Load In Peripheral Blood Mononuclear Cells Research Articles

Clinical value of plasma and peripheral blood mononuclear cells Epstein-Barr Virus DNA dynamics on prognosis of allogeneic stem cell transplantation.

The application of intracellular and extracellular Epstein–Barr virus (EBV) DNA in allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been poorly characterized. We conducted a combined prospective-retrospective study of 300 patients who underwent allo-HSCT between 2016 to 2019 in our center and monitored for EBV DNA within the first year after HSCT. Combining the optimal cut-off value of EBV DNA load (7.3×104 copies/106 cells) in peripheral blood mononuclear cells (PBMCs) and qualitative detection in plasma (400 copies/mL) allowed for the better differentiation of EBV-related posttransplant lymphoproliferative disorders (EBV-PTLD), with increased sensitivity (100%) and specificity (86%), and provided the effective risk stratification of EBV DNA level according to their impact on transplant outcomes. By multivariate analysis, patients with intermediate-level of EBV DNA load (low EBV DNA load in PBMCs or high load in PBMCs but negative in plasma) was associated with superior overall survival (HR 1.92, 95% CI 1.03-3.57, p=0.039) and lower transplant-related mortality (HR 3.35, 95% CI 1.31-8.58, p=0.012) compared to those with high-level (high load in PBMCs and positive in plasma). Notably, high EBV-level group had poor reconstitution of CD4+ and CD8+T cells, and both low and high EBV-level groups showed abnormally increase in IL-10 level within one year. Additionally, patients with peak EBV DNA load in PBMCs during 3-12 months had a higher incidence of chronic graft versus host disease (GVHD) than those within 3 months post transplantation (17.4% vs 13.7%, p=0.029). Collectively, EBV DNA in PBMCs can synergistically predict the risk of EBV-PTLD and GVHD. The intermediate-level of EBV DNA presented in plasma and PBMCs might contribute to a better reconstitution of T cells associated with favorable prognosis of allo-HSCT.

Prevalence of Occult Hepatitis C Virus Infection among Egyptian Haemodialysis Patients

Abstract Background Hepatitis C Virus (HCV) infection is a global health problem. OCI is characterized by presence of HCV-RNA in the liver tissue only and/or Peripheral Blood Mononuclear Cells (PBMCs) with undetectable HCV-RNA or antibodies in the serum. Support for presence of this entity comes from the observation that HCV, even though a hepatotropic virus, can replicate as well at extrahepatic sites, including peripheral blood mononuclear cells. Objectives The current study evaluated the hidden infection of hepatitis C virus among patients on maintenance hemodialysis with negative ELISA and PCR by using PCR in mononuclear cells as a marker in these patients. Our study was done in Sharkia Governorate. Patients and Methods In our study, the 60 included subjects had an average age of 48.27 years with range from 19 to 77 years and included 33 males (55%) and 27 females (45%). The subjects had average dialysis duration of 6.51 ± 4.10years with range from 7 months to 19 years. Results Our study showed no statistically significant differences between occult HCV cases and negative cases in frequency of blood transfusion. There were no statistically significant differences between occult HCV cases and negative cases in splenomegaly, hepatomegaly or coarse liver but there was a statistically significant increase in frequency of severe ascites, moderate LL edema and jaundice among occult HCV cases compared to negative cases. There were no statistically significant differences between occult HCV cases and negative cases in urea, creatinine, ALT, AST or albumin. In our study total bilirubin>1.7 and direct bilirubin >0.4 were significant predictors for the presence of occult HCV in hemodialysis patients. Our study showed that there were no statistically significant correlation between viral load in PBMCs and laboratory findings among occult HCV cases. Conclusion Hemodialysis patients are liable for occult HCV infections with a considerable impact on their health. Total and direct bilirubin are predictors of presence of occult hepatitis C infection at cut-off values of 1.7 mg/dl and 0.4 mg/dl respectively.

The application of quantitative analysis of EBV DNA loads in plasma and peripheral blood mononuclear cells in Epstein-Barr Virus infection-related diseases in children

