Epstein-Barr virus latency in transplant patients and healthy carriers

In this thesis, 1 studied the latency situation of Epstein- Barr virus (EBV) in bone marrow transplanted (BMT) patients and healthy virus carriers. Paper 1 Epstein-Barr virus genomes are found predominantly in lgA-positive B cells in the blood of healthy carriers. B lymphocytes have been identified as the main reservoir of latent Epstein-Barr virus (EBV) in healthy virus carriers. We have established a semi-quantitative PCR method (SQ-PCR) to estimate the EBV genome load in the blood B-cell subpopulation in healthy individuals. EBV DNA was detected in subfractionated IgM-, IgG- and IgApositive B cells. Between 80% and 90% of the viral DNA was found in the lgApositive compared with the IgA-negative fraction. Paper 2 Epstein-Barr virus (EBV) load in bone marrow transplant (BMT) recipients at risk to develop posttransplant lymphoproliferative disease: prophylactic infusion of EBV-specific cytotoxic T cells. We used same SQ-PCR as reported in paper 1 to monitor the blood levels of EBVDNA in 9 patients receiving allogeneic BMT. Four of 5 recipients of HLAmismatched T-cell-depleted grafts showed a 4- to 5-log increase of EBV-DNA within 1 to 3 months after BMT. Administration of 2 to 4 infusions of 107 EBV-specific cytotoxic Tlymphocytes (CTLS)/M2 starting from the time of maximal virus load resulted in a 2- to 3-log decrease of virus titers in 3 patients. One patient, who received a T-cell culture lacking a major EBV-specific component, progressed to fatal EBV-positive lymphoma. Administration of EBVCTLs before the onset of the EBV-DNA peak resulted in stabilization of the virus titers within 2 to 3 logs above the normal levels in the fifth patient. A moderate increase of virus titers was also detected in 3 of 4 patients receiving unmanipulated HLA-matched grafts, whereas one patient with WiskottAldrich syndrome (WAS) reached a 5-log increase of EBV-DNA load within 70 days after BMT. Our results suggest that a rapid increase of circulating EBV-DNA occurs in the absence of EBV-specific T-cell precursors or in the presence of congenital immune defects that prevent the reestablishment of virus-specific immunity. Prophylactic administration of EBV-CTLs early after BMT appears to provide the most effective protection against the development of EBV-associated lymphoproliferative disease. Paper 3 Circulating Epstein-Barr virus infected " resting" B lymphocytes in bone marrow transplant recipients at risk to develop post-transplant lymphoproliferative disease. EBV establishes a life long infection of humans where the proliferative potential of latently infected B blasts is kept in cheek by strong T-cell mediated rejection responses. A key feature of this virus host relationship is the capacity of the virus to establish a restricted latent infection in resting B cells that are insensitive to rejection and provide a reservoir for reactivation and spread to susceptible hosts. In immunosuppressed individuals the EBV infected blasts may give rise to lymphoproliferative disorders that are preceded by a dramatic increase of virus load in blood. We have used SQ-PCR assays and reverse transcriptase assisted (RT)-PCRs to investigate the EBV-DNA load and the pattern of viral gene expression in peripheral blood of immunosuppressed patients receiving T cell depleted or unmanipulated bone marrow grafts from healthy EBV carriers. Patients in both groups showed a significant increase of EBV-DNA load compared to healthy controls. Virus titers exceeding the normal levels by more than 4 logs were detected in recipients of T-cell depleted marrow and in one patient with Wiskott-Aldrich syndrome. Measurement of EBV-DNA in serum and limiting dilution analysis of EBV-DNA in PBMC demonstrated that the increased virus load is due to expansion of a latently infected cell compartment that contains less than 10 EBV genomes copies per cell and expresses EBERs, LMP-2A and occassionally LMP 1 and EBNA 1 but not other latency associated viral proteins that serve as targets for virus-specific immune responses. This is compatible with latency forms I-II in non proliferating B-cells, but not latency Ill. Administration of EBV-specific CTLs correlated with a slow decrease of EBV-DNA load followed by stabilization at levels significantly higher then in healthy controls. The results suggest that suppression of EBVspecific T cell responses allows the proliferation infected B blasts in lymphoid tissues and that these cells retain the capacity to differentiate into resting B cells that enter the circulation. Reconstitution of T cell immunity results in the establishment of a new virus hostbalance characterised by a significant expansion of the latent viral reservoir. The following main conclusions could be drawn from our results, · EBV can infect IgM-, IgG- and IgA-positive B cells. Between 80% and 90% of the viral DNA was found in the IgA-positive subfraction. · HLA-mismatched T-cell-depleted grafts showed a 4- to 5-log increase of EBV-DNA within 1 to 3 months after BMT. · Administration of EBV-CTLs early after BMT appears to provide the most effective protection against the development of EBV-associated lymphoproliferative disease. · In BMT patients, EBV is in latency forms I-II in non proliferating B-cells, neither latency 111 nor infected B cells in lytic cycle

