Upregulation of tumor necrosis factor-alpha gene by Epstein-Barr virus and activation of macrophages in Epstein-Barr virus-infected T cells in the pathogenesis of hemophagocytic syndrome.

A potentially fatal hemophagocytic syndrome has been noted in patients with malignant lymphomas, particularly in EBV-infected T cell lymphoma. Cytokines, such as interferon-gamma (IFN-gamma), TNF-alpha, and IL-1alpha, are elevated in patients' sera. To verify whether infection of T cells by EBV will upregulate specific cytokine genes and subsequently activate macrophages leading to hemophagocytic syndrome, we studied the transcripts of TNF-alpha, IFN-gamma, and IL-1alpha in EBV-infected and EBV-negative lymphoma tissues. By reverse transcription PCR analysis, transcripts of TNF-alpha were detected in 8 (57%) of 14 EBV-infected T cell lymphomas, higher than that detected in EBV-negative T cell lymphoma (one of six, 17%), EBV-positive B cell lymphoma (two of five, 40%) and EBV-negative B cell lymphomas (one of seven, 14%). Transcripts of IFN-gamma were consistently detected in T cell lymphoma and occasionally in B cell lymphoma, but were independent of EBV status. IL-1alpha expression was not detectable in any category. Consistent with these in vivo observations, in vitro EBV infection of T cell lymphoma lines caused upregulation of TNF-alpha gene, and increased secretion of TNF-alpha, but not IFN-gamma or IL-1alpha. Expression of TNF-alpha, IFN-gamma, and IL-1alpha was not changed by EBV infection of B cell lymphoma lines. To identify the specific cytokine(s) responsible for macrophage activation, culture supernatants from EBV-infected T cells were cocultured with a monocytic cell line U937 for 24 h. Enhanced phagocytosis and secretion of TNF-alpha, IFN-gamma, and IL-1alpha by U937 cells were observed, and could be inhibited to a large extent by anti-TNF-alpha (70%), less effectively by anti-IFN-gamma (31%), but almost completely by the combination of anti-TNF-alpha and anti-IFN-gamma (85%). Taken together, the in vivo and in vitro observations suggest that infection of T cells by EBV selectively upregulates the TNF-alpha expression which, in combination with IFN-gamma and probably other cytokines, can activate macrophages. This study not only highlights a probable pathogenesis for virus-associated hemophagocytic syndrome, but also suggests that anti-TNF-alpha will have therapeutic potential in the context of their fatal syndrome.

Secondary acute myelogenous leukemia following safe exposure to etoposide.

PURPOSE: To present two patients as illustrations of the risk of developing secondary acute myelogenous leukemia (sAML) when theoretically safe doses of etoposide (VP-16) are used. PATIENTS AND METHODS: Patient no. 1 was a 15-year-old white girl diagnosed with stage IIa Hodgkin's disease. She was treated with a combination of vincristine, doxorubicin, bleomycin, and VP-16 (2 g/m2 total) over 4 months, followed by 25.5 Gy of involved-field radiotherapy. Patient no. 2 was an 11-year-old white boy diagnosed with virus-associated hemophagocytic syndrome (VAHS). He was treated with VP-16 intravenously (IV) and orally (0.3 g and 2.8 g/m2, respectively). RESULTS: Patient no. 1 developed AML 16 months from the diagnosis of Hodgkin's disease. Patient no. 2 developed AML 26 months from diagnosis. Both bone marrows were consistent with French-American-British (FAB) M4 disease. Both patients had abnormalities of the long arm of chromosome 11. CONCLUSION: The use of low-dose or oral VP-16 can be associated with the development of sAML. Clinicians should be cautious in the use of VP-16 in low-risk diseases.

Requirement for etoposide in the treatment of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.

