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The specificity and affinity of polyclonal antibodies raised in rabbits against a hapten conjugated to rabbit serum albumin.

In this study, rabbit antisera to hapten-rabbit serum albumin conjugates were investigated regarding antibody titer, affinity, specificity, and affinity distribution. Methyl phosphonic acid p-amino-phenyl 1,2,2-trimethylpropyldiester (MATP) served as model hapten. Four MATP-rabbit serum albumin conjugates with various hapten densities (with and without spacer) were synthesized and used for immunization of rabbits. Antisera were collected over a 130 day-period and characterized with different ELISA methods. We found that immunogens with rabbit serum albumin gave antisera with lower titers, but similar affinity as compared to polyclonal or monoclonal antibodies obtained with non-rabbit protein as carrier protein. Immunogens with a low hapten density led to higher final titers without affecting antibody affinity or specificity. Immunogens containing a bridging group resulted in higher antibody affinity with a changed specificity. The pattern of antibody affinity distribution differed considerably among individual rabbits and showed a non-Gaussian subpopulation distribution.

Animals↗

Rabbit homocytotropic antibody. A unique rabbit immunoglobulin analogous to human IgE.

Rabbits immunized with egg albumin produce a homocytotropic antibody. The antibody is identified by its ability to produce passive anaphylaxis in rabbit skin. The time of appearance of this antibody, its persistence and recall after booster injections depends, in part, on the route of immunization and the adjuvant employed. The physicochemical characteristics of the homocytotropic antibody obtained was similar regardless of the immunization schedule used. The anaphylactic activity of these antisera showed some heterogeneity when chromatographed on diethylaminoethyl (DEAE)-cellulose, but all fractions were inactivated by heating and absorption with a specific antisera. The anaphylactic activity could be separated from rabbit IgG and IgA, and was not blocked by absorption with antisera specific for these classes of immunoglobulins. Anaphylactic activity was completely removed by absorption with a specific antiserum which did not react with any of the known rabbit immunoglobulins. The passive cutaneous anaphylaxis titer of a rabbit serum containing homocytotropic antibody was reduced by 50% after absorption with an antisera (anti-FcND) specific for human IgE. On the basis of these distinctive physicochemical characteristics, it is concluded that rabbit homocytotropic antibody represents a unique class of rabbit immunoglobulin, analogous to human IgE.

Absorption↗

Infection of human CD4+ rabbit cells with HIV-1: the possibility of the rabbit as a model for HIV-1 infection.

Although human T cell surface glycoprotein CD4 is the cellular receptor for human immunodeficiency virus 1 (HIV-1), the introduction of the human CD4 gene into murine cells does not render them susceptible to HIV-1 infection. Here we have established rabbit transfectant cell lines expressing human CD4 on the cell surface and demonstrated that the CD4+ rabbit transfectants could be readily infected by HIV-1 by co-cultivating with a HIV-1-infected human MOLT-4 T cell line (MOLT-4/HIV). Avid syncytia formation was observed upon co-cultivation and the syncytia abundantly produced HIV-1 mature particles, as revealed by electron microscopy. A significant increase of HIV-1 p24 antigen was also detected in the culture supernatant. The syncytia formation was blocked by pretreating the transfectant with anti-human CD4 or by pretreating the MOLT-4/HIV with anti-HIV-1 serum obtained from an infected individual, indicating that the syncytia formed as a result of the interaction of human CD4 on the rabbit transfectant with the HIV-1 envelope protein expressed on MOLT-4/HIV. In contrast, only a very small proportion of the rabbit transfectants expressed HIV-1-specific antigens upon infection with an HIV-1 stock. This may indicate that, although rabbit cells have partially acquired susceptibility to HIV-1 by transfection of human CD4 gene, rabbit cells may further require such a molecule as might be provided by MOLT-4 to become fully susceptible to HIV-1 infection. The possibility of the rabbit as a model for HIV-1 infection is also discussed.

Animals↗

Determinant differences between the rabbit and mouse immunoglobulin kappa enhancers impair the activity of the rabbit enhancer in mouse myeloma cells.

