Functional Immune Anatomy of the Liver-As an Allograft.

The liver is an immunoregulatory organ in which a tolerogenic microenvironment mitigates the relative "strength" of local immune responses. Paradoxically, necro-inflammatory diseases create the need for most liver transplants. Treatment of hepatitis B virus, hepatitis C virus, and acute T cell-mediated rejection have redirected focus on long-term allograft structural integrity. Understanding of insults should enable decades of morbidity-free survival after liver replacement because of these tolerogenic properties. Studies of long-term survivors show low-grade chronic inflammatory, fibrotic, and microvascular lesions, likely related to some combination of environment insults (i.e. abnormal physiology), donor-specific antibodies, and T cell-mediated immunity. The resultant conundrum is familiar in transplantation: adequate immunosuppression produces chronic toxicities, while lightened immunosuppression leads to sensitization, immunological injury, and structural deterioration. The "balance" is more favorable for liver than other solid organ allografts. This occurs because of unique hepatic immune physiology and provides unintended benefits for allografts by modulating various afferent and efferent limbs of allogenic immune responses. This review is intended to provide a better understanding of liver immune microanatomy and physiology and thereby (a) the potential structural consequences of low-level, including allo-antibody-mediated injury; and (b) how liver allografts modulate immune reactions. Special attention is given to the microvasculature and hepatic mononuclear phagocytic system.

Spontaneous dormancy of metastatic breast cancer cells in an all human liver microphysiologic system.

BACKGROUND: Metastatic outgrowth in breast cancer can occur years after a seeming cure. Existing model systems of dormancy are limited as they do not recapitulate human metastatic dormancy without exogenous manipulations and are unable to query early events of micrometastases. METHODS: Here, we describe a human ex vivo hepatic microphysiologic system. The system is established with fresh human hepatocytes and non-parenchymal cells (NPCs) creating a microenvironment into which breast cancer cells (MCF7 and MDA-MB-231) are added. RESULTS: The hepatic tissue maintains function through 15 days as verified by liver-specific protein production and drug metabolism assays. The NPCs form an integral part of the hepatic niche, demonstrated within the system through their participation in differential signalling cascades and cancer cell outcomes. Breast cancer cells intercalate into the hepatic niche without interfering with hepatocyte function. Examination of cancer cells demonstrated that a significant subset enter a quiescent state of dormancy as shown by lack of cell cycling (EdU(-) or Ki67(-)). The presence of NPCs altered the cancer cell fraction entering quiescence, and lead to differential cytokine profiles in the microenvironment effluent. CONCLUSIONS: These findings establish the liver microphysiologic system as a relevant model for the study of breast cancer metastases and entry into dormancy.

Hepatic B cells are readily activated by Toll-like receptor-4 ligation and secrete less interleukin-10 than lymphoid tissue B cells.

B cells perform various immunological functions that include production of antibody, presentation of antigens, secretion of multiple cytokines and regulation of immune responses mainly via their secretion of interleukin (IL)-10. While the liver is regarded both as an important immune organ and a tolerogenic environment, little is known about the functional biology of hepatic B cells. In this study we demonstrate that, following lipopolysaccharide (LPS) stimulation in vivo, normal mouse hepatic B cells rapidly increase their surface expression of CD39, CD40, CD80 and CD86, and produce significantly elevated levels of proinflammatory interferon (IFN)-γ, IL-6 and tumour necrosis factor (TNF)-α compared with splenic B cells. Moreover, LPS-activated hepatic B cells produce very low levels of IL-10 compared with activated splenic B cells that produce comparatively high levels of this immunosuppressive cytokine. Splenic, but not hepatic, B cells inhibited the activation of liver conventional myeloid dendritic cells (mDCs). Furthermore, compared with the spleen, the liver exhibited significantly smaller proportions of B1a and marginal zone-like B cells, which have been shown to produce IL-10 upon LPS stimulation. These data suggest that, unlike in the spleen, IL-10-producing regulatory B cells in the liver are not a prominent cell type. Consistent with this, when compared with liver conventional mDCs from B cell-deficient mice, those from B cell-competent wild-type mice displayed enhanced expression of the cell surface co-stimulatory molecule CD86, greater production of proinflammatory cytokines (IFN-γ, IL-6, IL-12p40) and reduced secretion of IL-10. These findings suggest that hepatic B cells have the potential to initiate rather than regulate inflammatory responses.

