Assessment of the in vitro acaricidal activity of Bravecto® (fluralaner) and a proposed orange oil-based formulation vehicle for the treatment of Sarcoptes scabiei.

BACKGROUND: Sarcoptic mange is a serious animal welfare concern in bare-nosed wombats (Vombatus ursinus). Fluralaner (Bravecto®) is a novel acaricide that has recently been utilised for treating mange in wombats. The topical 'spot-on' formulation of fluralaner can limit treatment delivery options in situ, but dilution to a volume for 'pour-on' delivery is one practicable solution. This study investigated the in vitro acaricidal activity of Bravecto, a proposed essential oil-based diluent (Orange Power®), and two of its active constituents, limonene and citral, against Sarcoptes scabiei. METHODS: Sarcoptes scabiei were sourced from experimentally infested pigs. In vitro assays were performed to determine the lethal concentration (LC50) and survival time of the mites when exposed to varying concentrations of the test solutions. RESULTS: All compounds were highly effective at killing mites in vitro. The LC50 values of Bravecto, Orange Power, limonene and citral at 1 h were 14.61 mg/ml, 4.50%, 26.53% and 0.76%, respectively. The median survival times of mites exposed to undiluted Bravecto, Orange Power and their combination were 15, 5 and 10 min, respectively. A pilot survival assay of mites collected from a mange-affected wombat showed survival times of < 10 min when exposed to Bravecto and Orange Power and 20 min when exposed to moxidectin. CONCLUSIONS: These results confirm the acaricidal properties of Bravecto, demonstrate acaricidal properties of Orange Power and support the potential suitability of Orange Power and its active constituents as a diluent for Bravecto. As well as killing mites via direct exposure, Orange Power could potentially enhance the topical delivery of Bravecto to wombats by increasing drug penetration in hyperkeratotic crusts. Further research evaluating the physiochemical properties and modes of action of Orange Power and its constituents as a formulation vehicle would be of value.

seco-Pregnane Glycosides from Australian Caustic Vine (Cynanchum viminale subsp. australe).

Cynanchum viminale subsp. australe, more commonly known as caustic vine, is a leafless succulent that grows in the northern arid zone of Australia. Toxicity toward livestock has been reported for this species, along with use in traditional medicine and its potential anticancer activity. Disclosed herein are novel seco-pregnane aglycones cynavimigenin A (5) and cynaviminoside A (6), together with new pregnane glycosides cynaviminoside B (7) and cynavimigenin B (8). Cynavimigenin B (8) contains an unprecedented 7-oxobicyclo[2.2.1]heptane moiety in the seco-pregnane series, likely arising from a pinacol-type rearrangement. Interestingly, these isolates displayed only limited cytotoxicity in cancer and normal human cell lines, in addition to low activity against acetylcholinesterase and Sarcoptes scabiei bioassays, suggesting that 5-8 are not associated with the reported toxicity of this plant species.

A unique group of scabies mite pseudoproteases promotes cutaneous blood coagulation and delays plasmin-induced fibrinolysis.

BACKGROUND: Scabies, a highly contagious skin disease affecting more than 200 million people worldwide at any time, is caused by the parasitic mite Sarcoptes scabiei. In the absence of molecular markers, diagnosis requires experience making surveillance and control challenging. Superficial microthrombi in the absence of vasculitis in scabies-affected skin are a recognised, yet unexplained histopathological differential of scabies infection. This study demonstrates that a family of Scabies Mite Inactivated Cysteine Protease Paralogues (SMIPP-Cs) excreted by the mites plays a role in formation of scabies-induced superficial microthrombi. METHODOLOGY/PRINCIPAL FINDINGS: A series of in vitro and ex vivo experiments involving two representative recombinant SMIPP-Cs was carried out. In the presence of SMIPP-Cs, the thrombin clotting time (TCT), fibrin formation and plasmin induced fibrinolysis were monitored in vitro. The ultrastructure of the SMIPP-C-modulated fibrin was analysed by Scanning Electron Microscopy (SEM). Immuno-histological analyses were performed ex vivo, to localise the SMIPP-C proteins within scabies infected skin biopsies. SMIPP-Cs displayed pro-coagulant properties. They bound calcium ions, reduced the thrombin clotting time, enhanced the fibrin formation rate and delayed plasmin-induced fibrinolysis. The SMIPP-Cs associated with fibrin clots during fibrinogen polymerisation and did not bind to preformed fibrin. Scanning electron microscopy revealed that the fibrin clots formed in the presence of SMIPP-Cs were aberrant and denser than normal fibrin clots. SMIPP-Cs were detected in microthrombi which are commonly seen in scabietic skin. CONCLUSIONS/SIGNIFICANCE: The SMIPP-Cs are the first scabies mite proteins found in sub-epidermal skin layers and their pro-coagulant properties promote superficial microthrombi formation in scabetic skin. Further research is needed to evaluate their potential as diagnostic or therapeutic target.