Enhancer activity of the rabbit immunoglobulin kappa light chain gene intron conserved region (KICR) was examined in mouse myeloma cells using transient expression experiments. Compared to the homologous region of the mouse kappa light chain gene, the rabbit KICR shows nearly no stimulatory effect on expression of the indicator gene, cat. Experiments with mouse-rabbit chimeric KICRs indicated that differences in the region around the NF-kappa B binding site are responsible for the impaired activity of the rabbit KICR whereas mouse sequences covering the kappa E2 and kappa E3 motifs can be replaced by the equivalent rabbit fragment without affecting enhancer function. Creation of a perfect mouse NF-kappa B target sequence in the rabbit gene only partially restores enhancer activity. Furthermore, mouse and rabbit DNA fragments encompassing the NF-kappa B target sequence behave in an identical manner in an electrophoretic mobility shift assay. The results indicate species-related functional differences in the immunoglobulin kappa light chain gene enhancer and suggest that although the NF-kappa B binding site plays a crucial role in enhancer activity surrounding gene elements are also necessary for full enhancer effect.

Animals↗

Infertility in female rabbits (Oryctolagus cuniculus) alloimmunized with the rabbit zona pellucida protein ZPB either as a purified recombinant protein or expressed by recombinant myxoma virus.

Development of immunocontraceptives for wild rabbit populations requires selection of both effective antigens and effective delivery systems. Recombinant rabbit zona pellucida glycoprotein B (ZPB) produced in eukaryotic cells in vitro was an effective antigen and induced sustained infertility in 70% of female rabbits. This required two boosts and serum antibody titers of 12 800 or greater. Antibody titers in females were low after the initial immunization, as might be expected with a self-antigen; however, male rabbits had a strong antibody response, indicating that the protein was immunologically foreign. To develop a delivery system, ZPB was delivered by infection with a recombinant myxoma virus. In contrast to the results with ZPB protein, infection of rabbits induced a similar serum antibody response to ZPB in both sexes. This indicated that presentation of ZPB in the context of a virus infection was able to overcome tolerance in females. However, the antibody titers were lower than 12 800, and only 25% of female rabbits were infertile. This antibody response was boosted by injections of recombinant ZPB protein, after which 80% of female rabbits were infertile. Infertility was associated with antibody binding to zonae and varying degrees of ovarian pathology characterized by follicular degeneration and substantial depletion of primordial follicles. Oocyte and follicular degeneration appeared to be the principal mechanism of infertility and may be primarily induced by antibodies to ZPB.

Animals↗

Pharmacological studies on prostanoid receptors in the rabbit isolated saphenous vein: a comparison with the rabbit isolated ear artery.

1. In isolated ring preparations of the rabbit saphenous vein, prostaglandin E2 (PGE2) caused well-defined, stable and concentration-dependent relaxations of KCl contracted tissues with a mean potency (p[A50]) of 9.39. 2. The prostanoid EP-receptor agonists, PGE1, 11-deoxy PGE1, 16,16-dimethyl PGE2 and misoprostol were all full agonists in this preparation. The EP2-receptor selective agonists, butaprost and AH13205, and the EP1/EP3-receptor selective agonist, sulprostone, also relaxed this tissue but were at least 300 times less potent than PGE2. 3. Prostaglandin D2 (PGD2), the DP-receptor agonist, BW245C, and the IP-receptor agonist, cicaprost, caused concentration-dependent relaxations of the rabbit saphenous vein but were at least 60 times less potent than PGE2. 4. The selective EP4-receptor antagonist, AH23848B (30 microM), antagonized the PGE2 concentration-effect (E/[A]) curves yielding a pA2 estimate of 4.96. The EP1/DP-receptor antagonist, AH6809 (10 microM), had no effect on the location of PGE2 E/[A] curves. 5. The stable thromboxane A2-mimetic, U46619, elicited concentration-dependent contractions of the rabbit saphenous vein (p[A50] = 8.01) however, PGE2 and prostaglandin F2 alpha (PGF2 alpha) were unable to produce a contractile response. The response to U46619 was competitively antagonized by the TP-receptor antagonist, GR32191B, yielding a pKB estimate of 7.08. 6. In the rabbit isolated ear artery, PGE2, misoprostol and AH13205 relaxed tissues pre-contracted with phenylephrine. PGE2 (p[A50] = 7.04) and misoprostol were equipotent, whereas AH13205 was some 40 fold less potent. AH23848B (30 microM) and AH6809 (1 and 10 microM) caused no significant shift in the location of PGE2 E/[A] curves. 7. These data suggest that the rabbit isolated saphenous vein contains prostanoid, EP-, DP-, IP- and TP-receptors. Based on antagonist affinity information and agonist potency orders, the rabbit saphenous vein contains an inhibitory prostanoid EP-receptor different from that in the rabbit ear artery, but comparable to the recently described EP4-receptor.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

