JAK2V617F-positive endothelial cells contribute to clotting abnormalities in myeloproliferative neoplasms

The Janus kinase 2 (JAK2) V₆₁₇F mutation is the primary pathogenic mutation in patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Although thrombohemorrhagic incidents are the most common causes of morbidity and mortality in patients with MPNs, the events causing these clotting abnormalities remain unclear. To identify the cells responsible for the dysfunctional hemostasis, we used transgenic mice expressing JAK2V₆₁₇F in specific lineages involved in thrombosis and hemostasis. When JAK2V₆₁₇F was expressed in both hematopoietic and endothelial cells (ECs), the mice developed a significant MPN, characterized by thrombocytosis, neutrophilia, and splenomegaly. However, despite having significantly higher platelet counts than controls, these mice showed severely attenuated thrombosis following injury. Interestingly, platelet activation and aggregation in response to agonists was unaltered by JAK2V₆₁₇F expression. Subsequent bone marrow transplants revealed the contribution of both endothelial and hematopoietic compartments to the attenuated thrombosis. Furthermore, we identified a potential mechanism for this phenotype through JAK2V₆₁₇F-regulated inhibition of von Willebrand factor (VWF) function and/or secretion. JAK2V₆₁₇F⁺ mice display a condition similar to acquired von Willebrand syndrome, exhibiting significantly less high molecular weight VWF and reduced agglutination to ristocetin. These findings greatly advance our understanding of thrombohemorrhagic events in MPNs and highlight the critical role of ECs in the pathology of hematopoietic malignancies.Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders, characterized by significant increases in one or more myeloid-cell lineages. Mutations in the Janus kinase 2 (JAK2) and MPL genes are common in the majority of Philadelphia chromosome-negative (Ph⁻) MPNs, which include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). By far the most frequent mutation in MPNs is JAK2V₆₁₇F (1–4), which occurs in the highly conserved autoinhibitory JAK homology (JH) 2 domain, causing hyperactive kinase activity and hyperproliferation of myeloid progenitor cells, leading to overproduction of red blood cells (RBCs), platelets, and leukocytes. Although ET and PV have a relatively benign clinical course, patients’ life expectancy can be severely reduced by bleeding or thrombosis, the manifestations of which are significantly more common than other MPN-related complications such as myelofibrosis and acute leukemic transformation (5). The frequency and nature of thrombotic and hemorrhagic events vary greatly depending on disease phenotype and patient history. Data taken from a number of previous studies indicate that the probability of major thrombosis ranges between 8–29% (ET) and 11–39% (PV) whereas the incidence of bleeding at initial presentation is less frequent than thrombosis, ranging between 3–18% (ET) and 3–8% (PV) (6–8).A number of abnormalities that could potentially contribute to this prothrombotic phenotype have been identified in the blood and vascular cells of JAK2V₆₁₇F⁺ MPN patients. Much work has focused on defining platelet abnormalities, including increased expression of membrane proteins such as P-selectin and tissue factor (TF), which would prime platelets for activation and increase levels of platelet-activation markers and platelet factor 4 (PF4) in the plasma (9–12). Interestingly, however, aggregation studies show a decreased response to ADP and epinephrine in platelets isolated from patients with ET and PV compared with controls (10). Furthermore, no correlation has been made between severity of thrombocytosis in ET patients and increased risk of thrombosis (6, 13). In contrast, extreme thrombocytosis (platelets >1,500 × 10⁹/L) is thought to contribute to a hemorrhagic phenotype in ET patients, and is commonly attributed to the development of acquired von Willebrand syndrome (AVWS) (11, 12, 14), where the increased platelets bind to highly prothrombotic, ultralarge von Willebrand factor (VWF) multimers, removing them from the plasma (15).Recent studies suggest that leukocytosis is a potential thrombotic risk factor in young PV and ET patients, possibly through the interactions of leukocytes, especially neutrophils, with platelets and endothelial cells (ECs) (16, 17) or the production of prothrombotic molecules such as TF. Increased basal activation of neutrophils has been shown in PV and ET patients, including elevated expression of CD11b and levels of neutrophil proteases in the plasma, both of which are prothrombotic (9, 18, 19). Studies have also shown increased activation of vascular ECs in JAK2V₆₁₇F⁺ MPN patients. Increased P- and E-selectin levels in the plasma, coupled with decreased levels of nitric oxide (NO), could conceivably contribute to a prothrombotic phenotype. Furthermore, JAK2V₆₁₇F⁺ ECs have recently been reported in a subpopulation of MPN patients, and EC expression was coupled with an increased risk of thrombosis (20, 21). Taken together, previous studies describe physiological abnormalities in a number of cell types in JAK2V₆₁₇F⁺ MPN patients, all of which could contribute toward increased thrombosis and/or bleeding. However, these data are often contradictory and fail to definitively explain the mechanism/s responsible for the development of thrombohemorrhagic disease.Here, we used FF1 transgenic mice (22) to express human JAK2V₆₁₇F in specific lineages to determine which cells are responsible for the thrombohemorrhagic manifestations seen in patients with MPNs. FF1 mice were crossed with Pf4-Cre or Tie2-Cre mice to express JAK2V₆₁₇F specifically in platelets alone, or in hematopoietic cells and ECs, respectively (23–28). These models have provided us with an unparalleled opportunity to determine the specific role/s of JAK2V₆₁₇F in pathological thrombosis and hemostasis.     

