Ileitis-associated tertiary lymphoid organs arise at lymphatic valves and impede mesenteric lymph flow in response to tumor necrosis factor.

Lymphangitis and the formation of tertiary lymphoid organs (TLOs) in the mesentery are features of Crohn's disease. Here, we examined the genesis of these TLOs and their impact on disease progression. Whole-mount and intravital imaging of the ileum and ileum-draining collecting lymphatic vessels (CLVs) draining to mesenteric lymph nodes from TNFΔARE mice, a model of ileitis, revealed TLO formation at valves of CLVs. TLOs obstructed cellular and molecular outflow from the gut and were sites of lymph leakage and backflow. Tumor necrosis factor (TNF) neutralization begun at early stages of TLO formation restored lymph transport. However, robustly developed, chronic TLOs resisted regression and restoration of flow after TNF neutralization. TNF stimulation of cultured lymphatic endothelial cells reprogrammed responses to oscillatory shear stress, preventing the induction of valve-associated genes. Disrupted transport of immune cells, driven by loss of valve integrity and TLO formation, may contribute to the pathology of Crohn's disease.

Lymph protects metastasizing melanoma cells from ferroptosis.

Cancer cells, including melanoma cells, often metastasize regionally through the lymphatic system before metastasizing systemically through the blood1-4; however, the reason for this is unclear. Here we show that melanoma cells in lymph experience less oxidative stress and form more metastases than melanoma cells in blood. Immunocompromised mice with melanomas derived from patients, and immunocompetent mice with mouse melanomas, had more melanoma cells per microlitre in tumour-draining lymph than in tumour-draining blood. Cells that metastasized through blood, but not those that metastasized through lymph, became dependent on the ferroptosis inhibitor GPX4. Cells that were pretreated with chemical ferroptosis inhibitors formed more metastases than untreated cells after intravenous, but not intralymphatic, injection. We observed multiple differences between lymph fluid and blood plasma that may contribute to decreased oxidative stress and ferroptosis in lymph, including higher levels of glutathione and oleic acid and less free iron in lymph. Oleic acid protected melanoma cells from ferroptosis in an Acsl3-dependent manner and increased their capacity to form metastatic tumours. Melanoma cells from lymph nodes were more resistant to ferroptosis and formed more metastases after intravenous injection than did melanoma cells from subcutaneous tumours. Exposure to the lymphatic environment thus protects melanoma cells from ferroptosis and increases their ability to survive during subsequent metastasis through the blood.

Effect of rumen-protected choline on fat digestibility and lymph metabolome in dairy cows.

Objectives were to determine the effects of supplementing rumen-protected choline (RPC) from an established source with low (L, 28.8%) or a prototype with less lipid coating protection and high (H, 60.0%) concentrations of choline chloride on digestibility of fat and supra-mammary lymph metabolome in feed-restricted cows. Pregnant, nonlactating Holstein cows (n = 33; 11/treatment) at mean (±standard deviation) 231 ± 4.7 days of gestation were blocked by body condition (4.23 ± 0.47) and assigned to receive 0 (CON) or 25.8 g/d of choline ion from L (L25.8) or H (H25.8). Cows were adapted to the diet and then fed-restricted to 42% of the net energy of lactation required for maintenance and pregnancy for 9 days. Intake of metabolizable methionine was maintained at 19 g/d. On Day 9, cows were fed 450 g of saturated fatty acids (SFA), and feces and blood were sampled continuously for 24 h. Supra-mammary lymph was sampled 6 h after feeding SFA and metabolome was characterized. Feeding RPC increased digestibility of fat (CON = 80.4 vs. RPC = 86.0 ± 1.9%) and reduced the concentration of haptoglobin in serum (CON = 174 vs. RPC = 77 ± 14 µg/ml) independent of source of RPC fed. Feeding RPC increased the concentrations of triacylglycerol in serum (CON = 15.1 vs. RPC = 17.8 ± 1.9 mg/dl) in feed-restricted cows after feeding SFA, and the increment tended to be greater for cows fed H25.8 than L25.8. Supplementing RPC tended to increase the concentrations of triacylglycerol (CON = 11.4 vs. RPC = 15.8 ± 3.4 mg/dl) in supra-mammary lymph. Feeding RPC increased the concentration of choline and affected the concentrations of analytes involved in metabolic pathways associated with amino acid metabolism and biosynthesis of phospholipids in lymph compared with CON. Feeding RPC, independent of source used, increased fat digestibility with some changes in lymph metabolome in cows under negative nutrient balance.

