Guinea pig Hageman factor as a vascular permeability enhancement factor.

Hageman factor was purified from guinea pig plasma by successive column chromatography. The guinea pig Hageman factor appeared homogeneous as a single-chain protein on polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS) and beta-mercaptoethanol. The apparent molecular weight was 76,000 daltons by SDS--polyacrylamide gel electrophoresis and 105,000 daltons by gel filtration with a Sephadex G-150 column. Amino acid composition of the guinea pig Hageman factor was similar to that reported for human, bovine, and rabbit Hageman factors. The purified guinea pig Hageman factor, as well as guinea pig plasma, showed strong clotting time correction activity in Hageman-factor--deficient human plasma. The activity could be blocked by the IgG fraction of antiserums against guinea pig Hageman factor raised in rabbits or a goat. The concentration of Hageman factor in guinea pig plasma was determined to be 120 microgram/ml by quantitative radial immunodiffusion assay. The 28,000-dalton active form of Hageman factor (beta-HFa) was prepared from guinea pig Hageman factor by treatment with plasma kallikrein. beta-HFa caused an increase in vascular permeability when injected into guinea pig skin at concentrations as low as 3 x 10(-10) M (0.8 ng). Native, or zymogen Hageman factor did not cause an increase in permeability at concentrations of up to 2 x 10(-7) M. The increased permeability induced by beta-HFa was short lasting, with about a 50% decrease in activity apparent within 6 minutes after intradermal injection. The permeability enhancement activity of beta-HFa was inhibited by pretreatment of beta-HFa with diisopropylfluorophosphate. It may be concluded that active Hageman factor in the interstitial space of guinea pigs acts as a vascular permeability factor of far greater potency than bradykinin.     

Development of a western blot method for detection of fish ectoparasite Argulus siamensis antigens

A study was undertaken to develop a western blot method for detection of immunogenic proteins of fish ectoparasite, Argulus siamensis for its further use as potential vaccine candidates. Argulus antigens were prepared by homogenization and injected to rohu (Labeo rohita) juveniles for development of immune serum. The serum was used to immunostain the antigens in western blot. The other reagents added in sequence were rabbit anti-rohu serum, goat anti-rabbit ALP conjugate and substrate (BCIP-NBT). However, similar banding patterns were observed with both control and immunized rohu serum. Hence, a possible cross-reaction was suspected and verified in number of western blot experiments. A typical cross-reaction observed was of rabbit serum reacting directly with Argulus antigens. Hence, the rabbit anti-rohu serum was replaced with guinea pig anti-rohu serum. Another cross-reaction of goat anti-guinea pig ALP conjugate with rohu serum was eliminated by using goat anti-rabbit ALP conjugate with guinea pig serum. Thus, the final western blot method consisting of Argulus antigens → rohu serum → guinea pig anti-rohu serum → goat anti-rabbit ALP conjugate → substrate, yielded distinguishing results between control and Argulus-immunized rohu serum samples. The developed test has tremendous downstream applications, particularly in immunoproteomic studies of Argulus antigens.     

Activation of the plasma kallikrein-kinin system by Vibrio vulnificus protease.

Vibrio vulnificus protease enhanced hypodermic vascular permeability when injected into the dorsal skin of a guinea pig. Enhancement of permeability was observed within 2 min, and the permeability-enhancing reaction terminated at about 10 min postinjection. The permeability-enhancing reaction was greatly augmented by simultaneous injection of a kininase II inhibitor, whereas the reaction was inhibited by soybean trypsin inhibitor, a well-known inhibitor of plasma kallikrein. Furthermore, in vitro activation of plasma prekallikrein to kallikrein by V. vulnificus protease was observed. These results indicate that V. vulnificus protease enhances vascular permeability through activation of the plasma kallikrein-kinin system which generates bradykinin, factor in edema formation.     

Preparation of antibodies to guinea pig IgE and its use for enzyme-linked immunosorbent assay of IgE antibodies

Guinea pigs were infected with Trichinella spiralis. A pooled serum from the infected guinea pigs was fractionated by DEAE-cellulose column chromatography and Sephadex G-200 gel filtration. An IgE-rich fraction was injected into rabbits. The antiserum from the rabbits, after appropriate absorption with a normal guinea pig plasma, formed a precipitin line in immunoelectrophoresis and in immunodiffusion against a guinea pig serum containing IgE antibodies to ovalbumin. Uptake of 125I ovalbumin was observed in radioimmunoelectrophoresis. This anti IgE could be used in enzyme-linked immunosorbent assay of IgE antibodies to ovalbumin.     

