You Can Breathe Easy: IFNλ Handles Flu without Triggering a Damaging Inflammatory Response.

Type III interferon (IFNλ) and type I IFN (IFNα/ß) have overlapping antiviral activities in the lung. In this issue of Immunity, Galani et al. (2017) identify a critical early role for IFNλ, not shared by IFNα/ß, in protection of the lung following influenza virus infection.

Limited Association Between Ascorbate Concentrations and Vitamin C Transporters in Renal Cell Carcinoma Cells and Clinical Samples.

BACKGROUND/AIMS: Maintenance of whole-body ascorbate levels and distribution is mediated via sodium-dependent vitamin C transporters (SVCTs). The kidney is one of a few organs that express both SVCT1 and SVCT2. Recent evidence suggests that accumulation of ascorbate may be different in tumour compared to normal tissue, but data on SVCT levels in tumours is sparse. METHODS: The role of the two SVCT isoforms in ascorbate uptake in renal cell carcinoma (RCC) was investigated in vitro and in clinical samples. In three human RCC cell lines, we investigated SVCT protein levels and cellular location in response to ascorbate supplementation and withdrawal. In clinical RCC samples (n=114), SVCT patterns of staining and protein levels were analysed and compared to ascorbate levels. RESULTS: In cell culture, transporter levels and cellular location were not modified by ascorbate availability at any time up to 8h, although basal SVCT2 levels governed maximal ascorbate accumulation. In clinical samples, SVCT1 protein levels in papillary RCC (pRCC) were similar to matched normal renal cortex, but were increased in clear-cell RCC (ccRCC). Native SVCT2 (72 kDa) was significantly decreased in both pRCC and ccRCC tissues compared to cortex (p<0.01), whereas a modified form of SVCT2 (100 kDa) was significantly increased (p<0.001). There was no association between the transporters (SVCT1, native or modified SVCT2) and ascorbate concentrations in either normal or tumour tissues. SVCT1 and SVCT2 displayed diffuse cytoplasmic staining in both pRCC and ccRCC tumour cells, with cortex showing distinct membrane staining for SVCT1. CONCLUSION: We observed a re-distribution of ascorbate transporters in tumour tissue compared to normal cortex and a shift from native to modified SVCT2 in cell culture and clinical samples. Data presented here show that SVCT protein levels do not appear to predict intracellular ascorbate accumulation in RCC.

Caspase-mediated cleavage of murine norovirus NS1/2 potentiates apoptosis and is required for persistent infection of intestinal epithelial cells.

Human norovirus (HNoV) is the leading cause of acute gastroenteritis and is spread by fecal shedding that can often persist for weeks to months after the resolution of symptoms. Elimination of persistent viral reservoirs has the potential to prevent outbreaks. Similar to HNoV, murine norovirus (MNV) is spread by persistent shedding in the feces and provides a tractable model to study molecular mechanisms of enteric persistence. Previous studies have identified non-structural protein 1 (NS1) from the persistent MNV strain CR6 as critical for persistent infection in intestinal epithelial cells (IECs), but its mechanism of action remains unclear. We now find that the function of CR6 NS1 is regulated by apoptotic caspase cleavage. Following induction of apoptosis in infected cells, caspases cleave the precursor NS1/2 protein, and this cleavage is prevented by mutation of caspase target motifs. These mutations profoundly compromise CR6 infection of IECs and persistence in the intestine. Conversely, NS1/2 cleavage is not strictly required for acute replication in extra-intestinal tissues or in cultured myeloid cells, suggesting an IEC-centric role. Intriguingly, we find that caspase cleavage of CR6 NS1/2 reciprocally promotes caspase activity, potentiates cell death, and amplifies spread among cultured IEC monolayers. Together, these data indicate that the function of CR6 NS1 is regulated by apoptotic caspases, and suggest that apoptotic cell death enables epithelial spread and persistent shedding.

Identifying the assembly intermediate in which Gag first associates with unspliced HIV-1 RNA suggests a novel model for HIV-1 RNA packaging.

