Lymph Protein Concentration Research Articles

Development and application of a simple pharmacokinetic model that quantitatively describes the distribution and elimination of the commonly measured proteins.

Increased plasma concentrations of a variety of cellular enzymes (alanine transaminase, aspartate aminotransferase, alkaline phosphatase, amylase, etc.) are commonly used as routine screening tests for a range of conditions. An increased concentration usually is assumed to result from an increased rate of delivery to the plasma. Factors such as decreased metabolism or excretion or altered extravascular distribution usually are ignored. As a prelude to a detailed analysis of all the factors producing altered plasma enzyme levels, we have reviewed the relevant literature describing the pharmacokinetics (PK) of 13 of the commonly measured plasma proteins and developed a PK model that provides a simple physiological description of all the data. Our model starts with the general 3-compartment, 6-parameter system previously developed for albumin and interprets the fluxes in terms of unidirectional sieved protein convectional volume flows from the plasma to the two tissue compartments and equal lymph flows returning to the plasma. This greatly constrains the model such that each protein is characterized by only two adjustable parameters (plasma clearance and sieving factor). In addition to accurately fitting the plasma kinetics, the model can accurately describe the tissue and lymph protein PK. For example, it can describe the thoracic duct lymph protein concentration following an intravenous infusion or the plasma concentration following a subcutaneous tissue injection. This simple model provides a satisfactory framework for the PK of 12 of the 13 proteins investigated. The glycoprotein intestinal alkaline phosphatase is the exception, requiring the addition of a liver recycling compartment involving the asialoglycoprotein receptor.

Development and application of a novel cervical lymph collection method to assess lymphatic transport in rats.

Background: Fluids, solutes and immune cells have been demonstrated to drain from the brain and surrounding structures to the cervical lymph vessels and nodes in the neck via meningeal lymphatics, nasal lymphatics and/or lymphatic vessels associated with cranial nerves. A method to cannulate the efferent cervical lymph duct for continuous cervical lymph fluid collection in rodents has not been described previously and would assist in evaluating the transport of molecules and immune cells from the head and brain via the lymphatics, as well as changes in lymphatic transport and lymph composition with different physiological challenges or diseases. Aim: To develop a novel method to cannulate and continuously collect lymph fluid from the cervical lymph duct in rats and to analyze the protein, lipid and immune cell composition of the collected cervical lymph fluid. Methods: Male Sprague-Dawley rats were cannulated at the carotid artery with or without cannulation or ligation at the cervical lymph duct. Samples of blood, whole lymph and isolated lipoprotein fractions of lymph were collected and analyzed for lipid and protein composition using commercial kits. Whole lymph samples were centrifuged and isolated pellets were stained and processed for flow cytometry analysis of CD3+, CD4+, CD8a+, CD45R+ (B220) and viable cell populations. Results: Flow rate, phospholipid, triglyceride, cholesterol ester, free cholesterol and protein concentrations in cervical lymph were 0.094 ± 0.014mL/h, 0.34 ± 0.10, 0.30 ± 0.04, 0.07 ± 0.02, 0.02 ± 0.01 and 16.78 ± 2.06mg/mL, respectively. Protein was mostly contained within the non-lipoprotein fraction but all lipoprotein types were also present. Flow cytometry analysis of cervical lymph showed that 67.1 ± 7.4% of cells were CD3+/CD4+ T lymphocytes, 5.8 ± 1.6% of cells were CD3+/CD8+ T lymphocytes, and 10.8 ± 4.6% of cells were CD3-/CD45R+ B lymphocytes. The remaining 16.3 ± 4.6% cells were CD3-/CD45- and identified as non-lymphocytes. Conclusion: Our novel cervical lymph cannulation method enables quantitative analysis of the lymphatic transport of immune cells and molecules in the cervical lymph of rats for the first time. This valuable tool will enable more detailed quantitative analysis of changes to cervical lymph composition and transport in health and disease, and could be a valuable resource for discovery of biomarkers or therapeutic targets in future studies.

