TNF superfamily members promote hepatitis C virus entry via an NF-κB and myosin light chain kinase dependent pathway.

Preventing virally induced liver disease begins with an understanding of the host factors that define susceptibility to infection. Hepatitis C virus (HCV) is a global health issue, with an estimated 170 million infected individuals at risk of developing liver disease including fibrosis and hepatocellular carcinoma. The liver is the major reservoir supporting HCV replication and this hepatocellular tropism is defined by HCV engagement of cellular entry receptors. Hepatocytes are polarized in vivo and this barrier function limits HCV entry. We previously reported that activated macrophages promote HCV entry into polarized hepatocytes via a TNF-α-dependent process; however, the underlying mechanism was not defined. In this study, we show that several TNF superfamily members, including TNF-α, TNF-ß, TWEAK and LIGHT, promote HCV entry via NF-κB-mediated activation of myosin light chain kinase (MLCK) and disruption of tight junctions. These observations support a model where HCV hijacks an inflammatory immune response to stimulate infection and uncovers a role for NF-κB-MLCK signalling in maintaining hepatocellular tight junctions.

Lentiviral hepatitis B pseudotype entry requires sodium taurocholate co-transporting polypeptide and additional hepatocyte-specific factors.

Hepatitis B virus (HBV) is one of the world's major unconquered infections, resulting in progressive liver disease, and current treatments rarely cure infection. A limitation to discovering new therapies is our limited knowledge of HBV entry and dissemination pathways that hinders the development of in vitro culture systems. To address this gap in our understanding we optimized the genesis of infectious lentiviral pseudoparticles (HBVpps). The recent discovery that the bile salt transporter sodium taurocholate co-transporting polypeptide (NTCP) acts as a receptor for HBV enabled us to assess the receptor dependency of HBVpp infection. HBVpps preferentially infect hepatoma cells expressing NTCP, whereas other non-liver cells engineered to express NTCP do not support infection, suggesting that additional hepatocyte-specific factors are required for HBVpp internalization. These results highlight the value of the HBVpp system to dissect the pathways of HBV entry and dissemination.

Hepatoma polarization limits CD81 and hepatitis C virus dynamics.

Many viruses target the polarized epithelial apex during host invasion. In contrast, hepatitis C virus (HCV) engages receptors at the basal surface of hepatocytes in the polarized liver parenchyma. Hepatocyte polarization limits HCV entry by undefined mechanism(s). Given the recent reports highlighting a role for receptor mobility in pathogen entry, we studied the effect(s) of hepatocyte polarization on viral receptor and HCV pseudoparticle (HCVpp) dynamics using real-time fluorescence recovery after photobleaching and single particle tracking. Hepatoma polarization reduced CD81 and HCVpp dynamics at the basal membrane. Since cell polarization is accompanied by changes in the actin cytoskeleton and CD81 links to actin via its C-terminus, we studied the dynamics of a mutant CD81 lacking a C-terminal tail (CD81(ΔC)) and its effect(s) on HCVpp mobility and infection. CD81(ΔC) showed an increased frequency of confined trajectories and a reduction of Brownian diffusing molecules compared to wild-type protein in non-polarized cells. However, these changes were notobserved in polarized cells. HCVpp showed a significant reduction in Brownian diffusion and infection of CD81(ΔC) expressing non-polarized cells. In summary, these data highlight the dynamic nature of CD81 and demonstrate a role for CD81 lateral diffusion to regulate HCV infection in a polarization-dependent manner.

Is laparoscopic appendicectomy a safe procedure for trainees in the peripheral hospital setting?

