In Vitro and in Vivo Stimulation of Murine Lymphocytes by Human Respiratory Syncytial Virus Strains

Respiratory syncytial virus (RSV) strains of subtype A (A2, WV9894, and WV12138) and of subtype B (WV1293, WV4843, and WV6873) are mitogenic in vitro for unprimed BALB/c spleen cells. The virus also triggered splenocytes in vitro to secrete immunoglobulins. Plaque-purified and UV-irradiated materials of both RSV subtypes produced comparable levels of DNA synthesis. Infectious materials of both subtypes also induced pronounced responses. Lymphocyte activation with UV-inactivated RSV strain A2 was dose-dependent and maximal responses occurred after 4-5 days of incubation. The virus preparations were mitogenic for spleen cells depleted of T lymphocytes by treatment with anti-Thy 1.2 and complement and for lymphocytes of congenitally athymic mice (nu-nu). They were also mitogenic for highly purified T lymphocytes separated by panning of spleen cells on anti-mouse Ig-coated Petri dishes, suggesting that both B and T lymphocytes respond to the mitogenic activity of RSV. Moreover, mice infected intranasally with RSV strain A2 generated local as well as peripheral cellular and humoral responses.     

Mouse Virulence of Human, Systemic, Pathogenic Escherichia coli Strains

Strains of Escherichia coli isolated from systemic infections of humans exhibited marked intraperitoneal and intracerebral virulence for mice. This marked virulence was not exhibited by enteropathogenic strains.     

A Micromethod for the Activation of Human Blood Lymphocytes In Vitro

A micro culture system is described in which 2.5 × 10⁴ human blood lymphocytes in aliquots of 100 μL are stimulated by PHA, Pokeweed, “Varidase” antigen, allogeneic small lymphocytes or mitomycin-C-treated allogeneic LCL cells. Careful regulation of the pH by a combination of bicarbonate and MOPS buffers seems to be important for detecting a response to weak stimuli. High and reproducible levels of activation by powerful stimuli (PHA and LCL cells) can be recorded from even smaller cultures (10⁴ responding cells in 40 μL aliquots). The technique allows large numbers of replicate cultures to be set up from a single blood sample so that the time course and/or dose-response relationships can be examined for a range of differen mitogens.     

A Micromethod for the Activation of Human Blood Lymphocytes In Vitro

A micro culture system is described in which 2.5 × 10⁴ human blood lymphocytes in aliquots of 100 μL are stimulated by PHA, Pokeweed, “Varidase” antigen, allogeneic small lymphocytes or mitomycin-C-treated allogeneic LCL cells. Careful regulation of the pH by a combination of bicarbonate and MOPS buffers seems to be important for detecting a response to weak stimuli. High and reproducible levels of activation by powerful stimuli (PHA and LCL cells) can be recorded from even smaller cultures (10⁴ responding cells in 40 μL aliquots). The technique allows large numbers of replicate cultures to be set up from a single blood sample so that the time course and/or dose-response relationships can be examined for a range of differen mitogens.     

In Vitro Activation of Human T Lymphocytes by Haemophilus influenzae Type b Capsular Polysaccharides

Polyribosilribitolphosphate (PRP), the capsular polysaccharide from Haemophilus influenzae type b, is a T-cell-independent type 2 antigen. In vitro culture of adult peripheral blood T cells with 15 micrograms/ml PRP leads to induction of interleukin-2 receptor (IL-2R) expression on up to 10% of T cells. These cells are CD4+ and carry the alpha beta T-cell receptor. PRP, at concentrations above 1-5 micrograms/ml, can also induce in vitro proliferation of both adult and neonatal T cells. We conclude that PRP acts as a human T-cell mitogen. The in vitro proliferative response as well as IL-2R expression was studied in T cells derived from adults after vaccination with native PRP, with PRP conjugated to a carrier protein, or with diphtheria toxoid. Vaccination with conjugated PRP decreased the doses of PRP required for in vitro induction of IL-2R expression and T-cell proliferation. This indicates that vaccination with PRP conjugated to a carrier protein improves the in vitro T-cell response to PRP activation.     

Specific and Nonspecific Immunoregulatory Events in the Clonal Response of Human Lymphocytes in Vitro

Stimulation of sensitive leucocyte populations with near optimal concentrations of soluble microbial antigens results in vigorous lymphocyte proliferation when ³H-thymidine incorporation is measured after 4–8 days. Lymphoblasts in these cultures revert to small lymphocytes after 10–14 days, at which time they are often refractory to any stimulant including the original incubating antigen. When these primed lymphocytes are irradiated with 500–1000 R to block their proliferation and added to fresh leucocyte culture from the same individual (autologous), they usually, but not invariably, reduce the proliferation of the unirradiated fresh leucocyte cultures. Exposure to 6000 R reduced the suppressor activity. Reduction was specific for the microbial antigen with which they were originally generated, but, more often, a combination of both specific and nonspecific suppression was observed. These data provide good evidence, with reciprocal specificity, for the generation of antigen specific suppression in vitro.     

