Eggi Arguni

A Role for c-fos/Activator Protein 1 in B Lymphocyte Terminal Differentiation

Expression of B lymphocyte-induced maturation protein 1 (Blimp-1) transcription factor is essential for promoting B cell differentiation into plasma cells. However, a critical transcription factor for Blimp-1 expression in activated B cells is unclear. When splenic B cells were stimulated with CD40 ligand (CD40L) and IL-4, terminal differentiation was induced in the B cells from c-fos transgenic (H2-c-fos) mice but barely in those from control littermates and from c-fos-deficient mice. AP-1 family and Blimp-1 mRNAs were transiently induced in the control B cells, and overexpression of c-Fos induced a sufficient amount of Blimp-1 for terminal differentiation in the H2-c-fos B cells. When normal and c-fos-deficient B cells were stimulated with LPS, a sufficient amount of Blimp-1 for terminal differentiation was induced in those B cells. However, expression of c-fos/AP-1 family mRNAs in LPS-stimulated normal B cells was similar to that of normal B cells stimulated with CD40L and IL-4. EMSA and chromatin immunoprecipitation assays using the AP-1-binding DNA sequence in the murine Blimp-1 promoter region demonstrated that AP-1-binding activity in nuclear protein of LPS-stimulated normal B cells was prolonged more than that in normal B cells stimulated with CD40L and IL-4. Furthermore, the percentage of CD138+ B cells within germinal center B cells in the spleen and the number of Ab-forming cells in the bone marrow of H2-c-fos mice was larger than that of control mice 12 days after immunization. Thus, although c-Fos is not essential for Blimp-1 expression, c-Fos/AP-1 positively regulates Blimp-1 expression and terminal differentiation of activated B cells.

Bcl6 controls granzyme B expression in effector CD8+ T cells

Bcl6, a sequence‐specific transcriptional repressor, is important for generation and maintenance of memory CD8+ T cells. Although memory CD8+ T cells are generated from effector CD8+ T cells, a role for Bcl6 in effector CD8+ T cells is largely unknown. We show here that Bcl6 expression was transiently induced in activated CD8+ T cells and continuously up‐regulated in effector CD8+ T cells. The amount of granzyme B mRNA among effector molecules produced by effector CD8+ T cells inversely correlated with the amount of Bcl6 mRNA in CD8+ T cells. Overexpression of Bcl6 in CD8+ T cells resulted in lower killing activity at their effector phase, supporting the reduction of granzyme B expression in effector CD8+ T cells by Bcl6. We identified a putative Bcl6‐binding DNA sequence in the promoter region of the granzyme B gene. Binding of Bcl6 to the Bcl6‐binding sequence was detected in naive CD8+ T cells but not in activated CD8+ T cells by chromatin immunoprecipitation assay. Furthermore, the Bcl6‐binding sequence was required for Bcl6 to repress the luciferase reporter gene expression controlled by the granzyme B promoter. Thus, the granzyme B gene is a molecular target of Bcl6 in effector CD8+ T cells.

Aedes aegypti has spatially structured and seasonally stable populations in Yogyakarta, Indonesia.

BackgroundDengue fever, the most prevalent global arboviral disease, represents an important public health problem in Indonesia. Control of dengue relies on the control of its main vector, the mosquito Aedes aegypti, yet nothing is known about the population history and genetic structure of this insect in Indonesia. Our aim was to assess the spatio-temporal population genetic structure of Ae. aegypti in Yogyakarta, a densely populated region on Java with common dengue outbreaks.MethodsWe used multiple marker systems (microsatellites, nuclear and mitochondrial genome-wide single nucleotide polymorphisms generated via Restriction-site Associated DNA sequencing) to analyze 979 Ae. aegypti individuals collected from the Yogyakarta city and the surrounding hamlets during the wet season in 2011 and the following dry season in 2012. We employed individual- and group-based approaches for inferring genetic structure.ResultsWe found that Ae. aegypti in Yogyakarta has spatially structured and seasonally stable populations. The spatial structuring was significant for the nuclear and mitochondrial markers, while the temporal structuring was non-significant. Nuclear markers identified three main genetic clusters, showing that hamlets have greater genetic isolation from each other and from the inner city sites. However, one hamlet experienced unrestricted mosquito interbreeding with the inner city, forming a single genetic cluster. Genetic distance was poorly correlated with the spatial distance among mosquito samples, suggesting stronger influence of human-assisted gene flow than active mosquito movement on spatial genetic structure. A star-shaped mitochondrial haplotype network and a significant R2 test statistic (R2 = 0.0187, P = 0.001) support the hypothesis that Ae. aegypti in Yogyakarta originated from a small or homogeneous source and has undergone a relatively recent demographic expansion.ConclusionWe report the first insights into the spatio-temporal genetic structure and the underlying processes in the dengue fever mosquito from Yogyakarta, Indonesia. Our results provide valuable information on the effectiveness of local control measures as well as guidelines for the implementation of novel biocontrol strategies such as release of Wolbachia-infected mosquitoes.

