Platelet function testing in patients with post-operative tonsillectomy bleeding may be a useful early identifier of inherited platelet function disorders.

BACKGROUND: Post-tonsillectomy bleeding is the most frequent complication of tonsillectomy. Inherited platelet function disorders have an estimated prevalence of 1 per cent. Any association between post-tonsillectomy bleeds and undiagnosed inherited platelet function disorders has not been investigated before. OBJECTIVES: To assess the prevalence of inherited platelet function disorders in a cohort of post-tonsillectomy bleed patients. METHODS: An observational cohort study was conducted using hospital digital records. Platelet function analyser 100 ('PFA-100') closure time was tested on post-tonsillectomy bleed patients who presented to hospital. RESULTS: Between 2013 and 2017, 9 of 91 post-tonsillectomy bleed patients who underwent platelet function analyser 100 testing (9.89 per cent) had positive results. Five patients (5.49 per cent) had undiagnosed inherited platelet function disorders. Four patients had false positive results secondary to a non-steroidal anti-inflammatory drug effect (specificity of 95.3 per cent) proven by repeat testing six weeks later, off medication. The false negative rate was 0 per cent. CONCLUSION: The prevalence of inherited platelet function disorders in our post-tonsillectomy bleed cohort is five-fold higher than in the general population. Platelet function analyser 100 testing when patients present with a post-tonsillectomy bleed allows management of their inherited platelet function disorder.

Short-term changes observed in multiparametric liver MRI following therapy with direct-acting antivirals in chronic hepatitis C virus patients.

METHODS: We applied multiparametric MRI to assess changes in liver composition, perfusion and blood flow in 17 patients before direct-acting antiviral (DAA) therapy and after treatment completion (within 12 weeks of last DAA tablet swallowed). RESULTS: We observed changes in hepatic composition indicated by a reduction in both liver longitudinal relaxation time (T1, 35 ± 4 ms), transverse relaxation time (T2, 2.5 ± 0.8 ms; T2* 3.0 ± 0.7 ms), and liver perfusion (28.1 ± 19.7 ml/100 g/min) which we suggest are linked to reduced pro-inflammatory milieu, including interstitial oedema, within the liver. No changes were observed in liver or spleen blood flow, splenic perfusion, or superior mesenteric artery blood flow. CONCLUSION: For the first time, our study has shown that treatment of HCV with DAAs in patients with cirrhosis leads to an acute reduction in liver T1, T2 and T2* and an increase in liver perfusion measured using MR parameters. The ability of MRI to characterise changes in the angio-architecture of patients with cirrhosis after intervention in the short term will enhance our understanding of the natural history of regression of liver disease and potentially influence clinical decision algorithms. KEY POINTS: • DAAs have revolutionised the treatment of hepatitis C and achieve sustained virological response in over 95% of patients, even with liver cirrhosis. • Currently available non-invasive measures of liver fibrosis are not accurate after HCV treatment with DAAs, this prospective single-centre study has shown that MRI can sensitively measure changes within the liver, which could reflect the reduction in inflammation with viral clearance. • The ability of MRI to characterise changes in structural and haemodynamic MRI measures in the liver after intervention will enhance our understanding of the progression/regression of liver disease and could potentially influence clinical decision algorithms.

Mannan binding lectin-associated serine protease 1 is induced by hepatitis C virus infection and activates human hepatic stellate cells.

Mannan binding lectin (MBL)-associated serine protease type 1 (MASP-1) has a central role in the lectin pathway of complement activation and is required for the formation of C3 convertase. The activity of MASP-1 in the peripheral blood has been identified previously as a highly significant predictor of the severity of liver fibrosis in hepatitis C virus (HCV) infection, but not in liver disease of other aetiologies. In this study we tested the hypotheses that expression of MASP-1 may promote disease progression in HCV disease by direct activation of hepatic stellate cells (HSCs) and may additionally be up-regulated by HCV. In order to do so, we utilized a model for the maintenance of primary human HSC in the quiescent state by culture on basement membrane substrate prior to stimulation. In comparison to controls, recombinant MASP-1 stimulated quiescent human HSCs to differentiate to the activated state as assessed by both morphology and up-regulation of HSC activation markers α-smooth muscle actin and tissue inhibitor of metalloproteinase 1. Further, the expression of MASP-1 was up-regulated significantly by HCV infection in hepatocyte cell lines. These observations suggest a new role for MASP-1 and provide a possible mechanistic link between high levels of MASP-1 and the severity of disease in HCV infection. Taken together with previous clinical observations, our new findings suggest that the balance of MASP-1 activity may be proinflammatory and act to accelerate fibrosis progression in HCV liver disease.

