Labeled Target Cells Research Articles

On-Chip Cell Sorting System Using Laser-Induced Heating of a Thermoreversible Gelation Polymer to Control Flow

We have developed a microfabricated fluorescence-activated cell sorter system using a thermoreversible gelation polymer (TGP) as a switching valve. The glass sorter chip has Y-shaped microchannels with one inlet and two outlets. A biological specimen containing fluorescently labeled cells is mixed with a solution containing a thermoreversible sol-gel polymer. The mixed solution is then introduced into the sorter chip through the inlet. The sol-gel transformation was locally induced by site-directed infrared laser irradiation to plug one of the outlets. The fluorescently labeled target cells were detected with sensitive fluorescence microscopy. In the absence of a fluorescence signal, the collection channel is plugged through laser irradiation of the TGP and the specimens are directed to the waste channel. Upon detection of a fluorescence signal from the target cells, the laser beam is then used to plug the waste channel, allowing the fluorescent cells to be channeled into the collection reservoir. The response time of the sol-gel transformation was 3 ms, and a flow switching time of 120 ms was achieved. Using this system, we have demonstrated the sorting of fluorescent microspheres and Escherichia coli cells expressing fluorescent proteins. These cells were found to be viable after extraction from the sorting system, indicating no damage to the cells.

The Fusion Activity of HIV-1 gp41 Depends on Interhelical Interactions

Infection by human immunodeficiency virus type I requires the fusogenic activity of gp41, the transmembrane subunit of the viral envelope protein. Crystallographic studies have revealed that fusion-active gp41 is a "trimer-of-hairpins" in which three central N-terminal helices form a trimeric coiled coil surrounded by three antiparallel C-terminal helices. This structure is stabilized primarily by hydrophobic, interhelical interactions, and several critical contacts are made between residues that form a deep cavity in the N-terminal trimer and the C-helix residues that pack into this cavity. In addition, the trimer-of-hairpins structure has an extensive network of hydrogen bonds within a conserved glutamine-rich layer of poorly understood function. Formation of the trimer-of-hairpins structure is thought to directly force the viral and target membranes together, resulting in membrane fusion and viral entry. We test this hypothesis by constructing four series of gp41 mutants with disrupted interactions between the N- and C-helices. Notably, in the three series containing mutations within the cavity, gp41 activity correlates well with the stability of the N-C interhelical interaction. In contrast, a fourth series of mutants involving the glutamine layer residue Gln-653 show fusion defects even though the stability of the hairpin is close to wild-type. These results provide evidence that gp41 hairpin stability is critical for mediating fusion and suggest a novel role for the glutamine layer in gp41 function.

Cell-to-cell interactions in changed gravity: Ground-based and flight experiments

Cell-to-cell interactions play an important role in all physiological processes and are mediated by humoral and mechanical factors. Mechanosensitive cells (e.g., osteocytes, chondrocytes, and fibroblasts) can be studied ex vivo to understand the effects of an altered gravity environment. In particular, cultured endothelial cells (EC) are very sensitive to a broad spectrum of mechanical and biochemical stimuli. Earlier, we demonstrated that clinorotation leads to cytoskeletal remodeling in cultured ECs. Long-term gravity vector changes also modulate the expression of surface adhesion molecules (ICAM-1, E-selectin, VCAM-1) on cultured ECs. To study the interactions of geterological cells, we cocultured endothelial monolayers and human lymphocytes, immune cells and myeloleucemic (K-560) cells. It was found that, although clinorotation did not alter the basal adhesion level of non-activated immune cells on endothelial monolayers, the adhesion of PMA-activated lymphocytes was increased. During flight experiments onboard the Russian segment of the International Space Station, we measured the cytotoxic activity of natural killer (NK) cells incubated with labeled target cells. It was found that immune cells in microgravity retained their ability to contact, recognize, and destroy oncogenic cells in vitro. Together, our data concerning the effects of simulated and real microgravity suggest that, despite changes in the cytoskeleton, cell motility, and expression of adhesion molecules, cell–cell interactions are not compromised, thus preserving the critical physiological functions of immune and endothelial cells.

