Lipolysis of Visceral Adipocyte Triglyceride by Pancreatic Lipases Converts Mild Acute Pancreatitis to Severe Pancreatitis Independent of Necrosis and Inflammation

Visceral fat necrosis has been associated with severe acute pancreatitis (SAP) for over 100 years; however, its pathogenesis and role in SAP outcomes are poorly understood. Based on recent work suggesting that pancreatic fat lipolysis plays an important role in SAP, we evaluated the role of pancreatic lipases in SAP-associated visceral fat necrosis, the inflammatory response, local injury, and outcomes of acute pancreatitis (AP). For this, cerulein pancreatitis was induced in lean and obese mice, alone or with the lipase inhibitor orlistat and parameters of AP induction (serum amylase and lipase), fat necrosis, pancreatic necrosis, and multisystem organ failure, and inflammatory response were assessed. Pancreatic lipases were measured in fat necrosis and were overexpressed in 3T3-L1 cells. We noted obesity to convert mild cerulein AP to SAP with greater cytokines, unsaturated fatty acids (UFAs), and multisystem organ failure, and 100% mortality without affecting AP induction or pancreatic necrosis. Increased pancreatic lipase amounts and activity were noted in the extensive visceral fat necrosis of dying obese mice. Lipase inhibition reduced fat necrosis, UFAs, organ failure, and mortality but not the parameters of AP induction. Pancreatic lipase expression increased lipolysis in 3T3-L1 cells. We conclude that UFAs generated via lipolysis of visceral fat by pancreatic lipases convert mild AP to SAP independent of pancreatic necrosis and the inflammatory response.Visceral fat necrosis has been noted to occur with pancreatitis for over 100 years.^1,2 This fat is typically located in or around the pancreas^3,4 and is a major component of necrotizing pancreatitis and peripancreatic necrosis.^5,6 Despite being a part of the criteria for staging the severity of acute pancreatitis (AP) in humans^7–9 and being included as a separate entity in the recently revised Atlanta criteria,⁶ the pathogenesis and role of fat necrosis, that is, whether it is a marker or mediator of severe AP (SAP), remains unclear.Clinical correlates of the relevance of fat necrosis include the several epidemiological studies that show individuals with increased intra-abdominal fat^10–13 or obese patients being at an increased risk for SAP.^11,14–20 Peripancreatic fat necrosis may occur independent of pancreatic necrosis⁵ and is associated with an increased risk for severe attacks.^21,22 Although there is basolateral leakage of pancreatic enzymes during pancreatitis,^23–26 and lipases have been noted in fat necrosis in human disease,^27,28 it is unclear whether these lipases cause fat necrosis or are a remnant of pancreatic damage.Triglycerides compose 80% to 90% of the volume of adipocytes^29–31 and can be hydrolyzed by lipases released basolaterally during pancreatitis.^23–26 Although in vitro studies done more than 2 decades ago showed lipase inhibition to reduce pancreatic acinar injury on co-incubation with pancreatic homogenates and triglycerides,³² in vivo lipase inhibition in SAP models showed no benefit³³ until recently, when this was studied in the context of increased visceral fat.^34,35Previous studies have shown that long-chain unsaturated fatty acids (UFAs) form the majority of the nonesterified fatty acids in necrotic collections^36,37 and are more toxic than are saturated fatty acids.^32,34,35,38 Since obese (ob/ob) mice and obese humans may have a similar visceral fat composition,^3,35 we chose to study whether a classically mild model of pancreatitis, that is, cerulein pancreatitis, would result in different outcomes in these mice versus lean mice and explore the role of fat necrosis in these outcomes. Our previous work has shown a benefit of pharmacological inhibition of pancreatic lipases in biliary, IL-12–induced, and IL-18–induced pancreatitis^34,35; we therefore used this as a tool to influence outcomes. Since obesity is also known to be associated with an exaggerated baseline inflammatory state, we went on to study whether the inflammatory response was different between the groups that had different outcomes. Additionally, since SAP may occur along with severe pancreatic necrosis or with multisystem organ failure (MSOF) with insignificant necrosis,^39–41 we also compared pancreatic necrosis between groups with different outcomes.Reasons for preferring a pharmacological approach^34,35 over a genetic one include: i) previous validation in mechanistically distinct models,^34,35 ii) the redundant roles of pancreatic lipases⁴² (which are supported by this study), iii) the deletion of the two lipases of interest being embryonically lethal,⁴³ and iv) the inability to make mice with a genetic deletion of lipases obese on a high-fat diet (unpublished data). Our results, in concert with those from previous studies,^34,35 show that pancreatic lipase–dependent visceral fat lipolysis worsens outcomes of AP, unrelated to the initiator of the disease, and suggest alternate ways to interpret the parameters of severity in AP, along with suggesting a potential approach to improving SAP outcomes.     

