Rabbit models of human diseases for diagnostics and therapeutics development.

This review presents some examples of studies using the European rabbit (Oryctolagus cuniculus) that have led to, and continue to, contribute to advancement of understanding of human diseases as well as therapeutics development. In addition, we tabulate FDA-approved rabbit polyclonal and rabbit monoclonal antibodies (mAbs) that are used for diagnostic applications, as well as an overview of some "humanized" or otherwise altered rabbit mAbs that are in initial phase I, II, or advanced to phase III clinical trials. Information about endogenous retriviruses learned from studies of rabbits and other members of the order Lagomorpha are summarized as this knowledge now applies to new therapeutics being developed for several human diseases including Multiple Sclerosis, Type 1 Diabetes and Cancer.

Rabbit genome analysis reveals a polygenic basis for phenotypic change during domestication.

The genetic changes underlying the initial steps of animal domestication are still poorly understood. We generated a high-quality reference genome for the rabbit and compared it to resequencing data from populations of wild and domestic rabbits. We identified more than 100 selective sweeps specific to domestic rabbits but only a relatively small number of fixed (or nearly fixed) single-nucleotide polymorphisms (SNPs) for derived alleles. SNPs with marked allele frequency differences between wild and domestic rabbits were enriched for conserved noncoding sites. Enrichment analyses suggest that genes affecting brain and neuronal development have often been targeted during domestication. We propose that because of a truly complex genetic background, tame behavior in rabbits and other domestic animals evolved by shifts in allele frequencies at many loci, rather than by critical changes at only a few domestication loci.

Accuracy and coverage assessment of Oryctolagus cuniculus (rabbit) genes encoding immunoglobulins in the whole genome sequence assembly (OryCun2.0) and localization of the IGH locus to chromosome 20.

We report on the analyses of genes encoding immunoglobulin heavy and light chains in the rabbit 6.51× whole genome assembly. This OryCun2.0 assembly confirms previous mapping of the duplicated IGK1 and IGK2 loci to chromosome 2 and the IGL lambda light chain locus to chromosome 21. The most frequently rearranged and expressed IGHV1 that is closest to IG DH and IGHJ genes encodes rabbit VHa allotypes. The partially inbred Thorbecke strain rabbit used for whole-genome sequencing was homozygous at the IGK but heterozygous with the IGHV1a1 allele in one of 79 IGHV-containing unplaced scaffolds and IGHV1a2, IGHM, IGHG, and IGHE sequences in another. Some IGKV, IGLV, and IGHA genes are also in other unplaced scaffolds. By fluorescence in situ hybridization, we assigned the previously unmapped IGH locus to the q-telomeric region of rabbit chromosome 20. An approximately 3-Mb segment of human chromosome 14 including IGH genes predicted to map to this telomeric region based on synteny analysis could not be located on assembled chromosome 20. Unplaced scaffold chrUn0053 contains some of the genes that comparative mapping predicts to be missing. We identified discrepancies between previous targeted studies and the OryCun2.0 assembly and some new BAC clones with IGH sequences that can guide other studies to further sequence and improve the OryCun2.0 assembly. Complete knowledge of gene sequences encoding variable regions of rabbit heavy, kappa, and lambda chains will lead to better understanding of how and why rabbits produce antibodies of high specificity and affinity through gene conversion and somatic hypermutation.

Molecular bases of genetic diversity and evolution of the immunoglobulin heavy chain variable region (IGHV) gene locus in leporids.

The rabbit has long been a model for studies of the immune system. Work using rabbits contributed both to the battle against infectious diseases such as rabies and syphilis, and to our knowledge, of antibodies' structure, function, and regulated expression. With the description of rabbit Ig allotypes, the discovery of different gene segments encoding immunoglobulins became possible. This challenged the "one gene-one protein" dogma. The observation that rabbit allotypic specificities of the variable regions were present on IgM and IgG molecules also led to the hypothesis of Ig class switching. Rabbit allotypes contributed to the documentation of phenomena such as allelic exclusion and imbalance in production of allelic gene products. During the last 30 years, the rabbit Ig allotypes revealed a number of unique features, setting them apart from mice, humans, and other mammals. Here, we review the most relevant findings concerning the rabbit IGHV. Among these are the preferential usage of one VH gene in VDJ rearrangements, the existence of trans-species polymorphism in the IGHV locus revealed by serology and confirmed by sequencing IGHV genes in Lepus, the unusually large genetic distances between allelic lineages and the fact that the antibody repertoire is diversified in this species only after birth. The whole genome sequence of a rabbit, plus re-sequencing of additional strains and related genera, will allow further evolutionary investigations of antibody variation. Continued research will help define the roles that genetic, allelic, and population diversity at antibody loci may play in host-parasite interactions.

