A phase I vaccination study with tyrosinase in patients with stage II melanoma using recombinant modified vaccinia virus Ankara (MVA-hTyr)

A significant percentage of patients with stage II melanomas suffer a relapse after surgery and therefore need the development of adjuvant therapies. In the study reported here, safety and immunological response were analyzed after vaccination in an adjuvant setting with recombinant modified vaccinia virus Ankara carrying the cDNA for human tyrosinase (MVA-hTyr). A total of 20 patients were included and vaccinated three times at 4-week intervals with 5×10⁸ IU of MVA-hTyr each time. The responses to the viral vector, to known HLA class I–restricted tyrosinase peptides, and to dendritic cells transfected with tyrosinase mRNA, were investigated by ELISpot assay on both ex vivo T cells and on T cells stimulated in vitro prior to testing. The delivery of MVA-hTyr was safe and did not cause any side effects above grade 2. A strong response to the viral vector was achieved, indicated by an increase in the frequency of MVA-specific CD4⁺ and CD8⁺ T cells and an increase in virus-specific antibody titers. However, no tyrosinase-specific T-cell or antibody response was observed with MVA-hTyr in any of the vaccinated patients. Although MVA-hTyr provides a safe and effective antigen-delivery system, it does not elicit a measurable immune response to its transgene product in patients with stage II melanoma after repeated combined intradermal and subcutaneous vaccination. We presume that modification of the antigen and/or prime-boost vaccination applying different approaches to antigen delivery may be required to induce an effective tyrosinase-specific immune response.     

Circadian differences in behavioral sensitization to cocaine: putative role of arylalkylamine N-acetyltransferase

Circadian rhythms might be involved in addictive behaviors. The pineal secretory product melatonin decreases cocaine sensitization in rats; mice mutant for the critical melatonin-synthesizing enzyme, arylalkylamine N-acetyltransferase (AANAT), exhibit altered behaviors. We hypothesized that AANAT/melatonin system, which is up-regulated at night, affects cocaine sensitization in mice. Intraperitoneal cocaine treatment (10 and 20 mg/kg) dose-dependently increased locomotor activity of both normal (C3H/HeJ) and AANAT mutant (C57BL/6J) mice; this effect was similar during the day and at night. Injections of cocaine during the day for three days resulted in behavioral sensitization in normal and AANAT mutant mice whereas treatment at night triggered sensitization in AANAT-deficient mice only. AANAT expression and synthesis of N-acetylserotonin/melatonin could play a role in addictive properties of cocaine.     

Intranasal ovalbumin immunotherapy with mycobacterial adjuvant promotes regulatory T cell accumulation in lung tissues

Allergen‐specific immunotherapy to induce T regulatory cells in the periphery has been used to treat allergic diseases. Mycobacteria can be used as an adjuvant for inducing T regulatory cells. However, it is unclear whether intranasal immunotherapy in combination with Mycobacteria adjuvant induces regulatory T cell differentiation and attenuates allergic responses in vivo. To investigate the role of intranasal ovalbumin (OVA) treatment alone and in combination with Mycobacteria vaccae, proportions of FoxP3⁺ regulatory T cells and anti‐inflammatory responses were evaluated in a murine model of asthma that was established in three groups of bicistronic Foxp3^EGFP reporter BALB/c mice. Before establishment of the asthma model, two groups of mice received intranasal OVA immunotherapy and one also received simultaneous s.c. M. vaccae. Expression of CD4⁺CD25⁺Foxp3^+EGFP+ T cells in the lung and spleen was analyzed by flow cytometry and the cytokine profiles of allergen‐stimulated lung and spleen lymphocytes assessed. The intranasal OVA immunotherapy group showed greater expression of CD4⁺CD25⁺Foxp3^+EGFP+ T cells in the spleen whereas in the group that also received M. vaccae such greater expression was demonstrated in the lung. Additionally, the proportion of IL‐10 and IFN‐γ‐secreting splenocytes was greater in the intranasal OVA + M. vaccae group. CD25 neutralization decreased CD4⁺Foxp3⁺ cells more than other groups. In parallel with this finding, production of IL‐10 and IFN‐γ was down‐regulated. Mucosal administration of OVA antigen results in a greater proportion of CD4⁺Foxp3⁺ T cells in the spleen. IL‐10 and IFN‐γ induced by intranasal OVA immunotherapy and M. vaccae administration is down‐regulated after CD25 neutralization.