Mitochondrial DNA variants help monitor the dynamics of Wolbachia invasion into host populations

Wolbachia is the most widespread endosymbiotic bacterium of insects and other arthropods that can rapidly invade host populations. Deliberate releases of Wolbachia into natural populations of the dengue fever mosquito, Aedes aegypti, are used as a novel biocontrol strategy for dengue suppression. Invasion of Wolbachia through the host population relies on factors such as high fidelity of the endosymbiont transmission and limited immigration of uninfected individuals, but these factors can be difficult to measure. One way of acquiring relevant information is to consider mitochondrial DNA (mtDNA) variation alongside Wolbachia in field-caught mosquitoes. Here we used diagnostic mtDNA markers to differentiate infection-associated mtDNA haplotypes from those of the uninfected mosquitoes at release sites. Unique haplotypes associated with Wolbachia were found at locations outside Australia. We also performed mathematical and qualitative analyses including modelling the expected dynamics of the Wolbachia and mtDNA variants during and after a release. Our analyses identified key features in haplotype frequency patterns to infer the presence of imperfect maternal transmission of Wolbachia, presence of immigration and possibly incomplete cytoplasmic incompatibility. We demonstrate that ongoing screening of the mtDNA variants should provide information on maternal leakage and immigration, particularly in releases outside Australia. As we demonstrate in a case study, our models to track the Wolbachia dynamics can be successfully applied to temporal studies in natural populations or Wolbachia release programs, as long as there is co-occurring mtDNA variation that differentiates infected and uninfected populations.

ADAR1 protein induces adenosine-targeted DNA mutations in senescent Bcl6 gene-deficient cells.

Background: Bcl6 is a potent inhibitor of cell senescence, and somatic mutations accumulate in senescent cells. Results: Mutation frequencies in senescent Bcl6-deficient cells were higher than senescent wild-type cells, and ADAR1 is overexpressed in Bcl6-deficient cells. Conclusion: Bcl6 negatively regulates ADAR1 expression, and ADAR1 overexpression induces adenosine-targeted DNA mutations. Significance: Bcl6 may protect senescent cells from accumulation of somatic mutations. Somatic mutations accumulate in senescent cells. Bcl6, which functions as a transcriptional repressor, has been identified as a potent inhibitor of cell senescence, but a role of Bcl6 in the accumulation of somatic mutations has remained unclear. Ig class-switch recombination simultaneously induces somatic mutations in an IgM class-switch (Ig-Sμ) region of IgG B cells. Surprisingly, mutations were detected in the Ig-Sμ region of Bcl6-deficient IgM B cells without class-switch recombination, and these mutations were mainly generated by conversion of adenosine to guanosine, suggesting a novel DNA mutator in the B cells. The ADAR1 (adenosine deaminase acting on RNA1) gene was overexpressed in Bcl6-deficient cells, and its promoter analysis revealed that ADAR1 is a molecular target of Bcl6. Exogenous ADAR1 induced adenosine-targeted DNA mutations in IgM B cells from ADAR1-transgenic mice and in wild-type mouse embryonic fibroblasts (MEFs). These mutations accumulated in senescent MEFs accompanied with endogenous ADAR1 expression, and the frequency in senescent Bcl6-deficient MEFs was higher than senescent wild-type MEFs. Thus, Bcl6 protects senescent cells from accumulation of adenosine-targeted DNA mutations induced by ADAR1.

