Publications by authors named "Hugh Reyburn"

146 Publications

Marked changes in innate immunity associated with a mild course of COVID-19 in identical twins with athymia and absent circulating T cells.

J Allergy Clin Immunol 2021 02 3;147(2):567-568. Epub 2020 Dec 3.

Department of Immunology, La Paz University Hospital, Madrid, Spain; Rare Disease Network Research Center (CIBERER U767), Madrid, Spain; Lymphocyte Pathophysiology Group, La Paz Institute of Biomedical Research, IdiPAZ, Madrid, Spain. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2020.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7713636PMC
February 2021

Deregulated cellular circuits driving immunoglobulins and complement consumption associate with the severity of COVID-19 patients.

Eur J Immunol 2021 03 22;51(3):634-647. Epub 2021 Jan 22.

Department of Immunology, Biomedical Research Institute La Princesa Hospital (IIS-IP), Madrid, Spain.

SARS-CoV-2 infection causes an abrupt response by the host immune system, which is largely responsible for the outcome of COVID-19. We investigated whether the specific immune responses in the peripheral blood of 276 patients were associated with the severity and progression of COVID-19. At admission, dramatic lymphopenia of T, B, and NK cells is associated with severity. Conversely, the proportion of B cells, plasmablasts, circulating follicular helper T cells (cTfh) and CD56 CD16 NK-cells increased. Regarding humoral immunity, levels of IgM, IgA, and IgG were unaffected, but when degrees of severity were considered, IgG was lower in severe patients. Compared to healthy donors, complement C3 and C4 protein levels were higher in mild and moderate, but not in severe patients, while the activation peptide of C5 (C5a) increased from the admission in every patient, regardless of their severity. Moreover, total IgG, the IgG1 and IgG3 isotypes, and C4 decreased from day 0 to day 10 in patients who were hospitalized for more than two weeks, but not in patients who were discharged earlier. Our study provides important clues to understand the immune response observed in COVID-19 patients, associating severity with an imbalanced humoral response, and identifying new targets for therapeutic intervention.
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http://dx.doi.org/10.1002/eji.202048858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7753288PMC
March 2021

SARS-CoV-2 Cysteine-like Protease Antibodies Can Be Detected in Serum and Saliva of COVID-19-Seropositive Individuals.

J Immunol 2020 12 4;205(11):3130-3140. Epub 2020 Nov 4.

Department of Immunology and Oncology, National Centre for Biotechnology, National Centre for Biotechnology-Spanish National Research Council, Madrid 28049, Spain; and

Currently, there is a need for reliable tests that allow identification of individuals that have been infected with SARS-CoV-2 even if the infection was asymptomatic. To date, the vast majority of the serological tests for SARS-CoV-2-specific Abs are based on serum detection of Abs to either the viral spike glycoprotein (the major target for neutralizing Abs) or the viral nucleocapsid protein that is known to be highly immunogenic in other coronaviruses. Conceivably, exposure of Ags released from infected cells could stimulate Ab responses that might correlate with tissue damage and, hence, they may have some value as a prognostic indicator. We addressed whether other nonstructural viral proteins, not incorporated into the infectious viral particle, specifically the viral cysteine-like protease, might also be potent immunogens. Using ELISA tests, coating several SARS-CoV-2 proteins produced in vitro, we describe that COVID-19 patients make high titer IgG, IgM, and IgA Ab responses to the Cys-like protease from SARS-CoV-2, also known as 3CLpro or Mpro, and it can be used to identify individuals with positive serology against the coronavirus. Higher Ab titers in these assays associated with more-severe disease, and no cross-reactive Abs against prior betacoronavirus were found. Remarkably, IgG Abs specific for Mpro and other SARS-CoV-2 Ags can also be detected in saliva. In conclusion, Mpro is a potent Ag in infected patients that can be used in serological tests, and its detection in saliva could be the basis for a rapid, noninvasive test for COVID-19 seropositivity.
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http://dx.doi.org/10.4049/jimmunol.2000842DOI Listing
December 2020

The impact of delayed treatment of uncomplicated P. falciparum malaria on progression to severe malaria: A systematic review and a pooled multicentre individual-patient meta-analysis.

PLoS Med 2020 10 19;17(10):e1003359. Epub 2020 Oct 19.

MRC Centre for Global Infectious Disease Analysis, Department of Infectious Disease Epidemiology, Imperial College London, London, United Kingdom.

Background: Delay in receiving treatment for uncomplicated malaria (UM) is often reported to increase the risk of developing severe malaria (SM), but access to treatment remains low in most high-burden areas. Understanding the contribution of treatment delay on progression to severe disease is critical to determine how quickly patients need to receive treatment and to quantify the impact of widely implemented treatment interventions, such as 'test-and-treat' policies administered by community health workers (CHWs). We conducted a pooled individual-participant meta-analysis to estimate the association between treatment delay and presenting with SM.

