Genetic basis for variation in plasma IL-18 levels in persons with chronic hepatitis C virus and human immunodeficiency virus-1 infections.

Inflammasomes are multi-protein complexes integrating pathogen-triggered signaling leading to the generation of pro-inflammatory cytokines including interleukin-18 (IL-18). Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections are associated with elevated IL-18, suggesting inflammasome activation. However, there is marked person-to-person variation in the inflammasome response to HCV and HIV. We hypothesized that host genetics may explain this variation. To test this, we analyzed the associations of plasma IL-18 levels and polymorphisms in 10 genes in the inflammasome cascade. About 1538 participants with active HIV and/or HCV infection in three ancestry groups are included. Samples were genotyped using the Illumina Omni 1-quad and Omni 2.5 arrays. Linear regression analyses were performed to test the association of variants with log IL-18 including HCV and HIV infection status, and HIV RNA in each ancestry group and then meta-analyzed. Eleven highly correlated single-nucleotide polymorphisms (r2=0.98-1) in the IL-18-BCO2 region were significantly associated with log IL-18; each T allele of rs80011693 confers a decrease of 0.06 log pg ml-1 of IL-18 after adjusting for covariates (rs80011693; rs111311302 ß=-0.06, P-value=2.7 × 10-4). In conclusion, genetic variation in IL-18 is associated with IL-18 production in response to HIV and HCV infection, and may explain variability in the inflammatory outcomes of chronic viral infections.

Phylogenetic analysis of full-length, early infection, hepatitis C virus genomes among people with intravenous drug use: the InC3 Study.

Cross-continental phylogenetic analysis is important to understand subtle molecular differences of currently circulating hepatitis C virus (HCV) subtypes. Existence of such differences can be crucial in pursuing a universal hepatitis C vaccine. We characterized molecular epidemiology of early HCV infections identified across nine cohorts [North America (n=4), Australia (n=4) and Europe (n=1)] in the International Collaborative of Incident HIV and Hepatitis C in Injecting Cohorts (InC3 ). One hundred and ninety-two full-length HCV genomes were amplified from plasma of incident infections and subjected to next generation sequencing to establish the largest cross-continental, full-length acute HCV genomic data set available to date. Genomes from the most common subtypes (1a: n=94, 2b: n=15 and 3a: n=68) were used in phylogenetic analysis. Using full genome trees, 78 sequences (44%) were found to lie within 29 phylogenetic clusters/pairs defined on the basis of molecular similarity of consensus sequences. Of these, 26 each had exclusively Australian or North American sequences indicating a strong geographical bias for molecular similarity. On further analysis of behavioural and demographic associations, binary logistic regression analysis showed that older age and non-Caucasian ethnicity were significantly associated with clustering. HCV probably evolves in micro-epidemics within geographically isolated communities.

Analysis of resistance-associated substitutions in acute hepatitis C virus infection by deep sequencing across six genotypes and three continents.

Several direct-acting antivirals (DAAs) have been approved for the treatment of chronic hepatitis C virus (HCV) infections, opening the door to highly effective interferon-free treatment regimens. Resistance-associated substitutions (RASs) have been reported both in treatment-naïve patients and following treatment with protease (NS3), phosphoprotein (NS5A) and polymerase (NS5B) inhibitors. The prevalence of naturally occurring RASs in untreated HCV-infected individuals has mostly been analysed in those infected with genotype 1 (GT1), in the late phase of infection, and only within limited regions of the genome. Furthermore, the geographic distribution of RASs remains poorly characterized. In this study, we used next-generation sequencing to analyse full-length HCV genomes for the prevalence of RASs in acute HCV infections identified in nine international prospective cohorts. RASs were analysed in 179 participants infected with all six major HCV genotypes (GT1-GT6), and the geographic distribution of RASs was assessed in 107 GT1a and GT3a samples. While RASs were detected at varied frequencies across the three genomic regions, and between genotypes, RASs relevant to multiple DAAs in the leading IFN-free regimens were rarely detected in combination. Low-frequency RASs (<10% of the viral population) were also shown to have a GT-specific distribution. The main RASs with geographic associations were NS3 Q80K in GT1a samples and NS5B N142T in GT3a. These data provide the backdrop for prospective surveillance of RASs during DAA treatment scale-up.

