Tumour associated lymphocytes in the pleural effusions of patients with mesothelioma express high levels of inhibitory receptors.

OBJECTIVE: Pleural effusion (PE) is a common feature of malignant pleural mesothelioma. These effusions typically contain lymphocytes and malignant cells. We postulated that the PE would be a source of lymphocytes for analysis of tumor immune milieu. The aim of this study was to compare the phenotype and T cell receptor usage of pleural effusion T cells with paired concurrently drawn peripheral blood lymphocytes. We used multi-parameter flow cytometry and high-throughput T cell receptor sequencing to analyse peripheral blood and pleural effusion mononuclear cells. RESULTS: Both CD8+ and CD4+ T cells from effusion showed increased expression of T cell inhibitory receptors PD-1, LAG-3 and Tim-3 compared to blood. Comprehensive T cell receptor sequencing on one of the patients showed a discordant distribution of clonotypes in the antigen-experienced (PD-1+) compartment between effusion and blood, suggesting an enrichment of antigen specific clonotypes in the effusion, with potential as an immunological response biomarker.

Prevalence of binary toxin positive Clostridium difficile in diarrhoeal humans in the absence of epidemic ribotype 027.

Virulence of Clostridium difficile is primarily attributed to the large clostridial toxins A and B while the role of binary toxin (CDT) remains unclear. The prevalence of human strains of C. difficile possessing only CDT genes (A-B-CDT+) is generally low (< 5%), however, this genotype is commonly found in neonatal livestock both in Australia and elsewhere. Zoonotic transmission of C. difficile has been suggested previously. Most human diagnostic tests will not detect A-B-CDT+ strains of C. difficile because they focus on detection of toxin A and/or B. We performed a prospective investigation into the prevalence and genetic characteristics of A-B-CDT+ C. difficile in symptomatic humans. All glutamate dehydrogenase or toxin B gene positive faecal specimens from symptomatic inpatients over 30 days (n = 43) were cultured by enrichment, and C. difficile PCR ribotypes (RTs) and toxin gene profiles determined. From 39 culture-positive specimens, 43 C. difficile isolates were recovered, including two A-B-CDT+ isolates. This corresponded to an A-B-CDT+ prevalence of 2/35 (5.7%) isolates possessing at least one toxin, 2/10 (20%) A-B- isolates, 2/3 CDT+ isolates and 1/28 (3.6%) presumed true CDI cases. No link to Australian livestock-associated C. difficile was found. Neither A-B-CDT+ isolate was the predominant A-B-CDT+ strain found in Australia, RT 033, nor did they belong to toxinotype XI. Previous reports infrequently describe A-B-CDT+ C. difficile in patients and strain collections but the prevalence of human A-B-CDT+ C. difficile is rarely investigated. This study highlights the occurrence of A-B-CDT+ strains of C. difficile in symptomatic patients, warranting further investigations of its role in human infection.

The dynamics of HCV-specific antibody responses in HIV/HCV patients on long-term antiretroviral therapy.

Antibody responses have not been fully characterised in chronically HIV/HCV patients receiving antiretroviral therapy (ART). Seventeen HIV/HCV patients receiving ART were followed for a median (range) interval of 597 (186-766) weeks. Prior to ART, HIV/HCV patients had lower levels of antibodies reactive with HCV core and JFH-1, and lower genotype cross-reactive neutralising antibodies (nAb) titres, than HCV patients. Levels of JFH-1 reactive antibody increased on ART, irrespective of CD4+ T-cell counts or changes in serum ALT levels. The appearance of nAb coincided with control of HCV viral replication in five HIV/HCV patients. In other patients, HCV viral loads remained elevated despite nAb responses. Sustained virological responses following HCV therapy were associated with reduced antibody responses to JFH-1 and core but elevated responses to non-structural proteins. We conclude that nAb responses alone may fail to clear HCV, but contribute to control of viral replication in some HIV/HCV patients responding to ART.

Primer ID ultra-deep sequencing reveals dynamics of drug resistance-associated variants in breakthrough hepatitis C viruses: relevance to treatment outcome and resistance screening.