On the Etiology of Type 1 Diabetes: A New Animal Model Signifying a Decisive Role for Bacteria Eliciting an Adverse Innate Immunity Response

The cause of type 1 diabetes (T1D) remains unknown; however, a decisive role for environmental factors is recognized. The increased incidence of T1D during the last decades, as well as regional differences, is paralleled by differences in the intestinal bacterial flora. A new animal model was established to test the hypothesis that bacteria entering the pancreatic ductal system could trigger β-cell destruction and to provide new insights to the immunopathology of the disease. Obtained findings were compared with those present in two patients dying at onset of T1D. Different bacterial species, present in the human duodenum, instilled into the ductal system of the pancreas in healthy rats rapidly induced cellular infiltration, consisting of mainly neutrophil polymorphonuclear cells and monocytes/macrophages, centered around the pancreatic ducts. Also, the islets of Langerhans attracted polymorphonuclear cells, possibly via release of IL-6, IL-8, and monocyte chemotactic protein 1. Small bleedings or large dilatations of the capillaries were frequently found within the islets, and several β-cells had severe hydropic degeneration (ie, swollen cytoplasm) but with preserved nuclei. A novel rat model for the initial events in T1D is presented, revealing marked similarities with the morphologic findings obtained in patients dying at onset of T1D and signifying a decisive role for bacteria in eliciting an adverse innate immunity response. The present findings support the hypothesis that T1D is an organ-specific inflammatory disease.Our understanding of the etiology of type 1 diabetes (T1D) remains limited and originates to a large extent from two animal models: the nonobese diabetic mouse and the BioBreeding-diabetes prone rat.¹ In both models a progressive T-cell–mediated destruction of the β-cells occurs; however, the immunopathologic findings reveal limited similarities with the human disease.^2–5 In human pancreatic specimens, insulitis is discrete, affects only a few islets, and is heterogeneously distributed within the gland. In a recent meta-analysis, insulitis was reported in only 29% of patients with onset between 15 and 39 years of age and with a disease duration of <1 month.⁶ At the time of diagnosis, autoantibodies were only present in approximately 70% to 80% of affected patients.⁷ Likewise, attempts to prevent disease progression with immunosuppression^8–11 or immunointerventions^12–14 cause no or only transient preservation of β-cell function.The fact that the exocrine pancreas gets affected in patients with T1D is underappreciated, and several studies have found autoantibodies in the exocrine cells before the onset of T1D.^15–18 Mild to moderate exocrine pancreatic insufficiency is an early event in T1D,¹⁹ and a substantial reduction (32%) in pancreatic volume is already present 3 to 4 months after onset.²⁰ Also, in the classic report by Gepts,⁴ lesions of the acinar tissue were reported to occur frequently in patients with recent onset of T1D. The findings comprised mostly focal or diffuse lesions of acute pancreatitis with accumulation of leukocytes, often centered around the excretory canals.^2–5 In a more recent study of patients with long-term T1D, 40% had periductal fibrosis and 60% of cases had periductal fibrosis that extended to the interlobular pancreas.²¹ Collectively, these observations suggest that the injurious process that causes T1D affects both the exocrine and endocrine components of the pancreas and challenge the view that T1D is a β-cell–specific autoimmune disease.The low concordance rate for the development of T1D in identical twins and the current rapid increase in incidence of T1D argue against a decisive role for genetic factors. Notably, there is a close to sixfold gradient in the incidence of T1D between Russian and Finland Karelia, although the population is homogenous and the predisposing HLA genotypes are equally frequent.²² In addition, children born in Finland by immigrants from Somalia, a low incidence country for T1D, acquire the same risk for T1D as the native Finish population.²³ On the basis of these and similar observations, it is generally assumed that environmental factors may act as triggers of T1D. For decades different enteroviruses have been implicated in the pathogenesis of T1D²⁴; however, evidence of causality remains missing.Bacterial colonization of the infantile gut is influenced by environmental factors and has changed markedly in developed countries during the last decades.²⁵ Interestingly, the increased incidence of T1D²⁶ and the difference in incidence of T1D in Sweden, Italy, and Africa^26–28 are paralleled by reported frequencies of intestinal Staphylococcus aureus.^29–32 Bacteria entering the ductal system of the pancreas would be exposed to the pancreatic juice–containing substances, with marked antibacterial effects initiating release of bacterial components, such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), and various toxins. Notably, these substances have been implicated in the etiology of neurogenerative diseases and neural cell death because they stimulate microglia to produce proinflammatory cytokines (IL-1b, IL-6, tumor necrosis factor-α), nitric oxide, and reactive oxygen species, causing significant cell death in neighboring neural cells.³³The present study was conducted to establish an animal model for the initial events in T1D to test the hypothesis that bacteria entering into the ductal system of the pancreas could elicit an adverse innate immunity response. Different bacterial species present transiently or continuously in the human duodenum were instilled into the ductal system of the pancreas in healthy rats. To examine the clinical relevance of the experimental model, obtained findings were compared with those present in the pancreases of two patients dying at onset of T1D.     

Fatty Acid Ethyl Esters Are Less Toxic Than Their Parent Fatty Acids Generated during Acute Pancreatitis

