A cGAS-mediated type I interferon response in human CD4+ T cells depends on productive infection and is conserved over HIV types and strains.

Human immunodeficiency virus (HIV) type 2 is known to be less pathogenic than HIV-1, possibly due to more effective immune control mechanisms. The mechanism of innate sensing of HIV-2 by T cells is at present unclear. In this study, we show that several primary isolates of HIV-2 (CBL20 and CI85) and HIV-1 (A8 and D2), similar to the molecular clone HIV-1 NL4.3-GFP-I, induce a significant type I interferon response in its main target, activated CD4+ T cells. However, they are unable to do so after shRNA-mediated knock-down of cGAS. In addition, both HIV-1- and HIV-2-induced type I interferon response in CD4+ T cells was dependent on productive infection and integration, as the presence of RT or integrase inhibitor dramatically suppressed the sensing. Our findings collectively showed that the cGAS-dependent type I interferon response of CD4+ T cells to HIV infection is conserved over HIV types and critically depends on productive infection.IMPORTANCEBy unveiling the role of cGAS in sensing Human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) across CD4+ T cells and highlighting its broader relevance that might be mirrored in other cell types, our research provides insights into the uniform mechanism of innate immune activation by different HIV isolates. By demonstrating the necessity of productive infection, we highlight the robust and specific nature of the observed cGAS-mediated innate response, dispelling concerns about contaminating plasmids triggering an immune response. Our preliminary data suggest that the lower pathogenicity of HIV-2 may not be directly correlated to superior innate immune control mediated by cGAS.

Nationwide quality assurance of high-throughput diagnostic molecular testing during the SARS-CoV-2 pandemic: role of the Belgian National Reference Centre.

Since the onset of the coronavirus disease (COVID-19) pandemic in Belgium, UZ/KU Leuven has played a crucial role as the National Reference Centre (NRC) for respiratory pathogens, to be the first Belgian laboratory to develop and implement laboratory developed diagnostic assays for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and later to assess the quality of commercial kits. To meet the growing demand for decentralised testing, both clinical laboratories and government-supported high-throughput platforms were gradually deployed across Belgium. Consequently, the role of the NRC transitioned from a specialised testing laboratory to strengthening capacity and coordinating quality assurance. Here, we outline the measures taken by the NRC, the national public health institute Sciensano and the executing clinical laboratories to ensure effective quality management of molecular testing throughout the initial two years of the pandemic (March 2020 to March 2022).

Rapid detection of S. pyogenes and S. pneumoniae in pleural fluid for diagnosis of parapneumonic empyema.

The aim of this study was to assess the reliability of rapid antigen detection tests (RADT) for Streptococcus pyogenes (GAS) and Streptococcus pneumoniae on pleural fluid samples for diagnosis of parapneumonic effusion/empyema (PPE) and their potential for improving pathogen identification rates. Sixty-three pleural samples were included from 54 patients on which GAS and S. pneumoniae RADT (BinaxNOW), culture, 16S rRNA PCR, and S. pneumoniae-specific PCR were performed. GAS RADT showed a sensitivity of 95.2% and a specificity of 100%. Pneumococcal RADT showed a sensitivity of 100% and specificity of 88.6%. Both RADT increased the pathogen identification rate in PPE compared to culture.

Emergence of the B.1.214.2 SARS-CoV-2 lineage with an Omicron-like spike insertion and a unique upper airway immune signature.

We investigate the emergence, mutation profile, and dissemination of SARS-CoV-2 lineage B.1.214.2, first identified in Belgium in January 2021. This variant, featuring a 3-amino acid insertion in the spike protein similar to the Omicron variant, was speculated to enhance transmissibility or immune evasion. Initially detected in international travelers, it substantially transmitted in Central Africa, Belgium, Switzerland, and France, peaking in April 2021. Our travel-aware phylogeographic analysis, incorporating travel history, estimated the origin to the Republic of the Congo, with primary European entry through France and Belgium, and multiple smaller introductions during the epidemic. We correlate its spread with human travel patterns and air passenger data. Further, upon reviewing national reports of SARS-CoV-2 outbreaks in Belgian nursing homes, we found this strain caused moderately severe outcomes (8.7% case fatality ratio). A distinct nasopharyngeal immune response was observed in elderly patients, characterized by 80% unique signatures, higher B- and T-cell activation, increased type I IFN signaling, and reduced NK, Th17, and complement system activation, compared to similar outbreaks. This unique immune response may explain the variant's epidemiological behavior and underscores the need for nasal vaccine strategies against emerging variants.