Measuring Melanoma-Specific Cytotoxic T Lymphocytes Elicited by Dendritic Cell Vaccines with a Tumor Inhibition Assay In Vitro

Improving cancer vaccines depends on assays measuring elicited tumor-specific T-cell immunity. Cytotoxic effector cells are essential for tumor clearance and are commonly evaluated using 51Cr release from labeled target cells after a short (4 hours) incubation with T cells. The authors used a tumor inhibition assay (TIA) that assesses the capacity of cytotoxic T lymphocytes (CTLs) to control the survival/growth of EGFP-labeled tumor cell lines. TIA was validated using CD8+ T cells primed in vitro against melanoma and breast cancer cells. TIA was then used to assess the CTL function of cultured CD8+ T cells isolated from patients with metastatic melanoma who underwent vaccination with peptide-pulsed CD34+ HPCs-derived DCs. After the DC vaccination, T cells from six of eight patients yielded CTLs that could inhibit the survival/growth of melanoma cells. The results of TIA correlated with killing of tumor cells in a standard 4-hour 51Cr release assay, yet TIA allowed detection of CTL activities that appeared marginal in the 51Cr release assay. Thus, TIA might prove valuable for measuring spontaneous and induced antigen-specific cytotoxic T cells.

Study on p53 tetramerization domain in improving functional affinity and biological activity of antibody

To fuse the genes of p53 tetramerization domain and anti-CD3/anti-prostate-cancer bispecific single-chain antibody (BsAb), and exploit a new way to improve the functional affinity and biological activity of antibody. Genes of p53 tetramerization domain and anti-CD3/anti-prostate-cancer BsAb was fused by technique of DNA sub-cloning. The fusion gene confirmed by sequencing was subcloned into the pSectag2-B plasmid. Then the recombinant plasmid was transfected into HeLa cells. The expression products, which were analyzed by both SDS-PAGE and western blotting, were purified with Ni(2+)-NTA superflow affinity chromatography. mBsAb-pSectag2-B plasmid was added into the suspensions of human peripheral blood mononuclear cells (PBMCs) and PC-3 cells respectively. Flow cytometry was used to examine the binding rate of multivalent anti-prostate-cander/anti-CD3 bispecific scFv with PBMCs and PC-3 cells. T cells were isolated from the PBMCs. PC-3 cells were labeled with Na(2)[(51)Cr]O(4) used as target cells. Labeled PC-3 cells, T cells, and different concentrations of mBsAb were mixed. Natural release control well with labeled target cells only and maximum release control well with labeled target cells and 10% SDS were prepared. The supernatants were extracted. gamma calculator was used to calculate the counts per minute (cpm) values to calculate the specific release rate of (51)Cr. Sequencing showed a fragment from mBsAb-pSectag2-B with the size of 1.7 kb corresponding to the predicted value. SDS-PAGE and Western blotting showed expression of 67 000 D protein in the supernatant of culture fluid of HeLa cells transfected with MBsAb-pSectag2-B plasmid. The binding rates of multivalent anti-prostate-cancer/anti-CD3 bispecific scFv with PBMC and PC-3 cells were 70.4% and 81% respectively, significantly higher than those of anti-prostate-cancer/anti-CD3 bispecific scFv. In the presence of mBsAb the activated T cells lysed PC-3 cells in positive correlation with the antibody concentration and effective cell/target cell ratio and with a lysis rate significantly higher than those of the control groups. Multivalent anti-CD3 x anti-prostate-cancer BsAb exhibits much higher functional affinity and biological activity than anti-CD3 x anti-prostate-cancer BsAb, which may break a new path to the improvement of functional affinity and biological activity of antibody.

