Proteasome Inhibition In Vivo Promotes Survival in a Lethal Murine Model of Severe Acute Respiratory Syndrome

Ubiquitination is a critical regulator of the host immune response to viral infection, and many viruses, including coronaviruses, encode proteins that target the ubiquitination system. To explore the link between coronavirus infection and the ubiquitin system, we asked whether protein degradation by the 26S proteasome plays a role in severe coronavirus infections using a murine model of SARS-like pneumonitis induced by murine hepatitis virus strain 1 (MHV-1). In vitro, the pretreatment of peritoneal macrophages with inhibitors of the proteasome (pyrrolidine dithiocarbamate [PDTC], MG132, and PS-341) markedly inhibited MHV-1 replication at an early step in its replication cycle, as evidenced by inhibition of viral RNA production. Proteasome inhibition also blocked viral cytotoxicity in macrophages, as well as the induction of inflammatory mediators such as IP-10, gamma interferon (IFN-γ), and monocyte chemoattractant protein 1 (MCP-1). In vivo, intranasal inoculation of MHV-1 results in a lethal pneumonitis in A/J mice. Treatment of A/J mice with the proteasome inhibitor PDTC, MG132, or PS-341 led to 40% survival (P < 0.01), with a concomitant improvement of lung histology, reduced pulmonary viral replication, decreased pulmonary STAT phosphorylation, and reduced pulmonary inflammatory cytokine expression. These data demonstrate that inhibition of the cellular proteasome attenuates pneumonitis and cytokine gene expression in vivo by reducing MHV-1 replication and the resulting inflammatory response. The results further suggest that targeting the proteasome may be an effective new treatment for severe coronavirus infections.Severe acute respiratory syndrome (SARS) was first introduced into the human population in the Guangdong Province in China and rapidly spread to several other countries (31). SARS is caused by infection with the SARS coronavirus (SARS-CoV) and is characterized by an atypical pneumonia and lymphopenia. Two-thirds of the SARS-infected patients developed a viral pneumonitis, of which 10% developed acute respiratory distress syndrome. During the outbreak in 2002 to 2003, 8,000 people were infected and 774 people died from respiratory failure (36; WHO, Summary of probable SARS cases with onset of illness from 1 November 2002 to 31 July 2003 [http://www.who.int]). At present there are no effective treatments for SARS as well as other coronavirus infections. Finding an effective treatment for coronavirus infections could be protective in the event of a reemergent coronavirus outbreak (7).We have recently reported that a rodent model of SARS mimics many of the features of severe clinical SARS pathology (11, 12). Intranasal infection of A/J mice with strain 1 of murine hepatitis virus (MHV-1) causes a lethal form of pneumonitis, characterized by marked innate immune inflammatory cytokine production and replication of the virus in pulmonary macrophages (11, 12). MHV-1 infection is uniformly fatal in infected A/J mice; the resultant disease serves as a model to understand the pathology of the most severe SARS cases. In mice, the pulmonary damage is histologically similar to that seen in human SARS and is similarly associated with a marked upregulation of inflammatory mediators, including monocyte chemoattractant protein 1 (MCP-1), IP-10, MIG, gamma interferon (IFN-γ), interleukin-8 (IL-8), and IL-6 (11, 12, 25). These innate immune mediators are likely to play roles in human SARS and MHV-1 SARS-like pathogenesis.A critical aspect of the host innate immune response to viral illness is the