During immature capsid assembly, HIV-1 genome packaging is initiated when Gag first associates with unspliced HIV-1 RNA by a poorly understood process. Previously, we defined a pathway of sequential intracellular HIV-1 capsid assembly intermediates; here we sought to identify the intermediate in which HIV-1 Gag first associates with unspliced HIV-1 RNA. In provirus-expressing cells, unspliced HIV-1 RNA was not found in the soluble fraction of the cytosol, but instead was largely in complexes ≥30S. We did not detect unspliced HIV-1 RNA associated with Gag in the first assembly intermediate, which consists of soluble Gag. Instead, the earliest assembly intermediate in which we detected Gag associated with unspliced HIV-1 RNA was the second assembly intermediate (~80S intermediate), which is derived from a host RNA granule containing two cellular facilitators of assembly, ABCE1 and the RNA granule protein DDX6. At steady-state, this RNA-granule-derived ~80S complex was the smallest assembly intermediate that contained Gag associated with unspliced viral RNA, regardless of whether lysates contained intact or disrupted ribosomes, or expressed WT or assembly-defective Gag. A similar complex was identified in HIV-1-infected T cells. RNA-granule-derived assembly intermediates were detected in situ as sites of Gag colocalization with ABCE1 and DDX6; moreover these granules were far more numerous and smaller than well-studied RNA granules termed P bodies. Finally, we identified two steps that lead to association of assembling Gag with unspliced HIV-1 RNA. Independent of viral-RNA-binding, Gag associates with a broad class of RNA granules that largely lacks unspliced viral RNA (step 1). If a viral-RNA-binding domain is present, Gag further localizes to a subset of these granules that contains unspliced viral RNA (step 2). Thus, our data raise the possibility that HIV-1 packaging is initiated not by soluble Gag, but by Gag targeted to a subset of host RNA granules containing unspliced HIV-1 RNA.

Activation of the hypoxia pathway in breast cancer tissue and patient survival are inversely associated with tumor ascorbate levels.

BACKGROUND: The transcription factor hypoxia inducible factor (HIF) -1 drives tumor growth and metastasis and is associated with poor prognosis in breast cancer. Ascorbate can moderate HIF-1 activity in vitro and is associated with HIF pathway activation in a number of cancer types, but whether tissue ascorbate levels influence the HIF pathway in breast cancer is unknown. In this study we investigated the association between tumor ascorbate levels and HIF-1 activation and patient survival in human breast cancer. METHODS: In a retrospective analysis of human breast cancer tissue, we analysed primary tumor and adjacent uninvolved tissue from 52 women with invasive ductal carcinoma. We measured HIF-1α, HIF-1 gene targets CAIX, BNIP-3 and VEGF, and ascorbate content. Patient clinical outcomes were evaluated against these parameters. RESULTS: HIF-1 pathway proteins were upregulated in tumor tissue and increased HIF-1 activation was associated with higher tumor grade and stage, with increased vascular invasion and necrosis, and with decreased disease-free and disease-specific survival. Grade 1 tumors had higher ascorbate levels than did grade 2 or 3 tumors. Higher ascorbate levels were associated with less tumor necrosis, with lower HIF-1 pathway activity and with increased disease-free and disease-specific survival. CONCLUSIONS: Our findings indicate that there is a direct correlation between intracellular ascorbate levels, activation of the HIF-1 pathway and patient survival in breast cancer. This is consistent with the known capacity of ascorbate to stimulate the activity of the regulatory HIF hydroxylases and suggests that optimisation of tumor ascorbate could have clinical benefit via modulation of the hypoxic response.

Quantifying BRCA1 and BRCA2 mRNA Isoform Expression Levels in Single Cells.

BRCA1 and BRCA2 spliceogenic variants are often associated with an elevated risk of breast and ovarian cancers. Analyses of BRCA1 and BRCA2 splicing patterns have traditionally used technologies that sample a population of cells but do not account for the variation that may be present between individual cells. This novel proof of concept study utilises RNA in situ hybridisation to measure the absolute expression of BRCA1 and BRCA2 mRNA splicing events in single lymphoblastoid cells containing known spliceogenic variants (BRCA1c.671-2 A>G or BRCA2c.7988 A>T). We observed a large proportion of cells (>42%) in each sample that did not express mRNA for the targeted gene. Increased levels (average mRNA molecules per cell) of BRCA2 ∆17_18 were observed in the cells containing the known spliceogenic variant BRCA2c.7988 A>T, but cells containing BRCA1c.671-2 A>G were not found to express significantly increased levels of BRCA1 ∆11, as had been shown previously. Instead, we show for each variant carrier sample that a higher proportion of cells expressed the targeted splicing event compared to control cells. These results indicate that BRCA1/2 mRNA is expressed stochastically, suggesting that previously reported results using RT-PCR may have been influenced by the number of cells with BRCA1/2 mRNA expression and may not represent an elevation of constitutive mRNA expression. Detection of mRNA expression in single cells allows for a more comprehensive understanding of how spliceogenic variants influence the expression of mRNA isoforms. However, further research is required to assess the utility of this technology to measure the expression of predicted spliceogenic BRCA1 and BRCA2 variants in a diagnostic setting.

