Analyses of tularemia cases and their long-term results.

Tularemia is a zoonotic disease and endemic in the northern hemisphere. The aim of this study was to evaluate the epidemiological, clinical and laboratory characteristics of tularemia patients, and to re-analyze their lymphadenopathy during the follow-up. The patients who were diagnosed with tularemia were reviewed. They were invited for the long term, physical and radiological evaluations. 69.8% patients had lived in rural areas. 54.7% patients were associated with animal husbandry, the 18.9% had contact with rodents. The most common form was the glandular type (62.3%). The frequency of granulomatous lymphadenitis was significantly higher in patients diagnosed later than 30 days from the onset of symptoms. Lymphadenopathy was undetectable in 61.5% patients, its severity was reduced in 38.4% patients compared to its state at the admission. In rural areas, avoiding contact with wild animals can ensure the protection from the pathogen. Public communities should be made aware of the disease.

Tetanus in adults: results of the multicenter ID-IRI study.

Tetanus is an acute, severe infection caused by a neurotoxin secreting bacterium. Various prognostic factors affecting mortality in tetanus patients have been described in the literature. In this study, we aimed to analyze the factors affecting mortality in hospitalized tetanus patients in a large case series. This retrospective multicenter study pooled data of tetanus patients from 25 medical centers. The hospitals participating in this study were the collaborating centers of the Infectious Diseases International Research Initiative (ID-IRI). Only adult patients over the age of 15 years with tetanus were included. The diagnosis of tetanus was made by the clinicians at the participant centers. Izmir Bozyaka Education and Research Hospital's Review Board approved the study. Prognostic factors were analyzed by using the multivariate regression analysis method. In this study, 117 adult patients with tetanus were included. Of these, 79 (67.5%) patients survived and 38 (32.5%) patients died. Most of the deaths were observed in patients >60 years of age (60.5%). Generalized type of tetanus, presence of pain at the wound area, presence of generalized spasms, leukocytosis, high alanine aminotransferase (ALT) and C-reactive protein (CRP) values on admission, and the use of equine immunoglobulins in the treatment were found to be statistically associated with mortality (p < 0.05 for all). Here, we describe the prognostic factors for mortality in tetanus. Immunization seems to be the most critical point, considering the advanced age of our patients. A combination of laboratory and clinical parameters indicates mortality. Moreover, human immunoglobulins should be preferred over equine sera to increase survival.

A novel approach to malignant-benign classification of pulmonary nodules by using ensemble learning classifiers.

Computer-aided detection systems can help radiologists to detect pulmonary nodules at an early stage. In this paper, a novel Computer-Aided Diagnosis system (CAD) is proposed for the classification of pulmonary nodules as malignant and benign. The proposed CAD system using ensemble learning classifiers, provides an important support to radiologists at the diagnosis process of the disease, achieves high classification performance. The proposed approach with bagging classifier results in 94.7 %, 90.0 % and 77.8 % classification sensitivities for benign, malignant and undetermined classes (89.5 % accuracy), respectively.

A new method for pulmonary nodule detection using decision trees.

A computer-aided detection (CAD) can help radiologists in diagnosing of lung diseases at an early level. In this study, a new CAD system for pulmonary nodule detection from CT imagery is presented by using morphological features and patient information properties. Decision trees are utilized for classification and overall detection performance is evaluated. Results are compared to similar techniques in the literature by using standard measures. Proposed CAD system with random forest classifier result in 90.5 % sensitivity and 87.6 % specificity of detection performance.

[New tools for preclinical pharmaceutic research]. / Nouveaux outils de la recherche pharmaceutique pré-clinique.

Despite constantly growing expenditures for pharmaceutical research, which have double in the last ten years, the number of new drugs marketed in the world has continued to decline. The last decade has nevertheless seen a series of major methodological advances in the fields including pharmacogenomics, pharamcogenetics, high-flow screening, and structural biology, all considered to contribute to the discovery of "drugs of the future". Systemic screening of compound banks has become the starting point for nearly all drug research. It is nevertheless only the first step towards a drug (typically lasting a few months compared with ten years before the final drug is marketed). For a compound to passé from a leading screening compound to a marketed drug, its often contradictory properties (solubility, permeability, duration of action, activity, selectivity, absence of toxicity) must undergo a long multidimensional optimization process. The development of easily interpreted low-cost in vitro tests allow prediction of these properties without animal experimentation, greatly facilitating the multidisciplinary process.