Spatial and Temporal Variation in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) Numbers in the Yogyakarta Area of Java, Indonesia, With Implications for Wolbachia Releases

Abstract Novel approaches to suppress dengue and other mosquito-borne diseases involve changing the composition of mosquito vector populations, particularly through Wolbachia endosymbionts. The success of these strategies depends on understanding the dynamics of vector populations. In preparation for Wolbachia releases around Yogyakarta, we have studied Aedes populations in five hamlets. Adult monitoring with BioGent-Sentinel (BG-S) traps indicated that hamlet populations had different dynamics across the year; while there was an increase in Aedes aegypti (L.) and Aedes albopictus (Skuse) numbers in the wet season, species abundance remained relatively stable in some hamlets but changed markedly (>2 fold) in others. Local rainfall a month prior to monitoring partly predicted numbers of Ae. aegypti but not Ae. albopictus. Site differences in population size indicated by BG-S traps were also evident in ovitrap data. Egg or larval collections with ovitraps repeated at the same location suggested spatial autocorrelation (<250 m) in the areas of the hamlets where Ae. aegypti numbers were high. Overall, there was a weak negative association (r < –0.43) between Ae. aegypti and Ae. albopictus numbers in ovitraps when averaged across collections. Ae. albopictus numbers in ovitraps and BG-S traps were positively correlated with vegetation around areas where traps were placed, while Ae. aegypti were negatively correlated with this feature. These data inform intervention strategies by defining periods when mosquito densities are high, highlighting the importance of local site characteristics on populations, and suggesting relatively weak interactions between Ae. aegypti and Ae. albopictus. They also indicate local areas within hamlets where consistently high mosquito densities may influence Wolbachia invasions and other interventions.

The AWED trial (Applying Wolbachia to Eliminate Dengue) to assess the efficacy of Wolbachia-infected mosquito deployments to reduce dengue incidence in Yogyakarta, Indonesia: study protocol for a cluster randomised controlled trial

BackgroundDengue and other arboviruses transmitted by Aedes aegypti mosquitoes, including Zika and chikungunya, present an increasing public health challenge in tropical regions. Current vector control strategies have failed to curb disease transmission, but continue to be employed despite the absence of robust evidence for their effectiveness or optimal implementation. The World Mosquito Program has developed a novel approach to arbovirus control using Ae. aegypti stably transfected with Wolbachia bacterium, with a significantly reduced ability to transmit dengue, Zika and chikungunya in laboratory experiments. Modelling predicts this will translate to local elimination of dengue in most epidemiological settings. This study protocol describes the first trial to measure the efficacy of Wolbachia in reducing dengue virus transmission in the field.Methods/designThe study is a parallel, two-arm, non-blinded cluster randomised controlled trial conducted in a single site in Yogyakarta, Indonesia. The aim is to determine whether large-scale deployment of Wolbachia-infected Ae. aegypti mosquitoes leads to a measurable reduction in dengue incidence in treated versus untreated areas. The primary endpoint is symptomatic, virologically confirmed dengue virus infection of any severity. The 26 km2 study area was subdivided into 24 contiguous clusters, allocated randomly 1:1 to receive Wolbachia deployments or no intervention. We use a novel epidemiological study design, the cluster-randomised test-negative design trial, in which dengue cases and arbovirus-negative controls are sampled concurrently from among febrile patients presenting to a network of primary care clinics, with case or control status classified retrospectively based on the results of laboratory diagnostic testing. Efficacy is estimated from the odds ratio of Wolbachia exposure distribution (probability of living in a Wolbachia-treated area) among virologically confirmed dengue cases compared to test-negative controls. A secondary per-protocol analysis allows for individual Wolbachia exposure levels to be assessed to account for movements outside the cluster and the heterogeneity in local Wolbachia prevalence among treated clusters.DiscussionThe findings from this study will provide the first experimental evidence for the efficacy of Wolbachia in reducing dengue incidence. Together with observational evidence that is accumulating from pragmatic deployments of Wolbachia in other field sites, this will provide valuable data to estimate the effectiveness of this novel approach to arbovirus control, inform future cost-effectiveness estimates, and guide plans for large-scale deployments in other endemic settings.Trial registrationClinicalTrials.gov, identifier: NCT03055585. Registered on 14 February 2017.

DC-SIGN (CD209) carbohydrate recognition domain is not polymorphic in dengue virus-infected Indonesian patients.

