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Immunological quantitation of rabbit plasminogen activator inhibitor-1 in biological samples: evidence that rabbit platelets do not contain PAI-1.

Two immunoassays for the specific quantitation of rabbit plasminogen activator inhibitor-1 (PAI-1) antigen and activity in biological samples were developed and applied for the evaluation of PAI-1 in rabbits. Levels of PAI-1 antigen in rabbit plasma were 9.8+/-4.6 ng/ml (mean +/- SD, n = 6), with a corresponding value of 20.5+/-13.5 ng/ml for PAI-1 activity. In rabbit serum PAI-1 antigen was 11.8+/-4.9 ng/ml (n = 6) and PAI-1 activity was 2.9+/-2.0 ng/ml (n = 6). Endotoxin injection (20 microg/kg, i.v.) induced a time-dependent increase of both PAI-1 antigen and PAI-1 activity levels in rabbit plasma, eventually resulting in a 40- to 90-fold increase (p<0.0001 vs. baseline). A linear correlation was found between PAI-1 antigen and activity levels in normal plasma (r = 0.90, n = 6, p<0.05) and in plasma from endotoxin-treated rabbits (r = 0.98, n = 20, p<0.001). Analysis of PAI-1 antigen and activity in lysates of washed rabbit platelets revealed the absence of PAI-1 (i.e. <0.03 ng/10(8) platelets). In conclusion, development of specific immunological assays allowed the quantitation of PAI-1 in rabbit samples. In striking contrast to other species (human, rat, mouse, pig) rabbit platelets lack detectable amounts of PAI-1 (i.e. >100-1000 fold lower vs other species studied). This observation may have important implications for the use of experimental rabbit models especially in studies on the role of platelets in various pathological conditions including thrombosis and atherosclerosis.

Animals↗

Pharmacokinetics of recombinant human basic fibroblast growth factor in rabbits and mice serum and rabbits aqueous humor.

AIM: To study the pharmacokinetics of recombinant human basic fibroblast growth factor (rhbFGF) in rabbits and mice after iv and postocular administration, and the changes of rhbFGF in rabbits aqueous humor after postocular administration. METHODS: After iv or postocular administration three doses of rhbFGF in rabbits and mice, rhbFGF concentration in serum and rabbit aqueous humor was determined by enzyme-linked immunosorbent assay. RESULTS: Serum concentration-time data of rabbits after iv administration of rhbFGF 1, 2, and 4 micro/kg were fitted to bi-exponential equations with half-lives of 0.9, 0.9, and 0.6 min for T1/2alpha and 7, 8, and 4.7 min for T1/2beta. Plasma concentration-time data of mice after iv administration of rhbFGF 2.5, 5 and 10 microg/kg were fitted to bi-exponential equations with half-lives of 0.4, 0.6, and 0.9 min for T1/2alpha and 6, 5, and 7 min for T1/2beta. The AUCs were linearly correlated to doses in both cases (r rabbit=0.997, r mouse=0.999). The serum concentrations of rhbFGF were very low, near to the background after postocular administration of 2 or 5 microg/kg, in both rabbits and mice. The rhbFGF levels in rabbits aqueous humor were higher than control 8 h postdose (P<0.01). CONCLUSION: rhbFGF within the examined doses had a linear pharmacokinetics in rabbits and mice. High concentration of rhbFGF was found in rabbits aqueous humor after postocular administration.

Administration, Topical↗

Effects of T-2 mycotoxin ingestion on phagocytosis of Aspergillus fumigatus conidia by rabbit alveolar macrophages and on hematologic, serum biochemical, and pathologic changes in rabbits.

