OBJECTIVE: To analyze the Epstein-Barr virus (EBV) load in different lymphocyte subsets, as well as clinical characteristics and outcomes in patients with hematologic malignancies experiencing EBV reactivation. METHODS: Peripheral blood samples from patients were collected. B, T, and NK cells were isolated sorting with magnetic beads by flow cytometry. The EBV load in each subset was quantitated by real-time quantitative polymerase chain reaction (RT-qPCR). Clinical data were colleted from electronic medical records. Survival status was followed up through outpatient visits and telephone calls. Statistical analyses were performed using SPSS 25.0. RESULTS: A total of 39 patients with hematologic malignancies were included, among whom 35 patients had undergone allogeneic hematopoietic stem cell transplantation (allo-HSCT). The median time to EBV reactivation was 4.8 months (range: 1.7-57.1 months) after allo-HSCT. EBV was detected in B, T, and NK cells in 20 patients, in B and T cells in 11 patients, and only in B cells in 4 patients. In the 35 patients, the median EBV load in B cells was 2.19×10⁴ copies/ml, significantly higher than that in T cells (4.00×10³ copies/ml, P <0.01) and NK cells (2.85×10² copies/ml, P <0.01). Rituximab (RTX) was administered for 32 patients, resulting in EBV negativity in 32 patients with a median time of 8 days (range: 2-39 days). Post-treatment analysis of 13 patients showed EBV were all negative in B, T, and NK cells. In the four non-transplant patients, the median time to EBV reactivation was 35 days (range: 1-328 days) after diagnosis of the primary disease. EBV was detected in one or two subsets of B, T, or NK cells, but not simultaneously in all three subsets. These patients received a combination chemotherapy targeting at the primary disease, with 3 patients achieving EBV negativity, and the median time to be negative was 40 days (range: 13-75 days). CONCLUSION In hematologic malignancy patients after allo-HSCT, EBV reactivation commonly involves B, T, and NK cells, with a significantly higher viral load in B cells compared to T and NK cells. Rituximab is effective for EBV clearance. In non-transplant patients, EBV reactivation is restricted to one or two lymphocyte subsets, and clearance is slower, highlighting the need for prompt anti-tumor therapy.
Humans ; Hematologic Neoplasms/virology* ; Herpesvirus 4, Human/physiology* ; Epstein-Barr Virus Infections ; Hematopoietic Stem Cell Transplantation ; Virus Activation ; Lymphocyte Subsets/virology* ; Flow Cytometry ; Killer Cells, Natural/virology* ; Male ; Female ; B-Lymphocytes/virology* ; Viral Load ; Adult ; T-Lymphocytes/virology* ; Middle Aged

OBJECTIVESevere Epstein-Barr (EB) virus infection is potentially a devastating process that often leads to death encountered in pediatrics recently. Inappropriate control of EB virus replication may cause severe infection resulting in multiple organ dysfunction. However, little information is available on pulmonary complications associated with EB virus infection. The aim of the present study was to investigate severe EB virus (EBV) infection complicated with lung injury in pediatric intensive care unit (PICU), including clinical characteristics, laboratory or imaging feature and outcomes.

METHODA total of 45 children with severe EBV infection seen in PICU of Shanghai Children's Hospital between January 2011 and December 2014 were retrospectively reviewed. According to clinical characteristics and imaging feature, 45 children were divided into non-lung injury group (n =27), lung injury without pulmonary fibrosis group(n = 12) and pulmonary fibrosis group (n = 6).

