RESUMO
BACKGROUND & AIMS: Although transient bacteremia is common during dental and endoscopic procedures, infections developing during sterile diseases like acute pancreatitis (AP) can have grave consequences. We examined how impaired bacterial clearance may cause this transition. METHODS: Blood samples from patients with AP, normal controls, and rodents with pancreatitis or those administered different nonesterified fatty acids (NEFAs) were analyzed for albumin-unbound NEFAs, microbiome, and inflammatory cell injury. Macrophage uptake of unbound NEFAs using a novel coumarin tracer were done and the downstream effects-NEFA-membrane phospholipid (phosphatidylcholine) interactions-were studied on isothermal titration calorimetry. RESULTS: Patients with infected AP had higher circulating unsaturated NEFAs; unbound NEFAs, including linoleic acid (LA) and oleic acid (OA); higher bacterial 16S DNA; mitochondrial DNA; altered ß-diversity; enrichment in Pseudomonadales; and increased annexin V-positive myeloid (CD14) and CD3-positive T cells on admission. These, and increased circulating dead inflammatory cells, were also noted in rodents with unbound, unsaturated NEFAs. Isothermal titration calorimetry showed progressively stronger unbound LA interactions with aqueous media, phosphatidylcholine, cardiolipin, and albumin. Unbound NEFAs were taken into protein-free membranes, cells, and mitochondria, inducing voltage-dependent anion channel oligomerization, reducing ATP, and impairing phagocytosis. These were reversed by albumin. In vivo, unbound LA and OA increased bacterial loads and impaired phagocytosis, causing infection. LA and OA were more potent for these amphipathic interactions than the hydrophobic palmitic acid. CONCLUSIONS: Release of stored LA and OA can increase their circulating unbound levels and cause amphipathic liponecrosis of immune cells via uptake by membrane phospholipids. This impairs bacterial clearance and causes infection during sterile inflammation.
Assuntos
Pancreatite , Humanos , Doença Aguda , Ácidos Graxos não Esterificados , Ácido Oleico , Inflamação , Albuminas , FosfatidilcolinasRESUMO
BACKGROUND & AIMS: Acute pancreatitis (AP) of different etiologies is associated with the activation of different signaling pathways in pancreatic cells, posing challenges to the development of targeted therapies. We investigated whether local pancreatic hypothermia, without systemic hypothermia, could lessen the severity of AP induced by different methods in rats. METHODS: A urethane balloon with 2 polyurethane tubes was placed inside the stomach of rats. AP was induced in Wistar rats by the administration of cerulein or glyceryl tri-linoleate (GTL). Then, cold water was infused into the balloon to cool the pancreas. Pancreatic temperatures were selected based on those found to decrease acinar cell injury. An un-perfused balloon was used as a control. Pancreatic and rectal temperatures were monitored, and an infrared lamp or heating pad was used to avoid generalized hypothermia. We collected blood, pancreas, kidney, and lung tissues and analyzed them by histology, immunofluorescence, immunoblot, cytokine and chemokine magnetic bead, and DNA damage assays. The effect of hypothermia on signaling pathways initiated by cerulein and GTL was studied in acinar cells. RESULTS: Rats with pancreatic cooling developed less severe GTL-induced AP compared with rats that received the control balloon. In acinar cells, cooling decreased the lipolysis induced by GTL, increased the micellar form of its fatty acid, lowered the increase in cytosolic calcium, prevented the loss of mitochondrial membrane potential (by 70%-80%), and resulted in a 40%-50% decrease in the uptake of a fatty acid tracer. In rats with AP, cooling decreased pancreatic necrosis by 48%, decreased serum levels of cytokines and markers of cell damage, and decreased markers of lung and renal damage. Pancreatic cooling increased the proportions of rats surviving 6 hours after induction of AP (to 90%, from <10% of rats that received the control balloon). In rats with cerulein-induced AP, pancreatic cooling decreased pancreatic markers of apoptosis and inflammation. CONCLUSIONS: In rats with AP, transgastric local pancreatic hypothermia decreases pancreatic necrosis, apoptosis, inflammation, and markers of pancreatitis severity and increases survival.
