Immune Control of Chlamydial Growth in the Human Epithelial Cell Line RT4 Involves Multiple Mechanisms That Include Nitric Oxide Induction,Tryptophan Catabolism and Iron Deprivation

The antimicrobial activity of T cell-derived cytokines, especially interferon (IFN)-γ, against intracellular pathogens, such as Chlamydia trachomatis, involves the induction of 3 major biochemical processes: tryptophan catabolism, nitric oxide (NO) induction and intracellular iron (Fe) deprivation. Since the epithelial cell is the natural target of chlamydial infection, the presence of these antimicrobial systems in the cell would suggest that they may be involved in T cell control of intracellular multiplication of Chlamydia. However, the controversy over whether these 3 antimicrobial processes are present in both mice and humans has precluded the assessment of the relative contribution of each of the 3 mechanisms to chlamydial inhibition in the same epithelial cell from either mice or humans. In the present study, we identified a Chlamydia-susceptible human epithelial cell line, RT4, that possesses the 3 antimicrobial systems, and we examined the role of nitric oxide (NO) induction, and deprivation of tryptophan or Fe in cytokine-induced inhibition of chlamydiae. It was found that the 3 antimicrobial systems contributed to cytokine-mediated inhibition of the intracellular growth of Chlamydia. NO induction accounted for ~20% of the growth inhibition; tryptophan catabolism contributed approximately 30%; iron deprivation was least effective; but the combination of the 3 systems accounted for greater than 60% of the inhibition observed. These results indicate that immune control of chlamydial growth in human epithelial cells may involve multiple mechanisms that include NO induction, tryptophan catabolism and Fe deprivation.     

The US national antimicrobial resistance monitoring system.

The use of antimicrobial agents in food animals can select for resistant bacterial pathogens that may be transmitted to humans via the commercial meat supply. In the USA, the FDA's Center for Veterinary Medicine regulatory duties require a determination that antimicrobial drugs are safe and effective for use in food animals. In addition, a qualitative assessment of risks to human health from antimicrobial resistance requires development. This risk assessment process is supported by data generated by the FDA's National Antimicrobial Resistance Monitoring System (NARMS) for enteric bacteria. NARMS data on antimicrobial susceptibility among Salmonella, Campylobacter, Escherichia coli and Enterococcus is collected. Research activities defining the genetic bases of resistance helps to understand the potential public health risks posed by the spread of antimicrobial resistance from food animal antimicrobial use. These activities help insure that antimicrobials are used judiciously to promote human and animal health.     

Trichinella spiralis: changes caused in the mouse's thymic, splenic, and lymph node cell populations.

Infections with the nematode Trichinella spiralis induce unresponsiveness in mice. A study was made to determine whether suppression could be due to a deficiency in the cells responsible for the immunological response. Mice were given low or moderate infections and were killed 7, 14, 28, or 56 days after inoculation; spleen macrophages and leucocytes, θ cells, and Con A- and LPS-sensitive cells were determined in the thymus, spleen, and the mesenteric and axillary lymph nodes. Spleen macrophages are diminished throughout the course of the infection, reaching significantly low levels on the 14th day. The thymus loses, whereas the spleen and the axillary node gain, cells bearing the θ antigen. In spite of the increase in leucocytes and θ cells in the secondary lymphoid tissue, the cells of these organs are insensitive to the blastogenic action of Con A in the heavier infections. In lower infections, however, spleen cells show an enhanced response to Con A and LPS; mesenteric cells, on the other hand, show an early enhanced susceptibility to LPS and a reduced susceptibility to Con A and, in the later phases of parasitism, an enhanced Con A and a reduced LPS susceptibility. It is suggested that these phenomena contribute to the immunosuppression phenomena which are characteristic of T. spiralis infections.     

