Recognition by human V gamma 9/V delta 2 T cells of a GroEL homolog on Daudi Burkitt's lymphoma cells.

All human gamma delta T cells coexpressing the products of the variable (V) region T cell receptor (TCR) gene segments V gamma 9 and V delta 2 recognize antigens from mycobacterial extracts and Daudi cells. Exogenous and endogenous ligands on the cell surface, homologous to the groEL heat shock family, induced reactivities that resembled superantigen responses in this major subset of human peripheral blood gamma delta T cells. Stimulation of human V gamma 9/V delta 2 T cells is not restricted by human leukocyte antigens (HLA), including nonpolymorphic beta 2-microglobulin (beta 2M)-associated class Ib molecules. These data may be important for understanding the role of gamma delta T cells in autoimmunity and in responses to microorganisms and tumors.

Regional differences in the incidence and growth of mouse tumors following intradermal or subcutaneous inoculation.

Tumor cells inoculated intradermally or s.c. into more cranial regions of the lateral trunk show strikingly greater tumor growth and development than do similar cells injected more caudally. At low tumor cell doses the incidence anteriorly may be double that found posteriorly and tumors become detectable more rapidly anteriorly; at higher cell doses the anterior:posterior ratio of tumor weight may be 4:1. The effect appears to be independent of the type of tumor used (mastocytoma, sarcoma, teratoma, lymphoma, or adenocarcinoma) and of the strain of mouse host; it does not appear to be influenced by the sex of the host animal, the immunogenicity of the tumor, or the immunological competence of the tumor recipient. The results are discussed both in terms of practical considerations for developing adequate tumor transplantation and treatment protocols and in terms of the biological significance in relation to spontaneous or induced oncogenesis.

Expression of organ-specific antigens on capillary endothelial cells.

Our central thesis is that the endothelial cells which line capillaries of various organs are not all alike. Using monoclonal and conventional antibodies we demonstrate that capillary endothelial cells express on their cell surface an array of antigens that manifest organ selectivity. Brain-derived endothelial cells possess brain-associated antigens, ovary-derived endothelial cells share antigenic markers with other ovarian cells, and lung-derived endothelium possesses antigens that are primarily expressed on cells of the lung. Our experiments lead us to suggest that organ-associated determinants on the endothelial cell surface may play a role in the selective adhesion of tumor cells during metastasis, in site-limited vascular pathology, and in the regionally limited release of angiogenesis-induced factors.

Heterogeneity of mouse vascular endothelium. In vitro studies of lymphatic, large blood vessel and microvascular endothelial cells.

Microvascular endothelial cell cultures have been established from mouse lung, liver, brain, heart, placenta, kidney, urinary bladder, mammary gland, ovary and epididymal fat pad. In addition, large vessel endothelial cells have been obtained from the mouse aorta and thoracic duct. The heterogeneity of these cells has been shown by flow cytometric determination of angiotensin-converting enzyme, by differential presence of the acetyl low density lipoprotein receptor, by the variable expression of cell surface antigens, and by differential binding of various plant lectins. The endothelial cell lines we have developed provide the means to examine in the mouse, long a key species for biomedical research, a wide range of biological functions and properties of the vascular endothelium.

Bivariate flow karyotyping, sorting, and peak assignment of all rat chromosomes.

A bivariate flow cytometric rat karyotype was established from second- and third-passage Copenhagen (Cop) rat embryo cell cultures. Chromosome suspensions from such cells (2n = 42 chromosomes) yielded bivariate flow karyotypes composed of 14-18 peaks, 10 of which were sortable into pools of single chromosome types. Conditions affecting resolution of peaks (including the length of colcemid treatment of cells and various combinations of fluorescent and nonfluorescent dyes) were optimized. Using chromosome suspensions from second-passage cultures of adult Cop male and female ear fibroblasts, peaks representing the X and Y chromosomes were identified. Assignment of chromosomes was accomplished by polymerase chain reactions of flow-sorted chromosomes using primers from mapped genes. Availability of this characterized rat flow karyotype should prove useful for assignment of genes to chromosomes as well as generation of chromosome-specific libraries in cloning assigned genes.

Forward scatter pulse width signals resolve multiple populations of endosomes.

The technique of pulse width analysis, developed to optimize cell size resolution in cell cycle kinetics, has not previously been applied to small particles such as endosomes. Offset is used to subtract a portion of the beam diameter from forward scatter pulse width signals to optimize visualization and discrimination of small particles. We identify multiple endosomal populations by offset pulse width of light scatter parameters. Specifically, linear forward scatter pulse width measurements reveal at least two populations of endosomes in the rat renal cortex, the rat renal papilla, and the luminal endothelium of the toad urinary bladder. Logarithmically amplified forward scatter pulse width measurements display the full dynamic range of these signals, resolving additional populations not manifest with linear amplification. To confirm that the endosomes observed were resolved from optical and electronic noise, we examined physiological function. The endosomes acidified after supplying ATP to the intrinsic membrane H(+)-ATPase present. Further, electron microscopy of sorted endosomal populations from the toad urinary bladder confirmed identity and homogeneity of the fraction. Flow cytometric analysis of endosomal populations by multiparametric techniques including pulse width analysis of structural parameters and pulse height analysis of fluorescence from entrapped fluorophores allows identification, isolation, and quantification of multiple endosomal populations.