Assuntos
Hipotermia Induzida/métodos , Pancreatite Necrosante Aguda/patologia , Pancreatite Necrosante Aguda/terapia , Animais , Biópsia por Agulha , Ceruletídeo/efeitos adversos , Ceruletídeo/farmacologia , Crioterapia/métodos , Modelos Animais de Doenças , Imuno-Histoquímica , Masculino , Pancreatite Necrosante Aguda/mortalidade , Distribuição Aleatória , Ratos , Ratos Wistar , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Estômago , Taxa de Sobrevida , Fatores de TempoRESUMO
BACKGROUND AND AIMS: Peripancreatic fat necrosis occurs frequently in necrotising pancreatitis. Distinguishing markers from mediators of severe acute pancreatitis (SAP) is important since targeting mediators may improve outcomes. We evaluated potential agents in human pancreatic necrotic collections (NCs), pseudocysts (PCs) and pancreatic cystic neoplasms and used pancreatic acini, peripheral blood mononuclear cells (PBMC) and an acute pancreatitis (AP) model to determine SAP mediators. METHODS: We measured acinar and PBMC injury induced by agents increased in NCs and PCs. Outcomes of caerulein pancreatitis were studied in lean rats coadministered interleukin (IL)-1ß and keratinocyte chemoattractant/growth-regulated oncogene, triolein alone or with the lipase inhibitor orlistat. RESULTS: NCs had higher fatty acids, IL-8 and IL-1ß versus other fluids. Lipolysis of unsaturated triglyceride and resulting unsaturated fatty acids (UFA) oleic and linoleic acids induced necro-apoptosis at less than half the concentration in NCs but other agents did not do so at more than two times these concentrations. Cytokine coadministration resulted in higher pancreatic and lung inflammation than caerulein alone, but only triolein coadministration caused peripancreatic fat stranding, higher cytokines, UFAs, multisystem organ failure (MSOF) and mortality in 97% animals, which were prevented by orlistat. CONCLUSIONS: UFAs, IL-1ß and IL-8 are elevated in NCs. However, UFAs generated via peripancreatic fat lipolysis causes worse inflammation and MSOF, converting mild AP to SAP.
Assuntos
Necrose Gordurosa/metabolismo , Ácidos Graxos Insaturados/metabolismo , Pancreatite Necrosante Aguda/patologia , Células Acinares/metabolismo , Células Acinares/patologia , Adulto , Idoso , Animais , Biomarcadores/metabolismo , Citocinas/administração & dosagem , Citocinas/metabolismo , Citocinas/farmacologia , Necrose Gordurosa/etiologia , Feminino , Humanos , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Lipólise , Masculino , Pessoa de Meia-Idade , Insuficiência de Múltiplos Órgãos/etiologia , Pâncreas/efeitos dos fármacos , Pseudocisto Pancreático/metabolismo , Pancreatite Necrosante Aguda/metabolismo , Ratos , Ratos Wistar , Índice de Gravidade de DoençaRESUMO
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.
Assuntos
Adipócitos/metabolismo , Gordura Intra-Abdominal/metabolismo , Lipase/metabolismo , Lipólise/fisiologia , Pancreatite/metabolismo , Triglicerídeos/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/patologia , Animais , Ceruletídeo , Inibidores Enzimáticos/farmacologia , Inflamação/metabolismo , Inflamação/patologia , Gordura Intra-Abdominal/efeitos dos fármacos , Gordura Intra-Abdominal/patologia , Lactonas/farmacologia , Lipase/antagonistas & inibidores , Lipólise/efeitos dos fármacos , Camundongos , Camundongos Obesos , Necrose/metabolismo , Necrose/patologia , Orlistate , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Pâncreas/patologia , Pancreatite/induzido quimicamente , Pancreatite/patologiaRESUMO
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.
Assuntos
Ácidos Graxos Insaturados/metabolismo , Lipólise , Pancreatite Necrosante Aguda/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Lipase/antagonistas & inibidores , Lipase/metabolismo , Masculino , Pancreatite Necrosante Aguda/patologia , Ratos , Ratos Wistar , Índice de Gravidade de DoençaRESUMO
BACKGROUND & AIMS: Acute pancreatitis (AP) and chronic pancreatitis (CP) share etiologies, but AP can be more severe and is associated with a higher rate of mortality. We investigated features of CP that protect against severe disease. The amount of intrapancreatic fat (IPF) is increased in obese patients and fibrosis is increased in patients with CP, so we studied whether fibrosis or fat regulate severity of AP attacks in patients with CP. METHODS: We reviewed records from the University of Pittsburgh Medical Center/Presbyterian Hospital Autopsy Database (1998-2008) for patients with a diagnosis of AP (n = 23), CP (n = 35), or both (AP-on-CP; n = 15). Pancreatic histology samples from these patients and 50 randomly selected controls (no pancreatic disease) were analyzed, and IPF data were correlated with computed tomography data. An adipocyte and acinar cell Transwell coculture system, with or without collagen type I, was used to study the effects of fibrosis on acinar-adipocyte interactions. We studied the effects of nonesterified fatty acids (NEFAs) and adipokines on acinar cells in culture. RESULTS: Levels of IPF were significantly higher in nonobese patients with CP than in nonobese controls. In patients with CP or AP-on-CP, areas of IPF were surrounded by significantly more fibrosis than in controls or patients with AP. Fat necrosis-associated peri-fat acinar necrosis (PFAN, indicated by NEFA spillage) contributed to most of the necrosis observed in samples from patients with AP; however, findings of peri-fat acinar necrosis and total necrosis were significantly lower in samples from patients with CP or AP-on-CP. Fibrosis appeared to wall off the fat necrosis and limit peri-fat acinar necrosis, reducing acinar necrosis. In vitro, collagen I limited the lipolytic flux between acinar cells and adipocytes and prevented increases in adipokines in the acinar compartment. This was associated with reduced acinar cell necrosis. However, NEFAs, but not adipokines, caused acinar cell necrosis. CONCLUSIONS: Based on analysis of pancreatic samples from patients with CP, AP, or AP-on-CP and in vitro studies, fibrosis reduces the severity of acute exacerbations of CP by reducing lipolytic flux between adipocytes and acinar cells.