Laparoscopic appendicectomy has become standard in the treatment of acute appendicitis in most hospitals in Ireland. Studies have shown that it is a safe procedure for trainees to perform. However, these studies were conducted in university teaching hospitals whereas a significant proportion of training in Ireland takes place in peripheral hospitals which provide a different training environment. The aim of this study was to determine whether laparoscopic appendicectomy is a safe procedure for surgical trainees to perform in a peripheral hospital setting. A retrospective analysis was performed of appendicectomies carried out at a peripheral hospital over a 12 month period. Comparisons were made between consultant surgeons and trainees for a variety of outcomes. Of 155 appendicectomies, 129 (83.2%) were performed laparoscopically, of which 10 (7.75%) were converted to open. Consultants performed 99 (77%) laparoscopic appendicectomies. There were no statistically significant differences between consultants and trainees in complication rates (19 (19.2%) vs. 4 (13.3%), p = 0.46), mean length of hospital stay (4.7 +/- 4.0 vs. 3.4 +/- 3.3 days, p = 0.13), or rate of conversion to open operation (9 (9.1%) vs. 1 (3.3%), p = 0.45). For cases of complicated appendicitis there were no significant differences between consultants and trainees in complication rates (12 vs. 2, p = 0.40) or length of hospital stay (6.4 +/- 3.9 vs. 4.7 +/- 5.6 days, p = 0.27). We conclude that laparoscopic appendicectomy is a safe procedure for trainees to perform in the peripheral hospital setting and should be incorporated into surgical training programs at an early stage of training.

Management of gastric lymphoma.

INTRODUCTION: The management of gastric lymphoma is controversial and a wide variety of unimodality or multimodality approaches have been used. The aim of this report is to highlight the variety of treatment regimens deployed, the outcomes achieved and to present a modern management approach for this enigmatic tumour. RESULTS: 42 cases of primary gastric lymphoma managed at one centre over a 15-year period were reviewed. Weight loss (52%), pain (41%) and anorexia (33%) were the most common presenting symptoms. Most patients (86%) had high-grade lymphoma. Primary treatment modalities included surgery (36%), chemotherapy (40%), supportive care only (22%) and H. pylori eradication (2%). Adjuvant therapies included chemotherapy (17%), radiotherapy (7%) and combined chemoradiotherapy (5%). The overall median survival was 53 months, with a five year survival of 46%. In the curative group, the median survival was 75 months and five year survival 58%. Curative surgery or chemotherapy +/- radiotherapy were similarly effective for stage IE and IIE disease. CONCLUSIONS: The prognosis for gastric lymphoma is grade- and stage-dependent. With equivalent outcomes for cure in localised gastric lymphoma for surgery and chemotherapy, the latter is preferred in this unit because of gastric preservation, with surgery being reserved for failed medical management or presentations with haemorrhage, perforation or obstruction refractive to steroid therapy.

Clinical utility of abdominal and pelvic ultrasound in the evaluation of right iliac fossa pain in females.

BACKGROUND: Ultrasound (US) is often the imaging modality of choice in women with acute right iliac fossa (RIF) pain, identifying the appendix in up to 99% of patients. The literature, however, lacks clear guidelines on how ultrasonography should be performed to maximise sensitivity and specificity in such patients. Many centres perform untargeted abdomino-pelvic scans, including organs such as the liver and spleen, which unlikely contribute to the presenting complaint. AIMS: We aimed to evaluate the clinical utility of unfocussed abdominal and pelvic US in women of reproductive age with acute RIF pain. METHODS: This multicentre study describes 501 women between the ages of 12 and 50, over a 3-year period from three institutions, presenting acutely with RIF pain and investigated with US abdomen and pelvis. RESULTS: 5.9% of cases confirmed appendicitis sonographically. A normal appendix was visualised in 0.2%. Over 10% identified gynaecological pathology, 41% relating to the right ovary. 10.4% incidental extra-pelvic findings were unrelated to the acute clinical presentation. 0.8% of patients had extra-pelvic findings meriting further clinical assessment. CONCLUSION: The results herein reflect findings from high volume emergency surgical departments, demonstrating that unfocussed abdominal and pelvic ultrasounds are not an appropriate use of resources in reproductive women with RIF pain. Clinically relevant extra-pelvic US findings account for less than 1%, rarely contributing to the acute presentation. The appendix was only visualised in 6% of patients, suggesting that a focussed appendiceal and pelvic US would better assist diagnosis with a higher yield and increased sensitivity and specificity.

