Dielectric properties of human leukocyte subpopulations determined by electrorotation as a cell separation criterion.
(57/9553)
The separation and purification of human blood cell subpopulations is an essential step in many biomedical applications. New dielectrophoretic fractionation methods have great potential for cell discrimination and manipulation, both for microscale diagnostic applications and for much larger scale clinical problems. To discover whether human leukocyte subpopulations might be separable by such methods, the dielectric characteristics of the four main leukocyte subpopulations, namely, B- and T-lymphocytes, monocytes, and granulocytes, were measured by electrorotation over the frequency range 1 kHz to 120 MHz. The subpopulations were derived from human peripheral blood by magnetically activated cell sorting (MACS) and sheep erythrocyte rosetting methods, and the quality of cell fractions was checked by flow cytometry. Mean specific membrane capacitance values were calculated from the electrorotation data as 10.5 (+/- 3.1), 12.6 (+/- 3.5), 15.3 (+/- 4.3), and 11.0 (+/- 3.2) mF/m2 for T- and B-lymphocytes, monocytes, and granulocytes, respectively, according to a single-shell dielectric model. In agreement with earlier findings, these values correlated with the richness of the surface morphologies of the different cell types, as revealed by scanning electron microscopy (SEM). The data reveal that dielectrophoretic cell sorters should have the ability to discriminate between, and to separate, leukocyte subpopulations under appropriate conditions. (+info)
Calcium currents during contraction and shortening in enzymatically isolated murine skeletal muscle fibres.
(58/9553)
1. Calcium currents (ICa) were monitored in enzymatically isolated murine toe muscle fibres using the two-microelectrode voltage-clamp technique. ICa was recorded (i) in hypertonic solution to suppress contraction, and (ii) in actively shortening fibres in isotonic solution. 2. In hypertonic solution the threshold potential for ICa was about -30 mV for both 2 and 10 mM external Ca2+ solution. Maximum peak currents measured -12.6 +/- 2.3 nA (mean +/- s.d.; n = 4) in 2 mM Ca2+ and -65 +/- 15 nA (n = 7) in 10 mM Ca2+. The time to peak (TTP) ICa was 96 +/- 22 ms (n = 4) in 2 mM Ca2+ and 132 +/- 13 ms (n = 7) in 10 mM Ca2+. The exponential decay of ICa was similar in 2 and 10 mM Ca2+ with rate constants (tau-1(V)) of 3.7 s-1 (2 mM) and 3.8 s-1 (10 mM) at +10 mV. 3. ICa in isotonic 10 mM Ca2+ solution was recorded by inserting the micropipettes exactly opposite to each other close to the centre of mass of the fibre where negligible contraction-induced movement occurs. 4. In isotonic 10 mM Ca2+ solution ICa had a smaller peak amplitude (-45 +/- 5 nA; n = 7) and faster TTP (82.8 +/- 22.1 ms; n = 7) than in hypertonic solution. The exponential decay of ICa showed a significantly larger tau-1(V) of 6.4 s-1 at +10 mV (P < 0.03). 5. To test for calcium depletion, extracellular Ca2+ was buffered by malic acid in isotonic solution to 9 mM. The decay of ICa had a time constant of 348 +/- 175 ms (n = 14) vs. 107 +/- 24 ms (n = 12; P < 0.001) at 0 mV in unbuffered 10 mM Ca2+ solution. 6. We conclude that calcium depletion from the transverse tubular system contributes significantly to the decay of calcium currents in murine toe muscle fibres under hypertonic as well as isotonic conditions. In the latter, depletion is even more prominent. (+info)
Human polymorphonuclear leukocytes produce IL-12, TNF-alpha, and the chemokines macrophage-inflammatory protein-1 alpha and -1 beta in response to Toxoplasma gondii antigens.
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The induction of a type 1 inflammatory cytokine response is a key event in the initiation of immunity to Toxoplasma gondii. Because polymorphonuclear leukocytes rapidly respond to infection by exiting the peripheral blood and accumulating at a site of infection, we sought to determine whether these cells produce cytokines in response to T. gondii. When human peripheral blood neutrophils were stimulated with parasite Ag, they produced both IL-12 (p70) and TNF-alpha. Similarly, up-regulated expression of macrophage-inflammatory protein-1 alpha (MIP-1 alpha) and MIP-1 beta gene transcripts was induced. Kinetic analysis of IL-12 and TNF-alpha production revealed distinct patterns following stimulation by T. gondii or LPS. Exogenous TNF-alpha alone also provided a potent stimulus of MIP-1 alpha and MIP-1 beta expression, and when neutralizing anti-TNF-alpha antiserum was included in cultures of parasite-stimulated cells, expression of these CC-family chemokines was partially blocked. These results establish that T. gondii possesses the ability of driving neutrophil proinflammatory cytokine production, and they suggest that parasite-induced MIP-1 alpha and MIP-1 beta partly results from autocrine stimulation through TNF-alpha. (+info)
Purine metabolites suppress proliferation of human NK cells through a lineage-specific purine receptor.
