Conservative versus liberal oxygenation targets in critically ill children (Oxy-PICU): a UK multicentre, open, parallel-group, randomised clinical trial.

BACKGROUND: The optimal target for systemic oxygenation in critically ill children is unknown. Liberal oxygenation is widely practiced, but has been associated with harm in paediatric patients. We aimed to evaluate whether conservative oxygenation would reduce duration of organ support or incidence of death compared to standard care. METHODS: Oxy-PICU was a pragmatic, multicentre, open-label, randomised controlled trial in 15 UK paediatric intensive care units (PICUs). Children admitted as an emergency, who were older than 38 weeks corrected gestational age and younger than 16 years receiving invasive ventilation and supplemental oxygen were randomly allocated in a 1:1 ratio via a concealed, central, web-based randomisation system to conservative peripheral oxygen saturations ([SpO2] 88-92%) or liberal (SpO2 >94%) targets. The primary outcome was the duration of organ support at 30 days following random allocation, a rank-based endpoint with death either on or before day 30 as the worst outcome (a score equating to 31 days of organ support), with survivors assigned a score between 1 and 30 depending on the number of calendar days of organ support received. The primary effect estimate was the probabilistic index, a value greater than 0·5 indicating more than 50% probability that conservative oxygenation is superior to liberal oxygenation for a randomly selected patient. All participants in whom consent was available were included in the intention-to-treat analysis. The completed study was registered with the ISRCTN registry (ISRCTN92103439). FINDINGS: Between Sept 1, 2020, and May 15, 2022, 2040 children were randomly allocated to conservative or liberal oxygenation groups. Consent was available for 1872 (92%) of 2040 children. The conservative oxygenation group comprised 939 children (528 [57%] of 927 were female and 399 [43%] of 927 were male) and the liberal oxygenation group included 933 children (511 [56%] of 920 were female and 409 [45%] of 920 were male). Duration of organ support or death in the first 30 days was significantly lower in the conservative oxygenation group (probabilistic index 0·53, 95% CI 0·50-0·55; p=0·04 Wilcoxon rank-sum test, adjusted odds ratio 0·84 [95% CI 0·72-0·99]). Prespecified adverse events were reported in 24 (3%) of 939 patients in the conservative oxygenation group and 36 (4%) of 933 patients in the liberal oxygenation group. INTERPRETATION: Among invasively ventilated children who were admitted as an emergency to a PICU receiving supplemental oxygen, a conservative oxygenation target resulted in a small, but significant, greater probability of a better outcome in terms of duration of organ support at 30 days or death when compared with a liberal oxygenation target. Widespread adoption of a conservative oxygenation saturation target (SpO2 88-92%) could help improve outcomes and reduce costs for the sickest children admitted to PICUs. FUNDING: UK National Institute for Health and Care Research Health Technology Assessment Programme.

Protocol for a Randomized Multiple Center Trial of Conservative Versus Liberal Oxygenation Targets in Critically Ill Children (Oxy-PICU): Oxygen in Pediatric Intensive Care.

OBJECTIVES: Oxygen administration is a fundamental part of pediatric critical care, with supplemental oxygen offered to nearly every acutely unwell child. However, optimal targets for systemic oxygenation are unknown. Oxy-PICU aims to evaluate the clinical effectiveness and cost-effectiveness of a conservative peripheral oxygen saturation (Sp o2 ) target of 88-92% compared with a liberal target of more than 94%. DESIGN: Pragmatic, open, multiple-center, parallel group randomized control trial with integrated economic evaluation. SETTING: Fifteen PICUs across England, Wales, and Scotland. PATIENTS: Infants and children age more than 38 week-corrected gestational age to 16 years who are accepted to a participating PICU as an unplanned admission and receiving invasive mechanical ventilation with supplemental oxygen for abnormal gas exchange. INTERVENTION: Adjustment of ventilation and inspired oxygen settings to achieve an Sp o2 target of 88-92% during invasive mechanical ventilation. MEASUREMENTS AND MAIN RESULTS: Randomization is 1:1 to a liberal Sp o2 target of more than 94% or a conservative Sp o2 target of 88-92% (inclusive), using minimization with a random component. Minimization will be performed on: age, site, primary reason for admission, and severity of abnormality of gas exchange. Due to the emergency nature of the treatment, approaching patients for written informed consent will be deferred to after randomization. The primary clinical outcome is a composite of death and days of organ support at 30 days. Baseline demographics and clinical status will be recorded as well as daily measures of oxygenation and organ support, and discharge outcomes. This trial received Health Research Authority approval on December 23, 2019 (reference: 272768), including a favorable ethical opinion from the East of England-Cambridge South Research Ethics Committee (reference number: 19/EE/0362). Trial findings will be disseminated in national and international conferences and peer-reviewed journals.

