The role of the Kinin-Kallikrein System in chronic lymphocytic leukaemia

Background:Chronic lymphocytic leukaemia (CLL) is an incurable heterogeneous disease. The identification of reliable and cost effective biomarkers is therefore imperative. A comparative proteomic approach was previously employed to study protein expression changes associated with in vitro BCR ligation. Kininogen, a critical protein of Kinin-Kallikrein System (KKS) was found to be upregulated (p≥2) in 3/3 “high risk” clinical samples upon BCR stimulation. Both High and Low Molecular Weight Kininogens (HMWK and LMWK, respectively) serve as a substrate from which Plasma and Tissue Kallikreins liberate Kinins, which in turn act upon B1 and B2 kinin receptors. This project aimed to investigate the role of the KKS in CLL and to identify novel proteins which may have clinical relevance in this disease.Materials and Methods:KKS was investigated using CLL clinical samples and a range of methods such as immunoblotting, reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and flow cytometry as appropriate.Results:The up regulation of LMWK upon in vitro BCR stimulation was confirmed by immunoblotting in 3/3 CLL samples previously used in a discovery phase proteomics. In a pilot series of 52 unselected CLL samples, 71% demonstrated basal LMWK expression. A total of 18 of these samples were also analysed for LMWK transcript, which was not detected in any of these samples. The expression of Kallikrein 6 was confirmed in 25 CLL samples. The B1 and B2 receptors were identified in 15 and 3 CLL samples, respectively. An elevated Bradykinin level was demonstrated in 27/36 (75%) plasma samples from CLL patients and was found to be associated with untreated (p=0.039) stage A (p=0.03) CLL and increased Plasma Kallikrein (p=0.001).Discussion:It has been demonstrated for the first time that CLL cells express the components for KKS signalling pathway, which can be further investigated for clinical relevance

Epstein-Barr virus encoded nuclear protein EBNA-3 binds a novel human uridine kinase/uracil phosphoribosyltransferase

BACKGROUND: Epstein-Barr virus (EBV) infects resting B-lymphocytes and transforms them into immortal proliferating lymphoblastoid cell lines (LCLs) in vitro. The transformed immunoblasts may grow up as immunoblastic lymphomas in immuno-suppressed hosts. RESULTS: In order to identify cellular protein targets that may be involved in Epstein-Barr virus mediated B-cell transformation, human LCL cDNA library was screened with one of the transformation associated nuclear antigens, EBNA-3 (also called EBNA-3A), using the yeast two-hybrid system. A clone encoding a fragment of a novel human protein was isolated (clone 538). The interaction was confirmed using in vitro binding assays. A full-length cDNA clone (F538) was isolated. Sequence alignment with known proteins and 3D structure predictions suggest that F538 is a novel human uridine kinase/uracil phosphoribosyltransferase. The GFP-F538 fluorescent fusion protein showed a preferentially cytoplasmic distribution but translocated to the nucleus upon co-expression of EBNA-3. A naturally occurring splice variant of F538, that lacks the C-terminal uracil phosphoribosyltransferase part but maintain uridine kinase domain, did not translocate to the nucleus in the presence of EBNA3. Antibody that was raised against the bacterially produced GST-538 protein showed cytoplasmic staining in EBV negative Burkitt lymphomas but gave a predominantly nuclear staining in EBV positive LCL-s and stable transfected cells expressing EBNA-3. CONCLUSION: We suggest that EBNA-3 by direct protein-potein interaction induces the nuclear accumulation of a novel enzyme, that is part of the ribonucleotide salvage pathway. Increased intranuclear levels of UK/UPRT may contribute to the metabolic build-up that is needed for blast transformation and rapid proliferation

Upregulation of the Chemokine Receptor CCR2B in Epstein‒Barr Virus-Positive Burkitt Lymphoma Cell Lines with the Latency III Program

