Baseline demographics, clinical features and predictors of conversion among 200 individuals in a longitudinal prospective psychosis-risk cohort.

BACKGROUND: DSM-5 proposes an Attenuated Psychosis Syndrome (APS) for further investigation, based upon the Attenuated Positive Symptom Syndrome (APSS) in the Structured Interview for Psychosis-Risk Syndromes (SIPS). SIPS Unusual Thought Content, Disorganized Communication and Total Disorganization scores predicted progression to psychosis in a 2015 NAPLS-2 Consortium report. We sought to independently replicate this in a large single-site high-risk cohort, and identify baseline demographic and clinical predictors beyond current APS/APSS criteria. METHOD: We prospectively studied 200 participants meeting criteria for both the SIPS APSS and DSM-5 APS. SIPS scores, demographics, family history of psychosis, DSM Axis-I diagnoses, schizotypy, and social and role functioning were assessed at baseline, with follow-up every 3 months for 2 years. RESULTS: The conversion rate was 30% (n = 60), or 37.7% excluding participants who were followed under 2 years. This rate was stable across time. Conversion time averaged 7.97 months for 60% who developed schizophrenia and 15.68 for other psychoses. Mean conversion age was 20.3 for males and 23.5 for females. Attenuated odd ideas and thought disorder appear to be the positive symptoms which best predict psychosis in a logistic regression. Total negative symptom score, Asian/Pacific Islander and Black/African-American race were also predictive. As no Axis-I diagnosis or schizotypy predicted conversion, the APS is supported as a distinct syndrome. In addition, cannabis use disorder did not increase risk of conversion to psychosis. CONCLUSIONS: NAPLS SIPS findings were replicated while controlling for clinical and demographic factors, strongly supporting the validity of the SIPS APSS and DSM-5 APS diagnosis.

Oxidative stress enables Epstein-Barr virus-induced B-cell transformation by posttranscriptional regulation of viral and cellular growth-promoting factors.

Infection of human B lymphocytes by Epstein-Barr virus (EBV) leads to the establishment of immortalized lymphoblastoid cell lines (LCLs) that are widely used as a model of viral oncogenesis. An early consequence of infection is the induction of DNA damage and activation of the DNA damage response, which limits the efficiency of growth transformation. The cause of the DNA damage remains poorly understood. We have addressed this question by comparing the response of B lymphocytes infected with EBV or stimulated with a potent B-cell mitogen. We found that although the two stimuli induce comparable proliferation during the first 10 days of culture, the EBV-infected blasts showed significantly higher levels of DNA damage, which correlated with stronger and sustained accumulation of reactive oxygen species (ROS). Treatment with ROS scavengers decreased DNA damage in both mitogen-stimulated and EBV-infected cells. However, while mitogen-induced proliferation was slightly improved, the proliferation of EBV-infected cells and the establishment of LCLs were severely impaired. Quenching of ROS did not affect the kinetics and magnitude of viral gene expression but was associated with selective downregulation of the viral LMP1 and phosphorylated cellular transcription factor STAT3 that have key roles in transformation. Analysis of the mechanism by which high levels of ROS support LMP1 expression revealed selective inhibition of viral microRNAs that target the LMP1 transcript. Our study provides novel insights into the role of EBV-induced oxidative stress in promoting B-cell immortalization and malignant transformation.

Telomere dysfunction and activation of alternative lengthening of telomeres in B-lymphocytes infected by Epstein-Barr virus.