RESULTIn totally 45 cases of severe EBV infection, 21 (46.7%) were male and 24 (53. 3%) were female, mean age was 2. 4 years; 18 cases were complicated with lung injury, including 8 male and 10 female, median age was 31. 2 months. All of 18 cases presented with fever and cough, 15 of them exhibited dyspnea,12 cases were complicated with gasping, and 6 cases with ARDS. Eight cases accepted mechanical ventilation for acute respiratory distress; 6 cases who developed pulmonary fibrosis had tachypnea, refractory hypoxemia and hypercapnia, severe pulmonary air leak. The average EBV-DNA level in peripheral blood was 4. 42 x 10(6) copies/ml (range: 3. 25 x 10(3) - 6.59 x 10(7) copies/ml). Anti-EBV antibodies were positive in 41 cases, 18 cases were positive (+) for VCA-IgM, 15 cases were VCA-IgG and EA-IgG (+), 8 cases VCA-IgM and VCA-IgG (+). The radiographic findings revealed pulmonary interstitial infiltrates in all 18 cases with lung injury, 4 cases with segmental consolidation and 2 cases showed pleural effusions. HRCT scanning found EBV associated fibrosis including multifocal patches and diffuse ground-glass attenuation in both lungs, reticular opacities and honeycombing changes were observed 4 weeks after illness onset. There were significant differences in respiratory failure, PICU stay (days), Pediatric risk of mortality III (PRISM III) and pediatric clinical illness score(PCIS), serum TNF-α, EBV-DNA levels, percentage of NK cells and CD4+/CD8+ T cell ratio among non-lung injury group, lung injury without pulmonary fibrosis group and pulmonary fibrosis group (X2 =27. 12, F = 85. 23, 78. 23, 88. 68, 323. 80, 7. 35, χ2 = 6. 71, 12. 15; all P < 0. 05). COX regression analysis revealed that EBV-DNA and serum TNF-α levels were correlated with pulmonary fibrosis significantly (OR = 3. 92, P = 0. 04; OR = 5. 95, P = 0. 01). The patients with EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) had higher incidence of pulmonary fibrosis compared with non-EB-HLH (70% vs. 13%, χ2 = 4. 82, P = 0. 03). Of 18 cases, 8 cases died, including 3 cases with pulmonary fibrosis. The surviving cases with pulmonary fibrosis needed longer additional oxygen. Chest HRCT imaging of 3 cases with pulmonary fibrosis was improved at 6 months and oxygen therapy was discontinued at 18 months after discharge.

CONCLUSIONEB virus infection complicated with lung injury had higher incidence of respiratory failure, pulmonary fibrosis with a fatal outcome. EBV-DNA and serum TNF-α level were found to be statistically significant indicators of pulmonary fibrosis. Pulmonary fibrosis associated with severe EB virus infection may be reversible.

Antibodies, Viral ; blood ; CD4-CD8 Ratio ; Child, Preschool ; China ; DNA, Viral ; blood ; Epstein-Barr Virus Infections ; pathology ; Female ; Herpesvirus 4, Human ; Humans ; Intensive Care Units, Pediatric ; Killer Cells, Natural ; Lung Injury ; virology ; Lymphohistiocytosis, Hemophagocytic ; pathology ; virology ; Male ; Pulmonary Fibrosis ; pathology ; virology ; Retrospective Studies ; Tumor Necrosis Factor-alpha ; blood

Killer cell immunoglobulin-like receptors (KIRs) which are mainly expressed on natural killer (NK) cells are implicated in many virus infections. However, it is unclear whether or not KIRs are associated with susceptibility to Epstein-Barr virus (EBV) infection related diseases. Therefore, the purpose of our study was to investigate possible correlation between polymorphisms of KIR genes and infectious mononucleosis (IM)/EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH). The polymorphisms of KIR genes were detected by polymerase chain reaction with sequence-specific primers (PCR-SSP). The results would contribute to clarify the association of KIRs with EBV induced diseases, and provide new insights into the role of NK cells and innate immune response against viral infections and/or subsequent progression.
Case-Control Studies ; Child ; Child, Preschool ; China ; Disease Progression ; Female ; Herpesvirus 4, Human ; physiology ; Humans ; Immunity, Innate ; Infectious Mononucleosis ; genetics ; immunology ; virology ; Killer Cells, Natural ; immunology ; metabolism ; Lymphohistiocytosis, Hemophagocytic ; genetics ; immunology ; virology ; Male ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Receptors, KIR ; genetics ; metabolism