Cell surface ATP synthase-released H+ and ATP play key roles in cocoa butter intake-mediated regulation of gut immunity through releases of cytokines in rat.

Proper food intake is important for maintaining good health in humans. Chocolate is known to exert anti-inflammatory effects; however, the mechanisms remain unclear. In this study, we aimed to investigate the effects of cocoa butter intake on gut immunity in rats and rabbits. Cocoa butter intake increased the lymph flow, cell density, and IL-1ß, IL-6 and IL-10 levels in mesenteric lymph. Clodronate, a macrophage depletion compound, significantly enhanced the release of all cytokines. The immunoreactivities of macrophage markers CD68 and F4/80 in the jejunal villi were significantly decreased with clodronate. Piceatannol, a selective cell surface ATP synthase inhibitor significantly reduced the cocoa butter intake-mediated releases of IL-1ß, IL-6 and IL-10. The immunoreactivities of cell surface ATP synthase were observed in rat jejunal villi. Shear stress stimulation on the myofibroblast cells isolated from rat jejunum released ATP and carbon dioxide depended with H+ release. In rabbit in vivo experiments, cocoa butter intake increased the concentrations of ATP and H+ in the portal vein. The in vitro experiments with isolated cells of rat jejunal lamina propria the pH of 3.0 and 5.0 in the medium released significantly IL-1ß and IL-6. ATP selectively released IL-10. These findings suggest that cocoa butter intake regulates the gut immunity through the release and transport of IL-1ß, IL-6, and IL-10 into mesenteric lymph vessels in a negative feedback system. In addition, the H+ and ATP released from cell surface ATP synthase in jejunal villi play key roles in the cocoa butter intake-mediated regulation of gut immunity.

Augmented experimental design for bioavailability enhancement: a robust formulation of abiraterone acetate.

Abiraterone acetate (ABRTA) is clinically beneficial in management of metastatic castration-resistant prostate cancer (PC-3). With highlighted low solubility and permeability, orally hampered treatment of ABRTA necessitate high dose to achieve therapeutic efficacy. To triumph these challenges, we aimed to develop intestinal lymphatic transport facilitating lipid-based delivery to enhance bioavailability. ABRTA-containing self-nano emulsified drug delivery (ABRTA-SNEDDS) was statistically optimized by D-optimal design using design expert. Optimized formulation was characterized for particle size, thermodynamic stability, in vitro release, in vivo bioavailability, intestinal lymphatic transport, in vitro cytotoxic effect, anti-metastatic activity, and apoptosis study. Moreover, hemolysis and histopathology studies have been performed to assess pre-clinical safety. Nano-sized particles and successful saturated drug loading were obtained for optimized formulation. In vitro release upto 98.61 ± 3.20% reveal effective release of formulation at intestinal pH 6.8. ABRTA-SNEDDS formulation shows enhanced in vivo exposure of Abiraterone (2.5-fold) than ABRTA suspension in Sprague-Dawley rats. In vitro efficacy in PC-3 cell line indicates 3.69-fold higher therapeutic potential of nano drug delivery system. Hemolysis and histopathology study indicates no significant toxicities to red blood cells and tissues, respectively. Apparently, an opportunistic strategy to increasing bioavailability of ABRTA via intestinal lymphatic transport will create a viable platform in rapidly evolving chemotherapy. Enhanced translational utility of delivery was also supported through in vitro therapeutic efficacy and safety assessments. HighlightsAbiraterone acetate is a prostate cancer drug, impeded with low bioavailability.ABRTA loaded in self nano emulsifying drug delivery enhanced its bioavailability.Intestinal lymphatic transport played role in enhanced bioavailability of ABRTA.ABRTA-SNEDDS enhanced in vitro cytotoxic activity of ABRTA.ABRTA-SNEDDS found safe in preclinical safety evaluations.