Comparative analysis of genome sequences of the Th2 cytokine region of rabbit (Oryctolagus cuniculus) with those of nine different species.

The regions encoding the coordinately regulated Th2 cytokines IL5, IL4 and IL13 are located on chromosomes 5 of man and 11 of mouse. They have been intensively studied because these interleukins have protective roles in helminth infections, but may lead to detrimental effects such as allergy, asthma, and fibrosis in lung and liver. We added to previous studies by comparing sequences of syntenic regions on chromosome 3 of the rabbit (Oryctolagus cuniculus) genome OryCun 2.0 assembly from a tuberculosis-susceptible strain, with the corresponding region of ENCODE ENm002 from a normal rabbit as well as with 9 other mammalian species. We searched for rabbit transcription factor binding sites in putative promoter and other non-coding regions of IL5, RAD50, IL13 and IL4. Although we identified several differences between the two donor rabbits in coding and non-coding regions of potential functional significance, confirmation awaits additional sequencing of other rabbits.

Gene expression profiles in a rabbit model of systemic lupus erythematosus autoantibody production.

We previously reported the establishment of a rabbit (Oryctolagus cuniculus) model in which peptide immunization led to production of lupus-like autoantibodies including anti-Sm, -RNP, -SS-A, -SS-B, and -dsDNA characteristic of those produced in systemic lupus erythematosus (SLE) patients. Some neurologic symptoms in the form of seizures and nystagmus were observed. The animals used in the previous and in the current study were from a National Institute of Allergy and Infectious Diseases colony of rabbits that were pedigreed, Ig-allotype defined, but not inbred. Their genetic heterogeneity may correspond to that found among patients of a given ethnicity. We extended the information about this rabbit model by microarray-based expression profiling. We first demonstrated that human expression arrays could be used with rabbit RNA to yield information on molecular pathways. We then designed a study evaluating gene expression profiles in eight groups of control and treated rabbits (47 rabbits in total). Genes significantly upregulated in treated rabbits were associated with NK cytotoxicity, Ag presentation, leukocyte migration, cytokine activity, protein kinases, RNA spliceosomal ribonucleoproteins, intracellular signaling cascades, and glutamate receptor activity. These results link increased immune activation with upregulation of components associated with neurologic and anti-RNP responses, demonstrating the utility of the rabbit model to uncover biological pathways related to SLE-induced clinical symptoms, including neuropsychiatric lupus. Our finding of distinct gene expression patterns in rabbits that made anti-dsDNA compared with those that only made other anti-nuclear Abs should be further investigated in subsets of SLE patients with different autoantibody profiles.

Generation and characterization of a chimeric rabbit/human Fab for co-crystallization of HIV-1 Rev.

Rev is a key regulatory protein of human immunodeficiency virus type 1. Its function is to bind to viral transcripts and effect export from the nucleus of unspliced mRNA, thereby allowing the synthesis of structural proteins. Despite its evident importance, the structure of Rev has remained unknown, primarily because Rev's proclivity for polymerization and aggregation is an impediment to crystallization. Monoclonal antibody antigen-binding domains (Fabs) have proven useful for the co-crystallization of other refractory proteins. In the present study, a chimeric rabbit/human anti-Rev Fab was selected by phage display, expressed in a bacterial secretion system, and purified from the media. The Fab readily solubilized polymeric Rev. The resulting Fab/Rev complex was purified by metal ion affinity chromatography and characterized by analytical ultracentrifugation, which demonstrated monodispersity and indicated a 1:1 molar stoichiometry. The Fab binds with very high affinity, as determined by surface plasmon resonance, to a conformational epitope in the N-terminal half of Rev. The complex forms crystals suitable for structure determination. The ability to serve as a crystallization aid is a new application of broad utility for chimeric rabbit/human Fab. The corresponding single-chain antibody (scFv) was also prepared, offering the potential of intracellular antibody therapeutics against human immunodeficiency virus type 1.

