Cyclosporin A Inhibits Induction but Not Production of Gamma Interferon Induced by Epstein–Barr Virus

Cyclosporin A (CsA) interferes with various T-cell functions in vitro and is a potent inhibitor of T-cell-dependent reactions in vivo, such as graft rejection and control of virus infections. Since human gamma interferon (Hu IFN-gamma) is synthesized by T cells and has a controlling role in regulation of Epstein-Barr virus (EBV) infection, we have studied the effects of CsA on EBV-induced cellular Hu IFN-gamma release. CsA inhibited dose-dependently the EBV-induced Hu IFN-gamma response, studied at the cellular level in human blood lymphocytes. These effects were not due to toxicity of CsA, since at inhibitory levels cellular EBV infection measured as polyclonal IgM production proceeded unaffected. CsA did not affect the number of spontaneous Hu IFN-gamma-secreting cells, nor did it have any inhibitory effect if added after virus exposure. It is concluded that CsA inhibits induction but not production of cellular Hu IFN-gamma.     

Immunoglobulin-Secreting Cells in Cord Blood: Effects of Epstein–Barr Virus and Interleukin-4

The contribution of cord blood B lymphocytes to the immune response has been under considerable investigation. Cord blood B cells produce almost no antibodies except of the immunoglobulin (Ig)M isotype, indicating immaturity of the cells or the environment they reside in. The aim of this study was to investigate the number of circulating IgA-, IgM-, IgG-, and IgE-producing cells in cord blood in comparison to adult peripheral blood using the ELISPOT method. Moreover, we studied the effect of transformation with the Epstein-Barr virus (EBV) on the proportion of cells producing different isotypes with or without interleukin (IL)-4. Cord blood had IgM-producing cells circulating predominantly, but also some IgA- and IgG-producing cells, whereas adult peripheral blood contained high amounts of circulating IgA-producing cells and some IgM- and IgG-producing cells. No circulating IgE-producing cells were found in either group. Transformation by EBV caused significant expansion of IgA-, IgM-, and IgG-producing cells in adult peripheral blood, but almost only of IgM-producing cells in cord blood. A low but detectable expansion of IgA- and IgG-producing cells was found. Cells producing IgE were still not found, even after EBV transformation. However, EBV transformation in the presence of IL-4 increased the numbers of IgE-producing cells significantly both in cord blood and adult peripheral blood. These findings indicate that cord blood contains some circulating IgA- and IgG-producing cells that are expanded to some extent after EBV infection. They also indicate that cord blood B cells have a similar capacity for IgE production to adult peripheral blood B cells when appropriately stimulated.     

Influence of Quillaja saponaria Triterpenoid Content on the Immunomodulatory Capacity of Epstein–Barr Virus Iscoms

The immune responses to immunostimulating complexes (iscoms) containing recombinant Epstein–Barr virus (EBV) gp340 envelope protein was evaluated in BALB/c (H-2^d) and CBA (H-2^k) mice. Gp340-iscoms were used either with a low content of Quillaja triterpenoid adjuvant (L-iscoms) or supplemented with additional Quillaja adjuvant in the form of iscomatrix (S-iscoms). Class and subclass distribution of anti-gp340 antibodies, EBV-neutralizing antibodies, antigen-specific T cell proliferation and cytokine production were determined and these results compared to those obtained by immunization with non-adjuvated gp340. The H-2^d and H-2^k mice were characterized as low or high responders in respect to the level of specific anti-gp340 antibodies, secretion of IgG2a isotype, antigen-specific lymphoproliferative capacity, interferon-γ (IFN-γ) and interleukin-10 (IL-10) production in the basic immunizations with gp340. While presentation of the antigen in iscom formulations with low levels of Quillaja triterpenoids induces a moderate enhancement of the immune responses in the low responder H-2^d mice, supplementation with high levels of iscomatrix immunomodulator was required to enhance the immune responses in the high responder H-2^k mice. In both mouse strains subcutaneous immunization with S-iscoms resulted in a significant increase of IgG1- and IgG2a-specific antibodies, as well as in strong antigen-specific proliferative response confirmed by the simultaneous cytokine production. The enhanced antigen-specific secretion of IL-2 and IFN-γ together with the abrogation of IL-10 and the absence of IL-4 indicates that the responses were driven towards a Th1-type rather than Th2-type immune response. The S-iscom formulations minimized the differences in immune responses between the two mouse strains, but the capacity of immune sera to neutralize EBV transformation in vitro remained completely strain-dependent. These data indicate that immune responses generated by iscoms can be manipulated by altering the triterpenoid composition of the iscoms and that the levels of triterpenoids can determine whether or not a Th1-type response is made.     