Methods And Findings: A search using Ovid MEDLINE and Embase was initially conducted to identify studies on severe Plasmodium falciparum malaria that included information on treatment delay, such as fever duration (inception to 22nd September 2017). Studies identified included 5 case-control and 8 other observational clinical studies of SM and UM cases. Risk of bias was assessed using the Newcastle-Ottawa scale, and all studies were ranked as 'Good', scoring ≥7/10. Individual-patient data (IPD) were pooled from 13 studies of 3,989 (94.1% aged <15 years) SM patients and 5,780 (79.6% aged <15 years) UM cases in Benin, Malaysia, Mozambique, Tanzania, The Gambia, Uganda, Yemen, and Zambia. Definitions of SM were standardised across studies to compare treatment delay in patients with UM and different SM phenotypes using age-adjusted mixed-effects regression. The odds of any SM phenotype were significantly higher in children with longer delays between initial symptoms and arrival at the health facility (odds ratio [OR] = 1.33, 95% CI: 1.07-1.64 for a delay of >24 hours versus ≤24 hours; p = 0.009). Reported illness duration was a strong predictor of presenting with severe malarial anaemia (SMA) in children, with an OR of 2.79 (95% CI:1.92-4.06; p < 0.001) for a delay of 2-3 days and 5.46 (95% CI: 3.49-8.53; p < 0.001) for a delay of >7 days, compared with receiving treatment within 24 hours from symptom onset. We estimate that 42.8% of childhood SMA cases and 48.5% of adult SMA cases in the study areas would have been averted if all individuals were able to access treatment within the first day of symptom onset, if the association is fully causal. In studies specifically recording onset of nonsevere symptoms, long treatment delay was moderately associated with other SM phenotypes (OR [95% CI] >3 to ≤4 days versus ≤24 hours: cerebral malaria [CM] = 2.42 [1.24-4.72], p = 0.01; respiratory distress syndrome [RDS] = 4.09 [1.70-9.82], p = 0.002). In addition to unmeasured confounding, which is commonly present in observational studies, a key limitation is that many severe cases and deaths occur outside healthcare facilities in endemic countries, where the effect of delayed or no treatment is difficult to quantify.

Conclusions: Our results quantify the relationship between rapid access to treatment and reduced risk of severe disease, which was particularly strong for SMA. There was some evidence to suggest that progression to other severe phenotypes may also be prevented by prompt treatment, though the association was not as strong, which may be explained by potential selection bias, sample size issues, or a difference in underlying pathology. These findings may help assess the impact of interventions that improve access to treatment.
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http://dx.doi.org/10.1371/journal.pmed.1003359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7571702PMC
October 2020

Non-malarial febrile illness: a systematic review of published aetiological studies and case reports from Africa, 1980-2015.

BMC Med 2020 09 21;18(1):279. Epub 2020 Sep 21.

Infectious Diseases Data Observatory, University of Oxford, New Richards Building,Old Road Campus,Headington, Oxford, OX3 7LG, UK.

Background: The availability of reliable point-of-care tests for malaria has heralded a paradigm shift in the management of febrile illnesses away from presumptive antimalarial therapy. In the absence of a definitive diagnosis, health care providers are more likely to prescribe empirical antimicrobials to those who test negative for malaria. To improve management and guide further test development, better understanding is needed of the true causative agents and their geographic variability.

Methods: A systematic review of published literature was undertaken to characterise the spectrum of pathogens causing non-malaria febrile illness in Africa (1980-2015). Literature searches were conducted in English and French languages in six databases: MEDLINE, EMBASE, Global Health (CABI), WHO Global Health Library, PASCAL, and Bulletin de la Société Française de Parasitologie (BDSP). Selection criteria included reporting on an infection or infections with a confirmed diagnosis, defined as pathogens detected in or cultured from samples from normally sterile sites, or serological evidence of current or past infection. A number of published articles (rather than incidence or prevalence) reporting a given pathogen were presented.

Results: A total of 16,523 records from 48 African countries were screened, of which 1065 (6.4%) met selection criteria. Bacterial infections were reported in 564 (53.0%) records, viral infections in 374 (35.1%), parasitic infections in 47 (4.4%), fungal infections in nine (0.8%), and 71 (6.7%) publications reported more than one pathogen group. Age range of the study population was not specified in 233 (21.9%) publications. Staphylococcus aureus (18.2%), non-typhoidal Salmonella (17.3%), and Escherichia coli (15.4%) were the commonly reported bacterial infections whereas Rift Valley fever virus (7.4%), yellow fever virus (7.0%), and Ebola virus (6.7%) were the most commonly reported viral infections. Dengue virus infection, previously not thought to be widespread in Africa, was reported in 54 (5.1%) of articles.

Conclusions: This review summarises the published reports of non-malaria pathogens that may cause febrile illness in Africa. As the threat of antimicrobial resistance looms, knowledge of the distribution of infectious agents causing fever should facilitate priority setting in the development of new diagnostic tools and improved antimicrobial stewardship.

Trial Registration: PROSPERO, CRD42016049281.
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http://dx.doi.org/10.1186/s12916-020-01744-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504660PMC
September 2020

Structural stability of SARS-CoV-2 3CLpro and identification of quercetin as an inhibitor by experimental screening.

Int J Biol Macromol 2020 Dec 1;164:1693-1703. Epub 2020 Aug 1.

