Expression of IgD by murine lymphocytes. Loss of surface IgD indicates maturation of memory B cells.

B lymphocytes capable of generating primary IgM and IgG plaque-forming cells (PFC) responses to burro erythrocytes have surface IgD, as do primary IgM PFC. IgG memroy cells arising after one injection of antigen are divided into two groups, one of which expresses surface IgD while the other has no detectable membrane IgD. PFC generated from the IgG memory cells lacking surface IgD show a higher average avidity than those arising from IgD-positive IgG memory cells, indicating that mature IgG memory cells do not have surface IgD. After more than one injection of antigen, few, if any, IgG memory cells have surface IgD. IgG PFC arising in primary or secondary immune response lack membrane-bound IgD. These data provide the outlines for a B-cell maturation pathway in which IgD marks unprimed and early memory B cells and is lost in mature memory cells. Studies presented here were conducted by isolating IgD+ and IgD- cells with the fluorescence-activated cell sorter and functional testing of the isolated populations in adoptive transfer experiments.

Sharing of endogenous lentiviral gene fragments among leporid lineages separated for more than 12 million years.

Lentiviruses are causal agents of severe pathologies of a variety of mammals, including cattle and humans (e.g., AIDS and different types of lymphoma). While endogenous forms of lentivirus do not occur in these species, A. Katzourakis and coworkers (A. Katzourakis, M. Tristem, O. G. Pybus, and R. J. Gifford, Proc. Natl. Acad. Sci. USA 104:6261-6265, 2007) recently reported the presence in the genome of the European rabbit (Oryctolagus cuniculus) of multiple sequences defining a lentiviral subgroup elegantly referred to as RELIK (rabbit endogenous lentivirus type K). Sequence comparisons indicated that the RELIK ancestor may have integrated into the rabbit lineage more than 7 million years ago. We have substantiated this by producing sequence data certifying the sharing of RELIK sequences among leporid lineages that diverged some 12 million years ago.

Evolution of rabbit haemorrhagic disease virus (RHDV) in the European rabbit (Oryctolagus cuniculus) from the Iberian Peninsula.

To date information on rabbit haemorrhagic disease virus (RHDV) in Spain and Portugal has been scarce, although the disease is endemic and continues to have a considerable impact on species conservation and hunting industry. We analysed RHDVs obtained between 1994 and 2007 at different geographic locations in Portugal (40 samples), Spain (3 samples) and France (4 samples) from wild European rabbits (Oryctolagus cuniculus) that succumbed to the disease. Phylogenetic analyses based on partial VP60 gene sequences allowed a grouping of these RHDVs into three groups, termed "Iberian" Groups IB1, IB2 and IB3. Interestingly, these three Iberian groups clustered separately, though not far from earlier RHDVs of Genogroup 1 (containing e.g., strain "AST89"), but clearly distinct from globally described RHDV strains of Genogroups 2-6. This result, supported by a bootstrap value of 76%, gives rise to the hypothesis that the virus evolved independently since its introduction to wild rabbit populations on the Iberian Peninsula, with the Pyrenees acting as a natural barrier to rabbit and hence to virus dispersal. No differences were observed in RHDV sequences obtained from geographic regions where the rabbit subspecies O. c. algirus prevails compared with those obtained from O. c. cuniculus.

Detection of positive selection in the major capsid protein VP60 of the rabbit haemorrhagic disease virus (RHDV).

Mutations were analysed in the major capsid protein VP60 of the rabbit haemorrhagic disease virus (RHDV), a calicivirus responsible for high mortality rates in both wild and domestic European rabbits (Oryctolagus cuniculus). Likelihood of positive selection was estimated using the PAML software applied to 43 non-identical complete sequences of the major capsid protein. Three codons showed signs of positive selection (with posterior probabilities over 95%), one of them is located in the region containing the major antigenic determinants (region E). The presence of positively selected codons (PSCs) in other regions may suggest the existence of other antigenic regions on the major capsid protein that stimulate protective immune responses. At all the 3 PSCs, variation contributes to putative N-glycosylation sites of the protein. An N-glycosylation site is deleted in the non-pathogenic strain RCV. Some of the substitutions at PSCs may alter the polarity and the charge of the protein with possible implications in the protein structure and host interaction. The detection of PSCs should allow a better understanding of the interaction between RHDV and the rabbit immune system.