Epstein-Barr virus latency in vivo and in vitro

In this thesis, I studied 1) The latency situation of Epstein- Barr virus (EBV) in bone marrow transplanted (BMT) patients and healthy virus carriers. 2) The role of EBNA1 and cellular transcriptional factors Oct and Grg/TLE family in EBV latency switch. B lymphocytes have been identified as the main reservoir of latent Epstein-Barr virus (EBV) in healthy virus carriers. We have established a semi-quantitative PCR method (SQ-PCR) to estimate the EBV genome load in the blood B-cell subpopulation in healthy individuals. EBV DNA was detected in subfractionated IgM-, IgG- and IgA-positive B cells. Between 80%. and 90% of the viral DNA was found in the lgA-positive compared with the lgA-negative fraction. We used SQ-PCR to monitor the blood levels of EBV-DNA in 9 patients receiving allogeneic BMT. Four of 5 recipients of HLA-mismatched T-cell-depleted grafts showed a 4- to 5-log increase of EBV-DNA within 1 to 3 months after BMT. Administration of 2 to 4 infusions of 107 EBV-specific cytotoxic T-lymphocytes (CTLs)/M2 starting from the time of maximal virus load resulted in a 2- to 3-log decrease of virus titers in 3 patients. A moderate increase of virus titers was also detected in 3 of 4 patients receiving unmanipulated HLA-matched grafts, whereas one patient with Wiskott-Aldrich syndrome (WAS) reached a 5-log increase of EBVDNA load within 70 days after BMT. Our results suggest that a rapid increase of circulating EBV-DNA occurs in the absence of EBV-specific T-cell precursors or in the presence of congenital immune defects that prevent the reestablishment of virus-specific immunity. Prophylactic administration of EBV-CTI-s early after 13MT appears to provide the most effective protection against the development of EBV-associated lymphoproliferative disease. We identified that Cp could also can be activated by octamer-binding factor (OCT) proteins. Physical binding to the FR by the cellular transcription factors OCT1 and OCT2 was demonstrated by using electrophoretic mobility shift assay (EMSA). Furthermore, OCT2 alone or OCTI in combination with co-regulator Bob-1could drive transcription of a heterologous thymidine kinase promoter liked to the FR both in B cells and epithelial cells. Cp controlled by the FR also activated by binding of OCT2 to FR. OCT proteins can recruit the Grg/TLE - farmly of transcription factor regulatory proteins (co-factors) and those proteins repress OCT2 promoter activity through FR. Although all Grg/TLE variants could repress the OCT2 induced activity from a FR-luciferase report vector, Grg-3 was most efficient. Grg/TLE proteins did not inhibit the EBNA1 activity on their own but when cotransfected with OCT2 also the EBNA I -induced FR enhancer effect was repressed. Addition of EBNA1 efficiently counteracted the transcriptional repression evoked by OCT-2 and Grg/TLE3. We also demonstrate that the levels of EBNA1 and OCT2 differ dramatically between latency III and I cells. Using competition and base substitutions of oligo-probes in EMSA1 we mapped in detail the binding of OCT-proteins to all octamer sequence three bp away from the EBNA1 core binding site. Different FR repeats vary in their ability to form complexes with OCTI, OCT2 and their cofactors. Direct physical binding of OCT and Grg/TLE to FR repeats was demonstrated in vitro by affinity adsorption with FR specific DNA as bait. Both OCT2 and EBNA I were shown to bind FR in vivo in EBV-positive cells representing latency I or III utilizing Chromatin immunoprecipitation assay (ChIP). Based on our results, a model is suggested that the on-off switch of the EBV C promoter in latency is controlled by competition between EBNA1 and OCT-proteins together with their co regulators

Numerical validation and physical explanation of the universal force theory of three-dimensional steady viscous and compressible flow

In a recent paper, Liu et al. ["Lift and drag in three-dimensional steady viscous and compressible flow", Phys. Fluids 29, 116105 (2017)] obtained a universal theory for the aerodynamic force on a body in three-dimensional steady flow, effective from incompressible all the way to supersonic regimes. In this theory, the total aerodynamic force can be determined solely with the vorticity distribution on a single wake plane locating in the steady linear far field. Despite the vital importance of this result, its validity and performance in practice has not been investigated yet. In this paper, we performed Reynolds-averaged Navier-Stokes simulations of subsonic, transonic, and supersonic flows over a three-dimensional wing. The aerodynamic forces obtained from the universal force theory are compared with those from the standard wall-stress integrals. The agreement between these two formulas confirms for the first time the validity of the theory in three-dimensional steady viscous and compressible flow. The good performance of the universal formula is mainly due to the fact that the turbulent viscosity in the wake is much larger than the molecular viscosity therein, which can reduce significantly the distance of the steady linear far field from the body. To further confirm the correctness of the theory, comparisons are made for the flow structures on the wake plane obtained from the analytical results and numerical simulations. The underlying physics relevant to the universality of the theory is explained by identifying different sources of vorticity in the wake

