Biochemical characterization of an Fc receptor of rabbit lymphocytes

Isolation of Fc-binding molecules was performed by complexing IgG antibodies and the corresponding antigens in lysates of radiolabeled rabbit lymphoid cells. A single-chain molecule of 110 000 apparent mol. wt. (unreduced) or 120 000 (reduced) was observed in sodium dodecyl sulfate polyacrylamide gel electrophoresis when the antibodies were in the IgG form but not as F(ab')2. This molecule was very susceptible to proteolysis, but the fragments thus produced remained associated by disulfide bridges, and the binding properties of the molecule were conserved. The molecule and its proteolytic fragments (mol. wts. 75 000, 45 000 and 20 000) were very similar to those obtained for the mouse Fc receptor under similar conditions, and therefore the molecule was designated as rabbit Fc receptor. Among several precipitating systems used, some Ig-anti-Ig complexes appeared to be the most efficient in coprecipitating the rabbit Fc receptor. Remarkably high titers of Fc receptor were found on lymphocyte membrane of infected rabbits, in agreement with a possible role of this molecule in immune regulation.     

Comparative evaluation of the new version of the INNO-LiPA Mycobacteria and genotype Mycobacterium assays for identification of Mycobacterium species from MB/BacT liquid cultures artificially inoculated with Mycobacterial strains

The performance of two DNA line probe assays, a new version of INNO-LiPA Mycobacteria (Innogenetics, Ghent, Belgium) and the GenoType Mycobacterium (Hain Diagnostika, Nehren, Germany), were evaluated for identification of mycobacterial species isolated from liquid cultures. Both tests are based on a PCR technique and designed for simultaneous identification of different mycobacterial species by reverse hybridization and line probe technology. The INNO-LiPA Mycobacteria v2 targeting the 16S-23S rRNA gene spacer region was developed for the simultaneous identification of 16 different mycobacterial species. The GenoType Mycobacterium, which targets the 23S rRNA gene, allows simultaneous identification of 13 mycobacterial species. Both tests were evaluated on 110 mycobacterial strains belonging to 22 different mycobacterial species (20 reference strains, 83 clinical strains, and 4 Mycobacterium kansasii strains isolated from tap water) that were previously inoculated into MB/BacT bottles. The sensitivity of both methods, defined as the number of positive results obtained with the Mycobacterium genus probe together with an interpretable result on the number of samples tested was 110 of 110 (100%) for INNO-LiPA and 102 of 110 (92.7%) for GenoType. For samples with interpretable results, INNO-LiPA was able to correctly identify 109 of 110 samples (99.1%), whereas the GenoType correctly identified 100 of 102 samples (98.0%). Both tests were easy to perform, rapid, and reliable when applied to mycobacterial identification directly from MB/BacT bottles.     

Impairment of a cortical event-related desynchronisation during a bimanual load-lifting task in children with autistic disorder

In autism, the abilities of communication are affected, associated with abnormalities of cognitive, sensorial and motor development. In a previous study based on a load-lifting task, we showed impairment of anticipation in children with autism as evidenced by kinematics and eletromyographic recordings [Neurosci. Lett. 348 (2003) 17]. In the present study, we assessed the cortical counterparts of the use of anticipatory postural adjustments in a group of control children and in a group of children with autism. The tasks required maintaining a stable forearm position despite imposed or voluntary lifting of an object placed either on the controlateral forearm or on a support. We investigated the differences between the two groups of children on the Event-Related Desynchronisation (ERD) which precedes movement onset in adults [Electroencephalogr. Clin. Neurophysiol. 46 (1979) 138]. Electroencephalogram (EEG) power evolution of a 6-8-Hz frequency band was averaged before and after imposed or voluntary movement onset. EEG reactivity of control and autistic children did not differ during the imposed unloading condition, but significant differences appeared in the voluntary unloading situations. Before lifting the object, control children showed an ERD above the left motor areas. An ERD also occurred above the right motor areas when the object was placed on their forearm. This indicates that the ERD can also translate the use of anticipatory postural adjustments. By contrast, children with autism did not show an ERD in the two voluntary situations. This suggests a central deficit of anticipation in both postural and motor control in children with autism.     