Mutations of Conserved Residues in the Major Homology Region Arrest Assembling HIV-1 Gag as a Membrane-Targeted Intermediate Containing Genomic RNA and Cellular Proteins.

UNLABELLED: The major homology region (MHR) is a highly conserved motif that is found within the Gag protein of all orthoretroviruses and some retrotransposons. While it is widely accepted that the MHR is critical for assembly of HIV-1 and other retroviruses, how the MHR functions and why it is so highly conserved are not understood. Moreover, consensus is lacking on when HIV-1 MHR residues function during assembly. Here, we first addressed previous conflicting reports by confirming that MHR deletion, like conserved MHR residue substitution, leads to a dramatic reduction in particle production in human and nonhuman primate cells expressing HIV-1 proviruses. Next, we used biochemical analyses and immunoelectron microscopy to demonstrate that conserved residues in the MHR are required after assembling Gag has associated with genomic RNA, recruited critical host factors involved in assembly, and targeted to the plasma membrane. The exact point of inhibition at the plasma membrane differed depending on the specific mutation, with one MHR mutant arrested as a membrane-associated intermediate that is stable upon high-salt treatment and other MHR mutants arrested as labile, membrane-associated intermediates. Finally, we observed the same assembly-defective phenotypes when the MHR deletion or conserved MHR residue substitutions were engineered into Gag from a subtype B, lab-adapted provirus or Gag from a subtype C primary isolate that was codon optimized. Together, our data support a model in which MHR residues act just after membrane targeting, with some MHR residues promoting stability and another promoting multimerization of the membrane-targeted assembling Gag oligomer. IMPORTANCE: The retroviral Gag protein exhibits extensive amino acid sequence variation overall; however, one region of Gag, termed the major homology region, is conserved among all retroviruses and even some yeast retrotransposons, although the reason for this conservation remains poorly understood. Highly conserved residues in the major homology region are required for assembly of retroviruses; however, when these residues are required during assembly is not clear. Here, we used biochemical and electron microscopic analyses to demonstrate that these conserved residues function after assembling HIV-1 Gag has associated with genomic RNA, recruited critical host factors involved in assembly, and targeted to the plasma membrane but before Gag has completed the assembly process. By revealing precisely when conserved residues in the major homology region are required during assembly, these studies resolve existing controversies and set the stage for future experiments aimed at a more complete understanding of how the major homology region functions.

A Rhesus Rhadinovirus Viral Interferon (IFN) Regulatory Factor Is Virion Associated and Inhibits the Early IFN Antiviral Response.

UNLABELLED: The interferon (IFN) response is the earliest host immune response dedicated to combating viral infection. As such, viruses have evolved strategies to subvert this potent antiviral response. Two closely related gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and rhesus macaque rhadinovirus (RRV), are unique in that they express viral homologues to cellular interferon regulatory factors (IRFs), termed viral IRFs (vIRFs). Cellular IRFs are a family of transcription factors that are particularly important for the transcription of type I IFNs. Here, we demonstrate a strategy employed by RRV to ensure rapid inhibition of virus-induced type I IFN induction. We found that RRV vIRF R6, when expressed ectopically, interacts with a transcriptional coactivator, CREB-binding protein (CBP), in the nucleus. As a result, phosphorylated IRF3, an important transcriptional regulator in beta interferon (IFN-ß) transcription, fails to effectively bind to the IFN-ß promoter, thus inhibiting the activation of IFN-ß genes. In addition, we found R6 within RRV virion particles via immunoelectron microscopy and, furthermore, that virion-associated R6 is capable of inhibiting the type I IFN response by preventing efficient binding of IRF3/CBP complexes to the IFN-ß promoter in the context of infection. The work shown here is the first example of a vIRF being associated with either the KSHV or RRV virion. The presence of this immunomodulatory protein in the RRV virion provides the virus with an immediate mechanism to evade the host IFN response, thus enabling the virus to effectively establish an infection within the host. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related rhesus macaque rhadinovirus (RRV) are the only viruses known to encode viral homologues to cellular interferon regulatory factors (IRFs), known as vIRFs. In KSHV, these proteins have been shown to play major roles in a variety of cellular processes and are particularly important in the evasion of the host type I interferon (IFN) response. In this study, we delineate the immunomodulatory mechanism of an RRV vIRF and its ability to assist the virus in rapid immune evasion by being prepackaged within the virion, thus providing evidence, for the first time, of a virion-associated vIRF. This work further contributes to our understanding of the mechanisms behind immunomodulation by the RRV vIRFs during infection.