Genetic characterization of the chemokine receptor CXCR4 gene in lagomorphs: comparison between the families Ochotonidae and Leporidae.

Chemokines receptors are transmembrane proteins that bind chemokines. Chemokines and their receptors are known to play a crucial role in the immune system and in pathogen entry. There is evidence that myxoma virus, the causative agent of myxomatosis, can use the chemokine receptor CXCR4 to infect cells. This virus causes a benign disease in its natural host, Sylvilagus, but in the European rabbit (Oryctolagus cuniculus) it causes a highly fatal and infectious disease known as myxomatosis. We have characterized the chemokine receptor CXCR4 gene in five genera of the order Lagomorpha, Ochotona (Ochotonidae), and Oryctolagus, Lepus, Bunolagus and Sylvilagus (Leporidae). In lagomorphs, the CXCR4 is highly conserved, with most of the protein diversity found at surface regions. Five amino acid replacements were observed, two in the intracellular loops, one in the transmembrane domain and two in the extracellular loops. Oryctolagus features unique amino acid changes at the intracellular domains, putting this genus apart of all other lagomorphs. Furthermore, in the 37 European rabbits analysed, which included healthy rabbits and rabbits with clinical symptoms of myxomatosis, 14 nucleotide substitutions were obtained but no amino acid differences were observed.

Variance analysis of immunoglobulin alleles in natural populations of rabbit (Oryctolagus cuniculus): the extensive interallelic divergence at the b locus could be the outcome of overdominance-type selection.

Population genetic data are presented which should contribute to evaluation of the hypothesis that the extraordinary evolutionary patterns observed at the b locus of the rabbit immunoglobulin light chain constant region can be the outcome of overdominance-type selection. The analysis of allele correlations in natural populations revealed an excess of heterozygotes of about 10% at the b locus while heterozygote excess was not observed at loci determining the immunoglobulin heavy chain. Data from the published literature, where homozygote advantage was suggested, were reevaluated and found in agreement with data here presented. Gene diversity was evenly distributed among populations and showed similarities with patterns reported for histocompatibility loci. Analysis of genotypic disequilibria revealed strong digenic associations between the leading alleles of heavy and light chain constant region loci in conjunction with trigenic disequilibria corresponding to a preferential association of b locus heterozygosity with the predominant allele of the heavy chain e locus. It is argued that this may indicate compensatory or nonadditive aspects of a putative heterozygosity enhancing mechanism, implying that effects at the light chain might be more pronounced in populations fixed for the heavy chain polymorphism.

Patterns of interallelic divergence at the rabbit b-locus of the immunoglobulin light chain constant region are in agreement with population genetical evidence for overdominant selection.

Population studies at the b-locus of the "constant" regions of the rabbit immunoglobulin kappa 1 light chain (c kappa 1) revealed patterns of gene diversity resembling those that mark the peculiar nature of the major histocompatibility complex, such as large number of alleles, high heterozygosity levels, consistent excess of heterozygous individuals and long allele coalescence times. This paper documents the evolutionary patterns at the b-locus as inferred from DNA sequence comparisons. Among alleles, synonymous substitutions outnumbered expectations for neutral alleles by an order of magnitude. They were distributed randomly throughout the c kappa 1 coding region while interallelic amino acid differences did cluster into segments overlapping with the regions exposed to the solvent. Within these regions, acceptance rates of mutation at amino acid replacement sites were even higher than those at synonymous sites (dr/ds = 1.6-3.0), while in the intervals between these regions the opposite was found (dr/ds approximately 0.3). Under the assumption that allelic variation is adaptive at the molecular surface, the divergence patterns at the b-locus are therefore very similar to those reported for the major histocompatibility complex. An analysis at the quasi silent bas-locus (c kappa 2), which is linked to the b-locus, and comparisons among genes of the "variable" region of the kappa 1 light chains (v kappa 1), revealed patterns of divergence which differed markedly from those observed at the c kappa 1 constant regions. It is suggested that allelic variability at immunoglobulin constant regions can be due to mechanisms similar to those enhancing diversity at histocompatibility loci.

Estimation of gene diversity at the b locus of the constant region of the immunoglobulin light chain in natural populations of European rabbit (Oryctolagus cuniculus) in Portugal, Andalusia and on the Azorean Islands.

