An Investigation of the Complexes Formed Between the Hepatitis C Virus E1 and E2 Glycoproteins

Hepatitis C virus (HCV) encodes two glycoproteins, termed El and E2, which are presumed to constitute the proteinaceous components of the virion envelope. From a number of studies, El and E2 associate to form a complex however, the nature of the types of interaction between the proteins is the subject of controversy. Two types of complex, termed the native and aggregated forms, have been identified. In native complexes, El and E2 associate by non-covalent interactions while disulphide bonds form intermolecular links between the glycoproteins in aggregated complexes. The aim of my project was to characterise the complex formed by El and E2 from a local genotype 1a strain, called strain Glasgow. Studies were later expanded to include analysis of the glycoproteins from strain H77, another genotype 1a strain that is infectious in chimpanzees. Due to the lack of efficient virus replication in tissue culture, the Semliki Forest virus vector was used for expression of the glycoproteins. This involved the introduction of in vitro transcribed RNA molecules into mammalian cells by electroporation and conditions to optimise the system were ascertained. In the absence of sufficient immunological reagents during the early phases of the project, a histidine tag was inserted at the N-terminus of El from strain Glasgow (El his) to provide a means for purification by metal chelate chromatography. This method co-eluted E2 with El his which was later verified by the ability of polyclonal sera against E2 to co-precipitate El with E2. Thus, a heteromeric complex formed between El and E2 from strain Glasgow, a finding which is in agreement with studies on the glycoproteins from other strains. Using the available reagents for identifying El and E2 and by analysis under reducing and non-reducing electrophoretic conditions, most of the complex was in the form of disulphide-linked aggregates with little detectable native complex. The requirement for disulphide bonds to enable El and E2 association was examined by treatment of cells synthesising the proteins with the reducing agent dithiothreitol (DTT). Addition of DTT to cells did not prevent cleavage and processing of the glycoproteins. Significantly, the intermolecular disulphide links in aggregates were disrupted by DTT but El and E2 continued to associate, indicating that both covalent and non-covalent intermolecular interactions occur in the aggregated complex. El and E2, synthesised in the presence of DTT, also formed a complex and therefore disulphide bond formation is not a prerequisite for interactions between the proteins. Comparisons between strain Glasgow and strain H77 showed that native E1E2 complexes were more readily detected from strain H77. It is proposed that this may be a consequence of alterations in the number of glycosylation sites in E2 between the strains as well as changes in amino acid sequences in El and E2. Moreover, El from strain Glasgow had reduced mobility as compared to strain H77 El protein which could be not be accounted by changes in the relative sizes of the nascent proteins or glycosylation patterns. Two deletion mutants in El from both strains also displayed higher apparent molecular weights than predicted. The behaviour of these mutant proteins may result from the deletion of hydrophobic segments in El. However, the apparent molecular weight difference between El from strain Glasgow and strain H77 could not be explained. In an attempt to identify regions of El and E2 in strain Glasgow that may account for the high level of aggregation by these proteins, deletion mutants were created in El and E2; for comparative purposes, an identical set of deletion mutants were created also for El from strain H77. Immunoprecipitation studies revealed that multiple regions were involved in interactions between El and E2. No single deletion was able to completely abolish E1E2 interactions although native complex formation in strain H77 was decreased on removal of sequences from El. Further analysis of complexes and the role of disulphide bond formation was undertaken by constructing a series of mutants in which the cysteine residues in El were replaced with either serine or alanine. With each of these mutants, El and E2 continued to associate but the amounts of native, non-aggregated E2 were reduced to undetectable levels. Hence, native complexes of El and E2 were highly sensitive to changes in either protein such that alteration even at a single cysteine residue was sufficient to reduce the abundance of native complex. Examination of El expressed in the presence and absence of E2 provided a set of criteria for assessing the folding status of the protein. In the absence of E2, El remained in the reduced state and formed homo-oligomeric molecules that could be identified under reducing electrophoretic conditions. The formation and stability of these homo-oligomers were not entirely dependent on disulphide bond formation. Moreover, there was an increase in the quantities of glycosylated forms of E1 in the absence of E2

