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AIMS/HYPOTHESIS: Infection with coxsackie B viruses (CVBs) can cause diseases ranging from mild common cold-type symptoms to severe life-threatening conditions. CVB infections are considered to be prime candidates for environmental triggers of type 1 diabetes. This, together with the significant disease burden of acute CVB infections and their association with chronic diseases other than diabetes, has prompted the development of human CVB vaccines. The current study evaluated the safety and immunogenicity of the first human vaccine designed against CVBs associated with type 1 diabetes in a double-blind randomised placebo-controlled Phase I trial. METHODS: The main eligibility criteria for participants were good general health, age between 18 and 45 years, provision of written informed consent and willingness to comply with all trial procedures. Treatment allocation (PRV-101 or placebo) was based on a computer-generated randomisation schedule and people assessing the outcomes were masked to group assignment. In total, 32 participants (17 men, 15 women) aged 18-44 years were randomised to receive a low (n=12) or high (n=12) dose of a multivalent, formalin-inactivated vaccine including CVB serotypes 1-5 (PRV-101), or placebo (n=8), given by intramuscular injections at weeks 0, 4 and 8 at a single study site in Finland. The participants were followed for another 24 weeks. Safety and tolerability were the primary endpoints. Anti-CVB IgG and virus-neutralising titres were analysed using an ELISA and neutralising plaque reduction assays, respectively. RESULTS: Among the 32 participants (low dose, n=12; high dose, n=12; placebo, n=8) no serious adverse events or adverse events leading to study treatment discontinuation were observed. Treatment-emergent adverse events considered to be related to the study drug occurred in 37.5% of the participants in the placebo group and 62.5% in the PRV-101 group (injection site pain, headache, injection site discomfort and injection site pruritus being most common). PRV-101 induced dose-dependent neutralising antibody responses against all five CVB serotypes included in the vaccine in both the high- and low-dose groups. Protective titres ≥8 against all five serotypes were seen in >90% of participants over the entire follow-up period. CONCLUSIONS/INTERPRETATION: The results indicate that the tested multivalent CVB vaccine is well tolerated and immunogenic, supporting its further clinical development. TRIAL REGISTRATION: ClinicalTrials.gov NCT04690426. FUNDING: This trial was funded by Provention Bio, a Sanofi company.
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Diabetes Mellitus Tipo 1 , Adolescente , Adulto , Femenino , Humanos , Masculino , Adulto Joven , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Método Doble Ciego , Vacunación , Vacunas CombinadasRESUMEN
Among the environmental factors associated with type 1 diabetes (T1D), viral infections of the gut and pancreas has been investigated most intensely, identifying enterovirus infections as the prime candidate trigger of islet autoimmunity (IA) and T1D development. However, the association between respiratory tract infections (RTI) and IA/T1D is comparatively less known. While there are significant amounts of epidemiological evidence supporting the role of respiratory infections in T1D, there remains a paucity of data characterising infectious agents at the molecular level. This gap in the literature precludes the identification of the specific infectious agents driving the association between RTI and T1D. Furthermore, the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections on the development of IA/T1D remains undeciphered. Here, we provide a comprehensive overview of the evidence to date, implicating RTIs (viral and non-viral) as potential risk factors for IA/T1D.
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COVID-19 , Diabetes Mellitus Tipo 1 , Islotes Pancreáticos , Infecciones del Sistema Respiratorio , Humanos , Islotes Pancreáticos/patología , COVID-19/patología , SARS-CoV-2 , Infecciones del Sistema Respiratorio/patologíaRESUMEN
This study investigated whether children with HLA-DQ-conferred risk for type 1 diabetes (T1D) have an altered immune response to the widely-used enterovirus vaccine, namely poliovirus vaccine, and whether initiation of autoimmunity to pancreatic islets modulates this response. Neutralizing antibodies induced by the inactivated poliovirus vaccine against poliovirus type 1 (Salk) were analysed as a marker of protective immunity at the age of 18 months in a prospective birth cohort. No differences were observed in antibody titers between children with and without genetic risk for T1D (odds ratio [OR] = 0.90 [0.83, 1.06], p = 0.30). In the presence of the genetic risk, no difference was observed between children with and without islet autoimmunity (OR = 1.00 [0.78, 1.28], p = 1.00). This did not change when only children with the autoimmunity before 18 months of age were included in the analyses (OR = 1.00 [0.85, 1.18], p = 1.00). No effect was observed when groups were stratified based on autoantigen specificity of the first-appearing autoantibody (IAA or GADA). The children in each comparison group were matched for sex, calendar year and month of birth, and municipality. Accordingly, we found no indication that children who are at risk to develop islet autoimmunity would have a compromised humoral immune response which could have increased their susceptibility for enterovirus infections. In addition, the proper immune response supports the idea of testing novel enterovirus vaccines for the prevention of T1D among these individuals.
