Collagenase synthesis by cloned rabbit pulp cells--molecular and immunological identity of pulp cell and fibroblast enzyme.

Cultures of cloned rabbit pulp (RP) cells without stimulation produced collagenase of a concentration as high as reference rabbit skin fibroblast cultures which were stimulated with phorbol myristate acetate (PMA, 100 ng/ml). The RP cell collagenase was compared with reference fibroblast collagenase in Western blot analysis using monoclonal antibodies prepared against RP cell collagenase and a polyclonal antibody prepared against rabbit fibroblast collagenase. Both enzyme preparations revealed, with either antibody, identical bands of approximate molecular masses 57,000, 52,500, and 45,000. These antibody preparations variously inhibited RP cell collagenase activity. Intracellular collagenase in RP cells in culture was demonstrated by the indirect immunofluorescence antibody technique using polyclonal anti-fibroblast collagenase antibody. RNA samples from RP cells hybridized with rabbit fibroblast collagenase cDNA (clone H9) and showed a distinct band at 2.7 kb. Both control and PMA-stimulated RP cells and PMA-stimulated reference skin fibroblasts demonstrated strong cytoplasmic hybridization between H9 and collagenase mRNA. The results indicate that RP cell collagenase is identical to rabbit fibroblast collagenase, and that the RP cell line provides a useful in vitro reference system for the study of collagenolysis in the rabbit model.     

Reiter protein complement fixation test; a preliminary comparison of the TPI test with the complement fixation test employing a soluble protein antigen derived from the Reiter strain

A comparative study of the specificity of the Reiter protein complement fixation (RPCF) test and the Treponema pallidum immobilization (TPI) test on 180 sera showed that the results in 178 instances or 98.9 per cent were in agreement. The sensitivity of the RPCF test, when compared to the TPI test, on 189 sera from patients known to have syphilis was in agreement in 182 or 96.3 per cent, assuming a correlation exists between positive TPI and both the "reactive" or "weakly reactive" RPCF test results. As to reproducibility of results, the RPCF test results agreed in 84 of the 87 sera tested (95.4 per cent). The sera were tested at least three times on different days. Anticomplementary reactions were observed in three of 91 normal sera.     

Some serological properties ofActinobacillus pleuropneumoniae strains of serotypes 1 through 5

Rabbits were hyperimmunized with live, formalin-killed, and heat-treated antigen preparations of the reference strains of serotypes 1 through 5 ofActinobacillus pleuropneumoniae in order to study the antibody response to both soluble and particulate antigens. The antibody response was studied by means of precipitation, agglutination, coagglutination, indirect hemagglutination, and complement fixation tests.Serotyping ofA. pleuropneumoniae strains was done by ring precipitation (RP) and coagglutination (CoA) tests with unheated and heated cell-saline extract as antigens and rabbit hyperimmune sera produced against either live cultures or formalin-killed whole-cell suspensions. The results showed that live cultures provoked more cross-reactive antibodies in rabbits, thus making the antisera unsuitable for use in serotyping by the RP test when unheated wholecell saline extract was used as antigen. Rabbit hyperimmune serum produced against formalinkilled bacterial suspension gave serotype-specific reactions in the RP test. Boiled or autoclaved cell-saline extracts gave serotype-specific reactions in the RP test even when rabbit anti-livecell sera were used. Serotype-specific reactions were obtained in the CoA test in both rabbit anti-live or anti-formalin-killed cell sera with either unheated or heated bacterial cell suspensions as antigens.Live and formalin-killed whole-cell suspensions as well as their saline extracts provoked a high antibody response in rabbits. Heating the cell suspension at 100°C for 1 h caused a significant reduction in their immunogenic potency, whereas autoclaving (121°C) of the cell suspension for 1 h almost completely destroyed their serotype-specific immunogenic properties, since the antibody response was either absent or very poor and not type-specific. However, neither boiling nor autoclaving of the cell suspensions caused significant reduction in their ability to react with preformed antibodies. Phenol-water-extracted antigens gave the highest degree of serotype specificity in the complement fixation test.     