PURPOSE: We sought to identify the clinical variables most critical to successful treatment of Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH). PATIENTS AND METHODS: Among the factors tested were age at diagnosis (< 2 years or > or = 2 years), time from diagnosis to initiation of treatment with or without etoposide-containing regimens, timing of cyclosporin A (CSA) administration during induction therapy, and the presence or absence of etoposide. RESULTS: By Kaplan-Meier analysis, the overall survival rate for the entire cohort of 47 patients, most of whom had moderately severe to severe disease, was 78.3% +/- 6.7% (SE) at 4 years. The probability of long-term survival was significantly higher when etoposide treatment was begun less than 4 weeks from diagnosis (90.2% +/- 6.9% v 56.5% +/- 12.6% for patients receiving this agent later or not at all; P <.01, log-rank test). Multivariate analysis with the Cox proportional hazards model demonstrated the independent prognostic significance of a short interval from EBV-HLH diagnosis to etoposide administration (relative risk of death for patients lacking this feature, 14.1; 95% confidence interval, 1.16 to 166.7; P =.04). None of the competing variables analyzed had significant predictive strength in the Cox model. However, concomitant use of CSA with etoposide in a subset of patients appears to have prevented serious complications from neutropenia during the first year of treatment. CONCLUSION: We conclude that early administration of etoposide, preferably with CSA, is the treatment of choice for patients with EBV-HLH.

Cytomegalovirus-associated hemophagocytic syndrome in a patient with adult onset Still's disease.

Reactive hemophagocytic syndrome (HPS) is characterized by hemophagocytosis by activated histiocytes, resulting in pancytopenia and liver dysfunction. We describe a patient with adult onset Still's disease (AOSD) in whom HPS developed. An 80-year-old Japanese woman with high fever, arthralgia, skin rash, and pleuritis was admitted to our hospital for further examination. She was diagnosed with AOSD and steroid therapy was initiated. During the course of steroid therapy, a re-elevation of serum ferritin levels and a marked increase in serum transaminase were observed. Bone marrow aspiration revealed an increase in the number of histiocytes with hemophagocytosis and cytomegalovirus (CMV)-positive leukocytes were detected. At this time we diagnosed the patient as having virus-associated hemophagocytic syndrome (VAHS) and elevated levels of trasaminase and ferritin were normalized by ganciclovir treatment. Reactive HPS occurs in cases of active AOSD. However, it should be noted that HPS may be accompanied by opportunistic infections during immunosuppressive therapy requiring prompt antibiotic therapy.

Spontaneous resolution of hemophagocytic syndrome and disseminated intravascular coagulation associated with parvovirus b19 infection in a previously healthy child.

A 10-year-old male with a brain abscess developed pancytopenia, liver dysfunction, disseminated intravascular coagulation (DIC) and decrease of immunoglobulin A (IgA) level during postoperative antibiotic and anticonvulsant therapy. A bone marrow examination revealed hemophagocytosis. Real-time PCR revealed parvovirus B19 infection. The hemophagocytic syndrome resolved without specific treatment. To our knowledge, this is the first report of a spontaneous resolution of parvovirus B19-associated hemophagocytic syndrome and DIC.

Virus-associated hemophagocytic syndrome in an international traveler as a differential diagnosis of SARS.

During the epidemic of severe acute respiratory syndrome in 2003, a 27-year- old Japanese woman presented a high fever and acute respiratory distress with pulmonary infiltrates after traveling to a high-risk area. An alternative diagnosis was made as Epstein-Barr virus-associated hemophagocytic syndrome, based on the proliferation of macrophages with hemophagocytosis in the bone marrow and Epstein-Barr viral marker profiles. Virus-associated hemophagocytic syndrome in an international traveler should be included in the differential diagnosis of severe acute respiratory syndrome.

Persistent cervical lymphadenopathy in an adolescent with Epstein-Barr induced hemophagocytic syndrome: manifestations of a rare but often fatal disease.

Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a non-malignant proliferative disorder characterized by histiocytic proliferation and hemophagocytosis following Epstein-Barr virus infection. Though quite rare, this condition represents an often fatal disease primarily affecting the pediatric population. We discuss the case of an adolescent female who presented initially with persistent cervical lymphadenopathy and the typical findings of tonsillar hypertrophy, pharyngitis, and splenomegaly associated with infectious mononucleosis. This case study outlines the pathogenesis, common clinical findings, diagnostic criteria, and a review of the HLH-94 treatment protocol. Early recognition and treatment is emphasized because of the fulminant course of the disorder.

