Exclusion of dextrans by meshworks of collagenous fibres.

Insoluble collagen from human dermis was equilibrated in a physiological medium with mixtures of 3H2O and fluorescein-conjugated dextrans of different molecular weights. Dextrans of mol.wts. greater than 10(5) were excluded from a volume of 3.82+/-0.87 ml(S.D.) per g of collagen; dextrans of lower molecular weight occupied a larger volume. The apparent excluded volume was proportional to the weight of the collagen. Dansylated albumin behaved similarly to dextran; the polymeric collagen from rat skin exhibited a much larger excluded volume than the insoluble collagen. These results indicated that the volume available to the plasma proteins in human dermis was limited by insoluble collagen as well as by the glycosaminoglycans of the tissue.     

Effect of 100% O2 on passage of uncharged dextrans from blood to lung lymph

Since charge as well as size may influence the passage of plasma proteins from blood to lung lymph, we used uncharged dextrans as tracers to study the effects of hyperoxic lung injury on the molecular sieving properties of the pulmonary microcirculation in unanesthetized sheep. Polydisperse [3H]dextran was infused intravenously into five sheep before and after the animals breathed 100% O2 until lymph flow increased threefold (66-84 h). Lymph-to-plasma concentration ratios (L/P) were determined for [3H]dextran fractions of graded molecular sizes (1.6-8.4 nm effective radius) from samples obtained during the infusions. Before hyperoxia the blood-lymph barrier was highly restrictive to transport of [3H]dextrans above 5.0 nm in radius; steady-state L/P for these molecules averaged 0.03 or less. After the sheep breathed 100% O2, [3H]dextrans as large as 8.4 nm radius appeared in the lymph. Posthyperoxia, the L/P were significantly increased relative to prehyperoxia base-line values for every [3H]dextran fraction larger than 2.0 nm radius (P less than 0.05). In contrast, neither the L/P for albumin or total protein changed significantly. At autopsy, electron microscopy showed widespread damage to the endothelium of the alveolar capillaries with infrequent gaps between endothelial cells. In two control sheep, inhalation of compressed air for 96 h had no effect on lymph flow or L/P for the [3H]dextrans. We conclude that O2 poisoning reduced the selective sieving of uncharged dextrans across the blood-lymph barrier of the lungs and allowed larger dextrans to enter the lymph. These larger molecules may have leaked from the pulmonary microcirculation via disruptions in the continuity of the endothelial lining.     

Preparation of benzoyl dextran and its use in aqueous two-phase systems.

The graft modification of dextran with benzoyl groups has been studied. The factors that affect the degree of substitution of benzoyl dextran were investigated. Phase diagrams for aqueous two-phase systems composed of polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran have been determined. Phase separation was also obtained in aqueous solution of two benzoyl dextran polymers with different degrees of substitution. A four-phase system was obtained with a mixture of polyethylene glycol, dextran and two kinds of benzoyl dextrans. The partitioning of methylene blue and a Procion yellow HE-3G dextran derivative were studied in polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran two-phase systems and in systems of two benzoyl dextrans differing in degree of substitution. The proteins bovine serum albumin and glucose-6-phosphate dehydrogenase were partitioned in polyethylene glycol/benzoyl dextran aqueous two-phase systems and the effect of the degree of substitution of benzoyl dextran was studied. Chlorella pyrenoidosa, thylakoid membrane vesicles, plasma membrane vesicles and chloroplasts were partitioned in polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran two-phase systems, and in a polyethylene glycol/dextran/benzoyl dextran four-phase system.     

Atriopeptin does not augment the transvascular flux of macromolecules in the hamster cheek pouch.

Natriuretic peptides elaborated by atrial myocytes promote marked renal sodium and water excretion as a mechanism for fluid and electrolyte balance. Recent evidence suggests that atriopeptin (ANP) also targets the non-renal vasculature as a site for enhanced fluid exchange. It remains unclear whether ANP alters microvascular integrity to facilitate the efflux of both plasma and proteins across the endothelial barrier, or if fluid exchange is selectively enhanced. This study evaluated the influence of ANP on macromolecular transport through the direct observation of microvessels in the hamster cheek pouch using fluorescent intravital microscopy. Fluorescein isothiocyanate conjugated to either bovine serum albumin or dextran 150,000 Mw was utilized as a permeability probe. Macromolecular efflux was quantified as fluorochrome clearance. The clearance of fluorescein-conjugated bovine serum albumin (57.94 +/- 7.03) or fluorescein-conjugated dextran 150 (4.09 +/- 1.35) remained unaltered by intravascular injection of 1 microgram/kg ANP. Topical application of 40 ng to cheek pouch microvessels produced similar results. All pouches demonstrated positive leakage response to histamine 2.5 x 10(-6) M, increasing fluorochrome clearance approximately 2- to 11-fold. Bolus injection of 1 microgram/kg ANP reduced mean arterial pressure, increased urine flow from 6.63 +/- 2.59 microliters/min to 8.20 +/- 6.13 microliters/min, and elevated sodium excretion from 1.37 +/- 0.49 microEq/min to 2.54 +/- 0.99 microEq/min. These results suggest that ANP fails to significantly alter the integrity of the protein-transporting channels in the microvascular exchange barrier.     