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Diabetes Mellitus Tipo 1 , Infecciones por Enterovirus , Enterovirus , Islotes Pancreáticos , Niño , Humanos , Lactante , Anticuerpos Neutralizantes , Estudios Prospectivos , Infecciones por Enterovirus/prevención & control , Autoanticuerpos , Vacuna Antipolio de Virus Inactivados , Antígenos HLA-DQ/genéticaRESUMEN
AIMS/HYPOTHESIS: The Diabetes Virus Detection (DiViD) study is the first study to laparoscopically collect pancreatic tissue and purified pancreatic islets together with duodenal mucosa, serum, peripheral blood mononuclear cells (PBMCs) and stools from six live adult patients (age 24-35 years) with newly diagnosed type 1 diabetes. The presence of enterovirus (EV) in the pancreatic islets of these patients has previously been reported. METHODS: In the present study we used reverse transcription quantitative real-time PCR (RT-qPCR) and sequencing to characterise EV genomes present in different tissues to understand the nature of infection in these individuals. RESULTS: All six patients were found to be EV-positive by RT-qPCR in at least one of the tested sample types. Four patients were EV-positive in purified islet culture medium, three in PBMCs, one in duodenal biopsy and two in stool, while serum was EV-negative in all individuals. Sequencing the 5' untranslated region of these EVs suggested that all but one belonged to enterovirus B species. One patient was EV-positive in all these sample types except for serum. Sequence analysis revealed that the virus strain present in the isolated islets of this patient was different from the strain found in other sample types. None of the islet-resident viruses could be isolated using EV-permissive cell lines. CONCLUSIONS/INTERPRETATION: EV RNA can be frequently detected in various tissues of patients with type 1 diabetes. At least in some patients, the EV strain in the pancreatic islets may represent a slowly replicating persisting virus.
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Diabetes Mellitus Tipo 1/virología , Infecciones por Enterovirus/virología , Enterovirus/aislamiento & purificación , Islotes Pancreáticos/virología , ARN Viral/genética , Adulto , Línea Celular , Diabetes Mellitus Tipo 1/diagnóstico , Enterovirus/genética , Heces/virología , Femenino , Humanos , Masculino , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto JovenRESUMEN
AIMS/HYPOTHESIS: Enteroviral infection has been implicated in the development of islet autoimmunity in type 1 diabetes and enteroviral antigen expression has been detected by immunohistochemistry in the pancreatic beta cells of patients with recent-onset type 1 diabetes. However, the immunohistochemical evidence relies heavily on the use of a monoclonal antibody, clone 5D8/1, raised against an enteroviral capsid protein, VP1. Recent data suggest that the clone 5D8/1 may also recognise non-viral antigens; in particular, a component of the mitochondrial ATP synthase (ATP5B) and an isoform of creatine kinase (CKB). Therefore, we evaluated the fidelity of immunolabelling by clone 5D8/1 in the islets of patients with type 1 diabetes. METHODS: Enteroviral VP1, CKB and ATP5B expression were analysed by western blotting, RT-PCR and immunocytochemistry in a range of cultured cell lines, isolated human islets and human tissue. RESULTS: Clone 5D8/1 labelled CKB, but not ATP5B, on western blots performed under denaturing conditions. In cultured human cell lines, isolated human islets and pancreas sections from patients with type 1 diabetes, the immunolabelling of ATP5B, CKB and VP1 by 5D8/1 was readily distinguishable. Moreover, in a human tissue microarray displaying more than 80 different cells and tissues, only two (stomach and colon; both of which are potential sites of enterovirus infection) were immunopositive when stained with clone 5D8/1. CONCLUSIONS/INTERPRETATION: When used under carefully optimised conditions, the immunolabelling pattern detected in sections of human pancreas with clone 5D8/1 did not reflect cross-reactivity with either ATP5B or CKB. Rather, 5D8/1 is likely to be representative of enteroviral antigen expression.
