Calcium-dependent enzyme activation and vacuole formation in the apical granular region of pancreatic acinar cells

The pancreatic acinar cell produces powerful digestive enzymes packaged in zymogen granules in the apical pole. Ca2+ signals elicited by acetylcholine or cholecystokinin (CCK) initiate enzyme secretion by exocytosis through the apical membrane. Intracellular enzyme activation is normally kept to a minimum, but in the often-fatal human disease acute pancreatitis, autodigestion occurs. How the enzymes become inappropriately activated is unknown. We monitored the cytosolic Ca2+ concentration ([Ca2+]i), intracellular trypsin activation, and its localization in isolated living cells with specific fluorescent probes and studied intracellular vacuole formation by electron microscopy as well as quantitative image analysis (light microscopy). A physiological CCK level (10 pM) eliciting regular Ca2+ spiking did not evoke intracellular trypsin activation or vacuole formation. However, stimulation with 10 nM CCK, evoking a sustained rise in [Ca2+]i, induced pronounced trypsin activation and extensive vacuole formation, both localized in the apical pole. Both processes were abolished by preventing abnormal [Ca2+]i elevation, either by preincubation with the specific Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N-N′,N′-tetraacetic acid (BAPTA) or by removal of external Ca2+. CCK hyperstimulation evokes intracellular trypsin activation and vacuole formation in the apical granular pole. Both of these processes are mediated by an abnormal sustained rise in [Ca2+]i.

Thymus transplantation, a critical factor for correction of autoimmune disease in aging MRL/+mice.

MRL/MP-+/+ (MRL/+) mice develop pancreatitis and sialoadenitis after they reach 7 months of age. Conventional bone marrow transplantation has been found to be ineffective in the treatment of these forms of apparent autoimmune disease. Old MRL/+ mice show a dramatic thymic involution with age. Hematolymphoid reconstitution is incomplete when fetal liver cells (as a source of hemopoietic stem cells) plus fetal bone (FB; which is used to recruit stromal cells) are transplanted from immunologically normal C57BL/6 donor mice to MRL/+ female recipients. Embryonic thymus from allogeneic C57BL/6 donors was therefore engrafted along with either bone marrow or fetal hematopoietic cells (FHCs) plus fragments of adult or fetal bone. More than seventy percent of old MRL/+ mice (> 7 months) that had been given a fetal thymus (FT) transplant plus either bone marrow or FHCs and also bone fragments survived more than 100 days after treatment. The mice that received FHCs, FB, plus FT from allogeneic donors developed normal T cell and B cell functions. Serum amylase levels decreased in these mice whereas they increased in the mice that received FHCs and FB but not FT. The pancreatitis and sialoadenitis already present at the time of transplantations were fully corrected according to histological analysis by transplants of allogeneic FHCs, FB and FT in the MRL/+ mice. These findings are taken as an experimental indication that perhaps stem cell transplants along with FT grafts might represent a useful strategy for treatment of autoimmune diseases in aged humans.

Canine and Feline Diabetes Mellitus: Nature or Nurture?

There is evidence for the role of genetic and environmental factors in feline and canine diabetes. Type 2 diabetes is the most common form of diabetes in cats. Evidence for genetic factors in feline diabetes includes the overrepresentation of Burmese cats with diabetes. Environmental risk factors in domestic or Burmese cats include advancing age, obesity, male gender, neutering, drug treatment, physical inactivity, and indoor confinement. High-carbohydrate diets increase blood glucose and insulin levels and may predispose cats to obesity and diabetes. Low-carbohydrate, high-protein diets may help prevent diabetes in cats at risk such as obese cats or lean cats with underlying low insulin sensitivity. Evidence exists for a genetic basis and altered immune response in the pathogenesis of canine diabetes. Seasonal effects on the incidence of diagnosis indicate that there are environmental influences on disease progression. At least 50% of diabetic dogs have type 1 diabetes based on present evidence of immune destruction of P-cells. Epidemiological factors closely match those of the latent autoimmune diabetes of adults form of human type 1 diabetes. Extensive pancreatic damage, likely from chronic pancreatitis, causes similar to28% of canine diabetes cases. Environmental factors such as feeding of high-fat diets are potentially associated with pancreatitis and likely play a role in the development of pancreatitis in diabetic dogs. There are no published data showing that overt type 2 diabetes occurs in dogs or that obesity is a risk factor for canine diabetes. Diabetes diagnosed in a bitch during either pregnancy or diestrus is comparable to human gestational diabetes.