Neutral and DEAE dextrans as tracers for assessing lung microvascular barrier permeability and integrity.

Steady-state lymph-to-plasma concentration ratios (L/Ps) of neutral dextrans, cationic DEAE dextrans, and endogenous proteins were determined under normal and increased permeability conditions in six unanesthetized yearling sheep prepared with chronic lung lymph fistulas. Fluorescent dextrans with radii ranging from 1 to 30 nm were intravenously infused, and after 24 h, perilla ketone (PK) was given to alter permeability while the dextran infusion was maintained. Plasma and lymph samples were collected before and after PK administration and analyzed for dextran and protein concentrations after high-performance liquid chromatography size separation. Under both baseline and increased permeability conditions, DEAE dextrans had higher L/Ps than neutral dextrans of similar size but lower L/Ps than proteins of similar size. Comparison of L/Ps before and after PK revealed that the percentage change in permeability for neutral and DEAE dextrans was significantly larger than that for proteins. These results suggest that 1) the pulmonary microvascular barrier behaves as a net negative barrier, 2) some transport mechanisms for proteins and dextrans are different, and 3) neutral and cationic dextrans are more sensitive markers than proteins of the same size for assessing changes in pulmonary capillary permeability.     

Erythrocytes sedimentation profiles under gravitational field as determined by He-Ne laser. VII. Influence of dextrans, albumin and saline on cellular aggregation and sedimentation rate

The erythrocytes sedimentation profiles (ESP) of normal blood and of blood mixed with saline, albumin (7%), and various molecular weight dextrans of different concentrations, at various height and widths of the sample holder are determined. These observations show that the sedimentation characteristics of the erythrocytes depend on the influence of these substitutes on the plasma and cellular constituents. The normalised aggregation and the sedimentation rate, as determined from these profiles, show that the dextran 40 and dextran 70 retard the erythrocytes sedimentation, for high molecular weight it is similar to that of normal blood and is the maximum for saline. This change for high molecular weight dextrans could be attributed to the enhanced aggregation tendency of erythrocytes and for saline to the enhanced sedimentation due to decrease in the viscosity and density of suspending medium. The influence of the various concentrations of dextrans on these parameters has been determined.     

Passage of uncharged dextrans from blood to lung lymph in awake sheep

To examine how molecular size alone influences the passage of macromolecules from the pulmonary microcirculation into lymph collected from the caudal mediastinal lymph node of the sheep, we infused polydisperse uncharged [3H]dextrans intravenously at a constant rate over a period of 7.5 h in nine awake sheep with lung lymph fistulas. Lymph and plasma were collected during hours 5.5-7.5 of the infusions, and the [3H]dextrans were separated by molecular sieve chromatography into fractions that ranged from 1.6 to 8.4 nm in effective molecular (Stokes-Einstein) radius. Lymph-to-plasma (L/P) ratios for [3H]dextrans were near 1.0 at 1.6-nm radius, decreased with increasing molecular size, and approached zero at radii above 5.0 nm. We confirmed that these L/P ratios represented steady-state values by extending the duration of the infusion to approximately 30 h in two of the nine sheep and finding that the L/P ratios remained unchanged. These results were consistent with molecular sieving through a homoporous membrane with cylindrical pores of 5.0-nm radius. We also found that the L/P ratio for albumin [0.76 +/- 0.13 (SE)] in five of the same sheep was much higher than that for the [3H]dextran fraction of the same effective molecular radius [0.11 +/- 0.02 (SE)]. These results suggest that the movement of macromolecules from the pulmonary microcirculation into pulmonary lymph collected from the caudal mediastinal node of the sheep is influenced by both molecular size and molecular charge and that, compared with uncharged dextrans, the steady-state passage of anionic endogenous proteins from plasma to lymph is enhanced.     

