Tonsillar cytotoxic CD4 T cells are involved in the control of EBV primary infection in children.

CD4 T cells play a key role in Epstein Barr virus (EBV) infection, by modulating latent antigen expression, and exhibiting cytotoxic and regulatory properties. Our aim was to evaluate the presence of Granzyme B (GZMB) and Foxp3 CD4 T cells at different EBV infection status and latency profiles. We examined CD4, GZMB, Foxp3, IL10, TGF-ß, CD4-GZMB and CD4-Foxp3 expression at the tonsils of pediatric patients with different infective status and EBV latency profiles. CD4+, GZMB+, Foxp3+, CD4-GZMB+ and CD4-Foxp3+ cell counts were higher at the interfollicular region. Higher expression of CD4-GZMB was found in primary infected patients compared to healthy carriers. In patients that expressed latency III antigens, we demonstrated lower CD4+, CD4-GZMB+, CD4-Foxp3+ expression; a negative correlation between the immunoregulatory cytokine IL-10+ and GZMB+ as well as a positive correlation of IL-10+ and CD4+. In patients expressing the lytic protein BMRF1, a positive correlation of TGF-ß+ with CD4-GZMB+ and CD4-Foxp3+ was observed. Our findings indicate that CD4-GZMB+ cells are involved in the restriction of primary EBV infection in pediatric patients, which could partially explain the lack of symptoms, whereas both CD4-GZMB+ and CD4-Foxp3+ cells could be involved in the modulation of latency.

EBV Impact in Peripheral Macrophages' Polarization Cytokines in Pediatric Patients.

Macrophages are exceptionally flexible cells. The presence of inflammatory cytokines such as IFN-γ and TNF-α results in an M1 (CD68) activation, while cytokines such as IL-10 or TGF-ß induce the M2 (CD163) activation. Our aim was to study the behavior of peripheral cytokines involved in macrophage polarization and relate them with tissue findings to further comprehend the role of macrophages in EBV pediatric infection. We studied cytokine expression in tonsils and peripheral blood samples of children in different stages of infection. Peripheral cytokines were compared with macrophage polarization markers and viral protein expression in tonsils. Only IL-10 showed a negative correlation between compartments, exclusively in patients undergoing viral reactivation (R). Higher expressions of peripheral IL-1ß, IL-23, and IL-12p40 in R children were observed. Lower expressions of local and peripheral TNF-α in patients with broader expressions of latent and lytic viral proteins were demonstrated. In healthy carrier (HC) patients, IL-23 positively correlated with CD163, and IP-10 positively correlated with CD68. Our results indicated that EBV might modulate antigen expression in the presence of TNF-α and influence peripheral cytokine expression differently in each stage of infection. Moreover, peripheral cytokines might have a particular role in macrophage polarization in HC.

MicroRNA signature from extracellular vesicles of HCV/HIV co-infected individuals differs from HCV mono-infected.

Hepatitis C virus (HCV) coinfection with human immunodeficiency virus (HIV) has a detrimental impact on disease progression. Increasing evidence points to extracellular vesicles (EVs) as important players of the host-viral cross-talk. The microRNAs (miRNAs), as essential components of EVs cargo, are key regulators of normal cellular processes and also promote viral replication, viral pathogenesis, and disease progression. We aimed to characterize the plasma-derived EVs miRNA signature of chronic HCV infected and HIV coinfected patients to unravel the molecular mechanisms of coinfection. EVs were purified and characterized from 50 plasma samples (21 HCV mono- and 29 HCV/HIV co-infected). EV-derived small RNAs were isolated and analyzed by massive sequencing. Known and de novo miRNAs were identified with miRDeep2. Significant differentially expressed (SDE) miRNA identification was performed with generalized linear models and their putative dysregulated biological pathways were evaluated. Study groups were similar for most clinical and epidemiological characteristics. No differences were observed in EVs size or concentration between groups. Therefore, HCV/HIV co-infection condition did not affect the concentration or size of EVs but produced a disturbance in plasma-derived EVs miRNA cargo. Thus, a total of 149 miRNAs were identified (143 known and 6 de novo) leading to 37 SDE miRNAs of which 15 were upregulated and 22 downregulated in HCV/HIV co-infected patients. SDE miRNAs regulate genes involved in inflammation, fibrosis, and cancer, modulating different biological pathways related to HCV and HIV pathogenesis. These findings may help to develop new generation biomarkers and treatment strategies, in addition to elucidate the mechanisms underlying virus-host interaction. KEY MESSAGES: HCV and HCV/HIV displayed similar plasma-EV size and concentration. EVs- derived miRNA profile was characterized by NGS. 37 SDE miRNAs between HCV and HCV/HIV were observed. SDE miRNAs regulate genes involved in inflammation, fibrosis and cancer.