Elevated levels of DNA-protein crosslinks and micronuclei in peripheral lymphocytes of tannery workers exposed to trivalent chromium

DNA-protein crosslinks (DPC) are a promising biomarker of exposure to hexavalent chromium, a known human carcinogen. Although trivalent chromium is considered to have much lower toxicity, the risk involved in chronic exposure is uncertain. DPC may be a useful tool in clarifying this risk, by signaling an exposure of body tissues to biologically active forms of chromium. DPC quantification was carried out in lymphocytes of a group of tannery workers exposed to trivalent chromium, a small group of manual metal arc stainless steel welders exposed to hexavalent chromium and a control group. This biomarker was compared with the frequency of micronuclei in cytokinesis blocked peripheral lymphocytes as a biomarker of cytogenetic lesions and total plasma and urine chromium levels as an index of exposure. The results indicate a significant increase in the formation of DPC in tannery workers compared with controls (0.88 +/- 0.19 versus 0.57 +/- 0.21%, P < 0.001, Mann-Whitney test) and an even higher level of DPC in welders (2.22 +/- 1.12%, P = 0.03). Tanners showed a significant increase in micronucleated cells compared with controls (6.35 +/- 2.94 versus 3.58 +/- 1.69 per thousand, P < 0.01), whereas in welders this increase was not significant (5.40 +/- 1.67 per thousand ). Urinary chromium was increased in both groups, with a greater increase observed in tanners compared with controls (2.63 +/- 1.62 versus 0.70 +/- 0.38 microg/g creatinine, P < 0.001) than in welders (1.90 +/- 0.37 microg/g creatinine, P < 0.005). Plasma chromium was also increased in both groups (tanners 2.43 +/- 2.11 microg/l, P < 0.001, welders 1.55 +/- 0.67 microg/l, P < 0.005 versus controls 0.41 +/- 0.11 microg/l). In summary, chronic occupational exposure to trivalent chromium can lead to a detectable increase in lymphocyte DNA damage which correlates with a significant exposure of the cells to the metal.     

Compilation of published comparative genomic hybridization studies

The power of comparative genomic hybridization (CGH) has been clearly proven since the first paper appeared in 1992 as a tool to characterize chromosomal imbalances in neoplasias. This review summarizes the chromosomal imbalances detected by CGH in solid tumors and in hemopathies. In May of 2001, we took a census of 430 articles providing information on 11,984 cases of human solid tumors or hematologic malignancies. Comparative generic hybridization has detected a number of recurrent regions of amplification or deletion that allows for identification of new chromosomal loci (oncogenes, tumor suppressor genes, or other genes) involved in the development, progression, and clonal evolution of tumors. When CGH data from different studies are combined, a pattern of nonrandom genetic aberrations appears. As expected, some of these gains and losses are common to different types of pathologies, while others are more tumor-specific.     

Use of conventional or replicating nucleic acid-based vaccines and recombinant Semliki forest virus-derived particles for the induction of immune responses against hepatitis C virus core and E2 antigens

Replicating and nonreplicating nucleic acid-based vaccines as well as Semliki Forest-recombinant Viruses (rSFVs) were evaluated for the development of a vaccine against hepatitis C virus (HCV). Replicating SFV-DNA vaccines (pSFV) and rSFVs expressing HCV core or E2 antigens were compared with classical CMV-driven plasmids (pCMV) in single or bimodal vaccine protocols. In vitro experiments indicated that all vaccine vectors produced the HCV antigens but to different levels depending on the antigen expressed. Both replicating and nonreplicating core-expressing plasmids induced, upon injection in mice, specific comparable CTL responses ranging from 10 to 50% lysis (E:T ratio 100:1). Comparison of different injection modes (intramuscular versus intraepidermal) and the use of descalating doses of DNA (1-100 microgram) did not show an increased efficacy of the core-SFV plasmid compared with the CMV-driven one. Surprisingly, rSFVs yielded either no detectable anticore CTL or very low anti-E2 antibody titers following either single or bimodal administration together with CMV-expressing counterparts. Prime-boost experiments revealed, in all cases, the superiority of DNA-based only vaccines. The anti-E2 antibody response was evaluated using three different assays which indicated that all generated anti-E2 antibodies were targeted at similar determinants. This study emphasizes the potential of DNA-based vaccines for induction of anti-HCV immune responses and reveals an unexpected and limited benefit of SFV-based vaccinal approaches in the case of HCV core and E2.     

