ZikaPLAN: addressing the knowledge gaps and working towards a research preparedness network in the Americas.

Zika Preparedness Latin American Network (ZikaPLAN) is a research consortium funded by the European Commission to address the research gaps in combating Zika and to establish a sustainable network with research capacity building in the Americas. Here we present a report on ZikaPLAN`s mid-term achievements since its initiation in October 2016 to June 2019, illustrating the research objectives of the 15 work packages ranging from virology, diagnostics, entomology and vector control, modelling to clinical cohort studies in pregnant women and neonates, as well as studies on the neurological complications of Zika infections in adolescents and adults. For example, the Neuroviruses Emerging in the Americas Study (NEAS) has set up more than 10 clinical sites in Colombia. Through the Butantan Phase 3 dengue vaccine trial, we have access to samples of 17,000 subjects in 14 different geographic locations in Brazil. To address the lack of access to clinical samples for diagnostic evaluation, ZikaPLAN set up a network of quality sites with access to well-characterized clinical specimens and capacity for independent evaluations. The International Committee for Congenital Anomaly Surveillance Tools was formed with global representation from regional networks conducting birth defects surveillance. We have collated a comprehensive inventory of resources and tools for birth defects surveillance, and developed an App for low resource regions facilitating the coding and description of all major externally visible congenital anomalies including congenital Zika syndrome. Research Capacity Network (REDe) is a shared and open resource centre where researchers and health workers can access tools, resources and support, enabling better and more research in the region. Addressing the gap in research capacity in LMICs is pivotal in ensuring broad-based systems to be prepared for the next outbreak. Our shared and open research space through REDe will be used to maximize the transfer of research into practice by summarizing the research output and by hosting the tools, resources, guidance and recommendations generated by these studies. Leveraging on the research from this consortium, we are working towards a research preparedness network

Evolving trends in the management of acute appendicitis during COVID-19 waves. The ACIE appy II study

Background: In 2020, ACIE Appy study showed that COVID-19 pandemic heavily affected the management of patients with acute appendicitis (AA) worldwide, with an increased rate of non-operative management (NOM) strategies and a trend toward open surgery due to concern of virus transmission by laparoscopy and controversial recommendations on this issue. The aim of this study was to survey again the same group of surgeons to assess if any difference in management attitudes of AA had occurred in the later stages of the outbreak. Methods: From August 15 to September 30, 2021, an online questionnaire was sent to all 709 participants of the ACIE Appy study. The questionnaire included questions on personal protective equipment (PPE), local policies and screening for SARS-CoV-2 infection, NOM, surgical approach and disease presentations in 2021. The results were compared with the results from the previous study. Results: A total of 476 answers were collected (response rate 67.1%). Screening policies were significatively improved with most patients screened regardless of symptoms (89.5% vs. 37.4%) with PCR and antigenic test as the preferred test (74.1% vs. 26.3%). More patients tested positive before surgery and commercial systems were the preferred ones to filter smoke plumes during laparoscopy. Laparoscopic appendicectomy was the first option in the treatment of AA, with a declined use of NOM. Conclusion: Management of AA has improved in the last waves of pandemic. Increased evidence regarding SARS-COV-2 infection along with a timely healthcare systems response has been translated into tailored attitudes and a better care for patients with AA worldwide

GBS after the Zika-outbreak in Colombia: the newcomer is gone but the problem continues! (p2.6-039).

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Granzyme B-induced neurotoxicity is mediated via activation of PAR-1 receptor and Kv1.3 channel.