PD-L1 is upregulated in CD163+ tonsillar macrophages from children undergoing EBV primary infection.

Epstein-Barr Virus (EBV) is a tumor associated virus that modulates not only the infected cells but also innate and adaptive immunity. Macrophages play a key role in tumor development and progression. Particularly, the M2 phenotype (CD163) with anti-inflammatory activity contributes to a favorable microenvironment for tumor development while the M1 (CD68) proinflammatory phenotype contributes to a restrictive one. In the context of pediatric EBV infection, little is known about macrophage contribution to PD-L1 expression, a molecule involved in immune exhaustion. We studied tonsils of primary infected (PI), healthy carriers (HC), reactivated (R), and not infected (NI) pediatric patients. Positive correlations were demonstrated for CD68+PD-L1+ in R and for CD163+PD-L1+ only in PI. Furthermore, CD163+PD-L1+ cell numbers were higher than PD-L1+CD68+ in PI patients. In addition, a positive correlation between PD-L1+CD163+ cells and LMP1 viral latent protein was observed in PI patients, and a positive correlation between PD-L1+CD68+ cells and BMRF1 lytic antigen was demonstrated. A positive correlation between TGF-ß and PD-L1 expression was demonstrated in HC patients. Our findings indicate that EBV's lytic and latent antigens might be regulating macrophages' PD-L1 expression, particularly in PI patients, whereas, surprisingly, only TGF-ß could be related to total PD-L1 upregulation. Given the relevance of macrophages and the PD-1/PD-L1 pathway in tumor progression and survival, more studies in early EBV infection could help to develop EBV-associated tumor therapies.

PD-1 and LAG-3 expression in EBV-associated pediatric Hodgkin lymphoma has influence on survival.

In pediatric Hodgkin lymphoma (HL), the inability of the cytotoxic microenvironment induced by EBV presence to eliminate tumor cells could reflect the fact that the virus might be able to induce the expression of exhaustion markers to evade an immune response. Therefore, the expression of exhaustion markers in pediatric EBV-associated HL was evaluated. A balance between cytotoxic GrB and Th1 Tbet markers with regulatory Foxp3 was proved in EBV+ cases. In addition, exclusively in EBV-associated cHL, a correlation between PD-1 and LAG-3 expression was observed. Furthermore, those cases also displayed a trend to worse survival when they expressed LAG-3 and inferior event-free survival when both PD-1 and LAG-3 molecules were present. Therefore, even though a cytotoxic and inflammatory environment was supposed to be triggered by EBV presence in pediatric cHL, it seems that the virus may also induce the synergic effect of inhibitory molecules LAG-3 and PD-1 in this series. These observations may reflect the fact that the permissive and exhausted immune microenvironment succeeds to induce lymphomagenesis.

Pediatric inborn errors of immunity causing hemophagocytic lymphohistiocytosis: Case report and review of the literature.

Inborn errors of immunity are a group of genetic disorders caused by mutations that affect the development and/or function of several compartments of the immune system, predisposing patients to infections, autoimmunity, allergy and malignancies. In this regard, mutations that affect proteins involved in trafficking, priming, docking, or membrane fusion will impair the exocytosis of lytic granules of effector NK and cytotoxic T lymphocytes. This may predispose patients to hemophagocytic lymphohistiocytosis, a life-threatening immune disorder characterized by systemic lymphocyte and macrophage activation, and increased levels of cytokines, which lead to an uncontrolled hyperinflammation state and progressive multiorgan damage. In this review, we will describe a clinical case and recent advances in inborn errors of immunity predisposing to hemophagocytic lymphohistiocytosis. Summary sentence: Review of recent advances in inborn errors of immunity predisposing to hemophagocytic lymphohistiocytosis.

The performance of soluble CD163 as a non-invasive biomarker of liver damage in chronically HCV and HCV/HIV infected subjects.