Although ethanol causes acute pancreatitis (AP) and lipolytic fatty acid (FA) generation worsens AP, the contribution of ethanol metabolites of FAs, ie, FA ethyl esters (FAEEs), to AP outcomes is unclear. Previously, pancreata of dying alcoholics and pancreatic necrosis in severe AP, respectively, showed high FAEEs and FAs, with oleic acid (OA) and its ethyl esters being the most abundant. We thus compared the toxicities of FAEEs and their parent FAs in severe AP. Pancreatic acini and peripheral blood mononuclear cells were exposed to FAs or FAEEs in vitro. The triglyceride of OA (i.e., glyceryl tri-oleate) or OAEE was injected into the pancreatic ducts of rats, and local and systemic severities were studied. Unsaturated FAs at equimolar concentrations to FAEEs induced a larger increase in cytosolic calcium, mitochondrial depolarization, and necro-apoptotic cell death. Glyceryl tri-oleate but not OAEE resulted in 70% mortality with increased serum OA, a severe inflammatory response, worse pancreatic necrosis, and multisystem organ failure. Our data show that FAs are more likely to worsen AP than FAEEs. Our observations correlate well with the high pancreatic FAEE concentrations in alcoholics without pancreatitis and high FA concentrations in pancreatic necrosis. Thus, conversion of FAs to FAEE may ameliorate AP in alcoholics.Although fat necrosis has been associated with severe cases of pancreatitis for more than a century,^{1, 2} and alcohol consumption is a well-known risk factor for acute pancreatitis (AP),³ only recently have we started understanding the mechanistic basis of these observations.^{4, 5, 6, 7} High amounts of unsaturated fatty acids (UFAs) have been noted in the pancreatic necrosis and sera of severe AP (SAP) patients by multiple groups.^{8, 9, 10, 11, 12} These high UFAs seem pathogenically relevant because several studies show UFAs can cause pancreatic acinar injury or can worsen AP.^{11, 12, 13, 14} Ethanol may play a role in AP by distinct mechanisms,³ including a worse inflammatory response to cholecystokinin,⁴ increased zymogen activation,¹⁵ basolateral enzyme release,¹⁶ sensitization to stress,⁷ FA ethyl esters (FAEEs),¹⁷ cytosolic calcium,¹⁸ and cell death.¹⁹Because the nonoxidative ethanol metabolite of fatty acids (FAs), FAEEs, were first noted to be elevated in the pancreata of dying alcoholics, they have been thought to play a role in AP.^{17, 19, 20, 21, 22} Conclusive proof of the role of FAEEs in AP in comparison with their parent UFAs is lacking. Uncontrolled release of lipases into fat, whether in the pancreas or in the peritoneal cavity, may result in fat necrosis, UFA generation, which has been associated with SAP.^{11, 12} Pancreatic homogenates were also noted to have an ability to synthesize FAEEs from FAs and ethanol,^{20, 23} and the putative enzyme for this was thought to be a lipase.^{24, 25} It has been shown that the FAEE synthase activity of the putative enzyme exceeds its lipolytic capacity by several fold.²⁵Triglyceride (TG) forms >80% of the adipocyte mass,^{26, 27, 28} oleic acid (OA) being the most enriched FA.^{9, 29} We recently showed that lipolysis of intrapancreatic TG worsens pancreatitis.^{11, 12} Therefore, after noting the ability of the pancreas to cause lipolysis of TG into FAs and also to have high FAEE synthase activity and FAEE concentrations, we decided to compare the relative ability of FAEEs and their parent FAs to initiate deleterious signaling in pancreatitis and to investigate their impact on the severity of AP.     

Acute Lipotoxicity Regulates Severity of Biliary Acute Pancreatitis without Affecting Its Initiation

Obese patients have worse outcomes during acute pancreatitis (AP). Previous animal models of AP have found worse outcomes in obese rodents who may have a baseline proinflammatory state. Our aim was to study the role of acute lipolytic generation of fatty acids on local severity and systemic complications of AP. Human postpancreatitis necrotic collections were analyzed for unsaturated fatty acids (UFAs) and saturated fatty acids. A model of biliary AP was designed to replicate the human variables by intraductal injection of the triglyceride glyceryl trilinoleate alone or with the chemically distinct lipase inhibitors orlistat or cetilistat. Parameters of AP etiology and outcomes of local and systemic severity were measured. Patients with postpancreatitis necrotic collections were obese, and 13 of 15 had biliary AP. Postpancreatitis necrotic collections were enriched in UFAs. Intraductal glyceryl trilinoleate with or without the lipase inhibitors resulted in oil red O–positive areas, resembling intrapancreatic fat. Both lipase inhibitors reduced the glyceryl trilinoleate–induced increase in serum lipase, UFAs, pancreatic necrosis, serum inflammatory markers, systemic injury, and mortality but not serum alanine aminotransferase, bilirubin, or amylase. We conclude that UFAs are enriched in human necrotic collections and acute UFA generation via lipolysis worsens pancreatic necrosis, systemic inflammation, and injury associated with severe AP. Inhibition of lipolysis reduces UFA generation and improves these outcomes of AP without interfering with its induction.The mystique of acute pancreatitis (AP) lies in its diverse origins, unpredictable course, and outcomes, ranging from resolution with minimal care to being a debilitating, protracted, and potentially lethal condition despite intensive care and complex interventions to manage its complications. The course AP takes seems unrelated to the origin in most cases, with differences in the predominant origin of AP reported in studies from different countries.^1–5 However, studies have repeatedly reported a higher body mass index (BMI) or obesity to be associated with severe AP (SAP).^1–8 SAP may result from severe pancreatic necrosis, in which >30% of the pancreas is necrosed,^9,10 or from persistent or multisystem organ failure, such as respiratory and renal failure. Obese patients have been reported to be more prone to both these types of complications of AP.¹⁻⁸In contrast to the clinical scenario, conventional animal models of AP differ in the initiating factor used, and the severity associated with these has been attributed to the inciting stimulus^11–13 or species in which the model has been executed.^12–15 For example, rat intraductal bile salt–induced pancreatitis has been classified as severe in contrast to the caerulein model, which is mild.^12,13 Interestingly, caerulein-induced AP is milder in rats than in mice, which have more pancreatic necrosis, and thus mouse caerulein pancreatitis is classified as severe.^14,15 However, in both these cases, the pancreas returns to normal a few days after cessation of the insult, with no residual necrotic areas or organ failure. On the basis of such models, a potential target is regarded as therapeutically relevant if it plays a role in mechanistically dissimilar models of AP. An example of this is phosphatidylinositol 3-kinases and associated trypsin generation,^11,16,17 which we and others have previously found to be relevant to AP of different causes.^11,16,17This discord (ie, the lack of association of outcomes to cause as noted clinically) and how animal models are interpreted have resulted in serious discrepancies between what is predicted to be beneficial in animal models of AP and the success of such interventions in clinical trials. The failure of serine protease and trypsin inhibition to improve outcomes of AP in >70 clinical trials performed during the last 5 decades is a classic example.^18–27Recently, the mechanistic proof of obesity being a modifier of AP outcomes has emerged, with the same model being mild in lean mice and severe in obese mice, associated with an exaggerated inflammatory response and mortality.²⁸ Our recent studies have found that lipolysis of visceral fat in obese mice may contribute to this severity.²⁹ However, obesity is also associated with a baseline proinflammatory state,^30–32 and because fatty acids (FAs) are proinflammatory,^29,33,34 it has yet to be decided whether short-term generation of FAs by the lipolysis of visceral fat or the preexistent inflammatory state associated with obesity determines the severity of AP in these models.We therefore analyzed human postpancreatitis necrotic collections (PPNCs) for the nature of FAs in them. We also noted the most common cause of AP in our patients. Because biliary AP was the most common type of AP and unsaturated FAs (UFAs) were abundant in PPNCs, we studied whether their acute lipolytic generation in rats, which are otherwise normal, results in the severe outcomes noted in SAP and whether inhibition of such lipolysis, using 2 distinct lipase inhibitors separately, alters the initiation of AP or the parameters of its severity. Interestingly, we realize that the beneficial effect of lipase inhibition, which decreases the generation of UFAs, is independent of the initiation of biliary AP. These findings have relevance to how we design and interpret animal models of AP in the context of human disease.     