Alogliptin after acute coronary syndrome in patients with type 2 diabetes

Background - To assess potentially elevated cardiovascular risk related to new antihyperglycemic drugs in patients with type 2 diabetes, regulatory agencies require a comprehensive evaluation of the cardiovascular safety profile of new antidiabetic therapies. We assessed cardiovascular outcomes with alogliptin, a new inhibitor of dipeptidyl peptidase 4 (DPP-4), as compared with placebo in patients with type 2 diabetes who had had a recent acute coronary syndrome. Methods - We randomly assigned patients with type 2 diabetes and either an acute myocardial infarction or unstable angina requiring hospitalization within the previous 15 to 90 days to receive alogliptin or placebo in addition to existing antihyperglycemic and cardiovascular drug therapy. The study design was a double-blind, noninferiority trial with a prespecified noninferiority margin of 1.3 for the hazard ratio for the primary end point of a composite of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke. Results - A total of 5380 patients underwent randomization and were followed for up to 40 months (median, 18 months). A primary end-point event occurred in 305 patients assigned to alogliptin (11.3%) and in 316 patients assigned to placebo (11.8%) (hazard ratio, 0.96; upper boundary of the one-sided repeated confidence interval, 1.16; P<0.001 for noninferiority). Glycated hemoglobin levels were significantly lower with alogliptin than with placebo (mean difference, -0.36 percentage points; P<0.001). Incidences of hypoglycemia, cancer, pancreatitis, and initiation of dialysis were similar with alogliptin and placebo. Conclusions - Among patients with type 2 diabetes who had had a recent acute coronary syndrome, the rates of major adverse cardiovascular events were not increased with the DPP-4 inhibitor alogliptin as compared with placebo. (Funded by Takeda Development Center Americas; EXAMINE ClinicalTrials.gov number, NCT00968708.)

New drugs for type 2 diabetes mellitus:what is their place in therapy?

Oral therapy for type 2 diabetes mellitus, when used appropriately, can safely assist patients to achieve glycaemic targets in the short to medium term. However, the progressive nature of type 2 diabetes usually requires a combination of two or more oral agents in the longer term, often as a prelude to insulin therapy. Issues of safety and tolerability, notably weight gain, often limit the optimal application of anti-diabetic drugs such as sulforylureas and thiazolidinediones. Moreover, the impact of different drugs, even within a single class, on the risk of long-term vascular complications has come under scrutiny. For example, recent publication of evidence suggesting potential detrimental effects of rosiglitazone on myocardial events generated a heated debate and led to a reduction in use of this drug. In contrast, current evidence supports the view that pioglitazone has vasculoprotective properties. Both drugs are contraindicated in patients who are at risk of heart failure. An additional recently identified safety concern is an increased risk of fractures, especially in postmenopausal women. Several new drugs with glucose-lowering efficacy that may offer certain advantages have recently become available. These include (i) injectable glucagonlike peptide-1 (GLP-1) receptor agonists and oral dipeptidyl peptidase-4 (DPP-4) inhibitors; (ii) the amylin analogue pramlintide; and (iii) selective cannabinoid receptor-1 (CB1) antagonists. GLP-1 receptor agonists, such as exenatide, stimulate nutrient-induced insulin secretion and reduce inappropriate glucagon secretion while delaying gastric emptying and reducing appetite. These agents offer a low risk of hypoglycaemia combined with sustained weight loss. The DPP-4 inhibitors sitagliptin and vildagliptin are generally weight neutral, with less marked gastrointestinal adverse effects than the GLP-1 receptor agonists. Potential benefits of GLP-1 receptor stimulation on P cell neogenesis are under investigation. Pancreatitis has been reported in exenatide-treated patients. Pramlintide, an injected peptide used in combination with insulin, can reduce insulin dose and bodyweight. The CB1 receptor antagonist rimonabant promotes weight loss and has favourable effects on aspects of the metabolic syndrome, including the hyperglycaemia of type 2 diabetes. However, in 2007 the US FDA declined approval of rimonabant, requiring more data on adverse effects, notably depression. The future of dual peroxisome proliferator-activated receptor-alpha/gamma agonists, or glitazars, is presently uncertain following concerns about their safety. In conclusion, several new classes of drugs have recently become available in some countries that offer new options for treating type 2 diabetes. Beneficial or neutral effects on bodyweight are an attractive feature of the new drugs. However, the higher cost of these agents, coupled with an absence of long-term safety and clinical outcome data, need to be taken into consideration by clinicians and healthcare organizations.

