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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.

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., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

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., Competing Interests: Conflict of Interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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 TNF ΔARE 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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Severe hemorrhagic shock leads to excessive inflammation and immune dysfunction, which results in high mortality related to mesenteric lymph return. A recent study showed that stellate ganglion block (SGB) increased the survival rate in rats suffering hemorrhagic shock. However, whether SGB ameliorates immune dysfunction induced by hemorrhagic shock remains unknown. The aim of the present study was to verify the favorable effects of SGB on the proliferation and function of splenic CD4 + T cells isolated from rats that underwent hemorrhagic shock and to investigate the mechanism related to the SGB interaction with autophagy and posthemorrhagic shock mesenteric lymph (PHSML). Male rats underwent SGB or sham SGB and conscious acute hemorrhage followed by resuscitation and multiple treatments. After 3 h of resuscitation, splenic CD4 + T cells were isolated to measure proliferation and cytokine production following stimulation with ConA in vitro. CD4 + T cells isolated from normal rats were treated with PHSML drained from SBG-treated rats, and proliferation, cytokine production, and autophagy biomarkers were detected. Hemorrhagic shock reduced CD4 + T cell proliferation and production of interleukin (IL)-2, IL-4, and tumor necrosis factor-α-induced protein 8-like 2 (TIPE2). SGB or administration of the autophagy inhibitor 3-methyladenine (3-MA) normalized these indicators. In contrast, administration of rapamycin (RAPA) autophagy agonist or intravenous injection of PHSML inhibited the beneficial effects of SGB on CD4 + T cells from hemorrhagic shocked rats. Furthermore, PHSML incubation decreased proliferation and cytokine production, increased LC3 II/I and Beclin-1 expression, and reduced p-PI3K and p-Akt expression in normal CD4 + T cells. These adverse effects of PHSML were also abolished by 3-MA administration, as well as incubation with PHSML obtained from SGB-treated rats. SGB improves splenic CD4 + T cell function following hemorrhagic shock, which is related to the inhibition of PHSML-mediated autophagy., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

By analogy with virions, the binding of biologically-inspired nanoparticles (NPs) with ligands to the cellular membrane containing receptors depends on the multivalent ligand-receptor interaction, membrane bending, and cytoskeleton deformation. The interplay of these factors results in the existence of the potential minimum and activation barrier on the pathway towards full absorption of a NP. Herein, I hypothesize and show theoretically that the interaction of a NP, bound to one cell, with another cell can stabilize the potential minimum and increase the corresponding activation barrier, i.e., NPs can mediate the formation of long-living pairs of cells and aggregates containing a few cells inside blood and lymphatic vessels., (Copyright © 2021 The Author. Published by Elsevier B.V. All rights reserved.)

The deposition of amyloid-β (Aβ) in the brain is a risk factor for Alzheimer's disease (AD). Therefore, new strategies for the stimulation of Aβ clearance from the brain can be useful in preventing AD. Transcranial photostimulation (PS) is considered a promising method for AD therapy. In our previous studies, we clearly demonstrated the PS-mediated stimulation of lymphatic clearing functions, including Aβ removal from the brain. There is increasing evidence that sleep plays an important role in Aβ clearance. Here, we tested our hypothesis that PS at night can stimulate Aβ clearance from the brain more effectively than PS during the day. Our results on healthy mice show that Aβ clearance from the brain occurs faster at night than during wakefulness. The PS course at night improves memory and reduces Aβ accumulation in the brain of AD mice more effectively than the PS course during the day. Our results suggest that night PS is a more promising candidate as an effective method in preventing AD than daytime PS. These data are an important informative platform for the development of new noninvasive and nonpharmacological technologies for AD therapy as well as for preventing Aβ accumulation in the brain of people with disorder of Aβ metabolism, sleep deficit, elderly age, and jet lag.