BACKGROUND: Use of direct-acting antiviral drugs (DAAs) that target HCV may be hampered by the rapid selection of viral strains that harbour drug resistance-associated variants (RAVs). These RAVs are often associated with a fitness cost and tend to occur on low-frequency strains within treatment-naive subjects. To address the clinical relevance of low frequency RAVs in the setting of DAAs, this study utilized a Primer ID ultra-deep sequencing approach to mitigate PCR errors and bias to accurately quantify viral sequences in subjects that failed DAA treatment. METHODS: Subjects were enrolled in the follow-up study P05063, and had previous treatment with boceprevir and all had detectable RAVs at virological failure (VF) based on Sanger-based population sequencing. Twelve subjects had three time points available: baseline, VF and follow-up (median 830.5 days). Viral RNA was amplified using unique primer identifiers (Primer IDs) and sequenced using 454 ultra-deep sequencing. RESULTS: The sequencing strategy used in this study improved the detection of clinically relevant low frequency strains bearing RAVs compared to population sequencing and showed that these strains can persist for up to 2 years post-treatment failure. Strains carrying multiple RAVs were common in breakthrough viruses. Putative compensatory mutations were identified. CONCLUSIONS: The Primer ID ultra-deep sequencing approach identifies RAVs that can reduce drug sensitivity at levels below the detection threshold for population sequencing. The approach also removes PCR errors and biases, suggesting this sequencing strategy should become the standard approach by which to perform temporal quasispecies studies and resistance screening. ClinicalTrials.gov NCT00689390.

Strong spontaneous tumor neoantigen responses induced by a natural human carcinogen.

A key to improving cancer immunotherapy will be the identification of tumor-specific "neoantigens" that arise from mutations and augment the resultant host immune response. In this study we identified single nucleotide variants (SNVs) by RNA sequencing of asbestos-induced murine mesothelioma cell lines AB1 and AB1-HA. Using the NetMHCpan 2.8 algorithm, the theoretical binding affinity of predicted peptides arising from high-confidence, exonic, non-synonymous SNVs was determined for the BALB/c strain. The immunoreactivity to 20 candidate mutation-carrying peptides of increased affinity and the corresponding wild-type peptides was determined using interferon-γ ELISPOT assays and lymphoid organs of non-manipulated tumor-bearing mice. A strong endogenous immune response was demonstrated to one of the candidate neoantigens, Uqcrc2; this response was detected in the draining lymph node and spleen. Antigen reactive cells were not detected in non-tumor bearing mice. The magnitude of the response to the Uqcrc2 neoantigen was similar to that of the strong influenza hemagglutinin antigen, a model tumor neoantigen. This work confirms that the approach of RNAseq plus peptide prediction and ELISPOT testing is sufficient to identify natural tumor neoantigens.

Decreased IP-10 and elevated TGFbeta1 levels are associated with viral clearance following therapy in patients with hepatitis C virus.

The role of pro-fibrogenic cytokines in the outcome of infections with hepatitis C virus (HCV) and the response to treatment with pegylated interferon-alpha (pegIFNalpha) and ribavirin remains unclear. To address this issue, we assessed hepatic fibrosis and plasma markers pertinent to T-cell mediated fibrogenesis and inflammation at the start of treatment. Levels of soluble (s)CD30, interleukin-13 receptor alpha 2 (IL-13Ralpha2), total and active transforming growth factor-beta 1 (TGFbeta1), interleukin-18 (IL-18) and interferon-gamma inducible protein-10 (IP-10, CXCL10) were correlated with the severity of fibrosis and with treatment outcome using multiple logistic regression modelling. The Hepascore algorithm was confirmed as a marker of fibrosis, but was a poor predictor of treatment outcome. Inclusion of all immunological markers improved prediction based on Hepascore alone (p=0.045), but optimal prediction was achieved with an algorithm ("TIPscore") based on TGFbeta1 (total), IP-10, age, sex and HCV genotype (p=0.003 relative to Hepascore). Whilst this was only marginally more effective than predictions based on HCV genotype age and sex (p=0.07), it associates high TGFbeta1 and low IP-10 levels with a failure of therapy.

Toll-like receptor (TLR) expression on CD4+ and CD8+ T-cells in patients chronically infected with hepatitis C virus.

Toll-like receptor (TLR) expression on T-cells and the signalling pathways that lead to the production of cytokines may limit antigen-specific T-cell responses. Here, expression of TLR and retinoic acid inducible gene I (RIG-I) on T-cells were evaluated in patients chronically infected with hepatitis C virus (HCV), before and during pegylated interferon-alpha and ribavirin therapy. Expression of TLR2,3,4,7,9 and retinoic acid inducible gene (RIG)-I on different CD4(+) and CD8(+) T-cell sub-populations (naïve: CD45RA(+)CD57(-); central memory: T(CM) CD45RA(-)CD57(-); effector memory: T(EM) CD45RA(-)CD57(+) and terminally differentiated effector memory: T(EMRA) CD45RA(+)CD57(+)) were measured by flow cytometry. TLR7, TLR9 and RIG-I expression on CD4(+) T-cells and RIG-I expression on CD8(+) T-cells was higher in patients than healthy controls. Therapy increased expression of TLR2, TLR4 and TLR9 and this was observed for all T-cell sub-populations. Evaluation of TLR expression at baseline did not identify patients able to achieve sustained virological response following therapy.