A temporospatial map that defines specific steps at which critical surfaces in the Gag MA and CA domains act during immature HIV-1 capsid assembly in cells.

UNLABELLED: During HIV-1 assembly, Gag polypeptides target to the plasma membrane, where they multimerize to form immature capsids that undergo budding and maturation. Previous mutational analyses identified residues within the Gag matrix (MA) and capsid (CA) domains that are required for immature capsid assembly, and structural studies showed that these residues are clustered on four exposed surfaces in Gag. Exactly when and where the three critical surfaces in CA function during assembly are not known. Here, we analyzed how mutations in these four critical surfaces affect the formation and stability of assembly intermediates in cells expressing the HIV-1 provirus. The resulting temporospatial map reveals that critical MA residues act during membrane targeting, residues in the C-terminal CA subdomain (CA-CTD) dimer interface are needed for the stability of the first membrane-bound assembly intermediate, CA-CTD base residues are necessary for progression past the first membrane-bound intermediate, and residues in the N-terminal CA subdomain (CA-NTD) stabilize the last membrane-bound intermediate. Importantly, we found that all four critical surfaces act while Gag is associated with the cellular facilitators of assembly ABCE1 and DDX6. When correlated with existing structural data, our findings suggest the following model: Gag dimerizes via the CA-CTD dimer interface just before or during membrane targeting, individual CA-CTD hexamers form soon after membrane targeting, and the CA-NTD hexameric lattice forms just prior to capsid release. This model adds an important new dimension to current structural models by proposing the potential order in which key contacts within the immature capsid lattice are made during assembly in cells. IMPORTANCE: While much is known about the structure of the completed HIV-1 immature capsid and domains of its component Gag proteins, less is known about the sequence of events leading to formation of the HIV-1 immature capsid. Here we used biochemical and ultrastructural analyses to generate a temporospatial map showing the precise order in which four critical surfaces in Gag act during immature capsid formation in provirus-expressing cells. Because three of these surfaces make important contacts in the hexameric lattices that are found in the completed immature capsid, these data allow us to propose a model for the sequence of events leading to formation of the hexameric lattices. By providing a dynamic view of when and where critical Gag-Gag contacts form during the assembly process and how those contacts function in the nascent capsid, our study provides novel insights into how an immature capsid is built in infected cells.

Phosphohistone H3 outperforms Ki67 as a marker of outcome for breast cancer patients.

AIMS: The proliferation marker Ki67 has been extensively investigated as a prognostic factor in breast cancer, but has not gained widespread clinical acceptance. Phosphohistone H3 is a new immunohistochemical marker for quantifying mitoses; however, there is limited information on its prognostic value in breast cancer. In this study, we performed a head-to-head comparison of Ki67 and phosphohistone H3 to establish the marker with the greatest prognostic value. METHODS AND RESULTS: Tissue microarrays from 108 breast cancer patients were immunohistochemically stained for Ki67 and phosphohistone H3. Our results showed that phosphohistone H3 had a greater prognostic value than Ki67 in a multivariable model that adjusted for traditional prognostic variables in breast cancer. Phosphohistone H3 staining was a stronger predictor of survival at 5 years after diagnosis [hazard ratio (HR) 4.35, P < 10(-5) ] than Ki67 (HR 2.44, P = 0.004), and better separated the risk of death in patients aged >45 years. Importantly, phosphohistone H3 consistently showed strong unequivocal staining, in contrast to the variable staining intensities associated with Ki67. CONCLUSIONS: Our study suggests that phosphohistone H3 staining is a stronger and more robust prognostic indicator than Ki67 staining in breast cancer patients, and has the potential for use in routine diagnostic laboratories.