REITER PROTEIN COMPLEMENT FIXATION TEST—A Preliminary Comparison of the TPI Test with the Complement Fixation Test Employing a Soluble Protein Antigen Derived from the Reiter Strain

A comparative study of the specificity of the Reiter protein complement fixation (RPCF) test and the Treponema pallidum immobilization (TPI) test on 180 sera showed that the results in 178 instances or 98.9 per cent were in agreement.The sensitivity of the RPCF test, when compared to the TPI test, on 189 sera from patients known to have syphilis was in agreement in 182 or 96.3 per cent, assuming a correlation exists between positive TPI and both the “reactive” or “weakly reactive” RPCF test results.As to reproducibility of results, the RPCF test results agreed in 84 of the 87 sera tested (95.4 per cent). The sera were tested at least three times on different days.Anticomplementary reactions were observed in three of 91 normal sera.     

MULTIPLE LABORATORY EVALUATION OF THE REITER PROTEIN COMPLEMENT FIXATION METHOD

Duplicate Reiter protein complement fixation tests were carried out on different days at the UCLA laboratory on 636 specimens entailing diagnostic problems. Results were in agreement in 94.2 per cent of cases.A comparison of the results obtained with the first RPCF test carried out on 639 diagnostic problem specimens submitted to four Los Angeles laboratories showed disagreement ranging from 10 to 13 per cent.Agreement between RPCF and TPI results in each of the four laboratories ranged from 81.2 to 82.7 per cent.A greater correlation was obtained between RPCF and TPI results than between TPI and STS or RPCF and STS.In the presence of nonreactive RPCF tests, from 5.6 to 7.1 per cent of the specimens tested in each laboratory were reactive to VDRL and TPI tests. Nonreactive VDRL and reactive TPI results ranged from 7.2 to 8.3 per cent of the specimens tested.In the presence of reactive TPI and RPCF results, 5.5 to 6.4 per cent of the specimens tested in each laboratory showed nonreactive VDRL tests.Twenty-nine or 4.6 per cent of 625 specimens tested showed nonreactive VDRL, reactive cardiolipin Kolmer and reactive TPI tests.Of the weakly reactive RPCF, VDRL or cardiolipin Kolmer tests, a higher percentage of the RPCF results gave reactive TPI tests than did either the VDRL or cardiolipin Kolmer procedures.The number of specimens showing discrepant RPCF results upon being retested in the four laboratories ranged from 33 to 68. Reactive TPI tests on those specimens ranged from 30.3 per cent to 54.4 per cent.     

Apoptotic cells of an epithelial cell line, AsPC-1, release monocyte chemotactic S19 ribosomal protein dimer

A pancreatic carcinoma cell line, AsPC-1, underwent apoptosis in vitro when heat-treated for 60 min at 43 degrees C. Apoptotic AsPC-1 cells liberated a monocyte chemotactic factor into the culture supernatant 24 to 30 h after the heat-treatment. This factor was immunologically identified as the cross-linked homodimer of S19 ribosomal protein (RP S19), since the majority of the chemotactic activity was absorbed by both anti--RP S19 rabbit antibodies and an anti--isopeptide bond monoclonal antibody immobilized on agarose beads. Intracellular transglutaminase activity increased during the apoptotic process, reaching the peak strength between 18 and 24 h after the heat-treatment. A recombinant RP S19 acquired the monocyte chemotactic capacity when incubated with the apoptotic cell extract obtained at the 18th hour. The chemotactic activity acquirement as well as the transglutaminase activity were blocked by treatment of the extract with anti--type II transglutaminase rabbit antibodies. When the recombinant RP S19 was treated with an authentic type II transglutaminase, the dimerization of RP S19 concomitant with the generation of the monocyte chemotactic activity was observed. Peptide-map analyses involving amino acid sequencing demonstrated that the inter-molecular isopeptide bond was heterogeneous: Gln12 or Gln137 and Lys29 or Lys122 were cross-linked. Site-directed mutagenic analysis indicated that the cross-linking of Gln137, but not other residues such as Gln12, Lys29, and Lys122, was essential for expression of the chemotactic activity.     

