A novel nucleotide-containing antigen for human blood γδ T lymphocytes

The stimulation of human γδ T cells by mycobacteria occurs through recognition of four distinct nonpeptide phosphorylated antigens termed TUBag1–4. Among these latter, TUBag4 has already been biochemically characterized as a γ-X derivative of 5′-deoxythymidine triphosphate (Constant, P., Davodeau, F., Peyrat, M. A., Poquet, Y., Puzo, G., Bonneville, M. and Fournié, J.-J., Science 1994. 264: 267). However, despite chemical synthesis of weakly stimulatory nucleotide-containing analogs, these mycobacterial compounds remained the sole nucleotide-containing antigens actually isolated from natural sources. Here, we present the complete isolation of the TUBag3 antigen from Mycobacterium fortuitum and demonstrate that this nonpeptide molecule contains a 5′-UTP nucleotide moiety. On selected Vγ9/Vδ2 clones, T cell responses can be triggered with nanomolar concentrations of TUBag3. Like crude mycobacterial extracts, this purified nucleotide conjugate elicits a strong polyclonal response of γδ PBL from healthy donors. Furthermore, we present evidence that this compound is distinct from the recently synthesized γ-isopentenyl 5′-UTP, a nucleotide conjugate of isopentenyl pyrophosphate that was found to be stimulatory for human γδ T cells (Tanaka, Y., Morita, C. T., Tanaka, Y., Nieves, E., Brenner, M. B. and Bloom, B. R., Nature 1995. 375: 155). Since it appears that both mycobacterial nucleotide antigens are molecules structurally related to peculiar precursors of nucleic acid synthesis, we propose that TUBag-reactive T cells might be specifically devoted to surveillance of proliferating cells.     

The modulation of the Ne-like wave on correct responses foreshadows errors

In reaction time (RT) tasks, event-related potentials (ERPs) reveal a response-locked negative wave when subjects commit errors. This wave, termed "error negativity" (Ne) or "error-related negativity" (ERN), is thought to index response-monitoring processes. With conventional monopolar recordings, this negativity is hardly seen on correct responses, likely overlapped by a large positive wave. Indeed, after Laplacian transformation (a spatial high-pass filter), a small Ne-like wave is unmasked. Recently, it has been shown that the positivity on monopolar recordings was larger for correct responses preceding an error than for correct responses preceding a correct trial. After Laplacian transformation, it appears that this effect is due, at least in part, to a decrease of the Ne-like wave on correct responses preceding an error. This result indicates that, as the Ne on errors, the Ne-like wave on correct responses is sensitive to performance and hence is likely related to response-monitoring processes.     

Investigations on the biology,epidemiology, pathology and control of<Emphasis Type="Italic"> Tunga penetrans</Emphasis> in Brazil: I. Natural history of tungiasis in man

Tungiasis is an important health problem in poor communities in Brazil and is associated with severe morbidity, particularly in children. The causative agent, the female flea Tunga penetrans, burrows into the skin of its host, where it develops, produces eggs and eventually dies. From the beginning of the penetration to the elimination of the carcass of the ectoparasite by skin repair mechanisms, the whole process takes 4-6 weeks. The present study is based on specimens from 86 patients, for some of whom the exact time of penetration was known. Lesions were photographed, described in detail and biopsied. Biopsies were examined histologically and by means of scanning electron microscopy (SEM). Based on clinical, SEM and histological findings, the "Fortaleza classification" was elaborated. This allows the natural history of tungiasis to be divided into five stages: (1) the penetration phase, (2) the phase of beginning hypertrophy, (3) the white halo phase, (4) the involution phase and (5) residues in the host's skin. Based on morphological and functional criteria, stages 3 and 4 are divided into further substages. The proposed Fortaleza classification can be used for clinical and epidemiological purposes. It allows a more precise diagnosis, enables the assessment of chemotherapeutic approaches and helps to evaluate control measures at the community level.     