Rabbits were given T-2 mycotoxin orally at 0, 0.25, 0.5, and 0.75 mg/kg of body weight/day for 21 days. Only rabbits in the 0.75 mg/kg/day group (4 of 5 rabbits) died. Alveolar macrophages were harvested on day 22 and used for in vitro phagocytosis of killed Aspergillus fumigatus conidia. Cultures included sera from untreated rabbits or rabbits treated with T-2. Phagocytosis was significantly (P less than 0.01) reduced in cultures that used serum from rabbits treated with 0.5 mg of T-2/kg/day and alveolar macrophages from untreated rabbits or rabbits treated with T-2. There was little reduction in phagocytosis when alveolar macrophages from rabbits treated with T-2 and normal serum were used. Ingestion of 0.5 mg of T-2 toxin/kg/day significantly (P less than 0.05) reduced weight gain, serum alkaline phosphatase activity, serum sorbitol dehydrogenase activity, and serum bacteriostasis. Similar changes were found in the 0.75 mg/kg/day group, as well as a significant (P less than 0.05) reduction in PCV, total WBC, and differential leukocyte counts. Neutrophil counts decreased, but not significantly (0.05 less than P less than 0.10). Significant changes were not detected in alanine transaminase activity, aspartate transaminase activity, blood urea nitrogen concentration, or complement hemolytic activity. Histopathologic changes consisting of centrilobular hepatocellular swelling, mild portal and periportal fibrosis and lymphocyte necrosis within secondary lymphoid tissues developed in most rabbits treated with T-2. Thymic atrophy, bile duct reduplication, and lymphocyte depletion of secondary lymphoid tissues developed in the group given 0.75 mg/kg/day.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Free cottontail rabbit papillomavirus DNA persists in warts and carcinomas of infected rabbits and in cells in culture transformed with virus or viral DNA.

We have compared warts and carcinomas from cottontail and domestic rabbits for the presence of cottontail rabbit papillomavirus (CRPV) and the status of the viral DNA genome. Our studies indicate that benign warts from cottontail rabbits, whether found naturally or induced in the laboratory, contain large amounts of virus and on the average 1000 copies of the virus genome per cell. Both benign warts and carcinomas from domestic rabbits contain significantly reduced levels of virus relative to cottontail rabbit warts and an average of 100 copies of the virus genome per cell. A single sample of a naturally occurring cottontail rabbit carcinoma contained approximately 80 copies of the viral genome per cell. None of the tumors that we have analyzed thus far appear to have integrated viral genomes by Southern blot analysis of undigested and restriction endonuclease-digested DNA samples. Furthermore, the CRPV genome present in domestic rabbit carcinomas and a cottontail rabbit carcinoma appears identical by restriction endonuclease mapping to that present in papillomas of cottontail and domestic rabbits indicating that no major deletions or rearrangements of the CRPV genome had occurred during the progression of benign to malignant tumors nor was a variant of wild-type CRPV responsible for this phenomenon. Finally, we have demonstrated morphological transformation in vitro of NIH 3T3 and C127 cells upon infection with purified CRPV and upon transfection with purified CRPV DNA. Furthermore, single cell clones derived from transformed foci contain free forms of CRPV DNA that persist through continued passage in culture. Cells transformed by CRPV grow in soft agar in vitro and produce tumors in athymic nude mice.

Animals↗

Immunization of rabbits with cottontail rabbit papillomavirus E1 and E2 genes: protective immunity induced by gene gun-mediated intracutaneous delivery but not by intramuscular injection.

We previously demonstrated that gene gun-based intracutaneous vaccination of rabbits with a combination of, but not with individual papillomavirus E1, E2, E6 and E7 genes provided complete protection against cottontail rabbit papillomavirus (CRPV) infection. In the present study, we tested whether vaccination of inbred and outbred rabbits with a combination of CRPV E1 and E2 genes could provide complete protection against virus infection. In the first experiment, gene gun-based intracutaneous vaccination with E1 and E2 genes prevented papilloma formation in the majority of inbred rabbits and promoted systemic papilloma regression in one non-protected rabbit. In contrast, needle-mediated intramuscular injection of E1 and E2 genes did not prevent papilloma formation nor promoted systemic papilloma regression, indicating an absence of strong protective immunity. In the second experiment, six outbred rabbits were immunized by gene gun-based intracutaneous administration of the E1 and E2 genes. Prevention of papilloma formation or systemic papilloma regression was observed in three vaccinated rabbits. Papillomas persisted on the remaining three rabbits, but were significantly smaller than that on control rabbits. These results suggested that gene gun-based intracutaneous vaccination with the combination of papillomavirus E1 and E2 genes induced strong protective antivirus immunity but may be insufficient for complete protection in an outbred population.