Low dose radiation induced immunomodulation: Effect on macrophages and CD8+ T cells

Purpose: The aim of the present investigation was to study the effect of fractionated whole body low dose ionizing radiation (LDR) on the functional responses of T lymphocytes, their subpopulations and macrophages.Materials and methods: C57BL/6 mice were exposed to 4 cGy from a 60Co source, at 0.31 cGy/min, at 24 h intervals for 5 days (total dose 20 cGy). Phagocytic activity was measured by flow cytometry using Bioparticles® and nitric oxide generation was estimated by spectrophotometry. Proliferation of lymphocytes in response to concanavalin A (con A) and alloantigens was measured by 3H thymidine incorporation. Expression of cell surface markers was assessed by flow cytometric analysis of antibody labeled cells. Target cell killing by cytotoxic T cells (CTL) generated against allogenic cells was assessed by flow cytometry using PKH26 labeled target cells. Cytokines were estimated by enzyme linked immunosorbent assay.Results: Exposure to LDR enhanced nitric oxide secretion and phagocytosis. The expression of early activation antigen, CD69, was enhanced in CD8+ T lymphocytes concomitant with enhanced proliferation in response to con A. In addition, mixed lymphocyte reaction (MLR) and CTL response were augmented and secretion of interferon gamma (IFN-γ) was suppressed following LDR exposure.Conclusions: LDR exposure enhanced the function of macrophages and responses of CD8+ T cells in C57BL/6 mice.

Paclitaxel probably enhances cytotoxicity of natural killer cells against breast carcinoma cells by increasing perforin production.

Paclitaxel, a semisynthetic taxane, is one of the most active chemotherapeutic agents for the treatment of patients with breast cancer. We focused on the effect of paclitaxel on the cytotoxicity of natural killer (NK) cells. NK cells were purified by negative selection with magnetic beads from peripheral blood mononuclear cells of healthy volunteers. A human breast carcinoma cell line BT-474 and an NK cell-sensitive erythroleukemia cell line K562 were used as targets. Cytotoxicity of NK cells was determined by 51Cr-release assay with labeled target cells. Paclitaxel (1-100 nM) did not affect cellular viability, and significantly enhanced cytotoxicity of NK cells in a dose-dependent manner. Although paclitaxel did not affect Fas-ligand expression of NK cells, paclitaxel induced mRNA and protein production of perforin, an effector molecule in NK cell-mediated cytotoxicity. Concanamycin A, a potent inhibitor of the perforin-mediated cytotoxic pathway, inhibited paclitaxel-dependent NK cell-mediated cytotoxicity. Furthermore, paclitaxel induced activation of nuclear factor kappa B (NF-kappa B) in NK cells. NF-kappa B inhibitor pyrrolidine dithiocarbamate significantly suppressed both paclitaxel-induced perforin expression and NK cell cytotoxicity. Our results show for the first time that paclitaxel enhances in vitro cytotoxicity of human NK cells. Moreover, our results suggest a significant association between enhanced NK cell cytotoxicity, increased perforin production, and NF-kappa B activation.

Retinal target cells of the centrifugal projection from the isthmo-optic nucleus.

Although the avian retina has long been known to receive projection from a midbrain nucleus, the isthmo-optic nucleus (ION), the output of its target cells has remained obscure. We labeled the isthmo-optic (IO) terminals in the Japanese quail retina, by using anterograde transport of fluorescent tracer injected into the ION, and then labeled target cells for these terminals by means of intracellular tracer injection under direct microscopic observation. Somata of the IO target cells (IOTCs) lie in the innermost zone of the inner nuclear layer of the ventral half of the retina and have no dendrites but an axon. The axons run in the inner plexiform layer (IPL) for up to 6 mm and terminate densely in a round or elliptical terminal field, about 90-290 microm in diameter, of the outermost zone of the IPL. Longer axons (> 2 mm) extend dorsally, but shorter ones (< 1 mm) project ventrally or horizontally, so the terminals are distributed widely in both dorsal and ventral halves of the retina. The IOTCs cannot be classified into any of the five conventional major classes of retinal cells, including amacrine cells, and are thought to be "slave" neurons whose output is controlled by the neurons in the brain. Topographic separation between input to and output from the IOTCs by the axons might be essential for the overall topographic organization of the centrifugal visual system in birds.

