[This corrects the article DOI: 10.1371/journal.pone.0009681.].

The first systematic investigation into the Baeyer-Villiger reaction of an anthraquinone is presented. The double Baeyer-Villiger reaction of quinizarin dimethyl ether is viable, directly providing the dibenzo[b,f][1,4]-dioxocin-6,11-dione ring-system, which is otherwise difficult to prepare. This methodology provides rapid access to 1,2,3,4-tetraoxygenated benzenes, and has been exploited by application to the total synthesis of a natural occurring benzodioxole and its biphenyl dimer, which both display noteworthy biological activity. Interestingly, the axially chiral biphenyl was found to be configurationally stable, but the resolved enantiomers exhibit no optical activity at the αD-line.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Immunology & Cell BiologyVolume 93, Issue 9 p. 765-766 Obituary Geoff Shellam (1942–2015) Alec Redwood, Corresponding Author Alec Redwood a.redwood@iiid.murdoch.edu.au Institute for Immunology and Infectious Diseases, Murdoch University, Murdoch, Western Australia, AustraliaAlec Redwood, E-mail: a.redwood@iiid.murdoch.edu.auSearch for more papers by this authorMegan Lloyd, Megan Lloyd School of Pathology and Laboratory Medicine, The University of Western Australia, Crawley, Western Australia, AustraliaSearch for more papers by this authorJane Allan, Jane Allan School of Medicine and Pharmacology, The University of Western Australia, Crawley, Western Australia, AustraliaSearch for more papers by this authorTony Scalzo, Tony Scalzo Botanic Gardens & Parks Authority, Kings Park and Botanic Garden, Fraser Avenue, Kings Park, Western Australia, AustraliaSearch for more papers by this author Alec Redwood, Corresponding Author Alec Redwood a.redwood@iiid.murdoch.edu.au Institute for Immunology and Infectious Diseases, Murdoch University, Murdoch, Western Australia, AustraliaAlec Redwood, E-mail: a.redwood@iiid.murdoch.edu.auSearch for more papers by this authorMegan Lloyd, Megan Lloyd School of Pathology and Laboratory Medicine, The University of Western Australia, Crawley, Western Australia, AustraliaSearch for more papers by this authorJane Allan, Jane Allan School of Medicine and Pharmacology, The University of Western Australia, Crawley, Western Australia, AustraliaSearch for more papers by this authorTony Scalzo, Tony Scalzo Botanic Gardens & Parks Authority, Kings Park and Botanic Garden, Fraser Avenue, Kings Park, Western Australia, AustraliaSearch for more papers by this author First published: 01 October 2015 https://doi.org/10.1038/icb.2015.83Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume93, Issue9October 2015Pages 765-766 Journal of the Australian and New Zealand Society for Immunology RelatedInformation

Toxoplasma gondii (T. gondii) causes serious infection, especially in immunocompromised hosts. The relevance of animal models of toxoplasmosis to human disease is unclear, but have indicated that the route of Toxoplasma infection may affect the outcome. A humanized model of toxoplasmosis of immunocompromised mice (i.e. hu-PBL SCID), using the intraperitoneal (IP) route demonstrated long-term engraftment of human cells and worsening of inflammation compared to controls. In this study, we examined the effect of route of infection on this hu-PBL SCID model using a Toxoplasma strain (i.e. DAG) isolated from an immunocompromised human. Oral infection led to an asymptomatic infection, whereas animals infected by the IP route succumbed more quickly to infection. Human cells, detected through species-specific β-actin mRNA, were not as prominent in IP-infected animals as compared to orally infected and uninfected animals. There was evidence of control of toxoplasmosis in some orally infected animals, and this was associated with the presence of human cells in multiple tissues. Thus, the route of infection dramatically affects the outcome of infection, either by affecting parasite replication or expansion of human immune cells. Further studies of oral Toxoplasma infection using hu-PBL SCID mice may help in developing chemotherapies and immunotherapeutic strategies for toxoplasmosis.

Neutralising antibodies to hepatitis C virus (HCV) are studied to characterise the determinants of HCV virulence and for vaccine and immunotherapy development. To discover useful antibodies, patients are screened for HCV neutralising activity by incubating their serum with a lab strain of HCV, and incubating this with susceptible cells. Days later the cultures are stained to reveal infected cells and the amount of infecting virus is expressed as focus forming units (FFU). This method is expensive and time-consuming. The Huh7-J20 reporter cell line, provided by Dr A Patel, may provide a cheaper and faster alternative. When this line is infected with HCV, the NS3/4A viral protease cleaves a reporter protein, releasing secreted alkaline phosphatase (SEAP) into the culture supernatant, which can be measured by spectrophotometry.

