[The Role of Notch Signaling Pathway in Adult Patients with Epstein-Barr Virus-induced Infectious Mononucleosis].

OBJECTIVE: To investigate the changes of Notch signaling molecules and Th22 cells in adult patients with infectious mononucleosis (IM), and assess the regulatory function of Notch signaling inhibition to Th22 cells. METHODS: Forty-two IM patients and twenty-one healthy controls were enrolled in this study. Their peripheral blood was collected, from which plasma and peripheral blood mononuclear cells (PBMCs) were isolated. Plasma interleukin (IL)-17 and IL-22 were measured by enzyme-linked immunosorbent assay. The percentages of CD3+ CD4+ IL-17+ Th17 cells and CD3+ CD4+ IL-22+ Th22 cells were investigated by flow cytometry. The mRNA relative levels corresponding to Th17 transcription factor retinoic acid related orphan receptor γt (RORγt), Th22 transcription factor aryl hydrocarbon receptor (AhR), and Notch signaling pathway molecules (including Notch receptors, Notch ligands, Notch downstream molecules) were semi-quantified by real-time PCR. CD4+ T cells were purified and stimulated with γ-secretase inhibitor (GSI). Cellular proliferation, Th17 and Th22 percentage, IL-17 and IL-22 secretion, transcription factor mRNA were measured in response to GSI stimulation. RESULTS: The relative expression levels of Notch1 and Notch2 mRNA in PBMCs of IM group were 13.58±3.18 and 4.73±1.16, respectively, which were significantly higher than 1.09±0.12 and 1.07±0.15 in PBMCs of control group (both P < 0.001). However, there were no significant differences in relative expression levels of Notch3 and Notch4 mRNA between IM group and control group (P >0.05). The relative expression levels of Notch ligands (including DLL1 and Jagged1 ) mRNA and Notch downstream molecules (including Hes1, Hes5, and Hey1 ) were increased in IM group compared with control group (all P < 0.001). In IM group, the Th17 and Th22 percentage were 5.03%±1.15% and 4.48%±1.29%, respectively, which were both higher than 4.36%±0.82% and 3.83%±0.55% in control group (both P < 0.05). In IM group, the IL-17 and IL-22 level were (301.1±53.82) and (101.2±16.45) pg/ml, respectively, which were both higher than (237.2±72.18) and (84.75±11.83) pg/ml in control group (both P < 0.001). In IM group, the relative expression levels of RORγt and AhR mRNA were 1.25±0.22 and 1.21±0.12, respectively, which were both higher than 0.99±0.15 and 1.04±0.11 in control group (both P < 0.001). There were no remarkable differences in CD4+ T cell proliferation, Th17 percentage, IL-17 secretion, and relative expression level of RORγt mRNA between cells with GSI stimulation and without GSI stimulation (P >0.05). GSI stimulation reduced Th22 percentage, IL-22 secretion, and relative expression level of AhR mRNA compared with non-stimulation (all P < 0.05). CONCLUSION: Notch signaling pathway regulates IL-22 secretion by CD4+ T cells via AhR in IM patients. Notch-AhR-Th22 pathway may take part in the pathogenesis of IM.

Interleukin-35 has a Protective Role in Infectious Mononucleosis-Induced Liver Inflammation Probably by Inhibiting CD8+ T Cell Function.

