Targeting the proteolytic processing of the viral glycoprotein precursor is a promising novel antiviral strategy against arenaviruses.

A crucial step in the arenavirus life cycle is the biosynthesis of the viral envelope glycoprotein (GP) responsible for virus attachment and entry. Processing of the GP precursor (GPC) by the cellular proprotein convertase site 1 protease (S1P), also known as subtilisin-kexin-isozyme 1 (SKI-1), is crucial for cell-to-cell propagation of infection and production of infectious virus. Here, we sought to evaluate arenavirus GPC processing by S1P as a target for antiviral therapy using a recently developed peptide-based S1P inhibitor, decanoyl (dec)-RRLL-chloromethylketone (CMK), and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). To control for off-target effects of dec-RRLL-CMK, we employed arenavirus reverse genetics to introduce a furin recognition site into the GPC of LCMV. The rescued mutant virus grew to normal titers, and the processing of its GPC critically depended on cellular furin, but not S1P. Treatment with the S1P inhibitor dec-RRLL-CMK resulted in specific blocking of viral spread and virus production of LCMV. Combination of the protease inhibitor with ribavirin, currently used clinically for treatment of human arenavirus infections, resulted in additive drug effects. In cells deficient in S1P, the furin-dependent LCMV variant established persistent infection, whereas wild-type LCMV underwent extinction without the emergence of S1P-independent escape variants. Together, the potent antiviral activity of an inhibitor of S1P-dependent GPC cleavage, the additive antiviral effect with ribavirin, and the low probability of emergence of S1P-independent viral escape variants make S1P-mediated GPC processing by peptide-derived inhibitors a promising strategy for the development of novel antiarenaviral drugs.

Melanoma patients respond to a cytotoxic T lymphocyte-defined self-peptide with diverse and nonoverlapping T-cell receptor repertoires.

HLA-A2+ melanoma patients develop naturally a strong CD8+ T cell response to a self-peptide derived from Melan-A. Here, we have used HLA-A2/peptide tetramers to isolate Melan-A-specific T cells from tumor-infiltrated lymph nodes of two HLA-A2+ melanoma patients and analyzed their TCR beta chain V segment and complementarity determining region 3 length and sequence. We found a broad diversity in Melan-A-specific immune T-cell receptor (TCR) repertoires in terms of both TCR beta chain variable gene segment usage and clonal composition. In addition, immune TCR repertoires selected in the patients were not overlapping. In contrast to previously characterized CD8+ T-cell responses to viral infections, this study provides evidence against usage of highly restricted TCR repertoire in the natural response to a self-differentiation tumor antigen.

Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium.

PURPOSE: The Cancer Vaccine Consortium of the Cancer Research Institute (CVC-CRI) conducted a multicenter HLA-peptide multimer proficiency panel (MPP) with a group of 27 laboratories to assess the performance of the assay. EXPERIMENTAL DESIGN: Participants used commercially available HLA-peptide multimers and a well characterized common source of peripheral blood mononuclear cells (PBMC). The frequency of CD8+ T cells specific for two HLA-A2-restricted model antigens was measured by flow cytometry. The panel design allowed for participants to use their preferred staining reagents and locally established protocols for both cell labeling, data acquisition and analysis. RESULTS: We observed significant differences in both the performance characteristics of the assay and the reported frequencies of specific T cells across laboratories. These results emphasize the need to identify the critical variables important for the observed variability to allow for harmonization of the technique across institutions. CONCLUSIONS: Three key recommendations emerged that would likely reduce assay variability and thus move toward harmonizing of this assay. (1) Use of more than two colors for the staining (2) collect at least 100,000 CD8 T cells, and (3) use of a background control sample to appropriately set the analytical gates. We also provide more insight into the limitations of the assay and identified additional protocol steps that potentially impact the quality of data generated and therefore should serve as primary targets for systematic analysis in future panels. Finally, we propose initial guidelines for harmonizing assay performance which include the introduction of standard operating protocols to allow for adequate training of technical staff and auditing of test analysis procedures.

Automatic top-down processing explains common left occipito-temporal responses to visual words and objects.

