Hypertonic sodium lactate reverses brain oxygenation and metabolism dysfunction after traumatic brain injury.

BACKGROUND: The mechanisms by which hypertonic sodium lactate (HSL) solution act in injured brain are unclear. We investigated the effects of HSL on brain metabolism, oxygenation, and perfusion in a rodent model of diffuse traumatic brain injury (TBI). METHODS: Thirty minutes after trauma, anaesthetised adult rats were randomly assigned to receive a 3 h infusion of either a saline solution (TBI-saline group) or HSL (TBI-HSL group). The sham-saline and sham-HSL groups received no insult. Three series of experiments were conducted up to 4 h after TBI (or equivalent) to investigate: 1) brain oedema using diffusion-weighted magnetic resonance imaging and brain metabolism using localized 1H-magnetic resonance spectroscopy (n = 10 rats per group). The respiratory control ratio was then determined using oxygraphic analysis of extracted mitochondria, 2) brain oxygenation and perfusion using quantitative blood-oxygenation-level-dependent magnetic resonance approach (n = 10 rats per group), and 3) mitochondrial ultrastructural changes (n = 1 rat per group). RESULTS: Compared with the TBI-saline group, the TBI-HSL and the sham-operated groups had reduced brain oedema. Concomitantly, the TBI-HSL group had lower intracellular lactate/creatine ratio [0.049 (0.047-0.098) vs 0.097 (0.079-0.157); P < 0.05], higher mitochondrial respiratory control ratio, higher tissue oxygen saturation [77% (71-79) vs 66% (55-73); P < 0.05], and reduced mitochondrial cristae thickness in astrocytes [27.5 (22.5-38.4) nm vs 38.4 (31.0-47.5) nm; P < 0.01] compared with the TBI-saline group. Serum sodium and lactate concentrations and serum osmolality were higher in the TBI-HSL than in the TBI-saline group. CONCLUSIONS: These findings indicate that the hypertonic sodium lactate solution can reverse brain oxygenation and metabolism dysfunction after traumatic brain injury through vasodilatory, mitochondrial, and anti-oedema effects.

[Assessment of an educational maze for major trauma care teaching]. / Évaluation d'un labyrinthe pédagogique pour l'enseignement de la traumatologie grave.

BACKGROUND: Assess efficacy, satisfaction and usefulness of an educational maze based on posters and audioguide for major trauma care teaching to medical students. The educational maze consists of posters with audio comments recorded in an audioguide. This tool was part of a larger educational program including medical simulation. STUDY DESIGN: Prospective, interventional, observational, monocentric study. STUDENT: Medical student of Grenoble University Hospital, in the four last years of medical school, following a training course in anesthesia, emergency medical services and intensive care units. METHOD: Forty essentials key messages for major trauma management were included in 10 posters and audioguides. A first assessment with short opened answers was handed to the students at the end of the educational maze to assess their memorization. A second assessment with simple choice answers regarding satisfaction and usefulness of this new educational tool was realized at the end of the entire program. RESULT: One hundred and eighty-four medical students attending the major trauma program were included in this study. On the first test, 75% of essential knowledge on major trauma management was memorized by more than 50% of the medical students. On the second test, 94% of medical students had a high satisfaction level of this educational maze. CONCLUSION: An educational maze based on posters and audioguides seems to be an efficient, useful tool for teaching essential knowledge on major trauma management to medical students.

Vaccination with experimental feline immunodeficiency virus vaccines, based on autologous infected cells, elicits enhancement of homologous challenge infection.

Cats were vaccinated with fixed autologous feline immunodeficiency virus (FIV)-infected cells in order to present viral proteins to the immune system of individual cats in an MHC-matched fashion. Upon vaccination, a humoral response against Gag was induced. Furthermore, virus-neutralizing antibodies were detected in a Crandell feline kidney cell-based neutralization assay, but not in a neutralization assay based on primary peripheral blood mononuclear cells. Despite the induction of these FIV-specific responses, vaccinated cats were not protected. Instead, accelerated virus replication was found, an observation similar to what previous experiments using other vaccine candidates have shown. Here, the results of the present study are discussed in the light of enhancement of lentivirus infections as a complicating factor in lentivirus vaccine development.