SerpinB2 Deficiency Results in a Stratum Corneum Defect and Increased Sensitivity to Topically Applied Inflammatory Agents.

SerpinB2 (plasminogen activator inhibitor type 2) is constitutively expressed at high levels by differentiating keratinocytes in mice and humans; however, the physiological function of keratinocyte SerpinB2 remains unclear. Herein, we show that SerpinB2(-/-) mice are more susceptible to contact dermatitis after topical application of dinitrofluorobenzene, and show enhanced inflammatory lesions after topical applications of phorbol ester. Untreated SerpinB2(-/-) mice showed no overt changes in epithelial structure, and we were unable to find evidence for a role for keratinocyte SerpinB2 in regulating immunity, apoptosis, IL-1ß production, proteasomal activity, or wound healing. Instead, the phenotype was associated with impaired skin barrier function and a defective stratum corneum, with SerpinB2(-/-) mice showing increased transepidermal water loss, increased overt loss of stratum corneum in inflammatory lesions, and impaired stratum corneum thickening after phorbol ester treatment. Immunoblotting suggested that SerpinB2 (cross-linked into the cornified envelope) is present in the stratum corneum and retains the ability to form covalent inhibitory complexes with urokinase. Data suggest that the function of keratinocyte SerpinB2 is protection of the stratum corneum from proteolysis via inhibition of urokinase, thereby maintaining the integrity and barrier function of the stratum corneum, particularly during times of skin inflammation. Implications for studies involving genetically modified mice treated with topical agents and human dermatological conditions, such as contact dermatitis, are discussed.

Chikungunya virus non-structural protein 2-mediated host shut-off disables the unfolded protein response.

The unfolded protein response (UPR) is a cellular defence mechanism against high concentrations of misfolded protein in the endoplasmic reticulum (ER). In the presence of misfolded proteins, ER-transmembrane proteins PERK and IRE1α become activated. PERK phosphorylates eIF2α leading to a general inhibition of cellular translation, whilst the expression of transcription factor ATF4 is upregulated. Active IRE1α splices out an intron from XBP1 mRNA, to produce a potent transcription factor. Activation of the UPR increases the production of several proteins involved in protein folding, degradation and apoptosis. Here, we demonstrated that transient expression of chikungunya virus (CHIKV) (family Togaviridae, genus Alphavirus) envelope glycoproteins induced the UPR and that CHIKV infection resulted in the phosphorylation of eIF2α and partial splicing of XBP1 mRNA. However, infection with CHIKV did not increase the expression of ATF4 and known UPR target genes (GRP78/BiP, GRP94 and CHOP). Moreover, nuclear XBP1 was not observed during CHIKV infection. Even upon stimulation with tunicamycin, the UPR was efficiently inhibited in CHIKV-infected cells. Individual expression of CHIKV non-structural proteins (nsPs) revealed that nsP2 alone was sufficient to inhibit the UPR. Mutations that rendered nsP2 unable to cause host-cell shut-off prevented nsP2-mediated inhibition of the UPR. This indicates that initial UPR induction takes place in the ER but that expression of functional UPR transcription factors and target genes is efficiently inhibited by CHIKV nsP2.

