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If one were to compare today's animal growth research to research from a mere 50 yr ago, one would see programs with few similarities. The evolution of this research from whole-animal through cell-based and finally molecular and genomic studies has been enhanced by the identification, isolation, and in vitro evaluation of adipose- and muscle-derived stem cells. This paper will highlight the struggles and the milestones that make this evolving area of research what it is today. The contribution of adipose and muscle stem cell research to development and growth, tissue regeneration, and final carcass composition are reviewed.

Tumor growth can increase the number of immature bone marrow-derived CD34+ cells that exhibit natural suppressor (NS) activity toward T-cell function. Using a metastatic Lewis lung carcinoma (LLC-LN7) tumor model, these CD34+ NS cells were shown to be present within the s.c. primary tumor tissue, but their levels declined after treatment with the inducer of myeloid cell differentiation, vitamin D3. Therefore, studies determined whether vitamin D3 treatment to diminish the CD34+ NS cell levels in LLC-LN7-bearing mice would enhance (a) intratumoral immune reactivity and (b) the antitumor activity of adoptive therapy consisting of tumor-reactive lymph node cells. The results showed that vitamin D3 treatment alone increased the intratumoral CD8+ cell content and the activity of the intratumoral infiltrate, as detected by production of interferon-gamma and expression of the p55 IL-2 receptor. Although vitamin D3 treatment had no effect on the size of the primary tumor, it lessened the extent of tumor metastasis. Treating mice with the combination of vitamin D3 and adoptive immunotherapy significantly reduced metastasis in mice with established tumors, and reduced both metastasis and locoregional recurrence after surgical excision of the primary tumor. These studies demonstrate that vitamin D3 treatment increases intratumoral T-cell immune reactivity, and that coupling vitamin D3 treatment to diminish levels of CD34+ NS cells with adoptive immunotherapy enhances the effectiveness of the adoptively transferred tumor-reactive lymph node cells at limiting both metastasis and locoregional tumor recurrence.

Patients with squamous cell carcinomas of the head and neck (HNSCC) have profound defects in their immune defenses. We have shown that among the mechanisms that contribute to this immune dysfunction are immune inhibitory CD34+ progenitor cells, whose levels become elevated in the peripheral blood and within the tumor tissue. One goal of our studies is to overcome the immune inhibitory activities of tumor-induced CD34+ progenitor cells by stimulating their differentiation into cells, such as dendritic or monocytic cells, that can stimulate immune reactivity to autologous cancer. Results of in vitro analyses with CD34+ suppressor cells of HNSCC patients and of in vivo studies in animal tumor models have shown the capacity of tumor-induced CD34+ cells to differentiate into cells that phenotypically resemble monocytic or dendritic cells. Whether these cells can differentiate into dendritic cells in HNSCC patients is currently being tested. Less clear is whether the pathway by which the tumor-induced CD34+ cells differentiate will result in cells having the full capacity to function as potent stimulators of immune reactivity to autologous tumor.

Progressive growth of metastatic Lewis lung carcinoma (LLC-LN7) tumors is associated with increased levels of bone-marrow-derived CD34+ cells having natural suppressor (NS) activity toward T cells. The present studies determined whether tumor-derived products are responsible for this induction of NS activity. Culturing normal bone marrow cells with LLC-LN7-conditioned medium (LLC-CM) or with recombinant granulocyte/macrophage-colony-stimulating factor (GM-CSF) resulted in the appearance of NS activity. The development of NS activity coincided with a prominent increase in the levels of CD34+ cells. That the CD34+ cells were responsible for the NS activity of the bone marrow cultures containing LLC-CM was shown by the loss of NS activity when CD34+ cells were depleted. The stimulation of CD34+ NS cells by LLC-CM was attributed to tumor production of GM-CSF, since neutralization of GM-CSF within the LLC-CM reduced its capacity to increase CD34+ cell levels. Studies also showed that the induction of CD34+ NS cells by LLC-CM and GM-CSF could be overcome by including in the cultures an inducer of myeloid differentiation, 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]. These results demonstrate that the mechanism by which the LLC-LN7 tumors stimulate increased levels of CD34+ NS cells from normal bone marrow is by their production of GM-CSF and that this can be blocked with the myeloid differentiation inducer 1,25(OH)2D3.