Attempt to modify the immune response developed against FIV gp120 protein by preliminary FIV DNA injection.

Following inactivated virus vaccination trials, the surface glycoprotein gp120 (SU) of the feline immunodeficiency virus (FIV) was considered as one of the determinants for protection. However, several vaccination trials using recombinant Env protein or some Env-derived peptides failed to induce protection. To study the influence of the environment in which the surface protein (SU) is injected. we analyzed the impact of a nucleocapsid (NC) DNA immunization on the presentation of the recSU protein to the immune system. Cats were vaccinated either with the recSU protein alone or with NC DNA followed by the recSU protein. Two routes of nucleocapsid DNA vaccination were tested: intramuscular and mucosal injections. Cats immunized with the recSU protein showed a facilitation of infection, since they presented the earliest and the highest humoral response correlating with the highest proviral load. They also showed an acceleration of the appearance of IL4 mRNA signal. Preliminary injection of the DNA coding for NC protein, regardless the route of inoculation, seemed to inhibit the facilitation induced by vaccination with the recSU protein alone. The previously nucleocapsid DNA immunized cats had infectious status similar to those of the control cats, but with lower proviral load and less developed anti-FIV humoral response. Cat No. 2, belonging to the group vaccinated with NC protein by the mucosal route, had a protected-like status which did not correlate with the humoral response. This cat was the only one to have a persisting IFN mRNA signal after challenge specific for the p10 nucleocapsid and recSU proteins. However, no NC specific cytotoxic cells were observed throughout the experiment in this cat. The role of nucleocapsid DNA vaccination is still unknown nevertheless we did demonstrate that the facilitation observed in vaccination trial with recombinant proteins could be modified and that recombinant proteins could be a component of an effective vaccine.

Partial protection by vaccination with recombinant feline immunodeficiency virus surface glycoproteins.

In an effort to induce a strong immune response that might protect against feline immunodeficiency virus (FIV) challenge infection, three groups of five specified pathogen-free (spf) cats each were immunized subcutaneously with different FIV antigen preparations. Immunizations were done at weeks 0, 2, and 4 with 100 microg of recombinant SU from an FIV Zurich 2 (FIV Z2) strain expressed by E. coli (group 1) or the baculovirus expression system (groups 2 and 3) adsorbed on aluminum hydroxyde and administered with QS-21 (groups 1 and 2) or Freund's adjuvant together with the recombinant nucleocapsid protein (protein NC) of rabies virus (group 3). Protein NC was described to act as an exogenous superantigen. Group 3 cats demonstrated the highest detectable antibody response to the vaccine antigen as determined by ELISA and Western blot analysis. All immunized cats together with seven control animals were challenged with 20 CID50 of cat lymphocyte-grown FIV Z2 3 weeks following the last immunization. Whereas virus was readily recovered from peripheral blood lymphocytes of seven of seven nonvaccinated control cats following this challenge dose, virus was not recovered from two cats of groups 1 and 2. All cats in groups 2 and 3 showed a provirus load significantly decreased to 3% of that of controls up to week 8 after challenge infection. Eleven of 15 vaccinated cats and 5 of 7 control cats developed virus-neutralizing antibodies by week 8 after challenge infection. The two cats negative on virus isolation remained seronegative, developed no detectable virus-neutralizing activities, but were repeatedly positive in provirus PCR. Moreover, starting at week 1 after challenge, both cats showed the lowest provirus load in their respective groups. These results indicate that immunization with recombinant FIV SU in conjunction with appropriate adjuvants may lead to partial protection against FIV challenge infection.

Enhancement of feline immunodeficiency virus (FIV) infection after DNA vaccination with the FIV envelope.

Despite intensive experimentation to develop effective and safe vaccines against the human immunodeficiency viruses and other pathogenic lentiviruses, it remains unclear whether an immune response that does not afford protection may, on the contrary, produce adverse effects. In the present study, the effect of genetic immunization with the env gene was examined in a natural animal model of lentivirus pathogenesis, infection of cats by the feline immunodeficiency virus (FIV). Three groups of seven cats were immunized by intramuscular transfer of plasmid DNAs expressing either the wild-type envelope or two envelopes bearing mutations in the principal immunodominant domain of the transmembrane glycoprotein. Upon homologous challenge, determination of plasma virus load showed that the acute phase of viral infection occurred earlier in the three groups of cats immunized with FIV envelopes than in the control cats. Genetic immunization, however, elicited low or undetectable levels of antibodies directed against envelope glycoproteins. These results suggest that immunization with the FIV env gene may result in enhancement of infection and that mechanisms unrelated to enhancing antibodies underlay the observed acceleration.