VAC Therapy in Large Infected Sacral Pressure Ulcer Grade IV—Can Be an Alternative to Flap Reconstruction?

Vacuum-assisted closure (VAC) therapy is a new entrant in wound care after growth factors and alginate or hydrocolloid dressing, in the treatment of pressure ulcers. We have been using this technique for diabetic foot ulcers. A young nondiabetic man presented with a large sacral bed sore after high doses of ionotropes in an intensive care unit for treating severe hypotension. His wound was debrided, and instead of flap surgery in such infected wound, he was treated with VAC therapy. The complete wound healing was achieved in 6 weeks and at half the cost of flap surgery. Moreover, the chances of flap failure and its related complications were eliminated.     

Increasing access to renal transplantation in India through our single‐center kidney paired donation program: a model for the developing world to prevent commercial transplantation

Because access to transplantation with HLA‐desensitization protocols and ABO incompatible transplantation is very limited due to high costs and increased risk of infections from more intense immunosuppression, kidney paired donation (KPD) promises hope to a growing number of end‐stage renal disease (ESRD) patient in India. We present a government and institutional ethical review board approved study of 56 ESRD patients [25 two‐way and 2 three‐way pairs] who consented to participate in KPD transplantation at our center in 2013, performed to avoid blood group incompatibility (n = 52) or positive cross‐match (n = 4). All patients had anatomic, functional, and immunologically comparable donors. The waiting time in KPD was short as compared to deceased donor transplantation. Laparoscopic donor nephrectomy was performed in 54 donors. Donor relationships were spousal (n = 40), parental (n = 13), others (n = 3), with median HLA match of 1. Graft survival was 97.5%. Three patients died with functioning graft. 16% had biopsy‐proven acute rejection. Mean serum creatinine was 1.2 mg/dl at 0.73 ± 0.32 months follow‐up. KPD is a viable, legal, and rapidly growing modality for facilitating LDRT for patients who are incompatible with their healthy, willing living donor. To our knowledge, this is the largest single‐center report from India.     

An optimized design of seizure detection system using joint feature extraction of multichannel EEG signals

The detection of seizure onset and events using electroencephalogram (EEG) signals are important tasks in epilepsy research. The literature available on seizure detection has discussed the implementation of advanced signal processing algorithms using tools accessed over the cloud. However, seizure monitoring application needs near sensor processing due to privacy and latency issues. In this paper, a real time seizure detection system has been implemented using an embedded system. The proposed system is based on ensemble empirical mode decomposition (EEMD) and tunable-Q wavelet transform (TQWT) algorithms. The analysis and classification of non-stationary EEG signals require the wavelet transform with high Q-factor. However, direct use of TQWT increases the computational complexity of feature extraction from multivariate EEG signals. In this paper, the first step is to process the signal by using EEMD to obtain 8 intrinsic mode functions (IMFs). The Kraskov (KraEn), sample (SampEn), and permutation (PermEn) entropy features of IMFs are extracted and based on optimum values, and 4 IMFs are decomposed using TQWT. Secondly, centered correntropy (CenCorrEn) features of the 1^st and 16^th sub-band of TQWT have been used as classifier inputs. The performance of multilayer perceptron neural networks (MLPNN), least squares support vector machine (LSSVM), and random forest (RF) classifiers has been tested on the multichannel EEG data recorded from a local hospital. The RF classifier has produced the highest accuracy of 96.2% in classifying the signals. The proposed scheme has been employed in developing an embedded seizure detection system to assist neurologists in making seizure diagnostic decisions.     

Evaluation of Efficacy of Microwave Irradiation in Disinfecting Dental Gypsum Casts: An Ex Vivo Study

The aim of this study is to assess the efficacy of microwave irradiation in disinfecting gypsum casts and also to compare its efficacy with validated method of chemical disinfection. The present study is an ex vivo study conducted on a sample of five irreversible hydrocolloid impressions in vitro and on ten patients gypsum casts in vivo following standard impression techniques to check the efficacy of microwave oven irradiation and compare its efficacy with standard chemical method of disinfection. Results were analysed using Mann–Whitney test and Wilcoxon signed rank test. Untreated gypsum casts showed cfu/ml counts with a median log value of 6, while microwave-irradiated ones had median cfu/ml counts of 0. Casts poured from chemically disinfected impressions demonstrated cfu/ml counts with a median log value of 5. Microwave irradiation was found to be effective in disinfecting gypsum casts when compared to chemical disinfectant in disinfecting dental impressions.