Clinical trial: a primary-care-based model for the delivery of anti-viral treatment to injecting drug users infected with hepatitis C.

BACKGROUND: Injecting drug use is the main risk factor for hepatitis C virus (HCV) infection. Secondary-care-based strategies for the management of HCV do not effectively target this vulnerable population. AIMS: To evaluate the feasibility, safety and efficacy of a primary-care-based model for the delivery of HCV services including anti-viral therapy to injecting drug users. METHODS: A partnership between a clinical nurse specialist employed by, and working under the supervision of, a secondary-care-based hepatitis service and drug workers and general practitioners. Three hundred and fifty-three clients attending opiate substitution clinics in primary care were evaluated. Outcomes were: number of new diagnoses of HCV infection, number of clients assessed as suitable for anti-viral treatment, and number of patients treated. RESULTS: 174 HCV antibody positive clients were identified. Of these, 124 were chronically infected with HCV of whom only six had been previously identified. Of 118 new chronically-infected individuals, 86 entered the care pathway, 43 were assessed as suitable for anti-viral treatment and 30 have so far been treated. Outcomes of anti-viral treatment are comparable with those obtained in secondary care settings. CONCLUSION: A primary-care-based model offers a new paradigm for the treatment of HCV in injecting drug users.

Response rates to combination therapy for chronic HCV infection in a clinical setting and derivation of probability tables for individual patient management.

Evidence for efficacy of established treatment guidelines for chronic hepatitis C virus (HCV) disease is based on multinational randomized controlled trials (RCTs). Strategies for managing HCV, however, require an assessment of the effectiveness of intervention in routine clinical practice. We report the outcomes of combination therapy in a large cohort of HCV-infected individuals in the UK. A total of 347 (113 genotype 1, 234 genotype non-1) patients were treated with pegylated interferon and ribavirin according to current guidelines. Forty-two (37.2%) of those with genotype 1 infection and 164 (70.1%) with genotype non-1 infection achieved sustained viral response (SVR). Thirty-nine (11%) patients withdrew from treatment. In addition to viral genotype, factors predictive of a response to therapy were age at start of treatment and disease stage on pretreatment liver biopsy. Multivariate regression analysis demonstrated that the effects of age [odds ratio 0.5; 95% confidence interval (0.31-0.82) per 10-year increment (P = 0.006)] were confined to genotype 1 disease. In order to further inform the management of the individual patient, a multivariate logistic model was used to predict the probability of SVR for subgroups defined by disease stage, genotype and age at commencement of therapy. This model revealed striking differences in predicted response rates between subgroups and provided a strong rationale for early treatment, particularly for those with genotype 1 disease. Our study demonstrates that results comparable with those of RCTs can be achieved in clinical practice, and suggests that prediction of response rates based on probability modelling will provide a valuable adjunct to individual patient management.

A closer look at water-related geologic activity on Mars.

Water has supposedly marked the surface of Mars and produced characteristic landforms. To understand the history of water on Mars, we take a close look at key locations with the High-Resolution Imaging Science Experiment on board the Mars Reconnaissance Orbiter, reaching fine spatial scales of 25 to 32 centimeters per pixel. Boulders ranging up to approximately 2 meters in diameter are ubiquitous in the middle to high latitudes, which include deposits previously interpreted as finegrained ocean sediments or dusty snow. Bright gully deposits identify six locations with very recent activity, but these lie on steep (20 degrees to 35 degrees) slopes where dry mass wasting could occur. Thus, we cannot confirm the reality of ancient oceans or water in active gullies but do see evidence of fluvial modification of geologically recent mid-latitude gullies and equatorial impact craters.

Clinical pathways for patients with newly diagnosed hepatitis C - what actually happens.