Activation of Human T Lymphocytes by Leishmania Lipophosphoglycan

This study describes Leishmania antigen-induced activation of lymphocytes isolated from Kenyan donors, previously treated for visceral leishmaniasis, and from Danish and Kenyan controls. Peripheral blood mononuclear cells (PBMC) from cured Kala-Azar patients proliferated and produced Interferon-gamma in vitro in response to lipophosphoglycan (LPG) isolated from Leishmania major. The proliferative response was mainly due to activation of CD2-positive T cells. PBMC from controls did not respond to LPG, but to sonicates prepared from both L. major and L. donovani promastigotes. The surface glycoprotein GP 63 failed to activate PBMC from any of the donors tested. These results show that the individuals cured from visceral leishmaniasis had expanded T-cell clones recognizing LPG, conceivably as a result of Leishmania infection. The LPG preparation was without detectable protein contamination. Thus, the results suggest that human T lymphocytes can respond to glycolipid antigens.     

Mitogen-Induced Activation of Stable E-Rosettes by Human Lymphocytes

Following on an earlier study which demonstrated that PHA can activate human lymphocytes to form temperature-stable (37°C.) E-rosettes with sheep red blood cells, a number of other mitogens were tested. These included Concanavalin A, pokeweed mitogen, soybean agglutinin, sodium periodate, and galactose oxidase. A11 except soybean agglutinin demonstrated the capablility to activate lymphocytes to form stable E-rosettes.     

In Vitro Modulation of Activation Antigens on Human Lymphocytes by β-Estradiol

PROBLEM: The possible in vitro immunomodulating effect of β-estradiol on phytohemagglutinin-stimulated human lymphocyte cultures was studied. METHOD OF STUDY: Lymphocyte cultures from 12 healthy men and women aged 25–35 years were set up for 12 hr in the presence and in the absence of β-estradiol, and the expression of the activation markers CD25, CD69, and CD71 was examined by flow cytometric analysis with specific fluorescent conjugated antibodies. RESULTS: Although the number of cases is small, in 10 of 12 cases in the presence of β-estradiol in two different concentrations, a significantly decreased expression of CD69 could be observed. A slight decrease could also be observed for the Interleukin-2 receptor expression; however, the difference, in the presence or absence of β-estradiol, was not significant. CONCLUSIONS: The results suggest that in vitro addition of β-estradiol can inhibit, to a certain degree, specific activation markers on phytohemagglutinin-stimulated lymphocytes from young men and women. The present study could not define the role of sex differences because of the small number of samples. A comparison between men and women at various ages in a greater number of cases, as well as studies on activation markers after treatments with estrogens, would be useful.     

单纯疱疹病毒体外诱导特异性抗体应答的实验研究

用灭活的单纯疱疹病毒（Ｈｅｒｐｅｓｓｉｍｐｌｅｘｖｉｒｕｓ，ＨＳＶ１）为致敏原，体外诱导成人外周血淋巴细胞产生特异性抗体应答，特异性抗体诱生水平与血清抗体无相关性（Ｒ＝０．４５，Ｐ＞０．０５），抗体类型为ＩｇＧ。同法致敬新生儿淋巴细胞则不能诱生任何类型的特异抗体，表明此体外抗体应答属继发性免疫应答。特异性抗体应答有明显的ＨＳＶ１抗原剂量依赖性，且需要Ｔ、Ｂ细胞的相互作用。ＨＳＶ１体外致敏的实验研究，为探讨正常和疾病状态下体外特异性抗体应答和免疫调节提供一个有用的实验模型。     

Stimulation of Human Lymphocytes by Allogeneic Endothelial Cells in Vitro

Endothelial cells separated from the vein of human umbilical cords were capable of stimulating allogeneic lymphocytes in mixed lymphocyte endothelial cultures. The degree of stimulation observed with endothelial cells was generally less than that for mitomycin treated lymphocytes, but of the same order of magnitude. Endothelial cells proved non-stimulatory to autologous lymphocytes separated from cord blood.     

