Native-like, long synthetic peptides as components of sub-unit vaccines: practical and theoretical considerations for their use in humans.

Vaccines have been used as a successful tool in medicine by way of controlling many major diseases. In spite of this, vaccines today represent only a handful of all infectious diseases. Therefore, there is a pressing demand for improvements of existing vaccines with particular reference to higher efficacy and undisputed safety profiles. To this effect, as an alternative to available vaccine technologies, there has been a drive to develop vaccine candidate polypeptides by chemical synthesis. In our laboratory, we have recently developed a technology to manufacture long synthetic peptides of up to 130 residues, which are correctly folded and biologically active. This paper discusses the advantages of the molecularly defined, long synthetic peptide approach in the context of vaccine design, development and use in human vaccination.

Reversible behavioural autistic-like regression: A manifestation of a special (new?) epileptic syndrome in a 28-month-old child. A 2-year longitudinal study .

A 28-month-old boy was referred for acute onset of abnormal head movements. History revealed an insidious progressive regression in behaviour and communication over several months. Head and shoulder 'spasms' with alteration of consciousness and on one occasion ictal laughter were seen. The electroencephalograph (EEG) showed repeated bursts of brief generalized polyspikes and spike-wave during the 'spasms', followed by flattening, a special pattern which never recurred after treatment. Review of family videos showed a single 'minor' identical seizure 6 months previously. Magnetic resonance imaging was normal. Clonazepam brought immediate cessation of seizures, normalization of the EEG and a parallel spectacular improvement in communication, mood and language. Follow-up over the next 10 months showed a new regression unaccompained by recognized seizures, although numerous seizures were discovered during the videotaped neuropsychological examination, when stereotyped subtle brief paroxysmal changes in posture and behaviour could be studied in slow motion and compared with the 'prototypical' initial ones. The EEG showed predominant rare left-sided fronto-temporal discharges. Clonazepam was changed to carbamazepin with marked improvement in behaviour, language and cognition which has been sustained up to the last control at 51 months. Videotaped home observations allowed the documentation of striking qualitative and quantitative variations in social interaction and play of autistic type in relation to the epileptic activity. We conclude that this child has a special characteristic epileptic syndrome with subtle motor and vegetative symptomatology associated with an insidious catastrophic 'autistic-like' regression which could be overlooked. The methods used to document such fluctuating epileptic behavioural manifestations are discussed.

A control flow prototype for a dose recommending device for chronic myeloid leukemia patients

In this paper we present a prototype of a control flow for an a posteriori drug dose adaptation for Chronic Myelogenous Leukemia (CML) patients. The control flow is modeled using Timed Automata extended with Tasks (TAT) model. The feedback loop of the control flow includes the decision-making process for drug dose adaptation. This is based on the outputs of the body response model represented by the Support Vector Machine (SVM) algorithm for drug concentration prediction. The decision is further checked for conformity with the dose level rules of a medical guideline. We also have developed an automatic code synthesizer for the icycom platform as an extension of the TIMES tool.

Allergic asthma to thaumatin : case report

Background: Thaumatin is a protein originally isolated from an African fruit (the "katemfe"), but various thaumatin-like proteins have been found in apples, grapes, kiwis and olives, etc. Thaumatin has natural sweetening properties and is about 2000 to 3000 times more potent than sucrose. It is therefore used in the food industry, particularly in the processing of low-calories sweeteners. Recently, thaumatin-like proteins have been described as a new family of allergens. Methods: We report the case of a 44-year-old woman occupationally exposed to thaumatin, who developed an allergy to it. The first symptom was an urticaria, which she presented two years after the factory she was working in (a sweeteners manufacturer), started to use thaumatin. Later on, she perceived an acute episode of a sensation of lump in the throat, persisting mild dyspnea, dysphonia and cough. Despite normal peak flow values and a normal chest examination, her general practitioner treated her with local corticoids and systemic antihistaminic drugs, which improved the symptoms. As occupational physicians, we were asked about the likelihood of an occupational disease. We visited her working place, and assessed the exposure to thaumatin. We concluded that the probability for a delayed asthma related to a thaumatin exposure was high, despite the lack of an exposure test. Results: The case was submitted to the insurance company as an occupational disease, and was accepted as such. It was therefore decided not to perform the exposure test, given the absence of true benefit for the patient, who was advised to switch job, and to avoid any exposure to thaumatin in the future. A few months later, the symptoms had completely disappeared. Conclusion: Some molecules only used in specific industry sectors, can cause health problems, such as allergy, but also toxic impairments, etc. The occupational physician, trained to find out which products and identify which molecules are involved, can contribute to the diagnosis, and help make possible a safe return to work for the patient.