Objective: To explore the correlation of EBV DNA load in two different types of plasma and peripheral blood mononuclear cells (PBMCs) in children with Epstein-Barr Virus (EBV) infection diseases. Methods: A retrospective evaluated was performed on EBV DNA quantification in plasma and PBMCs by qPCR between April, 2019 and December, 2020. The samples were collected from children of 456 cases with EBV infection and 2 306 healthy cases. In EBV infection group, boys were 253 and girls were 203, aged from 8 days to months to 16 years. In healthy group, boys were 1 267 and girls were 1 039, aged from 8 days to 16 years. Results: Infectious mononucleosis (IM) was the most common disease associated with EBV infection 73.68%(336/456). The detection rate of plasma and PBMCs in EBV infection group was 91.89% (419/456)and 99.34% (453/456)respectively, and was 100%(456/456) in plasma or PBMCs. The detection rate of plasma and PBMCs in healthy group was 1.13%(26/2 306) and 30.01%(715/2 306), respectively. Levels of EBV DNA in plasma and PBMCs in EBV infection group [IM, acute infections, pneumonia, post-transplantation lymphoproliferative disorder (PTLD), hemophagocytic lymphohistiocytosis, tonsillitis and lymphadenitis] was significantly higher than those in healthy group (In plasma, Z=-47.18,-34.41,-33.40,-31.71,-24.38,-20.86 and -20.59,respectively; In PBMCs, Z=-33.17,-16.45,-11.33,-9.45,-5.57,-5.16 and -5.45, respectively; P<0.05). In IM group, EBV DNA load in plasma and PBMCs in remission stage was significantly lower than those in infection stage (Z=-11.45, -8.53;P<0.05). In PTLD group, there was significant difference in EBV DNA load in plasma between infection and remission stage (Z=-4.13, P<0.05), while there was no significant difference in EBV DNA load in PBMCs (Z=-0.817, P>0.05). Conclusions: EBV infection was mainly caused by IM. Combined detection of plasma and PBMCs in EBV DNA is valuable for improving diagnosis ability of EBV infection-related diseases, and the load of EBV DNA could be used as a marker.

Relationship between biochemical markers and measles viral load in patients with immunologically naive cases and secondary vaccine failure: LDH is one of the potential auxiliary indicators for secondary vaccine failure.

This study investigated the correlation between biochemical markers and viral load among 38 measles cases, including 15 immunologically naive patients and 23 patients with secondary vaccine failure (SVF). We examined four biochemical markers, namely, aspartate aminotransferase, alanine aminotransferase, C-reactive protein, and lactate dehydrogenase (LDH) and their relationship between virus genome copy numbers in peripheral blood mononuclear cells (PBMCs) and throat swabs as well as the concentration of measles-specific IgG. Although viral genome copies in both clinical specimens showed a significant correlation with specific IgG concentration, they had a higher correlation in PBMCs (Pearson's product-moment correlation coefficient, -0.662; p < .0001) than in throat swabs (Spearman's rank correlation coefficient, -0.443; p = .0078). The viral load in PBMCs also significantly correlated with LDH values (correlation coefficient, 0.360; p = .036). Thus, the serum LDH level might be a potential auxiliary indicator to distinguish immunologically naive patients with measles from those with SVF.

Koala retrovirus (KoRV) subtypes and their impact on captive koala (Phascolarctos cinereus) health.

Koala retrovirus (KoRV), a major pathogen of koalas, exists in both endogenous (KoRV-A) and exogenous forms (KoRV-B to J). However, the impact of infection with multiple subtypes is not well understood. Accordingly, in this study, we surveyed a representative sample from a Japanese zoo population to determine the infection status for three KoRV subtypes (KoRV-A, B, and C) and to investigate the proviral and RNA load profiles in animals with single- and multiple-subtype infections, using peripheral blood mononuclear cells (PBMCs) and plasma. Six koalas were evaluated in the study; all were infected with KoRV-A, and two koalas were coinfected with non-A subtypes (KoRV-B and/or KoRV-C). The highest KoRV total RNA and viral loads in PBMCs and plasma were found in a koala infected with multiple subtypes (KoRV-A, -B and -C). The other koala infected with multiple subtypes (KoRV-A and B) showed the highest proviral PBMC load but the lowest RNA copy number in PBMC and plasma. PBMCs from this animal were cultured for further investigation, and KoRV RNA was detected in the cells and culture supernatant after 7 and/or 14 days. The koalas harboring multiple subtypes had a higher white blood cell count than those harboring only KoRV-A and were judged to be leukemic, and they subsequently died due to lymphoma. Accordingly, we conclude that coinfection with multiple KoRV subtypes may be linked to more-severe disease. In a sequence alignment, the detected KoRV-A env gene showed 100% sequence identity to the reference gene, whereas the KoRV-B and -C env genes varied from their reference sequences.

Illuminating the in vitro effects of Epstein-Barr virus and vitamin D on immune response in multiple sclerosis patients.