Epstein-Barr virus, a ubiquitous human herpesvirus, can induce both lytic and latent infections that result in a variety of human diseases, including lymphoproliferative disorders. The oncogenic potential of Epstein-Barr virus is related to its ability to infect and transform B lymphocytes into continuously proliferating lymphoblastoid cells. However, Epstein-Barr virus has also been implicated in the development of T/natural killer cell lymphoproliferative diseases. Epstein-Barr virus encodes a series of products that mimic several growth, transcription and anti-apoptotic factors, thus usurping control of pathways that regulate diverse homeostatic cellular functions and the microenvironment. However, the exact mechanism by which Epstein-Barr virus promotes oncogenesis and inflammatory lesion development remains unclear. Epstein-Barr virus-associated T/natural killer cell lymphoproliferative diseases often have overlapping clinical symptoms as well as histologic and immunophenotypic features because both lymphoid cell types derive from a common precursor. Accurate classification of Epstein-Barr virus-associated T/natural killer cell lymphoproliferative diseases is a prerequisite for appropriate clinical management. Currently, the treatment of most T/natural killer cell lymphoproliferative diseases is less than satisfactory. Novel and targeted therapies are strongly required to satisfy clinical demands. This review describes our current knowledge of the genetics, oncogenesis, biology, diagnosis and treatment of Epstein-Barr virus-associated T/natural killer cell lymphoproliferative diseases.
Cell Transformation, Viral ; Epstein-Barr Virus Infections/*complications ; Herpesvirus 4, Human/*physiology ; Humans ; Killer Cells, Natural/immunology/metabolism/*pathology/*virology ; Lymphoproliferative Disorders/diagnosis/*etiology/therapy ; T-Lymphocytes/immunology/metabolism/*pathology/*virology

The development of highly immunodeficient mouse strains has allowed the reconstitution of functional human immune system components in mice. New-generation humanized mice generated in this manner have been extensively used for modeling viral infections that are exclusively human tropic. Epstein-Barr virus (EBV)-infected humanized mice reproduce cardinal features of EBV-associated B-cell lymphoproliferative disease and EBV-associated hemophagocytic lymphohistiocytosis (HLH). Erosive arthritis morphologically resembling rheumatoid arthritis (RA) has also been recapitulated in these mice. Low-dose EBV infection of humanized mice results in asymptomatic, persistent infection. Innate immune responses involving natural killer cells, EBV-specific adaptive T-cell responses restricted by human major histocompatibility and EBV-specific antibody responses are also elicited in humanized mice. EBV-associated T-/natural killer cell lymphoproliferative disease, by contrast, can be reproduced in a distinct mouse xenograft model. In this review, recent findings on the recapitulation of human EBV infection and pathogenesis in these mouse models, as well as their application to preclinical studies of experimental anti-EBV therapies, are described.
Animals ; Disease Models, Animal ; Epstein-Barr Virus Infections/complications/immunology/*virology ; Herpesvirus 4, Human/*physiology ; Heterografts ; Humans ; Killer Cells, Natural/pathology/virology ; Lymphoproliferative Disorders/etiology ; Mice ; Mice, SCID ; T-Lymphocytes/pathology/virology

In vitro amplified human leukocyte antigen (HLA)-haploidentical donor immune cell infusion (HDICI) is not commonly used in children. Therefore, our study sought to evaluate its safety for treating childhood malignancies. Between September 2011 and September 2012, 12 patients with childhood malignancies underwent HDICI in Sun Yat-sen University Cancer Center. The median patient age was 5.1 years (range, 1.7-8.4 years). Of the 12 patients, 9 had high-risk neuroblastoma (NB) [7 showed complete response (CR), 1 showed partial response (PR), and 1 had progressive disease (PD) after multi-modal therapies], and 3 had Epstein-Barr virus (EBV)-positive lymphoproliferative disease (EBV-LPD). The 12 patients underwent a total of 92 HDICIs at a mean dose of 1.6×10(8) immune cells/kg body weight: 71 infusions with natural killer (NK) cells, 8 with cytokine-induced killer (CIK) cells, and 13 with cascade primed immune cells (CAPRIs); 83 infusions with immune cells from the mothers, whereas 9 with cells from the fathers. Twenty cases (21.7%) of fever, including 6 cases (6.5%) accompanied with chills and 1 (1.1%) with febrile convulsion, occurred during infusions and were alleviated after symptomatic treatments. Five cases (5.4%) of mild emotion changes were reported. No other adverse events occurred during and after the completion of HDIDIs. Neither acute nor chronic graft versus host disease (GVHD) was observed following HDICIs. After a median of 5.0 months (range, 1.0-11.5 months) of follow-up, the 2 NB patients with PR and PD developed PD during HDICIs. Of the other 7 NB patients in CR, 2 relapsed in the sixth month of HDICIs, and 5 maintained CR with disease-free survival (DFS) ranging from 4.5 to 11.5 months (median, 7.2 months). One EBV-LPD patient achieved PR, whereas 2 had stable disease (SD). Our results show that HDICI is a safe immunotherapy for childhood malignancies, thus warranting further studies.
Child ; Child, Preschool ; Cytokine-Induced Killer Cells ; immunology ; Epstein-Barr Virus Infections ; therapy ; Female ; Follow-Up Studies ; Graft vs Host Disease ; etiology ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Immunotherapy, Adoptive ; Infant ; Killer Cells, Natural ; immunology ; Lymphoproliferative Disorders ; therapy ; virology ; Male ; Neuroblastoma ; therapy ; Transplantation, Homologous ; Treatment Outcome