Blood Vitamin C Levels of Patients Receiving Immunotherapy and Relationship to Monocyte Subtype and Epigenetic Modification.

The treatment of metastatic melanoma has been revolutionised by immunotherapy, yet a significant number of patients do not respond, and many experience autoimmune adverse events. Associations have been reported between patient outcome and monocyte subsets, whereas vitamin C (ascorbate) has been shown to mediate changes in cancer-stimulated monocytes in vitro. We therefore investigated the relationship of ascorbate with monocyte subsets and epigenetic modifications in patients with metastatic melanoma receiving immunotherapy. Patients receiving immunotherapy were compared to other cancer cohorts and age-matched healthy controls. Ascorbate levels in plasma and peripheral blood-derived mononuclear cells (PBMCs), monocyte subtype and epigenetic markers were measured, and adverse events, tumour response and survival were recorded. A quarter of the immunotherapy cohort had hypovitaminosis C, with plasma and PBMC ascorbate levels significantly lower than those from other cancer patients or healthy controls. PBMCs from the immunotherapy cohort contained similar frequencies of non-classical and classical monocytes. DNA methylation markers and intracellular ascorbate concentration were correlated with monocyte subset frequency in healthy controls, but correlation was lost in immunotherapy patients. No associations between ascorbate status and immune-related adverse events or tumour response or overall survival were apparent.

Viral interferon regulatory factors decrease the induction of type I and type II interferon during rhesus macaque rhadinovirus infection.

Kaposi's sarcoma-associated herpesvirus and rhesus macaque rhadinovirus (RRV), two closely related gammaherpesviruses, are unique in their expression of viral homologs of cellular interferon regulatory factors (IRFs), termed viral IRFs (vIRFs). To assess the role of vIRFs during de novo infection, we have utilized the bacterial artificial chromosome clone of wild-type RRV(17577) (WT(BAC) RRV) to generate a recombinant virus with all 8 of the vIRFs deleted (vIRF-ko RRV). The infection of primary rhesus fibroblasts and peripheral blood mononuclear cells (PBMCs) with vIRF-ko RRV resulted in earlier and increased induction of type I interferon (IFN) (IFN-α/ß) and type II IFN (IFN-γ). Additionally, plasmacytoid dendritic cells maintained higher levels of IFN-α production in PBMC cultures infected with vIRF-ko RRV than in cultures infected with WT(BAC) RRV. Moreover, the nuclear accumulation of phosphorylated IRF-3, which is necessary for the induction of type I IFN, was also inhibited following WT(BAC) RRV infection. These findings demonstrate that during de novo RRV infection, vIRFs are inhibiting the induction of IFN at the transcriptional level, and one potential mechanism for this is the disruption of the activation and localization of IRF-3.

Viral interferon regulatory factors are critical for delay of the host immune response against rhesus macaque rhadinovirus infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related gamma-2 herpesvirus rhesus macaque (RM) rhadinovirus (RRV) are the only known viruses to encode viral homologues of the cellular interferon (IFN) regulatory factors (IRFs). Recent characterization of a viral IRF (vIRF) deletion clone of RRV (vIRF-knockout RRV [vIRF-ko RRV]) demonstrated that vIRFs inhibit induction of type I and type II IFNs during RRV infection of peripheral blood mononuclear cells. Because the IFN response is a key component to a host's antiviral defenses, this study has investigated the role of vIRFs in viral replication and the development of the immune response during in vivo infection in RMs, the natural host of RRV. Experimental infection of RMs with vIRF-ko RRV resulted in decreased viral loads and diminished B cell hyperplasia, a characteristic pathology during acute RRV infection that often develops into more severe lymphoproliferative disorders in immune-compromised animals, similar to pathologies in KSHV-infected individuals. Moreover, in vivo infection with vIRF-ko RRV resulted in earlier and sustained production of proinflammatory cytokines and earlier induction of an anti-RRV T cell response compared to wild-type RRV infection. These findings reveal the broad impact that vIRFs have on pathogenesis and the immune response in vivo and are the first to validate the importance of vIRFs during de novo infection in the host.