Although the general structure of the barrier between the gut and the blood is well known, many details are still missing. Here, we analyse the literature and our own data related to lipid transcytosis through adult mammalian enterocytes, and their absorption into lymph at the tissue level of the intestine. After starvation, the Golgi complex (GC) of enterocytes is in a resting state. The addition of lipids in the form of chyme leads to the initial appearance of pre-chylomicrons (ChMs) in the tubules of the smooth endoplasmic reticulum, which are attached at the basolateral plasma membrane, immediately below the 'belt' of the adhesive junctions. Then pre-ChMs move into the cisternae of the rough endoplasmic reticulum and then into the expansion of the perforated Golgi cisternae. Next, they pass through the GC, and are concentrated in the distensions of the perforated cisternae on the trans-side of the GC. The arrival of pre-ChMs at the GC leads to the transition of the GC to a state of active transport, with formation of intercisternal connections, attachment of cis-most and trans-most perforated cisternae to the medial Golgi cisternae, and disappearance of COPI vesicles. Post-Golgi carriers then deliver ChMs to the basolateral plasma membrane, fuse with it, and secret ChMs into the intercellular space between enterocytes at the level of their interdigitating contacts. Finally, ChMs are squeezed out into the interstitium through pores in the basal membrane, most likely due to the function of the actin-myosin 'cuff' around the interdigitating contacts. These pores appear to be formed by protrusions of the dendritic cells and the enterocytes per se. ChMs are absorbed from the interstitium into the lymphatic capillaries through the special oblique contacts between endothelial cells, which function as valves through the contraction-relaxation of bundles of smooth muscle cells in the interstitium. Lipid overloading of enterocytes results in accumulation of cytoplasmic lipid droplets, an increase in diameter of ChMs, inhibition of intra-Golgi transport, and fusion of ChMs in the interstitium. Here, we summarise and analyse recent findings, and discuss their functional implications., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Inflammatory lipid mediators in mesenteric lymph (ML), including arachidonic acid (AA), are considered to play an important role in the pathogenesis of multiple-organ dysfunction after hemorrhagic shock. A previous study suggested that vagus nerve stimulation (VNS) could relieve shock-induced gut injury and abrogate ML toxicity, resulting in the prevention of multiple-organ dysfunction. However, the detailed mechanism of VNS in lymph toxicity remains unclear. The study aimed to investigate the relationship between VNS and inflammatory lipid mediators in ML., Methods: Male Sprague-Dawley rats underwent laparotomy and superior mesenteric artery obstruction (SMAO) for 60 minutes to induce intestinal ischemia followed by reperfusion and observation. The ML duct was cannulated, and ML samples were obtained both before and after SMAO. The distal ileum was removed at the end of the observation period. In one group of animals, VNS was performed from 10 minutes before 10 minutes after SMAO (5 V, 0.5 Hz). Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of AA was performed for each ML sample. The biological activity of ML was examined using a monocyte nuclear factor κ-light-chain-enhancer of activated B cells activation assay. Western blotting of phospholipase A2 group IIA (PLA2-IIA) was also performed for ML and ileum samples., Results: Vagus nerve stimulation relieved the SMAO-induced histological gut injury. The concentration of AA and level of nuclear factor κ-light-chain-enhancer of activated B cells activation in ML increased significantly after SMAO, whereas VNS prevented these responses. Western blotting showed PLA2-IIA expression in the ML and ileum after SMAO; however, the appearance of PLA2-IIA band was remarkably decreased in the samples from VNS-treated animals., Conclusion: The results suggested that VNS could relieve gut injury induced by SMAO and decrease the production of AA in ML by altering PLA2-IIA expression in the gut and ML., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

T cell expression of sphingosine 1-phosphate (S1P) receptor 1 (S1PR1) enables T cell exit from lymph nodes (LNs) into lymph, while endothelial S1PR1 expression regulates vascular permeability. Drugs targeting S1PR1 treat autoimmune disease by trapping pathogenic T cells within LNs, but they have adverse cardiovascular side effects. In homeostasis, the transporter SPNS2 supplies lymph S1P and enables T cell exit, while the transporter MFSD2B supplies most blood S1P and supports vascular function. It is unknown whether SPNS2 remains necessary to supply lymph S1P during an immune response, or whether in inflammation other compensatory transporters are upregulated. Here, using a model of dermal inflammation, we demonstrate that SPNS2 supplies the S1P that guides T cells out of LNs with an ongoing immune response. Furthermore, deletion of Spns2 is protective in a mouse model of multiple sclerosis. These results support the therapeutic potential of SPNS2 inhibitors to achieve spatially specific modulation of S1P signaling., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

[Figure: see text].

