Immunogens of interest for the diagnosis of Campylobacter jejuni infections

In order to identify the C. jejuni immunogens of interest for the diagnosis of Campylobacter infections, we analyzed the humoral response of 153 patients by using complement fixation (CF) and western blot assays. A first group of 79 sera was from C. jejuni infected patients suffering from enteritis (n=16), Guillain-Barré syndrome (GBS) (n=40) and arthritis (n=23). A second group of 49 sera was from healthy blood donors and a third group consisted of 25 sera from children under 4 years old. Using the CF test, 88.6% of the C. jejuni infected patients were seropositive versus 28.5% of the healthy blood donors and none of the children. The Western blot assay allowed detection of antibodies directed against seven selected antigens ranging from 14 to 67 kDa. Three of these antigens with a molecular size of 29, 37 and 43 kDa were detected by 86.0%, 84.8% and 91.1% of the C. jejuni infected patients, respectively. These three antigens seem to be good candidates for the development of assays suitable for direct and indirect diagnosis of Campylobacter infections.     

Human Immunodeficiency Virus Type 1 Nef Protein Targets CD4 to the Multivesicular Body Pathway

The Nef protein of human immunodeficiency virus type 1 downregulates the CD4 coreceptor from the surface of host cells by accelerating the rate of CD4 endocytosis through a clathrin/AP-2 pathway. Herein, we report that Nef has the additional function of targeting CD4 to the multivesicular body (MVB) pathway for eventual delivery to lysosomes. This targeting involves the endosomal sorting complex required for transport (ESCRT) machinery. Perturbation of this machinery does not prevent removal of CD4 from the cell surface but precludes its lysosomal degradation, indicating that accelerated endocytosis and targeting to the MVB pathway are separate functions of Nef. We also show that both CD4 and Nef are ubiquitinated on lysine residues, but this modification is dispensable for Nef-induced targeting of CD4 to the MVB pathway.Primate immunodeficiency viruses infect helper T lymphocytes and cells of the macrophage/monocyte lineage by binding of their viral envelope glycoprotein, Env, to a combination of two host cell-specific surface proteins, CD4 and either the CCR5 or CXCR4 chemokine receptors (reviewed in reference 62). Ensuing fusion of the viral envelope with the host cell plasma membrane delivers the viral genetic material into the cytoplasm. Remarkably, the most highly transcribed viral gene in the early phase of infection does not encode an enzyme or structural protein but an accessory protein named Nef. Early expression of Nef is thought to reprogram the host cell for optimal replication of the virus. Indeed, Nef has been shown to enhance virus production (19, 24, 59, 74) and to promote progression to AIDS (23, 47, 48), making it an attractive candidate for pharmacologic intervention.Nef is an N-terminally myristoylated protein with a molecular mass of 27 kDa for human immunodeficiency virus type 1 (HIV-1) and 35 kDa for HIV-2 and simian immunodeficiency virus (27, 29, 50, 65). Nef has been ascribed many functions, the best characterized of which is the downregulation of the CD4 coreceptor from the surface of infected cells (28, 35, 57). CD4 downregulation is believed to prevent superinfection (8, 52) and to preclude the cellular retention of newly synthesized Env (8, 49), thus allowing the establishment of a robust infection (30, 71).The molecular mechanism by which Nef downregulates CD4 has been extensively studied. A consensus has emerged that Nef accelerates the endocytosis of cell surface CD4 (2, 64) by linking the cytosolic tail of CD4 to the heterotetrameric (α-β2-μ2-σ2) adaptor protein-2 (AP-2) complex (17, 25, 34, 45, 67). Determinants in the CD4 tail bind to a hydrophobic pocket comprising tryptophan-57 and leucine-58 on the folded core domain of Nef (34). On the other hand, a dileucine motif (i.e., ENTSLL, residues 160 to 165) (14, 22, 32) and a diacidic motif (i.e., DD, residues 174 and 175) (3) (residues correspond to the NL4-3 clone of HIV-1) within a C-terminal, flexible loop of Nef bind to the α and σ2 subunits of AP-2 (17, 18, 25, 51). AP-2, in turn, binds to clathrin, leading to the concentration of CD4 within clathrin-coated pits (15, 33). These pits eventually bud from the plasma membrane as clathrin-coated vesicles that deliver internalized CD4 to endosomes. In essence, then, Nef acts as a connector that confers on CD4 the ability to be rapidly internalized in a manner similar to endocytic receptors (75).Unlike typical endocytic recycling receptors like the transferrin receptor or the low-density lipoprotein receptor, however, CD4 that is forcibly internalized by Nef does not return to the cell surface but is delivered to lysosomes for degradation (4, 64, 68). Thus, expression of Nef decreases both the surface and total levels of CD4. What keeps internalized CD4 from returning to the plasma membrane? We hypothesized that Nef might additionally act on endosomes to direct CD4 to lysosomes. This is precisely the fate followed by signaling receptors, transporters, and other transmembrane proteins that undergo ubiquitination-mediated internalization and targeting to the multivesicular body (MVB) pathway (40, 46). This targeting involves the endosomal sorting complex required for transport (ESCRT), including the ESCRT-0, -I, -II, and -III complexes, which function to sort ubiquitinated cargoes into intraluminal vesicles of MVBs for eventual degradation in lysosomes (40, 46). Herein, we show that Nef indeed plays a novel role in targeting internalized CD4 from endosomes to the MVB pathway in an ESCRT-dependent manner. We also show that both Nef and CD4 undergo ubiquitination on lysine residues, but, strikingly, this modification is not required for CD4 targeting to the MVB pathway.     