Proceedings of resources for optimal care of acute care and emergency surgery consensus summit Donegal Ireland.

BACKGROUND: Opportunities to improve emergency surgery outcomes exist through guided better practice and reduced variability. Few attempts have been made to define optimal care in emergency surgery, and few clinically derived key performance indicators (KPIs) have been published. A summit was therefore convened to look at resources for optimal care of emergency surgery. The aim of the Donegal Summit was to set a platform in place to develop guidelines and KPIs in emergency surgery. METHODS: The project had multidisciplinary global involvement in producing consensus statements regarding emergency surgery care in key areas, and to assess feasibility of producing KPIs that could be used to monitor process and outcome of care in the future. RESULTS: Forty-four key opinion leaders in emergency surgery, across 7 disciplines from 17 countries, composed evidence-based position papers on 14 key areas of emergency surgery and 112 KPIs in 20 acute conditions or emergency systems. CONCLUSIONS: The summit was successful in achieving position papers and KPIs in emergency surgery. While position papers were limited by non-graded evidence and non-validated KPIs, the process set a foundation for the future advancement of emergency surgery.

Effective communication enhances the patients' endoscopy experience.

BACKGROUND: Undergoing an endoscopy is a stressful experience for patients. AIMS: To audit the endoscopy pathway to improve patient satisfaction. METHODS: A prospective survey of endoscopy patients to identify system improvements that were then implemented. RESULTS: The survey was performed before (N = 71) and after (N = 60) process improvements identified by the initial survey. Information provision and staff communication skills were identified for optimisation. Patient anxiety at home was significantly reduced (median 2 vs. 1, p < 0.01). Education of endoscopy staff significantly improved the quality of information provided before and after the procedure with regard to sedation (median 4 vs. 5, p < 0.01), discomfort (median 4 vs. 5, p < 0.01), complications (28 vs. 82 %, p < 0.01), findings (89 vs. 100 %, p < 0.01) and follow-up (73 vs. 90 %, p = 0.015). Gloucester Comfort Scores during endoscopy improved (median 1 vs. 0, p < 0.01) without increasing sedation levels. Patient feelings of invasion/trauma significantly decreased. Overall 95 % of patients were satisfied. CONCLUSION: Structured information leaflets and improved staff communication skills reduce anxiety and enhance patients' experiences. They are now standard operating procedures.

Three-dimensional (3D) simulation versus two-dimensional (2D) enhances surgical skills acquisition in standardised laparoscopic tasks: a before and after study.

INTRODUCTION: The aim of this study is to determine if simulated 3D vision improves the speed and accuracy of laparoscopic phantom tasks in laparoscopically naïve subjects. METHODS: Thirty laparoscopically naïve subjects were divided into matched groups according to age, sex, hand dominance and initial scores on a standardised visio-spatial test. Laprotrain(©) laparoscopic simulators were used, one attached to the standard 2D monitor and the other to a simulated 3D monitor and 3D glasses were worn by the subjects in this group. Five standardised laparoscopic tasks were developed and the subjects underwent testing on four separate occasions with more than 24 h between sessions. The subjects were timed for each task and errors were recorded by two independent observers. In the second part of the study, subjects switched to the opposite group and task times and errors were again recorded. Statistical differences between groups were calculated using student t-test and Fisher's exact test. RESULTS: There were fifteen subjects in each group with no significant difference in demographic or psychometric variables. The mean time to complete the tasks was faster in the 3D group compared with the 2D group. There was a lower rate of errors noted in the 3D group compared with the 2D group but this only reached statistical significance in two of the five laparoscopic tasks. In the crossover study, subjects who had trained on simulated 3D had better task times and fewer errors compared to those who had trained on 2D simulators. DISCUSSION & CONCLUSION: Training on a simulated 3D model (compared to standard 2D) allows trainees to reach proficiency sooner.