Tests for diagnosing and monitoring non-alcoholic fatty liver disease in adults

Non-alcoholic fatty liver disease (NAFLD) is a metabolic liver disease that encompasses a spectrum of progressive pathological conditions, ranging from non-alcoholic fatty liver (NAFL) to steatohepatitis (NASH), fibrosis, and cirrhosis. When hepatic steatosis occurs in the absence of excessive alcohol consumption and other recognised causes of liver fat, and with cardiometabolic risk factors, it is likely that the diagnosis is NAFLD as NAFLD is principally a diagnosis of exclusion. NAFLD is the commonest liver disease in high income countries, and is estimated to affect at least 25%-30% of adults in the general population and up to 70%-90% of persons with obesity or type 2 diabetes.1NAFLD is associated not only with liver related morbidity and mortality, but also with an increased risk of developing cardiovascular disease and type 2 diabetes.2 3 Liver biopsy remains the reference method for diagnosing NAFLD, as it provides the most accurate assessment of disease grade and stage.4 5 However, undertaking a liver biopsy is costly, risky, and potentially painful. Moreover, interpretation of NAFLD severity can be compromised by sampling errors in what can be a patchy disease.6 7 In this article, we discuss the diagnosis of NAFLD, testing for liver fibrosis in those with NAFLD, and monitoring of those most likely to develop advanced liver disease. We examine the evidence and guidelines from Europe, the United States, and the UK\u2019s National Institute for Health and Care Excellence (NICE)8-10 for and against the use of specific diagnostic tests. Our approach to the use of liver ultrasound in establishing a diagnosis of hepatic steatosis differs from the recent NICE guidelines,10 but complements British Society of Gastroenterology guidelines.11 Treatment options are beyond the scope of this article

Mycobacterial mutants with defective control of phagosomal acidifi -cation. PLoS Pathog

The pathogenesis of mycobacterial infection is associated with an ability to interfere with maturation of the phagosomal compartment after ingestion by macrophages. Identification of the mycobacterial components that contribute to this phenomenon will allow rational design of novel approaches to the treatment and prevention of tuberculosis. Microarray-based screening of a transposon library was used to identify mutations that influence the fate of Mycobacterium bovis bacille Calmette-Gué rin (BCG) following uptake by macrophages. A screen based on bacterial survival during a 3-d infection highlighted genes previously implicated in growth of Mycobacterium tuberculosis in macrophages and in mice, together with a number of other virulence genes including a locus encoding virulenceassociated membrane proteins and a series of transporter molecules. A second screen based on separation of acidified and non-acidified phagosomes by flow cytometry identified genes involved in mycobacterial control of early acidification. This included the KefB potassium/proton antiport. Mutants unable to control early acidification were significantly attenuated for growth during 6-d infections of macrophages. Early acidification of the phagosome is associated with reduced survival of BCG in macrophages. A strong correlation exists between genes required for intracellular survival of BCG and those required for growth of M. tuberculosis in mice. In contrast, very little correlation exists between genes required for intracellular survival of BCG and those that are up-regulated during intracellular adaptation of M. tuberculosis. This study has identified targets for interventions to promote immune clearance of tuberculosis infection. The screening technologies demonstrated in this study will be useful to the study of pathogenesis in many other intracellular microorganisms

Genetic determination of the effect of post-translational modification on the innate immune response to the 19 kDa lipoprotein of Mycobacterium tuberculosis

<p>Abstract</p> <p>Background</p> <p>The 19 kDa lipoprotein of <it>Mycobacterium tuberculosis </it>(MTB) is an important target of the innate immune response. To investigate the effect of post-translation modification of this protein on innate recognition in the context of the whole bacillus, we derived a recombinant <it>M. tuberculosis </it>H37Rv that lacked the 19 kDa gene (Δ19) and complemented this strain by reintroduction of the 19 kDa gene into the chromosome as a single copy to produce Δ19::19. We also reintroduced the 19 kDa gene in two modified forms that lacked motifs for acylation (Δ19::19NA) and <it>O</it>-glycosylation (Δ19::19NOG).</p> <p>Results</p> <p>Both acylation and <it>O</it>-glycosylation were necessary for the protein to remain within the cell. IL-1 Beta secretion from human monocytes was significantly reduced by deletion of the 19 kDa gene (p < 0.02). Complementation by the wild type, but not the mutagenised gene reversed this phenotype. The effect of deletion and complementation on IL-12p40 and TNF secretion was less marked with no statistically significant differences between strains. Although deletion of the 19 kDa reduced apoptosis, an effect that could also only be reversed by complementation with the wild type gene, the results were variable between donors and did not achieve statistical significance.</p> <p>Conclusion</p> <p>These results confirm in the context of the whole bacillus an important role for post-translational modification of the 19 kDa on both the cellular location and immune response to this protein.</p

