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.

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.

Immunological crossreactivity between the human immunodeficiency virus type 1 virion infectivity factor and a 170-kD surface antigen of Schistosoma mansoni.

A monoclonal antibody (mAb) directed against a synthetic peptide derived from the sequence of the human immunodeficiency virus type 1 (HIV-1) regulatory protein virion infectivity factor (vif) labeled the surface of Schistosoma mansoni schistosomula by indirect immunofluorescence. Western blotting showed that two S. mansoni proteins of 170 and 65 kD were recognized by the mAb. Sera from 20% of S. mansoni-infected HIV-seronegative individuals tested recognized the PS4 peptide in an ELISA as did sera from S. mansoni-infected rats. Sera from individuals seropositive for HIV-1, but without schistosomiasis, that reacted with the vif peptide also recognized a 170-kD S. mansoni protein. This crossreactive S. mansoni antigen appears to be a target of immunity in vivo since passive transfer of the mAb VIF-CD3 to naive rats had a protective effect against a challenge infection with S. mansoni cercariae.

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.

Trypanosoma cruzi infection inhibited by peptides modeled from a fibronectin cell attachment domain.

The mechanism by which Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, becomes attached to mammalian cells is not well understood. Fibronectin is thought to participate in the attachment, and in this study the region of fibronectin that interacts with the surface receptors of T. cruzi trypomastigotes was investigated by testing the binding of the amino acid sequence Arg-Gly-Asp-Ser, corresponding to the cell attachment site of fibronectin to T. cruzi trypomastigotes. Peptides with the sequence Arg-Gly-Asp-Ser, but not Arg-Phe-Asp-Ser, Arg-Phe-Asp-Ser-Ala-Ala-Arg-Phe-Asp, Ser-Lys-Pro, Glu-Ser-Gly, or Ala-Lys-Thr-Lys-Pro, bound to the parasite surface and inhibited cell invasion by the pathogen. Monoclonal antibodies to the cell attachment domain of fibronectin also inhibited cell infection by the parasite. The immunization of BALB/c mice with tetanus toxoid-conjugated peptide induced a significant protection against T. cruzi. The data support the notion that the sequence Arg-Gly-Asp-Ser of cell surface fibronectin acts as a recognition site for attachment of the parasites.

Replicative and cytopathic potential of HTLV-III/LAV with sor gene deletions.

The genome of the human T-lymphotropic virus type III (HTLV-III/LAV) has the potential to encode at least three polypeptides in addition to those encoded by the gag, pol, and env genes. In this study, the product of the sor (short open reading frame) region, which overlaps the 3' end of the pol gene, was found to be a protein with a molecular weight of 23,000. An assay was developed for testing the ability of cloned HTLV-III proviruses to produce viruses cytopathic for T4+ lymphocytes. In the cell line used, C8166, neither the HTLV-III sor gene product nor the complete 3'-orf gene product were necessary for the replication or cytopathic effects of the HTLV-III.

Diagnostic value of a synthetic peptide derived from Echinococcus granulosus recombinant protein.

A specific monoclonal antibody (MAb; EG 02 154/12) directed against a protein epitope of Echinococcus granulosus antigen 5 was used to screen a cDNA library constructed from E. granulosus protoscoleces RNA. One clone designated Eg14 was selected and shown to code for an amino acid sequence partially homologous to that of the clone Eg6 previously identified with the same MAb. Hydrophobic cluster analysis showed that both recombinant antigens may adopt a similar alpha-helical organization and share a common conformational epitope. A synthetic peptide (89-122) mimicking the conformational site of Eg6 and Eg14 was constructed and demonstrated to be able to inhibit binding of the MAb and human hydatid sera to the Eg6 fusion protein (FP6) or to native hydatid antigens. To assess the diagnostic value of the peptide 89-122, we tested sera from patients infected with different parasites for their antibody reactivity with this peptide in ELISA. A high binding sensitivity and specificity of IgG-A-M antibodies were obtained with E. granulosus-infected patient sera. Moreover, the peptide 89-122 was found to be specifically recognized by IgE antibodies from patients with hydatid disease. These results indicate the particular interest of this synthetic peptide as a standardized antigen in diagnosis and treatment surveillance of hydatidosis.

