Epstein-Barr Virus Reactivation Research Articles

EBV abortive lytic cycle promotes nasopharyngeal carcinoma progression through recruiting monocytes and regulating their directed differentiation.

Epstein-Barr virus (EBV) is associated with several types of human cancer including nasopharyngeal carcinoma (NPC). The activation of EBV to the lytic cycle has been observed in advanced NPC and is believed to contribute to late-stage NPC development. However, how EBV lytic cycle promotes NPC progression remains elusive. Analysis of clinical NPC samples indicated that EBV reactivation and immunosuppression were found in advanced NPC samples, as well as abnormal angiogenesis and invasiveness. To investigate the role of the EBV lytic cycle in tumor development, we established a system that consists of two NPC cell lines, respectively, in EBV abortive lytic cycle and latency. In a comparative analysis using this system, we found that the NPC cell line in EBV abortive lytic cycle exhibited the superior chemotactic capacity to recruit monocytes and polarized their differentiation toward tumor-associated macrophage (TAM)-like phenotype and away from DCs, compared to EBV-negative or EBV-latency NPC cells. EBV-encoded transcription activator ZTA is responsible for regulating monocyte chemotaxis and TAM phenotype by up-regulating the expression of GM-CSF, IL-8, and GRO-α. As a result, TAM induced by EBV abortive lytic cycle promotes NPC angiogenesis, invasion, and migration. Overall, this study elucidated the role of the EBV lytic life cycle in the late development of NPC and revealed a mechanism underlying the ZTA-mediated establishment of the tumor microenvironment (TME) that promotes NPC late-stage progression.

Ceramide promotes lytic reactivation of Epstein-Barr virus in gastric carcinoma.

Epstein-Barr virus (EBV) has a lifelong latency period after initial infection. Rarely, however, when the EBV immediate early gene BZLF1 is expressed by a specific stimulus, the virus switches to the lytic cycle to produce progeny viruses. We found that EBV infection reduced levels of various ceramide species in gastric cancer cells. As ceramide is a bioactive lipid implicated in the infection of various viruses, we assessed the effect of ceramide on the EBV lytic cycle. Treatment with C6-ceramide (C6-Cer) induced an increase in the endogenous ceramide pool and increased production of the viral product as well as BZLF1 expression. Treatment with the ceramidase inhibitor ceranib-2 induced EBV lytic replication with an increase in the endogenous ceramide pool. The glucosylceramide synthase inhibitor Genz-123346 inhibited C6-Cer-induced lytic replication. C6-Cer induced extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB phosphorylation, c-JUN expression, and accumulation of the autophagosome marker LC3B. Treatment with MEK1/2 inhibitor U0126, siERK1&2, or siCREB suppressed C6-Cer-induced EBV lytic replication and autophagy initiation. In contrast, siJUN transfection had no impact on BZLF1 expression. The use of 3-methyladenine (3-MA), an inhibitor targeting class III phosphoinositide 3-kinases (PI3Ks) to inhibit autophagy initiation, resulted in reduced beclin-1 expression, along with suppressed C6-Cer-induced BZLF1 expression and LC3B accumulation. Chloroquine, an inhibitor of autophagosome-lysosome fusion, increased BZLF1 protein intensity and LC3B accumulation. However, siLC3B transfection had minimal effect on BZLF1 expression. The results suggest the significance of ceramide-related sphingolipid metabolism in controlling EBV latency, highlighting the potential use of drugs targeting sphingolipid metabolism for treating EBV-positive gastric cancer.IMPORTANCEEpstein-Barr virus remains dormant in the host cell but occasionally switches to the lytic cycle when stimulated. However, the exact molecular mechanism of this lytic induction is not well understood. In this study, we demonstrate that Epstein-Barr virus infection leads to a reduction in ceramide levels. Additionally, the restoration of ceramide levels triggers lytic replication of Epstein-Barr virus with increase in phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB. Our study suggests that the Epstein-Barr virus can inhibit lytic replication and remain latent through reduction of host cell ceramide levels. This study reports the regulation of lytic replication by ceramide in Epstein-Barr virus-positive gastric cancer.

Understanding the Mode of Action of a Micro-Immunotherapy Formulation: Pre-Clinical Evidence from the Study of 2LEBV® Active Ingredients.

Epstein-Barr virus (EBV) is often kept silent and asymptomatic; however, its reactivation induces a chronic and/or recurrent infection that is associated with numerous diseases, including cancer and inflammation-related disorders. As no specific treatment is currently available, the immune factors-based micro-immunotherapy (MI) medicine 2LEBV® could be considered a valuable therapeutic option to sustain the immune system in EBV reactivation. The present work aimed to investigate, for the first time, the effect of 2LEBV® in several in vitro models of uninfected immune-related cells. 2LEBV® displayed phagocytosis-enhancing capabilities in granulocytes. In human peripheral blood mononuclear cells (PBMCs), it increased the intra- and extra-cellular expression of interleukin (IL)-2. Moreover, it modulated the secretion of other cytokines, increasing IL-4, IL-6, and tumor necrosis factor-α levels or lowering other cytokines levels such as IL-9. Finally, 2LEBV® reduced the expression of human leukocyte antigen (HLA)-II in endothelial cells and macrophages. Although these data are still preliminary and the chosen models do not consider the underlying EBV-reactivation mechanisms, they still provide a better understanding of the mechanisms of action of 2LEBV®, both at functional and molecular levels. Furthermore, they open perspectives regarding the potential targets of 2LEBV® in its employment as a therapeutic intervention for EBV-associated diseases.

Transcriptome analysis of Burkitt lymphoma cells treated with anti-convulsant drugs that are inhibitors of Epstein-Barr virus lytic reactivation.