Detection of Legionella DNA in Guinea Pig Peripheral Leukocytes, Urine and Plasma by the Polymerase Chain Reaction

Legionella DNA has been detected in respiratory tract, serum and urine samples from patients with pneumonia by the polymerase chain reaction. A guinea pig model was used to assess whether Legionella DNA could also be detected in peripheral leukocytes during active infection. Ten guinea pigs were infected intraperitoneally with Legionella pneumophila serogroup 1, and leukocyte, plasma and urine samples were collected immediately before inoculation and on days 1, 3, 7 and 14 thereafter. All samples were tested for Legionella DNA by the polymerase chain reaction. Overall, Legionella DNA was detected in 55% of leukocyte samples, 28% of urine samples and 21% of plasma samples collected after inoculation. The sensitivity of the polymerase chain reaction on leukocytes was highest for samples collected within 3 days of inoculation. Further studies testing leukocyte samples from human Legionella infection are required to confirm these observations.     

Intravascular coagulation in mice by the compact-colony-forming active substance (CCFAS) extracted from a strain of Staphylococcus aureus.

The compact-colony-forming active substance (CCFAS) extracted from a Staphylococcus aureus strain was capable of killing mice only when Staph. aureus, Staph. epidermidis or Escherichia coli was injected i.v. before the injection of CCFAS. In the mice killed 30 min after treatment with heat-killed Staph. aureus and CCFAS, remarkable congestion of the lung and thrombus-like lesions in the kidney were observed. In the mice killed 6 h after injection with CCFAS and living Staph. aureus congestion and inflammatory-cell filtration were found in the liver, especially within the Glisson''s capsule. However, when mice were killed 30 days after treatment with CCFAS and Staph. aureus, fibrin and hyalin thrombi were observed most frequently in the renal glomeruli but also in the liver and lung.     

Relationships Between β2-Microglobulin and Alloantigens Coded for by the Major Histocompatibility Complexes of the Rabbit and the Guinea Pig

Treatment of rabbit and guinea pig lymphocytes with Fab' fragments of anti-beta2-microglobulin completely inhibited the cytotoxic effects of alloantisera to RLA or GPLA antigens, respectively. Aggregation of beta2-microglobulin on the lymphocyte surface by successive incubations with goat anti-beta2-microglobulin on the lymphocyte surface by successive incubations with goat anti-beta2-microglobulin and F(ab')2 fragments of rabbit anti-goat IgG also made rabbit lymphocytes resistant to lysis by anti-RLA, and guinea pig lymphocytes resistant to lysis by anti-RLA, and guinea pig lymphocytes resistant to lysis by anti-GPLA. The two kinds of pretreatment of guinea pig lymphocytes did not affect the cytotoxicity of antisera directed against guinea pig Ia antigens. These results in conjunction with previous findings in the mouse and in man suggest that beta2-microglobulin on the lymphocyte surface in mammals is generally associated with major serologically defined histocompatibility antigens but not with I-region-associated antigens.     

A proteaselike permeability factor in guinea pig skin: contact activation of the latent permeability factor and its nature as a prekallikrein activator.

A proteaselike permeability factor in guinea pig skin, which was fractionated in a latent form into pseudoglobulin fractions, was activated by contact with kaolin particles at neutral pH. This contact activation was not prevented by the presence of 1 M KCl but was strongly inhibited by the simultaneous presence of hexadimethrine bromide. In this activation, the latent permeability factor was first bound to kaolin; later an active form permeability factor was released from the kaolin-bound parent molecule. Prekallikrein activator activity was also generated in this supernatant from the pretreated kaolin particle in the same time course as the permeability factor generation. Moreover, since the prekallikrein activator and permeability factor were always observed at the same fractions in every purification step, with DEAE-cellulose column chromatography, Sephadex G-75 gel filtration, and isoelectric focusing, these two molecules were recognized as identical. These results indicate that the latent permeability factor in the guinea pig skin has properties similar to those of the plasma Hageman factor.     