TIP47 is Required for the Production of Infectious HIV‐1 Particles from Primary Macrophages

Macrophages are among the major targets of HIV‐1 infection and play a key role in viral pathogenesis. Identification of the cellular cofactors involved in the production of infectious HIV‐1 from macrophages is thus crucial. Here, we investigated the role of the cellular cofactor TIP47 in HIV‐1 morphogenesis in primary macrophages. Using siRNA approach, we show that TIP47 is essential for HIV‐1 infectivity and propagation. TIP47 silencing disrupts Gag and Env colocalization in macrophages. Moreover, mutations in HIV‐1 Gag or Env, which abolish interaction with TIP47, impair HIV‐1 propagation and infectivity preventing colocalization of Gag and Env, Gag and Env coimmunoprecipitation. Interestingly, disruption of Gag‐TIP47 interaction by matrix mutation or TIP47 depletion also causes Gag to localize in scattered dots in the vicinity of the plasma membrane of macrophages. Therefore, TIP47 is required for the encounter between Gag and Env, and thus for the generation of infectious HIV‐1 particles from primary macrophages.     

Neuropilin-1 is involved in human T-cell lymphotropic virus type 1 entry

Human T-cell lymphotropic virus type 1 (HTLV-1) is transmitted through a viral synapse and enters target cells via interaction with the glucose transporter GLUT1. Here, we show that Neuropilin-1 (NRP1), the receptor for semaphorin-3A and VEGF-A165 and a member of the immune synapse, is also a physical and functional partner of HTLV-1 envelope (Env) proteins. HTLV-1 Env and NRP1 complexes are formed in cotransfected cells, and endogenous NRP1 contributes to the binding of HTLV-1 Env to target cells. NRP1 overexpression increases HTLV-1 Env-dependent syncytium formation. Moreover, overexpression of NRP1 increases both HTLV-1 and HTLV-2 Env-dependent infection, whereas down-regulation of endogenous NRP1 has the opposite effect. Finally, overexpressed GLUT1, NRP1, and Env form ternary complexes in transfected cells, and endogenous NRP1 and GLUT1 colocalize in membrane junctions formed between uninfected and HTLV-1-infected T cells. These data show that NRP1 is involved in HTLV-1 and HTLV-2 entry, suggesting that the HTLV receptor has a multicomponent nature.     

Study of the pectoral girdle and fins of the Late Carboniferous sibyrhynchid iniopterygians (Vertebrata,Chondrichthyes, Iniopterygia) from Kansas and Oklahoma (USA) by means of microtomography,with comments on iniopterygian relationships

The latest works on iniopterygians question their monophyly when considering only the neurocranium of the two families (Sibyrhynchidae and Iniopterygidae), which have different conditions of preservation. Some of the synapomorphies of the Iniopterygia concern the pectoral girdle and fins. However, the anatomy of these different elements is still poorly known in this taxon. Here we describe in details three dimensionally preserved cartilages of the pectoral girdle and fins of the sibyrhynchid Iniopera sp. These structures have been extracted virtually from phosphatised nodules thanks to conventional and synchrotron microtomography, using absorption and phase contrast based techniques in the later case. The pectoral girdle of Iniopera sp. consists of three elements, which are, from dorsal to ventral, a paired suprascapular cartilage, a pair of robust scapulocoracoids and an unpaired intercoracoid cartilage. The scapular part of the scapulocoracoids is extremely reduced and the suprascapular cartilages link the scapulcoracoids to the rear of the neurocranium. These characters may be iniopterygian synapomorphies. Iniopterygians, stem and crown-holocephalans share a basipterygium that articulates with the pectoral girdle and bears an enlarged first pectoral fin radial. Posteriorly, the basipterygium articulates with either a well-defined metapterygium (in crown-holocephalans) or a metapterygial axis (in stem-holocephalans).     

Beclin‐1 is required for chromosome congression and proper outer kinetochore assembly

The functions of Beclin‐1 in macroautophagy, tumorigenesis and cytokinesis are thought to be mediated by its association with the PI3K‐III complex. Here, we describe a new role for Beclin‐1 in mitotic chromosome congression that is independent of the PI3K‐III complex and its role in autophagy. Beclin‐1 depletion in HeLa cells leads to a significant reduction of the outer kinetochore proteins CENP‐E, CENP‐F and ZW10, and, consequently, the cells present severe problems in chromosome congression. Beclin‐1 associates with kinetochore microtubules and forms discrete foci near the kinetochores of attached chromosomes. We show that Beclin‐1 interacts directly with Zwint‐1—a component of the KMN (KNL‐1/Mis12/Ndc80) complex—which is essential for kinetochore–microtubule interactions. This suggests that Beclin‐1 acts downstream of the KMN complex to influence the recruitment of outer kinetochore proteins and promotes accurate kinetochore anchoring to the spindle during mitosis.     

