Reactivation of HIV gene expression in latently infected cells together with an efficient cART has been proposed as an adjuvant therapy aimed at eliminating/decreasing the reservoir size. Results ...from HIV clinical trials using deacetylase inhibitors (HDACIs) question the efficiency of these latency‐reversing agents (LRAs) used alone and underline the need to evaluate other LRAs in combination with HDACIs. Here, we evaluated the therapeutic potential of a demethylating agent (5‐AzadC) in combination with clinically tolerable HDACIs in reactivating HIV‐1 from latency first in vitro and next ex vivo. We showed that a sequential treatment with 5‐AzadC and HDACIs was more effective than the corresponding simultaneous treatment both in vitro and ex vivo. Interestingly, only two of the sequential LRA combinatory treatments tested induced HIV‐1 particle recovery in a higher manner than the drugs alone ex vivo and at concentrations lower than the human tolerable plasmatic concentrations. Taken together, our data reveal the benefit of using combinations of 5‐AzadC with an HDACI and, for the first time, the importance of treatment time schedule for LRA combinations in order to reactivate HIV.
Synopsis
Sequential administration of latency‐reversing agents (LRAs), namely deacetylase inhibitors (HDACIs) and demethylating agents, together with an efficient cART, could represent an adjuvant anti‐HIV‐1 therapy to induce viral production and possibly reduce the size of latent HIV‐1 reservoirs.
A sequential treatment with 5‐AzadC and HDACIs is more effective both in vitro and ex vivo at inducing HIV gene expression than the corresponding simultaneous treatments, highlighting for the first time the importance of treatment schedule for LRAs combinations.
Combining 5‐AzadC + panobinostat and 5‐AzadC + romidepsin show reactivation potentials at concentrations lower than the human tolerable plasmatic concentrations.
These findings are valuable for designing future anti‐latency therapeutic strategies and thereby constitute a step forward in achieving HIV remission.
Sequential administration of latency‐reversing agents (LRAs), namely deacetylase inhibitors (HDACIs) and demethylating agents, together with an efficient cART, could represent an adjuvant anti‐HIV‐1 therapy to induce viral production and possibly reduce the size of latent HIV‐1 reservoirs.
The persistence of latently infected cells in patients under combinatory antiretroviral therapy (cART) is a major hurdle to HIV-1 eradication. Strategies to purge these reservoirs are needed and ...activation of viral gene expression in latently infected cells is one promising strategy. Bromodomain and Extraterminal (BET) bromodomain inhibitors (BETi) are compounds able to reactivate latent proviruses in a positive transcription elongation factor b (P-TEFb)-dependent manner. In this study, we tested the reactivation potential of protein kinase C (PKC) agonists (prostratin, bryostatin-1 and ingenol-B), which are known to activate NF-κB signaling pathway as well as P-TEFb, used alone or in combination with P-TEFb-releasing agents (HMBA and BETi (JQ1, I-BET, I-BET151)). Using in vitro HIV-1 post-integration latency model cell lines of T-lymphoid and myeloid lineages, we demonstrated that PKC agonists and P-TEFb-releasing agents alone acted as potent latency-reversing agents (LRAs) and that their combinations led to synergistic activation of HIV-1 expression at the viral mRNA and protein levels. Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments. Importantly, we observed in ex vivo cultures of CD8+-depleted PBMCs from 35 cART-treated HIV-1+ aviremic patients that the percentage of reactivated cultures following combinatory bryostatin-1+JQ1 treatment was identical to the percentage observed with anti-CD3+anti-CD28 antibodies positive control stimulation. Remarkably, in ex vivo cultures of resting CD4+ T cells isolated from 15 HIV-1+ cART-treated aviremic patients, the combinations bryostatin-1+JQ1 and ingenol-B+JQ1 released infectious viruses to levels similar to that obtained with the positive control stimulation. The potent effects of these two combination treatments were already detected 24 hours post-stimulation. These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.
Noninvasive prenatal screening (NIPS) using cell-free DNA has transformed prenatal care. Belgium was the first country to implement and fully reimburse NIPS as a first-tier screening test offered to ...all pregnant women. A consortium consisting of all Belgian genetic centers report the outcome of two years genome-wide NIPS implementation.
The performance for the common trisomies and for secondary findings was evaluated based on 153,575 genome-wide NIP tests. Furthermore, the evolution of the number of invasive tests and the incidence of Down syndrome live births was registered.
Trisomies 21, 18, and 13 were detected in respectively 0.32%, 0.07%, and 0.06% of cases, with overall positive predictive values (PPVs) of 92.4%, 84.6%, and 43.9%. Rare autosomal trisomies and fetal segmental imbalances were detected in respectively 0.23% and 0.07% of cases with PPVs of 4.1% and 47%. The number of invasive obstetric procedures decreased by 52%. The number of trisomy 21 live births dropped to 0.04%.
