Highly sensitive detection of Epstein-Barr virus-infected cells by EBER flow FISH.

When Epstein-Barr virus (EBV) infection is suspected, identification of infected cells is important to understand the pathogenesis, determinine the treatment strategy, and predict the prognosis. We used the PrimeFlow™ RNA Assay Kit with a probe to detect EBV-encoded small RNAs (EBERs) and multiple surface markers, to identify EBV-infected cells by flow cytometry. We analyzed a total of 24 patients [11 with chronic active EBV disease (CAEBV), 3 with hydroa vacciniforme lymphoproliferative disorder, 2 with X-linked lymphoproliferative disease type 1 (XLP1), 2 with EBV-associated hemophagocytic lymphohistiocytosis, and 6 with posttransplant lymphoproliferative disorder (PTLD)]. We compared infected cells using conventional quantitative PCR methods and confirmed that infected cell types were identical in most patients. Patients with CAEBV had widespread infection in T and NK cells, but a small amount of B cells were also infected, and infection in patients with XLP1 and PTLD was not limited to B cells. EBV-associated diseases are believed to be complex pathologies caused by EBV infecting a variety of cells other than B cells. We also demonstrated that infected cells were positive for HLA-DR in patients with CAEBV. EBER flow FISH can identify EBV-infected cells with high sensitivity and is useful for elucidating the pathogenesis.

HLA-haploidentical T-cell receptor αßT/B-cell-depleted stem cell transplantation for Fanconi anemia.

HLA-haploidentical stem cell transplantation (haplo-SCT) using post-transplant high-dose cyclophosphamide (PT-CY) is an alternative choice when a suitable donors is unavailable. However, PT-CY is difficult in patients with Fanconi anemia (FA) due to their high vulnerability to alkylating agents. For FA, we prefer haplo-SCT by T-cell receptor αßT-cell and B-cell depletion (αßT/B-depleted haplo-SCT), which can reduce the risks of PT-CY-related complications and graft-versus-host disease (GVHD). An 11-year-old boy with diagnosed FA (FANCG mutation) and bone marrow failure was to receive αßT/B-depleted haplo-SCT from his father (HLA 4/8 allele matched) due to absence of an HLA-matched donors. αßT/B-depleted peripheral blood stem cells (CD34 + cell count, 1.17 × 107/kg; αß + T-cell count, 1.3 × 105/kg) were infused following conditioning consisting of fludarabine (150 mg/m2), cyclophosphamide (40 mg/kg), anti-thymocyte globulin (5 mg/kg), rituximab (375 mg/m2), and thoraco-abdominal irradiation (3 Gy). Tacrolimus was used for GVHD prophylaxis until day + 30. Neutrophil engraftment was achieved on day + 9, and complete chimerism was confirmed on days + 28 and + 96. At 12-month post-SCT, the patient was well without GVHD or any other complications. αßT/B-depleted haplo-SCT is a good choice not only for patients unsuitable for PT-CY, but also for all pediatric recipients to reduce SCT-related complications.

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.

Updated guidelines for chronic active Epstein-Barr virus disease.

Chronic active Epstein-Barr virus disease (CAEBV), formerly named chronic active Epstein-Barr virus infection, is characterized by systemic inflammation and clonal proliferation of Epstein-Barr virus (EBV)-infected T or NK cells. As CAEBV is a potentially life-threatening illness, appropriate diagnosis and therapeutic interventions are necessary for favorable clinical outcomes. Substantial evidence regarding the pathogenesis and treatment of CAEBV has been accumulated since previous guidelines for the diagnosis of CAEBV were proposed. To reflect this evidence, we updated the guidelines for the diagnosis and treatment of CAEBV to improve clinical management of the disease. The details of the updated guidelines are presented in this report. Diagnosis of CAEBV now requires confirmation of a high copy number of EBV genome and EBV-infected T or NK cells. An EBV DNA load ≥ 10,000 IU/mL in whole blood is proposed as the diagnostic cutoff value for CAEBV in this updated guideline. A standard treatment approach for CAEBV has not been established, and hematopoietic stem cell transplantation (HSCT) is considered the only curative treatment. Chemotherapy can be administered to control disease activity before HSCT.

No Additional Risk of Ampicillin Rash Among Pediatric Liver Transplant Recipients With Concurrent Epstein-Barr Virus Infection.

