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Biomedical subjects

P K Horan

Publications and source records attributed to P K Horan.

At least 19 recordsLinked to original sources
PubMed1993

Mechanisms of adoptive immunotherapy: improved methods for in vivo tracking of tumor-infiltrating lymphocytes and lymphokine-activated killer cells.

Adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) and lymphokine-activated killer cells has been demonstrated to mediate regression of tumors in murine models and in selected patients with advanced cancer. Improved methods for monitoring immune cell traffic, particularly to sites of tumor, are needed to elucidate mechanisms of antitumor activity and optimize treatment protocols. Traditional cell tracking methods such as fluorescent protein labeling and radiolabeling using 111In, 125I, or 51Cr are limited by isotope half-life, leakage or transfer of label from immune cells, and toxicity or altered cell function caused by the labeling process. Labeling with genetic markers allows long-term cell tracking but is laborious to perform and difficult to quantitate. We have used two recently described lipophilic cell tracking compounds (PKH26 and 125I-PKH95) which stably partition into lipid regions of the cell membrane to track immune cells in vivo. Concentrations of each tracking compound which had no adverse effects were determined for a variety of murine TIL and lymphokine-activated killer cell functions. Viability was unimpaired at labeling concentrations of up to 5 microM for PKH95 and 20 microM for PKH26. TIL proliferation was unaltered by labeling with up to 5 microM PKH95, 20 microM PKH26, or a combination of 15 microM PKH26 and 5 microM PKH95. In vivo cytotoxic effector function and in vivo therapeutic efficacy of lymphokine-activated killer cells and TIL were also unimpaired by labeling with 20 microM PKH26 or 1 microM 125I-PKH95. Subsequent studies in an adoptive transfer immunotherapy model used 125I-PKH95 to track the biodistribution of TIL in tumor and in non-tumor-bearing animals and PKH26 fluorescence to monitor microdistribution within tissues and distinguish TIL from host T-cells. The results suggest that differential accumulation, selective retention, or proliferation at the tumor site cannot account for the observed pattern of therapeutic efficacy. We hypothesize that a minimum number of TIL must reach the tumor site in order to achieve a demonstrable therapeutic effect.

Animals↗
PubMed1993

Specificity of gap junction communication among human mammary cells and connexin transfectants in culture.

In a previous paper (Lee et al., 1992), it was shown that normal human mammary epithelial cells (NMEC) express two connexin genes, Cx26 and Cx43, whereas neither gene is transcribed in a series of mammary tumor cell lines (TMEC). In this paper it is shown that normal human mammary fibroblasts (NMF) communicate and express Cx43 mRNA and protein. Transfection of either Cx26 or Cx43 genes into a tumor line, 21MT-2, induced the expression of the corresponding mRNAs and proteins as well as communication via gap junctions (GJs), although immunofluorescence demonstrated that the majority of Cx26 and Cx43 proteins present in transfected TMEC was largely cytoplasmic. Immunoblotting demonstrated that NMEC, NMF, and transfected TMEC each displayed a unique pattern of posttranslationally modified forms of Cx43 protein. The role of different connexins in regulating gap junction intercellular communication (GJIC) was examined using a novel two-dye method to assess homologous and heterologous communication quantitatively. The recipient cell population was prestained with a permanent non-toxic lipophilic dye that binds to membranes irreversibly (PKH26, Zynaxis); and the donor population is treated with a GJ-permeable dye Calcein, a derivative of fluorescein diacetate (Molecular Probes). After mixing the two cell populations under conditions promoting GJ formation, cells were analyzed by flow cytometry to determine the percentage of cells containing both dyes. It is shown here that Cx26 and Cx43 transfectants display strong homologous communication, as do NMEC and NMF. Furthermore, NMEC mixed with NMF communicate efficiently, Cx26 transfectants communicate with NMEC but not with NMF, and Cx43 transfectants communicate with NMF. Communication between Cx26 TMEC transfectants and NMEC was asymetrical with preferential movement of calcein from TMEC to NMEC. Despite the presence of Cx43 as well as Cx26 encoded proteins in the GJs of NMEC, few Cx43 transfectants communicated with NMEC. No heterologous GJIC was observed between Cx26- and Cx43-transfected TMEC suggesting that heterotypic GJs do not form or that Cx26/Cx43 channels do not permit dye transfer.

Blotting, Western↗
PubMed1992

Cell cycle analysis of asexual stages of erythrocytic malaria parasites.