Small molecule dual-inhibitors of TRPV4 and TRPA1 for attenuation of inflammation and pain.

TRPV4 ion channels represent osmo-mechano-TRP channels with pleiotropic function and wide-spread expression. One of the critical functions of TRPV4 in this spectrum is its involvement in pain and inflammation. However, few small-molecule inhibitors of TRPV4 are available. Here we developed TRPV4-inhibitory molecules based on modifications of a known TRPV4-selective tool-compound, GSK205. We not only increased TRPV4-inhibitory potency, but surprisingly also generated two compounds that potently co-inhibit TRPA1, known to function as chemical sensor of noxious and irritant signaling. We demonstrate TRPV4 inhibition by these compounds in primary cells with known TRPV4 expression - articular chondrocytes and astrocytes. Importantly, our novel compounds attenuate pain behavior in a trigeminal irritant pain model that is known to rely on TRPV4 and TRPA1. Furthermore, our novel dual-channel blocker inhibited inflammation and pain-associated behavior in a model of acute pancreatitis - known to also rely on TRPV4 and TRPA1. Our results illustrate proof of a novel concept inherent in our prototype compounds of a drug that targets two functionally-related TRP channels, and thus can be used to combat isoforms of pain and inflammation in-vivo that involve more than one TRP channel. This approach could provide a novel paradigm for treating other relevant health conditions.

Prospective Estimation of Radiation Dose and Image Quality for Optimized CT Performance

X-ray computed tomography (CT) is a non-invasive medical imaging technique that generates cross-sectional images by acquiring attenuation-based projection measurements at multiple angles. Since its first introduction in the 1970s, substantial technical improvements have led to the expanding use of CT in clinical examinations. CT has become an indispensable imaging modality for the diagnosis of a wide array of diseases in both pediatric and adult populations [1, 2]. Currently, approximately 272 million CT examinations are performed annually worldwide, with nearly 85 million of these in the United States alone [3]. Although this trend has decelerated in recent years, CT usage is still expected to increase mainly due to advanced technologies such as multi-energy [4], photon counting [5], and cone-beam CT [6].

Despite the significant clinical benefits, concerns have been raised regarding the population-based radiation dose associated with CT examinations [7]. From 1980 to 2006, the effective dose from medical diagnostic procedures rose six-fold, with CT contributing to almost half of the total dose from medical exposure [8]. For each patient, the risk associated with a single CT examination is likely to be minimal. However, the relatively large population-based radiation level has led to enormous efforts among the community to manage and optimize the CT dose.

As promoted by the international campaigns Image Gently and Image Wisely, exposure to CT radiation should be appropriate and safe [9, 10]. It is thus a responsibility to optimize the amount of radiation dose for CT examinations. The key for dose optimization is to determine the minimum amount of radiation dose that achieves the targeted image quality [11]. Based on such principle, dose optimization would significantly benefit from effective metrics to characterize radiation dose and image quality for a CT exam. Moreover, if accurate predictions of the radiation dose and image quality were possible before the initiation of the exam, it would be feasible to personalize it by adjusting the scanning parameters to achieve a desired level of image quality. The purpose of this thesis is to design and validate models to quantify patient-specific radiation dose prospectively and task-based image quality. The dual aim of the study is to implement the theoretical models into clinical practice by developing an organ-based dose monitoring system and an image-based noise addition software for protocol optimization.