Previous studies have demonstrated that a region in the left ventral occipito-temporal (LvOT) cortex is highly selective to the visual forms of written words and objects relative to closely matched visual stimuli. Here, we investigated why LvOT activation is not higher for reading than picture naming even though written words and pictures of objects have grossly different visual forms. To compare neuronal responses for words and pictures within the same LvOT area, we used functional magnetic resonance imaging adaptation and instructed participants to name target stimuli that followed briefly presented masked primes that were either presented in the same stimulus type as the target (word-word, picture-picture) or a different stimulus type (picture-word, word-picture). We found that activation throughout posterior and anterior parts of LvOT was reduced when the prime had the same name/response as the target irrespective of whether the prime-target relationship was within or between stimulus type. As posterior LvOT is a visual form processing area, and there was no visual form similarity between different stimulus types, we suggest that our results indicate automatic top-down influences from pictures to words and words to pictures. This novel perspective motivates further investigation of the functional properties of this intriguing region.

Auditory spatial deficits following hemispheric lesions : dissociation of explicit and implicit processing

Les déficits auditifs spatiaux se produisent fréquemment après une lésion hémisphérique ; un précédent case report suggérait que la capacité explicite à reconnaître des positions sonores, comme dans la localisation des sons, peut être atteinte alors que l'utilisation implicite d'indices sonores pour la reconnaissance d'objets sonores dans un environnement bruyant reste préservée. En testant systématiquement des patients avec lésion hémisphérique inaugurale, nous avons montré que (1) l'utilisation explicite et/ou implicite des indices sonores peut être perturbée ; (2) la dissociation entre l'atteinte de l'utilisation explicite des indices sonores versus une préservation de l'utilisation implicite de ces indices est assez fréquente ; et (3) différents types de déficits dans la localisation des sons peuvent être associés avec une utilisation implicite préservée de ces indices sonores. Conceptuellement, la dissociation entre l'utilisation explicite et implicite de ces indices sonores peut illustrer la dichotomie des deux voies du système auditif. Nos résultats parlent en faveur d'une évaluation systématique des fonctions auditives spatiales dans un contexte clinique, surtout quand l'adaptation à un environnement sonore est en jeu. De plus, des études systématiques sont nécessaires afin de mettre en lien les troubles de l'utilisation explicite versus implicite de ces indices sonores avec les difficultés à effectuer les activités de la vie quotidienne, afin d'élaborer des stratégies de réhabilitation appropriées et afin de s'assurer jusqu'à quel point l'utilisation explicite et implicite des indices spatiaux peut être rééduquée à la suite d'un dommage cérébral.

Combined clonotyping and gene signatures of individual tumor-reactive CD8 T-cells define their functional relevance following peptide vaccination in melanoma patients

Novel cancer vaccines are capableto efficiently induce and boost humantumor antigen specific T-cells. However,the properties of these CD8T-cells are only partially characterized.For in depth investigation ofT-cells following Melan-A/MART-1peptide vaccination in melanoma patients,we conducted a detailed prospectivestudy at the single cell level.We first sorted individual human naiveand effector CD8 T-cells from peripheralblood by flow cytometry, andtested a modified RT-PCR protocolincluding a global amplification ofexpressed mRNAs to obtain sufficientcDNAfromsingle cells.We successfullydetected the expression ofseveral specific genes of interest evendown to 106-fold dilution (equivalentto 10-5 cell). We then analyzed tumor-specific effector memory (EM)CD8T-cell subpopulations ex vivo, assingle cells from vaccinated melanomapatients. To elucidate the hallmarksof effective immunity the genesignatures were defined by a panel ofgenes related to effector functions(e.g. IFN-, granzyme B, perforin),and individual clonotypes were identifiedaccording to the expression ofdistinct T-cell receptors (TCR). Usingthis novel single cell analysis approach,we observed that T-cell differentiationis clonotype dependent,with a progressive restriction in TCRBV clonotype diversity from EMCD28pos to EMCD28neg subsets. However,the effector function gene imprintingis clonotype-independent,but dependent on differentiation,since it correlates with the subset oforigin (EMCD28pos or EMCD28neg). We also conducted a detailedcomparative analysis after vaccinationwith natural vs. analog Melan-Apeptide. We found that the peptideused for vaccination determines thefunctional outcome of individualT-cell clonotypes, with native peptideinducing more potent effector functions.Yet, selective clonotypic expansionwith differentiation was preservedregardless of the peptide usedfor vaccination. In summary, the exvivo single cell RT-PCR approach ishighly sensitive and efficient, andrepresents a reliable and powerfultool to refine our current view of molecularprocesses taking place duringT-cell differentiation.

