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.

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.

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.

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.

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.

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.

Influence of dimethyl myleran on tolerance induction and immune function in major histocompatibility complex-haploidentical murine bone-marrow transplantation.

To study murine major histocompatibility complex (MHC)-haploidentical bone-marrow transplantation (BMT), B6C3F1 mice (H-2b/k) underwent BMT using syngeneic [B6C3F1 (H-2b/k)], haploidentical [CB6F1 (H-2d/b)], or fully allogeneic [DBA/2 (H-2d)] donor mice. As pretreatment, dimethyl myleran (DMM), an alkylating agent that produces effective myeloablation but little immunosuppression, was used with total body irradiation (TBI). Four conditioning regimens were studied: TBI 800 rads (1 rad = 0.01 Gy), TBI 950 rads, TBI 800 rads plus DMM (0.2 mg per mouse), and TBI 950 rads plus DMM. Survival rates, chimerism, proliferative responses in mixed-lymphocyte culture, specific cell-mediated lympholysis, and in vivo plaque-forming cell responses to several antigens were compared. TBI 800 rads plus DMM was maximally effective. Haploidentical BMT was as successful in inducing long-term survival and immune and hematologic reconstitution as was syngeneic BMT. This regimen plus haploidentical BMT of T-cell-purged marrow yielded survivors tolerant of donor and recipient major histocompatibility complex. Such myeloablation and immunosuppression prevented graft rejection, immunodeficiency due to histoincompatibility, and damage to a radiosensitive cell population. A microenvironmental influence crucial to some antibody responses was thus revealed. Delayed recovery of antibody production after BMT in humans may be due partly to suboptimal myeloablation or excess irradiation.

Phenotypic markers for the feline monocyte: rosette formation with human erythrocytes and a monoclonal antibody which binds myeloid cells.

A small population of cells with the ability to form rosettes with human erythrocytes was found in feline peripheral blood leukocytes (PBL) (10%) and bone marrow (9%), but not in purified granulocyte preparations, thymus, and lymph node tissues. The morphologic appearance and ability to phagocytize latex beads indicated these cells were monocytes. A monoclonal antibody, CM277, with a binding specificity for feline peripheral blood phagocytes was also characterized. Immunofluorescent microscopy revealed CM277 to bind specifically to monocytes and polymorphonuclear neutrophils. The binding of CM277 to monocytes was also shown by human erythrocyte-rosette formation wherein there was a high degree of correlation between these two phenotypic markers for cells ingesting latex beads. Monocytes, polymorphonuclear neutrophils, and T lymphocytes of the cat rosette with guinea pig erythrocytes (GPE) and using CM277 we were able to determine the contribution of the former two cell types to the GPE-rosetting population. Monocytes and polymorphonuclear neutrophils comprised the majority of the GPE-rosetting cells in fresh PBL (greater than 60%), but after culturing overnight, there was a substantial decrease in these cells (less than 35%). In contrast, GPE-rosetting T lymphocytes comprised approximately 10% of the cells in fresh PBL, and after in vitro culture for 1 day they constituted 35-45% of all cells. The removal of monocytes by human erythrocyte-rosetting did not affect the pokeweed mitogen-induced synthesis of Ig, but did lead to an increased production of interleukin 2. Removal of the GPE-rosetting population from PBL resulted in a marked decrease in interleukin 2 production, pointing to a positive contribution of GPE-rosetting T lymphocytes to the synthesis of this lymphokine.

Two populations of guinea pig erythrocyte-rosetting cells in the cat: evidence for their T-helper function in mitogen-induced synthesis of Ig and interleukin-2.

