Cytophilic antibodies in mice immunized with sheep red cells

Antibodies cytophilic for homologous and heterologous (guinea pig, rat) peritoneal cells of the mononuclear phagocyte type were demonstrated in the sera of mice immunized with sheep red cells. The formation of these antibodies can be induced by immunization with red cells with and without complete Freund's adjuvant. They are also present, in varying titres, in the sera of non-immunized mice. The peritoneal cavity of normal and immunized mice contains cells which adsorb sheep red cells. This phenomenon is significantly reduced by preincubating the peritoneal cells for two hoursin vitro at 37° C. The cytophilic activity of mouse sera can be abolished by absorption by spleen homogenate, but not by pulverized guinea pig kidneys. The haemolysin and haemagglutinin titres before and after absorption are practically the same. The cytophilic component of some sera was inhibited by 2-mercaptoethanol. Tests of the sera of mice revaccinated with sheep red cells showed, after separation on Sephadex G 200, that cytophilic activity was present mainly in the 7 S fraction, but also in the 19 S fraction. Cells adsorbing cytophilic antibodies stain supravitally with neutral red; in fixed and stained preparations they are typical macrophages and monocytoid mononuclears with varying degrees of affinity for basic dyes.     

The dependence of the functional activity of "immune" macrophages on T-cells

The dependence of the functional activity of the peritoneal macrophages of mice immunized with Francisella tularensis vaccine strain on the presence of T-cells in the culture has been studied. The elimination of "immune" macrophages and sensitized T-lymphocytes by means of anti-Thy-1-2-serum has been shown to lead to a sharp decrease in both ingestive and digestive functions of the phagocytic mononuclears of peritoneal exudate to the level of the activity of macrophages isolated from intact animals.     

Acellular pertussis vaccine

Protective, immunogenic, toxic, and sensitizing properties of acellular pertussis vaccine (aPV) developed according to original technology were studied, aPV had marked protective activity which lasted more than 2 years. Sera of mice immunized by aPV also possess protective properties, and they were more prominent than in sera of mice immunized by pertussis bacteria suspension (PS). Immune sera to aPV neutralized cytopathogenic effect of pertussis toxin (PT) on ovarian Chinese hamster cells in 1:250 dilution, whereas neutralizing activity of sera to PS was very low. Level of antibodies to PT was higher in rabbits immunized, according to schedules and dosage recommended for children, by aPV than by PS. High immunogenicity of aPV was proved also by levels of IgG to PT in sera of mice immunized three times by aPV in human dosage. During experiments on mice and guinea pigs aPV had mild toxicity, did not induce autoimmune process, did not have anaphylactogenic properties compared with bacterial suspension characterized by high anaphylactogenic activity. Histamine-sensitizing abilityof aPVwas 40 times lower than that of PS. Assessment of pyrogenic properties of aPV and PS performed on rabbits showed that aPV was 1,000 times less pyrogenic than PS. Obtained results demonstrate high protective and immunogenic properties of domestic acellular pertussis vaccine and its low toxic and sensitizing characteristics.     

Effect of histamine on the rosette-forming capacity of T-lymphocytes in immunization with ADPT vaccine and its components

The results obtained in the study of the influence of histamine on the capacity of T-lymphocytes of guinea pigs immunized with DPT-vaccine and its components for spontaneous rosette formation are presented. Histamine at a concentration of 10(-3) M has been found to inhibit the capacity of blood and splenic lymphocytes of guinea pigs immunized with adsorbed DPT vaccine for spontaneous rosette formation. The inhibitory effect is more pronounced after the immunization of the animals with adsorbed DPT vaccine and Bordetella pertussis suspension.     

Induction and Characterization of Neutralizing Antibodies against a Human Immunodeficiency Virus Type 1 Primary Isolate

Chimpanzees infected with the primary isolate DH012 mount potent neutralizing antibodies. This DH012 neutralizing activity is highly strain specific. Immune sera from guinea pigs immunized with recombinant DH012 gp120 could also neutralize this primary isolate. The neutralizing activity in chimpanzee and guinea pig sera against wild-type DH012 appears to be independent of a linear epitope in the V3 region of gp120. Interestingly, the neutralization escape mutant derived from growing DH012 in the presence of the potent neutralizing chimpanzee serum is at least 50-fold more sensitive than wild-type DH012 to neutralization by guinea pig immune sera. The unusually potent neutralizing activity against the DH012 neutralization-resistant virus is due to the presence of anti-V3 antibodies in guinea pig sera. These results suggested that recombinant gp120 could induce neutralizing antibodies against primary isolate DH012. The V3 of wild-type DH012 is poorly immunogenic in infected chimpanzees and is not accessible to neutralizing V3 antibodies. It is likely that this cryptic V3 region became exposed when the virus escaped the neutralizing activity of the chimpanzee serum.     