Intra-erythrocytic Plasmodium species can be stained with the DNA binding dye, Hoechst 33342, and the distribution of DNA content determined for parasite populations by flow cytometric measurement of fluorescence. Analysis of this distribution will determine the parasitaemia (percentage of erythrocytes infected), and the percentages of trophozoite infected red blood cells, polyparasitized (trophozoite) red blood cells, and schizont/segmenter infected red blood cells. This analysis is based on the hypothesis that the asexual parasites cycle with single G1 period, and effectively, a single S phase with no significant G2/M period except at schizogony when the genome DNA content is equivalent to 8 N or higher, dependent on the species. Data are presented to support this model.

Animals↗
PubMed1991

Long-term tracking of lymphocytes in vivo: the migration of PKH-labeled lymphocytes.

Studies of in vivo cell migration using cell markers such as 51Cr, 111In, FITC, or XRITC have been limited to short time periods due to the elution, toxicity, or rapid loss of label detectability. We have labeled sheep lymphocytes in vitro with PKH-2, a new fluorescent cell membrane label, and, after their intravenous injection back into donor sheep, have been able to detect them in efferent lymph, using flow cytometry, for longer than 38 days. The PKH-2-labeled lymphocytes migrated with similar kinetics, efficiency, and tissue specificity as lymphocytes labeled with cell markers used previously. PKH-2-labeled cells mediated graft versus host reactions indistinguishable from those mediated by unlabeled cells, and cell surface antigens were equally detectable on the surface of labeled and unlabeled lymphocytes. According to the slow, consistent loss of fluorescence intensity of the labeled cells in vivo, we predict that labeled lymphocytes could remain detectable by flow cytometry for greater than 7 weeks with the labeling protocol used in these experiments.

Animals↗
PubMed1990

An improved clonal excess assay using flow cytometry and B-cell gating.

In humans with B-cell malignancies, the presence of monoclonal B lymphocytes (clonal proliferation) can be detected by comparing the fluorescence intensity distributions of lymphocytes stained with anti-kappa and anti-lambda reagents. The sensitivity of previously described single-color immunofluorescence techniques to low levels of clonal excess is limited by background from cytophilic immunoglobulins on non-B cells and by the low proportion of circulating B cells in individuals with minimal disease. We have used two-color immunofluorescence and B-cell gating to develop an improved assay that avoids false positives due to non-B cells, without requiring restrictive light scatter gates that may exclude true positives. This method is sensitive to 0.2% monoclonal B cells admixed with fresh normal lymphocytes, to 0.6% monoclonal B cells admixed with normal lymphocytes that have been stored for up to 72 hours, and readily detects 1% monoclonal cells in patient specimens. The two color B-cell gated assay offers sensitivity equivalent to the single-color assay and improved specificity for detection of low levels of clonal excess.

B-Lymphocytes↗
PubMed1989

Fluorescent in vivo tracking of hematopoietic cells. Part I. Technical considerations.

We report a new technology for in vivo tracking of hematopoietic cells, using fluorescent lipophilic probes. Because the probe is irreversibly bound in the lipids of the cell membrane; substantial numbers of dye molecules can be incorporated per cell and thus substantial signal to noise can be achieved. Although this technology can be used for all hematopoietic cells, these first findings are reported on red blood cells (RBCs) owing to the importance of the membrane to RBC function and integrity. We demonstrated that labeling 10% of the RBCs of a rabbit and reinjecting them into the animal makes possible the tracking of these cells at various times after injection. Furthermore, the labeling appears not to affect in vivo cell lifetime or cellular volume changes in response to hypotonic shock. The single cell fluorescence intensity of the labeled RBCs remains relatively constant for 60 days, and an immune response appears not to be generated against labeled cells. That labeled RBCs have lifetime kinetics in vivo, as shown in other studies, indicates that the membranes are functioning normally and are unaltered by the labeling technology. The technology we present is also applicable to white blood cells, bone marrow, and platelets.

Animals↗
PubMed1989

Stable cell membrane labelling.

Binding fluorescent or radioactive reporter molecules to the lipid bilayer of cell membranes allows cell growth and trafficking to be monitored in vivo.

Animals↗
PubMed1989

Cell-mediated cytotoxicity. A highly sensitive and informative flow cytometric assay.

Determination of target cell lysis by cytolytic effectors has typically been achieved by two methods: the release of various markers from the cell, as in 51chromium release assays and the uptake of markers into the cell, as in trypan blue uptake in single cell/conjugate binding assays. Problems associated with these assays might include: (1) poor uptake, (2) nonspecific release, (3) poor statistics, (4) length of assays, or (5) subjectivity. These difficulties prompted the development of a new sensitive flow cytometric assay employing two fluorochromes. PKH-1, a fluorochrome which fluoresces in the green, binds to the cytoplasmic membrane and does not leak or transfer, is used to identify the target cell population. Propidium iodide fluoresces in the red and is used to detect non-viable cells. Use of these two fluorochromes and two parameter analysis allows for identification of four subpopulations in the sample: live effectors, dead effectors, live targets and dead targets. By enumeration of these subpopulations the following information can be calculated: (1) the percent target lysis, (2) effector-to-target cell ratios, (3) viability of the effector cells at the termination of the assay, and (4) viable effector to target cell ratios. The results show that PKH-1 labeling of target cells had no effect on effector-target cell interactions. Excellent correlation was found between this method and the chromium assay, however, due to earlier detection of the lytic event, this method provides a distinct time advantage over current methods.