Radiation-induced alterations in binding of concanavalin A to cells and in their susceptibility to agglutination

Cell susceptibility to agglutination mediated by a plant lectin, concanavalin A (Con A), and the binding capacity of Con A to cells following gamma-irradiation have been examined in mouse myeloid leukaemia cells cultured in suspension. Irradiation caused an immediate decrease in the amount of Con A bound to the cell surface, whereas susceptibility of irradiated cells to agglutination by Con A was unchanged when compared to that of the unirradiated cells. Post-irradiation incubation of cells at 37 degrees C resulted in a temporary, more than 1.3-fold increase in cell susceptibility to agglutination 60 min after irradiation, whereas binding capacity of cells for Con A gradually recovered following irradiation, reaching a comparable level to that of unirradiated cells 3 h after irradiation. Cell susceptibility to agglutination by Con A does not depend strongly on its binding capacity.     

CD44-deficient mice exhibit enhanced hepatitis after concanavalin A injection: evidence for involvement of CD44 in activation-induced cell death

Administration of Con A induces severe injury to hepatocytes in mice and is considered to be a model for human hepatitis. In the current study, we investigated the role of CD44 in Con A-induced hepatitis. Intravenous administration of Con A (20 mg/kg) caused 100% mortality in C57BL/6 CD44-knockout (KO) mice, although it was not lethal in C57BL/6 CD44 wild-type (WT) mice. Administration of lower doses of Con A (12 mg/kg body weight) into CD44 WT mice induced hepatitis as evident from increased plasma aspartate aminotransferase levels accompanied by active infiltration of mononuclear cells and neutrophils, and significant induction of apoptosis in the liver. Interestingly, CD44 KO mice injected with similar doses of Con A exhibited more severe acute suppurative hepatitis. Transfer of spleen cells from Con A-injected CD44 KO mice into CD44 WT mice induced higher levels of hepatitis when compared with transfer of similar cells from CD44 WT mice into CD44 WT mice. The increased hepatitis seen in CD44 KO mice was accompanied by increased production of cytokines such as TNF-alpha, IL-2 and IFN-gamma, but not Fas or Fas ligand. The increased susceptibility of CD44 KO mice to hepatitis correlated with the observation that T cells from CD44 KO mice were more resistant to activation-induced cell death when compared with the CD44 WT mice. Together, these data demonstrate that activated T cells use CD44 to undergo apoptosis, and dysregulation in this pathway could lead to increased pathogenesis in a number of diseases, including hepatitis.     

Methicillin resistance study in clinical isolates of coagulase-negative staphylococci and determination of their susceptibility to alternative antimicrobial agents

AIMS: To achieve reliable detection of methicillin resistance in clinical isolates of coagulase-negative staphylococci. METHODS AND RESULTS: Strains (105) were evaluated by normatized antimicrobial susceptibility methods, and for the presence of the methicillin resistance-determining mecA gene, using the polymerase chain reaction. Correlation between phenotypic and genotypic methods was obtained in 87.6% of the samples. Six strains, classified as methicillin-susceptible by phenotypic assays, revealed the presence of the mecA gene, indicating that methicillin resistance expression was probably repressed. Another seven isolates failed to show mecA amplification after displaying methicillin resistance in phenotypic evaluations. The susceptibility of the methicillin-resistant isolates to other antimicrobial agents was variable. CONCLUSION: Genotypic determination of the mecA gene proved to be the most reliable method for detection of methicillin resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: Correct assessment of methicillin resistance, such as that attained through genotyping, is essential for defining therapeutic strategies, particularly when treating severely compromised patients.     