Factors associated with hepatitis C virus RNA levels in early chronic infection: the InC3 study.

Improved understanding of natural history of hepatitis C virus (HCV) RNA levels in chronic infection provides enhanced insights into immunopathogenesis of HCV and has implications for the clinical management of chronic HCV infection. This study assessed factors associated with HCV RNA levels during early chronic infection in a population with well-defined early chronic HCV infection. Data were from an international collaboration of nine prospective cohorts studying acute HCV infection (InC(3) study). Individuals with persistent HCV and detectable HCV RNA during early chronic infection (one year [±4 months] postinfection) were included. Distribution of HCV RNA levels during early chronic infection was compared by selected host and virological factors. A total of 308 individuals were included. Median HCV RNA levels were significantly higher among males (vs females; 5.15 vs 4.74 log IU/mL; P < 0.01) and among individuals with HIV co-infection (vs no HIV; 5.89 vs 4.86; P = 0.02). In adjusted logistic regression, male sex (vs female, adjusted odds ratio [AOR]: 1.93; 95%CI: 1.01, 3.69), interferon lambda 4 (IFNL4) rs12979860 CC genotype (vs TT/CT; AOR: 2.48; 95%CI: 1.42, 4.35), HIV co-infection (vs no HIV; AOR: 3.27; 95%CI: 1.35, 7.93) and HCV genotype G2 (vs G3; AOR: 5.40; 95%CI: 1.63, 17.84) were independently associated with high HCV RNA levels (>5.6 log IU/mL = 400 000 IU/mL). In conclusion, this study demonstrated that IFNL4 rs12979860 CC genotype, male sex, HIV co-infection and HCV genotype G2 are associated with high HCV RNA levels in early chronic infection. These factors exert their role as early as one year following infection.

Admixture analysis of spontaneous hepatitis C virus clearance in individuals of African descent.

Hepatitis C virus (HCV) infects an estimated 3% of the global population with the majority of individuals (75-85%) failing to clear the virus without treatment, leading to chronic liver disease. Individuals of African descent have lower rates of clearance compared with individuals of European descent and this is not fully explained by social and environmental factors. This suggests that differences in genetic background may contribute to this difference in clinical outcome following HCV infection. Using 473 individuals and 792,721 single-nucleotide polymorphisms (SNPs) from a genome-wide association study (GWAS), we estimated local African ancestry across the genome. Using admixture mapping and logistic regression, we identified two regions of interest associated with spontaneous clearance of HCV (15q24, 20p12). A genome-wide significant variant was identified on chromosome 15 at the imputed SNP, rs55817928 (P=6.18 × 10(-8)) between the genes SCAPER and RCN. Each additional copy of the African ancestral C allele is associated with 2.4 times the odds of spontaneous clearance. Conditional analysis using this SNP in the logistic regression model explained one-third of the local ancestry association. Additionally, signals of selection in this area suggest positive selection due to some ancestral pathogen or environmental pressure in African, but not in European populations.

Anti-inflammatory cytokines, pro-fibrogenic chemokines and persistence of acute HCV infection.

Chemokines and cytokines play a vital role in directing and regulating immune responses to viral infections. Persistent hepatitis C virus (HCV) infection is characterized by the loss of anti-HCV cellular immune responses, while control of HCV infection is associated with maintenance of anti-HCV cellular immune responses. To determine whether plasma concentrations of 19 chemokines and cytokines controlling T-cell trafficking and function differed based on infection outcome, we compared them in at-risk subjects followed prospectively for HCV infection. Levels were compared over time in subjects who controlled HCV infection (Clearance) and subjects who developed persistent HCV infection (Persistence) at two time points during acute infection: (i) first viraemic sample (initial viraemia) and (ii) last viraemic sample in Clearance subjects and time-matched samples in Persistence subjects. At initial viraemia, increased pro-inflammatory tumour necrosis factor α (TNFα) plasma concentrations were observed in the Clearance group, while the plasma levels of anti-inflammatory interleukin (IL)-2, IL-10 and IL-13 were higher in the Persistence group. IL-13 was positively correlated with IL-2 and IL-10 at initial viraemia in the Persistence group. At the time of last viraemia, plasma levels of eotaxin, macrophage chemoattractant protein-4 (MCP-4), IL-5 and IL-10 were higher in the Persistence group and IL-10 and IL-5 levels were positively correlated. Collectively, these results suggest that the development of persistent infection is associated with an anti-inflammatory and pro-fibrogenic chemokine and cytokine profile that is evident at the onset of infection and maintained throughout acute infection.

