Immunogenicity of synthetic peptides from circumsporozoite protein of Plasmodium falciparum.

In a study of recombinant proteins that might be useful in developing a vaccine against malaria, synthetic peptides from the circumsporozoite (CS) protein of Plasmodium falciparum were found to be immunogenic for mice and rabbits. Antibody to peptides from the repeating region of the CS protein recognized native CS protein and blocked sporozoite invasion of human hepatoma cells in vitro. Antibodies to peptides from regions I and II had no biologic activity, although antibody to region I recognized processed CS protein by Western blot analysis. These data support the feasibility of developing a vaccine against the sporozoite stage of the malaria parasite by using synthetic peptides of the repeating region of the CS protein conjugated to a carrier protein.

Regulation of water balance between blood and lymph in the frog, Rana pipiens.

To determine the relative importance of the lymphatic system and transcapillary reabsorption in maintaining water distribution between blood and lymph in Rana pipiens, the hematocrit (HCT) was measured (12 frogs) when each frog was: 1) under tricaine methanesulfonate (MS222) anesthesia, which inactivates the lymph hearts, 2) conscious at rest (control), and 3) conscious after activity. Arterial pressure (AP), lymph-heart rate (LHR), and plasma and lymph protein concentrations and colloid osmotic pressures (COP) were also measured. The average HCT during control state was 26.0 +/- 5.2% (SD), AP was 21.9 +/- 5.2 mmHg, and LHR was 86 +/- 13 bpm. MS222 increased HCT (42.6 +/- 7.9%), and did not affect AP. Experiments, in which plasma volumes were determined by intra-arterial injection of Evans blue-labeled albumin (EB-BSA) (16 frogs), showed that the increased HCT reflected a reduced plasma volume. Activity did not affect HCT, but increased AP (33.1 +/- 6.4 mmHg, p < 0.01), and LHR (115 +/- 26 bpm, p < 0.01). Thus lymph hearts are largely responsible for maintaining blood volume, and changes in transcapillary filtration rate are rapidly mirrored by changes in lymph heart function. Plasma and lymph COP values after MS222 or activity were not significantly different from control. This observation is consistent with the protein and fluid fluxes resulting from Starling forces if an average reflection coefficient to protein, sigma, of 0.5 is assumed, which is consistent with our EB-BSA data.

Fluid exchange across single capillaries in rat intestinal muscle.

Previous studies suggest that large differences exist among fluid exchange parameters in the mesenteric, muscle, and mucosal regions of the small intestine. However, few quantitative data from the separate regions, with the exception of the mesentery, are available for comparison. In this study, quantitative measurements of hydraulic conductivities (LP) and occluded effective pressures (P'e) were made on single capillaries in rat intestinal muscle. The microcirculation of longitudinal intestinal muscle was viewed through a microscope using the method of Bohlen and Gore. The single-occlusion method of Lee, Smaje, and Zweifach was used to estimate LP and P'e in single capillaries. Details of the procedures for analyzing the experimental data using the Lee analytical model are included in an APPENDIX. Measurements were made at four different sites in each capillary studied so gradients in LP could be quantitated and variations in P'e could be detected. The average LP at the venous end of the capillaries was nearly seven times greater than LP at the arterial end. The average LP at the arterial end [18.6 +/- 2.3(SE)% of the distance (%L/Lo) from the fifth-order arterioles] was 1.32 +/- 0.4 X 10(-2) micrometers.s-1.cmH2O-1. The average LP at the venous end (%L/Lo = 81.7 +/- 2.2%) was 9.15 +/- 0.34 X 10(-2) micrometers.s-1.cmH2O-1. Values of LP at intermediate capillary locations 39.9 +/- 2.3 and 61.3 +/- 3.2% were 2.17 +/- 0.34 and 4.48 +/- 0.62 X 10(-2) micrometers.s-1.cmH2O-1, respectively. The total mean LP for all the data (132 samples, 33 capillaries, 10 rats) was 4.19 +/- 0.61 X 10(-2) micrometers.s-1.cmH2O-1. Comparison of these data with results from other tissues indicates that LP of intestinal muscle capillaries is 1.7 times greater than LP of omental capillaries and three times greater than LP of mesenteric capillaries. Values of P'e were corrected for the unoccluded state and were used to calculate total transcapillary pressures (delta P). The results suggested that the intestinal muscle layers were well hydrated and that tissue hydrostatic pressures were positive. Transcapillary fluid fluxes (JVo) at different sites on the capillaries were estimated from the relationship, JVo = LP X delta P. The results imply that intestinal muscle capillaries are primarily an absorptive network when systemic arterial pressure and capillary pressures are normal.