High Prevalence of Cysticercosis in People with Epilepsy in Southern Rwanda

Background

Neurocysticercosis (NCC), the central nervous system infection by Taenia solium larvae, is a preventable and treatable cause of epilepsy. In Sub-Saharan Africa, the role of NCC in epilepsy differs geographically and, overall, is poorly defined. We aimed at contributing specific, first data for Rwanda, assessing factors associated with NCC, and evaluating a real-time PCR assay to diagnose NCC in cerebrospinal fluid (CSF).

Methodology/Principal findings

At three healthcare facilities in southern Rwanda, 215 people with epilepsy (PWE) and 51 controls were clinically examined, interviewed, and tested by immunoblot for cysticerci-specific serum antibodies. Additionally, CSF samples from PWE were tested for anticysticercal antibodies by ELISA and for parasite DNA by PCR. Cranial computer tomography (CT) scans were available for 12.1% of PWE with additional symptoms suggestive of NCC. The Del Brutto criteria were applied for NCC diagnosis. Cysticerci-specific serum antibodies were found in 21.8% of PWE and 4% of controls (odds ratio (OR), 6.69; 95% confidence interval (95%CI), 1.6–58.7). Seropositivity was associated with age and lack of safe drinking water. Fifty (23.3%) PWE were considered NCC cases (definitive, based on CT scans, 7.4%; probable, mainly based on positive immunoblots, 15.8%). In CSF samples from NCC cases, anticysticercal antibodies were detected in 10% (definitive cases, 25%) and parasite DNA in 16% (definitive cases, 44%). Immunoblot-positive PWE were older (medians, 30 vs. 22 years), more frequently had late-onset epilepsy (at age >25 years; 43.5% vs. 8.5%; OR, 8.30; 95%CI, 3.5–20.0), and suffered from significantly fewer episodes of seizures in the preceding six months than immunoblot-negative PWE.

Conclusions/Significance

NCC is present and contributes to epilepsy in southern Rwanda. Systematic investigations into porcine and human cysticercosis as well as health education and hygiene measures for T. solium control are needed. PCR might provide an additional, highly specific tool in NCC diagnosis.     

Éléments de physiologie et physiopathologie de la microcirculation et de rhéologie. Implications potentielles pour la circulation extracorporelle en chirurgie cardiaque de l’adulte

Cardiopulmonary bypass (CPB) is usually performed by defining therapeutic goals based on macro-hemodynamic parameters such as pump flow, mean arterial blood pressure, haemoglobin concentration or venous line saturation. Nevertheless, oxygen transport to tissues and cells is also dependent on the function of the microcirculation and the rheologic properties of blood. This articles summarizes for clinicians that perform CPB several aspects of physiology and pathophysiology of microcirculation and rheology. Physiological and pathophysiological changes of blood and plasma viscosities, red blood cell aggregation and deformability are explained. Potential clinical implications of changes of blood rheology are discussed.     

Structural and functional characterization of nonstructural protein 2 for its role in hepatitis C virus assembly

The hepatitis C virus (HCV) is a flavivirus replicating in the cytoplasm of infected cells. The HCV genome is a single-stranded RNA encoding a polyprotein that is cleaved by cellular and viral proteases into 10 different products. While the structural proteins core protein, envelope protein 1 (E1) and E2 build up the virus particle, most nonstructural (NS) proteins are required for RNA replication. One of the least studied proteins is NS2, which is composed of a C-terminal cytosolic protease domain and a highly hydrophobic N-terminal domain. It is assumed that the latter is composed of three trans-membrane segments (TMS) that tightly attach NS2 to intracellular membranes. Taking advantage of a system to study HCV assembly in a hepatoma cell line, in this study we performed a detailed characterization of NS2 with respect to its role for virus particle assembly. In agreement with an earlier report ( Jones, C. T., Murray, C. L., Eastman, D. K., Tassello, J., and Rice, C. M. (2007) J. Virol. 81, 8374-8383 ), we demonstrate that the protease domain, but not its enzymatic activity, is required for infectious virus production. We also show that serine residue 168 in NS2, implicated in the phosphorylation and stability of this protein, is dispensable for virion formation. In addition, we determined the NMR structure of the first TMS of NS2 and show that the N-terminal segment (amino acids 3-11) forms a putative flexible helical element connected to a stable alpha-helix (amino acids 12-21) that includes an absolutely conserved helix side in genotype 1b. By using this structure as well as the amino acid conservation as a guide for a functional study, we determined the contribution of individual amino acid residues in TMS1 for HCV assembly. We identified several residues that are critical for virion formation, most notably a central glycine residue at position 10 of TMS1. Finally, we demonstrate that mutations in NS2 blocking HCV assembly can be rescued by trans-complementation.