So-called antireceptor sera

Experiments in delayed type hypersensitivity transfer were carried out with the aim of studying the ability of rabbit antisera against peritoneal exudate cells of rats sensitized with bovine gamma globulin or rabbit kidney tissue antigen to block peritoneal exudate cells of guinea pigs. In the serological test the antisera prepared against the cells of sensitized rats and tentatively named "receptor antisera", reacted not only with the cells of these rats, respectively, but also with guinea pig cells. In hypersensitivity transfer experiments in guinea pigs receptor antisera showed a blocking effect on the transferred cells, making them incapable of transferring hypersensitivity, i. e. rabbit antisera against rat peritoneal exudate cells reacted with guinea pig cells. This interaction was specific: the blocking effect was manifested only when guinea pigs whose cells were used in the transfer were sensitized with the same antigen as the rats against whose cells the receptor antisera had been prepared. The control antisera, taken for the treatment of the transferred cells in the same doses as the receptor antisera, had no blocking effect on the cells.     

Blocking of delayed hypersensitivity by humoral antibody in an in vivo mouse transfer assay using sensitized guinea pig cells

Humoral antibody was shown to interfere specifically with the expression of cell-mediated immunity (delayed hypersensitivity) in an in vivo system. Mice that received peritoneal exudate cells obtained from guinea pigs sensitized to 1-chloro-2,4dinitrobenzene (DNCB) exhibited delayed hypersensitivity reactions after challenge with the sensitizing agent. While control groups that received either normal sera, saline, or anti-BSA (bovine serum albumin) in addition to peritoneal exudate cells from sensitized guinea pigs exhibited positive delayed reactons to challenge with DNCB, mice that received anti-DNP (dinitrophenyl group) in addition to the senstized cells were prevented from exhibiting a delayed reaction to DNCB.     

Studies on the mechanism of suppression of delayed hypersensitivity by the antischistosomal compund niridazole.

Niridazole given in a single oral dose of 100 mg/kg to guinea pigs sensitized to ortho-chlorobenzoyl chloride-bovine gamma-globulin (OCB-BGG) regularly abolished delayed cutaneous reactivity. Little effect was observed, however, when cells from these animals were tested in vitro with either direct or indirect assays for migration inhibitory factor (MIF). On the other hand, sera taken from nonsensitized guinea pigs after they had received 100 mg/kg of niridazole markedly diminished antigen-induced inhibition of migration of sensitized peritoneal exudate cells in vitro. The immunosuppressive effects of such sera could not be produced by niridazole itself, thereby suggesting an effect of niridazole metabolites. This suppressive activity was readily removed from the serum by dialysis. The active serum blocked the production of MIF by sensitized lymph node cells but did not affect the action of preformed MIF on macrophages. The effect of this serum was reversible; lymph node cells incubated for 24 hr with active serum, then washed and reincubated with antigen in normal serum, produced normal amounts of MIF. These studies suggest that metabolites of niridazole, but not the parent compound itslef, suppress delayed hypersensitivity in guinea pigs and prevent MIF production by lymphocytes without affecting the macrophage response to MIF.     

Reactivity of rabbit antiserum to guinea pig eosinophils.

Although the eosinophil has been recognized as a distinctive cell type for almost 100 years, the major functions of these cells remain unknown. As an approach to defining these functions we have treated guinea pigs with rabbit antiserum to eosinophils (AES) in an attempt to ablate these cells from tissues. Rabbits were immunized thrice with purified eosinophils and the antisera were absorbed with peripheral blood cells from guinea pigs made eosinopenic with methyprednisolone to remove antibodies reactive with serum proteins and erythrocytes. The resulting sera reacted strongly with eosinophils in cytotoxicity tests and had weak or no reactivity with neutrophils. However, absorption of AES with purified neutrophils removed antieosinophil activity. Intraperitoneal injection of potent AES into guinea pigs resulted in complete absence of eosinophils from the peripheral blood and from the peritoneal cavity with only transient or no reduction in circulating neutrophils. Eosinophils were also reduced in bone marrow, spleen, and intestine. The ability of neutrophils to absorb AES activity in spite of weak reactivity in cytotoxicity tests may reflect a quantitative difference in antigenic determinants between eosinophils and neutrophils.     

Leucocyte migration inhibition test as an index of immunological response to measles virus. IV. Blood, spleen, lymph nodes and peritoneal exudate cell reaction.

The migration inhibition test of leucocytes isolated both from the peripheral blood, spleen and lymph nodes as well as peritoneal exudate cells of guinea pigs, immunized with the various doses of measles virus was determined. For in vitro testing of cellular immunity to measles virus, viral antigens could be used in both infective and inactivated form.     