Malignant cells achieve replicative immortality by two alternative mechanisms, a common one dependent on de novo synthesis of telomeric DNA by telomerase, and a rare one based on telomere recombination known as alternative lengthening of telomeres (ALT). Epstein-Barr virus (EBV) transforms human B-lymphocytes into lymphoblastoid cell lines with unlimited growth potential in vitro and in vivo. Here we show that newly EBV-infected cells exhibit multiple signs of telomere dysfunction, including the occurrence of extra-chromosomal telomeres, telomere fusion and telomere length heterogeneity, and undergo progressive increase in telomere length without a parallel increase in telomerase activity. This phenotype is accompanied by the accumulation of telomere-associated promyelocytic leukemia nuclear bodies and telomeric-sister chromatid exchange, suggesting that EBV infection promotes the activation of ALT. Newly infected cells also display a significant reduction of telomere-associated TRF2 and express low levels of TRF1, TRF2, POT1 and ATRX, pointing to telomere de-protection as an important correlate of ALT activation. Collectively, these findings highlight the involvement of recombination-dependent mechanisms for maintenance of telomere homeostasis in EBV-induced B-cell immortalization.

The Epstein-Barr virus nuclear antigen-1 promotes telomere dysfunction via induction of oxidative stress.

The Epstein-Barr virus (EBV) nuclear antigen (EBNA)-1 promotes the accumulation of chromosomal aberrations in malignant B cells by inducing oxidative stress. Here we report that this phenotype is associated with telomere dysfunction. Stable or conditional expression of EBNA1 induced telomere abnormalities including loss or gain of telomere signals, telomere fusion and heterogeneous length of telomeres. This was accompanied by the accumulation of extrachromosomal telomeres, telomere dysfunction-induced foci (TIFs) containing phosphorylated histone H2AX and the DNA damage response protein 53BP1, telomere-associated promyelocytic leukemia nuclear bodies (APBs), telomeric-sister chromatid exchanges and displacement of the shelterin protein TRF2. The induction of TIFs and APBs was inhibited by treatment with scavengers of reactive oxygen species (ROS) that also promoted the relocalization of TRF2 at telomeres. These findings highlight a novel mechanism by which EBNA1 may promote malignant transformation and tumor progression.

The soluble form of urokinase receptor promotes angiogenesis through its Ser88-Arg-Ser-Arg-Tyr9² chemotactic sequence.

BACKGROUND: The urokinase plasminogen activator receptor (u-PAR) focuses the proteolytic activity of the urokinase plasminogen activator (u-PA) on the endothelial cell surface, thus promoting angiogenesis in a protease-dependent manner. The u-PAR may exist in a glycophosphatidylinositol-anchored and in a soluble form (soluble u-PAR [Su-PAR]), both including the chemotactic Ser88 -Arg-Ser-Arg-Tyr9² internal sequence. OBJECTIVE: To investigate whether Su-PAR may trigger endothelial cell signaling leading to new vessel formation through its chemotactic Ser88 -Arg-Ser-Arg-Tyr9² sequence. METHODS AND RESULTS: In this study, the formation of vascular-like structures by human umbilical vein endothelial cells was assessed by using a matrigel basement membrane preparation. First, we found that Su-PAR protein promotes the formation of cord-like structures, and that this ability is retained by the isolated Ser(88) -Arg-Ser-Arg-Tyr9² chemotactic sequence, the maximal effect being reached at 10 nmol L⁻¹ SRSRY peptide (SRSRY). This effect is mediated by the α(v) ß3 vitronectin receptor, is independent of u-PA proteolytic activity, and involves the internalization of the G-protein-coupled formyl-peptide receptor in endothelial cells. Furthermore, exposure of human saphenous vein rings to Su-PAR or SRSRY leads to a remarkable degree of sprouting. Finally, we show that Su-PAR and SRSRY promote a marked response in angioreactors implanted into the dorsal flank of nude mice, retaining 91% and 66%, respectively, of the angiogenic response generated by a mixture of vascular endothelial growth factor and fibroblast growth factor type 2. CONCLUSIONS: Our results show a new protease-independent activity of Su-PAR that stimulates in vivo angiogenesis through its Ser88 -Arg-Ser-Arg-Tyr9² chemotactic sequence.

Three Epstein-Barr virus latency proteins independently promote genomic instability by inducing DNA damage, inhibiting DNA repair and inactivating cell cycle checkpoints.