Increasing evidence supports a critical role of T cells in neurodegeneration associated with acute and subacute brain inflammatory disorders. Granzyme B (GrB), released by activated T cells, is a cytotoxic proteinase which may induce perforin-independent neurotoxicity. Here, we studied the mechanism of perforin-independent GrB toxicity by treating primary cultured human neuronal cells with recombinant GrB. GrBactivated the protease-activated receptor (PAR)-1 receptor on the neuronal cell surface leading to decreased intracellular cyclic AMP levels. This was followed by increased expression and translocation of the voltage gated potassium channel, Kv1.3 to the neuronal cell membrane. Similar expression of Kv1.3 was also seen in neurons of the cerebral cortex adjacent to active inflammatory lesions in patients with multiple sclerosis. Kv1.3 expression was followed by activation of Notch-1 resulting in neurotoxicity. Blocking PAR-1, Kv1.3 or Notch-1 activation using specific pharmacological inhibitors or siRNAs prevented GrB-induced neurotoxicity. Furthermore, clofazimine protected against GrB-induced neurotoxicity in rat hippocampus, in vivo. These observations indicate that GrB released from T cells induced neurotoxicity by interacting with the membrane bound Gi-coupled PAR-1 receptor and subsequently activated Kv1.3 and Notch-1. These pathways provide novel targets to treat T cell-mediated neuroinflammatory disorders. Kv1.3 is of particular interest since it is expressed on the cell surface, only under pathological circumstances, and early in the cascade of events making it an attractive therapeutic target

Clofazimine protected against GrB toxicity in hippocampal neurons <i>in vivo</i>.

<p>Eight-week-old female Sprague-Dawley rats were stereotaxically injected in the hippocampal dentate gyrus (DG). Group I (n = 5) received GrB injection in the DG and clofazimine (50 µg/kg/day) intraperitoneally for ten days (three days before and seven days after the GrB injection). Group II (n = 4) received GrB (1 µg/µl) injection in the DG and vehicle by intraperitoneal injection. Group III (n = 4) received control solution both by DG injection and intraperitoneally. Brain sections were then immunostained for DCX-positive cells (red) in rat DG. DCX-positive cells were then counted and presented as average ± SEM.</p

GrB activates Kv1.3 channel in neurons.

<p>(A) Human fetal neurons were treated with GrB (4 nM) for 24 hr. Cells were then fixed and immunostained for Kv1.3 and beta-III tubulin and analyzed by confocal microscopy. Representative photomicrographs from three independent experiments with similar results are shown. (B) Human fetal neurons were pretreated with cycloheximide (CHX, 100 µg/ml) or actinomycin D (Act D, 10 µM) for 30 min prior to GrB (4 nM) treatment. 24 hr later, cells were fixed and immunostained for Kv1.3 and analyzed by confocal microscopy. Representative photomicrographs from three independent experiments with similar results are shown. (C) Primary human neuronal cultures were first transfected with siRNA specific to Kv1.3 (KvSi). After 48 hr, GrB (4 nM) was used to treat the cells. Cells were fixed after 24 hr and immunostained for beta-III-tubulin. Neurite lengths were measured as detailed in Methods. Results represent average ± SEM from three independent experiments. (D) Human neuronal cells were transfected with PAR-1 specific siRNA (PARsi) or a nonspecific control siRNA (Nsi) 48 hr prior to GrB treatment and Western-blot analysis was used to detect Kv1.3 expression after 24 hr of GrB treatment. Representative blot is shown (Lane 1: control; lane 2: PARsi; lane 3: Nsi; lane 4: GrB; lane 5: GrB/PARsi: Lane 6: GrB/Nsi) and results are presented as average ± SEM from three independent experiments. (E) Primary human neuronal cultures were pretreated with corresponding inhibitors 30 min prior to GrB treatment (4 nM). Cell viability was determined using Cytoquantiblue assay 24 hr later. Results represent mean ± SEM. (F) Cells were incubated with a K free solution containing 5 uM PBFI AM for 2 hours. After washing, the cells were treated with GrB (10 nM) with/without MgTX (10 nM) pretreatment. Intracellular K+ concentration was determined by measuring the florescence at Ex 340 nM and Em 500 nM. Data represents mean ± SEM from five replicates.</p