Pia Klausen

On mouse and man : neutrophil gelatinase associated lipocalin is not involved in apoptosis or acute response

Abstract:  Neutrophil gelatinase‐associated lipocalin (NGAL) is a siderphore binding molecule present in the specific granules of neutrophils and induced in a variety of epithelial cells during inflammation. Its mouse orthologue, 24p3, is also an acute phase protein synthesized in the liver and adipose tissue during inflammation. 24p3 has recently been implicated in apoptosis of myeloid cells. We investigated whether similar features are characteristics of NGAL. First, isolated normal myeloid bone marrow cells were incubated with NGAL for 6 and 24 hr and analyzed for apoptosis by annexin V binding and by propidium iodide labeling. We found no indication that NGAL induces significant apoptosis in myeloid cells. Second, a human sepsis model where normal volunteers were given endotoxin 2 ng/kg intravenously, showed no evidence that NGAL is an acute phase protein. The plasma level of NGAL reflected the number of circulating neutrophils and was completely different from the kinetics of C‐reactive protein. We thus conclude that major differences exist between mouse and man with regards to the role of this lipocalin in myelopoiesis and inflammation.

Localization of serglycin in human neutrophil granulocytes and their precursors

Serglycin is a major proteoglycan of hematopoietic cells. It is thought to play a role in the packaging of granule proteins in human neutrophil granulocytes. The presence of serglycin in myeloid cells has been demonstrated only at the transcriptional level. We generated a polyclonal antibody against recombinant human serglycin. Here, we show the localization of serglycin in humans during neutrophil differentiation. Immunocytochemistry revealed serglycin immunoreactivity in the Golgi area of promyelocytes (PM) and myelocytes (MC), as well as in a few band cells and mature neutrophil granulocytes. Granular staining was detected near the Golgi apparatus in some of the PM, and the major part of the cytoplasm was negative. Immunoelectron microscopy showed serglycin immunoreactivity located to the Golgi apparatus and a few immature granules of PM and MC. The decreasing level of serglycin protein during myeloid differentiation coincided with a decrease of mRNA expression, as evaluated by Northern blotting. Subcellular fractions of neutrophil granulocytes were obtained. Serglycin immunoreactivity was detected in the fraction containing Golgi apparatus, plasma membrane, and secretory vesicles by Western blotting and enzyme‐linked immunosorbent assay. Serglycin was not detected in subcellular fractions containing primary, secondary, or tertiary granules. Together, these findings indicate that serglycin is located to the Golgi apparatus and a few immature granules during neutrophil differentiation. This is consistent with a function for serglycin in formation of granules in neutrophil granulocytes. Our findings contrast the view that native serglycin is present in mature granules and plays a role in packaging and regulating the activity of proteolytic enzymes there.

Confocal laser endomicroscopy: a novel method for prediction of relapse in Crohn's disease.

BACKGROUND AND STUDY AIMS Confocal laser endomicroscopy (CLE) has been shown to predict relapse in ulcerative colitis in remission, but little is currently known about its role in Crohns disease. The aim of this study was to identify reproducible CLE features in patients with Crohns disease and to examine whether these are risk factors for relapse. PATIENTS AND METHODS This was a single-center prospective feasibility study of CLE imaging in patients with Crohns disease. CLE imaging was performed in the terminal ileum and four colorectal sites, and was correlated with histopathology and macroscopic appearance. Clinical relapse, defined as the need for treatment escalation or surgical intervention, was recorded during follow-up. RESULTS The study included 50 patients: 39 with Crohns disease (20 in remission), and 11 controls. Ileal fluorescein leakage and microerosions were significantly more frequent in patients with endoscopically active Crohns disease compared with patients with inactive Crohns disease and controls (P = 0.005 and (P = 0.006, respectively). The same applied to colorectal fluorescein leakage and vascular alterations ((P = 0.043 and (P = 0.034, respectively). During a 12-month follow-up period, ileal fluorescein leakage and microerosions were significant risk factors for relapse in the subgroup of patients in remission (log rank (P = 0.009 and (P = 0.007, respectively) as well as in the entire group of patients with Crohns disease (log rank (P = 0.006 and (P = 0.01, respectively). Inter- and intraobserver reproducibility was almost perfect (κ > 0.80) or substantial (κ > 0.60) for the majority of CLE parameters. CONCLUSIONS CLE can identify reproducible microscopic changes in the terminal ileum that are risk factors for relapse in patients with otherwise inactive Crohns disease. TRIAL REGISTRATION ClinicalTrials.gov (NCT01738529).