Glomerular and postglomerular transcapillary exchange in dog kidney

The multiple-indicator dilution technique (MIDT) was used to study glomerular and postglomerular permselectivity to neutral dextran molecular weight markers in anesthetized dogs undergoing mannitol diuresis. Renal vein and urine outflow curves were obtained after an intraarterial pulse injection of 125I-labeled albumin (plasma reference), creatinine (extracellular reference), [14C]inulin, and chromatographically homogeneous 3H-labeled neutral dextrans. The urine recovery data reflect solute losses across the glomerulus. The renal vein outflow curves contain information about both glomerular and postglomerular extraction. For dextrans less than 13,500 daltons the urine transit pattern was superimposed on the glomerular markers creatinine and inulin. Relative to 125I-labeled albumin, the renal vein recoveries for creatinine, inulin, and dextrans (less than 13,500 daltons) were all equal. The renal vein mean transit time (tdextran) was greater than talbumin and less than tcreatinine. With increasing dextran size, tdextran progressively decreased. Eventually for dextrans greater than 15,500 daltons, tdextran became equal to talbumin in the renal vein, whereas urine recovery relative to inulin decreased with increasing size. Urine recovery of 3H-labeled dextran (ED) relative to inulin (Ei) provided a measure of unidirectional fractional glomerular extraction (ED/Ei). Constant infusion fractional clearance measurement of the same dextrans [U/P)D/(U/P)i) was found to equal ED/Ei obtained from the MIDT. Within the autoregulatory range of glomerular filtration, ED/Ei was invariant with reduction in renal plasma flow. Therefore, under the experimental conditions employed in the present study, diffusion across the glomerulus was negligible relative to convection. This permitted estimation of the reflection coefficients for the series of neutral dextrans. Postglomerular extraction was markedly flow dependent, which implies a major diffusion limitation. Application of a barrier-limited distributed model permitted quantitation of postglomerular capillary permeability coefficients for neutral markers of less than 5000 daltons.     

Assessment of red blood cell aggregation with dextran by ultrasonic interferometry.

Aggregation of human red blood cells (RBCs) induced by dextrans of various molecular weight has been studied by using a new ultrasonic interferometry method. This method, based on A-mode echography, allowed for the measurement of the accumulation rate of particles on a solid plate which is related to their sedimentation rate (i.e., to their mean size). The initial aggregation process, the mean and the maximum sedimentation rate of aggregates and the packing of the sedimented RBCs have been investigated. Effects of hematocrit, molecular weight of dextrans and inhibition by dextran 40 on the RBC aggregation induced by dextran of higher molecular weight have been determined by analysing variations of the aggregate size. Results obtained confirm the aggregation effect of dextrans of molecular weights equal or higher than 70,000 dalton and disaggregation effect of dextran 40,000 dalton on aggregation by dextrans of higher molecular weight.     

Decreased binding of cytotoxic antibody by developing Schistosoma mansoni. Evidence for a surface change independent of host antigen adsorption and membrane turnover.

Schistosoma mansoni schistosomula maintained in chemically defined culture media became increasingly resistant to the cytotoxic effects of infected guinea pig serum. Two- and 6-day-old schistosomula recovered from mice showed no uptake of IgG antibody from infected guinea pig serum, as revealed by the indirect fluorescent antibody test. Results from this test remained negative when the schistosomula were tested at 0 C, after exposure to drugs which inhibit synthetic and secretory processes, or after being killed by heat or formalin. In contrast, new schistosomula collected within 3 hr after skin penetration bound IgG from infection serum under all test conditions, and showed increased susceptibility to cytotoxicity after exposure to various drugs. It thus appears that soon after skin penetration schistosomula undergo surface changes which prevent binding of antibody from infection serum. These changes can apparently take place in the absence of host antigens, and once they have occurred, do not depend on worm physiological processes for their function.     

In vitro culture of Trichobilharzia ocellata

Cercariae and schistosomula of Trichobilharzia ocellata were cultured to the organogeny stage in vitro in a medium based on Earle's saline (gassed with 5% CO₂ in air) and containing lactalbumen hydrolysate and duck or chicken serum together with homologous red cells. Similar results were obtained irrespective of whether cercariae, schistosomula recovered from the skin of ducklings or chickens, or schistosomula recovered after cercariae had penetrated gelatine membranes were used to initiate cultures. A lipid coat developed around the body of each worm during the first 2–3 days in vitro, but subsequently it became dislodged. The worms ingested red cells, and the caeca became dark with haematin after a few days but by day 10 they were generally translucent. Maximum development was achieved at day 12; by this time males had attained a length of 2·1 mm and females 1·4 mm. Worms cultured for 7 and 9 days were injected into the leg veins of ducks and patent infections were established.     

Trypanosoma lewisi: accumulation of antigen-specific host IgG as a component of the surface coat during the course of infection in the rat.

