Modulation of human natural killer cell function by L-leucine methyl ester: monocyte-dependent depletion from human peripheral blood mononuclear cells

L-leucine methyl ester (Leu-OMe) causes lysosomal disruption and death of human monocytes (M phi). In addition, Leu-OMe removed natural killer cell (NK) activity from human peripheral mononuclear cells (PBM). Thus, a brief preincubation of PBM with Leu-OMe (greater than 1 mM) caused irreversible loss of NK function as assessed by the lysis of K562 targets. By contrast, a variety of other amino acid methyl esters, including L-glutamic dimethyl ester, L-valine methyl ester, and L-isoleucine methyl ester caused reversible inhibition of NK activity in a manner that was similar to other lysosomotropic agents such as chloroquine and ammonium chloride, but did not cause irreversible loss of all NK function. Leu-OMe appeared to cause actual removal of NK effector cells from PBM, because K562 target binding cells, Leu-11b+ lymphocytes, and OKM1+ lymphocytes were depleted. If M phi were removed from PBM before the incubation, Leu-OMe caused only reversible inhibition of NK function in a manner similar to that observed with other amino acid methyl esters. Upon the addition of freshly isolated M phi, polymorphonuclear leukocytes, or sonicates of these cells to M phi-depleted lymphocyte populations, irreversible ablation of NK function was again observed as a result of Leu-OMe exposure. After in vitro culture, M phi lost their susceptibility to Leu-OMe toxicity and the ability to mediate the irreversible deletion of NK cells resulting from Leu-OMe incubation. These results indicate that in the absence of M phi, Leu-OMe and a variety of other amino acid methyl esters are reversible inhibitors of NK function. However, Leu-OMe is unique in that it can interact with M phi or granulocytes to effect an irreversible loss of NK activity from human peripheral blood lymphocytes.     

The roles of T cell factors in activation, cell cycle progression, and differentiation of human B cells

The relationship of the T cell influences involved in human B cell activation and differentiation into immunoglobulin-secreting cells (ISC) was investigated. T cell supernatants (T supt) generated by stimulating T cells with phytohemagglutinin and phorbol myristate acetate contained activities capable of augmenting DNA synthesis and the growth of mitogen-stimulated B cells and supporting the differentiation of ISC. To examine the role of T supt in B cell activation and the progression through the cell cycle, T cell- and monocyte-depleted B cells were stimulated with formalinized Cowan I strain Staphylococcus aureus (SA), and the percentages of cells in G1, S, and G2 + M were determined by acridine orange staining and analysis. In all experiments, a similar percentage of cells entered G1 during the first 24 to 36 hr of culture when stimulated with SA or SA + T supt. Similar results were seen when B cell activation was analyzed by acquisition of a number of other markers of cell activation. Analysis of cell cycle progression with mithramycin staining of cellular DNA in the presence or absence of vinblastine to arrest mitosis indicated that SA-activated B cells were able to complete S and divide in the absence of T supt. Although an effect of T supt on the progression of B cells through the S phase was evident during the first cell cycle, the major effect only became apparent after the first round of cell division. Although T supt was not necessary for initial B cell activation, T cell influences were absolutely necessary for the differentiation of ISC. T supt did not need to be present during the initial 24 to 36 hr of incubation to permit subsequent generation of ISC. However, when T supt was present initially, an increased number of ISC were produced. Hydroxyurea elimination of cells traversing the G1-S interphase indicated that reception of the differentiation signal occurred before the S phase, but that the generation of ISC required subsequent DNA synthesis and/or cell division. Although precursors of ISC were entirely contained within the population triggered to divide by SA alone, there was no preferential expansion of such precursors as a result of SA stimulation. These results indicate that T cell signals are not absolutely necessary for initial B cell activation and progression through the first cell cycle, although T cell factors promote DNA synthesis by some activated B cells. In contrast, differentiation into ISC is completely dependent on T cell influences.(ABSTRACT TRUNCATED AT 400 WORDS)     