Instituto de Investigación Sanitaria de Aragón (IIS Aragon), 50009 Zaragoza, Spain; Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y Digestivas (CIBERehd), 28029 Madrid, Spain; Institute for Biocomputation and Physics of Complex Systems (BIFI), Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza, 50009 Zaragoza, Spain; Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, 50009 Zaragoza, Spain; Fundación ARAID, Gobierno de Aragón, 50018 Zaragoza, Spain. Electronic address:

The global health emergency generated by coronavirus disease 2019 (COVID-19) has prompted the search for preventive and therapeutic treatments for its pathogen, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). There are many potential targets for drug discovery and development to tackle this disease. One of these targets is the main protease, Mpro or 3CLpro, which is highly conserved among coronaviruses. 3CLpro is an essential player in the viral replication cycle, processing the large viral polyproteins and rendering the individual proteins functional. We report a biophysical characterization of the structural stability and the catalytic activity of 3CLpro from SARS-CoV-2, from which a suitable experimental in vitro molecular screening procedure has been designed. By screening of a small chemical library consisting of about 150 compounds, the natural product quercetin was identified as reasonably potent inhibitor of SARS-CoV-2 3CLpro (K ~ 7 μM). Quercetin could be shown to interact with 3CLpro using biophysical techniques and bind to the active site in molecular simulations. Quercetin, with well-known pharmacokinetic and ADMET properties, can be considered as a good candidate for further optimization and development, or repositioned for COVID-19 therapeutic treatment.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.07.235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395220PMC
December 2020

Tetraspanin-decorated extracellular vesicle-mimetics as a novel adaptable reference material.

J Extracell Vesicles 2019 4;8(1):1573052. Epub 2019 Mar 4.

Centro de Biología Molecular Severo Ochoa (CSIC-UAM) Departamento de Biología Molecular, Universidad Autónoma de Madrid (UAM), Madrid, Spain.

Features like small size, low refractive index and polydispersity pose challenges to the currently available detection methods for Extracellular Vesicles (EVs). In addition, the lack of appropriate standards to set up the experimental conditions makes it difficult to compare analyses obtained by different technical approaches. By modifying synthetic nanovesicles with recombinant antigenic regions of EV-enriched tetraspanins, we aimed to construct an EV-mimetic that can be used as a suitable standard for EV analyses. To this end, the sequences of the large extracellular loops of the tetraspanins CD9, CD63 and CD81 were tagged with a target sequence for the biotin ligase BirA, and co-transformed with a BirA expression plasmid into . GST fusion proteins were then isolated by affinity chromatography and released using thrombin. Biotinylated recombinant tetraspanin-loops were then coupled to (strept)avidin-coated synthetic nanovesicles and analysed and characterised by Dot-blot, Western-blot, Nanoparticle Tracking Analysis, Flow Cytometry and Transmission Electron Microscopy. With this method, we were able to efficiently produce tetraspanin-domain decorated nanovesicles that share biophysical properties with natural EVs, can be detected using specific antibodies against common EV markers such as tetraspanins, and can be used as robust reference materials for detection techniques that are often used in the EV field.
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http://dx.doi.org/10.1080/20013078.2019.1573052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6407598PMC
March 2019

Impaired control of multiple viral infections in a family with complete IRF9 deficiency.

J Allergy Clin Immunol 2019 07 28;144(1):309-312.e10. Epub 2019 Feb 28.

Department of Immunology, La Paz University Hospital, Madrid, Spain; Lymphocyte Pathophysiology Group, La Paz Institute of Biomedical Research, IdiPAZ, Madrid, Spain.

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http://dx.doi.org/10.1016/j.jaci.2019.02.019DOI Listing
July 2019

NKG2H-Expressing T Cells Negatively Regulate Immune Responses.

Front Immunol 2018 1;9:390. Epub 2018 Mar 1.

Department of Immunology and Oncology, National Centre for Biotechnology, CSIC, Madrid, Spain.

The biology and function of NKG2H receptor, unlike the better characterized members of the NKG2 family NKG2A, NKG2C, and NKG2D, remains largely unclear. Here, we show that NKG2H is able to associate with the signaling adapter molecules DAP12 and DAP10 suggesting that this receptor can signal for cell activation. Using a recently described NKG2H-specific monoclonal antibody (mAb), we have characterized the expression and function of lymphocytes that express this receptor. NKG2H is expressed at the cell surface of a small percentage of peripheral blood mononuclear cell (PBMC) and is found more frequently on T cells, rather than NK cells. Moreover, although NKG2H is likely to trigger activation, co-cross-linking of this receptor with an NKG2H-specific mAb led to decreased T cell activation and proliferation in polyclonal PBMC cultures stimulated by anti-CD3 mAbs. This negative regulatory activity was seen only after cross-linking with NKG2H, but not NKG2A- or NKG2C-specific monoclonal antibodies. The mechanism underlying this negative effect is as yet unclear, but did not depend on the release of soluble factors or recognition of MHC class I molecules. These observations raise the intriguing possibility that NKG2H may be a novel marker for T cells able to negatively regulate T cell responses.
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http://dx.doi.org/10.3389/fimmu.2018.00390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5837990PMC
May 2019

Novel genetic polymorphisms associated with severe malaria and under selective pressure in North-eastern Tanzania.