Antigenic variation of the Epstein–Barr virus nuclear antigen EBNA1 as revealed by monoclonal antibodies

The Epstein–Barr virus (EBV) nuclear antigen EBNA1 plays an essential role in the replication of EBV episomes in latently infected cells and is the only viral protein that is consistently expressed in all programs of latent EBV gene expression. In this study, four monoclonal antibodies (MoAbs) directed to a region (amino acid residues 442–530) of EBNA1 were generated. Competitive enzyme-linked immunosorbent assay (ELISA) experiments using biotinylated MoAbs showed that they recognized distinct epitopes. Reactivity of these MoAbs with various laboratory EBV strains and field EBV isolates was shown to be heterogeneous in that EBNA1 from certain strains (isolates) was recognized and that from others was not. All four MoAbs showed such heterogeneous reactivity, and moreover, each MoAb showed a distinct spectrum of reactivity with these EBV strains (isolates). These results demonstrate an extensive structural variation in this region of EBNA1 as predicted by previous sequencing studies. These MoAbs will be useful as probes to dissect this structural heterogeneity of EBNA1.     

Detection of Epstein–Barr virus small RNAs in routine paraffin sections using non-isotopic RNA/RNA in situ hybridization

The Epstein–Barr virus (EBV) is associated with an increasing range of reactive and neoplastic lesions. There is a need for a sensitive and specific method for detecting latent EBV in routine histological sections. We report the use of a highly sensitive paraffin section RNA/RNA in situ hybridization (ISH) technique using digoxigenin-labelled antisense riboprobes for demonstrating EBV encoded-small RNAs (EBERs), EBV gene products that are transcribed in abundance during latent EBV infection. We applied EBER-ISH to 846 paraffin embedded specimens, including cases of reactive lymphoid hyperplasia ( n = 28), infectious mononucleosis (16), Burkitt's lymphoma (44), immunodeficiency-associated lymphomas in transplant recipients (9) and AIDS patients (128), Hodgkin's disease (130), CD30 antigen positive lymphomas (106), peripheral T-cell lymphomas (104), sporadic B-cell non-Hodgkin's lymphomas (162), undifferentiated nasopharyngeal carcinoma (86), salivary gland lymphoepithelioma (11), and oral hairy leukoplakia (5). Strong, reproducible EBER staining was seen in EBV latently infected cells in archival surgical biopsy and autopsy specimens. EBER-ISH is specific, has a sensitivity comparable to that of the polymerase chain reaction, and is now the method of choice for the in situ detection of latent EBV infection.     

Human Immunodeficiency Virus Type 1 Replication,Immune Activation, and Circulating CytotoxicT Cells Against Uninfected CD4+ T Cells

Cytotoxic T lymphocytes (CTL) that kill uninfected activated CD4⁺ T cells can be induced in vitro by stimulating CD8⁺ T cells with activated autologous CD4⁺ T cells. Similar CTL have been detected in circulating T cells from human immunodeficiency virus type 1 (HIV)-infected individuals. To define the in vivo correlates of this CTL activity, we studied plasma -2 microglobulin and HIV RNA levels, T-lymphocyte subset counts, and expression of CD28 on CD8⁺ T cells concurrently with circulating CTL activity against uninfected CD4⁺ T cells in 75 HIV-infected individuals at different stages of disease progression. Mean values of each parameter were compared in subsets of this group of 75 segregated on the basis of this CTL activity. The group with CTL against uninfected activated CD4⁺ T lymphocytes had more CD8⁺ T cells, a higher percentage of CD28^– CD8⁺ T cells, and higher plasma levels of HIV RNA and -2 microglobulin. CTL against uninfected activated CD4⁺ T cells were predominantly CD28^– and in HIV-infected individuals were associated with immunological or virological evidence of progressive disease. In HIV infection, circulating CTL activity against uninfected activated CD4⁺ T lymphocytes is associated with immune activation, CD8⁺ T cell expansion, accumulation of CD28^– CD8⁺ T cells, and inadequate suppression of HIV replication.     