Is intra-operative blood flow predictive for early failure of radiocephalic arteriovenous fistula?

BACKGROUND: For over 50 years, radiocephalic wrist arteriovenous fistulae (RCAVF) have been the primary and best vascular access for haemodialysis. Nevertheless, early failure due to thrombosis or non-maturation is a major complication resulting in their abandonment. This prospective study was designed to investigate the predictive value of intra-operative blood flow on early failure of primary RCAVF before the first effective dialysis. METHODS: We enrolled patients undergoing creation of primary RCAVF for haemodialysis based on the pre-operative ultrasound vascular mapping discussed in a multidisciplinary approach. Intra-operative blood flow measurement was systematically performed once the anastomosis had been completed using a transit-time ultrasonic flowmeter. During the follow-up, blood flow was estimated by colour flow ultrasound at various intervals. Any events related to the RCAVF were recorded. RESULTS: Autogenous RCAVFs (n = 58) in 58 patients were constructed and followed up for an average of 30 days. Thrombosis and non-maturation occurred in eight (14%) and four (7%) patients, respectively. The intra-operative blood flow in functioning RCAVFs was significantly higher compared to non-functioning RCAVFs (230 vs 98 mL/min; P = 0.007), as well as 1 week (753 vs 228 mL/min; P = 0.0008) and 4 weeks (915 vs 245 mL/min, P < 0.0001) later. Blood flow volume measurements with a cut-off value of 120 mL/min had a sensitivity of 67%, specificity of 75% and positive predictive value of 91%. CONCLUSIONS: Blood flow <120 mL has a good predictive value for early failure in RCAVF. During the procedure, this cut-off value may be used to select appropriately which RCAVF should be investigated in the operation theatre in order to correct in real time any abnormality.

The sirtuin inhibitor cambinol impairs MAPK signaling, inhibits inflammatory and innate immune responses and protects from septic shock.

Sirtuins (SIRT1-7) are NAD(+)-dependent histone deacetylases (HDACs) that play an important role in the control of metabolism and proliferation and the development of age-associated diseases like oncologic, cardiovascular and neurodegenerative diseases. Cambinol was originally described as a compound inhibiting the activity of SIRT1 and SIRT2, with efficient anti-tumor activity in vivo. Here, we studied the effects of cambinol on microbial sensing by mouse and human immune cells and on host innate immune responses in vivo. Cambinol inhibited the expression of cytokines (TNF, IL-1β, IL-6, IL-12p40, and IFN-γ), NO and CD40 by macrophages, dendritic cells, splenocytes and whole blood stimulated with a broad range of microbial and inflammasome stimuli. Sirtinol, an inhibitor of SIRT1 and SIRT2 structurally related to cambinol, also decreased macrophage response to TLR stimulation. On the contrary, selective inhibitors of SIRT1 (EX-527 and CHIC-35) and SIRT2 (AGK2 and AK-7) used alone or in combination had no inhibitory effect, suggesting that cambinol and sirtinol act by targeting more than just SIRT1 and SIRT2. Cambinol and sirtinol at anti-inflammatory concentrations also did not inhibit SIRT6 activity in in vitro assay. At the molecular level, cambinol impaired stimulus-induced phosphorylation of MAPKs and upstream MEKs. Going well along with its powerful anti-inflammatory activity, cambinol reduced TNF blood levels and bacteremia and improved survival in preclinical models of endotoxic shock and septic shock. Altogether, our data suggest that pharmacological inhibitors of sirtuins structurally related to cambinol may be of clinical interest to treat inflammatory diseases.

Debris flow susceptibility mapping at a regional scale

Debris flow susceptibility mapping at a regional scale has been the subject of various studies. The complexity of the phenomenon and the variability of local controlling factors limit the use of process-based models for a first assessment. GISbased approaches associating an automatic detection of the source areas and a simple assessment of the debris flow spreading may provide a substantial basis for a preliminary susceptibility assessment at the regional scale. The use of a digital elevation model, with a 10 m resolution, for the Canton de Vaud territory (Switzerland), a lithological map and a land use map, has allowed automatic identification of the potential source areas. The spreading estimates are based on basic probabilistic and energy calculations that allow to define the maximal runout distance of a debris flow.

