Evaluation of Altona Diagnostics RealStar Zika Virus Reverse Transcription-PCR Test Kit for Zika Virus PCR Testing.

With the emerging Zika virus (ZIKV) epidemic, accessible real-time reverse transcription-PCR (rRT-PCR) assays are needed to streamline testing. The commercial Altona Diagnostics RealStar ZIKV rRT-PCR test kit (Altona PCR) has been approved for emergency use authorization by the U.S. FDA. Our aim was to verify the Altona PCR by comparing it to the CDC-designed dual-target ZIKV rRT-PCR reference assay (reference PCR) and describe the demographics of patients tested for ZIKV by rRT-PCR in Ontario, Canada. A large set of clinical specimens was tested for ZIKV by the Altona PCR and the reference PCR. Positive or equivocal specimens underwent PCR and Sanger sequencing targeting the ZIKV NS5 gene. A total of 671 serum specimens were tested by the reference PCR: 58 (8.6%) were positive, 193 (28.8%) were equivocal, and 420 (62.6%) were negative. Ninety percent of the reference PCR-positive patients were tested in the first 5 days after symptom onset. The Altona PCR was performed on 284/671 specimens tested by the reference PCR. The Altona PCR was positive for 53/58 (91%) reference PCR-positive specimens and 16/193 (8%) reference PCR-equivocal specimens; the ZIKV NS5 PCR was positive for all 68 Altona PCR-positive specimens and negative for all 181 Altona PCR-negative specimens that underwent the NS5 PCR. The Altona PCR has very good sensitivity (91%) and specificity (97%) compared to the reference PCR. The Altona PCR can be used for ZIKV diagnostic testing and has less extensive verification requirements than a laboratory-developed test.

The myxoma poxvirus protein, M11L, prevents apoptosis by direct interaction with the mitochondrial permeability transition pore.

M11L, an antiapoptotic protein essential for the virulence of the myxoma poxvirus, is targeted to mitochondria and prevents the loss of mitochondrial membrane potential that accompanies cell death. In this study we show, using a cross-linking approach, that M11L physically associates with the mitochondrial peripheral benzodiazepine receptor (PBR) component of the permeability transition (PT) pore. Close association of M11L and the PBR is also indicated by fluorescence resonance energy transfer (FRET) analysis. Stable expression of M11L prevents the release of mitochondrial cytochrome c induced by staurosporine or protoporphyrin IX (PPIX), a ligand of the PBR. Transiently expressed M11L also prevents mitochondrial membrane potential loss induced by PPIX, or induced by staurosporine in combination with PK11195, another ligand of the PBR. Myxoma virus infection and the associated expression of early proteins, including M11L, protects cells from staurosporine- and Fas-mediated mitochondrial membrane potential loss and this effect is augmented by the presence of PBR. We conclude that M11L regulates the mitochondrial permeability transition pore complex, most likely by direct modulation of the PBR.

Presence of Flavivirus Antibodies Does Not Lead to a Greater Number of Symptoms in a Small Cohort of Canadian Travelers Infected with Zika Virus.

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with a febrile illness as well as severe complications, including microcephaly and Guillain-Barré Syndrome. Antibody cross-reactivity between flaviviruses has been documented, and in regions where ZIKV is circulating, dengue virus (DENV) is also endemic, leaving the potential that previous exposure to DENV could alter clinical features of ZIKV infection. To investigate this, we performed a retrospective case-control study in which we compared Canadian travellers who had been infected with ZIKV and had serological findings indicating previous DENV or other flavivirus exposure (n = 16) to those without any previous exposure (n = 44). Patient samples were collected between February 2016 and September 2017 and submitted to Public Health Ontario for testing. ZIKV infection was determined using real-time RT-PCR and antibodies against DENV were identified by the plaque-reduction neutralization test. The mean time from symptom onset to sample collection was 5 days for both groups; the magnitude of viremia was not statistically different (Ct values: 35.6 vs. 34.9, p-value = 0.2). Clinical scores were also similar. Our findings indicate that previous DENV or other flavivirus exposure did not result in greater viremia or a higher illness score.

