The viability of cells obtained using the Reamer-Irrigator-Aspirator system and in bone graft from the iliac crest.

We hypothesised that cells obtained via a Reamer-Irrigator-Aspirator (RIA) system retain substantial osteogenic potential and are at least equivalent to graft harvested from the iliac crest. Graft was harvested using the RIA in 25 patients (mean age 37.6 years (18 to 68)) and from the iliac crest in 21 patients (mean age 44.6 years (24 to 78)), after which ≥ 1 g of bony particulate graft material was processed from each. Initial cell viability was assessed using Trypan blue exclusion, and initial fluorescence-activated cell sorting (FACS) analysis for cell lineage was performed. After culturing the cells, repeat FACS analysis for cell lineage was performed and enzyme-linked immunosorbent assay (ELISA) for osteocalcin, and Alizarin red staining to determine osteogenic potential. Cells obtained via RIA or from the iliac crest were viable and matured into mesenchymal stem cells, as shown by staining for the specific mesenchymal antigens CD90 and CD105. For samples from both RIA and the iliac crest there was a statistically significant increase in bone production (both p < 0.001), as demonstrated by osteocalcin production after induction. Medullary autograft cells harvested using RIA are viable and osteogenic. Cell viability and osteogenic potential were similar between bone grafts obtained from both the RIA system and the iliac crest.

Antimelanoma antibodies in swine with spontaneously regressing melanoma.

Sinclair swine provide a unique model for studying mechanisms of tumor regression because they are born with melanomas that spontaneously regress approximately 10 weeks after birth. To examine whether an antitumor immune response is present in these animals, and, if so, to study its relation to tumor regression, 38 sera specimens collected at different times from 13 swine born with melanomas were tested for melanoma antibodies by immunoprecipitation and SDS-PAGE analysis of 125I labelled swine melanoma macromolecules. Antibodies to melanoma were present in 13 (100%) of the swine versus 1 of 3 control swine. The antibodies were directed to antigens of approximately 45, 68-75, or 100 kDa. These antigens were also expressed on human melanomas and normal melanocytes but on only one of five unrelated tumors. The incidence and level of these antibodies increased with time. Antibodies to the 45, 68-75, and 100 kDa antigens were present in 36%, 55%, and 9%, respectively, of sera collected prior to 7 weeks of age, but in 80%, 100%, and 37% of sera collected between 7 and 20 weeks (P < 0.05). The rise in melanoma antibodies usually preceded or appeared together with tumor regression and loss of pigmentation. These findings indicate that Sinclair swine with melanomas have antibodies to antigens preferentially expressed on pigment cells, and support the hypothesis that the regression phenomenon and the vitiligo-like skin depigmentation result from immune responses to common antigens shared by normal and malignant swine pigment cells.

GM-CSF is required for the Pseudomonas exotoxin A-induced proliferation of immature T cells in athymic mice.

Previous studies have demonstrated that Pseudomonas exotoxin A stimulated the proliferation of immature T lymphocytes within the splenocytes of athymic mice. These studies were performed to determine which lymphokines were involved in the proliferation of the immature T cells. The results of this study indicate that exotoxin A does not induce the production of interleukin-2 or tumor necrosis factor from B cell-depleted splenotypes from athymic mice. However, exotoxin A does induce the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) from B cell-depleted splenocytes. Furthermore, the GM-CSF was shown to be produced by a Thy1+, CD4-, CD8- T lymphocyte. The addition of anti-GM-CSF antibody abrogates the exotoxin A-induced proliferation of B cell-depleted splenocytes from athymic mice. Thus, these data indicate that exotoxin A induces the production of GM-CSF from immature T lymphocytes within the splenocytes of athymic mice and the exotoxin A-induced proliferation of these immature T cells is dependent on the presence of GM-CSF.

Proliferation of immature T cells within the splenocytes of athymic mice by Pseudomonas exotoxin A.