Investigation of an international water polo tournament in Czechia as a potential source for early introduction of the SARS-CoV-2 Omicron variant into Belgium, Switzerland and Germany, November 2021.

BackgroundThe earliest recognised infections by the SARS-CoV-2 Omicron variant (Pango lineage B.1.1.529) in Belgium and Switzerland suggested a connection to an international water polo tournament, held 12-14 November 2021 in Brno, Czechia.AimTo study the arrival and subsequent spread of the Omicron variant in Belgium and Switzerland, and understand the overall importance of this international sporting event on the number of infections in the two countries.MethodsWe performed intensive forward and backward contact tracing in both countries, supplemented by phylogenetic investigations using virus sequences of the suspected infection chain archived in public databases.ResultsThrough contact tracing, we identified two and one infected athletes of the Belgian and Swiss water polo teams, respectively, and subsequently also three athletes from Germany. In Belgium and Switzerland, four and three secondary infections, and three and one confirmed tertiary infections were identified. Phylogenetic investigation demonstrated that this sporting event played a role as the source of infection, but without a direct link with infections from South Africa and not as a superspreading event; the virus was found to already be circulating at that time in the countries involved.ConclusionThe SARS-CoV-2 Omicron variant started to circulate in Europe several weeks before its identification in South Africa on 24 November 2021. Accordingly, it can be assumed that travel restrictions are usually implemented too late to prevent the spread of newly detected SARS-CoV-2 variants to other regions. Phylogenetic analysis may modify the perception of an apparently clear result of intensive contact tracing.

Fecal Microbiota Transplantation Reduces Symptoms in Some Patients With Irritable Bowel Syndrome With Predominant Abdominal Bloating: Short- and Long-term Results From a Placebo-Controlled Randomized Trial.

BACKGROUND & AIMS: Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder associated with intestinal dysbiosis. Given the reported promising results of open-label fecal microbiota transplantation (FMT) therapy in patients with predominant abdominal bloating, we studied efficacy of this treatment in a randomized, placebo-controlled trial. METHODS: Patients with refractory IBS, defined as failure of ≥3 conventional therapies, were randomly assigned to single-dose nasojejunal administration of donor stools (n = 43) or autologous stools (n = 19) in a double-blind study, performed from December 2015 through October 2017, and were followed up for 1 year. IBS-related symptoms were assessed by using a daily symptom diary to determine general abdominal discomfort, abdominal bloating, abdominal pain, and flatulence on a scale of 1-6. Number of daily bowel movements, consistency of the stools, and abdominal circumference were also recorded. Patients completed the IBS-specific quality of life questionnaire. Primary endpoints were improvement of IBS symptoms and bloating at 12 weeks (response). Secondary endpoints were changes in IBS symptom scores and quality of life. Stool samples were collected for microbiota amplicon sequencing. Open-label retransplantation was offered after the trial. RESULTS: At week 12, 56% of patients given donor stool reported improvement in both primary endpoints compared with 26% of patients given placebo (P = .03). Patients given donor stool had significant improvements in level of discomfort (mean reduction, 19%; median score before FMT, 3.98; range, 2.13-6.00; median score after FMT, 3.1; range, 951.29-5.90), stool frequency (mean reduction, 13%; median score before FMT, 2.10; range, 0.57-14.29; median score after FMT 1.7; range, 0.71-4.29), urgency (mean reduction, 38%; median score before FMT, 0.61; range, 0.00-1.00; median score after FMT, 0.37; range, 0.00-1.00), abdominal pain (mean reduction, 26%; median score before FMT, 3.88; range, 1.57-5.17; median score after FMT, 2.80; range, 1.14-4.94), flatulence (mean reduction, 10%; median score before FMT, 3.42; range, 0.71-6.00; median score after FMT, 3.07; range, 0.79-4.23), and quality of life (mean increase, 16%; median score before FMT 32.6; range, 11-119; median score after FMT, 43.1; range, 32.25-99). A significantly higher proportion of women given donor stool (69%) had a response than men (29%) (P = .01). Fecal samples from responders had higher diversity of microbiomes before administration of donor material than fecal samples from nonresponders (P = .04) and distinct baseline composition (P = .04), but no specific marker taxa were associated with response. After single FMT, 21% of patients given donor stool reported effects that lasted for longer than 1 year compared with 5% of patients given placebo stool. A second FMT reduced symptoms in 67% of patients with an initial response to donor stool but not in patients with a prior nonresponse. CONCLUSIONS: In a randomized trial of patients with treatment-refractory IBS with predominant bloating, FMT relieved symptoms compared with placebo (autologous transplant), although the effects decreased over 1 year. A second FMT restored the response patients with a prior response. Response was associated with composition of the fecal microbiomes before FMT; this might be used to as a biomarker to select patients for this treatment. ClinicalTrials.gov, Number: NCT02299973.