Management of hepatitis C virus (HCV)-infected individuals requires referral to specialist care. To determine whether patients newly diagnosed as anti-HCV positive are appropriately referred for further investigation and management, and if not, to determine why not. We studied patients tested for antibodies to HCV by Nottingham Public Health Laboratory in a 2-year period (2000-2002). The progress of newly diagnosed anti-HCV positive patients into specialist clinics for further management was documented. For patients not referred for specialist care, a questionnaire was sent to the clinician requesting the initial anti-HCV test, to identify reasons for nonreferral. Eleven thousand one hundred and seventy-seven patients were tested for anti-HCV. Two hundred and fifty-six (2.3%) were newly diagnosed as being anti-HCV positive. Two per cent of samples sent from primary care were anti-HCV positive, compared to 18.8, 18.9 and 1.3% sent from prison, drug and alcohol units, and secondary care, respectively. About 64.3% of positive patients diagnosed in primary care were referred to specialist care, compared to 18.4, 42.4 and 62.6% of patients diagnosed in the other three settings. One hundred and twenty-five (49%) newly diagnosed patients were referred appropriately for further management. 68 of these attended clinic, 45 underwent liver biopsy and 26 (10%) began treatment. One hundred and thirty-one patients (51%) were not referred. In 54 cases, there was no evidence that the anti-HCV positive result reached the patient. In 15, referral was considered but rejected, and 20 patients were referred to non-HCV-specialists (their general practitioners or to genito-urinary medicine). Hence less than 50% of newly diagnosed anti-HCV positive patients are referred to an appropriate clinic for further investigation and management. Reasons for this are multifarious and complex, reflecting both systems failure and patient choice. Unless these are understood and addressed, the Department of Health Hepatitis C Strategy (2002) and Action Plan for England (2004) will fail to achieve their intended objectives.

Caspase-independent killing of Burkitt lymphoma cell lines by rituximab.

Caspase-independent cell death may have a critical role to play in the therapeutic destruction of tumours. Recently it has been suggested that one of the mechanisms by which rituximab, a therapeutic anti-CD20 antibody, kills B cells is caspase-independent. In this study we show that rituximab can induce death in a variety of Burkitt lymphoma derived cell lines. Rituximab-treated cells show leakage of adenylate kinase, surface expression of phosphatidylserine, upregulation of the cellular stress protein HSP70, phosphorylation of the survival protein Akt, and depolarisation of the mitochondrial membrane but no loss of cytochrome c or apoptosis inducing factor. Caspase inhibitors do not block these events. In support of these data there is no cleavage of caspases 3, 8 and 9, poly(ADP-ribose) polymerase, BH3 interacting domain death agonist or genomic DNA. Morphologically, cells show nuclear enlargement and cytoplasmic vacuolisation. Triggering of receptor mediated death in CD95 responsive lines results in "classical" apoptosis indicating that the internal machinery necessary for apoptosis is intact in these lines. The results suggest that rituximab can kill human B cells via a caspase-independent form of programmed cell death that shares features of apoptosis and necrosis. This pathway may be relevant to the clinical efficacy of rituximab.

Hepatitis C virus infection.

Hepatitis C virus (HCV) infection is a major public health problem. Up to 3% of the world's population is infected with HCV, and at least 200 000 adults in the UK carry the virus. Of those exposed to HCV, 80% become chronically infected, and at least 30% of carriers develop chronic liver disease, including cirrhosis and hepatocellular carcinoma. This review provides an overview of selected features of the molecular biology and pathogenesis of HCV infection, and thereafter discusses in detail the epidemiology of HCV, the hepatic and extra-hepatic diseases caused by the virus, and the current treatment options for both acute and chronic virus infection. The special cases of healthcare workers, prison inmates and individuals coinfected with human immunodeficiency virus and HCV are considered in detail.

Loss of cytomegalovirus-specific immunological memory in a patient with thymoma.

Cytomegalovirus (CMV) retinitis is a re-activation infection associated with severely impaired T cell-mediated immunity. We describe a patient with long-standing Crohn's disease and thymoma who developed severe CMV retinitis. While thymoma can be associated with impaired humoral immunity and a quantitative CD4+ T helper cell deficiency, these were not evident in our patient. However, more detailed investigation of anti-CMV responses showed absence of specific T cell responses to CMV antigen. Normal CMV seropositive controls have detectable proliferation and interferon-gamma production by T cells in response to stimulation with CMV antigen, but this was absent in this patient both during the acute infection and in convalescence. Other measures of T cell function were normal. Since CMV retinitis is due to reactivation of latent CMV infection, it appears that selective loss of CMV-specific immunity had occurred, perhaps secondary to a thymoma. The causes of thymoma-associated immune impairment are not understood, but this case demonstrates that selective defects can occur in the absence of global T cell impairment. Opportunistic infections should therefore be suspected in patients with thymoma even in the absence of quantitative immune deficiencies.