Type I Interferon Contributes to Noncanonical Inflammasome Activation,Mediates Immunopathology,and Impairs Protective Immunity during Fatal Infection with Lipopolysaccharide-Negative Ehrlichiae

Ehrlichia species are intracellular bacteria that cause fatal ehrlichiosis, mimicking toxic shock syndrome in humans and mice. Virulent ehrlichiae induce inflammasome activation leading to caspase-1 cleavage and IL-18 secretion, which contribute to development of fatal ehrlichiosis. We show that fatal infection triggers expression of inflammasome components, activates caspase-1 and caspase-11, and induces host-cell death and secretion of IL-1β, IL-1α, and type I interferon (IFN-I). Wild-type and Casp1^−/− mice were highly susceptible to fatal ehrlichiosis, had overwhelming infection, and developed extensive tissue injury. Nlrp3^−/− mice effectively cleared ehrlichiae, but displayed acute mortality and developed liver injury similar to wild-type mice. By contrast, Ifnar1^−/− mice were highly resistant to fatal disease and had lower bacterial burden, attenuated pathology, and prolonged survival. Ifnar1^−/− mice also had improved protective immune responses mediated by IFN-γ and CD4⁺ Th1 and natural killer T cells, with lower IL-10 secretion by T cells. Importantly, heightened resistance of Ifnar1^−/− mice correlated with improved autophagosome processing, and attenuated noncanonical inflammasome activation indicated by decreased activation of caspase-11 and decreased IL-1β, compared with other groups. Our findings demonstrate that IFN-I signaling promotes host susceptibility to fatal ehrlichiosis, because it mediates ehrlichia-induced immunopathology and supports bacterial replication, perhaps via activation of noncanonical inflammasomes, reduced autophagy, and suppression of protective CD4⁺ T cells and natural killer T-cell responses against ehrlichiae.Ehrlichia chaffeensis is the causative agent of human monocytotropic ehrlichiosis, a highly prevalent life-threatening tickborne disease in North America.^{1, 2, 3} Central to the pathogenesis of human monocytotropic ehrlichiosis is the ability of ehrlichiae to survive and replicate inside the phagosomal compartment of host macrophages and to secrete proteins via type I and type IV secretion systems into the host-cell cytosol.⁴ Using murine models of ehrlichiosis, we and others have demonstrated that fatal ehrlichial infection is associated with severe tissue damage caused by TNF-α–producing cytotoxic CD8⁺ T cells (ie, immunopathology) and the suppression of protective CD4⁺ Th1 immune responses.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14} However, neither how the Ehrlichia bacteria trigger innate immune responses nor how these responses influence the acquired immunity against ehrlichiae is entirely known.Extracellular and intracellular pattern recognition receptors recognize microbial infections.^{15, 16, 17, 18} Recently, members of the cytosolic nucleotide-binding domain and leucine-rich repeat family (NLRs; alias NOD-like receptors), such as NLRP3, have emerged as critical pattern recognition receptors in the host defense against intracellular pathogens. NLRs recognize intracellular bacteria and trigger innate, protective immune responses.^{19, 20, 21, 22, 23} NLRs respond to both microbial products and endogenous host danger signals to form multimeric protein platforms known as inflammasomes. The NLRP3 inflammasome consists of multimers of NLRP3 that bind to the adaptor molecules and apoptosis-associated speck-like protein (ASC) to recruit pro–caspase-1 and facilitate cleavage and activation of caspase-1.^{15, 16, 24} The canonical inflammasome pathway involves the cleavage of immature forms of IL-1β and IL-18 (pro–IL-1β and pro–IL-18) into biologically active mature IL-1β and IL-18 by active caspase-1.^{25, 26, 27, 28} The noncanonical inflammasome pathway marked by the activation of caspase-11 has been described recently. Active caspase-11 promotes the caspase-1–dependent secretion of IL-1β/IL-18 and mediates inflammatory lytic host-cell death via pyroptosis, a process associated with the secretion of IL-1α and HMGB1.^{17, 29, 30, 31} Several key regulatory checkpoints ensure the proper regulation of inflammasome activation.^{16, 32} For example, blocking autophagy by the genetic deletion of the autophagy regulatory protein ATG16L1 increases the sensitivity of macrophages to the inflammasome activation induced by TLRs.³³ Furthermore, TIR domain-containing adaptor molecule 1 (TICAM-1; alias TRIF) has been linked to inflammasome activation via the secretion of type I interferons α and β (IFN-α and IFN-β) and the activation of caspase-11 during infections with Gram-negative bacteria.^{2, 34, 35, 36, 37, 38, 39}We have recently demonstrated that fatal ehrlichial infection induces excess IL-1β and IL-18 production, compared with mild infection,^{8, 12, 13, 14} and that lack of IL-18 signaling enhances resistance of mice to fatal ehrlichiosis.¹² These findings suggest that inflammasomes play a detrimental role in the host defense against ehrlichial infection. Elevated production of IL-1β and IL-18 in fatal ehrlichiosis was associated with an increase in hepatic expression of IFN-α.¹⁴ IFN-I plays a critical role in the host defense against viral and specific bacterial infections.^{28, 36, 37, 40, 41, 42, 43} However, the mechanism by which type I IFN contributes to fatal ehrlichial infection remains unknown. Our present results reveal, for the first time, that IFNAR1 promotes detrimental inflammasome activation, mediates immunopathology, and impairs protective immunity against ehrlichiae via mechanisms that involve caspase-11 activation, blocking of autophagy, and production of IL-10. Our novel finding that lipopolysaccharide (LPS)-negative ehrlichiae trigger IFNAR1-dependent caspase-11 activation challenges the current paradigm that implicates LPS as the major microbial ligand triggering the noncanonical inflammasome pathway during Gram-negative bacterial infection.     