Epstein-Barr virus (EBV) has been implicated in the pathogenesis of human malignancies, but its contribution to tumorigenesis is not well understood. EBV carriage is associated with increased genomic instability in Burkitt's lymphoma, suggesting that viral products may induce this tumor phenotype. Using a panel of transfected sublines of the B-lymphoma line BJAB expressing the viral genes associated with latent infection, we show that the EBV nuclear antigens, EBNA-1 and EBNA-3C, and the latent membrane protein 1, LMP-1, independently promote genomic instability, as detected by nonclonal chromosomal aberrations, DNA breaks and phosphorylation of histone H2AX. EBNA-1 promotes the generation of DNA damage by inducing reactive oxygen species (ROS), whereas DNA repair is inhibited in LMP-1-expressing cells through downregulation of the DNA damage-sensing kinase, ataxia telangiectasia mutated (ATM), reduction of phosphorylation of its downstream targets Chk2 and inactivation of the G(2) checkpoint. EBNA-3C enhances the propagation of damaged DNA through inactivation of the mitotic spindle checkpoint and transcriptional downregulation of BubR1. Thus, multiple cellular functions involved in the maintenance of genome integrity seem to be independently targeted by EBV, pointing to the induction of genomic instability as a critical event in viral oncogenesis.

Inhibition of serine-peptidase activity enhances the generation of a survivin-derived HLA-A2-presented CTL epitope in colon-carcinoma cells.

Cytotoxic T lymphocytes eliminate tumor cells expressing antigenic peptides in the context of MHC-I molecules. Peptides are generated during protein degradation by the proteasome and resulting products, surviving cytosolic amino-peptidases activity, may be presented by MHC-I molecules. The MHC-I processing pathway is altered in a large number of malignancies and modulation of antigen generation is one strategy employed by cells to evade immune control. In this study we analyzed the generation and presentation of a survivin-derived CTL epitope in HLA-A2-positive colon-carcinoma cells. Although all cell lines expressed the anti-apoptotic protein survivin, some tumors were poorly recognized by ELTLGEFLKL (ELT)-specific CTL cultures. The expression of MHC-I or TAP molecules was similar in all cell lines suggesting that tumors not recognized by CTLs may present defects in the generation of the ELT-epitope which could be due either to lack of generation or to subsequent degradation of the epitope. The cells were analyzed for the expression and the activity of extra-proteasomal peptidases. A significant overexpression and higher activity of TPPII was observed in colon-carcinoma cells which are not killed by ELT-specific CTLs, suggesting a possible role of TPPII in the degradation of the ELT-epitope. To confirm the role of TPPII in the degradation of the ELT-peptide, we showed that treatment of colon-carcinoma cells with a TPPII inhibitor resulted in a dose-dependent increased sensitivity to ELT-specific CTLs. These results suggest that TPPII is involved in degradation of the ELT-peptide, and its overexpression may contribute to the immune escape of colon-carcinoma cells.

Epstein-Barr virus promotes genomic instability in Burkitt's lymphoma.

Epstein-Barr virus (EBV) has been implicated in the pathogenesis of human malignancies but the mechanisms of oncogenesis remain largely unknown. Genomic instability and chromosomal aberrations are hallmarks of malignant transformation. We report that EBV carriage promotes genomic instability in Burkitt's lymphoma (BL). Cytogenetic analysis of EBV- and EBV+ BL lines and their sublines derived by EBV conversion or spontaneous loss of the viral genome revealed a significant increase in dicentric chromosomes, chromosome fragments and chromatid gaps in EBV-carrying cells. Expression of EBV latency I was sufficient for this effect, whereas a stronger effect was observed in cells expressing latency III. Telomere analysis by fluorescent in situ hybridization revealed an overall increase of telomere size and prevalence of telomere fusion and double strand-break fusion in dicentric chromosomes from EBV+ cells. Phosphorylated H2AX, a reporter of DNA damage and ongoing repair, was increased in virus-carrying cells in the absence of exogenous stimuli, whereas efficient activation of DNA repair was observed in both EBV+ and EBV- cells following treatment with etoposide. These findings point to induction of telomere dysfunction and DNA damage as important mechanisms for EBV oncogenesis.