Assuntos
Tecido Adiposo/patologia , Obesidade/patologia , Pâncreas/patologia , Pancreatite Necrosante Aguda/patologia , Pancreatite Crônica/patologia , Células Acinares/efeitos dos fármacos , Doença Aguda , Adipócitos/efeitos dos fármacos , Adipocinas/farmacologia , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Ácidos Graxos não Esterificados/farmacologia , Fibrose , Humanos , Pessoa de Meia-Idade , Necrose , Obesidade/complicações , Pancreatite Necrosante Aguda/complicações , Pancreatite Crônica/complicações , Estudos Retrospectivos , Índice de Gravidade de DoençaRESUMO
The role of obesity in relation to various disease processes is being increasingly studied, with reports over the last several years increasingly mentioning its association with worse outcomes in acute disease. Obesity has also gained recognition as a risk factor for severe acute pancreatitis (SAP).The mortality in SAP may be as high as 30% and is usually attributable to multi system organ failure (MSOF) earlier in the disease, and complications of necrotizing pancreatitis later [9-11]. To date there is no specific treatment for acute pancreatitis (AP) and the management is largely expectant and supportive. Obesity in general has also been associated with poor outcomes in sepsis and other pathological states including trauma and burns. With the role of unsaturated fatty acids (UFA) as propagators in SAP having recently come to light and with the recognition of acute lipotoxicity, there is now an opportunity to explore different strategies to reduce the mortality and morbidity in SAP and potentially other disease states associated with such a pathophysiology. In this review we will discuss the role of fat and implications of the consequent acute lipotoxicity on the outcomes of acute pancreatitis in lean and obese states and during acute on chronic pancreatitis.
Assuntos
Gordura Intra-Abdominal , Obesidade/complicações , Pâncreas/patologia , Pancreatite/etiologia , Doença Aguda , Humanos , Insuficiência de Múltiplos Órgãos/etiologia , Pancreatite/patologia , Pancreatite Necrosante Aguda/etiologia , Pancreatite Crônica/etiologia , Índice de Gravidade de DoençaRESUMO
With improvements in abdominal imaging, detection of incidental pancreatic cysts are becoming increasingly common. Analysis of pancreatic cyst fluid from fine-needle aspiration is particularly important in identifying intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs), which have significant implications in clinical intervention and follow-up. Previous controlled studies have shown that KRAS mutations in cyst fluid are highly specific for mucinous differentiation in pancreatic cysts; however, this has not been examined in the clinical setting. Over a 6-year study period, 618 pancreatic cyst fluids obtained by fine-needle aspiration at the time of endoscopic ultrasound were tested for KRAS mutations as part of routine evaluation for a cystic neoplasm. Of the 618 specimens, 603 (98%) from 546 patients were satisfactory for molecular analysis. Patients ranged in age from 17 to 90 years (mean, 63.9 years) and were predominantly female (68%). Pancreatic cysts were relatively evenly distributed throughout the pancreas and ranged in size from 0.6 to 11.0 cm (mean, 2.3 cm). Mutations in KRAS were detected in 232 of 603 (38%) aspirates. Although sufficient for molecular analysis, 320 of 603 (53%) specimens were either less than optimal (38%) or unsatisfactory (15%) for cytopathologic diagnosis. Surgical follow-up information was available for 142 (26%) patients and consisted of 53 KRAS-mutated and 89 KRAS-wild-type cysts. Overall, KRAS mutations had a specificity of 100%, but a sensitivity of 54% for mucinous differentiation. When stratified by cyst type, KRAS had a sensitivity of 67% and 14% for IPMNs and MCNs, respectively. In summary, KRAS mutations were highly specific for mucinous differentiation, but were inadequate in identifying MCNs. Future molecular studies and the combination of other fluid markers are required to improve the detection and classification of pancreatic mucinous neoplasms by endoscopic ultrasound fine-needle aspiration.
