In vitro effects of 1-(4-dimethylaminobenzylidene)-2-(2-hydroxybenzylidene) hydrazine and its copper complex on lymphocyte proliferation and function in the presence of free radical generator.

In this study, the in vitro effects of 1-(4-dimethylaminobenzylidene)-2-(2-hydroxybenzylidene) hydrazone (L1) and its corresponding copper complex [Cu(L1)], synthesized in our laboratory, were investigated on the proliferative responses, Th1 (interleukin-2 (IL-2), interferon-γ (INFγ)) and Th2 (IL-4) cytokine secretion, adenosine triphosphate (ATP) levels and intracellular redox status of T lymphocytes submitted to H2O2/FeSO4-mediated oxidative stress. T cells were isolated on histopaque density gradient by differential centrifugation, and were cultured with the mitogen concanavalin A (Con A), free radical generator (H2O2/FeSO4) and with different concentrations of L1 and [Cu(L1)] (1 - 100 µM). Proliferation (MTT assay), cytokines (Elisa kits), ATP levels, cytotoxic effect (micronucleus test) and oxidative markers (glutathione, catalase, superoxide dismutase, hydroperoxide and carbonyl protein contents) were investigated after 48-h incubation. Our results showed that H2O2/FeSO4 treatment induced a reduction in T lymphocyte proliferation, cytokine secretion and ATP levels associated to an evident intracellular oxidative stress, inflammatory profile and DNA damage. Addition of L1 at 100 µM was able to increase cell proliferation, IL-2, IL-4 and INFγ secretion and ATP contents and to reduce hydroperoxide and carbonyl protein contents, catalase activity and micronuclei number in lymphocytes under oxidative stress, with a partial protection. The [Cu(L1)] exhibited protective effects in T lymphocytes by inhibiting H2O2/FeSO4 - induced cell proliferation suppression, inflammatory status, ATP loss and oxidative stress generation, whatever the concentration used. In conclusion, in the situation of excessive oxidative stress, [Cu(L1)] treatment improved T lymphocyte proliferation, cytokine production, ATP contents and oxidant/antioxidant status. [Cu(L1)] could be effective at improving oxidative stress and T cell abnormalities.

[Treatment of complete traumatic avulsion of an incisor tooth in adults]. / Traitement des luxations complètes traumatiques du bloc incisif chez l'adulte.

It is possible to replant an incisor tooth completely avulsed after trauma in adults. These cases are relatively frequent among athletes. It is essential to conserve the tooth in saline solution. The time before replantation must be as short as possible. The simple technique described here, which requires a minimum of material and no dental chair, makes it possible to replant an avulsed incisor with a good success rate.

T cell diversity and TcR repertoires in teleost fish.

In vertebrates, the diverse and extended range of antigenic motifs is matched to large populations of lymphocytes. The concept of immune repertoire was proposed to describe this diversity of lymphocyte receptors--IG and TR--required for the recognition specificity. Immune repertoires have become useful tools to describe lymphocyte and receptor populations during the immune system development and in pathological situations. In teleosts, the presence of conventional T cells was first proposed to explain graft rejection and optimized specific antibody production. The discovery of TR genes definitely established the reality of conventional T cells in fish. The development of genomic and EST databases recently led to the description of several key T cell markers including CD4, CD8, CD3, CD28, CTLA4, as well as important cytokines, suggesting the existence of different T helper (Th) subtypes, similar to the mammalian Th1, Th2 and Th17. Over the last decade, repertoire studies have demonstrated that both public and private responses occur in fish as they do in mammals, and in vitro specific cytotoxicity assays have been established. While such typical features of T cells are similar in both fish and mammals, the structure of particular repertoires such as the one of gut intra-epithelial lymphocytes seems to be very different. Future studies will further reveal the particular characteristics of teleost T cell repertoires and adaptive responses.

Emerging vesiculo-type virus infections of freshwater fishes in Europe.