     

A Novel Laser-Doppler Flowmetry Assisted Murine Model of Acute Hindlimb Ischemia-Reperfusion for Free Flap Research

Suitable and reproducible experimental models of translational research in reconstructive surgery that allow in-vivo investigation of diverse molecular and cellular mechanisms are still limited. To this end we created a novel murine model of acute hindlimb ischemia-reperfusion to mimic a microsurgical free flap procedure. Thirty-six C57BL6 mice (n = 6/group) were assigned to one control and five experimental groups (subject to 6, 12, 96, 120 hours and 14 days of reperfusion, respectively) following 4 hours of complete hindlimb ischemia. Ischemia and reperfusion were monitored using Laser-Doppler Flowmetry. Hindlimb tissue components (skin and muscle) were investigated using histopathology, quantitative immunohistochemistry and immunofluorescence. Despite massive initial tissue damage induced by ischemia-reperfusion injury, the structure of the skin component was restored after 96 hours. During the same time, muscle cells were replaced by young myotubes. In addition, initial neuromuscular dysfunction, edema and swelling resolved by day 4. After two weeks, no functional or neuromuscular deficits were detectable. Furthermore, upregulation of VEGF and tissue infiltration with CD34-positive stem cells led to new capillary formation, which peaked with significantly higher values after two weeks. These data indicate that our model is suitable to investigate cellular and molecular tissue alterations from ischemia-reperfusion such as occur during free flap procedures.     

Idebenone Ameliorates Rotenone-Induced Parkinson’s Disease in Rats Through Decreasing Lipid Peroxidation

Neurochemical Research - Oxidative stress is considered one of the mechanisms responsible for neurodegenerative diseases, especially for Parkinson’s disease. Since oxidative stress causes...     

Host-interactor screens of Phytophthora infestans RXLR proteins reveal vesicle trafficking as a major effector-targeted process

Pathogens modulate plant cell structure and function by secreting effectors into host tissues. Effectors typically function by associating with host molecules and modulating their activities. This study aimed to identify the host processes targeted by the RXLR class of host-translocated effectors of the potato blight pathogen Phytophthora infestans. To this end, we performed an in planta protein–protein interaction screen by transiently expressing P. infestans RXLR effectors in Nicotiana benthamiana leaves followed by coimmunoprecipitation and liquid chromatography-tandem mass spectrometry. This screen generated an effector–host protein interactome matrix of 59 P. infestans RXLR effectors x 586 N. benthamiana proteins. Classification of the host interactors into putative functional categories revealed over 35 biological processes possibly targeted by P. infestans. We further characterized the PexRD12/31 family of RXLR-WY effectors, which associate and colocalize with components of the vesicle trafficking machinery. One member of this family, PexRD31, increased the number of FYVE positive vesicles in N. benthamiana cells. FYVE positive vesicles also accumulated in leaf cells near P. infestans hyphae, indicating that the pathogen may enhance endosomal trafficking during infection. This interactome dataset will serve as a useful resource for functional studies of P. infestans effectors and of effector-targeted host processes.

One-sentence summary In planta protein–protein interaction screen of Phytophthora infestans RXLR effectors by proteomics approach reveals host vesicle trafficking as a major effector-targeted process.     

The therapeutic effects of anti-oxidant and anti-inflammatory drug quercetin on aspiration-induced lung injury in rats

Aspiration pneumonitis refers to acute chemical lung injury caused by aspiration of sterile gastric contents. The aim of this study was to evaluate the role of quercetin (QC) in acid aspiration-induced lung injury in rats. Twenty-eight female Sprague–Dawley rats were used and divided into the following groups (n = 7): sham (aspirated normal saline, S), hydrochloric acid (aspirated HCl), S plus treatment with QC (S + QC), and HCl plus treatment with QC (HCl + QC). After aspiration, the treatment groups received QC 60 mg/kg/day intraperitoneally once a day for 7 days. As a result of acid aspiration, an increase was observed in the levels of serum clara cell protein-16 (CC-16) and advanced oxidation protein products, whereas there was a decrease in serum thiobarbituric acid-reactive substances, superoxide dismutase (SOD), and catalase levels. There was a significant decrease in peribronchial inflammatory cell infiltration, alveolar septal infiltration, alveolar edema, and alveolar exudate scores, except in the alveolar histiocytes in the HCl + QC group. The expression of nitric oxide synthase, which increased after aspiration in the HCl group, showed a statistically significant decrease after the QC treatment. After the treatment with QC, an increase in the serum SOD level was observed, whereas a significant decrease was determined in the serum CC-16 level relative to that of the aspiration group (HCl). The antioxidant QC is effective in the treatment of lung injury following acid aspiration and can be used as a serum CC-16 biomarker in predicting the severity of oxidative lung injury.     