[Successful treatment with liposomal doxorubicin and foscarnet in a patient with widespread Kaposi's sarcoma and human herpes virus 8-related, serious hemophagocytic syndrome, after renal transplantation]. / Efficacia del trattamento con doxorubicina liposomiale e foscarnet in un caso di sarcoma di Kaposi disseminato e grave sindrome emofagocitica herpes virus 8 correlate, nel post-trapianto di rene.

BACKGROUND: It is well known that the human herpes virus 8 (HHV8) is linked to several malignancies such as Kaposi's sarcoma (KS). Moreover, pancytopenia due to hemophagocytic syndrome could be associated with HHV8 infection. In renal transplant recipients affected by KS, the tapering of immunosuppression often leads to KS remission, but also results in graft loss in >50% of cases. Chemotherapy and antiviral therapy have also been used, mainly in the presence of visceral involvement. CASE REPORT: We describe a transplant recipient with widespread cutaneous and visceral KS HHV8 associated, complicated by hemophagocytic syndrome. At transplantation the patient's serology for HHV8 was negative, but thereafter it became positive. The first step in treatment (cyclosporine dose reduction until suspension) failed to improve the clinical course. Therefore, therapy combining liposomal doxorubicin and foscarnet was started. Clearance of HHV8 in the blood and complete resolution of the KS lesions were achieved. Immunosuppression with cyclosporine was resumed. No KS relapse has occurred, blood tests for HHV8 are negative, and graft function is good after a 5-yr follow-up. CONCLUSIONS: Therapy combining liposomal doxorubicin and foscarnet was effective in this renal transplant recipient with KS and HHV8 infection and enabled us to resume immunosuppressive therapy; therefore, reducing the risk of acute/chronic rejection.

Human herpesvirus 8-associated hemophagocytic lymphohistiocytosis in human immunodeficiency virus-infected patients.

We retrospectively reviewed 5 cases of hemophagocytic lymphohistiocytosis (HL) associated with human herpesvirus 8 (HHV-8) reactivation in human immunodeficiency virus (HIV)-infected patients. All patients had clinical and biological features characteristic of HL. Pulmonary symptoms were present in all patients and were frequently life threatening. The mean number of HL episodes was 6. Four patients had HL-associated Kaposi sarcoma, and 3 had multicentric Castleman disease. The mean CD4 cell count was 200 cells/mm(3). HIV loads were stable in all patients. All patients had high levels of HHV-8 in peripheral blood mononuclear cells during attacks, and a significant increase in this parameter before the attacks was seen in 3 patients. Although 2 patients died of HL, 3 are still alive and receiving etoposide therapy (mean follow-up, 3 years). HHV-8-related HL is associated with life-threatening symptoms and biological HHV-8 reactivation, and it may be controlled in the long term by etoposide therapy combined with highly active antiretroviral therapy.

Cell type specific infection of Epstein-Barr virus (EBV) in EBV-associated hemophagocytic lymphohistiocytosis and chronic active EBV infection.

While Epstein-Barr virus (EBV) tropism in B cells and nasopharygeal epithelial cells in the normal host has been demonstrated, recently the role of its infection into non-B cell populations has been suggested to play a pivotal role in the pathogenesis of several EBV-related hematological as well as non-hematological diseases. Ectopic EBV infection in T cells or natural killer (NK) cells has been reported in EBV-associated hematological diseases, such as acute fulminant EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) and chronic active EBV infection (CAEBV). Recent advances in the analysis of EBV infection in lymphocyte subpopulations have clarified the differential virus-cell interaction within these EBV-related disorders. EBV infection was predominantly found in CD8(+) T-cells from EBV-HLH, and in CD4(+) T-cells or NK cells from CAEBV, while the majority of EBV infected cells were found in B cells from acute infectious mononucleosis (IM). Different virus-cell interactions between acute EBV-HLH and CAEBV have indicated different pathogenic mechanisms against EBV infection between the two EBV-associated diseases, accounting for the difference in clinical manifestations between the two diseases.

Fatal primary infection due to human herpesvirus 6 variant A in a renal transplant recipient.