Abstract CCR2 is the cognate receptor to the chemokine CCL2. CCR2–CCL2 signaling mediates cancer progression and metastasis dissemination. However, the role of CCR2–CCL2 signaling in pathogenesis of B-cell malignancies is not clear. Previously, we showed that CCR2B was upregulated in ex vivo peripheral blood B cells upon Epstein‒Barr virus (EBV) infection and in established lymphoblastoid cell lines with the EBV latency III program. EBV latency III is associated with B-cell lymphomas in immunosuppressed patients. The majority of EBV-positive Burkitt lymphoma (BL) tumors are characterized by latency I, but the BL cell lines drift towards latency III during in vitro culture. In this study, the CCR2A and CCR2B expression was assessed in the isogenic EBV-positive BL cell lines with latency I and III using RT-PCR, immunoblotting, and immunostaining analyses. We found that CCR2B is upregulated in the EBV-positive BL cells with latency III. Consequently, we detected the migration of latency III cells toward CCL2. Notably, the G190A mutation, corresponding to SNP CCR2-V64I, was found in one latency III cell line with a reduced migratory response to CCL2. The upregulation of CCR2B may contribute to the enhanced migration of malignant B cells into CCL2-rich compartments

PRIMA-1MET induces nucleolar translocation of Epstein-Barr virus-encoded EBNA-5 protein

The low molecular weight compound, PRIMA-1MET restores the transcriptional transactivation function of certain p53 mutants in tumor cells. We have previously shown that PRIMA-1MET induces nucleolar translocation of p53, PML, CBP and Hsp70. The Epstein-Barr virus encoded, latency associated antigen EBNA-5 (also known as EBNA-LP) is required for the efficient transformation of human B lymphocytes by EBV. EBNA-5 associates with p53-hMDM2-p14ARF complexes. EBNA-5 is a nuclear protein that translocates to the nucleolus upon heat shock or inhibition of proteasomes along with p53, hMDM2, Hsp70, PML and proteasome subunits. Here we show that PRIMA-1MET induces the nucleolar translocation of EBNA-5 in EBV transformed B lymphoblasts and in transfected tumor cells. The PRIMA-1MET induced translocation of EBNA-5 is not dependent on the presence of mutant p53. It also occurs in p53 null cells or in cells that express wild type p53. Both the native and the EGFP or DSRed conjugated EBNA-5 respond to PRIMA-1MET treatment in the same way. Image analysis of DSRed-EBNA-5 expressing cells, using confocal fluorescence time-lapse microscopy showed that the nucleolar translocation requires several hours to complete. FRAP (fluorescence recovery after photobleaching) and FLIP (fluorescence loss in photobleaching) measurements on live cells showed that the nucleolar translocation was accompanied by the formation of EBNA-5 aggregates. The process is reversible since the aggregates are dissolved upon removal of PRIMA-1MET. Our results suggest that mutant p53 is not the sole target of PRIMA-1MET. We propose that PRIMA-1MET may reversibly inhibit cellular chaperons that prevent the aggregation of misfolded proteins, and that EBNA-5 may serve as a surrogate drug target for elucidating the precise molecular action of PRIMA-1MET

Impact of the Uridine–Cytidine Kinase Like-1 Protein and IL28B rs12979860 and rs8099917 SNPs on the Development of Hepatocellular Carcinoma in Cirrhotic Chronic Hepatitis C Patients—A Pilot Study

Background and objectives: The hepatitis C virus (HCV) is the major causative agent of hepatocellular carcinoma (HCC) in the western world. The efficacy of surveillance programs for early detection of HCC is not satisfactory: many tumors are diagnosed at the late, incurable stages. Therefore, there is a need in reliable prognostic markers for the proper follow-up of HCV-positive patients. The aim of the present study was to assess the prognostic value of the uridine–cytidine kinase-like protein 1 (UCKL-1), a putative oncoprotein, together with genetically determined polymorphisms in the interleukin 28B (IL28B) gene (rs12979860, rs8099917) in the development of HCC in HCV-positive cirrhotic patients. Materials and Methods: We included 32 HCV cirrhotic patients, 21 (65.6%) of whom had HCC. The expression of UCKL-1 was assessed in liver tissue sections, using immunohistochemistry. For IL28B rs12979860 and rs8099917 genotype analysis, the corresponding genomic regions were amplified by polymerase chain reaction (PCR) with appropriate primers. Results: We have found that UCKL-1 expression was significantly increased in HCC (p = 0.003). The presence of rs8099917 TT single-nucleotide polymorphism (SNP) elevated the chances of HCC manifestation more than sevenfold (OR = 7.3, p = 0.0273). The presence of rs12979860 CC SNP also heightened HCC chances more than sevenfold (OR = 7.5, p = 0.0765). Moreover, in the HCC group, a combination of IL28B rs12979860 non-TT and rs8099917 TT genotypes was observed more often, compared with the non-HCC group. Other combinations of IL28B rs12979860 and rs8099917 SNIPs were associated with a reduced risk of HCC development, approximately at the same extent. Conclusions: The presence of IL28B rs8099917 TT and rs12979860 CC SNPs, but not the intensity of UCKL-1 expression, is strongly associated with increased chances of HCC development in HCV-positive cirrhotic patients.This research was funded by Research Council of Lithuania, grant number TAP LU-1/2016