More specifically, Chapter 3 aims to develop an organ dose-prediction method for CT examinations of the body under constant tube current condition. The study effectively modeled the anatomical diversity and complexity using a large number of patient models with representative age, size, and gender distribution. The dependence of organ dose coefficients on patient size and scanner models was further evaluated. Distinct from prior work, these studies use the largest number of patient models to date with representative age, weight percentile, and body mass index (BMI) range.

With effective quantification of organ dose under constant tube current condition, Chapter 4 aims to extend the organ dose prediction system to tube current modulated (TCM) CT examinations. The prediction, applied to chest and abdominopelvic exams, was achieved by combining a convolution-based estimation technique that quantifies the radiation field, a TCM scheme that emulates modulation profiles from major CT vendors, and a library of computational phantoms with representative sizes, ages, and genders. The prospective quantification model is validated by comparing the predicted organ dose with the dose estimated based on Monte Carlo simulations with TCM function explicitly modeled.

Chapter 5 aims to implement the organ dose-estimation framework in clinical practice to develop an organ dose-monitoring program based on a commercial software (Dose Watch, GE Healthcare, Waukesha, WI). In the first phase of the study we focused on body CT examinations, and so the patient’s major body landmark information was extracted from the patient scout image in order to match clinical patients against a computational phantom in the library. The organ dose coefficients were estimated based on CT protocol and patient size as reported in Chapter 3. The exam CTDIvol, DLP, and TCM profiles were extracted and used to quantify the radiation field using the convolution technique proposed in Chapter 4.

With effective methods to predict and monitor organ dose, Chapters 6 aims to develop and validate improved measurement techniques for image quality assessment. Chapter 6 outlines the method that was developed to assess and predict quantum noise in clinical body CT images. Compared with previous phantom-based studies, this study accurately assessed the quantum noise in clinical images and further validated the correspondence between phantom-based measurements and the expected clinical image quality as a function of patient size and scanner attributes.

Chapter 7 aims to develop a practical strategy to generate hybrid CT images and assess the impact of dose reduction on diagnostic confidence for the diagnosis of acute pancreatitis. The general strategy is (1) to simulate synthetic CT images at multiple reduced-dose levels from clinical datasets using an image-based noise addition technique; (2) to develop quantitative and observer-based methods to validate the realism of simulated low-dose images; (3) to perform multi-reader observer studies on the low-dose image series to assess the impact of dose reduction on the diagnostic confidence for multiple diagnostic tasks; and (4) to determine the dose operating point for clinical CT examinations based on the minimum diagnostic performance to achieve protocol optimization.

Chapter 8 concludes the thesis with a summary of accomplished work and a discussion about future research.

Retinopatia de Purtscher-like e pancreatite aguda

Retinopatia de Purtscher-like é uma baixa súbita da visão associada à imagem de múltiplas áreas branco-amareladas (manchas algodonosas) e hemorragias no pólo posterior de ambos os olhos. O exato mecanismo da injúria ainda não é claro, mas provavelmente seria de natureza embólica. Tem sido descrita em uma variedade de condições, incluindo pancreatite aguda, síndrome de embolia gordurosa, insuficiência renal,nascimento (parto e pós-parto), desordens do tecido conectivo, entre outras. Serão relatados três casos de pancreatite aguda confirmada pelos exames laboratoriais e história clínica, associadas a alterações no exame do fundo de olho, compatíveis com esta retinopatia

Pancreatic pseudocyst: case report and short literature review

We report a case of pancreatic pseudocyst secondary to acute necrotizing pancreatitis treated with open cystogastrostomy. Following a literature review, we stress the enormous benefits offered by modern diagnostic techniques, and especially imaging techniques, for the diagnosis and monitoring of this disease. Treatment should be delayed for at least six weeks, following which the drainage by open surgery offers the best results and lowest morbidity and mortality, followed by laparoscopy and endoscopy, indicated in particular cases and in patients where open surgery is contraindicated.