Macrophage activation plays a key role in liver disease progression. Soluble CD163 (sCD163) is a specific macrophage activation biomarker useful for clinical estimating damage severity and predicting outcome in different liver conditions. sCD163 performance as a non-invasive marker of liver damage was evaluated in plasma samples at time of biopsy in 120 patients with different hepatic conditions (56 HCV, 20 HCV/HIV, 10 HBV and 34 MAFLD). sCD163 values were compared with those of healthy donors and analyzed related to histological damage. sCD163 together with other clinical parameters were used to create a logistical regression model to predict significant fibrosis. Only patients with viral hepatitis showed higher sCD163 values compared to the control group (HCV p<0.0001; HCV/HIV p<0.0001; HBV p = 0.0003), but no significant differences regarding fibrosis stages were observed. The proposed model predicts fibrosis severity using the logarithm sCD163 concentration, platelet count and age, it demonstrated to be a good marker for the HCV monoinfected group (AUROC 0.834) and an excellent one for the HCV/HIV co-infected group (AUROC 0.997). Moreover, the model displayed a diagnostic performance similar to FIB-4 in HCV cases and FIB-4 and APRI in HCV/HIV coinfected cases, and it even managed to correctly classify some cases that had been misclassified. The proposed model is able to determine, in a non-invasive way, the liver fibrosis stage of HCV and HCV/HIV patients, so after validation, it could be used in a complementary way in the clinical practice whenever APRI and FIB-4 failed to determine damage severity in HCV and HCV/HIV cases.

Molecular alterations in the integrated diagnosis of pediatric glial and glioneuronal tumors: A single center experience.

OBJECTIVES: Tumors of the central nervous system (CNS) are the most common pediatric solid tumors, where low grade (LGG) and high grade gliomas (HGG) represent up to 55% of CNS tumors. Current molecular classification of these tumors results in a more accurate diagnosis and risk stratification, which ultimately enables individualized treatment strategies. Identifying known alterations is a suitable approach, particularly in developing countries, where NGS approaches are not easily accessible. We sought to assess molecular alterations in BRAF and histone 3 genes. STUDY DESIGN: FISH, IHC and Sanger sequencing were performed in a series of 102 pediatric glial and glioneuronal tumors. We also correlated these results with clinical and histological findings to evaluate their usefulness as diagnostic and/or prognostic tools. RESULTS: We found that the KIAA1549-BRAF gene fusion was a relevant diagnostic tool for pilocytic astrocytoma, but not related to progression free survival (PFS) and overall survival (OS). BRAFV600E mutation was associated with a decreased OS in LGG, and with decreased PFS and OS among pilocytic astrocytomas. All HGG of the midline were H3K27M mutants, while H3G34R mutant cases were located in brain hemispheres. HGG harboring the H3K27M variant were associated with a decreased PFS and OS. CONCLUSIONS: Assessing druggable molecular markers with prognostic value is particularly important in those cases where complete resection or further radiation therapy is not possible. These potential diagnostic/prognostic markers may be suitable as further screening tests to reduce the requirement on NGS, which is not available in all laboratories. Furthermore, these results broaden data on BRAF and Histone 3 alterations in children from geographic regions, other than USA and Europe.

Intrahepatic immune infiltrate in chronic hepatitis B and chronic hepatitis C: Similar but not the same.

In chronic hepatitis B (CHB) and C (CHC) infections, the composition of the immune cell microenvironment at the site of infection is poorly understood. Thus, our aim was to characterize and compare liver infiltrates to identify shared and exclusive hepatic immune components. Immunohistochemistry was performed on 26 CHB and 42 CHC liver biopsies to determine Th (CD4+), Th1 (T-bet+), Th17 (IL-17A+), Treg (Foxp3+) and CTL (CD8+) cells frequency in portal/periportal and intralobular areas and relate them to liver damage. CHB and CHC cases shared a portal/periportal CD4+ lymphocyte predominance and a lobular CD8+ lymphocyte majority. However, CHC exhibited a concomitant lobular T-bet+ cell dominance while in CHB FoxP3+ cells prevail. CHC disclosed higher frequencies of P/P FoxP3+, IL-17A+ and T-bet+ cells and intralobular CD4+, IL-17A+ and T-bet+ lymphocytes. HBeAg+ chronic hepatitis and CHC cell frequencies were similar except for lobular T-bet+ that remained higher among CHC cases. Comparison among cases with less severe liver disease revealed lower lymphocyte frequencies in CHB samples, while no differences were observed between patients with more severe stages. Interestingly, in CHB portal/periportal CD4+ and lobular CD4+, CD8+ and IL-17A+ cells were associated with severe hepatitis. Even when all studied populations were identified in both infections preferential lymphocyte frequencies and prevalence at different areas along with their association with liver damage highlighted that CHB and CHC immune responses are not the same.