Heterologous desensitization of the glucagon-like peptide-1 receptor by phorbol esters requires phosphorylation of the cytoplasmic tail at four different sites.

Glucagon-like peptide-1 stimulates glucose-induced insulin secretion by binding to a specific G protein-coupled receptor that activates the adenylyl cyclase pathway. We previously demonstrated that heterologous desensitization of the receptor by protein kinase C correlated with phosphorylation in a 33-amino acid-long segment of the receptor carboxyl-terminal cytoplasmic tail. Here, we determined that the in vivo sites of phosphorylation are four serine doublets present at positions 431/432, 441/442, 444/445, and 451/452. In vitro phosphorylation of fusion proteins containing mutant receptor C-tails, however, indicated that whereas serines at position 431/432 were good substrates for protein kinase C (PKC), serines 444/445 and 451/452 were poor substrates, and serines 441/442 were not substrates. In addition, serine 416 was phosphorylated on fusion protein but not in intact cells. This indicated that in vivo a different PKC isoform or a PKC-activated kinase may phosphorylate the receptor. The role of phosphorylation on receptor desensitization was assessed using receptor mutants expressed in COS cells or Chinese hamster lung fibroblasts. Mutation of any single serine doublet to alanines reduced the extent of phorbol 12-myristate 13-acetate-induced desensitization, whereas substitution of any combination of two serine doublets suppressed it. Our data thus show that the glucagon-like peptide-1 receptor can be phosphorylated in response to phorbol 12-myristate 13-acetate on four different sites within the cytoplasmic tail. Furthermore, phosphorylation of at least three sites was required for desensitization, although maximal desensitization was only achieved when all four sites were phosphorylated.

Comparative cost-effectiveness analyses of cardiovascular magnetic resonance and coronary angiography combined with fractional flow reserve for the diagnosis of coronary artery disease.

BACKGROUND: According to recent guidelines, patients with coronary artery disease (CAD) should undergo revascularization if significant myocardial ischemia is present. Both, cardiovascular magnetic resonance (CMR) and fractional flow reserve (FFR) allow for a reliable ischemia assessment and in combination with anatomical information provided by invasive coronary angiography (CXA), such a work-up sets the basis for a decision to revascularize or not. The cost-effectiveness ratio of these two strategies is compared. METHODS: Strategy 1) CMR to assess ischemia followed by CXA in ischemia-positive patients (CMR + CXA), Strategy 2) CXA followed by FFR in angiographically positive stenoses (CXA + FFR). The costs, evaluated from the third party payer perspective in Switzerland, Germany, the United Kingdom (UK), and the United States (US), included public prices of the different outpatient procedures and costs induced by procedural complications and by diagnostic errors. The effectiveness criterion was the correct identification of hemodynamically significant coronary lesion(s) (= significant CAD) complemented by full anatomical information. Test performances were derived from the published literature. Cost-effectiveness ratios for both strategies were compared for hypothetical cohorts with different pretest likelihood of significant CAD. RESULTS: CMR + CXA and CXA + FFR were equally cost-effective at a pretest likelihood of CAD of 62% in Switzerland, 65% in Germany, 83% in the UK, and 82% in the US with costs of CHF 5'794, euro 1'517, £ 2'680, and $ 2'179 per patient correctly diagnosed. Below these thresholds, CMR + CXA showed lower costs per patient correctly diagnosed than CXA + FFR. CONCLUSIONS: The CMR + CXA strategy is more cost-effective than CXA + FFR below a CAD prevalence of 62%, 65%, 83%, and 82% for the Swiss, the German, the UK, and the US health care systems, respectively. These findings may help to optimize resource utilization in the diagnosis of CAD.