Pseudomonas aeruginosa exotoxin A has been shown to stimulate splenocytes from athymic nude mice. The present studies were performed to characterize the exotoxin A-responsive cells within the splenocytes of athymic nude mice. The results of these studies indicate that exotoxin A-responsive cells were represented in the nylon wool-adherent cell population and in the Ig-depleted splenocyte population (IgNA). Flow microfluorimetry analysis indicated that exotoxin A induced the expansion of Thy1+, CD3-, CD4-, and CD8- cells which also expressed the heat-stable antigen (HSA), an antigen expressed on immature T lymphocytes. Depletion of Thy1+ cells abrogated the exotoxin A-induced proliferation; however, depletion of CD4+ and CD8+ cells did not affect the exotoxin A-induced proliferation of IgNA cells. Thus, the results indicate that exotoxin A stimulates the proliferation of immature T cells within the splenocytes of nude mice that are Thy1+, HSA+, CD3-, CD4-, CD8-.

Sinclair miniature swine: an animal model of human melanoma.

Sinclair swine display cutaneous melanoma lesions and develop a generalized depigmentation subsequent to tumor regression. Sinclair swine represent a valuable animal model to study the factors influencing the development of melanoma and also the factors which lead to the development of vitiligo. Therefore, information obtained in studies of Sinclair swine should facilitate our understanding of the mechanisms by which melanoma and vitiligo develop and provide us with possible therapeutic treatments for these human diseases.

Partial cloning and sequencing of the gene encoding the porcine T-cell receptor delta-chain constant region.

The polymerase chain reaction (PCR) was utilized to clone the pig T-cell receptor (TCR) delta-chain constant region-encoding gene (C delta). A cDNA was generated from total RNA preparations of normal pig peripheral blood lymphocytes (PBLs) and a miniature pig peripheral blood cell line (PBLCL 62.G4). The cDNA was used to amplify the porcine TCR C delta gene by PCR using primers chosen by comparing other known C delta sequences for sequence identity. Clones were sequenced and used to determine the primary structure of the porcine TCR C delta chain. A comparison of the nucleotide and deduced amino acid (aa) sequences with the known human, mouse, sheep and cattle sequences revealed that the primary structure of the pig TCR C delta chain has been highly conserved. The immunoglobulin (Ig) domain has two conserved Cys residues and contains a high degree of sequence identity, whereas the hinge region is marked by a high level of diversity. The transmembrane and cytoplasmic regions are also highly conserved, including the presence of the two basic aa, Arg and Lys, in the transmembrane domain. Southern blot analysis has confirmed the presence of one TCR C delta gene in the porcine genome, consistent with similar findings in other species. Thus, the successful cloning and sequencing of the porcine TCR C delta gene should facilitate our understanding of the role of gamma delta T-lymphocytes in the swine immune system.

Isolation and characterization of gamma delta T lymphocyte cell lines from Sinclair swine peripheral blood.

Sinclair miniature swine represent a breed of miniature swine which display a significant incidence of inheritable melanoma which undergo a developmentally regulated spontaneous regression. In an attempt to characterize the host cellular immune response to the melanoma, lymphocyte cell lines have been generated from peripheral blood and designated as peripheral blood lymphocyte cell lines (PBLCLs). The cell lines were expanded in vitro without the addition of exogenous mediators, cloned by limiting dilution, and characterized by flow microfluorimetry, Western, and Northern blot analysis. The cell lines were shown to be CD2-, CD4-, CD8-, and slg-, a phenotype consistent with a null cell population described in swine. The null cell population in swine has been reported to consist of a subpopulation of cells which express the gamma delta T cell receptor (TCR) heterodimer, swine gamma delta T lymphocytes. The PBLCLs were further analyzed by flow microfluorimetry and observed to express the IL-2R, swine MHC Class II antigens, and the endothelial lymphocyte adhesion marker (CD44), which can function as a homing receptor for the skin. In addition, the PBLCLs were observed to express the antigen which is recognized by mAb 86D, an antibody that has been reported to recognize an external epitope on a subset of gamma delta TCR bearing swine T lymphocytes. Western blot analysis of Triton X-114 phase fractions of a PBLCL revealed a protein recognized by the W6 antibody, an antibody which recognizes a conserved region of the C delta chain. Furthermore, Southern and Northern blot analysis indicated that the PBLCL have rearranged the TCR gamma chain gene and express mRNA from the TCR gamma and delta chain genes prior to and following treatment with ionomycin or Concanavalin A. Therefore, the data indicates that the PBLCLs represent swine gamma delta T lymphocyte cell lines which should enable us to enhance our understanding of the role of gamma delta T lymphocytes in the porcine immune system.

Evaluation of the mitogen-induced proliferation and cell surface differentiation antigens of lymphocytes from pigs 1 to 30 days of age.