Murine and human mesothelioma tumors are susceptible to immunotherapy, particularly when immune adjuvants are delivered locally. We have shown that direct injection of IL-2 plus agonist anti-CD40 antibody induces regression of large mesothelioma tumors. These studies aimed to determine if NK cells contribute to IL-2/CD40 antibody-driven tumor eradication. We show that NK cells infiltrate developing mesothelioma tumors; however, their absence (in beige mice or in asialo GM(1) antibody-depleted C57BL/6J mice) does not alter tumor growth rates suggesting that they cannot function as effector cells in this microenvironment. Anti-CD40 antibody treatment did not alter the percent of NK cells in treated tumors or in draining lymph nodes (dLNs), and tumor resolution occurred in the absence of NK cells. However, a two-tumor model showed that NK cells contributed to CD40-driven systemic immunity leading to resolution of untreated distal tumors. IL-2 treatment led to increased proportions of NK cells in tumors and dLNs, and in the absence of NK cells, IL-2 lost its therapeutic effect. In contrast, the absence of NK cells did not reduce the anti-tumor activity of the IL-2/anti-CD40 antibody combination yet tumors recurred in NK-deficient mice and > 37% of tumor cell re-challenged mice were unable to provide protection, implying insufficient memory. Furthermore, untreated distal tumors in NK-depleted mice were less readily cured than in immunologically intact mice. These data show that NK cells infiltrate mesothelioma tumors, which, after local IL-2 and/or anti-CD40 antibody treatment, provide help for the acquisition and/or maintenance of systemic immunity and long-term effector/memory responses.

Background and Aims: Ultra-deep Pyrosequencing (UDPS) on the GS-FLX 454/Roche platform has been proposed as a powerful tool to study quasispecies dynamics.Here we have studied in depth the differences between the quasispecies of a chronic carrier and his partner who was infected by HCV through sexual route.Methods: Seven sets of overlapping primers were used to cover the complete NS3 protein (protease and helicase domain -1893nucleotides) from two G1a patients (chronic patient A [liver -L98A -and plasma from 1998 -p98A] who sexually transmitted infection to patient B [plasma 1998 -p98B] who resolved infection).High fidelity RT Accuscript and high fidelity Pfu UltraII Fusion HS polymerase has been used for RT-PCR-Nested amplifications.Results: Mutation frequency was similar in all subregions of acute patient B (master 85-92%).The acute infected patient B had a more homogeneous population than chronic patient A at the amino acid level.UDPS confirm that anti-HCV-NS3 protease resistant mutations were present at very low proportions (≥0.1) in both untreated patients.Massive sequencing analysis evidenced changes in CD4 and CD8 epitopes thus revealing differential selective pressures in the chronic patient compared to the acute one.Phylogenetic analysis of sequences revealed that infection of patient B could be initiated from a very few number of virus present in the chronic patient.IL28 genotype for the SNP rs12979860 and rs8099917 was CT-GT and CC-TT for patient A and B respectively.Interestingly the acute patient had the homozygous combinations CC and TT in both positions associated with the response to antiviral therapy.Conclusions: The transmission of a reduced number of viral particles (bottlenecking), the high homogeneity in the quasispecies of patient B together with the CC genotype in rs12979860 and TT in rs8099917 associated with higher probability to clear infection, may have facilitated self-resolution of HCV infection in patient B.

Cytomegalovirus (CMV) is a β-herpesvirus that establishes a lifelong latent or persistent infection. A hallmark of chronic CMV infection is the lifelong persistence of large numbers of virus-specific CD8+ effector/effector memory T cells, a phenomenon called "memory inflation". How the virus continuously stimulates these T cells without being eradicated remains an enigma. The prevailing view is that CMV establishes a low grade "smoldering" infection characterized by tiny bursts of productive infection which are rapidly extinguished, leaving no detectable virus but replenishing the latent pool and leaving the immune system in a highly charged state. However, since abortive reactivation with limited viral gene expression is known to occur commonly, we investigated the necessity for virus reproduction in maintaining the inflationary T cell pool. We inhibited viral replication or spread in vivo using two different mutants of murine CMV (MCMV). First, famcyclovir blocked the replication of MCMV encoding the HSV Thymidine Kinase gene, but had no impact on the CD8+ T cell memory inflation once the infection was established. Second, MCMV that lacks the essential glycoprotein L, and thus is completely unable to spread from cell to cell, also drove memory inflation if the virus was administered systemically. Our data suggest that CMV which cannot spread from the cells it initially infects can repeatedly generate viral antigens to drive memory inflation without suffering eradication of the latent genome pool.

