Laura Cuomo

Human herpesvirus 6 infection in neoplastic and normal brain tissue

The presence and variant distribution of human herpesvirus 6 (HHV‐6) was investigated by a nested polymerase chain reaction (PCR) in 118 biopsies from patients affected by nervous tissue tumor (115 primary tumors and 3 metastasis) and in 31 autopsy samples from the brain of healthy individuals. HHV‐6 DNA sequences were detected in normal and neoplastic nervous tissue at a frequency of 32% and 37%, respectively. In both tissues, variant A was three times more frequent than the variant B. Peripheral blood lymphocytes (PBLs) derived from seven tumor affected patients contained the same variant as their respective brain sample, as judged by PCR. The expression of HHV‐6 encoded immediate early protein p41 was detected by immunohistochemistry in neoplastic but not in normal brain. This may reflect viral reactivation from latency in immunocompromised patients. The seroepidemiological data indicated a frequency distribution of anti‐HHV‐6 antibodies in patients with brain tumors similar to that found in healthy donors. J. Med. Virol. 63:45–51, 2001.

Human herpesvirus 6 and multiple sclerosis: a study of T cell cross-reactivity to viral and myelin basic protein antigens.

Several reports have suggested an association of human herpesvirus 6 (HHV‐6) with multiple sclerosis. Autoreactive T lymphocytes directed against myelin components seem to contribute to the pathogenesis of the disease. It has been suggested that molecular mimicry between viral and self‐antigens might be one of the mechanisms that determine the onset of several autoimmune diseases. Following this hypothesis, the purpose of the present study was to evaluate if HHV‐6 could play a role in activating T cells capable of cross‐reaction with an important myelin component, the myelin basic protein. T cell lines were established from 22 multiple sclerosis patients and from 16 healthy controls, and their capability to react to both virus and myelin basic protein antigens was compared. The analysis of T cell cross‐reactivity in patients and controls did not show significant differences in the HHV‐6 ability to activate myelin basic protein‐reactive T cells. Similarly, the evaluation of the humoral immune response to HHV‐6 in patients and controls did not mirror any abnormality in the HHV‐6 status in multiple sclerosis patients. Therefore, although the findings of activity in vitro of T cell lines with dual specificity are consistent with the hypothesis of molecular mimicry, the lack of differences in cross‐reactivity between patients and controls do not support molecular mimicry as an important mechanism in the physiopathology of this disease. J. Med. Virol. 68: 268–272, 2002.

Suppression of dendritic cell differentiation through cytokines released by Primary Effusion Lymphoma cells

Functional impairment of dendritic cells (DC) appears to be one of the mechanisms responsible for tumor escape from the control of the immune system. DC isolated from tumor-bearing animals and cancer patients with solid or with hematological malignancies have phenotypic and functional abnormalities. In addition, supernatants from in vitro cultured tumor cells have been shown to interfere with DC differentiation from CD34+ and monocyte precursors. Primary effusion lymphoma (PEL) is a Human Herpesvirus-8 (HHV-8)-associated tumor, which releases several cytokines such as IL-6, IL-10 and VEGF and its growth seems to be dependent on them in vitro or in vivo. In the present study, we found that these cytokines released by PELs have also an important role in interfering with the in vitro differentiation of immature DC (iDC) from CD14+ monocytes. The iDC obtained in the presence of PEL supernatants showed reduction of FITC-dextran uptake, reduction of MLR allostimulatory activity and altered expression of surface molecules, suggesting that cytokines released by PEL adversely affect DC differentiation.

STAT3 activation by KSHV correlates with IL-10, IL-6 and IL-23 release and an autophagic block in dendritic cells

Kaposiss sarcoma associated herpesvirus (KSHV) has been reported to infect, among others, monocytes and dendritic cells DCs impairing their function. However, the underlying mechanisms remain not completely elucidated yet. Here we show that DC exposure to active or UV-inactivated KSHV resulted in STAT3 phosphorylation. This effect, partially dependent on KSHV-engagement of DC-SIGN, induced a high release of IL-10, IL-6 and IL-23, cytokines that in turn might maintain STAT3 in a phosphorylated state. STAT3 activation also correlated with a block of autophagy in DCs, as indicated by LC3II reduction and p62 accumulation. The IL-10, IL-6 and IL-23 release and the autophagic block could be overcome by inhibiting STAT3 activation, highlighting the role of STAT3 in mediating such effects. In conclusion, here we show that STAT3 activation can be one of the molecular mechanisms leading to KSHV-mediated DC dysfunction, that might allow viral persistence and the onset of KSHV-associated malignancies.