Epstein–Barr virus BZLF1 gene promoter variants in pediatric patients with acute infectious mononucleosis: Its comparison with pediatric lymphomas

Epstein–Barr virus genotypes can be distinguished by polymorphic variations in the genes encoding EBNA2, 3A, 3B, and 3C. The immediate early gene BZLF1 plays a key role in modulating the switch from latency to lytic replication and therefore enabling viral propagation. The aim of this study was to investigate and compare BZLF1 promoter sequence (Zp) variation in pediatric infectious mononucleosis (IM) and in pediatric EBV positive lymphoma biopsies. Zp was sequenced from peripheral blood mononuclear cells (PBMC) and throat swabs from 10 patients with IM at the time of diagnosis (D0) and during convalescence; and from 13 lymphoma biopsies. Zp ? P and Zp ? V3 variants were found in eight and one IM patients, as well as in five and six tumor biopsies, respectively. A correlation between viral genotype and Zp variant was found significant for Zp ? V3 and EBV2 (P = 0.0002). One IM patient harbored two concomitant Zp variants. Regardless of anatomical compartment or stage of disease all IM patients displayed the same Zp variant along the course of the study. No new infections or adaptative selection of different variants was evidenced. A new Zp variant (Zp ? V3 + 49) was described in two Hodgkin lymphomas, but not in IM. This is the first study to describe Zp variants compartmentalization in children with acute EBV infection and convalescence in a developing country; and comparing them with Zp variants in pediatric lymphomas from the same geographic area. J. Med. Virol. 81:1912–1917, 2009. © 2009 Wiley‐Liss, Inc.     

Activation of ferret erythrocyte Na+–K+–2Cl− cotransport by deoxygenation

Deoxygenation of ferret erythrocytes stimulates Na⁺–K⁺–2Cl⁻ cotransport by 111% ( s.d. , 46) compared to controls in air. Half-maximal activation occurs at a P _O2 of 24 mmHg ( s.d. , 2) indicating that physiological changes in oxygen tension can influence cotransport function. Approximately 25–35% of this stimulation can be attributed to the rise of intracellular free magnesium concentration that occurs on deoxygenation (from 0.82 ( s.d. , 0.07) to 1.40 m m ( s.d. , 0.17)). Most of the stimulation is probably caused by activation of a kinase which can be prevented or reversed by treating cells with the kinase inhibitors PP1 or staurosporine, or by reducing cell magnesium content to submicromolar levels. Stimulation by deoxygenation is comparable with that caused by calyculin A or sodium arsenite, compounds that cause a 2- to 3-fold increase in threonine phosphorylation of the cotransporter which can be detected with phospho-specific antibodies. However, the same approach failed to detect significant changes in threonine phosphorylation following deoxygenation. The results suggest that deoxygenation causes activation of a kinase that either phosphorylates the transporter, but probably not on threonine, or phosphorylates another protein that in turn influences cotransporter behaviour. They also indicate that more than one kinase and phosphatase are involved in cotransporter phosphorylation.     

Detection of Epstein–Barr virus nucleic acid sequences and protein in nodal T-cell lymphomas: relation between latent membrane protein-1 positivity and clinical course

Forty-six nodal T-cell lymphomas, classified according to the updated Kiel classification, were investigated for the presence of Epstein–Barr virus (EBV) DNA by polymerase chain reaction (PCR), EBER 1 and 2 (EBER 1/2) and latent membrane protein-1 (LMP-1) expression. A combination of RNA in situ hybridization and immunohistochemistry was used to establish the phenotype of the Epstein–Barr virus harbouring cells. In 21 of 45 cases Epstein–Barr virus DNA sequences could be detected with the polymerase chain reaction. In 15 cases (14 of 21 EBV PCR positive cases), EBER 1/2 positive cells could be demonstrated. As judged by morphology, EBER 1/2 expression was found in non neoplastic and neoplastic lymphoid cells. Double staining revealed that more than 80% of the EBER 1/2 harbouring cells, lacked B-, T- or histiocytic markers, suggesting down regulation of T- and B-cell markers by Epstein–Barr virus. In eight of 15 cases some EBER 1/2 positive T-cells (CD3, CD45RO, CD43) morphologically resembling tumour cells were found. In nine of 14 cases tested EBER 1/2 positive non-neoplastic B-cells (CD20) were seen. Based on in situ hybridization results, four patterns of EBER 1/2 positive cells were found, i.e. single cells (≤1 per medium power field (mpf), n = 3), scattered (1–25/mpf, n = 4), clustered (26–100/mpf, n = 5) and diffuse (≥100/mpf, n = 3). In eight of 15 cases a clustered or diffuse pattern of EBER 1/2 positive cells was found and these lymphomas were therefore considered to be strongly associated with Epstein–Barr virus. In these lymphomas LMP-1 expression was found to be associated with an aggressive clinical course and hepatosplenomegaly.     

