A method of purifying feline T lymphocytes from peripheral blood using the plant lectin from Pisum sativum.

The binding properties of several plant lectins on feline peripheral blood lymphocytes (PBL) were examined by flow cytometry to reveal if any could serve as probes for identifying specific lymphocyte subclasses. Most of the lectins bound > or = 90% of PBL in a uniform manner. The exceptions were the lectins from Glycine max, Phaseolus limensis, and Dolichos biflorus, which bound only a proportion of the PBL. The differential binding of the lectins to lymphocyte subclasses was examined by comparing the staining of control PBL to T cell-depleted PBL. The lectins from Pisum sativum, Lens culinaris, succinylated Triticum vulgaris, and Dolichos biflorus stained T-depleted PBL more brightly than control PBL. Extensive cytofluorometric analyses of the binding properties of Pisum sativum agglutinin (PSA) on feline PBL and mesenteric lymph node cells indicated that feline B lymphocytes express more higher affinity surface receptors for PSA than do T lymphocytes. The higher affinity binding of PSA to B lymphocytes was further demonstrated by PSA-mediated cellular affinity chromatography. With this procedure it was possible to deplete PBL of B lymphocytes and to obtain pure populations of feline T lymphocytes.

Sugar competition assays reveal high affinity receptors for Erythrina cristigalli lectin on feline monocytes.

An examination of fluorescein-labelled Erythrina cristigalli lectin (FITC-ECL) staining on feline mononuclear cells (MNC) using fluorescent microscopy and a novel sugar titration competition assay revealed that monocytes (MO) were stained brighter by FITC-ECL than were lymphocytes (LYM). When MNC were stained with FITC-ECL in the presence of 400 mM or greater D-galactose, analysis by flow cytometry revealed continued MO staining while LYM were negative. MO expressed a larger quantity of carbohydrate receptors (CHO-R) for ECL than did LYM. The CHO-R expressed on MO were mostly protease-insensitive and uncapped by sialic acid residues. All of the CHO-R on LYM were protease-sensitive and many were capped by sialic acid residues. A combined labelling of MNC for non-specific esterase staining, latex bead ingestion and FITC-ECL staining in the presence of 400 mM D-galactose confirmed that FITC-ECL specifically stains MO in the presence of high sugar competitor concentrations.

Polyclonal induction of immunoglobulin synthesis by feline leukocytes as identified in a reverse hemolytic plaque assay.

Optimal conditions of culture and assay for identification of feline immunoglobulin-secreting mononuclear cells were determined for the staphylococcal protein A-reverse hemolytic plaque assay (SpA-RHPA). Hemolytic plaques were most distinct and numerous when peripheral blood mononuclear cells were stimulated with 6.9 micrograms/ml pokeweed mitogen for 7 days. Immunoglobulin-secreting cells were identified morphologically within a zone of hemolysis utilizing a 1:5 dilution of rabbit anti-cat IgG and a 1:30 dilution of guinea pig complement as developing reagents. The SpA-RHPA system should contribute to an understanding of normal feline T- and B-lymphocyte interactions and will likely aid in the identification and understanding of immune cell dysfunctions associated with chronic feline leukemia virus infection.

Separation of feline bone marrow cells by counterflow centrifugal elutriation. Identification and isolation of presumptive early and late myeloid/erythroid progenitors.

Counterflow centrifugal elutriation (CCE) has been used to separate nucleated cells from mammalian bone marrow on the basis of size with the resultant isolation of hematopoietic cells in varying stages of lineage development. We examined the feasibility of identifying and isolating such cells from feline bone marrow. CCE was performed with a Beckman J6MI centrifuge and a Sanderson chamber, using a fixed rotor speed of 3000 rpm and collection of cells at (1) 16-, (2) 21-, (3) 25-, (4) 32 ml/min, and (5) a rotor off fraction. Recovery of the total input cells in four replicate experiments averaged 86%, with the maximum number of recovered cells in fraction 4. Analysis by flow cytometry and monoclonal antibodies revealed mononuclear cells in fractions 1 and 2 and early and late differentiating myeloid/erythroid cells in fractions 2 through 5. T lymphocytes and alloreactivity in a mixed lymphocyte reaction (MLR) were restricted to fractions 1 and 2; removal of T cells and MLR activity was accomplished by immunomagnetic depletion. In vitro cultures for clonogenic cells revealed CFU-GM and BFU-E colonies in fractions 2 through 5, with fraction 4 containing the greatest absolute number of myeloid colonies and fractions 3 and 4 the majority of the erythroid colonies. More important, in examining the plating efficiency for clonogenic cells in the different fractions it was found that this increased significantly in fractions 2 and 3 when the culture time was extended from 7 to 14 days; in contrast, fractions 4 and 5 reached their maximum plating efficiency within 7 days with no further increase on day 14. We interpret these findings to indicate the presence of late differentiating progenitors in the large-cell size fractions 4 and 5, while the smaller mononuclear cells in fractions 2 and 3 represent an earlier, more primitive population of hematopoietic cells requiring an extended time in culture for full colony development.