Lemongrass (Cymbopogon citratus) oil: A promising miticidal and ovicidal agent against Sarcoptes scabiei.

BACKGROUND: Essential oils may represent an alternative strategy for controlling scabies, a neglected tropical disease caused by the infestation of mite from the species Sarcoptes scabiei. Lemongrass (Cymbopogen citratus) oil is reported to possess pharmacological properties including antiparasitc, antioxidant, antimicrobial and anti-inflammatory. The aim of the present study was to assess the potential efficacy of lemongrass oil against the mites and eggs of Sarcoptes scabiei. METHODOLOGY/PRINCIPAL FINDINGS: Mass spectrometry analysis confirmed that the main component presented in lemongrass oil was citral. Lemongrass oil at concentrations of 10% and 5% killed all Sarcoptes mites within 10 and 25 min, respectively. The median lethal concentration value was 1.37%, 1.08%, 0.91%, 0.64%, and 0.48% at 1, 3, 6, 12, and 24 h, respectively. Lemongrass oil at all concentrations (10%, 5%, 1%, 0.5%, 0.1%) was able to significantly decrease the hatching rate of Sarcoptes eggs. CONCLUSIONS/SIGNIFICANCE: Lemongrass oil should be considered as a promising miticidal and ovicidal agent for scabies control.

Cytoplasmic and periplasmic expression of recombinant shark VNAR antibody in Escherichia coli.

Shark variable new antigen receptors (VNARs) are known to possess excellent heat-stability, and the long complementarity determining region 3 (CDR3) has permitted it to penetrate into the cleft region of antigens. The number of cysteine (Cys) residues contained within VNAR is greater than in conventional antibodies, entailing disulfide bond formation in both the inter- or intra-loop regions is required for interactions with the target protein antigens. Therefore, the selection of a suitable expression system is important to ensure the solubility and correct folding of functional VNAR protein production. Unlike higher organisms, the machinery for effecting posttranslational modifications of proteins in Escherichia coli (E. coli) are less sophisticated. To overcome this circumstance, a pDSB-28Y vector fusion with DsbA signal peptide was engineered for periplasmic H8VNAR production. Despite the periplasmic proteins showing a lower yield (62 µg/mL) than cytosolic proteins (468 µg/mL) that is obtained from pET-28a vector, it has demonstrated better performance than that of a cytosolic protein in terms of absorbance. However, these readings were still inferior to that of positive control mouse monoclonal antibody (mAb) C1-13 in this experiment. Therefore, further investigation is required to improve the binding affinity of selected recombinant VNAR towards malaria biomarkers.

Kunitz type protease inhibitor EgKI-1 from the canine tapeworm Echinococcus granulosus as a promising therapeutic against breast cancer.