Immunogenicity of Decellularized Porcine Liver for Bioengineered Hepatic Tissue

Liver disease affects millions of patients each year. The field of regenerative medicine promises alternative therapeutic approaches, including the potential to bioengineer replacement hepatic tissue. One approach combines cells with acellular scaffolds derived from animal tissue. The goal of this study was to scale up our rodent liver decellularization method to livers of a clinically relevant size. Porcine livers were cannulated via the hepatic artery, then perfused with PBS, followed by successive Triton X-100 and SDS solutions in saline buffer. After several days of rinsing, decellularized liver samples were histologically analyzed. In addition, biopsy specimens of decellularized scaffolds were seeded with hepatoblastoma cells for cytotoxicity testing or implanted s.c. into rodents to investigate scaffold immunogenicity. Histological staining confirmed cellular clearance from pig livers, with removal of nuclei and cytoskeletal components and widespread preservation of structural extracellular molecules. Scanning electron microscopy confirmed preservation of an intact liver capsule, a porous acellular lattice structure with intact vessels and striated basement membrane. Liver scaffolds supported cells over 21 days, and no increased immune response was seen with either allogeneic (rat-into-rat) or xenogeneic (pig-into-rat) transplants over 28 days, compared with sham–operated on controls. These studies demonstrate that successful decellularization of the porcine liver could be achieved with protocols developed for rat livers, yielding nonimmunogenic scaffolds for future hepatic bioengineering studies.Within the United States alone, tens of thousands of patients are awaiting a liver transplant, with only a few thousand donor organs available annually.¹ This widening mismatch has led physicians and researchers to pursue alternative therapies for chronic liver disease, including in situ cell-based therapies or xenotransplantation of organs.^2–4 The field of regenerative medicine offers another approach, in which elements of both would be combined for the bioengineering of neo-organs for transplantation.^5,6The concept of whole liver tissue engineering aims to combine patient-specific autologous hepatocytes or hepatic progenitor cells and a carrying platform, or scaffold, to allow for three-dimensional tissue growth and permit the complex cellularity of hepatic tissue. Use of decellularized organ matrices preserves the natural extracellular matrix (ECM) proteins and growth factors that guide cell attachment and proliferation in an organ-specific manner.⁷ Proper processing of the matrix scaffolds removes all cytotoxic chemicals from the decellularization process and performs complete degradation of donor nucleic acids to prevent an adverse host immune response.⁸ These bioengineered livers have the ultimate potential to surpass the current allograft gold standard.The process begins by removing the native cellular components from a donor tissue using detergents and enzymes and leaving behind an ECM scaffold with preserved vasculature and essential biological factors. The concept has been applied to many tissues, including the heart,^9,10 lungs,^11–14 bladder,¹⁵ blood vessels,^16,17 muscle,¹⁸ intestines,^19,20 trachea,^21–23 kidney,^7,24,25 and liver.^26–30 Each detergent, enzyme, washing buffer, and sterilization technique used to decellularize a tissue can have a direct influence on the host remodeling response and functional outcome.³¹ In a previous study, decellularized matrix scaffolds were immunologically favorable up until cells were added to the scaffolding material, where proinflammatory macrophages were activated.³² To evaluate whether a decellularized tissue represents a viable scaffold option, the generated matrices need to be implanted and evaluated over time, without cells, to allow a host’s immune cells to infiltrate and respond to the material.³³ The initial response can begin as early as 2 days and last for months.³⁴ During that time, the environment from both the host itself and the degrading matrix material can influence the phenotype of the host immune cells switching between activation states that will determine the future clinical viability of the biological matrix material.^35–37 Triggering of proinflammatory macrophage activation results in the release of cytokines, growth factors, proteolytic enzymes, and reactive oxygen and nitrogen intermediates that will greatly inhibit the integration of the biomaterial with the host tissue.³⁸ Studies on the immunogenicity of decellularized whole tissue are limited, and a key criterion of transplantation viability will be evaluating the activation of host macrophages toward the classically proinflammatory phenotype (M1) or the regenerative and repair phenotype (M2).The objectives of the current study were to generate a decellularized porcine liver by scaling up our previously established rodent perfusion protocol,²⁸ characterize the resultant scaffold, and compare the in vivo immunological response of a rodent host between allograft and xenograft decellularized liver matrices. We hypothesized that both tissues would elicit a similar host response as the result of the high levels of preservation the ECM protein structures share between species.³⁹ The generation of large-scale hepatic tissue platforms, and an understanding of the inherent immune response by a host species, will be vital in producing implantable bioengineered livers.     