upregulation of the antiviral type 1 IFN response. With respect to SARS, type 1 IFN responses have been reported to be suppressed by SARS-CoV in several models and in clinical cases (11, 39, 45, 52). In our model, MHV-1-infected A/J mice produce less type 1 IFN than resistant strains of mice and they respond poorly to IFN-β therapy (11). Type I IFN has been used clinically in the treatment of established SARS infections but has shown only limited efficacy (25). In the absence of an effective antiviral treatment, the innate immune pathways present a potential target for therapeutic intervention (7).Ubiquitination, the process by which cellular proteins are conjugated to the 7.5-kDa ubiquitin (Ub) protein, is a critical regulator of innate and adaptive immune pathways (40). There are several possible fates for ubiquitinated proteins: degradation by the 26S proteasome, trafficking to various subcellular sites, altered interactions with other proteins, and altered signal transduction functions (28). The fates of the ubiquitinated proteins, many of which overlap, can play a role in innate immunity. Since the first discovery that papillomavirus encodes an E3 ubiquitin ligase that targets p53, it has become widely appreciated that many viruses encode proteins that target or exploit ubiquitination pathways (37, 43). For example, Epstein-Barr virus and herpes simplex virus proteins interact with the host deubiquitinating (DUB) protein USP7 (13, 17). Ubiquitination of IRF3 has been implicated in the viral control of the innate immune system (22, 48, 49). DUB may also be important for viral functions, such as the assembly of viral replicase proteins with double-membrane vesicles at the site of replication, a process that parasitizes autophagy (39).All coronaviruses, including MHV (A59 and JHM), infectious bronchitis virus, and human CoV229E SARS coronavirus, encode one or more papain-like proteases (PLpros) (PL1pro and PL2pro) (3, 5, 19, 23, 50). One role for the PL2pro proteases is to cleave the coronavirus polyprotein into its component parts. This enzyme, isolated from the SARS-CoV, has also been shown to have DUB activity both in vitro and in HeLa cells (23), suggesting that it might also play a role in modulating the host ubiquitination pathways. PLpro proteases harbor an N-terminal Ub-like domain reported to mediate interactions between PLpro DUB activity and the cellular proteasome (35). Although there is no direct link between the proteasome and SARS-CoV DUB activity, the presence of the Ub1 domain and of SARS-CoV DUB activity suggests that the proteasome may be being exploited by the virus either to evade the immune response or to promote viral replication. These interactions also suggest that the ubiquitination system might be a target for antiviral therapeutic intervention.We explored the role of the cellular proteasome in MHV-1 replication and in the innate immune response to the virus by testing the effects of small-molecule proteasome inhibitors in both cell-based and murine models of SARS pneumonitis. We compared the results in the SARS model to a well-described model of lymphocytic choriomeningitis virus (LCMV) hepatitis in order to test for virus-specific effects. To control for nonspecific effects of the inhibitors, we used three different agents: pyrrolidine dithiocarbamate (PDTC), MG132, and PS-341 (bortezomib, Velcade). PDTC is a chelating agent that reversibly inhibits the proteasome complex, MG132 is a peptide aldehyde protease inhibitor, and PS-341 is a peptide boronic acid inhibitor (1, 20, 38). PS-341 is a clinically approved drug currently being used in the treatment of multiple myeloma.     

