Targeting burn prevention in the paediatric population : a prospective study of children's burns in the Lausanne area.

QUESTION UNDER STUDY: Domestic accidents are an important problem in paediatric medicine. This study was designed to gain a better understanding of burn mechanisms and target prevention. METHODS: Children treated for burn lesions in the Department of Paediatric Surgery between August 2004 and August 2005 were included in this prospective study. The burn mechanisms, the children's ages and the circumstances in which children were burned as well as their home environment variables were analyzed. RESULTS: The current study included eighty-nine patients, aged between 2 months and 15 years. Seventy-eight percent were less than 5 years old. More than half were boys. Hot liquid scalding was the most frequent mechanism. There does not seem to be an increased risk in the immigrant population or in low economic status families. In most cases, an adult person was present at time of injury. CONCLUSIONS: If we were to describe the highest "at risk" candidate for a burn in our region, it would be a boy aged 15 months to 5 years who is burned by a cup of hot liquid on his hand, at home, around mealtime, in the presence of one or both parents. Reduced attention in the safe domestic setting is probably responsible.

Vaccination with a Melan-A peptide selects an oligoclonal T cell population with increased functional avidity and tumor reactivity.

Both the underlying molecular mechanisms and the kinetics of TCR repertoire selection following vaccination against tumor Ags in humans have remained largely unexplored. To gain insight into these questions, we performed a functional and structural longitudinal analysis of the TCR of circulating CD8(+) T cells specific for the HLA-A2-restricted immunodominant epitope from the melanocyte differentiation Ag Melan-A in a melanoma patient who developed a vigorous and sustained Ag-specific T cell response following vaccination with the corresponding synthetic peptide. We observed an increase in functional avidity of Ag recognition and in tumor reactivity in the postimmune Melan-A-specific populations as compared with the preimmune blood sample. Improved Ag recognition correlated with an increase in the t(1/2) of peptide/MHC interaction with the TCR as assessed by kinetic analysis of A2/Melan-A peptide multimer staining decay. Ex vivo analysis of the clonal composition of Melan-A-specific CD8(+) T cells at different time points during vaccination revealed that the response was the result of asynchronous expansion of several distinct T cell clones. Some of these T cell clones were also identified at a metastatic tumor site. Collectively, these data show that tumor peptide-driven immune stimulation leads to the selection of high-avidity T cell clones of increased tumor reactivity that independently evolve within oligoclonal populations.

PchC thioesterase optimizes nonribosomal biosynthesis of the peptide siderophore pyochelin in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the antibiotic dihydroaeruginoate (Dha) and the siderophore pyochelin are produced from salicylate and cysteine by a thiotemplate mechanism involving the peptide synthetases PchE and PchF. A thioesterase encoded by the pchC gene was found to be necessary for maximal production of both Dha and pyochelin, but it was not required for Dha release from PchE and could not replace the thioesterase function specified by the C-terminal domain of PchF. In vitro, 2-aminobutyrate, a cysteine analog, was adenylated by purified PchE and PchF proteins. In vivo, this analog strongly interfered with Dha and pyochelin formation in a pchC deletion mutant but affected production of these metabolites only slightly in the wild type. Exogenously supplied cysteine overcame the negative effect of a pchC mutation to a large extent, whereas addition of salicylate did not. These data are in agreement with a role for PchC as an editing enzyme that removes wrongly charged molecules from the peptidyl carrier protein domains of PchE and PchF.

Immunization with synthetic peptides containing a defined malaria epitope induces a highly diverse cytotoxic T lymphocyte response. Evidence that two peptide residues are buried in the MHC molecule.

In the present study, we have explored ways of inducing a CTL response to a previously defined H-2Kd MHC class I restricted epitope in the circumsporozoite (CS) protein of Plasmodium berghei, and studied in detail the fine specificity of the response. We found that the s.c. injection of a variety of synthetic peptides emulsified in Freund's adjuvant efficiently induced a specific CTL response in (BALB/c x C57BL/6)F1 (H-2d x H-2b) mice. In contrast, BALB/c mice responded only marginally, consistent with the possible requirement for a concomitant Th response that would be provided by the C57BL/6 strain. Similar to our previous observations in analyzing CTL clones from sporozoite-immunized mice, the CTL response induced by peptide immunization was in part cross-reactive with an epitope from the Plasmodium yoelii species. The minimal P. berghei CS epitope, the octapeptide PbCS 253-260, was studied in detail by the analysis of a series of variant CS peptides containing single Ala substitutions. The relative antigenic activity for each variant peptide was calculated for 28 different CTL clones. Overall, the response to this P. berghei CTL epitope appeared to be extremely diverse in terms of fine specificity. This was evident among the CTL derived from sporozoite-immunized mice, as well as among those from peptide-immunized animals. The heterogeneity found at the functional level correlates with the highly diverse TCR repertoire that we have found for the same series of CTL clones in a study that is reported separately. The relative competitor activity for each Ala-substituted peptide was also determined in a quantitative functional competition assay. For the residues (Tyr253 and Ile260) within the 8-mer CS peptide, substitution with Ala reduced competitor activity by at least 40-fold, and for two others the reduction was 5- to 10-fold. When the relative antigenic activity for each CTL/peptide combination was normalized to the relative competitor activity of the peptide, a striking pattern emerged. The two residues that most affected competitor activity showed no additional effect on recognition beyond that observed for competition. In marked contrast, Ala substitutions at the other five positions tested varied widely, depending on the CTL/peptide combination. This pattern not only supports a model whereby the Tyr253 and Ile260 residues anchor the peptide to the Kd molecule, but also implies that they are virtually inaccessible to the TCR.