Herpesviruses have two distinct life cycle stages, latency and lytic replication. Epstein-Barr virus (EBV), a gamma-herpesvirus, establishes latency in vivo and in cultured cells. Cell lines harboring latent EBV can be induced into the lytic cycle by treatment with chemical inducing agents. In the Burkitt lymphoma cell line HH514-16 the viral lytic cycle is triggered by butyrate, a histone deacetylase (HDAC) inhibitor. Butyrate also alters expression of thousands of cellular genes. However, valproic acid (VPA), another HDAC inhibitor with global effects on cellular gene expression blocks EBV lytic gene expression in Burkitt lymphoma cell lines. Valpromide (VPM), an amide derivative of VPA, is not an HDAC inhibitor, but like VPA blocks induction of the EBV lytic cycle. VPA and VPM are the first examples of inhibitors of initial stages of lytic reactivation. We compared the effects of VPA and VPM, alone and in combination with butyrate, on host cellular gene expression using whole transcriptome analysis (RNA-seq). Gene expression was analyzed 6 h after addition of the compounds, a time before the first EBV lytic transcripts are detected. The results address two alternative, yet possibly complementary, mechanisms for regulation of EBV lytic reactivation. First, cellular genes that were up- or down-regulated by butyrate, but no longer altered in the presence of VPA or VPM, represent genes that correlated with EBV lytic reactivation. Second, genes regulated similarly by VPA and VPM in the absence and presence of butyrate are candidates for suppressors of EBV reactivation. Two genes upregulated by the lytic cycle inhibitors, CHAC1 and SLC7A11, are related to redox status and the iron-dependent cell death pathway ferroptosis. This study generates new hypotheses for control of the latency to lytic cycle switch of EBV and provides the first description of effects of the anti-convulsant drug VPM on global human cellular gene expression.

Clinical characteristics and outcomes of Epstein-Barr virus viral load after allogeneic hematopoietic stem cell transplantation

Epstein-Barr virus (EBV) reactivation can occur following allogenic hematopoietic stem cell transplantation (allo-HSCT). However, the clinical characteristics and outcomes of EBV-viral load are not well known. Thus, we retrospectively analyzed the clinical features and prognostic impact of the EBV viral load in 121 allo-HSCT recipients from our hospital. EBV DNA quantification was performed in whole blood after transplantation. Patients were grouped based on whether EBV DNA quantification reached > 1000 copies/mL during follow-up (N = 50) or not (N = 71). Patients with EBV > 1000 EBV copies/mL were relatively more common in the groups with graft versus host disease (GVHD) prophylaxis including ATG, haploidentical donor type, peripheral blood as a donor source, and acute GVHD II–IV. The 20-month OS and DFS were not significantly different between patients with < 1000 EBV copies/mL and patients with > 1000 EBV copies/mL (20-month OS, 56.0% vs. 60.6%; p = 0.503, 20-month DFS, 50.0% vs. 57.7%; p = 0.179). Immunosuppressant (ISS) dose reduction was achieved after the maximum increase in EBV in 41/50 (82%) patients. Additionally, 30/50 (60%) patients achieved a 50% dose reduction or no restarting of ISS within 3 months of the maximum EBV increase. Among cases wherein EBV DNA quantification reached > 1000 copies/mL, those that achieved rapid dose reduction of ISS tended to have longer overall survival (“not reached” vs 5.4 months, p < 0.001) and disease-free survival (88.4 months vs 5.3 months, p < 0.001) than those in patients who did not. Our data highlight the importance of rapid ISS reduction in post-transplant EBV reactivation.

Host factor KAP1 coordinates temporal control between transcription and replication

Temporal control of transcription and replication is necessary for efficient Epstein–Barr virus reactivation. Xu et al. identified the KAP1/EA-D/ATM axis as a critical regulator of these processes. This discovery illuminates the collaboration between host and viral factors as an essential interaction for viral reactivation.

Donor aKIR genes influence the risk of EBV and CMV reactivation after anti-thymocyte globulin-based haploidentical hematopoietic stem cell transplantation.

Hematopoietic stem cell transplantation (HSCT) offers the highest curative potential for patients with hematological malignancies. Complications including infection, graft-versus-host disease (GVHD), and relapse reflect delayed or dysregulated immune reconstitution. After transplantation, NK cells rapidly reconstitute and are crucial for immune surveillance and immune tolerance. NK cell function is tightly regulated by killer immunoglobin-like receptors (KIRs). Previous studies have revealed that donor KIRs, especially some activated KIRs (aKIRs) are closely related to transplant outcomes. Here, we performed a retrospective study, including 323 patients who received haploidentical (haplo) HSCT in our center. In univariate analysis, donor KIR2DS1, KIR2DS3 and KIR3DS1 gene protected patients with lymphoid disease from Epstein-Barr virus (EBV) and cytomegalovirus (CMV) reactivation, while donor KIR2DS1, KIR2DS5 and KIR3DS1 gene conferred a higher risk of CMV reactivation for patients with myeloid disease. Multivariate analysis confirmed that donor telomeric (Tel) B/x and KIR2DS3 gene best protected patients with lymphoid disease from EBV (p = 0.017) and CMV reactivation (p = 0.004). In myeloid disease, grafts lacking Tel B/x and KIR2DS5 gene correlated with the lowest risk of CMV reactivation (p = 0.018). Besides, donor aKIR genes did not influence the rates of GVHD, relapse, non-relapse mortality (NRM) and overall survival (OS) in this study. The reactivation of EBV and CMV was associated with poor prognosis of haplo-HSCT. In conclusion, we found that donor aKIR genes might have a synergistic effect on CMV and EBV reactivation after haplo-HSCT. Whether the influence of donor aKIR genes varies with disease types remained to be studied.

EBV Reactivation in Transplant Recipients following SARS-CoV-2 Infection: A Retrospective Study.