Investigations of a rabbit (Oryctolagus cuniculus) model of systemic lupus erythematosus (SLE), BAFF and its receptors.

B-cell activation factor belonging to the tumor necrosis factor family (BAFF) is a major contributor to survival of B lymphocytes during development and maturation. A relationship between circulating BAFF levels and disease activity has been reported in patients with the autoimmune disease Systemic Lupus Erythematosus (SLE). Clinical trials targeting BAFF or its receptors are currently in progress. In order to further characterize a rabbit (Oryctolagus cuniculus) model of SLE, we investigated the expression of BAFF and its receptors in non-inbred, pedigreed rabbits derived from breeding and selection based on autoantibody responses. We immunized rabbits related to previous groups that developed autoantibodies and inflammatory responses after immunizations with peptides synthesized on multiple antigen-branched polylysine backbones. Blood and sera collected before immunization and after boosts were used for health monitoring, analyses of serum autoantibody responses by ELISA and immunofluorescence. Peripheral blood mononuclear cells (PBMC) were studied by flow cytometry and were the source of mRNA for quantitative PCR analyses. We hypothesized that BAFF mRNA expression and serum BAFF levels measured indirectly through BAFF receptor binding might increase in autoantibody-producing rabbits. Immunized rabbits developed elevated levels of leucocyte populations, anti-nuclear, anti-dsDNA and other autoantibodies. BR3 mRNA levels in total PBMC decreased and BAFF levels remained low and unchanged in most immunized rabbits. By flow cytometry, percentages of BAFF positive cells decreased. Percentages of transmembrane activator and CAML interactor (TACI) decreased in most rabbits from all the immunized groups. The rabbit is an important model for human autoimmune and infectious diseases, and a high quality draft rabbit genome assembly was recently completed. Human disease models developed in non-inbred pedigreed animals are better able to reflect the complexities of diseases such as SLE with familial patterns of inheritance. Although no consistent pattern of elevated expression of BAFF mRNA or protein was found in the rabbits studied, the data collected and reported here build upon previous data to refine understanding of a rabbit model of SLE.

Characterization of rabbit CD5 isoforms.

Previously described polyclonal or monoclonal antibodies (mAb) to rabbit CD5, raised against expressed recombinant protein or peptides, recognize CD5 on most rabbit B cells. The mAb KEN-5 was originally reported to recognize rabbit CD5. However, KEN-5 binds almost exclusively to T cells and only to a minor population of B cells. We show here that by Enzyme-linked Immunosorbent Assay (ELISA), KEN-5 binds to recombinant rabbit CD5. This interaction is partially inhibited by polyclonal goat anti-CD5 antibody. In addition, immunoprecipitations from lysates of surface biotinylated rabbit lymphocytes with KEN-5 or our anti-CD5 mAb isolate molecules that migrate identically on gels with the same approximate relative molecular mass of 67,000 M(r). By flow cytometric analyses of individual cells from spleen, thymus and appendix, KEN-5 recognizes CD5-like molecules mainly on T cells and on 3-6% of IgM(+) B cells. Immunohistochemical staining of splenic and appendix tissues and confocal immunofluorescent imaging confirm and extend results from flow cytometric analyses. Quantitation of fluorescent colocalization indicates that staining by KEN-5 colocalizes with staining by anti-CD5 on small percentage lymphocytes in splenic tissue sections. As CD5 has both N- and O-linked glycosylation, we hypothesised that differential binding of KEN-5 to T cells and B-cells may be explained by different glycan structures on the CD5 present on T compared to B cells. This hypothesis is supported by ELISA data that show that deglycosylation diminishes the binding of KEN-5 to recombinant rabbit CD5. Screening KEN-5 on an array with 406 glycans was inconclusive. Although we did not identify a strongly binding glycan structure, the data are suggestive that the epitope recognized by KEN-5 may be influenced by glycan structures. The epitope this mAb recognizes may either be the glycan itself, or more likely, is influenced by neighboring glycan structure. Our findings suggest that development, selection and function of different B- and T-cell subsets or their preferential survival may be directly or indirectly dependent on different glycan structures associated with CD5 or CD5-like molecules expressed on T cells compared to B cells.