Increased proportion of the CD56(bright) NK cell subset in patients chronically infected with hepatitis C virus (HCV) receiving interferon-alpha and ribavirin therapy.

Natural killer (NK) cells are implicated in the regulation of a protective immune response in patients chronically infected with hepatitis C virus (HCV), but effects of interferon-alpha/ribavirin therapy on NK cell subsets and the consequences of viral clearance during therapy remain unclear. Samples were collected from chronically infected patients (n = 34) at baseline and from a subset after 3-10 months on pegylated interferon-alpha and ribavirin therapy (n = 19). NK cells present in cryopreserved PBMC were characterized by flow cytometry. Before therapy, the frequency of CD3-CD56+ NK cells was lower in patients than uninfected controls. Therapy increased proportions of CD56(bright) NK cells. Frequencies of CD56(dim) NK cells declined slightly while perforin and CD16 expression on CD56(dim) NK cells decreased compared to baseline samples. Evaluation of NK cell subsets at baseline did not identify patients able to achieve sustained virological response following therapy. However, therapy may promote the expansion of NK cells able to produce interferon-gamma, while minimizing cytotoxicity to limit liver damage.

A Neisseria meningitidis NMB1966 mutant is impaired for invasion of respiratory epithelial cells, survival in human blood and for virulence in vivo.

We sought to determine whether NMB1966, encoding a putative ABC transporter, has a role in pathogenesis. Compared to its isogenic wild-type parent strain Neisseria meningitidis MC58, the NMB1966 knockout mutant was less adhesive and invasive for human bronchial epithelial cells, had reduced survival in human blood and was attenuated in a systemic mouse model of infection. The transcriptome of the wild-type and the NMB1966 mutant was compared. The data are consistent with a previous functional assignment of NMB1966 being the ABC transporter component of a glutamate transporter operon. Forty-seven percent of all the differentially regulated genes encoded known outer membrane proteins or pathways generating complex surface structures such as adhesins, peptidoglycan and capsule. The data show that NMB1966 has a role in virulence and that remodelling of the outer membrane and surface/structures is associated with attenuation of the NMB1966 mutant.

Hepatitis C virus genotype and HIV coinfection affect cytokine mRNA levels in unstimulated PBMC but do not shift the T1/T2 balance.

Rapid progression of hepatitis C virus (HCV) disease in patients with HIV/HCV may reflect different cytokine responses and be influenced by HCV genotype. This is addressed by a study of patients with HIV/HCV coinfection and infection with HCV genotype 2 or 3 (2/3). They are compared with coinfected patients infected with genotype 1 and HCV monoinfected patients matched for HCV genotype. IFN-gamma, IL-10, IL-4 and IL-4delta2 mRNA were quantified by real-time PCR in unstimulated PBMC and after in vitro stimulation with HCV core or nonstructural 3/4A antigen. In unstimulated PBMC, levels of IFN-gamma and IL-4 mRNA were lowest in HIV/HCV genotype 1 patients, intermediate in HIV/HCV genotype 2/3 patients and highest in HCV genotype 2/3 patients. Neither HCV genotype nor HIV affected levels of IL-10 mRNA in unstimulated PBMC or IFN-gamma, IL-4 and IL-10 mRNA in PBMC stimulated with HCV antigens. Levels of IL-4 and IL-4delta2 mRNA correlated in mitogen-stimulated PBMC from all patient groups but both were low in HIV/HCV genotype 1 patients. Serum soluble CD30 levels (a putative marker of a T2 cytokine environment) did not differ between patient groups. The data do not suggest a shift in the T1/T2 balance driven by HIV coinfection or HCV genotype but either may affect IL-4 bioavailability.

Quantitation of defective and ecotropic viruses during LP-BM5 infection by real time PCR and RT-PCR.