Lack of serological evidence for venereal spirochaetosis in wild Victorian rabbits and the susceptibility of laboratory rabbits to Treponema paraluis-cuniculi.

Sera from 608 wild rabbits were examined using serological tests for syphilis as an indicator of infection with Treponema paraluis-cuniculi. Only eight sera gave positive or weakly positive results in the rapid plasma reagin (RPR) test, and none of these eight sera gave positive results in the Treponema pallidum haemagglutination assay (TPHA). Thus, it appears that wild rabbit populations in Victoria, Australia, are not naturally infected with T paraluis-cuniculi. Normal Australian laboratory rabbits however were readily infected with T paraluis-cuniculi, either by intradermal or intratesticular inoculation or by the venereal route. In the latter case, treponeme-containing lesions developed after about five months' cohabitation with infected mates. The disease was successfully transmitted from male to female and from female to male rabbits by the venereal route. In most cases infected rabbits became RPR-positive (17/19 rabbits) and in all cases TPHA-positive (19/19), indicating that serological tests for syphilis can be used to screen rabbits for this disease.

Animals↗

[Rearing of germfree rabbits and establishment of an SPF rabbit colony].

Baby rabbits hysterectomy-derived from conventional Japanese white rabbits were reared under aseptic condition by feeding with 4 types of artificial diets. Rabbit milk for the artificial diet was obtained from conventional dams at 7--25 days after delivery. The artificial diets was given by stomach tube twice a day. The total volumes of diet given (ml per day) were Y = 2.3X + a (1 to approximately 14 days of age), Y = 32.2 + a (l5 to approximately 25 days of age), or Y = (32.2 + a) - 37.5 (X - 25) (26 to approximately 34 days of age), (X = age in days, a = volumes fed at 0 day of age). After 14 days of age young animals were also fed freely sterilized commercial pellets and weaned at 35 days of age. Out of 155 rabbits, 130 were aseptically reared till 36 approximately 40 days of age, and no difference on weaning rate was seen between the 4 groups of rabbits. Thereafter, they were exposed to a barriered room of SPF rabbitry outside the isolators. The best growth was seen in animals given by artificial diet containing rabbit milk at 40%. The SPF breeding colony of rabbits was found to be free of Pasteurella pneumotropica, Bordetella bronchiseptica, Salmonella spp., Eimeria spp., Encephalitozoon cuniculi and Acaritic otistis.

Animal Feed↗

High density lipoprotein (HDL) polymorphisms in rabbit. I. A comparative study of rabbit and human serum high density lipoprotein.

Different classes of rabbit serum lipoprotein were prepared by ultracentrifugal flotation at densities 1-006, 1-063 and 1-21 g/ml. Agarose gel electrophoresis on rabbit whole serum and the serum fractions with different densities showed that this technique separates the different lipoprotein classes reasonably well. The electrophoretic mobility of the different lipoprotein classes of rabbit serum seems to be similar to that of the human lipoproteins, with the exception of alpha1-lipoprotein which had a greater mobility than human alpha1-lipoprotein. The chemical composition of rabbit high density lipoprotein (HDL)p was fairly similar to that of human HDL although the former seems to be richer in triglycerides. HDL was, after isolation by ultracentrifugal flotation at density 1-21, delipidated and submitted to gel filtration on Sephadex G-200 in 8 M urea. The major protein fraction of rabbit apo HDL corresponds in elution volume to that of the major fraction of human apo HDL, apoA-I. A protein fraction corresponding to human apoA-II does not seem to be present in rabbit HDL in demonstrable amounts. The rabbit protein fraction sometimes appearing in the area corresponding to human apoA-II could not be found to be affected by the reduction and alkylation method after which human poA-II splits into two identical chains.