Studies in our laboratory have shown that T-helper (T-H) and T-suppressor (T-S) cells in cat peripheral blood leukocytes (PBL) rosette with guinea pig (GP) and gerbil (G) erythrocytes (E), respectively. Removal of GE-rosetting cells leads to an enhanced (two- to threefold) synthesis of Ig in a pokeweed mitogen (PWM)-driven system as measured by plaque-forming cells (PFC) to protein A-coated sheep RBC, while depletion of GPE-rosetting cells yields a PFC response only 10-15% of the control. Surprisingly, removal of both GE- and GPE-rosetting cells gave a response equivalent to 40-100% of the control PBL. Analysis of the mixed GE/GPE rosette depleted cultures revealed the reappearance of GPE- but not GE-rosetting cells, reaching maximum values within 12-18 hr after in vitro culture. Cultures of control PBL and those following the mixed rosette depletion showed two populations of GPE-rosetting cells; the GPE-1 cells, present on Day 0 before culture, and the GPE-2 cells, those appearing on Day 1. Addition of cycloheximide prevented development of the GPE receptor while colchicine and mitomycin C were without effect. The development of PFC after the mixed GE/GPE rosette depletion was interpreted as being due to the GPE-2 cells functioning as T-H cells in the absence of any T-S (GE-rosetting) cells. This thesis was supported by showing a marked decrease in the PWM-induced Ig response when both the GPE-1 and GPE-2 populations were removed on Day 1. Additional evidence for functional T-H cells in the GPE-rosetting population was obtained by analyzing interleukin-2 (IL-2) production. Removal of the GPE-rosetting cells (GPE-1 and/or GPE-2) from PBL led to a marked decrease in Con A-induced IL-2 synthesis while removal of the GE-rosetting cells yielded a normal or slightly greater than normal response.

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.

Enrichment of natural suppressor activity in a wheat germ agglutinin positive hematopoietic progenitor-enriched fraction of monkey bone marrow.

Natural suppressor (NS) activity, the capacity of unprimed cells to suppress immunologic responses, is present in mouse, rabbit, and human bone marrow (BM). In this study we characterize NS activity in bone marrow cells of the rhesus monkey. Greatest NS activity was found in low-density cells (1.0600 to 1.0655 g/mL) obtained by density centrifugation on a discontinuous Percoll gradient. NS activity was further enriched when cells were separated by affinity for wheat germ agglutinin (WGA). Cells with high affinity to WGA demonstrated potent NS activity, whereas cells with low affinity to WGA had no NS activity. A significant relationship between NS activity and hematopoietic activity was demonstrated using in vitro assays of colony formation (CFU-GM and CFU-MIX). NS activity was not affected by treatment with monoclonal antibodies (MoAbs) to human Fc gamma receptors (Leu, 11a,b,c) or treatment with MoAbs to monkey natural killer cells. These findings extend our prior observations by showing that cells with NS activity, which apparently have WGA receptors, are present not only in murine BM but also in monkey bone marrow, and suggest that such cells may be involved in immunoregulation by primitive cells of BM.

Guinea pig and gerbil erythrocytes rosette with different cells in the blood, bone marrow, and thymus of the cat.

Cat thymocytes, bone marrow cells, and peripheral blood leukocytes (PBL) formed rosettes with guinea pig (GP) and gerbil (G) erythrocytes (E). In PBL from adult cats the frequency of rosettes was 27% with GPE and GE, while an average of 33% bone marrow cells formed rosettes with GPE and only 4% with GE. Thymocytes from kittens showed a high percentage of rosettes with both GPE and GE (35 to 81%), with the frequency of each type varying with the thymus tested. Fluorescein isothiocyanate labeling of one of the erythrocyte species revealed these cells to be rosetting with different nucleated cells; i.e., a low percentage (3-5%) of the rosettes formed with PBL and bone marrow had both labeled and unlabeled erythrocytes. In contrast, "mixed" rosettes were observed with a significant number of thymocytes, averaging 33% of thymocytes from six animals. A further distinction between the GE- and GPE-rosetting cells was revealed by a monoclonal antibody which blocked GE rosette formation without interfering with the binding of GPE to PBL and thymocytes. PBL could be depleted of either GPE- or GE-rosetting cells, with retention of IgG+ cells and cells capable of rosetting with the second erythrocyte species in the nonrosetting fractions. Stimulation of the latter nonrosetting fractions with pokeweed mitogen for induction of Ig synthesis revealed a T-lymphocyte specificity of the GE- and GPE-rosetting cells. PBL depleted of GE-rosetting cells yielded an increased Ig production, two- to threefold above the control; in contrast, depletion of GPE-rosetting cells from PBL resulted in a failure of the remaining cells to respond. These results suggest that T-suppressor cells of the cat are contained in the GE-rosetting fraction and T-helper cells are rosetted with GPE.

Natural killer cells in the blood and bone marrow of the rhesus monkey.