Fibroblastic reticular cells (FRCs), usually found and isolated from the T cell zone of lymph nodes, have recently been described as much more than simple structural cells. Originally, these cells were described to form a conduit system called the "reticular fiber network" and for being responsible for transferring the lymph fluid drained from tissues through afferent lymphatic vessels to the T cell zone. However, nowadays, these cells are described as being capable of secreting several cytokines and chemokines and possessing the ability to interfere with the immune response, improving it, and also controlling lymphocyte proliferation. Here, we performed a systematic review of the several methods employed to investigate the mechanisms used by fibroblastic reticular cells to control the immune response, as well as their ability in determining the fate of T cells. We searched articles indexed and published in the last five years, between 2016 and 2020, in PubMed, Scopus, and Cochrane, following the PRISMA guidelines. We found 175 articles published in the literature using our searching strategies, but only 24 articles fulfilled our inclusion criteria and are discussed here. Other articles important in the built knowledge of FRCs were included in the introduction and discussion. The studies selected for this review used different strategies in order to access the contribution of FRCs to different mechanisms involved in the immune response: 21% evaluated viral infection in this context, 13% used a model of autoimmunity, 8% used a model of GvHD or cancer, 4% used a model of Ischemic-reperfusion injury (IRI). Another four studies just targeted a particular signaling pathway, such as MHC II expression, FRC microvesicles, FRC secretion of IL-15, FRC network, or ablation of the lysophosphatidic acid (LPA)-producing ectoenzyme autotaxin. In conclusion, our review shows the strategies used by several studies to isolate and culture fibroblastic reticular cells, the models chosen by each one, and dissects their main findings and implications in homeostasis and disease.

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

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.

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society for Nutrition.)

The objective of this study was to obtain data on pathways of absorption of the synthetic pyrethroids deltamethrin (DLM) and cis-permethrin (CPM) following oral administration to rats. Adult male Sprague-Dawley rats with cannulated mesenteric lymph ducts and hepatic portal veins were given single doses of either 5 mg/kg DLM or 60 mg/kg CPM via the duodenum and lymph and portal blood samples collected for up to 300 min. The pyrethroid dosing vehicles (5 mL/kg body weight) were either corn oil or glycerol formal. Levels of DLM and CPM in lymph and portal blood samples were determined by high-performance liquid chromatography-mass spectrometry-mass spectrometry. Over the time period studied, levels of both DLM and CPM following administration in either corn oil or glycerol formal were greater in lymph than in portal blood. Lymphatic uptake of both DLM and CPM was enhanced following dosing in glycerol formal than in corn oil. The results of this study suggest that after oral administration to rats, these two pyrethroids are predominantly absorbed via the lymphatic system rather than via portal blood. The data obtained in this study thus support a recently developed physiologically-based pharmacokinetic (PBPK) model to evaluate age-related differences in pyrethroid pharmacokinetics in the rat, where it was assumed that absorption of pyrethroids was predominantly via lymphatic uptake., (Copyright © 2020. Published by Elsevier B.V.)

Vascular endothelial injury caused by post-hemorrhagic shock mesenteric lymph (PHSML) return is an important manifestation during refractory hemorrhagic shock. Using human umbilical vein endothelial cells (HUVECs) and transcriptome analysis, this study sought to investigate the molecular mechanism underlying the adverse effect of PHSML on vascular endothelium. Post-hemorrhagic shock mesenteric lymph was collected from male rats after they underwent hemorrhagic shock and following resuscitation, while normal mesenteric lymph (NML) was harvested from sham rats. Human umbilical vein endothelial cells were incubated with the culture medium containing either 10% phosphate buffered saline (Control), NML, or PHSML for 3 h, and then were harvested for RNA sequencing. In comparison with NML treated cells, 37 genes were differentially expressed in PHSML-treated HUVECs, including 32 upregulated genes and five downregulated genes. These differentially expressed genes were mainly enriched in inflammatory pathways, including signaling pathways for activation of the NOD-like receptors, NF-κB, and TNF. Furthermore, we found that C-C motif chemokine ligand 2 (CCL2) was increased significantly after PHSML treatment, and Bindarit, a CCL2 production inhibitor, attenuated the damage of HUVECs induced by PHSML. The results provide molecular evidence on vascular endothelium damage caused by PHSML. C-C motif chemokine ligand 2 might represent a new target for reducing vascular injury after severe hemorrhagic shock., (Copyright © 2020 Wang, Chi, Wei, Zhao, Zhang, Zhang, Liu, Kang, Zhao, Wang, Nie, Niu and Zhao.)

Hyaluronan (HA) has been widely used in medicines, cosmetics and supplements for health and beauty maintenance. Oral administration is the most desirable and convenient means for consumers. The intestine plays an important role in immune system. We hypothesized that orally administered HA would be transferred to both blood and lymph. In this study, we investigated how orally administered HA was absorbed from the gastrointestinal tract and how much HA was incorporated. Four HA formulations, HA-2,000, 8,000, 50,000 and 300,000, were administered to rats, and concentrations of HA in blood and lymph were determined. In the HA-2,000 group, the HA plasma concentration increased after oral administration. The highest lymph concentration of HA was also obtained by administration of HA-2,000. The plasma and lymph concentrations slightly increased after oral administration in the HA-8,000 group. On the other hand, little absorption was found in the HA-50,000 and 300,000 groups. It is speculated that smaller molecules of HA are more easily absorbed. HA-2,000 was absorbed mainly through the portal vein and through the lymph in gastrointestinal absorption. This is the first report showing that HAs, large molecular weight and water-soluble molecules, after oral administration are transferred not only into blood but also into lymph., Competing Interests: Declaration of Competing Interest The authors report no conflicts of interest in this work., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cancer cells, including melanoma cells, often metastasize regionally through the lymphatic system before metastasizing systemically through the blood 1-4 ; 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.

