. Oral administration of 30 mg/kg of PRT, indomethacin (IM), or ibuprofen (IB) significantly inhibited arachidonic acid-induced erythema in guinea pigs. Although 30 mg/kg of PRT significantly inhibited PGE2-induced erythema, IM and IB did not significantly inhibit it. PRT inhibited phospholipase A2 (PLA2) activity, and the IC50 value was 2.1 X 10-4 M. On the other hand, IM and IB exerted no effect on the PLA2 activity at 3 X 10-4 M. These results suggest that PRT possesses a broader pharmacological activity on the PG system than IM and IB. As for effects on the production of oxygen radicals, in order of relative inhibitory potency was PRT greater than metiazinic acid (MA) = IM greater than IB = phenylbutazone (PB) in the xanthine oxidase assay, PB great than IM greater than PRT greater than MA = IB in the rabbit neutrophil myeloperoxidase assay, and IM greater than PB greater than PRT greater than MA greater than IB in the guinea pig macrophage assay. In the rabbit neutrophil and aggregated IgG-bound micropore filter assay, the order was PRT greater than MA greater than PB greater than IM = IB. Thus, the inhibitory effects of PRT was verified in all experiments on the production of oxygen radicals in contrast to IB. In particular, it could be especially meaningful that PRT showed the most potent activity in the aggregated IgG-bound micropore filter assay which has been reported to be a good model for studying the pathogenesis of inflammatory diseases believed to be caused by immune complexes.

Animals↗

PubMed1989

Characterization of nonmotile neutrophil subpopulations in neonates and adults.

Previous studies have demonstrated that polymorphonuclear leukocytes (PMN) are not a homogeneous population of cells but differ significantly in their structure and function. PMN move at varying rates, and a fraction estimated from 20 to 70% do not move at all in response to chemotactic stimuli. To characterize this PMN subpopulation better, we studied PMN motility in neonates and adults using a polycarbonate micropore filter chemotactic assay and the 31D8 MAb. Most PMN strongly bind 31D8 MAb (31D8 "bright"), but a minority (31D8 "dull") weakly bind the antibody and in this respect are similar to immature PMN precursors. The 31D8 "dull" PMN have impaired function compared with 31D8 "bright" PMN. In the present study, a PMN subpopulation that failed to migrate using the micropore filter assay accounted for 58 +/- 7% of adult PMN and was similar to the migrating subpopulation in regard to viability and phagocytic function. The nonmotile subpopulation had a higher percentage of bands (5 +/- 3% versus 1 +/- 2%, p less than 0.01) and decreased binding of 31D8 MAb compared with the motile subpopulation. Neonates had a larger nonmigrating PMN subpopulation and 31D8 "dull" PMN subpopulation than those of adults (76 +/- 3% versus 58 +/- 7%, p = 0.04 and 26 +/- 11% versus 8 +/- 2%, p less than 0.01, respectively). These data indicate that although PMN appear morphologically as a homogeneous population of cells, there exists a viable, nonmotile PMN subpopulation that may be less mature than the motile PMN subpopulation. They also indicate that impaired neonatal PMN motility may be attributable in part to an increased size of the nonmotile PMN subpopulation.

Adult↗

PubMed1993

Effects of all-trans-retinoic acid on melanocyte adhesion and motility.

Human epidermal melanocytes were treated with all-trans-retinoic acid (RA) and examined for adhesion to bovine serum albumin-, fibronectin- and laminin-coated culture dishes. Control and treated cells were also examined for motility into micropore filters coated with the same proteins. Treatment of the cells with 3 x 10(-6) M RA for 3-4 days resulted in inhibition of attachment to all three substrates. Decreased attachment was observed within 1.5 h. Inhibition of attachment was not due to toxicity because differences between control and treated cells disappeared by 18 h, when most of the cells (approximately 75%) were attached and spread on all three substrates. The same treatment that inhibited adhesion also reduced migration into the interstices of micropore filters coated with the same three proteins. In additional experiments, human and mouse melanoma cell lines were examined in place of normal melanocytes. RA treatment also blocked adhesion and motility of these cells. The malignant melanoma cells were less sensitive to RA than normal melanocytes in the adhesion assay but were equally sensitive in the motility assay. The ability of RA to inhibit melanocyte adhesion and motility as well as melanocyte growth could explain, in part, the capacity of retinoids to modulate melanocyte function in hyperpigmented skin lesions.