EgKI-1, a member of the Kunitz type protease inhibitor family, is highly expressed by the oncosphere of the canine tapeworm Echinococcus granulosus, the stage that is infectious to humans and ungulates, giving rise to a hydatid cyst localized to the liver and other organs. Larval protoscoleces, which develop within the hydatid cyst, have been shown to possess anti-cancer properties, although the precise molecules involved have not been identified. We show that recombinant EgKI-1 inhibits the growth and migration of a range of human cancers including breast, melanoma and cervical cancer cell lines in a dose-dependent manner in vitro without affecting normal cell growth. Furthermore, EgKI-1 treatment arrested the cancer cell growth by disrupting the cell cycle and induced apoptosis of cancer cells in vitro. An in vivo model of triple negative breast cancer (MDA-MB-231) in BALB/c nude mice showed significant tumor growth reduction in EgKI-1-treated mice compared with controls. These findings indicate that EgKI-1 shows promise for future development as an anti-cancer therapeutic.

Phylogenetic relationships, stage-specific expression and localisation of a unique family of inactive cysteine proteases in Sarcoptes scabiei.

BACKGROUND: Scabies is worldwide one of the most common, yet neglected, parasitic skin infections, affecting a wide range of mammals including humans. Limited treatment options and evidence of emerging mite resistance against the currently used drugs drive our research to explore new therapeutic candidates. Previously, we discovered a multicopy family of genes encoding cysteine proteases with their catalytic sites inactivated by mutation (SMIPP-Cs). This protein family is unique in parasitic scabies mites and is absent in related non-burrowing mites. We postulated that the SMIPP-Cs have evolved as an adaptation to the parasitic lifestyle of the scabies mite. To formulate testable hypotheses for their functions and to propose possible strategies for translational research we investigated whether the SMIPP-Cs are common to all scabies mite varieties and where within the mite body as well as when throughout the parasitic life-cycle they are expressed. RESULTS: SMIPP-C sequences from human, pig and dog mites were analysed bioinformatically and the phylogenetic relationships between the SMIPP-C multi-copy gene families of human, pig and dog mites were established. Results suggest that amplification of the SMIPP-C genes occurred in a common ancestor and individual genes evolved independently in the different mite varieties. Recombinant human mite SMIPP-C proteins were produced and used for murine polyclonal antibody production. Immunohistology on skin sections from human patients localised the SMIPP-Cs in the mite gut and in mite faeces within in the epidermal skin burrows. SMIPP-C transcription into mRNA in different life stages was assessed in human and pig mites by reverse transcription followed by droplet digital PCR (ddPCR). High transcription levels of SMIPP-C genes were detected in the adult female life stage in comparison to all other life stages. CONCLUSIONS: The fact that the SMIPP-Cs are unique to three Sarcoptes varieties, present in all burrowing life stages and highly expressed in the digestive system of the infective adult female life stage may highlight an essential role in parasitism. As they are excreted from the gut in scybala they presumably are able to interact or interfere with host proteins present in the epidermis.

Acute Moraxella catarrhalis Airway Infection of Chronically Smoke-Exposed Mice Increases Mechanisms of Emphysema Development: A Pilot Study.

In chronic obstructive pulmonary disease (COPD), acute exacerbations and emphysema development are characteristics for disease pathology. COPD is complicated by infectious exacerbations with acute worsening of respiratory symptoms with Moraxella catarrhalis as one of the most frequent pathogens. Although cigarette smoke (CS) is the primary risk factor, additional molecular mechanisms for emphysema development induced by bacterial infections are incompletely understood. We investigated the impact of M. catarrhalis on emphysema development in CS exposed mice and asked whether an additional infection would induce a solubilization of pro-apoptotic and pro-inflammatory endothelial monocyte-activating-protein-2 (EMAPII) to exert its activities in the pulmonary microvas-culature and other parts of the lungs not exposed directly to CS. Mice were exposed to smoke (6 or 9 months) and/or infected with M. catarrhalis. Lungs, bronchoalveolar lavage fluid (BALF), and plasma were analyzed. CS exposure reduced ciliated area, caused rarefaction of the lungs, and induced apoptosis. EMAPII was increased independent of prior smoke exposure in BALF of infected mice. Importantly, acute M. catarrhalis infection increased release of matrixmetalloproteases-9 and -12, which are involved in emphysema development and comprise a mechanism of EMAPII release. Our data suggest that acute M. catarrhalis infection represents an independent risk factor for emphysema development in smoke-exposed mice.