Vascular perfusion limits mesenteric lymph flow during anaphylactic hypotension in rats

To determine fluid extravasation in the splanchnic vascular bed during anaphylactic hypotension, the mesenteric lymph flow (Q(lym)) was measured in anesthetized rats sensitized with ovalbumin, along with the systemic arterial pressure (P(sa)) and portal venous pressure (P(pv)). When the antigen was injected into the sensitized rats (n = 10), P(sa) decreased from 125 ± 4 to 37 ± 2 mmHg at 10 min with a gradual recovery, whereas P(pv) increased by 16 mmHg at 2 min and returned to the baseline at 10 min. Q(lym) increased 3.3-fold from the baseline of 0.023 ± 0.002 g/min to the peak levels of 0.075 ± 0.009 g/min at 2 min and returned to the baseline within 12 min. The lymph protein concentrations increased after antigen, a finding indicating increased vascular permeability. To determine the role of the P(pv) increase in the antigen-induced increase in Q(lym), P(pv) of the nonsensitized rats (n = 10) was mechanically elevated in a manner similar to that of the sensitized rats by compressing the portal vein near the hepatic hilus. Unexpectedly, P(pv) elevation alone produced a similar increase in Q(lym), with the peak comparable to that of the sensitized rats. This finding aroused a question why the antigen-induced increase in Q(lym) was limited despite the presence of increased vascular permeability. Thus the changes in splanchnic vascular surface area were assessed by measuring the mesenteric arterial flow. The mesenteric arterial flow was decreased much more in the sensitized rats (75%; n = 5) than the nonsensitized P(pv) elevated rats (50%; n = 5). In conclusion, mesenteric lymph flow increases transiently after antigen presumably due to increased capillary pressure of the splanchnic vascular bed via downstream P(pv) elevation and perfusion and increased vascular permeability in anesthetized rats. However, this increased extravasation is subsequently limited by decreases in vascular surface area and filtration pressure.

α-DEFENSIN-LIKE PRODUCT AND ASYMMETRIC DIMETHYLARGININE INCREASE IN MESENTERIC LYMPH AFTER HEMORRHAGE IN ANESTHETIZED RAT

Mesenteric lymph contains unidentified proinflammatory mediators that increase in concentration after hemorrhage. In the search for candidate mediators, we examined mesenteric lymph for the presence of proinflammatory substances that are known to be produced in the gut: (a) antimicrobial peptides and antimicrobial proteins produced in the Paneth cells of the intestine (alpha-defensin 4, secretory phospholipase A2 [sPLA2], and Reg 2 protein) and (b) asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NOS. Anesthetized male rats were hemorrhaged to 40 mmHg and maintained at that pressure by intermittent blood withdrawal until the pressure fell to less than 40 mmHg (decompensation) at which point they were resuscitated with three times the shed blood volume of Ringer's lactate solution administered over 1 h. Mesenteric lymph samples were analyzed for ADMA by enzyme-linked immunosorbent assay and for alpha-defensin 4, sPLA2, and Reg2 by Western blotting. Protein concentration in lymph was unchanged by hemorrhage, but alpha-defensin 4 increased significantly (12-fold greater than control) as did ADMA (2-fold greater than control). The sPLA2 could not be detected in lymph, and Reg 2 was unchanged during hemorrhage. During resuscitation, lymph flow tended to increase, but the concentration of ADMA and alpha-defensin 4 by volume did not increase. Reg 2 decreased during resuscitation. The results indicate that ADMA and immunoreactive product to alpha-defensin 4 may contribute to the increase in inflammatory activity of mesenteric lymph during hemorrhage, but they are unlikely to be the mediators responsible for the increase in the concentration of inflammatory mediators in postresuscitation lymph.

Functional and Molecular Adaptation of Lymphatic Vessels

We developed a bovine model of mesenteric venous hypertension (MVH)by surgically occluding mesenteric veins for 3 days to investigate the functional and molecular adaptations of the mesenteric lymphatic vessels. The MVH model was characterized by an increase in mesenteric vein pressure from 10 to 30 mm Hg, a decrease in lymph protein concentration from 6 to 5 g/dL and an increase in intestinal water content from 72 to 78%. After 3‐days of MVH, isolated mesenteric lymphatic vessels demonstrated decreased contraction frequency, decreased ejection fraction and decreased stroke volume at each transmural pressure tested. Microarray analysis revealed that 117 genes were upregulated and 79 genes were down regulated in MVH lymphatics. A large number of the regulated genes are muscle contractile proteins, consistent with the changes in function. We further characterized changes in the mRNA of actin isoforms expressed in lymphatic muscle in response to venous hypertension. The smooth muscle actins (alpha and gamma) and skeletal actin were increased and cardiac actin decreased in lymphatics from the MHV model. Actin protein levels were analyzed by western blot analysis and revealed similar directional changes in response to MHV. These data suggest that lymphatic vessels undergo functional and molecular adaptation in response to changes in lymphdynamics in a time frame that may be important in resolution of edema. This work was supported by a LSI grant from Texas A&M University and CDC grant 623086.