CD8+ T cells can be primed by peptides derived from endogenous proteins (direct presentation), or exogenously acquired protein (cross-presentation). However, the relative ability of these two pathways to prime CD8+ T cells during a viral infection remains controversial. Cytomegaloviruses (CMVs) can infect professional antigen presenting cells (APCs), including dendritic cells, thus providing peptides for direct presentation. However, the viral immune evasion genes profoundly impair recognition of infected cells by CD8+ T cells. Nevertheless, CMV infection elicits a very strong CD8+ T cell response, prompting its recent use as a vaccine vector. We have shown previously that deleting the immune evasion genes from murine cytomegalovirus (MCMV) that target class I MHC presentation, has no impact on the size or breadth of the CD8+ T cell response elicited by infection, suggesting that the majority of MCMV-specific CD8+ T cells in vivo are not directly primed by infected professional APCs. Here we use a novel spread-defective mutant of MCMV, lacking the essential glycoprotein gL, to show that cross-presentation alone can account for the majority of MCMV-specific CD8+ T cell responses to the virus. Our data support the conclusion that cross-presentation is the primary mode of antigen presentation by which CD8+ T cells are primed during MCMV infection.

The greater resistance of HCV genotype 1 infection to IFN therapy has been partially attributed to functional inhibition of the type 1 interferon induced anti-viral protein PKR in vitro. Whether PKR has antiviral activity against HCV in vivo is unknown. Whilst the ultra-structural localisation of PKR is known in vitro, it is not defined in chronic hepatitis C disease. Using a novel immuno-gold technique we characterised the expression of intrahepatic PKR protein at the ultra-structural level in four patients with chronic HCV disease compared to normal human PBMCs, HepG2 cells and a normal human liver biopsy. All four HCV patients labelled for PKR protein, localising to the nucleus, nucleolus and cytoplasm. Nuclear labelling was confined mainly to the nucleolus and euchromatin. Cytoplasmic labelling was evident within smooth vesicles. Strong immunogold labelling was also evident within the cisternae of the rough endoplasmic reticulum. A similar pattern of ultra-structural nuclear and cytoplasmic PKR protein labelling was seen in PBMCs from healthy donors, HepG2 cells and a normal liver biopsy. The mean nuclear and cytoplasmic count for PKR protein in the HCV group was 21 ± 4 and 18 ± 3 gold particles/μm2, respectively. This represented an increase, though not statistically significant, in nuclear and cytoplasmic labelling for PKR protein in HCV biopsies relative to normal liver tissue.

Differentiation of recurrent hepatitis C virus (HCV) disease from acute cellular rejection (ACR) following liver transplantation can be difficult. Previously, we have found that MxA protein, a specific and sensitive marker for type 1 interferon production, is strongly expressed in chronic HCV disease. Here, we investigate MxA expression as a marker for recurrent HCV disease in the livers of 14 adult HCV patients who underwent liver transplantation. Serial liver biopsies available for 12 of these patients were stained for MxA protein and scored using a semi-quantitative approach. Hepatocellular MxA protein levels were significantly up-regulated (p = 0.025) in recurrent HCV disease in comparison to ACR. In biopsies that showed histological changes consistent with recurrent HCV disease, strong hepatocellular MxA staining was present in 14/18 (78%). In the liver biopsies with histological evidence of ACR, strong MxA hepatocellular staining was present in only three of 10 (30%). Thus, assessment of hepatocellular MxA protein expression can contribute to the differential diagnosis of ACR and recurrent HCV disease following liver transplantation. In conclusion, analysis of intrahepatic MxA levels has the potential to reduce the inappropriate use with high-dose pulsing of steroids post-operatively.

