Phospho-CRKL monitoring for the assessment of BCR-ABL activity in imatinib-resistant chronic myeloid leukemia or Ph+ acute lymphoblastic leukemia patients treated with nilotinib

Actual BCR-ABL kinase inhibition in vivo as determined by phospho-CRKL (pCRKL) monitoring has been recognized as a prognostic parameter in patients with chronic myelogenous leukemia treated with imatinib. We report a biomarker sub-study of the international phase I clinical trial of nilotinib (AMN107) using the established pCRKL assay in imatinib-resistant chronic myeloid leukemia or Ph+ acute lymphoblastic leukemia. A minimum dose (200 mg) required for effective BCR-ABL inhibition in imatinib resistant/intolerant leukemia was determined. The pre-clinical activity profile of nilotinib against mutant BCR-ABL was largely confirmed. Substantial differences between peripheral blood baseline pCRKL/CRKL ratios were observed when comparing chronic myeloid leukemia with Ph+ acute lymphoblastic leukemia. Finally, rapid BCR-ABL-reactivation shortly after starting nilotinib treatment was seen in acute lymphoblastic leukemia patients with progressive disease carrying the P-loop mutations Y253H, E255K, or mutation T315I. Monitoring the actual BCR-ABL inhibition in nilotinib treated patients using pCRKL as a surrogate is a means to establish effective dosing and to characterize resistance mechanisms against nilotinib.     

Tropical calcific pancreatitis: strong association with SPINK1 trypsin inhibitor mutations

BACKGROUND & AIMS: Tropical calcific pancreatitis (TCP) is a chronic pancreatitis unique to developing countries in tropical regions. The cause of TCP is obscure. Whereas environmental factors, such as protein energy malnutrition and ingestion of cassava, have been implicated, a genetic predisposition to the disease also may be important. In the present study we report on mutations in the serine protease inhibitor, Kazal type 1 (SPINK1) gene in north Indian patients with TCP. METHODS: We studied 66 unrelated TCP patients (44 men, 49 with diabetes, and 6 with family history of TCP), 25 relatives, and 92 healthy control subjects. Samples were analyzed for SPINK1 variants (-53C>T, L14P, N34S, P55S, and 272T>C) and cationic trypsinogen (PRSS1) variants (A16V, K23R, N29I, and R122H) by melting curve analysis. RESULTS: Twenty-nine patients (44%) carried the N34S missense mutation, of whom 9 (14%) were homozygotes. In contrast, only 2 (2.2%) control subjects were N34S heterozygotes (prevalence ratio 20.2; 95% confidence interval 5.0-81.8; P < 0.0001 vs. TCP). The severity of pancreatitis did not differ between TCP patients with or without N34S, or among those heterozygous or homozygous for N34S. Among TCP patients with or without diabetes, the frequency of N34S carriers (43% vs. 47%) and N34S homozygotes (14% vs. 12%) was similar. CONCLUSIONS: TCP is highly associated with the SPINK1 N34S mutation. The high prevalence of N34S in TCP patients with and without diabetes suggests that these 2 subtypes have a similar genetic predisposition. The genetic predisposition to TCP resembles, at least in part, the idiopathic chronic pancreatitis found in industrialized countries.     

Comparison of dobutamine stress echocardiography with and without real-time perfusion imaging for detection of coronary artery disease

In a pilot study of 27 patients, those who presented with chest pain underwent 2 dobutamine stress echocardiographic studies, 1 with high mechanical index harmonic imaging to analyze wall motion without contrast and 1 with real-time low mechanical index perfusion imaging with intravenous Optison to assess myocardial perfusion and wall motion. All patients then underwent quantitative coronary angiography. Two independent reviewers demonstrated an improvement in sensitivity when analyzing myocardial perfusion. In the 21 patients who had significant coronary stenoses, 14 had abnormal myocardial perfusion detected at peak stress and 7 had abnormal wall motion detected by standard dobutamine stress echocardiography. There was decreased specificity with perfusion imaging by 1 reviewer. The addition of real-time perfusion imaging after intravenous contrast during dobutamine stress echocardiography has the potential to improve detection of coronary artery disease.     

Targeted Resequencing of 29 Candidate Genes and Mouse Expression Studies Implicate ZIC3 and FOXF1 in Human VATER/VACTERL Association

The VATER/VACTERL association describes the combination of congenital anomalies including vertebral defects, anorectal malformations, cardiac defects, tracheoesophageal fistula with or without esophageal atresia, renal malformations, and limb defects. As mutations in ciliary genes were observed in diseases related to VATER/VACTERL, we performed targeted resequencing of 25 ciliary candidate genes as well as disease‐associated genes (FOXF1, HOXD13, PTEN, ZIC3) in 123 patients with VATER/VACTERL or VATER/VACTERL‐like phenotype. We detected no biallelic mutation in any of the 25 ciliary candidate genes; however, identified an identical, probably disease‐causing ZIC3 missense mutation (p.Gly17Cys) in four patients and a FOXF1 de novo mutation (p.Gly220Cys) in a further patient. In situ hybridization analyses in mouse embryos between E9.5 and E14.5 revealed Zic3 expression in limb and prevertebral structures, and Foxf1 expression in esophageal, tracheal, vertebral, anal, and genital tubercle tissues, hence VATER/VACTERL organ systems. These data provide strong evidence that mutations in ZIC3 or FOXF1 contribute to VATER/VACTERL.     

