Force-induced globule-coil transition in laminin binding protein and its role for viral-cell membrane fusion.

The specific interactions of the pairs laminin binding protein (LBP)-purified tick-borne encephalitis viral surface protein E and certain recombinant fragments of this protein, as well as West Nile viral surface protein E and certain recombinant fragments of that protein, are studied by combined methods of single-molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves-based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human αVβ3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus-cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus-cell membrane fusion, SMDFS data reveal the existence of a force-induced (effective already for forces as small as 30-70 pN) sharp globule-coil transition for LBP and LBP-fragments of protein E complexes. We argue that this conformational transformation, being an analog of abrupt first-order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus-cell membrane fusion process. Copyright © 2014 John Wiley & Sons, Ltd.

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.

Expression of a functional single chain antibody on the surface of extracellular enveloped vaccinia virus as a step towards selective tumour cell targeting.

Recombinant vaccinia virus with tumour cell specificity may provide a versatile tool either for direct lysis of cancer cells or for the targeted transfer of genes encoding immunomodulatory molecules. We report the expression of a single chain antibody on the surface of extracellular enveloped vaccinia virus. The wild-type haemagglutinin, an envelope glycoprotein which is not required for viral infection and replication, was replaced by haemagglutinin fusion molecules carrying a single chain antibody directed against the tumour-associated antigen ErbB2. ErbB2 is an epidermal growth factor receptor-related tyrosine kinase overexpressed in a high percentage of human adenocarcinomas. Two fusion proteins carrying the single chain antibody at different NH2-terminal positions were expressed and exposed at the envelope of the corresponding recombinant viruses. The construct containing the antibody at the site of the immunoglobulin-like loop of the haemagglutinin was able to bind solubilized ErbB2. This is the first report of replacement of a vaccinia virus envelope protein by a specific recognition structure and represents a first step towards modifying the host cell tropism of the virus.

Human IgG responses against the N-terminal region of Merozoite Surface Protein 1 of Plasmodium vivax

The complete primary structure of the gene encoding the Merozoite Surface Protein 1 of Plasmodium vivax (PvMSP-1) revealed the existence of interspecies conserved regions among the analogous proteins of other Plasmodia species. Here, three DNA recombinant clones expressing 50, 200 and 500 amino acids from the N-terminal region of the PvMSP-1 protein were used on ELISA and protein immunoblotting assays to look at the IgG antibody responses of malaria patients from the Brasilian amazon region of Rondônia. The results showed the existance of P. vivax and P. falciparum IgG antibodies directed against PvMSP-1 antigenic determinants expressed in the clones containing the first 200 and the following 500 amino acids of the molecule, but not within the one expressing the most N-terminal 50 amino acids. Interestingly, there was no correlation between the levels of these IgG antibodies and the previous number of malaria infections.

T cell responses to repeat and non-repeat regions of the circumsporozoite protein detected in volunteers immunized with Plasmodium falciparum sporozoites

The design of malarial vaccine based on the circumsporozoite (CS) protein, a majuor surface antigen of the sporozoite stage of the malaria parasite, requires the identification of T and B cell epitopes for inclusion in recombinant or synthetic vaccine candidates. We have investigated the specificity and function of a series of T cell clones, derived from volunteers immunized with Plasmodium falciparum sporozoites in an effort to identify relevant epitopes in the immune response to the pre-erythrocytic stages of the parasite. CD4+ T cell clones were obtained wich specifically recognized a repetitive epitope located in the 5'repeat region of the CS protein. This epitope, when conjugated to the 3'repeat region in a synthetic MAPs construct, induced high titers of antisporozoite antibodies in C57B1 mice. A second T cell epitope, which mapped to aa 326-345 of the carboxy terminal, was recognized by lytic, as well as non-lytic, CD4+ T cells derived from the sporozoite-immunized volunteers. The demonstration of CD4+ CTL in the volunteers, and the recent studies inthe rodent model (Renia et al., 1991; Tsuji et al., 1990), suggested that CS-specific CD4+ T cells, in addition to their indirect role as helper cells in the induction of antibody and CD8 + effector cells, may also play a direct role in protection against sporozoite challenge by targeting EEF within the liver.

Distinct expression pattern of microtubule-associated protein-2 in human oligodendrogliomas and glial precursor cells.