BACKGROUND: Epstein-Barr virus (EBV) infection frequently develops in children undergoing liver transplantation (LT) because of mandated immunosuppressive therapy. There is a risk of ampicillin rash when penicillin derivatives are used in patients with EBV-associated infectious mononucleosis. Hence, the administration of penicillin derivatives may raise concerns about ampicillin rash in patients with high EBV loads. However, no studies confirmed the risk of administering penicillin derivatives to EBV-infected children after LT. METHODS: This retrospective study was conducted at the largest pediatric transplantation center in Japan. We investigated all pediatric liver transplant recipients who received penicillin derivatives within 2 years of LT between 2014 and 2020. We separated the cohort into EBV-positive and EBV-negative groups to assess the frequency of ampicillin and antibiotic-associated rash. RESULTS: Two hundred eighty-six liver transplant recipients were enrolled. There were 111 recipients in the EBV-positive group and 175 recipients in the EBV-negative group. In the EBV-positive group, 49 patients had high EBV DNA loads (≥1000 copies/µg DNA). None of the patients in either group developed ampicillin rash, and the frequency of antibiotic-associated rash did not differ [8/111 (7.2%) vs. 10/175 (5.7%), P = 0.797]. Additional subgroup analysis revealed no difference in the frequency of antibiotic-associated rashes regardless of the presence or absence of high EBV loads. CONCLUSIONS: In this study, ampicillin rash was not observed, and antibiotic-associated rash was not associated with concurrent EBV infection. Penicillin derivatives can be used safely, even in liver transplant recipients with persistent asymptomatic EBV infection.

Long-term remission of VEXAS syndrome achieved by a single course of CHOP therapy: A case report.

We herein describe the case of a 52-year-old male patient who presented with fever, arthritis, and neutrophilic dermatosis in 2013 and subsequently experienced macrophage activation syndrome treated with high-dose glucocorticoid therapy. Due to the persistent symptoms refractory to several immunomodulatory and immunosuppressive (IS) drug therapies with dapsone, methotrexate, tacrolimus, infliximab (IFX), and tocilizumab (TCZ), he received prednisolone (PSL) ≥20 mg/day to suppress disease activity. In 2017, Epstein-Barr virus (EBV)-associated haemophagocytic lymphohistiocytosis (HLH) was diagnosed and initially treated with immunochemotherapy consisting of dexamethasone, cyclosporine (CyA), and etoposide (ET). Because of the suboptimal response to the initial therapy, cytoreduction therapy consisting of CHOP (combination chemotherapy consisting of cyclophosphamide, doxorubicin, vincristine, and PSL) was administered. This regimen improved the EBV-associated HLH. Later, the patient's condition stabilised with methylprednisolone 1 mg/day and CyA 100 mg/day. In 2022, ubiquitylation-initiating E1 enzyme (UBA1) variant analysis using Sanger sequencing of peripheral blood leukocytes detected a previously reported somatic variant (NM_003334.3: c.118-1G>C), confirming the diagnosis of vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome. The clinical course in the present case suggested the possibility that CHOP could be a potential treatment option for VEXAS syndrome, in the pathophysiology of which the expansion of clones with UBA1 variant seems to play a pivotal role.

The hepatic niche leads to aggressive natural killer cell leukemia proliferation through the transferrin-transferrin receptor 1 axis.

Aggressive natural killer cell leukemia (ANKL) is a rare lymphoid neoplasm frequently associated with Epstein-Barr virus, with a disastrously poor prognosis. Owing to the lack of samples from patients with ANKL and relevant murine models, comprehensive investigation of its pathogenesis including the tumor microenvironment (TME) has been hindered. Here we established 3 xenograft mice derived from patients with ANKL (PDXs), which enabled extensive analysis of tumor cells and their TME. ANKL cells primarily engrafted and proliferated in the hepatic sinusoid. Hepatic ANKL cells were characterized by an enriched Myc-pathway and proliferated faster than those in other organs. Interactome analyses and in vivo CRISPR-Cas9 analyses revealed transferrin (Tf)-transferrin receptor 1 (TfR1) axis as a potential molecular interaction between the liver and ANKL. ANKL cells were rather vulnerable to iron deprivation. PPMX-T003, a humanized anti-TfR1 monoclonal antibody, showed remarkable therapeutic efficacy in a preclinical setting using ANKL-PDXs. These findings indicate that the liver, a noncanonical hematopoietic organ in adults, serves as a principal niche for ANKL and the inhibition of the Tf-TfR1 axis is a promising therapeutic strategy for ANKL.

[Epstein-Barr virus-associated lymphoproliferative disorders after BNT162b2 mRNA COVID-19 vaccination].