Assuntos
Carcinoma Ductal Pancreático/genética , Análise Mutacional de DNA , Testes Genéticos/métodos , Mutação , Neoplasias Císticas, Mucinosas e Serosas/genética , Cisto Pancreático/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Ductal Pancreático/patologia , Diferenciação Celular , Aspiração por Agulha Fina Guiada por Ultrassom Endoscópico , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Císticas, Mucinosas e Serosas/patologia , Cisto Pancreático/patologia , Neoplasias Pancreáticas/patologia , Fenótipo , Valor Preditivo dos Testes , Prognóstico , Proteínas Proto-Oncogênicas p21(ras) , Adulto JovemRESUMO
We compared three hospitalized patient cohorts and conducted mechanistic studies to determine if lipotoxicity worsens COVID-19. Cohort-1 (n = 30) compared COVID-19 patients dismissed home to those requiring intensive-care unit (ICU) transfer. Cohort-2 (n = 116) compared critically ill ICU patients with and without COVID-19. Cohort-3 (n = 3969) studied hypoalbuminemia and hypocalcemia's impact on COVID-19 mortality. Patients requiring ICU transfer had higher serum albumin unbound linoleic acid (LA). Unbound fatty acids and LA were elevated in ICU transfers, COVID-19 ICU patients and ICU non-survivors. COVID-19 ICU patients (cohort-2) had greater serum lipase, damage-associated molecular patterns (DAMPs), cytokines, hypocalcemia, hypoalbuminemia, organ failure and thrombotic events. Hypocalcemia and hypoalbuminemia independently associated with COVID-19 mortality in cohort-3. Experimentally, LA reacted with albumin, calcium and induced hypocalcemia, hypoalbuminemia in mice. Endothelial cells took up unbound LA, which depolarized their mitochondria. In mice, unbound LA increased DAMPs, cytokines, causing endothelial injury, organ failure and thrombosis. Therefore, excessive unbound LA in the circulation may worsen COVID-19 outcomes.
RESUMO
Pancreatic lymphoepithelial cysts are rare benign cysts that cannot be reliably differentiated from neoplastic mucinous cysts preoperatively. Although elevated cyst fluid carcinoembryonic antigen (CEA) levels support a diagnosis of a mucinous cyst, the finding of increased CEA levels in lymphoepithelial cysts prompted this study. Nine resected lymphoepithelial cysts were examined for expression of CEA, carbohydrate antigen (CA) 19-9, CK7, p63, PAS-D and a panel of mucins. The pathology data were correlated with clinical information, including serum, cyst fluid and imaging studies. By computed tomography scan, although most lymphoepithelial cysts appeared cystic, 23% were described as masses. The endoscopic ultrasound findings were variable, but the lymphoepithelial cysts tended to be hypoechoic cystic lesions or masses. On cytology, 44% of the cysts had squamous cells, 67% had glandular cells and 56% had atypical cells. The cysts were resected because of size ≥3 cm (89%), symptoms (44%) and/or elevated cyst fluid CEA levels (33%). The cyst fluid CEA levels in the three cysts tested were >450 ng/ml. Histopathologically, all cysts were lined by mature, stratified squamous-type cells and produced keratin. Mucous cells were present in 78% of the cysts. The immunohistochemical profile of the squamous lining was CK7+, p63+, MUC1+, MUC4+, MUC2-, MUC5AC- and MUC6-. Even though lymphoepithelial cysts are lined by squamous-type epithelium, all our resected lymphoepithelial cysts expressed CEA and/or CA19-9, many contained mucous cells, and three exhibited markedly elevated cyst fluid CEA levels. Although cyst fluid CEA levels >200 ng/ml support the diagnosis of mucinous neoplasms, this study emphasizes the need for clinicians and pathologists to recognize that lymphoepithelial cysts can mimic neoplastic mucinous cysts clinically, radiographically and on cyst fluid CEA analysis.