Mitochondrial ribosomal protein S18-2 is highly expressed in endometrial cancers along with free E2F1

ABSTRACT Endometrial cancer (EC) is one of the most frequent causes of cancer death among women in developed countries. Histopathological diagnosis and imaging techniques for EC are limited, thus new prognostic markers are needed to offer patients the best treatment and follow-up. In the present paper we showed that the level of mitochondrial ribosomal protein MRPS18-2 (S18-2) increased in EC compared with the normal endometrium and hyperplasia, based on a study of 42 patient biopsies. Importantly, high expression of free E2F1 in EC correlates well with high S18-2 expression. The EC cell line HEC- 1-A, which overexpresses S18-2 constitutively, showed an increased proliferation capacity in vitro and in vivo (in SCID mice). Moreover, pan-keratin, beta-catenin and E-cadherin signals are diminished in these cells, compared to the parental HEC-1-A line, in contrast to vimentin signal that is increased. This may be associated with epithelial-mesenchymal cell transition (EMT). We conclude that high expression of S18-2 and free E2F1, and low pan-keratin, beta-catenin, and E-cadherin signals might be a good set of prognostic markers for E

HIV-1-RNA Decay and Dolutegravir Concentrations in Semen of Patients Starting a First Antiretroviral Regimen

Background. The objective of this study was to quantify human immunodeficiency virus (HIV) type 1 RNA decay and dolutegravir (DTG) concentrations in the semen of HIV-infected patients receiving DTG-based first-line therapy

Persistent Roseoloviruses Infection in Adult Patients with Epilepsy

Background: Human herpesviruses (HHV)-6A, HHV-6B and HHV-7 are considered to be involved in the pathogenesis of epilepsy, a common neurological disorder. The objective of this study was to determine the association of roseoloviruses infection with epilepsy. Methods: 53 epilepsy patients and 104 ordinary blood donors were analyzed to determine presence of virus-specific antibodies by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), genomic sequences, viral load and gene expression by polymerase chain reactions (PCRs) and restriction analysis, HHV-6 protein expression by IFA and level of cytokines by ELISA. Results: Roseoloviruses genomic sequences in DNA samples from whole blood were found in 86.8% of patients versus 54.8% of controls and active infection was revealed only in patients with epilepsy (19.6% of roseolovirus-positive patients). Significantly higher viral load and more frequent gene expression was detected in patients compared to the controls. HHV-6-encoded protein expression was demonstrated in 53.3% of patients with previously detected HHV-6 DNA. Changes in level of cytokines were determined in patients with elevated viral load compared to the patients without elevated viral loads and to the controls. Conclusions: Results on frequent active HHV-6 and HHV-7 infection in epilepsy patient’ peripheral blood indicate on possible involvement of these viruses in the disease development

RMP-02/MTN-006: A Phase 1 Rectal Safety, Acceptability, Pharmacokinetic, and Pharmacodynamic Study of Tenofovir 1% Gel Compared with Oral Tenofovir Disoproxil Fumarate