The sterol 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] can inhibit T cell activation as well as restore the functional competence of suppressed T cells, The present studies determined whether 1,25(OH)2D3 had a differential effect on the activation of normal T cells or of suppressed T cells from mice bearing Lewis lung carcinoma tumors. Normal spleen cell proliferation in response to immobilized anti-CD3 was unaffected by the lower doses of 0.1-10 nM 1,25(OH)2D3, and was inhibited by the higher dose of 100 nM 1,25(OH)2D3. In contrast, 1,25(OH)2D3 increased proliferation and interferon gamma secretion by T cells of tumor bearers in response to stimulation through T cell receptor/CD3. Assessment of mechanisms associated with the 1,25(OH)2D3 stimulation of tumor-bearer T cells implicated protein phosphatase 2A (PP-2A). First, PP-2A activity of spleen cells from tumor bearers was reduced compared to that of normal spleen cells but was increased by 1,25(OH)2D3. Second, 1,25(OH)2D3 stimulation of tumor-bearer T cell proliferation was dependent on this PP-2A activity as it was blocked by doses of okadaic acid that selectively inhibit PP-2A. These results suggest that 1,25(OH)2D3 preferentially enhances the responsiveness of immunosuppressed T cells from tumor bearers to TCR/CD3 stimulation by restoring PP-2A activity.

The cytokine interleukin-6 (IL-6) has been implicated as a contributor to injury in several neurological disorders. The amounts of IL-6 released by the cerebral cortical tissue of mice of varying maturational age groups were measured and found to increase with age. Specifically, the basal level of IL-6 released from the tissue of infant (less than 2 weeks old) mice was low, although the tissue could be readily stimulated to secrete high levels of IL-6. Higher levels of IL-6 were released from young adult (2 month old) mice. Adult mice that were either 6 or 9 months of age secreted significantly higher levels of IL-6 compared to the tissue of either the young adult or infant animals, although the difference between levels secreted by the 6- or 9-month old groups was minimal. IL-6 production by adult cerebral cortical tissue could be further stimulated, but it was less readily achieved compared to the tissue of infants. In response to the negative regulator transforming growth factor-beta (TGF-beta), the levels of IL-6 released by stimulated cerebral cortical tissue of infants, as well as by unstimulated and stimulated tissue of adults, were reduced to the low basal levels of IL-6 produced by infant tissue. These results suggest that normal development and aging are correlated with an increase in IL-6 production that may be due to shifts in levels of stimulatory or inhibitory regulatory controls, but not to an inability of young tissue to produce IL-6 or to a lack of responsiveness of adult tissue to negative regulatory control by TGF-beta.

Lewis lung carcinoma (LLC-LN7) tumors stimulate myelopoiesis and increase the presence of granulocyte/macrophage (GM) progenitor cells having natural suppressor activity. Treatment of these tumor-bearing mice with interleukin-12 (IL-12) resulted in minimal immune modulation. The objective of this study was to determine whether eliminating natural suppressor activity would allow for immune stimulation by IL-12. Treatment of LLC-LN7 tumor-bearing mice with vitamin D3 eliminated natural suppressor activity. In mice that were first treated with vitamin D3 and then also with IL-12, there was stimulation of splenic T cell proliferation in response to immobilized anti-CD3 plus IL-2. In addition, spleen and lymph node cells from vitamin-D3/IL-12-treated tumor-bearing mice became stimulated in response to autologous tumor to produce interferon gamma (IFN gamma), although IL-2 production was not stimulated. A prominent effect of the combined vitamin-D3/IL-12 treatment regimen was the synergistic augmentation of autologous tumor-specific cytolytic activity within the regional lymph nodes. The generation of these tumor-specific effector cells required the presence of the tumor mass since such activity was not elicited in the lymph nodes of mice from which the tumors had been surgically excised. The results of this study show that, after treatment of tumor bearers with vitamin D3 to eliminate GM-suppressor cells, IL-12 can induce select regional antitumor immune responses, particularly IFN gamma production and cytolysis by regional lymph node cells of autologous tumor.