Administration, Cutaneous↗

Long-term survival of New Zealand rabbit haemorrhagic disease virus RNA in wild rabbits, revealed by RT-PCR and phylogenetic analysis.

Because Rabbit haemorrhagic disease virus (RHDV) is highly pathogenic for rabbits, farmers illegally introduced it as a bio-control agent onto New Zealand farms in 1997. The virus was dispersed rapidly, initially causing high fatality rates in rabbits. Nevertheless, many survived and these surviving rabbits have been investigated for evidence of infection by RHDV. Livers from healthy rabbits contained RHDV-specific RNA, as shown by nested RT-PCR sequencing. The sequences of the viral capsids were related closely to the released Czech strain of RHDV, although the sequence from one rabbit was related most closely to a Spanish strain of RHDV. Phylogenetic analysis of the capsid sequences of 38 samples implied that there have been at least two introductions of the Czech virus into New Zealand, probably corresponding firstly to the original illegal introduction by farmers and secondly to the introduction of the same virus under governmental control. Genomic length sequence of two samples was obtained, suggesting that they may have retained the potential to be infectious, although this has not yet been demonstrated. The detection of genomic-length RNA in the liver of healthy rabbits suggests that even though a highly virulent virus was introduced into New Zealand, it rapidly established persistent or latent infections in a proportion of rabbits. This might account for their ability to survive in the face of virulent released virus. Moreover, the co-circulation of other strains of RHDV in the same rabbit population, such as the Spanish strain, might also impact on their susceptibility to the bio-control agent.

Animals↗

Immunohistology of malignant rabbit fibroma virus--a comparative study with rabbit myxoma virus.

Malignant rabbit fibroma virus (MV) causes a syndrome that consists of disseminated malignant tumors and immunosuppression complicated by severe Pasteurella multocida infection and death. Tissues from rabbits given MV and rabbit myxoma virus were examined by direct immunofluorescence with the use of antibody against virus antigens. Primary and metastatic tumors caused by MV and rabbit myxoma virus were composed of soft tissue cells containing virus antigens. Skin appendages and epidermis overlying the respective tumors showed scant MV but abundant myxoma virus antigen. Both viruses were present systemically in the reticuloendothelial system. Epithelial cells from the liver, kidney, and lung of myxoma virus-infected rabbits contained virus, whereas in MV tumor-bearing rabbits, these cells were uninvolved. However, nasal mucosal and conjunctival epithelia, the locations of Pasteurella infection, showed squamous metaplasia and contained large amounts of MV and myxoma antigens. By analogy to other respiratory tract pathogens, these epithelial changes were probably etiologically significant for development of pasteurellosis in rabbits bearing virus-induced tumors. Thus by immunopathologic as well as clinical examination, MV produces a syndrome distinct from that seen with rabbit myxoma virus. MV induced severe immunosuppression despite T-lymphocyte hyperplasia in the lymphoid tissues observed. The combination of a systemic virus infection, epithelial alterations that impaired clearance mechanisms, and immunologic dysfunction is likely to contribute to the inability of rabbits given MV to survive their gram-negative infection.

Animals↗

Effect of KMD-3213, an alpha 1a-adrenoceptor-selective antagonist, on the contractions of rabbit prostate and rabbit and rat aorta.

KMD-3213, (-)-(R)-1-(3-hydroxypropyl)-5-[2-[[2-[2-(2,2,2-trifluoroethoxy) phenoxy]ethyl]amino]propyl]indoline-7-carboxamide, a newly synthesized alpha 1-adrenoceptor antagonist, has been shown to have potent action toward, and to be selective for human cloned and native alpha 1-adrenoceptors. In the present study, we characterized the inhibitory effect of KMD-3213 on the phenylephrine (alpha 1-adrenoceptor-selective agonist)-induced contraction of rabbit prostate, rabbit thoracic aorta and rat thoracic aorta to functionally confirm the tissue selectivity of KMD-3213. The mean pA2 value for KMD-3213 for the inhibition of the rabbit prostatic contraction was 10.05, whereas the values for the rabbit and rat aortic contractions were 9.36 and 8.13, respectively. The order of mean pA2 values for the inhibition of the rabbit prostatic contraction was KMD-3213 > or = tamsulosin >> prazosin, whereas that for the rabbit and rat aortic contractions was tamsulosin > KMD-3213 > prazosin and tamsulosin > or = prazosin >> KMD-3213, respectively. KMD-3213 produced a sigmoidal inhibition curve for single-dose phenylephrine-induced contractions of rabbit prostate, whereas it produced a non-sigmoidal curve for that of rabbit aorta. KMD-3213 had no effect on isoproterenol-induced chronotropic action in guinea-pig atria, and 5-hydroxytryptamine-, histamine- and acetylcholine-mediated contractions of rabbit aorta. These results indicate that the potency of the inhibitory activity of KMD-3213 depends on the tissue subtype expression and that KMD-3213 preferentially antagonizes prostatic contraction.