Tumor cell kinetics using two labels and flow cytometry.

A flow cytometry based, single sampling method employing sequential bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd) labeling with an intervening interval is examined. BrdUrd-specific and nonspecific monoclonal antibodies and flow cytometry are used to estimate the duration of DNA synthesis and the potential doubling time in tumor cell populations using a single sampling or biopsy. A correction for labeled, divided cells allows postlabel incubation intervals to exceed the length of the G2/M phase of the cell cycle. The method yields reliable results when tested in experimental tumor systems in vitro and in situ, as well as in nine human tumors labeled in situ. It uses a somewhat simpler analysis than the alternative relative movement method, requiring only labeling indices, rather than both a labeling index and relative movement, but doses require the administration of two, rather than one, label. It provides an independent verification of and a useful single sampling alternative to the relative movement method for the estimation of the length of S phase and, from this, the potential doubling time in experimental or clinical human tumors.

Detection of borreliacidal antibodies in hamsters by using flow cytometry.

Flow cytometry can be used to detect antibody that kills Borrelia burgdorferi. Borreliacidal activity was detected within 3 h of incubating B. burgdorferi with immune serum and complement. Right-angle light scatter and propidium iodide fluorescence were the cytometric parameters which correlated best with in vitro killing of B. burgdorferi. Flow cytometry is a rapid method for determining the presence of borreliacidal activity and may lead to a better serodiagnostic test for the detection of Lyme disease.

Heated lymphocytes express HLA-DR antigens despite their inability to stimulate in MLC.

We have utilized serological techniques and mixed lymphocyte culture (MLC) reactions to examine HLA-DR and HLA-D expression by heated (45 degrees C for 1 h) lymphocytes in order to study the functional relationship of these antigens. Heated lymphocytes do not stimulate proliferation of allogeneic lymphocytes in MLC, yet they express HLA-DR antigens. The fraction of peripheral blood lymphocytes (PBL) expressing DR is not altered by heating, nor is the staining intensity altered as detected by fluorescence microscopy. Alloantisera to "B cell alloantigens" recognize HLA-DR determinants on heated cells without any detectable change in either specificity or quantitative cytotoxic effects. Flow cytometry with monoclonal antibody demonstrates only minimal decrease in HLA-DR expression after heating. Thus stimulation in MLC requires more of the stimulating cell than the mere expression of HLA-DR.

Energy transfer assays of rat renal cortical endosomal fusion: evidence for superfusion.

The complex of components necessary to allow endosomal fusion includes both membrane-bound receptors and several soluble proteins. Although these factors have been isolated from cultured cell lines, and endosomal fusion has been reconstituted in vitro for vesicular systems from yeast to synaptosomes, there is a paucity of data from mammalian systems. To investigate fusion in rat renal cortical endosomes, we began by developing a fusion assay. As the immunoglobulin and avidin-based probes almost universally employed in fusion assays are excluded by the glomerular ultrafiltration barrier, it was necessary to begin by finding ultrafilterable probes which could serve as a fusion assay. We labeled the apical endosomal pathway of the renal proximal tubule by intravenous infusion of ultrafilterable fluorescent dextrans. Energy transfer from entrapped fluorescein-dextran to rhodamine-dextran had a narrow concentration dependence but allowed fluorometric assay of endosomal fusion. The "spectroscopic ruler" property of energy transfer, whereby it will only occur at < 60 A, makes fusion measurements unequivocal. The energy transfer efficiency of fluorometric (48 +/- 1%) and flow cytometry (57 +/- 1%) assays were close to the theoretical optimum (57%). Energy transfer is detected as a decrease in fluorescence of the fluorescein donor and an increase in fluorescence of the rhodamine acceptor. Our endosomal fusion assay was utilized to determine the optimal conditions for fusion of rat renal cortical light endosomes and heavy endosomes. Independent measurements of fluorescein-dextran and rhodamine-dextran on an endosome-by-endosome basis using dual-beam two-color flow cytometry demonstrated that each fusion event involves multiple endosomes rather than a single pair of endosomes. Electron microscopy analysis demonstrated that the average vesicle diameter was five times larger in the fused heavy endosomal fractions compared with control fractions without fusion. Hence, fusion of mammalian renal cortical endosomes reconstituted in vitro is consistent with multiple fusion events dubbed superfusion.

Monoclonal antibody against angiotensin-converting enzyme: its use as a marker for murine, bovine, and human endothelial cells.

A monoclonal antibody has been prepared against rat angiotensin-converting enzyme (ACE). By selection for antibody binding to endothelial cells of bovine rather than rat origin we have obtained a reagent that has broad cross-species binding properties and that can at the same time serve as a useful marker for the surface of endothelial cells. The IgM-producing clone that we have established, alpha-ACE 3.1.1, has been grown in ascites form to yield ascites fluid that binds selectively to immobilized ACE at a greater than 1:10,000 dilution. By use of enzyme-linked immunosorbent assays, immunofluorescence histology, and flow cytometry, we have demonstrated the presence of ACE on endothelial cells of murine, bovine, and human origin. By means of a fluorescence-activated cell sorter (FACS-IV) we have been able to selectively isolate viable endothelial cells from a mixture of endothelial cells and fibroblasts. We believe the antibody will be useful not only for the selection and in vitro cultivation of endothelial cells but also as a tool for the identification and pharmacological study of ACE.