Evaluation of the effect of cagPAI genes of Helicobacter pylori on AGS epithelial cell morphology and IL-8 secretion

Helicobacter pylori cagPAI genes play an important role in pathogenesis, however little is known about their functions in isolates from Turkish patients. We aimed to evaluate the intactness and the effect of the cagPAI genes (cagT, cagM, cagE, cagA) and cagA EPIYA motifs on the AGS morphological changes and IL-8 induction. Of 53 patients 38 were found infected with H. pylori. PCR amplification of the cagPAI genes showed 42.1 % intact, 39.5 % partially deleted and 18.4 % with complete deletions. Isolates from gastritis, duodenal and gastric ulcer patients with intact and partially deleted cagPAI genes induced higher IL-8 secretion than those with complete deletions. Isolates from gastritis patients had higher deletion frequencies of the cagT and cagM genes than the other two genes. Infection of AGS cells with isolates that possess intact cagPAI and EPIYA-ABC resulted in the formation of the hummingbird phenotype. The cagA positive isolates induced higher IL-8 secretion than cagA negative isolates. Isolates from DU patients with more than one EPIYA-C motif induced higher concentrations of IL-8 than those with EPIYA-ABC. In conclusion, the intactness of the cagPAI in our isolates from different patients was not conserved. An intact cagPAI was found to play an important role in the pathogenesis of DU but not GU or gastritis. The cagA gene, but not other cagPAI genes, was associated with the induction of IL-8 and the morphological changes of the AGS cells. An increase in the number of EPIYA-C motifs had noticeable effect on the formation of the hummingbird phenotype.     

The Plant Membrane-Associated REMORIN1.3 Accumulates in Discrete Perihaustorial Domains and Enhances Susceptibility to Phytophthora infestans