Differential Regulation of Epstein-Barr Virus (EBV) Latent Gene Expression in Burkitt Lymphoma Cells Infected with a Recombinant EBV Strain

ABSTRACT Epstein-Barr virus (EBV)-negative Burkitt lymphomas (BLs) can be infected in vitro with prototype EBV strains to study how the virus may affect the phenotype of tumor cells. Studies thus far have concentrated on the use of transforming B95-8 and nontransforming P3HR1 strains. Immunological and phenotypic differences between the sublines infected with these two strains were reported. The majority of these differences, if not all, can be attributed to the lack of EBNA-2 coding sequences in the P3HR1 strain. The recent development of a selectable Akata strain has opened up new possibilities for infecting epithelial and T cells as well. We infected five EBV-negative BL lines with the recombinant Akata virus. Our results indicate that the infected cell lines BL28, Ramos, and DG75 express EBNA-1, EBNA-2, and LMP1, the viral proteins associated with type III latency, and use both YUK and QUK splices. In contrast, two EBV-negative variants of Akata and Mutu when reinfected displayed restricted type I latency and expressed only EBNA-1. All clones of infected Mutu cells used the QUK splice exclusively. The usage of Qp was observed in a majority of Akata clones. Some Akata clones, however, were found to have double promoter usage (Qp and C/Wp) but at 4 months after infection did not express EBNA-2. The results demonstrate differential regulation of EBV latency in BLs with the same recombinant viral strain and suggest that the choice of latency type may be cell dependent. The restricted latency observed for infected Akata and Mutu cells indicates that a BL may opt for type I latency in the absence of immune pressure as well.

Epstein-Barr virus infection leads to partial phenotypic reversion of terminally differentiated malignant B cells.

The B cell lymphomas associated with Epstein-Barr virus (EBV) are not limited to any specific stage of B cell differentiation but covers widely different B cell phenotypes. In vitro infection of the virus negative tumors with a recombinant EBV strain has provided important insights into virus-tumor interaction. Here, we investigated the interaction between EBV and terminally differentiated tumor derived B cells, namely multiple myeloma (MM). The in vitro EBV infected MM expressed restricted viral latency. Acquisition of the virus was accompanied by a partial reprogramming to a mature B cell phenotype. Thus, the plasma cell markers syndecan-1 (CD138), Blimp1 and MUM1 were downregulated, while expression of HLADR, CIITA and TCL1, which are normally not expressed in plasmacytoid cells, was upregulated. The silenced transcription factor gene encoding Pax5 and its target BLNK were activated. Significantly, the free lambda light chains secreted in the medium were reduced in EBV infected MM clones. Collectively, these results suggest that the restricted EBV latency can cause at least partial phenotypic reversion of terminally differentiated B tumor cells. We suggest that the restricted EBV latent gene expression may not only be the consequence but the cause of the mature B cell phenotype, actively participating in the virus persistence.

Epstein‐Barr virus infection induces miR‐21 in terminally differentiated malignant B cells

The association of Epstein‐Barr virus (EBV) with plasmacytoid malignancies is now well established but how the virus influences microRNA expression in such cells is not known. We have used multiple myeloma (MM) cell lines to address this issue and find that an oncomiR, miR‐21 is induced after in vitro EBV infection. The PU.1 binding site in miR‐21 promoter was essential for its activation by the virus. In accordance with its noted oncogenic functions, miR‐21 induction in EBV infected MM cells caused downregulation of p21 and an increase in cyclin D3 expression. EBV infected MM cells were highly tumorigenic in SCID mice. Given the importance of miR‐21 in plasmacytoid malignancies, our findings that EBV could further exacerbate the disease by inducing miR‐21 has interesting implications both in terms of diagnosis and future miR based therapeutical approaches for the virus associated plasmacytoid tumors.

High glucose and hyperglycemic sera from type 2 diabetic patients impair DC differentiation by inducing ROS and activating Wnt/β-catenin and p38 MAPK.