In Situ Lymph Dynamic Characterization through Lymph Nodes in Rabbit Hind Leg: Special Reference to Nodal Inflammation

In some lymph nodes, water and water-soluble substances of smaller molecular weight are known to be absorbed into blood vessels, and consequently the protein concentration of lymph within the nodes increases. In this study, we examined pressure-flow relationships of lymph nodes in situ and exchange properties of water and water-soluble substances through the nodes with special reference to inflamed lymph nodes. A lymph perfusion model through the lymph node in situ was constructed by cannulating one of the afferent lymphatics and an efferent lymphatic. Increasing infusion pressure (0 to 150 cmH(2)O) or decreasing outflow pressure (10 to -5 cmH(2)O) in the model caused a significant increase of the lymph outflow rate through the node. This rate was also increased significantly with increases in both intranodal venous pressure (range: control, 20, 30, and 40 mmHg) and prenodal lymph albumin concentration (range: 0%, 2.6%, and 10%). When formyl-Met-Leu-Phe-OH (fMLP)-mediated acute inflammation was produced in the lymph nodes, the lymph outflow rate through the node was significantly decreased. These results indicate that colloid osmotic pressure and hydrostatic pressure within the lymph node may play important roles in the transport of water and water-soluble substances through the node. Acute fMLP-mediated inflammation of lymph nodes also produced a significant decrease of the lymph flow rate through lymph nodes.

Mass kinetics of apolipoprotein A-I in interstitial fluid after administration of intravenous apolipoprotein A-I/lecithin discs in humans

Apolipoprotein kinetics are customarily determined by modeling time curves of specific radioactivity or isotopic enrichment in plasma after intravenous infusion of radiolabeled lipoproteins or stable isotope-enriched amino acids. However, this provides no information on the fractional rate of transfer of the apolipoprotein from plasma to interstitial fluid (k(p-if)) or its mean residence time in interstitial fluid (MRT(if)). To determine these parameters for a pharmacologic dose of exogenous apolipoprotein A-I (apoA-I) given intravenously as apoA-I/lecithin discs, we measured apoA-I in plasma and prenodal leg lymph in five healthy men before, during, and after a 4 h infusion at 10 mg/kg/h. ApoA-I concentrations in plasma and lymph were modeled by linear compartmental models (SAAM II version 1.1), using lymph albumin to adjust for the effects of variations in lymph flow rate. k(p-if) averaged 0.75%/h (range, 0.33-1.32), and MRT(if) averaged 29.1 h (14.1-40.0). Neither parameter was correlated with the distribution volume (57-105 ml/kg) or the fractional elimination rate (1.44-2.91%/h) of apoA-I, determined by modeling plasma apoA-I concentration alone. Although used here to study the mass kinetics of apoA-I, if combined with infusion of a tracer, analysis of lymph could also expand the modeling of endogenous apolipoprotein kinetics.

Lung Fluid Balance During Development

After completing this article, readers should be able to: 1. Describe the major driving forces for liquid formation in and liquid removal from the lumen of the fetal lung. 2. Delineate the role of a normal volume of liquid within the lumen of the fetal lung for normal lung growth during development. 3. Describe the effects of labor on Na,K-ATPase activity in lung epithelial cells and clearance of lung liquid after birth. 4. Explain the route by which most residual liquid drains from the lung. 5. List the conditions associated with preterm birth that may contribute to delayed removal of fetal lung liquid. Curious students of mammalian development recognized more than a century ago that the lungs are filled with liquid during fetal life, (1) but the origin of that liquid remained obscure until 1948, when an unexpected discovery by two French scientists dispelled the prevailing view that fetal lung liquid derived from intrauterine inhalation of amniotic fluid. In studies designed to explore the ontogeny of the pituitary-adrenal axis, tracheal ligation of fetal rabbits for 9 days resulted in fluid distention of the lungs. (2) This serendipitous finding, subsequently confirmed and embellished by others, (3)(4)(5) established that the fetal lung itself, rather than the amniotic sac, is the source of the liquid that fills the lung during development. This liquid forms a slowly expanding structural template that prevents collapse and promotes growth of the fetal lung. Subsequent studies, conducted mostly with sheep, showed that liquid in the lumen of the fetal lung forms as a result of chloride secretion in the respiratory epithelium, (6)(7) a process that can be inhibited by diuretics that block Na,K-2Cl cotransport. (8)(9) In vitro experiments using cultured explants of lung tissue and monolayers of epithelial cells harvested from human fetal lung have …

Clearance of filtered fluid from the lung during exercise: role of hyperpnea.