Given the complexity of immune complex diseases including multiple sclerosis (MS) and the plausible interactions between different risk factors, delineating the interplay between them would be imperative. The current study aimed to evaluate the in vitro effects of Epstein-Barr virus (EBV) and vitamin D on immune response in MS patients and healthy controls. The status of vitamin D and EBV load was evaluated using multiple techniques. In vitro EBV-infected peripheral blood mononuclear cells (PBMCs), in the presence or absence of vitamin D, were checked for IL-10, IFN-γ, and vitamin D receptor. MS patients showed significantly higher plasma levels of 1,25-(OH)2D but not 25-OHD, increased EBV load, and lower levels of vitamin D receptor (VDR) expression compared with healthy controls. Interestingly, an inverse correlation was observed between VDR expression and EBV load in PBMCs. Indeed, the levels of IFN-γ and IL-10 production were significantly higher in supernatant collected from in vitro EBV-infected PBMCs in MS patients compared with controls. While all vitamin D-treated PBMCs showed reduced levels of IFN-γ production, in vitro treatment of vitamin D showed no influence in IL-10 production. EBV and vitamin D were found to exert opposite in vitro effects on immune dysregulation in these patients. Our results highlight the complex interactions of different risk factors with immune system.

INVESTIGATION OF EFFECTS OF PORCINE CIRCOVIRUS TYPE 2 (PCV2) VACCINATION ON CELLULAR IMMUNITY IN PIGS IN A TAIWAN PIG FARM

Porcine circovirus type 2 (PCV2) is the major cause of serious porcine circovirus-associated diseases (PCVADs) causing significant economic losses in the pig industry. The PCV2 vaccines have proved effective by lowering serum and tissue viral loads. However, little is known about its effects on the cellular functions of viral target cells in pigs. For this, blood samples from 38 vaccinated and 34 non-vaccinated, 12–14 week-old, clinically healthy and PCV2-infected pigs in a commercial farm were sampled and anticoagulated. The viral load in plasma and the cellular proliferation and mRNA expression levels of IL-1 [Formula: see text], IL-2, IL-4, IL-8, IL-10 and IFN-[Formula: see text] of peripheral blood mononuclear cells (PBMCs) with or without concanavalin A (Con A) and PCV2 stimulation in vitro were then investigated by real-time polymerase chain reaction and flow cytometry. Compared with the non-vaccinated pigs, the average viral loads in the plasma and cultivated PBMCs were reduced in the PCV2-vaccinated pigs. Significant differences in the aspects of increased cell proliferation, decreased average viral loads in PBMCs three days after Con A stimulation, increased mRNA expression levels of IL-2 and IFN-[Formula: see text] after re-exposure to PCV2, and decreased levels of IL-1 [Formula: see text], IL-4 and IL-8 after Con A or PCV2 stimulation were also noted in the PCV2-vaccinated pigs. The results suggested that the PCV2-vaccinated pigs were likely to develop better cell-mediated immunity than the non-vaccinated pigs.

Clinical application of Epstein-Barr virus DNA loads in Epstein-Barr virus-associated diseases: A cohort study

ObjectivesThis study evaluated the diagnostic value of Epstein-Barr virus (EBV) DNA load in blood samples of patients with EBV-associated diseases, and proposed a strategy for the interpretation of positive EBV DNA results. MethodsDerivation and validation cohorts were established to evaluate the clinical significance of EBV DNA loads in the peripheral blood mononuclear cells (PBMCs) and plasma from EBV-infected patients. EBV DNA loads were compared and receiver operating characteristic curves were employed to assess the optimal cutoff values of EBV DNA for identification of EBV-associated diseases. ResultsThe derivation and validation cohorts comprised 135 and 71 subjects, respectively. EBV DNA loads in the PBMCs of the EBV-associated diseases group was significantly higher than that of the EBV non-associated diseases group (5.8 × 104 vs 7.8 × 103 copies/106 cells, P<0.0001). The diagnostic cut-off value of viral load in PBMCs for EBV-associated diseases was determined to be 1.6 × 104 copies/106 cells. The combined EBV DNA load cutoff in PBMCs and positive EBV DNA qualitative detection in plasma (>500 copies/mL) allowed for the differentiation of EBV-associated and non-associated diseases; the sensitivity and specificity were 80.6 and 96.8%, respectively. ConclusionsThe strategy of combining EBV DNA loads in PBMCs and plasma will potentially help identify EBV-associated diseases.

Dissecting Multicellular Ecosystems of HTLV-1 Infection and ATL By Multi-Omics Single Cell Analysis

Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell malignancy, caused by human T-cell leukemia virus type-1 (HTLV-1) infection. To elucidate immune microenvironment and heterogeneity of HTLV-1-infected normal and leukemic cells, we performed multi-omics single cell analysis, evaluating whole-transcriptome, 101 surface marker proteins, and T/B-cell receptor repertoires in the same single cells. We analyzed 236,192 peripheral blood mononuclear cells (PBMCs) from 31 ATL patients (35 samples including 4 sequential samples), 11 HTLV-1-infected carriers, and 4 healthy donors. In our analysis, expression of HTLV-1-related genes, such as HBZ, clearly identified a distinct cluster of HTLV-1-infected cells within non-malignant CD4+ T cells. These cells are characterized by a CD45RO+CD62L-CD7-CCR4+CD25+CD73+ memory/effector phenotype. By contrast, malignant ATL cells were segregated into different clusters across patients, suggestive of inter-tumor heterogeneity. Transcriptome analysis of CD4+ T cells revealed up-regulation of interferon (IFN) responses and down-regulation of TNFa signaling in malignant ATL cells compared with HTLV-1-infected normal CD4+ T cells. Likewise, sequential sample analysis showed that progression from indolent to aggressive disease enhanced IFN responses, suggesting a pivotal role of this pathway in the ATL pathogenesis. Surface marker protein analysis demonstrated that HTLV-1 infection up-regulated the expression of stimulatory and inhibitory immune checkpoint molecules (such as OX40 and TIGIT, respectively), which was further augmented by ATL progression. Within malignant cells, we identified a fraction of cycling cells present across most ATL samples. This fraction showed an enhanced T-cell activation markers, such as CD25 and HLA-DR, and their frequency was increased in aggressive subtypes. On the other hand, in HTLV-1-infected carriers, HTLV-1-infected CD4+ T cells contained a small population of malignant-like cells showing clonal expansion. The degree of clonal expansion was significantly correlated with HTLV-1 viral load in PBMCs. These results clarify the heterogeneity within HTLV-1-infected cells and ATL malignant cells, pointing to its relevance during ATL initiation and progression. We also observed dynamic changes of the immune microenvironment in ATL. Although the relative frequencies of other cell types remained almost the same or reduced, only myeloid cells were increased in ATL patients compared with in HTLV-1-infected carriers. Re-clustering of myeloid cells identified a novel cluster of monocytes expressing FCGR1A, encoding CD64, a biomarker of IFN-stimulated gene levels. Transcriptome analysis revealed increased IFN signaling and decreased TNFa in myeloid cells from ATL patients compared with HTLV-1-infected carriers. Similar expression signatures changes were also observed in various immune cell types, such as B, CD8+ T, and NK cells, in ATL patients. In addition, substantial changes of surface marker proteins were also found in ATL patients. Particularly, T-cell activation markers, such as HLA-DR, and inhibitory immune checkpoint molecules, such as PD-1 and TIM-3, were up-regulated in CD8+ T cells from ATL patients. A co-culture experiment of ATL cell lines with PBMCs from healthy volunteers demonstrated that ATL cells induced immune-phenotypic changes of myeloid and CD8+ T cells, similar to those observed in ATL patient by our single-cell analysis, confirming the role of ATL cells in the modulation of the immune system. Taken together, the composition and function of immune microenvironment is dramatically altered in ATL patients, which may contribute to immunosuppression and disease progression in ATL. In summary, our multi-omics single-cell analysis comprehensively dissects the cellular and molecular architecture in HTLV-1-infected carriers and ATL patients. In particular, our approach clearly defines HTLV-1-infected cells by the expression of HTLV-1-related genes, leading to the detailed characterization of HTLV-1-infected cells and elucidation of their difference from ATL malignant cells. These findings will help to devise novel diagnostic and therapeutic strategies for HTLV-1-related disorders. Disclosures Kogure: Takeda Pharmaceutical Company Limited.: Honoraria. Shimoda:Japanese Society of Hematology: Research Funding; The Shinnihon Foundation of Advanced Medical Treatment Research: Research Funding; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company: Honoraria; Novartis: Honoraria, Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Kasei Medical: Research Funding; Shire plc: Honoraria; Celgene: Honoraria; Perseus Proteomics: Research Funding; PharmaEssentia Japan: Research Funding; AbbVie Inc.: Research Funding; Astellas Pharma: Research Funding; Merck &amp; Co.: Research Funding. Kataoka:CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Takeda Pharmaceutical Company: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Genomics: Current equity holder in private company.

The aim of the measurement of Epstein-Barr virus DNA in hydroa vacciniforme and hypersensitivity to mosquito bites.

Epstein-Barr virus (EBV) DNA load in the blood increases in posttransplant lymphoproliferative disorders and chronic active EBV infection. In this report, we analyzed the EBV DNA load in the peripheral blood mononuclear cells (PBMCs) and plasma of patients with hydroa vacciniforme (HV) and/or hypersensitivity to mosquito bites (HMB) to understand the clinical significance of EBV DNA load. All 30 patients showed high DNA loads in the PBMCs over the cut-off level. Of 16 plasma samples, extremely high in two samples obtained from patients with hemophagocytic lymphohistiocytosis (HLH). The amount of cell-free DNA in plasma was correlated to the serum levels of lactate dehydrogenase and inversely correlated to platelet counts. These results indicate that the EBV DNA load in PBMCs can provide one of the diagnostic indicators for HV and HMB and marked elevation of cell-free EBV DNA in plasma might be related to cytolysis such as that observed in HLH.