Molecular basis of the interactions of the Nogo-66 receptor and its homolog NgR2 with myelin-associated glycoprotein: development of NgROMNI-Fc, a novel antagonist of CNS myelin inhibition.

Myelin-associated glycoprotein (MAG) is a sialic acid-binding Ig-family lectin that functions in neuronal growth inhibition and stabilization of axon-glia interactions. The ectodomain of MAG is comprised of five Ig-like domains and uses neuronal cell-type-specific mechanisms to signal growth inhibition. We show that the first three Ig-like domains of MAG bind with high affinity and in a sialic acid-dependent manner to the Nogo-66 receptor-1 (NgR1) and its homolog NgR2. Domains Ig3-Ig5 of MAG are sufficient to inhibit neurite outgrowth but fail to associate with NgR1 or NgR2. Nogo receptors are sialoglycoproteins comprised of 8.5 canonical leucine-rich repeats (LRR) flanked by LRR N-terminal (NT) and C-terminal (CT)-cap domains. The LRR cluster is connected through a stalk region to a membrane lipid anchor. The CT-cap domain and stalk region of NgR2, but not NgR1, are sufficient for MAG binding, and when expressed in neurons, exhibit constitutive growth inhibitory activity. The LRR cluster of NgR1 supports binding of Nogo-66, OMgp, and MAG. Deletion of disulfide loop Cys(309)-Cys(336) of NgR1 selectively increases its affinity for Nogo-66 and OMgp. A chimeric Nogo receptor variant (NgR(OMNI)) in which Cys(309)-Cys(336) is deleted and followed by a 13 aa MAG-binding motif of the NgR2 stalk, shows superior binding of OMgp, Nogo-66, and MAG compared with wild-type NgR1 or NgR2. Soluble NgR(OMNI) (NgR(OMNI)-Fc) binds strongly to membrane-bound inhibitors and promotes neurite outgrowth on both MAG and CNS myelin substrates. Thus, NgR(OMNI)-Fc may offer therapeutic opportunities following nervous system injury or disease where myelin inhibits neuronal regeneration.

Expression and localization of rabbit B-cell activating factor (BAFF) and its specific receptor BR3 in cells and tissues of the rabbit immune system.

Rabbits are widely used for vaccine development, and investigations of human infectious and autoimmune diseases such as Systemic Lupus Erythematosus (SLE). For these applications, we cloned, sequenced and expressed rabbit B-cell Activating Factor (BAFF), and localized BAFF in cells and tissues of the rabbit immune system. The rabbit homolog of the human BAFF binding site (miniBR3 peptide) within the BAFF-specific receptor BR3 was synthesized. This 26-residue core domain binds to recombinant rabbit BAFF protein. Flow cytometric analyses using purified recombinant rabbit BAFF combined with real-time PCR findings revealed that BAFF detected on peripheral blood B-cells from normal rabbits is probably complexed to BAFF receptors rather than produced by the B-cells. BAFF was detected in developing appendix of young rabbits by immunohistochemical staining suggesting that BAFF plays a role during the period following birth when rabbit B-cell development and pre-immune antibody repertoire diversification and selection is occurring.

Genetic contributions to the autoantibody profile in a rabbit model of Systemic Lupus Erythematosus (SLE).