Interleukin (IL)-35 plays an immunosuppressive role in infectious diseases, autoimmune disorders, and cancers. However, IL-35 expression and its regulation of CD8+ T cells in infectious mononucleosis (IM) are not fully understood. In this study, three groups of participants were compared, including twenty-three patients of IM without liver inflammation, twenty-eight patients of IM with liver inflammation, and twenty-one controls. Plasma and peripheral blood mononuclear cells (PBMCs) were isolated. CD8+ T cells were purified. Plasma IL-35 was measured by ELISA. PBMCs and CD8+ T cells were stimulated with recombinant human IL-35 in vitro. Perforin and granzyme B secretion was assessed by ELISPOT. Immune checkpoint molecule expression was investigated by flow cytometry. CD8+ T cells were co-cultured with HepG2 cells in direct contact and indirect contact manner. The cytotoxicity of CD8+ T cells was calculated by measuring lactate dehydrogenase release and proinflammatory cytokine expression. There was no significant difference in plasma IL-35 levels between patients with IM without liver inflammation and the controls, but the IL-35 level was notably increased in patients with IM who presented with liver inflammation and negatively correlated with aminotransferase. CD8+ T cells in patients with IM with liver inflammation showed stronger cytotoxicity. IL-35 stimulation inhibited CD8+ T cell-induced target cell death in patients with IM, mainly through suppression of IFN-γ/TNF-α secretion and elevation of immune checkpoint molecule expression, but did not affect perforin or granzyme B secretion. The current data indicated that IL-35 dampened the cytotoxicity of CD8+ T cells in patients with IM probably via repression of cytokine secretion. Elevated IL-35 may protect against CD8+ T cell-induced liver inflammation in patients with IM.

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis.

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein-Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus. B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator. The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

Transient hypertriglyceridemia: a common finding during Epstein-Barr virus-induced infectious mononucleosis.

BACKGROUND: Hypertriglyceridemia can occur in lymphoproliferative disorders. Infectious mononucleosis is a self-limiting, benign lymphoproliferative disorder. This study aimed to investigate the serum triglyceride concentrations and their change over time in patients with infectious mononucleosis. METHODS: We evaluated an adult patient with severe hypertriglyceridemia (>1000 mg/dL) during infectious mononucleosis and reviewed the records of 360 patients admitted to our hospital because of infectious mononucleosis (median age, 19 years; range, 15-87 years; 51.4% male). We compared the serum triglyceride concentrations with those of a control sample from the general population (n=75). A second triglyceride measurement, obtained during convalescence (median of 30 days after the initial determination), was available for 160 patients. RESULTS: The triglyceride concentrations in the acute phase (median: 156 mg/dL) were significantly higher than those of the controls (median, 76 mg/dL; P<0.001). A total of 194 (53.9%) patients presented with hypertriglyceridemia (>150 mg/dL), which was more common in the patients older than 30 years than in the younger patients (78.6% vs. 50.6%; P<0.001). A significant correlation (P<0.005) was observed between the triglyceride levels and white blood cell counts, total cholesterol levels, and liver damage markers. The triglyceride concentrations decreased during convalescence (P<0.001) and were lower than the initial measurement in 83.7% of the cases. Conversely, the total cholesterol concentrations during the acute phase were lower than those of the controls and increased during convalescence (P<0.001). CONCLUSIONS: Patients with severe infectious mononucleosis frequently show mild, transient hypertriglyceridemia. Further studies are needed to elucidate the mechanisms underlying this finding.

Dynamically Expressed miR-BART16 Functions as a Suppressor of CAND1 in Infectious Mononucleosis Caused by Epstein-Barr Virus in Children.

OBJECTIVE: MiR-BART16 is a newly discovered Epstein-Barr Virus-encoded microRNA (miRNA). We aimed to explore the role of EBV-miR-BART16 in infectious mononucleosis (IM). METHODS: Peripheral blood lymphocyte subsets were analyzed in 30 IM and 10 healthy children by flow cytometry. MiR-BART16 and its targets were measured by real-time PCR, western blot, ELISA, and dual-luciferase assay. RESULTS: Serum miR-BART16 expression was significantly higher in the IM children than that in the healthy children, and was positively correlated with EBV copy number. Receiver operating characteristic analysis revealed serum miR-BART16 could differentiate IM and healthy individuals (P=0.0041). CAND1 was targeted and downregulated by miR-BART16 in an EBV infection-dependent way. CONCLUSIONS: These results highlight that EBV-miR-BART16 plays an important role in regulating the expression of CAND1 to affect pediatric IM.

Proteome-wide analysis of CD8+ T cell responses to EBV reveals differences between primary and persistent infection.