Rhabdoviruses were isolated from perch Perca fluviatilis and largemouth bass Micropterus salmoides exhibiting clinical signs of disease. Preliminary studies indicated that these viruses could be neutralised by antisera to perch rhabdovirus (Dorson et al. 1984) and may be similar to those previously isolated from grayling Thymallus thymallus and pike-perch Stizostedion stizostedion. The relationship between these viruses and the previously characterised fish rhabdoviruses, pike fry rhabdovirus (PFRV), spring viraemia of carp virus (SVCV) and lake trout rhabdovirus, was investigated. Viruses were propagated in bluegill fry (BF-2) cells and were characterised using electron microscopy, serum neutralisation tests, immunofluorescence tests, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and nucleotide sequence analysis. The bullet-shaped viral particles appeared to be compact, with spikes visible at the surface, a morphology similar to that of the vesiculovirus group of rhabdoviruses. Serum neutralisation tests showed that the viruses were antigenically closely related to the previously characterised perch rhabdovirus, but were not significantly neutralised by antisera to PFRV, SVCV or viral haemorrhagic septicaemia virus (VHSV). In immunofluorescence tests with perch rhabdovirus antisera, strong specific fluorescence was observed in cell cultures infected with the new rhabdovirus isolates, but no fluorescence was observed with antisera to PFRV, SVCV or VHSV. SDS-PAGE analysis revealed a polypeptide profile typical of vesiculoviruses, but the novel virus isolates had different relative mobilities of their P and M proteins compared to PFRV and SVCV. Nucleotide sequence analysis was carried out using reverse transcriptase-polymerase chain reaction (RT-PCR) and DNA sequencing of a 439 base-pair region of the viral L gene. The novel rhabdovirus isolates had <76% nucleotide sequence identity to PFRV, SVCV and lake trout rhabdovirus and >95% identity to perch rhabdovirus. Phylogenetic analysis using both maximum parsimony and neighbour-joining methods assigned the perch rhaboviruses to a separate group to that of PFRV, SVCV and lake trout rhabdovirus. These data are the initial characterisation of a group of emerging fish vesiculo-type viruses that are biochemically and genetically distinct from the PFRV, SVCV and lake trout rhabdoviruses.

Phylogenetic analysis of viral haemorrhagic septicaemia virus (VHSV) isolates from France (1971-1999).

The nucleotide sequences of a specific region of the glycoprotein gene were compared among 63 strains of viral haemorrhagic septicaemia virus (VHSV) isolated from fish in France between 1971 and 1999. The analysis was performed on a region corresponding to amino acids 238 to 331 of the glycoprotein gene, also designated the V2 region and previously shown to accumulate most of the mutations. The sequences of many VHSV isolates were found to be identical or very conserved. An isolate, designated L59X, obtained from elver in the Loire estuary, depicted a higher degree of divergence compared to the other French isolates. The deduced amino-acid sequences were analysed together with the results of neutralisation tests performed using monoclonal antibody 168m4 specific to serotype 1. Non-neutralised VHSV strains had mutations in the region corresponding to the previously described 168m4 epitope. Phylogenetic analysis showed that all the VHSV isolates studied, except L59X, belong to genotype I, previously described as containing VHSV strains isolated from continental Europe. Most of the VHSV isolates studied were found to be genetically related to one of the previously described VHSV strains representative of the major serotypes. Isolate L59X, which was the only French marine strain studied, was found to belong to genotype II, previously shown to encompass the VHSV strains isolated from the British Isles coastal waters. Overall there was a good correlation between the geographical origin of the studied isolates and their genetic characteristics.

Rhabdovirus infection induces public and private T cell responses in teleost fish.