Reactivation and primary infection with a high Epstein Barr Virus (EBV) DNA level in kidney transplant patients could cause severe complications, including the development of Post-Transplantation Lymphoproliferative Disease (PTLD). While in the general population the reactivation of EBV after SARS-CoV-2 infection has been reported, very few data are available in transplant recipients. Our retrospective study aimed to evaluate a possible EBV reactivation in kidney transplant patients following SARS-CoV-2 infection and a possible impairment of the immune system. In addition, the effects of changes in immunosuppressive therapy on EBV DNA reactivation and vaccination were also evaluated. A total of 166 kidney transplant patients followed at the Kidney-Pancreas Transplant Ambulatory Nephrology Unit at Padova University Hospital were retrospectively considered for an observation period of 6 months from January 2020 to April 2023. EBV DNA level was measured by Rt-PCR and evaluated 6 months before and after SARS-CoV-2 infection. Patients' serological states were established via quantification of anti-VCA and anti-EBNA (chemiluminescence). Patients' immune systems were characterized by CD4+/CD8+ lymphocyte ratio (flow cytometry). EBV DNA was reactivated in 50% of the 166 patients with COVID-19 who completed the study. Older patients with more severe forms of COVID-19 had higher EBV reactivation (p < 0.05). EBV reactivation significantly increased in patients with severe SARS-CoV-2 infection requiring hospitalization compared to patients managed at home (p < 0.001). CD4+/CD8+ lymphocyte ratio was reduced in patients with a younger age of transplant (p < 0.01) and on a higher dose of steroids (p < 0.01). The results of our study confirm the role of immunodepression, especially in recent transplant patients and those on high steroids, in EBV reactivation. These results combined with the few available in the literature might contribute to providing an optimal management of immunosuppressive treatment for these patients in order to obtain an immune state unfavorable to the activation of latent viruses, including EBV.

The Effect of Preemptive Rituximab Treatment in Patients with EBV Viremia after Solid Organ Transplantation on the Development of Post-Transplant Lymphoproliferative Disorder

The association between Epstein-Barr Virus (EBV) reactivation after solid organ transplantation (SOT) and Post-Transplant Lymphoproliferative Disorder (PTLD) is well described. There are currently no guidelines regarding the use of rituximab preemptively in patients with EBV viremia after SOT to reduce the risk of PTLD. We sought to determine whether rituximab in SOT patients with EBV viremia has benefit in preventing PTLD. This retrospective analysis of electronic medical record data identified SOT recipients who experienced EBV viremia post-transplant between January 2010 and February 2023 at Columbia University Irving Medical Center (CUIMC). Data queries were clinically reviewed. Logistic regression analysis was performed to investigate the relationship between predictor variables including rituximab administration, log-transformed EBV peak titer, duration of EBV viremia, and time from SOT to EBV reactivation, with the binary primary outcome of PTLD diagnosis status. The analysis encompassed both univariate and multivariate logistic regression. There were 2,168 patients who met the inclusion criteria of having EBV reactivation after SOT, out of 6,395 total SOT performed at CUIMC during this timeframe. Patient characteristics are described in table 1. The median peak EBV titer was 154 (range: not quantifiable - 5,335,816), the median duration of EBV viremia was 78 days (range: 1 - 6,980), and the median time from SOT to EBV reactivation was 688 days (range: 0-11103). Of the 2,168 patients, 185/2168 (8.5%) received rituximab for indications other than PTLD including EBV viremia, treatment of rejection, antibody desensitization therapy/immunosuppression peri-transplant, and other non-transplant related indications (e.g. lupus nephritis). Of the patients who received rituximab, 182/185 (98.4%) did not develop PTLD. The 3/185 (1.6%) patients who did develop PTLD were all ≤ 20 years-old at time of diagnosis, and had EBER(+), monomorphic PTLD, diffuse large B-cell lymphoma type, with a non-germinal center phenotype, involving the gastrointestinal tract. Two were CD20 positive, all 3 had partial weak CD30 positivity, and all 3 had clonal immunoglobulin heavy chain rearrangements. Among the patients who did not receive preemptive rituximab post-SOT, 195/1983 (9.8%) developed PTLD, whereas 1788/1983 (90.2%) did not. Prior rituximab therapy for any indication in patients with EBV viremia post-SOT was associated with lower risk of subsequent PTLD by univariate analysis (0.14% vs 8.39%, OR 0.15, p = 0.0013, 95% CI 0.048-0.48). Receipt of rituximab (OR 0.12, p=0.0002), log-transformed peak EBV titer (OR 1.10, p&amp;lt;0.0001), and duration of EBV viremia in months (OR 1.01, p&amp;lt;0.0001) were all independently associated with risk for PTLD by multivariate analysis (figure 1). The relationship between time from SOT to EBV viremia was not significantly associated with development of PTLD. This retrospective analysis suggests that administering preemptive rituximab in SOT patients with EBV viremia may lower the risk of developing PTLD. The fact that peak EBV titer and duration of EBV viremia remain associated with PTLD in multivariate analyses suggests that the benefit of rituximab may be independent of the degree or duration of EBV viremia. A prospective analysis of factors such as area under the curve of EBV exposure, HLA status, T cell clonotype diversity, organ type, and immunosuppression type, is needed to better understand individual risks for improved selection of those who would benefit from preemptive rituximab.