Microtubule-associated protein 2 (MAP2), a protein linked to the neuronal cytoskeleton in the mature central nervous system (CNS), has recently been identified in glial precursors indicating a potential role during glial development. In the present study, we systematically analyzed the expression of MAP2 in a series of 237 human neuroepithelial tumors including paraffin-embedded specimens and tumor tissue microarrays from oligodendrogliomas, mixed gliomas, astrocytomas, glioblastomas, ependymomas, as well as dysembryoplastic neuroepithelial tumors (DNT), and central neurocytomas. In addition, MAP2-immunoreactive precursor cells were studied in the developing human brain. Three monoclonal antibodies generated against MAP2A-B or MAP2A-D isoforms were used. Variable immunoreactivity for MAP2 could be observed in all gliomas with the exception of ependymomas. Oligodendrogliomas exhibited a consistently strong and distinct pattern of expression characterized by perinuclear cytoplasmic staining without significant process labeling. Tumor cells with immunoreactive bi- or multi-polar processes were mostly encountered in astroglial neoplasms, whereas the small cell component in neurocytomas and DNT was not labeled. These features render MAP2 immunoreactivity a helpful diagnostic tool for the distinction of oligodendrogliomas and other neuroepithelial neoplasms. RT-PCR, Western blot analysis, and in situ hybridization confirmed the expression of MAP2A-C (including the novel MAP2+ 13 transcript) in both oligodendrogliomas and astrocytomas. Double fluorescent laser scanning microscopy showed that GFAP and MAP2 labeled different tumor cell populations. In embryonic human brains, MAP2-immunoreactive glial precursor cells were identified within the subventricular or intermediate zones. These precursors exhibit morphology closely resembling the immunolabeled neoplastic cells observed in glial tumors. Our findings demonstrate MAP2 expression in astrocytic and oligodendroglial neoplasms. The distinct pattern of immunoreactivity in oligodendrogliomas may be useful as a diagnostic tool. Since MAP2 expression occurs transiently in migrating immature glial cells, our findings are in line with an assumed origin of diffuse gliomas from glial precursors.

Induction of synthesis of the rat cystatin S protein by the submandibular gland during the acute phase of experimental Chagas disease

Rats experimentally infected with Trypanosoma cruzi Y strain exhibited hypertrophy of the submandibular gland at 18 days after infection.SDS-PAGE of infected rats saliva revealed the presence of an additional band with an apparent molecular weight of about 13KDa. Electrophoresis of protein salivaand immunochemical analysis with antibody against rat cystatin S confirmed that the protein was identical to that induced by beta adrenergic stimulation.

Etiology of community-acquired pneumonia in hospitalized children based on WHO clinical guidelines