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Anticuerpos Monoclonales/metabolismo , Proteínas de la Cápside/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Infecciones por Enterovirus/metabolismo , Enterovirus/metabolismo , Páncreas/metabolismo , Antígenos Virales/metabolismo , Western Blotting , Proliferación Celular , Células Cultivadas , Reacciones Cruzadas , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/virología , Infecciones por Enterovirus/complicaciones , Infecciones por Enterovirus/inmunología , Femenino , Humanos , Inmunohistoquímica , Células Secretoras de Insulina/metabolismo , Masculino , Páncreas/inmunología , Páncreas/virología , Reproducibilidad de los Resultados , Replicación ViralRESUMEN
Coxsackievirus B (CVB) infection of pancreatic ß cells is associated with ß cell autoimmunity and type 1 diabetes. We investigated how CVB affects human ß cells and anti-CVB T cell responses. ß cells were efficiently infected by CVB in vitro, down-regulated human leukocyte antigen (HLA) class I, and presented few, selected HLA-bound viral peptides. Circulating CD8+ T cells from CVB-seropositive individuals recognized a fraction of these peptides; only another subfraction was targeted by effector/memory T cells that expressed exhaustion marker PD-1. T cells recognizing a CVB epitope cross-reacted with ß cell antigen GAD. Infected ß cells, which formed filopodia to propagate infection, were more efficiently killed by CVB than by CVB-reactive T cells. Our in vitro and ex vivo data highlight limited CD8+ T cell responses to CVB, supporting the rationale for CVB vaccination trials for type 1 diabetes prevention. CD8+ T cells recognizing structural and nonstructural CVB epitopes provide biomarkers to differentially follow response to infection and vaccination.
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Infecciones por Coxsackievirus , Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Humanos , Linfocitos T CD8-positivos , Anticuerpos , Epítopos , Péptidos , AntiviralesRESUMEN
Although the genetic basis and pathogenesis of type 1 diabetes have been studied extensively, how host responses to environmental factors might contribute to autoantibody development remains largely unknown. Here, we use longitudinal blood transcriptome sequencing data to characterize host responses in children within 12 months prior to the appearance of type 1 diabetes-linked islet autoantibodies, as well as matched control children. We report that children who present with insulin-specific autoantibodies first have distinct transcriptional profiles from those who develop GADA autoantibodies first. In particular, gene dosage-driven expression of GSTM1 is associated with GADA autoantibody positivity. Moreover, compared with controls, we observe increased monocyte and decreased B cell proportions 9-12 months prior to autoantibody positivity, especially in children who developed antibodies against insulin first. Lastly, we show that control children present transcriptional signatures consistent with robust immune responses to enterovirus infection, whereas children who later developed islet autoimmunity do not. These findings highlight distinct immune-related transcriptomic differences between case and control children prior to case progression to islet autoimmunity and uncover deficient antiviral response in children who later develop islet autoimmunity.
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Diabetes Mellitus Tipo 1 , Infecciones por Enterovirus , Islotes Pancreáticos , Humanos , Niño , Autoanticuerpos , Transcriptoma , Autoinmunidad/genética , Insulina/metabolismo , Infecciones por Enterovirus/genética , Islotes Pancreáticos/metabolismoRESUMEN
Coxsackievirus B (CVB) infection of pancreatic ß cells is associated with ß-cell autoimmunity. We investigated how CVB impacts human ß cells and anti-CVB T-cell responses. ß cells were efficiently infected by CVB in vitro, downregulated HLA Class I and presented few, selected HLA-bound viral peptides. Circulating CD8+ T cells from CVB-seropositive individuals recognized only a fraction of these peptides, and only another sub-fraction was targeted by effector/memory T cells that expressed the exhaustion marker PD-1. T cells recognizing a CVB epitope cross-reacted with the ß-cell antigen GAD. Infected ß cells, which formed filopodia to propagate infection, were more efficiently killed by CVB than by CVB-reactive T cells. Thus, our in-vitro and ex-vivo data highlight limited T-cell responses to CVB, supporting the rationale for CVB vaccination trials for type 1 diabetes prevention. CD8+ T cells recognizing structural and non-structural CVB epitopes provide biomarkers to differentially follow response to infection and vaccination.