Professional killer cell deficiencies and decreased survival in pulmonary arterial hypertension.

BACKGROUND AND OBJECTIVE: Increasing evidence implicates lymphocytes in pulmonary arterial hypertension (PAH) pathogenesis. Rats deficient in T-lymphocytes show increased propensity to develop PAH but when injected with endothelial progenitor cells are protected from PAH (a mechanism dependent on natural killer (NK) cells). A decreased quantity of circulating cytotoxic CD8+ T-lymphocytes and NK cells are now reported in PAH patients; however, the effect of lymphocyte depletion on disease outcome is unknown. METHODS: This prospective study analysed the lymphocyte profile and plasma brain natriuretic peptide (BNP) levels of patients with idiopathic PAH (IPAH), connective tissue disease-associated PAH (CTD-APAH) and matched healthy controls. Lymphocyte surface markers studied include: CD4+ (helper T-cell marker), CD8+ (cytotoxic T-cell marker), CD56/CD16 (NK cell marker) and CD19+ (mature B-cell marker). Lymphocyte deficiencies and plasma BNP levels were then correlated with clinical outcome. RESULTS: Fourteen patients with PAH (9 IPAH, 5CTD) were recruited. Three patients were deceased at 1-year follow-up; all had elevated CD4 : CD8 ratios and deficiencies of NK cells and cytotoxic CD8+ T-lymphocytes at recruitment. Patients with normal lymphocyte profiles at recruitment were all alive a year later, and none were on the active transplant list. As univariate markers, cytotoxic CD8+ T-cell and NK cell counts were linked to short-term survival. CONCLUSIONS: Deficiencies in NK cells and cytotoxic CD8+ T-cells may be associated with an increased risk of death in PAH patients. Further research is required in larger numbers of patients and to elucidate the mechanism of these findings.

Patients' and carers' priorities for cancer research in Aotearoa/New Zealand.

BACKGROUND: Discrepancies have been reported between what is being researched, and what patients/families deem important to be investigated. Our aim was to understand research priorities for those who live with cancer in Aotearoa/New Zealand, with emphasis on Maori. METHODS: Adult outpatients with cancer and their whanau/family completed a survey (demographics, selecting keywords, free-text comments) at Christchurch hospital. Quantitative and qualitative data were evaluated using standard statistical and thematic analyses, respectively. RESULTS: We recruited 205 participants, including both turoro/patients (n = 129) and their whanau/family/carer (n = 76). Partnership with Maori health workers enabled greater recruitment of Maori participants (19%), compared to the proportion of Maori in Canterbury (9%). Cancer research was seen as a priority by 96% of participants. Priorities were similar between Maori and non-Maori participants, with the keywords 'Cancer screening', 'Quality of Life' and 'Development of new drugs' chosen most often. Free-text analysis identified three themes; 'Genetics and Prevention', 'Early Detection and Treatment', and 'Service Delivery', with some differences by ethnicity. CONCLUSIONS: Cancer research is a high priority for those living with cancer. In addition, participants want researchers to listen to their immediate and practical needs. These findings may inform future cancer research in Aotearoa. MaORI TERMS AND TRANSLATION: Aotearoa (New Zealand) he aha o whakaaro (what are your thoughts) hui (gathering) mate pukupuku (cancer) mokopuna (descendent) Otautahi (Christchurch) rongoa (traditional healing) tane (male) te reo (Maori language) Te Whatu Ora (weaving of wellness, Health New Zealand) tikanga (methods, customary practices) turoro (patients) (alternative terms used: whanau affected by cancer or tangata whaiora (person seeking health)) wahine (female) Waitaha (Canterbury) whakapapa (genealogy) whanau ((extended) family, based on whakapapa, here also carer).

Characterisation of enzyme prodrug gene therapy combinations in coated spheroids and vascular networks in vitro.