The mechanics of arteriole-tissue interaction.

Arterioles are embedded in the extensive connective tissue matrix of the interstitium. Mechanical interactions with the interstitium may affect the length-tension characteristics of arterioles, and thus affect their reactivity. However, no studies have adequately characterized the coupling between arterioles and the interstitium or investigated how the interstitium might change the physiological expression of arterioles. Therefore, the goal of this project was to investigate the mechanical interactions between arterioles and the interstitium and then to predict the physiological consequences of these interactions. We measured in situ the mechanical coupling of arterioles to the interstitium, the mechanical properties of the interstitium, and the structure of the interstitium in the hamster cheek pouch. We demonstrated that there are mechanical interactions between arterioles and the interstitium that are mediated both through direct connections and through the movement of extracellular fluid through the connective tissue network. We also found that the elastic modulus of the interstitium increases in the vicinity of the arteriole. Finally, both the mechanical coupling of arterioles to the interstitium and the mechanical properties of the interstitium are explained by the structure of the connective tissue matrix. The arterioles appear to be connected to adjacent fibroblasts and fibrocytes by collagen fibrils. These cells are in turn connected to the fiber matrix of the interstitium. Furthermore, the presence of these cells may explain the mechanical heterogeneity of the interstitium. We propose that the physiological role of the interstitium surrounding arterioles is to protect arterioles from stretching and deformation of the tissue while allowing these vessels to constrict freely.

Determinants of cardiac function: simulation of a dynamic cardiac pump for physiology instruction.

A computer model is described that simulates the cardiac cycle of a mammalian heart. The model emphasizes the pressure-volume plot as a teaching tool to explain the behavior of the heart as a pump. It exhibits realistic responses to changes in preload, afterload, contractility, and heart rate while displaying time-dependent changes in pressure and volume in addition to the pressure versus volume plot. It differs from previous models by graphing these parameters on a beat-to-beat basis, allowing visualization of the dynamic adaptation of the pumping heart to various stimuli. A system diagram is also included to further promote student understanding of the physiology of cardiac function. The model is useful for teaching this topic to medical, graduate, or undergraduate students. It may also be used as a self-directed computer laboratory exercise.

Pressure regulation in the microcirculation.

The results of direct pressure measurements are described which demonstrate that pressures in a certain fraction of mesenteric capillaries remain remarkably constant during large changes in systemic pressure. The results of isogravimetric studies, reported in the literature, are also described which indicate that this phenomenon may also occur in the intestine. The question is raised whether capillary pressures may therefore be regulated. Pressures recorded from mesenteric arterioles and capillaries are shown which indicate that maintenance of a constant capillary pressure is primarily the consequence of the vascular architecture peculiar to this tissue, and is merely a secondary reflection of mechanisms associated with flow regulation. The results of direct pressure measurements recorded in the microcirculation of intestinal muscle are also shown. These data indicate that capillary pressures in innervated, denervated, and xylocaine-treated intestinal muscle change in direct proportion to variations in arterial pressure. It is concluded that capillary pressures in the intestinal muscle layers are therefore not regulated, so that the observation that capillary pressures may be maintained is probably a phenomenon unique to the mesentery. Pressures recorded from capillaries in the mucosal villi are also shown and compared to capillary pressures measured in the microvasculature of mesentery and intestinal muscle. When systemic pressure was normal (107 +/- 10 mm Hg), capillary pressure in the mesentery averaged 30 to 33 mm Hg; capillary pressures in the intestinal muscle averaged 22 to 24 mm Hg; and capillary pressures in the mucosal villi averaged 13 to 15 mm Hg. These data suggest that mesenteric capillaries are primarily a filtering network; intestinal muscle capillaries are normally in fluid balance; whereas at rest mucosal capillaries are primarily absorptive. These pressures, recorded from the three major regions of the rat intestine, were used to calculate a weighted average for the whole organ. The calculated value, based on assumed values for relative capillary densities, was 17 mm Hg. This result compares favorably with data from whole organ, isogravimetric studies, and may clarify some of the apparent discrepancies between previous isogravimetric and servopressure studies.