Objective: A non-lipolysis nanoemulsion (NNE) was designed to reduce the first-pass metabolism of raloxifene (RAL) by intestinal UDP-glucuronosyltransferases (UGTs) for increasing the oral absorption of RAL, coupled with in vitro and in vivo studies., Methods: In vitro stability of NNE was evaluated by lipolysis and the UGT metabolism system. The oral bioavailability of NNE was studied in rats and pigs. Finally, the absorption mechanisms of NNE were investigated by in situ single-pass intestinal perfusion (SPIP) in rats, Madin-Darby canine kidney (MDCK) cells model, and lymphatic blocking model., Results: The pre-NNE consisted of isopropyl palmitate, linoleic acid, Cremophor RH40, and ethanol in a weight ratio of 3.33:1.67:3:2. Compared to lipolysis nanoemulsion of RAL (RAL-LNE), the RAL-NNE was more stable in in vitro gastrointestinal buffers, lipolysis, and UGT metabolism system ( p < 0.05). The oral bioavailability was significantly improved by the NNE (203.30%) and the LNE (205.89%) relative to the suspension group in rats. However, 541.28% relative bioavailability was achieved in pigs after oral NNE intake compared to the suspension and had two-fold greater bioavailability than the LNE ( p < 0.05). The RAL-NNE was mainly absorbed in the jejunum and had high permeability at the intestine of rats. The results of both SPIP and MDCK cell models demonstrated that the RAL-NNE was absorbed via endocytosis mediated by caveolin and clathrin. The other absorption route, the lymphatic transport (cycloheximide as blocking agent), was significantly improved by the NNE compared with the LNE ( p < 0.05)., Conclusion: A NNE was successfully developed to reduce the first-pass metabolism of RAL in the intestine and enhance its lymphatic transport, thereby improving the oral bioavailability. Altogether, NNE is a promising carrier for the oral delivery of drugs with significant first-pass metabolism., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Ye et al.)

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 3 H-cholesteryl ester ( 3 H-CE) and/or 14 C-free cholesterol ( 14 C 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.

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.

The most abundant protein families in viper venoms are Snake Venom Metalloproteases (SVMPs), Snake Venom Serine Proteases (SVSPs) and Phospholipases (PLA 2 s). These are primarily responsible for the pathophysiology caused by the bite of pit-vipers; however, there are few studies that analyze the pharmacokinetics (PK) of whole venom (WV) and its protein families. We studied the pathophysiology, PK profile and differential absorption of representative toxins from venom of Neotropical Rattlesnake ( Crotalus simus ) in a large animal model (ovine). Toxins studied included crotoxin (the main lethal component), which causes moderate to severe neurotoxicity; SVSPs, which deplete fibrinogen; and SVMPs, which cause local tissue damage and local and systemic hemorrhage. We found that Whole Venom (WV) was highly bioavailable (86%) 60 h following intramuscular (IM) injection, and extrapolation suggests that bioavailability may be as high as 92%. PK profiles of individual toxins were consistent with their physicochemical properties and expected clinical effects. Lymph cannulated animals absorbed 1.9% of WV through lymph during the first 12 h. Crotoxin was minimally detectable in serum after intravenous (IV) injection; however, following IM injection it was detected in lymph but not in blood. This suggests that crotoxin is quickly released from the blood toward its tissue targets.

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., (© 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

In 2010, cytidine 5'-triphosphate synthase (CTPS) was reported to form the filamentous or serpentine structure in Drosophila, which we termed the cytoophidium. In the last decade, CTPS filaments/cytoophidia have been found in bacteria, budding yeast, human cells, mice, fission yeast, plants, and archaea, indicating that this mechanism is highly conserved in evolution. In addition to CTPS, other metabolic enzymes have been identified to have the characteristics of forming cytoophidia or similar advanced structures, demonstrating that this is a basic strategy of cells. Nevertheless, our understanding of the physiological function of the cytoophidium remains incomplete and elusive. Here, we took the larva of Drosophila melanogaster as a model to systematically describe the localization and distribution of cytoophidia in different tissues during larval development. We found that the distribution pattern of CTPS cytoophidia is dynamic and heterogenic in larval tissues. Our study provides a road map for further understanding of the function and regulatory mechanism of cytoophidia., (Copyright © 2020 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

