Induction of fusion in aggregated and nonaggregated liposomes bearing cationic detergents

The addition of polyanionic polymers such as poly(aspartic acid) (PASP), DNA or dextran sulfate to liposomes composed of phosphatidylcholine (PC) and cholesterol (CHOL) and bearing the quaternary ammonium detergent [[[(1,1,3,3-tetramethylbutyl)cresoxy]ethoxy]ethyl]dimethy lbe nzylammonium hydroxide (DEBDA[OH]) resulted in liposome aggregation and fusion. Liposome-liposome fusion was studied by using fluorescently labeled liposomes and fluorescence-dequenching (DQ) methods. Addition of monoanions, such as aspartate or acetate, to liposomes bearing DEBDA[OH] caused neither their aggregation nor liposome-liposome fusion. Aggregation of liposomes bearing DEBDA[OH] by the binding pair avidin-biotin did not result in their fusion. Fusion in such aggregated liposomes was observed by the addition of chaotropic anions, such as nitrate or thiocyanate, or by PASP. A variety of other quaternary ammonium detergents behaved similarly to DEBDA[OH] in their ability to confer fusogenic properties upon PC/chol liposomes. The relevance of these findings to the mechanism of liposome-liposome fusion is discussed.     

Reactivity of human lymphocytes to yeast-like and mold fungi

The capacity of the allergens of mold fungi (Rhizopus nigricans, Mucor pusillus, Alternaria tenuis, Cladosporum herbarum, Fusarium oxysporum) and yeast-like fungi (Candida albicans) to induce the production of lymphokins by human lymphocytes was studied. All these preparations were active in reactions with lymphocytes obtained from adult donors, but did not activate lymphocytes of newborns (obtained from umbilical blood). In equal doses (10 micrograms/ml) C. albicans allergen was more active than the preparations of mold fungi. The capacity of bacterial allergens to stimulate human lymphocytes was found to be either more pronounced (in Staphylococcus aureus, Streptococcus pyogenes) than that of the preparation of C. albicans or equal to it (in Streptococcus faecalis). The results thus obtained may be regarded as the manifestation of immunological contacts with the antigens of different microorganisms, as well as the evidence of the immunological nature of lymphocytic reactions to preparations intended for use in clinical allergology.     

Enhanced Ig production by human peripheral lymphocytes induced by aggregated C1q

Because B cells express receptors for C1q, we have investigated the role of C1q in the stimulation of B cells. When B cells were cultured in the presence of C1q that had been frozen, T cells, and suboptimal concentrations of PWM, there was a dose-dependent enhancement of IgM, IgG, and IgA by the B cells. No significant enhancement of Ig production by B cells was seen in the absence of T cells or PWM. The contribution of T cells or PWM could be replaced by supernatants of PMA and Con A-activated PBMC (T cell growth factor). C1q that had been frozen, in contrast with freshly isolated C1q, was at least 3 times more active in enhancement of the production of Ig by B cells in culture in the presence of suboptimal concentrations of T cell growth factor. The capability of C1q to stimulate B cells could be ascribed to aggregates of C1q. Monomeric C1q was only marginally active to stimulate B cell Ig production, whereas dimeric and tetrameric C1q were able to enhance Ig production by B cells in relation to their size. Furthermore, aggregation of C1q on soluble aggregates of rabbit IgM also increased its potential to enhance B cell Ig production. The interaction of C1q with the B cells occurs via the collagenous tail of C1q, as suggested by inhibition experiments with purified collagenous tails and globular heads of C1q. These results indicate that triggering of C1qR on B cells positively regulates Ig production in vitro.     

Reactivity of lymphocytes to a progesterone receptor-specific monoclonal antibody

In this study we present evidence for reactivity of pregnancy lymphocytes, but not nonpregnancy lymphocytes, with the progesterone receptor-specific monoclonal antibody mPRI. Using an avidin-biotin peroxidase detection system, we found a nuclear staining in 14.6 +/- 3.7% (mean +/- SEM, N = 27) of pregnancy lymphocytes, while only 0.47 +/- 0.33% (mean +/- SEM, N = 15) of nonpregnancy lymphocytes reacted with the antibody. To characterize the receptor-bearing subset, CD8+ and CD4+ cells were depleted by complement-dependent lysis. Depletion of CD8+ cells was accompanied by 62 +/- 18% loss of progesterone receptor-bearing cells, while depletion of CD4+ cells resulted in a twofold increase in the number of positively staining lymphocytes. In nonpregnancy lymphocytes a 3-day PHA treatment, as well as allogeneic stimulation, resulted in a significant increase in the number of receptor-containing cells. These results suggest that pregnancy, but not nonpregnancy, lymphocytes contain progesterone binding structures, and that these are inducible by mitogenic or alloantigenic stimuli.     

Reaction of human lymphocytes with aggregated IgG columns. Effect on antibody-dependent cytotoxicity and EAC rosette formation.