A single-day mouse mesenteric lymph surgery in mice: an updated approach to study dietary lipid absorption, chylomicron secretion, and lymphocyte dynamics.

The intestine plays a crucial role in regulating whole-body lipid metabolism through its unique function of absorbing dietary fat. In the small intestine, absorptive epithelial cells emulsify hydrophobic dietary triglycerides (TAGs) prior to secreting them into mesenteric lymphatic vessels as chylomicrons. Except for short- and medium-chain fatty acids, which are directly absorbed from the intestinal lumen into portal vasculature, the only way for an animal to absorb dietary TAG is through the chylomicron/mesenteric lymphatic pathway. Isolating intestinal lipoproteins, including chylomicrons, is extremely difficult in vivo because of the dilution of postprandial lymph in the peripheral blood. In addition, once postprandial lymph enters the circulation, chylomicron TAGs are rapidly hydrolyzed. To enhance isolation of large quantities of pure postprandial chylomicrons, we have modified the Tso group's highly reproducible gold-standard double-cannulation technique in rats to enable single-day surgery and lymph collection in mice. Our technique has a significantly higher survival rate than the traditional 2-day surgical model and allows for the collection of greater than 400 µl of chylous lymph with high postprandial TAG concentrations. Using this approach, we show that after an intraduodenal lipid bolus, the mesenteric lymph contains naïve CD4+ T-cell populations that can be quantified by flow cytometry. In conclusion, this experimental approach represents a quantitative tool for determining dietary lipid absorption, intestinal lipoprotein dynamics, and mesenteric immunity. Our model may also be a powerful tool for studies of antigens, the microbiome, pharmacokinetics, and dietary compound absorption.

Enteral glucose, absorbed and metabolized, potently enhances mesenteric lymph flow in chow- and high-fat-fed rats.

A portion of absorbed dietary triglycerides (TG) is retained in the intestine after the postprandial period, within intracellular and extracellular compartments. This pool of TG can be mobilized in response to several stimuli, including oral glucose. The objective of this study was to determine whether oral glucose must be absorbed and metabolized to mobilize TG in rats and whether high-fat feeding, a model of insulin resistance, alters the lipid mobilization response to glucose. Lymph flow, TG concentration, TG output, and apolipoprotein B48 (apoB48) concentration and output were assessed after an intraduodenal lipid bolus in rats exposed to the following intraduodenal administrations 5 h later: saline (placebo), glucose, 2-deoxyglucose (2-DG, absorbed but not metabolized), or glucose + phlorizin (intestinal glucose absorption inhibitor). Glucose alone, but not 2-DG or glucose + phlorizin treatments, stimulated lymph flow, TG output, and apoB48 output compared with placebo. The effects of glucose in high-fat-fed rats were similar to those in chow-fed rats. In conclusion, glucose must be both absorbed and metabolized to enhance lymph flow and intestinal lipid mobilization. This effect is qualitatively and quantitatively similar in high-fat- and chow-fed rats. The precise signaling mechanism whereby enteral glucose enhances lymph flow and mobilizes enteral lipid remains to be determined.NEW & NOTEWORTHY Glucose potently enhances mesenteric lymph flow in chow- and high-fat-fed rats. The magnitude of glucose effect on lymph flow is no different in chow- and high-fat-fed rats. Glucose must be absorbed and metabolized to enhance lymph flow and mobilize intestinal lipid.

Transport and lymphatic uptake of monoclonal antibodies after subcutaneous injection.