Identification of a peptide recognized by five melanoma-specific human cytotoxic T cell lines.

Of several thousand peptides presented by the major histocompatibility molecule HLA-A2.1, at least nine are recognized by melanoma-specific cytotoxic T lymphocytes (CTLs). Tandem mass spectrometry was used to identify and to sequence one of these peptide epitopes. Melanoma-specific CTLs had an exceptionally high affinity for this nine-residue peptide, which reconstituted an epitope for CTL lines from each of five different melanoma patients tested. Recognition by multiple CTL lines suggests that this may be a promising candidate for use in peptide-based melanoma vaccines.

Peptides presented to the immune system by the murine class II major histocompatibility complex molecule I-Ad.

Between 650 and 2000 different peptides are associated with the major histocompatibility complex class II molecule I-Ad. Sequences for nine of these were obtained by a combination of automated Edman degradation and tandem mass spectrometry. All of the peptides are derived from secretory or integral membrane proteins that are synthesized by the antigen-presenting cell itself. Peptides were 16 to 18 residues long, had ragged NH2-and COOH-termini, and contained a six-residue binding motif that was variably placed within the peptide chain. Binding data on truncated peptides suggest that the peptide binding groove on class II molecules can be open at both ends.

Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry.

Antigens recognized by T cells are expressed as peptides bound to major histocompatibility complex (MHC) molecules. Microcapillary high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry was used to fractionate and sequence subpicomolar amounts of peptides isolated from the MHC molecule HLA-A2.1. Of 200 different species quantitated, eight were sequenced and four were found in cellular proteins. All were nine residues long and shared a distinct structural motif. The sensitivity and speed of this approach should enhance the analysis of peptides from small quantities of virally infected and transformed cells as well as those associated with autoimmune disease states.

An investigation of auto-reactivity after head injury.

Murine models of CNS injury show auto-reactive T cell responses directed at myelin antigens, associated with improved neuronal survival and functional recovery. This pilot study shows, for the first time, that similar immune responses against myelin occur in human traumatic brain injury (TBI), with an expansion of lymphocytes recognising myelin basic protein observed in 40% of patients studied. "Reactive" patients did not have greater contusion volume on imaging, but were younger than the "unreactive" subgroup and tended towards a more favorable outcome. These findings are consistent with the concept of "beneficial autoimmunity".

[Diffuse panbronchiolitis in an Asian woman with severely obstructed pulmonary disease]. / Diffuse panbronchiolitis bij een Aziatische vrouw met een ernstig obstructief gestoorde longfunctie.

A 41-year-old woman was referred for severely obstructed pulmonary disease 4 years after she stopped smoking. Treatment for chronic obstructive pulmonary disease (COPD) had no effect and lung function worsened in the following years. The clinical, radiological and pathological characteristics led us to the diagnosis of diffuse panbronchiolitis. She was treated with clarithromycin. After 1 year, major improvement was seen in clinical, radiological and spirometric features. Diffuse panbronchiolitis is a rare obstructive disorder that is usually seen in people of Japanese descent. Patients respond well to treatment with low-dose macrolide treatment.

Cytotoxic T-lymphocyte response to autologous human squamous cell cancer of the lung: epitope reconstitution with peptides extracted from HLA-Aw68.