Delineation of the mechanism of inhibition of human T cell activation by PGE2

The capacity of PGE2 to inhibit human T cell responses was examined by investigating its effect on mitogen-induced IL-2 production and proliferation of highly purified CD4+ T cells. PGE2 inhibited both PHA and anti-CD3 induced proliferation and IL-2 production by an action directly on the responding T cell. Inhibition of IL-2 production reflected decreased accumulation of mRNA for IL-2. A variety of other cAMP elevating agents exerted similar inhibitory effects. Inhibition of proliferation could be overcome by supplemental IL-2, PMA, or the anti-CD28 mAb 9.3. Although PMA and 9.3 markedly increased the amount of IL-2 produced by mitogen-stimulated T cells, the percentage inhibition of IL-2 secretion caused by PGE2 and other cAMP elevating agents remained comparable in these costimulated cultures. Rescue of T cell DNA synthesis by these agents appeared to reflect the finding that, although PGE2 markedly inhibited IL-2 production, the absolute amount of IL-2 produced was increased sufficiently to sustain mitogen-induced proliferation. As anticipated, PGE2, forskolin, and cholera toxin increased T cell cAMP levels. The quantity of cellular cAMP generated in response to PGE2, cholera toxin, and forskolin could be inhibited by PMA or 2',5'-dideoxyadenosine. Using these reagents, the inhibitory effects of PGE2 were found to reflect intracellular cAMP levels, but only within a very narrow range. The results indicate that by elevating cAMP levels, PGE2 inhibits human T cell IL-2 production at a point that is common to both the CD3 and CD28 signaling pathways.     

Evolution of body condition‐dependent dispersal in metapopulations

Body condition‐dependent dispersal strategies are common in nature. Although it is obvious that environmental constraints may induce a positive relationship between body condition and dispersal, it is not clear whether positive body conditional dispersal strategies may evolve as a strategy in metapopulations. We have developed an individual‐based simulation model to investigate how body condition–dispersal reaction norms evolve in metapopulations that are characterized by different levels of environmental stochasticity and dispersal mortality. In the model, body condition is related to fecundity and determined either by environmental conditions during juvenile development (adult dispersal) or by those experienced by the mother (natal dispersal). Evolutionarily stable reaction norms strongly depend on metapopulation conditions: positive body condition dependency of dispersal evolved in metapopulation conditions with low levels of dispersal mortality and high levels of environmental stochasticity. Negative body condition‐dependent dispersal evolved in metapopulations with high dispersal mortality and low environmental stochasticity. The latter strategy is responsible for higher dispersal rates under kin competition when dispersal decisions are based on body condition reached at the adult life stage. The evolution of both positive and negative body condition‐dependent dispersal strategies is consequently likely in metapopulations and depends on the prevalent environmental conditions.     

Infrared Thermography and Ultrasonography to Indirectly Monitor the Influence of Liner Type and Overmilking on Teat Tissue Recovery

Eight Danish Holstein cows were milked with a 1-mm thick specially designed soft liner on their right rear teat and a standard liner mounted under extra high tension on their left rear teat. Four of the animals were overmilked for 5 min. Rear teats were subjected to ultrasound examination on the first day and to infrared thermography on the second day. Teats were submersed in ethanol 20 min post-milking on the second day. Ultrasonography measurements showed that teat canal length increased by 30–41% during milking. Twenty minutes after milking, teats milked with modified standard liners still had elongated teat canals while teats milked with the soft liner were normalized. Overmilking tended to increase teat wall thickness. Approximately 80% of variability in teat canal length, from before teat preparation to after milking, could be explained by changes during teat preparation. Thermography indicated a general drop in teat temperature during teat preparation. Teat temperature increased during milking and continued to increase until the ethanol challenge induced a significant drop. Temperatures approached pre-challenge rather than pre-milking temperatures within 10 minutes after challenge. Teat temperatures were dependent on type of liner. Mid-teat temperatures post-challenge relative to pre-teat preparation were dependent on overmilking. Thermography and ultrasound were considered useful methods to indirectly and non invasively evaluate teat tissue integrity.     