Assuntos
Antígeno Carcinoembrionário/análise , Líquido Cístico/química , Células Epiteliais/química , Tecido Linfoide/química , Mucinas/análise , Neoplasias Císticas, Mucinosas e Serosas/diagnóstico , Cisto Pancreático/diagnóstico , Neoplasias Pancreáticas/diagnóstico , Adulto , Idoso , Biópsia por Agulha Fina , Antígeno CA-19-9/análise , Diagnóstico Diferencial , Endossonografia , Células Epiteliais/patologia , Feminino , Humanos , Imuno-Histoquímica , Queratina-7/análise , Tecido Linfoide/patologia , Masculino , Proteínas de Membrana/análise , Pessoa de Meia-Idade , Neoplasias Císticas, Mucinosas e Serosas/química , Neoplasias Císticas, Mucinosas e Serosas/patologia , Neoplasias Císticas, Mucinosas e Serosas/cirurgia , Cisto Pancreático/química , Cisto Pancreático/patologia , Cisto Pancreático/cirurgia , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/cirurgia , Pennsylvania , Valor Preditivo dos Testes , Cuidados Pré-Operatórios , Tomografia Computadorizada por Raios XRESUMO
Bile acids (BA), with their large hydrophobic steroid nucleus and polar groups are amphipathic molecules. In bile, these exist as micelles above their critical micellar concentration (CMC). In blood at low concentrations, these exist as monomers, initiating cellular signals. This micellar to monomer transition may involve complex thermodynamic interactions between bile salts alone or with phospholipids, i.e. mixed micelles and the aqueous environment. We therefore went on to test if therapeutically relevant changes in temperature could influence micellar behavior of bile salts, and in turn whether this affected the biological responses in cells, and in vivo. Sodium taurocholate (STC) belongs to a major class of bile salts. STC has a CMC in the 5-8 mM range and its infusion into the pancreatic duct is commonly used to study pancreatitis. We thus studied micellar breakdown of STC using isothermal titration calorimetry (ITC), dynamic light scattering and cryogenic transmission electron microscopy. Under conditions relevant to the in vivo environment (pH 7.4, Na 0.15 M), ITC showed STC to have a U shaped reduction in micellar breakdown between 37 °C and 15 °C with a nadir at 25 °C approaching ≈90% inhibition. This temperature dependence paralleled pancreatic acinar injury induced by monomeric STC. Mixed micelles of STC and 1-palmitoyl, 2-oleyl phosphatidylcholine, a phospholipid present in high proportions in bile, behaved similarly, with ≈75% reduction in micellar breakdown at 25 °C compared to 37 °C. In vivo pancreatic cooling to 25 °C reduced the increase in circulating BAs after infusion of 120 mM (5%) STC into the pancreatic duct, and duct ligation. Lower BA levels were associated with improved cardiac function, reduced myocardial damage, shock, lung injury and improved survival independent of pancreatic injury. Thus micellar breakdown of bile salts is essential for their entry into the systemic circulation, and thermodynamic interference with this may reduce their systemic entry and consequent injury during cholestasis, such as from biliary pancreatitis.
Assuntos
Ácidos e Sais Biliares/metabolismo , Colestase/complicações , Inflamação/prevenção & controle , Lesão Pulmonar/prevenção & controle , Micelas , Contusões Miocárdicas/prevenção & controle , Choque/prevenção & controle , Animais , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Lesão Pulmonar/etiologia , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Masculino , Camundongos , Contusões Miocárdicas/etiologia , Contusões Miocárdicas/metabolismo , Contusões Miocárdicas/patologia , Choque/etiologia , Choque/metabolismo , Choque/patologia , Temperatura , TermodinâmicaRESUMO
Visceral adipose tissue plays a critical role in numerous diseases. Although imaging studies often show adipose involvement in abdominal diseases, their outcomes may vary from being a mild self-limited illness to one with systemic inflammation and organ failure. We therefore compared the pattern of visceral adipose injury during acute pancreatitis and acute diverticulitis to determine its role in organ failure. Acute pancreatitis-associated adipose tissue had ongoing lipolysis in the absence of adipocyte triglyceride lipase (ATGL). Pancreatic lipase injected into mouse visceral adipose tissue hydrolyzed adipose triglyceride and generated excess nonesterified fatty acids (NEFAs), which caused organ failure in the absence of acute pancreatitis. Pancreatic triglyceride lipase (PNLIP) increased in adipose tissue during pancreatitis and entered adipocytes by multiple mechanisms, hydrolyzing adipose triglyceride and generating excess NEFAs. During pancreatitis, obese PNLIP-knockout mice, unlike obese adipocyte-specific ATGL knockouts, had lower visceral adipose tissue lipolysis, milder inflammation, less severe organ failure, and improved survival. PNLIP-knockout mice, unlike ATGL knockouts, were protected from adipocyte-induced pancreatic acinar injury without affecting NEFA signaling or acute pancreatitis induction. Therefore, during pancreatitis, unlike diverticulitis, PNLIP leaking into visceral adipose tissue can cause excessive visceral adipose tissue lipolysis independently of adipocyte-autonomous ATGL, and thereby worsen organ failure.