Downregulation of a Chitin Deacetylase-Like Protein in Response to Baculovirus Infection and Its Application for Improving Baculovirus Infectivity

Several expressed sequence tags (ESTs) with homology to chitin deacetylase-like protein (CDA) were selected from a group of Helicoverpa armigera genes whose expression changed after infection with H. armigera single nucleopolyhedrovirus (HearNPV). Some of these ESTs coded for a midgut protein containing a chitin deacetylase domain (CDAD). The expressed protein, HaCDA5a, did not show chitin deacetylase activity, but it showed a strong affinity for binding to chitin. Sequence analysis showed the lack of any chitin binding domain, described for all currently known peritrophic membrane (PM) proteins. HaCDA5a has previously been detected in the H. armigera PM. Such localization, together with its downregulation after pathogen infection, led us to hypothesize that this protein might be responsible for the homeostasis of the PM structure and that, by reduction of its expression, the insect may reduce PM permeability, decreasing the entrance of baculovirus. To test this hypothesis, we constructed a recombinant nucleopolyhedrovirus to express HaCDA5a in insect cells and tested its influence on PM permeability as well as the influence of HaCDA5a expression on the performance of the baculovirus. The experiments showed that HaCDA5a increased PM permeability, in a concentration-dependent manner. Bioassays on Spodoptera frugiperda and Spodoptera exigua larvae revealed that NPV expressing HaCDA5a was more infective than its parental virus. However, no difference in virulence was observed when the viruses were injected intrahemocoelically. These findings support the downregulation of a midgut-specific CDA-like protein as a possible mechanism used by H. armigera to reduce susceptibility to baculovirus by decreasing PM permeability.Baculoviruses are a naturally occurring group of large double-stranded DNA viruses that are specific to arthropods and have potential for widespread use for insect pest management. It has already been proven that they can effectively replace chemical insecticides in the field, for example, in the case of Helicoverpa armigera single nucleopolyhedrovirus (HearNPV) sprayed on cotton fields in Australia (9) and China (38) to control one of the most widely spread polyphagous pests (10). Baculoviruses occur naturally, are nonpathogenic to humans or other vertebrates, and are relatively host specific, and no impact on nontarget organisms has been reported to date. These characteristics make them environmentally safe insecticides. Despite the environmental advantages of baculoviruses, their use as biocontrol agents is limited, mainly due to their slow action compared to that of other pesticides. Naturally occurring baculoviruses, although highly infectious, have adapted to their hosts during their evolution, therefore killing the hosts relatively slowly and achieving maximum viral propagation. It takes up to 10 days for the virus to stop insect feeding or to kill the infected larvae (34). For this reason, reduction in the time of killing has been the main focus of research to improve baculovirus performance, and several strategies have been used, such as coapplying synergistic chemicals or using genetic engineering to introducing foreign genes coding for toxins, hormones, or enzymes into their genomes (18, 19). The strategy of acquiring foreign genes has been used by viruses themselves. Most large cytoplasmic and nuclear DNA viruses have been shown to capture, by horizontal gene transfer, host genes related to ubiquitin signaling, defense against apoptosis, and immune responses (20). The average baculovirus genome contains more than 100 open reading frames (ORFs) encoding predicted proteins of more than 50 amino acids. Phylogenetic analyses suggest that during evolution, several baculovirus genes, such as the inhibitor of apoptosis (iap) and ecdysteroid UDP-glucosyltransferase (egt) genes, were acquired from their insect hosts by horizontal gene transfer (17). Access to the recently available genome of Bombyx mori enabled a survey of B. mori NPV (BmNPV) genes that might have been acquired from the host. The survey identified 35 insect homologs potentially encoded by 37 baculoviruses (22). Knockout studies of insect homologs in baculoviruses have shown that some host homologs are essential for complete in vivo pathogenicity (22). Their functions are maintained or modified in order to control host physiology and cell signaling pathways for better virus multiplication and vertical transmission in nature.To identify host genes whose expression could be advantageous for the baculovirus to increase its insecticidal characteristics, we checked the change in expression of host genes in response to baculovirus infection. DNA microarray experiments revealed a set of H. armigera midgut genes that were up- and downregulated due to infection with HearNPV (unpublished data). Among them, several expressed sequence tags (ESTs) coding for a chitin deacetylase-like protein (CDA) were found to be downregulated after virus infection, suggesting its possible role in the response to the infection. CDAs have been isolated from various fungi and bacteria, and their biological functions include softening of the insect cuticle to allow easier mycelial penetration (in the case of fungi) and evasion of lysozyme action (in the case of bacteria). They convert chitin, a β-1,4-linked N-acetylglucosamine polymer, into its deacetylated form, chitosan, a natural glucosamine polymer (42). Recently, CDAs were also identified in insects and appear to constitute one of two major classes of proteins recovered from the peritrophic membrane (PM) (4, 30). PM lining the insect midgut represents a major lepidopteran physical barrier against baculovirus infection (14, 35, 46). It consists of chitin and glycoproteins, and its physical role is to protect midgut epithelial cells from food particles, digestive enzymes, and pathogens. It also has a biochemical function, such as the inactivation of ingested toxins and enzyme recycling (3). Disruption of the link between chitin and the protein structure of the PM affects its functions in digestion and also leads to the collapse of the midgut defense against pathogens.In this work, H. armigera EST sequence analyses allowed the identification and full-length sequencing of three different CDA-like proteins, revealing that only one of them (HaCDA5a) was downregulated during the initial stages of baculovirus infection in larvae. HaCDA5a has been recombinantly expressed in insect cells, and its influence on PM permeability was checked. Given the natural ability of baculovirus to acquire insect host genes in order to improve survival and prevalence, we also analyzed the effects of CDA expression on the performance of baculovirus in insect bioassays. The results revealed that expression of CDA-like proteins by baculovirus may increase its infectivity and speed of kill and thus be applied for better pest control.     