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The incidence of immune-mediated diseases (IMDs) is increasing rapidly in the developed countries constituting a huge medical, economic, and societal challenge. The exposome plays an important role since genetic factors cannot explain such a rapid change. In the Human Exposomic Determinants of Immune Mediated Diseases (HEDIMED) project, altogether 22 academic and industrial partners join their multidisciplinary forces to identify exposomic determinants that are driving the IMD epidemic. The project is based on a combination of data and biological samples from large clinical cohorts constituting about 350,000 pregnant women, 30,000 children prospectively followed from birth, and 7,000 children from cross-sectional studies. HEDIMED focuses on common chronic IMDs that cause a significant disease burden, including type 1 diabetes, celiac disease, allergy, and asthma. Exposomic disease determinants and the underlying biological pathways will be identified by an exploratory approach using advanced omics and multiplex technologies combined with cutting-edge data mining technologies. Emphasis is put on fetal and childhood exposome since the IMD disease processes start early. Inclusion of several IMDs makes it possible to identify common exposomic determinants for the diseases, thus facilitating the development of widely operating preventive and curative treatments. HEDIMED includes data and samples from birth cohorts and clinical trials that have used exposomic interventions and cell and organ culture models to identify mechanisms of the observed associations. Importantly, HEDIMED generates a toolbox that offers science-based functional tools for key stakeholders to control the IMD epidemic. Altogether, HEDIMED aims at innovations, which become widely exploited in diagnostic, therapeutic, preventive, and health economic approaches.
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Enteroviruses, particularly the group B coxsackieviruses (CVBs), have been associated with the development of type 1 diabetes. Several CVB serotypes establish chronic infections in human cells in vivo and in vitro. However, the mechanisms leading to enterovirus persistency and, possibly, beta cell autoimmunity are not fully understood. We established a carrier-state-type persistent infection model in human pancreatic cell line PANC-1 using two distinct CVB1 strains and profiled the infection-induced changes in cellular transcriptome. In the current study, we observed clear changes in the gene expression of factors associated with the pancreatic microenvironment, the secretory pathway, and lysosomal biogenesis during persistent CVB1 infections. Moreover, we found that the antiviral response pathways were activated differently by the two CVB1 strains. Overall, our study reveals extensive transcriptional responses in persistently CVB1-infected pancreatic cells with strong opposite but also common changes between the two strains.
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Coxsackie B (CVB) viruses have been associated with type 1 diabetes. We have recently observed that CVB1 was linked to the initiation of the autoimmune process leading to type 1 diabetes in Finnish children. Viral persistency in the pancreas is currently considered as one possible mechanism. In the current study persistent infection was established in pancreatic ductal and beta cell lines (PANC-1 and 1.1B4) using four different CVB1 strains, including the prototype strain and three clinical isolates. We sequenced 5' untranslated region (UTR) and regions coding for structural and non-structural proteins and the second single open reading frame (ORF) protein of all persisting CVB1 strains using next generation sequencing to identify mutations that are common for all of these strains. One mutation, K257R in VP1, was found from all persisting CVB1 strains. The mutations were mainly accumulated in viral structural proteins, especially at BC, DE, EF loops and C-terminus of viral capsid protein 1 (VP1), the puff region of VP2, the knob region of VP3 and infection-enhancing epitope of VP4. This showed that the capsid region of the viruses sustains various changes during persistency some of which could be hallmark(s) of persistency.