Lymphoepithelioma-like salivary gland carcinoma in Taiwan: a clinicopathological study of nine cases demonstrating a strong association with Epstein–Barr virus

Aims : Lymphoepithelioma-like carcinoma (LELC) of the salivary glands is a rather rare tumour. Previous studies have shown its strong association with Epstein–Barr virus (EBV) among Chinese and Eskimos. We tested this observation with nine Chinese patients with salivary gland LELC in Taiwan including one with coexisting nasopharyngeal carcinoma (NPC) and studied the prognostic significance of their histopathological features. Methods and results : This series showed a predilection for female patients and parotid glands with a median age of 50 years. Three patients died 18.5–26 months after the diagnosis including the case with NPC. Six patients were alive without recurrence for 14–45 months with a median follow-up of 34.5 months. Histopathologically, the tumours showed either lobular or diffuse growth pattern. Granulomas and/or germinal centres were observed in most cases and both B- and T-cells were found in the lymphoid infiltrates, indicating that the salivary gland LELC was capable of inducing a strong host immune reaction. Microscopic growth pattern, lymph node metastasis, and presence or absence of granulomas and/or germinal centres seemed to be important prognostic factors. Both salivary gland LELC and NPC shared similar histopathological appearance and positive immunostaining for epithelial membrane antigen and cytokeratin AE1 but not AE3. Granulomas and amyloid might occur in both tumours. A nasopharyngeal examination is indicated in patients with salivary gland LELC to exclude the possibility of coexisting or metastatic NPC. All nine cases showed positive nuclear signals for EBV-encoded RNA by in situ hybridization including the case with NPC. Conclusions : Our study and the previously published studies show that the association of salivary gland LELC and EBV is strongly related to racial and geographical factors.     

A Short Noncoding Viral DNA Element Showing Characteristics of a Replication Origin Confers Bacteriophage Resistance toStreptococcus thermophilus

A 302-bp noncoding DNA fragment from the DNA replication module of phage φSfi21 was shown to protect theStreptococcus thermophilusstrainSfi1 from infection by 17 of 25 phages. The phage-inhibitory DNA possesses two determinants, each of which individually mediated phage resistance. The phage-inhibitory activity was copy number dependent and operates by blocking the accumulation of phage DNA. Furthermore, when cloned on a plasmid, the φSfi21 DNA acts as an origin of replication driven by phage infection. Protein or proteins in the φSfi21-infected cells were shown to interact with this phage-inhibitory DNA fragment, forming a retarded protein–DNA complex in gel retardation assays. A model in which phage proteins interact with the inhibitory DNA such that they are no longer available for phage propagation can be used to explain the observed bacteriophage resistance. Genome analysis of φSfi19, a phage that is insensitive to the inhibitory activity of the φSfi21-derived DNA, led to the characterisation of a variant putative phage replication origin that differed in 14 of 302 nucleotides from that of φSfi21. The variant origin was cloned and exhibited an inhibitory activity toward phages that were insensitive to the φSfi21-derived DNA.     

Activation of the FGFR1 signalling pathway by the Epstein–Barr virus‐encoded LMP1 promotes aerobic glycolysis and transformation of human nasopharyngeal epithelial cells