The antidepressant role of dietary long-chain polyunsaturated n-3 fatty acids in two phases in the developing brain

In this work we investigated the effect from fish oil (FO) supplementation, rich in n-3 fatty acids, on an antidepressant effect on adult rats in Phase A (supplementation during pregnancy and lactation) and phase B (supplementation during post-weaning until adulthood). During Phase A, female rats, used as matrix to obtain male rats, were divided in three groups: FO (daily supplemented), CF (coconut fat daily supplemented) and control (not supplemented). Our results showed that adult rats whose mothers were supplemented with FO during Phase A and rats supplemented during phase B demonstrated a significantly decreased immobility time when compared to control and CF groups. There was no difference in neither motor activity nor anxiety behavior in the three groups excluding false positive results. Our results suggest that n-3 fatty acids supplementation during Phases A and B had a beneficial effect on preventing the development of depression-like behavior in adult rats.     

Photodynamic effect of lanthanide derivatives of meso-tetra(N-methyl-4-pyridyl)porphine against Staphylococcus aureus

Photodynamic therapy (PDT), used for cancer treatment, is also an alternative method for eradication of drug-resistant bacteria. This method utilizes a nontoxic light-activated dye, called a photosensitizer, and visible light to produce reactive oxygen species that lead to bacterial cell death. The purpose of this study was to investigate the bactericidal effect of PDT using lanthanide derivatives of meso-tetra(N-methyl-4-pyridyl)porphine against Staphylococcus aureus strains. The new photosensitizers appeared to be photodynamically ineffective. No enhancement of antistaphylococcal activity of TMPyP was observed after the conjugation of the porphyrin with lanthanide ions. Additionally, a significant difference in the susceptibility of two bacterial strains to unmodified TMPyP was observed.     

Pathological effect of synthetic cereulide, an emetic toxin of Bacillus cereus, is reversible in mice

Cereulide is the causative toxin of the emetic type of food-borne illness caused by Bacillus cereus. This toxin was previously shown to be associated with fulminant liver failure in a human case. Mice were injected i.p. with synthetic cereulide and the development of histopathological changes was examined. Hepatocytes showed mitochondrial swelling with loss of cristae, and dose-dependent increase of small fatty droplets. These microsteatotic hepatocytes were distributed mainly in the pericentral area. At higher cereulide doses, massive degeneration of hepatocytes occurred. The serum values of hepatic enzymes were highest on days 2-3 after the inoculation of cereulide, and rapidly decreased thereafter. General recovery from the pathological changes and regeneration of hepatocytes was observed after 4 weeks.     

A new approach to oligonucleotide N3'-->P5' phosphoramidate building blocks

A new synthetic approach to 5-phosphoramidites of 3'-aminonucleosides was developed. The methodology relies upon the use of 3'-amino-2',3'-dideoxy nucleosides as the key starting materials. The final phosphoramidite products were obtained with high yields via 2-3-step efficient chemical transformations using selective introduction of orthogonal protective groups to the 3'-aminonucleoside sugar and base moieties.     

Mechanisms of ammonia-induced cell death in rat cortical neurons: roles of NMDA receptors and glutathione

The occurrence, nature and prevention of ammonia-induced cell death were assayed in cultured primary cortical neurons from newborn rats. Treatment with 1-10 mM ammonium chloride for 24 or 48 h, dose-dependently decreased neuronal survival (MTT assay) and GSH/GSSG ratio in the cultures, whereas total GSH content was significantly reduced only with 10mM ammonia. Treatment with a glutathione synthesis inhibitor, buthionyl sulfoximine (BSO) (10 microM), decreased the GSH content and GSH/GSSG ratio to a degree similar to that of 10 mM ammonia, but it did not decrease cell survival in control cells. This indicates that glutathione depletion per se is not a cause of ammonia-induced neuronal death. However, ammonia-induced decrease of cell viability was attenuated by incubation with glutathione diethyl ester (GEE), which transiently increased the intracellular GSH level in both control and ammonia-treated cells. Neuronal survival in the presence of ammonia was partly improved by the NMDA receptor antagonists MK-801 and APV. Morphological analysis revealed that ammonia treatment causes both apoptotic and non-apoptotic neuronal death, the former not being inhibited by MK-801. Apoptosis was the dominant type of cell death at 10mM ammonia, as concluded both from morphologic examination and the absence of survival improvement in the presence of GABA+nipecotic acid or taurine, model anti-excitotoxic treatments of cortical neurons. The mechanism underlying apoptosis may include inhibition of a survival kinase, Akt, whose activatory phosphorylation at Ser473 is reduced in neurons treated with 10 mM, but not 1 mM ammonia.     