The polymerase chain reaction in the diagnosis of vertically transmitted HIV infection.

The presence of HIV-1 DNA sequences in DNA from peripheral blood mononuclear cells (PBMCs) was investigated in a two-stage polymerase chain reaction ('double' PCR) using four sets of nested primers. The PBMCs tested were obtained from 46 children born to HIV-seropositive mothers, seven 'control' children born to HIV-seronegative mothers and seropositive fathers, and 45 healthy adult blood donors who were HIV seronegative. Nine of the children had symptomatic HIV infection and other laboratory features characteristic of HIV infection: all nine were PCR-positive with each set of primers in each of their 22 blood samples tested. The remaining 44 children had no clinical or laboratory evidence of HIV infection, and each of their 50 samples was PCR-negative with each set of primers, as were all blood donor samples. PCR-positive samples were tested in more detail using two of the sets of primers, which spanned hypervariable regions in the env gene. Polyacrylamide gel electrophoresis of DNA amplified from these regions yielded patterns of amplified DNA length variation which were characteristic for each child, and which changed little with time (in serial samples obtained over periods of 3-7 months). This excluded contamination as a cause of PCR positivity. This is the first report of the use of a double PCR for the diagnosis of HIV infection. The results demonstrate the specificity of this PCR method in diagnosis, with failure to reveal in this cohort any cases of vertically transmitted HIV-1 infection in addition to those already confirmed by conventional laboratory techniques.

Genetic hybrids of Plasmodium falciparum identified by amplification of genomic DNA from single oocysts.

Individual oocysts from Plasmodium falciparum-infected Anopheles gambiae and Anopheles stephensi mosquitoes have been examined by the PCR technique, after their removal from the midgut. The DNA obtained from these oocysts has been amplified using oligonucleotide primers specific for part of the merozoite surface antigen MSA-1 gene. This technique distinguishes oocysts which are the products of self-fertilisation events from those which are the products of cross-fertilisation between different parasite clones.

The role of the viral glycoprotein in HIV-1 persistence.

The study of the immunological defects which arise from HIV infection has led to a deeper understanding both of the normal immune system and of the mechanisms by which it is damaged in disease. The interactions between viral and host factors during the early stages of HIV infection leads to a post-seroconversion steady state or 'set point' of viral RNA load, which has been shown to be a prognostic marker for subsequent progression rates, further underlining the important role of early immunological responses in the disease process. The changing immune response during the course of infection, together with the changing patterns of antigenicity and tropism leads to a complex series of evolutionary interactions which can be monitored as a series of changes in the properties of the virus over time. Furthermore, significant differences may be seen between the antigenicity of viruses adapted to grow in tissue culture and viruses cultured only briefly in primary cells, and also between the antigenicity of monomeric and oligomeric subunit immunogens. The immunodominant, highly polymorphic and rapidly changing envelope glycoproteins of HIV remains the single biggest target for the design of successful candidate vaccines. The recent crystallisation of one HIV envelope, the proven existence of functionally conserved neutralisation targets and our increasing knowledge of the behaviour of the envelope glycoprotein in vivo offers the possibility that the next generation of vaccine candidates will have a far higher chance of success than has currently been achieved.

Immunogenicity of full length and truncated forms of the human immunodeficiency virus type I envelope glycoprotein.