Is the activity of partially agonistic MHC:peptide ligands dependent on the quality of immunological help?

CD8(+) cytotoxic T lymphocytes (CTL) are important for the immunological control of infections and tumours. Engagement of the T-cell receptor (TCR) with major histocompatibility complex (MHC) class I/peptide complexes on antigen-presenting cells (APC) is the key interaction, which initiates the process of T-cell activation. Depending on the affinity of this interaction, different arrays of signalling pathways and functional outcomes can be activated in the specific T cells. Molecular alterations in the peptide bound to the MHC class I can lead to a lower affinity of the MHC:TCR interaction resulting in incomplete or qualitatively different T-cell responses. Altered peptide ligands (APL) exhibiting such activity are referred to as partial agonists and often occur naturally through genetic instability, which affects T-cell epitopes derived from rapidly mutating viruses or tumour-associated cellular antigens. Partial agonists are usually viewed as peptide variants, which escape efficient CTL recognition. Our recent data suggest that APL can not only trigger incomplete activation but also induce and modulate intrinsic T-cell programmes leading to the shut-off of specific CTL responses. This APL-induced suppression appears to be more prominent in the absence of immunological help, suggesting that under conditions of immune deregulation APL may actively inhibit CTL responses against infectious agents or tumours. In this review, we discuss experimental data supporting this model and possible role of APL-induced immunosuppression in different pathological conditions.

The ubiquitin-specific protease USP25 interacts with three sarcomeric proteins.

The biological functions of the more than one hundred genes coding for deubiquitinating enzymes in the human genome remain mostly unknown. The USP25 gene, located at 21q11.2, encodes three protein isoforms produced by alternative splicing. While two of the isoforms are expressed nearly ubiquituously, the expression of the longer USP25 isoform (USP25m) is restricted to muscular tissues and is upregulated during myogenesis. USP25m interacts with three sarcomeric proteins: actin alpha-1 (ACTA1), filamin C (FLNC), and myosin binding protein C1 (MyBPC1), which are critically involved in muscle differentiation and maintenance, and have been implicated in the pathogenesis of severe myopathies. Biochemical analyses demonstrated that MyBPC1 is a short-lived proteasomal substrate, and its degradation is prevented by over-expression of USP25m but not by other USP25 isoforms. In contrast, ACTA1 and FLNC appear to be stable proteins, indicating that their interaction with USP25m is not related to their turnover rate.

Efficacy of the treatment of dogs with leishmaniosis with a combination of metronidazole and spiramycin.

Twenty-seven dogs infected naturally with Leishmania infantum were used in a randomised controlled trial to compare the clinical and parasitological efficacy of an oral treatment with a combination of metronidazole and spiramycin (13 dogs) with the efficacy of conventional treatment with meglumine antimonate and allopurinol (14 dogs) as controls. In the test group one dog had to be withdrawn from the treatment because it developed pemphigus foliaceus; 10 of the dogs were clinically responsive but none was cured parasitologically. In the control group four dogs were withdrawn from the treatment because of side effects; eight of the dogs were clinically responsive but none was cured parasitologically. The control group showed signs of improvement after an average of 30 days, whereas the test group did not show signs of improvement until after an average of 45 days.

Differential regulation of MHC class-I-restricted and unrestricted cytotoxicity by the Us3 protein kinase of herpes simplex virus-1.