Anchoring of an immunogenic Plasmodium falciparum circumsporozoite protein on the surface of Dictyostelium discoideum.

The circumsporozoite protein (CSP), a major antigen of Plasmodium falciparum, was expressed in the slime mold Dictyostelium discoideum. Fusion of the parasite protein to a leader peptide derived from Dictyostelium contact site A was essential for expression. The natural parasite surface antigen, however, was not detected at the slime mold cell surface as expected but retained intracellularly. Removal of the last 23 amino acids resulted in secretion of CSP, suggesting that the C-terminal segment of the CSP, rather than an ectoplasmic domain, was responsible for retention. Cell surface expression was obtained when the CSP C-terminal segment was replaced by the D. discoideum contact site A glycosyl phosphatidylinositol anchor signal sequence. Mice were immunized with Dictyostelium cells harboring CSP at their surface. The raised antibodies recognized two different regions of the CSP. Anti-sporozoite titers of these sera were equivalent to anti-peptide titers detected by enzyme-linked immunosorbent assay. Thus, cell surface targeting of antigens can be obtained in Dictyostelium, generating sporozoite-like cells having potentials for vaccination, diagnostic tests, or basic studies involving parasite cell surface proteins.

Improving binding affinity and stability of peptide ligands by substituting glycines with D-amino acids.

Improving the binding affinity and/or stability of peptide ligands often requires testing of large numbers of variants to identify beneficial mutations. Herein we propose a type of mutation that promises a high success rate. In a bicyclic peptide inhibitor of the cancer-related protease urokinase-type plasminogen activator (uPA), we observed a glycine residue that has a positive ϕ dihedral angle when bound to the target. We hypothesized that replacing it with a D-amino acid, which favors positive ϕ angles, could enhance the binding affinity and/or proteolytic resistance. Mutation of this specific glycine to D-serine in the bicyclic peptide indeed improved inhibitory activity (1.75-fold) and stability (fourfold). X-ray-structure analysis of the inhibitors in complex with uPA showed that the peptide backbone conformation was conserved. Analysis of known cyclic peptide ligands showed that glycine is one of the most frequent amino acids, and that glycines with positive ϕ angles are found in many protein-bound peptides. These results suggest that the glycine-to-D-amino acid mutagenesis strategy could be broadly applied.

Autophagy controls IL-1beta secretion by targeting pro-IL-1beta for degradation.

Autophagy is a key regulator of cellular homeostasis that can be activated by pathogen-associated molecules and recently has been shown to influence IL-1β secretion by macrophages. However, the mechanisms behind this are unclear. Here, we describe a novel role for autophagy in regulating the production of IL-1β in antigen-presenting cells. After treatment of macrophages with Toll-like receptor ligands, pro-IL-1β was specifically sequestered into autophagosomes, whereas further activation of autophagy with rapamycin induced the degradation of pro-IL-1β and blocked secretion of the mature cytokine. Inhibition of autophagy promoted the processing and secretion of IL-1β by antigen-presenting cells in an NLRP3- and TRIF-dependent manner. This effect was reduced by inhibition of reactive oxygen species but was independent of NOX2. Induction of autophagy in mice in vivo with rapamycin reduced serum levels of IL-1β in response to challenge with LPS. These data demonstrate that autophagy controls the production of IL-1β through at least two separate mechanisms: by targeting pro-IL-1β for lysosomal degradation and by regulating activation of the NLRP3 inflammasome.