Umbilical Cord Blood Stem Cells As Third-Party Adjuvant Infusion in Human Leukocyte Antigen Antibody-Positive Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation

Introduction: The presence of human leukocyte antigen (HLA) antibodies, even if not donor-specific antibodies (DSA), is an important risk factor for graft failure (GF) or poor graft function (PGF) in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, there is no ideal strategy for eradicating or descending HLA antibodies in this population. In the present study, we investigated the efficacy and safety of unrelated umbilical cord blood stem cells (CBCs) as a third-party adjuvant infusion in patients with HLA antibodies undergoing allo-HSCT. Methods:Patients undergoing allo-HSCT from July 2017 to June 2021 were enrolled in this study and HLA antibodies were routinely tested before transplantation. Patients were divided into three groups: Group A (patients with positive/weak positive HLA antibodies and with CBCs infusion), Group B (patients with HLA antibodies positive/weak positive without CBCs infusion), and Group C (patients without HLA antibody nor CBCs infusion). Single unrelated CBCs (HLA4/6 or more compatible ) were infused four hours before donor stem cells. The clinical characteristics of the patients and the paired donors were collected. The occurrence of granulocyte and platelet engraftment time, graft-versus-host disease (GVHD), cytomegalovirus (CMV), Epstein-Barr virus (EBV) reactivation, one-year and two-year relapse-free survival (RFS), transplantation-related mortality (TRM), and overall survival (OS) for all three groups of patients were also analyzed in this study. Results:A total of 90 patients were included in this study (17 in group A, 36 in group B, and 37 in group C). The baseline clinical characteristics of the patients were presented in Table 1. No obvious adverse effects were observed in the process of CBCs infusion. All the patients achieved 100% donor chimerism without CBCs engraftment. There were no significant differences in granulocyte and platelet engraftment time between the three groups. Two patients in Group A and C, and 3 patients in Group B developed PGF, respectively. There were no significant differences in the incidence and grades of acute or chronic GVHD among the three groups ( Figure A, B). In the first 3 months after allo-HSCT, the incidence of EBV reactivation was higher in patients in Group B than that in Group A and C, respectively ( Figure C). There were no significant differences in the CMV reactivation between the three groups in the first 3 months after transplantation ( Figure D). Patients in Group A and B both had lower 1-year RFS and higher 1-year TRM, compared with Group C, respectively ( Figure E, F). However, there were no significant statistical differences in 2-year RFS and TRM between the three groups. Although patients in Group A had longer 2-year OS than that in Group B, there were no significant differences between the two groups: Group A vs Group B (76.5% vs 55.6%, P = 0.194), Group B vs Group C (55.6% vs 85.5%, P &amp;lt; 0.05)( Figure G). A multifactorial Cox regression risk model was established to explore the risk factors affecting OS and suggested that patients' pre-transplantation status (with active malignant disease or minimal residual disease positive) and HLA antibodies pre-transplant were the most independent risk factors for OS (HR = 4.380, 95% CI = 1.544-12.427, P&amp;lt;0.05; HR = 0.196, 95% CI = 0.065-0.587, P&amp;lt;0.05). Conclusions: Patients with positive/weak positive HLA antibodies receiving CBCs as a third-party adjunctive infusion were safe, and there was a benefit in the incidence of EBV reactivation compared to such a population without CBCs infusion. The patients with negative HLA antibodies had higher 1-year RFS and lower 1-year TRM than those with positive/weakly positive HLA antibodies regardless CBCs infusion. Due to the small number of included cases, we will expand the sample number to further validate our results in the future.

Sitagliptin for Prevention of aGVHD in Patients Received Alternative Donor Transplantations: A Prospective, Multicenter, Open-Label, Randomized Controlled Trial

Background： Acute graft versus host disease (aGVHD) remains one of the most frequent complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT) with high mortality. Traditionally, alternative donor transplantation (ADT) from an unrelated donor or a haploidentical family donor has been reported in an increased incidence of aGVHD and severe GvHD. Sitagliptin is a selective inhibitor of dipeptidyl peptidase 4 (DPP-4; also known as CD26), a transmembrane receptor expressed on T cells which has a costimulatory function in activating T cells. This trial aims to evaluate the safety and efficacy of sitagliptin plus a calcineurin inhibitor, methotrexate (MTX), mycophenolate mofetil (MMF) and antithymocyte globulin (ATG) in the prevention of aGVHD for ADT. Methods： We are performing a prospective, multicenter, open-label, randomized controlled trial (NCT05149365). From 5 HSCT centers in China competitively, patients(pts) who underwent their first allo-HSCT in first or second complete remission (CR) state with hematologic malignancies were received busulfan and cyclophosphamide (Bu/Cy) myeloablative conditioning regimen followed by ADT. 190 eligible pts(18-60 years) were planned randomly assigned in a 1:1 ratio to either a sitagliptin group (600mg sitagliptin taken orally every 12 hours on day -1 to +14, plus conventional prophylaxis including Cyclosporin A(CsA)/ tacrolimus (FK506), MTX, MMF and ATG) and a conventional prophylaxis control group (CsA/FK506, MTX, MMF and ATG). The randomization was based on a computer-generated priori list of 190 binary numbers, with block-balanced with random variable block sizes of two or four pts, without stratification. The primary endpoint was grade II to IV aGvHD by day +100. Key secondary endpoints included severe (grade III or IV) aGvHD by day +100, transplant-related mortality (TRM), relapse-free survival (RFS) and GVHD-free, relapse-free survival (GRFS) from HSCT. At present we have completed pts enrollment and are at the stage of follow-up the study endpoints. Results : From December 2021 to May 2023, a total of 191 pts were enrolled. The ratio of male to female was 113 to 78 with a median age of 38 years (range, 18-60 years). Pts were randomly assigned to the sitagliptin group of 95 and the control group of 96. The clinical and transplantation characteristics at baseline of the intention to treat (ITT) population of the two groups were well balanced (Table1). The last follow-up was on May 30, 2023 and the median follow-up time was 164 (20-512) days. Comparison of the sitagliptin group and the control group by day +100, the cumulative incidence rate of grade II-IV aGVHD was 15.1% and 28.6%, respectively ( P=0.019), and the cumulative incidence rate of grade III or IV aGVHD was 7.6% and 15.9% ( P=0.068); median days of neutrophil engraftment were +11.48 and +11.65 days, respectively (P=0.545); TRM was 3.7% and 7.1% (P=0.454), RFS was 98.7% and 95.9% (P=0.889), respectively and OS of the two groups(96.7% vs 93.6%, P=0.341) was also the same as the RFS. There was no significant difference between the sitagliptin and control groups with regard to cytomegalovirus reactivation, Epstein-Barr virus reactivation, and transplant-related complications (Table 1, Figure 1). The regimen was well tolerated. Only 5(/95) pts discontinued sitagliptin treatment due to adverse events: 4 patients experienced abdominal pain (CTCAE Grade1-2) undergoing sitagliptin therapy 3-7 days and one of them also exhibited hypoglycemia (CTCAE Grade1), which were deemed by clinicians as probable (WHO-UMC) associated with sitagliptin. Symptoms resolved spontaneously within 2 days of cessation. Another patient discontinued treatment due to mild hematemesis initiating sitagliptin therapy only 2 days, which was determined by the clinician as unlikely (WHO-UMC) associated with sitagliptin. Conclusions : Our trial showed sitagliptin combined with conventional prophylaxis (CsA+MTX+MMF+ATG) resulted in a significant reduction of grade II-IV aGVHD in ADT. Furthermore, sitagliptin is ready available, easy of administration, safe, and relatively inexpensive. It remains to be followed up whether it would increase infection and relapse or improve the prognosis of patients after allo-HSCT.