Human herpesviruses are antigenically rich agents that induce strong CD8+T cell responses in primary infection yet persist for life, continually challenging T cell memory through recurrent lytic replication and potentially influencing the spectrum of antigen-specific responses. Here we describe the first lytic proteome-wide analysis of CD8+ T cell responses to a gamma1-herpesvirus, Epstein-Barr virus (EBV), and the first such proteome-wide analysis of primary versus memory CD8+ T cell responses to any human herpesvirus. Primary effector preparations were generated directly from activated CD8+ T cells in the blood of infectious mononucleosis (IM) patients by in vitro mitogenic expansion. For memory preparations, EBV-specific cells in the blood of long-term virus carriers were first re-stimulated in vitro by autologous dendritic cells loaded with a lysate of lytically-infected cells, then expanded as for IM cells. Preparations from 7 donors of each type were screened against each of 70 EBV lytic cycle proteins in combination with the donor's individual HLA class I alleles. Multiple reactivities against immediate early (IE), early (E) and late (L) lytic cycle proteins, including many hitherto unrecognised targets, were detected in both contexts. Interestingly however, the two donor cohorts showed a different balance between IE, E and L reactivities. Primary responses targeted IE and a small group of E proteins preferentially, seemingly in line with their better presentation on the infected cell surface before later-expressed viral evasins take full hold. By contrast, target choice equilibrates in virus carriage with responses to key IE and E antigens still present but with responses to a select subset of L proteins now often prominent. We infer that, for EBV at least, long-term virus carriage with its low level virus replication and lytic antigen release is associated with a re-shaping of the virus-specific response.

The Effects of Epstein-Barr Virus Nuclear Antigen 2 (EBNA2) on B-Lymphocytic Activity.

BACKGROUND: Epstein-Barr virus is a worldwide disease that can cause a wide range of human diseases, the person will become a lifelong carrier of the virus once infected. To investigate the mechanism of Epstein-Barr virus nuclear antigen 2 (EBNA2) on B-lymphocytic activity, sera from 35 patients with infectious mononucleosis and from 34 cases without Epstein-Barr virus infection are collected for experimental analysis. METHODS: Quantitative real-time PCR, western blot, and MTT assays were used to evaluate the relative expression level of EBNA2 and to examine its impact on cell vitality. RESULTS: Research found that the average EBNA2 mRNA and protein levels in 35 patients with infectious mononucleosis were higher than that 34 cases without Epstein-Barr virus infection. The MTT assay indicates that EBNA2 can promote the growth and proliferation of B lymphocytes. CONCLUSIONS: Combining the above implies that EBNA2 plays an important role in diseases that are induced by the Epstein-Barr virus. Other experiments reveal that ATO promotes the degradation of EBNA2 protein and induces the apoptosis of B lymphocytes which are EBNA2-positive.

DR3 regulation of apoptosis of naive T-lymphocytes in children with acute infectious mononucleosis.

Acute infectious mononucleosis (AIM) is a widespread viral disease that mostly affects children. Development of AIM is accompanied by a change in the ratio of immune cells. This is provided by means of different biological processes including the regulation of apoptosis of naive T-cells. One of the potential regulators of apoptosis of T-lymphocytes is a death receptor 3 (DR3). We have studied the role of DR3 in the regulation of apoptosis of naive CD4+ (nTh) and CD8+ (nCTL) T-cells in healthy children and children with AIM. In healthy children as well as in children with AIM, the activation of DR3 is accompanied by inhibition of apoptosis of nTh. In healthy children, the stimulation of DR3 resulted in the increase in apoptosis of nCTL. On the contrary, in children with AIM, the level of apoptosis of nCTL decreased after DR3 activation, which is a positive contribution to the antiviral immune response. In children with AIM, nCTL are characterized by reduced level of apoptosis as compared with healthy children. These results indicate that DR3 can be involved in the reduction of sensitivity of nCTL to apoptosis in children with AIM.