We have monitored the immunogenicity of a V1V2 sub-fragment of gp 120 in contrast to the full length protein and to a truncated form (PR12) where the V1, V2 and V3 regions were removed. In contrast to previously published work [1] these studies show that monomeric forms of envelope are capable of inducing antibodies specific for both linear and discontinuous epitopes. These antibodies are capable of neutralising HIV infectivity. The majority of neutralising antibodies were specific for epitopes within the V2 and V3 regions demonstrating the immunodominance of these regions in monomeric gp 120. Relatively few of the antibodies were specific for the CD4 binding site, suggesting that this region is poorly immunogenic. Immunisation of rats with the PR12 truncated protein did not significantly enhance the immunogenicity of the CD4 binding site. However, the immune response generated included antibodies capable of binding to diverse primary HIV-1 and HIV-2 envelope glycoproteins. We have shown that up to 30% of sera from HIV-1 infected individuals have antibodies that are capable of recognising conformation-dependent epitopes within the V1V2 region of the clone HXB10, suggesting the presence of conserved cross-reactive epitopes. Furthermore we have shown an association between the presence of V1V2 reactive antibodies and the neutralisation titre of the sera tested suggesting that antibodies to this region contribute to the cross-reactive neutralising response.

Sequence specificity of the human immunodeficiency virus type 2 (hiv-2) long terminal repeat u3 region in vivo allows subtyping of the principal hiv-2 viral subtypes a and b.

Sequences from the nef/LTR overlap region of the human immunodeficiency virus type 2 (HIV-2) genome were amplified from uncultured peripheral blood mononuclear cells (PBMCs) from 40 HIV-2-infected individuals in The Gambia, West Africa. Additional sequences from the plasma of three blood donors were also derived. Analysis of HIV-2 U3 LTR transcription factor elements (PuB-1, p-ets, PuB-2, peri-kappa B, and NF-kappa B sites) indicated a relatively high level of conservation in vivo. The region immediately 3' of the nef termination codon, which exhibits clade-dependent specificity, was targeted by PCR to differentiate HIV-2 subtype A from subtype B infections, the two principal clinical HIV-2 subtypes. All clinical samples analyzed (n = 43) from The Gambia were identified as HIV-2 subtype A by a combination of LTR sequence analysis and subtype-specific amplification of subtypes A and B. Differential PCR amplification of the HIV-2 U3 LTR region represents a rapid means of differentiating subtype A from subtype B infections, the two dominant HIV-2 subtypes that are important in human disease.

Cotransfection of HIV-1 molecular clones with restricted cell tropism may yield progeny virus with altered phenotype.

Seven infectious molecular clones were obtained from a human immunodeficiency virus type 1 isolate with rapid/high replicative capacity. Biological characterization of progeny viruses obtained after transfection of clones into peripheral blood mononuclear cells showed that six clones yielded virus with restricted cell tropism, whereas one clone yielded virus able to replicate in cell lines. Although transfection of each of the clones 12, 13, and 82 individually gave rise to viruses with restricted tropism, viruses recovered from cotransfection of the mixtures of these clones exhibited altered phenotype, inasmuch as they were able to replicate in cell lines. To test whether recombination and/or complementation has taken place in the mixture of clones 12 + 13 + 82, the progeny virus was diluted to end point in 15 parallel series. Viruses with diverse biological phenotypes were recovered. With the help of distinctive restriction enzyme markers in regions comprising the vpu/env junction and variable regions 4 and 5 (V4/V5) of the env gene, recombinant genotypes could be identified with high frequency. No particular biological phenotype could be linked to a certain genotype in this study. The results show that different coexisting variants may interact and thereby influence the biological phenotype of a viral population.

Relationship between V3 sequences of HIV type 1 from plasma, T cells, and dendritic cells suggests that plasma virus may arise from different cell sources.

Nucleotide sequences of HIV-1 from plasma virus RNA and proviral DNA extracted from blood dendritic cells (DCs) and from T cells were analyzed to determine whether blood DCs may harbor a restricted population of virus variants. The sequence of the V3 loop and 51 bases from the 3' flanking region were determined in four patients not receiving antiviral therapy. There was no evidence of a unique or more restricted population of variants in DCs for any of the four patients studied. However, for one patient there was evidence of differences between plasma virus and virus in the T cell population, with virus in the plasma showing a closer relationship to DC-derived sequences.