Natural killer (NK) cells in peripheral blood lymphocytes (PBL) and bone marrow (BM) cells of the rhesus monkey were detected by their functional activity against K562 cells. Animals could be grouped into "high" or "low" NK responders, a trait found to be consistent over a period of 2 years. NK active cells in PBL were in the nonadherent population, with the majority bearing Fc receptors and a further subdivision of these into CR+ (complement receptor) and CR- NK cells. Of 10 monoclonal antibodies directed against different epitopes of human lymphocytes, OKT11, OKT10, and Leu 11 showed reactivity with rhesus NK cells. Only OKT10 was reactive with the effector site of the cell, as shown by its capability to block NK function. Of the Leu 11 monoclonal antibodies (a, b, c), Leu 11c was nonreactive while Leu 11a and Leu 11b were shown by immunofluorescence to bind to 7 to 21% of PBL; Leu 11b was also cytotoxic to the NK cells. Leu 11b did not prevent binding of Leu 11a to PBL, suggesting reactivity of these antibodies with different epitopes. Percoll fractionation of PBL and BM revealed a greater enrichment of NK activity with BM; also, with PBL peak NK activity occurred in fractions 4 and 5 while this occurred in fraction 5 with BM. Although Percoll PBL fractions contained a higher percentage of Leu 11b cells, the NK activity of the BM fractions was proportionately greater. The majority of PBL cells with NK activity were FcR+ while significant activity could be attributed to FcR- cells of BM, in both the unseparated and Percoll fractions of each tissue. The data suggest NK active cells of BM may be distinct from those found in PBL.

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.

Suppression of humoral and cell-mediated immune responses in vitro by benzo(a)pyrene.

The effect of benzo(a)pyrene (BaP) at different molar (M) concentrations on the in vitro anti-sheep red blood cell (SRBC) plaque (antibody) forming cell (PFC) response and the one-way mixed lymphocyte response (MLR) was tested. Inhibition of the PFC response and the MLR occurred when spleen cells were exposed to a wide range of BaP concentrations from 10(-4) M to 10(-8) M. Maximum depression of the responses occurred at 10(-5) M for PFC production (47% of controls) and for the MLR (19% of controls) as measured by a stimulation index. No significant loss in cell viability was observed at this or lower molar concentrations of BaP. The non-carcinogenic analog of BaP, benzo(e)pyrene, did not suppress PFC responses at comparable concentrations. This in vitro system will facilitate manipulations of T and B lymphocytes and macrophages (adherent cells) in a controlled culture environment for precisely characterizing the sensitivity of these cells and their subpopulations on exposure to BaP.

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.

Subnormal expression of cell-mediated and humoral immune responses in progeny disposed toward a high incidence of tumors after in utero exposure to benzo[a]pyrene.

Pregnant mice were exposed to 150 micrograms benzo[a]pyrene (BaP) per gram of body weight during fetogenesis (d 11-17 of gestation) and the progeny were assayed for humoral and cell mediated immune responses at different time intervals after birth. Immature offspring (1-4 wk) were severely suppressed in their ability to produce antibody-(plaque-) forming cells (PFC) against sheep red blood cells (SRBC) and in the ability of their lymphocytes to undergo a mixed lymphocyte response (MLR). Lymphocytes from these progeny showed a moderate to weak capacity to inhabit production of colony-forming units (CFU) in host spleens following transfer with semiallogeneic bone marrow (BM) cells into lethally X-irradiated recipients syngeneic to the BM (in vivo graft-versus-host response, GVHR). A severe and sustained suppression in the MLR and the PFC response occurred from the fifth month up to 18 mo. The in vivo GVHR, also subnormal later in life, was not as severely suppressed as the other two parameters. Tumor incidence in the BP-exposed progeny was 8- to 10-fold higher than in those encountering corn oil alone from 18 to 24 mo of age. These data show that in utero exposure to the chemical carcinogen BaP alters development of components needed for establishing competent humoral and cell-mediated functions of the immune apparatus and leads to severe and sustained postnatal suppression of the defense mechanism. The immunodeficiency exhibited, particularly in the T-cell compartment (MLR, GVHR), before and during the increase in tumor frequency, may provide a favorable environment for the growth of nascent neoplasms induced by BaP.

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.