Chronic Hepatitis C Pathogenesis: Immune Response in the Liver Microenvironment and Peripheral Compartment.

Chronic hepatitis C (CHC) pathogenic mechanisms as well as the participation of the immune response in the generation of liver damage are still a topic of interest. Here, we evaluated immune cell populations and cytokines in the liver and peripheral blood (PB) to elucidate their role in CHC pathogenesis. B, CTL, Th, Treg, Th1, Th17, and NK cell localization and frequency were evaluated on liver biopsies by immunohistochemistry, while frequency, differentiation, and functional status on PB were evaluated by flow cytometry. TNF-α, IL-23, IFN-γ, IL-1ß, IL-6, IL-8, IL-17A, IL-21, IL-10, and TGF-ß expression levels were quantified in fresh liver biopsy by RT-qPCR and in plasma by CBA/ELISA. Liver CTL and Th1 at the lobular area inversely correlated with viral load (r = -0.469, p =0.003 and r = -0.384, p = 0.040). Treg correlated with CTL and Th1 at the lobular area (r = 0.784, p < 0.0001; r = 0.436, p = 0.013). Th17 correlated with hepatic IL-8 (r = 0.52, p < 0.05), and both were higher in advanced fibrosis cases (Th17 p = 0.0312, IL-8 p = 0.009). Hepatic cytokines were higher in severe hepatitis cases (IL-1ß p = 0.026, IL-23 p = 0.031, IL-8 p = 0.002, TGF-ß, p= 0.037). Peripheral NK (p = 0.008) and NK dim (p = 0.018) were diminished, while NK bright (p = 0.025) was elevated in patients vs. donors. Naïve Th (p = 0.011) and CTL (p = 0.0007) were decreased, while activated Th (p = 0.0007) and CTL (p = 0.0003) were increased. IFN-γ production and degranulation activity in NK and CTL were normal. Peripheral cytokines showed an altered profile vs. donors, particularly elevated IL-6 (p = 0.008) and TGF-ß (p = 0.041). Total hepatic CTLs favored damage. Treg could not prevent fibrogenesis triggered by Th17 and IL-8. Peripheral T-lymphocyte differentiation stage shift, elevated cytokine levels and NK-cell count decrease would contribute to global disease.

Comprehensive Evolutionary Analysis of Complete Epstein-Barr Virus Genomes from Argentina and Other Geographies.

The sequence variability of the Epstein-Barr virus has been extensively studied throughout previous years in isolates from various geographic regions and consequent variations at both genetic and genomic levels have been described. However, isolates from South America were underrepresented in these studies. Here, we sequenced 15 complete EBV genomes that we analyzed together with publicly available raw NGS data for 199 EBV isolates from other parts of the globe by means of a custom-built bioinformatic pipeline. The phylogenetic relations of the genomes, the geographic structure and variability of the data set, and the evolution rates for the whole genome and each gene were assessed. The present work contributes to overcoming the scarcity of complete EBV genomes from South America and is the most comprehensive geography-related variability study, which involved determining the actual contribution of each EBV gene to the geographic segregation of the entire genome. Moreover, to the best of our knowledge, we established for the first time the evolution rate for the entire EBV genome based on a host-virus codivergence-independent assumption and assessed their evolution rates on a gene-by-gene basis, which were related to the encoded protein function. Considering the evolution of dsDNA viruses with a codivergence-independent approach may lay the basis for future research on EBV evolution. The exhaustive bioinformatic analysis performed on this new dataset allowed us to draw a novel set of conclusions regarding the genome evolution of EBV.

Hepatic lymphocytes involved in the pathogenesis of pediatric and adult non-alcoholic fatty liver disease.