During strenuous exercise in sheep, lung lymph flow increases within seconds and rises to levels 7- to 10-fold over baseline. Concomitant with the flow increase, the lymph protein content rapidly decreases to levels consistent with severe capillary hypertension. This pattern of clearance of filtered fluid is quite different than is seen with the passive capillary hypertension that results from mechanical obstruction of the mitral valve. In passive capillary hypertension, the increase in lymph flow and reduction in lymph protein content develop over several hours. The purpose of this study was to discover if these observed differences in edema clearance are related to the hyperpnea that accompanies exercise. Sheep were instrumented for continuous measurement of pulmonary arterial, left atrial, and systemic pressures, cardiac output by ultrasound, lung lymph flow, and ventilation. First, hemodynamics, ventilatory, and lymph clearance variables were measured during moderate exercise at 2.8 mph on a treadmill. Second, on a separate occasion, sheep were induced to hyperventilate to the same minute ventilation as during exercise, using modest CO2 stimulation. Lymph flow and hemodynamics were unaffected by this hyperpnea. The third arm of the experiment was to raise pulmonary microvascular pressure at rest to the level seen with exercise by means of a balloon catheter placed in the mitral valve. Lymph flow rose and protein content decreased slowly and to a lower degree than seen with exercise despite a comparable microvascular pressure. Finally, left atrial hypertension and induced hyperpnea were combined in sheep at rest, and the resulting lymph flow and protein content were the same as seen with exercise at similar pressures and ventilation. We conclude that hyperpnea is a major mechanism of interstitial liquid clearance during exercise, and may be largely responsible for preventing pulmonary edema that might occur at the high microvascular pressures of strenuous exercise.

Protein washdown as a defense mechanism against myocardial edema.

Myocardial edema occurs in many pathological conditions. We hypothesized that protein washdown at the myocardial microvascular exchange barrier would change the distribution of interstitial proteins from large to small molecules and diminish the effect of washdown on the colloid osmotic pressure (COP) of interstitial fluid and lymph. Dogs were instrumented with coronary sinus balloon-tipped catheters and myocardial lymphatic cannulas to manipulate myocardial lymph flow and to collect lymph. Myocardial venous pressure was elevated by balloon inflation to increase transmicrovascular fluid flux and myocardial lymph flow. COP of lymph was measured directly and was also calculated from protein concentration. Decreases occurred in both protein concentration and COP of lymph. The proportion of lymph protein accounted for by albumin increased significantly, whereas that accounted for by beta-lipoprotein decreased significantly. The change in the calculated plasma-to-lymph COP gradient was significantly greater than the change in the measured COP gradient. We conclude that the change in the distribution of interstitial fluid protein species decreases the effect of protein washdown on interstitial fluid COP and limits its effectiveness as a defense mechanism against myocardial edema formation.

Modulation of microvascular permeability by 21-aminosteroids after burn injuries.

Burn injuries initiate lipid peroxidation in capillary endothelial cells and cause alterations in microvascular permeability, with subsequent leakage of fluid and protein from the plasma into the interstitium. We evaluated the effects of two lazaroid compounds (U74389F and U75412E) on alterations in microvascular permeability that resulted from burn injuries. A canine model was used for the evaluation of microvascular permeability at the site of the burn injury with the use of a measure of the reflection coefficient (sigma(d)). Hindpaw lymph flow, lymph and plasma total protein concentrations, and arterial, venous, and capillary pressures were measured before burn injuries and for 6 hours in 6 different groups. Footpaw weight gain was then calculated as the percentage of increase of experimental hindpaw relative to the contralateral paw. The damage was attenuated by 20 mg/kg of lazaroid U75412E given before the injuries, but a lower dose was not effective. This agent was also effective in limiting edema formation, as evidenced by changes in footpaw weight gain. However, the administration of either lazaroid compound produced no significant effect on the burn-induced changes in capillary permeability. We conclude that these lazaroids do not prevent burn-induced changes in permeability at the site of injury when administered after an injury. U75412E administered before the injury was effective in limiting the alterations in microvascular permeability.