DNA vaccines encoding membrane-bound or secreted forms of heat shock protein 70 exhibit improved potency.

Traditional vaccine strategies are inefficient against challenge with complex pathogens including HIV; therefore, novel vaccine technologies are required. DNA vaccines are attractive as they are relatively cheap and easy to manufacture, but a major limitation has been their lack of immunogenicity in humans, which may be overcome with the incorporation of an adjuvant. HSP70 is a recognised damage-associated molecular pattern, which is a potential adjuvant. We investigated the immunogenicity of a DNA vaccine encoding HIV gag and HSP70; the latter was genetically modified to produce cytoplasmic, secreted or membrane-bound HSP70, the expression of which was controlled by an independent promoter. The DNA was administered to C57BL/6 mice to evaluate gag-specific T-cell responses. Our results demonstrated the ability of membrane-bound and secreted HSP70 to significantly enhance gag-specific T-cell responses and increase the breadth of T-cell responses to include subdominant epitopes. Membrane-bound or secreted HSP70 also significantly improved the multifunctionality of HIV-specific T cells and T-cell proliferation, which is important for maintaining T-cell integrity. Most importantly, the inclusion of membrane-bound HSP70, secreted HSP70 or a combination significantly increased protection in mice challenged with EcoHIV, a chimeric virus that replicates in mouse leukocytes in vivo.

CCR2 deficiency promotes exacerbated chronic erosive neutrophil-dominated chikungunya virus arthritis.

UNLABELLED: Chikungunya virus (CHIKV) is a member of a globally distributed group of arthritogenic alphaviruses that cause weeks to months of debilitating polyarthritis/arthralgia, which is often poorly managed with current treatments. Arthritic disease is usually characterized by high levels of the chemokine CCL2 and a prodigious monocyte/macrophage infiltrate. Several inhibitors of CCL2 and its receptor CCR2 are in development and may find application for treatment of certain inflammatory conditions, including autoimmune and viral arthritides. Here we used CCR2(-/-) mice to determine the effect of CCR2 deficiency on CHIKV infection and arthritis. Although there were no significant changes in viral load or RNA persistence and only marginal changes in antiviral immunity, arthritic disease was substantially increased and prolonged in CCR2(-/-) mice compared to wild-type mice. The monocyte/macrophage infiltrate was replaced in CCR2(-/-) mice by a severe neutrophil (followed by an eosinophil) infiltrate and was associated with changes in the expression levels of multiple inflammatory mediators (including CXCL1, CXCL2, granulocyte colony-stimulating factor [G-CSF], interleukin-1ß [IL-1ß], and IL-10). The loss of anti-inflammatory macrophages and their activities (e.g., efferocytosis) was also implicated in exacerbated inflammation. Clear evidence of cartilage damage was also seen in CHIKV-infected CCR2(-/-) mice, a feature not normally associated with alphaviral arthritides. Although recruitment of CCR2(+) monocytes/macrophages can contribute to inflammation, it also appears to be critical for preventing excessive pathology and resolving inflammation following alphavirus infection. Caution might thus be warranted when considering therapeutic targeting of CCR2/CCL2 for the treatment of alphaviral arthritides. IMPORTANCE: Here we describe the first analysis of viral arthritis in mice deficient for the chemokine receptor CCR2. CCR2 is thought to be central to the monocyte/macrophage-dominated inflammatory arthritic infiltrates seen after infection with arthritogenic alphaviruses such as chikungunya virus. Surprisingly, the viral arthritis caused by chikungunya virus in CCR2-deficient mice was more severe, prolonged, and erosive and was neutrophil dominated, with viral replication and persistence not being significantly affected. Monocytes/macrophages recruited by CCL2 thus also appear to be important for both preventing even worse pathology mediated by neutrophils and promoting resolution of inflammation. Caution might thus be warranted when considering the use of therapeutic agents that target CCR2/CCL2 or inflammatory monocytes/macrophages for the treatment of alphaviral (and perhaps other viral) arthritides. Individuals with diminished CCR2 responses (due to drug treatment or other reasons) may also be at risk of exacerbated arthritic disease following alphaviral infection.

