Mary R. Cahill

Platelets circulate in an activated state in inflammatory bowel disease

BACKGROUND/AIMS Platelets show proinflammatory as well as prothrombotic properties. Patients with inflammatory bowel disease are at increased risk of systemic thromboembolism, and multifocal microvascular infarction has been proposed as a pathogenetic mechanism in Crohns disease. The aim of this study was to determine if inflammatory bowel disease is associated with abnormal platelet behavior. METHODS Platelet activation and aggregability were assessed using flow cytometry, Born aggregometry, and the modified method of Wu and Hoak. Serum beta-thromboglobulin was measured in patients with Crohns disease and ulcerative colitis and, as controls, in healthy volunteers and patients with active rheumatoid arthritis. RESULTS Platelet surface expression of P-selectin and GP53 (markers of activation) were increased in Crohns disease (13 of 30 patients abnormal for P-selectin; 9 of 28 abnormal for GP53) (P < 0.01) and ulcerative colitis (9 of 21 for P-selectin; 10 of 21 for GP53) (P < 0.01) compared with healthy controls. Increased circulating platelet aggregates (15 of 24 patients with Crohns disease and 8 of 16 with ulcerative colitis) (P < 0.01), platelet aggregability in vitro, and serum beta-thromboglobulin were detected in active inflammatory bowel disease compared with healthy controls. Platelet behavior in active rheumatoid arthritis resembled that in healthy controls. CONCLUSIONS Increased platelet activation and aggregation are features of inflammatory bowel disease and may contribute to the risk of systemic thromboembolism and the pathogenesis of mucosal inflammation. Therefore, antiplatelet agents may be valuable in the management of inflammatory bowel disease.

Fixation with formaldehyde induces expression of activation dependent platelet membrane glycoproteins, P selectin (CD62) and GP53 (CD63)

Summary. Platelet activation in vivo is important in the pathogenesis of thrombosis. Accurate measurement is difficult due to artefactual in vitro preparation related activation. This can be overcome by using whole blood techniques, such as flow cytometry. However, there is little consensus on methods of platelet preparation, procedures for use of fixation, or the optimal types/amounts of fixative. The use of unfixed platelets has received little attention.

Fatal thrombocytopaenia temporally related to the administration of alemtuzumab (MabCampath) for refractory CLL despite early discontinuation of therapy.

Abstract Alemtuzumab (Campath 1H -MabCampath), initially used for prophylaxis of graft versus host disease in allogenic transplantaion, is now increasingly used for refractory chronic lymphatic leukaemia (CLL). Its efficacy has been well documented in this--the commonest form of leukaemia. Alemtuzumab is associated with severe immunosuppression, allergic reactions and thrombocytopenia. Data sheet and information supplied by the manufacturer confirm the rare occurrence of serious immune thrombocytopenia, recommending discontinuation of therapy when platelet counts fall below 50×10 9 /l. We report a patient with refractory CLL in which relentless progressive cytopenia occurred despite the discontinuation of alemtuzumab therapy while the platelet count was over 97×10 9 /l. Marrow biopsy showed increased megakaryocytes, the patient bleed uncontrollably and died of cerebral haemorrhage with a platelet count <10×10 9 /l. Data on the predictive factors underlying this complication are few and deserve further study as this drug is increasingly used the treatment of CLL.

A multi-centre retrospective study of rituximab use in the treatment of relapsed or resistant warm autoimmune haemolytic anaemia.

This retrospective analysis assessed the response, safety and duration of response to standard dose rituximab 375 mg/m2 weekly for four weeks as therapy for patients with primary or secondary warm autoimmune haemolytic anaemia (WAIHA), who had failed initial treatment. Thirty‐four patients received rituximab for WAIHA in seven centres in the Republic of Ireland. The overall response rate was 70·6% (24/34) with 26·5% (9/34) achieving a complete response (CR). The time to response was 1 month post‐initiation of rituximab in 87·5% (21/24) and 3 months in 12·5% (3/24) of patients. The median duration of follow‐up was 36 months (range 6–90 months). Of the patients who responded, 50% (12/24) relapsed during follow up with a median time to next treatment of 16·5 months (range 6–60 months). Three patients were re‐treated with rituximab 375 mg/m2 weekly for four weeks at relapse and responded. There was a single episode of neutropenic sepsis. Rituximab is an effective and safe treatment for WAIHA but a significant number of patients will relapse in the first two years post treatment. Re‐treatment was effective in a small number of patients, suggesting that intermittent pulse treatment or maintenance treatment may improve long‐term response.

Induction of autophagy by Imatinib sequesters Bcr-Abl in autophagosomes and down-regulates Bcr-Abl protein.

Chronic Myeloid Leukemia (CML) is a disease of hematopoietic stem cells which harbor the chimeric gene Bcr‐Abl. Expression levels of this constitutively active tyrosine kinase are critical for response to tyrosine kinase inhibitor treatment and also disease progression, yet the regulation of protein stability is poorly understood. We have previously demonstrated that imatinib can induce autophagy in Bcr‐Abl expressing cells. Autophagy has been associated with the clearance of large macromolecular signaling complexes and abnormal proteins, however, the contribution of autophagy to the turnover of Bcr‐Abl protein in imatinib treated cells is unknown. In this study, we show that following imatinib treatment, Bcr‐Abl is sequestered into vesicular structures that co‐localize with the autophagy marker LC3 or GABARAP. This association is inhibited by siRNA mediated knockdown of autophagy regulators (Beclin 1/ATG7). Pharmacological inhibition of autophagy also reduced Bcr‐Abl/LC3 co‐localization in both K562 and CML patient cells. Bcr‐Abl protein expression was reduced with imatinib treatment. Inhibition of both autophagy and proteasome activity in imatinib treated cells was required to restore Bcr‐Abl protein levels to those of untreated cells. This ability to down‐regulate Bcr‐Abl protein levels through the induction of autophagy may be an additional and important feature of the activity of imatinib. 88:455–462, 2013.