We describe a fatal primary human herpesvirus 6 (HHV-6) variant A infection in a kidney transplanted adult woman. On day 20 post transplantation (TX), after rejection therapy, the patient presented an acute hemophagocytic syndrome with hepatitis and central nervous system involvement. HHV-6 IgG and IgM antibodies seroconversion was demonstrated. HHV-6 variant A was the sole pathogen detected by nested PCR and/or culture in blood, bone marrow aspiration, liver biopsy, cerebrospinal fluid and bronchoalveolar lavage. The graft was HHV-6 seropositive and the patient was not transfused before day 28 post TX, suggesting that the virus was transmitted by the graft. Despite immunoglobulins, ganciclovir and foscarnet therapy, the HHV-6 infection progressed and led to severe aplasia. The patient developed Aspergillus fumigatus pneumonia and died from fulminant candidemia. This case demonstrated for the first time that HHV-6 variant A primary infection can cause life-threatening disseminated infection in immunosuppressed patients.

Fatal hemophagocytic syndrome after living-related liver transplantation: a report of two cases.

BACKGROUND: Hemophagocytic syndrome (HPS) is a serious hematological disorder caused by activated T lymphocytes in immunologically compromised patients. There is no report of HPS in liver transplant recipients. METHODS: Among 135 patients who underwent living-related liver transplantation between June 1990 and October 2000, HPS developed in two pediatric patients (1.5%) on the 15th and 134th postoperative day, respectively. The courses of these patients were evaluated. RESULTS: The cause of HPS was unknown in patient 1 and suspected to be Epstein-Barr virus infection in patient 2. The course of patient 2 was also complicated by posttransplant lymphoproliferative disorder. Both patients had high fever, pancytopenia, coagulopathy, and marked elevation of serum-soluble interleukin 2 receptor, serum ferritin, and urine beta2-microglobulin levels. The diagnosis was established based on clinical findings, laboratory data, and bone marrow biopsy. Both patients died in an acute course despite intensive care. CONCLUSIONS: HPS should be recognized as a severe hematological complication in liver transplant patients. Prompt institution of adequate treatment is necessary to prevent fatality.

Polymerase chain reaction amplification of archival material for Epstein-Barr virus, cytomegalovirus, human herpesvirus 6, and parvovirus B19 in children with bone marrow hemophagocytosis.

Bone marrow hemophagocytosis may occur as an incidental finding, or it may be a manifestation of a systemic and potentially lethal disorder. When systemic, the proliferation is termed hemophagocytic lymphohistiocytosis (HLH), a clinicopathologic entity characterized by a widespread proliferation of benign hemophagocytic histiocytes, fever, pancytopenia, deranged liver function, and frequently coagulopathy and hepatosplenomegaly. A variety of infectious agents, including Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV6), and parvovirus B19 (PVB19), have been associated with HLH, but the relative frequency of each using one technique has not been evaluated. In addition, infectious causes of incidental bone marrow hemophagocytosis, not occurring in the setting of HLH, have not been evaluated. Review of bone marrow reports from bone marrow examinations done between December 1986 and June 1997 showed that 20 children aged 2 months to 15 years had bone marrow examinations that indicated hemophagocytosis. Archival materials from 19 patients were successfully retrieved, and DNA was extracted from archived unstained coverslips with subsequent polymerase chain reaction for EBV, CMV, HHV6, and PVB19 genomic DNA. DNA extracted from 16 bone marrow specimens of age-matched children was used as negative controls. Eleven of the 19 patients fulfilled the clinical and pathological criteria for HLH; the remaining eight patients had isolated hemophagocytosis without a systemic presentation. Viral DNA was detected in 8 of 11 patients with HLH but in none of eight patients with isolated hemophagocytosis. EBV was present in five of the bone marrows, followed in frequency by HHV6, CMV, and PVB19. Infection with more than one agent was present in three patients. Only one control patient was positive for HHV6 DNA; the remaining control patients were negative for all viruses. Viral infection, detected by PCR analysis of bone marrow, is a common finding in patients with HLH but not in patients with isolated bone marrow hemophagocytosis. This technique may provide another marker to aid in the diagnosis of HLH and suggests a different cause of hemophagocytosis occurring in patients with and without HLH.