Many viruses induce a strong T cell response that contributes to the elimination of infected cells presenting viral peptides by MHC molecules. The structure and expression of genes encoding molecules homologous to mammalian alphabeta TCRs have been recently characterized in rainbow trout and in several teleost species, but the alphabeta T cell response against pathogens has not been directly demonstrated. To study the modifications of the T cell repertoire during an acute viral infection in rainbow trout, we adapted the immunoscope methodology, which consists of spectratyping the complementarity-determining region 3 length of the TCRbeta chain. We showed that the naive T cell repertoire is polyclonal and highly diverse in the naive rainbow trout. Using viral hemorrhagic septicemia virus (VHSV), which provokes an acute infection in rainbow trout, we identified skewed complementarity-determining region 3 size profiles for several VbetaJbeta combinations, corresponding to T cell clonal expansions during primary and secondary response to VHSV. Both public and private T cell expansions were shown by immunoscope analysis of spleen cells from several infected individuals of a rainbow trout clone sharing the same genetic background. The public response to VHSV consisted of expansion of Vbeta4Jbeta1 T cell, which appeared early during the primary response and was strongly boosted during the secondary response.

Viral haemorrhagic septicaemia virus induces vig-2, a new interferon-responsive gene in rainbow trout.

An mRNA differential display methodology was used to study the rainbow trout response to viral infection. A new transcript (vig-2) induced by viral haemorrhagic septicaemia virus (VHSV) in rainbow trout leucocytes was identified from the head-kidney. vig-2 was also induced in vivo during experimental infection and following DNA immunisation with a plasmid containing a gene encoding the viral glycoprotein. Viral induction of vig-2 was blocked by cycloheximide (CHX), indicating its dependency on a newly synthesised intermediate protein. This intermediate protein is most probably related to interferon because treatment of cells with a conditioned medium displaying an interferon-like activity resulted in a strong vig-2 expression, which was not blocked by CHX treatment. The cDNA sequence of the vig-2 transcript displays several mRNA destabilisation motifs and two signals characteristic of immediate-early gene expression. Curiously, vig-2 has no evident encoding potential except for a small 51 amino acid putative polypeptide with no clear similarity to any sequence available in the databanks. Therefore, the complete vig-2 genomic sequence was determined from a lambda phage clone retrieved from a genomic DNA library of rainbow trout. The genomic organisation of vig-2 shows five exons delimited with typical splice acceptor and donor sites. A promoter with a canonical ISRE, confirming that vig-2 is an interferon-responsive gene, is also present 115 nt upstream of the first exon.

Mutations in the glycoprotein of viral haemorrhagic septicaemia virus that affect virulence for fish and the pH threshold for membrane fusion.

To study the molecular basis of virulence of viral haemorrhagic septicaemia virus (VHSV), we used a cross-reactive neutralizing MAb to select MAb-resistant (MAR) mutants with reduced pathogenicity for fish. From sequence determination of the G gene of MAR mutants, attenuated laboratory variant and avirulent field strains, we identified two distant regions of the glycoprotein associated with virulence: region I (aa 135-161), homologous to the putative fusion peptide of both rabies virus (RV) and vesicular stomatitis virus (VSV), and region II (surrounding aa 431-433), homologous to RV and VSV domains controlling the conformational changes necessary for the fusion process to take place. Simultaneous mutations in both regions resulted in the most attenuated phenotype and we obtained genetic evidence that regions I and II may be structurally linked. As the MAR mutants had mutations in or near domains involved in fusion, the fusion properties of VHSV and its variants were analysed. This work allowed us to postulate that the fusion domain of VHSV is probably constituted of two distinct regions of the protein connected through a disulfide bridge between cysteines 110 and 152. Finally, we obtained evidence suggesting that the pH threshold for fusion is a determinant for virulence: restriction of fusion to a more acidic pH was associated with attenuation for the variant tr25 which had a shift of the threshold for maximal fusion from pH 6.30 (for the parental strain) to pH 6.00; conversely, two field strains which had maximal fusion at pH 6.60 were the most virulent.

vig-1, a new fish gene induced by the rhabdovirus glycoprotein, has a virus-induced homologue in humans and shares conserved motifs with the MoaA family.