The minimal gene diversity at a locus of the antibody constant region, as estimated in natural populations of rabbit, revealed levels of heterozygosity similar to those reported for the major histocompatibility complex in human and murine populations. Sera of 416 wild rabbits were collected on the Iberian peninsula and on three islands of the Azorean archipelago and analyzed for the occurrence of the serological markers of the b locus of the immunoglobulin light chain. All four serotypes present in domestic rabbits were found in Portugal. They represented less than 50% of the gene pool. In Andalusia this was less than 15% and on the Azorean islands less than 10%. The pronounced and systematic hierarchy in allele frequencies, previously found in populations from the more recent distribution area of the species, was not observed. On the peninsula, the frequencies of the "domestic" alleles were similar, averaging 10%. The Portuguese sample revealed a total heterozygosity of at least 87%. This high value was supported by at least 11 serologically different alleles, none of them occurring at frequencies above 20%. These data are in agreement with an Iberian origin of the European rabbit and strongly suggest the coalescence of b locus allelic lines drawn from Iberian and western populations. The role of balancing selection in the evolution of the b locus polymorphism was further emphasized.

Compensatory aspects of allele diversity at immunoglobulin loci: gene correlations in rabbit populations devoid of light chain diversity (Oryctolagus cuniculus L.; Kerguelen Islands).

Is there a selective advantage of increased diversity at one immunoglobulin locus when diversity at another locus is low? A previous paper demonstrated excess heterozygosity at the rabbit light chain b locus when heterozygosity was low at the heavy chain constant region e locus. Here we consider the reverse situation by analyzing allele distributions at heavy chain loci in populations fixed for the light chain b locus. We analyzed the a locus that encodes the predominantly expressed heavy chain variable region, and the d and e loci that control different parts of the Ig gamma class constant region. While there was excess heterozygosity, genetic differentiation between localities was extensive and was most pronounced for females. This was in marked contrast with observations in areas where b-locus diversity was important and confirms a negative correlation between e- and b-locus heterozygosity. Trigenic disequilibria corresponded to a significant negative correlation between e- and a-locus heterozygosity due mainly to strong variation among localities within the context of pronounced (digenic) linkage disequilibria. Although substantial, the average increase in a/e-locus single heterozygosity implemented by higher order disequilibria within localities was not significant.

The quaternary Gs3 and Gs7 allotypes of the rabbit: generation of the determinants by interspecies molecular hybridization.

A number of allotypic markers of the rabbit immunoglobulins are present only on intact immunoglobulin molecules and not on the isolated heavy or light chains. Some of these markers, although determined by the kappa light chain, are limited to a single heavy chain class of Ig. They can be generated by in vitro hybridization of the kappa light chain with the appropriate heavy chain. The quaternary IgG allotypic determinants Gs3 and Gs7 which are determined respectively by the b4.1 and b4.2 allelic variants of the kappa b4 light chain can be generated not only by in vitro hybridization of these light chains with a rabbit gamma chain, but also to a certain extent by their hybridization to gamma chains derived from other species.

Clinimetric evaluation of the pain observation scale for young children in children aged between 1 and 4 years after ear, nose, and throat surgery.

This study assessed the reliability, validity, and responsiveness of a new pain measure for children aged 1 to 4 years that was developed from the Children's Hospital of Ontario Pain Scale and its Neonatal Infant Pain Scale. Pain in 311 children, aged 1 to 4 years, was measured by two observers at fixed intervals after adenotonsillectomy (n = 114), adenotomy (n = 109), or insertion of ventilation tubes (grommets) (n = 88) until discharge using a dichotomous pain scale of 9 behavioral and physiological categories. The scale proved to be strongly homogeneous. The interobserver agreement was substantial for 7 items. On these final 7 items, the ability to distinguish between patients with differing degrees of pain and the sensitivity to detect changes over time within each patient were substantial. The resulting Pain Observation Scale for Young Children is reliable and easy to use for assessment of short- and longer-lasting pain after ear, nose, and throat surgery and may be used for assessing pain with other conditions.

[Prevention and control of pain in (adeno)tonsillectomy]. / Pijnpreventie en pijnbestrijding bij (adeno)tonsillectomie.