The origin and life span of long-lived small lymphocytes in the bone marrow of mice have been evaluated by the use of radioautography, scintillation counting, and anti-theta serum. Thymus-deprived BALB/C mice and nude mice had a smaller percentage of long-lived lymphocytes in bone marrow and in thoracic duct lymph than sham-operated or normal littermates. Furthermore, the long-lived lymphocytes in the marrow of nudes have more varied—but generally shorter—life spans than long-lived lymphocytes from mice having a thymus. In thoracic duct lymph of nude mice a more homogeneous long-lived population—according to life span—was found.It was concluded that both long-lived T cells and long-lived B cells are normal residents in the bone marrow. Furthermore, it was concluded that cells of variable life spans comprise the B lymphocyte population: short-lived cells with life spans of 3–5 days and long-lived lymphocytes with life spans of weeks to months.     

Reactivity of B leukemic lymphocytes of patients with chronic lymphocytic leukemia to PWM and PHA

The ability of leukemic B lymphocytes to proliferate after in vitro stimulation with PWM and PHA was studied in 15 patients with chronic lymphocytic leukemia. Peripheral blood lymphocytes of five healthy subjects as well as purified normal B lymphocytes were used as controls. Leukemic lymphocytes of all donors expressed the same membrane phenotype, M receptor, and B7 and Ia antigens. The lymphocyte populations investigated were not completely free from myelomonocytic cells and contained small numbers of T lymphocytes. DNA synthesis was determined on days 3, 5, and 7 of culture by measuring the incorporation of tritiated thymidine. PWM-induced proliferation of leukemic B lymphocytes of nine patients was within normal limits, while the response of leukemic cells of six patients was very low. On the other hand, all CLL donors responded very well to PHA. Moreover, the response of leukemic B lymphocytes was significantly higher than the response of normal B cells. It was concluded that leukemic B lymphocytes of CLL patients are capable of proliferation after stimulation with PWM and PHA. The mechanisms underlying these responses to PWM and PHA are likely to be different.     

Temperature-sensitive binding of solid phase C1q to aggregated human immunoglobulin G

The first component of complement (C1q) coupled to Sepharose by cyanogen bromide was found not to bind aggregated human gamma-globulin or immune complexes at room temperature, whereas at 4 degrees C binding was nearly complete. The temperature sensitivity of solid phase C1q binding was reversible. Elution of aggregated human gamma-globulin bound at 42 degrees C was possible by raising the temperature to 23 degrees C. However, free C1q or C1q adsorbed onto polystyrene balls could bind immune complex-like material at both 23 and 4 degrees C. The conformational restraints of C1q covalently coupled to a solid support may not allow functional activity at elevated temperatures.     

The permeability of human lymphocytes to chloride

The normal amount of Cl in human lymphocytes is 82 mmoles/kg wet weight. Half of this undergoes rapid self-exchange with a half-time of 3 minutes, while the remainder exchanges slowly with a half-time of 180 minutes. The fast fraction of self-exchange of Cl also undergoes a rapid net loss into medium with a low concentration of Cl. Thus, exchange of Cl in lymphocytes has properties like that of K and Na with permeability constants on the order of 10(-6) cm/sec. The results are compatible with a simple model in which the fast fractions are dissolved within ordered cellular water at concentrations less than in the external medium and the slow fractions are adsorbed onto intracellular macromolecules.     

Chromosomal response of human lymphocytes to streptozotocin

The induction of chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) by the methylating agent streptozotocin (STZ) and the effect of this compound on mitotic index (MI) and cell cycle progression in human lymphocytes were investigated. Unstimulated (G(0)) or cycling lymphocytes derived from whole blood or purified lymphocyte cultures were pulse-treated with increasing doses of STZ for 0.5-24h. Induction of CAs by STZ was only observed in cycling lymphocytes derived from whole blood cultures (WBC) (P<0.05). On the contrary, STZ produced a significant and dose-response increase in the yield of SCEs in unstimulated as well as cycling lymphocytes (P<0.05). In addition, STZ induced a dose-dependent decrease in the MI but had a slight effect on cell cycle progression. These results suggest that SCEs are the most sensitive endpoint for evaluating the chromosomal effects of STZ on these cells.     

Binding of concanavalin-A to critical-point-dried and freeze-dried human lymphocytes

When human lymphocytes are treated with concanavalin-A (con A) and hemocyanin, the hemocyanin marker, which demonstrates con A binding sites, can be visualized by scanning (SEM) and transmission electron microscopy (TEM) on both critical-point-dried and freeze-dried cells. The ability to visualize the hemocyanin marker by SEM, its quantity and distribution, were all similar in lymphocytes prepared by both drying procedures. By TEM, hemocyanin was seen adjacent to the plasma membrane on critical-point-dried lymphocytes. In contrast, freeze-dried cells showed hemocyanin labeling at some distance from the plasma membrane (40-70 nm) as well as adjacent to it. The distribution of hemocyanin corresponded to the thickness of the amorphous coat seen on fixed, freeze-dried cells. Therefore, the extracellular coat on freeze-dried lymphocytes is a carbohydrate-containing glycocalyx.