Rapport de synthèse: Enjeux de la recherche : La pneumonie communautaire chez l'enfant est un problème de santé publique considérable. Elle est responsable de 2 millions de mort par année, 70% survenant dans les pays en voie de développement. Sous nos latitudes son incidence est de 40/1000 enfants par année, ce qui représente une morbidité importante. Deux difficultés surviennent lorsqu'on cherche à diagnostiquer une pneumonie. La première est de distinguer une pneumonie bactérienne d'une virale, particulièrement chez les petits enfants où les infections virales des voies respiratoires inférieures sont fréquentes. L'OMS a définit la pneumonie selon des critères exclusivement cliniques et une étude effectuée à Lausanne en 2000 a montré que ces critères peuvent être utilisés dans nos contrées. La seconde difficulté est de définir l'agent causal de la pneumonie, ceci pour plusieurs raisons : L'aspiration endotrachéale, seul examen fiable, ne peut être obtenue de routine chez l'enfant vu son caractère invasif, la culture des secrétions nasopharyngées reflète la flore physiologique de la sphère ORL et une bactériémie n'est présente que dans moins de 10% des pneumonies. L'étiologie de la pneumonie reste souvent inconnue, et de ce fait plusieurs enfants reçoivent des antibiotiques pour une infection non bactérienne ce qui contribue au développement de résistances. L'objectif de cette étude était d'effectuer une recherche extensive de l'agent causal de la pneumonie et de déterminer quels facteurs pourraient aider le clinicien à différencier une pneumonie virale de bactérienne, en corrélant l'étiologie avec la sévérité clinique et les marqueurs de l'inflammation. Contexte de la recherche : II s'agissait d'une étude prospective, multicentrique, incluant les enfants âgés de 2 mois à 5 ans hospitalisés pour une pneumonie, selon les critères de l'OMS, dans le service de pédiatrie de Lausanne et Genève entre mars 2003 et Décembre 2005, avant l'implantation de la vaccination antipneumococcique de routine. Chaque enfant, en plus des examens usuels, bénéficiait d'une recherche étiologique extensive : Culture virale et bactérienne, PCR (Mycoplasma Pneumoniae, Chlamydia Pneumoniae, Virus Influenza A et B, RSV A et B, Rhinovirus, Parainfluenza 1-3, enterovirus, human metapneumovirus, coronavirus OC43, E229 ; et NL 63) et détection d'AG viraux dans les sécrétions nasopharyngées ; sérologies virales et bactériennes à l'entrée et 3 semaines après la sortie (AG Influenza A et B, Parainfluenza 1,2 et 3, RSV, Adenovirus, M.Pneumoniae et S.Pneumoniae). Conclusions : Un agent pathogène a été découvert chez 86% des 99 patients retenus confirmant le fait que plus la recherche étiologique est étendue plus le pourcentage d'agent causal trouvé est élevé. Une infection bactérienne a été découverte chez 53% des patients dont 45% avaient une infection à S. Pneumoniae confirmant l'importance d'une vaccination antipneumococcique de routine. La déshydratation et les marqueurs de l'inflammation tels que la C-Reactive Protein et la Procalcitonine étaient significativement plus élevés dans les pneumonies bactériennes. Aucune corrélation n'a été trouvée entre le degré de sévérité de la pneumonie et l'étiologie. L'étude a confirmé la haute prévalence d'infections virales (67%) et de co-infection (33%) dans la pneumonie de l'enfant sans que l'on connaisse le rôle réel du virus dans la pathogenèse de la pneumonie. Perspectives : d'autres études à la suite de celle-ci devraient être effectuées en incluant les patients ambulatoires afin de déterminer, avec un collectif plus large de patient, une éventuelle corrélation entre sévérité clinique et étiologie. Abstract : Community-acquired pneumonia (CAP) is a major cause of death in developing countries and of morbidity in developed countries. The objective of the study was to define the causative agents among children hospitalized for CAP defined by WHO guidelines and to correlate etiology with clinical severity and surrogate markers. Investigations included an extensive etiological workup. A potential causative agent was detected in 86% of the 99 enrolled patients, with evidence of bacterial (53%), viral (67%), and mixed (33%) infections. Streptococcus pneumoniae was accounted for in 46% of CAP. Dehydration was the only clinical sign associated with bacterial pneumonia. CRP and PCT were significantly higher in bacterial infections. Increasing the number of diagnostic tests identifies potential causes of CAP in up to 86% of children, indicating a high prevalence of viruses and frequent co-infections. The high proportion of pneumococcal infections re-emphasizes the importance of pneumococcal immunization.

Dukes B colorectal cancer: distinct genetic categories and clinical outcome based on proximal or distal tumor location.

PURPOSE: The aim of this study was to determine whether tumor location proximal or distal to the splenic flexure is associated with distinct molecular patterns and can predict clinical outcome in a homogeneous group of patients with Dukes B (T3-T4, N0, M0) colorectal cancer. It has been hypothesized that proximal and distal colorectal cancer may arise through different pathogenetic mechanisms. Although p53 and Ki-ras gene mutations occur frequently in distal tumors, another form of genomic instability associated with defective DNA mismatch repair has been predominantly identified in the proximal colon. To date, however, the clinical usefulness of these molecular characteristics remains unproven. METHODS: A total of 126 patients with a lymph node-negative sporadic colon or rectum adenocarcinoma were prospectively assessed with the endpoint of death by cancer. No patient received either radiotherapy or chemotherapy. p53 protein was studied by immunohistochemistry using DO-7 monoclonal antibody, and p53 and Ki-ras gene mutations were detected by single strand conformation polymorphism assay. RESULTS: During a mean follow-up of 67 months, the overall five-year survival was 70 percent. Nuclear p53 staining was found in 57 tumors (47 percent), and was more frequent in distal than in proximal tumors (55 vs. 21 percent; chi-squared test, P < 0.001). For the whole group, p53 protein expression correlated with poor survival in univariate and multivariate analysis (log-rank test, P = 0.01; hazard ratio = 2.16; 95 percent confidence interval = 1.12-4.11, P = 0.02). Distal colon tumors and rectal tumors exhibited similar molecular patterns and showed no difference in clinical outcome. In comparison with distal colorectal cancer, proximal tumors were found to be statistically significantly different on the following factors: mucinous content (P = 0.008), degree of histologic differentiation (P = 0.012), p53 protein expression, and gene mutation (P = 0.001 and 0.01 respectively). Finally, patients with proximal tumors had a marginally better survival than those with distal colon or rectal cancers (log-rank test, P = 0.045). CONCLUSION: In this series of Dukes B colorectal cancers, p53 protein expression was an independent factor for survival, which also correlated with tumor location. Eighty-six percent of p53-positive tumors were located in the distal colon and rectum. Distal colon and rectum tumors had similar molecular and clinical characteristics. In contrast, proximal neoplasms seem to represent a distinct entity, with specific histopathologic characteristics, molecular patterns, and clinical outcome. Location of the neoplasm in reference to the splenic flexure should be considered before group stratification in future trials of adjuvant chemotherapy in patients with Dukes B tumors.