Epstein-Barr virus-associated lymphoproliferative disorders (EBV-LPD) is a rare disease characterized by persistent or recurrent inflammation accompanied by EBV infection of T or NK cells that is not self-limiting, and it is fatal, if untreated. After receiving the first dose of the BNT162b2 mRNA COVID-19 vaccine, a 79-year-old male presented to the hospital with a 2-week history of fever. Laboratory results indicated pancytopenia, elevated liver transaminase levels, hyperferritinemia, and hypofibrinogenemia. Computed tomography revealed hepatosplenomegaly, but lymphadenopathy was not observed. A bone marrow biopsy, a random skin biopsy, and a liver biopsy revealed no malignancy, but an infectious evaluation revealed EBV viremia (5.19 Log IU/ml). Flow cytometry and RT-PCR revealed that the EBV genome was localized in NK cells, suggesting the diagnosis of EBV-NK-LPD. We administered prednisolone, intravenous immunoglobulin, and etoposide, but the EBV-DNA load failed to decrease, and he died 2 months later. Recently, case reports of COVID-19 vaccination-related hemophagocytic lymphohistiocytosis have been published. Although the mechanisms and risk factors for EBV-LPD after BNT162b2 mRNA COVID-19 vaccination remain unknown, it is important to note the possibility of reactivation of EBV after COVID-19 vaccination to initiate early and targeted therapy.

Etiology of Severe Acute Hepatitis in a Pediatric Transplant Center in Japan.

The proportion of pediatric cases with severe acute hepatitis of unknown etiology in the coronavirus disease 2019 era was higher than that in the pre-coronavirus disease 2019 era in Japan's largest pediatric transplant center. Further research and monitoring are essential.

Development of Epstein-Barr virus-associated lymphoproliferative disorder and hemophagocytic lymphohistiocytosis during long-term lenalidomide maintenance therapy in multiple myeloma.

Reactivation of Epstein-Barr virus (EBV) has been considered a very rare event among patients on immunomodulatory drugs (IMiDs) such as lenalidomide, and an association between the two has not well been recognized. We have recently experienced a rare case of multiple myeloma in which the patient had suffered EBV reactivation during long-term lenalidomide maintenance therapy. The patient subsequently developed EBV-associated lymphoproliferative disease (LPD) as well as EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), which was fatal despite intensive treatment. Although rare, clinicians should be aware that such fatal EBV reactivation could occur as a minor yet critical complication of long-term maintenance therapy with IMiDs in multiple myeloma patients. Regular monitoring and early detection of EBV reactivation would be beneficial for these patients, so that proper diagnostic examinations can be initiated without delay.

Early Detection of Epstein-Barr Virus as a Risk Factor for Chronic High Epstein-Barr Viral Load Carriage at a Living-donor-dominant Pediatric Liver Transplantation Center.

BACKGROUND: Epstein-Barr virus (EBV) infection and posttransplant lymphoproliferative disorders (PTLDs) after pediatric liver transplantation (LT) account for significant morbidity and mortality. Knowledge of EBV kinetics, epidemiology, and outcomes among pediatric living-donor LT cases is largely lacking. This study aims to provide clinical information related to EBV infection, chronic high EBV load (CHL) carriage, and PTLD at a living-donor-dominant pediatric LT center. METHODS: A total of 5827 EBV load measurements from 394 LT recipients fulfilling inclusion criteria and their clinical data were analyzed. EBV loads >1000 copies/µg DNA (742 IU/µg DNA) were considered "high," and CHL was defined by persistence >6 mo. RESULTS: The highlighted results were as follows: (1) 94% of recipients underwent living-donor LT; (2) 80% of EBV seronegative recipients developed first EBV infection <2 y post-LT, and their EBV loads were consistently higher than those of seropositive recipients within <3 y post-LT but did not differ thereafter; (3) 61 (15%) recipients met CHL criteria, but none developed PTLD; (4) age <5 y, cytomegalovirus seronegative donors, and early development of EBV DNAemia <6 mo post-LT were independent risk factors for CHL; (5) the incidence of rejections after 1-y post-LT was comparably low among CHL carriers whose immunosuppression was minimized. CONCLUSIONS: Early detection of EBV following LT and CMV seronegative donors would facilitate risk stratification to prevent PTLD while titrating immunosuppression among pediatric LT recipients.

Chronic active Epstein-Bar virus infection complicated by pulmonary artery hypertension.

