The pursuit of mechanistic insights underpinning calcium-dependent inactivation of L-type calcium channels: All roads lead to calmodulin

L-type calcium channels (LTCCs) are critical conduits for Ca2+ entry into many excitable cells. In ventricular myocytes, they are responsible for shaping the action potential and triggering Ca2+ release from the sarcoplasmic reticulum leading to myocyte contraction. In the brain, these channels are vital for excitation-transcription coupling, synaptic plasticity, and neuronal firing. To perform their functions properly LTCCs employed two principal forms of feedback regulation which include voltage-dependent inactivation (VDI) and Ca2+-dependent inactivation (CDI). Disruptions in either of these two processes were linked to multiple disorders. For example, mutations in a Ca2+ sensor calmodulin (CaM), a known mediator of CDI, have been associated with long-QT syndrome (LQTS). Patients afflicted with these mutations (calmodulinopathies) suffer from life-threatening arrhythmias, refractory to conventional treatments. First, we dissected the mechanistic underpinnings of the LQTS form of calmodulinopathies. We found that disruption of CDI of CaV1.2 was a major culprit behind this disease. Leveraging off this knowledge, we created a customizable therapy for patients suffering from this group of diseases. Moreover, we investigated mechanisms underlying an autism-associated mutation A760G in CaV1.3 channels. Interestingly, this mutation disrupts both VDI and CDI of CaV1.3 which enables us to explore the intimate interplay between VDI and CDI. The knowledge gained from studying these two diseases extend beyond facilitation of therapy development and prove to be valuable in further our understanding in LTCC regulation under a physiologic state. Next, we utilized an in silico approach to complement our in vitro disease-based models of LTCC regulations. Although it is well known that each lobe of CaM is capable of responding spatially distinct Ca2+ sources, most CDI models fail to capture this unique property of CaM. Therefore, we developed the first kinetic model of CDI which truly captured the bi-lobal nature of CaM. From this model, we were able to gain deeper insight into the extent of interaction between the two lobes of CaM and explained the pathophysiology behind the LTQS form of calmodulinopathies. Once incorporated into a whole-cell or tissue level model, this novel CDI model could be an invaluable asset in understanding of both physiological and pathological states of the overall cardiac and neuronal electrical activities. Lastly, we developed an animal model as a new tool to investigate regulations of cardiac LTCCs in situ. Understanding of these channels’ modulation with high fidelity relies on examining LTCCs in their native environment with intact interacting proteins. Thus, such studies would benefit from genetic manipulation of endogenous LTCCs and their binding partners which often proves cumbersome in mammalian models. We have identified Drosophila as an alternative model to study LTCC regulation. Not only is Drosophila genetically pliable, but it also possesses conserved Ca2+ channels in its cardiomyocytes. Thus, this model may serve as a robust and effective platform for studying in situ LTCC regulation. In all, we utilized multiple approaches to dissect the mechanistic underpinnings of VDI and CDI modulation of LTCCs. We wish that this knowledge could help propel further scientific understanding of these channels’ regulations and brought hope to those suffering from dysregulations of these processes

Invariant NKT Cell Response to Dengue Virus Infection in Human

BACKGROUND:Dengue viral infection is a global health threat without vaccine or specific treatment. The clinical outcome varies from asymptomatic, mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF). While adaptive immune responses were found to be detrimental in the dengue pathogenesis, the roles of earlier innate events remain largely uninvestigated. Invariant natural killer T (iNKT) cells represent innate-like T cells that could dictate subsequent adaptive response but their role in human dengue virus infection is not known. We hypothesized that iNKT cells play a role in human dengue infection. METHODS:Blood samples from a well-characterized cohort of children with DF, DHF, in comparison to non-dengue febrile illness (OFI) and healthy controls at various time points were studied. iNKT cells activation were analyzed by the expression of CD69 by flow cytometry. Their cytokine production was then analyzed after α-GalCer stimulation. Further, the CD1d expression on monocytes, and CD69 expression on conventional T cells were measured. RESULTS:iNKT cells were activated during acute dengue infection. The level of iNKT cell activation associates with the disease severity. Furthermore, these iNKT cells had altered functional response to subsequent ex vivo stimulation with α-GalCer. Moreover, during acute dengue infection, monocytic CD1d expression was also upregulated and conventional T cells also became activated. CONCLUSION:iNKT cells might play an early and critical role in the pathogenesis of severe dengue viral infection in human. Targeting iNKT cells and CD1d serve as a potential therapeutic strategy for severe dengue infection in the future

Smartphone multiplex microcapillary diagnostics using Cygnus: development and evaluation of rapid serotype-specific NS1 detection with dengue patient samples

Laboratory diagnosis of dengue virus (DENV) infection including DENV serotyping requires skilled labor and well-equipped settings. DENV NS1 lateral flow rapid test (LFT) provides simplicity but lacks ability to identify serotype. A simple, economical, point-of-care device for serotyping is still needed. We present a gravity driven, smartphone compatible, microfluidic device using microcapillary film (MCF) to perform multiplex serotype-specific immunoassay detection of dengue virus NS1. A novel device–termed Cygnus–with a stackable design allows analysis of 1 to 12 samples in parallel in 40 minutes. A sandwich enzyme immunoassay was developed to specifically detect NS1 of all four DENV serotypes in one 60-μl plasma sample. This test aims to bridge the gap between rapid LFT and laboratory microplate ELISAs in terms of sensitivity, usability, accessibility and speed. The Cygnus NS1 assay was evaluated with retrospective undiluted plasma samples from 205 DENV infected patients alongside 50 febrile illness negative controls. Against the gold standard RT-PCR, clinical sensitivity for Cygnus was 82% in overall (with 78, 78, 80 and 76% for DENV1-4, respectively), comparable to an in-house serotyping NS1 microplate ELISA (82% vs 83%) but superior to commercial NS1-LFT (82% vs 74%). Specificity of the Cygnus device was 86%, lower than that of NS1-microplate ELISA and NS1-LFT (100% and 98%, respectively). For Cygnus positive samples, identification of DENV serotypes DENV2-4 matched those by RT-PCR by 100%, but for DENV1 capillaries false positives were seen, suggesting an improved DENV1 capture antibody is needed to increase specificity. Overall performance of Cygnus showed substantial agreement to NS1-microplate ELISA (κ = 0.68, 95%CI 0.58–0.77) and NS1-LFT (κ = 0.71, 95%CI 0.63–0.80). Although further refinement for DENV-1 NS1 detection is needed, the advantages of multiplexing and rapid processing time, this Cygnus device could deliver point-of-care NS1 antigen testing including serotyping for timely DENV diagnosis for epidemic surveillance and outbreak prediction