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Using immunohistochemistry, enterovirus capsid proteins were demonstrated in pancreatic islets of patients with type 1 diabetes. Virus proteins are mainly located in beta cells, supporting the hypothesis that enterovirus infections may contribute to the pathogenesis of type 1 diabetes. In samples of pancreatic tissue, enterovirus RNA was also detected, but in extremely small quantities and in a smaller proportion of cases compared to the enteroviral protein. Difficulties in detecting viral RNA could be due to the very small number of infected cells, the possible activity of PCR inhibitors, and the presence-during persistent infection-of the viral genome in unencapsidated forms. The aim of this study was twofold: (a) to examine if enzymes or other compounds in pancreatic tissue could affect the molecular detection of encapsidated vs. unencapsidated enterovirus forms, and (b) to compare the sensitivity of RT-PCR methods used in different laboratories. Dilutions of encapsidated and unencapsidated virus were spiked into human pancreas homogenate and analyzed by RT-PCR. Incubation of pancreatic homogenate on wet ice for 20 h did not influence the detection of encapsidated virus. In contrast, a 15-min incubation on wet ice dramatically reduced detection of unencapsidated forms of virus. PCR inhibitors could not be found in pancreatic extract. The results show that components in the pancreas homogenate may selectively affect the detection of unencapsidated forms of enterovirus. This may lead to difficulties in diagnosing persisting enterovirus infection in the pancreas of patients with type 1 diabetes.
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Proteínas de la Cápside/metabolismo , Diabetes Mellitus Tipo 1/virología , Infecciones por Enterovirus/complicaciones , Enterovirus/genética , ARN Viral/metabolismo , Diabetes Mellitus Tipo 1/etiología , Enterovirus Humano B/genética , Infecciones por Enterovirus/virología , Humanos , Células Secretoras de Insulina/enzimología , Células Secretoras de Insulina/virología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The current methods to study the distribution and dynamics of viral RNA molecules inside infected cells are not ideal, as electron microscopy and immunohistochemistry can only detect mature virions, and quantitative real-time PCR does not reveal localized distribution of RNAs. We demonstrated here the branched DNA in situ hybridization (bDNA ISH) technology to study both the amount and location of the emerging -RNA and +RNA during acute and persistent enterovirus infections. According to our results, the replication of the viral RNA started 2-3 h after infection and the translation shortly after at 3-4 h post-infection. The replication hotspots with newly emerging -RNA were located quite centrally in the cell, while the +RNA production and most likely virion assembly took place in the periphery of the cell. We also discovered that the pace of replication of -RNA and +RNA strands was almost identical, and -RNA was absent during antiviral treatments. ViewRNA ISH with our custom probes also showed a good signal during acute and persistent enterovirus infections in cell and mouse models. Considering these results, along with the established bDNA FISH protocol modified by us, the effects of antiviral drugs and the emergence of enterovirus RNAs in general can be studied more effectively.
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Anticuerpos Monoclonales/metabolismo , Proteínas de la Cápside/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Infecciones por Enterovirus/metabolismo , Enterovirus/metabolismo , Inmunohistoquímica/métodos , Oligopéptidos/inmunología , Páncreas/metabolismo , Femenino , Humanos , MasculinoRESUMEN
The group B Coxsackieviruses (CVB), belonging to the Enterovirus genus, can establish persistent infections in human cells. These persistent infections have been linked to chronic diseases including type 1 diabetes. Still, the outcomes of persistent CVB infections in human pancreas are largely unknown. We established persistent CVB infections in a human pancreatic ductal-like cell line PANC-1 using two distinct CVB1 strains and profiled infection-induced changes in cellular protein expression and secretion using mass spectrometry-based proteomics. Persistent infections, showing characteristics of carrier-state persistence, were associated with a broad spectrum of changes, including changes in mitochondrial network morphology and energy metabolism and in the regulated secretory pathway. Interestingly, the expression of antiviral immune response proteins, and also several other proteins, differed clearly between the two persistent infections. Our results provide extensive information about the protein-level changes induced by persistent CVB infection and the potential virus-associated variability in the outcomes of these infections.