Adhesion Molecules in Clinical Medicine

Cellular adhesion molecules (CAMs) are critical components in the processes of embryogenesis, tissue repair and organization, lymphocyte function, lymphocyte homing and tumor metastasis, as well as being central to the interactions between hemopoietic progenitors and bone marrow microenvironment, and between leukocytes and platelets with vascular endothelium. Expression of CAMs regulates normal hemopoiesis and migration and function of mature hemopoietic cells. CAMs are an important part of the inflammatory response and may regulate cytokine synthesis. In addition, CAM expression may be critical for tumorigenesis. Monoclonal antibodies to CAMs have been developed for clinical use; initial results suggest that these agents have great potential in the prevention and treatment of inflammation, thrombosis, reperfusion injury, and graft rejection.

Retinoid receptor signaling and autophagy in acute promyelocytic leukemia.

Retinoids are a family of signaling molecules derived from vitamin A with well established roles in cellular differentiation. Physiologically active retinoids mediate transcriptional effects on cells through interactions with retinoic acid (RARs) and retinoid-X (RXR) receptors. Chromosomal translocations involving the RARα gene, which lead to impaired retinoid signaling, are implicated in acute promyelocytic leukemia (APL). All-trans-retinoic acid (ATRA), alone and in combination with arsenic trioxide (ATO), restores differentiation in APL cells and promotes degradation of the abnormal oncogenic fusion protein through several proteolytic mechanisms. RARα fusion-protein elimination is emerging as critical to obtaining sustained remission and long-term cure in APL. Autophagy is a degradative cellular pathway involved in protein turnover. Both ATRA and ATO also induce autophagy in APL cells. Enhancing autophagy may therefore be of therapeutic benefit in resistant APL and could broaden the application of differentiation therapy to other cancers. Here we discuss retinoid signaling in hematopoiesis, leukemogenesis, and APL treatment. We highlight autophagy as a potential important regulator in anti-leukemic strategies.

Platelet surface activation antigen expression at baseline and during elective angioplasty in patients with mild to moderate coronary artery disease.

Platelet activation is an important pre-thrombotic event. The elucidation of its pathophysiology could contribute to a reduction in the mortality associated with coronary artery disease-the foremost cause of death in the UK. We examined the platelets of 27 patients with angiographically documented coronary artery disease. All patients had stable angina and were taking their regular medication-including aspirin. We demonstrated significantly increased expression of GP53 and activated GPIIb/IIIa on the platelet surface using a sensitive flow cytometric method of detection. Comparison was made with a control group of 35 patients. Seventeen of the patients had coronary angioplasty carried out. Serial studies of these patients demonstrate an immediate and sustained increase in platelet activation and this has important implications for prevention of restenosis after angioplasty.

A novel 33-Gene targeted resequencing panel provides accurate, clinical-grade diagnosis and improves patient management for rare inherited anaemias.

Accurate diagnosis of rare inherited anaemias is challenging, requiring a series of complex and expensive laboratory tests. Targeted next‐generation‐sequencing (NGS) has been used to investigate these disorders, but the selection of genes on individual panels has been narrow and the validation strategies used have fallen short of the standards required for clinical use. Clinical‐grade validation of negative results requires the test to distinguish between lack of adequate sequencing reads at the locations of known mutations and a real absence of mutations. To achieve a clinically‐reliable diagnostic test and minimize false‐negative results we developed an open‐source tool (CoverMi) to accurately determine base‐coverage and the ‘discoverability’ of known mutations for every sample. We validated our 33‐gene panel using Sanger sequencing and microarray. Our panel demonstrated 100% specificity and 99·7% sensitivity. We then analysed 57 clinical samples: molecular diagnoses were made in 22/57 (38·6%), corresponding to 32 mutations of which 16 were new. In all cases, accurate molecular diagnosis had a positive impact on clinical management. Using a validated NGS‐based platform for routine molecular diagnosis of previously undiagnosed congenital anaemias is feasible in a clinical diagnostic setting, improves precise diagnosis and enhances management and counselling of the patient and their family.

Induction of autophagy is a key component of all-trans-retinoic acid-induced differentiation in leukemia cells and a potential target for pharmacologic modulation

Acute myeloid leukemia (AML) is characterized by the accumulation of immature blood cell precursors in the bone marrow. Pharmacologically overcoming the differentiation block in this condition is an attractive therapeutic avenue, which has achieved success only in a subtype of AML, acute promyelocytic leukemia (APL). Attempts to emulate this success in other AML subtypes have thus far been unsuccessful. Autophagy is a conserved protein degradation pathway with important roles in mammalian cell differentiation, particularly within the hematopoietic system. In the study described here, we investigated the functional importance of autophagy in APL cell differentiation. We found that autophagy is increased during all-trans-retinoic acid (ATRA)-induced granulocytic differentiation of the APL cell line NB4 and that this is associated with increased expression of LC3II and GATE-16 proteins involved in autophagosome formation. Autophagy inhibition, using either drugs (chloroquine/3-methyladenine) or short-hairpin RNA targeting the essential autophagy gene ATG7, attenuates myeloid differentiation. Importantly, we found that enhancing autophagy promotes ATRA-induced granulocytic differentiation of an ATRA-resistant derivative of the non-APL AML HL60 cell line (HL60-Diff-R). These data support the development of strategies to stimulate autophagy as a novel approach to promote differentiation in AML.