640. E1 Bp-Specific CD8+ T Lymphocytes Are Highly Induced in the Liver and Spleen Eight Days after Adenoviral Infection

Top of pageAbstract The use of Adenovirus (Ad) as a gene delivery vector is limited by transient gene expression due to the host cytotoxic T lymphocyte (CTL) response and innate responses. We and others have shown that the cytotoxic response to Ad is only partially inhibited by administration of Ad to mutant mice deficient in producing a cytotoxic immune response, or by treatment with TNF inhibitors. However, analysis of the specific CTL response to Ad has not been possible due to the lack of reagents that recognize the rearranged T cell receptor expressed on Ad-specific cytotoxic T cells. We therefore developed a tetramer that recognizes the immunodominant epitope of E1B (192 VNIRNCCYI), referred to as E1Bp, in the context of class Ib MHC. The tetramer consists of H2-Db, β2-microglobulin and the peptide epitope, and is referred to as Db-E1Bp. First, to test the specificity of this peptide-MHC complex, C57BL/6 (H-2b; B6) mice were injected with 8×108i.u. of wild type (wt) Ad5. As a control, wt Ad5 injected DBA/2 (H-2d; D2) were analyzed. Lymphocytes (105 )isolated 8 days later from spleen or liver were restimulated with E1Bp in vitro and analyzed for IFN-γ production by an ELISPOT assay. There was a significant increase in spots (6 and 63 spots, respectively) compared to cells stimulated with human influenza virus peptide M1(58 GILGFVFTL) (0 spot, p 98%) of the Db-E1Bp+CD8+ T cells are CD44+. No Db-E1Bp+CD8+ T cells were found in immunized D2 or naive B6 mice. To determine peptide-specific lysis of target cells in vivo, we labeled target cells from syngeneic mice with either high (2μM; CFSEHi) or low (0.2μM; CFSELo) concentration of CFSE and then pulsed CFSEHi cells with E1Bp and CFSELo cells remained unpulsed. Target cells were adoptively transferred into B6 or D2 mice that were challenged 8 days earlier with wt Ad5, and the percentage of specific killing was determined 6 hours later. As a peptide specificity control, CFSEHi cells pulsed with M1 peptide and unpulsed CFSELo cells were transferred to identically immunized B6 mice. E1Bp-specific lysis was significantly increased in B6 mice compared to control mice or immunized D2 mice (p<0.001). There was a significant correlation between E1Bp-specific lysis and the frequency of Db-E1Bp+ CD8+ T cells in B6 mice (p<0.01). We next used these methods to measure the decreased Ad-specific CTL response in aged mice. Compared with 2-mo-old mice, there was a 30% decrease in killing efficiency in 18-mo-old mice and a 58% decrease in Db-E1Bp+CD8+ T cells. Our results indicate that this tetramer is novel, sensitive and specific tool to detect and follow the E1Bp-specific CTL response against Ad infection.

Cell-mediated cytotoxicity evaluation using monoclonal antibody staining for target or effector cells with annexinV/propidium iodide colabeling by fluorosphere-adjusted counts on three-color flow cytometry.

In addition to (51)chromium release assay, flow cytometric methods have been described to assess in vitro cell-mediated cytotoxicity. In this report, we describe a new flow cytometric approach for determination of in vitro cell-mediated cytotoxicity utilizing three-color flow cytometric assay. This method is based on monoclonal antibody staining of either effector or target cells to evaluate cytotoxicity with increased accuracy by utilizing fluorospheres for calibration. The basic strategy involves labeling effector or target cells with a specific fluorescent-conjugated monoclonal antibody, in addition to staining with annexinV-FITC and propidium iodide to identify apoptotic/dead cells. The effector and target cell populations as well as conjugates were clearly and easily identified by this approach. We obtained significant correlation between cytotoxicity calculated by this technique and (51)chromium release assay results. The integration of fluorospheres allowed us to determine the absolute number of events reflective of the cumulative cell death rather than a cross-sectional, percentage-based cytotoxicity assessment in the target cell population at the time of analysis. This method provides additional advantages to other methods and enables the study of target cell fate in more detail, as well as providing a potential contribution to understanding the mechanisms of cell elimination.

