Juraj Petrik

Hepatitis B virus genotypes and precore mutations in Scottish blood donors

Background and Objectives  This study was carried out to determine the frequency of hepatitis B virus (HBV) core promoter variants (nucleotide positions 1762, 1764) and precore variants (nucleotide position 1896) in hepatitis B surface antigen (HBsAg)‐positive Scottish blood donors. HBV genotypes present in this population were also indentified.

Hepatitis E virus in patients with decompensated chronic liver disease: a prospective UK/French study

In developed countries, hepatitis E is a porcine zoonosis caused by hepatitis E virus (HEV) genotype 3. In developing countries, hepatitis E is mainly caused by genotype 1, and causes increased mortality in patients with pre‐existing chronic liver disease (CLD).

Detection of HCV and HIV-1 antibody negative infections in Scottish and Northern Ireland blood donations by nucleic acid amplification testing.

Background and Objectives  To reduce the risk of transfusion‐transmissible viruses entering the blood supply, the nucleic acid amplification testing (NAT) was implemented to screen Scottish and Northern Irish blood donations in minipools. After 5 years of NAT for hepatitis C virus (HCV) and 2 years for human immunodeficiency virus‐1 (HIV‐1), the yield of serologically negative, nucleic acid positive ‘window donations’ and cost‐benefit of NAT is under review.

Polymers for the rapid and effective activation and aggregation of platelets

Platelets are responsible for plugging sites of vascular injury, where upon activation they spread out and become cross-linked, preventing further blood loss. It is desirable to control the activation process on demand for applications such as the rapid staunching of blood flow following trauma. Polymers are the material of choice in many biological areas, with physical properties that allow control of morphology as well as ease of functionalisation and production. Herein, polymer microarrays were used to screen a complex human fluid (platelet rich plasma) to identify polyacrylates that could be used to modulate platelet activation. Several polymers were identified which rapidly activated platelets as determined by CD61P binding and subsequent confirmation by scanning electron microcopy analysis. This approach enabled a direct comparison between the natural agonist collagen and synthetic polymers with respect to the activation status of the platelets as well as the number of bound platelets. Further investigations under physiological flow demonstrated that the static microarray experiments gave viable candidates for potential medical applications while specific protein binding to the polymers was identified as a possible mode of action. The approach demonstrates the ability of polymer microarrays to identify new polymers for specific biological activation events and in this case allowed the identification of materials that allowed higher levels of platelets to bind in advanced activation states than the natural standard collagen in static and flow studies.

A multiplexed protein microarray for the simultaneous serodiagnosis of human immunodeficiency virus/hepatitis C virus infection and typing of whole blood**

All donor blood samples must be tested pretransfusion to determine the donor blood type. Standard testing protocols require that assays be performed for important bloodborne pathogens such as hepatitis C, syphilis, hepatitis B, and human immunodeficiency virus. We have demonstrated proof of the concept that a protein microarray can type whole blood and detect antibody to significant pathogens simultaneously from the same donor blood sample. The data collected demonstrate the ability of the array to accurately type blood samples while also detecting the presence of antibodies against both human immunodeficiency virus and hepatitis C virus. In conclusion, we have successfully developed a platform capable of typing human whole blood samples, while at the same time testing for the presence of antibodies specific for human immunodeficiency virus/hepatitis C virus. The major benefits of this system are its amenability to expansion with additional assays, for example, rhesus typing and syphilis and/or hepatitis B virus detection, and also the adaptability of the assay to higher-throughput analysis, currently 16 individual samples per slide, but readily expandable to a 96-well format.

Sofosbuvir and Daclatasvir Anti–Viral Therapy Fails to Clear HEV Viremia and Restore Reactive T Cells in a HEV/HCV Co-Infected Liver Transplant Recipient