Detection of the toxin Hirsutellin A from Hirsutella thompsonii.

A total of 162 strains of Hirsutella thompsonii, isolated from infected mites collected worldwide, were examined for the production of Hirsutellin A (HtA). More than half of the broth filtrates exhibited mortality rates superior to 50% when assayed against Galleria mellonella. The presence of the gene coding for HtA, a previously characterized H. thompsonii protein exotoxin, was determined by PCR amplification using gene-specific primers. Most isolates (100 out of 162) were shown to possess the HtA gene. However, the presence of the gene could not be associated with enhanced insecticidal activity. Both isolate groups (with or without an amplifiable HtA gene) produced filtrates that caused the same average mortality rate (65%) when assayed against G. mellonella. The production and secretion of the HtA toxin were estimated by probing broth filtrates with an anti-HtA monoclonal antibody. Again, the detection of the HtA protein was poorly correlated with subsequent mortality rates induced by the broth filtrates of the various H. thompsonii strains. This study suggests that HtA is requisite for neither survival nor pathogenicity, and that H. thompsonii strains are likely to secrete other toxins that have yet to be characterized. Sequencing of a limited number of HtA genes showed that, when present, the gene is highly conserved, and it displays an interesting intronic polymorphism.

Characterization of an IL-2 mimetic with therapeutic potential.

Human interleukin-2 (IL-2) interacts with two types of functional receptors (IL-2R alpha betagamma and IL-2R betagamma) and acts on a broad range of target cells involved in inflammatory reactions and immune responses. IL-2 is also used in different clinical trials aimed at improving the treatment of some cancers and the recovery of CD4 lymphocytes by HIV patients. The therapeutic index of IL-2 is limited by various side effects dominated by the vascular leak syndrome. We have shown that a chemically synthesised fragment of the IL-2 sequence can fold into a helical tetramer likely mimicking the quatemary structure of an hemopoietin. Indeed, peptide p1-30 (containing amino acids 1 to 30, including the sequence corresponding to the entire alpha helix A of IL-2) spontaneously folds into an alpha-helical homotetramer and stimulates the growth of T-cell lines expressing human IL-2R beta, whereas shorter versions of the peptide lack helical structure and are inactive. At the cellular level, p1-30 induces lymphokine-activated killer (LAK) cells and preferentially activates CD8 low lymphocytes and natural killer cells, which constitutively express IL-2R beta. A significant IFN-gamma production is also detected following p1-30 stimulation. A mutant form of p1-30 (Asp20-->Lys) which is likely unable to induce vascular leak syndrome remains capable to generate LAK cells like the original p1-30 peptide. Altogether our data suggest that p1-30 has therapeutic potential.

Lipopeptides induce cell-mediated anti-HIV immune responses in seronegative volunteers.

OBJECTIVE: Test the efficacy of a mixture of six NEF (N1, N2, N3), GAG (G1, G2) and ENV (E) lipopeptides in the induction of B- and T-cell anti-HIV responses. DESIGN: A randomized phase I open-label dose-finding trial. Twenty-eight healthy seronegative volunteers received the lipopeptides, with or without the adjuvant QS21. METHODS: Anti-HIV-peptide antibodies were detected by enzyme-linked immunosorbent assay and Western blotting. Induction of cellulary responses was assessed by proliferative test and (51)Cr-release assay. RESULTS: Local and systemic adverse reactions were always mild or moderate. After three injections an antibody response was detected in 25 out of 28 volunteers (89%). T cells from 19 (79%) of the 24 volunteers proliferated in response to at least one peptide. The majority of the volunteers had induced a multispecific proliferative response; that is, cells from volunteers proliferated to two (five of 19), three (five of 19), four (three of 19) or five peptides (one of 19). Cytotoxic responses by anti-HIV CD8+ lymphocytes could be tested in 24 volunteers, 13 (54%) of whom had clear and reproducible responses, with strong activity in the remaining 12 (> 20% of specific lysis), and polyepitopic responses were detected in at least seven of the 13 responders. Cytotoxic responses were found against the whole NEF protein (clade B LAI) in three of four tested volunteers and cross-reactions with the proteins of clade B (MN) and clade A (Bangui) HIV-1 strains, and also HIV-2 ROD, were detected in one of two tested volunteers. CONCLUSIONS: Lipopeptides are promising immunogens for an AIDS vaccine.