[Lipid peroxidation, antioxidative system and platelet component of homeostasis in patients with infectious mononucleosis].

The authors consider parameters of lipid peroxidation, antioxidative system and platelet component of homeostasis in patients with infectious mononucleosis.

Epstein-Barr virus effect on frequency of functionally distinct T cell subsets in children with infectious mononucleosis.

PURPOSE: Epstein-Barr virus is a common human pathogen which infects the great majority of population worldwide. A striking proliferation of CD8⁺ T cells is an immune response to EBV invasion of B lymphocytes during infectious mononucleosis. The aim of the study was to analyze frequencies of CD28⁺CD95⁻, CD28⁺CD95⁺, CD28⁻CD95⁺ T cell subsets putative naïve (T(N)), central (T(CM)) and effector memory (T(EM)) T cells in children with infectious mononucleosis. MATERIAL/METHODS: Multiparameter flow cytometric analysis of CD4⁺ and CD8⁺ T cell subsets was performed in 19 children with acute infectious mononucleosis. RESULTS: The CD4⁺/CD8⁺ ratio was found to be decreased (0.53) in children with infectious mononucleosis. Median T(N), T(CM), T(EM) frequencies were estimated to be 3.7, 4.5, 15.1% of CD8⁺ and 23, 59.3, 5.5% of CD4⁺ T cells, respectively. In the present study we demonstrated negative correlations between CD8⁺CD28⁺CD95⁺ and CD8⁺CD28⁻CD95⁺ T cells and both VCA IgM antibody titers and disease duration. However, no such correlation was found when subset of CD4⁺ T cells or CD8⁺CD28⁺CD95⁻ cells was compared. CONCLUSIONS: We conclude that there is a rapid decrease in the number of memory CD8⁺ T cells in early acute stage of infectious mononucleosis.

[Changes of FoxP3, CD4(+)CD25(+) regulatory T cells, TLR2 and TLR9 in children with infectious mononucleosis].

The aim of this study was to investigate the effects of TLR2, TLR9, CD4(+)CD25(+) regulatory T cells (Treg) and transcription factor FoxP3 in the pathogenesis of children with infectious mononucleosis (IM). Thirty-five acute IM patients admitted in our hospital from April 2010 to January 2011 were enrolled in this study. Thirty-five healthy subjects were taken as control. The thirty-five patients before treatment were considered as patients in acute stage, after treatment and without clinical symptom they were thought as patients in recovery stage. The expression levels of TLR2, TLR9 and FoxP3 mRNA were detected by real time PCR using SYBR Green I. The expression of T lymphocyte subset CD4(+)CD25(+) in peripheral blood mononuclear cells was detected by flow cytometry. The results showed that the relative levels of TLR2 mRNA (4.03 ± 0.56), TLR9 mRNA (8.88 ± 1.56) in peripheral blood mononuclear cells of IM patients in acute stage were significantly higher than those of the controls [TLR2 mRNA (2.22 ± 0.57), TLR9 mRNA (3.63 ± 1.30)] and IM patients in recovery stage [TLR2 mRNA (2.76 ± 0.83), TLR9 mRNA (5.34 ± 1.60)] (P < 0.01). The result of CD4(+)CD25(+) (2.38 ± 1.32%) and relative level of FoxP3 mRNA(2.82 ± 0.90) in peripheral blood mononuclear cells of IM patients in acute stage were lower than those of the control [CD4(+)CD25(+) (7.85 ± 1.97%), FoxP3 mRNA (4.65 ± 1.23) ] (P < 0.01). There was no significant difference in CD4(+)CD25(+) (6.81 ± 1.84%), FoxP3 mRNA(4.11 ± 1.37) levels between IM patients in recovery stage and the controls (P > 0.05). It is concluded that the expression of CD4(+)CD25(+)regulatory T cells is reduced, and its special transcription factor FoxP3 mRNA is down-regulated, but expression levels of TLR2 mRNA, TLR9 mRNA are up-regulated in IM patients of acute stage.

Infectious mononucleosis mimicking lymphoma: distinguishing morphological and immunophenotypic features.