Protein a production in coagulase-negative or clumping factor (CF)-negative isolates of Staphylococcus aureus

The aim of study was to determine a production of proteinA in coagulase-negative Staphylococcus aureus (CNSA) or CF-negative S. aureus (CFNSA) strains. 59 CNSA and 18 CFNSA strains were isolated between 1997 and 2003 from different clinical specimens. The Protein A production was determined by immunoblotting method. The presence of protein A gene (spa) was investigated using the polymerase chain reaction (PCR). Two sets of phages and RFLP (Restriction Fragment Lenth Polymorphism) of coa gene method were used for typing strains. The results proved that the lack of ability of protein A production occurs more frequently in protein A-negative CFNSA strains with compare to the CNSA, which are protein A-positive for the majority of strains. Deficiencies of protein A, doesn't seem to be caused by the loss of spa gene. Protein A-negative CFNSA strains have phagotypes, RFLP and antibiotic resistant patterns which differ them from protein A-negative CNSA strains. Almost all of protein A-negative CFNSA and CNSA strains are resistant to methicillin.     

Methods of assessment and clinical relevance of QT dynamics

The dependence on heart rate of the QT interval has been investigated for many years and several mathematical formulae have been proposed to describe the QT interval/heart rate (or QT interval/RR interval) relationship. While the most popular is Bazett's formula, it overcorrects the QT interval at high heart rates and under-corrects it at slow heart rates. This formulae and many others similar ones, do not accurately describe the natural behaviour of the QT interval. The QT interval/RR interval relationship is generally described as QT dynamics. In recent years, several methods of its assessment have been proposed, the most popular of which is linear regression. An increased steepness of the linear QT/RR slope correlates with the risk of arrhythmic death following myocardial infarction. It has also been demonstrated that the QT interval adapts to heart rate changes with a delay (QT hysteresis) and that QT dynamics parameters vary over time. New methods of QT dynamics assessment that take into account these phenomena have been proposed. Using these methods, changes in QT dynamics have been observed in patients with advanced heart failure, and during morning hours in patients with ischemic heart disease and history of cardiac arrest. The assessment of QT dynamics is a new and promising tool for identifying patients at increased risk of arrhythmic events and for studying the effect of drugs on ventricular repolarisation.     

Middle-age male mice have increased severity of experimental autoimmune encephalomyelitis and are unresponsive to testosterone therapy

Treatment with sex hormones is known to protect against experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, little is known about how age affects the course of EAE or response to hormone treatment. This study demonstrates striking differences between middle-age vs young C57BL/6 male mice in the clinical course of EAE and response to both testosterone (T4) and estrogen (E2) hormone therapy. Unlike young males that developed an acute phase of EAE followed by a partial remission, middle-age males suffered severe chronic and unremitting EAE that was likely influenced by alterations in the distribution and function of splenic immunocytes and a significant reduction in suppressive activity of CD4+CD25+ regulatory T cells in the spleen and spinal cord. Middle-age males had reduced numbers of splenic CD4+ T cells that were generally hypoproliferative, but enhanced numbers of splenic macrophages and MHC class II-expressing cells, and increased secretion of the proinflammatory factors IFN-gamma and MCP-1. Surprisingly, middle-age males were unresponsive to the EAE-protective effects of T4 and had only a transient benefit from E2 treatment; young males were almost completely protected by both hormone treatments. T4 treatment of young males inhibited proliferation of myelin oligodendrocyte glycoprotein 35-55-specific T cells and secretion of TNF-alpha and IFN-gamma. The effects of T4 in vivo and in vitro were reversed by the androgen receptor antagonist, flutamide, indicating that the regulatory effects of T4 were mediated through the androgen receptor. These data are the first to define age-dependent differences in EAE expression and response to hormone therapy.