Nonspecific immunodepression and protective immunity in mice infected with Leishmania mexicana.

C57BL/6 mice infected with Leishmania mexicana showed depression of the in vitro immunoglobulin M-plaque-forming cell response to sheep erythrocytes. Immunodepression was present 3 weeks after inoculation and was maximal at 11 weeks. Thereafter, there was a gradual return to normal immunoresponsiveness correlated with the resolution of lesions. At the time of maximal immunodepression, spleen cells from infected mice diminished the plaque-forming cell response to sheep erythrocytes of normal spleen cells. On the other hand, specific responses, as exemplified by protective immunity to a challenge infection and delayed hypersensitivity responses to parasite antigens, were apparently unaffected. These responses were both present in mice bearing primary lesions and were maximal in recovered mice. The significance of these findings is discussed in relation to a current hypothesis on parasite-induced immunodepression.     

Coxsackievirus B3 replication and persistence in intestinal cells from mice infected orally and in the human CaCo-2 cell line

Although the transmission of coxsackievirus B3 occurs mainly via the oral route, little is known about the primary replication and persistence of this agent in the intestine. To address this question, BALB/c mice were inoculated by gavage with coxsackievirus B3, Nancy strain. The mice were killed from 1 hr to 90 days after infection. The viral markers were detected in the small intestine using RT-PCR, cell culture and detection of VP1 protein. Coxsackievirus B3 was detected positive by the three methods from hr 2 to day 45 after infection. By using monoclonal antibodies directed towards VP1, CD40 and CD26, the virus was shown to be present in the lymphocytes of the mucosa as soon as 2 hr after infection; in contrast, no virus was detected in the epithelial cells lining the intestinal lumen. Further experiments were performed to evaluate the capacity of coxsackievirus B3 to establish a persistent infection in two intestinal cell lines. In contrast to HT29 cells, the CaCo-2 cells were shown to develop a persistent infection for up to 20 passages, as demonstrated by the detection of viral RNA and VP1 protein. This study provides further evidence that, after infection by the oral route, the viral particles are concentrated in the lymphocytes of the mucosal layer. In addition, the results suggest that coxsackievirus B3 is capable of establishing a persistent infection in the small intestine that may act as a reservoir of viral particles for the delayed spread of the virus to other target organs.     

Contribution of the twin arginine translocation system to the virulence of enterohemorrhagic Escherichia coli O157:H7

Shiga toxin-producing Escherichia coli O157:H7 is a major food-borne infectious pathogen. In order to analyze the contribution of the twin arginine translocation (TAT) system to the virulence of E. coli O157:H7, we deleted the tatABC genes of the O157:H7 EDL933 reference strain. The mutant displayed attenuated toxicity on Vero cells and completely lost motility on soft agar plates. Further analyses revealed that the ΔtatABC mutation impaired the secretion of the Shiga toxin 1 (Stx1) and abolished the synthesis of H7 flagellin, which are two major known virulence factors of enterohemorrhagic E. coli O157:H7. Expression of the EDL933 stxAB₁ genes in E. coli K-12 conferred verotoxicity on this nonpathogenic strain. Remarkably, cytotoxicity assay and immunoblot analysis showed, for the first time, an accumulation of the holotoxin complex in the periplasm of the wild-type strain and that a much smaller amount of StxA₁ and reduced verotoxicity were detected in the ΔtatC mutant cells. Together, these results establish that the TAT system of E. coli O157:H7 is an important virulence determinant of this enterohemorrhagic pathogen.