Expanding the scope of NIPS beyond trisomy 21 fetal screening allows the implementation of personalized genomic medicine for the obstetric population. This genome-wide NIPS approach has been embedded successfully in prenatal genetic care in Belgium and might serve as a framework for other countries offering NIPS.
Reactivation of HIV-1 expression in persistent reservoirs together with an efficient HAART has been proposed as an adjuvant therapy aimed at reaching a functional cure for HIV. Previously, H3K9 ...methylation was shown to play a major role in chromatin-mediated repression of the HIV-1 promoter. Here, we evaluated the therapeutic potential of histone methyltransferase inhibitors (HMTIs) in reactivating HIV-1 from latency.
We evaluated the reactivation potential of two specific HMTIs (chaetocin and BIX-01294, two specific inhibitors of Suv39H1 and G9a, respectively) in ex-vivo cultures of resting CD4 T cells isolated from HIV-1-infected HAART-treated individuals.
We measured HIV-1 recovery in ex-vivo cultures treated with an HMTI alone or in combination with other HIV-1 inducers (in absence of IL-2 and of allogenic stimulation) of CD8-depleted peripheral blood mononuclear cells (PBMCs) or of resting CD4 T cells isolated from 67 HIV-infected, HAART-treated patients with undetectable viral load.
We demonstrated, for the first time, that chaetocin induced HIV-1 recovery in 50% of CD8-depleted PBMCs cultures and in 86% of resting CD4 T-cell cultures isolated from HIV-1-infected, HAART-treated patients, whereas BIX-01294 reactivated HIV-1 expression in 80% of resting CD4 T-cell cultures isolated from similar patients. Moreover, we showed that combinatory treatments including one HMTI and either the histone deacetylase inhibitor suberoylanilide hydroxamic acid or the non-tumor-promoting NF-κB inducer prostratin had a higher reactivation potential than these compounds alone.
Our results constitute a proof-of-concept for the therapeutic potential of HMTIs in strategies aiming at reducing the pool of latent reservoirs in HIV-infected, HAART-treated patient.
Objective
With the replacement of karyotyping by chromosomal microarray (CMA) in invasive prenatal diagnosis, new challenges have arisen. By building a national database, we standardize the ...classification and reporting of prenatally detected copy number variants (CNVs) across Belgian genetic centers. This database, which will link genetic and ultrasound findings with postnatal development, forms a unique resource to investigate the pathogenicity of variants of uncertain significance and to refine the phenotypic spectrum of pathogenic and susceptibility CNVs.
Methods
The Belgian MicroArray Prenatal (BEMAPRE) consortium is a collaboration of all genetic centers in Belgium. We collected data from all invasive prenatal procedures performed between May 2013 and July 2016.
Results
In this three‐year period, 13 266 prenatal CMAs were performed. By national agreement, a limited number of susceptibility CNVs and no variants of uncertain significance were reported. Added values for using CMA versus conventional karyotyping were 1.8% in the general invasive population and 2.7% in cases with an ultrasound anomaly. Of the reported CNVs, 31.5% would have remained undetected with non‐invasive prenatal test as the first‐tier test.
Conclusion
The establishment of a national database for prenatal CNV data allows for a uniform reporting policy and the investigation of the prenatal and postnatal genotype–phenotype correlation.
What's already known about this topic?
In 5%‐10% of pregnancies with a fetal structural anomaly and in 0.5%‐2% of pregnancies without ultrasound anomalies, CMA reveals cryptic, clinically relevant CNVs.
What does this study add?
This manuscript describes the establishment of a national database for prenatal microarray results in Belgium.
This database, which is one of the largest currently available, allows:
calculation of added values of microarray over karyotyping for different categories of indications;
determination of the most common syndromes, incidental findings, and susceptibility CNVs in the Belgian prenatal population;
evaluation of our national reporting policy; and
reflection on the effect of the implementation of NIPT.