Addition of Daclizumab to Standard Graft-Versus-Host Disease Prophylaxis in Patients with Thalassemia Major Undergoing Transplantation from Related Haploidentical Donor and Unrelated Donor Hematopoietic Stem-Cell Transplantation: A Single-Center Retrospective Analysis from China

Background: Previous studies have confirmed that the incidence of graft-versus-host disease (GVHD) remained still very high in patients with thalassemia major (TM) undergoing related haploidentical donor and unrelated donor hematopoietic stem-cell transplantation (HID-HSCT and MUD-HSCT), which seriously affected patients' overall survival and thalassemia-free survival, especially acute GVHD (aGVHD). This study aimed to investigate whether Daclizumab at a dose of 1 mg/Kg on days +7, +14, +28 and +42 reduced GVHD plus standard GVHD prophylaxis (tacrolimus [FK506] plus methotrexate [MTX] and mycophenolate mofetil [MMF]) in TM patients with HID-HSCT or MUD-HSCT. Methods: We retrospectively analyzed our data of TM patients who underwent HID-HSCT or MUD-HSCT at the First Affiliated Hospital of Guangxi Medical University in China from November 2022 to July 2023. TM patients enrolled all received the GVHD prophylaxis which included Daclizumab at a dose of 1 mg/kg on days +7, +14, +28 and +42 plus FK506 (0.03mg/kg/day), MTX (15mg/m 2 on days +1, 10mg/m 2 on days +3, +6, +11), and MMF (250mg/day × 90 days). The primary endpoint was grades 2-4 aGVHD on days +100. Results: Data of 37 patients' transplantation were evaluated. The ratio of male to female was 20 to 17 with a median age of 8 years (range, 3-17 years). Patients were assigned to the HID-HSCT of 24 and the MUD-HSCT of 13. Median follow-up period was 7 months (range, 3 to 11 months). The total of the cumulative incidence rate of grades 2-4 aGVHD was 18.9%, and the rate of grades 3-4 aGVHD was 10.8%. Subgroup analysis showed that the rate of grades 2-4 aGVHD was 20.8% in HID-HSCT and 15.4% in MUD-HSCT, respectively; the rate of grades 3-4 aGVHD was 8.3% in HID-HSCT and 15.4% in MUD-HSCT, respectively. The median days of neutrophil engraftment were +12 days, platelets Implantation days were +14 days. The cytomegalovirus reactivation, Epstein-Barr virus reactivation, fungal infection and septicemia were seen in 16(43.2%), 3(8.1%), 6(16.2%) and 5(13.5%) patients, respectively. One patient in MUD-HSCT died of severe pulmonary infection, but no deaths were attributable to HID-HSCT (Figure 1, Table 1). CONCLUSION: The results of our study analysis suggest that Daclizumab at a dose of 1 mg/kg on days +7, +14, +28 and +42 provides clinically meaningful benefits when added to standard GVHD prophylaxis in TM patients undergoing haploidentical donor and unrelated donor transplantation, including decrease in the incidence rate of aGVHD without sever infection and transplant-related complications. Four doses of Daclizumab should be included in the GVHD prophylaxis of patients with thalassemia major selected for related haploidentical donor and unrelated donor transplantation.

A Co-Reactivation Model of Cytomegalovirus and Epstein-Barr Virus after Allogeneic Hematopoietic Stem Cell Transplantation Was Constructed to Predict Mortality after Transplantation

Abstract Objective: The aim of this study was to create a predictive model for the occurrence of co-reactivation of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: The study included 373 patients who underwent allo-HSCT. The patient population was randomly divided into two groups: a training cohort comprising 70% of patients (n=261) and a validation cohort comprising 30% of patients (n=112). The predictive model was developed using several variables, including the time of reactivation of CMV and EBV, patient age and gender, presence of hepatitis B core antibody (HbcAb), type of transplant, use of anti-thymocyte globulin (ATG), and the occurrence of acute and chronic graft-versus-host disease (aGVHD and cGVHD, respectively). Results: The developed predictive model was expressed as y = -6.21338339 + 0.01923832 × (CMV reactivation time) + 0.00362628 × (EBV reactivation time) - 0.57604523 × (patient gender) + 0.01974136 × (patient age) + 0.85514424 × (presence of HbcAb) + 1.13782872 × (transplant type) + 0.95376138 × (use of ATG) - 0.62393602 × (aGVHD) - 1.09485262 × (cGVHD). The model demonstrated a g-mean value of 0.685 and a probability threshold of 0.297, allowing for the classification of patients into high-risk and low-risk groups. The incidence of co-reactivation of CMV and EBV over 100 days was significantly higher in the high-risk group compared to the low-risk group. In the high-risk group, the incidence was 49.3%, while in the low-risk group, it was 16.9% (P&amp;lt;0.0001, HR 95%CI 3.693(2.493,5.47)). For patients with aGVHD, the incidence was 41.7% in the high-risk group and 17.9% in the low-risk group (P=0.00026, HR 95%CI 2.7473(1.560,4.839)). In patients without aGVHD, the incidence was 53% in the high-risk group and 14.0% in the low-risk group (P&amp;lt;0.0001, HR 95%CI 4.9439(2.347,10.4)). Additionally, the model was able to predict post-transplant mortality and survival. Conclusion: Overall, the findings of this study suggest that the developed model is a valuable tool for predicting the risk of CMV and EBV co-reactivation following allo-HSCT. By identifying high-risk patients, clinicians can take proactive measures to prevent or manage viral infections, ultimately improving patient outcomes. Therefore, this model can potentially enhance clinical decision-making and optimize post-transplant care.