Causal mechanism behind the stall delay by airfoil’s pitching-up motion

Why the stall of an airfoil can be significantly delayed by its pitching-up motion? Various attempts have been proposed to answer this question over the past half century, but none is satisfactory. In this letter we prove that a chain of vorticity-dynamics processes at accelerating boundary is fully responsible for the causal mechanism underlying this peculiar phenomenon. The local flow behavior is explained by a simple potential-flow model

Spatiotemporal Pattern Analysis of Land Use Functions in Contiguous Coastal Cities Based on Long-Term Time Series Remote Sensing Data: A Case Study of Bohai Sea Region, China

The long-term accumulated remote sensing data and the emerging cloud-based geospatial processing platform Google Earth Engine (GEE) enable the mining of the spatiotemporal pattern of land-use (LU) functional changes in the contiguous area of large coastal cities. This study proposes a spatiotemporal pattern mining technique for land use function in a large area, which consists of two parts: (1) long-term time series land cover mapping based on the random forest (RF) classification algorithm in the GEE platform and a pixel-by-pixel temporal consistency correction, and (2) spatiotemporal pattern mining based on the constructed spatial temporal cubes (STCs). Specifically, for each LU functional series, we constructed the STC and applied change point detection, time series clustering, and emerging hot spot analysis to mine the spatiotemporal change patterns of LU functions. The study shows that (1) the construction land in the Bohai Sea region from 1990 to 2020 expanded significantly, with the development intensity increasing from 2.08% to 9.77%, having formed a contiguous area of large cities; at the same time, the arable land area decreased significantly, from 57.94% to 47.83%; (2) the emerged construction land experienced three periods: fluctuation, rise, and decline, with 2004 and 2014 being the change points during the period; and (3), the spatial and temporal pattern of the expansion of construction land shows a spatial gradient change in the scale and rate of expansion along the central cities and major axes. This study demonstrates the potential of using long-term time series remote sensing data towards cognizing the generation mechanisms of contiguous coastal big cities

Epstein-Barr virus latency switch in human B-cells: a physico-chemical model-3

<p><b>Copyright information:</b></p><p>Taken from "Epstein-Barr virus latency switch in human B-cells: a physico-chemical model"</p><p>http://www.biomedcentral.com/1752-0509/1/40</p><p>BMC Systems Biology 2007;1():40-40.</p><p>Published online 31 Aug 2007</p><p>PMCID:PMC2164963.</p><p></p>t time zero the system is stable in latency I, with an EBNA-1 level of 850 molecules, and an Oct-2 level of 15000 molecules. Transition to latency III is induced by lowering the Oct-2 level to 10000, activating the C promoter. Reaching the stable latency III level of EBNA-1 proteins thereafter take a few days. Induced switching back to resting latency I state demands a distinct increase in Oct-2, minimum a 10 fold change (green solid line). The greater increase in Oct-2 molecules the faster the cell is switched back to a stable latency I level of EBNA-1. The green solid and dashed line illustrate two different scenarios of elevated Oct-2 levels, where the red solid and dashed line are the corresponding resulting EBNA-1 levels

Both high and low levels of cellular Epstein-Barr virus DNA in blood identify failure after hematologic stem cell transplantation in conjunction with acute GVHD and type of conditioning

The level of Epstein-Barr virus DNA in blood has proven to be a biomarker with some predictive value in allogeneic hematopoietic stem cell transplantation patients (HSCT). We evaluated the impact of EBV load on survival of 51 patients (32M/19F, median age: 32 years, from < 1 to 68 years old), who had received HSCT for different types of malignancies (49 cases) or non-malignancies (2 cases). The overall survival [1]was compared between patients with extreme and moderate cell bound EBV DNA levels. Different sources of stem-cells (peripheral blood stem, n = 39; bone marrow, n = 9; or umbilical cord blood, n = 3) were used. Twenty patients received reduced-intensity conditioning regimen while the other 31 received myeloablative conditioning. Patients with high or very low level of cell bound EBV-DNA levels had a shorter OS than those with moderate EBV load: OS at 5 years was 67% vs 90% (p < 0.03). There was a conspicuous relationship between EBV load and the reconstitution dynamics of total and EBV-specific T cells, CD4+ and CD4- CD8- (double negative) T cells in the few patients where it was analyzed. This was not statistically significant. Two other factors were associated to early mortality in addition to high or low EBV load: acute GVHD II-IV (p < 0.02) and pre-transplant conditioning with total body irradiation (TBI) ≥6 Gy, (p < 0.03). All the patients meeting all three criteria died within two years after transplantation. This points to a subgroup of HSCT patients which deserve special attention with improvement of future, personalized treatment