Experimental Neonatal Status Epilepticus and the Development of Temporal Lobe Epilepsy with Unilateral Hippocampal Sclerosis

Hippocampal sclerosis is a common pathological finding in patients with temporal lobe epilepsy, including children, but a causal relationship to early-life seizures remains in question. Neonatal status epilepticus in animals can result in neuronal death within the hippocampus, although macroscopic features of hippocampal shrinkage are not evident at adulthood. Here, we examined electrophysiological and pathological consequences of focally evoked status epilepticus triggered by intra-amygdala microinjection of kainic acid in postnatal day 10 rat pups. Neonatal status epilepticus resulted in extensive neuronal death in the ipsilateral hippocampal CA1 and CA3 subfields and hilus, as assessed by DNA fragmentation and Fluoro-Jade B staining 72 hours later. The contralateral hippocampus was not significantly damaged. Histopathology at P55/P65 revealed unilateral hippocampal sclerosis (grade IV, modified Wyler/Watson scale) comprising >50% CA1 and CA3 neuron loss and astrogliosis. Additional features included hydrocephalus ex vacuo, modest dentate granule cell layer widening, and altered neuropeptide Y immunoreactivity indicative of synaptic rearrangement. Hippocampal atrophy was also evident on magnetic resonance imaging. Depth electrode recordings at adulthood detected spontaneous seizures that involved the ipsilateral hippocampus and amygdala. A significant positive correlation was found between hippocampal pathology grade and both frequency and duration of epileptic seizures at adulthood. The current study demonstrates that experimental neonatal status epilepticus can result in classical unilateral hippocampal sclerosis and temporal lobe epilepsy.Hippocampal sclerosis is the most common pathological finding in patients with temporal lobe epilepsy (TLE).¹ Characteristics include gross atrophy, selective and asymmetric neuron loss within CA1, CA3 and dentate hilus, and gliosis.^1–3 Additional features can include mossy fiber sprouting and granule cell layer dispersion.^1,4Hippocampal sclerosis is implicated as the cause of seizures and also associated cognitive deficits and seizure intractability, in patients with TLE.^3,5–8 Hippocampal sclerosis is less common in children than adults with TLE, but may nevertheless be present in up to half or more patients.^9–11 Pathology and neuroimaging studies have suggested seizures in children are the precipitant of hippocampal sclerosis, and hippocampal sclerosis the cause of TLE.^10,12–17 Nevertheless, a causal link between early-life seizures, development of hippocampal sclerosis, and TLE in humans remains uncertain.Animal models developed to address this question have demonstrated that hippocampal damage can result from early-life seizures.¹⁸ The immature rat brain is quite resistant to permanent seizure damage under 2 weeks of age, representing the time from newborn to 2 years of age in children.^19,20 Possible mechanisms accounting for this have been reviewed elsewhere,²¹ and developmental age, route, and means of seizure induction all have strong influence on outcome. Prolonged seizures induced by hyperthermia in postnatal (P) day 10 rats cause only transient hippocampal injury and no permanent cell loss.²² Similarly, systemic kainic acid (KA) elicits seizures but transient, limited or no permanent hippocampal injury in rats younger than P18.^23–25 In contrast, electrical stimulation of the perforant path in rat pups causes death of hilar and occasional pyramidal cells,²⁶ and pilocarpine-induced status epilepticus in P12–14 rats causes moderate CA1 damage, minor or occasional CA3 cell death, but not hilar cell loss.^27,28 Intrahippocampal or intraventricular KA injection in rat pups mainly damages the CA3 subfield.^29–31 Nevertheless, despite showing neonatal rats are vulnerable to seizure damage, these models provide little evidence that gross hippocampal atrophy later develops in adolescence or adulthood.The risk of epilepsy development after early-life status epilepticus in humans is estimated at 13 to 74%.³² Rates may be lower following febrile seizures.³³ Immature rats subjected to status epilepticus develop TLE, with model-dependent prevalence rates of 0 to 42%.^{24,27,34–36} However, because neuronal death is not a consistent feature of the precipitating injury, a causal relationship between hippocampal pathology and epileptogenesis in these models is not supported.The objective of our study was to investigate the outcome of status epilepticus induced by intra-amygdala KA in P10 rats. Focal generation of seizures in a brain region distant but nevertheless projecting to the hippocampus make this potentially relevant for addressing questions on the pathogenesis of hippocampal sclerosis. Our results indicate that neonatal status epilepticus in this model causes both unilateral hippocampal sclerosis and TLE.     