Treponema paraluis-cuniculi infection in a commercial rabbitry: epidemiology and serodiagnosis

The epidemiology of Treponema paraluis-cuniculi infection in a commercial rabbit breeding facility was described using several serologic tests. The Venereal Disease Research Laboratory, rapid plasma reagin, microhemagglutination and fluorescent treponemal antibody-absorption tests were used to detect antibodies to T paraluis-cuniculi. Young adult New Zealand white rabbits, tested prior to entry into the breeding program, were nearly always free of T paraluis-cuniculi infection. In adult females, the prevalence of T paraluis-cuniculi infection increased with parity; females para 6 or greater were usually seropositive. Most adult males seroconverted within 6 months of entering the breeding program; all males were seropositive after 12 months in the breeding program. This suggested that T paraluis-cuniculi spreads mainly by horizontal transmission during breeding in adult rabbits. Of the two nontreponemal antigen tests used, the Venereal Disease Research Laboratory test was more sensitive, whereas the rapid plasma reagin test was more specific in detecting T paraluis-cuniculi infection; the fluorescent treponemal antibody-absorption test was used as the confirmatory treponemal antigen test. However, neither nontreponemal antigen test was completely satisfactory. On the other hand, the sensitivity and specificity of the microhemagglutination test compared favorably with the fluorescent treponemal antibody-absorption test. Since the microhemagglutination test combines desirable features of both a screening and verification procedure, it should be the test of choice for detection of T paraluis-cuniculi infection.     

The learning ability and memory retention of broiler breeders: 2 transgenerational effects of reduced balanced protein diet on reward-based learning

The effect of reduced balanced protein (RP) diet in the F0 and F1 generation of broiler breeders on the learning ability and memory retention of the F2 generation was investigated by means of a reward v. no reward discrimination T-maze test. There were two treatments for the F0 generation: control (C) group, reared on standard commercial diets, and reduced balanced protein (RP) group, fed with RP diets (25% reduction in CP and amino acids). The female F0-progeny of each treatment was again separated into the two dietary treatments, resulting in four treatments for the F1 generation: C/C, C/RP, RP/C and RP/RP (breeder feed in F0/F1 generation). The RP diets fed breeders received on average 10% more feed than C diets fed breeders to achieve a similar target BW. The F2 generation was composed of four treatments coming from the female F1-progeny of the four treatments and were all fed with C diet (namely C/C/C, C/RP/C, RP/C/C and RP/RP/C). All four F2 generation groups were able to complete the T-maze learning test with a slight difference in success rate but a significant difference within groups was observed regarding the time needed to complete the test. In general, the RP/RP/C group needed more time for completing the test compared with the other three groups and the shortest time was recorded for the RP/C/C group. At similar ages, breeders with early learning experience spent significantly less time in completing the test compared with unexperienced breeders. Long-term memory retention was observed in all four groups whereas the learning ability in solving the test decreased with age. It took longer for the breeders to complete the test at older ages. In conclusion, under our experimental conditions, the RP dietary treatment in previous generations had no influence on the T-maze learning ability and memory retention of broiler breeders of the third generation, although it might have effects on the working performance in the T-maze learning test of F2 generation breeders.     

Enhancement of reaginic and hemagglutinating antibody production by an extract of Bordetella pertussis containing histamine sensitizing factor.

The effect of an extract containing the histamine-sensitizing factor (HSF) of Bordetella pertussis on the immune response of mice to ovalbumin was investigated with respect to dose of antigen and adjuvant. Of particular interest was the enhancement of reaginic antibody production. In comparison to the Al(OH)3 induced production of reaginic antibody where low doses of antigen and adjuvant yield high titers of reagin, the HSF extract demonstrated optimal adjuvant activity at high doses of both adjuvant and antigen. The reaginic antibody response was maximal usually by 2 to 3 weeks post-immunization and persisted for long periods of time. The hemagglutinating antibody response was maximal at 8 to 10 weeks post-immunization. The initial treatment of mice with HSF extract plus antigen resulted in the production of memory cells since a subsequent immunization with ovalbumin alone evoked a secondary reaginic response. These observations may have implications in clinical allergy since substances similar to the pertussis factor might be produced by other microbial organisms and these substances could modulate the immunologic response of individuals to common allergens.     