The first alpha helix of interleukin (IL)-2 folds as a homotetramer, acts as an agonist of the IL-2 receptor beta chain, and induces lymphokine-activated killer cells.

Interleukin (IL)-2 interacts with two types of functional receptors (IL-2Ralphabetagamma and IL-2Rbetagamma) and acts on a broad range of target cells involved in inflammatory reactions and immune responses. For the first time, we show that a chemically synthesized fragment of the IL-2 sequence can fold into a molecule mimicking the quaternary structure of a hemopoietin. Indeed, peptide p1-30 (containing amino acids 1-30, covering the entire alpha helix A of IL-2) spontaneously folds into an alpha-helical homotetramer and stimulates the growth of T cell lines expressing human IL-2Rbeta, whereas shorter versions of the peptide lack helical structure and are inactive. We also demonstrate that this neocytokine interacts with a previously undescribed dimeric form of IL-2Rbeta. In agreement with its binding to IL-2Rbeta, p1-30 activates Shc and p56(lck) but unlike IL-2, fails to activate Janus kinase (Jak)1, Jak3, and signal transducer and activator of transcription 5 (STAT5). Unexpectedly, we also show that p1-30 activates Tyk2, thus suggesting that IL-2Rbeta may bind to different Jaks depending on its oligomerization. At the cellular level, p1-30 induces lymphokine-activated killer (LAK) cells and preferentially activates CD8(low) lymphocytes and natural killer cells, which constitutively express IL-2Rbeta. A significant interferon gamma production is also detected after p1-30 stimulation. A mutant form of p1-30 (Asp20-->Lys), which is likely unable to induce vascular leak syndrome, remains capable of generating LAK cells, like the original p1-30 peptide. Altogether, our data suggest that p1-30 has therapeutic potential.

Multiepitopic B- and T-cell responses induced in humans by a human immunodeficiency virus type 1 lipopeptide vaccine.

We have attempted to develop an anti-human immunodeficiency virus (HIV) lipopeptide vaccine with several HIV-specific long peptides modified by C-terminal addition of a single palmitoyl chain. A mixture of six lipopeptides derived from regulatory or structural HIV-1 proteins (Nef, Gag, and Env) was prepared. A phase I study was conducted to evaluate immunogenicity and tolerance in lipopeptide vaccination of HIV-1-seronegative volunteers given three injections of either 100, 250, or 500 microg of each lipopeptide, with or without immunoadjuvant (QS21). This report analyzes in detail B- and T-cell responses induced by vaccination. The lipopeptide vaccine elicited strong and multiepitopic B- and T-cell responses. Vaccinated subjects produced specific immunoglobulin G antibodies that recognized the Nef and Gag proteins. After the third injection, helper CD4(+)-T-cell responses as well as specific cytotoxic CD8(+) T cells were also obtained. These CD8(+) T cells were able to recognize naturally processed viral proteins. Finally, specific gamma interferon-secreting CD8(+) T cells were also detected ex vivo.

Characterization of a multi-lipopeptides mixture used as an HIV-1 vaccine candidate.

A multi-component vaccine has been defined, which contains six different synthetic 24- to 32-amino acid lipopeptides derived from the sequence of HIV-1 proteins. The physicochemical properties of the lipopeptide components were compatible with multi-dimensional analysis, using RP-HPLC, Edman sequencing, electrospray mass spectrometry, and 2D-NMR. Detailed analysis of the impurity profiles led to the detection and evaluation of the relative proportions of most by-products: several contaminants resulted from the formation of acetylated fragments, transpeptidation reactions with succinimide or piperidide formation, or methionine and/or tryptophan mono-oxidations. The first batch to be produced underwent extensive pharmacotoxicological testings to confirm its safety; this vaccine candidate has now been used in phase I clinical trials. Despite the complexity of such multi-lipopeptide vaccines, our findings suggest the possibility of preparing a clear and precise assignment of by-products to toxicologically qualified impurities in the eventuality of a future production of several successive batches.

Convergent peptide libraries, or mixotopes, to elicit or to identify specific immune responses.