Direct ex vivo comparison of the breadth and specificity of the T cells in the liver and peripheral blood of patients with chronic HCV infection.

The role of intrahepatic lymphocytes in the control of hepatitis C virus (HCV) infection and the pathology associated with it is not understood; most studies of the immunology of this infection use peripheral blood lymphocyte populations. To address this further, we examined in detail the IHL from HCV-infected patients and controls, focusing on the antigen-specific CD8(+) T lymphocyte component. Individual T cells from needle liver biopsies and peripheral blood were isolated from patients with chronic HCV infection and examined directly ex vivo. We used RT-PCR spectratyping to compare the breadth of the T cell receptor usage in the liver in comparison with the peripheral blood, and applied MHC class I tetramer technology to investigate the numbers of HCV-specific CD8(+) cells in the two compartments. T cell receptor usage in the liver of HCV-infected patients was broad, comparable with that in the peripheral blood of the same patients. A much higher proportion of liver CD8(+) cells expressed receptors specific for HCV antigens compared with paired peripheral blood CD8(+) cells. A greater proportion of the liver tetramer-positive cells expressed the activation marker CD69, compared with those in the periphery or other CD8(+) cells in the liver. In the course of chronic HCV infection, HCV-specific CD8 cells, which have been recently activated, appear to accumulate specifically in the livers of infected patients but are present in much lower numbers in the peripheral circulation. Further studies are needed to determine the function of these cells and their role in protection and immunopathology.

Viruses and apoptosis.

Apoptosis, or programmed cell death, is essential in development and homeostasis in multi-cellular organisms. It is also an important component of the cellular response to injury. Many cells undergo apoptosis in response to viral infection, with a consequent reduction in the release of progeny virus. Viruses have therefore evolved multiple distinct mechanisms for modulating host cell apoptosis. Viruses may interfere with either the highly conserved 'effector' mechanisms of programmed cell death or regulatory mechanisms specific to mammalian cells. In addition to conferring a selective advantage to the virus, the capacity to prevent apoptosis has an essential role in the transformation of the host cell by oncogenic viruses. This article provides a focussed review of apoptosis and illustrates how the study of viruses has informed our understanding of this process. Selected mechanisms by which viral gene products interfere with cell death are discussed in detail and used to illustrate the general principles of the interactions between viruses and apoptosis.

Development of a strand-specific RT-PCR based assay to detect the replicative form of hepatitis C virus RNA.

The recent development of tagged RT-PCR and rTth RT-PCR has greatly improved strand-specific detection of hepatitis C virus (HCV) RNA but these assays are still prone to some false detection of the incorrect strand of RNA. In this study we aimed to address additional factors which contribute towards false detection of HCV RNA. Firstly the benefits of both tagged primers and the thermostable reverse transcriptase rTth during cDNA synthesis were combined and it was found that strand specificity was greatly improved without compromising sensitivity. The reliability of the assay was then optimised by addressing the following issues: control synthetic transcripts should be free of contaminating plasmid DNA, residual RT activity should be minimised in the presence of PCR primers and cDNA should be free of unincorporated tagged RT primer prior to PCR amplification. The alterations made to the assay eliminated completely false detection of the incorrect strand of RNA in the control assay whilst the correct strand was consistently detected at a cDNA dilution of 10(-3)-10(-4). Negative strand was not detected in RNA isolated from serum but was detected, at a ten-fold lower level than positive strand, in RNA isolated from liver tissue.

Possible ancient oceans on Mars: evidence from Mars Orbiter Laser Altimeter data.

High-resolution altimetric data define the detailed topography of the northern lowlands of Mars, and a range of data is consistent with the hypothesis that a lowland-encircling geologic contact represents the ancient shoreline of a large standing body of water present in middle Mars history. The contact altitude is close to an equipotential line, the topography is smoother at all scales below the contact than above it, the volume enclosed by this contact is within the range of estimates of available water on Mars, and a series of extensive terraces parallel the contact in many places.

TT virus sequence heterogeneity in vivo: evidence for co-infection with multiple genetic types.