DNA vaccination using expression vectors carrying FIV structural genes induces immune response against feline immunodeficiency virus.

Following inactivated virus vaccination trials, the surface glycoprotein gp120 of the feline immunodeficiency virus (FIV) was considered as one of the determinants for protection. However, several vaccination trials using recombinant Env protein or some peptides failed to induce protection. To understand the role of the gp120 protein in vivo, we vaccinated cats with naked DNA coding for FIV structural proteins gp120 and p10. We analyzed the ability of these vaccinations to induce immune protection and to influence the onset of infection. Injection in cat muscles of expression vectors coding for the FIV gp120 protein induced a humoral response. Cats immunized twice with the gp120 gene showed different patterns after challenge. Two cats were, like the control cats, infected from the second week after infection onwards. The two others maintained a low proviral load with no modification of their antibody pattern. The immune response induced by gp120 DNA injection could control the level of viral replication. This protective-like immune response was not correlated to the humoral response. All the cats immunized with the gp120 gene followed by the p10 gene were infected, like the control cats, from the second week but they developed a complete humoral response against viral proteins after challenge. Furthermore, they showed a sudden but transient drop of the proviral load at 4 weeks after infection. Under these conditions, one injection of the p10 gene after one injection of the gp120 gene was not sufficient to stimulate protection. On the contrary, after a period, it seems to facilitate virus replication.

Fetal versus adult PreB or B cells: the human VH repertoire.

At the preB stage, when only the IGH locus has rearranged, mu chains become expressed in association with the psi L chains, lambda-like and VpreB, thus forming the preB receptor. By the use of a monoclonal anti VpreB antibody, preB cells were isolated from two adult bone marrow samples, and the VH repertoire was analyzed and compared to fetal, XLA (X-linked agammaglobulinemia), and adult B repertoires. Most VH genes identified were also expressed in fetal liver, XLA bone marrow, and adult PBLs, with similar predominant usage of certain germline genes. Multiple D/D fusions, limited N diversity, and preferential use of JH4 with a low level of DQ52 usage were also identified. Few mutations could be observed, not specifically localized in CDR regions, that could be interpreted as not positively selected. Conversely, a shorter length of CDR3 appeared to be the hallmark of the preB step. Thus, the association of psi L chains with mu does not bring about a bias in the VH gene usage, but a first selection on the CDR3 region could be the result of recognition by given autoantigens or ligands different for preB cells and B cells.

Mechanisms that generate human immunoglobulin diversity operate from the 8th week of gestation in fetal liver.

The repertoire of immunoglobulin expressed very early in human development was approached by cloning and sequencing 55 rearranged and 11 germ-line VH transcripts, after amplification by polymerase chain reaction of cDNA libraries derived from two fetal livers at 8 and 13 weeks of gestation. All families with the exception of VH2, were expressed as soon as 8 weeks, with preferential usage of certain germ-line genes. Very few somatic mutations, randomly localized, were identified. By contrast, in a series of clones derived from the same VDJ rearrangement using the VH6 family, extensive mutations had taken place, mostly accumulated in the third complementarity-determining region (CDR3) suggesting that the specialized enzymatic machinery was at hand very early during human development. Some other characteristics of the fetal repertoire also emerged, namely increased usage of JH3 and JH2, as compared to the adult pattern, where JH4 is dominant and reduced length of the D/CDR3 regions. All D gene families were identified, and their usage frequently involved D-D fusions. N diversity was present very early, and increased with age. Identification of germ-line transcripts pertaining to all six VH families including pseudogenes, in the E55 library, revealed a population very different as compared to rearranged gene transcripts. This suggests that a large portion of VH locus is accessible for transcription, bringing no evidence of correlation between preferential rearrangement of a given VH gene and its localization in the locus.

IGM kappa/lambda EBV human B cell clone: an early step of differentiation of fetal B cells or a distinct B lineage?