Physiologically-based pharmacokinetic (PBPK) modelling provides an integrated framework to predict the disposition of small molecule drugs in children and is increasingly being used for dose recommendation and optimal design of paediatric studies and in regulatory submissions. Existing paediatric PBPK models can be adopted to describe the disposition of therapeutic proteins (TPs) in children by incorporating information on age-related changes of additional physiological and biological parameters (e.g. endogenous IgG, neonatal Fc receptor, lymph flow). In this study, physiological parameters were collated from literature and evaluated for any age-dependent trends. The age-dependent physiological parameters were used to construct a paediatric PBPK model for TPs. The model was then used to predict the pharmacokinetics of recombinant human erythropoietin (EPO), infliximab, etanercept, basiliximab, anakinra and enfuvirtide in paediatric subjects. The developed paediatric PBPK model predicted the drug concentration-time profiles reasonably well in full-term neonates (clinical PK data only available for EPO), infants, children and adolescents with the ratios of predicted over observed clearance values within 1.5-fold and 25 out of 26 clearance predictions were within 0.8- to 1.25-fold of the observed values. The clinically reported data are required to further assess the predictive accuracy of PK for Fc-containing proteins in term-born children younger than 2 months. This study demonstrates the ability of PBPK models accounting for age-dependent changes in relevant parameters to predict the pharmacokinetics of different types of TPs in paediatrics. The information gained from the PBPK models described here can facilitate our understanding of the complexities of TPs' disposition during growth and development.

BACKGROUND This study was carried out to evaluate the effects of a long-term high-fat diet on lipids and lipoproteins composition in thoracic duct lymph in pigs. MATERIAL AND METHODS We examined lymph taken from the thoracic duct from 24 female white sharp-ear pigs, divided into 3 experimental groups fed different diets for 12 months: (a) the control group, fed the standard balanced diet; (b) the HFD group, fed an unbalanced, high-fat diet, and (c) the reversal diet group (RD), fed an unbalanced, high-fat diet for 9 months and then a standard balanced diet for 3 months. RESULTS Lymph analysis after 12 months of fixed diets revealed significantly higher concentration of proteins in the HFD group in comparison to the control and RD groups. Examination of lymph lipoproteins fractions showed that the high-fat diet in the HFD group in comparison to control group caused an increase in cholesterol, phospholipids, and proteins content within HDL and chylomicrons. There were also more proteins within HDL in the HFD group in comparison to the RD group and more triglycerides within chylomicrons in the HFD group in comparison to the control group. CONCLUSIONS A long-term high-fat diet resulted in changed structure of HDL and chylomicrons in the thoracic duct lymph. Alterations in HDL composition suggest that a high-fat diet enhances reverses cholesterol transport. Changes in chylomicrons structure show the adaptation to more intense transport of dietary fat from the intestine to the liver under the influence of a high-fat diet. Reversal to a standard balanced diet had the opposite effects.

We studied the levels of serum and lymph cytokines involved in the pathogenesis of BC. BC was induced by injection of N-methyl-N-nitrosourea to Wistar rats. The animals underwent surgery, or received polychemotherapy (cyclophosphamide, methotrexate, and 5-fluorouracil), or surgical treatment was combined with polychemotherapy; in a special group, Panagen (fragmented DNA) was added to polychemotherapy. Cytokine concentration in the lymph was measured using Bio-Plex Pro Rat Cytokoness 24-Plex Assay test system (Bio-Rad). In rats with BC, the content of most studied cytokines (IL-1β, IL-2, IL-4, IL-6, IL-7, IL-12, IL-13, MIP-1α, MIP-3α, RANTES, TNFα, and MCP-1) in the lymph and blood was significantly higher, while the content of IL-10 and GRO/KC was lower than in intact animals. Surgical resection of the tumor led to a significant decrease in the content of both pro- and anti-inflammatory cytokines in the lymph. Polychemotherapy led to a significant decrease in the content of IL-1β, IL-4, IL-6, IL-7, MIP-1α, MIP-3α, and RANTES in the serum and lymph. Comparison of the cytokine content in the serum and lymph of operated animals after polychemotherapy with and without Panagen showed that the content of most cytokines (IL-5, IL-6, IL-7, IL-10, IL-13, IL-17A , IL-18, GRO/KC, IFNγ, and MIP-3α) was higher after Panagen administration.