Many recent studies have demonstrated the flexibility of epitope recognition by the immune system. This can be explored using a particular type of combinatorial peptide library, termed as 'convergent', consisting essentially of closely related molecular species; from this a fuzzy set can be constructed, which comprises several variants of a peptide that would act in synchrony to represent a model antigen and its recognition by the immune system.

Synthesis, folding, and structure of the beta-turn mimic modified B1 domain of streptococcal protein G.

The mechanism of beta-sheet formation remains a fundamental issue in our understanding of the protein folding process, but is hampered by the often encountered kinetic competition between folding and aggregation. The role of local versus nonlocal interactions has been probed traditionally by mutagenesis of both turn and strand residues. Recently, rigid organic molecules that impose a correct chain reversal have been introduced in several small peptides to isolate the importance of the long-range interactions. Here, we present the incorporation of a well-studied beta-turn mimic, designated as the dibenzofuran-based (DBF) amino acid, in the B1 domain of streptococcal protein G (B1G), and compare our results with those obtained upon insertion of the same mimic into the N-terminal beta-hairpin of B1G (O Melnyk et al., 1998, Lett Pept Sci 5:147-150). The DBF-B1G domain conserves the structure and the functional and thermodynamical properties of the native protein, whereas the modified peptide does not adopt a native-like conformation. The nature of the DBF flanking residues in the modified B1G domain prevents the beta-turn mimic from acting as a strong beta-sheet nucleator, which reinforces the idea that the native beta-hairpin formation is not driven by the beta-turn formation, but by tertiary interactions.

Branched synthetic peptide constructs mimic cellular binding and efflux of apolipoprotein AI in reconstituted high density lipoproteins.

This study investigates the suitability of the trimeric apolipoprotein (apo)AI(145-183) peptide that we recently described, to serve as a model to probe the relationship between apoAI structure and function. Three copies of the apoAI(145-183) unit, composed each of two amphipathic alpha-helical segments, were branched onto a covalent core matrix and the construct was recombined with phospholipids. A similar construct was made with the apoAI(102-140) peptide and used as a comparison with dimyristoylglycerophosphocholine (DMPC)-apoAI complexes. The DMPC-trimeric-apoAI(145-183) complexes had similar immunological reactivity with monoclonal antibodies directed against the 149-186 apoAI sequence (A44), suggesting that the A44 epitope is exposed similarly in both the synthetic peptide and the native apoAI complexes. The complexes generated with the trimeric-apoAI(145-183) bind specifically to HeLa cells with comparable affinity to the DMPC apoAI complexes; they are a good competitor for binding of apoAI to both HeLa cells and Fu5AH rat hepatoma cells; finally, these complexes promote cholesterol efflux from Fu5AH cells with an efficiency comparable with the apo AI/lipid complexes. To study LCAT activation by the trimeric apo AI(145-183) construct, complexes were prepared with dipalmitoylphosphatidylcholine (DPPC), cholesterol (C) and either the trimeric construct or apoAI. LCAT activation by the trimeric construct was much lower than by apo AI, possibly because the conformation of the trimeric 145-183 peptide in DPPC/C/peptide complexes does not mimic that of apoAI in the corresponding complexes. In comparison, the complexes generated with the multimeric apoAI(102-140) construct had a poor capacity to mimic the physico-chemical and biological properties of apoAI. The apoAI(102-140) construct had low affinity for lipid compared with the (145-183) construct. After association with lipids, it was a poor competitor of DMPC-apoAI complexes for cellular binding and had only limited capacity to promote cholesterol efflux. These results suggest trimeric constructs can serve as an appropriate models for apoAI, enabling further investigations and new experimental approaches to determine the structure-function relationship of apoAI.

Adjuvant is required when using Env lipopeptide construct to induce HIV type 1-specific neutralizing antibody responses in mice in vivo.