(60/9553)
NK cell proliferation is suppressed in some patients with cancer by unknown mechanisms. Because purine metabolites released into the extracellular space during cell lysis may affect cell function, we hypothesized that these metabolites could serve as feedback regulators of NK cell proliferation. Sorted NK (CD56+/CD3-) cells were incubated with IL-2 (1000 U/ml) in a 4-day thymidine uptake assay with or without 10-10,000 microM of nucleotides. Adenine nucleotides inhibited NK cell proliferation, with ATP = ADP > 5'-adenylylimidodiphosphate > AMP = adenosine; ADP-ribose and nicotinamide adenine dinucleotide, but not nicotinamide or UTP, caused a dose-dependent suppression of thymidine uptake. A total of 100 microM ATP, a concentration that induced a maximal (80%) inhibition of thymidine uptake, did not inhibit cytotoxic activity against K562 targets. Because NK cells retained the ability to lyse K562 targets 4 days after exposure to 500 microM ATP or 1000 microM adenosine, inhibition of thymidine uptake was not due to cell death. Incubation of NK cells with dibutyryl cAMP and forskolin also suppressed thymidine uptake. Cholera toxin and pertussis toxin suppressed NK cell proliferation. Pertussis toxin did not block the adenine nucleotide effects. Further, ATP, but not adenosine or other nucleotides, markedly increased intracellular cAMP in a dose-dependent manner. The ATP-induced increase in cAMP was specific to cytolytic cells, because CD19+ B cells and CD4+ T cells did not increase their intracellular cAMP. These studies demonstrate that NK proliferation is regulated through purine receptors by adenine nucleotides, which may play a role in decreased NK cell activity. The response to adenine nucleotides is lineage-specific. (+info)
Alcohol (ethanol) inhibits IL-8 and TNF: role of the p38 pathway.
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Acute ethanol (EtOH) intoxication has been identified as a risk factor for infectious complications in trauma and burn victims. However, the mechanism of this immune dysfunction has yet to be elucidated. The monocyte/macrophage production of cytokines, in particular IL-8 and TNF-alpha, is critical in the regulation of the acute inflammatory response to infectious challenge. IL-8 is a potent chemoattractant and activator of neutrophils. TNF-alpha, a proinflammatory cytokine, initiates expression of endothelial cell surface adhesion molecules and neutrophil migration. p38, a member of the mitogen-activated protein kinases, plays an important role in mediating intracellular signal transduction in endotoxin-induced inflammatory responses. We examined the effects of LPS and ethanol on p38 activation and the corresponding IL-8 and TNF-alpha production in human mononuclear cells. LPS-induced IL-8 and TNF-alpha production was inhibited in a similar pattern by pretreatment with either EtOH or SB202190 (1 microM), a specific inhibitor of p38 kinase. Western blot analysis, using a dual phospho-specific p38 mitogen-activated protein kinase Ab, demonstrated that EtOH pretreatment inhibited LPS-induced p38 activation. These results demonstrate that alcohol suppresses the normal host immune inflammatory response to LPS. This dysregulation appears to be mediated in part via inhibition of p38 activation. Inhibition of IL-8 and TNF-alpha production by acute EtOH intoxication may inhibit inflammatory focused neutrophil migration and activation and may be a mechanism explaining the increased risk of trauma- and burn-related infections. (+info)
Biased TCR repertoire in HIV-1-infected patients due to clonal expansion of HIV-1-reverse transcriptase-specific CTL clones.