Combined oropharyngeal/nasal swab is equivalent to nasopharyngeal sampling for SARS-CoV-2 diagnostic PCR.

BACKGROUND: Early 2020, a COVID-19 epidemic became a public health emergency of international concern. To address this pandemic broad testing with an easy, comfortable and reliable testing method is of utmost concern. Nasopharyngeal (NP) swab sampling is the reference method though hampered by international supply shortages. A new oropharyngeal/nasal (OP/N) sampling method was investigated using the more readily available throat swab. RESULTS: 35 patients were diagnosed with SARS-CoV-2 by means of either NP or OP/N sampling. The paired swabs were both positive in 31 patients. The one patient who tested negative on both NP and OP/N swab on admission, was ultimately diagnosed on bronchoalveolar lavage fluid. A strong correlation was found between the viral RNA loads of the paired swabs (r = 0.76; P < 0.05). The sensitivity of NP and OP/N analysis in hospitalized patients (n = 28) was 89.3% and 92.7% respectively. CONCLUSIONS: This study demonstrates equivalence of NP and OP/N sampling for detection of SARS-CoV-2 by means of rRT-PCR. Sensitivity of both NP and OP/N sampling is very high in hospitalized patients.

Prevalence of Cytoplasmic Actin Mutations in Diffuse Large B-Cell Lymphoma and Multiple Myeloma: A Functional Assessment Based on Actin Three-Dimensional Structures.

Mutations in actins have been linked to several developmental diseases. Their occurrence across different cancers has, however, not been investigated. Using the cBioPortal database we show that human actins are infrequently mutated in patient samples of various cancers types. Nevertheless, ranking these studies by mutational frequency suggest that some have a higher percentage of patients with ACTB and ACTG1 mutations. Within studies on hematological cancers, mutations in ACTB and ACTG1 are associated with lymphoid cancers since none have currently been reported in myeloid cancers. Within the different types of lymphoid cancers ACTB mutations are most frequent in diffuse large B-cell lymphoma (DLBCL) and ACTG1 mutations in multiple myeloma. We mapped the ACTB and ACTG1 mutations found in these two cancer types on the 3D-structure of actin showing they are in regions important for actin polymer formation or binding to myosin. The potential effects of the mutations on actin properties imply that mutations in cytoplasmic actins deserve dedicated research in DLBCL and multiple myeloma.

Improving timelines in reporting results from positive blood cultures: simulation of impact of rapid identification on therapy on a real-life cohort.