The expression profile of C19MC microRNAs in primary human trophoblast cells and exosomes.

The largest gene cluster of human microRNAs (miRNAs), the chromosome 19 miRNA cluster (C19MC), is exclusively expressed in the placenta and in undifferentiated cells. The precise expression pattern and function of C19MC members are unknown. We sought to profile the relative expression of C19MC miRNAs in primary human trophoblast (PHT) cells and exosomes. Using high-throughput profiling, confirmed by PCR, we found that C19MC miRNAs are among the most abundant miRNAs in term human trophoblasts. Hypoxic stress selectively reduced miR-520c-3p expression at certain time-points with no effect on other C19MC miRNAs. Similarly, differentiation in vitro had a negligible effect on C19MC miRNAs. We found that C19MC miRNAs are the predominant miRNA species expressed in exosomes released from PHT, resembling the profile of trophoblastic cellular miRNA. Predictably, we detected the similar levels of circulating C19MC miRNAs in the serum of healthy pregnant women at term and in women with pregnancies complicated by fetal growth restriction. Our data define the relative expression levels of C19MC miRNAs in trophoblasts and exosomes, and suggest that C19MC miRNAs function in placental-maternal signaling.

Treprostinil, a prostacyclin analog, ameliorates ischemia-reperfusion injury in rat orthotopic liver transplantation.

Prostaglandins have been evaluated for their ability to reduce IRI after liver transplantation; however, poor stability, side effects and the inability to show a significant difference in primary endpoint have limited their clinical application. Treprostinil, a prostacyclin (PGI(2) ) analog, has a higher potency and longer elimination half-life than other commercially available PGI(2) analogs. We examined the efficacy of treprostinil to prevent IRI during OLT. OLT was performed in syngeneic Lewis rats after 18 h of cold preservation (4°C) in the UW solution. IRI significantly increased serum ALT and AST levels, neutrophil infiltration, hepatic necrosis and mRNA levels of proinflammatory cytokines post-OLT, while treatment with treprostinil decreased all the parameters. Cold storage of liver grafts significantly reduced ATP levels and treprostinil restored energy levels in liver grafts early postreperfusion. In addition, treprostinil preserved the sinusoidal endothelial cell lining and reduced platelet deposition early post-transplantation compared to placebo. Hepatic tissue blood flow was significantly compromised in the placebo group, whereas treprostinil maintained blood-flow similar to normal levels. Treprostinil protected the liver graft against IRI during OLT. Treprostinil has the potential to serve as a therapeutic option to protect the liver graft against I/R injury in patients undergoing OLT.

Ex vivo application of carbon monoxide in UW solution prevents transplant-induced renal ischemia/reperfusion injury in pigs.

I/R injury is a major deleterious factor of successful kidney transplantation (KTx). Carbon monoxide (CO) is an endogenous gaseous regulatory molecule, and exogenously delivered CO in low concentrations provides potent cytoprotection. This study evaluated efficacies of CO exposure to excised kidney grafts to inhibit I/R injury in the pig KTx model. Porcine kidneys were stored for 48 h in control UW or UW supplemented with CO (CO-UW) and autotransplanted in a 14-day follow-up study. In the control UW group, animal survival was 80% (4/5) with peak serum creatinine levels of 12.0 +/- 5.1 mg/dL. CO-UW showed potent protection, and peak creatinine levels were reduced to 6.9 +/- 1.4 mg/dL with 100% (5/5) survival without any noticeable adverse event or abnormal COHb value. Control grafts at 14 days showed significant tubular damages, focal fibrotic changes and numerous infiltrates. The CO-UW group showed significantly less severe histopathological changes with less TGF-beta and p-Smad3 expression. Grafts in CO-UW also showed significantly lower early mRNA levels for proinflammatory cytokines and less lipid peroxidation. CO in UW provides significant protection against renal I/R injury in the porcine KTx model. Ex vivo exposure of kidney grafts to CO during cold storage may therefore be a safe strategy to reduce I/R injury.