The aim of this work was to study the bioavailability of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA), i.e. eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), carried by marine phospholipids (PL) and formulated in different supramolecular forms. Marine PL were administrated in rats either (1) in bulk form, or (2) as an oil-in-water emulsion, or (3) as liposomes. Each dietary formulation was characterized by a similar fatty acid (FA) profile and provided the same n-3 LC-PUFA amount. Intestinal bioavailability of n-3 LC-PUFA was monitored in the lymph compartment in a duct fistula model. On the one hand, the emulsification of plant oils with PL increased the overall intestinal absorption of dietary FA by 84% without affecting the lymph FA profile compared with the bulk form, suggesting that emulsification favoured the absorption of the total dietary FA derived from both triglycerides (TG) and PL. On the other hand, the liposome form did not modify the lymph lipid amount compared with the bulk form, but specifically increased the n-3 LC-PUFA levels. The dietary forms of PL influenced the position of some FA on the glycerol backbone of lymph TG and PL. In conclusion, using marine PL as an emulsifier promoted total FA absorption independently of the dietary lipid carrier (TG or PL) and the FA type. Structuring PL as liposomes specifically increased the intestinal bioavailability of FA esterifed in this lipid class, such as DHA, resulting in a higher incorporation into lymph lipids. Thus, using specific PL supramolecular forms would guide n-3 LC-PUFA towards total lipid absorption or specific FA absorption, according to the dietary needs.

Background: The discovery of intracranial lymphatic system provides a new theory about cerebrospinal fluid circulation. In order to investigate the role of lymphatic drainage in hydrocephalus after intraventricular hemorrhage (IVH), three parts of experiments were performed. Methods: IVH model was produced by 200ul autologous blood injection into lateral ventricle of SD male rats. Perls Prussian blue reaction was used to confirm the ability of iron drainage through deep cervical lymph nodes (DCLNs) in part I. The part II measured the volume of ventricles by MR scanner , and the part III compared the ferritin level of area surrounding ventricles by Western blotting after IVH. Results: The ability of iron drainage through DCLNs was confirmed. DCLNs-excised rats got a 19.6% higher incidence of hydrocephalus than DCLNs-preserved rats at day 28 after IVH. And DCLNs-excised group showed higher ferritin level than DCLNs-preserved group at day 3 after IVH. Conclusion: The whole experiments revealed the role of intracranial lymphatic system in discharging iron out of central nervous system at acute phase of IVH, and proved the function in alleviating and avoiding hydrocephalus at chronic phase after IVH.

Purpose: Inhaled delivery of pirfenidone to the lungs of patients with idiopathic pulmonary fibrosis holds promise to eliminate oral-observed side effects while enhancing efficacy. This study aimed to comprehensively describe the pulmonary pharmacokinetics of inhaled aerosol pirfenidone in healthy adult sheep., Methods: Pirfenidone concentrations were evaluated in plasma, lung-derived lymph and epithelial lining fluid (ELF) with data subjected to non-compartmental pharmacokinetic analysis., Results: Compartmental pharmacokinetic evaluation indicated that a 49 mg lung-deposited dose delivered an ELF Cmax of 62 ± 23 mg/L, and plasma Cmax of 3.1 ± 1.7 mg/L. Further analysis revealed that plasma pirfenidone reached Tmax faster and at higher concentrations than in lymph. These results suggested inhaled pirfenidone was cleared from the alveolar interstitium via blood faster than the drug could equilibrate between the lung interstitial fluid and lung lymphatics. However, the data also suggested that a 'reservoir' of pirfenidone feeds into lung lymph at later time points (after it has largely been cleared from plasma), prolonging lung lymphatic exposure., Conclusions: This study indicates inhaled pirfenidone efficiently deposits in ELF and is cleared from the lungs by initial absorption into plasma, followed by later equilibrium with lung interstitial and lymph fluid.