Animals↗

A reevaluation of rabbit anti-allotype antibody for the presence of cross-reactive idiotypes. I. A species-specific idiotype on rabbit anti-a1 antibody is recognized by guinea pig anti-IdX antibody.

Rabbit reagents previously thought to display specificity for a cross-reactive idiotype on anti-VHa allotype antibody from all tested rabbits have recently been shown to be contaminated with an induced (latent) molecule similar or identical to the original antigen (rabbit a1 or a2 allotype). In an attempt to circumvent this problem, we have immunized guinea pigs with rabbit anti-a1 allotype antibody to produce heterologous anti-idiotype antibody. The resulting guinea pig antibody (GP anti-R IdX) recognizes anti-a1 antibody from each of 17 immunized rabbits, and in four tested samples reacts with 22 to 100% of the molecules. Neither goat nor guinea pig anti-a1 reacts with the guinea pig anti-R IdX antibody, even though the goat, guinea pig, and rabbit anti-a1 Ab all recognize a similar set of a1 determinants. The reaction between IdX-bearing rabbit anti-a1 and guinea pig anti-R IdX is inhibited by the original antigen (a1 IgG), demonstrating that the IdX is at or near the antigen combining site of anti-a1 antibody. Immunoelectron microscopy of immune complexes supports this conclusion and demonstrates that the reactive site on the GP anti-R IdX is at or near its antigen combining site.

Animals↗

Accumulation of cholesterol ester in cultured smooth muscle cells of congenital hypercholesterolemic (WHHL) rabbits treated with normal and WHHL rabbit plasma LDL.

The DNA content, thymidine incorporation into DNA, and free and esterified cholesterol contents of cultured aortic smooth muscle cells (SMCs) of normal and WHHL rabbits were compared. In the first series of experiments, 5 groups of cultured normal rabbit aortic SMCs were compared: control cells and cells treated with LDL from normolipemic rabbit plasma (LDLN) and (DL from hypercholesterolemic rabbit plasma (LDLW), LDLN plus esterastin and LDLW plus esterastin. In the second series, the same groups of hereditable hypercholesterolemic WHHL rabbit aortic SMCs were compared. Results obtained with normal aortic SMCs showed that internalization of LDLW was higher than that of LDLN. LDLW was also more effective than LDLN for elevating thymidine incorporation into DNA. LDLW plus esterastin caused increases of 5, 7 and 3 times the control values in thymidine incorporation, and esterified and free cholesterol contents of the cells, respectively. In the control groups, thymidine incorporation into DNA of WHHL SMCs was 12 times more than that into the normal cells, and the free cholesterol content of WHHL cells was twice that of normal cells. Addition of LDLN caused further increases in thymidine incorporation and the esterified and free cholesterol contents of the cells. Esterastin had only a slight effect on these extremely high values. LDLW itself had no effect except that when added with esterastin it increased the cholesterol ester content of WHHL cells. The results are discussed with respect to metabolic differences between cultured aortic SMCs of normal and WHHL rabbits, and LDLs prepared from normal and hypercholesterolemic (WHHL) rabbit plasma.

Animals↗

Selective activation of rabbit ovarian protein kinase isozymes in rabbit ovarian follicles and corpora lutea.

The magnitude of activation of the type I and type II forms of cAMP-dependent protein kinase was investigated in estrous follicles and corpora lutea (CL) obtained from ovaries of control rabbits and rabbits injected acutely with human chorionic gonadotropin (hCG). To this end, a chromatographic technique which permitted quantitative evaluation of the in vivo activational state of the two forms of cAmP-dependent protein kinase was developed and verified. Results revealed that in follicles obtained from ovaries of untreated estrous rabbits, 15% of the soluble cAMP-dependent protein kinase, all of which exists as the type II isozyme, is activated. Intravenous administration of a single bolus of hCG promoted a concentration-dependent activation (in 10 min) of this protein kinase isozyme. In CL obtained from ovaries of control, 4-day pseudopregnant rabbits, 32% of the total soluble cAMP-dependent protein kinase exists as the type I form and 68% exists as the type II form. Both types of protein kinase are approximately 10% dissociated in CL from ovaries of untreated rabbits. Upon intravenous administration of hCG, only the type I form of cAMP-dependent protein kinase is further activated (in 10 min). Dissociation of this protein kinase is dependent upon the time and concentration of hCG. Preferential activation of the type I form of cAMP-dependent protein kinase in CL is also demonstrable in in vitro studies using exogenous cAMP. These data suggest that the physiological intracellular mediator of acute cAMP-regulated, hCG-triggered functions in rabbit ovarian follicles is the type II isozyme of cAMP-dependent protein kinase while in CL of 4-day pseudopregnant rabbits, it is the type I enzyme form.