PLoS Genet 2018 01 30;14(1):e1007172. Epub 2018 Jan 30.

Pathogen Molecular Biology Department, London School of Hygiene and Tropical Medicine, London, United Kingdom.

Significant selection pressure has been exerted on the genomes of human populations exposed to Plasmodium falciparum infection, resulting in the acquisition of mechanisms of resistance against severe malarial disease. Many host genetic factors, including sickle cell trait, have been associated with reduced risk of developing severe malaria, but do not account for all of the observed phenotypic variation. Identification of novel inherited risk factors relies upon high-resolution genome-wide association studies (GWAS). We present findings of a GWAS of severe malaria performed in a Tanzanian population (n = 914, 15.2 million SNPs). Beyond the expected association with the sickle cell HbS variant, we identify protective associations within two interleukin receptors (IL-23R and IL-12RBR2) and the kelch-like protein KLHL3 (all P<10-6), as well as near significant effects for Major Histocompatibility Complex (MHC) haplotypes. Complementary analyses, based on detecting extended haplotype homozygosity, identified SYNJ2BP, GCLC and MHC as potential loci under recent positive selection. Through whole genome sequencing of an independent Tanzanian cohort (parent-child trios n = 247), we confirm the allele frequencies of common polymorphisms underlying associations and selection, as well as the presence of multiple structural variants that could be in linkage with these SNPs. Imputation of structural variants in a region encompassing the glycophorin genes on chromosome 4, led to the characterisation of more than 50 rare variants, and individually no strong evidence of associations with severe malaria in our primary dataset (P>0.3). Our approach demonstrates the potential of a joint genotyping-sequencing strategy to identify as-yet unknown susceptibility loci in an African population with well-characterised malaria phenotypes. The regions encompassing these loci are potential targets for the design of much needed interventions for preventing or treating malarial disease.
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http://dx.doi.org/10.1371/journal.pgen.1007172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5806895PMC
January 2018

Impaired NK cell recognition of vemurafenib-treated melanoma cells is overcome by simultaneous application of histone deacetylase inhibitors.

Oncoimmunology 2018;7(2):e1392426. Epub 2017 Nov 6.

Department of Immunology and Oncology, National Centre for Biotechnology, CNB-CSIC, Madrid, Spain.

Therapy of metastatic melanoma advanced recently with the clinical implementation of signalling pathway inhibitors, such as vemurafenib, specifically targeting mutant BRAF. In general, patients experience remarkable clinical responses under BRAF inhibitor (BRAFi) treatment but eventually progress within 6-8 months due to resistance development. Responding metastases show an increased immune cell infiltrate, including also NK cells, that, however, is no longer detectable in BRAFi-resistant lesions, suggesting NK cell activity should be exploited to prevent disease progression. Here, we examined the effects of BRAFi on the expression of ligands targeting activating NK cells receptors immediately after treatment onset, prior to resistance development. We demonstrate that BRAF mutant melanoma cells cultured in the presence of vemurafenib, strongly decreased surface expression of ligands for NK activating receptors including the NKG2D-ligand, MICA, and the DNAM-1 ligand, CD155, and became significantly less susceptible to NK cell attack. NKG2D-ligand protein downregulation was due to a significant decrease in mRNA levels, already detectable 24 h after drug treatment. Interestingly, vemurafenib-induced MICA downregulation could be counteracted by treatment of melanoma cells with the histone deacetylase (HDAC) inhibitor (HDACi) sodium butyrate, that also upregulated the DNAM1-ligand, Nectin-2. HDACi treatment enhanced surface expression of NKG2D-ligands in the presence of BRAFi, accompanied by recovery of NK cell recognition, but only upon simultaneous drug application. These results suggest that co-administration of BRAFi and HDAC inhibitors as well as having direct effects on melanoma cell survival, could also synergise to improve NK cell recognition and avoid tumour immune evasion.
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http://dx.doi.org/10.1080/2162402X.2017.1392426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749663PMC
November 2017

Innate immune recognition of double-stranded RNA triggers increased expression of NKG2D ligands after virus infection.

J Biol Chem 2017 12 6;292(50):20472-20480. Epub 2017 Oct 6.

From the Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Científicas (CNB-CSIC), 28049 Madrid and

Self/non-self-discrimination by the innate immune system relies on germline-encoded, non-rearranging receptors expressed by innate immune cells recognizing conserved pathogen-associated molecular patterns. The natural killer group 2D (NKG2D) receptor is a potent immune-activating receptor that binds human genome-encoded ligands, whose expression is negligible in normal tissues, but increased in stress and disease conditions for reasons that are incompletely understood. Here it is not clear how the immune system reconciles receptor binding of self-proteins with self/non-self-discrimination to avoid autoreactivity. We now report that increased expression of NKG2D ligands after virus infection depends on interferon response factors activated by the detection of viral double-stranded RNA by pattern-recognition receptors (RIG-I/MDA-5) and that NKG2D ligand up-regulation can be blocked by the expression of viral dsRNA-binding proteins. Thus, innate immunity-mediated recognition of viral nucleic acids triggers the infected cell to release interferon for NK cell recruitment and to express NKG2D ligands to become more visible to the immune system. Finally, the observation that NKG2D-ligand induction is a consequence of signaling by pattern-recognition receptors that have been selected over evolutionary time to be highly pathogen-specific explains how the risks of autoreactivity in this system are minimized.
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http://dx.doi.org/10.1074/jbc.M117.818393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733586PMC
December 2017

"It is good to take her early to the doctor" - mothers' understanding of childhood pneumonia symptoms and health care seeking in Kilimanjaro region, Tanzania.