The antibody response in mice to carrier-free synthetic polymers of Plasmodium falciparum circumsporozoite repetitive epitope is I-Ab-restricted: possible implications for malaria vaccines.

The immunogenicity of a novel synthetic peptide consisting of an average of 40 (Asn-Ala-Asn-Pro) repeats of the circumsporozoite protein of Plasmodium falciparum, (NANP)40, was studied in mice without using any carrier proteins. First, high titers of anti-(NANP)40 antibodies could be obtained after immunization of C57BL/6 mice. These antibodies also reacted with an extract of mosquitoes infected with P. falciparum sporozoites. C57BL/6 nu/nu mice did not produce antibodies against (NANP)40. Secondly, when 14 strains of mice with nine different H-2 haplotypes were immunized with (NANP)40 without carrier, only H-2b mice were found to produce anti-(NANP)40 antibodies, whereas all non-H-2b mice were consistently unresponsive. This response was demonstrated to be I-A-linked by using recombinant and mutant mice. I-Ab [B10.A(5R)] mice produced anti-(NANP)40 antibodies as well as H-2b inbred mice. B6CH-2bm12 I-Ab-mutant mice showed only a very low response. Third, the antibody response against (NANP)40 could be induced in nonresponder mice by immunization with the peptide coupled to a carrier protein. In view of the existence of such an exceptional H-2b restriction in the response to sporozoite synthetic peptides in mice, the triggering of peptide-specific T cell responses in humans receiving sporozoite malaria vaccines might be difficult to achieve.

Naturally acquired antibodies against the major merozoite surface coat protein (MSP-1) of Plasmodium falciparum acquired by residents in an endemic area of Colombia

A preliminary baseline epidemiological malaria survey was conducted in the village of Punta Soldado, Colombia. Parasite prevalence and density as well as serological data were obtained from 151 asymptomatic children and adults. Fifty individuals were infected with Plasmodium falciparum. The mean parasite density was 184 parasites/mm3. Greater than 90 of the sample population were P. falciparum antibody positive as detected by the indirect immunofluorescent antibody test (IFAT). The enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against the major merozoite surface protein (MSP-1) of P. falciparum. In this population, anti-MSP-1 antibody concentration is acquired in an age dependent manner with equal immunogenicity to both the N- and C-terminal regions of the molecule. Infection at the time of sampling was associated with a higher anti-MSP-1 antibody concentration than that found in non-infected individuals. Further studies are planned to assess the role of immune and non-immune factors in limiting the number of cases of severe malaria seen in this population.

A novel merozoite surface antigen of Plasmodium falciparum (MSP-3) identified by cellular-antibody cooperative mechanism antigenicity and biological activity of antibodies

We report the identification of a 48kDa antigen targeted by antibodies which inhibit Plasmodium falciparum in vitro growth by cooperation with blood monocytes in an ADCI assay correlated to the naturally acquired protection. This protein is located on the surface of the merozoite stage of P. falciparum, and is detectable in all isolates tested. Epidemiological studies demonstrated that peptides derived from the amino acid sequence of MSP-3 contain potent B and T-cell epitopes recognized by a majority of individuals living in endemic areas. Moreover human antibodies either purified on the recombinant protein, or on the synthetic peptide MSP-3b, as well as antibodies raised in mice, were all found to promote parasite killing mediated by monocytes.