Dengue virus (DENV) infection is a significant burden in Indonesia and other tropical countries. DENV infection has a wide spectrum of clinical manifestations, i.e. asymptomatic, dengue fever, dengue hemorrhagic fever and dengue shock syndrome. The variety of clinical manifestations may be due to the diversity of genetic constitution of the host. The C-type lectin DC-SIGN (CD209) has been identified as the major dengue receptor on human dendritic cells. There are at least five polymorphisms in exon 5 and 6 of the DC-SIGN encoded gene which have been identified and recorded in dbSNP. The aim of this work is to measure the frequency of these polymorphisms among asymptomatic and hospitalized DENV-infected patients. We enrolled 23 hospitalized and 73 asymptomatic DENV-infected patients. Among the subjects, we performed PCR amplification and DNA direct seqencing for 23 hospitalized DENV-infected patients and 24 asymptomatic DENV-infected patients. The result showed that there were no polymorphic nucleotides in the CD209 encoded gene among the patients.

Baseline Characterization of Dengue Epidemiology in Yogyakarta City, Indonesia, before a Randomized Controlled Trial of Wolbachia for Arboviral Disease Control

Abstract. Dengue is endemic in Indonesia. Here, we describe the epidemiology of dengue in the city of Yogyakarta, Central Java, as a prelude to implementation of a cluster-randomized trial of Wolbachia for the biocontrol of arboviral transmission. Surveillance records from 2006 to 2016 demonstrate seasonal oscillations of dengue incidence with varying magnitude. Two lines of evidence demonstrate a high force of infection; the hospitalized case burden of patients diagnosed with dengue hemorrhagic fever or dengue shock syndrome over the last decade consisted predominantly of children/adolescents, and a serosurvey of 314 healthy children aged 1–10 years found 68% possessed dengue virus–neutralizing antibodies. Finally, a mobility survey indicated children aged 1–10 years, and particularly 1–5 year-olds, spent most of their daytime hours at home. These findings inform the design of clinical trials to measure the impact of novel vector control methods such as Wolbachia introgression into Aedes aegypti mosquitoes, by providing baseline data on disease incidence and identifying subpopulations for recruitment into prospective studies of dengue virus infection and disease. The mobility survey findings indicate that in cluster trials of interventions applied at the community level, young children can reasonably be expected to spend most of their exposure time, in epidemiological terms, within the treatment arm to which they were randomized.

Profil Klinis dan Laboratoris Ensefalopati Dengue pada Anak di RSUP Dr. Sardjito

Latar belakang . Ensefalopati dengue merupakan manifestasi neurologis infeksi dengue yang paling sering. Belum banyak penelitian yang menjelaskan gambaran ensefalopati dengue pada anak. Tujuan . Mengetahui gambaran klinis dan laboratoris anak dengan ensefalopati dengue. Metode . Penelitian menggunakan metode retrospektif. Data diambil dari rekam medis anak (1-18 tahun) dengan ensefalopati dengue dengan luaran hidup maupun meninggal yang dirawat di RSUP Dr. Sardjito sejak Januari 2010 hingga Desember 2016. Manifestasi yang diamati adalah perdarahan gastrointestinal, kejang, syok, hepatitis akut, gagal ginjal akut, pemanjangan prothrombin time (PT), activated partial thromboplastin time (APTT), kadar hematokrit, leukosit, dan trombosit. Rekam medis anak dengan riwayat gangguan fungsi ginjal, hati, dan koagulasi serta rekam medis yang tidak lengkap dieksklusi. Analisis data dengan uji independent t-test atau Mann Whitney. Hasil . Terdapat 81 anak dengan ensefalopati dengue dan 25 di antaranya meninggal. Mayoritas anak menunjukkan manifestasi perdarahan gastrointestinal, syok, sepsis, hepatitis ringan, dan gagal ginjal akut. Profil laboratoris memperlihatkan peningkatan SGOT yang lebih tinggi dari SGPT, pemanjangan PT dan kadar kreatinin yang lebih tinggi pada kelompok meninggal. Kesimpulan . Kelainan ensefalopati pada infeksi dengue merupakan suatu kondisi yang serius dengan angka kematian yang tinggi (30.86%). Perdarahan gastrointestinal, syok, sepsis, hepatitis ringan, gagal ginjal akut pada mayoritas pasien. Pemanjangan PT dan kadar kreatinin lebih tinggi pada kelompok meninggal.