Dermatitis herpetiforme como carta de presentación de la enfermedad celíaca

RESUMEN: La Dermatitis Herpetiforme (DH) es una enfermedad ampollar autoinmune (EAI) que corresponde a la manifestación cutánea de la Enfermedad Celíaca (EC), más precisamente de la intolerancia al gluten. Clínicamente se manifiesta como una erupción papulo-vesicular pruriginosa, topografiada fundamentalmente en superficies de extensión de extremidades. El diagnóstico se realiza mediante el estudio histopatológico de piel lesional e inmunofluorescencia directa (IFD) de piel perilesional, la cual muestra hallazgos característicos. En su patogenia intervienen factores genéticos, inmunológicos, y ambientales. El tratamiento de elección es la dieta libre de gluten (DLG) y la Dapsona. Se ha reportado una asociación cercana al 15% entre la afectación cutánea e intestinal, no existe hasta el momento ningún estudio prospectivo que muestre la frecuencia real de EC en pacientes con DH

Retinopatia de Purtscher-like e pancreatite aguda

Retinopatia de Purtscher-like é uma baixa súbita da visão associada à imagem de múltiplas áreas branco-amareladas (manchas algodonosas) e hemorragias no pólo posterior de ambos os olhos. O exato mecanismo da injúria ainda não é claro, mas provavelmente seria de natureza embólica. Tem sido descrita em uma variedade de condições, incluindo pancreatite aguda, síndrome de embolia gordurosa, insuficiência renal,nascimento (parto e pós-parto), desordens do tecido conectivo, entre outras. Serão relatados três casos de pancreatite aguda confirmada pelos exames laboratoriais e história clínica, associadas a alterações no exame do fundo de olho, compatíveis com esta retinopatia

Purtscher-like retinopathy

Purtscher-like retinopathy is associated with retinal hemorrhages and ischaemia probably due to the complement-mediated leukoembolization. It is a rare and severe angiopathy found in conditions such as acute pancreatitis. Case. We present a case of a 53-year-old man who presented with a Purtscher-like retinopathy associated with the development of acute pancreatitis in the context of a Klatskin tumour (a hilar cholangiocarcinoma). The ophthalmologic evaluation revealed the best corrected visual acuity (BCVA) of 20/32 in the right eye (RE) and of 20/40 in the left eye (LE); biomicroscopy of anterior segment showed scleral icterus and fundoscopy revealed peripapillary cotton-wool spots, optic disc edema, and RPE hypo- and hyperpigmentation in the middle peripheral retina in both eyes with an intraretinal hemorrhage in the LE. 15 months after the initial presentation, without ophthalmological treatment, there was an improvement of BCVA to 20/20 in both eyes and optical coherence tomography (OCT) revealed areas of reduction of retinal nerve fiber layer thickness corresponding to the previous cotton-wool spots. Conclusion. Purtscher-like retinopathy should not be neglected in complex clinical contexts. Its unclear pathophysiology determines an uncertain treatment strategy, but a meticulous follow-up is compulsory in order to avoid its severe complications.

Comportamiento de la patología biliar aguda y su manejo en el servicio de trauma y emergencia del Hospital "Vicente Corral Moscoso" Cuenca. Enero a junio de 2014