Chronic lung injury in preterm lambs: abnormalities of the pulmonary circulation and lung fluid balance.

Chronic lung disease of early infancy, or bronchopulmonary dysplasia, is a frequent complication of prolonged mechanical ventilation after premature birth. Pulmonary hypertension and edema are common features of this condition, which is often attributed to long-term, repetitive overinflation of incompletely developed lungs. The overall objective of this work was to examine the effects on the pulmonary circulation and lung fluid balance of different ventilation strategies using large versus small inflation volumes in an animal model of bronchopulmonary dysplasia. We studied 16 newborn lambs that were delivered prematurely (124+/-3 d gestation, term = 147 d) by cesarean section and mechanically ventilated for 3 to 4 wk. Ten lambs were ventilated at 20 breaths/min, yielding a tidal volume of 15+/-5 mL/kg, and six lambs were ventilated at 60 breaths/min, yielding a tidal volume of 6+/-2 mL/kg. All lambs received surfactant at birth and had subsequent surgery for closure of the ductus arteriosus and catheter placement to allow serial measurements of pulmonary vascular resistance and lung lymph flow. Chronic lung injury, documented by serial chest radiographs and postmortem pathologic examination, developed in all lambs irrespective of the pattern of assisted ventilation. Pulmonary vascular resistance, which normally decreases during the month after birth at term, did not change significantly from the first to the last week of study. Lung lymph flow, an index of net transvascular fluid filtration, increased with time in lambs that were ventilated at 20 breaths/min, but not in lambs ventilated at 60 breaths/min. Lymph protein concentration decreased with time, indicative of increased fluid filtration pressure, without evidence of a change in lung vascular protein permeability. Postmortem studies showed interstitial lung edema, increased pulmonary arteriolar smooth muscle and elastin, decreased numbers of small pulmonary arteries and veins, and decreased capillary surface density in distal lung of chronically ventilated lambs compared with control lambs that were killed either 1 d (same postconceptional age) or 3 wk (same postnatal age) after birth at term. Thus, chronic lung injury from prolonged mechanical ventilation after premature birth inhibits the normal postnatal decrease in pulmonary vascular resistance and leads to lung edema from increased fluid filtration pressure. These abnormalities of the pulmonary circulation may contribute to the abnormal respiratory gas exchange that often exists in infants with bronchopulmonary dysplasia.

Extracorporeal circulation exacerbates microvascular permeability after endotoxemia.

Extracorporeal life support without prior inflammatory stimuli results in a modest increase in microvascular permeability. Initiating ECLS after shock, sepsis, or hypoxia results in marked increases in interstitial fluid and total body water. We sought to determine whether an inflammatory stimulus prior to initiating ECLS increases microvascular permeability to protein. An anesthetized canine lymphatic fistula model was used to study Starling forces. LPS + ECLS received 1 mg/kg LPS 1 h prior to initiating ECLS, and the group ECLS received saline vehicle. To determine mesenteric microvascular permeability, mesenteric venous pressure was elevated to 32 +/- 1 mm Hg to reach a minimal lymph protein concentration (C(L)). With simultaneous measurement of plasma protein concentration (C(P)), the reflection coefficient, sigma, was calculated using the formula sigma = 1 - C(L)/C(P). Transvascular protein clearance and filtration coefficient (K(f)) were calculated from the measured Starling variables. After a steady state was achieved, normothermic right atrial-to-femoral artery ECLS was initiated and continued for 2 h and then discontinued. Measurements were continued for 30 min after ECLS was discontinued. Measurements were continued for 30 min after ECLS was discontinued. Within-group comparisons were made with ANOVA and Fisher's LSD, and between-group comparisons were made with Student's t tests where appropriate. The reflection coefficient (sigma) decreased significantly from 0.77 +/- 0.02 to 0.53 +/- 0.07 with LPS + ECLS and was lower compared to ECLS alone (0.77 +/- 0.02 to 0.65 +/- 0.03). Transvascular protein clearance increased to a greater extent in LPS + ECLS from 266 +/- 46 to 819 +/- 125 microl/min compared to ECLS alone (284 +/- 49 to 819 +/- 125 microl/min) (P = 0. 06). K(f) increased in both groups after initiation of ECLS, but to a significantly greater extent in LPS + ECLS at 60 min (30.7 +/- 2.2 microl/min/mm Hg/g in ECLS and 50.0 +/- 8.9 microl/min/mm Hg/g in LPS + ECLS). Ileal tissue water increased in both groups, but there were no differences between groups. Initiation of ECLS after exposure to LPS increases ileal microvascular permeability to protein and water to a greater extent than ECLS alone.