Activation of Human T Lymphocytes by Soluble Protein A

The proliferative response of highly purified human peripheral blood or tonsil lymphocytes in the presence of soluble protein A (SpA) was investigated. SpA was shown to be a potent mitogen for T lymphocytes from peripheral blood or tonsils. Conversely, the non-T lymphocyte population from peripheral blood responded poorly to SpA, and SpA did not stimulate non-T lymphocytes from tonsils. The addition of mitomycin-treated T lymphocytes from peripheral blood enhanced the response of blood non-T lymphocytes to protein A. This effect was not found when non-T lymphocytes isolated from tonsils were cultured with mitomycin-treated T lymphocytes. The proliferative response of unseparated cells or purified lymphocyte populations was enhanced when adherent cells were added to the cultures. It is concluded that the soluble form of protein A activates mainly T lymphocytes and does not induce B lymphocyte proliferation.     

Cell-Cycle Kinetics of Proliferating Mouse B Lymphocytes In Vitro

Resting mouse B lymphocytes were stimulated in vitro with Upopoiysaccharide, Sepharose-coupled anti-γ antibodies or a combination of the two. B lymphocytes stimulated with anti-γ entered the cell-cycle with more rapid kinetics and at a higher frequency than did the corresponding cell population stimulated with lipopolysaccharide. Using cell cycle analysis after DNA staining combined with an M phase block, the cell-cycle kinetics of in vitro cultured B-lymphocytes was studied. The labelling index of lipopoly saccharide stimulated B lymphocytes was 60% while that for anti-γ Sepharose stimulated cells was 85%. The generation time of the actively cycling population from both types of cultures was constant and was of the order of 18 h. Thus, the fraction of B lymphocytes induced to proliferate in vitro varies depending on the stimulus, while the growth kinetics of the actively proliferating populations are remarkably constant.     

Activation of Human T Lymphocytes by Phorbol-12,13-Dibutyrate and Ionomycin

The calcium ionophore ionomycin and the phorbol ester phorbol-12,13-dibutyrate (PDBu) are shown to have a synergistic effect upon interleukin 2 (IL-2) production, interleukin 2 receptor expression, and T-lymphocyte proliferation. The proliferative response was inhibited by addition of a monoclonal antibody directed against the IL-2R (Tac antigen) demonstrating that PDBu and ionomycin induce T-cell growth through an IL-2-dependent autocrine pathway. Sequential stimulation with PDBu and ionomycin failed to induce IL-2 production, IL-2R expression, and consequently proliferation of the T cells, indicating that T-cell activation requires simultaneous activation of protein kinase C (PKC) and elevation of cytosolic calcium. Exposure of T cells to both agents for different times resulted in IL-2 production, IL-2R expression, and proliferation in proportion to the duration of incubation with at least 4 h required for maximal T-cell activation. Further, in the presence of PDBu maximal T-cell activation was found to require stimulation with ionomycin for 4 h, indicating that a sustained increase in free cytoplasmic calcium of several hours' duration is essential for T-cell activation. In contrast T cells incubated with ionomycin were induced to produce IL-2 and express IL-2Rs upon brief exposure to PDBu with a 2-h incubation period being sufficient for maximal T-cell activation. Thus transient activation of PKC seems to be sufficient for activation of the IL-2 gene and IL-2R gene. However, maximal T-cell activation requires activation of PKC for at least 2 h.     

Expression of Activation and Costimulatory Elements by Human Intestinal Intraepithelial Lymphocytes

It is unclear whether human intestinal intraepithelial T lymphocytes (iIEL) are resting or activated cells. To address this question, an improved isolation procedure was developed for small bowel iIEL, which were analysed by two-colour flow cytometry and compared with resting and mitogen-activated peripheral blood lymphocytes. iIEL expression of CD44 isoforms, Bcl-2 and Ki67 antigen was also determined in tissue sections. iIEL expressed CD69 at levels comparable with 48-72 h phytohaemagglutinin blasts, but did not express CD25 or CD95. iIEL were Bcl-2+ but not Ki67+. alphaEbeta7 and alpha4B7 expression was relatively high, whereas alphaLbeta2, CD5 and CD28 were expressed at low density. Isolated iIEL expressed CD44 (core epitopes) at lower levels than peripheral blood lymphocytes, although almost all CD44 contained splice variant 6 (CD44v6). Peripheral blood lymphocytes expressed CD44 at very high density, but little CD44v6, even after activation. However, in tissue sections, iIEL showed differential labelling with CD44 core epitope antibodies and no detectable CD44v6, implying CD44 receptor occupancy or epitope masking in situ. Thus, normal iIEL express a quasi-activated phenotype with unusual patterns of adhesion receptors, which may act as costimulatory elements. These may permit iIEL to assume effector functions, with absence of CD25 preventing entry into the cell cycle, thereby maintaining an apoptosis-resistant phenotype.     