New Aplastic Anemia Preconditioned with Fludarabine, Thiotepa, Cyclophosphamide, and Anti-Thymocyte Globulin

Aplastic anemia (AA) is one of life-threatening hematological disorder that can be cured by hematopoietic stem cell transplantation. When the absence of a matched sibling donor, haploidentical stem cell transplantation (haplo-HSCT) becomes an alternative choice for AA patients. We used a prospective study aimed to confirm the feasibility of Thiotepa-based modified post-transplantation cyclophosphamide (PTCY) strategy in haplo-HSCT for AA patients. We analyzed the outcomes of 15 patients who had undergone haplo-HSCT between October 2021-May 2023 in our clinical center. The modified condition regimen consisted of anti-thymoglobulin/antilymphocyte globulin, fludarabine, thiotepa, busulfan and modified low and high-dose cyclophosphamide, mycophenolate mofetil (MMF) and tacrolimus (FK-506)/cyclosporin (CsA) were administered as graft versus host disease (GVHD) prophylaxis after transplantation. The median follow-up time was 267 (range 97-532) days. All 15 patients were successfully implanted (100%), the median times for neutrophil and platelet engraftment were 14 (range 12-19) days and 12 (range 8-25) days, respectively. But one patient developed secondary graft failure because of infected with COVID-19、cytomegalovirus and H1N1. Fortunately, she subsequently underwent a successful secondary haplo-HSCT. The most common regimen-related toxicity was canker sore, hemorrhagic cystitis occurred in 3 patients during transplantation. The cumulative incidence of grade Ⅰ-Ⅱ aGVHD and cGVHD were 13.3% and 6.7% respectively, whereas the incidence of grade Ⅲ-Ⅳ aGVHD was not found so far. All 15 patients are alive, with a failure-free survival rate of 100% and GVHD relapse-free survival rate of 100%. The efficacy evaluation of haplo-HSCT, 14 patients achieved CR and 1 patient achieved PR. Because of 7 patients received the prevention strategy of Letermovir since August 2022, the reactivation rate of cytomegalovirus (CMV) was only 20% and 60% of Epstein-Barr virus (EBV) reactivation, but the CMV diseases and post-transplantation lymphoproliferative disorder (PTLD) were rare. Our study using Thiotepa-based modified haplo-HSCT demonstrated an encouraging result with prolonged survival and reduced complications for aplastic anemia patients.

Epstein-Barr virus reactivation is not causative for post-COVID-19-syndrome in individuals with asymptomatic or mild SARS-CoV-2 disease course

PurposePost-COVID-19-Syndrome (PCS) frequently occurs after an infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, the understanding of causative mechanisms is still limited. Aim of this study was to determine the PCS rate among SARS-CoV-2 seropositive blood donors as representatives of supposedly healthy adults, who had experienced an asymptomatic or mild COVID-19 disease course, and to examine whether Epstein-Barr virus (EBV) is reactivated in individuals reporting PCS.MethodsThe PCS rate was determined using questionnaires that included questions about infection and persistent symptoms. Pre-pandemic blood samples and samples collected at regular, pre-defined times after a SARS-CoV-2 infection were analysed for neopterin, a marker for antiviral immune responses, by an enzyme-linked immunosorbent assay (ELISA). Additionally, we determined the rate of SARS-CoV-2 anti-N total antibodies using an electrochemiluminescence immunoassay (ECLIA). Furthermore, quantitative real-time polymerase chain reaction (qPCR) to detect EBV DNA and ECLIA screening for EBV viral capsid-antigen (VCA) IgM, IgG and EBV nuclear antigen 1 (EBNA) IgG were performed.ResultsOur data reveal that 18% of all infections result in PCS, with symptoms lasting for up to one year. In individuals reporting PCS, no elevated levels of neopterin were detected, indicating no persisting pro-inflammatory, antiviral immune response. SARS-CoV-2 antibody levels were declining in all participants in comparable manner over time, pointing to a successful virus clearance. In individuals with PCS, no EBV DNA could be detected. Furthermore, no differences in EBV specific antibody levels could be shown in PCS groups compared to non-PCS groups.ConclusionOur data suggest that PCS in per se healthy, immunocompetent adults cannot be ascribed to a reactivation of EBV.

Pre-Emptive Use of Rituximab in Epstein-Barr Virus Reactivation: Incidence, Predictive Factors, Monitoring, and Outcomes.