BACKGROUND: Enzyme prodrug gene therapy is designed as a targeted cancer treatment, destroying gene-modified and bystander cells via exogenous enzyme-generated cytotoxins. Targeting of tumour blood vessels using gene therapy is attractive, although optimal enzyme prodrug combinations have yet to be identified. METHODS: Seven enzyme prodrug combinations were ranked in two endothelial (HUVEC, HMEC-1) and one tumour cell line (T24) for their ability to reduce proliferation and viability. The ability to destroy bystander cells in two dimensions (2D) and three dimensions (3D), mode of cell kill, and the ability to disrupt vascular networks were measured. RESULTS: Endothelial cell proliferation (bromodeoxyuridine uptake) was reduced most effectively by Herpes simplex virus thymidine kinase (TK) with ganciclovir (GCV), followed by Escherichia coli nitroreductase NfsB (NTR) with CB1954; viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay] was reduced most efficiently by NTR/CB1954 followed by TK/GCV. Of the seven combinations, only NTR/CB1954 displayed measurable bystander effects in 2D monolayers, and none demonstrated bystander killing in coated spheroids, a 3D spatially distinct model with tissue-like cell density. NTR-expressing endothelial cells displayed increased apoptosis, necrosis and caspase-3 activity after CB1954 treatment. Despite good antiproliferative activity, TK/GCV was ineffective at disrupting vascular network-like structures of endothelial cells, whereas NTR/CB1954 was efficient. NTR/metronidazole and the vascular disrupting agent, combretastatin A-4 phosphate, were the only other effective agents. CONCLUSIONS: Collectively, these data demonstrate that cytotoxic rather than cytostatic activity is necessary for efficient vascular disruption in vitro, and bystander killing is not essential. We identify NTR/CB1954 and NTR/metronidazole as candidates for in vivo investigation of vascular-targeted gene therapy.

Deletion of the monkeypox virus inhibitor of complement enzymes locus impacts the adaptive immune response to monkeypox virus in a nonhuman primate model of infection.

Monkeypox virus (MPXV) is an orthopoxvirus closely related to variola virus, the causative agent of smallpox. Human MPXV infection results in a disease that is similar to smallpox and can also be fatal. Two clades of MPXV have been identified, with viruses of the central African clade displaying more pathogenic properties than those within the west African clade. The monkeypox inhibitor of complement enzymes (MOPICE), which is not expressed by viruses of the west African clade, has been hypothesized to be a main virulence factor responsible for increased pathogenic properties of central African strains of MPXV. To gain a better understanding of the role of MOPICE during MPXV-mediated disease, we compared the host adaptive immune response and disease severity following intrabronchial infection with MPXV-Zaire (n = 4), or a recombinant MPXV-Zaire (n = 4) lacking expression of MOPICE in rhesus macaques (RM). Data presented here demonstrate that infection of RM with MPXV leads to significant viral replication in the peripheral blood and lungs and results in the induction of a robust and sustained adaptive immune response against the virus. More importantly, we show that the loss of MOPICE expression results in enhanced viral replication in vivo, as well as a dampened adaptive immune response against MPXV. Taken together, these findings suggest that MOPICE modulates the anti-MPXV immune response and that this protein is not the sole virulence factor of the central African clade of MPXV.

Renal transplant recipients have elevated frequencies of circulating myeloid-derived suppressor cells.

BACKGROUND: Cancer, particularly cutaneous squamous cell carcinoma (SCC), is a major cause of mortality in renal transplant recipients (RTRs). Myeloid-derived suppressor cells (MDSC) play a central role in suppressing cancer immunosurveillance but their potential mobilisation in RTRs and levels relative to those of other immunoregulatory dendritic cell (DC) populations have not been analysed. METHODS: The circulating frequencies of MDSC and DC were analysed by multicolour flow cytometry in immunocompetent patients without (n = 13) or with (ICI-SCC(Pos), n = 14) current SCC, normal donors (NDs, n = 34), chronic kidney disease patients (CKD patients, n = 22) and RTRs (n = 31). RESULTS: Compared to NDs, RTRs had significantly elevated levels of both CD14(Neg) and CD14(Pos) MDSC subsets (P < 0.001), while CKD patients and ICI-SCC(Pos) had significantly elevated levels of only the CD14(Neg)-MDSC subset. DC frequencies were significantly decreased in RTRs and CKD patients but were at normal levels in ICI-SCC(Pos). The MDSC/DC ratio was significantly elevated (P < 0.05) in RTRs (median = 5.7), CKD patients (median = 3.2) and ICI-SCC(Pos) (median = 3.5) relative to NDs (median = 0.7). The use of immunosuppressive drugs in CKD patients and past/current occurrence of SCC in RTRs was associated with significantly increased CD14(Neg)-MDSC frequencies. MDSC enriched from RTRs, when co-cultured with activated NDs T cells significantly suppressed extracellular IL-10 levels and can, when activated with formyl-methionyl-leucyl-phenylalanine, inhibit T-cell proliferation. CONCLUSIONS: RTRs, CKD patients and ICI-SCC(Pos) have increased MDSC frequencies and MDSC/DC ratios. These changes may impact on cancer immunosurveillance. Therefore, MDSC represent both a potential therapeutic target and prognostic marker in these patients, with respect to the development of SCC and other malignancies.