Elevated levels of hepatocyte nuclear factor 3beta in mouse hepatocytes influence expression of genes involved in bile acid and glucose homeostasis.

The winged helix transcription factor, hepatocyte nuclear factor-3beta (HNF-3beta), mediates the hepatocyte-specific transcription of numerous genes important for liver function. However, the in vivo role of HNF-3beta in regulating these genes remains unknown because homozygous null HNF3beta mouse embryos die in utero prior to liver formation. In order to examine the regulatory function of HNF-3beta, we created transgenic mice in which the -3-kb transthyretin promoter functions to increase hepatocyte expression of the rat HNF-3beta protein. Postnatal transgenic mice exhibit growth retardation, depletion of hepatocyte glycogen storage, and elevated levels of bile acids in serum. The retarded growth phenotype is likely due to a 20-fold increase in hepatic expression of insulin-like growth factor binding protein 1 (IGFBP-1), which results in elevated levels in serum of IGFBP-1 and limits the biological availability of IGFs required for postnatal growth. The defects in glycogen storage and serum bile acids coincide with diminished postnatal expression of hepatocyte genes involved in gluconeogenesis (phosphoenolpyruvate carboxykinase and glycogen synthase) and sinusoidal bile acid uptake (Ntcp), respectively. These changes in gene transcription may result from the disruptive effect of HNF-3beta on the hepatic expression of the endogenous mouse HNF-3alpha,-3beta, -3gamma, and -6 transcription factors. Furthermore, adult transgenic livers lack expression of the canalicular phospholipid transporter, mdr2, which is consistent with ultrastructure evidence of damage to transgenic hepatocytes and bile canaliculi. These transgenic studies represent the first in vivo demonstration that the HNF-3beta transcriptional network regulates expression of hepatocyte-specific genes required for bile acid and glucose homeostasis, as well as postnatal growth.

Growth factor signal transduction immediately after two-thirds partial hepatectomy in the rat.

Liver regeneration after partial hepatectomy (PHx) of the liver serves as a model for studying normal growth factor signals that become aberrant in cancer. Growth factor signals that may play a role in initiating the proliferation of hepatocytes after 70% PHx in the rat were investigated immediately after surgical resection of the liver. Presumptive activity was evaluated by determining the tyrosine phosphorylation state of receptors for epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in the liver after PHx and after sham operation as a control. Under these conditions, it was determined that the EGF receptor was constitutively phosphorylated. EGF receptor tyrosine phosphorylation, however, was increased over basal levels by 60 min after resection. The HGF receptor, c-Met, was minimally phosphorylated in control livers, but a biphasic increase in phosphorylation was observed at 1-5 min after PHx and 60 min postsurgery. A slight increase in c-Met phosphorylation was observed in the sham-operated livers, but the signal was significantly less when compared with that in resected livers. Furthermore, 1 min after PHx, but not sham operation, urokinase-type plasminogen activator (u-PA) and u-PA receptor were observed in the immunoprecipitates of c-Met. Signaling downstream of growth factor receptor activation was also examined. There were no discernible phosphorylation changes in focal adhesion kinase during the early events after surgery in PHx; however, a rapid and sustained increase in the tyrosine phosphorylation of paxillin beginning 1 min after PHx, and a gradual increase in the phosphorylation beginning 5 min postsham operation, were observed. Changes in the activated state of the small GTP-binding protein Rho A and its associated proteins were seen but only after 3 h after PHx. The results indicate that HGF-related signal transduction cascades, which contribute to hepatocyte proliferation, are initiated within one min after PHx.