In Vitro Target Cell Lysis Mediated by Normal Human Lymphocytes

Lysis of DNP-coated chicken erythrocytes by human blood lymphocytes (K cells) was induced by means of rabbit anti-DNP antibodies. Antisera were prepared by injecting the animals with DNP-conjugated proteins emulsified in Freund's complete adjuvant An ammonium sulphate precipitation technique was used for assay of antibody concentration and affinity. Sephadex G-200 chromatography indicated that 90% of the DNP antibodies were 7S in the bleedings on days 10–16. whereas 99. 8% were 7S in later bleedings. 7S antibodies induced K-cell lysis at high dilutions, whereas 19S antibodies were essentially negative Antibody fractions obtained by DF.AE- or CM-cellulose chromatography were used to establish possible heterogeneities in the capacity of 7S antibodies to induce either K-cell- or complement-mediated target cell lysis. No such heterogeneities were found Fifteen IgG preparations containing antibodies of different affinities were compared with regard to their capacity to induce K-cell-mediated lysis. A statistically significant correlation was found between antibody affinity and efficiency in K-cell-mediated lysis. In a similar study of complement mediated lysis the correlation was not significant at the 5% level but was significant at the 10% level.     

Morphologic and Antigenic Maturation of Lymphocytes in the Mouse Thymus In Vitro

A simple technique for organ culture of the thymus of 14-day-old mouse embryos is decribed. It allowed a characteristic time-dependent development of the immature thymus into a lymphoid thymus with large numbers of nondividing small lymphocytes. Most of these cells carried the T-lymphocyte antigenic markers Thy-1 and TL, and all expressed H-2 antigens as determined in cytotoxicity assays. They probably developed from precursor cells without detectable Thy-1 and TL. This development appeared to be dependent on the thymic microenvironment, since it also occurred in serum-free organ cultures and in cultures with medium supplemented with serum from nude mice.     

Production of Specific Antisera to Human B Lymphocytes

Antisera have been prepared, in mice and rabbits, to membrane and sub-membrane fractions of human B lymphocyte derived lymphoid lines. Antisera to a protein subfraction were, after only minimal absorption, specific for human peripheral B lymphocytes, monocytes and B cell derived lymphoid lines. The antigen(s) recognised by these antisera were not the same as the previously described B-cell markers; immunoglobulin, Fc receptor, complement receptor and Ia antigens. The antigen(s) could not be removed from cells by lysostrip with anti- β₂ microglobulin.     

Identification of Regions of the Chromosome of Neisseria meningitidis and Neisseria gonorrhoeae Which Are Specific to the Pathogenic Neisseria Species

Neisseria meningitidis and Neisseria gonorrhoeae give rise to dramatically different diseases. Their interactions with the host, however, do share common characteristics: they are both human pathogens which do not survive in the environment and which colonize and invade mucosa at their port of entry. It is therefore likely that they have common properties that might not be found in nonpathogenic bacteria belonging to the same genetically related group, such as Neisseria lactamica. Their common properties may be determined by chromosomal regions found only in the pathogenic Neisseria species. To address this issue, we used a previously described technique (C. R. Tinsley and X. Nassif, Proc. Natl. Acad. Sci. USA 93:11109-11114, 1996) to identify sequences of DNA specific for pathogenic neisseriae and not found in N. lactamica. Sequences present in N. lactamica were physically subtracted from the N. meningitidis Z2491 sequence and also from the N. gonorrhoeae FA1090 sequence. The clones obtained from each subtraction were tested by Southern blotting for their reactivity with the three species, and only those which reacted with both N. meningitidis and N. gonorrhoeae (i.e., not specific to either one of the pathogens) were further investigated. In a first step, these clones were mapped onto the chromosomes of both N. meningitidis and N. gonorrhoeae. The majority of the clones were arranged in clusters extending up to 10 kb, suggesting the presence of chromosomal regions common to N. meningitidis and N. gonorrhoeae which distinguish these pathogens from the commensal N. lactamica. The sequences surrounding these clones were determined from the N. meningitidis genome-sequencing project. Several clones corresponded to previously described factors required for colonization and survival at the port of entry, such as immunoglobulin A protease and PilC. Others were homologous to virulence-associated proteins in other bacteria, demonstrating that the subtractive clones are capable of pinpointing chromosomal regions shared by N. meningitidis and N. gonorrhoeae which are involved in common aspects of the host interaction of both pathogens.