A novel micro-cell-mediated lympholytic assay for the evaluation of regulatory cells in human alloreactive CTL responses

Since cell-mediated lympholysis (CML), the most commonly used in vitro experimental cytotoxic method for the evaluation of regulatory cells, requires large numbers of cells that are often the limiting factor, we have developed a new micro-cell-mediated lympholytic (m-CML) assay. Various numbers of responding cells were stimulated with equivalent numbers of allogeneic irradiated stimulator cells in the presence of (fivefold) serial dilutions of regulatory cells. On the 8th day, 4-h 51Cr-release assays were performed by adding 5000 labeled target cells from the corresponding stimulators to the cultures. Even though results that were comparable to the macro- (bulk) CML and MLR modulation experiments were obtained with all the cell combinations tested in the m-CML, the combinations with 50,000 responder cells and stimulating cells and dilutions of 25,000 to 40 modulator (regulatory) cells were found to be the most reproducible for assaying regulatory cell potency in vitro. Similarly, expression of the results as percentage inhibition using percent specific lysis values was the simplest method of calculation. This assay was standardized for the evaluation of the inhibitory activity of a variety of regulatory cells, including long-term cultures of cadaver-donor vertebral body bone marrow cells (vDBMC-L), in vitro generated CD8 positive and CD28 negative suppressor T cells and donor chimeric cells isolated from renal transplant recipients who had been perioperatively infused with donor bone marrow cells (DBMC). The results indicate that the m-CML assay is a sensitive and reliable micromethod with at least 10-fold fewer responders, stimulator and modulator cell numbers needed than macro-CML assays for the evaluation of regulatory cells obtained from a variety of immune systems in vitro.

IMMUNOFLUORESCENCE DETECTION OF ESCHERICHIA COLI IN SEAWATER: A COMPARISON OF VARIOUS COMMERCIAL ANTISERA

ABSTRACT Through a microscopical method, relying on the interaction between fluorescent antibodies and target antigen, it is possible to detect and enumerate Escherichia coli in seawaters. Various commercial monoclonal and polyclonal antisera have been tested in an indirect immunofluorescence (IIF) assay developed for microbiological monitoring of coastal waters. Prior to use, they have been titrated and screened for cross-reactions with a collection of clinical and environmental isolates. A comparison among counts obtained on field samples showed higher performance for microscopical than for plate methods, due to the ability of all antisera to label target cells specifically, regardless of their viability. Because of their different specificities, polyclonal antisera yielded better quantitative results than monoclonal antisera. The study further suggested the usefulness of the immunofluorescence assay as a rapid alternative analytical tool for the specific detection of bacterial pathogens in aquatic environments.

A novel technique for the fluorometric assessment of T lymphocyte antigen specific lysis

The 51Cr release assay has traditionally been used to investigate effector cell cytotoxic function against labeled targets, but this method has inherent problems that include hazards associated with radioactivity, cell labeling and high spontaneous release. Here we describe a novel flow cytometric assay which addresses and improves upon the problems currently encountered with the 51Cr release assay. The fluorometric assessment of T lymphocyte antigen specific lysis (FATAL) assay employs dual staining (PKH-26 and CFSE) to identify and evaluate the target population. We found that the PKH-26/CFSE combination efficiently labeled target cells. Evaluation of the spontaneous leakage from dye labeled target cells was forty fold lower than the spontaneous leakage seen with the 51Cr release assay. The FATAL assay permitted a more accurate assessment of the effector: target ratio, and detected low levels of cytotoxic T lymphocyte (CTL) mediated lysis. There was a strong correlation between the 51Cr release and FATAL assays, when performed in parallel with identical effector and target cells ( r 2=0.998, P=<0.0001). This novel method of detecting cytolysis represents a qualitative and quantitative improvement over standard 51Cr release analysis. The FATAL assay will be of value to further investigate mechanisms of cytolysis by effector cell populations.