A new preparation for microcirculatory studies of the hamster cheek pouch.

Existing methods of preparing the hamster cheek pouch for observation under an intravital microscope have several disadvantages. The everted method, described by Duling (Microvasc. Res. 5: 423-429, 1973), appears to restrict blood flow by placing unnatural tension on the retractor muscle and by requiring an incision in the tip of the pouch. The method of Yamaki et al. (Microvasc. Res. 21: 299-301, 1981) requires an incision in the tip of the pouch and complete disconnection of the retractor muscle. The chamber method of Greenblatt et al. (Microvasc. Res. 1: 420-432, 1969) has a limited optical resolution because the tissue cannot easily be transilluminated with properly condensed light. We have devised a less traumatic method of preparing the pouch, which eliminates these disadvantages. The hamster is anesthetized, and a thin, glass support plate is inserted into the left cheek pouch. The plate is constructed and positioned so that it does not restrict flow to any part of the pouch. The free end of the plate is secured to the animal stage. An incision is made in the skin to expose the cheek pouch, and the loose, avascular connective tissue investing the pouch and the retractor muscle is removed. The pouch is positioned at an angle of 20 degrees from the hamster's body in a temperature-controlled chamber over a standard microscope condensor system. Throughout both surgical and experimental procedures, the pouch is superfused with Ringer-bicarbonate solution at 37.5 degrees C. This preparation minimizes surgical trauma and allows the entire vascular supply to the cheek pouch to be studied.

Capillary pressures in rat intestinal muscle and mucosal villi during venous pressure elevation.

Whole-organ experiments designed to estimate the capillary filtration coefficient require information about the numerical relationship between capillary pressure and venous pressure. Indirect estimates using isogravimetric and isovolumetric methods indicate that 62-85% of a step change in venous pressure reaches the intestinal capillaries, taken as a whole. We have made direct measurements of capillary pressure with a servo-null micropressure system in the microcirculation of both the intestinal muscle and the mucosal villi of rats during local elevation of venous pressure. Consistent regional differences in the relationship between capillary pressure and venous pressure were observed. During increased venous pressure, submucosal arterioles constricted, while muscularis arterioles dilated. The diameter changes of the small arterioles were consistent with blood flow redistribution from mucosa to muscle during venous pressure elevation, but inconsistent with a pure myogenic response. These data raise questions about the exact role for the expression of the myogenic response during venous pressure elevation in the intestine and about previous interpretations of whole-organ experiments concerned with intestinal blood flow and fluid exchange.

A comparison of capillary hydraulic conductivities in postural and locomotor muscle.

In a comparative skeletal muscle study Folkow and Halicka (Microvasc. Res. 1: 1-14, 1968) reported that the capillary filtration coefficient (CFC) of postural (red) muscle was two times the CFC of locomotor (white) muscle. It was concluded that the twofold difference in CFC was due solely to a difference in the perfused capillary surface areas (Sf) of red vs. white muscle. However, CFC is the product of capillary hydraulic conductivity (LP) and Sf. Hence their conclusion assumed that the average LP of red muscle capillaries is exactly equal to the average LP of white muscle capillaries. The following study was undertaken to test the validity of this assumption. The microocclusion procedures and analytical model described by Lee et al. (Circ. Res. 28: 358-370, 1971) and Gore [Am. J. Physiol. 242 (Heart Circ. Physiol. 11): H268-H287, 1982] were used to determine LP. Independent measurements of LP were recorded from single capillaries in red, anterior latissimus dorsi (ALD) and white, posterior latissimus dorsi (PLD) muscles of chickens anesthetized with L.A. Thesia. We found that the mean capillary hydraulic conductivity in postural muscle [(LP)ALD = 0.20 +/- 0.06 (SE) micrometers . s-1 . cmH2O-1 (n = 11)] was significantly different from the mean capillary hydraulic conductivity in locomotor muscle [(LP)PLD = 0.061 +/- 0.01 micrometers . s-1 . cmH2O-1 (n = 14)] (P less than 0.05). These results provide direct evidence that observed differences in red vs. white muscle CFC's may not be due solely to different perfused capillary surface areas but may also be due to differences in capillary hydraulic conductivity.

Double-barrel pipette system for microinjection.