Targeting Angiogenesis in Glioma - Challenges and Pitfalls

Malignant gliomas, notably glioblastoma are among the most vascularized and angiogenic cancers, and microvascular proliferation is one of the hallmarks for the diagnosis of glioblastoma. Angiogenesis is regulated by a balance of pro- and antiangiogenic signals; overexpression of VEGF and activation of its receptors, most notable VEGFR-2 and -3, results in endothelial cell proliferation and leaky vasculature. Heterogeneous perfusion and oxygenation, peritumoral edema and increased interstitial pressure are the consequence. Both endothelial and tumour cells are strongly dependent on integrin-mediated adhesion for cell proliferation, survival, migration and invasion.Strategies aiming at inhibition of cell signaling and angiogenesis, including integrin inhibitors, have been clinically investigated in gliomas over the last 5 years. Radiological responses, a decreased requirement of corticosteroids and temporary improvement in performance status have repeatedly been observed. Toxicity was mild-moderate and manageable, notably there was no evidence for a substantially increased incidence of intracranial bleeding. However definitive comparative (randomized !) investigation has failed to demonstrate improved outcome with singleagent inhibition of EGFR, or PDGFR or VEGF/VEGFRs pathways in recurrent glioblastoma. Definitive phase III trials combining the anti- VEGF monoclonal antibody bevacizumab, or cilengitide, a peptidic integrininhibitor, together with temozolomide and radiotherapy are ongoing (accrual completed).The integration of anti-angiogenic strategies in the management of malignant glioma also poses entirely new challenges in patient management: 1) Many agents are known for increasing the risk of thrombosis, embolism and intracranial bleeding. 2) Evaluation of treatment efficacy is difficult and new biomarkers of activity, including functional, metabolic or molecular imaging techniques are urgently needed. Normalization of vasculature leads to decrease in contrast enhancement without necessarily reflecting tumour shrinkage. Tumour heterogeneity, putative prognostic or predictive factors require early controlled trials, novel trial designs and endpoints.3) Activation of alternate pathways and tumour escape mechanisms may require combination of multiple agents, which is often not feasible due to regulatory restrictions and potential complex toxicities. Emerging clinical and experimental evidence suggests that anti-angiogenic drugs might need to be combined with drugs targeting tumour adaptive mechanisms in addition to cytotoxic chemotherapy and irradiation for a maximal antitumour effect.

Characterization of tumor reactive CD8 T cells induced by peptide vaccination in melanoma patients

ABSTRACT¦Naturally acquired tumor-specific T-cells can be detected in most advanced cancer patients.¦Yet, they often fail to control or eliminate the disease, in contrast to many virus-specific CD8¦T lymphocytes. Therapeutic vaccines aim at inducing and boosting specific T-cells mediated¦immunity to reduce tumor burden. The properties of CD8 T-cells required for protection from¦infectious disease and cancer are only partially characterized.¦The objectives of this study were to assess effector functions, stage of differentiation and¦clonotype selection of tumor-reactive T lymphocytes following peptide vaccination in¦melanoma patients over time. Results were compared to protective viral-specific T-cell¦responses found in healthy individuals. We also characterized dominant versus low/non¦dominant T-cell clonotypes with the aim to further understand the in vivo function of each set¦of frequency-based specific T-cells.¦Here we developed and applied a novel approach for molecular and functional analysis of¦single T lymphocytes ex vivo. T-cell receptor (TCR) clonotype mapping revealed rapid¦selection and expansion of co-dominant T-cell clonotypes, which made up the majority of the¦highly differentiated "effector" T-cells, but only 25% of the less differentiated "effectormemory"¦cells, mostly composed of non-dominant clonotypes. Moreover, we show that¦advanced effector cell differentiation was indeed clonotype-dependent. Surprisingly, however,¦the acquisition of effector functions (cytokine production, killing) was clonotype-independent.¦Vaccination of melanoma patients with native peptide induced competent effector function in¦both dominant and non-dominant clonotypes, suggesting that most if not all clonotypes¦participating in a T-cell response have the potential to develop equal functional competence.¦In contrast, many T-cells remained poorly functional after vaccination with analog peptide,¦despite similar clonotype-dependent differentiation. Our findings show that the type of¦peptide vaccine has a critical influence on the selection and functional activation of the¦clonotypic T-cell repertoire. They also show that systematic assessment of individual T-cells¦identifies the cellular basis of immune responses, contributing to the rational development of¦vaccines.

Active antiviral T-lymphocyte response can be redirected against tumor cells by antitumor antibody x MHC/viral peptide conjugates.