Interstitial, e.g. subcutaneous (SC) or intradermal (ID), administration of monoclonal antibodies (mAb) is less invasive than intravenous administration and leads to mAb uptake into both lymphatic and blood capillaries draining the injection site. Interstitial administration, however, is hindered by the presence of hyaluronan (HA), a glycosaminoglycan that is a major fluid barrier in the interstitial space. The transient removal of HA with recombinant human hyaluronidase (rHuPH20) helps facilitate the interstitial administration of often high therapeutic doses of mAb in the clinic. rHuPH20's impact on the systemic pharmacokinetics of several mAbs has been previously described, however effects on route of absorption (lymph vs blood) are unknown. The current study has therefore explored the lymphatic transport and bioavailability of cetuximab and trastuzumab after SC and ID coadministration in the presence and absence of rHuPH20 in rats. After SC administration cetuximab absolute bioavailability increased from 67 % to 80 % in the presence of rHuPH20. Cetuximab recovery in the lymphatics also increased after SC (35.8 % to 49.4 %) and ID (26.7 % to 58.8 %) administration in the presence of rHuPH20. When the injection volume (and therefore dose) was increased 10-fold in the presence of rHuPH20 cetuximab plasma exposure increased approximately linearly (12- and 8.9-fold respectively after SC and ID administration), although the proportional contribution of cetuximab lymphatic transport reduced slightly (6.2-fold increase for both administration routes). In contrast, co-administration with rHuPH20 did not lead to increases in plasma exposure for trastuzumab after SC or ID administration, most likely reflecting the fact that the reported absolute bioavailability of trastuzumab (in the absence of rHuPH20) is high (∼77-99 %). However, lymphatic transport of trastuzumab did increase when coadministered ID with rHuPH20 in spite of the lack of change to overall bioavailability. The data suggest that co-administration with rHuPH20 is able to increase the volume of mAb that can be administered interstitially, and in some instances can increase the amount absorbed into both the blood and the lymph. In the current studies the ability of rHuPH20 to enhance interstitial bioavailability was higher for cetuximab where intrinsic interstitial bioavailability was low, when compared to trastuzumab where interstitial bioavailability was high., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Doxorubicin is a risk factor for secondary lymphedema in cancer patients exposed to surgery or radiation. The risk is presumed to relate to its cytotoxicity. However, the present study provides initial evidence that doxorubicin directly inhibits lymph flow and this action appears distinct from its cytotoxic activity. We used real-time edge detection to track diameter changes in isolated rat mesenteric lymph vessels. Doxorubicin (0.5-20 μ mol/l) progressively constricted lymph vessels and inhibited rhythmic contractions, reducing flow to 24.2% ± 7.7% of baseline. The inhibition of rhythmic contractions by doxorubicin paralleled a tonic rise in cytosolic Ca 2+ concentration in lymphatic muscle cells, which was prevented by pharmacological antagonism of ryanodine receptors. Washout of doxorubicin partially restored lymph vessel contractions, implying a pharmacological effect. Subsequently, high-speed optical imaging was used to assess the effect of doxorubicin on rat mesenteric lymph flow in vivo. Superfusion of doxorubicin (0.05-10 μ mol/l) maximally reduced volumetric lymph flow to 34% ± 11.6% of baseline. Likewise, doxorubicin (10 mg/kg) administered intravenously to establish clinically achievable plasma concentrations also maximally reduced volumetric lymph flow to 40.3% ± 6.0% of initial values. Our findings reveal that doxorubicin at plasma concentrations achieved during chemotherapy opens ryanodine receptors to induce "calcium leak" from the sarcoplasmic reticulum in lymphatic muscle cells and reduces lymph flow, an event linked to lymph vessel damage and the development of lymphedema. These results infer that pharmacological block of ryanodine receptors in lymphatic smooth muscle cells may mitigate secondary lymphedema in cancer patients subjected to doxorubicin chemotherapy. SIGNIFICANCE STATEMENT: Doxorubicin directly inhibits the rhythmic contractions of collecting lymph vessels and reduces lymph flow as a possible mechanism of secondary lymphedema, which is associated with the administration of anthracycline-based chemotherapy. The inhibitory effects of doxorubicin on rhythmic contractions and flow in isolated lymph vessels were prevented by pharmacological block of ryanodine receptors, thereby identifying the ryanodine receptor family of proteins as potential therapeutic targets for the development of new antilymphedema medications., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Context: Historically, administration and dosing of antivenom (AV) have been guided primarily by physician judgment because of incomplete understanding of the envenomation process. As demonstrated previously, lymphatic absorption plays a major role in the availability and pharmacokinetics (PK) of coral snake venom injected subcutaneously, which suggests that absorption from subcutaneous tissue is the limiting step for venom bioavailability, supporting the notion that the bite site is an ongoing venom depot. This feature may underlie the recurrence phenomena reported in viperid envenomation that appear to result from a mismatch between venom and AV PK. The role of lymphatic absorption in neutralization of venom by AV administered intravenously remains unclear. Methods: The effect of AV on systemic bioavailability and neutralization of Micrurus fulvius venom was assessed using a central lymph-cannulated sheep model. Venom was administered by subcutaneous injection in eight sheep, four with and four without thoracic duct cannulation and drainage. Two hours after venom injection, AV was administered intravenously. Venom and AV concentrations in serum and lymph were determined by ELISA assay from samples collected over a 6-h period and in tissues harvested post-mortem. Results: After AV injection, venom levels in serum fell immediately to undetectable with a subsequent increase in concentration attributable to non-toxic venom proteins. In lymph, AV became detectable 6 min after treatment; venom levels dropped concurrently but remained detectable 4 h later. Post-mortem samples from the venom injection site confirmed the presence of venom near the point of injection. Neither venom nor AV was detected at significant concentrations in major organs or contralateral skin. Conclusions: Intravenous AV immediately neutralizes venom in the bloodstream and can extravasate to neutralize venom absorbed by lymph but this neutralization seems to be slow and incomplete. Residual venom in the inoculation site demonstrates that this site functions as a depot where it is not neutralized by AV, which allows the venom to remain active with slow delivery to the bloodstream for ongoing systemic distribution.

