Human cytomegalovirus-specific CD4+ and CD8+ T-cell reconstitution in adult allogeneic hematopoietic stem cell transplant recipients and immune control of viral infection

Background Human cytomegalovirus infection is the most frequent viral complication in patients undergoing hematopoietic stem cell transplantation. We investigated the development of human cytomegalovirus-specific T cells in adult recipients of hematopoietic stem cell transplants. Design and Methods From May 2003 through October 2006 a total of 45 patients were monitored for human cytomegalovirus-specific T-cell reconstitution. Human cytomegalovirus-infected autologous dendritic cells were used as a stimulus to detect interferon-γ-producing human cytomegalovirus-specific CD8+ and CD4+ T cells during the first year after transplantation. Interleukin-2 production by specific T cells was also determined. ![Figure 1.][1] Figure 1. Probability of HCMV infection development and HCMV-specific CD4+ and CD8+ T-cell immunity reconstitution. A: cumulative incidence curves of HCMV infection according to donor (D) and recipient (R) HCMV-serostatus. B: cumulative incidence curves of HCMV infection and HCMV-specific CD8+ and CD4+ T-cell reconstitution (i.e. corresponding to a specific T-cell number greater than 0.4 cells/μL blood). C: cumulative incidence curves of HCMV-specific CD8+ T-cell reconstitution according to D/R HCMV-serostatus. D: cumulative incidence curves of HCMV-specific CD4+ T-cell reconstitution according to D/R HCMV-serostatus. Results Human cytomegalovirus infection was detected in the blood of 39/45 patients at a median of 29 days after transplantation. Human cytomegalovirus-specific T-cell reconstitution followed reactivation of latent human cytomegalovirus infection at a median time of about 2 months after transplantation. Only donor human cytomegalovirus-seronegativity and bone marrow as a stem cell source were found to delay specific T-cell reconstitution significantly. Levels of three CD8+ and one CD4+ human cytomegalovirus-specific T-cells/μL blood had a positive predictive value of around 80% for identifying patients able to control human cytomegalovirus infection spontaneously. Five patients who received high doses of steroids for treatment of graft-versus-host disease developed human cytomegalovirus infection requiring pre-emptive treatment despite high levels of interferon-γ-producing T cells in response to human cytomegalovirus. Specific interleukin-2 production was not detected in patients with human cytomegalovirus infection requiring treatment, while 90% of patients who spontaneously controlled human cytomegalovirus infection had T cells that produced interleukin-2 and interferon-γ. Conclusions Pre-transplant human cytomegalovirus infection of the recipient is a major factor driving human cytomegalovirus-specific immune reconstitution. Control of human cytomegalovirus infection likely requires the presence of both interferon-γ and interleukin-2 producing T cells. Corticosteroid treatment may favor active viral replication even in patients with specific T cells. [1]: pending:ye

DOF-binding sites additively contribute to guard cell-specificity of AtMYB60 promoter

<p>Abstract</p> <p>Background</p> <p>We previously demonstrated that the <it>Arabidopsis thaliana </it>AtMYB60 protein is an R2R3MYB transcription factor required for stomatal opening. <it>AtMYB60 </it>is specifically expressed in guard cells and down-regulated at the transcriptional levels by the phytohormone ABA.</p> <p>Results</p> <p>To investigate the molecular mechanisms governing <it>AtMYB60 </it>expression, its promoter was dissected through deletion and mutagenesis analyses. By studying different versions of <it>AtMYB60 </it>promoter::GUS reporter fusions in transgenic plants we were able to demonstrate a modular organization for the <it>AtMYB60 </it>promoter. Particularly we defined: a minimal promoter sufficient to confer guard cell-specific activity to the reporter gene; the distinct roles of different DOF-binding sites organised in a cluster in the minimal promoter in determining guard cell-specific expression; the promoter regions responsible for the enhancement of activity in guard cells; a promoter region responsible for the negative transcriptional regulation by ABA. Moreover from the analysis of single and multiple mutants we could rule out the involvement of a group of DOF proteins, known as CDFs, already characterised for their involvement in flowering time, in the regulation of <it>AtMYB60 </it>expression.</p> <p>Conclusions</p> <p>These findings shed light on the regulation of gene expression in guard cells and provide new promoter modules as useful tools for manipulating gene expression in guard cells, both for physiological studies and future biotechnological applications.</p