For the development of rabbit models of Systemic Lupus Erythematosus (SLE), immunoglobulin allotype-defined pedigreed rabbits from the National Institute of Allergy and Infectious Diseases rabbit resource more closely approximate human populations due to their non-inbred pedigreed structure. In an initial study from this laboratory, peptides (SM and GR) from the spliceosomal Smith (Sm) and the NMDA glutamate receptor NR2b, on branched polylysine backbones (BB) elicited antinuclear and anti-dsDNA autoantibodies typical of SLE, as well as seizures and nystagmus sometimes observed as neurological manifestations in SLE patients. This suggested the feasibility of further selective breeding to develop a more reproducible rabbit model for investigations of SLE. Here we report the results of GR-MAP-8 and control BB immunization on autoantibody responses in a group of 24 rabbits specifically bred and developed from parents and ancestors tested for autoantibody responses. The changes in hematological profile and blood chemistry in the experimental rabbits were evaluated along with autoantibody responses. Elevations of total white blood cell (WBC), monocyte, eosinophil and basophil counts that developed following immunizations were moderately influenced by litter and presence of the antibody heavy chain allotype VH1a1. Autoantibody development followed a sequential pattern with anti-nuclear antibodies (ANA) followed by anti-dsDNA and subsequently anti-Sm and anti-RNP similar to SLE patients. High autoantibody levels to one autoantigen were not always associated with antibody response to another. Female rabbits had higher prevalence and levels of autoantibodies similar to human SLE. Higher autoantibody levels of anti-dsDNA and -ANA were observed among some full sibs and the presence of high responder ancestors in the pedigree was associated the augmented responses. We observed significant association between highest antibody responses to GR-MAP-8 and highest anti-dsDNA levels. Naturally occurring autoantibodies were found in some pre-immune sera and some unique ANA fluorescent staining patterns within the experimental group were observed. Background immunofluorescence in pre-immune sera, distinct patterns of programmed autoantibody responses unique among individual rabbits may have been modulated by genetic constitution, gender and environmental factors including exposure to antigens. The high incidence and intensity of autoantibody responses among descendants of high responders suggest that there may be an additive mode of inheritance with high heritability. It is conceivable that further rigorous pedigree selection for autoantibody responses could lead to development of rabbit models with spontaneous occurrence of SLE like serology and disease phenotypes.

Antibody elicited against the gp41 N-heptad repeat (NHR) coiled-coil can neutralize HIV-1 with modest potency but non-neutralizing antibodies also bind to NHR mimetics.

Following CD4 receptor binding to the HIV-1 envelope spike (Env), the conserved N-heptad repeat (NHR) region of gp41 forms a coiled-coil that is a precursor to the fusion reaction. Although it has been a target of drug and vaccine design, there are few monoclonal antibody (mAb) tools with which to probe the antigenicity and immunogenicity specifically of the NHR coiled-coil. Here, we have rescued HIV-1-neutralizing anti-NHR mAbs from immune phage display libraries that were prepared (i) from b9 rabbits immunized with a previously described mimetic of the NHR coiled-coil, N35(CCG)-N13, and (ii) from an HIV-1 infected individual. We describe a rabbit single-chain Fv fragment (scFv), 8K8, and a human Fab, DN9, which specifically recognize NHR coiled-coils that are unoccupied by peptide corresponding to the C-heptad repeat or CHR region of gp41 (e.g. C34). The epitopes of 8K8 and DN9 were found to partially overlap with that of a previously described anti-NHR mAb, IgG D5; however, 8K8 and DN9 were much more specific than D5 for unoccupied NHR trimers. The mAbs, including a whole IgG 8K8 molecule, neutralized primary HIV-1 of clades B and C in a pseudotyped virus assay with comparable, albeit relatively modest potency. Finally, a human Fab T3 and a rabbit serum (both non-neutralizing) were able to block binding of D5 and 8K8 to a gp41 NHR mimetic, respectively, but not the neutralizing activity of these mAbs. We conclude from these results that NHR coiled-coil analogs of HIV-1 gp41 elicit many Abs during natural infection and through immunization, but that due to limited accessibility to the corresponding region on fusogenic gp41 few can neutralize. Caution is therefore required in targeting the NHR for vaccine design. Nevertheless, the mAb panel may be useful as tools for elucidating access restrictions to the NHR of gp41 and in designing potential improvements to mimetics of receptor-activated Env.

Chimeric rabbit/human Fab and IgG specific for members of the Nogo-66 receptor family selected for species cross-reactivity with an improved phage display vector.