A peptide inhibitor of C-jun N-terminal kinase modulates hepatic damage and the inflammatory response after hemorrhagic shock and resuscitation

Hemorrhage and resuscitation (H/R) leads to phosphorylation of mitogen-activated stress kinases, an event that is associated with organ damage. Recently, a specific, cell-penetrating, protease-resistant inhibitory peptide of the mitogen-activated protein kinase c-JUN N-terminal kinase (JNK) was developed (D-JNKI-1). Here, using this peptide, we tested if inhibition of JNK protects against organ damage after H/R. Male Sprague-Dawley rats were treated with D-JNKI-1 (11 mg/kg, i.p.) or vehicle. Thirty minutes later, rats were hemorrhaged for 1 h to a MAP of 30 to 35 mmHg and then resuscitated with 60% of the shed blood and twice the shed blood volume as Ringer lactate. Tissues were harvested 2 h later. ANOVA with Tukey post hoc analysis or Kruskal-Wallis ANOVA on ranks, P < 0.05, was considered significant. c-JUN N-terminal kinase inhibition decreased serum alanine aminotransferase activity as a marker of liver injury by 70%, serum creatine kinase activity by 67%, and serum lactate dehydrogenase activity by 60% as compared with vehicle treatment. The histological tissue damage observed was blunted after D-JNKI-1 pretreatment both for necrotic and apoptotic cell death. Hepatic leukocyte infiltration and serum IL-6 levels were largely diminished after D-JNKI-1 pretreatment. The extent of oxidative stress as evaluated by immunohistochemical detection of 4-hydroxynonenal was largely abrogated after JNK inhibition. After JNK inhibition, activation of cJUN after H/R was also reduced. Hemorrhage and resuscitation induces a systemic inflammatory response and leads to end-organ damage. These changes are mediated, at least in part, by JNK. Therefore, JNK inhibition deserves further evaluation as a potential treatment option in patients after resuscitated blood loss.

Differential peptide binding to CD40 evokes counteractive responses.

The antigen-presenting cell-expressed CD40 is implied in the regulation of counteractive immune responses such as induction of pro-inflammatory and anti-inflammatory cytokines interleukin (IL)-12 and IL-10, respectively. The mechanism of this duality in CD40 function remains unknown. Here, we investigated whether such duality depends on ligand binding. Based on CD40 binding, we identifed two dodecameric peptides, peptide-7 and peptide-19, from the phage peptide library. Peptide-7 induces IL-10 and increases Leishmania donovani infection in macrophages, whereas peptide-19 induces IL-12 and reduces L. donovani infection. CD40-peptide interaction analyses by surface plasmon resonance and atomic force microscopy suggest that the functional differences are not associated with the studied interaction parameters. The molecular dynamic simulation of the CD40-peptides interaction suggests that these two peptides bind to two different places on CD40. Thus, we suggest for the first time that differential binding of the ligands imparts functional duality to CD40.

Lack of evidence of the interaction of the Aß peptide with the Wnt signaling cascade in Drosophila models of Alzheimer's disease.

Background Alzheimer's disease (AD) is the leading form of dementia worldwide. The Aß-peptide is believed to be the major pathogenic compound of the disease. Since several years it is hypothesized that Aß impacts the Wnt signaling cascade and therefore activation of this signaling pathway is proposed to rescue the neurotoxic effect of Aß. Findings Expression of the human Aß42 in the Drosophila nervous system leads to a drastically shortened life span. We found that the action of Aß42 specifically in the glutamatergic motoneurons is responsible for the reduced survival. However, we find that the morphology of the glutamatergic larval neuromuscular junctions, which are widely used as the model for mammalian central nervous system synapses, is not affected by Aß42 expression. We furthermore demonstrate that genetic activation of the Wnt signal transduction pathway in the nervous system is not able to rescue the shortened life span or a rough eye phenotype in Drosophila. Conclusions Our data confirm that the life span is a useful readout of Aß42 induced neurotoxicity in Drosophila; the neuromuscular junction seems however not to be an appropriate model to study AD in flies. Additionally, our results challenge the hypothesis that Wnt signaling might be implicated in Aß42 toxicity and might serve as a drug target against AD.

MART-1 peptide vaccination plus IMP321 (LAG-3Ig fusion protein) in patients receiving autologous PBMCs after lymphodepletion: results of a Phase I trial.