Adrenergic alpha-Antagonists↗

Bubonic plague from exposure to a rabbit: a documented case, and a review of rabbit-associated plague cases in the United States.

A 62-year-old woman developed bubonic plague with an epitrochlear bubo one to two days after skinning two cottontail rabbits. The implicated rabbits were later recovered from the patient's freezer, and Yersinia pestis was isolated from marrows of both rabbits. Although human plague cases in the United States have occasionally been traced to exposure to wild rabbits, this is the first documentation of plague infection in the actual rabbits to which the patient was exposed. All reported cases of rabbit-associated plague in the United States were then reviewed. Eight cases were characterized by direct exposure to rabbit tissues. Seven of the eight cases occurred in adult males who had hunted rabbits during winter months in plague-endemic areas. These patients had upper extremity buboes, and the case-fatality ration for the group was 50%. Three other cases in which rabbit exposure was indirect or its role less clear, were also analyzed.

Animals↗

Examination of the immunocontraceptive potential of recombinant rabbit fertilin subunits in rabbit.

Recombinant fertilin subunits produced in a bacterial expression systems were used to test fertilin as an immunocontraceptive antigen in the European rabbit (Oryctolagus cuniculus). Wild female rabbits (n = 40) were immunized with either recombinant rabbit fertilin alpha or beta subunits by the s.c. or intra-Peyer's patch route. High titers of serum anti-fertilin polyclonal IgG antibodies were achieved in all rabbits after repeated boosts, with fertilin-specific IgG but not IgA antibodies detected in vaginal lavages of all animals. The serum IgG antibodies recognized polypeptides in detergent extracts of rabbit sperm with relative molecular masses of 48, 53, and 85 kDa on reducing SDS-PAGE gels and were shown to bind to the head region of methanol-fixed and live caudal rabbit sperm. Preincubation of rabbit sperm with these anti-fertilin IgG antibodies at concentrations of 400 micrograms/ml blocked sperm binding to zona-intact oocytes and inhibited fertilization in vitro by 60-80%. However, despite the levels of circulating and vaginal IgG antibodies achieved, only 4 immunized does failed to become pregnant out of 33 that ovulated. The remaining animals either showed no effect on fertility (n = 29) relative to control animals or failed to ovulate (n = 7). All control animals ovulated and were either fully fertile (n = 15) or were mated to infertile males (n = 4). In addition, proven-fertile male domestic rabbits (n = 3) were immunized s.c. and boosted three times with fertilin beta. Only one animal subsequently showed impaired fertility. These results show that in the rabbit, high levels of circulating sperm-reactive anti-fertilin antibodies and the presence of vaginal IgG does not ensure infertility.

ADAM Proteins↗

Respiratory effects of progressive asphyxia in rabbit pups and adult rabbits.