Modulation of cytotoxic responses by viral immunoevasins plays an important role in the establishment of latent and persistent viral infections. Together with MHC class-I-restricted CD8T-lymphocytes, non-MHC-restricted natural killer (NK) and lymphokine-activated killer (LAK) cells participate in this anti-viral control. The Us3 protein kinase of herpes simplex virus-1 (HSV-1) inhibits CD8T-cell cytotoxicity, which correlates with the inhibition of granzyme-B (GrB)-induced activation of pro-apoptotic Bid. We have investigated the effect of Us3 on NK and LAK cytotoxicity, because these effectors are believed to share common mechanisms for inducing cell death. We show that, in contrast to their lower sensitivity to CD8T-cell lysis, HSV-1-infected cells are lysed by NK cells or LAK cells as efficiently as the uninfected controls. Both CD8T and NK/LAK effectors were dependent on the activity of GrB and were efficiently blocked by means of treatment with a GrB inhibitor. However, unlike CD8T cells, LAK cells and NK cells failed to induce Bid cleavage, suggesting that various GrB downstream targets be involved in the induction of cell lysis. This finding explains their various sensitivities to viral modulation, which is likely to be important for the respective role of MHC-restricted and non-restricted effectors in the control of HSV-1 infection.

The herpes simplex virus-1 Us3 protein kinase blocks CD8T cell lysis by preventing the cleavage of Bid by granzyme B.

The Us3 kinase is part of the antiapoptotic arsenal that salvages herpes simplex virus (HSV)-1-infected cells from damage caused by different stimuli. We demonstrate that Us3 protects HSV-1-infected cells from lysis by MHC class I-restricted CD8T cells without affecting antigen presentation. Expression of Us3 was associated with inhibition of caspase activation and reduced cleavage of the proapoptotic protein Bid. Recombinant granzyme B (GrB) failed to cleave Bid in cytosolic extracts from Us3 positive cells, while recombinant Bid served as substrate for Us3 phosphorylation, suggesting that modification of Bid by Us3 blocks its processing by GrB. Our data illustrate a new strategy of viral escape, where modification of a cellular proapoptotic substrate may prevent lysis of the infected cells without affecting other T-cell functions.

Pharmacological disintegration of lipid rafts decreases specific tetramer binding and disrupts the CD3 complex and CD8 heterodimer in human cytotoxic T lymphocytes.

Accumulating evidence strongly supports the role of lipid rafts in the regulation of T-lymphocyte activation, but the organization and molecular composition of these cholesterol- and sphingolipid-rich membrane microdomains in different subsets of T cells remain poorly investigated. Here, we show that pharmacological disruption of lipid rafts in human CD8+ cytotoxic T-lymphocyte (CTL) clones disturbs the integrity of CD3 complex and CD8 heterodimer, without affecting the reactivity with T-cell receptor (TCR)-specific antibodies. This demonstrates that interaction with completely assembled CD3 complex is not required for the stable expression of TCR at the cell surface. The effect of raft disruption on CD3 and CD8 expression correlates with failure to bind specific tetrameric complexes by a proportion of surface TCR molecules. However, the interaction of specific tetramer with the rest of surface TCR pools appears to be unaffected, demonstrating that TCR-signalling complexes may differ in their requirement for cholesterol to stably maintain their composition and to rearrange for efficient tetramer binding. Together with previously published data, our results support the existence of molecular and/or structural heterogeneity of lipid rafts that may play an important role in controlling distinct functional properties of T-cell subsets.

Avoiding proteasomal processing: the case of EBNA1.

Ubiquitin/proteasome-dependent proteolysis is involved in the regulation of a large variety of cellular processes including cell cycle progression, tissue development and atrophy, flux of substrates through metabolic pathways, selective elimination of abnormal proteins and processing of intracellular antigens for major histocompatibility complex (MHC) class I-restricted T-cell responses. Many viruses tamper with this proteolytic machinery by encoding proteins that interact with various components of the pathway. A particularly interesting example of a viral protein that interferes with proteasomal processing is the Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1). EBNA1 contains an internal repeat exclusively composed of glycines and alanines that inhibits in cis the presentation of MHC class I-restricted T-cell epitopes and prevents ubiquitin/proteasome-dependent proteolysis in vitro and in vivo. The glycine-alanine repeat acts as a transferable element on a variety of proteasomal substrates and may therefore provide a new approach to the modification of cellular proteins for therapeutic purposes.