The subcutaneous injection has emerged to become a feasible self-administration practice for biotherapeutics due to the patient comfort and cost-effectiveness. However, the available knowledge about transport and absorption of these agents after subcutaneous injection is limited. Here, a mathematical framework to study the subcutaneous drug delivery of mAbs from injection to lymphatic uptake is presented. A three-dimensional poroelastic model is exploited to find the biomechanical response of the tissue by taking into account tissue deformation during the injection. The results show that including tissue deformability noticeably changes tissue poromechanical response due to the significant dependence of interstitial pressure on the tissue deformation. Moreover, the importance of the amount of lymph fluid at the injection site and the injection rate on the drug uptake to lymphatic capillaries is highlighted. Finally, variability of lymphatic uptake due to uncertainty in parameters including tissue poromechanical and lymphatic absorption parameters is evaluated. It is found that interstitial pressure due to injection is the major contributing factor in short-term lymphatic absorption, while the amount of lymph fluid at the site of injection determines the long-term absorption of the drug. Finally, it is shown that the lymphatic uptake results are consistent with experimental data available in the literature.

Platelet Extracellular Vesicles: Beyond the Blood.

Extracellular vesicles (EVs) are a means of cell-to-cell communication and can facilitate the exchange of a broad array of molecules between adjacent or distant cells. Platelets are anucleate cells derived from megakaryocytes and are primarily known for their role in maintaining hemostasis and vascular integrity. Upon activation by a variety of agonists, platelets readily generate EVs, which were initially identified as procoagulant particles. However, as both platelets and their EVs are abundant in blood, the role of platelet EVs in hemostasis may be redundant. Moreover, findings have challenged the significance of platelet-derived EVs in coagulation. Looking beyond hemostasis, platelet EV cargo is incredibly diverse and can include lipids, proteins, nucleic acids, and organelles involved in numerous other biological processes. Furthermore, while platelets cannot cross tissue barriers, their EVs can enter lymph, bone marrow, and synovial fluid. This allows for the transfer of platelet-derived content to cellular recipients and organs inaccessible to platelets. This review highlights the importance of platelet-derived EVs in physiological and pathological conditions beyond hemostasis.

GLP-1 (Glucagon-Like Peptide-1) Is Physiologically Relevant for Chylomicron Secretion Beyond Its Known Pharmacological Role.

[Figure: see text].

Correlation of Cytokine Content in the Lymph with the Morphological Parameters of the Mesenteric Lymph Nodes in Adjuvant Therapy of Experimental Breast Cancer with the Use of Fragmented Human DNA.

We studied the relationship between the level of cytokines in the lymph of the thoracic duct and the morphometric parameters of the mesenteric lymph nodes after surgical treatment of breast cancer, chemotherapy, and administration of fragmented (double-stranded, dsDNA) human DNA. In comparison with surgical treatment and with chemotherapy alone, administration of a human dsDNA has a stimulating effect on the T-cell link of the immune response. In the paracortical zone, the relationship between the chemokine MCP-1 and increased content of small lymphocytes in this zone was revealed. Interrelations of IL-2 cytokines with small lymphocytes and of IL-4 with medium lymphocytes were revealed in germinal centers. We also observed interrelations of IL-7 with small lymphocytes and IL-4 with macrophages in the medullary cords, chemokine MIP-1α with immature and mature plasma cells (the number of these cells is reduced), and of MCP-1 with immunoblasts (the number of which is also reduced) in the medullary sinuses.

Intestinal Lymph Collection via Cannulation of the Mesenteric Lymphatic Duct in Mice.

BACKGROUND: We have optimized a technique for cannulation of mesenteric lymph duct (MLD) in mice. Mice have low rates of intestinal lymph production; the MLDs are smaller and associated with fragile vasculature. Previous protocols for lymph collection based on the open lymph fistula model were associated with low success rates in mice. Bariatric surgery procedures worsen success rates due to postoperative adhesions and GI rearrangement. We have used this procedure to collect mesenteric lymph from mice undergoing bile diversion from gall bladder to ileum (GB-IL). HYPOTHESIS: We hypothesize that peptide YY (PYY) levels in mesenteric lymph will increase following nutrient delivery in mice undergoing bile diversion from gall bladder to ileum (GB-IL). METHODS AND RESULTS: We observe that cannulation of the MLD using a needled-catheter maintains lymph vessel integrity, prevents excessive lymph leakage, and is less traumatic, leading to high success rates (>95%). PYY levels in mesenteric lymph after GB-IL were significantly higher post nutrient infusion. The procedure takes approximately 20 min; small rodent surgical experience and practice are required for success. CONCLUSIONS: Intestinal lymph can be collected from mice, including those undergoing bariatric surgical procedures with high success rates by cannulation of the mesenteric lymph duct.