ATP-sensitive K+ and Ca2+-activated K+ channels in lamprey ( Petromyzon marinus) red blood cell membrane

The patch-clamp technique was used to demonstrate the presence of ATP-sensitive K(+) channels and Ca(2+)-activated K(+) channels in lamprey ( Petromyzon marinus) red blood cell membrane. Whole-cell experiments indicated that the membrane current under isosmotic (285 mosmol l(-1)) conditions is carried by K(+). In the inside-out configuration an ATP-sensitive K(+) channel (70-80 pS inward, 35-40 pS outward) was present in 35% of patches. Application of ATP to the intracellular side reduced unitary current with half-maximal inhibition in the range 10-100 microM. A block was obtained with 100 microM lidocaine and inhibition was obtained with 0.5 mM barium acetate. A Ca(2+)-activated K(+) channel (25-30 pS inward, 10-15 pS outward) was present in 57% of patches. Inhibition was produced by 10 mM TEA and 500 nM apamin and sensitivity to Ba(2+) was lower than for ATP-sensitive channels. No spontaneous channel activity was recorded in the cell-attached configuration under isotonic conditions. With hypotonic saline 68% of patches showed spontaneous single-channel activity, and, of 75 active patches, 66 cell-attached patches showed channel activity corresponding to Ca(2+)-activated K(+) channels.     

Probing the coupling of Ca2+ and rigor activation of rabbit psoas myofibrillar ATPase with ethylene glycol

We have exploited solvent perturbation to probe the coupling of Ca²⁺ and rigor activation of the ATPase of myofibrils from rabbit psoas. Three techniques were used: overall myofibrillar ATPases by the rapid-flow quench method; kinetics of the interaction of ATP with myofibrils by fluorescence stopped-flow; and myofibrillar shortening by optical microscopy. Because of its extensive use with muscle systems, ranging from myosin subfragment-1 to muscle fibres, we chose 40% ethylene glycol as the relaxing agent. At 4°C, the glycol had little effect on the myofibrillar ATPase at low [Ca²⁺], but at high [Ca²⁺] the activity was reduced 50-fold, close to the level found under relaxing conditions, and there was no shortening. However, the ATPase of chemically cross-linked myofibrils (permanently activated even without Ca²⁺) was reduced only 3–4-fold. The lesser reduction of the ATPase of permanently activated myofibrils was also observed in single turnover experiments in which activation occurs by a few heads in the rigor state activating the remaining heads. The addition of ADP, which also promotes strong head-thin filament interactions, also activated the ATPase but only in the presence of Ca²⁺. Further experiments revealed that in 40% ethylene glycol, Ca²⁺ does initiate shortening but only with the aid of strong interactions and at temperatures above 15°C. This confirms that in the organized and intact myofibril, Ca²⁺ and rigor activation are coupled, as proposed previously for regulated actomyosin subfragment-1. This revised version was published online in September 2006 with corrections to the Cover Date.     

Frequent carriage of Panton-Valentine leucocidin genes by Staphylococcus aureus isolates from surgically drained abscesses

Between 1 February and 15 April 2002, 95 patients were admitted to Gaston Bourret Territorial Hospital (New Caledonia, France) for drainage of community-acquired soft tissue abscesses. Staphylococcus aureus was detected in 68 cases (72%). Two-thirds of the patients with S. aureus infection had furuncles, which were located on the limbs in 82% of cases. The median interval between symptom onset and hospital admission was 5.7 days. Three-quarters of the patients were Melanesians living in tribes. Fifty-four S. aureus isolates were screened for toxin genes. Panton-Valentine leucocidin (PVL) genes were detected in 48 isolates (89%), the exfoliative toxin A gene was detected in 1 isolate, and no toxin genes were detected in 4 isolates. S. aureus nasal carriage was detected in 39.7% of patients with S. aureus infections. Two infecting S. aureus strains and two nasal carriage strains were resistant to methicillin. Comparative pulsed-field gel electrophoresis, performed in 16 cases, showed that five of six patients with PVL-positive nasal carriage strains were infected by the same strains. In contrast, 8 of 10 patients with PVL-negative nasal carriage strains were infected by PVL-positive strains. PVL genes thus appear to be a major virulence factor in both primary and secondary S. aureus skin infections.     