Association between a defective CCR-5 gene and progression to disease in HIV infection.

We measured the effect(s) of CCR-5 genotype on disease progression by studying the frequency of a defective CCR-5 delta32 allele within a cohort of long-term infected individuals. An elevated frequency of CCR-5 delta32 heterozygotes within the cohort compared with a control population of blood donors was observed. An association between progression rate and CCR-5 delta32 heterozygosity was observed. Furthermore, analysis of proviral DNA V3 sequences from a subset of the cohort predicted that the majority of individuals (39 of 44) were infected with viruses predicted to utilize the beta-chemokine receptor CCR-5. The marked association between CCR-5 genotype and disease progression observed in this study may be a consequence of the predicted low frequency of CXCR-4-utilizing viruses present within the selected cohort.

A human monoclonal antibody specific for the V3 loop of HIV type 1 clade E cross-reacts with other HIV type 1 clades.

To ascertain the antigenic relationship between HIV-1 viruses belonging to various genetically defined subgroups (clades), shared epitopes need to be defined. Human monoclonal antibodies (MAbs) are particularly useful for this purpose because they can detect complex regions of viral proteins that may be missed by sequence analysis and because, by definition, they react with epitopes that stimulate the human immune system. Monoclonal antibodies derived from the cells of HIV-1 clade B-infected subjects have been used extensively for this purpose. Here we describe the first human MAb derived from a clade E-infected individual; the MAb is specific for the V3 loop, recognizing a core epitope represented by the amino acids TRTSVR on the N-terminal side of the crown of the V3 loop. The IgG1(kappa) MAb, designated 1324E, binds to the clade E consensus V3 loop, to rgp120 proteins from clade E and to peripheral blood mononuclear cells infected in vitro with the virus that infected the subject from whose cells the MAb-producing heterohybridoma was derived. Strong cross-reactivity of the MAb to the V3 peptides, rgp120 proteins, and native monomeric gp120s representing clades A and C, as well as to cells infected with a clade C primary isolate, revealed a shared V3 epitope between these clades. When tested for its neutralizing ability, MAb 1324E neutralized a clade E isolate that had been adapted for growth in H9 cells but failed to neutralize five clade E primary isolates.

Phylogenetic analysis of multiple heterosexual transmission events involving subtype b of HIV type 1.

Between 1996 and 1999 thirteen cases of HIV infection were detected in Doncaster, a small town in the north of England (population approximately 250,000). A complex network of shared sexual histories involving local nightclubs linked these cases, with the only known risk factor being heterosexual intercourse. A series of frozen blood samples was collected in 1998-1999 and amplified by PCR to generate full-length gp120 clones. Sequencing demonstrated that all the transmission events in this heterosexual group involved the B subtype of HIV-1. When relationships between the samples were assessed it became clear that these 13 cases represented at least three separate strains of HIV-1, indicating that HIV is well established in this community. Eleven of the 13 cases were related, forming two distinct groups. Further investigation revealed that one group contained five patients whose general health was good and who were not receiving HAART. In contrast, the second group of six patients, including the putative index case, were symptomatic, receiving HAART, and may have been infected with a CXCR-4-utilizing virus. Several of the cases that were linked by genetic criteria were not linked by contact tracing, implying that further undiagnosed cases may exist in this community. To our knowledge, this is the largest outbreak of HIV studied within the heterosexual community in the United Kingdom to date, suggesting that this route of infection is becoming more common within the United Kingdom.

A statistical method for the detection of false positives and false negatives in microtitre format PCR assays.

A method is described which can be used to determine whether a series of PCR reactions carried out in a microtitre plate are inherently unlikely to have occurred by chance, and hence to show 'false' results. The method is an extension of the familiar 'runs test' which can be used for tube-based PCRs. A Monte Carlo simulation program is discussed which can be used to generate expected probability distributions for either symmetric or asymmetric plate designs. In addition, systematic departures from a random pattern due to 'edge effects' can be detected.