(62/9553)
To study whether an expansion of HIV-1-specific CTL is contributing to the skewed TCR repertoire in HIV-1-infection, we characterized the TCR usage of CTL clones specific for a conserved epitope in HIV-1 reverse transcriptase (RT/476-484). CTL clones from three HIV-1-infected patients displayed highly similar TCR usage and used the identical Vbeta6.1 and Valpha2.5 gene segments. CTL clones from two patients showed a very high degree of similarity within the TCR complementarity-determining region-3 (CDR-3). In accordance with the similar molecular structure, all three CTL clones also exhibited a similar functional activity with regard to recognition of variant peptides and cytokine secretion pattern. In one subject clonal expansion of a single CTL specificity could be shown over a 10-mo period. TCR spectratyping of PBMC from two patients revealed a marked expansion of CDR-3 segments of a certain length within the Vbeta6-family. Sequence analysis of these CDR-3 yielded sequences identical to the RT/476-484-specific CTL previously isolated from the same patients. This analysis demonstrates that clonal expansion of HIV-1-specific CTL is contributing to the skewed TCR repertoire in HIV-1-infected patients. (+info)
Loss of fenamate-activated K+ current from epithelial cells during corneal wound healing.
(63/9553)
PURPOSE: The corneal epithelium provides a barrier between the external environment and the cornea. It also serves as an ion transporting epithelium. Because of its proximity with the external environment, the corneal epithelium is frequently injured through physical or chemical insult. The purpose of this study was to determine whether corneal epithelial cell whole-cell currents change during corneal wound healing as the author of the present study has previously reported for corneal keratocytes and endothelial cells. METHODS: Rabbit corneal epithelial cells were injured by scraping, heptanol exposure, or freezing. The epithelium was allowed to heal for 12 to 74 hours. Cells were dissociated from corneas, and whole-cell currents were examined using the amphotericin-perforated-patch technique. RESULTS: Cells from the wounded corneal groups had significantly increased capacitance values, indicating increased surface area compared with that of control cells. As previously reported, the primary control whole-cell current was a fenamate-activated K+ current. An inwardly rectifying K+ current and a Cl- current were also observed. In epithelial cells from heptanol-wounded corneas, these conductances were generally unchanged. In cells from scrape- and freeze-wounded corneas, however, the fenamate-activated current was absent or significantly attenuated. CONCLUSIONS: As they do in corneal keratocytes and endothelial cells, K+ channels disappear during some models of corneal epithelial wound healing. In addition, cell capacitance, a measurement of cell surface area, increases. These results suggest that substantial K+ channel activity is not required for in vivo epithelial cell proliferation during corneal wound healing. (+info)
Human fetal retinal pigment epithelial cells induce apoptosis in the T-cell line Jurkat.
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PURPOSE: To investigate the mechanism(s) involved in human fetal retinal pigment epithelium (HFRPE)-mediated T-cell death. METHODS: Pure HFRPE cells were isolated and cultured. Normal and interferon (IFN)-gamma-activated HFRPE from early and late in vitro passages were incubated with cells from the human T-cell leukemia line Jurkat (Jkt). Cultures were pulsed with [3H]-thymidine to measure Jkt cell proliferation. Jkt cells were evaluated for apoptosis either by staining with an ethidium bromide/acridine orange mixture (AO/EB) or with Annexin V-phycoerythrin. The role of Fas ligand (FasL) molecule in HFRPE-mediated apoptosis was assessed by using a mutant Jkt cell line (DD3), which is deficient in Fas-mediated signaling. The involvement of the antiapoptotic human gene bcl-xL was determined by using Jkt cells that were stably transfected with bcl-x(L). The role of cell- cell contact in the induction of apoptosis was evaluated in a transwell system in the presence or absence of neutralizing antibodies against IFN-gamma and tumor necrosis factor (TNF)-alpha. RESULTS: HFRPE cells inhibited the proliferation of Jkt cells by inducing apoptosis through a FasL-independent pathway. Passaging and IFN-gamma activation strengthened the inhibitory effect of HFRPE cells on the proliferation of Jkt cells. At lower HFRPE passages (P2), bcl-alphaL, overexpression rescued the HFRPE cell-mediated apoptosis. The separation of the cells by the transwell system did not affect the HFRPE cell-mediated suppression. This suppressive effect was not mediated by the secretion of IFN-gamma or TNF-alpha molecules. CONCLUSIONS: HFRPE cells suppressed the proliferation of Jkt cells by inducing apoptosis. HFRPE cells induced a stronger inhibitory effect on Jkt cells at higher in vitro passages. The HFRPE-induced apoptosis was not mediated through the FasL/Fas pathway or through the secretion of the apoptosis-inducing cytokines IFN-gamma and TNF-alpha. The bcl-xL gene may play a role in preventing HFRPE cell-induced apoptosis in Jkt cells. These combined results suggest that the HFRPE-mediated suppression of primary T cells may also be mediated by the induction of apoptosis. Therefore, the retinal pigment epithelium may play a role in the induction of immune privilege in the subretinal space. (+info)