The aim of this study was to analyze the clinical features and laboratory findings of adult Epstein-Barr virus associated T/NK cell lymphoproliferative disease (EBV+T/NK-LPD) and to investigate the early diagnosis and prognosis of EBV+T/NK-LPD. The clinical data of 19 adult patients with EBV+T/NK-LPD were retrospectively analyzed. The results indicated that there were 11 males and 8 females. The median age was 32 years (range: 20-70 years). The average duration from onset of symptoms to diagnosis was 3.5 months. The median survival time was 2.5 months. Unkown fever, hepatosplenomegaly, liver dysfunction and interstitial pneumonia were the main clinical features. High levels of β2-MG, LDH, TNF, IL-6 and significantly increased EBV-DNA level (median level > 10(6) copies/ml) were occurred in all the patients. Cytopenia was seen in 18 cases. Morphologically, atypical large granular lymphocytes and hemophagocytosis were common in bone marrow smears. Deletion of CD5 or CD7 were frequently observed in T/NK lymphocytes in bone marrow cells by flow cytometry. Bone marrow biopsy showed atypical lymphocyte interstitial infiltrated in 10 cases, while a few large cells infiltrated in 6 cases. Immunohistochemistry showed the expression of CD3(+)CD56(+) were seen in 2 cases, CD3(+)CD8(+) in 11 cases and CD3(+)CD4(+) in 3 cases. TIA-1 and EBER were positive in all biopsy specimens. Three cases underwent biopsy of lymph nodes showed reactive proliferations of lymphocytes. All the patients died of multiorgan failure. It is concluded that the fever, hepatosplenomegaly are the most common clinical features in adult EBV+T/NK-LPD, the bone marrow infiltration of EBV-infected T/NK lymphocytes and significantly increased EBV-DNA level can be observed in all cases, the clinical outcome of this disease is poor, these clinical and experimental features can be served as a reliable marker for the timely diagnosis of adult EBV+T/NK-LPD.
Adult ; Aged ; Epstein-Barr Virus Infections ; pathology ; Female ; Humans ; Immunophenotyping ; Killer Cells, Natural ; virology ; Lymphoproliferative Disorders ; pathology ; virology ; Male ; Middle Aged ; Retrospective Studies ; T-Lymphocytes ; virology ; Young Adult

OBJECTIVE: To observe the dynamic changes of 3 types of viral reservoir cells (NK cells, T lymphocytes and monocytes), and its relationship with treatment effect in Chinese HIV-1 infected patients receiving highly active antiretroviral treatment (HAART) for 2 years. METHODS: A total of 40 chronic HIV-1-infected adults who initiated HAART were enrolled in this study and followed up for 2 years. Peripheral whole blood was obtained from each patient at baseline (0 month), 6, 12, 18 and 24 months. Real-time fluorescent quantitative PCR was used to detect the HIV-1 RNA in the plasma and HIV-1 DNA in NK cells, T lymphocytes and monocytes. All the data were statistically analyzed. RESULTS: CD4 count increased with the decrease of the viral load during HAART. After HAART initiation, HIV-1 DNA showed a significant decrease in NK cells, T lymphocytes and monocytes. The HIV-1 DNA from T lymphocytes, NK cells and monocytes correlated positively with the HIV- 1 RNA (P<0.05) while NK cells and T lymphocytes correlated negatively with CD4+ T cell count. However we did not find significant correlation between CD4+ T cell count and HIV-1 DNA in monocytes at the baseline of HAART. CONCLUSION This study found that NK cell was an important HIV cellular reservoir besides T lymphocytes and monocytes. T lymphocytes may be the main long lasting HIV reservoir. HIV-1 proviral DNA may play an important role in the efficacy of treatment and monitoring the disease progression.
Adult ; Antiretroviral Therapy, Highly Active ; DNA, Viral ; analysis ; Female ; HIV Infections ; drug therapy ; virology ; HIV-1 ; genetics ; Humans ; Killer Cells, Natural ; virology ; Male ; Middle Aged ; Monocytes ; virology ; RNA, Viral ; blood ; T-Lymphocytes ; virology ; Viral Load