The immune response is critical in NAFLD pathogenesis, but the liver infiltrate's composition and the role of each T cell population is still up for debate. To characterize liver pathogenesis in pediatric and adult cases, frequency and localization of immune cell populations [Cytotoxic T Lymphocytes (CD8+), T helper Lymphocytes (CD4+), Regulatory T lymphocytes (Foxp3+) and Th17 (IL-17A+)] were evaluated. In portal/periportal (P/P) tracts, both age groups displayed a similar proportion of CD8+ and CD4+ lymphocytes. However, comparable Foxp3+ and IL-17A+ cell frequencies were observed in pediatric cases, meanwhile, in adults Foxp3+ was higher than IL-17A+ cells. Interestingly, IL-17A+ lymphocytes seemed to be nearly exclusive of P/P area in both age groups. In intralobular areas, both pediatric and adult cases showed CD8+ lymphocytes predominance with lower frequencies of CD4+ lymphocytes followed by Foxp3+ . Severe inflammation was associated with higher intralobular Foxp3+ lymphocytes (p = 0.026) in children, and lower P/P Foxp3+ and higher IL-17A+ lymphocytes in adults. All cases with fibrosis ≥ 2 displayed P/P low Foxp3+ and high IL-17A+ lymphocyte counts. Pediatric cases with worse steatosis showed high P/P CD4+ (p = 0.023) and intralobular CD8+ (p = 0.027) and CD4+ cells (p = 0.012). In NAFLD cases, the lymphocyte liver infiltrate composition differs between histological areas. Treg and Th17 balance seems to condition damage progression, denoting their important role in pathogenesis.

Differences in Epstein-Barr Virus Characteristics and Viral-Related Microenvironment Could Be Responsible for Lymphomagenesis in Children.

In Argentina, Epstein-Barr virus (EBV) presence is associated with Hodgkin lymphoma (HL) in patients younger than 10 years, suggesting a relationship between low age of EBV infection and HL. Given that HL is derived from germinal centers (GC), our aim was to compare EBV protein expression and microenvironment markers between pediatric HL patients and EBV+GC in children. METHODS: EBV presence and immune cell markers were assessed by in situ hybridization and immunohistochemistry (IHC). RESULTS: Viral latency II pattern was proved in all HL patients and in 81.8% of EBV+ tonsillar GCs. LMP1 and LMP2 co-expression were proved in 45.7% HL cases, but only in 7.7% EBV+ GC in pediatric tonsils. An increase in CD4+, IL10, and CD68+ cells was observed in EBV+ GC. In pediatric HL patients, only the mean of IL10+ cells was statistically higher in EBV+ HL. CONCLUSIONS: Our findings point us out to suggest that LMP1 expression may be sufficient to drive neoplastic transformation, that an immune regulatory milieu counteracts cytotoxic environment in EBV-associated Hodgkin lymphoma, and that CD4+ and CD68+ cells may be recruited to act in a local collaborative way to restrict, at least in part, viral-mediated lymphomagenesis in tonsillar GC.

Overexpression of survivin in pediatric Hodgkin lymphoma tumor cells: Characterization of protein expression and splice-variants transcription profile.

Survivin is abundantly expressed during fetal development but absent in most differentiated adult tissues; an exception being components of the immune system, such as B and T lymphocytes. Beyond acting as a master regulator of the cell cycle, survivin acts as an inhibitor of apoptosis and is overexpressed in almost all carcinoma types; however, its expression in lymphomas is lesser-explored. Survivin's role in carcinogenesis was subjected to its sub-cellular localization and splice transcripts expression, namely wild-type survivin, survivin-∆Ex3 and survivin-2B. To assess survivin's expression and sub-cellular localization in Epstein Barr virus positive and negative biopsies from treatment naïve pediatric patients with Hodgkin lymphoma (HL), samples were stained for survivin protein by immunofluorescence. The proportion of survivin+ cells was calculated, survivin sub-cellular localization assessed and its fluorescence intensity quantified. Transcription profile of survivin mRNA variants was studied by RT-qPCR. Survivin was overexpressed in the nucleus of tumor cells, and also in a greater proportion of tumor cells, in comparison with the non-tumoral infiltrating cells. Although a higher expression of survivin was observed in advanced clinical stages, no correlation was found between the expression level of survivin and a proliferation marker, or event-free survival. Instead, survivin was related to apoptosis inhibition in tumor cells. Additionally, survivin's transcriptional variants displayed similar expression levels. Present results suggest that although survivin is overexpressed in Hodgkin's tumor cells, it may not play a central role in the progression of classic HL, or act as a suitable progression biomarker, as suggested for most carcinomas.

The interplay between local immune response and Epstein-Barr virus-infected tonsillar cells could lead to viral infection control.