A study was conducted to characterize aspects of the immune system in the pig from 1 to 30 d of age. Pigs were killed on d 1 (n = 6), d 18 or 19 (n = 6), and d 27 to 30 (n = 7) of age. Lymphocytes were isolated from the blood, thymus, and spleen. Lymphocyte function was assessed for ability to proliferate as induced by concanavalin A (Con A), phytohemagglutinin (PHA), and pokeweed mitogen (PWM). Cell surface differentiation antigens on the lymphocytes were evaluated for percentages of cells expressing CD2+, CD4+, CD8+, and SLA class II molecules. Responses of lymphocytes from blood, thymus, and spleen to any of the mitogens were relatively low d 1 through 16; the greatest proliferation occurred by d 28. No detectable percentages of the cell surface differentiation antigens were found on d 1 and changes varied with age and organ. The results indicate that the porcine immune system is not fully developed at birth and the expression of cell surface differentiation antigens seems to occur before the lymphocytes have the ability to respond to mitogens.

Selective activation of murine V beta 8.2 bearing T cells by Pseudomonas exotoxin A.

We have determined that Pseudomonas aeruginosa exotoxin A (PE) can selectively stimulate the proliferation of V beta bearing T lymphocytes. Murine thymocytes were fractionated by selective agglutination with peanut agglutinin (PNA) and the PNA- thymocytes, which represent mature thymocytes, were shown to be responsive to PE stimulation. In addition, mature peripheral T lymphocytes (nylon wool nonadherent splenocytes) were also observed to respond to PE stimulation. Both CD4+ and CD8+ splenic T lymphocyte populations proliferated in response to PE. Flow microfluorimetry analysis of PNA- thymocytes stimulated with PE indicated that V beta 8.2 bearing T cells were preferentially expanded. Thus, our data indicate that PE represents a microbial super antigen which stimulates murine thymocytes which bear the V beta 8.2 element of the T cell receptor.

Dietary fat influences Ia antigen expression and immune cell populations in the murine peritoneum and spleen.

Peritoneal cells (PEC) and splenocytes were obtained from Listeria monocytogene (LM)-infected or noninfected mice fed a 20% fat diet rich in either (n-3) polyunsaturated fatty acids [(n-3) PUFA diet], linoleate [(n-6) PUFA diet], oleate (MONO diet), or saturated fatty acids (SAT diet) for 6 wk and were assessed for T cells, B cells, macrophages and Ia expression by flow cytometric analysis. In the peritoneum of noninfected mice, dietary fat did not affect total cell yield or the percentage of B cells, macrophages or Ia+ cells, but the (n-3) PUFA-fed group had a greater percentage of T cells than did the other groups. Among the LM-infected mice, the (n-3) PUFA-fed group generally had the highest percentage of B cells and the lowest percentages of T cells, macrophages and Ia+ cells in the peritoneum. Listeria monocytogene infection elevated peritoneal T cell numbers in all mice except the (n-3) PUFA-fed group. The density of Ia molecules on PEC was 40% lower in mice fed the (n-3) PUFA diet. In the spleen, dietary fat also influenced the immune cell populations and Ia+ cells. Two-color staining of spleen cells revealed that Ia+ splenocytes were predominately B cells. These data demonstrate that dietary fats influence Ia expression and immune cell populations and that the effects observed in one immune tissue or cell type may not be readily extrapolated to others.

Lymphoproliferative activity of Pseudomonas exotoxin A is dependent on intracellular processing and is associated with the carboxyl-terminal portion.

Pseudomonas aeruginosa exotoxin A (PE) represents a microbial superantigen that requires processing by accessory cells in order to induce the proliferation of V beta 8-bearing murine T lymphocytes. In this study, we have observed that PE requires intracellular processing by a protease in order to induce lymphoproliferation. Pepstatin A, an inhibitor of acid proteases, inhibited PE-induced lymphoproliferation, whereas leupeptin, an inhibitor of serine and thiol proteases, had no effect on PE-induced lymphoproliferation. A number of mutant forms of PE were examined for their ability to induce lymphoproliferation. The mutant form which lacks amino acids 5 to 224 of the receptor-binding domain, PE43, was capable of inducing murine thymocytes to proliferate in the presence of accessory cells. However, neither PEgly276, a mutant toxin which undergoes a different intracellular processing pattern than wild-type PE, nor PE589, a mutant toxin which lacks amino acids 590 to 613 at the carboxyl terminus, was able to induce thymocyte proliferation. In addition, the lymphoproliferation induced by the PE43 mutant form of PE could also be inhibited by pepstatin A. Therefore, our data indicate that intracellular processing by a proteolytic enzyme which is inhibited by pepstatin A is critical for PE-induced lymphoproliferation. Furthermore, the lymphoproliferative activity of PE is associated with the carboxyl-terminal portion of PE.