Cytotoxic T-lymphocytes (CTLs) specific for autologous human squamous cell cancer of the lung were generated by stimulation of peripheral blood lymphocytes with autologous tumor cells in vitro. The CTL line was >97% CD3+, CD8+, CD16- and produced tumor necrosis factor-alpha, gamma-interferon, and granulocyte-macrophage colony-stimulating factor after stimulation with autologous tumor. The CTLs lysed autologous tumor but failed to recognize autologous or histocompatibility leukocyte antigen-matched lymphoid cells, K562, or allogeneic tumor cells of several histological types. Antibody-blocking studies suggested that the CTLs recognized one or more antigens presented by the class I major histocompatibility complex molecule Aw68. To characterize these antigens further, histocompatibility leukocyte antigen Aw68 molecules were extracted from the squamous cell cancer of the lung tumor line by immunoaffinity chromatography, and the associated peptides were eluted in acid and separated by reversed-phase high-performance liquid chromatography. Reconstitution of the CTL epitope was evaluated by adding these peptides to autologous Epstein-Barr virus-transformed B-cells. Two peaks of reconstituting activity were observed, suggesting that these CTLs recognize at least two Aw68-associated peptides. This study confirms the existence of a CTL response against autologous human squamous cell cancer of the lung and suggests that this CTL response is directed against peptide epitopes presented by the class I major histocompatibility complex molecules. It is anticipated that this approach will permit identification of peptide epitopes for lung cancer-specific CTLs.

Infectious canine hepatitis in red foxes (Vulpes vulpes) in wildlife rescue centres in the UK.

Outbreaks of infectious canine hepatitis are described in red foxes ( ITALIC! Vulpes vulpes) at two wildlife rescue centres in the UK. Disease occurred in two-month-old to four-month-old juvenile foxes, which were held in small enclosures in groups of three to eight animals. The foxes died or were euthanased after a short clinical course, sometimes including neurological signs and jaundice, with a high case fatality rate. Four red foxes submitted for postmortem examination had enlarged, congested livers, with rounded borders and mild accentuation of the lobular pattern. On histological examination, there was random, multifocal to massive hepatic necrosis, along with multifocal vasculitis in the central nervous system (CNS) and mild, multifocal glomerulonephritis. Intranuclear inclusion bodies, typical of canine adenovirus type 1 (CAV-1) infection, were present in hepatocytes, vascular endothelial cells in the CNS, renal glomeruli and renal tubular epithelial cells. CAV-1 was detected in tissues from affected foxes by PCR and sequencing. Congregation of juvenile foxes in wildlife rescue centres is likely to be a risk factor for transmission of CAV-1. Preventive measures in wildlife centres should be implemented to prevent the spread of the virus among conspecifics and to other susceptible species.

Identification of the 1B vaccine strain of Chlamydia abortus in aborted placentas during the investigation of toxaemic and systemic disease in sheep.

CASE HISTORY: One hundred and forty Cheviot and 100 Suffolk cross Mule primiparous 1-2-year-old ewes, from a flock of about 700 ewes, were vaccinated with an attenuated live 1B strain Chlamydia abortus vaccine about 4 weeks before ram introduction (September 2011). Between 08 March and 01 April 2012, 50 2-year-old ewes aborted and 29 of these died, despite antimicrobial and anti-inflammatory treatment and supportive care. PATHOLOGICAL FINDINGS: Seven fetuses and three placentae from five 2-year-old ewes were submitted for pathological investigation. The aborted fetuses showed stages of autolysis ranging from being moderately fresh to putrefaction. Unusual, large multifocal regions of thickened membranes, with a dull red granular surface and moderate amounts of grey-white surface exudate were seen on each of the placentae. Intracellular, magenta-staining, acid fast inclusions were identified in Ziehl Neelsen-stained placental smears. Immunohistochemistry for Chlamydia-specific lipopolysaccharide showed extensive positive labelling of the placental epithelia. LABORATORY FINDINGS: Molecular analyses of the aborted placentae demonstrated the presence of the 1B vaccine-type strain of C. abortus and absence of any wild-type field strain. The vaccine strain bacterial load of the placental tissue samples was consistent with there being an association between vaccination and abortion. DIAGNOSIS: Initial laboratory investigations resulted in a diagnosis of chlamydial abortion. Further investigations led to the identification of the 1B vaccine strain of C. abortus in material from all three of the submitted aborted placentae. CLINICAL RELEVANCE: Timely knowledge and understanding of any potential problems caused by vaccination against C. abortus are prerequisites for sustainable control of chlamydial abortion. This report describes the investigation of an atypical abortion storm in sheep, and describes the identification of the 1B vaccine strain of C. abortus in products of abortion. The significance of this novel putative association between the vaccine strain of C. abortus and severe clinical disease is unknown. Aspects of the approach that is described are relevant to the investigation of all outbreaks of ovine abortion, irrespective of the diagnosis. Awareness of the changing role of C. abortus as a major global cause of abortion ought to reinforce the importance of monitoring of adequate biosecurity in those countries which are currently free from chlamydial abortion.