Comparative study on biological activities of various anaphylatoxins (C4a,C3a,C5a)

Several anaphylatoxic substances (human C3a, guinea pig C3a, human C4a, guinea pig C5a, and a synthetic C3a-related hexapeptide) were compared with regard to their ability to induce secretion of [³H]serotonin from guinea pig platelets. Functional identity of the C3a preparations, C4a, and the hexapeptide was demonstrated by the phenomenon of crossed desensitization. Whereas C3a of human and guinea pig origin proved to be qualitatively and quantitatively identical, C4a expressed only 3% of the activity of the C3 fragments on a molar basis. Investigations with goat anti-guinea pig C3a demonstrate that human and guinea pig C3a possess one antigenic determinant in common; however, this determinant is not the C-terminal amino acid sequence. Addition of the anaphylatoxins with low doses of thrombin led to a potentiation of [³H]serotonin release from the platelets. Under these conditions C3a concentrations of 1.5×10^–10 mol/liter (65 pg of C3a) could be detected. Thus the platelet system represents the most sensitive in vitro assay known for evaluation of biological activity of the C3a anaphylatoxins.Supported by grants of the DFG: SFB 107 (Mainz).     

Studies on sperm antigenicity. I. Delayed hypersensitivity to spermatozoa

Delayed hypersensitivity to human and guinea pig sperm was demonstrated in guinea pigs of the Rockefeller strain by immunization with H₃₇Ra adjuvant. The reaction in vivo was demonstrated by skin testing the animals and in vitro by the capillary method. It was found that the sensitivity is not only directed towards the sensitizing antigen, but also shows cross-reactivity. Thus, peritoneal exudate cells derived from guinea pigs sensitized to human sperm were inhibited by guinea pig sperm. This cross-reactivity revealed the possibility of a tissue specific antigen. In addition, supernatants obtained after incubation of the sensitized lymph node cells with the specific antigen were found to be spermatotoxic.     

Monoclonal antibodies specific for guinea pig eosinophil major basic protein: their use in an ELISA, immunocytochemistry and flow cytometry

Eosinophil, major basic protein (M BP), purified from guinea pig eosinophil granules was used to raise five monoclonal antibodies (MoAbs). Their reactivity with MBP was confirmed by immunoblotting and indirect ELISA. Two of the MoAbs were used to develop a sensitive and specific antigen capture (sandwich) ELISA for guinea pig eosinophil M BP which gives an accurate and reproducible standard curve over the range of 10-10000 ng/ml. The specificity of the ELISA for MBP was confirmed and its suitability for testing biological samples ascertained by measurement of MBP in bronchoalveolar lavage fluid (BALF) and plasma from guinea pigs sensitized and challenged with ovalbumin. The ELISA was also capable of detecting MBP in culture supernatants from purified eosinophil preparations challenged with calcium ionophore in vitro. One of the monoclonal could be used to strongly and specifically stain guinea pig eosinophils in immunocytochemistry, whilst all five could be used to visualize eosinophils in suspension in BALF or peritoneal lavage fluid by flow cytometry. There was no staining of other guinea pig leucocyte types, nor crossreactivity with human eosinophils by immunocytochemistry or Row cytometry.     

A high-molecular-weight trypsinlike protease in the skin sites of delayed hypersensitivity in guinea pigs.

A trypsinlike protease was extracted from the delayed hypersensitivity skin sites in guinea pigs. Extractable amounts of the enzyme were chronologically paralleled with the gross appearance of the inflammation, and the maximum activity from the inflamed sites at 24-36 hours was about 20 times stronger than that from normal skin, suggesting a potential role in the pathogenesis of the delayed hypersensitivity reaction. The enzyme, which suitably hydrolyzed t-butyloxycarbonyl-phenylalanyl-seryl-arginine 4-methylcoumaryl-7-amide, was partially purified by isoelectric focusing or by gel filtration. The enzyme demonstrated a single peak of activity on the former column with an apparent isoelectric point of 4.2, and in the latter it showed an apparent molecular weight of 600,000 (600K-protease). When incubated with 3H-diisopropylfluorophosphate, the enzyme lost all amidolytic activity and yielded a single band of radioactivity in polyacrylamide disk gel electrophoresis in the presence of sodium dodecyl sulfate, and a single peak of radioactivity in gel filtration, both having an apparent molecular weight of 31,000-33,000 (31K-protease). That the 600K-protease might be a complex with alpha 2-macroglobulin was ruled out. The 31K-protease was separated from the 600K-protease by gel filtration in the presence of 6 M guanidine hydrochloride, and was renatured to an active form. An apparent isoelectric point of the 31K-protease observed was 9.4, suggesting that the 600K-protease may be a complex of 31K-protease with an acidic carrier molecule(s). Both proteases, 31K- and 600K-protease, had identical substrate specificity, a pH profile of amidolytic activity, and susceptibility to exogenous protease inhibitors. However, when sensitivities to intrinsic protease inhibitors in guinea pig plasma, two kinds of trypsin inhibitor, and alpha 2-macroglobulin were compared, the 600K-protease was at least 100 times more resistant than the 31K-protease. It was supposed that one of the pathophysiologically significant functions of the complex formation might be to maintain the enzyme activity longer in vivo.     