The persistence of transcriptionally silent but replication-competent HIV-1 reservoirs in Highly Active Anti-Retroviral Therapy (HAART)-treated infected individuals, represents a major hurdle to ...virus eradication. Activation of HIV-1 gene expression in these cells together with an efficient HAART has been proposed as an adjuvant therapy aimed at decreasing the pool of latent viral reservoirs. Using the latently-infected U1 monocytic cell line and latently-infected J-Lat T-cell clones, we here demonstrated a strong synergistic activation of HIV-1 production by clinically used histone deacetylase inhibitors (HDACIs) combined with prostratin, a non-tumor-promoting nuclear factor (NF)- kappaB inducer. In J-Lat cells, we showed that this synergism was due, at least partially, to the synergistic recruitment of unresponsive cells into the expressing cell population. A combination of prostratin+HDACI synergistically activated the 5' Long Terminal Repeat (5'LTR) from HIV-1 Major group subtypes representing the most prevalent viral genetic forms, as shown by transient transfection reporter assays. Mechanistically, HDACIs increased prostratin-induced DNA-binding activity of nuclear NF-kappaB and degradation of cytoplasmic NF-kappaB inhibitor, IkappaBalpha . Moreover, the combined treatment prostratin+HDACI caused a more pronounced nucleosomal remodeling in the U1 viral promoter region than the treatments with the compounds alone. This more pronounced remodeling correlated with a synergistic reactivation of HIV-1 transcription following the combined treatment prostratin+HDACI, as demonstrated by measuring recruitment of RNA polymerase II to the 5'LTR and both initiated and elongated transcripts. The physiological relevance of the prostratin+HDACI synergism was shown in CD8(+)-depleted peripheral blood mononuclear cells from HAART-treated patients with undetectable viral load. Moreover, this combined treatment reactivated viral replication in resting CD4(+) T cells isolated from similar patients. Our results suggest that combinations of different kinds of proviral activators may have important implications for reducing the size of latent HIV-1 reservoirs in HAART-treated patients.
Objective
Belgian genetic centers established a database containing data on all chromosomal microarrays performed in a prenatal context. A study was initiated to evaluate postnatal development in ...children diagnosed prenatally with a non‐benign copy number variant (CNV).
Methods
All children diagnosed with a prenatally detected non‐benign CNV in a Belgian genetic center between May 2013 and February 2015 were included in the patient population. The control population consisted of children who had undergone an invasive procedure during pregnancy, with no or only benign CNVs. Child development was evaluated at 36 months using three (3) questionnaires: Ages and Stages Questionnaire Third edition, Ages and Stages Questionnaire Social‐Emotional Second Edition and a general questionnaire.
Results
A significant difference in communication and personal‐social development was detected between children with a reported susceptibility CNV and both children with an unreported susceptibility CNV and the control population. The outcome of children with a particular CNV is discussed in a case‐by‐case manner.
Conclusion
Our postnatal follow‐up project of children with a prenatally detected non‐benign CNV is the first nationwide project of its kind. A higher number of cases for each CNV category is however needed to confirm our findings.
The IkappaB kinases (IKKs) IKK-alpha and IKK-beta, and the IKK-related kinases TBK1 and IKK-epsilon, have essential roles in innate immunity through signal-induced activation of NF-kappaB, IRF3 and ...IRF7, respectively. Although the signaling events within these pathways have been extensively studied, the mechanisms of IKK and IKK-related complex assembly and activation remain poorly defined. Recent data provide insight into the requirement for scaffold proteins in complex assembly; NF-kappaB essential modulator coordinates some IKK complexes, whereas TANK, NF-kappaB-activating kinase-associated protein 1 (NAP1) or similar to NAP1 TBK1 adaptor (SINTBAD) assemble TBK1 and IKK-epsilon complexes. The different scaffold proteins undergo similar post-translational modifications, including phosphorylation and non-degradative polyubiquitylation. Moreover, increasing evidence indicates that distinct scaffold proteins assemble IKK, and potentially TBK1 and IKK-epsilon subcomplexes, in a stimulus-specific manner, which might be a mechanism to achieve specificity.
DiC14-amidine cationic liposomes were recently shown to promote Th1 responses when mixed with allergen. To further define the mode of action of diC14-amidine as potential vaccine adjuvant, we ...characterized its effects on mouse and human myeloid dendritic cells (DC). First, we observed that, as compared with two other cationic liposomes, only diC14-amidine liposomes induced the production of IL-12p40 and TNF-α by mouse bone marrow-derived DC. DiC14-amidine liposomes also activated human DC, as shown by synthesis of IL-12p40 and TNF-α, accumulation of IL-6, IFN-β and CXCL10 mRNA, and up-regulation of membrane expression of CD80 and CD86. DC stimulation by diC14-amidine liposomes was associated with activation of NF-κB, ERK1/2, JNK and p38 MAP kinases. Finally, we demonstrated in mouse and human cells that diC14-amidine liposomes use Toll-like receptor 4 to elicit both MyD88-dependent and Toll/IL-1R-containing adaptor inducing interferon IFN-β (TRIF)-dependent responses.Supporting Information for this article is available at www.wiley-vch.de/contents/jc_2040/2008/37998_s.pdf
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