Gene expression and splicing alterations analyzed by high throughput RNA sequencing of chronic lymphocytic leukemia specimens.

BackgroundTo determine differentially expressed and spliced RNA transcripts in chronic lymphocytic leukemia specimens a high throughput RNA-sequencing (HTS RNA-seq) analysis was performed.MethodsTen CLL specimens and five normal peripheral blood CD19+ B cells were analyzed by HTS RNA-seq. The library preparation was performed with Illumina TrueSeq RNA kit and analyzed by Illumina HiSeq 2000 sequencing system.ResultsAn average of 48.5 million reads for B cells, and 50.6 million reads for CLL specimens were obtained with 10396 and 10448 assembled transcripts for normal B cells and primary CLL specimens respectively. With the Cuffdiff analysis, 2091 differentially expressed genes (DEG) between B cells and CLL specimens based on FPKM (fragments per kilobase of transcript per million reads and false discovery rate, FDR q < 0.05, fold change >2) were identified. Expression of selected DEGs (n = 32) with up regulated and down regulated expression in CLL from RNA-seq data were also analyzed by qRT-PCR in a test cohort of CLL specimens. Even though there was a variation in fold expression of DEG genes between RNA-seq and qRT-PCR; more than 90 % of analyzed genes were validated by qRT-PCR analysis. Analysis of RNA-seq data for splicing alterations in CLL and B cells was performed by Multivariate Analysis of Transcript Splicing (MATS analysis). Skipped exon was the most frequent splicing alteration in CLL specimens with 128 significant events (P-value <0.05, minimum inclusion level difference >0.1).ConclusionThe RNA-seq analysis of CLL specimens identifies novel DEG and alternatively spliced genes that are potential prognostic markers and therapeutic targets. High level of validation by qRT-PCR for a number of DEG genes supports the accuracy of this analysis. Global comparison of transcriptomes of B cells, IGVH non-mutated CLL (U-CLL) and mutated CLL specimens (M-CLL) with multidimensional scaling analysis was able to segregate CLL and B cell transcriptomes but the M-CLL and U-CLL transcriptomes were indistinguishable. The analysis of HTS RNA-seq data to identify alternative splicing events and other genetic abnormalities specific to CLL is an added advantage of RNA-seq that is not feasible with other genome wide analysis

The research on the immuno-modulatory defect of Mesenchymal Stem Cell from Chronic Myeloid Leukemia patients

Overwhelming evidence from leukemia research has shown that the clonal population of neoplastic cells exhibits marked heterogeneity with respect to proliferation and differentiation. There are rare stem cells within the leukemic population that possess extensive proliferation and self-renewal capacity not found in the majority of the leukemic cells. These leukemic stem cells are necessary and sufficient to maintain the leukemia. While the hematopoietic stem cell (HSC) origin of CML was first suggested over 30 years ago, recently CML-initiating cells beyond HSCs are also being investigated. We have previously isolated fetal liver kinase-1-positive (Flk1+) cells carrying the BCR/ABL fusion gene from the bone marrow of Philadelphia chromosome-positive (Ph+) patients with hemangioblast property. Here, we showed that CML patient-derived Flk1+CD31-CD34-MSCs had normal morphology, phenotype and karyotype but appeared impaired in immuno-modulatory function. The capacity of patient Flk1+CD31-CD34- MSCs to inhibit T lymphocyte activation and proliferation was impaired in vitro. CML patient-derived MSCs have impaired immuno-modulatory functions, suggesting that the dysregulation of hematopoiesis and immune response may originate from MSCs rather than HSCs. MSCs might be a potential target for developing efficacious cures for CML

Biological properties of the venom of the Papuan black snake (Pseudechis papuanus): presence of a phospholipase A2 platelet inhibitor.

The whole venom of Pseudechis papuanus, in addition to its anticoagulant activity, powerfully inhibited platelet aggregation induced by ADP, adrenaline, collagen, ristocetin and thrombin. High levels of phospholipase A2 (PLA2) activity were detected. A mild procoagulant activity was also observed. Following exposure of platelets to P. papuanus venom, platelet factor 3 (procoagulant platelet phospholipid) showed decreased cofactor activity in factor X activation by Russell's viper, venom suggesting that the venom PLA2 plays a major role in the inhibition of the coagulation mechanism. In vivo rodent assays confirmed the inhibitory effect on platelets and the haemorrhagic and neurotoxic activities. It is possible that PLA2 is responsible for anticoagulation and that this, combined with the effect on platelet aggregation, a mild procoagulant and a moderately potent haemorrhagin, is responsible for the haemorrhagic diathesis observed in systemically envenomed patients. Polyvalent (Australia-Papua New Guinea) Commonwealth Serum Laboratories antivenom, currently used for clinical treatment of snakebite in Papua New Guinea, proved highly effective against P. papuanus venom in rodent and in vitro assays, despite the absence of this particular venom from the immunising mixture

Coagulopathy and haemorrhage in human victims of Bothrops jararaca envenoming in Brazil.