Studies in both cancer patients and in animal tumor models have shown that immune defenses can mediate destruction of tumor, but these defenses are often functioning at a suppressed or suboptimal level. Frequently, prostaglandins, mainly PGE2, have been implicated in this tumor-associated subversion of immune function, with immune reactivities to tumor typically being enhanced by prostaglandin synthesis inhibitor. Both the tumor and tumor-induced host immune suppressive macrophages have the capacity to suppress immune functions through their production of PGE2. Although the inhibitory functions have been more widely studied, recent evaluations of the effects of PGE2 have led to the surprising realization that not all of the PGE2's effects are inhibitory to immune function. Summarized below are some of the well characterized inhibitory effects of PGE2, as well as the lesser studied stimulatory effects of PGE2 toward the effector cells that are considered to be important in the immune defense against cancer.

By secreting granulocyte/macrophage colony-stimulating factor (GM-CSF), metastatic Lewis lung carcinoma (LLC-LN7) tumors induce the appearance of myelopoiesis-associated immune-suppressor cells that resemble granulocytic-macrophage (GM) progenitor cells. The presence of these GM-suppressor cells in mice bearing LLC-LN7 tumors was associated with a reduced capacity of splenic T cells to proliferate in response to interleukin-2 (IL-2). Administration of low doses of 100 U interferon gamma (IFN gamma) plus 10 U tumor necrosis factor alpha (TNF alpha) to the tumor bearers, a combination treatment that we previously showed to diminish the presence of GM-suppressor cells synergistically, restored proliferative responsiveness of the splenic T cells to IL-2. These LLC-LN7-bearing mice were also examined for whether cells that phenotypically resemble GM-progenitor cells (ER-MP12+ cells) infiltrate the tumor mass. ER-MP12+ cells composed approximately 10% of the cells isolated from dissociated tumors of mice that had been treated with placebo or with either IFN gamma or TNF alpha alone, but IFN gamma/TNF alpha therapy markedly reduced the number of tumor-infiltrating ER-MP12+ suppressor cells. The IFN gamma/TNF alpha treatment to eliminate GM-suppressor cells and restore T cell responsiveness to IL-2 was next coupled with low dose IL-2 therapy (100 U twice daily). Addition of IL-2 to the treatment regimen did not significantly influence the effectiveness of the IFN gamma/TNF alpha treatment in eliminating GM-suppressor cells from the LLC-LN7 tumor mass. However, inclusion of IL-2 with the IFN gamma/TNF alpha treatment regimen enhanced the CD8+, but not the CD4+, cell content within the tumor, and diminished the number of metastatic lung nodules within the mice. When these tumors were excised, dissociated, and bulk-cultured with a low dose of IL-2, an increased level of cytotoxic T lymphocyte (CTL) activity was generated in the TIL cultures from mice that had received IFN gamma/TNF alpha plus IL-2 treatments. A lesser but detectable level of CTL activity was generated in TIL cultures from mice that were treated with only IFN gamma/TNF alpha, while no CTL activity was generated in tumor cultures from mice receiving only placebo or low-dose IL-2. These results suggest the effectiveness of IFN gamma plus TNF alpha therapy in restoring IL-2 responsiveness in mice bearing GM-suppressor cell-inducing tumors and at enhancing both the intratumoral CD8+ cell content and the generation of CTL activity in bulk cultures of these tumors.

Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) by metastatic Lewis lung carcinoma cells (LLC-LN7) was previously shown to contribute to the maintenance of phenotypic characteristics associated with an increased capacity to metastasize. In the present study, pre-incubation of LLC-LN7 cells with neutralizing anti-GM-CSF antibodies diminished the capacity of the tumor cells to form experimental metastases after i.v. inoculation, while pre-incubation with recombinant GM-CSF (rGM-CSF) increased formation of metastases. In the presence of rGM-CSF, the LLC-LN7 cells exhibited an increased capacity to migrate, invade through a reconstituted basement membrane, and adhere to lung tissue. Studies to identify the signal transduction pathway through which GM-CSF enhanced the in vitro metastatic properties of the LLC-LN7 tumor cells implicated protein kinase A (PKA). Signaling through PKA was suggested by the demonstration that the stimulation of tumor-cell motility by GM-CSF was blocked in the presence of the adenylate cyclase inhibitor nicotinic acid, or the PKA inhibitors A3 or KT5720. In addition, the role of PKA as a signaling mechanism for GM-CSF was assessed by using REV-LN7 cells, which are LLC-LN7 cells that have been stably transfected with an expression vector encoding a mutant PKA RI alpha subunit and which, in turn, express a cAMP-resistant PKA. Adherence and invasion by the PKA-defective REV-LN7 cells were not stimulated by rGM-CSF, contrasting with the stimulation observed for wild-type LLC-LN7 cells. These data suggest that rGM-CSF can further enhance the in vitro metastatic characteristics of LLC-LN7 tumor cells and that this is dependent on signal transduction through PKA.