Evidence for Interference by Immune Complexes in the Serodiagnosis of Cryptococcosis

The latex agglutination test was used to compare cryptococcal antigen titers before and after protease treatment in 19 patients with pulmonary cryptococcosis. Antigen was detected by the LA test in 13 of 33 serum samples before protease treatment, and in an additional 13 samples following treatment. Of 26 antigen-positive serum samples, 22 (84.6%) showed an increased antigen titer after protease treatment. Using the cell slide agglutination test, antibody was detected in 3 of 19 cases. In one of these 3, antigen could only be detected after protease treatment. Soluble immune complex was prepared in vitro using anti-C. neoformans rabbit antiserum and polysaccharide antigen of C. neoformans, and the effects of immune complexes on the LA test were examined. In this experimental model, soluble immune complexes seemed to be observed in antibody excess region, because the antigen titers were increased after the protease treatment. We concluded that C. neoformans capsular polysaccharide antigen and anti-C. neoformans antibody formed soluble immune complexes in patients' sera, which interfered with antigen detection by the latex agglutination test without protease treatment.     

Inhibition of in vitro tumor cell growth by RP215 monoclonal antibody and antibodies raised against its anti-idiotype antibodies

RP215 monoclonal antibody (Mab) was shown to recognize carbohydrate-associated epitope(s) in the heavy chains of cancer cell-expressed immunoglobulins, designated in general as CA215 pan cancer marker. Growth inhibitions of tumor cells in vitro by RP215 Mab and antibodies against its anti-idiotype (anti-id) antibodies were investigated. Polyclonal rabbit anti-id antibodies and the corresponding rat anti-id Mabs were generated and characterized. Following immunizations in mice, antisera raised against anti-id antibodies were analyzed by typical immunoassays. It was observed that mouse anti-anti-id sera (Ab3) revealed binding affinity and specificity to CA215 that are comparable to those of RP215. Both RP215 and Ab3 were shown to induce apoptosis of cultured cancer cells in vitro by TUNEL and MTT assays. These experimental observations were consistent with that of in vivo tumor growth inhibition by RP215 in previous nude mouse experiments. Therefore, heterologous or homologous anti-id antibodies of RP215 that contain the internal image of its specific epitope in CA215 may serve as effective anti-cancer vaccines for therapeutic treatments of various cancers in humans. The relative stability of RP215-specific carbohydrate-associated epitope was compared to that of human IgG at extreme pH’s (≤2 or ≥12) or following NaIO₄ treatments. The major molecular forms of CA215 were further documented with various enzyme immunoassays and found to have similar secondary structures to those of normal human immunoglobulin G.     

Fluorescent Treponemal Antibody Tests—A Summary and Comparison

A comparison of current serologic tests for syphilis shows that treponemal tests are preferable to reagin tests in detecting specific antibodies, but that reagin tests are best for determining the response to treatment. The newly developed fta-absorption technique is suggested as a reliable, inexpensive test for treponemal antibodies.     

Estimation and Identification of Streptococcus dysgalactiae Protective Antibody in Sera of Rabbits and Cattle

Rabbit and cow anti-Streptococcus dysgalactiae sera were tested by bacterial agglutination, complement fixation, hemagglutination, and immunodiffusion for the presence of antibody. The results of these tests were compared with mouse-protection studies on the same serum to estimate which in vitro test would best reflect the in vivo protective capacity of serum. Identification of the antibody constituents responsible for the mouse protection, hemagglutination, and complement fixation titers were established by reacting whole and diluted antisera with mercaptoethanol before and after testing. Results indicate that the complement fixation test may be a more accurate indicator of IgG protective bovine and rabbit antibody, whereas the hemagglutination test may more readily reflect a wider range of protective antibody levels and IgM. The complement fixation test showed some shared responses to IgG and IgM in both the rabbit and cow, whereas the IgM components seemed to be the predominant factor influencing hemagglutination titers in the rabbit and more so in the bovine. Mouse protection tests with mercaptoethanol-treated cow and rabbit sera indicate that the protective capacity of these antisera is shared between IgM and IgG components.     