3D matrix‐embedding inhibits cycloheximide‐mediated sensitization to TNF‐alpha‐induced apoptosis of human endothelial cells

The programmed form of cell death (apoptosis) is essential for normal development of multicellular organisms. Dysregulation of apoptosis has been linked with embryonal death and is involved in the pathophysiology of various diseases. Others and we previously demonstrated endothelial biology being intertwined with biochemical and structural composition of the subendothelial basement membrane. We now demonstrate that a three‐dimensional growing environment significantly shields endothelial cells from cytokine‐induced apoptosis. Detailed analysis reveals differences in intracellular signaling pathways in naive endothelial cells and cytokine‐stimulated endothelial cells when cells are grown within a three‐dimensional collagen‐based matrix compared to cells grown on two‐dimensional tissue culture plates. Main findings are significantly reduced p53 expression and level of p38‐phosphorylation in three‐dimensional grown endothelial cells. Despite similar concentrations of focal adhesion kinase, three‐dimensional matrix‐embedded endothelial cells express significantly less tyrosine‐phosphorylated focal adhesion kinase. Pretreatment with antibodies against integrin α_vβ₃ partially reversed the protective effect of three‐dimensional matrix‐embedding on endothelial apoptosis. Our findings provide detailed insights into the mechanisms of endothelial apoptosis with respect to the spatial matrix environment. These results enhance our understanding of endothelial biology and may otherwise help in the design of tissue‐engineered materials. Furthermore, findings on focal adhesion kinase phosphorylation might enhance our understanding of clinical studies with tyrosine kinase inhibitors.     

Intravenous urokinase in acute myocardial infarction

To achieve reperfusion early, an intravenous bolus of 2 million units of urokinase was administered in 50 patients with transmural acute myocardial infarction (AMI) 1.8 +/- 2.5 hours after the onset of symptoms. Coronary angiography performed 1.1 +/- 0.6 hours after urokinase therapy revealed patent coronary arteries in 30 patients (60%), with no significant difference between those with anterior and those with inferior AMI. Reocclusion occurred in only 1 of 24 patients restudied. Failure to achieve reperfusion was not related to the degree of systemic fibrinolytic activity, which was equally high in patients who did and those who did not achieve reperfusion, as evident from serially obtained fibrinogen measurements (77 +/- 52 vs 84 +/- 24 mg/dl, difference not significant). Plasmin activity, measured serially from 15 minutes to 24 hours after urokinase in 7 patients, was maximal at 15 minutes and undetectable after 3 hours. Wall motion at the infarct site measured from contrast ventriculograms was significantly better at follow-up only in patients in whom reperfusion was achieved and who received urokinase within 2 hours after the onset of symptoms as compared with patients in whom reperfusion was not achieved (-1.2 +/- 1.4 vs -2.4 +/- 0.9 standard deviations from normal, p less than 0.05). Peak serum creatine kinase level was significantly lower in patients in whom reperfusion was achieved than in those in whom it was not or those who had rethrombosis (802 +/- 763 vs 1,973 +/- 1,071 U/liter, p less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)     

Pleural Effusions: A New Negative Prognostic Parameter for Acute Pancreatitis

Objective: To evaluate the incidence, localization, and size of pleural effusions in 133 patients with acute pancreatitis. Methods: A contrast-enhanced computed tomographic scan was prospectively obtained within 72 h after admission. Results: Patients with pleural effusions (66 = 50%) had significantly more severe morphological changes of the pancreas and necroses (independent of the size and localization of the effusions and etiology of the acute pancreatitis), more often had a pancreatic pseudocyst, and had a higher mortality rate than patients without this complication. Conclusion: Pleural effusions are indicative of severe acute pancreatitis and are a negative prognostic parameter for the course of the disease.     

Myocardial contrast echocardiography. Research instrument or already clinical application?

Increasing interest has been focused on myocardial contrast echocardiography (MCE) since latest imaging technologies allow the detection of echocontrast agents within myocardial structures following intravenous injections. Although numerous studies on different aspects of pre-clinical and clinical applications have been published, MCE has not yet become a standard clinical application. This paper summarises and estimates the clinical value of the recent developments in this evolving field of research, particularly with regard to the new real-time perfusion imaging technologies.     

Proteinase-activated receptor-2-mediated signaling and inhibition of DNA synthesis in human pancreatic cancer cells

Summary Conclusion Proteinase-activated receptor-2 (PAR-2)-mediated effects contribute to the intracellular signaling network in pancreatic tumor cells. A role of PAR-2 as negative regulator in human pancreatic tumor growth might be implied. Background Using the human pancreatic tumor cell line MIA PaCa-2, we evaluated cellular effects of trypsin and the PAR-2-activating peptide SLIGRL on [Ca²⁺]_i mobilization, Ins(1,4,5)P₃ level, and protein kinase (PKC) activation. Furthermore, PAR-2 involvement in the regulation of cell proliferation has been estimated by measurement of [³H]thymidine incorporation in MIA PaCa-2 cells. Results Trypsin and the PAR-2 synthetic peptide agonist SLIGRL induced [Ca²⁺]_i mobilization, transient increase in inositol (1,4,5) triphosphate level, and PKC translocation in MIA PaCa-2 cells. In addition, SLIGRL induced a decrease in DNA synthesis in MIA PaCa-2 cells.