Phenotype and specificity of T cells in primary human cytomegalovirus infection during pregnancy: IL-7Rpos long-term memory phenotype is associated with protection from vertical transmission

Congenital human cytomegalovirus (HCMV) infection is the major cause of birth defects and a precise definition of the HCMV-specific T-cell response in primary infection may help define reliable correlates of immune protection during pregnancy. In this study, a high throughput method was used to define the frequency of CD4+ and CD8+ T cells specific for four HCMV proteins in the naïve compartment of seronegative subjects and the effector/memory compartments of subjects with primary/remote HCMV infection. The naïve repertoire displayed comparable frequencies of T cells that were reactive with HCMV structural (pp65, gB and the pentamer gHgLpUL128L) and non-structural (IE-1) proteins. Whereas, following natural infection, the majority of effector/memory CD4+ and CD8+ T cells recognized either gB or IE-1, respectively, and pp65. The pattern of T cell reactivity was comparable at early and late stages of infection and in pregnant women with primary HCMV infection transmitting or not transmitting the virus to the fetus. At an early stage of primary infection, about 50% of HCMV-reactive CD4+ T cells were long- term IL-7Rpos memory cells, while 6–12 months later, the frequency of these cells increased to 70%, approaching 100% in remote infections. In contrast, only 10–20% of HCMV-specific CD8+ T cells were long-term memory cells up to 12 months after infection onset, thereafter increasing to 70% in remote infections. Interestingly, a significantly higher frequency of HCMV-specific CD4+ T cells with a long-term IL-7Rpos memory phenotype was observed in non-transmitting compared to transmitting women. These findings indicate that immunodominance in HCMV infection is not predetermined in the naïve compartment, but is the result of virus-host interactions and suggest that prompt control of HCMV infection in pregnancy is associated with the rapid development of long-term IL-7Rpos memory HCMV- specific CD4+ T cells and a low risk of virus transmission to the fetus

Detection of genotype-specific antibody responses to glycoproteins B and H in primary and non-primary human cytomegalovirus infections by peptide-based ELISA

Background: Strain-specific antibodies to human cytomegalovirus (HCMV) glycoproteins B and H (gB and gH) have been proposed as a potential diagnostic tool for identifying reinfection. We investigated genotype-specific IgG antibody responses in parallel with defining the gB and gH genotypes of the infecting viral strains. Methods: Subjects with primary (n = 20) or non-primary (n = 25) HCMV infection were studied. The seven gB (gB1-7) and two gH (gH1-2) genotypes were determined by real-time PCR and whole viral genome sequencing, and genotype-specific IgG antibodies were measured by a peptide-based enzyme-linked immunosorbent assay (ELISA). Results: Among subjects with primary infection, 73% (n = 8) infected by gB1-HCMV and 63% (n = 5) infected by gB2/3-HCMV had genotype-specific IgG antibodies to gB (gB2 and gB3 are similar in the region tested). Peptides from the rarer gB4-gB7 genotypes had nonspecific antibody responses. All subjects infected by gH1-HCMV and 86% (n = 6/7) infected by gH2-HCMV developed genotype-specific responses. Among women with non-primary infection, gB and gH genotype-specific IgG antibodies were detected in 40% (n = 10) and 80% (n = 20) of subjects, respectively. Conclusions: Peptide-based ELISA is capable of detecting primary genotype-specific IgG responses to HCMV gB and gH, and could be adopted for identifying reinfections. However, about half of the subjects did not have genotype-specific IgG antibodies to gB

Barriers and opportunities of soil knowledge to address soil challenges: Stakeholders? perspectives across Europe