The influence of genetic predisposition and autoimmune hepatitis inducing antigens in disease development.

Autoimmune hepatitis (AIH) is defined as a chronic liver inflammation with loss of tolerance against hepatocytes. The etiology and pathophysiology of AIH are still poorly understood because reliable animal models are limited. Therefore, we recently introduced a model of experimental murine AIH by a self-limited adenoviral infection with the AIH type 2 antigen formiminotransferase cyclodeaminase (FTCD). We could demonstrate that break of humoral tolerance towards liver specific autoantigens like FTCD and cytochrome P450 2D6 (CYP2D6) is not dependent on the genetic background. However, the development of AIH in autoantibody positive animals is determined by genetic background genes. We could also show that the break of humoral tolerance is necessary but not sufficient for the development of AIH. In contrast the break of tolerance against the ubiquitously expressed nuclear antigens (ANAs) is strictly dependent on genetic predisposition. Priming with the UGA suppressor tRNA-associated protein (soluble liver antigen; SLA) is a strong inducer of ANA reactivity, but not sufficient to cause AIH development thereby questioning the importance of anti-SLA immune response as an important driver in AIH. Monogenetic mutations such as Aire-deficiency can cause AIH in otherwise genetically resistant strains. CONCLUSION: The results have important implications for our understanding of the pathophysiology of AIH development and for the interpretation of humoral antibody responses in AIH.

Cloning and Characterization of Two Potent Kunitz Type Protease Inhibitors from Echinococcus granulosus.

The tapeworm Echinococcus granulosus is responsible for cystic echinococcosis (CE), a cosmopolitan disease which imposes a significant burden on the health and economy of affected communities. Little is known about the molecular mechanisms whereby E. granulosus is able to survive in the hostile mammalian host environment, avoiding attack by host enzymes and evading immune responses, but protease inhibitors released by the parasite are likely implicated. We identified two nucleotide sequences corresponding to secreted single domain Kunitz type protease inhibitors (EgKIs) in the E. granulosus genome, and their cDNAs were cloned, bacterially expressed and purified. EgKI-1 is highly expressed in the oncosphere (egg) stage and is a potent chymotrypsin and neutrophil elastase inhibitor that binds calcium and reduced neutrophil infiltration in a local inflammation model. EgKI-2 is highly expressed in adult worms and is a potent inhibitor of trypsin. As powerful inhibitors of mammalian intestinal proteases, the EgKIs may play a pivotal protective role in preventing proteolytic enzyme attack thereby ensuring survival of E. granulosus within its mammalian hosts. EgKI-1 may also be involved in the oncosphere in host immune evasion by inhibiting neutrophil elastase and cathepsin G once this stage is exposed to the mammalian blood system. In light of their key roles in protecting E. granulosus from host enzymatic attack, the EgKI proteins represent potential intervention targets to control CE. This is important as new public health measures against CE are required, given the inefficiencies of available drugs and the current difficulties in its treatment and control. In addition, being a small sized highly potent serine protease inhibitor, and an inhibitor of neutrophil chemotaxis, EgKI-1 may have clinical potential as a novel anti-inflammatory therapeutic.

Moraxella catarrhalis induces an immune response in the murine lung that is independent of human CEACAM5 expression and long-term smoke exposure.