A method for constructing double-barrel micropipettes is described. The pipettes are made from theta-tubing, which permits easy beveling of the tips. The two sides of the system operate independently and are suitable for simultaneous microinjection, microperfusion, or servo-null micropressure measurements.

Length-tension relationship of vascular smooth muscle in single arterioles.

Longitudinal response gradients in the microcirculation may in part be explained in terms of the length-tension relationship of vascular smooth muscle at different points along the vascular tree. To test this hypothesis, four branching orders of arterial vessels (20-80 microns ID) were dissected from the hamster cheek pouch and cannulated with concentric micropipettes. Intraluminal pressure was monitored with a servo-null micropipette, and arteriolar dimensions were measured using a videomicrometer. All arterioles developed spontaneous tone in physiological saline solution. Pressure-diameter curves were recorded for maximally activated vessels and for passive vessels. Maximal active wall tension varied nearly threefold, but maximal active medial wall stress (approximately 4 x 10(6) dyn/cm2) varied only approximately 20% between the different vessel orders. These data support the concept that smooth muscle cells from vessels of different sizes are mechanically similar but do not completely explain the longitudinal response gradients reported in the cheek pouch microcirculation. An analysis of the effect of arteriolar wall buckling suggests that the luminal folds that develop at short vessel radii may broaden the peak of the active stress-length curve and extend the pressure range over which arterioles are most sensitive to physical and chemical stimuli.

Simultaneous measurement of capillary distensibility and hydraulic conductance.

Capillaries are often assumed to be indistensible. Only recently has the error caused by capillary distensibility in the measurement of hydraulic conductance been considered. An oil-drop method was used to measure simultaneously distensibility and hydraulic conductance (Lp) of mesenteric capillaries of 40 cranially pithed Rana pipiens. All vessels studied were distensible in the range 5-80 mm Hg, with a mean equilibrium time, teq, of 12.4 +/- 0.5 sec (SEM, n = 7). Capillary distensibility decreased with increasing pressure. Mean capillary distensibilities at 10, 30, and 60 mm Hg were 18.9 +/- 4.9 X 10(-4) (mm Hg)-1, 13.5 +/- 1.2 X 10(-4) (mm Hg)-1 (mean +/- SEM, n = 9), and 7.6 +/- 2.7 X 10(-4) (mm Hg)-1 (mean +/- SEM, n = 7), respectively. Mean values of capillary Lp, uncorrected and corrected for distensibility, were 0.0113 +/- 0.0017 microns(sec.mm Hg)-1 and 0.0083 +/- 0.0016 microns(sec.mm Hg)-1 (SEM, n = 8 capillaries), respectively. A paired t test demonstrated that uncorrected and corrected values of Lp were statistically different from one another (P less than 0.005). Both distensibility and Lp varied widely between capillaries indicating the necessity of measuring and correcting for distensibility in each individual capillary when estimating Lp. The finding of a slow component of vessel distensibility demonstrates that previous filtration data taken not only from single vessels, but also from whole organs, should be reinterpreted.

A novel remote-sensing isometric force transducer for micromechanics studies.

We have developed an innovative transducer for measuring force with femtonewton-to-micronewton resolution in biological systems. A magnetic microsphere is attached to the specimen being studied and is positioned between two electromagnets. Video microscopy and edge detection are used to monitor small movements of the microsphere that occur when the specimen generates force. An automatic control system adjusts the current through the electromagnets to keep the microsphere stationary. Measured force is a linear function of this current. This transducer is unique in its combination of sensitivity and isometric properties and its ability to measure force without direct connections to the specimen. That is, the transducer is "remote sensing" and can measure force through intervening membrane or tissue. The transducer is isometric at steady state to the limit at which displacement of the microsphere can be resolved, which can be as low as 19 nm. The completed system is being used to study the mechanics of interstitial connective tissue but may also be used to study molecular generation of force.

Mechanics of smooth muscle in isolated single microvessels.

