Generation of cytolytic T lymphocytes in vitro. VIII. failure of anti-RS antisera to inhibit the generation of cytolytic T lymphocytes or cytolytic T lymphocyte activity.

Antibody reactive with "recognition structures" (RS) of mouse lymphoid cells for alloantigens (anti-RS) was prepared by immunization of F1 hybrid mice with parentalstrain lymphoid cells or with antibody produced in one parental strain against alloantigens of the other parental strain. Such antisera prevented generation of the "product of antigenic recognition" (PAR) that is produced within a few hours in cultures prepared with a mixture of lymphoid cells from genetically disparate mice. However, treatment of responding lymphoid cells with anti-RS sera and complement did not inhibit generation of cytolytic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC). Treatment of cells obtained from MLC with anti-RS sera and complement failed to inhibit cytolytic activity of such cells for specific alloantigens.     

Effector functions of CD8-positive guinea pig T lymphocytes

The response of guinea pig T lymphocytes to different stimuli was analysed with focus on the functions of CD8-positive T cells, which so far had been poorly defined in this animal model. For identification and purification of guinea pig cytotoxic T lymphocytes, three monoclonal antibodies, directed against the CD8 differentiation antigen were characterized and compared with respect to expression pattern and biochemical characteristics of the corresponding cell surface antigen. The antibodies were used for the identification of the cytotoxic T lymphocyte subpopulation within alloreactive T cell lines, and for the depletion of CD8-positive cells in in vitro assays. Purified CD4- and CD8-positive cells were tested for their ability to proliferate in response to antigen, mitogen or anti-guinea pig Thy-1 monoclonal antibodies. Both, CD4- and CD8-positive cells showed IL-2 release and subsequent proliferation after polyclonal stimulation. Cytotoxic activity in CD8-positive alloreactive T cells was expressed in vitro only after repeated stimulation.     

The activation of guinea pig T lymphocytes by anti-beta 2-microglobulin serum.

In order to study further the role of beta 2-m in the regulation of the immune response, we have examined the effects of a goat anti-guinea pig beta 2-m serum on a number of T lymphocyte functions in vitro. Anti-beta 2-m serum produced a marked inhibition of the response of peritoneal exudate T cells to antigen and mitogen stimulation. Surprisingly, a marked activation of lymph node T lymphocyte proliferation was observed in the absence of antigen or mitogen stimulation. This stimulatory effect of anti-beta 2-m serum was shown to be specific for beta 2-m and required the presence of macrophages. The T cell proliferative response induced by anti-beta 2-m could not be blocked by antisera to the antigens of the guinea pig MHC. These studies suggest that beta2-m may play some critical role in the immune response at the level of T cell activation.     

Antibacterial activity of stimulated guinea pig peritoneal exudate cell culture supernates

Guinea pigs infected with Mycobacterium bovis BCG at least 5 weeks previously yield peritoneal exudate cells which, when stimulated in culture for 2–3 days with specific antigen (PPD), elaborate into the culture medium a factor which is antibacterial for listeria and corynebacteria, but not against other organisms tested. Concentrations of colchicine and vinblastine above 10^?7M completely inhibited its production. The yield of the factor was also found to be dependent on the choice of culture medium, the source of serum, and whether or not the scrum was heated at 56 °C for 30 min. It was weakly antagonized by the anions, RNA, and cyclic and noncyclic nucleotides, but not by lysozyme. It did not lyse M. lysodcikticus and had at least 100 times more activity than egg white lysozyme against listeria. Nonadherent cells from BCG-infected guinea pigs, plus PPD, stimulated adherent monolayer cells from normal guinea pigs to produce this material, but they did not stimulate adherent, cells from rabbit peritoneal exudates to its production.     

Opiate-like activity of salsolinol on the electrically stimulated guinea pig ileum

Contractions elicited by electrical stimulation of the guinea pig ileum are reduced partially by the tetrahydroisoquinoline compound, salsolinol. This action is antagonized by pretreatment with the narcotic antagonist, naloxone, but not reversed by this agent once the effect is initiated. In addition, salsolinol can reduce the inhibitory activity of morphine. These results suggest that salsolinol may be a partial agonist on this opiate-sensitive preparation.     