Presence of urokinase in serum-free primary rat hepatocyte cultures and its role in activating hepatocyte growth factor.

Serum-free rat hepatocyte cultures can be stimulated to divide by the inactive, single-chain form of hepatocyte growth factor (scHGF), suggesting that hepatocytes contain a protein that can cleave scHGF to its biologically active, two-chain (tcHGF) form. We added radiolabeled scHGF to serum-free cultures and confirmed that tcHGF was being generated. Because scHGF can be cleaved to tcHGF by plasminogen activators (PAs), we next tested the cultures for active PA. Although little PA activity was initially present, the majority was of the urokinase type (u-PA) as determined by neutralization studies using either a polyclonal antibody against u-PA or, since u-PA functions in the context of its receptor (u-PAR), a monoclonal antibody against u-PAR. Considerable PA activity developed within 24 h, which was also neutralizable with antibody. To test whether the active, receptor-bound u-PA from the cell cultures was cleaving scHGF, iodinated scHGF was added to intact cells in the presence of the antibody against u-PAR. Comparison to control cultures determined that the antibody prevented scHGF cleavage. Analysis of cultures treated with HGF, epidermal growth factor, and transforming growth factor alpha (TGF-alpha) alpha showed these growth factors increased the hepatocyte PA activity in parallel with the mRNA for u-PA. TGF-beta had the opposite effect, and when TGF-beta was added to the culture system, conversion of scHGF to tcHGF was prevented in concert with the production of the type 1 PA inhibitor. When liver remnants from hepatectomized animals were assayed for active TGF-beta, elevated protein was found just prior to the appearance of PA inhibitor 1 message and protein. Collectively, our data show that in culture, active u-PA is present and cleaves scHGF to tcHGF in the context of its receptor. It also suggests that modulation of u-PA activity by various growth factors is relevant for regulating cleavage of scHGF to tcHGF both in vitro and in vivo.

Reactive nitrogen and oxygen radicals formed during hepatic ischemia-reperfusion kill weakly metastatic colorectal cancer cells.

Microscopic infarcts develop within the livers of athymic nude mice during the first 24 h after human colorectal carcinoma (CRC) cells arrest within hepatic sinusoids. Because these regions are reperfused, essentially all weakly metastatic clone A and MIP-101 CRC cells die, whereas many highly metastatic CX-1 CRC cells survive. Because hepatic sinusoidal endothelial cells kill tumor cells in vitro by producing nitric oxide, superoxide anion, and other reactive oxygen and nitrogen species, our purpose was to determine whether reoxygenation of ischemic hepatic cultures in vitro forms toxic oxygen and nitrogen radicals that kill weakly but not highly metastatic CRC cells. CRC cells (10(7)) were labeled with rhodamine-dextran and calcein AM, cultured with cells from one mouse liver in a rotating suspension culture system for up to 24 h, and the metabolic activity of the CRC cells was determined. Liver fragments oxygenated normally before harvest were not toxic to either CRC cell line, but coculture with liver made ischemic by a 3-min ligation of the portal vein and hepatic artery in vivo before harvest and then cultured in oxygenated medium killed 50-70% of weakly metastatic clone A and MIP-101 cells at 24 h but <15% of highly metastatic CX-1 cells. Inhibition of nitric oxide synthase, addition of exogenous superoxide dismutase, but not catalase or hypoxia, during coculture blocked the killing of weakly metastatic CRC cells. Thus, reoxygenation of hepatic parenchymal and nonparenchymal cells after ischemia may form toxic species that eliminate weakly metastatic CRCs within 24 h of their arrest in the liver.

A liver microphysiological system of tumor cell dormancy and inflammatory responsiveness is affected by scaffold properties.