Type 2 is the type of diabetes with higher prevalence in contemporary time, representing about 90% of the global cases of diabetes. In the course of diabetes, several complications can occur, mostly due to hyperglycemia and increased reactive oxygen species (ROS) production. One of them is represented by an increased susceptibility to microbial infections and by a reduced capacity to clear them. Therefore, knowing the impact of hyperglycemia on immune system functionality is of utmost importance for the management of the disease. In this study, we show that medium containing high glucose reduced the in-vitro differentiation of monocytes into functional DCs and their activation mediated by PAMPs or DAMPs. Most importantly, the same effects were mediated by the hyperglycemic sera derived by type 2 diabetic patients, mimicking a more physiologic condition. DC dysfunction caused by hyperglycemia may be involved in the inefficient control of infections observed in diabetic patients, given the pivotal role of these cells in both the innate and adaptive immune response. Searching for the molecular mechanisms underlying DC dysfunction, we found that canonical Wnt/β-catenin and p38 MAPK pathways were activated in the DCs differentiated either in the presence of high glucose or of hyper-glycemic sera. Interestingly, the activation of these pathways and the DC immune dysfunction were partially counteracted by the anti-oxidant quercetin, a flavonoid already known to exert several beneficial effects in diabetes.

Augmentation of leukocyte infiltration in murine tumors expressing B-cell derived but not nasopharyngeal carcinoma derived EBV membrane protein LMP1

The Epstein‐Barr virus (EBV) encoded latent membrane protein of B cell origin, B‐LMP1 (B95‐8 prototype) and nasopharyngeal carcinoma (NPC) derived C‐LMP1 (CAO prototype) were transfected individually in S6C adenocarcinoma cells of ACA (H‐2f) origin. We have shown previously that inoculation of B‐LMP1 expressing S6C cells led to tumor rejection in pre‐immunized, immunocompetent syngeneic ACA mice, whereas the C‐LMP1 transfectants were not immunogenic. Furthermore, B‐LMP1 but not C‐LMP1 expressing S6C cells grew with necrosis and extensive skin damage in non‐immunized mice. A study was carried out to determine whether the in vivo growth pattern of S6C cells expressing two different LMP1 isolates could be correlated to any immunomodulatory mechanism. An increased infiltration of CD45+ leukocytes was found in B‐LMP1 expressing S6C tumors originating in non‐immunized, syngeneic ACA mice. The C‐LMP1 expressors, vector transfectants and untransfected parental tumors had significantly lower number of infiltrating leukocytes. The immunoaccessory molecules ICAM‐1, B7‐1 and MHC Class I and II expression was unaltered in both B‐ and C‐LMP1 transfectants. The data suggest that B‐LMP1 but not C‐LMP1 induce anti‐tumor immune response. J. Med. Virol. 60:417–424, 2000.

Elevated antinuclear antibodies and altered anti-Epstein-Barr virus immune responses

It has been shown that Epstein-Barr virus (EBV) is able to alter the immune response towards self-antigens and may enhance risk of autoimmune diseases such as systemic lupus erythematosus (SLE) in genetically predisposed individuals. In this study, we evaluated the specific antibody immune response against EBV in patients with anti-nuclear autoantibodies (ANA) in comparison with ANA-negative healthy controls. For this purpose, 92 patients with an high anti-ANA reactivity with or without concomitant extractable nuclear antigen (ENA) or double stranded DNA (dsDNA) positivity were selected and compared with 146 healthy donors. We found that anti-EBV-VCA and EA IgG concentrations were significantly higher in ANA-positive patients in comparison to the controls (VCA P<0.0001 and EA P<0,03) as well as in those ANA-positive patients that showed a concomitant ENA positivity (P=0.0002). Interestingly, elevated anti-EBNA-1 IgG was found in a group of patients who had anti SSA/Ro antibodies. Anti-VCA IgM Abs were more frequently found in those patients with a very high titer of ANA (P=0.06); moreover detection of anti-VCA IgM/IgG in absence of anti-EBNA-1 IgG was more frequent in the patient than in the control group. Both these conditions correlate with a recent EBV infection or reactivation. The data suggest that EBV, particularly during acute infection or in its reactivation phase, could be involved in the ANA and ENA autoantibody formation.