La patología biliar afecta a un gran porcentaje de la población adulta, motivo por el cual su tratamiento en la actualidad ha cambiado hacia un nuevo paradigma de cuidado bajo el concepto de “Acute Care Surgery” (ACS) 1 el cual se caracteriza por priorizar la valoración integral del paciente e intervención precoz de la patología. En el Hospital Vicente Corral Moscoso (HVCM) bajo este modelo ACS, y mediante la utilización de protocolos estandarizados se ha logrado dar un giro importante en el tratamiento oportuno de la patología biliar mediante la utilización de herramientas habituales como pruebas de laboratorio, imagenología y si es el caso, la resolución quirúrgica mediante técnica mínimamente invasiva o por vía convencional. OBJETIVO: Describir el comportamiento de la patología biliar y su manejo en el servicio de Trauma y Emergencias del Hospital “Vicente Corral Moscoso”, durante el período de enero a junio de 2014, bajo el modelo ACS. MÉTODOS: Estudio descriptivo transversal, que analizó los casos de colecistitis aguda litiásica (CAL), coledocolitiasis, pancreatitis aguda biliar (PAB) y su manejo, registrado en la base de datos digital del servicio de Emergencias del Hospital Vicente Corral Moscoso, bajo criterios clínicos, de laboratorio e imagenológicos, durante el periodo de enero a junio del 2014. RESULTADOS: El estudio contó con un total de 240 pacientes atendidos en el servicio de Trauma y Emergencia del HVCM, durante el periodo de enero a junio de 2014. La patología en orden de frecuencia fue: en un 47%, la Coledocolitiasis; 35% colecistitis aguda y, pancreatitis aguda biliar 18%. La prevalencia fue mayor en el sexo femenino en un 85%, 67%, y 81% respectivamente y el tratamiento se adaptó a cada patología. 1 Acute Care Surgery” (ACS): si bien no existe una definición literal hace referencia a una disciplina tripartita que engloba la cirugía de trauma, general en emergencias y cuidados críticos quirúrgicos, y que prioriza la identificación y manejo de las patologías potencialmente letales y de alta morbilidad. En nuestro medio lo más próximo a la definición seria Cirugía de Trauma y Emergencias. El manejo de la pancreatitis aguda biliar (PAB) bajo el concepto de cuidado agudo de pacientes quirúrgicos o “Acute Care Surgery” hace indispensable una intervención oportuna y temprana, utilizando todos los recursos disponibles para un manejo integral. CONCLUSIONES: La implementación del modelo de Cirugía de Trauma y Emergencias en nuestra institución ha logrado un manejo integral de colecistitis aguda litiásica, pancreatitis aguda biliar y coledocolitiasis, disminuyendo las complicaciones asociadas y evitando las recidivas de cuadros de mayor gravedad.

Evaluación del efecto de la intoxicación aguda por Karwinskia humboldtiana en el páncreas de la rata Wistar

Propósito y método del estudio: Cuando se ingieren grandes cantidades de fruto de Karwinskia humboldtiana se produce una intoxicación aguda, que ocasiona daño en múltiples órganos, falla respiratoria y muerte en pocos días. En la intoxicación accidental y experimental con este fruto, se ha reportado daño histológico en pulmones, hígado y riñones. Se sabe que el daño histológico a estos órganos, además de la falla multiorgánica, son situaciones comunes cuando existe daño pancreático, sin embargo, hasta la fecha, el páncreas no ha sido estudiado en esta intoxicación. En este trabajo examinamos el efecto que ocasiona la intoxicación aguda con el fruto de Karwinskia humboldtiana en el páncreas en la rata Wistar. Contribuciones y conclusiones: En este trabajo se encontró daño progresivo confinado a la porción exocrina del páncreas, iniciando con reducción en el tamaño de los acinos pancreáticos, así como del número de gránulos de zimógeno, presencia de vesículas de apariencia autofágica y apoptosis, seguidos de edema, infiltrado inflamatorio, necrosis y pérdida completa de la arquitectura acinar. Cabe señalar que la morfología de los islotes de Langerhans se mantuvo conservada en todos los tiempos evaluados en este trabajo. Mediante las técnicas Western Blot e inmunofluorescencia, analizamos la expresión y localización de proteínas implicadas en la autofagia (LC3-I y LC3-II) y en la apoptosis (caspasa-3). Observamos que las proteínas LC3-I y LC3-II se encuentran expresadas en todos los tiempos experimentales, mientras que la proteína caspasa-3 se expresó únicamente a las 48 h de la intoxicación con Karwinskia humboldtiana. Mediante ensayos de histoquímica enzimática para la citocromo oxidasa c, comprobamos que desde las 24 h de intoxicación, existen cambios en la forma, tamaño y localización de las mitocondrias, organelos implicados en la muerte celular. Asimismo, realizamos la evaluación de la función pancreática mediante la determinación de la actividad de la amilasa sérica, sin encontrar diferencia significativa entre los grupos estudiados. Todos estos datos indican que el daño inducido por una dosis alta de fruto de Karwinskia humboldtiana en la rata Wistar, es consistente con una pancreatitis aguda necrotizante que afecta exclusivamente el páncreas exocrino. Este modelo de pancreatitis puede ser útil para el estudio de los mecanismos celulares y moleculares implicados en este padecimiento, así como para el ensayo de posibles tratamientos para esta y otras enfermedades pancreáticas.