NgR1, NgR2, and NgR3 which constitute the Nogo-66 receptor family are primarily expressed by neurons in the central nervous system (CNS) and believed to limit axonal growth and sprouting following CNS injury. In an attempt to define the expression and decipher the function of individual members of the Nogo-66 receptor family, we previously reported the generation of selective rabbit polyclonal antibodies. Here we exploit the same immune repertoires by phage display technology to generate rabbit monoclonal antibodies (mAbs) with nanomolar affinity to epitopes that are specific for NgR1 and NgR2, respectively, but at the same time conserved between mouse, rat, and human orthologs. Employing phage display vector pC3C, a newly designed phagemid optimized for the generation and selection of Fab libraries with human constant domains, rabbit mAbs were selected from chimeric rabbit/human Fab libraries, characterized in terms of specificity, affinity, and amino acid sequence, and finally converted to chimeric rabbit/human IgG. Using immunofluorescence microscopy and immunoprecipitation, we demonstrate strong and specific recognition of cell surface bound Nogo-66 receptor family members by chimeric rabbit/human IgG. The rabbit mAbs reported here together with their amino acid sequences constitute a defined panel of species cross-reactive reagents in infinite supply which will aid investigations toward a functional role of the Nogo-66 receptor family in and beyond the CNS.

De novo expression of MECA-79 glycoprotein-determinant on developing B lymphocytes in gut-associated lymphoid tissues.

Rabbit is one of several species that depend on development of B lymphocytes in gut-associated lymphoid tissues for primary immunoglobulin-repertoire diversification. The rabbit appendix is an important site of early B-lymphocyte development. We previously reported that peripheral lymph node addressin detected by monoclonal antibody (mAb) MECA-79 played a role in recruitment of immature blood-borne B cells into neonatal rabbit appendix. Here, we report expression of an approximately 127 000 MW O-linked sulphated proteoglycan on developing B cells in appendix and Peyer's patches recognized by the mAb MECA-79. Binding of the mAb to B lymphocytes was sensitive to enzyme treatment with O-sialoglycoprotease and expression was partially inhibited by sodium chlorate, a metabolic inhibitor of sulphation. The proportions of MECA-79(+) B lymphocytes gradually increased from < 0.5% at 3 days to > 70% at 6 weeks in appendix and Peyer's patches. The proportions of MECA-79(+) B lymphocytes in spleen and peripheral blood were very low (0.5-2%). However, the MECA-79 determinant was detected on B cells in splenic germinal centres after immunization. In situ labelling of appendix cells showed that the MECA-79 determinant was expressed on fluorescein-labelled B lymphocytes that migrated from appendix into mesenteric lymph nodes. B-cell MECA-79 may be involved in interactions with T cells and/or dendritic cells. Alternatively, because we found that lymphatic endothelium in the thymus-dependent area of appendix, a site for lymphocyte exit, expressed P-selectin (CD62P), interaction of the MECA-79 determinant on B cells with CD62P may have a role in the exit of B lymphocytes from rabbit appendix.

B cell and antibody repertoire development in rabbits: the requirement of gut-associated lymphoid tissues.

The antibody repertoire of rabbits has interested immunologists for decades, in part because of the ease with which large quantities of high affinity antibodies can be obtained in serum, and in part because of the presence of genetic variants, allotypes, within V(H), C(H) and C(L) regions. Studies of these allotypes led to the initial descriptions of allelic exclusion, and neonatal suppression of serum Ig production (allotype suppression), and were instrumental in demonstrating that V and C regions are encoded by separate genes and are usually expressed in cis. The immune system of rabbit continues to be of interest primarily because of the use of both gene conversion and somatic hypermutation to diversify rearranged heavy and light chain genes and the role that gut-associated lymphoid tissues (GALT) and intestinal flora play in developing the primary (preimmune) antibody repertoire.

CD5+ B cells are preferentially expanded in rabbit appendix: the role of CD5 in B cell development and selection.