The rational specimen for the quantitative detection of Epstein-Barr virus DNA load.

Background Epstein-Barr virus (EBV) DNA load monitoring in blood is essential for the diagnosis of EBV-associated diseases. However, the best-suited blood compartment for detection is still under discussion. The aim of this study was to evaluate the diagnostic value of EBV-DNA load in peripheral blood mononuclear cells (PBMC), plasma and whole blood (WB) samples. Methods A total of 156 patients, including 45 patients with infectious mononucleosis (IM), 57 patients with EBV-associated hemophagocytic lymphohistiocytosis (HLH) and 54 patients with post-transplant lymphoproliferative disorders (PTLD), were enrolled in this study. The EBV-DNA load in PBMC, plasma and WB samples were measured with real-time quantitative polymerase chain reaction (PCR). Results EBV-DNA load of patients with HLH showed no statistical difference in PBMC, plasma and WB samples, while patients with IM and PTLD showed a higher viral load in PBMC samples. The strongest correlation of EBV-DNA level was found between PBMC and WB samples among patients with IM, HLH and PTLD. The follow-up of EBV-DNA showed that the viral load became negative along with the recovery from the disease, while that in WB and PBMC would remain positive for a long time. Conclusions For the diagnosis and monitoring of EBV-DNA, the type of specimen should be chosen reasonably according to the disease. As for IM and HLH, plasma is recommended to quantify the EBV-DNA load, while PBMC and plasma are preferred in PTLD.

Mogamulizumab (Anti-CCR4) in HTLV-1–Associated Myelopathy

BackgroundHuman T-lymphotropic virus type 1 (HTLV-1) causes the debilitating neuroinflammatory disease HTLV-1–associated myelopathy–tropical spastic paraparesis (HAM–TSP) as well as adult T-cell leukemia–lymphoma (ATLL). In patients with HAM–TSP, HTLV-1 infects mainly CCR4+ T cells and induces functional changes, ultimately causing chronic spinal cord inflammation. We evaluated mogamulizumab, a humanized anti-CCR4 monoclonal antibody that targets infected cells, in patients with HAM–TSP.MethodsIn this uncontrolled, phase 1–2a study, we assessed the safety, pharmacokinetics, and efficacy of mogamulizumab in patients with glucocorticoid-refractory HAM–TSP. In the phase 1 dose-escalation study, 21 patients received a single infusion of mogamulizumab (at doses of 0.003 mg per kilogram of body weight, 0.01 mg per kilogram, 0.03 mg per kilogram, 0.1 mg per kilogram, or 0.3 mg per kilogram) and were observed for 85 days. Of those patients, 19 continued on to the phase 2a study and received infusions, over a period of 24 weeks, of 0.003 mg per kilogram, 0.01 mg per kilogram, or 0.03 mg per kilogram at 8-week intervals or infusions of 0.1 mg per kilogram or 0.3 mg per kilogram at 12-week intervals.ResultsThe side effects of mogamulizumab did not limit administration up to the maximum dose (0.3 mg per kilogram). The most frequent side effects were grade 1 or 2 rash (in 48% of the patients) and lymphopenia and leukopenia (each in 33%). The dose-dependent reduction in the proviral load in peripheral-blood mononuclear cells (decrease by day 15 of 64.9%; 95% confidence interval [CI], 51.7 to 78.1) and inflammatory markers in cerebrospinal fluid (decrease by day 29 of 37.3% [95% CI, 24.8 to 49.8] in the CXCL10 level and of 21.0% [95% CI, 10.7 to 31.4] in the neopterin level) was maintained with additional infusions throughout the phase 2a study. A reduction in spasticity was noted in 79% of the patients and a decrease in motor disability in 32%.ConclusionsMogamulizumab decreased the number of HTLV-1–infected cells and the levels of inflammatory markers. Rash was the chief side effect. The effect of mogamulizumab on clinical HAM–TSP needs to be clarified in future studies. (Funded by the Japan Agency for Medical Research and Development and the Ministry of Health, Labor, and Welfare; UMIN trial number, UMIN000012655.)

Diagnostic values for the viral load in peripheral blood mononuclear cells of patients with chronic active Epstein-Barr virus disease.