Exercise cardiorespiratory function before and one year after operation for pectus excavatum.

In 35 patients with pectus excavatum (aged 17.9 +/- 5.6 years) pulmonary function and maximal exercise test results were compared before and at 1 year after operation. The lower posteroanterior chest diameter on the lateral x-ray film was significantly smaller than normal (p < 0.0001) and increased significantly after operation (p < 0.0001). Preoperatively, total lung capacity (86.0% +/- 14.4%; p = 0.0001) and inspiratory vital capacity (79.7% +/- 16.2; p = 0.0001) were significantly smaller than predicted and further decreased after operation (-9.2% +/- 9.2%; p = 0.0001 and -6.6% +/- 10.7%; p = 0.0012, respectively). Arterial blood gas values displayed normal patterns with increasing exercise both before and after operation. Only the arterial pH decreased more after operation than before (p = 0.0026). After operation there was a significant increase in maximal oxygen uptake (oxygen uptake; p = 0.0002 and oxygen uptake per kilogram; p = 0.0025) and oxygen pulse (oxygen uptake/heart rate approximates an indirect parameter for stroke volume; p = 0.0333) during exercise, whereas the maximal work performed was unchanged. Efficiency of breathing (ratio of tidal volume/inspiratory vital capacity) at maximal exercise improved significantly after operation (p = 0.0005). Ventilatory limitation of exercise (defined by an increase in carbon dioxide tension during exercise) was found in 43.9% of the patients before operation. A tendency of improvement was noted (not significant) after operation (difference in carbon dioxide tension 0.6 +/- 0.4 kPa before versus 0.3 +/- 0.5 kPa after operation). However, the group with normal preoperative carbon dioxide elimination had a ventilatory limitation of exercise after operation (difference in carbon dioxide tension -0.4 +/- 0.3 kPa before versus -0.1 +/- 0.3 kPa after operation; p = 0.0128) with a significant increase in oxygen consumption (p = 0.0007). In conclusion the subjective physical improvement after operation is not explained by changes in cardiorespiratory function at exercise. The data suggest a higher work of breathing after operation.

FDG-PET in staging lung cancer: how does it change the algorithm?

BACKGROUND: In patients with lung cancer, positron emission tomography (PET) using fluor-18-fluorodesoxyglucose (FDG) may be used both to detect extrathoracic metastases (ETM) and for mediastinal lymph node staging (MLS), potentially reducing the need for mediastinoscopy. We assessed the added value of FDG-PET in detecting ETM and focused on the reliability of FDG-PET and mediastinoscopy for MLS. PATIENTS AND METHODS: In 72 consecutive patients with non-small cell lung cancer, the impact of adding FDG-PET to full conventional clinical staging was prospectively analyzed. The predictive value of FDG-PET findings and tumor location for pathologic mediastinal lymph node status were assessed in a logistic regression analysis. RESULTS: Unexpected extrathoracic metastases were detected by FDG-PET in 15% of patients. In MLS overall negative and positive predictive values were 71 and 83% for FDG-PET, and 92 and 100% for mediastinoscopy. However, the negative predictive value of FDG-PET was only 17% in case of FDG-PET positive N1 nodes and/or a centrally located primary tumor, whereas it was 96% in case of FDG-PET negative N1 nodes and a non-centrally located primary tumor. CONCLUSION: By incorporating FDG-PET in clinical staging, 15% of patients with lung cancer are upstaged due to unexpected extrathoracic metastases. In case of a negative mediastinal FDG-PET, mediastinoscopy can only be omitted in the presence of a non-centrally located primary tumor and without FDG-PET positive N1 nodes.