重组腺相关病毒2型转导人外周血单核细胞来源树突状细胞的研究

To find out the infection efficiency of recombinant adeno-as sociated virus 2-mediated exogenou s genes in human peripheral blood monocyte-derived dendritic cells(D Cs),the process of transfection wa s investigated by FITC-labeled rAA V2,and observed under confocal mic roscope.Newly separated dendritic cells were tranfected by rAAV2-luc and rAAV2-GFP at different MOI,and transfection efficiency were detec ted by luminometer for rAAV2-luc a nd flow cytometry for rAAV2-GFP.Th e results were elucidated in four different assay systems:(1)60min w as needed for rAAV2 to bind on den dritic cells,and got into cells in the following 10min;(2)the express ion of luc could be detected at th e MOI as low as 1×10~5v.g/cell,and the expression plateau was reached by the MOI of 10~6～10~7v.g/cell,furt her increase of MOI had no functio n on expression level;(3)transgene expression was detected after 48h, and maintained a higher expression level from 96h to 240h after infec tion;(4)7days postinfection of rAA V2-GFP,5%～18% dendritic cells were GFP positive. These data suggest th at rAAV2 vector can efficiently in fect monocyte-derived dendritic ce lls and mediate exogenous gene exp ression,and that the application o f rAAV2 as vector may be useful fo r gene transfer to dendritic cells in ex vivo immunotherapy.     

Patterns of costimulation of T cell clones by cross-linking CD3, CD4/CD8, and class I MHC molecules

The ability of mAb to class I MHC molecules, CD3, or CD4/CD8 to stimulate human T cell clones alone or in combination was examined. Cross-linking each of these surface Ag with appropriate mAb and goat anti-mouse Ig (GaMIg) resulted in a unique pattern of increase in intracellular free calcium ([Ca2+]i) and different degrees of functional activation. Cross-linking class I MHC molecules provided the most effective stimulus of IL-2 production and proliferation. Cross-linking more than one surface Ag induced a compound calcium signal with characteristics of each individual response. Cross-linking CD3 + HLA-A,B,C caused a rapid and prolonged increase in [Ca2+]i and synergistically increased IL-2 production and proliferation of all clones. Cross-linking CD3 + CD4/CD8 also generated a compound calcium signal and increased IL-2 production and DNA synthesis. Purposeful inclusion of CD3 was not required for costimulation as cross-linking HLA-A,B,C + CD4/CD8 also increased [Ca2+]i, IL-2 production, and proliferation. Cross-linking three surface Ag, CD3 + HLA-A,B,C + CD4/CD8, resulted in the greatest initial and sustained [Ca2+]i, IL-2 production, and DNA synthesis. Although there was a tendency for the various stimuli to increase both [Ca2+]i and functional responsiveness, neither the magnitude nor duration of the increased [Ca2+]i correlated with the amount of IL-2 produced or the ultimate proliferative response. To determine whether costimulation required that the various surface molecules were cross-linked together, experiments were carried out using isotype specific secondary antibodies. Augmentation of [Ca2+]i and costimulation of functional responses were noted when class I MHC molecules were cross-linked and CD3 was bound, but not cross-linked. Similarly, costimulation through CD3 and CD4/CD8 was observed when CD4/CD8 was cross-linked and the CD3 complex was engaged by an anti-CD3 mAb which was not further cross-linked. In contrast, costimulation by class I MHC molecules and CD4/CD8 was only observed when these molecules were cross-linked together. These data demonstrate that cross-linking class I MHC determinants or CD4/CD8 provides a direct signal to T cell clones that can be enhanced when CD3 is independently engaged. The results also indicate that T cell clones can be stimulated without engaging CD3 by the combination of signals delivered via class I MHC molecules and CD4/CD8, but only when these determinants were cross-linked together. These studies have demonstrated that these cell surface molecules differ in their capacity to deliver activation signals to T cell clones and also exhibit unique patterns of positive cooperativity in signaling potential.