In agreement with the clonal theory, one B lymphocyte synthesizes one antibody due to allelic and isotypic exclusion. We analyzed an EBV B-cell clone, E29.1, derived from an 11 week-old embryo, and secreting both IgM kappa and IgM lambda. Structural analysis of produced IgM, indicated that lambda-containing pentamers could be considered hybrid molecules, expressing both the kappa and lambda. chains, with a kappa/lambda ratio between 5 and 10. It was also found that 60% of the lambda chains were secreted in free form, presumably as a result of a better affinity of mu chains for kappa chains. The sequence of the three transcripts had an entirely ORF (Open Reading Frame), and were very close to germline sequences, with, however, an additional codon between V kappa and J kappa gene which has never been described in adult myeloma protein or cDNA human sequence. This observation is suggestive of N diversity taking place in kappa chains. The possible role of Kde (kappa deleting element) recombination onto kappa/lambda locus activation was analyzed on a collection of 23 lambda clones. The status of rearrangement of kappa genes indicated that 35% of these clones had retained, at least, one kappa allele without the Kde recombination, four lambda clones had one kappa allele in germline configuration. Different hypotheses of maturation from pre-B cell to B cell with activation of light chain genes are discussed.

Human immunoglobulin VH and VK repertoire revealed by in situ hybridization.

We report in this paper the first analysis of the expression pattern of Ig VH and VK families in human adult normal peripheral B lymphocytes, by in situ hybridization using specific VH1 to VH6 and VK1 to VK4 probes, which cover the known human V gene families reported to date. The major families were VH3 and VK1, with the respective gradient VH3 greater than VH4 greater than VH1 greater than VH5 greater than VH6 greater than VH2, and VK1 greater than VK3 greater than VK4 greater than VK2. Using a large sampling of EBV clones, we found that the pattern of VH and VK family usage was similar. The expression level correlated fairly with the estimated gene number for the VH, but diverged noticeably for the K chains. Taken together with the fact that the level of light chain expression (K + lambda) was about two-fold that of heavy chains, these results suggest that the VH and the VK repertoires are not regulated by a similar selective process.

Rapid expansion of human immunoglobulin repertoire (VH, V kappa, V lambda) expressed in early fetal bone marrow.

We have isolated pre-B- and B-cell clones after transformation by Epstein-Barr virus (EBV) of human fetal bone marrow cells between weeks 8 and 13 of gestation. These clones were characterized for immunoglobulin (Ig) chain synthesis, the status (rearranged versus germ line) of the heavy (H) chain, kappa, and lambda loci, and of the Ig mRNA transcripts by using specific variable (V), VH, V kappa, and V lambda probes covering the almost complete IgV repertoire. Mature B cells could already be identified in the 8-week-old bone marrow, with both kappa and lambda isotypes being present. Nevertheless, at this stage, most of the clones had Xhe characteristics of pre-B cells, as indicated by the presence of mu transcripts (either functional or sterile) in the absence of L chains. The kinetics of gene rearrangements were compatible with the classical scheme H----kappa----lambda. A rapid expansion of the expressed repertoire occurred between the weeks 8 and 11, with 95% of the EBV clones having the characteristics of mature B cells. V gene family usage was analyzed for the three loci and compared with the pattern of expression observed at 30 weeks of gestation and in adult clones. The "adult" pattern was rapidly acquired for the H and kappa loci, with the major subgroups being VH3 and V kappa 1. When the expression of the repertoire was "normalized," the pattern of V usage correlated fairly well with the estimated number of VH genes, but differed noticeably for the kappa chains, suggesting that the VH and V kappa repertoires are not regulated by similar processes.

Preferential expression of VH5 and VH6 immunoglobulin genes in early human B-cell ontogeny.

Analysis of Ig transcripts by dot blot hybridization revealed that VH5 and VH6 RNA first occurred during the 7th week of gestation in human fetal liver. At the same time, a C lambda or C lambda-like chain was also expressed, in the absence of detectable kappa chains. In adult B cells, VH3 appears to be the dominant family expressed. Because VH5 and VH6 were located as the most proximal V genes to the JH cluster, the orderly expression of human VH genes seems to resemble the situation reported for the mouse.