A real-time PCR antibiogram for drug-resistant sepsis

Current molecular diagnostic techniques for susceptibility testing of septicemia rely on genotyping for the presence of known resistance cassettes. This technique is intrinsically vulnerable due to the inability to detect newly emergent resistance genes. Traditional phenotypic susceptibility testing has always been a superior method to assay for resistance; however, relying on the multi-day growth period to determine which antimicrobial to administer jeopardizes patient survival. These factors have resulted in the widespread and deleterious use of broad-spectrum antimicrobials. The real-time PCR antibiogram, described herein, combines universal phenotypic susceptibility testing with the rapid diagnostic capabilities of PCR. We have developed a procedure that determines susceptibility by monitoring pathogenic load with the highly conserved 16S rRNA gene in blood samples exposed to different antimicrobial drugs. The optimized protocol removes heme and human background DNA from blood, which allows standard real-time PCR detection systems to be employed with high sensitivity (<100 CFU/mL). Three strains of E. coli, two of which were antimicrobial resistant, were spiked into whole blood and exposed to three different antibiotics. After real-time PCR-based determination of pathogenic load, a ΔC(t)<3.0 between untreated and treated samples was found to indicate antimicrobial resistance (P<0.01). Minimum inhibitory concentration was determined for susceptible bacteria and pan-bacterial detection was demonstrated with 3 gram-negative and 2 gram-positive bacteria. Species identification was performed via analysis of the hypervariable amplicons. In summary, we have developed a universal diagnostic phenotyping technique that assays for the susceptibility of drug-resistant septicemia with the speed of PCR. The real-time PCR antibiogram achieves detection, susceptibility testing, minimum inhibitory concentration determination, and identification in less than 24 hours.     

Perspectives for nano-biotechnology enabled protection and nutrition of plants

Indiscriminate use of pesticides and fertilizers causes environmental pollution, emergence of agricultural pests and pathogens, and loss of biodiversity. Nanotechnology, by virtue of nanomaterial related properties, has potential agro-biotechnological applications for alleviation of these problems. The literature pertaining to the role of nanotechnology in plant and soil systems demonstrates that nanomaterials may assist in a) the controlled release of agrochemicals for nutrition and protection against pests and pathogens, b) delivery of genetic material, c) sensitive detection of plant disease and pollutants and d) protection and formation of soil structure. For instance, porous silica (15 nm) and biodegradable, polymeric chitosan (78 nm) nanoparticles displayed slow release of encapsulated pesticide and fertilizer, respectively. Further, nanosized gold (5–25 nm) delivered DNA to plant cells while iron oxide (30 nm) based nanosensors detected pesticides at minute levels. These functions assist the development of precision farming by minimizing pollution and maximizing the value of farming practice.     

Variations in distribution of con A receptor sites and anionic groups during red blood cell differentiation in the rat

The distribution of receptors for concanavalin A (Con A) and anionic groups on plasma membranes of developing blood cells was investigated in the rat. Glutaraldehyde-fixed bone marrow and circulating blood cells were exposed to ferritin-conjugated Con A or positively chaged ferric oxide (CI) and processed for electro n microscopy. The frequency of Con A and CI binding sites varied during different erythroid developmental stages and amont different leukoid cell types. There was a constant inverse relationship between Con A and CI binding sites. Among leukoid cells, Con A binding was high on plasma cells and macrophages, lower on neutrophils and lymphocytes, and still lower on eosinophils and basophils; CI binding was highest in the latter and lowest in plasma cells and macrophages. Among erythroid cells, there was a progressive increase in Con A and a decrease in CI binding after successive divisions of erythroblasts, and a progressive decrease in Con A and an increase in CI binding upon maturation of the orthochromatic erythroblast to the reticulocyte. The most pronounced modification in distribution of these sites occurred during nuclear expulsion: that protion of the plasma membrane surrounding the extruded nucleus was heavily labeled by Con A (up to four times that of the orthochromatic erythroblast) whereas the reticulocyte had considerably fewer sites. The situation was reversed with CI. The results suggest that the concentration of nonsialated glycoproteins (to which Con A binds) varies inversely to that of sialoproteins (to which CI binds) in the membrane of the differentiating erythroid cell. The remarkable changes observed at the time of nuclear extrusion suggest that there is either local modification of glycosylated groups with removal of sialyl residues from the membrane surrounding the extruded nucleus of selective segregation of membrane glycoproteins leading to a high concentration of sialoproteins (glycophroin) in the membrane of the mature erythrocyte.     