Objective: Dietary triglycerides are partially retained in the intestine within intracellular or extracellular compartments, which can be rapidly mobilized in response to several stimuli, including glucose and GLP-2 (glucagon-like peptide-2). To elucidate the mechanism of intestinal lipid mobilization, this study examined the patterns and time course of lymph flow and triglycerides after glucose and GLP-2 treatment in rats. Approach and Results: Lymph flow, triglyceride concentration, and triglyceride output were assessed in mesenteric lymph duct-cannulated rats in response to an intraduodenal (i.d.) lipid bolus followed 5 hours later by either (1) i.d. saline+intraperitoneal (i.p.) saline (placebo), (2) i.d. glucose plus i.p. saline, (3) i.d. saline+i.p. GLP-2, or (4) i.d. glucose+i.p. GLP-2. GLP-2 and glucose administered alone or in combination stimulated total triglyceride output to a similar extent, but the timing and pattern of stimulation differed markedly. Whereas GLP-2 rapidly increased lymph flow with no effect on lymph triglyceride concentration or triglyceride:apoB48 (apolipoprotein B48) ratio (a surrogate marker of chylomicron size) compared with placebo, glucose transiently decreased lymph flow followed by delayed stimulation of lymph flow and increased lymph triglyceride concentration and triglyceride:apoB48 ratio., Conclusions: Glucose and GLP-2 robustly enhanced intestinal triglyceride output in rats but with different effects on lymph flow, lymph triglyceride concentration, and chylomicron size. GLP-2 stimulated triglyceride output primarily by enhancing lymph flow with no effect on chylomicron size, whereas glucose mobilized intestinal triglycerides, stimulating secretion of larger chylomicrons. This suggests that these 2 stimuli mobilize intestinal lipid by different mechanisms.

Secondary upper limb lymphoedema is usually caused by lymphatic system dysfunction. Diagnosis is primarily based on clinical features. However, there are no distinct diagnostic criteria for lymphoedema. Although conventional lymphoscintigraphy is a useful technique to diagnose the severity of lymphoedema, the resultant data are two-dimensional. In this study, we examined the pathology of lymphoedema using single photon emission computed tomography-computed tomography lymphoscintigraphy (SPECT-CT LSG), a new technique that provides 3-dimensional information on lymph flow. We observed lymph flow pathways in the subcutaneous and muscle layers of the upper limbs. A significant positive correlation was found between the dermal back flow (DBF) type and the visualization of lymph nodes around the clavicle (p = 0.000266), the type of lymph flow pathways and the visualization of lymph nodes around the clavicle (p = 0.00963), and the DBF type and the lymph flow pathway (p = 0.00766). As the severity of lymphoedema increased, the DBF appeared more distally in the upper limb and the flow into the lymph nodes around the clavicle decreased, whereas the lymph flow pathways in the muscle layer became dominant. These findings demonstrate the features of lymphoedema pathology and the functional anatomy and physiology of the lymphatic system without the need for cadaver dissection.

Liquid biopsies allow monitoring of cancer progression and detection of relapse, but reliable biomarkers in melanoma are lacking. Because secreted factors preferentially drain to lymphatic vessels before dilution in the blood, we hypothesized that lymph should be vastly enriched in cancer biomarkers. We characterized postoperative lymphatic exudate and plasma of metastatic melanoma patients after lymphadenectomy and found a dramatic enrichment in lymphatic exudate of tumor-derived factors and especially extracellular vesicles containing melanoma-associated proteins and miRNAs, with unique protein signatures reflecting early versus advanced metastatic spread. Furthermore, lymphatic exudate was enriched in memory T cells, including tumor-reactive CD137 + and stem cell-like types. In mice, lymph vessels were the major route of extracellular vesicle transport from tumors to the systemic circulation. We suggest that lymphatic exudate provides a rich source of tumor-derived factors for enabling the discovery of novel biomarkers that may reflect disease stage and therapeutic response., (© 2019 Broggi et al.)