Animals↗
PubMed1989

Kinetics of changes in peritoneal cell populations following acute inflammation.

The kinetics of macrophage (M phi) recruitment to the peritoneum following the induction of acute inflammation by thioglycollate broth (TG) was evaluated after prelabeling resident M phi with the fluorescent cell tracking dye, PKH-1. Most of the PKH-1-labeled resident M phi disappeared from the recoverable peritoneal cell population within the first hour after injection of TG. This disappearance coincided with the inflammatory influx of neutrophils (PMNs) and was sustained for at least 5 days after administration of TG, although the PMN number had returned to resident levels by this time. PKH-1-labeled peritoneal M phi were observed again in most animals at 7 days after injection of TG. The number of labeled M phi recovered at 7 days was approximately twice the number of resident peritoneal M phi in control animals which did not receive the TG broth. These additional M phi may include progeny of either the resident M phi or other local M phi precursors, such as omental M phi, which were labeled by the PKH-1 injection.

Animals↗
PubMed1988

In vivo labeling of resident peritoneal macrophages.

A novel method for labeling resident peritoneal macrophages (M phi) by injection of a dye into the peritoneal cavity is described. The dye, which fluoresces green, is selectively taken up by the resident M phi. Dye labeled cells can be further characterized by labeling of cell surface antigens with monoclonal antibodies (Mabs) and phycoerythrin conjugated second antibody. After such labeling with the Mabs F4/80 or Mac 1 the resident M phi were labeled by both the green dye and the red Mab markers, while recruited M phi or neutrophils were labeled with just the red Mab; the two populations of cells were readily distinguished by two-color flow cytometry. This technique enabled identification of resident and recruited M phi in each animal without the use of radioisotopes, irradiation, or bone marrow ablation. Sufficient numbers of cells can be analyzed from each animal so that individual animals could be evaluated. We found no adverse effects of this labeling technique on expression of cell surface antigens or M phi mediated cytotoxicity. We did find evidence that the i.p. injection induced a mild inflammation in the peritoneal cavities of animals injected with either the dye or the balanced salt solution vehicle. Examination of the intracellular staining pattern indicated that the label rapidly sequestered in the cytoplasm of the M phi, possibly in the lysosomes. Dye solubility studies showed that the dye was partially soluble at the concentration used for in vivo labeling. We hypothesize that the M phi labeling occurred by a combination of phagocytosis of dye aggregates and endocytosis of labeled plasma membrane.

Animals↗
PubMed1988

Maintenance of peritoneal macrophages in the steady state.

Resident peritoneal macrophages (M phi) were labeled in situ by intraperitoneal (i.p.) injection of the green fluorescent cell tracking dye PKH-1. After immunofluorescence staining with M phi specific monoclonal antibodies (Mabs) and phycoerythrin (PE) second antibody, the resident M phi were labeled with both the green dye and red Mab label, while recruited M phi were labeled only with the red Mab tag. These populations were distinguished by two-color flow cytometry. PKH-1 labeled resident peritoneal M phi were followed for 1-49 days in mice that received no further treatment (steady state). Dye labeled M phi were still detectable after 49 days in vivo, although their green fluorescence intensity had decreased steadily over time. The decrease in dye intensity was limited to M phi, as the fluorescence intensity of PKH-1 labeled peritoneal lymphocytes did not change. Resident M phi populations were clearly separated from recruited M phi by the intensity of their staining with PKH-1 for up to 28 days. No decrease in the number of resident (dye labeled) peritoneal M phi was observed over 1-28 days. These data indicate that resident peritoneal M phi were not replaced by recruited blood monocytes in the steady state.

Animals↗
PubMed1987

Antibody-bearing liposomes as multicolor immunofluorescence markers for flow cytometry and imaging.