Hemophagocytic syndrome associated with fulminant hepatitis A: a case report.

A 37-year-old man had a sore throat and pyrexia since January 1999. He was treated at a nearby hospital, but not improved. Jaundice was indicated there, and the patient was referred transferred to our hospital, where he was admitted for treatment with a diagnosis of severe acute hepatitis with acute renal failure. Thereafter the patient was revealed to have had a past history of heavy drinking, and he underwent the treatment with a diagnosis of acute fulminant hepatitis due to hepatitis A virus (HAV). He showed a tendency toward improvement. During the course, however, viral associated hemophagocytic syndrome (VAHS) developed. Various treatments were conducted, but it was not improved, and the patient died on Hospital Day 66. On pathologic autopsy, remarkable hepatosplenomegaly associated with marked bone marrow abnormalities compatible with VAHS was observed. Aspergillus abscesses were also observed in many organs, and they were considered as an adverse reaction to potent immunosuppressive therapy. Since there have been only a few reports on HAV-related VAHS, discussing VAHS related to HAV, the present case was considered valuable.

Virological and immunological characteristics of a 19-year-old Japanese female with fatal outcome with Epstein-Barr virus-associated hemophagocytic syndrome.

Hemophagocytic syndrome (HPS) is caused by the hyperactivation of T-cells and macrophages. The clinical characteristics associated with this disease result from overproduction of cytokines including interferon-gamma (INF-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6). HPS presents with fever, liver dysfunction, coagulation abnormalities, pancytopenia, and a benign histiocytic proliferation with prominent hemophagocytosis in bone marrow, lymph node, spleen, and liver. We describe a 19-year-old female with fatal HPS. She had been given corticosteroid every other day for systemic lupus erythematosus (SLE) without flare up. The causative underlying disease was acute primary Epstein-Barr virus (EBV) infection. EBV genomes were detected by the polymerase chain reaction (PCR). To measure the virus load we use a real-time PCR assay to quantify the amount of EBV DNA in peripheral blood lymphocytes, lung, kidney, brain and liver at autopsy. Further in situ hybridisation (ISH) and immunohistochemical studies demonstrated that Epstein-Barr virus encoded small RNA (EBER) was detected in CD8+ T-cells in bone marrow, lung, kidney, brain, liver and spleen. In each organ, mRNA levels of inflammatory cytokines (INF-gamma, TNF-alpha, IL-6) were highly detected compared with beta-Actin mRNA levels. These results suggest that EBV-infected CD8+ T-cells in each organ (peripheral blood lymphocytes, lung, kidney, brain and liver) may have an integral role in the pathophysiology of the HPS.

Longitudinal follow-up of patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.

BACKGROUND AND OBJECTIVES: Although immunochemotherapy has been reported to be an effective initial treatment for patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH), the long-term outcome of these patients remains unknown. The main purpose of this study was to determine the outcome of the EBV-HLH patients treated between 1992 and 2001. DESIGN AND METHODS: During this period, a total of 78 EBV-HLH patients were consecutively registered in 3 separate studies. The rates of initial response, reactivation, and survival as well as causes of death were analyzed. The outcome of the patients who received hematopoietic stem cell transplantation was also studied. RESULTS: With a median follow-up of 43 months, clinical reactivation was noted in 13 patients (19.4%) and a total of 12 patients needed hematopoietic stem cell transplantation, of whom 9 are alive and well. There had been 19 deaths: early deaths were due to hemorrhages and infections (n=11), while late deaths were related to late reactivation (n=4), transplant-associated causes (n=3) and secondary leukemia (n=1). Overall, after a median follow-up of 43 months, 59 (75.6%) of the 78 patients are alive and well. INTERPRETATION AND CONCLUSIONS: The majority of successfully treated EBV-HLH patients have a good outcome and remain disease-free.

Successful umbilical cord blood transplantation from an unrelated donor for a patient with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.