Molecular confocal laser endomicroscopy: a novel technique for in vivo cellular characterization of gastrointestinal lesions.

While flexible endoscopy is essential for macroscopic evaluation, confocal laser endomicroscopy (CLE) has recently emerged as an endoscopic method enabling visualization at a cellular level. Two systems are currently available, one based on miniprobes that can be inserted via a conventional endoscope or via a needle guided by endoscopic ultrasound. The second system has a confocal microscope integrated into the distal part of an endoscope. By adding molecular probes like fluorescein conjugated antibodies or fluorescent peptides to this procedure (either topically or systemically administered during on-going endoscopy), a novel world of molecular evaluation opens up. The method of molecular CLE could potentially be used for estimating the expression of important receptors in carcinomas, subsequently resulting in immediate individualization of treatment regimens, but also for improving the diagnostic accuracy of endoscopic procedures by identifying otherwise invisible mucosal lesions. Furthermore, studies have shown that fluorescein labelled drugs can be used to estimate the affinity of the drug to a target organ, which probably can be correlated to the efficacy of the drug. However, several of the studies in this research field have been conducted in animal facilities or in vitro, while only a limited number of trials have actually been carried out in vivo. Therefore, safety issues still needs further evaluations. This review will present an overview of the implications and pitfalls, as well as future challenges of molecular CLE in gastrointestinal diseases.

Endoscopic ultrasound-guided needle-based confocal laser endomicroscopy: a pilot study for use in focal pancreatic masses.

Copyright emerged in recent years as a novel method that enables in vivo microscopic analysis during ongoing endoscopy. Recently, CLE has gone beyond the superficial luminal indications with the development of a newmicroprobe (nCLE) (Cellvizio;Mauna Kea Technology, Paris, France). The aim of the present case series was to study the feasibility of EUS-guided nCLE and to correlate the findings with microscopy.

Endoscopic ultrasound guided needle-based confocal laser endomicroscopy in solid pancreatic masses – a prospective validation study

Background and study aims  Endoscopic ultrasound fine-needle aspiration (EUS-FNA) is a keystone in diagnosing and staging of pancreatic masses. Recently, a microfiber that can pass through a 19-gauge needle has been introduced for confocal laser endomicroscopy (nCLE). The aims of this study were to evaluate the diagnostic value and the reproducibility of nCLE criteria for solid malignant lesions. Patients and methods  This prospective dual-center study included patients with pancreatic masses suspicious of malignancy referred for EUS-FNA. Endomicroscopic imaging was performed under EUS-guidance until organ-specific structures were obtained. Afterwards, standard cytology was obtained and patients were followed for up to 12 months. All nCLE parameters included in former studies were correlated with the final diagnosis (dark lobular structures/normal acinar cells, dark cell aggregates > 40 µm, dilated irregular vessels with fluorescein leakage, fine white fibrous bands, small black cell movements, pseudoglandular structures). Finally, three CLE novices and three CLE experts assessed the unedited movies from all patients. Results  Twenty-eight patients were enrolled in the study. A final diagnosis was obtained in 24 patients (86 %). One patient (3 %) died before a diagnosis was obtained, while 3 were lost to follow-up (11 %). In 18/24 patients (74 %) the diagnosis was malignant. The mean sensitivity, specificity, and accuracy for the nCLE parameters ranged from 19 – 93 %, 0 – 56 %, 26 – 69 %, respectively. The inter-observer values ranged from κ = 0.20 – 0.41 for novices and κ = –0.02 – 0.38 for experts. Conclusions  The diagnostic value of nCLE in solid pancreatic masses is questionable and the inter-observer agreement for both novices and CLE experts appears limited.