Clinical deterioration during antituberculosis treatment in Africa: Incidence, causes and risk factors

BACKGROUND:HIV-1 and Mycobacterium tuberculosis cause substantial morbidity and mortality. Despite the availability of antiretroviral and antituberculosis treatment in Africa, clinical deterioration during antituberculosis treatment remains a frequent reason for hospital admission. We therefore determined the incidence, causes and risk factors for clinical deterioration. METHODS: Prospective cohort study of 292 adults who initiated antituberculosis treatment during a 3-month period. We evaluated those with clinical deterioration over the following 24 weeks of treatment. RESULTS: Seventy-one percent (209/292) of patients were HIV-1 infected (median CD4+: 129 cells/muL [IQR:62-277]). At tuberculosis diagnosis, 23% (34/145) of HIV-1 infected patients qualifying for antiretroviral treatment (ART) were receiving ART; 6 months later, 75% (109/145) had received ART. Within 24 weeks of initiating antituberculosis treatment, 40% (117/292) of patients experienced clinical deterioration due to co-morbid illness (n = 70), tuberculosis related illness (n = 47), non AIDS-defining HIV-1 related infection (n = 25) and AIDS-defining illness (n = 21). Using HIV-1 uninfected patients as the referent group, HIV-1 infected patients had an increasing risk of clinical deterioration as CD4+ counts decreased [CD4+>350 cells/muL: RR = 1.4, 95% CI = 0.7-2.9; CD4+:200-350 cells/muL: RR = 2.0, 95% CI = 1.1-3.6; CD4+<200 cells/muL: RR = 3.0, 95% CI = 1.9-4.7]. During follow-up, 26% (30/117) of patients with clinical deterioration required hospital admission and 15% (17/117) died. Fifteen deaths were in HIV-1 infected patients with a CD4+<200 cells/muL. CONCLUSIONS: In multivariate analysis, HIV-1 infection and a low CD4+ count at tuberculosis diagnosis were significant risk factors for clinical deterioration and death. The initiation of ART at a CD4+ count of <350 cells/muL will likely reduce the high burden of clinical deterioration

Tandem mass tag-based quantitative proteomic profiling identifies candidate serum biomarkers of drug-induced liver injury in humans

Diagnosis of drug-induced liver injury (DILI) and its distinction from other liver diseases are significant challenges in drug development and clinical practice. We used Tandem Mass Tag-labeled quantitative proteomics detecting 2323 proteins in a cohort comprising patients with DILI [at onset (DO) and follow-up (DF)], acute non-DILI [at onset (NDO) and follow-up (NDF)], and healthy volunteers (HV) to identify novel serum biomarkers. Thirteen candidates selected based on differential expression, liver-specific expression, and mechanistic relevance to liver pathology, were assessed in confirmatory and replication cohorts of HV (n=94), DO (n=123), DF (n=110), NDO (n=58) and NDF (n=37) using a targeted label-free SureQuant assay. Area under the receiver operating characteristic curve (AUC) ranging between 0.94 and 0.99 across cohorts for five of these biomarkers, reflected differentiation between DO and HV with high sensitivity and specificity. In addition, fructose-1,6-bisphosphatase 1 distinguished NDO from DO (AUC: 0.75 and 0.65) on its own or in combination with glutathione S-transferase A1 and leukocyte cell derived chemotaxin 2 (AUC: 0.78 and 0.68). These can potentially differentiate DILI and acute liver injury from non-drug etiologies

An Update on Clinical Trials and Potential Therapeutic Strategies in T-Cell Acute Lymphoblastic Leukemia