Extensive immunological studies on HIV-1 infection, the causative agent of AIDS in humans, have led to the conclusion that efficient protection against this infection should require early elicitation of neutralizing antibodies as well as cellular immune and particularly cytotoxic T lymphocyte responses. The use of synthetic peptides modified at one end by introduction of a lipidic tail is now well known to be an effective means of eliciting virus-specific cytotoxic T lymphocyte responses in vivo, both in mouse and humans. To ascertain that such a strategy can be used for vaccinal purposes, particularly against HIV-1 infection, it remains to be determined whether these molecules can also act as effective inducers of antibody responses, most of all of the neutralizing type. The present study set out to address this question by using a synthetic HIV-1 ENV lipopeptide construct, previously identified as a potent immunogen for in vivo induction of ENV-specific CTL responses in BALB/c mice. We first showed that V3 peptide-specific antibodies were effectively induced by the lipopeptide construct. However, we provided evidence that the biological activity of these antibodies, i.e., their ability to neutralize HIV-1 infectivity in vitro, was strongly influenced by the immunizing conditions and protocol, in that only those antibodies generated by the use of adjuvanted lipopeptide formulations were effective. Albeit at a slightly lower efficacy than by the intraperitoneal route, neutralizing antibodies could also be induced using the subcutaneous route. With the prospect of a human peptide vaccine in mind, we then studied the properties of different known or possibly clinically relevant adjuvants. We found that alum, the only relevant adjuvant for human use, not only provides inefficient help to the lipopeptide construct in generating neutralizing antibodies, but tends to have deleterious effects on the ability of the construct to induce CTL responses. The only protocol that gave satisfactory results in terms of the magnitude of the neutralizing antibody responses was a mineral oil-based lipopeptide formulation. When induced under those conditions, strong neutralizing activities were still present up to 8 months after the last injection.

Immunogenicity of four Plasmodium falciparum preerythrocytic antigens in Aotus lemurinus monkeys.

Aotus lemurinus monkeys were immunized with pools of either lipid-tailed peptides injected in PBS or peptides in Montanide ISA-51, all derived from four Plasmodium falciparum pre-erythrocytic antigens, namely, LSA1, LSA3, SALSA, and STARP. These formulations were well tolerated. Their immunogenicity was demonstrated by the induction of both B- and T-cell responses to most of the peptides studied (of the 12, 10 induced antibody production, 9 induced T-cell proliferative responses, and all 12 induced gamma interferon secretion). Immune responses proved to be long lasting, since some were still detectable 210 days after immunization. Of particular importance is the fact that B- and T-cell responses elicited in this way by synthetic peptides were specific for native parasite proteins on P. falciparum sporozoites and liver stage parasites.

Plasmodium falciparum sporozoite invasion is inhibited by naturally acquired or experimentally induced polyclonal antibodies to the STARP antigen.

Antibody(Ab)-mediated inhibition of sporozoite invasion of hepatocytes is a mechanism that has been clearly demonstrated to act upon Plasmodium falciparum pre-erythrocytic stages in humans. Consequently we have analyzed the Ab response to a recently identified P. falciparum sporozoite surface protein, STARP, in malaria-exposed individuals and tested the inhibitory effect of these Ab upon hepatocyte invasion in vitro. STARP-specific IgG were detected in 90 and 61% of sera from regions where individuals were exposed to 100 and 1-5 infectious bites per year, respectively. These IgG were predominantly of the cytophilic IgG1 or IgG3 type. STARP and the major sporozoite surface protein, CS, elicited equivalent IgG levels in adults. When affinity purified from either African immune sera or the serum of an individual experimentally protected by irradiated sporozoite immunization, STARP-specific Ab prevented up to 90% of sporozoites from invading human hepatocytes. The dose-dependent and reproducible inhibition was more pronounced than that observed with human CS-specific Ab affinity purified under identical conditions. Substantial reduction of sporozoite invasion was also observed with Ab induced by artificial immunization with recombinant STARP protein and reactive with the native protein. Taken together with recent findings of human cytotoxic T lymphocytes specific for this antigen, these results promote the interest of studying the efficacy of STARP as a target for immune effector mechanisms operating upon pre-erythrocytic stages.

Confronting the degeneracy of convergent combinatorial immunogens, or 'mixotopes', with the specificity of recognition of the target sequences.

Immunization by convergent combinatorial peptide libraries, or 'mixotopes' represents an interesting approach for inducing broadly cross-reactive immune response to hypervariable pathogens. The authors have immunized rabbits with a series of eight HIV-1 V3-loop derived constructs of increasing complexity, and analysed the reactivity of the corresponding antisera towards a set of V3-related peptides. Results were surprisingly homogeneous. Mixotopes containing as many as several billion closely related combinatorial sequences were immunogenic, and able to induce V3-specific antibodies. These results suggest that serological cross-reactivity depends on the sequential similarity of the antigen with the parent immunogen. Such 'mixotopes' could represent a useful approach to vaccination against hypervariable pathogens.