PURPOSE: To redirect an ongoing antiviral T-cell response against tumor cells in vivo, we evaluated conjugates consisting of antitumor antibody fragments coupled to class I MHC molecules loaded with immunodominant viral peptides. EXPERIMENTAL DESIGN: First, lymphochoriomeningitis virus (LCMV)-infected C57BL/6 mice were s.c. grafted on the right flank with carcinoembryonic antigen (CEA)-transfected MC38 colon carcinoma cells precoated with anti-CEA x H-2D(b)/GP33 LCMV peptide conjugate and on the left flank with the same cells precoated with control anti-CEA F(ab')(2) fragments. Second, influenza virus-infected mice were injected i.v., to induce lung metastases, with HER2-transfected B16F10 cells, coated with either anti-HER2 x H-2D(b)/NP366 influenza peptide conjugates, or anti-HER2 F(ab')(2) fragments alone, or intact anti-HER2 monoclonal antibody. Third, systemic injections of anti-CEA x H-2D(b) conjugates with covalently cross-linked GP33 peptides were tested for the growth inhibition of MC38-CEA(+) cells, s.c. grafted in LCMV-infected mice. RESULTS: In the LCMV-infected mice, five of the six grafts with conjugate-precoated MC38-CEA(+) cells did not develop into tumors, whereas all grafts with F(ab')(2)-precoated MC38-CEA(+) cells did so (P = 0.0022). In influenza virus-infected mice, the group injected with cells precoated with specific conjugate had seven times less lung metastases than control groups (P = 0.0022 and P = 0.013). Most importantly, systemic injection in LCMV-infected mice of anti-CEA x H-2D(b)/cross-linked GP33 conjugates completely abolished tumor growth in four of five mice, whereas the same tumor grew in all five control mice (P = 0.016). CONCLUSION: The results show that a physiologic T-cell antiviral response in immunocompetent mice can be redirected against tumor cells by the use of antitumor antibody x MHC/viral peptide conjugates.

Targeting the intragraft microenvironment and the development of chronic allograft rejection.

In this review, we discuss a paradigm whereby changes in the intragraft microenvironment promote or sustain the development of chronic allograft rejection. A key feature of this model involves the microvasculature including (a) endothelial cell (EC) destruction, and (b) EC proliferation, both of which result from alloimmune leukocyte- and/or alloantibody-induced responses. These changes in the microvasculature likely create abnormal blood flow patterns and thus promote local tissue hypoxia. Another feature of the chronic rejection microenvironment involves the overexpression of vascular endothelial growth factor (VEGF). VEGF stimulates EC activation and proliferation and it has potential to sustain inflammation via direct interactions with leukocytes. In this manner, VEGF may promote ongoing tissue injury. Finally, we review how these events can be targeted therapeutically using mTOR inhibitors. EC activation and proliferation as well as VEGF-VEGFR interactions require PI-3K/Akt/mTOR intracellular signaling. Thus, agents that inhibit this signaling pathway within the graft may also target the progression of chronic rejection and thus promote long-term graft survival.

Chronic Trypanosoma cruzi-elicited cardiomyopathy: from the discovery to the proposal of rational therapeutic interventions targeting cell adhesion molecules and chemokine receptors - how to make a dream come true

One hundred years ago, Carlos Chagas discovered a new disease, the American trypanosomiasis. Chagas and co-workers later characterised the disease's common manifestation, chronic cardiomyopathy, and suggested that parasitic persistence coupled with inflammation was the key underlying pathogenic mechanism. Better comprehension of the molecular mechanisms leading to clinical heart afflictions is a prerequisite to developing new therapies that ameliorate inflammation and improve heart function without hampering parasite control. Here, we review recent data showing that distinct cell adhesion molecules, chemokines and chemokine receptors participate in anti-parasite immunity and/or detrimental leukocyte trafficking to the heart. Moreover, we offer evidence that CC-chemokine receptors may be attractive therapeutic targets aiming to regain homeostatic balance in parasite/host interaction thereby improving prognosis, supporting that it is becoming a non-phantasious proposal.

Structural analysis of TCR-ligand interactions studied on H-2Kd-restricted cloned CTL specific for a photoreactive peptide derivative.

To study the interaction of the TCR with its ligand, the complex of a MHC molecule and an antigenic peptide, we modified a TCR contact residue of a H-2Kd-restricted antigenic peptide with photoreactive 4-azidobenzoic acid. The photoreactive group was a critical component of the epitope recognized by CTL clones derived from mice immunized with such a peptide derivative. The majority of these clones expressed V beta 1-encoded beta chains that were paired with J alpha TA28-encoded alpha chains. For one of these TCR, the photoaffinity labeled sites were mapped on the alpha chain as a J alpha TA28-encoded tryptophan and on the beta chain as a residue of the C' strand of V beta 1. Molecular modeling of this TCR suggested the presence of a hydrophobic pocket that harbors this tryptophan as well as a tyrosine on the C' strand of V beta 1 between which the photoreactive side chain inserts. It is concluded that this avid binding principle may account for the preferential selection of V beta 1 and J alpha TA28-encoded TCR.