Relationship between the concanavalin A-agglutinability and deformability of human erythrocytes

Cellular deformability has been proposed in the past as a major determinant of lectin-mediated agglutination of cells. In this paper we have evaluated the correlation between deformability and Con A-agglutinability of human erythrocytes by subjecting them to agents that alter either one of the properties and evaluating the effect on the other property. The following results have been obtained: (i) Treatment with pronase or trypsin, which makes the Con A-nonagglutinable normal red cells highly agglutinable, has practically no effect on deformability; while neuraminidase treatment, with a similar effect on agglutinability, produces a small but statistically significant reduction in deformability. (ii) Diamide treatment, on the other hand, produces a drastic reduction in the deformability of pronase-treated erythrocytes but has no effect on the Con A-agglutinability of the cells. Dinitrophenol also reduces deformability but without altering the agglutinability, (iii) Chlorpromazine, at 2 x 10(-5) M, does not have any effect on the deformability of trypsinized cells, but increases the agglutinability substantially. When the Con A-agglutinability of the cells and their deformability after these treatments are compared, a correlation coefficient r = -0.353 (P greater than 0.1) is obtained. This indicates the lack of any direct correlation between the two parameters, and rules out any significant role of deformability in the determination of Con A-agglutinability of erythrocytes. The agglutination with the lectin is completely reversed by methyl alpha-D-mannoside, the specific inhibitory sugar for Con A, also ruling out any secondary role for deformability in the non-lectin-mediated stabilization of clumps. Upon incubation of normal erythrocytes with Con A. a dose-dependent decrease in deformability is observed, with the deformability index falling to almost 25% of the normal value with 500 microgram/ml Con A. This indicates that Con A binding to its receptor produces changes in the membrane probably by altering properties of the membrane skeleton.     

Crucial role of lysosomal iron in the formation of dinitrosyl iron complexes in vivo

Dinitrosyl non-heme–iron complexes (DNIC) are found in many nitric oxide producing tissues. A prerequisite of DNIC formation is the presence of nitric oxide, iron and thiol/imidazole groups. The aim of this study was to investigate the role of the cellular labile iron pool in the formation of DNIC in erythroid K562 cells. The cells were treated with a nitric oxide donor in the presence of a permeable (salicylaldehyde isonicotinoyl hydrazone) or a nonpermeable (desferrioxamine mesylate) iron chelator and DNIC formation was recorded using electron paramagnetic resonance. Both chelators inhibited DNIC formation up to 50% after 6 h of treatment. To further investigate the role of lysosomal iron in DNIC formation, we prevented lysosomal proteolysis by pretreatment of whole cells with NH₄Cl. Pretreatment with NH₄Cl inhibited the formation of DNIC in a time-dependent manner that points to the importance of the degradation of iron metalloproteins in DNIC formation in vivo. Fractionation of the cell content after treatment with the nitric oxide donor revealed that DNIC is formed predominantly in the endosomal/lysosomal fraction. Taken together, these data indicate that lysosomal iron plays a crucial role in DNIC formation in vivo. Degradation of iron-containing metalloproteins seems to be important for this process.     

Reevaluation of colorimetric iron determination methods commonly used in geomicrobiology