Alterations in Glucose Homeostasis in a Murine Model of Chagas Disease

Chagas disease, caused by Trypanosoma cruzi, is an important cause of morbidity and mortality primarily resulting from cardiac dysfunction, although T. cruzi infection results in inflammation and cell destruction in many organs. We found that T. cruzi (Brazil strain) infection of mice results in pancreatic inflammation and parasitism within pancreatic β-cells with apparent sparing of α cells and leads to the disruption of pancreatic islet architecture, β-cell dysfunction, and surprisingly, hypoglycemia. Blood glucose and insulin levels were reduced in infected mice during acute infection and insulin levels remained low into the chronic phase. In response to the hypoglycemia, glucagon levels 30 days postinfection were elevated, indicating normal α-cell function. Administration of L-arginine and a β-adrenergic receptor agonist (CL316, 243, respectively) resulted in a diminished insulin response during the acute and chronic phases. Insulin granules were docked, but the lack of insulin secretion suggested an inability of granules to fuse at the plasma membrane of pancreatic β-cells. In the liver, there was a concomitant reduced expression of glucose-6-phosphatase mRNA and glucose production from pyruvate (pyruvate tolerance test), demonstrating defective hepatic gluconeogenesis as a cause for the T. cruzi-induced hypoglycemia, despite reduced insulin, but elevated glucagon levels. The data establishes a complex, multi-tissue relationship between T. cruzi infection, Chagas disease, and host glucose homeostasis.Chagas disease or American trypanosomiasis, a neglected tropical disease, is the result of a persistent infection with Trypanosoma cruzi.^1,2 Not only does it remain an important cause of morbidity and mortality in Latin America, but it is now also a global disease due to immigration to non-endemic areas.³ Furthermore, T. cruzi causes opportunistic infection in the setting of immunosuppression (eg, HIV/AIDS).² Infection of humans and experimental animals results in an intense inflammatory reaction accompanied by an upregulation of inflammatory mediators.^4,5 In the heart, this results in acute myocarditis and some patients eventually develop a chronic dilated cardiomyopathy accompanied by arrhythmias, congestive heart failure, and stroke.² T. cruzi infection also results in mega syndromes predominantly observed in the gastrointestinal tract.²In the 1980s, we became interested in the interface of host glucose homeostasis and parasitic diseases and observed that T. cruzi infection of mice pre-treated with streptozotocin, which destroys the insulin producing pancreatic β-cells, displayed increased parasitemia.⁶ We also demonstrated that obese diabetic mice, null for the leptin receptor (db/db)⁷ infected with T. cruzi displayed a high parasitemia and increased mortality. It has been reported that acute T. cruzi infection of mice resulted in hypoglycemia, which in some cases was predictive of increased mortality,^8,9 however, the etiology of T.cruzi-associated hypoglycemia remains unclear.There are also reports suggesting that diabetes may be more prevalent in individuals with Chagas disease, but these are based on small clinical case series^10–14 and often there are other confounding factors such as obesity, hyperlipidemia, and poverty.^15–17 The possibility that T. cruzi infection could contribute to the diabetic state is not surprising because pathological examination of the pancreas obtained from infected mice and humans revealed morphological and physiological alterations.^18–21 In addition, because of the interrelationship between the adipocyte and glucose metabolism, we examined the contributions of adipose tissue and the adipocyte in the pathogenesis of T. cruzi infection.^8,22,23 In this regard, we established that adipose tissue and adipocytes are important early targets, as well as a reservoir site for the parasite.^8,23,24 Infection of mice with T. cruzi resulted in inflammation of adipose tissue and an upregulation of inflammatory mediators,^8,22,23 suggesting that the adipocyte-pancreatic axis (ie, the adipo-insular axis) could also be impaired by T. cruzi infection.The role of the pancreas in T. cruzi infection has received limited attention in Chagas disease pathogenesis.^18,19,21 Herein, we report that T. cruzi-infection induces inflammation and parasitism of the pancreas, including the pancreatic β-cell. Furthermore, physiological studies strongly suggest that there is an impairment of both pancreatic function and hepatic gluconeogenesis. Alterations in glucose homeostasis in the setting of T. cruzi infection appear to be multifactorial and complex.     

PrPC,the Cellular Isoform of the Human Prion Protein,Is a Novel Biomarker of HIV-Associated Neurocognitive Impairment and Mediates Neuroinflammation