Liposomes covalently coupled to monoclonal antibodies retain the specificity of the antibody and bind only to cells bearing the appropriate determinant. As opposed to directly labeled antibodies which generally have fluorochrome to protein ratio of between 2-5, the entrapped space inside liposome can contain several hundred to several thousand molecules of fluorochromes in a space chemically isolated from the outside environment, thus providing the potential for an amplified fluorescence signal. We have prepared small unilamellar liposomes containing the soluble fluorochromes carboxyfluorescein (CF), which fluoresces in the green and sulforhodamine (SR), which fluoresces in the red, and covalently coupled a series of monoclonal antibodies using a heterobifunctional reagent. We were able to detect, on an Epics 753 flow cytometer equipped with an argon ion and a dye laser and by fluorescence microscopy, both single and double labeled mouse spleen lymphocyte subsets, fibroblast L cells and Raji cells. Complete color separation was obtained with CF-labeled cells being detected only by the green photomultiplier and SR-labeled cells by the red photomultiplier. Cells labeled with both were detected by both photomultipliers. Liposomes bearing anti-Ia antibodies bound only to B lymphocytes whereas those with anti-H-2K antibody bound both to T and B lymphocytes. In another system, single and dual color immunofluorescence made possible the simultaneous detection of HLA and H-2K molecules on transfected murine fibroblast L cells. The signal-to-noise ratio was more favorable for the liposome-labeled reagents than reagents labeled with fluorescein isothiocyanate. Cells labeled with antibody-bearing liposomes could be fixed with paraformaldehyde or glutaraldehyde without adversely affecting the original staining patterns. Apart from the two fluorochromes described above, other markers of choice could be encapsulated without any adverse effect on the antibody-liposome coupling procedure or on the specificity of the conjugated antibody. Since the fluorochrome is not directly coupled to the protein, there is no requirement for protein conjugation sites in order for it be usefully encapsulated inside liposomes. Therefore, this system provides new opportunities to exploit different, as yet untapped fluorochromes for use in flow cytometry and imaging.

Animals↗
PubMed1986

Improved flow cytometric analysis of leukocyte subsets: simultaneous identification of five cell subsets using two-color immunofluorescence.

Flow cytometric analysis of human peripheral blood leukocytes has typically been achieved by staining multiple aliquots of the same sample with fluorescent reagents specific for cell subsets of interest. Spectrally discrete fluorochrome tags have been developed for applications in which identification of multiple subsets (e.g., T and B cells) or of subsets not uniquely identified by a single reagent (e.g., activated T cells) requires use of multiple reagents per aliquot. Extension of this approach to more than two reagents per aliquot has led to multicolor methods requiring dual laser excitation and complex instrumentation. We describe an alternative two-color method using commercially available reagents that allows simultaneous identification of five discrete immune cell subsets using only a single excitation source. The technique uses dilution of commercial fluorochrome-labeled reagents with competing unlabeled reagents to selectively produce discrete fluorescence intensity profiles for cell subsets that would otherwise display overlapping or indistinguishable profiles when stained with reagents bearing the same fluorochrome. For example, the fluorescence intensity of phycoerythrin-labeled helper T (Th) cells can be adjusted to be distinct from that of phycoerythrin-labeled suppressor T (Ts) cells. Extending this technique to two colors, we have used a combination of seven different monoclonal antibodies to simultaneously quantify Th, Ts, B cells, natural killer cells, and monocytes in a single aliquot. An additional advantage of this approach is the ability to more accurately quantify "null" cells. Adjustment of fluorescence intensity profiles of different cell subsets by this method is applicable to flow cytometric analysis of a wide variety of cell types. The technique significantly extends the analytical capacity of flow cytometry without significantly increasing the complexity of the instrumentation required.

Antibodies, Monoclonal↗
PubMed1986

Methodological considerations for implementation of lymphocyte subset analysis in a clinical reference laboratory.

As the diagnostic utility of lymphocyte subset analysis has been recognized in the clinical research laboratory, a wide variety of reagents and cell preparation, staining and analysis methods have also been described. Methods that are perfectly suitable for analysis of smaller sample numbers in the biological or clinical research setting are not always appropriate and/or applicable in the setting of a high volume clinical reference laboratory. We describe here some of the specific considerations involved in choosing a method for flow cytometric analysis which minimizes sample preparation and data analysis time while maximizing sample stability, viability, and reproducibility. Monoclonal T- and B-cell reagents from three manufacturers were found to give equivalent results for a reference population of healthy individuals. This was true whether direct or indirect immunofluorescence staining was used and whether cells were prepared by Ficoll-Hypaque fractionation (FH) or by lysis of whole blood. When B cells were enumerated using a polyclonal anti-immunoglobulin reagent, less cytophilic immunoglobulin staining was present after lysis than after FH preparation. However, both preparation methods required additional incubation at 37 degrees C to obtain results concordant with monoclonal B-cell reagents. Standard reagents were chosen on the basis of maximum positive/negative separation and the availability of appropriate negative controls. The effects of collection medium and storage conditions on sample stability and reproducibility of subset analysis were also assessed. Specimens collected in heparin and stored at room temperature in buffered medium gave reproducible results for 3 days after specimen collection, using either FH or lysis as the preparation method. General strategies for instrument optimization, quality control, and biohazard containment are also discussed.

Antibodies, Monoclonal↗