Development of a vaccine strategy against human and bovine schistosomiasis: background and update

Schistosomiasis is a chronic and debilitating parasitic disease that affects over 200 million people throughout the world and causes about 500,000 deaths annually. Two specific characteristics of schistosome infection are of primordial importance to the development of a vaccine: schistosomes do not multiply within the tissues of their definitive hosts (unlike protozoan parasites) and a partial non-sterilizing immunity can have a marked effect on the incidence of pathology and on disease transmission. Since viable eggs are the cause of disease pathology, a reduction in worm fecundity whether or not accompanied by a reduction in parasite burden is a sufficient goal for vaccine induced immunity. We originally showed that IgE antibodies played in experimental models a pivotal role for the development of protective immunity. These laboratory findings have been now confirmed in human populations. Following the molecular cloning and expression of a protein 28 kDa protein of Schistosoma mansoni and its identification as a glutathion S-transferase, immunization experiments have been undertaken in several animal species (rats, mice, baboons). Together with a significant reduction in parasite burden, vaccination with Sm28 GST was recently shown to reduce significantly parasite fecundity and egg viability leading to a decrease in liver pathology. Whereas IgE antibodies were shown to be correlated with protection against infection, IgA antibodies have been identified as one of the factors affecting egg laying and viability. In human populations, a close association was found between IgA antibody production to Sm28 GST and the decrease of egg output. The use of appropriate monoclonal antibody probes has allowed the demonstration that the inhibition of parasite fecundity following immunization was related to the inhibition of enzymatic activity of the molecule. Epitope mapping of Sm28 GST has indicated the prominent role of the N and C terminal domains. Immunization with the corresponding synthetic peptides was followed by a decrease of 70% of parasite fecundity and egg viability. As a preliminary step towards phase I human trials, vaccination experiments have been performed in cattle, a natural model for Schistosoma bovis. Vaccination of calves with the S. bovis GST has led to a reduction of ever 80% of egg output and tissue egg count. Significant levels of protection were also observed in goats after immunization with the recombinant S. bovis GST. Increasing evidence of the participation of IgA antibodies in protective immunity has prompted us toward the development of mucosal immunization. Preliminary results indicate that significant levels of protection can be achieved following oral immunization with live attenuated vectors or liposomes. These studies seem to represent a promising approach towards the future development of a vaccine strategy against one of major human parasitic diseases.

Use of the 2,3-diacyl-trehalose and the purified protein derivative in the serodiagnosis of tuberculosis in AIDS

The effect of the human immunodeficiency virus (HIV) infection on IgG production against purified protein derivative (PPD) and 2,3-diacil-trehalose (SL-IV) was investigated by an enzyme-linked immunosorbent assay (ELISA) test. Comparison between the antigens showed that immunocompetent patients produce preferentially antibodies to SL-IV than to PPD (73.3% versus 63.3%). Combination of these results showed an increase of the sensitivity to 80%, which decreased over the spectrum of immunodepression caused by HIV. In the tuberculous HIV seropositive group the sensitivities of SL-IV and PPD were 36.4% versus 40% and 0% versus 22.2% in the tuberculosis/acquired immunodeficiency syndrome (TB/AIDS) group. Combination of these results gave respectively 54.5% and 20%, showing that serological tests have limited value for diagnosis of tuberculosis in HIV infected patients. High antibody levels were observed in HIV seropositive asymptomatic group, but only two individuals were positive for both antigens. In the follow up, one of them developed tuberculous lymphadenitis, indicating that further work is needed to access the value of serological tests in predicting tuberculosis in HIV infected individuals.

The serological differentiation of acute and chronic schistosomiasis japonica using IgA antibody to egg antigen

Two groups of Schistosoma japonicum infected patients (acute and chronic ) and non-infected individuals were studied using IgA antibody to egg antigen (SEA) and IgG and IgM antibodies to keyhole limpet haemocyanin (KLH). The means and standard deviation of the optical density in ELISA of acute, chronic and negative groups for IgA anti-SEA were 583±124.7, 98.2±78.8 and 82.2±39. 3, respectively. There was a statistically significance between acute patients and chronic patients (P<0.01). The means and standard deviation of IgG and IgM antibodies to KLH were 501.5±150.6, 113.0±79.1, 28.8±56.3 and 413.6±148.5, 70.2±14.8, 65.3±45.3, respectively. The detection results of IgA to SEA compared with the IgG and IgM to KLH did not demonstrate a significant difference (P>0.01). The sensitivities of IgA to SEA and IgG and IgM antibodies to KLH for the detection of acute infection were 95.24%, 90.48% and 85.71%, respectively. Therefore, this study showed that the detection of IgA to SEA is also a useful new method for the serological differentiation of acute and chronic schistosomiasis japonica in humans.