For patients with bloodstream infections, rapid initiation of the appropriate antimicrobial therapy is essential in reducing mortality and morbidity. New developments and automation in clinical microbiology labs speed up the identification and susceptibility results but are expensive. To gain insight in the added value of the new workflows, we simulated the possible impact of rapid identification and susceptibility tests on a real-life cohort of 158 positive blood culture episodes. Our routine workflow was theoretically challenged against two new workflows, one based on rapid identification with MALDI-TOF MS and one based on molecular testing. First, we observed an important role of the rapid communication of the gram stain results, as about one third of patients needed an adaptation of the antimicrobial therapy based on these results. Antibiotic adaptation based on the microorganism identification was necessary in 10% and in another 25% of cases after the availability of the susceptibility results. The added value of the newer workflow methods lies mainly in the field of the rapid identification and was rather limited in our cohort. In conclusion, for optimizing the blood culture workflow, each microbiology lab should critically scan its own workflow and know its own blood culture epidemiology, before investing in expensive or time-consuming processes.

Haematopoietic prolyl hydroxylase-1 deficiency promotes M2 macrophage polarization and is both necessary and sufficient to protect against experimental colitis.

Prolyl hydroxylase domain-containing proteins (PHDs) regulate the adaptation of cells to hypoxia. Pan-hydroxylase inhibition is protective in experimental colitis, in which PHD1 plays a prominent role. However, it is currently unknown how PHD1 targeting regulates this protection and which cell type(s) are involved. Here, we demonstrated that Phd1 deletion in endothelial and haematopoietic cells (Phd1f/f Tie2:cre) protected mice from dextran sulphate sodium (DSS)-induced colitis, with reduced epithelial erosions, immune cell infiltration, and colonic microvascular dysfunction, whereas the response of Phd2f/+ Tie2:cre and Phd3f/f Tie2:cre mice to DSS was similar to that of their littermate controls. Using bone marrow chimeras and cell-specific cre mice, we demonstrated that ablation of Phd1 in haematopoietic cells but not in endothelial cells was both necessary and sufficient to inhibit experimental colitis. This effect relied, at least in part, on skewing of Phd1-deficient bone marrow-derived macrophages towards an anti-inflammatory M2 phenotype. These cells showed an attenuated nuclear factor-κB-dependent response to lipopolysaccharide (LPS), which in turn diminished endothelial chemokine expression. In addition, Phd1 deficiency in dendritic cells significantly reduced interleukin-1ß production in response to LPS. Taken together, our results further support the development of selective PHD1 inhibitors for ulcerative colitis, and identify haematopoietic cells as their primary target. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Diaryl ethers with carboxymethoxyphenacyl motif as potent HIV-1 reverse transcriptase inhibitors with improved solubility.

In search of new non-nucleoside reverse transcriptase inhibitors (NNRTIs) with improved solubility, two series of novel diaryl ethers with phenacyl moiety were designed and evaluated for their HIV-1 reverse transcriptase inhibition potentials. All compounds exhibited good to excellent results with IC50 at low micromolar to submicromolar concentrations. Two most active compounds (7e and 7 g) exhibit inhibitory potency comparable or even better than that of nevirapine and rilpivirine. Furthermore, SupT1 and CD4+ cell infectivity assays for the most promising (7e) have confirmed its strong antiviral potential while docking studies indicate a novel binding interactions responsible for high activity.

The H3K27me3 demethylase UTX is a gender-specific tumor suppressor in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia that is mainly diagnosed in children and shows a skewed gender distribution toward males. In this study, we report somatic loss-of-function mutations in the X-linked histone H3K27me3 demethylase ubiquitously transcribed X (UTX) chromosome, in human T-ALL. Interestingly, UTX mutations were exclusively present in male T-ALL patients and allelic expression analysis revealed that UTX escapes X-inactivation in female T-ALL lymphoblasts and normal T cells. Notably, we demonstrate in vitro and in vivo that the H3K27me3 demethylase UTX functions as a bona fide tumor suppressor in T-ALL. Moreover, T-ALL driven by UTX inactivation exhibits collateral sensitivity to pharmacologic H3K27me3 inhibition. All together, our results show how a gender-specific and therapeutically relevant defect in balancing H3K27 methylation contributes to T-cell leukemogenesis.

Mimicking the tumour microenvironment of chronic lymphocytic leukaemia in vitro critically depends on the type of B-cell receptor stimulation.