The ferrozine and phenanthroline colorimetric assays are commonly applied for the determination of ferrous and total iron concentrations in geomicrobiological studies. However, accuracy of both methods depends on slight changes in their protocols, on the investigated iron species, and on geochemical variations in sample conditions. Therefore, we tested the performance of both methods using Fe(II)((aq)), Fe(III)((aq)), mixed valence solutions, synthetic goethite, ferrihydrite, and pyrite, as well as microbially-formed magnetite and a mixture of goethite and magnetite. The results were compared to concentrations determined with aqua regia dissolution and inductively coupled plasma atomic emission spectroscopy (ICP-AES). Iron dissolution prior to the photometric assays included dissolution in 1M or 6M HCl, at 21 or 60°C, and oxic or anoxic conditions. Results indicated a good reproducibility of quantitative total iron determinations by the ferrozine and phenanthroline assays for easily soluble iron forms such as Fe(II)((aq)), Fe(III)((aq)), mixed valence solutions, and ferrihydrite. The ferrozine test underestimated total iron contents of some of these samples after dissolution in 1M HCl by 10 to 13%, whereas phenanthroline matched the results determined by ICP-AES with a deviation of 5%. Total iron concentrations after dissolution in 1M HCl of highly crystalline oxides such as magnetite, a mixture of goethite and magnetite, and goethite were underestimated by up to 95% with both methods. When dissolving these minerals in 6M HCl at 60°C, the ferrozine method was more reliable for total iron content with an accuracy of ±5%, related to values determined with ICP-AES. Phenanthroline was more reliable for the determination of total pyritic iron as well as ferrous iron after incubation in 1M HCl at 21°C in the Fe(II)((aq)) sample with a recovery of 98%. Low ferrous iron concentrations of less than 0.5mM were overestimated in a Fe(III) background by up to 150% by both methods. Heating of mineral samples in 6M HCl increased their solubility and susceptibility for both photometric assays which is a need for total iron determination of highly crystalline minerals. However, heating also rendered a subsequent reliable determination of ferrous iron impossible due to fast abiotic oxidation. Due to the low solubility of highly crystalline samples, the determination of total iron is solely possible after dissolution in 6M HCl at 60°C which on the other hand makes determination of ferrous iron impossible. The recommended procedure for ferrous iron determination is therefore incubation at 21°C in 6M HCl, centrifugation, and subsequent measurement of ferrous iron in the supernatant. The different procedures were tested during growth of G. sulfurreducens on synthetic ferrihydrite. Here, the phenanthroline test was more accurate compared to the ferrozine test. However, the latter provided easy handling and seemed preferable for larger amounts of samples.     

Facile and rapid magnetic relaxation switch immunosensor for endocrine-disrupting chemicals

In order to develop facile, fast and sensitive detection methods for endocrine-disrupting chemicals (EDCs), we described a sensitive biosensing system involving magnetic relaxation switch, based on the assembly of cross-linked superparamagnetic iron oxide (CLIO) nanoparticles induced by the antigen-antibody biorecognition. The design of smart CLIO-based superparamagnetic iron oxide nanoparticles and antigen-OVA was described for the detection of bisphenol A [2,2-bis (4-hydroxyphenol) propane (BPA)]. The addition of BPA to the rapid magnetic relaxation switch immunosensor led to transverse relaxation time (T2) shortening compared to a blank control as shown by NMR relaxometry measurements. This process was also applied to the rapid and facile determination of concentrations of BPA in drinking water (tap water). Good linearity for all calibration curves was obtained, and the limit of detection (LOD) for BPA was 0.4 ng/mL in tap water.     

The co-ordinated regulation of iron homeostasis in murine macrophages limits the availability of iron for intracellular Salmonella typhimurium