Pulmonary capillary sieving of hetastarch is not altered by LPS-induced sepsis.

Gram-negative lipopolysaccharide (LPS) has been demonstrated to increase pulmonary capillary permeability as judged by the increased flow of protein-rich lymph from the lungs of sheep infused with LPS. This finding suggests that LPS-injured pulmonary capillaries might be less restrictive than uninjured capillaries to the filtration of large hetastarch molecules. Hetastarch has a broad molecular mass spectrum (35-1,500 kilodaltons (kDa)), and one way to test the restrictiveness of pulmonary capillaries is to measure the size of the largest hetastarch molecules that cross the microvascular barrier and enter the lymph. To evaluate the effects of LPS, we compared hetastarch molecular distributions in the lung lymph of normal and LPS-injured sheep. Adult sheep (38.2 +/- 0.8 kg) were surgically prepared for the collection of lung lymph, with study initiation after a 5- to 7-day recovery period. Hetastarch (6%) was infused (10 mL/kg) 24 hours before study to allow for stabilization of the hetastarch molecular distribution. On the day of study, LPS (Escherichia coli lipopolysaccharide, 2 microg/kg; n = 6) was infused, and plasma and lymph samples were collected for 12 hours. An additional group of animals not infused with LPS (n = 6) served as controls. Hetastarch molecular distributions in plasma and lymph were measured by using high performance size exclusion chromatography. In control sheep, the largest hetastarch molecules in lymph averaged 861 +/- 18 kDa (mean +/- SEM) (plasma, 1,065 +/- 18 kDa). In LPS-treated sheep, the largest hetastarch molecules in lymph averaged 845 +/- 19 kDa (not significant vs. normal) (plasma, 1,025 +/- 14 kDa). Hetastarch concentrations in plasma and lung lymph of normal sheep, respectively, were 0.61 +/- 0.05% and 0.34 +/- 0.07%. In LPS-treated sheep, hetastarch concentrations in plasma and lymph were 0.56 +/- 0.08 (not significant vs. normal) and 0.29 +/- 0.07, respectively (p < or = 0.05). Lymph concentrations were lower after LPS because of increased lymph flows (19.9 +/- 5.4 mL/30 min, compared with 3.6 +/- 0.8 mL/30 min in normal sheep). Our results suggest that LPS does not alter the diameter of the largest pores perforating the walls of pulmonary capillaries. Rather, the number of these pores in the capillary wall appears to be increased. This increase would explain why lymph flows rise after LPS with little change in the lymph protein concentration. Our results are also consistent with a filtration model in which capillaries are assumed to be perforated by small pores (protein reflection coefficient = 1) as well as large pores (protein reflection coefficient = 0).

Analysis of Intestinal Microvascular Permeability Associated with Cardiopulmonary Bypass

Background.Cardiopulmonary bypass (CPB) is associated with a generalized inflammatory response and splanchnic edema formation that are thought to be related to microvascular barrier injury. In particular, intestinal edema and dysfunction have been associated with sepsis and post-CPB complications. The purpose of this study was to measure the forces determining fluid flux induced by CPB across the intestinal microvascular barrier.Materials and methods.An anesthetized canine model was used for this study (n= 12). To determine mesenteric microvascular permeability, a mesenteric lymphatic was cannulated and mesenteric venous pressure was elevated to 33 ± 1 mm Hg to reach a minimal lymph protein concentration (CL). With simultaneous measurement of plasma protein concentrations (CP), the reflection coefficient, ς, was calculated using the formula: ς = 1 −CL/CP. Capillary pressures (PC), lymph flow (QL), lymph protein flux, transvascular protein flux, and intestinal tissue water were all measured using standard techniques. Normothermic cardiopulmonary bypass with flows of 75–80 ml/kg/min was initiated after a steady state was achieved, and CPB was continued for 2 h and then discontinued. Measurements were repeated 30 min after CPB was discontinued. A second group (n= 5) was studied without mesenteric venous pressure elevation to evaluate the role of capillary pressure on the increased fluid flux seen with the initiation of CPB.Results.Initiation of CPB was associated with an increase in intestinal microvascular permeability. ς decreased from 0.77 ± 0.01 to 0.68 ± 0.01 (P< 0.05) with the initiation of CPB. This corresponded with statistically significant increases in both transvascular protein flux from 310 ± 22 to 465 ± 39 ml/min at 30 min and intestinal tissue water from 82.8 ± 0.7 to 84.3 ± 0.5% after weaning from CPB. Capillary pressure did not significantly increase with the initiation of CPB.Conclusions.Initiation of CPB results in a moderate increase in intestinal microvascular permeability to protein and an increase in intestinal tissue water. The increases in tissue water are not due to increased capillary pressure. A better understanding of the microvascular changes associated with extracorporeal circulation will facilitate the search for clinical interventions to minimize the impact of CPB.