We report a case of a 5-year-old girl with EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) who underwent cord blood (CB) stem cell transplantation (CBSCT) from an unrelated donor. The patient presented with persistent high-grade fever and hepatosplenomegaly. Because the disease was refractory to immunochemotherapy according to the HLH94 protocol, she received 2.0 x 10(7) CB nucleated cells/kg body weight (BW) after conditioning with BU/CY/etoposide. No acute GVHD developed, using FK506 for prophylaxis. The neutrophil count reached >0.5 x 10(9)/l by day 21 and the platelet count reached >50 x 10(9)/l by day 84. The patient recovered well with sequelae of neurological deficits more than 10 months after receiving CBSCT, without showing evidence of HLH or chronic GVHD. Real-time PCR proved applicable for estimation of the EBV load in PBMC of the patient. We conclude that CBSCT may be indicated for some cases of refractory EBV-HLH, who have no HLA-matched siblings and are therefore dependent on unrelated marrow donors.

Virus-associated hemophagocytic syndrome characterized by clonal Epstein-Barr virus genome.

Virus-associated hemophagocytic syndromes are a heterogeneous group of disorders in which viral infection is associated with a proliferation of hemophagocytic histiocytes throughout the reticuloendothelial system. The authors report the case of a 24-year-old Vietnamese male who developed a hemophagocytic syndrome associated with Epstein-Barr virus (EBV) and who died following a rapidly progressive course. A proliferation of reactive-appearing lymphoid cells was associated with an extensive proliferation of erythrophagocytic histiocytes. Immunophenotypically, the lymphoid infiltrate consisted of CD56+ natural killer cells, predominantly CD8+ T-cells and rare B-cells (CD20+). Double-label immunohistochemical studies showed CD3+ T-cells and CD56+ natural killer cells to be distinct cell populations. Combined immunohistochemical-in situ hybridization studies localized EBV to CD43+, CD3-, CD68-, lymphoid-appearing cells, indicating the presence of EBV within natural killer cells. Southern hybridization analysis of EBV genomic termini revealed clonal EBV genome. However, there was no detectable immunoglobulin or T-cell receptor gene rearrangements. The findings indicate that this case of hemophagocytic syndrome represents a clonal proliferation of natural killer cells containing EBV and highlights the importance of the analysis of EBV genomic termini for determination of clonality in EBV-associated proliferations. It is possible that other cases of fulminant EBV-associated hemophagocytic syndromes represent clonal natural killer cell proliferations.

Treatment of EBV driven lymphoproliferation with erythrophagocytosis: 12 year follow up.

This is a report of a case of Epstein-Barr virus (EBV) associated haemophagocytic syndrome in a 17 year old woman with antibody deficiency. For two years before this presentation, serology showed abnormally high titres to EBV early antigen, suggestive of persistent infection with EBV. She became acutely unwell with clinical features consistent with virus associated haemophagocytic syndrome (VAHS). Histology showed lymphoproliferation with erythrophagocytosis and evidence of EBV encoded RNAs in liver, spleen, and lymph node. VAHS is often fatal, particularly when it occurs in patients with underlying immunodeficiencies. In this case, treatment with intravenous immunoglobulin, aciclovir, and alpha interferon was followed by a dramatic recovery. Twelve years later the patient remains relatively well on regular intravenous immunoglobulin.

Hemophagocytic syndrome in pregnancy.

BACKGROUND: Hemophagocytic syndrome is characterized by nonmalignant histiocytes that undergo uncontrolled phagocytosis of normal hematopoietic cells. Clinical severity ranges from complete recovery to rapid deterioration and death. CASE: Thrombocytopenia was discovered upon routine initial prenatal evaluation of a 24-year-old, gravida 2, para 1, at 29 weeks' gestation with a history of necrotizing lymphadenitis. Cytopenia and elevated transaminases developed, followed by hyperpyrexia. The patient delivered and her postpartum course was complicated by coagulopathy, multiorgan failure, and death. Bone marrow biopsy confirmed hemophagocytic syndrome. CONCLUSION: Early diagnosis of hemophagocytic syndrome during pregnancy might be helped by recognizing symptoms and signs, including a history of necrotizing lymphadenitis, and obtaining a bone marrow biopsy.