Effect of VEGFC on lymph flow and inflammation-induced alveolar bone loss.

The lymphatic system plays a crucial role in the maintenance of tissue fluid homeostasis and the immunological response to inflammation. The effects of lymphatic drainage dysfunction on periodontitis have not been well studied. Here we show that lymphatic vessel endothelial receptor 1 (LYVE1)+ /podoplanin (PDPN)+ lymphatic vessels (LVs) are increased in the periodontal tissues, with accumulation close to the alveolar bone surface, in two murine periodontitis models: rheumatoid arthritis (RA)-associated periodontitis and ligature-induced periodontitis. Further, PDPN+ /alpha-smooth muscle actin (αSMA)- lymphatic capillaries are increased, whereas PDPN+ /αSMA+ collecting LVs are decreased significantly in the inflamed periodontal tissues. Both mouse models of periodontitis have delayed lymph flow in periodontal tissues, increased TRAP-positive osteoclasts, and significant alveolar bone loss. Importantly, the local administration of adeno-associated virus for vascular endothelial growth factor C, the major growth factor that promotes lymphangiogenesis, increases the area and number of PDPN+ /αSMA+ collecting LVs, promotes local lymphatic drainage, and reduces alveolar bone loss in both models of periodontitis. Lastly, LYVE1+ /αSMA- lymphatic capillaries are increased, whereas LYVE1+ /αSMA+ collecting LVs are decreased significantly in gingival tissues of patients with chronic periodontitis compared with those of clinically healthy controls. Thus, our findings reveal an important role of local lymphatic drainage in periodontal inflammation-mediated alveolar bone loss. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Structure-dependent absorption of atypical sphingoid long-chain bases from digestive tract into lymph.

BACKGROUND: Dietary sphingolipids have various biofunctions, including skin barrier improvement and anti-inflammatory and anti-carcinoma properties. Long-chain bases (LCBs), the essential backbones of sphingolipids, are expected to be important for these bioactivities, and they vary structurally between species. Given these findings, however, the absorption dynamics of each LCB remain unclear. METHODS: In this study, five structurally different LCBs were prepared from glucosylceramides (GlcCers) with LCB 18:2(4E,8Z);2OH and LCB 18:2(4E,8E);2OH moieties derived from konjac tuber (Amorphophallus konjac), from GlcCers with an LCB 18(9Me):2(4E,8E);2OH moiety derived from Tamogi mushroom (Pleurotus cornucopiae var. citrinopileatus), and from ceramide 2-aminoethyphosphonate with LCB 18:3(4E,8E,10E);2OH moiety and LCB 18(9Me):3(4E,8E,10E);2OH moiety derived from giant scallop (Mizuhopecten yessoensis), and their absorption percentages and metabolite levels were analyzed using a lymph-duct-cannulated rat model via liquid chromatography tandem mass spectrometry (LC/MS/MS) with a multistage fragmentation method. RESULTS: The five orally administered LCBs were absorbed and detected in chyle (lipid-containing lymph) as LCBs and several metabolites including ceramides, hexosylceramides, and sphingomyelins. The absorption percentages of LCBs were 0.10-1.17%, depending on their structure. The absorption percentage of LCB 18:2(4E,8Z);2OH was the highest (1.17%), whereas that of LCB 18:3(4E,8E,10E);2OH was the lowest (0.10%). The amount of sphingomyelin with an LCB 18:2(4E,8Z);2OH moiety in chyle was particularly higher than sphingomyelins with other LCB moieties. CONCLUSIONS: Structural differences among LCBs, particularly geometric isomerism at the C8-C9 position, significantly affected the absorption percentages and ratio of metabolites. This is the first report to elucidate that the absorption and metabolism of sphingolipids are dependent on their LCB structure. These results could be used to develop functional foods that are more readily absorbed.