Post-transplant lymphoproliferative disease (PTLD) is a fatal complication of hematopoietic cell transplantation (HCT) associated with the Epstein-Barr virus (EBV). Multiple factors such as transplant type, graft-versus-host disease (GVHD), human leukocyte antigens (HLA) mismatch, patient age, and T-lymphocyte-depleting treatments increase the risk of PTLD. EBV reactivation in hematopoietic cell transplant recipients is monitored through periodic quantitative polymerase chain reaction (Q-PCR) tests. However, substantial uncertainty persists regarding the clinically significant EBV levels for these patients. Guidelines recommend initiating EBV monitoring no later than four weeks post-HCT and conducting it weekly. Pre-emptive therapies, such as the reduction of immunosuppressive therapy and the administration of rituximab to treat EBV viral loads are also suggested. In this study, we investigated the occurrence of EBV-PTLD in 546 HCT recipients, focusing on the clinical manifestations and risk factors associated with the disease. We managed to identify 67,150 viral genomic copies/mL as the cutoff point for predicting PTLD, with 80% sensitivity and specificity. Among our cohort, only 1% of the patients presented PTLD. Anti-thymocyte globulin (ATG) and GVHD were independently associated with lower survival rates and higher treatment-related mortality. According to our findings, prophylactic measures including regular monitoring, pre-emptive therapy, and supportive treatment against infections can be effective in preventing EBV-related complications. This study also recommends conducting EBV monitoring at regular intervals, initiating pre-emptive therapy when viral load increases, and identifying factors that increase the risk of PTLD. Our study stresses the importance of frequent and careful follow-ups of post-transplant complications and early intervention in order to improve survival rates and reduce mortality.

Effectiveness and Safety of Rabbit Anti-Thymocyte Globulin Versus Non-Anti-Thymocyte Globulin Regimens for Graft-Versus-Host Disease Prophylaxis: A Systematic Review and Meta-Analysis

Introduction:Graft-versus-host disease (GVHD) is a serious complication after allogeneic hematopoietic stem cell transplant (HSCT), limiting its success. Meta-analyses have confirmed clinical efficacy and safety of several types of anti-thymocyte globulins (ATGs) in GVHD prophylaxis; however, there are no meta-analyses assessing the efficacy of rabbit anti-thymocyte globulin (rATG; Thymoglobulin ®) in particular. This study assessed the effectiveness and safety of rATG relative to non-ATG prophylaxis regimens for acute GVHD (aGVHD) or chronic GVHD (cGVHD) based on randomized controlled trials (RCTs) and observational studies. Methods:A literature search was conducted in MEDLINE, Embase, Cochrane Central, and the Web of Science using the PICOS framework to identify studies published until June 2022 focusing on the prophylaxis use of rATG in GVHD. The United States and European Union clinical trial registry databases were also searched to identify unpublished data from studies. Efficacy outcomes included incidences of aGVHD (Grade II-IV and Grade III-IV), cGVHD (mild to severe and moderate to severe) and overall survival. Safety outcomes included the incidence of relapse, cytomegalovirus (CMV) infection, and Epstein-Barr virus (EBV) reactivation. Pre-specified subgroups of interest included donor type, and stem cell source (an unrelated peripheral blood [PB]/bone marrow [BM] transplant, a related PB/BM transplant, or a mixed unrelated and related PB/BM transplant). All analyses present the overall effect, the effect by subgroups, and the percentage contributing to the overall effect. Results: Overall, 4295 records were retrieved from the databases and hand searching (1000 from PubMed, 1700 from Embase, 1419 from the Web of Science, 155 from the Cochrane Central, 20 from the clinical trial registry databases and 1 from hand searching). After screening, 45 studies (6 RCTs, 35 retrospective studies and 4 prospective observational studies) published between 2001 and June 2022 were included in this review. Among the 45 studies, 39 reported aGVHD Grade II-IV, 33 reported aGVHD Grade III-IV, 33 reported mild to severe cGVHD, 31 reported moderate to severe cGVHD, and 41 reported overall survival ( Table). Use of rATG for GVHD prophylaxis resulted in a significant 24% reduction in Grade II-IV aGVHD (RR=0.76; 95% CI: 0.68-0.85) and a significant 43% reduction in Grade III-IV aGVHD (RR=0.57; 95% CI: 0.49-0.65) compared with non-ATG arm across all donor types. Similarly, a 35% reduction was observed in mild to severe cGVHD (RR=0.65; 95%CI: 0.57-0.74) and a significant 48% reduction (RR=0.52; 95% CI: 0.45-0.60) was observed in moderate to severe cGVHD across all donor types. Use of rATG decreased overall mortality by 9% (RR=0.91; 95% CI: 0.85-0.99). Among the subgroups, the decrease in mortality was statistically significant in unrelated PB/BM transplant donors. The incidence of relapse was reported in 36 studies, with an overall 18% increase (RR=1.18; 95% CI: 1.08-1.30) for patients treated with rATG. However, among the subgroups, the incidence of relapse was comparable between rATG and non-ATG prophylaxis arms for all types of donors. rATG and non-ATG prophylaxis regimens were comparable in terms of CMV infection (RR=1.00; 95% CI: 0.88-1.15). The incidence of EBV infection was significantly higher in the rATG arm (RR=2.53; 95% CI: 1.54-4.17) than that in the non-ATG arm. Conclusions: This meta-analysis provides useful information to support patients and health-care providers in making treatment decisions for the prevention of aGVHD or cGVHD after allogeneic HSCT. rATG demonstrated significant reductions in the incidence of both aGVHD and cGVHD and increased the overall survival across donor types and stem cell sources despite slightly increased risk of relapse in rATG arm versus non-ATG arm.