Tumor presence is detrimental to the development of antigen-presenting dendritic cells. Since dendritic cells can arise from CD34+ precursor cells, the present study assessed the capacity of bone marrow CD34+ cells from tumor bearers to develop into dendritic cells when cultured in the absence of either tumor cells or their products. Culturing bone marrow CD34+ cells from mice bearing Lewis lung carcinomas yielded a lower number of dendritic cells than arose from CD34+ cells of normal mice. This reduced yield of dendritic cells was associated with a shift to development of monocytic cells and a reduced antigen presenting capability by the cultures. When the CD34+ cell cultures from tumor bearers were supplemented with the differentiation-inducing hormone 1alpha,25-dihydroxyvitamin D3, there was the restoration of dendritic cell development and antigen presenting ability. These results show that CD34+ cells from tumor bearers remain defective in their development into dendritic cells even when cultured outside the tumor environment, but development of dendritic cells can be restored with 1alpha,25-dihydroxyvitamin D3.

Objective: To test whether irrigation during bipolar cautery confers thermoprotection from neuronal injury. Design: A rat animal model (15 rats for each treatment group) was used to test the thermoprotective effects of irrigation during bipolar cautery. In this model, the sciatic nerve was exposed, and a 1-second stimulus was applied using bipolar cautery forceps at 40 or 20 W placed directly on the nerve in the presence or absence of simultaneous irrigation. The effects of cautery were determined on the basis of clinical gait analysis by means of the Sciatic Functional Index, temperature response, and neuropathological findings. Results: The degree of paresis was reduced with irrigation. Neuropathological examination of the sciatic nerve after cautery showed significant axonal loss (more small than large fibers) with concomitant demyelination, which was partially inhibited by irrigation (x 2 ; P = .04). The mechanism of thermoprotection by irrigation was not the result of a reduction in the temperature spike that followed cautery, but resulted from a reduced temperature response during the 15 seconds that followed 40- or 20-W stimulation with bipolar cautery. Conclusions: Simultaneous irrigation and bipolar cautery enhance temperature recovery to basal levels and protect the peripheral nerve from the effects of cautery. Arch Otolaryngol Head Neck Surg. 2000;126:149-153

Metastatic Lewis lung carcinoma (LLC-LN7) tumors that secrete granulocyte/macrophage-colony-stimulating factor (GM-CSF) stimulate myelopoiesis and induce bone marrow-derived immunosuppressor cells that are homologous to granulocyte/macrophage progenitor cells. In vitro treatment of the LLC-LN7 cells with 1 alpha,25-dihydroxyvitamin D3 reduced tumor cell production of suppressor-inducing activity, although suppressor-inducing activity could be restored by reconstituting the tumor supernatants with recombinant GM-CSF. Treatment of mice having LLC-LN7 tumors with vitamin D3 reduced tumor production of GM-CSF and the frequency of myeloid progenitor cells. This was associated with a reduction in immunosuppressor activity and an increase in T cell function. Vitamin D3 treatment of mice having palpable tumors transiently retarded tumor growth, but caused a prominent reduction in tumor metastasis. Treating mice with vitamin D3 after tumor excision resulted in a reduction in the tumor-induced myelopoietic stimulation and associated immunosuppressive activity, and enhanced T cell function. These mice had a markedly reduced incidence of tumor recurrence. The results of this study suggest that vitamin D3 treatment of mice with GM-CSF-secreting tumors can interrupt the myelopoiesis-associated immunosuppressor cascade and, in turn, reduce tumor metastasis and recurrence.