Effects of prolonged airway occlusion were investigated in anesthetized and vagotomized 9-to 15-day-old pups and adult rabbits. The changes and temporal relationships between "integrated" phrenic activity, external intercostal electromyogram (INT), and esophageal pressure (Pes) were examined. Each occlusion resulted in hyperpnea, apnea, and gasping. Blood pressure recorded during the occlusion showed a marked decrease. During hyperpnea, the rate of changes and maximal amplitudes in Pes and INT were similar in both age groups. The increase in integrated phrenic activity (PHR) was significantly greater in young rabbits. In both age groups, changes in INT during gasping followed a similar time course and exceeded those in PHR. Maximal values of the three parameters were concurrent in adults, whereas the increase in INT peaked later than PHR and Pes in rabbit pups. In adult rabbits, PHR, INT, and Pes, during the last gasp, decreased to the values of the first hyperpnea breath. In rabbit pups, Pes of the last gasp decreased significantly below this value while INT was still elevated. This Pes decrease could result from inspiratory muscle dysfunction in the pups. Thus in rabbit pups, 1) greater changes in PHR were necessary to produce a given change in Pes than in adult rabbits; 2) activity of the external intercostal muscles was not efficient in developing pressure under conditions of asphyxia; and 3) the independent activation of diaphragmatic and intercostal motoneurons is not of vagal origin. Additionally, the results led us to conclude that Pes can serve as a close approximation of respiratory drive in adult rabbits. This parameter, however, cannot be used as an index of central inspiratory activity during gasping in rabbit pups.

Animals↗

Effects of HR780, a novel 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, in Watanabe heritable hyperlipidemic rabbits and cholesterol-fed rabbits.

The aim of this study was to evaluate the effects of (+)-(E)-6S-(2-(4-(4-fluorophenyl)-2-(1-methylethyl)-6-phenylpyridin-3-yl)ethenyl)-4R-hydroxy-3,4,5,6-tetrahydro-2H-pyran-2-one (HR780), a novel 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on hypercholesterolemia in Watanabe heritable hyperlipidemic (WHHL) rabbits and rabbits fed a diet with 1% cholesterol, in comparison with the effects of simvastatin. Each drug was administered orally to WHHL rabbits for 24 weeks and to 1% cholesterol-fed rabbits for 10 weeks. In WHHL rabbits, HR780 at doses of 1, 2.5 and 5 mg/kg/day reduced the plasma total cholesterol level by 15, 24 and 20%, respectively. In contrast, simvastatin at 5 mg/kg/day lowered the level by 23%. In 1% cholesterol-fed rabbits, HR780 (1 and 2.5 mg/kg/day) was found to inhibit the increases in the plasma total cholesterol and phospholipid levels and liver cholesterol contents in a dose-dependent manner. Simvastatin (5 mg/kg/day) also inhibited their increase. Neither HR780 nor simvastatin increased the contents of cholesterol and total bile acid in the gallbladder bile. In conclusion, long-term treatment with HR780 reduced the plasma cholesterol level in WHHL rabbits and 1% cholesterol-fed rabbits, and decreased the liver cholesterol contents in 1% cholesterol-fed rabbits.

Animals↗

Changes in vasoconstrictor and vasodilator responses of the basilar artery during maturation in the Watanabe heritable hyperlipidemic rabbit differ from those in the New Zealand White rabbit.

This investigation involved alterations in the local control of vascular tone in the isolated rabbit basilar artery in atherosclerosis, with Watanabe heritable hyperlipidemic (WHHL) rabbits as a model and New Zealand White (NZW) rabbits as controls. Vasoconstrictor responses to KCl in isolated preparations of the basilar artery at basal tone showed no differences at 4, 6, and 12 months of age in either WHHL or NZW rabbits. Contractile responses to both histamine and neuropeptide Y were significantly greater in 12-month-old WHHL rabbit preparations when compared with responses measured at 4 and 6 months. In NZW rabbit preparations, there was no change in maximum contractile responses to both histamine and neuropeptide Y over the same age range. Endothelium-dependent relaxations to acetylcholine in raised-tone preparations from WHHL rabbits were significantly greater at 6 months in comparison with responses measured at both 4 and 12 months of age. In contrast, endothelium-independent relaxations to calcitonin gene-related peptide and vasoactive intestinal polypeptide showed no change over the age range studied. In NZW rabbit preparations, both endothelium-dependent and endothelium-independent relaxations declined significantly between 4 and 12 months. The significance of these changes in the rabbit basilar artery in atherosclerosis is discussed in relation to the "protection" of intracranial arteries from atherosclerosis and their subsequent susceptibility to cerebral ischemia and stroke.