Animals↗

PubMed1998

[The effect of PMN adhesion mediated by CD11b/CD18 on the increasing permeability of microvascular endothelial monolayer after severe burn injury].

OBJECTIVE: To detect the effect of burn-activated PMN adhesion and its adhesion molecule CD11b/CD18 on microvascular endothelial permeability using an experimental model of endothelial monolayer on polycarbonate microporous filters. METHODS: An experimental model for in vitro study of endothelal monolayer for permeability analysis was established. Seven groups were divided into according to the treatment of microvascular endothelial monolayer. Fluid filtration coeffecient(Kf) and albumin reflection coeffecient(delta) were measured after endothelial monolayer was perfused with albumin labelled by FITC. RESULTS: Burn-activated PMN could increase the level of fluid filtration coeffecient(Kf) and decrease the albumin reflection coeffecient(delta). Monoclonal antibody sealing off CD11b/CD18 on PMN provented the change of delta induced by burn-activated PMN. Another microporous filter interposed between PMNs and endothelial monolayer corrected the changes of Kf and delta. CONCLUSION: The permeability enhancing effect of PMNs may be attributed mainly to the PMN-EC adhesion mediated by CD11b/CD18. Blocking the PMN-EC over-adhesion in moderation may be helpful in reducing the lung injury due to severe burn injury.

Animals↗

PubMed1996

Transduction of specific inhibition of HuT 78 human T cell chemotaxis by type I vasoactive intestinal peptide receptors.

The major immunoregulatory effects of vasoactive intestinal peptide (VIP) are mediated by structurally distinct types I (VIPR1) and II (VIPR2) G protein-associated receptors on some T cells, B cells, and macrophages. Identification of the separate immunologic activities of each type of VIPR has been complicated by the usual expression of only VIPR2 or of VIPR1 and VIPR2 together by most human T cells obtainable in sufficient number for functional analyses. The results of reverse-transcription PCR, Western blot, and [125I]VIP-binding studies have established that HuT 78 cultured human lymphoma T cells bear a mean of 75,000 VIPR1s per cell with a mean Kd of 3.3 nM, which transduce mean maximal increases in intracellular concentration of cAMP of 2.1-fold (ED50 = 72 nM), but no VIPR2s. HuT 78 T cells, in contrast to T cells that express VIPR2, did not respond to VIP by chemotaxis through micropore filters without or with a top layer of basement membrane-like Matrigel. Matrix metalloproteinase (MMP)-dependent in situ cleavage of [3H]type IV human collagen in the layer of Matrigel by HuT 78 T cells also was not stimulated by VIP. In contrast, IL-4 and TNF-alpha both stimulated HuT 78 T cell chemotaxis and in situ MMP activity at respective optimal concentrations ranging from 3 x 10(-10) M to 3 x 10(-9) M and 10(-10) M to 3 x 10(-10) M. VIP inhibited significantly HuT 78 T cell chemotaxis through Matrigel in response to both IL-4 and TNF-alpha, as a result of suppression of both chemotactic mobility, assessed by migration through micropore filters without Matrigel, and in situ MMP activity. The transduction of opposite effects of VIP on T cell migration through a model basement membrane by VIPR1 and VIPR2 suggests that the net chemotactic response of most T cells to VIP is determined by the VIPR2/VIPR1 ratio and that the predominant expression of VIPR1 would stabilize T cell populations in lymphoid follicles and tissue infiltrates.

Base Sequence↗

PubMed1993

Functional variation in endogenous and exogenous immunoglobulin binding to bovine neutrophils relative to parturition.