Hemagglutination with pseudorabies virus

Summary Pseudorabies virus grown in CPK cell cultures was tested for hemagglutination (HA) with erythrocytes of a variety of species at 4°C, 25°C and 37°C. HA was observed at all temperatures with mouse erythrocytes but not with cattle, sheep, goat, swine, cat, rabbit, guinea pig, rat, mongolian gerbil, chicken, and goose erythrocytes. Mice showed a strain variation in agglutinability of their erythrocytes, requiring selection of mice to obtain erythrocytes for HA. The HA reaction was inhibited by specific antiserum. Some factors involved in the HA and HA-inhibition (HI) were investigated and standard HA and HI tests were established. HI antibody titers of individual pig sera showed a significant positive correlation with their neutralizing antibody titers.     

Role of ribosomal protein S19-like plasma protein in blood coagulum resorption

Western blot analyses and monocyte chemoattraction analyses of guinea pig plasma and serum indicated the presence of a plasma protein indistinguishable from ribosomal protein S19 and the cross-linked dimerization of it gaining monocyte chemotactic capacity in association with blood coagulation as in the case of human. When coagula preformed in vitro were intraperitoneally inserted into guinea pigs, they were rapidly covered by macrophages within 24 h concomitant with an intra-coagulum macrophage infiltration. Differences were observed between the surface macrophages and the penetrating macrophages in ultrastructural, histochemical and immunohistochemical analyses. The inserted coagula were resorbed by day 7. When either anti-RP S19 antibodies or Gln137Asn-RP S19, a competitive inhibitor against RP S19, was premixed into the inserted coagulum, the attachment and penetration by macrophages decreased and the coagulum resorption retarded. These results indicate the role of the plasma RP S19-like molecule in coagulum resorption via macrophage recruitment.     

Hemagglutination with ovine rotavirus

Summary The virus was grown in MA 104 cells, a stable cell line derived from embryonic rhesus monkey kidney, and tested for hemagglutination (HA) with erythrocytes of a variety of species at 4°C, room temperature and 37°C. HA was observed at all temperatures with chicken, sheep, rabbit, guinea pig and human erythrocytes but not with horse, cattle, goat, swine and goose erythrocytes. HA reaction was inhibited by specific antiserum. Some factors involved in the HA and HA-inhibition (HI) were invetigated and standard HA and HI tests were worked out. The HI test as well as neutralization test clearly distinguished ovine rotavirus from bovine, human, simian, equine, porcine and lapine rotaviruses.With 2 Figures     

Sperm autoantigens and fertilization. III. Ultrastructural localization of guinea pig autoantigens

Guinea pig sperm autoantigens have been localized by direct and indirect immunoferritin techniques in (1) plasma membrane over the entire sperm head, (2) acrosomal contents, (3) fibrous sheath and outer dense fibers of the tail filament, and (4) the inner acrosomal membrane of 50% of acrosome reacted spermatozoa. These cellular structures are known to be involved in guinea pig sperm rouleaux formation, acrosome reaction, interaction of acrosome-reacted sperm with zona pellucida and with the vitellus of guinea pig ova. Since IgG and Fab of autoantiserum to guinea pig spermatozoa have been shown to interfere with these cellular reactions, this study provides further evidence, albeit indirect, that sperm autoantigens are involved in these cellular events.     

STUDIES ON TUBERCULIN GRANULOMA EXPERIMENTALLY PRODUCED ON THE GUINEA PIG MESENTERY

Tuberculin reaction in guinea pig mesentery has been studied as a model of delayed type hypersensitivity. The results obtained through these series of experiments are; 1) even in the delayed type hypersensitivity, the initial phase of the reaction consists of microclrculatory disturbances similar to those of the immediate type reaction, and 2) the capacity of lymphatic drainage after plasma exudation is the essential factor for the formation and absorption of granulomas which are located in rather far distant areas from the vascular tree.     