Modulation of Whole-Cell Currents in Plasmodium Falciparum-Infected Human Red Blood Cells by Holding Potential and Serum

Recent electrophysiological studies have identified novel ion channel activity in the host plasma membrane of Plasmodium falciparum -infected human red blood cells (RBCs). However, conflicting data have been published with regard to the characteristics of induced channel activity measured in the whole-cell configuration of the patch-clamp technique. In an effort to establish the reasons for these discrepancies, we demonstrate here two factors that have been found to modulate whole-cell recordings in malaria-infected RBCs. Firstly, negative holding potentials reduced inward currents (i.e. at negative potentials), although this result was highly complex. Secondly, the addition of human serum increased outward currents (i.e. at positive potentials) by approximately 4-fold and inward currents by approximately 2-fold. These two effects may help to resolve the conflicting data in the literature, although further investigation is required to understand the underlying mechanisms and their physiological relevance in detail.     

Strategy for diagnosis of congenital toxoplasmosis: evaluation of methods comparing mothers and newborns and standard methods for postnatal detection of immunoglobulin G, M, and A antibodies

In a study involving 14 laboratories supported by the European Community Biomed 2 program, we evaluated immunologic methods for the postnatal diagnosis of congenital toxoplasmosis (CT). Among babies born to mothers who seroconverted to positivity for toxoplasmosis during pregnancy, we analyzed 55 babies with CT on the basis of persistent anti-Toxoplasma immunoglobulin G (IgG) at 1 year of life and 50 control babies without anti-Toxoplasma IgG at 1 year of life in the absence of curative treatment with pyrimethamine-sulfonamides. We tested in-house methods such as the enzyme-linked immunofiltration assay (ELIFA) or Immunoblotting (IB) for the detection of IgG or IgM; these methods allowed comparison of the immunologic profiles of the mothers and the infants. We compared ELIFA and IB with a commercial enzyme immunoassay (EIA) or in-house immunosorbent agglutination assay (ISAGA) for the detection of IgM or IgA. The performances of combinations of methods were also assessed. A cumulative sensitivity of 98% during a 1-year follow-up was obtained with the ELIFA plus ISAGA combination. Only one case of CT was missed by the ELIFA plus ISAGA combination, whereas three cases were missed by the IB plus ISAGA combination, even though 48% of patients with CT were treated with pyrimethamine-sulfonamides, which are known to inhibit antibody neosynthesis. A similar performance was obtained with either ELIFA or IB in combination with EIA. The difference in performance between ELIFA plus ISAGA and IB plus ISAGA was not statistically significant (P = 0.31), and we conclude that both combinations of tests can be used for the diagnosis of CT in newborns.     

Human TLR-7-, -8-, and -9-mediated induction of IFN-alpha/beta and -lambda Is IRAK-4 dependent and redundant for protective immunity to viruses

Five TLRs are thought to play an important role in antiviral immunity, sensing viral products and inducing IFN-alpha/beta and -lambda. Surprisingly, patients with a defect of IRAK-4, a critical kinase downstream from TLRs, are resistant to common viruses. We show here that IFN-alpha/beta and -lambda induction via TLR-7, TLR-8, and TLR-9 was abolished in IRAK-4-deficient blood cells. In contrast, IFN-alpha/beta and -lambda were induced normally by TLR-3 and TLR-4 agonists. Moreover, IFN-beta and -lambda were normally induced by TLR-3 agonists and viruses in IRAK-4-deficient fibroblasts. We further show that IFN-alpha/beta and -lambda production in response to 9 of 11 viruses tested was normal or weakly affected in IRAK-4-deficient blood cells. Thus, IRAK-4-deficient patients may control viral infections by TLR-3- and TLR-4-dependent and/or TLR-independent production of IFNs. The TLR-7-, TLR-8-, and TLR-9-dependent induction of IFN-alpha/beta and -lambda is strictly IRAK-4 dependent and paradoxically redundant for protective immunity to most viruses in humans.