In vivo studies on frog mesenteric arterioles (4) indicate that segmental differences in the response of microvessels to physical and chemical stimuli can be explained simply in terms of the length-tension characteristics of vascular smooth muscle at different points along the vascular tree. Studies on single, isolated arterioles in vitro were initiated to examine more closely the validity of this explanation for regional response differences. This paper reports some of the results. First-, second-, and third-order arterioles (18-60 micron i.d.) were dissected from hamster cheek pouches. The vessels were cannulated with a modified Burg microperfusion system, and their mechanical properties studied using the methods described by Duling and Gore. Vessels were activated in four stages with K+ and norepinephrine. During activation, transmural pressures were adjusted to minimize vascular smooth-muscle shortening. Active pressure-diameter curves were recorded while adjusting transmural pressure through the range 5 to 400 cm H20 in 5-25 cm steps. Vessel dimensions were measured with a videomicrometer. Passive curves were obtained after equilibration overnight in Ca2+-free medium. The vessels were then fixed and prepared for histologic sectioning, and measurements of vessel-wall composition were made. The Laplace relationship was used to construct length-tension diagrams, and the histologic data were used to normalize the dimensional data to smooth-muscle lengths. Maximum active tension of second-order arterioles (1,170 dynes/cm) was two times previous values reported by Gore et al. This was due presumably to refinements in techniques and dissection procedures. Maximum active stress averaged 3.9 X 10(+6) dynes/cm2 for second-order arterioles. This number is identical to data obtained from hog carotid strips by Dillon et al.

Alteration of testicular microvascular pressures during venous pressure elevation.

We have addressed the hypothesis that varicocele-related infertility is caused in part by a pressure-induced disturbance of testicular convective transport that upsets the testicular hormonal environment and thus impairs spermatogenesis. The left testis of the hamster [pentobarbital sodium (Nembutal), 70 mg/kg ip] was prepared for microcirculatory observations. Testicular venous pressure was acutely elevated by ligating collateral routes of venous outflow and partially occluding, via a snare, the main venous outflow distal to the pampiniform plexus. Simultaneous direct pressure measurements (servo-null method) were made to monitor venous pressure elevation and quantify resulting pressure and diameter changes in the arterial feed to the testis and in postcapillary venules. The data show that over 90% of the venous pressure elevation (VPE) was transmitted to the postcapillary venules. VPE affected intravascular pressures throughout the testis microvasculature; on average, capsular artery pressure increased by 83% of the VPE, although part of this increase was due to a rise in systemic arterial pressure. Vasoconstriction helped to buffer the pressure rise in the capsular artery, probably at the expense of flow amplitude. Yet the vasoconstriction was ineffective in preventing a rise in exchange vessel pressure. These data suggest that microvascular fluid exchange may be dramatically altered in varicocele, upsetting the hormonal and paracrine environment of the testis, and hence, impairing physiological regulation of gametogenesis.

Vascular anatomy and hydrostatic pressure profile in the hamster cheek pouch.

The purpose of this study was to describe the vascular architecture and blood supply to the hamster cheek pouch and to measure the intravascular pressure distribution in the entire pouch. Previous anatomic descriptions have focused either on the vasculature of the facial region or on the microcirculation of the cheek pouch tip. Micropressures in cheek pouch capillaries, terminal arterioles, and small venules have not previously been measured. Cheek pouches were prepared for examination under an intravital microscope using both everted and noneverted methods. Microvessel diameters were measured through a video system using a video micrometer, and intravascular hydrostatic pressures were measured with a servo null micropressure system. Blood vessels in the face and pouch were traced after injection of Microfil into the external maxillary artery. The results indicate that the pouch is supplied directly or indirectly by six small arteries in the neck and face. Fifty percent of the total pressure drop across the cheek pouch vasculature occurs in the small cheek pouch arteries, suggesting that these vessels are potentially important in controlling cheek pouch blood flow. The measurements of microvascular pressures and diameters in this study help clarify apparent discrepancies in similar measurements from previous studies.

Locomotive forces produced by single leukocytes in vivo and in vitro.

We report here the first time-resolved measurements of the forces produced during the migration of single leukocytes in vivo and in vitro. Pulmonary macrophages from hamsters and mice, in vitro, and Nembutal (pentobarbital sodium)-anesthetized hamster neutrophils, in vivo, generated maximum locomotive forces ranging from 1.9 to 10.7 nN or tenths of microdynes. Force production was periodic and correlated with the length of the leading lamellipod but not with generalized cell ruffling. Although the extension of the leading lamella is critical to locomotive force generation, these direct measurements suggest that lamellar extension may not arise from the same contractile processes driving forward motion of the cell mass. Indeed, cell ruffling, lamellar extension, and locomotive force generation may be differentially controlled and have different origins. This technique may be extended to test numerous hypotheses of how these and other nonmuscle cells crawl.