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.

Antipeptide antibodies discriminate between different SAA proteins in human plasma.

Antipeptide antibodies raised against two distinct sequences of human serum amyloid A (SAA) discriminate between different plasma isoforms of this acute phase reactant. As different SAA gene products have meanwhile been identified in human plasma, the discrimination between these different isoforms may help to clarify further the time of appearance of these different forms in plasma and their potential amyloidogenicity.

Compared recognition of di- and trisulfide substrates by glutathione and trypanothione reductases.

Trypanothione trisulfide was synthesized according to two strategies. It was found to be recognized and reduced by trypanothione reductase as the natural disulfide substrate. At the difference with the mechanism observed for the reduction of glutathione trisulfide by glutathione reductase, the intermediate trypanothione persulfide was rapidly reduced. The enzymatic reduction of another trisulfide derived from an alternative substrate of trypanothione reductase was also studied. The structure of the trisulfide bridge of the substrate (intra- or intermolecular) appeared to be a determining factor in the enzymatic reduction pattern. Moreover, in the case of the alternative substrate of trypanothione reductase, differences of kinetics appeared for the first time between a di- and a trisulfide species. All kinetic parameters are given.

Characterization of a monoclonal antibody directed against the NH2 terminal area of interleukin-2 (IL-2) and inhibiting specifically the binding of IL-2 to IL-2 receptor beta chain (IL-2R beta).

An anti-human IL-2 mAb (19B11/beta) was found to selectively block the binding of IL-2 to TS1 beta cells expressing the interleukin-2 receptor beta (IL-2R beta) without affecting binding to TS1 alpha cells expressing the IL-2R alpha receptor. It also specifically inhibits the IL-2 driven cell proliferation in TS1 beta cells. These observations have lead to the hypothesis that its epitope is related to an IL-2 area involved in binding with IL-2R beta chain. This epitope was identified using various peptides covering the N-terminal half (including alpha helix A) of the 133 amino acids of IL-2. MAb 19B11/beta does not recognize peptides 30-54 and 44-54 but recognizes peptides 1-22 and 1-30 with a good affinity. Furthermore, threonine in position no. 3 was found to be critical for the binding of mAb 19B11/beta. A relationship between the epitope of mAb 19B11/beta and the glycosylation of the IL-2 molecule was observed. This further demonstrates that the NH2 terminal area of IL-2 is critical for IL-2/IL-2R beta interactions. Two other mAbs were studied during the course of this work. They served as control for the study of mAb 19B11/beta and provide some additional insight concerning the question of IL-2/IL-2R structure-function. MAb 16F11/alpha selectively blocks the IL-2 binding to TS1 alpha cells. The epitope of mAb 16F11 is conformational and it was not possible to study the corresponding IL-2/IL-2R alpha region of interaction. Epitope of mAb 3H9 is localized between residues 30 and 54 and does not affect the binding of IL-2 to IL-2R alpha.

Influence of helical organization on immunogenicity and antigenicity of synthetic peptides.

Using cooperative effects of different peptide structures synthesized in tandem, we have induced an alpha-helical structure in water solution on a peptide which, alone, is unorganized. This structure is particularly relevant in this case as the selected model protein (type 24 M protein of Streptococcus pyogenes) is an extended coiled-coil system. We were thus able to assess the importance of organization or unorganization of a unique amino acid sequence with regards to its immunogenicity and antigenicity. Although in a classical manner, antibodies cross-reacting with the protein can be obtained with the short, unorganized peptide, we demonstrate that conformation-specific antibodies are raised when longer, organized peptides are used as immunogens.

Influence of CONH2 or COOH as C-terminus groups on the antigenic characters of immunogenic peptides.