Anti-cancer immunotherapies aim to generate resolution of all existing tumors, including inaccessible ones, and provide long-term protection against recurrence. This is rarely achieved. Thus, we aimed to determine if the tumor microenvironment could be turned into a potent 'self'-vaccine site. Our target was to eradicate larger tumor burdens. Our models respond to single-agent immunotherapies; however, they fail at a precisely defined 'cut-off' tumor burden. Thus, this system was used to define the immune mechanisms required to mediate regression of larger tumors that are resistant to mono-immunotherapies. We report that direct injection of IL-2 with agonist anti-CD40 antibody into the tumor bed resulted in permanent resolution of treated and untreated distal tumors. Tumor-infiltrating CD8(+) T cells and neutrophils collaborated to eradicate treated tumors, IFNgamma was not critical and protective memory was preserved. This approach relied only on tumor antigens expressed within the tumor microenvironment. It also avoided systemic toxicities, did not require chemotherapy or surgery and is clinically useful because only one tumor site has to be accessible for treatment. We conclude that provoking intra-tumoral inflammation skews the tumor microenvironment from tumorigenic to immunogenic, resulting in the resolution of treated and untreated distal tumors, as well long-term protective memory.

Despite improved diagnostic skills and new chemotherapeutic regimens, malignant mesothelioma (MM) remains a pathological disease with survival expectations after diagnosis remaining < 1 year. As the incidence of this disease has yet to peak, there is a pressing need for new therapeutic approaches. One such approach is gene therapy, which inserts 'therapeutic' genes into (generally) tumour cells seeking to induce tumour regression via a number of different theoretical mechanisms. This approach may be particularly relevent for mesothelioma as it is localised to body cavities and is readily accessible for biopsy sampling or for gene delivery. Furthermore, as MM patients rarely die from distant metastases, treating the primary tumour site may result in significant symptomatic and survival benefit. Herein, the paper discusses past, present and future views on gene therapy in the treatment of MM.

At this stage, the only cell population that is directly implicated in the induction of fatal lymphocytic choriomeningitis is the class I MHC-restricted immune T cell. However, it is not clear that cell-mediated lysis is central to the development of neurological symptoms or that the T cells act alone to cause immunopathology.

UNLABELLED:Abstract Background and Aim: The presence of four or more amino acid substitutions within the interferon sensitivity determining region (ISDR) of the hepatitis C virus (HCV) genotype 1b NS5A gene determines sensitivity to interferon (IFN) monotherapy in Japanese patients. Resistance of HCV genotype 1 to IFN-alpha has been attributed to the functional inhibition of a RNA dependent protein kinase (PKR) by the HCV NS5A PKR binding domain (PKRBD), which includes the ISDR. The ability of the ISDR and PKRBD sequence to predict a response to IFN-alpha and ribavirin combination therapy was investigated in an Australian population. METHODS:The sequence of the PKRBD of NS5A, including the ISDR, for the dominant quasi-species of HCV was determined in 37 genotype 1 (genotype 1a: n = 26, genotype 1b: n = 11) and 13 genotype 3a infected patients. RESULTS:The number of PKRBD amino acid substitutions in HCV genotype 1 infected patients with a sustained virological response was significantly higher than that in patients with a non-response to treatment (P = 0.047). It was found that only 2/37 HCV genotype 1 infected patients had four or more amino acid substitutions relative to the prototype ISDR sequence (HCV-J). Importantly, a sustained virological response was not found in any of the HCV infected patients who had a prototype ISDR genotype 1 sequence (n = 5). CONCLUSIONS:There are relatively few amino acid mutations within the ISDR of this Western Australian patient population. Patients infected with a HCV genotype 1 prototype sequence should be counseled before receiving combination IFN-alpha and ribavirin therapy as they have a poor response to treatment.

The success of interferon-alpha and ribavirin combination therapy for the treatment of chronic hepatitis C viral infection differs between patients. In an attempt to identify predictors of host response to therapy, the levels of mRNA for interferon (IFN) stimulated genes: MxA, PKR, 2'5' OAS, ISG15, and interleukin 8 (IL-8), were examined in liver by real-time RT-PCR prior to commencement of therapy. The levels of intrahepatic classical IFN stimulated genes, but not IL-8, in chronic HCV disease (n = 44) were found to be significantly upregulated (P < 0.001) compared to the control cohort (n = 12). The genotype of the infecting HCV strain did not influence IFN stimulated gene expression. These results suggest that the endogenous type 1 IFN antiviral effector pathway is broadly activated during chronic HCV disease, although the levels of mRNA for any of the IFN-stimulated genes tested did not predict the outcome of combination therapy.