We used mRNA differential display methodology to analyze the shift of transcription profile induced by the fish rhabdovirus, viral hemorrhagic septicemia virus (VHSV), in rainbow trout leukocytes. We identified and characterized a new gene which is directly induced by VHSV. This VHSV-induced gene (vig-1) encodes a 348-amino-acid protein. vig-1 is highly expressed during the experimental disease in lymphoid organs of the infected fish. Intramuscular injection of a plasmid vector expressing the viral glycoprotein results in vig-1 expression, showing that the external virus protein is sufficient for the induction. vig-1 expression is also obtained by a rainbow trout interferon-like factor, indicating that vig-1 can be induced through different pathways. Moreover, vig-1 is homologous to a recently described human cytomegalovirus-induced gene. Accordingly, vig-1 activation may represent a new virus-induced activation pathway highly conserved in vertebrates. The deduced amino acid sequence of vig-1 is significantly related to sequences required for the biosynthesis of metal cofactors. This suggests that the function of vig-1 may be involved in the nonspecific virus-induced synthesis of enzymatic cofactors of the nitric oxide pathway.

Combined DNA immunization with the glycoprotein gene of viral hemorrhagic septicemia virus and infectious hematopoietic necrosis virus induces double-specific protective immunity and nonspecific response in rainbow trout.

Glycoprotein (G) of viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) contains several neutralizing epitopes. However, recombinant G protein never matches intact viral particles for immunogenicity. DNA immunization offers the possibility to deliver the antigen through the cellular machinery, thus mimicking natural infection. We constructed pCDNA gVHS and pCDNA gIHN plasmids with the G gene of VHSV and IHNV under the control of the CMV promoter, and we tested the plasmids for the accurate G protein expression prior to their use in fish immunization. Following intramuscular injection to adult rainbow trout, plasmid DNA was found inside the muscle cells shortly after injection and was still present 45 days later. mRNA of the G protein was detected in muscle tissue extracts, and the G protein was found within muscle cells at the site of injection. This resulted in the synthesis of high levels of specific neutralizing and protective antibodies. Fish injected with pCDNA gVHS and pCDNA gIHN in combination responded similarly to fish receiving one recombinant plasmid. In addition to the elicitation of a strong humoral response, DNA immunization was able to activate specialized cells of the immune system as well as nonspecific defense mechanisms, since mRNAs of MHC class II and Mx were strongly activated at the site of injection.

Sequence variation of the glycoprotein gene identifies three distinct lineages within field isolates of viral haemorrhagic septicaemia virus, a fish rhabdovirus.

To evaluate the genetic diversity of viral haemorrhagic septicaemia virus (VHSV), the sequence of the glycoprotein genes (G) of 11 North American and European isolates were determined. Comparison with the G protein of representative members of the family Rhabdoviridae suggested that VHSV was a different virus species from infectious haemorrhagic necrosis virus (IHNV) and Hirame rhabdovirus (HIRRV). At a higher taxonomic level, VHSV, IHNV and HIRRV formed a group which was genetically closest to the genus Lyssavirus. Compared with each other, the G genes of VHSV displayed a dissimilar overall genetic diversity which correlated with differences in geographical origin. The multiple sequence alignment of the complete G protein, showed that the divergent positions were not uniformly distributed along the sequence. A central region (amino acid position 245-300) accumulated substitutions and appeared to be highly variable. The genetic heterogeneity within a single isolate was high, with an apparent internal mutation frequency of 1.2 x 10(-3) per nucleotide site, attesting the quasispecies nature of the viral population. The phylogeny separated VHSV strains according to the major geographical area of isolation: genotype I for continental Europe, genotype II for the British Isles, and genotype III for North America. Isolates from continental Europe exhibited the highest genetic variability, with sub-groups correlated partially with the serological classification. Neither neutralizing polyclonal sera, nor monoclonal antibodies, were able to discriminate between the genotypes. The overall structure of the phylogenetic tree suggests that VHSV genetic diversity and evolution fit within the model of random change and positive selection operating on quasispecies.

Rapidly destructive disk degeneration. Report of a case documented by computed tomography and magnetic resonance imaging.

A case of rapidly destructive disk degeneration in which imaging studies were performed over a two-year period is reported. Rapidly destructive disk degeneration is a clinicopathological entity recently redefined by Revel et al.. It is analogous to conditions characterized by peripheral chondrolysis, such as rapidly destructive hip osteoarthritis. Its pathogenesis remains obscure.