Filamentous pathogens such as the oomycete Phytophthora infestans infect plants by developing specialized structures termed haustoria inside the host cells. Haustoria are thought to enable the secretion of effector proteins into the plant cells. Haustorium biogenesis, therefore, is critical for pathogen accommodation in the host tissue. Haustoria are enveloped by a specialized host-derived membrane, the extrahaustorial membrane (EHM), which is distinct from the plant plasma membrane. The mechanisms underlying the biogenesis of the EHM are unknown. Remarkably, several plasma membrane-localized proteins are excluded from the EHM, but the remorin REM1.3 accumulates around P. infestans haustoria. Here, we used overexpression, colocalization with reporter proteins, and superresolution microscopy in cells infected by P. infestans to reveal discrete EHM domains labeled by REM1.3 and the P. infestans effector AVRblb2. Moreover, SYNAPTOTAGMIN1, another previously identified perihaustorial protein, localized to subdomains that are mainly not labeled by REM1.3 and AVRblb2. Functional characterization of REM1.3 revealed that it is a susceptibility factor that promotes infection by P. infestans. This activity, and REM1.3 recruitment to the EHM, require the REM1.3 membrane-binding domain. Our results implicate REM1.3 membrane microdomains in plant susceptibility to an oomycete pathogen.Filamentous plant pathogens, including oomycetes of the genus Phytophthora, downy mildews and white rusts, as well as powdery mildews and rust fungi, are among the most devastating plant pathogens. These biotrophic parasites associate closely with plant cells through specialized infection structures called haustoria. Haustoria are specialized pathogen hyphal structures formed within host cells and enveloped by a perimicrobial membrane called the extrahaustorial membrane (EHM), a key interface between plant pathogens and the host cell. Haustoria are critical for successful parasitic infection by many filamentous plant pathogens and are a signature of the biotrophic lifestyle. In fungi, haustoria function as feeding structures (Voegele et al., 2001). In addition, haustoria are thought to enable the delivery of host-translocated virulence proteins, known as effectors, by both fungal and oomycete pathogens (Catanzariti et al., 2006; Whisson et al., 2007). However, little is known about the molecular mechanisms underlying the biogenesis and function of haustoria and EHM (Kemen and Jones, 2012; Lu et al., 2012).The EHM is thought to be continuous with the host plasma membrane (PM), yet it is a highly specialized membrane compartment that develops only in plant cells that accommodate haustoria (haustoriated cells; Coffey and Wilson, 1983). On the plant side, all eight PM proteins tested by Koh et al. (2005) were excluded from the EHM in Arabidopsis (Arabidopsis thaliana) cells infected with the powdery mildew fungus Golovinomyces cichoracearum. Conversely, the atypical Arabidopsis resistance protein Resistance to Powdery Mildew8.2 (RPW8.2) exclusively localizes to the EHM in this interaction (Wang et al., 2009). Ultrastructure analyses of the Golovinomyces orontii powdery mildew pathosystem revealed that the EHM is asymmetric, thicker and more electron opaque than the PM, and can be highly convoluted around mature haustoria (Micali et al., 2011). More recently, a survey of Arabidopsis and Nicotiana benthamiana plants infected by the oomycete pathogens Hyaloperonospora arabidopsidis and Phytophthora infestans, respectively, revealed that several integral host PM proteins are excluded from the EHM (Lu et al., 2012). Nevertheless, the remorin REM1.3 and the SYNAPTOTAGMIN1 (SYT1) peripheral membrane proteins localized to undetermined subcellular compartments around haustoria in P. infestans-plant interactions (Lu et al., 2012). Whether the differential accumulation of membrane proteins at the EHM is due to interference with the lateral diffusion of proteins from the PM or targeted secretion of specialized vesicles remains unclear (Lu et al., 2012).The subcellular distribution of effectors inside plant cells provides valuable clues about the host cell compartments they modify to promote disease, and effectors have emerged as useful molecular probes for plant cell biology (Whisson et al., 2007; Bozkurt et al., 2012). Heterologous expression of fluorescently tagged effectors in plant cells has been used to determine their subcellular localization in uninfected and infected tissue. This approach has been successful with the RXLR and CRINKLER (CRN) effectors, the two major classes of cytoplasmic (host-translocated) oomycete effectors (Bozkurt et al., 2012). The 49 H. arabidopsidis RXLR effectors studied by Caillaud et al. (2012) localized to the nucleus, the cytoplasm, or various plant membrane compartments. In contrast, CRN effectors from several oomycete species exclusively accumulate in the plant cell nucleus (Schornack et al., 2010; Stam et al., 2013). The P. infestans effectors AVRblb2 and AVR2 accumulate around haustoria when expressed in infected N. benthamiana cells, highlighting the PM and the EHM as important sites for effector activity (Bozkurt et al., 2011; Saunders et al., 2012). These effectors, therefore, can serve as useful probes for plant cell biology to dissect vesicular trafficking and focal immunity, processes that have proved difficult to study using standard genetic approaches (Bozkurt et al., 2011; Win et al., 2012).REM1.3 is one of two plant membrane-associated proteins detected around haustoria during the interaction between P. infestans and the model plant N. benthamiana (Lu et al., 2012). Therefore, we hypothesized that studying REM1.3 should prove useful for understanding the mechanisms governing the function and formation of perihaustorial membranes. REM1.3 belongs to a diverse family of plant-specific proteins containing a Remorin_C domain (PF03763) and has known orthologs in potato (Solanum tuberosum; StREM1.3), tomato (Solanum lycopersicum; SlREM1.2), tobacco (Nicotiana tabacum; NtREM1.2), and Arabidopsis (AtREM1.1–AtREM1.4; Raffaele et al., 2007). Several proteins from the remorin family, including REM1.3, are preferentially associated with membrane rafts, nanometric sterol- and sphingolipid-rich domains in PMs (Pike, 2006; Simons and Gerl, 2010). Indeed, StREM1.3 and NtREM1.2 are highly enriched in detergent-insoluble membranes (DIMs) and form sterol-dependent domains of approximately 75 nm in purified PMs (Mongrand et al., 2004; Shahollari et al., 2004; Raffaele et al., 2009). StREM1.3 directly binds to the cytoplasmic leaflet of the PM through a C-terminal anchor domain (RemCA) that folds into a hairpin of aliphatic α-helices in polar environments (Raffaele et al., 2009; Perraki et al., 2012). StREM1.3 is differentially phosphorylated upon the perception of polygalacturonic acid (Reymond et al., 1996). AtREM1.3 is differentially recruited to DIMs and differentially phosphorylated upon flg22 (for flagellin) peptide perception (Benschop et al., 2007; Keinath et al., 2010; Marín et al., 2012), suggesting a role in plant defense signaling. StREM1.3 and SlREM1.2 prevent Potato virus X spreading by interacting with the Triple Gene Block protein1 (TGBp1) viral movement protein, presumably in plasmodesmata or at the PM (Raffaele et al., 2009; Perraki et al., 2012). AtREM1.2 belongs to protein complexes formed by a negative regulator of immune responses, Resistance to Pseudomonas syringae pv maculicola1 (RPM1)-INTERACTING PROTEIN4, at the PM (Liu et al., 2009). Furthermore, Medicago truncatula MtSYMREM1 is enriched in root cell DIMs (Lefebvre et al., 2007) and localizes to patches at the peribacteroid membrane during symbiosis with Sinorhizobium meliloti (Lefebvre et al., 2010). MtSYMREM1 is important for nodule formation and interacts with the Lysin motif domain–containing receptor-like kinase3 (LYK3) symbiotic receptor (Lefebvre et al., 2010). Multiple lines of evidence, therefore, implicate several remorins in cell surface signaling and the accommodation of microbes during plant-microbe interactions (Raffaele et al., 2007; Jarsch and Ott, 2011; Urbanus and Ott, 2012). Nevertheless, little is known about REM1.3’s molecular function, and its role in immunity against filamentous plant pathogens has not been reported to date.In this study, we analyzed in detail the localization and function of REM1.3 during host colonization by P. infestans. We found that REM1.3 localizes exclusively to the vicinity of the PM and the EHM around noncallosic haustoria. Furthermore, our results suggest that the EHM is likely formed by multiple microdomains. REM1.3 silencing and overexpression experiments demonstrated that it promotes susceptibility to P. infestans in N. benthamiana and tomato. We also show that the REM1.3 membrane anchor domain is required for its localization at the EHM and for the promotion of susceptibility to P. infestans. This work demonstrates the importance of the dynamic reorganization of the PM in response to haustoria-forming pathogens. Our study also revealed that the effector AVRblb2 localizes to remorin-containing host membrane domains at the host-pathogen interface, possibly as a pathogen strategy to facilitate the accommodation of infection structures inside plant cells.     