An evaluation of the mixed lymphocyte culture reaction in marmosets.

Marmosets are unique in that all members may be considered to be natural blood chimeras because of the high frequency of fraternal twinning and placental vascular anastomoses between the fetuses. The mixed lymphocyte culture (MLC) reaction utilizing blood lymphocytes was evaluated to determine whether this in vitro test could detect histocompatibility differences among related and unrelated marmosets. It was found that the responses could be correlated with the probable immunogenetic relationships of these animals. Thus, an allogeneic MLC reaction in which the responding (R) and stimulating (S) cells were obtained from unrelated animals within one subspecies of marmosets (Saquinus fuscicollis illigeri) yielded a lower response than a semi-xenogeneic reaction involving R and S cells from two different subspecies of marmosets (S.f. illigeri (R) versus S.f. lagonotus (S). In contrast, MLC reactions between cells from chimeric marmoset co-twins were essentially negative, indicating specific immune tolerance. Under certain experimental conditions, however, it was suggested that the dual lymphocyte cell population from a chimeric marmoset could undergo "autostimulation" as measured by label incorporation studies. The necessity of further experiments was recognized before the data could be truly accepted as reflecting an in vitro manifestation of incompatibility between the two different genetic populations of blood lymphocytes from a chimera.

Effect of antithymocyte globulin pretreatment on immunological reconstitution of lethally irradiated animals.

Clinical and experimental studies have shown that antithymocyte globulin pretreatment will reduce the severity of the graft-versus-host reaction after allogeneic bone marrow transplantation. To determine whether this was attributable to a persisting cytotoxic factor in the recipients' sera or a result of the "masking" of foreign antigens by the antihymocyte globulin, an experimental schema utilizing a syngeneic transfer of immunocompetent cells was devised. Lethally irradiated mice that had been pretreated with rabbit antimouse thymocyte globulin (RAMTG) were injected with syngeneic spleen or bone marrow cells and their immunological competence was measured by the response to a test antigen, sheep red blood cells. It was found that such pretreatment had an adverse effect on the immunological potential of the infused cells. Thus, the plaque-forming cell response to sheep red blood cell antigen in RAMTG-pretreated recipients injected with spleen cells was reduced when compared to the saline or normal rabbit globulin-treated controls. The immunological recovery of lethally irradiated animals protected with syngeneic bone marrow was also delayed, but not permanently impaired, when the recipients had been pretreated with RAMTG. These effects were evident although the spleen or bone marrow cells had been injected into the RAMTG-pretreated recipients at a time when their sera were devoid of any cytotoxic antibodies. It is speculated that two mechanisms may contribute to this alteration in immune function of the infused cells: (1) that RAMTG exists in the serum in amounts sufficient to attach to receptor sites on lymphocytes or their precursors but insufficient to kill the cells, interfering with the antigen recognition mechanism or migration and homing patterns, or (2) that the RAMTG treatment creates a temporary defect in the microenvironment of the spleen and other hemopoietic tissues, thereby affecting the transplantation and proliferation kinetics of the infused cells.

Functional evaluation of T helper, T suppressor, and B lymphocytes in lethally irradiated rhesus monkeys injected with autologous bone marrow.