In patients with chronic obstructive pulmonary disease (COPD), Moraxella catarrhalis infection of the lower airways is associated with chronic colonization and inflammation during stable disease and acute exacerbations. Chronic smoke exposure induces chronic inflammation and impairs mucociliary clearance, thus contributing to bacterial colonization of the lower airways in COPD patients. The human-specific carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 5, expressed in human airways, has been shown to contribute to epithelial colonization of CEACAM-binding pathogens. To investigate the impact of CEACAM5 expression on pulmonary M. catarrhalis colonization, we infected mice transgenic for human CEACAM5 (hCEACAM5) and wild type mice intratracheally with M. catarrhalis with or without preceding smoke exposure and analyzed bacterial colonization and local and systemic inflammation. Our results show that airway infection with M. catarrhalis accelerated acute local but not systemic inflammation, albeit independent of hCEACAM5 expression. Long-term smoke exposure alone or prior to M. catarrhalis infection did not contribute to increased local or systemic inflammation. No difference was found in pulmonary clearance of M. catarrhalis in hCEACAM5-transgenic mice compared with wild-type mice. Smoke exposure neither altered time nor extent of persistence of M. catarrhalis in the lungs of both genotypes. In conclusion, M. catarrhalis induced a local acute immune response in murine airways. Neither hCEACAM5 expression nor chronic smoke exposure nor a combination of both was sufficient as prerequisites for the establishment of chronic M. catarrhalis colonization. Our results demonstrate the difficulties in mirroring conditions of chronic airways colonization of M. catarrhalis in a murine model.

Pseudoproteases: mechanisms and function.

Catalytically inactive enzymes (also known as pseudoproteases, protease homologues or paralogues, non-peptidase homologues, non-enzymes and pseudoenzymes) have traditionally been hypothesized to act as regulators of their active homologues. However, those that have been characterized demonstrate that inactive enzymes have an extensive and expanding role in biological processes, including regulation, inhibition and immune modulation. With the emergence of each new genome, more inactive enzymes are being identified, and their abundance and potential as therapeutic targets has been realized. In the light of the growing interest in this emerging field the present review focuses on the classification, structure, function and mechanism of inactive enzymes. Examples of how inactivity is defined, how this is reflected in the structure, functions of inactive enzymes in biological processes and their mode of action are discussed.

Autoantibodies to iron-binding proteins in pigs infested with Sarcoptes scabiei.

Despite the availability of effective treatments, Sarcoptes scabiei remains a major health problem in the pig industry. Unsuccessful control of the disease is often due to the lack of reliable detection methods, with current tests relying on skin scrapings and crude antigen ELISAs. A previous analysis of antigens in pig skin scrapings reported that anti-transferrin antibodies were present in S. scabiei infected animals and that this finding might be considered as a useful diagnostic tool. This paper confirms IgG autoantibodies against transferrin, including the first report of IgM autoantibodies, in both naturally and experimentally infected pigs using ELISA and dot blot assays. Autoantibodies were also detected in pigs to ferritin and to a lesser extent lactoferrin. Immunoblotting confirmed the presence of IgG and IgM autoantibodies in mange positive pigs, as well as IgM antibodies to transferrin and albumin in mange negative pigs. These findings suggest the presence of natural autoantibodies to transferrin and albumin in pigs. The development of the IgG autoimmune response may either be a host mechanism for limiting iron to the mite via antibody mediated clearance, the result of host exposure to mite iron-binding homologues or because of a mite-induced antigenic change to host transferrin. Further investigation into the formation of these autoantibodies may provide insights into the importance of iron in scabies infections and the development and perseverance of S. scabiei infections in pigs. The specificity and sensitivity of the anti-transferrin response reinforces its potential in the diagnosis of scabies in pigs.

Genetic predisposition and environmental danger signals initiate chronic autoimmune hepatitis driven by CD4+ T cells.