Role of Bile Acids and GLP-1 in Mediating the Metabolic Improvements of Bariatric Surgery.

BACKGROUND & AIMS: Bile diversion to the ileum (GB-IL) has strikingly similar metabolic and satiating effects to Roux-en-Y gastric bypass (RYGB) in rodent obesity models. The metabolic benefits of these procedures are thought to be mediated by increased bile acids, although parallel changes in body weight and other confounding variables limit this interpretation. METHODS: Global G protein-coupled bile acid receptor-1 null (Tgr5-/-) and intestinal-specific farnesoid X receptor null (FxrΔ/E) mice on high-fat diet as well as wild-type C57BL/6 and glucagon-like polypeptide 1 receptor deficient (Glp-1r-/-) mice on chow diet were characterized following GB-IL. RESULTS: GB-IL induced weight loss and improved oral glucose tolerance in Tgr5-/-, but not FxrΔ/E mice fed a high-fat diet, suggesting a role for intestinal Fxr. GB-IL in wild-type, chow-fed mice prompted weight-independent improvements in glycemia and glucose tolerance secondary to augmented insulin responsiveness. Improvements were concomitant with increased levels of lymphatic GLP-1 in the fasted state and increased levels of intestinal Akkermansia muciniphila. Improvements in fasting glycemia after GB-IL were mitigated with exendin-9, a GLP-1 receptor antagonist, or cholestyramine, a bile acid sequestrant. The glucoregulatory effects of GB-IL were lost in whole-body Glp-1r-/- mice. CONCLUSIONS: Bile diversion to the ileum improves glucose homeostasis via an intestinal Fxr-Glp-1 axis. Altered intestinal bile acid availability, independent of weight loss, and intestinal Akkermansia muciniphila appear to mediate the metabolic changes observed after bariatric surgery and might be manipulated for treatment of obesity and diabetes.

Rapeseed Lecithin Increases Lymphatic Lipid Output and α-Linolenic Acid Bioavailability in Rats.

BACKGROUND: Soybean lecithin, a plant-based emulsifier widely used in food, is capable of modulating postprandial lipid metabolism. With arising concerns of sustainability, alternative sources of vegetal lecithin are urgently needed, and their metabolic effects must be characterized. OBJECTIVES: We evaluated the impact of increasing doses of rapeseed lecithin (RL), rich in essential α-linolenic acid (ALA), on postprandial lipid metabolism and ALA bioavailability in lymph-cannulated rats. METHODS: Male Wistar rats (8 weeks old) undergoing a mesenteric lymph duct cannulation were intragastrically administered 1 g of an oil mixture containing 4% ALA and 0, 1, 3, 10, or 30% RL (5 groups). Lymph fractions were collected for 6 h. Lymph lipids and chylomicrons (CMs) were characterized. The expression of genes implicated in intestinal lipid metabolism was determined in the duodenum at 6 h. Data was analyzed using either sigmoidal or linear mixed-effects models, or one-way ANOVA, where appropriate. RESULTS: RL dose-dependently increased the lymphatic recovery (AUC) of total lipids (1100 µg/mL·h per additional RL%; P = 0.010) and ALA (50 µg/mL·h per additional RL%; P = 0.0076). RL induced a faster appearance of ALA in lymph, as evidenced by the exponential decrease of the rate of appearance of ALA with RL (R2 = 0.26; P = 0.0064). Although the number of CMs was unaffected by RL, CM diameter was increased in the 30%-RL group, compared to the control group (0% RL), by 86% at 3-4 h (P = 0.065) and by 81% at 4-6 h (P = 0.0002) following administration. This increase was positively correlated with the duodenal mRNA expression of microsomal triglyceride transfer protein (Mttp; ρ= 0.63; P = 0.0052). The expression of Mttp and secretion-associated, ras-related GTPase 1 gene homolog B (Sar1b, CM secretion), carnitine palmitoyltransferase IA (Cpt1a) and acyl-coenzyme A oxidase 1 (Acox1, beta-oxidation), and fatty acid desaturase 2 (Fads2, bioconversion of ALA into long-chain n-3 PUFAs) were, respectively, 49%, 29%, 74%, 48%, and 55% higher in the 30%-RL group vs. the control group (P < 0.05). CONCLUSIONS: In rats, RL enhanced lymphatic lipid output, as well as the rate of appearance of ALA, which may promote its subsequent bioavailability and metabolic fate.