BMC Int Health Hum Rights 2017 09 22;17(1):27. Epub 2017 Sep 22.

Department of Public Health Sciences, Global Health - Health System and Policy Research Group, Karolinska Institutet, SE-171 77, Stockholm, Sweden.

Background: Pneumonia is among the leading causes of avoidable deaths for young children globally. The main burden of mortality falls on children from poor and rural families who are less likely to obtain the treatment they need, highlighting inequities in access to effective care and treatment. Caretakers' illness perceptions and care-seeking practices are of major importance for children with pneumonia to receive adequate care. This study qualitatively explores the caretaker concepts of childhood pneumonia in relation to treatment seeking behaviour and health worker management in Moshi urban district, Tanzania.

Methods: In May - July 2013 data was gathered through different qualitative data collection techniques including five focus group discussions (FGDs) with mothers of children under-five years of age. The FGDs involved free listing of pneumonia symptoms and video presentations of children with respiratory symptoms done, these were triangulated with ten case narratives with mothers of children admitted with pneumonia and eleven in-depth interviews with hospital health workers. Transcripts were coded and analysed using qualitative content analysis.

Results: Mothers demonstrated good awareness of common childhood illnesses including pneumonia, which was often associated with symptoms such as cough, flu, chest tightness, fever, and difficulty in breathing. Mothers had mixed views on causative factors and treatments options but generally preferred modern medicine for persisting and severe symptoms. However, all respondent reported access to health facilities as a barrier to care, associated with transport, personal safety and economic constraints.

Conclusion: Local illness concepts and traditional treatment options did not constitute barriers to care for pneumonia symptoms. Poor access to health facilities was the main barrier. Decentralisation of care through community health workers may improve access to care but needs to be combined with strengthened referral systems and accessible hospital care for those in need.
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http://dx.doi.org/10.1186/s12914-017-0135-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5610440PMC
September 2017

Analysis of the recovery of CD247 expression in a PID patient: insights into the spontaneous repair of defective genes.

Blood 2017 09 25;130(10):1205-1208. Epub 2017 Jul 25.

Department of Immunology and Oncology, National Center for Biotechnology and Spanish National Research Council, Madrid, Spain.

Mutations in T-cell antigen receptor (TCR) subunit genes cause rare immunodeficiency diseases characterized by impaired expression of the TCR at the cell surface and selective T lymphopenia. Here, detailed analyses of spontaneously arising somatic mutations that recover CD247, and thus TCR expression, in a newly identified CD247-deficient patient are described. The recovery of CD247 expression in some patient T cells was associated with both reversion of the inactivating mutation and a variant with a compensating mutation that could reconstitute TCR expression, but not as efficiently as wild-type CD247. Multiple mutations were found in CD247 complementary DNAs (cDNAs) cloned from the patient as well as in cDNA and genomic DNA from other individuals, suggesting that genetic variation in this gene is frequent. Analyses of other genes mutated in primary immunodeficiency diseases (PIDs) where reversions have been described also revealed a higher rate of mutation than that observed for genes mutated in PIDs where revertants have not been identified or control genes. These data support the hypothesis that the occurrence of somatic mutations that may reconstitute genetic defects in PID is related to an increased propensity of those genes to mutate.
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http://dx.doi.org/10.1182/blood-2017-01-762864DOI Listing
September 2017

Transmembrane features governing Fc receptor CD16A assembly with CD16A signaling adaptor molecules.

Proc Natl Acad Sci U S A 2017 07 26;114(28):E5645-E5654. Epub 2017 Jun 26.

Department of Immunology and Oncology, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Madrid 28049, Spain;

Many activating immunoreceptors associate with signaling adaptor molecules like FcεR1γ or CD247. FcεR1γ and CD247 share high sequence homology and form disulphide-linked homodimers that contain a pair of acidic aspartic acid residues in their transmembrane (TM) domains that mediate assembly, via interaction with an arginine residue at a similar register to these aspartic acids, with the activating immunoreceptors. However, this model cannot hold true for receptors like CD16A, whose TM domains do not contain basic residues. We have carried out an extensive site-directed mutagenesis analysis of the CD16A receptor complex and now report that the association of receptor with the signaling adaptor depends on a network of polar and aromatic residues along the length of the TM domain. Molecular modeling indicates that CD16A TM residues F, D, and T form the core of the membrane-embedded trimeric interface by establishing highly favorable contacts to the signaling modules through rearrangement of a hydrogen bond network previously identified in the CD247 TM dimer solution NMR structure. Strikingly, the amino acid D also regulates the turnover and surface expression of CD16A in the absence of FcεR1γ or CD247. Modeling studies indicate that similar features underlie the association of other activating immune receptors, including CD64 and FcεR1α, with signaling adaptor molecules, and we confirm experimentally that equivalent F, D, and T residues in the TM domain of FcεR1α markedly influence the biology of this receptor and its association with FcεR1γ.
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http://dx.doi.org/10.1073/pnas.1706483114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5514760PMC
July 2017

Resistance to malaria through structural variation of red blood cell invasion receptors.