CD2 Antigens ; metabolism ; CD3 Complex ; metabolism ; Epstein-Barr Virus Infections ; Hematopoietic Stem Cell Transplantation ; Herpesvirus 4, Human ; isolation & purification ; Humans ; Killer Cells, Natural ; pathology ; virology ; Lymphoproliferative Disorders ; metabolism ; pathology ; therapy ; virology ; Poly(A)-Binding Proteins ; metabolism ; Prognosis ; T-Cell Intracellular Antigen-1

OBJECTIVETo study the clinicopathologic features, immunophenotype, clonality and Epstein-Barr virus (EBV) status of systemic EBV-positive T/NK-cell lymphoproliferative disease in adults (ASEBV(+)T/NK-LPD).

METHODSTwenty cases of ASEBV(+)T/NK-LPD were analyzed retrospectively with histopathologic review, immunohistochemistry and in-situ hybridization for EBV-encoded RNA (EBER). The follow-up data were collected.

RESULTSThere were altogether 15 males and 5 females. The median age of the patients was 34 years. The average duration from onset of symptoms to diagnosis was 8.7 months. Fever (18/20), hepatosplenomegaly (18/20) and lymphadenopathy (17/20) were the main clinical manifestations. Eleven of the 17 patients died during follow-up, with a mean survival of 2.9 months. Histologically, there was obvious expansion of T zone of the involved lymph nodes, associated with diminished lymphoid follicles. The interfollicular areas were widened and infiltrated by small to median-sized lymphoid cells which showed only mild atypia. Scattered large lymphoid cells were not uncommon. The nodal capsule was thickened in 6 cases. Focal necrosis was seen in 9 cases. Sinus histiocytic proliferation with erythrophagocytosis was observed in 3 cases. In addition, there were mild atypical lymphoid cells infiltrate into the liver, spleen, intestinal mucosa and bone marrow. Immunohistochemical study and in-situ hybridization showed that the EBER-positive cells were of T-cell lineage, with CD3 expression. They were also positive for cytotoxic molecules (granzyme B or TIA-1). Only 1 case was CD56 positive. A predominance of CD8-positive cells was demonstrated in 8 of the 14 cases studied, while CD4-positive cells predominated in the remaining 5 cases. One case showed similar proportion of CD8 and CD4-positive cells. The number of EBER-positive cells ranged from 30 to more than 300 per high-power fields. These EBER-positive cells were of small to large size and located mainly in the expanded T zone and occasionally in the germinal centers. Three of the 7 cases exhibited clonal rearrangement of T-cell receptor gamma gene, while the other 4 cases exhibited polyclonal rearrangement of T-cell receptor gamma gene.

CONCLUSIONSASEBV(+)T/NK-LPD is a systemic disease with a subacute or chronic clinical course. Most patients suffer from relapsing fever, lymphadenopathy and hepatosplenomegaly. The disease is characterized by proliferation of EBV-infected cytotoxic T cells. The T zone of the involved lymph nodes shows expansion by mildly atypical lymphoid cells. The disease is associated with poor clinical outcome and can be life-threatening. The patients often die of multiorgan failure and bleeding.

Adult ; Aged ; CD3 Complex ; metabolism ; Epstein-Barr Virus Infections ; pathology ; Female ; Follow-Up Studies ; Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor ; Granzymes ; metabolism ; Herpesvirus 4, Human ; isolation & purification ; Humans ; Killer Cells, Natural ; pathology ; Lymphoproliferative Disorders ; drug therapy ; genetics ; metabolism ; pathology ; virology ; Male ; Middle Aged ; Poly(A)-Binding Proteins ; metabolism ; RNA, Viral ; metabolism ; Retrospective Studies ; Survival Rate ; T-Cell Intracellular Antigen-1 ; T-Lymphocytes ; pathology ; Young Adult