High-Density Lipoprotein Composition Influences Lymphatic Transport after Subcutaneous Administration.

Interstitial administration (e.g., subcutaneous (SC) administration) of immunotherapies and vaccines within nanoparticles can improve access to lymph-resident immune cells, leading to enhanced efficacy and reduced off-target effects. Recently, endogenous high-density lipoproteins (HDLs) were found to return from peripheral tissue back to the systemic circulation via the lymphatic vessels and nodes. This suggests the potential utility of HDLs as biocompatible lymphatic-targeted therapeutic carriers. However, we have a limited understanding of the mechanisms that drive HDL uptake into peripheral lymphatics from the interstitium. This study investigated the influence of HDL physicochemical properties on lymphatic transport and lymph node (LN) retention of HDL after SC administration. A range of HDL particles was prepared and characterized. Sphere-shaped endogenous HDLs were isolated from biological fluids (rat lymph, rat plasma, and human plasma) and separated into two subclasses based on the density. Discoidal-shaped synthetic (reconstituted) HDLs (rHDLs) of similar sizes were assembled from lipids and apolipoprotein A-I. All HDLs had similar sizes of 10-20 nm and a slightly negative surface charge. All HDLs were radiolabeled with 3H-cholesteryl ester (3H-CE) and/or 14C-free cholesterol (14C FC) and administered SC into the hind leg of thoracic lymph-cannulated rats. The recovery of radiolabels in lymph, plasma, LN, and tissues was determined. From the interstitial injection site, all HDLs were preferentially transported into the lymph and not blood vessels as indicated by high lymph-to-plasma concentration ratios of the radiolabels (up to 100:1 during the absorption phase) and greater radiolabel recovery in LNs draining the injection site compared to the contralateral side. Several HDLs with unique composition demonstrated significantly higher lymphatic transport compared to other HDLs despite possessing similar physical properties, suggesting that HDL lymphatic transport is less influenced by physical properties. The LN retention of HDL was positively correlated to increasing the negative charge of HDL, which was related to surface composition. Overall, this study informs the optimal design of HDL-based nanoparticles to promote lymphatic targeting of immunotherapies and vaccines.

Mechanisms of Connexin-Related Lymphedema.

RATIONALE: Mutations in GJC2 and GJA1, encoding Cxs (connexins) 47 and 43, respectively, are linked to lymphedema, but the underlying mechanisms are unknown. Because efficient lymph transport relies on the coordinated contractions of lymphatic muscle cells (LMCs) and their electrical coupling through Cxs, Cx-related lymphedema is proposed to result from dyssynchronous contractions of lymphatic vessels. OBJECTIVE: To determine which Cx isoforms in LMCs and lymphatic endothelial cells are required for the entrainment of lymphatic contraction waves and efficient lymph transport. METHODS AND RESULTS: We developed novel methods to quantify the spatiotemporal entrainment of lymphatic contraction waves and used optogenetic techniques to analyze calcium signaling within and between the LMC and the lymphatic endothelial cell layers. Genetic deletion of the major lymphatic endothelial cell Cxs (Cx43, Cx47, or Cx37) revealed that none were necessary for the synchronization of the global calcium events that triggered propagating contraction waves. We identified Cx45 in human and mouse LMCs as the critical Cx mediating the conduction of pacemaking signals and entrained contractions. Smooth muscle-specific Cx45 deficiency resulted in 10- to 18-fold reduction in conduction speed, partial-to-severe loss of contractile coordination, and impaired lymph pump function ex vivo and in vivo. Cx45 deficiency resulted in profound inhibition of lymph transport in vivo, but only under an imposed gravitational load. CONCLUSIONS: Our results (1) identify Cx45 as the Cx isoform mediating the entrainment of the contraction waves in LMCs; (2) show that major endothelial Cxs are dispensable for the entrainment of contractions; (3) reveal a lack of coupling between lymphatic endothelial cells and LMCs, in contrast to arterioles; (4) point to lymphatic valve defects, rather than contraction dyssynchrony, as the mechanism underlying GJC2- or GJA1-related lymphedema; and (5) show that a gravitational load exacerbates lymphatic contractile defects in the intact mouse hindlimb, which is likely critical for the development of lymphedema in the adult mouse.