Climate-smart sustainable management of agricultural soil is critical to improve soil health, enhance food and water security, contribute to climate change mitigation and adaptation, biodiversity preservation, and improve human health and wellbeing. The European Joint Programme for Soil (EJP SOIL) started in 2020 with the aim to significantly improve soil management knowledge and create a sustainable and integrated European soil research system. EJP SOIL involves more than 350 scientists across 24 Countries and has been addressing multiple aspects associated with soil management across different European agroecosystems. This study summarizes the key findings of stakeholder consultations conducted at the national level across 20 countries with the aim to identify important barriers and challenges currently affecting soil knowledge but also assess opportunities to overcome these obstacles. Our findings demonstrate that there is significant room for improvement in terms of knowledge production, dissemination and adoption. Among the most important barriers identified by consulted stakeholders are technical, political, social and economic obstacles, which strongly limit the development and full exploitation of the outcomes of soil research. The main soil challenge across consulted member states remains to improve soil organic matter and peat soil conservation while soil water storage capacity is a key challenge in Southern Europe. Findings from this study clearly suggest that going forward climate-smart sustainable soil management will benefit from (1) increases in research funding, (2) the maintenance and valorisation of long-term (field) ex-periments, (3) the creation of knowledge sharing networks and interlinked national and European in-frastructures, and (4) the development of regionally-tailored soil management strategies. All the above -mentioned interventions can contribute to the creation of healthy, resilient and sustainable soil ecosystems across Europe

Fibroblast, Epithelial and Endothelial Cell-Derived Human Cytomegalovirus Strains Display Distinct Neutralizing Antibody Responses and Varying Levels of gH/gL Complexes

In sequential sera from pregnant women with HCMV primary infection (PI), the serum neutralizing activity is higher against virions produced in epithelial and endothelial cells than in fibroblasts. Immunoblotting shows that the pentamer complex/trimer complex (PC/TC) ratio varies according to the producer cell culture type used for the virus preparation to be employed in the neutralizing antibody (NAb) assay, and is lower in fibroblasts and higher in epithelial, and especially endothelial cells. The blocking activity of TC- and PC-specific inhibitors varies according to the PC/TC ratio of virus preparations. The rapid reversion of the virus phenotype following its back passage to the original cell culture (fibroblasts) potentially argues in favor of a producer cell effect on virus phenotype. However, the role of genetic factors cannot be overlooked. In addition to the producer cell type, the PC/TC ratio may differ in single HCMV strains. In conclusion, the NAb activity not only varies with different HCMV strains, but is a dynamic parameter changing according to virus strain, type of target and producer cells, and number of cell culture passages. These findings may have some important implications for the development of both therapeutic antibodies and subunit vaccines

Pitfalls in the Serological Diagnosis of Primary Human Cytomegalovirus Infection in Pregnancy Due to Different Kinetics of IgM Clearance and IgG Avidity Index Maturation

Primary infection occurs when seronegative women are infected by human cytomegalovirus (HCMV). Diagnosis of primary infection is based on the following: antibody seroconversion, presence of IgM and low IgG avidity index (AI), and presence of DNAemia. The kinetics of HCMV-specific IgM antibody and maturation of AI might be very rapid or long-lasting during primary infection, which makes serological diagnosis insidious. The aims of this study were as follows: (i) to report atypical kinetics of HCMV-specific IgM antibody and AI early after onset of primary HCMV infection in a population of pregnant women, and (ii) to assess the frequency of such results. Altogether, 1309 sequential serum samples collected from 465 pregnant women with primary HCMV infection were included in the study. As a general rule, using the LIAISON®CMVIgMII and LIAISON®CMVIgGAvidityII assays, virus-specific IgM antibody levels decreased, while IgG AI increased over time during the first three months after infection onset. However, early clearance of IgM antibody and/or early IgG AI maturation occurred in 46/426 (10.7%) women. In more details, 20/426 (4.7%) and 26/418 (6.2%) women had undetectable IgM antibody or high IgG AI, respectively, when tested within 1–3 months after well-defined infection onset. Twenty sera from as many women with high IgG AI by the LIAISON assay were further tested for IgG AI by VIDAS®CMVIgGAvidityII and Mikrogen recomLineCMVIgG Avidity assays. Comparable results were obtained with VIDAS, whereas 14/20 sera gave low AI with the Mikrogen assay. In conclusion, about 11% of pregnant women undergoing a primary HCMV infection showed misleading serological results. Additional and appropriate testing might help in reducing the risk of missing HCMV primary infection in pregnancy. Furthermore, preconceptional testing should be strongly recommended