The diagnosis of infectious mononucleosis (acute Epstein-Barr virus (EBV) infection) is usually made on the basis of clinical and laboratory findings. However, an atypical clinical presentation occasionally results in a lymph node or tonsillar biopsy. The morphological features of EBV-infected lymphoid tissue can easily mimic lymphoma. Furthermore, the immunophenotype of the immunoblasts has not been well characterized. To assess the morphological spectrum of acute EBV infection and the utility of immunohistochemistry in diagnosing difficult cases that resemble lymphoma, we reviewed 18 cases of acute EBV infection submitted in consultation to our institution with an initial diagnosis of/or suspicion for lymphoma. Patients included nine male and nine female individuals with a median age of 18 years (range 9-69). Biopsies were obtained from lymph nodes (3/18) or Waldeyer's ring (15/18). Infectious mononucleosis was confirmed by monospot or serological assays in 72% of cases (13/18). All cases featured architectural distortion by a polymorphous infiltrate with an immunoblastic proliferation, sometimes forming sheets. Reed-Sternberg-like cells were present in 8/18 (44%) of the cases. Infiltrates were often accompanied by necrosis (10/18) and mucosal ulceration (6/15). The majority of immunoblasts in all cases were CD20+ B cells with a post-germinal center immunophenotype (strongly positive for MUM1/IRF4 (18/18), CD10- (18/18 negative) and BCL-6- (16/18 negative; 2/18 faint BCL-6 expression in <10% of immunoblasts)). Immunoblasts showed variable weak expression of BCL-2 and polyclonal expression of κ and λ immunoglobulin light chains in 81% cases. Reed-Sternberg-like cells in 8/8 cases were CD30+, CD15-, BOB.1+ and OCT-2+. In conclusion, an atypical lymphoid infiltrate with numerous MUM1+, CD10-, BCL-6- immunoblasts should raise the suspicion of a reactive process, such as infectious mononucleosis, and warrants additional consideration before a diagnosis of lymphoma is made.

Tonsillar lesions of infectious mononucleosis resembling MALT type lymphoma. A report of two cases.

Infectious mononucleosis (IM) is an acute lymphoproliferative disorder that typically occurs in young patients and is usually caused by Epstein-Barr virus. We report here, two cases of tonsillar lesion of IM resembling marginal zone B-cell lymphoma mucosa-associated lymphoid tissue (MALT) type. The patients consisted of an 18-year-old Japanese woman and a 36-year-old Japanese man. Both patients presented with tonsillar mass. Histologically, in one case, the tonsil showed diffuse proliferation of medium-sized lymphocytes. The medium-sized lymphocytes had round or slightly indented nuclei with a small solitary nucleoli and abundant clear cytoplasm and somewhat resembled monocytoid B-cells. In the remaining one case, the lymphoid follicles had hyperplastic germinal centers with ill-defined borders surrounded by a sheet-like proliferation of polymorphous infiltration showing a marginal zone distribution pattern. On high-power field, the interfollicular area was diffusely infiltrated by a polymorphous infiltrate of medium-sized lymphocytes with angulated nuclei somewhat resembling centrocyte-like cells, immunoblasts, plasma cells, plasmacytoid cells and histiocytes with or without epithelioid cell feature. However, there were no CD43+ B-cells in either lesion. Moreover, the polytypic nature of the B-cells was demonstrated by immunohistochemistry or polymerase chain reaction. Although MALT type lymphoma rarely affected young adults, notably, a number of cases have been reported in the tonsil. The present two cases indicated that acute IM should be added to the differential diagnosis for MALT type lymphoma in young adults.

[Measurement of B-lymphocyte stimulator mRNA expression in children with infectious mononucleosis by real-time fluorescence quantitative method].