Acetylcholine↗

Pulmonary surfactant inhibits priming of rabbit alveolar macrophage. Evidence that surfactant suppresses the oxidative burst of alveolar macrophage in infant rabbits.

We have investigated the possible role of pulmonary surfactant in mediating a deficiency in alveolar macrophages (AM) from infant animals to produce chemiluminescent (CL) responses elicited by phorbol myristate acetate (PMA) or opsonized zymosan (Op-Zym). Freshly harvested AM from rabbits 7 to 28 days of age produced much reduced PMA-elicited CL responses compared to 5- to 6-month-old rabbits. Initially we observed that AM from both infant and adult rabbits exhibited increasing PMA- or Op-Zym-elicited CL responses in a time-dependent manner when they were incubated in RPMI 1640 medium without serum for more than 3 h. In addition, AM from infant rabbits acquired the capacity to become primed with macrophage activation factor (MAF) containing supernatants or fetal bovine serum (FBS) during an 18-h incubation period that was basically equivalent to that of AM from adult rabbits. Our studies reveal that natural and artificial surfactant inhibited "spontaneous" priming as well as priming of AM induced by MAF-containing supernatant or FBS for producing PMA- or OP-Zym-elicited CL responses from both normal infant and adult animals. In addition, these surfactant preparations also suppressed the response of AM from adult BCG-sensitized rabbits during the course of an 18-h incubation period. It is especially noteworthy that AM from infant rabbits appeared to be more susceptible to the inhibitory activity of surfactant than AM from adult animals. Our data indicate that freshly harvested AM from infant rabbits are under greater suppression than AM from adult rabbits in terms of their capacities to produce oxidative responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Rabbit placental relaxin: ultrastructural localization in secretory granules of the syncytiotrophoblast using rabbit placental relaxin antiserum.

Although relaxin has been isolated from the placenta of the human, rabbit, horse, and cat, this study represents the first ultrastructural localization of the hormone in placental tissue. Placentas were removed from rabbits on days 15, 23, and 30 of pregnancy, and the tissues were prepared for light and electron microscopies. The cytoplasm of the syncytiotrophoblast from all stages of pregnancy studied showed positive staining for the hormone at the light level using guinea pig antirabbit relaxin serum and the avidin-biotin technique. Ultrastructurally, the syncytiotrophoblast was found to contain membrane-bounded granules (150-400 nm in diameter) which formed at the Golgi and were seen in close association with the cell membrane. Exocytosis involving the incorporation of the granule membrane into the cell membrane was observed. These granules labeled positively for relaxin after treatment with guinea pig antirabbit relaxin serum and goat antiguinea pig immunoglobulin G-colloidal gold. Control sections in which the relaxin antiserum was absorbed with purified rabbit relaxin or substituted with normal guinea pig serum contained no gold-labeled granules. Cross-reactivity of the rabbit relaxin antiserum with porcine relaxin was demonstrated by labeling of the relaxin-containing granules in the pregnant pig corpus luteum with the rabbit relaxin antiserum and by inhibiting the labeling of rabbit placental and pig corpora luteal granules by absorbing the rabbit relaxin antiserum with porcine relaxin. We have previously described the labeling of rabbit placental relaxin with porcine relaxin antiserum. This study suggests that relaxin is synthesized and secreted from the syncytiotrophoblast of the rabbit placenta, with the subcellular site of storage being membrane-bounded granules.

Animals↗

The fibrinogen gamma chain dodecapeptide inhibits agonist-induced aggregation of rabbit platelets and fibrinogen binding to rabbit glycoprotein IIb-IIIa.