BACKGROUND: The B-cell receptor (BCR) has a key role in the cross-talk between chronic lymphocytic leukaemia (CLL) cells and the tissue microenvironment, which favours disease progression by promoting proliferation and drug resistance. In vitro studies on downstream signalling and functional effects of CLL BCR ligation often report contradictory results, in part owing to the lack of a standardised stimulation protocol. Our aim was to define a biologically relevant and robust in vitro stimulation method with regard to cellular phenotypic and transcriptional responses. METHODS: We evaluated mRNA (FOS, MYC, LPL) and protein (CD54, CD19, CD62L, CD184) expression of genes modulated by BCR triggering in immunoglobulin heavy-chain variable region genes (IGHV)-mutated and -unmutated CLL cells, after stimulation using soluble or immobilised anti-IgM antibodies from different suppliers. RESULTS: The effect of BCR stimulation on gene and protein expression was comparable in all CLL patients, irrespective of IGHV mutation status. However, immobilised anti-IgM stimulation elicited clear and robust changes in gene and protein expression, whereas the response to soluble anti-IgM was far less obvious. CONCLUSIONS: These data indicate that the method of BCR stimulation is of major importance regarding responsiveness of CLL cells in the context of the tumour microenvironment, whereas genetic differences in the BCR pathway are less critical.

The human immunodeficiency virus (HIV) Rev-binding protein (HRB) is a co-factor for HIV-1 Nef-mediated CD4 downregulation.

Human immunodeficiency virus type 1 (HIV-1)-mediated CD4 downregulation is an important determinant of viral replication in vivo. Research on cellular co-factors involved in this process could lead to the identification of potential therapeutic targets. We found that CD4 surface levels were significantly higher in HIV-1-infected cells knocked-down for the HIV Rev-binding protein (HRB) compared with control cells. HRB knock-down affected CD4 downregulation induced by Nef but not by HIV-1 Vpu. Interestingly, the knock-down of the related protein HRBL (HRB-like), but not of the HRB interaction partner EPS15 (epidermal growth factor receptor pathway substrate 15), increased CD4 levels in Vpu-expressing cells significantly. Both of these proteins are known to be involved in HIV-1-mediated CD4 downregulation as co-factors of HIV-1 Nef. These results identify HRB as a previously unknown co-factor for HIV-1 Nef-mediated CD4 downregulation and highlight differences with the related protein HRBL, which affects the CD4 downregulation in a dual role as co-factor of both HIV-1 Nef and Vpu.

Entry of equid herpesvirus 1 into CD172a+ monocytic cells.

Equid herpesvirus 1 (EHV-1) causes respiratory disease, abortion and neurological disorders in horses. Cells from the myeloid lineage (CD172a+) are one of the main target cells of EHV-1 during primary infection. Recently, we showed that EHV-1 restricts and delays its replication in CD172a+ cells as part of an immune-evasive strategy to disseminate to target organs. Here, we hypothesize that a low efficiency of EHV-1 binding to and entry in CD172a+ cells is responsible for this restriction. Thus, we characterized EHV-1 binding and entry into CD172a+ cells, and showed that EHV-1 only bound to 15-20 % of CD172a+ cells compared with 70 % of RK-13 control cells. Enzymic removal of heparan sulphate did not reduce EHV-1 infection, suggesting that EHV-1 does not use heparan sulphate to bind and enter CD172a+ cells. In contrast, we found that treatment of cells with neuraminidase (NA) reduced infection by 85-100 % compared with untreated cells, whilst NA treatment of virus had no effect on infection. This shows that sialic acid residues present on CD172a+ cells are essential in the initiation of EHV-1 infection. We found that αVß3 integrins are involved in the post-binding stage of CD172a+ cell infection. Using pharmacological inhibitors, we showed that EHV-1 does not enter CD172a+ cells via a clathrin- or caveolae-dependent endocytic pathway, nor by macropinocytosis, but requires cholesterol, tyrosine kinase, actin, dynamin and endosomal acidification, pointing towards a phagocytic mechanism. Overall, these results show that the narrow tropism of EHV-1 amongst CD172a+ cells is determined by the presence of specific cellular receptors.