Diagnosis of intraductal papillary mucinous neoplasm using endoscopic ultrasound guided microbiopsies: A case report

Pancreatic cysts are increasingly diagnosed due to expanding use of cross-sectional imaging, but current diagnostic modalities have limited diagnostic accuracy. Recently, a novel through-the-needle microbiopsy forceps has become available, offering the possibility of obtaining cyst-wall biopsies. We present a case of 41-year-old male with chronic pancreatitis and a 2-cm pancreatic cyst, initially considered a pseudocyst. Subsequently, endoscopic ultrasound guided microbiopsies were successfully obtained, which surprisingly revealed an intraductal papillary mucinous neoplasm of mixed subtype with low grade dysplasia. In conclusion, obtaining biopsies from the wall of the pancreatic cystic lesions with this novel instrument is feasible and, as demonstrated in this case, can possibly alter the clinical outcome. Microbiopsies offered enough cellular material, allowing supplemental gene mutation analysis, which combined with other modalities could lead to a more individual approach when treating pancreatic cysts. However, prospective studies are warranted before routine clinical implementation.

SMAD4 Protein Expression Is Downregulated in Ileal Epithelial Cells from Patients with Crohn’s Disease with Significant Inverse Correlation to Disease Activity

Background Small mothers against decapentaplegic (SMAD)4 and SMAD7 are key regulatory components in the immunosuppressive transforming growth factor- (TGF-) β signaling pathway, which is defective in inflammatory bowel disease (IBD). SMAD4 may play an important role in the pathogenesis of IBD as indicated in experimental models of colitis. Aims To examine the ileal expression levels of SMAD4 and to correlate these with CD disease activity. Methods The material comprised 29 CD patients (13 with active disease, 16 in remission) and 9 asymptomatic patients referred for ileocolonoscopy as part of an adenoma surveillance program serving as controls. Patients were examined with ileocolonoscopy. Corresponding ileal biopsies were obtained for histological analysis and assessment of SMAD4 and SMAD7 protein expression by immunohistochemistry (IHC). Results The protein expression of SMAD4 was significantly downregulated in ileal tissue sections from CD patients as compared to healthy controls (p < 0.001). Further, luminal SMAD4 expression was inversely correlated with endoscopic (r s = −0.315; p = 0.05) and histopathological activity (r s = −0.40; p = 0.013). Conclusions The SMAD4 epithelial protein level was markedly downregulated in CD patients and inversely correlated with disease activity. This may contribute to defective mucosal TGF-β signaling in active IBD.

Initial experience with EUS-guided microbiopsy forceps in diagnosing pancreatic cystic lesions: A multicenter feasibility study (with video)

Background and Objectives: Cystic lesions of the pancreas represent a diagnostic dilemma. Recently, a through-the-needle microbiopsy forceps has become available, enabling procurement of EUS-guided histological specimens from the pancreatic cyst wall. The aim of this study was to evaluate the use of this novel instrument in a multicenter clinical setting. Patients and Methods: Patients referred for EUS evaluation of pancreatic cysts and attempted EUS-guided microbiopsy was included retrospectively from six international tertiary centers. Patients demographics, EUS findings, technical and clinical success, and histopathological results were recorded. Results: A total of 28 patients were identified. We report a technical success rate of 85.7% (n = 24). Biopsies were generally of good quality and contributed to the diagnosis in 20 patients (clinical success of 71.4%). Three adverse events were recorded (10.7%). Conclusions: The use of the microbiopsy forceps is feasible with acceptable rates of technical and clinical success. Prospective studies are warranted to determine the diagnostic potential compared to the other modalities. However, the results from this preliminary study are promising.

Molecular biomarkers have the potential to improve the diagnostic work-up of pancreatic cystic lesions

Abstract Pancreatic cysts are increasingly diagnosed due to the widespread use of cross-sectional imaging, and some of these lesions harbor malignant potential. Mucinous cystic neoplasms and intraductal papillary mucinous neoplasms are the major premalignant cystic neoplasms of the pancreas. A variety of diagnostic tools are used to predict the malignant potential of these cysts, but specificity and sensitivity are limited. Thus, many patients undergo unnecessary operations for benign cysts. Balancing the risks of watchful waiting with those of operative management is key in managing these lesions. During the last decade, genetic changes of pancreatic cysts have been examined extensively to estimate their malignant potential. In this review, we provide an overview of the latest molecular and genetic aspects of pancreatic cysts and how they may contribute to the differential diagnosis in patients with pancreatic cystic neoplasms.