The HHLGGAKQAGDV (H12) sequence at the carboxyl termini of the y chains and the RGD sequences in the Aalpha chains of human fibrinogen are potential recognition sites for the binding of soluble fibrinogen to glycoprotein IIb-IIIa (GPIIb-IIIa) on activated human platelets. Thus, addition of either H12 or RGD-containing peptides inhibits aggregation of and fibrinogen binding to human platelets. In contrast, we reported previously that RGDS had relatively little inhibitory effect on these functions of rabbit platelets. In the present study, we found that H12 inhibited ADP- and thrombin-induced aggregation of rabbit platelets in a dose-dependent manner. Specificity was demonstrated by the failure of the variant HHLGGAKQAGEV peptide to inhibit ADP-induced aggregation. Furthermore, flow cytometric analyses demonstrated that H12 inhibited the binding of FITC-fibrinogen to ADP-activated rabbit platelets in a dose-dependent manner. To examine the direct interaction of H12 with rabbit GPIIb-IIIa, we performed affinity chromatography by applying an octylglucoside extract of rabbit platelet proteins onto an affinity matrix containing the fibrinogen gamma chain sequence. Proteins of approximately 135 kDa and approximately 95 kDa were specifically eluted by soluble H12, and the 95 kDa protein band was immunoblotted by anti-LIBS1, a monoclonal antibody against human GPIIIa. In control samples, no detectable protein from rabbit platelet lysates was eluted from an RGD affinity matrix by GRGDSP. Collectively, our results demonstrated that H12 inhibits aggregation of and fibrinogen binding to rabbit platelets by directly interacting with rabbit GPIIb-IIIa. These findings suggest that rabbit platelets would serve as a suitable thrombosis model for testing the efficacy of peptide mimetics derived from H12.

Animals↗

Establishment of specific pathogen-free rabbits by inoculation of hysterectomy-derived rabbits with fecal suspension and anaerobic bacteria.

Establishment of a specific pathogen-free (SPF) rabbit colony was attempted by transferring a total of 127 hysterectomy-derived rabbits directly into a barrier room where SPF rats had been reared. All rabbits except four died of mucoid enteritis-like diarrhea during 2 and 6 weeks of age. A second experiment consisted of four groups of hysterectomy-derived rabbits including: (1) noninoculated controls; (2) rabbits inoculated with suspension of feces from the survivors; (3) rabbits inoculated with a bacterial inoculum (Bacteroides spp. and Eubacterium sp. isolated from conventional rabbits); and (4) rabbits inoculated with Streptococcus faecalis in addition to the inoculum used for group 3. All rabbits in group 1 died of diarrhea by 3 weeks of age. Survival rates of groups 2, 3 and 4 are 53%, 38% and 40%, respectively. On 7 days after the inoculation, Bacteroides was not recovered from fecal samples of group 1, but was recovered from many samples of the inoculated groups. Populations of Enterobacteriaceae and Clostridium in the samples in group 1 were larger than each of those in the inoculated groups. The present results suggest that the lethal diarrhea, after transferring hysterectomy-derived animals directly into a barrier room, can be controlled by inoculation with non-pathogenic anaerobes, especially Bacteroides.

Animals↗

Allotype-defined mRNA for rabbit immunoglobulin H and L chains isolated from rabbit-mouse hybridomas.

Improved procedures have expedited the formation and propagation of stable rabbit-mouse hybridomas (RMH) that secrete rabbit immunoglobulin (Ig) chains and that serve as sources for allotype-defined mRNA, specifying both constant and variable regions of rabbit Ig. The Ig-secreting hybridomas were stabilized by multiple recloning steps and eventually attained a 90% frequency of cells secreting rabbit Ig chains. Stabilized RMH were propagated in vivo in nude (athymic) and in some instances in conventional BALB/c mice. Rabbit Ig products secreted by these RMH cell lines were isolated and were shown to have amino acid sequence characteristics of rabbit Ig chains and that are distinct from those of the mouse. Preparative amounts of polyadenylated RNA-(poly(A) RNA) encoding allotype-defined rabbit Ig chains were isolated from RMH grown as solid tumors. Poly(A) RNA encoding a b4 L chain from one RMH (12F2) and an a3 H chain from another (7D2) were purified 10-fold by sucrose density gradient centrifugation and were characterized in an in vitro translation system. The mRNA encoding the b4 allotype had an S value of 12 and directed the synthesis of a rabbit precursor L chain with m.w. 27,500. Radiochemical amino acid sequence analysis of the L chain precursor has shown it to include a 22 residue leader sequence with a leucine profile similar to that of murine L chain precursors. Analysis of the mRNA encoding the a3 H chain revealed it has an S value of 16 and directed the synthesis of an Ig chain with a m.w. identical to that of the rabbit H chain secreted by the RMH.

Amino Acid Sequence↗