Pain prevention and analgesia in (adeno)tonsillectomy OBJECTIVE. To study the efficacy of three analgesia protocols in children subjected to (adeno)tonsillectomy. SETTING. Twenteborg District Hospital, Almelo, the Netherlands. PATIENTS AND METHODS. In 64 children with body weights ranging from 15 to 25 kg (mean 20.4 kg) subjected to (adeno) tonsillectomy according to the procedure most current in the Netherlands (according to Sluder with administration of a volatile anaesthetic without intubation in day treatment), three protocols for preventing and treating postoperative pain were applied, with the analgetics most current in the Netherlands. Permission from the Medical Ethical Committee and written informed consent from the parents were obtained. According to protocol I 240 mg paracetamol was given, according to protocol II, 500 mg paracetamol and according to protocol III, 500 mg paracetamol plus 10 mg codeine, in suppository form, every 4 hours always starting 60 minutes before operation. Initially, protocol I was carried out, subsequently randomization of protocols II and III. After a pilot study use of a control group was omitted for ethical reasons because of the severe pain in a placebo group. MEASUREMENTS. The efficacy of the analgesia was evaluated by means of pain assessments by the children themselves using the Oucher test, 1, 3 and 6 hours after operation. The statistical processing of the children's pain scores was performed primarily in a descriptive sense, with simultaneous comparison of the three groups using a single variance analysis for comparison of several groups. Multiple comparisons were carried out according to Ducans' multiple range test. The level of significance was put at p less than or equal to 0.05. RESULTS. One hour postoperatively there was no significant difference among the mean pain scores (0: no pain and 10: very bad pain) of the three groups, viz. 5.2 (SD 3.0), 4.7 (SD 2.9) and 4.7 (SD 2.3), respectively; three hours postoperatively there was a significant difference between the mean pain scores of group I (4.5; SD 2.4) and group II (2.7; SD 2.4) but not between group I and group III (3.3; SD 1.5); six hours postoperatively there was a significant difference between the mean pain scores of group I (4.1; SD 2.1) on the one hand and of groups II (2.1; SD 1.5) and III (2.6; SD 1.6) on the other. CONCLUSION. The findings indicate existence of violent pain immediately after recovery from anaesthesia after (adeno)tonsillectomy as shown by the pain assessments by the children themselves one hour postoperatively, in spite of preventive administration of a high dose of a 'minor' analgetic according to each of the three protocols. Modification of the anaesthetic technique should be considered to effect adequate prevention or suppression of pain.

[Pediatric Pain Groups. Management related to pain in hospitalized children]. / Kinderpijngroepen. Het beleid ten aanzien van pijn bij kinderen in het ziekenhuis.

By means of multidisciplinary Pediatric Pain Groups in hospitals possibilities are created to implement recent knowledge about prevention of pain and relief of pain in children. The activities of these groups are amongst others: developing and testing pain protocols, applying systematic pain assessment and giving information about pediatric pain. These activities result in a reduction of pain in children and in prevention of needless suffering of pain in hospitalized children.

[Postoperative analgesia in 3 Dutch hospitals: a pilot study]. / Postoperatieve pijnbestrijding in drie Nederlandse ziekenhuizen: een pilotstudie.

Postoperative pain management is an area where preventive measures must be possible. This management will be linked to the expected pain in the postoperative period. To get a better insight in the expectancies and given treatment an inquiry was held in three Dutch hospitals. Of 48 often performed operative procedures in children only in 9 cases agreement was achieved. As a consequence treatment options were divers. Undertreatment is not unlikely. Postoperative pain scores by nurses was in 15 of 48 operative procedures significant higher than those by doctors.

Evidence for recombination in the major capsid gene VP60 of the rabbit haemorrhagic disease virus (RHDV).

Rabbit haemorrhagic disease (RHD) is a highly fatal disease caused by a virus of the family Caliciviridae. Whereas recombination is well documented in other members of this family, the extent of recombination has so far not been studied in RHDV. To reach a better evaluation of the possible role of recombination in the evolution of RHDV virulence, we have searched for recombination events in RHDV by analysing 43 complete sequences of the major capsid gene VP60. Phylogenetic analyses revealed two well separated groups. Clear evidence for recombination was found for the Hartmannsdorf strain which shows different phylogenetic profiles depending on the region of the capsid examined.