Ten healthy first- and second-lactation Holstein cows were observed from 1 week before to 1 week after calving and at postpartum day 30 to determine polymorphonuclear neutrophil (PMN) functional variation and immunoglobulin binding profiles. Blood and mammary PMN were obtained 3 times weekly and within 24 hours of calving. Functional traits measured included phagocytosis of Staphylococcus aureus and in vitro chemotaxis through micropore filters in a Boyden chamber. Additionally, PMN were evaluated for endogenous binding of IgG1, IgG2, IgA, and IgM before and after in vitro chemotaxis. Exogenous binding of the same isotypes was determined after incubation in pooled colostrum, purified immunoglobulin, and pooled sera. Phagocytosis results indicated a significant and transient increase in percentage of milk PMN with associated, rather than phagocytosed, bacteria for 1 week after calving. Blood PMN phagocytosis was not significantly different during this period. Though total chemotaxis was essentially unchanged, the percentage of PMN that were unable to complete migration increased substantially on the day of calving, an effect that disappeared by postpartum day 4. A significant (P < 0.01) positive correlation (r = 0.29) between percentage of PMN migrating completely through the micropore filter and percentage of blood PMN with associated bacteria was observed. Changes were not observed in endogenous immunoglobulin binding, with the exception of a peak in relative fluorescence intensity for IgG1 on the day of calving; this disappeared within 2 days after calving. Correlations between relative intensities of IgG2 and IgM, and percentage of mammary neutrophils phagocytosing were 0.37 and 0.70.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Effects of acute exposure to hyperbaric oxygen on the rheology and morphology of the red blood cells in the rat.

The effects of acute exposure to hyperbaric oxygen (HBO) on the rheology and morphology of red blood cells (RBC) were studied in three groups of Sprague-Dawley rats: a control (CON) group comprised rats not exposed to HBO, a second group was exposed to HBO at 2.8 atm for 6 hr and studied immediately after the exposure (HBO), and a third group was examined after being allowed to recover in room air for 24 hr after exposure to HBO (REC). RBC deformability was assessed by two different techniques, the ektacytometer and the micropore filters. While the ektacytometer did not detect a significant difference among the three groups, the filtration method showed that acute exposure to HBO causes a significant increase in the filtration index (FI), denoting a significant reduction in RBC deformability. However, this reduction returned to the control level after 24 hr of recovery in air (FI, 7.1 +/- 1.2 in CON and 9.5 +/- 2.6 in REC compared to 36.2 +/- 3.7 in HBO, P < 0.0001). The morphology of RBC was studied by scanning electron microscopy. Immediately after exposure to HBO there was a marked increase in the echinocytes (41.6 +/- 5.9%) compared to a control level of 16.7 +/- 4.8%, P < 0.05. The increase in the echinocytes became less pronounced after 24 hr of recovery (23.5 +/- 9.3%). In conclusion, acute exposure to HBO causes significant alterations in RBC rheology and morphology. Due to the sensitivity of the micropore filters, we hypothesize that the decrease in RBC deformability might affect their flow through small-caliber blood vessels. These alterations, however, are reversible after a relatively short period of recovery in air.

Animals↗

PubMed1992

Mastoparan activates apical chloride and potassium conductances, decreases cell volume, and increases permeability of cultured epithelial cell monolayers.

Mastoparan is a tetradecapeptide. Mastoparan added to the apical surface of monolayers of Madin-Darby canine kidney (MDCK) epithelial cells, cultured on micropore filters, activated ion transport and increased the permeability of the paracellular pathway across the monolayers. In monolayers of similar MDCK cells in which the basolateral membrane was permeabilized with Staphylococcus aureus alpha toxin (Staph. alpha toxin), the effects of mastoparan on apical membrane ion conductances were dependent on the presence of guanosine triphosphate (GTP). Mastoparan and GTP increased apical membrane chloride conductance more than potassium conductance, with very little change in sodium conductance. In intact monolayers, addition of barium to the apical bath prevented mastoparan activation of ion transport and the increase in paracellular permeability. Increasing bath potassium to 130 mM also reduced ion transport and prevented the increase in paracellular permeability. We hypothesized that these observations could be linked by mastoparan activation of apical chloride and potassium conductances, with consequent decreases in cell volume and resultant increases in paracellular permeability. Addition of 270 mM mannitol to isosmotic media to decrease cell volume decreased MDCK monolayer transepithelial resistance. Addition of mastoparan to monolayers of MDCK cells grown on micropore filters decreased cell volume to the same extent as addition of 270 mM mannitol to isosmotic media. Addition of the potassium channel inhibitor, barium, prevented the decrease in cell volume in response to mastoparan. Mastoparan activates apical membrane chloride and potassium conductances in MDCK cells. The loss of these ions from the cells decreases cell volume, and the decrease in cell volume increases the permeability of the paracellular pathway.