Isolation of monospecific antisera to guinea pig immunoglobulins

The results of the production and analysis of monospecific rabbit antisera to guinea pig IgG1, IgG2, IgA and IgM are presented. Isolated immunoglobulins of different isotypes, as well as immune precipitates obtained by immunoelectrophoresis, were used for immunization. After adsorption antisera of each type there formed one precipitation line with guinea pig serum in immunoelectrophoresis, thus indicating that they contained antibodies to immunoglobulins of the definite isotype.     

HIV-specific cytotoxic T lymphocytes directed against alveolar macrophages in HIV-infected patients

A CD8+ lymphocytic infiltration of the lungs is frequently observed in HIV-infected patients, even prior to the onset of opportunistic infections. In such patients, we could demonstrate that most of these CD8+ alveolar T lymphocytes displayed the D44 marker and were functional cytolytic T lymphocytes directed against autologous HIV-infected alveolar macrophages. This primary cytolytic activity was HLA-restricted and, at least partially, specific for the HIV envelope protein, since HLA-A2 alveolar T lymphocytes could specifically lyse cell lines expressing both the HLA-A2 and Env antigens. In contrast to data obtained in peripheral blood, no ADCC activity was observed against the Env antigen.HIV-specific alveolar T-lymphocyte cytolytic activity decreased with progression towards AIDS as shown by studies of a serie of 40 patients.Functional abnormalities of the lung epithelium could be associated with the specific lysis of alveolar macrophages, suggesting that local tissue injury could result from the in vivo immune conflict between alveolar HIV-specific CTL and HIV-infected macrophages.     

Characterization of an antiserum to guinea pig antithrombin III (AT III). I. Reactivity with guinea pig T lymphocytes

In the course of studies investigating the effects of antisera prepared against a variety of guinea pig proteins on lymphocyte function, a goat antiserum prepared against a guinea pig gamma-globulin preparation was found to react with guinea pig T lymphocytes. This antiserum, serum 592, contained a significant titer of antibodies that were cytotoxic for a subpopulation of lymph node cells and thymocytes, and mitogenic for lymph node T cells. Immunoelectrophoretic analysis and selective absorptions of the antiserum demonstrated that the antigen recognized on thymocytes was also present on an alpha 2 globulin in guinea pig plasma, which, on the basis of physiochemical characteristics and heparin-binding affinity, appeared to be guinea pig antithrombin III (AT III). Although the antiserum was shown to contain antibodies to both protein and carbohydrate determinants on the AT III molecule, studies comparing the effects of 7 M guanidine and periodate oxidation on the antigenicity of the AT III determinant also recognized on the thymocytes indicated that this shared antigenic determinant was carbohydrate in nature. The thymocyte membrane molecule bearing this determinant was also isolated and was found to be a 210,000-dalton macromolecule that was very sensitive to proteolytic and/or autolytic degradation. These data raise the interesting possibility that guinea pig lymphoid cells may have a membrane-associated protease inhibitor related to plasma AT III.     

Characterization of antisera directed against follistatin/activin-binding protein peptides.

An attempt was made to develop immunologic probes directed against follistatin/activin-binding protein (ABP), for use in investigating the distribution of ABP in various tissues. Five oligopeptides corresponding to different regions of the predicted ABP amino acid sequence (peptides 1-12, 28-43, 123-134, 270-281 and 300-315) were synthesized chemically, and coupled to Limulus polyphemus hemolymph hemocyanin. The peptide-hemocyanin conjugates were then used to immunize rabbits, and the immunoresponses were monitored by enzyme-linked immunosorbent assay. Reactivity of the antipeptide antisera with ABP was determined by SDS-polyacrylamide gel electrophoresis and immunoblotting analysis. All of the peptides produced immune responses. The antiserum to peptide 123-134 recognized all forms of ABP, whereas the antiserum to peptide 300-315 reacted specifically and sensitively with the long forms of ABP. These two antisera exhibited only a limited cross-reaction with other proteins or none at all. Therefore, they will be useful for studying the distribution of ABP in various tissues.     

Background activity pattern of guinea pig septal neurons in vitro

Activity of medial septum-diagonal band cells (MS-DB neurons) was investigated in slices of guinea pig septum. Four types of activity were distinguished on the basis of interspike interval distribution and coefficient of variation (CV): extremely regular (CV<0.3), regular (CV>0.3<0.7), irregular (CV>0.7), and rhythmic bursting patterns. Activity of cells belonging to the first group was resistant to superfusion with a medium low in Ca²⁺ and high in Mg²⁺ which produces blockade of synaptic effects. The same applied to a percentage of neurons with a rhythmic bursting pattern. Activity pattern of Mg²⁺-resistant bursting cells also remained unchanged by the effects of GABA and acetylcholine antagonists. It is concluded that cells with properties of regular and bursting endogenous pacemakers are found in the MS-DB.Institute of Biophysics, Academy of Sciences of the USSR, Pushchino-on-Oka. Translated from Neirofiziologiya, Vol. 19, No. 5, pp. 586–595, September–October, 1987.     