成都汉族群体五个短串联重复序列基因频率及其种属特异性研究

目的对成都汉族群体5个短串联重复序列（short tandem repeat，STR）基因座的等位基因频率及其种属特异性进行研究，探讨其在法医学中的应用价值。方法用PCR扩增、非变性聚丙烯酰胺凝胶电泳、硝酸银染色方法，对100名成都汉族无血缘关系健康个体的5个STR基因座的等位基因频率及其种属特异性进行研究。结果D18S979、D11S2014、D18S548、D1S1667和GATA164F07在成都汉族群体中等位基因个数分别为6、5、5、7、6；基因型个数分别为12、11、13、19、14；基因型分布符合Hardy-Weinberg平衡定律。通过种属特异性分析发现5个STR基因座中，猴在D18S979、D11S2014和D1S1667基因座均检测出扩增产物，但未在人类基因座分型区内；D18S979位点人类分型区内可检测到牛、狗、鳝鱼有扩增产物，猪、鸭、鼠、兔有微弱扩增产物；D18S548位点人类分型区内只检测到牛有微弱扩增产物；D1S1667位点人类分型区内检测到狗、羊、鳝鱼有扩增产物；GATA164F07位点人类分型区内只检测到狗有微弱扩增产物；泥鳅、鸡、豚鼠在5个基因座均未检测到扩增产物。结论5个短串联重复序列中D18S979、D18S548、D1S1667和GATA164F07在成都汉族群体中具有较好的遗传多态性，D11S2014、D18S548和GATA164F07具有较好的种属特异性，在法医学个人识别和亲子鉴定中有应用价值。     

Adenovirus-Mediated in Utero Gene Transfer in Mice and Guinea Pigs: Tissue Distribution of Recombinant Adenovirus Determined by Quantitative TaqMan–Polymerase Chain Reaction Assay

Fetal somatic cell gene therapy could become an attractive solution for some congenital genetic diseases or the disorders which manifest themselves during the fetal period. We performed adenovirus-mediated gene transfer to mice and guinea pig fetuses in utero and evaluated the efficiency of gene transfer by histochemical analysis and a quantitative TaqMan–polymerase chain reaction (TaqMan-PCR) assay. We first injected a replication-deficient recombinant adenovirus containing the Escherichia coli LacZ gene driven by a CAG promoter (AxCALacZ) into pregnant mice through the amniotic space, placenta, or intraperitoneal space of the fetus. Histochemical analysis showed limited transgene expression in fetal tissues. We then administered AxCALacZ to guinea pig fetuses in the late stage of pregnancy through the umbilical vein. The highest β-galactosidase expression was observed in liver followed by moderate expression in heart, spleen, and adrenal gland. The transgene expression was also present in kidney, intestine, and placenta to a lesser degree. No positively stained cells were observed in lung, muscle, or pancreas except in the vascular endothelium of these organs. Quantitative measurement of recombinant adenoviral DNA by the TaqMan-PCR assay showed that the vast majority of the injected viruses was present in liver. The current study indicated that adenovirus-mediated gene transfer into guinea pig fetus through the umbilical vein is feasible and results in efficient transgene expression in fetal tissues. The experimental procedures using pregnant guinea pigs might serve as a good experimental model for in utero gene transfer. Since our TaqMan-PCR assay detects the LacZ gene, one of the most widely used reporter genes, it may be generally applicable to adenovirus quantification in various gene transfer experiments.     

Development of an assay for the quantification of type I collagen synthesis in the guinea pig

There is a need for a reliable assay for the quantification of collagen type I synthesis in the guinea pig, an important model for many connective tissue diseases. Procollagen type I C-terminal propeptide (PICP) is the established marker of type I collagen synthesis but, to date, no assay has been developed to measure PICP in guinea pig tissue extracts. A monoclonal antibody, known to cross-react with intact guinea pig procollagen type I (anti-PICP), was tested for its ability to bind soluble guinea pig PICP in crude skin extracts using a biosensor. Anti-PICP was immobilised to the surface of a sensor chip and antibody-antigen binding was detected using the phenomenon of surface plasmon resonance (SPR). The binding component in the SPR-immunoassay was identified as PICP by purification and N-terminal sequencing. Guinea pig PICP was purified from skin by gel filtration, ion exchange chromatography and lectin affinity chromatography. Purified PICP was then biotinylated and used with anti-PICP to develop a competition ELISA that was able to selectively and sensitively measure PICP in extracts of guinea pig connective tissue.