Animals↗

PubMedDate not supplied

The effects of temperature on the filtration of diluted blood through 3 mu m and 5 mu m filters.

The effect of temperature on the flow of diluted blood [Hct = 0.21], through 5 mu m Nuclepore filters, is described by the Arrhenius equation with an energy of activation of 27.7 kJ/mol. Plasma, diluted with PBS, is almost three times less sensitive to temperature, with an energy of activation of 9.8 kJ/mol, while red cells are of intermediate sensitivity, with an energy of activation of 14.7 kJ/mol. The most sensitive elements to changes in temperature are leukocytes, with energies of activation of 31 kJ/mol and 35 kJ/mol for fast-flowing leukocytes (granulocytes and lymphocytes) and slow-flowing leukocytes (monocytes) respectively. Hence, the major determinants of the decline in filterability of blood through micropore filters are the leukocytes. This effect is compounded when blood is kept for 10 minutes or more at 10 degrees C due to activation of granulocytes, which leads to permanent pore blocking when the affected blood is filtered at room temperature. The combination of increased passage time of leukocytes through peripheral areas at abnormally low temperatures and subsequent activation might influence the flow of blood in non-affected tissues. The effect of temperature on the filterability of red blood cells through 3 mu m filters is not described by the Arrhenius equation and the deviations are seen as a gradual change of slope rather than a sharp break between two straight lines. The data are consistent with a gradual shift in rate limiting step away from the entry event into pores, which dominates at low temperature but becomes progressively less important at elevated temperatures. The changing parameter is probably the volume of the red cell, which is less important when flow is measured through 5 mu m pores.

Erythrocytes↗

PubMed1978

Concentration-time profile of phenytoin after admixture with small volumes of intravenous fluids.

A study was designed to determine if admixtures with small volumes of four intravenous fluids (0.45% sodium chloride, 0.9% sodium chloride, 5% dextrose in water and lactated Ringer's) maintain their phenytoin concentrations over a suitable period of time to allow intravenous infusion of the drug. Three phenytoin concentrations (4.6 mg/ml, 9.2 mg/ml and 18.4 mg/ml) were prepared by adding a sufficient volume of an i.v. fluid to the appropriate volume (10.0, 20.0 and 40.0 ml) of phenytoin sodium injection (46 mg of phenytoin acid/ml) to produce a total volume of 100 ml. Each admixture was visually inspected for crystallization, the pH of each solution was determined, and the solutions were filtered through a 0.22-micrometer micropore filter. Unfiltered and filtered aliquots of all solutions were collected at 0, 0.25, 0.5, 1, 4, 8 and 24 hours following admixture. Phenytoin concentrations did not decline systematically during this period nor was there a significant difference (p greater than 0.05) between unfiltered and filtered aliquots in any of the solutions studied, except for the 9.2 mg/ml concentration of 5% dextrose in water. The phenytoin concentrations in the 5% dextrose in water and lactated Ringer's solutions showed the greatest variability over the 24-hour period. The pH for all solutions ranged from 10.15 to 11.50. One-half normal saline and normal saline in small volumes appear to be suitable vehicles for intravenous infusion of phenytoin.

Chromatography, Gas↗

PubMed1991

[A severe epidemic of Streptococcus group G infection in hemodialysis: epidemiologic survey by molecular biology and preventive measures].