Chronic lymph flow responses to hyperproteinemia

The long-term responses of lymph flow, lymph protein transport, and the permeability-surface area (PS) product to hyperproteinemia have been studied in conscious dogs. Plasma protein concentration (PPC) was increased by daily intravenous infusion of previously collected autologous plasma for 9 days. Lymph flow was determined by collecting lymph chronically from a lymphatic afferent to the popliteal node in the hind leg. Compared with the average value during the normal-PPC period, the following changes occurred during 10 days of high PPC: lymph flow decreased from 12.3 +/- 1.1 to 3.8 +/- 0.6 microl/min, lymph protein transport decreased from 241 +/- 24 to 141 +/- 21 microg/min, PS product decreased from 4.7 +/- 0.5 to 3.0 +/- 0.5 microl/min, PPC increased from 7.1 +/- 0.1 to 8.8 +/- 0.4 g/dl, lymph protein concentration increased from 1.9 +/- 0.1 to 3.8 +/- 0.1 g/dl, plasma colloid osmotic pressure increased from 18. 6 +/- 0.8 to 24.2 +/- 2.1 mmHg, and lymph colloid osmotic pressure increased from 4.8 +/- 0.2 to 10.4 +/- 0.7 mmHg. In conclusion, long-term hyperproteinemia in dogs resulted in chronic decreases in lymph flow, lymph protein transport, and the PS product and chronic increases in lymph protein concentration and lymph colloid osmotic pressure. The marked decrease in lymph flow during hyperproteinemia decreased lymph protein transport and thus contributed to the increase in lymph protein concentration. In addition, the decreases in PS product and lymph protein transport suggest that transcapillary protein flux decreases during hyperproteinemia.

Pulmonary microvascular reflection coefficients estimated with modified lymphatic washdown technique.

Many investigators have used the lymphatic protein washdown technique to estimate the pulmonary microvascular membrane reflection coefficient to protein (sigma d). With that technique, the investigator causes a high microvascular filtration rate then estimates sigma d from the lymph and plasma protein concentrations. However the lymph may contain protein washed from the lung tissue, and the tissue protein may cause investigators to underestimate sigma d. Plasma protein osmotic pressure (IIc) may cause investigators to underestimate sigma d because IIc opposes fluid filtration. To minimize the effect of IIc, we decreased IIc to 5.6 +/- 1.1 mmHg in five anesthetized sheep. We increased the microvascular filtration rate by increasing pulmonary microvascular pressure to 22 +/- 3 mmHg. Then we tagged plasma protein with Evans blue dye and estimated sigma d from the lymph and plasma dye concentrations. Because tissue protein was not tagged, it did not interfere with our sigma d estimate. Our sigma d estimate (0.79 +/- 0.08) was much higher than previous estimates in anesthetized animals.

Changes in left thoracic duct lymph flow during progressive anemia in the ovine fetus

OBJECTIVE: The purpose of this study was to evaluate the extent to which increased lymph flow can return fluid and protein to the circulation in the chronically anemic fetus. STUDY DESIGN: Thoracic duct lymph flow rate over a range of outflow pressures was measured in 8 near-term fetal sheep 4 to 5 days after surgery and daily thereafter for 5 days. After each day's study 60 to 150 ml of blood was withdrawn at a rate of 1 ml per minute. Regression analysis was used to establish the lymph flow function curve. Lymph and plasma protein concentrations and lymph flow rate were compared by analysis of variance for repeated measures. RESULTS: As the hematocrit was reduced from 34.6% ± 1.3% (mean ± SE) to 14.4% ± 1.0%, thoracic duct lymph flow increased from 0.12 ± 0.01 to 0.28 ± 0.02 ml/min/kg. Plasma total protein concentration did not change, lymph protein concentration fell (2.6 ± 0.1 to 2.4 ± 0.1 gm/dl), and the difference between plasma and lymph protein concentrations increased (1.04 ± 0.05 to 1.34 ± 0.10 gm/dl). Protein returned to the circulation increased from 11.5 ± 0.3 to 23.7 ± 1.5 mg per minute. Central venous pressure did not change and remained less than the breakpoint pressure. Although the plateau lymph flow rate increased, neither the breakpoint or stopflow pressures of the lymph flow function curve were altered. CONCLUSIONS: Fetal lymph flow and thereby capillary filtration increased progressively as anemia became more severe. The increase in lymph flow did not appear to be limited by outflow pressure. By returning protein to the circulation, an increase in thoracic duct lymph flow helped to limit expansion of extravascular fluid volume during chronic fetal anemia. (AM J OBSTET GYNECOL 1996;174:1469-76.)