Science 2017 06 18;356(6343). Epub 2017 May 18.

Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.

The malaria parasite invades human red blood cells by a series of interactions between host and parasite surface proteins. By analyzing genome sequence data from human populations, including 1269 individuals from sub-Saharan Africa, we identify a diverse array of large copy-number variants affecting the host invasion receptor genes and We find that a nearby association with severe malaria is explained by a complex structural rearrangement involving the loss of and gain of two hybrid genes, which encode a serologically distinct blood group antigen known as Dantu. This variant reduces the risk of severe malaria by 40% and has recently increased in frequency in parts of Kenya, yet it appears to be absent from west Africa. These findings link structural variation of red blood cell invasion receptors with natural resistance to severe malaria.
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http://dx.doi.org/10.1126/science.aam6393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5575826PMC
June 2017

Use of Lentiviral Particles As a Cell Membrane-Based mFasL Delivery System for Treatment of Inflammatory Arthritis.

Front Immunol 2017 21;8:460. Epub 2017 Apr 21.

Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Madrid, Spain.

During budding, lentiviral particles (LVP) incorporate cell membrane proteins in the viral envelope. We explored the possibility of harnessing this process to generate LVP-expressing membrane proteins of therapeutic interest and studied the potential of these tools to treat different pathologies. Fas-mediated apoptosis is central to the maintenance of T cell homeostasis and prevention of autoimmune processes. We prepared LVP that express murine FasL on their surface. Our data indicate that mFasL-bearing LVP induce caspase 3 and 9 processing, cytochrome C release, and significantly more cell death than control LVP . This cytotoxicity is blocked by the caspase inhibitor Z-VAD. Analysis of the application of these reagents for the treatment of inflammatory arthritis suggests that FasL-expressing LVP could be useful for therapy in autoimmune diseases such as rheumatoid arthritis, where there is an excess of Fas-expressing activated T cells in the joint. LVP could be a vehicle not only for mFasL but also for other membrane-bound proteins that maintain their native conformation and might mediate biological activities.
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http://dx.doi.org/10.3389/fimmu.2017.00460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5399037PMC
April 2017

Impact of introduction of rapid diagnostic tests for malaria on antibiotic prescribing: analysis of observational and randomised studies in public and private healthcare settings.

BMJ 2017 Mar 29;356:j1054. Epub 2017 Mar 29.

London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK.

 To examine the impact of use of rapid diagnostic tests for malaria on prescribing of antimicrobials, specifically antibiotics, for acute febrile illness in Africa and Asia. Analysisof nine preselected linked and codesigned observational and randomised studies (eight cluster or individually randomised trials and one observational study). Public and private healthcare settings, 2007-13, in Afghanistan, Cameroon, Ghana, Nigeria, Tanzania, and Uganda. 522 480 children and adults with acute febrile illness. Rapid diagnostic tests for malaria. Proportions of patients for whom an antibiotic was prescribed in trial groups who had undergone rapid diagnostic testing compared with controls and in patients with negative test results compared with patients with positive results. A secondary aim compared classes of antibiotics prescribed in different settings. Antibiotics were prescribed to 127 052/238 797 (53%) patients in control groups and 167 714/283 683 (59%) patients in intervention groups. Antibiotics were prescribed to 40% (35 505/89 719) of patients with a positive test result for malaria and to 69% (39 400/57 080) of those with a negative result. All but one study showed a trend toward more antibiotic prescribing in groups who underwent rapid diagnostic tests. Random effects meta-analysis of the trials showed that the overall risk of antibiotic prescription was 21% higher (95% confidence interval 7% to 36%) in intervention settings. In most intervention settings, patients with negative test results received more antibiotic prescriptions than patients with positive results for all the most commonly used classes: penicillins, trimethoprim-sulfamethoxazole (one exception), tetracyclines, and metronidazole. Introduction of rapid diagnostic tests for malaria to reduce unnecessary use of antimalarials-a beneficial public health outcome-could drive up untargeted use of antibiotics. That 69% of patients were prescribed antibiotics when test results were negative probably represents overprescription.This included antibiotics from several classes, including those like metronidazole that are seldom appropriate for febrile illness, across varied clinical, health system, and epidemiological settings. It is often assumed that better disease specific diagnostics will reduce antimicrobial overuse, but they might simply shift it from one antimicrobial class to another. Current global implementation of malaria testing might increase untargeted antibiotic use and must be examined.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5370398PMC
http://dx.doi.org/10.1136/bmj.j1054DOI Listing
March 2017

Characterisation of the opposing effects of G6PD deficiency on cerebral malaria and severe malarial anaemia.

Elife 2017 01 9;6. Epub 2017 Jan 9.

Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is believed to confer protection against malaria, but the precise nature of the protective effecthas proved difficult to define as G6PD deficiency has multiple allelic variants with different effects in males and females, and it has heterogeneous effects on the clinical outcome of infection. Here we report an analysis of multiple allelic forms of G6PD deficiency in a large multi-centre case-control study of severe malaria, using the WHO classification of G6PD mutations to estimate each individual's level of enzyme activity from their genotype. Aggregated across all genotypes, we find that increasing levels of G6PD deficiency are associated with decreasing risk of cerebral malaria, but with increased risk of severe malarial anaemia. Models of balancing selection based on these findings indicate that an evolutionary trade-off between different clinical outcomes of infection could have been a major cause of the high levels of G6PD polymorphism seen in human populations.
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http://dx.doi.org/10.7554/eLife.15085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5222559PMC
January 2017

Point-of-care assessment of C-reactive protein and white blood cell count to identify bacterial aetiologies in malaria-negative paediatric fevers in Tanzania.

Trop Med Int Health 2017 03 28;22(3):286-293. Epub 2016 Dec 28.

Joint Malaria Programme, St Augustine's Hospital, Muheza, Tanzania.

Objective: To assess the role of point-of-care (PoC) assessment of C-reactive protein (CRP) and white blood cell (WBC) count to identify bacterial illness in Tanzanian children with non-severe non-malarial fever.

Methods: From the outpatient department of a district hospital in Tanzania, 428 patients between 3 months and 5 years of age who presented with fever and a negative malaria test were enrolled. All had a physical examination and bacterial cultures from blood and urine. Haemoglobin, CRP and WBC were measured by PoC devices.

Results: Positive blood cultures were detected in 6/428 (1.4%) children and urine cultures were positive in 24/401 (6.0%). Mean WBC was similar in children with or without bacterial illness (14.0 × 10 , 95% CI 12.0-16.0 × 109 vs. 12.0 × 10 , 95% CI 11.4-12.7 × 109), while mean CRP was higher in children with bacterial illness (41.0 mg/l, 95% CI 28.3-53.6 vs. 23.8 mg/l, 95% CI 17.8-27.8). In ROC analysis, the optimum cut-off value for CRP to identify bacterial illness was 19 mg/l but with an area under the curve of only 0.62. Negative predictive values exceeded 80%, while positive predictive values were under 40%.

Conclusion: WBC and CRP levels had limited value in identifying children with bacterial infections. The positive predictive values for both tests were too low to be used as single tools for treatment decisions.
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http://dx.doi.org/10.1111/tmi.12823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5336187PMC
March 2017

Variability of respiratory rate measurements in children suspected with non-severe pneumonia in north-east Tanzania.

Trop Med Int Health 2017 02 12;22(2):139-147. Epub 2016 Dec 12.

Kilimanjaro Christian Medical University College, Moshi, Tanzania.

Objective: Measurement of respiratory rate is an important clinical sign in the diagnosis of pneumonia but suffers from interobserver variation. Here, we assess the use of video recordings as a quality assurance tool that could be useful both in research and in training of staff.

Methods: Respiratory rates (RR) were recorded in children aged 2-59 months presenting with cough or difficulty breathing at two busy outpatient clinics in Tanzania. Measurements were repeated at 10-min intervals in a quiet environment with simultaneous video recordings that were independently reviewed by two paediatricians.

Results: Eight hundred and fifty-nine videos were sent to two paediatricians; 148 (17.2%) were considered unreadable by one or both. For the 711 (82.8%) videos that were readable by both paediatricians, there was perfect agreement for the presence of raised RR with a kappa value (κ) of 0.85 (P < 0.001); and in 476 (66.9%) cases, both paediatricians agreed on the RR within 2 breaths per minute (±2 bpm). A reported illness of 5 days or more was associated with unreadable video recordings (OR = 3.44, CI: 1.5-6.08; P < 0.001). The multilevel model showed that differences between observers accounted for only 13% of the variability in RR.

Conclusion: Video recordings are reliable tools for quality assurance of RR measurements in children with suspected pneumonia. Videos with a clear view of respiratory movements may also be useful in training primary healthcare staff.
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http://dx.doi.org/10.1111/tmi.12814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5299505PMC
February 2017

Examining Intervention Design: Lessons from the Development of Eight Related Malaria Health Care Intervention Studies.

Health Syst Reform 2016 Oct 15;2(4):373-388. Epub 2016 Sep 15.

Disease Control Department , London School of Hygiene & Tropical Medicine , London , UK.

-Rigorous evidence of "what works" to improve health care is in demand, but methods for the development of interventions have not been scrutinized in the same ways as methods for evaluation. This article presents and examines intervention development processes of eight malaria health care interventions in East and West Africa. A case study approach was used to draw out experiences and insights from multidisciplinary teams who undertook to design and evaluate these studies. Four steps appeared necessary for intervention design: (1) definition of scope, with reference to evaluation possibilities; (2) research to inform design, including evidence and theory reviews and empirical formative research; (3) intervention design, including consideration and selection of approaches and development of activities and materials; and (4) refining and finalizing the intervention, incorporating piloting and pretesting. Alongside these steps, projects produced theories, explicitly or implicitly, about (1) intended pathways of change and (2) how their intervention would be implemented.The work required to design interventions that meet and contribute to current standards of evidence should not be underestimated. Furthermore, the process should be recognized not only as technical but as the result of micro and macro social, political, and economic contexts, which should be acknowledged and documented in order to infer generalizability. Reporting of interventions should go beyond descriptions of final intervention components or techniques to encompass the development process. The role that evaluation possibilities play in intervention design should be brought to the fore in debates over health care improvement.
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http://dx.doi.org/10.1080/23288604.2016.1179086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176770PMC
October 2016

Primary T-cell immunodeficiency with functional revertant somatic mosaicism in CD247.