Using GFP retrovirus to label tumor cells and vascular endothelia cells

To prepare retrovirus which carry GFP gene and are able to label living cells simply and rapidly. The recombinant retroviral vector pLNCX-GFP was constructed by inserting 780?bp GFP cDNA fragment into the MCS site of retroviral plasmid pLNCX. Both ecotropic packaging cell line ΦX-Eco and amphotropic packaging cell line ΦX-Ampho and PA317 were transfected by pLNCX-GFP with liposome. The supernate collected from transfected packaging cells was used to infect a variety of tumor cell lines and vascular endothelia cell lines. When packaging cells were transfected by retroviral vector pLNCX-GFP, the GFP expression could be observed in 25%-40% of cells and GFP retrovirus then could be detected, however G418 resistant clones showed more stable GFP expression and higher retrovirus titer. The GFP retrovirus from different packaging cell line showed variant ability to infect tumor cell lines and vascular endothelia cell lines and the tumor cells infected by GFP retrovirus showed stable GFP expression in vitro. GFP transduced tumor cells could grow in syngenic animal and continue expressing GFP. Using GFP retrovirus to label target cells represent an important advantage over conventional plasmid because they can efficiently transfer GFP gene into target cells and GFP can be stably expressed in target cells no matter in vitro or in vivo.

Tumor-specific CD4+ T cells have a major "post-licensing" role in CTL mediated anti-tumor immunity.

A number of tumor studies have indicated a link between CD4 help and the magnitude and persistence of CTL activity; however, the mechanisms underlying this have been largely unclear. To evaluate and determine the mechanisms by which CD4(+) T cells synergize with CD8(+) T cells to prevent tumor growth, we used the novel technique of monitoring in vivo CTL by labeling target cells with CFSE. This approach was supported by the direct visualization of CTL using peptide-MHC tetramers to follow tumor-specific T cells. The data presented demonstrate that while cotransfer of Ag-specific CD4(+) T cells was not required for the generation of CTLs, because adoptive transfer of CD8(+) T cells alone was sufficient, CD4(+) T cells were required for the maintenance of CD8(+) T cell numbers. Our data suggest that there is a correlation among the number of CD8(+) T cells, in vivo CTL function, and IFN-gamma production, with no evidence of a partial or nonresponsive phenotype among tetramer-positive cells. We also show that CD4(+) T cells are required for CD8(+) T cell infiltration of the tumor.

Measurement of Cytotoxic Activity of NK/LAK Cells

In this unit, methodology is presented for measuring the capacity of human natural killer (NK) or lymphokine-activated killer (LAK) cells to lyse tumor cell targets. Cytotoxicity of these effector cells is evaluated in a short-term (51)Cr-release assay using NK-sensitive tumor cells as the targets for NK cells or NK-resistant tumor cells as the targets for LAK cells. In the basic protocol, a generic (51)Cr-release assay is described in which PBMC, purified NK cells or interleukin 2 (IL-2)-activated lymphocyte populations (LAK cells) are utilized as effector cells. Support protocols describe preparation of nonadherent tumor cells, cells obtained from malignant effusions, trypsinized tumor cells from adherent monolayer culture, or freshly isolated tumor cells from surgical specimens. All of these cell types can serve as the (51)Cr-labeled target cells in the basic protocol. The procedure for labeling target cells from any of these sources with (51)Cr is also provided.

RRNA probe‐based cell fishing of bacteria

We have developed a new, cultivation-independent, fast and flexible method for the rRNA-targeted probe-based enrichment of bacteria. The target cells were labelled by in situ hybridization with biotinylated polyribonucleotide probes. These probes were generated by in vitro transcription of amplified rDNA of a variable region in domain III of the 23S rRNA molecules. The probes were about 300 nucleotides in length and were labelled by incorporation of biotin-UTP during the transcription. Probes were hybridized with bacterial cells and incubated with paramagnetic streptavidin-coated particles. The labelled target cells can be separated in a column filled with steel wool inserted into the field of a permanent magnet. Unlabelled, non-target cells pass through the column, whereas labelled cells are retained. They were eluted from the column after removal of the magnetic field. Up to now, the method has been tested with mixtures of different pure cultures. For the first time, transcript probes have been used for the labelling of the target cells and for their specific separation. The enrichment of the target cells can be monitored by a streptavidin-fluorescein staining of the biotinylated target cells and/or by a subsequent in situ hybridization with fluorescently labelled oligonucleotide probes. Enrichment rates of up to 90-fold, depending on the original abundance of the cells of interest, could be determined. To demonstrate that the sorted cells were amenable to molecular analysis, we amplified and sequenced a part of the tuf gene of enriched Acinetobacter calcoaceticus cells.