This study was designed to assess the safety, acceptability, pharmacokinetic (PK), and pharmacodynamic (PD) responses to rectal administration of tenofovir (TFV) 1% vaginally formulated gel and oral tenofovir disoproxil fumarate (TDF). This study was designed as a phase 1, randomized, two-site (United States), double-blind, placebo-controlled study of sexually abstinent men and women. Eighteen participants received a single 300-mg exposure of oral TDF and were then randomized 2:1 to receive a single and then seven daily exposures of rectal TFV or hydroxyethyl cellulose (HEC) placebo gel. Safety endpoints included clinical adverse events (AEs) and mucosal safety parameters. Blood and colonic biopsies were collected for PK analyses and ex vivo HIV-1 challenge. No serious AEs were reported. However, AEs were significantly increased with 7-day TFV gel use, most prominently with gastrointestinal AEs (p=0.002). Only 25% of participants liked the TFV gel. Likelihood of use “if somewhat protective” was ∼75% in both groups. Indices of mucosal damage showed minimal changes. Tissue TFV diphosphate (TFV-DP) Cmax 30 min after single rectal exposure was 6–10 times greater than single oral exposure; tissue TFV-DP was 5.7 times greater following 7-day versus single rectal exposure. In vivo exposure correlated with significant ex vivo tissue infectibility suppression [single-rectal: p=0.12, analysis of covariance (ANCOVA) p=0.006; 7-day rectal: p=0.02, ANCOVA p=0.005]. Tissue PK–PD was significantly correlated (p=0.002). We conclude that rectal dosing with TFV 1% gel resulted in greater TFV-DP tissue detection than oral dosing with reduced ex vivo biopsy infectibility, enabling PK–PD correlations. On the basis of increased gastrointestinal AEs, rectally applied, vaginally formulated TFV was not entirely safe or acceptable, suggesting the need for alternative rectal-specific formulations

Identification of EBNA binding cellular proteins, using yeast two-hybrid system

Epstein-Barr virus (EBV) is a common herpesvirus that establishes life-long persistence in the human host through the elaborate regulation of different latency types. Latent EBV infection of resting B cells converts them into transformed cells that can develop into tumors in immune-compromised hosts. As such, EBV infection is a unique, well-defined in vitro system for malignant transformation. The latency- associated EBV proteins are the key factors for virus-induced cell transformation. To fully understand the molecular mechanisms that lead to virusinduced transformation, the cellular targets of the transforming viral proteins have to be identified. During the work summarized in this thesis, the following EBV encoded nuclear antigen (EBNA) binding proteins were identified: epsilon-subunit of the human chaperonin TCP- 1 complex; XAP2, the minor subunit of the arylhydrocarbon receptor (AhR) complex; a novel human uridine kinase/uracil phosphoribosyltransferase (for EBNA-3); and p14ARF (for EBNA-5). Epsilon-subunit of the human chaperonin TCP-1 complex is part of a huge protein complex that helps to fold the newly synthesized polypeptides. We mapped the interacting region to the apical domain of the epsilon-subunit the most likely site for recognition of newly translated polypeptides. We concluded that nascent EBNA-3 might receive help for its folding from the TCP-1 complex. Another protein that binds to EBNA-3 is XAP-2, which also interacts with the transformation associated X-protein encoded by the hepatitis B virus. We showed that EBNA-3 induces translocation of the cytoplasmic XAP-2 to the nucleus. We also found a previously unknown EBNA-3 binding protein, which was designated F538. We have shown that the predominantly cytoplasmic F538 relocated to the nucleus in the presence of EBNA-3, where these two proteins showed high levels of co-localization. A natural splice variant with the deleted C-terminus of F538 did not translocate to the nucleus. A SWISSModel 3D structure of F538 was constructed and compared with other known proteins. This raised the possibility that F538 is a novel human uridine kinase/uracil phosphoribosyltransferase (UK/UPRT). We suggested that EBNA-3 by direct protein- protein interaction induced the nuclear accumulation of this enzyme that is most likely part of the ribonucleotide salvage pathway. Increased intra-nuclear levels of UK/UPRT might contribute to the metabolic build-up that is needed for blast transformation and rapid proliferation. We found that EBNA-5 binds to p14ARF, one of the upstream regulators of the p53 pathway. We showed that EBNA-5 prolonged the survival of the p14ARFtransfected cells. We observed the accumulation of the p14ARF in extra-nuclear inclusions where it co-localized with p53, HDM2 and Hsp70. Formation of the p14ARF inclusions induced the translocation of PML bodies and 20S proteasome subunits. Co-expression of p14ARF and EBNA-5 led to the complete relocation of EBNA-5 into the p14ARF inclusions. We suggested that EBNA-5 might play a role in regulating the degradation of p14ARF-p53-HDM2 complexes. In conclusion, using the yeast two-hybrid system we found new cellular targets for EBV-encoded transformation associated latent proteins. Some of these target molecules participate in signal transduction (Xap-2); growth associated metabolic pathways (F538); cell cycle regulation and protein degradation (p14ARF)