Lysis of leukemia cells with a monoclonal antibody-cocktail (e.g. VIP-pool) and human complement

During the lysis of leukemic cells with a monoclonal antibody cocktail (the so-called VIB pool) and complement the attempt was made to replace rabbit serum as a complement source by human serum. For identifying the lysis of leukemic cells the complement-dependent in vitro cytotoxicity test was used and for excluding stem cell toxicity the CFU-c test according to PIKE and ROBINSON. In combination with the applied monoclonal antibody pool against B and c-ALL the human complement could be shown to be suitable to produce a lysis in the same manner as rabbit complement. Similarly to the pretested rabbit serum the treatment with the human complement had no impact on stem cell recovery. An optimal cytotoxic activity (95% against ALL blasts of patients, 100% against NALM) could be identified up to an antibody dilution of 1:32 with a volume percentage of 50% of human complement, an incubation temperature of at least 37 degrees C and an incubation time of 30 mins. With proved high reactivity against leukemic cells and lacking impairment of the haemopoietic power of the bone-marrow, this method can be recommended for "purging" protocol with the possibility of using human serum as a source of complement having advantages as far as clinical application is concerned.     

Capillaria hepatica: Granuloma formation to eggs: III. Anti-immunoglobulin augmentation and reagin activity in mice

Sera from mice with primary and secondary Capillaria hepatica egg granulomas were examined for 48-hr homologous PCA activity and for the presence of IgM, IgG₁, and IgG₂ using a microtiter anti-globulin augmentation assay following indirect hemagglutination testing. All three antibody types assayed were present. Secondary granuloma formation produced an antibody response characterized by the initial production of IgM followed by IgG₁ and IgG₂ during the latter phase of the test period, however primary granulomatous sera demonstrated a more varied antibody response with the presence of all three during the entire test period. Forty-eight-hour PCA tests demonstrated the presence of reagin activity in sera from granulomatous and infected mice. Reagin activity occurred more frequently in primary than secondary granulomatous mice. These studies thus confirm the presence of antibody during granuloma formation to C. hepatica eggs and conclusively demonstrate the presence of at least two classes of antibody.     

RP463: a stabilized technetium-99m complex of a hydrazino nicotinamide derivatized chemotactic peptide for infection imaging.

A HYNIC-conjugated chemotactic peptide (fMLFK-HYNIC) was labeled with (99m)Tc using tricine and TPPTS as coligands. The combination of fMLFK-HYNIC, tricine, and TPPTS with (99m)Tc produced a ternary ligand complex [(99m)Tc(fMLFK-HYNIC)(tricine)(TPPTS)] (RP463). RP463 was synthesized either in two steps, in which the binary ligand complex [(99m)Tc(fMLFK-HYNIC)(tricine)(2)] (RP469) was formed first and then reacted with TPPTS, or in one step by direct reduction of [(99m)Tc]pertechnetate with stannous chloride in the presence of fMLFK-HYNIC, tricine, and TPPTS. The radiolabeling yield for RP463 was usually >/=90% using 10 microg of fMLFK-HYNIC and 100 mCi of [(99m)Tc]pertechnetate. Unlike RP469, which decomposed rapidly in the absence of excess tricine coligand, RP463 was stable in solution for at least 6 h. [(99)Tc]RP463 was prepared and characterized by HPLC and electrospray mass spectrometry. In an in vitro assay, [(99)Tc]RP463 showed an IC(50) of 2 nM against binding of [(3)H]fMLF to receptors on PMNs. [(99)Tc]RP463 also induces effectively the superoxide release of polymorphonuclear leukocytes (PMNs) with an EC(50) value of 0.2 +/- 0.2 nM. The localization of RP463 in the infection foci was assessed in a rabbit infection model. RP463 was cleared from the blood faster than RP469 and was excreted mainly through the renal system. As a result of rapid blood clearance and increased uptake, the target-to-background ratios continuously increased from 1.5 +/- 0.2 at 15 min postinjection to 7.5 +/- 0.4 at 4 h postinjection. Visualization of the infected area could be as early as 2 h. A transient decrease in white blood cell count of 35% was observed during the first 30 min after injection of the HPLC-purified RP463 in the infected rabbit. This suggests that future research in this area should focus on developing highly potent antagonists for chemotactic peptide receptor or other receptors on PMNs and monocytes.     