Assuntos
Adipócitos/enzimologia , Gordura Intra-Abdominal/enzimologia , Lipase/metabolismo , Pancreatite/enzimologia , Transdução de Sinais , Doença Aguda , Adipócitos/patologia , Animais , Ácidos Graxos não Esterificados/genética , Ácidos Graxos não Esterificados/metabolismo , Feminino , Humanos , Inflamação/enzimologia , Inflamação/genética , Inflamação/patologia , Gordura Intra-Abdominal/patologia , Lipase/genética , Masculino , Camundongos , Camundongos Knockout , Pancreatite/genética , Pancreatite/patologiaRESUMO
The BRAF V600E mutation occurs in 15% of colorectal carcinomas (CRCs) and has important genetic, prognostic, and therapeutic implications. A monoclonal antibody (VE1) targeting the BRAF V600E mutant protein has become available with variable efficacy in literature reports. We investigated the utility of the VE1 antibody in detecting BRAF V600E mutant protein in two cohorts: (1) a retrospectively accrued series of 103 resected CRCs with (N = 57) and without (N = 46) known BRAF V600E mutation status by PCR and (2) a prospective series of 25 CRCs requiring BRAF analysis during routine screening for Lynch syndrome. All 74 cases with positive BRAF V600E mutation demonstrated cytoplasmic positivity with the VE1 antibody with most tumors (70/74, 95%) demonstrating moderate to strong staining. Of the 54 BRAF V600E-negative cases, 51/54 CRCs (94%) were negative with the VE1 antibody while 3 CRCs (6%) demonstrated weak cytoplasmic staining. The sensitivity and specificity of VE1 was 100% and 94%, respectively. Ten BRAF V600E-mutated CRCs had adjacent precursor lesions including 7 sessile serrated adenomas associated with CRCs with high-level microsatellite instability (MSI-H). All 10 precursor adenomas were positive for VE1 staining with the 7 sessile serrated adenomas maintaining preserved MLH1 expression. Our results indicate that VE1 immunohistochemistry is a useful surrogate for the detection of the BRAF V600E mutation in CRC, although weak staining must be evaluated by BRAF PCR analysis to exclude a false positive result. In addition, the BRAF V600E mutation appears to be an early event before the divergent development into MSS and MSI-H pathways.
Assuntos
Adenoma/diagnóstico , Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Neoplasias Colorretais/diagnóstico , Imuno-Histoquímica/métodos , Proteínas Mutantes/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Adenoma/metabolismo , Adenoma/patologia , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais Hereditárias sem Polipose/metabolismo , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Biologia MolecularRESUMO
CONTEXT: Novel chemotherapy regimens now provide the opportunity for "personalized" care in pancreatic cancer. Little is known about our ability to procure adequate cells for theranostic studies using standard ultrasound-guided fine-needle aspirations and cytologic techniques. OBJECTIVE: To assess cellularity of cytology material in patients with solid pancreatic lesions. DESIGN: One hundred sixty-nine endoscopic ultrasound-guided fine-needle aspirations with positive diagnoses of solid epithelial pancreatic neoplasms were evaluated for smear and cell block cellularity. Cellularity was scored on a scale of 1 to 4; scores of 3 or 4 (>100 cells) were deemed adequate for ancillary studies. Clinicopathologic variables were recorded. A 3-month prospective analysis was also performed using a new collection algorithm. RESULTS: Only 12.4% (21 of 169) of the positive cases had a cell block cellularity score that was adequate for theranostic studies. This score was not associated with on-site evaluation, needle gauge, or number of passes. Adenocarcinoma was the most common diagnosis (88%) but yielded fewer adequate cell blocks, P = .006. Cellularity showed correlation with endoscopists, P = .04. Tumor size and fibrosis score of resected tumors tended to correlate with cellularity, but only larger size in endocrine tumors was significantly associated with adequacy (P = .02). Standardized collection did not improve overall cell block cellularity. CONCLUSIONS: Changes in practice, such as obtaining dedicated passes for ancillary studies, may not be enough to improve the theranostic utility of endoscopic ultrasound-guided fine-needle aspiration in pancreatic neoplasia. Other methods to improve tumor cell yield, including modified cytologic techniques and new needle designs, need to be further investigated.