Normal bone marrow cells become immunosuppressive when cultured with supernatants of metastatic Lewis lung carcinoma (LLC-LN7) cells. The suppressor-inducing activities in the LLC-LN7 supernatants are interleukin-3 and granulocyte/macrophage-colony-stimulating factor. In the present study, the mechanisms by which these induced suppressor cells (LLCsup-BM) mediate their immunosuppression were investigated. The suppression by LLCsup-BM of splenic concanavalin CA blastogenesis was not dependent on cell contact since immunosuppression occurred regardless of whether the LLCsup-BM were separated from the responder spleen cells by a permeable membrane or if the LLCsup-BM were cocultured with the spleen cells. Culture supernatants of LLCsup-BM also inhibited T cell blastogenesis, being more suppressive than were supernatants of control bone marrow cells, which had been precultured with medium. The suppression by the soluble inhibitors elaborated from the LLCsup-BM was not restricted to the inhibition of T cell function as the supernatants also inhibited the natural killer activity of normal spleen cells. Studies to determine the identity of the suppressive activity produced by the LLCsup-BM showed increased levels of transforming growth factor beta (TGF beta) in their supernatants. Immunosuppressive bone marrow and spleen cells obtained from mice bearing metastatic LLC-LN7 tumors also secreted more TGF beta than did the cells obtained from normal mice. When anti-TGF beta antibodies were added to the LLCsup-BM supernatants, the suppressive activity was diminished. These results suggest that the LLCsup-BM mediate at least part of their immunosuppression through production of TGF beta.

The modulatory effects of serotonin on T-cell activity were investigated. T-cell blastogenesis of normal spleen cells was slightly stimulated by the addition of low doses (1 and 10 ng/ml) of the inducer of serotonin release, fenfluramine. In contrast to the stimulatory effects of low doses of fenfluramine, high doses of fenfluramine (1 and 10 ug/ml) or of exogenously added serotonin (> or = 0.1 ug/ml) inhibited T-cell activation. Both the stimulation by low dose fenfluramine and the inhibition by high dose fenfluramine were accentuated by pretreating mice with tryptophan to heighten intracellular stores of serotonin and then inducing serotonin release. Pretreatment of mice with the serotonin inhibitor p-chlorophenylalanine (PCPA) abolished the fenfluramine inhibition of T-cell activation indicating that the fenfluramine inhibitory effect was mediated via endogenous spleen cell-derived serotonin. However, the PCPA treatment diminished T-cell activation. These results suggest that endogenous serotonin causes a biphasic dose-response effect on T-cell activity with serotonin being required for optimal T-cell function, low doses being immune stimulatory and higher doses being suppressive.

The spontaneous tumoricidal abilities of alveolar and peritoneal macrophages from C57B1/6 mice bearing a metastatic or a nonmetastatic cloned variant of Lewis lung carcinoma (LLC) were measured. Cytotoxicity by alveolar macrophages was enhanced during the first few weeks after subcutaneous (s.c.) or intravenous (i.v.) injection of metastatic LLC-C3 cells, but not after injection of nonmetastatic LLC-C8 cells. Alveolar macrophages from mice with s.c.-lnjected metastatic tumors, but not with nonmetastatic tumors, could be further activated in vitro, but not beyond the maximal level of spontaneous cytotoxicity. Late in tumor growth, the spontaneous cytotoxicity by alveolar macrophages of metastatic LLC-C3 tumor bearers was suppressed and could not be increased by in vitro activation. The tumoricidal abilities of peritoneal macrophages from mice bearing either LLC-C3 or LLC-C8 tumors were modulated in a similar way, as were alveolar macrophages. The reduced cytotoxicity by alveolar macrophages from mice with nonmetastatic tumors or from mice bearing large metastatic tumors was not due to suppression by macrophage-derived prostaglandins. The loss of tumoricidal capabilities by macrophages from mice with large metastatic LLC-C3 tumors was not caused by elevated systemic prostaglandin E2 (PGE2) levels. These results suggest that alveolar and peritoneal macrophages are activated to be cytotoxic during development of pulmonary metastases and do not need to be functionally depressed for successful establishment of metastases.