Rôle de l'activation de c-Jun N-terminal kinase (JNK) dans un modèle de glaucome chez le rat et neuroprotection par inhibition de la voie de JNK

RESUME : Dans ce travail effectué chez le rat adulte, l'excitotoxicité rétinienne est élicitée par injection intravitréenne de NMDA. Les lésions en résultant sont localisées dans la rétine interne. Elles prennent la forme de pycnoses dans la couche des cellules ganglionnaires (corps cellulaires des cellules ganglionnaires et amacrines déplacées) et dans la partie interne de la couche nucléaire interne (cellules amacrines). Cette localisation est liée à la présence de récepteurs au glutamate de type NMDA sur ces cellules. L'activation de ces récepteurs entraîne un influx calcique et l'activation de diverses enzymes (phospholipase A, calpaïnes, calmoduline, synthase d'oxyde nitrique). La signalisation se poursuit en aval en partie par les voies des Mitogen Activated Protein Kinase (MAPK) : ERK, p38, ]NK. Dans les expériences présentées, toutes trois sont activées après l'injection de NMDA. Dans les cascades de signalisation de JNK, trois kinases s'ancrent sur une protéine scaffold. Les MAPKKK phosphorylent MKK4 et MKK7, qui phosphorylent JNK. JNK a de nombreuses cibles nucléaires (dont le facteur de transcription c-Jun) et cytoplasmiques. La voie de JNK est bloquée par l'inhibiteur peptidique D-JNKI-1 en empêchant l'interaction de la kinase avec son substrat. L'inhibiteur est formé de 20 acides aminés du domaine de liaison JBD et de 10 acides aminés de la partie TAT du virus HIV. L'injection intravitréenne de D-JNKI-1 permet une diminution des taux de JNK et c-Jun phosphorylés dans les lysats de rétine. L'effet prépondérant est la restriction importante des altérations histologiques des couches internes de la rétine. L'évaluation par électrorétinogramme met en sus en évidence une sauvegarde de la fonction cellulaire. Ce travail a ainsi permis d'établir la protection morphologique et fonctionnelle des cellules de la rétine interne par inhibition spécifique de la voie de JNK lors d'excitotoxicité. SUMMARY Excitotoxicity in the retina associates with several pathologies like retinal ischemia, traumatic optic neuropathy and glaucoma. In this study, excitotoxicity is elicited by intravitreal NMDA injection in adult rats. Lesions localise in the inner retina. They present as pyknotic cells in the ganglion cell layer (ganglion cells and displaced amacrines) and the inner nuclear layer (amacrine cells). These cells express NMDA glutamate receptors. The receptor activation leads to a calcium flow into the cell and hence enzyme activation (phospholipase, calpains, calmodulin, nitric oxide synthase). The subsequent signaling pathways can involve the Mitogen Activated Protein Kinases (MAPK): ERK, p38 end JNK. These were all activated in our experiments. The signaling cascade organises around several scaffold proteins. The various MAPKKK phosphorylate MKK4 and MKK7, which phosphorylate JNK. JNK targets are of nuclear (c-Jun transcription factor) or cytoplasmic localisation. The peptidic inhibitor D-JNKI-1, 20 amino acids from the JNK binding domain JBD coupled to 10 amino acids of the TAT transporter, disrupts the binding of JNK with its substrate. Intravitreal injection of the inhibitor lowers phosphorylated forms of JNK and c-Jun in retinal extracts. It protects strongly against histological lesions in the inner retina and allows functional rescue.

Tumor antigen-specific FOXP3+ CD4 T cells identified in human metastatic melanoma: peptide vaccination results in selective expansion of Th1-like counterparts.

We have previously shown that vaccination of HLA-A2 metastatic melanoma patients with the analogue Melan-A(26-35(A27L)) peptide emulsified in a mineral oil induces ex vivo detectable specific CD8 T cells. These are further enhanced when a TLR9 agonist is codelivered in the same vaccine formulation. Interestingly, the same peptide can be efficiently recognized by HLA-DQ6-restricted CD4 T cells. We used HLA-DQ6 multimers to assess the specific CD4 T-cell response in both healthy individuals and melanoma patients. We report that the majority of melanoma patients carry high frequencies of naturally circulating HLA-DQ6-restricted Melan-A-specific CD4 T cells, a high proportion of which express FOXP3 and proliferate poorly in response to the cognate peptide. Upon vaccination, the relative frequency of multimer+ CD4 T cells did not change significantly. In contrast, we found a marked shift to FOXP3-negative CD4 T cells, accompanied by robust CD4 T-cell proliferation upon in vitro stimulation with cognate peptide. A concomitant reduction in TCR diversity was also observed. This is the first report on direct ex vivo identification of antigen-specific FOXP3+ T cells by multimer labeling in cancer patients and on the direct assessment of the impact of peptide vaccination on immunoregulatory T cells.