TT virus (TTV) is a newly described DNA virus of humans that exhibits an unusually high degree of genetic heterogeneity. We have performed extensive analysis of the TTV populations present in samples, taken over a period of 2 to 6 years, from three individuals with persistent TTV infection. TTV DNA titres estimated for sequential samples were found to be quite stable over the entire study period in two patients, but fluctuated considerably in the third. DNA sequence analysis revealed different genetic diversity among TTV populations from samples from the three patients. In one case, absolute sequence homogeneity was observed among samples over a 3 year period. In a second, a limited amount of heterogeneity was found, including one sequence exhibiting G-->A hypermutation. TTV DNA sequences from the third patient exhibited quite remarkable genetic heterogeneity: evidence was found of seven distinct infecting viruses, representing four of the six TTV genotypes that have been described. In addition, minor variants of three of these seven sequences were observed. The heterogeneity of the viral population in this individual declined steadily over a 6 year period. This patient infected with a genetically diverse TTV population had the highest viral DNA titre.

TT virus infection in patients with hepatitis C: frequency, persistence, and sequence heterogeneity.

TT virus (TTV) was recently identified in the serum of a patient with hepatitis. The role of TTV in liver disease has not been established. Three polymerase chain reaction (PCR) protocols were used to detect TTV DNA in sera of persons infected with hepatitis C virus (HCV) and in blood donors. Sera from 11.5% of HCV-infected patients and 7.7% of blood donors were positive by protocols 1 or 2. In contrast, 48.7% and 57.7% of sera, respectively, were positive when tested by protocol 3. There was no difference in the severity of hepatitis in persons coinfected with TTV and HCV when compared with those infected with HCV alone, regardless of which TTV PCR protocol was used. TTV DNA persisted in serum samples taken up to 6 years apart in individual patients. Sequence analysis indicated that most viral sequences were distinct between patients, and there was evidence of genetic heterogeneity and viral evolution within individuals.

Low incidence of human herpesvirus 8 in stem cell collections from myeloma patients.

Human herpesvirus 8 is a gammaherpesvirus which may be implicated in the pathogenesis of multiple myeloma. Viral DNA sequences have been found in the bone marrow, peripheral blood and leukapheresis products of myeloma patients. These findings have significant implications for the use of leukapheresed cells in the transplantation and immunotherapy of myeloma. The studies suggest the cell which harbours the virus may be dendritic in origin. We have previously reported that dendritic cells cultured for use in the clinical setting do not harbour HHV-8. In this study, we examined the leukapheresis products of a larger cohort of myeloma patients for the presence of HHV-8 using a highly sensitive PCR technique. A strong association between HHV-8 and myeloma was not confirmed, with only 4% of the patient samples positive for viral sequences. While further study is needed, the current use of apheresis cells and their cultured progeny in the treatment of myeloma should not be compromised.

Dendritic cells cultured from mononuclear cells and CD34 cells in myeloma do not harbour human herpesvirus 8.

Dendritic cells (DC) are antigen-presenting cells with the potential to be a powerful adjuvant in the immunotherapy of haematological malignancy, including myeloma. Recently, human herpesvirus 8 (HHV-8) infection of dendritic cells in the long-term bone marrow stromal cultures of patients with myeloma has been reported. This finding is of great potential importance regarding oncogenesis in myeloma in addition to having significant implications for the use of DC in the immunotherapy of this disease. Therefore DC generated from mobilized blood mononuclear cells (MO-DC) and purified CD34+ cells (CD34-DC) of myeloma patients were examined for the presence of HHV-8 using a sensitive PCR technique. HHV-8 was not demonstrated in MO-DC or CD34-DC and we conclude that these cells remain a suitable vehicle for investigation in the immunotherapy of myeloma.

Q fever in pregnancy.

We describe a case of acute symptomatic infection with Coxiella burnetii acquired between the 16th and 28th week of pregnancy. Oral ciprofloxacin therapy was started on diagnosis, at the 28th week of pregnancy, but symptoms were unabated after 3 weeks treatment, suggesting persisting infection of the products of conception. Caesarean section was therefore performed at 32 weeks gestation when a healthy infant was delivered, and subsequent investigations showed no evidence of transplacental spread of infection. Infection control measures were applied at the time of delivery to minimize the risk of infection to obstetricians and midwives from potentially infectious products of conception.