The influence of the presence of a terminal COOH or CONH2 on the antigenic characters of synthetic immunogenic peptides has been studied on a streptococcal synthetic vaccine model. The obtained results show that when a peptide amide is used, the antibodies raised specifically against the amide group recognize neither free COOH nor the parent protein. The carboxamide group is thus unsuitable as was postulated for raising antibodies which recognize the peptide bond.

Immunogenicity and antigenicity of synthetic peptides derived from the mite allergen Der p I.

As an immunogen must contain both B- and T-cell epitopes, small peptides are usually reported as non-immunogenic unless coupled to a protein carrier. In this study, the immunogenicity of the Der p I synthetic uncoupled peptides (p52-71, p89-104, p117-133 and p176-187) previously reported as B-cell epitopes, was evaluated. Different schedules of immunization were used. Results indicated that by using the Vaitukaikis' method three injections of the same peptide without protein carrier was sufficient to induce an specific anti-peptide IgG antibody response (evaluated by ELISA). Indeed, the 16-20 amino-acid long peptides p52-71, p117-133 and p89-104 were revealed highly immunogenic in rabbits. Furthermore anti-peptide p52-71 and p117-133 antibodies were shown by Western-blotting or by neutralization assay to recognize the Der p I molecule either in denaturated or native form as well as Der f I (major allergen of Dermatophagoides farinae). Finally, taking into account the location of Der p I-derived peptides in the three-dimensional model of Der p I, the antigenicity and immunogenicity of peptides were discussed.

Analysis of antigenicity and immunogenicity of five different chemically defined constructs of a peptide.

The aim of this study was to compare the antigenicity and the immunogenicity of five constructs of a peptide, including the peptide in single copy, a tandem repeat containing three copies, a copolymer with glutaraldehyde and two constructs based on the MAP (Multiple Antigenic Peptide) model, one containing two copies (MAP-2) and the other, eight copies of the peptide (MAP-8). The peptide used in this test was the 115-131 sequence derived from the rSm28-GST antigen of Schistosoma mansoni. All constructs were recognized by rSm28-GST specific antibodies in solid phase immunoassays. However, the binding was higher when the MAP-8 was used as antigen at least partly because of its better coating on the microtiter plates. In vitro lymphoproliferative assays showed that polymer was mitogenic, repeat and MAP-2 did not stimulate rSm28-GST specific T cells while MAP-8 induced a slight response. The injection of MAP-8 to rats led to important antibody and T cell responses higher than those obtained with the other constructs. The IgG2a (cytotoxic antibody in schistosomiasis)/IgG2c (blocking antibody) ratio was independent of the immunogen. Taken together these results demonstrate that both the antigenicity and the immunogenicity of a peptide containing T and B cell epitope(s) are strongly related to the molecular form whereby it is presented and that the MAP-8 construct can be useful in serodiagnosis or in vaccination trials using synthetic peptides.

Specific histamine release capacity of peptides selected from the modelized Der p I protein, a major allergen of Dermatophagoides pteronyssinus.

Dust mite allergens are considered as a major cause of allergic disease and as a risk factor for asthma. Der p I, a 222 amino-acid residue globular glycoprotein, is one of the major allergens from Dermatophagoides pteronyssinus (Dpt) mites. In this study, we have used predictive conventional algorithms (i.e. hydrophilicity, mobility, accessibility) and a three-dimensional model of Der p I derived from comparison to actinidin and papain to select continuous amino acid sequences as potential B cell epitopes. Four peptides, 52-71, 117-133, 176-187, 188-199 were synthesized. Their antigenic reactivity was investigated, mainly by measuring their capacity to induce in vitro histamine release. Results indicated that only Dpt-sensitive patients react specifically to Der p I-derived peptides and more frequently to 52-71 and 117-133. For each peptide, the intensity of response was dependent on the patient tested and on the peptide concn. The capacity of peptides to induce histamine release was demonstrated to be correlated with the serum level of anti-Der p I IgE (r = 0.86; p less than 10(-2)). Taken together these data emphasize, in Dpt-sensitive patients, the heterogeneity of the specific response to synthetic Der p I-derived peptides and underline the possible variety of epitopes belonging to the allergen Der p I.