The superantigen Pseudomonas exotoxin A requires additional functions from accessory cells for T lymphocyte proliferation.

We have examined the functions required of accessory cells (AC) for murine thymocyte proliferation induced by Pseudomonas exotoxin A (PE) and have compared these functions to those required of a known superantigen, staphylococcal enterotoxin B (SEB). We demonstrate that PE, like SEB, preferentially stimulates PNA+ thymocytes expressing a specific V beta element within the T cell receptor. However, PE requires functions from AC that are distinct from those required by SEB. AC treated with paraformaldehyde (PCHO) prior to stimulation supported thymocyte proliferation induced by SEB but not PE. However, when AC were treated with PCHO subsequent to stimulation with PE, thymocyte proliferation was observed, which suggests that PE requires antigen processing in addition to presentation. Furthermore, treatment of AC with lysosomotropic agents abrogated thymocyte proliferation induced by PE but not SEB. Antibodies to MHC class II molecules inhibited thymocyte proliferation induced by both PE and SEB. In addition, we observed that interleukin 1 alpha (IL-1 alpha) participated in the proliferation of thymocytes induced by PE but not SEB. Thus, our data indicate that PE is a unique microbial superantigen that requires additional AC functions for T lymphocyte proliferation.

Induction of interleukin-1 from murine peritoneal macrophages by Pseudomonas aeruginosa exotoxin A.

Pseudomonas exotoxin A, an ADP-ribosylating toxin produced by Pseudomonas aeruginosa, has been shown to stimulate the proliferation of murine thymocytes, which requires the participation of accessory cells. This requirement for accessory cells can be replaced by supernatant from adherent peritoneal exudate cells that have been stimulated with exotoxin A. Antibody to exotoxin A inhibits the induction of the thymocyte mitogenic activity from adherent peritoneal macrophages. However, antibody to exotoxin A had no effect on the thymocyte proliferation if the antibody was added to supernatant which contained thymocyte mitogenic activity. The thymocyte mitogenic activity was associated with a protein or protein complex with a molecular mass of greater than 10,000 daltons. D10 bioassays indicated the presence of interleukin-1 (IL-1) in the supernatant. Antibody to IL-1 inhibited the ability of supernatant to induce thymocytes to proliferate. Therefore, these data suggest that Pseudomonas exotoxin A can stimulate the production of IL-1 from adherent peritoneal cells, which induces murine thymocytes to proliferate.

Association of uveal melanocyte destruction in melanoma-bearing swine with large granular lymphocyte cells.

Sinclair strain miniature swine spontaneously develop and regress malignant melanoma lesions, with uveitis and vitiligo occurring subsequent to tumor regression. Peripheral blood lymphocytes (PBL) of Sinclair swine undergoing tumor regression and melanocyte destruction demonstrated significant lytic activity against K562, porcine semiallogeneic uveal melanocytes, and melanoma cells in 4-h chromium release assays. The ability of porcine PBL to lyse these target cells appears to be an age-associated immune response, as evidenced by the relative inability of PBL of pigs less than 4 weeks old to lyse target cells. In young adult pigs, however, PBL cytotoxic activity significantly increases; piglets 6 weeks old and older demonstrate efficient killing of all three targets. Conjugate formation assays demonstrate that a lymphoid effector cell possessing large granular lymphocyte (LGL) morphology may be involved in melanocyte destruction. These findings suggest that a LGL subpopulation may participate in melanoma and melanocyte destruction which can induce a uveitic syndrome in Sinclair swine with melanoma.

Host environment as a modulating factor of swine natural killer cell activity.