Distant metastasis is the major cause of breast cancer-related mortality, commonly emerging clinically after 5 or more years of seeming 'cure' of the primary tumor, indicating a quiescent dormancy. The lack of relevant accessible model systems for metastasis that recreate this latent stage has hindered our understanding of the molecular basis and the development of therapies against these lethal outgrowths. We previously reported on the development of an all-human 3D ex vivo hepatic microphysiological system that reproduces several features of liver physiology and enables spontaneous dormancy in a subpopulation of breast cancer cells. However, we observed that the dormant cells were localized primarily within the 3D tissue, while the proliferative cells were in contact with the polystyrene scaffold. As matrix stiffness is known to drive inflammatory and malignant behaviors, we explored the occurrence of spontaneous tumor dormancy and inflammatory phenotype. The microphysiological system was retrofitted with PEGDa-SynKRGD hydrogel scaffolding, which is softer and differs in the interface with the tissue. The microphysiological system incorporated donor-matched primary human hepatocytes and non-parenchymal cells (NPCs), with MDA-MB-231 breast cancer cells. Hepatic tissue in hydrogel scaffolds secreted lower levels of pro-inflammatory analytes, and was more responsive to inflammatory stimuli. The proportion of tumor cells entering dormancy was markedly increased in the hydrogel-supported tissue compared to polystyrene. Interestingly, an unexpected differential response of dormant cells to varying chemotherapeutic doses was identified, which if reflective of patient pathophysiology, has important implications for patient dosing regimens. These findings highlight the metastatic microphysiological system fitted with hydrogel scaffolds as a critical tool in the assessment and development of therapeutic strategies to target dormant metastatic breast cancer.

A microscale in vitro physiological model of the liver: predictive screens for drug metabolism and enzyme induction.

In vitro models of the liver using isolated primary hepatocytes have been used as screens for measuring the metabolism, toxicity and efficacy of xenobiotics, for studying hepatocyte proliferation, and as bioartificial liver support systems. Yet, primary isolated hepatocytes rapidly lose liver specific functions when maintained under standard in vitro cell culture conditions. Many modifications to conventional culture methods have been developed to foster retention of hepatocyte function. Still, not all of the important functions -- especially the biotransformation functions of the liver -- can as yet be replicated at desired levels, prompting continued development of new culture systems. In the first part of this article, we review primary hepatocyte in vitro systems used in metabolism and enzyme induction studies. We then describe a scalable microreactor system that fosters development of 3D-perfused micro-tissue units and show that primary rat cells cultured in this system are substantially closer to native liver compared to cells cultured by other in vitro methods, as assessed by a broad spectrum of gene expression, protein expression and biochemical activity metrics. These results provide a foundation for extension of this culture model to other applications in drug discovery -- as a model to study drug-drug interactions, as a model for the assessment of acute and chronic liver toxicity arising from exposure to drugs or environmental agents; and as a disease model for the study of viral hepatitis infection and cancer metastasis.

Biochemical characterization of the soluble form of tumor antigen MUC1 isolated from sera and ascites fluid of breast and pancreatic cancer patients.

Transmembrane glycoprotein tumor antigen MUC1 that is overexpressed on pancreatic and breast tumor cells can be found in large amounts in soluble form in serum and ascites fluid. MUC1 has been identified as a target of human antitumor antibody and CTL responses that are generated in the absence of helper T cells. The soluble form of MUC1 should support generation of helper T cells, but we have found recently that this form, although effectively endocytosed by dendritic cells, remains trapped in early endosomes and is not trafficked to antigen-processing compartments. The exact biochemical structure of this form of MUC1 has not been elucidated to date, and it is thus not clear what structural characteristics may be responsible for its retention in early endosomes. We have purified soluble MUC1 from ascites fluid of breast/pancreatic cancer patients (ASC-MUC1) and quantitated O-linked carbohydrates. We have altered ASC-MUC1 by enzymatic treatment: trypsin or clostripain digestion, desialylation, and further in vitro glycosylation. We have found that desialylated ASC-MUC1 was further glycosylated by peptidyl N-acetylgalactosamine transferases and was not when sialic acid was present. These alterations created new forms of ASC-MUC1 that might be handled more efficiently by antigen-presenting cells to generate better tumor-specific immunity and used to identify structures that are directly involved in retention of this antigen in early endosomes.