Interleukin-2 Does Not Sequester Activated Lymphocytes into Lung Lymph of Sheep

Purpose of study: Interleukin-2 (IL-2) is a potent activator of lymphocytes, but its effectiveness as an anticancer agent is compromised by several adverse side effects including pulmonary edema. One explanation for the pulmonary toxicity of IL-2 is that activated lymphocytes directly induce the pulmonary vascular endothelium to become more leaky. Methods: To test this hypothesis the number of total lymphocytes, γδ T cells, and CD2-positive cells (αβ T cells and natural killer cells) in peripheral blood and lung lymph of sheep were compared before and after IL-2 infusion. Hemodynamic and lymph dynamic changes were also evaluated. Results: IL-2 decreased mean aortic pressure, increased cardiac output, lowered systemic vascular resistance, and doubled lung lymph flow (P≤ 0.05), but had no effect on plasma or lymph oncotic pressure. The lymph protein concentration and the lymph-to-plasma protein concentration ratio were not different after IL-2 infusion. IL-2 had no effect on the number of total lymphocytes, γδ T cells, or CD2-positive cells in the peripheral blood. In contrast, the number of total lymphocytes, γδ T cells, and CD2-positive cells in lung lymph decreased significantly (P≤ 0.05). Conclusions: The lymphocyte populations decreased more than could be explained by the increase in lymph flow, demonstrating that lung lymphocytes were not reduced simply by dilution. These results imply that the pulmonary edema associated with IL-2 is not caused by activated lymphocytes.

Thoracic lymphatic cannulation for the study of pulmonary immune responses in lung lymph of calves

Abstract Objective To develop a system for analysis of immune response variables in the lymph draining the lung and to establish baseline data for clinically normal calves. Design Surgery was performed on 6 calves to insert a cannula into the efferent lymphatic duct of the caudal mediastinal lymph node to create a long-term thoracic lymph fistula draining to the exterior. Lymph was collected daily, and on the fifth postoperative day, calves were exposed to an aerosol of cell culture medium (mock infection). For the next 10 days, lymph was collected for analysis and, on the tenth day, necropsy was performed. Animals Six 6- to 8-week-old Holstein bull calves. Procedures Daily lymph samples were evaluated for: flow rate; total and differential cell counts; and IgG, IgM, IgA, IgE, and protein concentrations. On days −4, −1, 1, 4, 7, and 10, cells were stained and quantitated by fluorescence-activated cell sorter analysis for T, B, CD4+, and CD8+ cells. Blood lymphocytes were evaluated on days -1 and 10 for comparison. Results Flow was established for up to 25 days, with a mean rate between 11 and 22 ml/h. Protein concentrations in lymph and plasma did not indicate a protein drain. Although mean lymphocyte counts reflected a slight gradual decrease in lymph lymphocytes, this effect was not apparent in every calf, nor was the effect seen in blood lymphocytes. There were no significant changes in IgG, IgM, IgA, or IgE concentration, with the exception of IgA concentration in 1 calf that developed an abscess at the cannulation site. The T-cell subset absolute numbers of CD4+ and CD8+ cells decreased slightly over time, but the CD4+-to-CD8+ cell ratio remained almost constant at near 2. Conclusions Creation of a thoracic lymphatic fistula appears to be a useful technique for studying effects of lung infection on immunologic variables, with potential application to bacterial and viral respiratory tract diseases. Clinical Relevance Thoracic lymphatic cannulation can be used in studies to determine pathogenic mechanisms in respiratory tract disease and to develop more effective vaccines against respiratory tract pathogens.