Lethally irradiated rhesus monkeys were treated with autologous bone marrow that had either been (1) nontreated, i.e., normal; (2) depleted of T lymphocytes with a monoclonal antibody directed against rhesus T lymphocytes; (3) fractionated with the soybean agglutinin (SBA- fraction); or (4) fractionated with SBA and further depleted of T cells by E-rosetting. There was no difference in hematologic reconstitution among the animals, but all showed a marked lowering of the T helper/T suppressor ratio during the first 10 months posttransplant and reduced capability of their peripheral blood leukocytes (PBL) to produce Ig upon stimulation with pokeweed mitogen. This subnormal ability of PBL to produce Ig, as measured by plaque-forming cells in a reverse hemolytic plaque assay, was not explained entirely by the altered T-H/T-S ratio but was correlated with the functional status of the T-H, T-S, and B lymphocytes. Isolated populations of the different lymphocyte subsets from PBL of the experimental animals were cocultured with normal cells of the appropriate subset to obtain Ig synthesis when stimulated with PWM. Animals treated with normal bone marrow showed recovery of T-H cell function after 5 months, but their T-S cells showed excessive suppressor activity that persisted for 20 months posttransplant. In contrast, those animals receiving treated marrow (mAb plus complement, or SBA) showed a much-delayed (12 months or more) return to normal T-H cell function and an earlier return of T-S cells expressing a normal level of suppressor activity. Since the SBA- fraction of marrow contains very few or no T-H cells and T cell depletion of marrow with mAb also removes these cells, it is suggested that the kinetics of immune recovery of the different lymphocyte subsets of PBL is influenced by the presence or absence of T-H cells in the marrow inocula.

Development and characterization of monoclonal antibodies to lymphocyte cell surface antigens of the rhesus monkey.

Three monoclonal antibodies directed against rhesus lymphocyte cell surface antigens are described. A pan-T mAb, T64, and a T suppressor mAb, T35, showed phenotypic and functional specificity for both human and rhesus cells. In contrast, a third mAb, N42, identifying natural killer cells in rhesus peripheral blood leukocytes, was not crossreactive with the corresponding homologous human cells. N42 reacted with the same cells identified by Leu 11a and Leu 11b in rhesus PBL and in a functional assay decreased NK activity by 80%. N42 precipitated a 50KD protein from rhesus PBL lysates and was reactive with the Fc receptor domain of the NK cell. The T-S functional activity of cells reactive with mAb T35 was demonstrated in a pokeweed-mitogen-driven system for Ig synthesis: removal of the T35 positive cells by complement-mediated lysis led to an enhanced production of Ig by rhesus PBL, and the addition of T35 positive cells to a culture of T helper and B cells resulted in a reduction of this response. T35 was determined to be an IgG2a immunoglobulin and precipitated a 34KD protein from rhesus cell lysates. An IgM immunoglobulin, mAb T64 delineated all T lymphocytes, inhibited E-rosette formation, interfered with the proliferation of cells stimulated with mitogens, and precipitated a 52KD membrane protein. The potential utilization of these mAbs in vivo for organ or tissue transplantation in the rhesus monkey is discussed.

Relative sedimentation of hematopoietic progenitors in human cord blood, peripheral blood, and bone marrow as determined by counterflow centrifugal elutriation.

BACKGROUND: The current use of cord blood (CB) and peripheral blood (PB) stem cells as alternatives or adjunctives to bone marrow (BM) for hematopoietic reconstitution in the treatment of various diseases prompted an examination of the progenitors of these tissues by counterflow centrifugal elutriation (CCE). METHODS: The cells, obtained from normal donors not primed with colony-stimulating factors, were centrifuged at 3000 rpm in a Beckman Sanderson Chamber. Fractions (Frs.) were collected at (1) 18 ml/min, (2) 25 ml/min, (3) 32 ml/min, (4) 40 ml/min, and (5) the rotor-off fraction. RESULTS: Clonogenic assays revealed differences in the fraction localizations for CB and PB when compared to BM, i.e., recovery of the colony-forming units for CB and PB was greater in the small-medium cell size CCE fractions, and those from BM were found primarily among the medium-large cell size fractions. Thus, although colony-forming unit granulocyte/macrophage colonies were distributed throughout Frs. 2-5 of BM, CB and PB showed 80% of the total to be in Frs. 2 and 3. Further, although burst-forming unit erythroid colonies of BM were distributed equally in Frs. 2 and 3, greater than 70% of the total burst-forming unit erythroid colonies in CB and PB were found in Fr. 2. Distribution of the CD34 cells in the fractions correlated with the colony-forming units in that these were found primarily in Frs. 2 and 3 of CB and PB, whereas they were present in significant numbers throughout Frs. 1-5 of BM. CONCLUSIONS: We interpret these findings to indicate CB and PB to be qualitatively similar in their hematopoietic lineage development and to contain a greater proportion of early versus late progenitors relative to those found in BM.