Chronic active Epstein-Barr virus disease (CAEBV) is a distinct EBV-associated lymphoproliferative disease with a poor prognosis. Although the viral load in blood samples has been widely used for diagnosing CAEBV, well-defined viral load thresholds to guide clinicians are currently lacking. The aim of the present study was to determine standardized diagnostic values for EBV load in blood samples of CAEBV patients using the World Health Organization international standard for reporting. Levels of EBV DNA in 103 peripheral blood mononuclear cells (PBMCs) and 95 plasma/serum samples from 107 cases with CAEBV were quantified and expressed in international units. Receiver operating characteristic curves were analyzed to assess the most appropriate cut-off values for levels of EBV DNA to distinguish CAEBV from EBV-associated infectious mononucleosis (IM) and controls with past EBV infection. Levels of EBV DNA in PBMCs were significantly higher in the CAEBV group (median, 10(4.2)IU/μgDNA) compared to the IM (median, 10(2.1)IU/μgDNA) and control groups. An inconsistent qualitative result was seen in 13 of 86 CAEBV patients; in these, EBV-DNA was positive in PBMCs, but negative in plasma. Diagnostic cut-off values for viral load in PBMCs from CAEBV patients, as compared to those of healthy controls and IM patients, were 10(2.0)IU/μgDNA and 10(3.2)IU/μgDNA, respectively. For diagnostic purposes, the viral load of PBMCs was better than of plasma/serum. A diagnostic cut-off EBV load for CAEBV may be useful for the management of CAEBV patients.

High Viral Loads of Epstein-Barr Virus DNA in Peripheral Blood of Patients with Chronic Lymphocytic Leukemia Associated with Unfavorable Prognosis.

Epstein-Barr virus (EBV) is a ubiquitous γ-herpesvirus that infects more than 90% of the world population. The potential involvement of EBV in the clinical course of chronic lymphocytic leukemia (CLL) remains unexplained. The aim of this study was to determine whether EBV-DNA load in the peripheral blood mononuclear cells (PBMCs) of CLL patients may influence heterogeneity in the course of the disease. The study included peripheral blood samples from 115 previously untreated patients with CLL (54 women and 61 men) and 40 healthy controls (16 women and 24 men). We analyzed the association between the EBV-DNA load in PBMCs and the stage of the disease, adverse prognostic factors, and clinical outcome. Detectable numbers of EBV-DNA copies in PBMCs were found in 62 out of 115 CLL patients (53.91%). The EBV-DNA copy number/μg DNA was significantly higher in patients who required early implementation of treatment, presented with lymphocyte count doubling time <12 months, displayed CD38-positive or ZAP-70-positive phenotype, and with the del(11q22.3) cytogenetic abnormality. Furthermore, the EBV-DNA copy number/μg DNA showed significant positive correlation with the concentrations of lactate dehydrogenase (LDH) and beta-2-microglobulin. We have shown that in CLL patients, higher EBV-DNA copy number predicted shorter survival and shorter time to disease progression, and it was associated with other established unfavorable prognostic factors. This suggests that EBV may negatively affect the outcome of CLL.

Human gammaherpesviruses viraemia in HIV infected patients

BackgroundThe Epstein–Barr virus (EBV) and Kaposi's sarcoma associated herpesvirus (KSHV) are consistently associated with lymphoproliferative diseases and cancers in humans, notably in patients with HIV.AimsOur aim was to evaluate whether...

Healthy rabbits are susceptible to Epstein-Barr virus infection and infected cells proliferate in immunosuppressed animals.

BackgroundEpstein-Barr virus (EBV) is an oncogenic virus implicated in the pathogenesis of several human malignancies. However, due to the lack of a suitable animal model, a number of fundamental questions pertaining to the biology of EBV remain poorly understood. Here, we explore the potential of rabbits as a model for EBV infection and investigate the impact of immunosuppression on viral proliferation and gene expression.MethodsSix healthy New Zealand white rabbits were inoculated intravenously with EBV and blood samples collected prior to infection and for 7 weeks post-infection. Three weeks after the last blood collection, animals were immunosuppressed with daily intramuscular injections of cyclosporin A at doses of 20 mg/kg for 15 days and blood collected twice a week from each rabbit. The animals were subsequently sacrificed and tissues from all major organs were collected for subsequent analysis.ResultsFollowing intravenous inoculation, all 6 rabbits seroconverted with raised IgG and IgM titres to EBV, but viral DNA in peripheral blood mononuclear cells (PBMCs) could only be detected intermittently. Following immunosuppression however, EBV DNA could be readily detected in PBMCs from all 4 rabbits that survived the treatment. Quantitative PCR indicated an increase in EBV viral load in PBMCs as the duration of immunosuppression increased. At autopsy, splenomegaly was seen in 3/4 rabbits, but spleens from all 4 rabbit were EBV PCR positive. EBER-in situ hybridization and immunoshistochemistry revealed the presence of a large number of EBER-positive and LMP-1 positive lymphoblasts in the spleens of 3/4 rabbits. To a lesser extent, EBER-positive cells were also seen in the portal tract regions of the liver of these rabbits. Western blotting indicated that EBNA-1 and EBNA-2 were also expressed in the liver and spleen of infected animals.ConclusionEBV can infect healthy rabbits and the infected cells proliferate when the animals are immunocompromised. The infected cells expressed several EBV-latent gene products which are probably driving the proliferation, reminiscent of what is seen in immunocompromised individuals. Further work is required to explore the potential of rabbits as an animal model for studying EBV biology and tumorigenesis.