Thrombospondin-1 restrains neutrophil granule serine protease function and regulates the innate immune response during Klebsiella pneumoniae infection.

Neutrophil elastase (NE) and cathepsin G (CG) contribute to intracellular microbial killing but, if left unchecked and released extracellularly, promote tissue damage. Conversely, mechanisms that constrain neutrophil serine protease activity protect against tissue damage but may have the untoward effect of disabling the microbial killing arsenal. The host elaborates thrombospondin-1 (TSP-1), a matricellular protein released during inflammation, but its role during neutrophil activation following microbial pathogen challenge remains uncertain. Mice deficient in TSP-1 (thbs1(-/-)) showed enhanced lung bacterial clearance, reduced splenic dissemination, and increased survival compared with wild-type (WT) controls during intrapulmonary Klebsiella pneumoniae infection. More effective pathogen containment was associated with reduced burden of inflammation in thbs1(-/-) mouse lungs compared with WT controls. Lung NE activity was increased in thbs1(-/-) mice following K. pneumoniae challenge, and thbs1(-/-) neutrophils showed enhanced intracellular microbial killing that was abrogated with recombinant TSP-1 administration or WT serum. Thbs1(-/-) neutrophils exhibited enhanced NE and CG enzymatic activity, and a peptide corresponding to amino-acid residues 793-801 within the type-III repeat domain of TSP-1 bridled neutrophil proteolytic function and microbial killing in vitro. Thus, TSP-1 restrains proteolytic action during neutrophilic inflammation elicited by K. pneumoniae, providing a mechanism that may regulate the microbial killing arsenal.

Rat mitochondrial ATP synthase ATP5G3: cloning and upregulation in pancreas after chronic ethanol feeding.

Individuals with chronic excessive alcohol ingestion are put at the risk of acute and chronic pancreatitis. Underlying molecular mechanisms are unknown. Differential gene expression in the pancreas was profiled using mRNA differential display by comparison between control and ethanol-consuming rats. Male Wistar rats were fed with diets containing 6.7% (vol/vol) ethanol for 4 wk. A cDNA tag that was overexpressed in the pancreas of rats fed ethanol was isolated. A 723-bp cDNA was cloned from a rat pancreatic cDNA library, which encodes a novel rat mitochondrial ATP synthase subunit 9, isoform 3 (ATP5G3), which is homologous to a human ATP5G3 gene. Real-time PCR demonstrated that all three nuclear gene isoforms (ATP5G1, ATP5G2, and ATP5G3) were consistently upregulated in the pancreas of alcohol-consuming rats, parallel with mitochondrial injury. The cellular response to mitochondrial damage and metabolic stress may reflect an adaptive process for mitochondrial repair in pancreatic acinar cells during chronic ethanol ingestion.

Toluene diisocyanate colocalizes with tubulin on cilia of differentiated human airway epithelial cells.

Toluene diisocyanate (TDI), a highly reactive industrial chemical with widespread use in the manufacture of polyurethane and plastics, is the leading cause of occupational asthma associated with chemical exposure. We report the effects of TDI vapor (20, 100, 500, 1000 ppb) in vitro on differentiated human bronchial epithelial cells. Increased mucus was observed by electron microscopy at all TDI concentrations. Cytotoxicity, as evidenced by cell pyknosis and DNA fragmentation, was detected following a 30-min exposure to TDI concentrations of 100 ppb or higher. At 1000 ppb, transepithelial resistance was lost. Using confocal microscopy and double staining, TDI was found colocalized with ciliary tubulin in cultures that had been exposed to 20 and 100 ppb. These findings are the first to identify TDI binding to human pulmonary epithelial cells and indicate extensive binding to the cilia of differentiated epithelial cells. The in vivo implications of these findings include decreased ciliary movement and longer retention of TDI and hence increased exposure. Altered cytoskeletal-derived signal transduction may be a consequence of tubulin involvement. The effects of such changes on respiratory sensitization remain to be explored.