Non‐keratinizing nasopharyngeal carcinoma (NPC) is closely associated with Epstein–Barr virus (EBV) infection. The EBV‐encoded latent membrane protein 1 (LMP1) is believed to play an important role in NPC pathogenesis by virtue of its ability to activate multiple cell signalling pathways which collectively promote cell proliferation, transformation, angiogenesis, and invasiveness, as well as modulation of energy metabolism. In this study, we report that LMP1 increases cellular uptake of glucose and glutamine, enhances LDHA activity and lactate production, but reduces pyruvate kinase activity and pyruvate concentrations. LMP1 also increases the phosphorylation of PKM2, LDHA, and FGFR1, as well as the expression of PDHK1, FGFR1, c‐Myc, and HIF‐1α, regardless of oxygen availability. Collectively, these findings suggest that LMP1 promotes aerobic glycolysis. With respect to FGFR1 signalling, LMP1 not only increases FGFR1 expression, but also up‐regulates FGF2, leading to constitutive activation of the FGFR1 signalling pathway. Furthermore, two inhibitors of FGFR1 (PD161570 and SU5402) attenuate LMP1‐mediated aerobic glycolysis, cellular transformation (proliferation and anchorage‐independent growth), cell migration, and invasion in nasopharyngeal epithelial cells, identifying FGFR1 signalling as a key pathway in LMP1‐mediated growth transformation. Immunohistochemical staining revealed that high levels of phosphorylated FGFR1 are common in primary NPC specimens and that this correlated with the expression of LMP1. In addition, FGFR1 inhibitors suppress cell proliferation and anchorage‐independent growth of NPC cells. Our current findings demonstrate that LMP1‐mediated FGFR1 activation contributes to aerobic glycolysis and transformation of epithelial cells, thereby implicating FGF2/FGFR1 signalling activation in the EBV‐driven pathogenesis of NPC. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Host ethnicity influences non-Hodgkin's lymphoma subtype frequency and Epstein–Barr virus association rate: the experience of a multi-ethnic patient population in Malaysia

AIMS: The pattern of malignant lymphoma is known to vary in different populations. This study aims to elucidate the effect of ethnicity on subtype frequency of non-Hodgkin's lymphoma and EBV association rate. METHODS AND RESULTS: A total of 232 reconfirmed lymphoma cases in Malaysian patients were retrieved from the archives in the Department of Pathology, University Hospital, Kuala Lumpur. There were 24 (10%) Hodgkin's and 208 (90%) non-Hodgkin's lymphomas, 173 of the latter were in adult group (aged > or = 15 years). The ethnic composition were 41 Malays, 107 Chinese, 21 Indians and four none of the above. A male : female ratio of 2.4 : 1 was observed. Complete immunohistochemical studies in 158 cases revealed 36 (23%) T-cell, 121 (76%) B-cell and one (1%) null-cell phenotype. Seventy-five percent of the T-cell lymphomas were peripheral T/NK-cell types. Among the classifiable lesions, low-grade/indolent lymphomas constituted 17%: 2% were the lymphocytic subtype and 10% were follicular lymphomas. Approximately one-third of the follicular lymphomas occurred in Indian patients. The largest group of high-grade lymphoma was diffuse large B-cell type (46%), followed by peripheral T/NK-cell (18%). A predominance of NK/T-cell lymphomas occurred in Chinese (5/7), and all were EBV associated. Burkitt's lymphoma accounted for 5% (eight cases), all were Chinese males, with a 38% EBV-association rate. The frequency of EBV-associated B-cell lymphoma is three times more common in Chinese than Malays. The EBV positivity rate among lymphomas in ethnic Malay, Chinese and Indian patients was 5%, 15% and 22%, respectively, and in T- and B-cell lymphomas was 36% and 7%, respectively. CONCLUSIONS: This Malaysian series reveals differences in the subtype frequencies of non-Hodgkin's lymphomas and EBV association rate amongst patients of various ethnic groups residing in the same environment.     

Activation of Human Lymphocyte Subpopulations by Rabbit Anti-Human β2-Microglobulin and by Lipopolysaccharide

Rabbit anti-human beta2-microglobulin (anti-beta2m) was found to increase DNA synthesis in peripheral blood lymphocytes (PBLs) and in cells from abdominal lymph nodes, spleen, tonsil, adenoid, appendix, and bone marrow. The response to anti-beta2m was highest in cells originating from abdominal lymph node, appendix, and spleen. These organs were shown to contain a high proportion of surface-Ig-positive cells. No response to anti-beta2m was seen in thymus cells or in B-cell-depleted lymphocyte populations. Lipopolysaccharide (LPS) increased DNA synthesis in spleen cells, bone marrow cells, tonsil cells, and, sometimes, in cells from abdominal lymph nodes but weakly or not at all in PBLs. To study whether anti-beta2m and LPS activated the same subpopulation of lymphocytes, cultures were exposed to both mitogens in various concentrations. The effect on DNA synthesis in spleen cells was almost additive. This may indicate that these two polyclonal B-cell activators (PBAs) stimulate mainly distinct subsets of B cells in spleen. On the other hand, these two mitogens have a synergistic effect on DNA synthesis in PBLs. Since anti-beta2m is the first described selective B-cell mitogen activating human PBLs, it might be of clinical importance in the functional characterization of lymphocyte subpopulations.