Live fish vaccines: history and perspectives.

In the development of live vaccines against enzootic fish viral diseases, the conventional approaches, although somewhat successful, failed finally to deliver efficient, safe, and tagged strains for vaccine application. The genetically-engineered vaccine approach also gave similarly disappointing results. Faced with these realities during our work with VHS, we turned our research effort towards understanding the molecular basis of virulence and antigenicity of the virus. Using sequence analysis of neutralization-escape mutants, we identified several amino acid positions on the glycoprotein which seemed to be involved in the pathological process. The attenuated phenotype was consistently associated with simultaneous mutations at two distant regions, 125-140 and 430-433 of the glycoprotein. We also demonstrated that reversion to virulence was accompanied by the loss of the concurrent mutations, which confirmed their involvement in virulence. The importance of these two regions of the glycoprotein was confirmed by the finding that laboratory or naturally attenuated variants had mutations within these regions. Using the same methodology, we selected mutants from an attenuated temperature resistant variant (tr25), which had previously been developed in our laboratory. Virulence, antigenicity and protective activity of the further attenuated mutants were evaluated in fish of different size by intramuscular (i.m.) or water bath administration. Strains having an additional mutation at position 139 were completely non-virulent for fish of 1000-1400 degree-days (dxd) by bath. These mutants retained their immunogenicity and had a thermo-resistant, an antigenic, and five genetic markers. Thus, they will constitute ideal candidates for live vaccine development, once their protective activity and containment have been confirmed in field trials.

Sarcoidosis and spondylarthropathy. Three case-reports.

Coexistent sarcoidosis and seronegative spondylarthropathy have rarely been reported. We add three new cases to the nine previously published. Two men and one woman with sarcoidosis met Amor's criteria for spondylarthropathy. The diagnosis of sarcoidosis was based on histologic findings in two cases and on roentgenographic and laboratory test findings in one case. The features of each of the two diseases were unremarkable. The two diagnoses were confirmed at about the same time. Osteoarticular manifestations of sarcoidosis are reviewed. Our case-reports illustrate the diagnostic difficulties raised by discovery of sacroiliitis in a patient with sarcoidosis: sarcoid osteitis, infection, or a spondyloarthropathy can be the cause of the sacroiliac lesions. Moreover, the pelvic and spinal manifestations of sarcoidosis can mimick a spondylarthropathy. Coexistence of sarcoidosis and spondylarthropathy is probably due to chance, since there are no shared predisposing genetic factors and the number of reported cases is small.

Immunodominant epitopes defined by a yeast-expressed library of random fragments of the rabies virus glycoprotein map outside major antigenic sites.

Nineteen yeast colonies secreting rabies virus glycoprotein (G) peptides immunoreactive with polyclonal anti-rabies virus sera were selected from a random expression library. The peptides, around 80 amino acids long, spanned amino acids 54-494 of the G protein. These peptides, together with two constructions including, respectively, immunodominant sites II and III, were analysed for their immunoreactivity with 40 anti-G protein monoclonal antibodies (MAbs) composed of 12 MAbs that reacted with SDS-treated protein in Western blot under reducing conditions (WB+) and 28 representative MAbs that did not react after denaturation (WB-). This last category represents 98% of anti-rabies virus G MAbs. None of the WB- MAbs bound peptides. Of the 12 WB+ MAbs, one bound two peptides situated before the transmembrane domain of the protein and six bound peptides overlapping a region situated between amino acids 223 and 276. These six MAbs define a new antigenic region that would be considered 'immunodominant' if the peptide strategy had been used to study the antigenicity of the protein; however, this region is only recognized by about 1% of our MAbs. Three of these WB+ MAbs had significant neutralizing activity; two were used for the selection of antigenic mutants (MAR mutants). Some mutants had a substitution within the region delimited by the peptides, confirming the pertinence of both the peptide and escape mutant approaches. However, a few mutants had a substitution outside the peptide-delimited region, suggesting that remote mutation(s) could affect epitope accessibility in the native protein.