Although only a small proportion of mouse and human B cells are CD5(+), most adult rabbit B cells express CD5. However, CD5 was not detectable on the majority of B cells in neonatal appendix 1 and 3days after birth. Cell trafficking studies demonstrated that CD5(+) and CD5(-) CD62L(+) B cells from bone marrow migrated into appendix. There, CD5(+) B cells were preferentially expanded and predominated by approximately 2weeks of age. In mutant ali/ali rabbits, VHa2(+) B cells develop through gene conversion-like alteration of rearranged VH genes upstream of deleted VH1a2. Correlated appearance of individual CD5(+) germinal centers and VHa2(+) B-cells in mutant appendix suggests that CD5 binding positively selects cells with a2(+) framework regions that bind CD5. Following negative and positive selection, cells with diversified rearranged heavy- and light-chain sequences exit appendix, migrate to peripheral tissues and constitute the preimmune repertoire of CD5(+) B cells that encounter foreign antigens.

Regulated expression of peripheral node addressin-positive high endothelial venules controls seeding of B lymphocytes into developing neonatal rabbit appendix.

Young rabbit appendix is a homologue of chicken bursa of Fabricius; both are crucial sites for preimmune B-cell repertoire diversification. Here, we report that appendix regulates precursor lymphocyte recruitment for further development by modulating the sites of extravasation. The total area of peripheral node addressin-positive (PNAd(+)) high endothelial venules (HEVs) increased from 1 day to 1 week after birth, remained constant up to 2 weeks and declined to a low and persistent amount by 3 weeks. In normal 1-week and manipulated 5-week appendix where growth of follicles was retarded, PNAd(+) HEVs were present in the basolateral sides of B-cell follicles whereas, in normal 5-wk-appendix these were restricted to T-cell areas. The PNAd was expressed on the lumenal surface of HEVs. The proportions of CD62L(+) B cells in appendix declined from approximately 40% at 3 days to 2-3% at 4 weeks. In lymphocyte transfer experiments, CD62L(+) B cells were preferentially recruited compared with CD62L(-) B cells, anti-PNAd antibody blocked migration of B cells by approximately 50%, and 100 times more B cells were recruited in 1-week compared to 6-week appendix. Thus, a unique spatiotemporal expression pattern of PNAd(+) HEVs is associated with development of B-cell follicles. This regulates migration of blood-borne B-lymphocytes into developing appendix by interacting with CD62L.

Models of systemic lupus erythematosus: development of autoimmunity following peptide immunizations of noninbred pedigreed rabbits.

Reported in this study are the initial results from studies to develop rabbit models of systemic lupus erythematosus (SLE) by immunizations using two distinct peptides on branched polylysine backbones (multiple Ag peptide)-peptides. Eleven rabbits received a peptide from the Sm B/B' spliceosomal complex previously shown to be immunogenic in rabbits, and 13 rabbits received a peptide from the rabbit N-methyl-d-aspartate receptor NR2b. All 24 animals in different generations of pedigreed, noninbred rabbits produced peptide-specific responses. Anti-nuclear autoantibody responses, including anti-dsDNA, were seen in 17 of 24 rabbits. To date, two rabbits have been observed to have seizure-like events and a third nystagmus. A model for eliciting development of SLE in genetically related yet heterogeneous rabbits may more closely resemble development of human SLE than do some models in inbred mice. Through selective breeding, it may also ultimately provide additional information about the genetics and etiology of SLE and serve as a model for assessing new treatment options.

Activation-induced deaminase cloning, localization, and protein extraction from young VH-mutant rabbit appendix.

Studies in mouse, human, and chicken suggest that activation-induced deaminase (AID) is involved in three known processes leading to antibody diversification: somatic hypermutation, gene conversion, and class-switch recombination. Developing rabbit appendix provides a particularly good site for studying all three of these B cell maturation events. We report here successful cloning of rabbit AID and isolation of AID protein from rabbit appendix-cell nuclear and cytoplasmic extracts. We succeeded in identifying and locating AID protein in cells by immunohistochemical and immunofluorescent staining techniques and examined colocalization of AID and other molecules important for Ab diversification. This report extends our knowledge about AID to a mammalian species that uses gene conversion to diversify rearranged Ig genes. Although much work remains to understand fully the mechanism of action of AID and its association with other cellular components, the rabbit system now offers a particularly useful model for future studies of these dynamics.