Induction of antigen-positive cell death by the expression of perforin, but not DTa, from a DNA vaccine enhances the immune response.

The failure of traditional protein-based vaccines to prevent infection by viruses such as HIV or hepatitis C highlights the need for novel vaccine strategies. DNA vaccines have shown promise in small animal models, and are effective at generating anti-viral T cell-mediated immune responses; however, they have proved to be poorly immunogenic in clinical trials. We propose that the induction of necrosis will enhance the immune response to vaccine antigens encoded by DNA vaccines, as necrotic cells are known to release a range of intracellular factors that lead to dendritic cell (DC) activation and enhanced cross-presentation of antigen. Here we provide evidence that induction of cell death in DNA vaccine-targeted cells provides an adjuvant effect following intradermal vaccination of mice; however, this enhancement of the immune response is dependent on both the mechanism and timing of cell death after antigen expression. We report that a DNA vaccine encoding the cytolytic protein, perforin, resulted in DC activation, enhanced broad and multifunctional CD8 T-cell responses to the HIV-1 antigen GAG and reduced viral load following challenge with a chimeric virus, EcoHIV, compared with the canonical GAG DNA vaccine. This effect was not observed for a DNA vaccine encoding an apoptosis-inducing toxin, DTa, or when the level of perforin expression was increased to induce cell death sooner after vaccination. Thus, inducing lytic cell death following a threshold level of expression of a viral antigen can improve the immunogenicity of DNA vaccines, whereas apoptotic cell death has an inhibitory effect on the immune response.

Gene profiling of Chikungunya virus arthritis in a mouse model reveals significant overlap with rheumatoid arthritis.

OBJECTIVE: Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes a chronic debilitating polyarthralgia/polyarthritis, for which current treatments are often inadequate. To assess whether new drugs being developed for rheumatoid arthritis (RA) might find utility in the treatment of alphaviral arthritides, we sought to determine whether the inflammatory gene expression signature of CHIKV arthritis shows any similarities with RA or collagen-induced arthritis (CIA), a mouse model of RA. METHODS: Using a recently developed animal model of CHIKV arthritis in adult wild-type mice, we generated a consensus CHIKV arthritis gene expression signature, which was used to interrogate publicly available microarray studies of RA and CIA. Pathway analyses were then performed using the overlapping gene signatures. RESULTS: Gene set enrichment analysis showed that there was a highly significant overlap in the differentially expressed genes in the CHIKV arthritis model and in RA. This concordance also increased with the severity of RA, as measured by the inflammation score. A highly significant overlap was also seen between CHIKV arthritis and CIA. Pathway analysis revealed that the overlap between these arthritides was spread over a range of different inflammatory processes. Involvement of T cells and interferon-γ (IFNγ) in CHIKV arthritis was confirmed in studies of MHCII-deficient mice and IFNγ-deficient mice, respectively. CONCLUSION: These results suggest that RA, a chronic autoimmune arthritis, and CHIKV disease, usually a self-limiting viral arthropathy, share multiple inflammatory processes. New drugs and biologic therapies being developed for RA may thus find application in the treatment of alphaviral arthritides.

The role of SerpinB2 in immunity.

SerpinB2 or plasminogen activator inhibitor type 2 (PAI-2) is widely described as an inhibitor of extracellular urokinase plasminogen activator. However, the evidence that this represents the physiological role of SerpinB2 is not overly compelling. SerpinB2 is substantially up-regulated under multiple inflammatory conditions, and dysregulated expression and polymorphisms are associated with several human inflammatory diseases. A bewildering array of diverse functions and activities have been attributed to SerpinB2, but despite ≈900 publications in the field, no coherent view of what SerpinB2 does in vivo has emerged. Although SerpinB2 is abundantly expressed by activated macrophages and a range of other haematopoietic and non-haematopoietic cells, SerpinB2(-/-) mice have surprisingly few phenotypes. However, SerpinB2(-/-) mice were recently shown to generate increased Th1 responses, suggesting that at least one function of SerpinB2 is sculpting of the adaptive immune response.

