Ponlapat Rojnuckarin

Molecular cloning, expression and characterization of albolamin: A type P-IIa snake venom metalloproteinase from green pit viper (Cryptelytrops albolabris)

Snake venom metalloproteinases (SVMPs) can damage vessel wall, degrade clotting factors, inhibit integrins and block platelet functions. Studying them not only gives us deeper insights in pathogenesis of snakebites, but also potentially yields novel therapeutic agents. Here, we discovered a clone of an RGD-containing SVMP from the green pit viper (Cryptelytrops albolabris) venom gland cDNA library. Sequence analysis revealed that it belonged to the P-IIa subclass of SVMP comprising signal peptide, prodomain, metalloproteinase and disintegrin. Compared with other P-II SVMPs, it contained 2 additional conserved cysteines that were predicted to prevent the release of disintegrin from the metalloproteinase domain in the mature protein. The N-terminal histidine-tagged construct of metalloproteinase and disintegrin domains of albolamin was inserted into the pPICZαA vector and expressed in Pichia pastoris. The recombinant protein molecular weight was approximately 35 kDa on Western blot probed with anti-polyhistidine antibody. The recombinant albolamin could digest human type IV collagen starting within 15 min after incubation. In addition, it dose-dependently inhibited collagen-induced platelet aggregation with the IC50 of 1.8 μM. However, there was no effect on ADP-induced platelet aggregation. Therefore, the inhibition mechanism is probably through blocking collagen receptor(s). Albolamin activities probably contributed to pathology of green pit viper bites. Its disintegrin domain deserves further studies for the potential to be a useful agent affecting platelet functions.

Snakebite-Induced Coagulopathy and Bleeding Disorders

Snake venoms target mainly neuromuscular and/or hemostatic systems. Each of them is a combination of several toxins. Therefore, coagulopathy is only a part of multi-systemic involvement from envenomation including muscular weakness, rhabdomyolysis, renal failure and hypotension. Kinetics studies reveal that viper venoms comprise long half-life components resulting in a delay onset and prolonged duration of bleeding in a subset of patients. On the other hand, elapid venoms are more rapidly cleared from the circulation showing faster recovery. Remarkably, snake venoms affect almost every component of hemostasis including vascular wall, platelets, coagulation factors, natural anticoagulants and fibrinolysis. They can be stimulatory or inhibitory through enzymatic or binding mechanisms. These effects can contribute to hemorrhagic, as well as thrombotic, manifestations of snakebites. The most prominent clinical syndrome is consumptive coagulopathy from the thrombin-like enzymes and/or coagulation factor activators in the venoms. In addition, anticoagulation syndrome, thromboembolism and thrombotic microangiopathy have been reported in victims of particular snake species. The key treatment of snakebites is antivenom that can promptly reverse coagulopathy in most situations.

Depleted nitric oxide and prostaglandin E2 levels are correlated with endothelial dysfunction in β-thalassemia/HbE patients

Mechanisms of vascular disorders in β-thalassemia/HbE patients remain poorly understood. In the present study, we aimed to determine the presence of endothelial dysfunction and its association with altered vascular mediators in this population. Forty-three β-thalassemia/HbE patients without clinically documented vascular symptoms and 43 age–sex-matched healthy controls were enrolled. Endothelial function was assessed using flow-mediated dilatation (FMD) before and after administration of nitroglycerine (NTG). β-Thalassemia/HbE patients showed a significant endothelial dysfunction using FMD. The percentage change in the brachial artery diameter before NTG was significantly lower in the thalassemia group compared to the control (5.0xa0±xa05.9 vs. 9.0xa0±xa04.0%, pxa0<xa00.01) while no significant differences after NTG (18.4xa0±xa08.3 vs. 17.8xa0±xa06.3%, pxa0=xa00.71). Plasma nitric oxide metabolites (NOx) and prostaglandin E2 (PGE2) levels were significantly decreased in β-thalassemia/HbE (117.2xa0±xa027.3 vs. 135.8xa0±xa011.3xa0µmol/L, pxa0<xa00.01) and (701.9xa0±xa0676.0 vs. 1374.7xa0±xa0716.5xa0pg/mL, pxa0<xa00.01), respectively, while a significant elevation in soluble thrombomodulin levels in β-thalassemia/HbE (3587.7xa0±xa01310.0 vs. 3093.9xa0±xa0583.8xa0pg/mL, pxa0=xa00.028). NOx and PGE2 levels were significantly correlated with FMD (rxa0=xa00.27, pxa0=xa00.025) and (rxa0=xa00.35, pxa0=xa00.003), respectively. These findings suggest roles for endothelial mediators and a new mechanism underlying endothelial dysfunction in β-thalassemia/HbE patients.