Fractionation of feline bone marrow with the soybean agglutinin lectin yields populations enriched for erythroid and myeloid elements: transplantation of soybean agglutinin-negative cells into lethally irradiated recipients.

Feline bone marrow cells treated with the soybean agglutinin (SBA) lectin are separated into two populations, the agglutinated SBA(+) fraction containing predominantly cells of myeloid origin and the nonagglutinated SBA(-) fraction consisting of cells primarily of the erythroid lineage. FACScan analyses revealed a clear distinction of the cells based on their light scattering properties, i.e., large cells and cells with high granularity were found in the SBA(+) fraction, whereas cells having a low forward light scatter and side light scatter were found in the SBA(-) fraction. Colony-forming assays showed colony-forming unit-granulocyte/monocyte (CFU-GM) cells to have a strong affinity for SBA because these were found almost entirely in the SBA(+) fraction; in contrast, burst-forming unit-erythroid (BFU-E)-forming cells were concentrated in the SBA(-) fraction. When the marrow was fractionated by counterflow centrifugal elutriation (CCE), a differential binding to SBA among the CFU-GM forming cells was found. The SBA(-) fractions of cells collected at 21 and 25 ml/min contained primarily BFU-E forming cells, similar to that observed with whole marrow; the later CCE fractions, those collected at 32 ml/min and the rotor off fraction, when treated with SBA showed a small but significant number of CFU-GM cells in the SBA(-) fraction. T lymphocytes were found predominantly in the SBA(+) fractions of whole bone marrow and the CCE fractions. Successful autologous marrow transplants were performed with the early CCE SBA(-) fractions. The latter cells were used for our initial transplant attempts because ongoing studies in our laboratory had shown these cells to be free of any viral-containing cells when the marrow had been obtained from animals infected with the feline immunodeficiency virus. In summary, although SBA treatment of feline marrow yields a marked separation of CFU-GM and BFU-E progenitors, select CCE SBA(-) fractions contain stem cells capable of providing hematopoietic reconstitution of lethally irradiated animals.

Radiation-induced hemopoietic death in mice as a function of photon energy and dose rate.

Radiation-induced hemopoietic death was measured in mice exposed to photons of four different energies: 250-kVp X rays, 60Co gamma rays (1.25 MeV), and 6- and 25-MV photons from a linear accelerator. For each radiation source, the lethal dose which killed 50% of the population in 30 days (LD50/30) associated with the hemopoietic syndrome was determined in groups of mice exposed to graded doses from 600 to 1150 cGy at dose rates of 20, 40, and 80 cGy/min. The calculated LD50/30 values for 25 and 6 MV were significantly different from each other at all exposure rates while no difference was observed between 6 MV and 60Co. Using 60Co gamma rays as the standard, the relative biologic effectiveness was as follows: 250 kVp greater than 25 MV greater than 6 MV = 60Co. The data suggest that there may be a greater damage to tissue within the marrow cavities following exposure to very high megavoltage radiation, a factor which must be considered with the increasing utilization of linear accelerators in the clinic and laboratory.

Development of monoclonal antibodies to erythroid progenitors in feline bone marrow.

Feline bone marrow cells can be enriched for erythroid and myeloid progenitors by counterflow centrifugal elutriation (CCE) and subsequent treatment of the CCE fractions with the soybean agglutinin (SBA) lectin. Separation of CCE fractions into SBA(-) and SBA(+) populations yielded cells enriched for BFU-E and CFU-GM progenitors, respectively. Differential analyses revealed a high percentage of erythroid lineage cells in the SBA(-) fractions and in the SBA(+) fractions a high concentration of myeloid cells of varying maturation stages. The latter cells, but not the CFU-GM progenitors, could be removed by immunomagnetic depletion from CCE fractions using a monoclonal antibody (MAb) specific for CD13 cells in feline bone marrow, resulting also in a population containing predominantly erythroid differentiating cells. Mice were immunized with CCE fractions enriched for erythroid lineage cells and the splenocytes fused with SP2/O cells for hybridoma development. Supernatant culture fluids from 400 hybridomas were analyzed by flow cytometry with whole bone marrow and select CCE/SBA fractions as the target cells. Those hybridomas suggestive of containing the desired antibodies as indicated by the percentage staining were subcloned and the MAbs utilized in clonogenic assays. Treatment of bone marrow cells or CCE fractions with the MAbs followed by immunomagnetic depletions led to identification of two, K-1 and Q-3, reactive with the BFU-E progenitor and one, K-7, reactive only with late-differentiating erythroid lineage cells. Thus, removal of cells from a CCE/SBA suspension reactive with K-1 or Q-3 led to greater than 80% reductions of the BFU-E progenitors and a population enriched for CFU-GM. Removal of cells reactive with MAb K-7, however, led to a marked enrichment, 5-8-fold, of both BFU-E and CFU-GM progenitors.