Chronic active Epstein-Bar virus infection (CAEBV) is known to cause various symptoms. Although pulmonary artery hypertension (PAH) has been reported as a cardiovascular complication of CAEBV, the mechanisms of PAH and the effects of treatment have not been fully elucidated. We experienced 4 adult patients with CAEBV complicated by PAH. All of them received treatment for PAH with a vasodilator followed by chemotherapy with or without allogeneic hematopoietic cell transplantation for CAEBV. In all of these patients, the transtricuspid pressure gradient improved under treatment with vasodilator, and further improvement was observed under treatment for CAEBV in 3 patients. Autopsy was performed in 2 patients, which revealed EBER-positive cells and a change in the pulmonary artery at each stage in the pathology. In conclusion, EBV-infected cells can cause vasculitis and finally PAH. However, PAH complicated with CAEBV can be improved by PAH medication and treatment of CAEBV.

Human antibody recognition and neutralization mode on the NTD and RBD domains of SARS-CoV-2 spike protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Variants of concern (VOCs) such as Delta and Omicron have developed, which continue to spread the pandemic. It has been reported that these VOCs reduce vaccine efficacy and evade many neutralizing monoclonal antibodies (mAbs) that target the receptor binding domain (RBD) of the glycosylated spike (S) protein, which consists of the S1 and S2 subunits. Therefore, identification of optimal target regions is required to obtain neutralizing antibodies that can counter VOCs. Such regions have not been identified to date. We obtained 2 mAbs, NIBIC-71 and 7G7, using peripheral blood mononuclear cells derived from volunteers who recovered from COVID-19. Both mAbs had neutralizing activity against wild-type SARS-CoV-2 and Delta, but not Omicron. NIBIC-71 binds to the RBD, whereas 7G7 recognizes the N-terminal domain of the S1. In particular, 7G7 inhibited S1/S2 cleavage but not the interaction between the S protein and angiotensin-converting enzyme 2; it suppressed viral entry. Thus, the efficacy of a neutralizing mAb targeting inhibition of S1/2 cleavage was demonstrated. These results suggest that neutralizing mAbs targeting blockade of S1/S2 cleavage are likely to be cross-reactive against various VOCs.

The Plasma Level of Interleukin-1ß Can Be a Biomarker of Angiopathy in Systemic Chronic Active Epstein-Barr Virus Infection.

Systemic chronic active Epstein-Barr virus infection (sCAEBV) is an EBV-positive T- or NK-cell neoplasm revealing persistent systemic inflammation. Twenty-five percent of sCAEBV patients accompany angiopathy. It is crucial to clarify the mechanisms of angiopathy development in sCAEBV because angiopathy is one of the main causes of death. Interleukin-1ß (IL-1ß) is reported to be involved in angiopathy onset. We investigated if IL-1ß plays a role as the inducer of angiopathy of sCAEBV. We detected elevated IL-1ß levels in four out of 17 sCAEBV patient's plasma. Interestingly, three out of the four had clinically associated angiopathy. None of the other patients with undetectable level of IL-1ß had angiopathy. In all patients with high plasma levels of IL-1ß and vascular lesions, EBV-infected cells were CD4-positive T cells. In one patient with high plasma IL-1ß, the level of IL-1ß mRNA of the monocytes was 17.2 times higher than the level of the same patient's EBV-infected cells in peripheral blood. In Ea.hy926 cells, which are the models of vascular endothelial cells, IL-1ß inhibited the proliferation and induced the surface coagulation activity. IL-1ß is a potent biomarker and a potent therapeutic target to treat sCAEBV accompanying angiopathy.

Mucosal-associated invariant T cells are activated in an interleukin-18-dependent manner in Epstein-Barr virus-associated T/natural killer cell lymphoproliferative diseases.

Mucosal-associated invariant T (MAIT) cells are a type of innate immune cells that protect against some infections. However, the involvement of MAIT cells in Epstein-Barr virus-associated T/natural killer cell lymphoproliferative diseases (EBV-T/NK-LPD) is unclear. In this study, we found that MAIT cells were highly activated in the blood of patients with EBV-T/NK-LPD. MAIT cell activation levels correlated with disease severity and plasma IL-18 levels. Stimulation of healthy peripheral blood mononuclear cells with EBV resulted in activation of MAIT cells, and this activation level was enhanced by exogenous IL-18. MAIT cells stimulated by IL-18 might thus be involved in the immunopathogenesis of EBV-T/NK-LPD.

Successful HLA-haploidentical stem cell transplantation with post-transplant cyclophosphamide in an older patient with chronic active Epstein-Barr virus infection.