BACKGROUND: Immunotherapy offers a promising novel approach for the treatment of cancer and both adoptive T-cell transfer and immune modulation lead to regression of advanced melanoma. However, the potential synergy between these two strategies remains unclear. METHODS: We investigated in 12 patients with advanced stage IV melanoma the effect of multiple MART-1 analog peptide vaccinations with (n = 6) or without (n = 6) IMP321 (LAG-3Ig fusion protein) as an adjuvant in combination with lymphodepleting chemotherapy and adoptive transfer of autologous PBMCs at day (D) 0 (Trial registration No: NCT00324623). All patients were selected on the basis of ex vivo detectable MART-1-specific CD8 T-cell responses and immunized at D0, 8, 15, 22, 28, 52, and 74 post-reinfusion. RESULTS: After immunization, a significant expansion of MART-1-specific CD8 T cells was measured in 83% (n = 5/6) and 17% (n = 1/6) of patients from the IMP321 and control groups, respectively (P < 0.02). Compared to the control group, the mean fold increase of MART-1-specific CD8 T cells in the IMP321 group was respectively >2-, >4- and >6-fold higher at D15, D30 and D60 (P < 0.02). Long-lasting MART-1-specific CD8 T-cell responses were significantly associated with IMP321 (P < 0.02). At the peak of the response, MART-1-specific CD8 T cells contained higher proportions of effector (CCR7⁻ CD45RA⁺/⁻) cells in the IMP321 group (P < 0.02) and showed no sign of exhaustion (i.e. were mostly PD1⁻CD160⁻TIM3⁻LAG3⁻2B4⁺/⁻). Moreover, IMP321 was associated with a significantly reduced expansion of regulatory T cells (P < 0.04); consistently, we observed a negative correlation between the relative expansion of MART-1-specific CD8 T cells and of regulatory T cells. Finally, although there were no confirmed responses as per RECIST criteria, a transient, 30-day partial response was observed in a patient from the IMP321 group. CONCLUSIONS: Vaccination with IMP321 as an adjuvant in combination with lymphodepleting chemotherapy and adoptive transfer of autologous PBMCs induced more robust and durable cellular antitumor immune responses, supporting further development of IMP321 as an adjuvant for future immunotherapeutic strategies.

Peptide substrate specificity of the membrane-bound metalloprotease of Leishmania.

The promastigote surface protease (PSP) of Leishmania is a neutral membrane-bound zinc enzyme. The protease has no exopeptidase activity and does not cleave a large selection of substrates with chromogenic and fluorogenic leaving groups at the P1' site. The substrate specificity of the enzyme was studied by using natural and synthetic peptides of known amino acid sequence. The identification of 11 cleavage sites indicates that the enzyme preferentially cleaves peptides at the amino side when hydrophobic residues are in the P1' site and basic amino acid residues in the P2' and P3' sites. In addition, tyrosine residues are commonly found at the P1 site. Hydrolysis is not, however, restricted to these residues. These results have allowed the synthesis of a model peptide, H2N-L-I-A-Y-L-K-K-A-T-COOH, which is cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 1.8 X 10(6) M-1 s-1. Furthermore, a synthetic nonapeptide overlapping the last four amino acids of the prosequence and the first five residues of mature PSP was found to be cleaved by the protease at the expected site to release the mature enzyme. This result suggests a possible autocatalytic mechanism for the activation of the protease. Finally, the hydroxamate-derivatized dipeptide Cbz-Tyr-Leu-NHOH was shown to inhibit PSP competitively with a KI of 17 microM.

Effect of metoclopramide on angiotensins, aldosterone, and atrial peptide during hypoxia.

The coupling of aldosterone with renin is altered during acute hypoxemia. We measured the various components of the renin-angiotensin system and the plasma levels of immunoreactive atrial natriuretic factor (iANF) during room air and hypoxic gas-mixture breathing before and after administration of metoclopramide, a competitive antagonist of dopamine. Seven resting volunteers were studied 1 wk apart under room air and hypoxic conditions (inspired O2 fraction 0.12). During hypoxemia, the release of aldosterone induced by metoclopramide was significantly smaller. This change was associated with a slight increase in iANF and with a decrease in plasma angiotensin II levels, without any change in immunoreactive blood angiotensin I concentrations. Plasma electrolytes and blood acid-base status did not show relevant changes, nor did blood pressure and heart rate. We conclude that the decreased aldosterone concentrations seen under hypoxemia are related to decreased angiotensin II levels. Other influences, such as elevated ANF, may also mediate this effect.

Transcriptomic and functional analysis of an autolysis-deficient, teicoplanin-resistant derivative of methicillin-resistant Staphylococcus aureus.