Comparative migration of guinea pig T and B lymphocytes from capillary tubes

Using a capillary tube migration technique, a comparison was made between the random mobility of separated guinea pig T- and B-lymph node lymphocyte subpopulations, and macrophage-rich peritoneal exudate cells. A decrease in random movement was found in that order, which would fit with the hypothesis that migration on a substrate is an adherence-dependent phenomenon. In order to characterize a possible dichotomy between T-and B-cell locomotion, the effects of several factors which might interfere with their migration were studied. These factors included drugs which affect cell metabolism, cell surface ligands, and some factors which may play a role in inflammatory foci (acidity, immune complexes, and lymphokines). The results emphasize the similarity in the mode of locomotion of T and B lymphocytes. However, a remarkable difference was found in the stronger inhibition of B-cell migration by pH values below pH 7.0. The relevance of these findings to the migration of T and B lymphocytes into inflammatory foci is discussed.     

Fusion of hamster and pig zona-free eggs stimulated by boar and guinea pig sperm at fertilization in vitro

In the course of in vitro fertilization of zona-free hamster and pig eggs by boar and guinea-pig spermatozoa it was observed that homologous and heterologous eggs fused together, forming cell hybrids between two or more cells. The fusogenic activity was attributed to spermatozoa and this was the hypothesis tested. The fusogenic activity (coinciding with sperm penetration activity) was dependent on the duration of sperm preincubation, which may be regarded as capacitation in vitro. Fusion occurred only after 3 hr of sperm preincubation and a narrow optimum was detected at 4–4.5 hr. Fusion of eggs was also dependent on sperm concentration. A relatively high proportion of fusions was observed at a sperm concentration of 4.0 × 10⁴ per ml and an optimum was attained at a concentration of 5.0 × 10⁵ per ml. The first fusions were observed at 90 min after semination. After 3 hr more than a half of the eggs reacted, and by 20 hr of incubation 80% of ova were fused. The fusability of eggs was tested and found to occur at 14 hr after ovulation. The fusion process was also studied using transmission electron microscopy. It is supposed that the process of egg fusion may be caused either by a similar mechanism to sperm-egg fusion, or by products released during the sperm acrosome reaction.     

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.     

In vitro characterization of anti-glucosylceramide rabbit antisera

Glucosylceramides (GlcCer) are biosynthetic precursors of glycosphingolipids. They are widely distributed in biological systems where they exhibit numerous biological functions. Studies on the localization of glucosylceramides in different tissues have used biochemical methods only since specific antibodies against GlcCer were not previously available. We have characterized two commercially available rabbit antisera which were prepared against GlcCer of plant origin (1-O-(beta-D-glucopyranosyl)-N-acyl-4-hydroxysphinganine; GlcCer-3) or human origin (1-O-(beta-D-glucopyranosyl)-N-acyl-sphingosine; GlcCer-2) and claimed to be specific for GlcCer. The antisera were also able to detect specifically GlcCer species in crude lipid extracts from human epidermis after separation by thin-layer chromatography. The reagents are sensitive since both antisera reacted at dilutions higher than 1:500 with their homologous antigen in the nanogram range in thin layer immunostaining or dot-blot assays. The antisera are specific for GlcCer although they did not differentiate between GlcCer-2 and GlcCer-3 containing sphingosine or 4-hydroxysphinganine. The antisera also reacted with N-stearoyl-DL-dihydroglucocere-broside indicating that the naturally occurring structural variations in the amino alcohol moiety are not determining the specificity. No crossreactivity was observed with other mono- or diglycosylceramides (galactosylceramides, lactosyl-ceramide), free ceramides or structurally unrelated lipids (cholesterol, sphingomyelin, or phospholipids). Therefore, the glycosylmoiety seems to represent the major antigenic determinant. Finally, the antisera also proved to be useful for the immunohistochemical localization of GlcCer in human epidermis by which earlier biochemical data on the distribution of GlcCer in the various epidermal layers were confirmed.