Naturally acquired host IgG, adsorbed to the surface of Trypanosoma lewisi during the course of infection in the rat, was labeled with fluorescein-conjugated rabbit IgG, or Fab fragments of this IgG, directed against rat IgG. The intensity of fluorescent labeling increases with time, concomitant with the increase in anti-T. lewisi activity of host plasma. Trypanosomes harvested from immunosuppressed hosts lack detectable surface IgG. Trypanosomes having little or no detectable surface IgG (harvested from immunosuppressed hosts or early in the infection from immunocompetent hosts) can adsorb IgG from serum with ablastic activity only (obtained from other infected rats between the first and second crises and adsorbed to remove trypanocidal antibodies), but not from normal serum. Therefore, the absence of detectable surface IgG on such cells is not caused by the parasites' inability to adsorb host IgG, but rather results from the immune state of the host. Hence surface IgG on T. lewisi is specific antibody. Host albumin is nonspecifically adsorbed, in contrast to IgG. Trypanosomes from immuno-suppressed and immunocompetent rats were positive and visually indistinguishable from each other when labeled with anti-rat albumin, and were equally agglutinable with anti-rat albumin serum.     

Schistosoma mansoni proteases Sm31 (cathepsin B) and Sm32 (legumain) are expressed in the cecum and protonephridia of cercariae.

Adult Schistosoma mansoni parasites live in the bloodstream of their vertebrate hosts where they consume red blood cells. Hemoglobin, released from the ingested red blood cells, is degraded by a variety of parasite proteases, including Sm31 (cathepsin B) and Sm32 (schistosome legumain). In this study the localization pattern of the Sm31 and Sm32 enzymes in cercariae (the infectious life cycle stage) was examined. Antibodies generated against recombinant Sm31 and Sm32 recognize their respective proteins in Western blots of soluble parasite extracts. Highest levels are seen in adult female extracts, whereas the level of both proteins is below detection in cercarial extracts. However, in fixed, whole cercariae, both proteins are seen in the cecum and protonephridia. In the cecum, the staining pattern has a granular appearance, suggesting that the proteins are packaged in vesicles. In the protonephridial system, Sm31 and Sm32 are detected in all 8 flame cells in the cercarial body and in both flame cells in the cercarial tail. The distribution of the 2 proteins differs in the flame cells. Examination of immunostained cercariae using laser scanning confocal microscopy shows that whereas Sm31 is located in the tubule cell, Sm32 is found in both the tubule cell and its adjoining cap cell. These findings suggest that the proteins are involved in the proposed excretory and osmoregulatory roles of flame cells.     

Target cell deformability determines the type of phagocytic mechanism used by Entamoeba histolytica-like,Laredo strain

Summary— Morphological study of red blood cell phagocytosis by Entamoeba histolytica-like (Laredo strain) has shown that this amoeba is able to ingest by two distinct mechanisms. One is classical phagocytosis and the other is by suction or microphagocytosis. Rigidification of red blood cells by treatment with glutaraldehyde shows that there is a correlation between the deformability of the ingested cell and the type of phagocytosis observed. Indeed, as the red cells become more rigid, less microphagocytosis is observed. To demonstrate that this shift in phagocytic mechanisms is not induced by the modification of a surface receptor by the glutaraldehyde treatment, the amoebas were fed with erythrocyte ghosts. Since these have lost most of their hemoglobin content, they are less rigid than the intact erythrocytes. The ghosts, even after glutaraldehyde treatment, are always ingested by microphagocytosis. These results have therefore led us to conclude that the type of erythrocyte phagocytosis used by E histolytica-like (Laredo strain) is determined by the deformability of the targetted red blood cells.     

Permselectivity of the glomerular capillary wall to macromolecules. II. Experimental studies in rats using neutral dextran.

To determine the permselectivity characteristics of the glomerular capillary wall, known molecular size fractions of [3H]dextran, prepared by gel chromatography, were infused into normally hydrated Wistar rats, thus permitting simultaneous measurement of Bowman's space/plasma water (BS/P) and urine/plasma water (U/P) concentration ratios, along with glomerular pressures and flows. Since (BS/P)inulin = 1.01 +/- 0.01 SE(n = 34, radius = approximately 14 A) and since (BS/P)dextran/(BS/P)inulin equaled (U/P)dextran/(U/P)inulin for dextrans ranging in molecular radius from 21 to 35 A, these findings validate that dextrans are neither secreted nor reabsorbed. For dextran radii of 20, 24, 28, 32, 36, 40, and 44 A, (U/P)dextran/(U/P)inulin averaged 0.99, 0.92, 0.69, 0.42, 0.19, 0.06, and 0.01, respectively. In accord with theoretical predictions that these fractional dextran clearances should vary appreciably with changes in glomerular transcapillary pressures and flows, an increase in glomerular plasma flow rate, induced in these same rats by plasma volume expansion, resulted in a highly significant lowering of fractional clearance of all but the smallest and largest dextrans studied. These findings emphasize that fractional solute clearances alone are inadequate to describe the permselective properties of the glomerular capillary wall unless glomerular pressures and flows are also known. This sensitivity of fractional dextran clearance to changes in plasma flow indicates that dextrans are transported across the capillary not only by bulk flow but also to an important extent by diffusion.     