Current therapies for T-cell acute leukemia are based on risk stratification and have greatly improved the survival rate for patients, but mortality rates remain high owing to relapsed disease, therapy resistance, or treatment-related toxicities/infection. Patients with relapsed disease continue to have poor outcomes. In the past few years, newer agents have been investigated to optimize upfront therapies for higher-risk patients in the hopes of decreasing relapse rates. This review summarizes the progress of chemo/targeted therapies using Nelarabine/Bortezomib/CDK4/6 inhibitors for T-ALL in clinical trials and novel strategies to target NOTCH-induced T-ALL. We also outline immunotherapy clinical trials using monoclonal/bispecific T-cell engaging antibodies, anti-PD1/anti-PDL1 checkpoint inhibitors, and CAR-T for T-ALL therapy. Overall, pre-clinical studies and clinical trials showed that applying monoclonal antibodies or CAR-T for relapsed/refractory T-ALL therapy is promising. The combination of target therapy and immunotherapy may be a novel strategy for T-ALL treatment

Multi-drug approaches to NASH: what’s in the development pipeline?

Introduction: The pandemic of obesity over the last two decades has triggered a rise in the prevalence of nonalcoholic fatty liver disease (NAFLD). NAFLD is associated with liver-related and cardiovascular complications. Despite this, the first licensed drug for NAFLD is yet to be approved. Given the scale of the problem and unmet needs, there is a myriad of agents in the development pipeline.Areas covered: We discuss promising agents in early phase clinical trials and categorize these agents based on their action on steatosis, steatohepatitis, and fibrosis. Furthermore, given the multisystemic nature of NAFLD, we consider the effects of these agents on the liver, their cardiometabolic effects, and the potential future strategies of combination therapies.Expert opinion: The paradigm for the ideal drug is the targeting of both steatohepatitis and fibrosis and the amelioration of cardiometabolic risk factors. New drugs that confer benefit in nonalcoholic steatohepatitis (NASH) must also be tested for their effects on type 2 diabetes mellitus and cardiovascular disease. The treatment of NASH will become analogous to the treatment of hypertension; it is very likely that multiple classes of drugs targeting different mechanistic pathways will be necessary because no single agent is likely to control all aspects of this complex liver disease

Update on cardiovascular risk in non-alcoholic fatty liver disease

PURPOSE OF REVIEW: To summarize recent evidence demonstrating increased cardiovascular disease (CVD) risk, and how CVD risk may be reduced, in patients with nonalcoholic fatty liver disease (NAFLD). RECENT FINDINGS: NAFLD is a multisystem disease, defined by a spectrum of liver fat-associated conditions extending from simple steatosis, to inflammation, fibrosis and cirrhosis. NAFLD not only increases the risk of liver morbidity and mortality but also increases the risk of CVD morbidity and mortality and is associated with recognized CVD risk factors such as hypertension, atherogenic dyslipidaemia, type 2 diabetes mellitus and chronic kidney disease. Evidence suggests that the liver fibrosis stage may be a strong CVD risk factor. Lifestyle measures (e.g. weight loss and increased physical activity) are effective in improving CVD risk factors. Hypoglycaemic agents, such as the peroxisome proliferator-activated receptor gamma agonist pioglitazone and the glucagon-like peptide-1 receptor agonist liraglutide, reduce cardiovascular risk and may improve liver histology. Statin and antihypertensive treatments are well tolerated and currently it is unclear whether novel antifibrotic drugs will reduce CVD risk. SUMMARY: Assessment and treatment of increased cardiovascular risk is important in patients with NAFLD. If not contra-indicated, pioglitazone or a glucagon-like peptide 1 agonist should be considered and may benefit both CVD risk and early liver disease.</p

Diabetes is associated with increased risk of hepatocellular carcinoma in non-alcoholic steatohepatitis with cirrhosis – implications for surveillance and future pharmacotherapy

Hepatocellular carcinoma (HCC) is the most common liver-related complication seen in patients with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) (1). NASH is strongly associated with type 2 diabetes mellitus (T2DM) but it has also been apparent for a number of years that T2DM is associated with an increased risk of HCC independent of the presence of NASH (2)