EBV- DNA Viral Load in Peripheral Blood Mononuclear Cells of Patients with Chronic Lymphocytic Leukemia Is Associated with Unfavorable Prognosis

Background: Epstein-Barr virus (EBV) is a ubiquitous γ-herpesvirus that infects more than 90% of the world population. Following primary infection in the oropharynx, EBV remains latently present in B cells. Asymptomatic EBV reactivations periodically occur in oral mucosa-associated lymphoid tissues in healthy carriers. B cells infected by EBV are suspected to be involved in the etiology of various human lymphoma. Previously, EBV reactivation has been diagnosed by serologic IgG, IgM, IgA profiles that suggested virus replication. However, serologic responses, in immunocompromised subjects, like patients with chronic lymphocytic leukemia (CLL), do not necessarily indicate ongoing replicative activity. CLL is the most frequent form of leukemia in adults in western countries. Despite by definition chronic character, CLL is characterized by marked heterogeneity. Potential involvement of EBV in the pathogenesis of CLL is still to be explain. Latent EBV infection in healthy carriers is controlled by a cell-mediated immune response that is impaired in CLL patients and might result in poor control of EBV infection. Since EBV may activate B cells, stimulate their proliferation and inhibit their apoptosis, we hypothesize that it could contribute to unfavorable clinical course of CLL and may be one of reason of its heterogeneity.Objective: The aim of this study was to determine whether EBV-DNA load in the peripheral blood mononuclear cells (PBMCs) of CLL patients may influence heterogeneity in clinical course of the disease. We analyzed the association between EBV-DNA load in PBMC and stage of the disease, adverse prognostic factors (including CD38 and ZAP-70 expression and cytogenetic abnormalities) and clinical outcome.Material and methods: Peripheral blood samples were obtained from 115 untreated CLL patients and 20 healthy individuals. Real-time PCR was used to quantitate EBV-DNA copy number in PBMCs (viral load). As the sensitivity of the system amounts to 10 copies per µl, all the samples with the EBV-DNA copy number below this detection threshold were considered EBV-negative. The immunophenotype of peripheral blood lymphocytes was analyzed by flow cytometer.Results: Detectable EBV-DNA load in PBMCs was found in 62 out of 115 CLL patients (53.91%). The median EBV-DNA copy number in these group was 184 per 1 µg DNA (range: 12.79-8957.69). The EBV-DNA copy number per 1 µg DNA was significantly higher in patients who required the treatment (p<0.0001) than in untreated ones, in patients with lymphocyte count doubling time <12 months than in than patients with lymphocyte count doubling time >12 months (p<0.0001), in patients with hepatomegaly (p=0.023) and, splenomegaly (p=0.005) than in patients without organomegaly. It was also significantly higher in patients with CD38 (p<0.0001) and ZAP-70 expression (p=0.008) comparing to the patients CD38- and ZAP-70-, respectively, as well as in the patients with del (11q22.3) as comparing to the patients without this cytogenetic abnormality. Furthermore, the EBV-DNA copy number per 1 µg DNA showed significant positive correlation with the concentrations of LDH (r=0.531, p=0.00001) and beta-2-microglobulin (r=0.425, p=0.00057). The presence of more than 12.79 copies of EBV DNA in 1 µg DNA isolated from PBMCs was associated with reduced time to first treatment (p=0.00001), and lymphocyte count doubling-free survival (p<0.000001).Conclusions: We proved for the first time, that higher of EBV-DNA copy number in PBMCs of patients with CLL is associated with adverse prognostic factors as well as more progressive clinical course of the disease expressed by reduced time to first treatment. These results suggest that higher of EBV-DNA copy number in peripheral blood mononuclear cells blood predicts poor clinical outcome of CLL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Duplex realtime PCR method for Epstein–Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detection