Animals↗

[A morphological study of the lesioned carotid artery--comparison with WHHL rabbit and Japanese white rabbit].

It is necessary for a neurosurgeon to know the course of repair of vascular injury caused by the procedure of an operation. And further more, it is very important to understand how existence of hypercholesteremia would affect it. Injuries of operations were caused to carotid arteries of Watanabe heritable hyperlipidemic (WHHL) rabbit and a morphological study of the course of repair was made. Japanese white rabbit (JWR) were treated exactly same as WHHL, and their course of repair was compared and examined as control. One week after operation, both WHHL rabbit and JWR showed edematous thickening of a intima, increase in grade of thickening which was continued until two of three months after operation was observed, however gradually decrease thereafter. In case of WHHL rabbit, thickened intima at suture portion was formed by smooth muscle cells migrated from media. Foamy cells were not observed here. The grade of thickening of WHHL rabbits was lower than that of JWR, and that WHHL rabbits tended to show slower change of elastofibrosis in thickened intima rather than JWR. It was considered that the smooth muscle cells of WHHL rabbit has a poor activity naturally.

Animals↗

Distribution of rabbit haemorrhagic disease virus RNA in experimentally infected rabbits.

Adult rabbits were inoculated with liver homogenate from a rabbit that died in a Japanese outbreak of rabbit haemorrhagic disease (RHD). All experimentally infected rabbits died with typical clinical, gross and histological findings of RHD. Distribution of RHD virus in tissues of the infected rabbits was studied by non-isotopic in-situ hybridization. Both viral plus- and minus-strand RNAs were detected within the cytoplasm of hepatocytes, Kupffer cells and splenic and alveolar macrophages, mainly in morphologically intact cells. Strand-specific reverse transcriptase-polymerase chain reaction also demonstrated viral minus-strand RNA as well as plus-strand RNA in the liver, lung and spleen of infected rabbits. These results suggest that viral replication occurs not only in hepatocytes but also in macrophages. The infected macrophages may contribute to viral dissemination in RHD.

Animals↗

Persistence of rabbit haemorrhagic disease virus genome in vaccinated rabbits after experimental infection.

Rabbit haemorrhagic disease (RHD) is usually a fatal disease in rabbits which has spread rapidly across the continents. While previous studies suggested persistence in rabbits to be an important factor in the epidemiology, the relevance of field virus infection of immune rabbits has not been investigated in experimentally infected animals before. This report describes for the first time the persistence of rabbit haemorrhagic disease virus (RHDV) genome for at least 15 weeks in rabbits immunized with an inactivated vaccine as well as a subunit vaccine and subsequently challenged with virulent RHDV. The viral RNA loads were determined by real-time reverse transcription-polymerase chain reaction. No conspicuous association of the detectable amount of RHDV RNA with the type of vaccine, the time after infection and--with one exception--the level of RHDV-specific antibodies in the immunized animals was observed. The results presented in this study are an urgent evidence for the existence of carrier animals as an important factor in the epidemiology of RHD.

Animals↗

Protection of rabbits from viral challenge by gene gun-based intracutaneous vaccination with a combination of cottontail rabbit papillomavirus E1, E2, E6, and E7 genes.