Figure 1. --Q4 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 Dear Editors: We read with great interest the recently published report ‘Sofosbuvir Inhibits Hepatitis E Virus Replication In Vitro and Results in an Additive Effect When CombinedWith Ribavirin’ by Dao Thi et al. The authors suggest that their important in vitro observations require a clinical proof-of-concept study to confirm that sofosbuvir might be considered as therapy for treatment of chronic hepatitis E. We have recently treated a patient with chronic hepatitis C (HCV genotype 3) and hepatitis E (HEV genotype 3) coinfection post liver transplantation (immunosuppressed with tacrolimus and azathioprine) with a 12-week course of daily sofosbuvir (400mg) and daclatasvir (60mg), in view of previous ribavirin intolerance. In contrast with the in vitro observations, we report that sofosbuvir without ribavirin was not effective in treating chronic HEV or restoring HEV specific reactive T cells in an immunocompromised patient. In our patient, after commencing treatment with the above regimen, serum alanine aminotransferase normalized after one week and HCV RNA was undetectable after 4 weeks of treatment. However, antiviral treatment had negligible effect on HEV viremia, as measured by quantitative HEV RNA in serum (Figure 1A). Variable T-cell immunity to HEV infection has been previously reported; although in one report HEV specific T cell responses could be detected in resolved HEV infection controls, no IFN-g response was observed in chronic HEV infection when cells were stimulated with ORF-2 and 3 peptides suggesting an impairment of adaptive responses. In our patient, HEV specific Tcell responses were monitored before and during antiviral treatment using HEV genotype 3 ORF-2 and 3 overlapping peptide stimulation of peripheral blood mononuclear cell (PBMC) cultures overnight and measurement of intracellular IL-2, TNF-a and IFN-g by flow cytometry. Modest CD4 and CD8 T-cell cytokine responses were detected in response to HEV peptide stimulation. In the pre-treatment samples the relative number of cells found reactive to HEV peptide stimulation was raised in TNF-a secreting CD4 andCD8T cells and in IL-2 producing CD8 cells. CD4 and CD8 T cells expressing elevated IFN-g and TNF-a were observed at 1 month of therapy. After 2 months of treatment, cells were non-reactive to HEV peptide stimulation. Alteration in the profile of cytokine response to HEV peptide stimulation over treatment may represent a change in tissue mobilization of reactive T cells with lymphopenia

Role of the HBx oncoprotein in carbonic anhydrase 9 induction

Carbonic anhydrase 9 (CA9), as one of the most hypoxia‐responsive genes, has been associated almost exclusively with hypoxic tumors. Its principal role is in pH regulation which helps tumor cells overcome intracellular acidosis and survive extended periods of time with low oxygen. Hypoxia‐inducible factor 1 (HIF‐1) is the main transcriptional activator of CA9. Hepatitis B virus X protein (HBx) has been shown to increase the transcriptional activity of HIF‐1. HBx is often expressed from the gene integrated in the hepatocytes infected persistently and contributes significantly to alterations in host gene expression that can lead to the development of hepatocellular carcinoma (HCC) associated with Hepatitis B virus (HBV). The aim of this study was to determine the effect of HBx on expression of CA9. Transient transfection of HBx led to an increase in the expression of CA9 as assessed by RT‐PCR and Western blotting. HBx was able to increase CA9 promoter activity significantly in several cell lines. The effect was mediated via HIF‐1 and a functional HRE element located −10/−3 bp upstream of the CA9 transcription initiation site. These data suggest that CA9 may be involved in the development of HCC by contributing to the survival of hepatocytes infected with HBV in liver tissue with fibrosis. J. Med. Virol. 82:32–40, 2010.

Detection of HTLV-I and -II in Scottish blood donor samples and archive donations

Background and Objectives  Positive samples identified during routine serological screening for HCV (hepatitis C virus), HBV (hepatitis B virus) and HIV (human immunodeficiency virus) are confirmed by nucleic acid testing in the SNBTS (Scottish National Blood Transfusion Service) PCR Reference laboratory. Serological screening for HTLV‐I (human T‐cell lymphotropic virus type I) and ‐II was implemented in Scotland in November 2002, at which time a PCR assay was not available for confirmation. Our aim was to develop a real‐time PCR assay that could be used for the confirmation of samples showing HTLV‐I serological positive or indeterminate reactivity and to investigate whether a serologically silent carrier status exists (‘Tax’ only) in the Scottish donor population.

Mesenchymal stromal cells as multifunctional cellular therapeutics - a potential role for extracellular vesicles.

Mesenchymal stromal cells (MSCs), multipotent cells present in tissues throughout the body, can reconstitute adipogenic, osteogenic and chondrogenic tissues, but are also of great interest as mediators of immune modulation and suppression. MSCs are able to improve transplant engraftment, treat graft versus host disease and suppress T cell responses and therefore have great potential as therapeutic agents. Their immune modulatory capacity is mediated through both cell-to-cell contact and cytokine secretion, but it is becoming clear that extracellular vesicles (EV) produced by MSC also possess immunomodulatory properties. These vesicles are easy to prepare and store, do not carry nuclear material and cannot form tumours, and therefore also represent a highly desirable therapeutic agent. This review outlines the formation and characterisation of extracellular vesicles, the reported function of MSC-EVs in vitro and in vivo, and addresses some of the emerging issues with nomenclature, EV therapeutic dose and tissue source. The development of GMP-grade production protocols and effective characterisation of MSC extracellular vesicles is essential to their successful use as immune modulating therapeutic agents, and this review outlines the current status of the research in this area.

Acute hepatitis C virus seroconversion in a Scottish blood donor: HCV antigen is not comparable with HCV nucleic acid amplification technology screening

Background and Objectives  This study was conducted to analyse the usefulness of hepatitis C virus (HCV) core antigen tests for the confirmation of HCV infection in a donor presenting as nucleic acid amplification technology (NAT) positive but negative for antibodies to HCV (anti‐HCV).