Legionella pneumophila intracellular growth in normal vs. Immune guinea pig macrophage cultures

Intracellular growth ofLegionella pneumophila, an opportunistic intracellular bacterium considered the cause of legionellosis, was assessed in peritoneal macrophages from normal and immunized guinea pigs. These bacteria grew exceedingly well in the normal guinea pig macrophages. Uptake of these bacteria was about the same by macrophages from either normal or immune guinea pigs, but their growth in immune macrophages was completely inhibited. Macrophages from normal guinea pigs stimulated with mezerin, a compound similar to diterpene ester, a known nonspecific stimulator of macrophages, or with specificLegionella vaccine released moderate or only small amounts of hydrogen peroxide, an indicator of macrophage activation to antimicrobicidal activity. In contrast, macrophages from immune guinea pigs produced much higher levels of hydrogen peroxide when stimulated with mezerein or theLegionella vaccine, and also showed a heightened response when cultured without a stimulator. These results indicate that macrophage activation related to the immune status of the host appears to have an important role in initial resistance toLegionella growth in susceptible individuals.     

The effect of histamine on the rosette-forming ability of T-lymphocytes upon immunization with DPT vaccine and its components

The influence exerted by histamine on the capacity of T-lymphocytes for spontaneous rosette formation in intact animals and in animals immunized with adsorbed DPT vaccine and its components has been studied. Histamine at a concentration of 10(-3) M has been found to produce an inhibitory effect on the capacity of lymphocytes in the blood and spleen of guinea pigs immunized with adsorbed DPT vaccine and its components for spontaneous rosette formation. This effect of histamine has proved to be even more pronounced after the immunization of the animals with adsorbed DPT vaccine and Bordetella pertussis suspension, which is probably due to their sensitizing action.     

The action of pertussis vaccines and GMDP on the change in the enzymatic activity of macrophages from peritoneal exudate

The influence of whole-cell and acellular pertussis vaccines, introduced both alone and in combination with N-acetylglucosaminylmuramyl-2-alanine-D-isoglutamine (GMDP) on the activity of two enzymes of peritoneal exudate macrophages (5'-nucleotidase and Na+K(+)-adenosine triphosphatase) was studied. The study revealed that both pertussis vaccines exhibited immunomodulating properties, these properties being most pronounced in whole-cell pertussis vaccine. The use of GMDP in combination with pertussis vaccines led to changes in the enzymatic activity of peritoneal exudate macrophages, which was indicative of a decrease in the immunomodulating action of pertussis preparations.     

A heterophile antigen associated with experimental toxoplasmosis

The biological characteristics of a heterophile protein (HP) in peritoneal exudate from mice, hamsters, rats, and guinea pigs infected with Toxoplasma gondii were studied by immunofluorescence, immunoelectrophoresis and immunodiffusion techniques using specific antisera raised in rabbits. HP of mice had the highest antigenicity, HP of hamsters and rats had intermediate antigenicity and HP of guinea pigs had the lowest antigenicity. HP was found in normal peritoneal exudates from mice, hamsters and rats inoculated with paraffin oil instead of T. gondii and in normal guinea pig serum. HP was detected by the fluorescent antibody technique on the surface of T. gondii in peritoneal exudates of mice, but not on mouse peritoneal cells, and by the indirect fluorescent antibody technique on L cells infected with T. gondii and on free Toxoplasma derived from them, but not on uninfected L cells. T. gondii could make host cells produce HP to cover its surface for protection. The relation between HP from host cells and T. gondii is discussed.     

The assessment of antitoxic anti-anthrax immunity

In experiments on guinea pigs immunized with avirulent noncapsular strains STI, Sterne (34F2) and the avirulent mutant of Bacillus anthracis strain 228/8 the relationship between the titers of serum antibodies to the preparations of purified protective antigens (PA) and purified lethal factor (LF) of B. anthracis toxin and the level of the antitoxic activity (ATA) of blood sera, as well as acquired resistance, was analyzed. The ATA of sera was evaluated in the primary culture of peritoneal macrophages affected by the mixture of PA and LF. The level of relationship (r) between individual ATA values and the titers of antibodies to PA and LF was shown to vary over a wide range, depending on the group of the animals and did not exceed, on the average, 0.19-0.37. At the same time the mean values of these characteristics, followed in their dynamics depending on the immunogenic properties of vaccine strains or the time elapsed after vaccination, were highly correlated (r = 0.76-0.87). The possibility of using these characteristics for the evaluation of acquired resistance are discussed.     

Some factors determining yields of peritoneal exudate macrophages from guinea pigs.