Assuntos
Aspiração por Agulha Fina Guiada por Ultrassom Endoscópico/métodos , Neoplasias Pancreáticas/diagnóstico , Adenocarcinoma/diagnóstico , Adenocarcinoma/patologia , Algoritmos , Carcinoma de Células Acinares/diagnóstico , Carcinoma de Células Acinares/patologia , Carcinoma Neuroendócrino/diagnóstico , Carcinoma Neuroendócrino/patologia , Aspiração por Agulha Fina Guiada por Ultrassom Endoscópico/estatística & dados numéricos , Humanos , Tumores Neuroendócrinos/diagnóstico , Tumores Neuroendócrinos/patologia , Neoplasias Pancreáticas/patologia , Estudos ProspectivosRESUMO
Several deleterious intra-acinar phenomena are simultaneously triggered on initiating acute pancreatitis. These culminate in acinar injury or inflammatory mediator generation in vitro and parenchymal damage in vivo. Supraphysiologic caerulein is one such initiator which simultaneously activates numerous signaling pathways including non-receptor tyrosine kinases such as of the Src family. It also causes a sustained increase in cytosolic calcium- a player thought to be crucial in regulating deleterious phenomena. We have shown Src to be involved in caerulein induced actin remodeling, and caerulein induced changes in the Golgi and post-Golgi trafficking to be involved in trypsinogen activation, which initiates acinar cell injury. However, it remains unclear whether an increase in cytosolic calcium is necessary to initiate acinar injury or if injury can be initiated at basal cytosolic calcium levels by an alternate pathway. To study the interplay between tyrosine kinase signaling and calcium, we treated mouse pancreatic acinar cells with the tyrosine phosphatase inhibitor pervanadate. We studied the effect of the clinically used Src inhibitor Dasatinib (BMS-354825) on pervanadate or caerulein induced changes in Src activation, trypsinogen activation, cell injury, upstream cytosolic calcium, actin and Golgi morphology. Pervanadate, like supraphysiologic caerulein, induced Src activation, redistribution of the F-actin from its normal location in the sub-apical area to the basolateral areas, and caused antegrade fragmentation of the Golgi. These changes, like those induced by supraphysiologic caerulein, were associated with trypsinogen activation and acinar injury, all of which were prevented by Dasatinib. Interestingly, however, pervanadate did not cause an increase in cytosolic calcium, and the caerulein induced increase in cytosolic calcium was not affected by Dasatinib. These findings suggest that intra-acinar deleterious phenomena may be initiated independent of an increase in cytosolic calcium. Other players resulting in acinar injury along with the Src family of tyrosine kinases remain to be explored.
Assuntos
Células Acinares/patologia , Cálcio/metabolismo , Citosol/metabolismo , Quinases da Família src/metabolismo , Animais , Dasatinibe/farmacologia , Ativação Enzimática , Imunofluorescência , Camundongos , Camundongos Endogâmicos ICR , Vanadatos/farmacologia , Quinases da Família src/antagonistas & inibidoresRESUMO
We assessed 103 resected gastroesophageal adenocarcinomas for HER2 amplification by fluorescence in situ hybridization (FISH) and 2 commercial immunohistochemical assays. Of 103, 30 (29%) were FISH-amplified. Both immunohistochemical assays had greater than 95% concordance with FISH. However, as a screening test for FISH amplification, the Ventana Medical Systems (Tucson, AZ) 4B5 antibody demonstrated superior sensitivity (87%) compared with the DAKO (Carpinteria, CA) A0485 (70%). Of the cases, 28 were immunohistochemically 3+ or immunohistochemically 2+/FISH-amplified with the 4B5 assay compared with only 22 cases with the A0485 assay, representing a large potential difference in patient eligibility for anti-HER2 therapy. Cases with low-level FISH amplification (HER2/CEP17, 2.2-4.0) express lower levels of HER2 protein compared with cases with high-level amplification (HER2/CEP17, ≥4.0), raising the possibility of a differential response to anti-HER2 therapy. The H score and digital image analysis may have a limited role in improving HER2 test performance.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias Esofágicas/metabolismo , Hibridização in Situ Fluorescente/métodos , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Idoso , Idoso de 80 Anos ou mais , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Receptor ErbB-2/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologiaRESUMO
Obesity increases the risk of adverse outcomes during acute critical illnesses such as burns, severe trauma, and acute pancreatitis. Although individuals with more body fat and higher serum cytokines and lipase are more likely to experience problems, the roles that these characteristics play are not clear. We used severe acute pancreatitis as a representative disease to investigate the effects of obesity on local organ function and systemic processes. In obese humans, we found that an increase in the volume of intrapancreatic adipocytes was associated with more extensive pancreatic necrosis during acute pancreatitis and that acute pancreatitis was associated with multisystem organ failure in obese individuals. In vitro studies of pancreatic acinar cells showed that unsaturated fatty acids were proinflammatory, releasing intracellular calcium, inhibiting mitochondrial complexes I and V, and causing necrosis. Saturated fatty acids had no such effects. Inhibition of lipolysis in obese (ob/ob) mice with induced pancreatitis prevented a rise in serum unsaturated fatty acids and prevented renal injury, lung injury, systemic inflammation, hypocalcemia, reduced pancreatic necrosis, and mortality. Thus, therapeutic approaches that target unsaturated fatty acid-mediated lipotoxicity may reduce adverse outcomes in obese patients with critical illnesses such as severe acute pancreatitis.