In being both, a modifier of cellular immune effector pathways and an essential nutrient for microbes, iron is a critical determinant in host-pathogen interaction. Here, we investigated the metabolic changes of macrophage iron homeostasis and immune function following the infection of RAW264.7 murine macrophages with Salmonella typhimurium. We observed an enhanced expression of the principal iron export protein, ferroportin 1, and a subsequent increase of iron efflux in Salmonella-infected phagocytes. In parallel, the expression of haem oxygenase 1 and of the siderophore-binding peptide lipocalin 2 was markedly enhanced following pathogen entry. Collectively, these modulations reduced both the cytoplasmatic labile iron and the ferritin storage iron pool within macrophages, thus restricting the acquisition of iron by intramacrophage Salmonella. Correspondingly, limitation of macrophage iron decreased microbial survival, whereas iron supplementation impaired immune response pathways in Salmonella-infected macrophages (nitric oxide formation and tumour necrosis factor-alpha production) and promoted intracellular bacterial proliferation. Our findings suggest that the enhancement of ferroportin 1-mediated iron efflux, the upregulation of the haem-degrading enzyme haem oxygenase 1 and the induction of lipocalin 2 following infection concordantly aim at withholding iron from intracellular S. typhimurium and to increase antimicrobial immune effector pathways thus limiting pathogen proliferation.     

Molecular basis of "suppressor" macrophages. Arginine metabolism via the nitric oxide synthetase pathway.

A molecular explanation for "suppressor" macrophage inhibition of lymphocyte proliferation is described. NG-monomethyl-L-arginine (NGMMA), a specific inhibitor of the nitric oxide synthetase pathway, markedly augments Con A-induced proliferation of rat splenic leukocytes. Macrophages are necessary and sufficient for NGMMA-releasable-suppression, as indicated by a loss of suppression after either pretreatment of isolated splenic macrophages with NGMMA or their depletion by plastic adherence or L-leucine methyl ester. L- (but not D-) arginine overrides NGMMA-releasable suppression, and suppression is blocked by RBC as would be expected if nitric oxide were the effector molecule. Unlike rats, NGMMA did not augment Con A-induced proliferation of normal mouse splenic leukocytes. However, NGMMA did augment Con A-induced proliferation of mouse splenic leukocytes induced to contain suppressor macrophages by intravenous injection of Corynebacterium parvum, which suggests a quantitative, not qualitative, difference in suppressor macrophages between rats and mice. Nitrite production, as an indicator of nitric oxide synthesis, correlated with suppressor macrophage activity in rats and mice and was inhibited by NGMMA. Finally, NGMMA also markedly enhanced proliferation with every other mitogen examined (PHA, protein A, PWM, and LPS). It is concluded that immunoregulation of lymphocyte proliferation by suppressor macrophages is mediated, in part, directly or indirectly by products of the nitric oxide synthetase pathway.     

Ontogeny of T-cell mitogen response in Lewis rats. III. Juvenile adherent suppressor cells block adult mitogen responses

Spleen cells from suckling female Lewis rats (4 to 20 days old) were able to suppress mitogenic responses to concanavalin A (Con A) and phytohemagglutinin (PHA) of spleen or thymus cells from adult female Lewis rats and thymus cells from suckling Lewis rats. Thymus cells from suckling rats were unable to suppress adult spleen cell mitogenic responses to Con A. Removal of carbonyl iron (cFe)-, plastic-, or nylon-wool-adherent cells removed the suppressive action of juvenile spleen cells, but irradiation did not. Separated plastic-adherent spleen cells from suckling animals suppressed adult mitogenic responses to Con A. at optimal Con A doses 2-mercaptoethanol (2-ME, 2 X 10(-5) M) abolished the suppressive effect of juvenile cells, however, at the hyperoptimal dose of Con A (125 micrograms/ml) even higher doses of 2-ME did not relieve suppression by juvenile cells. These suppressor cells in suckling pups were affected by early weaning which decreased suppression, resulting in enhanced mitogenic responses of juvenile cells and removal of the ability to suppress adult mitogenic response.     