Engagement of Posthemorrhagic Shock Mesenteric Lymph on CD4+ T Lymphocytes In Vivo and In Vitro.

BACKGROUND: Immune dysfunction is associated with posthemorrhagic shock mesenteric lymph (PHSML) return. To determine the proliferation and cytokine production capacity of CD4+ T lymphocytes, the effect of PHSML drainage on spleen CD4+ T lymphocytes in a mouse model of hemorrhagic shock was assessed. METHODS: The normal spleen CD4+ T lymphocytes were in vitro incubated with either drained normal mesenteric lymph (NML), PHSML during hypotension (PHSML-H), or PHSML from 0 h to 3 h after resuscitation (PHSML-R) to verify direct proliferation effects of PHSML. RESULTS: Hemorrhagic shock led to reduction of proliferation and mRNA expression of interleukin 2 (IL-2) and IL-2 receptor in CD4+ T lymphocytes and to decrease in IL-2 and interferon γ (IFN-γ) levels in supernatants. In contrast, the interleukin-4 levels were increased. These effects were reversed by PHSML drainage. Moreover, NML incubation promoted CD4+ T lymphocyte proliferation, whereas both PHSML-H and PHSML-R treatment had a biphasic effects on CD4+ T lymphocyte proliferation, exhibiting an enhanced effect at early stages and an inhibitory effect at later stages. Compared with NML, PHSML-H increased IL-2 expression at 12 h, but decreased expression of both IL-2 and IFN-γ at 24 h. By contrast, PHSML-R induced significant increases in IL-2 and IFN-γ levels at 24 h. Interleukin-4 expression in CD4+ T lymphocytes was reduced at 12 h, but augmented at 24 h after incubation with either PHSML-H or PHSML-R. CONCLUSIONS: The results indicate that PHSML has a direct inhibitory effect on CD4+ T lymphocyte proliferation that induces an inflammatory response, which is associated with cellular immune dysfunction.

Lymphatic Distribution of Etanercept Following Intravenous and Subcutaneous Delivery to Rats.

PURPOSE: The purpose of this work was to investigate the role of the lymphatic system in the pharmacokinetics of etanercept, a fusion protein. METHODS: Etanercept 1 mg/kg was administered intravenously (IV) and subcutaneously (SC) to thoracic lymph duct-cannulated and sham-operated control rats. Blood and lymph samples were obtained for up to 6 days. RESULTS: Model-based SC bioavailability of etanercept was 65.2% in the control group. In lymph-cannulated rats, etanercept concentration in the lymph was consistently lower than in serum following IV dosing; and the concentration in the lymph was significantly higher than in serum after SC injection. The absorption occurred predominantly through the lymphatic pathway (82.7%), and only 17.3% by direct uptake into the central compartment (blood pathway). Lymphatic cannulation reduced the area under the serum concentration-time curve by 28% in IV group and by 91% in SC group. A mechanistic pharmacokinetic model that combined dual absorption pathways with redistribution of the systemically available protein drug into lymph was developed. The model successfully captured serum and lymph data in all groups simultaneously, and all parameters were estimated with sufficient precision. CONCLUSIONS: Lymphatic system was shown to play an essential role in systemic disposition and SC absorption of etanercept.