High frequency of BCL2 translocation in Thai patients with follicular lymphomas.

Follicular lymphoma is characterized by chromosomal translocation involvingBCL2 and immunoglobulin heavy chain genes (IgH). That the incidence of follicular lymphoma and the previously reported frequency ofBCL2 translocation are lower in Asians than in Caucasians implies a different molecular pathology. The study ofBCL2 rearrangement will yield deeper insights into the pathogenesis of follicular lymphomas and into clinical applications of molecular diagnosis for Asian follicular lymphoma patients.BCL2 /IgH translocation was analyzed in paraffin-embedded tissues from follicular lymphoma patients by using polymerase chain reaction (PCR) analysis of the major breakpoint region (MBR), the intermediate cluster region (ICR), and the minor cluster region. In addition, fluorescence in situ hybridization (FISH) analysis with split-signalBCL2 probes was performed. PCR analysis revealedBCL2 rearrangement in 12 (23.5%) of 51 cases (10 MBR and 2 ICR breakpoints).This frequency is lower than the frequencies reported from Western countries (40%–60%). DNA sequencing of the breakpoints revealed nucleotide insertions suggesting V(D)J recombination-mediated mechanisms. On the other hand, FISH analysis revealed 11 (84.6%) of 13 cases with positive signals forBCL2 translocation. Our results suggest thatBCL2 translocation is essential for the pathogenesis of follicular lymphoma in Thai patients. In addition, the data demonstrate the low sensitivity of the PCR for diagnostic testing and suggest that split-signal FISH is the method of choice.

Balancing relapses versus cognitive impairment in primary central nervous system lymphoma: a single-center experience

ABSTRACT Objectives: The outcomes of primary central nervous system lymphoma (PCNSL) are much improved with multi-modality regimens. Unfortunately, in limited-resource countries, chemo-radiotherapy is the only option of curative-intent treatment. This study aimed to evaluate the effects of low-dose whole brain radiotherapy (WBRT) as a consolidation on disease control and long-term neurocognitive functions. Methods: We conducted a retrospective single-center study enrolling PCNSL patients from 2011 to May 2016 to evaluate the real-life treatment outcome and neurotoxicity from treatment especially radiotherapy. Results: Thirty-seven newly diagnosed immunocompetent PCNSL patients were treated with a high-dose methotrexate-based regimen with or without WBRT. The median age was 56 (range 16–78) years old. After chemotherapy, the overall response and complete response (CR) rates were 59.5% and 43.2%, respectively. All 6 partial response (PR) patients and 6 of 16 CR patients underwent radiotherapy. In 22 patients who achieved CR, the progression-free survival (PFS) of patients without WBRT was significantly inferior to the WBRT group with the hazard ratio of 4.7 (95% confidence interval 1.14–19.82, pu2009=u20090.03). The 3-year PFS were 35% and 78.75%, respectively, but there was no difference in overall survival. The serial Montreal Cognitive Assessment evaluations (20–72 months post chemotherapy) of 10 long-term CR patients revealed one dementia among three patients without WBRT and five mild cognitive impairments in seven patients with WBRT. Except for the dementia case, all the other patients can perform daily activities without assistance. Conclusion: The low-dose WBRT consolidation is associated with lower PCNSL relapses with only mild neurocognitive toxicity.

The first validated criteria for effective screening and a new simplified method for α-globin gene sequencing for diagnosis of uncommon α-globin mutations

No well-defined phenotypes that distinguish between unknown α- and β-globin mutations have been reported to date. Direct DNA sequencing of α-globin genes can be technically challenging, as α1- and α2-globin genes are nearly indistinguishable. To detect hemoglobin variants (HbXs) on Hb analysis, the entire β- and α-globin genes were directly sequenced using a newly developed sequencing protocol for α-globin genes. An algorithm to distinguish between α- and β-HbXs was constructed and subsequently validated in the independent validation group. Distinctive characteristics that can distinguish 39 α-HbXs from 24 β-HbXs were the presence of unidentifiable variants of HbA2 and/or HbX of <37% on isoelectric focusing and <31% on high-performance liquid chromatography. Another set of 67 HbXs was employed to validate our algorithm. This accurately predicted 33 α-HbXs with 100% sensitivity and 97.1% specificity. Our sequencing protocol for α-globin genes was able to identify 11 rare mutations among all exons of both α-globin genes from 72 subjects. Six of these variants were first discovered in Thais. This is the first well-characterized algorithm for distinguishing unknown Hb variants in a large cohort. Our validated criteria and DNA sequencing procedure are highly efficient for molecular characterization of rare Hb mutations.