Germline bias dictates cross-serotype reactivity in a common dengue-virus-specific CD8(+) T cell response.

Adaptive immune responses protect against infection with dengue virus (DENV), yet cross-reactivity with distinct serotypes can precipitate life-threatening clinical disease. We found that clonotypes expressing the T cell antigen receptor (TCR) β-chain variable region 11 (TRBV11-2) were 'preferentially' activated and mobilized within immunodominant human-leukocyte-antigen-(HLA)-A*11:01-restricted CD8(+) T cell populations specific for variants of the nonstructural protein epitope NS3133 that characterize the serotypes DENV1, DENV3 and DENV4. In contrast, the NS3133-DENV2-specific repertoire was largely devoid of such TCRs. Structural analysis of a representative TRBV11-2(+) TCR demonstrated that cross-serotype reactivity was governed by unique interplay between the variable antigenic determinant and germline-encoded residues in the second β-chain complementarity-determining region (CDR2β). Extensive mutagenesis studies of three distinct TRBV11-2(+) TCRs further confirmed that antigen recognition was dependent on key contacts between the serotype-defined peptide and discrete residues in the CDR2β loop. Collectively, these data reveal an innate-like mode of epitope recognition with potential implications for the outcome of sequential exposure to heterologous DENVs

Costs and effectiveness of ciprofloxacin and ceftriaxone in treatment of typhoid fever in children in Thailand.

The burden of typhoid fever remains high in impoverished settings, and increasing antibiotic resistance is making treatment costly. The purposes of this study were: to compare the costs and the effectiveness of typhoid programs between oral and injection treatments in pediatric patients at Songkhla Hospital

Complement alternative pathway genetic variation and Dengue infection in the Thai population

Dengue disease is a mosquito-borne infection caused by Dengue virus. Infection may be asymptomatic or variably manifest as mild Dengue fever (DF) to the most severe form, Dengue haemorrhagic fever (DHF). Mechanisms that influence disease severity are not understood. Complement, an integral component of the immune system, is activated during Dengue infection and the degree of activation increases with disease severity. Activation of the complement alternative pathway is influenced by polymorphisms within activation (factor B rs12614/rs641153, C3 rs2230199) and regulatory [complement factor H (CFH) rs800292] proteins, collectively termed a complotype. Here, we tested the hypothesis that the complotype influences disease severity during secondary Dengue infection. In addition to the complotype, we also assessed two other disease-associated CFH polymorphisms (rs1061170, rs3753394) and a structural polymorphism within the CFH protein family. We did not detect any significant association between the examined polymorphisms and Dengue infection severity in the Thai population. However, the minor allele frequencies of the factor B and C3 polymorphisms were less than 10%, so our study was not sufficiently powered to detect an association at these loci. We were also unable to detect a direct interaction between CFH and Dengue NS1 using both recombinant NS1 and DV2-infected culture supernatants. We conclude that the complotype does not influence secondary Dengue infection severity in the Thai population

Typhoid Outbreak in Songkhla, Thailand 2009–2011: Clinical Outcomes, Susceptibility Patterns, and Reliability of Serology Tests

<div><p>Objective</p><p>To determine the clinical manifestations and outcomes, the reliability of <i>Salmonella enterica</i> serotype Typhi (<i>S</i> ser. Typhi) IgM and IgG rapid tests, and the susceptibility patterns and the response to treatment during the 2009–2011 typhoid outbreak in Songkhla province in Thailand.</p><p>Method</p><p>The medical records of children aged <15 years with <i>S</i> ser. Typhi bacteremia were analysed. The efficacy of the typhoid IgM and IgG rapid tests and susceptibility of the <i>S</i> ser. Typhi to the current main antibiotics used for typhoid (amoxicillin, ampicillin, cefotaxime, ceftriaxone, co-trimoxazole, and ciprofloxacin), were evaluated.</p><p>Results</p><p><i>S</i> ser. Typhi bacteremia was found in 368 patients, and all isolated strains were susceptible to all 6 antimicrobials tested. Most of the patients were treated with ciprofloxacin for 7–14 days. The median time (IQR) of fever before treatment and duration of fever after treatment were 5 (4, 7) days and 4 (3, 5) days, respectively. Complications of ascites, lower respiratory symptoms, anemia (Hct <30%), and ileal perforation were found in 7, 7, 22, and 1 patients, respectively. None of the patients had recurrent infection or died. The sensitivities of the typhoid IgM and IgG tests were 58.3% and 25.6% respectively, and specificities were 74.1% and 50.5%, respectively.</p><p>Conclusion</p><p>Most of the patients were diagnosed at an early stage and treated with a good outcome. All <i>S</i> ser. Typhi strains were susceptible to standard first line antibiotic typhoid treatment. The typhoid IgM and IgG rapid tests had low sensitivity and moderate specificity.</p></div