Feline bone marrow transplantation: its use in FIV-infected cats.

The use of autologous and allogenic bone marrow transplantations (BMT) in FIV-infected and uninfected cats is a novel therapy for feline hematopoietic diseases and retroviral infections. A total of 13 specific pathogen-free (SPF) cats received either autologous or allogenic BMT and seven of these cats were also infected with FIV before autologous or allogenic BMT. All BMT recipients received total body irradiation of 900 cGy just before BMT. Two FIV-infected and four uninfected cats received autologous uninfected BM cells cryopreserved before BMT. Five infected and two uninfected cats received BM cells from allogenic uninfected donors (RBC-, MHC-, and cross-matched). MHC-matching was based on mixed leucocyte reaction (MLR) and the donor-recipient combination which was compatible by MLR analysis, was used in this study. Recipients were monitored for hematology, immunology, virology, and clinical signs. All FIV-infected and uninfected recipients of autologous BMT had complete engraftment with minimal complications. Uninfected recipients of allogenic BMT had a more severe clinical episode with slower rate of engraftment. None of these BMT groups had mortality. In contrast, only two of the five infected recipients of allogenic BMT survived for a significant period of time (23 and 50 weeks) and rest of the cats succumbed to transfusion reactions. Both infected BMT groups had persistent CD4/CD8 inversion, low CD4+ cell counts, and FIV infection of engrafted peripheral blood mononuclear cells (PBMC). Overall, successful autologous and allogenic BMTs were performed in FIV-free cats. All infected recipients of autologous BMT had compete engraftment and are currently alive, with thelongest survival time being over 1 year. Thus, BMT in combination with antiviral drug therapies may be an alternative therapy against retroviral infection.

Epoxide hydrolase: a marker for experimental hepatocarcinogenesis.

Rat liver chemical hepatocarcinogenesis, induced by interrupted feeding of 2-acetylaminofluorene, results in various cellular preneoplastic stages and finally in a hepatoma in about 70 to 90 percent of the rats. The putative precursors of hepatomas, called hyperplastic nodules, appear after 12 weeks of feeding and, after 16 weeks of feeding carcinogen, most of them are persistent. Epoxide hydrolase is a tightly bound endoplasmic reticulum enzyme which is strongly induced in hyperplastic nodules and hepatomas. This enzyme has been purified, high-titre rabbit antiserum prepared to it, and this antiserum used to search for epoxide hydrolase immunodeterminants in the sera from chemically induced nodule or hepatoma bearing rats. An enzyme linked immunosorbent assay (ELISA) assay using this antiserum showed significant titres of circulating microsomal epoxide hydrolase antigen, range 0.01 to 2.50 (mean 1.18 +/- 0.30) micrograms per ml, in all 24 hepatoma bearing rats tested. Eight sera from animals with large hyperplastic nodules were also significantly positive for this antigen, while sera from six normal controls were negative (less than 0.004 microgram per ml serum). A passive hemagglutination inhibition assay with sheep or normal rat red blood cells sensitized with pure rat microsomal epoxide hydrolase was capable of detecting 0.2 microgram hydrolase per ml serum. With this assay, sera from four rats with hepatomas were found to contain 0.8 to 1.6 micrograms epoxide hydrolase immunodeterminants per ml. Control rat sera had no detectable immunodeterminants. Thus epoxide hydrolase, a marker induced during experimental chemical hepatocarcinogenesis and called the preneoplastic antigen, has been shown to be circulating in the tumor bearing host.