A Modified Busulfan/Cyclophosphamide Preconditioning Regimen Incorporating Selinexor for Allogeneic Hematopoietic Stem Cell Transplantation in Relapsed/Refractory Hematologic Malignancies

Objective: To investigate the safety and efficacy of a modified busulfan (Bu)/cyclophosphamide (Cy) conditioning regimen incorporating Selinexor for allogeneic hematopoietic stem cell transplantation in relapsed/refractory hematologic malignancies. Methods: We retrospectively evaluated the clinical outcomes of patients with hematologic malignancies who had received allo-HSCT with conditioning incorporating Selinexor at the Institute of Hematology and Blood Disease Hospital. Results: Between September 2022 and June 2023, 13 adult patients with high-risk hematologic malignancies were enrolled including 6 males and 7 females with a median age of 49 (13-58) years. There were 7 patients with acute myeloid leukemia (AML), 4 with myelodysplastic syndromes (MDS)/AML, 1 with multiple myeloma (MM) and 1 with extramedullary plasmacytoma. Six patients were in complete remission (CR), four in CR1, two in ≥CR2, five in non-remission (NR), and two patients were in a progressive disease status (PD) prior to conditioning. Three patients received matched-sibling donors transplant, while ten received related haploid hematopoietic stem cell transplantation. All patients had received myeloablative conditioning regimen incorporating Selinexor. Eleven patients received the conditioning: Bu (3.2mg/kg/d*2d); Melphalan (Mel, 60mg/m 2/d*2d), Fludarabine(Flu 30mg/m 2/d*3d) or CLA (5mg/m 2/d*3d), Cy (40mg/kg/d*2d), Selinexor 60mg/d*2d. The other 2 patients received the conditioning: Decitabine (DAC, 20mg/m 2/d*5d), Bu (3.2mg/kg/d*3d), Cy (40mg/kg/d *2d), Flu 30mg/m2/d*3d, Selinexor 60mg/d*2d. The median amount of infused mononuclear cell and CD34 + stem cell were 11.72×10 8/kg(range, 9.66 to 14) and 2.92×10 6/kg(range, 2.35 to 9.61), respectively. All the 13 patients achieved neutrophil engraftment with a median time of 12 (range, 9 to 18) days and 9 patients achieved platelet engraftment with a median time of 13 (range, 11 to 30) days. Four patients were still experiencing prolonged isolated thrombocytopenia. All patients achieved complete donor chimerism and minimal residual disease (MRD) negative at 30 days post-transplantation.The cumulative incidence of cytomegalovirus (CMV) reactivation was 15.4%, while Epstein-Barr virus (EBV) reactivation was not observed. Six patients (46.2%) experienced mucosal barrier injury, comprising three cases of oral mucosal ulcer, one case of digestive tract hemorrhage, and two cases of hemorrhagic cystitis. The cumulative incidence of II-IV and III-IV acute GVHD was 23.1% and 15.4%. The median follow-up for all the participants was 93 (40-316) days after transplantation. Only one patient was dead due to Thrombotic Microangiopathy (TMA) 78 days after transplant. One patient experienced molecular relapse 156 days after transplant, and another patient experienced hematology relapse 20 days after transplant. The two relapsed patients had received donor lymphocyte infusions after chemotherapy. The other 10 patients were still in CR with complete donor chimerism and MRD negative. The cumulative transplant-related mortality (TRM) and relapse rates are 7.7% (1 case) and 15.4% (2 cases), respectively. Conclusions: Incorporating Selinexor into the modified BU/CY conditioning regimen for allogeneic HSCT in patients with relapsed/refractory hematologic malignancies demonstrated a favorable safety profile. Ongoing prospective trials will provide valuable insights into the potential benefits of Selinexor in patients undergoing HSCT.

Exploring the Significance of Immune Checkpoints and EBV Reactivation in Antibody Deficiencies with Near-Normal Immunoglobulin Levels or Hyperimmunoglobulinemia.

This study delves into the intricate landscape of primary immunodeficiencies, with a particular focus on antibody deficiencies characterized by near-normal immunoglobulin levels or hyperimmunoglobulinemia. Contrary to the conventional focus on genetic dysregulation, these studies investigate the key roles of immune checkpoints, such as PD-1/PD-L1, CTLA-4/CD86, and CD200R/CD200, on selected subpopulations of T and B lymphocytes and their serum concentrations of soluble forms in patients recruited for the studies in healthy volunteers. In addition, the studies also show the role of Epstein-Barr virus (EBV) reactivation and interactions with tested pathways of immune checkpoints involved in the immunopathogenesis of this disease. By examining the context of antibody deficiencies, this study sheds light on the nuanced interplay of factors beyond genetics, particularly the immune dysregulations that occur in the course of this type of disease and the potential role of EBV reactivation, which affects the clinical presentation of patients and may contribute to the development of cancer in the future, especially related to hematological malignancies.

Overview of microbes in hypertension

High blood pressure (BP), known as hypertension, is a major contributing factor to the development of cardiovascular disease. The development and pathogenesis of hypertension involve a wide array of factors including genetics, environment, hormones, hemodynamics, and inflammation. There is a significantly positive association between higher levels of colonization by Aggregatibacter actinomycetemcomitans , Porphyromonas gingivalis , Tannerella forsythia , and Treponema denticola (etiologic bacterial burden) below the gum line, and the presence of hypertension. The use of antibiotics during pregnancy, which is likely indicative of bacterial infections severe enough to require antibiotic treatment, is associated with a slight increase in average arterial BP. Cytomegalovirus infection is a risk factor for heightened arterial BP and acts as a co-factor in the development of aortic atherosclerosis. The relationship between hypertension and coronavirus disease 2019 involves endothelial dysfunction and dysregulation of the renin-angiotensin system. The effects of gut microbiota on BP, whether beneficial or harmful, are influenced by multiple factors including genetics, epigenetics, lifestyle choices, and antibiotic usage. These variables collectively contribute to overall BP levels and the control of hypertension. Several reports have examined the BP levels of patients infected with the Zika virus. In regions with a high incidence of nasopharyngeal carcinoma, hypertension has been linked to a higher risk of Epstein-Barr virus reactivation. Also, a potential causal link has been found between malaria and elevated BP. Also, the elevated prevalence of hypertension among dengue patients during their initial visit suggests that relying solely on BP measurements to predict severe infection may not be clinically reliable.