J Allergy Clin Immunol 2017 01 21;139(1):347-349.e8. Epub 2016 Aug 21.

Department of Immunology, Complutense University School of Medicine and Hospital, 12 de Octubre Health Research Institute, Madrid, Spain; Department of Microbiology and Immunology, The University of Melbourne, Melbourne, Australia.

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http://dx.doi.org/10.1016/j.jaci.2016.06.020DOI Listing
January 2017

Development of a rapid lateral flow immunoassay test for detection of exosomes previously enriched from cell culture medium and body fluids.

J Extracell Vesicles 2016 12;5:31803. Epub 2016 Aug 12.

Departamento de Química Física y Analítica, Universidad de Oviedo, Julián Clavería Oviedo, Spain;

Exosomes are cell-secreted nanovesicles (40-200 nm) that represent a rich source of novel biomarkers in the diagnosis and prognosis of certain diseases. Despite the increasingly recognized relevance of these vesicles as biomarkers, their detection has been limited due in part to current technical challenges in the rapid isolation and analysis of exosomes. The complexity of the development of analytical platforms relies on the heterogeneous composition of the exosome membrane. One of the most attractive tests is the inmunochromatographic strips, which allow rapid detection by unskilled operators. We have successfully developed a novel lateral flow immunoassay (LFIA) for the detection of exosomes based on the use of tetraspanins as targets. We have applied this platform for the detection of exosomes purified from different sources: cell culture supernatants, human plasma and urine. As proof of concept, we explored the analytical potential of this LFIA platform to accurately quantify exosomes purified from a human metastatic melanoma cell line. The one-step assay can be completed in 15 min, with a limit of detection of 8.54×10(5) exosomes/µL when a blend of anti-CD9 and anti-CD81 were selected as capture antibodies and anti-CD63 labelled with gold nanoparticles as detection antibody. Based on our results, this platform could be well suited to be used as a rapid exosome quantification tool, with promising diagnostic applications, bearing in mind that the detection of exosomes from different sources may require adaptation of the analytical settings to their specific composition.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4985618PMC
http://dx.doi.org/10.3402/jev.v5.31803DOI Listing
August 2016

The feasibility and acceptability of screening for hypertension in private drug retail outlets: a pilot study in Mwanza region, Tanzania.

Int Health 2016 09 11;8(5):360-6. Epub 2016 May 11.

Kilimanjaro Christian Medical University College (Tumaini University), Department of Epidemiology and Biostatistics, P.O. Box 2240, Moshi, Tanzania The London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK

Background: Hypertension is a major contributor to ill health in sub-Saharan Africa. Developing countries need to increase access for screening. This study assesses the feasibility and acceptability of using private sector drug retail outlets to screen for hypertension in Mwanza region, Tanzania.

Methods: A pilot study took place in eight drug retail outlets from August 2013 to February 2014. Customers ≥18 years were invited for screening. Socio-demographic characteristics, hypertension knowledge, hypertension screening and treatment history were collected. Subjects with systolic blood pressure over 140 mmHg were referred for follow up. Referral slips captured attendance. Mystery client visits and follow up phone calls were conducted to assess service quality.

Results: A total of 971 customers were screened, one person refused; 109 (11.2%) had blood pressure over 140/90 mmHg and were referred for ongoing assessment; 85/109 (78.0%) were newly diagnosed. Customers reported that the service was acceptable. Service providers were able to follow the protocol. Only 18/85 (21%) newly diagnosed participants visited the referral clinic within two weeks.

Conclusions: Blood pressure screening was feasible and acceptable to customers of private drug retail outlets. However many who were referred failed to attend at a referral centre and further research is needed in this area.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5039818PMC
http://dx.doi.org/10.1093/inthealth/ihw023DOI Listing
September 2016

Ionomycin Treatment Renders NK Cells Hyporesponsive.

PLoS One 2016 23;11(3):e0150998. Epub 2016 Mar 23.

Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, CNB-CSIC, Madrid, Spain.

Natural killer cells are cytotoxic lymphocytes important in immune responses to cancer and multiple pathogens. However, chronic activation of NK cells can induce a hyporesponsive state. The molecular basis of the mechanisms underlying the generation and maintenance of this hyporesponsive condition are unknown, thus an easy and reproducible mechanism able to induce hyporesponsiveness on human NK cells would be very useful to gain understanding of this process. Human NK cells treated with ionomycin lose their ability to degranulate and secrete IFN-γ in response to a variety of stimuli, but IL-2 stimulation can compensate these defects. Apart from reductions in the expression of CD11a/CD18, no great changes were observed in the activating and inhibitory receptors expressed by these NK cells, however their transcriptional signature is different to that described for other hyporesponsive lymphocytes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150998PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4805247PMC
July 2016