OBJECTIVE: B cell multiplication plays a key role in infections mononucleosis. The present study was designed to detect the expression of B-lymphocyte stimulator (BLyS) mRNA in peripheral blood using real-time fluorescence quantitative polymerase chain reaction (RFQ-PCR) in children with infectious mononucleosis in order to explore the role of BLys in this disorder. METHODS: Specific primers and TaqMan probes of BLyS were designed, and fluorescence of the PCR products were detected continuously during amplification. According to the standard curves created by plasmid DNA, the expression level of target genes in clinical samples were calculated using Stata Software version 8.0, and the results were presented as the ratio of copies of target gene mRNA to beta2 microglobulin (beta2M) mRNA copies. BLyS mRNA expression in peripheral blood was measured by RFQ-PCR in 18 children with infectious mononucleosis and the results were compared with those measured in 15 healthy controls. RESULTS: The range of target gene mRNA detected by REQ-PCR was from 109 ng/L to 101 ng/L. The coefficient of variation for intra-experimental and inter-experimental reproducibility ranged from 1.88% to 5.89% and 6.32% to 12.34%, respectively. BLyS mRNA expression in peripheral blood in children with infectious mononucleosis were significantly higher than that in controls (1.65+/-0.10 vs 0.56+/-0.08; P < 0.01). CONCLUSIONS: RFQ-PCR has a high sensitivity and reproducibility for the measurement of BLyS mRNA expression. BLyS may be involved in the development of infectious mononucleosis.

T-cell receptor triggering differentially regulates bim expression in human lymphocytes from healthy individuals and patients with infectious mononucleosis.

Bim, a proapoptotic member of the Bcl-2 protein family, is a major regulator of central and peripheral T-cell deletion. Regulation of Bim activity by T-cell receptor (TCR) triggering is not well understood. We investigated expression of Bim in different subpopulations of ex vivo isolated human T cells from healthy donors and patients with infectious mononucleosis (IM). Upregulation of Bim expression in response to TCR-triggering was observed only in a small proportion of analyzed samples of peripheral blood lymphocytes (PBLs) from healthy donors and only occasionally upon longitudinal analysis of cells isolated from the same individuals. Populations of naive or memory T cells enriched on the basis of CD45RO or CD45RA expression showed only slight and comparable Bim upregulation. In contrast, ex vivo isolated PBLs from IM patients in the acute stage of the disease with significant expansions of CD8+ cells expressed increased levels of Bim, and lymphocytes from the majority of analyzed IM patients exhibited significant upregulation of all major Bim isoforms in response to TCR triggering. These results demonstrate that at least some antigen-induced expansions of human CD8+ T cells are associated with increased levels of Bim, and TCR triggering in effector T lymphocytes may increase Bim activity by upregulation of its expression.

Different T-bet expression patterns characterize particular reactive lymphoid tissue lesions.

AIMS: To investigate T-bet expression profiles in various lymphoid tissue diseases caused by intracellular pathogens and to compare them in disorders without an infective aetiology. Murine and in vitro experiments have shown that the expression/induction of T-bet, the master regulator of Th1 differentiation, can be achieved by obligate intracellular pathogens and high interferon (IFN)-gamma levels. METHODS: Lymph node biopsies were analysed immunohistochemically employing single and double labelling for T-bet and CD20, CD4, CD8 and CD30 detection. RESULTS: In disorders associated with high IFN-gamma levels and intracellular pathogens (infectious mononucleosis, HIV-associated lymphadenopathy, cat-scratch disease, and toxoplasmic lymphadenitis), T-bet-expressing CD4 cells were accompanied by significant numbers of T-bet-positive CD8 and B cells. A similar profile was also found in histiocytic necrotizing (Kikuchi) lymphadenitis, a disease of unknown cause. In contrast, T-bet expression in disorders without an infective aetiology was observed in only a small portion of lymphocytes. CONCLUSIONS: Increased T-bet expression does not only identify intracellular infections in lymphoid tissue associated with high IFN-gamma levels, but also implies that, under these conditions, it becomes induced in B cells, which apparently support the Th1 response. T-bet expression in Kikuchi lymphadenitis underscores the hypothesis that it is caused by an intracellular microorganism.