The molecular basis of glycopeptide-intermediate S. aureus (GISA) isolates is not well defined though frequently involves phenotypes such as thickened cell walls and decreased autolysis. We have exploited an isogenic pair of teicoplanin-susceptible (strain MRGR3) and teicoplanin-resistant (strain 14-4) methicillin-resistant S. aureus strains for detailed transcriptomic profiling and analysis of altered autolytic properties. Strain 14-4 displayed markedly deficient Triton X-100-triggered autolysis compared to its teicoplanin-susceptible parent, although microarray analysis paradoxically did not reveal significant reductions in expression levels of major autolytic genes atl, lytM, and lytN, except for sle1, which showed a slight decrease. The most important paradox was a more-than-twofold increase in expression of the cidABC operon in 14-4 compared to MRGR3, which was correlated with decreased expression of autolysis negative regulators lytSR and lrgAB. In contrast, the autolysis-deficient phenotype of 14-4 was correlated with both increased expression of negative autolysis regulators (arlRS, mgrA, and sarA) and decreased expression of positive regulators (agr RNAII and RNAIII). Quantitative bacteriolytic assays and zymographic analysis of concentrated culture supernatants showed a striking reduction in Atl-derived, extracellular bacteriolytic hydrolase activities in 14-4 compared to MRGR3. This observed difference was independent of the source of cell wall substrate (MRGR3 or 14-4) used for analysis. Collectively, our results suggest that altered autolytic properties in 14-4 are apparently not driven by significant changes in the transcription of key autolytic effectors. Instead, our analysis points to alternate regulatory mechanisms that impact autolysis effectors which may include changes in posttranscriptional processing or export.

Syntaxin1a variants lacking an N-peptide or bearing the LE mutation bind to Munc18a in a closed conformation.

In neurons, soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins drive the fusion of synaptic vesicles to the plasma membrane through the formation of a four-helix SNARE complex. Members of the Sec1/Munc18 protein family regulate membrane fusion through interactions with the syntaxin family of SNARE proteins. The neuronal protein Munc18a interacts with a closed conformation of the SNARE protein syntaxin1a (Syx1a) and with an assembled SNARE complex containing Syx1a in an open conformation. The N-peptide of Syx1a (amino acids 1-24) has been implicated in the transition of Munc18a-bound Syx1a to Munc18a-bound SNARE complex, but the underlying mechanism is not understood. Here we report the X-ray crystal structures of Munc18a bound to Syx1a with and without its native N-peptide (Syx1aΔN), along with small-angle X-ray scattering (SAXS) data for Munc18a bound to Syx1a, Syx1aΔN, and Syx1a L165A/E166A (LE), a mutation thought to render Syx1a in a constitutively open conformation. We show that all three complexes adopt the same global structure, in which Munc18a binds a closed conformation of Syx1a. We also identify a possible structural connection between the Syx1a N-peptide and SNARE domain that might be important for the transition of closed-to-open Syx1a in SNARE complex assembly. Although the role of the N-peptide in Munc18a-mediated SNARE complex assembly remains unclear, our results demonstrate that the N-peptide and LE mutation have no effect on the global conformation of the Munc18a-Syx1a complex.

Viral envelope glycoprotein processing by proprotein convertases.

The proprotein convertases (PCs) are a family of nine mammalian enzymes that play key roles in the maintenance of cell homeostasis by activating or inactivating proteins via limited proteolysis under temporal and spatial control. A wide range of pathogens, including major human pathogenic viruses can hijack cellular PCs for their own purposes. In particular, productive infection with many enveloped viruses critically depends on the processing of their fusion-active viral envelope glycoproteins by cellular PCs. Based on their crucial role in virus-host interaction, PCs can be important determinants for viral pathogenesis and represent promising targets of therapeutic antiviral intervention. In the present review we will cover basic aspects and recent developments of PC-mediated maturation of viral envelope glycoproteins of selected medically important viruses. The molecular mechanisms underlying the recognition of PCs by viral glycoproteins will be described, including recent findings demonstrating differential PC-recognition of viral and cellular substrates. We will further discuss a possible scenario how viruses during co-evolution with their hosts adapted their glycoproteins to modulate the activity of cellular PCs for their own benefit and discuss the consequences for virus-host interaction and pathogenesis. Particular attention will be given to past and current efforts to evaluate cellular PCs as targets for antiviral therapeutic intervention, with emphasis on emerging highly pathogenic viruses for which no efficacious drugs or vaccines are currently available.