Epstein Barr virus (EBV) gains access to the host through tonsillar crypts. Our aim was to characterize microenvironment composition around EBV+ cells in tonsils from pediatric carriers, to disclose its role on viral pathogenesis. LMP1 expression, assessed by immunohistochemistry (IHC), was used to discriminate EBV + and - zones in 41 tonsil biopsies. Three regions were defined: Subepithelial (SE), interfollicular (IF) and germinal center (GC). CD8, GrB, CD68, IL10, Foxp3, PD1, CD56 and CD4 markers were evaluated by IHC; positive cells/100 total cells were counted. CD8+, GrB+, CD68+ and IL10+ cells were prevalent in EBV+ zones at the SE region (p < 0.0001, p = 0.03, p = 0.002 and p = 0.002 respectively, Wilcoxon test). CD4+ and CD68+ cell count were higher in EBV + GC (p = 0.01 and p = 0.0002 respectively, Wilcoxon test). Increment of CD8, GrB and CD68 at the SE region could indicate a specific response that may be due to local homing at viral entry, which could be counterbalanced by IL10, an immunosuppressive cytokine. Additionally, it could be hypothesized that CD4 augment at the GC may be involved in the EBV-induced B-cell growth control at this region, in which macrophages could also participate.

Recombination rates along the entire Epstein Barr virus genome display a highly heterogeneous landscape.

Epstein Barr virus (EBV) has a large DNA genome assumed to be stable, but also subject to mutational processes such as nucleotide substitution and recombination, the latter explored to a lesser extent. Moreover, differences in the extent of recombination events across herpes sub-families were recently reported. Given the relevance of recombination in viral evolution and its possible impact in pathogenesis, we aimed to fully characterize and quantify its extension in all available EBV complete genome by assessing global and local recombination rate values (⍴/bp). Our results provide the first EBV recombination map based on recombination rates assessment, both at a global and gene by gene level, where the mean value for the entire genome was 0.035 (HPDI 0.020-0.062) ⍴/bp. We quantified how this evolutionary process changes along the EBV genome, and proved it to be non-homogeneous, since regulatory regions depicted the lowest recombination rate values while repetitive regions the highest signal. Moreover, GC content rich regions seem not to be linked to high recombination rates as previously reported. At an intragenic level, four genes (EBNA3C, EBNA3B, BRRF2 and BBLF2-BBLF3) presented a recombination rate above genome average. We specifically quantified the signal strength among different recombination-initiators previously described features and concluded that those which elicited the greatest amount of changes in ⍴/bp, TGGAG and CCCAG, were two well characterized recombination inducing motifs in eukaryotic cells. Strikingly, although TGGAG was not the most frequently detected DNA motif across the EBV genome (697 hits), it still induced a significantly greater proportion of initiation events (0.025 events/hits) than other more represented motifs, p-value = 0.04; one tailed proportion test. Present results support the idea that diversity and evolution of herpesviruses are impacted by mechanisms, such as recombination, which extends beyond the usual consideration of point mutations.

Epstein-Barr virus lytic cycle involvement in diffuse large B cell lymphoma.

Epstein-Barr virus (EBV)-mediated B cell transformation is achieved predominantly through the action of latent proteins, but recent evidence suggests that lytic EBV replication has also a certain pathogenic role in lymphomagenesis, at least in the early phases of cell transformation. Particularly, in diffuse large B cell lymphoma (DLBCL), the EBV lytic cycle is by and large unexplored, so to disclose lytic cell contribution to lymphomagenesis, our aim was to evaluate viral early and late lytic gene expression in relation to several immune response markers in a series of EBV+ DLBCL from Argentina. An unexpected number of cells expressed lytic transcripts, being transcribed at the BZLF1, BHRF1, and BLLF1 locus, by real-time quantitative polymerase chain reaction. This lytic antigen expression was confirmed by immunohistochemical staining for BMRF1 early lytic protein, and a positive correlation between lytic and latent genes was confirmed, revealing a close link between their expressions in EBV+ DLBCL pathogenesis. Remarkably, BZLF1 displayed a negative correlation with CD4 cell counts, and this could be in part justified by the restriction of antigen presentation previously reported. The direct correlation for the late lytic gene BLLF1 and IFNγ in this series could represent a specific response directed towards this antigen. Interleukin 10 transcripts also displayed a positive correlation with lytic expression, indicating that regulatory mechanisms could be also involved on EBV-associated DLBCL pathogenesis in our series. Complete lytic reactivation in EBV-positive tumours could potentially kill EBV-positive malignant cells, providing a tool to promote tumour cell killing mediated by EBV as a complementary treatment strategy.