Murine AIDS (MAIDS) is a pathology induced by the LP-BM5 murine leukaemia virus mixture in susceptible strains of mice such as C57BL/6J resulting in lymphoproliferation and progressive immunodeficiency. The etiologic agent of this pathology is BM5d, a replication defective virus. BM5e is a replication competent virus in the viral mixture that functions as a helper virus. This paper describes real time PCR and RT-PCR assays for quantitation of the proviral DNA and viral RNA of BM5d and BM5e. Data is presented describing the change in BM5d and BM5e proviral DNA levels and viral RNA levels in both blood and spleen in the first 8 weeks of infection. Infected mice have increasing levels of BM5d and BM5e viral DNA and RNA detectable from as early as 2 weeks post infection. Similar levels of proviral DNA was found for BM5d and BM5e in PBMC and spleen, however higher levels of BM5e viral RNA were observed in both tissues throughout infection. The assays described can be used as both a diagnostic tool and to investigate the direct effect of treatments on the BM5d and BM5e viruses and MAIDS development.

Timed ablation of regulatory CD4+ T cells can prevent murine AIDS progression.

We describe successful immunotherapy of murine AIDS (MAIDS) in C57BL/6J mice based on the elimination of replicating CD4(+) regulator T cells. We demonstrate that a single injection of the antimitotic drug vinblastine (Vb) given 14 days postinfection (p.i.) with LP-BM5 can prevent MAIDS progression. Treatment with anti-CD4 mAb at 14 days p.i. is similarly able to prevent MAIDS. Treatment at other time points with Vb or anti-CD4 mAb is ineffective. The effect is based on ablation of a replicating dominantly suppressive CD4(+) T cell population, as indicated by adoptive transfer and in vivo depletion experiments using mAbs against CD4 as well as combinations of mAbs against the known regulatory cell surface markers CD25, GITR, and CTLA-4. Cell surface marker analysis shows a population of CD4(+)CD25(+) cells arising shortly before day 14 p.i. Cytokine analyses show a peak in IL-10 production from day 12 to day 16 p.i. MAIDS-infected mice also have CD4(+) T cells with significantly higher expression levels of CD38 and particularly CD69, which have been demonstrated to be regulator T cell markers in the Friend retroviral model. The immunotherapy appears to prevent disease progression, although no protection against reinfection with LP-BM5 is generated. These data define a new therapy for murine retroviral infection, which has potential for use in other diseases where T regulator cell-mediated immunosuppression plays a role in the disease process.

Interferon-gamma response by peripheral blood mononuclear cells to hepatitis C virus core antigen is reduced in patients with liver fibrosis.

Liver fibrosis was correlated with immunological parameters. Peripheral blood mononuclear cells (PBMCs) from patients with low fibrosis scores had more [corrected] interferon (IFN)-gamma-producing cells than did patients with higher fibrosis scores, when stimulated with hepatitis C virus (HCV) core antigen. Irrespective of liver fibrosis score, cells from all cytomegalovirus (CMV)-seropositive patients had similar IFN-gamma responses, when stimulated by CMV antigen, so patients with fibrosis did not have a broad-spectrum immunodeficiency. IFN-gamma response by PBMCs to HCV core antigen may provide a useful marker of the severity of liver disease in patients with hepatitis C.

Rhinovirus is not detectable in peripheral lung tissue after asthma death.

OBJECTIVE: Viral infections are associated with both mild and severe exacerbations of asthma and may therefore be associated with asthma death. As such we hypothesized that it might be possible to detect rhinovirus (RV), the virus most frequently implicated in acute asthma, in lung tissue from patients who died from asthma. METHODOLOGY: We studied archival, wax-embedded lung tissue obtained postmortem from: (i) patients who died from asthma (n = 12), (ii) asthma patients with non-asthma-related death (n = 3), and (iii) non-asthmatic individuals who died from unrelated causes (n = 3). A validated reverse transcription-polymerase chain reaction (RT-PCR) assay was used to detect RV. To confirm RNA preservation, RT-PCR was used to detect expression of the constitutive gene adenine-phosphoribosyl-transferase (APRT). Sensitivity of the assay was assessed using wax-embedded RV-infected cells. RESULTS: Sensitivity of RT-PCR for RV in wax-embedded sections was similar to previous studies (approximately 100 viral copies). Specimens used for study were predominantly of alveolar and small airway origin (< 2 mm). All tissues examined were negative for the presence of RV mRNA and positive for APRT mRNA. CONCLUSIONS: RV infection of the lower airway may be an uncommon cause of fatal asthma. Alternatively, RV may not extend to peripheral airways and more proximal tissue sampling or PCR assays for other viruses may be required to determine an association between viral respiratory tract infection and fatal asthma.