T helper cell epitopes of the human immunodeficiency virus (HIV-1) nef protein in rats and chimpanzees.

T helper cell antigenic and immunogenic determinants of the nef protein were investigated in the rat and chimpanzee models using recombinant nef protein and five synthetic peptides selected according to their amphipathic and alpha-helicity properties. The nef protein was shown to be immunogenic with both Freund's or aluminium hydroxide adjuvants. After immunization with the nef protein the 45-69 peptide was the most antigenic in rat and monkey models. In contrast, the 98-112 peptide, that required a carrier protein to induce in vitro rat T cell recall proliferation, was able to restimulate monkey T cells in the absence of a carrier. The amino acid sequence carrying the antigenic activity of the 45-69 peptide was further investigated by synthesizing short peptides overlapping this region. The antigenic sequence was precisely located in the middle of the peptide (region 50-59). This sequence was antigenic only when N alpha-acetylated. Circular dichroism analysis of the 45-69 peptide and the in vitro activity of the N-terminus group indicate in this case the involvement of the alpha-helical propensity for antigen presentation. However, the shorter sequence 50-64, able to induce a T cell reactivity, was determined as a beta-pleated sheet structure in aqueous solution. The 45-69 peptide was not only antigenic but also immunogenic and behaved in vivo as a functional T helper cell epitope. Indeed, the priming with the peptide or the transfer of peptide specific T cells to a naive recipient, followed by immunization with the nef protein, enhanced the subsequent antibody response to the nef protein. Together, these data indicate that the 45-69 peptide appears as a candidate for the in vivo elicitation of T cell immunity to the HIV-1 nef regulatory protein.

Determination of B-cell epitopes of nef HIV-I protein: immunogenicity related to their structure.

Determination of the B-cell epitopes of the nef molecule encoded by the human immunodeficiency virus type 1 (HIV-1) was undertaken using a set of six synthetic peptides. Sequences that were both antigenic and immunogenic and stimulated the production of antibodies recognizing the full length molecule, were considered as B-cell epitopes. Two peptidic sequences were antigenic both in rodents (mice and rats) and in non-human primates (chimpanzee). They were located in the regions 45-69 and 176-206 of the nef molecule. Two additional antigenic sequences were determined, one in chimpanzees, region 79-94, and the second in rodents, region 148-161. Immunogenicity was investigated in the rodents. Only the 45-69 and 176-206 sequences were immunogenic, and specific antibodies present in the sera of the immunized animals reacted with the nef protein. Therefore, each of these two sequences could be considered as containing at least one B-cell epitope. The fine epitopic specificity was determined using subfragments of these two sequences and it was shown that the antigenic determinants were contained in the C-terminal region of each sequence overlapping with the T-cell epitopes. These results raised the importance of vicinity of B- and T-cell determinants and their immunogenicity.

Amino acid sequence requirements for the epitope recognized by a monoclonal antibody reacting with the secreted antigen GP28.5 of Toxoplasma gondii.

We have characterized in detail, the epitope of the secreted antigen GP28.5 recognized by the mouse monoclonal antibody TG 17-179 using synthetic peptides and a truncated recombinant fusion protein. The screening of a T. gondii expression library with TG17-179 mAb led to the isolation of cDNAs clones, all encoding for the C-terminal region of GP28.5 and with one encoding for only the five last C-terminal residues. Competitive ELISA with longer peptides revealed that the immunoreactivity was retained for peptides of eight residues or longer, and lost when the peptide was reduced to the six last C-terminal residues or less. Experiments with the octapeptide lacking the carboxy-terminal glutamine residue showed it to be 64-fold less active. Moreover, neither addition of residues in the carboxy-end nor substitution of COOH function changed the immunoreactivity of the epitope. Competition experiments between TG17-179 mAb and sera from infected individuals demonstrates that the epitope defined by a mouse monoclonal probe is also a major epitope for human polyclonal antibodies. These results describe the sequence requirements within a probably linear epitope and give rise to some general question concerning experimental test for epitope mapping.

[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.