Adjuvant nodal field radiation in resected Stage III melanoma: A single-centre retrospective study in Christchurch, New Zealand.

INTRODUCTION: Stage III melanoma is associated with poor outcomes. We studied the characteristics and outcomes of patients with resected Stage III melanoma before the routine use of adjuvant immunotherapy. Some of these patients received adjuvant nodal radiation with modern radiation techniques. METHODS: We retrieved data of patients with resected Stage III melanoma treated in Christchurch over 10 years. Overall survival (OS), melanoma-specific survival (MSS), recurrence-free survival (RFS) and nodal recurrence-free rate (NRFR) were determined, and the association of these outcomes with tumour and treatment factors was investigated. RESULTS: We identified 178 patients (110 male and 68 female), of whom 61 received adjuvant radiation. The median age was 66.6 years, and the median follow-up was 2.7 years. First recurrences occurred in 108 (61%) patients. There were 42 (24%) nodal field relapses and 103 (58%) distant relapses. One-half of nodal relapses in patients treated with adjuvant radiation were infield. The 5-year OS, RFS, MSS and NRFR were 46.4%, 26.8%, 53.7% and 69.6%, respectively. Adjuvant radiation was associated with improved RFS and no OS benefit. T4 disease and extranodal spread were associated with poorer OS, while extranodal spread and >3 involved nodes were associated with worse RFS. CONCLUSION: Patients treated with adjuvant radiation remain at moderate risk of regional and high risk of distant relapse, despite the use of modern radiation techniques. Adjuvant radiation was associated with improved local control but infield recurrence rates remained a problem. The role of combined adjuvant radiation and immunotherapy in improving these outcomes requires further investigation.

Increased Ascorbate Content of Glioblastoma Is Associated With a Suppressed Hypoxic Response and Improved Patient Survival.

Glioblastoma multiforme is a challenging disease with limited treatment options and poor survival. Glioblastoma tumours are characterised by hypoxia that activates the hypoxia inducible factor (HIF) pathway and controls a myriad of genes that drive cancer progression. HIF transcription factors are regulated at the post-translation level via HIF-hydroxylases. These hydroxylases require oxygen and 2-oxoglutarate as substrates, and ferrous iron and ascorbate as cofactors. In this retrospective observational study, we aimed to determine whether ascorbate played a role in the hypoxic response of glioblastoma, and whether this affected patient outcome. We measured the ascorbate content and members of the HIF-pathway of clinical glioblastoma samples, and assessed their association with clinicopathological features and patient survival. In 37 samples (37 patients), median ascorbate content was 7.6 µg ascorbate/100 mg tissue, range 0.8 - 20.4 µg ascorbate/100 mg tissue. In tumours with above median ascorbate content, HIF-pathway activity as a whole was significantly suppressed (p = 0.005), and several members of the pathway showed decreased expression (carbonic anhydrase-9 and glucose transporter-1, both p < 0.01). Patients with either lower tumour HIF-pathway activity or higher tumour ascorbate content survived significantly longer than patients with higher HIF-pathway or lower ascorbate levels (p = 0.011, p = 0.043, respectively). Median survival for the low HIF-pathway score group was 362 days compared to 203 days for the high HIF-pathway score group, and median survival for the above median ascorbate group was 390 days, compared to the below median ascorbate group with 219 days. The apparent survival advantage associated with higher tumour ascorbate was more prominent for the first 8 months following surgery. These associations are promising, suggesting an important role for ascorbate-regulated HIF-pathway activity in glioblastoma that may impact on patient survival.