Chronic active Epstein-Barr virus infection (CAEBV) is a subtype of EBV-associated T/NK cell lymphoproliferative disease and is only curable by allogeneic hematopoietic stem cell transplantation. However, finding a human leukocyte antigen (HLA)-matched donor at a suitable time can sometimes be difficult. We report the case of a 60-year-old woman who received prednisolone (PSL) after being diagnosed with autoimmune hepatitis 3 years earlier. She suddenly developed high fever and impaired liver function. Based on a high EBV DNA load in the peripheral blood, CAEBV was diagnosed. The patient was started on cooling therapy with PSL, cyclosporine, and etoposide, which reduced symptoms. Subsequently, she received HLA-haploidentical stem cell transplantation (haplo-SCT) with reduced-intensity conditioning (fludarabine 25 mg/m2 for 5 days, melphalan 50 mg/m2 for 2 days, and total body irradiation at 2 Gy) and post-transplant cyclophosphamide (PTCy) because she lacked an HLA-matched donor. Liver function was restored, and EBV DNA load in peripheral white blood cells became undetectable. The patient is alive without relapse or severe complications over 1 year after transplantation. To our knowledge, this is the first report of successful haplo-SCT with PTCy for CAEBV. This approach may be an alternative therapeutic option for CAEBV patients lacking an HLA-matched donor.

Late-onset posttransplant Epstein-Barr virusrelated lymphoproliferative disease after cord blood transplantation for chronic active Epstein Barr virus infection: A case report.

INTRODUCTION: Posttransplant lymphoproliferative disease (PTLD) is a critical complication of hematopoietic stem cell transplantation (HSCT). PTLD is classified into early and late-onset PTLDs. In post-HSCT patients, late-onset PTLD is rare, particularly PTLD after HSCT for Epstein-Barr virus (EBV)-related lymphoproliferative disease. Here, we report the case of a patient diagnosed with late-onset EBV-related hemophagocytic lymphohistiocytosis (HLH), that of PTLD, after HSCT for chronic active EBV infection (CAEBV), that of EBV related lymphoproliferative disease, probably because of EBV reactivation. PATIENT CONCERNS AND DIAGNOSIS: A 22-year-old woman with abdominal fullness visited our hospital. Blood examination showed pancytopenia with atypical lymphocytes, liver dysfunction, and elevated lactate dehydrogenase level. In contrast, bone marrow aspiration showed slight hemophagocytosis with increased natural-killer cells (NK cells). As serum antibodies against EBV were atypical, we calculated the EBV-DNA level in peripheral blood and this level was significantly high. EBV was infected with NK cells, and EBV's monoclonality in NK cells was confirmed. Thus, the patient was diagnosed with CAEBV. INTERVENTIONS AND OUTCOMES: The patient received chemotherapy and cord blood cell transplantation (CBT); CAEBV was well controlled. Approximately 6years from CBT for CAEBV, she visited our hospital because of fever. Blood examination revealed pancytopenia with atypical lymphocytes, liver dysfunction, and elevated lactate dehydrogenase level. In contrast, bone marrow aspiration showed hemophagocytosis with increased B and T cell counts without increased NK cell count. Additionally, serum antibody titers against EBV were atypical, and the EBV-DNA level in the peripheral blood was high. EBV was infected with only B cells, and EBV's monoclonality was confirmed. A more detailed analysis indicated that EBV-specific cytotoxic T lymphocytes were inactive. Therefore, she was diagnosed with late-onset EBV-related HLH. She received extensive treatment, but EBV-related HLH did not improve. Finally, she died about 3 weeks after diagnosis. CONCLUSION: PTLD, including HLH, is a life-threatening complication after transplantation, including HSCT. To our knowledge, this is the first case of late-onset EBV-related HLH after CBT for CAEBV. Late-onset PTLD has an indolent clinical course, but our patient's disease course was extremely aggressive. Therefore, late-onset EBV-related PTLD may be life-threatening.

NK-cell post-transplant lymphoproliferative disease successfully treated by second allogenic hematopoietic stem cell transplantation in chronic active Epstein-Barr virus infection.

Chronic active Epstein-Barr virus infection (CAEBV) is a systemic T- or NK-lymphoproliferative disorder (LPD) caused by EBV. Allogenic hematopoietic stem cell transplantation (HSCT) is the only curative therapy for CAEBV, but relapse sometimes occurs. Relapse is generally attributed to proliferation of recipient-derived CAEBV cells. We herein report a case of donor-derived CAEBV-like NK-cell post-transplant lymphoproliferative disease (PTLD) in a 41-year-old female after the first allogenic HSCT for CAEBV from an HLA-matched sibling donor. A second HSCT from an HLA-matched unrelated donor successfully controlled the disease, but EBV infection of cells derived from the second donor continued to be detected. Although the mechanisms underlying CAEBV and CAEBV-like NK-cell PTLD have not yet been elucidated in detail, the findings of the present case imply that host genetic factors, including familial factors, may be important in disease development.