Lipoprotein lipase in chronic lymphocytic leukemia: function and prognostic implications.

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the accumulation of a clonal population of B cells in peripheral blood, bone marrow, and lymphoid organs. More than 10 years ago, lipoprotein lipase (LPL) mRNA was identified as being strongly expressed in patients experiencing a more aggressive phenotype, while CLL patients with an indolent disease course lack expression of this marker. Since then, several reports confirmed the capability of LPL to predict CLL disease evolution at the moment of diagnosis. In contrast, data on the functional implications of LPL in CLL are scarce. LPL exerts a central role in overall lipid metabolism and transport, but plays additional, non-catalytic roles as well. Which of those is more important in the pathogenesis of CLL remains largely unclear. Here, we review the current knowledge on the prognostic and biological relevance of LPL in CLL.

Accurate quantification of episomal HIV-1 two-long terminal repeat circles by use of optimized DNA isolation and droplet digital PCR.

Episomal HIV-1 two-long terminal repeat (2-LTR) circles are considered markers for ongoing viral replication. Two sample processing procedures were compared to accurately quantify 2-LTR in patients by using droplet digital PCR (ddPCR). Here, we show that plasmid isolation with a spiked non-HIV plasmid for normalization enables more accurate 2-LTR quantification than genomic DNA isolation.

Genome-wide shRNA screening identifies host factors involved in early endocytic events for HIV-1-induced CD4 down-regulation.

BACKGROUND: Down-modulation of the CD4 receptor is one of the hallmarks of HIV-1 infection and it is believed to confer a selective replicative advantage to the virus in vivo. This process is mainly mediated by three viral proteins: Env, Vpu and Nef. To date, the mechanisms that lead to CD4 depletion from the surface of infected cells during HIV-1 infection are still only partially characterized. In this study, we sought to identify and characterize cellular host factors in HIV-1-induced CD4 down-modulation. RESULTS: To identify host factors involved in CD4 down-regulation, we used a whole genome-targeting shRNA lentiviral library in HeLa CD4+ cells expressing Nef as an inducer of CD4 down-modulation. We identified 55 genes, mainly encoding for proteins involved in various steps of clathrin-mediated endocytosis. For confirmation and further selection of the hits we performed several rounds of validation, using individual shRNA lentiviral vectors with a different target sequence for gene knock-down in HIV-1-infected T cells. By this stringent validation set-up, we could demonstrate that the knock-down of DNM3 (dynamin 3), SNX22 (sorting nexin 22), ATP6AP1 (ATPase, H+ Transporting, Lysosomal Accessory Protein 1), HRBL (HIV-Rev binding protein Like), IDH3G (Isocitrate dehydrogenase), HSP90B1 (Heat shock protein 90 kDa beta member 1) and EPS15 (Epidermal Growth Factor Receptor Pathway Substrate 15) significantly increases CD4 levels in HIV-infected SupT1 T cells compared to the non-targeting shRNA control. Moreover, EPS15, DNM3, IDH3G and ATP6AP1 knock-down significantly decreases HIV-1 replication in T cells. CONCLUSIONS: We identified seven genes as cellular co-factors for HIV-1-mediated CD4 down-regulation in T cells. The knock-down of four out of seven of these genes also significantly reduces HIV-1 replication in T cells. Next to a role in HIV-mediated CD4 down-regulation, these genes might however affect HIV-1 replication in another way. Our findings give insights in the HIV-1-mediated CD4 down-regulation at the level of the plasma membrane and early endosomes and identify four possible new HIV-1 replication co-factors.

Replication competent virus as an important source of bias in HIV latency models utilizing single round viral constructs.

The central memory T cell (TCM) model forms a unique HIV-1 latency model based on primary cells that closely resemble in vivo TCM. The virus employed in this model is based on an engineered vector incapable of replication after initial infection. We show that despite this strategy, replication competent viral particles are released into the culture medium due to recombination between overlapping sequences of the env deleted HIV genome that is co-transfected with intact env. This finding emphasizes the need for careful data analysis and interpretation if similar constructs are employed and urges for additional caution during laboratory work.