Several features which affect the yields of guinea pig peritoneal exudate macrophages were investigated. Optimum yields were obtained from ex-breeding female guinea pigs, aged ca 18 mo, 10 da after injection with Marcol 80.     

Killing effect of normal peritoneal exudate cells in guinea pigs on microfilariae of Dirofilaria immitis evoked by passive transfer of immune humoral factor.

Parasiticidal activity of normal peritoneal exudate cells on microfilariae of Dirofilaria immitis in diffusion chambers implanted into normal guinea pigs was evoked by intraperitoneal passive transfer of anti-D. immitis serum. The immune serum was fractionated into supernatant and sediment by ammonium sulfate precipitation. The parasiticidal effect was reproduced with the supernatant and, to a less extent, with the sediment. Anti-D. immitis serum from the animals sensitized 5 days before was also effective in this respect. From these results it can be concluded that the factor responsible for the phenomenon is some other factor(s) than the antibody. In addition, the in vitro cytotoxicity test demonstrated an enhanced Mf-killing activity of sensitized peritoneal exudate cells by adding with anti-D. immitis serum.     

Use of the reaction of macrophage disappearance from peritoneal exudate for characterizing cellular immunity in Salmonella immunization

The test of macrophage disappearance from peritoneal exudate quite effectively shows the state of cell-mediated immunity in guinea pigs immunized with both live and killed S. typhimurium culture. The macrophages of the animals immunized with killed S. typhimurium culture react to the protein extract of these bacteria more actively than the macrophages of the animals immunized with killed S. sonnei cultures, which indicates the specificity of this test.     

Suppression of the intradermal hypersensitivity reaction of the delayed type by a serum fraction from patients containing leukocyte migration inhibition factor]

The blood serum of patients with active tuberculosis of the lungs and chronic pneumonia inhibited migration of donor's leukocytes and macrophages of the peritonal exudate of guinea pigs when compared with migration of similar cells in the medium with the serum of cattle or donors. After chromatography these sera were fractionated on the columns with Sephadex G-100. Fractions containing the leukocyte migration inhibition factor (LMIF) suppressed, up to complete abolition, the intradermal reaction to tuberculin in man and guinea pigs sensitized with BCG. The LMIF is supposed to act in the regulation of delayed hypersensitivity reaction.     

Evaluation of a guinea pig model to assess interference in the immunogenicity of different components of a combination vaccine comprising diphtheria, tetanus and acellular pertussis (DTaP) vaccine and haemophilus influenzae type b capsular polysaccharide conjugate vaccine.

A guinea pig model to assess the immunogenicity of a combination vaccine containing diphtheria, tetanus and acellular pertussis (DTaP) vaccine and Haemophilus influenzae type b (Hib) capsular polysaccharide conjugated to tetanus toxoid (HibT) was evaluated comparatively with the mouse immunogenicity test to study the effect of combining these antigens on the immunogenicity of various components. The immunogenicity test in mice was performed by subcutaneous injection of groups of 10 animals twice at an interval of four weeks with 1/10 of a single human dose of various formulations of combination vaccines, DTaP or HibT vaccine. The animals were bled at 4 and 6 weeks and IgG or total antibodies to various components were determined by ELISA or RIA. The guinea pig immunogenicity model included groups of animals injected subcutaneously twice at an interval of six weeks with 1.5 times the single human dose of various formulations. The animals were bled at 4, 6 and 8 weeks and serum samples were tested for antibodies to various components by ELISA, RIA and/or neutralization tests. Additionally, potency of tetanus and diphtheria components was assessed as per the US Food and Drug Administration's regulations. Aluminium phosphate (AIPO(4)) adsorbed HibT vaccine or HibT as a combination with AIPO(4)adsorbed DTaP vaccine showed significant increases in IgG antibodies to tetanus toxin in mice as well increased tetanus antitoxin levels in guinea pigs as compared to soluble HibT vaccine. In general, combining DTaP and HibT vaccines did not affect the antibody levels to tetanus and diphtheria toxoids whereas DTaP-HibT combination vaccine elicited significantly lower IgG antibodies to pertussis toxin and filamentous haemagglutinin than DTaP vaccine alone, particularly after first injection. Mice showed similar Hib antibody responses for the combination and HibT alone whereas guinea pigs consistently showed lower anamnestic responses to Hib for combination formulations than for HibT alone. Reducing the amount of HibT and/or tetanus toxoid in the combination formulations reduced this suppression of Hib antibody response in guinea pigs. Suppression of Hib antibody response in combination vaccines has also been reported from recent clinical trials. Based on the results from this study, it appears that the guinea pig model may be able to predict the human response to various components of combination vaccines.