Use of the vicinal element effect for regiochemical control of quinone substitutions and its implication for convergent mitomycin construction.

[reaction--see text] Nucleophilic substitution reactions of 2-methoxy-3-alkyl-p-benzoquinones are described as they relate to the construction of the mitomycin backbone. Normally controlled by activating groups attached to the olefin, the observed regioselection in these cases is determined by the deactivating substituent. Approximation of carbonyl activating ability would not have predicted the behavior of two systems investigated in which the poorer of two leaving groups is substituted in each case.

Prognosis of unsuspected but completely resectable N2 non-small cell lung cancer.

Of 111 patients with non-small cell lung cancer without clinically evident N2 disease 95 underwent mediastinoscopy between 1975 and 1985. In 63 cases mediastinoscopy was positive and in 32 negative. The patients with a positive mediastinoscopy were considered to have inoperable disease. Their 3- and 5-year survival rates were 5% and 0%, respectively. The patients with a negative mediastinoscopy and 16 patients in whom no mediastinoscopy was performed because of a peripheral tumor underwent operation. They underwent complete tumor resection and mediastinal lymph node dissection. Unsuspected N2 disease was found. Their 3- and 5-year survival rates were 19% and 10%, respectively. The better survival rate in the operated group was statistically significant and mainly due to a better survival of the lobectomy group. Multiple regression analysis showed no favorable prognostic factors in the nonoperated group, but in the operated group lobectomy and central location of the tumor significantly improved the prognosis. We conclude that patients with unsuspected stage IIIa non-small cell lung cancer discovered at thoracotomy benefit from complete tumor resection and mediastinal lymph node dissection, especially if the resection can be confined to lobectomy and if the tumor is located centrally.

Pectus excavatum. A clinical study with long-term postoperative follow-up.

Between 1972 and 1987, 192 patients have been operated upon for pectus excavatum of which 152 patients were included in the study (79%). Mean age at operation was 15.3 +/- 5.5 years; 117 were male. Mean follow-up was 8.1 +/- 3.6 years. The deformity was noted before the age of 5 in 90%. Type I symmetrical and localized deformity was seen in 33.2%, type II symmetrical but diffuse depression in 23.7% and type III localized or diffuse asymmetrical deformity in 43.1%. It was considered severe in 68.9%, fair in 16.9% and mild in 14.2%. There were significantly more asymmetrical defects in the older age groups. The operation consisted of subperichondral chondrectomy, transverse sternotomy and division of the intercostal bundles at the outer limit of the chondrectomy and suturing the edge of this broad sheet of muscle and perichondrium to the anterior surface of the chest wall more laterally and under tension, elevating and stabilizing the sternum. Results were satisfactory in 83.6% (excellent 44.1%, good 39.5%). Results were not significantly influenced by age, sex, severity, type, symmetry, the extent of cartilage resection or follow-up. Results were inversely influenced by the occurrence of wound problems. The optimal age for operation is considered to be between 5 and 10 years. Both physical as well as psychological cosmetic factors may serve as an indication for operation.

Surgical treatment of Pancoast tumor.

The treatment of superior pulmonary sulcus (Pancoast) tumor is not uniform and is still discussed. Literature data and our retrospective study are presented. Fourteen patients were operated for a Pancoast tumor. Nine patients underwent mediastinoscopy followed by preoperative radiotherapy. Five patients received adjuvant radiotherapy after incomplete resection. Five patients who did not have preoperative radiotherapy, received postoperative irradiation. All three patients who survived five years or more, had preoperative radiotherapy and two of them underwent a complete resection. Literature data are discussed and emphasis is laid on the importance of preoperative staging, including mediastinoscopy, preoperative radiotherapy and complete "en bloc" resection.