The immune response in the hamster. VII. Studies on cytophilic immunoglobulin.

Hamster 7S IgG1 and IgG2 antibodies to hen-egg albumin (HEA) were tested for their capacity to bind to macrophage cytophilic Ig receptors. Both IgG1 and IgG2 were cytophilic for hamster macrophages though the membrane receptor had a predominant specificity for IgG1. Hamster IgG1 bound primarily to homologous macrophages whereas IgG2 bound to macrophages from other rodent species as well. The binding of hamster Ig to hamster macrophages was inhibited by a wide range of heterologous rodent sera. The only exception was guinea pig serum since guinea pig IgG2 was found to bind only to homologous macrophages. Sheep red blood cells (SRBC) coated with hamster IgG2 were ingested by macrophages more readily than those coated with hamster IgG1. Thus, there appeared to be a paradoxical relationship between the apparently strong affinity of IgG1 for the hamster macrophage Ig receptor and its reactivity weak ingestion promoting activity. Implications of this phenomenon are discussed.     

Hydrolysis of fourteen native dextrans by arthrobacter isomaltodextranase and correlation with dextran structure

The isomaltodextranase (EC 3.2.1.94) from Arthrobacter globiformis T6 hydrolysed thirteen dextrans to various extents (11?64% after 13 days) at initially large but gradually decreasing rates. Dextran B-1355 fraction S was, unlike the other dextrans, hydrolysed by the dextranase initially at the lowest rate among the dextrans used, but the rate was maintained for a long period with little decrease, so that the hydrolysis reached as high as 85% after 13 days. Paper chromatography of these dextran digests revealed that this dextranase produces in addition to isomaltose, one or two trisaccharides [isomaltose residues substituted by (1 →2)-, (1→3)-, or (1→4)-α-D-glucopyranosyl groups at the non-reducing D-glucopyranosyl residues] from every dextran used. It is evident that the non-(1→6)-linkages of these trisaccharide products constitute the “anomalous” linkages of the corresponding dextrans. The relative amounts of these trisaccharide products appear to indicate the approxima te relative amounts of a particular linkage among the dextrans, or the relative amounts of two kinds of linkages of each dextran. The kinds and the relative amounts of “anomalous” linkages of some dextrans were established on the basis of the trisaccharides produced by isomaltodextranase.     

Size determination of red blood cell aggregates induced by dextran using ultrasound backscattering phenomenon.

Different methods are commonly used to study the red blood cell aggregation phenomenon. The major interest of the ultrasonic method presently discussed is to assess the mean size of red blood cell (RBC) aggregates by measuring ultrasonic intensity backscattered by blood. Applying Rayleigh theory of sound to blood medium, one can show that the scattered ultrasonic intensity is proportional to the 6th power of the size of the RBC aggregates. The ultrasonic method is used to evaluate the mean size of RBC aggregates induced by dextrans. RBCs are suspended at various hematocrits H, in solution of dextrans of different molecular weights M and at different weight concentrations Cw. Results are presented by using the ultrasonic backscattering coefficient chi which is a relevant quantity in a scattering experiment. For suspensions of RBCs aggregated with dextran of molecular weight 70,000 dalton (dextran 70) at concentration Cw = 40 g/l, variations of chi as a function of H are similar to those obtained for normal blood. At a fixed hematocrit, variation of chi versus Cw for dextran 70 exhibits a maximum at 40 g/l. In the case of RBCs suspended at hematocrit 20% and aggregated with dextrans of molecular weight M, 70,000 less than or equal to M less than or equal to 2,000,000, the variations of chi versus molar concentration Cm are similar to those of the microscopic aggregation index defined by Chien (1). Finally, a statistical model of the blood structure previously described (2) is applied to evaluate the mean size of the aggregates. According to this model, the mean size of aggregates is independent of hematocrit for H less than or equal to 40% and independent of the molecular weight of dextran for M greater than or equal to 150,000 dalton.