The large granular lymphocyte (LGL) population includes such heterologous effector cells as the natural killer (NK), lymphokine activated killer (LAK), antibody dependent cellular cytotoxic (ADCC) and non-MHC restricted T cells. These LGL subpopulations have all been associated with NK activity. In some species, enhanced NK activity is correlated with exposure to viral, bacterial and parasitic agents. Consequently, the host environment could serve as a modulatory factor of NK activity in laboratory animals. During our investigation of tumor regression in melanoma swine, we observed marked differences in the NK activity of peripheral blood lymphocytes collected from two separate groups of Sinclair melanoma miniature swine maintained under different conditions. Group A pigs were vaccinated and extensively treated for endo- and ectoparasites while group B swine were not. In addition, chronic exposure to infectious and parasitic diseases have been documented in the group B swine. Peripheral blood NK activity was assessed by standard in vitro 4-h chromium release assays. The NK activity of group B swine was markedly exaggerated when compared to group A swine. Thus, the significance of NK activity may be distorted as a result of the modulating effect of pathogen exposure.

Biological effects of Pseudomonas aeruginosa exotoxin A: lymphoproliferation of T lymphocytes in athymic mice.

Pseudomonas aeruginosa exotoxin A has been observed to exert modulatory effects on the immune response. The present study examines the ability of exotoxin A to induce proliferation of splenocytes from athymic nu/nu mice. We observed that exotoxin A induced the proliferation of athymic nude splenocytes which could be abrogated by heating the toxin at 70 degrees C or by preincubation of the toxin with rabbit anti-exotoxin A antiserum. Photoaffinity-labelled toxin significantly induced splenocyte proliferation although the relative activity was reduced. Maximum nude splenocyte proliferation was observed at a toxin dose of 100 ng. This same dose was shown previously for athymic splenocytes to induce an enhanced response to the thymus-dependent (TD) antigen, sheep red blood cells (SRBC). The increased [3H]-TdR uptake in athymic splenocytes stimulated by exotoxin A was initiated by 24 hours and continued to day 10. Nude splenocytes depleted of Ig+ and Ia+ cells were induced to proliferate by exotoxin A. Cyclosporin A addition abrogated the ability of exotoxin A to induce proliferation. These results suggest that Pseudomonas aeruginosa exotoxin A can stimulate the proliferation of splenic T lymphocytes in athymic nu/nu mice.

Variables which affect suppression of the immune response induced by Pseudomonas aeruginosa exotoxin A.

Pseudomonas exotoxin A has been shown previously to induce suppression of the murine immune response. In the present study, various parameters were examined which may have an effect on immunosuppression. The addition of 10(-4) ng of exotoxin A induced suppression of the immune response to trinitrophenylated Ficoll from days 3 to 10, while 10 ng of toxin exerted no suppressive effect over the same examination periods. When the toxin was administered 1 or 2 days before antigen stimulation, suppression of the response was observed with both 10 and 10(-4) ng. Priming splenocytes with toxin either in vivo or in vitro for 1 or 2 days suppressed the response of fresh cultured splenocytes to antigenic stimulation. Heated toxin, photoaffinity-labeled toxin, or preincubation of the toxin with rabbit anti-exotoxin A antiserum eliminated the toxin-induced suppression. These results suggest that Pseudomonas exotoxin A can generate multiple biological effects.

Induction of an immune response in athymic nude mice to thymus-dependent antigens by Pseudomonas aeruginosa exotoxin A.

Exposure of spleen cells from athymic nude mice to Pseudomonas aeruginosa exotoxin A induces these cells to respond to the thymus-dependent (TD) antigen sheep erythrocytes (SRBC). The response induced by toxin is dose dependent, antigen specific, and not due to polyclonal B-cell activation. Enhancement of the anti-SRBC response can be observed when toxin addition precedes antigen stimulation by 24-48 hr, which decreases when toxin administration follows antigen stimulation. A significant response is also observed when toxin and antigen are added simultaneously. A significant anti-SRBC response can be observed out to Day 10 postantigen and toxin stimulation after attaining a peak response at Day 5. Cultures exposed to toxin in the presence or absence of antigen exhibited a higher cell number and relative number of B cells as compared to control cultures. Exposure of T-cell depleted B cells from euthymic +/nu mice to toxin plus antigen does not result in an anti-SRBC response indicating that exotoxin A alone is not sufficient to induce B-cell responsiveness to T-dependent antigens and that other cells and/or factors are involved in the toxin-induced responsiveness of nude mice to T-dependent antigens.