Assuntos
Lipólise/fisiologia , Insuficiência de Múltiplos Órgãos/etiologia , Insuficiência de Múltiplos Órgãos/metabolismo , Obesidade/metabolismo , Pâncreas/metabolismo , Pâncreas/patologia , Pancreatite/metabolismo , Células Acinares/metabolismo , Células Acinares/patologia , Animais , Ácidos Graxos/metabolismo , Ácidos Graxos Insaturados/metabolismo , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , Camundongos Obesos , Necrose/etiologia , Necrose/metabolismo , Obesidade/fisiopatologia , Pancreatite/fisiopatologia , Pancreatite Necrosante Aguda/metabolismo , Pancreatite Necrosante Aguda/patologia , Pancreatite Necrosante Aguda/fisiopatologiaRESUMO
There is no clinical treatment that reduces acinar injury during pancreatitis. Human immunodeficiency virus (HIV) protease inhibitors (PI), including nelfinavir (NFV) and ritonavir (RTV), may reduce the rate of pancreatitis in HIV-infected patients. Since permeability transition pore (PTPC)-mediated mitochondrial dysfunction occurs during pancreatitis, and we have shown that PI prevents PTPC opening, we studied its effects in a model of pancreatitis. The effect of NFV plus RTV (NFV/RTV) or vehicle on caerulein-induced pancreatitis in mice was compared by measuring changes in mitochondrial membrane potential in vitro and cytochrome c leakage in vivo. Histological and inflammatory makers were also compared. NFV/RTV improved DiOC6 retention in acini exposed to caerulein in vitro. In vivo NFV prevented cytosolic leakage of cytochrome c and reduced pancreatic acinar injury, active caspase-3 staining, TUNEL-positive acinar cells, and serum amylase (P < 0.05). Conversely, trypsin activity, serum cytokine levels, and pancreatic and lung inflammation were unaffected. NFV/RTV reduces pancreatic injury and acinar cell death in experimental mouse caerulein-induced pancreatitis but does not impact inflammation.
Assuntos
Inibidores da Protease de HIV/farmacologia , Nelfinavir/farmacologia , Pâncreas/efeitos dos fármacos , Pancreatite/tratamento farmacológico , Ritonavir/farmacologia , Amilases/sangue , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Ceruletídeo , Citocromos c/metabolismo , Modelos Animais de Doenças , Quimioterapia Combinada , Mediadores da Inflamação/sangue , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Necrose , Pâncreas/metabolismo , Pâncreas/patologia , Pancreatite/induzido quimicamente , Pancreatite/metabolismo , Pancreatite/patologia , Tripsina/metabolismoRESUMO
BACKGROUND: Protease-activated receptor-2 (PAR-2) is present in the pancreas, where it has been shown to play a protective role during pancreatitis. However, the mechanism by which it protects against pancreatitis still remains to be elucidated. Acute pancreatitis is associated with premature zymogen activation and a blockage in digestive enzyme secretion. AIM: To examine the effects of PAR-2 activation on the severity of pancreatitis, and to determine whether its protective effects are mediated by affecting either premature activation or secretory blockage, or both. RESULTS: The results confirmed that PAR-2 -/- mice have more severe pancreatitis than wild-type mice. Interestingly, intrapancreatic trypsin levels in the PAR-2 knockouts remained high after 6 h of pancreatitis, whereas they reverted to normal in the wild types. During pancreatitis, PAR-2 mRNA levels were upregulated in wild-type mice in response to supramaximal caerulein administration. Further, after a single injection of supramaximal caerulein, PAR-2 mRNA levels were also elevated, reaching a peak at 3 h. Stimulating PAR-2 with trypsin or the PAR-2-activating peptide, serine-leucine-isoleucine-glycine-arginine-leucine (SLIGRL), induced significantly more secretion from the acini of these caerulein-sensitised mice compared with the controls. PAR-2 activation also reversed the inhibition of secretion observed in both the caerulein and arginine models. CONCLUSIONS: Trypsin released during the early stages of pancreatitis activates PAR-2 receptors on the acinar cells and stimulates secretion from these cells. Thus, PAR-2 activation may decrease pancreatic injury and limit the severity of pancreatitis by allowing extracellular trypsin to act as a secretagogue.