Of the 33 million people infected with the human immunodeficiency virus (HIV) worldwide, 40–60% of individuals will eventually develop neurocognitive sequelae that can be attributed to the presence of HIV-1 in the central nervous system (CNS) and its associated neuroinflammation despite antiretroviral therapy. PrP^C (protease resistant protein, cellular isoform) is the nonpathological cellular isoform of the human prion protein that participates in many physiological processes that are disrupted during HIV-1 infection. However, its role in HIV-1 CNS disease is unknown. We demonstrate that PrP^C is significantly increased in both the CNS of HIV-1–infected individuals with neurocognitive impairment and in SIV-infected macaques with encephalitis. PrP^C is released into the cerebrospinal fluid, and its levels correlate with CNS compromise, suggesting it is a biomarker of HIV-associated neurocognitive impairment. We show that the chemokine (c-c Motif) Ligand-2 (CCL2) increases PrP^C release from CNS cells, while HIV-1 infection alters PrP^C release from peripheral blood mononuclear cells. Soluble PrP^C mediates neuroinflammation by inducing astrocyte production of both CCL2 and interleukin 6. This report presents the first evidence that PrP^C dysregulation occurs in cognitively impaired HIV-1–infected individuals and that PrP^C participates in the pathogenesis of HIV-1–associated CNS disease.Approximately 33 million people are infected with the human immunodeficiency virus (HIV) worldwide.¹ Despite antiretroviral therapy, 40–60% of infected individuals develop neurocognitive sequelae that can be attributed to the presence of HIV-1 in the central nervous system (CNS) and its associated neuroinflammation.² HIV-associated neurocognitive disorder (HAND) represents a spectrum of disease ranging from subclinical to severe cognitive impairment.³ HAND is a significant morbidity among HIV-1–infected individuals^4,5 and is increasingly presenting as an AIDS (acquired immunodeficiency syndrome)-defining illness.^6,7PrP^C (protease resistant protein, cellular isoform) is the nonpathological cellular isoform of the human prion protein. It is most abundantly expressed in the CNS, where it is found constitutively on CNS cells,^8,9 the BBB,^10,11 and on infiltrating leukocytes.^11,12 PrP^C localizes to the cell membrane, where it is anchored by glycosylphosphatidylinositol¹³ and functions as both an adhesion molecule^14–18 and transducer of intracellular signaling.^17,19–21 Importantly, PrP^C plays a role in many of the physiological processes disrupted during HIV-1 infection. In the CNS these include monocyte transmigration across the BBB,¹⁶ macrophage phagocytosis,²² leukocyte^21,23 and microglia activation,²⁴ cellular taxis,²² glutamate metabolism,²⁵ neuronal synaptic plasticity,^15,17 and NMDA (N-methyl-D-aspartic acid) receptor-associated calcium signaling.²⁶ We therefore hypothesized that HIV-1 infection, and/or its associated neuroinflammation, dysregulate PrP^C, thereby contributing to the cognitive impairment observed in HIV-1–infected individuals.We compared CNS PrP^C expression in HIV-1–infected individuals with and without cognitive impairment to uninfected individuals and evaluated its soluble form as a potential biomarker of CNS dysfunction. We used the pigtail macaque model of neuroAIDS to investigate alterations in PrP^C during defined stages of an accelerated disease process. We found PrP^C is increased significantly in the brain of HIV-1–infected individuals with neurocognitive impairment, relative to infected and uninfected individuals who are unimpaired, and in SIV-infected macaques with encephalitis, as compared to infected and uninfected animals without encephalitis. We found that elevated soluble PrP^C (sPrP^C) cerebrospinal fluid (CSF) levels predict neurocognitive impairment in HIV-1–infected individuals, suggesting that CSF sPrP^C is a biomarker of HAND. We also showed that sPrP^C in the CSF reflects the extent of encephalitis in SIV-infected macaques. We demonstrated that CCL2, a chemokine that is elevated in the CNS of individuals with HAND and is associated with neuropathology,^27,28 significantly increases PrP^C release from CNS cells and that HIV-1 infection alters the release of PrP^C from peripheral blood mononuclear cells in vitro. We also showed that sPrP^C contributes to neuroinflammation by increasing the production of CCL2 and interleukin (IL)-6 by astrocytes. This is the first demonstration of PrP^C dysregulation in neurocognitively impaired individuals infected with HIV-1.     

Gastrointestinal Pathology in Juvenile and Adult CFTR-Knockout Ferrets

Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included steatosis, necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (<2 μg elastase-1 per gram of feces). These findings suggest that genetic factors likely influence the extent of exocrine pancreas disease in CF ferrets and have implications for the etiology of pancreatic sufficiency in CF patients. In summary, these studies demonstrate that the CF ferret model develops gastrointestinal pathology similar to CF patients.Cystic fibrosis (CF) is the most common life-threatening, autosomal recessive, genetic disorder among Caucasians, occurring in approximately 1 in 3500 births. Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that disrupt function of this chloride channel cause abnormalities in electrolyte and fluid movement across many epithelia of the body, leading to viscous, poorly hydrated, secretions.¹ Although chronic bacterial infections in the lung are the most significant cause of mortality in CF, pathology in multiple other organs contributes to the progression of disease and overall health of CF patients. These organs include the intestine, pancreas, liver, and gallbladder for which clinical and/or histological disease is seen in CF patients at frequencies of 10% to 90%.^1–4 In the current study, we evaluated gastrointestinal disease in juvenile and adult CFTR-knockout ferrets.Mouse models of CF have been critical to our understanding of CFTR function in several organs, however, CF mice fail to develop spontaneous disease in the lung and pancreas, and have relatively minor disease of the liver and gallbladder.^5,6 The recent creation of new CF pig and ferret models has provided the field with new tools to dissect CF disease pathophysiology and the factors that influence disease severity in CF patients.^7,8 Comparisons between CF mouse, pig, and ferret models have clearly demonstrated that species-specific differences in organ physiology and CFTR biology influence the extent of pathology in major organs affected in CF.^9,10 For example, all newborn CF models have intestinal pathology that manifests as meconium ileus at birth in the case of CF pigs (100% of animals)¹¹ and CF ferrets (75% of animals),¹² or as intestinal obstruction at weaning in the case of CF mice.⁵ By contrast, pancreatic phenotypes at birth are highly variable between species with disease being most severe in CF pigs,¹¹ less severe in CF ferrets,^12,13 and absent in CF mice.⁵ Similarly, CF pigs demonstrate histopathology in the gallbladder and liver at birth,¹¹ whereas disease in these organs is relative minor in newborn CF ferrets^12,13 and CF adult mice.^5,6 Elucidating the differences in the severity of CF gastrointestinal disease at birth and in disease progression between the various species may aid in dissecting genetic and environmental factors that influence gastrointestinal disease severity in CF patients.Here, we report the phenotype of gastrointestinal organs (pancreas, liver, gallbladder, stomach, and intestine) in older CF animals reared on antibiotics until 6 months of age, or the time at which they were euthanized due to severity of disease. We have also evaluated the extent of bacterial overgrowth in the CF intestine and the types of bacterial flora found in the intestine of non-CF and CF animals. Our findings demonstrate that juvenile and adult CF ferrets naturally acquire gastrointestinal disease at frequencies similar to that observed in CF patients. Of great interest, a small subset of CF animals was pancreatic sufficient from birth, implicating modifier genes that can compensate for the loss of CFTR in the exocrine pancreas. These studies suggest that the CF ferret model could be useful for testing therapies aimed at gastrointestinal organs and dissecting how genetic variation influences disease in CF.