UNLABELLED: Autoimmune hepatitis (AIH) is defined as a chronic liver disease with loss of tolerance against liver tissue eventually leading to cirrhosis if left untreated. 80%-90% of patients can be treated with a life-long immunosuppression. Unfortunately, there are strong drug-related side effects and steroid-refractory patients. Therefore, there is a need for a model system to investigate the complex immunopathogenesis of this chronic disease and subsequently to develop new therapeutic interventions. We developed a new model of experimental murine AIH (emAIH) by a self-limited adenoviral infection with the hepatic autoantigen formiminotransferase cyclodeaminase (FTCD). After an initial transient hepatitis there was a chronic evolving AIH, finally leading to portal and lobular fibrosis. We could show that the genetic predisposition provided by the NOD background was essential for creating a fertile field for the development of liver-specific autoimmunity. However, a strong environmental trigger was additionally necessary to initiate the disease. Besides the break of humoral tolerance, T-cell tolerance against hepatic self-antigens was also broken and CD4(+) T cells were identified as essential drivers of the disease. As the disease was successfully treated with prednisolone and budesonide, the model will be helpful to develop and test new therapeutic interventions. CONCLUSION: We developed a new murine AIH model closely resembling AIH in patients that explains the mechanisms of AIH pathophysiology. In addition, emAIH provides options to test therapeutic alternatives for patients not achieving remission, with reduced side effects of chronic nonspecific immunosuppression.

Malaria infection alters the expression of B-cell activating factor resulting in diminished memory antibody responses and survival.

Malaria is a major cause of morbidity worldwide with reports of over 200-500 million infected individuals and nearly 1 million deaths each year. Antibodies have been shown to play a critical role in controlling the blood stage of this disease; however, in malaria-endemic areas antibody immunity is slow to develop despite years of exposure to Plasmodium spp. the causative parasite. Using rodent Plasmodium yoelii YM, we provide evidence that malarial infections result in a decrease in the proportion of DCs that express the B-cell survival factor, BAFF, resulting in a decreased ability of these DCs to support memory B-cell differentiation into antibody secreting cells (ASCs) and/or the survival of ASCs. Further, compared with infected WT mice, ASC numbers were significantly increased in malaria-infected transgenic mice that either overexpressed BAFF or mice with BAFF-independent B-cell survival (B-cell-restricted TRAF3 deletion). Remarkably, BAFF-overexpressing mice were protected from lethal malaria infections, indicating the significance of the role BAFF plays in determining the outcome of malaria infections. These findings describe a previously unappreciated mechanism by which Plasmodium spp. can depress the generation of protective antibody responses.

Novel scabies mite serpins inhibit the three pathways of the human complement system.

Scabies is a parasitic infestation of the skin by the mite Sarcoptes scabiei that causes significant morbidity worldwide, in particular within socially disadvantaged populations. In order to identify mechanisms that enable the scabies mite to evade human immune defenses, we have studied molecules associated with proteolytic systems in the mite, including two novel scabies mite serine protease inhibitors (SMSs) of the serpin superfamily. Immunohistochemical studies revealed that within mite-infected human skin SMSB4 (54 kDa) and SMSB3 (47 kDa) were both localized in the mite gut and feces. Recombinant purified SMSB3 and SMSB4 did not inhibit mite serine and cysteine proteases, but did inhibit mammalian serine proteases, such as chymotrypsin, albeit inefficiently. Detailed functional analysis revealed that both serpins interfered with all three pathways of the human complement system at different stages of their activation. SMSB4 inhibited mostly the initial and progressing steps of the cascades, while SMSB3 showed the strongest effects at the C9 level in the terminal pathway. Additive effects of both serpins were shown at the C9 level in the lectin pathway. Both SMSs were able to interfere with complement factors without protease function. A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8). Direct binding was observed between SMSB4 and the complement proteases C1s and C1r. However no complex formation was observed between either mite serpin and the complement serine proteases C1r, C1s, MASP-1, MASP-2 and MASP-3. No catalytic inhibition by either serpin was observed for any of these enzymes. In summary, the SMSs were acting at several levels mediating overall inhibition of the complement system and thus we propose that they may protect scabies mites from complement-mediated gut damage.

Scabies: important clinical consequences explained by new molecular studies.