In this study, cottontail rabbit papillomavirus infection of domestic rabbits was used as an animal model to develop papillomavirus early gene-based vaccines. Groups of rabbits were intracutaneously vaccinated with single papillomavirus early genes E1, E2, E6, and E7 or with a combination of these four genes. Only a fraction of rabbits were protected from subsequent viral challenge when vaccinated with the E1 or E6 gene. Viral tumor growth in those rabbits vaccinated with the E1 or E2 gene was suppressed compared to that in controls. In contrast, seven of nine rabbits vaccinated with the combination of the E1, E2, E6, and E7 genes were completely protected against viral challenge. These data indicated that intracutaneous genetic vaccination with the combination of the E1, E2, E6, and E7 genes can be an effective strategy for immunoprophylaxis of papillomavirus infection.

Animals↗

Light microscopic distribution of some cholinergic markers in the rat and rabbit locus coeruleus and the nucleus angularis grisea periventricularis of the domestic pig (Sus scrofa): a correlative electron microscopic investigation of cholinergic receptor proteins in the rabbit.

Cholinergic modulation of locus coeruleus (LC) neurons evokes a variety of neuronal and behavioural effects. In an attempt to understand the LC cholinergic circuit, several markers has been investigated and compared. (Immuno)-histochemical and autoradiographic methods have been used on rat, rabbit, and pig tissue. To identify the boundaries of the LC in each of these species, sections through the entire brainstem have been stained for tyrosine hydroxylase. The results indicate that the pig does not possess a LC proper that conforms to the accepted features of this cell group. However, in this location fusiform cells reminiscent of LC interneurons are still present. This group of fusiform neurons has been named the nucleus angularis grisea periventricularis (NAGP). LC cells of the rat and rabbit show strong acetylcholinesterase (AChE) activity. In the pig the NAGP is markedly free from AChE staining. Muscarinic binding sites are densely distributed over the rabbit LC and adjacent region. The rat and rabbit LC neurons synthesise both muscarinic (mAChR) and nicotinic receptor protein (nAChR). In the pig NAGP region mAChR and nAChR positive cell bodies are almost absent, while some nAChR immunoreactive dendrites are present. The light microscopic data in the rabbit have been confirmed by electron microscopic analysis. It is concluded that the general concept of a noradrenergic LC that is present throughout mammals is questionable. At present, choline acetyltransferase immunoreactive terminals that closely correspond to the other cholinergic components in the rat or rabbit LC have not been observed. However, in these species the cholinergic sensitivity of LC cells is mediated via both muscarinic and nicotinic receptors on somata and dendrites.

Acetylcholinesterase↗

Differences in ligand binding profiles between cloned rabbit and human 5-HT1D alpha and 5-HT1D beta receptors: ketanserin and methiothepin distinguish rabbit 5-HT1D receptor subtypes.

The study of serotonin receptor function has been complicated by the extreme molecular diversity of serotonin receptor subtypes, the lack of selective agonists and antagonists for many of the subtypes, and divergence in the pharmacological properties of a single receptor subtype across different animal species. An example of this pharmacological diversity between species homologues is provided by the 5-HT1D receptor subfamily. To further advance the ability to characterize and pharmacologically compare functional responses mediated by native 5-HT1D receptors, we have cloned the 5-HT1D alpha and 5-HT1D beta receptor subtypes from the rabbit and evaluated their pharmacological profiles using radioligand binding assays. The deduced amino acid sequences of the rabbit 5-HT1D alpha and 5-HT1D beta receptor genes displayed 60% overall identity [75% transmembrane (TM) identity] to each other and > 90% overall identity (95% TM identity) to their corresponding human homologues. Two compounds were identified in binding assays which discriminated between the closely-related 5-HT1D receptors. Ketanserin exhibited high affinity (pKi = 7.66) and selectivity (> 20-fold) for the 5-HT1D alpha receptor while methiothepin displayed high affinity (pKi = 7.86) and selectivity (16-fold) for the 5-HT1D beta receptor subtype. The rabbit and human recombinant 5-HT1D receptors showed significant intraspecies (rabbit 5-HT1D alpha vs. 5-HT1D beta) and interspecies (i.e. rabbit vs. human 5-HT1D alpha) similarities in their ligand binding profiles. These data suggest that 5-HT1D-mediated responses in rabbit preparations may provide information relevant to the pharmacology of the 5-HT1D receptor subtypes in humans.

Amino Acid Sequence↗