A physiological function of inflammation-associated SerpinB2 is regulation of adaptive immunity.

SerpinB2 (plasminogen activator inhibitor-2) is widely described as an inhibitor of urokinase plasminogen activator; however, SerpinB2(-/-) mice show no detectable increase in urokinase plasminogen activator activity. In this study, we describe an unexpected immune phenotype in SerpinB2(-/-) mice. After immunization with OVA in CFA, SerpinB2(-/-) mice made approximately 6-fold more IgG2c and generated approximately 2.5-fold more OVA-specific IFN-gamma-secreting T cells than SerpinB2(+/+) littermate controls. In SerpinB2(+/+) mice, high inducible SerpinB2 expression was seen at the injection site and in macrophages low levels in draining lymph nodes and conventional dendritic cells, and no expression was seen in plasmacytoid dendritic, B, T, or NK cells. SerpinB2(-/-) macrophages promoted greater IFN-gamma secretion from wild-type T cells in vivo and in vitro and, when stimulated with anti-CD40/IFN-gamma or cultured with wild-type T cells in vitro, secreted more Th1-promoting cytokines than macrophages from littermate controls. Draining lymph node SerpinB2(-/-) myeloid APCs similarly secreted more Th1-promoting cytokines when cocultured with wild-type T cells. Regulation of Th1 responses thus appears to be a physiological function of inflammation-associated SerpinB2; an observation that may shed light on human inflammatory diseases like pre-eclampsia, lupus, asthma, scleroderma, and periodontitis, which are associated with SerpinB2 polymorphisms or dysregulated SerpinB2 expression.

Human papilloma virus transformed CaSki cells constitutively express high levels of functional SerpinB2.

Many malignant tissues, including human papilloma virus (HPV)-associated cancers, express SerpinB2, also known as plasminogen activator inhibitor type-2 (PAI-2). Whether SerpinB2 is expressed by the HPV-transformed cancer cells, and if so, whether SerpinB2 is mutated or behaves aberrantly remains unclear. Here we show that HPV-transformed CaSki cells express high levels of constitutive wild-type SerpinB2, with cellular distribution, glycosylation, secretion, cleavage, induction and urokinase binding similar to that reported for primary cells. Neutralization of secreted SerpinB2 failed to affect CaSki cell migration or growth. Lentivirus-based over-expression of SerpinB2 also had no effect on growth, and we were unable to confirm a role for SerpinB2 in binding or regulating expression of the retinoblastoma protein. CaSki cells thus emerge as a useful tool for studying SerpinB2, with the physiological function of SerpinB2 expression by tumor cells remaining controversial. Using CaSki cells as a source of endogenous SerpinB2, we confirmed that SerpinB2 efficiently binds the proteasomal subunit member ß1.

Chikungunya virus arthritis in adult wild-type mice.

Chikungunya virus is a mosquito-borne arthrogenic alphavirus that has recently reemerged to produce the largest epidemic ever documented for this virus. Here we describe a new adult wild-type mouse model of chikungunya virus arthritis, which recapitulates the self-limiting arthritis, tenosynovitis, and myositis seen in humans. Rheumatic disease was associated with a prolific infiltrate of monocytes, macrophages, and NK cells and the production of monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma). Infection with a virus isolate from the recent Reunion Island epidemic induced significantly more mononuclear infiltrates, proinflammatory mediators, and foot swelling than did an Asian isolate from the 1960s. Primary mouse macrophages were shown to be productively infected with chikungunya virus; however, the depletion of macrophages ameliorated rheumatic disease and prolonged the viremia. Only 1 microg of an unadjuvanted, inactivated, whole-virus vaccine derived from the Asian isolate completely protected against viremia and arthritis induced by the Reunion Island isolate, illustrating that protection is not strain specific and that low levels of immunity are sufficient to mediate protection. IFN-alpha treatment was able to prevent arthritis only if given before infection, suggesting that IFN-alpha is not a viable therapy. Prior infection with Ross River virus, a related arthrogenic alphavirus, and anti-Ross River virus antibodies protected mice against chikungunya virus disease, suggesting that individuals previously exposed to Ross River virus should be protected from chikungunya virus disease. This new mouse model of chikungunya virus disease thus provides insights into pathogenesis and a simple and convenient system to test potential new interventions.