An outbreak of group G streptococci infection affected 6 patients of an hemodialysis unit. Group G streptococci were isolated from patients and from numerous atmospheric specimens, different parts of two dialysis machines, and two blankets, but from only one nurse on the hospital staff. Typing of group G streptococci by an improved method of DNA fingerprinting showed that the isolates from one patient, the nurse and the two blankets differed from one another. The group G streptococci were probably transmitted to patients by dialysis machines with defective microporous filters. No further case of group G streptococci infection was reported three years later since microporous guard filters were systematically doubled.

Adult↗

PubMed1985

Effects of humic and fulvic acids on poliovirus concentration from water by microporous filtration.

Because naturally occurring organic matter is thought to interfere with virus adsorption to microporous filters, humic and fulvic acids isolated from a highly colored, soft surface water were used as model organics in studies on poliovirus adsorption to and recovery from electropositive Virosorb 1MDS and electronegative Filterite filters. Solutions of activated carbon-treated tap water containing 3, 10, and 30-mg/liter concentrations of humic or fulvic acid were seeded with known amounts of poliovirus and processed with Virosorb 1MDS filters at pH 7.5 or Filterite filters at pH 3.5 (with and without 5 mM MgCl2). Organic acids caused appreciable reductions in virus adsorption and recovery efficiencies with both types of filter. Fulvic acid caused greater reductions in poliovirus recovery with Virosorb 1MDS filters than with Filterite filters. Fulvic acid interference with poliovirus recovery by Filterite filters was overcome by the presence of 5 mM MgCl2. Although humic acid reduced poliovirus recoveries by both types of filter, its greatest effect was on virus elution and recovery from Filterite filters. Single-particle analyses demonstrated MgCl2 enhancement of poliovirus association with both organic acids at pH 3.5. The mechanisms by which each organic acid reduced virus adsorption and recovery appeared to be different for each type of filter.

Adsorption↗

PubMed2004

[Angioplasty of intra-stent restenosis of a saphenous graft complicated by massive embolism].

The main risk of angioplasty of saphenous vein aortocoronary bypass grafts is myocardial infarction by distal embolism, explaining the introduction of systems of distal protection with encouraging results. Although embolism of an atheromatous stenosis is classical, that of intra-stent restenosis is exceptional. The authors report a very unusual case of atheromatous and/or thrombotic embolism occurring during angioplasty of an intra-stent restenosis which was recovered by a micropore filter system.

Aged↗

PubMed1979

Effect of micropore filtration on pulmonary function after massive transfusion.

1. This study demonstrates a positive correlation between the number and size of infused microaggregates and the subsequent abnormality in pulmonary function as measured by oxygenation and dead space. 2. No such correlation between the severity of injury and the altered pulmonary function or transfusion volume was demonstrated. 3. We were unable to demonstrate an advantage to the use of a 40 mu micropore filter in preventing the adult respiratory distress syndrome (ARDS) or in improving pulmonary function in our patients. 4. One explanation for the failure to demonstrate such an advantage is the low efficiency of the filter used.

Adolescent↗

PubMedDate not supplied

Observations on the removal of microaggregates from stored blood by centrifugation and filtration through a standard 170 micron transfusion filter.

The effect of centrifugation (5 minutes, 6900 X g) on the amount and particle size of blood microaggregates was studied in order to assess whether centrifugation and filtration by a standard (170 micron) transfusion set filter could be used for aggregate removal instead of microfiltration. Aggregates were quantitated using a method based on nylon screens with graded pore sizes. At the beginning of storage, centrifugation increased the particle size of aggregates only slightly. In 7-day-old blood, aggregates capable of passing a 160 micron screen amounted to 308 +/- 107 (SD) mg of debris protein per liter of blood before and 55 +/- 26 (SD) mg of protein (n = 10) after centrifugation. In 14-day-old blood centrifugation increased the size of aggregates even more effectively: 311 +/- 81 (SD) before and 29 +/- 14 (SD) mg of protein following centrifugation. Microaggregates can thus be removed effectively by the centrifugation and filtration method from blood stored for more than 1 week. A standard transfusion set filter can be used instead of a micropore filter.

Blood↗