In 2004, we reviewed the status of disease caused by the scabies mite Sarcoptes scabiei at the time and pointed out that very little basic research had ever been done. The reason for this was largely the lack of availability of mites for experimental purposes and, to a degree, a consequent lack of understanding of its importance, resulting in the trivial name 'itch mite'. Scabies is responsible for major morbidity in disadvantaged communities and immunocompromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by bacterial pathogens such as Streptococcus pyogenes and Staphylococcus aureus via skin lesions, resulting in severe downstream disease such as in a high prevalence of rheumatic fever/heart disease in affected communities. We now have further evidence that in disadvantaged populations living in tropical climates, scabies rather than 'Strep throat' is an important source of S. pyogenes causing rheumatic fever and eventually rheumatic heart disease. In addition, our work has resulted in two fundamental research tools that facilitate much of the current biomedical research efforts on scabies, namely a public database containing ~45,000 scabies mite expressed sequence tags and a porcine in vivo model. Here we will discuss novel and unexpected proteins encountered in the database that appear crucial to mite survival with regard to digestion and evasion of host defence. The mode(s) of action of some of these have been at least partially revealed. Further, newly discovered molecules that may well have a similar role, such as a family of inactivated cysteine proteases, are yet to be investigated. Hence, there are now whole families of potential targets for chemical inhibitors of S. scabiei. These efforts put today's scabies research in a unique position to design and test small molecules that may specifically interfere with mite-derived molecules, such as digestive proteases and mite complement inhibitors. The porcine scabies model will be available to trial in vivo treatment with potential inhibitors. New therapies for scabies may be developed from these studies and may contribute to reduce the spread of scabies and the subsequent prevalence of bacterial skin infections and their devastating sequelae in the community.

Induction of peroxiredoxin I gene expression by LPS involves the Src/PI3K/JNK signalling pathway.

Peroxiredoxin I (Prx I) belongs to a family of proteins with thiol-dependent peroxidase activity and is involved in the cellular protection against oxidative stress, the modulation of intracellular signalling cascades as well as the regulation of cell proliferation and apoptosis. In RAW 264.7 mouse macrophage cells Prx I was up-regulated on the mRNA and protein level by lipopolysaccharide (LPS). Treatment of cells with LPS increased the phosphorylation of c-Jun-NH(2) terminal kinase (JNK) and protein kinase B (PKB). Both SP600125, an inhibitor of JNK, and LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), dose-dependently decreased LPS-induced Prx I mRNA expression. Furthermore, up-regulation of Prx I mRNA by LPS was diminished by the Src tyrosine kinase inhibitor PP2 and the iNOS inhibitor L-NMMA. LPS-dependent induction of Prx I is likely mediated by an activator protein-1 site within the Prx I promoter region binding JunB and c-Fos. In contrast, NFkappaB was not involved in the activation of Prx I transcription. Our results suggest that the up-regulation of Prx I gene expression by LPS is part of the cellular response to stress and may protect against oxidative stress-related injury in RAW 264.7 cells.

Collateral bystander damage by myelin-directed CD8+ T cells causes axonal loss.

Permanent disability of patients suffering from central nervous system (CNS) inflammation such as multiple sclerosis, the most common chronic inflammatory disorder of the CNS, originates mainly from demyelination and axonal damage. Although many studies in the past focused on the role of CD4(+) T cells, several recent findings postulate the relevance of autoaggressive, cytotoxic CD8(+) T cells in the effector phase of multiple sclerosis. Yet, it remains unresolved whether axonal injury is the result of a CD8(+) T cell-targeted hit against the axon itself or the consequence of an attack against the myelin structure. To address this issue of CD8-mediated tissue damage in CNS inflammation, we performed continuous confocal imaging of autoaggressive, cytotoxic CD8(+) T cells in living organotypic cerebellar brain slices. We observed that loading brain slices with the cognate peptide antigen caused CD8-mediated damage of myelinated axons. To exclude the possibility that the cognate peptide loaded onto the brain slices was presented by axons directly, we restricted the cognate antigen expression exclusively to the cytosol of oligodendrocytes. Aside from vast myelin damage, extensive axonal bystander injury occurred. Using this model system of inflammatory CNS injury, we visualize that axonal loss can be the consequence from "collateral bystander damage" by autoaggressive, cytotoxic CD8(+) T cells, targeting their cognate antigen processed and presented by oligodendrocytes.