Thirty-four patients envenomed by Bothrops jararaca in Brazil were studied. Of these, 20 (59%) had incoagulable blood associated with local and/or systemic bleeding and 10 of the 20 were thrombocytopenic. Among 14 patients with coagulable blood, 6 (43%) had bleeding symptoms and 3 (21%) were thrombocytopenic. High levels of von Willebrand factor (vWF), plasminogen activator inhibitor type 1 (PAI-1) and tissue type plasminogen activator (t-PA) antigens were also recorded in some patients with systemic bleeding with or without incoagulable blood. These substances may have been released from endothelial cells. Admission serum venom antigen levels were similar in both groups. The study indicated that systemic haemorrhage may occur in patients with coagulable blood and thrombocytopenia and that coagulopathy is not therefore the primary cause of haemorrhage

The inhibition of platelet aggregation and blood coagulation by Micropechis ikaheka venom.

Uncoagulable blood and life-threatening bleeding can result from the action of some snake venom toxins on haemostatic components of blood and vessel walls. Although envenoming by Micropechis ikaheka primarily affects neurones and muscle cells causing post-synaptic neuromuscular blockade and rhabdomyolysis, disturbances of haemostasis also occur. Therefore, the present study explored the effects of M. ikaheka venom on platelets and endothelium, which are important components of the haemostatic mechanism. The venom inhibited platelet aggregation in response to ADP and collagen, and also delayed clotting dependent on platelet activation or endothelial cell tissue factor expression. Some of these effects were reduced by the incubation of venom with a phospholipase A2 (PLA2) inhibitor and could be reproduced by a 17 kDa venom fraction containing a PLA2. In addition, an 11 kDa fraction containing a long-chain neurotoxin reduced ADP-induced aggregation. The venom was also found to reduce endothelial cell adherence to vitronectin-, fibronectin- and collagen-coated surfaces. These results suggest that, by inhibiting procoagulant activities of platelets and endothelial cells, a 17 kDa PLA2 plays an important role in the anticoagulant action of M. ikaheka venom

Reliability of the simple 20 minute whole blood clotting test (WBCT20) as an indicator of low plasma fibrinogen concentration in patients envenomed by Bothrops snakes. Butantan Institute Antivenom Study Group.

Reliability of the simple 20 minute whole blood clotting test (WBCT20) as an indicator of low plasma fibrinogen concentration in patients envenomed by Bothrops snakes. Toxicon 32, 1045-1050, 1994.--A simple whole blood clotting test (WBCT20) was assessed for its efficacy in determination of severe defibrinogenation in patients envenomed by Bothrops snakes in Brazil. There was a close relationship between the results of the WBCT20 and plasma fibrinogen levels in 69 moderately envenomed patients. The advantage of the WBCT20 over estimation of plasma fibrinogen concentrations in patients is that it is a simpler, faster and more reliable test. It is also of use in assessing the effectiveness of antivenom therapy in relation to the restoration of blood coagulability

Fatores associados à incoagulabilidade sangüínea no envenenamento por serpentes do gênero Bothrops Risk factors associated with coagulation abnormalities in Bothrops envenoming

Com o objetivo de conhecer fatores associados à incoagulabilidade sangüínea no envenenamento botrópico, foram obtidas informações de 2.991 prontuários médicos de pacientes atendidos no Instituto Butantan de 1981 a 1990. Associaram-se positivamente à incoagulabilidade sangüínea (p<0,05): picadas no final do ano e em extremidades dos membros inferiores; dor, edema e equimose local; hemorragia e choque; dose de antiveneno; tempo do acidente à chegada ao Instituto Butantan. Associaram-se negativamente à incoagulabilidade (p<0,05): tamanho de Bothrops jararaca; uso de torniquete; tempo entre a chegada ao Instituto Butantan e o início da administração do antiveneno. Não se associaram à incoagulabilidade (p>0,05): horário do acidente; presença de presa recém-deglutida no tubo digestivo da serpente; sexo e idade do paciente; ocorrência de bolha, necrose, abscesso e incisão local, amputação, insuficiência renal e óbito. Pode-se concluir que, embora a incoagulabilidade sangüínea apresente associação com manifestações precoces do envenenamento, não tem boa associação com a evolução clínica do paciente.<br>This study aimed at assessing, in the envenoming by Bothrops, factors that are associated with blood incoagulability. Information was obtained from the charts of 2,991 patients admitted to Instituto Butantan, from 1981 to 1990. Factors positively associated with blood incoagulability (p<0.05) were: snake bite in the late months of the year; bites in the distal segments of the lower limbs; pain, edema, and bruising at the bite site; systemic bleeding and shock; dose of antivenom administered; time between bite and admission to Instituto Butantan. Size of the snake Bothrops jararaca; use of a tourniquet; and time between arrival to Instituto Butantan and start of the antivenom administration were negatively associated with blood incoagulability (p<0,05). Factors not associated with blood incoagulability (p>0.05) were: time of the bite; presence of recently swallowed prey in the snake gut; gender and age of the patient; blister, necrosis, and abscess at the bite site; occurrence of amputation, renal failure and death; presence of an incision at the bite site. We conclude that although blood incoagulability is associated with early manifestations of Bothrops envenoming, it is not associated with the clinical outcome