Autoantibodies against the replication protein A complex in systemic lupus erythematosus and other autoimmune diseases

Replication protein A (RPA), a heterotrimer with subunits of molecular masses 70, 32, and 14 kDa, is a single-stranded-DNA-binding factor involved in DNA replication, repair, and recombination. There have been only three reported cases of anti-RPA in systemic lupus erythematosus (SLE) and Sjögren syndrome (SjS). This study sought to clarify the clinical significance of autoantibodies against RPA. Sera from 1,119 patients enrolled during the period 2000 to 2005 were screened by immunoprecipitation (IP) of ³⁵S-labeled K562 cell extract. Antigen-capture ELISA with anti-RPA32 mAb, immunofluorescent antinuclear antibodies (ANA) and western blot analysis with purified RPA were also performed. Our results show that nine sera immunoprecipitated the RPA70–RPA32–RPA14 complex and all were strongly positive by ELISA (titers 1:62,500 to 1:312,500). No additional sera were positive by ELISA and subsequently confirmed by IP or western blotting. All sera showed fine speckled/homogeneous nuclear staining. Anti-RPA was found in 1.4% (4/276) of SLE and 2.5% (1/40) of SjS sera, but not in rheumatoid arthritis (0/35), systemic sclerosis (0/47), or polymyositis/dermatomyositis (0/43). Eight of nine patients were female and there was no racial predilection. Other positive patients had interstitial lung disease, autoimmune thyroiditis/hepatitis C virus/pernicious anemia, or an unknown diagnosis. Autoantibody specificities found in up to 40% of SLE and other diseases, such as anti-nRNP, anti-Sm, anti-Ro, and anti-La, were unusual in anti-RPA-positive sera. Only one of nine had anti-Ro, and zero of nine had anti-nRNP, anti-Sm, anti-La, or anti-ribosomal P antibodies. In summary, high titers of anti-RPA antibodies were found in nine patients (1.4% of SLE and other diseases). Other autoantibodies found in SLE were rare in this subset, suggesting that patients with anti-RPA may form a unique clinical and immunological subset.