In vitro reduction of virus infectivity by antibody-dependent cell-mediated immunity

Antibody-dependent cellular cytotoxicity (ADCC), an important defense against viral infections, is generally measured in 51 Cr -release assays. However, the effect of ADCC on viral burden is more relevant in vivo. An assay was developed to determine the impact of antibody and cytotoxic cells on reducing the amount of measles virus cultured from infected cells. Although the components of this assay are the same as those involved in ADCC, the endpoint is a reduction in virus infectivity rather than cytotoxicity. The immune function measured in the assay has therefore been termed antibody-dependent cell-mediated immunity (ADCMI). Measles virus-infected Raji cells and blood mononuclear cells served as target and effector cells, respectively. Effector cells were incubated with antibody-labeled or unlabeled target cells for 24 h, and virus infectivity determined. Adding effector cells to unlabeled target cells reduced virus titer by 81.8%. Labeling target cells with measles-seronegative serum had little further effect. However, labeling target cells with measles-seropositive serum reduced infectivity an additional 96.5%. By allowing serum to remain in the supernatant fluid after labeling target cells, neutralizing and cell-mediated antibody functions were simultaneously measured. Finally, arming cytokine-activated effector cells with measles-seropositive serum also reduced virus infectivity. This novel assay provides an important tool for evaluating the anti-viral effects mediated by antibody and effector cells.

A Non-Radioisotopic Human Natural Killer Cell Assay Using Rhodamine-123 Fluorescent Dye as Labelling Probe

A non-radioisotopic method for assessment of human natural killer (NK) cell activity in peripheral blood mononuclear cells (PBMC) was established by labelling K562 erythroleukemia target cells with a fluorescent dye, rhodamine-123 (Rh-123). The labelling and assay conditions were determined for minimizing spontaneous release (SR). In order to investigate whether NK activity assessed by measuring Rh-123 release agrees with the activity determined by a 51Cr release assay, the NK activity of PBMC was measured simultaneously by both assay methods. Statistical analysis demonstrates that NK activities determined by Rh-123 release correlate well with those measured by 51Cr release. The Rh-123 release assay under the conditions determined was found to be applicable to measurement of the enhanced NK activity resulting from pretreatment of effector leukocytes with interferon-α It is concluded that the Rh-123 release assay with use of K-562 labelled target cells is practical for the assessment of human NK activity in laboratories where use of radioisotopes is not permitted or undesirable.

Identification of a Streptococcus gordonii SspB domain that mediates adhesion to Porphyromonas gingivalis.

Porphyromonas gingivalis, a primary pathogen in adult periodontitis, may establish itself in the oral cavity by adhering to early plaque bacteria such as Streptococcus gordonii. Our previous studies (R. J. Lamont et al., Microbiology 140:867-872, 1994) suggested that this interaction is mediated by the SspB polypeptide, a member of the antigen I/II family of streptococcal surface proteins. S. gordonii was recently shown to express a second Ssp polypeptide (SspA) that resembles SspB and the structurally homologous antigen I/II polypeptide (Pac) of Streptococcus mutans. To determine if all of these related antigen I/II proteins interacted with P. gingivalis, SspA, SspB, and Pac were tested for adhesion to P. gingivalis cells. Both of the S. gordonii Ssp proteins bound labeled target cells, whereas the S. mutans Pac polypeptide did not, suggesting that antigen I/II-mediated binding of P. gingivalis by streptococci may be species specific. To investigate the molecular basis for this functional difference, the P. gingivalis binding domain of SspB was mapped. The binding properties of a family of truncated SspB polypeptides lacking C-terminal sequences were determined. In addition, the lack of binding activity exhibited by the Pac protein was exploited to construct and analyze chimeric SspB-Pac polypeptides. Both approaches revealed that the region defined by residues 1167 to 1250 of SspB was essential for P. gingivalis binding. This region of SspA and SspB is entirely conserved, consistent with the binding properties determined for these proteins. However, the corresponding region of Pac differs in both the primary sequence and predicted secondary structure, suggesting that the overall structure of this domain may define its functional activity.