Lectin-stimulated cellular iron uptake and toxin generation in the freshwater cyanobacterium Microcystis aeruginosa

The lectin family is composed of mono- and oligosaccharide binding proteins that could activate specific cellular activities, such as cell-cell attachment and toxin production. In the present study, the effect of the external addition of lectins to culture media containing the freshwater cyanobacterium Microcystis aeruginosa on its metabolic activities, such as iron uptake and toxin production was investigated. Among the three lectins examined in this study (concanavalin A [Con A], wheat germ agglutinin [WGA] and peanut agglutinin [PNA]), PNA substantially increased the accumulated intracellular and extracellular iron content. The binding of PNA and Con A to M. aeruginosa cells was visualized via fluorescence microscopy using a lectin adjunct with fluorescein isothiocyanate, and resulted in carbohydrate and protein accumulation in the cellular capsule. Given that the highest carbohydrate accumulation was seen in the Con A system (where iron accumulation was relatively lower), carbohydrate quality is likely important factor that influences cellular iron accumulation. Since PNA specifically binds to sugars such as galactose and N-acetylgalactosamine, these saccharide species could be important candidates for intracellular and extracellular iron accumulation and transport. Microcystin biosynthesis was stimulated in the presence of PNA and WGA, whereas cellular iron uptake increased only in the presence of PNA. Thus, the iron uptake was not necessarily congruent with the upregulation of microcystin synthesis, which suggested that the positive effect of lectin on iron uptake is probably attributable to the PNA-assisted iron accumulation around the cell surface. Overall, the present study provides insights into the interactions of lectin that influence cellular metabolic activities such as iron uptake, extracellular polymeric substance accumulation, and toxin production.     

Requirement for three distinct lymphokines for the induction of cytotoxic T lymphocytes from thymocytes

The induction of cytotoxic T lymphocytes (CTL) from CTL precursors requires a combination of antigen and lymphokine signals. To investigate lymphokine requirements for CTL generation, we used an assay in which helper T cell and accessory cell-depleted spleen cells or whole thymocytes were cultured with lectin (Con A) and lymphokines. This culture was followed by assessment of lectin-dependent cytolysis. High concentrations of recombinant interleukin 2 (R-IL 2) (100 U/ml) alone were not sufficient for lectin-mediated CTL induction from thymocytes, whereas 20 to 100 U/ml of R-IL 2 alone could induce a significant lectin-mediated CTL response from accessory cell-depleted spleen cells. Using thymocytes as responders, we found purified or recombinant interferon-gamma (IFN-gamma) did not cause cytolytic activity either in the absence of or in the presence of R-IL 2. However, supernatant from Con A-stimulated rat spleen cells (rat Con A SN) in combination with R-IL 2 could induce cytolytic activity, suggesting that several factors are required for CTL induction. Con A SN was fractionated by gel filtration and the fractions were tested for ability to induce CTL. In the presence of a low level of R-IL 2 (5 U/ml), fractions with a Mr of approximately 31,000 could induce CTL, and this activity was referred to as CTL differentiation factor (CDF). The peak fractions containing CDF activity did not have detectable IL 1, IL 2, IFN-gamma, or CSF activity. However, by add-back experiments and the use of blocking antibodies, a monoclonal antibody against the IL 2 receptor or antibodies against murine IFN-gamma, we demonstrated that CTL induction from mature thymocytes (L3T4-, Lyt-2+) requires CDF activity in addition to IL 2 and IFN-gamma.     

Flow cytometric measurement of fluorescence resonance energy transfer on cell surfaces. Quantitative evaluation of the transfer efficiency on a cell-by-cell basis

A method has been developed for the determination of the efficiency (E) of the fluorescence resonance energy transfer between moieties on cell surfaces by use of a computer-controlled flow cytometer capable of dual wavelength excitation. The absolute value of E may be calculated on a single-cell basis. The analysis requires the measurement of samples stained with donor and acceptor conjugated ligands alone as well as together. In model experiments HK 22 murine lymphoma cells labeled with fluorescein-conjugated concanavalin A (Con A) and/or rhodamine conjugated Con A were used to determine energy transfer histograms. Using the analytic solution to energy transfer in two dimensions, a high surface density of Con A binding sites was found that suggests that the Con A receptor sites on the cell surface are to a degree preclustered . We call this technique flow cytometric energy transfer ( FCET ).