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Enteroviruses (EVs) are common RNA viruses that cause diseases ranging from rash to paralytic poliomyelitis. For example, EV-A and EV-C viruses cause hand-foot and mouth disease and EV-B viruses cause encephalitis and myocarditis, which can result in severe morbidity and mortality. While new vaccines and treatments for EVs are under development, methods for studying and diagnosing EV infections are still limited and therefore new diagnostic tools are required. Our aim was to produce and characterize new antibodies that work in multiple applications and detect EVs in tissues and in vitro. Rats were immunized with Coxsackievirus B1 capsid protein VP1 and hybridomas were produced. Hybridoma clones were selected based on their reactivity in different immunoassays. The most promising clone, 3A6, was characterized and it performed well in multiple techniques including ELISA, immunoelectron microscopy, immunocyto- and histochemistry and in Western blotting, detecting EVs in infected cells and tissues. It recognized several EV-Bs and also the EV-C representative Poliovirus 3, making it a broad-spectrum EV specific antibody. The 3A6 rat monoclonal antibody can help to overcome some of the challenges faced with commonly used EV antibodies: it enables simultaneous use of mouse-derived antibodies in double staining and it is useful in murine models.
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Anticuerpos Monoclonales/inmunología , Proteínas de la Cápside/inmunología , Enterovirus Humano B/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Proteínas de la Cápside/química , Enterovirus Humano B/clasificación , Enterovirus Humano B/ultraestructura , Infecciones por Enterovirus/inmunología , Infecciones por Enterovirus/virología , Ensayo de Inmunoadsorción Enzimática , Epítopos , Humanos , Inmunohistoquímica , Ratones , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios Proteicos/inmunología , RatasRESUMEN
Enteroviruses, specifically of the Coxsackie B virus family, have been implicated in triggering islet autoimmunity and type 1 diabetes, but their presence in pancreata of patients with diabetes has not been fully confirmed.To detect the presence of very low copies of the virus genome in tissue samples from T1D patients, we designed a panel of fluorescently labeled oligonucleotide probes, each of 17-22 nucleotides in length with a unique sequence to specifically bind to the enteroviral genome of the picornaviridae family.With these probes enteroviral RNA was detected with high sensitivity and specificity in infected cells and tissues, including in FFPE pancreas sections from patients with T1D. Detection was not impeded by variations in sample processing and storage thereby overcoming the potential limitations of fragmented RNA. Co-staining of small RNA probes in parallel with classical immunstaining enabled virus detection in a cell-specific manner and more sensitively than by viral protein.
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Diabetes Mellitus Tipo 1/virología , Hibridación Fluorescente in Situ/métodos , Sondas de Oligonucleótidos , Páncreas/virología , Enterovirus , Infecciones por Enterovirus/diagnóstico , Colorantes Fluorescentes , Humanos , Inmunohistoquímica , Reacción en Cadena de la Polimerasa , ARN Viral/análisis , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Enteroviruses (EVs) have been linked to the pathogenesis of several diseases and there is a collective need to develop improved methods for the detection of these viruses in tissue samples. OBJECTIVES: This study evaluates the relative sensitivity of immunohistochemistry (IHC), proteomics, in situ hybridization (ISH) and RT-PCR to detect one common EV, Coxsackievirus B1 (CVB1), in acutely infected human A549 cells in vitro. STUDY DESIGN: A549 cells were infected with CVB1 and diluted with uninfected A549 cells to produce a limited dilution series in which the proportion of infected cells ranged from 10(-1) to 10(-8). Analyses were carried out by several laboratories using IHC with different anti-EV antibodies, ISH with both ViewRNA and RNAScope systems, liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM/MS/MS), and two modifications of RT-PCR. RESULTS: RT-PCR was the most sensitive method for EV detection yielding positive signals in the most diluted sample (10(-8)). LC/MRM/MS/MS detected viral peptides at dilutions as high as 10(-7). The sensitivity of IHC depended on the antibody used, and the most sensitive antibody (Dako clone 5D8/1) detected virus proteins at a dilution of 10(-6), while ISH detected the virus at dilutions of 10(-4). CONCLUSIONS: All methods were able to detect CVB1 in infected A549 cells. RT-PCR was most sensitive followed by LC/MRM/MS/MS and then IHC. The results from this in vitro survey suggest that all methods are suitable tools for EV detection but that their differential sensitivities need to be considered when interpreting the results from such studies.