Draft Genome Sequences of Four Clinical Legionella pneumophila Isolates from Ontario, Canada.

Legionella pneumophila outbreak investigations require the development of reliable typing methods to better understand the genetic relationships of the isolates involved. Here, we report the draft genome sequences of four clinical Legionella pneumophila isolates obtained between 2000 and 2012 in Ontario, Canada.

Comparative evaluation of three JAK2V617F mutation detection methods.

The correlation of JAK2V617F with a proportion of chronic myeloproliferative disorders has generated numerous studies focused on the development of molecular-based assays for JAK2V617F detection. The current parallel study comparatively evaluated 3 JAK2V617F molecular detection methods. Genomic DNA from blood or bone marrow was assayed by 3 laboratories using allele-specific polymerase chain reaction (AS-PCR) or kit-based restriction fragment length polymorphism methods, which used polyacrylamide gel or capillary electrophoresis analysis. In addition, samples were sequenced in 2 of the laboratories. Results found 100% concordance among the 3 methods, with analytic sensitivities of 5% for both kit methods and 0.01% for AS-PCR. The kitbased assays detect JAK2V617F with equal sensitivity regardless of analysis method, and, despite greater sensitivity of AS-PCR, all 3 methods yielded 100% concordant results for this 36-sample set. Consistent with other reports, direct sequencing was insufficiently sensitive to serve as an initial diagnostic tool for JAK2V617F detection.

Transformation of polycythemia vera to chronic myelogenous leukemia.

Transformation of polycythemia vera to chronic myelogenous leukemia is a rare event. We report 2 women with long-standing polycythemia vera who developed chronic myelogenous leukemia. Both patients had no BCR/ABL1 fusion at the time of polycythemia vera diagnosis but were positive for the fusion at chronic myelogenous leukemia onset. Most patients with polycythemia vera have JAK2(V617F) mutation. Analysis of an archival bone marrow aspirate sample from 1 patient showed a heterozygous mutation status. The blood and bone marrow samples from the other patient showed the presence of homozygous JAK2(V617F) mutation and BCR/ABL1 fusion. The possible pathogenesis of such an event is discussed in the light of current literature.

JSI-124 (cucurbitacin I) inhibits Janus kinase-3/signal transducer and activator of transcription-3 signalling, downregulates nucleophosmin-anaplastic lymphoma kinase (ALK), and induces apoptosis in ALK-positive anaplastic large cell lymphoma cells.

JSI-124 (cucurbitacin I) has been recently described as a specific inhibitor of signal transducer and activator of transcription-3 (STAT3). As STAT3 activation is pathogenetically important in anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ ALCL), we investigated whether JSI-124 can mediate significant inhibitory effects in this cell type. In two ALK+ ALCL cell lines (Karpas 299 and SU-DHL-1), JSI-124 significantly reduced the number of viable cells to 50% of that of negative controls at a dose of 5-10 micromol/l at 24 h and 1-1.25 micromol/l at 48 h. This decrease in viability was associated with apoptosis, as confirmed by the increase in the subG(0/1) fraction, poly(ADP-ribose)polymerase cleavage and expression of active caspase 3. JSI-124 decreased the phosphorylated-STAT3 and -Janus kinase-3 (JAK3) levels in a dose-dependent fashion, and these changes were coupled with significant decreases in several STAT3 downstream targets, including mcl-1, bcl-2, bcl-xL and cyclin D3. Interestingly, JSI-124 also dramatically decreased the protein levels of JAK3 and nucleophosmin (NPM)-ALK, and these effects were reversible by MG132. Our data support that JSI-124 is a potentially useful therapeutic agent for ALK+ ALCL. In addition to its role as a tyrosine kinase inhibitor, JSI-124 appears to be involved in regulating proteosome degradation for proteins such as JAK3 and NPM-ALK.

Loss of SHP1 enhances JAK3/STAT3 signaling and decreases proteosome degradation of JAK3 and NPM-ALK in ALK+ anaplastic large-cell lymphoma.