In vitro inhibition of Salmonella enterica serovars choleraesuis and typhimurium,Escherichia coli F-18, and Escherichia coli O157:H7 by a porcine continuous-flow competitive exclusion culture

A competitive exclusion (CE) culture of porcine cecal bacteria was developed as a continuous-flow culture in chemostats, was designated RPCF, and was used as a model to determine its usefulness against in vitro colonization by Salmonella enterica serovars Typhimurium and Choleraesuis, Escherichia coli strain F-18, and E. coli serotype O157:H7 (933). Chemostats with or without RPCF were inoculated with 10⁶ colony-forming units (CFU)/ml of Typhimurium, Choleraesuis, F-18, or O157:H7. Chemostats were sampled for salmonellae and E. coli at 15 min, 7 h, and every 24 h thereafter. In control chemostats without RPCF, Typhimurium, Choleraesuis, F-18, and O157:H7 rapidly established colonization and had concentrations of 10⁶ CFU/ml for 96–120 h post-inoculation. In the chemostats that contained RPCF, reductions (P < 0.05) of Choleraesuis, F-18, and O157:H7 were observed at 24 h post-inoculation. Typhimurium was decreased (P < 0.05) at 48 h post-inoculation, and by 120 h post-inoculation, all chemostats were negative for the four challenge microorganisms. These results demonstrate that RPCF cultures were able to inhibit the growth of Typhimurium, Choleraesuis, and E. coli strains F-18 and O157:H7 in vitro and suggest the potential for the use of CE in swine to prevent disease induced by these microorganisms. Received: 2 October 2001 / Accepted: 31 December 2001     

兔抗麻雀IgY酶标抗体的制备

目的制备辣根过氧化物酶（HRP）标记的兔抗麻雀IgY抗体,为禽类血清学检测体系的建立提供技术储备。方法硫酸铵盐析法粗提麻雀血清IgY,进一步在SDS-PAGE上分离后,切下带有目的条带的凝胶作为免疫原,免疫实验兔制备抗血清,Protein-A柱亲和纯化兔抗IgY血清IgG,,使用改良过碘酸钠法制备酶结合物。ELISA检测酶标抗体的工作浓度,western blotting检测酶标抗体的特异性。结果硫酸铵盐析法粗提IgY,可去除部分杂蛋白,SDS-PAGE上分离后切下带有目的条带的凝胶,可以得到足够纯度的抗原,将带有IgY的凝胶作为抗原免疫后获得的抗血清经Protein-A纯化后,二抗在SDS-PAGE上鉴定,纯度达到99%以上。改良的过碘酸钠法标记获得的抗体浓度为1.008 mg/mL,ELISA检测酶标抗体效价为1∶1000。Western blotting鉴定抗体具有特异性。结论获得了优质可靠的兔抗麻雀IgY酶标抗体。     

Fluorescence microscopic localization of actin in pollen tubes: comparison of actin antibody and phalloidin staining

A comparison of actin localization in pollen tubes of Nicotiana has been made using a monoclonal actin antibody and rhodamine-phalloidin (RP). The monoclonal antiactin, based on Western blotting of pollen tube extract, labels a polypeptide at 45 kD that comigrates with muscle actin. A 51-kD unknown protein and three bands less than 45 kD, presumed to be proteolytic fragments of actin, are also observed. Structural observations using this antibody reveal a network of axially oriented strands of microfilaments (MFs). The MFs are distributed throughout the length of the pollen tube except at the very tip, where diffuse staining is usually observed. A similar pattern of MFs is evident after RP staining. When pollen tubes are treated with cytochalasins (CB or CD) cytoplasmic streaming is inhibited, as is tube elongation. Microscopic analysis reveals that the microfilament (MF) pattern is markedly altered; however, the antibody and RP produce different staining patterns. The antibody reveals many MF strands that distribute throughout the tube length and extend into the very tip. In contrast, RP shows mostly a diffuse staining pattern with only a few short clumps of filamentous material. Immunogold labelling of sections of pollen tubes prepared by rapid-freeze fixation and freeze substitution reveals that actin MF bundles are indeed present after cytochalasin treatment. Our results thus question reports in the literature, based on phalloidin staining, asserting that cytochalasin fragments or destroys actin MFs.