Lyophilization of cationic lipid-protamine-DNA (LPD) complexes.

Cationic lipid-based gene delivery systems have shown promise in transfecting cells both in vitro and in vivo. However, these systems tend to form aggregates in liquid formulation during storage, which has limited their clinical applications. As a result, lyophilization of these systems has recently become a subject of increasing interest. In this paper, lyophilization of LPD, a novel cationic lipid-based gene delivery system, was studied. Both particle size and transfection efficiency could be preserved in the presence of sufficient amount of appropriate lyoprotectant. A series of monosaccharides and disaccharides, including dextrose, galactose, mannose, lactose, maltose, sucrose and trehalose, were evaluated for their lyoprotective effect and disaccharides showed more superior protection to monosaccharides. The effect of different freezing protocols for lyophilization was also evaluated and no significant difference was found. However, for freeze-thawing, fast freezing caused less aggregation. Finally, nonlyophilized LPD and LPD lyophilized with 10% sucrose were stored at different temperatures and their stability was followed for eight weeks. Lyophilized LPD could be stored at room temperature without significant change in particle size or loss of transfection efficiency.

Thrombospondin-1 triggers macrophage IL-10 production and promotes resolution of experimental lung injury.

Mononuclear phagocyte recognition of apoptotic cells triggering suppressive cytokine signaling is a key event in inflammation resolution from injury. Mice deficient in thrombospondin (TSP)-1 (thbs1⁻/⁻), an extracellular matrix glycoprotein that bridges cell-cell interactions, are prone to lipopolysaccharide-induced lung injury and show defective macrophage interleukin (IL)-10 production during the resolution phase of inflammation. Reconstitution of IL-10 rescues thbs1⁻/⁻ mice from persistent neutrophilic lung inflammation and injury and thbs1⁻/⁻ alveolar macrophages show defective IL-10 production following intratracheal instillation of apoptotic neutrophils despite intact efferocytosis. Following co-culture with apoptotic neutrophils, thbs1⁻/⁻ macrophages show a selective defect in IL-10 production, whereas prostaglandin E2 and transforming growth factor beta 1 responses remain intact. Full macrophage IL-10 responses require the engagement of TSP-1 structural repeat 2 domain and the macrophage scavenger receptor CD36 LIMP-II Emp sequence homology (CLESH) domain in vitro. Although TSP-1 is not essential for macrophage engulfment of apoptotic neutrophils in vivo, TSP-1 aids in the curtailment of inflammatory responses during the resolution phase of injury in the lungs by providing a means by which apoptotic cells are recognized and trigger optimal IL-10 production by macrophages.

Structural impact of hydrodynamic injection on mouse liver.

The impact of hydrodynamic injection on liver structure was evaluated in mice using various microscopic techniques. Upon hydrodynamic injection of approximately 9% of body weight by volume, the liver rapidly expanded, reaching maximal size at the end of the injection and returned to its original size in 30 min. Histological analysis revealed a swollen appearance in the peri-central region of the liver where delivery of genes and fluorescence-labeled markers was observed. Scanning and transmission electron microscopy showed enlargement and rupture of endothelium that in about 24-48 h regains its morphology and normal function as a barrier against infection by adenovirus viral particles. At the cellular level in hydrodynamically treated animals, four types of hepatocytes were seen: cells with normal appearance; cells with enriched vesicles in the cytoplasm; cells with lightly stained cytosol; and cells with significant dilution of the cytoplasm. In addition, red blood cells and platelets were observed in the space of Disse and even inside hepatocytes. Vesicle formation is triggered by hydrodynamic injection and resembles the process of macropinocytosis. These results, whereas confirming the physical nature of hydrodynamic delivery, are important for a better understanding of this efficient method for intrahepatic gene and small interfering RNA delivery.