Loss of long term protection with the inclusion of HIV pol to a DNA vaccine encoding gag.

Traditional vaccine strategies that induce antibody responses have failed to protect against HIV infection in clinical trials, and thus cell-mediated immunity is now an additional criterion. Recent clinical trials that aimed to induce strong T cell responses failed to do so. Therefore, to enhance induction of protective T cell responses, it is crucial that the optimum antigen combination is chosen. Limited research has been performed into the number of antigens selected for an HIV vaccine. This study aimed to compare DNA vaccines encoding either a single HIV antigen or a combination of two antigens, using intradermal vaccination of C57BL/6 mice. Immune assays were performed on splenocytes, and in vivo protection was examined by challenge with a chimeric virus, EcoHIV, able to infect mouse but not human leukocytes, at 10 days (short term) and 60 days (long term) post final vaccination. At 60 days there was significantly lower frequency of induced antigen-specific CD8(+) T cells in the spleens of pCMVgag-pol-vaccinated mice compared with mice which received pCMVgag only. Most importantly, short term viral control of EcoHIV was similar for pCMVgag and pCMVgag-pol-vaccinated mice at day 10, but only the pCMVgag-vaccinated significantly controlled EcoHIV at day 60 compared with pCMV-vaccinated mice, showing that control was reduced with the inclusion of the HIV pol gene.

Tumor cell-expressed SerpinB2 is present on microparticles and inhibits metastasis.

Expression of SerpinB2 (plasminogen activator inhibitor type 2/PAI-2) by certain cancers is associated with a favorable prognosis. Although tumor-associated host tissues can express SerpinB2, no significant differences in the growth of a panel of different tumors in SerpinB2(-/-) and SerpinB2(+/+) mice were observed. SerpinB2 expression by cancer cells (via lentiviral transduction) also had no significant effect on the growth of panel of mouse and human tumor lines in vivo or in vitro. SerpinB2 expression by cancer cells did, however, significantly reduce the number of metastases in a B16 metastasis model. SerpinB2-expressing B16 cells also showed reduced migration and increased length of invadopodia-like structures, supporting the classical view that that tumor-derived SerpinB2 is inhibiting extracellular urokinase. Importantly, although SerpinB2 is usually poorly secreted, we found that SerpinB2 effectively reaches the extracellular milieu on the surface of 0.5-1 µm microparticles (MPs), where it was able to inhibit urokinase. We also provide evidence that annexins mediate the binding of SerpinB2 to phosphatidylserine, a lipid characteristically exposed on the surface of MPs. The presence of SerpinB2 on the surface of MPs provides a physiological mechanism whereby cancer cell SerpinB2 can reach the extracellular milieu and access urokinase plasminogen activator (uPA). This may then lead to inhibition of metastasis and a favorable prognosis.

Induction of SerpinB2 and Th1/Th2 modulation by SerpinB2 during lentiviral infections in vivo.