IntroductionThe quantification of circulating Epstein–Barr virus (EBV) DNA is used to monitor transplant patients as an early marker of Post-Transplant Lymphoproliferative Disorders (PTLD). So far no standardized methodology exists for such determination. ObjectiveOur purpose was to develop and validate a real-time PCR assay to quantify EBV DNA in clinical samples from transplant recipients. MethodsA duplex real-time PCR method was developed to amplify DNA from EBV and from a human gene. The EBV load was determined in peripheral blood mononuclear cells (PBMC), plasma and oropharyngeal tissue from 64 non-transplanted patients with lymphoid-hypertrophy (Non-Tx), 47 transplant recipients without PTLD (Tx), 54 recipients with PTLD (Tx-PTLD), and 66 blood donors (BD). WinPEPI, version 11.14 software was used for statistical analysis. ResultsAnalytical validation: the intra and inter-assays variation coefficients were less than 4.5% (EBV-reaction) and 3% (glyceraldehyde 3-phosphate dehydrogenase – GAPDH reaction). Linear ranges comprised 107–10 EBV genome equivalents (gEq) (EBV-reaction) and 500,000–32 human gEq (GAPDH-reaction). The detection limit was 2.9 EBV gEq (EBV-reaction). Both reactions showed specificity. Application to clinical samples: higher levels of EBV were found in oropharyngeal tissue from transplanted groups with and without PTLD, compared to Non-Tx (p<0.05). The EBV load in PBMC from the groups of BD, Non-Tx, Tx and Tx-PTLD exhibited increasing levels (p<0.05). In BD, PBMC and plasma, EBV loads were undetectable. ConclusionsThe performance of the assay was suitable for the required clinical application. The assay may be useful to monitor EBV infection in transplant patients, in particular in laboratories from low-income regions that cannot afford to use commercial assays.

Impact of Epstein–Barr virus load, virus genotype, and frequency of the 30 bp deletion in the viral BNLF‐1 gene in patients harboring the human immunodeficiency virus

Patients infected with the human immunodeficiency virus (HIV) are at higher risk of developing Epstein-Barr Virus (EBV)-associated lymphomas. The usefulness of monitoring EBV in peripheral blood mononuclear cells (PBMCs) of patients infected with HIV has not been established. The aim of this study was to evaluate the EBV viral load in PBMCs, the frequency of viral genotypes, and the presence of the 30-bp deletion in the BNLF-1 gene. DNA samples from 156 patients attending the HIV/AIDS Day Clinic at Botucatu School of Medicine, Sao Paulo State University were evaluated. The EBV viral load was detectable by real time PCR in 123/156 (78.8%) cases and was higher in patients not receiving antiretroviral treatment or under therapeutic failure than in patients under successful highly active antiretroviral therapy (HAART) (P = 0.0076). Overall, the profile of patients with high EBV viral load included elevated HIV viremia (P = 0.0005), longer time of HIV diagnosis (P = 0.0026), and increased levels of T CD8 (+) lymphocytes (P = 0.0159). The successful amplification of the EBNA-2 gene by nested-PCR was achieved in 95 of 123 (77.2%) cases, of which 75.8% were EBV-1, 9.5% EBV-2, and 14.7% were co-infected with both EBV-1 and -2. The analysis of the BNLF-1 gene was possible in 99 of 123 (80.5%) cases, of which 50.5% had the 30-bp deletion. EBV-1 was more common than EBV-2, which may reflect the fact that the cohort was predominantly Caucasian and heterosexual.

Selective reactivation of human herpesvirus 6 in patients with autoimmune connective tissue diseases

Viral infections have been associated with autoimmune connective tissue diseases. To evaluate whether active infection by Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus (HHV)-6, -7, -8, as well as parvovirus B19 (B19V) occur in patients with autoimmune connective tissue diseases, viral DNA loads were assessed in paired samples of serum and peripheral blood mononuclear cells (PBMCs) of 115 patients affected by different disorders, including systemic sclerosis, systemic, and discoid lupus erythematosus, rheumatoid arthritis, and dermatomyositis. Two additional groups, patients affected by inflammatory diseases (n=51) and healthy subjects (n=58) were studied as controls. The titers of anti-HHV-6 and anti-EBV antibodies were also evaluated. Cell-free HHV-6 serum viremia was detected in a significantly higher proportion of connective tissue diseases patients compared to controls (P<0.0002); a significant association between HHV-6 reactivation and the active disease state was found only for lupus erythematosus (P=0.021). By contrast, the rate of cell-free EBV viremia was similar in patients and controls groups. Cell-free CMV, HHV-8, and B19V viremia was not detected in any subject. Anti-HHV-6 and anti-EBV early antigen IgG titers were both significantly higher in autoimmune diseases patients as compared to healthy controls, although they were not associated with the presence of viremia. EBV, HHV-6, -7 prevalence and viral load in PBMCs of patients with connective tissue diseases and controls were similar. These data suggest that HHV-6 may act as a pathogenic factor predisposing patients to the development of autoimmune connective tissue diseases or, conversely, that these disorders may predispose patients to HHV-6 reactivation.