Chronic hepatitis C liver microenvironment: role of the Th17/Treg interplay related to fibrogenesis.

The role of the different lymphocyte populations in liver microenvironment of chronic hepatitis C (CHC) patients is still matter of debate. Since Th17 and Treg have opposite functions, their balance could affect disease progression. The aim was to explore liver microenvironment and its peripheral blood counterpart in adult CHC patients. CD4+ lymphocytes were predominant in the liver, with high Foxp3+ but low IL-17A+ frequency. IL-17A+ lymphocytes and IL-17A+/Foxp3+ ratio displayed association with advanced fibrosis (p = 0.0130; p = 0.0236, respectively), while Foxp3+ lymphocytes and IL-10 expression level inversely correlated with fibrosis severity (p = 0.0381, p = 0.0398, respectively). TGF-ß/IL-6 ratio correlated with IL-17A+/Foxp3+ ratio (p = 0.0036, r = 0.5944) and with IL-17A+ lymphocytes (p = 0.0093; r = 0.5203). TNF-α and TGF-ß were associated with hepatitis severity (p = 0.0409, p = 0.0321). Peripheral blood lymphocyte frequency was not associated with liver damage. There are functionally different immune cell populations actively involved in liver damage, but the liver cytokine milieu actually drives the pathogenesis. The intrahepatic Foxp3+ lymphocytes predominance beside the low IL-17A+ lymphocytes frequency, delineate a skewed IL-17A+/Foxp3+ balance towards Foxp3+ lymphocytes. However, the IL-17A+ lymphocytes association with advanced fibrosis denotes their role in the pathogenesis. Therefore, the interplay between Th17 and Treg conditions liver fibrogenesis.

A novel recombinant variant of latent membrane protein 1 from Epstein Barr virus in Argentina denotes phylogeographical association.

AIM: To study LMP1 variants distribution among children with EBV+ malignant and benign conditions as well as in healthy carriers. METHODS: Oral secretions and blood cells from 31 children with IM, and biopsies from 14 EBV+ reactive lymphoid hyperplasia and 33 EBV+ lymphomas were included. LMP1 was amplified by nested PCR and sequenced. Phylogenetic reconstructions were made under Maximun Likelihood, Bayesian and coalescent algorithms. RESULTS: Six clades were defined (China1, China2, Med-, Alaskan, B95.8 and Argentine). Argentine variants, the most prevalent (46%), harbored 3 distinctive mutations and were a recombination between Raji and China1. Despite no pathology or compartment associations were observed for LMP1, the Argentine clade showed a phylogeographic association with our region. LMP1 estimated evolution rate was 8.591x10-5s/s/y and the estimated tMRCA for Raji and Argentine was 136ybp. CONCLUSIONS: An LMP1 Argentine clade was defined. LMP1 evolutionary rate was higher than expected for herpesviruses. The tMRCA for Raji and the Argentine agrees with African immigration and could explain the recombinant nature of the Argentine variant.

Analysis of Epstein-Barr virus infection models in a series of pediatric carriers from a developing country.

Epstein-Barr virus (EBV) is a B lymphotropic human herpesvirus. Two models, germinal center (GC) and direct infection, describe how EBV infects B-cells. Since in Argentina primary infection is mostly subclinical at young ages, children represent an interesting population where to analyze EBV infection, especially considering that most studies are usually performed in adults. Tonsil biopsies from pediatric carriers were studied to describe infection characteristics. EBV+ lymphocytes at the interfollicular region were mainly observed. Latency III pattern in subepithelial (SubEp) lymphocytes was observed at young ages, probably indicating a recent infection. In older patients EBV was mostly detected in epithelial cells, suggesting that they could have been infected some time ago. This finding was sustained by tonsillar viral load, which was higher in cases with LMP1+SubEp cells vs. LMP1+nonSubEp cells (p = 0.0237, Mann-Whiney test). Latency III was prevalent and related to the GC, while latency II was associated with non-GC (p = 0.0159, χ2 test). EBERs+/IgD+ cells were statistically prevalent over EBERs+/CD27+ cells (p = 0.0021, χ2 test). These findings indicated that both EBV infection models are not mutually exclusive and provide some basis for further understanding of EBV infection dynamics. Moreover, we provide a more accurate explanation of EBV infection in pediatric asymptomatic carriers from a developing country.