Previous studies showed that most cases of ALK(+) anaplastic large-cell lymphoma (ALK(+)ALCL) do not express SHP1, a tyrosine phosphatase and an important negative regulator for cellular signaling pathways such as that of JAK/STAT. To fully assess the biologic significance of loss of SHP1 in ALK(+)ALCL, we transfected SHP1 plasmids into 2 SHP1(-), ALK(+)ALCL cell lines, Karpas 299 and SU-DHL-1. After 24 hours of transfection, pJAK3 and pSTAT3 were decreased, and these changes correlated with down-regulation of STAT3 downstream targets including cyclin D3, mcl-1, and bcl-2. Expression of SHP1 in these 2 cell lines also resulted in marked decreases in the protein levels of JAK3 and NPM-ALK, and these effects were reversible by proteosome inhibitor MG132. Conversely, when SHP1 expression in SUP-M2 (a SHP1(+) ALK(+)ALCL cell line) was inhibited using siRNA, pSTAT3, pJAK3, JAK3, and NPM-ALK were all up-regulated. Coimmunoprecipitation studies showed that SHP1 was physically associated with JAK3 and NPM-ALK. SHP1 expression in Karpas 299 and SU-DHL-1 led to significant G(1) cell cycle arrest but not apoptosis. To conclude, loss of SHP1 contributes to the pathogenesis of ALK(+)ALCL by 2 mechanisms: (1) it leaves the tyrosine phosphorylation and activation of JAK3/STAT3 unchecked and (2) it decreases proteosome degradation of JAK3 and NPM-ALK.

Signal transducers and activators of transcription-3 up-regulates tissue inhibitor of metalloproteinase-1 expression and decreases invasiveness of breast cancer.

Signal transducers and activators of transcription (STAT)-3 is an oncogenic protein that is constitutively activated in many human cancers, including 30 to 60% of primary breast cancer. The biological significance of STAT3 activation in breast cancer is not fully understood. We have previously shown that STAT3 up-regulates tissue inhibitors of metalloproteinase (TIMP)-1, a cytokine known to block metalloproteinases and decrease invasiveness in certain cancer cell types. We hypothesize that STAT3 activation may modulate tumor invasiveness of breast cancer by regulating TIMP1 expression. Using MCF-7 cells transfected with tetracycline-off STAT3C (constitutively active STAT3), we generated an in vitro system in which STAT3C levels can be tightly controlled in breast cancer cells. Increasing tetracycline levels gradually decreased STAT3C and TIMP1 in a dose-dependent manner, and down-regulation of these proteins led to a reciprocal decrease in invasiveness of these cells in Matrigel. Addition of a neutralizing anti-TIMP1 antibody increased invasiveness in the same experimental system. Using immunohistochemistry and 142 primary breast tumors, we found a significant association between the expression of the phosphorylated/active form of STAT3 (pSTAT3) and that of TIMP1. Importantly, STAT3 activation correlated significantly with a lower frequency of vascular and lymphatic invasion (P = 0.015 and P = 0.0002, respectively). Our data support the concept that STAT3 activation significantly modulates the biological and clinical behavior of breast cancer.

The granzyme B-serglycin complex from cytotoxic granules requires dynamin for endocytosis.

Cytotoxic T lymphocytes and natural killer cells destroy target cells via the directed exocytosis of lytic effector molecules such as perforin and granzymes. The mechanism by which these proteins enter targets is uncertain. There is ongoing debate over whether the most important endocytic mechanism is nonspecific or is dependent on the cation-independent mannose 6-phosphate receptor. This study tested whether granzyme B endocytosis is facilitated by dynamin, a key factor in many endocytic pathways. Uptake of and killing by the purified granzyme B molecule occurred by both dynamin-dependent and -independent mechanisms. However most importantly, serglycin-bound granzyme B in high-molecular-weight degranulate material from cytotoxic T lymphocytes predominantly followed a dynamin-dependent pathway to kill target cells. Similarly, killing by live cytotoxic T lymphocytes was attenuated by a defect in the dynamin endocytic pathway, and in particular, the pathways characteristically activated by granzyme B were affected. We therefore propose a model where degranulated serglycin-bound granzymes require dynamin for uptake.