SerpinB2, also known as plasminogen activator inhibitor type 2, is a major product of activated monocytes/macrophages and is often strongly induced during infection and inflammation; however, its physiological function remains somewhat elusive. Herein we show that SerpinB2 is induced in peripheral blood mononuclear cells following infection of pigtail macaques with CCR5-utilizing (macrophage-tropic) SIVmac239, but not the rapidly pathogenic CXCR4-utilizing (T cell-tropic) SHIVmn229. To investigate the role of SerpinB2 in lentiviral infections, SerpinB2(-/-) mice were infected with EcoHIV, a chimeric HIV in which HIV gp120 has been replaced with gp80 from ecotropic murine leukemia virus. EcoHIV infected SerpinB2(-/-) mice produced significantly lower anti-gag IgG1 antibody titres than infected SerpinB2(+/+) mice, and showed slightly delayed clearance of EcoHIV. Analyses of published microarray studies showed significantly higher levels of SerpinB2 mRNA in monocytes from HIV-1 infected patients when compared with uninfected controls, as well as a significant negative correlation between SerpinB2 and T-bet mRNA levels in peripheral blood mononuclear cells. These data illustrate that SerpinB2 can be induced by lentiviral infection in vivo and support the emerging notion that a physiological role of SerpinB2 is modulation of Th1/Th2 responses.

Potent inhibition of HIV-1 replication by a Tat mutant.

Herein we describe a mutant of the two-exon HIV-1 Tat protein, termed Nullbasic, that potently inhibits multiple steps of the HIV-1 replication cycle. Nullbasic was created by replacing the entire arginine-rich basic domain of wild type Tat with glycine/alanine residues. Like similarly mutated one-exon Tat mutants, Nullbasic exhibited transdominant negative effects on Tat-dependent transactivation. However, unlike previously reported mutants, we discovered that Nullbasic also strongly suppressed the expression of unspliced and singly-spliced viral mRNA, an activity likely caused by redistribution and thus functional inhibition of HIV-1 Rev. Furthermore, HIV-1 virion particles produced by cells expressing Nullbasic had severely reduced infectivity, a defect attributable to a reduced ability of the virions to undergo reverse transcription. Combination of these inhibitory effects on transactivation, Rev-dependent mRNA transport and reverse transcription meant that permissive cells constitutively expressing Nullbasic were highly resistant to a spreading infection by HIV-1. Nullbasic and its activities thus provide potential insights into the development of potent antiviral therapeutics that target multiple stages of HIV-1 infection.

Ticks associated with macquarie island penguins carry arboviruses from four genera.

Macquarie Island, a small subantarctic island, is home to rockhopper, royal and king penguins, which are often infested with the globally distributed seabird tick, Ixodes uriae. A flavivirus, an orbivirus, a phlebovirus, and a nairovirus were isolated from these ticks and partial sequences obtained. The flavivirus was nearly identical to Gadgets Gully virus, isolated some 30 year previously, illustrating the remarkable genetic stability of this virus. The nearest relative to the orbivirus (for which we propose the name Sandy Bay virus) was the Scottish Broadhaven virus, and provided only the second available sequences from the Great Island orbivirus serogroup. The phlebovirus (for which we propose the name Catch-me-cave virus) and the previously isolated Precarious Point virus were distinct but related, with both showing homology with the Finnish Uukuniemi virus. These penguin viruses provided the second and third available sequences for the Uukuniemi group of phleboviruses. The nairovirus (for which we propose the name Finch Creek virus) was shown to be related to the North American Tillamook virus, the Asian Hazara virus and Nairobi sheep disease virus. Macquarie Island penguins thus harbour arboviruses from at least four of the seven arbovirus-containing genera, with related viruses often found in the northern hemisphere.

Human papillomavirus E7 requires the protease calpain to degrade the retinoblastoma protein.

Cervical cancers transformed by high risk human papilloma virus (HPV) express the E7 oncoprotein, which accelerates the degradation of the retinoblastoma protein (Rb). Here we show that the E7-mediated degradation of Rb requires the calcium-activated cysteine protease, calpain. E7 bound and activated mu-calpain and promoted cleavage at Rb(810), with mutation of this residue preventing E7-mediated degradation. The calpain cleavage product, Rb(1-810), was unable to mediate cell cycle arrest but retained the ability to repress E6/E7 transcription. E7 also promoted the accelerated proteasomal degradation of Rb(1-810). Calpain inhibitors reduced the viability of HPV-transformed cells and synergized with cisplatin. Calpain, thus, emerges as a central player in E7-mediated degradation of Rb and represents a potential new drug target for the treatment of HPV-associated lesions.